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, 40 sec each, every 20 min, in 8 hr cycles for 0, 3, 6, or 9 days). 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.

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 three months prior to SIV infection and continued throughout the duration of study. Gene transcriptomes of SKM excised at necropsy (~10 mo. post-SIV) from healthy naive control (Control), sucrose-administered, SIV-infected (SUC-SIV), and CBA-administered, SIV-infected (CBA-SIV) macaques were evaluated in microarray datasets. The Protein Analysis Through Evolutionary Relationships (PANTHER) 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, 1124 genes were differentially regulated between SUC-SIV and controls, 2022 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, MMP2 and MMP9 mRNA expression and TGF-β 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 (SD) but not in metabolic syndrome (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/Results Rats underwent transient, repetitive LAD occlusion (RI) for 0-10 days. CCG was measured in the collateral-dependent (CZ) and normal (NZ) 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 SD rats on days 6 and 9 of RI. Increased endostatin and angiostatin correlated with increased MMP12 (∼4 fold) activation in JCR but not in SD 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 (CZ flow was ∼66% of NZ flow vs. ∼12% for JCR RI). Conclusions Compromised CCG in the metabolic syndrome is in large part due to increased MMP12 activation and consequent increased generation of endostatin and angiostatin, which inhibits late-stage collateral remodeling.

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.