Coicis semen protects against focal cerebral ischemia-reperfusion injury by inhibiting oxidative stress and promoting angiogenesis via the TGFß/ALK1/Smad1/5 signaling pathway.

BACKGROUND: Ischemic stroke is a devastating disease that causes long-term disability. However, its pathogenesis is unclear, and treatments for ischemic stroke are limited. Recent studies indicate that oxidative stress is involved in the pathological progression of ischemic stroke and that angiogenesis participates in recovery from ischemic stroke. Furthermore, previous studies have shown that Coicis Semen has antioxidative and anti-inflammatory effects in a variety of diseases. In the present study, we investigated whether Coicis Semen has a protective effect against ischemic stroke and the mechanism of this protective effect. RESULTS: Coicis Semen administration significantly decreased the infarct volume and mortality and alleviated neurological deficits at 3, 7 and 14 days after MCAO. In addition, cerebral edema at 3 days poststroke was ameliorated by Coicis Semen treatment. DHE staining showed that ROS levels in the vehicle group were increased at 3 days after reperfusion and then gradually declined, but Coicis Semen treatment reduced ROS levels. The levels of GSH and SOD in the brain were increased by Coicis Semen treatment, while MDA levels were reduced. Furthermore, Coicis Semen treatment decreased the extravasation of EB dye in MCAO mouse brains and elevated expression of the tight junction proteins ZO-1 and Occludin. Double immunofluorescence staining and western blot analysis showed that the expression of angiogenesis markers and TGFß pathway-related proteins was increased by Coicis Semen administration. Consistent with the in vivo results, cytotoxicity assays showed that Coicis Semen substantially promoted HUVEC survival following OGD/RX in vitro. Additionally, though LY2109761 inhibited the activation of TGFß signaling in OGD/RX model animals, Coicis Semen cotreatment markedly reversed the downregulation of TGFß pathway-related proteins and increased VEGF levels. METHODS: Adult male wild-type C57BL/6J mice were used to develop a middle cerebral artery occlusion (MCAO) stroke model. Infarct size, neurological deficits and behavior were evaluated on days 3, 7 and 14 after staining. In addition, changes in superoxide dismutase (SOD), GSH and malondialdehyde (MDA) levels were detected with a commercial kit. Blood-brain barrier (BBB) permeability was assessed with Evans blue (EB) dye. Western blotting was also performed to measure the levels of tight junction proteins of the BBB. Additionally, ELISA was performed to measure the level of VEGF in the brain. The colocalization of CD31, angiogenesis markers, and Smad1/5 was assessed by double immunofluorescent staining. TGFß pathway-related proteins were measured by western blotting. Furthermore, the cell viability of human umbilical vein endothelial cells (HUVECs) following oxygen-glucose deprivation/reoxygenation (OGD/RX) was measured by Cell Counting Kit (CCK)-8 assay. CONCLUSIONS: Coicis Semen treatment alleviates brain damage induced by ischemic stroke through inhibiting oxidative stress and promoting angiogenesis by activating the TGFß/ALK1 signaling pathway.

Activin type II receptor ligand signaling inhibition after experimental ischemic heart failure attenuates cardiac remodeling and prevents fibrosis.

Myostatin (MSTN) is a transforming growth factor (TGF)-ß superfamily member that acts as a negative regulator of muscle growth and may play a role in cardiac remodeling. We hypothesized that inhibition of activin type II receptors (ACTRII) to reduce MSTN signaling would reduce pathological cardiac remodeling in experimental heart failure (HF). C57BL/6J mice underwent left anterior descending coronary artery ligation under anesthesia to induce myocardial infarction (MI) or no ligation (sham). MI and sham animals were each randomly divided into groups (n ≥ 10 mice/group) receiving an ACTRII or ACTRII/TGFß receptor-signaling inhibiting strategy: 1) myo-Fc group (weekly 10 mg/kg Myo-Fc) or 2) Fol + TGFi group (daily 12 µg/kg follistatin plus 2 mg/kg TGFß receptor inhibitor), versus controls. ACTRII/TGFBR signaling inhibition preserved cardiac function by echocardiography and prevented an increase in brain natriuretic peptide (BNP). ACTRII/TGFBR inhibition resulted in increased phosphorylation (P) of Akt and decreased P-p38 mitogen-activated protein kinase (MAPK) in MI mice. In vitro, Akt contributed to P-SMAD2,3, P-p38, and BNP regulation in cardiomyocytes. ACTRII/TGFBR inhibition increased sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) levels and decreased unfolded protein response (UPR) markers in MI mice. ACTRII/TGFBR inhibition was associated with a decrease in cardiac fibrosis and fibrosis markers, connective tissue growth factor (CTGF), type I collagen, fibronectin, α-smooth muscle actin, and matrix metalloproteinase (MMP)-12 in MI mice. MSTN exerted a direct regulation on the UPR marker eukaryotic translation initiation factor-2α (eIf2α) in cardiomyocytes. Our study suggests that ACTRII ligand inhibition has beneficial effects on cardiac signaling and fibrosis after ischemic HF.NEW & NOTEWORTHY Activin type II receptor ligand inhibition resulted in preserved cardiac function, a decrease in cardiac fibrosis, improved SERCA2a levels, and a prevention of the unfolded protein response in mice with myocardial infarction.

Restriction of spontaneous and prednisolone-induced leptin production to dedifferentiated state in human hip OA chondrocytes: role of Smad1 and ß-catenin activation.

OBJECTIVE: The aetiology of OA is not fully understood although several adipokines such as leptin are known mediators of disease progression. Since leptin levels were increased in synovial fluid compared to serum in OA patients, it was suggested that joint cells themselves could produce leptin. However, exact mechanisms underlying leptin production by chondrocytes are poorly understood. Nevertheless, prednisolone, although displaying powerful anti-inflammatory properties has been recently reported to be potent stimulator of leptin and its receptor in OA synovial fibroblasts. Therefore, we investigated, in vitro, spontaneous and prednisolone-induced leptin production in OA chondrocytes, focusing on transforming growth factor-ß (TGFß) and Wnt/ß-catenin pathways. DESIGN: We used an in vitro dedifferentiation model, comparing human freshly isolated hip OA chondrocytes cultivated in monolayer during 1 day (type II, COL2A1 +; type X, COL10A1 + and type I collagen, COL1A1 -) or 14 days (COL2A1 -; COL10A1 - and COL1A1+). RESULTS: Leptin expression was not detected in day1 OA chondrocytes whereas day14 OA chondrocytes produced leptin, significantly increased with prednisolone. Activin receptor-like kinase 1 (ALK1)/ALK5 ratio was shifted during dedifferentiation, from high ALK5 and phospho (p)-Smad2 expression at day1 to high ALK1, endoglin and p-Smad1/5 expression at day14. Moreover, inactive glycogen synthase kinase 3 (GSK3) and active ß-catenin were only found in dedifferentiated OA chondrocytes. Smad1 and ß-catenin but not endoglin stable lentiviral silencing led to a significant decrease in leptin production by dedifferentiated OA chondrocytes. CONCLUSIONS: Only dedifferentiated OA chondrocytes produced leptin. Prednisolone markedly enhanced leptin production, which involved Smad1 and ß-catenin activation.

A small molecule targeting ALK1 prevents Notch cooperativity and inhibits functional angiogenesis.

Activin receptor-like kinase 1 (ALK1, encoded by the gene ACVRL1) is a type I BMP/TGF-ß receptor that mediates signalling in endothelial cells via phosphorylation of SMAD1/5/8. During angiogenesis, sprouting endothelial cells specialise into tip cells and stalk cells. ALK1 synergises with Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2 and thereby represses tip cell formation and angiogenic sprouting. The ALK1-Fc soluble protein fusion has entered clinic trials as a therapeutic strategy to sequester the high-affinity extracellular ligand BMP9. Here, we determined the crystal structure of the ALK1 intracellular kinase domain and explored the effects of a small molecule kinase inhibitor K02288 on angiogenesis. K02288 inhibited BMP9-induced phosphorylation of SMAD1/5/8 in human umbilical vein endothelial cells to reduce both the SMAD and the Notch-dependent transcriptional responses. In endothelial sprouting assays, K02288 treatment induced a hypersprouting phenotype reminiscent of Notch inhibition. Furthermore, K02288 caused dysfunctional vessel formation in a chick chorioallantoic membrane assay of angiogenesis. Such activity may be advantageous for small molecule inhibitors currently in preclinical development for specific BMP gain of function conditions, including diffuse intrinsic pontine glioma and fibrodysplasia ossificans progressiva, as well as more generally for other applications in tumour biology.

Estrogen therapy for hereditary haemorrhagic telangiectasia (HHT): Effects of raloxifene, on Endoglin and ALK1 expression in endothelial cells.

Hereditary haemorrhagic telangiectasia (HHT), or Rendu-Osler-Weber syndrome, is an autosomal dominant vascular disease. The clinical manifestations are epistaxis, mucocutaneous and gastrointestinal telangiectases, and arteriovenous malformations. There are two predominant types of HHT caused by mutations in Endoglin (ENG) and activin receptor-like kinase 1 (ALK1) (ACVRL1) genes, HHT1 and HHT2, respectively. No cure for HHT has been found and there is a current need to find new effective drug treatments for the disease. Some patients show severe epistaxis which interferes with their quality of life. We report preliminary results obtained with Raloxifene to treat epistaxis in postmenopausal HHT women diagnosed with osteoporosis. We tried to unravel the molecular mechanisms involved in the therapeutic effects of raloxifene. ENG and ACVRL1 genes code for proteins involved in the transforming growth factor beta pathway and it is widely accepted that haploinsufficiency is the origin for the pathogenicity of HHT. Therefore, identification of drugs able to increase the expression of those genes is essential to propose new therapies for HHT. In vitro results show that raloxifene increases the protein and mRNA expression of ENG and ALK1 in cultured endothelial cells. Raloxifene also stimulates the promoter activity of these genes, suggesting a transcriptional regulation of ENG and ALK1. Furthermore, Raloxifene improved endothelial cell functions like tubulogenesis and migration in agreement with the reported functional roles of Endoglin and ALK1. Our pilot study provides a further hint that oral administration of raloxifene may be beneficial for epistaxis treatment in HHT menopausal women. The molecular mechanisms of raloxifene involve counteracting the haploinsufficiency of ENG and ALK1.

Narrative review: the enigma of pulmonary arterial hypertension: new insights from genetic studies.

Pulmonary arterial hypertension (PAH) occurs as an idiopathic disease (formerly called primary pulmonary hypertension) and as a consequence of other illnesses. These illnesses include connective tissue diseases, portal hypertension, diet and stimulant drug use, HIV infection, and congenital heart disease. Inherited susceptibility to PAH occurs in families and is almost always due to mutations in genes of the TGF-beta family of receptors. The most common mutation leading to PAH is in bone morphogenetic protein receptor type 2 (BMPR2), originally discovered to be involved in bone healing. Mutations in BMPR2 have also been found in patients with idiopathic PAH, although the true prevalence of this susceptibility has not been determined. About 20% of individuals with a BMPR2 mutation develop symptomatic pulmonary hypertension. Evidence is growing that imbalanced activation of other TGF-beta receptors coupled with reduced activity of mutated BMPR2 increases the likelihood of development of PAH. Many signaling systems have been found to participate in PAH, including K channels, serotonin, angiopoietin, and cyclooxygenases. An interaction of these signaling systems with BMPR2 is a focus of research in PAH. Approaches to altering the imbalance of activation of BMPR2 and other TGF-beta receptors may yield future therapies for PAH.

Activin type 2 receptor restoration in MSI-H colon cancer suppresses growth and enhances migration with activin.

BACKGROUND & AIMS: Colon cancers with high-frequency microsatellite instability (MSI-H) develop frameshift mutations in tumor suppressors as part of their pathogenesis. ACVR2 is mutated at its exon 10 polyadenine tract in >80% of MSI-H colon cancers, coinciding with loss of protein. ACVR2 transmits the growth effects of activin via phosphorylation of SMAD proteins to affect gene transcription. The functional effect of activin in colon cancers has not been studied. We developed and characterized a cell model in which we studied how activin signaling affects growth. METHODS: hMLH1 and ACVR2 mutant HCT116 cells were previously stably transferred with chromosome 2 (HCT116+chr2), restoring a single regulated copy of wild-type ACVR2 but not hMLH1. Both HCT116+chr2 and parental HCT116 cells (as well as HEC59 and ACVR2 and hMSH2 complemented HEC59+chr2 cells) were assessed for genetic complementation and biologic function. RESULTS: HCT116+chr2 cells and HEC59+chr2 cells, but not ACVR2-mutant HCT116 or HEC59 cells, acquired wild-type ACVR2 as well as expression of ACVR2 wild-type messenger RNA. Complemented ACVR2 protein complexed with ACVR1 with activin treatment, generating nuclear phosphoSMAD2 and activin-specific gene transcription. ACVR2-restored cells showed decreased growth and reduced S phase but increased cellular migration following activin treatment. ACVR2 small interfering RNA reversed these effects in complemented cells. CONCLUSIONS: ACVR2-complemented MSI-H colon cancers restore activin-SMAD signaling, decrease growth, and slow their cell cycle following ligand stimulation but show increased cellular migration. Activin is growth suppressive and enhances migration similar to transforming growth factor beta in colon cancer, indicating that abrogation of the effects of activin contribute to the pathogenesis of MSI-H colon cancers.

Possible endocrine control by follistatin 315 during liver regeneration based on changes in the activin receptor after a partial hepatectomy in rats.

BACKGROUND/AIMS: Activin A is an autocrine inhibitor of cell growth in the liver. The biological activity of activin A is mediated by a heteromeric receptor complex. Follistatin (FS) binds to activin and inhibits its biological effects, and acts as a negative regulator of muscle cells. The role of activin receptors during liver regeneration following a hepatectomy has not been fully assessed. This study investigates the mechanism underlying how activin receptors regulate hepatocyte growth, and the effects of intravenous administration of FS during liver regeneration. METHODOLOGY: The expression of both activins and activin receptors in the liver after a 70% partial hepatectomy (HT) was assessed by RT-PCR and immunohistochemistry. FS 315 or 288 was infused for different periods of time based on changes in hepatocyte activin receptor expression after HT. RESULTS: Activin receptor expression peaked between 48 and 72 hours after HT. 72 hours after HT, an injection of FS 315 resulted in a more potent stimulation of DNA synthesis and produced a greater increase in body weight compared with the control rats. CONCLUSIONS: The expression of activin receptors after peak DNA synthesis might be a key component in the downregulation of DNA synthesis. Intravenous administration of FS 315 might promote liver regeneration and have anabolic actions.