Contributions
Abstract: EP1060
Type: E-Poster Presentation
Session title: Myeloproliferative neoplasms - Biology & Translational Research
Background
Thrombotic complications across the vascular tree are frequent in patients with polycythemia vera (PV). These patients also have an increased risk of second primary malignancies. Although genetic predisposition may partially explain these links, whether and how the presence of PV-clonal cells induce pro-thrombotic and oncogenic environment is poorly understood. Secreted by all types of cells, exosomes are nano-scaled particles which carry a molecular cargo that reflects, at least partially the molecular makeup to their cell of origin. Exosomes travel in blood, may reach distant sites away from their cell of origin and affect various physiological and pathological processes. These features led us think that PV-derived exosomes (hereafter PV-exosomes) contribute to the thrombotic manifestations and oncogenic potential of our patients. Since mutated JAK2 is present in virtually all patients with PV, we hypothesized that exosomes that are derived from a PV clone, carry the mutated JAK2 oncogene and contribute to the increased incidence of thrombogenic complications and oncogenic risk.
Aims
1) Determine whether PV-exosomes harbor mutated JAK2 transcripts.
2) Determine whether PV-exosomes are pro-thrombotic.
3) Explore the oncogenic potential of PV-exosomes.
Methods
We isolated microparticles by serial ultracentrifugation, confirmed the presence of exosomes by NTA (Malvern) and imaged them by EM. We extracted RNA, generated cDNA (Invitrogen) and amplified a 465 bp long DNA segment within exon14 of JAK2 by PCR. Sanger sequencing was used to determine the JAK2 mutational status. We used thrombin generation assay to assess the thrombotic potential of PV-exosomes and Trans Epithelial Electric Resistance (TEER) assay to assess endothelial dysfunction. We measured exosomal uptake by HUVECS and keratinocytes by flow cytometry and assessed the oncogenic potential of these exosomes by the NIH/3T3 assay.
Results
To determine whether PV-exosomes carry mutated JAK2 transcripts, we harvested exosomes from the peripheral blood of 21 patients with PV and 20 healthy individuals. Reverse-transcriptase PCR revealed the presence of mutated JAK2 mRNA in all patients and the presence of wild type JAK2 in all healthy individuals. Because mutated JAK2 induces endothelial dysfunction, we wondered whether JAK2mutated-exosomes are taken up by endothelial cells and induce endothelial dysfunction. We stained PV-exosomes with the FM-1-43 dye and showed by flow cytometry that these exosomes are taken up by Human-Umbilical-Vein-derived-Endothelial-Cells (HUVECs) in a dose- and time- dependent manner, peaking at 24 hours. To assay the functional status of the endothelium, we measured the membranal resistance of HUVECS prior to and after exposure to PV-exomes for 24 hours. The resistance was dramatically decreased in the presence of PV-exosomes, indicating that the tight junction between adjacent cells was impaired implicating that PV-exosomes induce endothelial dysfunction. To assess the thrombogenic potential of PV-exomes, we measured the rate of thrombin generation in platelets poor plasma and recorded an increase in the peak thrombin concentration in the presence of PV-exosomes compared with exosomes from healthy individuals. Finally, using the NIH/3T3 platform we found that PV-exosome have the capacity to transform normal cells from various tissues.
Conclusion
Like their parental cells, PV-derived exosomes carry mutant JAK2 transcripts. These exosomes are pro thrombotic and have transforming potential both directly and indirectly through endothelial dysfunction.
Keyword(s): Endothelial dysfunction, Microvesicles, Oncogene, Polycythemia vera
Abstract: EP1060
Type: E-Poster Presentation
Session title: Myeloproliferative neoplasms - Biology & Translational Research
Background
Thrombotic complications across the vascular tree are frequent in patients with polycythemia vera (PV). These patients also have an increased risk of second primary malignancies. Although genetic predisposition may partially explain these links, whether and how the presence of PV-clonal cells induce pro-thrombotic and oncogenic environment is poorly understood. Secreted by all types of cells, exosomes are nano-scaled particles which carry a molecular cargo that reflects, at least partially the molecular makeup to their cell of origin. Exosomes travel in blood, may reach distant sites away from their cell of origin and affect various physiological and pathological processes. These features led us think that PV-derived exosomes (hereafter PV-exosomes) contribute to the thrombotic manifestations and oncogenic potential of our patients. Since mutated JAK2 is present in virtually all patients with PV, we hypothesized that exosomes that are derived from a PV clone, carry the mutated JAK2 oncogene and contribute to the increased incidence of thrombogenic complications and oncogenic risk.
Aims
1) Determine whether PV-exosomes harbor mutated JAK2 transcripts.
2) Determine whether PV-exosomes are pro-thrombotic.
3) Explore the oncogenic potential of PV-exosomes.
Methods
We isolated microparticles by serial ultracentrifugation, confirmed the presence of exosomes by NTA (Malvern) and imaged them by EM. We extracted RNA, generated cDNA (Invitrogen) and amplified a 465 bp long DNA segment within exon14 of JAK2 by PCR. Sanger sequencing was used to determine the JAK2 mutational status. We used thrombin generation assay to assess the thrombotic potential of PV-exosomes and Trans Epithelial Electric Resistance (TEER) assay to assess endothelial dysfunction. We measured exosomal uptake by HUVECS and keratinocytes by flow cytometry and assessed the oncogenic potential of these exosomes by the NIH/3T3 assay.
Results
To determine whether PV-exosomes carry mutated JAK2 transcripts, we harvested exosomes from the peripheral blood of 21 patients with PV and 20 healthy individuals. Reverse-transcriptase PCR revealed the presence of mutated JAK2 mRNA in all patients and the presence of wild type JAK2 in all healthy individuals. Because mutated JAK2 induces endothelial dysfunction, we wondered whether JAK2mutated-exosomes are taken up by endothelial cells and induce endothelial dysfunction. We stained PV-exosomes with the FM-1-43 dye and showed by flow cytometry that these exosomes are taken up by Human-Umbilical-Vein-derived-Endothelial-Cells (HUVECs) in a dose- and time- dependent manner, peaking at 24 hours. To assay the functional status of the endothelium, we measured the membranal resistance of HUVECS prior to and after exposure to PV-exomes for 24 hours. The resistance was dramatically decreased in the presence of PV-exosomes, indicating that the tight junction between adjacent cells was impaired implicating that PV-exosomes induce endothelial dysfunction. To assess the thrombogenic potential of PV-exomes, we measured the rate of thrombin generation in platelets poor plasma and recorded an increase in the peak thrombin concentration in the presence of PV-exosomes compared with exosomes from healthy individuals. Finally, using the NIH/3T3 platform we found that PV-exosome have the capacity to transform normal cells from various tissues.
Conclusion
Like their parental cells, PV-derived exosomes carry mutant JAK2 transcripts. These exosomes are pro thrombotic and have transforming potential both directly and indirectly through endothelial dysfunction.
Keyword(s): Endothelial dysfunction, Microvesicles, Oncogene, Polycythemia vera