Immature Endothelial Progenitor Cells Research Articles

The GG genotype of the -152 G/A vascular endothelial growth factor (VEGF) polymorphism predisposes to hypertension-related chronic kidney disease.

Our purpose was to determine whether the VEGF -152 G/A polymorphism could be associated with chronic kidney disease and endothelial dysfunction in hypertensive patients. There were 100 healthy volunteers enrolled into the control group. The group of patients was constituted by 99 consecutively admitted hypertensive patients referred to our Institution by their general practitioner. All patients were treated with anti-hypertensive polytherapy. Presented study revealed that the hypertensive patients bearing the GG genotype were characterized by the highest values of diastolic blood pressure and markers of endothelial damage such as Angiogenin, Endostatin, CRP as well as von Willebrandt factor. In addition, higher number of immature endothelial progenitor cells with CD34(+)CD133(+), CD34(+)CD133(-) markers was observed in GG hypertensive carriers while in normotensive individuals no differences were found. Such phenomenon may indicate an increased mobilization of bone-marrow derived endothelial progenitors. It may testify to the preserved compensatory mechanism in chronic kidney disease (CKD) patients until the G3a stage of the disease. Moreover, patients with higher estimated glomerular filtration rate (eGFR) level had lower of vWf and Endostatin values, and higher level of VEGF. Taken together our findings clearly indicate the -152 GG hypertensive carriers as more prone to develop CKD. We can suspect that the VEGF -152 GG genotype is strongly associated with hypertension-dependent CKD.

Vascular endothelial growth factor polymorphism (–460 T/C) is related to hypertension-associated chronic kidney disease

ABSTRACTOur aim was to characterize the endothelial progenitor cells (EPCs) in normotensive controls and treated hypertensive individuals within the vascular endothelial growth factor (VEGF) –460 C/T polymorphism as well as to investigate whether this polymorphism predisposes to hypertension-related chronic kidney disease. The hypertensive patients bearing the TT genotype had the highest levels of immature EPC with the following phenotypes: CD34+, CD34+CD45dim, CD34+CD133+CD45dim. The study showed the estimated glomerular filtration rate values significantly lower and creatinine and BUN parameters higher among the TT hypertensive patients. We presume that the highest mobilization of EPCs from bone marrow may signalize more severe renal hypertension-related complications in the VEGF –460 TT genotype.

Correlation between osteocalcin-positive endothelial progenitor cells and spotty calcification in patients with coronary artery disease.

Immature endothelial progenitor cells (EPC) carrying osteocalcin (OCN) might mediate vascUlar calcification in coronary artery disease (CAD). Spotty calcification within atherosclerotic plaque is associated with cardiovascular events. The aim of the present study was to assess the correlation between immature EPC levels and spotty calcification in CAD patients. In the 224 CAD patients studied, 76 had acute myocardial infarction (AMI), 102 had unstable angina pectoris (UAP), and 46 had stable angina pectoris (SAP). The levels of OCN-positive (OCN+) EPC were analysed by flow cytometry. The status of spotty calcification was determined by cardiac computed tomography angiography. OCN+ EPC and calcium deposits were significantly increased in acute coronary artery syndrome (ACS) when compared with those in SAP patients. Positive correlation was also revealed between the number of OCN+ EPC and the frequency of spotty calcification and levels of serum high-sensitivity C-reactive protein (hs-CRP) and serum alkaline phosphatase in AMI and UAP patients. In summary, the number of OCN+ EPC is positively related to the frequency of spotty calcification in ACS patients. Serum hs-CRP and serum alkaline levels are thought to contribute to the elevation of OCN+ EPC.

Fluid shear stress induces differentiation of circulating phenotype endothelial progenitor cells

Endothelial progenitor cells (EPCs) are mobilized from bone marrow to peripheral blood, and contribute to angiogenesis in tissue. In the process, EPCs are exposed to shear stress generated by blood flow and tissue fluid flow. Our previous study showed that shear stress induces differentiation of mature EPCs in adhesive phenotype into mature endothelial cells and, moreover, arterial endothelial cells. In this study we investigated whether immature EPCs in a circulating phenotype differentiate into mature EPCs in response to shear stress. When floating-circulating phenotype EPCs derived from ex vivo expanded human cord blood were exposed to controlled levels of shear stress in a flow-loading device, the bioactivities of adhesion, migration, proliferation, antiapoptosis, tube formation, and differentiated type of EPC colony formation increased. The surface protein expression rate of the endothelial markers VEGF receptor 1 (VEGF-R1) and -2 (VEGF-R2), VE-cadherin, Tie2, VCAM1, integrin α(v)/β(3), and E-selectin increased in shear-stressed EPCs. The VEGF-R1, VEGF-R2, VE-cadherin, and Tie2 protein increases were dependent on the magnitude of shear stress. The mRNA levels of VEGF-R1, VEGF-R2, VE-cadherin, Tie2, endothelial nitric oxide synthase, matrix metalloproteinase 9, and VEGF increased in shear-stressed EPCs. Inhibitor analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signal transduction pathway is a potent activator of adhesion, proliferation, tube formation, and differentiation in response to shear stress. Western blot analysis revealed that shear stress activated the VEGF-R2 phosphorylation in a ligand-independent manner. These results indicate that shear stress increases differentiation, adhesion, migration, proliferation, antiapoptosis, and vasculogenesis of circulating phenotype EPCs by activation of VEGF-R2 and the PI3K/Akt/mTOR signal transduction pathway.

SUPPRESSED INCREASE IN BLOOD ENDOTHELIAL PROGENITOR CELL CONTENT AS RESULT OF SINGLE EXHAUSTIVE EXERCISE BOUT IN MALE REVASCULARISED CORONARY ARTERY DISEASE PATIENTS

Endothelial progenitor cells (EPCs) significantly affect endothelial repair capacity and, hence, cardiovascular disease incidence. In healthy subjects, blood EPC content increases significantly as result of a single maximal exercise test, hereby stimulating endothelial repair capacity. It remains to be shown whether a single exercise positively affects blood EPCs in revascularised coronary artery disease (CAD) patients. From male revascularised CAD patients (n = 60) and healthy volunteers (n = 25) blood samples were collected before and immediately after a maximal cardiopulmonary exercise test. Blood samples were analyzed by optimised flow cytometry methodology for EPC content (CD34+, CD34+ CD133+, CD34+VEGFR2+, CD34+CD133+VEGFR2+, and CD34+CD133-VEGFR2+ cells) and compared between groups. CFU-Hill colonies were additionally assessed. As a result of a maximal exercise test, blood CD34+, CD34+VEGFR2+ (all EPCs), CD34+CD133+, and CD34+ CD133-VEGFR2+ (mature EPCs) cells increased significantly in CAD patients (p < 0.05), but less than in healthy subjects (p < 0.05, and p = 0.06 for CD34+VEGFR2+). CD34+CD133+VEGFR2+ cells (immature EPCs) did not change as result of exercise (p > 0.05). No changes in CFUHill colonies as result of exercise were observed. This study shows that blood mature EPCs (CD34+CD133- VEGFR2+) increase significantly as result of a single exercise bout in revascularised CAD patients, but with smaller magnitude compared to healthy subjects. Blood immature EPCs (CD34+CD133+VEGFR2+) did not change significantly as result of exercise.

Identification of CD133+ Cells in Pituitary Adenomas

Stem-like cells in tumors are capable of self-renewal and pluri-differentiation; they are thought to play important roles in tumor initiation and maintenance. Stem-like cells in malignant glioma express CD133. We examined samples from human pituitary adenoma, a generally benign neoplasm, for CD133 expression using routine immunohistochemical and biochemical methods. Our study of 70 pituitary adenomas (clinically nonfunctioning adenomas and growth hormone-, prolactin-, adrenocorticotropic hormone-, and thyroid-stimulating hormone-producing adenomas) showed that 18 (25.7%) expressed CD133. This rate was higher in clinically nonfunctioning (33.3%) than functioning adenomas (12.0%) (p = 0.085). Real-time PCR assay revealed the expression of CD133 mRNA in samples immunohistochemically positive for CD133. Neither the patient age and gender, nor the tumor size or postoperative recurrence rate correlated with CD133 positivity. CD133+ cells ubiquitously coexpressed CD34, nestin, and VEGFR2 (KDL1). S-100 and GFAP were not coexpressed with CD133. Chromogranin A, Pit-1, SF-1, and NeuroD1 were immune-negative, indicating that CD133+ cells did not have the potential to differentiate into functional endocrine cells. Our data suggest that the expression of CD133 in pituitary adenomas is related to immature endothelial progenitor cells that may play a role in the neovascularization of pituitary adenomas. Further studies are needed to elucidate the significance of CD133+ cells with respect to neovascularization and their sustainable growth in pituitary adenomas.

Polyelectrolyte multilayer films promote human cord blood stem cells differentiation into mature endothelial cells exhibiting a stable phenotype

recent studies in bio-engineering have showed the influence of Polyelectrolyte Multilayer (PEM) films on endothelial cells (ECs), especially poly(sodium-4-styrene-sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). They were tested either with human mature ECs or rabbit immature endothelial progenitor cells (EPCs), but never on human EPCs. In view to obtain an EC covered surface, human cord blood (HCB) EPCs were cultivated on PSS/PAH films. PEMs were obtained by 7 alternate depositions of cationic PAH and anionic PSS layers. HCB mononuclear cells were isolated by centrifugation through density gradient. 7 days after seeding on PEM, unattached cells were removed and adherent EPCs were cultivated in endothelial specific medium until P6. Appearance of CD31 and vWF was evaluated by confocal microscopy. EPCs not only successfully adhered on PEM, but also spread and proliferated. Moreover, cells differentiated into a typical endothelial cobblestone monolayer within 2 weeks. Immunostaining of CD31 and vWF confirmed the formation of an EC-like confluent monolayer. Furthermore, these cells showed after 6 passages a good phenotypic stability while reseeded on the PEM film. these results show an easy way to obtain mature ECs from human stem cells, which may open new applications for a scaffold cellularization in tissue bio-engineering.

Wnt5a Is Required for Endothelial Differentiation of Embryonic Stem Cells and Vascularization via Pathways Involving Both Wnt/β-Catenin and Protein Kinase Cα

In this study, we examined the signaling pathways activated by Wnt5a in endothelial differentiation of embryonic stem (ES) cells and the function of Wnt5a during vascular development. We first found that Wnt5a(-/-) mouse embryonic stem (mES) cells exhibited a defect in endothelial differentiation, which was rescued by addition of Wnt5a, suggesting that Wnt5a is required for endothelial differentiation of ES cells. Involvement of both beta-catenin and protein kinase (PK)Calpha pathways in endothelial differentiation of mES cells requiring Wnt5a was indicated by activation of both beta-catenin and PKCalpha in Wnt5a(+/-) but not in Wnt5a(-/-) mES cells. We also found that beta-catenin or PKCalpha knockdowns inhibited the Wnt5a-induced endothelial differentiation of ES cells. Moreover, the lack of endothelial differentiation of Wnt5a(-/-) mES cells was rescued only by transfection of both beta-catenin and PKCalpha, indicating that both genes are required for Wnt5a-mediated endothelial differentiation. Wnt5a was also found to be essential for the differentiation of mES cells into immature endothelial progenitor cells, which are known to play a role in repair of damaged endothelium. Furthermore, a defect in the vascularization of the neural tissue was detected at embryonic day 14.5 in Wnt5a(-/-) mice, implicating Wnt5a in vascular development in vivo. Thus, we conclude that Wnt5a is involved in the endothelial differentiation of ES cells via both Wnt/beta-catenin and PKC signaling pathways and regulates embryonic vascular development.

Link between erythropoietin release and mobilization of endothelial progenitor cells in acute myocardial infarction The opinions expressed in this article are not necessarily those of the Editors of the European Heart Journal or of the European Society of Cardiology.

Myocardial infarction (MI) is associated with the increase in plasma levels of inflammatory and haematopoietic cytokines and mobilization of a heterogenous population of cells which consists predominantly of committed lineages (monocytes, polymorphonuclear granulocytes, and lymphocytes), as well as numerous types of stem/progenitor cells [endothelial progenitor cells (EPCs), haematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs)]. The number of circulating EPCs may have a prognostic value; however, this concept remains to be proved in large prospective studies.1,2 The number of circulating EPCs and mature endothelial cells (ECs) supposedly reflects the vascular endothelial injury and the repair mechanisms activated to restore the endothelial integrity. The article by Ferrario et al. 3 investigates the haemoglobin-independent increase of the plasma levels of erythropoietin (Epo) in patients with acute MI and seeks to confirm the hypothesis that changes of Epo levels are associated with the mobilization of EPCs and ECs from the bone marrow. The paper by Ferrario et al. confirms previous work by the same group, published in 2005, describing the mobilization of heterogenous population of progenitor cells in patients with acute MI.4 In the present paper the authors identify two populations of immature EPCs (CD34+CD133+VEGFR2+ and CD34+CD117+VEGFR2+), more mature EPCs, and mature ECs (CD34+VEGFR2+). This study continues the search for humoral factors involved in EPC mobilization following acute myocardial ischaemia and vascular injury. One of the important issues … *Corresponding author. Tel: +48 604188669; fax: +48 32 2523930. E-mail address: wojwoj{at}mp.pl

Colony Forming Unit-Endothelial Cells (CFU-EC) Are Derived from CD14+ Cells and Need Support by CD14− Cells.

Since endothelial progenitor cells (EPC) were found in adult blood, active interferences of vascular diseases by cellular therapy with EPC were initiated. However, controversies exist on the identity of the EPC, e.g. concerning phenotype before and after culture, in vitro and in vivo behavior, as well as on quantities and differentiation. EPC can be quantified in vitro by culturing CFU-EC. In order to clarify which cells are essential and which are facilitating the formation of CFU-EC, we determined the contribution of several cell types to the formation of colonies. Peripheral blood mononuclear cells were isolated and were either positively selected or depleted for several cell populations (CD34+/KDR+/CD146+, CD3+, CD14+, CD19+ or CD56+). Subsequently the cells were labeled with PKH2 or irradiated (40Gy) to determine the contribution to colony formation. The purity of the cell populations was determined by flow cytometry. From these cell populations, CFU-EC were cultured on fibronectin-coated tissue culture plates, in endothelial selective medium EndoCultTM (Stem Cell Technologies). After 2 days of culture, non-adherent cells were subsequently cultured for an additional 3 days to form colonies. To determine the contribution of soluble factors to colony formation, transwells (0.8 μm pore size) were used to separate the cell populations during culture. CFU-EC are not derived from mature endothelial cells nor from immature EPC, as similar number of colonies per well were found when the starting population was depleted for CD146+/CD34+/KDR+ cells (18±9 vs. 20±14 in controls; n=4). In contrast, when CD14+ cells were depleted from the starting population, no CFU-EC were formed (0±0 vs. 29±23 in controls; n=7, p<0.02). After depletion of CD3+, CD19+ or CD56+ cells, CFU-EC were formed similarly as compared to controls. To investigate whether the CD14+ cells were the clonogenic cells, or whether they are facilitating the formation of colonies, monocytes were labeled with phagocyte specific PKH2 or irradiated and combined with untreated CD14− cells. Labeled cells were found in the colonies and among the spindle shaped cells surrounding the colonies. Hardly any colony formation was found after irradiating CD14+ cells (4.5±1 vs. 34±9 in controls), indicating that proliferating monocytes are necessary for CFU-EC formation. Culturing CD14+ cells alone did not result in a similar number of colonies as compared to the control (4±4 vs. 29±23, in controls; n=7, p<0.03). This indicates that CD14− cells are important in outgrowth of CFU-EC from monocytes. To test if this is due to paracrine factors, CD14− cells were cultured in a transwell insert above the >95% CD14+ fraction. This resulted in an increase in colony formation (>95% monocytes: 2±1; with insert: 10±9, n=2). In conclusion, CFU-EC are derived from CD14+ cells, but need the presence of CD14− cells, which is in part due to paracrine factors secreted by CD14− cells. This study further clarifies the identity of the EPC as determined by CFU-EC, and may give more insight to the role of several cell populations in vascular repair.