Gene therapy with Pellino-1 improves perfusion and decreases tissue loss in Flk-1 heterozygous mice but fails in MAPKAP Kinase-2 knockout murine hind limb ischemia model.

OBJECTIVES: In the United States, over 8.5 million people suffer from peripheral arterial disease (PAD). Previously we reported that Pellino-1(Peli1) gene therapy reduces ischemic damage in the myocardium and skin flaps in Flk-1 [Fetal Liver kinase receptor-1 (Flk-1)/ Vascular endothelial growth factor receptor-2/VEGFR2] heterozygous (Flk-1+/--) mice. The present study compares the angiogenic response and perfusion efficiency following hind limb ischemia (HLI) in, Flk-1+/- and, MAPKAPKINASE2 (MK2-/-) knockout (KO) mice to their control wild type (WT). We also demonstrated the use of Peli1 gene therapy to improve loss of function following HLI. STUDY DESIGN AND METHODS: Femoral artery ligation (HLI) was performed in both Flk-1+/- and MK2-/- mice along with their corresponding WT. Another set of Flk-1+/- and MK2-/- were injected with either Adeno-LacZ (Ad.LacZ) or Adeno-Peli1 (Ad.Peli1) after HLI. Hind limb perfusion was assessed by laser doppler imaging at specific time points. A standardized scoring scale is used to quantify the extent of ischemia. Histology analysis performed includes capillary density, fibrosis, pro-angiogenic and anti-apoptotic proteins. RESULTS: Flk-1+/- and MK2-/- had a slower recovery of perfusion efficiency in the ischemic limbs than controls. Both Flk-1+/- and MK2-/- KO mice showed decreased capillary density and capillary myocyte ratios with increased fibrosis than their corresponding wild types. Ad.Peli1 injected ischemic Flk-1+/- limb showed improved perfusion, increased capillary density, and pro-angiogenic molecules with reduced fibrosis compared to Ad.LacZ group. No significant improvement in perfusion was observed in MK2-/- ischemic limb after Ad. Peli1 injection. CONCLUSION: Deletion of Flk-1 and MK2 impairs neovascularization and perfusion following HLI. Treatment with Ad. Peli1 results in increased angiogenesis and improved perfusion in Flk-1+/- mice but fails to rectify perfusion in MK2 KO mice. Overall, Peli1 gene therapy is a promising candidate for the treatment of PAD.

Evaluation of dermal tissue regeneration using resveratrol loaded fibrous matrix in a preclinical mouse model of full-thickness ischemic wound.

The complete loss of dermal tissue due to ischemia is a serious challenge facing clinicians. Frequently, the failure of wound healing is due to ischemic conditions prevailing at the site of damaged tissue. Restoration of lost vasculature at the ischemic site can be achieved by supplementing proangiogenic stimuli through an engineered scaffold mimicking dermal extracellular matrix. Towards this objective, we have developed an electrospun scaffold loaded with the pro-angiogenic molecule resveratrol. The physical and chemical changes in the polymeric scaffold before and after loading of resveratrol were characterized using field emission scanning electron microscopy (FE-SEM), Fourier-transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), coherence scanning interferometry (CSI) and X-ray diffraction (XRD). A sustained release of resveratrol from the scaffold was elucidated by UV-spectrophotometer analysis. The enhancement in cell-matrix interaction was studied using human umbilical vein endothelial cells (HUVECs) seeded on the scaffolds. The biocompatibility analysis of resveratrol loaded scaffolds was evaluated through a subcutaneous implantation study in mice. The therapeutic potential of resveratrol loaded scaffolds to accelerate tissue repair was analyzed in a full-thickness ischemic wound model in mice. Wound closure and H&E staining analysis showed rapid closure of ischemic wound area and re-epithelialization in resveratrol loaded scaffold treated groups compared to collagen and negative control groups. The immunostaining analysis further revealed the activation of thioredoxin-1 (Trx-1), heme oxygenase-1 (HO-1) mediated vascular endothelial growth factor (VEGF) signaling in resveratrol loaded scaffold treated group. The expression of Bcl-2 in healing wound edges post-treatment with resveratrol loaded scaffold confirmed the anti-apoptotic effect mediated by resveratrol. From this study, we explored a synergistic effect mediated by resveratrol and fibrous scaffolds to aid the ischemic wound healing process through effective vascularization.

Overexpression of Thioredoxin1 enhances functional recovery in a mouse model of hind limb ischemia.

BACKGROUND: There is keen interest in finding nonsurgical treatments for peripheral vascular disease (PVD). Previously, we demonstrated that selective activation of Thioredoxin1 (Trx1), a 12-kDa cytosolic protein, initiates redox-dependent signaling and promotes neovascularization after ischemic heart disease. Therefore, Trx1 might possess immense potential to not only treat murine hind limb ischemia (HLI) through effective angiogenesis but also provide PVD patients with nonsurgical therapy to enhance neovascularization and improve blood perfusion. METHODS: To determine whether activation of Trx1 increases blood perfusion in HLI, two different strategies were used-gene therapy and transgenic model system. In adenoviral-mediated gene therapy, 8- to 12-wk-old mice were divided into two groups: (1) control Adeno-LacZ (Ad-LacZ) and (2) Adeno-Thiroedoxin1 (Ad-Trx1). The mice underwent surgical intervention to induce right HLI followed by injection with Ad-LacZ or Ad-Trx1, respectively. In the second strategy, we used wild-type and transgenic mice overexpressing Trx1 (Trx1Tg/+). All the animals underwent Doppler imaging for the assessment of limb perfusion followed by immunohistochemistry and Western blot analysis. RESULTS: Significant increases in perfusion ratio were observed in all the Trx1 overexpressed groups compared with their corresponding controls. Expressions of heme oxygenase-1, vascular endothelial growth factor, and the vascular endothelial growth factor receptors Flk-1 and Flt-1 were increased in Trx1 overexpressed mice compared with their respective controls. Blood perfusion in the ischemic limb gradually improved and significantly recovered in Trx1Tg/+ and Ad-Trx1 groups compared with their corresponding controls. The capillary and arteriolar density in the ischemic zone were found to be higher in Trx1Tg/+ group compared with wild type. CONCLUSIONS: The overall outcomes of our study demonstrate that Trx1 enhances blood perfusion and increases angiogenic protein expression in a rodent HLI model. These results suggest that Trx1 is a potential target for clinical trials and drug therapy for the treatment of PVD.

Trimodal rescue of hind limb ischemia with growth factors, cells, and nanocarriers: fundamentals to clinical trials.

Peripheral artery disease is a severe medical condition commonly characterized by critical or acute limb ischemia. Gradual accumulation of thrombotic plaques in peripheral arteries of the lower limb may lead to intermittent claudication or ischemia in muscle tissue. Ischemic muscle tissue with lesions may become infected, resulting in a non-healing wound. Stable progression of the non-healing wound associated with severe ischemia might lead to functional deterioration of the limb, which, depending on the severity, can result in amputation. Immediate rescue of ischemic muscles through revascularization strategies is considered the gold standard to treat critical limb ischemia. Growth factors offer multiple levels of protection in revascularization of ischemic tissue. In this review, the basic mechanism through which growth factors exert their beneficial properties to rescue the ischemic limb is extensively discussed. Moreover, clinical trials based on growth factor and stem cell therapy to treat critical limb ischemia are considered. The clinical utility of stem cell therapy for the treatment of limb ischemia is explained and recent advances in nanocarrier technology for selective growth factor and stem cell supplementation are summarized.