Vasile I. Pavlov

Mannose-binding lectin and its associated proteases (MASPs) mediate coagulation and its deficiency is a risk factor in developing complications from infection, including disseminated intravascular coagulation.

The first line of host defense is the innate immune system that includes coagulation factors and pattern recognition molecules, one of which is mannose-binding lectin (MBL). Previous studies have demonstrated that MBL deficiency increases susceptibility to infection. Several mechanisms are associated with increased susceptibility to infection, including reduced opsonophagocytic killing and reduced lectin complement pathway activation. In this study, we demonstrate that MBL and MBL-associated serine protease (MASP)-1/3 together mediate coagulation factor-like activities, including thrombin-like activity. MBL and/or MASP-1/3 deficient hosts demonstrate in vivo evidence that MBL and MASP-1/3 are involved with hemostasis following injury. Staphylococcus aureus infected MBL null mice developed disseminated intravascular coagulation (DIC), which was associated with elevated blood IL-6 levels (but not TNF-α and multi-organ inflammatory responses). Infected MBL null mice also develop liver injury. These findings suggest that MBL deficiency may manifest into DIC and organ failure during infectious diseases.

Myocardial ischemia and reperfusion injury is dependent on both IgM and mannose-binding lectin

Complement activation has been shown to play an important role in the inflammation and tissue injury following myocardial ischemia and reperfusion (MI/R). Several recent studies from our laboratory demonstrated the importance of mannose-binding lectin (MBL) as the initiation pathway for complement activation and the resulting pathological effects following MI/R. However, other studies from the past suggest an important role of the classical pathway and perhaps natural antibodies. In the present study, we used newly generated genetically modified mice that lack secreted IgM (sIgM), MBL-A, and MBL-C (sIgM/MBL null) in a plasma reconstitution mouse model of MI/R. Following 30 min of ischemia and 4 h of reperfusion, left ventricular ejection fractions were significantly higher in sIgM/MBL null mice reconstituted with MBL null or sIgM/MBL null plasma compared with reconstitution with wild-type (WT) plasma or WT mice reconstituted with WT plasma following MI/R. Serum troponin I concentration, myocardial polymorphonuclear leukocyte infiltration, and C3 deposition were dependent on the combined presence of sIgM and MBL. These results demonstrate that MI/R-induced complement activation, inflammation, and subsequent tissue injury require both IgM and MBL. Thus MBL-dependent activation of the lectin pathway may not be completely antibody independent in I/R models.

Targeting Mannose-Binding Lectin Confers Long-Lasting Protection With a Surprisingly Wide Therapeutic Window in Cerebral Ischemia

Background— The involvement of the complement system in brain injury has been scarcely investigated. Here, we document the pivotal role of mannose-binding lectin (MBL), one of the recognition molecules of the lectin complement pathway, in brain ischemic injury. Methods and Results— Focal cerebral ischemia was induced in mice (by permanent or transient middle cerebral artery occlusion) and rats (by 3-vessel occlusion). We first observed that MBL is deposited on ischemic vessels up to 48 hours after injury and that functional MBL/MBL-associated serine protease 2 complexes are increased. Next, we demonstrated that (1) MBL−/− mice are protected from both transient and permanent ischemic injury; (2) Polyman2, the newly synthesized mannosylated molecule selected for its binding to MBL, improves neurological deficits and infarct volume when given up to 24 hours after ischemia in mice; (3) anti-MBL-A antibody improves neurological deficits and infarct volume when given up to 18 hours after ischemia, as assessed after 28 days in rats. Conclusions— Our data show an important role for MBL in the pathogenesis of brain ischemic injury and provide a strong support to the concept that MBL inhibition may be a relevant therapeutic target in humans, one with a wide therapeutic window of application.

Mannose-Binding Lectin-Associated Serine Protease-1 Is a Significant Contributor to Coagulation in a Murine Model of Occlusive Thrombosis

Bleeding disorders and thrombotic complications constitute a major cause of death and disability worldwide. Although it is known that the complement and coagulation systems interact, no studies have investigated the specific role or mechanisms of lectin-mediated coagulation in vivo. FeCl3 treatment resulted in intra-arterial occlusive thrombogenesis within 10 min in wild-type (WT) and C2/factor B-null mice. In contrast, mannose-binding lectin (MBL)-null and MBL-associated serine protease (MASP)-1/-3 knockout (KO) mice had significantly decreased FeCl3-induced thrombogenesis. Reconstitution with recombinant human (rh) MBL restored FeCl3-induced thrombogenesis in MBL-null mice to levels comparable to WT mice, suggesting a significant role of the MBL/MASP complex for in vivo coagulation. Additionally, whole blood aggregation demonstrated increased MBL/MASP complex-dependent platelet aggregation. In vitro, MBL/MASP complexes were captured on mannan-coated plates, and cleavage of a chromogenic thrombin substrate (S2238) was measured. We observed no significant differences in S2238 cleavage between WT, C2/factor B-null, MBL-A−/−, or MBL-C−/− sera; however, MBL-null or MASP-1/-3 KO mouse sera demonstrated significantly decreased S2238 cleavage. rhMBL alone failed to cleave S2238, but cleavage was restored when rMASP-1 was added to either MASP-1/-3 KO sera or rhMBL. Taken together, these findings indicate that MBL/MASP complexes, and specifically MASP-1, play a key role in thrombus formation in vitro and in vivo.

Endogenous and Natural Complement Inhibitor Attenuates Myocardial Injury and Arterial Thrombogenesis

Background— Coagulation disorders and reperfusion of ischemic myocardium are major causes of morbidity and mortality. Lectin pathway initiation complexes are composed of multimolecular carbohydrate recognition subcomponents and 3 lectin pathway–specific serine proteases. We have recently shown that the lectin pathway–specific carbohydrate recognition subcomponent mannose-binding lectin plays an essential role in the pathophysiology of thrombosis and ischemia/reperfusion injury. Thus, we hypothesized that the endogenous mannose-binding lectin (MBL)/ficolin-associated protein-1 (MAP-1) that inhibits complement activation in vitro also could be an in vivo regulator by attenuating myocardial schema/reperfusion injury and thrombogenesis when used at pharmacological doses in wild-type mice. Methods and Results— In 2 mouse models, MAP-1 preserves cardiac function, decreases infarct size, decreases C3 deposition, inhibits MBL deposition, and prevents thrombogenesis. Furthermore, we demonstrate that MAP-1 displaces MBL/ficolin-associated serine protease (MASP)-1, MASP-2, and MASP-3 from the MBL complex. Conclusions— Our results suggest that the natural, endogenous inhibitor MAP-1 effectively inhibits lectin pathway activation in vivo. MAP-1 at pharmacological doses represents a novel therapeutic approach for human diseases involving the lectin pathway and its associated MASPs.

Absence of Mannose-Binding Lectin Prevents Hyperglycemic Cardiovascular Complications

Diabetes, stress, pharmaceuticals, surgery, and physical trauma can lead to hyperglycemic conditions. A consistent relationship has been found between chronic inflammation and the cardiovascular complications of hyperglycemia. We hypothesized that cardiomyopathy and vasculopathy resulting from acute hyperglycemia are dependent on mannose-binding lectin (MBL) and lectin complement pathway activation. Hyperglycemia was induced in wild-type (WT) C57BL/6 and MBL-null mice after streptozotocin administration. Echocardiographic data and tissue samples were collected after 4, 7, or 14 days of acute hyperglycemia. Hyperglycemic WT mice demonstrated dilated cardiomyopathy with significantly increased short and long axis area measurements during systole and diastole compared to hyperglycemic MBL-null mice. The EC(50) for acetylcholine-induced relaxation of mesenteric arterioles in WT mice after 4 days of hyperglycemia demonstrated a significant loss of nitric oxide-mediated relaxation compared to normoglycemic WT or hyperglycemic MBL-null mice. Myocardial histochemistry and Western blot analysis revealed a significant influx of macrophages, altered morphology, and increased elastin and collagen deposition in hyperglycemic WT hearts compared to MBL-null hearts. Serum transforming growth factor-β1 levels were significantly lower in hyperglycemic MBL-null compared to WT mice, suggesting decreased profibrotic signaling. Together, these data suggest that MBL and the lectin complement pathway play a significant role in vascular dysfunction and cardiomyopathy after acute hyperglycemia.

Complement 3 is involved with ventilator-induced lung injury

Humoral molecules can trigger injury on mechanically stressed and damaged tissue. We have studied the role of complement 3 (C3) in a mouse model of ventilator-induced lung injury (VILI). Compared with sham-treated wild type (WT) mice, ventilated WT mice have reduced total bronchoalveolar lavage (BAL) cells; and elevated activities of thrombin and matrix metalloproteinases (MMPs), such as gelatinase/collagenase in the BAL fluid. In contrast, these parameters in ventilated C3 null mice are not significantly different from sham-treated WT and C3 null mice. In mechanically ventilated mice, thrombin activity and MMPs are lower in C3 null mice than in WT mice and are inversely correlated with total single BAL cells. C3 activation is associated with MMP activation in vitro. Pretreatment of WT mice with humanized cobra venom factor, which inactivates C3, reduces C3 deposition in the lung and increases total BAL cells in VILI. We propose that C3 is involved with VILI and inhibition of complement activation may be a potential therapeutic strategy.

Human mannose-binding lectin inhibitor prevents myocardial injury and arterial thrombogenesis in a novel animal model.

Myocardial infarction and coagulation disorders are leading causes of disability and death in the world. An important role of the lectin complement pathway in myocardial infarction and coagulation has been demonstrated in mice genetically deficient in lectin complement pathway proteins. However, these studies are limited to comparisons between wild-type and deficient mice and lack the ability to examine reversal/inhibition of injury after disease establishment. We developed a novel mouse that expresses functional human mannose-binding lectin (MBL) 2 under the control of Mbl1 promoter. Serum MBL2 concentrations averaged approximately 3 μg/mL in MBL2(+/+)Mbl1(-/-)Mbl2(-/-) [MBL2 knock in (KI)] mice. Serum MBL2 level in MBL2 KI mice significantly increased after 7 (8 μg/mL) or 14 (9 μg/mL) days of hyperglycemia compared to normoglycemic mice (P < 0.001). Monoclonal antibody 3F8 inhibited C3 deposition on mannan-coated plates in MBL2 KI, but not wild-type, mice. Myocardial ischemia/reperfusion in MBL2 KI mice revealed that 3F8 preserved cardiac function and decreased infarct size and fibrin deposition in a time-dependent manner. Furthermore, 3F8 prevented ferric chloride-induced occlusive arterial thrombogenesis in vivo. MBL2 KI mice represent a novel animal model that can be used to study the lectin complement pathway in acute and chronic models of human disease. Furthermore, these novel mice demonstrate the therapeutic window for MBL2 inhibition for effective treatment of disease and its complications.

Murine hyperglycemic vasculopathy and cardiomyopathy: whole-genome gene expression analysis predicts cellular targets and regulatory networks influenced by mannose binding lectin

Hyperglycemia, in the absence of type 1 or 2 diabetes, is an independent risk factor for cardiovascular disease. We have previously demonstrated a central role for mannose binding lectin (MBL)-mediated cardiac dysfunction in acute hyperglycemic mice. In this study, we applied whole-genome microarray data analysis to investigate MBL’s role in systematic gene expression changes. The data predict possible intracellular events taking place in multiple cellular compartments such as enhanced insulin signaling pathway sensitivity, promoted mitochondrial respiratory function, improved cellular energy expenditure and protein quality control, improved cytoskeleton structure, and facilitated intracellular trafficking, all of which may contribute to the organismal health of MBL null mice against acute hyperglycemia. Our data show a tight association between gene expression profile and tissue function which might be a very useful tool in predicting cellular targets and regulatory networks connected with in vivo observations, providing clues for further mechanistic studies.