Ling Tu

Epoxyeicosatrienoic acids protect rat hearts against tumor necrosis factor-α-induced injury

Epoxyeicosatrienoic acids (EET), the primary arachidonic acid metabolites of cytochrome P450 2J (CYP2J) epoxygenases, possess potent vasodilatory, anti-inflammatory, antiapoptotic, and mitogenic effects. To date, little is known about the role of CYP2J2 and EETs in tumor necrosis factor (TNF)-α–induced cardiac injury. We utilized cell culture and in vivo models to examine the effects of exogenously applied EETs or CYP2J2 overexpression on TNF-α–induced cardiac apoptosis and cardiac dysfunction. In neonatal rat cardiomyocytes, TNF-α–induced apoptosis was markedly attenuated by EETs or CYP2J2 overexpression, leading to significantly improved cell survival. Further studies showed that TNF-α decreased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL, decreased IκBα and PPARγ, and also inhibited PI3K-dependent Akt and EGFR signaling. Both EETs and CYP2J2 overexpression reversed the effects of TNF-α on these pathways. Furthermore, overexpression of CYP2J2 in rats prevented the decline in cardiac function that is normally observed in TNF-α-challenged animals. These results demonstrate that EETs or CYP2J2 overexpression can prevent TNF-α–induced cardiac cell injury and cardiac dysfunction by inhibiting apoptosis, reducing inflammation, and enhancing PPARγ expression. Targeting the CYP2J2 epoxygenase pathway may represent a novel approach to mitigate cardiac injury in diseases such as heart failure, where increased TNF-α levels are known to occur.

CYP2J2 overexpression increases EETs and protects against angiotensin II-induced abdominal aortic aneurysm in mice

Cytochrome P450 epoxygenase 2J2 (CYP2J2) metabolizes arachidonic acids to form epoxyeicosatrienoic acids (EETs), which possess various beneficial effects on the cardiovascular system. However, whether increasing EETs production by CYP2J2 overexpression in vivo could prevent abdominal aortic aneurysm (AAA) remains unknown. Here we investigated the effects of recombinant adeno-associated virus (rAAV)-mediated CYP2J2 overexpression on angiotensin (Ang) II-induced AAA in apoE-deficient mice. rAAV-CYP2J2 delivery led to an abundant aortic CYP2J2 expression and increased EETs generation. It was shown that CYP2J2 overexpression attenuated matrix metalloproteinase expression and activity, elastin degradation, and AAA formation, which was associated with reduced aortic inflammation and macrophage infiltration. In cultured vascular smooth muscle cells (VSMCs), rAAV-mediated CYP2J2 overexpression and EETs markedly suppressed Ang II-induced inflammatory cytokine expression. Moreover, overexpressed CYP2J2 and EETs inhibited Ang II-induced macrophage migration in a VSMC-macrophage coculture system. We further indicated that these protective effects were mediated by peroxisome proliferator-activated receptor (PPAR)γ activation. Taken together, these results provide evidence that rAAV-mediated CYP2J2 overexpression prevents AAA development which is likely via PPARγ activation and anti-inflammatory action, suggesting that increasing EETs levels could be considered as a potential strategy to prevent and treat AAA.

EETs and CYP2J2 inhibit TNF-α-induced apoptosis in pulmonary artery endothelial cells and TGF-β1-induced migration in pulmonary artery smooth muscle cells

Cytochrome P450 epoxygenase-derived epoxyeicosatrienoic acids (EETs) have multiple biological functions in cardiovascular homeostasis. The antiinflammatory, anti-migratory and pro-proliferative effects of EETs suggest a possible beneficial role for EETs in the apoptosis, proliferation and migration of pulmonary vascular cells. In this study, we investigated the effects of exogenous EETs and cytochrome P450 2J2 (CYP2J2) overexpression on tumor necrosis factor-α (TNF-α)-induced pulmonary artery endothelial cell (PAEC) apoptosis, and transforming growth factor-β1 (TGF-β1)-induced pulmonary artery smooth muscle cell (PASMC) proliferation and migration. PAECs and PASMCs were cultured from porcine pulmonary arteries. Our findings indicated that EETs or CYP2J2 overexpression significantly protected the PAECs from TNF-α-induced apoptosis, as evaluated by cell viability and flow cytometry. Two mechanisms were found to be involved in these important protective effects: firstly, EETs and CYP2J2 overexpression inhibited the decrease in the expression of the antiapoptotic proteins, Bcl-2 and Bcl-xL, as well as the increase in the expression of the pro-apoptotic protein, Bax, mediated by TNF-α; secondly, they activated the phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) signaling pathways. We also found that 11,12-EET and 14,15-EET significantly inhibited TGF-β1-stimulated PASMC migration. However, EETs did not suppress TGF-β1-induced PASMC proliferation in vitro. These data may represent a novel approach to mitigate pulmonary vascular remodeling in diseases, such as pulmonary arterial hypertension.

CYP2J3 Gene Delivery Up-Regulated Adiponectin Expression via Reduced Endoplasmic Reticulum Stress in Adipocytes

Ample evidences demonstrate that cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play crucial and diverse roles in cardiovascular homeostasis. We and others have identified that EETs exert a beneficial role on insulin resistance and diabetes. This study investigated the effects of CYP2J3 epoxygenase gene delivery on adiponectin expression in rats treated with high-fat (HF) diet. CYP2J3 gene delivery in vivo increased EET generation, enhanced adiponectin expression and secretion and accompanied by activation of adiponectin downstream signaling, and decreased insulin resistance as determined by plasma insulin levels, insulin resistance index and glucose tolerance test, as well as phosphorylation of protein kinase B in both liver and muscle. Furthermore, CYP2J3 overexpression prevented HF diet-induced endoplasmic reticulum (ER) stress in adipose tissue of rats. Also, CYP2J3 gene transfection and exogenous administration of EETs inhibited thapsigargin-induced ER stress with increased adiponectin expression and secretion in differentiated 3T3-L1 adipocytes. Thus, CYP2J3 gene delivery up-regulated adiponectin expression and excretion in adipose tissue of rats treated with HF diet through inhibition of ER stress, which can decrease adiponectin expression. These results further highlight the beneficial roles of the CYP epoxygenase 2J3 and its metabolites EETs on adiponectin expression and secretion.

Bradykinin Inhibits Oxidative Stress-Induced Cardiomyocytes Senescence via Regulating Redox State

Background Cell senescence is central to a large body of age related pathology, and accordingly, cardiomyocytes senescence is involved in many age related cardiovascular diseases. In consideration of that, delaying cardiomyocytes senescence is of great importance to control clinical cardiovascular diseases. Previous study indicated that bradykinin (BK) protected endothelial cells from senescence induced by oxidative stress. However, the effects of bradykinin on cardiomyocytes senescence remain to be elucidated. In this study, we investigated the effect of bradykinin on H2O2-induced H9C2 cells senescence. Methods and Results Bradykinin pretreatment decreased the senescence induced by H2O2 in cultured H9C2 cells in a dose dependent manner. Interestingly, 1 nmol/L of BK almost completely inhibited the increase in senescent cell number and p21 expression induced by H2O2. Since H2O2 induces senescence through superoxide-induced DNA damage, we also observed the DNA damage by comet assay, and BK markedly reduced DNA damage induced by H2O2, and moreover, BK treatment significantly prevented reactive oxygen species (ROS) production in H9C2 cells treated with H2O2. Importantly, when co-incubated with bradykinin B2 receptor antagonist HOE-140 or eNOS inhibitor N-methyl-L-arginine acetate salt (L-NAME), the protective effects of bradykinin on H9C2 senescence were totally blocked. Furthermore, BK administration significantly prevented the increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity characterized by increased ROS generation and gp91 expression and increased translocation of p47 and p67 to the membrane and the decrease in superoxide dismutase (SOD) activity and expression induced by H2O2 in H9C2 cells, which was dependent on BK B2 receptor mediated nitric oxide (NO) release. Conclusions Bradykinin, acting through BK B2 receptor induced NO release, upregulated antioxidant Cu/Zn-SOD and Mn-SOD activity and expression while downregulating NADPH oxidase activity and subsequently inhibited ROS production, and finally protected against cardiomyocytes senescence induced by oxidative stress.

Effect of simvastatin on IL-6 and adiponectin secretion and mRNA expression in 3T3-L1 adipocytes

In order to investigate the effects of simvastatin on secretion and mRNA expression of interleukin-6 (IL-6) and adiponectin in 3T3-L1 adipocytes, mouse 3T3-L1 adipocytes were stimulated with lipopolysaccharide (LPS). Production and mRNA expression of IL-6 and adiponectin in 3T3-L1 adipocytes were measured using enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. The results showed that simvastatin could significantly suppress LPS-induced IL-6 production and mRNA expression in adipocytes (P<0.05), but increase the LPS-induced adiponectin secretion and mRNA expression in a dose-dependent manner (P<0.05). It was suggested that simvastatin could exert beneficial effects on prevention of obesity-induced metabolic changes in adipocytes.

Exogenous Bradykinin Inhibits Tissue Factor Induction and Deep Vein Thrombosis via Activating the eNOS/Phosphoinositide 3-Kinase/Akt Signaling Pathway.

Background/Aims: Bradykinin has been shown to exert a variety of protective effects against vascular injury, and to reduce the levels of several factors involved in the coagulation cascade. A key determinant of thrombin generation is tissue factor (TF). However, whether bradykinin can regulate TF expression remains to be investigated. Methods: To study the effect of bradykinin on TF expression, we used Lipopolysaccharides (LPS) to induce TF expression in human umbilical vein endothelial cells and monocytes. Transcript levels were determined by RT-PCR, protein abundance by Western blotting. In the in vivo study, bradykinin and equal saline were intraperitoneally injected into mice for three days ahead of inferior cava vein ligation that we took to induce thrombus formation, after which bradykinin and saline were injected for another two days. Eventually, the mice were sacrificed and tissues were harvested for tests. Results: Exogenous bradykinin markedly inhibited TF expression in mRNA and protein level induced by LPS in a dose-dependent manner. Moreover, the NO synthase antagonist L-NAME and PI3K inhibitor LY294002 dramatically abolished the inhibitory effects of bradykinin on tissue factor expression. PI3K/Akt signaling pathway activation induced by bradykinin administration reduced the activity of GSK-3ß and MAPK, and reduced NF-κB level in the nucleus, thereby inhibiting TF expression. Consistent with this, intraperitoneal injection of C57/BL6 mice with bradykinin also inhibited the thrombus formation induced by ligation of inferior vena cava. Conclusion: Bradykinin suppressed TF protein expression in human umbilical vein endothelial cells and monocytes in vitro; in line with this, it inhibits thrombus formation induced by ligation of inferior vena cava in vivo.

Effects and mechanism of irbesartan on tubulointerstitial fibrosis in 5/6 nephrectomized rats

SummaryTubulointerstitial fibrosis (TIF) is a common pathological feature of end-stage kidney disease. Previous studies showed that upregulation of TGFβ1 notably contributed to the chronic renal injury and irbesartan halted the development of TIF in rats with 5/6 renal mass reduction. This study was to investigate the effects of irbesartan on chronic TIF and the mechanism involved TGFβ1 in the rodent model of chronic renal failure involving 5/6 nephrectomy. The results showed that irbesartan significantly attenuated the rise in blood pressure and tubulointerstitial injury observed in this model. Masson staining of the renal tissue revealed that there appeared severe renal tubule atrophy and fibrosis in operation group, but the lesion was attenuated mostly in irbesartan-treated group. Immunohistochemistry showed that irbesartan treatment apparently decreased the protein expression of TGFβ1 which was up-regulated in operation groups. Western blot showed that irbesartan treatment down-regulated the expression of TGFβ1, phosphorylated smad2 (p-smad2), AT1R and phosphorylated p38 (p-p38) MAPK, but significantly up-regulated the protein expression of smad6 as compared with operation group. These findings suggest that irbesartan attenuates hypertension and reduces the development of TIF in rats with 5/6 renal mass reduction via changes in the expression of these proteins at least including smad6, TGF-β1, p-smad2, AT1 and p-p38 MAPK.Tubulointerstitial fibrosis (TIF) is a common pathological feature of end-stage kidney disease. Previous studies showed that upregulation of TGFβ1 notably contributed to the chronic renal injury and irbesartan halted the development of TIF in rats with 5/6 renal mass reduction. This study was to investigate the effects of irbesartan on chronic TIF and the mechanism involved TGFβ1 in the rodent model of chronic renal failure involving 5/6 nephrectomy. The results showed that irbesartan significantly attenuated the rise in blood pressure and tubulointerstitial injury observed in this model. Masson staining of the renal tissue revealed that there appeared severe renal tubule atrophy and fibrosis in operation group, but the lesion was attenuated mostly in irbesartan-treated group. Immunohistochemistry showed that irbesartan treatment apparently decreased the protein expression of TGFβ1 which was up-regulated in operation groups. Western blot showed that irbesartan treatment down-regulated the expression of TGFβ1, phosphorylated smad2 (p-smad2), AT1R and phosphorylated p38 (p-p38) MAPK, but significantly up-regulated the protein expression of smad6 as compared with operation group. These findings suggest that irbesartan attenuates hypertension and reduces the development of TIF in rats with 5/6 renal mass reduction via changes in the expression of these proteins at least including smad6, TGF-β1, p-smad2, AT1 and p-p38 MAPK.

Increased Age-Related Cardiac Dysfunction in Bradykinin B2 Receptor–Deficient Mice

Experimental evidence indicates that the kinin peptide binds to bradykinin B2 receptor (B2R) to trigger various beneficial effects on the cardiovascular system. However, the effects and underlying mechanisms of B2R in cardiac aging remain unknown. A significant age-dependent decrease in B2R expression in the myocardium was observed in C57BL/6J mice. Echocardiographic measurements showed that aging caused a significant cardiac dysfunction in C57BL/6J mice, and importantly B2R deficiency augmented this dysfunction in aging mice. The deficiency of B2R expression in the aging heart repressed p53-pGC-1α-induced mitochondria renewal, increased reactive oxygen species production, and destroyed mitochondrial ultrastructure. Age-related decrease or lack of B2R increased oxidative stress, macrophage infiltration, and inflammatory cytokine expression and compromised antioxidant enzyme expression. Moreover, the inflammatory signals were mainly mediated by the activation of p38 MAPK, JNK, and subsequent translocation of nuclear factor-kappa B to the nucleus. In summary, our data provide evidence that B2R deficiency contributes to the aging-induced cardiac dysfunction, which is likely mediated by increased mitochondrial dysfunction, oxidative stress, and inflammation. This study indicates that preventing the loss of cardioprotective B2R expression may be a novel approach for the prevention and treatment of age-related cardiac dysfunction.

Telmisartan protects against insulin resistance by attenuating inflammatory response in rats

SummaryThis study investigated the effects of telmisartan on insulin resistance in high-fat diet-treated rats and the possible mechanism. A total of 40 male Sprague-Dawley rats enrolled in the study were divided into 4 groups at random: ND group (n=10) and HD group (n=10), in which the rats were given a normal chow diet or a high-fat diet for 20 weeks following a one-week adaptation; ND+telmisartan (n=10) group and HD+telmisartan group (n=10), in which the rats were initially administered in the same way as the ND or HD group, and then they were orally gavaged with telmisartan (5 mg/kg daily) additionally for 5 weeks. Related inflammatory factors were measured by ELISA. Monocyte chemotactic protein 1 (MCP-1), phosphorylated JNK and IκB-α expressions in both adipose and liver were detected by Western blotting. CRP and angiotensin II receptor 1 (AT1) mRNA expressions in both adipose and liver were determined by RT-PCR. The results showed that telmisartan administration in vivo reversed insulin resistance as evidenced by a decrease in plasma fasting glucose levels, plasma fasting insulin levels and homeostasis model of assessment-insulin resistance (HOMA-IR). Furthermore, telmisartan administration significantly reduced serum CRP, TNF-α and IL-1β levels, and elevated serum IL-10 levels. It was also found to hamper the high-fat diet-induced increase in CRP mRNA, AT1 mRNA and MCP-1, and decrease in IκB-α in both adipose and liver. It was concluded that telmisartan administration in vivo may improve insulin resistance through attenuated inflammatory response pathways.This study investigated the effects of telmisartan on insulin resistance in high-fat diet-treated rats and the possible mechanism. A total of 40 male Sprague-Dawley rats enrolled in the study were divided into 4 groups at random: ND group (n=10) and HD group (n=10), in which the rats were given a normal chow diet or a high-fat diet for 20 weeks following a one-week adaptation; ND+telmisartan (n=10) group and HD+telmisartan group (n=10), in which the rats were initially administered in the same way as the ND or HD group, and then they were orally gavaged with telmisartan (5 mg/kg daily) additionally for 5 weeks. Related inflammatory factors were measured by ELISA. Monocyte chemotactic protein 1 (MCP-1), phosphorylated JNK and IκB-α expressions in both adipose and liver were detected by Western blotting. CRP and angiotensin II receptor 1 (AT1) mRNA expressions in both adipose and liver were determined by RT-PCR. The results showed that telmisartan administration in vivo reversed insulin resistance as evidenced by a decrease in plasma fasting glucose levels, plasma fasting insulin levels and homeostasis model of assessment-insulin resistance (HOMA-IR). Furthermore, telmisartan administration significantly reduced serum CRP, TNF-α and IL-1β levels, and elevated serum IL-10 levels. It was also found to hamper the high-fat diet-induced increase in CRP mRNA, AT1 mRNA and MCP-1, and decrease in IκB-α in both adipose and liver. It was concluded that telmisartan administration in vivo may improve insulin resistance through attenuated inflammatory response pathways.