Endothelial Progenitor Cells Apoptosis Research Articles

Apoptosis of endothelial progenitor cells in a metabolic syndrome experimental model

Aim:This study tests the hypothesis postulating that metabolic syndrome induced by chronic administration of fructose to spontaneously hypertensive rats (FFHR) generates impairment in vascular repair by endothelial progenitor cells (EPC).Materials and Methods:To characterize the vascular adverse environment present in this experimental model we measured: NAD(P)H oxidase activity, eNOS activity, presence of apoptosis in the arterial wall, all these parameters were most affected in the FFHR group. Also, we found decreased level and proliferative capacity of EPC measured by flow cytometry and colonies forming units assay in cultured cells, respectively, in both groups treated with fructose; FFHR (SHR fructose fed rats) and FFR (WKY fructose fed rats) compared with their controls; SHR and WKY.Results:The fructose-fed groups FFR and SHR also showed an incremented number of apoptotic (annexinV+/7AADdim) EPC measured by flow cytometry that returns to almost normal values after eliminating fructose administration.Conclusion:Our findings suggest that increased apoptosis levels of EPC generated in this experimental model could bein part the underlying cause for the impaired vascular repair by in EPC.

416 Inhibition of Four- and- a- Half LIM Domain Protein 2 Increases Survival and Migratory Capacity of Human Early Outgrowth Endothelial Progenitor Cells Through Upregulation of Sphingosin Kinase- 1: Implications for Endothelial Regeneration

Inhibition of Four-and-a-half LIM domain protein-2 (FHL-2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Endothelial progenitor cells (EPCs) substantially contribute to endothelial repair. We investigated the role of FHL-2 in the regulation of early outgrowth EPC number and function. Early outgrowth EPCs were obtained from human peripheral blood. FHL-2 knockdown in EPCs by small-interfering RNA (siRNA) resulted in a significant increase in EPC number and a reduction of apoptosis (by 40%), as indicated by a decrease of cleaved caspase-3, through activation and translocation to the membrane, of sphingosine kinase-1 (SK-1), enzyme that metabolizes sphingosine- 1 phosphate (s1p). Furthermore, FHL-2 siRNA increased significantly (2 fold) stromal derived factor (SDF) -1- induced EPC migration; through upregulation of α-v/β-3 and α-v/β-5 integrins; this was associated with an increase of the F-actin binding protein cortactin, known to promote migration. Interestingly, increased SDF-1- induced EPC migration and upregulation of cortactin by FHL-2 siRNA were totally prevented by CAY10621, a specific inhibitor of SK-1. In addition stimulation of EPCs with exogenous s1p peptide significantly decreased apoptosis and increased SDF-1- induced migration. These results were confirmed In vivo using FHL-2 knockout (FHL-2 −/−) mice. Moreover, apoptosis was significantly decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL-2 −/− vs. WT EPCs. These effects were abolished by VPC23019, an antagonist of sphingosine- 1- phosphate receptor- 1 and 3. Finally, reendothelialization after focal carotid endothelial electric injury in WT mice was significantly increased after application of spleen-derived progenitor cells from FHL-2 −/− mice vs. WT mice. Our findings suggest that FHL-2 negatively regulates early outgrowth EPC function and secretion of paracrine factors. FHL-2 inhibition reduces apoptosis, enhances survival and migratory capacity of EPCs and ECs by upregulating SK-1/s1p pathway, integrin subunits and cortactin; which results in the improvement of endothelial regeneration.

The effects of low-dose Nepsilon-(carboxymethyl)lysine (CML) and Nepsilon-(carboxyethyl)lysine (CEL), two main glycation free adducts considered as potential uremic toxins, on endothelial progenitor cell function

BackgroundPatients with chronic kidney disease (CKD) are at high risk of cardiovascular disease (CVD). Endothelial progenitor cell (EPCs) dysfunction plays a key role in this pathogenesis. Uremic retention toxins have been reported to be in associated with EPC dysfunction. Advanced glycation end-products (AGEs) free adducts, including Nepsilon-(carboxymethyl)lysine (CML) and Nepsilon-(carboxyethyl)lysine (CEL), are formed by physiological proteolysis of AGEs and released into plasma for urinary excretion. They are retained in CKD patients and are considered to be potential uremic toxins. Though AGEs have been demonstrated to impair EPC function in various ways, the effect of AGE free adducts on EPC function has not been studied. Thus, we examined the role of CML and CEL in the regulation of growth-factor-dependent function in cultured human EPCs and the mechanisms by which they may affect EPC function.MethodsLate outgrowth EPCs were incubated with different concentrations of CML or CEL for up to 72 hours. Cell proliferation was determined using WST-1 and BrdU assays. Cell apoptosis was tested with annexin V staining. Migration and tube formation assays were used to evaluate EPC function.ResultsThough CML and CEL were determined to have anti-proliferative effects on EPCs, cells treated with concentrations of CML and CEL in the range found in CKD patients had no observable impairment on migration or tube formation. CML and CEL did not induce EPC apoptosis. The reduced growth response was accompanied by significantly less phosphorylation of mitogen-activated protein kinases (MAPKs).ConclusionsOur study revealed that CML and CEL at uremic concentrations have low biological toxicity when separately tested. The biologic effects of AGE free adducts on the cardiovascular system merit further study.

Abstract 200: Inhibition of Four-and-a-Half Lim Domain Protein 2 Increases Survival and Migratory Capacity of Human Early Outgrowth Endothelial Progenitor Cells Through Upregulation of Sphingosine Kinase-1: Implications for Endothelial Regeneration

Background: Inhibition of Four-and-a-half LIM domain protein-2 (FHL-2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Endothelial progenitor cells (EPCs) substantially contribute to endothelial repair. We investigated the role of FHL-2 in the regulation of early outgrowth EPC number and function. Methods and Results: Early outgrowth EPCs were obtained from human peripheral blood. FHL-2 knockdown in EPCs by small-interfering RNA (siRNA) resulted in a significant increase in EPC number and a reduction of apoptosis (by 40%), as indicated by a decrease of cleaved caspase-3, through activation and translocation to the membrane, of sphingosine kinase-1 (SK-1), enzyme that metabolizes sphingosine- 1 phosphate (s1p). Furthermore, FHL-2 siRNA increased significantly (2 fold) stromal derived factor (SDF) -1- induced EPC migration; through upregulation of α-v/β-3 and α-v/β-5 integrins; this was associated with an increase of the F-actin binding protein cortactin, known to promote migration. Interestingly, increased SDF-1- induced EPC migration and upregulation of cortactin by FHL-2 siRNA were totally prevented by CAY10621 , a specific inhibitor of SK-1. In addition stimulation of EPCs with exogenous s1p peptide significantly decreased apoptosis and increased SDF-1- induced migration. These results were confirmed In vivo using FHL-2 knockout (FHL-2 -/-) mice. Moreover, apoptosis was significantly decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL-2 -/- vs. WT EPCs. These effects were abolished by VPC23019 , an antagonist of sphingosine- 1- phosphate receptor- 1 and 3. Finally, reendothelialization after focal carotid endothelial electric injury in WT mice was significantly increased after application of spleen-derived progenitor cells from FHL-2 -/- mice vs. WT mice. Conclusions: Our findings suggest that FHL-2 negatively regulates early outgrowth EPC function and secretion of paracrine factors. FHL-2 inhibition reduces apoptosis, enhances survival and migratory capacity of EPCs and ECs by upregulating SK-1/s1p pathway, integrin subunits and cortactin; which results in the improvement of endothelial regeneration.

Tissue Kallikrein Promotes Cardiac Neovascularization by Enhancing Endothelial Progenitor Cell Functional Capacity

Tissue kallikrein (TK) has been demonstrated to improve neovasculogenesis after myocardial infarction (MI). In the present study, we examined the role and underlying mechanisms of TK in peripheral endothelial progenitor cell (EPC) function. Peripheral blood-derived mononuclear cells containing EPCs were isolated from rat. The in vitro effects of TK on EPC differentiation, apoptosis, migration, and vascular tube formation capacity were studied in the presence or absence of TK, kinin B(2) receptor antagonist (icatibant), and phosphatidylinositol-3 kinase inhibitor (LY294002). Apoptosis was evaluated by flow-cytometry analysis using Annexin V-FITC/PI staining, as well as western-blot analysis of Akt phosphorylation and cleaved caspase-3. Using an MI mouse model, we then examined the in vivo effects of human TK gene adenoviral vector (Ad.hTK) administration on the number of CD34(+)Flk-1(+) progenitors in the peripheral circulation, heart tissue, extent of vasculogenesis, and heart function. Administration of TK significantly increased the number of Dil-LDL/UEA-lectin double-positive early EPCs, as well as their migration and tube formation properties in vitro. Transduction of TK in cultured EPCs attenuated apoptosis induced by hypoxia and led to an increase in Akt phosphorylation and a decrease in cleaved caspase-3 levels. The beneficial effects of TK were blocked by pretreatment with icatibant and LY294002. The expression of recombinant human TK in the ischemic mouse heart significantly improved cardiac contractility and reduced infarct size 7 days after gene delivery. Compared with the Ad.Null group, Ad.hTK reduced mortality and preserved left ventricular function by increasing the number of CD34(+)Flk-1(+) EPCs and promoting the growth of capillaries and arterioles in the peri-infarct myocardium. These data provide direct evidence that TK promotes vessel growth by increasing the number of EPCs and enhancing their functional properties through the kinin B(2) receptor-Akt signaling pathway.

Downregulation of microRNA-126 in endothelial progenitor cells from diabetes patients, impairs their functional properties, via target gene Spred-1

Diabetes mellitus (DM) adversely affects the number and function of circulating endothelial progenitor cells (EPCs). Consequently, there is also a reduction in the repair mechanism of these cells, which is a critical and initiating factor in the development of diabetic vascular disease. The aim of the present study was to analyze miR expression profiles in EPCs from patients with DM and choose the most significantly regulated miR to study its possible role on EPC dysfunction and elucidate its mechanism of action. EPCs were collected from subjects with Type II DM and non-diabetic control subjects. Total RNA was harvested from EPCs, and a total of 5 candidate miRNAs were identified by microarray screening and were quantified by TaqMan real-time PCR. Lentiviral vectors expressing miR-126 and miR-126 inhibitor (anti-miR-126) were transfected into EPCs, and the EPC colony-forming capacity, proliferation activity, migratory activity, differentiation capacity, and apoptotic susceptibility were determined and Western Blotting and mRNA real-time PCR analyses were performed. To study the mechanisms, lentiviral vectors expressing Spred-1 and a short interfering RNA (siRNA) targeting Spred-1 were prepared. Five miRs were aberrantly downregulated in EPCs from DM patients. These miRs included miR-126, miR-21, miR-27a, miR-27b and miR-130a. Anti-miR-126 inhibited EPC proliferation, migration, and enhanced apoptosis. Restored miR-126 expression in EPCs from DM promoted EPC proliferation, migration, and inhibited EPC apoptosis ability. Despite this, miR-126 had no effect on EPC differentiation. miR-126 overexpression significantly downregulated Spred-1 in EPCs. The knockdown of Spred-1 expression in EPCs from DM promoted proliferation, migration, and inhibited apoptosis of the cells. The signal pathway of miR-126 effecting on EPCs is partially mediated through Ras/ERK/VEGF and PI3K/Akt/eNOS regulation. This study provides the first evidence that miR-126 is downregulated in EPCs from diabetic patients, and impairs EPCs-mediated function via its target, Spred-1, and through Ras/ERK/VEGF and PI3K/Akt/eNOS signal pathway.

Endothelin 1 Activation of Endothelin A Receptor/NADPH Oxidase Pathway and Diminished Antioxidants Critically Contribute to Endothelial Progenitor Cell Reduction and Dysfunction in Salt-Sensitive Hypertension

Circulating endothelial progenitor cells (EPCs) are reduced in hypertension, which inversely correlates with its mortality. Deoxycorticosterone acetate (DOCA)-salt hypertension features elevated endothelin (ET) 1 and oxidative stress. We tested the hypothesis that ET-1 induces EPC dysfunction by elevating oxidative stress through the ET(A)/NADPH oxidase pathway in salt-sensitive hypertension. Both ET(A) and ET(B) receptors were expressed in EPCs, but only ET(A) receptors were significantly increased in EPCs of DOCA-salt rats. EPC number and function were reduced in DOCA-salt rats compared with sham controls, and both were reversed by in vivo blockade of ET(A) receptors or NADPH oxidase. The enzymatic activities of NAPDH oxidase and its subunits gp91(phox), p22(phox), and Rac1 were augmented in EPCs of DOCA-salt rats, with concomitantly decreased antioxidant enzymes manganese superoxide dismutase, copper-zinc superoxide dismutase, and glutathione peroxidase 1. Reactive oxygen species level was elevated in EPCs from DOCA-salt rats, accompanied by increased EPC telomerase inactivation, senescence, and apoptosis, which were rescued by ET(A) or NADPH oxidase blockade. Cell therapy of normal or treated DOCA EPCs, but not untreated DOCA EPCs, significantly increased capillary density and blood perfusion in ischemic hindlimbs of DOCA-salt rats. p53 and Bax/Bcl-2 ratios were increased in EPCs of DOCA-salt rats, which were reversed by ET(A) antagonist, NADPH oxidase inhibitor, or polyethylene glycol-superoxide dismutase. Finally, in ET(B)-deficient rats, plasma ET-1 was elevated, and EPC number and telomerase activity were diminished. These results demonstrate, for the first time, that both ET-1 activation of ET(A)/NADPH oxidase pathway and diminished antioxidants critically contribute to EPC reduction and dysfunction via increased oxidative stress in salt-sensitive hypertension.

Fast reduction of peripheral blood endothelial progenitor cells in healthy humans exposed to acute systemic hypoxia

There are hints that hypoxia exposure may affect the number of circulating endothelial progenitor cells (EPCs) in humans. To test this hypothesis, the concentration of EPCs was determined by flow cytometry in the peripheral blood of 10 young healthy adults before (0 h), at different times (0.5 h, 1 h, 2 h and 4 h) during a 4 h normobaric hypoxic breathing simulating 4100 m altitude, and in the following recovery breathing room air. Results were interpreted mainly on the basis of the changes in surface expression of CXC chemokine receptor-4 (CXCR-4, a chemokine receptor essential for EPC migration and homing) and the percentage of apoptotic cells, the plasmatic levels of markers of oxidative stress induced by hypoxic breathing. Compared to 0 h, the concentration of EPCs, identified as either CD45(dim)/CD34(+)/KDR(+) or CD45(dim)/CD34(+)/KDR(+)/CD133(+) cells, decreased from 337 ± 83 ml(-1) (mean ± SEM) to 223 ± 52 ml(-1) (0.5 h; P < 0.005) and 100 ± 37 ml(-1) (4 h; P < 0.005), and from 216 ± 91 to 161 ± 50 ml(-1) (0.5 h; P < 0.05) and 45 ± 23 ml(-1) (4 h; P < 0.005), respectively. Upon return to normoxia, their concentration increased slowly, and after 4 h was still lower than at 0 h (P < 0.05). During hypoxia, CXCR-4 expression and plasmatic stromal derived cell factor-1 (SDF-1) increased abruptly (0.5 h: +126% and +13%, respectively; P < 0.05), suggesting cell marginalization as a possible cause of the rapid hypoxia-induced EPC reduction. Moreover, hypoxia exposure induced an increase in EPC apoptosis and markers of oxidative stress, which was significantly evident only starting from 2 h and 4 h after hypoxia offset, respectively, suggesting that EPC apoptosis may contribute to the later phase of hypoxia-induced EPC reduction. Overall, these observations may provide new insights into the understanding of the mechanisms operated by EPCs to maintain endothelial homeostasis.

C-reactive protein down-regulates endothelial nitric oxide synthase expression and promotes apoptosis in endothelial progenitor cells through receptor for advanced glycation end-products

ObjectiveC-reactive protein (CRP), the prototypic marker of inflammation, has been shown to be an independent predictor of atherosclerosis. CRP can regulate receptor for advanced glycation end-products (RAGE) expression in endothelial progenitor cells (EPCs). Endothelial nitric oxide synthase (eNOS) deficiency is a pivotal event in atherogenesis. It is believed that decreased eNOS bioactivity occurs early in atherogenesis. Therefore, we tested the hypothesis that CRP can alter eNOS expression and promote apoptosis in EPCs through RAGE. Methods and resultsEPCs, isolated from bone marrow, were cultured in the presence or absence of LPS-free CRP (5, 10, 15, 20, and50μg/ml). RAGE protein expression and siRNA were measured by flow cytometric analysis. PCR was used to detect eNOS mRNA expression. eNOS protein expression was measured by Western blot analysis. A spectrophotometer was used to assess eNOS activity. A modified Boyden's chamber was used to assess the migration of EPCs and the number of recultured EPCs was counted to measure adhesiveness. A MTT assay was used to determine proliferation. Apoptosis was evaluated by annexin V immunostaining and TUNEL staining. Co-culturing with CRP caused a significant down-regulation of eNOS expression, inhibited the proliferation, migration, and adhesion of EPCs, and induced EPC apoptosis. In addition, these effects were attenuated during RAGE protein expression blockade by siRNA. ConclusionsCRP, at concentrations known to predict cardiovascular event, directly quenches the expression of eNOS and diminishes NO production, and may serve to impair EPC function and promote EPC apoptosis through RAGE. These data further support a direct role of CRP in the development and/or progression of atherosclerosis and indicate a new pathophysiologic mechanism of disturbed vascular adaptation in atherosclerosis.

Sun Ginseng Protects Endothelial Progenitor Cells From Senescence Associated Apoptosis

Endothelial progenitor cells (EPC) are a population of cells that circulate in the blood stream. They play a role in angiogenesis and, therefore, can be prognostic markers of vascular repair. Ginsenoside Rg3 prevents endothelial cell apoptosis through the inhibition of the mitochondrial caspase pathway. It also affects estrogen activity, which reduces EPC senescence. Sun ginseng (SG), which is heat-processed ginseng, has a high content of ginsenosides. The purpose of this study was to investigate the protective effects of SG on senescence-associated apoptosis in EPCs. In order to isolate EPCs, mononuclear cells of human blood buffy coats were cultured and characterized by their uptake of acetylated low-density lipoprotein (acLDL) and their binding of Ulex europaeus agglutinin I (ulex-lectin). Flow cytometry with annexin-V staining was performed in order to assess early and late apoptosis. Senescence was determined by β-galactosidase (β-gal) staining. Staining with 4′-6-Diamidino-2-phenylindole verified that most adherent cells (93±2.7%) were acLDL-positive and ulex-lectin-positive. The percentage of β-gal-positive EPCs was decreased from 93.8±2.0% to 62.5±3.6% by SG treatment. A fluorescence-activated cell sorter (FACS) analysis showed that 4.9% of EPCs were late apoptotic in controls. Sun ginseng decreased the apoptotic cell population by 39% in the late stage of apoptosis from control baseline levels. In conclusion, these results show antisenescent and antiapoptotic effects of SG in human-derived EPCs, indicating that SG can enhance EPC-mediated repair mechanisms.

Effects of endothelin-1 on endothelial progenitor cell function

Circulating endothelial progenitor cells (EPCs) contribute to vascular endothelial repair. Endothelin (ET)-1 is associated with endothelial damage and atherogenesis. The experimental aim of this study was to determine, in vitro, the effects of ET-1 on the ability of EPCs to form colonies, migrate, release angiogenic growth factors and resist apoptosis. Peripheral blood samples were collected from 10 healthy adult humans. Cells with phenotypic EPC characteristics were isolated and EPC colony-forming capacity (CFU assay), migratory activity (Boyden chamber), release of angiogenic growth factors (enzyme immunoassay) and apoptosis (TUNEL assay) were determined in the absence and presence of ET-1 (100 pmol). EPC colony-forming units (42±12 vs. 39±11), migratory capacity (910±146 vs. 936±148 AU) and release of vascular endothelial growth factor (202.8±68.1 vs. 204.8±69.8 pg/mL) and granulocyte-colony stimulating factor (1294.4±378.3 vs. 1136.1±310.3 pg/mL) were not significantly affected by ET-1. EPCs treated with ET-1 demonstrated a 20% increase (p<0.05) in cellular apoptosis. The proapoptotic effect of ET-1 was abolished with ET receptor blockade as well as with apocynin, a nicotinamide adenine dinucleotide phosphate (NADPH) inhibitor. These results indicate that ET-1 does not affect EPC colony formation, migratory capacity or angiogenic growth factor release, but does increase EPC susceptibility to apoptosis through an NADPH-dependent mechanism. Increased EPC apoptosis may contribute to the proatherogenic effects of ET-1.

Shear stress and interleukin-8 (IL-8) on the proliferation, differentiation and tube formation of endothelial progenitor cells (EPCs)

Endothelial progenitor cells (EPCs) derived from bone marrow, are also found in circulation and involved in both tumor vasculogenesis and wound healing. During the process of EPCs incorporation into tissues and neovascularization, the cells are exposed to fluid shear stress. Interleukin-8 (IL-8) has been shown to play an important role in tumor growth, angiogenesis and metastasis. In our previous study, IL-8 gene expression of cultured human umbilical vein endothelial cells was significantly increased after stimulation by low shear stress for 1 to 2 h. Hence, there may be some connection between the IL-8 and shear stress in the EPCs incorporation into tissues and neovascularization. This study aimed to investigate whether the shear stress and IL-8 played important role in this process. After the cells were pretreated by shear stress, the cells were stimulated by IL-8, and then the expressions of CD133, VWF, VE-cadherins and VEGFR-2 in EPCs were measured. The proliferation, apoptosis, cell cycle, migration and tube formation of EPCs were detected after the cells expose to shear stress stimulated by IL-8. Results show that IL-8 or shear stress alone can induce EPCs proliferation decreased. 5 and 10 dyne/cm 2 shear stress can both induce EPCs differentiation into the mature ECs.

Abstract 2: IL-10 Modulates Mobilization of Bone Marrow Endothelial Progenitor Cells and Enhances Their Survival and Angiogenic Properties in Ischemic Myocardium

Endothelial progenitor cells (EPC) transplantation has been shown to enhance neovascularization and improve myocardial infarction (MI)-induced ventricular dysfunctions. However, persistent inflammation in the ischemic myocardium, adversely affect EPC survival and function, thereby compromising full benefits of EPC-mediated vascular repair. We hypothesized that modulation of IL-10 signaling in EPCs enhances their mobilization, survival and function in ischemic myocardium after MI. GFP-labeled EPC were transplanted intramyocardially after induction of MI, and the mice were treated with either saline or recombinant IL-10. EPC survival and EPC-mediated neovascularization and myocardial repair were evaluated. IL-10-treated mice showed increased number of GFP+EPCs retention that was associated with reduced EPC apoptosis in the myocardium (P&lt;0.05). The engraftment of EPC into the vascular structures and the associated capillaries density was significantly higher in IL-10-treated mice (P&lt;0.05). The above findings were corroborated with reduced infarct size, fibrosis and enhanced LV function (echocardiography) in IL-10+EPC group as compared to EPC+saline group. Invitro, IL-10-deficient EPCs showed higher LPS-induced apoptosis compared to WT-EPCs (P&lt;0.05). IL-10 treatment induced VEGF expression in WT-EPCs which was abrogated by STAT3 inhibition (using curcurbitacin I). Furthermore, microRNA (miR) profile experiments identified significant increases in a number of pro-apoptotic and anti-angiogenic-related miRs in EPCs from IL-10 deficient mice. Interestingly, IL-10-deficient mice showed impaired MI-induced mobilization of bone marrow EPCs (Sca1+Flk1+ cells) into the circulation and the associated SDF-1 mRNA expression in the myocardium. Bone marrow transplantation studies involving replacement of IL-10-deficient marrow with WT marrow attenuated these effects. Invitro, LPS-induced CXCR4 expression was lower in IL-10-deficient EPCs as compared to WT-EPC. Taken together, our studies suggest that IL-10 enhances EPC mobilization, possibly in an SDF1-CXCR4 dependent manner and increased their survival and neovascularization and the associated myocardial repair, in part via activation of STAT3 signaling cascades.

Tissue kallikrein increase endothelial progenitor cells via B2-PI 3-kinase/Akt pathway

ObjectiveTissue kallikrein (TK) has been demonstrated to improve neovasculogenesis after myocardial infarction (MI). In the present study, we examined whether TK plays a role in the peripheral endothelial progenitor cells...

Interleukin-10 Deficiency Impairs Bone Marrow–Derived Endothelial Progenitor Cell Survival and Function in Ischemic Myocardium

Endothelial progenitor cell (EPC) survival and function in the injured myocardium is adversely influenced by hostile microenvironment such as ischemia, hypoxia, and inflammatory response, thereby compromising full benefits of EPC-mediated myocardial repair. We hypothesized that interleukin-10 (IL-10) modulates EPC biology leading to enhanced survival and function after transplantation in the ischemic myocardium. Myocardial infarction (MI)-induced mobilization of bone marrow EPC (Sca-1+Flk1+cells) into the circulation was significantly impaired in IL-10 knockout (KO) mice. Bone marrow transplantation to replace IL-10 KO marrow with wild-type (WT) marrow attenuated these effects. Impaired mobilization was associated with lower stromal cell-derived factor (SDF)-1 expression levels in the myocardium of KO mice. Interestingly, SDF-1 administration reversed mobilization defect in KO mice. In vitro, hypoxia-mediated increases in CXCR4 expression and cell survival were lower in IL-10-deficient EPCs. Furthermore, SDF-1-induced migration of WT EPCs was inhibited by AMD3100, an inhibitor of CXCR4. To further study the effect of IL-10 on in vivo EPC survival and engraftment into vascular structures, GFP-labeled EPC were injected intramyocardially after induction of MI, and the mice were treated with either saline or recombinant IL-10. The IL-10-treated group showed increased retention of transplanted EPCs in the myocardium and was associated with significantly reduced EPC apoptosis after MI. Interestingly, increased EPC retention and their association with the vascular structures was observed in IL-10-treated mice. Increased EPC survival and angiogenesis in the myocardium of IL-10-treated mice corroborated with improved left ventricular function, reduced infarct size, and fibrosis in the myocardium. In vitro, IL-10-induced increase in VEGF expression in WT EPC was abrogated by STAT3 inhibitor, suggesting IL-10 signals through STAT3 activation. Taken together, our studies demonstrate that MI-induced EPC mobilization was impaired in IL-10 KO mice and that IL-10 increases EPC survival and function possibly through activation of STAT3/VEGF signaling cascades, leading to attenuation of MI-induced left ventricular dysfunction and remodeling.

Thymosin β4 activates integrin‐linked kinase and decreases endothelial progenitor cells apoptosis under serum deprivation

Thymosin β4 (Tβ4) has been suggested to regulate multiple cell signal pathways and a variety of cellular functions such as cell migration, proliferation, survival, and angiogenesis. Here, we investigated the effect of Tβ4 on endothelial progenitor cells (EPCs) apoptosis induced by serum deprivation and the corresponding signal transduction pathways involved in this process. Circulating EPCs, isolated from healthy volunteers, were cultured in the absence or presence of Tβ4 and various signal cascade inhibitors. Apoptosis was evaluated with Annexin V immunostaining and cytosolic cytochrome c expression. Incubation of EPCs with Tβ4 caused a concentration dependent increase in cell viability and proliferation activity. It also caused an inhibitory effect on EPCs apoptosis, which was abolished by PI3K inhibitors (either LY294002 or Wortmannin) or JNK MAPK inhibitor SP600125. In addition, the expression and activity of caspase-3 and -9 were decreased by Tβ4, which markedly increased the Bcl-2/Bax ratio within EPCs. Furthermore, Tβ4 was immunoprecipitated with integrin-linked kinase (ILK), accompanied by augmentation of ILK activity. Transfection of EPCs with ILK-siRNA resulted in abolishment of the activation of ILK-Akt and the ameliorative effect on apoptosis by Tβ4. Together, Tβ4 mediated inhibitory effect on EPCs apoptosis under serum deprivation can be attributed, at least in part, to ILK-Akt activation. The activation of JNK MAPK might also be involved in this process.

Angiotensin II Impairs Endothelial Progenitor Cell Number and Function In Vitro and In Vivo

Endothelial progenitor cells (EPCs) contribute to endothelial regeneration. Angiotensin II (Ang II) through Ang II type 1 receptor (AT(1)-R) activation plays an important role in vascular damage. The effect of Ang II on EPCs and the involved molecular mechanisms are incompletely understood. Stimulation with Ang II decreased the number of cultured human early outgrowth EPCs, which express both AT(1)-R and Ang II type 2 receptor, mediated through AT(1)-R activation and induction of oxidative stress. Ang II redox-dependently induced EPC apoptosis through increased apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase phosphorylation; decreased Bcl-2 and increased Bax expression; and activation of caspase 3 but had no effect on the low cell proliferation. In addition, Ang II impaired colony-forming and migratory capacities of early outgrowth EPCs. Ang II infusion diminished numbers and functional capacities of EPCs in wild-type (WT) but not AT(1)a-R knockout mice (AT(1)a(-/-)). Reendothelialization after focal carotid endothelial injury was decreased during Ang II infusion. Salvage of reendothelialization by intravenous application of spleen-derived progenitor cells into Ang II-treated WT mice was pronounced with AT(1)a(-/-) cells compared with WT cells, and transfusion of Ang II-pretreated WT cells into WT mice without Ang II infusion was associated with less reendothelialization. Transplantation of AT(1)a(-/-) bone marrow reduced atherosclerosis development in cholesterol-fed apolipoprotein E-deficient mice compared with transplantation of apolipoprotein E-deficient or WT bone marrow. Randomized treatment of patients with stable coronary artery disease with the AT(1)-R blocker telmisartan significantly increased the number of circulating CD34/KDR-positive EPCs. Ang II through AT(1)-R activation, oxidative stress, and redox-sensitive apoptosis signal-regulating kinase 1-dependent proapoptotic pathways impairs EPCs in vitro and in vivo, resulting in diminished vascular regeneration.

Homocysteine-Induced Caspase-3 Activation by Endoplasmic Reticulum Stress in Endothelial Progenitor Cells from Patients with Coronary Heart Disease and Healthy Donors

Previous studies have suggested an association of hyperhomocysteinemia-induced vascular pathology with enhanced apoptotic potential of endothelial progenitor cells in patients with coronary heart disease. Our results indicate that 500 µmol/L homocysteine induced endothelial progenitor cell apoptosis and activation of caspase-3, both of which were abolished by 100 µmol/L and 200 µmol/L salubrinal, an agent that prevents endoplasmic reticulum stress-induced apoptosis. The addition of 500 µmol/L homocysteine caused a release of Ca(2+) from intracellular stores, and enhanced phosphor-eukaryotic initiation factor 2α phosphorylation at Ser51 and the expression of a glucose-regulated protein of 78 kDa and a C/EBP homologous protein independently of extracellular Ca(2+). These effects of homocysteine on endothelial progenitor cells were significantly greater in patients with coronary heart disease than in healthy donors. These findings suggest that homocysteine induces endoplasmic reticulum stress-mediated activation of caspase-3 in endothelial progenitor cells, an event that is enhanced in patients with coronary heart disease. Furthermore, enhanced endoplasmic reticulum stress-mediated activation of caspase-3 in endothelial progenitor cells might be involved in hyperhomocysteinemia-associated vascular pathology.

Effects of tumour necrosis factor-alpha on activity and nitric oxide synthase of endothelial progenitor cells from peripheral blood

The aim of this investigation was to determine whether tumour necrosis factor-alpha (TNF-α) has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with tumour necrosis factor-α (final concentrations: 0, 10, 20, 50 and 100 mg/l) for 0, 6, 12, 24 and 48 h. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding, by direct fluorescence staining. EPC proliferation and migration were assayed using the MTT assay and modified Boyden chamber assay, respectively. EPC adhesion assay was performed by re-plating those cells on fibronectin-coated dishes, and adherent cells were counted. Tube formation activity was assayed using a tube formation kit. Levels of apoptosis were revealed using an annexin V apoptosis detection kit. Vascular endothelial growth factor Receptor-1 (VEGF-R1) and stromal derived factor-1 (SDF-1) mRNA, assessed by real-time RT-PCR inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were assayed by western blot analysis. Incubation of EPCs with tumour necrosis factor-α reduced EPC proliferation, migration, adhesion, tube formation capacity, iNOS and eNOS in concentration- and time-dependent manners. Tumour necrosis factor-α reduced proliferation, migration, adhesion and tube formation capacity of EPCs. TNF-α increased EPC apoptosis level, reduced VEGF-R1 and SDF-1 mRNA expression; tumour necrosis factor-α also reduced iNOS and eNOS in the EPCs.

Blood Levels, Apoptosis, and Homing of the Endothelial Progenitor Cells After Skin Burns and Escharectomy

Skin burns are an acute trauma involving an extensive vascular damage and an intense inflammatory response. Bone marrow-derived circulating endothelial progenitor cells (EPC) are known to migrate to sites of neovascularization in response to mediators (vascular endothelial growth factor and stromal cell-derived factor-1) released after trauma and ischemia, to contribute to wound healing, and to increase neovascularization of animal prefabricated flaps. Recent data showed an increase in EPC number in burned patients and a positive correlation between EPC number and total body surface area (TBSA) burnt, but data were limited to the first 5 days after thermal injury. By using flow cytometry, we studied EPC (CD34, CD133, CD45, and KDR cells) blood levels, apoptosis, and homing (stromal cell-derived factor-1 receptor expression and CXC chemokine receptor 4) in a 1-month follow-up postburn in 25 patients with ≥15% TBSA burnt, at least grade II burns and escharectomy performed at days 5 to 6, with respect to 31 controls. EPC count at admission showed a positive linear correlation with TBSA burnt. The EPC blood levels of the patients were low (50.7 cells/mL±61.8 cells/mL) immediately after thermal injury, then increased with two peaks, at day 1 (188.3 cells/mL±223.2 cells/mL) and day 12 (253.1 cells/mL±430.7 cells/mL) with respect to controls (95.2 cells/mL±28.5 cells/mL, p<0.05), and then returned to normal levels in 1 month. EPC apoptotic rate and inflammatory parameters paralleled EPC blood count. No significant variations were found in CXC chemokine receptor 4 expression. Thermal injury and escharectomy seem to induce an intense response in EPC production. In particular, escharectomy could improve physiologic wound repair by increasing EPC levels.