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Abstract Current treatments to prevent thrombosis, namely anticoagulants and platelets antagonists, remain complicated by the persistent risk of bleeding. Improved therapeutic strategies that diminish this risk would have a huge clinical impact. Antithrombotic agents that neutralize and inhibit polyphosphate (polyP) can be a powerful approach towards such a goal. Here, we report a design concept towards polyP inhibition, termed macromolecular polyanion inhibitors (MPI), with high binding affinity and specificity. Lead antithrombotic candidates are identified through a library screening of molecules which possess low charge density at physiological pH but which increase their charge upon binding to polyP, providing a smart way to enhance their activity and selectivity. The lead MPI candidates demonstrates antithrombotic activity in mouse models of thrombosis, does not give rise to bleeding, and is well tolerated in mice even at very high doses. The developed inhibitor is anticipated to open avenues in thrombosis prevention without bleeding risk, a challenge not addressed by current therapies.

Abstract Deep venous thrombosis and residual thrombus burden correlates with circulating IL-6 levels in humans. To investigate the cellular source and role of IL-6 in thrombus resolution, Wild type C57BL/6J (WT), and IL-6−/− mice underwent induction of VT via inferior vena cava (IVC) stenosis or stasis. Vein wall (VW) and thrombus were analyzed by western blot, immunohistochemistry, and flow cytometry. Adoptive transfer of WT bone marrow derived monocytes was performed into IL6-/- mice to assess for rescue. Cultured BMDMs from WT and IL-6−/− mice underwent quantitative real time PCR and immunoblotting for fibrinolytic factors and matrix metalloproteinase activity. No differences in baseline coagulation function or platelet function were found between WT and IL-6−/− mice. VW and thrombus IL-6 and IL-6 leukocyte-specific receptor CD126 were elevated in a time-dependent fashion in both VT models. Ly6Clo Mo/MØ were the predominant leukocyte source of IL-6. IL-6−/− mice demonstrated larger, non-resolving stasis thrombi with less neovascularization, despite a similar number of monocytes/macrophages (Mo/MØ). Adoptive transfer of WT BMDM into IL-6−/− mice undergoing stasis VT resulted in phenotype rescue. Human specimens of endophlebectomized tissue showed co-staining of Monocyte and IL-6 receptor. Thrombosis matrix analysis revealed significantly increased thrombus fibronectin and collagen in IL-6−/− mice. MMP9 activity in vitro depended on endogenous IL-6 expression in Mo/MØ, and IL-6−/− mice exhibited stunted matrix metalloproteinase activity. Lack of IL-6 signaling impairs thrombus resolution potentially via dysregulation of MMP-9 leading to impaired thrombus recanalization and resolution. Restoring or augmenting monocyte-mediated IL-6 signaling in IL-6 deficient or normal subjects, respectively, may represent a non-anticoagulant target to improve thrombus resolution.

Objective: Deep vein thrombosis (DVT) and its sequela, post-thrombotic syndrome (PTS), remain a clinically significant problem. Interleukin-6 (IL-6) is a proinflammatory cytokine that is elevated in patients who develop PTS. We hypothesized that genetic deletion of IL-6 and the use of anti–IL-6 pharmacologic agents would be associated with decreased late vein wall injury. Methods: Wild-type C57BL/6J (WT) and IL-6−/− mice underwent induction of stasis venous thrombosis by ligation of the infrarenal IVC. Vein wall inferior vena cava and thrombus were harvested at 21 days after ligation and analyzed by Western blot and immunohistochemistry of the vein wall using monocyte markers CCR2 and arginase 1, the endothelial marker CD31, and fibroblast markers DDR2 and FSP-1. Two anti–IL-6 pharmacologic agents (gp130 [glycoprotein 130] and tocilizumab) were tested and compared with low-molecular-weight heparin (LMWH) as the reference standard in WT mice. Plasma was collected at 4 and 48 hours to confirm the pharmacologic agents’ effects. Results: Less fibrosis but no increase in luminal endothelialization was found in IL-6−/− mice compared with WT mice at 21 days. The IL-6−/− mice had fewer DDR2- and arginase 1-positive cells in the vein wall compared with the WT mice. However, no difference was found in the CCR2+ cells. Despite documented in vivo activity, exogenous gp130 and tocilizumab were not associated with decreased vein wall fibrosis or increased endothelial luminal coverage at 21 days. LMWH therapy, both before and after treatment, was not associated with decreased vein wall fibrosis at 21 days. Conclusions: IL-6 genetic deletion was associated with less fibrotic vein wall injury at a late time point, consistent with the PTS timeframe. However, neither the standard of care LMWH nor two available anti–IL-6 agents showed antifibrotic biologic effects in this model. : Clinical Relevance: Post-thrombotic syndrome (PTS) has no direct therapy. Also, although anticoagulation therapy can prevent recurrent deep vein thrombosis (DVT), it cannot prevent PTS. PTS is a fibrotic disease, and interleukin-6 is a biomarker for incident PTS. We examined, in a stasis mouse model of DVT, the effect of genetically deleting interleukin-6 (IL-6−/−) and tested anti–IL-6 agents. Although IL-6−/− was associated with less vein wall injury, the anti–IL-6 agents did not confer a similar effect and were not associated with vein wall healing.

Background Atherectomy has become the fastest growing catheter‐based peripheral vascular intervention performed in the United States, and overuse has been linked to increased reimbursement, but the patterns of use have not been well characterized. Methods and Results We used Blue Cross Blue Shield of Michigan Preferred Provider Organization and Medicare fee‐for‐service professional claims data from the Michigan Value Collaborative for patients undergoing office‐based laboratory atherectomy in 2019 to calculate provider‐specific rates of atherectomy use, reimbursement, number of vessels treated, and number of atherectomies per patient. We also calculated the rate that each provider converted a new patient visit to an endovascular procedure within 90 days. Correlations between parameters were assessed with simple linear regression. Providers completing ≥20 office‐based laboratory atherectomies and ≥20 new patient evaluations during the study period were included. A total of 59 providers performing 4060 office‐based laboratory atherectomies were included. Median professional reimbursement per procedure was $4671.56 (interquartile range [IQR], $2403.09–$7723.19) from Blue Cross Blue Shield of Michigan and $14 854.49 (IQR, $9414.80–$18 816.33) from Medicare, whereas total professional reimbursement from both payers ranged from $2452 to $6 880 402 per year. Median 90‐day conversion rate was 5.0% (IQR, 2.5%–10.0%), whereas the median provider‐level average number of vessels treated per patient was 1.20 (IQR, 1.13–1.31) and the median provider‐level average number of treatments per patient was 1.38 (IQR, 1.26–1.63). Total annual reimbursement for each provider was directly correlated with new patient‐procedure conversion rate (R2=0.47; P

Background: Quantitative methods for evaluating microstructure of arterial specimens typically rely on histologic techniques that involve random sampling, which cannot account for the unique spatial distribution of features in three dimensions. Methods: To overcome this limitation, we demonstrate a nondestructive method for three-dimensional imaging of intact human blood vessels using microcomputed tomography (microCT). Human artery segments were dehydrated and stained in an iodine solution then imaged with a standard laboratory microCT scanner. Image visualization and segmentation was performed using commercially available and open source software. Results: Staining of cadaveric vessels with iodine enabled clear visualization of the arterial wall with microCT, preserved tissue morphology, and generated high-resolution images with a voxel size of 5.4 μm. Various components of the arterial wall were segmented using a combination of manual and automatic thresholding algorithms. Conclusions: Our approach allows for spatial mapping of human artery tissue samples that can guide targeted histologic analysis of smaller tissue segments, provide geometric data to inform finite element models, quantify degree of atherosclerosis, and help to evaluate the foreign body response to intravascular medical implants. (JVS–Vascular Science 2021;2:13-19.) Clinical Relevance: In this article, we describe a powerful technique for whole artery analysis of pathologic human tissue specimens that provides high-resolution spatial detail regarding composition of the blood vessel wall. The protocol described here is a valuable adjunct that can be used as a research tool to inform finite element modeling of arteries, quantify pathologic response (ie, neointimal hyperplasia and vascular calcification), and evaluate the tissue/device interface of implanted medical devices.

Abstract Venous thromboembolism (VTE) is a major cause of morbidity and mortality. The impact of the Surgeon General’s Call to Action in 2008 has been lower than expected given the public health impact of this disease. This scientific statement highlights future research priorities in VTE, developed by experts and a crowdsourcing survey across 16 scientific organizations. At the fundamental research level (T0), researchers need to identify pathobiologic causative mechanisms for the 50% of patients with unprovoked VTE and better understand mechanisms that differentiate hemostasis from thrombosis. At the human level (T1), new methods for diagnosing, treating, and preventing VTE will allow tailoring of diagnostic and therapeutic approaches to individuals. At the patient level (T2), research efforts are required to understand how foundational evidence impacts care of patients (eg, biomarkers). New treatments, such as catheter‐based therapies, require further testing to identify which patients are most likely to experience benefit. At the practice level (T3), translating evidence into practice remains challenging. Areas of overuse and underuse will require evidence‐based tools to improve care delivery. At the community and population level (T4), public awareness campaigns need thorough impact assessment. Large population‐based cohort studies can elucidate the biologic and environmental underpinings of VTE and its complications. To achieve these goals, funding agencies and training programs must support a new generation of scientists and clinicians who work in multidisciplinary teams to solve the pressing public health problem of VTE.

Venous thromboembolism (VTE) is a pathology encompassing deep vein thrombosis (DVT) and pulmonary embolism (PE) associated with high morbidity and mortality. Because patients often present after a thrombus has already formed, the mechanisms that drive DVT resolution are being investigated in search of treatment. Herein, we review the current literature, including the molecular mechanisms of fibrinolysis and collagenolysis, as well as the critical cellular roles of macrophages, neutrophils, and endothelial cells. We propose two general models for the operation of the immune system in the context of venous thrombosis. In early thrombus resolution, neutrophil influx stabilizes the tissue through NETosis. Meanwhile, macrophages and intact neutrophils recognize the extracellular DNA by the TLR9 receptor and induce fibrosis, a complimentary stabilization method. At later stages of resolution, pro-inflammatory macrophages police the thrombus for pathogens, a role supported by both T-cells and mast cells. Once they verify sterility, these macrophages transform into their pro-resolving phenotype. Endothelial cells both coat the stabilized thrombus, a necessary early step, and can undergo an endothelial-mesenchymal transition, which impedes DVT resolution. Several of these interactions hold promise for future therapy.

Macrophages are essential immune cells necessary for regulated inflammation during wound healing. Recent studies have identified that Notch plays a role in macrophage-mediated inflammation. Thus, we investigated the role of Notch signaling on wound macrophage phenotype and function during normal and diabetic wound healing. We found that Notch receptor and ligand expression are dynamic in wound macrophages during normal healing. Mice with a myeloid-specific Notch signaling defect (DNMAMLfloxedLyz2Cre+) demonstrated delayed early healing (days 1–3) and wound macrophages had decreased inflammatory gene expression. In our physiologic murine model of type 2 diabetes (T2D), Notch receptor expression was significantly increased in wound macrophages on day 6, following the initial inflammatory phase of wound healing, corresponding to increased inflammatory cytokine expression. This increase in Notch1 and Notch2 was also observed in human monocytes from patients with T2D. Further, in prediabetic mice with a genetic Notch signaling defect (DNMAMLfloxedLyz2Cre+ on a high-fat diet), improved wound healing was seen at late time points (days 6–7). These findings suggest that Notch is critical for the early inflammatory phase of wound healing and directs production of macrophage-dependent inflammatory mediators. These results identify that canonical Notch signaling is important in directing macrophage function in wound repair and define a translational target for the treatment of non-healing diabetic wounds.

OBJECTIVE:: To characterize the national epidemiology of adult osteomyelitis (OM) and, using a single institutions’ experience, test the hypothesis that early surgical therapy as compared with antibiotics alone results in an improved chance of wound healing and limb salvage.BACKGROUND:: Foot and digit OM is a very common problem for which management is variable and for which few guidelines exist.METHODS:: The Nationwide Inpatient Sample (NIS) and a single institution review from 1993 to 2000 form the basis of this study, using ICD-9CM codes for lower extremity foot and digit OM. Demographics, risk factors, and treatments were analyzed against the outcomes of a healed wound, limb salvage, and death.RESULTS:: The NIS included 51,875 patients (incidence = 9/10,000 patients per year) with a mean age of 60 years, and 59% were men. The median length of stay decreased from 9 to 6 days (P < 0.001), but the average admission charge of $19,000 did not significantly decrease over 7 years. Of these patients, 23% underwent a digit amputation and 8.5% suffered proximal limb loss. Single-institution analysis of 237 consecutive patients with OM confirmed a similar mean age (58 years), gender (67% men), and most presented with a foot or digit ulcer (56%). Wound healing was achieved in 56% and overall limb salvage was 80%. Decreased wound healing was associated with peripheral vascular occlusive disease (odds ratio, 0.4; 95% confidence interval, 0.2–0.8, P = 0.006) and preadmission antibiotic use (odds ratio, 0.2; 95% confidence interval, 0.05–1.1, P=0.07), while surgical debridement (odds ratio, 2.2; 95% confidence interval, 1.2–4.2, P = 0.02) was associated with increased healing. Limb salvage was improved with an arterial bypass (odds ratio, 3.9; 95% confidence interval, 1.1–14, P = 0.04), while preadmission solid organ transplant (odds ratio, 0.37; 95% confidence interval, 0.14–0.96, P = 0.04), peripheral vascular occlusive disease (odds ratio, 0.25; 95% confidence interval, 0.12–0.5, P = 0.001), and preadmission antibiotic use (odds ratio, 0.34; 95% confidence interval, 0.15–0.77, P = 0.009) were associated with greater limb loss.CONCLUSION:: Digit OM is an expensive and morbid disease. Aggressive surgical debridement/digit amputation and selected use of arterial bypass should improve wound healing and limb salvage, respectively. In contrast, antibiotic therapy alone is associated with decreased wound healing and limb salvage. [ABSTRACT FROM AUTHOR]

SUMMARY: OBJECTIVE To determine the contemporary clinical relevance of acute lower extremity ischemia and the factors associated with amputation and in-hospital mortality.SUMMARY BACKGROUND DATA Acute lower extremity ischemia is considered limb- and life-threatening and usually requires therapy within 24 hours. The equivalency of thrombolytic therapy and surgery for the treatment of subacute limb ischemia up to 14 days duration is accepted fact. However, little information exists with regards to the long-term clinical course and therapeutic outcomes in these patients.METHODS Two databases formed the basis for this study. The first was the National Inpatient Sample (NIS) from 1992 to 2000 of all patients (N = 23,268) with a primary discharge diagnosis of acute embolism and thrombosis of the lower extremities. The second was a retrospective University of Michigan experience from 1995 to 2002 of matched ICD-9-CM coded patients (N = 105). Demographic factors, atherosclerotic risk factors, the need for amputation, and in-hospital mortality were assessed by univariate and multivariate logistic regression analysis.RESULTS In the NIS, the mean patient age was 71 years, and 54% were female. The average length of stay (LOS) was 9.4 days, and inflation-adjusted cost per admission was $25,916. The amputation rate was 12.7%, and mortality was 9%. Decreased amputation rates accompanied: female sex (0.90, 0.81-0.99), age less than 63 years (0.47, 0.41-0.54), angioplasty (0.46, 0.38-0.55), and embolectomy (0.39, 0.35-0.44). Decreased mortality accompanied: angioplasty (0.79, 0.64-0.96), heparin administration (0.50, 0.29-0.86), and age less than 63 years(0.27, 0.23-0.33).The University of Michigan patients'' mean age was 62 years, and 57% were men. The LOS was 11 days, with a 14% amputation rate and a mortality of 12%. Prior vascular bypasses existed in 23% of patients, and heparin use was documented in 16%. Embolectomy was associated with decreased amputation rates (0.054, 0.01-0.27) and mortality (0.07, 0.01-0.57).CONCLUSIONS In patients with acute limb ischemia, the more widespread use of heparin anticoagulation and, in select patients, performance of embolectomy rather than pursuing thrombolysis may improve patient outcomes. [ABSTRACT FROM AUTHOR]

BackgroundCigarette smoking, hypertension, dyslipidemia, diabetes, and obesity are conventional risk factors (RFs) for coronary artery disease (CAD). Population trends for these RFs have varied in recent decades. Consequently, the risk factor profile for patients presenting with a new diagnosis of CAD in contemporary practice remains unknown.ObjectivesTo examine the prevalence of RFs and their temporal trends among patients without a history of myocardial infarction or revascularization who underwent their first percutaneous coronary intervention (PCI).MethodsWe examined the prevalence and temporal trends of RFs among patients without a history of prior myocardial infarction, PCI, or coronary artery bypass graft surgery who underwent PCI at 47 non-federal hospitals in Michigan between 1/1/2010 and 3/31/2018.ResultsOf 69,571 men and 38,930 women in the study cohort, 95.5% of patients had 1 or more RFs and nearly half (55.2% of women and 48.7% of men) had ≥3 RFs. The gap in the mean age at the time of presentation between men and women narrowed as the number of RFs increased with a gap of 6 years among those with 2 RFs to ConclusionsModifiable RFs are widely prevalent among patients undergoing their first PCI. Smoking and obesity are associated with an earlier age of presentation. Population-level interventions aimed at preventing obesity and smoking could significantly delay the onset of CAD and the need for PCI.

To determine the predictors of periprocedural blood transfusion and the association of transfusion on outcomes in high risk patients undergoing endoluminal percutaneous vascular interventions (PVI) for peripheral arterial disease.Between 2010-2014 at 47 hospitals participating in a statewide quality registry, 4.2% (n = 985) of 23,273 patients received a periprocedural blood transfusion. Transfusion rates varied from 0 to 15% amongst the hospitals in the registry. Using multiple logistic regression, factors associated with increased transfusion included female gender (OR = 1.9; 95% CI: 1.6-2.1), low creatinine clearance (1.3; 1.1-1.6), pre-procedural anemia (4.7; 3.9-5.7), family history of CAD (1.2; 1.1-1.5), CHF (1.4; 1.2-1.6), COPD (1.2; 1.1-1.4), CVD or TIA (1.2; 1.1-1.4), renal failure CRD (1.5; 1.2-1.9), pre-procedural heparin use (1.8; 1.4-2.3), warfarin use (1.2; 1.0-1.5), critical limb ischemia (1.7; 1.5-2.1), aorta-iliac procedure (1.9; 1.5-2.5), below knee procedure (1.3; 1.1-1.5), urgent procedure (1.7; 1.3-2.2), and emergent procedure (8.3; 5.6-12.4). Using inverse weighted propensity matching to adjust for confounders, transfusion was a significant risk factor for death (15.4; 7.5-31), MI (67; 29-150), TIA/stroke (24; 8-73) and ARF (19; 6.2-57). A focused QI program was associated with a 28% decrease in administration of blood transfusion (p = 0.001) over 4 years.In a large statewide PVI registry, post procedure transfusion was highly correlated with a specific set of clinical risk factors, and with in-hospital major morbidity and mortality. However, using a focused QI program, a significant reduction in transfusion is possible.

Despite anticoagulation therapy, up to one-half of patients with deep vein thrombosis (DVT) will develop the post-thrombotic syndrome (PTS). Improving the long-term outcome of DVT patients at risk for PTS will therefore require new approaches. Here we investigate the effects of statins--lipid-lowering agents with anti-thrombotic and anti-inflammatory properties--in decreasing thrombus burden and decreasing vein wall injury, mediators of PTS, in established murine stasis and non-stasis chemical-induced venous thrombosis (N = 282 mice). Treatment of mice with daily atorvastatin or rosuvastatin significantly reduced stasis venous thrombus burden by 25% without affecting lipid levels, blood coagulation parameters, or blood cell counts. Statin-driven reductions in VT burden (thrombus mass for stasis thrombi, intravital microscopy thrombus area for non-stasis thrombi) compared similarly to the therapeutic anticoagulant effects of low molecular weight heparin. Blood from statin-treated mice showed significant reductions in platelet aggregation and clot stability. Statins additionally reduced thrombus plasminogen activator inhibitor-1 (PAI-1), tissue factor, neutrophils, myeloperoxidase, neutrophil extracellular traps (NETs), and macrophages, and these effects were most notable in the earlier timepoints after DVT formation. In addition, statins reduced DVT-induced vein wall scarring by 50% durably up to day 21 in stasis VT, as shown by polarized light microscopy of picrosirius red-stained vein wall collagen. The overall results demonstrate that statins improve VT resolution via profibrinolytic, anticoagulant, antiplatelet, and anti-vein wall scarring effects. Statins may therefore offer a new pharmacotherapeutic approach to improve DVT resolution and to reduce the post-thrombotic syndrome, particularly in subjects who are ineligible for anticoagulation therapy.

Venous thromboembolism is a major cause of death during and immediately post-sepsis. Venous thrombosis (VT) is mediated by cell adhesion molecules and leukocytes, including neutrophil extracellular traps (NETs). Sepsis, or experimentally, endotoxaemia, shares similar characteristics and is modulated via toll like receptor 4 (TLR4). This study was undertaken to determine if endotoxaemia potentiates early stasis thrombogenesis, and secondarily to determine the role of VT TLR4, ICAM-1 and neutrophils (PMNs). Wild-type (WT), ICAM-1 -/- and TLR4 -/- mice underwent treatment with saline or LPS (10 mg/kg i. p.) alone, or followed by inferior vena cava (IVC) ligation to generate stasis VT. In vivo microscopy of leukocyte trafficking was performed in non-thrombosed mice, and tissue and plasma were harvested during early VT formation. Pre-thrombosis, circulating ICAM-1 was elevated and increased leukocyte adhesion and rolling occurred on the IVC of LPS-treated mice. Post-thrombosis, endotoxaemic mice formed larger, platelet-poor thrombi. Endotoxaemic TLR4 -/- mice did not have an augmented thrombotic response and exhibited significantly decreased circulating ICAM-1 compared to endotoxaemic WT controls. Endotoxaemic ICAM-1 -/- mice had significantly smaller thrombi compared to controls. Hypothesising that PMNs localised to the inflamed endothelium were promoting thrombosis, PMN depletion using anti-Ly6G antibody was performed. Paradoxically, VT formed without PMNs was amplified, potentially related to endotoxaemia induced elevation of PAI-1 and circulating FXIII, and decreased uPA. Endotoxaemia enhanced early VT occurs in a TLR-4 and ICAM-1 dependent fashion, and is potentiated by neutropenia. ICAM-1 and/or TLR-4 inhibition may be a unique strategy to prevent sepsis-associated VT.

Deep-vein thrombosis (DVT) resolves via a sterile inflammatory response. Defining the inflammatory response of DVT may allow for new therapies that do not involve anticoagulation. Previously, we have shown that Toll-like receptor 9 (Tlr9) gene deleted mice had impaired venous thrombosis (VT) resolution. Here, we further characterise the role of Tlr9 signalling and sterile inflammation in chronic VT and vein wall responses. First, we found a human precedent exists with Tlr9+ cells present in chronic post thrombotic intraluminal tissue. Second, in a stasis VT mouse model, endogenous danger signal mediators of uric acid, HMGB-1, and neutrophil extracellular traps marker of citrullinated histone-3 (and extracellular DNA) were greater in Tlr9-/- thrombi as compared with wild-type (WT), corresponding with larger VT at 8 and 21 days. Fewer M1 type (CCR2+) monocyte/macrophages (MØ) were present in Tlr9-/- thrombi than WT controls at 8 days, suggesting an impaired inflammatory cell influx. Using bone marrow-derived monocyte (BMMØ) cell culture, we found decreased fibrinolytic gene expression with exposure to several endogenous danger signals. Next, adoptive transfer of cultured Tlr9+/+ BMMØ to Tlr9-/- mice normalised VT resolution at 8 days. Lastly, although the VT size was larger at 21 days in Tlr9-/- mice and correlated with decreased endothelial antigen markers, no difference in fibrosis was found. These data suggest that Tlr9 signalling in MØ is critical for later VT resolution, is associated with necrosis clearance, but does not affect later vein wall fibrosis. These findings provide insight into the Tlr9 MØ mechanisms of sterile inflammation in this disease process., Competing Interests: There are no conflicts-of-interest to report for any authors of this work.