Measure Matrix Metalloproteinase Research Articles

Cluster analysis of extracellular matrix biomarkers predicts the development of impaired systolic function within 1 year of acute myocardial infarction

The clinical utility of combining extracellular matrix (ECM) biomarkers to predict the development of impaired systolic function following acute myocardial infarction (AMI) remains largely undetermined. A combination of ELISA and multiplexing assays were performed to measure matrix metalloproteinase (MMP)-2, MMP-3, MMP-8, MMP-9, periostin, N-terminal type I procollagen (PINP) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in plasma samples from 120 AMI patients. All patients had an echocardiogram within 1 year of AMI, and were divided into impaired (n = 37, LVEF < 50%) and preserved (n = 83, LVEF ≥ 50%) systolic function groups. Exploratory factor analysis was performed on log-transformed biomarkers using principle axis analysis with Oblimin rotation. Cluster analysis was performed on log-transformed and normalised biomarkers using Ward’s method of minimum variance and the squared Euclidean distance metric. Upon univariate analysis, current smoking, prescription of ACE inhibitors at discharge, peak hsTnT > 610 ng/L (median), MMP-8 levels, Factor 1 scores and Cluster One assignment were predictive of impaired systolic function. Upon multivariate analysis, Cluster One assignment (odds ratio [95% CI], 2.74 [1.04–7.23], p = 0.04) remained an independent predictor of systolic dysfunction in combination with clinical variables. These observations support the usefulness of combining ECM biomarkers using cluster analysis for predicting the development of impaired systolic function in AMI patients.

Superoxide Dismutase Mimic, MnTE-2-PyP Enhances Rectal Anastomotic Strength in Rats after Preoperative Chemoradiotherapy.

Background Rectal cancer is one of the malignant diseases with high morbidity and mortality in the world. Currently, surgical resection is the main treatment method, and preoperative chemoradiotherapy (CRT) is widely used in clinical application to increase resectability and decrease the local recurrence rate. However, CRT increases the risk of colon anastomotic leak, and currently, there are no FDA approved treatments against this side effect. It is essential to develop new drugs to reduce postoperative anastomotic leak after preoperative CRT. Methods 90 rats underwent standard resection and intestine anastomosis treatment and were divided into six groups for different treatments. During the relaparotomy, bursting pressure of anastomosis was measured and intestinal segments were taken for histopathologic examination and biochemical analyses. RT-PCR and ELISA were applied to measure matrix metalloproteinase (MMP) mRNA and protein levels. Blood vessels were observed by immunohistochemistry, and collagen deposition was observed by Picrosirius Red staining. Results Preoperative CRT reduced the postoperative anastomotic strength. MnTE-2-PyP increased the bursting pressure and hydroxyproline levels of intestine anastomosis after CRT treatment. Mechanically, MnTE-2-PyP decreased the MMP levels and increased microvessel density (MVD) and collagen deposition. The MMP inhibitor doxycycline had a positive effect on anastomosis healing, but was inferior to MnTE-2-PyP. Conclusions MnTE-2-PyP enhanced intestine anastomotic strength in rats with preoperative CRT. Specifically, MnTE-2-PyP decreased MMP levels and increased MVD in anastomosis. Therefore, MnTE-2-PyP may be helpful in the prevention of anastomotic leak after preoperative CRT.

Lung Adenocarcinoma Syndecan-2 Potentiates Cell Invasiveness.

Altered expression of syndecan-2 (SDC2), a heparan sulfate proteoglycan, has been associated with diverse types of human cancers. However, the mechanisms by which SDC2 may contribute to the pathobiology of lung adenocarcinoma have not been previously explored. SDC2 levels were measured in human lung adenocarcinoma samples and lung cancer tissue microarrays using immunohistochemistry and real-time PCR. To understand the role of SDC2 in vitro, SDC2 was silenced or overexpressed in A549 lung adenocarcinoma cells. The invasive capacity of cells was assessed using Matrigel invasion assays and measuring matrix metalloproteinase (MMP) 9 expression. Finally, we assessed tumor growth and metastasis of SDC2-deficient A549 cells in a xenograft tumor model. SDC2 expression was upregulated in malignant epithelial cells and macrophages obtained from human lung adenocarcinomas. Silencing of SDC2 decreased MMP9 expression and attenuated the invasive capacity of A549 lung adenocarcinoma cells. The inhibitory effect of SDC2 silencing on MMP9 expression and cell invasion was reversed by overexpression of MMP9 and syntenin-1. SDC2 silencing attenuated NF-κB p65 subunit nuclear translocation and its binding to the MMP9 promoter, which were restored by overexpression of syntenin-1. SDC2 silencing in vivo reduced tumor mass volume and metastasis. These findings suggest that SDC2 plays an important role in the invasive properties of lung adenocarcinoma cells and that its effects are mediated by syntenin-1. Thus, inhibiting SDC2 expression or activity could serve as a potential therapeutic target to treat lung adenocarcinoma.

Rapid, Automated, and Specific Immunoassay to Directly Measure Matrix Metalloproteinase-9-Tissue Inhibitor of Metalloproteinase-1 Interactions in Human Plasma Using AlphaLISA Technology: A New Alternative to Classical ELISA.

The protocol describes a novel, rapid, and no-wash one-step immunoassay for highly sensitive and direct detection of the complexes between matrix metalloproteinases (MMPs) and their tissue inhibitor of metalloproteinases (TIMPs) based on AlphaLISA® technology. We describe two procedures: (i) one approach is used to analyze MMP-9–TIMP-1 interactions using recombinant human MMP-9 with its corresponding recombinant human TIMP-1 inhibitor and (ii) the second approach is used to analyze native or endogenous MMP-9–TIMP-1 protein interactions in samples of human plasma. Evaluating native MMP-9–TIMP-1 complexes using this approach avoids the use of indirect calculations of the MMP-9/TIMP-1 ratio for which independent MMP-9 and TIMP-1 quantifications by two conventional ELISAs are needed. The MMP-9–TIMP-1 AlphaLISA® assay is quick, highly simplified, and cost-effective and can be completed in less than 3 h. Moreover, the assay has great potential for use in basic and preclinical research as it allows direct determination of native MMP-9–TIMP-1 complexes in circulating blood as biofluid.

Micronized progesterone pretreatment affects the inflammatory response of human gestational tissues and the cervix to lipopolysaccharide stimulation

IntroductionVaginal administration of micronized progesterone (utrogestan capsule, UG) reduces the risk of preterm birth (PTB) in asymptomatic women with a sonographic short cervix at mid-trimester or with a prior history of spontaneous PTB; however, its exact mechanisms remain unclear. We hypothesized that UG limits the inflammatory processes within the gestational tissues and the cervix. MethodsFetal membranes and villous tissues were obtained from normal term placentas from women with cesarean delivery before labor onset. Ectocervical tissues were obtained from premenopausal women undergoing hysterectomies for uterine fibroids. Explant tissue cultures were pretreated with UG for 24 h and then exposed to UG with or without lipopolysaccharide (LPS) for 48 h. Tumor necrosis factor-α, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein-1, interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, IL-13, interferon-γ, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 levels in tissue homogenates and culture medium were measured by enzyme-linked immunosorbent assays. Real-time quantitative PCR, Western blot, and gelatine zymography were used to measure matrix metalloproteinase (MMP)-9 and MMP-2 mRNA, protein, and activity levels, respectively. ResultsUG pretreatment did not cause a significant change in basal levels or in LPS-induced production and secretion of cytokines, chemokines, and TIMPs in the three tissues. However, UG pretreatment significantly reduced MMP-9 and MMP-2 expression and activity in fetal membranes stimulated with LPS but not in villous or cervical tissues. DiscussionUG pretreatment significantly reduced MMP-9 and MMP-2 expression and activity in fetal membranes stimulated with LPS, suggesting a possible protective mechanism of micronized progesterone in preventing infection-associated PTB.

Trans-chalcone added in topical formulation inhibits skin inflammation and oxidative stress in a model of ultraviolet B radiation skin damage in hairless mice

Trans-chalcone (TC) is a common precursor of flavonoids. However, the pharmacological properties of TC remain to be fully understood. The present study investigated whether topical formulation containing TC (TFcTC) presents therapeutic effect in UVB radiation-induced skin damage using disease, enzyme activity, antioxidant activity, protein and mRNA parameters. Control topical formulation (CTF) and TFcTC were applied in hairless mice before and after exposure to UVB radiation. Dorsal skin samples were collected after UVB exposure to evaluate: i) skin edema (weight) was measured by punch biopsy; ii) spectrophotometric assays were used to measure myeloperoxidase (MPO) and catalase activities, ferric (FRAP) and ABTS cation reducing antioxidant power, superoxide anion production and levels of reduced glutathione (GSH); iii) enzymography was used to measure matrix metalloproteinase-9 (MMP-9) activity; iv) chemiluminescence was used to measure the lipid peroxidation (LPO); v) enzyme-linked immunosorbent assay (ELISA) was used to measure tumor necrosis factor alpha (TNF-α) levels; vi) reverse transcription quantitative PCR (RT-qPCR) was used to measure cyclooxygenase-2 (COX-2), gp91phox (NADPH oxidase sub-unity), glutathione peroxidase-1 (Gpx1), glutathione reductase (Gr), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) mRNA expression. TFcTC inhibited UVB-induced skin edema, MPO activity, MMP-9 activity, TNF-α production, and COX-2 mRNA expression. TFcTC inhibited UVB-induced LPO, down-regulated superoxide anion levels and gp91phox mRNA expression, and improved antioxidant potential and GSH skin levels. The mRNA expression of detoxification systems such as Nrf2, HO-1, Gpx1 and Gr, and catalase activity were also enhanced by treatment with TFcTC. In conclusion, TFcTC protects mice skin from UVB radiation by inhibiting inflammation, and improving antioxidant and detoxification systems. Therefore, topical treatment with TC is a novel therapeutic approach for the treatment of UVB radiation skin damages, which merits further pre-clinical and clinical investigation.

LINC00312 inhibits the migration and invasion of bladder cancer cells by targeting microRNA-197-3p

To investigate the influence of the long non-coding RNA LINC00312 on bladder cancer (BC) cell invasion and metastasis by targeting miR-197-3p. BC and corresponding adjacent tissues were collected. LINC00312 and miR-197-3p were measured, and their correlation was detected through quantitative real-time PCR (qRT-PCR). BC cell line T24 was transfected and grouped (five groups) according to different transfection conditions. A scratch test was applied to analyze cell migration, and a Transwell assay was used to test cell invasion ability. Western blotting was to measure matrix metalloproteinase (MMP)-2, MMP-9, and the tissue inhibitor of metalloproteinase 2 (TIMP2) protein levels. qRT-PCR indicated that LINC00312 expression was lower but miR-197-3p expression was higher in BC tissues compared with adjacent tissues; LINC00312 was negatively correlated with miR-197-3p. The migration test revealed that the downregulation of miR-197-3p and overexpression of LINC00312 inhibited cell migration and invasion abilities, while the overexpression of miR-197-3p and the upregulation of LINC00312 promoted cell migration and invasion. BC cells with downregulated miR-197-3p or upregulated LINC00312 had low MMP-2 and MMP-9 levels but high TIMP2. LINC00312 inhibited BC cell invasion and metastasis through mediating miR-197-3p.

Establishment of brain edema model of rats with intracerebral hemorrhage by IV collagenase combined with autologous blood

Objective To explore the model of IV combined with autologous blood to build a model of compound brain edema in rats, and provide experimental evidence for the related research of brain edema. Methods 180 SD rats were randomly divided into 6 groups: Group A (IV collagenase + autologous coagulation blood + autologous blood coagulation), group B (autologous coagulation), group C (IV collagenase), group D (autologous anti coagulation), E group (blank control), F group (sham operation). Group A, B, C, D were modeled, Group E was not given treatment, Group F was injected with normal saline.Neurological deficit was evalated by longa/Bederson and physical symmetry test score.The water content in brain tissue was detected by wet and dry weight method.K+ , Na+ , Ca2+ were detected by atomic absorption spectrometry.The ultrastructural changes of glial cells were observed by transmission electron microscope (TEM). Glial cells were identified by immunofluorescence assay.FCM and TUNEL tested the apoptosis of glial cells.CCK-8 and EDU tested the proliferation of glial cells.Real time quantitative polymerase chain reaction and Western blot were used to measure matrix metalloproteinase-9(MMP-9) in glial cells and water channel protein 4(AQP4) mRNA and protein expression.The scratch test and Transwell assayed glial cell migration and invasion. Results Comparison among groups: Longa /Bederson score of Neurologic impairment, Brain tissue water content, electrolyte Na+ was the highest level of group A, electrolyte K+ , Ca2+ at least.The apoptosis of group A was the most(32.27±3.33), the least proliferation(5.33±1.14), migration and invasion ability were the weakest(85.35±8.11; 8.08±1.96); mRNA(9.31±0.84; 15.69±1.77) and protein(4.25±0.27; 4.34±0.33) of MMP-9 and AQP4 were the most significant in group A (P<0.05). Conclusion IV collagenase combined with autologous blood to build a model of brain edema animal model makes the brain edema markers MMP-9 and AQP4 stronger, and the functional changes of cytology is more significant. Key words: Cerebral hemorrhage; Brain edema; Experiment; MMP-9; AQP4

Matrix Metalloproteinase 9 Testing in Dry Eye Disease Using a Commercially Available Point-of-Care Immunoassay

To measure matrix metalloproteinase 9 (MMP-9) in the tear film of patients with dry eye disease (DED) compared with controls and to correlate clinical findings. In a prospective study, 101 patients and controls underwent MMP-9 testing of the tear film. Thereafter, they were evaluated for symptoms and signs of DED. Included patients were those who showed 3 of the following 4 dry eye criteria: ocular surface disease index (OSDI) score of more than 12, tear film break-up time (TBUT) of 10 seconds or less, Schirmer test results without anesthesia of less than 10 mm/5 minutes, and corneal staining results of 1 or more. Fifty-four healthy eyes and 47 eyes fulfilling diagnostic criteria for DED of various levels of severity were included in this study. The tear film was analyzed for MMP-9 by a commercially available test (InflammaDry; Rapid Pathogen Screening, Inc, Sarasota, FL) detecting MMP-9 levels of more than 40 ng/ml. Symptoms and signs of DED were evaluated using the OSDI questionnaire, TBUT, conjunctival and corneal staining, Schirmer test results without anesthesia, and meibomian gland examination. These findings were correlated to results of the MMP-9 test in tears. Positive MMP-9 results in tears. In 19 of 47 patients confirmed with dry eye (40.4%) and in 3 of 54 controls (5.6%), the MMP-9 results were positive. This difference was statistically significant (P < 0.001). Thus, the MMP-9 results indicated a clinically significant inflammation in 40% of dry eye patients. Positive results correlated well with subjective symptoms of DED evaluated by OSDI (P= 0.001), TBUT of less than 5 seconds (P < 0.013), Schirmer test results (P < 0.001), conjunctival staining (P < 0.001), and corneal staining (P= 0.007). Moreover, MMP-9 results correlated with the number of obstructed meibomian ducts (P= 0.005) and a pathologic meibomian gland secretion (P= 0.001). The MMP-9 results were increased significantly in women (P < 0.001) and in patients with autoimmune disease (P= 0.005), especially Sjögren's syndrome (P= 0.001) and thyroid disease (P= 0.012). Matrix metalloproteinase 9 testing in DED is a valuable new diagnostic tool. It correlated well with other dry eye tests and identified the presence of ocular surface inflammation in 40% of confirmed dry eyepatients. It may be especially helpful to identify patients with ocular surface inflammation and autoimmune disease and may facilitate the decision to institute anti-inflammatory treatment in these patients.

LINC00312 inhibits the migration and invasion of bladder cancer cells by targeting miR-197-3p.

To investigate the influence of the long non-coding RNA LINC00312 on bladder cancer (BC) cell invasion and metastasis by targeting miR-197-3p. BC and corresponding adjacent tissues were collected. LINC00312 and miR-197-3p were measured, and their correlation was detected through quantitative real-time PCR (qRT-PCR). BC cell line T24 was transfected and grouped (five groups) according to different transfection conditions. A scratch test was applied to analyze cell migration, and a Transwell assay was used to test cell invasion ability. Western blotting was to measure matrix metalloproteinase (MMP)-2, MMP-9, and the tissue inhibitor of metalloproteinase 2 (TIMP2) protein levels. qRT-PCR indicated that LINC00312 expression was lower but miR-197-3p expression was higher in BC tissues compared with adjacent tissues; LINC00312 was negatively correlated with miR-197-3p. The migration test revealed that the downregulation of miR-197-3p and overexpression of LINC00312 inhibited cell migration and invasion abilities, while the overexpression of miR-197-3p and the upregulation of LINC00312 promoted cell migration and invasion. BC cells with downregulated miR-197-3p or upregulated LINC00312 had low MMP-2 and MMP-9 levels but high TIMP2. LINC00312 inhibited BC cell invasion and metastasis through mediating miR-197-3p.

Transient expression of the diseased phenotype of osteoarthritic chondrocytes in engineered cartilage.

Due to the degradation of osteoarthritic (OA) cartilage in post-traumatic OA (PTOA), these tissues are challenging to study and manipulate in vitro. In this study, chondrocytes isolated from either PTOA (meniscal-release (MR) model) or normal (contralateral limb) cartilage of canine knee joints were used to form micropellets to assess the maintenance of the OA chondrocyte phenotype in vitro. Media samples from the micropellet cultures were used to measure matrix metalloproteinase (MMP), chemokine, and cytokine concentrations. Significant differences in matrix synthesis were observed as a function of disease with OA chondrocytes generally synthesizing more extracellular matrix with increasing time in culture. No donor dependent differences were detected. Luminex multiplex analysis of pellet culture media showed disease and time-dependent differences in interleukin (IL)-8, keratinocyte chemoattractant (KC)-like protein, MMP-1, MMP-2, and MMP-3, which are differentially expressed in OA. This memory of their diseased phenotype persists for the first 2 weeks of culture. These results demonstrate the potential to use chondrocytes from an animal model of OA to study phenotype alterations during the progression and treatment of OA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:829-836, 2017.

Effects of azithromycin on gene expression profiles of proinflammatory and anti-inflammatory mediators in the eyelid margin and conjunctiva of patients with meibomian gland disease.

Topical application of azithromycin suppresses expression of proinflammatory mediators while restoring transforming growth factor β1 (TGF-β1) levels as evaluated by eyelid margin and conjunctival impression cytology. To explore the effects of azithromycin therapy on expression of proinflammatory and anti-inflammatory mediators in meibomian gland disease (MGD). Case-control study performed in a clinic setting from August 17, 2010, to December 31, 2010. Sixteen patients with posterior blepharitis and conjunctival inflammation due to MGD were treated with azithromycin, 1%, drops for 4 weeks. Impression cytology of the lower eyelid margin and tarsal conjunctiva to measure cytokine expression by quantitative real-time polymerase chain reaction as well as tear collection to measure matrix metalloproteinase 9 (MMP-9) activity were performed once in 8 asymptomatic healthy control participants and 5 times in the 16 symptomatic patients (every 2 weeks for 8 weeks), before, during, and after azithromycin treatment. Azithromycin, 1%, drops for 4 weeks. Cytokine expression in the eyelid margin and conjunctiva, and MMP-9 activity in tears. Compared with a 1-time measurement of 8 healthy participants, among 16 symptomatic patients, the mean (SD; 95% CI) fold change of expression of proinflammatory mediators interleukin 1β (IL-1β), IL-8, and MMP-9 increased to 13.26 (4.33; 11.14-15.38; P < .001), 9.38 (3.37; 7.73-11.03; P < .001), and 13.49 (4.92; 11.08-15.90; P < .001), respectively, in conjunctival cells and to 11.75 (3.96; 9.81-13.69; P < .001), 9.31 (3.28; 7.70-10.92; P < .001), and 11.52 (3.50; 9.81-13.24; P < .001), respectively, in the eyelid margin of patients with MGD. In contrast, the mean (SD; 96% CI) fold change of expression of TGF-β1 messenger RNA (mRNA) decreased to 0.58 (0.25; 0.46-0.70; P = .02) and 0.63 (0.14; 0.56-0.70; P = .02) in conjunctival and eyelid margin cells, respectively, of patients with MGD. Azithromycin, 1%, caused a change in the expression pattern of these mediators toward normal levels during 4 weeks of treatment. Levels of IL-1β, IL-8, and MMP-9 mRNA remained suppressed, although they rebounded toward pretreatment values 4 weeks after azithromycin withdrawal. Expression of TGF-β1 increased during treatment and remained at levels similar to the healthy controls after drug withdrawal. Change in tear MMP-9 activity was similar to the pattern of MMP-9 transcripts. While the study did not control for potential confounding factors over time independent of the intervention that may have contributed to the results, topical azithromycin suppressed expression of proinflammatory mediators and increased expression of TGF-β1 to normal levels. Increased TGF-β1 expression may contribute to the anti-inflammatory activity of azithromycin in MGD.

On-chip assay of matrix metalloproteinase-3 activity using fluorescence-conjugated gelatin arrays

We developed a new high-throughput approach to measure matrix metalloproteinase (MMP)-3 activity using Cy3-conjugated gelatin arrays. We fabricated Cy3-conjugated gelatin arrays by immobilizing Cy3-conjugated gelatin onto well-type amine arrays. The ratio of Cy3-NHS to gelatin was optimized to improve the efficiency of the arrays. MMP-3 activity was determined by the decrease of fluorescence intensity due to MMP-3-mediated degradation of Cy3-conjugated gelatin. The gelatinolytic activity of MMP-3 increased in the presence of Brij-35, a non-ionic detergent, in a dose-dependent manner. Cy3-conjugated gelatin arrays were successfully applied for the analyses of MMP-3 activity, which showed a dose-dependent degradation of the Cy3-conjugated substrate. This chip-based activity assay using a Cy3-conjugated gelatin array is simple, rapid, cost-effective, and has a potential for activity-based proteomic researches and screening of MMP-3 inhibitors.

In vivo priming and ex vivo activation of equine neutrophils in black walnut extract-induced equine laminitis is not attenuated by systemic lidocaine administration

Laminitis is a crippling disease of horses characterized by an inflammatory response in the tissue that suspends the axial skeleton within the hoof. Pain is a common feature of laminitic pathology and its management is an important component of the treatment regime for this disease. Systemic lidocaine administration is commonly utilized to manage pain in equine laminitis; however, the potential anti-inflammatory effects of this drug during the treatment of equine laminitis have not been investigated. Here, we sought to determine if lidocaine concentrations achieved in the plasma (therapeutic concentrations) of horses systemically administered lidocaine are capable of attenuating neutrophil activation and associated inflammation. To identify markers of activation, purified neutrophils were stimulated in vitro with LPS or recombinant equine IL-8 (reqIL-8) and surface expression of CD13 and CD18 was ascertained by immunofluorescent staining. Activation with LPS or reqIL-8 in vitro induced an elevated expression of CD13 as well as a putative conformational change in CD18 detected by elevated staining with a sub-saturating concentration of anti-CD18 mAb. Lidocaine attenuated the activation-induced changes in CD13 and CD18 expression only when used at 30–70 times therapeutic concentrations. For in vivo analyses, horses were administered black walnut extract (BWE) to induce laminitis and either systemic lidocaine ( n = 6) or saline ( n = 6) as a control. Whole blood was collected and incubated with or without reqIL-8. Following which, leukocytes were stained for CD13 and CD18. Protein was extracted from laminar tissue and subjected to gelatin zymography to measure matrix metalloproteinase-9 (MMP-9) accumulation. Results obtained show that changes in neutrophil size, granularity/complexity, CD13 surface expression and CD18 staining intensity occurred over time post BWE administration irrespective of lidocaine treatment in response to incubation alone or with 100 ng/ml of reqIL-8. The mean fluorescence intensities of neutrophils stained for either CD13 or CD18 did not differ between lidocaine treated and saline controls, nor did lamellar MMP-9 content measured by gelatin zymography. Thus, using changes in surface expression of CD13 and CD18 as markers of neutrophil activation in the horse we have shown that BWE treatment activates neutrophils in vivo and this is not affected by systemic administration of lidocaine at levels used to manage pain.

Folic acid mitigated homocysteine‐mediated decrease in bone blood flow and bone remodeling

Treatment with folic acid has been shown to decrease the risk of osteoporosis. Although homocysteine (Hcy) accumulated collagen in bone and contributed to decrease in bone strength and density, the mechanism of Hcy‐induced bone loss and collagen remodeling was unclear. To test the hypothesis that homocysteine‐mediated decrease in bone strength is in part due to decrease in bone blood flow and remodeling, we used wild type (WT) and hyperhomocysteinemia (cystathionine beta synthase heterozygote knockout, CBS−/+) mice treated with and without folic acid (FA 300 mg/kg in drinking water). The tibial arterial bone blood flow was measured by laser Doppler and ultrasonic flow probes. The tibial bone density was measured by a digital micrometer. The bone homogenates were analyzed for bone remodeling by measuring matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP). The results suggested that there was decrease in tibial arterial blood flow in CBS−/+ mice. The bone density was also reduced in CBS−/+ mice. There was increase in MMP‐2 and ‐9 protein level in CBS−/+ tibia. The levels of TIMP‐3 were increased, where TIMP‐4 was decreased in CBS−/+ tibia. Interestingly, the treatment with FA mitigated the decrease in TIMP‐4 in CBS−/+ tibia. We concluded that folic acid treatment mitigated the decrease in bone blood flow by increasing TIMP‐4 and strengthening the bone.

Endothelial Cell Culture of Intramuscular Venous Malformation and Its Invasive Behavior Related to Matrix Metalloproteinase-9

Intramuscular venous malformation is characterized by abnormal morphology of blood vessels. In certain cases, it displays an invasion of surrounding tissues. The in vitro model of its endothelial cell culture has not been reported, and the pathogenesis of its development is not clear. Matrix metalloproteinase-9 is identified to facilitate cell migration and tumor progression. The aim of this study was to establish the primary cell culture from intramuscular venous malformation and explore its invasive mechanism related to matrix metalloproteinase-9. Explant culture was adopted to isolate endothelial cells from intramuscular venous malformation. In contrast, with human umbilical vein endothelial cells collected by collagenase digestion, the biological behavior of cell migration and invasion was explored. Western blot, reverse-transcription polymerase chain reaction, and immunohistochemical study were performed to measure matrix metalloproteinase-9 expression of endothelial cells or tissue sections. The primary endothelial cell culture from intramuscular venous malformation was established successfully by using explant culture. Compared with human umbilical vein endothelial cells, the established endothelial cells indicated more powerful capability of migration and invasion and expressed more matrix metalloproteinase-9 at both mRNA and protein levels. Matrix metalloproteinase-9 mRNA expression in intramuscular venous malformation was significantly higher than in normal subcutaneous tissues. The intensity of matrix metalloproteinase-9 demonstrated no statistically significant differences with varying courses, locations, dimensions, and infiltration degrees. The positive expression of matrix metalloproteinase-9 of intramuscular venous malformation tissues and endothelial cells provided circumstantial evidence that it may be involved in the pathogenesis of invasive biological behavior of the lesions.

Altered expression of tight junction proteins and matrix metalloproteinases in thiamine-deficient mouse brain

Wernicke's encephalopathy (WE) in humans is a metabolic disorder caused by thiamine deficiency (TD). In both humans and experimental animals, TD leads to selective neuronal cell death in diencephalic and brainstem structures. Neuropathologic features of WE include petechial hemorrhagic lesions, and blood–brain barrier (BBB) breakdown has been suggested to play an important role in the pathogenesis of TD. The goal of the present study was to examine expression of the tight junction (TJ) protein occludin, its associated scaffolding proteins zona occludens (ZO-1 and ZO-2), and to measure matrix metalloproteinase (MMP) levels as a function of regional BBB permeability changes in thiamine-deficient mice. TD was induced in 12-week-old male C57Bl/6 mice by feeding a thiamine-deficient diet and administration of the central thiamine antagonist pyrithiamine. BBB permeability was measured by IgG extravasation; expression of occludin, ZO-1 and ZO-2 was measured by Western blot analysis and RT-PCR, structural integrity of the BBB was assessed using occludin and ZO-1 immunostaining, and MMPs levels were measured by gelatin zymography and immunohistochemistry. Studies were performed in vulnerable (medial thalamus) versus spared (frontal cortex) regions of the brain. Hemorrhagic lesions, selective increases in brain IgG extravasation, a concomitant loss in protein expression of occludin, ZO-1 and ZO-2, as well as decreased and disrupted patterns of occludin and ZO-1 immunostaining were observed in the medial thalamus of thiamine-deficient mice. MMP-9 levels were also selectively increased in the medial thalamus of these animals, and were found to be localized in the vascular endothelium, as well as in cells with an apparent polymorphonuclear morphology. No changes of TJ gene expression were observed. These results indicate that alterations in TJ proteins occur in TD, and offer a plausible explanation for the selective increase in BBB permeability in thiamine-deficient animals. They also suggest a role for MMP-9 in the initiation of changes to BBB integrity in TD.

Role of Copper and Homocysteine in Pressure Overload Heart Failure

Elevated levels of homocysteine (Hcy) (known as hyperhomocysteinemia HHcy) are involved in dilated cardiomyopathy. Hcy chelates copper and impairs copper-dependent enzymes. Copper deficiency has been linked to cardiovascular disease. We tested the hypothesis that copper supplement regresses left ventricular hypertrophy (LVH), fibrosis and endothelial dysfunction in pressure overload DCM mice hearts. The mice were grouped as sham, sham + Cu, aortic constriction (AC), and AC + Cu. Aortic constriction was performed by transverse aortic constriction. The mice were treated with or without 20 mg/kg copper supplement in the diet for 12 weeks. The cardiac function was assessed by echocardiography and electrocardiography. The matrix remodeling was assessed by measuring matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinases (TIMPs), and lysyl oxidase (LOX) by Western blot analyses. The results suggest that in AC mice, cardiac function was improved with copper supplement. TIMP-1 levels decreased in AC and were normalized in AC + Cu. Although MMP-9, TIMP-3, and LOX activity increased in AC and returned to baseline value in AC + Cu, copper supplement showed no significant effect on TIMP-4 activity after pressure overload. In conclusion, our data suggest that copper supplement helps improve cardiac function in a pressure overload dilated cardiomyopathic heart.

Role of Copper and Homocysteine in Pressure Overload Heart Failure

Elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) causes dilated cardiomyopathy (DCM). Hcy chelates copper and impairs copper‐dependent enzymes. To test the hypothesis that copper supplement regresses left ventricle hypertrophy (LVH), fibrosis and endothelial dysfunction in pressure overload DCM hearts (Figure 1), we created aortic constriction (AC) in mice and treated with or without 20 ppm copper supplements in diet for 16 weeks. The mice were grouped as sham; AC, sham+Cu; and AC+Cu. The cardiac function was assessed by ECG and Echocardiography. The levels of fibrosis was assessed by measuring matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinase (TIMP) and lysl oxidase (LOX) by zymography and Western blot analyses. The results suggest that cardiac function was restored with copper supplement. Fractional shortening was 51.3±0.9% in AC; 62.7±3.7% in sham; 60.9±4.7% in sham+Cu; and 62.8±3.1% in AC+Cu. The QRS complexes from ECG were: 13.0±1.4 ms in sham; 14.0±2.8 ms in AC; and 13.0±2.1 ms in AC+Cu. Western blot analyses show that MMP‐13, TIMP‐3, and TIMP‐4 activity decreases in AC and then is improved in AC+Cu. TIMP‐1 and LOX activity increases in AC and returns to sham value in AC+Cu. The data suggests that copper supplement restores cardiac function in a pressure overload DCM heart.

Mucin and Leukocyte Interactions In Vitro

To measure matrix metalloproteinase 9 (MMP-9) in the tear film of patients with dry eye disease (DED) compared with controls and to correlate clinical findings.In a prospective study, 101 patients and controls underwent MMP-9 testing of the tear film. Thereafter, they were evaluated for symptoms and signs of DED.Included patients were those who showed 3 of the following 4 dry eye criteria: ocular surface disease index (OSDI) score of more than 12, tear film break-up time (TBUT) of 10 seconds or less, Schirmer test results without anesthesia of less than 10 mm/5 minutes, and corneal staining results of 1 or more. Fifty-four healthy eyes and 47 eyes fulfilling diagnostic criteria for DED of various levels of severity were included in this study.The tear film was analyzed for MMP-9 by a commercially available test (InflammaDry; Rapid Pathogen Screening, Inc, Sarasota, FL) detecting MMP-9 levels of more than 40 ng/ml. Symptoms and signs of DED were evaluated using the OSDI questionnaire, TBUT, conjunctival and corneal staining, Schirmer test results without anesthesia, and meibomian gland examination. These findings were correlated to results of the MMP-9 test in tears.Positive MMP-9 results in tears.In 19 of 47 patients confirmed with dry eye (40.4%) and in 3 of 54 controls (5.6%), the MMP-9 results were positive. This difference was statistically significant (P < 0.001). Thus, the MMP-9 results indicated a clinically significant inflammation in 40% of dry eye patients. Positive results correlated well with subjective symptoms of DED evaluated by OSDI (P = 0.001), TBUT of less than 5 seconds (P < 0.013), Schirmer test results (P < 0.001), conjunctival staining (P < 0.001), and corneal staining (P = 0.007). Moreover, MMP-9 results correlated with the number of obstructed meibomian ducts (P = 0.005) and a pathologic meibomian gland secretion (P = 0.001). The MMP-9 results were increased significantly in women (P < 0.001) and in patients with autoimmune disease (P = 0.005), especially Sjögren's syndrome (P = 0.001) and thyroid disease (P = 0.012).Matrix metalloproteinase 9 testing in DED is a valuable new diagnostic tool. It correlated well with other dry eye tests and identified the presence of ocular surface inflammation in 40% of confirmed dry eye patients. It may be especially helpful to identify patients with ocular surface inflammation and autoimmune disease and may facilitate the decision to institute anti-inflammatory treatment in these patients.