The Efficacy of HGF/VEGF Gene Therapy for Limb Ischemia in Mice with Impaired Glucose Tolerance: Shift from Angiogenesis to Axonal Growth and Oxidative Potential in Skeletal Muscle

Background: Combined non-viral gene therapy (GT) of ischemia and cardiovascular disease is a promising tool for potential clinical translation. In previous studies our group has developed combined gene therapy by vascular endothelial growth factor 165 (VEGF165) + hepatocyte growth factor (HGF). Our recent works have demonstrated that a bicistronic pDNA that carries both human HGF and VEGF165 coding sequences has a potential for clinical application in peripheral artery disease (PAD). The present study aimed to test HGF/VEGF combined plasmid efficacy in ischemic skeletal muscle comorbid with predominant complications of PAD-impaired glucose tolerance and type 2 diabetes mellitus (T2DM). Methods: Male C57BL mice were housed on low-fat (LFD) or high-fat diet (HFD) for 10 weeks and metabolic parameters including FBG level, ITT, and GTT were evaluated. Hindlimb ischemia induction and plasmid administration were performed at 10 weeks with 3 weeks for post-surgical follow-up. Limb blood flow was assessed by laser Doppler scanning at 7, 14, and 21 days after ischemia induction. The necrotic area of m.tibialis anterior, macrophage infiltration, angio- and neuritogenesis were evaluated in tissue sections. The mitochondrial status of skeletal muscle (total mitochondria content, ETC proteins content) was assessed by Western blotting of muscle lysates. Results: At 10 weeks, the HFD group demonstrated impaired glucose tolerance in comparison with the LFD group. HGF/VEGF plasmid injection aggravated glucose intolerance in HFD conditions. Blood flow recovery was not changed by HGF/VEGF plasmid injection either in LFD or HFD conditions. GT in LFD, but not in HFD conditions, enlarged the necrotic area and CD68+ cells infiltration. However, HGF/VEGF plasmid enhanced neuritogenesis and enlarged NF200+ area on muscle sections. In HFD conditions, HGF/VEGF plasmid injection significantly increased mitochondria content and ETC proteins content. Conclusions: The current study demonstrated a significant role of dietary conditions in pre-clinical testing of non-viral GT drugs. HGF/VEGF combined plasmid demonstrated a novel aspect of potential participation in ischemic skeletal muscle regeneration, through regulation of innervation and bioenergetics of muscle. The obtained results made HGF/VEGF combined plasmid a very promising tool for PAD therapy in impaired glucose tolerance conditions

Angiogenic and pleiotropic effects of VEGF165 and HGF combined gene therapy in a rat model of myocardial infarction

<div><p>Since development of plasmid gene therapy for therapeutic angiogenesis by J. Isner this approach was an attractive option for ischemic diseases affecting large cohorts of patients. However, first placebo-controlled clinical trials showed its limited efficacy questioning further advance to practice. Thus, combined methods using delivery of several angiogenic factors got into spotlight as a way to improve outcomes. This study provides experimental proof of concept for a combined approach using simultaneous delivery of VEGF165 and HGF genes to alleviate consequences of myocardial infarction (MI). However, recent studies suggested that angiogenic growth factors have pleiotropic effects that may contribute to outcome so we expanded focus of our work to investigate potential mechanisms underlying action of VEGF165, HGF and their combination in MI. Briefly, Wistar rats underwent coronary artery ligation followed by injection of plasmid bearing VEGF165 or HGF or mixture of these. Histological assessment showed decreased size of post-MI fibrosis in both—VEGF165- or HGF-treated animals yet most prominent reduction of collagen deposition was observed in VEGF165+HGF group. Combined delivery group rats were the only to show significant increase of left ventricle (LV) wall thickness. We also found dilatation index improved in HGF or VEGF165+HGF treated animals. These effects were partially supported by our findings of c-kit+ cardiac stem cell number increase in all treated animals compared to negative control. Sporadic Ki-67+ mature cardiomyocytes were found in peri-infarct area throughout study groups with comparable effects of VEGF165, HGF and their combination. Assessment of vascular density in peri-infarct area showed efficacy of both–VEGF165 and HGF while combination of growth factors showed maximum increase of CD31+ capillary density. To our surprise arteriogenic response was limited in HGF-treated animals while VEGF165 showed potent positive influence on a-SMA+ blood vessel density. The latter hinted to evaluate infiltration of monocytes as they are known to modulate arteriogenic response in myocardium. We found that monocyte infiltration was driven by VEGF165 and reduced by HGF resulting in alleviation of VEGF-stimulated monocyte taxis after combined delivery of these 2 factors. Changes of monocyte infiltration were concordant with a-SMA+ arteriole density so we tested influence of VEGF165 or HGF on endothelial cells (EC) that mediate angiogenesis and inflammatory response. In a series of <i>in vitro</i> experiments we found that VEGF165 and HGF regulate production of inflammatory chemokines by human EC. In particular MCP-1 levels changed after treatment by recombinant VEGF, HGF or their combination and were concordant with NF-κB activation and monocyte infiltration in corresponding groups <i>in vivo</i>. We also found that both–VEGF165 and HGF upregulated IL-8 production by EC while their combination showed additive type of response reaching peak values. These changes were HIF-2 dependent and siRNA-mediated knockdown of HIF-2α abolished effects of VEGF165 and HGF on IL-8 production. To conclude, our study supports combined gene therapy by VEGF165 and HGF to treat MI and highlights neglected role of pleiotropic effects of angiogenic growth factors that may define efficacy via regulation of inflammatory response and endothelial function.</p></div

Quantitative analysis of c-kit+ CSC and Ki-67+ cardiomyocyte density.

<p>(A) Representative images of sections co-stained for c-kit/troponin-I (upper panel) and Ki-67 (lower panel); (B) Statistical analysis of c-kit+ CSC and Ki-67+ cardiomyocyte counts. n = 4-5/group, data presented as Mean±S.D.; *p<0.05 vs “pC4W” negative control (Mann-Whitney’s U-test). No significant differences were found in comparison of “VEGF+HGF” vs. “VEGF” or “HGF” groups.</p

Effects of human VEGF165 and HGF on IL-8 and MCP-1 production by endothelial cells <i>in vitro</i>.

<p>A—MCP-1 production by HUVEC after stimulation with VEGF165, HGF or both factors over 4–12 hrs. Student’s t-test (Mean±S.D.; n = 4): * increased MCP-1 compared vs. HSA (p<0.05); # decreased MCP-1 compared vs. HSA or VEGF (p<0.01); ## decreased MCP-1 compared vs. VEGF (p<0.03). B—Autoradiograms of membrane stained for pIκBα и IκBα after Western blotting of extracts from HUVEC stimulated by VEGF165 (25 ng/ml), HGF (25 ng/ml) or VEGF165+HGF (25 ng/ml total); HSA (25 ng/ml) served as a negative, and TNF-α (5 ng/ml) as a positive control. C—IL-8 production by HUVEC after stimulation with VEGF165, HGF or both factors over 4–12 hrs. Student’s t-test (Mean±S.D.; n = 3–4): * increased IL-8 compared vs. HSA (p<0.05); ** increased IL-8 compared vs. VEGF or HGF (p<0.001); *** increased IL-8 compared vs. VEGF or HGF (p<0.01). D—Activation of HIF and IL-8 promoter in EA.hy926 after treatment with VEGF, HGF and their combination. Data of luciferase-based reporter assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197566#sec002" target="_blank">Materials and methods</a> for detailed description) Mann-Whitney U-test (Mean±S.D.; n = 3–4): *p<0.05 vs HSA; **p<0.025 vs. VEGF or HGF.</p

Morphometry of LV sections obtained from study groups.

<p>A—Representative images of stained sections of myocardium obtained at day 14 of experiment (fibrotic depositions stain blue); B—Results of morphometry in experimental groups treated by VEGF165, HGF or combined gene delivery; Student’s t-test (n = 4-5/group, presented as Mean±S.E.M.). LV fibrosis size graph: *p<0.05 vs “pC4W” negative control; **p<0.05 vs. “pC4W” negative control and vs. “VEGF” or “HGF” group. LV thickness graph: N.S.–not significant vs. “pC4W”; # p<0.05 vs. “pC4W”. Dilatation index graph: N.S.–not significant vs. “pC4W”; $ p<0.05 vs. “pC4W”. C—Representative images of myocardium cross-section stained for laminin (left panel) and results of cardiomyocyte length analysis in respective study groups (right graph). No significant intergroup variability was found (p>0.05).</p

Influence of VEGF165 and HGF gene delivery on monocyte infiltration of peri-infarct area.

<p>A—Photomicrographs of immunohistochemical visualization of monocytes in myocardium samples obtained at day 3 after MI and injection of pDNA; B—Results of evaluation showing changes of CD68+ cells density per mm² of tissue at days 3 and 7 of experiment (data presented as Mean±S.E.M.). Student’s t-test (n = 4/group): *significant increase of CD68+ cells density (p<0.05 vs. “pC4W” negative control); **significant decrease of CD68+ cells density (p<0.05 vs. “pC4W” at corresponding time point).</p

Detection of human VEGF165 and HGF in rat myocardium explants.

<p>Primary tissue was harvested at days 3 or 7 after DNA injection (A) or homogenates of left ventricle harvested at day 3 after DNA injection (B); n = 2 animals per column, ELISA data presented as Mean±S.D.</p

Influence of HIF-1/2α «knockdown» on IL-8 production by endothelial cells (in response to VEGF, HGF or their combination.

<p>EA.hy926 cells were transfected with respective siRNA and after 24 hrs were incubated with human serum albumin (HSA), VEGF165, HGF or their combination for 6 hrs. Student’s t-test (mean±S.D, n = 4): p-values are given at the graph.</p