Proteolytic Activity Of Matrix Metalloproteinases Research Articles

Cytolethal Distending Toxin Modulates Cell Differentiation and Elicits Epithelial to Mesenchymal Transition.

The bacterial genotoxin, cytolethal distending toxin (CDT), causes DNA damage in host cells, a risk factor for carcinogenesis. Previous studies have shown that CDT induces phenotypes reminiscent of epithelial to mesenchymal transition (EMT), a process involved in cancer initiation and progression. We investigated different steps of EMT in response to Helicobacter hepaticus CDT and its active CdtB subunit using in vivo and in vitro models. Most of the steps of the EMT process were induced by CDT/CdtB and observed throughout the study in murine and epithelial cell culture models. CdtB induced cell-cell junction disassembly, causing individualization of cells and acquisition of a spindle-like morphology. The key transcriptional regulators of EMT (SNAIL and ZEB1) and some EMT markers were upregulated at both RNA and protein levels in response to CDT/CdtB. CdtB increased the expression and proteolytic activity of matrix metalloproteinases, as well as cell migration. A range of these results were confirmed in Helicobacter hepaticus-infected and xenograft murine models. In addition, colibactin, a genotoxic metabolite produced by Escherichia coli, induced EMT-like effects in cell culture. Overall, these data show that infection with genotoxin-producing bacteria elicits EMT process activation, supporting their role in tumorigenesis.

Factors Influencing Venous Remodeling in the Development of Varicose Veins of the Lower Limbs.

One of the early symptoms of chronic venous disease (CVD) is varicose veins (VV) of the lower limbs. There are many etiological environmental factors influencing the development of chronic venous insufficiency (CVI), although genetic factors and family history of the disease play a key role. All these factors induce changes in the hemodynamic in the venous system of the lower limbs leading to blood stasis, hypoxia, inflammation, oxidative stress, proteolytic activity of matrix metalloproteinases (MMPs), changes in microcirculation and, consequently, the remodeling of the venous wall. The aim of this review is to present current knowledge on CVD, including the pathophysiology and mechanisms related to vein wall remodeling. Particular emphasis has been placed on describing the role of inflammation and oxidative stress and the involvement of extracellular hemoglobin as pathogenetic factors of VV. Additionally, active substances used in the treatment of VV were discussed.

Irisin: A Possible Marker of Adipose Tissue Dysfunction in Obesity.

Adipose tissue (AT) secretes pro- and anti-inflammatory cytokines involved in AT homeostasis, including tumor necrosis factor-α (TNFα) and irisin. The functionality of AT is based on a regulated equilibrium between adipogenesis and extracellular matrix (ECM) remodeling. We investigated the contributions of adipose progenitors (ASCs) and adipocytes (AMCs) to TNFα-induced ECM remodeling and a possible implication of irisin in AT impairment in obesity. ASCs and AMCs were exposed to TNFα treatment and nuclear factor-kappa (NF-kB) pathway was investigated: Tissue Inhibitor of Metalloproteinase (TIMP-1), Twist Family Transcription Factor 1 (TWIST-1), and peroxisome proliferator-activated receptor-γ (PPARγ) expression levels were analyzed. The proteolytic activity of matrix metalloproteinases (MMPs) -2 and -9 was analyzed by zymography, and the irisin protein content was measured by ELISA. In inflamed AMCs, a TIMP-1/TWIST-1 imbalance leads to a drop in PPARγ. Adipogenesis and lipid storage ability impairment come with local tissue remodeling due to MMP-9 overactivation. In vitro and ex vivo measurements confirm positive correlations among inflammation, adipose secreting irisin levels, and circulating irisin levels in patients with visceral obesity. Our findings identify the NF-kB downstream effectors as molecular initiators of AT dysfunction and suggest irisin as a possible AT damage and obesity predictive factor.

The invariant chain CD74 protein is a cell surface binding partner of TIMP-1 in breast cancer cells.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the proteolytic activity of matrix metalloproteinases (MMPs), playing an important role in the homeostasis of the extracellular matrix. Beyond its well-known role in tissue maintenance, TIMP-1 has been associated with multiple MMP-independent cytokine-like functions. The protein structure of TIMP-1, with two distinct domains, one interacting with MMPs and another able to bind multiple partners, provides a rationale for this multi-functionality. The identification of CD63 as a cell surface receptor for TIMP-1, able to mediate intracellular signaling through the Erk/MAPK axis, provided a molecular basis for the role of TIMP-1 in cellular signaling. However, several lines of evidence suggest that TIMP-1 may be able to associate with many interaction partners, thus attaining multiple functions. To enable the identification of previously unknown interaction partners that may underpin the core cellular functions of TIMP-1, known as well as unknown, we performed a yeast two-hybrid screening using a mammary gland complementary DNA (cDNA) library. We report here the identification of multiple interactors, including MHC class II-associated invariant chain γ (CD74). We verified that CD74 interacts with TIMP-1 in breast cancer cells and that this interaction contributes to cellular internalization of TIMP-1 and mediates intracellular signaling through the Akt signaling axis in breast cancer cells. These data provide new insights into the complex nature of the functions of TIMP-1 and their potential mechanistic basis.

Omeprazole prevents stress induced gastric ulcer by direct inhibition of MMP-2/TIMP-3 interactions

The healing of damaged tissues in gastric tract starts with the extracellular matrix (ECM) remodeling by the action of matrix metalloproteinases (MMPs). Particularly, MMP-2 (gelatinase-A) maintains ECM structure and function by degrading type IV collagen, the major component of basement membranes and by clearing denatured collagen. The proteolytic activities of MMPs are critically balanced by endogenous tissue inhibitors of metalloproteinases (TIMPs) and disruption of this balance results in several diseases. The well-known drug omeprazole is a proton pump inhibitor used for curing gastric ulcer. However, the action of omeprazole in ECM remodeling on gastroprotection has never been explored. Herein, using rat model of gastric ulcer, we report that restraint cold stress caused increase apoptosis to surface epithelia of gastric tissues along with TIMP-3 upregulation and inhibition of MMP-2 activity thereon. In contrast, omeprazole treatment suppressed TIMP-3 while increasing MMP-2 activity and thereby, restoring MMP-2/TIMP-3 balance. Additionally, nanomolar binding constant (Kd = 318 nM) of omeprazole with purified MMP-2 indicates a direct effect of omeprazole in restoring MMP-2 activity. Further in silico simulations revealed a plausible mechanism of action of omeprazole for TIMP-3 deactivation. Altogether, omeprazole restores MMP-2 activity and reduces apoptosis while preventing acute stress-induced gastric ulcer that occurs via suppression of nuclear factor kappa B (NF-κB) activity and peroxisome proliferator-activated receptor gamma activity (PPAR-γ). This represents an unprecedented correlation between physical docking of drug molecule to a protease and the severity of organ injury and provides a novel therapeutic approach to prevent stress induced tissue damage.

Neutrophil Gelatinase-associated Lipocalin: A Potential Predictor of Embolic Events in Endocarditis

BackgroundAs the complication of infective endocarditis (IE), embolic events are associated with increased mortality and morbidity. However, there are no reliable indicators to predict embolism. The aim of this study was to evaluate neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker to identify IE patients at high risk of embolism. MethodsWe conducted a prospective observational study of IE patients from January 2018 to December 2020. Plasma NGAL levels were measured in 88 IE patients (37 patients with embolic events and 51 patients without embolic events), 91 noninfectious heart valve disease patients, and 20 healthy blood donors by enzyme-linked immunosorbent assay. Native valve tissue was obtained from 16 IE patients and 16 noninfectious heart valve disease patients. Western blot and immunohistochemical staining were performed to detect NGAL and matrix metalloproteinase-9. ResultsHigher levels of NGAL were observed in IE patients than in noninfectious heart valve disease patients (P < .001) or healthy blood donors (P < .01). In addition, NGAL levels were higher in IE patients with embolic events compared with patients not having embolic events (P < .001). Receiver-operating characteristics analysis demonstrated that NGAL acted as a potential embolic events predictor with the cutoff value of 166.78 ng/mL. The IE patients with higher NGAL levels had significantly more severe native valve morphologic changes. The NGAL was colocalized with matrix metalloproteinase-9, and their expression in the valves of IE patients was higher than in those of noninfectious heart valve disease patients. ConclusionsNeutrophil gelatinase-associated lipocalin is a potential predictor of embolic events in IE. That may be attributed to its potency of increasing the proteolytic activity of matrix metalloproteinase-9, which leads to valve morphologic impairment.

The Emerging Role of Epigenetic Mechanisms in the Causation of Aberrant MMP Activity during Human Pathologies and the Use of Medicinal Drugs.

Matrix metalloproteinases (MMPs) cleave extracellular matrix proteins, growth factors, cytokines, and receptors to influence organ development, architecture, function, and the systemic and cell-specific responses to diseases and pharmacological drugs. Conversely, many diseases (such as atherosclerosis, arthritis, bacterial infections (tuberculosis), viral infections (COVID-19), and cancer), cholesterol-lowering drugs (such as statins), and tetracycline-class antibiotics (such as doxycycline) alter MMP activity through transcriptional, translational, and post-translational mechanisms. In this review, we summarize evidence that the aforementioned diseases and drugs exert significant epigenetic pressure on genes encoding MMPs, tissue inhibitors of MMPs, and factors that transcriptionally regulate the expression of MMPs. Our understanding of human pathologies associated with alterations in the proteolytic activity of MMPs must consider that these pathologies and their medicinal treatments may impose epigenetic pressure on the expression of MMP genes. Whether the epigenetic mechanisms affecting the activity of MMPs can be therapeutically targeted warrants further research.

Mechanisms of Lower Extremity Vein Dysfunction in Chronic Venous Disease and Implications in Management of Varicose Veins.

Chronic venous disease (CVD) is a common venous disorder of the lower extremities. CVD can be manifested as varicose veins (VVs), with dilated and tortuous veins, dysfunctional valves and venous reflux. If not adequately treated, VVs could progress to chronic venous insufficiency (CVI) and lead to venous leg ulcer (VLU). Predisposing familial and genetic factors have been implicated in CVD. Additional environmental, behavioral and dietary factors including sedentary lifestyle and obesity may also contribute to CVD. Alterations in the mRNA expression, protein levels and proteolytic activity of matrix metalloproteinases (MMPs) have been detected in VVs and VLU. MMP expression/activity can be modulated by venous hydrostatic pressure, hypoxia, tissue metabolites, and inflammation. MMPs in turn increase proteolysis of different protein substrates in the extracellular matrix particularly collagen and elastin, leading to weakening of the vein wall. MMPs could also promote venous dilation by increasing the release of endothelium-derived vasodilators and activating potassium channels, leading to smooth muscle hyperpolarization and relaxation. Depending on VVs severity, management usually includes compression stockings, sclerotherapy and surgical removal. Venotonics have also been promoted to decrease the progression of VVs. Sulodexide has also shown benefits in VLU and CVI, and recent data suggest that it could improve venous smooth muscle contraction. Other lines of treatment including induction of endogenous tissue inhibitors of metalloproteinases (TIMPs) and administration of exogenous synthetic inhibitors of MMPs are being explored, and could provide alternative strategies in the treatment of CVD.

Mesenchymal stem cells promote metastasis through activation of an ABL-MMP9 signaling axis in lung cancer cells.

Mesenchymal stem cells (MSCs) are recruited and activated by solid tumors and play a role in tumor progression and metastasis. Here we show that MSCs promote metastasis in a panel of non-small cell lung cancer (NSCLC) cells. MSCs elicit transcriptional alterations in lung cancer cells leading to increased expression of factors implicated in the epithelial-to-mesenchymal transition (EMT) and secreted proteins including matrix metalloproteinase-9 (MMP9). MSCs enhance secretion of enzymatically active MMP9 in a panel of lung adenocarcinoma cells. High expression of MMP9 is linked to low survival rates in lung adenocarcinoma patients. Notably, we found that ABL tyrosine kinases are activated in MSC-primed lung cancer cells and functional ABL kinases are required for MSC-induced MMP9 expression, secretion and proteolytic activity. Importantly, ABL kinases are required for MSC-induced NSCLC metastasis. These data reveal an actionable target for inhibiting MSC-induced metastatic activity of lung adenocarcinoma cells through disruption of an ABL kinase-MMP9 signaling axis activated in MSC-primed lung cancer cells.

Thymoquinone Suppresses Migration of Human Renal Carcinoma Caki-1 Cells through Inhibition of the PGE2-Mediated Activation of the EP2 Receptor Pathway.

Renal cell carcinoma (RCC) is likely to metastasize to other organs, and is often resistant to conventional chemotherapies. Thymoquinone (TQ), a phytochemical derived from the seeds of Nigella sativa, has been shown to inhibit migration and metastasis in various cancers. In this study, we assessed the effect of TQ on the migratory activity of human RCC Caki-1 cells. We found that treatment with TQ reduced the proteolytic activity of matrix metalloproteinase-9 (MMP-9) in Caki-1 cells. TQ significantly repressed prostaglandin E2 (PGE2) production, its EP2 receptor expression as well as the activation of Akt and p38, the well-known upstream signal proteins of MMP-9. In addition, treatment with butaprost, a PGE2 agonist, also induced MMP-9 activity and migration/invasion in Caki-1 cells. Moreover, pharmacological inhibitors of PI3K/Akt and p38 remarkably attenuated butaprost-induced Caki-1 cell migration and invasion, implying that activation of PI3K/Akt and p38 is a bridge between the PGE2-EP2 axis and MMP-9-dependent migration and invasion. Taken together, these data suggest that TQ is a promising anti-metastatic drug to treat advanced and metastatic RCC.

Investigation of MMP-9 rs3918242 and TIMP-2 rs8179090 polymorphisms in renal cell carcinoma tissues

Abstract Background The proteolytic activities of matrix metalloproteinases (MMP), cell surface enzymes degrading extracellular matrix, is inhibited by matrix metalloproteinase tissue inhibitors (TIMP). We aim to detect the effects of MMP-9 rs3918242 and TIMP-2 rs8179090 gene variations in renal cell cancer transformation. Methods One hundred tumor and 100 adjacent healthy tissues were obtained from the patients with renal cell cancer. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) was performed and the products carried out in agarose gel electrophoresis were visualized under UV light. Statistical analyses were performed using SPSS 22 and p-values of less than 0.05 were considered as statistically significant. Results MMP-9 rs3918242 T allele was higher in tumor tissues (36.5%) than adjacent tissues (17%) and odds ratio was found 2.8 in T allele (p&lt;0.001). Odds ratio values were found 3.325 in the carriers of TT genotype and 3.5 in the carriers of CT genotype compared to the carriers of CC genotype (p&lt;0.01). The polymorphism of TIMP-2 rs8179090 was not found statically significant in tumor and adjacent tissues (p&gt;0.05). Conclusion MMP-9 rs3918242 T allele, TT and CT genotypes can be used as biomarkers in determining of renal cell carcinoma.

Mongolian Chelidonium majus Suppresses Metastatic Potential of Hepatocellular Carcinoma Cells through TIMP Up-regulation and MMP Down-regulation

In this study, the antimetastatic effect of Chelidonium majus ethanol extract on hepatocellular carcinoma in vitro was investigated. The viability of SK-Hep1 and normal liver cells was determined using a cell counting kit-8 assay. Wound healing assays were performed to investigate SK-Hep1 cell migration and various metastatic characteristics including adhesion, aggregation and invasion were also measured using these cells. Furthermore, the proteolytic activity of extracellular matrix metalloproteinase-9 was measured using gelatin zymography. Expression levels of matrix metalloproteinase-2, matrix metalloproteinase-9, membrane type 1-matrix metalloproteinase and tissue inhibitor of metalloproteinase-1 were measured using reverse transcription-polymerase chain reaction and Western blotting. Chelidonium majus ethanol extract significantly inhibited the proliferation of SK-Hep1 hepatocellular carcinoma cells in a dose-dependent manner. Moreover, metastatic characteristics including adhesion, migration, aggregation and invasion were significantly suppressed by Chelidonium majus ethanol extract treatment. Further, this preparation downregulated the expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and membrane type 1-matrix metalloproteinase, but upregulated tissue inhibitor of metalloproteinase-1 and tissue inhibitor of metalloproteinase-2, in a dose-dependent manner. Additionally, the proteolytic activity of matrix metalloproteinase-9 was greatly diminished with 400 µg/ml of Chelidonium majus ethanol extract. Taken together, it was suggested that Chelidonium majus ethanol extract might exert an antimetastatic effect on hepatocellular carcinoma cells by inhibiting proliferation, adhesion, migration, aggregation and invasion through the downregulation of matrix metalloproteinases and upregulation of tissue inhibitor of metalloproteinases. Thus, this extract could represent a promising therapeutic agent for this disease.

Effect of sodium ascorbate on bond strength and metalloproteinases activity in bleached dentin.

AimThis study evaluated the effect of sodium ascorbate (SA) on the proteolytic activity of matrix metalloproteinases (MMPs) and investigated the related effects on the bond strength of bleached dentin.Materials and methodsEighty freshly extracted human third molars were randomly divided according to treatment (bleaching or SA application), type of analysis (microshear or measuring MMP activity), and post-bleaching time to assess bond strength (24 hrs or 30 days). Data from both analyses were subjected to one-way analysis of variance to detect differences among groups, followed by Tukey’s multiple comparison test (p≤0.05).ResultsDental bleaching significantly reduced bond strength values when the adhesive strategy was performed after 24 hrs (despite the SA treatment) or 30 days after the bleaching procedure. However, after 30 days, the bond strength values of the groups who received bleaching or SA application were similar to those of the unbleached group. Dental bleaching caused the activation of MMPs, and SA did not influence this activity.ConclusionIt was concluded that SA does not affect the activity of MMPs or the bond strength in bleached dentin immediately after the bleaching treatment.

EZH2-mediated epigenetic silencing of TIMP2 promotes ovarian cancer migration and invasion

Enhancer of zeste homolog 2 (EZH2) is often increased in malignant tumors and is involved in metastasis. EZH2 silences gene expression by tri-methylating the lysine 27 residue of histone H3 (H3K27me3). However, the mechanism underlying EZH2 promotion of ovarian cancer metastasis remains elusive. Here, we showed that EZH2 is up-regulated in ovarian cancer and is associated with tumor metastasis and poor survival by mRNA sequencing and microarray results from databases. Tissue microarray and immunohistochemistry results revealed that EZH2 was negatively correlated with the expression of tissue inhibitor of metalloproteinases 2 (TIMP2). EZH2 overexpression inhibited TIMP2 expression and promoted proteolytic activities of matrix metalloproteinases 2 and 9 and vice versa. EZH2 promoted ovarian cancer invasion and migration, which could be largely reversed by TIMP2 down-regulation in vitro and in vivo. Both H3K27me3 inhibition and demethylation could reduce methylation of the TIMP2 promoter and finally reactivate TIMP2 transcription. The presence of EZH2 and H3K27me3 at the TIMP2 promoter was confirmed by chromatin immunoprecipitation. H3K27me3 and DNA methyltransferases at the promoter were significantly increased by EZH2 overexpression. These results suggest that EZH2 inhibits TIMP2 expression via H3K27me3 and DNA methylation, which relieve the repression of MMP and facilitate ovarian cancer invasion and migration.

Baicalein, unlike 4-hydroxytamoxifen but similar to G15, suppresses 17β-estradiol-induced cell invasion, and matrix metalloproteinase-9 expression and activation in MCF-7 human breast cancer cells.

Estrogen performs an important role in the growth and development of breast cancer. There are at least three major receptors, including estrogen receptor (ER)α and β, and G protein-coupled receptor 30 (GPR30), which mediate the actions of estrogen through using transcriptional and rapid non-genomic signaling pathways. Flavonoids have been considered candidates for chemopreventive agents in breast cancer. Baicalein, the primary flavonoid derived from the root of Scutellaria baicalensis Georgi, has been reported to exert an anti-estrogenic effect. In the present study, the effects of baicalein on 17β-estradiol (E2)-induced cell invasion, and matrix metalloproteinase-9 (MMP-9) expression and activation were investigated. Furthermore, its effects were compared with that of the active form of the ER modulator tamoxifen 4-hydroxytamoxifen (OHT) and the GPR30 antagonist G15 in ERα- and GPR30-positive MCF-7 breast cancer cells. The results demonstrated that OHT failed to prevent E2-induced cell invasion, upregulation and proteolytic activity of MMP-9. However, baicalein was able to significantly suppress these E2-induced effects. Furthermore, E2-stimulated invasion, and MMP-9 expression and activation were significantly attenuated following G15 treatment. In addition, baicalein significantly inhibited G-1, a specific GPR30 agonist, induced invasion, and reduced G-1 promoted expression and activity of MMP-9, consistent with effects of G15. The results of the present study suggest that baicalein is a therapeutic candidate for GPR30-positive breast cancer treatment, and besides ERα targeting the GPR30 receptor it may achieve additional therapeutic benefits in breast cancer.

Fucus vesiculosus extract inhibits the proteolytic activity and gene expression of matrix metalloproteinases in Atlantic salmon (Salmo salar L.)

Fucoidans are sulfated fucose-rich polysaccharides found in macroalgae. Multiple biological functions have been attributed to fucoidans including inhibitory activity against matrix metalloproteinases (MMPs). A recent study showed that kidney and blood remains left in the abdominal cavity after gutting had a significant negative effect on gaping and fillet firmness in Atlantic salmon (Salmo salar L.). The authors hypothesized that one reason could be that MMPs from kidney and blood remains break down the connective tissue in Atlantic salmon fillets. The objectives of this study were to investigate the presence of MMPs in Atlantic salmon kidney and the inhibitory effect of a Fucus vesiculosus extract (FVE) on the proteolytic activity and gene expression of MMPs in Atlantic salmon. The inhibitory effect of FVE was compared to the inhibitory effect of the synthetic broad-spectrum MMP inhibitor marimastat. In this study, we found that FVE inhibits the proteolytic activity of MMP-1, MMP-2, MMP-9, and MMP-13 in Atlantic salmon kidney as well as MMP-2 gene expression in Atlantic salmon liver. Although the inhibitory effect of FVE on the proteolytic activity of MMPs is small compared to the inhibitory effect of marimastat, both FVE and marimastat inhibit 80% of the DQ-gelatin breakdown caused by Atlantic salmon kidney at a concentration of 0.1 mg mL−1. This indicates that FVE may also be capable of inhibiting other proteinases present in Atlantic salmon kidney. Overall, our findings suggest that FVE could be used in the fish processing industry to limit collagenase and gelatinase activity, thus reducing the connective tissue degradation in Atlantic salmon fillets.

Ruptured abdominal aortic aneurysm-epidemiology, predisposing factors, and biology.

Abdominal aortic aneurysm is a common degenerative vascular disorder associated with sudden death due to aortic rupture. This review describes epidemiology, predisposing factors, and biology of ruptured abdominal aortic aneurysms (rAAAs). Based on a selective literature search in Medline (PubMed), original publications, meta-analyses, systematic reviews, and Cochrane reviews were evaluated for rAAA. The hospital admission rate for rAAA is decreasing and is now in the range of approximately 10 per 100,000 population in men. Smoking contributes to about 50% of population risk for rupture or surgically treated AAA. AAA rupture is a multifaceted biological process involving biochemical, cellular, and proteolytic influences, in addition to biomechanical factors. AAA rupture occurs when the stress (force per unit area) on the aneurysm wall exceeds wall strength. Proteolytic activities of matrix metalloproteinases have been implicated in aneurysm wall weakening and rupture. Aneurysm diameter is the most prominent predisposing factor for aneurysm growth and rupture. Wall stress, aneurysm shape and geometry, intraluminal thrombus, wall thickness, calcification, and metabolic activity influence the rupture risk. The best conservative option to avoid AAA rupture consists in smoking cessation and control of hypertension. Many biological factors influence rupture risk.

Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy.

ObjectivesMarfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis.MethodsWe performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM).ResultsIHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1 (compared to untreated Marfan aorta: Ad.hTIMP-1 p = 0.902; control Ad.β-Gal. p = 0.165). The virus-untreated and not transplanted mgR/mgR aorta revealed a significant increase of albumin diffusion through the endothelial barrier (p = 0.037). TEM analysis of adenovirus-exposed mgR/mgR aortas displayed disruption of the basement membrane and basolateral space.ConclusionsMurine Marfan aortic grafts developed severe inflammation after adenoviral contact. We demonstrated that fibrillin-1 deficiency is associated with relevant dysfunction of the endothelial barrier that enables adenovirus to induce vessel-harming inflammation. Endothelial dysfunction may play a pivotal role in the development of the vascular phenotype of Marfan syndrome.

The Effects of Platelet-Rich Plasma on Halting the Progression in Porcine Intervertebral Disc Degeneration.

Disc degeneration and the subsequent herniation and/or rupture of the intervertebral disc (IVD) are due to a failure of the extracellular matrix of the annulus to contain the contents of the nucleus. This results from inadequate maintenance of the matrix components as well as the proteolytic activity of matrix metalloproteinases (MMPs) that degrade matrix molecules. Arresting progression of disc degeneration in the annulus holds greater clinical potential at this point than prevention of its onset in the nucleus. Therefore, in this study, we have therapeutic aims that would decrease levels of the cytokines and growth factors that indirectly lead to disc degeneration via stimulating MMP and increase levels of several beneficial growth factors, such as transforming growth factor-β, with the addition of platelet-rich plasma (PRP) that would stimulate cell growth and matrix synthesis. For this study, we attempted to address these imbalances of metabolism by using tumor necrosis factor-α treated annulus fibrosus cells isolated from porcine IVD tissue and incubating the cells in a growth factor rich environment with PRP. These results indicate that the PRP in vitro increased the production of the major matrix components (type II collagen and aggrecan) and decreased the inhibitory collagenase MMP-1. This application will address a therapeutic approach for intervening early in the degenerative process.

Ubiquitin-specific peptidase 22 inhibits colon cancer cell invasion by suppressing the signal transducer and activator of transcription 3/matrix metalloproteinase 9 pathway.

Colon cancer is associated with increased cell migration and invasion. In the present study, the role of ubiquitin-specific peptidase 22 (USP22) in signal transducer and activator of transcription 3 (STAT3)-mediated colon cancer cell invasion was investigated. The messenger RNA levels of STAT3 target genes were measured by reverse transcription-quantitative polymerase chain reaction, following USP22 knockdown by RNA interference in SW480 colon cancer cells. The matrix metalloproteinase 9 (MMP9) proteolytic activity and invasion potential of SW480 cells were measured by zymography and Transwell assay, respectively, following combined USP22 and STAT3 short interfering (si)RNA treatment or STAT3 siRNA treatment alone. Similarly, a cell counting kit-8 assay was used to detect the proliferation potential of SW480 cells. The protein expression levels of USP22, STAT3 and MMP9 were detected by immunohistochemistry in colon cancer tissue microarrays (TMAs) and the correlation between USP22, STAT3 and MMP9 was analyzed. USP22/STAT3 co-depletion partly rescued the MMP9 proteolytic activity and invasion of SW480 cells, compared with that of STAT3 depletion alone. However, the proliferation of USP22/STAT3si-SW480 cells was decreased compared with that of STAT3si-SW480 cells. USP22 expression was positively correlated with STAT3 and MMP9 expression in colon cancer TMAs. In conclusion, USP22 attenuated the invasion capacity of colon cancer cells by inhibiting the STAT3/MMP9 signaling pathway.