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1
Aref, Zeen, and Paul H. A. Quax. "In Vivo Matrigel Plug Assay as a Potent Method to Investigate Specific Individual Contribution of Angiogenesis to Blood Flow Recovery in Mice." International Journal of Molecular Sciences 22, no. 16 (August 18, 2021): 8909. http://dx.doi.org/10.3390/ijms22168909.
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Neovascularization restores blood flow recovery after ischemia in peripheral arterial disease. The main two components of neovascularization are angiogenesis and arteriogenesis. Both of these processes contribute to functional improvements of blood flow after occlusion. However, discriminating between the specific contribution of each process is difficult. A frequently used model for investigating neovascularization is the murine hind limb ischemia model (HLI). With this model, it is difficult to determine the role of angiogenesis, because usually the timing for the sacrifice of the mice is chosen to be optimal for the analysis of arteriogenesis. More importantly, the occurring angiogenesis in the distal calf muscles is probably affected by the proximally occurring arteriogenesis. Therefore, to understand and subsequently intervene in the process of angiogenesis, a model is needed which investigates angiogenesis without the influence of arteriogenesis. In this study we evaluated the in vivo Matrigel plug assay in genetic deficient mice to investigate angiogenesis. Mice deficient for interferon regulatory factor (IRF)3, IRF7, RadioProtective 105 (RP105), Chemokine CC receptor CCR7, and p300/CBP-associated factor (PCAF) underwent the in vivo Matrigel model. Histological analysis of the Matrigel plugs showed an increased angiogenesis in mice deficient of IRF3, IRF7, and RP105, and a decreased angiogenesis in PCAF deficient mice. Our results also suggest an involvement of CCR7 in angiogenesis. Comparing our results with results of the HLI model found in the literature suggests that the in vivo Matrigel plug assay is superior in evaluating the angiogenic response after ischemia.
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Meng, Shu, Jie Lv, Palas K. Chanda, Iris Owusu, Kaifu Chen, and John P. Cooke. "Reservoir of Fibroblasts Promotes Recovery From Limb Ischemia." Circulation 142, no. 17 (October 27, 2020): 1647–62. http://dx.doi.org/10.1161/circulationaha.120.046872.
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Background: The angiogenic response to ischemia restores perfusion so as to preserve tissue. A role for mesenchymal-to-endothelial transition in the angiogenic response is controversial. This study is to determine if resident fibroblasts contribute to angiogenesis. Methods: We utilized the murine model of hindlimb ischemia, and in vivo Matrigel plug assay together with lineage tracing studies and single cell RNA-sequencing to examine the transcriptional and functional changes in fibroblasts in response to ischemia. Results: Lineage tracing using Fsp1-Cre: R26R-EYFP mice revealed the emergence within the ischemic hindlimb of a small subset of YFP + CD144 + CD11b − fibroblasts (E* cells) that expressed endothelial cell (EC) genes. Subcutaneous administration of Matrigel in Fsp1-Cre: R26R-EYFP mice generated a plug that became vascularized within 5 days. Isolation of YFP + CD11b - cells from the plug revealed a small subset of YFP + CD144 + CD11b − E* cells which expressed EC genes. Pharmacological or genetic suppression of innate immune signaling reduced vascularity of the Matrigel plug and abrogated the generation of these E* cells. These studies were repeated using human fibroblasts, with fluorescence-activated cell sorting analysis revealing that a small percentage of human fibroblasts that were induced to express EC markers in Matrigel plug assay. Pharmacological suppression or genetic knockout of inflammatory signaling abolished the generation of E* cells, impaired perfusion recovery and increased tissue injury after femoral artery ligation. To further characterize these E* cells, single cell RNA-sequencing studies were performed and revealed 8 discrete clusters of cells expressing characteristic fibroblast genes, of which 2 clusters (C5 and C8) also expressed some EC genes. Ischemia of the hindlimb induced expansion of clusters C5 and C8. The C8 cells did not express CD144, nor did they form networks in Matrigel, but did generate angiogenic cytokines. The C5 fibroblasts most resembled E* cells in their expression of CD144 and their ability to form EC-like networks in Matrigel. Conclusions: Together, these studies indicate the presence of subsets of tissue fibroblasts which seem poised to contribute to the angiogenic response. The expansion of these subsets with ischemia is dependent on activation of innate immune signaling and contributes to recovery of perfusion and preservation of ischemic tissue.
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Kwon, Oh Sung, Myoung Seok Jeong, Bonglee Kim, and Sung-Hoon Kim. "Antiangiogenic Effect of Ethanol Extract ofVigna angularisvia Inhibition of Phosphorylation of VEGFR2, Erk, and Akt." Evidence-Based Complementary and Alternative Medicine 2015 (2015): 1–9. http://dx.doi.org/10.1155/2015/371368.
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Though dietary azuki bean (Vigna angularis) seed containing antioxidant proanthocyanidins was known to have multibiological activities including antioxidant, hypotensive, anti-inflammatory, and immunomodulatory activities, the antiangiogenic activity of ethanol extract ofVigna angularis(EVA) was never reported so far. In the present study, the antiangiogenic mechanism of EVA was examined in human umbilical vein endothelial cells (HUVECs). EVA showed weak cytotoxicity in HUVECs, while it significantly suppressed the VEGF induced proliferation of HUVECs. Consistently, wound healing assay revealed that EVA inhibited the VEGF induced migration of HUVECs. Also, EVA abrogated the VEGF induced tube formation of HUVECs in a concentration dependent fashion. Furthermore, Matrigel plug assay showed that EVA significantly reduced the hemoglobin level of Matrigel plug in mice compared to untreated control. Of note, EVA effectively attenuated the phosphorylation of VEGFR2, Erk, and Akt in VEGF-treated HUVECs. Overall, our findings suggest that EVA inhibits angiogenesis in VEGF-treated HUVECs via inhibition of phosphorylation of VEGFR2, ERK, and Akt.
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Coltrini, Daniela, Emanuela Di Salle, Roberto Ronca, Mirella Belleri, Chiara Testini, and Marco Presta. "Matrigel plug assay: evaluation of the angiogenic response by reverse transcription-quantitative PCR." Angiogenesis 16, no. 2 (November 11, 2012): 469–77. http://dx.doi.org/10.1007/s10456-012-9324-7.
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Lopes, Fláavia Cristine Mascia, Raquel Costa, Sara T. Olalla Saad, Raquel Soares, Fernando Ferreira Costa, and Nicola Conran. "Proangiogenic Effects of Plasma From Sickle Cell Disease Patients and Antiangiogenic Effects of Hydroxyurea: Evaluation of Invasion and Proliferation of Human Endothelial Cells and Effects of Hydroxyurea in a Mouse Matrigel Plug Neovascularization Assay." Blood 120, no. 21 (November 16, 2012): 377. http://dx.doi.org/10.1182/blood.v120.21.377.377.
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Abstract Abstract 377 Sickle Cell Disease (SCD) results from mutations in the β hemoglobin chain and is associated with a complex pathophysiology that often involves recurrent vasoocclusion in association with vascular inflammation, oxidative stress, ischemia-reperfusion injury and endothelial activation. Dysregulation of angiogenesis occurs in various pathologies and a newly recognized proangiogenic state is associated with SCD. Central to the process of angiogenesis are the endothelial cells (EC) that are activated by angiogenic signals and release degrading enzymes that allow EC to migrate, proliferate and finally differentiate to form new vessels. In keeping with this proposed proangiogenic state, we previously found that plasma from SCD patients increases human umbilical vein endothelial cell (HUVEC) tube formation on Matrigel. In contrast, plasma from SCD patients treated with hydroxyurea (HU; a drug that has been used successfully for SCD therapy) inhibited the ability of HUVEC to form branching and thick anastomosing capillaries in the same model. In the present study, we aimed to determine the effects of plasma from SCD patients on additional HUVEC functions associated with key steps of angiogenesis, including invasion and proliferation, as well as further evaluating the direct antiangiogenic effects of HU with a mouse Matrigel plug neovascularization assay. Steady-state HbSS (SS), steady-state HbSS treated with HU (SSHU, 20–30mg/kg/day HU) and healthy control individuals (CON) were recruited for the study. Invasive cell behavior in the presence of 10 % plasma was quantified in vitro using a double-chamber assay. Under the same conditions, cell proliferation analyses were carried out using cellular incorporation of bromodeoxyuridine. Interestingly, an effective increase of 39.66 ± 29.54% in EC invasion was observed in the presence of SS plasma (P<0.05, N=5) compared to basal cell invasion. In contrast, SSHU plasma resulted in a significant decrease in cell invasive ability (51.94 ± 7.82% reduction; N=5, P<0.001). Accordingly, when the proliferative activity of SS plasma was investigated, increased cell proliferation was observed (13.73 ± 3.41%, compared to basal EC proliferation; N=5, P<0.05). Plasma from SSHU individuals significantly reduced HUVEC proliferation by 24.90 ± 3.45% (P<0.0, N=5). In contrast, CON plasma did not modify either the invasive or proliferative activities of HUVECs. For the Matrigel plug assay, C57BL/6 mice received subcutaneous Matrigel plugs supplemented, or not, with 100μM HU in the presence or absence of vascular endothelial growth factor (VEGF). After seven days, the plugs were removed and Matrigel hemoglobin content measured, using Drabkin's method. The positive control group (VEGF) presented extensive neovascularization of the Matrigel, as shown by the red color distributed in the whole plug. In contrast, Matrigel implants treated with both VEGF and HU demonstrated a strong inhibition of vascular development (67.53 ± 6.68% reduction in neovascularization; N=6, P<0.05) that was similar to that of negative controls (Matrigel not treated with VEGF). Data presented herein show that important features of the angiogenic process, endothelial cell invasion and proliferation, can be upregulated by plasma from SCD patients, confirming the apparent proangiogenic status of these individuals. In contrast, plasma from patients treated with HU exerted antiangiogenic effects by inhibiting the same angiogenic steps. Furthermore, HU was found to have direct antiangiogenic effects in in vivo assays. To our knowledge, this is the first report of the antiangiogenic activity of HU in a mouse model. Balancing angiogenesis is essential for SCD individuals, as enhanced angiogenesis may increase the incidence of manifestations such as proliferative retinopathy and pulmonary hypertension. On the other hand, angiogenesis is essential for mechanisms such as ulcer recovery, neovascularization of ischemic tissues and tissue regeneration and it may be that HU therapy may retard such processes. In conclusion, this study finds further evidence for a proangiogenic state in SCD. HU inhibits key steps in angiogenic mechanisms, demonstrating a possible use for this drug in the treatment of pathological angiogenesis in this and other diseases. Disclosures: No relevant conflicts of interest to declare.
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Cattaneo, Maria Grazia, Sandra Pola, Valeria Dehò, Anna Maria Sanguini, and Lucia Maria Vicentini. "Alprostadil suppresses angiogenesis in vitro and in vivo in the murine Matrigel plug assay." British Journal of Pharmacology 138, no. 2 (January 2003): 377–85. http://dx.doi.org/10.1038/sj.bjp.0705051.
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M, Thiruselvi, and Brindha Durairaj. "IN VITRO AND IN VIVO ANTIANGIOGENIC EFFECT OF ARTOCARPUS HETEROPHYLLUS SEED EXTRACT." Asian Journal of Pharmaceutical and Clinical Research 11, no. 9 (September 7, 2018): 268. http://dx.doi.org/10.22159/ajpcr.2018.v11i9.27488.
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Objective: Angiogenesis the formation of new blood vessels from the pre-existing vasculature plays a major role in tumor growth, invasion, and metastasis of cancer diseases. The current research was designed for the inhibition of angiogenesis, which can provide a novel way to inhibit tumor growth and metastasis in cancer.Methods: The antiangiogenic properties of serial concentrations of the hydroethanolic extract of Artocarpus heterophyllus were examined in human umbilical vein endothelial cells (HUVECs) using a tube formation assay in vitro and in a Matrigel plug assay as in vivo model.Results: Hydroethanolic extract of A. heterophyllus significantly inhibited vascular endothelial growth factor (VEGF)-mediated angiogenesis in the HUVECs in culture dose-dependently. Further, the new blood vessel formation was observed to be inhibited by the extract at 100 mg/kg p.o. in Matrigel plug model in C57BL/6 mice. However, the effect was enhanced in higher concentration (500 mg/kg p.o.) demonstrating the in vivo antiangiogenic activity of the extract.Conclusion: This study demonstrated that the hydroethanolic seed extract of A. heterophyllus strongly inhibited the angiogenesis in HUVECs. Moreover, the extract significantly inhibited the VEGF production in HUVECs, confirming their possible antiangiogenic mechanism.
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Cheranov, Sergey Y., Dong Wang, Venkatesh Kundumani-Sridharan, Manjula Karpurapu, Qiuhua Zhang, Koteswara R. Chava, and Gadiparthi N. Rao. "The 15(S)-hydroxyeicosatetraenoic acid-induced angiogenesis requires Janus kinase 2-signal transducer and activator of transcription-5B–dependent expression of interleukin-8." Blood 113, no. 23 (June 4, 2009): 6023–33. http://dx.doi.org/10.1182/blood-2008-10-183210.
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Abstract To understand the molecular basis underlying 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE)–induced angiogenesis, we have studied the role of the Janus kinase-signal transducer and activator of transcription (Jak-STAT) signaling. The 15(S)-HETE stimulated tyrosine phosphorylation of Jak2 in a time-dependent manner in human retinal microvascular endothelial cells (HRMVECs). Inhibition of Jak2 activation via adenovirus-mediated expression of its dominant-negative mutant attenuated 15(S)-HETE–induced HRMVEC migration and tube formation and Matrigel plug angiogenesis. Similarly, 15(S)-HETE activated tyrosine phosphorylation of STAT-5B in a time-dependent manner. Dominant-negative mutant-mediated interference of STAT-5B activation suppressed 15(S)-HETE–induced HRMVEC migration and tube formation and Matrigel plug angiogenesis. The 15(S)-HETE induced interleukin-8 (IL-8) expression in Jak2-STAT-5B–dependent manner in HRMVECs. In addition, neutralizing anti–IL-8 antibodies reduced 15(S)-HETE–induced HRMVEC migration and tube formation and Matrigel plug angiogenesis. Cloning and Transfac analysis of IL-8 promoter revealed the presence of 1 putative STAT-binding sequence at −476 nt, and electrophoretic mobility shift assay and chromatin immunoprecipitation analysis showed the binding of STAT-5B to this site in response to 15(S)-HETE. Mutational analysis showed that STAT binding site is essential for 15(S)-HETE–induced IL-8 promoter activity. Together, these observations suggest that 15(S)-HETE–induced angiogenesis requires Jak2-STAT-5B–dependent expression of IL-8.
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Sun, Cheng, Shi-Bin Feng, Zheng-Wang Cao, Jun-Jie Bei, Qiang Chen, Xian-Jie Xu, Zhou Zhou, Zheng-Ping Yu, and Hou-Yuan Hu. "Up-Regulated Expression of Matrix Metalloproteinases in Endothelial Cells Mediates Platelet Microvesicle-Induced Angiogenesis." Cellular Physiology and Biochemistry 41, no. 6 (2017): 2319–32. http://dx.doi.org/10.1159/000475651.
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Background/Aims: Platelet microvesicles (PMVs) contribute to angiogenesis and vasculogenesis, but the mechanisms underlying these contributions have not been fully elucidated. In the present study, we investigated whether PMVs regulate the angiogenic properties of endothelial cells (ECs) via mechanisms extending beyond the transport of angiogenic regulators from platelets. Methods: In vitro Matrigel tube formation assay and in vivo Matrigel plug assay were used to evaluate the pro-angiogenic activity of PMVs. The effects of PMVs on the migration of human umbilical vein endothelial cells (HUVECs) were detected by transwell assay and wound-healing assay. Real-time PCR and western blot were conducted to examine mRNA and protein expression of pro-angiogenic factors in HUVECs. Matrix metalloproteinase (MMP) activity was assayed by gelatin zymography. Moreover, the effects of specific MMP inhibitors were tested. Results: PMVs promoted HUVEC capillary-like network formation in a dose-dependent manner. Meanwhile, PMVs dose-dependently facilitated HUVEC migration. Levels of MMP-2 and MMP-9 expression and activity were up-regulated in HUVECs stimulated with PMVs. Inhibition of MMPs decreased their pro-angiogenic and pro-migratory effects on HUVECs. Moreover, we confirmed the pro-angiogenic activity of PMVs in vivo in mice with subcutaneous implantation of Matrigel, and demonstrated that blockade of MMPs attenuated PMV-induced angiogenesis. Conclusion: The findings of our study indicate that PMVs promote angiogenesis by up-regulating MMP expression in ECs via mechanism extending beyond the direct delivery of angiogenic factors.
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Miyake, Makito, Steve Goodison, Evan Gomes, Wasia Rizwani, Shanti Ross, Ge Zhang, and Charles Joel Rosser. "Induction of endothelial proliferation and angiogenesis through activating the ERK1/2/EGF pathway mediate by CXC chemokine receptor 2 by chemokine (C-X-C motif) ligand 1." Journal of Clinical Oncology 31, no. 6_suppl (February 20, 2013): 138. http://dx.doi.org/10.1200/jco.2013.31.6_suppl.138.
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138 Background: Endothelial cell growth and proliferation are critical for tumoral angiogenesis. We report here that blockade of Chemokine (C-X-C motif) ligand 1 (CXCL1) results in reduction of human endothelial cell proliferation and its ability to induce angiogenesis. Methods: Two human endothelial cell lines, HUVEC and HDMEC, were used in the in vitro assays. Proliferation assay and matrigel tube formation assay were performed to test the inhibitory effect of anti-CXCL antibody on the activity of endothelial cells in vitro. Matrigel plug assay in nude mice was performed to test the in vivo angiogenic activity of CXCL1. Results: CXCL1 interacts with its receptor CXC chemokine Receptor 2 and induces endothelial cell proliferation, whereas blockade of CXCL1 is associated with reduction in cellular proliferation through a decrease in levels of cyclin D and cdk4 and inhibition of angiogenesis through EGF and ERK 1/2. Targeting CXCL1 inhibits neoangiogenesis but has no effect on disrupting established vasculature. Furthermore targeting CXCL1 is associated with reduction in migration of human endothelial cells in an in vitro model. Additionally, neutralizing antibody against CXCL1 in a xenograft angiogenesis model resulted in inhibition of angiogenesis. Conclusions: CXCL1-induced regulation of angiogenesis has not been studied extensively in human cancers, thus these findings illustrate a novel contribution of CXCL1 interactions in pathological angiogenesis. Therefore, the ability to selectively modulate CXCL1, specifically in tumoral angiogenesis, may promote the development of novel oncologic therapeutic strategies.
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Kato, Makoto, Shin Tsunekawa, Nobuhisa Nakamura, Emiri Miura-Yura, Yuichiro Yamada, Yusuke Hayashi, Hiromi Nakai-Shimoda, et al. "Secreted Factors from Stem Cells of Human Exfoliated Deciduous Teeth Directly Activate Endothelial Cells to Promote All Processes of Angiogenesis." Cells 9, no. 11 (October 31, 2020): 2385. http://dx.doi.org/10.3390/cells9112385.
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Diabetes is a major risk factor for atherosclerosis and ischemic vascular diseases. Recently, regenerative medicine is expected to be a novel therapy for ischemic diseases. Our previous studies have reported that transplantation of stem cells promoted therapeutic angiogenesis for diabetic neuropathy and ischemic vascular disease in a paracrine manner, but the precise mechanism is unclear. Therefore, we examined whether secreted factors from stem cells had direct beneficial effects on endothelial cells to promote angiogenesis. The soluble factors were collected as conditioned medium (CM) 48 h after culturing stem cells from human exfoliated deciduous teeth (SHED) in serum-free DMEM. SHED-CM significantly increased cell viability of human umbilical vein endothelial cells (HUVECs) in MTT assays and accelerated HUVECs migration in wound healing and Boyden chamber assays. In a Matrigel plug assay of mice, the migrated number of primary endothelial cells was markedly increased in the plug containing SHED-CM or SHED suspension. SHED-CM induced complex tubular structures of HUVECs in a tube formation assay. Furthermore, SHED-CM significantly increased neovascularization from the primary rat aorta, indicating that SHED-CM stimulated primary endothelial cells to promote comprehensive angiogenesis processes. The angiogenic effects of SHED-CM were the same or greater than the effective concentration of VEGF. In conclusion, SHED-CM directly stimulates vascular endothelial cells to promote angiogenesis and is promising for future clinical application.
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Nam, Hyun, Gee-Hye Kim, Yoon-Kyung Bae, Da-Eun Jeong, Kyeung-Min Joo, Kyunghoon Lee, and Sun-Ho Lee. "Angiogenic Capacity of Dental Pulp Stem Cell Regulated by SDF-1α-CXCR4 Axis." Stem Cells International 2017 (2017): 1–10. http://dx.doi.org/10.1155/2017/8085462.
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Previously, the perivascular characteristics of dental pulp stem cells (DPSCs) were reported, which suggested the potential application of DPSCs as perivascular cell source. In this study, we investigated whether DPSCs had angiogenic capacity by coinjection with human umbilical vein endothelial cells (HUVECs) in vivo; in addition, we determined the role of stromal cell-derived factor 1-α(SDF-1α) and C-X-C chemokine receptor type 4 (CXCR4) axis in the mutual interaction between DPSCs and HUVECs. Primarily isolated DPSCs showed mesenchymal stem cell- (MSC-) like characteristics. Moreover, DPSCs expressed perivascular markers such as NG2,α-smooth muscle actin (α-SMA), platelet-derived growth factor receptorβ(PDGFRβ), and CD146. In vivo angiogenic capacity of DPSCs was demonstrated by in vivo Matrigel plug assay. We could observe microvessel-like structures in the coinjection of DPSCs and HUVECs at 7 days postinjection. To block SDF-1αand CXCR4 axis between DPSCs and HUVECs, AMD3100, a CXCR4 antagonist, was added into Matrigel plug. No significant microvessel-like structures were observed at 7 days postinjection. In conclusion, DPSCs have perivascular characteristics that contribute to in vivo angiogenesis. The findings of this study have potential applications in neovascularization of engineered tissues and vascular diseases.
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Takeda, Yoshito, Alexander R. Kazarov, Catherine E. Butterfield, Benjamin D. Hopkins, Laura E. Benjamin, Arja Kaipainen, and Martin E. Hemler. "Deletion of tetraspanin Cd151 results in decreased pathologic angiogenesis in vivo and in vitro." Blood 109, no. 4 (October 5, 2006): 1524–32. http://dx.doi.org/10.1182/blood-2006-08-041970.
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Abstract Tetraspanin protein CD151 is abundant on endothelial cells. To determine whether CD151 affects angiogenesis, Cd151-null mice were prepared. Cd151-null mice showed no vascular defects during normal development or during neonatal oxygen-induced retinopathy. However, Cd151-null mice showed impaired pathologic angiogenesis in other in vivo assays (Matrigel plug, corneal micropocket, tumor implantation) and in the ex vivo aortic ring assay. Cd151-null mouse lung endothelial cells (MLECs) showed normal adhesion and proliferation, but marked alterations in vitro, in assays relevant to angiogenesis (migration, spreading, invasion, Matrigel contraction, tube and cable formation, spheroid sprouting). Consistent with these functional impairments, and with the close, preferential association of CD151 with laminin-binding integrins, Cd151-null MLECs also showed selective signaling defects, particularly on laminin substrate. Adhesion-dependent activation of PKB/c-Akt, e-NOS, Rac, and Cdc42 was diminished, but Raf, ERK, p38 MAP kinase, FAK, and Src were unaltered. In Cd151-null MLECs, connections were disrupted between laminin-binding integrins and at least 5 other proteins. In conclusion, CD151 modulates molecular organization of laminin-binding integrins, thereby supporting secondary (ie, after cell adhesion) functions of endothelial cells, which are needed for some types of pathologic angiogenesis in vivo. Selective effects of CD151 on pathologic angiogenesis make it a potentially useful target for anticancer therapy.
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Tanaka, Aya, Fumiko Itoh, Koichi Nishiyama, Toshiaki Takezawa, Hiroki Kurihara, Susumu Itoh, and Mitsuyasu Kato. "Inhibition of endothelial cell activation by bHLH protein E2-2 and its impairment of angiogenesis." Blood 115, no. 20 (May 20, 2010): 4138–47. http://dx.doi.org/10.1182/blood-2009-05-223057.
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E2-2 belongs to the basic helix-loop-helix (bHLH) family of transcription factors. E2-2 associates with inhibitor of DNA binding (Id) 1, which is involved in angiogenesis. In this paper, we demonstrate that E2-2 interacts with Id1 and provide evidence that this interaction potentiates angiogenesis. Mutational analysis revealed that the HLH domain of E2-2 is required for the interaction with Id1 and vice versa. In addition, Id1 interfered with E2-2–mediated effects on luciferase reporter activities. Interestingly, injection of E2-2–expressing adenoviruses into Matrigel plugs implanted under the skin blocked in vivo angiogenesis. In contrast, the injection of Id1-expressing adenoviruses rescued E2-2–mediated inhibition of in vivo angiogenic reaction. Consistent with the results of the Matrigel plug assay, E2-2 could inhibit endothelial cell (EC) migration, network formation, and proliferation. On the other hand, knockdown of E2-2 in ECs increased EC migration. The blockade of EC migration by E2-2 was relieved by exogenous expression of Id1. We also demonstrated that E2-2 can perturb VEGFR2 expression via inhibition of VEGFR2 promoter activity. This study suggests that E2-2 can maintain EC quiescence and that Id1 can counter this effect.
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Auerbach, Robert, Rachel Lewis, Brenda Shinners, Louis Kubai, and Nasim Akhtar. "Angiogenesis Assays: A Critical Overview." Clinical Chemistry 49, no. 1 (January 1, 2003): 32–40. http://dx.doi.org/10.1373/49.1.32.
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Abstract Background: Angiogenesis, the formation of new blood vessels, is an integral part of both normal developmental processes and numerous pathologies, ranging from tumor growth and metastasis to inflammation and ocular disease. Angiogenesis assays are used to test efficacy of both pro- and antiangiogenic agents. Methods: Most studies of angiogenesis inducers and inhibitors rely on various models, both in vitro and in vivo, as indicators of efficacy. In this report we describe the principal methods now in use: the in vivo Matrigel plug and corneal neovascularization assays, the in vivo/in vitro chick chorioallantoic membrane (CAM) assay, and the in vitro cellular (proliferation, migration, tube formation) and organotypic (aortic ring) assays. We include description of two new methods, the chick aortic arch and the Matrigel sponge assays. Conclusions: In vitro tests are valuable, can be carried out expeditiously, and lend themselves to quantification, but must be interpreted with extreme caution. In vitro tests are best viewed as providing initial information, subject to confirmation by in vivo assays. Multiple tests should be used to obtain maximum benefit from in vitro tests. In vivo tests are more difficult and time-consuming to perform, thereby limiting the number of tests that can run at any one time. Quantification is generally more difficult as well. However, in vivo assays are essential because of the complex nature of vascular responses to test reagents, responses that no in vitro model can fully achieve.
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Dissanayaka, Waruna L., Yuanyuan Han, Lili Zhang, Ting Zou, and Chengfei Zhang. "Bcl-2 Overexpression and Hypoxia Synergistically Enhance Angiogenic Properties of Dental Pulp Stem Cells." International Journal of Molecular Sciences 21, no. 17 (August 26, 2020): 6159. http://dx.doi.org/10.3390/ijms21176159.
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Post-implantation cell survival and angio-/vasculogenesis are critical for the success of cell-based regenerative strategies. The current study aimed to overexpress B-cell lymphoma 2 (Bcl-2) gene in dental pulp stem cells (DPSCs) and examine the anti-apoptotic and angio-/vasculogenic effects both in-vitro and in-vivo. DPSCs were transduced with Bcl-2-green fluorescent protein (GFP) lentiviral particles and examined for cell proliferation and apoptosis. The cells were cultured under normoxic or hypoxic (0.5 mM CoCl2) conditions and examined for the expression of angiogenic factors and effects on endothelial cell proliferation, migration and vessel morphogenesis. Cells with or without hypoxic preconditioning were used in in-vivo Matrigel plug assay to study the post-implantation cell survival and angio-/vasculogenesis. Bcl-2-overexpressing-DPSCs showed significantly lower apoptosis than that of null-GFP-DPSCs under serum-free conditions. Under hypoxia, Bcl-2-overexpressing-DPSCs expressed significantly higher levels of vascular endothelial growth factor compared to that under normoxia and null-GFP-DPSCs. Consequently, Bcl-2-overexpressing-DPSCs significantly enhanced endothelial cell proliferation, migration and vascular tube formation on Matrigel. Immunohistological assessment of in-vivo transplanted Matrigel plugs showed significantly higher cell survival and vasculature in hypoxic preconditioned Bcl-2-overexpressing-DPSC group compared to null-GFP-DPSC group. In conclusion, Bcl-2 overexpression and hypoxic-preconditioning could be synergistically used to enhance post-implantation cell survival and angio-/vasculogenic properties of DPSCs.
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Guo, Ting, Hongyuan Song, Zichang Zhao, Zhongtian Qi, and Shihong Zhao. "Overexpression of Annexin A2 Receptor Inhibits Neovascularization via the Promotion of Krüppel-Like Transcription Factor 2." Cellular Physiology and Biochemistry 46, no. 4 (2018): 1617–27. http://dx.doi.org/10.1159/000489209.
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Background/Aims: Annexin A2 receptor (AX2R) can mediate annexin A2 signalling and induce apoptosis in a variety of cells, but its role in neovascularization (NV) remains unclear. Krüppel-like transcription factor 2 (KLF2) is known to be expressed in a range of cell types and to participate in a number of processes during development and disease, such as endothelial homeostasis, vasoregulation and vascular growth/remodelling. The aim of our study was to investigate the role of AX2R in NV and the plausible molecular mechanism. Methods: We constructed a eukaryotic overexpression plasmid for AX2R (Lenti-AX2R) by using polymerase chain reaction (PCR). The full-length human AX2R gene was transfected into human retinal endothelial cells (HRECs) and human umbilical vein endothelial cells (HUVECs) using lentivirus vectors to overexpress AX2R. All experiments were divided into three groups: control, negative control (Lenti-EGFP), and Lenti-AX2R.Cell proliferation, cell migration, tube formation, mouse aortic ring assays and mouse matrigel plug assay were applied to analyse the effect of AX2R in NV. Furthermore, we conducted flow cytometry to evaluate whether AX2R could influence the cell cycle. A series of cell cycle-related proteins including cyclin A1, cyclin B1, cyclin D1, cyclin E1, CDK1, and p-CDC2 were detected by WB. The mRNA and protein levels of KLF2, vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) were further quantified by RT-PCR and WB to reveal the possible mechanism. Results: Overexpression of AX2R significantly inhibited cell proliferation, migration and tube formation in both types of endothelial cells (ECs), HRECs and HUVECs. It also suppressed vessel sprouting in the mouse aortic ring assay and NV in mouse matrigel plug assay. Furthermore, infection with Lenti-AX2R lentivirus arrested the cell cycle in S/G2 and influenced the expression of a series of cell cycle-related proteins. We also found that the overexpression of AX2R increased the expression of KLF2, mediating VEGF and VEGFR2. Conclusions: Overexpression of AX2R contributes to the inhibition of NV via suppressing KLF2 ubiquitin-dependent protein degradation, which might therefore be a therapeutic option for NV. It could be considered more broadly as an anti-angiogenic agent in the treatment of neovascular-related diseases in the future.
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Wake, Hidenori, Shuji Mori, Keyue Liu, Hideo K. Takahashi, and Masahiro Nishibori. "Histidine-rich glycoprotein inhibited high mobility group box 1 in complex with heparin-induced angiogenesis in matrigel plug assay." European Journal of Pharmacology 623, no. 1-3 (November 2009): 89–95. http://dx.doi.org/10.1016/j.ejphar.2009.09.010.
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Mihai, Maria Cristina, Mirel Adrian Popa, Viorel Iulian Șuică, Felicia Antohe, Edwin K. Jackson, Brigitte Leeners, Maya Simionescu, and Raghvendra K. Dubey. "Proteomic Analysis of Estrogen-Mediated Enhancement of Mesenchymal Stem Cell-Induced Angiogenesis In Vivo." Cells 10, no. 9 (August 24, 2021): 2181. http://dx.doi.org/10.3390/cells10092181.
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Therapeutic use of mesenchymal stem cells (MSCs) for tissue repair has great potential. MSCs from multiple sources, including those derived from human umbilical matrix, namely Wharton’s jelly, may serve as a resource for obtaining MSCs. However, low in vivo engraftment efficacy of MSCs remains a challenging limitation. To improve clinical outcomes using MSCs, an in-depth understanding of the mechanisms and factors involved in successful engraftment is required. We recently demonstrated that 17β-estradiol (E2) improves MSCs in vitro proliferation, directed migration and engraftment in murine heart slices. Here, using a proteomics approach, we investigated the angiogenic potential of MSCs in vivo and the modulatory actions of E2 on mechanisms involved in tissue repair. Specifically, using a Matrigel® plug assay, we evaluated the effects of E2 on MSCs-induced angiogenesis in ovariectomized (OVX) mice. Moreover, using proteomics we investigated the potential pro-repair processes, pathways, and co-mechanisms possibly modified by the treatment of MSCs with E2. Using RT-qPCR, we evaluated mRNA expression of pro-angiogenic molecules, including endoglin, Tie-2, ANG, and VEGF. Hemoglobin levels, a marker for blood vessel formation, were increased in plugs treated with E2 + MSCs, suggesting increased capillary formation. This conclusion was confirmed by the histological analysis of capillary numbers in the Matrigel® plugs treated with E2 + MSC. The LC-MS screening of proteins obtained from the excised Matrigel® plugs revealed 71 proteins that were significantly altered following E2 exposure, 57 up-regulated proteins and 14 down-regulated proteins. A major result was the association of over 100 microRNA molecules (miRNAs) involved in cellular communication, vesicle transport, and metabolic and energy processes, and the high percentage of approximately 25% of genes involved in unknown biological processes. Together, these data provide evidence for increased angiogenesis by MSCs treated with the sex hormone E2. In conclusion, E2 treatment may increase the engraftment and repair potential of MSCs into tissue, and may promote MSC-induced angiogenesis after tissue injury.
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Ye, Qing, Shukui Qin, Yanhong Liu, Jundong Feng, Qiong Wu, Wenshu Qu, and Xiaojin Yin. "Inhibitory Effect of Endostar on Specific Angiogenesis Induced by Human Hepatocellular Carcinoma." Gastroenterology Research and Practice 2015 (2015): 1–9. http://dx.doi.org/10.1155/2015/957574.
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To investigate the effect of endostar on specific angiogenesis induced by human hepatocellular carcinoma, this research systematically elucidated the inhibitory effect on HepG2-induced angiogenesis by endostar from 50 ng/mL to 50000 ng/mL. We employed fluorescence quantitative Boyden chamber analysis, wound-healing assay, flow cytometry examination using a coculture system, quantitative analysis of tube formation, andin vivoMatrigel plug assay induced by HCC conditioned media (HCM) and HepG2 compared with normal hepatocyte conditioned media (NCM) and L02. Then, we found that endostar as a tumor angiogenesis inhibitor could potently inhibit human umbilical vein endothelial cell (HUVEC) migration in response to HCM after four- to six-hour action, inhibit HCM-induced HUVEC migration to the lesion part in a dose-dependent manner between 50 ng/mL and 5000 ng/mL at 24 hours, and reduce HUVEC proliferation in a dose-dependent fashion. Endostar inhibited HepG2-induced tube formation of HUVECs which peaked at 50 ng/mL.In vivoMatrigel plug formation was also significantly reduced by endostar in HepG2 inducing system rather than in L02 inducing system. It could be concluded that, at cell level, endostar inhibited the angiogenesis-related biological behaviors of HUVEC in response to HCC, including migration, adhesion proliferation, and tube formation. At animal level, endostar inhibited the angiogenesis in response to HCC in Matrigel matrix.
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Nowak, Witold N., Hevidar Taha, Joanna Markiewicz, Neli Kachamakova-Trojanowska, Jacek Stępniewski, Damian Klóska, Urszula Florczyk-Soluch, et al. "Atorvastatin and Conditioned Media from Atorvastatin-Treated Human Hematopoietic Stem/Progenitor-Derived Cells Show Proangiogenic Activity In Vitro but Not In Vivo." Mediators of Inflammation 2019 (July 16, 2019): 1–15. http://dx.doi.org/10.1155/2019/1868170.
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Myeloid angiogenic cells (MAC) derive from hematopoietic stem/progenitor cells (HSPCs) that are mobilized from the bone marrow. They home to sites of neovascularization and contribute to angiogenesis by production of paracrine factors. The number and function of proangiogenic cells are impaired in patients with diabetes or cardiovascular diseases. Both conditions can be accompanied by decreased levels of heme oxygenase-1 (HMOX1), cytoprotective, heme-degrading enzyme. Our study is aimed at investigating whether precursors of myeloid angiogenic cells (PACs) treated with known pharmaceuticals would produce media with better proangiogenic activity in vitro and if such media can be used to stimulate blood vessel growth in vivo. We used G-CSF-mobilized CD34+ HSPCs, FACS-sorted from healthy donor peripheral blood mononuclear cells (PBMCs). Sorted cells were predominantly CD133+. CD34+ cells after six days in culture were stimulated with atorvastatin (AT), acetylsalicylic acid (ASA), sulforaphane (SR), resveratrol (RV), or metformin (Met) for 48 h. Conditioned media from such cells were then used to stimulate human aortic endothelial cells (HAoECs) to enhance tube-like structure formation in a Matrigel assay. The only stimulant that enhanced PAC paracrine angiogenic activity was atorvastatin, which also had ability to stabilize endothelial tubes in vitro. On the other hand, the only one that induced heme oxygenase-1 expression was sulforaphane, a known activator of a HMOX1 inducer—NRF2. None of the stimulants changed significantly the levels of 30 cytokines and growth factors tested with the multiplex test. Then, we used atorvastatin-stimulated cells or conditioned media from them in the Matrigel plug in vivo angiogenic assay. Neither AT alone in control media nor conditioned media nor AT-stimulated cells affected numbers of endothelial cells in the plug or plug’s vascularization. Concluding, high concentrations of atorvastatin stabilize tubes and enhance the paracrine angiogenic activity of human PAC cells in vitro. However, the effect was not observed in vivo. Therefore, the use of conditioned media from atorvastatin-treated PAC is not a promising therapeutic strategy to enhance angiogenesis.
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Wang, L., W. Tan, F. Wang, and M. Zhang. "POS0398 ADIPONECTIN INDUCES SYNOVIAL ANGIOGENESIS IN RHEUMATOID ARTHRITIS THROUGH METABOLIC REMODELING." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 428.2–428. http://dx.doi.org/10.1136/annrheumdis-2021-eular.3585.
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Background:Our team have previously reported that Adiponectin correlates well with synovial inflammation and progressive bone erosion in rheumatoid arthritis (RA). Angiogenesis is another important part, which plays a critical role in the pathogenesis of RA.Objectives:We hypothesized that adiponectin induces synovial angiogenesis in RA.Methods:Single-cell RNA sequencing (scRNA-Seq) was used to screen cellular changes in local knee joint of collagen-induced arthritis (CIA) after intraarticularly injected of adiponectin. Chimera models of synovium-cartilage-NOD/SCID mice, matrigel plug assay and rat aortic ring assay were performed to demonstrate the pro-angiogenesis role of adiponectin. Cellular experiment, including proliferation, migration, apoptosis, tube formation and angiogenesis related gene expression profile, were detected with Human Umbilical Vein Endothelial Cells (HUVEC) and Mice Lung Microvessel Endothelial Cell (MLMEC) after adiponectin stimulation. Seahorse was performed to clear the influence of adiponectin to cell metabolism.Results:The synovium and pannus hyperplasia worse in CIA model after intraarticularly injected of adiponectin, along with more serious synovitis and bone erosion. ScRNA-Seq of synovial tissues separated from CIA reminded that endothelial cell barbarically grows via metabolic remodeling after stimulated with adiponectin. Synovial chimera, matrigel plug and rat aortic ring shows adiponectin accelerates angiogenesis significantly in different background conditions. In vitro, endothelial cell proliferation detecting by RCTA and CCK8, migration by wound healing and transwell, apoptosis by FACS, tube formation and angiogenesis related gene expression profile by PCR-ARRAY were promoted by adiponectin in both HUVEC and MLMEC. Seahorse showed HUVEC made more use of glycolysis after co-cultured with adiponectin, a method of cell energy supply that tumor cells possess called warburg effect, that drives endothelial cell hyperplasia in severe environment.Conclusion:As a classic metabolic regulator, adiponectin exacerbates CIA by promoting angiogenesis through metabolic remodeling. The findings not only provide a novel insight into the pathogenic role of adiponectin, but also reveals a potential therapeutical strategy to attenuate revascularization in RA.Disclosure of Interests:None declared
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Cheng, Shu-Yu, Nan-Fu Chen, Pi-Yu Lin, Jui-Hsin Su, Bing-Hung Chen, Hsiao-Mei Kuo, Chun-Sung Sung, Ping-Jyun Sung, Zhi-Hong Wen, and Wu-Fu Chen. "Anti-Invasion and Antiangiogenic Effects of Stellettin B through Inhibition of the Akt/Girdin Signaling Pathway and VEGF in Glioblastoma Cells." Cancers 11, no. 2 (February 14, 2019): 220. http://dx.doi.org/10.3390/cancers11020220.
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Angiogenesis and invasion are highly related with tumor metastatic potential and recurrence prediction in the most aggressive brain cancer, glioblastoma multiforme (GBM). For the first time, this study reveals that marine-sponge-derived stellettin B reduces angiogenesis and invasion. We discovered that stellettin B reduces migration of glioblastoma cells by scratch wound healing assay and invasion via chamber transwell assay. Further, stellettin B downregulates Akt/Mammalian Target of Rapamycin (Akt/mTOR) and Signal transducer and activator of transcription 3 (Stat3) signaling pathways, which are essential for invasion and angiogenesis in glioblastoma. This study further demonstrates that stellettin B affects filamentous actin (F-actin) rearrangement by decreasing the cross-linkage of phosphor-Girdin (p-Girdin), which attenuates glioblastoma cell invasion. Moreover, stellettin B blocks the expression and secretion of a major proangiogenic factor, vascular endothelial growth factor (VEGF), in glioblastoma cells. Stellettin B also reduces angiogenic tubule formation in human umbilical vein endothelial cells (HUVECs). In vivo, we observed that stellettin B decreased blood vesicle formation in developmental zebrafish and suppressed angiogenesis in Matrigel plug transplant assay in mice. Decreased VEGF transcriptional expression was also found in stellettin B–treated zebrafish embryos. Overall, we conclude that stellettin B might be a potential antiangiogenic and anti-invasion agent for future development of therapeutic agents for cancer therapy.
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Di Carlo, Anna, Sara Beji, Silvia Palmerio, Mario Picozza, Marco D’Agostino, Vincenzo Petrozza, Roberta Melchionna, et al. "The Nucleolar Protein Nucleophosmin Is Physiologically Secreted by Endothelial Cells in Response to Stress Exerting Proangiogenic Activity Both In Vitro and In Vivo." International Journal of Molecular Sciences 22, no. 7 (April 1, 2021): 3672. http://dx.doi.org/10.3390/ijms22073672.
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Nucleophosmin (NPM), a nucleolar multifunctional phosphoprotein, acts as a stress sensor in different cell types. NPM can be actively secreted by inflammatory cells, however its biology on endothelium remains unexplored. In this study, we show for the first time that NPM is secreted by human vein endothelial cells (HUVEC) in the early response to serum deprivation and that NPM acts as a pro-inflammatory and angiogenic molecule both in vitro and in vivo. Accordingly, 24 h of serum starvation condition induced NPM relocalization from the nucleus to cytoplasm. Interestingly, NPM was increasingly excreted in HUVEC-derived conditioned media in a time dependent fashion upon stress conditions up to 24 h. The secretion of NPM was unrelated to cell necrosis within 24 h. The treatment with exogenous and recombinant NPM (rNPM) enhanced migration as well as the Intercellular Adhesion Molecule 1 (ICAM-1) but not Vascular cell adhesion protein 1 (VCAM-1) expression and it did not affect cell proliferation. Notably, in vitro tube formation by Matrigel assay was significantly increased in HUVEC treated with rNPM compared to controls. This result was confirmed by the in vivo injection of Matrigel plug assay upon stimulation with rNPM, displaying significant enhanced number of functional capillaries in the plugs. The stimulation with rNPM in HUVEC was also associated to the increased expression of master genes regulating angiogenesis and migration, including Vascular Endothelial Growth Factor-A (VEGF-A), Hepatocyte Growth Factor (HGF), Stromal derived factor-1 (SDF-1), Fibroblast growth factor-2 (FGF-2), Platelet Derived Growth Factor-B (PDGF-B), and Matrix metallopeptidase 9 (MMP9). Our study demonstrates for the first time that NPM is physiologically secreted by somatic cells under stress condition and in the absence of cell necrosis. The analysis of the biological effects induced by NPM mainly related to a pro-angiogenic and inflammatory activity might suggest an important autocrine/paracrine role for NPM in the regulation of both phenomena.
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Kumar, Pawan, Mohammad A. Amin, Lisa A. Harlow, Peter J. Polverini, and Alisa E. Koch. "Src and phosphatidylinositol 3–kinase mediate soluble E-selectin–induced angiogenesis." Blood 101, no. 10 (May 15, 2003): 3960–68. http://dx.doi.org/10.1182/blood-2002-04-1237.
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Abstract Angiogenesis plays an important role in a variety of pathophysiologic processes, including tumor growth and rheumatoid arthritis. We have previously shown that soluble E-selectin (sE-selectin) is an important angiogenic mediator. However, the mechanism by which sE-selectin mediates angiogenesis is still unknown. In this study, we show that sE-selectin is a potent mediator of human dermal microvascular endothelial cell (HMVEC) chemotaxis, which is predominantly mediated through the Src and the phosphatidylinositiol 3–kinase (PI3K) pathways. Further, sE-selectin induced a 2.2-fold increase in HMVEC tube formation in the Matrigel in vitro assay. HMVECs pretreated with the Src inhibitor (PP2) and the PI3K inhibitor (LY294002) or transfected with Src antisense oligonucleotides or Akt dominant-negative mutants significantly inhibited sE-selectin–mediated HMVEC tube formation. In contrast, HMVECs transfected with an extracellular signal-related kinase 1/2 (ERK1/2) mutant or pretreated with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 failed to show sE-selectin–mediated HMVEC tube formation. Similarly, in the Matrigel-plug in vivo assay, sE-selectin induced a 2.2-fold increase in blood vessel formation, which was significantly inhibited by PP2 and LY294002 but not by PD98059. sE-selectin induced a marked increase in Src, ERK1/2, and PI3K phosphorylation. PI3K and ERK1/2 phosphorylation was significantly inhibited by PP2, thereby suggesting that both of these pathways may be activated via Src kinase. Even though the ERK1/2 pathway was activated by sE-selectin in HMVECs, it seems not to be essential for sE-selectin–mediated angiogenesis. Taken together, our data clearly show that sE-selectin–induced angiogenesis is predominantly mediated through the Src-PI3K pathway.
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Li, Jing, Youming Zhu, Na Li, Tao Wu, Xianyu Zheng, Boon chin Heng, Duohong Zou, and Jianguang Xu. "Upregulation of ETV2 Expression Promotes Endothelial Differentiation of Human Dental Pulp Stem Cells." Cell Transplantation 30 (January 1, 2021): 096368972097873. http://dx.doi.org/10.1177/0963689720978739.
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The lack of vasculogenesis often hampers the survivability and integration of newly engineered tissue grafts within the host. Autologous endothelial cells (ECs) are an ideal cell source for neovascularization, but they are limited by their scarcity, lack of proliferative capacity, and donor site morbidity upon isolation. The objective of this study was to determine whether differentiation of human dental pulp stem cells (DPSCs) into the endothelial lineage can be enhanced by recombinant ETV2 overexpression. DPSCs were extracted from fresh dental pulp tissues. ETV2 overexpression in DPSCs was achieved by lentiviral infection and cellular morphological changes were evaluated. The mRNA and protein expression levels of endothelial-specific markers were assessed through quantitative real-time polymerase chain reaction, western blot, immunofluorescence staining, and flow cytometry. The tube formation assay and Matrigel plug assay were also performed to evaluate the angiogenic potential of the ETV2-transduced cells in vitro and in vivo, respectively. Additionally, proteomic analysis was performed to analyze global changes in protein expression following ETV2 overexpression. After lentiviral infection, ETV2-overexpressing DPSCs showed endothelial-like morphology. Compared with control DPSCs, significantly higher mRNA and protein expression levels of endothelial-specific genes, including CD31, VE-Cadherin, VEGFR1, and VEGFR2, were detected in ETV2-overexpressing DPSCs. Moreover, ETV2 overexpression enhanced capillary-like tube formation on Matrigel in vitro, as well as neovascularization in vivo. In addition, comparative proteomic profiling showed that ETV2 overexpression upregulated the expression of vascular endothelial growth factor (VEGF) receptors, which was indicative of increased VEGF signaling. Taken together, our results indicate that ETV2 overexpression significantly enhanced the endothelial differentiation of DPSCs. Thus, this study shows that DPSCs can be a promising candidate cell source for tissue engineering applications.
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Webler, Anke C., U. Ruth Michaelis, Rüdiger Popp, Eduardo Barbosa-Sicard, Andiappan Murugan, John R. Falck, Beate Fisslthaler, and Ingrid Fleming. "Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis." American Journal of Physiology-Cell Physiology 295, no. 5 (November 2008): C1292—C1301. http://dx.doi.org/10.1152/ajpcell.00230.2008.
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Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5( Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.
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Fan, Junqiang, Guanxin Xu, Zhibo Chang, Ling Zhu, and Jie Yao. "miR-210 transferred by lung cancer cell-derived exosomes may act as proangiogenic factor in cancer-associated fibroblasts by modulating JAK2/STAT3 pathway." Clinical Science 134, no. 7 (April 2020): 807–25. http://dx.doi.org/10.1042/cs20200039.
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Abstract It has been generally believed that cancer-associated fibroblasts (CAFs) have the ability to increase the process of tumor angiogenesis. However, the potential mechanisms by which cancer-derived exosomes in lung cancer (LC) remains to be investigated. LC-derived exosomes were administrated to NIH/3T3 cells. A variety of experiments were conducted to investigate the proangiogenic factors of CAFs, including Western blot, RT-PCR, colony formation assay, tube formation assay, Matrigel plug assay et al. In addition, the impact of JAK2/STAT3 signaling pathway were also explored. The role of hsa-miR-210 was identified with microarray profiling and validated in vitro and in vivo assays. The target of miR-210 was screened by RNA pull down, RNA-sequencing and then verified. It was shown that LC-derived exosomes could induce cell reprogramming, thus promoting the fibroblasts transferring into CAFs. In addition, the exosomes with overexpressed miR-210 could increase the level of angiogenesis and vice versa, which suggested the miR-210 secreted by the LC-derived exosomes may initiate the CAF proangiogenic switch. According to our analysis, the miR-210 had the ability of elevating the expression of some proangiogenic factors such as MMP9, FGF2 and vascular endothelial growth factor (VEGF) a (VEGFa) by activating the JAK2/STAT3 signaling pathway, ten-eleven translocation 2 (TET2) was identified as the target of miR-210 in CAFs which has been involved in proangiogenic switch. miR-210 was overexpressed in serum exosomes of untreated non-small cell LC (NSCLC) patients. We concluded that the promotion effect of exosomal miR-210 on proangiogenic switch of CAFs may be explained by the modulation of JAK2/STAT3 signaling pathway and TET2 in recipient fibroblasts.
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Chabot, Sophie, Nabila Jabrane-Ferrat, Karine Bigot, Julie Tabiasco, Alexandra Provost, Muriel Golzio, Muhammad Zaeem Noman, et al. "A novel antiangiogenic and vascular normalization therapy targeted against human CD160 receptor." Journal of Experimental Medicine 208, no. 5 (April 11, 2011): 973–86. http://dx.doi.org/10.1084/jem.20100810.
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Angiogenesis plays an essential role in several diseases of the eye and in the growth of solid tumors, but existing antiangiogenic therapies have limited benefits in several cases. We report the antiangiogenic effects of a monoclonal antibody, CL1-R2, in several animal models of neovascularization. CL1-R2 recognizes human CD160, a membrane receptor which is conserved in various mammal species. We show that CD160 is expressed on the endothelial cells of newly formed blood vessels in human colon carcinoma and mouse B16 melanoma but not in vessels of healthy tissues. CL1-R2 reduced fibroblast growth factor 2–induced neovascularization in the rabbit cornea, in a mouse model of oxygen-induced retinopathy, and in a mouse Matrigel plug assay. Treatment of B16 melanoma-bearing mice with CL1-R2 combined with cyclophosphamide chemotherapy caused regression of the tumor vasculature and normalization of the remaining vessels as shown by Doppler ultrasonography, intravital microscopy, and histology. These studies validate CD160 as a potential new target in cases of human pathological ocular and tumor neoangiogenesis that do not respond or become resistant to existing antiangiogenic drugs.
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Sweet, Daniel Timothy, Zhongming Chen, David M. Wiley, Victoria L. Bautch, and Ellie Tzima. "The adaptor protein Shc integrates growth factor and ECM signaling during postnatal angiogenesis." Blood 119, no. 8 (February 23, 2012): 1946–55. http://dx.doi.org/10.1182/blood-2011-10-384560.
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Abstract Angiogenesis requires integration of cues from growth factors, extracellular matrix (ECM) proteins, and their receptors in endothelial cells. In the present study, we show that the adaptor protein Shc is required for angiogenesis in zebrafish, mice, and cell-culture models. Shc knockdown zebrafish embryos show defects in intersegmental vessel sprouting in the trunk. Shc flox/flox; Tie2-Cre mice display reduced angiogenesis in the retinal neovascularization model and in response to VEGF in the Matrigel plug assay in vivo. Functional studies reveal a model in which Shc is required for integrin-mediated spreading and migration specifically on fibronectin, as well as endothelial cell survival in response to VEGF. Mechanistically, Shc is required for activation of the Akt pathway downstream of both integrin and VEGF signaling, as well as for integration of signals from these 2 receptors when cells are grown on fibronectin. Therefore, we have identified a unique mechanism in which signals from 2 critical angiogenic signaling axes, integrins and VEGFR-2, converge at Shc to regulate postnatal angiogenesis.
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Amin, Mohammad A., Phillip L. Campbell, Jeffrey H. Ruth, Takeo Isozaki, Bradley J. Rabquer, W. Alex Stinson, Martin O'Brien, et al. "A key role for Fut1-regulated angiogenesis and ICAM-1 expression in K/BxN arthritis." Annals of the Rheumatic Diseases 74, no. 7 (March 24, 2014): 1459–66. http://dx.doi.org/10.1136/annrheumdis-2013-204814.
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ObjectivesAngiogenesis contributes to the pathogenesis of rheumatoid arthritis. Fucosyltransferases (Futs) are involved in angiogenesis and tumour growth. Here, we examined the role of Fut1 in angiogenesis and K/BxN serum transfer arthritis.MethodsWe examined Fut1 expression in human dermal microvascular endothelial cells (HMVECs) by quantitative PCR. We performed a number of angiogenesis assays to determine the role of Fut1 using HMVECs, Fut1 null (Fut1−/−), and wild type (wt) endothelial cells (ECs) and mice. K/BxN serum transfer arthritis was performed to determine the contribution of Fut1-mediated angiogenesis inFut1−/−and wt mice. A static adhesion assay was implemented with RAW264.7 (mouse macrophage cell line) and mouse ECs. Quantitative PCR, immunofluorescence and flow cytometry were performed withFut1−/−and wt ECs for adhesion molecule expression.ResultsTumour necrosis factor-α induced Fut1 mRNA and protein expression in HMVECs. HMVECs transfected with Fut1 antisense oligodeoxynucleotide andFut1−/−ECs formed significantly fewer tubes on Matrigel.Fut1−/−mice had reduced angiogenesis in Matrigel plug and sponge granuloma angiogenesis assays compared with wt mice.Fut1−/−mice were resistant to K/BxN serum transfer arthritis and had decreased angiogenesis and leucocyte ingress into inflamed joints. Adhesion of RAW264.7 cells to wt mouse ECs was significantly reduced when Fut1 was lacking.Fut1−/−ECs had decreased intercellular adhesion molecule-1 (ICAM-1) expression at mRNA and protein levels compared with wt ECs. ICAM-1 was also decreased inFut1−/−arthritic ankle cryosections compared with wt ankles.ConclusionsFut1 plays an important role in regulating angiogenesis and ICAM-1 expression in inflammatory arthritis.
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Min, Jeong-Ki, Young-Lai Cho, Jae-Hoon Choi, Yonghak Kim, Jeong Hun Kim, Young Suk Yu, Jaerang Rho, et al. "Receptor activator of nuclear factor (NF)–κB ligand (RANKL) increases vascular permeability: impaired permeability and angiogenesis in eNOS-deficient mice." Blood 109, no. 4 (October 12, 2006): 1495–502. http://dx.doi.org/10.1182/blood-2006-06-029298.
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Abstract Receptor activator of nuclear factor (NF)–κB ligand (RANKL) is emerging as an important regulator of vascular pathophysiology. Here, we demonstrate a novel role of RANKL as a vascular permeability factor and a critical role of endothelial nitric oxide synthase (eNOS) in RANKL-induced endothelial function. RANKL increased the vascular permeability and leukocyte infiltration in vivo and caused the breakdown of the blood-retinal barrier in wild-type mice but not in eNOS-deficient mice. In vitro, it increased endothelial permeability and reduced VE-cadherin–facilitated endothelial cell-cell junctions in a NO-dependent manner. RANKL also led to the activation of Akt and eNOS and to NO production in endothelial cells (ECs). These effects were suppressed by the inhibition of TRAF6, phosphoinositide 3′-kinase (PI3K), Akt, or NOS by genetic or pharmacologic means. Inhibition of the TRAF6-mediated NO pathway reduced EC migration and capillary-like tube formation in response to RANKL. Moreover, the effects of RANKL on ECs sprouting from the aorta, and neovessel formation in both the mouse Matrigel plug assay and corneal micropocket assay, were impaired in eNOS-deficient mice. These results demonstrate that RANKL promotes vascular permeability and angiogenesis by stimulating eNOS by a TRAF6-PI3K-Akt–dependent mechanism. These properties may be relevant to the pathogenesis of angiogenesis-dependent and inflammatory vascular diseases.
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Zhu, Kui, Mohammed Asif Amin, Yuanyuan Zha, Lisa A. Harlow, and Alisa E. Koch. "Mechanism by which H-2g, a glucose analog of blood group H antigen, mediates angiogenesis." Blood 105, no. 6 (March 15, 2005): 2343–49. http://dx.doi.org/10.1182/blood-2004-08-3140.
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AbstractThe 4A11 antigen is a unique cytokine-inducible antigen up-regulated on rheumatoid arthritis (RA) synovial endothelial cells (ECs) compared with normal ECs. Previously, we showed that in soluble form, this antigen, Lewisy-6/H-5-2 (Ley/H) or its glucose analog, 2-fucosyl lactose (H-2g), induced the expression of EC intercellular adhesion molecule-1 (ICAM-1) and leukocyte-endothelial adhesion through the Janus kinase 2 (JAK2)–signal transducer and activator of transcription 3 (STAT3) pathway. Currently, we show that H-2g induces release of EC angiogenic basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), an effect inhibited by decoy nuclear factor κB (NFκB) oligodeoxynucleotide (ODN). JAK2 and phosphoinositide-3 kinase (PI3K) are 2 upstream kinases of NFκB activated by H-2g, as confirmed by an inhibitor of kappa B kinase (IKKβ) assay. In vitro, H-2g induces vascular sprouting in the rat aortic ring model, whereas blockade of JAK2, PI3K, or NFκB inhibits sprouting. Likewise, in the in vivo mouse Matrigel plug angiogenesis assay, chemical inhibitors and antisense or decoy ODNs of JAK2, PI3K, or NFκB decrease angiogenesis, confirming the importance of these pathways in H-2g–induced EC signaling. The critical role of Ley/H involvement in angiogenesis and its signaling pathways may provide new targets for therapy of diseases characterized by pathologic neovascularization.
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Oike, Yuichi, Yasuhiro Ito, Hiromitsu Maekawa, Tohru Morisada, Yoshiaki Kubota, Masaki Akao, Takashi Urano, Kunio Yasunaga, and Toshio Suda. "Angiopoietin-related growth factor (AGF) promotes angiogenesis." Blood 103, no. 10 (May 15, 2004): 3760–65. http://dx.doi.org/10.1182/blood-2003-04-1272.
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Abstract We report here the identification of angiopoietin-related growth factor (AGF) as a positive mediator for angiogenesis. To investigate the biologic function of AGF in angiogenesis, we analyzed the vasculature in the dermis of transgenic mice expressing AGF in mouse epidermal keratinocytes (K14-AGF). K14-AGF transgenic mice were grossly red, especially in the ears and snout, suggesting that hypervascularization had occurred in their skin. Histologic examination of ear skin from K14-AGF transgenic mice revealed increased numbers of microvessels in the dermis, whereas the expression of several angiogenic factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factors (VEGFs), and angiopoietin-1 (Ang-1), was decreased. We showed that AGF is a secreted protein and does not bind to tyrosine kinase with immunoglobulin and EGF-homology domain (Tie1) or Tie2 receptors. An in vitro chamber assay revealed that AGF directly promotes chemotactic activity of vascular endothelial cells. Both mouse corneal and matrigel plug assays showed that AGF induces neovascularization in vivo. Furthermore, we found that plasma leakage occurred after direct injection of AGF into the mouse dermis, suggesting that AGF directly induces a permeability change in the local vasculature. On the basis of these observations, we propose that AGF is a novel angiogenic factor and that handling of its biologic functions could lead to novel therapeutic strategies for control of angiogenesis.
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Gangadaran, Prakash, Ramya Lakshmi Rajendran, Ji Min Oh, Eun Jung Oh, Chae Moon Hong, Ho Yun Chung, Jaetae Lee, and Byeong-Cheol Ahn. "Identification of Angiogenic Cargo in Extracellular Vesicles Secreted from Human Adipose Tissue-Derived Stem Cells and Induction of Angiogenesis In Vitro and In Vivo." Pharmaceutics 13, no. 4 (April 5, 2021): 495. http://dx.doi.org/10.3390/pharmaceutics13040495.
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Angiogenesis is defined as the generation of new blood vessels or the sprouting of endothelial cells from a pre-existing vascular network. Angiogenesis occurs during the growth and development of an organism, the response of organs or tissues to injury, and during cancer development and progression. The majority of studies on stem-cell-derived extracellular vesicles (EVs) have used cell lines, and have primarily focused on well-known solitary proteins. Here, we isolated stem cells from human adipose tissue (ADSCs), and we isolated EVs from them (ADSC-EVs). The ADSC-EVs were characterised and 20 angiogenic proteins were analysed using an angiogenic antibody array. Furthermore, we analysed the ability of ADSC-EVs to induce angiogenesis in vitro and in vivo. ADSC-EVs were positive for CD81 and negative for GM130, calnexin, and cytochrome-C. ADSC-EVs showed typical EV spherical morphology and were ~200 nm in size. ADSC-EVs were found to contain angiogenic proteins as cargo, among which interleukin 8 (IL-8) was the most abundant, followed by chemokine (C-C motif) ligand 2 (CCL2), a tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, and vascular endothelial growth factor-D (VEGF-D). ADSC-EVs treatment increased the proliferation, migration, total vessel length, total number of junctions, and junction density of endothelial cells in vitro. The results of an in vivo Matrigel plug assay revealed that ADSC-EVs induced more blood vessels in the Matrigel compared with the control. These results demonstrate that ADSC-EVs contain angiogenic proteins as cargo and promote angiogenesis in vitro and in vivo. Therefore, ADSC-EVs have potential for therapeutic use in ischaemia.
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da Silva, Sabrina Daniela, Fabio Albuquerque Marchi, Jie Su, Long Yang, Ludmila Valverde, Jessica Hier, Krikor Bijian, et al. "Co-Overexpression of TWIST1-CSF1 Is a Common Event in Metastatic Oral Cancer and Drives Biologically Aggressive Phenotype." Cancers 13, no. 1 (January 5, 2021): 153. http://dx.doi.org/10.3390/cancers13010153.
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Invasive oral squamous cell carcinoma (OSCC) is often ulcerated and heavily infiltrated by pro-inflammatory cells. We conducted a genome-wide profiling of tissues from OSCC patients (early versus advanced stages) with 10 years follow-up. Co-amplification and co-overexpression of TWIST1, a transcriptional activator of epithelial-mesenchymal-transition (EMT), and colony-stimulating factor-1 (CSF1), a major chemotactic agent for tumor-associated macrophages (TAMs), were observed in metastatic OSCC cases. The overexpression of these markers strongly predicted poor patient survival (log-rank test, p = 0.0035 and p = 0.0219). Protein analysis confirmed the enhanced expression of TWIST1 and CSF1 in metastatic tissues. In preclinical models using OSCC cell lines, macrophages, and an in vivo matrigel plug assay, we demonstrated that TWIST1 gene overexpression induces the activation of CSF1 while TWIST1 gene silencing down-regulates CSF1 preventing OSCC invasion. Furthermore, excessive macrophage activation and polarization was observed in co-culture system involving OSCC cells overexpressing TWIST1. In summary, this study provides insight into the cooperation between TWIST1 transcription factor and CSF1 to promote OSCC invasiveness and opens up the potential therapeutic utility of currently developed antibodies and small molecules targeting cancer-associated macrophages.
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Birdsey, Graeme M., Nicola H. Dryden, Valerie Amsellem, Frank Gebhardt, Kapil Sahnan, Dorian O. Haskard, Elisabetta Dejana, Justin C. Mason, and Anna M. Randi. "Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin." Blood 111, no. 7 (April 1, 2008): 3498–506. http://dx.doi.org/10.1182/blood-2007-08-105346.
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Abstract Tight regulation of the balance between apoptosis and survival is essential in angiogenesis. The ETS transcription factor Erg is required for endothelial tube formation in vitro. Inhibition of Erg expression in human umbilical vein endothelial cells (HUVECs), using antisense oligonucleotides, resulted in detachment of cell-cell contacts and increased cell death. Inhibition of Erg expression by antisense in HUVECs also lowered expression of the adhesion molecule vascular endothelial (VE)–cadherin, a key regulator of endothelial intercellular junctions and survival. Using chromatin immunoprecipitation, we showed that Erg binds to the VE-cadherin promoter. Furthermore, Erg was found to enhance VE-cadherin promoter activity in a transactivation assay. Apoptosis induced by inhibition of Erg was partly rescued by overexpression of VE-cadherin–GFP, suggesting that VE-cadherin is involved in the Erg-dependent survival signals. To show the role of Erg in angiogenesis in vivo, we used siRNA against Erg in a Matrigel plug model. Erg inhibition resulted in a significant decrease in vascularization, with increase in caspase-positive endothelial cells (ECs). These results identify a new pathway regulating angiogenesis and endothelial survival, via the transcription factor Erg and the adhesion molecule VE-cadherin.
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Peng, XinZhi, Jinsong Wang, Roberta M. Lassance-Soares, Amir H. Najafi, Subeena Sood, Nima Aghili, Lee O. Alderman, et al. "Gender differences affect blood flow recovery in a mouse model of hindlimb ischemia." American Journal of Physiology-Heart and Circulatory Physiology 300, no. 6 (June 2011): H2027—H2034. http://dx.doi.org/10.1152/ajpheart.00004.2011.
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Blood flow restoration to ischemic tissue is affected by various risk factors. The aim of this study was to examine gender effects on arteriogenesis and angiogenesis in a mouse ischemic hindlimb model. C57BL/6J mice were subjected to unilateral hindlimb ischemia. Flow recovery was less and hindlimb use impairment was greater in females. No gender difference in vessel number was found at baseline, although 7 days postsurgery females had fewer α-smooth muscle actin-positive vessels in the midpoint of the adductor region. Females had higher hindlimb vascular resistance, were less responsive to vasodilators, and were more sensitive to vasoconstrictors postligation. Western blotting showed that females had higher baseline levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the calf, while 7 days postligation males had higher levels of VEGF, eNOS, and phosphorylated vasodilator stimulated phosphoprotein. Females had less angiogenesis in a Matrigel plug assay and less endothelial cell proliferation in vitro. Females have impaired recovery of flow, a finding presumably caused by multiple factors including decreased collateral remodeling, less angiogenesis, impaired vasodilator response, and increased vasoconstrictor activity; our results also suggest the possibility that new collateral formation, from capillaries, is impaired in females.
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Langer, Harald F., Kyoung-Jin Chung, Valeria V. Orlova, Eun Young Choi, Sunil Kaul, Michael J. Kruhlak, Markella Alatsatianos, et al. "Complement-mediated inhibition of neovascularization reveals a point of convergence between innate immunity and angiogenesis." Blood 116, no. 22 (November 25, 2010): 4395–403. http://dx.doi.org/10.1182/blood-2010-01-261503.
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Abstract Beyond its role in immunity, complement mediates a wide range of functions in the context of morphogenetic or tissue remodeling processes. Angiogenesis is crucial during tissue remodeling in multiple pathologies; however, the knowledge about the regulation of neovascularization by the complement components is scarce. Here we studied the involvement of complement in pathological angiogenesis. Strikingly, we found that mice deficient in the central complement component C3 displayed increased neovascularization in the model of retinopathy of prematurity (ROP) and in the in vivo Matrigel plug assay. In addition, antibody-mediated blockade of C5, treatment with C5aR antagonist, or C5aR deficiency in mice resulted in enhanced pathological retina angiogenesis. While complement did not directly affect angiogenesis-related endothelial cell functions, we found that macrophages mediated the antiangiogenic activity of complement. In particular, C5a-stimulated macrophages were polarized toward an angiogenesis-inhibitory phenotype, including the up-regulated secretion of the antiangiogenic soluble vascular endothelial growth factor receptor-1. Consistently, macrophage depletion in vivo reversed the increased neovascularization associated with C3- or C5aR deficiency. Taken together, complement and in particular the C5a-C5aR axes are potent inhibitors of angiogenesis.
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Mousa, Shaker S., Dhruba Bahrali, Shaymaa S. Mousa, Emmy Dier, Murat Yalcin, and Patricia Phillips. "Site Directed Delivery of Chemotherapy and Non-Anticoagulant Sulfated Heparin in Breast Cancer." Blood 112, no. 11 (November 16, 2008): 5050. http://dx.doi.org/10.1182/blood.v112.11.5050.5050.
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Abstract There is substantial literature support for the use of low molecular weight heparins (LMWH) for treating coagulation disorders in cancer patients. Recent prospective and retrospective clinical trials have also demonstrated that they provide significant advantages in terms of progression-free and overall survival in certain cancers and in certain subgroups of patients. LMWH treatments are often associated with increased bleeding, constituting a dose-limiting effect. We have developed novel non-anticoagulant heparin (NACH) compounds that have minimal effects on hemostasis (El-Naggar and Mousa, US Patents 6,908,907 B2, (2005), and 10, 667,216, (2003). We have evaluated them for efficacy vs. tumor growth and metastasis and have begun to investigate the mechanisms involved in anti-tumor activities. Modified sulfated LMWH with weak or no anticoagulant activities were still highly effective in inhibiting angiogenesis and metastasis, demonstrating that anticoagulation is not essential for attenuation of angiogenesis or metastasis. The modified heparins were characterized with respect to their ability to release endothelial tissue factor pathway inhibitor (TFPI) and inhibit selectin-mediated interactions, molecular components that have been shown to modulate tumor growth, tumor angiogenesis and metastasis. One of these modified heparin compounds that showed significant activity in a selectin-mediated tumor cell adhesion assay was also highly effective in reducing tumor burden in mice with MC-38 colon carcinoma and B16-BL6 melanoma (>70%) and in reducing the number of metastatic foci (>65%) in these animals. We also investigated the efficacy of NACH compounds on growth factor-induced angiogenesis in a mouse Matrigel model in which new vessel growth was quantified by measuring hemoglobin concentration extracted from the Matrigel plug. Values are Means ± SEM. Matrigel alone: 0.57± 0.12; bFGF + Matrigel: 7.27 ± 1.18; NAC-S-S: 0.86 ± 0.10. This sulfated compound which demonstrated no anticoagulant activity in aPTT and TEG assays, reduced capillary formation to baseline levels. These data demonstrate that non-anticoagulant heparin compounds exhibit a profile of anti-tumor activities without disrupting normal hemostasis. Site-directed therapy with non-anticoagulant heparins (NACH) and chemotherapy would allow for optimization of treatments in the tumor microenvironment. In studies that are currently underway, we are targeting the sites of tumor neovascularization using a biodegradable nanoparticulate system made up of a blend of MPEG-PLGA (methoxy-polyethyleneglycol-poly (lactide-co-glycolide) and maleimide-PEG-PLGA. These nanoparticles have their surfaces conjugated to alpha-v beta-3 antibody and contain chemotherapeutic agent Doxorubicin, with or without NACH. Preliminary data indicate that repeated administration of sulfated non-anticoagulant heparin compound at 10 mg/kg S.C. daily for up to 14 days in conjunction with doxorubicin caused no bruising at the injection site, whereas Enoxaparin showed injection site bruising in >50% of the mice. The use of NACH agents that are co-encapsulated with chemotherapeutic agents could minimize the toxic side effects of the chemotherapy while delivering a combination of effective therapeutic agents directly to the tumor.
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Guda, Maheedhara R., Kiran K. Velpula, Swapna Asuthkar, Charlie P. Cain, and Andrew J. Tsung. "Targeting RGS4 Ablates Glioblastoma Proliferation." International Journal of Molecular Sciences 21, no. 9 (May 7, 2020): 3300. http://dx.doi.org/10.3390/ijms21093300.
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Glioblastoma (GBM) is the most common type of adult primary brain tumor with a median survival rate of less than 15 months, regardless of the current standard of care. Cellular heterogeneity, self-renewal ability and tumorigenic glioma cancer stem cell (GSC) populations contribute to the difficulty in treating GBM. G-protein-coupled receptors (GPCRs) are the largest group of membrane proteins and mediate many cellular responses. Regulators of G-protein signaling 4 (RGS4) are negative regulators of G-protein signaling, and elevated levels of RGS4 are reportedly linked with several human diseases, including cancer. This study investigates the effect of silencing RGS4, resulting in inhibition of GSC growth, invasion and migration. Data obtained from The Cancer Genome Atlas (TCGA) demonstrated poor patient survival with high expression of RGS4. Immunohistochemistry and immunoblot analysis conducted on GBM patient biopsy specimens demonstrated increased RGS4 expression correlative with the TCGA data. RNA sequencing confirmed a significant decrease in the expression of markers involved in GSC invasion and migration, particularly matrix metalloproteinase-2 (MMP2) in knockout of RGS4 using CRISPR plasmid (ko-RGS4)-treated samples compared to parental controls. Gelatin zymography confirmed the reduced activity of MMP2 in ko-RGS4-treated samples. Silencing RGS4 further reduced the invasive and migratory abilities and induction of apoptosis of GSCs as evidenced by Matrigel plug assay, wound healing assay and human apoptosis array. Collectively, our results showed that the silencing of RGS4 plays an important role in regulating multiple cellular functions, and is an important therapeutic target in GBM.
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Lahat, Nitza, Haim Bitterman, Miri Engelmayer-Goren, Doron Rosenzweig, Lea Weiss-Cerem, and Michal A. Rahat. "Reduced TIMP-2 in hypoxia enhances angiogenesis." American Journal of Physiology-Cell Physiology 300, no. 3 (March 2011): C557—C566. http://dx.doi.org/10.1152/ajpcell.00177.2010.
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Hypoxia, which characterizes ischemia, trauma, inflammation, and solid tumors, recruits monocytes, immobilizes them, and alters their function, leading to an anti-inflammatory and proangiogenic phenotype. Monocyte extravasation from the circulation and their migration in tissues are partially mediated by the balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). The mechanisms evoked by hypoxia that regulate monocyte migration and activation are not entirely clear. Specifically, the effect of hypoxia on TIMPs in these cells has hardly been investigated. We show that hypoxia reduces TIMP-2 secretion from human primary monocytes and from the monocyte-like cell lines U937 and THP-1 by three- to fourfold ( P < 0.01), by inhibiting TIMP-2 transcription through mechanisms that involve the transcription factor SP-1. Hypoxia also lowers TIMP-2 protein secretion from human endothelial cells (by 2-fold, P < 0.05). TIMP-2 levels do not influence the reduced migration of THP-1 cells in hypoxia; however, low TIMP-2 levels enhance endothelial cell migration/proliferation, their ability to form tubelike structures in vitro, and the appearance of mature blood vessels in a Matrigel plug assay in vivo. Thus we conclude that reduced TIMP-2 levels secreted from both hypoxic monocytes and endothelial cells are proangiogenic.
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Abéngozar, María Angeles, Sergio de Frutos, Sergio Ferreiro, Joaquím Soriano, Manuel Perez-Martinez, David Olmeda, Marco Marenchino, et al. "Blocking ephrinB2 with highly specific antibodies inhibits angiogenesis, lymphangiogenesis, and tumor growth." Blood 119, no. 19 (May 10, 2012): 4565–76. http://dx.doi.org/10.1182/blood-2011-09-380006.
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Abstract Membrane-anchored ephrinB2 and its receptor EphB4 are involved in the formation of blood and lymphatic vessels in normal and pathologic conditions. Eph/ephrin activation requires cell-cell interactions and leads to bidirectional signaling pathways in both ligand- and receptor-expressing cells. To investigate the functional consequences of blocking ephrinB2 activity, 2 highly specific human single-chain Fv (scFv) Ab fragments against ephrinB2 were generated and characterized. Both Ab fragments suppressed endothelial cell migration and tube formation in vitro in response to VEGF and provoked abnormal cell motility and actin cytoskeleton alterations in isolated endothelial cells. As only one of them (B11) competed for binding of ephrinB2 to EphB4, these data suggest an EphB-receptor–independent blocking mechanism. Anti-ephrinB2 therapy reduced VEGF-induced neovascularization in a mouse Matrigel plug assay. Moreover, systemic administration of ephrinB2-blocking Abs caused a drastic reduction in the number of blood and lymphatic vessels in xenografted mice and a concomitant reduction in tumor growth. Our results show for the first time that specific Ab-based ephrinB2 targeting may represent an effective therapeutic strategy to be used as an alternative or in combination with existing antiangiogenic drugs for treating patients with cancer and other angiogenesis-related diseases.
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Yoon, Chang Min, Bok Sil Hong, Hyung Geun Moon, Seyoung Lim, Pann-Ghill Suh, Yoon-Keun Kim, Chi-Bom Chae, and Yong Song Gho. "Sphingosine-1-phosphate promotes lymphangiogenesis by stimulating S1P1/Gi/PLC/Ca2+ signaling pathways." Blood 112, no. 4 (August 15, 2008): 1129–38. http://dx.doi.org/10.1182/blood-2007-11-125203.
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Abstract The lymphatic system plays pivotal roles in mediating tissue fluid homeostasis and immunity, and excessive lymphatic vessel formation is implicated in many pathological conditions, which include inflammation and tumor metastasis. However, the molecular mechanisms that regulate lymphatic vessel formation remain poorly characterized. Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that is implicated in a variety of biologic processes such as inflammatory responses and angiogenesis. Here, we first report that S1P acts as a lymphangiogenic mediator. S1P induced migration, capillary-like tube formation, and intracellular Ca2+ mobilization, but not proliferation, in human lymphatic endothelial cells (HLECs) in vitro. Moreover, a Matrigel plug assay demonstrated that S1P promoted the outgrowth of new lymphatic vessels in vivo. HLECs expressed S1P1 and S1P3, and both RNA interference–mediated down-regulation of S1P1 and an S1P1 antagonist significantly blocked S1P-mediated lymphangiogenesis. Furthermore, pertussis toxin, U73122, and BAPTA-AM efficiently blocked S1P-induced in vitro lymphangiogenesis and intracellular Ca2+ mobilization of HLECs, indicating that S1P promotes lymphangiogenesis by stimulating S1P1/Gi/phospholipase C/Ca2+ signaling pathways. Our results suggest that S1P is the first lymphangiogenic bioactive lipid to be identified, and that S1P and its receptors might serve as new therapeutic targets against inflammatory diseases and lymphatic metastasis in tumors.
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Lee, Hye Min, Soon Bin Kwon, Ahyun Son, Doo Hyun Kim, Kyun-Hwan Kim, Jonghyo Lim, Young-Guen Kwon, et al. "Stabilization of Intrinsically Disordered DKK2 Protein by Fusion to RNA-Binding Domain." International Journal of Molecular Sciences 20, no. 11 (June 11, 2019): 2847. http://dx.doi.org/10.3390/ijms20112847.
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Intrinsic disorders are a common feature of hub proteins in eukaryotic interactomes controlling the signaling pathways. The intrinsically disordered proteins (IDPs) are prone to misfolding, and maintaining their functional stability remains a major challenge in validating their therapeutic potentials. Considering that IDPs are highly enriched in RNA-binding proteins (RBPs), here we reasoned and confirmed that IDPs could be stabilized by fusion to RBPs. Dickkopf2 (DKK2), Wnt antagonist and a prototype IDP, was fused with lysyl-tRNA synthetase (LysRS), with or without the fragment crystallizable (Fc) domain of an immunoglobulin and expressed predominantly as a soluble form from a bacterial host. The functional competence was confirmed by in vitro Wnt signaling reporter and tube formation in human umbilical vein endothelial cells (HUVECs) and in vivo Matrigel plug assay. The removal of LysRS by site-specific protease cleavage prompted the insoluble aggregation, confirming that the linkage to RBP chaperones the functional competence of IDPs. While addressing to DKK2 as a key modulator for cancer and ischemic vascular diseases, our results suggest the use of RBPs as stabilizers of disordered proteinaceous materials for acquiring and maintaining the structural stability and functional competence, which would impact the druggability of a variety of IDPs from human proteome.
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Pore, Nabendu, Shuang Liu, Hui-Kuo Shu, Bin Li, Daphne Haas-Kogan, David Stokoe, Julie Milanini-Mongiat, et al. "Sp1 Is Involved in Akt-mediated Induction of VEGF Expression through an HIF-1–independent Mechanism." Molecular Biology of the Cell 15, no. 11 (November 2004): 4841–53. http://dx.doi.org/10.1091/mbc.e04-05-0374.
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Increased expression of vascular endothelial growth factor (VEGF) contributes to the growth of many tumors by increasing angiogenesis. Although hypoxia is a potent inducer of VEGF, we previously showed that epidermal growth factor receptor amplification and loss of PTEN, both of which can increase phosphatidylinositol-3-kinase (PI3K) activity, increase VEGF expression. Using both adenoviral vectors and a cell line permanently expressing constitutively active myristoylated Akt (myrAkt), we show that activation of Akt, which is downstream of PI3K, increases VEGF expression in vitro and increases angiogenesis in a Matrigel plug assay. Transient transfection experiments using reporter constructs containing the VEGF promoter showed that up-regulation of VEGF by Akt is mediated through Sp1 binding sites located in the proximal promoter. Small interfering RNA directed against Sp1 prevented the induction of VEGF mRNA in response to myrAkt but not to hypoxia. Expression of myrAkt is associated with increased phosphorylation of Sp1 and its increased binding to a probe corresponding to the -88/-66 promoter region. In conclusion, our results indicate that Sp1 is required for transactivation of the VEGF by Akt. Others have proposed that the PI3K/Akt pathway can increase VEGF expression via the hypoxia-inducible factor 1 (HIF-1); however, our results suggest an alternative mechanism can also operate.
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Singla, Bhupesh, Hui-Ping Lin, WonMo Ahn, Joseph White, and Gábor Csányi. "Oxidatively Modified LDL Suppresses Lymphangiogenesis via CD36 Signaling." Antioxidants 10, no. 2 (February 23, 2021): 331. http://dx.doi.org/10.3390/antiox10020331.
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Arterial accumulation of plasma-derived LDL and its subsequent oxidation contributes to atherosclerosis. Lymphatic vessel (LV)-mediated removal of arterial cholesterol has been shown to reduce atherosclerotic lesion formation. However, the precise mechanisms that regulate LV density and function in atherosclerotic vessels remain to be identified. The aim of this study was to investigate the role of native LDL (nLDL) and oxidized LDL (oxLDL) in modulating lymphangiogenesis and underlying molecular mechanisms. Western blotting and immunostaining experiments demonstrated increased oxLDL expression in human atherosclerotic arteries. Furthermore, elevated oxLDL levels were detected in the adventitial layer, where LV are primarily present. Treatment of human lymphatic endothelial cells (LEC) with oxLDL inhibited in vitro tube formation, while nLDL stimulated it. Similar results were observed with Matrigel plug assay in vivo. CD36 deletion in mice and its siRNA-mediated knockdown in LEC prevented oxLDL-induced inhibition of lymphangiogenesis. In addition, oxLDL via CD36 receptor suppressed cell cycle, downregulated AKT and eNOS expression, and increased levels of p27 in LEC. Collectively, these results indicate that oxLDL inhibits lymphangiogenesis via CD36-mediated regulation of AKT/eNOS pathway and cell cycle. These findings suggest that therapeutic blockade of LEC CD36 may promote arterial lymphangiogenesis, leading to increased cholesterol removal from the arterial wall and reduced atherosclerosis.
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Gu, Wenduo, Xuechong Hong, Alexandra Le Bras, Witold N. Nowak, Shirin Issa Bhaloo, Jiacheng Deng, Yao Xie, Yanhua Hu, Xiong Z. Ruan, and Qingbo Xu. "Smooth muscle cells differentiated from mesenchymal stem cells are regulated by microRNAs and suitable for vascular tissue grafts." Journal of Biological Chemistry 293, no. 21 (April 11, 2018): 8089–102. http://dx.doi.org/10.1074/jbc.ra118.001739.
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Tissue-engineered vascular grafts with long-term patency are greatly needed in the clinical settings, and smooth muscle cells (SMCs) are a critical graft component. Human mesenchymal stem cells (MSCs) are used for generating SMCs, and understanding the underlying regulatory mechanisms of the MSC-to-SMC differentiation process could improve SMC generation in the clinic. Here, we found that in response to stimulation of transforming growth factor-β1 (TGFβ1), human umbilical cord–derived MSCs abundantly express the SMC markers α-smooth muscle actin (αSMA), smooth muscle protein 22 (SM22), calponin, and smooth muscle myosin heavy chain (SMMHC) at both gene and protein levels. Functionally, MSC-derived SMCs displayed contracting capacity in vitro and supported vascular structure formation in the Matrigel plug assay in vivo. More importantly, SMCs differentiated from human MSCs could migrate into decellularized mouse aorta and give rise to the smooth muscle layer of vascular grafts, indicating the potential of utilizing human MSC-derived SMCs to generate vascular grafts. Of note, microRNA (miR) array analysis and TaqMan microRNA assays identified miR-503 and miR-222-5p as potential regulators of MSC differentiation into SMCs at early time points. Mechanistically, miR-503 promoted SMC differentiation by directly targeting SMAD7, a suppressor of SMAD-related, TGFβ1-mediated signaling pathways. Moreover, miR-503 expression was SMAD4-dependent. SMAD4 was enriched at the miR-503 promoter. Furthermore, miR-222-5p inhibited SMC differentiation by targeting and down-regulating ROCK2 and αSMA. In conclusion, MSC differentiation into SMCs is regulated by miR-503 and miR-222-5p and yields functional SMCs for use in vascular grafts.
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Vinci, Paola, Antonio Bastone, Silvia Schiarea, Erica Dander, Mario Salmona, Silvano Sozzani, Andrea Biondi, and Giovanna D'Amico. "Chemerin Produced By Mesenchymal Stromal Cells (MSC) Is an Important Factor for In Vivo macrophage Migration." Blood 126, no. 23 (December 3, 2015): 1195. http://dx.doi.org/10.1182/blood.v126.23.1195.1195.
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Abstract Mesenchymal Stromal Cells (MSC) are multipotent cells currently used for treating several inflammatory disorders thanks to their ability to modulate the immune response. However, the mechanisms by which MSC are able to suppress the immune response have not been fully understood. Chemerin has been recently identified as a chemotactic protein, secreted as a precursor, named Prochemerin, and converted into its active form through the proteolitic cleavage of the last six-seven amino acids at the C-terminal domain by different serine and cysteine proteases derived from the fibrinolitic, coagulation and inflammatory cascade. In particular, we observed that both human and mouse bone marrow-derived MSC were able to produce Chemerin under basal conditions and its production was strongly increased after stimulation with inflammatory cytokines. The aim of this study was to understand whether Chemerin produced by MSC is involved in their potent immune-modulatory activity. Chemerin was immune-purified from supernatant of human MSC (MSC-Chem) and utilized for measuring in vitro migration index (MI) of pre-B cells expressing the human ChemR23 receptor (L1.2-ChemR23). MSC-Chem was able to induce the migration of ChemR23-expressing cells in a dose-depend manner (MI 1nM=85, MI 5nM=480, MI 10nM=1131). However, recombinant human (rh)-chemerin induced higher migration of L1.2-ChemR23 cells compared to MSC-Chem (MI 5nM=1938), suggesting that only a fraction of MSC-Chem was converted into its active form by MSC themselves. In accordance, LC/MS mass spectrometry analysis on purified MSC-Chem did not identify the active form of the protein. Interestingly, pre-incubation of MSC-Chem with Neutrophil Elastase and Cathepsin L induced a strong migration of L1.2-ChemR23 cells compared to MSC-Chem alone (MI MSC-Chem alone 1 nM=23.33; MI MSC-Chem 1 nM + Neutrophil Elastase=328; MI MSC-Chem 1 nM + Cathepsin L=4950; p=0.002), suggesting that MSC-Chem were converted in its active form, after cleavage by proteases. Starting from these data, we established an in vivo migration assay by injecting under the abdominal skin of C57BL6 mice a mix of matrigel and murine (m)MSC (secreting or not Chemerin). After 5 days, the matrigel plug was excided, digested and infiltrating immune cells were analyzed by FACS analysis. Chemerin production by mMSC was totally abrogated by using RNA interference approach (sChem-MSC). Interestingly, mMSC features, such as phenotype and differentiation ability, were not affected by the gene-silencing process. Preliminary results showed that 5 days after injection, scramble Chem-MSC were able to recruit macrophages (CD45+CD11b+F4/80+ cells) into the matrigel plug. On the other hand, sChem-MSC drastically decreased their ability to induce macrophages migration, (sChem-MSC mean=2.38%, range=0.8%-6.4%; scramble MSC mean=8.2%; range=4%-11.5%; p=0.01; n=3). These findings identify a new mechanism by which MSC, through Chemerin production, attract macrophages in vivo. Further studies are needed to understand whether recruited macrophages are also affected by the immunomodulatory activity of MSC Disclosures No relevant conflicts of interest to declare.
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Saretzki, Erika, Franziska Pankratz, Bianca Engert, Sebastian Grundmann, Christoph Bode, Martin Moser, Qian Zhou, and Jennifer Esser. "Bone morphogenetic protein 4 regulates microRNAs miR-494 and miR-126–5p in control of endothelial cell function in angiogenesis." Thrombosis and Haemostasis 117, no. 04 (2017): 734–49. http://dx.doi.org/10.1160/th16-08-0643.
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SummaryMicroRNAs are small non-coding RNAs that negatively regulate posttranscriptional gene expression. Several microRNAs have been described to regulate the process of angiogenesis. Previously, we have shown that bone morphogenetic protein 4 (BMP4) increased the proangiogenic activity of endothelial cells. In this project, we now investigated how the pro-angiogenic BMP4 effect is mediated by microRNAs. First, we performed a microRNA array with BMP4-stimulated human umbilical vein endothelial cells (HUVECs). Among the topregulated microRNAs, we detected a decreased expression of miR-494 and increased expression of miR-126–5p. Next, we analysed the canonical Smad and alternative signalling pathways, through which BMP4 would regulate miR-126–5p and miR-494 expression. Furthermore, the functional effect of miR-494 and miR-126–5p on endothelial cells was investigated. MicroRNA-494 overexpression decreased endothelial cell proliferation, migration and sprout formation. Consistently, miR-494 inhibition increased endothelial cell function. As potential miR-494 targets, bFGF and BMP endothelial cell precursorderived regulator (BMPER) were identified and confirmed by western blot. Luciferase assays showed direct miR-494 binding in BMPER 3’UTR. In contrast, miR-126–5p overexpression increased pro-angiogenic endothelial cell behaviour and, accordingly, miR-126–5p inhibition decreased endothelial cell function. As a direct miR-126–5p target we identified the anti-angiogenic thrombospondin-1 which was confirmed by western blot analysis and luciferase assays. In the Matrigel plug assay application of antagomiR-494 increased endothelial cell ingrowth, whereas antagomiR-126–5p treatment decreased cell ingrowth in vivo. Taken together, through differential regulation of the anti-angiomiR-494 and the angiomiR-126–5p by BMP4 both microRNAs contribute to the pro-angiogenic BMP4 effect on endothelial cells.Supplementary Material to this article is available online at www.thrombosis-online.com.