Importance of ß2AR elevation for re-endothelialization capacity mediated by late endothelial progenitor cells in hypertensive patients.

Dysfunction of late endothelial progenitor cells (EPCs) has been suggested to be associated with hypertension. ß2-Adrenergic receptor (ß2AR) is a novel and key target for EPC homing. Here, we proposed that attenuated ß2AR signaling contributes to EPCs dysfunction, whereas enhanced ß2AR signaling restores EPCs' functions in hypertension. EPCs derived from hypertensive patients exhibited reduced cell number, impaired in vitro migratory and adhesion abilities, and impaired re-endothelialization after transplantation in nude mice with carotid artery injury. ß2AR expression of EPCs from hypertensive patients was markedly downregulated, whereas the phosphorylation of the p38 mitogen-activated protein kinase (p38-MAPK) was elevated. The cleaved caspase-3 levels were elevated in EPCs. The overexpression of ß2AR in EPCs from hypertensive patients inhibited p38-MAPK signaling, whereas it enhanced in vitro EPC proliferation, migration, and adhesion and in vivo re-endothelialization. The ß2AR-mediated effects were attenuated by treating the EPCs with a neutralizing monoclonal antibody against ß2AR, which could be partially antagonized by the p38-MAPK inhibitor SB203580. Moreover, shear stress stimulation, a classic nonpharmacological intervention, increased the phosphorylation levels of ß2AR and enhanced the in vitro and in vivo functions of EPCs from hypertensive patients. Collectively, the current investigation demonstrated that impaired ß2AR/p38-MAPK/caspase-3 signaling at least partially reduced the re-endothelialization capacity of EPCs from hypertensive patients. Restoration of ß2AR expression and shear stress treatment could improve their endothelial repair capacity by regulating the p38-MAPK/caspase-3 signaling pathway. The clinical significance of ß2AR in endothelium repair still requires further investigation.NEW & NOTEWORTHY Impaired ß2-adrenergic receptor (ß2AR) expression with an elevation of p38-MAPK/caspase-3 signaling at least partially contributes to the decline of re-endothelialization capacity of late endothelial progenitor cells (EPCs) from hypertensive patients. ß2AR gene transfer and shear stress treatment improve the late EPC-mediated enhancement of the re-endothelialization capacity in hypertensive patients through activating ß2AR/p38-MAPK/caspase-3 signaling. The present study is the first to reveal the potential molecular mechanism of the impaired endothelium-reparative capacity of late EPCs in hypertension after vascular injury and strongly suggests that ß2AR is a novel and crucial therapeutic target for increasing EPC-mediated re-endothelialization capacity in hypertension.

CD276 (B7-H3) Maintains Proliferation and Regulates Differentiation in Angiogenic Function in Late Endothelial Progenitor Cells.

Endothelial progenitor cells (EPCs) provide an important source of recovery from blood vessel dysfunction. Late EPCs (LEPCs) are circulating blood cells that are capable of promoting vascular repair. Using transcriptome analysis, we identified distinctive LEPC profiles and found that CD276 (B7-H3) mRNA is strongly expressed in LEPCs. CD276 protein is present abundantly on the cell surface of LEPC when analyzed by fluorescence-activated cell sorter and immunocytochemistry. CD276, a B7 family member, is a type I transmembrane glycoprotein. The role of CD276 in LEPCs remains unknown. CD276 knockdown by lentivirus transduction in LEPCs significantly decreased proliferation and increased apoptosis of LEPCs in vitro. After CD276 silencing, the cell cycle of LEPCs was prone to remain at the G0/G1 phase, and the cell migration rates as well as transwell and wound-healing migration were decreased. CD276 knockdown in LEPCs increased the G1 phase regulators cyclin D2/D3/E1-cyclin-dependent kinases (CDK2/4/6), but decreased the S-G2-M phase regulators cyclin A/B-CDK1. However, LEPCs with CD276 knockdown resulted in increased tube formation in vitro and angiogenesis in a Matrigel plug assay in vivo. FoxC1/C2, an upstream signal of Notch in arterial cell proliferation, and Hey1/2, which is known to promote arterial differentiation in the vasculature, were upregulated in CD276 knockdown LEPCs. In LEPCS, CD276 has a positive effect on proliferation and migration of endothelial cells, but negative effects on angiogenesis, particularly endothelial cell differentiation. Our data indicate, for therapeutic purpose, that CD276 can be used to acquire and maintain cell populations of LEPCs and blocking CD276 will promote angiogenetic differentiation. We found that CD276 (B7-H3) is enriched on the cell membrane of LEPCs. CD276 knockdown reduced proliferation and migration of LEPCs by increasing cell cycle inhibitors such as p21cip1 and pRb and decreasing pErk1/2 and pAkt but promoted angiogenesis and endothelial cell differentiation by elevating vascular endothelial growth factor-vascular endothelial growth factor receptor 1 and p-p38. Stem Cells 2019;37:382-394.

Systemic microvascular rarefaction is correlated with dysfunction of late endothelial progenitor cells in mild hypertension: a substudy of EXCAVATION-CHN1.

BACKGROUND: Hypertension often presents with microvascular rarefaction (MVR), which is closely associated with impaired angiogenesis. Early detection of MVR is essential for systemic assessment in patient with hypertension. We aimed to determine the systemic MVR through both optical coherence tomography angiography (OCTA) and intravital capillaroscopy, and to investigate their respective efficacies and related mechanisms associated with late endothelial progenitor cells (LEPCs) dysfunction. METHODS: Seventy-one hypertensive and sixty-nine age-match normotensive subjects were included in this study. All subjects received intravital capillaroscopy for skin capillary density (SCD) and OCTA for retinal capillary density (RCD) and non-perfused areas (R-NPA). Subsequently, correlation of LEPCs activities and microvascular rarefaction were examined. RESULTS: Compared with normotensive subjects, hypertensive patients had significantly lower RCD [(52.9 ± 2.9)% vs. (57.8 ± 1.6)%, P < 0.01] and higher R-NPA [(0.12 ± 0.07) mm2 vs. (0.053 ± 0.020) mm2, P < 0.01]. SCD correlated positively with RCD but negatively with R-NPA [(RCD: OR = 0.40, 95% CI 0.25-0.67, P < 0.01); (R-NPA: OR = 0.39, 95% CI - 0.0029 to 0.0011, P < 0.01)]. The discriminative powers of RCD performed best (AUC 0.79 versus SCD AUC 0.59, P < 0.001) followed by R-NPA (AUC 0.73 versus SCD AUC 0.59, P < 0.001) for systolic blood pressure. Similar pattern is also found for diastolic blood pressure (RCD AUC 0.80 versus SCD AUC 0.54, P < 0.001; R-NPA AUC 0.77 versus SCD AUC 0.54, P < 0.001). Furthermore, LEPCs tube formation was impaired in hypertensive patients (36.8 ± 2.3 vs. 28 ± 3.7, P < 0.01). After multivariate adjustments, positive correlation existed between RCD or R-NPA with LEPCs tube formation (RCD: ß = 0.64, 95% CI 0.34-0.91, P < 0.01; R-NPA: ß = - 24.67, 95% CI - 43.14 to - 4.63, P < 0.05) but not with SCD (ß = 0.082, 95% CI 0.01-0.18, P = 0.085). CONCLUSION: Compared to intravital capillaroscopy, OCTA is a more precise technique for early detection of hypertensive microvascular rarefaction, which is associated with the fall in LEPC-mediated angiogenesis. Both of OCTA and LEPCs function can help identify hypertension-related capillary abnormality. Trail Registration The trial is a substudy of EXCAVATION-CHN1, registered at clinicaltrials.gov as NCT02817204 (June 26, 2016).

Impact of transduction towards the proliferation and migration as well as the transduction efficiency of human umbilical cord-derived late endothelial progenitor cells with nine recombinant adeno-associated virus serotypes.

OBJECTIVES: To evaluate the transduction efficiency of human umbilical cord-derived, late endothelial progenitor cells late (HUCB-late EPCs) with nine recombinant adeno-associated virus (rAAV) serotypes and the ability of proliferation and migration of the cells after transduction. RESULTS: rAAV2 and rAAV6 showed a greater ability than other serotypes to transduce late EPCs (P < 0.05). After transduction, cell proliferation ability weakened (P < 0.05), but the ability of migration to stromal cell-derived factor (SDF-1) unchanged. CONCLUSION: There is an advantage of choosing the optimal rAAV serotype as a gene vector to alter the biologic characteristics of late EPCs.

Platelets of Healthy Origins Promote Functional Improvement of Atherosclerotic Endothelial Progenitor Cells.

The purpose was to evaluate the effect of platelets on functional properties of late endothelial progenitor cells (EPCs), in the direct co-culture conditions, and to investigate the involved mediators, in experimental induced atherosclerosis. The late EPCs obtained from two animal groups, hypertensive-hyperlipidemic (HH) and control (C) hamsters, named late EPCs-HH and late EPCs-C, were co-incubated with or without platelets isolated from both groups. Our results have showed that exposure to platelets from control animals: (i) promoted the late EPCs-C capacity to form colonies and capillary-like structures, and also to proliferate and migrate; (ii) improved the functional properties of late EPCs-HH; (iii) strengthened the direct binding EPCs-platelets; (iv) increased SDF-1α,VEGF, PDGF, and reduced CD40L, IL-1ß,-6,-8 levels; and (v) enhanced miR-223 and IGF-1R expressions. Platelets from HH group diminished functional abilities for both EPC types and had opposite effects on these pro-angiogenic and pro-inflammatory molecules. Furthermore, testing the direct effect of miR-223 and IGF-1R on late EPCs disclosed that these molecular factors improve late EPC functional properties in atherosclerosis in terms of stimulation of the proliferation and migration abilities. In conclusion, in vitro exposure to platelets of healthy origins had a positive effect on functional properties of atherosclerotic late EPCs. The most likely candidates mediating EPC-platelet interaction can be SDF-1α, VEGF, CD40L, PDGF, IL-1ß,-6,-8, miR-223, and IGF-1R. The current study brings evidences that the presence of healthy origin platelets is of utmost importance on functional improvement of EPCs in atherosclerosis.

IFN-γ enhances the wound healing effect of late EPCs (LEPCs) via BST2-mediated adhesion to endothelial cells.

Circulating late endothelial progenitor cells (LEPCs) home to injured vessels, initiating blood vessel regeneration. This process requires the initial adhesion of LEPCs to endothelial cells within the wounded site. In this study, treating LEPCs with IFN-γ enhanced wound healing through BST2-mediated adhesion to endothelial cells. We found that IFN-γ significantly upregulated BST2 expression in both LEPCs and ECs and increased tube formation in LEPCs. Upregulated BST2 increased LEPC adhesion to ECs through a tight homophilic interaction of its extracellular domain. Finally, when the IFN-γ-treated LEPCs were injected into the wounded mouse tail vein, superior therapeutic effects of wound closure were observed. This study provides a useful application to enhance the adhesion of LEPCs for vessel regeneration and wound closure.

Resveratrol protects late endothelial progenitor cells from TNF-α-induced inflammatory damage by upregulating Krüppel-like factor-2.

Cardiovascular risk factors can negatively influence late endothelial progenitor cell (EPCs) number and functions, thus EPCs biology is a clinical implications for cardiovascular diseases. The present study aimed to investigate the potential protective effects of resveratrol (RES) on tumor necrosis factor (TNF)­α­induced inflammatory damage in late endothelial progenitor cells (EPCs) and to elucidate the underlying mechanisms. Late EPCs at passages 3­5 were pretreated with RES at a concentration of 20 µmol/l for 12 h and subsequently incubated with TNF­α (10 ng/ml) for 24 h. The adhesion, migration, proliferation and vasculogenesis of EPCs were subsequently detected. Furthermore, the mRNA expression levels of intercellular adhesion molecule­1 (ICAM­1) and monocyte chemoattractant protein­1 (MCP­1) were measured by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). Nitric oxide (NO) levels in the supernatant were determined using a colorimetric assay kit. Additionally, the mRNA and protein expression of Krüppel­like factor­2 (KLF2) was determined by RT­qPCR and western blot analysis, respectively. The results indicated that TNF­α markedly inhibited the proliferation, adhesion, migration and vasculogenesis of late EPCs. However, RES ameliorated the effects induced by TNF­α. Furthermore, exposure of EPCs to TNF­α decreased the levels of NO secretion and KLF2 expression at the mRNA and protein levels, but upregulated the levels of inflammatory factors, including ICAM­1 and MCP­1, compared with the control group. RES significantly inhibited TNF­α­induced inflammatory damage through upregulation of KLF2 expression and downregulation of the expression of ICAM­1 and MCP­1. In conclusion, RES may exert protective effects on the cardiovascular system, as demonstrated by the amelioration of TNF-α-induced inflammation in EPCs following RES treatment, and may therefore be used in the future for the prevention of cardiovascular disease.