Objective Transient, repetitive occlusion stimulates collateral growth (CCG) in normal animals. Vascular smooth muscle cells (VSMCs) switch to synthetic phenotype early in CCG then return to contractile phenotype. CCG is impaired in the metabolic syndrome. We determined whether impaired CCG was due to aberrant VSMC phenotypic modulation by miR-145-mediated mechanisms and if restoration of physiological miR-145 levels in metabolic syndrome (JCR rat) improved CCG. Approach/Results CCG was stimulated by transient, repetitive LAD occlusion and evaluated after 9 days by coronary blood flow measurements (microspheres). miR-145 was delivered to JCR VSMCs via adenoviral vector (miR-145-Adv). In JCR rats, miR-145 was decreased late in CCG (~2 fold day 6; ~4 fold day 9 vs. SD), which correlated with decreased expression of SM-specific contractile proteins (~5 fold day 6; ~10 fold day 9 vs. SD) indicative of VSMCs’ failure to return to the contractile phenotype late in CCG. miR-145 expression in JCR rats (miR-145-Adv) on days 6–9 of CCG completely restored VSMCs contractile phenotype and CCG (CZ/NZ flow ratio was 0.93±0.09 JCR+miR-145-Adv vs. 0.12±0.02 JCR vs. 0.87±0.02 SD). Conclusions Restoration of VSMC contractile phenotype through miR-145 delivery is a highly promising intervention for restoration of CCG in the metabolic syndrome.

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.

We have recently shown that the inability of repetitive ischemia (RI) to activate p38 MAPK (p38) and Akt in metabolic syndrome [JCR:LA-cp (JCR)] rats was associated with impaired coronary collateral growth (CCG). Furthermore, Akt and p38 activation correlated with optimal O 2 Ϫ ⅐ levels and were altered in JCR rats, and redox-sensitive p38 activation was required for CCG. Here, we determined whether the activation of Src, a possible upstream regulator, was altered in JCR rats and whether redox-dependent Src and Akt activation were required for CCG. CCG was assessed by myocardial blood flow (microspheres) and kinase activation was assessed by Western blot analysis in the normal zone and collateral-dependent zone (CZ). RI induced Src activation (ϳ3-fold) in healthy [Wistar-Kyoto (WKY)] animals but not in JCR animals. Akt inhibition decreased (ϳ50%), and Src inhibition blocked RI-induced CCG in WKY rats. Src inhibition decreased p38 and Akt activation. Myocardial oxidative stress (O 2 Ϫ ⅐ and oxidized/reduced thiols) was measured quantitatively (X-band electron paramagnetic resonance). An antioxidant, apocynin, reduced RI-induced oxidative stress in JCR rats to levels induced by RI in WKY rats versus the reduction in WKY rats to very low levels. This resulted in a significant restoration of p38 (ϳ80%), Akt (ϳ65%), and Src (ϳ90%) activation in JCR rats but decreased the activation in WKY rats (p38: ϳ45%, Akt: ϳ65%, and Src: ϳ100%), correlating with reduced CZ flow in WKY rats (ϳ70%), but significantly restored CZ flow in JCR rats (ϳ75%). We conclude that 1) Akt and Src are required for CCG, 2) Src is a redox-sensitive upstream regulator of RI-induced p38 and Akt activation, and 3) optimal oxidative stress levels are required for RI-induced p38, Akt, and Src activation and CCG. signal transduction; oxidative stress; coronary artery disease; syndrome X CARDIAC ISCHEMIA-REPERFUSION INJURY is a biphasic process where the prolonged reduction in blood flow (ischemia) initiates myocardial cell death followed by further injury upon reperfusion, leading to stunning or necrosis. The massive amounts of ROS released during reperfusion have been implicated as the major cause of myocardial tissue death (25). In contrast, transient repetitive ischemia (RI) renders the myocardium tolerant to ischemia-reperfusion (16,20). The definitive driving force for coronary collateral growth (CCG) is still debated. Ischemia is a well-accepted stimulus for angiogenesis, but elevated fluid shear stress, resulting from occlusion-induced pressure gradients across the coronary circulation, has been proposed to drive collateral remodeling (13,27,34,35). None of these studies have definitively excluded ischemia as the contributing factor to collateral growth. The suggestion that ischemia is not a driving force for CCG rests on a single study (24) in a model of canine coronary occlusion, which observed only epicardial vascular growth while the subendocardium became ischemic.Several studies now support the view that RI is an important driving force for...

Inadequate cell proliferation is considered a major causative factor for impaired coronary collateral growth (CCG). Proangiogenic growth factors (GFs) stimulate cell proliferation, but their administration does not promote CCG in patients. These GFs are increased in patients with metabolic syndrome and in animal models, where CCG is impaired. Here, we investigated whether excessive cell proliferation underlies impaired CCG in metabolic syndrome. Normal [Sprague-Dawley (SD)] and metabolic syndrome [James C. Russell (JCR)] rats underwent repetitive ischemia (RI; transient, repetitive coronary artery occlusion and myocardial ischemia). We have shown that CCG was maximal at d 9 of RI in SD rats but did not occur in JCR rats. The increase in cell proliferation (PCNA, Ki-67, cyclin A, phospho- cdc2, p21Waf, p27Kip) was transient (∼4-fold, d 3 RI) in SD rats but greater and sustained in JCR rats (∼8- to 6-fold, d 3-9 RI). In JCR rats, this was associated with increased and sustained miR-21 expression and accumulation of proliferating synthetic vascular smooth muscle cells in the lumen of small arterioles, which failed to undergo outward expansion. Administration of anti-miR-21 blocked RI-induced cell proliferation and significantly improved CCG in JCR rats (∼60%). miR-21-dependent excessive cell proliferation in the later stages of collateral remodeling correlates with impaired CCG in metabolic syndrome.