Disrupted BMP-9 Signaling Impairs Pulmonary Vascular Integrity in Hepatopulmonary Syndrome.

Rationale: Hepatopulmonary syndrome (HPS) is a severe complication of liver diseases characterized by abnormal dilation of pulmonary vessels, resulting in impaired oxygenation. Recent research highlights the pivotal role of liver-produced BMP-9 (bone morphogenetic protein-9) in maintaining pulmonary vascular integrity. Objectives: This study aimed to investigate the involvement of BMP-9 in human and experimental HPS. Methods: Circulating BMP-9 levels were measured in 63 healthy control subjects and 203 patients with cirrhosis with or without HPS. Two animal models of portal hypertension were employed: common bile duct ligation with cirrhosis and long-term partial portal vein ligation without cirrhosis. Additionally, the therapeutic effect of low-dose BMP activator FK506 was investigated, and the pulmonary vascular phenotype of BMP-9-knockout rats was analyzed. Measurements and Main Results: Patients with HPS related to compensated cirrhosis exhibited lower levels of circulating BMP-9 compared with patients without HPS. Patients with severe cirrhosis exhibited consistently low levels of BMP-9. HPS characteristics were observed in animal models, including intrapulmonary vascular dilations and an increase in the alveolar-arterial gradient. HPS development in both rat models correlated with reduced intrahepatic BMP-9 expression, decreased circulating BMP-9 level and activity, and impaired pulmonary BMP-9 endothelial pathway. Daily treatment with FK506 for 2 weeks restored the BMP pathway in the lungs, alleviating intrapulmonary vascular dilations and improving gas exchange impairment. Furthermore, BMP-9-knockout rats displayed a pulmonary HPS phenotype, supporting its role in disease progression. Conclusions: The study findings suggest that portal hypertension-induced loss of BMP-9 signaling contributes to HPS development.

Large-scale phosphoproteomics reveals activation of the MAPK/GADD45ß/P38 axis and cell cycle inhibition in response to BMP9 and BMP10 stimulation in endothelial cells.

BACKGROUND: BMP9 and BMP10 are two major regulators of vascular homeostasis. These two ligands bind with high affinity to the endothelial type I kinase receptor ALK1, together with a type II receptor, leading to the direct phosphorylation of the SMAD transcription factors. Apart from this canonical pathway, little is known. Interestingly, mutations in this signaling pathway have been identified in two rare cardiovascular diseases, hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension. METHODS: To get an overview of the signaling pathways modulated by BMP9 and BMP10 stimulation in endothelial cells, we employed an unbiased phosphoproteomic-based strategy. Identified phosphosites were validated by western blot analysis and regulated targets by RT-qPCR. Cell cycle analysis was analyzed by flow cytometry. RESULTS: Large-scale phosphoproteomics revealed that BMP9 and BMP10 treatment induced a very similar phosphoproteomic profile. These BMPs activated a non-canonical transcriptional SMAD-dependent MAPK pathway (MEKK4/P38). We were able to validate this signaling pathway and demonstrated that this activation required the expression of the protein GADD45ß. In turn, activated P38 phosphorylated the heat shock protein HSP27 and the endocytosis protein Eps15 (EGF receptor pathway substrate), and regulated the expression of specific genes (E-selectin, hyaluronan synthase 2 and cyclooxygenase 2). This study also highlighted the modulation in phosphorylation of proteins involved in transcriptional regulation (phosphorylation of the endothelial transcription factor ERG) and cell cycle inhibition (CDK4/6 pathway). Accordingly, we found that BMP10 induced a G1 cell cycle arrest and inhibited the mRNA expression of E2F2, cyclinD1 and cyclinA1. CONCLUSIONS: Overall, our phosphoproteomic screen identified numerous proteins whose phosphorylation state is impacted by BMP9 and BMP10 treatment, paving the way for a better understanding of the molecular mechanisms regulated by BMP signaling in vascular diseases.

BMP9 and BMP10: Two close vascular quiescence partners that stand out.

Bone morphogenetic proteins (BMPs) are dimeric transforming growth factor ß (TGFß) family cytokines that were first described in bone and cartilage formation but have since been shown to be involved in many pleiotropic functions. In human, there are 15 BMP ligands, which initiate their cellular signaling by forming a complex with two copies of type I receptors and two copies of type II receptors, both of which are transmembrane receptors with an intracellular serine/threonine kinase domain. Within this receptor family, ALK1 (activin receptor-like kinase 1), which is a type I receptor mainly expressed on endothelial cells, and BMPRII (BMP Receptor type II), a type II receptor also highly expressed on endothelial cells, have been directly linked to two rare vascular diseases: hereditary hemorrhagic telangiectasia (HHT), and pulmonary arterial hypertension (PAH), respectively. BMP9 (gene name GDF2) and BMP10, two close members of the BMP family, are the only known ligands for the ALK1 receptor. This specificity gives them a unique role in physiological and pathological angiogenesis and tissue homeostasis. The aim of this current review is to present an overview of what is known about BMP9 and BMP10 on vascular regulation with a particular emphasis on recent results and the many questions that remain unanswered regarding the roles and specificities between BMP9 and BMP10.

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.

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.

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.

Sulfamoylated Estradiol Analogs Targeting the Actin and Microtubule Cytoskeletons Demonstrate Anti-Cancer Properties In Vitro and In Ovo.

The microtubule-disrupting agent 2-methoxyestradiol (2-ME) displays anti-tumor and anti-angiogenic properties, but its clinical development is halted due to poor pharmacokinetics. We therefore designed two 2-ME analogs in silico-an ESE-15-one and an ESE-16 one-with improved pharmacological properties. We investigated the effects of these compounds on the cytoskeleton in vitro, and their anti-angiogenic and anti-metastatic properties in ovo. Time-lapse fluorescent microscopy revealed that sub-lethal doses of the compounds disrupted microtubule dynamics. Phalloidin fluorescent staining of treated cervical (HeLa), metastatic breast (MDA-MB-231) cancer, and human umbilical vein endothelial cells (HUVECs) displayed thickened, stabilized actin stress fibers after 2 h, which rearranged into a peripheral radial pattern by 24 h. Cofilin phosphorylation and phosphorylated ezrin/radixin/moesin complexes appeared to regulate this actin response. These signaling pathways overlap with anti-angiogenic, extra-cellular communication and adhesion pathways. Sub-lethal concentrations of the compounds retarded both cellular migration and invasion. Anti-angiogenic and extra-cellular matrix signaling was evident with TIMP2 and P-VEGF receptor-2 upregulation. ESE-15-one and ESE-16 exhibited anti-tumor and anti-metastatic properties in vivo, using the chick chorioallantoic membrane assay. In conclusion, the sulfamoylated 2-ME analogs displayed promising anti-tumor, anti-metastatic, and anti-angiogenic properties. Future studies will assess the compounds for myeloproliferative effects, as seen in clinical applications of other drugs in this class.

BMP9 is a key player in endothelial identity and its loss is sufficient to induce arteriovenous malformations.

AIMS: BMP9 is a high affinity ligand of ALK1 and endoglin receptors that are mutated in the rare genetic vascular disorder hereditary hemorrhagic telangiectasia (HHT). We have previously shown that loss of Bmp9 in the 129/Ola genetic background leads to spontaneous liver fibrosis via capillarization of liver sinusoidal endothelial cells (LSEC) and kidney lesions. We aimed to decipher the molecular mechanisms downstream of BMP9 to better characterize its role in vascular homeostasis in different organs. METHODS AND RESULTS: For this, we performed an RNA-seq analysis on LSEC from adult WT and Bmp9-KO mice and identified over 2000 differentially expressed genes. Gene ontology analysis showed that Bmp9 deletion led to a decrease in BMP and Notch signalling, but also LSEC capillary identity while increasing their cell cycle. The gene ontology term 'glomerulus development' was also negatively enriched in Bmp9-KO mice vs. WT supporting a role for BMP9 in kidney vascularization. Through different imaging approaches (electron microscopy, immunostainings), we found that loss of Bmp9 led to vascular enlargement of the glomeruli capillaries associated with alteration of podocytes. Importantly, we also showed for the first time that the loss of Bmp9 led to spontaneous arteriovenous malformations (AVMs) in the liver, gastrointestinal tract, and uterus. CONCLUSION: Altogether, these results demonstrate that BMP9 plays an important role in vascular quiescence both locally in the liver by regulating endothelial capillary differentiation markers and cell cycle but also at distance in many organs via its presence in the circulation. It also reveals that loss of Bmp9 is sufficient to induce spontaneous AVMs, supporting a key role for BMP9 in the pathogenesis of HHT.

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.

Future treatments for hereditary hemorrhagic telangiectasia.

Hereditary Hemorrhagic Telangiectasia (HHT), also known as Rendu-Osler syndrome, is a genetic vascular disorder affecting 1 in 5000-8000 individuals worldwide. This rare disease is characterized by various vascular defects including epistaxis, blood vessel dilations (telangiectasia) and arteriovenous malformations (AVM) in several organs. About 90% of the cases are associated with heterozygous mutations of ACVRL1 or ENG genes, that respectively encode a bone morphogenetic protein receptor (activin receptor-like kinase 1, ALK1) and a co-receptor named endoglin. Less frequent mutations found in the remaining 10% of patients also affect the gene SMAD4 which is part of the transcriptional complex directly activated by this pathway. Presently, the therapeutic treatments for HHT are intended to reduce the symptoms of the disease. However, recent progress has been made using drugs that target VEGF (vascular endothelial growth factor) and the angiogenic pathway with the use of bevacizumab (anti-VEGF antibody). Furthermore, several exciting high-throughput screenings and preclinical studies have identified new molecular targets directly related to the signaling pathways affected in the disease. These include FKBP12, PI3-kinase and angiopoietin-2. This review aims at reporting these recent developments that should soon allow a better care of HHT patients.

High-Resolution Shortwave Infrared Imaging of Vascular Disorders Using Gold Nanoclusters.

We synthesized a generation of water-soluble, atomically precise gold nanoclusters (Au NCs) with anisotropic surface containing a short dithiol pegylated chain (AuMHA/TDT). The AuMHA/TDT exhibit a high brightness (QY ∼ 6%) in the shortwave infrared (SWIR) spectrum with a detection above 1250 nm. Furthermore, they show an extended half-life in blood (t1/2ß = 19.54 ± 0.05 h) and a very weak accumulation in organs. We also developed a non-invasive, whole-body vascular imaging system in the SWIR window with high-resolution, benefiting from a series of Monte Carlo image processing. The imaging process enabled to improve contrast by 1 order of magnitude and enhance the spatial resolution by 59%. After systemic administration of these nanoprobes in mice, we can quantify vessel complexity in depth (>4 mm), allowing to detect very subtle vascular disorders non-invasively in bone morphogenetic protein 9 (Bmp9)-deficient mice. The combination of these anisotropic surface charged Au NCs plus an improved SWIR imaging device allows a precise mapping at high-resolution and an in depth understanding of the organization of the vascular network in live animals.

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.

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.

Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis.

BACKGROUND: Angiogenesis assays are important tools for the identification of regulatory molecules and the potential development of therapeutic strategies to modulate neovascularization. Although numerous in vitro angiogenesis models have been developed in the past, they exhibit limitations since they do not recapitulate the entire angiogenic process or correspond to multi-step procedures that are not easy to use. Convenient, reliable, easily quantifiable and physiologically relevant assays are still needed for pharmacological screenings of angiogenesis. RESULTS: Here, we have optimized an angiogenesis model based on ES cell differentiation for screening experiments. We have established conditions leading to angiogenic sprouting of embryoid bodies during ES cell differentiation in type I three-dimensional collagen gels. Immunostaining experiments carried out during these cultures showed the formation of numerous buds comprising CD31 positive cells, after 11 days of culture of ES cells. Moreover, this one-step model has been validated in response to activators and inhibitors of angiogenesis. Sprouting was specifically stimulated in the presence of VEGF and FGF2. Alternatively, endothelial sprouting induced by angiogenic activators was inhibited by angiogenesis inhibitors such as angiostatin, TGFbeta and PF4. Sprouting angiogenesis can be easily quantified by image analysis after immunostaining of endothelial cells with CD31 pan-endothelial marker. CONCLUSION: Taken together, these data clearly validate that this one-step ES differentiation model constitutes a simple and versatile angiogenesis system that should facilitate, in future investigations, the screening of both activators and inhibitors of angiogenesis.

Antagonistic functions of tetradecanoyl phorbol acetate-inducible-sequence 11b and HuR in the hormonal regulation of vascular endothelial growth factor messenger ribonucleic acid stability by adrenocorticotropin.

Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is up-regulated by a variety of factors including hypoxia, growth factors, and hormones. In the adrenal cortex, regulation of VEGF expression by the pituitary hormone ACTH ensures the maintenance of the organ vasculature. We have previously shown that ACTH evokes a rapid and transient increase in VEGF mRNA levels in primary adrenocortical cells through transcription-independent mechanisms. We further demonstrated that the zinc finger RNA-binding protein Tis11b (tetradecanoyl phorbol acetate-inducible-sequence 11b) destabilizes VEGF mRNA through its 3'-untranslated region (3'-UTR) and that Tis11b is involved in the decay phase of ACTH-induced VEGF mRNA expression. In the present study, we attempted to determine the mechanisms underlying ACTH-elicited increase in VEGF mRNA levels in adrenocortical cells. We show that ACTH triggers an increase in the levels of the mRNA-stabilizing protein HuR in the cytoplasm and a concomitant decrease in the levels of HuR in the nucleus. This process is accompanied by an increased association of HuR with the nucleocytoplasmic shuttling protein pp32, indicating that ACTH induces HuR translocation from the nuclear to the cytoplasmic compartment. Leptomycin B, a specific inhibitor of CRM1-dependent nuclear export of pp32, significantly reduced ACTH-induced VEGF mRNA levels. Furthermore, RNA interference-mediated depletion of HuR in adrenocortical cells abrogated ACTH-induced VEGF mRNA expression. Finally, we show that Tis11b and HuR exert antagonistic effects on VEGF 3'-UTR in vitro. Although both proteins could bind simultaneously on VEGF 3'-UTR, Tis11b markedly decreases HuR-binding to this RNA sequence. Altogether, these results suggest that the RNA-stabilizing protein HuR is instrumental to ACTH-induced expression of VEGF mRNA and that the nuclear export of HuR is a rate-limiting step in this process. HuR appears to transiently stabilize VEGF transcripts after ACTH stimulation of adrenocortical cells, and Tis11b appears to subsequently trigger their degradation.

ACTH Action on Messenger RNA Stability Mechanisms.

The regulation of mRNA stability has emerged as a critical control step in dynamic gene expression. This process occurs in response to modifications of the cellular environment, including hormonal variations, and regulates the expression of subsets of proteins whose levels need to be rapidly adjusted. Modulation of messenger RNA stability is usually mediated by stabilizing or destabilizing RNA-binding proteins (RNA-BP) that bind to the 3'-untranslated region regulatory motifs, such as AU-rich elements (AREs). Destabilizing ARE-binding proteins enhance the decay of their target transcripts by recruiting the mRNA decay machineries. Failure of such mechanisms, in particular misexpression of RNA-BP, has been linked to several human diseases. In the adrenal cortex, the expression and activity of mRNA stability regulatory proteins are still understudied. However, ACTH- or cAMP-elicited changes in the expression/phosphorylation status of the major mRNA-destabilizing protein TIS11b/BRF1 or in the subcellular localization of the stabilizing protein Human antigen R have been reported. They suggest that this level of regulation of gene expression is also important in endocrinology.

Indolizine-Based Scaffolds as Efficient and Versatile Tools: Application to the Synthesis of Biotin-Tagged Antiangiogenic Drugs.

We describe the design and optimization of polyfunctional scaffolds based on a fluorescent indolizine core derivatized with various orthogonal groups (amines, esters, oximes, alkynes, etc.). To show one application as tools in biology, the scaffold was used to prepare drug-biotin conjugates that were then immobilized onto avidin-agarose for affinity chromatography. More specifically, the antiangiogenic drug COB223, whose mechanism of action remained unclear, was chosen as a proof-of-concept drug. The drug-selective discrimination of proteins observed after elution of the cell lysates through the affinity columns, functionalized either with the biologically active COB223 or a structurally related inactive analogue (COB236), is a clear indication that the presence of the indolizine core does not limit drug-protein interaction and confirms the usefulness of the indolizine scaffold. Furthermore, the separation of COB223-interacting proteins from human placental extracts unveiled unanticipated protein targets belonging to the family of regulatory RNA-binding proteins, which opens the way to new hypotheses on the mode of action of this antiangiogenic drug.

The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1.

TPA-inducible sequence 11b/butyrate response factor 1 (TIS11b/BRF1) belongs to the tristetraprolin (TTP) family of zinc-finger proteins, which bind to mRNAs containing AU-rich elements in their 3'-untranslated region and target them for degradation. Regulation of TTP family function through phosphorylation by p38 MAP kinase and Akt/protein kinase B signaling pathways has been extensively studied. In contrast, the role of cAMP-dependent protein kinase (PKA) in the control of TTP family activity in mRNA decay remains largely unknown. Here we show that PKA activation induces TIS11b gene expression and protein phosphorylation. Site-directed mutagenesis combined with kinase assays and specific phosphosite immunodetection identified Ser-54 (S54) and Ser-334 (S334) as PKA target amino acids in vitro and in vivo. Phosphomimetic mutation of the C-terminal S334 markedly increased TIS11b half-life and, unexpectedly, enhanced TIS11b activity on mRNA decay. Examination of protein-protein interactions between TIS11b and components of the mRNA decay machinery revealed that mimicking phosphorylation at S334 enhances TIS11b interaction with the decapping coactivator Dcp1a, while preventing phosphorylation at S334 potentiates its interaction with the Ccr4-Not deadenylase complex subunit Cnot1. Collectively our findings establish for the first time that cAMP-elicited phosphorylation of TIS11b plays a key regulatory role in its mRNA decay-promoting function.

A new chemical inhibitor of angiogenesis and tumorigenesis that targets the VEGF signaling pathway upstream of Ras.

The efficacy of anti-angiogenic therapies on cancer patients is limited by the emergence of drug resistance, urging the search for second-generation drugs. In this study, we screened an academic chemical library (DCM, University of Grenoble-Alpes) and identified a leader molecule, COB223, that inhibits endothelial cell migration and proliferation. It inhibits also Lewis lung carcinoma (LLC/2) cell proliferation whereas it does not affect fibroblast proliferation. The anti-angiogenic activity of COB223 was confirmed using several in vitro and in vivo assays. In a mouse LLC/2 tumor model, ip administration of doses as low as 4 mg/kg COB223 efficiently reduced the tumor growth rate. We observed that COB223 inhibits endothelial cell ERK1/2 phosphorylation induced by VEGF, FGF-2 or serum and that it acts downstream of PKC and upstream of Ras. This molecule represents a novel anti-angiogenic and anti-tumorigenic agent with an original mechanism of action that deserves further development as an anti-cancer drug.

Azaindole derivatives are inhibitors of microtubule dynamics, with anti-cancer and anti-angiogenic activities.

BACKGROUND AND PURPOSE: Drugs targeting microtubules are commonly used for cancer treatment. However, the potency of microtubule inhibitors used clinically is limited by the emergence of resistance. We thus designed a strategy to find new cell-permeable microtubule-targeting agents. EXPERIMENTAL APPROACH: Using a cell-based assay designed to probe for microtubule polymerization status, we screened a chemical library and identified two azaindole derivatives, CM01 and CM02, as cell-permeable microtubule-depolymerizing agents. The mechanism of the anti-tumour effects of these two compounds was further investigated both in vivo and in vitro. KEY RESULTS: CM01 and CM02 induced G2/M cell cycle arrest and exerted potent cytostatic effects on several cancer cell lines including multidrug-resistant (MDR) cell lines. In vitro experiments revealed that the azaindole derivatives inhibited tubulin polymerization and competed with colchicines for this effect, strongly indicating that tubulin is the cellular target of these azaindole derivatives. In vivo experiments, using a chicken chorioallantoic xenograft tumour assay, established that these compounds exert a potent anti-tumour effect. Furthermore, an assay probing the growth of vessels out of endothelial cell spheroids showed that CM01 and CM02 exert anti-angiogenic activities. CONCLUSIONS AND IMPLICATIONS: CM01 and CM02 are reversible microtubule-depolymerizing agents that exert potent cytostatic effects on human cancer cells of diverse origins, including MDR cells. They were also shown to inhibit angiogenesis and tumour growth in chorioallantoic breast cancer xenografts. Hence, these azaindole derivatives are attractive candidates for further preclinical investigations.