Microvascular Experimentation in the Chick Chorioallantoic Membrane as a Model for Screening Angiogenic Agents including from Gene-Modified Cells

The chick chorioallantoic membrane (CAM) assay model of angiogenesis has been highlighted as a relatively quick, low cost and effective model for the study of pro-angiogenic and anti-angiogenic factors. The chick CAM is a highly vascularised extraembryonic membrane which functions for gas exchange, nutrient exchange and waste removal for the growing chick embryo. It is beneficial as it can function as a treatment screening tool, which bridges the gap between cell based in vitro studies and in vivo animal experimentation. In this review, we explore the benefits and drawbacks of the CAM assay to study microcirculation, by the investigation of each distinct stage of the CAM assay procedure, including cultivation techniques, treatment applications and methods of determining an angiogenic response using this assay. We detail the angiogenic effect of treatments, including drugs, metabolites, genes and cells used in conjunction with the CAM assay, while also highlighting the testing of genetically modified cells. We also present a detailed exploration of the advantages and limitations of different CAM analysis techniques, including visual assessment, histological and molecular analysis along with vascular casting methods and live blood flow observations.

Micro-RNA 92a as a Therapeutic Target for Cardiac Microvascular Dysfunction in Diabetes

Microvascular dysfunction is a pathological hallmark of diabetes, and is central to the ethology of diabetes-associated cardiac events. Herein, previous studies have highlighted the role of the vasoactive micro-RNA 92a (miR-92a) in small, as well as large, animal models. In this study, we explore the effects of miR-92a on mouse and human cardiac microvascular endothelial cells (MCMEC, HCMEC), and its underlying molecular mechanisms. Diabetic HCMEC displayed impaired angiogenesis and a pronounced inflammatory phenotype. Quantitative PCR (qPCR) showed an upregulation of miR-92a in primary diabetic HCMEC. Downregulation of miR-92a by antagomir transfection in diabetic HCMEC rescued angiogenesis and ameliorated diabetic endothelial bed inflammation. Furthermore, additional analysis of potential in silico-identified miR-92a targets in diabetic HCMEC revealed the miR-92a dependent downregulation of an essential metalloprotease, ADAM10. Accordingly, downregulation of ADAM10 impaired angiogenesis and wound healing in MCMEC. In myocardial tissue slices from diabetic pigs, ADAM10 dysregulation in micro- and macro-vasculature could be shown. Altogether, our data demonstrate the role of miR-92a in cardiac microvascular dysfunction and inflammation in diabetes. Moreover, we describe for the first time the metalloprotease ADAM10 as a novel miR-92a target, mediating its anti-angiogenic effect.

Recombinant TAT-Thymosin β-4 Promotes Angiogenesis by Activating VEGFR2 Signaling Pathway

Thymosin β-4 is a 43-amino acid intracellular polypeptide that was originally isolated from bovine thymus. Of the 16 known thymosin families, thymosin β-4 is the most common type found in all tissues. Thymosin β-4 regulates angiogenesis, cell differentiation, morphogenesis, migration, and organogenesis and is linked to a dynamic equilibrium between G-actin and F-actin. In particular, thymosin β-4 is well-known for its angiogenic and anti-apoptotic functions. In this study, we synthesized thymosin β-4 linked with the well-known cell-penetrating peptide TAT (YGRKKRRRQRRR). TAT-thymosin β-4 promotes angiogenesis and cell migration in vitro via the VEGFR2 signaling pathway and reduces apoptosis. To examine angiogenic potential in vivo, a Matrigel Plus assay was conducted that revealed the angiogenic effect of TAT-thymosin β-4. In conclusion, TAT-thymosin β-4 promotes blood vessels and is expected to be applicable in regenerative medicine for all organs requiring blood vessels.

Tropoelastin Promotes the Formation of Dense, Interconnected Endothelial Networks

Tropoelastin, the soluble precursor of elastin, has been used for regenerative and wound healing purposes and noted for its ability to accelerate wound repair by enhancing vascularization at the site of implantation. However, it is not clear whether these effects are directly due to the interaction of tropoelastin with endothelial cells or communicated to endothelial cells following interactions between tropoelastin and neighboring cells, such as mesenchymal stem cells (MSCs). We adapted an endothelial tube formation assay to model in vivo vascularization with the goal of exploring the stimulatory mechanism of tropoelastin. In the presence of tropoelastin, endothelial cells formed less tubes, with reduced spreading into capillary-like networks. In contrast, conditioned media from MSCs that had been cultured on tropoelastin enhanced the formation of more dense, complex, and interconnected endothelial tube networks. This pro-angiogenic effect of tropoelastin is mediated indirectly through the action of tropoelastin on co-cultured cells. We conclude that tropoelastin inhibits endothelial tube formation, and that this effect is reversed by pro-angiogenic crosstalk from tropoelastin-treated MSCs. Furthermore, we find that the known in vivo pro-angiogenic effects of tropoelastin can be modeled in vitro, highlighting the value of tropoelastin as an indirect mediator of angiogenesis.

HIF-1α overexpression in adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia-induced dysfunction and inflammation in HUVECs

Background Increasing evidence suggests that ADSCs execute their paracrine function via the secretion of exosomes, especially under hypoxic conditions. However, the mechanisms by which ADSCs-derived exosomes (ADSC-exos) enhance angiogenesis under hypoxia remain unclear. Methods Exosomes were isolated from HIF-1α-modified ADSCs culture supernatants. To investigate the effects HIF-1α-ADSC-exos on HUVECs, cell growth, apoptosis, and tube formation assay were performed with or without HIF-1α-ADSC-exos. Moreover, to determine the function of HIF-1α-ADSC-exos, the therapeutic effects of ADSC-exos and HIF-1α-ADSC-exos were examined in PAH rats. Results Exosomes released by HIF-1α-modified ADSCs rescued the impaired angiogenic ability, migratory function, and inflammatory factors of hypoxia-injured HUVECs, with increased SDF-1α, Rac1, Rac2, VEGF and IL-10 expression. Furthermore, exos-HIF-1α activated SIRT3 to enhance angiogenesis in HUVECs and induced IL-10 expression to inhibit inflammatory response. Block SIRT3 or SDF-1α abolished the angiogenic effect in HUVECs. Conclusion Our findings indicated that the SIRT3 contributed a crucial role in HIF-1α-ADSC-exos in tissue repair under hypoxia.