Urokinase-type Plasminogen Activator (uPA) Promotes Angiogenesis by Attenuating Proline-rich Homeodomain Protein (PRH) Transcription Factor Activity and De-repressing Vascular Endothelial Growth Factor (VEGF) Receptor Expression

Urokinase-type plasminogen activator (uPA) regulates angiogenesis and vascular permeability through proteolytic degradation of extracellular matrix and intracellular signaling initiated upon its binding to uPAR/CD87 and other cell surface receptors. Here, we describe an additional mechanism by which uPA regulates angiogenesis. Ex vivo VEGF-induced vascular sprouting from Matrigel-embedded aortic rings isolated from uPA knock-out (uPA(−/−)) mice was impaired compared with vessels emanating from wild-type mice. Endothelial cells isolated from uPA(−/−) mice show less proliferation and migration in response to VEGF than their wild type counterparts or uPA(−/−) endothelial cells in which expression of wild type uPA had been restored. We reported previously that uPA is transported from cell surface receptors to nuclei through a mechanism that requires its kringle domain. Intranuclear uPA modulates gene transcription by binding to a subset of transcription factors. Here we report that wild type single-chain uPA, but not uPA variants incapable of nuclear transport, increases the expression of cell surface VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) by translocating to the nuclei of ECs. Intranuclear single-chain uPA binds directly to and interferes with the function of the transcription factor hematopoietically expressed homeodomain protein or proline-rich homeodomain protein (HHEX/PRH), which thereby lose their physiologic capacity to repress the activity of vehgr1 and vegfr2 gene promoters. These studies identify uPA-dependent de-repression of vegfr1 and vegfr2 gene transcription through binding to HHEX/PRH as a novel mechanism by which uPA mediates the pro-angiogenic effects of VEGF and identifies a potential new target for control of pathologic angiogenesis

Эпикардиальная трансплантация пластов из мезенхимальных стромальных клеток жировой клетчатки способствует активации эпикарда и стимулирует ангиогенез при инфаркте миокарда (экспериментальное исследование)

Aim: to evaluate the impact of tissue-engineered structures (TES) transplantation based on mesenchymal stromal cell (MSC) sheets in myocardial infarction on the activation of the epicardial cell pool and vascularization of the damaged zone.Materials and methods. Mesenchymal stromal cells were obtained from samples of subcutaneous fat of Wistar rats and C57Bl/6 mice. Tissue engineering structures were obtained by culturing cell sheets on thermosensitive plates (Nunc Dishes with UpCell Surface). Transplantation of TESs was performed after myocardial infarction modeling in rats by ligation of the anterior descending coronary artery. Transplant cells and damaged zones were assessed using immunofluorescent staining of myocardial cryosections. The impact of MSC secretion products on the migration activity of epicardial cells in vitro was evaluated using the explant culture method.Results. MSCs in TESs after transplantation remain viable and induce activation of the epicardial cell pool and local increase of the damaged zone vascularization. The in vitro experiments showed that the conditioned environment of MSCs stimulates the migratory activity of epicardial cells and initiates the formation of activated Wt1/POD1 precursor cells.Conclusion. TES transplantation on the basis of MSC sheets seems to be a promising approach for effective delivery of viable cells into myocardium to activate the epicardial cellular niche and reparative angiogenesis.Цель исследования: оценить влияние трансплантации тканеинженерных конструкций (ТИК) на основе пластов мезенхимальных стромальных клеток (МСК) при инфаркте миокарда на активацию эпикардиального пула клеток и васкуляризацию зоны повреждения.Материалы и методы. МСК получили из образцов подкожной жировой клетчатки крыс линии Wistar и мышей линии C57Bl/6. ТИК получили путем культивирования пластов клеток на чашках с термочувствительным покрытием (Nunc Dishes with UpCell Surface). Трансплантацию ТИК проводили после моделирования инфаркта миокарда у крысы путем перевязки передней нисходящей коронарной артерии. Оценку состояния клеток трансплантата и зоны повреждения проводили с использованием иммунофлуоресцентного окрашивания криосрезов миокарда. Для оценки влияния продуктов секреции МСК на миграционную активность клеток эпикарда in vitro использовали метод эксплантной культуры.Результаты. МСК в составе ТИК после трансплантации сохраняют жизнеспособность и вызывают активацию эпикардиального пула клеток и локальное повышение васкуляризации зоны повреждения. Эксперименты in vitro показали, что кондиционированная среда МСК оказывает стимулирующее воздействие на миграционную активность клеток эпикарда и вызывает образование активированных Wt1/POD1 клеток-предшественниц.Заключение. Трансплантация ТИК на основе пластов МСК представляется рациональным подходом для эффективной доставки жизнеспособных клеток в миокард с целью активирующего воздействия на эпикардиальную клеточную нишу и репаративный ангиогенез

UK–Russia Researcher Links Workshop: extracellular vesicles – mechanisms of biogenesis and roles in disease pathogenesis, M.V. Lomonosov Moscow State University, Moscow, Russia, 1–5 March 2015

The UK–Russia extracellular vesicles (EVs) workshop was held at the Medical Center of the M.V. Lomonosov Moscow State University, Moscow, Russia, with 56 attendees from UK and Russian universities and research institutes. The program consisted of 6 research sessions and was focused on studies of EVs isolated from in vitro model systems or biological fluids, including blood and urine. The multidisciplinary program included presentations on mechanisms of EV biogenesis, the role of EVs in disease pathogenesis, the diagnostic value of EVs, including their quantitation and cargo load, as well as the clinical use of EVs in regenerative medicine. Methodological challenges imposed by the nanoscale size of EVs as well as targeted delivery approaches for therapeutics were considered in a separate session on technologies. The main aim of the workshop was to overview challenges confronting EV researchers and to facilitate knowledge exchange between researchers with different backgrounds and skills. Given the lack of definitive EV nomenclature, specific terms (exosomes or microvesicles) were only applied in the meeting report to studies that carried out full EV characterization, including differential ultracentrifugation isolation approaches, comprehensive protein marker characterization, and single vesicle analysis (electron microscopy and nanoparticle analysis), to ascertain EV size and morphology following the International Society for Extracellular Vesicles standardization recommendations (1,2). In studies where characterization was not conclusive, the term EV is used

Epicardial Transplantation of Adipose Mesenchymal Stromal Cell Sheets Promotes Epicardial Activation and Stimulates Angiogenesis in Myocardial Infarction (Experimental Study)

Aim: to evaluate the impact of tissue-engineered structures (TES) transplantation based on mesenchymal stromal cell (MSC) sheets in myocardial infarction on the activation of the epicardial cell pool and vascularization of the damaged zone.Materials and methods. Mesenchymal stromal cells were obtained from samples of subcutaneous fat of Wistar rats and C57Bl/6 mice. Tissue engineering structures were obtained by culturing cell sheets on thermosensitive plates (Nunc Dishes with UpCell Surface). Transplantation of TESs was performed after myocardial infarction modeling in rats by ligation of the anterior descending coronary artery. Transplant cells and damaged zones were assessed using immunofluorescent staining of myocardial cryosections. The impact of MSC secretion products on the migration activity of epicardial cells in vitro was evaluated using the explant culture method.Results. MSCs in TESs after transplantation remain viable and induce activation of the epicardial cell pool and local increase of the damaged zone vascularization. The in vitro experiments showed that the conditioned environment of MSCs stimulates the migratory activity of epicardial cells and initiates the formation of activated Wt1/POD1 precursor cells.Conclusion. TES transplantation on the basis of MSC sheets seems to be a promising approach for effective delivery of viable cells into myocardium to activate the epicardial cellular niche and reparative angiogenesis

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