Administration of Low-Dose Cyclophosphamide Reduces Progression of Atherosclerosis in Mice by Controlling the Cellular Microenvironment.

Atherosclerosis develops as a result of multiple inflammatory-fibroproliferative responses and is the primary cause of heart disease and stroke in Western countries. Circulating BM-derived progenitor cells (CFU-Cs or EPCs) can regenerate injured vasculature by accelerating reendothelialization and limiting atherosclerotic lesion formation. Risk factors for coronary artery disease like age and diabetes reduce the number and functional activity of these cells, thus limiting the regenerative capacity. The impairment of stem/progenitor cells by risk factors may contribute to atherosclerotic disease progression. High leukocyte counts, and especially neutrophils, a marker of inflammation have been shown to be an independent risk factor and prognostic indicator of future cardiovascular outcomes. Cyclophosphamide (CY) is a synthetic antineoplastic drug, which reduces white blood cell counts (WBC), especially neutrophils, and under certain circumstances can promote hematopoietic progenitor (colony forming unit-cells, CFU-C) mobilization from the bone marrow (BM) into the circulation. We hypothesized that administration of CY limits atherosclerotic lesion formation by decreasing inflammation-associated cells, and by increasing circulating BM-derived progenitors cells. Apolipoprotein E knockout (ApoE−/ −) mice were fed a high cholesterol diet and received different doses of CY in their drinking water (37.5mg/kg/day, 18.75mg/kg/day, 9.375mg/kg/day or nothing). To control for possible effects on progenitor release a control experiment was set up in which C57/B6 received the highest CY in their drinking water (37.5mg/kg/day) or remained untreated. Peripheral blood was drawn from mice every other week. Peripheral blood mononuclear cells (PBMCs) were isolated and subjected to cultures to detect EPCs and CFU-Cs. In CY-treated mice, WBC and neutrophil counts decreased within the first 10 days after the start of the treatment as compared to non-treated controls, and stayed low over the course of the experiment (until day 70). When apoE mice receiving a high-cholesterol diet were treated with CY, the number of circulating CFU-Cs doubled, whereas no major change was found in the c57Bl/6 control group receiving only Cy. EPCs were not detectable at any time point in the CY or control apoE group. Aortic atherosclerotic tree lesion areas were approximately 50% smaller in apoE-KO mice receiving CY in their drinking water after 12 weeks on a high-cholesterol diet than control animals. Most strikingly, apoE-KO mice treated with CY showed a prolonged survival rate as compared to untreated controls. The presence of macrophage-derived foam cells is a hallmark of the atherosclerotic lesion, and an increase in their content promotes plaque instability. Immunohistochemical analysis revealed that the number of macrophages was reduced in plaques of CY-treated animals, indicating that plaques in these might be more stable. Vascular endothelial growth factor (VEGF) is expressed in atherosclerotic plaques from local macrophages and can induce metalloproteinase-9 (MMP-9). Plasma VEGF levels were lower in the CY-treatment group, coinciding with a decrease in MMP-9 plasma levels. This study demonstrates the potential clinical use of a myelosuppressive agent for the treatment of a benign, but not less “malignant” deadly disease like atherosclerosis.