Miao medicine Gu Yan Xiao tincture inhibits mTOR to stimulate chondrocyte autophagy in a rabbit model of osteoarthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: The Gu Yan Xiao tincture, a blend of traditional Chinese herbs, is traditionally used for osteoarthritis and related pain. This study investigated its mechanism of action in order to rationalize and validate its therapeutic use. AIM OF THE STUDY: This study analyzed, in a rabbit model of knee osteoarthritis, whether and how Gu Yan Xiao tincture exerts therapeutic benefits by modulating chondrocyte autophagy. MATERIALS AND METHODS: The active constituents within the GYX tincture were identified using liquid chromatography-mass spectrometry. The rabbit model was established by injecting animals with type II collagenase intra-articularly, and the effects of topically applied tincture were examined on osteoarthritis lesions of the knee using histopathology, micro-computed tomography and x-ray imaging. Effects of the tincture were also evaluated on levels of inflammatory cytokines, matrix metalloproteases, and autophagy in chondrocytes. As a positive control, animals were treated with sodium diclofenac. RESULTS: The tincture mitigated the reduction in joint space, hyperplasia of the synovium and matrix metalloproteases in serum that occurred after injection of type II collagenase in rabbits. These therapeutic effects were associated with inhibition of mTOR and activation of autophagy in articular chondrocytes. Inhibiting mTOR with rapamycin potentiated the therapeutic effects of the tincture, while inhibiting autophagy with 3-methyladenine antagonized them. CONCLUSIONS: Gu Yan Xiao tincture mitigates tissue injury in a rabbit model of osteoarthritis, at least in part by inhibiting mTOR and thereby promoting autophagy in chondrocytes. These results rationalize the use of the tincture not only against osteoarthritis but also potentially other diseases involving inhibition of autophagy in bones and joints.

Fermented black ginseng extract prevents UVB-induced inflammation by regulating the nc886-PKR pathway in human keratinocytes.

BACKGROUND: Continuous exposure of the skin to ultraviolet B (UVB) rays can cause inflammation and photodamage. In previous studies, we observed that the upregulation of nc886, a noncoding RNA (ncRNA), can alleviate UVB-induced inflammation through suppression of the protein kinase RNA (PKR) pathway. We aim to investigate the effect of fermented black ginseng extract (FBGE), which has been shown to increase the expression of nc886, on UVB-induced inflammation in keratinocytes. METHODS: To confirm the cytotoxicity of FBGE, MTT assay was performed, and no significant cytotoxicity was found on human keratinocytes. The efficacies of FBGE were assessed through qPCR, Western blotting, and ELISA analysis which confirmed regulation of UVB-induced inflammation. RESULTS: The analysis results showed that FBGE inhibited the decrease in nc886 expression and the increase in the methylated nc886 caused by UVB. It also prevented the UVB-induced increase of metalloproteinase-9 (MMP-9), metalloproteinase-1 (MMP-1), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α). Additionally, FBGE suppressed the PKR-MAPK pathways activated by UVB. CONCLUSION: These results implicate that FBGE can alleviate UVB-induced inflammation through regulation of the nc886-PKR pathway.

Novel marine metalloprotease-new approaches for inhibition of biofilm formation of Stenotrophomonas maltophilia.

Many marine organisms produce bioactive molecules with unique characteristics to survive in their ecological niches. These enzymes can be applied in biotechnological processes and in the medical sector to replace aggressive chemicals that are harmful to the environment. Especially in the human health sector, there is a need for new approaches to fight against pathogens like Stenotrophomonas maltophilia which forms thick biofilms on artificial joints or catheters and causes serious diseases. Our approach was to use enrichment cultures of five marine resources that underwent sequence-based screenings in combination with deep omics analyses in order to identify enzymes with antibiofilm characteristics. Especially the supernatant of the enrichment culture of a stony coral caused a 40% reduction of S. maltophilia biofilm formation. In the presence of the supernatant, our transcriptome dataset showed a clear stress response (upregulation of transcripts for metal resistance, antitoxins, transporter, and iron acquisition) to the treatment. Further investigation of the enrichment culture metagenome and proteome indicated a series of potential antimicrobial enzymes. We found an impressive group of metalloproteases in the proteome of the supernatant that is responsible for the detected anti-biofilm effect against S. maltophilia. KEY POINTS: • Omics-based discovery of novel marine-derived antimicrobials for human health management by inhibition of S. maltophilia • Up to 40% reduction of S. maltophilia biofilm formation by the use of marine-derived samples • Metalloprotease candidates prevent biofilm formation of S. maltophilia K279a by up to 20.

Intramuscular Bleeding and Formation of Microthrombi during Skeletal Muscle Damage Caused by a Snake Venom Metalloprotease and a Cardiotoxin.

The interactions between specific snake venom toxins and muscle constituents are the major cause of severe muscle damage that often result in amputations and subsequent socioeconomic ramifications for snakebite victims and/or their families. Therefore, improving our understanding of venom-induced muscle damage and determining the underlying mechanisms of muscle degeneration/regeneration following snakebites is critical to developing better strategies to tackle this issue. Here, we analysed intramuscular bleeding and thrombosis in muscle injuries induced by two different snake venom toxins (CAMP-Crotalus atrox metalloprotease (a PIII metalloprotease from the venom of this snake) and a three-finger toxin (CTX, a cardiotoxin from the venom of Naja pallida)). Classically, these toxins represent diverse scenarios characterised by persistent muscle damage (CAMP) and successful regeneration (CTX) following acute damage, as normally observed in envenomation by most vipers and some elapid snakes of Asian, Australasian, and African origin, respectively. Our immunohistochemical analysis confirmed that both CAMP and CTX induced extensive muscle destruction on day 5, although the effects of CTX were reversed over time. We identified the presence of fibrinogen and P-selectin exposure inside the damaged muscle sections, suggesting signs of bleeding and the formation of platelet aggregates/microthrombi in tissues, respectively. Intriguingly, CAMP causes integrin shedding but does not affect any blood clotting parameters, whereas CTX significantly extends the clotting time and has no impact on integrin shedding. The rates of fibrinogen clearance and reduction in microthrombi were greater in CTX-treated muscle compared to CAMP-treated muscle. Together, these findings reveal novel aspects of venom-induced muscle damage and highlight the relevance of haemostatic events such as bleeding and thrombosis for muscle regeneration and provide useful mechanistic insights for developing better therapeutic interventions.

The Effect of Metformin on Triple-Negative Breast Cancer Cells and Nude Mice.

Triple-negative breast cancer (TNBC) presents the most adverse prognosis due to its pronounced invasive and metastatic features. Existing research has highlighted that metformin, a prevalent diabetes medication, possesses strong anti-tumor properties, particularly in inhibiting tumor invasion and metastasis. This study delves deeper into the impact of metformin on TNBC by examining changes in proliferation, apoptosis, invasion, migration, and adhesion of TNBC cells, specifically MDA-MB-231, post-metformin exposure. The treatment of MDA-MB-231 with metformin in immunodeficient nude mice led to discernible changes in tumor metrics such as size, weight, lymph node engagement, and angiogenesis. Post-treatment, MDA-MB-231 cells exhibited a marked decline in proliferation, invasion, migration, and adhesion, alongside a significant rise in apoptosis. In the in vivo model with nude mice, tumors displayed notable reductions in size and weight post-metformin exposure. Furthermore, there was a pronounced decline in lymph node plasma cell proliferation and tumor angiogenesis. Through the use of both Enzyme-Linked Immunosorbent Assay and Real-Time Fluorescence Quantification, it was ascertained that the expression of Signal Transducer and Activator of Transcription 3 (STAT3) saw significant augmentation, while expressions of Matrix Metallopeptidase-2 (MMP-2), Matrix Metallopeptidase-9 (MMP-9), Interleukin-6 (IL-6), and Interleukin-7 (IL-7) decreased markedly. This suggests metformin's potential efficacy against TNBC, potentially mediated via the STAT3 signaling pathway and interleukins 6 and 7.

Suramin analogues protect cartilage against osteoarthritic breakdown by increasing levels of tissue inhibitor of metalloproteinases 3 (TIMP-3) in the tissue.

Osteoarthritis is a chronic degenerative joint disease affecting millions of people worldwide, with no disease-modifying drugs currently available to treat the disease. Tissue inhibitor of metalloproteinases 3 (TIMP-3) is a potential therapeutic target in osteoarthritis because of its ability to inhibit the catabolic metalloproteinases that drive joint damage by degrading the cartilage extracellular matrix. We previously found that suramin inhibits cartilage degradation through its ability to block endocytosis and intracellular degradation of TIMP-3 by low-density lipoprotein receptor-related protein 1 (LRP1), and analysis of commercially available suramin analogues indicated the importance of the 1,3,5-trisulfonic acid substitutions on the terminal naphthalene rings for this activity. Here we describe synthesis and structure-activity relationship analysis of additional suramin analogues using ex vivo models of TIMP-3 trafficking and cartilage degradation. This showed that 1,3,6-trisulfonic acid substitution of the terminal naphthalene rings was also effective, and that the protective activity of suramin analogues depended on the presence of a rigid phenyl-containing central region, with para/para substitution of these phenyl rings being most favourable. Truncated analogues lost protective activity. The physicochemical characteristics of suramin and its analogues indicate that approaches such as intra-articular injection would be required to develop them for therapeutic use.

A Triazaspirane Derivative Inhibits Migration and Invasion in PC3 Prostate Cancer Cells.

Cancer is a serious health problem due to the complexity of establishing an effective treatment. The purpose of this work was to evaluate the activity of a triazaspirane as a migration and invasion inhibitor in PC3 prostatic tumor cells through a possible negative regulation of the FAK/Src signal transduction pathway and decreased secretion of metalloproteinases 2 and 9. Molecular docking analysis was performed using Moe 2008.10 software. Migration (wound-healing assay) and invasion (Boyden chamber assay) assays were performed. In addition, the Western blot technique was used to quantify protein expression, and the zymography technique was used to observe the secretion of metalloproteinases. Molecular docking showed interactions in regions of interest of the FAK and Src proteins. Moreover, the biological activity assays demonstrated an inhibitory effect on cell migration and invasion, an important suppression of metalloproteinase secretion, and a decrease in the expression of p-FAK and p-Src proteins in treated PC3 cells. Triazaspirane-type molecules have important inhibitory effects on the mechanisms associated with metastasis in PC3 tumor cells.

Mouse skin peptidomic analysis of the hemorrhage induced by a snake venom metalloprotease.

Hemorrhage induced by snake venom metalloproteases (SVMPs) results from proteolysis, capillary disruption, and blood extravasation. HF3, a potent SVMP of Bothrops jararaca, induces hemorrhage at pmol doses in the mouse skin. To gain insight into the hemorrhagic process, the main goal of this study was to analyze changes in the skin peptidome generated by injection of HF3, using approaches of mass spectrometry-based untargeted peptidomics. The results revealed that the sets of peptides found in the control and HF3-treated skin samples were distinct and derived from the cleavage of different proteins. Peptide bond cleavage site identification in the HF3-treated skin showed compatibility with trypsin-like serine proteases and cathepsins, suggesting the activation of host proteinases. Acetylated peptides, which originated from the cleavage at positions in the N-terminal region of proteins in both samples, were identified for the first time in the mouse skin peptidome. The number of peptides acetylated at the residue after the first Met residue, mostly Ser and Ala, was higher than that of peptides acetylated at the initial Met. Proteins cleaved in the hemorrhagic skin participate in cholesterol metabolism, PPAR signaling, and in the complement and coagulation cascades, indicating the impairment of these biological processes. The peptidomic analysis also indicated the emergence of peptides with potential biological activities, including pheromone, cell penetrating, quorum sensing, defense, and cell-cell communication in the mouse skin. Interestingly, peptides generated in the hemorrhagic skin promoted the inhibition of collagen-induced platelet aggregation and could act synergistically in the local tissue damage induced by HF3.

Inhibition of the extracellular enzyme A disintegrin and metalloprotease with thrombospondin motif 4 prevents cardiac fibrosis and dysfunction.

AIMS: Heart failure is a condition with high mortality rates, and there is a lack of therapies that directly target maladaptive changes in the extracellular matrix (ECM), such as fibrosis. We investigated whether the ECM enzyme known as A disintegrin and metalloprotease with thrombospondin motif (ADAMTS) 4 might serve as a therapeutic target in treatment of heart failure and cardiac fibrosis. METHODS AND RESULTS: The effects of pharmacological ADAMTS4 inhibition on cardiac function and fibrosis were examined in rats exposed to cardiac pressure overload. Disease mechanisms affected by the treatment were identified based on changes in the myocardial transcriptome. Following aortic banding, rats receiving an ADAMTS inhibitor, with high inhibitory capacity for ADAMTS4, showed substantially better cardiac function than vehicle-treated rats, including ∼30% reduction in E/e' and left atrial diameter, indicating an improvement in diastolic function. ADAMTS inhibition also resulted in a marked reduction in myocardial collagen content and a down-regulation of transforming growth factor (TGF)-ß target genes. The mechanism for the beneficial effects of ADAMTS inhibition was further studied in cultured human cardiac fibroblasts producing mature ECM. ADAMTS4 caused a 50% increase in the TGF-ß levels in the medium. Simultaneously, ADAMTS4 elicited a not previously known cleavage of TGF-ß-binding proteins, i.e. latent-binding protein of TGF-ß and extra domain A-fibronectin. These effects were abolished by the ADAMTS inhibitor. In failing human hearts, we observed a marked increase in ADAMTS4 expression and cleavage activity. CONCLUSION: Inhibition of ADAMTS4 improves cardiac function and reduces collagen accumulation in rats with cardiac pressure overload, possibly through a not previously known cleavage of molecules that control TGF-ß availability. Targeting ADAMTS4 may serve as a novel strategy in heart failure treatment, in particular, in heart failure with fibrosis and diastolic dysfunction.

Enhanced protection against hypoxia/reoxygenation-induced apoptosis in H9c2 cells by puerarin-loaded liposomes modified with matrix metalloproteinases-targeting peptide and triphenylphosphonium.

Based on the inhibition of mitochondrial permeability transition pore (mPTP) opening, puerarin (PUE) has a good potential to reduce myocardial ischemia/reperfusion injury (MI/RI). However, the lack of targeting of free PUE makes it difficult to reach the mitochondria. In this paper, we constructed matrix metalloproteinase-targeting peptide (MMP-TP) and triphenylphosphonium (TPP) cation co-modified liposomes loaded with PUE (PUE@T/M-L) for mitochondria-targeted drug delivery. PUE@T/M-L had a favorable particle size of 144.9 ± 0.8 nm, an encapsulation efficiency of 78.9 ± 0.6%, and a sustained-release behavior. The results of cytofluorimetric experiments showed that MMP-TP and TPP double-modified liposomes (T/M-L) enhanced intracellular uptake, escaped lysosomal capture, and promoted drug targeting into mitochondria. In addition, PUE@T/M-L enhanced the viability of hypoxia-reoxygenation (H/R) injured H9c2 cells by inhibiting mPTP opening and reactive oxygen species (ROS) production, reducing Bax expression and increasing Bcl-2 expression. It was inferred that PUE@T/M-L delivered PUE into the mitochondria of H/R injured H9c2 cells, resulting in a significant increase in cellular potency. Based on the ability of MMP-TP to bind the elevated expression of matrix metalloproteinases (MMPs), T/M-L had excellent tropism for Lipopolysaccharide (LPS) -stimulated macrophages and can significantly reduce TNF-α and ROS levels, thus allowing both drug accumulation in ischemic cardiomyocytes and reducing inflammatory stimulation during MI/RI. Fluorescence imaging results of the targeting effect using a DiR probe also indicated that DiR@T/M-L could accumulate and retain in the ischemic myocardium. Taken together, these results demonstrated the promising application of PUE@T/M-L for mitochondria-targeted drug delivery to achieve maximum therapeutic efficacy of PUE.

Matrix-Metalloproteinase Inhibitory Study of Novel Tetrahydroisoquinoline-4-carboxylates: Design, Synthesis, and Molecular Docking Studies.

The design, molecular docking, synthesis and structure-activity relationship (SAR) of a series of novel methyl 1-oxo-2-(propan-2-yl)-3-(pyridin-3-yl)-1,2,3,4-tetrahydroisoquinoline-4-carboxylates, were investigated for antiproliferative and cytotoxic studies by screening against cancer cell lines of different origin by MTT, LDH and Trypan Blue Assay. Irrespective of cell lines, among the synthesized nonpeptido-mimetic analogs 5a-e, 5c has executed potent bio-potency with IC50 value of 7.00 to 7.21 µM, which further expressed in-vivo anti-tumor activity against murine T-cell lymphoma cell lines (Daltons Lymphoma-DLA) by regressing tumor growth. The formation of neovessels from the vasculogenesis was diminished reflecting the antitumor activity. The neovessel formation is directly relied on expression of matrix meteloproteases (MMP's) level which was drastically reduced by 5c treatment as evaluated by immonoblot assays. This is further supported by in-silico ADMET studies performed by ACD I-Lab 2.0 were in agreement with Lipinski rule of five. Reporting results were assessed as a positive parameter for further validation of the compound for therapeutic potential of cancer by 5c for preclinical studies in near future.

Novel biochemical aspects of lugdulysin, a Staphylococcus lugdunensis metalloprotease that inhibits formation and disrupts protein biofilm of methicillin-resistant Staphylococcus aureus.

Staphylococcus lugdunensis produces lugdulysin, a metalloprotease that may contribute to its virulence. This study aimed to evaluate the biochemical aspects of lugdulysin and investigate its effect on Staphylococcus aureus biofilms. The protease was isolated and characterized for its optimal pH and temperature, hydrolysis kinetics, and influence of metal cofactor supplementation. The protein structure was determined via homology modeling. The effect on S. aureus biofilms was assessed by the micromethod technique. The protease optimal pH and temperature were 7.0 and 37 °C, respectively. EDTA inhibited protease activity, confirming it as a metalloprotease. Lugdulysin activity was not recovered by divalent ion supplementation post-inhibition, and supplementation with divalent ions did not change enzymatic activity. The isolated enzyme was stable for up to 3 h. Lugdulysin significantly inhibited the formation and disrupted preestablished protein-matrix MRSA biofilm. This preliminary study indicates that lugdulysin has a potential role as a competition mechanism and/or modulation of staphylococcal biofilm.

Pressure does not affect fibrotic function of dermal fibroblast through an in vitro 3D hydrogel culture model.

Pressure therapy has been used for the prevention and treatment of hypertrophic scars for decades. However, the cellular and molecular mechanisms of this treatment modality have not been fully elaborated, leading to long-lasting controversies regarding its clinical effectiveness. In this current study, we adopted an in vitro 3D culture and compression model to explore the effect of pressure force on fibroblasts, in order to further explain the working mechanism of compression force during pressure treatment. Human dermal fibroblasts were cultured in the 3D culture hydrogel and treated with 1.5 atm of external compression force through a syringe tube device, for 4, 8, and 20 h respectively. RNA-seq identified 437 differentially regulated genes after an 8-h compression intervention compared with control cells, among which 256 genes were up-regulated and 181 genes were down-regulated. Further q-PCR analysis confirmed that early growth response 1(EGR1) and c-fos were down-regulated after an 8-h compression intervention. However, the down-regulation of EGR1 and c-fos at the mRNA level does not lead to altered protein synthesis through western blot, for both 8 and 20-h time points after pressure intervention. Genes closely related to the fibrotic function of fibroblasts including type I collagen (COL1), type III collagen (COL3), transforming growth factor ß1(TGF-ß1), matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 1 (TIMP1), connective tissue growth factor (CTGF), α smooth muscle actin (α-SMA), and fibronectin 1 (FN1), were also unaffected after pressure treatment for 8 h. The current study indicated that in our 3D hydrogel culture model, pressure does not directly affect the fibrotic function of dermal fibroblast in vitro. Indirect regulation including reducing oedema, blood perfusion, and tension could be a more possible mechanism of pressure therapy.

Brain-Derived Neurotrophic Factor Precursor Contributes to a Proinflammatory Program in Monocytes/Macrophages After Acute Myocardial Infarction.

Background The imbalance of monocyte/macrophage polarization toward the preferential proinflammatory phenotype and a lack of normal inflammation resolution are present in acute myocardial infarction (AMI). Our previous study showed that upregulation of brain-derived neurotrophic factor precursor (proBDNF) in M2-like monocytes may contribute to the proinflammatory response in the Stanford type-A acute aortic dissection. The present study aimed to investigate the role of proBDNF signaling in monocytes/macrophages in the progress of AMI. Methods and Results We observed the upregulation of proBDNF in the proinflammatory monocytes of patients with AMI. The upregulation of proBDNF was also observed in the circulating proinflammatory Ly6Chigh monocytes and cardiac F4/80+CD86+ macrophages 3 days after AMI in a mice model. To neutralize proBDNF, the mice subjected to AMI were injected intraperitoneally with a monoclonal anti-proBDNF antibody. Echocardiography, 2,3,5-triphenyltetrazolium chloride staining, and positron emission tomography/computed tomography results demonstrate that monoclonal anti-proBDNF antibody treatment further impaired cardiac functions, increased infarct size, and exacerbated the proinflammatory state. Moreover, the level of proinflammatory Ly6Chigh in the blood and F4/80+CD86+ in the heart was further increased in monoclonal anti-proBDNF antibody mice. RNA sequencing revealed that matrix metalloprotease-9 protein level was dramatically increased, along with the activated proinflammatory-related cytokines. Matrix metalloprotease-9 inhibitor treatment attenuated the deteriorated effect of monoclonal anti-proBDNF antibody on cardiac function and infarct areas. Conclusions Our study shows that endogenous proBDNF in monocytes/macrophages may exert protective roles in cardiac remodeling after AMI by regulating matrix metalloprotease-9 activity.

The Impact of JWH-133 on Articular Cartilage Regeneration in Osteoarthritis Via Metalloproteinase 13-Dependent Mechanism.

Objective: Osteoarthritis (OA) is common degenerative joint disease, mostly characterized by gradual cartilage breakdown. Currently there are no disease-modifying drugs available, therefore, there is an increasing need for basic research to focus on cartilage function in OA. Changes in cannabinoid receptor 2 (CB2) expression were observed in the OA-affected joints, although its action on cartilage chondrocytes remain unclear. We studied the action of dimethylbutyl-deoxy-delta-8-THC (JWH-133), selective CB2 agonist, on chondrocytes metabolism using both in vitro and in vivo studies. Design: Intraarticular (i.a.) injection of monoiodoacetate (MIA) was used to induce OA in rats. OA-related pain symptoms were assessed by pressure application measurements (PAMs). Primary human chondrocytes treated with MIA were used to investigate action of JWH-133 on chondrocytes viability, proliferation, and motility. Cannabinoid system components, inflammatory cytokines and metalloproteinases (MMPs) expression was measured on messenger RNA and protein levels in chondrocytes and animal cartilage. Results: Repeated, i.a. administration of JWH-133 showed antinociceptive potential in PAM, as well as decreased levels of MMPs, which suggests that CB2 agonism may modify degradation of cartilage. JWH-133 administration partially reduced toxicity, increased proliferation, and chondrocytes' migration. Moreover, our data suggest that CB2 agonism leads to alleviation of MMPs expression both in vitro and in vivo. Conclusions: In this study, we demonstrate modifying effect of JWH-133 local administration on cartilage metabolism and MMP13 expression that was shown to be involved in cartilage degradation. CB2 receptors' activation may be of benefit for chondrocytes' proliferation, therefore delaying disease progression. Our results propose direction of studies on OA-modifying treatment that can benefit in management of human OA.

Effect of Dexamethasone on Adhesion of Human Neutrophils and Concomitant Secretion.

Neutrophils play a dual role in protecting the body. They are able to penetrate infected tissues and destroy pathogens there by releasing aggressive bactericidal substances. While into the surrounding tissues, the aggressive products secreted by neutrophils initiate development of inflammatory processes. Invasion of neutrophils into tissues is observed during the development of pneumonia in the patients with lung diseases of various etiologies, including acute respiratory distress syndrome caused by coronavirus disease. Synthetic corticosteroid hormone dexamethasone has a therapeutic effect in treatment of lung diseases, including reducing mortality in the patients with severe COVID-19. The acute (short-term) effect of dexamethasone on neutrophil adhesion to fibrinogen and concomitant secretion was studied. Dexamethasone did not affect either attachment of neutrophils to the substrate or their morphology. Production of reactive oxygen species (ROS) and nitric oxide (NO) by neutrophils during adhesion also did not change in the presence of dexamethasone. Dexamethasone stimulated release of metalloproteinases in addition to the proteins secreted by neutrophils during adhesion under control conditions, and selectively stimulated release of free amino acid hydroxylysine, a product of lysyl hydroxylase. Metalloproteinases play a key role and closely interact with lysyl hydroxylase in the processes of modification of the extracellular matrix. Therapeutic effect of dexamethasone could be associated with its ability to reorganize extracellular matrix in the tissues by changing composition of the neutrophil secretions, which could result in the improved gas exchange in the patients with severe lung diseases.

BmooMPα-I, a Metalloproteinase Isolated from Bothrops moojeni Venom, Reduces Blood Pressure, Reverses Left Ventricular Remodeling and Improves Cardiac Electrical Conduction in Rats with Renovascular Hypertension.

BmooMPα-I has kininogenase activity, cleaving kininogen releasing bradykinin and can hydrolyze angiotensin I at post-proline and aspartic acid positions, generating an inactive peptide. We evaluated the antihypertensive activity of BmooMPα-I in a model of two-kidney, one-clip (2K1C). Wistar rats were divided into groups: Sham, who underwent sham surgery, and 2K1C, who suffered stenosis of the right renal artery. In the second week of hypertension, we started treatment (Vehicle, BmooMPα-I and Losartan) for two weeks. We performed an electrocardiogram and blood and heart collection in the fourth week of hypertension. The 2K1C BmooMPα-I showed a reduction in blood pressure (systolic pressure: 131 ± 2 mmHg; diastolic pressure: 84 ± 2 mmHg versus 174 ± 3 mmHg; 97 ± 4 mmHg, 2K1C Vehicle, p < 0.05), improvement in electrocardiographic parameters (Heart Rate: 297 ± 4 bpm; QRS: 42 ± 0.1 ms; QT: 92 ± 1 ms versus 332 ± 6 bpm; 48 ± 0.2 ms; 122 ± 1 ms, 2K1C Vehicle, p < 0.05), without changing the hematological profile (platelets: 758 ± 67; leukocytes: 3980 ± 326 versus 758 ± 75; 4400 ± 800, 2K1C Vehicle, p > 0.05), with reversal of hypertrophy (left ventricular area: 12.1 ± 0.3; left ventricle wall thickness: 2.5 ± 0.2; septum wall thickness: 2.3 ± 0.06 versus 10.5 ± 0.3; 2.7 ± 0.2; 2.5 ± 0.04, 2K1C Vehicle, p < 0.05) and fibrosis (3.9 ± 0.2 versus 7.4 ± 0.7, 2K1C Vehicle, p < 0.05). We concluded that BmooMPα-I improved blood pressure levels and cardiac remodeling, having a cardioprotective effect.

Leptolysin, a Leptospira secreted metalloprotease of the pappalysin family with broad-spectrum activity.

Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a first characterization of leptolysin, a Leptospira metalloprotease of the pappalysin family identified in a previous exoproteomic study. Comparative molecular analysis of leptolysin with two other pappalysins from prokaryotes, ulilysin and mirolysin, reveals similarities regarding calcium, zinc, and arginine -binding sites conservation within the catalytic domain, but also discloses peculiarities. Variations observed in the primary and tertiary structures may reflect differences in primary specificities. Purified recombinant leptolysin of L. interrogans was obtained as a ~50 kDa protein. The protease exhibited maximal activity at pH 8.0 and 37°C, and hydrolytic activity was observed in the presence of different salts with maximum efficiency in NaCl. Substrate specificity was assessed using a small number of FRET peptides, and showed a marked preference for arginine residues at the P1 position. L. interrogans leptolysin proteolytic activity on proteinaceous substrates such as proteoglycans and plasma fibronectin was also evaluated. All proteins tested were efficiently degraded over time, confirming the protease´s broad-spectrum activity in vitro. In addition, leptolysin induced morphological alterations on HK-2 cells, which may be partially attributed to extracellular matrix (ECM) degradation. Hemorrhagic foci were observed in the dorsal skin of mice intradermally injected with leptolysin, as a plausible consequence of ECM disarray and vascular endothelium glycocalyx damage. Assuming that leptospiral proteases play an important role in all stages of the infectious process, characterizing their functional properties, substrates and mechanisms of action is of great importance for therapeutic purposes.

Initiation of the SGLT2 inhibitor canagliflozin to prevent kidney and heart failure outcomes guided by HbA1c, albuminuria, and predicted risk of kidney failure.

BACKGROUND: Sodium glucose co-transporter-2 (SGLT2) inhibitors reduce the risk of kidney and heart failure events independent of glycemic effects. We assessed whether initiation of the SGLT2 inhibitor canagliflozin guided by multivariable predicted risk based on clinical characteristics and novel biomarkers is more efficient to prevent clinical outcomes compared to a strategy guided by HbA1c or urinary-albumin-creatinine ratio (UACR) alone. METHODS: We performed a post-hoc analysis of the CANVAS trial including 3713 patients with available biomarker measurements. We compared the number of composite kidney (defined as a sustained 40% decline in eGFR, chronic dialysis, kidney transplantation, or kidney death) and composite heart failure outcomes (defined as heart failure hospitalization or cardiovascular (CV) death) prevented per 1000 patients treated for 5 years when canagliflozin was initiated in patients according to HbA1c ≥ 7.5%, UACR, or multivariable risk models consisting of: (1) clinical characteristics, or (2) clinical characteristics and novel biomarkers. Differences in the rates of events prevented between strategies were tested by Chi2-statistic. RESULTS: After a median follow-up of 6.1 years, 144 kidney events were recorded. The final clinical model included age, previous history of CV disease, systolic blood pressure, UACR, hemoglobin, body weight, albumin, estimated glomerular filtration rate, and randomized treatment assignment. The combined biomarkers model included all clinical characteristics, tumor necrosis factor receptor-1, kidney injury molecule-1, matrix metallopeptidase-7 and interleukin-6. Treating all patients with HbA1c ≥ 7.5% (n = 2809) would prevent 33.0 (95% CI 18.8 to 43.3 ) kidney events at a rate of 9.6 (95% CI 5.5 to 12.6) events prevented per 1000 patients treated for 5 years. The corresponding rates were 5.8 (95% CI 3.4 to 7.9), 16.6 (95% CI 9.5 to 22.0) (P < 0.001 versus HbA1c or UACR approach), and 17.5 (95% CI 10.0 to 23.0) (P < 0.001 versus HbA1c or UACR approach; P = 0.54 versus clinical model). Findings were similar for the heart failure outcome. CONCLUSION: Initiation of canagliflozin based on an estimated risk-based approach prevented more kidney and heart failure outcomes compared to a strategy based on HbA1c or UACR alone. There was no apparent gain from adding novel biomarkers to the clinical risk model. These findings support the use of risk-based assessment using clinical markers to guide initiation of SGLT2 inhibitors in patients with type 2 diabetes.

Evaluation of the Anti-Inflammatory and Chondroprotective Effect of Celecoxib on Cartilage Ex Vivo and in a Rat Osteoarthritis Model.

OBJECTIVE: The potential chondroprotective effect of celecoxib, a nonsteroidal anti-inflammatory drug and selective cyclooxygenase-2 inhibitor used to reduce pain and inflammation in knee osteoarthritis patients, is disputed. This study aimed at investigating the chondroprotective effects of celecoxib on (1) human articular cartilage explants and (2) in an in vivo osteoarthritis rat model. DESIGN: Articular cartilage explants from 16 osteoarthritis patients were cultured for 24 hours with celecoxib or vehicle. Secreted prostaglandins (prostaglandin E2, prostaglandin F2α, prostaglandin D2) and thromboxane B2 (TXB2) concentrations were determined in medium by ELISA, and protein regulation was measured with label-free proteomics. Cartilage samples from 7 of these patients were analyzed for gene expression using real-time quantitative polymerase chain reaction. To investigate the chondroprotective effect of celecoxib in vivo, 14 rats received an intra-articular injection of celecoxib or 0.9% NaCl after osteoarthritis induction by anterior cruciate ligament transection and partial medial meniscectomy (ACLT/pMMx model). Histopathological scoring was used to evaluate osteoarthritis severity 12 weeks after injection. RESULTS: Secretion of prostaglandins, target of Nesh-SH3 (ABI3BP), and osteonectin proteins decreased, whereas tissue inhibitor of metalloproteinase 2 (TIMP-2) increased significantly after celecoxib treatment in the human (ex vivo) explant culture. Gene expression of a disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS4/5) and metalloproteinase 13 (MMP13) was significantly reduced after celecoxib treatment in human cartilage explants. Cartilage degeneration was reduced significantly in an in vivo osteoarthritis knee rat model. CONCLUSIONS: Our data demonstrated that celecoxib acts chondroprotective on cartilage ex vivo and a single intra-articular bolus injection has a chondroprotective effect in vivo.