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Deep vein thrombosis and common complications, including pulmonary embolism and post-thrombotic syndrome, represent a major source of morbidity and mortality worldwide. Experimental models of venous thrombosis have provided considerable insight into the cellular and molecular mechanisms that regulate thrombus formation and subsequent resolution. Here, we critically appraise the ex vivo and in vivo techniques used to assess venous thrombosis in these models. Particular attention is paid to imaging modalities, including magnetic resonance imaging, micro–computed tomography, and high-frequency ultrasound that facilitate longitudinal assessment of thrombus size and composition.

Introduction: The poor retention of cells delivered into ischemic tissue is a hurdle that needs to be addressed for cell therapy to be effective. We have previously shown that CD16+ monocytes expressing upregulated cell adhesion markers are mobilized in critical limb ischemia (CLI) and salvage limbs in pre-clinical studies of hindlimb ischemia (HLI). We hypothesized that these cells are well retained following delivery into ischemic muscle. Methods and Results: CD16+ and CD16- monocytes were isolated from CLI patients, fluorescently labeled with QuantumDots and injected into the adductor muscle of nude athymic mice following HLI (n=15/group). Flow cytometric analysis of cell suspensions from injected muscle showed higher 3 and 7 day retention of CD16+ compared with CD16- monocytes (P7 ) were radiolabeled with Indium 111 and injected into ischemic legs. Cells were tracked at 30mins, 2, 24 and 72 hours using whole-body γ-camera imaging: radiodecay-corrected retention percentages were 60.0, 37.4, 29.9 and 22.0% respectively with evidence of “washout” into the liver. Analysis of muscle biopsies showed >85% viability of the cells at 10 days and areas of increased arteriogenesis compared with remote, non-injected muscle. Conclusion: CD16+ Mo are preferentially retained within ischemic muscle in experimental HLI. This first in man study shows these cells remain viable in ischemic tissue. Isolation of sufficient numbers of these cells for delivery directly into poorly perfused muscle may provide a more effective cell therapy for CLI patients.

Introduction: Cell therapy using unselected mononuclear cell populations has had modest benefits in patients with critical limb ischemia (CLI). We hypothesized that tissue-remodeling monocytes, identified by their expression of CD16 (CD16+ Mo), may be a novel cellular therapy for CLI. Methods and Results: Flow cytometry showed that the proportion of circulating CD16+ Mo was greater in CLI patients (n=25) compared with matched controls (n=15, P6 cells injected into the adductor muscles of nude athymic mice following femoral artery excision. More ischemic hindlimbs were salvaged when treated with CD16+ compared with CD16- Mo (83% [10/12] vs 17% [2/12] limbs, P Conclusion: Circulating CD16+ Mo from CLI patients have increased expression of adhesion markers, are preferentially retained within ischemic muscle and promote robust arteriogenesis and limb salvage in experimental HLI. This monocyte subset may be an effective cellular therapy for CLI.

Rationale : Abdominal aortic aneurysms constitute a degenerative process in the aortic wall. Both the miR-29 and miR-15 families have been implicated in regulating the vascular extracellular matrix. Objective : Our aim was to assess the effect of the miR-15 family on aortic aneurysm development. Methods and Results : Among the miR-15 family members, miR-195 was differentially expressed in aortas of apolipoprotein E–deficient mice on angiotensin II infusion. Proteomics analysis of the secretome of murine aortic smooth muscle cells, after miR-195 manipulation, revealed that miR-195 targets a cadre of extracellular matrix proteins, including collagens, proteoglycans, elastin, and proteins associated with elastic microfibrils, albeit miR-29b showed a stronger effect, particularly in regulating collagens. Systemic and local administration of cholesterol-conjugated antagomiRs revealed better inhibition of miR-195 compared with miR-29b in the uninjured aorta. However, in apolipoprotein E–deficient mice receiving angiotensin II, silencing of miR-29b, but not miR-195, led to an attenuation of aortic dilation. Higher aortic elastin expression was accompanied by an increase of matrix metalloproteinases 2 and 9 in mice treated with antagomiR-195. In human plasma, an inverse correlation of miR-195 was observed with the presence of abdominal aortic aneurysms and aortic diameter. Conclusions : We provide the first evidence that miR-195 may contribute to the pathogenesis of aortic aneurysmal disease. Although inhibition of miR-29b proved more effective in preventing aneurysm formation in a preclinical model, miR-195 represents a potent regulator of the aortic extracellular matrix. Notably, plasma levels of miR-195 were reduced in patients with abdominal aortic aneurysms suggesting that microRNAs might serve as a noninvasive biomarker of abdominal aortic aneurysms.

Peripheral arterial disease (PAD) affects 27 million individuals in Europe and North America.1 This condition causes a severe restriction of blood flow that can lead to critical limb ischemia (CLI), a condition characterized by rest pain, ulceration, or gangrene, which is associated with limb loss in ≈25% of cases.2 The ischemic limb can be revascularized by endovascular techniques (angioplasty/stenting) or surgical bypass. Imaging modalities, including duplex ultrasound, MR angiography, computer tomographic angiography, or intra-arterial angiography, are used to assess the extent of disease, plan intervention, and confirm the patency of major blood vessels after intervention. These techniques do not provide information about perfusion at the tissue level. An early relatively crude method for assessing ischemic limbs, venous occlusion plethysmography3 relies on the principle that the volume of the limb is dependent on the arterial inflow when venous drainage is occluded. Under resting conditions, ≤70% of blood flow to the limb is directed to skeletal muscle, with the remainder to the skin circulation.4 Venous occlusion plethysmography provides a global indication of limb perfusion but gives no anatomic information and cannot delineate segmental perfusion deficits. It is influenced by numerous factors, such as ambient temperature and arteriovenous shunting that may lead to erroneous readings. Objective measurement of limb perfusion would particularly benefit patients with CLI but most attempts at addressing this have used patients with claudication. The latter can lead to CLI but is a less severe form of PAD that is often treated conservatively and is characterized by pain only when walking as opposed to pain at rest or gangrene. An accurate, noninvasive method of measuring limb perfusion would aid the diagnosis and treatment of PAD and could be combined with conventional angiography to provide both functional …

Background— The magnetic resonance longitudinal relaxation time (T 1 ) changes with thrombus age in humans. In this study, we investigate the possible mechanisms that give rise to the T 1 signal in venous thrombi and whether changes in T 1 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 T 1 relaxation time correlated with thrombus composition. The mean T 1 relaxation time of thrombus was shortest at 7days following thrombus induction and returned to that of blood as the thrombus resolved. T 1 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 Fe 3+ , which causes thrombus T 1 relaxation time shortening. Studies using chemokine receptor-2–deficient mice ( Ccr2 −/− ) revealed that the return of the T 1 signal to that of blood is regulated by removal of Fe 3+ by macrophages that accumulate in the thrombus during its resolution. Quantification of T 1 relaxation time was a good predictor of successful thrombolysis with a cutoff point of Conclusions— The source of the T 1 signal in the thrombus results from the oxidation of iron (released from the lysis of trapped erythrocytes in the thrombus) to its paramagnetic Fe 3+ form. Quantification of T 1 relaxation time appears to be a good predictor of the success of thrombolysis.

Background— Endothelial dysfunction promotes atherosclerosis and precedes acute cardiovascular events. We investigated whether in vivo magnetic resonance imaging with the use of an albumin-binding contrast agent, gadofosveset, could detect endothelial damage associated with atherosclerosis in apolipoprotein E–deficient (ApoE −/− ) mice. Furthermore, we tested whether magnetic resonance imaging could noninvasively assess endothelial function by measuring the endothelial-dependent vasodilation in response to acetylcholine. Methods and Results— ApoE −/− mice were imaged at 4, 8, and 12 weeks after commencement of a high-fat diet. Statin-treated ApoE −/− mice were scanned after 12 weeks of a high-fat diet. Wild-type mice were imaged before and 48 hours after injection of Russell's viper venom, an endothelial toxin. Delayed enhancement magnetic resonance imaging and T1 mapping of the brachiocephalic artery, 30 minutes after injection of gadofosveset, showed increased vessel wall enhancement and relaxation rate (R 1 ) with progression of atherosclerosis in ApoE −/− (R 1 [s −1 ]: R 4 weeks 2.42±0.35, R 8 weeks 3.45±0.54, R 12 weeks 3.83±0.52) and Russell's viper venom–injected wild-type mice (R 1 =4.57±0.86). Conversely, wild-type (R 1 =2.15±0.34) and statin-treated ApoE −/− (R 1 =3.0±0.65) mice showed less enhancement. Uptake of gadofosveset correlated with Evans blue staining, morphological changes of endothelial cells, and widening of the cell-cell junctions, suggesting that uptake occurs in regions of increased vascular permeability. Endothelial-dependent vasomotor responses showed vasoconstriction of the arteries of the ApoE −/− (−22.22±7.95%) and Russell's viper venom–injected (−10.37±17.60%) mice compared with wild-type mice (32.45±12.35%). Statin treatment improved endothelium morphology and function (−8.12±8.22%). Conclusions— We demonstrate the noninvasive assessment of endothelial permeability and function with the use of an albumin-binding magnetic resonance contrast agent. Blood albumin leakage could be a surrogate marker for the in vivo evaluation of interventions that aim to restore the endothelium.

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 P P P P l -mimosine compared with controls. The levels of 13 other HIF1-mediated angiogenic factors were also increased. Thrombus weight ( P P l -mimosine–treated mice compared with controls, whereas vein recanalization ( P P Conclusion— 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.

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.

Background— Mutations in the FOXC2 gene cause lymphedema distichiasis, an inherited primary lymphedema in which a significant number of patients have varicose veins. Because lymphedema distichiasis is believed to be caused by lymphatic valve failure (reflux), and FOXC2 is highly expressed on venous valves in mouse embryos, we tested the hypothesis that FOXC2 mutations may be linked to venous valve failure and reflux. Methods and Results— The venous system of the leg was investigated with Duplex ultrasound. Pathological reflux was recorded by color Duplex ultrasound in all 18 participants with a FOXC2 mutation, including 3 without lymphedema. Every participant with a mutation in FOXC2 showed reflux in the great saphenous vein (n=18), compared with only 1 of 12 referents (including 10 family members; P Conclusions— FOXC2 is the first gene in which mutations have been strongly associated with primary venous valve failure in both the superficial and deep veins in the lower limb. This gene appears to be important for the normal development and maintenance of venous and lymphatic valves.

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 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.

Introduction: We have previously demonstrated that non-contrast MRI using magnetisation transfer rate (MTR), apparent diffusion coefficient (ADC) and T1 mapping can characterise different aspects of organisation in a resolving venous thrombus. We now investigate whether the combination of these non-contrast agent MRI sequences can be used to identify thrombi suitable for lysis in an experimental model, and whether multi-sequence thrombus imaging (MSTI) can be translated to man. Methods: Magnetisation transfer, diffusion weighted images and T1 relaxation times were measured at days 2, 4, 7, 10, 14, 21 and 28 after venous thrombus induction in 8-10wk old male BALB/C mice (n=8/gp). Tissue plasminogen activator (10mg/kg) was administered through tail vein injection immediately after imaging at each time point and mice scanned 24hrs later to evaluate the effect of lysis. This was considered successful if more than 50% of the vein recanalised. Murine imaging sequences were combined and optimised to image the pelvic veins in man using healthy volunteers in order to produce a clinically useable imaging card. MSTI sequences were validated using phantoms before application to patients with iliofemoral deep vein thrombosis (DVT) undergoing lysis. Results: ROC curve analysis shows that the combination of MTR smaller than 2,900(%/cm3), ADC larger than 0.93(x10-3 mm2/s) and T1 shorter than 784ms has a sensitivity of 88% and specificity of 97% to identify experimental thrombi amenable to lysis. MSTI is feasible in man, with optimisation leading to successful characterisation of iliofemoral DVT in under 25mins (Figure 1). Conclusions: Non-contrast MR imaging, using a combination of MTR, ADC and T1 mapping, accurately identifies experimental venous thrombi susceptible to lysis. These MSTI sequences can also be readily translated to man where may find utility in characterising the age and structure of thrombus, and to stratify patients undergoing thrombolysis.

Introduction: Abdominal aortic aneurysm (AAA) is a degenerative disease of the aorta. Apart from the miR-29 family, miR-195 has been implicated in the regulation of the vascular extracellular matrix (ECM). Our aim was to assess the effect of the miR-15 family in aneurysm formation. Methods and Results: Expression of the miR-15 and miR-29 families was assessed in human aortic tissue. In human aneurysmal tissue, miR-195 expression was reduced to the same extent as miR-29b. In plasma, an inverse correlation of miR-195 was observed with the presence of AAA and aortic diameter. To evaluate the effect of miR-195 in ECM deposition, proteomics analysis was performed on the secretome of murine aortic smooth muscle cells (SMCs) following miRNA manipulation. MiR-195 targeted a cadre of ECM proteins, including collagens, proteoglycans, elastin and proteins associated with elastic microfibrils, albeit miR-29b showed a stronger effect. Systemic administration of cholesterol bound antagomiRs revealed better targeting efficacy of miR-195 compared to miR-29b in the uninjured aorta. A significant increase in aortic elastin expression was observed in wildtype animals injected with antagomiR-195. Yet, in apolipoprotein E-deficient mice receiving angiotensin II, inhibition of miR-29b but not miR-195 led to a significant inhibition of aortic aneurysm formation. Conclusions: We provide the first evidence that miR-195 may contribute to the pathogenesis of aneurysmal disease. Levels of miR-195 were reduced in human aneurysmal tissue and in plasma of patients with AAA. Although, miR-195 emerged as a potent regulator of aortic ECM, its inhibition was less effective than miR-29b in preventing aneurysm formation in preclinical models.

Rationale: Extracellular matrix (ECM) remodeling is a key function of vascular smooth muscle cells (SMCs). MicroRNAs (miRNAs), in particular the miR-29 family and miR-195, have been implicated in the control of ECM secretion. Objective: To perform a proteomics comparison of miRNA effects on ECM production by vascular SMCs. Methods and Results: Murine SMCs were transfected with miRNA mimics and antimiRs of miR-29b and miR-195, and their conditioned medium was analyzed by mass spectrometry. Both miRNAs targeted a cadre of ECM proteins, including proteoglycans, collagens, proteases, elastin and proteins associated with elastic microfibrils, albeit miR-29 showed a stronger effect. The proteomics findings were subsequently validated at the transcription level using quantitative polymerase chain reaction. Similar to miR-29, in vivo inhibition of miR-195 by intraperitoneal injection of cholesterol bound antagomiRs led to significant alterations of elastin expression in murine aortas. Since elastin degradation is a key event in aortic aneurysm formation, we investigated miR-195 expression in patients. In human aortic aneurysmal tissue, miR-195 expression was reduced compared to non-aneurysmal tissue. In plasma, a comparison between male patients with abdominal aortic aneurysms and controls matched for diabetes and hypertension returned a panel of five highly correlated miRNAs: miR-195, miR-125b, miR-148a, miR-20a and miR-340 showed significant inverse associations with the presence of abdominal aortic aneurysms and aortic diameter, with miR-195 dominating in terms of association strength. Conclusions: Using proteomic analysis, we compared the effect of miR-29 and miR-195 on ECM secretion by vascular SMCs and identified novel miRNA targets. Findings in patients support an important role for miR-195 in vascular remodeling as evidenced by reduced miR-195 expression in human aneurysmal tissue and an inverse correlation between plasma miR-195 levels and aortic diameter.

An investigation of restriction fragment length polymorphisms (RFLPs) and association with blood pressure was carried out for the candidate genes coding for renin (REN) and atrial natriuretic peptide (ANP). The relationship between blood pressure, REN and ANP gene RFLPs was tested in a population of black Afro-Caribbean and white European subjects sampled randomly from family practice registers. Upper and lower quintiles for diastolic blood pressure were selected for analysis. Digests of DNA were prepared from leucocytes and RFLPs were determined using Southern blotting analysis with REN and ANP gene probes. There were highly significant ethnic differences found for BglI, Bg/lI and TaqI polymorphisms with 5'REN probe and for BglI polymorphism with an ANP probe. There was also an association between blood pressure and the BglI/REN polymorphism in Afro-Caribbeans but not with any other polymorphisms studied in either ethnic group. The findings show significant ethnic RFLP differences at the gene loci for both renin and ANP and provide evidence for a possible link between variations within or close to the renin gene and elevated blood pressure in Afro-Caribbeans.