Endothelial SRSF1 regulates hematopoietic stem cell function in the bone marrow microenvironment Free

The bone marrow (BM) microenvironment is a dynamic cellular network composed of endothelial cells (ECs) and EC-associated perivascular stromal cells that support hematopoietic stem cell (HSC) activity to facilitate daily blood production. Collectively, the vascular niche instructs HSC quiescence, self-renewal, and differentiation through the expression of paracrine factors, including Kitl, Cxcl12, and Jag1. BM niche dysfunction can result in the impairment of hematopoietic output, delayed recovery from myelosuppressive injuries, chronic inflammation, and increased susceptibility to hematological malignancies. Understanding the BM niche-derived signals and molecular mechanisms that dictate HSC function will enable the development of targeted therapies to improve hematopoietic outcomes. To date, studies evaluating BM niche biology have largely focused on elucidating the individual paracrine factors that modulate HSC function. However, little is known about the alternatively spliced isoforms emanating from the vascular niche and their relative impact on hematopoietic health. In this context, alternative splicing of exon 6 within the pro-HSC paracrine factor KITL dictates membrane-bound retention or release of the soluble signaling peptide, demonstrating altered signaling activities. However, a comprehensive analysis of how alternative splicing affects the instructive function of BM-derived ECs (BMECs) remains unexplored. Serine/Arginine (SR) splicing factor 1 (SRSF1) is the prototypical member of the SR family of RNA binding proteins that regulate RNA splicing. SRSF1 has been shown to regulate a discrete number of alternatively spliced endothelial genes and has been implicated in vascular aging and dysfunction. Moreover, SRSF1 relocalization to the cytoplasm has been implicated in endothelial senescence in cell culture. Herein, we aim to explore the functional alternative splicing landscape within the instructive vascular niche that maintains HSPC activity and healthy hematopoietic output. To this end, we employed a conditional murine model with EC-specific deletion of Srsf1 (Cdh5-creER^T2; Srsf1^fl/fl mice) and assessed their hematopoietic parameters. Tamoxifen-induced EC-specific Srsf1 knockout mice (Srsf1^iECKO) displayed a significant increase in white blood cells (WBCs) and a decrease in red blood cells and platelets. Myeloid frequency in the peripheral blood was also increased at the expense of both B cells and T cells. BM analyses revealed a significant decrease in total cellularity with no changes in immunophenotypic HSC frequency. To assess HSC activity in Srsf1^iECKO mice, whole BM was competitively transplanted and assessed for long-term multi-lineage engraftment. Srsf1^iECKO donors revealed a significant decrease in hematopoietic engraftment with no changes in lineage output; secondary transplantations to assess HSC self-renewal are ongoing. To evaluate vascular function in Srsf1^iECKO mice, we utilized a modified Evan's blue dye (EBD) assay to assess vascular permeability and non-invasive blood pressure tail-cuffs to measure blood pressure parameters. Notably, EBD extravasation revealed no significant vascular leakiness in the BM. Moreover, systolic and diastolic blood pressure in Srsf1^iECKO mice were comparable with control littermates. Peripheral tissue perfusion, as measured by mean arterial pressure, remained consistent; total body weight was also unaffected. To account for the observed increase in WBC counts and decrease in BM cellularity in Srsf1^iECKO mice, we examined the peripheral blood for the presence of circulating stem cells, revealing a 24-fold increase in immunophenotypic HSCs, suggestive of cellular relocalization. Accordingly, spleens also displayed a significant increase in both frequency and total numbers of HSCs in Srsf1^iECKO mice with no differences in their spleen weights. Taken together, Srsf1^iECKO mice display HSC mobilization and impaired long-term hematopoietic engraftment, extramedullary hematopoiesis, and an increase in peripheral WBCs, in the absence of obvious changes in vascular permeability. These findings suggest an instructive role for endothelial SRSF1-regulated spliced isoforms in modulating HSC homeostasis and localization. Gene expression and alternative splicing analysis of in vivo and in vitro sources of Srsf1^iECKO BMECs are ongoing and will be the focus of understanding the SRSF1 alternative splicing regulons that instruct hematopoiesis in the BM microenvironment.