Outgrowth Endothelial Progenitor Cells Research Articles

In vitro dynamic perfusion of prevascularized OECs-DBMs (outgrowth endothelial progenitor cell - demineralized bone matrix) complex fused to recipient vessels in an internal inosculation manner

ABSTRACT The current research on seed cells and scaffold materials of bone tissue engineering has achieved milestones. Nevertheless, necrosis of seed cells in center of bone scaffold is a bottleneck in tissue engineering. Therefore, this study aimed to investigate the in vivo inosculation mechanism of recipient microvasculature and prevascularized outgrowth endothelial progenitor cells (OECs)-demineralized bone matrix (DBM) complex. A dorsal skinfold window-chamber model with tail vein injection of Texas red-dextran was established to confirm the optimal observation time of microvessels. OECs-DBM complex under static and dynamic perfusion culture was implanted into the model to analyze vascularization. OECs-DBM complex was harvested on 12th day for HE staining and fluorescent imaging. The model was successfully constructed, and the most appropriate time to observe microvessels was 15 min after injection. The ingrowth of recipient microvessels arcoss the border of OECs-DBM complex increased with time in both groups, and more microvessels across the border were observed in dynamic perfusion group on 3rd, 5th, 7th day. Fluorescent integrated density of border in dynamic perfusion group was higher at all-time points, and the difference was more significant in central area. Fluorescent imaging of OECs-DBM complex exhibited that no enhanced green fluorescent protein-positive cells were found beyond the verge of DBM scaffold in both groups. In vitro prevascularization by dynamic perfusion culture can increase and accelerate the blood perfusion of OECs-DBM complex obtained from recipient microvasculature by internal inosculation. Accordingly, this approach may markedly contribute to the future success of tissue engineering applications in clinical practice.

Outgrowth endothelial progenitor cells restore cerebral barrier function following ischaemic damage: The impact of NOX2 inhibition.

Disruption of blood-brain barrier (BBB), formed mainly by human brain microvascular endothelial cells (HBMECs), constitutes the major cause of mortality following ischaemic stroke. This study investigates whether OECs (outgrowth endothelial cells) can restore BBB integrity and function following ischaemic damage and how inhibition of NOX2, a main source of vascular oxidative stress, affects the characteristics of BBB established with OECs and HBMECs in ischaemic settings. In vitro models of human BBB were constructed by co-culture of pericytes and astrocytes with either OECs or HBMECs before exposure to oxygen-glucose deprivation (OGD) alone or followed by reperfusion (OGD + R) in the absence or presence of NOX2 inhibitor, gp91ds-tat. The function and integrity of BBB were assessed by measurements of paracellular flux of sodium fluorescein (NaF) and transendothelial electrical resistance (TEER), respectively. Treatment with OECs during OGD + R effectively restored BBB integrity and function. Compared to HBMECs, OECs possessed lower NADPH oxidase activity, superoxide anion levels and had greater total antioxidant capacity during OGD and OGD + R. Inhibition of NADPH oxidase during OGD and OGD + R restored the integrity and function of BBB established by HBMECs. This was evidenced by reductions in NADPH oxidase activity and superoxide anion levels. In contrast, treatment with gp91ds-tat aggravated ischaemic injury-induced BBB damage constructed by OECs. In conclusion, OECs are more resistant to ischaemic conditions and can effectively repair cerebral barrier following ischaemic damage. Suppression of oxidative stress through specific targeting of NOX2 requires close attention while using OECs as therapeutics.

Endothelial Colony Forming Cells from Individuals with Low VWF Levels Demonstrate Impairment in Stimulated VWF Release

BackgroundVon Willebrand factor (VWF) is an essential hemostatic protein and deficiencies of VWF predispose to mucocutaneous bleeding (MCB) such as seen in von Willebrand disease (VWD). VWD type 1 is characterized by incomplete penetrance and variable expressivity even in individuals and families with the same mutation in VWF . In addition to those patients with VWD type 1, there is a large subset of individuals that have VWF levels below 50 IU/dL, who suffer from MCB but do not meet the full criteria for VWD (VWF levels less than 30 IU/dL). The mechanisms for low VWF levels in these patients are poorly understood but are thought to be due to decreased synthesis and/or decreased secretion, increased clearance, or altered proteolysis. Endothelial colony forming cells (ECFCs, also known as blood outgrowth endothelial cells or endothelial progenitor cells) are a source of donor-specific endothelial cells and have demonstrated defects in VWF release and packaging in VWD. ECFCs have been previously studied in patients with VWD, but have not been evaluated in individuals with low VWF levels.Hypothesis/ObjectiveWe hypothesize that the study of ECFCs from individuals with low VWF levels will reveal deficiencies of abnormal VWF release and shed light on the mechanisms of low VWF.MethodsECFC Derivation: Patients with low VWF levels and MCB (30-50 IU/dL) were enrolled in a IRB-approved study and whole blood was collected into sodium heparin vacutainers, layered on Ficoll and centrifuged to isolate the mononuclear cell layer, which is then plated in a twelve-well collagen coated plate. After extended incubation, the presence of phenotypically ECFCs was confirmed by visual morphology and by single stain flow cytometry with appropriate (positive/negative) staining for the endothelial cells markers CD146, CD105, CD31 and negative non-endothelial cell markers CD34, CD90, CD133, and CD45.Stimulated Release of VWF from ECFC: ECFCs were plated in 12 well tissue culture plates. For stimulated release, the cells were exposed to 1mL serum-free media ± phorbol-12-myristate-13-acetate (PMA) (50ng/mL) for 1 hour. The conditioned media samples were then collected and assayed via the VWF:Ag ELISA.Enzyme-Linked ImmunoSorbant Assay (VWF:Ag ELISA): A 96-well plate was be coated with anti-VWF antibodies and after blocking and incubation with respective samples VWF levels were determined anti-VWF primary and HRP-linked secondary antibodies; values were compared to a standard curve and normalized to cell surface area. Results are analyzed via ANOVA with correction for multiple comparisons.ResultsECFC Derivation: A total of seven ECFC lines were created, 5 from individuals with MCB and VWF levels between 30-50 IU/dL (4:1 female: male ratio, age range 11-54 years) and 2 from healthy controls (2 female, age range 22-39 years, control ECFC 1, control ECFC 2) without symptoms of MCB.VWF release from patient derived endothelial cells: When treated with 0ng/mL of PMA, Low VWF derived ECFCs showed no demonstrable differences in VWF release into the conditioned media (Figure A). However, when treated with 50ng/mL of PMA, there is a noticeable trend of increased VWF release in the HUVEC and Control 1 and 2 ECFCs in comparison to the Low VWF ECFCs 1-5, although the results were not statistically significant (Figure B). When respective cohorts are grouped, the low VWF cohort demonstrates a statistically significant decrease in VWF at 50ng/mL as compared to HUVEC (p=0.0001) or control ECFC (p=0.0147) (Figure C).ConclusionsWe have demonstrated the ability to derive ECFCs from patients with Low VWF levels and MCB. Patient derived ECFCs from individuals with Low VWF levels demonstrate impairments in VWF release when stimulated with PMA and as a group, those individuals with low VWF levels demonstrate a significant decrease in VWF release. This decrease in stimulated VWF release is seen with several different individual patient samples and suggests that individuals with low VWF levels may have defects in VWF synthesis and or release that correspond to their lower plasma levels of VWF. [Display omitted] DisclosuresNg:Shire: Consultancy; CSL Behring Heimburger Award: Research Funding.

MicroRNA-126 protects against vascular injury by promoting homing and maintaining stemness of late outgrowth endothelial progenitor cells

BackgroundEndothelial progenitor cells (EPCs) contribute to reendothelialization and neovascularization and protect against vascular injury and ischemia of various organs. We have previously shown downregulation of microRNA (miR)-126 in EPCs from diabetic patients, which contributes to dysfunction of EPCs including impaired migratory ability. The aims of the present study were to examine (1) in vitro the effects of miR-126 on the homing and stemness of late outgrowth EPCs (LOCs), along with relevant signaling pathways, and (2) in vivo the effects of modulating LOCs by manipulating miR-126 expression on LOC homing and reendothelialization of injured arteries in GK rats (a non-obese diabetes model).MethodsRat bone marrow-derived LOCs were transfected with miR-126 inhibitor or lentiviral vectors expressing miR-126. LOC migration was determined by transwell migration assay. CXCR4 expression was measured by real-time PCR, Western blotting, and confocal microscopy while related signaling pathway proteins were measured by Western Blotting. Stemness gene expression, and gene and protein expression and promoter activity of KLF-8 were also measured. LOCs transfected with lenti-miR-126 or miR-126 inhibitor were injected into GK rats with carotid artery injury, and then vascular reendothelialization and the extent of intimal hyperplasia were examined.ResultsLenti-miR-126 increased while miR-126 inhibitor decreased LOC migration and CXCR4 expression on LOCs. miR-126 positively regulated p-ERK, VEGF, p-Akt, and eNOS protein expression, and inhibitors of these proteins blocked miR-126-induced CXCR4 expression and also reduced LOC migration. Overexpression of miR-126 promoted while inhibition of miR-126 suppressed stemness gene expression in LOCs. miR-126 also inhibited gene and protein expression and promoter activity of KLF-8 while shRNA-mediated knockdown of KLF-8 increased stemness gene expression. Upregulation of stemness gene expression by miR-126 overexpression was completely abrogated by co-transfection of lenti-KLF-8 and lenti-miR-126 into LOCs. In GK rats, transplantation of LOCs overexpressing miR-126 enhanced LOC homing and reendothelialization and decreased intimal hyperplasia of injured arteries.ConclusionOur results indicate that miR-126 protects against vascular injury by promoting CXCR4 expression and LOC homing via ERK/VEGF and Akt/eNOS signaling pathways and maintaining stemness via targeting KLF-8.

BMPR2-expressing bone marrow-derived endothelial-like progenitor cells alleviate pulmonary arterial hypertension in vivo.

Pulmonary arterial hypertension (PAH) is characterized by increased resistance in the distal pulmonary arteries, ultimately leading to right heart failure and, despite the available therapeutics, survival remains poor. Reduced expression of bone morphogenetic protein receptor type 2 (BMPR2) is strongly associated with PAH. Cell therapies are of interest in PAH, but whether this approach can upregulate BMPR2 is not known. Our objective was to evaluate a preclinical cell therapy approach based on upregulation of BMPR2. We assessed the therapeutic effect of intravenously injected BMPR2-augmented rat bone marrow-derived endothelial-like progenitor cells (BMPR2-BM-ELPC) on PAH in the rat monocrotaline (MCT) model. The cells accumulate in the lungs with negligible systemic distribution, but the vast majority are lost from the lungs by 24 h. Lungs from rats treated with BMPR2-BM-ELPC exhibited an immediate increase in BMPR2 and related intracellular signalling proteins. Treatment with BMPR2-BM-ELPC attenuated PAH as demonstrated by a reduction in right ventricular hypertrophy as well as right ventricular systolic and mean pulmonary arterial pressures. In addition, this treatment reversed PAH-induced vascular remodelling with a significant reduction in vessel thickness and muscularization. In view of the short retention time of injected cells in the lungs, the mechanism for the effects seen may be intracellular communication via exosomes. In support of this hypothesis, we demonstrate that BMPR2-transduced outgrowth endothelial progenitor cells (OECs) release BMPR2-expressing exosomes. BMPR2-augmented ELPC demonstrate therapeutic benefits in the rat model and may have clinical translation potential.

Telbivudine Reduces Parvovirus B19-Induced Apoptosis in Circulating Angiogenic Cells.

Aims: Human parvovirus B19 (B19V) infection directly induces apoptosis and modulates CXCR4 expression of infected marrow-derived circulating angiogenic cells (CACs). This leads to dysfunctional endogenous vascular repair. Treatment for B19V-associated disease is restricted to symptomatic treatment. Telbivudine, a thymidine analogue, established in antiviral treatment for chronic hepatitis B, modulates pathways that might influence induction of apoptosis. Therefore, we tested the hypothesis of whether telbivudine influences B19V-induced apoptosis of CAC. Methods and Results: Pretreatment of two CAC-lines, early outgrowth endothelial progenitor cells (eo-EPC) and endothelial colony-forming cells (ECFC) with telbivudine before in vitro infection with B19V significantly reduced active caspase-3 protein expression (−39% and −40%, both p < 0.005). Expression of Baculoviral Inhibitor of apoptosis Repeat-Containing protein 3 (BIRC3) was significantly downregulated by in vitro B19V infection in ECFC measured by qRT-PCR. BIRC3 downregulation was abrogated with telbivudine pretreatment (p < 0.001). This was confirmed by single gene PCR (p = 0.017) and Western blot analysis. In contrast, the missing effect of B19V on angiogenic gene expression postulates a post-transcriptional modulation of CXCR4. Conclusions: We for the first time show a treatment approach to reduce B19V-induced apoptosis. Telbivudine reverses B19V-induced dysregulation of BIRC3, thus, intervening in the apoptosis pathway and protecting susceptible cells from cell death. This approach could lead to an effective B19V treatment to reduce B19V-related disease.

ENDOTHELIAL PROGENITOR CELLS ENCAPSULATED IN MATRIX-SUPPLEMENTED MICROGEL IMPROVES CELL RETENTION AND THERAPEUTIC EFFICACY IN PULMONARY ARTERIAL HYPERTENSION

Late outgrowth (L) endothelial progenitor cells (EPC) represent a uniform, highly endothelial-like progenitor cell population; however, their therapeutic benefits in models of pulmonary arterial hypertension (PAH) are limited by poor cell persistence, due to rapid cell loss by apoptosis (anoikis) and redistribution to non-target organs. Temporary single-cell microencapsulation (i.e. cocooning) provides a portable stem cell niche, that can promote cell survival and retention in animal models of organ lung and heart injury. We hypothesize that microencapsulation of L-EPC with an agarose hydrogel supplemented with integrin-binding proteins will increase survival and retention of L-EPCs injected into the jugular vein and result in greater therapeutic benefits compared to non-cocooned cells in a rat monocrotaline (MCT) model of PAH. L-EPCs were encapsulated by vortex-emulsion using various concentrations of agarose, together with fibronectin and fibrinogen, and capsule size and initial cell viability were assessed. Encapsulated and non-encapsulated L-EPCs were transduced with luciferase and administered to SD rats three days after injection of MCT. L-EPCs were tracked in vivo by bioluminescence imaging (BLI) to assess cell persistence and bio-distribution for up to three weeks post cell injection. At end-study, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) were assessed for therapeutic efficacy. The initial BLI signals at 15 minutes after delivery were similar in non-cocooned and cocooned L-EPCs; however, only cocooned cells could be detected by BLI after four and 24 hours (28±12% and 12±8% of baseline signal, respectively; p< 0.0001 and 0.05, n=11). Microencapsulation of L-EPCs led to significant improvement in RVSP three weeks after delivery compared to MCT alone (56±24 vs. 80±7 mmHg, respectively; p<0.05), whereas no improvement in pulmonary hemodynamics was seen with delivery of non-encapsulated cells. These results demonstrate that single-cell cocooning can significantly increase retention of L-EPCs within the lungs. Furthermore, even a modest increase in L-EPC persistence over 24 hours can provide an important therapeutic benefit, not seen with non-encapsulated L-EPCs in the rat MCT model of PAH.

Endothelial Progenitor Cells and Cardiovascular Correlates.

Cardiovascular disease is cited as the underlying cause of death in one out of every three deaths within the United States; this burden on the health care system percolates down to affect patients on an individual level. In part, the problem arises from the low regenerative capacity of cardiovascular system cells, for example, cardiac myocytes, and from oxidative stressors to the human body.Endothelial progenitor cells (EPCs) are a type of stem cell, and various clinical conditions including hypertension and renal failure underlie their dysfunction. EPCs are classified as either early or late outgrowth endothelial progenitor cells depending on the time they appear in circulation and at the site of injury after an inciting event. Their function is paracrine through the release of cytokines, growth factors and chemokines such as interleukin-6 and vascular endothelial growth factor, and they are involved in transdifferentiation into vascular smooth muscle cells and potentially cardiac myocytes. They are beneficial to the modification of cardiovascular cell apoptosis, fibrosis, and contractility. In times of stress, the normal function of endothelial progenitor cells is altered; this creates a maladaptive cycle where stress and failed coping mechanisms enhance each other toward the culmination of cardiovascular disease.The development of the cardiovascular system follows gastrulation in the embryonic period, and the cells that form the system are derived from the mesoderm; being mesoderm, the vascular cells exhibit heterogeneity in their origin and function. The need to understand the molecular and cellular regulatory pathways during development can amalgamate efforts of endothelial cell and cardiovascular system pathophysiology for the advancement of patient cardiovascular reserve and function.

337 - Single-cell matrix-supplemented hydrogel cocooning of endothelial progenitor cells improves retention and therapeutic efficacy in pulmonary arterial hypertension

Background: Late outgrowth (L) endothelial progenitor cells (EPC) represent a uniform, highly endothelial-like progenitor cell population; however, their therapeutic benefits in models of pulmonary arterial hypertension (PAH) are limited by poor cell persistence, due to rapid cell loss by apoptosis (anoikis) and redistribution to non-target organs. Temporary microencapsulation (i.e. cocooning) provides a portable stem cell niche, that can promote cell survival and retention in animal models of organ lung and heart injury.

Synthetic microparticles conjugated with VEGF165 improve the survival of endothelial progenitor cells via microRNA-17 inhibition

Several cell-based therapies are under pre-clinical and clinical evaluation for the treatment of ischemic diseases. Poor survival and vascular engraftment rates of transplanted cells force them to work mainly via time-limited paracrine actions. Although several approaches, including the use of soluble vascular endothelial growth factor (sVEGF)—VEGF165, have been developed in the last 10 years to enhance cell survival, they showed limited efficacy. Here, we report a pro-survival approach based on VEGF-immobilized microparticles (VEGF-MPs). VEGF-MPs prolong VEGFR-2 and Akt phosphorylation in cord blood-derived late outgrowth endothelial progenitor cells (OEPCs). In vivo, OEPC aggregates containing VEGF-MPs show higher survival than those treated with sVEGF. Additionally, VEGF-MPs decrease miR-17 expression in OEPCs, thus increasing the expression of its target genes CDKN1A and ZNF652. The therapeutic effect of OEPCs is improved in vivo by inhibiting miR-17. Overall, our data show an experimental approach to improve therapeutic efficacy of proangiogenic cells for the treatment of ischemic diseases.

Evaluation of late outgrowth endothelial progenitor cell and umbilical vein endothelial cell responses to thromboresistant collagen-mimetic hydrogels.

Bioactive coatings which support the adhesion of late-outgrowth peripheral blood endothelial progenitor cells (EOCs) are actively being investigated as a means to promote rapid endothelialization of "off-the-shelf," small-caliber arterial graft prostheses following implantation. In the present work, we evaluated the behavior of EOCs on thromboresistant graft coatings based on the collagen-mimetic protein Scl2-2 and poly(ethylene glycol) (PEG) diacrylate. Specifically, the attachment, proliferation, migration, and phenotype of EOCs on PEG-Scl2-2 hydrogels were evaluated as a function of Scl2-2 concentration (4, 8, and 12 mg/mL) relative to human umbilical vein endothelial cells (HUVECs). Results demonstrate the ability of each PEG-Scl2-2 hydrogel formulation to support EOC and HUVEC adhesion, proliferation, and spreading. However, only the 8 and 12 mg/mL PEG-Scl2-2 hydrogels were able to support stable EOC and HUVEC confluence. These PEG-Scl2-2 formulations were, therefore, selected for evaluation of their impact on EOC and HUVEC phenotype relative to PEG-collagen hydrogels. Cumulatively, both gene and protein level data indicated that 8 mg/mL PEG-Scl2-2 hydrogels supported similar or improved levels of EOC maturation relative to PEG-collagen controls based on evaluation of CD34, VEGFR2, PECAM-1, and VE-Cadherin. The 8 mg/mL PEG-Scl2-2 hydrogels also appeared to support similar or improved levels of EOC homeostatic marker expression relative to PEG-collagen hydrogels based on von Willebrand factor, collagen IV, NOS3, thrombomodulin, and E-selectin assessment. Combined, the present results indicate that PEG-Scl2-2 hydrogels warrant further investigation as "off-the-shelf" graft coatings. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1712-1724, 2017.

Preconditioned Endothelial Progenitor Cells as Biomarker of Vascular Reparation?

The endothelial progenitor cells (EPCs) have defined as cells positively labeled with both hematopoietic stem cells (CD34) and endothelial cell markers predominantly VEGF receptor-2 (VEGFR2) cumulatively. Therefore, there are at least two types of EPCs labelled as early outgrowth EPCs and late outgrowth EPCs probably distinguished their vascular protective ability. Recent animal and clinical studies have shown that reduced number and weak function of EPCs may not only indicate to higher CV risk, but contribute to the impaired heart and vessels reparation. Interestingly, there are some subpopulations of EPCs especially recruited from peripheral blood cells, which may exhibit very variable pro-angiogenic effect and endothelial repair capacity and they are called “preconditioned” EPCs. The aim of the short commentary is depicted the possibilities to use of measurement of traditionally labeled EPCs as biomarker of cardiovascular risk.

Dynamic Perfusion Culture of Human Outgrowth Endothelial Progenitor Cells on Demineralized Bone Matrix In Vitro.

BackgroundThe aim of this study was to investigate the proliferation, differentiation, and tube formation of human outgrowth endothelial progenitor cells (OECs) cultured with porous demineralized bone matrix (DBM) under a dynamic perfusion system in vitro.Material/MethodsOECs were isolated, expanded, characterized, eGFP-transfected and seeded on DBM scaffold and cultured under static or dynamic perfusion conditions, and continuously observed under fluorescence microscope. DBM scaffolds were harvested on day six for RT-PCR and western blot assay to analyze the mRNA and protein expression level of CD34, VE-cadherin, and VEGF. Scanning electron microscope (SEM) was used to observe the tube formation of OECs seeded on DBM scaffolds.ResultsThe results showed the cell density of OECs on DBM was higher when exposed to shear stress generated by a dynamic perfusion system. Shear stress also markedly increased the expression level of VE-cadherin and VEGF and decreased the expression of CD34, at both mRNA and protein levels. SEM showed that the shear-stressed OECs formed tube-like structures inside the pores of DBM scaffolds.ConclusionsA dynamic perfusion system can be used as an innovative method for the rapid vascularization in tissue engineering, which can accelerate the proliferation and differentiation of OECs and the vascularization of implanted scaffolds.

Establishment of an Early Vascular Network Promotes the Formation of Ectopic Bone.

Vascularization is crucial for the induction of bone formation. In this study, we investigated the application of two subtypes of peripheral blood-derived endothelial progenitor cells (EPCs) to stimulate vessel formation in ectopic bone constructs. Early and late outgrowth EPCs (E-EPC and L-EPC, respectively) were characterized for their ability to form network structures in vitro and perfused vessels subcutaneously in mice. Only L-EPCs showed the formation of fully connected networks on Matrigel two-dimensional (2D) angiogenesis assays. The presence of multipotent stromal cells (MSCs) inhibited network formation in 2D assays, but stimulated network formation in three-dimensional plugs. In vivo studies revealed that at 2 weeks, the highest incidence of formed perfused vessels was reached by implanted E-EPC/MSC constructs and this could be attributed to the presence of E-EPCs. L-EPCs displayed a significantly lower frequency of blood vessel formation than E-EPCs and this was accompanied by a lowering of total luminal area densities. Nevertheless, combined E-EPC/L-EPC application somewhat increased the percentage incidence of perfused vessels. After 6 weeks, differences in vascularization were still obvious as all three EPC-based constructs contained higher numbers of perfused vessels than constructs containing MSCs alone. Bone was formed in all constructs at an incidence that coincided with high density of perfused vessels after 2 weeks. Altogether, our findings suggest the differential establishment of vascular networks by E-EPCs and L-EPCs and suggest the importance of early vasculogenesis in ectopic bone formation.

Progenitor Endothelial Cell Dysfunction in Obese Patients: Possibilities for Cardiovascular Risk Prediction

Obesity is recognized a leading factor contributing in diabetes and cardiovascular (CV) disease development worldwide. Among obese individuals at least two phenotypes are determined, i.e. metabolically healthy obese and metabolically non-healthy obese. Subjects with one or other phenotype appear to be distinguished in CV risk and diabetes. Although lack of strong definition of metabolically healthy obese as a transient age- and ethnic-related phenotype accompanied to some behavioral and environmental factors, the role of co-existing metabolic abnormalities in translation of metabolically healthy obese to metabolically non-healthy obese is under debates. Outgrowth endothelial progenitor cells (EPCs) have protective abilities to vasculature and they are posed as a central key of endogenous repair system. In this context, weak functionality and reduced number of circulating EPCs determined as EPC dysfunction might link phenotypes of obese and CV risk. The editorial is considered a role of EPC dysfunction as a predictive biomarker in CV diseases and events in patients with different phenotypes of obese.

Disrupted Endothelial Cell Layer and Exposed Extracellular Matrix Proteins Promote Capture of Late Outgrowth Endothelial Progenitor Cells.

Late outgrowth endothelial progenitor cells (LO-EPC) possess a high proliferative potential, differentiate into vascular endothelial cells (EC), and form networks, suggesting they play a role in vascular repair. However, due to their scarcity in the circulation there is a requirement for ex vivo expansion before they could provide a practical cell therapy and it is currently unclear if they would home and engraft to an injury site. Using an in vitro flow system we studied LO-EPC under simulated injury conditions including EC activation, ischaemia, disrupted EC integrity, and exposed basement membrane. Perfused LO-EPC adhered to discontinuous EC paracellularly at junctional regions between adjacent cells under shear stress 0.7 dyn/cm2. The interaction was not adhesion molecule-dependent and not enhanced by EC activation. LO-EPC expressed high levels of the VE-Cadherin which may explain these findings. Ischaemia reperfusion injury decreased the interaction with LO-EPC due to cell retraction. LO-EPC interacted with exposed extracellular matrix (ECM) proteins, fibronectin and vitronectin. The interaction was mediated by integrins α5β3, αvβ1, and αvβ3. This study has demonstrated that an injured local environment presents sufficient adhesive signals to capture flow perfused LO-EPC in vitro and that LO-EPC have properties consistent with their potential role in vascular repair.

The regulation of endothelial nitric oxide synthase by extracellular matrix in human late outgrowth endothelial progenitor cells

The regulation of endothelial nitric oxide synthase by extracellular matrix in human late outgrowth endothelial progenitor cells

Therapeutic effects of late outgrowth endothelial progenitor cells or mesenchymal stem cells derived from human umbilical cord blood on infarct repair

BackgroundThis study sought to systematically investigate the derivation of late outgrowth endothelial progenitor cells (late EPC) and mesenchymal stem cells (MSC) from umbilical cord blood (UCB) and to examine their therapeutic effects on myocardial infarction (MI). MethodsThe expression of angiogenic genes was determined by qRT-PCR. Myocardial infarction (MI) was induced in rats, and cells were directly transplanted into the border regions of ischemic heart tissue. ResultsCulture of UCB mononuclear cells yielded two distinct types of cells by morphology after 2weeks in the same culture conditions. These cells were identified as late EPC and MSC, and each was intramyocardially injected into rat hearts after induction of MI. Echocardiography and histologic analyses demonstrated that both EPC and MSC improved cardiac function and enhanced vascularization, although fibrosis was reduced only in the EPC transplanted hearts. Different paracrine factors were enriched in EPC and MSC. However, once injected into the hearts, they induced similar types of paracrine factors in the heart. Transplanted EPC or MSC were mostly localized at the perivascular areas. This study demonstrated that EPC and MSC can be simultaneously derived from UCB under the same initial culture conditions, and that common paracrine factors are involved in the repair of MI. ConclusionLate EPC and MSC are effective for infarct repair, apparently mediated through common humoral mechanisms.

RHO-ASSOCIATED KINASE (ROCK) REGULATES ENDOTHELIAL NITRIC OXIDE SYNTHASE IN BLOOD OUTGROWTH ENDOTHELIAL CELLS

Human blood outgrowth endothelial progenitor cells (BOECs), derived from peripheral blood mononuclear cells express endothelial protein profiles (i.e. CD31, 144, 146, KDR, and vWF). Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular tone and loss of eNOS activity is a hallmark of endothelial dysfunction. Previous studies have demonstrated that the extracellular matrix (ECM) proteins negatively affect eNOS expression and activity, and a recent study has shown that BOECs deposit extensive amounts of ECM proteins. Rho-associated kinase (ROCK) plays a critical role in the regulation of cytoskeleton and ECM production. In this study, we have investigated the role of ROCK on eNOS expression and activity in BOECs. Human BOECs were cultured with ROCK inhibitors (Y27632 or Fasudil) and ECM proteins (type I collagen (Col. I) and fibronectin (FN)) deposition were measured. After 24 hours of culture, BOECs deposit an organized mesh-like structure of Col. I. This collagen structure was not seen in cells treated with Y27632 or fasudil. To explore the regulation of ROCK on eNOS, we have measured eNOS expression (Western Blot), nitric oxide (4-Amino-5-Methylamino-2',7'-Difluorofluorescein), nitrotyrosine (Western Blot) and superoxide (Dihydroethidium) levels after Y27632 treatment. We found a significant increase in eNOS protein expression (1.37 ± 0.11 fold change, p < 0.01) in BOECs after incubating with Y27632. Interestingly, the nitrotyrosine and superoxide levels were both markedly reduced (0.60 ± 0.10 fold change in Nitrotyrosine, p < 0.01) to a similar level as superoxide dismutase treated group. Taken together, this result suggests that ROCK inhibition may negatively regulate ECM production and potentially enhance eNOS expression while at the same time limiting peroxynitrite mediated nitrosylation, suggesting an enhanced eNOS bioactivity in BOECs. Human blood outgrowth endothelial progenitor cells (BOECs), derived from peripheral blood mononuclear cells express endothelial protein profiles (i.e. CD31, 144, 146, KDR, and vWF). Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular tone and loss of eNOS activity is a hallmark of endothelial dysfunction. Previous studies have demonstrated that the extracellular matrix (ECM) proteins negatively affect eNOS expression and activity, and a recent study has shown that BOECs deposit extensive amounts of ECM proteins. Rho-associated kinase (ROCK) plays a critical role in the regulation of cytoskeleton and ECM production. In this study, we have investigated the role of ROCK on eNOS expression and activity in BOECs. Human BOECs were cultured with ROCK inhibitors (Y27632 or Fasudil) and ECM proteins (type I collagen (Col. I) and fibronectin (FN)) deposition were measured. After 24 hours of culture, BOECs deposit an organized mesh-like structure of Col. I. This collagen structure was not seen in cells treated with Y27632 or fasudil. To explore the regulation of ROCK on eNOS, we have measured eNOS expression (Western Blot), nitric oxide (4-Amino-5-Methylamino-2',7'-Difluorofluorescein), nitrotyrosine (Western Blot) and superoxide (Dihydroethidium) levels after Y27632 treatment. We found a significant increase in eNOS protein expression (1.37 ± 0.11 fold change, p < 0.01) in BOECs after incubating with Y27632. Interestingly, the nitrotyrosine and superoxide levels were both markedly reduced (0.60 ± 0.10 fold change in Nitrotyrosine, p < 0.01) to a similar level as superoxide dismutase treated group. Taken together, this result suggests that ROCK inhibition may negatively regulate ECM production and potentially enhance eNOS expression while at the same time limiting peroxynitrite mediated nitrosylation, suggesting an enhanced eNOS bioactivity in BOECs.

Impaired vascular remodeling after endothelial progenitor cell transplantation in MMP9-deficient mice suffering cortical cerebral ischemia.

Endothelial progenitor cells (EPCs) are being investigated for advanced therapies, and matrix metalloproteinase 9 (MMP9) has an important role in stroke recovery. Our aim was to determine whether tissue MMP9 influences the EPC-induced angiogenesis after ischemia. Wild-type (WT) and MMP9-deficient mice (MMP9/KO) were subjected to cerebral ischemia and treated with vehicle or outgrowth EPCs. After 3 weeks, we observed an increase in the peri-infarct vessel density in WT animals but not in MMP9/KO mice; no differences were found in the vehicle-treated groups. Our data suggest that tissue MMP9 has a crucial role in EPC-induced vascular remodeling after stroke.