Jin-Rui Chang

Ghrelin protects heart against ERS-induced injury and apoptosis by activating AMP-activated protein kinase

Ghrelin, the endogenous ligand of growth hormone secretagogue receptor (GHS-R), is a cardioprotective peptide. In our previous work, we have revealed that ghrelin could protect heart against ischemia/reperfusion (I/R) injury by inhibiting endoplasmic reticulum stress (ERS), which contributes to many heart diseases. In current study, using both in vivo and in vitro models, we investigated how ghrelin inhibits myocardial ERS. In the in vivo rat heart injury model induced by isoproterenol (ISO), we found that exogenous ghrelin could alleviate heart dysfunction, reduce myocardial injury and apoptosis and inhibit the excessive myocardial ERS induced by ISO. More importantly, the activation of AMP-activated protein kinase (AMPK) was observed. To explore the role of AMPK activation in ERS inhibition by ghrelin, we set up two in vitro ERS models by exposing cultured rat cardiomyocytes to tunicamycin(Tm) or dithiothreitol (DTT). In both models, compared with Tm or DTT treatment alone, pre-incubation cardiomyocytes with ghrelin significantly activated AMPK, reversed the upregulation of the ERS markers, C/EBP-homologous protein (CHOP) and cleaved caspase-12, and reduced apoptosis of cardiomyocytes. Further, we found that the ERS inhibitory and anti-apoptotic actions induced by ghrelin were blocked by an AMPK inhibitor. To investigate how ghrelin activates AMPK, selective antagonist of GHS-R1a and inhibitor of Ca(2+)/Calmodulin-dependent protein kinase kinase (CaMKK) were added, respectively, before ghrelin pre-incubation, and we found that AMPK activation was prevented and the ERS inhibitory and anti-apoptotic actions of ghrelin were blocked. In conclusion, ghrelin could protect heart against ERS-induced injury and apoptosis, at least partially through a GHS-R1a/CaMKK/AMPK pathway.

Activating transcription factor 4 is involved in endoplasmic reticulum stress-mediated apoptosis contributing to vascular calcification

Our previous work reported that endoplasmic reticulum stress (ERS)-mediated apoptosis was activated during vascular calcification (VC). Activating transcription factor 4 (ATF4) is a critical transcription factor in osteoblastogenesis and ERS-induced apoptosis. However, whether ATF4 is involved in ERS-mediated apoptosis contributing to VC remains unclear. In the present study, in vivo VC was induced in rats by administering vitamin D3 plus nicotine. Vascular smooth muscle cell (VSMC) calcification in vitro was induced by incubation in calcifying media containing β-glycerophosphate and CaCl2. ERS inhibitors taurine or 4-phenylbutyric acid attenuated ERS and VSMC apoptosis in calcified rat arteries, reduced calcification and retarded the VSMC contractile phenotype transforming into an osteoblast-like phenotype in vivo. Inhibition of ERS retarded the VSMC phenotypic transition into an osteoblast-like cell phenotype and reduced VSMC calcification and apoptosis in vitro. Interestingly, ATF4 was activated in calcified aortas and calcified VSMCs in vitro. ATF4 knockdown attenuated ERS-induced apoptosis in calcified VSMCs. ATF4 deficiency blocked VSMC calcification and negatively regulated the osteoblast phenotypic transition of VSMCs in vitro. Our results demonstrate that ATF4 was involved at least in part in the process of ERS-mediated apoptosis contributing to VC.

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.

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.

Cardiac sympathetic afferent reflex response to intermedin microinjection into paraventricular nucleus is mediated by nitric oxide and γ-amino butyric acid in hypertensive rats.

Intermedin (IMD) is a member of calcitonin/calcitonin gene-related peptide (CGRP) and involves in the regulation of cardiovascular function in both peripheral tissues and central nervous system (CNS). Paraventricular nucleus (PVN) of hypothalamus is an important site in the control of cardiac sympathetic afferent reflex (CSAR) which participates in sympathetic over-excitation of hypertension. The aim of this study is to investigate whether IMD in the PVN is involved in the inhibition of CSAR and its related mechanism in hypertension. Rats were subjected to two-kidney one-clip (2K1C) surgery to induce renovascular hypertension or sham-operation (Sham). Acute experiments were carried out four weeks later under anesthesia. The CSAR was evaluated with the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to the epicardial application of capsaicin. The RSNA and MAP were recorded in sinoaortic-denervated, cervical-vagotomized and anesthetized rats. Bilateral PVN microinjection of IMD (25 pmol) caused greater decrease in the CSAR in 2K1C rats than in Sham rats, which was prevented by pretreatment with adrenomedullin (AM) receptor antagonist AM22-52, non-selective nitric oxide (NO) synthase (NOS) inhibitor l-NAME or γ-amino butyric acid (GABA)B receptor blocker CGP-35348. PVN pretreatment with CGRP receptor antagonist CGRP8-37 or GABAA receptor blocker gabazine had no significant effect on the CSAR response to IMD. AM22-52, l-NAME and CGP-35348 in the PVN could increase CSAR in Sham and 2K1C rats. These data indicate that IMD in the PVN inhibits CSAR via AM receptor, and both NO and GABA in the PVN involve in the effect of IMD on CSAR in Sham and renovascular hypertensive rats.

Intermedin1-53 attenuates abdominal aortic aneurysm by inhibiting oxidative stress

Abdominal aortic aneurysm (AAA), a progressive aortic dilation, is a common vascular disease associated with high mortality. Although conventional treatments including surgery or percutaneous intervention are performed in highrisk patients, effective medical therapies for AAA have not been developed. The hallmark pathological features of AAA are typified by intense oxidative stress, inflammation, matrix degradation, and apoptosis of vascular smooth muscle cells (VSMCs). Reactive oxygen species (ROS) are thought to be a common link between inflammation, matrix degradation, and VSMC apoptosis, so reducing ROS may be a therapeutic target of AAA. In animal models of AAA, genetic and pharmacological inhibition of ROS suppressed aneurysm formation. A major source of ROS in vascular tissue is the membrane-bound nicotinamide vadenine dinucleotide phosphate (NADPH) oxidase, which contains transmembrane subunits such as Nox1–5, Duox1 and Duox2, and p22phox and cytosolic subunits such as p47phox, p67phox, Rac1, and optionally p40phox (for Nox2) or the respective homologs of p47phox and p67phox, NOXO1 and NOXA1 (for Nox1). These subunits assemble to form a functional oxidase. NADPH oxidase activity was markedly upregulated in human aneurysmal aortas. Inhibition of NADPH oxidase attenuated AAA formation, which suggests that NADPH oxidase plays a critical role in AAA formation. Nox1 and Nox4 are the 2 Nox isoforms expressed in VSMCs, where ROS are mainly generated in AAA. Nox4 has been shown to produce hydrogen peroxide (H 2 O 2 ), which induced apoptosis in VSMCs. Nox4-mediated oxidative stress may participate in the pathogenesis of AAA. Paracrine/autocrine factors are involved in maintaining circulatory homeostasis and mediating the pathogenesis of cardiovascular diseases. Some of these factors such as adrenomedullin (ADM) can antagonize oxidative stress and have properties of anti-inflammation, suppression of proliferation, and migration of VSMCs as well as promotion of growth and reparation of endothelium. Thus, investigation of endogenous peptides may help reveal new mechanisms and therapeutic strategies for AAA.