BMP9 signaling has been implicated in hereditary hemorrhagic telangiectasia (HHT) and vascular remodeling, acting via the HHT target genes, endoglin and ALK1. This study sought to identify endothelial BMP9-regulated proteins that could affect the HHT phenotype. Gene ontology analysis of cDNA microarray data obtained after BMP9 treatment of primary human endothelial cells indicated regulation of chemokine, adhesion, and inflammation pathways. These responses included the up-regulation of the chemokine CXCL12/SDF1 and down-regulation of its receptor CXCR4. Quantitative mass spectrometry identified additional secreted proteins, including the chemokine CXCL10/IP10. RNA knockdown of endoglin and ALK1 impaired SDF1/CXCR4 regulation by BMP9. Because of the association of SDF1 with ischemia, we analyzed its expression under hypoxia in response to BMP9 in vitro, and during the response to hindlimb ischemia, in endoglin-deficient mice. BMP9 and hypoxia were additive inducers of SDF1 expression. Moreover, the data suggest that endoglin deficiency impaired SDF1 expression in endothelial cells in vivo. Our data implicate BMP9 in regulation of the SDF1/CXCR4 chemokine axis in endothelial cells and point to a role for BMP9 signaling via endoglin in a switch from an SDF1-responsive autocrine phenotype to an SDF1 nonresponsive paracrine state that represses endothelial cell migration and may promote vessel maturation. IntroductionEndoglin directly interacts with the TGF-␤ receptors, 1 including ALK1, 2 and modulates TGF-␤ and bone morphogenetic protein (BMP) signaling. 3 Mutations in either endoglin 4 or ALK1 5 increase the risk of hereditary hemorrhagic telangiectasia (HHT1 and HHT2, respectively), whose symptoms include arteriovenous malformation, tissue ischemia, and reperfusion defects. 6 The ALK1-endoglin signaling complex in endothelial cells is activated by BMP9, 7 a circulating cytokine produced in the liver reticuloendothelium 8 and endothelial cells, including those lining the mouse aorta. 9 BMP9 interacts with endoglin and ALK1 to activate signaling pathways 7 that promote endothelial cell quiescence 10 and vessel maturation. 11 Several endothelial cell-derived factors, including BMP9, are known to regulate vessel maturation via paracrine recruitment of other cell types. 12 Moreover, our recent work using nonendothelial cells implicates endoglin in the regulation of tumor neoangiogenesis via the secreted insulin-like growth factor binding protein 4. 13 Therefore, elucidation of the role of BMP9 signaling, specifically in terms of its effects on the expression of endothelial cell-secreted factors, is needed to better understand the mechanisms by which BMP9 affects vessel maturation, integrity, the vascular response to injury, and how deficiency in either endoglin or ALK1 impacts vessel integrity and cause HHT.Stromal-derived factor 1 (SDF1, CXCL12) is a chemokine that signals via the chemokine receptor, CXCR4, to modulate hypoxiainduced angiogenesis. 14 SDF1 regulates both endothelial cellmediated paracrine signaling ...

Extracellular matrix (ECM) remodeling regulates angiogenesis. However, the precise mechanisms by which structural changes in ECM proteins contribute to angiogenesis are not fully understood. Integrins are molecules with the ability to detect compositional and structural changes within the ECM and integrate this information into a network of signaling circuits that coordinate context-dependent cell behavior. The role of integrin ␣v␤3 in angiogenesis is complex, as evidence exists for both positive and negative functions. The precise downstream signaling events initiated by ␣v␤3 may depend on the molecular characteristics of its ligands. Here, we identified an RGD-containing cryptic collagen epitope that is generated in vivo. Surprisingly, rather than inhibiting ␣v␤3 signaling, this collagen epitope promoted ␣v␤3 activation and stimulated angiogenesis and inflammation. An antibody directed to this RGDKGE epitope but not other RGD collagen epitopes inhibited angiogenesis and inflammation in vivo. The selective ability of this RGD epitope to promote angiogenesis and inflammation depends in part on its flanking KGE motif. Interestingly, a subset of macrophages may represent a physiologically relevant source of this collagen epitope. Here, we define an endothelial cell mechano-signaling pathway in which a cryptic collagen epitope activates ␣v␤3 leading to an Src and p38 MAPK-dependent cascade that leads to nuclear accumulation of Yes-associated protein (YAP) and stimulation of endothelial cell growth. Collectively, our findings not only provide evidence for a novel mechano-signaling pathway, but also define a possible therapeutic strategy to control ␣v␤3 signaling by targeting a proangiogenic and inflammatory ligand of ␣v␤3 rather than the receptor itself.

Background:We examined the impact of insulin-like growth factor binding protein-4 (IGFBP-4) on growth factor-induced angiogenesis in vivo. Results: IGFBP-4 inhibited IGF-1 and FGF-2, but not VEGF-induced angiogenesis, and this inhibition depended on p38 MAPK activity. Conclusion:The anti-angiogenic activity of IGFBP-4 depends in part on p38 MAPK. Significance: New insight is provided into how blood vessels respond to endogenous inhibitors during growth factor-stimulated angiogenesis.

Endoglin is a type III TGFβ auxiliary receptor that is upregulated in endothelial cells during angiogenesis and, when mutated in humans, results in the vascular disease hereditary hemorrhagic telangiectasia (HHT). Though endoglin has been implicated in cell adhesion, the underlying molecular mechanisms are still poorly understood. Here we show endoglin expression in endothelial cells regulates subcellular localization of zyxin in focal adhesions in response to BMP9. RNA knockdown of endoglin resulted in mislocalization of zyxin and altered formation of focal adhesions. The mechanotransduction role of focal adhesions and their ability to transmit regulatory signals through binding of the extracellular matrix are altered by endoglin deficiency. BMP/TGFβ transcription factors, SMADs, and zyxin have recently been implicated in a newly emerging signaling cascade, the Hippo pathway. The Hippo transcription coactivator, YAP1 (yes-associated protein 1), has been suggested to play a crucial role in mechanotransduction and cell-cell contact. Identification of BMP9-dependent nuclear localization of YAP1 in response to endoglin expression suggests a mechanism of crosstalk between the two pathways. Suppression of endoglin and YAP1 alters BMP9-dependent expression of YAP1 target genes CCN1 (cysteine-rich 61, CYR61) and CCN2 (connective tissue growth factor, CTGF) as well as the chemokine CCL2 (monocyte chemotactic protein 1, MCP-1). These results suggest a coordinate effect of endoglin deficiency on cell matrix remodeling and local inflammatory responses. Identification of a direct link between the Hippo pathway and endoglin may reveal novel mechanisms in the etiology of HHT.