Natalia I. Kalinina

Platelet-derived growth factor regulates the secretion of extracellular vesicles by adipose mesenchymal stem cells and enhances their angiogenic potential

BackgroundSeveral studies demonstrate the role of adipose mesenchymal stem cells (ASCs) in angiogenesis. The angiogenic mechanism has been ascribed to paracrine factors since these cells secrete a plenty of signal molecules and growth factors. Recently it has been suggested that besides soluble factors, extracellular vesicles (EVs) that include exosomes and microvesicles may play a major role in cell-to-cell communication. It has been shown that EVs are implicated in the angiogenic process.ResultsHerein we studied whether EVs released by ASCs may mediate the angiogenic activity of these cells. Our results demonstrated that ASC-derived EVs induced in vitro vessel-like structure formation by human microvascular endothelial cells (HMEC). EV-stimulated HMEC when injected subcutaneously within Matrigel in SCID mice formed vessels. Treatment of ASCs with platelet-derived growth factor (PDGF) stimulated the secretion of EVs, changed their protein composition and enhanced the angiogenic potential. At variance of EVs released in basal conditions, PDGF-EVs carried c-kit and SCF that played a role in angiogenesis as specific blocking antibodies inhibited in vitro vessel-like structure formation. The enhanced content of matrix metalloproteinases in PDGF-EVs may also account for their angiogenic activity.ConclusionsOur findings indicate that EVs released by ASCs may contribute to the ASC-induced angiogenesis and suggest that PDGF may trigger the release of EVs with an enhanced angiogenic potential.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 3. Inhibitory effect of SkQ1 on tumor development from p53-deficient cells

It was proposed that increased level of mitochondrial reactive oxygen species (ROS), mediating execution of the aging program of an organism, could also be critical for neoplastic transformation and tumorigenesis. This proposal was addressed using new mitochondria-targeted antioxidant SkQ1 (10-(6′-plastoquinonyl) decyltriphenylphosphonium) that scavenges ROS in mitochondria at nanomolar concentrations. We found that diet supplementation with SkQ1 (5 nmol/kg per day) suppressed spontaneous development of tumors (predominantly lymphomas) in p53-/- mice. The same dose of SkQ1 inhibited the growth of human colon carcinoma HCT116/p53-/- xenografts in athymic mice. Growth of tumor xenografts of human HPV-16-associated cervical carcinoma SiHa was affected by SkQ1 only slightly, but survival of tumor-bearing animals was increased. It was also shown that SkQ1 inhibited the tumor cell proliferation, which was demonstrated for HCT116 p53-/- and SiHa cells in culture. Moreover, SkQ1 induced differentiation of various tumor cells in vitro. Coordinated SkQ1-initiated changes in cell shape, cytoskeleton organization, and E-cadherin-positive intercellular contacts were observed in epithelial tumor cells. In Ras- and SV40-transformed fibroblasts, SkQ1 was found to initiate reversal of morphological transformation of a malignant type, restoring actin stress fibers and focal adhesion contacts. SkQ1 suppressed angiogenesis in Matrigel implants, indicating that mitochondrial ROS could be important for tumor angiogenesis. This effect, however, was less pronounced in HCT116/p53-/- tumor xenografts. We have also shown that SkQ1 and related positively charged antioxidants are substrates of the P-glycoprotein multidrug resistance pump. The lower anti-tumor effect and decreased intracellular accumulation of SkQ1, found in the case of HCT116 xenografts bearing mutant forms of p53, could be related to a higher level of P-glycoprotein. The effects of traditional antioxidant N-acetyl-L-cysteine (NAC) on tumor growth and tumor cell phenotype were similar to the effects of SkQ1 but more than 1,000,000 times higher doses of NAC than those of SkQ1 were required. Extremely high efficiency of SkQ1, related to its accumulation in the mitochondrial membrane, indicates that mitochondrial ROS production is critical for tumorigenesis at least in some animal models.

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.

Viability and angiogenic activity of mesenchymal stromal cells from adipose tissue and bone marrow under hypoxia and inflammation in vitro

Progenitor stromal cells derived from adipose tissue (ADSC) and bone marrow (BMDSC) hold great promise for use in the cell-based therapy of ischemic diseases. It was demonstrated that these cells secrete a number of angiogenic cytokines that stimulate vascularization. It was demonstrated that ADSC or BMDSC injected intramuscularly or intravenously into the animals with experimental hind-limb ischemia improve vascularization. However, low oxygen levels and inflammation may impair the viability and functional activity of transplanted cells. We have examined ADSC and BMDSC properties in vitro under hypoxic and inflammatory conditions. ADSC and BMDSC derived from Balb/c mice have been cultivated under hypoxia or in the presence of inflammatory cytokines. The viability of cells assessed by annexin V-PE binding and 7AAD storage, as well as by the quantitative TUNEL method, was not changed under hypoxic conditions Cell exposure to inflammatory cytokines induced apoptosis in 70% of cells. Inflammatory cytokines did not stimulate gene expression of angiogenic growth factors. Under hypoxia conditions up-regulation of genes for pro-angiogenic factors and down-regulation of anti-angiogenic genes were more apparent in ADSC. Using angiogenesis models in vitro and in vivo, we demonstrated that stromal cell maintenance under hypoxic conditions increased their ability to stimulate the growth of blood vessels.

In Vitro Neuronal Induction of Adipose-Derived Stem Cells and their Fate after Transplantation into Injured Mouse Brain

The effect of substances known as inducers of neuronal differentiation on cultured human and mouse adipose-derived mesenchymal stem cells (ASCs) and their fate after transplantation into the injured and ischemic mouse brains were studied. ASCs were isolated from the human and mouse adipose tissue. Inducers of neuronal differentiation included β-mercaptoethanol, glial cell line-derived neurotrophic factor (GNDF), brain-derived neurotrophic factor (BDNF), retinoic acid (RA), 5-azacytidine, as well as their combinations. Three days after the induction, the phenotype of the induced cells was analyzed using immunocytochemistry and real-time PCR assay for differential expression of specific genes. The induction efficiency was evaluated by the increased transcription of neuronal differentiation markers: nestin, β-III-tubulin (Tub-B), microtubule-associated protein 2 (MAP2), and neuron-specific enolase (ENO2). The expression of marker genes was tested by immunocytochemical analysis. ASC cultivation in the medium with RA or BDNF in combination with 5- azacytidine for a week increased the mRNA and protein levels of nestin, Tub-B, and ENO2. The transplantation of induced mouse ASCs into the mouse brain increased the lifespan of the cells relative to control uninduced cells and promoted their migration from the transplantation site to the recipient cerebral parenchyma. The transplantation of the induced cells into the mouse brain pre-exposed to endothelin- 1 promoted a more active cell migration into the surrounding ischemic brain tissue. Thus, ASC exposure to RA or BDNF in combination with 5-azacytidine elevated the transcription of the neuronal differentiation markers and improved the viability and integration of ASCs grafted into the mouse brain.

Non-viral transfer of BDNF and uPA stimulates peripheral nerve regeneration

Peripheral nerves connect brain and spinal cord with the extremities and inner organs, and nerves injury can lead the disability and social exclusion. Growth factors and other natural stimulators of regeneration processes look very promising as future medicines. In our study, we tested the influence of genetic constructions that contain genes of brain-derived neurotrophic factor and urokinase plasminogen activator on nerves structure and function after traumatic and ischemic injuries. Injection of pVax1-hBDNF and pVax1-muPA after traumatic injury led to better restoration of nerves structure and function compared to similar parameters of control group mice. In ischemic injury model pVax1-hBDNF and pVax1-muPA slowed and reduced the damage progression and stimulated nerve regeneration as well. However, the treatment with pVax1-muPA was less effective after the traumatic injury. As we chose a non-viral method of gene delivery during our study the optimal conditions of plasmid intramuscular delivery were also determined.

Local angiotensin II promotes adipogenic differentiation of human adipose tissue mesenchymal stem cells through type 2 angiotensin receptor

Obesity is often associated with high systemic and local activity of renin-angiotensin system (RAS). Mesenchymal stem cells of adipose tissue are the main source of adipocytes. The aim of this study was to clarify how local RAS could control adipose differentiation of human adipose tissue derived mesenchymal stem cells (ADSCs). We examined the distribution of angiotensin receptor expressing cells in human adipose tissue and found that type 1 and type 2 receptors are co-expressed in its stromal compartment, which is known to contain mesenchymal stem cells. To study the expression of receptors specifically in ADSCs we have isolated them from adipose tissue. Up to 99% of cultured ADSCs expressed angiotensin II (AngII) receptor type 1 (AT1). Using the analysis of Ca2+ mobilization in single cells we found that only 5.2±2.7% of ADSCs specifically respond to serial Ang II applications via AT1 receptor and expressed this receptor constantly. This AT1const ADSCs subpopulation exhibited increased adipose competency, which was triggered by endogenous AngII. Inhibitory and expression analyses showed that AT1const ADSCs highly co-express AngII type 2 receptor (AT2), which was responsible for increased adipose competency of this ADSC subpopulation.

Activation of β-adrenergic receptors is required for elevated α1A-adrenoreceptors expression and signaling in mesenchymal stromal cells

Sympathetic neurons are important components of mesenchymal stem cells (MSCs) niche and noradrenaline regulates biological activities of these cells. Here we examined the mechanisms of regulation of MSCs responsiveness to noradrenaline. Using flow cytometry, we demonstrated that α1A adrenergic receptors isoform was the most abundant in adipose tissue-derived MSCs. Using calcium imaging in single cells, we demonstrated that only 6.9 ± 0.8% of MSCs responded to noradrenaline by intracellular calcium release. Noradrenaline increases MSCs sensitivity to catecholamines in a transitory mode. Within 6 hrs after incubation with noradrenaline the proportion of cells responding by Ca2+ release to the fresh noradrenaline addition has doubled but declined to the baseline after 24 hrs. Increased sensitivity was due to the elevated quantities of α1A-adrenergic receptors on MSCs. Such elevation depended on the stimulation of β-adrenergic receptors and adenylate cyclase activation. The data for the first time clarify mechanisms of regulation of MSCs sensitivity to noradrenaline.

T-Cadherin Expression in Melanoma Cells Stimulates Stromal Cell Recruitment and Invasion by Regulating the Expression of Chemokines, Integrins and Adhesion Molecules

T-cadherin is a glycosyl-phosphatidylinositol (GPI) anchored member of the cadherin superfamily involved in the guidance of migrating cells. We have previously shown that in vivo T-cadherin overexpression leads to increased melanoma primary tumor growth due to the recruitment of mesenchymal stromal cells as well as the enhanced metastasis. Since tumor progression is highly dependent upon cell migration and invasion, the aim of the present study was to elucidate the mechanisms of T-cadherin participation in these processes. Herein we show that T-cadherin expression results in the increased invasive potential due to the upregulated expression of pro-oncogenic integrins, chemokines, adhesion molecules and extracellular matrix components. The detected increase in chemokine expression could be responsible for the stromal cell recruitment. At the same time our previous data demonstrated that T-cadherin expression inhibited neoangiogenesis in the primary tumors. We demonstrate that T-cadherin overexpression leads to the increase in the expression of anti-angiogenic molecules and reduction in pro-angiogenic factors. Thus, T-cadherin plays a dual role in melanoma growth and progression: T-cadherin expression results in anti-angiogenic effects in melanoma, however, this also stimulates transcription of genes responsible for migration and invasion of melanoma cells.

T-Cadherin Stimulates Melanoma Cell Proliferation and Mesenchymal Stromal Cell Recruitment, but Inhibits Angiogenesis in a Mouse Melanoma Model

Melanocytes are special pigment cells that reside predominantly in the skin and eyes. In the skin, melanocytes are located in the bottom layer (the stratum basale) of the skins epidermis and in the hair follicles (Gray-Schopfer et al., 2001). Melanocytes produce melanins responsible for skin and hair color and perform protection function of the basal keratinocytes from ultraviolet light through synthesis and donation of melanin (Gray-Schopfer et al., 2001). Melanocytes maintain constant contact with the basal layer of the epidermis through direct interaction with basal keratinocytes and via secretion of soluble factors. Upon ultraviolet radiation, keratinocytes produce factors that control melanocyte proliferation, differentiation and motility (Gray-Schopfer et al., 2007). Melanocytes maintain during a lifetime a stable-ratio of 1:5 with basal keratinocytes (Fitzpatrick et al., 1979).