ALBERT

Description: Bacterial sepsis triggers robust activation of the complement system with subsequent generation of anaphylatoxins (C3a, C5a) and the terminal complement complex (TCC) that together contribute to organ failure and death. Here we tested the effect of RA101295, a 2-kDa macrocyclic peptide inhibitor of C5 cleavage, using in vitro whole-blood assays and an in vivo baboon model of Escherichia coli sepsis. RA101295 strongly inhibited E. coli-induced complement activation both in vitro and in vivo by blocking the generation of C5a and the soluble form of TCC, sC5b-9. RA101295 reduced the E. coli-induced “oxidative burst,” as well as leukocyte activation, without affecting host phagocytosis of E. coli. RA101295 treatment reduced plasma LPS content in E. coli-challenged baboons, implying reduced complement-mediated bacteriolysis, whereas treated animals showed slightly improved bacterial clearance during the bacteremic stage compared with controls. Treatment with RA101295 also improved consumptive coagulopathy and preserved endothelial anticoagulant and vascular barrier functions. RA101295 abolished sepsis-induced surges in proinflammatory cytokines and attenuated systemic circulatory and febrile responses, likely reflecting decreased systemic levels of LPS and C5a. Overall, RA101295 treatment was associated with significant organ protection and markedly reduced mortality compared with nontreated controls (four of five animals survived in a 100% lethal model). We therefore conclude that inhibition of C5 cleavage during the bacteremic stage of sepsis could be an important therapeutic approach to prevent sepsis-induced inflammation, consumptive coagulopathy, and subsequent organ failure and death.

Description: Thrombosis and cardiovascular disease (CVD) represent major causes of morbidity and mortality. Low androgen correlates with higher incidence of CVD/thrombosis. Tissue Factor Pathway Inhibitor (TFPI) is the major inhibitor of tissue factor-factor VIIa (TF-FVIIa)–dependent FXa generation. Because endothelial cell (EC) dysfunction leading to vascular disease correlates with low EC-associated TFPI, we sought to identify mechanisms that regulate the natural expression of TFPI. Data mining of NCBI's GEO microarrays revealed strong coexpression between TFPI and the uncharacterized protein encoded by C6ORF105, which is predicted to be multispan, palmitoylated and androgen-responsive. We demonstrate that this protein regulates both the native and androgen-enhanced TFPI expression and activity in cultured ECs, and we named it androgen-dependent TFPI-regulating protein (ADTRP). We confirm ADTRP expression and colocalization with TFPI and caveolin-1 in ECs. ADTRP-shRNA reduces, while over-expression of ADTRP enhances, TFPI mRNA and activity and the colocalization of TF-FVIIa–FXa-TFPI with caveolin-1. Imaging and Triton X-114–extraction confirm TFPI and ADTRP association with lipid rafts/caveolae. Dihydrotestosterone up-regulates TFPI and ADTRP expression, and increases FXa inhibition by TFPI in an ADTRP- and caveolin-1-dependent manner. We conclude that the ADTRP-dependent up-regulation of TFPI expression and activity by androgen represents a novel mechanism of increasing the anticoagulant protection of the endothelium.

Description: Caveolin-1 (CAV1) is a scaffolding protein that is essential for the formation of caveolae membrane domains, functions as a master-regulator of signaling molecules in caveolae, and has major role in the regulation of endothelial nitric oxide synthase (eNOS). Although it is thought that caveolins play some immunomodulatory roles, the actual function of CAV1 with respect to innate immunity in response to bacterial challenge is not clear. The aim of our study was to examine the in vivo role of CAV1 in a mouse model of endotoxemia. CAV1 knock out (ko) and wild-type (WT) mice were intravenously injected with LD80 of E. coli lipopolysaccharide (LPS, 1 mg/kg). Assessment of mortality during 7 days showed that the survival rate of CAV1 ko mice was significantly higher than WT mice (46% vs. 19%). Another group of mice similarly injected with LPS were sacrificed after 2, 8 and 24 hrs, at which times we analyzed multiple parameters of the inflammatory, NO production and coagulation responses in plasma and tissues. Non-challenged CAV1 ko mice have slightly increased numbers of leukocytes comparing to WT mice. After LPS challenge, the amount of circulating leukocytes decreased at 2 hrs in both genotypes, but were significantly lower in CAV1 ko vs. WT mice and correspondingly increased into the tissues (e.g. lung). Interestingly, non-challenged CAV1 ko mice displayed slightly but significantly higher number of neutrophils in the lung than WT mice. After LPS challenge, neutrophils migration increased dramatically at 2 and 24 hrs in WT, but not in CAV1 ko mice. This was paralleled by an increase in myeloperoxidase (a neutrophil product) in WT vs. CAV1 ko mice at 2 hrs and 24 hrs post-challenge. The levels of several cytokines (GM-CSF, IL-1b, IL-5, IL-6, IL-12) were significantly lower in LPS-treated CAV1 ko versus the corresponding WT mice. Only TNF-alpha peaked in CAV1 ko vs. WT mice at 2 hrs post challenge, then decreased at 24 hrs. Several chemokines (KC, IP-10, MCP1, MIG, MIP1a) were transiently upregulated at 2 hrs and diminished at 24 hrs during endotoxemia, but no significant difference between groups was observed. NO generation in response to LPS was evaluated by measuring nitrite/nitrate production in plasma and tissues. While CAV1 ko mice generated higher NO levels during the early stages of sepsis, likely due to increased eNOS function, WT mice displayed 4-fold increase in nitrite/nitrate at 24 hrs, due to a significant upregulation of inducible NOS (iNOS) in tissue leukocytes, as visualized by immunocytochemistry. We observed a temporal correlation between WT mice morbidity and NO generation during the late stages of endotoxemia. Consistent with their increased survival, CAV1 ko mice had lower nitrite/nitrate levels. Non-challenged CAV1 ko mice already have increased tissue factor activity in the lung comparing to WT mice, which further increased at 8 hrs post LPS injection. The resulting procoagulant state was also reflected by elevated levels of thrombin-antithrombin complexes, decreased fibrinogen in plasma, increased fibrin deposition in the lung, increased D-dimmer (peak at 24 hrs after LPS administration) and soluble thrombomodulin plasma levels, as compared with the WT animals. In conclusion, our data reveal that CAV1 ko mice have a more procoagulant but less inflammatory phenotype, which may confer them better survival during endotoxemic challenge. The mechanisms underlying the increased procoagulant and anti-inflammatory functions in CAV1-deficient mice remain to be determined.

Description: The assembly of tissue factor-factor VIIa (TF-FVIIa) complex results in the proteolytic activation of factors X (FX) and IX and ultimate thrombin generation. The inhibition of this pathway by the Kunitz-type inhibitor, tissue factor pathway inhibitor (TFPI), involves the formation of a stable TF-FVIIa-FXa-TFPI complex. TFPI in endothelial cells (EC) locates primarily in rafts and caveolae, which are membrane microdomains enriched in cholesterol, glycosphingolipids (GSL) and caveolins, and which regulate the function of TFPI. Since caveolin-1 supports the TFPI-dependent inhibition of TF-FVIIa, we aimed to decipher the role played by the individual components of rafts in the anticoagulant function of cell surface TFPI. To this end, we studied the distribution of TFPI, TF and caveolin-1 by immunofluorescence microscopy, and we assayed the functional activity of TFPI after cholesterol-complexation on EC (EA. hy926 and HUVEC) and HEK293 expressing TFPI or TFPI+caveolin-1, or we used GSL-deficient CHO mutant cell lines. In EC, cholesterol complexation with filipin led to patching of TFPI over the cell surface and reduced inhibition of TF-FVIIa. Extraction of cholesterol from the external leaflet of the membrane with methyl-β-cyclodextrin (M-β-CD) shifted the partition of TFPI from predominantly raft-associated to the non-raft cellular fractions isolated through temperature-induced phase separation of Triton X-114 lysates. Although activation of FX by TF-FVIIa was significantly enhanced by M-β-CD and reversed after cholesterol replenishment, the effect was only modestly affected by the TFPI activity reduction. By immunofluorescence we observed that M-β-CD produced redistribution of both TFPI and TF over the EC and 293 cell surface with apparent segregation into separate domains and complete lack of co-localization. Such accumulations of TF will likely promote strong procoagulant activity when not inhibited by TFPI. Since M-β-CD selectively disrupts the glycerophospholipid-rich regions of the membrane while leaving the caveolar cholesterol virtually intact, we also tested progesterone, which extracts cholesterol specifically from caveolae. Treatment of HEK293 cells with progesterone for 2 hrs reduced significantly the inhibition of TF-FVIIa-dependent activation of FX by TFPI for TFPI+Cav+ cells but not for TFPI+ cells, suggesting that the process was specific for cells that have caveolae. To study the role of GSL for the activity of TFPI, we used Ly-B cells, a GSL-deficient mutant derived from CHO-K1, which have a defect in the LCB1 subunit of serine palmitoyltransferase. Characterization of endogenous TFPI in CHO-K1, Ly-B and its genetically corrected revertant Ly-B/cLCB1 (cLCB) revealed strong similarities between CHOs and EC with regard to the expression and function of TFPI. Whereas not affecting cLCB cells, incubation of Ly-B for 2 days in sphingolipid-deficient medium shifted the partition of cellular TFPI from the detergent-soluble (rafts) to the water-soluble (non-raft) fraction, which suggests that GSL play a major role in the distribution and function of the membrane TFPI. The fundamental knowledge developed by these studies will improve our understanding of the mechanisms by which TFPI functions against TF-FVIIa procoagulant activity on cell surfaces. In the long term, they may guide novel therapeutic approaches to prevent inflammation and thrombosis.

Description: Severe sepsis leads to massive activation of coagulation and complement cascades that could contribute to multiple organ failure and death. To investigate the role of the complement and its crosstalk with the hemostatic system in the pathophysiology and therapeutics of sepsis, we have used a potent inhibitor (compstatin) administered early or late after Escherichia coli challenge in a baboon model of sepsis-induced multiple organ failure. Compstatin infusion inhibited sepsis-induced blood and tissue biomarkers of complement activation, reduced leucopenia and thrombocytopenia, and lowered the accumulation of macrophages and platelets in organs. Compstatin decreased the coagulopathic response by down-regulating tissue factor and PAI-1, diminished global blood coagulation markers (fibrinogen, fibrin-degradation products, APTT), and preserved the endothelial anticoagulant properties. Compstatin treatment also improved cardiac function and the biochemical markers of kidney and liver damage. Histologic analysis of vital organs collected from animals euthanized after 24 hours showed decreased microvascular thrombosis, improved vascular barrier function, and less leukocyte infiltration and cell death, all consistent with attenuated organ injury. We conclude that complement-coagulation interplay contributes to the progression of severe sepsis and blocking the harmful effects of complement activation products, especially during the organ failure stage of severe sepsis is a potentially important therapeutic strategy.

Description: We used in vitro and in vivo models to characterize the physiological role of the novel protein encoded by C6ORF105. This gene's expression is androgen-responsive, and the encoded protein is predicted to be palmitoylated and membrane multi-spanning. Previously we showed that C6ORF105 expression co-regulates with tissue factor pathway inhibitor (TFPI)in human endothelial cells (EC); hence we named this protein "androgen-dependent TFPI-regulating protein" (ADTRP). Using in vitro cell-based TOP-Flash reporter assay we identified ADTRP as a negative regulator of canonical Wnt signaling in human cells. Overexpressing ADTRP in HEK293T cells inhibited the activity of beta-catenin/TCF-dependent transcriptional reporter, while silencing ADTRP increased the expression of Wnt target genes LEF-1, AXIN-2, IL-8 and DKK-2 in EA.hy926 EC line and HUVEC. Addition of LiCl showed that the effect of ADTRP was upstream of GSK3, therefore we focused the investigations on the Wnt signalosome proteins. ADTRP expression in HEK293T cells led to decreased phosphorylation of Wnt co-receptor LRP6, suggesting that ADTRP can affect this critical membrane-located event of Wnt signaling. Furthermore, ADTRP expression in reporter cells transfected with a constitutively phosphorylated form of LRP6 (LRP6DN mutant) inhibited Wnt3a- induced signaling, which suggests that ADTRP can interfere with events downstream of LRP6 phosphorylation, such as Axin-2 binding. Altogether, these data indicate that the Wnt signaling inhibitory activity of ADTRP takes place at the plasma membrane level. Site directed mutagenesis of the predicted palmitoylation site Cys61 showed that Wnt inhibitory effects of ADTRP require palmitoyl-mediated anchoring, highlighting the importance of proper membrane location of ADTRP for Wnt pathway inhibition. In vivo morpholino-based knockdown of adtrp in zebrafish embryos produced aberrant angiogenesis, defective branching and ruptured vessels, hemorrhage spots, pericardial edema and slow heart-beat, all reminiscent of defects caused by activation of canonical Wnt signaling. Indeed, adtrp knock down increased Wnt mediated lef-1 and pax-2a as well as mmp2 and mmp9 mRNA expression. Co-injection of ADTRP mRNA partially recovered the adtrp morpholino- induced morphologic abnormalities. Also, knock down of adtrp in a Wnt reporter zebrafish showed increased expression of ectopic Wnt signaling. Furthermore, our recently established Adtrp-/- mice also display some typical Wnt-mediated vascular defects, including: (i) abnormal patterning, increased capillary tortuosity, abnormal branching and increased density of the capillary network; (ii) dilated vessels, especially venules and veins; (iii) increased leakeage of permeability tracers (Evans blue and fluorescent dextran) without evident changes in endothelial junctions; (iv) hemorrhage spots in the skin, meningeal layers, heart, bladder and kidneys; (v) intravascular and interstitial fibrin deposition in the lung, liver and kidney. ADTRP deficiency decreased plasma TFPI antigen by ~2-times. Furthermore, TFPI antigen and anticoagulant activity in lung extracts and isolated lung EC were similarly decreased, which confirms our previous in vitro data. We aslo noticed increased tail bleeding time (〉500 sec vs. 200 sec in WT littermates) and blood volume loss, which likely was caused by increased dilation of the tail vein. Gene expression analysis of whole organs showed upregulation of Wnt target genes involved in vascular contractility (Nos3), and extracellular matrix remodeling (Mmp2). Similarly, skin fibroblasts and lung EC isolated from Adtrp-/- mice showed increased expression of Wnt target genes (Lef-1, Cyclin D, Dkk2, c-Myc), which indicates constitutive activation of canonical Wnt signaling. In conclusion, we used genetic animal models and cell culture systems to show for the first time that the novel protein ADTRP plays major roles in vascular development and function. Lack of, or low levels of ADTRP associate with activation of coagulation and vascular development defects, which may be due, at least in part, to intrinsic high levels of ectopic canonical Wnt signaling. Disclosures No relevant conflicts of interest to declare.

Description: Bacterial sepsis induces strong activation of coagulation, complement and fibrinolytic systems that contribute to disseminated intravascular coagulation, organ damage and death. While the contact of the blood with pathogens or pathogen-associated molecular patterns (PAMPs) can trigger the activation of both systems, a bidirectional complement-coagulation crosstalk is believed to occur. Although the role of complement activation products as positive regulators of coagulation is documented, direct activation of the complement proteins by thrombin or other hemostatic proteases was alluded but not demonstrated in vivo. Here we aimed to: (i) determine if in vivo generation of thrombin and other hemostatic proteases can activate the complement proteins and (ii) discriminate between the direct effect of the pathogen/PAMPs vs. hemostatic proteases on complement activation in a clinically relevant model of sepsis. We have compared the time-course of complement activation markers (C3b, C5a and C5b-9 terminal complex) in plasma of baboons exposed to 1010 cfu/kg (LD100) E. coli vs. intravenous infusion of factor Xa/PC:PS, a potent procoagulant stimulus. In baboons challenged with LD100 E. coli, complement activation markers C3b, C5a and C5b-9 reached maximum levels after 2 hrs (see figure). Complement activation coincided with the peak of bacteremia and LPS, but not with markers of thrombin generation (TAT and fibrinogen consumption; see figure) or fibrinolysis (FDP, D-dimers), which reached peak levels after 6 hours. Differently, infusion of FXa/PC:PS (36.6 pmol/L FXa and 56.3 nmol/L PC/PS per kg body weight) induced a rapid burst of thrombin and almost full consumption of fibrinogen during the first 10 min post-infusion, with no increase of complement activation markers. Based on these data we conclude that in vivo activation of the coagulation cascade does not support complement activation as was postulated by previous in vitro studies. Therefore, we conclude that pathogens and PAMPs are the main activators of the complement during sepsis while direct activation by hemostatic proteases is minor or absent. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Description: Key Points In vivo–generated thrombin and plasmin do not contribute to complement activation in nonhuman primates. Bacteria and lipopolysaccharide are the main drivers of in vivo complement activation in E coli sepsis in baboons.

Description: Abstract 348 Cardiovascular disease (CVD) and thrombotic complications (deep vein thrombosis/venous thromboembolism, DVT/VTE) represent major health problems, with men having higher rates of clinical events than women. Tissue Factor Pathway Inhibitor (TFPI) is the key natural inhibitor of coagulation: it neutralizes factor Xa (FXa) and inhibits tissue factor-factor VIIa (TF-FVIIa) in the presence of FXa. In vivo most of TFPI is in endothelial cells (EC), reversibly bound to yet unidentified receptors, and glycosyl phosphatidylinositol-floated in caveolae and/or lipid raft microdomains. Intravascular thrombosis occurs frequently in older people, especially associated with cancer, diabetes, or CVD. TF is directly involved in tumor hypercoagulability, angiogenesis and metastasis. Cell-associated TFPI is the most physiologically significant inhibitor of the TF-FVIIa- triggered coagulation pathway; nevertheless, very few mechanisms/factors that could regulate the natural expression of TFPI have been identified so far. Here we describe androgen treatment of EC as a novel way to preserve and/or enhance a healthy vascular function, particularly related to the regulation of TFPI-dependent anticoagulant function of the endothelium. Our hypothesis is that a yet uncharacterized protein encoded by C6orf105 is a novel regulator of TFPI expression and function in EC, both in native conditions and during androgen stimulation. “In silico” data mining using global meta-analysis of publicly available NCBI's Gene Expression Omnibus 2-channel human microarray datasets identified C6orf105 as highly co-expressed with TFPI and following a parallel co-regulation. The uncharacterized protein has 230-aa, Mr ∼27 kDa, 5–6 predicted transmembrane domains and has sequence similarities with members of the androgen-inducible genes family. We tentatively named it TFPI-Regulating Factor (TFPI-RF). Real-time qPCR and western blot confirmed robust expression of TFPI-RF in EC in culture (HUVEC and EA.hy926 hybrid cell line). By immunofluorescence (IMF) TFPI-RF appears both on the cell surface and intracellularly co-localizing with TFPI and caveolin-1 (cav-1). Post-transcriptional (siRNA) down-regulation of TFPI-RF decreased TFPI, both as protein (∼2-times) and as anticoagulant activity (∼3-fold), apparently by reducing the co-localization of the TF-FVIIa-FXa-TFPI complex with cav-1. Over-expression of TFPI-RF in HUVEC and EA.hy926 led to enhanced co-localization of TFPI-RF with TFPI, and increased TFPI mRNA and anticoagulant activity (∼2-times). Western blot of cellular fractions after extraction with Triton X-114 and temperature-induced phase separation revealed the presence of TFPI and TFPI-RF in detergent-insoluble fractions, which suggests predominant lipid raft association. IMF illustrates TFPI-RF co-clustering with TFPI and cav-1 or GM1 (raft marker) in live EC incubated with anti-TFPI antibody or Cholera Toxin-B, respectively. The effect of androgens was studied by incubating EC with 30 nM dehydrotestosterone (DHT) or equivalent testosterone-BSA (cell-impermeable). 1-h incubation led to 2-times enhanced TFPI activity, increased co-localization of the quaternary complex with cav-1 and TFPI-RF, and enhanced exposure of TFPI and TFPI-RF on the cell surface. 24-h treatment with DHT up-regulates the expression of both TFPI (2-fold) and TFPI-RF (3-fold), as well as the TFPI inhibitory activity against FXa. DHT failed to enhance TFPI activity in TFPI-RF siRNA EC. Our results reveal a novel mechanism of up-regulation of the anticoagulant activity of endogenous TFPI in response to physiological levels of androgen. While the precise role of androgens in the ageing process is unclear, it is believed that androgen replacement could have beneficial influence on the declining functions in the elderly. Our data could expand on the effects of androgens on the haemostatic function of the endothelium and discover new roles for novel proteins like C6orf105/TFPI-RF in enhancing the endothelial anticoagulant function. These may open possibilities to manipulate the cellular endogenous TFPI and/or other intrinsic factors to counteract pro-thrombotic states associated with CVD, DVT/VTE, sepsis and cancer. Disclosures: No relevant conflicts of interest to declare.