Roberta M. Lassance-Soares

Metallothionein Enhances Angiogenesis and Arteriogenesis by Modulating Smooth Muscle Cell and Macrophage Function

Objective—In a previous study we identified metallothionein (MT) as a candidate gene potentially influencing collaterogenesis. In this investigation, we determined the effect of MT on collaterogenesis and examined the mechanisms contributing to the effects we found. Methods and Results—Collateral blood flow recovery was assessed using laser Doppler perfusion imaging, and angiogenesis was measured using a Matrigel plug assay. Smooth muscle cells were isolated from MT knockout (KO) mice for functional assays. Gene expression of matrix metalloproteinase-9, platelet-derived growth factor, vascular endothelial growth factor, and Fat cadherin in smooth muscle cells was measured by real-time polymerase chain reaction, and protein levels of vascular endothelial growth factor and matrix metalloproteinase-9 were determined using enzyme-linked immunosorbent assay and Western blot. CD11b+ macrophages were tested for invasiveness using a real-time impedance assay. Both flow recovery and angiogenesis were impaired in MT KO mice. Proliferation, migration, and invasion were decreased in MT KO smooth muscle cells, and matrix metalloproteinase-9, platelet-derived growth factor, and vascular endothelial growth factor expression were also decreased, whereas FAT-1 cadherin expression was elevated. MT KO CD11b+ cells were more invasive than wild-type cells. Conclusion—MT plays an important role in collateral flow recovery and angiogenesis, an activity that appears to be mediated, in part, by the effects of MT on the functionality of 3 cell types essential for these processes: endothelial cells, smooth muscle cells, and macrophages.

Vascular Regeneration in Ischemic Hindlimb by Adeno‐Associated Virus Expressing Conditionally Silenced Vascular Endothelial Growth Factor

Background Critical limb ischemia (CLI) is the extreme manifestation of peripheral artery disease, a major unmet clinical need for which lower limb amputation is the only option for many patients. After 2 decades in development, therapeutic angiogenesis has been tested clinically via intramuscular delivery of proangiogenic proteins, genes, and stem cells. Efficacy has been modest to absent, and the largest phase 3 trial of gene therapy for CLI reported a worsening trend of plasmid fibroblast growth factor. In all clinical trials to date, gene therapy has used unregulated vectors with limited duration of expression. Only unregulated extended expression vectors such as adeno‐associated virus (AAV) and lentivirus have been tested in preclinical models. Methods and Results We present preclinical results of ischemia (hypoxia)‐regulated conditionally silenced (CS) AAV–human vascular endothelial growth factor (hVEGF) gene delivery that shows efficacy and safety in a setting where other strategies fail. In a BALB/c mouse model of CLI, we show that gene therapy with AAV‐CS‐hVEGF, but not unregulated AAV or plasmid, vectors conferred limb salvage, protection from necrosis, and vascular regeneration when delivered via intramuscular or intra‐arterial routes. All vector treatments conferred increased capillary density, but organized longitudinal arteries were selectively generated by AAV‐CS‐hVEGF. AAV‐CS‐hVEGF therapy reversibly activated angiogenic and vasculogenic genes, including Notch,SDF1, Angiopoietin, and Ephrin‐B2. Reoxygenation extinguished VEGF expression and inactivated the program with no apparent adverse side effects. Conclusions Restriction of angiogenic growth factor expression to regions of ischemia supports the safe and stable reperfusion of hindlimbs in a clinically relevant murine model of CLI.

Loss of c-Kit function impairs arteriogenesis in a mouse model of hindlimb ischemia

Background. Arteriogenesis is a process whereby collateral vessels remodel usually in response to increased blood flow and/or wall stress. Remodeling of collaterals can function as a natural bypass to alleviate ischemia during arterial occlusion. Here we used a genetic approach to investigate possible roles of tyrosine receptor c‐Kit in arteriogenesis. Methods. Mutant mice with loss of c‐Kit function (KitW/W‐v), and controls were subjected to hindlimb ischemia. Blood flow recovery was evaluated pre‐, post‐, and weekly after ischemia. Foot ischemic damage and function were assessed between days 1 to 14 post‐ischemia while collaterals remodeling were measured 28 days post‐ischemia. Both groups of mice also were subjected to wild type bone marrow cells transplantation 3 weeks before hindlimb ischemia to evaluate possible contributions of defective bone marrow c‐Kit expression on vascular recovery. Results. KitW/W‐v mice displayed impaired blood flow recovery, greater ischemic damage and foot dysfunction after ischemia compared to controls. KitW/W‐v mice also demonstrated impaired collateral remodeling consistent with flow recovery findings. Because arteriogenesis is a biological process that involves bone marrow‐derived cells, we investigated which source of c‐Kit signaling (bone marrow or vascular) plays a major role in arteriogenesis. KitW/W‐v mice transplanted with bone marrow wild type cells exhibited similar phenotype of impaired blood flow recovery, greater tissue ischemic damage and foot dysfunction as nontransplanted KitW/W‐v mice. Conclusion. This study provides evidence that c‐Kit signaling is required during arteriogenesis. Also, it strongly suggests a vascular role for c‐Kit signaling because rescue of systemic c‐Kit activity by bone marrow transplantation did not augment the functional recovery of KitW/W‐v mouse hindlimbs.

c-Kit modifies the inflammatory status of smooth muscle cells

Background c-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis. Methods High-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (KitW/W–v) and control (Kit+/+) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations. Results The microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner. Discussion Our study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation.