Matrix Metalloproteinase-9 Protein Research Articles

Matrix Metalloproteinase 9 (MMP9) Inhibitor Prevents Mustard Induced Microvesication and Promotes Cutaneous Wound Healing

Skin exposure to the chemical warfare agent, sulfur mustard (bis‐2‐chloroethylsulfide, SM), is characterized by severe blister formation and a prolonged inflammatory response. The mechanisms of these SM toxic effects are still poorly defined, and effective medical countermeasures against SM‐induced cutaneous injury have yet to be established. Animal models demonstrate disruption of the skin architecture at the dermal‐epidermal junction (DEJ) when exposed to the vesicant, SM; the damage may be further exacerbated by overexpression of MMP9. Studies show MMP9 degrades basement membrane (BM) components and regulates inflammation in tissue injuries; inhibition of MMP9 suppresses inflammation and promotes wound repairs in tumors and various wound models. In the present studies, ([N‐hydroxy‐3‐phenyl‐2‐(4‐phenylbenzenesulfonamido) propanamide], BiPS), a specific MMP9 inhibitor, was evaluated for its ability to prevent microvesication, suppress inflammation, and promote wound healing following SM exposure using the mouse ear vesicant model. Treatment of male CD1 mouse ear skin with SM (0.08 mg) caused a characteristic SM induced injury including edema, inflammatory cell infiltration, and the formation of microvesicles at the DEJ. Expression of MMP9 mRNA and protein in the skin progressively increased with time 24–168 h following SM exposure. Immunofluorescence studies showed disruption of the BM molecule type IV collagen (COL4), increased expression of inflammation marker, COX2, and upregulation of the skin wound marker keratin 6 (K6) in SM induced skin wounds. SM also caused macrophage (F4/80) accumulation in the tissue which persisted up to 168 h post exposure. Pretreatment of ear skin with BiPS significantly reduced SM‐induced dermal edema and maintained the integrity of the DEJ‐BM as evidenced by contiguous expression of COL4. At 168 h post SM exposure, mRNA and protein expression of MMP9 was significantly downregulated in BiPS treated mice. BiPS treatment also reduced the number of macrophages accumulating in the tissue, and suppressed expression of COX2, and downregulated expression of K6. Taken together, these data indicate that BiPS treatment was able to suppress both inflammation and degradation of BM zone in skin due to SM. This compound was effective in targeting MMP9 to protect against SM induced microvesication and promote wound repairs. MMP9 inhibitors may be useful as medical countermeasure against SM‐induced cutaneous injury.Support or Funding InformationSupported by NIH grants ES005022 and AR055073.

Regulation of the Phenotypic Switch of Vascular Smooth Muscle Cells by Adiponectin

Vascular smooth muscle cells (VSMCs) play an essential role in the development and progression of atherosclerosis. The phenotypic switch from contractile to synthetic cells induces excess proliferation and migration of VSMCs into the intima contributing to plaque formation. Adiponectin (Adpn) is an adipokine that regulates glucose metabolism through AMPK and plays beneficial roles in atherosclerosis. We hypothesize that Adpn promotes the contractile phenotype of VSMC reducing proliferation and migration of VSMCs, a process that is protective against plaque formation.Differentiation, proliferation, and signaling through AMPK‐dependent pathways were assessed in VSMCs isolated from aortas of wild type (WT) and Adpn−/− mice. Proliferation markers, including cyclin D1, were increased in Adpn−/− cells, and AMPK activity and differentiation markers were reduced. Cells were analyzed for matrix metalloproteinases (MMPs) which are responsible for extracellular matrix (ECM) remodeling, a contributing factor to the VSMC phenotypic switch. We found that MMP protein expression and activity, measured by zymography, were reduced in Adpn−/− cells. To test whether the phenotypic switch is regulated by AMPK, we treated cells with Metformin, an AMPK activator, or AdipoRon, an activator of Adpn receptors. Metformin and AdipoRon reduced Cyclin D1 and MAPK signaling in Adpn−/− cells, suggesting that AMPK prevents VSMC proliferation; however, none of the treatments yielded an effect on markers of differentiation.These data illustrated that Adpn regulates proliferation through an AMPK‐dependent signaling pathway, while regulating differentiation by an AMPK‐independent mechanism. The role of MMPs in VSMC differentiation in response to Adpn remains to be elucidated.

Curcumin attenuates endothelial cell fibrosis through inhibiting endothelial-interstitial transformation.

Curcumin (Cur) has various pharmacological activities, including anti‐inflammatory, antiapoptotic and anticancer effects. However, there is no report on the effect of Cur on endothelial cell fibrosis. This study was designed to investigate the effect and mechanism of Cur on endothelial cell fibrosis. An endothelial cell fibrosis model was established by using transforming growth factor (TGF) induction. Proliferation assays, qRT‐PCR, western blotting and immunostaining were performed to investigate the effects and mechanism of Cur on endothelial cell fibrosis. We found that in human umbilical vein endothelial cells (HUVECs), TGF‐β1 treatment significantly decreased the expression of nuclear factor erythroid‐2‐related factor 2 (NRF‐2), dimethylarginine dimethylaminohydrolase‐1 (DDAH1), and VE‐cadherin, the secretion of cellular nitric oxide (NO) and the activity of nitrous oxide synthase (NOS), while asymmetric dimethylarginine (ADMA) and the release of inflammatory factors were elevated. Immunofluorescence showed decreased CD31 and increased α‐smooth muscle actin (α‐SMA). Overexpression of NRF‐2 significantly attenuated the effects of TGF‐β1, while downregulation of DDAH1 potently counteracted the effect of NRF‐2. In addition, ADMA treatment resulted in similar results to those of TGF‐β1, and Cur significantly attenuated the effect of TGF‐β1, accompanied by increased VE‐cadherin, DDAH1 and NRF‐2 and decreased matrix metalloproteinase‐9 (MMP‐9) and extracellular regulated protein kinases 1/2 (ERK1/2) phosphorylation. The NRF‐2 inhibitor ML385 had the opposite effect as that of Cur. These results demonstrated that Cur inhibits TGF‐β1‐induced endothelial‐to‐mesenchymal transition (EndMT) by stimulating DDAH1 expression via the NRF‐2 pathway, thus attenuating endothelial cell fibrosis.

Human Serum Albumin-Based Dual-Agent Delivery Systems for Combination Therapy: Acting against Cancer Cells and Inhibiting Neovascularization in the Tumor Microenvironment

To cause tumor regression by acting against cancer cells and inhibiting neovascularization in the tumor microenvironment, we constructed human serum albumin (HSA)-based delivery systems of 2-acetylpyridine-4,4-dimethyl-3-thiosemicarbazone-copper(II) [Cu(Ap44mT)]Cl and paclitaxel to improve both the therapeutic efficacy and the targeting ability in vivo. X-ray crystallography and matrix-assisted laser desorption/ionization time-of-flight mass spectra confirmed that [Cu(Ap44mT)]Cl complexed with HSA, whereas paclitaxel was tethered to the HSA complex by a linker sensitive to the active matrix metalloproteinase 2 (MMP2) protein. Up to 78% of paclitaxel was released from HSA within 2 h owing to MMP2 protein cleavage. In addition, a large amount of Cu(Ap44mT) was released from HSA in a pH 4.7 buffer. In vivo results revealed the following: (1) the tumor inhibitory rates of the HSA conjugate and the two-agent combination were 72.1 and 50.7%, respectively; (2) the inhibition rate of tumor angiogenesis of the HSA conjugate (73.3%) was higher than that of the two-agent combination (52.4%); (3) the increased amount of Cu in the tumor treated with the HSA conjugate was about 2-fold that in the tumor treated with the two-agent combination. Obviously, the HSA conjugate not only possessed a stronger capacity to inhibit neovascularization and the growth of liver tumors but also improved the targeting ability compared to the combination of the two agents alone.

Recombinant Matrix Metalloproteinase-9 Induces Ventricular Remodeling By Up-Regulating Angiotensin II

Abstract Objective To study the effect of matrix metalloproteinase-9 (MMP-9) on ventricular remodeling and involvement of angiotensin II (Ang II). Methods Thirty-six adult male Wistar rats were randomly divided into control (n = 12), MMP-9 (n = 12), and MMP-9 + olmesartan (n = 12) groups. Recombinant MMP-9 (2.1 ng/g) was injected intraperitoneally twice a week. Olmesartan (3 mg/kg) was given by oral gavage once daily. Animals were treated for 4 or 8 weeks. Cardiac function was assessed by echocardiography followed by histological analysis. The messenger RNA (mRNA) and protein levels of MMP-9 in myocardial tissues were analyzed by reverse transcription polymerase chain reaction and Western blotting, respectively. Serum levels of Ang I, II, angiotensin converting enzyme (ACE) and MMP-9 were determined by enzyme-linked immunosorbent assay (ELISA). Results MMP9 administration for 4 or 8 weeks increased mRNA and protein levels of MMP-9 in myocardial tissues, serum levels of MMP-9, Ang I, II and ACE, and left ventricular mass index (LVMI) but decreased collagen volume fraction (CVF) compared with the control group (all P < 0.05). Cardiomyocyte cell size was significantly enlarged and disorganized with cytoplasmic lysis and necrosis in the MMP-9 group. After olmesartan treatment, myocardial MMP-9 mRNA and protein levels, serum levels of MMP-9 and Ang II, and LVMI were lower, and CVF was higher with significant improvement in myocardial morphology and cardiac function (all P<0.05). Conclusion Recombinant MMP-9 treatment induces ventricular remodeling and cardiac dysfunction by up-regulating Ang II.

The Biological Potential Hidden in Inclusion Bodies.

Inclusion bodies (IBs) are protein nanoclusters obtained during recombinant protein production processes, and several studies have demonstrated their potential as biomaterials for therapeutic protein delivery. Nevertheless, IBs have been, so far, exclusively sifted by their biological activity in vitro to be considered in further protein-based treatments in vivo. Matrix metalloproteinase-9 (MMP-9) protein, which has an important role facilitating the migration of immune cells, was used as model protein. The MMP-9 IBs were compared with their soluble counterpart and with MMP-9 encapsulated in polymeric-based micelles (PM) through ionic and covalent binding. The soluble MMP-9 and the MMP-9-ionic PM showed the highest activity values in vitro. IBs showed the lowest activity values in vitro, but the specific activity evolution in 50% bovine serum at room temperature proved that they were the most stable format. The data obtained with the use of an air-pouch mouse model showed that MMP-9 IBs presented the highest in vivo activity compared to the soluble MMP-9, which was associated only to a low and a transitory peak of activity. These results demonstrated that the in vivo performance is the addition of many parameters that did not always correlate with the in vitro behavior of the protein of interest, becoming especially relevant at evaluating the potential of IBs as a protein-based nanomaterial for therapeutic purposes.

Punicalagin inhibits the viability, migration, invasion, and EMT by regulating GOLPH3 in breast cancer cells

Breast cancer (BC) is one of the most common malignancies worldwide. Punicalagin (PN), which is a type of polyphenol, has been reported to act as a tumor suppressor. This study aimed to investigate the effects of PN on cellular process in BC and its molecular mechanism. The effects of various doses of PN on cell viability, migration, and invasion capacities of MCF-7 and MDA-MB-231 cells were detected by CCK-8, wound-healing, and Transwell assays. Golgi phosphoprotein 3 (GOLPH3) was then transfected into the cells with or without PN treatment, and GPLPH3 expression level was examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, and expressions of epithelial–mesenchymal transition (EMT)-related protein matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), E-Cadherin, and N-Cadherin were measured by Western blot. High dose of PN treatment (50 μM or higher) significantly inhibited viability, migration, and invasion of MCF-7 and MDA-MB-231 cells, while overexpressed GOLPH3 promoted cell viability, migration, and invasion, and partially reversed the effects of PN treatment on the BC cells. PN inhibited the expressions of GOLPH3, MMP-2, MMP-9, and N-Cadherin, and promoted E-Cadherin expression, while overexpression of GOLPH3 partly reversed above effects attributing to PN. Thus, PN suppresses cell viability and metastasis via regulating GOLPH3 in BC, which provides a possible therapeutic direction to the treatment of BC.

Expressions of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases 1 in skeletal muscles of aged rats with sarcopenia

To investigate the changes of skeletal muscle mass and strength and the expressions of matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinases-1 (TIMP-1) and collagen-1 in the skeletal muscle of aged rats with sarcopenia. With 11 young (6-month-old) SD rats as control group, 18 aged (25-month-old) SD rats were divided into two groups (n=9) according to the relative lean mass determined dual X-ray absorptiometry (DXA), namely aged control group and aged sarcopenia group (the relative lean mass was 2SD higher in aged control than in aged sarcopenia group. The forelimb grip strength of the rats was measured using an electronic grip strength meter. The extracellular matrix (ECM) of the rat's gastrocnemius was observed with HE staining and sirius Red staining, and the protein expressions of collagen-1, MMP-1, and TIMP-1 in the muscular tissues were detected with Western blotting. Compared with the young rats, the aged control rats had significantly lower relative grip strength (P < 0.01) and increased expressions of collagen-1 and TIMP-1 (P < 0.05) and ECM content in the skeletal muscles, but the relative lean mass and MMP-1 protein expression were comparable between the two groups (P>0.05). Compared with the aged control rats, the aged sarcopenic rats had significantly lowered relative lean mass (P < 0.01) and MMP-1 expressions of (P < 0.05) and increased expressions of collagen-1 and TIMP-1 proteins and ECM content in the muscular tissues (P < 0.05) without significant changes in the relative grip strength (P>0.05). MMP-1/TIMP-1 imbalance in the skeletal muscle during aging affects ECM metabolism and leads to increased collagen fibers, which in turn affects the skeletal muscle mass and function and contribute to the onset of sarcopenia.

Role of the p53‑TRPM1/miR‑211‑MMP9 axis in UVB‑induced human melanocyte migration and its potential in repigmentation.

Clinical studies have proven that ultraviolet B (UVB) based phototherapy can induce perifollicular and marginal repigmentation patterns in the skin of vitiligo patients. It is, however, difficult to conceive how melanocytes can easily exit from their tightly interconnected epidermal microenvironment to re-enter a different location in the skin to establish a new network with neighboring keratinocytes. While it is known that matrix metalloprotease 9 (MMP9) is involved in the degradation of the extracellular matrix in physiological or pathological processes, little is known about whether MMP9 affects melanocyte migration in vitiligo repigmentation. To investigate the effects of the p53- transient receptor potential cation channel subfamily M member 1 (TRPM1)/microRNA (miR/miRNA)-211-MMP9 axis to regulate melanocyte migration following exposure to UVB, the expression profile of MMP9 in cultured human melanocytes transfected with or without the miR-211-mimic and p53-GFP lentiviral vector, respectively were determined. Quantitative polymerase chain reaction and western blotting were used to examine p53, TRPM1 and MMP9 mRNA and protein levels in UVB-exposed and unexposed cells. The capacity of melanocytes to migrate on collagen IV substrate was estimated using a Transwell migration assay. Interestingly, the upregulation of p53 and MMP9 at the mRNA and protein levels was evident in melanocytes treated with single or repeat exposures to UVB, whereas levels of TRPM1 and miR-211 were significantly suppressed in UVB-exposed melanocytes compared with the UVB-unexposed control cells. These results indicate that the p53-TRPM1/miR-211-MMP9 axis is significantly activated in melanocytes exposed to UVB. Notably, the ability of melanocyte migration was altered by the overexpression of p53 using a lentiviral vector and by the upregulation of miR-211 using an miRNA mimic. That altered migration could be neutralized by co-treatment with GM6001 (a broad-spectrum MMP inhibitor). Overall, these results show that the MMP9-mediated migration of melanocytes is regulated by a novel mechanism driven by the p53-TRPM1/miR-211-MMP9 axis. Activation of the p53-TRPM1/miR-211-MMP9 axis potentially represents an attractive therapeutic target to improve repigmentation outcomes in vitiligo patients.

Marein protects human nucleus pulposus cells against high glucose-induced injury and extracellular matrix degradation at least partly by inhibition of ROS/NF-κB pathway

Intervertebral disc degeneration (IDD), a major cause of discogenic low back pain, is a musculoskeletal disorder involving the apoptosis of nucleus pulposus cells (NPCs) and extracellular matrix (ECM) degradation. Marein is a major active flavonoid ingredient extracted from the hypoglycemic plant Coreopsis tinctoria with several beneficial biological activities including anti-diabetic effects. Nevertheless, there are no reports concerning the effects of marein on IDD. Our study aimed to evaluate the effects of marein on high glucose (HG)-induced injury and ECM degradation in human NPCs (HNPCs). CCK-8 assay was applied to evaluate cell viability. Flow cytometry analysis, a cell death detection ELISA, and caspase-3 activity assay were used to assess apoptosis. The mRNA expression of ECM-related proteins matrix metalloproteinase (MMP)-3, MMP-13, Collagen II, and aggrecan were determined by qRT-PCR. The changes of the nuclear factor-kappa B (NF-κB) pathway were examined by western blot. Stimulation with HG significantly reduced cell viability and induced apoptosis in HNPCs. Moreover, HG exposure increased MMP-3 and MMP-13 expression and decreased Collagen II and aggrecan expression in HNPCs. Notably, marein effectively alleviated HG-induced viability reduction, apoptosis and ECM degradation in HNPCs. We also found that marein inhibited HG-induced ROS generation and NF-κB activation in HNPCs. Inhibition of NF-κB pathway reinforced HG-induced injury and ECM degradation in HNPCs. In summary, marein protected HNPCs against HG-induced injury and ECM degradation at least partly by inhibiting the ROS/NF-κB pathway.

Naringenin sensitizes lung cancer NCI-H23 cells to radiation by downregulation of akt expression and metastasis while promoting apoptosis

Pharmacognosy Magazine,2020,16,70,229-235.DOI:10.4103/pm.pm_535_19Published:August 2020Type:Original ArticleAuthors:Taranga Jyoti Baruah, Kimrose Hauneihkim, and Lakhon Kma Author(s) affiliations:Taranga Jyoti Baruah, Kimrose Hauneihkim, Lakhon Kma Department of Biochemistry, North-Eastern Hill University, Shillong, Meghalaya, India Abstract:Background: Naringenin (NGN) is a flavonoid that has shown anticancer activities, but the ability of NGN to radiosensitize cells of the non-small cell variety of lung cancer has not been looked into yet. Objectives: The objective of the study was to check for the ability of NGN to promote radiosensitization of the lung cancer cell line – NCI-H23 (H23). Materials and Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue exclusion assay, and colony-forming assay were performed to check the effect of NGN, with and without X-ray treatment, on the viability of cancer cells. Protein carbonylation, DNA fragmentation, and caspase-3 activity were determined. The levels of pAkt, Akt, matrix metalloproteinase-2 (MMP-2), RAD50, and p21 proteins were looked at by Western blotting. Messenger RNA (mRNA) levels of AKT1, CASPASE3, BAX, BCL2, and BCLXL were also checked. Results: When combined with radiation, NGN lowered the survivability of H23 cells. NGN showed a prooxidant effect while no DNA fragmentation was observed. mRNA levels of CASPASE3 and caspase-3 activity also showed an increase. pAkt, Akt, MMP-2, and p21 protein levels were lowered in the NGN-treated groups, while the RAD50 protein levels showed an increase. The mRNA levels of BCL2 and BCLXL were lowered, while BAX mRNA levels were elevated in response to NGN treatment. Conclusion: The results showed that NGN, used singularly or in combination with radiation, can lower the procancerous and prometastatic Akt, p21, and MMP-2 proteins while upregulating the apoptotic process. Keywords:Akt, Caspase-3, Matrix metalloproteinase-2, naringenin, Non-small cell lung cancer, RadiosensitizationView:PDF (1.56 MB)

Combination of serum matrix metalloproteinase-3 activity and EBV antibodies improves the diagnostic performance of nasopharyngeal carcinoma.

Objective: Nasopharyngeal carcinoma (NPC) is a malignant head and neck tumor that is highly prevalent in Southeast Asia. The two traditional NPC markers VCA-IgA (EBV viral capsid antigen) and EA-IgA (EBV early antigen) are limited in the screening and diagnosis of NPC. The purpose of present study is to evaluate the diagnostic value of matrix metalloproteinase-3 (MMP3) in NPC.Methods: The levels of 23 secretory MMPs in serum samples from 15 healthy controls and 26 NPC patients were detected by Cytokine Antibody Array 2000. Immunohistochemistry, Real-time PCR and western bolt were used to detect MMP3 mRNA and protein levels in NPC tissues and cell lines. The serum protein levels of MMP3 were further measured by ELISA in healthy control individuals (n = 200) and NPC patients (n = 206).Results: MMP3 can be expressed and secreted by both NPC and fibroblast cell lines, suggesting that the higher expression of MMP3 protein in both tumor nests and stromal of NPC tissues may be the source of circulating MMP3 in NPC patients. Furthermore, we found out both MMP3 concentration and enzymatic activity were significantly increased in the NPC group (n = 206) than the healthy control group (n = 200) (P < 0.001). However, serum MMP3 enzymatic activity, but not MMP3 concentration, was significantly associated with the progression of NPC. In addition, serum MMP3 activity was more valuable in diagnosis of NPC than its concentration (0.86 vs. 0.78, AUC), and MMP3 activity can improve the diagnosis of NPC by combining with EBV-infection biomarkers VCA-IgA and EA-IgA with a sensitivity of 91.5% and a specificity of 92.3%.Conclusions: This study suggested the combination of MMP3 activity and EBV antibodies may be a useful biomarker for screening and diagnosis of NPC.

Associations of Changes in Serum Inflammatory Factors, MMP-3, 25(OH)D and Intestinal Flora with Osteoporosis and Disease Activity in Rheumatoid Arthritis Patients.

To explore the associations of changes in serum inflammatory factors, matrix metalloproteinase-3 (MMP-3), 25-hydroxy vitamin D [25(OH)D], and intestinal flora with osteoporosis and disease activity in rheumatoid arthritis (RA) patients, so as to provide references for clinical diagnosis and treatment. A total of 98 RA patients were selected as the objects of study (RA group), and divided into active-stage group (n = 56) and remission-stage group (n = 42) according to the disease activity score (DAS28). Another 50 healthy people receiving physical examination in our hospital during the same period were selected as the control group. The changes in serum inflammatory factors, MMP-3, 25(OH)D, and intestinal flora were compared among the three groups, and the osteoporosis of the subjects was analyzed in each group. Moreover, the associations of changes in serum inflammatory factors, MMP-3, 25(OH)D, and intestinal flora with osteoporosis and disease activity in RA patients were analyzed using the Pearson's method. Compared with those in the control group, the levels of serum MMP-3, interleukin-6 (IL-6), IL-10, and C-reactive protein (CRP). The Escherichia coli count were significantly increased, while the level of serum 25(OH)D, bone mineral density (BMD), and Lactobacillus and Bifidobacterium counts were significantly decreased in the active-stage group and remission-stage group, more obviously in active-stage group (p < 0.05). The osteoporosis and disease activity in RA patients were positively correlated with serum IL-6, IL-10, CRP, MMP-3, Escherichia coli and BMD, but negatively correlated with 25(OH)D, Lactobacillus and Bifidobacterium (p < 0.05), and not correlated with the sharp score (p > 0.05). There are certain associations of changes in serum inflammatory factors, MMP-3, 25(OH)D, and in-testinal flora with osteoporosis and disease activity in RA patients, showing certain value in clinical application.

Network Pharmacology-Based Approach to Revealing Biological Mechanisms of Qingkailing Injection against IschemicStroke: Focusing on Blood-Brain Barrier.

Ischemic stroke is the most common type of cerebrovascular accident worldwide. It causes long-term disability and death. Qingkailing (QKL) injection is a traditional Chinese patent medicine which has been clinically applied in the treatment of ischemic stroke for nearly thirty years. In the present study, network pharmacology combined with experimentation was used to elucidate the mechanisms of QKL. ADME screening and target prediction identified 62 active compounds and 275 targets for QKL. Topological screening of the protein-protein interaction (PPI) network was used to build a core PPI network consisting of 408 nodes and 17,830 edges. KEGG enrichment indicated that the main signaling pathway implicated in ischemic stroke involved hypoxia-inducible factor-1 (HIF-1). Experimentation showed that QKL alleviated neurological deficits, brain infraction, blood-brain barrier (BBB) leakage, and tight junction degeneration in a mouse ischemic stroke model. Two-photon laser scanning microscopy was used to evaluate BBB permeability and cerebral microvessel structure in living mice. HIF-1α, matrix metalloproteinase-9 (MMP-9), and tight junction proteins such as occludin, zonula occludins-1 (ZO-1), claudin-5, and VE-Cadherin were measured by western blotting. QKL upregulated ZO-1 and downregulated HIF-1α and MMP-9. QKL has a multiapproach, multitarget, and synergistic effect against ischemic stroke.

MicroRNA-33b inhibits liver cancer cell proliferation, migration and invasion via down-regulation of Fli-1 and MMP-2 protein expressions

Purpose: To study the influence of microRNA-33b (miR-33b) on liver cancer cell proliferation, migration and invasiveness, and the mechanism involved. Methods: MicroRNA-33b or Fli-1 overexpression plasmid was transfected into liver cancer (SMMC7721) cells. Cell proliferation, migration, and invasiveness were determined using cell counting kit 8 (CCK-8), scratch test, and Transwell invasion assay, respectively. The amounts of miR-33b and Fli-1 in liver cancer tissues, paracancerous normal tissues, and miR-33b overexpression and control groups were measured using qRT-PCR, while protein concentration of matrix metalloproteinase 2 (MMP-2) was assayed using Western blotting. Results: Fli-1 protein was markedly upregulated in liver cancerous cells, relative to paracancerous normal tissues (p < 0.05). MicroRNA-33b protein expression was also significantly upregulated in miR33b overexpression group, but the corresponding Fli-1 expression was downregulated in miR-33b overexpression group, relative to control (p < 0.05). MicroRNA-33b overexpression significantly and time-dependently inhibited SMMC7721 cell proliferation and migration, but it reduced the degree of apoptosis (p < 0.05). Liver cancer (SMMC7721) cells in miR-33b overexpression group were less invasive than the control group (p < 0.05). Similarly, miR-33b overexpression significantly downregulated MMP-2 protein expression in SMMC7721cells (p < 0.05). Conclusion: Overexpression of miR-33b suppresses the proliferation, migratory and invasive potential of hepatic cancer cells via down-regulation of Fli-1 and MMP-2 protein expression. This finding may be useful in the identification of new liver cancer drugs.

Role of serum matrix metalloproteinase in the diagnosis of gastric cancer.

Objective:To determine the clinical value of a matrix metalloproteinase (MMP) antibody array in diagnosing gastric cancer (GC).Methods:In this prospective study, serum samples of patients with GC (n=66) and non-neoplastic gastric disease (NGD; n=34) were collected between November 2017 and July 2018. The quantitative measurement of 10 MMP-related proteins was done using MMP arrays and compared between the two groups.Results:The serum levels of MMPs 3, 8, 9 and tissue inhibitor of metalloproteinases (TIMPs) 1 and 2 were significantly higher in the GC group than in the NGD group (p<0.05). The area under curve (AUC) of the 10 MMP proteins for the diagnosis of GC varied between 0.500 and 0.658. The total AUC of all MMPs was 0.897 (95% CI: 0.837-0.957). The total AUC of the five MMPs (MMPs 3, 8, 9, and TIMPs 1 and 2) was 0.821 (95% CI: 0.733-0.909) for diagnosing GC. Also, the 10-factor and 5-factor predictive models had good diagnostic ability for early GC with an AUC of 0.865 (95% CI: 0.753-0.977) and 0.749 (95% CI: 0.600-0.898), respectively.Conclusions:The detection of multiple serum MMPs with protein biochip technology is promising to be used as a novel non-invasive tool for facilitating early diagnosis or screening of GC.

Combination of tanshinone IIA and astragaloside IV attenuate atherosclerotic plaque vulnerability in ApoE(-/-) mice by activating PI3K/AKT signaling and suppressing TRL4/NF-κB signaling

Tanshinone IIA (TS IIA) and Astragaloside IV (AS IV) are natural herbal products which exert anti-inflammatory and anti-oxidant effects in order to eliminate unstable plaque in atherosclerosis. However, the combined effect of these two drugs on atherosclerotic plaque vulnerability and its molecular mechanism remains unclear. In the current study, we evaluate the effects of TS IIA and AS IV on atherosclerotic unstable plaque stability, and then further explore the mechanism of TS IIA and AS IV intervention on unstable plaque in vivo and in vitro. Histological characterization of atherosclerotic plaques was measured by Hematoxylin-Eosin (HE), Masson’s Trichrome and Oil Red O staining. Cellular lipid droplet was measured by Oil Red O staining. The size of atherosclerotic lesion areas and content of lipids and collagen in the right common carotid arteries of apoE-/- mice were examined by Hematoxylin-Eosin (HE), Oil-red O, and Masson staining, respectively. The protein expression levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α and C-reactive protein (CRP) in ApoE−/− mice and RAW264.7 cells were determined by enzyme-linked immunosorbent assay. The protein expression levels of matrix metalloproteinase-9 (MMP-9) and endothelial nitric oxide synthase (eNOS) in ApoE−/− mice and RAW264.7 cells were determined by western blotting. In addition, the PI3K/AKT and TRL4/NF-κB signaling were determined by western blotting. Our results revealed that the combination of TS IIA and AS IV significantly decreased lipid areas, increased collagen content and thickened fibrous cap in the right common carotid arteries compared with ApoE (-/-) mice model group. TS IIA and AS IV visibly reduced the cytoplasmic lipid droplet accumulation induced by oxLDL in RAW 264.7 macrophages. The ApoE−/− mice model group and oxLDL -stimulated RAW 264.7 macrophages treated with TS IIA and AS IV showed a downregulation in IL-6, MMP-9, TNF-α and CRP protein expression and upregulation in eNOS protein expression. Furthermore, TSIIA and AS IV may activate PI3K/AKT signaling and suppress TLR4/NF-κB signaling in vivo and in vitro. Additionally, blocking the PI3K/Akt signaling enhanced the translocation of NF-κB to the nucleus, TLR4, IL-6, MMP-9, TNF-α and CRP expression and inhibited eNOS expression in TS IIA and AS IV-treated RAW 264.7 macrophages. Therefore, the present study implicates that TS IIA and AS IV reinforces plaque stability via PI3K/AKT and TLR4/NF-κB signaling. TS IIA and AS IV administration may provide the basis for a potential therapeutic approach for the inhibition of vulnerable atherosclerotic plaques.

MicroRNA‑15a‑5p induces pulmonary artery smooth muscle cell apoptosis in a pulmonary arterial hypertension model via the VEGF/p38/MMP‑2 signaling pathway.

The aim of the present study was to investigate the role of microRNA-15a-5p (miR-15a-5p) in pulmonary arterial hypertension (PAH) and elucidate the underlying pro-apoptotic mechanism. Reverse transcription-quantitative PCR analysis and gene microarray hybridization were used to measure the expression of miR-15a-5p in the lung tissues of rats with monocrotaline (MCT)-induced PAH. Flow cytometry and caspase-3/9 activity assays were adopted to measure the apoptosis of pulmonary artery smooth muscle cells (PASMCs). The expression of apoptosis-related proteins was analyzed using western blotting. The results demonstrated that the expression of miR-15a-5p was significantly increased in the lung tissues of rats with MCT-induced PAH. In addition, the overexpression of miR-15a-5p reduced PASMC proliferation, induced apoptosis, promoted the activity of caspase-3/9, induced the protein expression of B-cell lymphoma 2-associated X protein (Bax), decreased the expression of B-cell lymphoma 2 (Bcl-2), increased inflammation, as indicated by the expression of tumor necrosis factor-α (TNF)-α and interleukin (IL)-1β, IL-6 and IL-18, suppressed the protein expression of vascular endothelial growth factor (VEGF), and promoted the protein expression levels of phosphorylated (p)-p38 mitogen-activated protein kinase (p38) and matrix metalloproteinase (MMP)-2 in the PASMCs of rats with MCT-induced PAH. By contrast, the downregulation of miR-15a-5p increased cell proliferation, decreased apoptosis, reduced the activity of caspase-3/9 and the protein expression of Bax, increased the expression of Bcl-2, inhibited inflammation (as suggested by the expression of TNF-α, IL-1β, IL-6 and IL-18), induced the protein expression of VEGF, and suppressed the protein expression of p-p38 and MMP-2 in the PASMCs of rats with MCT-induced PAH. The inhibition of VEGF attenuated the effects of the overexpression of miR-15a-5p on the inhibition of cell proliferation, apoptotic rate, caspase-3/9 activity and protein expression of Bax, and it attenuated the increased inflammation, as indicated by the protein expression of p38 and MMP-2 in the PASMCs. In conclusion, the data of the present study demonstrated that miR-15a-5p induced the apoptosis of PASMCs in an animal model of PAH via the VEGF/p38/MMP-2 signaling pathway. However, further research is required to fully elucidate the role of miR-15a-5p in the development of PAH.

An immuno-MALDI mass spectrometry assay for the oral cancer biomarker, matrix metalloproteinase-1, in dried saliva spot samples

Oral cavity cancer is a common cancer type that presents an increasingly serious global problem. Oral squamous cell carcinoma (OSCC) accounts for >90% oral cancer cases. No biomarker tests are currently available for management of this cancer type in clinical practice. Previously, we validated matrix metalloproteinase-1 (MMP1) as one of the most promising salivary biomarkers for OSCC detection. Development of a convenient, rapid and high-throughput assay should further facilitate application of salivary MMP1 measurement for early detection of OSCC. The present study aimed to develop a workflow comprising dry saliva spot (DSS) sampling and immunoenrichment-coupled MALDI-TOF MS (immuno-MALDI) analysis to quantify salivary MMP1. We generated recombinant MMP1 protein and anti-peptide antibodies against MMP1, which were used to optimize the procedures of the entire workflow, including DSS sampling, on-paper protein digestion and elution, KingFisher magnetic particle processor-assisted immuno-enrichment and MALDI-TOF MS analysis. The established workflow was applied to measure salivary MMP1 levels in DSS samples from 5 healthy donors and 9 OSCC cases. The newly developed workflow showed good precision (intra-day and inter-day variations <10%) and accuracy (80–100%) in quantification of MMP1 in DSS samples, with the limit of quantification at 3.07 ng/ml. Using this assay, we successfully detected elevated salivary MMP1 levels (ranging from 5.95 to 242.52 ng/ml) in 7 of 9 OSCC cases while MMP1 was not detectable in samples from the 5 healthy donors. In comparison, the traditional immunoassay was not effective in measuring MMP1 in DSS samples, highlighting the significant advantage of our immuno-MALDI assay. The DSS sampling format confers high flexibility and convenience of collection, storage and delivery of saliva specimens and the KingFisher-assisted immuno-MALDI analysis renders the assay as suitable for high-throughput screening. By combining the two features, the workflow developed in this study should facilitate improvement of molecular diagnostic tests for OSCC using salivary MMP1 as a biomarker.

MMP3 Is a Non-invasive Biomarker of Rejection in Skin-Bearing Vascularized Composite Allotransplantation: A Multicenter Validation Study.

Background: There is unmet need for non-invasive immunomonitoring to improve diagnosis and treatment of acute rejection in vascularized composite allotransplantation (VCA). Circulating matrix metalloproteinase 3 (MMP3) was described as a candidate non-invasive biomarker to predict treatment response to acute rejection in clinical VCA. However, larger validation studies are yet to be reported to allow for more definitive conclusions.Methods: We retrospectively measured MMP3 levels using ELISA in a total of 140 longitudinal serum samples from six internal and three external face transplant recipients, as well as three internal and seven external upper extremity transplant recipients. The control groups comprised serum samples from 36 kidney transplant recipients, 14 healthy controls, and 38 patients with autoimmune skin disease. A linear mixed model was used to study the effect of rejection state (pre-transplant, no-rejection, non-severe rejection (NSR), and severe rejection) on MMP3 levels.Results: In VCA, MMP3 levels increased significantly (p < 0.001) between pre- and post-transplant no-rejection states. A further increase occurred during severe rejection (p < 0.001), while there was no difference in MMP3 levels between non-severe and no-rejection episodes. A threshold of 5-fold increase from pre-transplant levels could discriminate severe from NSR with 76% sensitivity and 81% specificity (AUC = 0.79, 95% CI = 0.65–0.92, p < 0.001). In kidney transplantation, the MMP3 levels were significantly (p < 0.001) elevated during antibody-mediated rejection but not during T-cell mediated rejection (TCMR) (p = 0.547). MMP3 levels in healthy controls and autoimmune skin disease patients were comparable with either pre-transplant or no-rejection/NSR episodes of VCA patients.Conclusion: The results of this study suggest that serum MMP3 protein is a promising marker for stratifying patients according to severity of rejection, complementary to biopsy findings.