Pravastatin Promotes Endothelial Colony-Forming Cell Function, Angiogenic Signaling and Protein Expression In Vitro

Abstract

Endothelial dysfunction is a primary feature of several cardiovascular diseases. Endothelial colony-forming cells (ECFCs) represent a highly proliferative subtype of endothelial progenitor cells (EPCs), which are involved in neovascularization and vascular repair. Statins are known to improve the outcome of cardiovascular diseases via pleiotropic effects. We hypothesized that treatment with the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor pravastatin increases ECFCs' functional capacities and regulates the expression of proteins which modulate endothelial health in a favourable manner. Umbilical cord blood derived ECFCs were incubated with different concentrations of pravastatin with or without mevalonate, a key intermediate in cholesterol synthesis. Functional capacities such as migration, proliferation and tube formation were addressed in corresponding in vitro assays. mRNA and protein levels or phosphorylation of protein kinase B (AKT), endothelial nitric oxide synthase (eNOS), heme oxygenase-1 (HO-1), vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and endoglin (Eng) were analyzed by real time PCR or immunoblot, respectively. Proliferation, migration and tube formation of ECFCs were enhanced after pravastatin treatment, and AKT- and eNOS-phosphorylation were augmented. Further, expression levels of HO-1, VEGF-A and PlGF were increased, whereas expression levels of sFlt-1 and Eng were decreased. Pravastatin induced effects were reversible by the addition of mevalonate. Pravastatin induces beneficial effects on ECFC function, angiogenic signaling and protein expression. These effects may contribute to understand the pleiotropic function of statins as well as to provide a promising option to improve ECFCs' condition in cell therapy in order to ameliorate endothelial dysfunction.

Keywords: angiogenesis; cardiovascular disease; cell therapy; endothelial colony-forming cells; endothelial dysfunction; endothelial progenitor cells; pravastatin; preeclampsia; statins; vascular repair.

Figure 1

Pravastatin increased endothelial colony-forming cells’ (ECFCs’) migration. (A) Representative images of 4′,6-diamidino-2-phenylindole (DAPI)-stained migrated ECFCs treated with pravastatin (2, 20, 200 and 2000 µM) in a chemotaxis assay, scale bar 150 µm. (B) ECFCs showed a significantly higher directional migration in the presence of pravastatin after 4 h. (C) Mevalonate (200 µM) decreased directional migration and, in combination with pravastatin (2000 µM), significantly decreased the pravastatin induced increase in directional migration. Further representative images can be found in Supplemental Figure S1. Numbers of DAPI-stained migrated cells on the lower side of the membranes were counted in each picture, n = 15–20. (D) Pravastatin treatment (2, 20, 200 and 2000 µM) of ECFCs enhanced wound closure assessed as remigrated area after 18 h compared to control. (E) Mevalonate (200 µM) alone had no significant effect on ECFCs’ migration, but its addition to pravastatin (200 µM) reduced the pravastatin effect significantly. (F) Representative images of monolayers with scratch wounds at 18 h of incubation with culture medium only (a), 200 µM mevalonate (b), 200 µM pravastatin (c) and combination of 200 µM pravastatin and 200 µM mevalonate (d), scale bar 1000 µm. Cell-free area after 18 h was subtracted from cell-free area at start to calculate remigrated area. n = 14–16. Con, control; Prava, pravastatin; Mev, mevalonate. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control, # p < 0.05 compared to 200 µM pravastatin, ## p < 0.01 compared to 2000 µM pravastatin; (B–E) control group set as 1.

Figure 2

Pravastatin enhanced ECFCs’ angiogenesis. (A) Representative images from ECFCs treated with medium only (a), 20 µM (b), 200 µM (c) or 2000 µM (d) pravastatin for 6 h, scale bar 500 µm. (B) Pravastatin treatment (2, 20 and 200 µM) led to a higher number of branching points in ECFCs after 6 h of incubation. A total of 2000 µM pravastatin, in contrast, reduced the number of branch points. (C) Pravastatin treatment (2, 20 and 200 µM) of ECFCs significantly increased tube length after 6 h of incubation, whereas 2000 µM pravastatin significantly impaired tube formation ability. Mevalonate (200 µM) did not affect tube formation ability in ECFCs but diminished pravastatin induced effects. In comparison to pravastatin treatment, only tube length was significantly less after pravastatin (2 µM, 20 µM and 200 µM) treatment in the presence of mevalonate. Mevalonate (200 µM) also attenuated the inhibitory effect on tube formation of high-dose pravastatin (2000 µM). Additional representative images of combined treatment can be found in Supplemental Figure S2. n = 12. Con, control; Prava, pravastatin; Mev, mevalonate. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control, ## p < 0.01 compared to 2 µM Prava, °°° p < 0.001 compared to 20 µM pravastatin, + p < 0.05 compared to 200 µM pravastatin, ^xx p < 0.01 compared to 2000 µM pravastatin; control group set as 1.

Figure 3

Pravastatin had a biphasic impact on ECFC proliferation. (A) Overlay of growth curves of ECFCs treated with pravastatin (2 µM, 20 µM, 200 µM or 2000 µM). ECFC proliferation was significantly increased after treatment with 2 µM or 20 µM pravastatin, but significantly decreased after treatment with 200 µM or 2000 µM pravastatin after 24 h, 48 h and 72 h. (B) Overlay of growth curves of ECFCs treated with 20 µM or 200 µM pravastatin in the presence or absence of 200 µM mevalonate. Mevalonate reduced the proliferative effect of 20 µM pravastatin and lessened the antiproliferative effect of 200 µM pravastatin significantly after 72 h. n = 15–20; control group set as 1. (C) High dose pravastatin led to apoptosis. Representative measurement of apoptosis and necrosis in ECFCs after 48 h treatment with pravastatin at 2 µM, 20 µM, 200 µM or 2000 µM. Viable cells are located in the lower left field (Annexin V neg./PI neg). Pravastatin (2000 µM) caused a significantly higher rate of non-viable cells. n = 5. Con, control; Prava, pravastatin; Mev, mevalonate. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Pravastatin increased phosphorylation of protein kinase B (AKT) and endothelial nitric oxide synthase (eNOS). (A) Representative immunoblot of AKT phosphorylation after treatment with pravastatin (200 µM) in the presence or absence of mevalonate (200 µM). (B) Pravastatin significantly induced AKT phosphorylation, whereas mevalonate abrogates the pravastatin effect. (C) Representative immunoblot of eNOS phosphorylation after treatment with 200 µM pravastatin with or without the addition of mevalonate. (D) eNOS phosphorylation was significantly induced after incubation with pravastatin. Mevalonate reversed the pravastatin effect. The effect of pravastatin was investigated in 5 and of mevalonate at least in 2 independent experiments. Con, control; Prava, pravastatin; Mev, mevalonate. * p < 0.05; control group set as 1.

Figure 5

Pravastatin increased heme oxygenase-1 (HO-1) expression in real time PCR and immunoblot. (A) Pravastatin (20 µM) significantly induced HO-1 mRNA expression. The addition of mevalonate (200 µM) significantly reduced the pravastatin induced HO-1 mRNA expression. The increase in mRNA expression at intermediate pravastatin concentrations (200 µM) was not modulated by mevalonate at all. (B) Representative immunoblot of HO-1 protein expression after pravastatin (200 µM) treatment with or without additional mevalonate (200 µM). (C) Pravastatin significantly increased HO-1 protein expression in ECFCs, whereas mevalonate reversed the pravastatin induced effect. Real time PCR was performed in triplicates for at least 2 independent runs. Immunoblots were performed at least 4 times. Con, control; Prava, pravastatin; Mev, mevalonate. ** p < 0.01 compared to control, ## p < 0.01 compared to 20 µM pravastatin; control group set as 1.

Figure 6

Pravastatin affected mRNA expression of pro-angiogenic and anti-angiogenic molecules. Pravastatin (20 µM) significantly induced mRNA expression of pro-angiogenic molecules vascular endothelial growth factor A (VEGF-A) (A) and placental growth factor (PlGF) (B), whereas mevalonate alone had no effect. In immunoblot (C), treatment with pravastatin (200 µM) increased VEGF-A protein expression (D). Co-treatment with mevalonate (200 µM) reduced pravastatin induced VEGF-A and PlGF mRNA expression. mRNA expression of the anti-angiogenic molecule soluble fms-like tyrosine kinase-1 (sFlt-1) (E) and the angiogenesis-related protein endoglin (Eng) (F) was significantly reduced by pravastatin (200 µM). Mevalonate (200 µM) did not significantly affect sFlt-1 mRNA expression but, in combination with pravastatin, reversed the pravastatin effect on sFlt-1 mRNA expression in ECFCs. Results are from at least 3 independent runs. All runs were performed in triplicates. Con, control; Prava, pravastatin; Mev, mevalonate. * p < 0.05, ** p < 0.01; control group set as 1.