Abstract 17949: Recruitment of Pro-Inflammatory Ly6ChighCCR2+ Monocytes to the Myocardium is Critical to the Development of Hypertension Related Myocardial Fibrosis

Background: Monocytes/macrophages (Mφ) are key regulators of myocardial inflammation and fibrosis. It is known that Ly6C high CCR2 + monocytes are the primary infiltrating cell in hypertension-dependent myocardial fibrosis, with recent evidence identifying the spleen as an additional source. Changes in monocyte/Mφ subsets between spleen, circulation, and heart in hypertension-dependent myocardial fibrosis remains poorly understood. Here, we characterized this interrelationship in a well-described model of hypertension. Method: C57BL/6 mice were infused with saline or Angiotensin-II (Ang-II; 2.8 mg/kg/d) via osmotic mini-pumps for 3d, after which tissues and blood were harvested and purified over a Ficoll gradient for mononuclear cell isolation, then counted and stained (CD11b, Ly6C, CCR2, CX 3 CR1) for flow cytometry. Results: We identified a CX 3 CR1 + CCR2 + Mφ population that are Ly6C low in control hearts, suggestive of resident cardiac Mφ (210.6 ± 30.2 cells/mg). Ang-II exposure led to an influx of CX 3 CR1 + CCR2 + Mφ (1035 ± 197.3 cells/mg, p ≤ 0.01) with a phenotypic shift to pro-inflammatory CD11b high Ly6C high (1676 ± 343.0 vs. 617 ± 61.9 Ly6C MFI, p ≤ 0.05). In the Ang-II spleen, a 2.7-fold reduction was observed in isolated cells compared to control (7.9x10 7 ± 1.6x10 7 vs. 2.9x10 7 ± 6.1x10 6 cells/tissue p ≤ 0.05). We observed two subpopulations of splenic CD11b + monocytes – (1) CD11b high , primarily Ly6C high CCR2 + , and (2) CD11b low , primarily Ly6C low CCR2 low . The ratio of CD11b low Ly6C low CCR2 low to CD11b high Ly6C high CCR2 + cells was reduced in the Ang-II spleen compared to controls (3.7 ± 1.4 vs 8.9 ± 1.1, p ≤ 0.05), suggesting a pro-inflammatory phenotypic shift. No significant differences in circulating CD11b + subpopulations were seen at 3d, suggesting prior splenic deployment and recovery from bone marrow/spleen. Conclusion: We provide evidence that a significant influx of cardiac Mφ in the context of hypertension arise from splenic reservoir. Furthermore, we demonstrate significant changes in myocardial Mφ composition towards pro-inflammatory Ly6C high CCR2 + Mφ at the expense of immunomodulatory Ly6C low Mφ. This offers novel insight into therapies that would prevent the recruitment of splenic monocytes to control myocardial inflammation.