WO2021013942A1

WO2021013942A1 - Use of myeloperoxidase inhibitors for the treatment of cardiovascular diseases in patients suffering from myeloproliferative neoplasms

Info

Publication number: WO2021013942A1
Application number: PCT/EP2020/070827
Authority: WO
Inventors: Pierre-Emmanuel RAUTOU; Chantal BOULANGER-ROBERT; Johanne POISSON
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Assistance Publique-Hôpitaux De Paris (Aphp); Université de Paris
Priority date: 2019-07-24
Filing date: 2020-07-23
Publication date: 2021-01-28

Abstract

Arterial cardiovascular events, i.e. the leading cause of death in patients with JAK2 ^V617F myeloproliferative neoplasms (MPN), are poorly understood. The inventors demonstrated that Jak2 ^V617Fmice display a strong increase in arterial contraction with disturbed endothelial nitric oxide pathway and increased endothelial oxidative stress. This augmented arterial contraction was reproduced by circulating microvesicles isolated from patients carrying JAK2 ^V617F and by microvesicles derived from JAK2 ^V617Ferythrocytes. Using proteomics, the inventors identified a high expression of myeloperoxidase in microvesicles derived from JAK2 ^V617F erythrocytes that could account for this effect. To assess the role of myeloperoxidase in this effect, the inventors then directly inhibited myeloperoxidase in microvesicles derived from Jak2 ^V617F erythrocytes and observed that it completely reversed the increase in endothelial oxidative stress induced by microvesicles derived from Jak2 ^V617F.The results prompt the inventors to conclude that JAK2 ^V617FMPN induce a potent increase in arterial contraction with increased endothelial oxidative stress, mediated by erythrocytes microvesicles and that myeloperoxidase (MPO) inhibitors would be suitable for preventing cardiovascular diseases in patient suffering from MPN.

Description

USE OF MYELOPEROXIDASE INHIBITORS FOR THE TREATMENT OF CARDIOVASCULAR DISEASES IN PATIENTS SUFFERING FROM MYELOPROLIFERATIVE NEOPLASMS

FIELD OF THE INVENTION:

The present invention is in the field of medicine.

BACKGROUND OF THE INVENTION:

Bcr/Abl-negative myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by the proliferation of particular hematopoietic lineages without blockage in cell maturation. They include polycythemia vera, essential thrombocythemia, and primary myelofibrosis (1). JAK2 is the most common MPN driver gene. JAK2^V617F is a gain of function mutation leading to growth factors hypersensitivity, detected in around 70% of MPNs (95% in polycythemia vera and 50% to 60% in essential thrombocythemia and pre-primary myelofibrosis / primary myelofibrosis) (1). JAK2^V617F appears in pluripotent hematopoietic progenitor cells and is present in all erythroid and myeloid lineages (1). In addition, several groups described JAK2^V617F in endothelial cells in the liver and the spleen of patients with splanchnic vein thrombosis (2, 3) and in circulating endothelial progenitor cells (4 6).

Cardiovascular diseases (CVD) reveal MPNs in about 30% of the patients and are the first cause of morbidity and mortality in these patients (7). Arterial events represent 60-70% of these cardio-vascular events (7). Interestingly, myocardial infarction without significant coronary stenosis by angiography was observed in 21% of patients with MNP (8) versus only 3% in a similar population without MPN (9). This observation prompted the European society of cardiology to recommend searching for MPNs in case of myocardial infarction without obstructive coronary artery disease (10). The mechanism underlying this link between myocardial infarction without obstructive coronary artery disease and MPNs is unknown, but vasoactive phenomenon (local intense vasoconstriction) can be suspected (11, 12).

SUMMARY OF THE INVENTION:

As defined by the claims, the present invention relates to the use of myeloperoxidase inhibitors for the treatment of cardiovascular diseases in patients suffering from myeloproliferative neoplasms. DETAILED DESCRIPTION OF THE INVENTION:

Arterial cardiovascular events, i.e. the leading cause of death in patients with JAK2^V617F myeloproliferative neoplasms (MPN), are poorly understood. The inventors demonstrated that Jak2^V617F mice display a strong increase in arterial contraction with disturbed endothelial nitric oxide pathway and increased endothelial oxidative stress. This augmented arterial contraction was reproduced by circulating microvesicles isolated from patients carrying JAK2^V617F zhά by microvesicles derived from JAK2^V617F erythrocytes. Using proteomics, the inventors identified a high expression of myeloperoxidase in microvesicles derived from JAK2^V617F erythrocytes that could account for this effect. To assess the role of myeloperoxidase in this effect, the inventors then directly inhibited myeloperoxidase in microvesicles derived from Jak2^V6I7F erythrocytes and observed that it completely reversed the increase in endothelial oxidative stress induced by microvesicles derived from Jak2^V6I7F. The results prompt the inventors to conclude that JAK2^V6I7F MPN induce a potent increase in arterial contraction with increased endothelial oxidative stress, mediated by erythrocytes microvesicles and that myeloperoxidase (MPO) inhibitors would be suitable for preventing cardiovascular diseases in patient suffering from MPN.

Accordingly, the first object of the present invention relates to a method of preventing the occurrence of cardiovascular event in a patient suffering from a myeloproliferative neoplasm comprising administering to the patient a therapeutically effective amount of a myeloperoxidase inhibitor.

As used herein, the term“myeloproliferative neoplasm” or“MPN” has its general meaning in the art and refers to an acquired clonal hematopoietic stem cell disorder, characterized by an increase in one or more myeloid lineages. MPNs typically include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). They are a diverse but inter-related group of clonal disorders of pluripotent hematopoietic stem cells that share a range of biological, pathological and clinical features including the relative overproduction of one or more cell types from myeloid origin with growth factor independency/hypersensitivity, marrow hypercellularity, extramedullary hematopoiesis, spleno- and hepatomegaly, and thrombotic and/or hemorrhagic diathesis. An international working group for myeloproliferative neoplasms research and treatment (IWG-MRT) has been established to delineate and define these conditions (see for instance Vannucchi et al, CA Cancer J. Clin., 2009, 59: 171-191), and those disease definitions are to be applied for purposes of this specification. In some embodiments, the patient harbours one mutation in JAK2. As used herein the term“JAK2” has its general meaning in the art and refers to the Janus Kinase 2 protein. The amino acid sequence of human JAK2 is well known in the art. Human JAK2 sequences are, for example, represented in the NCBI database (www.ncbi.orgwww.ncbi.nlm.nih.gov/), for example, under accession number NP 004963. Typical MPD associated mutation is the Jak2^V6I7F mutation which refers to the point mutation (1849 G for T) in exon 14, which causes the substitution of phenylalanine for valine at codon 617 in the JAK homology JH2 domain. Other examples of JAK2 mutations include exon 12 mutations which can be substitutions, deletions, insertions and duplications, and all occur within a 44 nucleotides region in the JAK2 gene which encompasses amino acids 533-547 at the protein level. The most commonly reported mutations are small in-frame deletions of 3-12 nucleotides with a six nucleotides deletion being the most frequent. Complex mutations are present in one-third of cases with some mutations occurring outside this hotspot region. The N542-E543del is the most common mutation (23-30%), the K537L, E543-D544del and F537-K39delinsL represent 10-14%, and R541-E543delinsK comprise less than 10% of these mutations. JAK2 exon 12 mutations are located in a region close to the pseudo-kinase domain which acts as a linker between this domain and the Src homology 2 domain of JAK2.

As used herein, the term "cardiovascular event" as used herein refers to any disorder of the cardiovascular system including preferably any acute cardiovascular event. Acute cardiovascular events are, preferably, stable angina pectoris (SAP) or acute coronary syndrome (ACS). ACS patients can show unstable angina pectoris (UAP) or myocardial infarction (MI). MI can be an ST-elevation MI (STEMI) or a non-ST-elevation MI (NSTEMI). NSTE-ACS as used herein encompasses UAP and NSTEMI. The occurring of an MI can be followed by a left ventricular dysfunction (LVD), development of heart failure or even mortality. Further preferred cardiovascular events encompass cardiac brady- or tachyarrhythmias including sudden cardiac death and stroke (cerebrovascular events or accidents). Also, mortality can also refer to the death rate or the ratio of number of deaths to a given population of subjects.

As used herein the term“Myeloperoxidase” or MPO has its general meaning in the art and refers to a heme-containing enzyme. The enzyme uses hydrogen peroxide to oxidize chloride to hypochlorous acid. Other halides and pseudohalides (like thiocyanate) are also physiological substrates to MPO.

As used herein, a“MPO inhibitor” refers to any compound natural or not which is capable of inhibiting the activity of MPO, in particular MPO kinase activity. MPO inhibitors are well known in the art. The term encompasses any MPO inhibitor that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition or down-regulation of a biological activity associated with activation of the MPO. The term also encompasses inhibitor of expression. The MPO inhibition of the compounds may be determined using various methods well known in the art.

Examples of compounds that can be used as MPO-inhibitors are compounds described in WO 2006/062465, WO 2006/062465, WO 2003/089430, WO 2003/089430, or WO 2003/089430.

In some embodiments, the MPO inhibitor of the present invention is selected from the group consisting of:

l-butyl-2-thioxo-l,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimidin-4-one;

l-isobutyl-2-thioxo-l,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimidin-4-one;

l-(pyridin-2-ylmethyl)-2-thioxo- 1,2,3, 5-tetrahydro-pyrrolo[3,2-d]pyrimidin-4- one;

l-(2-fluoro-benzyl)-2-thioxo- 1,2,3, 5-tetrahydro-pyrrolo[3,2-d]pyrimidin-4- one;

l-[2-(2-methoxyethoxy)-3-propoxybenzyl]-2-thioxo-l,2,3,5-tetrahydro- pyrrolo[3,2-d]pyrimidin-4-one;

l-(6-ethoxy-pyridin-2-ylmethyl)-2-thioxo-l,2,3,5-tetrahydro-pyrrolo[3,2- d]pyrimidin-4-one;

1 -piperidin-3 -ylmethyl-2-thioxo- 1 ,2,3 , 5 -tetrahydro-pyrrolo[3 ,2-d]pyrimidin-4- one;

l-butyl-4-thioxo-l,3,4,5-tetrahydro-2H-pyrrolo[3,2-d]pyrimidin-2-one;

l-(2-isopropoxyethyl)-2-thioxo-l,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimidin-4- one;

1 -(2-methoxy-2-methylpropyl)-2-thioxo- 1 ,2,3 , 5 -tetrahydro-pyrrolo[3 ,2- d]pyrimidin-4-one;

l-(2-ethoxy-2-methylpropyl)-2-thioxo-l,2,3,5-tetrahydro-pyrrolo[3,2- d]pyrimidin-4-one;

l-(piperi din-4-ylmethyl)-2-thioxo- 1,2,3, 5-tetrahydro-pyrrolo[3,2-d]pyrimidin- 4-one;

l-[(l-methylpiperidin-3-yl)methyl]-2-thioxo-l,2,3,5-tetrahydro-pyrrolo[3,2- d]pyrimidin-4-one; l-[2-hydroxy-2-(4-methoxyphenyl)ethyl]-2-thioxo- 1,2,3, 5-tetrahydro- pyrrolo[3,2-d]pyrimidin-4-one;

1 -(2-methoxybenzyl)-2-thioxo- 1 ,2,3 , 5-tetrahydro-pyrrolo [3 ,2-d]pyrimidin-4- one;

1 -(3 -methoxybenzyl)-2-thioxo- 1 ,2,3 , 5-tetrahydro-pyrrolo[3 ,2-d]pyrimidin-4- one;

l-(2,4-dimethoxybenzyl)-2-thioxo-l,2,3,5-tetrahydro-pyrrolo[3,2-d]pyrimidin-

4-one;

l-[(3-chloropyridin-2-yl)methyl]-2-thioxo-l,2,3,5-tetrahydro-pyrrolo[3,2- d]pyrimidin-4-one;

l-{ [3-(2-ethoxyethoxy)pyridin-2-yl]methyl}-2 -thioxo- 1,2, 3, 5-tetrahydro- pyrrolo[3,2-d]pyrimidin-4-one;

l-[(6-oxo-l,6-dihydropyridin-2-yl)methyl]-2-thioxo-l,2,3,5-tetrahydro- pyrrolo[3,2-d]pyrimidin-4-one;

l-(lH-indol-3-ylmethyl)-2 -thioxo- 1,2,3, 5-tetrahydro-pyrrolo[3,2-d]pyrimidin- 4-one;

1 -( 1 H-benzimidazol-2-ylmethyl)-2-thioxo- 1 ,2,3 , 5 -tetrahydro-pyrrolo[3 ,2- d]pyrimidin-4-one;

l-[(5-chloro-lH-indol-2-yl)methyl]-2 -thioxo- 1,2,3, 5-tetrahydro-pyrrolo[3, 2- d]pyrimidin-4-one;

1 - [(5 -fluoro- 1 H-indol-2-yl)methyl] -2-thioxo- 1,2,3 , 5-tetrahydro-pyrrolo[3 ,2- d]pyrimidin-4-one;

l-(lH-indol-6-ylmethyl)-2 -thioxo- 1,2,3, 5-tetrahydro-pyrrolo[3,2-d]pyrimidin- 4-one;

l-(lH-indol-5-ylmethyl)-2 -thioxo- 1,2,3, 5-tetrahydro-pyrrolo[3,2-d]pyrimidin- 4-one;

1 - [(5 -fluoro- 1 H-indol-3 -yl)methyl] -2-thioxo- 1,2,3 , 5-tetrahydro-pyrrolo[3 ,2- d]pyrimidin-4-one;

l-(lH-imidazol-5-ylmethyl)-2-thioxo-l,2,3,5-tetrahydro-pyrrolo[3,2- d]pyrimidin-4-one;

l-(lH-imidazol-2-ylmethyl)-2-thioxo-l,2,3,5-tetrahydro-pyrrolo[3,2- d]pyrimidin-4-one;

l-[(5-chloro-lH-benzimidazol-2-yl)methyl]-2-thioxo- 1,2,3, 5-tetrahydro- pyrrolo[3,2-d]pyrimidin-4-one; 1 - [(4, 5 -dimethyl- 1 H-benzimidazol-2-yl)methyl] -2-thioxo- 1 ,2,3 , 5-tetrahydro- pyrrolo[3,2-d]pyrimidin-4-one;

7-bromo- 1 -isobutyl-2 -thioxo- 1 ,2,3 ,5-tetrahydro-pyrrolo[3 ,2-d]pyrimidin-4- one; and

1 -(3 -chlorophenyl)-2-thioxo- 1,2,3 , 5-tetrahydro-pyrrolo[3 ,2-c]pyrimidin-4-one.

1.3-diisobutyl-8-methyl-6-thioxanthine;

1.3-dibutyl-8-methyl-6-thioxanthine;

3-isobutyl- 1 , 8-dimethyl-6-thioxanthine;

3-(2-methylbutyl)-6-thioxanthine;

3-isobutyl-8-methyl-6-thioxanthine;

3-isobutyl-2-thioxanthine;

3-isobutyl-2,6-dithioxanthine;

3-isobutyl-8-methyl-2-thioxanthine;

3-isobutyl-7-methyl-2-thioxanthine;

3-cyclohexylmethyl-2-thioxanthine;

3-(3-methoxypropyl)-2-thioxanthine;

3-cyclopropylmethyl-2-thioxanthine;

3-isobutyl- 1 -methyl-2-thioxanthine;

3-(2-tetrahydrofuryl-methyl)-2-thioxanthine;

3-(2-methoxy-ethyl)-2-thioxanthine;

3 -(3 -( 1 -morpholinyl)-propyl)-2-thioxanthine;

3-(2-furyl-methyl)-2-thioxanthine;

3-(4-methoxybenzyl)-2-thioxanthine;

3-(4-fluorobenzyl)-2-thioxanthine;

3 -phenethyl-2-thioxanthine;

(+)-3 -(2-tetrahydrofuryl-methyl)-2-thioxanthine;

(-)-3-(2-tetrahydrofuryl-methyl)-2-thioxanthine; and

3 -n-butyl-2-thioxanthine .

3-(pyridin-2-ylmethyl)-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one;

3-(pyridin-3-ylmethyl)-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one;

3-(pyridin-4-ylmethyl)-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one;

3-{[3-ethoxy-4-(2-ethoxyethoxy)pyridin-2-yl]methyl}-2-thioxo-l,2,3,7- tetrahydro-6H-purin-6-one; 3-[(5-fhioro-lH-indol-2-yl)methyl]-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6- one;

3-[(5-fluoro-lH-indol-2-yl)methyl]-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6- one;

3-[(2-butyl-4-chloro-lH-imidazol-5-yl)methyl]-2-thioxo-l,2,3,7-tetrahydro-

6H-purin-6-one;

3-(lH-benzimidazol-2-ylmethyl)-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one; 3-[l-(lH-benzimidazol-2-yl)ethyl]-2-thioxo- 1,2,3, 7-tetrahydro-6H-purin-6- one;

3-[(5-chloro-lH-indol-3-yl)methyl]-2 -thioxo- 1,2,3, 7-tetrahydro-6H-purin-6- one

3-[(4-fluoro-lH-indol-3-yl)methyl]-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6- one;

3 - [2-( 1 H-Benzimidazol-2-yl)ethyl] -2-thioxo- 1 ,2,3 , 7 -tetrahydro-6H-purin-6- one;

3 -( 1 H-Pyrazol-3 -ylmethyl)-2-thioxo- 1,2,3 , 7-tetrahydro-6H-purin-6-one;

3-[(5-Methylpyrazin-2-yl)methyl)-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one; 3 - [(3 -Isopropyli soxazol-5 -yl)methyl] -2-thioxo- 1 ,2,3 , 7-tetrahydro-6H-purin-6- one;

3-[(4-Methyl-l,2,5-oxadiazol-3-yl)methyl]-2-thioxo-l,2,3,7-tetrahydro-6H- purin-6-one;

3-[(6-Butoxypyridin-2-yl)methyl]-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one;

3-[(4-Butoxypyridin-2-yl)methyl]-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one;

3-[(3-Butoxypyridin-2-yl)methyl]-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one;

3-[2-(Pyridin-2-ylmethoxy)propyl]-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6- one;

3-[(3,5-Dimethylisoxazol-4-yl)methyl]-2-thioxo-l,2,3,7-tetrahydro-6H-purin-

6-one;

3-[(l -Methyl- lH-indol-2-yl)methyl]-2-thioxo- 1,2,3, 7-tetrahydro-6H-purin-6- one;

3-(2-Phenyl-2-pyridin-2-ylethyl)-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one;

3-(Quinolin-4-ylmethyl)-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one;

3-[(6-Phenoxypyridin-3-yl)methyl]-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6- one; 3-{2-[(Quinolin-4-ylmethyl)amino]ethyl}-2-thioxo-l,2,3,7-tetrahydro-6H- purin-6-one;

3 -(2- { [( 1 -Methyl- 1 H-indol-3 -yl)methyl]amino } ethyl)-2-thioxo- 1 ,2,3 , 7- tetrahydro-6H-purin-6-one;

3-{2-[Methyl(quinolin-4-ylmethyl)amino]ethyl}-2 -thioxo- 1,2, 3, 7-tetrahydro- 6H-purin-6-one;

3-(2-Aminopropyl)-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one

trifluoroacetate;

3-{2-[(Pyridin-2-ylmethyl)amino]propyl}-2-thioxo- 1,2,3, 7-tetrahydro-6H- purin-6-one trifluoroacetate;

3-{2-[(Pyridin-3-ylmethyl)amino]propyl}-2-thioxo- 1,2,3, 7-tetrahydro-6H- purin-6-one;

3-{2-[(Pyridin-4-ylmethyl)amino]propyl}-2-thioxo- 1,2,3, 7-tetrahydro-6H- purin-6-one;

3-(2-{[(6-Chloropyridin-3-yl)methyl]amino}propyl)-2-thioxo-l,2,3,7- tetrahydro-6H-purin-6-one trifluoroacetate;

3 -[2-({ [6-(Trifluoromethyl)pyri din-3 -yl] methyl }amino)propyl] -2-thioxo-

1.2.3.7-tetrahydro-6H-purin-6-one trifluoroacetate;

3-(2-{[(4,6-Dichloropyrimidin-5-yl)methyl]amino}propyl)-2-thioxo-l,2,3,7- tetrahydro-6H-purin-6-one;

3-[2-({[2-(Dimethylamino)pyrimidin-5-yl]methyl}amino)propyl]-2-thioxo-

1.2.3.7-tetrahydro-6H-purin-6-one;

3-{2-[(Quinolin-2-ylmethyl)amino]propyl}-2-thioxo- 1,2,3, 7-tetrahydro-6H- purin-6-one trifluoroacetate;

3-{2-[(Quinolin-3-ylmethyl)amino]propyl}-2-thioxo- 1,2,3, 7-tetrahydro-6H- purin-6-one;

3-(2-{[(l-tert-Butyl-3,5-dimethyl-lH-pyrazol-4-yl)methyl]amino}propyl)-2- thioxo-l,2,3,7-tetrahydro-6H-purin-6-one;

3-[2-({[l-(l,l-Dioxidotetrahydro-3-thienyl)-3,5-dimethyl-lH-pyrazol-4- yl]methyl }amino)propyl]-2-thioxo- 1 ,2,3,7-tetrahydro-6H-purin-6-one;

3-{2-[(lH-Benzoimidazol-2-ylmethyl)amino]propyl}-2 -thioxo- 1,2, 3,7- tetrahydro-6H-purin-6-one;

3-[2-({[l-(Phenylsulfonyl)-lH-pyrrol-2-yl]methyl}amino]propyl]-2-thioxo-

1.2.3.7-tetrahydro-6H-purin-6-one trifluoroacetate; 3-{2-[({ l-[(4-methylphenyl)sulfonyl]-lH-pyrrol-2-yl}methyl)amino]propyl}-

2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one trifluoroacetate;

3 -(2- { [( 1 -methyl- 1 H-py rrol-2-yl)methyl]amino } propyl)-2-thioxo- 1 ,2,3 , 7- tetrahydro-6H-purin-6-one;

3 - [2-( { [ 1 -(4-sec-Butylphenyl)- 1 H-pyrrol-2-yl] methyl } amino)propyl)-2-thioxo- l,2,3,7-tetrahydro-6H-purin-6-one;

3-[2-({[l-(3 -Methoxyphenyl)- lH-pyrrol-2-yl] methyl } amino)propyl] -2-thioxo- l,2,3,7-tetrahydro-6H-purin-6-one;

3 - [2-( { [2, 5-Dimethyl- 1 -( 1 , 3 -thiazol-2-yl)- lH-pyrrol-3 - yl]methyl}amino)propyl]-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one;

3-[2-({[4-(3-Chlorobenzoyl)-l-methyl-lH-pyrrol-2-yl]methyl}amino)propyl]-

2-thioxo- 1,2,3 , 7-tetrahydro-6H-purin-6-one;

3-{2-[(lH-Imidazol-2-ylmethyl)amino]propyl}-2-thioxo-l,2,3,7-tetrahydro- 6H-purin-6-one;

3-(2-{ [(1 -Methyl- lH-imidazol-2-yl)methyl]amino}propyl)-2-thioxo-l, 2,3,7- tetrahydro-6H-purin-6-one;

3-(2-{ [(4-Bromo-l -methyl- lH-imi dazol-5-yl)methyl]amino}propyl)-2 -thioxo- l,2,3,7-tetrahydro-6H-purin-6-one;

3-(2-{ [(1 -Methyl- lH-indol-3-yl)methyl]amino}propyl)-2-thioxo- 1,2, 3,7- tetrahydro-6H-purin-6-one;

2-Thioxo-3-{2-[(lH-l,2,3-triazol-5-ylmethyl)amino]propyl}-l,2,3,7- tetrahydro-6H-purin-6-one;

3-[2-({[l-(Benzyloxy)-lH-imidazol-2-yl]methyl}amino)propyl]-2-thioxo- l,2,3,7-tetrahydro-6H-purin-6-one;

3-(2-{[(6-Bromo-2-methylimidazo[l,2-a]pyridin-3-yl)methyl]amino}propyl}-

2-thioxo- 1,2,3 , 7-tetrahydro-6H-purin-6-one;

3-{2-[({ l-[2-(2-Methoxyphenoxy)ethyl]-lH-pyrrol-2- yl}methyl)amino]propyl]-2-thioxo-l,2,3,7-tetrahydro-6H-purin-6-one;

N-[ 1 -Methyl-2-(6-oxo-2-thioxo- 1 ,2,6,7-tetrahydro-3H-purin-3- yl)ethyl]pyridine-2-carboxamide;

N-[ 1 -Methyl-2-(6-oxo-2-thioxo- 1 ,2,6,7-tetrahydro-3H-purin-3- yl)ethyl]nicotinamide;

N-[ 1 -Methyl-2-(6-oxo-2-thioxo- 1 ,2,6,7-tetrahydro-3H-purin-3-yl)- ethyl ] i soni cotinamide; N-[l-methyl-2-(6-oxo-2-thioxo-l,2,6,7-tetrahydro-3H-purin-3-yl)ethyl]-l,8- naphthyridine-2-carboxamide;

N-[ 1 -Methyl-2-(6-oxo-2-thioxo- 1 ,2,6,7-tetrahydro-3H-purin-3- yl)ethyl]quinoline-2-carboxamide;

N-[ 1 -Methyl-2-(6-oxo-2-thioxo- 1 ,2,6,7-tetrahydro-3H-purin-3- yl)ethyl]pyrimidine-2-carboxamide; and

N-[ 1 -Methyl-2-(6-oxo-2-thioxo- 1 ,2,6,7-tetrahydro-3H-purin-3-yl)ethyl]- 1H- imidazole-2-carboxamide trifluroaceate.

In some embodiment, the MPO inhibitor is AZD5904 which has the formula of:

In some embodiments, the MPO inhibitor is an inhibitor of MPO expression. An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In a preferred embodiment of the invention, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti- sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of MPO mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of MPO, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding MPO can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. MPO gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that MPO gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing MPO. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

By a "therapeutically effective amount" of the inhibitor as above described is meant a sufficient amount to provide a therapeutic effect. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Typically, the inhibitor of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Typically, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Sterile injectable solutions are prepared by incorporating the inhibitor at the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

A further object of the present invention relates to a method of determining whether a patient suffering from a myeloproliferative neoplasm is at risk of having a cardiovascular event comprising the steps of determining the level of microvesicles derived from Jak2^V6!7F erythrocytes in a blood sample obtained from the patient wherein the level correlates with the risk of having a cardiovascular disease. As used herein, the term“risk" relates to the probability that an event will occur over a specific time period, as in the conversion to a cardiovascular event, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. Alternative continuous measures which may be assessed in the context of the present invention include time to a cardiovascular event conversion and therapeutic a cardiovascular event conversion risk reduction ratios.

Thus the expression "determining whether a patient is at risk of having a cardiovascular event" as used herein means that the patient to be analyzed by the method of the present invention is allocated either into the group of patients of a population having an elevated risk, or into a group having a reduced risk of having a cardiovascular event. An elevated risk as referred to in accordance with the present invention, preferably, means that the risk of developing a cardiovascular event within a predetermined predictive window is elevated significantly (i.e. increased significantly) for a patient with respect to the average risk for a cardiovascular event or cardiac mortality in a population of patients. A reduced risk as referred to in accordance with the present invention, preferably, means that the risk of developing a cardiovascular event within a predetermined predictive window is reduced significantly for a patient with respect to the average risk for a cardiovascular event or cardiac mortality in a population of patients. Particularly, a significant increase or reduction of a risk is an increase or reduction or a risk of a size which is considered to be significant for prognosis, particularly said increase or reduction is considered statistically significant. The terms "significant" and "statistically significant" are known by the person skilled in the art. Thus, whether an increase or reduction of a risk is significant or statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools.

As used herein the term“blood sample” means a whole blood, serum, or plasma sample obtained from the patient. Preferably the blood sample, according to the invention, is a plasma sample. A plasma sample may be obtained using methods well known in the art. For example, blood may be drawn from the patient following standard venipuncture procedure on tri-sodium citrate buffer. Plasma may then be obtained from the blood sample following standard procedures including but not limited to, centrifuging the blood sample at about 2500*g for about 15 minutes (room temperature), followed by pipetting of the plasma layer. Platelet-free plasma (PFP) will be obtained following a second centrifugation at about 2500*g for 15 min. Analyses can be performed directly on this PFP. Alternatively, microvesicles may be more specifically isolated by further centrifuging the PFP at about 15,000 to about 25,000*g at 4°C. Different buffers may be considered appropriate for resuspending the pelleted cellular debris which contains the MPs. Such buffers include reagent grade (distilled or deionized) water and phosphate buffered saline (PBS) pH 7.4 or NaCl 0.9%. Preferably, PBS buffer (Sheath fluid) is used.

As used herein the term“microvesicle” or“MV” or“extracellular vesicles” has its general meaning in the art and denotes a plasma membrane vesicle shed from an apoptotic or activated cell. The size of microvesicles / extracellular vesicles ranges from 0.1 pm to 1 pm in diameter. The surface markers of microvesicles are the same as the cells from they originated.

Standard methods for determining the level of microvesicles in a blood sample are well known in the art and typically involve the methods described in the EXAMPLE.

In some embodiments, the level of the microvesicles derived from Jak2^V617F erythrocytes is compared to a predetermined reference value. Typically, the predetermined reference value is a threshold value or a cut-off value. Typically, a "threshold value" or "cut off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of expression levels in properly banked historical patient samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after quantifying the expression level in a group of reference, one can use algorithmic analysis for the statistic treatment of the determined levels in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is Receiver Operator Characteristic Curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWERSAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

In some embodiments, the predetermined reference value was established in a population of patients who did not have a cardiovascular event when blood was drawn. Accordingly when the level of microvesicles is higher than the predetermined reference value, it is concluded that the patient is at risk of having a cardiovascular event. On contrary, when the level of microvesicles is lower than the predetermined reference value, then is it concluded that the patient is not at risk of having a cardiovascular event.

In some embodiments, high statistical significance values (e.g. low P values) are obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value. Thus, in some embodiments, instead of using a definite predetermined reference value, a range of values is provided. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P value) are retained, so that a range of quantification values is provided. This range of quantification values includes a "cut-off¹ value as described above. For example, according to this specific embodiment of a "cut-off¹ value, the outcome can be determined by comparing the expression level with the range of values which are identified. In some embodiments, a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum p value which is found). For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6. For example, a patient may be assessed by comparing values obtained by determining the level of microvesicles, where values greater than 5 reveal that the patient is at risk of having a cardiovascular event and values less than 5 reveal that the patient is not at risk of having a cardiovascular event. In some embodiments, a patient may be assessed by comparing values obtained by measuring the level of microvesicles and comparing the values on a scale, where values above the range of 4-6 indicate that the patient is at risk of having a cardiovascular event and values below the range of 4-6 indicate that the patient is not at risk of having a cardiovascular event, with values falling within the range of 4-6 indicating an intermediate risk.

The result given by the method of the invention may be used as a guide in determining how frequently a cardiovascular event should be screened, in selecting a therapy or treatment regimen for the patient. For example, when the patient has been determined as having a high risk of a cardiovascular event, he can be eligible for a therapy with a MPO inhibitor as described herein, and/or with a statin. As used herein the term“Statin” has its general meaning in the art and refers to a class of drugs that are inhibitors of HMG-CoA reductase. Examples of statin include but are not limited to pravastatin, fluvastatin, atorvastatin, lovastatin, simvastatin, rosuvastatin, and cerivastatin. Statins may be in the form of a salt, hydrate, solvate, polymorph, or a co-crystal. Statins may also be in the form of a hydrate, solvate, polymorph, or a co-crystal of a salt. Statins may also be present in the free acid or acetone form.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. Jak2^V617F in hematopoietic and endothelial cells increases arterial contraction in an endothelial-dependent manner.

Representative picture of the spleen (A). Haemoglobin (B), platelet (C) and white blood cell count (D) of 8 to 12 weeks old control mice (Jak2WT, n=13) and Jak2^V617F Flex/WT ;VE- Cadherin-cre mice ( Jak2^V6I7F HC-EC, n=13). Cumulative dose-response curves to phenylephrine (E) (Jak2WT, n=13; Jak2^V6I7F HC-EC, n=13), to angiotensin II (G) (Jak2WT, n=3; Jak2^l '^>!71 HC-EC, n=4) and contraction response to potassium chloride (80 mmol/L) (F) (Jak2WT, n=13; Jak2^V6I7F HC-EC, n=13) of aortas with endothelium. Cumulative dose- response curves to phenylephrine of aortas without endothelium (H) (Jak2WT, n=6; Jak2^V617F HC-EC, n=6). Diameter change of femoral artery after phenylephrine injection (10-3 mol/L) (I) (Jak2WT, n=8; Jak2^V6I7F HC-EC, n=8). Electrocardiogram recording before and after intravenous phenylephrine injection (3 mg/kg; Jak2WT, n=13; Jak2^V6I7F HC-EC, n=6) (J), with representative images of the changes observed in 5/6 Jak2^V617F HC-EC vs. only 4/13 Jak2WT (p=0.057) (K). Quantitative data are expressed as median with interquartile range and cumulative dose-response curves are expressed as mean with standard error of the mean. Abbreviations: * p<0.05, *** p< 0.001; ns, not significant. Cumulative dose response curves and electrocardiogram recording were compared using an analysis of variance for repeated measures and other data were compared using the Mann- Whitney U-test. All tests were 2 sided.

Figure 2. Jak2^V617F specifically expressed in hematopoietic cells, but not in endothelial cells, increases arterial contraction

Representative picture of the spleen (A and F). Blood cell count of 10 to 13 weeks old control mice (Jak2WT, n=7) and .Iak2^{1 6171} Flex/WT; VE-Cadherin-cre-ERT2 mice (, Jak2^V617F EC, n=7) (B, C, D) and of 13 to 15 weeks old chimeric C57BL/6 mice transplanted with a bone marrow of wild-type mice (Jak2WT, n=5) or of.lak2^{l 7 /7/} HC-F.C mice (Jak2^V617F HC, n=5) (G, H, I). Data are expressed as median with interquartile range. Cumulative dose-response curve to phenylephrine of aortas from Jak2WT (n=7) and Jak2^V6!7F EC (n=7) (E) and from Jak2WT (n=5) and Jak2^V6I7F HC (n=5) (J). Quantitative data are expressed as median with interquartile range and cumulative dose-response curves are expressed as mean with standard error of the mean. Abbreviations: * p<0.05, ** p< 0.01; ns, not significant. Cumulative dose response curves were compared using an analysis of variance for repeated measures and other data were compared using the Mann- Whitney U-test. All tests were 2 sided.

Figure 3. Microvesicles derived from Jak2^V617F red blood cells are responsible for an increased arterial contraction

Cumulative dose-response curves to phenylephrine of aortas from WT mice incubated with microvesicles isolated from Jak2^V617F patients (n=7) and controls (n=5) at their circulating concentration (A). Cumulative dose-response curves to phenylephrine of aortas from WT mice incubated with microvesicles generated from platelets (n=5 and n=5, respectively) (B), PBMC (n=5 and n=6, respectively) (C), PMNC (n=5 and n=5) (D) and red blood cells (n=9 and n=4, respectively) (E) from Jak2^V617F HC-EC mice (Jak2V617F) or littermate control mice (WT). Change in the diameter of femoral artery induced by phenylephrine injection (10-3 mol/L) in control mice previously injected with control erythrocyte-derived microvesicles (JAK2WT MV GR WT, n=10) or with Jak2^V617F erythrocyte-derived microvesicles (JAK2WT MV GR Jak2^V617F n=10) (F). Allelic discrimination plot of ARN isolated from microvesicles derived from Jak2\VT and Jak2^V6!7F erythrocytes (n=3 per group) (no template control, black) (G). Quantification (H) of the uptake by endothelial cells (HUVEC) of erythrocyte-derived microvesicles from JAK2^V617F mice (n=5) or JAK2^m mice (n=5) or respective 20500 g supernatant (n=3 for each group). Cumulative dose-response curve to phenylephrine of aortas from WT mice injected with vehicle (n 5) or with epoietin (n=8) (I). Quantitative data are expressed as median with interquartile range and cumulative dose-response curves are expressed as mean with standard error of the mean. Abbreviations: *p<0.05; ** p< 0.01; EPO, epoietin, HUVEC, Human umbilical vein endothelial cells, MVs, microvesicles; ns, not significant; NTC, no template control; PBMC, peripheral blood mononuclear cells; PMNC, polymorphonuclear cells; RBC, Red blood cells; SNT, Supernatant; WT, wild type. Cumulative dose response curves were compared using an analysis of variance for repeated measures and other data were compared using the Mann- Whitney U-test. All tests were 2 sided

Figure 4. Disturbed endothelial NO pathway and increased oxidative stress status.

Cumulative dose-response curve of aortas from Jak2^V617F Flex/WT ;VE-Cadherin-cre mice (Jak2^V6I7F HC-EC mice) and littermate controls (Jak2WT) to acetylcholine (n=l l and n=l l, respectively) (A), to S-Mtroso-N-Acetylpenicillamine (SNAP) (n=5 and n=6, respectively) (C) and to phenylephrine after L-NAME incubation (n=l 1 and n=7, respectively) (E). Diameter change of femoral arteries after injection of acetylcholine (10-2 mol/L) (Jak2WT, n=8; Jak2^V617F HC-EC, n=8 (B) and SNAP (10-3 mol/L) (Jak2WT, n=4, .VA2^{, 7,/7/} HC-EC, n=4) (D). Quantification of reactive oxygen species (ROS) generation per endothelial cell in: control mice (Jak2WT) vs. Jak2^V617F HC-EC mice (F); control mice (Jak2WT) and Jak2^{i r}'^r/I Flex/WT : VE-Cadherin-cre-ERT2 mice (Jak2^V6I7F EC) (G); control mice injected with microvesicles derived from control (JAK2WT MV GR WT, n=6) or Jak2^V617F erythrocytes (JAK2WT MV GR Jak2^V617F n=6) (H). Bar scale 10 pm. Cumulative dose-response curve to phenylephrine of aortas from Jak2^V617F HC-EC mice treated with vehicle (n=5) and with NAC (n=7) (I). Quantitative data are expressed as median with interquartile range and compared using the Mann-Whitney U-test and cumulative dose-response curves are expressed as mean with standard error of the mean and compared using an analysis of variance for repeated measures. Abbreviations: * p<0.05, *** p< 0.001; CY24A, NAC, N-Acetyl-cysteine; ns, not significant.

Figure 5. Myeloperoxidase carried by erythrocyte-derived microvesicles from Jak2V617F mice is responsible for increased endothelial oxidative stress.

Volcano plot obtained by using quantitative label-free mass spectrometry analysis of proteins isolated from microvesicles derived from Jak2^V6i7F (n=6) and JAK2WT (n=4) erythrocytes (ratio JAK2^V617FMAK2^WT) (A), only proteins involved in cellular oxidant detoxification (GO 0098869) and ROS metabolic process (GO 0072593) are presented (line corresponds to p-value< 0.05). Respective quantification of GSTT1 (B) and MPO (C) western blots performed on erythrocyte-derived microvesicles (JAK2WT erythrocyte microvesicles, n=l l; Jak2^V617F erythrocyte microvesicles, n=l l). Quantification of reactive oxygen species (ROS) generation per endothelial cell (HUVEC) after exposition of erythrocyte-derived microvesicles from control mice (Jak2WT) (n=4) and Jak2^{i >171} HC-EC mice and without (n=4) and with (n=4) preincubation with a myeloperoxidase inhibitor (MPOi, PF06281355, Sigma, 5 pmol/L) (D). Bar scale 10 pm. Quantitative data are expressed as median with interquartile range and compared using the Mann- Whitney U-test and Kruskal- Wallis test for multiple comparison. Abbreviations: * p<0.05, *** p< 0.001; CY24A, Cytochrome b-245 light chain; CY24B, Cytochrome-b245 Heavy chain; GSTT1, glutathione s transferase theta 1; MPO, myeloperoxidase; ns, not significant.

Figure 6. Simvastatin improves the increased arterial contraction induced by

Jak2^V617F

Spleen to body weight ratio (A), haemoglobin level (B), platelet (C) and white blood cell count (D) in Jak2^V6I7F Flex/WT ;VE-Cadherin-cre mice (Jak2^V617F HC-EC) treated with vehicle {Jak2^V617F HC-EC vehicle, n=4) or with hydroxyurea ( Jak2^V6I7F HC-EC HU, n=7). Spleen to body weight ratio (F), haemoglobin level (G), platelet (H) and white blood cell count (I) in Jak2^V617F HC-EC mice treated with vehicle (Jak2^V6I7F HC-EC vehicle, n=5) or with ruxolitinib {Jak2^V6I7F HC-EC ruxolitinib, n=4). Spleen to body weight ratio (K), haemoglobin level (L), platelet (M) and white blood cell count (N) in Jak2^V6I7F HC-EC mice treated with vehicle (n=10) or with simvastatin ( Jak2^V617F HC-EC simvastatin, n=7). Cumulative dose- response curves to phenylephrine of aortas from Jak2^V617F Jak2^V617F HC-EC mice treated with vehicle or hydroxyurea (./ak2^{i r}'¹⁷¹ HC-EC vehicle n=4; and Jak2^V6I7F HC-EC HU, n=7) (E), with vehicle or ruxolitinib Uak2} ^{6 ! 71} HC -EC vehicle, n= 5; and Jak2^V617F HC-EC Ruxolitinib n=4) (J), and with vehicle or simvastatin (Jak2^V617F HC-EC vehicle, n=10; and Jak2^V6I7F HC- EC simvastatin in, n=7) (O). Data are expressed as mean with standard error of the mean for cumulative curve and median with interquartile range for spleen weight and blood cell count. Abbreviations: * p<0.05, ** p< 0.01, HU hydroxyurea; ns, not significant. Cumulative dose response curves were compared using an analysis of variance for repeated measures and other data were compared using the Mann- Whitney U-test. All tests were 2 side

EXAMPLE: Methods

Experimental design

The objective of our study was to analyse endothelial reactivity in MPN. We first noticed a major increase in arterial contraction in Jak2^V617F HC-EC mice, a model with Jak2^V617F expression both in hematopoietic and endothelial cells, that mimics the human disease. We then created mouse models specifically mutated in endothelial or in hematopoietic cells. We then searched for the mediators responsible for the increased response to vasoconstrictors when Jak2^V6I7F was present in hematopoietic cells and tested the hypothesis that circulating blood might convey biological information from hematopoietic cells to the vascular wall and focused on microvesicles. We identified that erythrocyte-derived microvesicles were responsible for this effect and performed a mass spectrography analysis to highlight the proteins involved. Sample size was chosen based on previous works using the same technique (myography) and microvesicles, published by our team (48, 49).

Mouse breeding occurred in our animal facility in accordance with the local recommendations. Control mice were littermate, appropriate, age, sex and genetic background, matched to account for any variation in data. Institutional animal care and use committee at INSERM (Descartes university, Paris, France) approved all animal experiments (CEEA- 17053).

Number of experimental replicates is provided in each figure legend and included at least 3 independent experiments. For each myography experiment, duplicates with the same aorta were used, averaged and counted as n=l . There was no randomization in these experiments. We did not exclude any other sample than those not fulfilling the quality criteria detailed in the corresponding methods section. Only aorta with a viable endothelium were used for myography (see corresponding methods section for criteria).

For human samples, inclusion and exclusion criteria were defined prior to sample collection (see corresponding methods section for criteria). No outlier was excluded. Investigators were not blinded to group allocation during collection and analysis of the data. All patients (carrying Jak2^V617F with a past history of splanchnic vein thrombosis, not receiving any specific treatments other than vitamin-k antagonists) and healthy volunteers gave writing consent to the study. Human study was performed in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the institutional review board Bichat- Claude-Bemard (Paris; France). Murine models

All mice were on a C57BL/6 background. Mice carrying constitutive Jak2^{l Y}'¹⁷¹ mutation in endothelial and hematopoietic cells were obtained by crossing VE-cadherin-Cre transgenic mice provided by M. Souyri (13) with J 2^(/577 Flex/WT mice provided by M. Villeval (50). Mice carrying inducible Jak2^V617F mutation specifically in endothelial cells were obtained by crossing VE-Cadherin-cre-ERT2 transgenic mice provided by R. Adams (51) with Jak2^V6I7F Flex/WT mice provided by M. Villeval (50). The Flex (for Flip-Excision) strategy allows the expression of a mutated gene in adulthood, in a temporal and tissue-specific manner (52). It allows an efficient and reliable Cre-mediated genetic switch: the expression of a given gene is turned on by inversion, while expression of another one is simultaneously turned off by excision. In all experiments, male and female mice were used.

For organ chamber experiments and femoral in vivo experiments, mice were euthanized between the ages of 8 and 17 weeks. For induction of Cre recombinase expression in Jak2^V6!7F Flex/WT; VE-Cadherin-cre-ERT2 mice, mice were injected intraperitoneally with tamoxifen (Sigma, T5648), 1 mg/mouse/day for 5 consecutive days over 2 consecutive weeks (10 mg in total per mouse) between the ages of 5 to 7 weeks. Experiments were performed between 4 to 6 weeks after the last tamoxifen injection. Both female and male were used for each experiment.

Experiments were conducted according to the French veterinary guidelines and those formulated by the European community for experimental animal use (L358-86/609EEC) and were approved by the French ministry of agriculture (n° A75-15-32).

Verification of the efficient endothelial recombination in mouse models

All mice were on a C57BL/6 background. Mice with the mTmG reporter provided by C. James (Inserm 1034) were crossed with VE-cadherin-Cre transgenic mice provided by M. Souyri or VE-Cadherin-cre-ERT2 transgenic mice provided by R. Adams (51). For induction of mTmG;VE-Cadherin-cre-ERT2 model, mice were injected intraperitoneally with tamoxifen (Sigma, T5648), 1 mg/mice/day for 5 consecutive days over 2 consecutive weeks (10 mg in total per mice) between the ages of 5 to 7 weeks, and experiments were performed 2 weeks after the last injection of tamoxifen. Aortas and femurs were harvested under isoflurane anaesthesia and fixed in 4% PFA. Aorta were mounted“en face” on glass slides, while femurs were cryosectioned. All tissues were imaged using a Leica SP5 confocal microscope (Leica) at 400 X magnification. For flow cytometry analysis in Cre;mT/mG mice, bone marrow cells were stained with TER - 119 APC and Gr - 1 APC (553673, Becton Dickinson) and analyzed on an Accuri C6 flow cytometer (BD Biosciences). Data were interpreted using BD Accuri C6 Analysis Software.

Patient's inclusion

All patients fulfilling inclusion criteria were prospectively included at the Hepatology department, Beaujon Hospital, Clichy, France, between May 2016 and July 2016. Only patients carrying Jak2^V617F without specific treatment for MPNs were included. All patients had a past history of Budd-Chiari syndrome or portal vein thrombosis and were receiving vitamin K antagonists. Controls were healthy voluntaries. All patients and controls gave written consent to the study. This study was performed in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by our institutional review board (CPP lie de France IV, Paris; France).

Organ Chamber Experiments

Thoracic aortas from adult mice were isolated after animal sacrifice under 2% isoflurane anaesthesia. The aortic rings were mounted immediately in organ chambers (Multi WireMyograph system, model 610 M; Danish Myo Technology, Aarhus, Denmark) filled with Krebs-Ringer solution (NaCl 118.3 mmol/L, KC1 4.7 mmol/L, MgS04 1.2 mmol/L, KH2P04 1.2 mmol/L, CaC12 1.25 mmol/L, NaHC03 25.0 mmol/L and glucose 5 0 mmol/L) gassed with a mixture of 02 95% and C02 5% (pH 7.4). The presence of functional endothelial cells was confirmed by the relaxation to acetylcholine chloride (Sigma, A6625) (10-5 mol/L) following a contraction evoked by phenylephrine (10-7 mol/L) and was defined as a relaxation > 70% of the precontraction as previously described (49). After extensive washout and equilibration, contraction to phenylephrine hydrochloride (concentration-response curve, 10-9 to 10-4 mol/L) (Sigma, P1250000) or angiotensin II (concentration-response curve, 10-9 to 10-6 mol/L) (Sigma, A9525) or KCL (80 mmol/L) and relaxation to acetylcholine chloride (concentration-response curve, 10-9 to 10-4 mol/L) or SNAP (S-Nitroso-N-acetyl-DL- penicillamine, Sigma, N3398) (concentration-response curve, 10-10 to 10-5 mol/L) were studied. For NO synthase inhibition, aorta rings were preincubated for 45 min with L-NAME 10-4 mol/L (Cayman, 80210) prior to concentration-response curve to phenylephrine without washout. In some experiments, the endothelium was mechanically removed by inserting the tip of a pair of forceps within the lumen and by gently rubbing the ring back and forth on a piece of wet tissue. For the N- Acetyl- Cysteine experiment (NAC, commercial HIDONAC®, Zambon), NAC was added to the Krebs-Ringer solution at a final concentration of 20 mmol/L. In vivo femoral reactivity

The femoral artery was carefully exposed from adult mice under 2% isoflurane anaesthesia.

Krebs-Ringer solution (cf organ chamber experiment) gassed with a mixture of 02 95% and C02 5% (pH 7.4) at 37° was permanently superfuse (2 mL/min) on the exposed artery. After a 15-minute equilibration, arterial responses were determined by addition of phenylephrine (10-3 mol/L) for 2 min then acetylcholine (10-1 mol/L) for 1 min (Sigma Chemical Co.). On the controlateral leg, with the same protocol, KCL (80 mmol/L) then SNAP (S-Nitroso-N-acetyl-DL-penicillamine, 10-3 mol/L) were used. All dilutions were prepared just before application. Changes in vessel diameter were continuously recorded on a videotape recorder. Subsequently, images were exported and vessel outer diameters were analyzed using Image J Software.

Isolation and characterization of patients’ circulating microvesicles

Circulating microvesicles from patients or healthy control were isolated from platelet- free plasma obtained by successive centrifugations of venous blood, as reported previously (53). Briefly, citrated venous blood (15 mL) was centrifuged twice at 2500g for 15 minutes (at room temperature) to remove cells and cell debris and to obtain platelet-free plasma (PFP). A portion of this PFP was then aliquoted and stored at -80°C. The rest was centrifuged at 20500g for 2 hours (4°C). Supernatant of this 20500g centrifugation was then discarded and the resulting microvesicles pellet was resuspended in a minimal volume of supernatant, aliquoted and stored at -80°C. For each patient, concentrations of annexin V positive microvesicles were analysed in the PFP and the resuspended pellet of microvesicles.

Circulating levels of annexin V+ microvesicles (IM3614, BD) were determined on a Gallios flow cytometer (Beckman Coulter, Villepinte, France) using a technique previously described in detail (49, 53).

Generation of microvesicles from mice

Blood samples were collected from the inferior vena cava of Jak2^V617F HC-EC mice or littermate controls using a 25 G x G needle in a 1 mL syringe pre-coated with 3.8% sodium citrate. PFPs were generated as described above for patients and used to measure plasma annexin V positive microvesicles in mice. The pelleted cells obtained following the first 2500g centrifugation were resuspended in PBS to a final volume of 5 mL for control mice and 10 mL for Jak2^V6I7F HC-EC mice. PBMC, PMNC and erythrocytes were separated using a double percoll gradient (63% and 72% for control mice and 63% and 66% for Jak2^{V l7!} HC-EC mice) using a 700g centrifugation for 25 min, without brake. The slight differences between the protocols used for control and Jak2^V617F HC-EC mice are the results of the preliminary experiments we did to obtain pure isolation of each cell type. Cells were subsequently washed with PBS, then incubated with 5 gmol/L ionomycin TBS for 30min at 37°C to induce microvesicles generation. 5 mmol/L EDTA was then added to chelate free calcium. Cells were then discarded by centrifugations at 15000g for 1 min and the supernatants were collected. Microvesicles were isolated, as described above using a 20500g centrifugation during 45min. Concentrations of annexin V positive microvesicles (as described above) were analysed in the PFP and the 20500g microvesicles pellet for each mouse.

To isolate platelets, 500 pL of whole blood were diluted in 10 mL of PBS. A 1.063 g/mL density barrier was created by combining 5 mL of 1.320 g/mL 60% iodixanol stock solution (OptiPrep density gradient medium, Sigma-Aldrich, Saint Louis, MO, USA) with 22 mL of diluent (0.85% NaCl, 20 mM HEPES-NaOH, pH 7.4, 1 mM EDTA). For platelet separation, 10 mL of each diluted blood were layered over 10 mL of density barrier and centrifuged at 350 g for 15 minutes at 20°C with the brake turned off. The interface between the density barrier and the blood contained platelets. Residual contaminating erythrocytes were removed by magnetic sorting. Briefly, the cell suspension was labelled with Anti-Ter-119 MicroBeads (Miltenyi Biotec ref 130-049-901) and erythrocytes (Ter- 119+) were negatively sorted using a MACS® Separator. The remaining cells (platelets) were subsequently washed with PBS and exposed to 5 pmol/L ionomycin in TBS for 30 minutes at 37°C. 5 mM EDTA was then added to chelate free calcium. Finally, cells were discarded by centrifugation at 15000g for 1 minute, the supernatant was collected and microvesicles isolated, as previously described.

Vascular reactivity following exposure to microvesicles

For organ chamber experiments, thoracic aortas from adult C57BL/6 mice (8 to 10 weeks old) were isolated after sacrifice under isoflurane anaesthesia. Mouse aortic rings were incubated for 24 hrs; 37°C in a 5% C02 incubator, with filtered DMEM supplemented with antibiotics (100 IU/mL streptomycin, 100 IU/mL penicillin (Gibco, Invitrogen, Paisley, Scotland), and 10 pg/mL polymyxin B (Sigma, St Louis, MO) in the presence of microvesicles. Aortic rings were then mounted in organ chambers and concentration-response curves to pharmacological agents were performed. For femoral artery in vivo experiment, C57BL/6 mice were injected intravenously (retro-orbital injection) with microvesicles (100 pL final volume with 2 pL heparin sodium (5000 IU/mL)). Experiments were performed 2 hours after injection.

Microvesicles from patients and healthy controls were incubated at their respective individual plasma concentration (Annexin-V positive microvesicles). Microvesicles generated from mice were incubated or injected at the same final concentration for Juki^{1 6171} HC-EC mice and control mice, namely 7000 Annexin V positive microvesicles / pL for erythrocyte and platelet-derived microvesicles and 700 Annexin V positive microvesicles / pL for PBMC and PMNC-derived microvesicles. We chose these concentrations since we found in preliminary experiments that the majority of mice have concentrations of Annexin V positive microvesicles between 1000 and 10000 / pL, and because PBMC and PMNC-derived microvesicles are consistently found less abundant in the blood than erythrocyte and platelet-derived microvesicles (27, 32).

Bone marrow transplantation

We subjected 6 to 8 weeks old C57B1/6J mice to medullar aplasia following 9.5 gray lethal total body irradiation. We repopulated the mice with an intravenous injection of bone marrow cells isolated from femurs and tibias of age matched Jak2^V617F HC-EC and of littermate control mice. Medullar reconstitution was allowed for 8 weeks before experiments.

Treatments

Hydroxyurea (Sigma, H8627), or the same volume of vehicle (NaCl 0.9%), was administrated for 10 consecutive days (100 mg/kg/day BID) by intra-peritoneal injections.

Ruxolitinib (Novartis) was administered for 21 consecutive days (30 mg/kg 2 times per day) by oral gavage (54). Ruxolitinib was prepared from 15-mg commercial tablets in PEG300/5% dextrose mixed at a 1 :3 ratio, as previously reported (55). Control mice were administered the same volume of vehicle (PEG300/dextrose 5%).

Simvastatin (Sigma S6196) was administered for 14 days (20 mg/kg/day, once a day) by intra-peritoneal injections. Activation by hydrolysis was first achieved by dissolving 50 mg in 1 mL of pure ethanol and adding 0.813 ml of 1 mol/L NaOH. pH was adjusted to 7.2 by adding small quantities of 1 mol/L HC1 and dilution was then performed in PBS (56). Control mice were injected with the same volume of vehicle. Human recombinant Epoietin alfa (5000 UEkg, diluted in 0.2% BSA in PBS) or vehicle (0.2% BSA in PBS) was administered to wild type mice every 2 days for 3 weeks by intraperitoneal injection, as previously described (57).

N-Acetyl-Cysteine (commercial HIDONAC, Zambon) diluted in NaCl 0.9% or the same volume of vehicle (NaCl 0.9%), was administrated for 14 consecutive days (500 mg/kg/day) by intraperitoneal injections.

Blood Cell count analysis

Blood was collected on the day of sacrifice from the inferior vena cava using a 25G x 1’ needle in a 1 mL syringe pre-coated with 3.8% sodium citrate. Blood counts analyses were performed using a Hemavet 950FS analyser (Drew scientific).

Quantification of reactive oxygen species generation

Thoracic aortas from adult mice were isolated after animal sacrifice under 2% isoflurane anaesthesia, longitudinally opened and placed directly in HBSS (Hanks' balanced salt solution, Sigma, 14025-092). For each set of experiments, all aortas were processed immediately after removal, at the same time, with the same reagents and in the same manner. No plasma factor or blood cells were added during the ROS generation assessment. For positive and negative controls, 2 pieces of wild type aortas were incubated with H202 (100 pmol/L final concentration) for 20 min at 37°C. For negative controls, N-Acetyl-Cysteine (5 mmol/L final concentration) was incubated together with H202 for 20 min at 37°C. All aortas were then incubated with 5 pmol/L CellROX® (Fisher scientific, C10422) for 30 min at 37°C. CellROX® Deep Red Reagent is a fluorogenic probe designed to reliably measure reactive oxygen species inside living cells. The cell-permeable CellROX® Deep Red dye is nonfluorescent while outside of the cell and in a reduced state and, upon oxidation, exhibits excitation/emission maxima at 640/665 nm. After rinsing and fixation (Paraformaldehyde 4%, 20 min), samples were costained with DAPI (0.1 pg mL, Sigma) in order to identify cell nuclei. After staining, aortas were washed with PBS, mounted“en face” on glass slides and imaged using a bright field Zeiss Axio Imager Z1 (Zeiss) microscope. Images were acquired in the 2 hours following staining at 400 X magnification. CellROX® positive surface (in red) and the number of cells were quantified using Image J Software.

Myeloperoxidase inhibition in microvesicles Erythrocyte-derived microvesicles from Jak2^V617F HC-EC mice were incubated for 1 hr with an irreversible MPO inhibitor (MPOi, PF06281355, resuspended in DMSO, Sigma), diluted in PBS (5 Dmol/L final concentration). Then, the same amount of annexin V positive erythrocyte-derived microvesicles (JAK2WT, Jak2^V617F and Jak2^V617F with MPOi) were washed in PBS and centrifuged at 20500g for 2 hrs. The pellet containing the microvesicles was then resuspended in endothelial cell basic medium (Promocell). HUVECs (single donor, C-12200, lot 445Z011, PromoCell) were then incubated for 2 hrs at 37°C with these microvesicles. At the end of the incubation, and without washing cells, reactive oxygen species generation was assessed using CELLROX® (Fisher scientific, Cl 0422), as described above. After rinsing with medium and paraformaldehyde (4%, 5 min), HUVECs were costained with DAPI (0.1 pg/mL, Sigma) in order to identify nuclei. Images were acquired using a Confocal microscope, Leica SP8 at 400 X magnification.

Electrocardiography

Electrocardiograms were recorded from mice using the non-invasive ecgTUNNEL (Emka Technologies) with minimal filtering Electrocardiogram was continuously monitored for 3 min (baseline). Waveforms were recorded using Iox Software and heart rate and intervals were measured with ECG Auto from recording traces. Following baseline determination, the animals received a single administration of phenylephrine (bolus, 3mg/Kg) by intravenous route at the caudal vein and electrocardiogram were recording 3-5 minutes more.

RNA gene allelic discrimination

Erythrocytes microvesicles were lysed with Qiazol lysis reagent (Qiagen) and RNA was extracted with RNeasy micro Kit (Qiagen) according to manufacturer’s instructions. Dosage of RNA was performed with Qubit HS RNA assay Kit (ThermoFisher Scientitic). cDNA synthesis was performed with QuantiTect Reverse Transcription Kit (Qiagen). RNA gene allelic discrimination was performed by Taqman analysis with the ABI Prism GeneAmp 7500 Sequence Detection System (Applied Biosystem, Invitrogen) using as primers: TTTACAAATTCTTGAACCAGAATGTTC (JAK2 forward - SEQ ID NO: 1) and TTCTCACAAGCATTTGGTTTTGAAT (JAK2 reverse SEQ ID NO: 2) and as probes: VIC- CTCCACAGACACAGAC-MGB for JAK2WT (SEQ ID NO: 3) and 6-FAM- TCTCCACAGAAACAGAGA-MGB for Jak2^Vf'^m (SEQ ID NO: 4)

Mass spectrometry analysis Size-exclusion chromatography of microvesicles was then performed in order to separate microvesicles from soluble proteins. Successive aliquot of 150 pL were collected and measurement of protein absorbance was performed. Fractions contained in tubes 6 to 11, containing microvesicles, were selected and then centrifuged at 20 500g for two hours. To finish, microvesicles were lysed using a 1% triton buffer.

For mass spectrometry analysis, proteins were precipitated overnight at -20°C with 0.1 mol/L Ammonium Acetate glacial in 80% methanol (buffer 1). After centrifugation at 14000xg and 4°C for 15 min, the resulting pellets were washed twice with 100 pL of buffer 1 and further dried under vacuum (Speed-Vac concentrator). Proteins were then reduced by incubation with 10 pL of 5 mmol/L dithiotreitol (DTT) at 57°C for one hour and alkylated with 2 pL of 55 mmol/L iodoacetamide for 30 min at room temperature in the dark. Trypsin/LysC (Promega) was added twice at 1 : 100 (wt:wt) enzyme: substrate, at 37°C for 2 hrs first and then overnight. Samples were then loaded onto a homemade Cl 8 StageTips for desalting. Peptides were eluted using 40/60 MeCN/H20 + 0.1% formic acid and vacuum concentrated to dryness. Online chromatography was performed with an RSLCnano system (Ultimate 3000, Thermo Scientific) coupled online to a Q Exactive F1F-X with a Nanospay Flex ion source (Thermo Scientific) Peptides were first trapped on a Cl 8 column (75 pm inner diameter x 2 cm; nanoViper Acclaim PepMapTM 100, Thermo Scientific) with buffer A (2/98 MeCN/H20 in 0.1% formic acid) at a flow rate of 2.5 pL/min over 4 min. Separation was then performed on a 50 cm x 75 pm Cl 8 column (nanoViper Acclaim PepMapTM RSLC, 2 pm, lOOA, Thermo Scientific) regulated to a temperature of 50°C with a linear gradient of 2% to 30% buffer B (100% MeCN in 0.1% formic acid) at a flow rate of 300 nL/min over 91 min. MS full scans were performed in the ultrahigh-field Orbitrap mass analyzer in ranges m/z 375-1500 with a resolution of 120 000 at m/z 200. The top 20 intense ions were subjected to Orbitrap for further fragmentation via high energy collision dissociation (HCD) activation and a resolution of 15 000 with the intensity threshold kept at 1.3 x 105. We selected ions with charge state from 2+ to 6+ for screening. Normalized collision energy (NCE) was set at 27 and the dynamic exclusion of 40s.

For identification, data were searched against the Mus Musculus one gene one protein (UP000000589_10090) UniProt database and a databank of the common contaminants using Sequest F1F through proteome discoverer (version 2.2). Enzyme specificity was set to trypsin and a maximum of two-missed cleavage sites were allowed. Oxidized methionine and N- terminal acetylation were set as variable modifications. Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.02 Da for MS MS peaks. The resulting files were further processed using myProMS (58) v3.6. FDR calculation used Percolator and was set to 1% at the peptide level for the whole study. The label free quantification was performed by peptide Extracted Ion Chromatograms (XICs) computed with MassChroQ version 2.2 (59). For protein quantification, XICs from proteotypic peptides shared between compared conditions (TopN matching) with two-missed cleavages were used. Median and scale normalization was applied on the total signal to correct the XICs for each biological replicate. To estimate the significance of the change in protein abundance, a linear model (adjusted on peptides and biological replicates) was performed and p-values were adjusted with a Benjamini-Hochberg FDR procedure with a control threshold set to 0.05.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (60) partner repository with the dataset identifier PXD014451.

Western Blot on erythrocyte-derived microvesicles

Erythrocyte-derived microvesicles generated as mentioned above were centrifuged at 20 500g for 2 hrs, and then lysed in 100 DL RIPA buffer containing 150 mmol/L NaCl, 50 mmol/L TrisHCl, pH 7.4, 2 mmol/L EDTA, 0.5% sodium deoxycholate, 0.2% sodium dodecyl sulfate, 2 mmol/L activated orthovanadate, complete protease inhibitor cocktail tablet (Complet mini, Roche, France) and complete phosphatase inhibitor cocktail tablet (Roche, France). Protein content was quantified using the Micro BCA Protein Assay Kit (Thermo Scientific). Equal loading was checked using Ponceau red solution. Membranes were incubated with primary antibodies (1/1000) (Anti-GP91, BD611415; Anti-GSTTl, Abcaml99337; AntiMPO, Abcam45977). After secondary antibody incubation (anti-rat, Cell Signaling, 1/1000; anti rabbit or anti-mouse, Amersham, GE Healthcare, UK 1/3000), immunodetection was performed using an enhanced chemiluminescence kit (Immun-Star Western C kit, Bio-Rad). Bands were revealed using the LAS-4000 imaging system. Values reported from Western blots were obtained by band density analysis using with ImageJ software and expressed as the ratio protein of interest compared to Ponceau.

Uptake of microvesicles by endothelial cells

Erythrocyte-derived microvesicles were stained with PKH26 dye (Sigma Aldrich) diluted in PBS, following manufacturer’s instructions, washed in PBS and then centrifuged at 20500g for 2 hrs. The 20500g supernatant served for control experiments. Murine endothelial cells (the cell line called SVEC4-10, CRL-2181, lot 70008729, ATCC) were then incubated with these stained microvesicles or an equal volume of the 20500g supernatant. After 2 hrs at 37°C, cells were washed 3 times with DMEM (Gibco). Cells were then fixed in PFA 4% for 5 min, and then costained with DAPI (0.1 Dg/mL, Sigma) in order to identify cell nuclei. Images were acquired using a Confocal microscope, Leica SP8 at 600 X magnification.

Statistics

For cumulative dose response curves, data were expressed as mean with standard error of the mean and compared using an analysis of variance for repeated measures. Other data were expressed as median with interquartile range (blood cell count and spleen weight) and compared using the Mann- Whitney U-test. All tests were 2 sided and used a significance level of 0.05. Data handling and analysis were performed with GraphPad Software, Inc.

Study approval

Institutional animal care and use committee at INSERM (Descartes university, Paris, France) approved all animal experiments (CEEA- 17-053).

For human samples, inclusion and exclusion criteria were defined prior to sample collection (see corresponding methods section for criteria). All patients (carrying Jak2^V617F with a past history of splanchnic vein thrombosis, not receiving any specific treatments other than vitamin-k antagonists) and healthy volunteers gave writing consent to the study. Human study was performed in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the institutional review board (CPP He de France IV, Paris; France).

Results

Increased arterial contraction in mice carrying Jak2^V6I7F in hematopoietic and endothelial cells

As Jak2^V617Fis present in both hematopoietic and endothelial cells in patients with MPN (2, 3), we first investigated vasoactive response in a mouse model mimicking the human disease. We generated mice expressing Jak2^V617F in hematopoietic and in endothelial cells by crossing Jak2^V6I7F Flex/WT mice with VE-Cadherin-cre mice. VE-Cadherin being expressed during early embryonic life in a precursor of both endothelial and hematopoietic cells (13), Jak2^V6I7F Flex/WT; VE-Cadherin-cre, thereafter referred to as Jak2^V6I7F HC-EC, developed as expected a MPN, attested by higher spleen weight (2.3 to 5.7 % of body weight vs. 0.3 to 0.6 % for littermate controls, pO.OOOl), and higher haemoglobin level, platelet and white blood cell counts than littermate controls (Figures 1A-D). The endothelial and hematopoietic progenitor cells recombination was verified by crossing VE-Cadherin-cre with mTmG mice. By performing myography assay, we observed ex vivo that aortas from Jak2^V617F HC- EC mice displayed a major increase in the response to phenylephrine (Figure IE), but also to potassium chloride (Figure IF) and to angiotensin II (Figure 1G), as compared with littermate controls. Removing the endothelium suppressed this increased arterial contraction (Figure 1H). Likewise, we observed in vivo that femoral arteries from Jak2^V617F HC-EC mice displayed an increased response to phenylephrine as compared with littermate controls (Figure II). Thus, Jak2^V617F in endothelial and hematopoietic cells strongly increases arterial response to vasoconstrictors in an endothelium-dependent manner.

Because of the high rate of myocardial infarction without significant coronary stenosis reported in patients with MPN (8), we investigated cardiac vascular bed by performing electrocardiography in Jak2^V6!7F HC-EC mice and their littermate controls. After intravenous injection of phenylephrine Jak2^V617F HC-EC mice displayed electrocardiogram modifications including bradycardia and arrhythmia, that are indirect signs of coronary spasm (14) (Figures 1J-K)

Increased arterial contraction in mice with Jak2^V617F specifically expressed in hematopoietic cells but not in endothelial cells

To determine if this increased arterial contraction was due to Jak2^V617F in endothelial cells or in hematopoietic cells, we first generated mice expressing Jak2^V6I7F only in endothelial cells. We crossed Jak2^V6I7F Flex/WT mice with inducible VE-Cadherin-cre-ERT2 mice expressing the ere recombinase after tamoxifen injection only in endothelial cells. As expected, Jak2^V6I7F Flex/WT; VE-Cadherin-cre-ERT2 (thereafter referred to as ./ak2^{i 6l /}' EC) mice did not develop MPN (Figures 2A-D). Adequate endothelial recombination was verified by crossing VE-Cadherin-cre-ERT2 mice with mTmG mice. We previously demonstrated the absence of hematopoietic recombination in this model (15). We observed no difference in arterial response to phenylephrine between the Jak2^V617F EC mice and littermate controls (Jak2WT) (Figure 2E).

To assess the implication of Jak2^V617F in hematopoietic cells, we generated mice expressing Jak2^V617F only in hematopoietic cells, by transplanting lethally irradiated C57BL/6 mice with Jak2^V617F bone marrow cells obtained from Jak2^V617F HC-EC mice. Irradiated C56BL/6 mice transplanted with Jak2WT BM were used as controls. Hematopoietic expression of Jak2^V617F induced a MPN (Figures 2F-I) and an increased arterial response to phenylephrine (Figure 2J). Taken altogether, these findings demonstrate that presence of the Jak2^V617F mutation in hematopoietic, but not in endothelial cells, is responsible for the increase in arterial contraction in response to vasoconstrictors we observed in Jak2^V617F HC-EC mice (Figures 1E- G)

Increased arterial contraction induced by microvesicles from Jak2^V6I7F patients

We then sought to identify the mediators responsible for the increased response to vasoconstrictors when Jak2^V617F was present in hematopoietic cells and tested the hypothesis that circulating blood might convey biological information from hematopoietic cells to the vascular wall. Circulating microvesicles, i.e. extracellular vesicles having a size ranging from 0.1 to 1 pm, are now recognized as triggers of various types of vascular dysfunction (16). We therefore examined the effect of circulating microvesicles isolated from the blood of patients with MPN on vascular responses to vasoactive agents. We isolated plasma microvesicles from 7 patients carrying Jak2^V617F (2 males, 5 females; blood drawn before introduction of cytoreductive therapy), and from 5 healthy controls (2 males, 3 females; age not significantly different from patients). We incubated these microvesicles at their plasma concentration with aortic rings from wild type mice and observed that plasma microvesicles from patients carrying Jak2^V617F reproduced the increased response to phenylephrine (Figure 3A). Plasma without microvesicles from the same patients and controls had no effect (data not shown).

Increased arterial contraction induced by erythrocytes-derived microvesicles from Jak2^V617F mice

We then sought to determine the subpopulation of microvesicles responsible for this increased arterial contraction. We generated microvesicles from each type of blood cells from Jak2^V617F HC-EC mice or littermate controls and incubated these microvesicles, at the same concentration, with aortic rings from wild type mice. Erythrocyte-derived microvesicles generated from Jak2^V617F HC-EC mice reproduced the increased response to phenylephrine on aortic rings ex vivo (Figure 3E), while platelet, peripheral blood mononuclear cell and polynuclear neutrophil microvesicles did not (Figures 3B-D). Likewise, in vivo, femoral arteries from wild-type mice injected with microvesicles derived from Jak2^V617F erythrocytes displayed an increased response to phenylephrine as compared with wild-type mice injected with microvesicles derived from littermate controls’ erythrocytes (Figure 3F). Microvesicles generated from Jak2^V617F HC-EC mice erythrocytes carried Jak2^V617F mRNA (Figure 3G). To investigate the interaction of erythrocyte-derived microvesicles with endothelial cells, we labeled microvesicles with the fluorescent dye PKH-26, incubated them with endothelial cells and then performed confocal microscopy on endothelial cells. Fluorescence was detected in endothelial cells, suggesting that erythrocyte-derived microvesicles were taken-up by endothelial cells (17). No difference in uptake was observed between microvesicles from Jak2^V617F HC-EC erythrocytes and their wild-type counterparts (Figure 3H).

We then wanted to determine whether the increased number of erythrocytes could in itself explain this effect or if qualitative changes were involved. We generated a mouse model of polycythaemia without Jak2^V6!7F , caused by chronic epoietin injections. After 3 weeks of epoietin treatment, haemoglobin reached a level similar to that of Jak2^V6I7F HC-EC mice (18.5 g/dL, interquartile range 16.5-19.5, vs. 17.6 g/dL, interquartile range 15.7-19.7, respectively; n=5 and n=13 respectively; p=0.67). However, this model with high number of circulating erythrocytes failed to reproduce the increased response to phenylephrine observed in Jak2^V6,7F HC-EC mice aortas (Figure 31). Thus, the presence of the Jak2^V617F mutation in erythrocyte- derived microvesicles is required to cause increased arterial contraction.

NO pathway inhibition and endothelial increased oxidative stress status

We then investigated how microvesicles derived from Jak2^V617F erythrocytes increase response to vasoconstrictive agents.

We examined first the nitric oxide (NO) pathway. We observed ex vivo on aortas and in vivo on femoral arteries that Jak2^V617F HC-EC mice, reproducing the human disease, display an impaired dilatation to acetylcholine (Figures 4A, B). This impaired dilatation capacity was not due to decreased sensitivity to NO of vascular smooth muscle cells, as the response to a direct NO-donor (SNAP) was not different between Jak2^V617F HC-EC mice and littermate controls, both ex vivo and in vivo (Figures 4C, D). We also observed that, after pre-incubation with the NO synthase (NOS) inhibitor L-Name, aorta from Jak2^V617F HC-EC mice had a similar response to phenylephrine as littermate controls (Figure 4E). Therefore, these results demonstrate that the increased arterial contraction observed in Jak2^V617F HC-EC mice results from a dysfunctional endothelial NO pathway.

Because previous works showed that heme in erythrocytes microvesicles can scavenge NO (18, 19), we quantified heme in microvesicles derived from Jak2^V617F HC-EC mice and control mice erythrocytes, but observed no difference.

We then investigated generation of reactive oxygen species, i.e. inhibitors of NOS activity and NO bioavailability (20) and demonstrated 4 times more reactive oxygen species in the aortic endothelium of Jak2^V6I7F HC-EC mice than in littermate controls (Figure 4F). Conversely, reactive oxygen species generation was normal in the aortic endothelium of Jak2^V6I7F EC mice, expressi ng Jak2^{1 7} ' ⁷ only in endothelial cells (Figure 4G). Likewise, aortic endothelium from wild-type mice injected with microvesicles derived from Jak2^V6I7F erythrocytes displayed more reactive species generation than aortic endothelium of mice injected with microvesicles derived from littermate controls erythrocytes (Figure 4H). There was no reactive oxygen species generation in underlying smooth muscle cells in any of these experiments (data not shown). Altogether, these results show that microvesicles derived from JAK2^V617F erythrocytes induce an excessive oxidative stress in endothelial cells leading to a decreased availability of NO.

To ascertain the implication of the increased oxidative stress in the increased arterial contraction in Jak2^V617F HC-EC mice, we treated these mice with N-Acetyl-Cysteine (NAC, activator of glutathione pathway and anti-oxidant) for 14 days intraperitoneally. This treatment had no effect on blood cell count or spleen weight but normalized arterial contraction to phenylephrine (Figure 41).

Increased endothelial oxidative stress status by myeloperoxidase in erythrocyte- derived microvesicles from Jak2^V617F mice

To shed light on the mechanisms underlying this increased oxidative stress induced by microvesicles derived from Jak2^V6I7F erythrocytes, we performed a proteomic analysis of these microvesicles (data are available via ProteomeXchange with identifier PXD014451). All proteins involved in reactive oxygen species detoxification or generation are shown in the volcano-plot shown in Figure 5A. We found one protein significantly deregulated (glutathione S transferase theta 1, GSTT1) and one protein only detected in microvesicles derived from Jak2^V6I7F erythrocytes (myeloperoxidase, MPO) that could explain the observed effect (Figure 5A). We also considered cytochrome b-245 heavy and light chain (NOX2) although only few peptides were detected in microvesicles derived from Jak2^V617F erythrocytes (1 peptide of heavy chain in 1/6 samples and 1 peptide of light chain in 2/6 samples), since NOX plays a key role in oxidative stress and since no NOX2 peptide was detected in microvesicles derived from JAK2WT erythrocytes. Western blot analyses were then performed on microvesicles derived from Jak2^V617F and control erythrocytes to verify proteomic results. By western blot, NOX2 expression evaluated by Gp91 was not significantly different between Jak2^V6I7F and controls erythrocyte-derived microvesicles. GSTT1, which has an anti-oxidant effect (21), was significantly lower in Jak2^V617F than in control erythrocyte-derived microvesicles (Figure 5B). Expression of MPO, a protein with a strong pro-oxidant effect (22) was much higher in Jak2^V617F than in control erythrocyte-derived microvesicles (Figure 5C). We then directly inhibited MPO in microvesicles derived from erythrocytes of Jak2^V617F HC-EC mice before incubation with endothelial cells (HUVEC) in vitro. We observed that the MPO inhibitor (PF06281355) completely reversed the increase in endothelial oxidative stress induced by Jak2^V617F erythrocyte-derived microvesicles (Figure 5D).

In conclusion, Jak2^V6I7F erythrocyte-derived microvesicles carry MPO that confers a pro-oxidant phenotype to endothelial cells, leading to increased arterial contraction observed in Jak2^V6]7F ¥LC-EC mice.

Statins as a potential new treatment in myeloproliferative neoplasms

We then tested if available treatments for MPNs, namely hydroxyurea and ruxolitinib, affect this increased arterial contraction. ln Jak2^V617FY£-EC mice, best representing the human disease, hydroxyurea for 10 consecutive days decreased spleen weight, haemoglobin level and white blood cell count (Figures 6A, 6B, 6D). However, platelet count was not affected by this short time hydroxyurea treatment (Figure 6C). Hydroxyurea significantly improved contraction in response to phenylephrine as compared to vehicle (Figure 6E).

We then treated Jak2^V617F HC-EC mice with ruxolitinib for 21 consecutive days and observed a significant decrease in the spleen weight and white blood cell count (Figures 6F, 61), but no effect on the haemoglobin level and on platelet count (Figures 6G, 6H) Ruxolitinib had no effect on arterial response to phenylephrine (Figure 6J).

Beyond its lowering cholesterol effect, simvastatin also improves endothelial function through NO pathway and by preventing oxidative stress damage (23, 24). Thus, we tested its effect on arterial response to phenylephrine in Jak2^V617F HC-EC mice. Fourteen days of treatment with simvastatin did not change spleen weight, haemoglobin level or platelet count (Figures 6 K-M). There was only a slight decrease in white blood cells count following simvastatin treatment (Figure 6N). Interestingly, simvastatin significantly improved aortic response to phenylephrine as compared to vehicle (Figure 60).

Discussion:

This study demonstrated that Jak2^V617F erythrocyte-derived microvesicles carrying MPO, are responsible for an increased oxidative stress in arterial endothelium and a decreased availability of NO, which strongly increased arterial contraction to vasoconstrictive agents, possibly accounting for arterial events associated with MPNs. Simvastatin, a drug with anti oxidant properties, improved arterial contraction.

The first major finding of our study is the demonstration that Jak2^V617F MPN induces a considerable increase in arterial contraction. This finding suggests a vasospastic phenomenon associated with MPN and thus represents a paradigm shift in MPNs where arterial events were only seen as a result of a thrombotic process (7). Our results obtained ex vivo and in vivo could explain this higher incidence of arterial events in patients with polycythemia vera than in the general population and the high prevalence of myocardial infarction without significant coronary stenosis by angiography in patients with MPN (8). Arterial spasm is an underdiagnosed phenomenon that can happen in patients without atherosclerosis but is also favoured by underlying non-stenotic atherosclerotic plaques. This suggests that the effect we observed might not only account for myocardial infarction without significant coronary stenosis reported in patients with MPN, but might also more widely favour arterial events in patients with atherosclerotic plaques and MPN (11, 12). Moreover, arterial spasm not only occurs in coronary arteries, but also in brain arteries (25). We also found an impairment in arterial dilatation, which is in line with the altered endothelial dependant flow mediated vasodilatation reported in patients with polycythemia vera, in the absence of overt arterial disease (26).

The second major finding of our work is the contribution of Jak2^V617F erythrocyte- derived microvesicles to this increased arterial contraction associated with MPN. Importantly, we observed this effect with Jak2^V617F erythrocyte microvesicles from mice, but also with microvesicles isolated from patients carrying Jak2^V6I7F. We thus highlight here a crucial vascular role of microvesicles in MPNs, beyond their so far described implication in coagulation in this setting (27-31). Although patients with MPNs have higher circulating levels of microvesicles than healthy individuals, we assessed vascular reactivity using the same concentrations of microvesicles for both groups, suggesting that microvesicles composition, and not concentration, accounts for the observed vascular effect (27, 28, 32-35). We cannot rule out the fact that Jak2^V6I7F erythrocytes themselves, in addition to microvesicles, could also directly increase arterial contraction associated with MPNs.

Finally, in our work we demonstrated that NO pathway inhibition and increased endothelial oxidative stress are implicated in this increased arterial contraction in MPN. Several groups reported high levels of circulating reactive oxygen species products (36-38) and low antioxidant status in MPN (37, 39), but endothelial oxidative stress had never been investigated. Erythrocyte microvesicles have already been linked to vascular dysfunction in different settings, such as erythrocytes storage or sickle cell disease (18, 19, 40), but never in the context of MPN. Thanks to proteomics approaches we were able to identify a defect in GSTT1 and an over expression of MPO in microvesicles derived from Jak2^V617F erythrocytes. Using a direct and irreversible inhibition of MPO, we were able to ascertain the role of MPO carried by microvesicles derived from Jak2^V617F erythrocytes in the increase endothelial oxidative stress. MPO is a polycationic heme-containing glycoprotein stored mainly in the azurophilic granules of neutrophils, but up to 30% of total cellular MPO can be released as active enzyme into the extracellular space. Interestingly, extra-cellular MPO can bind to red blood cells membrane and favours endothelial dysfunction in the context of ischemic heart disease (41-45). Our results demonstrate that MPO binds to erythrocyte-derived microvesicles, increases endothelial oxidative stress and vascular response to vasoconstrictors.

The role of GSTT1 in the increased endothelial oxidative stress status and vascular reactivity we observed with microvesicles derived from Jak2^V617F erythrocytes remains uncertain, because we could not restore a normal level of GSTT1 only in microvesicles. The normalisation of vascular reactivity induced by NAC could be explained by the glutathione inducer activity of NAC, but could also just be due to the potent anti-oxidant activity of this drug.

In addition to demonstrating how Jak2^V617F induces this increased arterial contraction, our results open new potential therapeutic perspectives to prevent cardio-vascular events in patients with MPN. We demonstrated that simvastatin, a well-known and easily accessible drug, strongly improves arterial response to vasoconstrictive agent in our MPN mouse model. These results thus pave the way for testing simvastatin to prevent arterial events in patients with MPN We also tested available treatments for MPN and observed that hydroxyurea, but not ruxolitinib, improved arterial contraction. This difference might be explained by the fact that hydroxyurea decreased erythrocyte count in our mouse model whereas ruxolitinib did not (46, 47).

In conclusion, our study showed that microvesicles derived from erythrocytes are responsible for an increased arterial contraction in Jak2^V617F MPNs. This effect is due to an overexpression of MPO in Jak2^V6I7F erythrocyte-derived microvesicles, which is responsible for an increased endothelial oxidative stress and a NO pathway inhibition. Simvastatin appears as an original new approach to prevent arterial events in MPN and warrants further studies.

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Claims

CLAIMS:

1. A method of preventing the occurrence of cardiovascular event in a patient suffering from a myeloproliferative neoplasm comprising administering to the patient a therapeutically effective amount of a myeloperoxidase inhibitor.

2. The method of claim 1 wherein the patient suffers from polycythemia vera (PV), essential thrombocythemia (ET) or primary myelofibrosis (PMF).

3. The method of claim 1 wherein the patient harbors one mutation in JAK2.

4. The method of claim 3 wherein the mutation is the Jak2^V617F mutation.

5. A method of determining whether a patient suffering from a myeloproliferative neoplasm is at risk of having a cardiovascular event comprising the steps of determining the level of microvesicles derived from Jak2^V6I7F erythrocytes in a blood sample obtained from the patient wherein the level correlates with the risk of having a cardiovascular disease.

6. The method of claim 5 wherein the level of the microvesicles derived from Jak2^V6I7F erythrocytes is compared to a predetermined reference value.

7. The method of claim 5 wherein the patient is treated with a statin when it is concluded that the patient has been determined as having a high risk of a cardiovascular event.