Publications by authors named "Cristina Lupu"

24 Publications

  • Page 1 of 1

Complement C5 inhibition protects against hemolytic anemia and acute kidney injury in anthrax peptidoglycan-induced sepsis in baboons.

Proc Natl Acad Sci U S A 2021 Sep;118(37)

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104;

Late-stage anthrax infections are characterized by dysregulated immune responses and hematogenous spread of , leading to extreme bacteremia, sepsis, multiple organ failure, and, ultimately, death. Despite the bacterium being nonhemolytic, some fulminant anthrax patients develop a secondary atypical hemolytic uremic syndrome (aHUS) through unknown mechanisms. We recapitulated the pathology in baboons challenged with cell wall peptidoglycan (PGN), a polymeric, pathogen-associated molecular pattern responsible for the hemostatic dysregulation in anthrax sepsis. Similar to aHUS anthrax patients, PGN induces an initial hematocrit elevation followed by progressive hemolytic anemia and associated renal failure. Etiologically, PGN induces erythrolysis through direct excessive activation of all three complement pathways. Blunting terminal complement activation with a C5 neutralizing peptide prevented the progressive deposition of membrane attack complexes on red blood cells (RBC) and subsequent intravascular hemolysis, heme cytotoxicity, and acute kidney injury. Importantly, C5 neutralization did not prevent immune recognition of PGN and shifted the systemic inflammatory responses, consistent with improved survival in sepsis. Whereas PGN-induced hemostatic dysregulation was unchanged, C5 inhibition augmented fibrinolysis and improved the thromboischemic resolution. Overall, our study identifies PGN-driven complement activation as the pathologic mechanism underlying hemolytic anemia in anthrax and likely other gram-positive infections in which PGN is abundantly represented. Neutralization of terminal complement reactions reduces the hemolytic uremic pathology induced by PGN and could alleviate heme cytotoxicity and its associated kidney failure in gram-positive infections.
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http://dx.doi.org/10.1073/pnas.2104347118DOI Listing
September 2021

Disseminated intravascular coagulation and its immune mechanisms.

Blood 2021 Aug 24. Epub 2021 Aug 24.

United States.

Disseminated intravascular coagulation (DIC) is a syndrome triggered by infectious and non-infectious pathologies characterized by excessive generation of thrombin within the vasculature and widespread proteolytic conversion of fibrinogen. Despite diverse clinical manifestations ranging from thrombo-occlusive damage to bleeding diathesis, DIC etiology commonly involves excessive activation of blood coagulation and overlapping dysregulation of anticoagulants and fibrinolysis. Initiation of blood coagulation follows intravascular expression of tissue factor or activation of contact pathway in response to pathogen-associated or host derived damage-associated molecular patterns. The process is further amplified through inflammatory and immuno-thrombotic mechanisms. Consumption of anticoagulants and disruption of endothelial homeostasis lower the regulatory control and disseminate microvascular thrombosis. Clinical DIC development in patients associates with worsening morbidities and increased mortality regardless of the underlying pathology, therefore timely recognition of DIC is critical to reduce the pathologic burden. Due to diversity of triggers and pathogenic mechanisms leading to DIC, diagnosis is based on algorithms that quantify hemostatic imbalance, thrombocytopenia and fibrin/ogen conversion. Since current diagnosis primarily assesses overt consumptive coagulopathies, there is a critical need for better recognition of non-overt DIC and/or pre-DIC states. Therapeutic strategies for DIC patients involve resolution of the eliciting triggers and supportive care for the hemostatic imbalance. Despite medical care, mortality in DIC patients remains high and new strategies, tailored to the underlying pathologic mechanisms, are needed.
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http://dx.doi.org/10.1182/blood.2020007208DOI Listing
August 2021

Acceleration of Small Intestine Development and Remodeling of the Microbiome Following Hyaluronan 35 kDa Treatment in Neonatal Mice.

Nutrients 2021 Jun 12;13(6). Epub 2021 Jun 12.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.

The beneficial effects of human milk suppressing the development of intestinal pathologies such as necrotizing enterocolitis in preterm infants are widely known. Human milk (HM) is rich in a multitude of bioactive factors that play major roles in promoting postnatal maturation, differentiation, and the development of the microbiome. Previous studies showed that HM is rich in hyaluronan (HA) especially in colostrum and early milk. This study aims to determine the role of HA 35 KDa, a HM HA mimic, on intestinal proliferation, differentiation, and the development of the intestinal microbiome. We show that oral HA 35 KDa supplementation for 7 days in mouse pups leads to increased villus length and crypt depth, and increased goblet and Paneth cells, compared to controls. We also show that HA 35 KDa leads to an increased predominance of Clostridiales Ruminococcaceae, Lactobacillales Lactobacillaceae, and Clostridiales Lachnospiraceae. In seeking the mechanisms involved in the changes, bulk RNA seq was performed on samples from the terminal ileum and identified upregulation in several genes essential for cellular growth, proliferation, and survival. Taken together, this study shows that HA 35 KDa supplemented to mouse pups promotes intestinal epithelial cell proliferation, as well as the development of Paneth cells and goblet cell subsets. HA 35 KDa also impacted the intestinal microbiota; the implications of these responses need to be determined.
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http://dx.doi.org/10.3390/nu13062030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231646PMC
June 2021

Is the AO spine thoracolumbar injury classification system reliable and practical? a systematic review.

Acta Orthop Belg 2021 Mar;87(1):181-190

Controversy surrounding the classification of thoracolumbar injuries has given rise to various classification systems over the years, including the most recent AOSpine Thoracolumbar Injury Classification System (ATLICS). This systematic review aims to provide an up-to-date evaluation of the literature, including assessment of a further three studies not analysed in previous reviews. In doing so, this is the first systematic review to include the reliability among non-spine subspecialty professionals and to document the wide variety between reliability across studies, particularly with regard to sub-type classification. Relevant studies were found via a systematic search of PubMed, EBESCO, Cochrane and Web of Science. Data extraction and quality assessment were conducted in line with Cochrane Collaboration guidelines. Twelve articles assessing the reliability of ATLICS were included in this review. The overall inter-observer reliability varied from fair to substantial, but the three additional studies in this review, compared to previous reviews, presented on average only fair reliability. The greatest variation of results was seen in A1 and B3 subtypes. Least reliably classified on average was A4 subtype. This systematic review concludes that ATLICS is reliable for the majority of injuries, but the variability within subtypes suggests the need for further research in assessing the needs of users in order to increase familiarity with ATLICS or perhaps the necessity to include more subtype-specific criteria into the system. Further research is also recommended on the reliability of modifiers, neurological classification and the application of ATLICS in a paediatric context.
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March 2021

Insights into the Functional Role of ADTRP (Androgen-Dependent TFPI-Regulating Protein) in Health and Disease.

Int J Mol Sci 2021 Apr 24;22(9). Epub 2021 Apr 24.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.

The novel protein ADTRP, identified and described by us in 2011, is androgen-inducible and regulates the expression and activity of Tissue Factor Pathway Inhibitor, the major inhibitor of the Tissue Factor-dependent pathway of coagulation on endothelial cells. Single-nucleotide polymorphisms in ADTRP associate with coronary artery disease and myocardial infarction, and deep vein thrombosis/venous thromboembolism. Some athero-protective effects of androgen could exert through up-regulation of expression. We discovered a critical role of ADTRP in vascular development and vessel integrity and function, manifested through Wnt signaling-dependent regulation of matrix metalloproteinase-9. ADTRP also hydrolyses fatty acid esters of hydroxy-fatty acids, which have anti-diabetic and anti-inflammatory effects and can control metabolic disorders. Here we summarize and analyze the knowledge on ADTRP and try to decipher its functions in health and disease.
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http://dx.doi.org/10.3390/ijms22094451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123165PMC
April 2021

Factor XII plays a pathogenic role in organ failure and death in baboons challenged with Staphylococcus aureus.

Blood 2021 07;138(2):178-189

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK.

Activation of coagulation factor (F) XI promotes multiorgan failure in rodent models of sepsis and in a baboon model of lethal systemic inflammation induced by infusion of heat-inactivated Staphylococcus aureus. Here we used the anticoagulant FXII-neutralizing antibody 5C12 to verify the mechanistic role of FXII in this baboon model. Compared with untreated control animals, repeated 5C12 administration before and at 8 and 24 hours after bacterial challenge prevented the dramatic increase in circulating complexes of contact system enzymes FXIIa, FXIa, and kallikrein with antithrombin or C1 inhibitor, and prevented cleavage and consumption of high-molecular-weight kininogen. Activation of several coagulation factors and fibrinolytic enzymes was also prevented. D-dimer levels exhibited a profound increase in the untreated animals but not in the treated animals. The antibody also blocked the increase in plasma biomarkers of inflammation and cell damage, including tumor necrosis factor, interleukin (IL)-1β, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor, nucleosomes, and myeloperoxidase. Based on clinical presentation and circulating biomarkers, inhibition of FXII prevented fever, terminal hypotension, respiratory distress, and multiorgan failure. All animals receiving 5C12 had milder and transient clinical symptoms and were asymptomatic at day 7, whereas untreated control animals suffered irreversible multiorgan failure and had to be euthanized within 2 days after the bacterial challenge. This study confirms and extends our previous finding that at least 2 enzymes of the contact activation complex, FXIa and FXIIa, play critical roles in the development of an acute and terminal inflammatory response in baboons challenged with heat-inactivated S aureus.
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http://dx.doi.org/10.1182/blood.2020009345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8288658PMC
July 2021

CD14 inhibition improves survival and attenuates thrombo-inflammation and cardiopulmonary dysfunction in a baboon model of Escherichia coli sepsis.

J Thromb Haemost 2021 02 3;19(2):429-443. Epub 2020 Dec 3.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.

Background: During sepsis, gram-negative bacteria induce robust inflammation primarily via lipopolysacharride (LPS) signaling through TLR4, a process that involves the glycosylphosphatidylinositol (GPI)-anchored receptor CD14 transferring LPS to the Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) complex. Sepsis also triggers the onset of disseminated intravascular coagulation and consumptive coagulopathy.

Objectives: We investigated the effect of CD14 blockade on sepsis-induced coagulopathy, inflammation, organ dysfunction, and mortality.

Methods: We used a baboon model of lethal Escherichia (E) coli sepsis to study two experimental groups (n = 5): (a) E coli challenge; (b) E coli challenge plus anti-CD14 (23G4) inhibitory antibody administered as an intravenous bolus 30 minutes before the E coli.

Results: Following anti-CD14 treatment, two animals reached the 7-day end-point survivor criteria, while three animals had a significantly prolonged survival as compared to the non-treated animals that developed multiple organ failure and died within 30 hours. Anti-CD14 reduced the activation of coagulation through inhibition of tissue factor-dependent pathway, especially in the survivors, and enhanced the fibrinolysis due to strong inhibition of plasminogen activator inhibitor 1. The treatment prevented the robust complement activation induced by E coli, as shown by significantly decreased C3b, C5a, and sC5b-9. Vital signs, organ function biomarkers, bacteria clearance, and leukocyte and fibrinogen consumption were all improved at varying levels. Anti-CD14 reduced neutrophil activation, cell death, LPS levels, and pro-inflammatory cytokines (tumor necrosis factor, interleukin (IL)-6, IL-1β, IL-8, interferon gamma, monocyte chemoattractant protein-1), more significantly in the survivors than non-surviving animals.

Conclusions: Our results highlight the crosstalk between coagulation/fibrinolysis, inflammation, and complement systems and suggest a protective role of anti-CD14 treatment in E coli sepsis.
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http://dx.doi.org/10.1111/jth.15162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8312235PMC
February 2021

Neutrophil extracellular trap inhibition increases inflammation, bacteraemia and mortality in murine necrotizing enterocolitis.

J Cell Mol Med 2020 Jun 8. Epub 2020 Jun 8.

Oklahoma Medical Research Foundation, Cardiovascular Biology Research Program, Oklahoma City, OK, USA.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease affecting primarily premature infants. The disease is characterized by intestinal inflammation and leucocyte infiltration, often progressing to necrosis, perforation, systemic inflammatory response and death. Neutrophil extracellular traps (NETs), denoting nuclear DNA, histone and antimicrobial protein release, have been suggested to play a role in NEC. This study aimed to determine the role of NETs in NEC and explore the effect of chloramidine, a NET inhibitor, on a murine NEC-like intestinal injury model. Blood and intestinal tissues were collected from infants diagnosed with ≥ Stage II NEC, and levels of nucleosomes and NETs, respectively, were compared with those of case-matched controls. In mice, NEC was induced with dithizone/Klebsiella, and mice in the treatment group received 40 mg/kg chloramidine. Bacterial load, intestinal histology, plasma myeloperoxidase and cytokine levels, and immunofluorescent staining were compared with controls. Nucleosomes were significantly elevated in both human and mouse NEC plasma, whereas NET staining was only present in NEC tissue in both species. Chloramidine treatment increased systemic inflammation, bacterial load, organ injury and mortality in murine NEC. Taken together, our findings suggest that NETs are critical in the innate immune defence during NEC in preventing systemic bacteraemia.
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http://dx.doi.org/10.1111/jcmm.15338DOI Listing
June 2020

Fondaparinux pentasaccharide reduces sepsis coagulopathy and promotes survival in the baboon model of Escherichia coli sepsis.

J Thromb Haemost 2020 01 16;18(1):180-190. Epub 2019 Oct 16.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma.

Background: Sepsis triggers dysfunction of coagulation and fibrinolytic systems leading to disseminated intravascular coagulation (DIC) that contributes to organ failure and death. Fondaparinux (FPX) is a synthetic pentasaccharide that binds to antithrombin (AT) and selectively inhibits factor (F) Xa and other upstream coagulation proteases but not thrombin (T).

Objectives: We used a baboon model of lethal Escherichia coli sepsis to investigate the effects of FPX treatment on DIC, organ function, and outcome.

Methods: Two experimental groups were studied: (a) E. coli challenge (n = 4); and (b) E coli plus FPX (n = 4). Bacteremia was modeled by intravenous infusion of pathogen (1-2 × 10  CFU/kg). Fondaparinux (0.08 mg/kg) was administered subcutaneously, 3 h prior to and 8 h after bacteria infusion.

Results: Bacteremia rapidly increased plasma levels of inhibitory complexes of AT with coagulation proteases. Activation markers of both intrinsic (FXIa-AT), and extrinsic (FVIIa-AT) pathways were significantly reduced in FPX-treated animals. Factor Xa-AT and TAT complexes were maximal at 4 to 8 h post challenge and reduced >50% in FPX-treated animals. Fibrinogen consumption, fibrin generation and degradation, neutrophil and complement activation, and cytokine production were strongly induced by sepsis. All parameters were significantly reduced, while platelet count was unchanged by the treatment. Fondaparinux infusion attenuated organ dysfunction, prolonged survival, and saved two of four challenged animals (log-rank Mantel-Cox test, P = .0067).

Conclusion: Our data indicate that FPX-mediated inhibition of coagulation prevents sepsis coagulopathy; protects against excessive complement activation, inflammation, and organ dysfunction; and provides survival benefit in E. coli sepsis.
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http://dx.doi.org/10.1111/jth.14642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940562PMC
January 2020

Inhibition of contact-mediated activation of factor XI protects baboons against -induced organ damage and death.

Blood Adv 2019 02;3(4):658-669

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK.

infections can produce systemic bacteremia and inflammation in humans, which may progress to severe sepsis or septic shock, even with appropriate antibiotic treatment. Sepsis may be associated with disseminated intravascular coagulation and consumptive coagulopathy. In some types of mouse infection models, the plasma coagulation protein factor XI (FXI) contributes to the pathogenesis of sepsis. We hypothesize that FXI also contributes to the pathogenesis of sepsis in primates, and that pharmacological interference with FXI will alter the outcome of -induced lethality in a baboon model. Pretreatment of baboons with the anti-FXI antibody 3G3, a humanized variant of the murine monoclonal 14E11 that blocks FXI activation by FXIIa, substantially reduced the activation of coagulation, as reflected by clotting times and plasma complexes of coagulation proteases (FXIIa, FXIa, FIXa, FXa, FVIIa, and thrombin) with serpins (antithrombin or C1 inhibitor) following infusion of heat-inactivated 3G3 treatment reduced fibrinogen and platelet consumption, fibrin deposition in tissues, neutrophil activation and accumulation in tissues, cytokine production, kininogen cleavage, cell death, and complement activation. Overall, 3G3 infusion protected the structure and function of multiple vital organs, including lung, heart, liver, and kidney. All treated animals reached the end point survival (7 days), whereas all nontreated animals developed terminal organ failure within 28 hours. We conclude that FXI plays a role in the pathogenesis of -induced disseminated intravascular coagulation and lethality in baboons. The results provide proof of concept for future therapeutic interventions that may prevent sepsis-induced organ failure and save lives in certain forms of sepsis.
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http://dx.doi.org/10.1182/bloodadvances.2018029983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391670PMC
February 2019

Role of ADTRP (Androgen-Dependent Tissue Factor Pathway Inhibitor Regulating Protein) in Vascular Development and Function.

J Am Heart Assoc 2018 11;7(22):e010690

1 Cardiovascular Biology Research Program Oklahoma Medical Research Foundation Oklahoma City OK.

Background The physiological function of ADTRP (androgen-dependent tissue factor pathway inhibitor regulating protein) is unknown. We previously identified ADTRP as coregulating with and supporting the anticoagulant activity of tissue factor pathway inhibitor in endothelial cells in vitro. Here, we studied the role of ADTRP in vivo, specifically related to vascular development, stability, and function. Methods and Results Genetic inhibition of Adtrp produced vascular malformations in the low-pressure vasculature of zebrafish embryos and newborn mice: dilation/tortuosity, perivascular inflammation, extravascular proteolysis, increased permeability, and microhemorrhages, which produced partially penetrant lethality. Vascular leakiness correlated with decreased endothelial cell junction components VE -cadherin and claudin-5. Changes in hemostasis in young adults comprised modest decrease of tissue factor pathway inhibitor antigen and activity and increased tail bleeding time and volume. Cell-based reporter assays revealed that ADTRP negatively regulates canonical Wnt signaling, affecting membrane events downstream of low-density lipoprotein receptor-related protein 6 ( LRP 6) and upstream of glycogen synthase kinase 3 beta. ADTRP deficiency increased aberrant/ectopic Wnt/β-catenin signaling in vivo in newborn mice and zebrafish embryos, and upregulated matrix metallopeptidase ( MMP )-9 in endothelial cells and mast cells ( MCs ). Vascular lesions in newborn Adtrp pups displayed accumulation of MCs , decreased extracellular matrix content, and deficient perivascular cell coverage. Wnt-pathway inhibition reversed the increased mmp9 in zebrafish embryos, demonstrating that mmp9 expression induced by Adtrp deficiency was downstream of canonical Wnt signaling. Conclusions Our studies demonstrate that ADTRP plays a major role in vascular development and function, most likely through expression in endothelial cells and/or perivascular cells of Wnt-regulated genes that control vascular stability and integrity.
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http://dx.doi.org/10.1161/JAHA.118.010690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404433PMC
November 2018

In vivo-generated thrombin and plasmin do not activate the complement system in baboons.

Blood 2017 12 11;130(24):2678-2681. Epub 2017 Oct 11.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK.

Sepsis concurrently activates both coagulation and complement systems. Although complement activation by bacteria is well documented, work in mice and in vitro suggests that coagulation proteases can directly cleave complement proteins. We aimed to determine whether generation of coagulation proteases in vivo can activate the complement cascade in 2 highly coagulopathic models. We compared temporal changes in activation biomarkers of coagulation (thrombin-antithrombin [TAT]), fibrinolysis (plasmin-antiplasmin [PAP]), and complement (C3b, C5a, C5b-9) in baboons infused with factor Xa (FXa) and phospholipids (FXa/phosphatidylcholine-phosphatidylserine [PCPS]) vs LD100 We found that, albeit with different timing, both FXa/PCPS and infusion led to robust thrombin and plasmin generation. Conversely, only challenge activated the complement system, reaching a maximum at 2 hours postchallenge during the peaks of lipopolysaccharide and bacteremia but not of TAT and PAP. Despite inducing a strong burst of thrombin and plasmin, FXa/PCPS infusion did not produce measurable levels of complement activation in vivo. Similarly, ex vivo incubation of baboon serum with thrombin, plasmin, or FXa did not show noticeable complement cleavage unless supraphysiologic amounts of enzymes were used. Our results suggest that in vivo-generated thrombin and plasmin do not directly activate the complement in nonhuman primates.
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http://dx.doi.org/10.1182/blood-2017-06-788216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5731087PMC
December 2017

Inhibition of complement C5 protects against organ failure and reduces mortality in a baboon model of sepsis.

Proc Natl Acad Sci U S A 2017 Aug 18;114(31):E6390-E6399. Epub 2017 Jul 18.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104;

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 sepsis. RA101295 strongly inhibited 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 induced oxidative burst," as well as leukocyte activation, without affecting host phagocytosis of RA101295 treatment reduced plasma LPS content in 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.
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http://dx.doi.org/10.1073/pnas.1706818114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547645PMC
August 2017

Complement inhibition decreases early fibrogenic events in the lung of septic baboons.

J Cell Mol Med 2015 Nov 3;19(11):2549-63. Epub 2015 Sep 3.

Programs in Cardiovascular Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.

Acute respiratory distress syndrome (ARDS) induced by severe sepsis can trigger persistent inflammation and fibrosis. We have shown that experimental sepsis in baboons recapitulates ARDS progression in humans, including chronic inflammation and long-lasting fibrosis in the lung. Complement activation products may contribute to the fibroproliferative response, suggesting that complement inhibitors are potential therapeutic agents. We have been suggested that treatment of septic baboons with compstatin, a C3 convertase inhibitor protects against ARDS-induced fibroproliferation. Baboons challenged with 10(9) cfu/kg (LD50) live E. coli by intravenous infusion were treated or not with compstatin at the time of challenge or 5 hrs thereafter. Changes in the fibroproliferative response at 24 hrs post-challenge were analysed at both transcript and protein levels. Gene expression analysis showed that sepsis induced fibrotic responses in the lung as early as 24 hrs post-bacterial challenge. Immunochemical and biochemical analysis revealed enhanced collagen synthesis, induction of profibrotic factors and increased cell recruitment and proliferation. Specific inhibition of complement with compstatin down-regulated sepsis-induced fibrosis genes, including transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinase 1 (TIMP1), various collagens and chemokines responsible for fibrocyte recruitment (e.g. chemokine (C-C motif) ligand 2 (CCL2) and 12 (CCL12)). Compstatin decreased the accumulation of myofibroblasts and proliferating cells, reduced the production of fibrosis mediators (TGF-β, phospho-Smad-2 and CTGF) and inhibited collagen deposition. Our data demonstrate that complement inhibition effectively attenuates collagen deposition and fibrotic responses in the lung after severe sepsis. Inhibiting complement could prove an attractive strategy for preventing sepsis-induced fibrosis of the lung.
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http://dx.doi.org/10.1111/jcmm.12667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627561PMC
November 2015

Acute lung injury and fibrosis in a baboon model of Escherichia coli sepsis.

Am J Respir Cell Mol Biol 2014 Feb;50(2):439-50

1 Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma.

Sepsis-induced inflammation of the lung leads to acute respiratory distress syndrome (ARDS), which may trigger persistent fibrosis. The pathology of ARDS is complex and poorly understood, and the therapeutic approaches are limited. We used a baboon model of Escherichia coli sepsis that mimics the complexity of human disease to study the pathophysiology of ARDS. We performed extensive biochemical, histological, and functional analyses to characterize the disease progression and the long-term effects of sepsis on the lung structure and function. Similar to humans, sepsis-induced ARDS in baboons displays an early inflammatory exudative phase, with extensive necrosis. This is followed by a regenerative phase dominated by proliferation of type 2 epithelial cells, expression of epithelial-to-mesenchymal transition markers, myofibroblast migration and proliferation, and collagen synthesis. Baboons that survived sepsis showed persistent inflammation and collagen deposition 6-27 months after the acute episodes. Long-term survivors had almost double the amount of collagen in the lung as compared with age-matched control animals. Immunostaining for procollagens showed persistent active collagen synthesis within the fibroblastic foci and interalveolar septa. Fibroblasts expressed markers of transforming growth factor-β and platelet-derived growth factor signaling, suggesting their potential role as mediators of myofibroblast migration and proliferation, and collagen deposition. In parallel, up-regulation of the inhibitors of extracellular proteases supports a deregulated matrix remodeling that may contribute to fibrosis. The primate model of sepsis-induced ARDS mimics the disease progression in humans, including chronic inflammation and long-lasting fibrosis. This model helps our understanding of the pathophysiology of fibrosis and the testing of new therapies.
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http://dx.doi.org/10.1165/rcmb.2013-0219OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3930952PMC
February 2014

Novel protein ADTRP regulates TFPI expression and function in human endothelial cells in normal conditions and in response to androgen.

Blood 2011 Oct 25;118(16):4463-71. Epub 2011 Aug 25.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.

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.
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http://dx.doi.org/10.1182/blood-2011-05-355370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3204913PMC
October 2011

Complement inhibition decreases the procoagulant response and confers organ protection in a baboon model of Escherichia coli sepsis.

Blood 2010 Aug 13;116(6):1002-10. Epub 2010 May 13.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.

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.
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http://dx.doi.org/10.1182/blood-2010-02-269746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924221PMC
August 2010

Extracellular protein disulfide isomerase regulates coagulation on endothelial cells through modulation of phosphatidylserine exposure.

Blood 2010 Aug 6;116(6):993-1001. Epub 2010 May 6.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.

Tissue factor (TF) is the cellular receptor for plasma protease factor VIIa (FVIIa), and the TF-FVIIa complex initiates coagulation in both hemostasis and thrombosis. Cell surface-exposed TF is mainly cryptic and requires activation to fully exhibit the procoagulant potential. Recently, the protein disulfide isomerase (PDI) has been hypothesized to regulate TF decryption through the redox switch of an exposed disulfide in TF extracellular domain. In this study, we analyzed PDI contribution to coagulation using an in vitro endothelial cell model. In this model, extracellular PDI is detected by imaging and flow cytometry. Inhibition of cell surface PDI induces a marked increase in TF procoagulant function, whereas exogenous addition of PDI inhibits TF decryption. The coagulant effects of PDI inhibition were sensitive to annexin V treatment, suggesting exposure of phosphatidylserine (PS), which was confirmed by prothrombinase assays and direct labeling. In contrast, exogenous PDI addition enhanced PS internalization. Analysis of fluorescent PS revealed that PDI affects both the apparent flippase and floppase activities on endothelial cells. In conclusion, we identified a new mechanism for PDI contribution to coagulation on endothelial cells, namely, the regulation of PS exposure, where PDI acts as a negative regulator of coagulation.
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http://dx.doi.org/10.1182/blood-2009-10-249607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924232PMC
August 2010

Role of PDI in regulating tissue factor: FVIIa activity.

Thromb Res 2010 Apr 16;125 Suppl 1:S38-41. Epub 2010 Feb 16.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.

Cell exposed tissue factor (TF) is generally in a low procoagulant ("cryptic") state, and requires an activation step (decryption) to exhibit its full procoagulant potential. Recent data suggest that TF decryption may be regulated by the redox environment through the oxidoreductase activity of protein disulfide isomerase (PDI). In this article we review PDI contribution to different models of TF decryption, namely the disulfide switch model and the phosphatidylserine dynamics, and hypothesize on PDI contribution to TF self-association and association with lipid domains. Experimental evidence debate the disulfide switch model of TF decryption and its regulation by PDI. More recently we showed that PDI oxidoreductase activity regulates the phosphatidylserine equilibrium at the plasma membrane. Interestingly, PDI reductase activity could maintain TF in the reduced monomeric form, while also maintaining low exposure of PS, both states correlated with low procoagulant function. In contrast, PDI inhibition or oxidants may promote the adverse effects with a net increase in coagulation. The relative contribution of disulfide isomerization and PS exposure needs to be further analyzed to understand the redox control of TF procoagulant function. For the moment however TF regulation remains cryptic.
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http://dx.doi.org/10.1016/j.thromres.2010.01.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2839035PMC
April 2010

Sepsis-induced coagulation in the baboon lung is associated with decreased tissue factor pathway inhibitor.

Am J Pathol 2007 Sep 19;171(3):1066-77. Epub 2007 Jul 19.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104, USA.

Increased tissue factor (TF)-dependent procoagulant activity in sepsis may be partly due to decreased expression or function of tissue factor pathway inhibitor (TFPI). To test this hypothesis, baboons were infused with live Escherichia coli and sacrificed after 2, 8, or 24 hours. Confocal and electron microscopy revealed increased leukocyte infiltration and fibrin deposition in the intravascular and interstitial compartments. Large amounts of TF were detected by immunostaining in leukocytes and platelet-rich microthrombi. TF induction was documented by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and coagulation assays. Lung-associated TFPI antigen and mRNA decreased during sepsis, and TFPI activity diminished abruptly at 2 hours. Blocking antibodies against TFPI increased fibrin deposition in septic baboon lungs, suggesting that TF-dependent coagulation might be aggravated by reduced endothelial TFPI. Decreased TFPI activity coincided with the release of tissue plasminogen activator and the peak of plasmin generation, suggesting that TFPI could undergo proteolytic inactivation by plasmin. Enhanced plasmin produced in septic baboons by infusion of blocking antibodies against plasminogen activator inhibitor-1 led to decreased lung-associated TFPI and unforeseen massive fibrin deposition. We conclude that activation of TF-driven coagulation not adequately countered by TFPI may underlie the widespread thrombotic complications of sepsis.
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http://dx.doi.org/10.2353/ajpath.2007.070104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1959475PMC
September 2007

Temporal dynamics of gene expression in the lung in a baboon model of E. coli sepsis.

BMC Genomics 2007 Feb 26;8:58. Epub 2007 Feb 26.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.

Background: Bacterial invasion during sepsis induces disregulated systemic responses that could lead to fatal lung failure. The purpose of this study was to relate the temporal dynamics of gene expression to the pathophysiological changes in the lung during the first and second stages of E. coli sepsis in baboons.

Results: Using human oligonucleotide microarrays, we have explored the temporal changes of gene expression in the lung of baboons challenged with sublethal doses of E. coli. Temporal expression pattern and biological significance of the differentially expressed genes were explored using clustering and pathway analysis software. Expression of selected genes was validated by real-time PCR. Cytokine levels in tissue and plasma were assayed by multiplex ELISA. Changes in lung ultrastructure were visualized by electron microscopy. We found that genes involved in primary inflammation, innate immune response, and apoptosis peaked at 2 hrs. Inflammatory and immune response genes that function in the stimulation of monocytes, natural killer and T-cells, and in the modulation of cell adhesion peaked at 8 hrs, while genes involved in wound healing and functional recovery were upregulated at 24 hrs.

Conclusion: The analysis of gene expression modulation in response to sepsis provides the baseline information that is crucial for the understanding of the pathophysiology of systemic inflammation and may facilitate the development of future approaches for sepsis therapy.
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http://dx.doi.org/10.1186/1471-2164-8-58DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1819384PMC
February 2007

Adenovirus-mediated expression of tissue factor pathway inhibitor-2 inhibits endothelial cell migration and angiogenesis.

Arterioscler Thromb Vasc Biol 2007 Feb 30;27(2):310-6. Epub 2006 Nov 30.

Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.

Objective: Extracellular matrix (ECM) remodeling during angiogenesis is accomplished through plasmin-dependent pericellular proteolysis and through the action of matrix metalloproteinases (MMPs). Because tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type protease inhibitor with prominent ECM localization, inhibits plasmin and MMPs activity, we investigated the role of TFPI-2 in endothelial cell (EC) migration and angiogenesis.

Methods And Results: Real-time polymerase chain reaction and immunostaining showed that the expression of TFPI-2 mRNA and protein was upregulated in migrating ECs. The effect of TFPI-2 on angiogenesis was studied in mouse models of Matrigel and polyvinylalcohol sponge implants by overexpressing TFPI-2 through infection with a replication-deficient adenovirus (AdTFPI-2). Using (immuno)fluorescence and confocal microscopy we observed that TFPI-2 reduced neovascularization and promoted ECM deposition. Lateral cell migration and capillary tube formation in vitro also were impaired by TFPI-2, a process reversed by anti-TFPI-2 antibodies. Increased apoptosis occurred both in AdTFPI-2-treated ECs and in the mouse implants. Zymography and assays in the absence of plasminogen confirmed plasmin inhibition as a main mechanism through which TFPI-2 inhibits EC migration.

Conclusions: Our data suggest that TFPI-2 may be an important regulator of aberrant angiogenesis associated with tumor growth/metastasis, cardiovascular diseases, chronic inflammation, or diabetes.
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http://dx.doi.org/10.1161/01.ATV.0000254147.89321.cfDOI Listing
February 2007

Tissue factor-dependent coagulation is preferentially up-regulated within arterial branching areas in a baboon model of Escherichia coli sepsis.

Am J Pathol 2005 Oct;167(4):1161-72

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 NE 13 St., Oklahoma City, OK 73104, USA.

Endothelium plays a critical role in the pathobiology of sepsis by integrating systemic host responses and local rheological stimuli. We studied the differential expression and activation of tissue factor (TF)-dependent coagulation on linear versus branched arterial segments in a baboon sepsis model. Animals were injected intravenously with lethal doses of Escherichia coli or saline and sacrificed after 2 to 8 hours. Whole-mount arterial segments were stained for TF, TF-pathway inhibitor (TFPI), factor VII (FVII), and markers for endothelial cells (ECs), leukocytes, and platelets, followed by confocal microscopy and image analysis. In septic animals, TF localized preferentially at branches, EC surface, leukocytes, and platelet aggregates and accumulated in large amounts in the subendothelial space. FVII strongly co-localized with TF on ECs and leukocytes but less so with subendothelial TF. TFPI co-localized with TF and FVII on endothelium and leukocytes but not in the subendothelial space. Focal TF increases correlated with fibrin deposition and increased endothelial permeability to plasma proteins. Biochemical analysis confirmed that aortas of septic baboons expressed more TF mRNA and protein than controls. Branched segments contained higher TF protein levels and coagulant activity than equivalent linear areas. These data suggest that site-dependent endothelial heterogeneity and rheological factors contribute to focal procoagulant responses to E. coli.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1415276PMC
http://dx.doi.org/10.1016/S0002-9440(10)61204-7DOI Listing
October 2005

Caveolin-1 enhances tissue factor pathway inhibitor exposure and function on the cell surface.

J Biol Chem 2005 Jun 6;280(23):22308-17. Epub 2005 Apr 6.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.

Tissue factor pathway inhibitor (TFPI) blocks tissue factor-factor VIIa (TF-FVIIa) activation of factors X and IX through the formation of the TF-FVIIa-FXa-TFPI complex. Most TFPI in vivo associates with caveolae in endothelial cells (EC). The mechanism of this association and the anticoagulant role of caveolar TFPI are not yet known. Here we show that expression of caveolin-1 (Cav-1) in 293 cells keeps TFPI exposed on the plasmalemma surface, decreases the membrane lateral mobility of TFPI, and increases the TFPI-dependent inhibition of TF-FVIIa. Caveolae-associated TFPI supports the co-localization of the quaternary complex with caveolae. To investigate the significance of these observations for EC we used RNA interference to deplete the cells of Cav-1. Functional assays and fluorescence microscopy revealed that the inhibitory properties of TFPI were diminished in EC lacking Cav-1, apparently through deficient assembly of the quaternary complex. These findings demonstrate that caveolae regulate the inhibition by cell-bound TFPI of the active protease production by the extrinsic pathway of coagulation.
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http://dx.doi.org/10.1074/jbc.M503333200DOI Listing
June 2005
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