Dissertations / Theses on the topic 'Circulating progenitor cells'

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2006.
Vito, Raymond, Committee Member ; Nerem, Robert, Committee Chair ; Eskin, Suzanne, Committee Member ; Hanson, Stephen, Committee Member ; Gibbons, Gary, Committee Member.

Background Endothelial progenitor cells (EPCs) are circulating progenitor cells involved in vascular repair and regeneration. I investigated the role of EPCs in cardiac transplant recipients, to establish whether reduced EPCs are associated with cardiac allograft vasculopathy (CAV), and to investigate whether patients receiving older donor hearts have lower EPCs than those receiving hearts from younger donors. I carried out a study to 'determine whether controlled vascular damage occurring at percutaneous coronary intervention (pCI) results in EPC release. Finally I assessed whether EPCs show diurnal variation in healthy individuals. Methods and Results I used flow cytometry quantification of absolute EPC counts with all the commonly used phenotypes from whole peripheral blood. The methods underwent extensive optimisation stages and reproducibility studies. I did not find any difference EPC numbers between cardiac transplant patients with and without CAV. There was no reduction in EPCs in patients with older donor hearts. There was no evidence of EPC mobilisation in the first 24 hours after PCI, rather there was a fall in EPCs in the first 6 hours. This is similar to the pattern ofdiurnal variation found in healthy volunteers sampled at 3 time points who showed a fall in EPCs between 8am and 3pm and a subsequent rise at lOpm. Conclusions My work has shown that EPCs are not reduced in CAV which highlights the pathophysiological differences from coronary disease. It provides evidence supporting the theory that age related decline in EPCs is not due to aging of the target tissues. also challenge the existence of a vascular injury specific mobilisation ofEPCs following PCI. Finally I have demonstrated a significant diurnal variation in EPCs which is important both in underst&lding EPC kinetics and also for the interpretation and conduction of future clinical studies.

Background: Thrombocytopenia is common in sick preterm babies in the first day of life. Despite this, platelet production in thrombocytopenic preterm babies has rarely been assessed. Methods: To address this problem I have developed miniaturised assays to study circulating megakaryocyte (MK) progenitors (BFU-MK and CFU-MK), total cultured MK precursors and mature MK, by culturing mononuclear cells purified from 0.5-1ml of preterm peripheral blood. MK lineage colonies and cells are identified by a MK specific anti-glycoprotein IIb/IIIa antibody (CD61) by APAAP. Results: i) Normal values for these cells at birth were established in cord blood from healthy term and preterm babies. ii) Circulating BFU-MK/CFU-MK, total cultured MK precursors and mature MK were then prospectively studied in 63 preterm babies (gestational age 24-34 wks). iii) At birth 18 (69%) of the thromocytopenic babies were also neutropenic. Conclusion: These data indicate that the principal cause of the thrombocytopenia and neutropenia in the preterm babies studied was reduced platelet and neutrophil production occurring as a consequence of reduced numbers of MK and neutrophil progenitors respectively. Taken together these data suggest the haematological abnormalities characteristic of new-born born to mothers with PIH or with IUGR (thrombocytopenia, neutropenia and polycythaemia) are a consequence of dysregulation of fetal haemopoiesis occurring proximal to committed MK and neutrophil progenitors, most likely at the level of the primitive multipotent haemopoietic stem cell (CFU-GEMM). Finally the value of miniaturised MK progenitor and precursor assays in evaluating rare or unusual cases of neonatal thrombocytopenia has been demonstrated. In the three examples investigated this approach provided unique insights into the pathogenesis of the thromocytopenia in each case.

Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice with rapidly rising prevalence and incidence predominantly due to advancing age in Western populations. Of particular concern however is the strong relationship between AF and stroke. This relates to a number of factors, but there is an emerging body of evidence to suggest that AF confers a hypercoagulable state. Disruption of endothelial homeostasis (damage vs. repair) is thought to be central to this process. The endothelium appears to be damaged both by AF and various other vascular diseases (e.g. hypertension) that frequently co-exist with the arrhythmia, with similar disruption to endothelial repair (normally effected by endothelial progenitor cells). Endothelial damage seems to be an essential prerequisite to thrombogenesis in AF. Significantly, the endothelium also links a number of processes including inflammation, growth factors, the renin-angiotensin-aldosterone system among others, which may directly or indirectly lead to activation of the coagulation cascade. This thesis investigates the relationship between the temporal pattern of AF (paroxysmal, persistent, permanent) and established markers of endothelial dysfunction (vonWillebrand factor, vWf; soluble E-selectin, sEsel), angiogenesis (vascular Endothelial Growth Factor, VEGF), apoptosis (soluble Fas/Fas ligand, sFas/sFasL) and inflammation (C-reactive protein, CRP; Interleukin-6, IL-6) in AF with particular reference to circulating progenitor cells (CPCs) as a novel marker of endothelial health/angiogenesis. Additionally the impact of restoration of sinus rhythm using electrical cardioversion on these indices and the relevance of the AF arrhythmia burden in influencing these markers is investigated. In conclusion, the endothelium seems to be a central link through which all three components of Virchow’s triad interact in AF. This thesis finds a possible link for CPCs to interact with various other reported aberrancies of the hypercoagulable state in this process. Also reported is a modest alteration in CPC counts following restoration of sinus rhythm, however, only limited numbers of patients were assessed and this requires examination with a more in depth study. Finally, the thesis has also examined the role of paroxysmal AF in influencing surrogate markers of the hypercoagulable state, but failed to find any significant differences on the basis of the arrhythmia burden. These findings must however been considered in light of numerous study limitations, the most notable of which is limited statistical power.

Endometriosis is a common cause of subfertility and pelvic pain, affecting up to 10% of women of reproductive age. It is characterised by the presence of endometrial-like tissue outside the uterus. The development of the disease is still poorly understood and, currently, the diagnosis relies on visualisation of typical lesions during surgery. There is great interest in identifying biomarkers to assist in diagnosis and disease management. Blood vessel development is known to be a crucial feature of endometriosis, but the mechanisms involved in angiogenesis are not well described for this disease. Most vessel development relies on the proliferation and migration of pre-existing endothelial cells. However, there may also be roles for cells derived from peripheral blood (circulating angiogenic cells) and surrounding stromal cells. In this thesis, the contribution of these different cell types to vessel development in endometriosis is assessed. In chapter 2, a robust protocol was optimised to identify circulating angiogenic cells (CACs) with flow cytometry. The reliability of the protocol was verified, and the level of these cells was found not to fluctuate with the menstrual cycle in healthy women (P=0.279, F=1.359, 3 d.f.). In chapter 3, levels of CACs in women with and without endometriosis were found to be equivalent (0.0835% ± 0.0422 compared to 0.0724% ± 0.0414), demonstrating that they have no use as a disease biomarker. In chapter 4, isolation and culture of endothelial cells from the endometrium was attempted. However, a pure culture of endometrial endothelial cells could not be obtained, which may be due to contamination by other cell types or cellular transdifferentiation. Finally, in chapter 5, the contribution of endometrial stromal cells to vessel development was considered. Stromal cells were found not to differentiate towards an endothelial cell phenotype, but were able to participate in tube formation assays. However, the presence of endometriosis did not influence this behaviour.

Mestrado em Biologia Molecular e Celular
Endothelial dysfunction and impaired endothelial regenerative capacity play a key role in the pathogenesis of cardiovascular disease, which is one of the major causes of mortality in chronic kidney disease (CKD) patients. Circulating endothelial cells (CEC) may be an indicator of vascular damage, while circulating endothelial progenitor cells (EPC) may be a biomarker for vascular repair. However, the simultaneously evaluation of CEC and EPC circulating levels and its relation were not previously examined in CKD population. A blood sample (18ml) of healthy subjects (n=10), early CKD (n=10) and advanced CKD patients (n=10) was used for the isolation of early and late EPCs, CECs, and hematopoietic cells, identified by flow cytometry (BD FACSCanto™ II system) using a combination of fluorochrome-conjugated primary antibodies: CD31-PE, CD45-APC Cy7, CD34-FITC, CD117-PerCp Cy5.5, CD133-APC, CD146-Pacific Blue, and CD309-PECy7. Exclusion of dead cells was done according to a fixable viability dye staining. This eightcolor staining flow cytometry optimized protocol allowed us to accurate simultaneously identify EPCs, CECs and hematopoietic cells. In addition, it was also possible to distinguish the two subpopulations of EPCs, early and late EPCs subpopulation, by CD45intCD31+CD34+CD117-CD133+CD309-CD146- and CD45intCD31+CD34+CD117-CD133-CD309+CD146- multiple labeling, respectively. Moreover, the identification of CECs and hematopoietic cells was performed by CD45-CD31+CD34-/lowCD117-CD133-CD309-CD146+ and CD34+CD117+, respectively. The levels of CECs were non-significantly increased in early CKD (312.06 ± 91.34) and advanced CKD patients (191.43±49.86) in comparison with control group (103.23±24.13). By contrast, the levels of circulating early EPCs were significantly reduced in advanced CKD population (17.03±3.23) in comparison with early CKD (32.31±4.97), p=0.04 and control group (36.25 ± 6.16), p=0.03. In addition the levels of late EPCs were significantly reduced in both advanced (6.60±1.89), p=0.01, and early CKD groups (8.42±2.58), p=0.01 compared with control group (91.54±29.06). These results were accompanied by a dramatically reduction in the recruitment, differentiation and regenerative capacity indexes in CKD population. Taken together, these results suggest an imbalance in the process of endothelial repairment in CKD population, and further propose that the indexes of recruitment, differentiation and regenerative capacity of EPCs, may help to select the patients to benefit from guiding intervention strategies to improve cardiovascular health by inducing vascular protection.
A disfunção endotelial e as alterações nos processos de regeneração endotelial podem desempenhar um papel determinante na patogénese da doença cardiovascular, que é uma das principais causas de mortalidade na doença renal crónica (DRC). As células endoteliais circulantes (CEC) podem ser um indicador de dano vascular, enquanto que as células progenitoras endoteliais circulantes (CPEC) pode ser um biomarcador de reparação vascular. No entanto, a avaliação simultânea dos níveis de CECs e de CPECs e sua relação não foram previamente avaliados numa população de doentes renais crónicos. Amostras de sangue (18 mL) foram recolhidas a partir de indivíduos saudáveis (n = 10), e a partir de doentes renais crónicos em estadios precoces (n=10) e em estádios avançados (n=10), para se proceder ao isolamento de populações de CPECs imaturas e maduras, CECs e células hematopoiéticas. Estas populações de células foram identificadas por citometria de fluxo (sistema BD FACS Canto II) usando uma combinação de anticorpos primários conjugados com fluorocromos: CD31-PE, CD45-APC Cy7, CD34-FITC, CD117-PerCp Cy5.5, CD133-APC, CD309-PE Cy7 e CD146-Paciific blue. Para a exclusão das células mortas recorreu-se a um marcador de viabilidade (“fixable viability dye”). Este protocolo otimizado de citometria de fluxo de oito cores permitiu identificar simultaneamente e com precisão as subpopulações de CECs, CPECs e células hematopoiéticas. Além disso, também foi possível distinguir as duas subpopulações de CPECs, imaturas e maduras, por marcação múltipla CD45intCD31+ CD34+ CD117-CD133+ CD309-CD146- e CD45intCD31+ CD34+CD117- CD133-CD309+ CD146-, respetivamente. Adicionalmente, a identificação de CECs e células hematopoiéticas foi realizada por CD45-CD31+ CD34-/lowCD117- CD133-CD309- CD146+ e CD34+ CD117+, respetivamente. Os níveis de CECs foram mais elevados em pacientes em estadios precoces de DRC (312,1±91,3) e em estadios avançados (191,4±49,9) comparativamente com o grupo controlo (103,23±24,13), n.s. Para além disso, os níveis de CPECs imaturas foram significativamente diminuídos em estadios avançados de DRC (17,1±3,2) em comparação com estadios precoces (32,3±4,9), p=0,04, e com o grupo controlo (36,3±6,2), p=0,03. Os níveis de CPECs maduras foram significativamente reduzidos em estadios avançados de DRC (6,6±1,9), p=0,01 e em estadios precoces (8,4±2,6), p=0,01, em comparação com o grupo controlo (91,5±29,1). Estes resultados foram acompanhados por uma diminuição acentuada nos índices de capacidade de recrutamento, diferenciação e regeneração na população de doentes renais crónicos. Globalmente, estes resultados sugerem um desequilíbrio no processo de reparação endotelial na DRC, e sugerem ainda, que os índices de recrutamento, diferenciação e regeneração podem ajudar na seleção de pacientes que possam beneficiar de estratégias de intervenção para melhorar a saúde cardiovascular induzindo proteção vascular.

Nel 1997 venne isolata una popolazione cellulare con caratteristiche appartenenti a cellule endoteliali mature e a cellule progenitrici ; le cellule appartenenti a queste popolazione furono denominate EPCs (cellule endoteliali progenitrici circolanti) e fu messa in evidenza la loro capacità di dare origine a vasculogenesi postnatale. Lo scopo dello studio è stata la caratterizzazione di tale popolazione cellulare in termini biologici e la valutazione delle differenze delle EPCs in soggetti sani e nefropatici in emodialisi. È stata infine valutata l’eventuale capacità della Vitamina D di influenzare le capacità delle Late EPCs in termini di formazione di colonie in vitro e di attività anticalcifica in soggetti in insufficienza renale cronica.

Angiogenic cell therapy is currently being developed as a treatment for coronary artery disease (CAD); however, endothelial dysfunction (ED), commonly found in patients with CAD, impairs the ability for revascularization to occur. We hypothesized that culture on a collagen matrix will improve survival and function of circulating progenitor cells (CPCs) isolated from a mouse model of ED. Overall, ED decreased the expression of endothelial markers in CPCs and impaired their function, compared to normal mice. Culture of CPCs from ED mice on collagen was able to increase cell marker expression, and improve migration and adhesion potential, compared to CPCs on fibronectin. Nitric oxide production was reduced for CPCs on collagen for the ED group; however, CPCs on collagen had better viability under conditions of serum deprivation and hypoxia, compared to fibronectin. This study suggests that a collagen matrix may improve the function of therapeutic CPCs that have been exposed to ED.

L’endothélium joue un rôle primordial dans le développement et le maintien des multiples fonctions vasculaires. Il est ainsi largement impliqué dans des situations pathologiques comme les maladies cardio-vasculaires. La description de marqueurs endothéliaux circulants a permis une exploration non invasive de l'endothélium. Notre équipe s’est intéressée principalement aux cellules endothéliales circulantes (CEC), dont le taux reflète la lésion ou l’activation de l’endothélium, et aux progéniteurs endothéliaux circulants (PEC), marqueurs de régénération endothéliale. La découverte en 1997 par Asahara de la présence chez l’adulte de ces PEC, participant à la formation de nouveaux vaisseaux par vasculogenèse, a ouvert de nouvelles perspectives, notamment pour la thérapie cellulaire des pathologies ischémiques. Ce travail a consisté à développer les méthodes d’étude de ces cellules dans plusieurs contextes. Tout d’abord, nous avons exploré l’utilité de ces marqueurs dans la physiopathologie de l’hypertension artérielle pulmonaire (HTAP). Puis nous avons analysé le potentiel de mobilisation des progéniteurs endothéliaux à partir de la paroi vasculaire lors d’une ischémie locale chez des volontaires sains dans le cadre du développement d’un produit de thérapie cellulaire autologue. Une partie de ce projet a été de mettre en place et d’optimiser les techniques d’étude de ces marqueurs. Les CEC ont été quantifiées par immunoséparation magnétique (IMS), technique mise au point en 1992 (Dignat-George 1992) et transférée dans notre laboratoire. La quantification des PEC a été réalisée par cytométrie en flux et par culture cellulaire. En culture, deux types de PEC sont décrits : les PEC précoces, dont l’origine est monocytaire et pour lesquels la culture est déjà standardisée, et les « Endothelial Colony Forming Cells » (ECFC), seules cellules présentant des caractéristiques de cellules endothéliales progénitrices et pouvant être proposées comme produit de thérapie cellulaire. Nous avons optimisé la quantification des ECFC en culture en étudiant l’effet de diverses matrices et de la densité d’ensemencement des cellules mononucléées issues du sang total sur l’obtention de ces cellules et leurs propriétés angiogènes. La dysfonction endothéliale a été décrite comme un élément central dans le développement de l’HTAP dont le diagnostic repose sur la mesure de la pression artérielle pulmonaire par cathétérisme cardiaque droit. En l’absence de marqueur biologique non invasif dans cette maladie, nous avons quantifié les CEC et les progéniteurs circulants dans deux études. Une étude réalisée chez des patients adultes a montré une augmentation spécifique des CEC dans l’HTAP et non dans l’hypertension pulmonaire thromboembolique chronique. Ainsi les CEC semblent être le reflet des lésions endothéliales pulmonaires et non de la sévérité clinique des patients. L’autre étude a montré l’intérêt de la quantification des CEC dans la prise en charge thérapeutique des enfants souffrant d’HTAP secondaire à une cardiopathie congénitale, dont les formes irréversibles présentaient des taux élevés de CEC. Nous avons ainsi défini un nouveau marqueur non invasif à utilité diagnostique et pronostique. Les PEC sont des cellules rares dans le sang circulant, difficiles à expandre, et dont les essais de mobilisation médullaire se sont révélés insuffisants. L’hypothèse récente d’une réserve vasculaire des progéniteurs endothéliaux nous a conduits à étudier l’effet d’un processus d’ischémie locale sur la mobilisation de ces cellules chez des volontaires sains. Deux groupes d'âge ont été inclus afin d'évaluer l'impact du vieillissement sur la méthode de mobilisation étudiée. Malgré un effet de cette ischémie sur la dilatation endothéliale cette méthode n’a pas permis de mobiliser significativement les PEC issus de la paroi endothéliale, quel que soit l'âge des sujets. A l’inverse, l’hypoxie a eu un effet délétère sur les capacités angiogènes des ECFC
The endothelium plays a key role in the development and the homeostasis of vascular functions. It is also well involved in pathological situations like cardiovascular diseases. Thanks to the description of circulating endothelial markers, non invasive study of the endothelium is now possible. Our group was particularly interested in circulating endothelial cells (CECs), the level of which reflects an endothelial activation or lesion, and to circulating endothelial progenitors cells (EPCs), markers of endothelial repair. EPC description by Asahara in 1997 in adult blood, involved in new blood vessel formation by vasculogenesis, offered new perspectives, specially for cell therapy in ischemic diseases. This work consisted in the development of methods to study these markers in different contexts. First, we explored the interest of these markers in the physiopathology of pulmonary arterial hypertension (PAH). Then we evaluated endothelial progenitors mobilization from the vascular wall by a local ischemia process in healthy volunteers, in the perspective of an autologous cell therapy product development. One part of this project was the implementation and optimization of the methods to study CEC and EPC. CEC were quantified by magnetic immunoseparation. This technique was developped in 1992 by F. Dignat-George's group and transferred in our laboratory. EPC were quantified by flow cytometry and cell culture. Two types of EPC are described in culture: the early EPC, which originate from monocyte lineage and which culture is standardized, and the « Endothelial Colony Forming Cells » (ECFC), the only cells presenting endothelial progenitor cell properties and which use as a cell therapy product can be considered. ECFC quantification by culture was optimized by assessment of the impact of diverse matrices and seeding concentrations of mononuclear cells isolated from whole blood, on ECFC commitment and their angiogenic properties. Endothelial dysfunction was described as a central element in the development of PAH, which diagnosis is based on the use of right heart catheterization. Due to the lack of noninvasive marker for this disease, CEC and circulating progenitors were quantified in two studies. One of them realized in adult patients showed a specific increase of CEC in PAH and not in post-embolic PH. CEC would then reflect the presence of specific endothelial lesions and not the clinical state of the patients. The other study demonstrated the interest of CEC quantification in the therapeutic care of children with PAH secondary to congenital heart disease, for whom patients in irreversible state had a higher level of CEC. We then defined a new noninvasive biomarker.that can be used for the diagnosis and prognosis of PAH. EPC are rare events in whole blood, difficult to expand and for which, mobilization protocols revealed insufficient. The recent hypothesis of a vascular reservoir for endothelial progenitor led us to study the effect of a local ischemia procedure on the mobilization of these cells in healthy volunteers. Two age groups were included to assess the impact of aging on this procedure. Despite a significant endothelial dilation with the local ischemia, no EPC were mobilized, whatever the age group. Ischemia even altered ECFC angiogenic properties

Orientadores: Cristina Pontes Vicente, Patrícia Muniz Mendes Freire de Moura
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-26T00:13:01Z (GMT). No. of bitstreams: 1 Guimaraes_TaniaMariaRocha_D.pdf: 3499853 bytes, checksum: 2767ae0d1bab61dda96ce4765cd63588 (MD5) Previous issue date: 2014
Resumo: As células progenitoras endoteliais (EPCs) estão envolvidas em neovasculogênese e na manutenção da homeostase vascular, sua deficiência pode ter papel na patogênese da hipertensão. Este estudo teve como objetivo analisar o perfil de expressão das EPCs circulantes e diferentes fatores de risco cardiovascular em mulheres hipertensas, na pré-menopausa, em comparação com mulheres normotensas saudáveis. Realizou-se um estudo caso-controle com 45 mulheres voluntárias, faixa etária de 30 a 50 anos (41 ± 6) no Ambulatório do Pronto Socorro Cardiológico de Pernambuco. As EPCs definidas como CD45-/CD34+/KDR+ foram coletadas em sangue venoso periférico e analisadas por citometria de fluxo. As mulheres foram classificadas como controles (CT) saudáveis normotensas com PAS (pressão arterial sistólica) < 130 mmHg e PAD (pressão arterial diastólica) < 85 mmHg (n=15), com hipertensão primária: a) Leve (HL) PAS=140-159mm Hg e PAD=90-99 mmHg (n=15) e b) Severa (HS) PAS > 180 mmHg e PAD > 110 mmHg (n=15). Os grupos foram entrevistados quanto aos hábitos de fumo, prática de exercícios físicos e Índice de Massa Corporal (IMC), sendo aferido o nível da PA em repouso. Realizou-se análise nos prontuários dos resultados dos exames séricos de colesterol total, lipoproteínas de alta densidade-colesterol (HDL-c), lipoproteínas de baixa densidade-colesterol (LDL-c), triglicerídeos e glicemia de jejum, no mês da coleta das amostras sanguíneas. Os resultados comprovaram redução significativa ao número de EPCs no HL (74%) e HS (88%) versus CT; e redução de 67% no HS versus HL, evidenciando relação inversa entre o número de células e o estágio da hipertensão. O grupo HS apresentou aumento de 49% de células CD45+ demonstrando padrão inflamatório e redução de 61% de CD45-/CD34+. Quanto aos níveis séricos verificou-se: HDL-c [HL (52±7); HS (48±5)]; LDL-c [HL (130±8); HS (143±15)]; triglicerídeos [HL (138±19); HS(153 ±40)]; glicemia de jejum [HL(95±7);HS(121±39)] e IMC [HL(31±4);HS(29±3)]; revelando que 67% das mulheres com hipertensão severa apresentavam síndrome metabólica (SM). O desenvolvimento da hipertensão e da SM foi diretamente correlacionado com a diminuição das EPCs. Portanto, a contagem de EPCs pode ser considerada um marcador biológico adequado para indicar a gravidade do estado hipertensivo em mulheres
Abstract: Endothelial progenitor cells (EPCs) are involved in neovasculogenesis and maintenance of vascular homeostasis and their impairment may have a role in the pathogenesis of hypertension. This study aimed analyzes the expression profile of circulating EPCs and different cardiovascular risk factors in hypertensive premenopausal women compared with healthy normotensive women. A case-control study was conducted enrolling 45 women volunteers, aged from 30- 50 years (41 ± 6) in Ambulatory of the Cardiologic Emergency Hospital of Pernambuco. EPCs numbers were determined by flow cytometry in peripheral blood as the CD45-/CD34+/KDR+ cells. The women were classified as healthy normotensive controls (CT) with SBP (systolic blood pressure) <130 mmHg and DBP (diastolic blood pressure) < 85 mmHg (n=15), and with essential hypertension; a) mild (MH), SBP=140-159 mmHg and DBP=90-99 mmHg (n=15); and b) severe (SH), SBP>180 mmHg and DBP>110 mmHg (n=15). The group were interviewed regarding smoking habits, physical exercise and body mass index (BMI), and measured the level of blood pressure at quiescent. An analysis in records of test results cholesterol, high density lipoprotein-cholesterol (HDL-c), low density lipoprotein-cholesterol (LDL-c), triglycerides and fasting glucose in the month of collection of blood samples. The results found a significant reduction in circulating EPCs numbers in MH (74%) and SH (88%) when compared to the CT and reduction of 67% in SH when compared to MH, an inverse relationship between the number of cells and the stage of hypertension. SH group showed an increase of 49% CD45+ cells demonstrating inflammation and reduction of 61% CD45-/ CD34+ cells. Regarding the biochemical serum was found: HDL-c [MH (52±7); SH (48±5)]; LDL-c [MH (130±8); SH (143±15)]; triglycerides [MH (138±19); SH (153±40)]; fasting glucose [MH (95±7); SH (121±39)] and BMI [MH (31±4); SH (29±3)]; revealing that 67% of women with severe hypertension had metabolic syndrome (MS). Development of hypertension and the parameters related to MS are directly correlated with a decrease of circulating EPCs. Therefore, the EPCs counting may be considered a suitable biological marker to follow up the evolution of the hypertensive state in women
Doutorado
Biologia Celular
Doutora em Biologia Celular e Estrutural

Malgré l'efficacité thérapeutique avérée des agents anti-angiogéniques et des agents anti-vasculaires (VDA), le mécanisme d'action précis des stratégies ciblant les vaisseaux sanguins tumoraux, les raisons de leur efficacité ainsi que les mécanismes de résistance à ces drogues sont encore mal compris. Il est rapidement apparu essentiel d'identifier des biomarqueurs capables de refléter l'angiogénèse tumorale ou les effets sur la vascularisation tumorale de ces traitements. Compte tenu de leur importance dans des pathologies vasculaires, les cellules endothéliales matures circulantes (CEC) et les progéniteurs endothéliaux circulants (CEP) ont d'emblée été pressenties comme des candidats intéressants pour être des biomarqueurs de réponse aux stratégies ciblant la vascularisation tumorale. Nous avons exploré l'intérêt de ces cellules en tant que biomarqueurs de l'angiogénèse dans des tumeurs pédiatriques, et leur rôle en tant que biomarqueurs de traitement par des agents anti-angiogéniques chez des sujets adultes atteints de cancer. Ces travaux ont mis en lumière l'intérêt des CEP et ont été à la source d'un travail plus " mécanistique " où nous avons étudié dans différents modèles murins le rôle des CEC et CEP dans le mécanisme d'action des agents anti-vasculaires et plus particulièrement le rôle fonctionnel des CEP dans la résistance à ces molécules. Par des stratégies d'association d'agents anti-angiogéniques aux VDA destinées à inhiber les CEP, nous montrons l'augmentation de l'activité anti-tumorale des VDA et offrons un rationnel mécanistique pour optimiser les schémas thérapeutiques actuels des traitements anti-vasculaires. Nos données apportent des arguments en faveur du rôle potentiel de ces cellules en tant que biomarqueurs de l'angiogénèse, des traitements anti-angiogéniques et de la résistance aux traitements anti-vasculaires.

Cell therapy has been shown to improve the process of revascularization in ischemic myocardial tissue; however, the search for a cell population that can achieve optimal vasculogenic and/or angiogenic potential is ongoing. This research compared the in vitro functional properties of different circulating progenitor cell (CPC) subpopulations from the peripheral blood and determined whether interactions between subsets of CPCs are involved in mediating their function. Of all populations investigated, a novel population referred to as derived CD133+ cells seemed to exhibit superior functional potential. Furthermore, the migratory ability of the different CPC populations was significantly enhanced in the presence of cytokines/growth factors secreted by cultured monocytes. Lastly, we observed that CPC tubule formation in a pro-angiogenic environment required the support of a mature endothelial cell population. Taken together, these results indicate that specific phenotype and intercellular interactions may be necessary for CPCs to achieve their optimal functional potential for new blood vessel growth.

La présence synchrone de métastases hépatiques (MH) et carcinose péritonéale (CP)d'origine colorectale (CRC) est associée à une survie globale médiocre et est traditionnellement considérée comme une contre-indication à l’approche chirurgicale curative. Cependant, suite aux résultats encourageants après traitement chirurgicale, quelques séries ont rapporté une survie prolongée atteignant 3 ans chez des patients sélectionnés, ce qui suggère qu’un traitement chirurgicale curative est possible. À ce jour, en cas de chirurgie majeure hépatique et péritonéale associée, aucune stratégie thérapeutique n'a été établie, Nous avons postulé que la régénération hépatique après une résection hépatique pourrait favoriser la croissance de la CP. Nous avons construit un modèle animal immunnocompetent de CP limitée. L'objectif de l’étude était d'analyser les effets de l’hépatectomie majeure et de la régénération hépatique dans notre modèle murin de PC et le processus d'angiogenèse associé. En outre, nous avons analysée une cohorte prospective multicentrique de patients ayant eu une résection hépatique et une chirurgie cytoréductive avec HIPEC synchrone. L'objectif de cette étude était d'évaluer les outcomes et identifier les facteurs pronostiques afin d'optimiser la sélection des candidats au traitement chirurgical et de déterminer la stratégie chirurgicale optimale
The synchronous presence of liver metastases (LM) and peritoneal carcinomatosis (PC)from colorectal cancer (CRC) is associated with poor outcome and is traditionally considered acontraindication to any surgical approach. However, few series reported a prolonged survival aftersurgical management, reaching 3 years in selected patients thus suggesting that a curative surgicalmanagement may be possible. To date, no standard management pathway has been established,especially if a major liver and peritoneal surgery has to be performed. We postulated that liverregeneration after liver resection could promote PC growth. We constructed an immunocompetentanimal model of limited PC. The objective of our study was to analyze the effects of major LR andliver regeneration after hepatectomy on peritoneal carcinomatosis growth and the associatedangiogenesis process. Furthermore, we have analyzed a prospective international cohort of patientsundergoing synchronous liver resection and cytoreductive surgery with HIPEC. The aim of this studywas to describe the outcomes, to identify variables potentially related to poor outcome, in order toestablish future guideline for the management of these patients, to optimize the selection of candidatesfor surgical treatment and determine the best surgical strategy

碩士
國立陽明大學
生醫光電工程研究所
98
Colorectal cancer is sencend common cause of cancer-related death. Angiogenesis is the key steps in tumor growth and Metastasis. Circulating endothelial cell (CEC) and circulating progenitor cell (EPC) is very important in angiogenesis. Circulating endothelial cell (CEC) and circulating progenitor cell (EPC) numbers are present in cancer, but their relationship with angiogenesis, vascular biology, and prognosis is unclear. Increases in the number of circulating endothelial cells (CECs) and progenitors (EPCs) have been reported in various pathological conditions including cancer. At the clinical level, evidence is emerging that CEC kinetics and viability might correlate with clinical outcomes in cancer patients who undergo anti-angiogenic treatment. We studied 144 patients with colorectal cancer , measuring EPCs .(CD45dim-CD31+-CD146++(或KDR+)-CD133+) and CPCs (CD45dim-CD31+-CD146++(或KDR+)-CD133-)by flow cytometry. In the result circulating endothelial progenitor cells noncorrelate with stage in colorectal cancer, then Stage II EPC and CEC levels higher than another stage. CECs and EPCs number are different after surgery. the CECs and EPCs number decrease after surgery.

The objective of the present thesis was to compare the levels of circulating endothelial progenitor cells (EPCs), circulating endothelial cells (CECs), and hematopoietic stem cells (HSCs) between patients with heart failure with reduced ejection fraction (HFrEF) and a group of subjects with cardiovascular risk factors. We also compared the levels of circulating EPCs, CECs, and HSCs between subgroups regarding the presence of cardiovascular risk factors (e.g. diabetes mellitus) and the etiology of heart failure (HF). To achieved this, whole peripheral blood was drawn from patients previously diagnosed with HFrEF (n = 42) and age-matched subjects presenting similar cardiovascular risk factors but without established cardiovascular disease (n = 42). Then, a combination of markers was used in peripheral blood samples in order to assess the number of circulating EPCs, CECs, and HSCs via flow cytometry analysis. Patients with HFrEF had significantly decreased levels of circulating EPCs (5.28 x 10-3 ± 6.83 x 10-4 % vs 7.76 x 10-3 ± 4.91 x 10-4 %, P ≤ 0.001) and CECs (5.11 x 10-3 ± 7.87 x 10-4 % vs 6.51 x 10-3 ± 5.21 x 10-4 %, P = 0.005) compared to subjects with cardiovascular risk factors. However, levels of HSCs were not significantly different between the two groups (P = 0.590). Additionally, CECs (6.69 x 10-3 ± 6.38 x 10-3 % vs 3.61 x 10-3 ± 2.71 x 10-3 %, P = 0.057) tended to circulate in higher number in patients with ischemic HF compared to patients with non-ischemic HF. Patients with HFrEF and diagnosed as overweight/obese had significantly higher levels of circulating EPCs (6.10 x 10-3 ± 4.78 x 10-3 % vs 4.13 x 10-3 ± 3.55 x 10-3 %, P = 0.043) and CECs (6.27 x 10-3 ± 5.66 x 10-3 % vs 3.47 x 10-3 ± 3.54 x 10-3 %, P = 0.019) when compared to patients with HFrEF presenting a normal weight. Lastly, when comparing subjects from the age-matched group, subjects with dyslipidemia had significantly higher levels of CECs (7.74 x 10-3 ± 3.64 x 10-3 % vs 5.34 x 10-3 ± 2.59 x 10-3 %, P = 0.042) compared to subjects without dyslipidemia. In conclusion, the main result of this study is that the circulating levels of EPCs and CECs were significantly decreased in patients with HFrEF in comparison to subjects with cardiovascular risk factors. The current observations regarding cardiovascular risk factors suggest that EPCs, CECs, and HSCs play an important role in the detection and repair of vascular damage and endothelial dysfunction.
O presente trabalho teve como principal objetivo comparar os níveis de células endoteliais progenitoras (CEPs), células endoteliais circulantes (CECs) e células estaminais hematopoiéticas (CEHs) em circulação entre doentes com insuficiência cardíaca com fração de ejeção reduzida (ICFEr) e um grupo de adultos com fatores de risco cardiovasculares. Adicionalmente, os níveis das CEPs, CECs e CEHs foram comparados entre subgrupos em função da presença de fatores de risco (ex. diabetes) e da etiologia da insuficiência cardíaca. Inicialmente foram recolhidas amostras de sangue periférico de doentes com ICFEr (n = 42) e indivíduos da mesma faixa etária com fatores de risco cardiovasculares, mas sem qualquer doença cardiovascular estabelecida (n = 42). Em seguida, foi utilizada uma combinação de anticorpos nas amostras de sangue periférico para quantificação do número de CEPs, CECs e CEHs por citometria de fluxo. Doentes com ICFEr apresentaram níveis de CEPs (5.28 x 10-3 ± 6.83 x 10-4 % vs 7.76 x 10-3 ± 4.91 x 10-4 %, P ≤ 0.001) e CECs (5.11 x 10- 3 ± 7.87 x 10-4 % vs 6.51 x 10-3 ± 5.21 x 10-4 %, P = 0.005) significativamente inferiores aos indivíduos com fatores de risco cardiovasculares. Contudo, não foram encontradas diferenças significativas nos níveis de CEHs entre os dois grupos (P = 0.590). Adicionalmente, observou-se que as CECs (6.69 x 10-3 ± 6.38 x 10-3 % vs 3.61 x 10-3 ± 2.71 x 10-3 %, P = 0.057) tendem a circular em maior número em doentes com ICFEr com etiologia isquémica comparativamente a doentes com ICFEr não isquémica. Doentes com ICFEr e com sobrepeso/obesidade apresentaram níveis de CEPs (6.10 x 10-3 ± 4.78 x 10-3 % vs 4.13 x 10-3 ± 3.55 x 10-3 %, P = 0.043) e CECs (6.27 x 10-3 ± 5.66 x 10- 3 % vs 3.47 x 10-3 ± 3.54 x 10-3 %, P = 0.019) significativamente superiores comparativamente a doentes com ICFEr e com peso normal. Por último, dentro do grupo de indivíduos com fatores de risco cardiovasculares, indivíduos com dislipidemia apresentaram níveis de CECs (7.74 x 10-3 ± 3.64 x 10-3 % vs 5.34 x 10-3 ± 2.59 x 10-3 %, P = 0.042) significativamente superiores em comparação a indivíduos sem dislipidemia. Em conclusão, os principais resultados deste estudo indicam que o número de CECs e CEPs em circulação encontra-se significativamente reduzido em doentes com ICFEr comparativamente a indivíduos com fatores de risco para doenças cardiovasculares. As observações atuais em relação aos fatores de risco para doenças cardiovasculares sugerem que CEPs, CECs e CEHs desempenham um papel fundamental na sinalização e reparação do dano vascular e disfunção endotelial.
Mestrado em Biomedicina Molecular

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Introdução: Estudos recentes sugerem que as células progenitoras endoteliais (EPCs) circulantes podem participar na resposta à terapêutica de ressincronização cardíaca (TRC). O objetivo deste estudo foi avaliar o efeito da TRC nos níveis de EPCs circulantes e avaliar o impacto das EPCs no prognóstico a longo prazo. População e métodos: Estudo prospetivo de 50 doentes submetidos a TRC. Antes da implantação, foram quantificadas 2 populações de EPCs circulantes por citometria de fluxo: células CD34+KDR+ e CD133+KDR+. Os níveis destas EPCs foram reavaliados 6 meses após TRC. Os endpoints durante o seguimento a longo prazo foram mortalidade por todas as causas, transplantação cardíaca e hospitalização por insuficiência cardíaca (IC). Resultados: A proporção de não respondedores à TRC foi de 42%, tendendo a ser maior nos doentes com etiologia isquémica versus não isquémica (64% vs 35%, p = 0.098). Os doentes com miocardiopatia isquémica (MCI) apresentavam níveis significativamente mais baixos de EPCs CD34+KDR+ quando comparados aos doentes com miocardiopatia dilatada não isquémica (MCD) (0.0010 ± 0.0007 vs 0.0030 ± 0.0024 células/100 leucócitos, p = 0.032). Não se verificaram diferenças significativas nos níveis basais de EPCs entre sobreviventes e não sobreviventes, nem entre doentes com ou sem necessidade de internamento para tratamento da IC durante o seguimento. Aos 6 meses de seguimento, os níveis de EPCs circulantes eram significativamente maiores do que os níveis basais (0.0024 ± 0.0023 vs 0.0047 ± 0.0041 CD34+KDR+/100 leucócitos, p = 0.010 e 0.0007 ± 0,0004 vs 0.0016 ± 0.0013 CD133+KDR+/100 leucócitos, p = 0.007). Conclusões: Os doentes com MCI apresentam níveis basais de EPCs circulantes significativamente mais baixos que os seus homólogos. A TRC parece melhorar o pool endógeno de EPCs circulantes e níveis basais reduzidos de EPCs não parecem influenciar os outcomes a longo prazo após a TRC.
Aims: Recent studies suggest that circulating endothelial progenitor cells (EPCs) may influence the response to cardiac resynchronization therapy (CRT). The aim of this study was to evaluate the effect of CRT on EPCs levels and to assess the impact of EPCs on long-term clinical outcomes. Population and methods: Prospective study of 50 patients submitted to CRT. Two populations of circulating EPCs were quantified previously to CRT implantation: CD34+KDR+ and CD133+KDR+ cells. EPCs levels were reassessed 6 months after CRT. Endpoints during the long-term follow-up were all-cause mortality, heart transplantation and hospitalization for heart failure (HF) management. Results: The proportion of non-responders to CRT was 42% and tended to be higher in patients with an ischemic vs non-ischemic etiology (64% vs 35%, p = 0.098). Patients with ischemic cardiomyopathy (ICM) showed significantly lower CD34+KDR+ EPCs levels when compared to non-ischemic dilated cardiomyopathy patients (DCM) (0.0010 ± 0.0007 vs 0.0030 ± 0.0024 cells/100 leukocytes, p = 0.032). There were no significant differences in baseline EPCs levels between survivors and non-survivors nor between patients who were rehospitalized for HF management during follow-up or not. At 6-month follow-up, circulating EPCs levels were significantly higher than baseline levels (0.0024 ± 0.0023 vs 0.0047 ± 0.0041 CD34+KDR+ cells/100 leukocytes, p = 0.010 and 0.0007 ± 0.0004 vs 0.0016 ± 0.0013 CD133+KDR+ cells/100 leukocytes, p = 0.007). Conclusions: Patients with ICM showed significantly lower levels of circulating EPCs when compared to their counterparts. CRT seems to improve the pool of endogenously circulating EPCs and reduced baseline EPCs levels seem not influence long-term outcomes after CRT.

碩士
長庚大學
物理治療學系
100
Background and Purpose: Bone marrow-derived, circulating progenitor cells (CPCs) is contributing to the maintenance of endothelial function by mechanisms that endothelial repair to postnatal angiogenesis and vasculogenesis. Although exercise has been suggested that can upregulate CPCs number in blood, the effect of different mode of exercise on CPCs subsets remains unclear. High intensity aerobic interval training (AIT) has been shown to have a more profound influence on cardiovascular function and aerobic capacity than isocaloric moderate intensity continue training (MCT). The aim of this study was to investigate how AIT influences the mobilization of CPCs subsets into peripheral blood compartment. A second objective was to compare MCT with AIT that the adaptation of ischemic and hypoxic hypoxia changing in cell number and function. Methods: Thirty-two sedentary healthy men were participating in the study, which were randomly distributed to AIT exercise group (reciprocal 40%, 80%VO2max on a bicycle ergometer for 30min per day, 5 days per week, total 25 times) or MCT exercise group (60%VO2max). Acute severity hypoxia exercise testing was performed at pre and post chronic exercise training (60% VO2 max exercise intensity for 40 min under 12%O2 in air). At rest and immediately after severity hypoxia exercise, total of stem cells (SCs, CD34+ cells), circulating angiogenesis cells (CACs, CD34+/KDR+ cells), hematopoietic stem cells (HSCs, CD34+/KDR+/CD117+ cells), endothelial progenitor cells (EPCs, CD34+/KDR+/CD133+ cells), circulation endothelial cells (CECs, CD34+/KDR+/CD31+ cells), and the apoptosic cells number of CACs (CD34+/KDR+/phosphotidylserine exposed cells) were measured by three-color flow cytometry. Subsequently, we measured the ability of CPCs and subtypes cell proliferation by cell culture. Result：Exercise training improved cardiopulmonary fitness in AIT and MCT group. In blood analysis, acute hypoxia exercise testing could improve CACs and EPCs in first time. After five weeks training, AIT group increased the number of SCs, CACs and EPCs in peripheral blood compartment and MCT group significantly increased the number of CACs in the blood. Conclusion：Aerobic interval training markedly induced SCs differentiation to the functional CPCs into peripheral blood. Clinic Application and Future Research ：This study clarify the relationships between AIT and MCT modulated redistribution of CPCs. In future, require more research relevant to CPCs differentiation and proliferation capacity and improve angiogenesis ability in vivo by physical therapy method. We expect to provide a safe and effective strategy of exercise therapy for patients with endothelial dysfunction.

博士
國立成功大學
臨床醫學研究所
105
Pulmonary hypertension is a progressive disease and leading to right heart failure and death. Although newer treatments for pulmonary arterial hypertension have emerged, only modest functional improvement with minimal change in hemodynamic measurements have been achieved. Based on the current knowledge of PAH, the major problem is the poor prognosis and unclear mechanism. Established an adequate predictive model to adjust treatment strategy and an experimental model for deeper understanding of mechanism and new therapies are important. In experimental models, I focus on the functional and molecular remodeling in pulmonary circulation following cessation of excessive pulmonary flow. We enrolled patients with pulmonary hypertension related to congenital left to right shunt to evaluate dynamic change following cessation of excessive flow. We demonstrated that hemodynamic, right ventricular function and daily performance improve following volume reduction. I also create an A-V fistula rat model demonstrated that normalization of pulmonary blood flow in subjects with flow-induced pulmonary hypertension reverses the remodeling in the right ventricle and pulmonary arterial circulation, and potentiates the vascular reactivity of pulmonary artery. The remodeling process of flow-induced pulmonary hypertension is most likely reversible and these changes are closely related to differentiation and switching of vascular smooth muscle cells in the pulmonary artery and modulation of tissue inflammatory cytokines. In predictive model, my study reassessed the predictive power of the REVEAL prognostic equation using exercise treadmill test in place of six-minute walk distance. The predictive power of the equation improved and supports exercise treadmill test for predicting survival in PAH using the REVEAL risk calculator. The study allows centers to utilize exercise treadmill test in place of the six-minute walk distance allow better prognostic information for their patients. From our results, we believe that the regression of remodeling caused by pulmonary hypertension is possible. Another novel finding of this study is that qualitative enhancement of colony formation in the circulating EPCs and simultaneous and positively correlated with the patients’ daily performance. Molecular and functional changes of endothelial progenitor cells may play an important role for pulmonary artery remodeling. We adjust treatment strategy according to predictive model and aggressively control volume status in daily practice and critical care leading to improve patient’s outcome. At the same time, deeper investigation to obtain a deeper understanding of the mechanism of remodeling regression is ongoing with our experimental models.

Endometriosis affects 5-10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Treatment for endometriosis primarily focuses on symptom relief, is short term with severe side effects and often leads to recurrence of the condition. Establishing new blood supply is a fundamental requirement for endometriosis lesions growth. This has led to the idea that antiangiogenic therapy may be a successful approach for inhibiting endometriosis. Recent evidence indicates that endothelial progenitor cells (EPCs) contribute to neoangiogenesis of endometriotic lesions. These EPCs are recruited to the lesion site by stromal cell-derived factor-1 (SDF-1). We hypothesize that SDF-1 is central to the neoangiogenesis and survival of endometriotic lesions and that administration of SDF-1 blocking antibody will inhibit lesion growth by inhibiting angiogenesis in a murine model of endometriosis. Immunohistochemistry for SDF-1 and CD34 was performed on human endometriosis and normal endometrial samples. Quantification of SDF-1 and EPCs was performed in the blood of endometriosis patients and controls using ELISA and flow cytometry, respectively. A new mouse model of endometriosis was developed using BALB/c-Rag2-/-/IL2rg-/- mice to investigate role of SDF-1 in neoangiogenesis. Either SDF-1 blocking antibody or an isotype control was administered on a weekly basis for four weeks. Weekly samples of peripheral blood from mice were analyzed for SDF-1, other cytokines of interest and EPCs. Mice were euthanized at seven weeks to observe lesion growth and blood vessel development. Our results indicate overabundance of SDF-1 and CD34+ progenitor cells in human endometriotic lesions compared to eutopic endometrium. In the mouse model, SDF-1 and circulating EPC levels decreased from pre-treatment levels after one week, and remained constant over the course of the treatment in both SDF-1 blocking antibody and isotype control groups. In the SDF-1 blocking group, reduced vascularity of lesions, identified by immunofluorescence staining for CD31, was revealed compared to isotype controls. These findings suggest that SDF-1 may be responsible for CD34+ progenitor cell recruitment to the neoangiogenic sites in endometriosis. Blocking of SDF-1 reduces neovascularization of human endometriotic lesions in a mouse model. Further studies on blocking SDF-1 in combination with other antiangiogenic agents are needed.
Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2011-12-21 19:34:43.054

碩士
國立陽明大學
臨床醫學研究所
102
Background: Phytosterols, plant sterols, which have a similar structure to cholesterol, have cholesterol lowering and anti-inflammatory effects. We previously reported that nonalcoholic fatty liver disease (NAFLD) had decreased circulating endothelial progenitor cells (EPCs) number, and this has been suggested as one of the mechanisms behind atherosclerotic disease progression and enhanced cardiovascular risk in patients with NAFLD. Objective: This study tested the hypothesis that treatment with phytosterols in patients with NAFLD may suppress systemic inflammation and increase circulating EPC levels. Design: Forty consecutive patients with an abdominal ultrasonographic diagnosis of NAFLD were randomly assigned to phytosterols powder treatment at 1.8g/day for 4 weeks (n=20) or a control group (n=20), with crossover to the alternate therapy for another 4 weeks after 2-week wash-out period. Flow cytometry with quantification of EPC markers (defined as CD34+, CD34+KDR+, and CD34+KDR+CD133+) in peripheral blood samples was used to assess circulating EPC levels. Results: The mean age of the 40 study patients (21 males, 53%) was 51±2 years (range 25-78 years). Four weeks of phytosterols treatment significantly decreased levels of total cholesterol, non high-density lipoprotein cholesterol (non HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting glucose, and hemoglobin A1c (HBA1c), but did not change levels of high-density lipoprotein cholesterol (HDL-C) and triglycerides. Treatment with phytosterols for 4 weeks in patients with NAFLD markedly suppressed high sensitivity C-reactive protein, an inflammatory marker (p=0.0069), and enhanced superoxide dismutase (SOD),an anti-oxidative marker (p=0.0005). We also showed that administration of phytosterols significantly increased the insulin-like growth factor-1 (IGF-1) concentrations (p<0.0001), but failed to have any significant effects on nitric oxide or stromal derived factor-1 levels. Moreover, intake of phytosterols significantly enhanced circulating EPC levels (including: CD34+, CD34+KDR+, CD34+KDR+CD133+, all p<0.05) in NAFLD patients. Conclusion: Four weeks treatment with phytosterols in NAFLD patients significantly improved insulin resistance, suppressed systemic inflammation, enhanced anti-oxidant capacity, and increased circulating EPC levels. Despite beneficial effect on lipids, glucose and EPCs, short term phytosterols did not seem to improve endothelial function in NAFLD patients. Trial Registration: clinicaltrials.gov number NCT01875978 completed

碩士
長庚大學
物理治療學系
98
Background and Purpose: Bone marrow-derived, circulating endothelial progenitor cells (EPC) is contributing to the maintenance of endothelial function and organ perfusion by mechanisms ranging from endothelial repair to postnatal angiogenesis and vasculogenesis. Although exercise or hypoxia has been observed to modulate EPC number in blood, the effect of exercise combined with hypoxia on the redistribution of circulating EPC subsets remains unclear. This study investigates how hypoxic exercise influences the mobilization of EPC subsets into the peripheral blood compartment. Methods: Forty-one sedentary healthy men were participated in the study, who were randomly distributed to hypoxic exercise group (60%VO2max exercise intensity for 40 min under 15%O2 in air on a bicycle ergometer 5days per week, total training 25 times) or normoxia group (under 21%O2 in air). Acute severity hypoxia exercise testing was performed at pre and post chronic exercise training (60%VO2max exercise intensity for 30 min under 12%O2 in air). At rest and immediately after severity hypoxia exercise, total of stem cells (SC, CD34+ cells) and overall the cell of associated with angiogenesis (CD34+/KDR+ cells), hematopoietic stem cells (HSC, CD34+/KDR+/CD117+ cells), endothelial progenitor cells (EPC, CD34+/KDR+/CD133+ cells), circulation endothelial precursor cells (CEP, CD34+/KDR+/CD133- cells), circulation endothelial cells (CEC, CD34+/KDR+/CD31+ cells), and shedding endothelial cells from vessels (SEC, KDR+/CD31+/phosphotidylserine exposed cells) were measured by three-color flow cytometry. Subsequently, we measured EPCs and subtypes cell proliferation ability by cell culture. Results: Exercise training improved cardiopulmonary fitness in normoxia and hypoxia groups. In blood analysis, acute hypoxia exercise testing could improve lymphocyte, stem cells, CD34 with KDR double positive cells, EPCs and CEPs in the first time. Through five weeks training, normoxia exercise increased the count of EPCs in peripheral blood compartment and hypoxia exercise significantly increased the number of CEP in the blood. Moreover, the amounts of SC, EPC and CEP in blood and EPC proliferation rate were significantly higher followed by normoxia exercise training. Additionally, hypoxia exercise improved more capability of resistance severely hypoxia. Conclusion: Hypoxic exercise markedly induced stem cell differentiation to the functional CEPs into peripheral blood, but that decreased count of EPC colony and proliferation rate by oxidation stress. Clinic Application and Future Research: This study clarify the relationships between hypoxic and normoxia exercise modulated redistribution of hematopoietic progenitor cells. In future, require more research relevant to EPC differentiation and proliferation capacity and improve angiogenesis ability in vivo by physical therapy method. We expect to provide a safe and effective strategy of physical therapy for patients with endothelial dysfunction.

碩士
國立陽明大學
微生物及免疫學研究所
104
Endothelial progenitor cells (EPCs) plays important role in vascular repair and angiogenesis. Repair of endothelial cell layer could be accomplished by induction EPCs origin from bone marrow. EPCs maintains the function and the integrity of blood vessels. There and many risk factor that may reduce the number and function of EPCs. Dysfunction and reduction of EPCs in peripheral blood is also correlated with the onset of cardiovascular disorders, including coronary artery diasease(CAD). Previous published paper has demonstrated cell function of CAD-EPCs were reduced compare to Healthy-EPCs. Previous member of our lab has performed several group of CAD plasma small RNA sequencing (smRNA-seq). We found there are 83 miRNA elevated in plasma from patients with CAD and 84 miRNA elevated in CAD-EPCs. We are curious about the effect of miRNA that were elevated in both CAD plasma & EPCs. We are hypothesis these miRNA will play a role in EPCs function. Then we cross these two list of miRNA and found 12 miRNA are elevated both in CAD plasma & EPCs We validated top 5 miRNA expression with patients with CAD, and found miR-26a-5p,miR-146a-5p and miR-146b-5p were enriched in CAD plasma furthermore miR-146a-5p and miR-146b-5p were also really high. Using lenti-virus packed with anti-miR-146a-5p and anti-miR-146b-5p to knockdown the expression of both microRNAs. We further discover knockdown of miR-146a-5p and miR-146b-5p improves the function of EPCs. Performing microarray to find out the gene that were regulated by these two microRNAs. We found 15 gene that could improve angiogenesis ability of EPCs, but there are only 8 genes that could be potential target by miR-146a-5p that is CXCL12, PRKCE, SKP2, CSF1, S1PR3, FLNA, CAV1 and RHOJ and also 8 genes that could be potential target of miR-146b-5p that is CXCL12, MMP2 ,PRKCE ,S1PR3 ,FLNA ,CAV1 ,NRG1 and RHOJ. We observe the expression of these genes in miR-146a-5p and miR-146b-5p separately, and we found the expression of S1PR3, FLNA, CAV1 and RHOJ were elevated relative to control. Further we compared these genes in CAD & Healthy EPCs and discover only RHOJ and CAV1 has significant difference between these two groups. For further confirm the binding of microRNA to the target site we perform reporter assay, and found the expression of RHOJ could by miR-146b-5p.So we use shRNA to knockdown RHOJ and found reduced tubeformation but not migration. Collectively, we discover miR-146a-5p & miR-146b-5p could regulated the expression of EPCs that could help developed new therapeutic not only for cardiovascular disease but also tumor angiogenesis.

碩士
長庚大學
傳統中國醫學研究所
96
The prevalence of obesity increased markedly in recent years, and many studies revealed that obesity increased the incidences of steatosis, insulin resistance (IR), diabetes mellitus, hypertension, and cardiovascular diseases. Oxidative stress and inflammation have been recognized as the important factors in the development of obesity-related liver injury. Adipokines and adipocytokines secreted by abdominal fat tissue promoted inflammatory cells infiltration and oxidative stress in the liver. Otherwise, a relationship between IR and vascular endothelial dysfunction has been identified as an important factor in the development of atherosclerosis. Circulating endothelial progenitor cell (EPC) of bone marrow origin contributes to repair damaged vascular endothelium, and it has been identified as one of the prognostic factors of cardiovascular diseases. Yin-Chen-Hao-Tang (YCHT) has been used for the treatment of jaundice traditionally, and the previous studies revealed that it could inhibit hepatocyte apoptosis, liver oxidative stress, and liver fibrosis. One of the aims of this study was to determine whether obesity induce liver oxidative stress and inflammation to suppress circulating EPC, and the other was to determine whether YCHT can ameliorate obesity-related liver oxidative stress and inflammation. Female hamsters were fed normal chow (N group, n=6) or liquid high fat diet (HFD group, n=5) for 15 weeks, and the treatment group (YCHT group, n=5) was fed liquid high fat diet for 15 weeks with YCHT 250mg/kg/day at the last three weeks. Body weight was monitored once a week. Blood samples were collected for biochemistry analysis and flow cytometry at the end of the experimental period, and the thoracic aortas were collected for histopathology. Liver samples were collected for histopathology, glutathione (GSH) analysis and proteomics. Two-dimensional gel electrophoresis was used to analyze proteome changes. Protein spots were detected from pH4 to pH7 and identified with MALDI-TOF mass spectrometry. Compared with N group, HFD group and YCHT group exhibited higher body weight and serum triglyceride. Flow cytometry analysis revealed that YCHT could reverse HFD-induced circulating EPC decreasing, and it inhibited collagen deposition at the vessel wall of thoracic aorta due to obesity. Histopathology of liver showed that HFD-induced fat accumulation in the hepatocyte around the central vein and granulocyte infiltration around the portal triad were both attenuated by YCHT. GSH/GSSG ratio in liver also decreased significantly in HFD group, and it was reversed by YCHT. Twenty four different proteins down-regulated significantly in HFD group identified by proteomic analysis, included oxidative stress-related proteins such as peroxiredoxin-6, glutathione S-transferase, and regucalcin. The expression of proteins related to glucose metabolism, such as glucose-regulated protein and fructose-1,6-bisphosphatase, were also decreased in obese hamsters. YCHT reversed the expressions of proteins in the liver. In conclusion, YCHT attenuated obesity-induced liver oxidative stress to reverse the decreasing of circulating EPC, and it may influence the metabolism of glucose and lipid.