Tuesday, April 23, 2024
4/23/2024
Cardiovascular disease (CVD) accounts for more annual deaths in the US than cancer and lung disease combined highlighting the pressing need for novel therapeutic interventions in this setting. Recent success of the CANTOS trial supports the notion that interventions to attenuate CVD-induced inflammation may hold the key to future therapeutics. A body of evidence from us and others have demonstrated that CVD induces emergency hematopoiesis in the bone marrow (BM) inciting the release of leukocytes which travel to infarct or atherosclerotic tissue worsening pathology, a phenomenon that has been described in humans. Our unpublished work found that models of CVD induce a pro-angiogenic phenotype in the BM vasculature which drives leukocyte release into the blood in an incompletely understood process. We observed elevated circulating vascular endothelial growth factor (VEGF) levels in mice with CVD. Interestingly, vascular endothelial growth factor receptor 3 (VEGFR3) is highly expressed in the sinusoidal niche of the marrow, the vascular compartment where hematopoietic cells and leukocytes reside. VEGF-C/VEGFR3 signaling has been reported to regulate transendothelial migration of cells across lymphatic endothelium during cancer-induced inflammation, a process reminiscent of the gate-keeping function of BM sinusoids. My preliminary data examining neutrophil intravasation by time-lapse intravital microscopy in a sepsis-like model of BM inflammatory stress found that neutrophil entry increases at specific sites which we term “EC gates” with no change in the number of gates suggesting that leukocyte intravasation likely occurs at specialized sites of the endothelium. Thus, we hypothesize that endothelial VEGFR3 signaling regulates leukocyte trafficking through “gates” within the BM sinusoidal endothelium during inflammatory stress. We will test this hypothesis by investigating the relationship between inflammatory stress and EC gate activity using time-lapse intravital microscopy to track neutrophil intravasation frequency, the frequency of distinct “gates” of entry and the ratio of intravasation events to gates across LPS and control groups. We will measure VEGFR3 signaling in sorted BM endothelial cells and test whether inhibition of VEGFR3 inhibits EC gate activity following inflammatory stress. These experiments build the foundation for testing the hypothesis that myocardial infarction (MI) induces similar changes to EC gate activity. We will perform sham or MI-surgery on adult mice and examine EC gate activity by time-lapse intravital microscopy, VEGFR3 signaling in sorted BM endothelial cells and correlate these findings with flow cytometric analysis of circulating leukocyte levels. We finally propose to test the contributions of VEGFR3 signaling in the context of MI by investigating EC gate activity in mice lacking endothelial VEGFR3 or mice pre-treated with VEGFR3 inhibitor. This work proposes to investigate novel VEGFR3-dependent mechanisms that regulate leukocyte release from the BM following CVD-induced inflammatory stress and to test whether modulation of this pathway may inform future therapeutic interventions.
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