MMP-2 Activation Research Articles

Gelatinolytic activity in dentin upon adhesive treatment

In this multi-parameter study, the effect of diverse factors related to adhesive application on the activation of host-derived gelatinases was investigated by gelatin zymography, in-situ zymography, fluorogenic DQ-gelatin assay and micro-tensile bond-strength (μTBS) testing. Gelatin zymography disclosed the presence of gelatinases in phosphoric acid-etched dentin powder, while two gold-standard adhesives generated no measurable MMP activation. In-situ zymography revealed that the interfacial gelatinolytic activity from specimens treated with the two adhesives appeared similar as that of the EDTA negative control, indicating no detectable gelatinases were activated upon adhesive treatment. In solution, MMP-2/9 activity significantly decreased upon interaction with both adhesives (two-way linear mixed effects model [LMEM]: p < 0.05); gelatinases were almost completely deactivated upon 1-week incubation at 37 °C (general linear model: p < 0.05); light-curing adhesives increased temperature up to 55 °C, which appeared sufficient to dramatically decrease MMP-2/9 activity (two-way ANOVA: p < 0.05). Finally, challenging adhesive-dentin interfaces with highly concentrated MMP-9 (at a much higher concentration than present in saliva) for 1 m did not significantly affect μTBS (two-way LMEM: p > 0.05). Taken together, the two adhesives did not activate but rather inhibited the release and activation of dentinal gelatinases.

Inhibition of phosphodiesterase 10A mitigates neuronal injury by modulating apoptotic pathways in cold-induced traumatic brain injury

Brain injury develops from a complex series of pathophysiological phases, resulting in acute necrotic or delayed apoptotic cell death after traumatic brain injury (TBI). Inhibition of apoptotic cell death is critical for the treatment of acute neurodegenerative disorders, such as TBI. Here, we investigated the role of phosphodiesterase 10A (PDE10A) in the development of neuronal injury, particularly in apoptotic cell death. Using the PDE10A inhibitor TAK-063, we found that PDE10A inhibition is associated with decreased brain injury, brain swelling, and blood brain barrier disruption 48 h after cold-induced TBI. Furthermore, a particularly notable result was observed with 3 mg/kg TAK-063, which reduced disseminated neuronal injury. Protein abundance analysis revealed that PDE10A inhibition activates survival kinases AKT and ERK-1/-2, which were associated with the decreased activation of MMP-9 and PTEN. Additionally, iNOS and nNOS levels significantly reduced in the TAK-063 group, playing roles in inflammation and apoptosis. A planar surface immunoassay was performed for in-depth analyses of the apoptotic signaling pathways. We observed that inhibition of PDE10A resulted in the decreased expression of TNFRSF1A, TNFRSF10B, and TNFRSF6 receptors, particularly inducing apoptotic cell death. Moreover, these findings correlated with reduced levels of pro-apoptotic proteins, including PTEN, p27, Cytochrome-c, cleaved Caspase-3, Bad, and p53. Interestingly, TAK-063 treatment reduced levels of anti-apoptotic proteins or enzymes, including XIAP, Claspin, and HIF1α, without affecting Bcl-x, MCL-1, SMAC, HO-1, HO-2, HSP27, HSP60, and HSP70. The findings suggest that PDE10A regulates cellular signaling predominantly pro-apoptotic pathways, and inhibition of this protein is a promising approach for the treatment of acute brain injury.

Radiofrequency Currents Modulate Inflammatory Processes in Keratinocytes.

Keratinocytes play an essential role in the inflammatory phase of wound regeneration. In addition to migrating and proliferating for tissue regeneration, they produce a large amount of cytokines that modulate the inflammatory process. Previous studies have shown that subthermal treatment with radiofrequency (RF) currents used in capacitive resistive electric transfer (CRET) therapy promotes the proliferation of HaCat keratinocytes and modulates their cytokine production. Although physical therapies have been shown to have anti-inflammatory effects in a variety of experimental models and in patients, knowledge of the biological basis of these effects is still limited. The aim of this study was to investigate the effect of CRET on keratinocyte proliferation, cytokine production (IL-8, MCP-1, RANTES, IL-6, IL-11), TNF-α secretion, and the expression of MMP9, MMP1, NF-κB, ERK1/2, and EGFR. Human keratinocytes (HaCat) were treated with an intermittent 448 kHz electric current (CRET signal) in subthermal conditions and for different periods of time. Cell proliferation was analyzed by XTT assay, cytokine and TNF-α production by ELISA, NF-κB expression and activation by immunofluorescence, and MMP9, MMP1, ERK1/2, and EGF receptor expression and activation by immunoblot. Compared to a control, CRET increases keratinocyte proliferation, increases the transient release of MCP-1, TNF-α, and IL-6 while decreasing IL-8. In addition, it modifies the expression of MMPs and activates EGFR, NF-κB, and ERK1/2 proteins. Our results indicate that CRET reasonably modifies cytokine production through the EGF receptor and the ERK1/2/NF-κB pathway, ultimately modulating the inflammatory response of human keratinocytes.

Isoliquiritigenin: A natural compound with a promising role in inhibiting breast cancer lung metastasis

Background and aimIsoliquiritigenin (ISL) is a dietary bioactive compound that is derived from the roots of licorice and various other plants. Accumulating evidence suggests that ISL has noteworthy anticancer activity in a variety of cancer cells, but it is currently unknown how ISL affects the invasion and migration of breast cancer (BC) cells. Hence, the purpose of this study to determine the inhibitory effect of ISL on cell invasion, migration, and metastasis of BC cells in 4T1-treated lung metastatic animal models. Experimental ProcedureISL was found to inhibit BC lung metastasis, as confirmed by MTT assay, clonogenic assay, wound healing assay, transwell assay, histological analysis, western blot and bioluminescence assay in the xenograft model. Results and conclusionThe in vitro studies revealed that ISL treatment significantly prevented BC cell invasion and migration for 24 h. ISL decreased the protein expressions of PI3K, Akt, p-Akt, mTOR, p-mTOR, matrix metalloproteinase (MMP)-2, MMP-9, and N-cadherin , thereby inhibiting BC cell invasion. In addition, ISL decreased MMP-2/9 and N-cadherin expression in lung tissues and reduced mouse 4T1 BC cell lung metastasis. Based on these findings, it suggests that ISL inhibits cell migration and invasion in BC cells by inhibiting the PI3K/Akt signaling pathway and decreasing the activation of MMP-2 and MMP-9 expression. Therefore, ISL may be useful in managing BC invasion and metastasis.

Possibility of correction of the blood-heart barrier damage as a consequence of the impact of the SARS-CoV-2 virus on the cardiovascular system in patients with coronary artery disease in combination with COVID-19.

damage to the blood-heart barrier (BHB) and endothelial dysfunction is a characteristic feature of congestive (cardiopulmonary) chronic heart failure (CHF), the main cause of death in elderly people with CHF caused by new coronavirus variants (SARS-CoV-2), but the mechanism of this phenomenon remains unclear. The aim of this project is to determine the mechanism of BHB damage in coronary artery disease (CAD) with COVID-19, as well as the possibility of its correction with the use of sulodexide. The endocardial endothelium (EE) is a barrier that prevents blood leakage from the endocardium to the interstitium; however, this barrier is impaired during the course of COVID-19 in patients with CAD. Previous studies have shown that one of the pathophysiological mechanisms is the activation of matrix metalloproteinases (MMPs) in CAD with CHF. MMP-9 degrades connexins, which leads to EE dysfunction. One study [Rubens P. et al. 2021] found a juxtacrine connection of EE with the myocyte and mitochondria (Mito), but how this works is still an open question. Materials and methods. We examined 65 patients with coronary artery disease diagnosed with COVID-19. Patients were divided into two groups: Group I (n=35) - patients who had been taking sulodexide at a dose of 500 LE x 2p/day for 6 months against the background of standard therapy of coronary artery disease; Group II (n=30) - patients without sulodexide. Echocardiography and laser Doppler flowmetry were performed at baseline and 6 months later. Echocardiography at the time of inclusion in the study revealed that 30 (50%) patients in group I and 14 (47%) patients in group II had reduced left ventricular ejection fraction (LV EF) values of 40 to 50%. After 6 months of treatment with sulodexide, a tendency to improve LV systolic function and decrease in left ventricular myocardial mass index (LFMMI) was noted in patients of group I. There were no differences between the groups at the time of inclusion in the study in terms of the level of the capillary flow reserve – occlusion test (CFRo) and the capillary flow reserve – nitroglycerin test (CFRn). A repeated study of CFR revealed a significant increase in CFRo and CFRn levels only in group I. In patients of group II, no significant changes in CFRo and CFRn were found. An inverse relationship between the level of CFRo and C-reactive protein (CRP) was found in patients of group I (r=0.52, p&lt;0.05). After 6 months of treatment, plasma CRP concentrations decreased significantly: from 17.7 [1.3; 50.1] to 5.7 [1.0; 12.0] mg/L in group I (p=0.01) and from 14.2 [1.2; 27.0] to 4.2 [1.0; 11.0] mg/L in group II (p=0.01). No significant correlations between CRP level and CFRo after 6 months of treatment were found. There were correlations of CFRo and CFRn with left ventricular systolic function, as well as inverse relationships with the size of the left and right ventricles and systolic pressure in the pulmonary artery. There was a tendency to improve systolic and diastolic left ventricular function in the first group, where sulodexide was used, and no significant changes in echocardiography were noted in the second group. At the initial examination of patients, the ratio of CFRo and CFRn had no significant differences. After 6 months of treatment, a significant increase in the ratio of CFRo and CFRn was observed only in patients taking sulodexide, and no positive dynamics of this ratio was observed in group II. The results indicate that MMP-9 activation, endothelial damage, endothelial-myocyte (E-M) uncoupling, and mitochondrial-myocyte uncoupling in heart failure in patients with CAD combined with COVID-19 were detected to a significant extent; however, treatment with sulodexide successfully mitigated the destructive changes in the heart in CAD with CHF. The results obtained are directly relevant to the range of cardiac manifestations and phenotypes arising from COVID-19 complications in people with CAD. Conclusion. The obtained results confirm the improvement of microcirculation, as well as a tendency to improve systolic function and left ventricular myocardial mass index after 6 months of sulodexide treatment in patients with coronary artery disease with preserved and moderately reduced left ventricular ejection fraction (LV EF) who have undergone COVID-19. The described effects of improving endothelial function, as well as improving the state of the blood-brain barrier due to the use of sulodexide, make it possible to recommend the use of this drug in the category of patients with CAD to reduce the negative impact of COVID-19 on the cardiovascular system.

Hepatocyte MMP14 mediates liver and inter-organ inflammatory responses to diet-induced liver injury.

The matrix metalloproteinase MMP14 is a ubiquitously expressed, membrane-bound, secreted endopeptidase that proteolyzes substrates to regulate development, signaling, and metabolism. However, the spatial and contextual events inciting MMP14 activation and its metabolic sequelae are not fully understood. Here, we introduce an inducible, hepatocyte-specific MMP14-deficient model (MMP14LKO mice) to elucidate cell-intrinsic and systemic MMP14 function. We show that hepatocyte MMP14 mediates diet-induced body weight gain, peripheral adiposity, and impaired glucose homeostasis and drives diet-induced liver triglyceride accumulation and induction of hepatic inflammatory and fibrotic gene expression. Single-nucleus RNA sequencing revealed that hepatocyte MMP14 mediates Kupffer cell and T-cell accumulation and promotes diet-induced hepatocellular subpopulation shifts toward protection against lipid absorption. MMP14 co-immunoprecipitation and proteomic analyses revealed MMP14 substrate binding across both inflammatory and cytokine signaling, as well as metabolic pathways. Strikingly, hepatocyte MMP14 loss-of-function suppressed skeletal muscle and adipose inflammation in vivo, and in a reductionist adipose-hepatocyte co-culture model. Finally, we reveal that trehalose-type glucose transporter inhibitors decrease hepatocyte MMP14 gene expression and nominate these inhibitors as translatable therapeutic metabolic agents. We conclude that hepatocyte MMP14 drives liver and inter-organ inflammatory and metabolic sequelae of obesogenic dietary insult. Modulating MMP14 activation and blockade thus represents a targetable node in the pathogenesis of hepatic inflammation.

CCL28 promotes progression of hepatocellular carcinoma through PDGFD-regulated MMP9 and VEGFA pathways

Hepatocellular carcinoma (HCC) remains a major global health concern with limited therapeutic options and poor prognosis. Chemokines have emerged as critical regulators in the progression and metastasis of HCC. This study aims to investigate the mechanisms involved in CCL28-promoted progression of HCC and provide novel therapeutic targets for HCC treatment. Relationship between CCL28 expression and HCC progression were investigated by bioinformatic analysis and immunohistochemical staining assays. CCK-8, Transwell, and colony formation assay were conducted to explore the impact of CCL28 on the growth, migration and invasion of HCC cells. Quantitative real-time PCR and western blotting assays were performed to learn potential molecular mechanisms underlying the transformation of HCC driven by CCL28. The results showed that there was a direct link between increased CCL28 levels and the advancement of HCC, leading to a worse outcome. CCL28 significantly augmented malignant transformation of HCC cells, containing proliferation, migration, invasion, and clonogenicity, via activation of PDGFD-regulated MMP9 and VEGFA pathways. CCL28 emerges as a pivotal contributor to HCC tumorigenesis, propelling HCC development through the PDGFD signaling pathway. Our findings unveil potential therapeutic targets for HCC treatment.

Multiphysics modeling of 3D traction force microscopy with application to cancer cell-induced degradation of the extracellular matrix

Abstract3D Traction Force Microscopy (3DTFM) constitutes a powerful methodology that enables the computation of realistic forces exerted by cells on the surrounding extracellular matrix (ECM). The ECM is characterized by its highly dynamic structure, which is constantly remodeled in order to regulate most basic cellular functions and processes. Certain pathological processes, such as cancer and metastasis, alter the way the ECM is remodeled. In particular, cancer cells are able to invade its surrounding tissue by the secretion of metalloproteinases that degrade the extracellular matrix to move and migrate towards different tissues, inducing ECM heterogeneity. Typically, 3DTFM studies neglect such heterogeneity and assume homogeneous ECM properties, which can lead to inaccuracies in traction reconstruction. Some studies have implemented ECM degradation models into 3DTFM, but the associated degradation maps are defined in an ad hoc manner. In this paper, we present a novel multiphysics approach to 3DTFM with evolving mechanical properties of the ECM. Our modeling considers a system of partial differential equations based on the mechanisms of activation of diffusive metalloproteinase MMP2 by membrane-bound metalloproteinase MT1-MMP. The obtained ECM density maps in an ECM-mimicking hydrogel are then used to compute the heterogeneous mechanical properties of the hydrogel through a multiscale approach. We perform forward and inverse TFM simulations both accounting for and omitting degradation, and results are compared to ground truth reference solutions in which degradation is considered. The main conclusions resulting from the study are: (i) the inverse methodology yields results that are significantly more accurate than those provided by the forward methodology; (ii) ignoring ECM degradation results in a considerable overestimation of tractions and non negligible errors in all analyzed cases.

Complement regulatory protein CD46 promotes bladder cancer metastasis through activation of MMP9.

CD46, a transmembrane protein known for protecting cells from complement‑mediated damage, is frequently dysregulated in various types of cancer. Its overexpression in bladder cancers safeguards the cancer cells against both complement and antibody‑mediated cytotoxicity. The present study explored a new role of CD46 in facilitating cancer cell invasion and metastasis, examining its regulatory effect on matrix metalloproteases (MMPs) and their effect on the metastatic capability of bladder cancer cells. Specifically, CD46 alteration positively influenced MMP9 expression, but not MMP2, in several bladder cancer cell lines. Furthermore, CD46 overexpression triggered phosphorylation of p38 MAPK and protein kinase B (AKT), leading to enhanced activator protein 1 (AP‑1) activity via c‑Jun upregulation. The inhibition of p38 or AKT pathways attenuated the CD46‑induced MMP9 and AP‑1 upregulation, indicating that the promotion of MMP9 by CD46 involved activating both p38 MAPK and AKT. Functionally, the upregulation of MMP9 by CD46 translated to increased migratory and invasive capabilities of bladder cancer cells, as well as enhanced in vivo metastasis. Overall, the present study revealed a novel role for CD46 as a metastasis promoter through MMP9 activation in bladder cancers and highlighted the regulatory mechanism of CD46‑mediated MMP9 promotion via p38 MAPK and AKT activation.

The role of zinc and matrix metalloproteinases in myofibrillar protein degradation in critical illness myopathy

Due to an unexpected activation of different zinc (Zn) transporters in a recent prospective clinical study, we have revisited the role of Zn homeostasis and the activation of matrix metalloproteinases (MMPs) in skeletal muscle exposed to the intensive care unit (ICU) condition (immobilization and mechanical ventilation). ICU patients exposed to 12 days ICU condition were followed longitudinally with six repeated muscle biopsies while they showed a progressive preferential myosin loss, i.e., the hallmark of Critical Illness Myopathy (CIM), in parallel with the activation of Zn-transporters. In this study, we have revisited the expression of Zn-transporters and the activation of MMPs in clinical as well as in experimental studies using an established ICU model. MMPs are a group Zn-dependent endopeptidases which do not only target and cleave extracellular proteins but also intracellular proteins including multiple sarcomeric proteins. MMP-9 is of specific interest since the hallmark of CIM, the preferential myosin loss, has also been reported in dilated cardiomyopathy and coupled to MMP-9 activation. Transcriptional activation of Zn-transporters was observed in both clinical and experimental studies as well as the activation of MMPs, in particular MMP-9, in various limb and respiratory muscles in response to long-term exposure to the ICU condition. The activation of Zn-transporters was paralleled by increased Zn levels in skeletal muscle which in turn showed a negative linear correlation with the preferential myosin loss associated with CIM, offering a potential intervention strategy. Thus, activation of Zn-transporters, increased intramuscular Zn levels, and activation of the Zn-dependent MMPs are forwarded as a probable mechanism involved in CIM pathophysiology. These effects were confirmed in different rat strains subjected to a model of CIM and exacerbated by old age. This is of specific interest since old age and muscle wasting are the two factors most strongly associated with ICU mortality.

Isoflurane increases the activity of the vascular matrix metalloproteinase-2 in non-pregnant rats and increases the nitric oxide metabolites in pregnancy.

Surgeries that require general anesthesia occur in 1.5-2% of gestations. Isoflurane is frequently used because of its lower possibility of affecting fetal growth. Therefore, we examined the isoflurane anesthesia-induced effects on maternal hemodynamic and vascular changes. We hypothesized that isoflurane would enhance endothelium-dependent vasodilation as a consequence of increased nitric oxide and decreased metalloproteinases (MMPs). Female rats (n=28) were randomized into 4 groups (7 rats/group): conscious (non-anesthetized) non-pregnant group, non-pregnant anesthetized group, conscious pregnant group, and pregnant anesthetized group. Anesthesia was performed on the 20th pregnancy day, and hemodynamic parameters were monitored. Nitric oxide metabolites, gelatinolytic activity of MMP-2 and MMP-9, and the vascular function were assessed. Isoflurane caused no significant hemodynamic changes in pregnant compared with non-pregnant anesthetized group. Impaired acetylcholine-induced relaxations were observed only in conscious non-pregnant group (by approximately 62%) versus 81% for other groups. Phenylephrine-induced contractions were greater in endothelium-removed aorta segments of both pregnant groups (with or without isoflurane) compared with non-pregnant groups. Higher nitric oxide metabolites were observed in anesthetized pregnant in comparison with the other groups. Reductions in the 75 kDa activity and concomitant increases in 64 kDa MMP-2 isoforms were observed in aortas of pregnant anesthetized (or not) groups compared with conscious non-pregnant group. Isoflurane anesthesia shows stable effects on hemodynamic parameters and normal MMP-2 activation in pregnancy. Furthermore, there were increases in nitric oxide bioavailability, suggesting that isoflurane provides protective actions to the endothelium in pregnancy.

Establishing the Inhibition of the Serine Protease Plasmin as a Skin Anti-Aging Pathway

Plasmin is a serine protease induced by UV-irradiation in skin that contributes to inflammation. We showed that plasmin is upregulated in photo-exposed facial skin and that this correlates with increased transepidermal water loss (TEWL). Plasmin activity upregulates downstream pathways such as pro-inflammatory cytokines and matrix-metalloproteinases (MMPs). In addition, the plasminogen system modulates cutaneous melanogenesis. In this study, we investigated potential skin-aging effects of plasmin with a dual inhibitor of plasmin and its activator urokinase (uPA). We established a range of in vitro and ex vivo assays to investigate inflammation, MMP-9 activation, and collagen modulation, and the melanogenesis modulation activity of plasmin. A specific plasmin inhibitor, Amidinobenzyl Benzylsulfonyl D-Seryl Homophenylalaninamide Acetate (ABSHA), was used in these assays to downregulate these effects. We found that ABSHA was able to down-regulate UV-irradiation-induced MMP-9 expression, and subsequent collagen IV degradation, ex vivo. In addition, the increased melanin synthesis in epidermal melanocytes was reduced significantly by ABSHA. Furthermore, dermal fibroblasts treated with the plasmin inhibitor showed increased collagen I synthesis. We further investigated these effects in a two-month, monocentric, placebo-controlled human study on female Chinese volunteers. We found a significant increase in collagen density by ultrasound measurement and an increase in elasticity by cutometer assessment in the group using a formulation consisting of a 10 ppm ABSHA solution. This resulted in decreased wrinkle volumes on both the forehead and crow’s feet as shown by Primos CR. Looking at age spots, there was a decrease in overall ITA° and melanin density as well as in the total age spot area. Our results establish plasmin as a skin-aging enzyme. Using specific inhibitors against plasmin shows promise against age-induced skin conditions.

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 &amp;gt;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.

Cardiac Fibroblasts Enhance MMP2 Activity to Suppress Gap Junction Function in Cardiomyocytes.

Although it is crucial to promptly restore blood perfusion to revive the ischemic myocardium, reperfusion itself can paradoxically contribute to the electrical instability and arrhythmias of the myocardium. Several studies have revealed that cardiac fibroblasts can impact cardiac electrophysiology through various mechanisms including the deposition of extracellular matrix, release of chemical mediators, and direct electrical coupling with myocytes. Previously, we have shown that hypoxia/reoxygenation (H/R)-treated rat fibroblasts conditional medium (H/R-FCM) could decrease the spontaneous beating frequency of rat neonatal cardiomyocytes and downregulate the expression of gap junction proteins. However, the specific mechanism by which H/R-FCM affects the gap junctions requires further investigation. H/R-FCM was obtained by culturing confluent rat cardiac fibroblasts (RCF) for 4h under hypoxic conditions. Gap junction function, hemichannel activity, and expression of Cx43 were examined upon treatment with H/R-FCM. Gelatin zymography was performed to detect matrix metalloproteinase (MMP) activity in the conditioned medium. The effect of H/R-FCM and MMP2 inhibitors on cardiac electrophysiology and arrhythmias was investigated with an isolated rat ischemia/reperfusion (I/R) model. H/R-FCM treatment impaired gap junction function, downregulated Cx43 expression, and increased hemichannel activity in rat cardiomyocytes (H9c2). The adverse effect of H/R-FCM on gap junction, which was confirmed by the cardiomyocyte H/R model, was involved in the activation of MMP2. MMP2 inhibition could partially attenuate the detrimental effects of I/R on myocardial electrophysiological indices and arrhythmia susceptibility. Our study indicates that inhibition of MMP2 may be a promising therapeutic target for the treatment of reperfusion arrhythmia.

Effects of Lithium Ions on tPA-Induced Hemorrhagic Transformation under Stroke.

Thrombolytic therapy with the tissue plasminogen activator (tPA) is a therapeutic option for acute ischemic stroke. However, this approach is subject to several limitations, particularly the increased risk of hemorrhagic transformation (HT). Lithium salts show neuroprotective effects in stroke, but their effects on HT mechanisms are still unknown. In our study, we use the models of photothrombosis (PT)-induced brain ischemia and oxygen-glucose deprivation (OGD) to investigate the effect of Li+ on tPA-induced changes in brain and endothelial cell cultures. We found that tPA did not affect lesion volume or exacerbate neurological deficits but disrupted the blood-brain barrier. We demonstrate that poststroke treatment with Li+ improves neurological status and increases blood-brain barrier integrity after thrombolytic therapy. Under conditions of OGD, tPA treatment increased MMP-2/9 levels in endothelial cells, and preincubation with LiCl abolished this MMP activation. Moreover, we observed the effect of Li+ on glycolysis in tPA-treated endothelial cells, which we hypothesized to have an effect on MMP expression.

Protective effect of Albizia chinensis saponin on ethanol-induced gastric mucosal injury in rats focus on mucus and mucosal barriers

This study aims to explore the protective effect of Albizia chinensis saponin on ethanol-induced acute gastric ulcer in rats and elucidate its mechanisms. SD rats were deprived of water for 24 hours before the experiment. The control group and model group were administered water by gavage, and the positive drug group received rabeprazole sodium solution(40 mg·kg~(-1)) by gavage. The experimental groups were given different doses of Albizia chinensis saponin solution(3, 10, and 30 mg·kg~(-1)). After 30 minutes, the control group received 1.5 mL of water by gavage, while the other groups were administered an equal volume of 95% ethanol for modeling. After six hours, the rats were killed by cervical dislocation, and the stomachs were collected. The ulcer area was measured, and the ulcer index was calculated. Hematoxylin-eosin(HE) staining was performed to assess histopathological changes in gastric tissue. Periodic acid-Schiff(PAS) staining was used to evaluate the distribution of gastric mucosal surface mucus. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of phospholipids and aminohexose in the gastric mucosa. Western blot was performed to determine the expression levels of the bicarbonate transporter, matrix metalloproteinase, and tight junction-associated proteins in gastric tissue. Immunohistochemistry(IHC) staining was conducted to quantify the number of positive cells for secreted mucin and tight junction-associated proteins. The results showed that the gastric tissue surface of rats in the control group was smooth without ulceration, and the gastric ulcer index of rats in the model group was 35±11. Albizia chinensis saponin at doses of 3, 10, and 30 mg·kg~(-1) resulted in inhibition rates of gastric ulcer of 46%(P&lt;0.01), 85%(P&lt;0.001), and 100%(P&lt;0.001), respectively. Severe disruption of gastric mucosal structure and absence of the mucus layer were observed in the model group. Compared with the model group, the Albizia chinensis saponin group showed intact gastric mucosal surface mucus layer, significantly increased levels of phospholipids and aminohexose in the mucus, increased number of MUC5AC positive cells, and upregulated expression levels of the bicarbonate transporter SLC26A3 and CFTR. It also showed decreased phosphorylation of JNK and c-Jun, reduced expression levels of MMP-8, elevated expression of TIMP-1, and increased expression levels of Occludin and ZO-1. In conclusion, Albizia chinensis saponin enhances the function of the mucus-bicarbonate barrier by upregulating the content of MUC5AC, phospholipids, and aminohexose and increasing the expression levels of the bicarbonate transporter SLC26A3 and CFTR. Moreover, Albizia chinensis saponin exerts its protective effects on gastric ulcers by inhibiting the JNK signaling pathway to prevent excessive activation of MMP-8, thereby reducing the degradation of Occludin and ZO-1 and enhancing the mucosal barrier function. In summary, Albizia chinensis saponin exerts its anti-gastric ulcer effects by simultaneously enhancing the mucus barrier and the mucosal barrier.

Enhanced expression of miR-20a driven by nanog exacerbated the degradation of extracellular matrix in thoracic aortic dissection

Thoracic aortic dissection (TAD) is a life-threatening vascular disease manifested as intramural bleeding in the medial layers of the thoracic aorta. The key histopathologic feature of TAD is medial degeneration, characterized by depletion of vascular smooth muscle cells (VSMCs) and degradation of extracellular matrix (ECM). MicroRNA, as essential epigenetic regulators, can inhibit the protein expression of target genes without modifying the sequences. This study aimed to elucidate the role and underlying mechanism of miR-20a, a member of the miR-17-92 cluster, in regulating ECM degradation during the pathogenesis of TAD. The expression of the miR-17-92 cluster was significantly increased in synthetic VSMCs derived from TAD lesions compared to contractile VSMCs isolated from normal thoracic aortas. Notably, the expression of miR-20a was increased in VSMCs in response to serum exposure and various stimuli. In TAD lesions, the expression of miR-20a was significantly negatively correlated with that of elastin. Elevated expression of miR-20a was also observed in thoracic aortas of TAD mice induced by β-aminopropionitrile fumarate and angiotensin II. Overexpression of miR-20a via mimic transfection enhanced the growth and invasive capabilities of VSMCs, with no significant impact on their migratory activity or the expression of phenotypic markers (α-SMA, SM22, and OPN). Silencing of miR-20a with inhibitor transfection mitigated the hyperactivation of MMP2 in VSMCs stimulated by PDGF-bb, as evidenced by reduced levels of active-MMP2 and increased levels of pro-MMP2. Subsequently, TIMP2 was identified as a novel target gene of miR-20a. The role of miR-20a in promoting the activation of MMP2 was mediated by the suppression of TIMP2 expression in VSMCs. In addition, the elevated expression of miR-20a was found to be directly driven by Nanog in VSMCs. Collectively, these findings indicate that miR-20a plays a crucial role in maintaining the homeostasis of the thoracic aortic wall during TAD pathogenesis and may represent a potential therapeutic target for TAD.

Human Umbilical Cord Mesenchymal Stromal Cell-Derived Extracellular Vesicles Induce Fetal Wound Healing Features Revealed by Single-Cell RNA Sequencing.

The potential of human umbilical cord mesenchymal stromal cell-derived extracellular vesicles (hucMSC-EVs) in wound healing is promising, yet a comprehensive understanding of how fibroblasts and keratinocytes respond to this treatment remains limited. This study utilizes single-cell RNA sequencing (scRNA-seq) to investigate the impact of hucMSC-EVs on the cutaneous wound microenvironment in mice. Through rigorous single-cell analyses, we unveil the emergence of hucMSC-EV-induced hematopoietic fibroblasts and MMP13+ fibroblasts. Notably, MMP13+ fibroblasts exhibit fetal-like expressions of MMP13, MMP9, and HAS1, accompanied by heightened migrasome activity. Activation of MMP13+ fibroblasts is orchestrated by a distinctive PIEZO1-calcium-HIF1α-VEGF-MMP13 pathway, validated through murine models and dermal fibroblast assays. Organotypic culture assays further affirm that these activated fibroblasts induce keratinocyte migration via MMP13-LRP1 interactions. This study significantly contributes to our understanding of fibroblast heterogeneities as well as intercellular interactions in wound healing and identifies hucMSC-EV-induced hematopoietic fibroblasts as potential targets for reprogramming. The therapeutic targets presented by these fibroblasts offer exciting prospects for advancing wound healing strategies.

Triphenylamine‐appended tri‐organotin complexes: Synthesis, anticancer activities, and targeted research

Mitochondria play a crucial role in mediating apoptosis and have emerged as a promising therapeutic target for the treatment of cancer. Nevertheless, exploration of mitochondrial apoptosis as a mechanism for anticancer activity in vitro remains notably sparse. Herein, the ligands 4′,4″′‐(phenylazanediyl)bis(([1,1′‐biphenyl]‐4‐carboxylic acid)) (H2L1) and 4′,4″′‐(phenylazanediyl)bis(3‐fluoro‐[1,1′‐biphenyl]‐4‐carboxylic acid) (H2L2) were synthesized and further combined with organotin precursors and auxiliary ligands (4,4′‐bipy = 4,4′‐bipyridine, 4,4′‐bpe = 4,4′‐vinylenedipyridine) to give eight organotin complexes [(Ph3Sn)2L] (1a and 2a), [(Et3Sn)2L]n (1b and 2b), [(n‐Bu3Sn)2L(4,4′‐bipy)]n (1c and 2c), [(n‐Bu3Sn)2L(4,4′‐bpec)]n (1d and 2d). The complexes were characterized by X‐ray single crystal diffraction, NMR (1H, 13C, and 119Sn), IR, and UV–Vis spectroscopy. The complexes exhibited strong anticancer activity against the four cancer cells and a certain degree of broad‐spectrum with the IC50 ranging from 0.25 to 22.43 μM. Furthermore, complexes 2a and 2b were selected as representatives to further investigate their anticancer mechanisms. The results suggested that these complexes could target mitochondria after cellular uptake, thereby inducing intracellular ROS production, leading to MMP collapse and activation of caspase‐3, that is, these complexes potentially exert their anticancer effects through the mitochondria‐mediated apoptotic pathway.

Molecular mechanisms of the effects of mulberry (Morus Rubra) extracts on UV-radiation and oxidative stress-induced skin cell damage

Solar ultraviolet (UV) radiation, while beneficial for the homeostasis of human skin at proper doses, causes skin cell damage under extreme conditions, with a result of photoaging and skin cancer. With the depletion of the ozone layer, the prevalence of UV-induced skin cancer including melanoma has increased dramatically worldwide. Unfortunately, that poses a public health challenge for almost all countries. Over the past two decades, we have been working extensively on understanding the cellular and molecular mechanisms through which UV radiation induces skin aging and skin cancer. We have observed that UV radiation activates cell surface receptors, more pronouncedly EGFR, leading to a cascade of activation of MAP kinase pathways, with a result of MMP activation and in turn matrix degradation, and apoptosis. We have also found that UV radiation induces the downregulation of water channel aquaporins and skin cell dehydration. This novel discovery together with other reports has resulted in the emergence of numerous skin care products. Moreover, UV radiation induces the generation of reactive oxygen species, which exacerbates skin cell damage. With this important line of discovery, more skin care products with added antioxidants have been developed. Mulberries ( Morus Rubra) have been known to be rich in antioxidants and applied to cosmetic products in Asian countries with reported effcacy although the cellular and molecular mechanisms of its actions remain elusive. The objective of this study was to investigate these mechanisms from various perspectives. We hypothesize that the mulberry extracts affect cell signaling pathways that lead to protection against UV radiation or oxidative stressed-induced cell damage. First, we extracted the antioxidants with methanol from two types of mulberries, red and white. Human skin keratinocytes (HaCaT cells) were treated with UV radiation and hydrogen peroxide (H2O2), with and without mulberry extracts. Confocal microscopy and Western blot analysis were employed to measure various cell signaling components. The results showed that mulberry extracts protect against UV radiation and H2O2 induced cell death, with red extracts showing better protection than white extracts. Mulberry extracts attenuate H2O2-induced mitochondrial activity as tested by MitoTracker™ Red CMXRos. Our previous studies have shown that UV radiation induces mTOR pathway activation evidenced by elevation of phosphorylated S6. Interestingly, in this study, Western blot analysis data indicated that mulberry extracts potentiate UV-induced S6 phosphorylation and H2O2-induced MEK activation. Furthermore, confocal data demonstrated that mulberry extract pretreatment inhibits IL1β induced NFκB p65 translocation from the cytoplasm to the nucleus. Taken all together, our data suggest that mulberry extracts containing abundant antioxidants affect MAPK kinase and mTOR pathways and modulate cell signaling pathways associated with skin cell survival and inflammation under UV radiation and oxidative stress. Funding from the Committee on Aid to Faculty Research (CAFR) from Providence College. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.