Konstantin V. Dergilev

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.

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.

Urokinase-type plasminogen activator (uPA) is critical for progression of tuberous sclerosis complex 2 (TSC2)-deficient tumors

Lymphangioleiomyomatosis (LAM) is a fatal lung disease associated with germline or somatic inactivating mutations in tuberous sclerosis complex genes (TSC1 or TSC2). LAM is characterized by neoplastic growth of smooth muscle-α-actin–positive cells that destroy lung parenchyma and by the formation of benign renal neoplasms called angiolipomas. The mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin slows progression of these diseases but is not curative and associated with notable toxicity at clinically effective doses, highlighting the need for better understanding LAMs molecular etiology. We report here that LAM lesions and angiomyolipomas overexpress urokinase-type plasminogen activator (uPA). Tsc1−/− and Tsc2−/− mouse embryonic fibroblasts expressed higher uPA levels than their WT counterparts, resulting from the TSC inactivation. Inhibition of uPA expression in Tsc2-null cells reduced the growth and invasiveness and increased susceptibility to apoptosis. However, rapamycin further increased uPA expression in TSC2-null tumor cells and immortalized TSC2-null angiomyolipoma cells, but not in cells with intact TSC. Induction of glucocorticoid receptor signaling or forkhead box (FOXO) 1/3 inhibition abolished the rapamycin-induced uPA expression in TSC-compromised cells. Moreover, rapamycin-enhanced migration of TSC2-null cells was inhibited by the uPA inhibitor UK122, dexamethasone, and a FOXO inhibitor. uPA-knock-out mice developed fewer and smaller TSC2-null lung tumors, and introduction of uPA shRNA in tumor cells or amiloride-induced uPA inhibition reduced tumorigenesis in vivo. These findings suggest that interference with the uPA-dependent pathway, when used along with rapamycin, might attenuate LAM progression and potentially other TSC-related disorders.

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.

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.

C-Kit Cardiac Progenitor Cell Based Cell Sheet Improves Vascularization and Attenuates Cardiac Remodeling following Myocardial Infarction in Rats

The adult heart contains small populations of multipotent cardiac progenitor cells (CPC) that present a convenient and efficient resource for treatment of myocardial infarction. Several clinical studies of direct CPC delivery by injection have already been performed but showed low engraftment rate that limited beneficial effects of procedure. «Cell sheet» technology has been developed to facilitate longer retention of grafted cells and show new directions for cell-based therapy using this strategy. In this study we hypothesized that СPC-based cell sheet transplantation could improve regeneration after myocardial infarction. We demonstrated that c-kit+ CPC were able to form cell sheets on temperature-responsive surfaces. Cell sheet represented a well-organized structure, in which CPC survived, retained ability to proliferate, expressed progenitor cell marker Gata-4 formed connexin-43+ gap junctions, and were surrounded by significant amount of extracellular matrix proteins. Transplantation of cell sheets after myocardial infarction resulted in CPC engraftment as well as their proliferation, migration, and differentiation; cell sheets also stimulated neovascularization and cardiomyocyte proliferation in underlining myocardium and ameliorated left ventricular remodeling. Obtained data strongly supported potential use of CPC sheet transplantation for repair of damaged heart.

447. Notch Activation Enchances Vascular Lineage Commitment of Cardiac Stem Cells

Notch signaling pathway is an evolutionary conserved system that control heart development and could be important for cardiac tissue renewal and regeneration after injury by controlling the maintenance and commitment of a cardiac stem cell compartment. However the precise cell targets of Notch signaling in the mammalian damaged heart remain poorly defined. OBJECTIVE: To investigate the functional role of Notch signaling in the regulation of cardiac stem cells (CSCs) commitment in ischemic mice myocardium. METHODS AND RESULTS: Notch 1 receptor, their ligands and markers of cardiomyocyte, SMC, and endothelial cell lineages were examined in undamaged and infarcted C57BL/6 mice hearts by immunofluorescence staining. We have found that the majority of c-kit+ CSCs were nested in the interstitium between cardiomyocytes, and expressed Notch 1 receptor. Ligand Jagged 1 is presented on cardiomyocyte and cardiac fibroblasts, localized near c-kit+Notch1+ CSCs. Conversely, Delta-like4 ligand expressed on the endothelial and smooth muscle cells of coronary vessels. No signs of Notch1 signaling activation (such as nuclear NICD localization) were detected in undamaged mice hearts. Acute myocardial infarction was accompanied by the increase in the total amount of c-kit + Notch1 + CSCs and the appearance of nuclear NICD localization in a number of CSCs showing an activation of Notch signaling. Cultivation of CSC in vitro on dishes coated with Jagged 1 ligand released NICD and activated expression of Notch target genes (Hes, Hey). Activation of Notch signaling upregulated expression of endothelial cell transcription factors Gata 4 and Vezf1 in CSCs but γ-secretase inhibitor prevented Notch signaling activation and endothelial cell commitment of CSCs. CONCLUSIONS: These results revealed that Notch signaling activation promote CSCs commitment toward vascular cells. These findings suggest that modulation of Notch signaling can be promising therapeutic strategy for the treatment of myocardial ischemic damage.

448. Therapeutic Angiogenesis by Subcutaneous Cell Sheet Delivery Is Superior to Cell Injection: A Study of ADSC Efficacy in a Model of Hind Limb Ischemia

Engineering of cell sheets (CS) is an effective approach for delivery of cells to induce angiogenesis and tissue regeneration. Basis for increased CS efficacy is better engraftment and cell survival due to absence of anoikis and intact cell-to-cell interaction in the transplant. Still question to be addressed is whether CS are superior to injection route in terms of efficacy/cell engraftment and how can we improve therapeutic output of CS delivery for stimulation of tissue repair. We conducted a comparative study of adipose-derived stromal cells (ADSC) delivery in a model of hind-limb ischemia. C57/B6 male mice with unilateral limb ischemia (n=8-10/group) were injected with 106 of passage 3 syngeneic ADSC or transplanted with equivalent amount of cells in CS shape. After that animals were monitored for limb perfusion by laser-Doppler for 2 wks and then euthanized for histology studies of vessel density and ADSC detection using a PKH26 or CMFDA fluorescent label. Obtained samples were stained for macrophage invasion, endothelial cell markers and proliferation/apoptosis to evaluate cell fate. Our animal test data has revealed that by Day 14 delivery of ADSC by means of injection induced restoration of limb perfusion compared to negative control group (41.5±4.7% vs. 29.7±3.0% respectively; p=0.01) indicating well-known pro-angiogenic properties of these cells. Still, subcutaneous transplantation of CS was found to be superior to injection in terms of perfusion. CS-treated animals had the highest (55.3±7.3; p=0.03 vs. injected ADSC) perfusion by the end of experiment. This data was supported by vascular density assessment, which revealed increased capillary counts in both ADSC-treated groups with significantly higher values after CS delivery compared to injection (220.9±11.4 vs. 191.3±8.8 respectively; p=0.01). Analysis of necrotic tissue span in hematoxylin/eosin-stained section found a significant decrease of necrosis in ADSC-treated animals and also found CS to have better performance in terms of tissue protection compared to injection. Furthermore, we also evaluated ADSC engraftment and found that after injection pre-labeled cells reside as scattered mass and found their number to decrease over time by Day 14. Whilst after CS transplantation the cells were compactly localized in the site of application. CS were found to be vascularized by capillary vessels and infiltrated by CD68+ macrophage indicating graft-host interaction. Interestingly, certain cells within CS were found to show signs of proliferation (Ki67+) with sporadic apoptosis (cleaved caspase-3+) with overall transplant staying intact and viable by Day 14 after delivery. Overall, our data indicates that transplantation of CS is superior to injection of equivalent amount of cells. We may speculate that this is not limited to ADSC and can be utilized for novel treatment methods. In an attempt to enhance the CS efficacy we turned our attention to hybrid constructs consisting of ADSC and endothelial cells to generate pre-vascularized constructs. Our preliminary data revealed “tube-like” behavior of HUVEC seeded on top of a CS from ADSC and may be the way to overcome the diffusion distance issue and generate CS with vascularized structure, which have a closer resemblance to native tissue structures. Moreover, this unveils the possibility to generate multi-layered constructs and imitate cell-to-cell interaction for basic and applied studies.

Regulatory Effects of Urokinase on Mesenchymal Stromal Cell Migration, Proliferation, and Matrix Metalloproteinase Secretion

We studied the effect of urokinase, its recombinant forms, and domain fragments on migration and proliferation of adipose tissue mesenchymal stromal cells (MSCs) and MMP secretion by these cells. Urokinase, but not its recombinant forms, slightly induced directed migration of MSCs. Spontaneous migration of MSCs increased under the action of urokinase or its isolated kringle domain. Migration induced by platelet-derived growth factor was inhibited by proteolytically inactive form of urokinase, the kringle domain, and blocking antibody to urokinase receptor. Urokinase, its proteolytically inactive form, and kringle domain produced no effect on MSC proliferation. In contrast to platelet-derived growth factor, all urokinase forms induced secretion of MMP-9 by MSCs.

657. Delivery of Genetically Engineered Adipose-Derived Cell Sheets for Treatment of Ischemic Disorders – Development of Application in Animal Models

Cell sheet (CS) approach is extensively developed in recent years for a number of applications in regenerative medicine including lesion treatment, tissue-engineered constructs and organoplastics. In this study we have targeted ischemic diseases – myocardial infarction (MI) and peripheral artery disease (PAD) using CS from differetnt sources transplanted as CS to the impaired region.We established a protocol for generation of CS from c-kit+ cardiac primitive cells and cardiosphere cells for delivery to rats with induced MI using clinical-grade fibrin glue for CS adhesion subepicardially. We found CS to be vascularized after transplantation and used ultrasound assessment to detect improvement of left ventricle function and significant positive changes of tissue status in histology studies: capillary density and reduction of fibrosis). Moreover, we conducted a comparative analysis of this approach vs. CS from adipose-derived stomal cells (ADSC) and VEGF-expressing ADSC. The latter were obtained via baculovirus transduction of CS, which enabled significant production of human VEGF165 by murine cells and, thus, enhanced their therapeutic potential.Our previous application of ADSC for therapeutic angiogenesis and established methods for genetic modification of cells allowed us to develop a protocol in limb ischemia model. We have demonstrated, that subcutaneous implantation of CS or VEGF-expressing CS from mouse ADSC resulted in significant improvement of limb perfusion with modified cells to be superior to mock-transduced. Increased blood flow was supported by higher CD31+ capillary density, reduction of necrosis and detection of viable CS subcutaneously with focal proliferation of mADSC and minimal (app. 10%) prevalence of apoptotic cells at 14 days post transplantation. Paracrine mechanism for CS mode of action is suggested due to the fact, that ww found vessel growth within CS mass, which indicates possibility of cells’ nutrition and uptake of secreted proteins from the site of transplantation besides diffusion.Thus, our findings indicate, that CS-based delivery for treatment of MI or PAD is a potential method in regenerative medicine in this field and furthermore genetic modification of cells used for CS generation may be a way to “tune up” the cells’ paracrine activity and efficacy of their application.