Activator Protein-2α Mediates Carbon Monoxide–Induced Stromal Cell–Derived Factor-1α Expression and Vascularization in Ischemic Heart

Abstract

Objective-Increased cardiac stromal cell-derived factor-1α (SDF-1α) expression promotes neovascularization and myocardial repair after ischemic injury through recruiting stem cells and reducing cardiomyocyte death. Previous studies have shown that heme oxygenase-1 and its reaction byproduct, carbon monoxide (CO), induce SDF-1α expression in ischemic heart. However, the mechanism underlying heme oxygenase-1/CO-induced cardiac SDF-1α expression remains elusive. This study aims to investigate the signaling pathway and the transcriptional factor that mediate CO-induced SDF-1α gene expression and cardioprotection. Approach and Results-CO gas and a CO-releasing compound, tricarbonyldichlororuthenium (II) dimer, dosedependently induced SDF-1α expression in primary neonatal cardiomyocytes and H9C2 cardiomyoblasts. Promoter luciferase-reporter assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation demonstrated that the activator protein 2α (AP-2α) mediated tricarbonyldichlororuthenium (II) dimer-induced SDF-1α gene transcription. Tricarbonyldichlororuthenium (II) dimer induced AP-2α expression via protein kinase B (AKT)-dependent signaling.AKT inhibition or AP-2α knockdown reduced tricarbonyldichlororuthenium (II) dimer-induced SDF-1α expression. Coronary ligation induced transient increases of cardiac AP-2α and SDF-1α expression, which were declined at 1 week postinfarction in mice. Periodic exposure of coronary-ligated mice to CO (250 ppm for 1 hour/day, 6 days) resumed the induction of AP-2α and SDF-1α gene expression in infarcted hearts. Immunohistochemistry and echocardiography performed at 4 weeks after coronary ligation revealed that CO treatment enhanced neovascularization in the myocardium of peri-infarct region and improved cardiac function. CO-mediated SDF-1α expression and cardioprotection was ablated by intramyocardial injection of lentivirus bearing specific short hairpin RNA targeting AP-2α. Conclusions-Our data demonstrate that AKT-dependent upregulation of AP-2α is essential for CO-induced SDF-1α expression and myocardial repair after ischemic injury. (Arterioscler Thromb Vasc Biol. 2013;33:785-794.) Key Words: activator protein 2α ◼ carbon monoxide ◼ heme oxygenase-1 ◼ ischemic heart ◼ stromal cell-derived factor-1α ◼ vascularization Activator Protein-2α Mediates Carbon MonoxideInduced Stromal Cell-Derived Factor-1α Expression and Vascularization in Ischemic HeartHeng-Huei Lin, Yen-Hui Chen, Ming-Tsai Chiang, Pei-Ling Huang, Lee-Young Chau Materials and MethodsMaterials and Methods are available in the online-only Supplement. Results HO-1/CO Induces SDF-1α ExpressionTo confirm our previous finding that HO-1 overexpression promotes SDF-1α gene induction in myocardium in vivo, 14 we performed HO-1 gene transduction experiment in primary neonatal cardiomyocytes and H9C2 cardiomyoblasts. As shown in Figure 1A and 1B, when cells were infected with adenovirus bearing HO-1 gene for 48 hours, SDF-1α gene expression examined by quantitative real-time PCR was significantly higher, as comp...

Heme Oxygenase-1 and Carbon Monoxide in the Heart

“…12;164 Work by Lin et al showed that treatment with CO gas or CORM-2 promoted neovascularization and myocardial repair after coronary artery ligation. 125 CO increased the activation of SDF-1 via an Akt-dependent AP-2α expression. Blockade of AP-2α abrogated the beneficial effects of CO treatment.…”

“…HO-1 and its products have not been directly examined in the regeneration of heart vessels after IR but can confer protection against vascular injury and inflammation in models of atherosclerosis and vessel injury 113;123 and significantly contribute to neoangiogenesis and neovascularisation 124;125 (see below). Therefore, we can foresee a multifunctional implication of the HO-1 system in IR: 1) as a sensor of cardiac injury and DAMPs; 2) as a modulator of inflammation and the immune response and; 3) as a mediator that aids the repair and reconstruction of cardiac tissue.…”

Chemokines

“…24 It has been shown that heme oxygenase-1 and its reaction byproduct carbon monoxide can induce CXCL12 expression in the hearts of infarcted mice. 25 A study by Lin et al 26 further elucidated the mechanism behind this carbon monoxide-induced CXCL12 expression and cardioprotection. Applying several in vitro approaches revealed that CXCL12 upregulation by carbon monoxide was mediated by protein kinase B-dependent stimulation of the transcription factor activator protein-2α in cardiomyocytes.…”

Regulation of the HMOX1 gene by the transcription factor AP-2δ with unique DNA binding site

“…Ap-2 proteins have been reported to bind to a highly conserved GC-rich consensus sequence present in numerous cellular and viral promoters and enhancers. An in vitro binding site selection assay identified GCCN3/4GGC and GCCN3/4GGG as the preferred sequence motifs bound by Ap-2 transcription factors (7,8). Two multiple protein-binding sites were identified in the HMOX1 upstream promoter regions, which were HMOX1-1, 5'-agttcctgatgttgcccaccaggctattgctctgagcagc-3' (underlined bases represent similar AP-2-binding sequence) and HMOX1-2, 5'-ctcctctccaccccacactggcccggggcgggctgggcgc-3' (underlined bases represent consensus AP-2-binding sequence).…”

“…The transactivation domains of AP-2 proteins are significantly more divergent, although certain critical residues and a PY motif (XPPXY) are conserved (1)(2)(3)(4)(5)(6). A number of functional AP-2 binding sites, consensus to a palindromic core sequence, 5'-GCCN 3 GGC-3', have been identified in cellular and viral enhancers, and preferred binding to the sequence motifs GCCN 3 GGC, GCCN 4 GGC and GCCN 3/4 GGG was observed in an in vitro binding site selection assay (7,8). The PY motif and critical residues in the transactivation domain, which are considered necessary for transactivation, were divergent in Ap-2δ.…”