Tracking of SDF-1+ and CXCR4+ cells in the ischemic heart utilizing novel (BAC)-EGFP reporter mice

Abstract

Objective: Recently published data from our lab indicate that stabilization of the cardiac SDF-1/CXCR4 axis preserves myocardial function and attenuates ischemic cardiomyopathy. However, the basic mechanisms of SDF-1/CXCR4 mediated cellular repair are barely understood. Here, we aimed to track the fate of SDF-1+ and CXCR4+ cell populations in the bone marrow, peripheral blood and the heart under normoxaemic and ischemic conditions utilizing novel SDF-1 and CXCR4 (BAC)-EGFP reporter mice. Methods: In order to track the fate of SDF-1+ and CXCR4+ cells, genetically tagged EGFP BAC reporter mice were used. These mice carry an EGFP sequence downstream of the translational (ATG) start side of a corresponding ca. 150-200kb large bacterial artificial chromosome (BAC), including all regulatory sequences of the SDF-1 and CXCR4 promoter. FACS and immunhistochemical analyses of SDF-1 and CXCR4-EGFP+ bone marrow (BM), peripheral blood (PB) and heart cells were performed under normoxaemic and ischemic conditions. Results: SDF-1-EGFP+ cells target to vascular structures in coronary arteries. Moreover, SDF-1-EGFP cells seem to participate in a connecting network of vascular and/or perivascular cells within the ischemic heart. FACS analyses of transgenic CXCR4-EGFP BM cells revealed that CXCR4+ was most frequently expressed on CD11b+ monocytes, angiogenic CD31+, CD34+, c-kit+, and Flk1+ cells, as well as stem cell populations like ACC133+ and Lin-/c-kit+/Sca-1+. After ischemia CXCR4+/CD11b+ monocytes cells were upregulated. In the heart CXCR4 expressing CD11b+ monocytes, angiogenic CD31+, CD34+, c-kit+, and Flk1+ cells, as well as stem cell populations like ACC133+, and Lin-/c-kit+/Sca-1+ cells were significantly upregulated after ischemia. Immunofluorescent staining revealed only few CXCR4-EGFP+ cells in non-injured hearts, whereas tissue ischemia strongly increased the number of CXCR4-EGFP+ cells in the heart. Staining of CXCR4-EGFP+ expressing cells with PEACAM (CD31) revealed significant co-expression of a subpopulation of CXCR4+ cells in capillaries after ischemia. In summary, SDF-1 is expressed in smooth muscle cells and perivascular cells in the heart. CXCR4 is highly expressed on angiogenic and on stem cell populations in the BM, PB and the myocardium. In the heart, CXCR4-EGFP+ cells are upregulated after ischemia and target to vascular structures.