Peroxisome proliferator-activated receptor γ regulates angiotensin II-induced catalase downregulation in adventitial fibroblasts of rats

Peroxisome proliferator-activated receptor (PPAR) γ ligands oppose the effect induced by angiotensin II (Ang II) to reduce oxidative stress and improve antioxidant status. In this study, Ang II inhibited catalase (CAT) and peroxisome proliferator-activated receptor γ (PPAR γ) protein and mRNA expressions. Transfection with PPAR γ small-interfering RNA (siRNA) led to a reduction in CAT expression. PPAR γ ligands enhanced CAT expression and inhibited extracellular signal-regulated kinase 1/2 activation. We further reveal that Ang II type 1 receptor is not involved in the inhibitory effects of PPAR γ ligands on Ang II stimulatory events.     

The cardioprotective effect of dihydromyricetin prevents ischemia–reperfusion-induced apoptosis in vivo and in vitro via the PI3K/Akt and HIF-1α signaling pathways

Reperfusion therapy is widely used to treat acute myocardial infarction (AMI). However, further injury to the heart induced by rapidly initiating reperfusion is often encountered in clinical practice. A lack of pharmacological strategies in clinics limits the prognosis of patients with myocardial ischemia-reperfusion injury (MIRI). Dihydromyricetin (DMY) is one of the most abundant components in vine tea, commonly known as the tender stems and leaves of Ampelopsis grossedentata. The aim of this study was to evaluate the cardioprotection of DMY against myocardial ischemia-reperfusion (I/R) injury and to further investigate the underlying mechanism. An I/R injury was induced by left anterior descending coronary artery occlusion in adult male rats in vivo and a hypoxia–reoxygenation (H/R) injury in H9c2 cardiomyocytes in vitro. We found that DMY pretreatment provided significant protection against I/R-induced injury, including enhanced antioxidant capacity and inhibited apoptosis in vivo and in vitro. This effect correlated with the activation of the PI3K/Akt and HIF-1α signaling pathways. Conversely, blocking Akt activation with the PI3K inhibitor LY294002 effectively suppressed the protective effects of DMY against I/R-induced injury. In addition, the PI3K inhibitor partially blocked the effects of DMY on the upregulation of Bcl-2, Bcl-xl, procaspase-3, -8, and -9 protein expression and the downregulation of HIF-1α, Bnip3, Bax, Cyt-c, cleaved caspase-3, -8, and -9 protein expression. Collectively, these results showed that DMY decreased the apoptosis and necrosis by I/R treatment, and PI3K/Akt and HIF-1α plays a crucial role in protection during this process. These observations indicate that DMY has the potential to exert cardioprotective effects against I/R injury and the results might be important for the clinical efficacy of AMI treatment.     

Moderate activation of Wnt/β-catenin signaling promotes the survival of rat nucleus pulposus cells via regulating apoptosis,autophagy, and senescence

This study aimed to investigate the specific role of Wnt/β-catenin signaling in compression-induced apoptosis, autophagy, and senescence in rat nucleus pulposus (NP) cells. Initially, the cells underwent various periods of exposure to 1.0 MPa compression. Wnt/β-catenin signaling associated molecules were assessed in detail, and then 0, 24 and 48 hours exposure periods were selected. The cells were then divided into control, Wnt/β-catenin inhibitor (IWP-2), Wnt/β-catenin activator (LiCl), and β-catenin overexpression groups. After 0, 24, and 48 hours of compression, apoptosis, autophagy, and senescence were evaluated by Western blot analysis and real-time polymerase chain reaction and were visually observed by Hoechst33258, monodansylcadaverine, and SA-β-gal stainings, respectively. Additionally, the regulatory effect of Wnt/β-catenin signaling on cell morphology, viability, cell cycle, death ratio, and ultrastructure was detected to thoroughly evaluate the survival capacity of NP cells. The results established that compression elicited a time-dependent activation of Wnt/β-catenin signaling. The IWP-2 treatment decreased cell survival rate, which corresponded to downregulation of autophagy as well as increases in apoptosis and senescence. LiCl treatment enabled more efficient of cell survival accompanied by increased autophagy and downregulated apoptosis and senescence; however, in contrast to LiCl, overexpression of β-catenin aggravated compression-induced NP cells death. In conclusion, moderate activation of Wnt/β-catenin signaling enables more efficient of NP cells survival via downregulation of apoptosis, senescence, and upregulation of autophagy, and overactivation of Wnt/β-catenin signaling achieved the opposite effect. Treatment strategies that aim to regulate Wnt/β-catenin signaling might be a novel target for improving compression-induced NP cells death and potential treatment of intervertebral disc degeneration.     

MicroRNA-101 protects cardiac fibroblasts from hypoxia-induced apoptosis via inhibition of the TGF-β signaling pathway

Cardiac fibroblasts (CFs) are the most numerous cells in the heart and are recognized primarily for their ability to maintain both the structural integrity and the physiological functions of the heart. The transforming growth factor beta (TGF-β) signaling pathway is reportedly involved in the modulation of CF functions, including apoptosis. Recent studies have indicated that microRNA-101 (miR-101) attenuates the TGF-β signaling pathway, either by inhibiting the expression of TGFβ1 or by targeting transforming growth factor-β receptor type I (TGFβRI). The present study aimed to determine whether miR-101 protects CFs from hypoxia-induced apoptosis and to investigate the mechanisms underlying its protective effects. The CCK-8 test, electron microscopy and TUNEL assay results demonstrated that miR-101a/b significantly inhibited hypoxia-induced CF apoptosis. The results of Western blotting, quantitative RT-PCR and immunofluorescence assays indicated that miR-101a dramatically inhibited the hypoxia-induced up-regulation of both TGFβRI and p-Smad 3 but not TGFβ1 in CFs. Additionally, miR-101a significantly reversed the hypoxia-induced up-regulation of Bax and Caspase-3, the down-regulation of Bcl-2 and the activation of Caspase-3 in CFs. Moreover, miR-101a markedly inhibited the intracellular Ca²⁺ ([Ca²⁺]_i) overload caused by hypoxia. Taken together, our results suggest that miR-101a protects CFs against hypoxia-induced apoptosis by inhibiting the TGF-β signaling pathway, which may be a potential therapeutic target for heart injury.     

Ascending aortic adventitial remodeling and fibrosis are ameliorated with Apelin-13 in rats after TAC via suppression of the miRNA-122 and LGR4-β-catenin signaling

Apelin has been proved to be a critical mediator of vascular function and homeostasis. Here, we investigated roles of Apelin in aortic remodeling and fibrosis in rats with transverse aortic constriction (TAC). Male Sprague-Dawley rats were subjected to TAC and then randomized to daily deliver Apelin-13 (50 μg/kg) or angiotensin type 1 receptor (AT1) blocker Irbesartan (50 mg/kg) for 4 weeks. Pressure overload resulted in myocardial hypertrophy, systolic dysfunction, aortic remodeling and adventitial fibrosis with reduced levels of Apelin in ascending aortas of rat after TAC compared with sham-operated group. These changes were associated with marked increases in levels of miRNA-122, TGFβ1, CTGF, NFAT5, LGR4, and β-catenin. More importantly, Apelin and Irbesartan treatment strikingly prevented TAC-mediated aortic remodeling and adventitial fibrosis in pressure overloaded rats by blocking AT1 receptor and miRNA-122 levels and repressing activation of the CTGF-NFAT5 and LGR4-β-catenin signaling. In cultured primary rat adventitial fibroblasts, exposure to angiotensin II (100 nmol L⁻¹) led to significant increases in cellular migration and levels of TGFβ1, CTGF, NFAT5, LGR4 and β-catenin, which were effectively reversed by pre-treatment with Apelin (100 nmol L⁻¹) and miRNA-122 inhibitor (50 nmol L⁻¹). In conclusion, Apelin counterregulated against TAC-mediated ascending aortic remodeling and angiotensin II-induced promotion of cellular migration by blocking AT1 receptor and miRNA-122 levels and preventing activation of the TGFβ1-CTGF-NFAT5 and LGR4-β-catenin signaling, ultimately contributing to attenuation of aortic adventitial fibrosis. Our data point to Apelin as an important regulator of aortic remodeling and adventitial fibrosis and a promising target for vasoprotective therapies.     

PAF enhances MMP-2 production in rat aortic VSMCs via a β-arrestin2-dependent ERK signaling pathway

Platelet-activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, is a potent phospholipid mediator and has been reported to be localized in atherosclerotic plaque. However, its role in the progression of atherosclerosis remains unclear. In the present study, we investigated the role of PAF in the production of matrix metalloproteinase (MMP) in primary vascular smooth muscle cells (VSMCs). When rat aortic primary VSMCs were stimulated with PAF (1 nmol/l), the expressions of MMP-2 mRNA and protein, but not of MMP-9, were significantly increased, and these upregulations were markedly attenuated by inhibiting extracellular signal-regulated kinases (ERKs) using molecular and pharmacological inhibitors, but not by using inhibitors of p38 mitogen-activated protein kinase or c-Jun N-terminal kinase. Likewise, ERK phosphorylation was markedly enhanced in PAF-stimulated VSMCs, and this was attenuated by WEB2086, but not by EGF receptor inhibitor, demonstrating the specificity of PAF receptor (PAFR) in PAF-induced ERK phosphorylation. In immunofluorescence studies, β-arrestin2 in PAF-stimulated VSMCs colocalized with PAFR and phosphorylated ERK (P-ERK). Coimmunoprecipitation results suggest that β-arrestin2-bound PAFRs existed as a complex with P-ERK. In addition, PAF-induced ERK phosphorylation and MMP-2 production were significantly attenuated by β-arrestin2 depletion. Taken together, the study shows that PAF enhances MMP-2 production in VSMCs via a β-arrestin2-dependent ERK signaling pathway.     

Grb14 inhibits FGF receptor signaling through the regulation of PLCγ recruitment and activation

To decipher the mechanism involved in Grb14 binding to the activated fibroblast growth factor receptor (FGFR), we used the bioluminescence resonance energy transfer (BRET) technique and the Xenopus oocyte model. We showed that Grb14 was recruited to FGFR1 into a trimeric complex containing also phospholipase C gamma (PLCγ). The presence of Grb14 altered FGF-induced PLCγ phosphorylation and activation. Grb14-FGFR interaction involved the Grb14-SH2 domain and the FGFR pY766 residue, which is the PLCγ binding site. Our data led to a molecular model whereby Grb14 binding to the phosphorylated FGFR induces a conformational change that unmasks a PLCγ binding motif on Grb14, allowing trapping and inactivation of PLCγ.

Structured summary

MINT-8019680: Grb14 (uniprotkb:O88900) physically interacts (MI:0915) with FGFR1 (uniprotkb:P11362) by anti tag coimmunoprecipitation (MI:0007)MINT-8019693, MINT-8019727: Grb14 (uniprotkb:O88900) physically interacts (MI:0915) with FGFR1 (uniprotkb:P11362) by bioluminescence resonance energy transfer (MI:0012)MINT-8019714, MINT-8019746: PLC gamma1 (uniprotkb:P19174) physically interacts (MI:0915) with FGFR1 (uniprotkb:P11362) by bioluminescence resonance energy transfer (MI:0012)     

Structure–function analysis of VEGF receptor activation and the role of coreceptors in angiogenic signaling

Vascular endothelial growth factors (VEGFs) constitute a family of six polypeptides, VEGF-A, -B, -C, -D, -E and PlGF, that regulate blood and lymphatic vessel development. VEGFs specifically bind to three type V receptor tyrosine kinases (RTKs), VEGFR-1, -2 and -3, and to coreceptors such as neuropilins and heparan sulfate proteoglycans (HSPG). VEGFRs are activated upon ligand-induced dimerization mediated by the extracellular domain (ECD). A study using receptor constructs carrying artificial dimerization-promoting transmembrane domains (TMDs) showed that receptor dimerization is necessary, but not sufficient, for receptor activation and demonstrates that distinct orientation of receptor monomers is required to instigate transmembrane signaling. Angiogenic signaling by VEGF receptors also depends on cooperation with specific coreceptors such as neuropilins and HSPG. A number of VEGF isoforms differ in binding to coreceptors, and ligand-specific signal output is apparently the result of the specific coreceptor complex assembled by a particular VEGF isoform. Here we discuss the structural features of VEGF family ligands and their receptors in relation to their distinct signal output and angiogenic potential.     

Sufentanil protects the rat myocardium against ischemia–reperfusion injury via activation of the ERK1/2 pathway

Sufentanil, a lipophilic opioid, is the most frequently used clinical drug for ischemic heart disease. The effects of sufentanil on MAPK signaling in ischemic heart disease were explored. The effects of sufentanil on ischemia–reperfusion (IR)-induced myocardial injury in a rat model were examined. The serum levels of CK, LDH, MDA and SOD, and the activities of Na⁺–K⁺-ATPase and Ca²⁺–Mg²⁺-ATPase were measured. The levels of total and phosphorylated ERK1/2, JNK, and p38 were measured by western blotting in the heart, and the myocardial H9C2 cell line was studied. Using the Cell Counting Kit-8, the growth rate of H9C2 cells affected by sufentanil was studied. The serum levels of CK, LDH and MDA were higher in the IR group than in the SO and SUF groups. The SOD level, as well as the activities of Na⁺–K⁺-ATPase and Ca²⁺–Mg²⁺-ATPase, were lower in the SO and SUF groups than in the IR group. The phosphorylated ERK1/2 level was lower in the IR group than in the SO and SUF groups. The growth rate of H9C2 cells increased with the concentration of sufentanil and the exposure time. The phosphorylated ERK level was upregulated by 4–12 h of sufentanil exposure, indicating that the effects were time-dependent. Furthermore, an inhibition of ERK signaling by chemical inhibition suppressed the sufentanil-mediated increase in the growth rate of H9C2 cells. Sufentanil appears to be beneficial for cases of worsening ischemic heart disease. Further studies are necessary before a clinical application is considered.     

Further elucidation of a pertussis toxin-sensitive transmembrane signaling mechanism involved in central α2-adrenoceptor activation in the rat

In adult male Sprague-Dawley rats anesthetized with pentobarbital sodium, we elucidated the molecular consequence of central ₂-adrenoceptor activation. The hypotensive and negative chronotropic and inotropic actions of the ₂-adrenoceptor agonist guanabenz were used as our experimental index. Intracerebroventricular administration of pertussis toxin (2.5 µg) significantly attenuated the cardiovascular suppressant effects of the aminoguanidine compound (100 µg/kg i.v.). However, application of N-ethylmaleimide (0.125 or 0.250 µg), phorbol 12-myristate 13-acetate (1.25 or 2.50 µg), cholera toxin (1.25 or 2.50 µg) or forskolin (12.5 or 25.0 µg) into the lateral cerebral ventricle elicited no appreciable blunting effect on the circulatory depression produced by guanabenz. These results were essentially duplicated when pertussis toxin (0.125 or 0.250 µg), N-ethylmaleimide (0.0125 or 0.05 µg), phorbol 12-myristate 13-acetate (0.125 or 0.25 µg), cholera toxin (0.125 or 0.25 µg) or forskolin (1.25 or 2.50 µg) was microinjected bilaterally to the nucleus reticularis gigantocellularis, a medullary site believed to be intimately related to the antihypertensive action of guanabenz. These findings suggest that stimulation of the ₂-adrenoceptors in the medulla oblongata may result in the activation of a pertussis toxin-sensitive GTP-binding regulatory protein. They further suggest that the biologic signals subsequent to this action may not be linked to Gs, Gi or Gp but possibly Go.     

ACE2 – From the renin–angiotensin system to gut microbiota and malnutrition

The renin–angiotensin system (RAS) is a complex network that regulates blood pressure, electrolyte and fluid homeostasis, as well as the function of several organs. Angiotensin-converting enzyme 2 (ACE2) was identified as an enzyme that negatively regulates the RAS by converting Ang II, the main bioactive molecule of the RAS, to Ang 1–7. Thus, ACE2 counteracts the role of angiotensin-converting enzyme (ACE) which generates Ang II from Ang I. ACE and ACE2 have been implicated in several pathologies such as cardiovascular and renal disease or acute lung injury. In addition, ACE2 has functions independent of the RAS: ACE2 is the receptor for the SARS coronavirus and ACE2 is essential for expression of neutral amino acid transporters in the gut. In this context, ACE2 modulates innate immunity and influences the composition of the gut microbiota, which can explain diarrhea and intestinal inflammation observed in Hartnup disorder, Pellagra, or under conditions of severe malnutrition. Here we review and discuss the diverse functions of ACE2 and its relevance to human pathologies.     

Obesity and malnutrition similarly alter the renin–angiotensin system and inflammation in mice and human adipose

The main goal of the present study was to evaluate the metabolic profile, inflammatory markers and the gene expression of the renin–angiotensin system (RAS) components in the visceral adipose tissue of eutrophic, obese and malnourished individuals and mice models of obesity and food restriction. Male Swiss mice were divided into eight groups and fed different levels of food restriction (20%, 40%, or 60%) using standard or high-fat diet. Metabolic profile and adipose tissues were assessed. The expression of AGT (Angiotensinogen), ACE (Angiotensin-converting enzyme), ACE2 (Angiotensin-converting enzyme 2), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the mice epididymal adipose tissue and the human visceral adipose tissue was assessed. The main findings showed reduced body weight, improved metabolism, decreased adipose tissues weight and reduced adipocyte area in mice submitted to food restriction. Diminished expression of IL-6, TNF-α, AGT, AT1 and ACE was detected in the 20% and 40% food restriction animal groups, although they were increased in the 60% malnourished group. Increased expression of IL-6, TNF-α, AGT and ACE in obese and malnourished individuals was observed. Adipocytes size was increased in obese individuals and reduced in malnutrition. In conclusion, we found that food restriction of 20% and 40% improved the metabolic profile, ameliorated the inflammatory status and down-regulated the RAS in mice. Severe 60% food restriction (malnutrition), however, stimulated a proinflammatory state and increased AGT and ACE expression in the adipose tissue of mice. A similar profile was observed in the adipose tissue of obese and malnourished humans, supporting the critical role of inflammation and RAS as mediators of metabolic disorders.     

Heat shock treatment suppresses angiotensin II-induced activation of NF-kappaB pathway and heart inflammation: a role for IKK depletion by heat shock?

Heat shock (HS) proteins (Hsps) function in tissue protection through their chaperone activity and by interacting with cell signaling pathways to suppress apoptosis. Here, we investigated the effect of HS treatment on the nuclear factor (NF)-kappaB signaling pathway in the angiotensin II (ANG II) model of inflammation. Male Sprague-Dawley rats were divided into sham and HS-, ANG II-, and HS + ANG II-treated groups. HS treatment was administered 24 h before the initiation of ANG II infusion. HS treatment (42 degrees C for 15 min) decreased 7-day ANG II-induced hypertension from 191 +/- 4 to 147 +/- 3 mmHg (P < 0.01). Histological staining of hearts showed that HS treatment reduced ANG II-induced leukocyte infiltration, perivascular and interstitial inflammation, and fibrosis. Heart NF-kappaB nuclear translocation and activity, examined by Western blot analysis and electrophoretic mobility shift assay, was suppressed by HS treatment. HS treatment depleted IkappaB kinase-alpha (IKK-alpha) and phosphorylated IKK-alpha and suppressed the depletion of IkappaB-alpha and the accumulation of phosphorylated IkappaB-alpha. HS treatment blocked ANG II induced expression of IL-6 and ICAM-1 in the heart. ANG II and HS treatment induced high-level expression of Hsp27 and Hsp70 and their phosphorylation. Phosphorylated isoforms of Hsp27 and Hsp70 may play an important role in protecting the heart against ANG II-induced inflammation.     

Mechanical stretch induces mitochondria-dependent apoptosis in neonatal rat cardiomyocytes and G2／M accumulation in cardiac fibroblasts

Heart remodeling is associated with the loss of cardiomyocytes and increase of fibrous tissue owing to abnormal mechanical load in a number of heart disease conditions. In present study, a well-described in vitro sustained stretch model was employed to study mechanical stretch-induced responses in both neonatal cardiomyocytes and cardiac fibroblasts. Cardiomyocytes, but not cardiac fibroblasts, underwent mitochondria-dependent apoptosis as evidenced by cytochrome c (cyto c) and Smac/DIABLO release from mitochondria into cytosol accompanied by mitochondrial membrane potential (△ψm) reduction, indicative of mitochondrial permeability transition pore (PTP)opening. Cyclosporin A, an inhibitor of PTP, inhibited stretch-induced cyto c release, △ψm reduction and apoptosis,suggesting an important role of mitochondrial PTP in stretch-induced apoptosis. The stretch also resulted in increased expression of the pro-apoptotic Bcl-2 family proteins, including Bax and Bad, in cardiomyocytes, but not in fibroblasts. Bax was accumulated in mitochondria following stretch. Cell permeable Bid-BH3 peptide could induce and facilitate stretch-induced apoptosis and △ψm reduction in cardiomyocytes. These results suggest that Bcl-2 family proteins play an important role in coupling stretch signaling to mitochondrial death machinery, probably by targeting to PTP. Interestingly, the levels of p53 were increased at 12 h after stretch although we observed that Bax upregulation and apoptosis occurred as early as 1 h. Adenovirus delivered dominant negative p53 blocked Bax upregulation in cardiomyocytes but showed partial effect on preventing stretch-induced apoptosis, suggesting that p53 was only partially involved in mediating stretch-induced apoptosis. Furthermore, we showed that p21 was upregulated and cyclin B 1 was downregulated only in cardiac fibroblasts, which may be associated with G2/M accumulation in response to mechanical stretch.