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

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.

Protease-activated receptor (PAR)2, but not PAR1, is involved in collateral formation and anti-inflammatory monocyte polarization in a mouse hind limb ischemia model.

AIMS: In collateral development (i.e. arteriogenesis), mononuclear cells are important and exist as a heterogeneous population consisting of pro-inflammatory and anti-inflammatory/repair-associated cells. Protease-activated receptor (PAR)1 and PAR2 are G-protein-coupled receptors that are both expressed by mononuclear cells and are involved in pro-inflammatory reactions, while PAR2 also plays a role in repair-associated responses. Here, we investigated the physiological role of PAR1 and PAR2 in arteriogenesis in a murine hind limb ischemia model. METHODS AND RESULTS: PAR1-deficient (PAR1-/-), PAR2-deficient (PAR2-/-) and wild-type (WT) mice underwent femoral artery ligation. Laser Doppler measurements revealed reduced post-ischemic blood flow recovery in PAR2-/- hind limbs when compared to WT, while PAR1-/- mice were not affected. Upon ischemia, reduced numbers of smooth muscle actin (SMA)-positive collaterals and CD31-positive capillaries were found in PAR2-/- mice when compared to WT mice, whereas these parameters in PAR1-/- mice did not differ from WT mice. The pool of circulating repair-associated (Ly6C-low) monocytes and the number of repair-associated (CD206-positive) macrophages surrounding collaterals in the hind limbs were increased in WT and PAR1-/- mice, but unaffected in PAR2-/- mice. The number of repair-associated macrophages in PAR2-/- hind limbs correlated with CD11b- and CD115-expression on the circulating monocytes in these animals, suggesting that monocyte extravasation and M-CSF-dependent differentiation into repair-associated cells are hampered. CONCLUSION: PAR2, but not PAR1, is involved in arteriogenesis and promotes the repair-associated response in ischemic tissues. Therefore, PAR2 potentially forms a new pro-arteriogenic target in coronary artery disease (CAD) patients.

Molecular imaging of bone marrow mononuclear cell survival and homing in murine peripheral artery disease.

OBJECTIVES: This study aims to provide insight into cellular kinetics using molecular imaging after different transplantation methods of bone marrow-derived mononuclear cells (MNCs) in a mouse model of peripheral artery disease (PAD). BACKGROUND: MNC therapy is a promising treatment for PAD. Although clinical translation has already been established, there is a lack of knowledge about cell behavior after transplantation and about the mechanism whereby MNC therapy might ameliorate complaints of PAD. METHODS: MNCs were isolated from F6 transgenic mice (FVB background) that express firefly luciferase (Fluc) and green fluorescence protein (GFP). Male FVB and C57Bl6 mice (n = 50) underwent femoral artery ligation and were randomized into 4 groups receiving the following: 1) single intramuscular (IM) injection of 2 × 10(6) MNCs; 2) 4 weekly IM injections of 5 × 10(5) MNCs; 3) 2 × 10(6) MNCs intravenously; and 4) phosphate-buffered saline as control. Cells were characterized by flow cytometry and in vitro bioluminescence imaging (BLI). Cell survival, proliferation, and migration were monitored by in vivo BLI, which was validated by ex vivo BLI, post-mortem immunohistochemistry, and flow cytometry. Paw perfusion and neovascularization was measured with laser Doppler perfusion imaging (LDPI) and histology, respectively. RESULTS: In vivo BLI revealed near-complete donor cell death 4 weeks after IM transplantation. After intravenous transplantation, BLI revealed that cells migrated to the injured area in the limb, as well as to the liver, spleen, and bone marrow. Ex vivo BLI showed presence of MNCs in the scar tissue and adductor muscle. However, no significant effects on neovascularization were observed, as monitored by LDPI and histology. CONCLUSIONS: This is one of the first studies to assess kinetics of transplanted MNCs in PAD using in vivo molecular imaging. MNC survival is short-lived, MNCs do not preferentially home to injured areas, and MNCs do not significantly stimulate perfusion in this particular model.