Intermedin alleviates diabetic vascular calcification by inhibiting GLUT1 through activation of the cAMP/PKA signaling pathway.

BACKGROUND AND AIMS: Vascular calcification (VC) is regarded as an independent risk factor for cardiovascular events in type 2 diabetic patients. Glucose transporter 1 (GLUT1) involves VC. Intermedin/Adrenomedullin-2 (IMD/ADM2) is a cardiovascular protective peptide that can inhibit multiple disease-associated VC. However, the role and mechanism of IMD in diabetic VC remain unclear. Here, we investigated whether IMD inhibits diabetic VC by inhibiting GLUT1. METHODS AND RESULTS: It was found that plasma IMD concentration was significantly decreased in type 2 diabetic patients and in fructose-induced diabetic rats compared with that in controls. Plasma IMD content was inversely correlated with fasting blood glucose level and VC severity. IMD alleviated VC in fructose-induced diabetic rats. Deficiency of Adm2 aggravated and Adm2 overexpression attenuated VC in high-fat diet-induced diabetic mice. In vitro, IMD mitigated high glucose-induced calcification of vascular smooth muscle cells (VSMCs). Mechanistically, IMD reduced advanced glycation end products (AGEs) content and the level of receptor for AGEs (RAGE). IMD decreased glucose transporter 1 (GLUT1) levels. The inhibitory effect of IMD on RAGE protein level was blocked by GLUT1 knockdown. GLUT1 knockdown abolished the effect of IMD on alleviating VSMC calcification. IMD receptor antagonist IMD17-47 and cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) inhibitor H89 abolished the inhibitory effects of IMD on GLUT1 and VSMC calcification. CONCLUSIONS: These findings revealed that IMD exerted its anti-calcification effect by inhibiting GLUT1, providing a novel therapeutic target for diabetic VC.

Intermedin Alleviates Vascular Calcification in CKD through Sirtuin 3-Mediated Inhibition of Mitochondrial Oxidative Stress.

Vascular calcification (VC) is a common pathophysiological process of chronic kidney disease (CKD). Sirtuin 3 (Sirt3), a major NAD+-dependent protein deacetylase predominantly in mitochondria, is involved in the pathogenesis of VC. We previously reported that intermedin (IMD) could protect against VC. In this study, we investigated whether IMD attenuates VC by Sirt3-mediated inhibition of mitochondrial oxidative stress. A rat VC with CKD model was induced by the 5/6 nephrectomy plus vitamin D3. Vascular smooth muscle cell (VSMC) calcification was induced by CaCl2 and ß-glycerophosphate. IMD1-53 treatment attenuated VC in vitro and in vivo, rescued the depressed mitochondrial membrane potential (MMP) level and decreased mitochondrial ROS levels in calcified VSMCs. IMD1-53 treatment recovered the reduced protein level of Sirt3 in calcified rat aortas and VSMCs. Inhibition of VSMC calcification by IMD1-53 disappeared when the cells were Sirt3 absent or pretreated with the Sirt3 inhibitor 3-TYP. Furthermore, 3-TYP pretreatment blocked IMD1-53-mediated restoration of the MMP level and inhibition of mitochondrial oxidative stress in calcified VSMCs. The attenuation of VSMC calcification by IMD1-53 through upregulation of Sirt3 might be achieved through activation of the IMD receptor and post-receptor signaling pathway AMPK, as indicated by pretreatment with an IMD receptor antagonist or AMPK inhibitor blocking the inhibition of VSMC calcification and upregulation of Sirt3 by IMD1-53. AMPK inhibitor treatment reversed the effects of IMD1-53 on restoring the MMP level and inhibiting mitochondrial oxidative stress in calcified VSMCs. In conclusion, IMD attenuates VC by improving mitochondrial function and inhibiting mitochondrial oxidative stress through upregulating Sirt3.

Intermedin1-53 Inhibits NLRP3 Inflammasome Activation by Targeting IRE1α in Cardiac Fibrosis.

Intermedin (IMD), a paracrine/autocrine peptide, protects against cardiac fibrosis. However, the underlying mechanism remains poorly understood. Previous study reports that activation of nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cardiac fibrosis. In this study, we aimed to investigate whether IMD mitigated cardiac fibrosis by inhibiting NLRP3. Cardiac fibrosis was induced by angiotensin II (Ang II) infusion for 2 weeks in rats. Western blot, real-time PCR, histological staining, immunofluorescence assay, RNA sequencing, echocardiography, and hemodynamics were used to detect the role and the mechanism of IMD in cardiac fibrosis. Ang II infusion resulted in rat cardiac fibrosis, shown as over-deposition of myocardial interstitial collagen and cardiac dysfunction. Importantly, NLRP3 activation and endoplasmic reticulum stress (ERS) were found in Ang II-treated rat myocardium. Ang II infusion decreased the expression of IMD and increased the expression of the receptor system of IMD in the fibrotic rat myocardium. IMD treatment attenuated the cardiac fibrosis and improved cardiac function. In addition, IMD inhibited the upregulation of NLRP3 markers and ERS markers induced by Ang II. In vitro, IMD knockdown by small interfering RNA significantly promoted the Ang II-induced cardiac fibroblast and NLRP3 activation. Moreover, silencing of inositol requiring enzyme 1 α (IRE1α) blocked the effects of IMD inhibiting fibroblast and NLRP3 activation. Pre-incubation with PKA pathway inhibitor H89 blocked the effects of IMD on the anti-ERS, anti-NLRP3, and anti-fibrotic response. In conclusion, IMD alleviated cardiac fibrosis by inhibiting NLRP3 inflammasome activation through suppressing IRE1α via the cAMP/PKA pathway.

Intermedin1-53 attenuates atherosclerotic plaque vulnerability by inhibiting CHOP-mediated apoptosis and inflammasome in macrophages.

Atherosclerotic plaque vulnerability and rupture increase the risk of acute coronary syndromes. Advanced lesion macrophage apoptosis plays important role in the rupture of atherosclerotic plaque, and endoplasmic reticulum stress (ERS) has been proved to be a key mechanism of macrophage apoptosis. Intermedin (IMD) is a regulator of ERS. Here, we investigated whether IMD enhances atherosclerotic plaque stability by inhibiting ERS-CHOP-mediated apoptosis and subsequent inflammasome in macrophages. We studied the effects of IMD on features of plaque vulnerability in hyperlipemia apolipoprotein E-deficient (ApoE-/-) mice. Six-week IMD1-53 infusion significantly reduced atherosclerotic lesion size. Of note, IMD1-53 lowered lesion macrophage content and necrotic core size and increased fibrous cap thickness and vascular smooth muscle cells (VSMCs) content thus reducing overall plaque vulnerability. Immunohistochemical analysis indicated that IMD1-53 administration prevented ERS activation in aortic lesions of ApoE-/- mice, which was further confirmed in oxidized low-density lipoproteins (ox-LDL) induced macrophages. Similar to IMD, taurine (Tau), a non-selective ERS inhibitor significantly reduced atherosclerotic lesion size and plaque vulnerability. Moreover, C/EBP-homologous protein (CHOP), a pro-apoptosis transcription factor involved in ERS, was significantly increased in advanced lesion macrophages, and deficiency of CHOP stabilized atherosclerotic plaques in AopE-/- mice. IMD1-53 decreased CHOP level and apoptosis in vivo and in macrophages treated with ox-LDL. In addition, IMD1-53 infusion ameliorated NLRP3 inflammasome and subsequent proinflammatory cytokines in vivo and in vitro. IMD may attenuate the progression of atherosclerotic lesions and plaque vulnerability by inhibiting ERS-CHOP-mediated macrophage apoptosis, and subsequent NLRP3 triggered inflammation. The inhibitory effect of IMD on ERS-induced macrophages apoptosis was probably mediated by blocking CHOP activation.

Inhibition of Notch1-mediated inflammation by intermedin protects against abdominal aortic aneurysm via PI3K/Akt signaling pathway.

The Notch1-mediated inflammatory response participates in the development of abdominal aortic aneurysm (AAA). The vascular endogenous bioactive peptide intermedin (IMD) plays an important role in maintaining vascular homeostasis. However, whether IMD inhibits AAA by inhibiting Notch1-mediated inflammation is unclear. In this study, we found Notch intracellular domain (NICD) and hes1 expression were higher in AAA patients' aortas than in healthy controls. In angiotensin II (AngII)-induced AAA mouse model, IMD treatment significantly reduced AAA incidence and maximal aortic diameter. IMD inhibited AngII-enlarged aortas and -degraded elastic lamina, reduced NICD, hes1 and inflammatory factors expression, decreased infiltration of CD68 positive macrophages and the NOD-like receptor family pyrin domain containing 3 protein level. IMD inhibited lipopolysaccharide-induced macrophage migration in vitro and regulated macrophage polarization. Moreover, IMD overexpression significantly reduced CaCl2-induced AAA incidence and down-regulated NICD and hes1 expression. However, IMD deficiency showed opposite results. Mechanically, IMD treatment significantly decreased cleavage enzyme-a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) level. Pre-incubation with IMD17-47 (IMD receptors blocking peptide) and the phosphatidylinositol 3-kinase/protein kinase b (PI3K/Akt) inhibitor LY294002 reversed ADAM10 level. In conclusion, exogenous and endogenous IMD could inhibit the development of AAA by inhibiting Notch1 signaling-mediated inflammation via reducing ADAM10 through IMD receptor and PI3K/Akt pathway.

STAT3 Promotes Schistosome-Induced Liver Injury by Inflammation, Oxidative Stress, Proliferation, and Apoptosis Signal Pathway.

Schistosomiasis is a parasitic helminth disease that can cause organ lesions leading to health damage. During a schistosome infection, schistosome eggs can flow into the liver along the portal vein. Numerous inflammatory cells gather around the eggs, causing granulomas and fibrosis in the liver. In this process, many molecules are involved in the initiation and regulation of the fibrous scar formation. However, the precise molecular mechanisms responsible for the progression of granuloma formation and fibrosis initiation caused by schistosome infection have not been extensively studied. In this study, C57BL/6 wild-type mice and Stat3flox/flox Alb-Cre mice were infected with cercariae of Schistosoma japonicum Liver injury, effector molecule levels, and RNA transcriptome resequencing of liver tissue were detected at 4, 5, and 6 weeks postinfection. We investigated the role of STAT3 (signal transducer and activator of transcription 3) in Schistosoma-induced liver injury in mice. After 6 weeks postinfection, there was obvious liver fibrosis. A sustained pathological process (inflammation, oxidative stress, proliferation, and apoptosis) occurred in S. japonicum-induced liver fibrosis initiation. Meanwhile, we observed activation of the STAT3 pathway in hepatic injury during S. japonicum infection by RNA transcriptome resequencing. Liver deficiency of phospho-STAT3 alleviated infection-induced liver dysfunction, hepatic granuloma formation, and fibrosis initiation. It also promoted STAT3-dependent apoptosis and reduced liver inflammation, oxidative stress, and proliferation. Our results suggest that STAT3 signal pathway and its mediating inflammation, oxidative stress, proliferation, and apoptosis are involved in S. japonicum-induced liver injury and may be a new potential guideline for the treatment of schistosomiasis.

STAT3: A key regulator in liver fibrosis.

Janus protein tyrosine kinase (JAK) has the ability to activate signal transducer and activator of transcription (STAT). STAT3 is a valued member of the JAK/STAT signaling pathway. In recent years, several studies have documented that STAT3 is closely related to the occurrence and development of liver fibrosis caused by various factors. Activation of STAT3 can play anti- or pro-inflammatory roles in the pathogenesis of liver fibrosis. This article reviewed the recent studies on STAT3 in the development of various liver fibrosis to find a more effective method to relieve and cure liver diseases, such as hepatitis B virus (HBV), non-alcoholic fatty liver disease (NAFLD), schistosomiasis, and chemical liver injury.

Intermedin alleviates pathological cardiac remodeling by upregulating klotho.

Cardiac remodeling is accompanied by cardiac hypertrophy, fibrosis, dysfunction, and eventually leading to heart failure. Intermedin (IMD), as a paracrine/autocrine peptide, has a protective effect in cardiovascular diseases. In this study, we elucidated the role and the underlying mechanism of IMD in pathological remodeling. Pathological remodeling mouse models were induced by abdominal aorta constriction for 4 weeks or angiotensin II (Ang II) infusion for 2 weeks in wildtype, IMD-overexpression, IMD-knockout and klotho-knockdown mice. Western blot, real-time PCR, histological staining, echocardiography and hemodynamics were used to detect the role of IMD in cardiac remodeling. Cardiac hypertrophy, fibrosis and dysfunction were significantly aggravated in IMD-knockout mice versus wildtype mice, and the expression of klotho was downregulated. Conversely, cardiac remodeling was alleviated in IMD-overexpression mice, and the expression of klotho was upregulated. Hypertension induced by Ang II infusion rather than abdominal aorta constriction was mitigated by IMD. However, the cardioprotective effect of IMD was blocked in klotho-knockdown mice. Similar results were found in cultured neonatal rat cardiomyocytes, which was pretreated with IMD before Ang II stimulation. Mechanistically, IMD inhibited the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the activity of calcineurin to protect against cardiac hypertrophy through upregulating klotho in vivo and in vitro. Furthermore, peroxisome proliferator-activated receptor γ (PPARγ) might mediate IMD upregulating klotho. In conclusion, pathological remodeling may be alleviated by endogenous IMD, which inhibits the expression of calcineurin and p-CaMKII by upregulating klotho via the PPARγ pathway. It suggested that IMD might be a therapeutic target for heart disease.

Intermedin1-53 attenuates aging-associated vascular calcification in rats by upregulating sirtuin 1.

Vascular calcification is a common phenomenon in older adults. Intermedin (IMD) is a cardiovascular bioactive peptide inhibiting vascular calcification. In this study, we aimed to investigate whether IMD1-53 attenuates aging-associated vascular calcification. Vascular calcification was induced by vitamin D3 plus nicotine (VDN) in young and old rats. The calcification in aortas was more severe in old rats treated with VDN than young control rats, and IMD expression was lower. Exogenous administration of IMD1-53 significantly inhibited the calcium deposition in aortas and the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) in VDN-treated old rats. Moreover, levels of aging-related p16, p21 and ß-galactosidase were all greatly decreased by IMD1-53. These results were further confirmed in rat and human VSMCs in vitro. In addition, IMD-deficient mouse VSMCs showed senescence features coinciding with osteogenic transition as compared with wild-type mouse VSMCs. Mechanistically, IMD1-53 significantly increased the expression of the anti-aging factor sirtuin 1 (sirt1); the inhibitory effects of IMD1-53 on calcification and senescence were blocked by sirt1 knockdown. Furthermore, preincubation with inhibitors of PI3K, AMPK or PKA efficiently blunted the upregulatory effect of IMD1-53 on sirt1. Consequently, IMD1-53 could attenuate aging-associated vascular calcification by upregulating sirt1 via activating PI3K/Akt, AMPK and cAMP/PKA signaling.

Intermedin1-53 Ameliorates Homocysteine-Promoted Atherosclerotic Calcification by Inhibiting Endoplasmic Reticulum Stress.

AIM: Vascular calcification (VC) is thought to be an independent predictor of cardiovascular morbidity and mortality. Intermedin1-53 (IMD) is a cardiovascular protective peptide and can inhibit vascular medial calcification in rats. In this study, we investigated the effect of IMD on atherosclerotic calcification induced by a high-fat diet plus homocysteine (Hcy) and the potential mechanisms. METHODS: ApoE-/- mice were fed a high-fat diet with Hcy in drinking water to induce atherosclerotic calcification. RESULTS: As compared to the high-fat diet alone, Hcy treatment significantly increased atherosclerotic lesion areas and the number of calcified nodules in aortic roots and was reduced by IMD infusion or 4-phenylbutyric acid (PBA) treatment. In vitro, as compared to calcifying medium alone, Hcy treatment further increased alkaline phosphatase activity, calcium content, and calcium nodule number in human aorta vascular smooth muscle cells (HA-VSMCs), all blocked by IMD or PBA pretreatment. Mechanistically, IMD or PBA significantly alleviated endoplasmic reticulum stress (ERS) activation compared with Hcy treatment. In parallel, IMD or PBA attenuated the messenger RNA levels of osteogenic markers and inflammatory cytokines in aortas and their protein levels in lesions of aortic roots. In vitro, Hcy treatment significantly increased the protein levels of osteoblast-like cell markers in primary rat VSMCs and inflammation markers in mouse peritoneal macrophages, all decreased with IMD or PBA pretreatment. Intermedin1-53 pretreatment also markedly reduced the protein levels of ERS markers in rat VSMCs and mouse peritoneal macrophages. CONCLUSIONS: Intermedin1-53 protects against Hcy-promoted atherosclerotic calcification in ApoE-/- mice by inhibiting ERS.

Taurine Alleviates Schistosoma-Induced Liver Injury by Inhibiting the TXNIP/NLRP3 Inflammasome Signal Pathway and Pyroptosis.

Schistosomiasis is a parasitic helminth disease that can cause severe inflammatory pathology, leading to organ damage, in humans. During a schistosomal infection, the eggs are trapped in the host liver, and products derived from eggs induce a polarized Th2 cell response, resulting in granuloma formation and eventually fibrosis. Previous studies indicated that the nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is involved in schistosomiasis-associated liver fibrosis and that taurine could ameliorate hepatic granulomas and fibrosis caused by Schistosoma japonicum infection. Nevertheless, the precise role and molecular mechanism of the NLRP3 inflammasome and the protective effects of taurine in S. japonicum infection have not been extensively studied. In this study, we investigated the role of the NLRP3 inflammasome and the hepatoprotective mechanism of taurine in schistosoma-induced liver injury in mice. NLRP3 deficiency ameliorated S. japonicum-infection-induced hepatosplenomegaly, liver dysfunction, and hepatic granulomas and fibrosis; it also reduced NLRP3-dependent liver pyroptosis. Furthermore, taurine suppressed hepatic thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation in mice with S. japonicum infections, thereby inhibiting the activation of downstream inflammatory mediators such as interleukin-1ß and subsequent pyroptosis. Our results suggest that the TXNIP/NLRP3 inflammasome pathway and mediating pyroptosis are involved in S. japonicum-induced liver injury and may be a potential therapeutic target for schistosomiasis treatment. In addition, taurine may be useful to alleviate or to prevent the occurrence of schistosomiasis-associated liver fibrosis.

Endogenous intermedin protects against intimal hyperplasia by inhibiting endoplasmic reticulum stress.

Extensive proliferation of vascular smooth muscle cell (VSMC) contributes to intimal hyperplasia following vascular injury, in which endoplasmic reticulum stress (ERS) plays a critical role. Intermedin (IMD) is a vascular paracrine/autocrine peptide exerting numerous beneficial effects in cardiovascular diseases. IMD overexpression could alleviate intimal hyperplasia. Here, we investigated whether endogenous IMD protects against intimal hyperplasia by inhibiting endoplasmic reticulum stress. The mouse left common carotid-artery ligation-injury model was established to induce intimal hyperplasia using IMD-/-mice and C57BL/6 J wild-type (WT) mice. Platelet-derived growth factor-BB (PDGF-BB) was used to stimulate the proliferation of VSMC. IMD-/- mice displayed exacerbated intimal hyperplasia induced by complete ligation of the left carotid artery at 14 d and 28 d compared to WT mice. However, IMD-deficiency had no effect on blood pressure, plasma triglyceride, and fasting blood glucose levels in mice. Furthermore, VSMCs derived from IMD-/- mice showed increased cell proliferation and dramatically elevated levels of glucose regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), ATF6 mRNA under PDGF-BB treatment compared to WT mice-derived VSMCs. In addition, exogenous administration of IMD significantly attenuated PDGF-BB-induced cell proliferation and GRP78, phosphorylase-inositol requiring enzyme 1α, ATF4, and ATF6 protein levels. Thus, endogenous IMD may counteract ERS to exert protective role in response to vascular injury and IMD is expected to be a therapeutic target for the prevention and treatment of restenosis.

Inhibition of endoplasmic reticulum stress by intermedin1-53 attenuates angiotensin II-induced abdominal aortic aneurysm in ApoE KO Mice.

Endoplasmic reticulum stress (ERS) is involved in the development of abdominal aortic aneurysm (AAA). Since bioactive peptide intermedin (IMD)1-53 protects against AAA formation, here we investigated whether IMD1-53 attenuates AAA by inhibiting ERS. AAA model was induced by angiotensin II (AngII) in ApoE KO mouse background. AngII-treated mouse aortas showed increased ERS gene transcription of caspase12, eukaryotic translation initiation factor 2a (eIf2a) and activating transcription factor 4(ATF4).The protein level of ERS marker glucose regulated protein 94(GRP94), ATF4 and C/EBP homologous protein 10(CHOP) was also up-regulated by AngII. Increased ERS levels were accompanied by severe VSMC apoptosis in human AAA aorta. In vivo administration of IMD1-53 greatly reduced AngII-induced AAA and abrogated the activation of ERS. To determine whether IMD inhibited AAA by ameliorating ERS, we used 2 non-selective ERS inhibitors phenyl butyrate (4-PBA) and taurine (TAU). Similar to IMD, PBA, and TAU significantly reduced the incidence of AAA and AAA-related pathological disorders. In vitro, AngII infusion up-regulated CHOP, caspase12 expression and led to VSMC apoptosis. IMD siRNA aggravated the CHOP, caspase12-mediated VSMC apoptosis, which was abolished by ATF4 silencing. IMD infusion promoted the phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) in aortas in ApoE KO mice, and the AMPK inhibitor compound C abolished the protective effect of IMD on VSMC ERS and apoptosis induced by AngII. In conclusion, IMD may protect against AAA formation by inhibiting ERS via activating AMPK phosphorylation.

Intermedin reduces neointima formation by regulating vascular smooth muscle cell phenotype via cAMP/PKA pathway.

BACKGROUND AND AIMS: Vascular smooth muscle cell (VSMC) dedifferentiation contributes to neointima formation, which results in various vascular disorders. Intermedin (IMD), a cardiovascular paracrine/autocrine polypeptide, is involved in maintaining circulatory homeostasis. However, whether IMD protects against neointima formation remains largely unknown. The purpose of this study is to investigate the role of IMD in neointima formation and the possible mechanism. METHODS: IMD1-53 (100ng/kg/h) or saline water was used on rat carotid-artery balloon-injury model. The mouse left common carotid-artery ligation-injury model was established using IMD-transgenic and C57BL/6J mice. Immunohistochemistry and immunofluorescence staining was used to detect the protein expression in rat carotid arteries. Radioimmunoassay was used to determine the serum IMD level. The hematoxylin andeosin staining was used for carotid arteries morphological testing. In vitro, for rat primary cultured VSMC phenotype transition, proliferation and migration assays, platelet-derived growth factor-BB (PDGF-BB) reagent and IMD1-53 peptide were added to the culture media at the final concentration of 20 ng/mL and 10-7mol/L respectively. Quantification of VSMC proliferation involved MTT and BrdU assay and migration was detected by wound-healing assay. Western blot and realtime PCR were used to detect the protein and mRNA levels of tissues or cells. RESULTS: With the rat carotid-artery balloon-injury model, IMD was significantly downregulated in injured arteries and plasma. Exogenous IMD1-53 greatly inhibited neointima formation and prevented VSMC from switching to a synthetic phenotype. With the left common carotid-artery ligation-injury model, IMD-transgenic mice showed less neointima formation than C57BL/6J mice. PDGF-BB reduced IMD mRNA expression in rat primary cultured VSMCs but increased that of its receptors, calcitonin receptor-like receptor or receptor activity-modifying proteins. Furthermore, PDGF-BB promoted VSMC proliferation and migration and transformed VSMCs to the synthetic phenotype, which was reversed with IMD1-53 treatment. Mechanistically, IMD1-53 maintained the contractile VSMC phenotype via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway. CONCLUSIONS: IMD attenuated neointima formation both in the rat model of carotid-artery balloon injury and mouse model of common carotid-artery ligation injury. IMD protection may be mediated by maintaining a VSMC contractile phenotype via the cAMP/PKA pathway.

Intermedin1-53 Attenuates Abdominal Aortic Aneurysm by Inhibiting Oxidative Stress.

OBJECTIVE: Oxidative stress plays a critical role in the development of abdominal aortic aneurysm (AAA). Intermedin (IMD) is a regulator of oxidative stress. Here, we investigated whether IMD reduces AAA by inhibiting oxidative stress. APPROACH AND RESULTS: In angiotensin II-induced ApoE-/- mouse and CaCl2-induced C57BL/6J mouse model of AAA, IMD1-53 significantly reduced the incidence of AAA and maximal aortic diameter. Ultrasonography, hematoxylin, and eosin staining and Verhoeff-van Gieson staining showed that IMD1-53 significantly decreased the enlarged aortas and elastic lamina degradation induced by angiotensin II or CaCl2. Mechanistically, IMD1-53 attenuated oxidative stress, inflammation, vascular smooth muscle cell apoptosis, and matrix metalloproteinase activation. IMD1-53 inhibited the activation of redox-sensitive signaling pathways, decreased the mRNA and protein expression of nicotinamide adenine dinucleotide phosphate oxidase subunits, and reduced the activity of nicotinamide adenine dinucleotide phosphate oxidase in AAA mice. Expression of Nox4 was upregulated in human AAA segments and in angiotensin II-treated mouse aortas and was markedly decreased by IMD1-53. In vitro, vascular smooth muscle cells with small-interfering RNA knockdown of IMD showed significantly increased angiotensin II-induced reactive oxygen species, and small-interfering RNA knockdown of Nox4 markedly inhibited the reactive oxygen species. IMD knockdown further increased the apoptosis of vascular smooth muscle cells and inflammation, which was reversed by Nox4 knockdown. Preincubation with IMD17-47 and protein kinase A inhibitor H89 inhibited the effect of IMD1-53, reducing Nox4 protein levels. CONCLUSIONS: IMD1-53 could have a protective effect on AAA by inhibiting oxidative stress.

Intermedin1-53 Protects Against Myocardial Fibrosis by Inhibiting Endoplasmic Reticulum Stress and Inflammation Induced by Homocysteine in Apolipoprotein E-Deficient Mice.

AIM: Endoplasmic reticulum stress (ERS) and inflammation participate in cardiac fibrosis. Importantly, a novel paracrine/autocrine peptide intermedin1-53 (IMD1-53) in the heart inhibits myocardial fibrosis in rats. However, the mechanisms are yet to be fully elucidated. METHODS: Myocardial fibrosis in apolipoprotein E-deficient (ApoE -/-) mice and neonatal rat cardiac fibroblasts (CFs) were induced using homocysteine (Hcy). RESULTS: IMD1-53 inhibited myocardial fibrosis in vivo and in vitro. Picrosirius red staining showed that IMD1-53 reduced myocardial interstitial collagen deposition in ApoE-/- mice treated with Hcy and decreased the expression of myocardial collagen I and III, which was further verified in rat CFs. IMD1-53 attenuated myocardial hypertrophy, as shown by cardiomyocyte cross-sectional area, ratio of heart weight to body weight, and mRNA levels of atrial natriuretic peptide and brain natriuretic peptide. IMD1-53 inhibited the upregulation of ERS hallmarkers such as glucose-regulated protein 78 (GRP78), GRP94, activating transcription factor 6 (ATF6), ATF4, inositol-requiring enzyme 1α, spliced-X-box-binding protein-1, protein kinase receptor-like ER kinase, and eukaryotic translation initiation factor 2α in mouse myocardium and rat CFs treated with Hcy. In addition, IMD1-53 decreased the production of inflammatory factors such as tumor necrosis factor-α, monocyte chemotactic protein-1, interleukin-6 (IL-6), and IL-1ß in the mouse myocardium and rat CFs treated with Hcy. Concurrently, IMD1-53 ameliorated the expression of nuclear factor-κB, transforming growth factor-ß1, and c-Jun N-terminal kinase in the mouse myocardium and rat CFs treated with Hcy. CONCLUSIONS: IMD potentially protects against myocardial fibrosis induced by Hcy in ApoE-/- mice, possibly via attenuating myocardial ERS and inflammation.

Taurine drinking ameliorates hepatic granuloma and fibrosis in mice infected with Schistosoma japonicum.

In schistosomiasis, egg-induced hepatic granuloma formation is a cytokine-mediated, predominantly CD4(+) Th2 immune response that can give rise to hepatic fibrosis. Hepatic fibrosis is the main cause of increased morbidity and mortality in humans with schistosome infection. Taurine has various physiological functions and hepatoprotective properties as well as anti-inflammatory and immunomodulatory activity. However, little is known about the role of taurine in schistosome egg-induced granuloma formation and fibrosis. We aimed to evaluate the therapeutic potential of taurine as preventative treatment for Schistosoma japonicum infection. Mice infected with S. japonicum cercariae were supplied with taurine drinking water (1% w/v) for 4 weeks starting at 4 weeks post-infection. Taurine supplementation significantly improved the liver pathologic findings, reduced the serum levels of aminotransferases and area of hepatic granuloma, and prevented fibrosis progression. In addition, taurine decreased the expression of the granulomatous and fibrogenic mediators transforming growth factor ß1, tumor necrosis factor α, monocyte chemotactic protein 1α and macrophage inflammatory protein 1α as well as the endoplasmic reticulum stress marker glucose-regulated protein 78. Thus, taurine can significantly attenuate S. japonicum egg-induced hepatic granuloma and fibrosis, which may depend in part on the downregulation of some relevant cytokine/chemokines and reducing the endoplasmic reticulum stress response.

Control of Methicillin-Resistant Staphylococcus aureus Pneumonia Utilizing TLR2 Agonist Pam3CSK4.

The spread of methicillin-resistant Staphylococcus aureus (MRSA) is a critical health issue that has drawn greater attention to the potential use of immunotherapy. Toll-like receptor 2 (TLR2), a pattern recognition receptor, is an essential component in host innate defense system against S. aureus infection. However, little is known about the innate immune response, specifically TLR2 activation, against MRSA infection. Here, we evaluate the protective effect and the mechanism of MRSA murine pneumonia after pretreatment with Pam3CSK4, a TLR2 agonist. We found that the MRSA-pneumonia mouse model, pretreated with Pam3CSK4, had reduced bacteria and mortality in comparison to control mice. As well, lower protein and mRNA levels of TNF-α, IL-1ß and IL-6 were observed in lungs and bronchus of the Pam3CSK4 pretreatment group. Conversely, expression of anti-inflammatory cytokine IL-10, but not TGF-ß, increased in Pam3CSK4-pretreated mice. Our additional studies showed that CXCL-2 and CXCL1, which are necessary for neutrophil recruitment, were less evident in the Pam3CSK4-pretreated group compared to control group, whereas the expression of Fcγ receptors (FcγⅠ/Ⅲ) and complement receptors (CR1/3) increased in murine lungs. Furthermore, we found that increased survival and improved bacterial clearance were not a result of higher levels of neutrophil infiltration, but rather a result of enhanced phagocytosis and bactericidal activity of neutrophils in vitro and in vivo as well as increased robust oxidative activity and release of lactoferrin. Our cumulative findings suggest that Pam3CSK4 could be a novel immunotherapeutic candidate against MRSA pneumonia.

Rutaecarpine Reverses the Altered Connexin Expression Pattern Induced by Oxidized Low-density Lipoprotein in Monocytes.

Adhesion of monocytes to the vascular endothelium is crucial in atherosclerosis development. Connexins (Cxs) which form hemichannels or gap junctions, modulate monocyte-endothelium interaction. We previously found that rutaecarpine, an active ingredient of the Chinese herbal medicine Evodia, reversed the altered Cx expression induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells, and consequently decreases the adhesive properties of endothelial cells to monocytes. This study further investigated the effect of rutaecarpine on Cx expression in monocytes exposed to ox-LDL. In cultured human monocytic cell line THP-1, ox-LDL rapidly reduced the level of atheroprotective Cx37 but enhanced that of atherogenic Cx43, thereby inhibiting adenosine triphosphate release through hemichannels. Pretreatment with rutaecarpine recovered the expression of Cx37 but inhibited the upregulation of Cx43 induced by ox-LDL, thereby improving adenosine triphosphate-dependent hemichannel activity and preventing monocyte adhesion. These effects of rutaecarpine were attenuated by capsazepine, an antagonist of transient receptor potential vanilloid subtype 1. The antiadhesive effects of rutaecarpine were also attenuated by hemichannel blocker 18α-GA. This study provides additional evidence that rutaecarpine can modulate Cx expression through transient receptor potential vanilloid subtype 1 activation in monocytes, which contributes to the antiadhesive properties of rutaecarpine.

Intermedin1-53 attenuates vascular calcification in rats with chronic kidney disease by upregulation of α-Klotho.

Deficiency in α-Klotho is involved in the pathogenesis of vascular calcification. Since intermedin (IMD)1-53 (a calcitonin/calcitonin gene-related peptide) protects against vascular calcification, we studied whether IMD1-53 inhibits vascular calcification by upregulating α-Klotho. A rat model of chronic kidney disease (CKD) with vascular calcification induced by the 5/6 nephrectomy plus vitamin D3 was used for study. The aortas of rats with CKD showed reduced IMD content but an increase of its receptor, calcitonin receptor-like receptor, and its receptor modifier, receptor activity-modifying protein 3. IMD1-53 treatment reduced vascular calcification. The expression of α-Klotho was greatly decreased in the aortas of rats with CKD but increased in the aortas of IMD1-53-treated rats with CKD. In vitro, IMD1-53 increased α-Klotho protein level in calcified vascular smooth muscle cells. α-Klotho knockdown blocked the inhibitory effect of IMD1-53 on vascular smooth muscle cell calcification and their transformation into osteoblast-like cells. The effect of IMD1-53 to upregulate α-Klotho and inhibit vascular smooth muscle cell calcification was abolished by knockdown of its receptor or its modifier protein, or treatment with the protein kinase A inhibitor H89. Thus, IMD1-53 may attenuate vascular calcification by upregulating α-Klotho via the calcitonin receptor/modifying protein complex and protein kinase A signaling.