ARPE-19-derived VEGF-containing exosomes promote neovascularization in HUVEC: the role of the melanocortin receptor 5

ARPE-19 retinal pigment epithelial cells cultured in a medium containing 35 mM D-glucose led to an augmented ROS formation and release of vascular endothelial factor (VEGF)-containing exosomes compared to ARPE-19 cells cultured in a medium containing 5 mM D-glucose (standard medium). Exposing these cells to the melanocortin 5 receptor agonist (MCR₅) PG-901 (10^?10M), for 9 d reduced ROS generation, the number of exosomes released and their VEGF content. In contrast, incubating the cells with the melanocortin receptor MCR₁ agonist BMS-470539 (10^?5 M) or with the mixed MCR_3/4 agonist MTII (0.30 nmol) did not produce any significant decrease in ROS levels. ARPE-19-derived VEGF-containing exosomes promoted neovascularization in human umbilical vein endothelial cells (HUVEC), an effect that was markedly reduced by PG-901 (10^?10M) but not by the MCR_3/4 agonist MTII (0.30 nmol) or the MCR₁ agonist BMS-470539 (10^?5 M). The MCR₅-related action in the ARPE-19 cells was accompanied by the increased expression of two coupled factors, cytochrome p4502E1 (CYP2E1) and nuclear factor kappa b (Nf-κB). These are both involved in high glucose signalling, in ROS generation and, interestingly, were reduced by the MCR₅ agonist in the ARPE-19 cells. Altogether, these data suggest that MCR₅ is a modulator of the responses stimulated by glucose in ARPE-19 cells, which might possibly be translated into a modulation of the retinal pigment epithelium response to diabetes in vivo.     

Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding

Neuropilins (Nrps) are co-receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.     

Vasculogenesis, angiogenesis, and arteriogenesis: mechanisms of blood vessel formation and remodeling

In this review, the concept of oxygen homeostasis will be presented as an organizing principle for discussion of the phylogeny, ontogeny, physiology, and pathology of blood vessel formation and remodeling, with a focus on molecular mechanisms and potential therapeutic applications.     

Phosphatidylcholine-specific phospholipase C and ROS were involved in chicken blastodisc differentiation to vascular endothelial cells

To find the key factors that were involved in the survival and vascular endothelial differentiation of chick blatodisc induced by fibroblast growth factor 2 (FGF-2), we built a chick vasculogenesis model in vitro. Subsequently, the activities of phosphatidylcholine-specific phospholipase C (PC-PLC), including Ca(2+)-dependent and -independent PC-PLC, and the level of reactive oxygen species (ROS) were evaluated during the endothelial differentiation of chick blastodisc. The results showed that Ca(2+)-indepentent PC-PLC underwent a remarkable increase in 24 h (P < 0.01), then it decreased gradually with the cell differentiation, while the Ca(2+)-depentent PC-PLC was nearly not changed in the whole process. At the same time, ROS level dramatically decreased during the cell differentiation. To understand the role of PC-PLC and how it performs its function in the vascular endothelial differentiation induced by FGF-2, we suppressed PC-PLC activity by its specific inhibitor D609 (tricyclodecan-9-yl potassium xanthate) at 24 h during the cell differentiation. As a result, the cell differentiation could not progress and the intracellular level of ROS was elevated. The data suggested that PC-PLC and ROS were involved in chicken blastodisc differentiation to vascular endothelial cells. PC-PLC was an important factor in the blastodisc cell survival and differentiation, and it might perform its function associated with ROS.     

Regulation of vascular endothelial junction stability and remodeling through Rap1-Rasip1 signaling

The ability of blood vessels to sense and respond to stimuli such as fluid flow, shear stress, and trafficking of immune cells is critical to the proper function of the vascular system. Endothelial cells constantly remodel their cell–cell junctions and the underlying cytoskeletal network in response to these exogenous signals. This remodeling, which depends on regulation of the linkage between actin and integral junction proteins, is controlled by a complex signaling network consisting of small G proteins and their various downstream effectors. In this commentary, we summarize recent developments in understanding the small G protein RAP1 and its effector RASIP1 as critical mediators of endothelial junction stabilization, and the relationship between RAP1 effectors and modulation of different subsets of endothelial junctions.     

Hypoxia-Induced Endothelial Progenitor Cell Function Is Blunted in Angiotensinogen Knockout Mice

Angiotensinogen (AGT), the precursor of angiotensin I, is known to be involved in tumor angiogenesis and associated with the pathogenesis of coronary atherosclerosis. This study was undertaken to determine the role played by AGT in endothelial progenitor cells (EPCs) in tumor progression and metastasis. It was found that the number of EPC colonies formed by AGT heterozygous knockout (AGT^+/−) cells was less than that formed by wild-type (WT) cells, and that the migration and tube formation abilities of AGT^+/− EPCs were significantly lower than those of WT EPCs. In addition, the gene expressions of vascular endothelial growth factor (VEGF), Flk1, angiopoietin (Ang)-1, Ang-2, Tie-2, stromal derived factor (SDF)-1, C-X-C chemokine receptor type 4 (CXCR4), and of endothelial nitric oxide synthase (eNOS) were suppressed in AGT^+/− EPCs. Furthermore, the expressions of hypoxia-inducible factor (HIF)-1α and -2α were downregulated in AGT^+/− early EPCs under hypoxic conditions, suggesting a blunting of response to hypoxia. Moreover, the activation of Akt/eNOS signaling pathways induced by VEGF, epithelial growth factor (EGF), or SDF-1α were suppressed in AGT^+/− EPCs. In AGT^+/− mice, the incorporation of EPCs into the tumor vasculature was significantly reduced, and lung tumor growth and melanoma metastasis were attenuated. In conclusion, AGT is required for hypoxia-induced vasculogenesis.     

An affordable method to obtain cultured endothelial cells from peripheral blood

The culture of endothelial progenitor cells (EPC) provides an excellent tool to research on EPC biology and vascular regeneration and vasculogenesis. The use of different protocols to obtain EPC cultures makes it difficult to obtain comparable results in different groups. This work offers a systematic comparison of the main variables of most commonly used protocols for EPC isolation, culture and functional evaluation. Peripheral blood samples from healthy individuals were recovered and mononuclear cells were cultured. Different recovery and culture conditions were tested: blood volume, blood anticoagulant, coating matrix and percentage of foetal bovine serum (FBS) in culture media. The success of culture procedure, first colonies of endothelial cells appearance time, correlation with number of circulating EPC (cEPC) and functional comparison with human umbilical vein endothelial cells (HUVEC) were studied. The use of heparin, a minimum blood volume of 30 ml, fibronectin as a coating matrix and endothelial growing media‐2 supplemented with 20% FBS increased the success of obtaining EPC cultures up to 80% of the processed samples while reducing EPC colony appearance mean time to a minimum of 13 days. Blood samples exhibiting higher cEPC numbers resulted in reduced EPC colony appearance mean time. Cells isolated by using this combination were endothelial cell‐like EPCs morphological and phenotypically. Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC. Thus, above‐mentioned conditions allow the isolation and culture of EPC with smaller blood volumes and shorter times than currently used protocols.