Plasma-derived and recombinant C1 esterase inhibitor: Binding profiles and neuroprotective properties in brain ischemia/reperfusion injury.

C1 esterase inhibitor (C1INH) is known to exert its inhibitory effect by binding to several target proteases of the contact and complement systems. One of C1INH's targets comprise mannose-binding lectin (MBL), a critical player in post-stroke pathophysiology. We therefore explored the effects of recombinant human (rh) and plasma derived (pd) C1INH in C57BL/6J mice subjected to transient occlusion of the middle cerebral artery (tMCAo), receiving 15U/mouse of pd or rhC1INH intravenously, at reperfusion. We analyzed the compounds' (i)neuroprotective effects, (ii) plasma presence, (iii)effects on circulating and brain MBL, (iv)time course of endothelial deposition, and (v) effects on the formation of active complement products. rhC1INH-treated mice had neuroprotective effects, including reduced behavioral deficits and neuronal loss, associated with decreased MBL brain deposition and decreased formation of complement C4b active fragments. In contrast, pdC1INH did not show these neuroprotective effects despite its longer plasma residence time. We also analyzed the response to tMCAo in C1INH-deficient mice, observing a poorer ischemic outcome compared to the wild type mice, which could be partially prevented by rhC1INH administration. In conclusion, we show that rhC1INH exhibits stronger neuroprotective effects than the corresponding plasma-derived protein after experimental ischemia/reperfusion injury in the brain, placing it as a promising drug for stroke. Differential effects are likely related to more effective MBL inhibition which further confirms it as a useful pharmacological target for stroke.

Targeted donor complement blockade after brain death prevents delayed graft function in a nonhuman primate model of kidney transplantation.

Delayed graft function (DGF) in renal transplant is associated with reduced graft survival and increased immunogenicity. The complement-driven inflammatory response after brain death (BD) and posttransplant reperfusion injury play significant roles in the pathogenesis of DGF. In a nonhuman primate model, we tested complement-blockade in BD donors to prevent DGF and improve graft survival. BD donors were maintained for 20 hours; kidneys were procured and stored at 4°C for 43-48 hours prior to implantation into ABO-compatible, nonsensitized, MHC-mismatched recipients. Animals were divided into 3 donor-treatment groups: G1 - vehicle, G2 - rhC1INH+heparin, and G3 - heparin. G2 donors showed significant reduction in classical complement pathway activation and decreased levels of tumor necrosis factor α and monocyte chemoattractant protein 1. DGF was diagnosed in 4/6 (67%) G1 recipients, 3/3 (100%) G3 recipients, and 0/6 (0%) G2 recipients (P = .008). In addition, G2 recipients showed superior renal function, reduced sC5b-9, and reduced urinary neutrophil gelatinase-associated lipocalin in the first week posttransplant. We observed no differences in incidence or severity of graft rejection between groups. Collectively, the data indicate that donor-management targeting complement activation prevents the development of DGF. Our results suggest a pivotal role for complement activation in BD-induced renal injury and postulate complement blockade as a promising strategy for the prevention of DGF after transplantation.

Inhibition of complement improves graft outcome in a pig model of kidney autotransplantation.

BACKGROUND: Ischemia reperfusion injury (IRI) induced immune response is a critical issue in transplantation. Complement and contact system activation are among its key mechanisms. STUDY DESIGN: We investigated the benefits of pre-reperfusion treatment with recombinant human C1INH (rhC1INH), inhibitor of both complement and contact activation, in a pig model of kidney autotransplantation, subjecting the organ to 60 min warm ischemia prior to 24 h static preservation to maximize damage. RESULTS: Serum creatinine measurement showed that treated animals recovered glomerular function quicker than the Vehicle group. However, no difference was observed in tubular function recovery, and elevated level of urinary NGal (Neutrophil gelatinase-associated lipocalin) and plasma AST (Aspartate Aminotransferase) were detected, indicating that treatment did not influence IRI-mediated tubular cell necrosis. Regarding chronic graft outcome, rhC1INH significantly prevented fibrosis development and improved function. Immunohistochemistry and western blot showed decreased invasion by macrophages and T lymphocytes, and reduction of epithelial to mesenchymal transition. We determined the effect of treatment on complement activation with immunofluorescence analyses at 30 min post reperfusion, showing an inhibition of C4d deposition and MBL staining in treated animals. CONCLUSIONS: In this model, the inhibition of complement activation by rhC1INH at reperfusion, while not completely counteracting IRI, limited immune system activation, significantly improving graft outcome on the short and long term.

Endothelial-to-mesenchymal transition and renal fibrosis in ischaemia/reperfusion injury are mediated by complement anaphylatoxins and Akt pathway.

BACKGROUND: Increasing evidence demonstrates a phenotypic plasticity of endothelial cells (ECs). Endothelial-to-mesenchymal transition (EndMT) contributes to the development of tissue fibrosis. However, the pathogenic factors and signalling pathways regulating this process in ischaemia/reperfusion (I/R) injury are still poorly understood. METHODS: We investigated the possible role of complement in the induction of this endothelial dysfunction in a swine model of renal I/R injury by using recombinant C1 inhibitor in vivo. RESULTS: Here, we showed that I/R injury reduced the density of renal peritubular capillaries and induced tissue fibrosis with generation of CD31(+)/α-SMA(+) and CD31(+)/FPS-1(+) cells indicating EndMT. When we inhibited complement, the process of EndMT became rare, with preserved density of peritubular capillaries and significant reduction in renal fibrosis. When we activated ECs by anaphylatoxins in vitro, C3a and C5a led to altered endothelial phenotype with increased expression of fibroblast markers and decrease expression of specific endothelial markers. The activation of Akt pathway was pivotal for the C3a and C5a-induced EndMT in vitro. In accordance, inhibition of complement in vivo led to the abrogation of Akt signalling, with hampered EndMT and tissue fibrosis. CONCLUSIONS: Our data demonstrate a critical role for complement in the acute induction of EndMT via the Akt pathway. Therapeutic inhibition of these systems may be essential to prevent vascular damage and tissue fibrosis in transplanted kidney.

Population pharmacokinetics of recombinant human C1 inhibitor in patients with hereditary angioedema.

AIMS: To characterize the pharmacokinetics (PK) of recombinant human C1 inhibitor (rhC1INH) in healthy volunteers and hereditary angioedema (HAE) patients. METHODS: Plasma levels of C1INH following 294 administrations of rhC1INH in 133 subjects were fitted using nonlinear mixed-effects modelling. The model was used to simulate maximal C1INH levels for the proposed dosing scheme. RESULTS: A one-compartment model with Michaelis-Menten elimination kinetics described the data. Baseline C1INH levels were 0.901 [95% confidence interval (CI): 0.839-0.968] and 0.176 U ml(-1) (95% CI: 0.154-0.200) in healthy volunteers and HAE patients, respectively. The volume of distribution of rhC1INH was 2.86 l (95% CI: 2.68-3.03). The maximal rate of elimination and the concentration corresponding to half this maximal rate were 1.63 U ml(-1) h(-1) (95% CI: 1.41-1.88) and 1.60 U ml(-1) (95% CI: 1.14-2.24), respectively, for healthy volunteers and symptomatic HAE patients. The maximal elimination rate was 36% lower in asymptomatic HAE patients. Peak C1INH levels did not change upon repeated administration of rhC1INH. Bodyweight was found to be an important predictor of the volume of distribution. Simulations of the proposed dosing scheme predicted peak C1INH concentrations above the lower level of the normal range (0.7 U ml(-1)) for at least 94% of all patients. CONCLUSIONS: The population PK model for C1INH supports a dosing scheme on a 50 U kg(-1) basis up to 84 kg, with a fixed dose of 4200 U above 84 kg. The PK of rhC1INH following repeat administration are consistent with the PK following the first administration.

Complement Blockade in Recipients Prevents Delayed Graft Function and Delays Antibody-mediated Rejection in a Nonhuman Primate Model of Kidney Transplantation.

BACKGROUND: Complement activation in kidney transplantation is implicated in the pathogenesis of delayed graft function (DGF). This study evaluated the therapeutic efficacy of high-dose recombinant human C1 esterase inhibitor (rhC1INH) to prevent DGF in a nonhuman primate model of kidney transplantation after brain death and prolonged cold ischemia. METHODS: Brain death donors underwent 20 h of conventional management. Procured kidneys were stored on ice for 44-48 h, then transplanted into ABO-compatible major histocompatibility complex-mismatched recipients. Recipients were treated with vehicle (n = 5) or rhC1INH 500 U/kg plus heparin 40 U/kg (n = 8) before reperfusion, 12 h, and 24 h posttransplant. Recipients were followed up for 120 d. RESULTS: Of vehicle-treated recipients, 80% (4 of 5) developed DGF versus 12.5% (1 of 8) rhC1INH-treated recipients (P = 0.015). rhC1INH-treated recipients had faster creatinine recovery, superior urinary output, and reduced urinary neutrophil gelatinase-associated lipocalin and tissue inhibitor of metalloproteinases 2-insulin-like growth factor-binding protein 7 throughout the first week, indicating reduced allograft injury. Treated recipients presented lower postreperfusion plasma interleukin (IL)-6, IL-8, tumor necrosis factor-alpha, and IL-18, lower day 4 monocyte chemoattractant protein 1, and trended toward lower C5. Treated recipients exhibited less C3b/C5b-9 deposition on day 7 biopsies. rhC1INH-treated animals also trended toward prolonged mediated rejection-free survival. CONCLUSIONS: Our results recommend high-dose C1INH complement blockade in transplant recipients as an effective strategy to reduce kidney injury and inflammation, prevent DGF, delay antibody-mediated rejection development, and improve transplant outcomes.

Complement inhibition attenuates acute kidney injury after ischemia-reperfusion and limits progression to renal fibrosis in mice.

The complement system is an essential component of innate immunity and plays a major role in the pathogenesis of ischemia-reperfusion injury (IRI). In this study, we investigated the impact of human C1-inhibitor (C1INH) on the early inflammatory response to IRI and the subsequent progression to fibrosis in mice. We evaluated structural damage, renal function, acute inflammatory response, progression to fibrosis and overall survival at 90-days post-injury. Animals receiving C1INH prior to reperfusion had a significant improvement in survival rate along with superior renal function when compared to vehicle (PBS) treated counterparts. Pre-treatment with C1INH also prevented acute IL-6, CXCL1 and MCP-1 up-regulation, C5a release, C3b deposition and infiltration by neutrophils and macrophages into renal tissue. This anti-inflammatory effect correlated with a significant reduction in the expression of markers of fibrosis alpha smooth muscle actin, desmin and picrosirius red at 30 and 90 days post-IRI and reduced renal levels of TGF-ß1 when compared to untreated controls. Our findings indicate that intravenous delivery of C1INH prior to ischemic injury protects kidneys from inflammatory injury and subsequent progression to fibrosis. We conclude that early complement blockade in the context of IRI constitutes an effective strategy in the prevention of fibrosis after ischemic acute kidney injury.

C1 Inhibitor Limits Organ Injury and Prolongs Survival in Swine Subjected to Battlefield Simulated Injury.

Complement system activation is recognized as a deleterious component of the mammalian physiological response to traumatic injury with severe hemorrhage (TH). Female Yorkshire swine were subjected to a simulated austere prehospital battlefield scenario. Each animal underwent controlled hemorrhage of 22 mL/kg at 100 mL/min rate for approximately 10 min followed by soft tissue injury, femur fracture, and spleen injury. Subsequent blood loss was uncontrolled. Twenty-eight minutes postinjury the animals were randomized into treatment or no treatment with recombinant human C1 esterase inhibitor (C1INH) (500 IU/kg, n = 11) and into receiving or not permissive hypotensive resuscitation (n = 14) with infusion of 45 mL/kg lactated Ringer's solution (2× blood lost). Observation and animal maintenance continued for 6 h at which time the animals had either expired or were euthanized. Heart, lung, and small intestine tissue samples were collected. Pharmacokinetic, hemodynamic, and metabolic parameters as well as survival time, plasma complement activity and tissue deposition, cytokine levels, and tissue injury were determined. We found that administration of C1INH protected tissues from damage, reduced the levels of inflammatory cytokines, and improved blood chemistry. Immunohistochemical analyses revealed that C1INH administration following TH markedly reduced complement activation and deposition in tissues. Importantly, C1INH administration prolonged survival of animals particularly in those which received resuscitation fluid infusion. Our data urge early administration of C1INH to limit organ damage and prolong survival of those injured in the battlefield.

Recombinant C1-inhibitor: effects on coagulation and fibrinolysis in patients with hereditary angioedema.

BACKGROUND: Recombinant human C1-inhibitor (rhC1INH; Ruconest®) has been developed for treatment of acute angioedema attacks in patients with hereditary angioedema (HAE) due to heterozygous deficiency of C1INH. Previous reports suggest that administration of plasma-derived C1INH products may be associated with an increased risk for thromboembolic complications. OBJECTIVES: Our aim is to evaluate the effects of rhC1INH on coagulation and fibrinolysis in symptomatic HAE patients. METHODS: Levels of various coagulation and fibrinolytic parameters were determined in pre- and post-exposure plasma samples from HAE patients included in a randomized clinical trial. Patients were treated with either saline, or 50 or 100 U/kg rhC1INH for an acute angioedema attack. RESULTS: Prior to rhC1INH treatment, the majority of patients had low to normal activated partial thromboplastin times (aPTT) and increased levels of prothrombin fragment 1+2, thrombin-antithrombin complexes, D-dimers and plasmin-antiplasmin complexes, all of which indicate activation of both coagulation and fibrinolysis. Infusion of rhC1INH at doses up to 100 U/kg did not affect these parameters except for a dose-dependent prolongation of aPTT, confirming that rhC1INH is an inhibitor of the contact system, and that F1+2 levels decreased. CONCLUSION: Coagulation and fibrinolytic systems are activated in HAE patients suffering from an acute angioedema attack. Treatment with rhC1INH at 50 or 100 U/kg had no effect on parameters reflecting activation of these systems except for a significant effect on aPTT, which likely reflects a pharmacodynamic effect of rhC1INH, and a reduction on plasma levels of the prothrombin activation fragment F1+2. We conclude that these results argue against a prothrombotic effect of treatment with this rhC1INH product in HAE patients.

Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock.

Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins.

Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues.

The ATP-binding cassette transporter 1 (ABCA1) has recently been identified as a key regulator of high-density lipoprotein (HDL) metabolism, which is defective in familial HDL-deficiency syndromes such as Tangier disease. ABCA1 functions as a facilitator of cellular cholesterol and phospholipid efflux, and its expression is induced during cholesterol uptake in macrophages. To assess the role of macrophage ABCA1 in atherosclerosis, we generated low-density lipoprotein (LDL) receptor knockout (LDLr(-/-)) mice that are selectively deficient in leukocyte ABCA1 (ABCA1(-/-)) by using bone marrow transfer (ABCA1(-/-) --> LDLr(-/-)). Here we demonstrate that ABCA1(-/-) --> LDLr(-/-) chimeras develop significantly larger and more advanced atherosclerotic lesions compared with chimeric LDLr(-/-) mice with functional ABCA1 in hematopoietic cells. Targeted disruption of leukocyte ABCA1 function did not affect plasma HDL cholesterol levels. The amount of macrophages in liver and spleen and peripheral blood leukocyte counts is increased in the ABCA1(-/-) --> LDLr(-/-) chimeras. Our results provide evidence that leukocyte ABCA1 plays a critical role in the protection against atherosclerosis, and we identify ABCA1 as a leukocyte factor that controls the recruitment of inflammatory cells.