Matrix Metalloproteinase Activity Research Articles

Localized Microrobotic Delivery of Enzyme-Responsive Hydrogel-Immobilized Therapeutics to Suppress Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC), characterized by its aggressive metastatic propensity and lack of effective targeted therapeutic options, poses a major challenge in oncological management. A proof-of-concept neoadjuvant strategy aimed at inhibiting TNBC tumor growth and mitigating metastasis through a localized delivery of chemotherapeutics is reported in this paper. This approach addresses the limitations in payload capacity and stimuli responsiveness commonly associated with microrobotics in oncology. A hydrogel-based system is developed for the immobilization of chemotherapeutic agents, subsequently encapsulated within magnetically responsive microrobots. This design leverages external magnetic fields to facilitate the precise navigation and localization of the therapeutic agents directly to the tumor site. The efficacy of this approach is demonstrated in an animal model, in which a significant 14-fold reduction in tumor size and suppression of metastasis to critical organs such as the liver and lungs are observed. Crucially, the drug release mechanism is engineered to be responsive to the tumor microenvironment and is regulated by the overexpression of the enzymatic activity of matrix metalloproteinases (MMP2 and MMP9) in TNBC tumors, triggering the degradation of the hydrogel matrix, leading to controlled release of the immobilized therapeutic drug. This ensures that the therapeutic action is localized, reducing systemic toxicity and enhancing treatment efficacy. These findings suggest that this neoadjuvant approach holds promise for broader applications in other cancer types.

Eucalyptol Attenuates Cerebral Ischemia/Reperfusion Injury in Mice

Background One of the leading causes of acute brain injury is cerebral ischemia/reperfusion (I/R) injury. It is an ailment that occurs when the brain does not receive enough oxygen-rich blood supply, which damages brain cells. Oxidative stress is recognized as a significant mechanism that causes I/R injury and is a critical focal point for therapy. Apoptosis and inflammation are all pathogenic processes underpinning I/R damage. Eucalyptol (EU), a plant-derived substance, protects neuronal damage. We postulate that EU inhibits apoptosis and oxidative stress after focal cerebral I/R damage in mice. Purpose Thus, the present work sought to examine the neurological protective benefits of EU in mice models of cerebral ischemia/reperfusion (CI/R) injury. Methods The measurement of neurological function, infarct volume, and the amount of water in the brain were evaluated subsequently. The brain tissue was examined to assess the activities of oxidative stress, inflammatory cytokines, and matrix metalloproteinases (MMP) levels, and histopathology was determined. Using enzyme-linked immunosorbent assay (ELISA), apoptotic marker expression was used to measure the levels of B-cell lymphoma 2 (Bcl-2), caspase-3, and Bcl-2 associated X-protein (Bax) proteins that exist as indicators of apoptosis. Results EU treatment malondialdehyde (MDA) levels decreased while glutathione (GSH), superoxide dismutase (SOD), A-TOC, and catalase (CAT) activity increased. Following EU therapy dramatically reduced cerebral dysfunction, which was supported by reduced histological damage in I/R injury. Furthermore, with lower levels of MMP-2 and 9, EU treatment could be a novel approach for preventing CI/R injury. EU-treated mice had a notable reduction in cleaved Bax, caspase-3, and increased Bcl-2 protein activities. EU therapy shields the brain against CI/R injury by inhibiting cell death. Conclusion According to the present study’s findings, EU is a promising neuroprotective agent that can be used to treat CI/R injury in mice. By focusing on these aspects, nurses can provide comprehensive care to patients involved in studies on the effects of EU on CI/R injury, promoting better outcomes through careful monitoring, supportive interventions, and education.

Differential modulation of inflammatory cytokines by recombinant IL-10 in IL-1β and TNF-α ̶ stimulated equine chondrocytes and synoviocytes: impact of washing and timing on cytokine responses

Osteoarthritis (OA) remains a challenging joint disorder necessitating effective anti-inflammatory interventions. In this study, our primary objective was to establish an in vitro protocol that replicates the clinical investigation of anti-inflammatory drugs intended for OA management. Focusing on recombinant IL-10 (r.IL-10) as a potential anti-inflammatory treatment, we designed and implemented two distinct protocols to evaluate the efficacy of r.IL-10 in modulating chondrocyte and synoviocyte inflammation.The experimental design involved sequential stimulation with IL-1β and TNF-α for 24 h, followed by washing (model 1) or not washing (model 2) the cells before r.IL-10 treatment. Samples were collected after 6–24 h of treatment. Cellular responses were evaluated by quantifying gene expression and synthesis of key inflammatory cytokines and proteases.The expression and synthesis of inflammatory cytokines and proteases was significantly affected by washing and treatment time. The expression of IL-1β, TNF-α, IL-8, MMP-13, and ADAMTS5 were effectively reduced in r.IL-10-treated chondrocytes and synoviocytes in model 2 after 24 h, particularly at concentrations of 10 and 20 ng/mL. r.IL-10 treatment significantly increased IL-6 gene expression in chondrocytes at all time points. However, in synoviocytes, IL-6 expression was significantly lower in model 2 after 24 h of r.IL-10 treatment. r.IL-10 treatment significantly decreased IL-1β and TNF-α content in synoviocyte supernatants, particularly in model 2 at concentrations of 10 and 20 ng/mL after 6 and 24 h. r.IL-10 treatment in chondrocytes led to a significant decrease in IL-1β supernatant concentrations in model 2 after 24 h only.This study demonstrated that r.IL-10 treatment effectively reduces key inflammatory markers and matrix metalloproteinase activity in both chondrocytes and synoviocytes, particularly in model 2 where cells were not washed prior to treatment. These findings highlight r.IL-10’s potential as a robust anti-inflammatory agent for OA management and suggest its critical role in developing effective therapeutic strategies for OA.

Acute Three-Dimensional Hypoxia Regulates Angiogenesis.

Hypoxia elicits a multitude of tissue responses depending on the severity and duration of the exposure. While chronic hypoxia is shown to impact development, regeneration, and cancer, the understanding of the threats of acute (i.e., short-term) hypoxia is limited mainly due to its transient nature. Here, a novel gelatin-dextran (Gel-Dex) hydrogel is established that decouples hydrogel formation and oxygen consumption and thus facilitates 3D sprouting from endothelial spheroids and, subsequently, induces hypoxia "on-demand." The Gel-Dex platform rapidly achieves acute moderate hypoxic conditions without compromising its mechanical properties. Acute exposure to hypoxia leads to increased endothelial cell migration and proliferation, promoting the total length and number of vascular sprouts. This work finds that the enhanced angiogenic response is mediated by reactive oxygen species, independently of hypoxia-inducible factors. Reactive oxygen species-dependent matrix metalloproteinases activity mediated angiogenic sprouting is observed following acute hypoxia. Overall, the Gel-Dex hydrogel offers a novel platform to study how "on-demand" acute moderate hypoxia impacts angiogenesis, with broad applicability to the development of novel sensing technologies.

Disruption of PCSK9 Suppresses Inflammation and Attenuates Abdominal Aortic Aneurysm Formation.

Abdominal aortic aneurysm (AAA) is a chronic vascular inflammatory disease without effective medications. PCSK9 (proprotein convertase subtilisin/kexin 9), a serine protease from the proprotein convertase family, has recently been associated with AAA in human genome-wide association studies. However, its role in AAA is unknown. Transcriptional and histological expression of PCSK9 was examined in AAA tissues and healthy controls. The impact of PCSK9 deletion and inhibition on AAA formation was assessed in mice with hyperlipidemia and Ang II (angiotensin II) overproduction. AAA lesion morphology was assessed by tissue staining. MMP (matrix metalloproteinase) activity was evaluated by gelatin zymography, and leukocyte-vessel wall interaction was monitored by intravital microscopy. RNA sequencing was used to characterize the downstream signaling of PCSK9. PCSK9 expression was upregulated and colocalized with macrophages in human and mouse AAAs. Pcsk9 deletion attenuated AAA formation, improved survival, and decreased systemic inflammation, without altering circulating cholesterol levels. Pcsk9 deficiency reduced aortic infiltration of macrophages and elastin degradation, without affecting vascular smooth muscle cell apoptosis and proliferation. Mechanistically, PCSK9 was essential in leukocyte-endothelium adhesion and expression of proinflammatory cytokines and MMP9 by macrophages. RNA sequencing of stimulated macrophages revealed that Pcsk9 deficiency upregulated histone deacetylase SIRT1 (sirtuin-1) and suppressed NF-κB (nuclear factor-κB) inflammatory signaling. SIRT1 inhibition attenuated the proinflammatory actions of PCSK9. Furthermore, administration of PCSK9 small interfering RNA or antibody constrained AAA formation/progression and inhibited vascular inflammation. PCSK9 critically mediates macrophage inflammation and elastin degradation, promoting AAA formation. PCSK9 inhibitors bear a promise to curtail AAA, beyond being used as cholesterol-lowering drugs.

Retinol and Hydroxyasiaticoside Synergistically Relieve Histamine-Induced Atopic Dermatitis Activity by Repressing TRPV1, L1R1, and CD130 Targets

Background: Retinol, an important bioactive substance with multiple physiological functions such as promoting collagen synthesis, inhibiting matrix metalloproteinase activity, alleviating oxidative stress, regulating gene expression, and promoting epidermal cell proliferation, has a significant effect on skin damage recovery. Hydroxyasiaticoside, a triterpenoid saponin derived from Centella asiatica (L.) Urb., is closely related to the secretion of collagen types I and III, and possesses multiple biological activities, including moisturizing, antioxidants, anti-apoptosis, neuroprotection, anti-inflammation, and the promotion of wound healing. It plays a particularly prominent role in reducing oxidative stress in wounds and inducing vasodilatation. Objective: The aim of this study was to investigate the therapeutic efficacy of retinol combined with hydroxyasiaticoside in histamine-induced atopic dermatitis. Materials and Methods: The experiment was carried out using three different concentrations of a retinol and hydroxyasiaticoside mixed solution: low, medium, and high concentrations. After inducing atopic dermatitis in mice through histamine administration, these solutions were applied to the skin surface of the mice, and a comparative analysis was conducted with both the control group and the model group. The effect of combination therapy on atopic dermatitis was evaluated through histopathology, immunohistochemistry, and transcriptomic analysis. Results: The combination of retinol and hydroxyasiaticoside significantly attenuated histamine-induced scratching behaviors, alleviated the phenomenon of epidermal hyperplasia, and effectively reduced the proliferation, infiltration, and degranulation of mast cells. In addition, the combination inhibited the expression of relevant pro-inflammatory cytokines. Quantitative RNA-seq analysis revealed that the gene expression patterns were similar in different concentration groups. However, the medium dose group may be able to regulate skin inflammation by regulating upstream genes to inhibit autophagy-related pathways. Further GO analysis revealed that the low-dose group mainly affected metabolism-related genes, the medium-dose group affected more genes related to body systems, and the high-dose group was dominated by genes related to human diseases.

The dual effect of fiber density and matrix stiffness on A549 tumor multicellular migration

The tumor microenvironment features dynamic biomechanical interactions between extracellular matrix physics and tumor progression. Tumor growth compresses the supportive matrix, and the stiffness-gradient guides tumor invasion. From the mechanical perspective, the complexity of the matrix topology involving durotaxis-driven metastasis remains lacking in a comprehensive description. In this study, A549 adenocarcinoma spheroids were exposed to a stiffness-and fiber-adjusted collagen matrix to examine the influence of collective motility. Centrifugated compression on the collagen constructs was adopted to mimic the matrix deformation in response to solid tumor development. Centrifugated compression physically stiffened and condensed collagen constructs simultaneously. Cultured with A549 spheroids for 7 days, compressed collagen constructs disadvantaged spheroid expansion without the effect of tumor proliferation potency but promoted matrix metalloproteinase activity corresponding to softened rigidity. Results suggested that the fibrous structure may counterbalance the matrix stiffness-induced motility.

Diabetes-induced alteration of metal ion levels declines the activity of MMPs to decrease aortic aneurysm risk

AimsDiabetes mellitus (DM) links the risk of cardiovascular diseases. Inverse to the enhanced expression of matrix metalloproteinases (MMPs), the development of aortic aneurysm is lower in diabetic population. We examined the hypothesis that DM-induced alteration of metal ion levels declines the activity of MMPs to decrease aortic aneurysm risk. Methods & ResultsBy culturing vascular smooth muscle cells (VSMCs) or macrophages with different concentrations of glucose in the medium, we confirmed that high glucose significantly increased the expression of fibronectin and CTGF in VSMCs, and induced MMP2 expression and MMP9 secretion in macrophages. We also established an abdominal aortic aneurysm model in streptozotocin-induced diabetic mice and evaluated aneurysm development six weeks later. Compared to the healthy controls, diabetic mice had significantly lower levels of Zn2+ and Mg2+ in serum and developed significantly smaller sizes of aneurysms with higher expression of fibronectin and CTGF; but dietary zinc supplementation to diabetic mice effectively neutralized these differences. Gelatin zymography assay indicated that the enzymatic digestion activity of MMP2 was changed under different concentrations of ZnSO4 and MgSO4. Clinical data analysis also confirmed that DM, serum Zn2+ level, and aortic aneurysm risk closely correlated with each other. ConclusionIt seems that DM-induced alteration of metal ion levels declines the activity of MMPs to negate aortic aneurysm development. Our data provide novel mechanistical insight and therapeutic strategy for aortic aneurysms.

Role of MMPs in connective tissue breakdown and periodontal disease: A Review

Matrix metalloproteinases (MMPs) are a group of zinc-dependent enzymes that play a critical role in the degradation of extracellular matrix components, including collagen, elastin, and proteoglycans, making them central to tissue remodeling processes. In the context of periodontal disease, MMPs are key mediators of connective tissue breakdown and alveolar bone destruction, driven by chronic inflammation. This review focuses on the role of MMPs in periodontal disease, highlighting their regulation, activation mechanisms, and the interplay with inflammatory cytokines such as interleukins and tumor necrosis factor-alpha (TNF-α). Furthermore, the review discusses the contribution of specific MMPs, including MMP-1, MMP-8, and MMP-9, in periodontal tissue degradation and explores potential therapeutic approaches to inhibit MMP activity to prevent periodontal disease progression. By understanding the molecular basis of MMP involvement in periodontal pathology, this review aims to provide insights into novel therapeutic strategies aimed at mitigating connective tissue breakdown in periodontal disease.

Abstract A028: Precise monitoring of osteosarcoma patients by noninvasive assessment of matrix metalloproteinases activities in plasma extracellular vesicles

Abstract Osteosarcoma (OS) is the most prevalent bone cancer in children, characterized by aggressive primary tumors and high metastatic potential. Current treatment protocols involve a combination of neoadjuvant chemotherapy, surgery, and adjuvant chemotherapy, tailored based on histological grade and metastatic risk. Identifying metastatic lesions through routine imaging significantly impacts treatment outcomes, highlighting the need for innovative diagnostic tools for timely monitoring. Noninvasive in vitro diagnostics, analyzing circulating tumor components like tumor-derived extracellular vesicles (EVs), would offer promise for monitoring treatment responses in pediatric sarcomas. Matrix metalloproteinases (MMPs) play a crucial role in tumor progression by degradation of the extracellular matrix; thus, recent research has focused on understanding their functional roles by profiling their proteolytic activities. Tumor EVs are crucial in transporting MMPs to facilitate invasion and metastasis, making them suitable surrogate for noninvasive profiling of MMP activities. Herein, we introduce an innovative OS EV MMP Activity Assay to assess MMP activities in OS patients noninvasively. This assay evaluates six different combinations of surface markers on OS EVs and OS-associated MMPs, generating a unique profile for each patient. Using a logistic regression model, an OS EV MMP Activity Score is generated from the most effective combinations. This Score has shown excellent performance in distinguishing between metastatic and localized OS. In a longitudinal study of six OS patients undergoing treatment, the Score strongly correlated with treatment response as measured by routine imaging. The assay represents a significant advancement in harnessing tumor EVs for diagnostics, providing a noninvasive, quantitative, and reliable approach to assess MMP activities in solid tumors. Citation Format: Junseok Lee, You-Ren Ji, Hyoyong Kim, Zhao Chen, Ryan Zhang, Diego Banuet, Yusef Mathkour, Kenny Vo, Steven Jonas, Yazhen Zhu, Hsian-Rong Tseng. Precise monitoring of osteosarcoma patients by noninvasive assessment of matrix metalloproteinases activities in plasma extracellular vesicles [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A028.

Serum integrin accumulation during asthma exacerbation: The role of matrix metalloproteinases.

The inflammation caused by asthma exacerbation can lead to permanent changes in the airways and loss of lung function. Integrins are membrane receptors that interact with components of the extracellular matrix and cell adhesion molecules. It is known that these receptors can be found in soluble form in some conditions such as asthma, but it is unknown if exacerbation during asthma leads to soluble integrins. Our results indicated that asthma patients showed higher levels of soluble α1, α2, and β2 integrin subunits in their serum compared to controls, as confirmed by both ELISA and western blot. During asthma exacerbation, the levels of α2 and β2 integrin subunits increased even more compared to non-exacerbation and controls, while the α1 integrin subunit decreased. Western blot analysis identified two β2 integrin subunits, one at 75 kDa and another at 120 kDa; the 120 kDa subunit increased during asthma exacerbation. The activity of matrix metalloproteinase 9 (MMP9) increased during exacerbation, while MMP2 remained unchanged. Lower forced expiratory volume in 1 second (FEV1) values were associated with higher expression levels of α2, β1, and β2 integrin subunits. Active and latent MMP9 were correlated with the levels of the β2 integrin subunit, which means that at low levels of active and latent MMP9, there are lower levels of β2 integrin subunit. In conclusion, asthma exacerbation leads to the presence of soluble integrins, particularly the β2 subunit, most likely due to MMP9-induced proteolytic cleavage.

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.

Abnormal proteolytic activity profile in plasma of blood donors according to anti-SARS-CoV-2 IgG titer

This work aims to study whether there is a relationship between titer values of anti-SARS-CoV-2 IgG and changes in proteolytic processes. To confirm this hypothesis, we analyzed the content and activity of Matrix Metalloproteinases (MMPs) as well as the concentration and composition of circulating peptide pools in the plasma of blood donors divided into groups on the basis of anti-SARS-CoV-2 IgG titers. The results of gelatin zymography showed the presence of active MMP-2 in donors’ plasma who recovered from COVID-19. In contrast, collagenases and their complexes were detected in the plasma of donors with no anti-SARS-CoV-2 IgG, while their activity was undetectable in some groups of COVID-19 convalescent individuals. The content of MMPs also differed among the donors with different titers of anti-SARS-CoV-2 IgG. Plasma peptide content was identical among the donors' groups, but there were more peptide fractions in plasma peptide pools of COVID-19 convalescent individuals; furthermore, they all were characterized by the presence of peptides with molecular weights less than 920 Da and greater than 1530 Da. We hypothesized a link between proteolytic alterations and peptide fraction composition. Our data need further validation to confirm the relationship between the titer values of anti-SARS-CoV-2 IgG and the severity of the proteolytic imbalance.

Interpenetrating Polymer Network Hydrogels with Tunable Viscoelasticity and Proteolytic Cleavability to Direct Stem Cells In Vitro.

The dynamic nature of cellular microenvironments, regulated by the viscoelasticity and enzymatic cleavage of the extracellular matrix, remains challenging to emulate in engineered synthetic biomaterials. To address this, a novel platform of cell-instructive hydrogels is introduced, composed of two concurrently forming interpenetrating polymer networks (IPNs). These IPNs consist of the same basic building blocks - four-armed poly(ethylene glycol) and the sulfated glycosaminoglycan (sGAG) heparin - are cross-linked through either chemical or physical interactions, allowing for precise and selective tuning of the hydrogel's stiffness, viscoelasticity, and proteolytic cleavability. The studies of the individual and combined effects of these parameters on stem cell behavior revealed that human mesenchymal stem cells exhibited increased spreading and Yes-associated protein transcriptional activity in more viscoelastic and cleavable sGAG-IPN hydrogels. Furthermore, human induced pluripotent stem cell (iPSC) cysts displayed enhanced lumen formation, growth, and pluripotency maintenance when cultured in sGAG-IPN hydrogels with higher viscoelasticity. Inhibition studies emphasized the pivotal roles of actin dynamics and matrix metalloproteinase activity in iPSC cyst morphology, which varied with the viscoelastic properties of the hydrogels. Thus, the introduced sGAG-IPN hydrogel platform offers a powerful methodology for exogenous stem cell fate control.

Endogenous/exogenous stimuli‐responsive smart hydrogels for diabetic wound healing

AbstractDiabetes significantly impairs the body's wound‐healing capabilities, leading to chronic, infection‐prone wounds. These wounds are characterized by hyperglycemia, inflammation, hypoxia, variable pH levels, increased matrix metalloproteinase activity, oxidative stress, and bacterial colonization. These complex conditions complicate effective wound management, prompting the development of advanced diabetic wound care strategies that exploit specific wound characteristics such as acidic pH, high glucose levels, and oxidative stress to trigger controlled drug release, thereby enhancing the therapeutic effects of the dressings. Among the solutions, hydrogels emerge as promising due to their stimuli‐responsive nature, making them highly effective for managing these wounds. The latest advancements in mono/multi‐stimuli‐responsive smart hydrogels showcase their superiority and potential as healthcare materials, as highlighted by relevant case studies. However, traditional wound dressings fall short of meeting the nuanced needs of these wounds, such as adjustable adhesion, easy removal, real‐time wound status monitoring, and dynamic drug release adjustment according to the wound's specific conditions. Responsive hydrogels represent a significant leap forward as advanced dressings proficient in sensing and responding to the wound environment, offering a more targeted approach to diabetic wound treatment. This review highlights recent advancements in smart hydrogels for wound dressing, monitoring, and drug delivery, emphasizing their role in improving diabetic wound healing. It addresses ongoing challenges and future directions, aiming to guide their clinical adoption.

LKB1 and STRADα Promote Epithelial Ovarian Cancer Spheroid Cell Invasion.

Late-stage epithelial ovarian cancer (EOC) involves the widespread dissemination of malignant disease throughout the peritoneal cavity, often accompanied by ascites. EOC metastasis relies on the formation of multicellular aggregates, called spheroids. Given that Liver Kinase B1 (LKB1) is required for EOC spheroid viability and LKB1 loss in EOC cells decreases tumor burden in mice, we investigated whether the LKB1 complex controls the invasive properties of human EOC spheroids. LKB1 signalling was antagonized through the CRISPR/Cas9 genetic knockout of LKB1 and/or the RNAi-dependent targeting of STE20-related kinase adaptor protein (STRAD, an LKB1 activator). EOC spheroids expressing nuclear GFP (green) or mKate2 (red) constructs were embedded in Matrigel for real-time live-cell invasion monitoring. Migration and invasion were also assessed in spheroid culture using Transwell chambers, spheroid reattachment, and mesothelial clearance assays. The loss of LKB1 and STRAD signalling decreased cell invasion through Matrigel and Transwell membranes, as well as mesothelial cell clearance. In the absence of LKB1, zymographic assays identified a loss of matrix metalloproteinase (MMP) activity, whereas spheroid reattachment assays found that coating plates with fibronectin restored their invasive potential. A three-dimensional EOC organoid model demonstrated that organoid area was greatly reduced by LKB1 loss. Overall, our data indicated that LKB1 and STRAD facilitated EOC metastasis by promoting MMP activity and fibronectin expression. Given that LKB1 and STRAD are crucial for EOC metastasis, targeting LKB1 and/or STRAD could disrupt the dissemination of EOC, making inhibitors of the LKB1 pathway an alternative therapeutic strategy for EOC patients.

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.

Epoxytiglianes induce keratinocyte wound healing responses via classical protein kinase C activation to promote skin re-epithelialization

Epoxytiglianes are a novel class of diterpene esters. The prototype epoxytigliane, EBC-46 (tigilanol tiglate), is a potent anti-cancer agent in clinical development for local treatment of a range of human and animal tumors. EBC-46 also consistently promotes wound re-epithelialization at the treatment sites, mediated via activation of classical protein kinase C (PKC) isoforms. We have previously shown that epoxytiglianes stimulate proliferative and wound repopulation responses in immortalized human skin keratinocytes (HaCaTs) in vitro, abrogated by pan-PKC inhibitor, bisindolylmaleimide-1. In this study, we further investigate the specific PKC isoforms responsible for inducing such wound healing responses, following HaCaT treatment with 1.51 nM-15.1 µM EBC-46 or analogue, EBC-211. Classical PKC inhibition by GӦ6976 (1 μM), significantly attenuated epoxytigliane induced, HaCaT proliferation and wound repopulation at all epoxytigliane concentrations. PKC-βI/-βII isoform inhibition by enzastaurin (1 μM), significantly inhibited HaCaT proliferation and wound repopulation responses induced by both epoxytiglianes, especially at 1.51–151 nM. PKC-α inhibitor, Ro 31–8220 mesylate (10 nM), exerted lesser inhibitory effects on HaCaT responses. Epoxytigliane changes in key keratin (KRT17) and cell cycle (cyclin B1, CDKN1A) protein levels were partly attenuated by GӦ6976 and enzastaurin. GӦ6976 also inhibited increases in matrix metalloproteinase (MMP-1, MMP-7, MMP-10) activities. Phospho-PKC (p-PKC) studies confirmed that epoxytiglianes transiently activated classical PKC isoforms (p-PKCα, p-PKC-βI/-βII, p-PKCγ) in a dose- and time-dependent manner. By identifying how epoxytiglianes stimulate classical PKCs to facilitate keratinocyte healing responses and re-epithelialization, these findings support further epoxytigliane development as topical therapeutics for clinical situations involving impaired re-epithelialization, such as non-healing wounds in skin.

Endogenous Tissue Inhibitor of Metalloproteinase-2 Levels Are Associated With High-Quality Neat Semen but Unrelated to Sperm Cryoresistance in Bulls.

Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) is part of the tissue inhibitors of the metalloproteinases (TIMPs) family. Its primary function is to regulate the activity of matrix metalloproteinases (MMPs) across various tissues, including those of the reproductive system. This study aimed to quantify the natural levels of TIMP-2 in seminal plasma (SP) and sperm membrane (SM) of bulls, explore potential associations between TIMP-2 levels and semen quality parameters, and examine the relationship between TIMP-2 levels and sperm cryoresistance in bulls. Thirty semen samples from Frieswal breeding bulls were categorized into two groups based on their initial progressive motility (IPM): Good (IPM ≥ 70%; n = 21) and Poor (IPM ≤ 40%; n = 9). The samples were evaluated for their quality parameters at the fresh stage, and TIMP-2 levels were measured in SP and SM using a bovine-specific ELISA kit. Following cryopreservation of Good samples (n = 21), post-thaw motility (PTM) was used to further classify samples into Freezeable (PTM ≥ 50%; n = 14) and Non-Freezable (PTM < 50%; n = 7) groups. In frozen-thawed samples, sperm attributes, kinetics, and functional parameters were assessed, and the results were correlated with retrospective TIMP-2 levels of SP/SM. Our study revealed that the quantified levels of TIMP-2 ranged from 100.27 to 535.95 ng/L in SP and from 0 to 115.78 ng/10 million spermatozoa in SM. TIMP-2 levels in both SP and SM were significantly higher in Good ejaculates compared to Poor ejaculates (p < 0.01). Furthermore, total TIMP-2 levels in the SP/SM of semen samples from bulls showed a positive correlation with fresh semen attributes. However, SP/SM TIMP-2 levels in the Freezeable group did not show any significant differences compared to the Non-Freezable group in post-thaw semen quality attributes, kinetic parameters, and functional tests, except for a significant positive correlation (r = 0.530, p < 0.05) between sperm DNA integrity and SP-TIMP-2 levels. In conclusion, the findings suggested that TIMP-2 can be a positive regulator of semen quality at the neat stage. However, when it comes to the resilience of sperm to cryopreservation, the levels of TIMP-2 do not seem to exert any significant influence in breeding bulls.

Whole Blood Transcriptome Analysis in Dairy Ewes Fed a Dietary Grape Pomace Supplementation.

The present study aims to evaluate the effect of a dietary supplementation with 10% grape pomace (GP) on the whole blood transcriptome of lactating ewes. By applying a log2FC higher than 0.5 or lower than -0.5 and a false discovery rate (FDR) <0.05, the down-regulation of genes coding for plexin C1, ethanolamine kinase 1, tax1-binding protein 1, transmembrane 9 superfamily member 2, and Beclin-1 was observed in animals that received the dietary supplementation. This aspect was also accompanied by a reduction in the blood activity of matrix metalloproteinase 9 (MMP-9; p < 0.05), a gelatinase commonly involved in both acute and chronic pathological events. The ELISA test on other factors involved in inflammatory processes, interleukin 1 (IL-1) and tumor necrosis factor α (TNF-α), as well as in the antioxidant response, glutathione peroxidase (GPx), and catalase (CAT), did not reveal any significant changes (p > 0.05). Overall, the introduction of GP in the diet of ewes gave indications of greater efficacy in preserving animal welfare, with interesting cues regarding the valorization of a by-product with a high biological value.