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Myeloproliferative neoplasms are associated with increased risk for thrombotic complications. These conditions most commonly involve somatic mutations in genes that lead to constitutive activation of the Janus-associated kinase signaling pathway (eg, Janus kinase 2, calreticulin, myeloproliferative leukemia protein). Acquired gain-of-function mutations in these genes, particularly Janus kinase 2, can cause a spectrum of disorders, ranging from clonal hematopoiesis of indeterminate potential, a recently recognized age-related promoter of cardiovascular disease, to frank hematologic malignancy. Beyond thrombosis, patients with myeloproliferative neoplasms can develop other cardiovascular conditions, including heart failure and pulmonary hypertension. The authors review the pathophysiologic mechanisms of cardiovascular complications of myeloproliferative neoplasms, which involve inflammation, prothrombotic and profibrotic factors (including transforming growth factor-beta and lysyl oxidase), and abnormal function of circulating clones of mutated leukocytes and platelets from affected individuals. Anti-inflammatory therapies may provide cardiovascular benefit in patients with myeloproliferative neoplasms, a hypothesis that requires rigorous evaluation in clinical trials.

BACKGROUND: CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury. METHODS: IDO-1 expression in mice and human vessels was examined. IDO-1(−/−) mice, IDO-1 inhibitors, an adenine-induced CKD, and carotid artery injury models were used. RESULTS: Both global IDO-1(−/−) CKD mice and IDO-1 inhibitor in wild-type CKD mice showed reduced blood Kyn levels, TF expression in their arteries, and thrombogenicity compared with respective controls. Several advanced IDO-1 inhibitors downregulated TF expression in primary human aortic vascular smooth muscle cells specifically in response to uremic serum. Further mechanistic probing of arteries from an IS-specific mouse model, and CKD mice, showed upregulation of IDO-1 protein, which was due to inhibition of its polyubiquitination and degradation by IS in vascular smooth muscle cells. In two cohorts of patients with advanced CKD, blood IDO-1 activity was significantly higher in sera of study participants who subsequently developed thrombosis after endovascular interventions or vascular surgery. CONCLUSION: Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.

Coronavirus disease-2019 (COVID-19) provokes a hypercoagulable state with increased incidence of thromboembolism and mortality. Platelets are major effectors of thrombosis and hemostasis. Suitable animal models are needed to better understand COVID-19-associated coagulopathy (CAC) and underlying platelet phenotypes. Here, we assessed K18-hACE2 mice undergoing a standardized SARS-CoV-2 infection protocol to study dynamic platelet responses via mass spectrometry-based proteomics. In total, we found significant changes in >1,200 proteins. Strikingly, protein alterations occurred rapidly by 2 days post-infection (dpi) and preceded outward clinical signs of severe disease. Pathway enrichment analysis of 2dpi platelet proteomes revealed that SARS-CoV-2 infection upregulated complement-coagulation networks (F2, F12, CFH, CD55/CD59), platelet activation-adhesion-degranulation proteins (PF4, SELP, PECAM1, HRG, PLG, vWF), and chemokines (CCL8, CXCL5, CXCL12). When mice started to lose weight at 4dpi, pattern recognition receptor signaling (RIG-I/MDA5, CASP8, MAPK3), and interferon pathways (IFIT1/IFIT3, STAT1) were predominant. Interestingly, SARS-CoV-2 spike protein in the lungs was observed by immunohistochemistry, but in platelets was undetected by proteomics. Similar to patients, K18-hACE2 mice during SARS-CoV-2 infection developed progressive lymphohistiocytic interstitial pneumonia with platelet aggregates in the lungs and kidneys. In conclusion, this model recapitulates activation of coagulation, complement, and interferon responses in circulating platelets, providing valuable insight into platelet pathology during COVID-19.Key PointsSARS-CoV-2-infected humanized ACE2 mice recapitulate platelet reprogramming towards activation-degranulation-aggregation.Complement/coagulation pathways are dominant in platelets at 2 days post-infection (dpi), while interferon signaling is dominant at 4dpi.

Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm (MPN) that usually portends a poor prognosis with limited therapeutic options available. Currently only allogeneic stem cell transplantation is curative in those who are candidates, while administration of the JAK1/2 inhibitor ruxolitonib carries a risk of worsening cytopenia. The limited therapeutic options available highlight the need for the development of novel treatments for PMF. Lysyl oxidase (LOX), an enzyme vital for collagen cross-linking and extracellular matrix stiffening, has been found to be up-regulated in PMF. Herein, we evaluate two novel LOX inhibitors, PXS-LOX_1 and PXS-LOX_2, in two animal models of PMF (GATA1low and JAK2V617F-mutated mice). Specifically, PXS-LOX_1 or vehicle was given to 15-16-week-old GATA1low mice via intraperitoneal injection at a dose of 15 mg/kg four times a week for nine weeks. PXS-LOX_1 was found to significantly decrease the bone marrow fibrotic burden and megakaryocyte number compared to vehicle in both male and female GATA1low mice. Given these results, PXS-LOX_1 was then tested in 15-17-week-old JAK2V617F-mutated mice at a dose of 30 mg/kg four times a week for eight weeks. Again, we observed a significant decrease in bone marrow fibrotic burden. PXS-LOX_2, a LOX inhibitor with improved oral bioavailability, was next evaluated in 15 to 17-week-old JAK2V617F-mutated mice at a dose of 5 mg/kg p.o. four times a week for eight weeks. This inhibitor also resulted in a significant decrease in bone marrow fibrosis, albeit with a more pronounced amelioration in female mice. Taking these results together, PXS-LOX_1 and PXS-LOX_2 appear to be promising new candidates for the treatment of fibrosis in PMF.

Highlights •Clonal hematopoiesis can develop from JAK2V617F mutant cells, but mouse models harboring this mutation are confounded by myeloproliferative disease phenotypes. •To establish a model of JAK2V617F clonal hematopoiesis, a lentivirus vector was used to transduce hematopoietic stem and progenitor cells with a construct that expresses this mutation from a myeloid-specific promoter. •When transduced hematopoietic stem and progenitor cells were implanted into mice, JAK2V617F chimerism was achieved in monocytes and neutrophils in the absence of changes in blood cell counts, and these mice exhibited greater myocardial inflammation and accelerated heart failure when subjected to models of cardiac injury. •These data suggest that clonal hematopoiesis can arise from the acquisition of JAK2V617F mutations in a progenitor cell subpopulation that gives rise to circulating myeloid cells, and that this condition can promote cardiovascular disease through proinflammatory mechanisms.

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of chronic hematological diseases that arise from the clonal expansion of abnormal hematopoietic stem cells, of which polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) have been extensively reviewed in the context of control of clonal expansion, fibrosis, and other phenotypes. Herein, we review current knowledge on the influence of different forms of MPN on bone health. In studies, murine models and human data have implicated various degrees of effect of different forms of MPN on bone density and on osteoblast proliferation and differentiation. Most results have shown that bone volume is generally increased in patients with PMF, whereas it is slightly decreased or not altered in patients with ET or PV, although possible differences between male and female phenotypes were not fully explored in most MPN forms. Osteosclerosis in patients with PMF is a serious complication that can lead to bone marrow failure, and the loss of bone reported in some patients with ET or PV can lead to osteoporotic fractures. Some MPN forms are associated with an increased number of megakaryocytes (MKs), and several of the MK-associated factors in MPN are known to affect bone development. We review known mechanisms involved in these processes, with a focus on the role of MKs and secreted factors. Understanding MPN-associated changes in bone health could improve early intervention and treatment of this side effect of the pathology.

Adenosine is an extracellular signaling molecule that is particularly relevant in times of cellular stress, inflammation and metabolic disturbances when the levels of the purine increase. Adenosine acts on two G-protein-coupled stimulatory and on two G-protein-coupled inhibitory receptors, which have varying expression profiles in different tissues and conditions, and have different affinities for the endogenous ligand. Studies point to significant roles of adenosine and its receptors in metabolic disease and bone health, implicating the receptors as potential therapeutic targets. This review will highlight our current understanding of the dichotomous effects of adenosine and its receptors on adipogenesis versus osteogenesis within the bone marrow to maintain bone health, as well as its relationship to obesity. Therapeutic implications will also be reviewed.

The biomechanical properties of the bone marrow microenvironment emerge from a combination of interactions between various extracellular matrix (ECM) structural proteins and soluble factors. Matrix stiffness directs stem cell fate, and both bone marrow stromal and hematopoietic cells respond to biophysical cues. Within the bone marrow, the megakaryoblasts and erythroblasts are thought to originate from a common progenitor, giving rise to fully mature magakaryocytes (the platelet precursors) and erythrocytes. Erythroid and megakaryocytic progenitors sense and respond to the ECM through cell surface adhesion receptors such as integrins and mechanosensitive ion channels. While hematopoietic stem progenitor cells remain quiescent on stiffer ECM substrates, the maturation of the erythroid and megakaryocytic lineages occurs on softer ECM substrates. This review surveys the major matrix structural proteins that contribute to the overall biomechanical tone of the bone marrow, as well as key integrins and mechanosensitive ion channels identified as ECM sensors in context of megakaryocytosis or erythropoiesis.

Primary myelofibrosis (PMF) is a neoplasm prone to leukemic transformation, for which limited treatment is available. Among individuals diagnosed with PMF, the most prevalent mutation is the JAK2V617F somatic point mutation that activates the Janus kinase 2 (JAK2) enzyme. Our earlier reports on hyperactivity of β1 integrin and enhanced adhesion activity of the α2β1 complex in JAK2V617F megakaryocytes (MKs) led us to examine the new hypothesis that this mutation leads to posttranslational modification via changes in glycosylation. Samples were derived from immunoprecipitation of MKs obtained from Vav1-hJAK2

Individuals with CKD are particularly predisposed to thrombosis after vascular injury. Using mouse models, we recently described indoxyl sulfate, a tryptophan metabolite retained in CKD and an activator of tissue factor (TF) through aryl hydrocarbon receptor (AHR) signaling, as an inducer of thrombosis across the CKD spectrum. However, the translation of findings from animal models to humans is often challenging. Here, we investigated the uremic solute-AHR-TF thrombosis axis in two human cohorts, using a targeted metabolomics approach to probe a set of tryptophan products and high-throughput assays to measure AHR and TF activity. Analysis of baseline serum samples was performed from 473 participants with advanced CKD from the Dialysis Access Consortium Clopidogrel Prevention of Early AV Fistula Thrombosis trial. Participants with subsequent arteriovenous thrombosis had significantly higher levels of indoxyl sulfate and kynurenine, another uremic solute, and greater activity of AHR and TF, than those without thrombosis. Pattern recognition analysis using the components of the thrombosis axis facilitated clustering of the thrombotic and nonthrombotic groups. We further validated these findings using 377 baseline samples from participants in the Thrombolysis in Myocardial Infarction II trial, many of whom had CKD stage 2-3. Mechanistic probing revealed that kynurenine enhances thrombosis after vascular injury in an animal model and regulates thrombosis in an AHR-dependent manner. This human validation of the solute-AHR-TF axis supports further studies probing its utility in risk stratification of patients with CKD and exploring its role in other diseases with heightened risk of thrombosis.

Team science has been recognized as critical to solving increasingly complex biomedical problems and advancing discoveries in the prevention, diagnosis, and treatment of human disease. In 2009, the Evans Center for Interdisciplinary Biomedical Research (ECIBR) was established in the Department of Medicine at Boston University School of Medicine as a new organizational paradigm to promote interdisciplinary team science. The ECIBR is made up of affinity research collaboratives (ARCs), consisting of investigators from different departments and disciplines who come together to study biomedical problems that are relevant to human disease and not under interdisciplinary investigation at the university. Importantly, research areas are identified by investigators according to their shared interests. ARC proposals are evaluated by a peer review process, and collaboratives are funded annually for up to three years.Initial outcomes of the first 12 ARCs show the value of this model in fostering successful biomedical collaborations that lead to publications, extramural grants, research networking, and training. The most successful ARCs have been developed into more sustainable organizational entities, including centers, research cores, translational research projects, and training programs.To further expand team science at Boston University, the Interdisciplinary Biomedical Research Office was established in 2015 to more fully engage the entire university, not just the medical campus, in interdisciplinary research using the ARC mechanism. This approach to promoting team science may be useful to other academic organizations seeking to expand interdisciplinary research at their institutions.

Patients with malignancy are at 4- to 7-fold higher risk of venous thromboembolism (VTE), a potentially fatal, yet preventable complication. Although general mechanisms of thrombosis are enhanced in these patients, malignancy-specific triggers and their therapeutic implication remain poorly understood. Here we examined a colon cancer-specific VTE model and probed a set of metabolites with prothrombotic propensity in the inferior vena cava (IVC) ligation model. Athymic mice injected with human colon adenocarcinoma cells exhibited significantly higher IVC clot weights, a biological readout of venous thrombogenicity, compared with the control mice. Targeted metabolomics analysis of plasma of mice revealed an increase in the blood levels of kynurenine and indoxyl sulfate (tryptophan metabolites) in xenograft-bearing mice, which correlated positively with the increase in the IVC clot size. These metabolites are ligands of aryl hydrocarbon receptor (AHR) signaling. Accordingly, plasma from the xenograft-bearing mice activated the AHR pathway and augmented tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) levels in venous endothelial cells in an AHR-dependent manner. Consistent with these findings, the endothelium from the IVC of xenograft-bearing animals revealed nuclear AHR and upregulated TF and PAI-1 expression, telltale signs of an activated AHR-TF/PAI-1 axis. Importantly, pharmacological inhibition of AHR activity suppressed TF and PAI-1 expression in endothelial cells of the IVC and reduced clot weights in both kynurenine-injected and xenograft-bearing mice. Together, these data show dysregulated tryptophan metabolites in a mouse cancer model, and they reveal a novel link between these metabolites and the control of the AHR-TF/PAI-1 axis and VTE in cancer.

Thrombosis is at the heart of cardiovascular complications observed in specific diseases. A heightened thrombosis risk above that in general population in diseases such as myelofibrosis and chronic kidney disease implicates disease-specific mediators of thrombosis. This relative lack of information regarding the mechanisms of thrombosis in specific organ pathologies hitherto has remained limited. Evolving literature implicates some soluble factors in the blood of patients with discrete disorders, inflicting fundamental changes in the components of thrombosis. In this era of precision medicine, integrating these disease-specific factors in a comprehensive thrombotic risk assessment of patients is imperative in guiding therapeutic decisions. A complex network of mechanisms regulates each organ pathology and resultant thrombotic phenotypes. This review surveys different effectors of thrombogenicity associated with two pathologically fibrotic organs used as model systems, the bone marrow and kidney, as well as focuses attention to a common inducer of fibrosis and thrombosis, lysyl oxidase.

A2BAR (A2B adenosine receptor) has been implicated in several physiological conditions, such as allergic or inflammatory disorders, vasodilation, cell growth and epithelial electrolyte secretion. For mediating the protein–protein interactions of A2BAR, the receptor's C-terminus is recognized to be crucial. In the present study, we unexpectedly found that two point mutations in the A2BAR C-terminus (F297A and R298A) drastically impaired the expression of A2BAR protein by accelerating its degradation. Thus we tested the hypothesis that these two point mutations disrupt A2BAR's interaction with a protein essential for A2BAR stability. Our results show that both mutations disrupted the interaction of A2BAR with actinin-1, an actin-associated protein. Furthermore, actinin-1 binding stabilized the global and cell-surface expression of A2BAR. By contrast, actinin-4, another non-muscle actinin isoform, did not bind to A2BAR. Thus our findings reveal a previously unidentified regulatory mechanism of A2BAR abundance.

To the editor: Primary myelofibrosis (PMF) is characterized by the presence of bone marrow fibrosis associated with increased abundance of megakaryocytes and atypia in bone marrow histology.[1][1][⇓][2]-[3][3] The main driver mutations found in PMF are JAK2 (50% to 60% of patients), MPL (3% to 5

BACKGROUND: Myeloproliferative neoplasms (MPNs) are clonal stem cell neoplasms characterized by terminal expansion of the myeloid cell lineage and include essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF). MF carries the worst prognosis and is characterized by reactive bone marrow fibrosis. Currently bone marrow transplant is the only known therapy that can reverse fibrosis and alter the disease course. Lysyl oxidases (LOX, LOXL1-4) are copper amine oxidase enzymes that facilitate the cross-linking of collagen and elastin through deamination and oxidization of lysine residues, yielding highly reactive aldehydes. This is essential for fibrotic tissue formation. An earlier study identified an important role for LOX in the development of MF in mice (Eliades et al, J. Biol. Chem. 2011; PMID: 21665949). Furthermore, small molecule pan-LOX inhibitors reduced spleen size and bone marrow fibrosis in mouse models of MF (Leiva et al, Int J. Hemat. 2019; PMID: 31637674). This provides clinical rationale for the use of pan-lysyl oxidase inhibition in MF. A Phase 1 study was conducted to establish the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of PXS-5505, a pan-lysyl oxidase inhibitor. Plasma levels of LOX and LOXL2 were investigated in a separate cohort of MPN patients, with the ultimate goal to develop PXS-5505-based therapy in MF patients. METHODS: PXS-5505 was dosed orally in a Phase I, randomized, placebo-controlled, single ascending (Part A) and multiple ascending dose (Part B) double-blind study in healthy male volunteers. Plasma LOX and LOXL2 levels were also measured in a separate cohort of MPN patients using ELISA-based Single Molecule Array technology (Simoa; Quanterix). RESULTS: Forty subjects were enrolled in Part A and 16 were enrolled in Part B. In Part A, there were 5 cohorts consisting of 8 subjects (6 active, 2 placebo) with doses of 10, 50, 100, 200 or 300 mg administered once. In Part B, there were 2 cohorts of 8 subjects each (6 active, 2 placebo) at doses of 100 or 200 mg daily for 14 days. There were no significant treatment-related adverse events. Mean age in Part A was 32.0 years (SD 11.14) and 32.1 years (SD 12.14) in Part B. Cmax and AUC increased linearly across the dose range of 10 to 300 mg for single dose administration. In multiple dosing at 200 mg daily, Cmax was 916 ng/mL and AUC0-24 was 7421 hr*ng/mL on Day 14, with median Tmax of 1 hour and t1/2 of 7 hours. When measured in plasma, LOX was dose-dependently inhibited and achieved very strong inhibition (median 80%) with a 200 mg dose around Tmax. The 300 mg dosing did not significantly increase inhibition. Multiple daily doses of 100-200 mg resulted in 60-70% and 50-60% inhibition of plasma LOX at 12 and 24 hours. Average LOX plasma levels were 3.61 ng/mL (range: 3.20-13.42 ng/mL; SD: 2.92 ng/mL). We measured plasma LOX and LOXL2 levels in a separate MPN cohort of 9 ET, 8 PV, and 13 MF patients (mean age 61.4, range:24-84, 65% males). LOXL2 levels were higher in MF (mean 415 pg/mL) compared to ET (mean 209 pg/mL) and PV (mean 322 pg/ml), although this was not significant. However, LOXL2 levels in all MPN patients (mean 333 pg/mL) were significantly higher than LOXL2 levels in normal controls (mean 152 pg/mL, p DISCUSSION: PXS-5505 demonstrated an excellent safety profile and was well tolerated in healthy human subjects. PK/PD properties are consistent with preclinical data and support once or twice daily >100 mg dosing over 14 days. PXS-5505 achieves long-lasting, strong inhibition of lysyl oxidases. Plasma LOXL2 levels are higher in MF patients compared to healthy controls, and we found no significant disease associations of LOX or LOXL2 among MPN subtypes in our small cohort. LOXL2 is likely a more sensitive MF biomarker as it is present at low concentrations in the blood, while LOX is constantly produced from major organs making detection due to disease more difficult. Based on previous mouse studies, it is possible that LOX levels would be higher in MF patients when compared to age-matched controls, which we will investigate further. We will open a Phase IB/II study of PXS-5505 in MF patients resistant to ruxolitinib. Disclosures Neuberg: Madrigak Pharmaceuticals: Current equity holder in publicly-traded company; Celgene: Research Funding; Pharmacyclics: Research Funding. Ravid:Pharmaxis: Research Funding. Jarolimek:Pharmaxis Ltd: Current Employment. Charlton:Pharmaxis Ltd: Current Employment. Hobbs:Novartis: Honoraria; Constellation: Honoraria, Research Funding; Jazz: Honoraria; Celgene/BMS: Honoraria; Merck: Research Funding; Incyte: Research Funding; Bayer: Research Funding.

Emerging evidence in animal models of chronic kidney disease (CKD) implicates Aryl Hydrocarbon Receptor (AHR) signaling as a mediator of uremic toxicity. However, details about its tissue-specific and time-dependent activation in response to various renal pathologies remain poorly defined. Here, a comprehensive analysis of AHR induction was conducted in response to discrete models of kidney diseases using a transgenic mouse line expressing the AHR responsive-promoter tethered to a β-galactosidase reporter gene. Following validation using a canonical AHR ligand (a dioxin derivative), the transgenic mice were subjected to adenine-induced and ischemia/reperfusion-induced injury models representing CKD and acute kidney injury (AKI), respectively, in humans. Indoxyl sulfate was artificially increased in mice through the drinking water and by inhibiting its excretion into the urine. Adenine-fed mice showed a distinct and significant increase in β-galactosidase in the proximal and distal renal tubules, cardiac myocytes, hepatocytes, and microvasculature in the cerebral cortex. The pattern of β-galactosidase increase coincided with the changes in serum indoxyl sulfate levels. Machine-learning–based image quantification revealed positive correlations between indoxyl sulfate levels and β-galactosidase expression in various tissues. This pattern of β-galactosidase expression was recapitulated in the indoxyl sulfate–specific model. The ischemia/reperfusion injury model showed increase in β-galactosidase in renal tubules that persisted despite reduction in serum indoxyl sulfate and blood urea nitrogen levels. Thus, our results demonstrate a relationship between AHR activation in various tissues of mice with CKD or AKI and the levels of indoxyl sulfate. This study demonstrates the use of a reporter gene mouse to probe tissue-specific manifestations of uremia in translationally relevant animal models and provide hypothesis-generating insights into the mechanism of uremic toxicity that warrant further investigation.

Highlights • GATA1 and FLI1 switch on the endogenous platelet factor 4 (Pf4) gene in fibroblasts. • Pf4 expression is maintained and increases over time. • PF4 protein is secreted and readily collected. • A new technology for the g0065neration of important biomolecules in heterologous cells., The ability of transcriptional regulators to drive lineage conversion of somatic cells offers great potential for the treatment of human disease. To explore the concept of switching on specific target genes in heterologous cells, we developed a model system to screen candidate factors for their ability to activate the archetypal megakaryocyte-specific chemokine platelet factor 4 (PF4) in fibroblasts. We found that co-expression of the transcriptional regulators GATA1 and FLI1 resulted in a significant increase in levels of PF4, which became magnified over time. This finding demonstrates that such combinations can be used to produce potentially beneficial chemokines in readily available heterologous cell types.

In addition to various physiologic roles, emerging evidence strongly points to pathogenic roles of tryptophan and of its metabolites, especially in diseases such as renal failure. Accurate estimation of levels of these metabolites in blood is important to mechanistically probe their contribution to disease pathogenesis, while clinically, such a panel can be used to risk stratify patients for a clinical phenotype. Herein, we describe a comprehensive liquid chromatography-mass spectrometry (LC/MS)-based method to determine the level of tryptophan and its metabolites (kynurenine, kynurenic acid, xanthurenic acid, anthranilic acid, indoxyl sulfate and indoxyl acetate). Human sera samples were processed through a C18 column followed by application of a binary gradient and quantitation by MS/MS. The linearity, lower limit of detection, inter- and intraassay variabilities and recovery were determined, yielding a precise, reproducible method for all the metabolites. Unlike previous studies, we further validated these methods in a well-characterized set of human sera from end stage renal disease patients compared to age-, gender- and ethnic-background matched human controls. Overall, we report an optimized LC/MS-based estimation of a comprehensive panel of tryptophan-derived metabolites with quality features within FDA standards, underscoring their readiness for translational use.

The priming of macrophages with IFN-γ prior to TLR stimulation results in enhanced and prolonged inflammatory cytokine production. In this study, we demonstrate that, following TLR stimulation, macrophages upregulate the adenosine 2b receptor (A2bR) to enhance their sensitivity to immunosuppressive extracellular adenosine. This upregulation of A2bR leads to the induction of macrophages with an immunoregulatory phenotype and the downregulation of inflammation. IFN-γ priming of macrophages selectively prevents the induction of the A2bR in macrophages to mitigate sensitivity to adenosine and to prevent this regulatory transition. IFN-γ–mediated A2bR blockade leads to a prolonged production of TNF-α and IL-12 in response to TLR ligation. The pharmacologic inhibition or the genetic deletion of the A2bR results in a hyperinflammatory response to TLR ligation, similar to IFN-γ treatment of macrophages. Conversely, the overexpression of A2bR on macrophages blunts the IFN-γ effects and promotes the development of immunoregulatory macrophages. Thus, we propose a novel mechanism whereby IFN-γ contributes to host defense by desensitizing macrophages to the immunoregulatory effects of adenosine. This mechanism overcomes the transient nature of TLR activation, and prolongs the antimicrobial state of the classically activated macrophage. This study may offer promising new targets to improve the clinical outcome of inflammatory diseases in which macrophage activation is dysregulated.

Modulation of the low affinity adenosine receptor subtype, the A2b adenosine receptor (A2bAR), has gained interest as a therapeutic target in various pathologic areas associated with cardiovascular disease. The actions of the A2bAR are diverse and at times conflicting depending on cell and tissue type and the timing of activation or inhibition of the receptor. The A2bAR is a promising and exciting pharmacologic target, however, a thorough understanding of A2bAR action is necessary to reach the therapeutic potential of this receptor. This review will focus on the role of the A2bAR in various cardiovascular and metabolic pathologies in which the receptor is currently being studied. We will illustrate the complexities of A2bAR signaling and highlight areas of research with potential for therapeutic development.

Perlecan is a major heparan sulfate (HS) proteoglycan in the arterial wall. Previous studies have linked it to atherosclerosis. Perlecan contains a core protein and three HS side chains. Its core protein has five domains (DI-DV) with disparate structures and DII is highly homologous to the ligand-binding portion of LDL receptor (LDLR). The functional significance of this domain has been unknown. Here, we show that perlecan DII interacts with LDL. Importantly, the interaction largely relies on O-linked glycans that are only present in the secreted DII. Among the five repeat units of DII, most of the glycosylation sites are from the second unit, which is highly divergent and rich in serine and threonine, but has no cysteine residues. Interestingly, most of the glycans are capped by the negatively charged sialic acids, which are critical for LDL binding. We further demonstrate an additive effect of HS and DII on LDL binding. Unlike LDLR, which directs LDL uptake through endocytosis, this study uncovers a novel feature of the perlecan LDLR-like DII in receptor-mediated lipoprotein retention, which depends on its glycosylation. Thus, perlecan glycosylation may play a role in the early LDL retention during the development of atherosclerosis.

Chronic kidney disease (CKD/uremia) remains vexing because it increases the risk of atherothrombosis and is also associated with bleeding complications on standard antithrombotic/antiplatelet therapies. Although the associations of indolic uremic solutes and vascular wall proteins [such as tissue factor (TF) and aryl hydrocarbon receptor (AHR)] are being defined, the specific mechanisms that drive the thrombotic and bleeding risks are not fully understood. We now present an indolic solute-specific animal model, which focuses on solute-protein interactions and shows that indolic solutes mediate the hyperthrombotic phenotype across all CKD stages in an AHR- and TF-dependent manner. We further demonstrate that AHR regulates TF through STIP1 homology and U-box-containing protein 1 (STUB1). As a ubiquitin ligase, STUB1 dynamically interacts with and degrades TF through ubiquitination in the uremic milieu. TF regulation by STUB1 is supported in humans by an inverse relationship of STUB1 and TF expression and reduced STUB1-TF interaction in uremic vessels. Genetic or pharmacological manipulation of STUB1 in vascular smooth muscle cells inhibited thrombosis in flow loops. STUB1 perturbations reverted the uremic hyperthrombotic phenotype without prolonging the bleeding time, in contrast to heparin, the standard-of-care antithrombotic in CKD patients. Our work refines the thrombosis axis (STUB1 is a mediator of indolic solute-AHR-TF axis) and expands the understanding of the interconnected relationships driving the fragile thrombotic state in CKD. It also establishes a means of minimizing the uremic hyperthrombotic phenotype without altering the hemostatic balance, a long-sought-after combination in CKD patients., National Institutes of Health (U.S.) (Grant R01HL132325), National Institutes of Health (U.S.) (Grant R01CA175382), National Institute of General Medical Sciences (U.S.) (Grant R01GM49039), American Heart Association (Grant 12FTF12080241)

The extracellular matrix (ECM) is a key acellular structure in constant remodeling to provide tissue cohesion and rigidity. Deregulation of the balance between matrix deposition, degradation, and crosslinking results in fibrosis. Bone marrow fibrosis (BMF) is associated with several malignant and nonmalignant pathologies severely affecting blood cell production. BMF results from abnormal deposition of collagen fibers and enhanced lysyl oxidase-mediated ECM crosslinking within the marrow, thereby increasing marrow stiffness. Bone marrow stiffness has been recently recognized as an important regulator of blood cell development, notably by modifying the fate and differentiation process of hematopoietic or mesenchymal stem cells. This review surveys the different components of the ECM and their influence on stem cell development, with a focus on the impact of the ECM composition and stiffness on the megakaryocytic lineage in health and disease. Megakaryocyte maturation and the biogenesis of their progeny, the platelets, are thought to respond to environmental mechanical forces through a number of mechanosensors, including integrins and mechanosensitive ion channels, reviewed here.

Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in Primary Myelofibrosis (PMF). Myelofibrosis was long regarded as a bystander of disease in PMF. However, as cells have the ability to sense the surrounding ECM through integrin receptors, we examined the hypothesis that abnormal ECM in myelofibrosis, mediated by integrin activation, contributes to BM megakaryocytosis in JAK2V617F+ PMF. Due to its complex genetic landscape, modeling PMF in animals is challenging. While there is no ideal model to replicate the human disease, Vav1-hJAK2V617F transgenic mice (JAK2V617F+) (Xing S et al. Blood. 2008; 111:5109-5117) are most appropriate for our studies because they express the most frequent mutation in PMF and show megakaryocytosis in BM and a predictable development of myelofibrosis throughout the animal's life. Fibronectin (FN) is a dimeric ECM glycoprotein produced by a variety of cells in BM. FN secretion by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Analysis of myelofibrotic BM from JAK2V617F+ mice revealed elevated levels of FN in BM ECM. Integrins are heterodimeric cell adhesion receptors of α and β subunits that connect the cell cytoskeleton to ECM. Integrin engagement and subsequent signaling affect cell differentiation, proliferation, migration and survival. Integrins α5β1, α4β1, αIIbβ3, and αυβ3 are FN-binding integrins known to be expressed in megakaryocytes. Expression analysis detected significantly elevated expression of α5, αIIb, αυ and β3 subunits in CD41+ megakaryocytes fromJAK2V617F+ mice in vitro and in vivo. Expression of β1 integrin was unchanged in megakaryocytes fromJAK2V617F+ mice, but expression of 9EG7, an antibody clone that detects the high-affinity conformation of β1 integrin, was elevated in JAK2V617F+ CD41+ megakaryocytes. Levels of the high-affinity form of β1 integrin decrease in response to inhibition of α5 integrin function (using Hmα5-1 antibody), indicating a high degree of correlation between affinity to FN and β1 activation in megakaryocytes fromJAK2V617F+ mice. As suggested by expression studies, in-vitro differentiated JAK2V617F+ megakaryocytes showed increased affinity to FN in adhesion assays. To ascertain the contribution of adhesion to FN in development of megakaryocytosis, megakaryocytes were differentiated in vitro on FN, with and without the presence of α5 integrin inhibitory antibody Hmα5-1. Culture on FN had a positive effect on the number of CD41+ megakaryocytes after four days of culture, especially in JAK2V617F+ BM. Importantly, inhibition of α5 integrin decreased the number of CD41+ megakaryocytes in JAK2V617F+ BM to nearly wild-type levels. To determine the effect of in vivo inhibition of α5 integrin on megakaryocyte numbers, the antibody clone 5H10-27, or MFR-5, previously reported to be effective in vivo, was used. Effective targeting in vivo of BM megakaryocytes by the 5H10-27 antibody was confirmed using a fluorochrome-labelled secondary antibody specific to the isotype of the injected clone (Rat IgG2a, κ). The same method was used to determine minimum required dose and interval of administration. JAK2V617F+ mice were treated with three doses of 5H10-27 or isotype control antibody (1mg/Kg IV every 48 hours). Analysis of treated animals on day 5 detected a decrease in megakaryocyte α5 integrin expression in animals treated with 5H10-27 antibody relative to control isotype antibody-treated animals. Platelet counts in peripheral blood and percentage of BM CD41+ megakaryocytes were consistently, albeit non-significantly, lower in 5H10-27 antibody-treated than in isotype antibody-treated animals. Corroborating our findings in mice, analysis of megakaryocytes from patients carrying the JAK2V617F mutation revealed elevated cell surface expression of α5 integrin subunit and increased adhesion to FN, which was dampened by an anti-α5 integrin antibody (Sam-1). Our results uncovered a global de-regulation of FN-binding integrin expression in megakaryocytes carrying the JAK2V617F mutation and the central role of FN-α5 integrin in development of megakaryocytosis in PMF. These results challenge the current paradigm by bringing ECM and myelofibrosis from a bystander position to center stage in the pathology of PMF. Disclosures No relevant conflicts of interest to declare.

G-protein coupled receptors (GPCRs) are a large family of proteins that coordinate extracellular signals to produce physiologic outcomes. Adenosine receptors (AR) are one class of GPCRs that have been shown to regulate functions as diverse as inflammation, blood flow, and cellular differentiation. Adenosine signals through four GPCRs that either inhibit (A1AR and A3AR) or activate (A2aAR and A2bAR) adenylyl cyclase. This review will focus on the role of GPCRs, and in particular, adenosine receptors, in adipogenesis. Preadipocytes differentiate to mature adipocytes as the adipose tissue expands to compensate for the consumption of excess nutrients. These newly generated adipocytes contribute to maintaining metabolic homeostasis. Understanding the key drivers of this differentiation process can aid the development of therapeutics to combat the growing obesity epidemic and associated metabolic consequences. Although much literature has covered the transcriptional events that culminate in the formation of an adipocyte, less focus has been on receptor-mediated extracellular signals that direct this process. This review will highlight GPCRs and their downstream messengers as significant players controlling adipocyte differentiation.

Adenosine is an endogenous metabolite that is released from all tissues and cells including liver, pancreas, muscle and fat, particularly under stress, intense exercise, or during cell damage. The role of adenosine in glucose homeostasis has been attributed to its ability to regulate, through its membrane receptors, processes such as insulin secretion, glucose release and clearance, glycogenolysis, and glycogenesis. Additionally, adenosine and its multiple receptors have been connected to lipid metabolism by augmenting insulin-mediated inhibition of lipolysis, and the subsequent increase in free fatty acids and glycerol levels. Furthermore, adenosine was reported to control liver cholesterol synthesis, consequently affecting plasma levels of cholesterol and triglycerides, and the amount of fat tissue. Alterations in the balance of glucose and lipid homeostasis have implications in both cardiovascular disease and diabetes. The ability of different adenosine receptors to activate and inhibit the same signaling cascades has made it challenging to study the influence of adenosine, adenosine analogs and their receptors in health and disease. This review focuses on the role and significance of different adenosine receptors in mediating the effect of adenosine on glucose and lipid homeostasis. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Throughout the plant and animal kingdoms specific cell types become polyploid, increasing their DNA content to attain a large cell size. In mammals, megakaryocytes (MKs) become polyploid before fragmenting into platelets. The mammalian trophoblast giant cells (TGCs) exploit their size to form a barrier between the maternal and embryonic tissues. The mechanism of polyploidization has been investigated extensively in Drosophila , in which a modified cell cycle—the endocycle, consisting solely of alternating S and gap phases—produces polyploid tissues. During S phase in the Drosophila endocycle, heterochromatin and specific euchromatic regions are underreplicated and reduced in copy number. Here we investigate the properties of polyploidization in murine MKs and TGCs. We induced differentiation of primary MKs and directly microdissected TGCs from embryonic day 9.5 implantation sites. The copy number across the genome was analyzed by array-based comparative genome hybridization. In striking contrast to Drosophila , the genome was uniformly and integrally duplicated in both MKs and TGCs. This was true even for heterochromatic regions analyzed by quantitative PCR. Underreplication of specific regions in polyploid cells is proposed to be due to a slower S phase, resulting from low expression of S-phase genes, causing failure to duplicate late replicating genomic intervals. We defined the transcriptome of TGCs and found robust expression of S-phase genes. Similarly, S-phase gene expression is not repressed in MKs, providing an explanation for the distinct endoreplication parameters compared with Drosophila . Consistent with TGCs endocycling rather than undergoing endomitosis, they have low expression of M-phase genes.

Megakaryocytes (MKs), the platelet precursors, undergo an endomitotic cell cycle that leads to polyploidy. Lysyl oxidase propeptide (LOX-PP) is generated from lysyl oxidase (LOX) pro-enzyme after proteolytical cleavage. We recently reported that LOX, a known matrix cross-linking enzyme, contributes to MK lineage expansion. In addition, LOX expression levels are ploidy-dependent, with polyploidy MKs having minimal levels. This led us to test the effects of LOX-PP on the number and ploidy of primary MKs. LOX-PP significantly decreases mouse bone marrow MK ploidy coupled with a reduction in MK size. MK number is unchanged upon LOX-PP treatment. Analysis of LOX-PP- or vehicle-treated MKs by western blotting revealed a reduction in ERK1/2 phosphorylation and in the levels of its downstream targets, cyclin D3 and cyclin E, which are known to play a central role in MK endomitosis. Pull-down assays and immunochemistry staining indicated that LOX-PP interacts with α-tubulin and the mictotubules, which can contribute to decreased MK ploidy. Thus, our findings defined a role for LOX-PP in reducing MK ploidy. This suggests that high-level expression of LOX in aberrantly proliferating MKs could play a part in inhibiting their polyploidization via LOX-PP.

Thrombotic microangiopathy (TMA) is characterized by the presence of microangiopathic hemolytic anemia and thrombocytopenia along with organ dysfunction, and pathologically, by the presence of microthrombi in multiple microvascular beds. Delays in diagnosis and initiation of therapy are common due to the low incidence, variable presentation, and poor awareness of these diseases, underscoring the need for interdisciplinary approaches to clinical care for TMA. We describe a new approach to improve clinical management via a TMA team that originally stemmed from an Affinity Research Collaborative team focused on thrombosis and hemostasis. The TMA team consists of clinical faculty from different disciplines who together are charged with the responsibility to quickly analyze clinical presentations, guide laboratory testing, and streamline prompt institution of treatment. The TMA team also includes faculty members from a broad range of disciplines collaborating to elucidate the pathogenesis of TMA. To this end, a clinical database and biorepository have been constructed. TMA leaders educate front-line providers from other departments through presentations in various forums across multiple specialties. Facilitated by an Affinity Research Collaborative mechanism, we describe an interdisciplinary team dedicated to improving both clinical care and translational research in TMA.

Megakaryocytes (MK) in the bone marrow (BM) are immersed in a network of extracellular matrix components that regulates platelet release into the circulation. Combining biological and bioengineering approaches, we found that the activation of transient receptor potential cation channel subfamily V member 4 (TRPV4), a mechano-sensitive ion channel, is induced upon MK adhesion on softer matrices. This response promoted platelet production by triggering a cascade of events that lead to calcium influx, β1 integrin activation and internalization, and Akt phosphorylation, responses not found on stiffer matrices. Lysyl oxidase (LOX) is a physiological modulator of BM matrix stiffness via collagen crosslinking. In vivo inhibition of LOX and consequent matrix softening lead to TRPV4 activation cascade and increased platelet levels. At the same time, in vitro proplatelet formation was reduced on a recombinant enzyme-mediated stiffer collagen. These results suggest a novel mechanism by which MKs, through TRPV4, sense extracellular matrix environmental rigidity and release platelets accordingly.

Megakaryocytes (MKs), the platelet precursors, are capable of accumulating DNA greater than a diploid content as part of their cell cycle. MKs have been recognized as mediating fibrosis in a subset of hematologic malignancies, including acute megakaryoblastic leukemia and a subset of myeloproliferative neoplasms. The mechanisms responsible for fibrosis remain only partially understood. Past studies highlighted the role of growth factors in such pathologies, and recently, the protein lysyl oxidase (LOX) has been implicated in proliferation of MKs, ploidy and deposition of fibers. LOX was initially characterized as a protein responsible for the intermolecular cross-linking of elastin and collagen, and in recent years it has been identified as regulator of various pathologies, such as cancer and inflammation. Here, we review recent advances in the understanding of the contribution of MKs to the progression of myelofibrosis, highlighting the newly identified role of LOX.

Adenosine is an endogenous metabolite that has an anti-inflammatory effect across the vasculature. Extracellular adenosine activates 4 G-protein coupled receptors (A1, A3, A2A, and A2B) whose expression varies in different cells and tissues, including the vasculature and blood cells. Higher levels of adenosine are generated during stress, inflammation, and upon tissue damage. Some of the adenosine receptors (AR), such as the A2BAR, are further up-regulated following such stresses. This review discusses the role of adenosine and adenosine receptors in the development of atherosclerosis and some of the risk factors associated with this pathology. These include adenosine receptor-regulated changes in atherosclerosis, blood pressure, thrombosis, and myocardial infarction. Potential therapeutic applications are reviewed, as well as reasons for phenotypic differences occasionally observed between receptor knockout and pharmacological inhibition via drug administration.

Reactive oxygen species (ROS), generated as a result of various reactions, control an array of cellular processes. The role of ROS during megakaryocyte (MK) development has been a subject of interest and research. The bone marrow niche is the major site of MK differentiation and maturation. In this environment, a gradient of oxygen tension, from normoxia to hypoxia results in different levels of ROS, impacting cellular physiology. This article provides an overview of major sources of ROS, their implication in different signaling pathways, and their effect on cellular physiology, with a focus on megakaryopoiesis. The importance of ROS-generating oxidases in MK biology and pathology, including myelofibrosis, is also described.