Our data establish, for the first time, that Ang-1 plays a critical role in the recruitment of hematopoietic stem cell CD133+/c-kit+ and CD45+ cells into ischemic areas of the diabetic infarcted heart. This is accompanied with a significant reduction in myocardial/endothelial apoptosis and cardiac hypertrophy as well as fibrosis formation in diabetic heart. Our study also demonstrates that upregulation of SDF-1α/CXCR-4 expression in the diabetic infarcted hearts is a novel downstream signaling pathway of Ang-1-mediated recruitment of CD133+ cells. Further, our studies reveal that overexpression of Ang-1 results in increases in capillary density and arteriole formation together with a dramatic improvement of cardiac functional recovery in the diabetic infarcted hearts. These studies strongly suggest that Ang-1 gene therapy protects the diabetic heart from ischemic injury by promoting CD133+ and c-kit+ stem cell recruitment, and enhancing angiogenesis via a mechanism involving SDF-1α/CXCR-4 signaling.
Increasing evidence reveals that Ang-1 has a burgeoning role in heart disease and possesses cardioprotective effects against myocardial ischemia ,
. Previously, we showed that overexpression 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 
. The present studies further reveal that overexpression 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, 
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.
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) 
. 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 
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 
. Intriguingly, local therapy with CD133+
cells promotes angiogenesis and significantly accelerates healing of ischemic hind-limb skin wounds in a diabetes mellitus model 
. To our knowledge, the present study is the first demonstration showing that overexpression of Ang-1 significantly increases the recruitment of CD133+
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 
. 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-1α, 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-1α +
cells in the area of myocardial ischemia. In addition, SDF-1α/CXCR-4 has been shown to protect the hearts after myocardial infarction 
. These findings suggest that activation of SDF-1α/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 
. 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 
. We have demonstrated 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 
. 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 
. Jagged1 plays a critical role in the regulation of VSMC recruitment/maturation via Notch3 during early embryonic development 
. Deficiency of Notch3 has been shown to disrupt VSMC differentiation and to increase infarct size in ischemic stroke 
. 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 
. The present data show that overexpression of Ang-1 significantly increased Jagged 1 and Notch3 expression in the db/db mouse hearts. Immunohistochemical analysis further confirmed that the Notch3 expression was localized to the wall of larger arterioles. Apelin is an endogenous ligand of the human orphan G-protein-coupled receptor APJ 
. Apelin exerts a variety of cardiovascular effects and particularly acts as an activator of angiogenesis 
. A recent study also reported that the apelin/APJ system was involved in the regulation of blood vessel diameter during angiogenesis 
. Our present data also demonstrated that treatment with Ad-Ang-1 significantly increase apelin expression. These data suggest potential role of Jagged1/Notch3 and apelin in Ang-1-mediated angiogenesis. Most intriguingly, overexpression of Ang-1 increases the number of BM-SMA+
cells as well as Ang-1+
cells in the diabetic infarcted hearts, suggesting that Ang-1 over-expressing SMA+
cells in the BM may also be recruited to the ischemic myocardium rather than their homing to Ang-1+
cells in the heart itself.
cells represent a rich source of hematopoietic stem cell cytokines in the ischemic heart.
Our data showed that CD45+
cells were dramatically increased in diabetic infarcted hearts after Ad-Ang-1 treatment. Furthermore, the abundance Tie-2-expressing CD45+
cell was increased implying that these cells may contribute to the Ang-1-mediated angiogenic response in diabetic infarcted hearts. Our data also demonstrate that overexpression of Ang-1 significantly increased BM-derived CD31+
cells and upregulated VEGF expression in BM. Recent studies showed that BM-CD31+
cells were enriched with remarkably higher levels of angiogenic and hematopoietic genes as compared to BM-CD31−
cells. Furthermore, BM-CD31+
cells induced the activation of angiogenic, anti-apoptotic and chemo-attractant factors in ischemic hind-limbs via a paracrine effect. Transplantation with BM-CD31+
cells significantly increased angiogenesis and improved recovery from ischemia 42
. Jagged1 has been shown to drive immature BM progenitor cells to differentiate into functional EPCs 
. Jagged1 and Notch3 were increased during the differentiation of BM-derived mononuclear cells into VSMC and EC 
. Further studies are needed to elucidate the molecular mechanisms and crosstalk between the Jagged1/Notch3 and Ang-1/Tie-2 pathways in the regulation of BM/stem cell recruitment/differentiation into VSMC/EC and vascular maturation.
The present study demonstrates that following myocardial ischemia, Ang-1 gene therapy promotes hematopoietic stem cell recruitment. These changes are associated with an increase in CXCR-4/SDF-1α expression and myocardial angiogenesis and with a reduction in cardiac hypertrophy and improvement of cardiac functional recovery after ischemia. Given the roles of Ang-1/Tie-2 signaling in hematopoietic stem cell development as well as EC/VSMCs differentiation, our data provide novel insights into the intracellular pathways that regulate the formation of mature vasculature in the diabetic infarcted heart and mechanisms that promote cardiac repair. Our studies may aid in the development of novel therapeutic avenues to ameliorate the impaired stem cell function and insufficient angiogenesis that are characteristic of the diabetic state.