Identification and validation of novel candidate protein biomarkers for the detection of human gastric cancer.

The timely detection of gastric cancer will contribute significantly towards effective treatment and is aided by the availability and reliability of appropriate biomarkers. A combination of several biomarkers can improve the sensitivity and specificity of cancer detection and this work reports results from a panel of 4 proteins. By combining a validated preclinical mouse model with a proteomic approach we have recently discovered novel biomarkers for the detection of gastric cancer. Here, we investigate the specificity of four of those biomarkers (afamin, clusterin, VDBP and haptoglobin) for the detection of gastric cancer using two independent methods of validation: ELISA, and a non antibody based method: Multiple Reaction Monitoring with High Resolution Mass Spectrometry (MRM-HR). All four biomarkers reliably differentiated GC from benign patient serum, and also in a small cohort of 11 early stage cases. We also present a novel isoform specific biomarker alpha-1-antitrypsin (A1AT) that was identified using a mouse model for gastric cancer. This isoform is distinct in charge and mobility in a pH gradient and was validated using human samples by isoelectric focussing and Western-blot (IEF-WB). This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Antiangiogenic therapy inhibits venous thrombus resolution.

OBJECTIVE: Venous thromboembolism is a common complication in patients with cancer, resulting in significant morbidity and mortality. Clinical studies suggest that the incidence of venous thromboembolic events increased after treatment of these patients with antiangiogenic agents. Thrombi resolve through a process of remodeling, involving the formation of microvascular channels within the thrombus. Our aim was to determine whether inhibiting angiogenesis affects venous thrombus resolution. APPROACH AND RESULTS: Thrombus was induced in the inferior vena cava of mice. These mice were treated with axitinib (50 mg/kg per day), 2-methoxyestradiol (2ME, 150 mg/kg per day), or vehicle control. Thrombus size, recanalization, neovascularization, inflammatory cell content, and collagen content were assessed after axitinib (days 3, 10, 17) and 2ME (day 10 only) treatment (n=6/group). Axitinib treatment resulted in reduced thrombus resolution (P<0.002) and vein recanalization (P<0.001) compared with vehicle-treated controls. This was associated with inhibition of organization as seen through reduced thrombus neovascularization (P<0.0001) and collagen (P<0.0001) content, as well as reduced macrophage accumulation in the thrombus (P<0.001). Treatment with a second antiangiogenic agent, 2ME, mirrored these findings, with a similar order of magnitude of effect of treatment over vehicle control in all of the parameters measured, with the exception of neutrophil content, which was significantly reduced after 2ME treatment but not affected by axitinib. CONCLUSIONS: Antiangiogenic therapy (using axitinib and 2ME) inhibits the resolution of venous thrombi, which could lead to persistent venous obstruction and the possibility of thrombus extension. This potential prolongation of venous occlusion by antiangiogenic agents should therefore be taken into consideration in trials of these agents and when managing the complications of venous thromboembolic events in patients with cancer.

Magnetic resonance T1 relaxation time of venous thrombus is determined by iron processing and predicts susceptibility to lysis.

BACKGROUND: The magnetic resonance longitudinal relaxation time (T1) changes with thrombus age in humans. In this study, we investigate the possible mechanisms that give rise to the T1 signal in venous thrombi and whether changes in T1 relaxation time are informative of the susceptibility to lysis. METHODS AND RESULTS: Venous thrombosis was induced in the vena cava of BALB/C mice, and temporal changes in T1 relaxation time correlated with thrombus composition. The mean T1 relaxation time of thrombus was shortest at 7 days following thrombus induction and returned to that of blood as the thrombus resolved. T1 relaxation time was related to thrombus methemoglobin formation and further processing. Studies in inducible nitric oxide synthase (iNOS(-/-))-deficient mice revealed that inducible nitric oxide synthase mediates oxidation of erythrocyte lysis-derived iron to paramagnetic Fe3+, which causes thrombus T1 relaxation time shortening. Studies using chemokine receptor-2-deficient mice (Ccr2(-/-)) revealed that the return of the T1 signal to that of blood is regulated by removal of Fe3+ by macrophages that accumulate in the thrombus during its resolution. Quantification of T1 relaxation time was a good predictor of successful thrombolysis with a cutoff point of <747 ms having a sensitivity and specificity to predict successful lysis of 83% and 94%, respectively. CONCLUSIONS: The source of the T1 signal in the thrombus results from the oxidation of iron (released from the lysis of trapped erythrocytes in the thrombus) to its paramagnetic Fe3+ form. Quantification of T1 relaxation time appears to be a good predictor of the success of thrombolysis.

Hematopoietic progenitor cells and restenosis after carotid endarterectomy.

BACKGROUND AND PURPOSE: Hematopoietic progenitor cells (HPCs) may attenuate the response to vascular injury by maintaining endothelial integrity and function. Our aim was to determine whether circulating HPC number and function correlate with restenosis after carotid endarterectomy. METHODS: HPC number (CD34(+)/CD133(+) cells), early colony-forming units, migratory capacity, and senescence were analyzed in blood collected preoperatively, 1 day, and 6 weeks postoperatively. Mobilizing cytokine levels were also measured. Stenosis was assessed by duplex scanning. RESULTS: HPC numbers (P<0.001) and early colony-forming unit count (P=0.001) fell rapidly 24 hours postoperatively. Restenosis at 6 months correlated negatively with the magnitude of postoperative falls in HPC numbers (R=-0.38, P=0.013) and early colony-forming unit counts (R=-0.42, P=0.008). The migratory capacity of preoperative HPCs correlated negatively with restenosis (R=-0.48, P=0.007). Preoperative SDF1 levels correlated with falls in HPC number (R=0.42, P=0.044) and early colony-forming unit counts (R=0.56, P=0.004). CONCLUSIONS: HPC function appears to be linked to the development of carotid artery restenosis after endarterectomy. These data support the concept that HPCs have a role in regulating remodeling of the injured arterial wall.

Leukocytes and the natural history of deep vein thrombosis: current concepts and future directions.

Observational studies have shown that inflammatory cells accumulate within the thrombus and surrounding vein wall during the natural history of venous thrombosis. More recent studies have begun to unravel the mechanisms that regulate this interaction and have confirmed that thrombosis and inflammation are intimately linked. This review outlines our current knowledge of the complex relationship between inflammatory cell activity and venous thrombosis and highlights new areas of research in this field. A better understanding of this relationship could lead to the development of novel therapeutic targets that inhibit thrombus formation or promote its resolution.

Nox activator 1: a potential target for modulation of vascular reactive oxygen species in atherosclerotic arteries.

BACKGROUND: Despite a concerted effort by many laboratories, the critical subunits that participate in vascular smooth muscle cell (VSMC) NADPH oxidase function have yet to be elucidated. Given the potential therapeutic importance of cell-specific inhibition of NADPH oxidase, we investigated the role of Nox activator 1 (NoxA1), a homolog of p67phox, in VSMC NADPH oxidase function and atherosclerosis. METHODS AND RESULTS: The presence of NoxA1 in mouse aortic VSMCs was confirmed by reverse-transcription polymerase chain reaction and sequencing. NoxA1/p47phox interaction after thrombin treatment was observed by immunoprecipitation/Western analysis of lysates from p47phox(-/-) VSMCs transfected with adenoviral HA-NoxA1 and Myc-p47phox. Infection with adenoviral NoxA1 significantly enhanced thrombin-induced reactive oxygen species generation in wild-type but not in p47phox(-/-) and Nox1(-/-) VSMCs. Thrombin-induced reactive oxygen species production and VSMC proliferation were significantly reduced after downregulation of NoxA1 with shRNA. Infection with NoxA1 shRNA but not scrambled shRNA significantly decreased thrombin-induced activation of the redox-sensitive protein kinases (Janus kinase 2, Akt, and p38 mitogen-activated protein kinase) in VSMCs. Adenovirus-mediated overexpression of NoxA1 in guidewire-injured mouse carotid arteries significantly increased superoxide production in medial VSMCs and enhanced neointimal hyperplasia. NoxA1 expression was significantly increased in aortas and atherosclerotic lesions of ApoE(-/-) mice compared with age-matched wild-type mice. Furthermore, in contrast to p67phox, immunoreactive NoxA1 is present in intimal and medial SMCs of human early carotid atherosclerotic lesions. CONCLUSIONS: NoxA1 is the functional homolog of p67phox in VSMCs that regulates redox signaling and VSMC phenotype. These findings support the potential for modulation of NoxA1 expression as a viable approach for the treatment of vascular diseases.

Hypoxia and upregulation of hypoxia-inducible factor 1{alpha} stimulate venous thrombus recanalization.

OBJECTIVE: Angiogenic factors are expressed within thrombus during resolution, but the primary stimulus for neovascularization is unknown. Our aims were to determine whether (1) hypoxia and hypoxia-inducible factor 1α (HIF1α) are induced in resolving thrombus, (2) this stimulates angiogenic factor production, and (3) upregulating HIF1α enhances thrombus resolution and vein recanalization. METHODS AND RESULTS: Oxygen tension in the thrombus was negatively correlated with HIF1α levels (Spearman correlation [RS] = -0.77, P<0.0001), whereas HIF1α levels positively correlated with vascular endothelial growth factor (VEGF) expression (Pearson correlation [R] = 0.85, P<0.0005), during resolution in a murine model. HIF1α (P<0.005), VEGF (P<0.005), and VEGF receptor 1 (VEGFR1) (P<0.05) expression was 2-fold greater in the thrombus of mice treated with the prolyl hydroxylase domain inhibitor L-mimosine compared with controls. The levels of 13 other HIF1-mediated angiogenic factors were also increased. Thrombus weight (P<0.001) and volume (P<0.05) were reduced by a third in l-mimosine-treated mice compared with controls, whereas vein recanalization (P<0.005) and thrombus neovascularization (P<0.001) were 2-fold greater, and this was associated with increased inflammatory cell content. CONCLUSIONS: Hypoxia and HIF1α are induced in the naturally resolving thrombus and correlate with increased angiogenic factor expression. Upregulation of HIF1α enhances thrombus resolution and vein recanalization. HIF1α may represent a novel target for treatments that promote resolution and recanalization and reduce the incidence of post-thrombotic syndrome.

The monocyte/macrophage as a therapeutic target in atherosclerosis.

It is now clear that the monocyte/macrophage has a crucial role in the development of atherosclerosis. This cell appears to be involved in all stages of atherosclerotic plaque development and is increasingly seen as a candidate for therapeutic intervention and as a potential biomarker of disease progression and response to therapy. The main mechanisms related to the activity of the monocyte/macrophage that have been targeted for therapy are those that facilitate recruitment, cholesterol metabolism, inflammatory activity and oxidative stress. There is also increasing evidence that there is heterogeneity within the monocyte/macrophage population, which may have important implications for plaque development and regression. A better insight into how specific phenotypes may influence plaque progression should facilitate the development of novel methods of imaging and more refined treatments.

Monocyte urokinase-type plasminogen activator up-regulation reduces thrombus size in a model of venous thrombosis.

BACKGROUND: Our previous studies showed that the direct injection of an adenovirus construct expressing urokinase-type plasminogen activator (uPA) into experimental venous thrombi significantly reduces thrombus weight. The systemic use of adenovirus vectors is limited by inherent hepatic tropism and inflammatory response. As macrophages are recruited into venous thrombi, it is reasonable to speculate that these cells could be used to target the adenovirus uPA (ad-uPA) gene construct to the thrombus. The aims of this study were to determine whether macrophages transduced with ad-uPA have increased fibrinolytic activity and whether systemic injection of transduced cells could be used to target uPA expression to the thrombus and reduce its size. METHODS: The effect of up-regulating uPA was examined in an immortalized macrophage cell line (MM6) and macrophages differentiated from human blood monocyte-derived macrophages (HBMMs). Cells were infected with ad-uPA or blank control virus (ad-blank). Fibrinolytic mediator expression, cell viability, and cytokine expression were measured by activity assays and enzyme-linked immunosorbent assays. Monocyte migration was measured using a modified Boyden chamber assay. A model of venous thrombosis was developed and characterized in mice with severe combined immunodeficiency (SCID). This model was used to study whether systemically administered macrophages over-expressing uPA reduced thrombus size. Uptake of HBMMs into the thrombus induced in these mice was confirmed by a combination of PKH2-labeled cell tracking and colocalization with human leukocyte antigen (HLA) by immunohistology. RESULTS: Compared with ad-blank, treated HBMMs transduction with ad-uPA increased uPA production by >1000-fold (P = .003), uPA activity by 150-fold (P = .0001), and soluble uPA receptor (uPAR) by almost twofold (P = .043). Expression of plasminogen activator inhibitor (PAI-1) and PAI-2 was decreased by about twofold (P = .011) and threefold (P = .005), respectively. Up-regulation of uPA had no effect on cell viability or inflammatory cytokine production compared with ad-blank or untreated cells. Ad-uPA transduction increased the migration rate of HBMMs (about 20%, P = .03) and MM6 cells (>twofold, P = .005) compared with ad-blank treated controls. Human macrophage recruitment into the mouse thrombus was confirmed by the colocalization of HLA with the PKH2-marked cells. Systemic injection of uPA-up-regulated HBMMs reduced thrombus weight by approximately 20% compared with ad-blank (P = .038) or sham-treated controls (P = .0028). CONCLUSION: Transduction of HBBM with ad-uPA increases their fibrinolytic activity. Systemic administration of uPA up-regulated HBBMs reduced thrombus size in an experimental model of venous thrombosis. Alternative methods of delivering fibrinolytic agents are worth exploring.

Bone marrow lesions detected by specific combination of MRI sequences are associated with severity of osteochondral degeneration.

BACKGROUND: Bone marrow lesions (BMLs) are useful diagnostic and prognostic markers in knee osteoarthritis (OA), but what they represent at the tissue level remains unclear. The aim of this study was to provide comprehensive tissue characterization of BMLs detected using two specific MRI sequences. METHODS: Tibial plateaus were obtained from 60 patients (29 females, 31 males), undergoing knee arthroplasty for OA. To identify BMLs, MRI was performed ex vivo using T1 and PDFS-weighted sequences. Multi-modal tissue level analyses of the osteochondral unit (OCU) were performed, including cartilage volume measurement, OARSI grading, micro-CT analysis of bone microstructure, routine histopathological assessment and quantitation of bone turnover indices. RESULTS: BMLs were detected in 74 % of tibial plateaus, the remainder comprising a No BML group. Of all BMLs, 59 % were designated BML 1 (detected only by PDFS) and 41 % were designated BML 2 (detected by both PDFS + T1). The presence of a BML was related to degeneration of the OCU, particularly within BML 2. When compared to No BML, BML 2 showed reduced cartilage volume (p = 0.008), higher OARSI scores (p = 0.004), thicker subchondral plate (p = 0.002), increased trabecular bone volume and plate-like structure (p = 0.0004), increased osteoid volume (p = 0.002) and thickness (p = 0.003), more bone marrow oedema (p = 0.03), fibrosis (p = 0.002), necrosis (p = 0.01) and fibrovascular cysts (p = 0.04). For most measures, BML 1 was intermediate between No BML and BML 2. CONCLUSIONS: BMLs detected by specific MRI sequences identify different degrees of degeneration in the OCU. This suggests that MRI characteristics of BMLs may enable identification of different BML phenotypes and help target novel approaches to treatment and prevention of OA.