GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.

Growth differentiation factor 15 (GDF15) as a stress response cytokine is involved in the development and progression of several diseases associated with metabolic disorders. However, the regulatory role and the underlying mechanisms of GDF15 in sepsis remain poorly defined. Our study analyzed the levels of GDF15 and its correlations with the clinical prognosis of patients with sepsis. In vivo and in vitro models of sepsis were applied to elucidate the role and mechanisms of GDF15 in sepsis-associated lung injury. We observed strong correlations of plasma GDF15 levels with the levels of C-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), and lactate as well as Sequential Organ Failure Assessment (SOFA) scores in patients with sepsis. In the mouse model of lipopolysaccharide-induced sepsis, recombinant GDF15 inhibited the proinflammatory responses and alleviated lung tissue injury. In addition, GDF15 decreased the levels of cytokines produced by alveolar macrophages (AMs). The anti-inflammatory effect of glycolysis inhibitor 2-DG on AMs during sepsis was mediated by GDF15 via inducing the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) and the expression of activating transcription factor 4 (ATF4). Furthermore, we explored the mechanism underlying the beneficial effects of GDF15 and found that GDF15 inhibited glycolysis and mitogen-activated protein kinases (MAPK)/nuclear factor-κB (NF-κB) signaling via promoting AMPK phosphorylation. This study demonstrated that GDF15 inhibited glycolysis and NF-κB/MAPKs signaling via activating AMP-activated protein kinase (AMPK), thereby alleviating the inflammatory responses of AMs and sepsis-associated lung injury. Our findings provided new insights into novel therapeutic strategies for treating sepsis.

Novel RNA polymerase I inhibitor CX-5461 suppresses imiquimod-induced experimental psoriasis.

Clinical treatment of psoriasis remains challenging because of possible long-term drug toxicities and loss of therapeutic effects over time. CX-5461 is a novel selective inhibitor of RNA polymerase I. Our previous studies have shown that CX-5461 has potent anti-inflammatory effects. Here we investigated whether CX-5461 could inhibit the development of imiquimod-induced experimental psoriasis in mice. Adult male C57BL/6 mice were used, and psoriasis-like lesions were induced by topical imiquimod treatment. In vivo, we demonstrated that topical application of CX-5461 prevented the development of imiquimod-induced psoriasis, with decreases in keratinocyte proliferation, T-cell infiltration and pathological angiogenesis. CX-5461 also reversed existing skin inflammation induced imiquimod and retarded the development of 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia and inflammation. In vitro, CX-5461 induced cell cycle arrest in keratinocytes, inhibited expressions of interleukin-17, interleukin-23 receptor and retinoic acid receptor-related orphan receptor-γt in activated T cells, and reduced angiogenic functions of endothelial cells. In conclusion, CX-5461 exhibits therapeutic effects on experimental psoriasis in mice, likely via multiple mechanisms including anti-proliferative, anti-inflammatory and anti-angiogenic activities.

CX-5461 is a potent immunosuppressant which inhibits T cell-mediated alloimmunity via p53-DUSP5.

CX-5461 is a first-in-class selective RNA polymerase I inhibitor. Previously we found that CX-5461 had anti-inflammatory activities. In this study we characterized potential immunosuppressive effects of CX-5461 and explored the underlying mechanisms. Allogeneic skin transplantation model (BALB/c to C57BL/6 mice) and heterotopic heart transplantation model (F344 to Lewis rats) were used. We showed that CX-5461 was a potent inhibitor of alloimmunity which prevented acute allograft rejections. CX-5461 treatment was invariably associated with expansion of the regulatory T cell population. In vitro, CX-5461 inhibited agonists-induced T cell activation. CX-5461 consistently inhibited the expression of interferon-γ and interleukin - 2, key mediators of T cell-mediated alloimmunity. Mechanistically, CX-5461-induced immunosuppression was, at least partly, dependent on the p53-DUSP5 (dual-specificity phosphatase 5) axis and subsequent antagonism of the Erk1/2 mitogen-activated protein kinase pathway. In conclusion, our results suggest that CX-5461 is a promising candidate of a novel class of immunosuppressant which may be used as an alternative to the currently approved anti-rejection therapies.

Amino acid starvation-induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance.

Mammalian cells utilize Akt-dependent signaling to deploy intracellular Glut4 toward cell surface to facilitate glucose uptake. Low-density lipoprotein receptor (LDLR) is the cargo receptor mediating endocytosis of apolipoprotein B-containing lipoproteins. However, signaling-controlled regulation of intracellular LDLR trafficking remains elusive. Here, we describe a unique amino acid stress response, which directs the deployment of intracellular LDLRs, causing enhanced LDL endocytosis, likely via Ca2+ and calcium/calmodulin-dependent protein kinase II-mediated signalings. This response is independent of induction of autophagy. Amino acid stress-induced increase in LDL uptake in vitro is comparable to that by pravastatin. In vivo, acute AAS challenge for up to 72 h enhanced the rate of hepatic LDL uptake without changing the total expression level of LDLR. Reducing dietary amino acids by 50% for 2 to 4 weeks ameliorated high fat diet-induced hypercholesterolemia in heterozygous LDLR-deficient mice, with reductions in both LDL and VLDL fractions. We suggest that identification of signaling-controlled regulation of intracellular LDLR trafficking has advanced our understanding of the LDLR biology, and may benefit future development of additional therapeutic strategies for treating hypercholesterolemia.

Anti-fibrotic effects of p53 activation induced by RNA polymerase I inhibitor in primary cardiac fibroblasts.

Several lines of studies have indicated that the p53 pathway may have important anti-fibrotic functions. Previously we found that the novel selective RNA polymerase I inhibitor CX-5461 induced a robust response of p53 phosphorylation and activation in vascular smooth muscle cells. In the present study, we characterized the anti-fibrotic effects of CX-5461 in primary cardiac fibroblasts. We showed that CX-5461 suppressed spontaneous and mitogen-stimulated activation, proliferation, and myofibroblast differentiation, at a concentration (1 µM) with no cytotoxicity. The inhibitory effects of CX-5461 were primarily mediated by activation of the p53 pathway rather than limiting the rate of ribosome biogenesis. It was also shown that CX-5461 triggered a non-canonical DNA damage response in cardiac fibroblasts, which acted as the upstream signal leading to p53 activation. Taking these together, we suggest that p53 activation by pharmacological inhibition of Pol I may represent a viable approach to repress the development of cardiac fibrosis.

The p53 pathway in vasculature revisited: A therapeutic target for pathological vascular remodeling?

Pathological vascular remodeling contributes to the development of restenosis following intraluminal interventions, transplant vasculopathy, and pulmonary arterial hypertension. Activation of the tumor suppressor p53 may counteract vascular remodeling by inhibiting aberrant proliferation of vascular smooth muscle cells and repressing vascular inflammation. In particular, the development of different lines of small-molecule p53 activators ignites the hope of treating remodeling-associated vascular diseases by targeting p53 pharmacologically. In this review, we discuss the relationships between p53 and pathological vascular remodeling, and summarize current experimental data suggesting that drugging the p53 pathway may represent a novel strategy to prevent the development of vascular remodeling.

Therapeutic efficacy of the novel selective RNA polymerase I inhibitor CX-5461 on pulmonary arterial hypertension and associated vascular remodelling.

BACKGROUND AND PURPOSE: CX-5461 is a novel selective RNA polymerase I (Pol I) inhibitor. Previously, we found that CX-5461 could inhibit pathological arterial remodelling caused by angioplasty and transplantation. In the present study, we explored the pharmacological effects of CX-5461 on experimental pulmonary arterial hypertension (PAH) and PAH-associated vascular remodelling. EXPERIMENTAL APPROACH: PAH was induced in Sprague-Dawley rats by monocrotaline or Sugen/hypoxia. KEY RESULTS: We demonstrated that CX-5461 was well tolerated for in vivo treatments. CX-5461 prevented the development of pulmonary arterial remodelling, perivascular inflammation, pulmonary hypertension, and improved survival. More importantly, CX-5461 partly reversed established pulmonary hypertension. In vitro, CX-5461 induced cell cycle arrest in human pulmonary arterial smooth muscle cells. The beneficial effects of CX-5461 in vivo and in vitro were associated with increased activation (phosphorylation) of p53. CONCLUSION AND IMPLICATIONS: Our results suggest that pharmacological inhibition of Pol I may be a novel therapeutic strategy to treat otherwise drug-resistant PAH.

Nucleolar stress induces a senescence-like phenotype in smooth muscle cells and promotes development of vascular degeneration.

Senescence of smooth muscle cells (SMCs) has a crucial role in the pathogenesis of abdominal aortic aneurysm (AAA), a disease of vascular degeneration. Perturbation of cellular ribosomal DNA (rDNA) transcription triggers nucleolar stress response. Previously we demonstrated that induction of nucleolar stress in SMCs elicited cell cycle arrest via the ataxia-telangiectasia mutated (ATM)/ATM- and Rad3-related (ATR)-p53 axis. However, the specific roles of nucleolar stress in vascular degeneration remain unexplored. In the present study, we demonstrated for the first time that in both human and animal AAA tissues, there were non-coordinated changes in the expression of RNA polymerase I machinery components, including a downregulation of transcription initiation factor-IA (TIF-IA). Genetic deletion of TIF-IA in SMCs in mice (smTIF-IA-/-) caused spontaneous aneurysm-like lesions in the aorta. In vitro, induction of nucleolar stress triggered a non-canonical DNA damage response, leading to p53 phosphorylation and a senescence-like phenotype in SMCs. In human AAA tissues, there was increased nucleolar stress in medial cells, accompanied by localized DNA damage response within the nucleolar compartment. Our data suggest that perturbed rDNA transcription and induction of nucleolar stress contribute to the pathogenesis of AAA. Moreover, smTIF-IA-/- mice may be a novel animal model for studying spontaneous AAA-like vascular degenerations.

Long noncoding RNAs: Important participants and potential therapeutic targets for myocardial ischaemia reperfusion injury.

Myocardial ischaemia reperfusion (I/R) injury is one of the leading causes of coronary artery disease-associated morbidity and mortality. While different strategies have been used to limit I/R injuries, cardiac functions often do not recover to the normal level as anticipated. Recent studies have pointed to important roles of long noncoding RNAs (lncRNAs) in the development of myocardial I/R injury. LncRNA is a class of RNA molecules of more than 200 nucleotides in length which are not translated into proteins. I/R causes dysregulation of lncRNA expression in cardiomyocytes, thereby affecting multiple cellular functions including mitochondrial homeostasis, apoptosis, necrosis and autophagy, suggesting that manipulating lncRNAs may be of great potential in counteracting I/R injury-induced myocardial dysfunctions. In this review, we provide an updated summary on our knowledge about contributions of lncRNAs to the development of I/R injury, with an emphasis on the functional links between several well established cardiac lncRNAs and regulation of cellular outcomes post I/R.

Pharmacological preconditioning with the cellular stress inducer thapsigargin protects against experimental sepsis.

Previous studies have shown that pretreatment with thapsigargin (TG), a cellular stress inducer, produced potent protective actions against various pathologic injuries. So far there is no information on the effects of TG on the development of bacterial sepsis. Using lipopolysaccharides- and cecal ligation/puncture-induced sepsis models in mice, we demonstrated that preconditioning with a single bolus administration of TG conferred significant improvements in survival. The beneficial effects of TG were not mediated by ER stress induction or changes in Toll-like receptor 4 signaling. In vivo and in cultured macrophages, we identified that TG reduced the protein production of pro-inflammatory cytokines, but exhibited no significant effects on steady state levels of their transcriptions. Direct measurement on the fraction of polysome-bound mRNAs revealed that TG reduced the translational efficiency of pro-inflammatory cytokines in macrophages. Moreover, we provided evidence suggesting that repression of the mTOR (the mammalian target of rapamycin) signaling pathway, but not activation of the PERK (protein kinase R-like endoplasmic reticulum kinase)-eIF2α (eukaryotic initiation factor 2α) pathway, might be involved in mediating the TG effects on cytokine production. In summary, our results support that pharmacological preconditioning with TG may represent a novel strategy to prevent sepsis-induced mortality and organ injuries.

Adventitial Activation in the Pathogenesis of Injury-Induced Arterial Remodeling: Potential Implications in Transplant Vasculopathy.

Transplant vasculopathy is one of the major causes of chronic rejection after solid organ transplantation. The pathogenic mechanisms of transplant vasculopathy are still poorly understood. Herein, we summarize current evidence suggesting that activation of the tunica adventitia may be involved in the pathogenesis of transplant vasculopathy. Adventitia is an early responder to various vascular injuries and plays an integral role in eliciting vascular inflammation and remodeling. Accumulation of macrophages in the adventitia promotes the development of vascular remodeling by releasing a variety of paracrine factors that have profound impacts on vascular mural cells. Targeting adventitial macrophages has been shown to be effective for repressing transplantation-induced arterial remodeling in animal models. Adventitia also fosters angiogenesis, and neovascularization of the adventitial layer may facilitate the transport of inflammatory cells through the arterial wall. Further investigations are warranted to clarify whether inhibiting adventitial oxidative stress and/or adventitial neovascularization are better strategies for preventing transplant vasculopathy.

Protective Effects of Live Combined B. subtilis and E. faecium in Polymicrobial Sepsis Through Modulating Activation and Transformation of Macrophages and Mast Cells.

Aims: Clinical studies showed that the use of probiotics during critical illness reduced nosocomial infection and improved clinical outcome. However, the functional mechanisms of probiotics is remains unclear. Therefore the aim of current study is to explore the protective effects and understand the underlying mechanisms for the beneficial effects of live combined Bacillus subtilis and Enterococcus faecium (LCBE) in cecal ligation puncture (CLP)-induced sepsis. Methods and Results: Seven-week-old C57BL/6J mice were divided into three groups: sham group (6 mice), CLP-control group (20 mice, pretreatment with saline for 7 days before CLP surgery) and CLP-probiotics group (14 mice, pretreatment with LCBE enteric-coated capsules for 7 days before CLP surgery). In survival experiment, mice were monitored for 7 days after CLP. After the procedure, mice were sacrificed, and, serum, and peritoneal lavage fluid were collected and intestinal ileal samples were harvested. Results: Our results showed that the mortality was significantly reduced in mice CLP-probiotics group vs. CLP-control group (P < 0.05). Also, treatment CLP-probiotics group decreased the injury scores CLP-probiotics group when compared to CLP-control group. Additionally, levels of pro-inflammatory cytokines IL-6 and TNF-α levels in the serum and intestinal ileal tissues of CLP-probiotics group were reduced when compared to CLP-control group (P < 0.05). However, no significant differences in anti-inflammatory levels of IL-10 and TGF-ß1 were observed between CLP-control and CLP-probiotic groups. Furthermore, our experiments showed that that probiotic treatment suppressed the macrophage activation and transformation from M-type to M1-type, inhibited the mast cells (MCs) degranulation, and activation of AKT (kinase B) pathway. Conclusion: In conclusion, our data shows that probiotics have a protective role in CLP septic mice through reducing intestinal inflammation, altering macrophage polarization and MCs degranulation, and regulating AKT signaling. Significance and Impact of Study: This study demonstrated the protective effects and mechanisms involved in the protective role of live combined Bacillus subtilis and Enterococcus faecium (LCBE) in CLP-induced septic mice model.

Co-expression of podoplanin and fibroblast growth factor 1 predicts poor prognosis in patients with lung squamous cell carcinoma.

Podoplanin and fibroblast growth factor (FGF) 1 have been detected more frequently in lung squamous cell carcinoma (SQCC) compared with lung adenocarcinoma. Furthermore, it has been previous demonstrated that FGF1 is located on the edge of tumor nests in certain lung SQCC sections, which resembles the characteristic expression pattern of podoplanin. Podoplanin and FGF1 have roles in lymphangiogenesis and angiogenesis. Based on their consistently specific expression in lung SQCC and similar localization patterns, the present study aimed to investigate whether the expression of podoplanin in tumor cells is correlated with FGF1 expression in lung SQCC and whether their co­expression has clinicopathological significance, particularly for lymphangiogenesis/angiogenesis. The correlation between podoplanin and FGF1 expression in tumor cells of 82 lung SQCC cases was investigated by immunohistochemical staining and the association between the co­expression of podoplanin and FGF1, and clinicopathological factors such as microvessel density (MVD), was examined in these samples. In addition, the prognostic value of co­expression of podoplanin and FGF1 in tumor cells was determined, and the regulation of FGF1 expression and angiogenesis by podoplanin was examined in vitro in a human lung SQCC cell line. Immunohistochemical analysis demonstrated that there was a significant correlation between podoplanin and FGF1 expression in lung SQCC tumor cells (R=0.591; P<0.0001). Co­expression of podoplanin and FGF1 was significantly associated with larger primary tumor size, advanced TNM stage and higher intratumoral MVD. Survival analysis demonstrated that cases with podoplanin and FGF1 double­positive staining had a significantly lower survival rate compared with cases with double­negative staining. In vitro experiments revealed that podoplanin regulated FGF1 expression and affected tube formation of human umbilical vein endothelial cells. Combined, the results demonstrated that podoplanin was co­expressed with FGF1 in lung SQCC and this co­expression was correlated with poor prognosis.

Progranulin promotes colorectal cancer proliferation and angiogenesis through TNFR2/Akt and ERK signaling pathways.

Progranulin (PGRN) has been shown to be involved in the process of inflammation, wound healing, and cartilage development; and its role in the progression of breast and ovarian cancer is also well established. However, the expression status of PGRN in colorectal cancers (CRCs) and its molecular mechanisms responsible for tumorigenesis have not been addressed so far. Herein, we demonstrated that PGRN was highly expressed and had clinical relevance with CRCs since its overexpression was associated with advanced stages of CRCs, poorer patients' prognosis, and increased expression of proliferation and angiogenesis markers. PGRN up-regulation significantly promoted the expression of Ki67 and vascular endothelial growth factor A (VEGF-A) as well as the growth rate in CRC cell lines, while PGRN down-regulation had the opposite effects. Strikingly, PGRN derived from CRCs could directly induce proliferation, migration, tubule formation, as well as VEGF-A expression in human umbilical vein endothelial cells (HUVECs). Furthermore, we provided mechanistic evidences that the regulation of Ki67 and VEGF-A expression by PGRN was mediated by tumor necrosis factor receptor 2 (TNFR2)/Akt and the ERK signaling pathways in both CRC cells and HUVECs. Taken together, these findings suggested that PGRN could promote proliferation and angiogenesis through TNFR2/Akt and ERK signaling pathways in CRCs, providing the new insight into the mechanism of PGRN in tumor proliferation and angiogenesis.

Immunoglobulin-like transcript 4 promotes tumor progression and metastasis and up-regulates VEGF-C expression via ERK signaling pathway in non-small cell lung cancer.

Immunoglobulin-like transcript (ILT) 4 has long been thought to be cell-surface molecule in certain immune cells and negatively regulates immune response. Recently, overexpression of ILT4 has been observed in a few cancers with unknown function. Here, we showed manipulation of ILT4 affected non-small cell lung cancer (NSCLC) cell proliferation, migration and invasion in vitro analyses. In vivo, ILT4 promoted the tumor growth and metastasis. Furthermore, the phosphorylation of extracellular regulated protein kinases (ERK1/2) was enhanced in ILT4 overexpressing NSCLC cells. ERK1/2 specific inhibitor U0126 suppressed the proliferation, migration and invasion of those cells. Stepwise investigations demonstrated that vascular endothelial growth factor C (VEGF-C) was the downstream effector of ILT4 and ERK1/2. Silence of VEGF-C attenuated the migration and invasion activity of ILT4 overexpressing cells. Moreover, Kaplan-Meier survival analysis indicated that NSCLC patients with ILT4 positive expression had a poor patient survival. ILT4 and VEGF-C expression had notable positive correlation in cancer cells, and their co-expression was significantly associated with adverse prognostic factors. Our findings suggest that ILT4 drives NSCLC development in part on activation of ERK signaling which in turn upregulates VEGF-C. ILT4 could be a novel cancer therapeutic target for NSCLC.

Co-expression of immunoglobulin-like transcript 4 and angiopoietin-like proteins in human non-small cell lung cancer.

The development of strategies for the inhibition of non­small cell lung cancer (NSCLC) progression and metastasis have been mainly unsuccessful, in part due to insufficient mechanistic understanding of the disease. In the current study, the critical role of the co­expression of immunoglobulin­like transcript 4 (ILT4) and its ligands, angiopoietin­like proteins (ANGPTLs), in the development of NSCLC was demonstrated. ILT4 and ANGPTL2 or ANGPTL5 were found to be co­expressed in the five NSCLC cell lines that were investigated at the mRNA and protein level. Upon up­ or downregulation of ILT4, the expression of ANGPTL2 was increased or reduced, respectively, while the expression of ANGPTL5 was unaffected. The co­expression of ILT4 and ANGPTL2/ANGPTL5 was detected in human primary NSCLC tissues using immunohistochemical analysis. In total, 114 lung cancer specimens were included in the study; high expression of ILT4, ANGPTL2 and ANGPTL5 was observed in 58.8, 45.6 and 55.3%, respectively. The expression of ILT4 was found to be significantly correlated with a high expression level of ANGPTL2 (R=0.466, P=0.004); however, it was not correlated with the expression of ANGPTL5 (R=0.142, P=0.131). In ILT4­positive samples, cases with ANGPTL2­positive expression levels presented greater levels of lymph node metastasis (P=0.011) and shorter overall survival times (P=0.045). In addition, cases with ANGPTL5­positive expression presented poor overall survival rates (P=0.040). By contrast, in the ILT4­negative cases, no statistically significant differences were identified in the overall survival rates between samples with high and low expression of ANGPTL2 or ANGPTL5. In conclusion, the present study demonstrated the presence of interaction among ILT4 and ANGPTLs, which may be important in NSCLC progression. Therefore, the blockade of ANGPTLs or ILT4 may be an effective therapeutic approach for NSCLC treatment.

Insulin inhibits lipopolysaccharide-induced nitric oxide synthase expression in rat primary astrocytes.

Excessive production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) from reactive astrocytes and microglia may contribute to the development of many types of neurological diseases. Insulin has been shown to inhibit the expression of iNOS, in several organs and cell types. Although insulin and its receptors are present in the central nervous system, the effects of insulin on the iNOS pathway in the brain have not been determined. In this study, using lipopolysaccharide (LPS)-stimulated astrocytes as a model of reactive astrocytes, we investigated the effects of insulin on iNOS expression in activated astrocytes and the mechanism involved. The expression of iNOS was significantly upregulated by LPS in astrocytes. Insulin applied prior to LPS, dose-dependently inhibited LPS-induced iNOS gene expression and iNOS protein levels. In agreement with the suppressive effects of insulin on iNOS expression, insulin also inhibited LPS-induced iNOS activity and NO production. Moreover, insulin was found to significantly inhibit LPS-induced IκB-α phosphorylation and degradation, which led to a decrease in levels of the p65 subunit of NF-κB in the nuclear fraction. Therefore, insulin inhibited LPS-induced iNOS expression via suppressing NF-κB pathway in astrocytes. In addition, treatment with insulin had no effect on LPS-induced PKB phosphorylation. Based on our results, it is plausible to speculate that insulin in the brain may play a neuroprotective role in neurological disorders by controlling the release of NO via the regulation of iNOS expression in astrocytes.

Calcineurin mediates the protective effect of postconditioning on skeletal muscle.

We aimed to investigate whether ischemic postconditioning (I-postC) protects skeletal muscle against ischemia-reperfusion (I/R) injury through the calcineurin (CaN) pathway. Male Wistar rats underwent 4 h of right-hind-limb ischemia induced by clamping the femoral artery, then reperfusion for 2 h (I/R-2 h), 12 h (I/R-12 h), or 24 h (I/R-24 h) with or without I-postC. Ischemic postconditioning was induced by three cycles of 1-min reperfusion and 1-min ischemia at the onset of reperfusion after prolonged ischemia. The I-postC-24 h group was treated with or without cyclosporine A (a CaN inhibitor) 10 mg/kg per day for 3 days before artery occlusion. Cultured skeletal muscle cells (SMCs) from neonatal rats were exposed to 2-h hypoxia then 24-h reoxygenation (H/R), then postconditioned with two cycles of 10-min reoxygenation and 10-min hypoxia after prolonged hypoxia (hypoxia postconditioning [H-postC]) in the presence or absence of cyclosporine A. We observed the effects of activated CaN overexpression on apoptosis and viability of SMCs under H-postC. Ischemic postconditioning attenuated the increase in the level of malondialdehyde in skeletal muscle induced by I/R-2 h and I/R-24 h (P < 0.05) and lactate dehydrogenase in plasma induced by I/R-12 h and I/R-24 h (P < 0.05). Cyclosporine A abolished the protective role of I-postC in malondialdehyde level and lactate dehydrogenase leakage (P < 0.05, vs. I-postC group). Hypoxia postconditioning suppressed SMC apoptosis induced by H/R (P < 0.05, vs. H/R), which was accompanied by increased CaN expression. Cyclosporine A abolished the antiapoptotic effect of H-postC on SMCs (P < 0.05, vs. H-postC group). Overexpression of activated CaN strengthened the cytoprotection of H-postC (P < 0.05, vs. H-postC group). Ischemic postconditioning may protect skeletal muscle against I/R injury through the CaN pathway.

[C/EBP homologous protein-mediated endoplasmic reticulum stress-related apoptosis pathway is involved in abdominal aortic constriction-induced myocardium hypertrophy in rats.].

Endoplasmic reticulum stress (ERS) is an adaptive process in response to circumstantial changes, but excessive and/or prolonged ERS can induce cell apoptosis. C/EBP homologous protein (CHOP) is a very important marker participating in ERS-associated cell apoptosis, while the role of the myocyte apoptosis induced by CHOP remains unclear in the development of hypertrophy. The present study aimed to investigate the effect of CHOP-mediated ERS-associated apoptosis on myocardial hypertrophy induced by abdominal aortic constriction in rats. Healthy male Wistar rats were randomly divided into model group (n=45) and control group (n=40). The rats in model group received abdominal aortic constriction. Hemodynamic changes, whole heart weight/body weight (HW/BW) and left ventricular weight/body weight (LVW/BW) were measured on 1 d, 3 d, 7 d, 14 d and 28 d after surgery, respectively. The mRNA expression of glucose-regulated protein 78 (GRP78), calreticulin (CRT) and CHOP, which are important markers of ERS, were detected by RT-PCR, and Western blot was used to assess the protein level of GRP78, CRT, CHOP, and apoptosis-associated proteins, Bax and Bcl-2. The results obtained were as follows. Compared with control group, the blood pressure, LVW/BW, and HW/BW of rats in model group increased significantly and cardiac function enhanced compensatively on 7 d after surgery, and increased progressively during the experiment. As early as 1 d after surgery, the mRNA level of CRT in model group increased by 136% (P< 0.01) compared with control, while the protein expression increased by 69.2% on 7 d after surgery (P<0.01). Both mRNA and protein expression of GRP78 increased by 20% and 186% (P<0.01) respectively on 7 d after surgery, and the expression sustained high level during the experiment afterwards. Correlation analysis indicated a positive correlation between +dp/dt(max) and CRT protein expression (r=0.780, P<0.01) as well as GRP78 protein expression (r=0.694, P<0.01). Prolonged ERS triggered myocyte apoptosis, as both the mRNA and protein level of CHOP in model group increased by 22.2% (P<0.01) and 76.0% (P<0.01) respectively compared with control on 7 d after hypertrophy (14 d after surgery), and meanwhile, the protein expression of pro-apoptotic Bax increased by 41.1% (P<0.01) and anti-apoptotic Bcl-2 protein expression decreased by 25.5% (P<0.01). Correlation analysis indicated a positive correlation between CHOP and Bax expression (r=0.654, P<0.01), and a negative correlation between CHOP and Bcl-2 expression (r=-0.671, P<0.01). These results suggest that abdominal aortic constriction induces a significant up-regulation in ER molecular chaperones at early stage of post-surgery, indicating that ERS response is activated in the rat heart; while prolonged ERS could lead to myocyte apoptosis, and CHOP-mediated ERS-associated apoptosis may contribute to myocardial hypertrophy. We speculate that cell apoptosis may take part in the regulation of myocardial hypertrophy and heart failure, and determine the progression of decompensated hypertrophy.

[Calreticulin is involved in ischemic postconditioning-induced attenuation of ischemia/reperfusion injury in rat skeletal muscle].

The present study was aimed to investigate the effect of ischemic postconditioning (I-postC) on ischemia/reperfusion (I/R) injury and whether calreticulin (CRT) is involved in its intracellular signal transduction both in vivo and in cultured skeletal muscle cells. I/R injury in the right hind limb of healthy male Wistar rats was induced by clamping the right femoral artery, and the rats were randomly divided into 3 groups (n=16): I/R group (4-hour ischemia/12- or 24-hour reperfusion), ischemic preconditioning (IPC) group (3 cycles of 1-minute ischemia/1-minute reperfusion) and I-postC group (3 cycles of 5-minute reperfusion/5-minute ischemia). The left hind limb was used as control. Lactate dehydrogenase (LDH) activity in blood plasma, wet/dry weight ratio (W/D) and ultramicrostructure of skeletal muscle were detected 12 h or 24 h after reperfusion. Cultured skeletal muscle cells from neonatal Sprague-Dawley (SD) rat were divided into 6 groups: hypoxia/reoxygenation (H/R) group, hypoxic postconditioning (H-postC) group, hypoxic preconditioning (HPC) group, cyclosporine A (CsA) + H-postC group, CsA + H/R group and control group. H/R was produced by 2-hour hypoxia/24-hour reoxygenation. The survival rate and apoptotic rate of skeletal muscle cells in each group were measured. Western blot was used to detect the expressions of CRT and calcineurin (CaN). The results were as follows: (1) During in vivo experiment, compared with I/R, I-postC significantly decreased LDH activity and W/D, attenuated the ultramicrostructure injury of skeletal muscle and the apoptosis of nucleolus. 12 h and 24 h after reperfusion, compared with that in I/R group, the expression of CRT in I-postC group increased by 439% and 102%, respectively (P<0.05), and the expression of CaN increased by 196% and 63%, respectively (P<0.05). Correlation analysis indicated a positive correlation between CRT and CaN expressions (r=0.865, P<0.01). (2) In cultured skeletal muscle cells, H-postC attenuated cell injury induced by H/R. Compared with those in H/R group, CRT and CaN expressions in H-postC increased by 31.8% (P<0.05) and 6.02%, respectively. The protection of H-postC and CaN up-regulation were eliminated when CsA, the inhibitor of CaN, was added before H-postC. Both in vivo and in vitro results indicate that I-postC, similar as IPC, can protect the skeletal muscle against I/R injury, and its effects may be mediated by CRT and CaN up-regulation. The inhibition of CaN expression may also attenuate the protective effects of I-postC.