Ang-1 Reduces Myocardial Infarction in db/db Mice Figure 8. Overexpression of Ang-1 improves cardiac functional recovery of post-MI in db/db mice. A. Cardiac output (CO) was significantly improved in the db/db mice treated with Ad-Ang-1 (n = 5) compare to the Ad-b-gal treated db/db mice (n = 4) at 14 days of ischemia. Sham control (n = 4). All data represent mean 6 SD; *p,0.05. B. Myocardial ejection fraction (EF%) was significantly increased in the db/db mice treated with Ad-Ang-1 (n = 5) compare to the Ad-b-gal treated db/db mice (n = 4) at 14 days of ischemia. Sham control (n = 4). All data represent mean 6 SD; *p,0.05. doi:10.1371/journal.pone.0035905.g008 CD133+ and c-kit+ stem cell recruitment, and enhancing angiogenesis via a mechanism involving SDF-1a/CXCR-4 signaling. Increasing evidence reveals that Ang-1 has a burgeoning role in heart disease and possesses cardioprotective effects against myocardial ischemia [18],[19]. Previously, we showed that over- expression of Ang-1 increases myocardial angiogenesis and decreases myocardial infarction in STZ mouse model, indicating a potential link between angiogenesis and reduction of MI in diabetes [12]. The present studies further reveal that over- expression of Ang-1 in db/db mouse hearts results in a significant reduction in myocardial apoptosis as well as endothelial cell apoptosis. This was accompanied by significant increase in capillary density and cardiac functional recovery. Although overexpression of Ang-1 alleviates myocardial ischemic injury in the STZ and db/db diabetic animal models, [4,12] the underlying mechanisms by which overexpression of Ang-1 attenuates myocardial apoptosis and promotes cardiac repair are not completely understood. Previous studies showed that Tie-2 expression was detected in hematopoietic stem cell and Tie-2 signaling was crucial for the maintenance of the hematopoietic microenvironment. Ang-1 actives Tie-2 on hematopoietic stem cell in the niche, maintains the in vivo repopulation ability of hematopoietic stem cell.[13] Recent evidence suggests that hematopoietic stem cell CD133+ cells are involved in healing the ischemic myocardium and thus may be important in functional recovery of the myocardium following acute infarction (AMI) [20–22]. In non-diabetic patients with AMI, there is often a surge of CD133+ cells in the circulation. However, in diabetic patients experiencing AMI, CD133+ cell recruitment and function was significantly impaired [20]. CD133+ cells have been reported to release large amounts of VEGF, which activates PI3K/Akt and exerts pro-angiogenic and pro-survival effects. Transplantation of a low number of CD133+ human fetal aorta-derived vascular progenitor cells promotes reparative neovascularization and skeletal myocyte regeneration in a non- diabetic ischemic hind-limb model [22]. Intriguingly, local therapy with CD133+ cells promotes angiogenesis and significantly accelerates healing of ischemic hind-limb skin wounds in a diabetes mellitus model [21]. To our knowledge, the present study is the first demonstration showing that overexpression of Ang-1 significantly increases the recruitment of CD133+ and c-kit+ into myocardial ischemic areas in diabetic db/db mice. Previously, we have shown that overexpression of Ang-1 significantly increases myocardial VEGF, Akt and eNOS expression in db/db mice [4]. Based upon these findings, we postulate that Ang-1 promotes CD133+ cell and c-kit+ recruitment into ischemic areas, leading to secretion of VEGF and activation of the Akt/eNOS signaling pathway, which exert beneficial effects on the diabetic infarcted hearts. This notion is supported by our findings that CXCR-4 and SDF-1a, the chemokine for hematopoietic progenitor cells, were significantly increased within the ischemic myocardium of Ang-1 treated db/db mouse. Furthermore, our data reveal that the increased CD133+ co-localize with CXCR-4+ and SDF-1a + cells in the area of myocardial ischemia. In addition, SDF-1a/CXCR-4 has been shown to protect the hearts after myocardial infarction [14,23,24]. These findings suggest that activation of SDF-1a/ CXCR-4 signaling mediates Ang-1-induced recruitment of CD133+ progenitor cells into the infarcted ischemic heart leading to attenuation of myocardial apoptosis. In addition, our present study shows that Ang-1 protects myocardial endothelial cell against ischemia-induced endothelial apoptosis. Our previous study showed that Ang-1 increases myocardial angiogenesis via activation of Tie-2/Akt/eNOS pathway in coronary endothelial cells [25]. These direct protective effects of Ang-1 on endothelial cells may also contribute to the attenuation of myocardial ischemic injury and increase in myocardial angiogenesis and cardiac function in diabetes. Ang-1/Tie-2 signaling plays a predominant role in controlling both VSMC and pericyte maturation and is essential for the maintenance of vascular stabilization [1,26]. We have demon- strated that overexpression of Ang-1 increases VSMC recruitment and promotes mature neovessel formation; this is accompanied by a significant increase in capillary density in db/db mouse hearts [4]. However, the intracellular molecular mechanisms by which Ang-1 increases VSMC recruitment and maturation remains unexplored. The Notch ligand Jagged1 is essential for vascular remodeling and has been linked to congenital heart failure in humans [27]. Jagged1 plays a critical role in the regulation of VSMC recruitment/maturation via Notch3 during early embry- onic development [28]. Deficiency of Notch3 has been shown to disrupt VSMC differentiation and to increase infarct size in ischemic stroke [29–31]. Recent studies reveal that ethanol and cyclic strain stimulate endothelial cell angiogenic activity via a Notch/Ang-1/Tie-2 pathway suggesting potential cross-talk between Notch signaling and the Ang-1/Tie-2 pathway [32,33]. The present data show that overexpression of Ang-1 significantly increased Jagged 1 and Notch3 expression in the db/db mouse PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e35905