Ashar Wadoodi

Hypoxia and Upregulation of Hypoxia-Inducible Factor 1α 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 1&agr; (HIF1&agr;) are induced in resolving thrombus, (2) this stimulates angiogenic factor production, and (3) upregulating HIF1&agr; enhances thrombus resolution and vein recanalization. Methods and Results—Oxygen tension in the thrombus was negatively correlated with HIF1&agr; levels (Spearman correlation [RS]=−0.77, P<0.0001), whereas HIF1&agr; levels positively correlated with vascular endothelial growth factor (VEGF) expression (Pearson correlation [R]=0.85, P<0.0005), during resolution in a murine model. HIF1&agr; (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. Conclusion—Hypoxia and HIF1&agr; are induced in the naturally resolving thrombus and correlate with increased angiogenic factor expression. Upregulation of HIF1&agr; enhances thrombus resolution and vein recanalization. HIF1&agr; may represent a novel target for treatments that promote resolution and recanalization and reduce the incidence of post-thrombotic syndrome.

Adenovirus-Mediated VEGF Gene Therapy Enhances Venous Thrombus Recanalization and Resolution

Objective—Rapid thrombus recanalization reduces the incidence of post–thrombotic complications. This study aimed to discover whether adenovirus-mediated transfection of the vascular endothelial growth factor gene (ad.VEGF) enhanced thrombus recanalization and resolution. Methods and Results—In rats, thrombi were directly injected with either ad.VEGF (n=40) or ad.GFP (n=37). Thrombi in SCID mice (n=12) were injected with human macrophages transfected with ad.VEGF or ad.GFP. Thrombi were analyzed at 1 to 14 days. GFP was found mainly in the vein wall and adventitia by 3 days, but was predominantly found in cells within the body of thrombus by day 7. VEGF levels peaked at 4 days (376±299 pg/mg protein). Ad.VEGF treatment reduced thrombus size by >50% (47.7±5.1 mm2 to 22.0±4.0 mm2, P=0.0003) and increased recanalization by >3-fold (3.9±0.69% to 13.6±4.1%, P=0.024) compared with controls. Ad.VEGF treatment increased macrophage recruitment into the thrombus by more than 50% (P=0.002). Ad.VEGF-transfected macrophages reduced thrombus size by 30% compared with controls (12.3±0.89 mm2 to 8.7±1.4 mm2, P=0.04) and enhanced vein lumen recanalization (3.39±0.34% to 5.07±0.57%, P=0.02). Conclusion—Treatment with ad.VEGF enhanced thrombus recanalization and resolution, probably as a consequence of an increase in macrophage recruitment.

The role of endothelial cells and their progenitors in intimal hyperplasia

Intimal hyperplasia leading to restenosis is the major process that limits the success of cardiovascular intervention. The emergence of vascular progenitor cells and, in particular, endothelial progenitor cells has led to great interest in their potential therapeutic value in preventing intimal hyperplasia. We review the mechanism of intimal hyperplasia and highlight the important attenuating role played by a functional endothelium. The role of endothelial progenitor cells in maintaining endothelial function is reviewed and we describe how reduced progenitor cell number and function and reduced endothelial function lead to an increased risk of intimal hyperplasia. We review other potential sources of endothelial cells, including monocytes, mesenchymal stem cells and tissue resident stem cells. Endothelial progenitor cells have been used in clinical trials to reduce the risk of restenosis with varied success. Progenitor cells have huge therapeutic potential to prevent intimal hyperplasia but a more detailed understanding of vascular progenitor cell biology is necessary before further clinical trials are commenced.

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.

Local accumulation of hypoxia-inducible factor 2 alpha during venous thrombus resolution.

Venous thrombus resolution occurs by a process of organisation, which includes the infiltration of neutrophils and macrophages, and the formation of new vascular channels within and around the thrombus [1]. Rapid thrombus resolution is associated with reductions in the incidence of post-thrombotic syndrome [2,3]. Characterisation of the cellular andmolecularmechanisms that control venous thrombus resolution could therefore lead to the development of novel therapies for patients with deep vein thrombosis. The tissue remodelling response to hypoxia is controlled primarily by activation of hypoxia-inducible factors (HIFs) 1 and 2 [4]. Accumulation of HIF1α and 2α (the hypoxia-dependent subunits of HIF1 and HIF2 respectively) leads to HIF activation, and subsequent upregulation of a variety of factors that mediate vascular remodelling [4]. We previously showed that: (i) naturally resolving venous thrombus is hypoxic compared with venous blood; (ii) HIF1α is expressed in distinct spatial and temporal patterns throughout resolution; and (iii) this process is accelerated when HIF1α levels are enhanced in the thrombus and surrounding vein [5–7]. Although it is known that the 2 HIFα isoforms regulate an over-lapping but distinct catalogue of target genes, and that these isoforms can act in cooperation or opposition (depending on the cell type, tissue type, and condition studied) [8], the role of HIF2α in venous thrombus resolution is unknown. Our primary aim was to determine whether HIF2α is expressed in the newly formed and naturally resolving thrombus and surrounding vein. Given that thrombus resolution is increased by treatment with a HIF agonist, L-mimosine [6,7], we also wished to determine whether these increases could be partly mediated by HIF2.

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.

Protein fragments from the VEGF binding domain of fibronectin are expressed in distinct spatial and temporal patterns during venous thrombus resolution

Deep vein thrombosis (DVT) affects 1-2% of the general population [1,2] and can lead to chronic and debilitating complications including leg pain, swelling, lipodermatosclerosis, and ulceration [3,4]. Clinical outcome is improved in patients whose thrombi resolve rapidly by a process of vein recanalisation and organisation that is similar to wound healing [5,6]. This process is enhanced by infiltration of inflammatory and endothelial cells, which mediate resolution by releasing a catalogue of angiogenic chemokines, proteases, and growth factors [5]. These factors include the potent angiogenic chemokine vascular endothelial growth factor (VEGF), which is highly expressed in resolving thrombus and accelerates this process [7,8]. Although spatial and temporal patterns of VEGF expression have been identified during venous thrombus resolution [8], the mechanisms that control natural resolution remain unclear. Fibronectin is found in blood plasma, extracellular matrix, and platelets, and this angiogenic glyocoprotein has roles in cell adhesion, migration, differentiation, and proliferation [9]. Fibronectin specifically binds with VEGF or VEGF receptor 1 (VEGFR1) and in doing so enhances processes that contribute to venous thrombus resolution including macrophage and endothelial cell migration [10,11]. VEGF binds with fibronectin at the N-terminal collagen binding domain and the C-terminal Hep II binding domain, the latter of which enhances endothelial cellmigration andmitogen-activated protein kinase activity [10,11]. The role of the N-terminal VEGF binding domain in fibronectin, however, is unclear. Proteolytic fragments of fibronectin, for example, can enhance monocyte migration in response to VEGF and placental growth factor (PLGF) via VEGFR1 [12], suggesting that the N-terminal VEGF/VEGFR1 binding region of fibronectin could mediate VEGFdependent tissue remodelling responses during thrombus resolution. It follows that fibronectin-VEGF binding could amplify VEGF activity and accelerate venous thrombus resolution. It was therefore our aim to identify spatial and temporal expression patterns of fibronectin and its proteolytic fragments during natural thrombus resolution in an established mouse model of venous thrombosis. We show that fibronectin and several of its proteolytic fragments are expressed in distinct temporal patterns in the thrombus and surrounding vein throughout natural resolution, suggesting that fibronectin could amplify the angiogenic and chemotactic effects of VEGF during thrombus resolution and in doing so accelerate this process.

Abstract B7: Hypoxia-inducible factor 1 mediates thrombosis-associated cancer progression

Background. Cancer patients are up to 50 times more likely to suffer from lethal venous thromboembolism compared with non-cancer patients, but mechanisms that regulate the positive correlation between these conditions are unclear. Our primary aim was to test whether hypoxia within cells contained in the thrombus and adjacent vein leads to upregulation of hypoxia-inducible factor 1 (HIF1), which in turn stimulates the production of factors that mediate tumor growth and metastasis. Methods. Systemic or endothelial and myeloid HIF1 levels were altered (via administration of HIF1 agonist L-mimosine or via cre-Tie2 driven HIF1 deletion respectively) in established mouse models of thrombosis and breast cancer (inferior vena cava stenosis or polyoma middle T mutation driven by mouse mammary tumour virus respectively). Protein arrays were used to quantify the expression of 25 factors that regulate coagulation, thrombosis, and cancer progression. Adhesion and migration assays were used to characterise tumor and endothelial cell behaviour on thrombotic surfaces; while endothelial and tumor cell oxygen consumption rate was measured before and after thrombin treatment. Image analysis was also used to quantify intra-tumor thrombus formation. Results. Thrombus formation or upregulation of HIF1 increased the circulating expression of 10 factors that mediate tumor growth and remodelling including vascular endothelial growth factor (VEGF, 2±1 vs 1±0.2pg/ml in controls, n=6/group, P<0.05), hepatocyte growth factor (HGF, 10±5 vs 7±2% in controls, n=8/group, P<0.05), and stromal cell-derived factor 1 (SDF1, 14±6 vs 2±1% in controls, n=8/group, P<0.05). Hypoxia (1% oxygen) tended to attenuate endothelial cell migration on fibrin (100±13 vs 64±10 cells in controls, n=6/group, P=0.05); while HIF1 deletion in Lewis lung cancer cells abolished their hypoxia-induced increase in adhesion onto fibrin (n=5/group, P<0.05). Thrombin administration reduced the oxygen consumption rate of Lewis lung cancer cells (59±13 vs 79±16 pMol/min in controls, n=5/group, P<0.005) but not endothelial cells (P=0.06). Deposition of fibrin in mammary tumours was reduced following endothelial and myeloid HIF1 deletion (5±1 vs 13±3% in controls, n=7/group, P<0.05) and this was associated with reduced tumour size (n=14, R=0.7, P<0.01). Conclusions. HIF1 regulates the progression of venous thrombosis and cancer, and could also be responsible for the positive association between these conditions. Cell and tissue-specific HIF1 is an attractive therapeutic target for treatments that aim to reduce thrombosis-associated cancer progression. Citation Format: Colin E. Evans, Cristina Branco-Price, Julia Humphries, Ashar Wadoodi, Prakash Saha, Jung-whan Kim, Mattias Belting, Alberto Smith, Randall S. Johnson. Hypoxia-inducible factor 1 mediates thrombosis-associated cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B7.

Bone marrow mononuclear cells drive thrombus resolution

Objective: ESCT and NASCET established the role of CEA in appropriate patients but reported a 5–7% 30-day stroke/death risk. Strategies reducing this would be important. The GALA Trial was conceived following analysis of non-randomised and randomised studies suggesting a 50% risk reduction for LA CEA. Method: A total of 3526 patients (symptomatic or asymptomatic disease) were randomised to GA or LA (95 centres, 24 countries). Primary outcome events were stroke, myocardial infarction or death (randomisation −30 days post-surgery). The data were analysed by intention-to-treat analysis. Results: Events occurred (99·9% follow-up) in 84/1752 (4·8%) GA and 80/1771 (4·5%) LA patients (not significant: three events prevented per 1000 LA patients [95% CI −11, +17]; risk ratio 0·94 [95% CI 0·70, 1·27]). There were no differences for individual outcome events: stroke 70 (4·0%) GA versus 66 (3·7%) LA (three prevented per 1000 LA patients [95% CI −10 to +16]); death 26 (1·5%) GA versus 19 (1·1%) LA (four prevented per 1000 [95% CI −3 to +12]); myocardial infarction LA 9 (0·5%) versus GA 4 (0·2%) (three more per 1000 LA patients [95% CI −2 to +8]). In patients with contralateral carotid occlusion (pre-defined sub-group), outcome events occurred in 15/150 (10%) GA versus 8/160 (5%) LA, p = 0·098. Further 1-year survival data indicate fewer subsequent events (stroke, death, MI) in LA patients (p = 0·094). Conclusion: These data show that CEA outcomes have improved by up to a third since earlier trials and that both LA and GA are safe. For patients with contralateral carotid occlusion, LA might offer a benefit and trends suggesting improved 1-year survival following LA surgery require further analysis.