Phenotypic characterisation of SMAD4 variant carriers.

BACKGROUND: Both hereditary haemorrhagic telangiectasia (HHT) and juvenile polyposis syndrome (JPS) are known to be caused by SMAD4 pathogenic variants, with overlapping symptoms for both disorders in some patients. Additional connective tissue disorders have also been reported. Here, we describe carriers of SMAD4 variants followed in an HHT reference centre to further delineate the phenotype. METHODS: Observational study based on data collected from the Clinical Investigation for the Rendu-Osler Cohort database. RESULTS: Thirty-three participants from 15 families, out of 1114 patients with HHT, had an SMAD4 variant (3%).Regarding HHT, 26 out of 33 participants (88%) had a definite clinical diagnosis based on Curaçao criteria. Complication frequencies were as follows: epistaxis (n=27/33, 82%), cutaneous telangiectases (n=19/33, 58%), pulmonary arteriovenous malformations (n=17/32, 53%), hepatic arteriovenous malformations (AVMs) (n=7/18, 39%), digestive angiodysplasia (n=13/22, 59%). No cerebral AVMs were diagnosed.Regarding juvenile polyposis, 25 out of 31 participants (81%) met the criteria defined by Jass et al for juvenile polyposis syndrome. Seven patients (21%) had a prophylactic gastrectomy due to an extensive gastric polyposis incompatible with endoscopic follow-up, and four patients (13%) developed a digestive cancer.Regarding connective tissue disorders, 20 (61%) had at least one symptom, and 4 (15%) participants who underwent echocardiography had an aortic dilation. CONCLUSION: We describe a large cohort of SMAD4 variant carriers in the context of HHT. Digestive complications are frequent, early and diffuse, justifying endoscopy every 2 years. The HHT phenotype, associating pulmonary and hepatic AVMs, warrants systematic screening. Connective tissue disorders broaden the phenotype associated with SMAD4 gene variants and justify systematic cardiac ultrasound and skeletal complications screening.

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 dilatation of pulmonary vessels, resulting in impaired oxygenation. Recent research highlights the pivotal role of liver-produced bone morphogenetic protein (BMP)-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 controls and 203 cirrhotic patients, with or without HPS. Two animal models of portal hypertension were employed: common bile duct ligation (CBDL) with cirrhosis and long-term partial portal vein ligation (PPVL) 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 demonstrated lower levels of circulating BMP-9 compared to patients without HPS. Severe cirrhosis patients exhibited consistently low levels of BMP-9. In animal models, HPS characteristics, including intrapulmonary vascular dilations (IPVDs) and alveolo-arterial gradient enlargement, were observed. 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 BMP pathway in the lungs, alleviating IPVDs, and improving gas exchange impairment. Furthermore, BMP-9 knockout rats displayed a pulmonary HPS phenotype, supporting its role in disease progression. CONCLUSION: 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.

Hereditary hemorrhagic telangiectasia: from signaling insights to therapeutic advances.

Hereditary hemorrhagic telangiectsia (HHT) is an inherited vascular disorder with highly variable expressivity, affecting up to 1 in 5,000 individuals. This disease is characterized by small arteriovenous malformations (AVMs) in mucocutaneous areas (telangiectases) and larger visceral AVMs in the lungs, liver, and brain. HHT is caused by loss-of-function mutations in the BMP9-10/ENG/ALK1/SMAD4 signaling pathway. This Review presents up-to-date insights on this mutated signaling pathway and its crosstalk with proangiogenic pathways, in particular the VEGF pathway, that has allowed the repurposing of new drugs for HHT treatment. However, despite the substantial benefits of these new treatments in terms of alleviating symptom severity, this not-so-uncommon bleeding disorder still currently lacks any FDA- or European Medicines Agency-approved (EMA-approved) therapies.

Warning regarding hematological toxicity of tamoxifen activated CreERT2 in young Rosa26CreERT2 mice.

The Cre-lox system is a versatile and powerful tool used in mouse genetics. It allows spatial and/or temporal control of the deletion of a target gene. The Rosa26-CreERT2 (R26CreERT2) mouse model allows ubiquitous expression of CreERT2. Once activated by tamoxifen, CreERT2 will enter into the nuclei and delete floxed DNA sequences. Here, we show that intraperitoneal injection of tamoxifen in young R26CreERT2 mice leads to morbidity and mortality within 10 days after the first injection, in the absence of a floxed allele. Activation of CreERT2 by tamoxifen led to severe hematological defects, with anemia and a strong disorganization of the bone marrow vascular bed. Cell proliferation was significantly reduced in the bone marrow and the spleen resulting in the depletion of several hematopoietic cells. However, not all cell types or organs were affected to the same extent. We realized that many research groups are not aware of the potential toxicity of Cre recombinases, resulting in misinterpretation of the observed phenotype and in a waste of time and resources. We discuss the necessity to include tamoxifen injected CreERT2 controls lacking a floxed allele in experimental designs and to improve communication about the limitations of Cre-lox mouse models among the scientific community.

In Chagas disease, transforming growth factor beta neutralization reduces Trypanosoma cruzi infection and improves cardiac performance.

Chronic Chagasic cardiomyopathy (CCC), a progressive inflammatory and fibrosing disease, is the most prominent clinical form of Chagas disease, a neglected tropical disease caused by Trypanosoma cruzi infection. During CCC, the parasite remains inside the cardiac cells, leading to tissue damage, involving extensive inflammatory response and irregular fibrosis. Among the fibrogenic factors is transforming growth factor-ß (TGF-ß), a key cytokine controlling extracellular matrix synthesis and degradation. TGF-ß is involved in CCC onset and progression, with increased serum levels and activation of its signaling pathways in the cardiac tissue, which crucially contributes to fibrosis. Inhibition of the TGF-ß signaling pathway attenuates T. cruzi infection and prevents cardiac damage in an experimental model of acute Chagas disease. The aim of this study was to investigate the effect of TGF-ß neutralization on T. cruzi infection in both in vitro and in vivo pre-clinical models, using the 1D11 monoclonal antibody. To this end, primary cultures of cardiac cells were infected with T. cruzi trypomastigote forms and treated with 1D11. For in vivo studies, 1D11 was administered in different schemes for acute and chronic phase models (Swiss mice infected with 104 parasites from the Y strain and C57BL/6 mice infected with 102 parasites from the Colombian strain, respectively). Here we show that the addition of 1D11 to cardiac cells greatly reduces cardiomyocyte invasion by T. cruzi and the number of parasites per infected cell. In both acute and chronic experimental models, T. cruzi infection altered the electrical conduction, decreasing the heart rate, increasing the PR interval and the P wave duration. The treatment with 1D11 reduced cardiac fibrosis and reversed electrical abnormalities improving cardiac performance. Taken together, these data further support the major role of the TGF-ß signaling pathways in T. cruzi-infection and their biological consequences on parasite/host interactions. The therapeutic effects of the 1D11 antibody are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-ß neutralization.

Transforming growth factor-ß as a therapeutic target for the cardiac damage of Chagas disease.

Transforming growth factor beta (TGF-ß) is deeply involved on the pathogenesis of Chagas disease. Our group has been investigating the participation of this pleiotropic cytokine in different aspects of Chagas disease over the last 20 years. Important observations have been made, such as: (i) the ability of Trypanosoma cruzi in activating latent TGF-ß; (ii) the potential involvement of TGF-ß pathway on T. cruzi invasion of host cells; (iii) association of TGF-ß with parasite intracellular replication; (iv) cardiac fibrosis development and maintenance; (v) disruption of Connexin-43 plaque structures and (vi) inflammation and immune response. In this perspective article we intend to discuss the advances of the potential use of new therapies targeting TGF-ß to treat the cardiac alterations of Chagas disease-affected patients.

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.

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.

Transforming growth factor-ß as a therapeutic target for the cardiac damage of Chagas disease

Transforming growth factor beta (TGF-β) is deeply involved on the pathogenesis of Chagas disease. Our group has been investigating the participation of this pleiotropic cytokine in different aspects of Chagas disease over the last 20 years. Important observations have been made, such as: (i) the ability of Trypanosoma cruzi in activating latent TGF-β; (ii) the potential involvement of TGF-β pathway on T. cruzi invasion of host cells; (iii) association of TGF-β with parasite intracellular replication; (iv) cardiac fibrosis development and maintenance; (v) disruption of Connexin-43 plaque structures and (vi) inflammation and immune response. In this perspective article we intend to discuss the advances of the potential use of new therapies targeting TGF-β to treat the cardiac alterations of Chagas disease-affected patients.

The quiescent endothelium: signalling pathways regulating organ-specific endothelial normalcy.

Endothelial cells are at the interface between circulating blood and tissues. This position confers on them a crucial role in controlling oxygen and nutrient exchange and cellular trafficking between blood and the perfused organs. The endothelium adopts a structure that is specific to the needs and function of each tissue and organ and is subject to tissue-specific signalling input. In adults, endothelial cells are quiescent, meaning that they are not proliferating. Quiescence was considered to be a state in which endothelial cells are not stimulated but are instead slumbering and awaiting activating signals. However, new evidence shows that quiescent endothelium is fully awake, that it constantly receives and initiates functionally important signalling inputs and that this state is actively regulated. Signalling pathways involved in the maintenance of functionally quiescent endothelia are starting to be identified and are a combination of endocrine, autocrine, paracrine and mechanical inputs. The paracrine pathways confer a microenvironment on the endothelial cells that is specific to the perfused organs and tissues. In this Review, we present the current knowledge of organ-specific signalling pathways involved in the maintenance of endothelial quiescence and the pathologies associated with their disruption. Linking organ-specific pathways and human vascular pathologies will pave the way towards the development of innovative preventive strategies and the identification of new therapeutic targets.

The Search for Biomarkers and Treatments in Chagas Disease: Insights From TGF-Beta Studies and Immunogenetics.

The anti-inflammatory cytokine transforming growth factor beta (TGF-ß) plays an important role in Chagas disease (CD), a potentially life-threatening illness caused by Trypanosoma cruzi. In this review we revisited clinical studies in CD patients combined with in vitro and in vivo experiments, presenting three main sections: an overview of epidemiological, economic, and clinical aspects of CD and the need for new biomarkers and treatment; a brief panorama of TGF-ß roles and its intracellular signaling pathways, and an update of what is known about TGF-ß and Chagas disease. In in vitro assays, TGF-ß increases during T. cruzi infection and modulates heart cells invasion by the parasite fostering its intracellular parasite cycle. TGF-ß modulates host immune response and inflammation, increases heart fibrosis, stimulates remodeling, and slows heart conduction via gap junction modulation. TGF-ß signaling inhibitors reverts these effects opening a promising therapeutic approach in pre-clinical studies. CD patients with higher TGF-ß1 serum level show a worse clinical outcome, implicating a predictive value of serum TGF-ß as a surrogate biomarker of clinical relevance. Moreover, pre-clinical studies in chronic T. cruzi infected mice proved that inhibition of TGF-ß pathway improved several cardiac electric parameters, reversed the loss of connexin-43 enriched intercellular plaques, reduced fibrosis of the cardiac tissue, restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Finally, TGF-ß polymorphisms indicate that CD immunogenetics is at the base of this phenomenon. We searched in a Brazilian population five single-nucleotide polymorphisms (-800 G>A rs1800468, -509 C>T rs1800469, +10 T>C rs1800470, +25 G>C rs1800471, and +263 C>T rs1800472), showing that CD patients frequently express the TGF-ß1 gene genotypes CT and TT at position -509, as compared to noninfected persons; similar results were observed with genotypes TC and CC at codon +10 of the TGF-ß1 gene, leading to the conclusion that 509 C>T and +10 T>C TGF-ß1 polymorphisms are associated with Chagas disease susceptibility. Studies in genetically different populations susceptible to CD will help to gather new insights and encourage the use of TGF-ß as a CD biomarker.

Eye lymphatic defects induced by bone morphogenetic protein 9 deficiency have no functional consequences on intraocular pressure.

Aqueous humor drainage is essential for the regulation of intraocular pressure (IOP), a major risk factor for glaucoma. The Schlemm's canal and the non-conventional uveoscleral pathway are known to drain aqueous humor from the eye anterior chamber. It has recently been reported that lymphatic vessels are involved in this process, and that the Schlemm's canal responds to some lymphatic regulators. We have previously shown a critical role for bone morphogenetic protein 9 (BMP9) in lymphatic vessel maturation and valve formation, with repercussions in drainage efficiency. Here, we imaged eye lymphatic vessels and analyzed the consequences of Bmp9 (Gdf2) gene invalidation. A network of lymphatic vessel hyaluronan receptor 1 (LYVE-1)-positive lymphatic vessels was observed in the corneolimbus and the conjunctiva. In contrast, LYVE-1-positive cells present in the ciliary bodies were belonging to the macrophage lineage. Although enlarged conjunctival lymphatic trunks and a reduced valve number were observed in Bmp9-KO mice, there were no morphological differences in the Schlemm's canal compared to wild type animals. Moreover, there were no functional consequences on IOP in both basal control conditions and after laser-induced ocular hypertonia. Thus, the BMP9-activated signaling pathway does not constitute a wise target for new glaucoma therapeutic strategies.

Efficacy and Safety of a 0.1% Tacrolimus Nasal Ointment as a Treatment for Epistaxis in Hereditary Hemorrhagic Telangiectasia: A Double-Blind, Randomized, Placebo-Controlled, Multicenter Trial.

Hereditary hemorrhagic telangiectasia is a rare but ubiquitous genetic disease. Epistaxis is the most frequent and life-threatening manifestation and tacrolimus, an immunosuppressive agent, appears to be an interesting new treatment option because of its anti-angiogenic properties. Our objective was to evaluate, six weeks after the end of the treatment, the efficacy on the duration of nosebleeds of tacrolimus nasal ointment, administered for six weeks to patients with hereditary hemorrhagic telangiectasia complicated by nosebleeds, and we performed a prospective, multicenter, randomized, placebo-controlled, double-blinded, ratio 1:1 phase II study. Patients were recruited from three French Hereditary Hemorrhagic Telangiectasia (HHT) centers between May 2017 and August 2018, with a six-week follow-up, and we included people aged over 18 years, diagnosed with hereditary hemorrhagic telangiectasia and epistaxis (total duration > 30 min/6 weeks prior to inclusion). Tacrolimus ointment 0.1% was self-administered by the patients twice daily. About 0.1 g of product was to be administered in each nostril with a cotton swab. A total of 50 patients was randomized and treated. Mean epistaxis duration before and after treatment in the tacrolimus group were 324.64 and 249.14 min, respectively, and in the placebo group 224.69 and 188.14 min, respectively. Epistaxis duration improved in both groups, with no significant difference in our main objective comparing epistaxis before and after treatment (p = 0.77); however, there was a significant difference in evolution when comparing epistaxis before and during treatment (p = 0.04). Toxicity was low and no severe adverse events were reported. In conclusion, tacrolimus nasal ointment, administered for six weeks, did not improve epistaxis in HHT patients after the end of the treatment. However, the good tolerance, associated with a significant improvement in epistaxis duration during treatment, encouraged us to perform a phase 3 trial on a larger patient population with a main outcome of epistaxis duration during treatment and a longer treatment time.

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.

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.

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.

TGF-ß inhibitor therapy decreases fibrosis and stimulates cardiac improvement in a pre-clinical study of chronic Chagas' heart disease.

TGF-ß involvement in Chagas disease cardiomyopathy has been clearly demonstrated. The TGF-ß signaling pathway is activated in the cardiac tissue of chronic phase patients and is associated with an increase in extracellular matrix protein expression. The aim of this study was to investigate the effect of GW788388, a selective inhibitor of TßR1/ALK5, on cardiac function in an experimental model of chronic Chagas' heart disease. To this end, C57BL/6 mice were infected with Trypanosoma cruzi (102 parasites from the Colombian strain) and treated orally with 3mg/kg GW788388 starting at 120 days post-infection (dpi), when 100% of the infected mice show cardiac damage, and following three distinct treatment schedules: i) single dose; ii) one dose per week; or iii) three doses per week during 30 days. The treatment with GW788388 improved several cardiac parameters: reduced the prolonged PR and QTc intervals, increased heart rate, and reversed sinus arrhythmia, and atrial and atrioventricular conduction disorders. At 180 dpi, 30 days after treatment interruption, the GW3x-treated group remained in a better cardiac functional condition. Further, GW788388 treatment reversed the loss of connexin-43 enriched intercellular plaques and reduced fibrosis of the cardiac tissue. Inhibition of the TGF-ß signaling pathway reduced TGF-ß/pSmad2/3, increased MMP-9 and Sca-1, reduced TIMP-1/TIMP-2/TIMP-4, and partially restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Moreover, GW788388 administration did not modify cardiac parasite load during the infection but reduced the migration of CD3+ cells to the heart tissue. Altogether, our data suggested that the single dose schedule was not as effective as the others and treatment three times per week during 30 days seems to be the most effective strategy. The therapeutic effects of GW788388 are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-ß inhibitors.

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.