[Anticancer effect of SN-38 combined with sorafenib on hepatocellular carcinoma in vitro and its mechanism].

OBJECTIVE: To investigate the anticancer effect and its mechanism of SN-38 combined with sorafenib on hepatocellular cancer cell lines HepG-2 and BEL-7402. METHODS: SRB colorimetry was employed to measure the viability of HepG-2 and BEL-7402 cells after the treatment of SN-38 with sorafenib. Propidium iodide flow cytometric assay and DAPI staining were used to evaluate the apoptosis of HCC cells. Western blotting was conducted to detect the expression level of apoptosis-related and DNA damage-related proteins. RESULTS: SRB colorimetry showed the synergistic anticancer activities of SN-38 combined with sorafenib, with a combination index of <0.9. The apoptotic rates of HepG-2 cells in control, 60 nmol/L SN-38, 2.5µmol/L sorafenib and combination groups were 4.25%±2.45%, 28.95%±10.75%, 3.49%±2.49% and 53.19%±11.21%, respectively(P<0.05). Western blotting showed that the combination of these two drugs increased the enzymolysis of PARP, Caspase-8 and Caspase-3, and promoted the expression levels of p53, p21 and γ-H2AX significantly. CONCLUSION: SN-38 and sorafenib have synergistic anticancer activity on hepatocellular carcinoma cells in vitro with the augmentation of apoptosis.

Cardiac mTOR protects the heart against ischemia-reperfusion injury.

Cardiac mammalian target of rapamycin (mTOR) is necessary and sufficient to prevent cardiac dysfunction in pathological hypertrophy. However, the role of cardiac mTOR in heart failure after ischemic injury remains undefined. To address this question, we used transgenic (Tg) mice with cardiac-specific overexpression of mTOR (mTOR-Tg mice) to study ischemia-reperfusion (I/R) injury in two animal models: 1) in vivo I/R injury with transient coronary artery ligation and 2) ex vivo I/R injury in Langendorff-perfused hearts with transient global ischemia. At 28 days after I/R, mortality was lower in mTOR-Tg mice than littermate control mice [wild-type (WT) mice]. Echocardiography and MRI demonstrated that global cardiac function in mTOR-Tg mice was preserved, whereas WT mice exhibited significant cardiac dysfunction. Masson's trichrome staining showed that 28 days after I/R, the area of interstitial fibrosis was smaller in mTOR-Tg mice compared with WT mice, suggesting that adverse left ventricular remodeling is inhibited in mTOR-Tg mice. In the ex vivo I/R model, mTOR-Tg hearts demonstrated improved functional recovery compared with WT hearts. Perfusion with Evans blue after ex vivo I/R yielded less staining in mTOR-Tg hearts than WT hearts, indicating that mTOR overexpression inhibited necrosis during I/R injury. Expression of proinflammatory cytokines, including IL-6 and TNF-α, in mTOR-Tg hearts was lower than in WT hearts. Consistent with this, IL-6 in the effluent post-I/R injury was lower in mTOR-Tg hearts than in WT hearts. These findings suggest that cardiac mTOR overexpression in the heart is sufficient to provide substantial cardioprotection against I/R injury and suppress the inflammatory response.

mTOR attenuates the inflammatory response in cardiomyocytes and prevents cardiac dysfunction in pathological hypertrophy.

Previous studies have suggested that inhibition of the mammalian target of rapamycin (mTOR) by rapamycin suppresses myocardial hypertrophy. However, the role of mTOR in the progression of cardiac dysfunction in pathological hypertrophy has not been fully defined. Interestingly, recent reports indicate that the inflammatory response, which plays an important role in the development of heart failure, is enhanced by rapamycin under certain conditions. Our aim in this study was to determine the influence of mTOR on pathological hypertrophy and to assess whether cardiac mTOR regulates the inflammatory response. We generated transgenic mice with cardiac-specific overexpression of wild-type mTOR (mTOR-Tg). mTOR-Tg mice were protected against cardiac dysfunction following left ventricular pressure overload induced by transverse aortic constriction (TAC) (P < 0.01) and had significantly less interstitial fibrosis compared with littermate controls (WT) at 4 wk post-TAC (P < 0.01). In contrast, TAC caused cardiac dysfunction in WT. At 1 wk post-TAC, the proinflammatory cytokines interleukin (IL)-1ß and IL-6 were significantly increased in WT mice but not in mTOR-Tg mice. To further characterize the effects of mTOR activation, we exposed HL-1 cardiomyocytes transfected with mTOR to lipopolysaccharide (LPS). mTOR overexpression suppressed LPS-induced secretion of IL-6 (P < 0.001), and the mTOR inhibitors rapamycin and PP242 abolished this inhibitory effect of mTOR. In addition, mTOR overexpression reduced NF-κB-regulated transcription in HL-1 cells. These data suggest that mTOR mitigates adverse outcomes of pressure overload and that this cardioprotective effect of mTOR is mediated by regulation of the inflammatory reaction.

MicroRNAs Associated With Reverse Left Ventricular Remodeling in Humans Identify Pathways of Heart Failure Progression.

BACKGROUND: Plasma extracellular RNAs have recently garnered interest as biomarkers in heart failure (HF). Most studies in HF focus on single extracellular RNAs related to phenotypes and outcomes, and few describe their functional roles. We hypothesized that clusters of plasma microRNAs (miRNAs) associated with left ventricular (LV) remodeling in human HF would identify novel subsets of genes involved in HF in animal models. METHODS AND RESULTS: We prospectively measured circulating miRNAs in 64 patients with systolic HF (mean age, 64.8 years; 91% men; median LV ejection fraction, 26%) with serial echocardiography (10 months apart) during medical therapy. We defined LV reverse remodeling as a 15% reduction in LV end-systolic volume index. Using principal components analysis, we identified a component associated with LV reverse remodeling (odds ratio=3.99; P=0.01) that provided risk discrimination for LV reverse remodeling superior to a clinical model (C statistic, 0.58 for a clinical model versus 0.71 for RNA-based model). Using network bioinformatics, we uncovered genes not previously widely described in HF regulated simultaneously by >2 miRNAs. We observed increased myocardial expression of these miRNAs during HF development in animals, with downregulation of target gene expression, suggesting coordinate miRNA-mRNA regulation. Target mRNAs were involved in autophagy, metabolism, and inflammation. CONCLUSIONS: Plasma miRNAs associated with LV reverse remodeling in humans are dysregulated in animal HF and target clusters of genes involved in mechanisms implicated in HF. A translational approach integrating human HF, bioinformatics, and model systems may uncover novel pathways involved in HF. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT00351390.

Augmented cardiac hypertrophy in response to pressure overload in mice lacking the prostaglandin I2 receptor.

BACKGROUND: In the heart, the expressions of several types of prostanoid receptors have been reported. However, their roles in cardiac hypertrophy in vivo remain unknown. We intended to clarify the roles of these receptors in pressure overload-induced cardiac hypertrophy using mice lacking each of their receptors. METHODS AND RESULTS: We used a model of pressure overload-induced cardiac hypertrophy produced by banding of the transverse aorta in female mice. In wild-type mice subjected to the banding, cardiac hypertrophy developed during the observation period of 8 weeks. In mice lacking the prostaglandin (PG) I2 receptor (IP(-/-)), however, cardiac hypertrophy and cardiomyocyte hypertrophy were significantly greater than in wild-type mice at 2 and 4 weeks but not at 8 weeks, whereas there was no such augmentation in mice lacking the prostanoid receptors other than IP. In addition, cardiac fibrosis observed in wild-type hearts was augmented in IP(-/-) hearts, which persisted for up to 8 weeks. In IP(-/-) hearts, the expression level of mRNA for atrial natriuretic peptide, a representative marker of cardiac hypertrophy, was significantly higher than in wild-type hearts. In vitro, cicaprost, an IP agonist, reduced platelet-derived growth factor-induced proliferation of wild-type noncardiomyocytes, although it could not inhibit cardiotrophin-1-induced hypertrophy of cardiomyocytes. Accordingly, cicaprost increased cAMP concentration efficiently in noncardiomyocytes. CONCLUSIONS: IP plays a suppressive role in the development of pressure overload-induced cardiac hypertrophy via the inhibition of both cardiomyocyte hypertrophy and cardiac fibrosis. Both effects have been suggested as originating from the action on noncardiomyocytes rather than cardiomyocytes.

A 6 gene signature identifies the risk of developing cirrhosis in patients with chronic hepatitis B.

Clinical factors and liver biopsy cannot accurately predict the risk of developing cirrhosis in chronic hepatitis B (CHB).This study was to develop a predictive gene signature for cirrhosis in CHB patients. A total of 183 untreated CHB patients were enrolled. GeneChip, significant analysis of microarray (SAM) and prediction analysis of microarray (PAM) were used to select predictor genes (PGs) in liver tissues. The Cirrhosis Risk Score (CRS) was calculated based on 6 PG variables and the predictive value of CRS was evaluated. Firstly differentially expressed genes were filtered from a genome scan and SAM, and 87 significant genes were selected for the signature building. Secondly a signature consisting of 6 PGs (CD24, CXCL6, EHF, ITGBL1, LUM and SOX9) most predictive for cirrhosis risk in CHB patients was developed in the selection set (n=40) by use of PAM and PCR approach. Finally the CRS was calculated to estimate the risk of developing cirrhosis and then tested in validation cohort (n=143). The area under the ROC curves (AUROC) of the CRS was 0.944 and exceeded to 6 PGs and clinical factors. A low CRS cutoff of 6.43 to identify low-risk patients would misclassify only 8.16% of high-risk patients, while a high cutoff of 8.32 to identify high-risk patients would misclassify 0% of low-risk patients. So CRS is a better predictor than clinical factors in differentiating high-risk versus low-risk for cirrhosis and application of CRS in clinical practice could help to reduce the rate of liver biopsy in patients with CHB.

Prostaglandin E2 protects the heart from ischemia-reperfusion injury via its receptor subtype EP4.

BACKGROUND: In the heart with acute myocardial infarction, production of prostaglandin (PG) E2 increases significantly. In addition, several subtypes of PGE2 receptors (EPs) have been reported to be expressed in the heart. The role of PGE2 in cardiac ischemia-reperfusion (I/R) injury, however, remains unknown. We intended to clarify the role of PGE2 via EP4, an EP subtype, in I/R injury using mice lacking EP4 (EP4-/- mice). METHODS AND RESULTS: In murine cardiac ventricle, competitive reverse transcription-polymerase chain reaction revealed the highest expression level of EP4 mRNA among EP mRNAs. EP4-/- mice had larger infarct size than wild-type mice in a model of I/R; the left anterior descending coronary artery was occluded for 1 hour, followed by 24 hours of reperfusion. In addition, isolated EP4-/- hearts perfused according to the Langendorff technique had greater functional and biochemical derangements in response to I/R than wild-type hearts. In vitro, AE1-329, an EP4 agonist, raised cAMP concentration remarkably in noncardiomyocytes, whereas the action was weak in cardiomyocytes. When 4819-CD, another EP4 agonist, was administered 1 hour before coronary occlusion, it reduced infarct size significantly in wild-type mice. Notably, a similar cardioprotective effect was observed even when it was administered 50 minutes after coronary occlusion. CONCLUSIONS: Both endogenous PGE2 and an exogenous EP4 agonist protect the heart from I/R injury via EP4. The potent cardioprotective effects of 4819-CD suggest that the compound would be useful for treatment of acute myocardial infarction.

Thromboxane A2 regulates vascular tone via its inhibitory effect on the expression of inducible nitric oxide synthase.

BACKGROUND: Circulatory failure in sepsis arises from vascular hyporesponsiveness, in which nitric oxide (NO) derived from inducible NO synthase (iNOS) plays a major role. Details of the cross talk between thromboxane (TX) A2 and the iNOS-NO system, however, remain unknown. We intended to clarify the role of TXA2, via the cross talk, in vascular hyporesponsiveness. METHODS AND RESULTS: We examined cytokine-induced iNOS expression and NO production in cultured vascular smooth muscle cells (VSMCs) and cytokine-induced hyporesponsiveness of the aorta from mice lacking the TXA2 receptor (TP-/- mice). The cytokine-induced iNOS expression and NO production observed in wild-type VSMCs were significantly augmented in TP-/- VSMCs, indicating an inhibitory effect of endogenous TXA2 on iNOS expression. Furthermore, in indomethacin-treated wild-type VSMCs, U-46619, a TP agonist, inhibited cytokine-induced iNOS expression and NO production in a concentration-dependent manner, effects absent from TP-/- VSMCs. In an ex vivo system, the cytokine-induced hyporesponsiveness of aortas to phenylephrine was significantly augmented in TP-/- aorta but was almost completely canceled by aminoguanidine, an iNOS inhibitor. Accordingly, cytokine-induced NO production was significantly higher in TP-/- aorta than in wild-type aorta. Moreover, U-46619 significantly suppressed lipopolysaccharide-induced NO production in vivo only in wild-type mice. CONCLUSIONS: These results suggest that TXA2 has a protective role against the development of vascular hyporesponsiveness via its inhibitory action on the iNOS-NO system under pathological conditions such as sepsis.

Mitochondrial-to-nuclear translocation of apoptosis-inducing factor in cardiac myocytes during oxidant stress: potential role of poly(ADP-ribose) polymerase-1.

OBJECTIVE: Oxidant stress-induced activation of poly(ADP-ribose) polymerase (PARP) plays a role in the pathogenesis of various cardiovascular diseases. We have now investigated the role of PARP in the death of cardiac myocytes in response to oxidant stress induced by hydrogen peroxide, with focus on the mitochondrial function. METHODS AND RESULTS: Using wild-type and PARP-1-deficient murine myocytes challenged with hydrogen peroxide, we found that mitochondrial respiration and mitochondrial membrane potential were better preserved in PARP-deficient myocytes and cellular NAD+ levels were maintained. The release of the mitochondrial cell death factor cytochrome c, and the mitochondrial-to-nuclear translocation of apoptosis-inducing factor (AIF) were also attenuated in the PARP-deficient myocytes. CONCLUSION: PARP-1, directly or indirectly, regulates the translocation of AIF in myocytes subjected to oxidative stress. The current results are consistent with the view that PARP-1 activation, via induction of mitochondrial dysfunction and promotion of mitochondrial cell death pathways, plays a deleterious pathophysiological role under conditions of oxidative stress.

FOXP1 and SPINK1 reflect the risk of cirrhosis progression to HCC with HBV infection.

BACKGROUND: Hepatocellular carcinoma (HCC) deriving from cirrhosis with HBV infection harbors higher morbidity and poor prognosis. The diagnosis of HCC at its early stage is essential for improving the effect of treatment and survival rate of patients. METHOD: Affymetrix GeneChip was practiced to establish gene expression profile and significance analysis of microarray (SAM) as well as prediction analysis of microarray (PAM) was utilized to screen candidate marker genes in tissue of carcinoma and para-cancerous with cirrhosis from 15 hepatitis B virus (HBV) related HCC patients. RESULT: Total 497 differential genes were selected by microarray (fold change >2; P value<0.01). Then 162 significant genes were determined by SAM (fold change -1.46 to 1.28). A number of 8-genes showing "poor risk signature" was validated with threshold of 6.2, which was associated with cirrhosis progressing to HCC. Only 3 down-regulated and 2 up-regulated predictor genes had statistical difference in HCC and cirrhosis groups by RT-PCR (P value<0.01). Forkhead box protein 1 (FOXP1) and serine protease inhibitor Kazal-type 1 (SPINK1) proteins were found significantly increased in carcinoma tissues than para-cancerous cirrhotic tissues by IH and WB. CONCLUSION: Over-expression of FOXP1 and SPINK1 may participate in the carcinogenesis of HBV related cirrhosis. They could use as potential biomarkers for diagnosing early HCC.

Effects of the prostanoids on the proliferation or hypertrophy of cultured murine aortic smooth muscle cells.

Effects of the prostanoids on the growth of cultured aortic vascular smooth muscle cells (VSMCs) were examined using mice lacking prostanoid receptors. Proliferation of VSMCs was assessed by measuring [(3)H]-thymidine incorporation and the cell number, and their hypertrophy by [(14)C]-leucine incorporation and protein content. In VSMCs from wild-type mice, expressions of mRNAs for the EP(4) and TP were most abundant, followed by those for the IP, EP(3) and FP, when examined by competitive reverse transcriptase-PCR. Those for the EP(1), EP(2) and DP, however, could not be detected. AE1-329, an EP(4) agonist, and cicaprost, an IP agonist, inhibited platelet derived growth factor (PDGF)-induced proliferation of VSMCs from wild-type mice; these inhibitory effects disappeared completely in VSMCs from EP(4)(-/-) and IP(-/-) mice, respectively. In accordance with these effects, AE1-329 and cicaprost stimulated cAMP production in VSMCs from wild-type mice, which were absent in VSMCs from EP(4)(-/-) and IP(-/-) mice, respectively. Effects of PGE(2) on cell proliferation and adenylate cyclase were almost similar with those of AE1-329 in VSMCs from wild-type mice, which disappeared in VSMCs from EP(4)(-/-) mice. PGD(2) inhibited PDGF-induced proliferation of VSMCs from both wild-type and DP(-/-) mice to a similar extent. This action of PGD(2) was also observed in VSMCs from EP4(-/-) and IP(-/-) mice. In VSMCs from wild-type mice, I-BOP, a TP agonist, showed potentiation of PDGF-induced hypertrophy. I-BOP failed to show this action in VSMCs from TP(-/-) mice. The specific agonists for the EP(1), EP(2) or EP(3), and PGF(2)alpha showed little effect on the growth of VSMCs. These results show that PGE(2), PGI(2) and TXA(2) modulate PDGF-induced proliferation or hypertrophy of VSMCs via the EP(4), IP and TP, respectively, and that the inhibitory effect of PGD(2) on PDGF-induced proliferation is not mediated by the DP, EP(4) or IP.

Activation of poly(ADP-ribose) polymerase in circulating leukocytes during myocardial infarction.

Myocardial ischemia-reperfusion can lead to increased oxidative stress both locally and in circulating leukocytes. Oxidant-mediated DNA single strand breaks are known to activate the nuclear enzyme poly(ADP-ribose) polymerase (PARP) in various forms of shock, inflammation, and ischemia-reperfusion injury. The aim of the current study was to investigate whether a local insult such as myocardial ischemia-reperfusion is sufficient to lead to activation of PARP in circulating leukocytes. In anesthetized rats myocardial ischemia-reperfusion was induced by transient ligation of the left anterior descending coronary artery. There was a marked increase in poly(ADP-ribosyl)ation of proteins in homogenates of leukocytes isolated from rats at the end of the reperfusion period. Poly(ADP-ribosyl)ation was inhibited by administration of the pharmacologic PARP inhibitor INO-1001 (30 mg/kg) to the rats. We conclude that local insults, such as myocardial reperfusion injury, are sufficient to activate PARP in circulating leukocytes. PARP activation in circulating cells may mediate certain systemic effects of local ischemia-reperfusion injury such as inflammatory mediator production and remote organ injury.

Three-dimensional myocardial scarring along myofibers after coronary ischemia-reperfusion revealed by computerized images of histological assays.

Adverse left ventricular (LV) remodeling after acute myocardial infarction is characterized by LV dilatation and development of a fibrotic scar, and is a critical factor for the prognosis of subsequent development of heart failure. Although myofiber organization is recognized as being important for preserving physiological cardiac function and structure, the anatomical features of injured myofibers during LV remodeling have not been fully defined. In a mouse model of ischemia-reperfusion (I/R) injury induced by left anterior descending coronary artery ligation, our previous histological assays demonstrated that broad fibrotic scarring extended from the initial infarct zone to the remote zone, and was clearly demarcated along midcircumferential myofibers. Additionally, no fibrosis was observed in longitudinal myofibers in the subendocardium and subepicardium. However, a histological analysis of tissue sections does not adequately indicate myofiber injury distribution throughout the entire heart. To address this, we investigated patterns of scar formation along myofibers using three-dimensional (3D) images obtained from multiple tissue sections from mouse hearts subjected to I/R injury. The fibrotic scar area observed in the 3D images was consistent with the distribution of the midcircumferential myofibers. At the apex, the scar formation tracked along the myofibers in an incomplete C-shaped ring that converged to a triangular shape toward the end. Our findings suggest that myocyte injury after transient coronary ligation extends along myofibers, rather than following the path of coronary arteries penetrating the myocardium. The injury pattern observed along myofibers after I/R injury could be used to predict prognoses for patients with myocardial infarction.

Regulation of the mPTP by SIRT3-mediated deacetylation of CypD at lysine 166 suppresses age-related cardiac hypertrophy.

Cardiac failure is a leading cause of age-related death, though its root cause remains unknown. Mounting evidence implicates a decline in mitochondrial function due to increased opening of the mitochondrial permeability transition pore (mPTP). Here we report that the NAD+-dependent deacetylase SIRT3 deacetylates the regulatory component of the mPTP, cyclophilin D (CypD) on lysine 166, adjacent to the binding site of cyclosporine A, a CypD inhibitor. Cardiac myocytes from mice lacking SIRT3 exhibit an age-dependent increase in mitochondrial swelling due to increased mPTP opening, a phenotype that is rescued by cyclosporine A. SIRT3 knockout mice show accelerated signs of aging in the heart including cardiac hypertrophy and fibrosis at 13 months of age. SIRT3 knockout mice are also hypersensitive to heart stress induced by transverse aortic constriction (TAC), as evidenced by cardiac hypertrophy, fibrosis, and increased mortality. Together, these data show for the first time that SIRT3 activity is necessary to prevent mitochondrial dysfunction and cardiac hypertrophy during aging and shed light on new pharmacological approaches to delaying aging and treating diseases in cardiac muscle and possibly other post-mitotic tissues.

Myocyte injury along myofibers in left ventricular remodeling after myocardial infarction.

Left ventricular (LV) remodeling following myocardial infarction (MI) is considered to contribute to cardiac dysfunction. Though myofiber organization is a key component of cardiac structure, functional and anatomical features of injured myofiber during LV remodeling have not been fully defined. We investigated myocyte injury after acute MI in a mouse model. Mice were subjected to surgical coronary occlusion/reperfusion by left anterior descending coronary artery (LAD) ligation and examined at 1 week and 4 weeks post-MI. Magnetic resonance imaging (MRI) analysis demonstrated a significant decrease in systolic regional wall thickening (WT) in the border and remote zones at 4 weeks post-MI compared to that at 1 week post-MI (-86% in border zone, P<0.05, and -77% in remote zone, P<0.05). Histological assays demonstrated that a broad fibrotic scar extended from the initial infarct zone to the remote zone along mid-circumferential myofibers. Of particular note was the fact that no fibrosis was found in longitudinal myofibers in the epi- and endo-myocardium. This pattern of the scar formation coincided with the helical ventricular myocardial band (HVMB) model, introduced by Torrent-Guasp. MRI analysis demonstrated that the extension of the fibrotic scar along the band might account for the progression in cardiac dysfunction during LV remodeling.

Sphingosine 1-phosphate S1P2 and S1P3 receptor-mediated Akt activation protects against in vivo myocardial ischemia-reperfusion injury.

Sphingosine 1-phosphate (S1P) is released at sites of tissue injury and effects cellular responses through activation of G protein-coupled receptors. The role of S1P in regulating cardiomyocyte survival following in vivo myocardial ischemia-reperfusion (I/R) injury was examined by using mice in which specific S1P receptor subtypes were deleted. Mice lacking either S1P(2) or S1P(3) receptors and subjected to 1-h coronary occlusion followed by 2 h of reperfusion developed infarcts equivalent to those of wild-type (WT) mice. However, in S1P(2,3) receptor double-knockout mice, infarct size following I/R was increased by >50%. I/R leads to activation of ERK, JNK, and p38 MAP kinases; however, these responses were not diminished in S1P(2,3) receptor knockout compared with WT mice. In contrast, activation of Akt in response to I/R was markedly attenuated in S1P(2,3) receptor knockout mouse hearts. Neither S1P(2) nor S1P(3) receptor deletion alone impaired I/R-induced Akt activation, which suggests redundant signaling through these receptors and is consistent with the finding that deletion of either receptor alone did not increase I/R injury. The involvement of cardiomyocytes in S1P(2) and S1P(3) receptor mediated activation of Akt was tested by using cells from WT and S1P receptor knockout hearts. Akt was activated by S1P, and this was modestly diminished in cardiomyocytes from S1P(2) or S1P(3) receptor knockout mice and completely abolished in the S1P(2,3) receptor double-knockout myocytes. Our data demonstrate that activation of S1P(2) and S1P(3) receptors plays a significant role in protecting cardiomyocytes from I/R damage in vivo and implicate the release of S1P and receptor-mediated Akt activation in this process.

Poly(ADP-Ribose) polymerase promotes cardiac remodeling, contractile failure, and translocation of apoptosis-inducing factor in a murine experimental model of aortic banding and heart failure.

Oxidant stress-induced activation of poly(ADP-ribose) polymerase (PARP) plays a role in the pathogenesis of various cardiovascular diseases. We have now investigated the role of PARP in the process of cardiac remodeling and heart failure in a mouse model of heart failure induced by transverse aortic constriction (banding). The catalytic activity of PARP was inhibited by the potent isoindolinone-based PARP inhibitor INO-1001 or by PARP-1 genetic deficiency. PARP inhibition prevented the pressure overload-induced decrease in cardiac contractile function, despite the pressure gradient between both carotid arteries being comparable in the two experimental groups. The development of hypertrophy, the formation of collagen in the hearts, and the mitochondrial-to-nuclear translocation of the cell death factor apoptosis-inducing factor (AIF) were attenuated by PARP inhibition. The ability of the inhibitor to block the catalytic activity of PARP was confirmed by immunohistochemical detection of poly(ADP-ribose), the product of the enzyme in the heart. Plasma levels of INO-1001, as measured at the end of the experiments, were in the concentration range sufficient to block the oxidant-mediated activation of PARP in murine cardiac myocytes in vitro. Myocardial hypertrophy and AIF translocation was also reduced in PARP-1-deficient mice undergoing aortic banding, compared with their wild-type counterparts. Overall, the current results demonstrate the importance of poly(ADP-ribos)ylation in the pathogenesis of banding-induced heart failure.

Poly(ADP-ribose) polymerase contributes to the development of myocardial infarction in diabetic rats and regulates the nuclear translocation of apoptosis-inducing factor.

Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP)-1 by oxidant-mediated DNA damage is an important pathway of cell dysfunction and tissue injury during myocardial infarction. Because diabetes mellitus can substantially alter cellular signal transduction pathways, we have now investigated whether the PARP pathway also contributes to myocardial ischemia/reperfusion (MI/R) injury in diabetes mellitus in rodents. Myocardial ischemia/reperfusion in control and streptozotocin-diabetic rats was induced by transient ligation of the left anterior descending coronary artery. PARP activation was inhibited by the isoindolinone derivative PARP inhibitor INO-1001. In diabetic rats, a more pronounced degree of myocardial contractile dysfunction developed, which also was associated with a larger infarct size, and significant mortality compared with nondiabetic rats. Inhibition of PARP provided a similar degree of myocardial protective effect in diabetic and nondiabetic animals and reduced infarct size and improved myocardial contractility. In diabetic rats, PARP inhibition reduced mortality during the reperfusion phase. There was marked activation of PARP in the ischemic/reperfused myocardium, which was blocked by INO-1001. In addition, there was a significant degree of mitochondrial-to-nuclear translocation of the cell death effector apoptosis-inducing factor (AIF) in myocardial infarction, which was blocked by pharmacological inhibition of PARP. The role of PARP in regulating AIF translocation in myocytes also was confirmed in an isolated perfused heart preparation. Overall, the current results demonstrate the importance of the PARP pathway in diabetic rats subjected to myocardial infarction and demonstrate the role of PARP in regulating AIF translocation in MI/R.