Yan-Rong Yu

Extracellular signal-regulated kinase 1/2 activation is involved in intermedin1–53 attenuating myocardial oxidative stress injury induced by ischemia/reperfusion

Intermedin (IMD)(1-53) is a novel member of the calcitonin gene-related peptide superfamily and has potent cardioprotective effects against myocardial injury induced by ischemia-reperfusion (I/R). To explore the mechanism of the IMD(1-53) cardioprotective effect, we studied the anti-oxidant effects of IMD(1-53) on myocardial injury induced by I/R in vivo in rat and H(2)O(2) treatment in vitro in rat cardiomyocytes. Compared with sham treatment, I/R treatment induced severe lipid peroxidation injury in rat myocardium: plasma malondialdehyde (MDA) content and myocardial LDH activity was increased by 34% and 85% (all P<0.01); Mn-superoxide dismutase (Mn-SOD) and catalase (CAT) activity was reduced 80% and 86% (all P<0.01), respectively, and the protein levels of the NADPH oxidase complex subunits gp91(phox) and p47(phox) were markedly increased, by 86% (P<0.05) and 95% (P<0.01), respectively; IMD(1-53) treatment ameliorated lipid peroxidation injury: plasma MDA content and myocardial LDH activity was decreased by 30% (P<0.05) and 36% (P<0.01); Mn-SOD and CAT activity was elevated 1.0- and 4.3-fold (all P<0.01), respectively; and the protein levels of gp91(phox) and p47(phox) were reduced, by 28% and 36% (both P<0.05), respectively. Concurrently, IMD(1-53) treatment markedly promoted cell viability and inhibited apoptosis in cardiomyocytes as compared with H(2)O(2) treatment alone. Furthermore, IMD(1-53) increased the ratio of p-ERK to ERK by 66% (P<0.05) as compared with I/R alone, and the protective effect of IMD(1-53) on H(2)O(2)-induced apoptosis was abolished by preincubation with PD98059, a MEK inhibitor. IMD(1-53) may improve the oxidative stress injury induced by I/R via inhibiting the production of reactive oxygen species and enhancing ERK phosphorylation.

Endogenous aldosterone is involved in vascular calcification in rat

Aldosterone (Aldo) is an important active hormone in the renin–angiotensin–aldosterone system and plays a vital role in the development of hypertension, heart failure and other cardiovascular diseases. We aimed to explore the role of endogenous Aldo in aortic calcification in rats. We induced arterial calcification in rats by intramuscular administration of vitamin D3 plus oral nicotine (VDN) and determined calcium content, 45Ca2+ accumulation and activity of alkaline phosphatase (ALP). The mRNA level of osteopontin (OPN) was measured by semi-quantitative reverse transcriptase polymerase chain reaction. Deposition of collagen in the aorta wall was measured by Sirius red staining. The content of angiotensin II (Ang II) and Aldo in plasma and myocardial and vascular tissue was determined by radioimmunoassay. In rats with VDN treatment, von Kossa staining showed calcification in vascular smooth muscle cells and extracellular matrix, and the content of calcium in calcified arteries was 5.8-fold of that in control arteries (P < 0.01). The accumulation of 45Ca2+ and activity of ALP in calcified aortic tissue was three- and 2.5-fold, respectively, that in control tissue (P < 0.01). The mRNA expression of OPN was significantly higher, by 58%, in calcified than control tissue (P < 0.01). Vascular fibrosis was greater in rats with calcified tissue than in control rats. The level of Ang II and Aldo was 58% and 80% higher, respectively, in calcified than control tissue (both P < 0.01). The changes could be significantly improved by treatment with captopril, an angiotensin-converting enzyme inhibitor, and the Aldo receptor antagonist spironolactone. These results suggest that Aldo is an endogenous bioactive factor involved in vascular calcification.

Intermedin1-53 attenuates vascular smooth muscle cell calcification by inhibiting endoplasmic reticulum stress via cyclic adenosine monophosphate/protein kinase A pathway

We previously reported that endoplasmic reticulum (ER) stress-mediated apoptosis participated in vascular calcification. Importantly, a novel paracrine/autocrine peptide intermedin1–53 (IMD1–53) in the vasculature inhibited vascular calcification in rats. But the mechanisms needed to be fully elucidated. Vascular smooth muscle cells (VSMCs) calcification was induced by CaCl2 and β-glycerophosphate. Tunicamycin (Tm) or dithiothreitol (DTT) was used to induce ER stress. We found that IMD1–53 (10−7 mol/L) treatment significantly alleviated the protein expression of ER stress hallmarks activating transcription factor 4 (ATF4), ATF6, glucose-regulated protein 78 (GRP78) and GRP94 induced by Tm or DTT. ER stress occurred in early and late calcification of VSMCs but was inhibited by IMD1–53. These inhibitory effects of IMD1–53 were abolished by treatment with the protein kinase A (PKA) inhibitor H89. Pretreatment with IMD1–53 decreased the number of apoptotic VSMCs and downregulated protein expression of cleaved caspase 12 and C/EBP homologous protein (CHOP) in calcified VSMCs. Concurrently, IMD1–53 restored the loss of VSMC lineage markers and ameliorated calcium deposition and alkaline phosphatase activity in calcified VSMCs as well. The observation was further verified by Alizarin Red S staining, which showed that IMD1–53 reduced positive red nodules among calcified VSMCs. In conclusion, IMD1–53 attenuated VSMC calcification by inhibiting ER stress through cAMP/PKA signalling.

Angiotensin-(1-7) inhibits vascular calcification in rats

Angiotensin-(1-7) [Ang-(1-7)] is a new bioactive heptapeptide in the renin-angiotensin-aldosterone system (RAAS) with potent protective effects in cardiovascular diseases, opposing many actions of angiotensin II (Ang II) mediated by Ang II type 1 (AT1) receptor. It is produced mainly by the activity of angiotensin-converting enzyme 2 (ACE2) and acts through the Mas receptor. However, the role of Ang-(1-7) in vascular calcification (VC) is still unclear. In this study, we investigated the protective effects of Ang-(1-7) on VC in an in vivo rat VC model induced by vitamin D3 plus nicotine. The levels of ACE2 and the Mas receptor, as well as ACE, AT1 receptor, Ang II type 2 receptor and angiotensinogen, were significantly increased in calcified aortas, and Ang-(1-7) reversed the increased levels. Ang-(1-7) restored the reduced expression of lineage markers, including smooth muscle (SM) α-actin, SM22α, calponin and smoothelin, in vascular smooth muscle cells (VSMCs) and retarded the osteogenic transition of VSMCs by decreasing the expression of bone-associated proteins. It reduced alkaline phosphatase activity and calcium deposition in VC and alleviated the hemodynamic disorders of rats with VC. We provide the first in vivo evidence that Ang-(1-7) can inhibit the development of VC by inhibiting the osteogenic transition of VSMCs, at least in part by decreasing levels of the ACE/Ang II/AT1 axis. The increased expression of ACE2 and the Mas receptor in calcified aortas suggests the involvement of the ACE2/Ang-(1-7)/Mas axis during VC. Ang-(1-7) might be an efficient endogenous vasoprotective factor for VC.

Adrenomedullin protects against fructose-induced insulin resistance and myocardial hypertrophy in rats

Adrenomedullin (ADM) has been recognized as a multipotent multifunctional peptide. To explore the pathophysiological roles of ADM in insulin resistance (IR), we studied the changes in ADM mRNA level in the myocardium and vessels and the effect of ADM supplementation on rats with IR induced by fructose feeding. Rats were fed 4% fructose in drinking water for 8 weeks, and ADM was administered subcutaneously in pure water through an Alzet Mini-osmotic Pump at 300 ng/kg/h for the last 4 weeks. Compared with controls, rats with IR showed increased levels of fasting blood sugar and serum insulin, by 95% and 67%, respectively (all P<0.01), and glycogen synthesis and glucose transport activity of the soleus decreased by 54% and 55% (all P<0.01). mRNA level and content of brain natriuretic peptide (BNP) in myocardial were all increased significantly. Fructose-fed rats showed increased immunoreactive-ADM content in plasma by 110% and in myocardia by 55% and increased mRNA level in myocardia and vessels (all P<0.01). ADM administration ameliorated the induced IR and myocardial hypertrophy. The glycogen synthesis and glucose transport activity of the soleus muscle increased by 41% (P<0.01) and 32% (P<0.05). ADM therapy attenuated myocardial and soleus lipid peroxidation injury and enhanced the antioxidant ability. Our results showed upregulation of endogenous ADM during fructose-induced IR and the protective effect of ADM on fructose-induced IR and concomitant cardiovascular hypertrophy probably by its antioxidant effect, which suggests that ADM could be an endogenous protective factor in IR.

Intermedin1-53 protects against cardiac hypertrophy by inhibiting endoplasmic reticulum stress via activating AMP-activated protein kinase.

Objective: Intermedin (IMD), a novel member of the calcitonin/calcitonin gene-related peptide family, is involved in maintaining circulatory homeostasis and is a protective factor of heart and vessel. Here, we investigated the effects of IMD on cardiac hypertrophy in vivo and in vitro and explored the mechanisms involved. Methods and results: IMD1–53 (100 ng/kg/h) was systemically administered to rats with cardiac hypertrophy induced by abdominal aortic constriction (AAC) by a mini-osmotic pump the next day after surgery continuously for 4 weeks. The AAC-treated rats before IMD infusion showed increased IMD content and expression of its receptors in the hearts. In-vivo administration of IMD1–53 greatly attenuated the cardiac hypertrophy as shown by heart weight to body weight ratio (HW/BW), haemodynamics, echocardiography, histological analyses and expression of hypertrophic markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) induced by AAC. IMD1–53 treatment significantly reduced the myocardial protein expression of endoplasmic reticulum stress (ERS) markers such as glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP) and caspase-12, whereas the protein level of phosphorylated AMP-activated protein kinase (p-AMPK) was upregulated with IMD1–53 treatment, which was further confirmed in cultured cardiomyocytes. Concurrently, cardiomyocyte apoptosis in vivo and in vitro was ameliorated by IMD1–53 treatment. The inhibitory effects of IMD1–53 on ERS and apoptosis were eliminated on pretreatment with compound C, an AMPK inhibitor. Conclusion: IMD1–53 could exert its cardioprotective effect on cardiac hypertrophy by inhibiting myocardial ERS and apoptosis, possibly via activation of AMPK signalling.

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-1β in the mouse myocardium and rat CFs treated with Hcy. Concurrently, IMD1–53 ameliorated the expression of nuclear factor-κB, transforming growth factor-β1, 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.

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.

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 β1, 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.

Endoplasmic reticulum stress-mediated apoptosis is activated in intestines of mice with Trichinella spiralis infection.

Gastrointestinal helminth infection, including Trichinella spiralis, initiates a series of intestinal structural, cellular and physiological changes. Intestinal invasion is an important stage of trichinellosis because it determines the development and subsequent course of the disease and its consequences. Apoptosis mediated by endoplasmic reticulum stress (ERS) plays a key role in infectious diseases, but the effect of T. spiralis infection on inducing apoptosis in the small intestine has been neglected. We investigated apoptosis and changes in ERS-associated apoptosis molecules in the intestine of mice with T. spiralis infection. TUNEL staining and detection of the apoptotic marker cleaved caspase 3 revealed that apoptosis occurred in the mouse intestine at days 3 and 7 post-infection. The ER chaperone 78-kDa glucose-regulated protein (GRP78) was upregulated at days 3 and 7 post-infection. The ERS-associated apoptosis molecules C/EBP homologous protein, cleaved caspase 12 and c-Jun NH2-terminal kinase were upregulated at days 3 and 7, days 3, 7 and 10 and days 7 and 10 post-infection, respectively. Thus, apoptosis occurred in the intestine of mice with T. spiralis infection, and the ERS-mediated apoptosis pathway was activated by infection with this small intestine dwelling nematode.