[Show abstract][Hide abstract] ABSTRACT: Recent attempts to fi nd an effective therapeutical for ischemic disease treatment led to a concept that precise screening is required to trigger drug research and put basis for novel growth factor based medicine that will influence crucial mechanisms involved in angiogenesis and disease pathogenesis. Initially we have conducted a screening study aimed to find which growth factors and cytokines are increased in patients with post-MI
heart failure (N=94). We have examined plasma level of a panel of 12 growth factors and cytokines and found, that VEGF and HGF were significantly correlated and increased in post-MI patients and HGF was independently correlated with heart failure onset. We have concluded, that this increase is, most likely, a compensatory mechanism for cardiovascular system to induce angiogenesis and could be linked to systemic hypoxia. Thus, we suggested that HGF and VEGF might be an attractive combination to be delivered to ischemic
myocardium as far as these factors take part it physiological perfusion restoration and homeostasis support in post-infarction period. As a proof-of concept we tried to evaluate VEGF+HGF gene delivery by plasmid injection into ischemic mouse skeletal muscle and rat myocardium. We found, that within 14-21 days post injection combination of VEGF+HGF was superior to single VEGF or HGF in terms of vascular density increase (both capillary and arteriole), but was unable to signifi cantly reduce fi brosis, yet certain trend was observed. In VEGF+HGF group we also found certain reduction of cellular infi ltration to peri-infarction zone, which might indicate decrement of infl ammatory response in myocardium. In further in vitro studies using HUVEC and TIME endothelial cells we found that production of pro-inflammatory chemokines MCP-1 and IL-6 is increased by VEGF, and reduced after addition of HGF. Combination of factors resulted in average levels of MCP-1 and IL-6. Thus, HGF seems to augment propro-infl ammatory action known for VEGF. As for IL-8 both factors had stimulating effect on its production and their combination yielded maximum amount of IL-8 from endothelial cell cultures, which suggests an alternative way for IL-8 regulation involved.
As a result of the study we can conclude that combination of VEGF and HGF has good translational promise. Deeper in-sights into their mechanism of action might show non-canonical properties and modes of action of that combination that may drive its way into practice.
[Show abstract][Hide abstract] ABSTRACT: Background
Modified 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 methods
Human 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.
We 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.
Transplantation 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.
Full-text · Article · Jun 2013 · Journal of Translational Medicine
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: In the past few years, it has been established that the heart contains a reservoir of stem and progenitor cells. These cells are able to differentiate in cardiomyogenic, endothelial, and smooth muscle lineages in vitro and in vivo and, following injection into an infarcted myocardium, these cells prevent the remodeling of the left ventricle and improve cardiac function. The aim of the present study was the immunophenotypic characterization of resident stem cells in the tissue of a chronic left aneurism, an analysis of their localization, and distribution in various aneurism areas.
Fifteen samples of aneurism tissue obtained surgically were analyzed. The number of cells positive for c-kit stem-cell marker was about 3327 ± 234 cells per 1 cm3 tissue. Single cells were found in fibrous, muscle, and adipose parts of aneurism tissue. Most of the cells were revealed in fibrous tissue near large vessels; however, the coexpression of c-kit and markers of endothelial cells was not observed. C-kit-positive cells did not express CD34 and CD45 markers and, thereby, were not of hematopoietic origin. The majority of c-kit-positive cells expressed MDR1, another stem-cell marker. These c-kit-positive cells did not proliferate, since they did not express Ki67. About 20% of these cells expressed an inhibitor of cyclin-dependent kinase p21cip/Waf1. C-kit cells did not express markers of cardiomyocytes (Nkx 2.5, Gata 4, Mef2c, α-actinin), endothelium (Ets 1, CD105, wW), and smooth muscle cells (Gata 6, SMA). Single-cell clusters made up of 70–100 c-kit-positive cells (in cell niches) were observed; however, no markers of cardiomyogenic differentiation were expressed in these cells. Thus, aneurism tissue of the left ventricle contains c-kit-positive cells that, are most likely resident cardiac stem cells; however, their regenerative capacity should be further explored.
No preview · Article · Dec 2010 · Cell and Tissue Biology
[Show abstract][Hide abstract] ABSTRACT: Peripheral artery disease (PAD) is one of leading causes of
disability in Western societies. Gene and cell therapy can be
used to induce angiogenesis in ischemic skeletal muscle to
prevent major necrosis and amputations in affected patients.
Benefit from gene transfer and cell transplantation was
shown in many experimental and clinical studies but development
of a novel combined approach in this field is still an
important point in medical science.
Our group focused on animal testing of several plasmid
constructs with human VEGF165, HGF, angiopoietin-1 and
mouse uPA to find combinations which could amplify angiogenic
efficacy of gene transfer. We found that co-transfer
of 2 growth factors (VEGFþHGF, VEGFþAng-1 or
HGFþAng-1) renders a higher angiogenic effect than sole
plasmids, which was shown by laser Doppler imaging and
Another important point assessed in our studies was use of
AAV-transduced adipose derived stromal cells (ADSC) for
induction of angiogenesis in mouse ischemic limb and Matrigel
plug model. Recombinant AAV was used to induce
overexpression of VEGF165 in human ADSC and modified
cells showed higher angiogenic efficacy than GFP-transduced
or unmanipulated ones. Cell transplantation gave a higher
and more stable increase in limb perfusion than plasmid gene
transfer. Our findings indicate that gene and cell therapy efficacy
in PAD might be increased using combined methods:
1) gene transfer of several growth factors, 2) modified cells
transplantation or, possibly, using both which is still to be
tested in appropriate animal models.
[Show abstract][Hide abstract] ABSTRACT: Stromal cells from subcutaneous adipose tissue (adipose derived stromal cells - ASCs) are perspective for cell therapy of ischemic states because of ability to stimulate growth of vessels. For the elucidation of mechanisms of angiogenic action of ASCs we used the model of co-cultivation of ASCs with cells isolated from postnatal hearts (fraction of cardiomyocutes - CMC). CMC fraction contained mature cardiomyocytes, endothelial and progenitor cells. On the 2-nd day spontaneously beating colonies of CMC with growing from them CD31-positive capillary-like structures were formed in CMC culture. Observed structures were unstable and came apart after 5 days of cultivation. At co-cultivation of CMC with ASCs formation of stable ramified CD31-positive structures was observed. Using the method of co-cultivation of CMC with mitomycin C treated ASCs and the method of immune magnetic depletion for removal of epithelial cells from the CMC fraction we found that ASCs stimulates formation of capillary like structure at the account of secretion of angiogenic factors, stabilization of forming CD31-positive structures at the account of intercellular contacts and stimulation of endothelial differentiation of progenitor cells present in CMC fraction.
[Show abstract][Hide abstract] ABSTRACT: In the past few years it has been established that the heart contains a reservoir of stem and progenitor cells that have the ability to differentiate in vitro and in vivo toward vascular and cardiac lineages and that show cardiac regeneration potential in vivo following injection into the infracted myocardium. The aim of the present study was to characterize cardiac stem cells in the tissue of chronic left ventricular aneurism. It was shown that human c-kit positive cells were scattered in fibrous, muscle and adipose parts of aneurism tissue. C-kit positive cells localized mainly in fibrous tissue nearby large vessels, however, c-kit positive cells did not express endothelial, smooth muscle or cardiomyocyte cell markers. Co-localization experiments demonstrated that all c-kit positive cells were of non-hematopoietic origin, since they did not express markers such as CD34 and CD45. Majority of c-kit positive cells expressed MDR1, but showed no proliferation activity (Ki67). It thus appears that aneurism tissue could be an alternative source of autologous cardiac stem cells. However, their regeneration capacity should be further explored.
[Show abstract][Hide abstract] ABSTRACT: Adipose-derived stromal cells (ASCs) are suggested to be potent candidates for cell therapy of ischemic conditions due to their ability to stimulate blood vessel growth. ASCs produce many angiogenic and anti-apoptotic growth factors, and their secretion is significantly enhanced by hypoxia. Utilizing a Matrigel implant model, we showed that hypoxia-treated ASCs stimulated angiogenesis as well as maturation of the newly formed blood vessels in vivo. To elucidate mechanisms of ASC angiogenic action, we used a co-culture model of ASCs with cells isolated from early postnatal hearts (cardiomyocyte fraction, CMF). CMF contained mature cardiomyocytes, endothelial cells, and progenitor cells. On the second day of culture CMF cells formed spontaneously beating colonies with CD31+ capillary-like structures outgrowing from those cell aggregates. However, these vessel-like structures were not stable, and disassembled within next 5 days. Co-culturing of CMF with ASCs resulted in the formation of stable and branched CD31+ vessel-like structures. Using immunomagnetic depletion of CMF from vascular cells as well as incubation of CMF with mitomycin C-treated ASCs, we showed that in co-culture ASCs enhance blood vessel growth not only by production of paracrine-acting factors but also by promoting the endothelial differentiation of cardiac progenitor cells. All these mechanisms of actions could be beneficial for the stimulation of angiogenesis in ischemic tissues by ASCs administration.
Full-text · Article · May 2009 · Tissue Engineering Part A
[Show abstract][Hide abstract] ABSTRACT: Urokinase plasminogen activator (uPA) is required for both endogenous and vascular endothelial growth factor (VEGF)-augmented angiogenesis in normal tissues, leading us to hypothesize that uPA augmentation by gene transfer might promote angiogenesis in ischemic tissues. Overexpression of uPA was studied in rat myocardial infarction (MI) and mouse hind limb ischemia models and compared with VEGF overexpression effects. Animals were divided into control and three experimental groups (n = 6), receiving intramuscular injections of plasmids as follows: (i) control (empty vector or expressing beta-galactosidase); (ii) uPA; (iii) VEGF(165); (iv) a 1:1 mixture of uPA and VEGF(165). The capillary densities in both ischemic models were greater (P < 0.05) in tissues treated with uPA, VEGF, or a combination of both than in controls. Infarct size was reduced in hearts from uPA and VEGF experimental groups compared with controls (P < 0.05). Local overexpression of uPA induced a marked increase in the number of macrophages and myofibroblasts present within infarcts. Hind limb blood flow was greater in all experimental groups by day 10 (P < 0.05). Overall, the effects of uPA and VEGF were uniformly comparable. Additional analysis revealed association of local edema with VEGF but not with uPA treatment. This study established that uPA gene therapy effectively induces functionally significant angiogenesis in models of acute MI and hind limb ischemia.
Full-text · Article · Nov 2007 · Molecular Therapy
[Show abstract][Hide abstract] ABSTRACT: Hypoxia induces angiogenesis in ischemized tissues by means of pro-angiogenic factor expression. The key role in the growth processes and blood vessel functioning belongs to the matrix metalloproteinases, plasminogen, and its activator systems. Effect of hypoxia on expression of the urokinase activating agent plasminogen and its receptor in endothelium was studied in human umbilical vein endothelial cell model. Incubation of the endothelial cells under the conditions of hypoxia proved to reduce both urokinase formation in these cells and its secreting into the culture medium. The hypoxia-induced reduction of urokinase contents was accompanied by enhancement of expression of the urokinase receptor. The hypoxia also entailed reduction of the adenylate cyclase activity and cAMP contents in the endothelial cells. The data obtained suggest that reduction of the adenylate cyclase activity and cAMP contents under the conditions of hypoxia provide basis for suppression of the urokinase expression by the endothelial cells and, consequently, inhibition of blood vessel formation in the ischemized tissue.
No preview · Article · Jul 2005 · Rossiĭskii fiziologicheskiĭ zhurnal imeni I.M. Sechenova / Rossiĭskaia akademiia nauk
[Show abstract][Hide abstract] ABSTRACT: The role of plasminogen activators in the regulation of key processes of atherosclerosis progression stays unclear. The aim of this study was to evaluate the expression of urokinase plasminogen activator (uPA), its receptor (uPAR) and the plasminogen activator inhibitor type 1 (PAI-1) in human aorta, and to balance them with the stage of atherosclerotic lesion. We have shown that uPA and uPAR in normal aorta are mostly expressed by intimal smooth muscle cells. The expression of these proteins was up-regulated in diseased aorta compared to normal artery. The most part of cells in both fatty streak and fibro-fatty lesion were monocytes/macrophages, and about 60% of these cells expressed uPA and its receptor. PAI-1 was mostly localized on the lumonal part of the aorta and in the extracellular matrix of the intima. We observed a moderate increase of PAI-1 expression in atherosclerotic lesion. Thus, our data indicate participation of plasminogen system in atherogenesis.