Anastasia Yu. Efimenko

Adipose Stromal Cells Stimulate Angiogenesis via Promoting Progenitor Cell Differentiation, Secretion of Angiogenic Factors, and Enhancing Vessel Maturation

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.

Angiogenic properties of aged adipose derived mesenchymal stem cells after hypoxic conditioning

BackgroundMesenchymal stem cells derived from adipose tissue (ADSC) are multipotent stem cells, originated from the vascular-stromal compartment of fat tissue. ADSC are used as an alternative cell source for many different cell therapies, however in ischemic cardiovascular diseases the therapeutic benefit was modest. One of the reasons could be the use of autologous aged ADSC, which recently were found to have impaired functions. We therefore analysed the effects of age on age markers and angiogenic properties of ADSC. Hypoxic conditioning was investigated as a form of angiogenic stimulation.MethodsADSC were harvested from young (1-3 month), adult (12 month) and aged (18-24 month) mice and cultured under normoxic (20%) and hypoxic (1%) conditions for 48 h. Differences in proliferation, apoptosis and telomere length were assessed in addition to angiogenic properties of ADSC.ResultsProliferation potential and telomere length were decreased in aged ADSC compared to young ADSC. Frequency of apoptotic cells was higher in aged ADSC. Gene expression of pro-angiogenic factors including vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and hepatic growth factor (HGF) were down-regulated with age, which could be restored by hypoxia. Transforming growth factor (TGF-β) increased in the old ADSC but was reduced by hypoxia.Expression of anti-angiogenic factors including thrombospondin-1 (TBS1) and plasminogen activator inhibitor-1 (PAI-1) did increase in old ADSC, but could be reduced by hypoxic stimulation. Endostatin (ENDS) was the highest in aged ADSC and was also down-regulated by hypoxia. We noted higher gene expression of proteases system factors like urokinase-type plasminogen activator receptor (uPAR), matrix metalloproteinases (MMP2 and MMP9) and PAI-1 in aged ADSC compared to young ADSC, but they decreased in old ADSC. Tube formation on matrigel was higher in the presence of conditioned medium from young ADSC in comparison to aged ADSC.ConclusionsADSC isolated from older animals show changes, including impaired proliferation and angiogenic stimulation. Angiogenic gene expression can be partially be improved by hypoxic preconditioning, however the effect is age-dependent. This supports the hypothesis that autologous ADSC from aged subjects might have an impaired therapeutic potential.

Adipose-Derived Mesenchymal Stromal Cells From Aged Patients With Coronary Artery Disease Keep Mesenchymal Stromal Cell Properties but Exhibit Characteristics of Aging and Have Impaired Angiogenic Potential

Tissue regeneration is impaired in aged individuals. Adipose‐derived mesenchymal stromal cells (ADSCs), a promising source for cell therapy, were shown to secrete various angiogenic factors and improve vascularization of ischemic tissues. We analyzed how patient age affected the angiogenic properties of ADSCs. ADSCs were isolated from subcutaneous fat tissue of patients with coronary artery disease (CAD; n = 64, 43–77 years old) and without CAD (n = 31, 2–82 years old). ADSC phenotype characterized by flow cytometry was CD90+/CD73+/CD105+/CD45−/CD31− for all samples, and these cells were capable of adipogenic and osteogenic differentiation. ADSCs from aged patients had shorter telomeres (quantitative reverse transcription polymerase chain reaction) and a tendency to attenuated telomerase activity. ADSC‐conditioned media (ADSC‐CM) stimulated capillary‐like tube formation by endothelial cells (EA.hy926), and this effect significantly decreased with the age of patients both with and without CAD. Angiogenic factors (vascular endothelial growth factor, placental growth factor, hepatocyte growth factor, angiopoetin‐1, and angiogenin) in ADSC‐CM measured by enzyme‐linked immunosorbent assay significantly decreased with patient age, whereas levels of antiangiogenic factors thrombospondin‐1 and endostatin did not. Expression of angiogenic factors in ADSCs did not change with patient age (real‐time polymerase chain reaction); however, gene expression of factors related to extracellular proteolysis (urokinase and its receptor, plasminogen activator inhibitor‐1) and urokinase‐type plasminogen activator receptor surface expression increased in ADSCs from aged patients with CAD. ADSCs from aged patients both with and without CAD acquire aging characteristics, and their angiogenic potential declines because of decreasing proangiogenic factor secretion. This could restrict the effectiveness of autologous cell therapy with ADSCs in aged patients.

Disturbed angiogenic activity of adipose-derived stromal cells obtained from patients with coronary artery disease and diabetes mellitus type 2

BackgroundMultipotent mesenchymal stem/stromal cells (MSC) including adipose-derived stromal cells (ADSC) have been successfully applied for cardiovascular diseases treatment. Their regenerative potential is considered due to the multipotency, paracrine activity and immunologic privilege. However, therapeutic efficacy of autologous MSC for myocardial ischemia therapy is modest. We analyzed if ADSC properties are attenuated in patients with chronic diseases such as coronary artery disease (CAD) and diabetes mellitus type 2 (T2DM).Methods and resultsADSC were isolated from subcutaneous fat tissue of patients without established cardiovascular diseases and metabolic disorders (control group, n = 19), patients with CAD only (n = 32) and patients with CAD and T2DM (n = 28). ADSC phenotype (flow cytometry) was CD90+/CD73+/CD105+/CD45−/CD31− and they were capable of adipogenic and osteogenic differentiation. ADSC morphology and immunophenotype were similar for all patients, but ADSC from patients with CAD and T2DM had higher proliferation activity and shorter telomeres compared to control patients.ADSC conditioned media stimulated capillary-like tubes formation by endothelial cells (EA.hy926), but this effect significantly decreased for patients with CAD (p = 0.03) and with CAD + T2DM (p = 0.017) compared to the control group. Surprisingly we revealed significantly higher secretion of some pro-angiogenic factors (ELISA) by ADSC: vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) for patients with CAD and HGF and placental growth factor (PlGF) for patients with CAD + T2DM. Among angiogenesis inhibitors such as thrombospondin-1, endostatin and plasminogen activator inhibitor-1 (PAI-1) level of PAI-1 in ADSC conditioned media was significantly higher for patients with CAD and CAD + T2DM compared to the control group (p < 0.01). Inhibition of PAI-1 in ADSC conditioned media by neutralizing antibodies partially restored ADSC angiogenic activity (p = 0.017).ConclusionsADSC angiogenic activity is significantly declined in patients with CAD and T2DM, which could restrict the effectiveness of autologous ADSC cell therapy in these cohorts of patients. This impairment might be due to the disturbance in coordinated network of pro- and anti-angiogenic growth factors secreted by ADSC. Changes in ADSC secretome differ between patients with CAD and T2DM and further investigation are necessary to reveal the MSC-involved mechanisms of cardiovascular and metabolic diseases and develop novel approaches to their correction using the methods of regenerative medicine.

Autologous Stem Cell Therapy: How Aging and Chronic Diseases Affect Stem and Progenitor Cells

Abstract During recent years different types of adult stem/progenitor cells have been successfully applied for the treatment of many pathologies, including cardiovascular diseases. The regenerative potential of these cells is considered to be due to their high proliferation and differentiation capacities, paracrine activity, and immunologic privilege. However, therapeutic efficacy of the autologous stem/progenitor cells for most clinical applications remains modest, possibly because of the attenuation of their regenerative potential in aged patients with chronic diseases such as cardiovascular diseases and metabolic disorders. In this review we will discuss the risk factors affecting the therapeutic potential of adult stem/progenitor cells as well as the main approaches to mitigating them using the methods of regenerative medicine.

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.

587. MiRNA-92a Is Involved in the Regulation of Adipose-Derived Stromal Cell (ADSC) Angiogenic Properties

Reparative and regenerative processes after tissue damage are strongly dependent on successful restoration of vascular network. Mesenchymal stem/stromal cells (MSC) including ADSC located within the vessel wall and closely interacted with endothelial cells appear to be key regulators of angiogenesis and regeneration in adult tissues. Mechanisms of MSC ability to stimulate blood vessel growth and remodeling are extensively studying during last decades. microRNAs (miRs), small noncoding RNAs that regulate gene expression by binding to target mRNAs reducing their stability and/or inhibiting translation, play an important role in MSC regulation. It was demonstrated that miR-92a expressed in endothelial cells was induced in ischemia and involved in the regulation of angiogenesis and vessel patterning. We studied miR-92a impact in ADSC angiogenic properties regulation.ADSC were isolated from subcutaneous fat tissue of healthy young donors (n=4) and cultured up to 2-3 passages. ADSC phenotype characterized by flow cytometry was CD90+/CD73+/CD105+/CD45-/CD31- for all samples and these cells were capable of adipogenic and osteogenic differentiation. We analyzed miR profile in ADSC using Illumina microarrays and found that miR-92a was one of the highly expressed miRs in all ADSC samples. ADSC transfection by pre-miR-92a or anti-miR-92a led to the coordinated changes of miR-92a well-known target genes expression (integrin alpha 5, ITGA5, and mitogen activated protein kinase, MEK4). ADSC with over-expression of miR-92a completely lost the ability to stimulate capillary-like tube formation by endothelial cells (HUVEC) in vitro compared to ADSC transfected by the control gene. However, transfection of ADSC by anti-miR-92a did not significantly increase their stimulating effect in tube formation assay. As paracrine mechanisms are considered to be the most important for realizing of MSC angiogenic potential, we analyzed gene expression (qRT-PCR) and secretion (Bio-Plex assay and ELISA) of some key angiogenic factors in ADSC. We found that mRNA level of vascular endothelial growth factor (VEGF), angiogenin and leptin was slightly increased in ADSC transfected by anti-miR-92a and this tendency was also observed for VEGF secretion. When miR-92a was over-expressed in ADSC secretion of hepatocyte growth factor (HGF) and angiopoetin-1 by these cells significantly decreased compared to the control cells.We conclude that miR-92a might play an important role in the regulation of ADSC ability to stimulate angiogenesis and key angiogenic factors such as VEGF, HGF and angiopoetin-1 are involved in this process. The underlying mechanisms of miR-92a-mediated direct or indirect effects in ADSC need to be further investigated. Anyway, miR-92a could be considered as a promising target for the modulation of ADSC angiogenic potential.

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.