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499 Unlocking the Role of Endothelial MPL Receptor and JAK2V617F Mutation: Insights into Cardiovascular Dysfunction in MPNs and Chip

Program: Oral and Poster Abstracts
Type: Oral
Session: 301. Vasculature, Endothelium, Thrombosis and Platelets: Basic and Translational: Insights Into the Role of Endothelium in Cardiovascular Disease and Thrombosis
Sunday, December 10, 2023: 12:00 PM

Huichun Zhan, MD1,2, Haotian Zhang3, Nicholas Kafeiti4*, Sandy Lee5,6, Kyla Masarik4* and Haoyi Zheng7*

1Department of Medicine, Stony Brook School of Medicine, Stony Brook
2Northport VA Medical Center, Northport
3Stony Brook University, Stony Brook, NY
4Stony Brook School of Medicine, Stony Brook, NY
5Graduate Program in Molecular & Cellular Pharmacology, Stoney Brook University, Stony Brook, NY
6Graduate Program in Molecular & Cellular Pharmacology, Stoney Brook University, Setauket, NY
7Saint Francis Hospital, Roslyn, NY


Patients with JAK2V617F-positive myeloproliferative neoplasms (MPNs) and clonal hematopoiesis of indeterminate potential (CHIP) are at a significantly higher risk of cardiovascular diseases (CVDs). Vascular endothelial cells (ECs) carrying the JAK2V617F mutation can be detected in many MPN patients. The thrombopoietin receptor MPL is expressed on vascular ECs, though its role in endothelial function is unclear. In this study, we investigated the mechanisms through which the JAK2V617F mutation induces endothelial dysfunction to accelerate CVDs, shedding light on endothelial MPL as a potential therapeutic target for preventing or mitigating cardiovascular complications in JAK2V617F-positive MPNs.


JAK2V617F Flip-Flop mice (FF1; provided by Dr. Radek Skoda) were crossed with VEcadherin-creERT2 mice (CDH5) to generate CDH5+/-FF1+/- mice, which can be induced by tamoxifen to express human JAK2V617F specifically in vascular ECs but not in blood cells. MPLfl mice (provided by Dr. Warren Alexander) were crossed with CDH5+FF1+ mice to generate CDH5+FF1+MPLfl/fl mice. To validate findings in transgenic murine models in human cells, we utilized a pair of isogenic induced pluripotent stem (iPS) cell lines derived from the same MPN patient, one carrying the JAK2V617F mutation (PVB1.4) and the other being JAK2 wild-type (PVB1.11). These iPS cell lines were differentiated into human ECs using a modified iPS differentiation protocol.


CDH5+FF1+ mice exhibited mild thrombocytosis but no cardiac dysfunction. However, when challenged with a high-fat diet, these mice developed dilated cardiomyopathy with significant decreases in LV ejection fraction and increases in LV end-diastolic and end-systolic volumes. Pathological evaluation revealed the presence of thromboses in segment pulmonary arteries and scattered coronary capillaries. There was intimal thickening, smooth muscle cell thickening, and perivascular fibrosis of scattered coronary arterioles in CDH5+FF1+ mice compared to control mice. These findings, along with impaired cardiac EC function (measured by angiogenesis in vitro) and loss of EC markers, as well as significant enrichment of gene expression signatures associated with inflammatory response and epithelial-mesenchymal transition in JAK2V617F mutant cardiac ECs, suggest that the JAK2V617F mutation induces endothelial-to-mesenchymal transition (EndMT) in cardiac ECs from the CDH5+FF1+ mice. Similarly, JAK2 wild-type ECs derived from PVB1.11 iPS cell line maintained the expression of EC markers and the characteristic cobblestone endothelial morphology throughout serial passages in vitro; in contrast, JAK2V617F mutant ECs derived from PVB1.4 exhibited a gradual loss of EC marker expression, decreased EC function, and underwent a phenotypical transition toward a fibroblastoid morphology, providing compelling evidence of EndMT induction by the JAK2V617F mutation in human ECs.

We noticed that the thrombopoietin receptor MPL is abundantly expressed in the heart’s vascular endothelium, compared to other tissues (e.g., lung, marrow, spleen). MPL expression is upregulated in JAK2V617F mutant ECs compared to wild-type ECs, and Inhibiting JAK-STAT signaling using Ruxolitinib abolished this MPL upregulation. To investigate the role of endothelial TPO/MPL signaling in JAK2V617F-induced cardiovascular dysfunction, we generated CDH5+FF1+MPLfl/fl mice to knock down MPL in JAK2V617F-bearing vascular ECs. The CDH5+FF1+MPLfl/fl mice maintained normal blood counts and cardiac function even after a high-fat diet challenge. In JAK2V617F mutant human ECs derived from PVB1.4 iPS cell line, treatment with AMM2, an anti-MPL neutralizing antibody, preserved EC markers and endothelial morphology compared to untreated mutant ECs. These results demonstrated that inhibiting the endothelial MPL can suppress JAK2V617F-induced EndMT and prevent cardiovascular dysfunction caused by mutant ECs. Notably, the endothelial MPL receptor is not indispensable for the normal physiological regulation of blood cell counts and cardiac function.


Our study provides valuable insights into the increased risk of CVDs observed in patients with JAK2V617F-positive CHIP and MPNs. Further investigations into the role of EndMT in other CHIP-associated mutations may shed light on the diverse CVD risks associated with different CHIP mutations.

Disclosures: No relevant conflicts of interest to declare.

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