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3 promotes the initiation of the alternative pathway activation
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Abstracts / Immunobiology 217 (2012) 1129–1222
161 MASP levels in cardio- and cerebrovascular diseases: A pilot study Vera Frauenknecht 1 , Steffen Thiel 2 , Marcel Arnold 3 , Jean-Paul Schmid 4 , Hugo Saner 4 , Verena Schroeder 1 1
University Clinic of Haematology, Haemostasis Research Laboratory, University Hospital & University of Bern, Bern, Switzerland 2 Department of Biomedicine, Aarhus University, Aarhus, Denmark 3 Department of Neurology, University Hospital & University of Bern, Bern, Switzerland 4 Division of Cardiovascular Prevention, Rehabilitation, and Sports Cardiology, Department of Cardiology, University Hospital & University of Bern, Bern, Switzerland Chronic inflammation and changes in blood coagulation and fibrinolysis result in a proinflammatory and prothrombotic state that contributes to development of cardio- and cerebrovascular diseases (CVD). The complement system is an important player in inflammation and there is growing evidence for a prominent role in the pathogenesis of CVD. Mannan-binding lectin-associated serine proteases (MASPs) 1 and 2 of the complement lectin pathway may contribute to fibrin clot formation. MASPs may therefore represent an important link between inflammation and thrombosis. However, there are no data available on MASP plasma levels in CVD. The aim of our study was to investigate for the first time plasma levels of MASP-1, 2 and 3 in patients with coronary artery disease (CAD), myocardial infarction (MI) and acute ischaemic stroke. We performed a pilot study in 50 healthy volunteers, in chronic CAD patients with 1-vessel disease and 3-vessel disease and agematched controls with normal coronary arteries (53 each), in 49 subacute MI patients, and in 66 patients with acute ischaemic stroke. We measured MASP-1 levels by in-house ELISA using a novel specific antibody. MASP-2 and MASP-3 levels were measured using commercial ELISA kits (Hycult Biotech, NL). Result: MASP-1 levels were highest in MI patients and lowest in stroke patients. MASP-2 levels were lower in MI and stroke patients compared to controls and CAD patients. MASP-3 levels did not differ between groups. MASP levels were not associated with severity of disease. However, MASP levels were associated with several cardiovascular risk factors including dyslipidaemia, body mass index, diabetes and hypertension. For the first time we have determined MASP plasma levels in patients with CAD, MI and ischaemic stroke. Our results suggest that MASP levels may be altered in vascular diseases, but larger studies are needed to confirm our pilot study and to elucidate the underlying mechanisms.
roles of MASP-1 and MASP-3, we generated a MASP-1- and MASP-3-deficient (M1/3 KO) mouse, and found that the deficient mice lacked alternative pathway activation, because factor D (Df) remained as a proenzyme in the serum. MASP-1 was able to convert the proenzyme of Df to an active form in vitro, and also to activate MASP-2 and MASP-3 as C1r activates C1s. In addition, we investigated the mechanism of MASP-3 activation and its substrate using the recombinant mouse MASP-3 (rMASP-3). The obtained rMASP3 was a proenzyme form, and it was activated by incubation with Staphylococcus aureus in the presence of MBL-A but not MBL-C. The in vitro experiments showed that rMASP-3 directly activated factors B and D, suggesting that the activated MASP-3 triggered the initial activation step of the alternative complement pathway. Furthermore, in vivo expression of MASP-3 converted proenzyme form of Df to the active form in M1/3 KO mouse. Thus, MASP-1 and MASP-3 play an important role in activation of both the lectin and alternative pathways. http://dx.doi.org/10.1016/j.imbio.2012.08.164 163 MBL/ficolin associated protein-1 inhibits the complex formation between MASPs and MBL Anne Rosbjerg, Mikkel-Ole Skjoedt, Peter Garred Laboratory of Molecular Medicine, Department of Clinical Immunology, Sect. 7631, Rigshospitalet, Copenhagen, Denmark
MBL-associated serine protease (MASP)-1/3 promotes the initiation of the alternative pathway activation
The recognition molecules in the lectin complement pathway (LCP) are the mannose-binding lectin (MBL) and the ficolins. Activation of the LCP is mediated by the MBL/ficolin-associated serine proteases (MASPs) named MASP-1, MASP-2 and MASP-3. The MASPs initiate a protease cascade generating opsonizing and inflammatory molecules. Recently, a transcription variant derived from the MASP-1 gene named MBL/ficolin associated protein-1 (MAP-1) was discovered, which has the capability to inhibit LCP activation. However, the mechanisms by which MAP-1 effectuates this, and the biological function of MAP-1 have yet to be established. To address this, we asked the question whether MAP-1 can inhibit the interaction between MBL and MASP-1, MASP-2 and MASP-3, respectively. The interaction patterns were investigated using recombinant MBL and recombinant MAP-1, MASP-1, -2 and 3. MAP-1 was co-incubated with MASP-1, -2 and -3, respectively, on rMBL bound to mannan. Detection of MASP binding was made with monoclonal antibodies against the different MASP molecules. The results show, that a dose-dependent inhibition of the interaction between MBL and MASP-1, -2, -3, respectively, occurs in the presence of increasing amounts of MAP-1. MAP-1 has a clear inhibitory effect on the complex formation between MBL and each of the three MASPs, which can explain the observed inhibitory effect of MAP-1 on complement activation. Thus, MAP-1 could be a promising antiinflammatory molecule in treatment of complement dependent diseases.
Toshihisa Kodama 1 , Minoru Takahashi 1 , Daisuke Iwaki 1 , Hideharu Sekine 1 , Yuichi Endo 1 , Teizo Fujita 1,2
http://dx.doi.org/10.1016/j.imbio.2012.08.165
http://dx.doi.org/10.1016/j.imbio.2012.08.163 162
1 2
Department of Immunology, Fukushima Medical University, Japan Fukushima Prefectural General Hygiene Institute, Japan
In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as pattern recognition molecules for pathogens, resulting in the activation of MBL-associated serine proteases (MASPs; MASP-1, MASP-2 and MASP-3). Among these proteases, MASP-2 is a key enzyme that cleaves C4 and C2 to assemble a C3 convertase (C4b2a). However, a physiological function of MASP-1 and MASP-3 remains unclear. To investigate the
Abstracts / Immunobiology 217 (2012) 1129–1222
161 MASP levels in cardio- and cerebrovascular diseases: A pilot study Vera Frauenknecht 1 , Steffen Thiel 2 , Marcel Arnold 3 , Jean-Paul Schmid 4 , Hugo Saner 4 , Verena Schroeder 1 1
University Clinic of Haematology, Haemostasis Research Laboratory, University Hospital & University of Bern, Bern, Switzerland 2 Department of Biomedicine, Aarhus University, Aarhus, Denmark 3 Department of Neurology, University Hospital & University of Bern, Bern, Switzerland 4 Division of Cardiovascular Prevention, Rehabilitation, and Sports Cardiology, Department of Cardiology, University Hospital & University of Bern, Bern, Switzerland Chronic inflammation and changes in blood coagulation and fibrinolysis result in a proinflammatory and prothrombotic state that contributes to development of cardio- and cerebrovascular diseases (CVD). The complement system is an important player in inflammation and there is growing evidence for a prominent role in the pathogenesis of CVD. Mannan-binding lectin-associated serine proteases (MASPs) 1 and 2 of the complement lectin pathway may contribute to fibrin clot formation. MASPs may therefore represent an important link between inflammation and thrombosis. However, there are no data available on MASP plasma levels in CVD. The aim of our study was to investigate for the first time plasma levels of MASP-1, 2 and 3 in patients with coronary artery disease (CAD), myocardial infarction (MI) and acute ischaemic stroke. We performed a pilot study in 50 healthy volunteers, in chronic CAD patients with 1-vessel disease and 3-vessel disease and agematched controls with normal coronary arteries (53 each), in 49 subacute MI patients, and in 66 patients with acute ischaemic stroke. We measured MASP-1 levels by in-house ELISA using a novel specific antibody. MASP-2 and MASP-3 levels were measured using commercial ELISA kits (Hycult Biotech, NL). Result: MASP-1 levels were highest in MI patients and lowest in stroke patients. MASP-2 levels were lower in MI and stroke patients compared to controls and CAD patients. MASP-3 levels did not differ between groups. MASP levels were not associated with severity of disease. However, MASP levels were associated with several cardiovascular risk factors including dyslipidaemia, body mass index, diabetes and hypertension. For the first time we have determined MASP plasma levels in patients with CAD, MI and ischaemic stroke. Our results suggest that MASP levels may be altered in vascular diseases, but larger studies are needed to confirm our pilot study and to elucidate the underlying mechanisms.
roles of MASP-1 and MASP-3, we generated a MASP-1- and MASP-3-deficient (M1/3 KO) mouse, and found that the deficient mice lacked alternative pathway activation, because factor D (Df) remained as a proenzyme in the serum. MASP-1 was able to convert the proenzyme of Df to an active form in vitro, and also to activate MASP-2 and MASP-3 as C1r activates C1s. In addition, we investigated the mechanism of MASP-3 activation and its substrate using the recombinant mouse MASP-3 (rMASP-3). The obtained rMASP3 was a proenzyme form, and it was activated by incubation with Staphylococcus aureus in the presence of MBL-A but not MBL-C. The in vitro experiments showed that rMASP-3 directly activated factors B and D, suggesting that the activated MASP-3 triggered the initial activation step of the alternative complement pathway. Furthermore, in vivo expression of MASP-3 converted proenzyme form of Df to the active form in M1/3 KO mouse. Thus, MASP-1 and MASP-3 play an important role in activation of both the lectin and alternative pathways. http://dx.doi.org/10.1016/j.imbio.2012.08.164 163 MBL/ficolin associated protein-1 inhibits the complex formation between MASPs and MBL Anne Rosbjerg, Mikkel-Ole Skjoedt, Peter Garred Laboratory of Molecular Medicine, Department of Clinical Immunology, Sect. 7631, Rigshospitalet, Copenhagen, Denmark
MBL-associated serine protease (MASP)-1/3 promotes the initiation of the alternative pathway activation
The recognition molecules in the lectin complement pathway (LCP) are the mannose-binding lectin (MBL) and the ficolins. Activation of the LCP is mediated by the MBL/ficolin-associated serine proteases (MASPs) named MASP-1, MASP-2 and MASP-3. The MASPs initiate a protease cascade generating opsonizing and inflammatory molecules. Recently, a transcription variant derived from the MASP-1 gene named MBL/ficolin associated protein-1 (MAP-1) was discovered, which has the capability to inhibit LCP activation. However, the mechanisms by which MAP-1 effectuates this, and the biological function of MAP-1 have yet to be established. To address this, we asked the question whether MAP-1 can inhibit the interaction between MBL and MASP-1, MASP-2 and MASP-3, respectively. The interaction patterns were investigated using recombinant MBL and recombinant MAP-1, MASP-1, -2 and 3. MAP-1 was co-incubated with MASP-1, -2 and -3, respectively, on rMBL bound to mannan. Detection of MASP binding was made with monoclonal antibodies against the different MASP molecules. The results show, that a dose-dependent inhibition of the interaction between MBL and MASP-1, -2, -3, respectively, occurs in the presence of increasing amounts of MAP-1. MAP-1 has a clear inhibitory effect on the complex formation between MBL and each of the three MASPs, which can explain the observed inhibitory effect of MAP-1 on complement activation. Thus, MAP-1 could be a promising antiinflammatory molecule in treatment of complement dependent diseases.
Toshihisa Kodama 1 , Minoru Takahashi 1 , Daisuke Iwaki 1 , Hideharu Sekine 1 , Yuichi Endo 1 , Teizo Fujita 1,2
http://dx.doi.org/10.1016/j.imbio.2012.08.165
http://dx.doi.org/10.1016/j.imbio.2012.08.163 162
1 2
Department of Immunology, Fukushima Medical University, Japan Fukushima Prefectural General Hygiene Institute, Japan
In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as pattern recognition molecules for pathogens, resulting in the activation of MBL-associated serine proteases (MASPs; MASP-1, MASP-2 and MASP-3). Among these proteases, MASP-2 is a key enzyme that cleaves C4 and C2 to assemble a C3 convertase (C4b2a). However, a physiological function of MASP-1 and MASP-3 remains unclear. To investigate the