Extracellular Matrix Proteolysis Research Articles

Unveiling Biomarkers in Head and Neck Squamous Cell Carcinoma through Bioinformatics: The Role of SPP1 and KRT78.

Head and neck squamous cell carcinoma (HNSCC) is the most common form of head and neck cancer, ranking sixth in global cancer incidence. Identifying molecular drivers of tumorigenesis and metastasis is essential for early detection and treatment. This study analyzed gene expression profiles from three datasets (GSE6791, GSE29330, and GSE58911) to identify differentially expressed genes (DEGs) in HNSCC. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were employed to functionally annotate these DEGs. A protein-protein interaction (PPI) network was constructed for selecting hub genes using the STRING database. Finally, hub gene and protein expression levels were evaluated in patients with HNSCC, along with their association with overall survival. Our analysis identified twenty-eight co-DEGs comprising eight up-regulated and twenty down-regulated genes, primarily involved in extracellular matrix (ECM) organization, proteolysis, ECM disassembly, and keratinization processes. Furthermore, the PPI network revealed eight hub genes based on their high degree of connectivity. Notably, SPP1 demonstrated up-regulation, while KRT78 was down-regulated in HNSCC. Remarkably, the expression levels of these hub genes correlated with tumor grade, clinical cancer stage, and poor prognosis in HNSCC. Our findings hold significant clinical potential for early diagnosis and the development of novel therapeutic targets for patients with HNSCC.

Identification of extracellular matrix proteins in plasma as a potential biomarker for intervertebral disc degeneration.

Recently, there has been significant focus on extracellular matrix proteolysis due to its importance in the pathological progression of intervertebral disc degeneration (IVDD). The present study investigates the circulating levels of extracellular matrix proteins in the plasma of IVDD and determines their potential relevance as biomarkers in disc degeneration. Global proteomic analysis was performed in the plasma samples of 10 healthy volunteers (HV) and 10 diseased subjects (DS) after depletion of highly abundant proteins such as albumin and IgG. We identified 144 and 135 matrix-associated proteins in plasma samples from healthy volunteers (HV) and patients with disc degeneration (DS), respectively. Among these, 49 of the matrix-associated proteins were identical to the proteins found in intervertebral disc (IVD) tissues retrieved from the in-house library. Applying stringent parameters, we selected 28 proteins, with 26 present in DS and 21 in HV. 19 proteins were found common between the groups, two of which-aggrecan (ACAN) and fibulin 1 (FBLN1) - showed statistically significant differences. Specifically, ACAN was up-regulated and FBLN1 was down-regulated in the DS-plasma. In particular, DS-plasma exhibited specific expression of collagen type 2a1 (COL2A1), native to the nucleus pulposus. The distinct presence of collagen type 2a1 and the elevated expression of aggrecan in IVDD plasma may serve as the basis for the development of a potential biomarker for monitoring the progression of disc degeneration.

Active interfacial degradation/deposition of an elastic matrix by a fluid inclusion: Theory and pattern formation

During collective invasion in 3D, cohesive cellular tissues migrate within a fibrous extracellular matrix (ECM). This process requires significant remodeling of the ECM by cells, notably proteolysis at the cell–ECM interface by specialized molecules. Motivated by this problem, we develop a theoretical framework to study the dynamics of a fluid inclusion (modeling the cellular tissue) embedded in an elastic matrix (the ECM), which undergoes surface degradation/deposition. To account for the active nature of this process, we develop a continuum theory based on irreversible thermodynamics, leading to a kinetic relation for the degradation front that locally resembles the force–velocity relation of a molecular motor. We further study the effect of mechanotransduction on the stability of the cell–ECM interface, finding a variety of self-organized dynamical patterns of collective invasion. Our work identifies ECM proteolysis as an active process possibly driving the self-organization of cellular tissues.

A Novel Strategy for the Treatment of Aneurysms: Inhibition of MMP-9 Activity through the Delivery of TIMP-1 Encoding Synthetic mRNA into Arteries.

Aneurysms pose life-threatening risks due to the dilatation of the arteries and carry a high risk of rupture. Despite continuous research efforts, there are still no satisfactory or clinically effective pharmaceutical treatments for this condition. Accelerated inflammatory processes during aneurysm development lead to increased levels of matrix metalloproteinases (MMPs) and destabilization of the vessel wall through the degradation of the structural components of the extracellular matrix (ECM), mainly collagen and elastin. Tissue inhibitors of metalloproteinases (TIMPs) directly regulate MMP activity and consequently inhibit ECM proteolysis. In this work, the synthesis of TIMP-1 protein was increased by the exogenous delivery of synthetic TIMP-1 encoding mRNA into aortic vessel tissue in an attempt to inhibit MMP-9. In vitro, TIMP-1 mRNA transfection resulted in significantly increased TIMP-1 protein expression in various cells. The functionality of the expressed protein was evaluated in an appropriate ex vivo aortic vessel model. Decreased MMP-9 activity was detected using in situ zymography 24 h and 48 h post microinjection of 5 µg TIMP-1 mRNA into the aortic vessel wall. These results suggest that TIMP-1 mRNA administration is a promising approach for the treatment of aneurysms.

The β-arrestin1/endothelin axis bolsters ovarian fibroblast-dependent invadosome activity and cancer cell metastatic potential

Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix (ECM) remodeling, invasion, and metastasis, highlighting the need to investigate the molecular mechanisms driving CAF function. Endothelin-1 (ET-1) regulates the communication between cancer and stroma and facilitates the progression of serous ovarian cancer (SOC). By binding to Endothelin A (ETA) and B (ETB) receptors, ET-1 enables the recruitment of β-arrestin1 (β-arr1) and the formation of signaling complexes that coordinate tumor progression. However, how ET-1 receptors might “educate” human ovarian fibroblasts (HOFs) to produce altered ECM and promote metastasis remains to be elucidated. This study identifies ET-1 as a pivotal factor in the activation of CAFs capable of proteolytic ECM remodeling and the generation of heterotypic spheroids containing cancer cells with a propensity to metastasize. An autocrine/paracrine ET-1/ETA/BR/β-arr1 loop enhances HOF proliferation, upregulates CAF marker expression, secretes pro-inflammatory cytokines, and increases collagen contractility, and cell motility. Furthermore, ET-1 facilitates ECM remodeling by promoting the lytic activity of invadosome and activation of integrin β1. In addition, ET-1 signaling supports the formation of heterotypic HOF/SOC spheroids with enhanced ability to migrate through the mesothelial monolayer, and invade, representing metastatic units. The blockade of ETA/BR or β-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ETA/BR using bosentan (BOS) significantly reduces the metastatic potential of combined HOFs/SOC cells, associated with enhanced apoptotic effects on tumor cells and stromal components. These findings support a model in which ET-1/β-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ETA/BR antagonists.

Tumor Microenvironment Modulates Invadopodia Activity of Non-Selected and Acid-Selected Pancreatic Cancer Cells and Its Sensitivity to Gemcitabine and C18-Gemcitabine.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with high mortality due to early metastatic dissemination and high chemoresistance. All these factors are favored by its extracellular matrix (ECM)-rich microenvironment, which is also highly hypoxic and acidic. Gemcitabine (GEM) is still the first-line therapy in PDAC. However, it is quickly deaminated to its inactive metabolite. Several GEM prodrugs have emerged to improve its cytotoxicity. Here, we analyzed how the acidic/hypoxic tumor microenvironment (TME) affects the response of PDAC cell death and invadopodia-mediated ECM proteolysis to both GEM and its C18 prodrug. For this, two PDAC cell lines, PANC-1 and Mia PaCa-2 were adapted to pHe 6.6 or not for 1 month, grown as 3D organotypic cultures and exposed to either GEM or C18 in the presence and absence of acidosis and the hypoxia inducer, deferoxamine. We found that C18 has higher cytotoxic and anti-invadopodia activity than GEM in all culture conditions and especially in acid and hypoxic environments. We propose C18 as a more effective approach to conventional GEM in developing new therapeutic strategies overcoming PDAC chemoresistance.

Serpine1 mRNA confers mesenchymal characteristics to the cell and promotes CD8+ T cells exclusion from colon adenocarcinomas

Serine protease inhibitor clade E member 1 (SERPINE1) inhibits extracellular matrix proteolysis and cell detachment. However, SERPINE1 expression also promotes tumor progression and plays a crucial role in metastasis. Here, we solve this apparent paradox and report that Serpine1 mRNA per se, independent of its protein-coding function, confers mesenchymal properties to the cell, promoting migration, invasiveness, and resistance to anoikis and increasing glycolytic activity by sequestering miRNAs. Expression of Serpine1 mRNA upregulates the expression of the TRA2B splicing factor without affecting its mRNA levels. Through transcriptional profiling, we found that Serpine1 mRNA expression downregulates through TRA2B the expression of genes involved in the immune response. Analysis of human colon tumor samples showed an inverse correlation between SERPINE1 mRNA expression and CD8+ T cell infiltration, unveiling the potential value of SERPINE1 mRNA as a promising therapeutic target for colon tumors.

Proteolytic remodeling of 3D bioprinted tumor microenvironments

In native tissue, remodeling of the pericellular space is essential for cellular activities and is mediated by tightly regulated proteases. Protease activity is dysregulated in many diseases, including many forms of cancer. Increased proteolytic activity is directly linked to tumor invasion into stroma, metastasis, and angiogenesis as well as all other hallmarks of cancer. Here we show a strategy for 3D bioprinting of breast cancer models using well-defined protease degradable hydrogels that can facilitate exploration of the multifaceted roles of proteolytic extracellular matrix remodeling in tumor progression. We designed a set of bicyclo[6.1.0]nonyne functionalized hyaluronan (HA)-based bioinks cross-linked by azide-modified poly(ethylene glycol) (PEG) or matrix metalloproteinase (MMP) degradable azide-functionalized peptides. Bioprinted structures combining PEG and peptide-based hydrogels were proteolytically degraded with spatial selectivity, leaving non-degradable features intact. Bioprinting of tumor-mimicking microenvironments using bioinks comprising human breast cancer cells (MCF-7) and fibroblast in hydrogels with different susceptibilities to proteolytic degradation shows that MCF-7 proliferation and spheroid size were significantly increased in protease degradable hydrogel compartments, but only in the presence of fibroblasts. In the absence of fibroblasts in the stromal compartment, cancer cell proliferation was reduced and did not differ between degradable and nondegradable hydrogels. The interactions between spatially separated fibroblasts and MCF-7 cells consequently resulted in protease-mediated remodeling of the bioprinted structures and a significant increase in cancer cell spheroid size, highlighting the close interplay between cancer cells and stromal cells in the tumor microenvironment and the influence of proteases in tumor progression.

ADAMTS Proteases: Their Multifaceted Role in the Regulation of Cancer Metastasis.

Cancer leads to nearly 10 million deaths worldwide per year. The tumour microenvironment (TME) is fundamental for tumour growth and progression. A key component of the TME, the extracellular matrix (ECM) has recently become a focus of interest in cancer research. Dysregulation of ECM synthesis and proteolysis leads to uncontrolled tumour growth and metastasis. Matrix remodelling enzymes, secreted by cancer cells and stromal cells, modify the overall structure and organisation of ECM proteins, therefore influencing biochemical interactions, tissue integrity and tissue turnover. While A Disintegrin and Metalloproteinases (ADAMs)' and matrix metalloproteinases' role in cancer has been deeply investigated, other proteolytic enzymes, like ADAMs with thrombospondin(-like) motifs (ADAMTSs) have been gaining interest due to their roles in modulating cancer cell-ECM interactions and oncogenic signalling pathways. In this review, we will discuss the dysregulation of ADAMTSs in cancer and their roles in regulating cancer development and progression, via ECM remodelling and cell signalling modulation.

Expression, intracellular localization and maturation of cysteine cathepsins in renal embryonic and cancer cell lines

Cysteine cathepsins play an important role in tumor development and metastasis. The expression of these enzymes is often increased in many types of tumor cells. Cysteine cathepsins contribute to carcinogenesis through a number of mechanisms, including proteolysis of extracellular matrix and signaling molecules on the cell surface, as well as degradation of transcription factors and disruption of signaling cascades in the cell nucleus. Distinct oncogenic functions have been reported for several members of the cysteine cathepsin family in various types of cancer, but a comparative study of all eleven cysteine cathepsins in one experimental model is still missing. In this work, we assessed and compared the expression, localization, and maturation of all eleven cysteine cathepsins in embryonic kidney cells HEK293 and kidney cancer cell lines 769-P and A-498. We found that the expression of cathepsins V, B, Z, L, and S was 3- to 9-fold higher in kidney tumor cells than in embryonic cells. We also showed that all cysteine cathepsins were present in varying amounts in the nucleus of both embryonic and tumor cells. Notably, more than half of the cathepsin Z or K and over 88% of cathepsin F were localized in tumor cell nuclei. Moreover, mature forms of cysteine cathepsins were more prevalent in tumor cells than in embryonic cells. These results can be further used to develop novel diagnostic tools and may assist in the investigation of cysteine cathepsins as potential therapeutic targets.

In vitro dose-dependent effects of matrix metalloproteinases on ECM hydrogel biodegradation

Matrix metalloproteinases (MMPs) cause proteolysis of extracellular matrix (ECM) in tissues affected by stroke. However, little is known about how MMPs degrade ECM hydrogels implanted into stroke cavities to regenerate lost tissue. To establish a structure-function relationship between different doses of individual MMPs and isolate their effects in a controlled setting, an in vitro degradation assay quantified retained urinary bladder matrix (UBM) hydrogel mass as a measure of degradation across time. A rheological characterization indicated that lower ECM concentrations (<4 mg/mL) did not cure completely at 37 °C and had a high fraction of mobile proteins that were easily washed-out. Hydrolysis by dH2O caused a steady 2 % daily decrease in hydrogel mass over 14 days. An acceleration of degradation to 6 % occurred with phosphate buffered saline and artificial cerebrospinal fluid. MMPs induced a dose-dependent increase and within 14 days almost completely (>95 %) degraded the hydrogel. MMP-9 exerted the most significant biodegradation, compared to MMP-3 and -2. To model the in vivo exposure of hydrogel to MMPs, mixtures of MMP-2, -3, and -9, present in the cavity at 14-, 28-, or 90-days post-stroke, revealed that 14- and 28-days mixtures achieved an equivalent biodegradation, but a 90-days mixture exhibited a slower degradation. These results revealed that hydrolysis, in addition to proteolysis, exerts a major influence on the degradation of hydrogels. Understanding the mechanisms of ECM hydrogel biodegradation is essential to determine the therapeutic window for bioscaffold implantation after a stroke, and they are also key to determine optimal degradation kinetics to support tissue regeneration. Statement of significanceAfter implantation into a stroke cavity, extracellular matrix (ECM) hydrogel promotes tissue regeneration through the degradation of the bioscaffold. However, the process of degradation of an ECM hydrogel remains poorly understood. We here demonstrated in vitro under highly controlled conditions that hydrogel degradation is very dependent on its protein concentration. Lower protein concentration hydrogels were weaker in rheological measurements and particularly susceptible to hydrolysis. The proteolytic degradation of tissue ECM after a stroke is caused by matrix metalloproteinases (MMPs). A dose-dependent MMP-driven biodegradation of ECM hydrogel exceeded the effects of hydrolysis. These results highlight the importance of in vitro testing of putative causes of degradation to gain a better understanding of how these factors affect in vivo biodegradation.

Expression, Intracellular Localization, and Maturation of Cysteine Cathepsins in Renal Embryonic and Cancer Cell Lines

Cysteine cathepsins play an important role in tumor development and metastasis. The expression of these enzymes is often increased in many types of tumor cells. Cysteine cathepsins contribute to carcinogenesis through a number of mechanisms, including proteolysis of extracellular matrix and signaling molecules on the cell surface, as well as degradation of transcription factors and disruption of signaling cascades in the cell nucleus. Distinct oncogenic functions have been reported for several members of the cysteine cathepsin family in various types of cancer, but a comparative study of all eleven cysteine cathepsins in one experimental model is still missing. In this work, we assessed and compared the expression, localization, and maturation of all eleven cysteine cathepsins in embryonic kidney cells HEK293 and kidney cancer cell lines 769-P and A-498. We found that the expression of cathepsins V, B, Z, L, and S was 3- to 9-fold higher in kidney tumor cells than in embryonic cells. We also showed that all cysteine cathepsins were present in varying amounts in the nucleus of both embryonic and tumor cells. Notably, more than half of the cathepsin Z or K and over 88% of cathepsin F were localized in tumor cell nuclei. Moreover, mature forms of cysteine cathepsins were more prevalent in tumor cells than in embryonic cells. These results can be further used to develop novel diagnostic tools and may assist in the investigation of cysteine cathepsins as potential therapeutic targets.

Degradomic Identification of Membrane Type 1-Matrix Metalloproteinase as an ADAMTS9 and ADAMTS20 Substrate

The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.

Crataegus monogyna and Centella asiatica Extracts as Inhibitors of Cathepsin S

Cathepsin cysteine proteases such as cathepsin S are responsible for the unfavorable and irreversible dysregulation of extracellular matrix (ECM) proteolysis found in cardiomyopathy, heart valve diseases and atherosclerosis. Cathepsin S plays a central role in ECM remodeling, which has been implicated in the development and progression of cardiovascular diseases. The current study aimed to evaluate the aqueous and ethanol extracts of the berries of Crataegus monogyna and the leaves of Centella asiatica for inhibition of cathepsin S activity using an in vitro fluorescent assay. Dried and ground berries of Crataegus monogyna and the dried and ground leaves of Centella asiatica were used to prepare the aqueous and organic extracts. A fluorometric enzyme assay was used to evaluate the inhibition of cathepsin S by the plant extracts. The aqueous extracts of Crataegus monogyna and Centella asiatica produced a maximum enzyme inhibition of 6% and 9.6% respectively, at a maximum of 200 μg/ml. The organic extract of Centella asiatica produced a maximum inhibition of 14%, occurring with the 100 μg/ml concentration. The ethanol extract of Crataegus monogyna produced a concentration-dependent inhibition of cathepsin S enzyme activity for all concentrations, with a maximum inhibition of 71.7% obtained with the highest concentration of 200 μg/ml. This demonstrates that the organic extract of Crataegus monogyna plays a crucial role in the inhibition of cathepsin S in vitro and could serve as a viable source for the isolation of cathepsin S inhibitors.

The Repertoire of Tissue Inhibitors of Metalloproteases: Evolution, Regulation of Extracellular Matrix Proteolysis, Engineering and Therapeutic Challenges.

Tissue inhibitors of metalloproteases (TIMPs) belong to a fascinating protein family expressed in all Metazoa. They act as regulators of the turnover of the extracellular matrix, and they are consistently involved in essential processes. Herein, we recapitulate the main activities of mammalian TIMPs (TIMP1–4) in the control of extracellular-matrix degradation and pathologies associated with aberrant proteostasis. We delineate the activity of TIMPs in the control of extracellular matrix (ECM) homeostasis and discuss the diversity of TIMPs across metazoans taking into account the emergence of the components of the ECM during evolution. Thus, the TIMP repertoire herein analysed includes the homologues from cnidarians, which are coeval with the origins of ECM components; protostomes (molluscs, arthropods and nematodes); and deuterostomes (echinoderms and vertebrates). Several questions, including the maintenance of the structure despite low sequence similarity and the strategies for TIMP engineering, shed light on the possibility to use recombinant TIMPs integrating unique features and binding selectivity for therapeutic applications in the treatment of inflammatory pathologies.

P-523 rs2277698 Single nucleotides polymorphisms in tissue inhibitors of metalloproteinases 2 (TIMP2) affected clinical outcomes of women undergoing in vitro fertilization

Abstract Study question Is there any association between clinical outcomes of women undergoing in vitro fertilization (IVF) and the polymorphic variants of matrix metalloproteinases (MMPs) or tissue inhibitors of metalloproteinases (TIMPs)? Summary answer Our findings suggested that clinical outcomes of women undergoing IVF were associated with rs2277698 SNPs in TIMP2. What is known already The extracellular matrix proteolytic machinery is one of the most important mechanisms for regulating trophoblast invasion. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), which are involved in the extracellular matrix proteolysis process, have the ability to regulate the degradation of the extracellular matrix and play a critical role in trophoblast invasion. Study design, size, duration A prospective study was composed of 1014 women undergoing their first fresh IVF cycle with non-donor from January 2014 to December 2015. Participants/materials, setting, methods The present study was carried out in Lee Women’s Hospital, Taiwan. DNA was extracted from the peripheral blood of all participants, and the SNPs were genotyped by real-time polymerase chain reaction. The effects of the following three single nucleotide polymorphisms (SNPs) on IVF outcomes were explored: TIMP1 (rs4898 C/T), TIMP2 (rs2277698 C/T) and MMP2 (rs243865 C/T). The SNP genotype, correlation with clinical pregnancy, embryo implantation, abortion and live birth rates of IVF were analyzed. Main results and the role of chance In the analysis of 1014 patients attempting their first cycle of IVF, the genotypes frequencies of the study population showed a statistically significant association between TIMP2 (rs2277698 C/T) and clinical outcome. For TIMP2 polymorphisms, women with wild genotype (CC) hadhigher clinical pregnancy rate (34.8% v.s 28.0%; p = 0.032), embryo implantation rate (19.4% v.s 15.4%; p = 0.009), live birth rate (29.8% v.s 22.1%; p = 0.006), and lower abortion rate (7.0% v.s 17.1%; p = 0.005) compared women with CT/TT genotype. On the other hand, the clinical outcomes in different TIMP1 or MMP2 gene polymorphisms groups were no statistically significant difference. Limitations, reasons for caution The size number of the study samples. Wider implications of the findings This study showed that the TIMP2 SNP polymorphisms CT/TT was associated with low trend of clinical outcomes. The mechanism of TIMP2 gene T allele affected the outcomes of IVF remains to be determined. Further studies should focus on the mechanism of these associations in a larger, more heterogeneous cohort. Trial registration number CS13194

PROTEOMIC PROFILE OF CYSTEINE OXIDATION IN VASCULAR SMOOTH MUSCLE CELLS IN HUMAN HYPERTENSION

Objective: Phenotypic switching of vascular smooth muscle cells (VSMCs) is a fundamental process in vascular dysfunction and remodeling associated with hypertension. Mechanisms underlying this process involve reactive oxygen species (ROS) and redox-dependent signaling pathways. However, the proteomic signature and effects of ROS on VSMC proteome in human hypertension are unknown. Using high fidelity proteomic analysis, we characterized the proteome of VSMC in human hypertension. Design and method: VSMC from resistance arteries from normotensive (NT) and hypertensive (HT) subjects were studied. Protein expression and cell migration were assessed by immunoblotting and wound healing assay respectively. VSMC proteins were labelled with isobaric tandem mass tags and identified by liquid chromatography tandem mass spectrometry. The oxidative proteome was assessed using stable isotope-labelled iodoacetamide to target cysteine thiols. Results: VSMCs from HT subjects exhibit reduced expression of alpha-SMA (0.05+0.01 vs NT:0.20 + 0.03, p &lt; 0.05), increased expression of the proliferation marker, PCNA (0.162 + 0.3 vs NT:051 + 0.004, p &lt; 0.05) and increased migration (54.68 + 2.86 vs NT:23.37 + 8.36, p &lt; 0.05). The proteomic analysis identified 207 proteins upregulated in HT subjects (fold change&gt; 1.5, p &lt; 0.05). Gene ontology enrichment of upregulated proteins in HT showed that most proteins belong to extracellular space and plasma membrane compartments and were involved in biological processes such as extracellular matrix organization, immune response, proteolysis and cell proliferation. Extracellular matrix (ECM) proteins COL1A1, COL9A1, COL10A1, FBN1, FBLN1 were increased in cells from HT (fold change&gt; 1.5, p &lt; 0.05), suggesting a switch to a fibroblast-like phenotype in hypertension. Expression of proteins related to the interferon and IL-1beta pathways (IFIT1, IFIT2, IFIT3, MX1, MX2, ABCA1, ABCA2, IL1RAP, CD36, ICAM1) were also increased in cells from HT subjects (fold change&gt; 1.5, p &lt; 0.05). Considering the importance of oxidative stress in hypertension, we assessed the VSMC oxidative proteome. Results identified 130 significant cysteine-containing peptides, 88 showed increased oxidation in HT (fold change&gt; 1.5, p &lt; 0.05). Among the highly oxidized proteins in HT were ECM proteins, COL11A1, COL16A1, FBLN1 and FBLN2 (fold change&gt; 1.5, p &lt; 0.05). Conclusions: Our study provides new insights into the proteomic changes related to vascular phenotype in hypertension and highlights differentially oxidised candidate targets that may drive phenotypic switching associated with vascular injury and remodeling in hypertension.

The Proteolysis of ECM in Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration (IDD) is a pathological process that commonly occurs throughout the human life span and is a major cause of lower back pain. Better elucidation of the molecular mechanisms involved in disc degeneration could provide a theoretical basis for the development of lumbar disc intervention strategies. In recent years, extracellular matrix (ECM) homeostasis has received much attention due to its relevance to the mechanical properties of IVDs. ECM proteolysis mediated by a variety of proteases is involved in the pathological process of disc degeneration. Here, we discuss in detail the relationship between the IVD as well as the ECM and the role of ECM proteolysis in the degenerative process of the IVD. Targeting ECM proteolysis-associated proteases may be an effective means of intervention in IDD.

Downregulation of metallothionein 2A reduces migration, invasion and proliferation activities in human squamous cell carcinoma cells.

The invasive behaviour of squamous cell carcinoma (SCC), a common malignant tumour of the mouth, is a process mediated by cell proliferation, extracellular matrix proteolysis and other factors. Studies have shown a potential relationship between growth factors, metallothionein 2A (MT2A) and matrix metalloproteinase (MMP) activation in malignant tumours. The aim of this study was to downregulate MT2A in cells (Cal27) derived from human squamous cell carcinoma. Cal27 cells with reduced MT2A were subjected to proliferation, migration and invasion assays. Immunofluorescence and western blot confirmed MT2A depletion by siRNA. Growth curve assays assessed cell proliferation. Indirect immunofluorescence analysed the expression of MT2A, MMP-2, MMP-9, epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), tumour necrosis factor alpha (TNF-α) and Ki67. Zymography evaluated the effects of MT2A silencing on MMP-2 and -9 expression. Migration and invasion activities were evaluated using migration and invasion assays. CAL27 cells displayed MT2A, MMP-2, MMP-9, EGF, TGF-α, TNF-α and Ki67. MT2A depletion decreased MMP-9, EGF, TGF-α and Ki67 protein levels, while increasing TNF-α. MT2A downregulation reduced cell proliferation, migration and invasion activities. Therefore, MT2A has an important role in cell proliferation, migration and invasion in human oral SCC cells.

The ameliorating effect of Terminalia muelleri extract on oxidative stress–related factors in induced hepatocellular carcinoma rat model

The present study investigates the efficacy of Terminalia muelleri ethanol extract (TME) in alleviating the oxidative stress and inflammation that could be the main player of hepatocellular carcinoma (HCC) development and progression. Forty-eight male albino rats were divided into two main groups (32 rats for DENA administration; 16 rats for control groups); after the induction period of DENA administration (6 weeks), the HCC-induced rats were treated with TME, DOX, or their combination for eight weeks. The obtained results revealed that the DENA induced HCC resulted in remarkable dysregulation in the circulatory levels of TNF-α and IL-10 that accompanied by the initiation of free radicals with the generation of the oxidative stress, in addition to an increase in the mRNA expression levels of NFkB, MMP 9 and downregulation of mRNA expression level of TIMP1. The treatment of HCC induced rats with DOX, TME, or their combination alleviated and suppressed the progression of HCC in the following manner TME + DOX followed by TME then DOX. The obtained results revealed that TME was more valuable as an anti-cancer agent than DOX, employed in the current study as reference chemotherapy. T. muelleri extract attenuates inflammation, oxidative stress, and extracellular matrix proteolysis in hepatocellular carcinoma-induced rats.