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SIRT3 is a longevity factor that acts as the primary deacetylase in mitochondria. Although ubiquitously expressed, previous global SIRT3 knockout studies have shown primarily a cardiac-specific phenotype. Here, we sought to determine how specifically knocking out SIRT3 in cardiomyocytes (SIRTcKO mice) temporally affects cardiac function and metabolism. Mice displayed an age-dependent increase in cardiac pathology, with 10-month-old mice exhibiting significant loss of systolic function, hypertrophy, and fibrosis. While mitochondrial function was maintained at 10 months, proteomics and metabolic phenotyping indicated SIRT3 hearts had increased reliance on glucose as an energy substrate. Additionally, there was a significant increase in branched-chain amino acids in SIRT3cKO hearts without concurrent increases in mTOR activity. Heavy water labeling experiments demonstrated that, by 3 months of age, there was an increase in protein synthesis that promoted hypertrophic growth with a potential loss of proteostasis in SIRT3cKO hearts. Cumulatively, these data show that the cardiomyocyte-specific loss of SIRT3 results in severe pathology with an accelerated aging phenotype.

Apart from autopsy, tissue correlates of coronavirus disease 2019 (COVID-19) clinical stage are lacking. In the current study, cutaneous punch biopsy specimens of 15 individuals with severe/critical COVID-19 and six with mild/moderate COVID-19 were examined. Evidence for arterial and venous microthrombi, deposition of C5b-9 and MASP2 (representative of alternative and lectin complement pathways, respectively), and differential expression of interferon type I-driven antiviral protein MxA (myxovirus resistance A) versus SIN3A, a promoter of interferon type I-based proinflammatory signaling, were assessed. Control subjects included nine patients with sepsis-related acute respiratory distress syndrome (ARDS) and/or acute kidney injury (AKI) pre-COVID-19. Microthrombi were detected in 13 (87%) of 15 patients with severe/critical COVID-19 versus zero of six patients with mild/moderate COVID-19 (P 0.001) and none of the nine patients with pre-COVID-19 ARDS/AKI (P 0.001). Cells lining the microvasculature staining for spike protein of severe acute respiratory syndrome coronavirus 2, the etiologic agent of COVID-19, also expressed tissue factor. C5b-9 deposition occurred in 13 (87%) of 15 patients with severe/critical COVID-19 versus zero of six patients with mild/moderate COVID-19 (P 0.001) and none of the nine patients with pre-COVID-19 ARDS/AKI (P 0.001). MASP2 deposition was also restricted to severe/critical COVID-19 cases. MxA expression occurred in all six mild/moderate versus two (15%) of 13 severe/critical cases (P 0.001) of COVID-19. In contrast, SIN3A was restricted to severe/critical COVID-19 cases co-localizing with severe acute respiratory syndrome coronavirus 2 spike protein. SIN3A was also elevated in plasma of patients with severe/critical COVID-19 versus control subjects (P ≤ 0.02). In conclusion, the study identified premortem tissue correlates of COVID-19 clinical stage using skin. If validated in a longitudinal cohort, this approach could identify individuals at risk for disease progression and enable targeted interventions.

Transforming growth factor β1 (TGF-β1) regulates a wide variety of events in adult bone marrow (BM), including quiescence of hematopoietic stem cells, via undefined mechanisms. Because megakaryocytes (MKs)/platelets are a rich source of TGF-β1, we assessed whether TGF-β1 might inhibit its own production by comparing mice with conditional inactivation of Tgfb1 in MKs (PF4Cre;Tgfb1flox/flox) and control mice. PF4Cre;Tgfb1flox/flox mice had ∼30% more MKs in BM and ∼15% more circulating platelets than control mice (P < .001). Thrombopoietin (TPO) levels in plasma and TPO expression in liver were approximately twofold higher in PF4Cre;Tgfb1flox/flox than in control mice (P < .01), whereas TPO expression in BM cells was similar between these mice. In BM cell culture, TPO treatment increased the number of MKs from wild-type mice by approximately threefold, which increased approximately twofold further in the presence of a TGF-β1–neutralizing antibody and increased the number of MKs from PF4Cre;Tgfb1flox/flox mice approximately fourfold. Our data reveal a new role for TGF-β1 produced by MKs/platelets in regulating its own production in BM via increased TPO production in the liver. Additional studies are required to determine the mechanism.

BackgroundCardiac valve disease (CVD) is observed in 2.5% of the general population and 10% of the elderly people. Effective pharmacological treatments are currently not available, and patients with severe CVD require surgery. PROX1 and FOXC2 are transcription factors that are required for the development of lymphatic and venous valves. We found that PROX1 and FOXC2 are expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 and FOXC2 regulate cardiac valve development and disease is not known.MethodsWe used histology, electron microscopy and echocardiography to investigate the structure and functioning of heart valves fromProx1ΔVECmice in whichProx1was conditionally deleted from VECs. Isolated valve endothelial cells and valve interstitial cells were used to identify the molecular mechanismsin vitro, which were testedin vivoby RNAScope, additional mouse models and pharmacological approaches. The significance of our findings was tested by evaluation of human samples of mitral valve prolapse (MVP) and aortic valve insufficiency.ResultsHistological analysis revealed that the aortic and mitral valves ofProx1ΔVECmice become progressively thick and myxomatous. Echocardiography revealed that the aortic valves ofProx1ΔVECmice are stenotic. FOXC2 was downregulated and platelet-derived growth factor-B (PDGF-B) was upregulated in the VECs ofProx1ΔVECmice. Conditional knockdown of FOXC2 and conditional overexpression of PDGF-B in VECs recapitulated the phenotype ofProx1ΔVECmice. PDGF-B was also increased in mice lacking FOXC2 and in human MVP and insufficient aortic valve samples. Pharmacological inhibition of PDGF-B signaling with imatinib partially ameliorated the valve defects ofProx1ΔVECmice.ConclusionPROX1 antagonizes PDGF-B signaling partially via FOXC2 to maintain the extracellular matrix composition and prevent myxomatous degeneration of cardiac valves.Novelty and SignificanceWhat Is Known?The transcription factors PROX1 and FOXC2 are critical regulators of lymphatic and venous valve development.PROX1 and FOXC2 are expressed in the downstream valvular endothelial cells of heart valves.What Is New?Deletion ofProx1from the valvular endothelial cells of mice results in enlarged and myxomatous aortic and mitral valves. Aortic valves of the mutant (Prox1ΔVEC) mice were stenotic.FOXC2 is partially responsible for the phenotype ofProx1ΔVECmice.PROX1 and FOXC2 inhibit the expression of the cytokine PDGF-B in heart valves.Hyperactivation of PDGF-B signaling results in aortic and mitral valve thickening.Inhibition of PDGF-B signaling ameliorates aortic valve stenosis inProx1ΔVECmice.PDGFBis overexpressed andPROX1is downregulated in human mitral valve prolapse (MVP) samples.Our findings suggest that PROX1 is an inhibitor of myxomatous valve disease that afflicts ~10% of the elderly population. We have also identified PDGF-B as a potential target for treating myxomatous valve disease.

Vaso-occlusive episode (VOE) is a common and critical complication of sickle cell disease (SCD). Its pathogenesis is incompletely understood. von Willebrand factor (VWF), a multimeric plasma hemostatic protein synthesized and secreted by endothelial cells and platelets, is increased during a VOE. However, whether and how VWF contributes to the pathogenesis of VOE is not fully understood. In this study, we found increased VWF levels during tumor necrosis factor (TNF)–induced VOE in a humanized mouse model of SCD. Deletion of endothelial VWF decreased hemolysis, vascular occlusion, and organ damage caused by TNF-induced VOE in SCD mice. Moreover, administering ADAMTS13, the VWF-cleaving plasma protease, reduced plasma VWF levels, decreased inflammation and vaso-occlusion, and alleviated organ damage during VOE. These data suggest that promoting VWF cleavage via ADAMTS13 may be an effective treatment for reducing hemolysis, inflammation, and vaso-occlusion during VOE.

BACKGROUND: A substantial proportion of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe/critical coronavirus disease 2019 (COVID-19) characterized by acute respiratory distress syndrome (ARDS) with thrombosis. OBJECTIVES: We tested the hypothesis that SARS-CoV-2--induced upregulation of tissue factor (TF) expression may be responsible for thrombus formation in COVID-19. METHODS: We compared autopsy lung tissues from 11 patients with COVID-19--associated ARDS with samples from 6 patients with ARDS from other causes (non-COVID-19 ARDS) and 11 normal control lungs. RESULTS: Dual RNA in situ hybridization for SARS-CoV-2 and TF identified sporadic clustered SARS-CoV-2 with prominent co-localization of SARS-CoV-2 and TF RNA. TF expression was 2-fold higher in COVID-19 than in non-COVID-19 ARDS lungs (P = .017) and correlated with the intensity of SARS-CoV-2 staining (R2 = .36, P = .04). By immunofluorescence, TF protein expression was 2.1-fold higher in COVID-19 versus non-COVID-19 ARDS lungs (P = .0048) and 11-fold (P

The role of the lectin pathway (LP) of complement has not been explored in the thrombotic microangiopathies (TMA). We examined levels of MASP2, the effector enzyme of the LP, in three major forms of TMA and assessed the effect of the anti‐MASP2 human monoclonal antibody narsoplimab on TMA plasma‐induced activation of caspase 8 in microvascular endothelial cells. MASP2 levels were highly elevated in all TMA patients versus controls. Caspase 8 activation was suppressed by clinically relevant levels of narsoplimab. In conclusion, we found that the LP of complement is activated in TMAs of diverse etiology and LP inhibition may be of therapeutic benefit in these disorders., Summary Involvement of the alternative complement pathway (AP) in microvascular endothelial cell (MVEC) injury characteristic of a thrombotic microangiopathy (TMA) is well documented. However, the role of the lectin pathway (LP) of complement has not been explored. We examined mannose‐binding lectin associated serine protease (MASP2), the effector enzyme of the LP, in thrombotic thrombocytopenic purpura, atypical hemolytic uremic syndrome and post‐allogeneic hematopoietic stem cell transplantation (alloHSCT) TMAs. Plasma MASP2 and terminal complement component sC5b‐9 levels were assessed by enzyme‐linked immunosorbent assay (ELISA). Human MVEC were exposed to patient plasmas, and the effect of the anti‐MASP2 human monoclonal antibody narsoplimab on plasma‐induced MVEC activation was assessed by caspase 8 activity. MASP2 levels were highly elevated in all TMA patients versus controls. The relatively lower MASP2 levels in alloHSCT patients with TMAs compared to levels in alloHSCT patients who did not develop a TMA, and a significant decrease in variance of MASP2 levels in the former, may reflect MASP2 consumption at sites of disease activity. Plasmas from 14 of the 22 TMA patients tested (64%) induced significant MVEC caspase 8 activation. This was suppressed by clinically relevant levels of narsoplimab (1·2 μg/ml) for all 14 patients, with a mean 65·7% inhibition (36.8–99.4%; P

Objective Aortic stenosis (AS) is characterized by narrowing of aortic valve opening, resulting in peak blood flow velocity that induces high wall shear stress (WSS) across the valve. Severe AS leads to heart failure and death. There is no treatment available for AS other than valve replacement. Platelet-derived TGF-β1 partially contributes to AS progression in mice, and WSS is a potent activator of latent TGF-β1. N-acetylcysteine (NAC) inhibits WSS-induced TGF-β1 activation in vitro. We hypothesize that NAC will inhibit AS progression by inhibiting WSS-induced TGF-β1 activation. Approach We treated a cohort of Ldlr(-/-)Apob(100/100) (LDLR) mice fed a high fat diet with N-acetylcysteine (NAC, 2% in drinking water) at different stages of disease progression and measured its effect on AS progression and TGF-β1 activation. Results Short-term NAC treatment inhibited AS progression in mice with moderate and severe AS relative to controls, but not in LDLR mice lacking platelet-derived TGF-β1 (TGF-β1platlet-KO-LDLR). NAC treatment reduced TGF-β signaling, p-Smad2 and collagen levels, and mesenchymal transition from Isolectin-B4 and CD45-positive cells in LDLR mice. Mechanistically, NAC treatment resulted in plasma NAC concentrations ranging from 75.5 to 449.2 ng/ml, which was sufficient to block free thiol labeling of plasma proteins and reduce active TGF-β1 levels without substantially affecting reactive oxygen species-modified products in valvular cells. Conclusions Short-term treatment with NAC inhibits AS progression, by inhibiting WSS-induced TGF-β1 activation in the LDLR mouse model of AS, motivating a clinical trial of NAC and/or other thiol-reactive agent(s) as a potential therapy for AS.

Background: Heart valves regulate the unidirectional forward flow and prevent retrograde backflow of blood during the cardiac cycle. Cardiac valve disease (CVD) is observed in approximately 2.5% of the general population and the incidence increases to ~10% in elderly people. Patients with severe CVD require surgery and effective pharmacological treatments are currently not available. PROX1 is a transcription factor that regulates the development of lymphatic, venous, and lymphovenous valves (vascular valves). We identified that PROX1 is also expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 regulates cardiac valve development and disease is not known. Method and Results: We have discovered that mice lacking Prox1 in their VECs ( Prox1 ΔVEC ) develop enlarged aortic and mitral valves in which the expression of proteoglycans is increased (control, N=10; Prox1 ΔVEC , N=9, p Prox1 ΔVEC mice (control, N=5; Prox1 ΔVEC , N=9, p Prox1 ΔVEC mice. Specific knockdown of FOXC2 in VECs results in the thickening of aortic valves (control, N=10; shFoxc2 ΔVEC , N=8, p Foxc2 OE-VEC ) ameliorates the thickening of the aortic valves of Prox1 ΔVEC mice ( Prox1 ΔVEC , N=9; Foxc2 OE-VEC ; Prox1 ΔVEC , N=8, p Pdgfb ) is increased in the valve tissue of Prox1 ΔVEC mice and in PROX1 deficient sheep mitral valve VECs (MVECs) (siCtrl , N=4; siProx1 , N=4, p Prox1 ΔVEC mice (control , N=4; Pdgfb GOF , N=3, p Conclusion: Together these data suggest that PROX1 maintains the extracellular matrix composition of cardiac valves by regulating the expressions of FOXC2 and PDGF-B in VECs.

Aortic stenosis (AS) is a progressive aortic valve disease that can lead to heart failure, syncope and sudden death if not treated. Severe AS currently affects more than 2 million people over 65 years of age in the USA. The only treatment option for AS is valve replacement by either surgical aortic valve replacement (AVR) or the less invasive transcatheter aortic valve replacement (TAVR), also called transcatheter aortic valve implantation (TAVI). AVR requires intensive care unit (ICU) admission, whereas TAVR can be performed in a non-ICU setting. Both procedures are commonly associated with complications, such as stroke.1 Bleeding and thrombosis are also potential complications of these procedures. Given the considerable morbidity and mortality, patients with these postoperative complications are admitted to the ICU for management. SARS-CoV-2, which causes COVID-19, has become a pandemic and has caused high mortality rates among the elderly. Currently, there is no vaccine against SARS-CoV-2, and there is no effective treatment to treat patients with severe COVID-19. More than 730 000 people worldwide and over 160 000 in the USA have died from COVID-19 as of 10 August 2020 according to Johns Hopkins Coronavirus Resource Center; these figures are expected to continue to rise until there are effective vaccines and therapies. Treating patients with COVID-19 during this pandemic requires many medical resources. Importantly, acute respiratory distress syndrome and multiorgan failures, believed to be due to systemic and pulmonary thrombosis,2 3 are common symptoms of severe COVID-19 that require treatment in the ICU. Therefore, government officials have recommended ceasing almost all elective surgeries until the COVID-19 pandemic is under control.4 Although many patients with symptomatic AS can delay procedures intended to alleviate their symptoms, some may require life-saving procedures. In cases where life-saving procedures (AVR or TAVR) are required, …

Inflammation is a major contributor to deep vein thrombosis (DVT). Flow restriction of the inferior vena cava (IVC) in mice induces DVT like that in humans. In this model, P-selectin–dependent adhesion of neutrophils and monocytes leads to release of neutrophil extracellular traps (NETs) and expression of tissue factor. However, it is not known what signals cause myeloid cells to generate these procoagulant effectors. Using ultrasonography and spinning-disk intravital microscopy in genetically engineered mice, we found that engagement of P-selectin glycoprotein ligand-1 (PSGL-1) and the chemokine receptor CXCR2 on rolling neutrophils propagated signals that cooperated to induce β2 integrin–dependent arrest in flow-restricted IVCs. Unlike previous reports, PSGL-1 signaling in neutrophils did not require L-selectin, and it used tyrosine 145 rather than tyrosines 112 and 128 on the adaptor Src homology domain-containing leukocyte phosphoprotein of 76 kDa. PSGL-1 and CXCR2 signaling cooperated to increase the frequency and size of thrombi, in part by stimulating release of NETs. Unlike in neutrophils, blocking PSGL-1 or CXCR2 signaling in monocytes did not affect their recruitment into thrombi or their expression of tissue factor. Our results demonstrate that neutrophils cooperatively signal through PSGL-1 and CXCR2 to promote DVT.

Transforming growth factor-β1 (TGF-β1) signaling in hepatic stellate cells (HSCs) plays a primary role in liver fibrosis, but the source of TGF-β1 is unclear. Because platelets are rich in TGF-β1, we examined the role of platelet TGF-β1 in liver fibrosis by challenging wild-type (WT) mice and mice deficient in platelet TGF-β1 (PF4CreTgfb1f/f) with carbon tetrachloride (CCl4), an inducer of acute hepatic injury and chronic fibrosis. CCl4 elicited equivalent hepatic injury in WT and PF4CreTgfb1f/f mice based on loss of cytochrome P450 (Cyp2e1) expression, observed at 6 hours and peaking at 3 days after CCl4 challenge; PF4CreTgfb1f/f mice exhibited less liver fibrosis than control mice. Activated platelets were observed during acute liver injury (6 hours), and WT mice with transient platelet depletion (thrombocytopenia) were partially protected from developing fibrosis compared with control mice (P = .01), suggesting an association between platelet activation and fibrosis. Transient increases in TGF-β1 levels and Smad2 phosphorylation signaling were observed 6 hours and 3 days, respectively, after CCl4 challenge in WT, but not PF4CreTgfb1f/f , mice, suggesting that increased TGF-β1 levels originated from platelet-released TGF-β1 during the initial injury. Numbers of collagen-producing HSCs and myofibroblasts were higher at 3 days and 36 days, respectively, in WT vs PF4CreTgfb1f/f mice, suggesting that platelet TGF-β1 may have stimulated HSC transdifferentiation into myofibroblasts. Thus, platelet TGF-β1 partially contributes to liver fibrosis, most likely by initiating profibrotic signaling in HSCs and collagen synthesis. Further studies are required to evaluate whether blocking platelet and TGF-β1 activation during acute liver injury prevents liver fibrosis.

Transforming growth factor-β1 (TGF-β1) has been used as a biomarker in disorders associated with pathologic fibrosis. However, plasma TGF-β1 assessment is confounded by the significant variation in reported normal values, likely reflecting variable release of the large pool of platelet TGF-β1 after blood drawing. Moreover, current assays measure only total TGF-β1, which is dominated by the latent form of TGF-β1 rather than the biologically active form. To address these challenges, we developed methodologies to prevent ex vivo release of TGF-β1 and to quantify active TGF-β1. We then used these techniques to measure TGF-β1 in healthy controls and patients with heart failure (HF) before and after insertion of left ventricular assist devices (LVAD). Total plasma TGF-β1 was 1.0 ± 0.60 ng/mL in controls and 3.76 ± 1.55 ng/mL in subjects with HF (P < 0.001), rising to 5.2 ± 2.3 ng/mL following LVAD placement (P = 0.006). These results were paralleled by the active TGF-β1 values; controls had 3-16 pg/mL active TGF-β1, whereas levels were 2.7-fold higher in patients with HF before, and 4.2-fold higher after, LVAD implantation. Total TGF-β1 correlated with levels of the platelet-derived protein thrombospondin-1 (r = 0.87; P < 0.001), suggesting that plasma TGF-β1 may serve as a surrogate indicator of in vivo platelet activation. von Willebrand factor high molecular weight multimers correlated inversely with TGF-β1 levels (r = -0.63; P = 0.023), suggesting a role for shear forces in loss of these multimers and platelet activation. In conclusion, accurate assessment of circulating TGF-β1 may provide a valuable biomarker for in vivo platelet activation and thrombotic disorders.

This review evaluates the role of platelet-derived transforming growth factor (TGF)-β1 in oxidative stress-linked pathologic fibrosis, with an emphasis on the heart and kidney, by using ionizing radiation as a clinically relevant stimulus. Current radiation-induced organ fibrosis interventions focus on pan-neutralization of TGF-β or the use of anti-oxidants and anti-proliferative agents, with limited clinical efficacy. Recent Advances: Pathologic fibrosis represents excessive accumulation of collagen and other extracellular matrix (ECM) components after dysregulation of a balance between ECM synthesis and degradation. Targets based on endogenous carbon monoxide (CO) pathways and the use of redox modulators such as N-acetylcysteine present promising alternatives to current therapeutic regimens.Ionizing radiation leads to direct DNA damage and generation of reactive oxygen species (ROS), with TGF-β1 activation via ROS, thrombin generation, platelet activation, and pro-inflammatory signaling promoting myofibroblast accumulation and ECM production. Feed-forward loops, as TGF-β1 promotes ROS, amplify these profibrotic signals, and persistent low-grade inflammation insures their perpetuation. We highlight differential roles for platelet- versus monocyte-derived TGF-β1, establishing links between canonical and noncanonical TGF-β1 signaling pathways in relationship to macrophage polarization and autophagy, and define points where pharmacologic agents can intervene.Additional studies are needed to understand mechanisms underlying the anti-fibrotic effects of current and proposed therapeutics, based on limiting platelet TGF-β1 activity, promotion of macrophage polarization, and facilitation of collagen autophagy. Models incorporating endogenous CO and selective TGF-β1 pathways that impact the initiation and progression of pathologic fibrosis, including nuclear factor erythroid 2-related factor (Nrf2) and redox, are of particular interest. Antioxid. Redox Signal. 27, 977-988.

Cardiovascular mechanical stresses trigger physiological and pathological cellular reactions including secretion of Transforming Growth Factor β1 ubiquitously in a latent form (LTGF-β1). While complex shear stresses can activate LTGF-β1, the mechanisms underlying LTGF-β1 activation remain unclear. We hypothesized that different types of shear stress differentially activate LTGF-β1. We designed a custom-built cone-and-plate device to generate steady shear (SS) forces, which are physiologic, or oscillatory shear (OSS) forces characteristic of pathologic states, by abruptly changing rotation directions. We then measured LTGF-β1 activation in platelet releasates. We modeled and measured flow profile changes between SS and OSS by computational fluid dynamics (CFD) simulations. We found a spike in shear rate during abrupt changes in rotation direction. OSS activated TGF-β1 levels significantly more than SS at all shear rates. OSS altered oxidation of free thiols to form more high molecular weight protein complex(es) than SS, a potential mechanism of shear-dependent LTGF-β1 activation. Increasing viscosity in platelet releasates produced higher shear stress and higher LTGF-β1 activation. OSS-generated active TGF-β1 stimulated higher pSmad2 signaling and endothelial to mesenchymal transition (EndoMT)-related genes PAI-1, collagen, and periostin expression in endothelial cells. Overall, our data suggest variable TGF-β1 activation and signaling occurs with competing blood flow patterns in the vasculature to generate complex shear stress, which activates higher levels of TGF-β1 to drive vascular remodeling.

Background: Chronic kidney disease (CKD) characterized by tubular injury and fibrosis occurs in HIV infection treated with certain protease inhibitor (PI)-based antiretroviral therapies (ART). The pathophysiology is unclear. Methods: We hypothesized that fibrosis, mediated by platelet-derived transforming growth factor (TGF)-β1 and macrophage polarization to an inflammatory subtype underlies PI-associated CKD. We induced this in mice exposed to the PI ritonavir (RTV) in the absence of HIV, and intervened with low-dose inhaled carbon monoxide (CO), to activate erythroid 2- related factor (Nrf2)-associated anti-oxidant pathways. C57BL/6 mice, wild-type and deficient in platelet TGF-β1, were given RTV (10mg/kg) or vehicle daily for 8 weeks. Select groups were exposed to CO (250ppm) for 4 hours after RTV or vehicle injection. Renal pathology, fibrosis, and TGF-β1- and Nrf2-based signaling were examined by histology, immunofluorescence, and flow cytometry. Renal damage and dysfunction were assessed by KIM-1 and cystatin C ELISAs. Clinical correlations were sought among HIV-infected individuals. Findings: RTV induced glomerular and tubular injury, elevating urinary KIM-1. It enhanced TGF-β1-related signaling, accompanied by kidney fibrosis, macrophage polarization to an inflammatory phenotype, and renal dysfunction with cystatin C elevation. Mice with platelet TGF-β1 deletion were partially protected from these abnormalities. CO inhibited RTVinduced fibrosis and macrophage polarization in association with upregulation of Nrf2 and heme oxygenase-1 (HO-1). RTV use correlated with elevated cystatin C and HO-1 levels in HIV-infected individuals vs. controls and HIV-infected subjects receiving no therapy or non-PI-based regimens. Interpretation: Platelet TGF-β1 contributes to RTV-induced kidney fibrosis and dysfunction, which may be amenable to anti-oxidant-based interventions. Funding Statement: This work was supported by NIH grants R21 HL125044 (J.L. and J.A.), R01 HL123605 (J.A.), RO1 HL133801 and RO1 HL060234 (M.E.C.), COBRE grant GM114731, and funding from PHF (J.A.) and the Angelo Donghia Foundation (J.L.). Declaration of Interests: The spouse of M.E.C is a co-founder, shareholder and serves on the Scientific Advisory Board of Proterris, Inc. The remaining authors have no conflicts. Ethics Approval Statement: Animal studies were approved by the Institutional Animal Care and Use Committee of OMRF and WCMC. Patient samples were obtained through study approval by the Institutional Review Board of WCMC.

Highlights • Circuit thrombosis complicates CRRT in COVID-19 despite standard heparin-based anticoagulation regimens. • 5 cases of CRRT thrombosis despite heparin-based anticoagulation resolved using a direct thrombin inhibitor, argatroban. • Changes in fibrinogen levels better reflected response to anticoagulation than did changes in D-dimer levels. • High fibrinogen levels and decreased anti-thrombin III activity may relate to argatroban superiority in these cases.

HIV infection elevates the incidence of cardiovascular disease (CVD) independent of traditional risk factors. Autopsy series document cardiac inflammation and endomyocardial fibrosis in the HIV+ treatment naive, and gadolinium enhancement magnetic resonance imaging has identified prominent myocardial fibrosis in the majority of HIV+ individuals despite use of suppressive antiretroviral therapies (ART). The extent of such disease may correlate with specific ART regimens. For example, HIV-infected patients receiving ritonavir (RTV)-boosted protease inhibitors have the highest prevalence of CVD, and RTV-exposed rodents exhibit cardiac dysfunction coupled with cardiac and vascular fibrosis, independent of RTV-mediated lipid alterations. We recently showed that platelet transforming growth factor (TGF)-β1 is a key contributor to cardiac fibrosis in murine models. We hypothesize that in the HIV+/ART naive, cardiac fibrosis is a consequence of proinflammatory cytokine and/or ART-linked platelet activation with release of TGF-β1. Resultant TGF-β1/Smad signaling would promote collagen synthesis and organ fibrosis. We document these changes in a pilot immunohistochemical evaluation of cardiac tissue from two ART-naive pediatric AIDS patients. In terms of ART, we showed that RTV inhibits immunoproteasome degradation of TRAF6, a nuclear adapter signaling molecule critical to the regulation of proinflammatory cytokine signaling pathways involved in osteoclast differentiation and accelerated osteoporosis. We now present a model illustrating how RTV could similarly amplify TGF-β1 signaling in the promotion of cardiac fibrosis and accelerated CVD. Supportive clinical data correlate RTV use with elevation of NT-proBNP, a biomarker for CVD. We discuss potential interventions involving intrinsic modulators of inflammation and collagen degradation, including carbon monoxide-based therapeutics.

Megakaryocytes (MK) and platelets contain a high concentration of transforming growth factor β1 (TGFβ1). Mice with conditional deletion of Tgfb1 in megakaryocytes (PF4Cre/Tgfb1flox/flox) resulted in >90% reduction of TGFβ1 in platelets and 50% reduction of TGFβ1 in plasma. TGFβ1 has been shown to play an inhibitory role in megakaryopoiesis in vitro, and inhibiting TGFβ1 increased megakaryopoiesis in vivo. However, the source of TGFβ1 in megakaryopoiesis is unknown. In this study, we tested whether megakaryocyte-derived TGFβ1 contributes to megakaryopoiesis in bone marrow (BM) by comparing three groups of mice: PF4Cre/Tgfb1flox/flox, littermate control Tgfb1flox/flox, and WTC57Bl/6 mice. Bones (femurs) from these mice (n=12) (age 15-30 weeks, males 60% and females 40%) were harvested, fixed, decalcified, sectioned, and H&E stained. Whole stained BM areas of the sectioned femurs were scanned with an Aperio slide scanner to quantify the number of megakaryocytes and the demarcation membrane system (DMS) and ploidy (nucleus size) of the megakaryocytes were quantified by manually counting megakaryocytes and tracing their DMS and nucleus. The percentage of MK among total BM cells was calculated by dividing total numbers of BM cells in the total area of a BM section with the number of MK in the section. Freshly isolated BM cells were cultured in vitro in culture medium (DMEM+10%FBS) in the presence of thrombopoietin (TPO, 100 ng/ml) with and without TGFβ1 (20 ng/ml) or with a neutralized antibody against the active form of TGFβ1 (AF-101; 2 ug/ml). TGFβ1 and TPO levels in plasma, BM exudates, and cells were measured by ELISA. PF4Cre/Tgfb1flox/floxmice had >50% reduction in TGFβ1 levels in BM cells and exudates (TGFβ1 levels in BM exudates were 1.4 ± 0.033 ng in WT and 0.68 ± 0.065 ng in PF4CreTgfb1flox/floxmice, p We conclude that MK-derived TGFβ1 negatively regulates megakaryopoiesis in mice. Further investigation is needed to determine the mechanism by which TGFβ1 regulates TPO-induced megakaryopoiesis. Our study may be important in megakaryocyte generation in vitro and may have important implications in vivo under normal and stress-inducing conditions where variable megakaryopoiesis is observed, such as essential thrombocythemia and primary myelofibrosis. Disclosures No relevant conflicts of interest to declare.

A substantial proportion of patients with severe COVID-19 pneumonia develop thrombosis (both venous and arterial) via an undefined mechanism. Systemic elevation of high levels of D-dimer, a marker of coagulation activation and thrombolysis, is evident in almost all advanced COVID-19 patients and is associated with disease severity and mortality. However, the extrinsic factors that initiate blood coagulation in COVID-19 is not clear. Because tissue factor (TF) is the prime initiator of the extrinsic coagulation cascade, and because it is expressed and exposed under inflammatory conditions leading to vascular damage, we tested the hypothesis that higher TF expression is responsible for thrombi formation in the lungs of patients with severe COVID-19. To this end, we examined autopsy lung tissues from five COVID-19 pneumonia patients with acute respiratory syndrome (ARDS) who required ICU hospitalization, with 4 of the 5 patients requiring mechanical ventilation before they died, and five controls with acute ARDS caused by bacterial pneumonia, one with a prior influenza infection, and all on mechanical ventilation. Histological findings revealed all the COVID-19 lungs had characteristics of ARDS, including DAD with hyaline deposition and inflammatory cell invasion. Immunofluorescence staining showed TF expression throughout all lung tissues and in many blood vessels that were filled with thrombi with either fibrin, activated platelets, or both. TF expression was significantly higher in COVID-19 than control ARDS lung tissues (1.2 ± 0.3% in COVID-19 vs. 0.52 ± 0.2% in ARDS controls (p=0.004). Fibrin-enriched thrombi areas were higher in COVID-19 cases than in controls (1.6 ± 0.36% vs. 0.94 ± 0.4%; p=0.008) and correlated with TF expression (R2=0.4, p=0.02). Platelet factor 4 (PF4)-enriched thrombi areas were also higher in COVID-19 lung, but this trend was not statistically significant (0.94 ± 0.4% in COVID-19 and 0.54 ± 0.3% in controls; p=0.09), although it did, however, correlate with TF expression (R2=0.4, p=0.02). Many thrombi were in close proximity to TF-expressing areas in both COVID-19 and ARDS pneumonia controls. Dual RNA in situ hybridization with SARS-CoV-2 and TF fluorescence probes showed variable viral and TF mRNA expression. Increased TF mRNA expression was seen in COVID-19 vs. control lung (0.77 ± 0.4 % vs. 0.31 ± 0.15 %, p=0.05). TF mRNA expression correlated with viral mRNA in COVID-19 patients (R2=0.78, p=0.01). High-resolution images identified both sporadic and clustered SARS-CoV-2, and some areas co-localized with TF mRNA expression. We conclude that higher TF expression might be responsible for fibrin formation and platelet activation in the lungs of both COVID-19 and ARDS controls. Our observation of higher TF expression in COVID-19 patients was documented by two very sensitive methods, RNA in situ hybridization and immunostaining with very specific antibodies against TF and mRNA probes. Its correlation with SARS-CoV-2 mRNA suggests that SARS-CoV-2 infection induces both de novo gene transcription and protein synthesis in the lungs of COVID-19 patients. Thus, TF-initiated extrinsic coagulation might be responsible for the critical thrombi formation observed in many COVID-19 cases, rendering TF a potential therapeutic target. Disclosures Laurence: Alexion Pharmaceuticals: Honoraria, Research Funding.

Objective— Aortic valve stenosis (AS) is characterized by fibrosis and calcification of valves leading to aortic valve narrowing, resulting in high wall shear stress across the valves. We previously demonstrated that high shear stress can activate platelet-derived transforming growth factor-β1 (TGF-β1), a cytokine that induces fibrosis and calcification. The aim of this study was to investigate the role of shear-induced platelet release of TGF-β1 and its activation in AS. Approach and Results— We studied hypercholesterolemic Ldlr − /− Apob 100/100 / Mttp fl/fl / Mx1Cre +/+ (Reversa) mice that develop AS on Western diet and a surgical ascending aortic constriction mouse model that acutely simulates the hemodynamics of AS to study shear-induced platelet TGF-β1 release and activation. Reversa mice on Western diet for 6 months had thickening of the aortic valves, increased wall shear stress, and increased plasma TGF-β1 levels. There were weak and moderate correlations between wall shear stress and TGF-β1 levels in the progression and reversed Reversa groups and a stronger correlation in the ascending aortic constriction model in wild-type mice but not in mice with a targeted deletion of megakaryocyte and platelet TGF-β1 ( Tgfb1 flox ). Plasma total TGF-β1 levels correlated with collagen deposition in the stenotic valves in Reversa mice. Although active TGF-β1 levels were too low to be measured directly, we found (1) canonical TGF-β1 (phosphorylated small mothers against decapentaplegic 2/3) signaling in the leukocytes and canonical and noncanonical (phosphorylated extracellular signal–regulated kinases 1/2) TGF-β1 signaling in aortic valves of Reversa mice on a Western diet, and (2) TGF-β1 signaling of both pathways in the ascending aortic constriction stenotic area in wild-type but not Tgfb1 flox mice. Conclusions— Shear-induced, platelet-derived TGF-β1 activation may contribute to AS.

Although survival and quality of life have improved in patients with advanced heart failure (HF) after implantation of left ventricular assist devices (LVADs), they still pose risks of hemocompatibility-related complications, including thrombosis and bleeding. Development of biomarkers predictive of these LVAD-associated complications could guide decision making for both clinicians and patients. Recently, we showed higher plasma TGF-β1 levels within one-week after implantation with a miniaturized mechanical-bearing axial-flow pump HeartMate II (HM-II), and reasoned that platelet activation by the rotor may have caused the release of TGF-β1 in plasma in HF patients (Mancini et al. Transl. Res. 2018; 192:15-29). Recent clinical trials with the newest LVAD, the Heartmate 3 (HM-3), which uses a fully magnetically-levitated pump, showed superior clinical outcomes, including significantly reduced incidences of pump thrombosis and stroke (Mehra et al. N Engl J Med. 2019; 380:1618-1627). In this study, we evaluated release of TGF-β1 in plasma following implantation of HM-II and HM-3 LVADs compared to either coronary artery bypass graft (CABG) surgery or extracorporeal membrane oxygenation (ECMO) therapy. We measured serial total TGF-β1 levels in 38 Stage-D HF patients (11 received HM-II and 27 received HM-3). As a control, we collected blood samples from 10 patients undergoing CABG surgery, and 10 patients receiving ECMO therapy following acute onset cardio-pulmonary failure. Blood samples were collected before and 4-8 hours after procedures, and thereafter daily for up to one week. Plasma was prepared by centrifuging blood at 12,000 rpm for 5 min at 4°C within 10 min of blood drawing, which reduces in vitro release of TGF-β1 from platelets and thus allows accurate measurement of plasma TGF-β1. Total TGF-β1 levels were measured after acidification and neutralization of samples using DUO-ELISA kit (R&D Systems). Baseline total plasma TGF-β1 levels were higher in HF patients before LVAD implantation than in healthy controls [4.7 ± 1.9 ng/mL in HF patients (n= 38); 3.3 ± 0.8 ng/mL in healthy controls (n= 6); p=0.006)]. Total TGF-β1 levels surged transiently to 14.6 ± 6.1 ng/mL within 4-8 hours after LVAD implantation [(p We conclude that LVAD implantation causes a transient surge in total plasma TGF-β1 within a few hours after the procedure, presumably due to platelet activation by LVAD, not the surgery itself, as CABG or circulating blood through ECMO did not cause the surge. The observation that a reduced initial surge and lower levels of TGF-β1 in HM-3 vs. HM-II recipients needs further investigation to determine whether these differences are due to LVAD-specific factors (different rotors causing variable shear effects) or to confounding differences in implantation procedures, such as, by-pass time, cardiac tissue injury, number of platelet transfusions, blood suction with catheters etc. or other unknown factors. Our data suggest that serial TGF-β1 measurements after LVAD implantation may serve as a surrogate biomarker for platelet activation in association with hemocompatibility-related adverse events (Uriel et al. Circ. 2017; 135:2003-2012). Disclosures No relevant conflicts of interest to declare.

Combination antiretroviral therapies (cART) have markedly reduced mortality in HIV infection. However, cardiovascular disease (CVD), including heart failure linked to fibrosis, remains a major cause of morbidity and mortality in HIV/cART patients. The magnitude of this risk increases with use of certain protease inhibitors (PI), but the underlying mechanism remains unclear. We showed that the PI ritonavir leads to increased plasma levels of the pro-fibrotic cytokine TGF-β1, cardiac dysfunction, and pathologic cardiac fibrosis in wild-type (wt) C57BL/6 mice. Mice with targeted depletion of platelet TGF-β1 had reduced cardiac fibrosis and partially preserved cardiac function following ritonavir exposure (Laurence, et al. PLoS One 2017;12:e0187185). Several groups have examined the effects of a variety of cART agents on agonist-induced platelet aggregation, but correlations with clinical CVD are weak. Since platelets are a rich source of TGF-β1, we hypothesized that ritonavir and other PIs linked clinically to an increased CVD risk directly activate platelets to release TGF-β1 and activate latent (L)TGF-β1 to initiate signaling for organ fibrosis. We examined the impact of clinically relevant doses of ritonavir, alone and in combination with two other contemporary PIs, atazanavir and darunavir, which are currently used along with low dose ritonavir in so-called PI-boosted cART regimens. We incubated human platelet-rich plasma and washed platelets with PIs alone or in combinations at various doses for 10 min at 37°C in a platelet aggregometer (BioData. Corp). Total and active TGF-β1 levels were measured by ELISA. For in vivo assessment, we treated wt mice with a low dose of ritonavir, as used in PI-boosted cART, and measured the levels of plasma TGF-β1 by ELISA, and TGF-β1 signaling in tissues by immunofluorescence imaging for pSmad2. We found that ritonavir dose-dependently increased total TGF-β1 release from freshly-isolated platelet-rich plasma and washed human platelets. This release was blocked by ceefurin-1 and MK517, potent inhibitors of the ATP binding cassette transporter ABCC4. Darunavir alone did not cause release of TGF-β1, and did not alter significantly ritonavir-induced TGF-β1 release (Figure-1A). Atazanavir alone did induce release of TGF-β1 from platelets and did not affect the extent of such release induced by ritonavir (Figure-1A). Since total TGF-β1 released from platelets must be activated in order to signal, we tested whether these PIs could activate LTGF-β1. Ritonavir alone, in low dose, activated TGF-β1 by 4-5-fold (Fig-1B). Darunavir alone did not activate LTGF-β1, and had only a minor effect on ritonavir-induced TGF-β1 activation (Fig-1B). In marked contrast, while atazanavir also did not activate LTGF-β1, it significantly inhibited ritonavir-induced LTGF-β1 activation (Fig-1B). For in vivo assessment, wt mice were injected daily for 8 weeks with ritonavir, which dose-dependently increased plasma TGF-β1 levels (mean levels with vehicle 2.1 ng/ml; 6.4 ng/ml with 5 mg/kg ritonavir; 8.5 ng/ml with 10 mg/kg ritonavir). Increased TGF-β1 levels correlated with development of pathologic fibrosis and increased phosphorylated Smad signaling in hearts of ritonavir-treated vs. vehicle-treated mice. Clinical correlations with these in vitro and in vivo mouse studies are important. The fact that ritonavir effected both release and activation of platelet TGF-β1 is consistent with its ability to induce cardiac fibrosis and dysfunction in mice, and its association with accelerated CVD in HIV-infected individuals. Our findings that low dose ritonavir in combination with darunavir induced release and activation of platelet TGF-β1, whereas atazanavir blocked TGF-β1 activation, are consistent with the strong association of ritonavir-boosted darunavir, but not ritonavir-boosted atazanavir, with CVD in the setting of HIV (Ryom, et al. Lancet-HIV 2018;5:e291-e300). Future work will examine the effects of other contemporary cART agents, including cobicistat, which is currently replacing ritonavir in many PI-boosted therapies and some integrase-boosted regimens, on TGF-β1 release and activation, for which correlations with clinical CVD are not yet available. Identification of the mechanism of pathologic fibrosis in the heart, and potentially other organs affected by certain cART regimens, such as the kidney, may suggest specific therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Introduction: 8500 adult allogeneic hematopoietic stem cell transplants (alloHSCT) are performed annually in the U.S. and 17,000 in Europe. HSCT-associated thrombotic microangiopathy (TMA), defined by thrombocytopenia, microangiopathic hemolytic anemia, and organ dysfunction in the absence of disseminated intravascular coagulation, complicates some 20% of these procedures. About half of post-alloHSCT TMAs may resolve when GvHD immunoprophylaxis is modified, but three year survival rates for those with severe HSCT-TMAs are a dismal 11%. Clinical manifestations are similar to other TMAs, but their pathophysiology may be distinct. Damage to vascular endothelium, independent of loss of ADAMTS13 activity, is thought to be critical. Fibrin-rich microthrombi, often accompanied by C4d and C5b-9 (membrane attack complex) deposition, occur in the microvasculature of multiple organs (Clin Adv Hematol Oncol 2014;12:565-573; Transplantation. 2013;96:217-223). This supports a role for complement activation in HSCT-TMA, but the importance of the various complement activation pathways is unclear. Recently, narsoplimab (OMS721), a monoclonal antibody inhibitor of MBL-associated serine protease-2 (MASP-2), a principal component of lectin-dependent complement activation, received Breakthrough Therapy Designation for HSCT-TMA, based on improved survival compared to historical controls in a phase 2 study. A phase 3 program is ongoing. Chemotherapy in association with autologous HSCT is linked to a marked increase in serum MASP-2 levels, persisting for about 4 weeks post-transplant (Front Immunol 2018;9:2153). However, TMAs are rare in autologous transplants, and circulating levels of MASP-2 following alloHSCT, and the impact of TMA development on those levels, are unknown. Methods: All individuals >18 years of age scheduled to undergo alloHSCT for hematologic malignancy at New York Presbyterian Hospital-Cornell were approached to participate in this study (NCT02604420). 100 of the first 101 subjects, age 58.3 +14 yrs, were enrolled and followed for >1.5 years post-transplant. This interval is consistent with the median time to HSCT-TMA diagnosis in adults of 90 days (range 32-733 days). Plasma was obtained at baseline (defined as between the time of consent and beginning of conditioning regimen), and at each regularly scheduled visit post-transplant-day 28 +5 days; day 100 +28 days; day 180 +28 days; and day 365 +28 days-as well as at the time of TMA diagnosis, based on the Consensus Criteria of Cho et al. (Transplantation 2010;90:918-926). A commercial ELISA (MyBioSource) was used to assess MASP-2 concentrations. Results: 20 subjects met study criteria for a HSCT-TMA diagnosis, occurring a median of 69 days (range 33-289) post-transplant. Three resolved following discontinuation of GvHD prophylaxis (mTOR or calcineurin inhibitor) and switch to mycophenolate and increased corticosteroid doses, and 7 had an intercurrent infection, 6 of whom expired with ongoing severe TMA despite a change in GvHD prophylaxis (Figure). TMAs persisted in the remaining 10 subjects. Median MASP-2 levels were significantly elevated in all subjects post-transplant, assessed at the time of TMA development or, in those not developing a TMA, at day 100 + 28 days post-transplant vs. controls (n=36, 86.2ng/ml (23.3-210.9): persistent TMA (n=9 (one plasma unavailable), 154ng/ml (range 82-209)); alloHSCT subjects who did not experience a TMA (n=40 evaluated to date, 113.5ng/ml (56-430.3)). (Figure). Lack of a significant rise in MASP-2 levels in patients with persistent TMAs vs. those who did not develop a TMA, combined with a significant decrease in variance of MASP-2 levels in the former group (p=0.005), may reflect consumption of product at sites of disease activity, i.e., the microvasculature. Conclusions: There is a significant increase in MASP-2 levels, with a wide variance, in post-alloHSCT patients evaluated at a time post-transplant typical of HSCT-TMA development. At time of development of a HSCT-TMA that persists despite withdrawal of GvHD prophylaxis, MASP-2 levels remain elevated over controls, but with a significantly lower variance vs. those not developing TMA. A study of additional samples, including longitudinal specimens, from this cohort is underway to determine if a change in MASP-2 levels correlates with HSCT-TMA development post-alloHSCT. Disclosures Van Besien: Miltenyi Biotec: Research Funding.

TGF-β1 is a disulfide-bonded homodimeric protein produced by platelets and other cells that plays a role in many physiologic and pathologic processes. TGF-β1 is secreted as an inactive large latent complex (LLC) comprised of TGF-β1, latency-associated peptide, and latent TGF-β binding protein 1. We previously demonstrated that shear force can activate LLC and that thiol-disulfide exchange contributes to the process. We have now investigated the role of thiol isomerases in the activation of LLC in platelet releasates (PR) and recombinant LLC. The wasp venom peptide mastoparan, which inhibits the chaperone activity of PDI, inhibited stirring- and shear-induced activation of latent TGF-β1 by 90 and 75% respectively. To identify the proteins that bind to mastoparan either directly or indirectly, PR were chromatographed on a mastoparan affinity column. Latent TGF-β binding protein 1, latency-associated peptide, TGF-β1, clusterin, von Willebrand factor, multimerin-1, protein disulfide isomerase (PDI), ERp5, ERp57, and ERp72 eluted specifically from the column. Anti-PDI RL90 attenuated the inhibitory effect of mastoparan on LLC activation. Furthermore, reduced PDI inhibited activation of PR LLC, whereas oxidized PDI had no effect. We conclude that thiol isomerases and thiol-disulfide exchange contribute to TGF-β1 activation and identify a number of molecules that may participate in the process.

Introduction: Although ruptured atherosclerotic plaques have been extensively analyzed, the composition of thrombi causing arterial occlusion in patients with ST-segment elevation acute myocardial infarction has been less thoroughly investigated. We sought to investigate whether coagulant active tissue factor can be retrieved in thrombi of patients with STEMI undergoing primary percutaneous coronary intervention. Methods: Nineteen patients with ST-segment elevation acute myocardial infarction referred for primary percutaneous coronary intervention were enrolled in this study. Coronary thrombi aspirated from coronary arteries were routinely processed for paraffin embedding and histological evaluation (4 patients) or immediately snap frozen for evaluation of tissue factor activity using a modified aPTT test (15 patients). Immunoprecipitation followed by immunoblotting was also performed in 12 patients. Results: Thrombi aspirated from coronary arteries showed large and irregular areas of tissue factor staining within platelet aggregates, and in close contact with inflammatory cells. Some platelet aggregates stained positive for tissue factor, whereas others did not. Monocytes consistently stained strongly for tissue factor, neutrophils had a more variable and irregular tissue factor staining, and red blood cells did not demonstrate staining for tissue factor. Median clotting time of plasma samples containing homogenized thrombi incubated with a monoclonal antibody that specifically inhibits tissue factor-mediated coagulation activity (mAb 5G9) were significantly longer than their respective controls (88.9 seconds versus 76.5 seconds, respectively; p

HIV infection is a risk factor for cardiovascular disease (CVD). This risk is accentuated by certain combination antiretroviral therapies (cARTs), independent of their effects on lipid metabolism and insulin sensitivity. We sought to define potential mechanisms for this association through systematic review of clinical and preclinical studies of CVD in the setting of HIV/cART from the English language literature from 1989 to March 2018. We used PubMed, Web of Knowledge and Google Scholar, and conference abstracts for the years 2015–March 2018. We uncovered three themes: (1) a critical role for the HIV protease inhibitor (PI) ritonavir and certain other PI-based regimens. (2) The importance of platelet activation. Virtually all PIs, and one nucleoside reverse transcriptase inhibitor, abacavir, activate platelets, but a role for this phenomenon in clinical CVD risk may require additional postactivation processes, including: release of platelet transforming growth factor-β1; induction of oxidative stress with production of reactive oxygen species from vascular cells; suppression of extracellular matrix autophagy; and/or sustained proinflammatory signalling, leading to cardiac fibrosis and dysfunction. Cardiac fibrosis may underlie an apparent shift in the character of HIV-linked CVD over the past decade from primarily left ventricular systolic to diastolic dysfunction, possibly driven by cART. (3) Recognition of the need for novel interventions. Switching from cART regimens based on PIs to contemporary antiretroviral agents such as the integrase strand transfer inhibitors, which have not been linked to clinical CVD, may not mitigate CVD risk assumed under prior cART. In conclusion, attention to the effects of specific antiretroviral drugs on platelet activation and related profibrotic signalling pathways should help: guide selection of appropriate anti-HIV therapy; assist in evaluation of CVD risk related to novel antiretrovirals; and direct appropriate interventions.

The serine protease zymogen factor X is converted to its catalytically active form factor Xa by the binary complex of factor VIIa bound to its cell surface receptor tissue factor (TF) or by the intrinsic Xase complex, which consists of active factors VIII (VIIIa), IX (IXa), factor X, and Ca2+. Factor Xa has procoagulant activity by conversion of prothrombin to thrombin and also induces signal transduction, either alone or in the ternary TF:VIIa:factor Xa coagulation initiation complex. Factor Xa cleaves and activates protease activated receptor (PAR)1 or -2, but factor Xa signaling efficiency varies among cell types. We show here that annexin 2 acts as a receptor for factor Xa on the surface of human umbilical vein endothelial cells and that annexin 2 facilitates factor Xa activation of PAR-1 but does not enhance coagulant function of factor Xa. Overexpression of TF abolishes annexin 2 dependence on factor Xa signaling and diminishes binding to cell surface annexin 2, whereas selectively abolishing TF promotes the annexin 2/factor Xa interaction. We propose that annexin 2 serves to regulate factor Xa signaling specifically in the absence of cell surface TF and may thus play physiological or pathological roles when factor Xa is generated in a TF-depleted environment.

Coagulation activation by tissue factor (TF) is implicated in cancer progression, cancer-associated thrombosis and metastasis. The role of direct TF signaling pathways in cancer, however, remains incompletely understood. Here we address how TF contributes to primary tumor growth by using a unique pair of isotype-matched antibodies that inhibit either coagulation (monoclonal antibody [Mab]-5G9) or direct signaling (Mab-10H10). We demonstrate that the inhibitory antibody of direct TF-VIIa signaling not only blocks TF-VIIa mediated activation of PAR2, but also disrupts the interaction of TF with integrins. In epithelial and TF-expressing endothelial cells, association of TF with β1 integrins is regulated by TF extracellular ligand binding and independent of PAR2 signaling or proteolytic activity of VIIa. In contrast, α3β1 integrin association of TF is constitutive in breast cancer cells and blocked by Mab-10H10 but not by Mab-5G9. Mab-5G9 has antitumor activity in vivo, but we show here that Mab-10H10 is at least as effective in suppressing human xenograft tumors in 2 different models. Breast tumor growth was also attenuated by blocking PAR2 signaling. These results show that tumor cell TF-PAR2 signaling is crucial for tumor growth and suggest that anti-TF strategies can be applied in cancer therapy with minor impairment of TF-dependent hemostatic pathways.

Tissue factor (TF) is the primary initiator of coagulation, and the TF pathway mediates signaling through protease-activated receptors (PARs). In sepsis, TF is up-regulated as part of the proinflammatory response in lipopolysaccharide (LPS)–stimulated monocytes leading to systemic coagulation activation. Here we demonstrate that TF cytoplasmic domain–deleted (TFΔCT) mice show enhanced and prolonged systemic coagulation activation relative to wild-type upon LPS challenge. However, TFΔCT mice resolve inflammation earlier and are protected from lethality independent of changes in coagulation. Macrophages from LPS-challenged TFΔCT mice or LPS-stimulated, in vitro–differentiated bone marrow–derived macrophages show increased TF mRNA and functional activity relative to wild-type, identifying up-regulation of macrophage TF expression as a possible cause for the increase in coagulation of TFΔCT mice. Increased TF expression of TFΔCT macrophages does not require PAR2 and is specific for toll-like receptor, but not interferon γ receptor, signaling. The presence of the TF cytoplasmic domain suppresses ERK1/2 phosphorylation that is reversed by p38 inhibition leading to enhanced TF expression specifically in wild-type but not TFΔCT mice. The present study demonstrates a new role of the TF cytoplasmic domain in an autoregulatory pathway that controls LPS-induced TF expression in macrophages and procoagulant responses in endotoxemia.

Transmethylation affects several cellular events, including T cell activation, and blockade of this pathway may curtail inflammatory/autoimmune responses. Here, we demonstrate that transmethylation inhibition by a novel reversible S-adenosyl-l-homocysteine hydrolase inhibitor leads to immunosuppression by reducing phosphorylation of several key proteins involved in TCR signaling, including Akt, Erk1/2, and NF-κB. Remarkably, this effect was largely restricted to CD4 T cells and correlated with reduced arginine methylation of Vav1, an essential guanine nucleotide exchange factor in T cell stimulation. Treatment with the transmethylation inhibitor averted, and even ameliorated, the CD4-mediated autoimmune disease, experimental autoimmune encephalomyelitis. The data suggest that transmethylation is required for CD4 T cell activation, and its inhibition may be a novel approach in the treatment of multiple sclerosis, and other CD4-mediated autoimmune diseases.

Cell-surface tissue factor (TF) binds the serine protease factor VIIa to activate coagulation or, alternatively, to trigger signaling through the G protein-coupled, protease-activated receptor 2 (PAR2) relevant to inflammation and angiogenesis. Here we demonstrate that TF·VIIa-mediated coagulation and cell signaling involve distinct cellular pools of TF. The surface-accessible, extracellular Cys 186 –Cys 209 disulfide bond of TF is critical for coagulation, and protein disulfide isomerase (PDI) disables coagulation by targeting this disulfide. A TF mutant (TF C209A) with an unpaired Cys 186 retains TF·VIIa signaling activity, and it has reduced affinity for VIIa, a characteristic of signaling TF on cells with constitutive TF expression. We further show that PDI suppresses TF coagulant activity in a nitric oxide-dependent pathway, linking the regulation of TF thrombogenicity to oxidative stress in the vasculature. Furthermore, a unique monoclonal antibody recognizes only the noncoagulant, cryptic conformation of TF. This antibody inhibits formation of the TF·PAR2 complex and TF·VIIa signaling, but it does not prevent coagulation activation. These experiments delineate an upstream regulatory mechanism that controls TF function, and they provide initial evidence that TF·VIIa signaling can be specifically inhibited with minimal effects on coagulation.

Tissue Factor (TF) is the mammalian plasma membrane cofactor responsible for initiation of blood coagulation. Binding of blood coagulation factor VIIa to TF activates the serine proteinase zymogens factors IX and X by limited proteolysis leading to the formation of a thrombin and fibrin meshwork that stabilizes the thrombus. TF on the plasma membrane of cells resides mostly in a cryptic configuration, which rapidly transforms into an active configuration in response to certain stimuli. The extracellular part of TF consists of two fibronectin type III domains. The disulfide bond in the membrane proximal domain (Cys186-Cys209) is atypical for domains of this type in that it links adjacent strands in the same beta sheet, what we have called an allosteric bond. Ablation of the allosteric disulfide by mutating both cysteine residues severely impairs procoagulant activity. The thiol-alkylating agents N-ethylmaleimide and methyl methanethiolsulfonate block TF activation by ionomycin, while the thiol-oxidizing agent HgCl2 and dithiol cross-linkers promote activation. TF activation could not be explained by exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Cryptic TF contained unpaired cysteine thiols that were depleted upon activation, and de-encryption was associated with a change in the conformation of the membrane-proximal domain. These findings imply that the Cys186-Cys209 disulfide bond is reduced in the cryptic form of TF and that activation involves formation of the disulfide. It is likely that formation of this disulfide bond changes the conformation of the domain that facilitates productive binding of factors IX and X.

Objective— To test the hypothesis that Grp78 negatively regulates cell surface tissue factor (TF) procoagulant activity and whether this is mediated by physical interaction. Methods and Results— Biopanning with phage-displayed peptidyl libraries has identified peptide probes that bind selectively in vivo to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds 78-kDa glucose regulated protein (Grp78). Grp78 participates in numerous pathological processes, including the regulation of the coagulation cascade, but the mechanism of Grp78 regulation of coagulation is unknown. To characterize this function, we analyzed the effect of Grp78 on TF-mediated procoagulant activity on murine brain endothelial cells (bEND.3) and macrophage-like (RAW) cells, which are relevant in mediation of atherothrombosis. We show that Grp78 is present on the surface of endothelium and monocyte/macrophage-like cells in atherosclerotic lesions. Inhibition of Grp78 resulted in increased procoagulant activity. We demonstrate that Grp78 negatively regulates procoagulant activity by interacting physically with the TF extracellular domain on the cell surface. Conclusions— The evidence indicates that Grp78 negatively regulates TF functional activity via direct binding to and functional inhibition of TF. Identification of the mechanism by which Grp78 regulates TF function may advance insight into the pathobiology of atherosclerosis and associated arterial thrombosis.

Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activation and a more robust Ca2+ mobilization in RBL-2H3 cells expressing carboxyl terminus deletion, phosphorylation-deficient mutant of PAF receptor (mPAFR) when compared with the wild-type receptor (PAFR). However, PAF did not provide sufficient signal for CC chemokine receptor ligand 2 (CCL2) production in cells expressing mPAFR. Based on these findings, we hypothesized that receptor phosphorylation provides a G protein-independent signal that synergizes with Ca2+ mobilization to induce CCL2 production. Here, we show that a mutant of PAFR (D289A), which does not couple to G proteins, was resistant to agonist-induced receptor phosphorylation. Unexpectedly, we found that when this mutant was coexpressed with mPAFR, it restored NF-kappaB activation and CCL2 production. PAF caused translocation of beta-arrestin from the cytoplasm to the membrane in cells expressing PAFR but not a phosphorylation-deficient mutant in which all Ser/Thr residues were replaced with Ala (DeltaST-PAFR). Interestingly, PAF induced significantly higher NF-kappaB and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing DeltaST-PAFR than those expressing PAFR. Furthermore, a Ca2+/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-kappaB activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-kappaB activation, which synergizes with G protein-mediated Ca2+/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and beta-arrestin recruitment inhibit CCL2 production by blocking both NF-kappaB activation and Ca2+/calcineurin-dependent signaling pathways.

Tissue factor (TF) is the physiological activator of the coagulation cascade that plays pathophysiological roles in metastasis, angiogenesis, and inflammation. Downstream in coagulation, thrombin is the central protease that signals through G protein-coupled, protease-activated receptors (PARs). However, the TF-VIIa-Xa complex upstream in coagulation also activates PAR1 and 2. Here, we address the question of whether signaling of the TF initiation complex is a relevant pathway that leads to TF cytoplasmic domain phosphorylation. In heterologous expression systems and primary endothelial cells, we demonstrate that the ternary TF-VIIa-Xa complex induces TF phosphorylation specifically by activating PAR2 but not through PAR1 signaling. In addition, TF cytoplasmic domain phosphorylation is induced only by TF-dependent signaling but not by other coagulation factors in endothelial cells. Phosphorylation of the Pro-directed kinase target site Ser258 is dependent on prior phosphorylation of Ser253 by protein kinase C (PKC) alpha. TF phosphorylation is somewhat delayed and coincides with sustained PKCalpha activation downstream of PAR2 but not PAR1 signaling. Phosphatidylcholine-dependent phospholipase C is the major pathway that leads to prolonged PKCalpha recruitment downstream of PAR2. Thus, PAR2 signaling specifically phosphorylates TF in a receptor cross-talk that distinguishes upstream from downstream coagulation protease signaling.

Objective—Leukotriene B4(LTB4), a potent leukocyte chemoattractant, is known to promote several inflammatory diseases, including atherosclerosis. We sought to determine mechanisms through which LTB4modulates atherosclerosis in cell lines expressing LTB4receptors, BLT-1, and in mice deficient in BLT-1 as well as macrophage cell lines derived from BLT-1+/+and BLT-1−/−mice.Methods and Results—Analysis of global changes in gene expression induced by LTB4in rat basophilic leukemia cells (RBL-2H3) expressing the human BLT-1 showed highest-fold increase in expression of fatty acid translocase/CD36 and the chemokine MCP1/JE/CCL2 , which are critical in atherogenesis. To determine the importance of BLT-1 in atherogenesis, we crossed BLT-1-null mice with apolipoprotein (apo)-E-deficient mice, which develop severe atherosclerosis. Deletion of BLT-1 significantly reduced the lesion formation in apo-E−/−mice only during initiating stages (4 and 8 weeks) but had no effect on the lesion size in mice fed atherogenic diet for 19 weeks. Macrophage cell lines from BLT-1-deficient mice expressed the low-affinity LTB4receptor, BLT-2, and exhibited chemotaxis to LTB4.Conclusions—The effects of LTB4in atherosclerosis are likely mediated through the high-affinity BLT-1 and the low-affinity BLT-2 receptors. LTB4promotes atherosclerosis by chemo-attracting monocytes, by providing an amplification loop of monocyte chemotaxis via CCL2 production, and by converting monocytes to foam cells by enhanced expression of CD36 and fatty acid accumulation.

Platelet activating factor (PAF) interacts with cell surface G protein-coupled receptors on leukocytes to induce degranulation, leukotriene C(4) (LTC(4)) generation, and chemokine CCL2 production. Using a basophilic leukemia RBL-2H3 cell line expressing wild-type PAF receptor (PAFR) and a phosphorylation-deficient mutant (mPAFR), we have previously demonstrated that receptor phosphorylation mediates desensitization of PAF-induced degranulation. Here, we sought to determine the role of receptor phosphorylation on PAF-induced LTC(4) generation and CCL2 production. We found that PAF caused a significantly enhanced LTC(4) generation in cells expressing mPAFR when compared with PAFR cells. In contrast, PAF-induced CCL2 production was greatly reduced in mPAFR cells. Pertussis toxin and U0126, which inhibit G(i) and p44/42 mitogen-activated protein kinase (ERK) activation, respectively, caused very little inhibition of PAF-induced CCL2 production (approximately 20% inhibition). In contrast, these inhibitors almost completely blocked both PAF-induced ERK phosphorylation and LTC(4) generation in PAFR cells. However, in mPAFR cells pertussis toxin only partially inhibited PAF-induced ERK phosphorylation. A Ca(2+)/calmodulin inhibitor had no effect on PAF-induced ERK phosphorylation in PAFR cells but completely blocked the response in mPAFR cells. These data demonstrate that receptor phosphorylation, which serves to desensitize PAF-induced LTC(4) generation, is required for chemokine CCL2 production. They also indicate a previously unrecognized selectivity in G protein usage and ERK activation for PAF-induced responses. Whereas PAF-induced CCL2 production is, in large part, mediated independently of G(i) activation or ERK phosphorylation, LTC(4) generation requires ERK phosphorylation, which is mediated by different G proteins depending on the phosphorylation status of the receptor.

Human immunodeficiency virus (HIV) infection is an independent risk factor for cardiovascular disease. This risk is magnified by certain antiretrovirals, particularly the protease inhibitor ritonavir, but the pathophysiology of this connection is unknown. We postulated that a major mechanism for antiretroviral-associated cardiac disease is pathologic fibrosis linked to platelet activation with release and activation of transforming growth factor (TGF)-β1, and that these changes could be modeled in a murine system. We also sought to intervene utilizing inhaled carbon monoxide (CO) as proof-of-concept for therapeutics capable of regulating TGF-β1 signaling and collagen autophagy. We demonstrate decreased cardiac function indices, including cardiac output, ejection fraction and stroke volume, and prominent cardiac fibrosis, in mice exposed to pharmacological doses of ritonavir. Cardiac output and fibrosis correlated with plasma TGF-β1 levels. Mice with targeted deletion of TGF-β1 in megakaryocytes/platelets (PF4CreTgfb1flox/flox) were partially protected from ritonavir-induced cardiac dysfunction and fibrosis. Inhalation of low dose CO (250ppm), used as a surrogate for upregulation of inducible heme oxygenase/endogenous CO pathways, suppressed ritonavir-induced cardiac fibrosis. This occurred in association with modulation of canonical (Smad2) and non-canonical (p38) TGF-β1 signaling pathways. In addition, CO treatment suppressed the M1 pro-inflammatory subset of macrophages and increased M2c regulatory cells in the hearts of RTV-exposed animals. The effects of CO were dependent upon autophagy as CO did not mitigate ritonavir-induced fibrosis in autophagy-deficient LC3-/- mice. These results suggest that platelet-derived TGF-β1 contributes to ritonavir-associated cardiac dysfunction and fibrosis, extending the relevance of our findings to other antiretrovirals that also activate platelets. The anti-fibrotic effects of CO are linked to alterations in TGF-β1 signaling and autophagy, suggesting a proof-of-concept for novel interventions in HIV/antiretroviral therapy-mediated cardiovascular disease.

Bone marrow-derived inflammatory cells, including platelets, may contribute to the progression of pressure overload-induced left ventricular hypertrophy (LVH). However, the underlying mechanisms for this are still unclear. One potential mechanism is through release of granule cargo. Unc13-d Jinx (Jinx) mice, which lack Munc13-4, a limiting factor in vesicular priming and fusion, have granule secretion defects in a variety of hematopoietic cells, including platelets. In the current study, we investigated the role of granule secretion in the development of LVH and cardiac remodeling using chimeric mice specifically lacking Munc13-4 in marrow-derived cells. Pressure overload was elicited by transverse aortic constriction (TAC). Chimeric mice were created by bone marrow transplantation. Echocardiography, histology staining, immunohistochemistry, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and mass spectrometry were used to study LVH progression and inflammatory responses. Wild-type (WT) mice that were transplanted with WT bone marrow (WT→WT) and WT mice that received Jinx bone marrow (Jinx→WT) developed LVH and a classic fetal reprogramming response early (7 days) after TAC. However, at late times (5 wk), mice lacking Munc13-4 in bone marrow-derived cells (Jinx→WT) failed to sustain the cardiac hypertrophy observed in WT chimeric mice. No difference in cardiac fibrosis was observed at early or late time points. Reinjection of WT platelets or platelet releasate partially restored cardiac hypertrophy in Jinx chimeric mice. These results suggest that sustained LVH in the setting of pressure overload depends on one or more factors secreted from bone marrow-derived cells, possibly from platelets. Inhibiting granule cargo release may represent a novel target for preventing sustained LVH.