BACKGROUND:Chronic binge alcohol (CBA) administration exacerbates skeletal muscle (SKM) wasting at the terminal stage of simian immunodeficiency virus (SIV) infection in rhesus macaques. This is associated with a pro-inflammatory and oxidative milieu which we have previously shown to be associated with a disrupted balance between anabolic and catabolic mechanisms. In this study, we attempted to characterize the SKM gene expression signature in CBA-administered SIV-infected macaques, using the same animals from the previous study.METHODS:Administration of intragastric alcohol or sucrose to male rhesus macaques began 3 months prior to SIV infection and continued throughout the duration of study. Gene transcriptomes of SKM excised at necropsy (~10 months post-SIV) from healthy na\xEFve control (Control), sucrose-administered, SIV-infected (SUC-SIV), and CBA-administered, SIV-infected (CBA-SIV) macaques were evaluated in microarray data sets. The Protein Analysis Through Evolutionary Relationships classification tool was used to filter differentially regulated genes based on their predicted function into select biological processes relevant to SKM wasting which were inflammation, extracellular matrix (ECM) remodeling, and metabolism.RESULTS:In total, 1,124 genes were differentially regulated between SUC-SIV and Controls, 2,022 genes were differentially expressed between the CBA-SIV and Controls, and 836 genes were differentially expressed between CBA-SIV and SUC-SIV animals. The relevance of altered gene expression was reflected in the up-regulation of pro-inflammatory CCL-2, CCL-8, CX3CL1, SELE, HP, and TNFRS10A mRNA expression. In addition, ECM remodeling was reflected in the up-regulation of TIMP-1, MMP 2, and MMP 9 mRNA expression and transforming growth factor-beta 1 protein expression. In addition, hydroxyproline content and picrosirius staining reflected increased collagen deposition in the CBA-SIV muscle tissue.CONCLUSIONS:The results of the study demonstrate SKM inflammation as an important underlying mechanism for muscle wasting. In addition, the study provides evidence of SKM fibrotic transformation as a factor in CBA-induced accentuation of SIV-associated muscle wasting.

Objective—We have previously shown that transient coronary artery occlusion stimulated coronary collateral growth (CCG) in healthy (Sprague Dawley) but not in metabolic syndrome (JCR:LA-cp [JCR] ) rats. Here, we sought to determine whether matrix metalloproteinases (MMPs) negatively regulate CCG in the metabolic syndrome via release of endostatin and angiostatin. Approach and Results—Rats underwent transient, repetitive left anterior descending occlusion and resultant myocardial ischemia (RI) for 0 to 10 days. CCG was measured in the collateral-dependent and normal zones using microspheres, MMP activation by Western blot, and endostatin and angiostatin by ELISA on days 0, 3, 6, 9, or 10 of RI. Endostatin and angiostatin were increased in JCR but not in Sprague Dawley rats on days 6 and 9 of RI. Increased endostatin and angiostatin correlated with increased MMP12 (≈4-fold) activation in JCR but not in Sprague Dawley rats on days 6 and 9 of RI. Inhibition of MMP12 in JCR rats nearly completely blocked endostatin (≈85%) and angiostatin (≈90%) generation and significantly improved CCG (collateral-dependent zone flow was ≈66% of normal zone flow versus ≈12% for JCR RI). Conclusions—Compromised CCG in the metabolic syndrome is, in large part, because of increased MMP12 activation and consequent increased generation of endostatin and angiostatin, which inhibits late-stage collateral remodeling.

Following percutanous transluminal coronary angioplasty (PTCA) restenosis is markedly increased in metabolic syndrome patients. We hypothesized that increased restenosis in the metabolic syndrome was due to enhanced activation of p38 MAPK (p38) and subsequent p38‐dependent activation of MMP2 and 9. Carotid arteries were injured with a Fogarty 2F catheter in a rat model mimicking the human metabolic syndrome (JCR rat) and normal, healthy controls (SD rats) and neointima formation was quantified. p38 phosphorylation and MMP2 and 9 expression were assessed by immunoblotting and MMP2 and 9 activity by zymography in injured and contralateral control carotids at 4, 7, 10 and 14 days post injury. No basal differences were observed between JCR and SD rats. Maximal increase in p38 activation of similar magnitude occurred at 4 days post injury in both rat phenotypes. p38 activation rapidly returned to baseline in SD but remained sustained in JCR rats. MMP2 and 9 expression and activation mirrored that of p38. Thus, while the absolute magnitude of activation in response to vascular injury is similar, our findings suggest that the sustained activation of p38 and MMP2 and 9 in the metabolic syndrome contribute to increased neointima formation and may provide targets for pharmacological therapies devised for prevention of restenosis following PTCA. Support: NIH HL 084159 and HL 093052

Transient, repetitive ischemia (RI) stimulates coronary collateral growth (CCG) in normal, healthy (SD) rats, which requires p38 MAPK activation. In contrast, RI does not induce CCG in the metabolic syndrome (JCR) rats, which is associated with lack of p38 MAPK activation. The functional consequences of p38 MAPK activation in CCG remain unknown. Theoretically, effective collateral growth would require extracellular matrix remodeling; however, direct assessment as well as identification of proteases responsible for this degradation are lacking. In this study, we investigated the role of p38 MAPK in the regulation of matrix metalloproteinases 2 and 9 (MMPs 2 and 9) and their requirement for CCG in SD vs. JCR rats. The rats underwent the RI protocol (8 LAD occlusions, 40s each, every 20min, in 8h cycles for 0, 3, 6, or 9days). MMP expression was measured in the ischemic, collateral-dependent zone (CZ) and the normal zone (NZ) by Western blot, and MMP activity by zymography. Expression and activation of MMP 2 and 9 were significantly increased (~3.5 fold) on day 3 of RI in the CZ of SD rats. In vivo p38 MAPK inhibition completely blocked RI-induced MMP 2 and 9 expression and activation. MMP activation correlated with increased degradation of components of the basement membrane and the vascular elastic laminae: elastin (~3 fold), laminin (~3 fold) and type IV collagen (~2 fold). This was blocked by MMP 2 and 9 inhibition, which also abolished RI-induced CCG. In contrast, in JCR rats, RI did not induce expression or activation of MMP 2 or 9 and there was no associated degradation of elastin, laminin or type IV collagen. In conclusion, MMP 2 and 9 activation is essential for CCG and is mediated, in part, by p38 MAPK. Furthermore, compromised CCG in the metabolic syndrome may be partially due to the lack of p38 MAPK-dependent activation of MMP 2 and 9 and resultant decreased extracellular matrix degradation.

We have previously demonstrated that Akt was required for repetitive ischemia (RI)-induced coronary collateral growth (CCG) in healthy rats but was not activated by RI in the metabolic syndrome (JCR:LA-cp rats) where CCG was impaired. Here we hypothesized that failure of angiotensin type I receptor (AT₁R) blockers to restore Akt activation is a key determinant of their inability to completely restore CCG in the metabolic syndrome. Therefore, we investigated whether adenovirus-mediated delivery of constitutively active Akt (MyrAkt-Adv) in conjunction with AT₁R blockade (candesartan) was able to restore RI-induced CCG in JCR:LA-cp rats. Successful myocardial MyrAkt-Adv delivery was confirmed by a >80% transduction efficiency and an approximately fourfold increase in Akt expression and activation. CCG was assessed by myocardial blood flow measurements in the normal and collateral-dependent zones. MyrAkt-Adv alone significantly increased RI-induced CCG in JCR:LA-cp rats (~30%), but it completely restored CCG in conjunction with administration of candesartan. In contrast, dominant negative Akt (DN-Akt-Adv) reversed the beneficial effect of candesartan on CCG in JCR:LA-cp rats. We conclude that optimal restoration of coronary collateral growth in JCR:LA-cp rats requires a combination of AT₁R blockade with constitutive Akt activation. These findings may carry implications for metabolic syndrome patients in need of coronary revascularization.