ABSTRACT
OBJECTIVE: The immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. Here, we studied the role of lymphocytes in a murine model of hindlimb ischemia. METHODS AND RESULTS: Lymphocytes, detected with markers for NK1.1, CD3, and CD4, invaded the collateral vessel wall. Arteriogenesis was impaired in C57BL/6 mice depleted for Natural Killer (NK)-cells by anti-NK1.1 antibodies and in NK-cell-deficient transgenic mice. Arteriogenesis was, however, unaffected in J alpha281-knockout mice that lack NK1.1+ Natural Killer T (NKT)-cells, indicating that NK-cells, rather than NKT-cells, are involved in arteriogenesis. Furthermore, arteriogenesis was impaired in C57BL/6 mice depleted for CD4+ T-lymphocytes by anti-CD4 antibodies, and in major histocompatibility complex (MHC)-class-II-deficient mice that more selectively lack mature peripheral CD4+ T-lymphocytes. This impairment was even more profound in anti-NK1.1-treated MHC-class-II-deficient mice that lack both NK- and CD4+ T-lymphocytes. Finally, collateral growth was severely reduced in BALB/c as compared with C57BL/6 mice, 2 strains with different bias in immune responsiveness. CONCLUSIONS: These data show that both NK-cells and CD4+ T-cells modulate arteriogenesis. Promoting lymphocyte activation may represent a promising method to treat ischemic disease.
Subject(s)
Arterial Occlusive Diseases/immunology , CD4-Positive T-Lymphocytes/immunology , Collateral Circulation/immunology , Killer Cells, Natural/immunology , Neovascularization, Physiologic/immunology , Animals , Arterial Occlusive Diseases/physiopathology , CD4-Positive T-Lymphocytes/metabolism , Disease Models, Animal , Femoral Artery/growth & development , Hindlimb/blood supply , Ischemia , Killer Cells, Natural/metabolism , Mice , Mice, Inbred BALB C , Mice, KnockoutABSTRACT
In previous work we have demonstrated increased expression of NOX2 in cardiomyocytes of infarcted human hearts. In the present manuscript we investigated the functional role of NOX2 in ischemically challenged H9c2 cells, a rat cardiomyoblast cell line, and adult rat cardiomyocytes. Expression of NOX2 in H9c2 cells was confirmed by RT-PCR. In Western-blot experiments, increased NOX2 expression was detected during ischemia, which was inhibited by transcription and translation inhibitors. Surprisingly, under ischemia, in addition to an increased cytosolic expression, NOX2 was localized mainly in the nucleus of apoptotic cardiomyocytes, where it colocalized with nitrotyrosine residues and activated caspase 3. Inhibition of reactive-oxygen-species generation with the flavoenzyme inhibitor diphenylene iodonium (DPI) and the NADPH-oxidase inhibitor apocynin led to a significantly decreased induction of apoptosis as assessed by quantification of caspase-3 activity and by TUNEL analysis. These results demonstrate that NOX2 is expressed in the nucleus of cardiomyocytes during apoptosis and that it likely participates in proapoptotic signaling. To the best of our knowledge, this is the first demonstration of nuclear NOX2 expression and its involvement in cardiomyocyte apoptosis.
