ABSTRACT
Diabetes is an important risk factor for ischemic stroke (IS). Tissue-type plasminogen activator (tPA) has been associated with less successful revascularization and poor functional outcome in diabetes. We assessed whether a new thrombolytic strategy based on MMP10 was more effective than tPA in a murine IS model of streptozotocin (STZ)-induced diabetes. Wild-type mice were administered a single dose of streptozotocin (STZ) (180 mg/kg) to develop STZ-induced diabetes mellitus. Two weeks later, IS was induced by thrombin injection into the middle cerebral artery and the effect of recombinant MMP10 (6.5 µg/kg), tPA (10 mg/kg) or tPA/MMP10 on brain damage and functional outcome were analysed. Motor activity was assessed using the open field test. Additionally, we studied plasminogen activator inhibitor-1 (PAI-1) and thrombin-antithrombin complex levels (TAT) by ELISA and oxidative stress and blood-brain barrier (BBB) integrity by immunohistochemistry and western blot. MMP10 treatment was more effective at reducing infarct size and neurodegeneration than tPA 24 h and 3 days after IS in diabetic mice. Locomotor activity was impaired by hyperglycemia and ischemic injury, but not by the thrombolytic treatments. Additionally, TAT, oxidative stress and BBB permeability were reduced by MMP10 treatment, whereas brain bleeding or PAI-1 expression did not differ between treatments. Thrombolytic treatment with MMP10 was more effective than tPA at reducing stroke and neurodegeneration in a diabetic murine model of IS, without increasing haemorrhage. Thus, we propose MMP10 as a potential candidate for the clinical treatment of IS in diabetic patients.
Subject(s)
Brain Ischemia/drug therapy , Diabetes Mellitus, Experimental/drug therapy , Matrix Metalloproteinase 10/administration & dosage , Thrombolytic Therapy/methods , Administration, Intravenous , Animals , Brain Ischemia/blood , Brain Ischemia/pathology , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/pathology , Fibrinolytic Agents/administration & dosage , Male , Mice , Random Allocation , Stroke , Tissue Plasminogen Activator/administration & dosageABSTRACT
BACKGROUND AND AIMS: Matrix metalloproteinases (MMPs) have been implicated in atherosclerosis and vascular calcification. Among them, we reported that MMP10 is present in human atheroma, associated with atherosclerosis. However, it remains unclear whether MMP10 is involved in atherogenesis and vascular calcification. METHODS: MMP10 was measured in serum from patients with subclinical atherosclerosis and analyzed in carotid endarterectomies by immunostaining. ApoE-deficient mice (Apoe-/-) were crossed to MMP10-deficient (Mmp10-/-) mice and followed up to 20 months. Plaque area and composition were assessed by histology and immunohistochemistry. Inflammatory markers were measured in atherosclerotic plaques by RT-qPCR, and leukocyte subpopulations were analyzed by flow cytometry. In vitro calcification assays were performed in aortic vascular smooth muscle cells (VSMC). RESULTS: MMP10 serum levels were associated with coronary calcification in subjects with subclinical atherosclerosis. Immunostaining revealed MMP10 expression in human atheromas, spatially associated with calcification areas, and complicated plaques released higher amounts of MMP10 than non-diseased segments. Interestingly, vascular MMP10 expression was confined to the atherosclerotic lesion in Apoe-/- mice, and Apoe-/-Mmp10-/- showed a substantial reduction in atherosclerotic lesion size, macrophage content and plaque calcification. Reduced local and systemic inflammatory markers could be demonstrated in Apoe-/-Mmp10-/- by gene expression and flow cytometry analysis. Calcium phosphate deposition and vascular calcification markers were downregulated in VSMC from Apoe-/-Mmp10-/- mice. CONCLUSIONS: Delayed plaque progression and altered cellular composition in the absence of MMP10 suggests that MMP10 plays a role in atherosclerosis, favoring inflammation, development and complication of the plaque.
Subject(s)
Matrix Metalloproteinase 10/deficiency , Matrix Metalloproteinase 10/physiology , Plaque, Atherosclerotic/metabolism , Aged , Animals , CD11b Antigen/metabolism , Disease Progression , Endarterectomy, Carotid , Female , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Humans , Inflammation , Male , Matrix Metalloproteinase 10/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout, ApoE , Middle Aged , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Vascular Calcification/pathologyABSTRACT
In an effort to find novel chemical series as antifibrinolytic agents, we explore α-phenylsulfonyl-α-spiropiperidines bearing different zinc-binding groups (ZBGs) to target those metalloproteinases involved in the fibrinolytic process: MMP3 and MMP10. Surprisingly, all these new chemical series were inactive against these metalloproteinases; however, several new molecules retained the antifibrinolytic activity in a phenotypic functional assay using thromboelastometry and human whole blood. Further optimization led to compound 38 as a potent antifibrinolytic agent in vivo, three times more efficacious than the current standard-of-care (tranexamic acid, TXA) at 300 times lower dose. Finally, in order to decipher the underlying mode-of-action leading to this phenotypic response, an affinity-based probe 39 was successfully designed to identify the target involved in this response: a potentially unknown mechanism-of-action in the fibrinolytic process.
ABSTRACT
Discovery of potent and safe therapeutics that improve upon currently available antifibrinolytics, e.g., tranexamic acid (TXA, 1) and aprotinin, has been challenging. Matrix metalloproteinases (MMPs) participate in thrombus dissolution. Then we designed a novel series of optimized MMP inhibitors that went through phenotypic screening consisting of thromboelastometry and mouse tail bleeding. Our optimized lead compound, CM-352 (2), inhibited fibrinolysis in human whole blood functional assays and was more effective than the current standard of care, 1, in the tail-bleeding model using a 30â¯000 times lower dose. Moreover, 2 reduced blood loss during liver hepatectomy, while 1 and aprotinin had no effect. Molecule 2 displayed optimal pharmacokinetic and safety profiles with no evidence of thrombosis or coagulation impairment. This novel mechanism of action, targeting MMP, defines a new class of antihemorrhagic agents without interfering with normal hemostatic function. Furthermore, 2 represents a preclinical candidate for the acute treatment of bleeding.
Subject(s)
Benzamides/pharmacology , Drug Evaluation, Preclinical/methods , Hemorrhage/prevention & control , Hemostatics/chemistry , Hemostatics/pharmacology , Hydroxamic Acids/pharmacology , Animals , Antifibrinolytic Agents/chemistry , Antifibrinolytic Agents/pharmacology , Benzamides/chemistry , Caco-2 Cells/drug effects , Drug Discovery/methods , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Hemorrhage/drug therapy , Hemorrhage/metabolism , Humans , Hydroxamic Acids/chemistry , Matrix Metalloproteinase 10/metabolism , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase Inhibitors/chemistry , Matrix Metalloproteinase Inhibitors/pharmacology , Mice, Inbred C57BL , Molecular Structure , Molecular Targeted Therapy/methodsABSTRACT
Growing evidence suggests that matrix metalloproteinases (MMP) are involved in thrombus dissolution; then, considering that new therapeutic strategies are required for controlling hemorrhage, we hypothesized that MMP inhibition may reduce bleeding by delaying fibrinolysis. Thus, we designed and synthesized a novel series of MMP inhibitors to identify potential candidates for acute treatment of bleeding. Structure-based and knowledge-based strategies were utilized to design this novel chemical series, α-spiropiperidine hydroxamates, of potent and soluble (>75 µg/mL) pan-MMP inhibitors. The initial hit, 12, was progressed to an optimal lead 19d. Racemic 19d showed a remarkable in vitro phenotypic response and outstanding in vivo efficacy; in fact, the mouse bleeding time at 1 mg/kg was 0.85 min compared to 29.28 min using saline. In addition, 19d displayed an optimal ADME and safety profile (e.g., no thrombus formation). Its corresponding enantiomers were separated, leading to the preclinical candidate 5 (described in Drug Annotations series, J. Med. Chem. 2015, ).
Subject(s)
Benzamides/chemical synthesis , Benzamides/pharmacology , Drug Design , Hemorrhage/drug therapy , Hydroxamic Acids/chemistry , Matrix Metalloproteinase Inhibitors/chemistry , Matrix Metalloproteinase Inhibitors/pharmacology , Matrix Metalloproteinases/chemistry , Animals , Cell Membrane Permeability/drug effects , Humans , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/pharmacology , Male , Matrix Metalloproteinases/metabolism , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
Fresh adipose-derived cells have been shown to be effective in the treatment of acute myocardial infarction (MI), but their role in the chronic setting is unknown. We sought to determine the long-term effect of the adipose derived-stromal vascular fraction (SVF) cell transplantation in a rat model of chronic MI. MI was induced in 82 rats by permanent coronary artery ligation and 5 weeks later rats were allocated to receive an intramyocardial injection of 10(7) GFP-expressing fresh SVF cells or culture media as control. Heart function and tissue metabolism were determined by echocardiography and (18)F-FDG-microPET, respectively, and histological studies were performed for up to 3 months after transplantation. SVF induced a statistically significant long-lasting (3 months) improvement in cardiac function and tissue metabolism that was associated with increased revascularization and positive heart remodeling, with a significantly smaller infarct size, thicker infarct wall, lower scar fibrosis, and lower cardiac hypertrophy. Importantly, injected cells engrafted and were detected in the treated hearts for at least 3 months, directly contributing to the vasculature and myofibroblasts and at negligible levels to cardiomyocytes. Furthermore, SVF release of angiogenic (VEGF and HGF) and proinflammatory (MCP-1) cytokines, as well as TIMP1 and TIMP4, was demonstrated in vitro and in vivo, strongly suggesting that they have a trophic effect. These results show the potential of SVF to contribute to the regeneration of ischemic tissue and to provide a long-term functional benefit in a rat model of chronic MI, by both direct and indirect mechanisms.
Subject(s)
Adipocytes/cytology , Myocardial Infarction/therapy , Paracrine Communication , Stromal Cells/transplantation , Ventricular Remodeling , Angiogenic Proteins/metabolism , Animals , Cell Differentiation , Chronic Disease , Cytokines/metabolism , Disease Models, Animal , Echocardiography , Female , Heart Ventricles/physiopathology , Myocardial Infarction/pathology , Myocardial Revascularization , Phenotype , Positron-Emission Tomography , Rats , Rats, Sprague-Dawley , Stromal Cells/cytology , Tissue Inhibitor of Metalloproteinase-1/metabolism , Tissue Inhibitor of Metalloproteinases/metabolism , Tissue Inhibitor of Metalloproteinase-4ABSTRACT
Objetivo. Las metaloproteasas (MMP) son una familia de endopeptidasas que degradan la matriz extracelular y desempeñan un papel clave en la aterogénesis. La MMP-10 (estromelisina-2) se asocia con inflamación en sujetos asintomáticos con factores de riesgo cardiovascular, pero se desconoce si se puede relacionar con la gravedad de la aterosclerosis. Este estudio analiza la relación entre MMP-10, factores de riesgo aterosclerótico y aterosclerosis subclínica. Métodos y resultados. Se midieron los valores circulantes de MMP-1, 9 y 10, y marcadores inflamatorios en 400 sujetos (edad media, 54,3 años; 77,7% varones) con factores de riesgo, pero sin clínica cardiovascular. La aterosclerosis subclínica se evaluó mediante ultrasonidos en todos los sujetos y se midió tanto el espesor íntima-media (EIM) de la carótida, como la presencia de placas ateroscleróticas. Los valores de MMP-10 correlacionaron positivamente con el fibrinógeno, hs-proteína C reactiva (PCR) y EIM de la carótida. La asociación de MMP-10 y EIM se mantuvo significativa en el análisis de regresión múltiple (p < 0,02) cuando se ajustó por los factores de riesgo aterosclerótico tradicionales y los marcadores inflamatorios. Los sujetos en el tercil superior de MMP-10 presentaron un incremento del EIM carotídeo (odds ratio ajustada 6,3 [1,3-31,4]; p = 0,024). Además, los valores de MMP-10 fueron mayores en pacientes con placas en la carótida (n = 78) que en los que no tenían placas, tras ajustar para edad y sexo (p < 0,01). Conclusión. Valores séricos elevados de MMP-10 se asociaron con un incremento en los marcadores inflamatorios, el EIM carotídeo y con la presencia de placas ateroscleróticas en sujetos asintomáticos. La MMP-10 puede ser útil para identificar aterosclerosis subclínica en sujetos sin enfermedad cardiovascular (AU)
Objective. MMPs are a family of endopeptidase which play a key role in atherogenesis through ECM degradation. MMP-10 (stromelysin-2) has been associated with inflammation in asymptomatic subjects with cardiovascular risk factors. Whether MMP-10 is associated with the severity of atherosclerosis remains to be determined. We examined the relationship of systemic MMP-10 levels with cardiovascular risk factors and subclinical atherosclerosis. Methods and results. MMP-1, -9, and -10 levels and markers of inflammation were measured in 400 subjects (mean age 54.3 years; 77.7% men) with cardiovascular risk factors but free of clinical cardiovascular disease. Severity of carotid atherosclerosis was evaluated by carotid intima-media thickness (IMT) and the presence of atherosclerotic plaques with the use of B-mode ultrasound. MMP-10 levels correlated with fibrinogen, hs-C reactive protein (CRP) and carotid IMT. The association of MMP-10 and IMT remained significant in multiple regression analysis (P<.02) when controlling for traditional risk factors and inflammatory markers. Subjects in the highest MMP-10 tertile had significantly higher carotid IMT (adjusted OR 6.3 [1.3-31.4]; P=.024). In addition, MMP-10 levels were elevated in subjects with higher carotid IMT (P<.01) and in patients with carotid plaques (n = 78, P<.01). Conclusions. Higher systemic MMP-10 levels were associated with inflammation, increased carotid IMT and atherosclerotic plaques in asymptomatic subjects. Circulating MMP-10 may be useful marker for subclinical atherosclerosis in subjects free of cardiovascular disease (AU)
Subject(s)
Male , Female , Adult , Middle Aged , Humans , Metalloproteases/immunology , Arteriosclerosis/immunology , Metalloproteases/genetics , Metalloproteases , Metalloproteases/blood , Arteriosclerosis/diagnosis , BiomarkersABSTRACT
OBJECTIVE: Data suggest that matrix metalloproteinase-9 (MMP-9) has a role in atherosclerosis. The phagocytic NADPH oxidase has been also associated with atherosclerosis. This study aimed to investigate the association between phagocytic NADPH oxidase and MMP-9 in human atherosclerosis. METHODS AND RESULTS: In vitro experiments performed in human monocytes showed that NADPH oxidase activation enhanced MMP-9 secretion and activity, determined by enzyme-linked immunosorbent assay and zymography, respectively. Immunohistochemical study showed that phagocytic NADPH oxidase localized with MMP-9 in endarterectomies from patients with carotid stenosis. In addition, a positive relationship (P<0.001) was found between phagocytic NADPH oxidase-dependent superoxide production determined with lucigenin and plasma MMP-9 levels in 188 asymptomatic subjects free of overt clinical atherosclerosis. In multivariate analysis, this association remained significant after adjustment for cardiovascular risk factors. Interestingly, subjects in the upper quartile of superoxide production exhibited the highest values of MMP-9, oxidized low-density lipoprotein, nitrotyrosine, carotid intima media thickness, and an increased presence of carotid plaques. CONCLUSIONS: Enhanced NADPH oxidase-dependent *O2(-) production stimulates MMP-9 in monocytes and this relationship may be relevant in the atherosclerotic process. Moreover, MMP-9 emerges as an important mediator of the phagocytic NADPH oxidase-dependent oxidative stress in atherosclerosis.
Subject(s)
Carotid Artery Diseases/enzymology , Carotid Artery, Common/enzymology , Matrix Metalloproteinase 9/metabolism , NADPH Oxidases/metabolism , Superoxides/metabolism , Adult , Analysis of Variance , Carotid Artery Diseases/physiopathology , Carotid Artery, Common/pathology , Case-Control Studies , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Humans , Leukocytes, Mononuclear/enzymology , Male , Middle Aged , Multivariate Analysis , Oxidative Stress/physiology , Oxygen/metabolism , Phagocytes/enzymology , Probability , Sensitivity and SpecificityABSTRACT
OBJECTIVE: Our aim was to compare the efficacy of surgical versus percutaneous administration of skeletal myoblasts (SkM) in a swine model of chronic myocardial infarction and to determine the mechanism(s) involved in their beneficial effect. METHODS: Two months after induction of myocardial infarction (MI), Goettingen miniature pigs underwent autologous SkM transplant either by direct surgical injection (n=6) or percutaneous access and intramyocardial delivery under fluoroscopic and echocardiographic guidance (n=6). Control animals received media alone (n=4). Functional analysis was performed by 2D echocardiography. Myoblast engraftment, in vivo cell differentiation, vessel formation, fibrosis, and the ratio between collagen type I/III deposition were analyzed in the infarct (IA) and non-infarct area (NIA) by immunohistochemistry. RESULTS: Animals received a median of 407.55+/-115x10(6) BrdU-labeled autologous SkM. Myoblast transplant was associated with a statistically significant increase in left ventricular ejection fraction (p<0.01), increased vasculogenesis and decreased fibrosis (p<0.05), and reduced collagen type I/III ratio in the IA and NIA areas as compared with control animals. No differences were found between groups receiving SkM by percutaneous or surgical access. CONCLUSIONS: Our results indicate that increased vasculogenesis and changes in matrix remodeling with decreased fibrosis are associated with the beneficial effect of SkM transplant in chronic MI. The equivalent benefit observed from surgical and percutaneous delivery has important clinical implications.
