- Email: [email protected]
Flowmotion in microcirculation among elderly patients with heart failure
Abstracts a
Institute of Human Genetics Polish Academy of Sciences in Poznan, Poland b Department of General and Vascular Surgery, Poznan University of Medical Sciences, Poznan, Poland E-mail address: [email protected] (E. Strauss) Abdominal aortic aneurysm (AAA) is an age-related vascular disease, occurring most commonly among men older than 65 years. The pathogenesis of AAA is multifactorial with a significant genetic component. Previous studies show that polymorphisms in genes involved in homocysteine metabolism may contribute to the risk, however up to now there are very few appropriately powered association studies considering the role of specific genetic variants. Paraoxonase 1 gene (PON1) codes a HDL-associated multifunctional protein, which detoxifies homocysteine thiolactone in vascular wall. The other substrates of this enzyme include oxidized lipids, sex hormones (estrogen esters) and specific drugs, such as aspirin and statins. PON1 gene is highly polymorphic. The aim of this study was to evaluate the effects of three functional polymorphisms in paraoxonase 1 gene (PON1) on AAA risk. Potential gender differences were also analyzed. The study was performed in groups of 525 AAA cases, 425 control persons and in random population sample of 220 subjects. PON1 −108C N T (rs705379), L55M (162T NA; rs854560) and Q192R (575A NG, rs662) SNPs were analyzed using PCR-RFLP or multiplex PCR-RFLP methods. Haploview software was used for analysis of LD across the gene and for the haplotype reconstruction. PON1 coding region polymorphisms L55M and Q192R were in strong linkage disequilibrium (D′ = 0,76), which results in relatively low frequency of 55M-192R haplotype in Polish population (2.3%). The frequency of this rare haplotype in patients (0.8%) was significantly lower as compared to that in controls (1.8%, p = 0.04). This association was enhanced in men (0.5% and 1.9%; respectively; p = 0.01). In women, the frequencies of this rare haplotype were not differing between the cases (2.3%) and controls (1.8%; p = 0.8). Contrary to the protective effect of the above described PON1 haplotype, the 5.6-fold higher risk of AAA was noted in the homozygotes of PON1 55L-192R haplotype heterozygous for PON1 −108 CT (PON1 −108CT/55LL/192RR genotype, p = 0.003). This association was strong in men (OR = 5.7; p = 0.0097), and unsignificant in women (OR = 2.3; p = 0.5). In conclusion, our results show that functional variants of PON1 gene influence AAA risk in men. Observed sex related differences in the impact of PON1 functional SNPs on the AAA risk support the concept of the sex specific differences in AAA pathogenesis. Supported by Polish Ministry of Sciences grant nos.: PO5C03828, NN403_250440.
doi:10.1016/j.vph.2011.08.203
SESSION 15 Systems biology applied to cardiovascular disease P.15.1 Liver sinusoidal endothelium: A microenvironment-dependent differentiation program Cyrill Géraud, Kai Schledzewski, Alexandra Demory, Diana Klein, Miriam Kaus, Francis Peyre, Carsten Sticht, Konstantin Evdokimov, Shun Lu, Astrid Schmieder, Sergij Goerdt Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany E-mail address: [email protected] (C. Géraud)
381
Liver sinusoidal endothelium (LSEC) is a prime example of organspecific microvascular differentiation and functions. Disease-associated capillarization of LSEC in vivo and dedifferentiation of LSEC in vitro indicate the importance of the hepatic microenvironment. To identify the LSEC-specific molecular differentiation program in the rat we used a two-sided gene expression profiling approach comparing LSEC freshly isolated ex vivo with both lung microvascular endothelial cells (LMEC) and with LSEC cultured for 42 h. The LSEC signature consisted of 48 genes both down-regulated in LMEC and in LSEC upon culture (fold change N7 in at least one comparison); quantitative reverse-transcription polymerase chain reaction confirmation of these genes included numerous family members and signaling pathway-associated molecules. The LSEC differentiation program comprised distinct sets of growth (Wnt2, Fzd4, 5, 9, Wls, vascular endothelial growth factors [VEGFR] 1, 2, 3, Nrp2) and transcription factors (Gata4, Lmo3, Tcfec, Maf) as well as endocytosisrelated (Stabilin-1/2, Lyve1, and Ehd3) and cytoskeleton-associated molecules (Rnd3/RhoE). Specific gene induction in cultured LSEC versus freshly isolated LSEC as well as LMEC (Esm-1, Aatf) and upregulation of gene expression to LMEC levels (CXCR4, Apelin) confirmed true transdifferentiation of LSEC in vitro. Conclusion: Comparative microvascular analysis in rat identified a hepatic microenvironmentdependent LSEC-specific differentiation program. Reference 1. Géraud, C., Schledzewski, K., Demory, A., Klein, D., Kaus, M., Peyre, F., Sticht, C., Evdokimov, K., Lu, S., Schmieder, A. and Goerdt, S. (2010), Liver sinusoidal endothelium: A microenvironment-dependent differentiation program in rat including the novel junctional protein liver endothelial differentiation-associated protein-1. Hepatology, 52: 313–326. doi: 10.1002/hep.23618. doi:10.1016/j.vph.2011.08.204
P.15.2 Flowmotion in microcirculation among elderly patients with heart failure Barbara Gryglewska, Magdalena Fedyk-Lukasik, Anna Skalska, Tomasz Grodzicki Department of Internal Medicine and Gerontology, Jagielonian University, Medical College, Krakow, Poland E-mail address: [email protected] (B. Gryglewska) Objective: The aim of the study was to assess the influence of heart failure presence on flow and flowmotion in skin microcirculation at rest, during ischemia and post occlusive hyperaemia. Material and methods: Studied population consisted of patients over 65 years with diagnosed heart failure. Body mass index (BMI) and blood pressure (BP) measurements, pulse wave velocity (PWVComplior), echocardiography (General Electrics Vivid 3) and Nterminal pro-B-type natriuretic peptide (NT proBNP) were performed. Skin microcirculation was assessed by laser Doppler flowmetry (PeriFlux PF 5000). Rest flow (RF), minimal flow during ischemia (biological zero — BZ) and post occlusive reactive hyperaemia (PORH) were registered. Fast Fourier's transformation of RF, BZ and PORH were also performed. Power frequency oscillations were evaluated in five frequency intervals of endothelial, neurogenic, myogenic, respiratory and heart origin. Data were analyzed in 2 groups: with BP values lower than 140/90 mmHg (I) and equal or higher than 140/90 mmHg (II). Statistical analysis was performed with U Mann–Whitney test, and Spearman correlation. Results: Study population consisted of 55 persons (I — 35 and II — 20 subjects) aged 70.6 ± 8.8 years, 60% men. Both groups were similar according to age, gender, PWV and ejection fraction (EF), mean RF, BZ and PORH flows. Group I had lower BP than group II (120.5 ± 11.2/73.5 ± 8.2 vs 153.4 ± 11.4/85.4 ± 8.1 mmHg, p b 0.001),
382
Abstracts
but higher levels of NT proBNP (1945 ± 2808 vs 727 ± 1420 pg/ml, p b 0.01) and myogenic flowmotion of RF, BZ and PORH (0.28 ± 0.23 vs 0.20 ± 0.21, 0.18 ± 0.20 vs 0.07 ± 0.09, 0.50 ± 0.30 vs 0.33 ± 0.27, respectively, p b 0.05). Myogenic oscillations of RF and PORH correlated positively with EF (r = 0.39, r = 0.36, respectively) and that oscillations of BZ with NT proBNP (r = 0.4). Conclusions: Myogenic oscillations of flow in microcirculation were higher in heart failure with lower BP values and significantly correlated with ejection fraction and NT proBNP. doi:10.1016/j.vph.2011.08.205
P.15.3 The comparison of antithrombotic properties of CORM-3 and CORM-A1 in arterial thrombosis in rats Karol Kramkowskia, Agnieszka Leszczynskaa, Andrzej Mogielnickia, Roberto Motterlinib, Stefan Chlopickic, Wlodzimierz Buczkoa a Department of Pharmacodynamics, Medical University of Bialystok, Poland b Department of Drug Discovery and Development, Italian Institute of Technology, Genova, Italy c Experimental Pharmacology, Jagiellonian University Medical College, Krakow, Poland E-mail address: [email protected] (K. Kramkowski) It has been shown that water-soluble CORM-3 inhibits platelet aggregation in vitro, due to the release of CO. Newly-synthesized CORM-A1, which does not contain transition metal — ruthenium (Ru) and its structure is based on sodium boranocarbonate liberates CO at a much slower rate than CORM-3 under physiological conditions. The aim of this work was to estimate in vivo the effects of new CORM-A1 in comparison to CORM-3 on electrically-stimulated arterial thrombosis in rats. CORM-3 inhibited electrically induced arterial thrombosis only at dose of 30 μmol/kg (0.7 ± 0.04 mg, p b 0.5 vs. 0.81 ± 0.02 mg in the VEH treated group), while the effect of CORM-A1 was strictly dosedependent (0.69 ± 0.07 mg, p b 0.05 and 0.62 ± 0.03 mg, p b 0.001 for rats treated with 10 and 30 μmol/kg CORM-A1, respectively; vs. 0.81 ± 0.02 mg in the VEH treated group. iCORM-3 and iCORM-A1 (compounds deprived of CO) failed to influence on thrombus weight (at doses of 30 μmol/kg). In thrombotic animals CORM-3 at doses of 10 and 30 μmol/kg did not inhibit collagen-stimulated platelet aggregation while CORM-A1 did (74.67 ± 19.47%, and 57.08 ± 10.33%, respectively vs. 100 ± 6.36% in VEH treated animals). The antiplatelet effect of CORM-A1, but not CORM-3 correlated with decrease in thrombus weight (r = 0.60227; p b 0.01). In thrombotic animals activity of PAI-1 was inhibited by treatment with CORM-A1 (30 μmol/kg; 1.28 ± 0.08 ng/ml vs. 1.60 ± 0.08 ng/ml in rats treated with VEH, p b 0.05) but not by CORM-3. Fibrin generation was profoundly inhibited by treatment with CORM-3 (10 and 30 μmol/kg, 405.0 ± 69.0 units and 162.3 ± 115.6 units vs. 919.5 ± 36.4 units for VEH, p b 0.01) while CORM-A1 had no effect. Also CORM-3 administered in dose of 30 μmol/kg decreased prothrombin time (23.06 ± 0.75 s vs. 21.10 ± 1.02 s for VEH treated animals, p b 0.05), while CORM-A1 did not. Neither of CORMs and iCORMs changed blood cell count and gasometry parameters in thrombotic and in healthy animals. In conclusion, we have shown that both CORM-3 and CORM-A1 possess antithrombotic activity in vivo. However, CORM-A1 releasing CO at a slower rate than CORM-3 rate possessed stronger antithrombotic, anti-platelet and profibrinolytic activities at in vivo conditions. In contrast, CORM-3 inhibited thrombosis mainly due to decreasing of fibrin production. doi:10.1016/j.vph.2011.08.206
P.15.4 A SILAC approach to MT1-MMP degradome in inflammatory angiogenesis Agnieszka Martínez-Koziola, Pilar Gonzaloa, Ángela Pollána, Alba Motaa, Nuria Coloméb, David Montanerc, Joaquín Dopazoc, Joaquín Arribasb, Francesc Canalsb, Alicia G. Arroyoa a Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain b Proteomics Laboratory and Medical Oncology Research Program Vall d'Hebron University Hospital Research Institute, Barcelona, Spain c Bioinformatics Department, Centro de Investigacion Príncipe Felipe, Valencia, Spain E-mail address: [email protected] (A. Martínez-Koziol) Angiogenesis, the formation of new capillaries from pre-existing vessels, is often coupled to inflammation in adult organisms. MT1MMP is a key protease required for angiogenesis induced by certain inflammatory factors. We have aimed at identifying the degradome (collection of substrates) of MT1-MMP in this context. To this end, we have performed quantitative proteomics, SILAC (stable isotope labelling of amino acids in cell culture), in TNFα-stimulated immortalized mouse lung endothelial cells (iMLEC) from wildtype and MT1-MMP-deficient mice. Glycoproteins identified in the supernatant as putative MT1-MMP substrates include extracellular matrix and related proteins some of them relevant to the angiogenic response; notably, a few seem to be unique substrates of MT1MMP. System biology analysis of this MT1-MMP endothelial degradome performed with Babelomics and FatiGO platforms revealed a combinatorial MT1-MMP substrate proteolytic program that governs endothelial cell adhesion, motility and chemotaxis finally leading to angiogenesis. Validation of selected substrates in 2D and 3D-endothelial cell models in vitro and in mouse retinas ex vivo underscores the relevance of MT1-MMP-mediated protein processing. In sum, SILAC identification of MT1-MMP degradome in TNFαactivated endothelial cells provides a system biology understanding of MT1-MMP contribution to inflammation-driven angiogenesis that might help to define surrogate markers in chronic inflammatory disorders such as psoriasis, rheumatoid arthritis and atherosclerosis. doi:10.1016/j.vph.2011.08.207
P.15.5 Anti-atherosclerotic action of agmatine in apoE-knockout mice Anna Gebska, Anna Niepsuj, Justyna Toton-Zuranska, Rafal Olszanecki, Ryszard Korbut Chair of Pharmacology, Jagiellonian University Medical College, Krakow, Poland E-mail address: [email protected] (A. Niepsuj) Atherosclerosis is an inflammatory disease, in which disturbances in lipid metabolism as well as enhanced apoptosis of various cells within vessel wall play important role. Importantly, the pathways of metabolism of fatty acids and apoptosis intersect in mitochondria. Agmatine is an endogenous polyamine produced by decarboxylation of l-arginine showing a wide array of biologic effects. It has been demonstrated that agmatine may regulate blood pressure, stimulate fatty acid oxidation and exert protective effect on mitochondria along with anti-inflammatory and neuroprotective action. We investigated anti-atherosclerotic action of exogenous agmatine (administrated p.o. for 4 months in dose of 20 mg/kg/day) in mouse model of atherosclerosis — apoE-knockout mice. As evidenced by en face and cross section methods, agmatine by 40% inhibited formation of atherosclerotic plaques in aorta of apoE knockout mice. Importantly, action of agmatine was associated with beneficial changes of the levels of
Institute of Human Genetics Polish Academy of Sciences in Poznan, Poland b Department of General and Vascular Surgery, Poznan University of Medical Sciences, Poznan, Poland E-mail address: [email protected] (E. Strauss) Abdominal aortic aneurysm (AAA) is an age-related vascular disease, occurring most commonly among men older than 65 years. The pathogenesis of AAA is multifactorial with a significant genetic component. Previous studies show that polymorphisms in genes involved in homocysteine metabolism may contribute to the risk, however up to now there are very few appropriately powered association studies considering the role of specific genetic variants. Paraoxonase 1 gene (PON1) codes a HDL-associated multifunctional protein, which detoxifies homocysteine thiolactone in vascular wall. The other substrates of this enzyme include oxidized lipids, sex hormones (estrogen esters) and specific drugs, such as aspirin and statins. PON1 gene is highly polymorphic. The aim of this study was to evaluate the effects of three functional polymorphisms in paraoxonase 1 gene (PON1) on AAA risk. Potential gender differences were also analyzed. The study was performed in groups of 525 AAA cases, 425 control persons and in random population sample of 220 subjects. PON1 −108C N T (rs705379), L55M (162T NA; rs854560) and Q192R (575A NG, rs662) SNPs were analyzed using PCR-RFLP or multiplex PCR-RFLP methods. Haploview software was used for analysis of LD across the gene and for the haplotype reconstruction. PON1 coding region polymorphisms L55M and Q192R were in strong linkage disequilibrium (D′ = 0,76), which results in relatively low frequency of 55M-192R haplotype in Polish population (2.3%). The frequency of this rare haplotype in patients (0.8%) was significantly lower as compared to that in controls (1.8%, p = 0.04). This association was enhanced in men (0.5% and 1.9%; respectively; p = 0.01). In women, the frequencies of this rare haplotype were not differing between the cases (2.3%) and controls (1.8%; p = 0.8). Contrary to the protective effect of the above described PON1 haplotype, the 5.6-fold higher risk of AAA was noted in the homozygotes of PON1 55L-192R haplotype heterozygous for PON1 −108 CT (PON1 −108CT/55LL/192RR genotype, p = 0.003). This association was strong in men (OR = 5.7; p = 0.0097), and unsignificant in women (OR = 2.3; p = 0.5). In conclusion, our results show that functional variants of PON1 gene influence AAA risk in men. Observed sex related differences in the impact of PON1 functional SNPs on the AAA risk support the concept of the sex specific differences in AAA pathogenesis. Supported by Polish Ministry of Sciences grant nos.: PO5C03828, NN403_250440.
doi:10.1016/j.vph.2011.08.203
SESSION 15 Systems biology applied to cardiovascular disease P.15.1 Liver sinusoidal endothelium: A microenvironment-dependent differentiation program Cyrill Géraud, Kai Schledzewski, Alexandra Demory, Diana Klein, Miriam Kaus, Francis Peyre, Carsten Sticht, Konstantin Evdokimov, Shun Lu, Astrid Schmieder, Sergij Goerdt Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany E-mail address: [email protected] (C. Géraud)
381
Liver sinusoidal endothelium (LSEC) is a prime example of organspecific microvascular differentiation and functions. Disease-associated capillarization of LSEC in vivo and dedifferentiation of LSEC in vitro indicate the importance of the hepatic microenvironment. To identify the LSEC-specific molecular differentiation program in the rat we used a two-sided gene expression profiling approach comparing LSEC freshly isolated ex vivo with both lung microvascular endothelial cells (LMEC) and with LSEC cultured for 42 h. The LSEC signature consisted of 48 genes both down-regulated in LMEC and in LSEC upon culture (fold change N7 in at least one comparison); quantitative reverse-transcription polymerase chain reaction confirmation of these genes included numerous family members and signaling pathway-associated molecules. The LSEC differentiation program comprised distinct sets of growth (Wnt2, Fzd4, 5, 9, Wls, vascular endothelial growth factors [VEGFR] 1, 2, 3, Nrp2) and transcription factors (Gata4, Lmo3, Tcfec, Maf) as well as endocytosisrelated (Stabilin-1/2, Lyve1, and Ehd3) and cytoskeleton-associated molecules (Rnd3/RhoE). Specific gene induction in cultured LSEC versus freshly isolated LSEC as well as LMEC (Esm-1, Aatf) and upregulation of gene expression to LMEC levels (CXCR4, Apelin) confirmed true transdifferentiation of LSEC in vitro. Conclusion: Comparative microvascular analysis in rat identified a hepatic microenvironmentdependent LSEC-specific differentiation program. Reference 1. Géraud, C., Schledzewski, K., Demory, A., Klein, D., Kaus, M., Peyre, F., Sticht, C., Evdokimov, K., Lu, S., Schmieder, A. and Goerdt, S. (2010), Liver sinusoidal endothelium: A microenvironment-dependent differentiation program in rat including the novel junctional protein liver endothelial differentiation-associated protein-1. Hepatology, 52: 313–326. doi: 10.1002/hep.23618. doi:10.1016/j.vph.2011.08.204
P.15.2 Flowmotion in microcirculation among elderly patients with heart failure Barbara Gryglewska, Magdalena Fedyk-Lukasik, Anna Skalska, Tomasz Grodzicki Department of Internal Medicine and Gerontology, Jagielonian University, Medical College, Krakow, Poland E-mail address: [email protected] (B. Gryglewska) Objective: The aim of the study was to assess the influence of heart failure presence on flow and flowmotion in skin microcirculation at rest, during ischemia and post occlusive hyperaemia. Material and methods: Studied population consisted of patients over 65 years with diagnosed heart failure. Body mass index (BMI) and blood pressure (BP) measurements, pulse wave velocity (PWVComplior), echocardiography (General Electrics Vivid 3) and Nterminal pro-B-type natriuretic peptide (NT proBNP) were performed. Skin microcirculation was assessed by laser Doppler flowmetry (PeriFlux PF 5000). Rest flow (RF), minimal flow during ischemia (biological zero — BZ) and post occlusive reactive hyperaemia (PORH) were registered. Fast Fourier's transformation of RF, BZ and PORH were also performed. Power frequency oscillations were evaluated in five frequency intervals of endothelial, neurogenic, myogenic, respiratory and heart origin. Data were analyzed in 2 groups: with BP values lower than 140/90 mmHg (I) and equal or higher than 140/90 mmHg (II). Statistical analysis was performed with U Mann–Whitney test, and Spearman correlation. Results: Study population consisted of 55 persons (I — 35 and II — 20 subjects) aged 70.6 ± 8.8 years, 60% men. Both groups were similar according to age, gender, PWV and ejection fraction (EF), mean RF, BZ and PORH flows. Group I had lower BP than group II (120.5 ± 11.2/73.5 ± 8.2 vs 153.4 ± 11.4/85.4 ± 8.1 mmHg, p b 0.001),
382
Abstracts
but higher levels of NT proBNP (1945 ± 2808 vs 727 ± 1420 pg/ml, p b 0.01) and myogenic flowmotion of RF, BZ and PORH (0.28 ± 0.23 vs 0.20 ± 0.21, 0.18 ± 0.20 vs 0.07 ± 0.09, 0.50 ± 0.30 vs 0.33 ± 0.27, respectively, p b 0.05). Myogenic oscillations of RF and PORH correlated positively with EF (r = 0.39, r = 0.36, respectively) and that oscillations of BZ with NT proBNP (r = 0.4). Conclusions: Myogenic oscillations of flow in microcirculation were higher in heart failure with lower BP values and significantly correlated with ejection fraction and NT proBNP. doi:10.1016/j.vph.2011.08.205
P.15.3 The comparison of antithrombotic properties of CORM-3 and CORM-A1 in arterial thrombosis in rats Karol Kramkowskia, Agnieszka Leszczynskaa, Andrzej Mogielnickia, Roberto Motterlinib, Stefan Chlopickic, Wlodzimierz Buczkoa a Department of Pharmacodynamics, Medical University of Bialystok, Poland b Department of Drug Discovery and Development, Italian Institute of Technology, Genova, Italy c Experimental Pharmacology, Jagiellonian University Medical College, Krakow, Poland E-mail address: [email protected] (K. Kramkowski) It has been shown that water-soluble CORM-3 inhibits platelet aggregation in vitro, due to the release of CO. Newly-synthesized CORM-A1, which does not contain transition metal — ruthenium (Ru) and its structure is based on sodium boranocarbonate liberates CO at a much slower rate than CORM-3 under physiological conditions. The aim of this work was to estimate in vivo the effects of new CORM-A1 in comparison to CORM-3 on electrically-stimulated arterial thrombosis in rats. CORM-3 inhibited electrically induced arterial thrombosis only at dose of 30 μmol/kg (0.7 ± 0.04 mg, p b 0.5 vs. 0.81 ± 0.02 mg in the VEH treated group), while the effect of CORM-A1 was strictly dosedependent (0.69 ± 0.07 mg, p b 0.05 and 0.62 ± 0.03 mg, p b 0.001 for rats treated with 10 and 30 μmol/kg CORM-A1, respectively; vs. 0.81 ± 0.02 mg in the VEH treated group. iCORM-3 and iCORM-A1 (compounds deprived of CO) failed to influence on thrombus weight (at doses of 30 μmol/kg). In thrombotic animals CORM-3 at doses of 10 and 30 μmol/kg did not inhibit collagen-stimulated platelet aggregation while CORM-A1 did (74.67 ± 19.47%, and 57.08 ± 10.33%, respectively vs. 100 ± 6.36% in VEH treated animals). The antiplatelet effect of CORM-A1, but not CORM-3 correlated with decrease in thrombus weight (r = 0.60227; p b 0.01). In thrombotic animals activity of PAI-1 was inhibited by treatment with CORM-A1 (30 μmol/kg; 1.28 ± 0.08 ng/ml vs. 1.60 ± 0.08 ng/ml in rats treated with VEH, p b 0.05) but not by CORM-3. Fibrin generation was profoundly inhibited by treatment with CORM-3 (10 and 30 μmol/kg, 405.0 ± 69.0 units and 162.3 ± 115.6 units vs. 919.5 ± 36.4 units for VEH, p b 0.01) while CORM-A1 had no effect. Also CORM-3 administered in dose of 30 μmol/kg decreased prothrombin time (23.06 ± 0.75 s vs. 21.10 ± 1.02 s for VEH treated animals, p b 0.05), while CORM-A1 did not. Neither of CORMs and iCORMs changed blood cell count and gasometry parameters in thrombotic and in healthy animals. In conclusion, we have shown that both CORM-3 and CORM-A1 possess antithrombotic activity in vivo. However, CORM-A1 releasing CO at a slower rate than CORM-3 rate possessed stronger antithrombotic, anti-platelet and profibrinolytic activities at in vivo conditions. In contrast, CORM-3 inhibited thrombosis mainly due to decreasing of fibrin production. doi:10.1016/j.vph.2011.08.206
P.15.4 A SILAC approach to MT1-MMP degradome in inflammatory angiogenesis Agnieszka Martínez-Koziola, Pilar Gonzaloa, Ángela Pollána, Alba Motaa, Nuria Coloméb, David Montanerc, Joaquín Dopazoc, Joaquín Arribasb, Francesc Canalsb, Alicia G. Arroyoa a Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain b Proteomics Laboratory and Medical Oncology Research Program Vall d'Hebron University Hospital Research Institute, Barcelona, Spain c Bioinformatics Department, Centro de Investigacion Príncipe Felipe, Valencia, Spain E-mail address: [email protected] (A. Martínez-Koziol) Angiogenesis, the formation of new capillaries from pre-existing vessels, is often coupled to inflammation in adult organisms. MT1MMP is a key protease required for angiogenesis induced by certain inflammatory factors. We have aimed at identifying the degradome (collection of substrates) of MT1-MMP in this context. To this end, we have performed quantitative proteomics, SILAC (stable isotope labelling of amino acids in cell culture), in TNFα-stimulated immortalized mouse lung endothelial cells (iMLEC) from wildtype and MT1-MMP-deficient mice. Glycoproteins identified in the supernatant as putative MT1-MMP substrates include extracellular matrix and related proteins some of them relevant to the angiogenic response; notably, a few seem to be unique substrates of MT1MMP. System biology analysis of this MT1-MMP endothelial degradome performed with Babelomics and FatiGO platforms revealed a combinatorial MT1-MMP substrate proteolytic program that governs endothelial cell adhesion, motility and chemotaxis finally leading to angiogenesis. Validation of selected substrates in 2D and 3D-endothelial cell models in vitro and in mouse retinas ex vivo underscores the relevance of MT1-MMP-mediated protein processing. In sum, SILAC identification of MT1-MMP degradome in TNFαactivated endothelial cells provides a system biology understanding of MT1-MMP contribution to inflammation-driven angiogenesis that might help to define surrogate markers in chronic inflammatory disorders such as psoriasis, rheumatoid arthritis and atherosclerosis. doi:10.1016/j.vph.2011.08.207
P.15.5 Anti-atherosclerotic action of agmatine in apoE-knockout mice Anna Gebska, Anna Niepsuj, Justyna Toton-Zuranska, Rafal Olszanecki, Ryszard Korbut Chair of Pharmacology, Jagiellonian University Medical College, Krakow, Poland E-mail address: [email protected] (A. Niepsuj) Atherosclerosis is an inflammatory disease, in which disturbances in lipid metabolism as well as enhanced apoptosis of various cells within vessel wall play important role. Importantly, the pathways of metabolism of fatty acids and apoptosis intersect in mitochondria. Agmatine is an endogenous polyamine produced by decarboxylation of l-arginine showing a wide array of biologic effects. It has been demonstrated that agmatine may regulate blood pressure, stimulate fatty acid oxidation and exert protective effect on mitochondria along with anti-inflammatory and neuroprotective action. We investigated anti-atherosclerotic action of exogenous agmatine (administrated p.o. for 4 months in dose of 20 mg/kg/day) in mouse model of atherosclerosis — apoE-knockout mice. As evidenced by en face and cross section methods, agmatine by 40% inhibited formation of atherosclerotic plaques in aorta of apoE knockout mice. Importantly, action of agmatine was associated with beneficial changes of the levels of