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.

Effects of long-term rapamycin treatment on glial scar formation after cryogenic traumatic brain injury in mice.

Glial scar impedes axon regeneration and functional recovery following traumatic brain injury (TBI). Although it has been shown that rapamycin (a specific inhibitor of mammalian target of rapamycin) can reduce astrocyte reactivation in the early stage of TBI, its effect on glial scar formation has not been characterized in TBI and other acute brain injury models. To test this, ICR mice received daily administration of rapamycin (0.5 or 1.5 mg/kg, i.p.) beginning at 1 h after cryogenic TBI (cTBI). The results showed that at 3 d post-injury, 1.5 mg/kg rapamycin increased cTBI-induced motor functional deficits and infarct size, and attenuated astrocyte reactivation in the ipsilateral cortex, while 0.5 mg/kg rapamycin did not worsen brain damage and only slightly attenuated astrocyte reactivation. Furthermore, at 7 and 14 d after cTBI, 0.5 mg/kg rapamycin group showed a better motor functional performance than cTBI group. At 14 d post-injury, 0.5 mg/kg rapamycin significantly reduced the area and thickness of glial scar and chondroitin sulfate proteoglycan expression, accompanied by decreased expression of p-S6 and enhanced expression of growth associated protein 43 (an axon regeneration marker) in the region of glial scar. Our data suggest that long-term treatment with rapamycin can inhibit glial scar formation after cTBI, which may be involved in the mechanisms of increased axon regeneration and improved neurological functional recovery, and low-dose rapamycin may be more beneficial for such a therapy.

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.

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.

Attenuation of inflammatory response and reduction in infarct size by postconditioning are associated with downregulation of early growth response 1 during reperfusion in rat heart.

Early growth response 1 (EGR-1) works as a master regulator that plays a key role in triggering inflammation-induced tissue injury after ischemia and reperfusion. This study tested the hypothesis that postconditioning (Postcon) or anti-inflammatory compound, curcumin, ameliorates inflammatory responses and further reduces infarct size by normalizing EGR-1 expression during reperfusion. In the control group, male Sprague-Dawley rats were subjected to 30-min ischemia and 180-min reperfusion. Postcon with four cycles of 10-s/10-s reperfusion/ischemia was applied at the onset of reperfusion. Curcumin (150 mg/kg per day) was fed 5 days before ischemia. Relative to the control, Postcon reduced expression of EGR-1 mRNA and protein, as further identified by less EGR-1 immunoreactivity in myocardial nuclei and microvessels during reperfusion. Along with EGR-1 downregulation, levels of plasma and myocardial tumor necrosis factor α and interleukin 6 (IL-6) were significantly decreased. Upregulated P-selectin and intercellular adhesion molecule 1 mRNA and protein as well as their immunoreactivity at area at risk myocardium were significantly attenuated. Neutrophil extravasation identified by myeloperoxidase immunohistochemical staining was inhibited. Infarct size, determined with triphenyltetrazolium chloride staining, was smaller in the Postcon group than that in the control. The protection achieved with pretreatment with curcumin was comparable to the benefits gained by Postcon in all end points measured. In H9C2 rat cardiomyoblast cell line, EGR-1 siRNA downregulated hydrogen peroxide-induced EGR-1 mRNA expression and subsequently reduced tumor necrosis factor α mRNA level. These results suggest that EGR-1 seems to play a critical role in myocardial reperfusion injury because downregulation of EGR-1 either by Postcon or the use of pharmacological intervention reduces infarct size, most likely through an inhibition of inflammation-mediated processes.