Prolylcarboxypeptidase Promotes Endothelial Cell Proliferation and Vascular Repair

The S28 serine protease, prolylcarboxypeptidase (PRCP) degrades bradykinin, angiotensin II, alpha melanocyte stimulating hormone and actives plasma prekallikrein. Additionally our studies indicate that PRCP depletions in vivo and in cultured cells are associated with increased reactive oxygen species (ROS) and loss of constitutive anticoagulant function of endothelium (Blood 2011; 117:3929). PRCP-depleted mice are prothrombotic and hypertensive. We observed that PRCP-depleted cells in culture have reduced growth. We posited that PRCP promotes vascular health by influencing cell proliferation, angiogenesis, and wound repair.

Initial investigations determined that PRCP influences vascular endothelial cell proliferation. Bovine aortic endothelial cells (BAEC) were depleted of PRCP by siRNA knockdown resulting in 5% residual mRNA. After transfecting equal numbers of BAEC, at 24 h, the PRCP siRNA transfected cells have reduced proliferation, −18±3 change in cells/high power field (HPF) (mean±SEM), compared to the sham transfected cells, +23±8 cells/HPF, p<0.05. Additionally, PRCP siRNA-treated BAEC demonstrate less proliferation as measured by the MTS assay (Promega) (0.23±0.01 OD490 nm in PRCP-depleted cells vs 0.31±0.01 OD490 nm in sham transfected cells, p<0.02). Alternatively, when BAEC are transfected with full-length PRCP cDNA, at 24 h there is increased proliferation, +58±9 cells/HPF, vs +31±2 of sham-transfected cells, p<0.05. On a BAEC scratch assay, the degree of endothelial cell migration at 5 h in PRCP siRNA-knocked down cells is only 69% of that seen with sham-transfected cells (38±4% scratch coverage in PRCP knockdown BAEC vs 55±5% in sham knockdowns). These combined studies indicate that the content of PRCP in endothelial cells directly correlates with the degree of cell migration and proliferation. Studies next determined the influence of PRCP on angiogenesis. PRCP-depleted mice (PRCPgt/gt) have reduced new vessel growth into sub-cutaneous matrigel plugs containing FGF and VEGF. Matrigel plugs from the PRCP gt/gt mice show 3.5±0.5 Hgb mg/dL/mg-matrigel vs 6.7±1.2 Hgb mg/dL/mg matrigel in plugs in littermate wild type (WT) mice (p<0.03). When sections from the matrigel plugs are stained for the vascular marker CD31, the percent area of new vessels in the PRCPgt/gt (5.5±0.9%), as determined by ImageJ analysis, is significantly less (p<0.04) than that seen (11.9±2.1%) in WT plugs. These data indicate that host PRCP levels influence induced angiogenesis in the whole animal. Additional studies examined if PRCPgt/gt have reduced wound repair angiogenesis. Punch biopsies (5 mm) were performed on PRCPgt/gt. At day 7, no wound healed in PRCPgt/gt but 5/10 wounds healed in WT. The mean size of the PRCPgt/gt wounds is 5.5±1.1 mm2 vs 1.4±0.7 mm2 for WT, p<0.05. Also, at day 7, the wounds of PRCPgt/gt have 11.6±1.0 % area of CD31 stained vessels vs 15.0±1.0 % area of CD31 stained vessels in control wounds, p<0.03. Since there is no difference in the number of vessels in unwounded skin biopsies in PRCPgt/gt vs WT, the reduced vessel growth and delayed wound closure indicates that PRCPgt/gt mice have reduced repair angiogenesis.

These combined studies indicate that PRCP levels in endothelial cells influence cell proliferation and growth. In the whole animal this cell biology observation translates into less induced and wound repair angiogenesis. Since PRCP-depleted endothelial cells and vessels from PRCPgt/gt have increased ROS with loss of anticoagulant properties and PRCPgt/gt have higher thrombosis risk, the finding that PRCP also influences endothelial cell growth and angiogenesis suggests that PRCP promotes vascular health and injury repair.

No relevant conflicts of interest to declare.