Matrix Metalloproteinase Inhibitor Research Articles

Comparative evaluation of effect of three different MMP inhibitors on flexural bond strength of resin composite used to restore NCCL - In vitro study

Interface between resin composite and dentin is critical and important for the success of restoration. Application of MMP inhibitor may alter surface characteristic of dentin that improves bond strength of resin composite. This study evaluated and compared the effect of three different Matrix Metalloproteinase (MMP) inhibitors on flexural bond strength of resin composite restorations; done to restore non carious cervical lesions (NCCL).: Standardized NCCL was simulated on 41 freshly extracted and selected human premolars. The teeth were randomly assigned into four groups i.e. Group I: Control group (no MMP inhibitor). Group II: Chlorhexidine. Group III: Chitosan and group IV: Proanthocyanidin. NCCL were pretreated with MMP inhibitor of respective group prior to its restoration with nanofilled resin composite. Restorations in each group were finished and polished with an appropriate finishing and polishing kit. The flexural bond strength was evaluated using the universal testing machine. Data were analysed using one-way analysis of variance and Tukey's test. Among the tested MMP inhibitors; Group II (90.73 Mpa) and Group III (90.17 Mpa) showed highest mean flexural bond strength with insignificant difference ( P = 1.0) between them. Group IV showed higher but insignificantly different (P= 0.768) flexural bond strength to that of group I. Group II and Group III worked best to increase the flexural bond strength of resin composite indirectly representing its higher MMP inhibition and collagen stabilizing activity than Group IV and Group I.

MMP9: Link between neuropathy and colorectal cancer?

As chemotherapy is still a cornerstone of colorectal cancer (CRC) treatment, chemotherapy-induced peripheral neuropathy (CIPN) presents significant clinical challenges, affecting millions worldwide. A subset of colon cancer patients (approximately 30%) develop chronic CIPN, with detrimental, untreatable neuropathic pain symptoms. The risk factors of such intractable chronic CIPN are unknown. However, there is growing literature data investigating the intriguing interplay of neurons and cancer (cancer neuroscience). Recent data shows that this interplay might have a key role in the development and severity of CIPN. Given its vast (patho)physiological roles in both colon cancer and neuropathy, MMP9 seems to be a key factor that might drive the development of neuronal damage in colon cancer patients. This review investigates the role of matrix metalloproteinase 9 (MMP9) in linking CRC to neuropathy, aiming to uncover shared mechanisms that could offer new therapeutic targets. By synthesizing insights from a broad range of studies published over the last 20 years, we explore MMP9’s involvement in CRC progression, its role in CIPN, and the interconnected pathways influencing both conditions. These studies reveal MMP9 as a pivotal mediator in ECM remodeling, inflammation, and signal transduction pathways, emphasizing its modulation by macrophages. These shared mechanisms of colon cancer and CIPN pathophysiology suggest MMP9’s potential contribution to neuropathic conditions in CRC patients, positioning it as a critical factor in disease progression and a promising therapeutic target. Future research should focus on longitudinal studies to assess MMP9’s impact on neuropathy outcomes in CRC patients, exploring MMP9 inhibitors, and developing targeted interventions to mitigate the detrimental symptoms of CIPN. MMP9 also seems to be a feasible driving factor in the development of chronic CIPN in colon cancer patients.

Effects of Acute Maximum-Intensity Exercise on Matrix Metalloproteinase-2, -9, and Tissue Inhibitor of Metalloproteinase-1 Levels in Adult Males with Type 1 Diabetes Mellitus Treated with Insulin Pumps

Background: Dysregulation of matrix metalloproteinases (MMPs) activity is considered one of the potential causes of vascular complications in diabetic patients. Since training volume may influence MMPs levels in varying ways, the aim of our study was to evaluate changes in MMPs levels following acute maximum-intensity exercise in male patients with type 1 diabetes mellitus (T1DM). Methods: This study included 24 male T1DM patients and 10 healthy controls. Aerobic capacity was evaluated with a treadmill test. Levels of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured both before the aerobic capacity test and 60 min after its completion utilizing enzyme-linked immunosorbent assay (ELISA) system kits. Results: Before the aerobic capacity test only, MMP-9 serum levels were significantly elevated in the T1DM group compared to the controls. Following maximum-intensity exercise, the levels of MMP-2, MMP-9, and TIMP-1 were significantly higher in T1DM patients than in the control group. Between-group comparisons revealed that maximum-intensity exercise induced a statistically significant increase in MMP-2 serum levels from baseline in T1DM patients compared to controls. Conclusions: Our findings suggest that high-intensity exercise in T1DM patients leads to dysregulation of MMPs, as manifested by a significant increase in MMP-2 levels. This dysregulation may play a role in the development of vascular complications in diabetic patients.

In silico analysis of Arbacia lixula-derived peptides and plasmid construction for recombinant anti-aging therapies

Skin aging is one of the degenerative processes influenced by tyrosinase, elastase, collagenase, hyaluronidase, and matrix metalloproteinase-9 (MMP9) activity. One promising avenue for discovering antiaging therapeutics is the peptides from the Arbacia lixula spine. The aim of this study was to explore the potential of peptides from A. lixula spine as a multitarget inhibitor for recombinant antiaging therapies through in silico approaches. The crystal structure of peptides previously identified in A. lixula spine was visualized using the UCSF Chimera. The protein data bank (PDB) database was used to obtain the crystal structures of protein targets. The webservers Innovagen, AllerTop, and ToxinPred were utilized to predict the peptide's water solubility, toxicity, and allergenicity. MOE application was used to prepare all ligands and proteins, molecular docking, and visualization. Molecular dynamics simulations were carried out on the protein-ligand complexes on Yasara Dynamics application. The Benchling website was used to perform virtual electrophoresis and reconstruct the recombinant plasmid (Psb1c3). Based on the molecular docking results, peptide REGSPDLLE has the potential as a multitarget inhibitor of tyrosinase (-9.07 kcal/mol), hyaluronidase (-10.57 kcal/mol), elastase (-9.32 kcal/mol), collagenase (-10.57 kcal/mol), and MMP9 (-10.43 kcal/mol). Peptide REGSPDLLE was selected due to its strong binding affinity on the active site of each target protein and exhibits non-toxic, non-allergenic, and good water-soluble as indicated by Support Vector Machine score <0. Molecular dynamics simulations confirmed stable interactions with receptor proteins. Peptide REGSPDLLE was successfully inserted into the recombinant pSB1C3 plasmid, confirmed by virtual electrophoresis with bands at ~2000 bp and ~150 bp. Further in vitro and in vivo studies are necessary to verify the anti-aging efficacy of peptide REGSPDLLE.

Ionizing radiation inhibits zebrafish embryo hatching through induction of tissue inhibitors of metalloproteinases (TIMPs) expression.

Ionizing radiation (IR) has garnered growing attention because of its biological effects on aquatic organisms and humans. Here, we identify the most impacted organs and uncover the molecular mechanisms causing the changes in the context of vertebrate development using single-cell RNA sequencing. Alterations in cellular composition and biological functions were explored using transcriptomic profiling of zebrafish embryos exposed to 5 Gy. Single-cell RNA sequencing analyses unveiled notable shifts in the proportions of brain/central nervous system and hatching gland clusters. Although IR exposure led to increased expression of hatching enzymes, a significant but mild delay in hatching was observed following 5 Gy IR exposure. Gene Ontology analysis showed an increased expression of tissue inhibitors of metalloproteinases (TIMPs), known as matrix metalloproteinase inhibitors, which was confirmed via whole-mount in situ hybridization. Correlation analysis linked TIMPs to transcription factors cebpb and cebpd, which were significantly correlated post-IR exposure. Although no morphological changes were observed in some organs, including the brain, the study reveals substantial alterations in developing vertebrates. Notably, despite increased hatching enzymes, elevated TIMPs in the hatching gland suggest a regulatory mechanism impacting hatching activity. This research contributes to comprehending the ecological repercussions of IR exposure, emphasizing the importance of safety measures for aquatic ecosystems and overall environmental health.

Abstract 4137688: Inhibition of Cardiac Fibrosis and Pro-Fibrotic Collagen Hormone Endotrophin by VS-041, a Novel Drug Candidate for Heart Failure with Preserved Ejection Fraction

Introduction: Endotrophin, a collagen type VI–derived signaling peptide, has been linked to cardiac metabolic dysregulation, inflammation, and fibrosis. Several matrix metalloproteinases (MMPs), incl. MMP2 and MMP9, have been shown to cleave collagen 6A3 and release endotrophin. Baseline plasma endotrophin levels in heart failure with preserved ejection fraction (HFpEF) patients were shown to be strongly and independently associated with increased risk of poor outcomes (death or HF-admissions). VS-041 is a novel drug candidate for HFpEF that inhibits key MMPs implicated in cardiac fibrosis and diastolic dysfunction. Previously, VS-041 demonstrated robust efficacy in the Dahl Salt Sensitive rat model of hypertension and HFpEF, improving diastolic function and dose-proportionally reducing left ventricle (LV) fibrosis. Hypothesis: Inhibition of endotrophin release by VS-041 could contribute to its anti-fibrotic mechanism and serve as a potential biomarker of MMP inhibition (target engagement) in patients. Methods and Results: The effect of VS-041 on production of endotrophin was investigated in a “Scar-in-a-Jar” model using primary human cardiac fibroblasts isolated from adult healthy ventricles. Fibroblasts were treated with 10 ng/mL platelet-derived growth factor (PDGF) AB in the presence of 0.03, 0.3, or 3 µM VS-041, or DMSO control. The concentration of endotrophin in the medium was assessed via the ELISA nordicPRO-C6 TM (Nordic Bioscience A/S). After 4 days of treatment, VS-041 at 0.3 µM and 3 µM significantly inhibited PDGF AB-stimulated production of endotrophin. Cumulative inhibitory effects of VS-041 could still be observed after 12 days of culture. The antifibrotic activity of VS-041 was also tested in a model of extensive cardiac fibrosis in 16-months old C57BL/6 mice exacerbated by angiotensin II infusion at 1 mg/kg/day for 28 days. Oral treatment with VS-041 at 75 mg/kg BID resulted in a 62% reduction (p<0.01) of LV fibrosis as assessed by morphometry of Masson trichrome stained sections. VS-041 also improved the diastolic echocardiography parameters IVRT and E/a ratio. Conclusions: The production of endotrophin by fibroblasts is diminished by VS-041 and, therefore, its plasma levels could be explored as a biomarker of in vivo MMP inhibition by VS-041. Additionally, the inhibition of endotrophin release by VS-041 could be causatively linked to reduction of cardiac fibrosis and potentially translate into improved clinical outcomes in HFpEF patients.

Unveiling the depths of pelvic organ prolapse: From risk factors to therapeutic methods (Review).

Pelvic organ prolapse (POP) is a condition where one or more pelvic organs (such as the uterus, bladder and rectum) descend from their normal anatomical positions into the vagina, primarily due to the weakening of the pelvic floor support structures. While not life-threatening, POP can substantially diminish the patient's quality of life and lead to serious social and psychological complications. Researchers have explored novel directions regarding the etiology, mechanism and treatment of POP. However, existing literature on the subject often lacks comprehensive and systematic overviews. To address this gap and enhance researchers' understanding of POP, the present study reviewed the risk factors and molecular mechanisms of POP [including matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs, transforming growth factor β, advanced glycation end products (AGEs)/receptor for AGE, phosphoinositide 3-kinase/protein kinase B, fibulin, lysyl oxidase-like 1, homeobox A11, collagen α-1 (XVIII) chain, Wnt signaling pathways and estrogen receptor α], as well as therapeutic approaches, such as lifestyle interventions, physical methods, pharmacotherapy, stem cell transplantation and surgical techniques. The present review aims to provide new insights for future research and contribute to the advancement of diagnosis and treatment strategies for POP.

Metalloproteinases are involved in the regulation of prenatal tooth morphogenesis.

During development, tooth germs undergo various morphological changes resulting from interactions between the oral epithelium and ectomesenchyme. These processes are influenced by the extracellular matrix, the composition of which, along with cell adhesion and signalling, is regulated by metalloproteinases. Notably, these include matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), and a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTSs). Our analysis of previously published scRNAseq datasets highlights that these metalloproteinases show dynamic expression patterns during tooth development, with expression in a wide range of cell types, suggesting multiple roles in tooth morphogenesis. To investigate this, Marimastat, a broad-spectrum inhibitor of MMPs, ADAMs, and ADAMTSs, was applied to ex-vivo cultures of mouse molar tooth germs. The treated samples exhibited significant changes in tooth germ size and morphology, including an overall reduction in size and an inversion of the typical bell shape. The cervical loop failed to extend, and the central area of the inner enamel epithelium protruded. Marimastat treatment also disrupted proliferation, cell polarization and organization compared to control tooth germs. Additionally, a decrease in laminin expression was observed, leading to a disruption in continuity of the basement membrane at the epithelial-mesenchymal junction. Elevated Hif-1α expression correlated with a disruption to blood vessel development around the tooth germs. These results reveal the crucial role of metalloproteinases in tooth growth, shape, cervical loop elongation, and the regulation of blood vessel formation during prenatal tooth development.

Metalloproteinase inhibitors in the adhesion process dental

Matrix metalloproteinases (MMPs) are enzymes involved in tooth physiological development, caries processes, and hybrid layer degradation, in addition to being associated with dentin collagen breakdown. The interplay of MMP activity, masticatory forces, and biofilm action may, over time, compromise composite resin restorations. To reduce hybrid layer degradation by endogenous proteases and extend the longevity of resin restorations, MMP inhibitors and collagen cross-linking agents have been extensively studied. This work aims to identify, through a literature review, agents capable of inactivating MMPs at the dentin adhesive interface. This integrative review, based on studies retrieved from PUBMED/MEDLINE, LILACS, BBO, and VHL databases, identified 19 agents able to inhibit MMP activity at the adhesive-dentin interface in studies published between January 2018 and June 2023. It can be concluded that several agents can partially or completely block MMP activity, thereby enhancing restoration longevity. However, further studies are required to facilitate the clinical use of these agents, beyond chlorhexidine, in dental practice, with the application techniques suited to dentists’ daily clinical routines.

Muscle Tissue Response to Vipera berus berus and Vipera berus nikolskii Venoms: A Study on Matrix Metalloproteinases and Cytokine Modulation

Vipera berus, also known as the common adder, is a snake species widely distributed in Europe and parts of Asia. Its venom is a mix of enzymatic and non-enzymatic components causing both local and systemic effects including edema, hemorrhage, myonecrosis, gastrointestinal disorders, and rarely – rhabdomyolysis, coagulopathy and hypovolemic shock. Matrix metalloproteinases (MMPs) and cytokines, being inflammatory mediators, participate both in the development of these reactions and in tissue regeneration after acute damage. The current study was aimed at analyzing the concentrations of important mediators of tissue damage, inflammation and regeneration, namely, MMP-1, -2, -3, -8, -10, TIMP-1 (a tissue inhibitor of MMPs), and pro- and anti-inflammatory cytokines, in rat muscle after intraperitoneal injection of venoms from two subspecies of adders – V. berus berus and V. berus nikolskii — to clarify their role in the local response of skeletal muscle to the studied venoms. Changes in muscle tissue were revealed, namely, significant increase in almost all MMPs including MMP-2, as well as TIMP-1, IFN-γ and anti-inflammatory IL-4 and IL-10 content. Moreover, a tendency for some pro-inflammatory cytokines levels to decline 24 h after venom administration was observed, which may be a sign of inflammation suppression and the beginning of reparative processes associated with extracellular matrix remodeling and formation of an optimal environment for muscle regeneration. However, to establish the exact mechanisms of the changes we found, further studies are needed. The ability of V. berus nikolskii venom to induce more pronounced changes was also shown. Our results may be useful for the development of differentiated and more effective treatment strategies for the consequences of bites by these snakes, which include myonecrosis

MMP inhibition, marginal integrity and cytotoxicity of zinc-releasing GIC

ObjectivesThe degradation of the restorative-dentin interface due to endogenous dentin enzymes, such as matrix metalloproteinases (MMPs), is a significant issue that accelerates the deterioration of the dentin matrix and leads to the failure of restorative treatments. Caredyne Restore (CR), a novel glass ionomer cement (GIC) with zinc ions in its formulation, represents the latest effort to mitigate this issue. This investigation aimed to evaluate the MMPs inhibitory effect, marginal integrity, and cytotoxicity of CR compared to a conventional GIC, Fuji IX (FIX). MethodsThe inhibitory effect of CR on MMPs activity was evaluated using in-situ zymography to visualize the endogenous gelatinolytic activity in the GIC-dentin interface. Additionally, CR's sealing properties were investigated using dye-assisted confocal laser microscopy (CLSM) to assess marginal leakage across the GIC-dentin interface. Both in-situ zymography and CLSM observations were conducted 1 day and seven days after pH cycling. Finally, the cytotoxicity of the GIC eluates was examined on rat dental pulp cells (RPCC2A). Assay measurements were performed after 24 and 48 h of incubation with test solutions prepared using various GIC eluate concentrations. ResultsThe MMPs activity of the CR specimens was significantly lower than that of FIX specimens after seven days of pH cycling. Signs of marginal leakage were also lower in the CR specimens. Comparison of CR and FIX eluates at the same concentration showed no significant difference in terms of biocompatibility. ConclusionsCaredyne restore is capable of inhibiting endogenous enzyme activity and improving sealing properties, while maintaining sufficient biocompatibility. Clinical significanceZinc ions released from Caredyne Restore offer a safe way to improve the quality of dental restorations and promote minimally invasive treatment, especially in lesions where the dentin matrix is susceptible to enzymatic degradation and recurrent caries.

A comparative evaluation of the effect of matrix metalloproteinase inhibitor on the fracture resistance of endodontically treated teeth restored with Everstick-reinforced composite resin: An in vitro study

Aim: To evaluate the effect of applying a matrix metalloproteinase (MMP) inhibitor on the fracture resistance of root-filled teeth restored with Everstick fiber-reinforced composite resin. Subjects and Methods: After the selection of 60 freshly extracted human mandibular first molar, root canal access and standard uniformly sized mesio-occluso-distal (MOD) cavities were made and the teeth were randomly assigned into three groups (n = 20 each): Group I, the MOD cavity was first lined with flowable composite resin and then restored with composite resin. In Group II, Everstick fiber was placed into the bed of flowable composite in buccal–pulpal–lingual direction before the composite restoration was placed. In Group III, after etching of the cavity, a 2% chlorhexidine MMP inhibitor was applied. Then, the MOD cavity was restored same as group II. A universal testing machine was employed to compressively load the teeth at a cross-head speed of 0.5 mm/min till fracture. The maximum fracture loads were recorded in Newtons (N) and data were analyzed with one-way ANOVA and post-hoc Tukey tests. Results: Group III exhibited significantly higher fracture resistance compared to all other groups (P < 0.001), whereas Group I demonstrated the lowest fracture resistance. Conclusion: The utilization of Everstick glass fiber, combined with MMP inhibitor treatment, yielded the greatest fracture resistance. Hence, this method may be prioritized over conventional restoration techniques for strengthening root canal-treated teeth with structurally compromised crowns.

The Dual-edged Role of MMPs and MMP Inhibitors in Restorative Dentistry: Pros, Cons, and Clinical Implications: A Comprehensive Review

Matrix metalloproteinases (MMPs) and their inhibitors take on a multifaceted impact on restorative dentistry, influencing both the integrity of dental materials and the healing processes of dental tissues. This review explores the dual-edged nature of MMPs and MMP inhibitors, highlighting their beneficial effects in promoting dental adhesion in restorative materials and compromised bond strength. We discuss the clinical implications of manipulating MMP activity, evaluating strategies for optimizing their roles in restorative procedures. This article reviews the role of MMPs in the breakdown of collagen within dentin and enamel, highlighting their contribution to dental caries and post-restorative failures. We discuss various MMP inhibitors, including synthetic compounds and natural agents, and their potential applications in enhancing the longevity and efficacy of dental restorations. The synergistic effects of these inhibitors with modern adhesive systems are explored, demonstrating their ability to improve bond strength and resistance to hydrolytic degradation. By integrating recent studies, research and clinical findings, this paper seeks to offer an in-depth insight into MMPs and their treypes of inhibitors can be effectively leveraged in restorative dentistry, while also addressing the challenges they pose to long-term treatment outcomes. Ultimately, our findings underscore the necessity for careful consideration of MMP dynamics to enhance both material performance and patient care in dental practice.

Morin, a matrix metalloproteinase 9 inhibitor, attenuates endothelial-to-mesenchymal transition in atherosclerosis by downregulating Notch-1 signaling

ObjectiveAtherosclerotic cardiovascular disease poses a significant health challenge globally. Recent findings highlight the pivotal role of the endothelial-to-mesenchymal transition (EndMT) in atherosclerosis. Morin is a bioflavonoid mainly extracted from white mulberry, a traditional Chinese herbal medicine with anti-inflammatory and antioxidant properties. This study examines whether morin can alleviate atherosclerosis by suppressing EndMT and seeks to elucidate the underlying mechanism. MethodsWe induced an in vitro EndMT model in human umbilical vein endothelial cells (HUVECs) by stimulating the cells with transforming growth factor β1 (TGF-β1) (10 ng/mL) for 48 h. The in vivo experiments were performed in an atherosclerosis model using apolipoprotein E (ApoE)–/– mice fed with a high-fat diet (HFD). Mice in the intervention group were given morin (50 mg/kg) orally for 4 weeks. Molecular docking and microscale thermophoresis were assayed to understand the interactions between morin and matrix metalloproteinase 9 (MMP-9). ResultsMorin inhibited the expression of EndMT markers in a dose-dependent manner in TGF-β1-treated HUVECs. Administering 50 μmol/L morin suppressed the upregulation of MMP-9 and Notch-1 signaling in TGF-β1-induced EndMT. Moreover, the overexpression of MMP-9 activated Notch-1 signaling, thereby reversing morin’s inhibitory effect on EndMT. In the HFD-induced atherosclerotic ApoE–/– mice, morin notably reduced aortic intimal hyperplasia and plaque formation by suppressing EndMT. Furthermore, morin demonstrated a strong binding affinity for MMP-9. ConclusionMorin acts as an MMP-9 inhibitor to disrupt EndMT in atherosclerosis by limiting the activation of Notch-1 signaling. This study underscores morin’s potential utility in the development of anti-atherosclerotic medication.

Diabetes mellitus exacerbates inflammation in a murine model of ligature-induced peri-implantitis: A histological and microtomographic study.

To investigate the influence of diabetes mellitus (DM) in a murine model of peri-implantitis (PI). Twenty-seven 4-week-old C57BL/6J male mice had their first and second maxillary left molars extracted. Eight weeks later, one machined implant was placed in each mouse. Four weeks after osseointegration, the mice were divided into three groups: (a) control (C), (b) PI and (c) DM + PI. DM was induced by streptozotocin (STZ) administration. After DM induction, PI was induced using ligatures for 2 weeks. The hemimaxillae were collected for micro-CT and histological analyses. The primary outcomes consisted of linear (mm) and volumetric (mm3) bone loss. Secondary outcomes were based on histological analysis and included inflammatory infiltrate, osteoclastic activity, matrix organization, composition and remodelling. Data are presented as means ± SEM. Statistical analyses were performed using one-way ANOVA, followed by Tukey's test. Gingival tissue oedema was detected in the PI and DM + PI groups. Micro-CT showed significantly increased linear and volumetric bone loss in the DM + PI group compared to the C and PI groups. H&E staining showed greater inflammatory response and bone resorption in the PI and DM + PI groups than in the C group. The DM + PI group had significantly higher osteoclast numbers than the C and PI groups. Picrosirius red stained less for types I and III collagen in the PI and DM + PI groups than in the C group. There was a significant increase in monocyte/macrophage (CD-11b) counts and matrix metalloproteinases (MMP-2 and MMP-8) marker levels and a significant decrease in the matrix metalloproteinases inhibition marker (TIMP-2) levels in the DM + PI group compared to the C and PI groups. DM exacerbates PI-induced soft-tissue inflammation, matrix degradation and bone loss.

Genetic removal of Nlrp3 protects against sporadic and R345W Efemp1-induced basal laminar deposit formation.

Chronic, unresolved inflammation has long been speculated to serve as an initiating and propagating factor in numerous neurodegenerative diseases, including a leading cause of irreversible blindness in the elderly, age-related macular degeneration (AMD). Intracellular multiprotein complexes called inflammasomes in combination with activated caspases facilitate production of pro-inflammatory cytokines such as interleukin 1 beta. Specifically, the nucleotide-binding oligomerization (NOD)-like receptor protein 3 (NLRP3) has received heightened attention due to the wide range of stimuli to which it can respond and its potential involvement in AMD. In this study, we directly tested the role of Nlrp3 and its downstream effector, caspase 1 (Casp1) in mediating early AMD-like pathology (i.e., basal laminar deposits [BLamDs]) in wild-type (WT) mice and the Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) mouse model (p.R345W mutation in Efemp1). Compared to aged-matched controls, R345W+/+ knockin mice demonstrated increased Muller cell gliosis, subretinal Iba-1+ microglial cells, higher Nlrp3 immunoreactivity in the retina, as well as significant transcriptional upregulation of complement component 3, Nlrp3, pro-Il1b, pro-caspase-1, and tissue inhibitor of matrix metalloproteinase 3 in the retinal pigmented epithelium (RPE)/choroid. These findings were accompanied by an age-related increase in BLamD formation in the R345W+/+ mice. Genetic elimination of either Nlrp3 or Casp1 significantly reduced both the size and coverage of BLamDs in the R345W+/+ background, highlighting an important and underappreciated pathway that could affect ML/DHRD onset and progression. Moreover, Nlrp3 knockout reduced spontaneous, idiopathic BLamDs in WT mice, suggesting translatability of our findings not only to rare inherited retinal dystrophies, but also potentially to AMD itself.

Engineered TIMP2 with narrow MMP-9 specificity is an effective inhibitor of invasion and proliferation of triple-negative breast cancer cells

Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade extracellular matrix proteins, functioning in various physiological processes such as tissue remodeling, embryogenesis, and morphogenesis. Dysregulation of these enzymes is linked to multiple diseases. Specific inhibition of particular MMPs is crucial for anti-MMP drug development as some MMPs have shown antidisease properties. In this study, we aimed to design a highly specific inhibitor of MMP-9, that plays a crucial role in cell invasion and metastasis, using tissue inhibitor of metalloproteinases 2 (TIMP2s), an endogenous broad-family MMP inhibitor, as a prototype. In our earlier work, we were able to narrow down the specificity of the N-terminal domain of TIMP2 (N-TIMP2) toward MMP-9, yet at the expense of lowering its affinity to MMP-9. In this study, a library of N-TIMP2 mutants based on previous design with randomized additional positions was sorted for binding to MMP-9 using yeast surface display. Two selected N-TIMP2 mutants were expressed, purified, and their inhibitory activity against a panel of MMPs was measured. The best engineered N-TIMP2 mutant (REY) exhibited a 2-fold higher affinity to MMP-9 than that of the WT N-TIMP2, and 6- to 1.1 x 104-fold increase in binding specificity toward MMP-9 compared to five alternative MMPs. Moreover, REY demonstrated a significant increase in inhibition of cell invasion and proliferation compared to the WT N-TIMP2 in MDA-MB-231 breast cancer cells. Therefore, our engineered N-TIMP2 mutant emerges as a promising candidate for future therapeutic development, offering precise targeting of MMP-9 in MMP-9-driven diseases.

Determining key residues of engineered scFv antibody variants with improved MMP-9 binding using deep sequencing and machine learning

Given the crucial role of specific matrix metalloproteinases (MMPs) in the extracellular matrix, an imbalance in the regulation of activation of matrix metalloproteinase-9 (MMP-9) zymogen and inhibition of the enzyme can result in various diseases, such as cancer, neurodegenerative, and gynecological diseases. Thus, developing novel therapeutics that target MMP-9 with single-chain antibody fragments (scFvs) is a promising approach. We used fluorescent-activated cell sorting (FACS) to screen a synthetic scFv antibody library displayed on yeast for enhanced binding to MMP-9. The screened scFv mutants demonstrated improved binding to MMP-9 compared to the natural inhibitor of MMPs, tissue inhibitor of metalloproteinases (TIMPs). To identify the molecular determinants of these engineered scFv variants that affect binding to MMP-9, we used next-generation DNA sequencing and computational protein structure analysis. Additionally, a deep-learning language model was trained on the screened scFv library of variants to predict the binding affinities of scFv variants based on their CDR-H3 sequences.

Clinical and morphological features of the myometrium in patients with placental adherent and invasive pathology

BACKGROUND: The fast increase in the frequency of placental adherent and invasive pathology worldwide accounts for the growing interest in studying the pathogenesis of placenta accreta. According to the literature, the clinical and morphological features of the myometrium from the placental attachment area in placental adherent and invasive pathology are described in single articles. Study of morphological features using proteolytic markers in the myometrium from the placenta accreta area could help in understanding the pathogenesis of placental adherent and invasive pathology. For the first time in this study, the clinical and morphological features of the myometrium from the placental attachment area in patients with uterine scar and placental adherent and invasive pathology are compared with the myometrium of women with uterine scar without placenta accreta and intact myometrium. AIM: The aim of this study was to evaluate the expression of matrix metalloproteinase 2 and tissue inhibitors of matrix metalloproteinases 1 and 2 in myometrial biopsies in placental adherent and invasive pathology. MATERIALS AND METHODS: This study included 15 myometrial biopsies from the placental site, which were divided into three groups according to the clinical diagnosis of the patients: group 1 (main group), with a uterine scar after cesarean section and placental adherent and invasive pathology (n = 5); group 2 (comparison group), with a uterine scar after cesarean section (n = 5); group 3 (control group), with a normal pregnancy without a uterine scar (n = 5). Histological examination was carried out using the standard procedure. Immunohistochemical study was performed using antibodies to matrix metalloproteinase 2 and tissue inhibitors of matrix metalloproteinases 1 and 2 (Abcam, USA). Morphometry was carried out using the VideoTesT-Morphology 5.2 program (Videotest Ltd., Russia). The statistical analysis was performed using the IBM SPSS Statistics 26.0 software. RESULTS: In the biopsies of the main group, we verified terminal chorionic villi with hypervascularization and uneven plethora of the vascular bed among hypertrophied muscle fibers, a basal plate with lumen ectasia of unevenly full-blooded vessels, and the absence of a decidual membrane, in contrast to groups 2 and 3. The matrix metalloproteinase 2 expression area in the main group was higher than in the comparison and control groups (p = 0.008; p1–2 = 0.049*, p1–3 = 0.011), the matrix metalloproteinase 2 optical density not differing between the groups (p = 0.122). The tissue inhibitors of matrix metalloproteinases 1 expression area was higher in the comparison group compared to the main group (p = 0.035; p2–3 = 0.032), and the tissue inhibitors of matrix metalloproteinases 1 and 2 optical density was higher in the comparison group compared to control (p = 0.008; p2–3 = 0.005). CONCLUSIONS: In myometrial biopsies of patients with a uterine scar and placental adherent and invasive pathology, we verified pathology of the basal plate of the placenta, increased matrix metalloproteinase 2 expression and decreased tissue inhibitors of matrix metalloproteinases 1 expression, which may indicate the peculiarities of the inflammatory response in the myometrium in placental adherent and invasive pathology.

Elevated MMP-9, Survivin, TGB1 and Downregulated Tissue Inhibitor of TIMP-1, Caspase-3 Activities are Independent of the Low Levels miR-183 in Endometriosis.

This study aimed to measure the correlation between miR-183 and gene expression that regulates apoptosis and adhesion mechanism that may be linked to the pathogenesis of endometriosis. Forty-four subjects, including 22 control subjects, participated in this study. We collected ectopic endometriosis and endometrial samples. For the control, the sample was taken from endometrial tissue through pipelle biopsy. RNA was extracted from all tissues using RNA mini kit, and the expression was assessed using quantitative-real time PCR. Relative mRNA and miRNA expression were presented using the formula of the Livak method. The data were statistically analyzed using GraphPad Prism 8. The expression of Caspase-3, Survivin, Integrin β1 (ITGB1), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) (adhesion- and apoptosis-related gene) were calculated using the relative expression method. We found significant differences in Caspase-3, Survivin, ITGB1, MMP-9, and TIMP-1 expression between ectopic endometriosis tissues of women with endometriosis compared to healthy endometrium. MMP-9, Survivin, and ITGB1 was significantly increased in the endometriosis group, while Caspase-3, TIMP-1, and miR-183 were significantly reduced in the endometriosis group. No correlation was found between the expression level of miR-183 and Caspase3, Survivin, ITGB1, and Cadherin in both tissue types. Despite the difference in expression levels of miR-183 and associated adhesion- and apoptosis-related genes, there was no significant association between miR-183 with specific adhesion and apoptosis genes in endometriosis tissue.