Decoding Endothelial MPL and JAK2V617F Mutation: Insight into Cardiovascular Dysfunction in Myeloproliferative Neoplasms

Background: Patients with JAK2V617F-positive myeloproliferative neoplasms (MPNs) and clonal hematopoiesis of indeterminate potential (CHIP) face a significantly elevated risk of cardiovascular diseases (CVDs). Endothelial cells (ECs) carrying the JAK2V617F mutation have been detected in many MPN patients. In this study, we investigated the molecular basis for the high incidence of cardiovascular complications in MPN patients. Methods: We investigated the impact of endothelial JAK2V617F mutation on CVD development using both transgenic murine models and MPN patient-derived induced pluripotent stem cell lines. Results and Conclusions: Our investigations revealed that JAK2V617F mutant ECs promote CVDs by impairing endothelial function and undergoing endothelial-to-mesenchymal transition (EndMT). Importantly, we discovered that inhibiting the endothelial thrombopoietin receptor MPL suppressed JAK2V617F-induced EndMT and prevented cardiovascular dysfunction caused by mutant ECs. Notably, the endothelial MPL receptor is not essential for the normal physiological regulation of blood cell counts and cardiac function, rendering it a promising therapeutic target for preventing or ameliorating cardiovascular complications in patients with MPNs. Overall design: JAK2 wild-type and JAK2V617F mutant cardiac ECs (CD45-CD31+ ) were isolated from Tie2+FF1+ or Tie2-cre mice (n = 3 mice in each group) and their transcriptomic profiles were previously assessed using RNA sequencing1 . RNA sequencing was also conducted on JAK2 wild-type cardiac ECs (one pooled sample from 3 mice) and JAK2V617F mutant cardiac ECs (one pooled sample from 3 mice) isolated from CDH5+FF1+ mice and CDH5+FF1- control mice following high-fat diet treatment, and on human iPS-derived JAK2 wild-type and JAK2V617F mutant ECs treated with or without AMM2 between passage 3 and passage 5 (~15 days). Briefly, total RNA was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany). The isolated RNA samples were sent to Novogene, Inc. for bulk RNA sequencing. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads and used to generate sequencing libraries using NEBNext? Ultra™ RNA Library Prep Kit for Illumina® (New England BioLabs, MA, USA). The library preparations were sequenced on an Illumina platform. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome. featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. Differential expression analysis between two groups was performed using the edgeR R package (3.22.5). The resulting P-values were adjusted using the Benjamini and Hochberg's method for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 found by edgeR were assigned as differentially expressed. Gene set enrichment analysis (GSEA) was performed using the complete list of genes according to their differential expression. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the ClusterProfiler R package. GO terms with corrected P-value less than 0.05 were considered significantly enriched.