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In early blood vessel development, trafficking programs, such as those using Rab GTPases, are tasked with delivering vesicular cargo with high spatiotemporal accuracy. However, the function of many Rab trafficking proteins remain ill-defined in endothelial tissue; therefore, their relevance to blood vessel development is unknown. Rab35 has been shown to play an enigmatic role in cellular behaviors which differs greatly between tissue-type and organism. Importantly, Rab35 has never been characterized for its potential contribution in sprouting angiogenesis; thus, our goal was to map Rab35's primary function in angiogenesis. Our results demonstrate that Rab35 is critical for sprout formation; in its absence, apicobasal polarity is entirely lost in vitro and in vivo. To determine mechanism, we systematically explored established Rab35 effectors and show that none are operative in endothelial cells. However, we find that Rab35 partners with DENNd1c, an evolutionarily divergent guanine exchange factor, to localize to actin. Here, Rab35 regulates actin polymerization through limiting Rac1 and RhoA activity, which is required to set up proper apicobasal polarity during sprout formation. Our findings establish that Rab35 is a potent brake of actin remodeling during blood vessel development., (© 2022. The Author(s).)

Functional blood vessel growth depends on generation of distinct but coordinated responses from endothelial cells. Bone morphogenetic proteins (BMP), part of the TGFβ superfamily, bind receptors to induce phosphorylation and nuclear translocation of SMAD transcription factors (R-SMAD1/5/8) and regulate vessel growth. However, SMAD1/5/8 signalling results in both pro- and anti-angiogenic outputs, highlighting a poor understanding of the complexities of BMP signalling in the vasculature. Here we show that BMP6 and BMP2 ligands are pro-angiogenic in vitro and in vivo, and that lateral vessel branching requires threshold levels of R-SMAD phosphorylation. Endothelial cell responsiveness to these pro-angiogenic BMP ligands is regulated by Notch status and Notch sets responsiveness by regulating a cell-intrinsic BMP inhibitor, SMAD6, which affects BMP responses upstream of target gene expression. Thus, we reveal a paradigm for Notch-dependent regulation of angiogenesis: Notch regulates SMAD6 expression to affect BMP responsiveness of endothelial cells and new vessel branch formation.

Objective: To investigate whether adiposity influences endothelial progenitor cell (EPC) number and colony-forming capacity., Design: Cross-sectional study of normal weight, overweight and obese adult humans., Participants: Sixty-seven sedentary adults (aged 45-65 years): 25 normal weight (body mass index (BMI) or=30 kg/m(2); 18 males/6 females). All participants were non-smokers and free of overt cardiometabolic disease., Measurements: Peripheral blood samples were collected and circulating EPC number was assessed by flow cytometry. Putative EPCs were defined as CD45(-)/CD34(+)/VEGFR-2(+)/CD133(+) or CD45(-)/CD34(+) cells. EPC colony-forming capacity was measured in vitro using a colony-forming unit (CFU) assay., Results: Number of circulating putative EPCs (either CD45(-)/CD34(+)/VEGFR-2(+)/CD133(+) or CD45(-)/CD34(+) cells) was lower (P<0.05) in obese (0.0007+/-0.0001%; 0.050+/-0.006%) compared with overweight (0.0016+/-0.0004%; 0.089+/-0.019%) and normal weight (0.0015+/-0.0003%; 0.082+/-0.008%) adults. There were no differences in EPC number between the overweight and normal weight groups. EPC colony formation was significantly less in the obese (6+/-1) and overweight (4+/-1) compared with normal weight (9+/-2) adults., Conclusion: These results indicate that: (1) the number of circulating EPCs is lower in obese compared with overweight and normal weight adults; and (2) EPC colony-forming capacity is blunted in overweight and obese adults compared with normal weight adults. Impairments in EPC number and function may contribute to adiposity-related cardiovascular risk.