Maria A. Boldyreva

Genetic variation in the morphology of the septo-hippocampal cholinergic and GABAergic system in mice. I. Cholinergic and GABAergic markers.

In the present study, variations of cholinergic and GABAergic markers in the medial septum/vertical limb of the diagonal band of Broca (MS/vDB) and the hippocampus of eight different inbred mouse strains were investigated. By means of immunocytochemistry against the acetylcholine‐synthesizing enzyme choline acetyltransferase (ChAT), the cholinergic neurons were visualized and the number of ChAT‐positive neuronal profiles in the MS/vDB was counted. Cholinergic and GABAergic septo‐hippocampal projection neurons were detected with a combined retrograde tracing and immunocytochemical approach. In order to quantify the cholinergic innervation of various hippocampal subregions, we estimated the density of acetylcholinesterase (AChE)‐containing fibers as visualized by AChE histochemistry. Additionally, the densities of muscarinic receptors (mainly the subtypes M1 and M2) in different hippocampal areas of seven inbred strains were measured by means of quantitative receptor autoradiography.

Genetic variation in the morphology of the septo-hippocampal cholinergic and GABAergic systems in mice: II. Morpho-behavioral correlations

We investigated the contribution of the septo‐hippocampal cholinergic and GABAergic system to spatial and nonspatial aspects of learning and memory that had previously been found to correlate with the extent of the hippocampal intra‐ and infrapyramidal mossy fiber projection in different inbred mouse strains. The following cholinergic and GABAergic markers were measured in the septi and hippocampi of male mice: the number of cholinergic and parvalbumin‐containing neurons in the medial septum/vertical limb of the diagonal band of Broca (MS/vDB), the number of septo‐hippocampal cholinergic and GABAergic projection neurons, the density of cholinergic fibers in different hippocampal subfields, and the density of muscarinic receptors (predominantly M1 and M2) in the hippocampus. In addition, animals were behaviorally tested for spatially dependent and activity‐dependent variables in a water maze and spatial and nonspatial working and reference memory in different experimental set‐ups in an eight‐arm radial maze.

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.

Cholinergic and GABAergic septo-hippocampal projection neurons in mice: a retrograde tracing study combined with double immunocytochemistry for choline acetyltransferase and parvalbumin

The present experiments were directed to determine the proportions of the cholinergic and GABAergic septo-hippocampal projection neurons in NMRI mice. For the labeling of the septal neurons we used a double immunocytochemical method combined with retrograde transport of wheatgerm agglutinin apo-horseradish peroxidase-gold (WAHG) injected unilaterally into the hippocampus. Monoclonal antibodies against choline acetyltransferase (ChAT) and parvalbumin (PARV) were used as markers for cholinergic and GABAergic neurons in the medial septum/diagonal band complex (MS/DB). Both antibodies were visualized in the same section using the ABC detection system with diaminobenzidine and 4-chloro-1-naphtol as chromogens. Cholinergic and PARV-containing neurons are coexisting in the MS/DB. About 38% of all retrogradely labeled neurons were ChAT-positive whereas only 10% of all retrogradely labeled cells were immunostained for PARV. On the other hand, 40% of all ChAT-positive neurons and about 24% of all PARV-positive neurons were retrogradely labeled. No double immunolabeled neurons were detected. The proportion of GABAergic neurons may have been underestimated because immunostaining for PARV only labels a subpopulation of GABAergic neurons. The present results were compared with those of previous studies in rats. They may serve as a basis of further comparative studies in mice.

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.

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.

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.