Molecular Mechanisms of Desensitization Underlying the Differential Effects of Formyl Peptide Receptor 2 Agonists on Cardiac Structure-Function Post Myocardial Infarction.

Formyl peptide receptor 2 (FPR2) plays an integral role in the transition of macrophages from a pro-inflammatory program to one that is pro-resolving. FPR2-mediated stimulation of resolution post myocardial infarction has demonstrated efficacy in rodent models and is hypothesized to reduce progression into heart failure. FPR2 agonists that promote long-lasting receptor internalization can lead to persistent desensitization and diminished therapeutic benefits. In vitro signaling profiles and propensities for receptor desensitization of two clinically studied FPR2 agonists, namely, BMS-986235 and ACT-389949, were evaluated. In contrast to BMS-986235, pre-stimulation with ACT-389949 led to a decrease in its potency to inhibit cAMP production. Moreover, ACT-389949 displayed greater efficacy for ß-arrestin recruitment, while efficacy of Gi activation was similar for both agonists. Following agonist-promoted FPR2 internalization, effective recycling to the plasma membrane was observed only with BMS-986235. Use of G protein-coupled receptor kinase (GRK) knock-out cells revealed a differential impact of GRK2 versus GRK5/6 on ß-arrestin recruitment and Gi activation promoted by the two FPR2 agonists. In vivo, decreases of granulocytes in circulation were greatly diminished in mice treated with ACT-389949 but not for BMS-986235. With short-term dosing, both compounds induced a pro-resolution polarization state in cardiac monocyte/macrophages post myocardial infarction. By contrast, with long-term dosing, only BMS-986235 preserved the infarct wall thickness and increased left ventricular ejection fraction in a rat model of myocardial infarction. Altogether, the study shows that differences in the desensitization profiles induced by ACT-389949 and BMS-986235 at the molecular level may explain their distinct inflammatory/pro-resolving activities in vivo.

Spatial correlates of COVID-19 first wave across continental Portugal.

The first case of COVID-19 in continental Portugal was documented on the 2nd of March 2020 and about seven months later more than 75 thousand infections had been reported. Although several factors correlate significantly with the spatial incidence of COVID-19 worldwide, the drivers of spatial incidence of this virus remain poorly known and need further exploration. In this study, we analyse the spatiotemporal patterns of COVID-19 incidence in the at the municipality level and test for significant relationships between these patterns and environmental, socioeconomic, demographic and human mobility factors to identify the mains drivers of COVID-19 incidence across time and space. We used a generalized liner mixed model, which accounts for zero inflated cases and spatial autocorrelation to identify significant relationships between the spatiotemporal incidence and the considered set of driving factors. Some of these relationships were particularly consistent across time, including the 'percentage of employment in services'; 'average time of commuting using individual transportation'; 'percentage of employment in the agricultural sector'; and 'average family size'. Comparing the preventive measures in Portugal (e.g., restrictions on mobility and crowd around) with the model results clearly show that COVID-19 incidence fluctuates as those measures are imposed or relieved. This shows that our model can be a useful tool to help decision-makers in defining prevention and/or mitigation policies.

Discovery of Heteroaryl Urea Isosteres for Formyl Peptide Receptor 2 Agonists.

Formyl peptide receptor 2 (FPR2) agonists have shown efficacy in inflammatory-driven animal disease models and have the potential to treat a range of diseases. Many reported synthetic agonists contain a phenylurea, which appears to be necessary for activity in the reported chemotypes. We set out to find isosteres for the phenylurea and focused our efforts on heteroaryl rings. The wide range of potencies with heterocyclic isosteres demonstrates how electronic effects of the heteroatom placement impact molecular recognition. Herein, we report our discovery of benzimidazole and aminophenyloxadiazole FPR2 agonists with low nanomolar activity.

Formyl peptide receptor 2 and heart disease.

Formyl peptide receptor type 2 (FPR2) regulates the initiation and resolution phases of the inflammatory response. In the setting of heart injury and disease, dysregulated inflammation can potentiate maladaptive healing and pathological remodeling of the heart leading to cardiac dysfunction and failure. The potential to regulate and resolve adverse inflammation is postulated to improve outcome in the setting of heart disease. This review covers emerging concepts on the role of FPR2 in heart disease and strategies to activate pro-resolution processes to limit disease progression. We summarize key preclinical studies that support use of FPR2 agonists in heart disease. Finally, we briefly discuss the status of FPR2 agonists under evaluation in the clinic.

Selective FPR2 Agonism Promotes a Proresolution Macrophage Phenotype and Improves Cardiac Structure-Function Post Myocardial Infarction.

Dysregulated inflammation following myocardial infarction (MI) leads to maladaptive healing and remodeling. The study characterized and evaluated a selective formyl peptide receptor 2 (FPR2) agonist BMS-986235 in cellular assays and in rodents undergoing MI. BMS-986235 activated G proteins and promoted ß-arrestin recruitment, enhanced phagocytosis and neutrophil apoptosis, regulated chemotaxis, and stimulated interleukin-10 and monocyte chemoattractant protein-1 gene expression. Treatment with BMS-986235 improved mouse survival, reduced left ventricular area, reduced scar area, and preserved wall thickness. Treatment increased macrophage arginase-1 messenger RNA and CD206 receptor levels indicating a proresolution phenotype. In rats following MI, BMS-986235 preserved viable myocardium, attenuated left ventricular remodeling, and increased ejection fraction relative to control animals. Therefore, FPR2 agonism improves post-MI healing, limits remodeling and preserves function, and may offer an innovative therapeutic option to improve outcomes.

Adaptive laboratory evolution of native methanol assimilation in Saccharomyces cerevisiae.

Utilising one-carbon substrates such as carbon dioxide, methane, and methanol is vital to address the current climate crisis. Methylotrophic metabolism enables growth and energy generation from methanol, providing an alternative to sugar fermentation. Saccharomyces cerevisiae is an important industrial microorganism for which growth on one-carbon substrates would be relevant. However, its ability to metabolize methanol has been poorly characterised. Here, using adaptive laboratory evolution and 13C-tracer analysis, we discover that S. cerevisiae has a native capacity for methylotrophy. A systems biology approach reveals that global rearrangements in central carbon metabolism fluxes, gene expression changes, and a truncation of the uncharacterized transcriptional regulator Ygr067cp supports improved methylotrophy in laboratory evolved S. cerevisiae. This research paves the way for further biotechnological development and fundamental understanding of methylotrophy in the preeminent eukaryotic model organism and industrial workhorse, S. cerevisiae.

A comprehensive approach to understanding flood risk drivers at the municipal level.

During the period 1998-2017, floods were responsible for 11% of the loss of life and 23% of the economic loss caused by climate-related and geophysical-related disasters worldwide. An integrated and effective definition of flood risk management strategies therefore still requires synthesized and comprehensive knowledge about the driving forces of flood risk. In this study, 278 Portuguese municipalities are analyzed and classified according to flood hazard, exposure, and vulnerability. After evaluating the three components that describe risk, an index of the flood risk is calculated and a cluster analysis is further performed to understand the role of the risk drivers (hazard, exposure, and vulnerability) in each municipality. The proposed approach therefore provides flood risk indexes on a municipal basis, which are built upon different sources of both cell-by-cell data and an aggregation of municipal-level data that has been statistically validated. Municipalities both in the NW part of the country and along the valleys of major rivers demonstrate a significant superimposition of high levels of exposure and hazard, while vulnerability presents a disperse pattern throughout the country. The results obtained using this approach should contribute to the diversification of flood risk management strategies. This is still lacking in the majority of the national-level flood risk governance processes, namely those strategies that focus on the contingency of daily activities and those aiming at a long-term reduction of the exposure, vulnerability, and hazard components that shape flood disasters.

Engineering Escherichia coli for propionic acid production through the Wood-Werkman cycle.

Native to propionibacteria, the Wood-Werkman cycle enables propionate production via succinate decarboxylation. Current limitations in engineering propionibacteria strains have redirected attention toward the heterologous production in model organisms. Here, we report the functional expression of the Wood-Werkman cycle in Escherichia coli to enable propionate and 1-propanol production. The initial proof-of-concept attempt showed that the cycle can be used for production. However, production levels were low (0.17 mM). In silico optimization of the expression system by operon rearrangement and ribosomal-binding site tuning improved performance by fivefold. Adaptive laboratory evolution further improved performance redirecting almost 30% of total carbon through the Wood-Werkman cycle, achieving propionate and propanol titers of 9 and 5 mM, respectively. Rational engineering to reduce the generation of byproducts showed that lactate (∆ldhA) and formate (∆pflB) knockout strains exhibit an improved propionate and 1-propanol production, while the ethanol (∆adhE) knockout strain only showed improved propionate production.

Preservation of Post-Infarction Cardiac Structure and Function via Long-Term Oral Formyl Peptide Receptor Agonist Treatment.

Dysregulated inflammation following myocardial infarction (MI) promotes left ventricular (LV) remodeling and loss of function. Targeting inflammation resolution by activating formyl peptide receptors (FPRs) may limit adverse remodeling and progression towards heart failure. This study characterized the cellular and signaling properties of Compound 43 (Cmpd43), a dual FPR1/FPR2 agonist, and examined whether Cmpd43 treatment improves LV and infarct remodeling in rodent MI models. Cmpd43 stimulated FPR1/2-mediated signaling, enhanced proresolution cellular function, and modulated cytokines. Cmpd43 increased LV function and reduced chamber remodeling while increasing proresolution macrophage markers. The findings demonstrate that FPR agonism improves cardiac structure and function post-MI.

The BCR-ABL1 Inhibitors Imatinib and Ponatinib Decrease Plasma Cholesterol and Atherosclerosis, and Nilotinib and Ponatinib Activate Coagulation in a Translational Mouse Model.

Treatment with the second and third generation BCR-ABL1 tyrosine kinase inhibitors (TKIs) increases cardiovascular risk in chronic myeloid leukemia (CML) patients. We investigated the vascular adverse effects of three generations of TKIs in a translational model for atherosclerosis, the APOE*3Leiden.CETP mouse. Mice were treated for sixteen weeks with imatinib (150 mg/kg BID), nilotinib (10 and 30 mg/kg QD) or ponatinib (3 and 10 mg/kg QD), giving similar drug exposures as in CML-patients. Cardiovascular risk factors were analyzed longitudinally, and histopathological analysis of atherosclerosis and transcriptome analysis of the liver was performed. Imatinib and ponatinib decreased plasma cholesterol (imatinib, -69%, p < 0.001; ponatinib 3 mg/kg, -37%, p < 0.001; ponatinib 10 mg/kg-44%, p < 0.001) and atherosclerotic lesion area (imatinib, -78%, p < 0.001; ponatinib 3 mg/kg, -52%, p = 0.002; ponatinib 10 mg/kg, -48%, p = 0.006), which were not affected by nilotinib. In addition, imatinib increased plaque stability. Gene expression and pathway analysis demonstrated that ponatinib enhanced the mRNA expression of coagulation factors of both the contact activation (intrinsic) and tissue factor (extrinsic) pathways. In line with this, ponatinib enhanced plasma levels of FVII, whereas nilotinib increased plasma FVIIa activity. While imatinib showed a beneficial cardiovascular risk profile, nilotinib and ponatinib increased the cardiovascular risk through induction of a pro-thrombotic state.

Genome-scale model guided design of Propionibacterium for enhanced propionic acid production.

Production of propionic acid by fermentation of propionibacteria has gained increasing attention in the past few years. However, biomanufacturing of propionic acid cannot compete with the current oxo-petrochemical synthesis process due to its well-established infrastructure, low oil prices and the high downstream purification costs of microbial production. Strain improvement to increase propionic acid yield is the best alternative to reduce downstream purification costs. The recent generation of genome-scale models for a number of Propionibacterium species facilitates the rational design of metabolic engineering strategies and provides a new opportunity to explore the metabolic potential of the Wood-Werkman cycle. Previous strategies for strain improvement have individually targeted acid tolerance, rate of propionate production or minimisation of by-products. Here we used the P. freudenreichii subsp. shermanii and the pan-Propionibacterium genome-scale metabolic models (GEMs) to simultaneously target these combined issues. This was achieved by focussing on strategies which yield higher energies and directly suppress acetate formation. Using P. freudenreichii subsp. shermanii, two strategies were assessed. The first tested the ability to manipulate the redox balance to favour propionate production by over-expressing the first two enzymes of the pentose-phosphate pathway (PPP), Zwf (glucose-6-phosphate 1-dehydrogenase) and Pgl (6-phosphogluconolactonase). Results showed a 4-fold increase in propionate to acetate ratio during the exponential growth phase. Secondly, the ability to enhance the energy yield from propionate production by over-expressing an ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK) and sodium-pumping methylmalonyl-CoA decarboxylase (MMD) was tested, which extended the exponential growth phase. Together, these strategies demonstrate that in silico design strategies are predictive and can be used to reduce by-product formation in Propionibacterium. We also describe the benefit of carbon dioxide to propionibacteria growth, substrate conversion and propionate yield.

Self-Assembling Cyclic d,l-α-Peptides as Modulators of Plasma HDL Function. A Supramolecular Approach toward Antiatherosclerotic Agents.

There is great interest in developing new modes of therapy for atherosclerosis to treat coronary heart disease and stroke, particularly ones that involve modulation of high-density lipoproteins (HDLs). Here, we describe a new supramolecular chemotype for altering HDL morphology and function. Guided by rational design and SAR-driven peptide sequence enumerations, we have synthesized and determined the HDL remodeling activities of over 80 cyclic d,l-α-peptides. We have identified a few distinct sequence motifs that are effective in vitro in remodeling human and mouse plasma HDLs to increase the concentration of lipid-poor pre-beta HDLs, which are key initial acceptors of cholesterol in the reverse cholesterol transport (RCT) process, and concomitantly promote cholesterol efflux from macrophage cells. Functional assays with various control peptides, such as scrambled sequences, linear and enantiomeric cyclic peptide variants, and backbone-modified structures that limit peptide self-assembly, provide strong support for the supramolecular mode of action. Importantly, when the lead cyclic peptide c[wLwReQeR] was administered to mice (ip), it also promoted the formation of small, lipid-poor HDLs in vivo, displayed good plasma half-life (∼6 h), did not appear to have adverse side effects, and exerted potent anti-inflammatory effects in an acute in vivo inflammation assay. Given that previously reported HDL remodeling peptides have been based on α-helical apoA-I mimetic architectures, the present study, involving a new structural class, represents a promising step toward new potential therapeutics to combat atherosclerosis.

P2Y6 receptor potentiates pro-inflammatory responses in macrophages and exhibits differential roles in atherosclerotic lesion development.

BACKGROUND: P2Y(6), a purinergic receptor for UDP, is enriched in atherosclerotic lesions and is implicated in pro-inflammatory responses of key vascular cell types and macrophages. Evidence for its involvement in atherogenesis, however, has been lacking. Here we use cell-based studies and three murine models of atherogenesis to evaluate the impact of P2Y(6) deficiency on atherosclerosis. METHODOLOGY/PRINCIPAL FINDINGS: Cell-based studies in 1321N1 astrocytoma cells, which lack functional P2Y(6) receptors, showed that exogenous expression of P2Y(6) induces a robust, receptor- and agonist-dependent secretion of inflammatory mediators IL-8, IL-6, MCP-1 and GRO1. P2Y(6)-mediated inflammatory responses were also observed, albeit to a lesser extent, in macrophages endogenously expressing P2Y(6) and in acute peritonitis models of inflammation. To evaluate the role of P2Y(6) in atherosclerotic lesion development, we used P2Y(6)-deficient mice in three mouse models of atherosclerosis. A 43% reduction in aortic arch plaque was observed in high fat-fed LDLR knockout mice lacking P2Y(6) receptors in bone marrow-derived cells. In contrast, no effect on lesion development was observed in fat-fed whole body P2Y(6)xLDLR double knockout mice. Interestingly, in a model of enhanced vascular inflammation using angiotensin II, P2Y(6) deficiency enhanced formation of aneurysms and exhibited a trend towards increased atherosclerosis in the aorta of LDLR knockout mice. CONCLUSIONS: P2Y(6) receptor augments pro-inflammatory responses in macrophages and exhibits a pro-atherogenic role in hematopoietic cells. However, the overall impact of whole body P2Y(6) deficiency on atherosclerosis appears to be modest and could reflect additional roles of P2Y(6) in vascular disease pathophysiologies, such as aneurysm formation.

Myocardial matrix metalloproteinase-2: inside out and upside down.

Since their inaugural discovery in the early 1960s, matrix metalloproteinases (MMPs) have been shown to mediate multiple physiological and pathological processes. In addition to their canonical function in extracellular matrix (ECM) remodeling, research in the last decade has highlighted new MMP functions, including proteolysis of novel substrates beyond ECM proteins, MMP localization to subcellular organelles, and proteolysis of susceptible intracellular proteins in those subcellular compartments. This review will provide a comparison of the extracellular and intracellular roles of MMPs, illustrating that MMPs are far more interesting than the one-dimensional view originally taken. We focus on the roles of MMP-2 in cardiac injury and repair, as this is one of the most studied MMPs in the cardiovascular field. We will highlight how understanding all dimensions, such as localization of activity and timing of interventions, will increase the translational potential of research findings. Building upon old ideas and turning them inside out and upside down will help us to better understand how to move the MMP field forward.

Dynamic changes in myocardial matrix and relevance to disease: translational perspectives.

The cardiac extracellular matrix (ECM) provides the architectural scaffold to support efficient contraction and relaxation of cardiomyocytes. The elegant design of the ECM facilitates optimal force transduction, electric transmission, intercellular communication, and metabolic exchange within the myocardial microenvironment. In the setting of increased wall stress, injury, or disease, the ECM can undergo a series of dynamic changes that lead to favorable chamber remodeling and functional adaptation. Over time, sustained matrix remodeling can impair diastolic and systolic function caused by excess deposition of interstitial fibrous tissue. These pathological alterations in ECM structure/function are considered central to the evolution of adverse cardiac remodeling and the development of heart failure. This review discusses the complex dynamics of the cardiac ECM in the setting of myocardial infarction, pressure overload, and volume overload. We also summarize the current status of ECM biomarkers that may have clinical value in prognosticating cardiac disease progression in patients. Finally, we discuss the most current status of drugs under evaluation for use in cardiac fibrosis.

11ß-hydroxysteroid dehydrogenase type 1 gene knockout attenuates atherosclerosis and in vivo foam cell formation in hyperlipidemic apoE⁻/⁻ mice.

BACKGROUND: Chronic glucocorticoid excess has been linked to increased atherosclerosis and general cardiovascular risk in humans. The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) increases active glucocorticoid levels within tissues by catalyzing the conversion of cortisone to cortisol. Pharmacological inhibition of 11ßHSD1 has been shown to reduce atherosclerosis in murine models. However, the cellular and molecular details for this effect have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: To examine the role of 11ßHSD1 in atherogenesis, 11ßHSD1 knockout mice were created on the pro-atherogenic apoE⁻/⁻ background. Following 14 weeks of Western diet, aortic cholesterol levels were reduced 50% in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice vs. 11ßHSD1⁺/⁺/apoE⁻/⁻ mice without changes in plasma cholesterol. Aortic 7-ketocholesterol content was reduced 40% in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice vs. control. In the aortic root, plaque size, necrotic core area and macrophage content were reduced ∼30% in 11ßHSD1⁻/⁻/apoE⁻/⁻mice. Bone marrow transplantation from 11ßHSD1⁻/⁻/apoE⁻/⁻ mice into apoE⁻/⁻ recipients reduced plaque area 39-46% in the thoracic aorta. In vivo foam cell formation was evaluated in thioglycollate-elicited peritoneal macrophages from 11ßHSD1⁺/⁺/apoE⁻/⁻ and 11ßHSD1⁻/⁻/apoE⁻/⁻ mice fed a Western diet for ∼5 weeks. Foam cell cholesterol levels were reduced 48% in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice vs. control. Microarray profiling of peritoneal macrophages revealed differential expression of genes involved in inflammation, stress response and energy metabolism. Several toll-like receptors (TLRs) were downregulated in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice including TLR 1, 3 and 4. Cytokine release from 11ßHSD1⁻/⁻/apoE⁻/⁻-derived peritoneal foam cells was attenuated following challenge with oxidized LDL. CONCLUSIONS: These findings suggest that 11ßHSD1 inhibition may have the potential to limit plaque development at the vessel wall and regulate foam cell formation independent of changes in plasma lipids. The diminished cytokine response to oxidized LDL stimulation is consistent with the reduction in TLR expression and suggests involvement of 11ßHSD1 in modulating binding of pro-atherogenic TLR ligands.

Pharmacological therapies for raising HDL cholesterol beyond synthetic small molecules.

Epidemiological studies support the hypothesis that HDL particles possess inherent atheroprotective properties. The protective properties of HDL are primarily attributed to its known involvement in cholesterol mobilization from peripheral tissues and reverse cholesterol transport for hepatic excretion of cholesterol; removal of excess cholesterol load from the arterial wall is essential for prevention or reversal of unstable plaque development. Other known protective properties of HDL include its anti-inflammatory, antioxidant, antithrombotic and vasoprotective activities, which have been demonstrated in preclinical models of disease. Pharmacological therapies aimed at increasing HDL are predicted to offer tremendous clinical benefit for the prevention and treatment of cardiovascular disease. Advances in biologicals as therapeutics provide new opportunities for drug discovery. This review discusses some of the potential benefits of therapeutic approaches designed to raise HDL beyond that of traditional synthetic small molecules.

Apolipoprotein A-IMilano/POPC complex attenuates post-ischemic ventricular dysfunction in the isolated rabbit heart.

Irreversible myocardial injury is a potential consequence of coronary artery revascularization. Reperfusion leads to the production of oxidized products that can damage myocardium. High-density lipoproteins (HDL) are effective at removing oxidized lipids. We hypothesized that a synthetic HDL preparation, comprising recombinant apolipoprotein A-I(Milano) (apoA-I(M)) complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) (apoA-I(M)/POPC) would protect the heart from reperfusion injury. The ex vivo model consisted of rabbit hearts perfused by the Langendorff method. Hearts were equilibrated with Krebs-Henseleit buffer (10 min), pretreated with either apoA-I(M)/POPC (0.45 mg/mL) or vehicle (10 min), subjected to global ischemia (30 min) and reperfused for 60 min. ApoA-I(M)/POPC (n=7) prevented the left ventricular end-diastolic pressure elevation observed in the vehicle group (n=6) at the end of reperfusion (p<0.05). During reperfusion, coronary artery perfusion pressure increased in the controls (p<0.001), but not with apoA-I(M)/POPC. ApoA-I(M)/POPC reduced the release of creatine kinase at the end of the ischemic period (p<0.001). It also reduced cardiac left ventricle muscle lipid hydroperoxides by 46% (p<0.05). Direct comparison of the antioxidant potential indicated that recombinant apoA-I(M) was much more potent than apoA-I in attenuating low-density lipoprotein oxidation. Electron microscopy showed that apoA-I(M)/POPC prevented mitochondrial granulation, disorganization and sarcomere contraction band formation indicative of reperfusion injury. The apoA-I(M)/POPC complex thus appears to reduce reperfusion injury under global ischemic conditions, and may therefore have therapeutic application in the reduction of myocardial ischemia.

Effects of timed administration of doxycycline or methylprednisolone on post-myocardial infarction inflammation and left ventricular remodeling in the rat heart.

The development of strategies to ameliorate post-myocardial infarction (MI) remodeling and improve function continues to be an area of clinical importance. Use of steroids for this purpose is controversial since the effects of timed treatment on relevant inflammatory, biochemical and structure/function endpoints are unclear. In a previous report, we demonstrated that use of doxycycline pre-treatment improves post-MI remodeling and passive left ventricular (LV) function. However, the effects of timed doxycycline post-MI treatment are unknown. To examine these issues, we performed a study using a rat MI model. Animals were administered one of the following: doxycycline (DOX), the corticosteroid methylprednisolone (MP), or aqueous vehicle. Treatment was given early, short-term (at time of MI to 24 h post-MI) or late, long term (2-7 days post-MI). Animals were sacrificed at 3, 7 or 42 days post-surgery. We assessed LV hemodynamics, pressure-volume, and pressure-scar strains, histomorphometry, inflammation via measurements of myeloperoxidase activity, and matrix metalloproteinase (MMP) activity. Late MP treatment yielded a robust right-shifted pressure-volume curve, which was accompanied by increased scar strains. Late DOX treatment yielded reduced average heart weight and size and preserved scar thickness. DOX treatment did not suppress inflammation, which contrasts with the suppressive effects of MP. Use of early or late MP yielded increased MMP activity in infarcted and non-infarcted regions. Early and late treatment with DOX yielded infarct-associated MMP activity levels comparable to those of vehicle-treated animals. In conclusion, results indicate that late use of MP yields adverse post-MI structure/function outcomes that correlate with suppression of inflammation and increased MMP activity. These observations contrast with those of DOX, in particular, late treatment where improved outcomes were observed in LV structure and were accompanied by the lack of suppression of inflammation.

Molecular interactions between matrilysin and the matrix metalloproteinase inhibitor doxycycline investigated by deuterium exchange mass spectrometry.

Matrix metalloproteinases (MMPs) play an essential role in normal and pathological extracellular matrix degradation. Deuterium exchange mass spectrometry (DXMS) was used to localize the binding regions of the broad-spectrum MMP inhibitor doxycycline on the active form of matrilysin (residues 95-267) and to assess alterations in structure induced by doxycycline binding. DXMS analyses of inhibitor-bound versus inhibitor-free forms of matrilysin reveal two primary sites of reduced hydrogen/deuterium exchange (residues 145-153; residues 193-204) that flank the structural zinc binding site. Equilibrium dialysis studies of doxycycline-matrilysin binding yielded a K(d) of 73 microM with a binding stoichiometry of 2.3 inhibitor molecules per protein, which compares well with DXMS results that show principal reduction in deuterium exchange at two sites. Lesser changes in deuterium exchange evident at the amino and carboxyl termini are attributed to inhibitor-induced structural fluctuations. Tryptophan fluorescence quenching experiments of matrilysin with potassium iodide suggest changes in conformation induced by doxycycline binding. In the presence of doxycycline, tryptophan quenching is reduced by approximately 17% relative to inhibitor-free matrilysin. Examination of the X-ray crystal structure of matrilysin shows that the doxycycline-binding site at residues 193 to 204 is positioned within the structural metal center of matrilysin, adjacent to the structural zinc atom and near both calcium atoms. These results suggest a mode of matrilysin inhibition by doxycycline that could involve interactions with the structural zinc atom and/or calcium atoms within the structural metal center of the protein.