VE-cadherin facilitates BMP-induced endothelial cell permeability and signaling.

Several vascular disorders, such as aberrant angiogenesis, atherosclerosis and pulmonary hypertension, have been linked to dysfunctional BMP signaling. Vascular hyperpermeability via distortion of endothelial cell adherens junctions is a common feature of these diseases, but the role of BMPs in this process has not been investigated. BMP signaling is initiated by binding of ligand to, and activation of, BMP type I (BMPRI) and type II (BMPRII) receptors. Internalization of VE-cadherin as well as c-Src kinase-dependent phosphorylation have been implicated in the loosening of cell-cell contacts, thereby modulating vascular permeability. Here we demonstrate that BMP6 induces hyperpermeabilization of human endothelial cells by inducing internalization and c-Src-dependent phosphorylation of VE-cadherin. Furthermore, we show BMP-dependent physical interaction of VE-cadherin with the BMP receptor ALK2 (BMPRI) and BMPRII, resulting in stabilization of the BMP receptor complex and, thereby, the support of BMP6-Smad signaling. Our results provide first insights into the molecular mechanism of BMP-induced vascular permeability, a hallmark of various vascular diseases, and provide the basis for further investigations of BMPs as regulators of vascular integrity, both under physiological and pathophysiological conditions.

Role of bone morphogenetic proteins in sprouting angiogenesis: differential BMP receptor-dependent signaling pathways balance stalk vs. tip cell competence.

Before the onset of sprouting angiogenesis, the endothelium is prepatterned for the positioning of tip and stalk cells. Both cell identities are not static, as endothelial cells (ECs) constantly compete for the tip cell position in a dynamic fashion. Here, we show that both bone morphogenetic protein 2 (BMP2) and BMP6 are proangiogenic in vitro and ex vivo and that the BMP type I receptors, activin receptor-like kinase 3 (ALK3) and ALK2, play crucial and distinct roles in this process. BMP2 activates the expression of tip cell-associated genes, such as delta-like ligand 4 (DLL4) and kinase insert domain receptor (KDR), and p38-heat shock protein 27 (HSP27)-dependent cell migration, thereby generating tip cell competence. Whereas BMP6 also triggers collective cell migration via the p38-HSP27 signaling axis, BMP6 induces in addition SMAD1/5 signaling, thereby promoting the expression of stalk cell-associated genes, such as hairy and enhancer of split 1 (HES1) and fms-like tyrosine kinase 1 (FLT1). Specifically, ALK3 is required for sprouting from HUVEC spheroids, whereas ALK2 represses sprout formation. We demonstrate that expression levels and respective complex formation of BMP type I receptors in ECs determine stalk vs. tip cell identity, thus contributing to endothelial plasticity during sprouting angiogenesis. As antiangiogenic monotherapies that target the VEGF or ALK1 pathways have not fulfilled efficacy objectives in clinical trials, the selective targeting of the ALK2/3 pathways may be an attractive new approach.-Benn, A., Hiepen, C., Osterland, M., Schütte, C., Zwijsen, A., Knaus, P. Role of bone morphogenetic proteins in sprouting angiogenesis: differential BMP receptor-dependent signaling pathways balance stalk vs. tip cell competence.

BMP2-induced chemotaxis requires PI3K p55γ/p110α-dependent phosphatidylinositol (3,4,5)-triphosphate production and LL5ß recruitment at the cytocortex.

BACKGROUND: BMP-induced chemotaxis of mesenchymal progenitors is fundamental for vertebrate development, disease and tissue repair. BMP2 induces Smad and non-Smad signalling. Whereas signal transduction via Smads lead to transcriptional responses, non-Smad signalling induces both, transcriptional and immediate/early non-transcriptional responses. However, the molecular mechanisms by which BMP2 facilitates planar cell polarity, cortical actin rearrangements, lamellipodia formation and chemotaxis of mesenchymal progenitors are poorly understood. Our aim was to uncover the molecular mechanism by which BMP2 facilitates chemotaxis via the BMP2-dependent activation of PI3K and spatiotemporal control of PIP3 production important for actin rearrangements at the mesenchymal cell cytocortex. RESULTS: We unveiled the molecular mechanism by which BMP2 induces non-Smad signalling by PI3K and the role of the second messenger PIP3 in BMP2-induced planar cell polarity, cortical actin reorganisation and lamellipodia formation. By using protein interaction studies, we identified the class Ia PI3K regulatory subunit p55γ to act as a specific and non-redundant binding partner for BMP receptor type II (BMPRII) in concert with the catalytic subunit p110α. We mapped the PI3K interaction to a region within the BMPRII kinase. Either BMP2 stimulation or increasing amounts of BMPRI facilitated p55γ association with BMPRII, but BMPRII kinase activity was not required for the interaction. We visualised BMP2-dependent PIP3 production via PI3K p55γ/p110α and were able to localise PIP3 to the leading edge of intact cells during the process of BMP2-induced planar cell polarity and actin dependent lamellipodia formation. Using mass spectrometry, we found the highly PIP3-sensitive PH-domain protein LL5ß to act as a novel BMP2 effector in orchestrating cortical actin rearrangements. By use of live cell imaging we found that knock-down of p55γ or LL5ß or pharmacological inhibition of PI3K impaired BMP2-induced migratory responses. CONCLUSIONS: Our results provide evidence for an important contribution of the BMP2-PI3K (p55γ/p110α)- PIP3-LL5ß signalling axis in mesenchymal progenitor cell chemotaxis. We demonstrate molecular insights into BMP2-induced PI3K signalling on the level of actin reorganisation at the leading edge cytocortex. These findings are important to better understand BMP2-induced cytoskeletal reorganisation and chemotaxis of mesenchymal progenitors in different physiological or pathophysiological contexts.

Endothelial SMAD1/5 signaling couples angiogenesis to osteogenesis in juvenile bone.

Skeletal development depends on coordinated angiogenesis and osteogenesis. Bone morphogenetic proteins direct bone formation in part by activating SMAD1/5 signaling in osteoblasts. However, the role of SMAD1/5 in skeletal endothelium is unknown. Here, we found that endothelial cell-conditional SMAD1/5 depletion in juvenile mice caused metaphyseal and diaphyseal hypervascularity, resulting in altered trabecular and cortical bone formation. SMAD1/5 depletion induced excessive sprouting and disrupting the morphology of the metaphyseal vessels, with impaired anastomotic loop formation at the chondro-osseous junction. Endothelial SMAD1/5 depletion impaired growth plate resorption and, upon long-term depletion, abrogated osteoprogenitor recruitment to the primary spongiosa. Finally, in the diaphysis, endothelial SMAD1/5 activity was necessary to maintain the sinusoidal phenotype, with SMAD1/5 depletion inducing formation of large vascular loops and elevated vascular permeability. Together, endothelial SMAD1/5 activity sustains skeletal vascular morphogenesis and function and coordinates growth plate remodeling and osteoprogenitor recruitment dynamics in juvenile mouse bone.

Endothelial SMAD1/5 signaling couples angiogenesis to osteogenesis during long bone growth.

Skeletal development depends on coordinated angiogenesis and osteogenesis. Bone morphogenetic proteins direct bone development by activating SMAD1/5 signaling in osteoblasts. However, the role of SMAD1/5 in skeletal endothelium is unknown. Here, we found that endothelial cell-conditional SMAD1/5 depletion in juvenile mice caused metaphyseal and diaphyseal hypervascularity, resulting in altered cancellous and cortical bone formation. SMAD1/5 depletion induced excessive sprouting, disrupting the columnar structure of the metaphyseal vessels and impaired anastomotic loop morphogenesis at the chondro-osseous junction. Endothelial SMAD1/5 depletion impaired growth plate resorption and, upon long term depletion, abrogated osteoprogenitor recruitment to the primary spongiosa. Finally, in the diaphysis, endothelial SMAD1/5 activity was necessary to maintain the sinusoidal phenotype, with SMAD1/5 depletion inducing formation of large vascular loops, featuring elevated endomucin expression, ectopic tip cell formation, and hyperpermeability. Together, endothelial SMAD1/5 activity sustains skeletal vascular morphogenesis and function and coordinates growth plate remodeling and osteoprogenitor recruitment dynamics during bone growth.

BMP-SMAD1/5 Signaling Regulates Retinal Vascular Development.

Vascular development is an orchestrated process of vessel formation from pre-existing vessels via sprouting and intussusceptive angiogenesis as well as vascular remodeling to generate the mature vasculature. Bone morphogenetic protein (BMP) signaling via intracellular SMAD1 and SMAD5 effectors regulates sprouting angiogenesis in the early mouse embryo, but its role in other processes of vascular development and in other vascular beds remains incompletely understood. Here, we investigate the function of SMAD1/5 during early postnatal retinal vascular development using inducible, endothelium-specific deletion of Smad1 and Smad5. We observe the formation of arterial-venous malformations in areas with high blood flow, and fewer and less functional tip cells at the angiogenic front. The vascular plexus region is remarkably hyperdense and this is associated with reduced vessel regression and aberrant vascular loop formation. Taken together, our results highlight important functions of SMAD1/5 during vessel formation and remodeling in the early postnatal retina.

Sex-specific metabolic and functional differences in human umbilical vein endothelial cells from twin pairs.

BACKGROUND AND AIMS: Gonadal hormones are mainly thought to account for sex and gender differences in the incidence, clinical manifestation and therapy of many cardiovascular diseases. However, intrinsic sex differences at the cellular level are mostly overlooked. Here, we assessed sex-specific metabolic and functional differences between male and female human umbilical vein endothelial cells (HUVECs). METHODS: Cellular metabolism was investigated by bioenergetic studies (Seahorse Analyser) and a metabolomic approach. Protein levels were determined by Western blots and proteome analysis. Vascular endothelial growth factor (VEGF)-stimulated cellular migration was assessed by gap closure. HUVECs from dizygotic twin pairs were used for most experiments. RESULTS: No sex differences were observed in untreated cells. However, sexual dimorphisms appeared after stressing the cells by serum starvation and treatment with VEGF. Under both conditions, female cells had higher intracellular ATP and metabolite levels. A significant decline in ATP levels was observed in male cells after serum starvation. After VEGF, the ratio of glycolysis/mitochondrial respiration was higher in female cells and migration was more pronounced. CONCLUSIONS: These results point to an increased stress tolerance of female cells. We therefore propose that female cells have an energetic advantage over male cells under conditions of diminished nutrient supply. A more favourable energy balance of female HUVECs after serum starvation and VEGF could potentially explain their stronger migratory capacity.

Does cellular sex matter? Dimorphic transcriptional differences between female and male endothelial cells.

OBJECTIVE: Significant sex differences exist in cardiovascular diseases. Although an impact of gonadal hormones is presumed, it is largely unknown whether sexually dimorphic gene expression also plays a role and whether cells themselves show intrinsic sex differences. METHODS: We performed whole genome expression analyses in human umbilical vein endothelial cells (HUVEC) from 20 male and 20 female donors and compared levels of gene transcription between the sexes. To further assess whether there is a sex-specific response to stress, we subjected male and female HUVEC to shear for 24 h and analysed changes in gene expression. RESULTS: Genes indicative for greater immune responsiveness were stronger expressed in female compared to male HUVEC. There was a significant enrichment of 77 immune-related genes in female HUVEC. These increased transcriptional levels in female cells were verified for 20 genes by real-time RT-PCR. 6.7% of all mRNAs were regulated by shear stress. Female HUVEC showed a more pronounced transcriptional response to shear than did their male counterparts. In addition to quantitative differences, a number of genes were regulated in the opposite direction between the two sexes by shear stress. Functionally, female HUVEC showed a higher cell viability after serum starvation and an increased tube formation capacity compared to male cells. CONCLUSION: These findings underscore the importance for differentiation between male and female cells in cell culture experiments. This may apply not only to endothelial cells but might be generalized to other cell types as well. The observed sexual dimorphisms in gene expression in endothelial cells may contribute to sex differences between males and females in endothelial function.