Thrombotic, Vascular, and Bleeding Complications of the Myeloproliferative Neoplasms.

Thrombotic, vascular, and bleeding complications are the most frequent causes of morbidity and mortality in myeloproliferative neoplasms (MPNs). The interplay and reciprocal amplification between two factors are considered to lead to thrombosis in MPNs: (1) circulating blood cell-intrinsic abnormalities caused by an MPN driver mutation in their hematopoietic progenitor/stem cells, interacting with vascular endothelial cells, show prothrombotic and proadhesive phenotypes; and (2) a state of usually subclinical systemic inflammation that fuels the thrombotic tendency. Prevention and treatment require maintenance of hematocrit less than 45% and cytoreductive therapy in patients with a high risk for thrombotic and vascular complications.

The pathobiology of thrombosis, microvascular disease, and hemorrhage in the myeloproliferative neoplasms.

Thrombotic, vascular, and bleeding complications are the most common causes of morbidity and mortality in the Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). In these disorders, circulating red cells, leukocytes, and platelets, as well as some vascular endothelial cells, each have abnormalities that are cell-intrinsic to the MPN driver mutations they harbor (eg, JAK2 V617F). When these cells are activated in the MPNs, their interactions with each other create a highly proadhesive and prothrombotic milieu in the circulation that predisposes patients with MPN to venous, arterial, and microvascular thrombosis and occlusive disease. Bleeding problems in the MPNs are caused by the MPN blood cell-initiated development of acquired von Willebrand disease. The inflammatory state created by MPN stem cells in their microenvironment extends systemically to amplify the clinical thrombotic tendency and, at the same time, preferentially promote further MPN stem cell clonal expansion, thereby generating a vicious cycle that favors a prothrombotic state in these diseases.

Incremental Utility of Right Ventricular Dysfunction in Patients With Myeloproliferative Neoplasm-Associated Pulmonary Hypertension.

BACKGROUND: Myeloproliferative neoplasm (MPN) has been associated with pulmonary hypertension (PH) on the basis of small observational studies, but the mechanism and clinical significance of PH in MPN are not well established. The aims of this study were to expand understanding of PH in a well-characterized MPN cohort via study of PH-related symptoms, mortality risk, and cardiac remodeling sequalae of PH using quantitative echocardiographic methods. METHODS: The population comprised a retrospective cohort of patients with MPN who underwent transthoracic echocardiography: Doppler-derived pulmonary arterial systolic pressure applied established cutoffs for PH (≥35 mm Hg) and advanced PH (≥50 mm Hg); right ventricular (RV) performance was assessed via conventional indices (tricuspid annular plane systolic excursion [TAPSE], S') and global longitudinal strain. Symptoms and mortality were discerned via standardized review. RESULTS: Three hundred one patients were studied; 56% had echocardiography-demonstrated PH (20% advanced) paralleling a high prevalence (67%) among patients with invasively quantified PASP. PH was associated with adverse left ventricular (LV) remodeling indices, including increased myocardial mass and diastolic dysfunction (P ≤ .001 for all): LV mass and filling pressure (P < .01) were associated with PH independent of LV ejection fraction. RV dysfunction by strain and TAPSE and S' increased in relation to PH (P ≤ .001) and was about threefold greater among patients with advanced PH compared with those without PH. Patients with RV dysfunction were more likely to report dyspnea, as were those with advanced PH (P < .05). During median follow-up of 2.2 years, all-cause mortality was 27%. PH grade (hazard ratio, 1.9; 95% CI, 1.1-3.0; P = .012) and TAPSE- and S'-demonstrated RV dysfunction (hazard ratio, 3.3; 95% CI, 1.3-8.2; P = .01) were independently associated with mortality; substitution of global longitudinal strain for TAPSE and S' yielded similar associations of RV dysfunction with death (hazard ratio, 3.2; 95% CI, 1.5-6.7; P = .003) independent of PH. CONCLUSIONS: PH is highly prevalent in patients with MPN and is linked to LV diastolic dysfunction; echocardiography-quantified RV dysfunction augments risk for mortality independent of PH.

Paraneoplastic thrombocytosis: the secrets of tumor self-promotion.

Paraneoplastic thrombocytosis is associated with many solid tumors and often correlates with reduced survival. Recent studies suggest that a pathogenic feed back loop may be operative between platelets and tumor cells, with reciprocal interactions between tumor growth/metastasis and thrombocytosis/platelet activation. Specific molecular pathways have been identified in which tumors can stimulate platelet production and activation; activated platelets can, in turn, promote tumor growth and metastasis. Taken together, these findings provide exciting new potential targets for therapeutic intervention.

In vivo two-photon excited fluorescence microscopy reveals cardiac- and respiration-dependent pulsatile blood flow in cortical blood vessels in mice.

Subtle alterations in cerebral blood flow can impact the health and function of brain cells and are linked to cognitive decline and dementia. To understand hemodynamics in the three-dimensional vascular network of the cerebral cortex, we applied two-photon excited fluorescence microscopy to measure the motion of red blood cells (RBCs) in individual microvessels throughout the vascular hierarchy in anesthetized mice. To resolve heartbeat- and respiration-dependent flow dynamics, we simultaneously recorded the electrocardiogram and respiratory waveform. We found that centerline RBC speed decreased with decreasing vessel diameter in arterioles, slowed further through the capillary bed, and then increased with increasing vessel diameter in venules. RBC flow was pulsatile in nearly all cortical vessels, including capillaries and venules. Heartbeat-induced speed modulation decreased through the vascular network, while the delay between heartbeat and the time of maximum speed increased. Capillary tube hematocrit was 0.21 and did not vary with centerline RBC speed or topological position. Spatial RBC flow profiles in surface vessels were blunted compared with a parabola and could be measured at vascular junctions. Finally, we observed a transient decrease in RBC speed in surface vessels before inspiration. In conclusion, we developed an approach to study detailed characteristics of RBC flow in the three-dimensional cortical vasculature, including quantification of fluctuations in centerline RBC speed due to cardiac and respiratory rhythms and flow profile measurements. These methods and the quantitative data on basal cerebral hemodynamics open the door to studies of the normal and diseased-state cerebral microcirculation.

Hyperhomocysteinemia impairs endothelium-derived hyperpolarizing factor-mediated vasorelaxation in transgenic cystathionine beta synthase-deficient mice.

Hyperhomocysteinemia (HHcy) is associated with endothelial dysfunction (ED), but the mechanism is largely unknown. In this study, we investigated the role and mechanism of HHcy-induced ED in microvasculature in our newly established mouse model of severe HHcy (plasma total homocysteine, 169.5 µM). We found that severe HHcy impaired nitric oxide (NO)- and endothelium-derived hyperpolarizing factor (EDHF)-mediated, endothelium-dependent relaxations of small mesenteric arteries (SMAs). Endothelium-independent and prostacyclin-mediated endothelium-dependent relaxations were not changed. A nonselective Ca(2+)-activated potassium channel (K(Ca)) inhibitor completely blocked EDHF-mediated relaxation. Selective blockers for small-conductance K(Ca) (SK) or intermediate-conductance K(Ca) (IK) failed to inhibit EDHF-mediated relaxation in HHcy mice. HHcy increased the levels of SK3 and IK1 protein, superoxide (O(2)(-)), and 3-nitrotyrosine in the endothelium of SMAs. Preincubation with antioxidants and peroxynitrite (ONOO(-)) inhibitors improved endothelium-dependent and EDHF-mediated relaxations and decreased O(2)(-) production in SMAs from HHcy mice. Further, EDHF-mediated relaxation was inhibited by ONOO(-) and prevented by catalase in the control mice. Finally, L-homocysteine stimulated O(2)(-) production, which was reversed by antioxidants, and increased SK/IK protein levels and tyrosine nitration in cultured human cardiac microvascular endothelial cells. Our results suggest that HHcy impairs EDHF relaxation in SMAs by inhibiting SK/IK activities via oxidation- and tyrosine nitration-related mechanisms.

Regulation of homocysteine metabolism and methylation in human and mouse tissues.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine (Hcy) metabolism involves multiple enzymes; however, tissue Hcy metabolism and its relevance to methylation remain unknown. Here, we established gene expression profiles of 8 Hcy metabolic and 12 methylation enzymes in 20 human and 19 mouse tissues through bioinformatic analysis using expression sequence tag clone counts in tissue cDNA libraries. We analyzed correlations between gene expression, Hcy, S-adenosylhomocysteine (SAH), and S-adenosylmethionine (SAM) levels, and SAM/SAH ratios in mouse tissues. Hcy metabolic and methylation enzymes were classified into two types. The expression of Type 1 enzymes positively correlated with tissue Hcy and SAH levels. These include cystathionine beta-synthase, cystathionine-gamma-lyase, paraxonase 1, 5,10-methylenetetrahydrofolate reductase, betaine:homocysteine methyltransferase, methionine adenosyltransferase, phosphatidylethanolamine N-methyltransferases and glycine N-methyltransferase. Type 2 enzyme expressions correlate with neither tissue Hcy nor SAH levels. These include SAH hydrolase, methionyl-tRNA synthase, 5-methyltetrahydrofolate:Hcy methyltransferase, S-adenosylmethionine decarboxylase, DNA methyltransferase 1/3a, isoprenylcysteine carboxyl methyltransferases, and histone-lysine N-methyltransferase. SAH is the only Hcy metabolite significantly correlated with Hcy levels and methylation enzyme expression. We established equations expressing combined effects of methylation enzymes on tissue SAH, SAM, and SAM/SAH ratios. Our study is the first to provide panoramic tissue gene expression profiles and mathematical models of tissue methylation regulation.

The academic job offer: what to expect and how to negotiate.

In contrast to the familiar college, medical school, residency and fellowship application mechanisms, securing the first academic faculty position has no ground rules and no timelines. The open-ended and idiosyncratic nature of securing a job can be frustrating. Negotiating a competitive start-up package when continuing on the faculty of one's training institution can be particularly challenging. Deciding which offer to accept should be determined less by the "best deal" that can be negotiated and more by the short-term and long-term opportunities for career success.

Hyperhomocysteinemia promotes inflammatory monocyte generation and accelerates atherosclerosis in transgenic cystathionine beta-synthase-deficient mice.

BACKGROUND: Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Monocytes display inflammatory and resident subsets and commit to specific functions in atherogenesis. In this study, we examined the hypothesis that HHcy modulates monocyte heterogeneity and leads to atherosclerosis. METHODS AND RESULTS: We established a novel atherosclerosis-susceptible mouse model with both severe HHcy and hypercholesterolemia in which the mouse cystathionine beta-synthase (CBS) and apolipoprotein E (apoE) genes are deficient and an inducible human CBS transgene is introduced to circumvent the neonatal lethality of the CBS deficiency (Tg-hCBS apoE(-/-) Cbs(-/-) mice). Severe HHcy accelerated atherosclerosis and inflammatory monocyte/macrophage accumulation in lesions and increased plasma tumor necrosis factor-alpha and monocyte chemoattractant protein-1 levels in Tg-hCBS apoE(-/-) Cbs(-/-) mice fed a high-fat diet. Furthermore, we characterized monocyte heterogeneity in Tg-hCBS apoE(-/-) Cbs(-/-) mice and another severe HHcy mouse model (Tg-S466L Cbs(-/-)) with a disease-relevant mutation (Tg-S466L) that lacks hyperlipidemia. HHcy increased monocyte population and selective expansion of inflammatory Ly-6C(hi) and Ly-6C(mid) monocyte subsets in blood, spleen, and bone marrow of Tg-S466L Cbs(-/-) and Tg-hCBS apoE(-/-) Cbs(-/-) mice. These changes were exacerbated in Tg-S466L Cbs(-/-) mice with aging. Addition of l-homocysteine (100 to 500 micromol/L), but not l-cysteine, maintained the Ly-6C(hi) subset and induced the Ly-6C(mid) subset in cultured mouse primary splenocytes. Homocysteine-induced differentiation of the Ly-6C(mid) subset was prevented by catalase plus superoxide dismutase and the NAD(P)H oxidase inhibitor apocynin. CONCLUSIONS: HHcy promotes differentiation of inflammatory monocyte subsets and their accumulation in atherosclerotic lesions via NAD(P)H oxidase-mediated oxidant stress.

The cyclic GMP modulators YC-1 and zaprinast reduce vessel remodeling through antiproliferative and proapoptotic effects.

Guanosine-specific cyclic nucleotide signaling is suggested to serve protective actions in the vasculature; however, the influence of selective pharmacologic modulation of cyclic guanosine monophosphate- synthesizing soluble guanylate cyclase or cyclic guanosine monophosphate-degrading phosphodiesterase on vessel remodeling has not been thoroughly examined. In this study, rat carotid artery balloon injury was performed and the growth-modulating effects of the soluble guanylate cyclase stimulator YC-1 or the cyclic guanosine monophosphate-dependent phosphodiesterase-V inhibitor zaprinast were examined. YC-1 or zaprinast elevated vessel cyclic guanosine monophosphate content, reduced medial wall and neointimal cell proliferation, stimulated medial and neointimal cellular apoptosis, and markedly attenuated neointimal remodeling in comparable fashion. Interestingly, soluble guanylate cyclase inhibition by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one failed to noticeably alter neointimal growth, and concomitant zaprinast with YC-1 did not modify any parameter compared to individual treatments. These results provide novel in vivo evidence that YC-1 and zaprinast inhibit injury-induced vascular remodeling through antimitogenic and proapoptotic actions and may offer promising therapeutic approaches against vasoproliferative disorders.

Inhibition of plasminogen activator inhibitor-1 expression in vascular smooth muscle cells by protoporphyrins through a heme oxygenase-independent mechanism.

Heme oxygenase-1 (HO-1), a rate-limiting enzyme in heme catabolism, has been shown to play a regulatory role in the expression of plasminogen activator inhibitor-1 (PAI-1), a risk factor for vascular disease. Accordingly, we examined the effect of protoporphyrins, both HO inhibitors and activators, on PAI-1 expression in human vascular smooth muscle cells (VSMCs). Tin-protoporphyrin (SnPP) markedly inhibited the transforming growth factor beta1 (TGFbeta1)-induced expression of PAI-1 protein. Protoporphyrins, whether they are inhibitors or activators of HO, produced a similar inhibitory effect. However, SnPP had no effect on the level of PAI-1 mRNA transcripts. Knockdown of human HO-1 with a specific siRNA did not reduce the PAI-1 protein level in TGFbeta1-treated cells. In addition, the proteasome inhibitor lactacystin reversed the inhibitory effect of SnPP on PAI-1 protein expression. Both cobalt-protoporphyrin (CoPP) and CoCl2 markedly induced HO-1 expression. However, CoPP did not affect PAI-1 gene expression, whereas CoCl2 upregulated PAI-1 mRNA in a dose-dependent manner. Our results demonstrate that protoporphyrins can block the TGFbeta1-mediated induction of PAI-1 protein in VSMCs and that this inhibitory effect is independent of HO activity.

Hemin upregulates Egr-1 expression in vascular smooth muscle cells via reactive oxygen species ERK-1/2-Elk-1 and NF-kappaB.

Reactive oxygen species (ROS) and oxidant stress are important mediators of cardiovascular pathologies including atherosclerosis. One source of ROS in the vasculature is free heme released from hemoglobin. Because Egr-1, the regulator of cell proliferation and apoptosis, is also induced by oxidant stress and is likewise implicated in atherosclerosis, we examined the regulation of Egr-1 by heme in vascular smooth muscle cells (SMCs). Hemin increased Egr-1 expression (mRNA, protein) within 30 minutes and ERK-1/2 phosphorylation and nuclear translocation within 5 minutes. Inhibiting hemin-induced ERK-1/2 activation by U0126 (MAPK-inhibitor), the antioxidant N-acetyl cysteine, the NADPH oxidase inhibitors apocynin and diphenyleneiodonium chloride, the superoxide scavenger tiron, or tricarbonyldichlororuthenium(II)-dimer (carbon-monoxide donor; CORM-2) blocked hemin-induced Egr-1 expression. Hemin activated Elk-1, SRF, and NF-kappaB and promoted their interaction with the Egr-1 promoter. Downregulating Elk-1 (via siRNA) or blocking NF-kappaB activation (via BAY-11-7082) abolished hemin induction of Egr-1. Finally, hemin-induced Egr-1 bound the promoters of tissue factor (TF), Plasminogen Activator Inhibitor (PAI)-1, and NGF-1A Binding (NAB)-2, upregulating their expression, and increased the biochemical activity of TF and PAI-1. Upregulation of Egr-1 and its target genes by heme-induced oxidant stress may be an important event in the initiation and progression of inflammatory vascular diseases such as atherosclerosis.

Homocysteine inhibits endothelial cell growth via DNA hypomethylation of the cyclin A gene.

We reported previously that homocysteine (Hcy) inhibits endothelial cell (EC) growth by transcriptional inhibition of the cyclin A gene via a hypomethylation-related mechanism. In this study, we examined the effect of Hcy on epigenetic modification of the cyclin A gene and its biologic role in human ECs. Cyclin A mRNA levels were significantly suppressed by Hcy and a DNA methyltransferase inhibitor. The cyclin A promoter contains a CpG island spanning a 477-bp region (-277/200). Bisulfite sequencing followed by polymerase chain reaction (PCR) amplification of the cyclin A promoter (-267/37) showed that Hcy eliminated methylation at 2 CpG sites in the cyclin A promoter, one of which is located on the cycle-dependent element (CDE). Mutation of CG sequence on the CDE leads to a 6-fold increase in promoter activity. Hcy inhibited DNA methyltransferase 1 (DNMT1) activity by 30%, and reduced the binding of methyl CpG binding protein 2 (MeCP2) and increased the bindings of acetylated histone H3 and H4 in the cyclin A promoter. Finally, adenovirus-transduced DNMT1 gene expression reversed the inhibitory effect of Hcy on cyclin A expression and EC growth inhibition. In conclusion, Hcy inhibits cyclin A transcription and cell growth by inhibiting DNA methylation through suppression of DNMT1 in ECs.

Differential regulation of homocysteine transport in vascular endothelial and smooth muscle cells.

OBJECTIVE: We previously reported that homocysteine (Hcy) inhibits endothelial cell (EC) growth and promotes vascular smooth muscle cell (VSMC) proliferation. This study characterized and directly compared Hcy transport in cultured human aortic ECs (HAECs) and smooth muscle cells (HASMCs). METHODS AND RESULTS: Hcy (10 micromol/L) was transported into both cell types in a time-dependent fashion but was approximately 4-fold greater in HASMCs, and is nonstereoenantiomer specific. Hcy transport in HAECs had a Michaelis-Menten constant (Km) of 39 micromol/L and a maximal transport velocity (Vmax) of 873 pmol/mg protein/min. In contrast, Hcy transport in HASMCs had a lower affinity (Km = 106 micromol/L) but a higher transport capacity (Vmax = 4192 pmol/mg protein/min). Competition studies revealed that the small neutral amino acids tyrosine, cysteine, glycine, serine, alanine, methionine, and leucine inhibited Hcy uptake in both cell types, but the inhibition was greater for tyrosine, serine, glycine, and alanine in HAECs. Sodium-depletion reduced Hcy transport to 16% in HAECs and 56% in HASMCs. Increases in pH from 6.5 to 8.2 or lysosomal inhibitors blocked Hcy uptake only in HAECs. In addition, Hcy shares carrier systems with cysteine, in a preferable order of alanine-serine-cysteine (ASC) > aspartate and glutamate (X(AG)) = large branched-chain neutral amino acids (L) transporter systems in HAECs and ASC > L > X(AG) in HASMCs. The sodium-dependent system ASC plays a predominant role for Hcy transport in vascular cells. CONCLUSIONS: Transport system ASC predominantly mediates Hcy transport in EC and is lysosomal dependent.

Thrombocytosis: When is an incidental finding serious?

For most patients, elevated platelet counts are benign and require no treatment, but for some, severe complications or death may ensue. This article discusses how to classify thrombocytosis and identify which patients require treatment, describes the characteristic complications that may arise, and provides an algorithm for management.