Gelatinolytic Activity Of MMP-2 Research Articles

53 - Pentoxifylline treatment decreases MMP-2 activity, oxidative stress and vascular hypertrophy in renovascular hypertension

Introduction Matrix metalloproteinases (MMP) contributes to hypertensive vascular remodeling. Angiotensin II and tumoral necrose factor (TNF)-α are involved in reactive oxygen species (ROS) formation, which is responsible for in vitro MMP-2 activation. Pentoxifylline (PTX), a non-selective inhibitor of phosphodiesterases, has been reported to possess antioxidants and anti-inflammatory effects. Thus, we hypothesized that PTX decreases vascular hypertrophy in 2-kidney and 1-Clip (2K1C) rats through reduction of TNF-α, oxidative stress and MMP-2 activity. Etanercept (ETN), an TNF-α inhibitor, was used as the positive control in this study. Methods Male 2K1C and Sham rats were treated with Vehicle, PTX (100mg/Kg/day) or ETN 0.3 mg/Kg (three times/week). Systolic blood pressure (SBP) was measured weekly by tail cuff plethysmography. Vascular hypertrohy were examined in aortas sections stained with hematoxylin/eosin. Aortic MMP-2 levels and gelatinolytic activity were determined using gelatin and in situ zymography assays, respectively. Vascular oxidative stress was evaluated using dihydroethidium (DHE). TNF-α levels were analyzed using the enzyme-linked immunosorbent assay (ELISA). ResuIts 2K1C rats exhibited high SBP (191±4 mmHg vs Sham) and significant vascular hypertrophy when compared to Sham group (p 0.05). MMP-2 levels and activity were augmented in 2K1C rats when compared to sham group (p Conclusion PTX but not Etanercept treatment, ameliorates high blood pressure, vascular remodeling, oxidative stress and elevated MMP-2 activity in 2K1C rats. Financial support: FAPESP.

Electronegative LDL induces MMP-9 and TIMP-1 release in monocytes through CD14 activation: Inhibitory effect of glycosaminoglycan sulodexide

ObjectiveElectronegative LDL (LDL(−)) is involved in atherosclerosis through the activation of the TLR4/CD14 inflammatory pathway in monocytes. Matrix metalloproteinases (MMP) and their inhibitors (tissue inhibitors of metalloproteinase [TIMP]) are also crucially involved in atherosclerosis, but their modulation by LDL(−) has never been investigated. The aim of this study was to examine the ability of LDL(−) to release MMPs and TIMPs in human monocytes and to determine whether sulodexide (SDX), a glycosaminoglycan-based drug, was able to affect their secretion. Approach and resultsNative LDL (LDL(+)) and LDL(−) separated by anion-exchange chromatography were added to THP1-CD14 monocytes in the presence or absence of SDX for 24 h. A panel of 9 MMPs and 4 TIMPs was analyzed in cell supernatants with multiplex immunoassays. The gelatinolytic activity of MMP-9 was assessed by gelatin zymography. LDL(−) stimulated the release of MMP-9 (13-fold) and TIMP-1 (4-fold) in THP1-CD14 monocytes, as well as the gelatinolytic activity of MMP-9. Co-incubation of monocytes with LDL(−) and SDX for 24 h significantly reduced both the release of MMP-9 and TIMP-1 and gelatinase activity. In THP1 cells not expressing CD14, no effect of LDL(−) on MMP-9 or TIMP-1 release was observed. The uptake of DiI-labeled LDL(−) was higher than that of DiI-LDL(+) in THP1-CD14 but not in THP1 cells. This increase was inhibited by SDX. Experiments in microtiter wells coated with SDX demonstrated a specific interaction of LDL(−) with SDX. ConclusionsLDL(−) induced the release of MMP-9 and TIMP-1 in monocytes through CD14. SDX affects the ability of LDL(−) to promote TIMP-1 and MMP-9 release by its interaction with LDL(−).

Tear biomarkers in latanoprost and bimatoprost treated eyes.

PurposeProstaglandin analogues (PGA’s) are the mainstay and first line of treatment in current glaucoma practise. Though latanoprost and bimatoprost are the most commonly used PGA’s with minimal side effects at lower concentrations like bimaotoprost 0.01%, direct comparison of their cytokine/MMP profile in tears has not been evaluated earlier. The study intends to ascribe PGA to the upregulation of MMPs, Cytokines and Chemokines mediating varied pathways to result in side effects of the drugs.MethodsTear sample collection was done from outer canthus of 30 eyes of 30 patients (primary open angle glaucoma (n = 26 and n’ = 20), normal tension glaucoma (n = 4 and n’ = 10), in latanoprost (n) 0.005% and bimatoprost (n’) 0.01% group respectively, with a mean age of 62±10.5 years) on >6 months of PGA use using Tear floTM Schirmer filter strip. Tear samples from 30 eyes of 30 cataract patients without drug treatment were used as the control. Gelatinolytic activity of MMP-9 and MMP-2 were examined by substrate gelatine zymography MMP-1 and TIMP-1 concentrations from tears samples with PGAs were evaluated by ELISA while cytokine concentration in the eluted tears was evaluated using a convenient bioplex kit assay (Milliplex MAP kit, HCYTMAG-60K-PX41, Millipore, Massachusetts, United States). The mean duration of use of PGA in both groups did not differ significantly (median 1.3 years in bimatoprost and 1.1 years in latanoprost eyes, p = 0.6).ResultsThe tear MMP-9 expression was higher in eyes receiving latanoprost while the MMP-2 expression was higher in eyes receiving bimatoprost with MMP1 protein levels being higher in the former. Latanoprost treated eyes had marginally elevated tear cytokines involved in tissue remodelling while bimatoprost eyes showed elevated cytokines regulating allergic pathways.ConclusionDifferential cytokine and MMP expression indicates differential signalling pathways mediating different cellular effects (evident as clinical and side effects) with the two drugs which can be explored further.

Ischemic-Reperfusion Injury Increases Matrix Metalloproteinases and Tissue Metalloproteinase Inhibitors in Fetal Sheep Brain

Hypoxic-ischemic brain injury is a leading cause of neurodevelopmental morbidities in preterm and full-term infants. Blood-brain barrier dysfunction represents an important component of perinatal hypoxic-ischemic brain injury. The extracellular matrix (ECM) is a vital component of the blood-brain barrier. Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) are important ECM components. They contribute to brain development, blood-brain barrier maintenance, and to regenerative and repair processes after hypoxic-ischemic brain injury. We hypothesized that ischemia at different durations of reperfusion affects the ECM protein composition of MMPs and TIMPs in the cerebral cortex of fetal sheep. Cerebral cortical samples were snap-frozen from sham control fetuses at 127 days of gestation and from fetuses after exposure to 30-min carotid occlusion and 4-, 24-, and 48-h of reperfusion. Protein expression of MMP-2, -8, -9, and -13 and TIMP-1, -2, -3, and -4 was measured by Western immunoblotting along with the gelatinolytic activity of MMP-2 and MMP-9 by zymography. The expression of MMP-8 was increased (Kruskal-Wallis, p = 0.04) in fetuses 48 h after ischemia. In contrast, changes were not observed in the protein expression of MMP-2, -9, or -13. The gelatinolytic activity of pro-MMP-2 was increased (ANOVA, p = 0.02, Tukey HSD, p = 0.05) 24 h after ischemia. TIMP-1 and -3 expression levels were also higher (TIMP-1, ANOVA, p = 0.003, Tukey HSD, p = 0.01; TIMP-3, ANOVA, p = 0.006, Tukey HSD, p = 0.01) 24 h after ischemia compared with both the sham controls and with fetuses exposed to 4 h of reperfusion. The changes in the expression of TIMP-1, -2, and -3 correlated with the changes in the MMP-8 and -13 protein expression. We speculate that regulation of MMP-8, MMP-13, and TIMPs contributes to ECM remodeling after is chemic-reperfusion injury in the fetal brain.

ΔFosB regulates rosiglitazone-induced milk fat synthesis and cell survival.

Rosiglitazone induces adipogenesis in adipocyte and regulates cell survival and differentiation in number of cell types. However, whether PPARγ regulates the synthesis of milk fat and cell survival in goat mammary gland remains unknown. Rosiglitazone strongly enhanced cellular triacylglycerol content and accumulation of lipid droplet in goat mammary epithelial cells (GMEC). Furthermore, ΔFosB decreased the expression of PPARγ at both mRNA and protein levels, and rosiglitazone-induced milk fat synthesis was abolished by ΔFosB overexpression. ΔFosB reduced milk fat synthesis and enhanced saturated fatty acid concentration. Rosiglitazone increased the number of GMEC in G0/G1 phase and inhibited cell proliferation, and these effects were improved by overexpression of ΔFosB. ΔFosB was found to promote the expression of Bcl-2 and suppress the expression of Bax, and protected GMEC from apoptosis induced by rosiglitazone. Intracellular calcium trafficking assay revealed that rosiglitazone markedly increased intracellular calcium concentration. ΔFosB protected GMEC from apoptosis induced by intracellular Ca2+ overload. ΔFosB increased MMP-9 gelatinolytic activity. SB-3CT, an MMP-9 inhibitor, suppressed the expression of Bcl-2, and increased intracellular calcium levels, and this effect was abolished by ΔFosB overexpression. SB-3CT induced GMEC apoptosis and this effect was inhibited by ΔFosB overexpression. These findings suggest that ΔFosB regulates rosiglitazone-induced milk fat synthesis and cell survival. Therefore, ΔFosB may be an important checkpoint to control milk fat synthesis and cell apoptosis.

Dual Effects of Cell Free Supernatants from Lactobacillus acidophilus and Lactobacillus rhamnosus GG in Regulation of MMP-9 by Up-Regulating TIMP-1 and Down-Regulating CD147 in PMADifferentiated THP-1 Cells.

Objective Recent studies have reported dysregulated expression of matrix metalloproteinases (MMPs), especially MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, TIMP-2), and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in activated macrophages of patients with inflammatory diseases. Therefore, MMP-2, MMP-9, and their regulators may represent a new target for treatment of inflammatory diseases. Probiotics, which are comprised of lactic acid bacteria, have the potential to modulate inflammatory responses. In this experimental study, we investigated the anti-inflammatory effects of cell-free supernatants (CFS) from Lactobacillus acidophilus (L. acidophilus) and L. rhamnosus GG (LGG) in phorbol myristate acetate (PMA)-differentiated THP-1 cells. Materials and Methods In this experimental study, PMA-differentiated THP-1 cells were treated with CFS from L. acidophilus, LGG and uninoculated bacterial growth media (as a control). The expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNAs were determined using real-time quantitative reverse transcription polymerase chain reaction (RT- PCR). The levels of cellular surface expression of CD147 were assessed by flow cytometry, and the gelatinolytic activity of MMP-2 and MMP-9 were determined by zymography. Results Our results showed that CFS from both L. acidophilus and LGG significantly inhibited the gene expression of MMP-9 (P=0.0011 and P=0.0005, respectively), increased the expression of TIMP-1 (P<0.0001), decreased the cell surface expression of CD147 (P=0.0307 and P=0.0054, respectively), and inhibited the gelatinolytic activity of MMP-9 (P=0.0003 and P<0.0001, respectively) in PMA-differentiated THP-1 cells. Although, MMP-2 expression and activity and TIMP-2 expression remained unchanged. ConclusionOur results indicate that CFS from L. acidophilus and LGG possess anti-inflammatory properties and can modulate the inflammatory response.

Expression of extracellular matrix metalloproteinase inducer and matrix metalloproteinase-2 and -9 in horses with chronic airway inflammation.

OBJECTIVE To examine whether expression of extracellular matrix metalloproteinase inducer (EMMPRIN) can be detected in equine lungs and whether it correlates with matrix metalloproteinase (MMP)-2 and -9 expression in bronchoalveolar lavage fluid (BALF) of horses with chronic inflammation of the lungs (ie, lower airway inflammation [LAI]). ANIMALS 29 horses with signs of chronic respiratory tract disease, which were classified as the LAI (n = 17) and LAI with respiratory distress (RDLAI [12]) groups, and 15 control horses. PROCEDURES BALF, tracheal aspirate, and blood samples were obtained, and EMMPRIN expression was determined from BALF cells and RBCs by use of western blotting. Activities of MMP-2 and -9 were determined with zymography. RESULTS Expression of EMMPRIN protein was identified in BALF cells of all horses. Expression of EMMPRIN protein was highest for the RDLAI group and was correlated with MMP-2 and -9 protein expression, MMP-9 gelatinolytic activity, and airway neutrophilia. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that EMMPRIN was involved in the pathophysiologic processes of asthma in horses. However, additional studies of horses and other species are warranted to elucidate the regulation of EMMPRIN expression in asthmatic lungs.

Lipopolysaccharides induce Smad2 phosphorylation through PI3K/Akt and MAPK cascades in HSC-T6 hepatic stellate cells

AimsEndotoxemia and its pro-fibrogenic signaling play a significant role in the development of hepatic fibrosis. This study investigated whether lipopolysaccharide (LPS) directly activate cultured HSC-T6 hepatic stellate cells (HSCs) through triggering Smad-dependent pro-fibrogenic signaling pathway. Main methodsDirect cell counting and assays for cell proliferation and migration were used to measure the effects of LPS on HSC behaviors. Quantitative PCR, Western blot, and gelatin zymography were used to quantify the molecular effects of LPS on expression of HSC activation markers and signaling activity. Key findingsLong-term exposure to LPS exhibited moderately stimulatory effect on HSC cell growth. A wound-healing cell migration assay showed that LPS suppressed HSC-T6 cell migration. qPCR and Western blotting detection indicated that LPS treatment induced upregulation of type I and IV collagens, α-smooth muscle actin (α-SMA), and matrix metalloproteinase-9 (MMP-9). Gelatin zymography confirmed that LPS elevated MMP-9, but not MMP-2 gelatinolytic activity. Moreover, LPS immediately stimulated Akt, EKR1/2, JNK, p38 MAPK, and Smad2 hyperphosphorylation, supporting that LPS directly triggers pro-fibrogenic Smad signaling cascade without TGF-β1 stimulation. Kinase blockade experiments demonstrated the involvement of PI3K/Akt, JNK, p38 MAPK, but not ERK1/2 signaling activation in the LPS-elicited Smad2 phosphorylation as well as the overexpression of type I collagen and α-SMA in HSC-T6 cells. SignificanceThese findings demonstrate that LPS exerts pro-fibrogenic effect through activation and transformation of HSCs. The tissue-remodeling effect of LPS may be attributable to its ability to activate non-canonical Smad pathway through PI3K/Akt and MAPK signaling cascades.

Abstract 2225: Green tea polyphenols suppress tumor growth and invasion by targeting matrix metalloproteinases, RECK and TIMP-3, in a mouse model implanted with prostate tumors

Abstract Green tea polyphenols (GTP) and its major constituent, epigallocatechin-3-gallate (EGCG) reactivate epigenetically silenced genes in cancer cells that reduces invasiveness and migration capabilities; however, the mechanisms whereby these effects occur are not well understood. RECK, a novel tumor suppressor and the tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) genes negatively regulates matrix metalloproteinases (MMPs) and inhibits tumor invasion, angiogenesis and metastasis. In the present study, we demonstrate that GTP mediate epigenetic induction of RECK and TIMP-3 thereby playing a key role in suppressing invasiveness and gelatinolytic activity of MMP-2 and MMP-9 in vivo in an orthotopic implantation model of human prostate cancer. Athymic nude mice were implanted with human prostate cancer LNCaP tumor in the ventral prostate for 2 weeks and later treated DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (AZA), histone deacetylase inhibitor, trichostatin A (TSA) and histone methyltransferase inhibitor, 3-Deazaneplanocin A (DZNep) individually at 0.1 mg/kg body weight intraperitoneally at alternate days/week; and combination of AZA+TSA and DZNep+TSA at similar doses and times; whereas GTP was provided peroral by gavage at 7.5 and 15.0 mg/kg body weight freshly prepared in 100µl PBS. Treatment of mice with GTP resulted in marked decrease in tumor growth and its local invasion in dose dependent manner, compared to treatment with epigenetic inhibitors and their combination after 8 weeks of intervention. GTP treatment significantly reduced serum levels of MMP-2, MMP-9 and VEGF, compared to treatment with epigenetic inhibitors alone. Combination of AZA+TSA exhibited similar effect which was equivalent to the lower dose of GTP treatment. Furthermore, GTP treatment significantly reduced EZH2 and H3K27me3 and class I HDAC protein levels in tumors. GTP partially reversed the hypermethylation status of RECK and TIMP3 gene and significantly enhanced their protein expression in the tumor tissue. Inhibition of VEGF, MMP-2 and MMP-9 levels were also noted after GTP treatment in tumor tissue in dose-dependent manner. Our findings suggest that induction of RECK and TIMP-3 are key epigenetic events modulated by GTP that results in suppression of matrix degradation and angiogenesis to delay prostate cancer invasion and its subsequent progression. Citation Format: Eswar Shankar, Natarajan Bhaskaran, Rajnee Kanwal, Sanjay Gupta. Green tea polyphenols suppress tumor growth and invasion by targeting matrix metalloproteinases, RECK and TIMP-3, in a mouse model implanted with prostate tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2225. doi:10.1158/1538-7445.AM2017-2225

Inhibition of Matrix Metalloproteinase 9 Enhances Rod Survival in the S334ter-line3 Retinitis Pigmentosa Model.

Retinitis Pigmentosa (RP) is one of the most common forms of inherited visual loss with the initial degeneration of rod photoreceptors, followed by a progressive cone photoreceptor deterioration. Coinciding with this visual loss, the extracellular matrix (ECM) is reorganized, which alters matrix metalloproteinase (MMP) activity levels. A potential pathological role of MMPs, MMP-9 in particular, involves an excitotoxicity-mediated physiological response. In the current study, we examine the MMP-9 and MMP-2 expression levels in the rhodopsin S334ter-line3 RP rat model and investigate the impact of treatment with SB-3CT, a specific MMP-9 and MMP-2 inhibitor, on rod cell survival was tested. Retinal MMP-9 and MMP-2 expression levels were quantified by immunoblot analysis from S334ter-line3 rats compared to controls. Gelatinolytic activities of MMP-9 and MMP-2 by zymography were examined. The geometry of rod death was further evaluated using Voronoi analysis. Our results revealed that MMP-9 was elevated while MMP-2 was relatively unchanged when S334ter-line 3 retinas were compared to controls. With SB-3CT treatment, we observed gelatinolytic activity of both MMPs was decreased and diminished clustering associated with rod death, in addition to a robust preservation of rod photoreceptors. These results demonstrate that up-regulation of MMP-9 in retinas of S334ter-line3 are associated with rod death. The application of SB-3CT dramatically interferes with mechanisms leading to apoptosis in an MMP-9-dependent manner. Future studies will determine the feasibility of using SB-3CT as a potential therapeutic strategy to slow progression of vision loss in genetic inherited forms of human RP.

Low-Dose and Short-Duration Matrix Metalloproteinase 9 Inhibition Does Not Affect Adhesion Formation during Murine Flexor Tendon Healing.

After flexor tendon injury and repair, adhesion formation is a substantial concern, as it can result in loss of motion and functional disability. Matrix metalloproteinase 9 (Mmp9) is a gelatinase that contributes to degradation of extracellular matrix and is expressed during flexor tendon healing. Mmp9(-/-) mice have accelerated remodeling of adhesions during flexor tendon healing, relative to wild-type mice. The purpose of this study was to investigate whether Ro 32-3555, an Mmp9 inhibitor, can improve flexor tendon healing by limiting adhesion formation or enhancing remodeling of scar tissue during murine flexor tendon healing. Flexor digitorum longus laceration and repair was performed in female C57BL/6J mice. Mice were treated with vehicle or the Mmp9 inhibitor Ro 32-3555 for 8 days. Analysis was performed for digit range of motion and gliding function, biomechanics, gene expression, and Mmp9 activity. An Mmp9 activity assay and zymography confirmed suppression of Mmp9 activity in mice treated with Ro 32-3555. There was no significant difference in tendon gliding or range of motion between vehicle and Ro 32-3555-treated mice. There was also no difference in tendon biomechanical properties between the two groups. Local inhibition of Mmp9 gelatinolytic activity at the flexor tendon repair site is insufficient to alter adhesion formation, remodeling of adhesions, or mechanical properties of healing murine flexor tendons.

Sodium nitrite attenuates MMP-9 production by endothelial cells and may explain similar effects of atorvastatin.

Imbalanced matrix metalloproteinase (MMP) activity promotes cardiovascular alterations that are attenuated by statins. These drugs exert pleiotropic effects independent of cholesterol concentrations, including upregulation of nitric oxide (NO) formation and MMP downregulation. However, statins also increase tissue concentrations of nitrites, which activate new signaling pathways independent of NO. We examined whether atorvastatin attenuates MMP-9 production by human umbilical vein endothelial cells (HUVEC) stimulated with phorbol 12-myristate 13-acetate (PMA) by mechanisms possibly involving increased nitrite, and whether this effect results of NO formation. We also examined whether such an effect is improved by sildenafil, an inhibitor of phosphodiesterase-5 which potentiates NO-induced increases in cyclic GMP. MMP activity and nitrite concentrations were measured by gelatin zymography and ozone-based reductive chemiluminescence, respectively, in the conditioned medium of HUVECs incubated for 24 h with these drugs. Phospho-NFκB p65 concentrations were measured in cell lysate to assess NFκB activation. Atorvastatin attenuated PMA-induced MMP-9 gelatinolytic activity by mechanisms not involving NO, although it increased nitrite concentrations, whereas sildenafil had no effects. Combining both drugs showed no improved responses compared to atorvastatin alone. While sodium nitrite attenuated MMP-9 production by HUVECs, adding hemoglobin (NO scavenger) did not affect the responses to nitrite. Neither atorvastatin nor nitrite inhibited PMA-induced increases in phospho-NFκB p65 concentrations. These findings show that sodium nitrite attenuates MMP-9 production by endothelial cells and may explain similar effects exerted by atorvastatin. With both drugs, the inhibitory effects on MMP-9 production are not dependent on NO formation or on inhibition of NFκB activation. Our findings may help to elucidate important new nitrite-mediated mechanisms by which statins affect imbalanced MMP activity in a variety of cardiovascular disease.

Cytotoxicity and gelatinolytic activity of a new silicon-based endodontic sealer.

To compare the cytotoxicity, gelatinolytic activity, and protein levels (MMP-2 and MMP-9) produced by 3T3 fibroblasts cells after stimulation with GuttaFlow 2 and AH Plus. 3T3 fibroblasts were incubated with elutes of GuttaFlow 2 and AH Plus for 24 h. The cytotoxicity of tested materials was determined using the MTT and the LDH assay. Supernatants of cell cultures incubated with sealers were collected to determine the levels of MMP-2 and MMP-9 gelatinolytic activity by gelatin zymography. Cell lysates were used to determine MMP-2 and MMP-9 protein levels by Western Blot. Data were analyzed using ANOVA and Tukey test (P&lt;0.05). AH Plus showed significantly less cell viability (mitochondrial activity of cells) than GuttaFlow 2 (P&lt;0.01). Moreover, GuttaFlow 2 was noncytotoxic, showing no statistically significant difference in LDH leakage levels compared to the control group (P&gt;0.05). Specific characterization of MMPs demonstrated that GuttaFlow 2 seemed not to affect MMP-2 levels compared with the control group, while AH Plus had elevated gelatinolytic activity and protein levels of MMP-2 as confirmed by quantitative measurements. No detectable gelatinolytic activity or protein levels of MMP-9 (92 kDa) was observed in any tested group. GuttaFlow 2 did not showed cytotoxic effects and did not induce MMP-2 or MMP-9 expression.

Selective inactivation of NADPH oxidase 2 causes regression of vascularization and the size and stability of atherosclerotic plaques

BackgroundA variety of NADPH oxidase (Nox) isoforms including Noxs 1, 2, 4 and 5 catalyze the formation of reactive oxygen species (ROS) in the vascular wall. The Nox2 isoform complex has arguably received the greatest attention in the progression of atherogenesis in animal models. Thus, in the current study we postulated that specific Nox2 oxidase inhibition could reverse or attenuate atherosclerosis in mice fed a high-fat diet. MethodsWe evaluated the effect of isoform-selective Nox2 assembly inhibitor on the progression and vascularization of atheromatous plaques. Apolipoprotein E-deficient mice (ApoE−/−) were fed a high fat diet for two months and treated over 15 days with Nox2ds-tat or control sequence (scrambled); 10 mg/kg/day, i.p. Mice were sacrificed and superoxide production in arterial tissue was detected by cytochrome C reduction assay and dihydroethidium staining. Plaque development was evaluated and the angiogenic markers VEGF, HIF1-α and visfatin were quantified by real time qRT-PCR. MMP-9 protein release and gelatinolytic activity was determined as a marker for vascularization. ResultsNox2ds-tat inhibited Nox-derived superoxide determined by cytochrome C in carotid arteries (2.3 ± 0.1 vs 1.7 ± 0.1 O2•− nmol/min*mg protein; P < 0.01) and caused a significant regression in atherosclerotic plaques in aorta (66 ± 6 μm2 vs 37 ± 1 μm2; scrmb vs. Nox2ds-tat; P < 0.001). Increased VEGF, HIF-1α, MMP-9 and visfatin expression in arterial tissue in response to high-fat diet were significantly attenuated by Nox2ds-tat which in turn impaired both MMP-9 protein expression and activity. ConclusionGiven these results, it is quite evident that selective Nox inhibitors can reverse vascular pathology arising with atherosclerosis.

In vitro effect of caffeic acid phenethyl ester on matrix metalloproteinases (MMP-1 and MMP-9) and their inhibitor (TIMP-1) in lipopolysaccharide-activated human monocytes

ObjectiveThe role of matrix metalloproteinases (MMPs) in tissue degradation has become evident in many diseases and great interest therefore exists in the pharmacological control of the activity of these enzymes. This study evaluated the effect of caffeic acid phenethyl ester (CAPE) on the production of MMPs and their inhibitor (TIMP) in monocytes activated by lipopolysaccharide (LPS). DesignThe human monocytic cell line (THP-1) was treated with non-cytotoxic concentrations of CAPE (10 and 60μM) combined with 1μg/mL of LPS. The gene expression of MMP-1, MMP-9 and TIMP-1 was evaluated by quantitative real-time polymerase chain reaction. The protein secretion into the culture medium was assessed via enzyme-linked immunosorbent assay and the gelatinolytic activity of MMP-9 by zymography. ResultsCAPE, especially at the highest concentration, down-regulated MMP-1 and MMP-9 gene expression but up-regulated the gene expression of TIMP-1. Furthermore, CAPE reduced the secreted protein level of MMP-1 and MMP-9 as well as the gelatinolytic activity of MMP-9. ConclusionCAPE was able to inhibit the gene expression, production and the activity of MMPs induced by LPS and also increased the gene expression of TIMP-1. The present observations suggest that CAPE exerted a positive effect on the regulatory mechanism between MMPs and TIMP, which is important for the control of different diseases.

Mechanical stretch promotes matrix metalloproteinase-2 and prolyl-4-hydroxylase α1 production in human aortic smooth muscle cells via Akt-p38 MAPK-JNK signaling

Hypertension can increase mechanical stretch on the vessel wall, an important stimulus that induces collagen remodeling. Prolyl-4-hydroxylaseα1 (P4Hα1) and matrix metalloproteinases (MMPs) are essential for collagen synthesis and degradation. However, the effect of mechanical strain and collagen synthesis remains largely unknown. This study aimed to identify the effect of stretch on MMPs and P4Hα1 and the involved signaling pathways. Human aortic smooth muscle cells (HASMCs) were stimulated with mechanical stretch (0, 10% and 18% strain), and production of P4Hα1 as well as production and gelatinolytic activity of MMP-2 was force-dependently increased. Mechanical stretch at 18% also increased the expression of type I and III collagen and the phosphorylation of Akt, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). MMP-2 production and activity enhanced by 18% stretch were inhibited by the PI3K/Akt inhibitor LY294002. Blockade of p38 MAPK or JNK inhibited the promoting effect of stretch on P4Hα1. The in vivo model of aortic banding showed increased protein levels of MMP-2, P4Hα1 and collagen I and III in the aorta. Thus, mechanical stretch increased MMP-2 and P4Hα1 expression in HASMCs via AKT-P38 MAPK-JNK signaling, thereby inducing vascular remodeling.

In vitro response of human peripheral blood mononuclear cells to AISI 316L austenitic stainless steel subjected to nitriding and collagen coating treatments.

Surface modification treatments can be used to improve the biocompatibility of austenitic stainless steels. In the present research two different modifications of AISI 316L stainless steel were considered, low temperature nitriding and collagen-I coating, applied as single treatment or in conjunction. Low temperature nitriding produced modified surface layers consisting mainly of S phase, which enhanced corrosion resistance in PBS solution. Biocompatibility was assessed using human peripheral blood mononuclear cells (PBMC) in culture. Proliferation, lactate dehydrogenase (LDH) levels, release of cytokines (TNF-α, IL-1β, IL-12, IL-10), secretion of metalloproteinase (MMP)-9 and its inhibitor TIMP-1, and the gelatinolytic activity of MMP-9 were determined. While the 48-h incubation of PBMC with all the sample types did not negatively influence cell proliferation, LDH and MMP-9 levels, suggesting therefore a good biocompatibility, the release of the pro-inflammatory cytokines was always remarkable when compared to that of control cells. However, in the presence of the nitrided and collagen coated samples, the release of the pro-inflammatory cytokine IL-1β decreased, while that of the anti-inflammatory cytokine IL-10 increased, in comparison with the untreated AISI 316L samples. Our results suggest that some biological parameters were ameliorated by these surface treatments of AISI 316L.

Abstract T P401: Human Albumin Confers Neurovascular Protection And Long-term Neurobehavioral Improvement After Subarachnoid Hemorrhage

Introduction: Subarachnoid hemorrhage (SAH) patients suffer from high mortality and worsening of their daily functioning. Currently no clinically effective agents have been recognized yet. Hypothesis: This study aimed to investigate whether albumin (Alb) confers a sustained improvement in SAH, and whether the salutary effects are associated with neurovascular preservation. Methods: Endovascular perforation method was used to induce SAH. Alb (0.63 g/kg or 1.25 g/kg) was immediately intravenously administered after surgery. Cerebral blood flow and early vasospasm-induced arterial morphology changes were measured to evaluate vascular dysfunction. Fluoro-Jade C staining and TUNEL staining were used to evaluate neuronal injury. To determine the blood-brain barrier (BBB) integrity, EB extravasation, IgG leakage, and tight junction protein levels were assessed. Gelatin zymography was also used to gelatinolytic activity of MMP-2 and MMP-9. Neurological deficits were assessed up to post-operative day 42. Results: SAH grade and mortality were not significantly different among groups. Alb prevented early cerebral blood flow reduction and vasospasm with decreased arterial luminal perimeter, wall thickness and increased arterial diameter as compared with vehicle group. Upon SAH, neuronal degeneration and cell apoptosis were noticed in the hippocampal and cortical areas, which was significantly attenuated by Alb. This neurovascular protection was accompanied with lowered IgG leakage and EB extravasation. Mechanically, the expression levels of tight junction proteins (ZO-1, occludin, claudin-5) and adherent junction protein (VE-cadherin), which maintain the BBB structure and function, were remarkably preserved after treatment of Alb. The expression and activity of gelatinase were also conserved. As for neurological deficits, SAH led to a sensorimotor dysfunction up to 28 days after the insult, which could be improved by Alb. Alb also improved the long-term cognitive outcomes, as assessed by novel object recognition and Morris water maze. Conclusions: In conclusion, Alb preserves neurovascular functions and long-term neurobehavioral outcomes in experimental SAH.

Root canal content from primary endodontic infection and upregulation of gelatinases in fibroblast cells.

To investigate endotoxin levels from primary endodontic infections before and after chemomechanical preparation (CMP) and to determine their antigenicity against 3T3 fibroblasts through gelatinolytic activity of matrix metalloproteinases (MMPs). Twenty-four root canals with primary endodontic infection and apical periodontitis were selected. Samples were collected using paper points before (S1) and after chemomechanical preparation (CMP) (S2). The limulus amebocyte lysate assay was used for endotoxin measurement. Fibroblasts were stimulated with root canal contents for 24h. Supernatants of cell cultures stimulated with root canal contents were collected after 24h to determine the levels of MMP-2 and MMP-9 gelatinolytic activity using the zymography technique. Friedman and Wilcoxon tests were used to compare the amount of endotoxin before (S1) and after CMP (S2) (P<0.05). Data obtained from gelatinolytic activity were analysed using anova and Tukey's tests (P<0.05). Endotoxin was recovered in 100% of the samples. There was a significant reduction in endotoxin levels after CMP (P<0.05). A correlation was found between the levels of endotoxins and MMP-2 expression (P<0.05). Root canal contents of initial samples (S1) induced significantly greater MMP-2 expression by fibroblasts when compared to S2 and the nonstimulated group (P<0.05). No gelatinolytic activity of MMP-9 was observed in S1, S2 and control group. Root canal contents from primary endodontic infections had gelatinolytic activity for MMP-2. Moreover, CMP was effective in reducing endotoxin levels and their antigenicity against fibroblasts on gelatinolytic activity.

English

Metastasis is responsible for the majority of cancer-related deaths. Tumour invasion and metastasis result from processes that include the proteolytic degradation of the extracellular matrix adjacent to the tumour. The matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have prognostic influence in human cancers after they cleave the main structural components of the basal membrane. These actions make MMPs an attractive target for cancer and metastasis studies. This study evaluated the inhibitory potency of extracts of Bauhinia ungulata L . (BU) on the gelatinolytic activity of MMP-2 and -9 and recognized the group of secondary compounds responsible for this property. The zymographic analysis of the BU stem revealed that the ethyl acetate partition (D) caused a higher inhibition of MMP-2 and MMP-9. The phytochemical study of D showed the presence of steroids, tannins, and coumarins and the significant presence of alkaloids and flavonoids. The phytochemical study of the fractions obtained through the column chromatography of partition D revealed a significant presence of flavonoids and alkaloids in the fractions that showed better inhibition of the gelatinolytic activity of MMPs. In conclusion, these results suggest that the stem fraction of BU has the potential to inhibit MMP-2 and MMP-9 and should be used in studies on the recognition of active biomolecules.