Abstract
ObjectiveCoronary collateral circulation plays a protective role in patients with coronary artery disease (CAD). We investigated whether thrombospondin-1(TSP-1) has an inhibitory effect on angiogenesis potential in endothelial progenitor cells(EPCs) and tested whether TSP-1 are altered in plasma of patients who had chronic total occlusion (CTO) in at least one coronary artery and with different collateral stages(according to Rentrop grading system).Methods and ResultsWe isolated early and late EPCs from human cord blood and investigated a dose-dependent effect of TSP-1 on their angiogenesis potential by Matrigel angiogenesis assay. We found that TSP-1 (5 µg/ml) inhibited early EPCs incorporation into tubules after pretreatment for 1, 6 and 12 hours, respectively (83.3±11.9 versus 50.0±10.1 per field for 1 hour,161.7±12.6 versus 124.0±14.4 for 6 hours, 118.3±12.6 versus 68.0±20.1 for 12 hours, p<0.05). TSP-1 also inhibited late EPCs tubule formation at 1 µg/ml (6653.4±422.0 µm/HPFversus 5552.8±136.0 µm/HPF, p<0.05), and the inhibition was further enhanced at 5 µg/ml (6653.4±422.0 µm/HPF versus 2118.6±915.0 µm/HPF p<0.01). To explore the mechanism involved, a small interfering RNA was used. In vitro, CD47 siRNA significantly attenuated TSP-1's inhibition of angiogenesis on late EPCs and similar results were obtained after functional blocking by anti-CD47 antibody. Then we investigated pathways downstream of CD47 and found TSP-1 regulated VEGF-induced VEGFR2 phosphorylation via CD47. Furthermore, we examined plasma TSP-1 levels in patients with CTO who developed different stages of collaterals and found a paradoxical higher level of TSP-1 in patients with good collaterals compared with bad ones (612.9±554.0 ng/ml versus 224.4±132.4 ng/ml, p<0.05).ConclusionTSP-1 inhibited angiogenesis potential of early and late EPCs in vitro. This inhibition may be regulated by TSP-1's interaction with CD47, resulting in down regulation of VEGFR2 phosphorylation. In patients with CTO, there may be a self-adjustment mechanism in bad collaterals which is shown as low level of angiogenesis inhibitor TSP-1, and thus favoring collateral formation.
Highlights
In patients with chronic total occlusion (CTO), there may be a self-adjustment mechanism in bad collaterals which is shown as low level of angiogenesis inhibitor TSP-1, and favoring collateral formation
Coronary collateral circulation plays a protective role in patients with coronary artery disease(CAD) for they provide an alternative source of blood supply to myocardium that has been jeopardized by occlusive coronary artery disease, and help to preserve myocardial function[1]
Endothelial progenitor cells (EPCs) did not form tube-like structures on Matrigel, they can integrate and incorporate into vascular structure when co-cultured with HUVECs on Matrigel[21]
Summary
Coronary collateral circulation plays a protective role in patients with coronary artery disease(CAD) for they provide an alternative source of blood supply to myocardium that has been jeopardized by occlusive coronary artery disease, and help to preserve myocardial function[1]. Collaterals are formed by microvascular blood vessel formation like capillary growth which occurs through both angiogenesis–the sprouting and proliferation of endothelial cells located in pre-existing blood vessels and vasculogenesis–de novo vessel growth or macrovascular vessels development through arteriogenesis–the transformation of small arterioles into larger conductance arteries. Endothelial progenitor cells (EPCs) residing in the bone marrow are mobilized in response to ischemia and promote vascularization by differentiating into tissue-specific vascular cells or by paracrine effect[2]. Thrombospondin-1 (TSP-1) has been shown to have a negative effect on endothelial colony forming cells (ECFC) angiogenic potential in patients with peripheral arterial disease. The role of TSP-1 in angiogenesis of EPCs in patients with CAD is still unknown
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