O-GlcNAc modification of GSDMD attenuates LPS-induced endothelial cells pyroptosis.

OBJECTIVE: Increased O-linked ß-N-acetylglucosamine (O-GlcNAc) stimulation has been reported to protect against sepsis associated mortality and cardiovascular derangement. Previous studies, including our own research, have indicated that gasdermin-D(GSDMD)-mediated endothelial cells pyroptosis contributes to sepsis-associated endothelial injury. This study explored the functions and mechanisms of O-GlcNAc modification on lipopolysaccharide (LPS)-induced pyroptosis and its effects on the function of GSDMD. METHODS: A LPS-induced septic mouse model administrated with O-GlcNAcase (OGA) inhibitor thiamet-G (TMG) was used to assess the effects of O-GlcNAcylation on sepsis-associated vascular dysfunction and pyroptosis. We conducted experiments on human umbilical vein endothelial cells (HUVECs) by challenging them with LPS and TMG to investigate the impact of O-GlcNAcylation on endothelial cell pyroptosis and implications of GSDMD. Additionally, we identified potential O-GlcNAcylation sites in GSDMD by utilizing four public O-GlcNAcylation site prediction database, and these sites were ultimately established through gene mutation. RESULTS: Septic mice with increased O-GlcNAc stimulation exhibited reduced endothelial injury, GSDMD cleavage (a marker of pyroptosis). O-GlcNAc modification of GSDMD mitigates LPS-induced pyroptosis in endothelial cells by preventing its interaction with caspase-11 (a human homologous of caspases-4/5). We also identified GSDMD Serine 338 (S338) as a novel site of O-GlcNAc modification, leading to decreased association with caspases-4 in HEK293T cells. CONCLUSIONS: Our findings identified a novel post-translational modification of GSDMD and elucidated the O-GlcNAcylation of GSDMD inhibits LPS-induced endothelial injury, suggesting that O-GlcNAc modification-based treatments could serve as potential interventions for sepsis-associated vascular endothelial injury.

Phosphorylated PKM2 regulates endothelium-dependent vasodilation in diabetes. / ç£·é ¸åŒ–PKM2è°ƒæŽ§ç³–å°¿ç— å† çš®ä¾èµ–æ€§è¡€ç®¡èˆ’å¼ åŠŸèƒ½.

OBJECTIVES: Endothelium-dependent vasodilation dysfunction is the pathological basis of diabetic macroangiopathy. The utilization and adaptation of endothelial cells to high glucose determine the functional status of endothelial cells. Glycolysis pathway is the major energy source for endothelial cells. Abnormal glycolysis plays an important role in endothelium-dependent vasodilation dysfunction induced by high glucose. Pyruvate kinase isozyme type M2 (PKM2) is one of key enzymes in glycolysis pathway, phosphorylation of PKM2 can reduce the activity of pyruvate kinase and affect the glycolysis process of glucose. TEPP-46 can stabilize PKM2 in its tetramer form, reducing its dimer formation and phosphorylation. Using TEPP-46 as a tool drug to inhibit PKM2 phosphorylation, this study aims to explore the impact and potential mechanism of phosphorylated PKM2 (p-PKM2) on endothelial dependent vasodilation function in high glucose, and to provide a theoretical basis for finding new intervention targets for diabetic macroangiopathy. METHODS: The mice were divided into 3 groups: a wild-type (WT) group (a control group, C57BL/6 mice) and a db/db group (a diabetic group, db/db mice), which were treated with the sodium carboxymethyl cellulose solution (solvent) by gavage once a day, and a TEPP-46 group (a treatment group, db/db mice+TEPP-46), which was gavaged with TEPP-46 (30 mg/kg) and sodium carboxymethyl cellulose solution once a day. After 12 weeks of treatment, the levels of p-PKM2 and PKM2 protein in thoracic aortas, plasma nitric oxide (NO) level and endothelium-dependent vasodilation function of thoracic aortas were detected. High glucose (30 mmol/L) with or without TEPP-46 (10 µmol/L), mannitol incubating human umbilical vein endothelial cells (HUVECs) for 72 hours, respectively. The level of NO in supernatant, the content of NO in cells, and the levels of p-PKM2 and PKM2 protein were detected. Finally, the effect of TEPP-46 on endothelial nitric oxide synthase (eNOS) phosphorylation was detected at the cellular and animal levels. RESULTS: Compared with the control group, the levels of p-PKM2 in thoracic aortas of the diabetic group increased (P<0.05). The responsiveness of thoracic aortas in the diabetic group to acetylcholine (ACh) was 47% lower than that in the control group (P<0.05), and that in TEPP-46 treatment group was 28% higher than that in the diabetic group (P<0.05), while there was no statistically significant difference in the responsiveness of thoracic aortas to sodium nitroprusside (SNP). Compared with the control group, the plasma NO level of mice decreased in the diabetic group, while compared with the diabetic group, the phosphorylation of PKM2 in thoracic aortas decreased and the plasma NO level increased in the TEPP-46 group (both P<0.05). High glucose instead of mannitol induced the increase of PKM2 phosphorylation in HUVECs and reduced the level of NO in supernatant (both P<0.05). HUVECs incubated with TEPP-46 and high glucose reversed the reduction of NO production and secretion induced by high glucose while inhibiting PKM2 phosphorylation (both P<0.05). At the cellular and animal levels, TEPP-46 reversed the decrease of eNOS (ser1177) phosphorylation induced by high glucose (both P<0.05). CONCLUSIONS: p-PKM2 may be involved in the process of endothelium-dependent vasodilation dysfunction in Type 2 diabetes by inhibiting p-eNOS (ser1177)/NO pathway.

HMGB1 mediates homocysteine-induced endothelial cells pyroptosis via cathepsin V-dependent pathway.

Endothelial cells injury and pro-inflammation cytokines release are the initial steps of hyperhomocysteinemia (HHcy)-associated vascular inflammation. Pyroptosis is a newly identified pro-inflammation form of programmed cell death, causing cell lysis and IL-1ß release, and characterized by the caspases-induced cleavage of its effector molecule gasdermins (GSDMs). However, the effect of homocysteine (Hcy) on endothelial cells pyroptosis and the underlying mechanisms have not been fully defined. We have previously reported that Hcy induces vascular endothelial inflammation accompanied by the increase of high mobility group box-1 protein (HMGB1) and lysosomal cysteine protease cathepsin V in endothelial cells, and other studies have shown that HMGB1 or cathepsins are involved in activation of NLRP3 inflammasome and caspase-1. Here, we investigated the role of HMGB1 and cathepsin V in the process of Hcy-induced pyroptosis. We observed an increase in plasma IL-1ß levels in HHcy patients and mice models, cathepsin V inhibitor reduced the plasma IL-1ß levels and cleavage of GSDMD full-length into GSDMD N-terminal in the thoracic aorta of hyperhomocysteinemia mice. Using cultured HUVECs, we observed that Hcy promoted GSDMD N-terminal expression, silencing GSDMD or HMGB1 rescued Hcy-induced pyroptosis. HMGB1 also increased GSDMD N-terminal expression, and silencing cathepsin V reversed HMGB1-induced pyroptosis. HMGB1 could increase lysosome permeability, and silencing cathepsin V attenuated HMGB1-induced activation of caspase-1. In conclusion, this study has delineated a novel mechanism that HMGB1 mediated Hcy-induced endothelial cells pyroptosis partly via cathepsin V-dependent pathway.

NRP1 regulates HMGB1 in vascular endothelial cells under high homocysteine condition.

Endothelial inflammation plays an important role in hyperhomocysteinemia (HHcy)-associated vascular diseases. High mobility group box 1 (HMGB1) is a pro-inflammatory danger molecule produced by endothelial cells. However, whether HMGB1 is involved in vascular endothelial inflammation of HHcy is poorly understood. Neuropilin-1 (NRP1) mediates inflammatory response and activates mitogen-activated protein kinases (MAPKs) pathway that has been reported to be involved in regulation of HMGB1. The aim of this study was to determine the alteration of HMGB1 in HHcy, and the role of NRP1 in regulation of endothelial HMGB1 under high homocysteine (Hcy) condition. In the present study, we first observed that the plasma level of HMGB1 was elevated in HHcy patients and an experimental rat model, and increased HMGB1 was also observed in the thoracic aorta of an HHcy rat model. HMGB1 was induced by Hcy accompanied with upregulated NRP1 in vascular endothelial cells. Overexpression of NRP1 promoted expression and secretion of HMGB1 and endothelial inflammation; knockdown of NRP1 inhibited HMGB1 and endothelial inflammation induced by Hcy, which partially regulated through p38 MAPK pathway. Furthermore, NRP1 inhibitor ATWLPPR reduced plasma HMGB1 level and expression of HMGB1 in the thoracic aorta of HHcy rats. In conclusion, our data suggested that Hcy requires NRP1 to regulate expression and secretion of HMGB1. The present study provides the evidence for inhibition of NRP1 and HMGB1 to be the novel therapeutic targets of vascular endothelial inflammation in HHcy in the future. NEW & NOTEWORTHY This study shows for the first time to our knowledge that the plasma level of high mobility group box 1 (HMGB1) is elevated in hyperhomocysteinemia (HHcy) patients, and homocysteine promotes expression and secretion of HMGB1 partially regulated by neuropilin-1 in endothelial cells, which is involved in endothelial inflammation. Most importantly, these new findings will provide a potential therapeutic strategy for vascular endothelial inflammation in HHcy.

Novel role of PKR in palmitate-induced Sirt1 inactivation and endothelial cell senescence.

Endothelial cell senescence is regarded as a vital characteristic of cardiovascular diseases. Elevated palmitate (PA) is an independent risk factor of cardiovascular diseases, but its role in endothelial cell senescence is currently unknown. During the course of studying the prosenescent role of PA, we discovered a key role of dsRNA-dependent protein kinase [protein kinase R (PKR)] in endothelial senescence. Exposure of human umbilical vein endothelial cells (HUVECs) to PA-induced cell senescence is characterized by increased levels of senescence-associated ß-galactose glucosidase activity, excessive production of reactive oxygen species production, impaired cellular proliferation, and G1 phase arrest. This phenomenon is associated with an increase of PKR autophosphorylation and decreased activity of sirtuin 1 (Sirt1), a pivotal antisenescent factor. PKR inactivation by PKR siRNA or its phosphorylation inhibitor 2-aminopurine significantly attenuated PA-induced HUVEC senescence by reversing Sirt1 activity and its downstream signaling. Moreover, to study the regulatory mechanism between PKR and Sirt1, we found that PKR promotes JNK activation to inhibit Sirt1 activity and that this effect could be reversed by the JNK inhibitor SP600125. These findings provide evidence that PKR mediates PA-induced HUVEC senescence by inhibiting Sirt1 signaling. Our study provides novel insights into the actions and mechanisms of PKR in endothelial senescence. NEW & NOTEWORTHY This study first provides a novel observation that dsRNA-dependent protein kinase (PKR) mediates palmitate-induced sirtuin 1 inactivation and subsequent human umbilical vein endothelial cell senescence. Most importantly, these new findings will provide a potential therapeutic strategy to improve free fatty acid-induced endothelial senescence by targeting PKR in cardiovascular diseases.

What is the impact of PCSK9 rs505151 and rs11591147 polymorphisms on serum lipids level and cardiovascular risk: a meta-analysis.

BACKGROUND: PCSK9 rs505151 and rs11591147 polymorphisms are identified as gain- and loss-of-function mutations, respectively. The effects of these polymorphisms on serum lipid levels and cardiovascular risk remain to be elucidated. METHODS: In this meta-analysis, we explored the association of PCSK9 rs505151 and rs11591147 polymorphisms with serum lipid levels and cardiovascular risk by calculating the standardized mean difference (SMD) and odds ratios (OR) with 95% confidence intervals (CI). RESULTS: Pooled results analyzed under a dominant genetic model indicated that the PCSK9 rs505151 G allele was related to higher levels of triglycerides (SMD: 0.14, 95% CI: 0.02 to 0.26, P = 0.021, I2 = 0) and low-density lipoproteins cholesterol (LDL-C) (SMD: 0.17, 95% CI: 0.00 to 0.35, P = 0.046, I2 = 75.9%) and increased cardiovascular risk (OR: 1.50, 95% CI: 1.19 to 1.89, P = 0.0006, I2 = 48%). The rs11591147 T allele was significantly associated with lower levels of total cholesterol (TC) and LDL-C (TC, SMD: -0.45, 95% CI: -0.57 to -0.32, P = 0.000, I2 = 0; LDL-C, SMD: -0.44, 95% CI: -0.55 to -0.33, P = 0.000, I2 = 0) and decreased cardiovascular risk (OR: 0.77, 95% CI: 0.60 to 0.98, P = 0.031, I2 = 59.9) in Caucasians. CONCLUSIONS: This study indicates that the variant G allele of PCSK9 rs505151 confers increased triglyceride (TG) and LDL-C levels, as well as increased cardiovascular risk. Conversely, the variant T allele of rs11591147 protects carriers from cardiovascular disease susceptibility and lower TC and LDL-C levels in Caucasians. These findings provide useful information for researchers interested in the fields of PCSK9 genetics and cardiovascular risk prediction not only for designing future studies, but also for clinical and public health applications.

ASGR1 promotes liver injury in sepsis by modulating monocyte-to-macrophage differentiation via NF-κB/ATF5 pathway.

AIMS: Liver is a pivotal organ for sepsis-induced injury and approximately 40 % of liver injury results from sepsis. During hepatic injury, monocyte-to-macrophage differentiation is a key event because it results in the regulation of immune response. Asialoglycoprotein receptor 1 (ASGR1) is enriched in classical monocyte of peripheral blood mononuclear cells (PBMCs). We aimed to explore the effect of ASGR1 on monocyte-to-macrophage differentiation and the modulation of sepsis-induced liver injury. MAIN METHODS: ASGR1-knockdown/overexpression THP-1 cells and mice bone marrow-derived macrophages (BMDMs) induced by PMA and 30 % L929-cell conditioned medium were utilized to test the impact of ASGR1 on monocyte-to-macrophage differentiation and molecular mechanism respectively. Expression of differentiation specific factors were assessed via flow cytometry and real-time quantitative PCR. RNA-sequencing (RNA-seq) analysis revealed the effect of ASGR1 on monocyte-to-macrophage differentiation. Further, differentiation specific factors ATF5 and NF-κB pathways were examined via Western blot. The interaction between ASGR1 and ATF5 was further examined by co-IP. Finally, LPS-induced ASGR1-knockdown mice sepsis was used to investigate the effect of ASGR1 on monocyte-to-macrophage differentiation, liver injury and survival. KEY FINDINGS: ASGR1 promoted monocyte-to-macrophage differentiation via up-regulating CD68, F4/80 and CD86. Additionally, inhibited-ASGR1 decreased ATF5 expression by suppressing phosphorylation of NF-κB and IKBa in vitro and in vivo. ASGR1-knockdown mice suppressed Ly6Chi inflammatory monocytes in PBMCs, and restrained CD45+CD11bhiF4/80+Ly6Clo monocyte-derived macrophages and CD45+CD11b+F4/80+Ly6C+ inflammatory macrophages in livers. It also suppressed the level of IL-1ß, IL-6, TNF-α and alleviated liver injury and improved survival after sepsis. SIGNIFICANCE: ASGR1 is a negative regulator for sepsis-induced liver injury and survival.

A Mendelian randomization study to assess the genetic liability of type 1 diabetes mellitus for IgA nephropathy.

Background: The prevalence of immunoglobulin A nephropathy (IgAN) seems to be higher in patients with type 1 diabetes mellitus (T1DM) than that in the general population. However, whether there exists a causal relationship between T1DM and IgAN remains unknown. Methods: This study conducted a standard two-sample Mendelian randomization (MR) analysis to assess the causal inference by four MR methods, and the inverse variance-weighted (IVW) approach was selected as the primary method. To further test the independent causal effect of T1DM on IgAN, multivariable MR (MVMR) analysis was undertaken. Sensitivity analyses incorporating multiple complementary MR methods were applied to evaluate how strong the association was and identify potential pleiotropy. Results: MR analyses utilized 81 single-nucleotide polymorphisms (SNPs) for T1DM. The evidence supports a significant causal relationship between T1DM and increased risk of IgAN [odds ratio (OR): 1.39, 95% confidence interval (CI): 1.10-1.74 for IVW, p < 0.05]. The association still exists after adjusting for triglyceride (TG), fasting insulin (FI), fasting blood glucose (FBG), homeostasis model assessment of beta-cell function (HOMA-B) and insulin resistance (HOMA-IR), and glycated hemoglobin (HbA1c). MVMR analysis indicated that the effect of T1DM on IgAN vanished upon accounting for low-density lipoprotein cholesterol (LDL-c; OR: 0.97, 95% CI: 0.90-1.05, p > 0.05). Conclusions: This MR study provided evidence that T1DM may be a risk factor for the onset of IgAN, which might be driven by LDL-c. Lipid-lowering strategies targeting LDL-c should be enhanced in patients with T1DM to prevent IgAN.

Caspase-4/11-Mediated Pulmonary Artery Endothelial Cell Pyroptosis Contributes to Pulmonary Arterial Hypertension.

BACKGROUND: Endothelial dysfunction enhances vascular inflammation, which initiates pulmonary arterial hypertension (PAH) pathogenesis, further induces vascular remodeling and right ventricular failure. Activation of inflammatory caspases is an important initial event at the onset of pyroptosis. Studies have shown that caspase-1-mediated pyroptosis has played a crucial role in the pathogenesis of PAH. However, the role of caspase-11, another inflammatory caspase, remains to be elucidated. Therefore, the purpose of this study was to clarify the role of caspase-11 in the development of PAH and its mechanism on endothelial cell function. METHODS: The role of caspase-11 in the progression of PAH and vascular remodeling was assessed in vivo. In vitro, the effect of caspase-4 silencing on the human pulmonary arterial endothelial cells pyroptosis was determined. RESULTS: We confirmed that caspase-11 and its human homolog caspase-4 were activated in PAH animal models and TNF (tumor necrosis factor)-α-induced human pulmonary arterial endothelial cells. Caspase-11-/- relieved right ventricular systolic pressure, right ventricle hypertrophy, and vascular remodeling in Sugen-5416 combined with chronic hypoxia mice model. Meanwhile, pharmacological inhibition of caspase-11 with wedelolactone exhibited alleviated development of PAH on the monocrotaline-induced rat model. Moreover, knockdown of caspase-4 repressed the onset of TNF-α-induced pyroptosis in human pulmonary arterial endothelial cells and inhibited the activation of pyroptosis effector GSDMD (gasdermin D) and GSDME (gasdermin E). CONCLUSIONS: These observations identified the critical role of caspase-4/11 in the pyroptosis pathway to modulate pulmonary vascular dysfunction and accelerate the progression of PAH. Our findings provide a potential diagnostic and therapeutic target in PAH.

Contribution of endogenous inhibitor of nitric oxide synthase to hepatic mitochondrial dysfunction in streptozotocin-induced diabetic rats.

AIMS: Mitochondrial dysfunction plays important roles in the development of diabetes. Elevated nitric oxide (NO) synthase inhibitor asymmetric dimethylarginine (ADMA) has been shown to be closely related to diabetes. But the relationship between them in diabetes has not been determined. This study was to explore the role of ADMA in hepatic mitochondrial dysfunction and its potential mechanisms in diabetic rats and hepatocytes. METHODS: Respiratory enzymes activities, mitochondrial transmembrane potential and ATP content were measured to evaluate mitochondrial function. The copy number ratio of mitochondrial gene to nuclear gene was used to represent mitochondrial biogenesis. The activity of superoxide dismutase and malondialdehyde content were detected to reflect oxidative stress. Furthermore, changes in ADMA and NO contents, uncoupling protein 2 (UCP2) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) transcriptions were determined. RESULTS: Elevated ADMA levels in serum of diabetic rats were found to be associated with hepatic mitochondrial dysfunction reflected by reductions of respiratory enzyme activities, mitochondrial membrane potential and ATP contents. Similar mitochondrial dysfunction also occurred in ADMA-treated hepatocytes. The mitochondrial dysfunction observed in diabetic rats or hepatocytes was accompanied with suppressions of mitochondrial biogenesis, PGC-1α transcription and NO synthesis as well as enhances of UCP 2 transcription and oxidative stress. These effects of ADMA could be attenuated by treatments with antioxidant or NO donor. CONCLUSIONS: These results indicate that elevated endogenous ADMA contributes to hepatic mitochondrial dysfunction in diabetic rats, and underlying mechanisms may be related to the suppression of mitochondrial biogenesis and mitochondrial uncoupling via inhibiting NO synthesis and enhancing oxidative stress.

Endothelial cell-specific deficiency of the adenosine deaminase ADAR1 aggravates LPS-induced lung injury in mice via an MDA5-independent pathway.

Adenosine deaminase acting on RNA 1 (ADAR1) has been shown to participate in the regulation of endothelial cells (ECs), as well as local and systemic inflammatory responses. Here, we find that bacterial lipopolysaccharide (LPS)-induced upregulation of ADAR1 in lung ECs is impaired in aged mice, an animal model with high rates of sepsis and mortality. Endothelial cell-specific ADAR1 knockout (ADAR1ECKO ) mice suffer from higher mortality rates, aggravated lung injury, and increased vascular permeability under LPS challenge. In primary ADAR1 knockout ECs, expression of the melanoma differentiation-associated gene 5 (MDA5), a downstream effector of ADAR1, is significantly elevated. MDA5 knockout completely rescues the postnatal offspring death of ADAR1ECKO mice. However, there is no reduction in mortality or apoptosis in lung cells of ADAR1ECKO /MDA5-/- mice challenged with LPS, indicating the involvement of an MDA5-independent mechanism in this process.

Gene Regulatory Effect of Pyruvate Kinase M2 is Involved in Renal Inflammation in Type 2 Diabetic Nephropathy.

BACKGROUND AND AIMS: The inflammation of glomerular endothelial cells induces and promotes the activation of macrophages and contributes to the development of diabetic nephropathy. Thus, this study aimed to investigate the gene regulatory effect and potential role of pyruvate kinase M2 (PKM2) in inflammatory response in diabetic nephropathy. METHODS: The plasma PKM2 levels of patients with diabetes were evaluated. Eight-week-old mice were divided into three groups (WT, db/db mice, and db/db mice treated with TEPP-46) and raised for 12 weeks. Blood and kidney samples were collected at the end of the experiment. Endothelial cells were stimulated with high glucose with or without TEPP-46. The expression of intercellular adhesion molecule 1 (ICAM-1), interleukin 6 (IL-6), interleukin 1 beta (IL-1ß), phospho-PKM2, PKM2, phospho-STAT3(signal transducer and activator of transcription), STAT3, nuclear factor kappa B (NF-kB), and phospho-NF-kB in vivo and in vitro were determined using Western blot. The activation of macrophages (CD68+CD86+) in the glomeruli was assessed via fluorescent double staining. Moreover, immune endothelial adhesion experiments were performed. RESULTS: The plasma PKM2 levels of patients with type 2 diabetes increased. P-PKM2 was up-regulated in vivo and in vitro. TEPP-46 decreased inflammatory cell infiltration and ICAM-1 expression in vivo and in vitro and inhibited the differentiation of macrophages to M1 cells in db/db mice with diabetic nephropathy. PKM2 regulated the phosphorylation of STAT3 and NF-kB. Furthermore, high glucose levels induced the transition from tetramer to dimer and the nuclear translocation of PKM2. CONCLUSION: The gene regulatory effect of PKM2 is involved in renal inflammation in type 2 diabetic nephropathy by promoting the phosphorylation of STAT3 and NF-kB and the expression of intercellular adhesion molecule 1. Thus, the down-regulation of phosphorylated PKM2 may have protective effects against diabetic nephropathy by inhibiting renal inflammation.

l-Homocysteine-induced cathepsin V mediates the vascular endothelial inflammation in hyperhomocysteinaemia.

BACKGROUND AND PURPOSE: Vascular inflammation, including the expression of inflammatory cytokines in endothelial cells, plays a critical role in hyperhomocysteinaemia-associated vascular diseases. Cathepsin V, specifically expressed in humans, is involved in vascular diseases through its elastolytic and collagenolytic activities. The aim of this study was to determine the effects of cathepsin V on l-homocysteine-induced vascular inflammation. EXPERIMENTAL APPROACH: A high methionine diet-induced hyperhomocysteinaemic mouse model was used to assess cathepsin V expression and vascular inflammation. Cultures of HUVECs were challenged with l-homocysteine and the cathepsin L/V inhibitor SID to assess the pro-inflammatory effects of cathepsin V. Transfection and antisense techniques were utilized to investigate the effects of cathepsin V on the dual-specificity protein phosphatases (DUSPs) and MAPK pathways. KEY RESULTS: Cathepsin L (human cathepsin V homologous) was increased in the thoracic aorta endothelial cells of hyperhomocysteinaemic mice; l-homocysteine promoted cathepsin V expression in HUVECs. SID suppressed the activity of cathepsin V and reversed the up-regulation of inflammatory cytokines (IL-6, IL-8 and TNF-α), adhesion and chemotaxis of leukocytes and vascular inflammation induced by l-homocysteine in vivo and in vitro. Increased cathepsin V promoted the degradation of DUSP6 and DUSP7, phosphorylation and subsequent nuclear translocation of ERK1/2, phosphorylation of STAT1 and expression of IL-6, IL-8 and TNF-α. CONCLUSIONS AND IMPLICATIONS: This study has identified a novel mechanism, which shows that l-homocysteine-induced upregulation of cathepsin V mediates vascular endothelial inflammation under high homocysteine condition partly via ERK1/2 /STAT1 pathway. This mechanism could represent a potential therapeutic target in hyperaemia-associated vascular diseases. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Involvement of increased endogenous asymmetric dimethylarginine in the hepatic endoplasmic reticulum stress of type 2 diabetic rats.

OBJECTIVE: Increasing evidence suggested that endoplasmic reticulum (ER) stress contributes to insulin resistance, which plays an important role in the development of type 2 diabetes mellitus (T2DM). Accumulation of endogenous nitric oxide synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA), is associated with insulin resistance, T2DM, and diabetic cardiovascular complications, although the mechanisms have not been elucidated. This study was to determine whether elevated endogenous ADMA is involved in hepatic ER stress of type 2 diabetic rats, verify their causal relationship, and elucidate the potential mechanism underlying ADMA induced ER stress in rat hepatocytes. METHODS: Immunoglobulin binding protein (Bip) transcription, eukaryotic initiation factor 2α kinase (eIF2α) phosphorylation, X box-binding protein-1 (XBP-1) mRNA splicing and C/EBP homologues protein (CHOP) expression were measured to reflect ER stress. Contents of ADMA and nitrite/nitrate as well as activities or expression of NOS and dimethylarginine dimethylaminohydrolase (DDAH) were detected to show the changes in DDAH/ADMA/NOS/NO pathway. The lipid peroxidation product malondialdehyde content and antioxidant enzyme superoxide dismutase activity were analyzed to evaluate oxidative stress. RESULTS: ER stress was provoked in the liver of type 2 diabetic rats, as expressed by increases of Bip transcription, eIF2α phosphorylation, XBP-1 splicing and CHOP expression, all of which were in parallel with the elevation of serum ADMA, suppression of NO generation, NOS and DDAH activities in the liver. Exposure of hepatocytes to ADMA or hydrogen peroxide also induced ER stress, which was associated with the inhibition of NO production and increase of oxidative stress. Treatment of hepatocytes with antioxidant pyrrolidine dithiocarbamate not only decreased ADMA-induced oxidative stress and inhibition of NO production but also reduced ADMA-triggered ER stress. CONCLUSIONS: These results indicate that increased endogenous ADMA contributes to hepatic ER stress in type 2 diabetic rats, and the mechanism underlying ADMA-induced ER stress may relate to oxidative stress via NOS uncoupling.