Tissue Inhibitors Of Matrix Metalloproteinases Research Articles

Human Levels of MMP-1 in Degenerated Disks Can Be Mitigated by Signaling Peptides from Mesenchymal Stem Cells

Degradation of extracellular matrix (ECM) in intervertebral disks (IVDs) during IVD degeneration plays a vital role in low back pain (LBP). In healthy IVDs, synthesis and degradation of ECM are kept in balance by matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs. MMPs are enzymes responsible for ECM degradation, and their expression levels are known to increase in degenerated disks. However, the exact pathophysiological concentration of MMP-1 in the degenerated disks of patients with chronic LBP has not been reported previously. Factors secreted by human mesenchymal stem cells (hMSCs) have shown positive results in cell therapy of degenerated disks. The aim of this study was to investigate the pathophysiological MMP-1 concentration (in ng/mL) in degenerated disk tissue and to evaluate if conditioned media (CM) from hMSCs could mitigate the effects of MMP-1 at the detected levels in a 3D in vitro disk cell (DC) pellet model. Tissue levels of MMP-1 were quantified in disk tissue collected from 6 chronic LBP patients undergoing surgery. DC pellet cultures were performed to investigate the effects of MMP-1 alone and the effects of conditioned media (CM) in the presence of MMP-1. MMP-1 was introduced in the pellets on day 14 at concentrations of 5, 50, or 100 ng/mL. The pellets were harvested on day 28 and evaluated for cell viability, proliferation, and ECM production. The mean concentration of MMP-1 in disk tissue was 151 ng/mL. Results from pellet cultures demonstrated a higher number of viable cells, glycosaminoglycan production, and ECM accumulation in the CM group even in the presence of MMP-1 compared to the controls. However, the level decreased with increasing MMP-1 concentration. The results demonstrated that CM has the ability to mitigate matrix degradation property of MMP-1 up to 50 ng/mL suggesting that CM could potentially be used to treat early stages of disk degeneration.

Markers of remodeling in subcutaneous adipose tissue are strongly associated with overweight and insulin sensitivity in healthy non-obese men

Alteration in extracellular matrix (ECM) in adipose tissues (AT) has been associated with insulin resistance, diabetes and obesity. We investigated whether selected biomarkers of ECM remodeling in AT in healthy subjects associated with the amount and distribution of AT and with glucometabolic variables. Subcutaneous AT and fasting blood samples from 103 middle-aged healthy non-obese men were used. AT gene expression and circulating levels of the biomarkers were quantified. Distribution of AT was assessed by computed tomography, separated into subcutaneous, deep subcutaneous and visceral AT. Insulin sensitivity was measured by glucose clamp technique. Metalloproteinase (MMP)-9, tissue inhibitor of MMP (TIMP)-1 and plasminogen activator inhibitor (PAI)-1 expression in AT correlated significantly to the amount of AT in all compartments (rs = 0.41–0.53, all p ≤ 0.01), and to insulin sensitivity, insulin, C-peptide, waist circumference and body mass index (BMI) (rs = 0.25–0.57, all p ≤ 0.05). MMP-9 was 5.3 fold higher in subjects with insulin sensitivity below median (p = 0.002) and 3.1 fold higher in subjects with BMI above median level (p = 0.013). In our healthy non-obese middle-aged population AT-expressed genes, central in remodeling of ECM, associated strongly with the amount of abdominal AT, overweight and insulin sensitivity, indicating AT-remodeling to play a role also in non-obese individuals. The remodeling process seems furthermore to associate significantly with glucometabolic disturbances.Trial registration: ClinicalTrials.gov, NCT01412554. Registered 9 August 2011, https://clinicaltrials.gov/ct2/show/NCT01412554?term=NCT01412554.

Effect of Regulating the Expression of HSP47 on Collagen Metabolism in Scleral Fibroblasts

ABSTRACT Purpose To explore the effect of heat shock protein 47 (HSP 47) on collagen (types I, III, and V) metabolism in scleral fibroblasts. Methods Scleral fibroblasts with over- or low-expression of HSP 47 were constructed by plasmid transfection. The mRNA and intracellular proteins expression of HSP 47, collagen (types I, III, and V) and α-smooth muscle actin (α-SMA) were detected by quantitative real-time polymerase chain reaction and western blot. The proteins expression of collagen (types I, III, and V), matrix metalloproteinase 2 (MMP-2), and tissue inhibitor of matrix metalloproteinases 1 and 2 (TIMP-1 and −2) in extracellular matrix (ECM) were detected by ELISA. The migration and proliferation activities of cells were detected by scratch-wound assay and MTS. The internal structure of scleral fibroblasts was observed by transmission electron microscopy (TEM). Results The results obtained demonstrated significant increases in the expression of the mRNA and protein expression of collagen I in HSP47 up-regulated cells. Overexpression of HSP 47 promotes the expression of α-SMA and cell migration. Down-regulated expression of HSP 47 results in decreased mRNA and protein expression of collagen. Low expression of HSP 47 significantly inhibits cell migration and proliferation, and affects the internal structure of cells. Conclusion HSP 47 affects collagen metabolism in scleral fibroblasts. It appears to promote the synthesis and secretion of collagen I as well as inhibit degradation.

Galectin-3: role in the formation of various hemodynamic phenotypes of heart failure and interaction with some neurohumoral factors

Aim. To study the interaction of serum galectin-3 (Gal-3) with fibrogenic factors, cardiac remodeling, as well as parameters of central hemodynamics in various hemodynamic phenotypes of heart failure (HF). Material and methods . A total of 210 male and female patients with HF were examined (mean age 64,1±1,4 years). HF was established on the basis of patient complaints, medical history, physical examination and laboratory tests using the 2016 ESC Chronic Heart Failure Guidelines. In addition to standard diagnostic tests, enzyme immunoassays were performed: serum Gal-3, aldosterone, matrix metalloproteinase 1 (MMP1), tissue inhibitor of matrix metalloproteinase 1 (TIMP1). Results. Patients were divided into two groups: group I (n=59) — patients with HF with reduced ejection fraction (LVEF <40%), group II (n=56) — patients with HF with midrange EF (LVEF 41-49%), group III (n=95) — patients with HF with preserved EF (LVEF >50%). Reference values of studied substances were as follows: Gal-3 — 8,6 [3,7; 11,7] ng/ml; aldosterone — 86,8 [47,8; 199,1] pg/ml; MMP1 — 14,5 [8,5; 18,7] ng/ml; TIMP1 — 87,4 [68,6; 115,2] ng/ml. In patients with HF, the levels of Gal-3, aldosterone and TIMP1 increased with the disease functional class (FC). In patients of group I, it significantly increased by 1,7-2,5 (p<0,01), 4,1-5,9 (p<0,05) and 4,1-5,7 (p<0,05); in group II, 1,8-2,8 (p<0,01), 5,6-6,8 (p<0,01) and 6,1-8,3 (p<0,01); in group III, by 2,1-3,1 (p<0,01), 6,1-6,9 (p<0,01) and 6,8-9,3 times (p<0,01), respectively. A positive correlation was established with FC: in groups I and II, Gal-3 had a significant negative relationship with LVEF (p<0,001; p<0,01, respectively); in group III, Gal-3 had a moderate positive relationship with LV posterior wall thickness (p<0,05), interventricular septum thickness (p<0,05), the left ventricle mass index (p<0,01) and LV relative wall thickness (p<0,01). Conclusion. Levels of Gal-3 and aldosterone increased with HF FC and had a relevant relationship with the activation of some other neurohumoral factors. Gal-3 can be used as an early biomarker of myocardial fibrosis and cardiac remodeling, in predicting and evaluating risk factors, clinical course and outcome of the disease, as well as to assess the effectiveness of treatment in patients with these HF phenotypes.

Effects of interleukin-6 and tumor necrosis factor-α on the expression of angiogenic and collagenolytic factors in premature and mature adipocytes

ObjectiveThe remodeling of the vascular network and collagen in the extracellular matrix is closely associated with the expansion and dysfunction of adipose tissue. In the present study, we investigated the effects of interleukin (IL)-6 and tumor necrosis factor (TNF)-α on the expression of angiogenic factors, collagen, and collagenase and its endogenous inhibitor in premature and mature adipocytes. MethodsPremature and mature adipocytes were differentiated from 3T3-L1 cells and stimulated with IL-6 or TNF-α to mimic the early and late phases of obesity development. The levels of expression of angiogenic factors, including vascular endothelial cell growth factor a (Vegfa), hepatocyte growth factor (Hgf), angiopoietin (Angpt)1, and Angpt2, as well as type I collagen, matrix metallopeptidase (Mmp) 13, and tissue inhibitor of Mmp (Timp) 1, were determined using real-time reverse transcription polymerase chain reaction or enzyme-linked immunosorbent assay. Human umbilical vein endothelial cells were grown with the culture supernatant of adipocytes stimulated with/without IL-6 or TNF-α, and the formation of tube structures was evaluated. ResultsIL-6 and TNF-α induced the expression of Vegfa, Hgf, and Angpt2 and decreased the expression of Angpt1 in premature adipocytes, whereas, they decreased the expression of Vegfa and Hgf in mature adipocytes. The culture supernatant of IL-6- or TNF-α-stimulated premature adipocytes induced the formation of tube structures. IL-6 and TNF-α had no effects on type I collagen expression in both premature and mature adipocytes but suppressed the expression of Mmp13 and Timp1 in mature and premature adipocytes, respectively. ConclusionThe effects of IL-6 and TNF-α on the expression of angiogenic and collagenolytic factors differed between premature and mature adipocytes. This finding suggests that these inflammatory cytokines induce expansion and dysfunction of adipose tissue via angiogenesis and collagen turnover in premature and mature adipocytes.

Baihe Wuyao decoction ameliorates CCl4-induced chronic liver injury and liver fibrosis in mice through blocking TGF-β1/Smad2/3 signaling, anti-inflammation and anti-oxidation effects

Ethnopharmacological relevanceBaihe Wuyao decoction (BWD), a prescription of Traditional Chinese Medicines, composed of Lilium brownii var. viridulum Baker.(Lilii Bulbus) and Lindera aggregata (Sims) Kosterm. (Linderae Radix), has been used to treat epigastric pain and superficial gastritis for hundreds of years in China. Recently, some compounds obtained from Lilii Bulbus and Linderae Radix had active effects of hepatic protection or liver fibrosis alleviation. Thus, we aim to evaluate the effects of BWD on treatment of chronic liver injury and liver fibrosis induced by carbon tetrachloride (CCl4) and to elucidate the possible molecular mechanism. Materials and methodsMice were treated with BWD (low, medium and high dose), diammonium glycyrrhizinate or vehicle by oral gavage once daily, simultaneously intraperitoneal injected with a single dose of CCl4 (1 μl/g body weight) twice a week for consecutive 6 weeks. Next, all mice were sacrificed after fasted 12 h, and serums and liver tissues were harvested for analysis. The hepatic injury was detected by serum biomarker assay, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The hepatic histology and collagen were illustrated by hematoxylin-eosin staining and Sirius red staining respectively. The antioxidant capacity of liver tissues was evaluated by the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in liver homogenization. The mRNA gene or protein expressions related to fibrosis, oxidative stress and inflammation molecules were performed by real-time quantitative PCR (RT-PCR) or Western-blot. ResultsBWD exhibited a good hepatic protection with ameliorating liver histological changes, decreasing serum AST and ALT contents, and reducing hepatic fibrosis with stimulation ECMs (such as Collagen1 and Collagen3) degradation. BWD inhibited hepatic stellate cells (HSCs) activation, promoted matrix metalloproteinase-2 (MMP2), MMP9, and MMP12 while suppressing tissue inhibitors of matrix metalloproteinase-1 (TIMP1) expression, and blocked traditional fibrosis TGF-β1/Smad2/3 signal pathway. Moreover, BWD exhibited anti-inflammation effect proved by the reduction of liver Interleukin-1β (IL-1β), TNF-α, IL-11 mRNA levels and promoted anti-oxidation effects determined by inhibition of liver MDA and iNOS levels while promoting liver SOD and Mn-SOD. ConclusionBWD ameliorates CCl4-induced CLI and liver fibrosis which is correlated to its blocking TGF-β1/Smad2/3 signaling, anti-inflammation, and anti-oxidation effects. BWD, as a small traditional prescription, is a promising treatment for CLI and liver fibrosis through multiple pharmacological targets.

METRONIDAZOLE GEL EFFECT ON RATS WITH BACTERIA-INDUCED PERIODONTITIS

Background: Periodontitis is an inflammatory disease in the periodontal tissue and causes progressive tissue damage. Tissue Inhibitor of Matrix Metalloproteinases-1 (TIMP-1) is directly responsible for the pathogenesis of periodontal diseases. Topical application of the metronidazole gel as a therapy for periodontal diseases can be used for additional therapy. This study aimed to analyze the effect of Metronidazole gel on periodontitis based on TIMP-1 levels in bacteria-induced periodontitis in rats. Methods: Twenty Sprague Dawley rats weighing 170-200 gram aged two months. Rats were divided into two groups: the control group and Metronidazole gel (Ti-es Metronidazole gel, 0.45 mg). Metronidazole gel was topically applied to the gingival sulcus. The gingival crevicular fluid sample was taken on day 3 with periopaper subjected to the Tissue Inhibitor of Matrix Metalloproteinase-1 level evaluation using the ELISA Method. Results: Normal reference Tissue Inhibitor of Matrix Metalloproteinase 1 levels gingival crevicular fluid 0.156-10 ng/ml. Tissue Inhibitor of Matrix Metalloproteinase 1 gingiva crevicular fluid in control and metronidazole gel was 1.34 ng/ ml and 3.57 ng /ml. There was a significant difference between control and metronidazole gel group (p<0.005) Conclusion: The Application of Metronidazole gel affects TIMP-1 levels in periodontitis.

The renin-angiotensin system in cutaneous hypertrophic scar and keloid formation.

Hypertrophic scar and keloid are two types of fibroproliferative conditions that result from excessive extracellular matrix production. The underlying pathological mechanism is not entirely clear. Activation of the renin-angiotensin system (RAS) is associated with fibrosis in various organs. RAS components including angiotensin II (Ang II), angiotensin AT1 and AT2 receptors, and angiotensin-converting enzyme (ACE) are expressed in the skin and act independently from the plasma RAS. AT1 receptors, which are usually the dominating receptor subtype, promote fibrosis and scar formation, while AT2 receptors inhibit the aforementioned AT1 receptor-coupled effects. Elevated angiotensin II (Ang II) levels acting on the AT1 receptor contribute to skin scar formation through increased expression of inflammatory factors such as interleukin-6 (IL-6), angiogenic factors such as vascular endothelial growth factor (VEGF) and fibrinogenic factors such as transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF), while at the same time suppressing the anti-fibrotic tissue inhibitors of matrix metalloproteinase (TIMPs). First, small clinical trials have provided evidence that inhibition of the ACE/Ang II/ AT1 receptor axis may be effective in the treatment of hypertrophic scars/keloids. This review provides a detailed overview of the current literature on the RAS in skin, wound healing and scar formation and discusses the translational potential of targeting this hormonal system for treatment and prevention of hypertrophic scars and keloids.

MAb NJ001 inhibits lung adenocarcinoma invasiveness by directly regulating TIMP-3 promoter activity via FOXP1 binding sites.

BackgroundPreviously, we developed a monoclonal antibody (mAb) NJ001 that binds to the antigen SP70 in human non‐small cell lung cancer (NSCLC) cells and showed it could inhibit lung adenocarcinoma (AD) growth. Here, we investigated the effect and mechanisms of NJ001 in lung AD metastasis.MethodsHuman lung AD cells (SPC‐A1 and A549) were treated with different concentrations of mAb NJ001, and the effects of NJ001 on cell migration and invasive activity were investigated using wound‐healing and Matrigel assays, respectively. The molecular mechanism of this inhibition was explored by microarrays, qRT‐PCR, western blot, luciferase assays and electrophoretic mobility shift assays (EMSA).ResultsMAb NJ001 markedly suppressed lung AD cell migration; and the invasiveness of SPC‐A1 and A549 cells treated with mAb NJ001 was diminished by 65%. Tissue inhibitor of matrix metalloproteinase‐3 (TIMP‐3) was highly expressed in SPC‐A1 cells treated with mAb NJ001, whereas knockdown of TIMP‐3 by shRNA significantly increased SPC‐A1 and A549 invasiveness. MAb NJ001 affects lung AD by inhibiting TIMP‐3 through direct transcriptional regulation of FOXP1 binding sites in the TIMP‐3 promoter region, as shown in luciferase assays and EMSA.ConclusionsMAb NJ001 inhibits invasiveness and metastasis in lung AD through the FOXP1 binding sites in the TIMP‐3 promoter region. It may have clinical applications in preventing and treating metastatic lung AD.

TLR4-mediated hippocampal MMP/TIMP imbalance contributes to the aggravation of perioperative neurocognitive disorder in db/db mice

Although type 2 diabetes is an important predictor of perioperative neurocognitive disorder (PND), little is currently known about its mechanism of action. Adult male db/db and db/m mice were subjected to four different treatments, including either sham or tibial fracture surgery as well as intraperitoneal injection of vehicle or TAK-242 (the selective inhibitor of TLR4) at 1, 24, and 48 h after surgery. The fear conditioning test was performed to detect cognitive impairment on post-operative day (POD) 3. The hippocampus was collected on POD 1 for western-blots and on POD 3 for western-blots, transmission electron microscopy, and electrophysiological experiments. Toll-like receptor 4 (TLR4) inhibition reversed more profound decline in the freezing behavior of db/db mice on POD 3. The surgery reduced the slope of hippocampal field excitatory postsynaptic potentials, and induced blood-brain barrier (BBB) damage in db/db mice on POD 3. The surgery also increased protein levels of TLR4, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, albumin, matrix metalloproteinase (MMP)-2, and MMP-9, and decreased protein levels of claudin-5, occludin, tissue inhibitor of matrix metalloproteinase (TIMP)-1, and TIMP-2 in the hippocampus of db/db and db/m mice. These changes were all reversed by TAK-242 treatment. At last, compared with those in post-operative db/m mice, the surgery increased protein levels of TLR4, TNF-α, and IL-1β, decreased protein levels of claudin-5 and occludin, and sustained the MMP/TIMP imbalance in the hippocampus of db/db mice on POD 3. Our results suggest that TLR4-mediated aggravated hippocampal MMP/TIMP imbalance, BBB disruption, sustained inflammatory cytokine release, and impairment of long-term potentiation play a key role in tibial fracture surgery-induced persistent PND in db/db mice.

TIMP3/TGF‑β1 axis regulates mechanical loading‑induced chondrocyte degeneration and angiogenesis.

Chondrocytes in injured cartilage tissue are susceptible to mechanical loading; mechanical overloading can induce cartilage degeneration. The aim of the present study was to investigate whether mechanical loading can regulate chondrocyte degeneration and angiogenesis via the tissue inhibitor of matrix metalloproteinase-3 (TIMP3)/transforming growth factor (TGF)-β1 axis. Primary human chondrocytes were obtained from knee articular cartilage of a healthy donor. Then, normal chondrocytes or TIMP3 lentivirus-transfected (LV-TIMP3) chondrocytes were subjected to mechanical loading (10 MPa compression). Then, chondrocytes were stimulated with 1 µg/ml lipopolysaccharide (LPS) or treated with LDN-193189 (inhibitor of TGF-β1 signaling pathway). In addition, human umbilical vein endothelial cells (HUVECs) were co-cultured with chondrocytes or LV-TIMP3 chondrocytes. The expression levels of collagen-I, proteoglycan, TIMP3, TGF-β1, Smad2 and Smad3 were detected by reverse transcription-quantitative PCR and western blotting. Moreover, cell apoptosis and viability were determined using flow cytometry and MTT analysis, while cell migration was observed by Transwell assays. In addition, the vascular endothelial growth factor (VEGF)/VEGF receptor (R)2 binding rate in HUVECs was detected by a solid-phase binding assay. It was demonstrated that mechanical loading significantly inhibited the expression levels of collagen-I and proteoglycan in chondrocytes, as well as reducing cell proliferation and promoting cell apoptosis. In addition, the expression levels of TIMP3, TGF-β1, phosphorylated (p)-Smad2 and p-Smad3 were significantly decreased in degenerated chondrocytes that were induced by LPS, as well as in chondrocytes treated with LDN-193189. Furthermore, TIMP3 overexpression suppressed cell migration and reduced the VEGF/VEGFR2 binding rate in HUVECs. Mechanical loading significantly inhibited the expression levels of TIMP3, TGF-β1, p-Smad2 and p-Smad3 in chondrocytes, and also increased cell migration of HUVECs; TGF-β1 treatment or TIMP3 overexpression reversed these effects. Thus, the TIMP3/TGF-β1 axis may be a vital signaling pathway in mechanical loading-induced chondrocyte degeneration and angiogenesis.

Effect of Sacubitril/Valsartan on Biomarkers of Extracellular Matrix Regulation in Patients With HFpEF

BackgroundMyocardial fibrosis may contribute to the pathophysiology of heart failure with preserved ejection fraction. Given the biochemical targets of sacubitril/valsartan, this study hypothesized that circulating biomarkers reflecting the mechanisms that determine extracellular matrix homeostasis are altered by sacubitril/valsartan compared with valsartan alone. ObjectivesThis study investigated the effects of sacubitril/valsartan on biomarkers of extracellular matrix homeostasis and the association between biomarkers and the primary endpoint (total heart failure hospitalizations and cardiovascular death). MethodsN-terminal propeptide of collagen I and III, tissue inhibitor of matrix metalloproteinase 1, carboxyl-terminal telopeptide of collagen type I, and soluble ST2 were measured at baseline (n = 1,135) and 16 (n = 1,113) and 48 weeks (n = 1,016) after randomization. The effects of sacubitril/valsartan on these biomarkers were compared with those of valsartan alone. Baseline biomarker values and changes from baseline to 16 weeks were related to primary endpoint. ResultsAt baseline, all 5 biomarkers were higher than published referent control values. Sixteen weeks after randomization, sacubitril/valsartan decreased tissue inhibitor of matrix metalloproteinase 1 by 8% (95% confidence interval [CI]: 6% to 10%; p < 0.001), soluble ST2 by 4% (95% CI: 1% to 7%; p = 0.002), and N-terminal propeptide of collagen III by 3% (95% CI: 0% to 6%; p = 0.04) and increased carboxyl-terminal telopeptide of collagen type I by 4% (95% CI: 1% to 8%; p = 0.02) compared with valsartan alone, consistently in men and women and patients with left ventricular ejection fraction above or below the median of 57%. Higher levels of tissue inhibitor of matrix metalloproteinase 1 and soluble ST2 at baseline and increases in these markers at 16 weeks were associated with higher primary endpoint event rates. ConclusionsBiomarkers reflecting extracellular matrix homeostasis are elevated in heart failure with preserved ejection fraction, favorably altered by sacubitril/valsartan, and have important prognostic value. (Prospective Comparison of ARNI With ARB Global Outcomes in HF With Preserved Ejection Fraction [PARAGON-HF]; NCT01920711)

Circulating TIMP-1 is associated with hematoma volume in patients with spontaneous intracranial hemorrhage

Matrix metalloproteinases (MMPs) are proteolytic zinc-endopeptidases regulated by tissue Inhibitors of matrix metalloproteinases (TIMPs). We evaluated the potential of MMPs and TIMPs as clinical tools for Intracranial Haemorrhage (ICH). Spontaneous non‐traumatic ICH patients were recruited from two hospitals: Complejo Hospitalario de Navarra (CHN = 29) and Vall d´Hebron (VdH = 76). Plasmatic levels of MMP-1, −2, −7, −9, −10 and TIMP-1 and their relationship with clinical, radiological and functional variables were evaluated. We further studied the effect of TIMP-1 (0.05–0.2 mg/Kg) in an experimental tail-bleeding model. In CHN, TIMP-1 was associated with admission-hematoma volume and MMP-7 was elevated in patients with deep when compared to lobar hematoma. In VdH, admission-hematoma volume was associated with TIMP-1 and MMP-7. When data from both hospitals were combined, we observed that an increase in 1 ng/ml in TIMP-1 was associated with an increase of 0.14 ml in haemorrhage (combined β = 0.14, 95% CI = 0.08–0.21). Likewise, mice receiving TIMP-1 (0.2 mg/Kg) showed a shorter bleeding time (p < 0.01). Therefore, the association of TIMP-1 with hematoma volume in two independent ICH cohorts suggests its potential as ICH biomarker. Moreover, increased TIMP-1 might not be sufficient to counterbalance MMPs upregulation indicating that TIMP-1 administration might be a beneficial strategy for ICH.

Influence of plasma matrix metalloproteinase levels on longitudinal changes in Alzheimer's disease (AD) biomarkers and cognitive function in patients with mild cognitive impairment due to AD registered in the Alzheimer's Disease Neuroimaging Initiative database

ObjectiveThe present study investigated the effects of plasma matrix metalloproteinases (MMPs) on longitudinal changes in Alzheimer's disease (AD)-related biomarkers in cerebrospinal fluid (CSF), brain atrophy, and cognitive function in patients with mild cognitive impairment due to AD (MCI-AD). MethodsWe used data from the Alzheimer's Disease Neuroimaging Initiative database. We included 95 ApoE4-positive patients with MCI-AD who were confirmed to have low Aβ42 and/or high phosphorylated-tau (p-tau) in CSF. We obtained baseline demographic data, plasma MMP levels, including MMP-1, −2, −7, −9, −10, and tissue inhibitor of MMP-1 (TIMP-1), longitudinal annual data on Aβ42, total tau, and p-tau in CSF, MRI-measured hippocampal volumes, and cognitive function evaluated by the Mini-Mental State Examination (MMSE) and AD Assessment Scale-11 (ADAS-11) over 4 years. We examined the effects of baseline MMP levels on longitudinal changes in CSF AD biomarkers, hippocampal volumes, and cognitive function using a linear mixed regression analysis. ResultsNo significant differences were observed in baseline plasma MMP levels between MCI-AD patients and control subjects, except for MMP-10, which was significantly lower in MCI-AD than in controls. The baseline levels of MMPs did not correlate with longitudinal changes in CSF biomarkers. Declines in hippocampal volumes and cognitive function evaluated by MMSE and ADAS-11 were significantly faster in MCI-AD patients with high-MMP-9 levels at baseline than in those with middle and low MMP-9 levels at baseline. ConclusionHigh plasma MMP-9 levels in MCI-AD patients might enhance neurodegeneration and cognitive decline.

Irisin Recovers Osteoarthritic Chondrocytes In Vitro.

Physical exercise favors weight loss and ameliorates articular pain and function in patients suffering from osteoarthritis. Irisin, a myokine released upon muscle contraction, has demonstrated to yield anabolic effects on different cell types. This study aimed to investigate the effect of irisin on human osteoarthritic chondrocytes (hOAC) in vitro. Our hypothesis was that irisin would improve hOAC metabolism and proliferation. Cells were cultured in growing media and then exposed to either phosphate-buffered saline (control group) or human recombinant irisin (experimental group). Cell proliferation, glycosaminoglycan content, type II/X collagen gene expression and protein quantification as well as p38/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK), protein kinase B (Akt), c-Jun N-terminal kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) involvement were evaluated. Furthermore, gene expression of interleukin (IL)-1 and -6, matrix metalloproteinase (MMP)-1 and -13, inducible nitric oxide synthase (iNOS), and tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -3 were investigated following irisin exposure. Irisin increased hOAC cell content and both type II collagen gene expression and protein levels, while decreased type X collagen gene expression and protein levels. Moreover, irisin decreased IL-1, IL-6, MMP-1, MMP-13 and iNOS gene expression, while increased TIMP-1 and TIMP-3 levels. These effects seemed to be mediated by inhibition of p38, Akt, JNK and NFκB signaling pathways. The present study suggested that irisin may stimulate hOAC proliferation and anabolism inhibiting catabolism through p38, Akt, JNK, and NFκB inactivation in vitro, demonstrating the existence of a cross-talk between muscle and cartilage.

TIMP1 down-regulation enhances gemcitabine sensitivity and reverses chemoresistance in pancreatic cancer

The therapeutic effect of gemcitabine (GEM) in pancreatic ductal adenocarcinoma (PDAC) is limited due to low drug sensitivity and high drug resistance. Tissue inhibitor of matrix metalloprotease 1 (TIMP1) is reportedly associated with GEM resistance in PDAC. However, the effect of TIMP1 down-regulation in combination with GEM treatment is unknown. We analyzed the expression of TIMP1 in human PDAC tissue using western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry. TIMP1 was highly expressed in PDAC specimens. Kaplan-Meier survival analysis suggested that a higher level of TIMP1 was correlated with poorer overall survival in 103 PDAC patients. The mRNA and protein expression profiles of TIMP1 were explored in the HTERT-HPNE human pancreatic ductal epithelium cell line, five PDAC cell lines (MIA PaCa-2, PANC-1, BxPC-3, Capan2, and SW1990), and two GEM-resistant PDAC cell lines (MIA PaCa-2R and PANC-1R). Compared with HTERT-HPNE, TIMP1 was highly expressed in the PDAC cell lines. In addition, TIMP1 was upregulated in GEM-resistant PDAC cell lines compared with their parental cells. When TIMP1 was knocked-down using short hairpin RNA, GEM-induced cytotoxicity and apoptosis were increased, while colony formation was repressed in MIA PaCa-2, PANC-1, and their GEM-resistant cells. When Bax was activated by BAM7 or Bcl-2 was inhibited by venetoclax, CCK-8 assays demonstrated that GEM sensitivity was restored in GEM-resistant cells. When Bax was down-regulated by siRNA, CCK-8 assays verified that GEM sensitivity was decreased in PDAC cells. The observations that TIMP1 knockdown enhanced GEM sensitivity and reversed chemoresistance by inducing cells apoptosis indicated cooperative antitumor effects of shTIMP1 and GEM therapy on PDAC cells. The combination may be a potential strategy for PDAC therapy.

Three Tiaobu Feishen formulae reduces cigarette smoke-induced inflammation in human airway epithelial cells.

To investigate the therapeutic efficacy of Tiaobu Feishen formulae (TBFS) on cigarette smoke-induced inflammation in vitro using lipopolysaccharide (LPS)-induced and cigarette smoke extract (CSE)-induced NCI-H292 cells. We evaluated the inhibitory effects of Bufei Jianpi formula (BJF), Bufei Yishen formula (BYF), and Yiqi Zishen formula (YZF) on the expressions of inflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-8, matrix metalloproteinase (MMP)-9, tissue inhibitor of matrix metalloprotease (TIMP)-1, and superoxide dismutase (SOD) in H292 cells stimulated with LPS or CSE. Their related transcription factors and signaling pathways were also analyzed. BJF, BYF, and YZF significantly inhibited the LPS- or CSE-induced expressions of TNF-α, IL-8, MMP-9, TIMP-1, and SOD in H292 cells, and suppressed the activation of transcription factors including nuclear transcription factor (NF)-κB, activator protein (AP)-1, and signal transducers and activators of transcription (STAT) 3 and their corresponding pathways, including NF-κB, mitogen-activated protein kinase (MAPK), STAT3, and peroxisome proliferator-activated receptor (PPAR). BJF, BYF, and YZF effectively suppressed inflammatory responses, protease-antiprotease imbalance, and oxidative stress induced by LPS and CSE, an effect that was closely associated with the inhibition of the NF-κB, MAPK, STAT3, and PPAR pathways.

1448-P: Enhanced Liver Fibrosis (ELF) Test Can Accurately Predict Advanced Fibrosis in Patients with Type 2 Diabetes and Nonalcoholic Fatty Liver Disease (NAFLD)

Background: The ELF test (score calculated from markers of fibrosis: hyaluronic acid, procollagen III amino-terminal peptide, and tissue inhibitor of matrix metalloproteinase 1) is a non-invasive blood test used to estimate fibrosis stage in chronic liver disease. Since NAFLD patients with advanced fibrosis are at the highest risk for adverse long-term outcomes, the aim is to assess performance of ELF for identification of advanced fibrosis in patients with NAFLD. Methods: Patients with biopsy-proven NAFLD were included. ELF scores were calculated using an ADVIA Centaur XP analyzer. Results: There were 409 patients with NAFLD: 47 ± 12.9 years, 30% male, 70% white, 34% type 2 diabetes (T2DM), 56% hyperlipidemia, 54% hypertension, mean (SD) BMI 41.0 ± 10.0 kg/m2, ALT 48.4 ± 42.0 U/L, mean ELF score was 9.0 ± 1.3. Of the study cohort, 23% patients (N=93) had advanced fibrosis. Patients with advanced fibrosis had significantly higher ELF: 10.2 ± 1.3 vs. 8.6 ± 1.0, p&amp;lt;0.0001. There was a correlation between ELF and liver enzymes: rho=0.23 for ALT, 0.34 for AST (p&amp;lt;0.0001). ELF negatively correlated with BMI: rho = -0.28 (p&amp;lt;0.0001). Across all NAFLD patients, ELF had good performance for identifying patients with advanced fibrosis: area under curve (AUC) = 0.83 (0.79 - 0.87). The performance of ELF was similar in NAFLD patients with [AUC = 0.81 (0.73 - 0.87)] and without [AUC = 0.83 (0.77 - 0.87)] T2DM. With the commonly used cut-off of ELF ≥9.8 for prediction of advanced fibrosis, observed accuracy was as follows: sensitivity 62.4% (51.7% - 72.2%), specificity 89.6% (85.6% - 92.7%) positive predictive value (PPV) 63.7% (55.1% - 71.6%), negative predictive value (NPV) 89.0% (86.1% - 91.3%). Cut-off of 11.3 returned almost perfect specificity 99.0% (97.3% - 99.8%) with a sensitivity of 22.6% (14.6% - 32.4%), PPV 87.5% (68.1% - 95.8%), NPV 81.3% (79.6% - 82.9%). Conclusions: ELF test performs well in identifying high risk NAFLD patients with or without T2DM. Disclosure Z. Younossi: Consultant; Self; AbbVie Inc., Bristol-Myers Squibb, Gilead Sciences, Inc., Intercept Pharmaceuticals, Inc., Novo Nordisk Inc., Terns and Viking. S. Felix: None. T. Jeffers: None. E. Younossi: None. F. Nader: None. H. Pham: None. A. Afendy: None. R. Cable: None. A. Racila: None. Z. Younoszai: None. B.P. Lam: None. P. Golabi: None. L. Henry: None. S. Clement: None. M. Stepanova: None.

P1613INFLUENCE OF GENETIC POLYMORPHISM IN MMPS AND TIMPS ON ALLOGRAFT OUTCOME IN RENAL TRANSPLANT RECIPIENTS

Abstract Background and Aims Renal transplantation (RT) is currently the most effective replacement therapy and believed as a boon for patients with end-stage renal disease (ESRD). Allograft rejection remains to be one of the crucial impediments in successful renal transplantation. Matrix metalloproteinases (MMPs) and their natural inhibitors (tissue inhibitors of matrix metalloproteinases; TIMPs) play an important role in immune-mediated tissue destruction of the allograft by allowing influx of leucocytes and mononuclear cells into the graft. The study of MMPs and TIMPs in transplant rejection may also lead to novel approaches in the therapy of rejection processes. To investigate the association of functional polymorphisms in MMP-1 (-1607 1G/2G), (-519 A/G); MMP-3(5356 A/G), (1161 A/G); MMP-9 (1721G/C), (Q279R A/G), (R668Q G/A); TIMP1(+536 C/T) and TIMP3(-1298 C/T gene with risk of allograft rejection in renal transplant recipients of North India. Method A total of two hundred live related renal transplant donors and recipients pairs blood sample was collected and were genotyped for MMP-1 (-1607 1G/2G), (-519 A/G); MMP-3(5356 A/G), (1161 A/G); MMP-9 (1721G/C), (Q279R A/G), (R668Q G/A); TIMP1(+536 C/T) and TIMP3(-1298 C/T) gene polymorphisms by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) methodology. All two hundred renal transplant recipients were categorized into 162 non-rejecters and 32 rejecters and were analyzed further. All the statistical analysis was done using SPSS software. Results The genotype frequencies of MMP1 (-1607 1G/2G) [2G/2G; odds ratio (OR) = 2.97, 95% confidence intervals (95% CI) = 1.69-5.20, P ≤ 0.05]; MMP9 (Q279R) [RQ; odds ratio (OR) = 0.47, 95% confidence intervals (95% CI) = 0.25-0.99, P = 0.02 &amp; QQ; odds ratio (OR) = 0.49, 95% confidence intervals (95% CI) = 0.25-0.94, P = 0.03]; MMP9 (P574R) [PR; odds ratio (OR) = 2.70, 95% confidence intervals (95% CI) = 1.77-4.13, P ≤ 0.05 &amp; RR; odds ratio (OR) = 2.70, 95% confidence intervals (95% CI) = 1.09-6.71, P = 0.03]; TIMP1(+536 C/T) [CT; odds ratio (OR) = 15.41, 95% confidence intervals (95% CI) = 9.12-26.05, P ≤ 0.05 &amp; TT; odds ratio (OR) = 7.01, 95% confidence intervals (95% CI) = 3.39-14.49, P ≤ 0.05] and TIMP3(-1298 C/T) [CT; odds ratio (OR) = 0.31, 95% confidence intervals (95% CI) = 0.19-0.50, P ≤ 0.05] was significantly associated with allograft rejection. On comparison of genotypic frequencies for MMP-1, MMP-3, MMP-9, TIMP1 and TIMP3 gene polymorphisms between rejecters and non-rejecters no significant difference was found while comparing genotypic frequencies of non-rejecters with donors a significant association was observed in MMP1 (-1607 1G/2G) [P ≤ 0.05]; MMP9 (Q279R) [P = 0.02]; MMP9 (P574R) [P ≤ 0.05]; TIMP1 [P ≤ 0.05] and TIMP 3 [P ≤ 0.05] respectively. Therefore, MMP1 (-1607 1G/2G), MMP9 (Q279R), MMP9 (P574R), TIMP1 and TIMP3 demonstrated significantly reduced risk for allograft rejection. MMP1 (-1607 1G/2G) [P ≤ 0.02]; MMP9 (P574R) [P ≤ 0.05]; TIMP1 [P ≤ 0.05] and TIMP3 [P =0.01] was also found significantly associated on comparison between rejecters and donors. Conclusion Mutant genotypes/ alleles for MMP1 (-1607 1G/2G); MMP9 (Q279R); MMP9 (P574R); TIMP1 (+536 C/T) and TIMP3 (-1298 C/T) are associated with reduced risk for allograft rejection and improved allograft survival in North Indian transplant recipients and could serve as an ideal marker to predict pre-transplant allograft outcome.

New Markers of Renal Failure in Multiple Myeloma and Monoclonal Gammopathies.

Multiple myeloma (MM) is a common plasma cell malignancy, which is responsible for significant mortality, often related to severe renal impairment (RI). Kidney injury can limit therapeutic choices and may often translate into poor outcomes, but it remains potentially reversible in a proportion of patients. The most accessible, conventional markers of RI are subject to several shortfalls, among which are the delayed onset following kidney insult, multiple interfering factors, and lesser sensitivity to mild changes in glomerular filtration. Neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C have accumulated large interest in MM-RI due to being very sensitive markers of renal injury, as well as indicators of tubular-glomerular axis impairment. Of interest, recent data suggest that prediction of acute kidney injury may be aided by urinary tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7), which both act to induce G1 cell cycle arrest, reflective of a state of pre-injury, and thus may be superior to other measures of kidney insult (NGAL, kidney injury molecule ((KIM-1)). Moreover, TIMP-2 seems to be a biomarker dedicated to distal tubular cells, whereas insulin-like growth factor-binding protein 7 (IGFBP7) secretion has been found in proximal tubule cells. IGFBP7 can also identify a subsection of the normal proximal nephron, even, maybe the one that is responding to insult. They may be adopted into a conceptual screening panel for MM-RI. Unfortunately, no biomarker is ideal (influence of non-renal, biologic factors), and novel measures are limited by economic constraints, availability, lack of standardization. With the emergence of more advanced diagnostic and prognostic MM models, markers reflective of disease processes (including RI) are of high interest. Candidate molecules also include peptidome markers.