Miriam Belzunce

Different expression of MMPs/TIMP-1 in human atherosclerotic lesions. Relation to plaque features and vascular bed

BACKGROUND Proteolytic imbalance might determine arterial remodeling and plaque destabilization in atherosclerotic vessels. The aim of this study was to examine differences in the patterns of metalloproteinases (MMPs) and MMP inhibitor (TIMP-1) expression in advanced human atheromas, both in relation to the plaque features and the vascular bed involved. METHODS AND RESULTS Immunohistochemistry for MMP-1, -3, -9 and TIMP-1 as well as the collagen content were measured in vascular sections from patients undergoing peripheral revascularization (carotid n=11, femoral n=23) and aorto-coronary bypass surgery (mammary arteries n=20, as controls). Increased expression of all MMPs was detected in atherosclerotic as compared with control sections (P<0.01). Aneurysmal plaques showed a significant increase of MMP-1 and-3 and a reduction in total collagen (P<0.05) in relation to occlusive lesions. Calcification areas in atherosclerotic plaques were consistently associated with increased TIMP-1 expression (P<0.01). Finally, MMP-9 expression was higher in occlusive lesions from carotid than femoral arteries (P<0.01). CONCLUSIONS Aneurysm lesions expressed higher MMP-1 and-3 expression than occlusive plaques, and MMP-9 was mainly detected in carotid as compared with femoral arteries. TIMP-1 was associated with arterial calcification. These differences in the MMPs/TIMP-1 expression might determine the evolution of advanced atherosclerotic plaques and contribute to its vulnerability.

Phagocytic NADPH Oxidase-Dependent Superoxide Production Stimulates Matrix Metalloproteinase-9: Implications for Human Atherosclerosis

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.

Antioxidant vitamins increase the collagen content and reduce MMP-1 in a porcine model of atherosclerosis: implications for plaque stabilization

Degradation of extracellular matrix, particularly interstitial collagen, promotes plaque instability and contributes to restenosis after vascular injury. We have explored the effects of vitamins C and E on the collagen content and metalloproteinase-1 (MMP-1) expression after angioplasty in hypercholesterolemic pigs. Iliac angioplasty was performed on 18 minipigs divided into three diet groups: a normal-cholesterol (NC), a high-cholesterol (HC) and a high-cholesterol plus vitamins C+E (HCV). Four weeks later, after sacrifice, the vascular collagen content and MMP-1 protein expression, along with the plasma caseinolytic activity and lipid peroxidation, were measured. MMP-1 was also determined in arterial rings stimulated with native low-density lipoproteins (LDL) isolated from experimental groups. Cholesterol-rich diet augmented plasma lipid peroxidation (P<0.05), reduced the collagen content and increased vascular MMP-1 expression after injury (P<0.05). Enhanced caseinolytic activity (identified as MMP-1) was also observed in HC plasma samples and in supernatants from arterial rings incubated with HC-LDL. Vitamins C and E markedly increased neointimal collagen content (P<0.01), reduced the hypercholesterolemia-induced changes in vascular MMP-1 (P<0.05) and diminished plasma and ex vivo caseinolytic activity. Vitamins C and E may help stabilize atherosclerotic plaque after angioplasty and favor vascular remodeling by increasing collagen content and reducing vascular MMP-1 expression in porcine hypercholesterolemia.

Adipose stromal vascular fraction improves cardiac function in chronic myocardial infarction through differentiation and paracrine activity.

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 107 GFP-expressing fresh SVF cells or culture media as control. Heart function and tissue metabolism were determined by echocardiography and 18F-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.

The 4G/5G PAI-1 polymorphism influences the endothelial response to IL-1 and the modulatory effect of pravastatin

Summary.  Background: Increased plasminogen activator inhibitor (PAI‐1) levels lead to impaired fibrinolytic function associated with higher cardiovascular risk. PAI‐1 expression may be regulated by different inflammatory cytokines such as interleukin‐1α (IL‐1). Several polymorphisms have been described in the PAI‐1 gene. Aim: We examined the influence of the 4G/5G polymorphism in the promoter region on IL‐1α‐induced PAI‐1 expression by human umbilical vein endothelial cells (HUVEC) in presence or absence of pravastatin. Methods and results: Genotyped HUVEC were incubated with IL‐1α (500 U mL−1) in presence or absence of pravastatin (1–10 μm). PAI‐1 expression was analyzed by real time polymerase chain reaction (PCR), and PAI‐1 antigen measured in supernatants by ELISA. IL‐1α increased PAI‐1 secretion in a genotype‐dependent manner, and higher values were observed for 4G/4G compared with both 4G/5G and 5G/5G cultures (P < 0.05). Preincubation of HUVEC with 10 μm pravastatin significantly reduced IL‐1‐induced PAI‐1 expression in 4G/4G HUVEC compared with untreated cultures (177.5% ± 24.5% vs. 257.9% ± 39.0%, P < 0.05). Pravastatin also attenuated the amount of secreted PAI‐1 by 4G/4G HUVEC after IL‐1 stimulation (5020.6 ± 165.7 ng mL−1 vs. 4261.1 ± 309.8 ng mL−1, P < 0.05). This effect was prevented by coincubation with mevalonate, indicating a dependence on HMG‐CoA reductase inhibition. Conclusions: The endothelial 4G/5G PAI‐1 genotype influences the PAI‐1 response to IL‐1α and the modulatory effect of pravastatin. As increased PAI‐1 levels have been linked to cardiovascular disease the observed endothelial modulation by pravastatin may have potential clinical implications.

Design, Synthesis, and Biological Evaluation of Novel Matrix Metalloproteinase Inhibitors As Potent Antihemorrhagic Agents: From Hit Identification to an Optimized Lead

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, ).

Discovery and Safety Profiling of a Potent Preclinical Candidate, (4-[4-[[(3R)-3-(Hydroxycarbamoyl)-8-azaspiro[4.5]decan-3-yl]sulfonyl]phenoxy]-N-methylbenzamide) (CM-352), for the Prevention and Treatment of Hemorrhage

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 30 000 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.

Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke.

BACKGROUND Thrombin-activatable fibrinolysis inhibitor (TAFI) plays an important role in coagulation and fibrinolysis. Whereas TAFI deficiency may lead to a haemorrhagic tendency, data from TAFI knockout mice (TAFI-/-) are controversial and no differences have been reported in these animals after ischemic stroke. There are also no data regarding the role of circulating microparticles (MPs) in TAFI-/-. OBJECTIVES to examine the effect of tPA on the rate of intracranial haemorrhage (ICH) and on MPs generated in a model of ischemic stroke in TAFI-/- mice. METHODS Thrombin was injected into the middle cerebral artery (MCA) to analyse the effect of tPA (10mg/Kg) on the infarct size and haemorrhage in the absence of TAFI. Immunofluorescence for Fluoro-Jade C was performed on frozen brain slides to analyse neuronal degeneration after ischemia. MPs were isolated from mouse blood and their concentrations calculated by flow cytometry. RESULTS Compared with saline, tPA significantly increased the infarct size in TAFI-/- mice (p<0.05). Although plasma fibrinolytic activity (fibrin plate assay) was higher in these animals, no macroscopic or microscopic ICH was detected. A positive signal for apoptosis and degenerating neurons was observed in the infarct area, being significantly higher in tPA treated TAFI-/- mice (p<0.05). Interestingly, higher numbers of MPs were found in TAFI-/- plasma as compared to wild type, after stroke (p<0.05). CONCLUSIONS TAFI deficiency results in increased brain damage in a model of thrombolysis after ischemic stroke, which was not associated with bleeding but with neuronal degeneration and MP production.

Phenotypic Screening To Discover Novel Chemical Series as Efficient Antihemorrhagic Agents

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.

Metaloproteasa-10 (estromelisina-2): un nuevo marcador de aterosclerosis subclínica

Objetivo Las metaloproteasas (MMP) son una familia de endopeptidasas que degradan la matriz extracelular y desempenan un papel clave en la aterogenesis. La MMP-10 (estromelisina-2) se asocia con inflamacion en sujetos asintomaticos con factores de riesgo cardiovascular, pero se desconoce si se puede relacionar con la gravedad de la aterosclerosis. Este estudio analiza la relacion entre MMP-10, factores de riesgo aterosclerotico y aterosclerosis subclinica. Metodos y resultados Se midieron los valores circulantes de MMP-1, 9 y 10, y marcadores inflamatorios en 400 sujetos (edad media, 54,3 anos; 77,7% varones) con factores de riesgo, pero sin clinica cardiovascular. La aterosclerosis subclinica se evaluo mediante ultrasonidos en todos los sujetos y se midio tanto el espesor intimamedia (EIM) de la carotida, como la presencia de placas ateroscleroticas. Los valores de MMP-10 correlacionaron positivamente con el fibrinogeno, hs-proteina C reactiva (PCR) y EIM de la carotida. La asociacion de MMP-10 y EIM se mantuvo significativa en el analisis de regresion multiple (p Conclusion Valores sericos elevados de MMP-10 se asociaron con un incremento en los marcadores inflamatorios, el EIM carotideo y con la presencia de placas ateroscleroticas en sujetos asintomaticos. La MMP-10 puede ser util para identificar aterosclerosis subclinica en sujetos sin enfermedad cardiovascular.