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.

Suppression of angiogenic response in local vein wall is associated with reduced thrombus resolution.

INTRODUCTION: The formation of new vascular channels within and around venous thrombus contributes to its resolution. Neovascularisation arising from the surrounding vein may facilitate this process. Treatment of cancer patients with anti-angiogenic agents can lead to increased incidence of venous thromboembolic events, but the effect of these agents on the processes that govern thrombus resolution are unclear. The aim of this study was to determine the effect of anti-angiogenic treatment with 2-methoxyestradiol (2ME) on (i) angiogenic response in the thrombosed vein and (ii) venous thrombus resolution. MATERIALS AND METHODS: Venous thrombus was induced in the inferior vena cava (IVC) of 36 adult male BALB/C mice. Thrombosed mice received either the anti-angiogenic agent, 2ME (150 mg/kg/day, i/p), or vehicle control (n=18/group). In the thrombosed IVC of both groups: hypoxia-inducible factor (HIF) 1α, and its angiogenic targets, vascular endothelial growth factor (VEGF) and placental growth factor (PLGF), were quantified using enzyme-linked immunosorbent assays at days 1 and 10 post-thrombus induction (n=6/group); and inflammatory cell content, cell proliferation, and vein recanalisation were quantified using immunostaining and image analysis at day 10 (n=6/group). RESULTS: In the IVC of mice treated with 2ME compared with control: HIF1α (P<0.005 and P<0.02), VEGF (P<0.005 and P<0.02), and PLGF levels (P<0.01 and P<0.001) were reduced at days 1 and 10 post-thrombus induction respectively, and macrophage content (P<0.005), neutrophil content (P<0.01), vein recanalistion (P<0.05), and thrombus resolution (P<0.001) were also reduced at day 10. CONCLUSIONS: Anti-angiogenic treatment with 2ME supressed the HIF1-mediated angiogenic drive in local vein wall and attenuated venous thrombus resolution. The potential pro-thrombotic effect of anti-angiogenic agents should be carefully considered when managing venous thromboembolic events in cancer patients.

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.

TIE2-expressing monocytes/macrophages regulate revascularization of the ischemic limb.

A third of patients with critical limb ischemia (CLI) will eventually require limb amputation. Therapeutic neovascularization using unselected mononuclear cells to salvage ischemic limbs has produced modest results. The TIE2-expressing monocytes/macrophages (TEMs) are a myeloid cell subset known to be highly angiogenic in tumours. This study aimed to examine the kinetics of TEMs in patients with CLI and whether these cells promote neovascularization of the ischemic limb. Here we show that there are 10-fold more circulating TEMs in CLI patients, and removal of ischemia reduces their numbers to normal levels. TEM numbers in ischemic muscle are two-fold greater than normoxic muscle from the same patient. TEMs from patients with CLI display greater proangiogenic activity than TIE2-negative monocytes in vitro. Using a mouse model of hindlimb ischemia, lentiviral-based Tie2 knockdown in TEMs impaired recovery from ischemia, whereas delivery of mouse macrophages overexpressing TIE2, or human TEMs isolated from CLI patients, rescued limb ischemia. These data suggest that enhancing TEM recruitment to the ischemic muscle may have the potential to improve limb neovascularization in CLI patients.

Encapsulation of angiogenic monocytes using bio-spraying technology.

Therapeutic neovascularisation using angiogenic cells has been hampered by the loss of cells from the target tissue. Encapsulation of these cells within a semi-permeable membrane could improve their retention within the ischaemic tissue without affecting the excretion of the angiogenic growth factors produced. Bio-spraying is a novel cell-handling technique that does not adversely affect cell viability. We used this technique to encapsulate human peripheral blood monocytes and found that cell viability, cell phenotype and functional downstream angiogenic signalling were preserved. Encapsulation of monocytes with macrophage-colony stimulating factor resulted in increased vascular-endothelial growth factor production and enhanced angiogenic function. Bio-spraying/encapsulation has the potential to enhance the efficacy of current angiogenic cell therapy strategies and merits further investigation.

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.

Application of in vivo micro-computed tomography in the temporal characterisation of subchondral bone architecture in a rat model of low-dose monosodium iodoacetate-induced osteoarthritis.

INTRODUCTION: Osteoarthritis (OA) is a complex, multifactorial joint disease affecting both the cartilage and the subchondral bone. Animal models of OA aid in the understanding of the pathogenesis of OA and testing suitable drugs for OA treatment. In this study we characterized the temporal changes in the tibial subchondral bone architecture in a rat model of low-dose monosodium iodoacetate (MIA)-induced OA using in vivo micro-computed tomography (CT). METHODS: Male Wistar rats received a single intra-articular injection of low-dose MIA (0.2 mg) in the right knee joint and sterile saline in the left knee joint. The animals were scanned in vivo by micro-CT at two, six, and ten weeks post-injection, analogous to early, intermediate, and advanced stages of OA, to assess architectural changes in the tibial subchondral bone. The articular cartilage changes in the tibiae were assessed macroscopically and histologically at ten weeks post-injection. RESULTS: Interestingly, tibiae of the MIA-injected knees showed significant bone loss at two weeks, followed by increased trabecular thickness and separation at six and ten weeks. The trabecular number was decreased at all time points compared to control tibiae. The tibial subchondral plate thickness of the MIA-injected knee was increased at two and six weeks and the plate porosity was increased at all time points compared to control. At ten weeks, histology revealed loss of proteoglycans, chondrocyte necrosis, chondrocyte clusters, cartilage fibrillation, and delamination in the MIA-injected tibiae, whereas the control tibiae showed no changes. Micro-CT images and histology showed the presence of subchondral bone sclerosis, cysts, and osteophytes. CONCLUSIONS: These findings demonstrate that the low-dose MIA rat model closely mimics the pathological features of progressive human OA. The low-dose MIA rat model is therefore suitable to study the effect of therapeutic drugs on cartilage and bone in a non-trauma model of OA. In vivo micro-CT is a non-destructive imaging technique that can track structural changes in the tibial subchondral bone in this animal model, and could also be used to track changes in bone in preclinical drug intervention studies for OA treatments.

Upregulation of hypoxia-inducible factor 1 alpha in local vein wall is associated with enhanced venous thrombus resolution.

INTRODUCTION: Venous thrombus resolution may be regulated by an angiogenic process that involves the surrounding vein wall. The aims of this study were to determine whether: (i) thrombosis stimulates activation of the angiogenic transcription factor, hypoxia-inducible factor (HIF) 1α, and downstream expression of growth factors in vein wall; and (ii) upregulation of HIF1α in vein wall leads to increased growth factor expression and enhanced thrombus resolution. MATERIALS AND METHODS: HIF1α, vascular endothelial growth factor (VEGF), and placental growth factor (PLGF) were quantified in mouse inferior vena cava (IVC) at days 1, 3, 7, and 14 after thrombus formation (n = 10-13 per group). An additional group of thrombosed mice were treated with the prolyl-hydroxylase domain (PHD) inhibitor, L-mimosine (L-mim) or vehicle control. HIF1α, VEGF, and PLGF in IVC were measured at days 1 and 7; and vein recanalisation and thrombus resolution were measured at days 7 and 10 (n = 6-7 per group). RESULTS: HIF1α was expressed in thrombosed IVC and its levels remained relatively constant throughout natural resolution. The levels of VEGF in thrombosed IVC were elevated at days 1 (P < 0.0001) and 3 (P < 0.05); and PLGF at days 1 (P < 0.0001), 3 (P < 0.0001), and 7 (P < 0.0001). Treatment with L-mim led to: increased HIF1α (P<0.05), VEGF (P < 0.005), and PLGF (P < 0.001) levels in the IVC; decreased thrombus size (P < 0.01); and increased vein recanalisation (P < 0.001). CONCLUSIONS: HIF1α levels in vein wall are not affected by thrombosis and it appears that the angiogenic drive in the vein surrounding resolving thrombus is regulated independently of HIF1α. Stimulating HIF1α levels in the vein wall leads to an increased angiogenic drive and promotes vein recanalisation and thrombus resolution.

Comparative lipidomics profiling of human atherosclerotic plaques.

BACKGROUND: We sought to perform a systematic lipid analysis of atherosclerotic plaques using emerging mass spectrometry techniques. METHODS AND RESULTS: A chip-based robotic nanoelectrospray platform interfaced to a triple quadrupole mass spectrometer was adapted to analyze lipids in tissue sections and extracts from human endarterectomy specimens by shotgun lipidomics. Eighteen scans for different lipid classes plus additional scans for fatty acids resulted in the detection of 150 lipid species from 9 different classes of which 24 were detected in endarterectomies only. Further analyses focused on plaques from symptomatic and asymptomatic patients and stable versus unstable regions within the same lesion. Polyunsaturated cholesteryl esters with long-chain fatty acids and certain sphingomyelin species showed the greatest relative enrichment in plaques compared to plasma and formed part of a lipid signature for vulnerable and stable plaque areas in a systems-wide network analysis. In principal component analyses, the combination of lipid species across different classes provided a better separation of stable and unstable areas than individual lipid classes. CONCLUSIONS: This comprehensive analysis of plaque lipids demonstrates the potential of lipidomics for unraveling the lipid heterogeneity within atherosclerotic lesions.

Molecular characterization of dermal lymphatic endothelial cells from primary lymphedema skin.

BACKGROUND: Lymphatic endothelial cells from primary lymphedema skin have never been cultured nor characterized. A subgroup of patients with primary lymphedema undergo surgery to bring about an improvement in their quality of life. The aim of this study was to culture and characterize LECs from the skin of these patients. METHODS AND RESULTS: Lymphatic endothelial cells were isolated and cultured from the skin of patients with primary lymphedema and from normal skin. The isolated cells were compared in their ability to form microvascular networks in a three-dimensional culture medium, and in their response to treatment with vascular endothelial growth factors A, C, and D. Whole tissue transcriptional profiling was carried out on two pools of isolated lymphatic endothelial cells--one from primary lymphedema skin and the other from normal skin. Lymphatic endothelial cells from primary lymphedema skin form tubule-like structures when cultured in three-dimensional media. They respond in a similar fashion to stimulation with the vascular endothelial growth factors A, C, and D. Comparative analysis between lymphedema tissue and normal tissue (fold change >2) showed differential expression of 2793 genes (5% of all transcripts), 2184 upregulated, and 609 downregulated. Genes involved in cellular apoptosis (vascular endothelial growth inhibitor, zinc finger protein), extracellular matrix turnover (matrix metalloproteinase inhibitor-16), and type IV collagen deposition were upregulated. Various pro-inflammatory genes (interleukin-6, interleukin-8, interleukin-32, E-selectin) were downregulated. CONCLUSION: Cellular adhesion, apoptosis, and increased extracellular matrix turnover play a more prominent role in primary lymphedema than previously thought. In addition, the acute inflammatory response is attenuated as evidenced by the downregulation of various pro-inflammatory genes.This sheds further light on the interplay of the various pathological processes taking place in primary lymphedema.

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.

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.

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.