A Novel Mitochondrial Complex of Aldosterone Synthase, Steroidogenic Acute Regulatory Protein, and Tom22 Synthesizes Aldosterone in the Rat Heart.

Aldosterone, which regulates renal salt retention, is synthesized in adrenocortical mitochondria in response to angiotensin II. Excess aldosterone causes myocardial injury and heart failure, but potential intracardiac aldosterone synthesis has been controversial. We hypothesized that the stressed heart might produce aldosterone. We used blue native gel electrophoresis, immunoblotting, protein crosslinking, coimmunoprecipitations, and mass spectrometry to assess rat cardiac aldosterone synthesis. Chronic infusion of angiotensin II increased circulating corticosterone levels 350-fold and induced cardiac fibrosis. Angiotensin II doubled and telmisartan inhibited aldosterone synthesis by heart mitochondria and cardiac production of aldosterone synthase (P450c11AS). Heart aldosterone synthesis required P450c11AS, Tom22 (a mitochondrial translocase receptor), and the intramitochondrial form of the steroidogenic acute regulatory protein (StAR); protein crosslinking and coimmunoprecipitation studies showed that these three proteins form a 110-kDa complex. In steroidogenic cells, extramitochondrial (37-kDa) StAR promotes cholesterol movement from the outer to inner mitochondrial membrane where cholesterol side-chain cleavage enzyme (P450scc) converts cholesterol to pregnenolone, thus initiating steroidogenesis, but no function has previously been ascribed to intramitochondrial (30-kDa) StAR; our data indicate that intramitochondrial 30-kDa StAR is required for aldosterone synthesis in the heart, forming a trimolecular complex with Tom22 and P450c11AS. This is the first activity ascribed to intramitochondrial StAR, but how this promotes P450c11AS activity is unclear. The stressed heart did not express P450scc, suggesting that circulating corticosterone (rather than intracellular cholesterol) is the substrate for cardiac aldosterone synthesis. Thus, the stressed heart produced aldosterone using a previously undescribed intramitochondrial mechanism that involves P450c11AS, Tom22, and 30-kDa StAR. SIGNIFICANCE STATEMENT: Prior studies of potential cardiac aldosterone synthesis have been inconsistent. This study shows that the stressed rat heart produces aldosterone by a novel mechanism involving aldosterone synthase, Tom22, and intramitochondrial steroidogenic acute regulatory protein (StAR) apparently using circulating corticosterone as substrate. This study establishes that the stressed rat heart produces aldosterone and for the first time identifies a biological role for intramitochondrial 30-kDa StAR.

Steroidogenic acute regulatory protein/aldosterone synthase mediates angiotensin II-induced cardiac fibrosis and hypertrophy.

Aldosterone produced in adrenal glands by angiotensin II (Ang II) is known to elicit myocardial fibrosis and hypertrophy. This study was designed to test the hypothesis that Ang II causes cardiac morphological changes through the steroidogenic acute regulatory protein (StAR)/aldosterone synthase (AS)-dependent aldosterone synthesis primarily initiated in the heart. Sprague-Dawley rats were randomized to following groups: Ang II infusion for a 4-week period, treatment with telmisartan, spironolactone or adrenalectomy during Ang II infusion. Sham-operated rats served as control. Relative to Sham rats, Ang II infusion significantly increased the protein levels of AT1 receptor, StAR, AS and their tissue expression in the adrenal glands and heart. In coincidence with reduced aldosterone level in the heart, telmisartan, an AT1 receptor blocker, significantly down-regulated the protein level and expression of StAR and AS. Ang II induced changes in the expression of AT1/StAR/AS were not altered by an aldosterone receptor antagonist spironolactone. Furthermore, Ang II augmented migration of macrophages, protein level of TGFß1, phosphorylation of Smad2/3 and proliferation of myofibroblasts, accompanied by enhanced perivascular/interstitial collagen deposition and cardiomyocyte hypertrophy, which all were significantly abrogated by telmisartan or spironolactone. However, adrenalectomy did not fully suppress Ang II-induced cell migration/proliferation and fibrosis/hypertrophy, indicating a role of aldosterone synthesized within the heart in pathogenesis of Ang II induced injury. These results indicate that myocardial fibrosis and hypertrophy stimulated by Ang II is associated with tissue-specific activation of aldosterone synthesis, primarily mediated by AT1/StAR/AS signaling pathways.

Postconditioning attenuates coronary perivascular and interstitial fibrosis through modulating angiotensin II receptors and angiotensin-converting enzyme 2 after myocardial infarction.

BACKGROUND: Postconditioning (Postcon) is known to reduce infarct size. This study tested the hypothesis that Postcon attenuates the perivascular and interstitial fibrosis after myocardial infarction through modulating angiotensin II-activated fibrotic cascade. MATERIALS AND METHODS: Male Sprague-Dawley rats were subjected to 45-min coronary occlusion followed by 1 and 6 wk of reperfusion. Postcon was applied at the onset of reperfusion with four cycles of 10/10-s reperfusion-ischemia at the onset of reperfusion. Preconditioning (Precon) with two cycles of 5/5-min ischemia-reperfusion was applied before coronary occlusion. RESULTS: Postcon reduced angiotensin-converting enzyme protein and expression in the perivascular area and intermyocardium, coincident with the less-expressed angiotensin II receptor, type 1, enhanced angiotensin II receptor, type 2, and angiotensin converting enzyme 2. Postcon lowered the monocyte chemoattractant protein-1 and inhibited the populations of interstitial macrophages (60 ± 12 versus 84 ± 9.5 number per high-powered field [HPF] in control, P < 0.05). Along with these modulations, Postcon also downregulated transforming growth factor ß1 protein and inhibited proliferation of α-smooth muscle actin expressing myofibroblasts (41 ± 11 versus 79 ± 8.2 number per HPF in control, P < 0.05), consistent with downregulated phospho-Smad2 and phospho-Smad3. Furthermore, the synthesis of collagen I and III was attenuated, and the perivascular-interstitial fibrosis was inhibited by Postcon as demonstrated by reduced perivascular fibrosis ratio (0.6 ± 0.6 versus 1.6 ± 0.5 per HPF in control, P < 0.05) and smaller collagen-rich area (16 ± 4.7 versus 34 ± 9.2% per HPF in control, P < 0.05). Precon conferred a comparable level of protection as Postcon did in all parameters measured, suggesting protection trigged by this endogenous stimulation can be achieved when it was applied either before ischemia or after reperfusion. CONCLUSIONS: These results suggest that Postcon could be selected as an adjunctive intervention with other existing therapeutic drugs to treat the fibrosis-derived heart failure patients after myocardial infarction.

Preservation of Glucagon-Like Peptide-1 Level Attenuates Angiotensin II-Induced Tissue Fibrosis by Altering AT1/AT 2 Receptor Expression and Angiotensin-Converting Enzyme 2 Activity in Rat Heart.

PURPOSE: The glucagon-like peptide-1 (GLP-1) has been shown to exert cardioprotective effects in animals and patients. This study tests the hypothesis that preservation of GLP-1 by the GLP-1 receptor agonist liraglutide or the dipeptidyl peptidase-4 (DPP-4) inhibitor linagliptin is associated with a reduction of angiotensin (Ang) II-induced cardiac fibrosis. METHODS AND RESULTS: Sprague-Dawley rats were subjected to Ang II (500 ng/kg/min) infusion using osmotic minipumps for 4 weeks. Liraglutide (0.3 mg/kg) was subcutaneously injected twice daily or linagliptin (8 mg/kg) was administered via oral gavage daily during Ang II infusion. Relative to the control, liraglutide, but not linagliptin decreased MAP (124 ± 4 vs. 200 ± 7 mmHg in control, p < 0.003). Liraglutide and linagliptin comparatively reduced the protein level of the Ang II AT1 receptor and up-regulated the AT2 receptor as identified by a reduced AT1/AT2 ratio (0.4 ± 0.02 and 0.7 ± 0.01 vs. 1.4 ± 0.2 in control, p < 0.05), coincident with the less locally-expressed AT1 receptor and enhanced AT2 receptor in the myocardium and peri-coronary vessels. Both drugs significantly reduced the populations of macrophages (16 ± 6 and 19 ± 7 vs. 61 ± 29 number/HPF in control, p < 0.05) and α-SMA expressing myofibroblasts (17 ± 7 and 13 ± 4 vs. 66 ± 29 number/HPF in control, p < 0.05), consistent with the reduction in expression of TGFß1 and phospho-Smad2/3, and up-regulation of Smad7. Furthermore, ACE2 activity (334 ± 43 and 417 ± 51 vs. 288 ± 19 RFU/min/µg protein in control, p < 0.05) and GLP-1 receptor expression were significantly up-regulated. Along with these modulations, the synthesis of collagen I and tissue fibrosis were inhibited as determined by the smaller collagen-rich area and more viable myocardium. CONCLUSION: These results demonstrate for the first time that preservation of GLP-1 using liraglutide or linagliptin is effective in inhibiting Ang II-induced cardiac fibrosis, suggesting that these drugs could be selected as an adjunctive therapy to improve clinical outcomes in the fibrosis-derived heart failure patients with or without diabetes.

Postconditioning promotes the cardiac repair through balancing collagen degradation and synthesis after myocardial infarction in rats.

Postconditioning (Postcon) reduces infarct size. However, its role in modulation of cardiac repair after infarction is uncertain. This study tested the hypothesis that Postcon inhibits adverse cardiac repair by reducing degradation of extracellular matrix (ECM) and synthesis of collagens via modulating matrix metalloproteinase (MMP) activity and transforming growth factor (TGF) ß1/Smad signaling pathway. Sprague-Dawley rats were subjected to 45 min ischemia followed by 3 h, 7 or 42 days of reperfusion, respectively. In acute studies, four cycles of 10/10 s Postcon significantly reduced infarct size, which was blocked by administration of a mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate (5-HD) at reperfusion. In chronic studies, Postcon inhibited MMP activity and preserved ECM from degradation as evidenced by reduced extent of collagen-rich scar and increased mass of viable myocardium. Along with a reduction in collagen synthesis and fibrosis, Postcon significantly down-regulated expression of TGFß1 and phospho-Smad2/3, and up-regulated Smad7 as compared to the control, consistent with a reduction in the population of α-smooth muscle actin expressing myofibroblasts within the infarcted myocardium. At 42 days of reperfusion, echocardiography showed significant improvements in left ventricular end-diastolic volume and ejection fraction. The wall thickness of the infarcted middle anterior septum in the Postcon was also significantly greater than that in the control. The beneficial effects of Postcon on cardiac repair were comparable to preconditioning and still evident after a blockade with 5-HD. These data suggest that Postcon is effective to promote cardiac repair and preserve cardiac function; protection is potentially mediated by inhibiting ECM degradation and collagen synthesis.

Suppression of angiotensin II-activated NOX4/NADPH oxidase and mitochondrial dysfunction by preserving glucagon-like peptide-1 attenuates myocardial fibrosis and hypertension.

This study aims to investigate whether stabilization of glucagon-like peptide-1 (GLP-1) level reduces angiotensin II (Ang II)-induced cardiac fibrosis and -elevated blood pressure accompanying with inhibition of NADPH oxidase (NOX) expression and preservation of mitochondrial integrity. The study was performed in Sprague-Dawley rat model of Ang II infusion (500 ng/kg/min) using osmotic minipumps for 4 weeks. GLP-1 receptor agonist liraglutide (0.3 mg/kg, injected subcutaneously twice daily) and dipeptidyl peptides-4 inhibitor, linagliptin (8 mg/kg, administered via oral gavage) were selected to preserve GLP-1 level. Blood pressure was measured noninvasively. Heart and aorta were saved for histological analysis. Relative to the animals with Ang II infusion, in the heart, liraglutide and linagliptin comparatively reduced the protein levels of NOX4 and TGFß1 and expression of monocyte chemoattractant protein 1, and attenuated the proliferation of myofibroblasts (15 ± 4 and 13 ± 3 vs. 42 ± 22/HPF in Ang II group). The number of distorted mitochondria in both groups was significantly reduced (8 ± 4 and 10 ± 6 vs. 27 ± 13/HPF in Ang II group), in company with a significant reduction in cardiac fibrosis. In the aorta, treatment with liraglutide and linagliptin significantly downregulated the expression of NOX4 and intercellular adhesion molecule 1, and enhanced endothelial NOS expression. Aortic wall thickness was reduced comparatively (267 ± 22 and 286 ± 25 vs. 339 ± 40 µm in Ang II group). The area of fibrotic aorta was also reduced (13 ± 6 and 14 ± 5 vs. 38 ± 24 mm2 in Ang II group), respectively, in coincidence with a significant reduction in mean blood pressure. Taken together, these results suggest that the conservation of GLP-1 level with exogenous supply of liraglutide or the prevention of endogenous degradation of GLP-1 with linagliptin protects against Ang II-induced injury in the heart and aorta, potentially associated with inhibition of NOX4 expression and preservation of mitochondrial integrity.

Factor Xa induces tissue factor expression in endothelial cells by P44/42 MAPK and NF-κB-dependent pathways.

BACKGROUND: Tissue factor (TF) is an initiator of coagulation. The serine protease factor Xa (FXa) is the convergence point of the extrinsic and intrinsic components of the coagulation cascade. In addition to its hemostatic function, FXa elicits inflammatory responses in endothelial cells that may be important in surgical procedures in which inflammation is triggered. This study tested the hypothesis that FXa can up-regulate TF on vascular endothelial cells by a mitogen-activated protein kinase (MAPK)- and NF-κB-dependent pathway. METHODS AND RESULTS: Incubation of cultured human umbilical vein endothelial cells (HUVECs) with FXa increased TF protein expression and activity in a dose-dependent manner. Pre-incubation of HUVECs with the serine protease inhibitor antithrombin, which targets not only thrombin but also FXa and FIXa, inhibited FXa-induced TF expression, but the selective thrombin inhibitor hirudin did not inhibit FXa-induced TF expression, ruling out a thrombin-mediated pathway. After 10 min incubation with HUVECs, FXa rapidly induced P44/42 MAPK activation (immunoblotting of phosphorylated P44/42 MAPK) with a peak at 30 min. The MEK 1/2 inhibitor PD98059 partially reduced FXa-induced TF expression and activity (3.82 ± 0.11 vs 6.54 ± 0.08 fmol/min/cm(2), P < 0.05). NF-κB was activated by FXa, confirmed by cytoplasmic IkBα degradation and increased NF-κB P65 nuclear translocation. Interruption of the NF-κB pathway by the IkBα phosphorylation inhibitor Bay 11-7802 abrogated FXa-induced TF protein expression and activity (1.93 ± 0.02 versus 6.54 ± 0.08 fmol/min/cm(2), P < 0.05). However, inhibition of PI3 kinase by LY 294002 did not attenuate FXa-induced TF protein expression and activity. CONCLUSIONS: (1) FXa up-regulates TF protein expression and activity in HUVECs, (2) FXa-induced up-regulation of TF is independent of the thrombin-PAR1 pathway, and (3) the MAPK and NF-κB pathways, but not PI3 kinase pathway, are involved in FXa-induced TF expression on human umbilical endothelial cells. FXa may be a feed-forward alternative mechanism of activating TF expression and activity, thereby increasing a procoagulant state or inflammation. This mechanism may be important in the pro-inflammatory state initiated by cardiac surgical procedures.

Attenuation of renal ischemia-reperfusion injury by postconditioning involves adenosine receptor and protein kinase C activation.

SUMMARY: Significant organ injury occurs after transplantation and reflow (i.e., reperfusion injury). Postconditioning (PoC), consisting of alternating periods of reperfusion and re-occlusion at onset of reperfusion, attenuates reperfusion injury in organs including heart and brain. We tested whether PoC attenuates renal ischemia-reperfusion (I/R) injury in the kidney by activating adenosine receptors (AR) and protein kinase C (PKC). The single kidney rat I/R model was used. Groups: (1) sham: time-matched surgical protocol only. In all others, the left renal artery (RA) was occluded for 45 min and reperfused for 24 h. (2) CONTROL: I/R with no intervention at R. All antagonists were administered 5 min before reperfusion. (3) PoC: I/R + four cycles of 45 s of R and 45 s of re-occlusion before full R. (4) PoC + ARi: PoC plus the AR antagonist 8-rho-(sulfophenyl) theophylline (8-SPT). (5) PoC + PKCi: PoC plus the PKC antagonist chelerythrine (Che). In shams, plasma blood urea nitrogen (BUN mg/dl) at 24 h averaged 23.2 +/- 5.3 and creatinine (Cr mg/dl) averaged 1.28 +/- 0.2. PoC reduced BUN (87.2 +/- 10 in CONTROL vs. 38.8 +/- 9, P = 0.001) and Cr (4.2 +/- 0.6 in CONTROL vs. 1.5 +/- 0.2, P < 0.001). 8-SPT and Che reversed renal protection indices after PoC. I/R increased apoptosis, which was reduced by PoC, which was reversed by 8-SPT and Che. Postconditioning attenuates renal I/R injury by adenosine receptor activation and PKC signaling.

Conservation of glucagon like peptide-1 level with liraglutide and linagilptin protects the kidney against angiotensin II-induced tissue fibrosis in rats.

This study tested the hypothesis that the enhancement of glucagon-like peptide-1 (GLP-1) level through either exogenous supply of GLP-1 agonist, liraglutide or prevention of endogenous GLP-1 degradation with dipeptidyl peptidease-4 inhibitor, lingaliptin ameliorates angiotensin II (Ang II)-induced renal fibrosis. Sprague-Dawley rats were randomly divided into four groups: 0.9% saline or Ang II (500 ng/kg/min) was infused with osmotic minipumps for 4 weeks, defined as sham and Ang II groups. In drug treated groups, liraglutide (0.3 mg/kg) was injected subcutaneously twice daily or linagliptin (8 mg/kg) was administered daily via oral gavage during Ang II infusion. Compared with Ang II stimulation, liraglutide or linagliptin comparatively down-regulated the protein level of the AT1 receptor, and up-regulated the AT2 receptor, as identified by a reduced AT1/AT2 ratio (all p < 0.05), consistent with less locally-expressed AT1 receptor and enhanced AT2 receptor in the glomerular capillaries and proximal tubules of the renal cortex. Furthermore, both drugs significantly increased the expression of GLP-1 receptor and attenuated the protein levels of TLR4, NOX4 and IL-6. The populations of macrophages and α-SMA expressing myofibroblasts decreased with treatment of liraglutide and linagliptin, in coincidence with the reduced expression of phosphor-Smad2/3, Smad4, TGFß1, and up-regulated Smad7. Along with these modulations, renal morphology was preserved and synthesis of fibronectin/collagen I was down-regulated, as identified by small collagen-rich area in the renal cortex. These results suggest that the preservation of GLP-1 level using liraglutide or linagliptin might be considered as an add-on therapeutic option for inhibiting Ang II induced renal fibrosis and failure.

Liraglutide attenuates cardiac remodeling and improves heart function after abdominal aortic constriction through blocking angiotensin II type 1 receptor in rats.

OBJECTIVE: Angiotensin II (Ang II) is known to contribute to the pathogenesis of heart failure by eliciting cardiac remodeling and dysfunction. The glucagon-like peptide-1 (GLP-1) has been shown to exert cardioprotective effects in animals and patients. This study investigates whether GLP-1 receptor agonist liraglutide inhibits abdominal aortic constriction (AAC)-induced cardiac fibrosis and dysfunction through blocking Ang II type 1 receptor (AT1R) signaling. METHODS: Sprague-Dawley rats were subjected to sham operation and abdominal aortic banding procedure for 16 weeks. In treated rats, liraglutide (0.3 mg/kg) was subcutaneously injected twice daily or telmisartan (10 mg/kg/day), the AT1R blocker, was administered by gastric gavage. RESULTS: Relative to the animals with AAC, liraglutide reduced protein level of the AT1R and upregulated the AT2R, as evidenced by reduced ratio of AT1R/AT2R (0.59±0.04 vs. 0.91±0.06, p<0.05). Furthermore, the expression of angiotensin converting enzyme 2 was upregulated, tissue levels of malondialdehyde and B-type natriuretic peptide were reduced, and superoxide dismutase activity was increased. Along with a reduction in HW/BW ratio, cardiomyocyte hypertrophy was inhibited. In coincidence with these changes, liraglutide significantly decreased the populations of macrophages and myofibroblasts in the myocardium, which were accompanied by reduced protein levels of transforming growth factor beta1, Smad2/3/4, and upregulated smad7. The synthesis of collagen I and III was inhibited and collagen-rich fibrosis was attenuated. Consistent with these findings, cardiac systolic function was preserved, as shown by increased left ventricular systolic pressure (110±5 vs. 99±2 mmHg, p<0.05), ejection fraction (83%±2% vs. 69%±4%, p<0.05) and fraction shortening (49%±2% vs. 35%±3%, p<0.05). Treatment with telmisartan provided a comparable level of protection as compared with liraglutide in all the parameters measured. CONCLUSION: Taken together, liraglutide ameliorates cardiac fibrosis and dysfunction, potentially via suppressing the AT1R-mediated events. These data indicate that liraglutide might be selected as an add-on drug to prevent the progression of heart failure.

CD44 Deficiency in Mice Protects the Heart Against Angiotensin Ii-Induced Cardiac Fibrosis.

This study tested the hypothesis that CD44 is involved in the development of cardiac fibrosis via angiotensin II (Ang II) AT1 receptor-stimulated TNFα/NFκB/IκB signaling pathways. Study was conducted in C57BL/6 wild type and CD44 knockout mice subjected to Ang II infusion (1,000 ng/kg/min) using osmotic minipumps up to 4 weeks or with gastric gavage administration of the AT1 receptor blocker, telmisartan at a dose of 10 mg/kg/d. Results indicated that Ang II enhances expression of the AT1 receptor, TNFα, NFκB, and CD44 as well as downregulates IκB. Further analyses revealed that Ang II increases macrophage migration, augments myofibroblast proliferation, and induces vascular/interstitial fibrosis. Relative to the Ang II group, treatment with telmisartan significantly reduced expression of the AT1 receptor and TNFα. These changes occurred in coincidence with decreased NFκB, increased IκB, and downregulated CD44 in the intracardiac vessels and intermyocardium. Furthermore, macrophage migration and myofibroblast proliferation were inhibited and fibrosis was attenuated. Knockout of CD44 did not affect Ang II-stimulated AT1 receptor and modulated TNFα/NFκB/IκB signaling, but significantly reduced macrophage/myofibroblast-mediated fibrosis as identified by less extensive collagen-rich area. These results suggest that the AT1 receptor is involved in the development of cardiac fibrosis by stimulating TNFα/NFκB/IκB-triggered CD44 signaling pathways. Knockout of CD44 blocked Ang II-induced cell migration/proliferation and cardiac fibrosis. Therefore, selective inhibition of CD44 may be considered as a potential therapeutic target for attenuating Ang II-induced deleterious cardiovascular effects.

Intravenous infusion of mesenchymal stem cells enhances regional perfusion and improves ventricular function in a porcine model of myocardial infarction.

Transplantation of stem cells may improve regional perfusion and post-infarct ventricular function, but the optimal dose and efficacy of cell delivery via the intravenous route has not been determined. This study tested the hypothesis that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) enhances regional perfusion and improves ventricular function after myocardial infarction. In a closed-chest pig model, the LAD coronary artery was occluded for 75 min by angioplasty balloon inflation followed by 12 weeks of reperfusion. After 15 min of reperfusion, pigs randomly received 1 of 4 treatments: (1) Vehicle (Control, n = 10); (2) 1 x 10(6) MSCs/kg (1 mill, n = 7); (3) 3 x 10(6) MSCs/kg (3 mill, n = 8) and (4) 10 x 10(6) MSCs/kg (10 mill, n = 8). Angiogenesis was demonstrated by immunohistochemical staining, myocardial blood flow (steady state and vasodilator reserve) was measured using 15 microm neutron-activated microspheres, and cardiac function was determined by contrast left ventriculography (ejection fraction) and pressure-volume relationships. After 12 week of reperfusion, von Willebrand Factor-positive vessels and tissue vascular endothelial growth factor (VEGF) expression in the scar zone was significantly greater in all MSCs-treated animals relative to Control. Steady state myocardial blood flow in the scar tissue was comparable among groups. However, adenosine recruited vasodilator reserve in the scar zone induced by intracoronary adenosine was significantly higher in the MSC-treated animals compared to Control. Furthermore, preload-recruitable stroke work and systolic performance were significantly greater compared to Control. In conclusion, these data demonstrate that intravenous delivery of MSCs during early reperfusion augments vasculogenesis, enhances regional perfusion, and improves post-infarct ventricular function. The results suggest that intravenous infusion of MSCs is an effective modality for the treatment of ischemia/reperfusion induced myocardial injury.

Inhibition of myocardial apoptosis by postconditioning is associated with attenuation of oxidative stress-mediated nuclear factor-kappa B translocation and TNF alpha release.

Oxidative stress-stimulated nuclear factor-kappa B (NF-kappa B) activation has been associated with rapid transcription of TNF-alpha and induction of apoptosis. This study tested the hypothesis that postconditioning (Postcon) reduces myocardial apoptosis and inhibits translocation of NF-kappa B and release of TNF-alpha secondary to an attenuation of oxidant generation during reperfusion. Anesthetized rats were subjected to 30 min of ischemia and 3 h of reperfusion and divided randomly to Control or Postcon (three cycles of 10-s reperfusion and 10-s reocclusion applied at the onset of reperfusion) group, respectively. Relative to Control, Postcon reduced the plasma malondialdehyde (1.21 +/- 0.08 vs. 0.8 +/- 0.06* microM/mL) and decreased the generation of superoxide radical in area at risk myocardium (dihydroethidium staining). Compared with Control, Postcon also inhibited translocation of NF-kappa B to nuclei (167% +/- 21% vs. 142% +/- 18%*), decreased the level of plasma TNF-alpha (1,994 +/- 447 vs. 667 +/- 130* pg/mL), and inhibited caspase-3 activity (0.57% +/- 0.1% vs. 0.21% +/- 0.1%*). The number of apoptotic cells (percent total nuclei) in ischemic myocardium was reduced (20% +/- 1% vs. 11% +/- 2%*), consistent with reduced appearance of DNA fragmentation. To support whether oxidant generation is important in the triggering of cytokine release and apoptosis, N-acetylcysteine (NAC), a potent antioxidant agent, was administered before ischemia and at reperfusion. Treatment with NAC inhibited superoxide radical generation and decreased plasma malondialdehyde to a comparable level to that in Postcon, concomitant with an inhibition of NF-kappa B expression (42% +/- 8%*) and reduction of release of TNF-alpha (231 +/- 72* pg/mL). Caspase-3 activity (0.33% +/- 0.1%*) and apoptotic cells (12% +/- 1%*) were also comparably reduced by NAC. These data suggest that Postcon attenuates myocardial apoptosis, reduces caspase-3 activity, and is potentially mediated by inhibiting oxidant-activated NF-kappa B-TNF-alpha signaling pathway. *P < 0.05 Postcon and NAC vs. Control.

Recruitment of macrophages from the spleen contributes to myocardial fibrosis and hypertension induced by angiotensin II.

INTRODUCTION: The purpose of this study was to determine whether macrophages migrated from the spleen are associated with angiotensin II-induced cardiac fibrosis and hypertension. METHODS: Sprague-Dawley rats were subjected to angiotensin II infusion in vehicle (500 ng/kg/min) for up to four weeks. In splenectomy, the spleen was removed before angiotensin II infusion. In the angiotensin II AT1 receptor blockade, telmisartan was administered by gastric gavage (10 mg/kg/day) during angiotensin II infusion. The heart and aorta were isolated for Western blot analysis and immunohistochemistry. RESULTS: Angiotensin II infusion caused a significant reduction in the number of monocytes in the spleen through the AT1 receptor-activated monocyte chemoattractant protein-1. Comparison of angiotensin II infusion, splenectomy and telmisartan comparatively reduced the recruitment of macrophages into the heart. Associated with this change, transforming growth factor ß1 expression and myofibroblast proliferation were inhibited, and Smad2/3 and collagen I/III were downregulated. Furthermore, interstitial/perivascular fibrosis was attenuated. These modifications occurred in coincidence with reduced blood pressure. At week 4, invasion of macrophages and myofibroblasts in the thoracic aorta was attenuated and expression of endothelial nitric oxide synthase was upregulated, along with a reduction in aortic fibrosis. CONCLUSIONS: These results suggest that macrophages when recruited into the heart and aorta from the spleen potentially contribute to angiotensin II-induced cardiac fibrosis and hypertension.

Angiotensin II AT1 receptor alters ACE2 activity, eNOS expression and CD44-hyaluronan interaction in rats with hypertension and myocardial fibrosis.

AIM: This study tested the hypothesis that angiotensin II (Ang II) AT1 receptor is involved in development of hypertension and cardiac fibrosis via modifying ACE2 activity, eNOS expression and CD44-hyaluronan interaction. MAIN METHODS: Male Sprague-Dawley rats were subjected to Ang II infusion (500ng/kg/min) using osmotic minipumps up to 4weeks and the AT1 receptor blocker, telmisartan was administered by gastric gavage (10mg/kg/day) during Ang II infusion. KEY FINDINGS: Our results indicated that Ang II enhances AT1 receptor, downregulates AT2 receptor, ACE2 activity and eNOS expression, and increases CD44 expression and hyaluronidase activity, an enzyme for hyaluronan degradation. Further analyses revealed that Ang II increases blood pressure and augments vascular/interstitial fibrosis. Comparison of the Ang II group, treatment with telmisartan significantly increased ACE2 activity and eNOS expression in the intracardiac vessels and intermyocardium. These changes occurred in coincidence with decreased blood pressure. Furthermore, the locally-expressed AT1 receptor was downregulated, as evidenced by an increased ratio of the AT2 over AT1 receptor (1.4±0.4% vs. 0.4±0.1% in Ang II group, P<0.05). Along with these modulations, telmisartan inhibited membrane CD44 expression and hyaluronidase activity, decreased populations of macrophages and myofibroblasts, and reduced expression of TGFß1 and Smads. Collagen I synthesis and tissue fibrosis were attenuated as demonstrated by the less extensive collagen-rich area. SIGNIFICANCE: These results suggest that the AT1 receptor is involved in development of hypertension and cardiac fibrosis. Selective activating ACE2/eNOS and inhibiting CD44/HA interaction might be considered as the therapeutic targets for attenuating Ang II induced deleterious cardiovascular effects.

Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion.

OBJECTIVE: We previously showed that brief intermittent ischemia applied during the onset of reperfusion (i.e., postconditioning) is cardioprotective in a canine model of ischemia-reperfusion. This study tested the hypothesis that the early minutes of reperfusion (R) during which postconditioning (Post-con) is applied are critical to its cardioprotection. METHODS: In anesthetized open-chest rats, the left coronary artery (LCA) was occluded for 30 min and reperfused for 3 h. All rats were randomly divided into six groups: Control (n=8): no intervention at R; Ischemic preconditioning (IPC) (n=8): the LCA was occluded for 5 min followed by 10 min of R before the index occlusion; Post-con 1 (n=8): after LCA occlusion, three cycles of 10 s R followed by 10 s LCA re-occlusion were applied during the first minute of R; Post-con 2 (n=8): Six cycles of 10 s R and 10 s re-occlusion were applied during the first 2 min of R; Delayed Post-con (n=8): the ligature was loosened for full reflow for the first minute of R, after which the three-cycle Post-con algorithm was applied; Sham (n=6): the surgical procedure was identical to other groups, but the LCA ligature was not ligated. RESULTS: Infarct size (TTC staining) was 23% smaller in Post-con 1 (40+/-2%*) than in Control (52+/-3%), confirmed by plasma creatine kinase activity (18+/-2* vs. 46+/-6 IU/g protein). There was no further reduction in infarct size with 6 cycles of Post-con (40+/-2.9%, p>0.05 vs. Post-con 1). Meanwhile, infarct size reduction was significantly greater in the IPC group (17+/-3%) than in Post-con1 (p<0.01). The plasma lipid peroxidation product malondialdehyde (MDA, microM/ml) was less after R in IPC and Post-con 1 (0.8+/-0.07* and 0.8+/-0.06*) vs. Control (1.21+/-0.08), consistent with a visual decrease in superoxide anion generation (dihydroethidium staining) in the AAR myocardium after 3 h of reperfusion. Neutrophil accumulation (myeloperoxidase activity, MPO, U/100 g tissue) in the AAR was less in IPC (1.4+/-0.3*) and Post-con 1 (2.5+/-0.3*) vs. Control (5.5+/-0.6). The reductions in infarct size, creatine kinase, MDA and DHE staining were lost with delayed Post-con, while MPO activity remained lower than in Control (3.2+/-0.4*). CONCLUSIONS: (1) Post-con at onset of R reduces myocardial injury; (2) cardioprotection may be mediated, in part, by inhibiting oxidant generation and oxidant mediated injury; (3) the first minute of R in the rat model is critical to cardioprotection by Post-con; and (4) cardioprotection by Post-con may be independent of neutrophil accumulation in AAR. *p<0.05 Post-con vs. Control.

Inhibition of myocardial apoptosis reduces infarct size and improves regional contractile dysfunction during reperfusion.

OBJECTIVE: Myocardial apoptosis is primarily triggered during reperfusion (R) through various mechanisms that may involve endonuclease to cleavage genomic DNA in the internucleosomal linker regions. However, the relative contribution of myocardial apoptosis to development of myocardial injury during R remains unknown. In the present study, we examined whether inhibition of apoptosis with aurintricarboxylic acid (ATA), an endonuclease inhibitor, during R reduces infarct size and improves regional contractile function. METHODS AND RESULTS: In two groups of chronically-instrumented dogs, 1 h of left anterior descending (LAD) coronary occlusion was followed by 24 h of R with infusion of saline (control, n=8) or ATA (1 mg/kg/h, n=8) into the left atrium starting 5 min before R and continuing for 2 h. ATA significantly reduced apoptotic cells (TUNEL staining) in the peri-necrotic myocardium (12+/-1%* vs. 36+/-4%), consistent with the absence of DNA laddering. To confirm inhibition of apoptosis with ATA, densitometrically, Bcl-2 (% of normal myocardium) was significantly increased vs. control (102+/-12* vs. 68+/-9) and Bax as well as the activated caspase-3 were significantly reduced vs. control (108+/-17* vs. 194+/-42 and -29+/-4* vs. 174+/-43, respectively). ATA significantly improved segmental shortening (3.3+/-1.2* vs. -1.8+/-0.7%) and segmental work (79.3+/-11.3* vs. 7.1+/-5.8 mmHg/mm) in area at risk myocardium, and reduced infarct size (TTC staining, 27+/-0.2* vs. 37+/-0.5%), confirmed by lower plasma creatine kinase activity. In addition, myocardial blood flow (0.9+/-0.1* vs. 0.4+/-0.1 ml/min/g) and endothelial-dependent maximal vascular relaxation (119+/-6* vs. 49+/-8%) were significantly improved. Myeloperoxidase activity in area at risk myocardium, a marker for neutrophil accumulation, was also significantly reduced (17+/-4* vs. 138+/-28 Delta Abs/min). CONCLUSIONS: These data suggest that the inhibition of apoptosis during R is associated with a reduction in infarction, improvement in regional contractile and vascular endothelial functions as well as augmentation in myocardial blood flow. *P<0.05 vs. control group.

Attenuation of myocardial fibrosis with curcumin is mediated by modulating expression of angiotensin II AT1/AT2 receptors and ACE2 in rats.

Curcumin is known to improve cardiac function by balancing degradation and synthesis of collagens after myocardial infarction. This study tested the hypothesis that inhibition of myocardial fibrosis by curcumin is associated with modulating expression of angiotensin II (Ang II) receptors and angiotensin-converting enzyme 2 (ACE2). Male Sprague Dawley rats were subjected to Ang II infusion (500 ng/kg/min) using osmotic minipumps for 2 and 4 weeks, respectively, and curcumin (150 mg/kg/day) was fed by gastric gavage during Ang II infusion. Compared to the animals with Ang II infusion, curcumin significantly decreased the mean arterial blood pressure during the course of the observation. The protein level of the Ang II type 1 (AT1) receptor was reduced, and the Ang II type 2 (AT2) receptor was up-regulated, evidenced by an increased ratio of the AT2 receptor over the AT1 receptor in the curcumin group (1.2±0.02%) vs in the Ang II group (0.7±0.03%, P<0.05). These changes were coincident with less locally expressed AT1 receptor and enhanced AT2 receptor in the intracardiac vessels and intermyocardium. Along with these modulations, curcumin significantly decreased the populations of macrophages and alpha smooth muscle actin-expressing myofibroblasts, which were accompanied by reduced expression of transforming growth factor beta 1 and phosphorylated-Smad2/3. Collagen I synthesis was inhibited, and tissue fibrosis was attenuated, as demonstrated by less extensive collagen-rich fibrosis. Furthermore, curcumin increased protein level of ACE2 and enhanced its expression in the intermyocardium relative to the Ang II group. These results suggest that curcumin could be considered as an add-on therapeutic agent in the treatment of fibrosis-derived heart failure patient who is intolerant of ACE inhibitor therapy.

Dual ACE-inhibition and angiotensin II AT1 receptor antagonism with curcumin attenuate maladaptive cardiac repair and improve ventricular systolic function after myocardial infarctionin rat heart.

Curcumin has been shown to improve cardiac function by reducing degradation of extracellular matrix and inhibiting synthesis of collagen after ischemia. This study tested the hypothesis that attenuation of maladaptive cardiac repair with curcumin is associated with a dual ACE-inhibition and angiotensin II AT1 receptor antagonism after myocardial infarction. Sprague-Dawley rats were subjected to 45min ischemia followed by 7 and 42 days of reperfusion, respectively. Curcumin was fed orally at a dose of 150mg/kg/day only during reperfusion. Relative to the control animals, dietary treatment with curcumin significantly reduced levels of ACE and AT1 receptor protein as determined by Western blot assay, coincident with less locally-expressed ACE and AT1 receptor in myocardium and coronary vessels as identified by immunohistochemistry. Along with this inhibition, curcumin significantly increased protein level of AT2 receptor and its expression compared with the control. As evidenced by less collagen deposition in fibrotic myocardium, curcumin also reduced the extent of collagen-rich scar and increased mass of viable myocardium detected by Masson׳s trichrome staining. Echocardiography showed that the wall thickness of the infarcted anterior septum in the curcumin group was significantly greater than that in the control group. Cardiac contractile function was improved in the curcumin treated animals as measured by fraction shortening and ejection fraction. In cultured cardiac muscle cells, curcumin inhibited oxidant-induced AT1 receptor expression and promoted cell survival. These results suggest that curcumin attenuates maladaptive cardiac repair and enhances cardiac function, primarily mediated by a dual ACE-inhibition and AT1 receptor antagonism after myocardial infarction.