Tatiana Lopatina

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.

Cross Talk between Cancer and Mesenchymal Stem Cells through Extracellular Vesicles Carrying Nucleic Acids

Extracellular vesicles (EVs) are considered to be a novel complex mechanism of cell communication within the tumor microenvironment. EVs may act as vehicles for transcription factors and nucleic acids inducing epigenetic changes in recipient cells. Since tumor EVs may be present in patient biological fluids, it is important to investigate their function and molecular mechanisms of action. It has been shown that tumor cells release EVs, which are capable of regulating cell apoptosis, proliferation, invasion, and epithelial–mesenchymal transition, as well as to suppress activity of immune cells, to enhance angiogenesis, and to prepare a favorable microenvironment for metastasis. On the other hand, EVs derived from stromal cells, such as mesenchymal stem cells (MSCs), may influence the phenotype of tumor cells through reciprocal cross talk greatly influenced by the transcription factors and nucleic acids they carry. In particular, non-coding RNAs (ncRNAs), including microRNAs and long ncRNAs, have recently been identified as the main candidates for the phenotypic changes induced in the recipient cells by EVs. ncRNAs, which are important regulators of mRNA and protein expression, can function either as tumor suppressors or as oncogenes, depending on their targets. Herein, we have attempted to revise actual evidence reported in the literature on the role of EVs in tumor biology with particular regard to the cross talk of ncRNAs between cancer cells and MSCs.

Extracellular vesicles as new players in angiogenesis.

Growing evidence suggests that small vesicles actively released from cells may encapsulate transcriptional regulators and RNA molecules. Their ability to interact with neighbouring cells and/or with distant cells through biological fluids, makes them a medium through which intercellular exchange of information can happen. Recently, membrane vesicles, which include exosomes and microvesicles, gained a place amongst the vast group of angiogenic mediators. In the present review we discuss the potential relevance of these vesicles in physiological and pathological situations of angiogenesis as well as their mechanism of action.

Extracellular vesicles derived from mesenchymal stem cells induce features of diabetic retinopathy in vitro.

AimsLoss of pericytes in the early phases of diabetic retinopathy (DR) may disrupt their stable association with endothelial cells (EC), leading to EC proliferation and, eventually, angiogenesis. Extracellular vesicles (EV) are small membrane particles derived from different cells which contain biologically active proteins and RNA and are known to promote phenotypic changes in target cells. In diabetic-like conditions, EV derived from MSC may play a role in vessel destabilization by interfering with the strict interactions between EC/pericytes and pericyte/extracellular matrix.MethodsWe examined the behaviour of retinal pericytes exposed to EV derived from MSC cultured in physiological and diabetic-like conditions (high glucose and/or hypoxia).ResultsMSC-derived EV are able to enter the pericytes, cause their detachment and migration from the substrate, and increase blood-barrier permeability. Moreover, EV added to EC/pericytes co-cultures in Matrigel promote in vitro angiogenesis. These effects may be mediated by matrix metalloproteinase-2, expressed by both EV and EV-stimulated pericytes, and are exacerbated if MSC are previously cultured in conditions (high glucose and/or hypoxia) mimicking the diabetic microvascular milieu.ConclusionsWe confirm that MSC-derived EV contribute to angiogenesis, showing that they may not only exert a direct stimulus to EC proliferation, but also induce pericyte detachment, thus leaving EC free to proliferate. In addition, we demonstrate a possible link between EV and the early stages of the pathogenesis of DR. Diabetic-like conditions may influence vessel remodelling during angiogenesis through EV paracrine signalling.

The Angiogenic Potential of Adipose Mesenchymal Stem Cell-derived Extracellular Vesicles is modulated by Basic Fibroblast Growth Factor

Objective: The aim of the present study was to investigate the effects of basic fibroblast growth factor (bFGF) on the angiogenic properties of extracellular vesicles (EVs) secreted by adipose-derived mesenchymal stem cells (ASCs) in culture. Methods: We isolated EVs from ASC (ASC-EVs) cultured with bFGF (FGF-EVs) or without bFGF (b-EVs). We compared the EV angiogenic action on human microvascular endothelial cells (HMEC) in vitro by capillarylike structure formation assay and in vivo by subcutaneous injection of HMEC-containing Matrigel in SCID mice. We analysed micro-RNA composition of EVs by PCR array and we selected two mi-RNAs changed after bFGF stimulation: miR-223 and miR-21. We transfected HMEC with short antisense anti-miR-223 or with mimic miR- 21 to compensate the action of EVs. Results: In vitro, the total length and the number of branches of vessel-like structures formed by FGF-EVstimulated HMEC were significantly reduced in respect to b-EV stimulation and the structures were significantly larger than after b-EV stimulation. In vivo, vessels formed by FGF-EV-stimulated HMEC were significantly lower in number in respect to those formed by b-EV-stimulated HMEC, but they were significantly larger in size and contained cells positive for mouse smooth muscle actin. We found that bFGF led to molecular changes in ASCEVs characterized by decreased expression of angiogenic factors. Moreover, bFGF stimulation up-regulated the expression of the anti-angiogenic miR-223 and decreased the level of pro-angiogenic miR-21. The effects of FGFEVs were antagonized by the inhibition of miR-233 and by the miR-21 mimic Conclusions: The results indicate that culture conditions may modify the pro-angiogenic activity of ASCderived EVs, changing their protein and RNA contents. In particular, bFGF induces production of EVs that stimulate vessel stabilization rather than an increase in vessel number.

Effects of the neuroprotective drugs somatostatin and brimonidine on retinal cell models of diabetic retinopathy

AimsDiabetic retinopathy is considered a microvascular disease, but recent evidence has underlined early involvement of the neuroretina with interactions between microvascular and neural alterations. Topical administration of somatostatin (SST), a neuroprotective molecule with antiangiogenic properties, prevents diabetes-induced retinal neurodegeneration in animals. The α2-adrenergic receptor agonist brimonidine (BRM) decreases vitreoretinal vascular endothelial growth factor and inhibits blood–retinal barrier breakdown in diabetic rats. However, SST and BRM effects on microvascular cells have not yet been studied. We investigated the behaviour of these drugs on the crosstalk between microvasculature and neuroretina.MethodsExpression of SST receptors 1–5 in human retinal pericytes (HRP) was checked. We subsequently evaluated the effects of diabetic-like conditions (high glucose and/or hypoxia) with/without SST/BRM on HRP survival. Endothelial cells (EC) and photoreceptors were maintained in the above conditions and their conditioned media (CM) used to culture HRP. Vice versa, HRP-CM was used on EC and photoreceptors. Survival parameters were assessed.ResultsHRP express the SST receptor 1 (SSTR1). Glucose fluctuations mimicking those occurring in diabetic subjects are more damaging for pericytes and photoreceptors than stable high glucose and hypoxic conditions. SST/BRM added to HRP in diabetic-like conditions decrease EC apoptosis. However, neither SST nor BRM changed the response of pericytes and neuroretina–vascular crosstalk under diabetic-like conditions.ConclusionsRetinal pericytes express SSTR1, indicating that they can be a target for SST. Exposure to SST/BRM had no adverse effects, direct or mediated by the neuroretina, suggesting that these molecules could be safely evaluated for the treatment of ocular diseases.

Prevalence of retinopathy in patients with type 1 diabetes diagnosed before and after puberty.

AbstractAims There is conflicting evidence to support the concept that the years with diabetes preceding puberty may not contribute to the development of vascular complications. In this paper, duration-related prevalence of retinopathy was compared in patients who developed type 1 diabetes before and after pubertal age.MethodsRetrospective analysis of prospectively collected data of 1,483 patients was screened for retinopathy in 1991–2010, with diabetes onset at age ≤29, who were on insulin treatment and aged ≤60. Prepubertal age was defined as 0–12 in males and 0–11 in females.ResultsA total of 647 patients had developed diabetes before and 836 after puberty. Cumulative prevalence of retinopathy was initially lower among those with prepubertal onset diabetes but rates became superimposable after 20-year duration. Patients with prepubertal onset diabetes had higher lifetime HbA1c and lower blood pressure than those who became diabetic after puberty.Conclusions/interpretationRetinopathy is infrequent during childhood and develops later than in patients with post-pubertal onset diabetes. After 20-year duration, however, retinopathy becomes just as prevalent suggesting that, in the long term, prepubertal years do contribute to the development of retinopathy. In this series, higher blood pressure may have played a role in the earlier appearance of retinopathy in patients with diabetes onset after puberty, whereas worse metabolic control may have contributed to the late “catch-up” effect in those with prepubertal onset disease.

Extracellular vesicles from human liver stem cells inhibit tumor angiogenesis: HLSC extracellular vesicles inhibit tumor angiogenesis

Human liver stem‐like cells (HLSC) and derived extracellular vesicles (EVs) were previously shown to exhibit anti‐tumor activity. In our study, we investigated whether HLSC‐derived EVs (HLSC‐EVs) were able to inhibit tumor angiogenesis in vitro and in vivo, in comparison with EVs derived from mesenchymal stem cells (MSC‐EVs). The results obtained indicated that HLSC‐EVs, but not MSC‐EVs, inhibited the angiogenic properties of tumor‐derived endothelial cells (TEC) both in vitro and in vivo in a model of subcutaneous implantation in Matrigel. Treatment of TEC with HLSC‐EVs led to the down‐regulation of pro‐angiogenic genes. Since HLSC‐EVs carry a specific set of microRNAs (miRNAs) that could target these genes, we investigated their potential role by transfecting TEC with HLSC‐EV specific miRNAs. We observed that four miRNAs, namely miR‐15a, miR‐181b, miR‐320c and miR‐874, significantly inhibited the angiogenic properties of TEC in vitro, and decreased the expression of some predicted target genes (ITGB3, FGF1, EPHB4 and PLAU). In parallel, TEC treated with HLSC‐EVs significantly enhanced expression of miR‐15a, miR‐181b, miR‐320c and miR‐874 associated with the down‐regulation of FGF1 and PLAU. In summary, HLSC‐EVs possess an anti‐tumorigenic effect, based on their ability to inhibit tumor angiogenesis.

Molecular and functional characterization of circulating extracellular vesicles from diabetic patients with and without retinopathy and healthy subjects

&NA; Diabetic retinopathy is a sight‐threatening complication of diabetes, characterized by loss of retinal pericytes and abnormal angiogenesis. We previously demonstrated that extracellular vesicles (EVs) derived from mesenchymal stem cells cultured in diabetic‐like conditions are able to enter the pericytes, causing their detachment and migration, and stimulating angiogenesis in vitro. The purpose of this work was the molecular and functional characterization of EVs derived from diabetic subjects with or without diabetic retinopathy, compared with healthy controls. Characterization of EVs extracted from serum/plasma of diabetic patients with or without retinopathy, and healthy controls, was performed by FACS and microarray analysis of microRNA (miRNA) content. Relevant miRNA expression was validated through qRT‐PCR. EV influence on pericyte detachment, angiogenesis and permeability of the blood‐retinal barrier was also investigated. Diabetic subjects had a 2.5 fold higher EV concentration than controls, while expression of surface molecules was unchanged. Microarray analysis revealed 11 differentially expressed miRNAs. Three of them (miR‐150‐5p, miR‐21‐3p and miR‐30b‐5p) were confirmed by qRT‐PCR. Plasma EVs from subjects with diabetic retinopathy induced pericyte detachment and pericyte/endothelial cell migration, increased the permeability of pericyte/endothelial cell bilayers and the formation of vessel‐like structures, when compared with EVs from controls. In conclusion, circulating EVs show differences between diabetic patients and healthy subjects. EVs extracted from plasma of diabetic retinopathy patients are able to induce features of retinopathy in in vitro models of retinal microvasculature. Our data suggest a role for miR‐150‐5p, miR‐21‐3p and miR‐30b‐5p as potential biomarkers of the onset of diabetic retinopathy. HighlightsCirculating extracellular vesicles are different in diabetic and healthy subjects.Vesicles from diabetic retinopathy patients induce features of retinopathy in vitro.MiRNAs shuttled by extracellular vesicles are involved.miR‐150‐5p, miR‐21‐3p, miR‐30b‐5p are potential biomarkers of diabetic retinopathy.