Antagonism and synergy between extracellular BMP modulators Tsg and BMPER balance blood vessel formation.

Growth and regeneration of blood vessels are crucial processes during embryonic development and in adult disease. Members of the bone morphogenetic protein (BMP) family are growth factors known to play a key role in vascular development. The BMP pathway is controlled by extracellular BMP modulators such as BMP endothelial cell precursor derived regulator (BMPER), which we reported previously acts proangiogenically on endothelial cells in a concentration-dependent manner. Here, we explore the function of other BMP modulators, especially Tsg, on endothelial cell behaviour and compare them to BMPER. In Matrigel assays, BMP modulators chordin and noggin had no stimulatory effect; however, gremlin and Tsg enhanced human umbilical vein endothelial cell (HUVEC) sprouting. As the activation dynamics of Tsg were similar to those of BMPER, we further investigated the proangiogenic effect of Tsg on endothelial cells. Tsg enhanced endothelial cell ingrowth in the mouse Matrigel plug assay as well as HUVEC sprouting, migration and proliferation in vitro, dependent on Akt, Erk and Smad signalling pathway activation in a concentration-dependent manner. Surprisingly, silencing of Tsg also increased HUVEC sprouting, migration and proliferation, which is again associated with Akt, Erk and Smad signalling pathway activation. Furthermore, we reveal that Tsg and BMPER interfere with each other to enhance proangiogenic events. However, in vivo the presence of Tsg as well as of BMPER is mandatory for regular development of the zebrafish vasculature. Taken together, our results suggest that BMPER and Tsg maintain a fine-tuned equilibrium that controls BMP pathway activity and is necessary for vascular cell homeostasis.

Inhibition of BMP activity protects epithelial barrier function in lung injury.

Epithelial injury is a central finding in pulmonary disease and is accompanied by disruption of epithelial barrier function, leading to pulmonary oedema and inflammation. Injured epithelial cells lose their properties and gain mesenchymal characteristics, a phenotypic switch that contributes to lung remodelling after injury. Here we studied bone morphogenetic protein (BMP) signalling and, in particular, the role of BMP2 and the BMP modulator BMPER in injured lung epithelium. Increased BMP activity, reflected by up-regulation of the Smad1/5-Id1 axis, is detected after injury of lung epithelium in vitro and in vivo. Two members of the BMP family, BMP2 and BMPER, have opposing effects. BMP2 is up-regulated after epithelial injury and causes epithelial dysfunction and hyperpermeability, mediated by the Smad1/5-Id1-dependent down-regulation of E-cadherin. In contrast, BMPER expression is decreased following injury, which in turn impairs epithelial integrity, characterized by reduction of E-cadherin and epithelial leakage in vitro and in vivo. High levels of BMPER antagonized BMP2-Smad5-Id1 signalling and prevented BMP2-mediated decrease of E-cadherin and hyperpermeability, suggesting that BMPER restores epithelial homeostasis. Supporting this notion, pharmacological inhibition of BMP signalling by LDN193189 prevented reduction of E-cadherin and disruption of epithelial barrier function. Inhibition of excessive BMP activation could be a new approach to restore epithelial integrity and prevent disruption of epithelial barrier function after lung injury.

BMP activity controlled by BMPER regulates the proinflammatory phenotype of endothelium.

The endothelium plays a pivotal role in vascular inflammation. Here we study bone morphogenetic protein (BMP) signaling in endothelial inflammation and in particular the role of BMPER, an extracellular BMP modulator that is important in vascular development and angiogenesis. Using the BMP antagonist dorsomorphin or BMP2 as an agonist we show that BMP signaling is essential for the inflammatory response of vascular endothelial cells as demonstrated by intravital microscopy. We found that BMPER is decreased in inflammation similar to vascular protective genes like KLF2 and eNOS. Using in vitro and in vivo models we show that BMPER is down-regulated through the TNFα-NFκB-KLF2 signaling pathway. Functionally, lack of BMPER induced by siRNA or in BMPER(+/-) mice confers a proinflammatory endothelial phenotype with reduced eNOS levels and enhanced expression of adhesion molecules leading to increased leukocyte adhesion and extravasation in ex vivo and in vivo experiments. Vice versa, addition of BMPER exerts endothelium protective functions and antagonizes TNFα induced inflammation. Mechanistically, we demonstrate that these effects of BMPER are dependent on BMP signaling because of enhanced NFκB activity. In conclusion, the BMP modulator BMPER is a new protective regulator of vascular inflammation that modulates leukocyte adhesion and migration in vitro and in vivo.

MicroRNA-100 regulates neovascularization by suppression of mammalian target of rapamycin in endothelial and vascular smooth muscle cells.

BACKGROUND: The adaptive growth of blood vessels is an important protective mechanism in cardiovascular disease. However, the underlying regulatory mechanisms of this process are only partly understood. Recently, small endogenous RNAs (microRNAs [miRNAs]) were found to play an important role in embryonic and postnatal vascular development. Here, we used miRNA transcriptome analysis after induction of hind-limb ischemia in mice to screen for miRNAs involved in adaptive blood vessel growth following arterial occlusion. METHODS AND RESULTS: Using miRNA arrays, we explored the miRNA expression profile during adaptive neovascularization. We describe specific changes in miRNA expression patterns and show that miRNA-100 is significantly downregulated after induction of hind-limb ischemia in mice. Our data demonstrate that miR-100 modulates proliferation, tube formation, and sprouting activity of endothelial cells and migration of vascular smooth muscle cells and functions as an endogenous repressor of the serine/threonine protein kinase mammalian target of rapamycin (mTOR). Whereas miR-100 inhibition increased mTOR levels in endothelial cells, overexpression of miR-100 reduced mTOR expression and consequently attenuated cellular proliferation. Supporting this notion, overexpression of an mTOR construct lacking the miRNA binding site rescued the inhibitory effect of miR-100 on cell proliferation. Accordingly, miR-100 inhibition by specific antagomirs in vivo stimulated angiogenesis and resulted in functional improvement of perfusion after femoral artery occlusion in mice. In contrast, treatment with the mTOR inhibitor rapamycin had the opposite effect. CONCLUSIONS: Our data demonstrate that miR-100 has an antiangiogenic function and represses mTOR signaling in endothelial and vascular smooth muscle cells. Inhibition of miR-100 could be a novel approach for the modulation of blood vessel growth and other mTOR-dependent processes.

BMPER is upregulated by statins and modulates endothelial inflammation by intercellular adhesion molecule-1.

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 Yersinia 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. CONCLUSIONS: 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.

BMPER is an endothelial cell regulator and controls bone morphogenetic protein-4-dependent angiogenesis.

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.

Life is a pattern: vascular assembly within the embryo.

The formation of the vascular system is one of the earliest and most important events during organogenesis in the developing embryo because the growing organism needs a transportation system to supply oxygen and nutrients and to remove waste products. Two distinct processes termed vasculogenesis and angiogenesis lead to a complex vasculature covering the entire body. Several cellular mechanisms including migration, proliferation, differentiation and maturation are involved in generating this hierarchical vascular tree. To achieve this aim, a multitude of signaling pathways need to be activated and coordinated in spatio-temporal patterns. Understanding embryonic molecular mechanism in angiogenesis further provides insight for therapeutic approaches in pathological conditions like cancer or ischemic diseases in the adult. In this review, we describe the current understanding of major signaling pathways that are necessary and active during vascular development.

Krüppel-like factor 15 regulates BMPER in endothelial cells.

AIMS: Bone morphogenetic proteins (BMPs) are involved in embryonic and adult blood vessel formation in health and disease. Previous studies have shown that BMP endothelial cell precursor-derived regulator (BMPER) plays an important role in endothelial cell function and blood vessel formation. BMPER is a key regulator of BMP4 activity and a prerequisite for BMP pathway activation by BMP4 in endothelial cells. Here, we characterize the BMPER promoter and elucidate mechanisms of BMPER regulation. METHODS AND RESULTS: To investigate transcriptional mechanisms of BMPER expression, the murine BMPER promoter was cloned and characterized. A series of 5' deletions of the BMPER promoter revealed that the proximal promoter contains activating cis-elements. By overexpression or siRNA-based knockdown, we demonstrate that BMPER expression is activated by Krüppel-like factor (KLF) 15. As determined by gelshift analyses, KLF15 binds directly to a predicted KLF-binding element at -284 bp within the BMPER promoter. Co-expression experiments show that Sp1 acts as an antagonist for KLF15-induced promoter activation. Endothelin-1 was identified as a potent inhibitor of KLF15 and BMPER expression in endothelial cells, suggesting that KLF15 is a transducer of endothelin-1 activity on BMPER expression. The selective ET(B) endothelin receptor antagonist BQ788 abolished the downregulation of BMPER expression by endothelin-1. CONCLUSION: Mechanistically, we found that KLF15 is a strong and direct activator of the BMPER expression. BMPER is downregulated by endothelin-1 in a dose-dependent fashion and in parallel to KLF15. As KLF15 deficiency is accompanied by a vascular phenotype and BMPER is necessary for proper blood vessel formation, we suggest a chain of events in which the effects of endothelin-1 on BMPER are mediated by KLF15.

HoxB5 induces endothelial sprouting in vitro and modifies intussusceptive angiogenesis in vivo involving angiopoietin-2.

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSION: HoxB5 has an activating effect on Ang2 that is essential for endothelial cell sprouting and coordinated vascular growth.

Human CMV immediate-early enhancer: a useful tool to enhance cell-type-specific expression from lentiviral vectors.

BACKGROUND: Lentiviral vectors are attractive delivery tools for gene therapy, especially in terminally differentiated target cells. While restriction of gene expression to specific cell populations is of particular importance, highly efficient cell-type-specific gene expression after viral gene transfer so far has been hampered by low levels of transgene expression. METHODS: Addressing this problem, we have integrated the human cytomegalovirus (CMV) immediate-early enhancer into an 'advanced' generation lentiviral vector. Expression cassettes with the reporter gene green fluorescent protein (GFP), combined with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) under control of a ubiquitous phosphoglycerate kinase (mouse PGK), cardiomyocyte- (human atrial natriuretic factor (ANF), human ventricular myosin light chain (MLC2v)), or type II alveolar epithelial cell (AT-2)-specific human surfactant protein C (SP-C) promoter, were introduced. As insertion of an enhancing element can interfere with the promoter's specificity, expression levels conferred by our enhancer/promoter constructs were evaluated in target and non-target cells. RESULTS: Transduction of target cells with human CMV enhancer containing lentiviral vectors resulted in a multiple-log increase in GFP expression compared to corresponding vectors lacking the human CMV enhancer. In the case of the ANF, the MLC2v, and the SP-C promoters, tissue-specific reporter gene expression in cardiomyocytes and in lung AT-2 cells was maintained, as expression in non-target cells increased only up to 7-fold. CONCLUSIONS: The results of this study indicate that lentiviral vectors with the human CMV enhancer conferring efficient cell-type-specific gene expression may be useful tools for gene therapy purposes or cell tracing, e.g. to analyze stem cell differentiation in transplantation and co-culture settings.

ERK signaling is a central regulator for BMP-4 dependent capillary sprouting.

OBJECTIVE: Bone Morphogenetic Protein-4 (BMP-4) and Extracellular-Signal Regulated Kinases (ERK) play crucial roles in vascular diseases. Here, we demonstrate that BMP-4 not only signals through the classical Smad cascade but also activates ERK phosphorylation as an alternative pathway in human umbilical vein endothelial cells (HUVEC) and that Smad and ERK pathways communicate through signal crosstalk. METHODS: HUVECs were treated with BMP-4 and/or MEK inhibitors. Smad 6 and constitutively active (ca) MEK1 were overexpressed. Loss of function of Smad 4 and Smad 6 was achieved by specific siRNA transfection. Cell lysates were analyzed by western blotting for Smad and ERK phosphorylation. HUVEC spheroids were generated for angiogenesis quantification. RESULTS: Treatment with BMP-4 results in a dose- and time-dependent activation of the MEK-ERK 1/2 pathway in addition to activation of the Smad pathway and is blocked by MEK inhibitors. Quantitative in-gel angiogenesis assays in the presence or absence of MEK inhibitors demonstrate that ERK signals are necessary for BMP-4 induced capillary sprouting. Furthermore sprouting is not blocked by inhibition of the Smad signaling pathway. Overexpression of the inhibitory Smad 6 inhibits ERK phosphorylation and ERK-induced capillary sprouting, whereas loss of function of Smad 4 has no effect. CONCLUSIONS: We demonstrate that ERK1/2 functions as an alternative pathway in BMP-4 signaling in HUVECs. Capillary sprouting induced by BMP-4 is dependent on ERK phosphorylation. ERK is essential for efficient transduction of BMP signals and serves as a positive feedback mechanism. On the other hand, stimulation of Smad 6 inhibits ERK activation and thus results in a negative feedback loop to fine-tune BMP signaling in HUVECs.