Pavel I. Makarevich

Transplantation of modified human adipose derived stromal cells expressing VEGF165 results in more efficient angiogenic response in ischemic skeletal muscle.

BackgroundModified cell-based angiogenic therapy has become a promising novel strategy for ischemic heart and limb diseases. Most studies focused on myoblast, endothelial cell progenitors or bone marrow mesenchymal stromal cells transplantation. Yet adipose-derived stromal cells (in contrast to bone marrow) are abundantly available and can be easily harvested during surgery or liposuction. Due to high paracrine activity and availability ADSCs appear to be a preferable cell type for cardiovascular therapy. Still neither genetic modification of human ADSC nor in vivo therapeutic potential of modified ADSC have been thoroughly studied. Presented work is sought to evaluate angiogenic efficacy of modified ADSCs transplantation to ischemic tissue.Materials and methodsHuman ADSCs were transduced using recombinant adeno-associated virus (rAAV) serotype 2 encoding human VEGF165. The influence of genetic modification on functional properties of ADSCs and their angiogenic potential in animal models were studied.ResultsWe obtained AAV-modified ADSC with substantially increased secretion of VEGF (VEGF-ADSCs). Transduced ADSCs retained their adipogenic and osteogenic differentiation capacities and adhesion properties. The level of angiopoetin-1 mRNA was significantly increased in VEGF-ADSC compared to unmodified cells yet expression of FGF-2, HGF and urokinase did not change. Using matrigel implant model in mice it was shown that VEGF-ADSC substantially stimulated implant vascularization with paralleling increase of capillaries and arterioles. In murine hind limb ischemia test we found significant reperfusion and revascularization after intramuscular transplantation of VEGF-ADSC compared to controls with no evidence of angioma formation.ConclusionsTransplantation of AAV-VEGF- gene modified hADSC resulted in stronger therapeutic effects in the ischemic skeletal muscle and may be a promising clinical treatment for therapeutic angiogenesis.

Combined Transfer of Human VEGF165 and HGF Genes Renders Potent Angiogenic Effect in Ischemic Skeletal Muscle

Increased interest in development of combined gene therapy emerges from results of recent clinical trials that indicate good safety yet unexpected low efficacy of “single-gene” administration. Multiple studies showed that vascular endothelial growth factor 165 aminoacid form (VEGF165) and hepatocyte growth factor (HGF) can be used for induction of angiogenesis in ischemic myocardium and skeletal muscle. Gene transfer system composed of a novel cytomegalovirus-based (CMV) plasmid vector and codon-optimized human VEGF165 and HGF genes combined with intramuscular low-voltage electroporation was developed and tested in vitro and in vivo. Studies in HEK293T cell culture, murine skeletal muscle explants and ELISA of tissue homogenates showed efficacy of constructed plasmids. Functional activity of angiogenic proteins secreted by HEK293T after transfection by induction of tube formation in human umbilical vein endothelial cell (HUVEC) culture. HUVEC cells were used for in vitro experiments to assay the putative signaling pathways to be responsible for combined administration effect one of which could be the ERK1/2 pathway. In vivo tests of VEGF165 and HGF genes co-transfer were conceived in mouse model of hind limb ischemia. Intramuscular administration of plasmid encoding either VEGF165 or HGF gene resulted in increased perfusion compared to empty vector administration. Mice injected with a mixture of two plasmids (VEGF165+HGF) showed significant increase in perfusion compared to single plasmid injection. These findings were supported by increased CD31+ capillary and SMA+ vessel density in animals that received combined VEGF165 and HGF gene therapy compared to single gene therapy. Results of the study suggest that co-transfer of VEGF and HGF genes renders a robust angiogenic effect in ischemic skeletal muscle and may present interest as a potential therapeutic combination for treatment of ischemic disorders.

Regulation of Adipose Tissue Stem Cells Angiogenic Potential by Tumor Necrosis Factor-Alpha

Tissue regeneration requires coordinated “teamwork” of growth factors, proteases, progenitor and immune cells producing inflammatory cytokines. Mesenchymal stem cells (MSC) might play a pivotal role by substituting cells or by secretion of growth factors or cytokines, and attraction of progenitor and inflammatory cells, which participate in initial stages of tissue repair. Due to obvious impact of inflammation on regeneration it seems promising to explore whether inflammatory factors could influence proangiogenic abilities of MSC. In this study we investigated effects of TNF‐α on activity of adipose‐derived stem cells (ADSC). We found that treatment with TNF‐α enhances ADSC proliferation, F‐actin microfilament assembly, increases cell motility and migration through extracellular matrix. Exposure of ADSC to TNF‐α led to increased mRNA expression of proangiogenic factors (FGF‐2, VEGF, IL‐8, and MCP‐1), inflammatory cytokines (IL‐1β, IL‐6), proteases (MMPs, uPA) and adhesion molecule ICAM‐1. At the protein level, VEGF, IL‐8, MCP‐1, and ICAM‐1 production was also up‐regulated. Pre‐incubation of ADSC with TNF‐α‐enhanced adhesion of monocytes to ADSC but suppressed adherence of ADSC to endothelial cells (HUVEC). Stimulation with TNF‐α triggers ROS generation and activates a number of key intracellular signaling mediators known to positively regulate angiogenesis (Akt, small GTPase Rac1, ERK1/2, and p38 MAP‐kinases). Pre‐treatment with TNF‐α‐enhanced ADSC ability to promote growth of microvessels in a fibrin gel assay and accelerate blood flow recovery, which was accompanied by increased arteriole density and reduction of necrosis in mouse hind limb ischemia model. These findings indicate that TNF‐α plays a role in activation of ADSC angiogenic and regenerative potential. J. Cell. Biochem. 117: 180–196, 2016.

Non-viral transfer of BDNF and uPA stimulates peripheral nerve regeneration

Peripheral nerves connect brain and spinal cord with the extremities and inner organs, and nerves injury can lead the disability and social exclusion. Growth factors and other natural stimulators of regeneration processes look very promising as future medicines. In our study, we tested the influence of genetic constructions that contain genes of brain-derived neurotrophic factor and urokinase plasminogen activator on nerves structure and function after traumatic and ischemic injuries. Injection of pVax1-hBDNF and pVax1-muPA after traumatic injury led to better restoration of nerves structure and function compared to similar parameters of control group mice. In ischemic injury model pVax1-hBDNF and pVax1-muPA slowed and reduced the damage progression and stimulated nerve regeneration as well. However, the treatment with pVax1-muPA was less effective after the traumatic injury. As we chose a non-viral method of gene delivery during our study the optimal conditions of plasmid intramuscular delivery were also determined.

The role of urokinase in vascular cell migration and in regulation of growth and branching of capillaries

The urokinase system, represented by a plasminogen activator of urokinase type (urokinase, uPA), urokinase receptor (uPAR), and inhibitors of plasminogen activator (PAI-1 and PAI-2), plays an important role in the regulation of vascular wall functioning. Urokinase signaling initiates proteolytic cascade and degradation of the extracellular matrix; and also activates intracellular signaling in vascular cells. This study is the first to reveal a urokinase-mediated fundamental mechanism that regulates the growth trajectory and branching morphogenesis of blood vessels. This mechanism may be of particular importance during vessel growth in early embryogenesis and in the adult during tissue regeneration.

Comparison of cardiac stem cell sheets detached by Versene solution and from thermoresponsive dishes reveals similar properties of constructs

Cell sheets (CS) from c-kit+ cardiac stem cell (CSC) hold a potential for application in regenerative medicine. However, manufacture of CS may require thermoresponsive dishes, which increases cost and puts one in dependence on specific materials. Alternative approaches were established recently and we conducted a short study to compare approaches for detachment of CS from c-kit+ CSC. Our in-house developed method using chelation by Versene solution was compared to UpCell™ thermoresponsive plates in terms of CSC proliferation, viability, gap junction formation and engraftment in a model of myocardial infarction. Use of Versene solution instead of thermoresponsive dishes resulted in comparable CS thickness (approximately 100mcm), cell proliferation rate and no signs of apoptosis detected in both types of constructs. However, we observed a minor reduction of gap junction count in Versene-treated CS. At day 30 after delivery to infarcted myocardium both types of CS retained at the site of transplantation and contained comparable amounts of proliferating cells indicating engraftment. Thus, we may conclude that detachment of CS from c-kit+ CSC using Versene solution followed by mechanical treatment is an alternative to thermoresponsive plates allowing use of routinely available materials to generate constructs for cardiac repair.

Isolation and characterization of cardiac progenitor cells from myocardial right atrial appendage tissue

Resident cardiac stem cells, known as “cardiogenic progenitor cells” (CPCs), are a heterogeneous population of immature cells residing in the myocardium and capable of self-renewal and differentiation into cardiomyocyte-like and vascular-like cells. CPCs are usually isolated by long enzymatic digestion of heart tissue and selection with stem cell markers. However, long exposure to enzymatic digestion and the small size of a myocardial sample significantly hinder acquiring a large number of viable cells. To avoid these problems, we developed a method based on CPC growth ex vivo and subsequent immunomagnetic selection.

Recombinant human alpha-fetoprotein as a regulator of adipose tissue stromal cell activity

Recombinant human alpha-fetoprotein (rhAFP), expressed in the yeast system as a glycoprotein, has been isolated and purified to 98% by the multistep method. The testing of rhAFP in the culture of adipose tissue stromal cells (hASC) has revealed its ability to enhance hASC proliferation and migration, as well as the vascular endothelial growth factor production, with no reliable influence on cell invasion and secretion of matrix metalloproteinases-2 and -9. It has also been established that rhAFP is internalized in hASC via a clathrin-dependent mechanism. The study in the murine experimental model of hindlimb ischemia has shown the ability of rhAFP to enhance blood flow recovery. These data suggest that rhAFP is a promising agent for enhancement of the hASC regenerative ability.

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

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 in vitro 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 in vivo. 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.

Menstrual-Blood Serum Displays an Antifibrotic Effect on Human Endometrial Mesenchymal Stromal Cells

The endometrium of reproductive-age women is capable of epimorphic regeneration, which is unique to human beings. Its key feature is a wound-healing stage, which ends not in scar formation, but in complete restoration of the normal structure of uterine mucosa. The participation of endometrial mesenchymal stromal cells (eMSC) in wound healing may be due to specific environment formed during menstruation. Menstrual blood is secreted from the wound throughout the entire healing process. We decided to use serum to model the effects of the environment-soluble part on eMSC. Peripheral-blood serum (PBs) served as a control. We found that menstrual-blood serum (MBs) affected eMSC proliferation and morphology and reduced expression of α-smooth-muscle actin. Unlike fetal bovine serum (FBs), both MBs and PBs induced expression of VE-cadherin in eMSC. Expression of E-cadherin was independent of the sera used. These findings suggest antifibrotic effect of menstrual blood on eMSC.