PROJECT 2: Cardiovascular disease (CVD) and the stromal bone marrow niche

Project 2 investigators study the role of the hematopoietic niche in CVD-associated leukocyte oversupply. In the past funding cycle, we learned that CVD reaches the vascular bone marrow niche, inducing endothelial dysfunction. Moreover, MI and hypertension give rise to angiogenesis near newly forming leukocyte clusters and their progenitors. Stopping post-MI angiogenesis via endothelial Vegfr2 deletion reduced the hematopoietic response, systemic leukocyte numbers and cardiovascular inflammation. In aim 1 of our renewal application, we follow up on our angiogenesis observations. We will investigate the Vegf pathway by using a VegfGfp mouse to identify Vegf sources after MI. We will further evaluate bone marrow angiogenesis' functional consequences for persistently elevated myelopoiesis and aggravated atherosclerosis by subjecting Vegfr-/- and Vegfr+/+ atherosclerosis-prone mice to MI. Collaborating with P1, P4 and the imaging core, we will follow leukocyte traffic and clonal expansion of myeloid progenitor cells in vascular niches that were newly assembled post-MI. Deploying image-seq, we will compare transcriptomes of cells in new niches, testing the hypothesis that these new niches generate leukocytes with inflammatory phenotypes which contribute to accelerated atherosclerosis after MI. In aim 2, we propose to expand our studies beyond endothelial cells (EC) and investigate all bone marrow niche cells, based on our hypothesis that they assume regulatory roles that influence inflammatory leukocyte supply in CVD. Among others, we will study bone marrow mesenchymal stromal cells (MSC), which influence HSPC and their progeny. In the previous funding cycle, we reported that hyperlipidemia and hypertension increase HSPC proliferation in mice and humans. In preparation for this renewal, we collected scRNA-seq data on the marrow stromal cell compartment from obese mice with hypertension, thus combining two prevalent CVD risk factors. Our unpublished pan-niche whole transcriptome data obtained in these mice provide leads which we now propose to examine with cell-specific gene deletion. Specifically, IFNɣ signaling in MSC emerged as a top-ranked pathway; accordingly, we propose to delete the IFNɣ receptor in MSC to investigate how this affects hematopoiesis and inflammation in mice with CVD. Using gene editing in HSPC, supported by the gene editing core, we will then disrupt HSPC-MSC crosstalk, which IFNɣ receptor deletion alters, to probe niche signals regulated by IFNɣ. Using an IFNɣ reporter mouse, we will define this cytokine's sources in CVD. Ultimately, the proposed work, catalyzed by expertise from collaborators in all P01 components, may uncover pathways that are active during CVD and relayed by the hematopoietic niche; such results could become the basis for therapeutically limiting leukocyte overproduction and inflammatory cardiovascular complications. The program will result in a comprehensive understanding of bone marrow niche pathologies during CVD and uncover the niche-relayed mechanisms that lead to systemic leukocyte oversupply.