Bone morphogenetic proteins (BMPs) are involved in embryonic and adult blood vessel formation in health and disease. BMPER (BMP endothelial cell precursor–derived regulator) is a differentially expressed protein in embryonic endothelial precursor cells. In earlier work, we found that BMPER interacts with BMPs and when overexpressed antagonizes their function in embryonic axis formation. In contrast, in a BMPER-deficient zebrafish model, BMPER behaves as a BMP agonist. Furthermore, lack of BMPER induces a vascular phenotype in zebrafish that is driven by disarray of the intersomitic vasculature. Here, we investigate the impact of BMPER on endothelial cell function and signaling and elucidate its role in BMP-4 function in gain- and loss-of-function models. As shown by Western blotting and immunocytochemistry, BMPER is an extracellular matrix protein expressed by endothelial cells in skin, heart, and lung. We show that BMPER is a downstream target of FoxO3a and consistently exerts activating effects on endothelial cell sprouting and migration in vitro and in vivo. Accordingly, when BMPER is depleted from endothelial cells, sprouting is impaired. In terms of BMPER related intracellular signaling, we show that BMPER is permissive and necessary for Smad 1/5 phosphorylation and induces Erk1/2 activation. Most interestingly, BMPER is necessary for BMP-4 to exert its activating role in endothelial function and to induce Smad 1/5 activation. Vice versa, BMP-4 is necessary for BMPER activity. Taken together, BMPER is a dose-dependent endothelial cell activator that plays a unique and pivotal role in fine-tuning BMP activity in angiogenesis.

The mechanisms of the progression of aortic valve stenosis are unknown. The involvement of mononuclear cells and of chronic systemic inflammation has been suggested by analysis of pathological specimens. We hypothesize that shear stress caused by the constricted aortic orifice contributes to systemic proinflammation by activation of circulating blood cells and thereby generation of microparticles. Using flow cytometry we analyzed 22 patients with severe aortic valve stenosis (AVS) and 18 patient controls for the generation of circulating microparticles from platelet-(PMPs: CD31⁺/CD61⁺ or CD62P⁺), leukocyte-(LMPs: CD11b⁺) and endothelial cell (EMPs: CD62E⁺) origin. Apart from the constricted valve orifice groups were similar. PMPs were increased in AVS patients and their number correlated with valvular shear stress. Monocytes were activated in AVS patients, an observation that was also reflected by increased numbers of LMPs and by the detection of PMP-monocyte conjugates. Furthermore, EMPs reflecting the activation of endothelial cells but also conferring systemic inflammatory activity were increased in AVS patients and correlated with the number of activated monocytes. In conclusion, we show that AVS is accompanied by increased levels of microparticles and that shear stress can induce the formation of microparticles. Based on our results and histologic findings of other investigators the speculation that shear stress related to aortic valve stenosis induces a vicious circle including the generation of PMPs, the subsequent activation of monocytes and LMPs and finally the activation of endothelial cells contributing to the progress of aortic valve stenosis appears to be justified.

Homeobox (Hox) proteins are transcriptional regulators in embryonic patterning, cell differentiation, proliferation, and migration in vertebrates and invertebrates. A growing body of evidence suggests that Hox proteins are involved in endothelial cell regulation. We have shown earlier that HoxB5 upregulates vascular endothelial growth factor receptor-2 and thereby contributes to enhanced endothelial precursor cell differentiation. Here we aim to elucidate the role of HoxB5 in angiogenesis. Endothelial cell sprouting was investigated in the human umbilical vein endothelial cell spheroid assay. We investigated in vivo angiogenesis in the chick (Gallus gallus) chorioallantoic membrane assay. Expression profiling of proangiogenic factors was done by quantitative PCR. The angiopoietin-2 (Ang2) promoter and deletion fragments thereof were cloned into the pGL3 reporter system for analysis of transcriptional activity. We observed that HoxB5 enhances endothelial cell sprouting and modulates the expression of adhesion molecules in vitro. Accordingly, we observed a modification of vascular growth by HoxB5 in vivo. The HoxB5 effect is reminiscent of the effects of angiopoietins. We demonstrate that Ang2 is upregulated upon HoxB5 overexpression and that the HoxB5 effect is abolished by the angiopoietin antagonist soluble Tie-2. HoxB5 has an activating effect on Ang2 that is essential for endothelial cell sprouting and coordinated vascular growth.

Objective— In addition to lowering cholesterol, statins exert pleiotropic effects on endothelial cells. Bone morphogenetic proteins (BMPs) have recently been implicated in vascular inflammation and disease. We set out to investigate the effect of statins on BMP endothelial cell precursor–derived regulator (BMPER), a novel member of the BMP pathway. Methods and Results— Mevastatin enhanced BMPER expression in cultured endothelial cells in a time- and concentration-dependent manner as determined by immunocytochemistry, RT-PCR, and Western blotting. Similar effects were observed in vitro and in vivo using simvastatin. Actinomycin D chase analysis and BMPER promoter reporter assays revealed that this is mostly a posttranscriptional event resulting in prolonged BMPER RNA half-life. We confirmed that the RhoA/Rho-associated coiled-coil containing protein kinase Rho kinase (Rock)/actin pathway is involved using the specific pathway activator cytotoxic necrotizing factor of Y ersinia pseudotuberculosis , which prevented upregulation of BMPER expression by mevastatin and pathway inhibitors (C3-toxin, RhoAN19 mutant, fasudil, and cytochalasin D) that enhanced BMPER expression. Increasing concentrations of BMPER exert antiinflammatory features in endothelial cells as reflected by intercellular adhesion molecule–1 downregulation. Accordingly, silencing of BMPER enhances intercellular adhesion molecule–1 expression. Furthermore, mevastatin reduced the expression of proinflammatory BMP4, a well-known direct interaction partner of BMPER. Conclusion— Mevastatin modulates the BMP pathway by enhancing BMPER via the RhoA/Rock/actin pathway, as well as by reducing BMP4 expression. BMP4 downregulation and BMPER upregulation contribute to the antiinflammatory pleiotropic effects of statins.