miR-21-mediated decreased neutrophil apoptosis is a determinant of impaired coronary collateral growth in metabolic syndrome.

Coronary collateral growth (CCG) is impaired in metabolic syndrome. microRNA-21 (miR-21) is a proproliferative and antiapoptotic miR, which we showed to be elevated in metabolic syndrome. Here we investigate whether impaired CCG in metabolic syndrome involved miR-21-mediated aberrant apoptosis. Normal Sprague-Dawley (SD) and metabolic syndrome [J. C. Russel (JCR)] rats underwent transient, repetitive coronary artery occlusion [repetitive ischemia (RI)]. Antiapoptotic Bcl-2, phospho-Bad, and Bcl-2/Bax dimers were increased on days 6 and 9 RI, and proapoptotic Bax and Bax/Bax dimers and cytochrome-c release concurrently decreased in JCR versus SD rats. Active caspases were decreased in JCR versus SD rats (~50%). Neutrophils increased transiently on day 3 RI in the collateral-dependent zone of SD rats but remained elevated in JCR rats, paralleling miR-21 expression. miR-21 downregulation by anti-miR-21 induced neutrophil apoptosis and decreased Bcl-2 and Bcl-2/Bax dimers (~75%) while increasing Bax/Bax dimers, cytochrome-c release, and caspase activation (~70, 400, and 400%). Anti-miR-21 also improved CCG in JCR rats (~60%). Preventing neutrophil infiltration with blocking antibodies resulted in equivalent CCG recovery, confirming a major role for deregulated neutrophil apoptosis in CCG impairment. Neutrophil and miR-21-dependent CCG inhibition was in significant part mediated by increased oxidative stress. We conclude that neutrophil apoptosis is integral to normal CCG and that inappropriate prolonged miR-21-mediated survival of neutrophils plays a major role in impaired CCG, in part via oxidative stress generation.

miR-21 normalizes vascular smooth muscle proliferation and improves coronary collateral growth in metabolic syndrome.

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.

MMPs 2 and 9 are essential for coronary collateral growth and are prominently regulated by p38 MAPK.

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.

Slingshot isoform-specific regulation of cofilin-mediated vascular smooth muscle cell migration and neointima formation.

OBJECTIVE: We hypothesized that cofilin activation by members of the slingshot (SSH) phosphatase family is a key mechanism regulating vascular smooth muscle cell (VSMC) migration and neoinitima formation following vascular injury. METHODS AND RESULTS: Scratch wound and modified Boyden chamber assays were used to assess VSMC migration following downregulation of the expression of cofilin and each SSH phosphatase isoform (SSH1, SSH2, and SSH3) by small interfering RNA (siRNA), respectively. Cofilin siRNA greatly attenuated the ability of VSMC migration into the "wound," and platelet-derived growth factor (PDGF)-induced migration was virtually eliminated versus a 3.5-fold increase in nontreated VSMCs, establishing a critical role for cofilin in VSMC migration. Cofilin activation (dephosphorylation) was increased in PDGF-stimulated VSMCs. Thus, we assessed the role of the SSH family of phosphatases on cofilin activation and VSMC migration. Treatment with either SSH1 or SSH2 siRNA attenuated cofilin activation, whereas SSH3 siRNA had no effect. Only SSH1 siRNA significantly reduced wound healing and PDGF-induced VSMC migration. Both SSH1 expression (4.7-fold) and cofilin expression (3.9-fold) were increased in balloon injured versus noninjured carotid arteries, and expression was prevalent in the neointima. CONCLUSION: These studies demonstrate that the regulation of VSMC migration by cofilin is SSH1 dependent and that this mechanism potentially contributes to neointima formation following vascular injury in vivo.

Angiotensin type I receptor blockade in conjunction with enhanced Akt activation restores coronary collateral growth in the metabolic syndrome.

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.

Physiological status of male and female Popillia japonica (Coleoptera: Scarabaeidae) affects mating and grouping behavior.

Because mating may be costly, sexually active males or females are predicted to be in relatively good physiological condition and may preferentially direct their mating behavior toward relatively high-quality mates. We tested this hypothesis in Japanese beetles (Popillia japonica Newman), a pest species in which males and females may be either isolated or in aggregations while feeding on host plants. We examined male size and lipid content and female size and egg load with respect to both their pairing status and whether they were isolated or in aggregations. Males that were paired had the highest lipid levels, and single, isolated males had the lowest. Paired females had the highest egg loads and single, isolated females had the lowest. Neither male nor female size was related to pairing status. Females captured during the times of relatively high pairing frequency (i.e., morning and evening) had higher egg loads than females captured at times of lower pairing frequency (i.e., afternoon). These results suggest that mating and aggregative behaviors in Japanese beetles are dependent on the physiological status of males and the reproductive condition of females.

High-performance thin-layer chromatographic determination of rabeprazole sodium and domperidone in combined dosage form.

A new, simple, high-performance thin-layer chromatographic method for determination of rabeprazole sodium (RAB) and domperidone (DOM) in combined tablet dosage form has been developed and validated. The mobile phase was toluene-acetone-methanol (4.5 + 4.5 + 0.5, v/v/v) with UV detection at 285 nm. The retention factors for RAB and DOM were found to be 0.53 +/- 0.12 and 0.32 +/- 0.20. The method was validated with respect to linearity, accuracy, precision, and robustness. Beer's law was obeyed in the concentration range of 50-800 ng/band for both RAB and DOM. The method has been successfully applied for the analysis of drugs in a pharmaceutical formulation.

Redox-sensitive Akt and Src regulate coronary collateral growth in metabolic syndrome.

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)(-). 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 (approximately 3-fold) in healthy [Wistar-Kyoto (WKY)] animals but not in JCR animals. Akt inhibition decreased (approximately 50%), and Src inhibition blocked RI-induced CCG in WKY rats. Src inhibition decreased p38 and Akt activation. Myocardial oxidative stress (O(2)(-). 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 (approximately 80%), Akt (approximately 65%), and Src (approximately 90%) activation in JCR rats but decreased the activation in WKY rats (p38: approximately 45%, Akt: approximately 65%, and Src: approximately 100%), correlating with reduced CZ flow in WKY rats (approximately 70%), but significantly restored CZ flow in JCR rats (approximately 75%). 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.