Active MMP-2 Research Articles

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.

Assessment of circulating biomarkers and hemodynamic response during stress to detect early cardiopulmonary complications in systemic sclerosis

Abstract Background Systemic sclerosis (SSc) is characterised by persistent immune-inflammatory system activation and excessive extracellular matrix (ECM) accumulation leading to fibrosis. Exercise Doppler Echocardiography (EDE) is a promising method to detect the early changes of fibrosis-related pulmonary and myocardial complications, like elevated left ventricular filling pressure or stress-induced pulmonary hypertension (PH). Aims The study aims to assess the linkage between the activity of fibrosis markers and abnormal exercise haemodynamics during stress in patients with SSc. Methods A total of 18 patients with SSc were enrolled in the single-centre prospective study (age 54±15,5years, 94,4 % women). SSc patients with resting PH and moderate to severe pulmonary fibrosis were excluded. According to a standardised protocol, all subjects underwent resting and exercise echocardiography on a semi-recumbent cycle ergometer with an incremental (25 watts) workload up to the maximally tolerated workload. Measurements focused on the left ventricular diastolic function (E/e’) and right ventricular haemodynamics (estimated systolic pulmonary pressure: PASP, right ventricular coupling: TAPSE/PASP, pulmonary vascular resistance: PVR). Blood samples were taken immediately before the stress test, and the Matrix Metalloproteinase-9 and 2 (MMP-9, MMP-2) levels were assessed with gelatin zymography method. Results During the EDE, PASP from baseline increased significantly (24,6mmHg±11 vs 38±16mmHg, p <0,0001), also PVR (1,2±0,49 vs 1,5±0,15 Wood unit, p<0,001) RV coupling was decreased significantly (1,2±0,68 vs 0,93±0,5, p<0,001). Left ventricular E/e’ was also increased during the stress (8,2±2 vs 10,5±5,1, p<0,05). Enzymatically active MMP-9 level was significantly correlated with RV coupling at rest and stress (p<0,05, r 0,63 at rest and r 0,52 at stress) and negatively correlated with PASP (p<0,05, r:-0,62 at rest and r -0,5 at stress). MMP-2 level only negatively correlated with resting PASP (p<0,05, r:-0,527). The left ventricular parameters did not show any relationship with MMP levels. Conclusions The level of circulating MMP-9 predicts haemodynamic behaviour during stress in SSc. MMPs could be a promising marker for detecting early changes in the right ventricle-pulmonary circulation unit.

Non-small cell lung cancer sensitisation to platinum chemotherapy via new thiazole-triazole hybrids acting as dual T-type CCB/MMP-9 inhibitors

Cisplatin remains the unchallenged standard therapy for NSCLC. However, it is not completely curative due to drug resistance and oxidative stress-induced toxicity. Drug resistance is linked to overexpression of matrix metalloproteinases (MMPs) and aberrant calcium signalling. We report synthesis of novel thiazole-triazole hybrids as MMP-9 inhibitors with T-type calcium channel blocking and antioxidant effects to sensitise NSCLC to cisplatin and ameliorate its toxicity. MTT and whole cell patch clamp assays revealed that 6d has a balanced profile of cytotoxicity (IC50 = 21 ± 1 nM, SI = 12.14) and T-type calcium channel blocking activity (⁓60% at 10 μM). It exhibited moderate ROS scavenging activity and nanomolar MMP-9 inhibition (IC50 = 90 ± 7 nM) surpassing NNGH with MMP-9 over −2 and MMP-10 over −13 selectivity. Docking and MDs simulated its receptor binding mode. Combination studies confirmed that 6d synergized with cisplatin (CI = 0.69 ± 0.05) lowering its IC50 by 6.89 folds. Overall, the study introduces potential lead adjuvants for NSCLC platinum-based therapy.

Moscatilin Reverses EMT Progression and its Resulting Enhanced Invasion and Migration by Affecting the TGF-β Signaling Pathway in Bladder Cancer.

Bladder cancer metastasis is an essential process in the progression of muscle-invasive bladder cancer. EMT plays a crucial role in facilitating the spread of cancer cells. Identifying compounds that can inhibit these abilities of cancer cells is a significant international endeavor. To explore the migration and invasion effect of Moscatilin on the bladder and clarify the mechanism of action Methods: The anti-bladder cancer effect of Moscatilin was observed by a cell proliferation experiment. The migration and invasion of bladder cancer cells inhibited by Moscatilin were detected by Transwell and Wound healing. The effects of Moscatilin on EMT-related proteins E-cadherin, N-cadherin, Snail1, Vimentin, and TGF-β signaling pathways were detected by Western blot, and nucleic acid levels were verified by qPCR. Our study revealed that Moscatilin reduced the viability of bladder cancer cells in vitro and impeded their migration and invasion in experimental settings. Furthermore, we observed that Moscatilin decreased the activation levels of active proteins, specifically Smad3, Samd2, and MMP2. Additionally, we found that moscatilin significantly reduced the expression level of TGF-β and was also capable of reversing the overexpression effect of TGF-β. Treatment with Moscatilin also led to significant inhibition of interstitial cell markers Ncadherin and Snail1, which are associated with EMT. These findings indicate that Moscatilin impedes the migration and invasion of bladder cancer cells by influencing cell survival, modulating TGF-β/Smad signaling, and inhibiting EMT.

MDB-44. MIR-106B-5P DEPLETION IN GROUP 4 METASTATIC MEDULLOBLASTOMA SEVS FINE-TUNES MMP-2 ACTIVATION VIA DIFFERENTIAL TIMP-2 PROTEIN ABUNDANCE

Abstract BACKGROUND Small Extracellular Vesicles (sEVs) are major intercellular communicators, with growing evidence of a role in establishing favourable metastatic niches during cancer progression. We have shown that, relative to their paired primary counterpart, Group 4 (Grp4) metastatic Medulloblastoma (MB) cell-lines release significantly more sEVs with increased functionally active MMP-2 on their surface, resulting in enhanced extracellular matrix degradation and subsequently increased cell invasiveness. METHODS Transcriptomic analysis of sEV microRNA cargoes derived from metastatic/recurrent and primary cell-line and patient CSF samples, followed by microRNA target prediction of differentially abundant microRNAs using TargetScan and miRDB was conducted. Patient dataset analysis was used to confirm microRNA target mRNA expression levels. Finally, microRNA inhibition followed by RT-qPCR or invasion assays were used to quantify changes at the gene and functional level. RESULTS Transcriptomic analysis demonstrated >75% miR-106b-5p depletion in Grp4 MB metastatic sEVs compared to their primary sEV counterparts derived from cell-line and patient CSF samples. This microRNA is predicted to target TIMP-2, which plays a crucial role in MMP-2 activation. We confirmed TIMP-2 to be upregulated in paired metastatic Grp4 tumours relative to their primary counterpart using a publicly available patient dataset. Following miR-106b-5p inhibition in the primary cell-line, RT-qPCR analysis and invasion assays determined the existence of a complex functional response, with increasing TIMP-2 gene expression leading to decreasing invasiveness. This suggests a delicately balanced process where TIMP-2 overabundance can ultimately impede MMP-2 activation. In support of this, analysis of patient datasets showed intermediate TIMP-2 gene expression amongst Grp3/4 patients compared to less frequently metastatic subgroups. CONCLUSIONS Our data support the hypothesis that sEV-derived miR-106b-5p regulates enhanced ECM degradation through fine-tuning MMP-2 activation via differential TIMP-2 abundance. Future work will quantify changes in cell/sEV surface-bound TIMP-2 and MMP-2 alongside MMP-2 activation via flow cytometry and gelatin zymography respectively.

NF-κB/RelA signaling in secretoglobin progenitors mediates plasticity and MMP-induced barrier disruption in house dust mite-induced allergic asthma.

The mechanisms how aeroallergens induce sensitization are incompletely understood. The house dust mite (HDM) Dermatophagoides pteronyssius (Der p) is a ubiquitous aeroallergen that represents a major cause of allergic rhinitis and asthma. Herein, we tested whether HDM-induced aeroallergen exposure sensitivity is caused by the innate-immune response in small airway epithelial cells. HDM exposure is a rapid activator of NF-κB/RelA in the Secretoglobin (Scgb1a1+) lineage associated with upregulation of NF-κB/RelA-dependent markers of epithelial plasticity. To determine the effect of epithelial NF-κB signaling, NF-κB was depleted in a tamoxifen (TMX)-inducible Scgb1a1-CreERTM mouse within a CL57B/L6 background. Corn oil or TMX-treated/RelA-depleted [RelA knockdown (KD)] mice were repetitively exposed to airway HDM challenges to induce airway hyperresponsiveness (AHR). Strikingly, we observed that HDM induces hallmarks of epithelial plasticity through upregulation of the mesenchymal core factors SNAI1 and ZEB1 and production of metalloproteinase (MMP)9 that are RelA-dependent. Downstream, HDM-induced mucous metaplasia, Th2 polarization, allergen sensitivity, and airway hyperreactivity were all reduced in the RelA-depleted mice. Mechanistically, HDM-induced functional and structural barrier disruption was dependent on RelA signaling and associated with active MMP secretion into the bronchoalveolar lavage fluid. To establish the role of MMP2/9 in barrier disruption, we observe that a small-molecule MMP inhibitor (SB-3CT) blocked HDM-induced barrier disruption and activation of plasticity in naïve wild-type (WT) mice. Loss of functional barrier was associated with MMP disruption of zona occludens (ZO)-1 containing adherens junctions. Overall, this data indicates that host innate signaling in the Scgb1a1+ progenitors is directly linked to epithelial plasticity, MMP9 secretion, and enhanced barrier permeability that allows allergen penetration, sensitization producing allergic asthma (AA) in vivo. We propose that maintenance of epithelial integrity may reduce allergic sensitization and AA.NEW & NOTEWORTHY Allergic asthma from house dust mite (HDM) allergy causes substantial morbidity. This study examines the dynamic changes in small airway epithelial cells in a mouse model of HDM exposure. Our findings indicate that NF-κB/RelA signaling mediates matrix metalloproteinase production, disrupting the epithelial barrier resulting in allergic sensitization. Our findings bring new insight into mechanisms for epithelial cell-state change in the allergen response, creating a potential therapeutic pathway for maintaining barrier function in asthma.

Abstract 3015: Matrix Metalloproteinase Inhibition In Rat Abdominal Aortic Aneurysm

Background: Matrix metalloproteinases (MMPs) have a robust proteolytic activity and play a fundamental role in degradation of extracellular matrix proteins. This degradation is a hallmark of the initiation, progression, and rupture of abdominal aortic aneurysms (AAAs). Hypothesis: We hypothesized that broad inhibition of MMPs with a 3 rd generation MMP inhibitor (GM6001) can reduce AAA inflammation and rupture using an established rat model. Aims: Our study aimed to investigate the potential of GM6001 in reducing AAA-related inflammation using a rat model, filling a critical gap in AAA research. Methods: Male Sprague-Dawley rats underwent intraluminal infrarenal perfusion of abdominal aorta using porcine pancreatic elastase (PPE), and daily β-aminopropionitrile (BAPN) to promote AAA rupture. Starting on postoperative day 3 (POD3) rats were treated daily either with or without GM6001 via intraperitoneal injections. On POD6, treated and untreated groups, underwent PET/CT imaging using a pan-MMP radiotracer 64Cu-RYM2 and in vivo aortic diameter measurement using ultrasound. Rate of rupture of treated and untreated groups was observed until POD14 (Fig 1A). Results: Treated rats demonstrated no evidence of toxicity or weight loss. Compared to untreated rats, treated rats had lower AAA rupture rates (P<0.01; Fig 1B), smaller AAA diameters (P<0.05; Fig 1C), and lower AAA MMP radiotracer uptake (P<0.05; Fig 1D,1G). Total and active MMP-9 were significantly lower in treated rats (Fig 1E-F). In addition, IL-1B, IL-17A, IL-18, IFN-y, and RANTAS were significantly lower in treated rats, but TNF-a was higher (Fig 1H-M). VVG staining demonstrated a higher content of preserved elastin in treated rats (Fig 1N). Conclusion: Inhibition of MMPs with a 3 rd generation MMP inhibitor appears to be a promising therapeutic approach to impede the progression, inflammation, and rupture of AAAs in rats. This may represent a renewed pathway for MMP inhibition in human subjects with AAAs.

Circulating levels of potential markers of ischemic stroke in patients with the different forms of atrial fibrillation and chronic heart failure

Atrial fibrillation (AF) is the most common abnormal type of heart rhythm (cardiac arrhythmia), which is considered the leading cause of stroke. There have been limited studies on the prognostic markers for atrial disease and AF-associated ischemic stroke, despite the high demand for this procedure in daily clinical practice to monitor disease course and assess risk of stroke in patients with AF and chronic heart failure (CHF). Thus, the aim of the present study was to evaluate the levels of serum biomarkers related to ischemic stroke in CHF patients with the different forms of AF. Forty-six patients with various types of AF (paroxysmal, persistent and permanent) with or without ischemic stroke were enrolled in the study, 36 clinically healthy donors served as a control. The levels of inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF) and angiostatins (AS) were evaluated by western blot analysis in the serum. The levels of active matrix metalloproteinases (MMPs) were analysed by gelatin zymography. Elevated levels of iNOS were shown in patients with all AF forms as compared with control, but iNOS levels in post-ischemic patients were significantly higher than that in paroxysmal AF individuals. However, the levels of VEGF and AS did not differ from the baseline value in patients with paroxysmal AF, while dramatic increase of their contents was shown in post-stroke patients with persistent and permanent types of AF. Elevated active MMP-9 levels were shown to be associated with the diagnosis of all AF forms, regardless of the occurrence of stroke. Taken together, our findings­ demonstrate that tested proteins can be considered as valuable biomarkers of AF forms transformation and potentially useful for ischemic stroke risk stratification in patients with AF and CHF. Observed changes in regulatory protein levels may expand our understanding of pathological roles of endothelial function dysregulation, disrupted angiogenesis balance and abnormal tissue remodeling in AF and associated ischemic events. Keywords: angiostatins, atrial fibrillation, biomarkers, iNOS, ischemic stroke, MMP-9, VEGF

NOS3 prevents MMP-9, and MMP-13 induced extracellular matrix proteolytic degradation through specific microRNA-targeted expression of extracellular matrix metalloproteinase inducer in hypertension-related atherosclerosis.

Endothelial nitric oxide synthase (NOS3) elicits atheroprotection by preventing extracellular matrix (ECM) proteolytic degradation through inhibition of extracellular matrix metalloproteinase inducer (EMMPRIN) and collagenase MMP-13 by still unknown mechanisms. C57BL/6 mice lacking ApoE , NOS3, and/or MMP13 were fed with a high-fat diet for 6 weeks. Entire aortas were extracted and frozen to analyze protein and nucleic acid expression. Atherosclerotic plaques were detected by ultrasound imaging, Oil Red O (ORO) staining, and Western Blot. RNA-seq and RT-qPCR were performed to evaluate EMMPRIN, MMP-9, and EMMPRIN-targeting miRNAs. Mouse aortic endothelial cells (MAEC) were incubated to assess the role of active MMP-13 over MMP-9. One-way ANOVA or Kruskal-Wallis tests were performed to determine statistical differences. Lack of NOS3 in ApoE null mice fed with a high-fat diet increased severe plaque accumulation, vessel wall widening, and high mortality, along with EMMPRIN-induced expression by upregulation of miRNAs 46a-5p and 486-5p. However, knocking out MMP-13 in ApoE/NOS3 -deficient mice was sufficient to prevent mortality (66.6 vs. 26.6%), plaque progression (23.1 vs. 8.8%), and MMP-9 expression, as confirmed in murine aortic endothelial cell (MAEC) cultures, in which MMP-9 was upregulated by incubation with active recombinant MMP-13, suggesting MMP-9 as a new target of MMP-13 in atherosclerosis. We describe a novel mechanism by which the absence of NOS3 may worsen atherosclerosis through EMMPRIN-induced ECM proteolytic degradation by targeting the expression of miRNAs 146a-5p and 485-5p. Focusing on NOS3 regulation of ECM degradation could be a promising approach in the management of atherosclerosis.

Mechanical force increases tooth movement and promotes remodeling of alveolar bone defects augmented with bovine bone mineral

BackgroundOrthodontic tooth movement (OTM) in a region containing alveolar bone defects with insufficient height and width is hard to achieve. Bovine bone mineral (Bio-Oss) is available to restore the alveolar defect; however, whether the region augmented with a bovine bone mineral graft (BG) is feasible for OTM, and the mechanisms by which macrophages remodel the BG material, is uncertain under the mechanical force induced by OTM.Material and methodsRats were divided into three groups: OTM (O), OTM + BG material (O + B), and Control (C). First molars were extracted to create bone defects in the O and O + B groups with bovine bone mineral grafting in the latter. Second molars received OTM towards the bone defects in both groups. After 28 days, maxillae were analyzed using microfocus-computed tomography (μCT) and scanning-electron-microscopy (SEM); and macrophages (M1/M2) were stained using immunofluorescence. THP-1 cell-induced macrophages were cultured under mechanical force (F), BG material (B), or both (F + B). Phagocytosis-related signaling molecules (cAMP/PKA/RAC1) were analyzed, and conditioned media was analyzed for MMP-9 and cytokines (IL-1β, IL-4).ResultsOur study demonstrated that alveolar defects grafted with BG materials are feasible for OTM, with significantly increased OTM distance, bone volume, and trabecular thickness in this region. SEM observation revealed that the grafts served as a scaffold for cells to migrate and remodel the BG materials in the defect during OTM. Moreover, the population of M2 macrophages increased markedly both in vivo and in cell culture, with enhanced phagocytosis via the cAMP/PKA/RAC1 pathway in response to mechanical force in combination with BG particles. By contrast, M1 macrophage populations were decreased under the same circumstances. In addition, M2 macrophage polarization was also indicated by elevated IL-4 levels, reduced IL-1β levels, and less active MMP-9 in cell culture.ConclusionThis study explored the mechanisms of mechanical force-induced alveolar bone remodeling with bovine bone mineral grafts during OTM. The results might provide molecular insights into the related clinical problems of whether we can move teeth into the grafted materials; and how these materials become biologically remodeled and degraded under mechanical force.

Developmental Regulation of Matrix Metalloproteinases in Response to Multifactorial, Severe Traumatic Brain Injuries during Immaturity

Introduction: A striking pattern in young children after severe TBI is when the entire cortical ribbon displays tissue damage: hemispheric hypodensity (HH). HH is often a result of abusive head trauma (AHT). We previously reported a model of HH in a gyrencephalic species where a combination of injuries consisting of (1) cortical impact, (2) midline shift, (3) subdural hematoma/subarachnoid hemorrhage, (4) traumatic seizures, and (5) brief apnea and hypoventilation resulted in extensive, hypoxic-ischemic-type injury. Importantly, this mechanism closely resembles that seen in children, with relative sparing of the contralateral cortex, thus ruling out a pure asphyxia mechanism. In this model, piglets of similar developmental stage to human toddlers (postnatal day 30, PND30) have extensive hypoxic-ischemic damage to the cortical ribbon with sparing of the contralateral hemisphere and deep gray matter areas. However, piglets of similar developmental stage to human infants (postnatal day 7, PND7) have less hypoxic-ischemic damage that is notably bilateral and patchy. We therefore sought to discover whether the extensive tissue damage observed in PND30 was due to a greater upregulation of matrix metalloproteinases (MMPs). Materials and Methods: In PND7 or PND30 piglets receiving AHT injuries (cortical impact, midline shift, subdural hematoma/subarachnoid hemorrhage, traumatic seizures, and brief apnea and hypoventilation) or a sham injury, the pattern of albumin extravasation and MMP-9 upregulation throughout the brain was determined via immunohistochemistry, brain tissue adjacent to the cortical impact where the tissue damage spreads was collected for Western blots, and the gelatinase activity was determined over time in peripheral plasma. EEG was recorded, and piglets survived up to 24 h after injury administration. Results: The pattern of albumin extravasation, indicating vasogenic edema, as well as increase in MMP-9, were both present at the same areas of hypoxic-ischemic tissue damage. Evidence from immunohistochemistry, Western blot, and zymogens demonstrate that MMP-2, -3, or -9 are constitutively expressed during immaturity and are not different between developmental stages; however, active forms are upregulated in PND30 but not PND7 after in response to AHT model injuries. Furthermore, peripheral active MMP-9 was downregulated after model injuries in PND7. Conclusions: This differential response to AHT model injuries might confer protection to the PND7 brain. Additionally, we find that immature gyrencephalic species have a greater baseline and array of MMPs than previously demonstrated in rodent species. Treatment with an oral or intravenous broad-spectrum matrix metalloproteinase inhibitor might reduce the extensive spread of injury in PND30, but the exposure to metalloproteinase inhibitors must be acute as to not interfere with the homeostatic role of matrix metalloproteinases in normal postnatal brain development and plasticity as well as post-injury synaptogenesis and tissue repair.

Comparative analysis of matrix metalloproteinases by zymography in patients with colorectal carcinoma.

Zymography is an electrophoretic method in which proteins are separated in a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE). This method is used for the detection of enzymatic activity and molecular characterization of proteins. In contrast to the standard SDS-PAGE method, a substrate is incorporated into the gel during zymography, which is subsequently cleaved by target proteases. Many studies have focused on the development and progression of inflammatory diseases affecting the gastrointestinal tract, emphasizing the role of the largest group of proteases, matrix metalloproteinases (MMPs). The most used classification of this group of enzymes (by researchers in MMP biology) is based in part on the historical evaluation of the substrate specificity of MMPs and in part on the cellular localization of MMPs. MMPs are thus classified into the groups of collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs (MT-MMPs), and others. An important group of MMPs are gelatinases which are involved in the breakdown of collagen type IV and gelatin of extracellular matrix and participate in the regulation of various physiological or pathological processes such as morphogenesis, angiogenesis, tissue repair, cirrhosis, arthritis, and metastasis. The present study's objective was to determine the amount of active MMP-9 and MMP-2 forms in tissue samples using zymography. The patient group was according to histology findings divided into the benign tumor (control) group (8 patients), and the malignant tumor group (24 patients). The respondents in the malignant tumor group were further divided according to the standard TNM classification. The results of this study confirmed that MMP-2, unlike MMP-9, can be used as a prognostic biomarker of CRC, because only the expression of active MMP-2 confirmed statistically significant differences between individual stages of CRC. Moreover, MMP-2 seems to play a more important role in higher stages of CRC. Substantial disparities in the determination of active MMPs between the observed groups support the assumption for the integration of zymography into clinical diagnostics of CRC together with molecular and other studies.

Induction of Collagenolytic MMP-8 and -9 Tissue Destruction Cascade in Mouth by Head and Neck Cancer Radiotherapy: A Cohort Study.

The effect of head and neck cancer (HNC) radiotherapy (RT) on biomarkers is not known but there is a lot of potential for gaining more precise cancer treatments and less side effects. This cohort study investigated the levels and molecular forms of the matrix metalloproteinase (MMP) -8 and -9, tissue inhibitor of metalloproteinase (TIMP)-1, myeloperoxidase (MPO) and interleukin (IL)-6 in mouth-rinse samples as well as the clinical periodontal status in HNC patients (n = 21) receiving RT. Complete periodontal examinations were performed pre-RT and one month after RT. Mouth-rinse samples (pre-RT, after six weeks of RT and one month after RT) were assayed using a point-of-care-kit (PerioSafe®/ORALyzer® (Dentognostics GmbH, Jena, Germany)) for active MMP-8 and ELISA analysis for total MMP-8 and -9, MPO, TIMP-1, and IL-6 levels. Molecular forms of MMP-9 were assessed by gelatinolytic zymography and MMP-8 by western immunoblot. Significant changes were observed between the three time points in the mean levels of active and total MMP-8, active MMP-9, and IL-6. Their levels increased during the RT and decreased after the RT period. The aMMP-8 levels stayed elevated even one month after RT compared to the pre-RT. Clinical attachment loss, probing depths, and bleeding on probing were increased between pre- and post-calculations in periodontal status. Elevated inflammatory biomarker levels together with clinical recordings strongly suggest that RT eventually increases the risk to the periodontal tissue destruction by inducing the active proteolytical MMP-cascade, and especially by prolonged activity of collagenolytic aMMP-8. Eventually, the aMMP-8 point-of-care mouth-rinse test could be an easy, early detection tool for estimating the risk for periodontal damage by the destructive MMP-cascade in HNC patients with RT treatment.

Expression and Purification of Active Monomeric MMP7.

MMP7 is the smallest member of the MMP family and plays multiple physiological and pathological roles through interaction with a variety of molecules. Purified MMP7 would be beneficial for studying its function and for the development of inhibitors, which could be potential therapeutics. Due to low levels of endogenously produced MMP7, its recombinant expression and purification using E. coli have been established. Here, we describe an effective method to express and purify an active form of MMP7. Our recent discovery is that adding high concentration of CaCl2 during refolding process prevents nonspecific binding of MMP7 to plastic and its aggregation, significantly improving the yield of active monomeric forms of MMP7.

Mechanism of pro-MMP9 activation in co-culture of pro-inflammatory macrophages and cardiomyocytes

ObjectiveA wide range of cardiac diseases is associated with inflammation. “Inflamed” heart tissue is infiltrated with pro-inflammatory macrophages which extensively secrete matrix metalloproteinase 9 (MMP9), a regulator of extracellular matrix turnover. As MMP9 is released from macrophages in a latent form, it requires activation. The present study addresses the role of cardiomyocytes in the course of this activation process. Methods and resultsIn mono- and co-cultures of pro-inflammatory rat macrophages (bone marrow-derived and peritoneal) and cardiomyocytes (H9C2 cell line) gelatin zymography demonstrated that activated macrophages robustly secreted latent pro-MMP9, whereas cardiomyocytes could not produce the enzyme. Co-culturing of the two cell species was critical for pro-MMP9 activation and was also accompanied by processing of cardiomyocyte-secreted pro-MMP2. A cascade of pro-MMP9 activation was initiated on macrophage membrane with pro-MMP2 cleavage. Namely, pro-inflammatory macrophages expressed an active membrane type 1 MMP (MT1MMP), which activated pro-MMP2, which in turn converted pro-MMP9. Downregulation of MT1MMP in macrophages by siRNA abolished activation of both pro-MMP2 and pro-MMP9 in co-culture. In addition, both cell species secreted MMP13 as a further pro-MMP9 activator. In co-culture, activation of pro-MMP13 occurred on membranes of macrophages and was enhanced in presence of active MMP2. Using incubations with recombinant MMPs and isolated macrophage membranes, we demonstrated that while both MMP2 and MMP13 individually had the ability to activate pro-MMP9, their combined action provided a synergistic effect. ConclusionActivation of pro-MMP9 in a co-culture of pro-inflammatory macrophages and cardiomyocytes was the result of a complex interaction of several MMPs on the cell membrane and in the extracellular space. Both cell types contributed critically to pro-MMP9 processing.

Effects of SGLT2 inhibition on cardiac remodeling and heart failure development in rats with aortic stenosis

Abstract Introduction Aortic stenosis prevalence has been increasing as population ages. Currently, no medical therapies influence the natural history of aortic stenosis. Sodium glucose co-transporter 2 inhibitors (SGLT2i) have reduced heart failure hospitalization and cardiovascular mortality in heart failure patients with both reduced and preserved ejection fraction. The aim of this study was to evaluate the effects of SGLT2 inhibitor empagliflozin (EMPA) on cardiac remodeling and heart failure development in aortic stenosis rats. Methods Eighteen weeks after supra-aortic banding surgery, male Wistar rats were divided in four groups: Sham (n=15); sham + EMPA (Sham-EMPA, n=16); aortic stenosis (AS, n=27); and aortic stenosis + EMPA (AS-EMPA, n=27). Empagliflozin (10 mg/kg/day) was added to rat chow for 8 weeks. Echocardiogram was performed to evaluate cardiac structure and function. Myocardial collagen content was quantified by histology; activity of antioxidant enzymes and enzymes involved in energy metabolism was assessed by spectrophotometry. Protein expression was quantified by Western blot, and matrix metalloproteinase (MMP) activity by zymography. Statistical analysis: ANOVA and Bonferroni or Kruskal-Wallis and Dunn (p<0.05). Results AS-EMPA group had a lower frequency of anatomopathological heart failure features, a lower left ventricular (LV) dilatation (LV diastolic diameter: Sham 8.39±0.57; Sham-EMPA 8.25±0.38; AS 9.80±0.53; AS-EMPA 9.08±0.87 mm; LV systolic diameter: Sham 4.24±0.68; Sham-EMPA 4.19±0.57; AS 5.72±0.74; AS-EMPA 5.09±1.08 mm) and LV mass (Sham 0.85±0.11; Sham-EMPA 0.82±0.07; AS 2.19±0.27; AS-EMPA 1.76±0.34 g), and better diastolic function (isovolumetric relaxation time: Sham 28.6±2.28; Sham-EMPA 29.8±3.04; AS 18.9±2.59; AS-EMPA 22.9±5.02 ms) than AS. Systolic function was impaired in both AS and AS-EMPA compared to their respective controls and did not differ between AS-EMPA and AS. AS and AS-EMPA had larger myocyte diameter, higher interleukin-6 protein expression and lower catalase activity than Sham and Sham-EMPA, respectively. AS and AS-EMPA groups had lower enzyme activity from energy metabolism than their respective controls. AS presented higher lipid hydroperoxide concentration, collagen III and p65 NF-kB protein expression, and lower glutathione peroxidase activity than Sham. AS-EMPA had higher collagen I expression and inactive MMP-2 than Sham-EMPA. AS-EMPA presented higher glutathione peroxidase and intermediary active MMP-2, and lower interstitial collagen fraction (Sham 2.68±0.59; Sham-EMPA 3.16±0.33; AS 5.68±0.97; AS-EMPA 3.55±0.70 %) than AS. Conclusion Empagliflozin improves cardiac remodeling, diastolic function and myocardial oxidative stress, and reduces heart failure feature frequency and interstitial collagen fraction in aortic stenosis rats.

Abstract P127: Protective Effects Of Protocatechuic Acid In Murine Model Of UUO-induced Renal Injury

Chronic kidney disease (CKD) is a global public health problem, affecting millions of people worldwide. CKD can result from obstructive nephropathy; a condition that arises from urinary flow obstruction in the kidneys, leading to renal inflammation and injury. Protocatechuic acid (PCA) is a natural phenolic compound that showed a promising protective effect in cardiovascular diseases. In the current study, we investigated the reno-protective effects of PCA in mice model of unilateral ureteral obstruction (UUO). Male C57BL/6J mice (10-12 weeks old) were divided into 4 groups (n=6-8/group). Sham control, Sham/P (sham plus PCA (50 mg/kg IP daily for two weeks), UUO, and UUO plus PCA. Induction of UUO significantly decreased creatinine clearance (CrCl) and increased pulse wave velocity (PWV), a marker of arterial stiffness, relative to sham control (CrCl; sham control: 60.6 ± 14 vs. UUO: 24.5 ± 6.0 ml/day and PWV; sham control: 1.58 ± 0.1 vs. UUO: 3.35 ± 0.22 m/s). Immuno-histochemical analysis revealed that KIM-1 expression, a marker of tubular injury, was elevated in UUO mice vs. sham control and PCA reduced this elevation, however there was no significance difference in urinary KIM-1 excretion. Moreover, UUO significantly increased renal oxidative stress and inflammation as evidenced by elevation in renal TBARs, IL-6, active caspase-1 and active MMP2 compared to sham control (TBARs; sham control: 8.3 ± 0.9 vs. UUO: 30.5 ± 3.33 nmol/mg protein, renal IL-6; sham control: 1 ± 0.07 vs. UUO: 24.35 ± 8.55 pg/mg protein, active caspase-1; sham control: 1.7 x 10 3 ± 95 vs. UUO: 2.79 x 10 3 ± 145 cpm/μg protein and MMP2; sham control: 1.6 ± 0.4 vs. UUO: 6 ± 0.6 ng/μg protein). PCA treatment significantly improved the UUO-induced decline in creatinine clearance (52.5 ± 10.2 ml/day) and attenuated UUO-induced elevation in renal PWV (1.95 ± 0.1 m/s). Furthermore, PCA treatment significantly decreased renal TBARs (5.2 ± 1.12 nmol/mg protein), IL-6 (5.39 ± 1.4 pg/mg protein) and active MMP2 (1.1 ± 0.3 ng/μg protein) in UUO-induced renal injury. Collectively, these data suggests that PCA provides renal protection in murine model of UUO via reduction of renal oxidative stress, inflammation and fibrosis.

Prognostic value of different forms of serum MMP-2 and MMP-9 in dogs with mammary tumors; a longitudinal study

Gelatinases (MMP-2 and MMP-9) are related to tumor invasion and metastasis. In humans, the diagnostic value of serum levels of gelatinases has been confirmed in breast cancer, but their diagnostic value in canine mammary tumors is still unknown. This study aimed to determine the serum level of gelatinases in dogs with mammary tumors in order to determine their value in the diagnosis of malignancy or benign tumors and also in predicting the possibility of metastasis and recurrence. Frequent measurement of MMP-2 and MMP-9 by gelatin zymography in serum before and after surgical treatment has not previously been studied for monitoring mammary tumors in dogs. Thus, the serum levels of MMP-2 and MMP-9 in 26 dogs with mammary tumors before surgical treatment and then 1, 4 and 12 months after surgery were evaluated by gelatin zymography. Serum samples of 26 healthy dogs with normal conditions were used as control.Dogs with benign and malignant mammary tumors showed bands of pro-MMP-2, pro-MMP-9 and active MMP-9. However, only pro-MMP-2 and pro-MMP-9 bands appeared in the serum of control group. Our results showed that the presence of active MMP-9, regardless of its level, was prognostically important for metastasis and or recurrence (M/R). However, the presence of active MMP-2 band was more important for M/R than active MMP-9, as its presence coincides with definitive M/R. It seems that serum gelatin zymography could possibly be used at regular intervals before and after surgery to evaluate the probability of M/R in dogs with mammary tumors. More research is needed in this regard.

Effect of low-dose hydrocortisone and inhaled nitric oxide on inflammatory mediators and local pulmonary metalloproteinases activity in LPS-induced sepsis in piglets

Hospital mortality in sepsis varies between 30–45%. It has been shown that administration of inhaled nitric oxide (iNO) and intravenous corticosteroid in a porcine endotoxemia model attenuated the systemic inflammatory response. We explored the anti-inflammatory effect of a double-treatment strategy (iNO + low-dose steroid) on the lungs in a long-term porcine endotoxic shock model. As metalloproteinases (MMPs) are involved in the initiation of multiple organ dysfunction in septic shock, we evaluated the influence of this combination therapy on MMP2 and MMP9 activity and proIL-1β maturation. A shock-like condition was established in 23 animals by continuous infusion of E. coli lipopolysaccharide (LPS) for 10 h. Then the animals were observed for 10 h. Twelve pigs received iNO and hydrocortisone (iNO treatment started 3 h after the initial LPS infusion and continued until the end of the experiment). Eleven pigs were controls. Pigs treated with iNO and hydrocortisone displayed less inflammatory infiltrates in the lungs than the controls and a lower level of IL-1β. The proMMP2 was significantly decreased in the iNO and hydrocortisone group. The amount of an active MMP9 (~ 60 kDa) was decreased in the iNO and hydrocortisone group. Total gelatinolytic activity was lower in the iNO and hydrocortisone group. Reduced MMP activity was accompanied by a 2.5-fold decrease of the active IL-1β form (17 kDa) in the pulmonary tissue of iNO combined with hydrocortisone exposed pigs. We demonstrated that in a porcine endotoxemia model the NO inhalation combined with intravenous hydrocortisone led to the attenuation of the inflammatory cascade induced by bacterial LPS. The decrease in pulmonary MMPs activities was accompanied by reduced proIL-1β processing.

Increased Matrix Metalloproteinase-1 Activation Enhances Disruption and Regression of k-RasV12–Expressing Arteriovenous Malformation-Like Vessels

This study sought to identify potential mechanisms by which k-RasV12-expressing endothelial cell (EC) tubes demonstrate an increased propensity to regress compared with controls. Activated k-Ras mutations play a role in a variety of pathological conditions, including arteriovenous malformations, which are prone to bleed, causing serious hemorrhagic complications. ECs expressing active k-RasV12 demonstrate markedly excessive lumen formation with widened and shortened tubes accompanied by reduced pericyte recruitment and basement membrane deposition, leading to deficient capillary network assembly. The current study showed that active k-Ras-expressing ECs secreted greater amounts of MMP-1 proenzyme compared with control ECs, and readily converted it to increased active MMP-1 levels through the action of plasmin or plasma kallikrein (generated from their added zymogens). Active MMP-1 degraded three-dimensional collagen matrices, leading to more rapid and extensive regression of the active k-Ras-expressing EC tubes, in conjunction with matrix contraction, compared with control ECs. Under conditions where pericytes protect control EC tubes from plasminogen- and MMP-1-dependent tube regression, this failed to occur with k-RasV12 ECs, due to reduced pericyte interactions. In summary, k-RasV12-expressing EC vessels showed an increased propensity to regress in response to serine proteinases through accentuated levels of active MMP-1, a novel pathogenic mechanism that may underlie hemorrhagic events associated with arteriovenous malformation lesions.