Circulating angiogenic cells (CACs), represent a potential new therapeutic tool for the treatment of cardiovascular diseases, but their regenerative function is impaired in patients with coronary artery disease (CAD) and cardiac risk factors. The objective of this study is to assess the effect of lentiviral overexpression of endothelial nitric oxide synthase (eNOS) on the activity of CACs from patients with CAD and cardiac risk factors. In vitro and in vivo assays were employed to evaluate the regenerative capacity of the cells compared to CACs derived from healthy volunteers. Lentiviral eNOS transduction of cells from CAD patients significantly improved chemotactic migration compared with sham transduction, and increased the ability of CACs to induce angiogenic tube formation when cocultured with human umbilical vein endothelial cells (HUVECs) on Matrigel. In addition, eNOS transduction restored the ability of patient-derived CACs to enhance neovascularization and improve ischemic hind limb perfusion, approaching the efficacy of cells from healthy donors. These data indicate that CAC dysfunction seen in high-risk patients can be partially reversed by eNOS overexpression, suggesting that ex vivo gene delivery may improve the efficacy of autologous cell therapy for cardiovascular disease.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder. Circulating angiogenic cells (CACs) play an important role in vascular repair and regeneration. This study was designed to examine the function of CACs derived from patients with HHT. Peripheral blood mononuclear cells (PBMNCs) isolated from patients with HHT and age- and gender-matched healthy volunteers were assessed for expression of CD34, CD133 and VEGF receptor 2 by flow cytometry. PBMNCs were cultured to procure early outgrowth CACs. Development of endothelial cell (EC) phenotype in CACs was analyzed by fluorescence microscopy. CAC apoptosis was assayed with Annexin V staining, and CAC migration assessed by a modified Boyden chamber assay. mRNA expression of endoglin (ENG), activin receptor-like kinase-1 (ACVLR1 or ALK1) and endothelial nitric oxide synthase (eNOS) in CACs was measured by real time RT-PCR. The percentage of CD34+ cells in PBMNCs from HHT patients was significantly higher than in PBMNCs of healthy controls. CACs derived from patients with HHT not only showed a significant reduction in EC-selective surface markers following 7-day culture, but also a significant increase in the rate of basal apoptosis and blunted migration in response to vascular endothelial growth factor and stromal cell-derived factor-1. CACs from HHT patients expressed significantly lower levels of ENG, ALK1 and eNOS mRNAs. In conclusion, CACs from patients with HHT exhibited various functional impairments, suggesting a reduced regenerative capacity of CACs to repair the vascular lesions seen in HHT patients.

Endothelial progenitor cells (EPCs) have been used in clinical trials to treat ischemic heart disease. Monocyte infiltration plays an important role in inflammation, angiogenesis, and tissue repair during tissue ischemia. It is important to understand the interactions between EPCs and monocytes. In this study, a human EPC/THP-1 monocytic cell coculture system was used to examine EPC effect on IL-1α, IL-1β, and TNF-α expression in THP-1 cells. Late, but not early, EPCs upregulated IL-1β expression at both mRNA and protein levels. In contrast, neither early nor late EPCs affected IL-1α or TNF-α expression. Coculture with human umbilical vein endothelial cells did not alter IL-1β expression. It has been shown that activation of integrin β2 in human neutrophils augments IL-1β synthesis; however integrin β2 was not involved in IL-1β expression in THP-1 cells. Addition of late EPC conditioned medium to THP-1 cell culture led to a modest increase of IL-1β mRNA levels, indicating that late EPCs upregulate IL-1β expression partly through a paracrine pathway. IL-1β, an important inflammation mediator, has been shown to promote EPC function. Our data therefore suggest that late EPCs can exert self-enhancement effects by interacting with monocytes and that EPCs might modulate inflammatory reactions by regulating IL-1β expression in monocytes.

Endothelial cell (EC) dysfunction has been implicated in a variety of pathological conditions. The collection of ECs from patients is typically conducted postmortem or through invasive procedures, such as surgery and interventional procedures, hampering efforts to clarify the role of ECs in disease onset and progression. In contrast, endothelial colony-forming cells (ECFCs), also termed late endothelial progenitor cells, late outgrowth endothelial cells, blood outgrowth endothelial cells, or endothelial outgrowth cells, are obtained in a minimally invasive manner, namely, by the culture of human peripheral blood mononuclear cells in endothelial growth medium. ECFCs resemble mature ECs phenotypically, genetically, and functionally, making them excellent surrogates for ECs. Numerous studies have been performed that examined ECFC function in conditions such as coronary artery disease, diabetes mellitus, hereditary hemorrhagic telangiectasia, congenital bicuspid aortic valve disease, pulmonary arterial hypertension, venous thromboembolic disease, and von Willebrand disease. Here, we provide an updated review of studies using ECFCs that were performed to better understand the pathophysiology of disease. We also discuss the potential of ECFCs as disease biomarkers and the standardized methods to culture, quantify, and evaluate ECFCs and suggest the future direction of research in this field.

Background: Thrombosis is a common cause of vascular prosthesis failure. Antibody coating of prostheses to capture circulating endothelial progenitor cells to aid endothelialization on the device surface appears a promising solution to prevent thrombus formation. Compared with random antibody immobilization, oriented antibody coating (OAC) increases antibody-antigen binding capacity and reduces antibody immunogenicity in vivo. Currently, few OAC methods have been documented, with none possessing clinical application potential. Results: Dopamine and the linker amino-PEG8-hydrazide-t-boc were successfully deposited on the surface of cobalt chromium (CC) discs, CC stents and expanded polytetrafluoroethylene (ePTFE) grafts under a slightly basic condition. CD34 antibodies were immobilized through the reaction between aldehydes in the Fc region created by oxidation and hydrazides in the linker after t-boc removal. CD34 antibody-coated surfaces were integral and smooth as shown by scanning electron microscopy (SEM), had significantly reduced or no substrate-specific signals as revealed by X-ray photoelectron spectroscopy, were hospitable for HUVEC growth as demonstrated by cell proliferation assay, and specifically bound CD34 + cells as shown by cell binding testing. CD34 antibody coating turned hydrophobic property of ePTFE grafts to hydrophilic. In a porcine carotid artery interposition model, a confluent monolayer of cobblestone-shaped CD31 + endothelial cells on the luminal surface of the CD34 antibody coated ePTFE graft were observed. In contrast, thrombi and fibrin fibers on the bare graft, and sporadic cells on the graft coated by chemicals without antibodies were seen. Conclusion: A universal, OAC method was developed. Our in vitro and in vivo data suggest that the method can be potentially translated into clinical application, e.g., modifying ePTFE grafts to mitigate their thrombotic propensity and possibly provide for improved long-term patency for small-diameter grafts.