Pitavastatin nanoparticle-engineered endothelial progenitor cells repair injured vessels

Abstract

Endothelial progenitor cells (EPC) participate in vessel recovery and maintenance of normal endothelial function. Therefore, pitavastatin-nanoparticles (NPs)-engineered EPC may be effective in repairing injured vasculature. Pitavastatin-loaded poly(lactic-co-glycolic) acid (PLGA) NPs were obtained via ultrasonic emulsion solvent evaporation with PLGA as the carrier encapsulating pitavastatin. The effects and mechanism of pitavastatin-NPs on EPC proliferation in vitro were evaluated. Then, EPC that internalized pitavastatin-NPs were transplanted into rats after carotid artery injury. EPC homing, re-endothelialization, and neointima were evaluated by fluorescence labeling, evans Blue and hematoxylin/eosin (H&E) staining. Pitavastatin-NPs significantly improved EPC proliferation compared with control and pitavastatin group. Those effects were blocked by pretreatment with the pharmacological phosphoinositide 3-kinase (PI3K) blockers LY294002. After carotid artery injury, more transplanted EPC were detected in target zone in Pitavastatin-NPs group than pitavastatin and control group. Re-endothelialization was promoted and intimal hyperplasia was inhibited as well. Thus, pitavastatin-NPs promote EPC proliferation via PI3K signaling and accelerate recovery of injured carotid artery.

Figure 1

Characterization of pitavastatin-NPs. (A) Micrographs of pitavastatin-NPs obtained by TEM. (B) Micrographs of pitavastatin-NPs obtained by SEM. (C) Average particle size distribution profile of pitavastatin-NPs. (D) Zeta potential of pitavastatin-NPs. (E) In vitro cumulative drug release profile of pitavastatin-NPs in PBS and plasma (pH 7.4) incubated at 37 °C (n = 3).

Figure 2

Cellular uptake of PLGA nanoparticle:Distribution of FITC-PLGA NPs in the cytoplasm of EPC after (A) 2 h and (B) 4 h of incubation;The cellular uptake kinetics of Pitavastatin in EPC (C).

Figure 3

EPC viability and proliferation were analyzed by CCK8. (A) Effect of blank PLGA NPs on EPC viability (n = 9). (B) Effect of pitavastatin-NPs on EPC proliferation: *P < 0.05 vs. control, **P < 0.01 vs. control; ^&P < 0.05 vs. 0.01 μM pitavastatin, ^&&P < 0.01 vs. 0.1 μM pitavastatin; NS = not significant (n = 9).

Figure 4

Role of PI3K/Akt signaling in pitavastatin-NP-induced EPC proliferation. (A) Effect of PI3K inhibitor treatment on pitavastatin-NP-induced EPC proliferation. **P < 0.01 vs. control-NP; ^##P < 0.01 vs. pitavastatin-NP (n = 9). (B) Effect of PI3K inhibitor treatment on pitavastatin-NP-induced Akt phosphorylation in EPC. *P < 0.01 vs. control, **P < 0.01 vs. control; ^#P < 0.05 vs. pitavastatin; ^&&P < 0.01 vs. pitavastatin-NP (n = 4).

Figure 5

Effect of pitavastatin-NPs on the homing of EPC in damaged blood vessels. (A) EPC group, (B) 0.01 μM pitavastatin-EPC; (C) 0.01 μM pitavastatin-NP-EPC, (D) 0.1 μM pitavastatin-EPC, and (E) 0.1 μM pitavastatin-NP-EPC (n = 3). (F) Histogram of the number of EPC exhibiting homing in each group. ^#P < 0.05 vs. EPC group, ^##P < 0.01 vs. EPC group; ^&P < 0.05 vs. 0.1 μM pitavastatin-EPC group; NS = not significant.

Figure 6

Effect of pitavastatin-NPs on re-endothelialization of damaged blood vessels. (A) Control, (B) EPC, (C) 0.01 μM pitavastatin-EPC, (D) 0.01 μM pitavastatin-NP-EPC, (E) 0.1 μM pitavastatin-EPC, and (F) 0.1 μM pitavastatin-NP-EPC (n = 3). (G) Histogram of re-endothelialization rate. *P < 0.05 vs. control; ^##P < 0.01 vs. EPC group; ^&P < 0.05 vs. pitavastatin-EPC group; NS = not significant.

Figure 7

Effect of pitavastatin-NPs on intimal hyperplasia in injured blood vessels. (A) Control, (B) EPC, (C) 0.01 μM pitavastatin-EPC, (D) 0.01 μM pitavastatin-NP-EPC, (E) 0.1 μM pitavastatin-EPC, and (F) 0.1 μM pitavastatin-NP-EPC (n = 6). (G) I/M ratio. **P < 0.01 vs. control; ^#P < 0.05 vs. EPC group; ^##P < 0.01 vs. EPC group; ^&P < 0.05 vs. pitavastatin-EPC group; NS = not significant.