Short hairpin RNA gene silencing of prolyl hydroxylase-2 with a minicircle vector improves neovascularization of hindlimb ischemia

Abstract

In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×10(5)±3.3×10(5) (MC-Luc) versus 0.4×10(5)±0.3×10(5) (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31(+) expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.

FIG. 1.

In vitro BLI of irradiated C2C12 cells after transfection with MC-Luc or PL-Luc. (A) Graphical representation of BLI signals as mean maximum radiance (Max Rad) in p/s/cm²/sr in irradiated C2C12 cells after transfection (*p<0.05). (B) Representative in vitro BLI images of irradiated C2C12 cells up to 48 hr after transfection with MC-Luc or PL-Luc, respectively. BLI, bioluminescence imaging; Luc, luciferase; MC, minicircle; PL, plasmid.

FIG. 2.

In vivo BLI of the transfection efficiency of MC-Luc compared with PL-Luc in C57Bl6 mice. (A) Graphical representation of the mean maximum radiance in p/s/cm²/sr up to 28 days after transfection with MC-Luc in the left paw and PL-Luc in the right paw (*p<0.05). (B) Representative in vivo BLI images of the mice after transfection. First image shows both the MC-Luc signal and the PL-Luc signal using a lower scale.

FIG. 3.

Paw perfusion as measured by LDPI. (A) Graphical representation of the mean blood flow recovery of mice subjected to hindlimb ischemia and treated with MC-shPHD2 as compared with PL-shPHD2 or PBS control (*p<0.05). (B) Representative LDPI images of the differences in blood flow recovery after unilateral hindlimb ischemia of the left paw between the study groups. LDPI, laser Doppler perfusion imaging; PBS, phosphate-buffered saline; shPHD2, short hairpin RNA interference therapy targeting prolyl hydroxylase-2.

FIG. 4.

Immunohistochemical analyses of the adductor (SMA) and calf (CD31⁺) muscles harvested 10 days after surgery. (A) Graphical representation of the mean number of collaterals and (B) mean surface per collateral as quantified by SMA staining after MC-shPHD2 (Minicircle) injection compared with PL-shPHD2 (Plasmid) or PBS injection (*p<0.05). (C) Representative images of the SMA staining after PBS (left), PL-shPHD2 (middle), or MC-shPHD2 (right) therapy. (D) Graphical representation of the mean CD31⁺-capillary density after MC-shPHD2 injection compared with PL-shPHD2 or PBS injection (*p<0.05). (E) Representative images of the CD31⁺ staining after PBS (left), PL-shPHD2 (middle), or MC-shPHD2 (right) therapy. SMA, smooth muscle actin.

FIG. 5.

Representative example of angiographies showing collateral formation after (A) PBS injection, (B) PL-shPHD2 injection, and (C) MC-shPHD2 injection.

FIG. 6.

Graphical representation of the mean relative PHD2 mRNA levels (mean ΔCt) in the gastrocnemius muscles of mice 7 days after surgery and treated with MC-shPHD2, PL-shPHD2, or PBS, respectively (*p<0.05).

FIG. 7.

(A) Representative Western blot image of the difference in HIF-1-alpha protein levels among the study groups MC-shPHD2 (MC), PL-shPHD2 (PL), and PBS control. The HIF-1-alpha subunits are shown at 100–120 kDa, and actin served as a loading control at 43 kDa, as represented by the protein standard ladder (LAD). (B) Graphical representation of the Western blot shown in (A), relative to β-actin protein expression (*p<0.05).