Prognostic Biomarkers for Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome in Myeloablative Allogeneic Hematopoietic Cell Transplantation: Results from the Blood and Marrow Transplant Clinical Trials Network 1202 Study.

Hepatic veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) is a potentially life-threatening complication of hematopoietic cell transplantation (HCT). This study aimed to determine a blood biomarker signature early post-HCT that identifies patients at high risk for VOD/SOS. A set of 23 plasma biomarkers, selected from the VOD/SOS literature, was measured on days 0, 7, and 14 after myeloablative HCT using blood samples from patients enrolled in the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) Protocol 1202. Eligible cases were diagnosed with VOD/SOS in BMT CTN 1202 using the Baltimore criteria. Controls (without VOD/SOS) were matched to cases for conditioning regimen and age. Significant biomarkers were identified using the Bonferroni-adjusted Wilcoxon rank-sum test (P ≤ .002). Thirty-three patients with mild or severe VOD/SOS were identified (cases) and matched to 107 controls. Two, 8, and 5 biomarkers measured from the plasma of these patients were significantly associated with the development of VOD/SOS at days 0, 7, and 14, respectively, with the strongest associations on days 7 and 14. Biomarker associations were stronger for severe VOD/SOS risk and were stronger prognostic markers for VOD/SOS cases occurring within 28 days of HCT. Hyaluronan was most strongly associated with VOD/SOS risk, with an area under the receiver operating characteristic curve (AUC) of .81 on day 7 and .79 on day 14. Multivariate models of up to 5 biomarkers generated AUCs ranging from .82 to .85. All associations with VOD/SOS risk were independent of clinical risk factors. This study confirms previously identified biomarkers of VOD/SOS risk and identified novel prognostic biomarker signatures that identify patients at risk for VOD/SOS shortly after HCT. Multivariate analysis suggests that a combination of up to 5 of these protein biomarkers may provide a prognostic tool for identifying patients at risk for VOD/SOS.

Gene variants associated with deep vein thrombosis.

CONTEXT: The genetic causes of deep vein thrombosis (DVT) are not fully understood. OBJECTIVE: To identify single-nucleotide polymorphisms (SNPs) associated with DVT. DESIGN, SETTING, AND PATIENTS: We used 3 case-control studies of first DVT. A total of 19 682 gene-centric SNPs were genotyped in 443 cases and 453 controls from the Leiden Thrombophilia Study (LETS, 1988-1992). Twelve hundred six SNPs associated with DVT were reinvestigated in the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis study (MEGA-1, 1999-2004) in a subset of 1398 cases and 1757 controls. Nine SNPs associated with DVT in both LETS and MEGA-1 were investigated a third time in 1314 cases and 2877 controls from MEGA-2, a second subset of MEGA. Additional SNPs close to one SNP in CYP4V2 were genotyped in LETS and MEGA-1. MAIN OUTCOME MEASURE: Odds ratios (ORs) for DVT were estimated by logistic regression. False discovery rates served to investigate the effect of multiple hypothesis testing. RESULTS: Of 9 SNPs genotyped in MEGA-2, 3 were strongly associated with DVT (P < .05; false discovery rate < or =.10): rs13146272 in CYP4V2 (risk allele frequency, 0.64), rs2227589 in SERPINC1 (risk allele frequency, 0.10), and rs1613662 in GP6 (risk allele frequency, 0.84). The OR for DVT per risk allele was 1.24 (95% confidence interval [95%CI], 1.11-1.37) for rs13146272, 1.29 (95% CI, 1.10-1.49) for rs2227589, and 1.15 (95% CI, 1.01-1.30) for rs1613662. In the region of CYP4V2, we identified 4 additional SNPs (in CYP4V2, KLKB1, and F11) that were also associated with both DVT (highest OR per risk allele, 1.39; 95% CI, 1.11-1.74) and coagulation factor XI level (highest increase per risk allele, 8%; 95% CI, 5%-11%). CONCLUSIONS: We identified SNPs in several genes that were associated with DVT. We also found SNPs in the region around the SNP in CYP4V2 (rs13146272) that were associated with both DVT and factor XI levels. These results show that common genetic variation plays an important role in determining thrombotic risk.

Association of the Trp719Arg polymorphism in kinesin-like protein 6 with myocardial infarction and coronary heart disease in 2 prospective trials: the CARE and WOSCOPS trials.

OBJECTIVES: We asked whether 35 genetic polymorphisms, previously found to be associated with cardiovascular disease, were associated with myocardial infarction (MI) in the CARE (Cholesterol and Recurrent Events) trial and with coronary heart disease (CHD) in the WOSCOPS (West of Scotland Coronary Prevention Study) trial and whether the risk associated with these polymorphisms could be reduced by pravastatin treatment. BACKGROUND: Identification of genetic polymorphisms associated with CHD may improve assessment of CHD risk and understanding of disease pathophysiology. METHODS: We tested the association between genotype and recurrent MI in the CARE study and between genotype and primary CHD in the WOSCOPS trial using regression models that adjusted for conventional risk factors: Cox proportional hazards models for the CARE study and conditional logistic regression models for a nested case-control study of the WOSCOPS trial. RESULTS: We found that Trp719Arg (rs20455) in KIF6 was associated with coronary events. KIF6 encodes kinesin-like protein 6, a member of the molecular motor superfamily. In placebo-treated patients, carriers of the KIF6 719Arg allele (59.4% of the CARE trial cohort) had a hazard ratio of 1.50 (95% confidence interval [CI] 1.05 to 2.15) in the CARE trial and an odds ratio of 1.55 (95% CI 1.14 to 2.09) in the WOSCOPS trial. Among carriers, the absolute risk reduction by pravastatin was 4.89% (95% CI 1.81% to 7.97%) in the CARE trial and 5.49% (95% CI 3.52% to 7.46%) in the WOSCOPS trial. CONCLUSIONS: In both the CARE and the WOSCOPS trials, carriers of the KIF6 719Arg allele had an increased risk of coronary events, and pravastatin treatment substantially reduced that risk.

Association of gene variants with incident myocardial infarction in the Cardiovascular Health Study.

OBJECTIVE: We asked whether single nucleotide polymorphisms (SNPs) that had been nominally associated with cardiovascular disease in antecedent studies were also associated with cardiovascular disease in a population-based prospective study of 4522 individuals aged 65 or older. METHODS AND RESULTS: Based on antecedent studies, we prespecified a risk allele and an inheritance model for each of 74 SNPs. We then tested the association of these SNPs with myocardial infarction (MI) in the Cardiovascular Health Study (CHS). The prespecified risk alleles of 8 SNPs were nominally associated (1-sided P<0.05) with increased risk of MI in White CHS participants. The false discovery rate for these 8 was 0.43, suggesting that about 4 of these 8 are likely to be true positives. The 4 of these 8 SNPs that had the strongest evidence for association with cardiovascular disease before testing in CHS (association in 3 antecedent studies) were in KIF6 (CHS HR=1.29; 90%CI 1.1 to 1.52), VAMP8 (HR=1.2; 90%CI 1.02 to 1.41), TAS2R50 (HR=1.13; 90%CI 1 to 1.27), and LPA (HR=1.62; 90%CI 1.09 to 2.42). CONCLUSIONS: Although most of the SNPs investigated were not associated with MI in CHS, evidence from this investigation combined with previous studies suggests that 4 of these SNPs are likely associated with MI.

Integrin alpha9beta1 directly binds to vascular endothelial growth factor (VEGF)-A and contributes to VEGF-A-induced angiogenesis.

Vascular endothelial growth factor A (VEGF-A) is a potent inducer of angiogenesis. We now show that VEGF-A-induced adhesion and migration of human endothelial cells are dependent on the integrin alpha9beta1 and that VEGF-A is a direct ligand for this integrin. Adhesion and migration of these cells on the 165 and 121 isoforms of VEGF-A depend on cooperative input from alpha9beta1 and the cognate receptor for VEGF-A, VEGF receptor 2 (VEGF-R2). Unlike alpha3beta1or alphavbeta3 integrins, alpha9beta1 was also found to bind the 121 isoform of VEGF-A. This interaction appears to be biologically significant, because alpha9beta1-blocking antibody dramatically and specifically inhibited angiogenesis induced by VEGF-A165 or -121. Together with our previous findings that alpha9beta1 directly binds to VEGF-C and -D and contributes to lymphangiogenesis, these results identify the integrin alpha9beta1 as a potential pharmacotherapeutic target for inhibition of pathogenic angiogenesis and lymphangiogenesis.

Obstetric complications in patients with hereditary thrombophilia identified using the LCx microparticle enzyme immunoassay: a controlled study of 5,000 patients.

Factor V Leiden (FVL) and prothrombin (PT) G20210A mutations are associated with increased risk of deep venous thrombosis, pulmonary embolism, and obstetric complications. The development of inexpensive and reliable screening methods will assist in defining subpopulations of patients at risk who should undergo testing. We used a method, developed by Abbott Laboratories (Abbott Park, IL), to study 5,000 pregnant women and evaluated the association of obstetric complications with the presence of the FVL and PT G20210A mutations. We found a statistically significant association between FVL and stillbirth. There were also trends toward an association between FVL and placental abruption and between PT G20210A and intrauterine growth retardation. In addition, an association may exist between PT G20210A and preterm delivery for white women. All other parameters studied, including miscarriage and preeclampsia, did not show a statistically significant association with FVL or PT G20210A. These results confirm the association between genetic thrombophilia and selected obstetric complications.

Asp92Asn polymorphism in the myeloid IgA Fc receptor is associated with myocardial infarction in two disparate populations: CARE and WOSCOPS.

OBJECTIVE: Statins reduce inflammation and risk of myocardial infarction (MI). Because the myeloid IgA Fc receptor encoded by FCAR mediates inflammation, we hypothesized that the FCAR Asp92Asn polymorphism is associated with risk of MI and that this risk would be modified by pravastatin. METHODS AND RESULTS: In the placebo arm of the Cholesterol and Recurrent Events (CARE) study, male carriers of the 92Asn allele had an adjusted hazard ratio for incident MI of 1.68 (95% CI 1.10 to 2.57); relative risk reduction by pravastatin was 69% in carriers and 12% in noncarriers (P(interaction)=0.007). In the placebo arm of the all-male West of Scotland Coronary Prevention Study (WOSCOPS), carriers had an adjusted odds ratio for incident coronary heart disease (CHD) of 1.46 (90% CI 1.05 to 2.03); for pravastatin compared with placebo treatment, the adjusted odds ratios were 0.55 (95% CI 0.32 to 0.93) in carriers and 0.65 (95% CI 0.51 to 0.83) in noncarriers (P(interaction)=0.55). CONCLUSIONS: Carriers of 92Asn had increased risk of MI in CARE and increased odds of CHD in WOSCOPS. Pravastatin significantly reduced risk in carriers in both CARE and WOSCOPS. A genotype by treatment interaction was observed in CARE but not in WOSCOPS.

Gene variants of VAMP8 and HNRPUL1 are associated with early-onset myocardial infarction.

OBJECTIVE: Identify gene variants associated with early-onset myocardial infarction (MI). METHODS AND RESULTS: We tested 11 647 single-nucleotide polymorphisms (SNPs) for association with early-onset MI in a case-control study (study 1 200 cases, 262 controls). To reduce the number of false positives among the 666 SNPs that were nominally associated with early-onset MI (P<0.05) in study 1, we tested these SNPs in study 2 (434 cases, 504 controls). We found that 8 of the 666 SNPs were associated with early-onset MI in study 2 (P<0.05) and had the same risk alleles as in study 1. These 8 SNPs were then tested for association with early-onset MI in study 3 (187 cases, 434 controls). We found that a VAMP8 variant (P = 0.025; odds ratio [OR], 1.75; CI, 1.17 to 2.62) and an HNRPUL1 variant (P = 0.0043; OR, 1.92; CI, 1.28 to 2.86) were associated with early-onset MI (nominal P<0.05; false discovery rate <10%) and had the same risk alleles in all 3 studies. CONCLUSIONS: Variants in 2 genes were associated with early-onset MI: VAMP8, which is involved in platelet degranulation, and HNRPUL1, which encodes a ribonuclear protein. The identification of these variants could improve understanding of disease mechanisms and suggest novel drug targets.

Receptor protein tyrosine phosphatase micro regulates the paracellular pathway in human lung microvascular endothelia.

The pulmonary vascular endothelial paracellular pathway and zonula adherens (ZA) integrity are regulated, in part, through protein tyrosine phosphorylation. ZA-associated protein tyrosine phosphatase (PTP)s are thought to counterregulate tyrosine phosphorylation events within the ZA multiprotein complex. One such receptor PTP, PTPmu, is highly expressed in lung tissue and is almost exclusively restricted to the endothelium. We therefore studied whether PTPmu, in pulmonary vascular endothelia, associates with and/or regulates both the tyrosine phosphorylation state of vascular endothelial (VE)-cadherin and the paracellular pathway. PTPmu was expressed in postconfluent human pulmonary artery and lung microvascular endothelial cells (ECs) where it was almost exclusively restricted to EC-EC boundaries. In human lung microvascular ECs, knockdown of PTPmu through RNA interference dramatically impaired barrier function. In immortalized human microvascular ECs, overexpression of wild-type PTPmu enhanced barrier function. PTPmu-VE-cadherin interactions were demonstrated through reciprocal co-immunoprecipitation assays and co-localization with double-label fluorescence microscopy. When glutathione S-transferase-PTPmu was incubated with purified recombinant VE-cadherin, and when glutathione S-transferase-VE-cadherin was incubated with purified recombinant PTPmu, PTPmu directly bound to VE-cadherin. Overexpression of wild-type PTPmu decreased tyrosine phosphorylation of VE-cadherin. Therefore, PTPmu is expressed in human pulmonary vascular endothelia where it directly binds to VE-cadherin and regulates both the tyrosine phosphorylation state of VE-cadherin and barrier integrity.

The lymphangiogenic vascular endothelial growth factors VEGF-C and -D are ligands for the integrin alpha9beta1.

Mice homozygous for a null mutation of the integrin alpha9 subunit die 6-12 days after birth from bilateral chylothoraces suggesting an underlying defect in lymphatic development. However, until now the mechanisms by which the integrin alpha9beta1 modulates lymphangiogenesis have not been described. In this study we show that adhesion to and migration on the lymphangiogenic vascular endothelial growth factors (VEGF-C and -D) are alpha9beta1-dependent. Mouse embryonic fibroblasts and human colon carcinoma cells (SW-480) transfected to express alpha9beta1 adhered and/or migrated on both growth factors in a concentration-dependent fashion, and both adhesion and migration were abrogated by anti-alpha9beta1 function-blocking antibody. In SW-480 cells, which lack cognate receptors for VEGF-C and -D, both growth factors induced alpha9beta1-dependent Erk and paxillin phosphorylation. Human microvascular endothelial cells, which express both alpha9beta1 and VEGF-R3, also adhered to and migrated on both growth factors, and both responses were blocked by anti-alpha9beta1 antibody. Furthermore, in a solid phase binding assay recombinant VEGF-C and -D bound to purified alpha9beta1 integrin in a dose- and cation-dependent fashion showing that VEGF-C and VEGF-D are ligands for the integrin alpha9beta1. The interaction between alpha9beta1 and VEGF-C and/or -D may begin to explain the abnormal lymphatic phenotype of the alpha9 knock-out mice.

Spermidine/spermine N1-acetyltransferase specifically binds to the integrin alpha9 subunit cytoplasmic domain and enhances cell migration.

The integrin alpha9beta1 is expressed on migrating cells, such as leukocytes, and binds to multiple ligands that are present at sites of tissue injury and inflammation. alpha9beta1, like the structurally related integrin alpha4beta1, mediates accelerated cell migration, an effect that depends on the alpha9 cytoplasmic domain. alpha4beta1 enhances migration through reversible binding to the adapter protein, paxillin, but alpha9beta1-dependent migration is paxillin independent. Using yeast two-hybrid screening, we identified the polyamine catabolizing enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) as a specific binding partner of the alpha9 cytoplasmic domain. Overexpression of SSAT increased alpha9beta1-mediated migration, and small interfering RNA knockdown of SSAT inhibited this migration without affecting cell adhesion or migration that was mediated by other integrin cytoplasmic domains. The enzyme activity of SSAT is critical for this effect, because a catalytically inactive version did not enhance migration. We conclude that SSAT directly binds to the alpha9 cytoplasmic domain and mediates alpha9-dependent enhancement of cell migration, presumably by localized effects on acetylation of polyamines or of unidentified substrates.

Protein tyrosine phosphatase activity regulates endothelial cell-cell interactions, the paracellular pathway, and capillary tube stability.

Protein tyrosine phosphorylation is tightly regulated through the actions of both protein tyrosine kinases and protein tyrosine phosphatases. In this study, we demonstrate that protein tyrosine phosphatase inhibition promotes tyrosine phosphorylation of endothelial cell-cell adherens junction proteins, opens an endothelial paracellular pathway, and increases both transendothelial albumin flux and neutrophil migration. Tyrosine phosphatase inhibition with sodium orthovanadate or phenylarsine oxide induced dose- and time-dependent increases in [14C]bovine serum albumin flux across postconfluent bovine pulmonary artery endothelial cell monolayers. These increases in albumin flux were coincident with actin reorganization and intercellular gap formation in both postconfluent monolayers and preformed endothelial cell capillary tubes. Vanadate (25 microM) increased tyrosine phosphorylation of endothelial cell proteins 12-fold within 1 h. Tyrosine phosphorylated proteins were immunolocalized to the intercellular boundaries, and several were identified as the endothelial cell-cell adherens junction proteins, vascular-endothelial cadherin, and beta-, gamma-, and p120-catenin as well as platelet endothelial cell adhesion molecule-1. Of note, these tyrosine phosphorylation events were not associated with disassembly of the adherens junction complex or its uncoupling from the actin cytoskeleton. The dose and time requirements for vanadate-induced increases in phosphorylation were comparable with those defined for increments in transendothelial [14C]albumin flux and neutrophil migration, and pretreatment with the tyrosine kinase inhibitor herbimycin A protected against these effects. These data suggest that protein tyrosine phosphatases and their substrates, which localize to the endothelial cell-cell boundaries, regulate adherens junctional integrity, the movement of macromolecules and cells through the endothelial paracellular pathway, and capillary tube stability.

Increased circulating concentrations of the counteradhesive proteins SPARC and thrombospondin-1 in systemic sclerosis (scleroderma). Relationship to platelet and endothelial cell activation.

OBJECTIVE: To determine whether circulating concentrations of the counteradhesive proteins SPARC (secreted protein acidic and rich in cysteine) and thrombospondin-1 (TSP-1) are elevated in scleroderma (systemic sclerosis, SSc). The relationship of these counteradhesive proteins to measures of platelet and endothelial cell activation was examined. METHODS: Plasma from 45 patients with SSc (26 limited form, 19 diffuse) and 22 age and sex matched controls was assayed for SPARC, TSP-1, beta-thromboglobulin (betaTG), and platelet factor 4 (PF4), 2 distinct platelet a-granule products, and soluble E-selectin, a marker of endothelial cell activation. RESULTS: The mean (+/- SE) SPARC concentration was greater in patients with limited SSc (124.0 +/- 9.6 ng/ml) compared to controls (66.8 +/- 8.0 ng/ml) (p = 0.0005), whereas in patients with diffuse SSc (74.1 +/- 7.9 ng/ml) it was not. Elevated SPARC concentrations in the limited SSc group could not be ascribed to either platelet or endothelial cell activation. TSP-1 concentrations were also increased in SSc patients (n = 29) compared to controls (n = 11) (2.98 +/- 0.12 vs 2.4 +/- 0.21 log transformed ng/ml; p < 0.02). Unlike SPARC, TSP-1 concentrations correlated with both betaTG (r = 0.57, p = 0.0014) and PF4 (r = 0.41, p = 0.026) levels, indicating that increased TSP-1 could, in part, be explained through elevated platelet a-granule release in SSc patients. Plasma levels of betaTG, PF4, and E-selectin were each similarly elevated (p < 0.003) in patients with both limited and diffuse SSc compared to controls. CONCLUSION: That circulating SPARC and TSP-1 are elevated in patients with SSc raises the possibility that counteradhesive proteins, which regulate vascular organization and remodeling, might contribute to the pathogenesis of SSc vasculopathy.