Different cardiovascular and pulmonary phenotypes for single- and double-knock-out mice deficient in BMP9 and BMP10.

AIMS: BMP9 and BMP10 mutations were recently identified in patients with pulmonary arterial hypertension, but their specific roles in the pathogenesis of the disease are still unclear. We aimed to study the roles of BMP9 and BMP10 in cardiovascular homeostasis and pulmonary hypertension using transgenic mouse models deficient in Bmp9 and/or Bmp10. METHODS AND RESULTS: Single- and double-knockout mice for Bmp9 (constitutive) and/or Bmp10 (tamoxifen inducible) were generated. Single-knock-out (KO) mice developed no obvious age-dependent phenotype when compared with their wild-type littermates. However, combined deficiency in Bmp9 and Bmp10 led to vascular defects resulting in a decrease in peripheral vascular resistance and blood pressure and the progressive development of high-output heart failure and pulmonary hemosiderosis. RNAseq analysis of the lungs of the double-KO mice revealed differential expression of genes involved in inflammation and vascular homeostasis. We next challenged these mice to chronic hypoxia. After 3 weeks of hypoxic exposure, Bmp10-cKO mice showed an enlarged heart. However, although genetic deletion of Bmp9 in the single- and double-KO mice attenuated the muscularization of pulmonary arterioles induced by chronic hypoxia, we observed no differences in Bmp10-cKO mice. Consistent with these results, endothelin-1 levels were significantly reduced in Bmp9 deficient mice but not Bmp10-cKO mice. Furthermore, the effects of BMP9 on vasoconstriction were inhibited by bosentan, an endothelin receptor antagonist, in a chick chorioallantoic membrane assay. CONCLUSIONS: Our data show redundant roles for BMP9 and BMP10 in cardiovascular homeostasis under normoxic conditions (only combined deletion of both Bmp9 and Bmp10 was associated with severe defects) but highlight specific roles under chronic hypoxic conditions. We obtained evidence that BMP9 contributes to chronic hypoxia-induced pulmonary vascular remodelling, whereas BMP10 plays a role in hypoxia-induced cardiac remodelling in mice.

Differential Consequences of Bmp9 Deletion on Sinusoidal Endothelial Cell Differentiation and Liver Fibrosis in 129/Ola and C57BL/6 Mice.

The aim of the present work was to address the role of BMP9 in different genetic backgrounds (C57BL/6, BALB/c, and 129/Ola) of mice deleted for Bmp9. We found that Bmp9 deletion led to premature mortality only in the 129/Ola strain. We have previously shown that Bmp9 deletion led to liver sinusoidal endothelial cells (LSEC) capillarization and liver fibrosis in the 129/Ola background. Here, we showed that this is not the case in the C57BL/6 background. Analysis of LSEC from Wild-type (WT) versus Bmp9-KO mice in the C57BL/6 background showed no difference in LSEC fenestration and in the expression of differentiation markers. Comparison of the mRNA expression of LSEC differentiation markers between WT C57BL/6 and 129/Ola mice showed a significant decrease in Stabilin2, Plvap, and CD209b, suggesting a more capillary-like phenotype in WT C57BL/6 LSECs. C57BL/6 mice also had lower BMP9 circulating concentrations and hepatic Vegfr2 mRNA levels, compared to the 129/Ola mice. Taken together, our observations support a role for BMP9 in liver endothelial cell fenestration and prevention of fibrosis that is dependent on genetic background. It also suggests that 129/Ola mice are a more suitable model than C57BL/6 for the study of liver fibrosis subsequent to LSEC capillarization.

Bone Morphogenetic Protein 9 Is a Paracrine Factor Controlling Liver Sinusoidal Endothelial Cell Fenestration and Protecting Against Hepatic Fibrosis.

Bone morphogenetic protein 9 (BMP9) is a circulating factor produced by hepatic stellate cells that plays a critical role in vascular quiescence through its endothelial receptor activin receptor-like kinase 1 (ALK1). Mutations in the gene encoding ALK1 cause hereditary hemorrhagic telangiectasia type 2, a rare genetic disease presenting hepatic vessel malformations. Variations of both the circulating levels and the hepatic mRNA levels of BMP9 have been recently associated with various forms of hepatic fibrosis. However, the molecular mechanism that links BMP9 with liver diseases is still unknown. Here, we report that Bmp9 gene deletion in 129/Ola mice triggers hepatic perisinusoidal fibrosis that was detectable from 15 weeks of age. An inflammatory response appeared within the same time frame as fibrosis, whereas sinusoidal vessel dilation developed later on. Proteomic and mRNA analyses of primary liver sinusoidal endothelial cells (LSECs) both revealed that the expression of the LSEC-specifying transcription factor GATA-binding protein 4 was strongly reduced in Bmp9 gene knockout (Bmp9-KO) mice as compared with wild-type mice. LSECs from Bmp9-KO mice also lost the expression of several terminal differentiation markers (Lyve1, Stab1, Stab2, Ehd3, Cd209b, eNos, Maf, Plvap). They gained CD34 expression and deposited a basal lamina, indicating that they were capillarized. Another main characteristic of differentiated LSECs is the presence of permeable fenestrae. LSECs from Bmp9-KO mice had a significantly reduced number of fenestrae. This was already observable in 2-week-old pups. Moreover, we could show that addition of BMP9 to primary cultures of LSECs prevented the loss of their fenestrae and maintained the expression levels of Gata4 and Plvap. Conclusion: Taken together, our observations show that BMP9 is a key paracrine regulator of liver homeostasis, controlling LSEC fenestration and protecting against perivascular hepatic fibrosis.

Selective BMP-9 Inhibition Partially Protects Against Experimental Pulmonary Hypertension.

RATIONALE: Although many familial cases of pulmonary arterial hypertension exhibit an autosomal dominant mode of inheritance with the majority having mutations in essential constituents of the BMP (bone morphogenetic protein) signaling, the specific contribution of the long-term loss of signal transduction triggered by the BMPR2 (type 2 BMP receptor) remains poorly characterized. OBJECTIVE: To investigate the role of BMP9, the main ligand of ALK1 (Activin receptor-like kinase 1)/BMPR2 heterocomplexes, in pulmonary hypertension. METHOD AND RESULTS: The absence of BMP9 in Bmp9-/- mice and its inhibition in C57BL/6 mice using neutralizing anti-BMP9 antibodies substantially prevent against chronic hypoxia-induced pulmonary hypertension judged by right ventricular systolic pressure measurement, right ventricular hypertrophy, and pulmonary distal arterial muscularization. In agreement with these observations, we found that the BMP9/BMP10 ligand trap ALK1ECD administered in monocrotaline or Sugen/hypoxia (SuHx) rats substantially attenuate proliferation of pulmonary vascular cells, inflammatory cell infiltration, and regresses established pulmonary hypertension in rats. Our data obtained in human pulmonary endothelial cells derived from controls and pulmonary arterial hypertension patients indicate that BMP9 can affect the balance between endothelin-1, apelin, and adrenomedullin. We reproduced these in vitro observations in mice chronically exposed to hypoxia, with Bmp9-/- mice exhibiting lower mRNA levels of the vasoconstrictor peptide ET-1 (endothelin-1) and higher levels of the 2 potent vasodilator factors apelin and ADM (adrenomedullin) compared with Bmp9+/+ littermates. CONCLUSIONS: Taken together, our data indicate that the loss of BMP9, by deletion or inhibition, has beneficial effects against pulmonary hypertension onset and progression.

BMP9, but not BMP10, acts as a quiescence factor on tumor growth, vessel normalization and metastasis in a mouse model of breast cancer.

BACKGROUND: Angiogenesis has become an attractive target for cancer therapy. However, despite the initial success of anti-VEGF (Vascular endothelial growth factor) therapies, the overall survival appears only modestly improved and resistance to therapy often develops. Other anti-angiogenic targets are thus urgently needed. The predominant expression of the type I BMP (bone morphogenetic protein) receptor ALK1 (activin receptor-like kinase 1) in endothelial cells makes it an attractive target, and phase I/II trials are currently being conducted. ALK1 binds with strong affinity to two ligands that belong to the TGF-ß family, BMP9 and BMP10. In the present work, we addressed their specific roles in tumor angiogenesis, cancer development and metastasis in a mammary cancer model. METHODS: For this, we used knockout (KO) mice for BMP9 (constitutive Gdf2-deficient), for BMP10 (inducible Bmp10-deficient) and double KO mice (Gdf2 and Bmp10) in a syngeneic immunocompetent orthotopic mouse model of spontaneous metastatic breast cancer (E0771). RESULTS: Our studies demonstrate a specific role for BMP9 in the E0771 mammary carcinoma model. Gdf2 deletion increased tumor growth while inhibiting vessel maturation and tumor perfusion. Gdf2 deletion also increased the number and the mean size of lung metastases. On the other hand, Bmp10 deletion did not significantly affect the E0771 mammary model and the double deletion (Gdf2 and Bmp10) did not lead to a stronger phenotype than the single Gdf2 deletion. CONCLUSIONS: Altogether, our data show that in a tumor environment BMP9 and BMP10 play different roles and thus blocking their shared receptor ALK1 is maybe not appropriate. Indeed, BMP9, but not BMP10, acts as a quiescence factor on tumor growth, lung metastasis and vessel normalization. Our results also support that activating rather than blocking the BMP9 pathway could be a new strategy for tumor vessel normalization in order to treat breast cancer.

A heterodimer formed by bone morphogenetic protein 9 (BMP9) and BMP10 provides most BMP biological activity in plasma.

Bone morphogenetic protein 9 (BMP9) and BMP10 are the two high-affinity ligands for the endothelial receptor activin receptor-like kinase 1 (ALK1) and are key regulators of vascular remodeling. They are both present in the blood, but their respective biological activities are still a matter of debate. The aim of the present work was to characterize their circulating forms to better understand how their activities are regulated in vivo First, by cotransfecting BMP9 and BMP10, we found that both can form a disulfide-bonded heterodimer in vitro and that this heterodimer is functional on endothelial cells via ALK1. Next, we developed an ELISA that could specifically recognize the BMP9-BMP10 heterodimer and which indicated its presence in both human and mouse plasma. In addition to using available Bmp9-KO mice, we generated a conditional Bmp10-KO mouse strain. The plasma from Bmp10-KO mice, similarly to that of Bmp9-KO mice, completely lacked the ability to activate ALK1-transfected 3T3 cells or phospho-Smad1-5 on endothelial cells, indicating that the circulating BMP activity is mostly due to the BMP9-BMP10 heterodimeric form. This result was confirmed in human plasma that had undergone affinity chromatography to remove BMP9 homodimer. Finally, we provide evidence that hepatic stellate cells in the liver could be the source of the BMP9-BMP10 heterodimer. Together, our findings demonstrate that BMP9 and BMP10 can heterodimerize and that this heterodimer is responsible for most of the biological BMP activity found in plasma.

BMP9 and BMP10 are necessary for proper closure of the ductus arteriosus.

The transition to pulmonary respiration after birth requires rapid alterations in the structure of the mammalian cardiovascular system. One dramatic change that occurs is the closure of the ductus arteriosus (DA), an arterial connection in the fetus that directs blood flow away from the pulmonary circulation. Two members of the TGFß family, bone morphogenetic protein 9 (BMP9) and BMP10, have been recently involved in postnatal angiogenesis, both being necessary for remodeling of newly formed microvascular beds. The aim of the present work was to study whether BMP9 and BMP10 could be involved in closure of the DA. We found that Bmp9 knockout in mice led to an imperfect closure of the DA. Further, addition of a neutralizing anti-BMP10 antibody at postnatal day 1 (P1) and P3 in these pups exacerbated the remodeling defect and led to a reopening of the DA at P4. Transmission electron microscopy images and immunofluorescence stainings suggested that this effect could be due to a defect in intimal cell differentiation from endothelial to mesenchymal cells, associated with a lack of extracellular matrix deposition within the center of the DA. This result was supported by the identification of the regulation by BMP9 and BMP10 of several genes known to be involved in this process. The involvement of these BMPs was further supported by human genomic data because we could define a critical region in chromosome 2 encoding eight genes including BMP10 that correlated with the presence of a patent DA. Together, these data establish roles for BMP9 and BMP10 in DA closure.

Functional analysis of endoglin mutations from hereditary hemorrhagic telangiectasia type 1 patients reveals different mechanisms for endoglin loss of function.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant inheritable vascular dysplasia caused by mutations in genes encoding either endoglin or activin receptor-like kinase-1 (ALK1). Functional significance of endoglin missense mutations remains largely unknown leading to a difficult discrimination between polymorphisms and pathogenic mutations. In order to study the functional significance of endoglin mutations and to help HHT1 diagnosis, we developed a cellular assay based on the ability of endoglin to enhance ALK1 response to bone morphogenetic protein 9 (BMP9). We generated and characterized 31 distinct ENG mutants reproducing human HHT1 missense mutations identified in patients of the Molecular Genetics Department in Lyon. We found that 16 mutants behaved like wild-type (WT) endoglin, and thus corresponded to benign rare variants. The 15 other variants showed defects in BMP9 response and were identified as pathogenic mutations. Interestingly, two mutants (S278P and F282V) had lost their ability to bind BMP9, identifying two crucial amino acids for BMP9 binding to endoglin. For all the others, the functional defect was correlated with a defective trafficking to the cell surface associated with retention in the endoplasmic reticulum. Further, we demonstrated that some intracellular mutants dimerized with WT endoglin and impaired its cell-surface expression thus acting as dominant-negatives. Taken together, we show that endoglin loss-of-function can result from different mechanisms in HHT1 patients. We also provide a diagnostic tool helping geneticists in screening for novel or conflicting ENG mutations.

Bone morphogenetic protein 9 (BMP9) controls lymphatic vessel maturation and valve formation.

Lymphatic vessels are critical for the maintenance of tissue fluid homeostasis and their dysfunction contributes to several human diseases. The activin receptor-like kinase 1 (ALK1) is a transforming growth factor-ß family type 1 receptor that is expressed on both blood and lymphatic endothelial cells (LECs). Its high-affinity ligand, bone morphogenetic protein 9 (BMP9), has been shown to be critical for retinal angiogenesis. The aim of this work was to investigate whether BMP9 could play a role in lymphatic development. We found that Bmp9 deficiency in mice causes abnormal lymphatic development. Bmp9-knockout (KO) pups presented hyperplastic mesenteric collecting vessels that maintained LYVE-1 expression. In accordance with this result, we found that BMP9 inhibited LYVE-1 expression in LECs in an ALK1-dependent manner. Bmp9-KO pups also presented a significant reduction in the number and in the maturation of mesenteric lymphatic valves at embryonic day 18.5 and at postnatal days 0 and 4. Interestingly, the expression of several genes known to be involved in valve formation (Foxc2, Connexin37, EphrinB2, and Neuropilin1) was upregulated by BMP9 in LECS. Finally, we demonstrated that Bmp9-KO neonates and adult mice had decreased lymphatic draining efficiency. These data identify BMP9 as an important extracellular regulator in the maturation of the lymphatic vascular network affecting valve development and lymphatic vessel function.

BMP9 and BMP10 are critical for postnatal retinal vascular remodeling.

ALK1 is a type I receptor of the TGF-ß family that is involved in angiogenesis. Circulating BMP9 was identified as a specific ligand for ALK1 inducing vascular quiescence. In this work, we found that blocking BMP9 with a neutralizing antibody in newborn mice significantly increased retinal vascular density. Surprisingly, Bmp9-KO mice did not show any defect in retinal vascularization. However, injection of the extracellular domain of ALK1 impaired retinal vascularization in Bmp9-KO mice, implicating another ligand for ALK1. Interestingly, we detected a high level of circulating BMP10 in WT and Bmp9-KO pups. Further, we found that injection of a neutralizing anti-BMP10 antibody to Bmp9-KO pups reduced retinal vascular expansion and increased vascular density, whereas injection of this antibody to WT pups did not affect the retinal vasculature. These data suggested that BMP9 and BMP10 are important in postnatal vascular remodeling of the retina and that BMP10 can substitute for BMP9. In vitro stimulation of endothelial cells by BMP9 and BMP10 increased the expression of genes involved in the Notch signaling pathway (Jagged1, Dll4, Hey1, Hey2, Hes1) and decreased apelin expression, suggesting a possible cross-talk between these pathways and the BMP pathway.

BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain.

Bone Morphogenetic Protein 9 (BMP9) has been recently found to be the physiological ligand for the activin receptor-like kinase 1 (ALK1), and to be a major circulating vascular quiescence factor. Moreover, a soluble chimeric ALK1 protein (ALK1-Fc) has recently been developed and showed powerful anti-tumor growth and anti-angiogenic effects. However, not much is known concerning BMP9. This prompted us to investigate the human endogenous sources of this cytokine and to further characterize its circulating form(s) and its function. Analysis of BMP9 expression reveals that BMP9 is produced by hepatocytes and intrahepatic biliary epithelial cells. Gel filtration analysis combined with ELISA and biological assays demonstrate that BMP9 circulates in plasma (1) as an unprocessed inactive form that can be further activated by furin a serine endoprotease, and (2) as a mature and fully active form (composed of the mature form associated with its prodomain). Analysis of BMP9 circulating levels during mouse development demonstrates that BMP9 peaks during the first 3 weeks after birth and then decreases to 2 ng/mL in adulthood. We also show that circulating BMP9 physiologically induces a constitutive Smad1/5/8 phosphorylation in endothelial cells. Taken together, our results argue for the role of BMP9 as a hepatocyte-derived factor, circulating in inactive (40%) and active (60%) forms, the latter constantly activating endothelial cells to maintain them in a resting state.

Functional analysis of the BMP9 response of ALK1 mutants from HHT2 patients: a diagnostic tool for novel ACVRL1 mutations.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetically inheritable vascular dysplasia caused by mutations in genes encoding receptors of the transforming growth factor-beta (TGF-beta) family: ENG, encoding endoglin (HHT1), and ACVRL1, encoding activin receptor-like kinase-1 (ALK1; HHT2). Our recent discovery of bone morphogenetic protein 9 (BMP9) as the specific ligand for ALK1 allowed us to reevaluate the functional significance of ACVRL1 mutations. We generated 19 ALK1 mutants reproducing HHT2 mutations (4 were novel mutations) found throughout the protein. We show that all ALK1 mutant proteins were expressed by transfected cells; most of them were present at the cell surface and retained their ability to bind BMP9 (except for the extracellular mutants). However, most were defective in BMP9 signaling. None of the ALK1 mutants had a dominant negative effect on wild-type ALK1 activity. These data demonstrate that mutations of ACVRL1 fit with a functional haploinsufficiency model affecting BMP9 signaling. Our study also identified 4 ACVRL1 mutations (D179A, R386C, R454W, and A482V) that did not alter the BMP9 responses that are polymorphisms and 2 novel mutations that are pathogenic (L381P and I485F). This demonstrates that the analysis of BMP9 responses can be used as a diagnostic tool by geneticists confronted with novel or conflicting ACVRL1 mutations.

Bone morphogenetic protein-9 is a circulating vascular quiescence factor.

Angiogenesis is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals. ALK1 (activin receptor like-kinase 1) is an endothelial-specific type 1 receptor of the transforming growth factor-beta receptor family. Heterozygotes with mutations in the ALK1 gene develop hereditary hemorrhagic telangiectasia type 2 (HHT2). Recently, we reported that bone morphogenetic protein (BMP)9 and BMP10 are specific ligands for ALK1 that potently inhibit microvascular endothelial cell migration and growth. These data lead us to suggest that these factors may play a role in the control of vascular quiescence. To test this hypothesis, we checked their presence in human serum. We found that human serum induced Smad1/5 phosphorylation. To identify the active factor, we tested neutralizing antibodies against BMP members and found that only the anti-BMP9 inhibited serum-induced Smad1/5 phosphorylation. The concentration of circulating BMP9 was found to vary between 2 and 12 ng/mL in sera and plasma from healthy humans, a value well above its EC(50) (50 pg/mL). These data indicated that BMP9 is circulating at a biologically active concentration. We then tested the effects of BMP9 in 2 in vivo angiogenic assays. We found that BMP9 strongly inhibited sprouting angiogenesis in the mouse sponge angiogenesis assay and that BMP9 could inhibit blood circulation in the chicken chorioallantoic membrane assay. Taken together, our results demonstrate that BMP9, circulating under a biologically active form, is a potent antiangiogenic factor that is likely to play a physiological role in the control of adult blood vessel quiescence.

Activin receptor-like kinase 1 inhibits human microvascular endothelial cell migration: potential roles for JNK and ERK.

Activin receptor-like kinase 1 (ALK1) is an endothelial-specific type I receptor of the TGFbeta receptor family that is implicated in angiogenesis and in the pathogenesis of the vascular disease, hereditary hemorrhagic telangiectasia (HHT). In the absence of a specific ligand, ALK1 cellular functions have been mainly studied through the use of a constitutively active form of this receptor (ALK1ca) and are still debated. We previously reported that ALK1ca inhibits proliferation and migration of human endothelial cells suggesting that ALK1 plays an important role in the maturation phase of angiogenesis (Lamouille et al., 2002, Blood 100: 4495-4501). In the present work, we further analyzed the role of ALK1 in the migration of human dermal microvascular endothelial cell (HMVEC-d) and observed that silencing endogenous ALK1 expression with siRNAs accelerates endothelial cell migration in the wound assay. Further, we demonstrate that ALK1-induced inhibition of migration is Smad-independent. Using a panel of kinase inhibitors, we found that HMVEC-d wound closure was completely inhibited by a JNK inhibitor and to a lower degree by an ERK kinase inhibitor. Further, HMVEC-d wounding induced activation of both JNK and ERK, and these were inhibited by ALK1ca expression. Taken together, these results support a significant role for ALK1 as a negative regulator of endothelial cell migration and suggest the implication of JNK and ERK as mediators of this effect.

Identification of BMP9 and BMP10 as functional activators of the orphan activin receptor-like kinase 1 (ALK1) in endothelial cells.

ALK1 is an endothelial-specific type I receptor of the TGFbeta receptor family whose heterozygous mutations cause hereditary hemorrhagic telangiectasia type 2. Although TGFbeta1 and TGFbeta3 have been shown to bind ALK1 under specific experimental conditions, they may not represent the physiological ligands for this receptor. In the present study, we demonstrate that BMP9 induces the phosphorylation of Smad1/5/8 in microvascular endothelial cells, and this phosphorylation lasts over a period of 24 hours. BMP9 also activates the ID1 promoter-derived BMP response element (BRE) in a dose-dependent manner (EC50 = 45 +/- 27 pg/mL), and this activation is abolished by silencing ALK1 expression or addition of ALK1 extracellular domain. Overexpression of endoglin increases the BMP9 response, whereas silencing of both BMPRII and ActRIIA expressions completely abolishes it. BMP10, which is structurally close to BMP9, is also a potent ALK1 ligand. Finally, we demonstrate that BMP9 and BMP10 potently inhibit endothelial cell migration and growth, and stimulate endothelial expression of a panel of genes that was previously reported to be activated by the constitutively active form of ALK1. Taken together, our results suggest that BMP9 and BMP10 are two specific ALK1 ligands that may physiologically trigger the effects of ALK1 on angiogenesis.

TGFbeta1 induces vasculogenesis and inhibits angiogenic sprouting in an embryonic stem cell differentiation model: respective contribution of ALK1 and ALK5.

Transforming growth factor-beta1 (TGFbeta1) is a multipotent cytokine that is involved in the regulation of vasculogenesis and angiogenesis. However, the actions of TGFbeta1 on vascular cells in vitro and in vivo are extremely complex and still incompletely understood. The aim of the present study was to investigate the role of TGFbeta1 and its two type I receptors, activin receptor-like kinase-1 (ALK1) and ALK5, in an embryonic stem cell (ESC) differentiation model that recapitulates the developmental steps of vasculogenesis and sprouting angiogenesis. We show that TGFbeta1 increases endothelial cell differentiation in a vascular endothelial growth factor (VEGF)-independent manner and inhibits endothelial tube formation. Furthermore, we demonstrate that undifferentiated ESCs express ALK5 but do not express ALK1, with ALK1 being expressed only after day 5 of differentiation. Finally, we demonstrate that constitutively active forms of ALK1 and ALK5 both inhibit growth factor-induced endothelial sprouting from embryoid bodies. In conclusion, the use of this ESC differentiation model allowed us to propose the following model: at early stages of development, TGFbeta1, through the ALK5 receptor, is provasculogenic in a VEGF-independent manner. Later, in differentiated endothelial cells in which both ALK1 and ALK5 are expressed, both receptors are implicated in inhibition of sprouting angiogenesis.

ACTH depletion represses vascular endothelial-cadherin transcription in mouse adrenal endothelium in vivo.

Vascular endothelial-cadherin (VE-cadherin) is an endothelial cell-specific adhesion protein that is localised at cell-cell contacts. This molecule is an important determinant of vascular architecture and endothelial cell survival. In the adrenal cortex, steroidogenic and endothelial cells form a complex architecture. The adrenocorticotrophin hormone (ACTH) regulates gland homeostasis whose secretion is subjected to a negative feedback by adrenocorticosteroids. The aim of the present study was to determine whether VE-cadherin expression in the adrenal gland was regulated by hormonal challenge. We demonstrated that VE-cadherin protein levels were dramatically decreased (23.5+/-3.7%) by dexamethasone injections in the mouse and were restored by ACTH within 7 days (94.9+/-18.6%). Flow cytometry analysis of adrenal cells showed that the ratios of endothelial versus total adrenal cells were identical (35%) in dexamethasone- or ACTH-treated or untreated mice, suggesting that VE-cadherin expression could be regulated by ACTH. We demonstrate the existence of a transcriptional regulation of the VE-cadherin gene using transgenic mice carrying the chloramphenicol acetyl transferase gene under the control of the VE-cadherin promoter. Indeed, the promoter activity in the adrenals, but not in the lung or liver, was decreased in response to dexamethasone treatment (40+/-1.3%) and was partially restored after gland regeneration by ACTH injection (82+/-3%). In conclusion, our results show that transcription of a specific endothelial gene is controlled by the hypothalamo-pituitary axis and the data expand the knowledge regarding the role of ACTH in the regulation of the adrenal vascular network.

Expressional regulation of the angiopoietin-1 and -2 and the endothelial-specific receptor tyrosine kinase Tie2 in adrenal atrophy: a study of adrenocorticotropin-induced repair.

Angiopoietin-1 (Ang-1), a newly discovered ligand of the endothelial-specific tyrosine kinase receptor Tie-2, has been found to promote cell survival, vascular maturation, and stabilization, and to function in concert with vascular endothelial growth factor. Adrenal gland has an intense capillary network that regulation remains to be documented. Recently, we demonstrated that vascular endothelial growth factor, and its receptors are expressed in mouse adrenal in vivo, but no detailed study on Ang expression in the adrenal has been reported. The present study shows the expression of Tie2 receptors, Ang-1, and its endogenous antagonist, Ang-2 in mouse adrenal in vivo. Immunohistochemistry disclosed that Tie2 colocalized with platelet-endothelial-cell-adhesion-molecule in endothelial cells from normal mouse adrenal. Daily administration of dexamethasone (DEX) (0.5 mg/100 g body weight.d) for 6 d in mice, decreased steroidogenic function of adrenal as shown by inhibition of the 36-kDa ACTH receptor protein expression, and decreased plasma corticosterone level [control from 465 +/- 35 ng/ml to 114 +/- 18 ng/ml in DEX group (P < 0.001)]. Using semiquantitative RT-PCR, we demonstrate that DEX treatment down regulates Ang-1 mRNA levels by 3- to 4-fold. No significant changes in Ang-2 were detected between control and DEX groups, resulting in an altered Ang-2 to Ang-1 relative ratio. The Tie2 receptor was also found to be down-regulated in DEX group at both mRNA and protein level. ACTH was found to play a causal role in DEX-induced decrease in Ang-1/Tie2 system, because 7 d treatment with long acting 1-39 ACTH (30 IU/kg x d) increased Ang-1, Tie2 expression, and plasma corticosterone back to control levels. These results reinforce the role of ACTH in the regulation of angiogenic factors in adrenal gland and suggest that the Ang/Tie2 system might represent a key player for stabilization of adrenal endothelium.

Differential expression of VEGF receptors in adrenal atrophy induced by dexamethasone: a protective role of ACTH.

Although ACTH is important to adrenal growth and steroidogenesis, its role in vascular development and function has not been established in vivo. In the present study, we demonstrate the expression of mRNA for all four VEGF isoforms (mVEGF(120,144,164,188)) and for Flk-1/KDR and Flt-1 receptors in the mouse adrenal in vivo. Suppression of the pituitary adrenocortical axis by dexamethasone (0.5 mg x 100 g body wt(-1) x day(-1) during 6 days) induced a decrease in corticosterone levels, adrenal weights by 50% (P < 0.001), VEGF(188) mRNA, and Flk-1/KDR mRNA, whereas Flt-1 remained consistent during steroid treatment. A daily injection of ACTH-(1-39) restored the transcript for Flk-1/KDR and both VEGF(188) and plasma corticosterone to control levels. To gain further insights into the effects of ACTH, cultured endothelial cells (ECs) were treated with forskolin, which increases cAMP, the second messenger in ACTH action. We demonstrate that Flk-1/KDR protein expression was markedly increased by forskolin within 24-48 h of treatment in a dose-dependent manner (0.1-10 microM). The biological effect of ACTH on ECs was then tested by use of coincubations of fasciculata cells and ECs in 3D-collagen assay. Within 5-7 days of culture, ECs organized into multicellular structures that resemble networks of microvasculature, which characterize angiogenesis in vitro.

Activin receptor-like kinase 1 is implicated in the maturation phase of angiogenesis.

Activin receptor-like kinase 1 (ALK-1) is an orphan type I receptor of the transforming growth factor beta (TGF-beta) receptor family. In vivo studies have demonstrated that this endothelial-specific receptor is implicated in angiogenesis. In this study, we addressed the cellular function of ALK-1 in cultured human microvascular endothelial cells from the dermis (HMVEC-d's) using adenoviral expression of a constitutively active form of ALK-1 (ALK-1QD). We observed that ALK-1QD expression inhibits cell proliferation through an arrest in the G1 phase in the cell cycle. ALK-1QD expression also inhibited migration. This inhibition was also observed in other endothelial cells (human microvascular endothelial cells [HMEC-1's], HMVECs from the lung, and human umbilical vein endothelial cells [HUVECs]). Finally, ALK-1QD expression decreased re-adhesion and spreading to different matrices. This led us to examine the dynamic formation of adhesion complexes. We demonstrated that while beta-gal-infected cells reorganized actin stress fibers and focal adhesion complexes at the edge of a wound, ALK-1QD-infected cells did not. To identify downstream genes implicated in ALK-1 cellular responses, we next performed a cDNA array analysis of the expressed genes. There were 13 genes found to be significantly induced or suppressed by ALK-1QD. Among them, 2 genes encoded cell cycle-related proteins (c-myc and p21/waf1), 3 encoded components of the cytoskeleton-focal adhesion complex (beta-actin, paxillin, and zyxin), and 2 encoded members of the TGF-beta family (BMPRII and GDF-15). Taken together, our results suggest that ALK-1 is implicated in the maturation phase of angiogenesis. Disruption of this latter phase of angiogenesis may be an important step in the development of hereditary hemorrhagic telangiectasia.