Circulating endothelial progenitor cell and platelet microparticle impact on platelet activation in hypertension associated with hypercholesterolemia

Abstract

Aim: The purpose of this project was to evaluate the influence of circulating endothelial progenitor cells (EPCs) and platelet microparticles (PMPs) on blood platelet function in experimental hypertension associated with hypercholesterolemia.

Methods: Golden Syrian hamsters were divided in six groups: (i) control, C; (ii) hypertensive-hypercholesterolemic, HH; (iii) 'prevention', HHin-EPCs, HH animals fed a HH diet and treated with EPCs; (iv) 'regression', HHfin-EPCs, HH treated with EPCs after HH feeding; (v) HH treated with PMPs, HH-PMPs, and (vi) HH treated with EPCs and PMPs, HH-EPCs-PMPs.

Results: Compared to HH group, the platelets from HHin-EPCs and HHfin-EPCs groups showed a reduction of: (i) activation, reflected by decreased integrin 3β, FAK, PI3K, src protein expression; (ii) secreted molecules as: SDF-1, MCP-1, RANTES, VEGF, PF4, PDGF and (iii) expression of pro-inflammatory molecules as: SDF-1, MCP-1, RANTES, IL-6, IL-1β; TFPI secretion was increased. Compared to HH group, platelets of HH-PMPs group showed increased activation, molecules release and proteins expression. Compared to HH-PMPs group the combination EPCs with PMPs treatment induced a decrease of all investigated platelet molecules, however not comparable with that recorded when EPC individual treatment was applied.

Conclusion: EPCs have the ability to reduce platelet activation and to modulate their pro-inflammatory and anti-thrombogenic properties in hypertension associated with hypercholesterolemia. Although, PMPs have several beneficial effects in combination with EPCs, these did not improve the EPC effects. These findings reveal a new biological role of circulating EPCs in platelet function regulation, and may contribute to understand their cross talk, and the mechanisms of atherosclerosis.

Figure 1. The flow cytometric detection on platelet activated Integrin β- 3

(1): control group, C (2): hypertensive- hypercholesterolemic (HH) group; (3): prevention group, HHin-EPCs (4) regression group, HHfin-EPCs (5) HH treated with PMPs group, HH-PMPs and (6) HH treated with EPCs and PMPs, HH-EPCs-PMPs. The left panel (A): representative unmarked sample; the right panel (B): representative sample marked with Integrin β3 antibody. The marked events for Integrin β3 are illustrated in gates R7.

Figure 2. Representative immunoblots and densitometric data for the platelets from hamster groups: control (C), hypertensive-hypercholesterolemic (HH), prevention (HHin-EPCs), regression (HHfin-EPCs), HH treated with PMPs (HH-PMPs) and HH treated with EPCs and PMPs (HH-EPCs-PMPs).

(A): pFAK, FAK, (B): p85 subunit of PI3K, β- actin, (C): p-src, src. (*) Groups vs Control: p≤0.05. (**) Groups vs HH: p≤0.05.

Figure 3. Representative immunoblots and quantification by densitometric analysis for pro-inflammatory molecules released by platelets isolated from the hamsters groups: control (C), hypertensive-hypercholesterolemic (HH), prevention (HHin-EPCs), regression (HHfin-EPCs), HH treated with PMPs (HH-PMPs) and HH treated with EPCs and PMPs (HH-EPCs-PMPs).

(A): SDF-1, RANTES, MCP-1, β- actin, (B): Il-6, Il-1β, β- actin. (*) Groups vs Control: p≤0.05. (**) Groups vs HH: p≤0.05.