Angiotensin II induces TIMP-1 production in rat heart endothelial cells

AbstractAngiotensin II (AII) was found to upregulate tissue inhibitor of metalloproteineses-1 (TIMP-1) gene expression in rat heart endothelial cells in a dose and time-dependent manner. The maximal stimulation of TIMP-1 mRNA was achieved by 2 h after the addition of AII. This effect was blocked by losartan, an AT1 receptor antagonist and by calphostin C, a protein kinase C inhibitor. Addition of cycloheximide superinduced and actinomycin D abolished the induction. These results suggest that AII stimulates TIMP-1 production by a protein kinase C dependent pathway which is dependent upon de novo RNA synthesis. Immunoprecipitation experiment showed an enhanced band of 28 kDa from the conditioned medium of AII-treated cultures. Immunoblot analysis revealed that TIMP-1 was detectable in the conditioned medium 4 h after AII stimulation. Since endothelial cells line the blood vessels and sense the rise in AII associated with hypertension, the TIMP-1 released by these cells may provide an initial trigger leading to cardiac fibrosis in angiotensin-renin dependent hypertension

Attenuation of doxorubicin-induced cardiac injury by mitochondrial glutaredoxin 2

AbstractWhile the cardiotoxicity of doxorubicin (DOX) is known to be partly mediated through the generation of reactive oxygen species (ROS), the biochemical mechanisms by which ROS damage cardiomyocytes remain to be determined. This study investigates whether S-glutathionylation of mitochondrial proteins plays a role in DOX-induced myocardial injury using a line of transgenic mice expressing the human mitochondrial glutaredoxin 2 (Glrx2), a thiotransferase catalyzing the reduction as well as formation of protein–glutathione mixed disulfides, in cardiomyocytes. The total glutaredoxin (Glrx) activity was increased by 76% and 53 fold in homogenates of whole heart and isolated heart mitochondria of Glrx2 transgenic mice, respectively, compared to those of nontransgenic mice. The expression of other antioxidant enzymes, with the exception of glutaredoxin 1, was unaltered. Overexpression of Glrx2 completely prevents DOX-induced decreases in NAD- and FAD-linked state 3 respiration and respiratory control ratio (RCR) in heart mitochondria at days 1 and 5 of treatment. The extent of DOX-induced decline in left ventricular function and release of creatine kinase into circulation at day 5 of treatment was also greatly attenuated in Glrx2 transgenic mice. Further studies revealed that heart mitochondria overexpressing Glrx2 released less cytochrome c than did controls in response to treatment with tBid or a peptide encompassing the BH3 domain of Bid. Development of tolerance to DOX toxicity in transgenic mice is also associated with an increase in protein S-glutathionylation in heart mitochondria. Taken together, these results imply that S-glutathionylation of heart mitochondrial proteins plays a role in preventing DOX-induced cardiac injury

Attenuation of Doxorubicin-Induced Cardiac Injury by Mitochondrial Glutaredoxin 2

While the cardiotoxicity of doxorubicin (DOX) is known to be partly mediated through the generation of reactive oxygen species (ROS), the biochemical mechanisms by which ROS damage cardiomyocytes remain to be determined. This study investigates whether S-glutathionylation of mitochondrial proteins plays a role in DOX-induced myocardial injury using a line of transgenic mice expressing the human mitochondrial glutaredoxin 2 (Glrx2), a thiotransferase catalyzing the reduction as well as formation of protein–glutathione mixed disulfides, in cardiomyocytes. The total glutaredoxin (Glrx) activity was increased by 76% and 53 fold in homogenates of whole heart and isolated heart mitochondria of Glrx2 transgenic mice, respectively, compared to those of nontransgenic mice. The expression of other antioxidant enzymes, with the exception of glutaredoxin 1, was unaltered. Overexpression of Glrx2 completely prevents DOX-induced decreases in NAD- and FAD-linked state 3 respiration and respiratory control ratio (RCR) in heart mitochondria at days 1 and 5 of treatment. The extent of DOX-induced decline in left ventricular function and release of creatine kinase into circulation at day 5 of treatment was also greatly attenuated in Glrx2 transgenic mice. Further studies revealed that heart mitochondria overexpressing Glrx2 released less cytochrome c than did controls in response to treatment with tBid or a peptide encompassing the BH3 domain of Bid. Development of tolerance to DOX toxicity in transgenic mice is also associated with an increase in protein S-glutathionylation in heart mitochondria. Taken together, these results imply that S-glutathionylation of heart mitochondrial proteins plays a role in preventing DOX-induced cardiac injury

β-arrestin2/miR-155/GSK3β Regulates Transition of 5\u27-Azacytizine-Induced Sca-1-Positive Cells to Cardiomyocytes

Stem-cell antigen 1-positive (Sca-1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5\u27-azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. b-arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of b-arrestin2 in Sca-1+ CSC differentiation, we used b-arrestin2-knockout mice and overexpression strategies. Real-time PCR revealed that b-arrestin2 promoted 5\u27-azacytizine-induced Sca-1+ CSC differentiation in vitro. Because the microRNA 155 (miR-155) may regulate b-arrestin2 expression, we detected its role and relationship with b-arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR-155. Real-time PCR revealed that miR-155, inhibited by b-arrestin2, impaired 5\u27-azacytizine-induced Sca-1+ CSC differentiation. On luciferase report assay, miR-155 could inhibit the activity of b-arrestin2 and GSK3β, which suggests a loop pathway between miR-155 and b-arrestin2. Furthermore, b-arrestin2-knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in b-arrestin2-Knockout mice, so the activity of GSK3β was regulated by b-arrestin2 not Akt. We transplanted Sca-1+ CSCs from b-arrestin2-knockout mice to mice with myocardial infarction and found similar protective functions as in wild-type mice but impaired arterial elastance. Furthermore, low level of b-arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β-arrestin2/miR-155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease

β-arrestin 2 attenuates cardiac dysfunction in polymicrobial sepsis through gp130 and p38

AbstractSepsis is an exaggerated systemic inflammatory response to persistent bacteria infection with high morbidity and mortality rate clinically. β-arrestin 2 modulates cell survival and cell death in different systems. However, the effect of β-arrestin 2 on sepsis-induced cardiac dysfunction is not yet known. Here, we show that β-arrestin 2 overexpression significantly enhances animal survival following cecal ligation and puncture (CLP)-induced sepsis. Importantly, overexpression of β-arrestin 2 in mice prevents CLP-induced cardiac dysfunction. Also, β-arrestin 2 overexpression dramatically attenuates CLP-induced myocardial gp130 and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CLP. Therefore, β-arrestin 2 prevents CLP-induced cardiac dysfunction through gp130 and p38. These results suggest that modulation of β-arrestin 2 might provide a novel therapeutic approach to prevent cardiac dysfunction in patients with sepsis

Upregulation of Endothelin-1 Production by Lysophosphatidic Acid in Rat Aortic Endothelial Cells

Addition of lysophosphatidic acid (LPA) to rat aorta-derived endothelial cells significantly induced preproendothelin-1 (preproET-1) mRNA expression. PreproET-1 mRNA levels reached a plateau within 1 h after the addition of 0.5 μM LPA and declined after 2 h. The induction was superinduced by cycloheximide and was blocked by actinomycin D. Suramin, an LPA receptor antagonist, abolished the induction of preproET-1 mRNA by LPA. Protein kinase C inhibitors, H7 and bisindolylmaleimide, were able to block the induction. Transient transfection experiment revealed that the elevated preproET-1 mRNA was a result of the activation of ET-1 gene activity. Electrophoretic mobility shift assay revealed that LPA stimulated the binding of AP-1. The secreted level of ET-1 was elevated 2.3-fold after 12 h of stimulation with LPA. Our results suggest that the upregulation of preproET-1 by LPA may serve to augment and prolong the vasoconstriction action of LPA

Upregulation of Vascular Endothelial Growth Factor by H\u3csub\u3e2\u3c/sub\u3eO\u3csub\u3e2\u3c/sub\u3e in Rat Heart Endothelial Cells

Hydrogen peroxide (H2O2) is a reactive oxygen species generated by several metabolic pathways in mammalian cells. Endothelial cells are extremely susceptible to oxidative stress. H2O2 has been reported to increase the permeability in these cells. Using rat heart endothelial cell culture as a model system, we examined the effect of H2O2 on the gene expression of vascular endothelial growth factor (VEGF), a potent mitogen of endothelial cells and a vascular permeability factor. By Northern blot analysis we found that VEGF mRNA responded to H2O2 in a dose-and time- dependent manner. The induction was superinduced by cycloheximide and blocked by actinomycin D. N-Acetylcysteine, a synthetic antioxidant, was able to suppress the induction. H7, a protein kinase C inhibitor, could also block the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors, AP-1 and NF-κB. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after H2O2 stimulation. Our results demonstrate that VEGF gene expression is upregulated by H2O2 in these endothelial cells

Mechanism of Transforming Growth Factor-β1-Induced Expression of Vascular Endothelial Growth Factor in Murine Osteoblastic MC3T3-E1 Cells

Transforming growth factor-β1 (TGF-β1), an abundant growth factor in bone matrix, has been shown to be involved in bone formation and fracture healing. The mechanism of action of the osteogenic effect of TGF-β1 is not clearly understood. In this study, we found that the addition of TGF-β1 to murine osteoblastic MC3T3-E1 cells induced vascular endothelial growth factor (VEGF) mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of TGF-β1. The induction was superinduced by cycloheximide and blocked by actinomycin D. Ro 31-8220, a protein kinase C inhibitor, abrogated the induction. In addition, curcumin, an inhibitor for transcription factor AP-1, also blocked the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-κB. Transient transfection experiment showed that VEGF promoter activity increased 3.6-fold upon TGF-β1 stimulation. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after TGF-β1 stimulation. Our results therefore suggest that at least part of the osteogenic activity of TGF-β1 may be attributed to the production of VEGF

Upregulation of Vascular Endothelial Growth Factor by Angiotensin II in Rat Heart Endothelial Cells

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and a vascular permeability factor. In this study we found that the addition of angiotensin II (AII) to rat heart endothelial cells induced VEGF mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 4 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT1 receptor antagonist, abolished the induction of VEGF mRNA by AII, whereas PD 123319, an AT2 receptor antagonist, had no effect on VEGF mRNA induction. H7, a protein kinase C inhibitor, blocked the induction. RT-PCR experiments showed two mRNA species (VEGF 120 and VEGF 164) in these cells and both species were stimulated by AII. Transient transfection experiment showed that VEGF promoter activity was increased 2.2-fold upon AII stimulation. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-KB. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 8 h after AII stimulation. Our results demonstrate for the first time that the upregulation of VEGF by AII may play a significant role in AII-induced hyperpermeability

Regulation of Endothelin-1 Production by a Thromboxane a\u3csub\u3e2\u3c/sub\u3e Mimetic in Rat Heart Smooth Muscle Cells

Thromboxane A2 (TXA2) and ET-1 have been known to play important roles in modulating vascular contraction and growth. The present study was undertaken to examine the effect of TXA2 on the induction of endothelin-1 (ET-1) mRNA and protein levels in smooth muscle cells derived from rat heart. U-46619, a stable TXA2 mimetic, superinduced preproET-1 mRNA in the presence of cycloheximide in these cells. This effect could be blocked by SQ-29548, a TXA2/prostaglandin H2 receptor antagonist and by actinomycin D, an RNA synthesis inhibitor. In addition, H7, a protein kinase C inhibitor, could abolish the induction. Transient transfection experiment revealed that the elevated ET-1 mRNA level after U-46619 treatment was a result of the activation of ET-1 gene activity. The elevated ET-1 message level was accompanied by increased ET-1 release into the cultured medium. These results show that the short-lived TXA2 can induce potent and long-lived ET-1. These findings support a potential role for ET-1 in the pathogenesis of coronary atherosclerosis and hypertension evoked by TXA2