Plasma Tissue Inhibitor Of Metalloproteinases-1 Research Articles

Estrogen-dependent gene regulation: Molecular basis ofTIMP-1 as a sex-specific biomarker for acute lung injury.

Increased circulating tissue inhibitor of metalloproteinases-1 (TIMP-1) levels have been observed in patients with acute lung injury (ALI). However, the sex-specific regulation of TIMP-1 and the underlying molecular mechanisms have not been well elucidated. In this study, we found that plasma TIMP-1 levels were significantly higher in COVID-19 and H1N1 patients compared with those in healthy subjects (n = 25). TIMP-1 concentrations were significantly different between males and females in each disease group. Among female but not male patients, TIMP-1 levels significantly correlated with the PaO2/FiO2 ratio and hospital length of stay. Using the mouse model of ALI induced by the H1N1 virus, we found that TIMP-1 is strikingly induced in PDGFRα-positive cells in the murine lungs. Moreover, female mice showed a higher Timp-1 expression in the lungs on day 3 postinfection. Mechanistically, we observed that estrogen can upregulate TIMP-1 expression in lung fibroblasts, not epithelial cells. In addition, overexpression of estrogen receptor α (ERα) increased the TIMP-1 promoter activity. In summary, TIMP-1 is an estrogen-responsive gene, and its promoter activity is regulated by ERα. Circulating TIMP-1 may serve as a sex-specific marker, reflecting the severity and worst outcomes in female patients with SARS-CoV2- and IAV-related ALI.

Determining the Effect of L-arginine and Physical Activity on the Plasma Levels of Endothelin-1 and Tissue Inhibitor of Metalloproteinase-1 in Postmenopausal Women with Hypertension

Background and Objectives Exercise and L-arginine supplementation are known as effective strategies for treating hypertension. The aim of the present study was to determine the effect of aerobic exercise and L-arginine consumption on tissue inhibitor of metalloproteinase-1 (TIMP-1) and endothelin-1 (ET-1) levels in postmenopausal women with hypertension. Subjects and Methods In this quasi-experimental research, 40 postmenopausal women with hypertension were divided into four equal groups including exercise, exercise + L-arginine, L-arginine, and placebo groups. Interventions of aerobic exercise (60-75% of maximum heart rate) and L-arginine supplementation (6 g/day) were completed for 12 weeks. Forty-eight hours after the last intervention session, blood samples similar to the pre-test were collected to measure TIMP-1 and ET-1 levels using ELISA method. Results Plasma ET-1 levels in the training and training + L-arginine groups were significantly reduced compared to the placebo and L-arginine groups. In addition, plasma TIMP-1 increased significantly in the exercise group and exercise + L-arginine group compared to the placebo group. No synergistic effect was observed on TIMP-1 and ET-1 levels, despite the greater effect of exercise combined with L-arginine supplementation. Conclusion According to the results of the present research, improved blood pressure in adaptation to aerobic exercises is associated with reduced ET-1 and increased TIMP-1. L-arginine supplement, either alone or in combination with aerobic exercise, has no significant effect on TIMP-1 and ET-1 changes.

Plasma TIMP-1 as a sex-specific biomarker for acute lung injury.

Acute respiratory distress syndrome (ARDS) confers high morbidity and mortality, with a death rate reaching 40%. Pre-clinical and clinical studies have cited sex-specific sex hormones as a critical contributor to divergent immunologic responses. Therefore, exploration of sex and sex hormone roles following lung injury and ARDS development is needed. Tissue inhibitor of metalloproteinase-1 (TIMP-1) was the first-discovered natural collagenase inhibitor and is located exclusively on the X chromosome. This study aimed to evaluate the prognostic role of circulating TIMP-1, and if concentration differences between males and females correlate with the mortality of ARDS patients. Human plasma samples from 100 ARDS patients enrolled in Albuterol to Treat Acute Lung Injury (ALTA) trial on the day of randomization were evaluated. The amount of TIMP-1 was measured using an enzyme-linked immunoassay (ELISA). Area under the receiver operating characteristic (AUROC) was computed to assess the predictive power of TIMP-1 for 30 and 90-day mortality. Chi-squared tests and Kaplan-Meier curves were computed to assess different variables and survival. AUROC analysis of TIMP-1 and 30-day mortality among females showed that TIMP-1 exhibited an AUC of 0.87 (95% confidence interval [CI] 0.78 to 0.97; P = 0.0014) with an optimal cut-off value of 159.7ng/mL producing a 100% sensitivity and 74% specificity. For 90-day mortality, AUROC analysis showed an AUC of 0.82 (95% confidence interval [CI] 0.67 to 0.97; P = 0.0016) with a similar cut-off value producing a 90% sensitivity and 76.47% specificity. Stratifying subjects by TIMP-1 concentration as high (≥ 159.7ng/mL) or low (< 159.7ng/mL) indicated that high TIMP-1 was associated with increased 30 and 90-day mortality rates (all P < 0.0001). Lastly, high TIMP-1 group was associated with worse other outcomes including ventilator-free days (VFDs) and ICU-free days (all P < 0.05). Circulating TIMP-1 appeared to be a promising biomarker for mortality among females with ARDS. The high TIMP-1 group showed worse VFDs and ICU-free days. Circulating TIMP-1 may be a sex-specific biomarker in the setting of ARDS and could improve ARDS phenotyping as well as provide a novel therapeutic target in females.

Plasma Levels of Matrix Metalloprotease MMP-9 and Tissue Inhibitor TIMP-1 in Caucasian Patients with Polypoidal Choroidal Vasculopathy

Background: Matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1) are regulating enzymes of the extracellular matrix. A systemic imbalance of MMP-9 and TIMP-1, thought to reflect an imbalance of the extracellular matrix homeostasis, is previously associated with polypoidal choroidal vasculopathy (PCV) in Asian patients. Previous studies suggest inter-ethnical differences in the genetic background and etiology of PCV. To further explore this issue, we studied the plasma levels of MMP-9 and TIMP-1 in Caucasian patients with PCV and compared to healthy age-matched controls. Methods: For this prospective case-control study, 60 participants were recruited who were either patients with PCV (n = 26) or healthy controls (n = 34). All participants underwent detailed clinical examination. We sampled fresh venous blood, isolated plasma, and quantified plasma concentrations of the extracellular matrix regulators MMP-9 and TIMP-1 using electrochemiluminescence immunoassays. Results: Plasma levels of MMP-9 (p = 0.4), TIMP-1 (p = 0.9), and MMP-9/TIMP-1 ratio (p = 0.4) did not differ significantly between patients with PCV and healthy controls. No differences appeared after adjusting for influencing co-variates in multivariate analyses. Conclusion: We demonstrate that Caucasian patients with PCV do not have altered levels of plasma MMP-9 or plasma TIMP-1. These findings suggest no strong evidence of a systemic imbalance of the extracellular matrix homeostasis in Caucasian patients with PCV. Our findings are in line with studies of other aspects of PCV that are also subject to significant inter-ethnical differences.

Plasma MMP-9, TIMP-1, and TGF-β1 Responses to Exercise-Induced Muscle Injury.

The purpose of this study was to analyze changes in the levels of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), and transforming growth factor-β1 (TGF-β1) in response to strength recovery after eccentric exercise in humans. In this study, 16 healthy males participated and were divided into a faster recovery (FR) group (n = 8, >80% of baseline) and a slower recovery (SR) group (n = 8, <40% of baseline) on the basis of their recovery of maximal isometric strength (MIS) 96 h after eccentric exercise. For both groups, measurements were taken of muscle soreness, creatine kinase (CK) activity, and MMP-9, TIMP-1, and TGF-β1 levels during the 24- to 96-h period after eccentric muscle contraction of their non-dominant elbow flexor. Muscle soreness (p < 0.001), CK activity (p < 0.01), and TGF-β1 level (p < 0.01) were significantly lower in the FR group compared with SR group, whereas no significant differences in MMP-9 and TIMP-1 levels were found between the two groups (p > 0.05). These results suggest that scar tissue formation caused by the pro-fibrotic activity of growth factors such as TGF-β1 is a potential cause of delay in strength recovery after exercise-induced muscle injury.

Procollagen type I carboxy-terminal propeptide (PICP) and MMP-2 are potential biomarkers of myocardial fibrosis in patients with hypertrophic cardiomyopathy.

Whether current proposed biomarkers of myocardial fibrosis (BMFs) actually reflect the changes in fibrous characteristics of myocardial tissue remains unclear. The relation between peripheral BMFs and histological myocardial fibrosis in patients with hypertrophic cardiomyopathy (HCM) has been unknown. We studied 52 HCM patients who underwent a transaortic extended septal myectomy. Complete medical history was collected, and related examinations were performed. Echocardiography and cardiovascular magnetic resonance were employed to characterize cardiac morphology and function. Procollagen type I carboxy-terminal propeptide (PICP), C-terminal telopeptide of type I collagen (CITP), matrix metalloproteinases (total MMP-2 and total MMP-9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) levels in both plasma and myocardial tissues were determined and compared. Myocardial fibrosis was detected with Masson's trichrome staining, and collagen volume fraction (CVF) was calculated. There was a significant correlation between plasma PICP levels and myocardial PICP contents (r=0.382, P=.007). Besides, plasma PICP (r=0.332, P=.020) levels correlated positively with CVF. In addition, plasma TIMP-1 levels were significantly correlated with myocardial TIMP-1 contents (r=0.282, P=.043). Plasma MMP-2 levels correlated positively with CVF (r=0.379, P=.006). Patients who took calcium channel blockers (CCBs; diltiazem or verapamil) had significantly lower plasma PICP levels, myocardial PICP content, and CVF in comparison with those who did not take CCBs. In patients with HCM, plasma PICP and MMP-2 levels quantitatively reflect myocardial fibrosis, suggesting that PICP and MMP-2 may be used as reliable BMFs. CCBs may attenuate cardiac fibrosis in patients with HCM.

Chromosome 4q25 variants and biomarkers of myocardial fibrosis in patients with atrial fibrillation.

Little is known about how genetic predisposition and fibrosis relate in atrial fibrillation (AF). Hence, we sought to determine whether the genetic variants and biomarkers for fibrosis enhance prediction of outcomes after catheter ablation. Consecutive patients who underwent catheter ablation of AF (paroxysmal, 158; nonparoxysmal, 137) or supraventricular tachycardia without AF (n = 70) were studied retrospectively. Plasma levels of transforming growth factorβ1 (TGF-β1), tissue inhibitor of metalloproteinase 1 (TIMP-1), and 4q25 single-nucleotide polymorphisms (SNPs) (rs10033464 and rs220073) were measured. Mean plasma levels of both TGF-β1 and TIMP-1 were higher in patients with AF than in the control (all P < .001). Plasma levels of TIMP-1 were higher in patients with recurrence compared with those without recurrence (P = .039). Patients with variant alleles of rs10033464 showed increased recurrence after catheter ablation in patients with paroxysmal AF including after adjustment (P = .027). Patients with TIMP-1 < 107 ng/mL and no variant allele (GG) at rs10033464 had lower recurrence rates compared with other groups in those with paroxysmal AF (logrank; P = .007), whereas there was no significant difference among those patients with persistent forms of AF. Inclusion of biomarkers and genotype improved discrimination of AF recurrence in patients with paroxysmal AF (C-statistic .499 vs .600). The combination of plasma TIMP-1 concentrations less than 107 ng/mL and the absence of a variant allele at rs10033464 was associated with lower recurrence rates in patients with paroxysmal AF. This study suggests that 4q25 SNPs and biomarkers for fibrosis may provide additive value in risk stratification for AF recurrence after catheter ablation.

Association of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 levels with diabetic foot ulcer in Egyptians with type 2 diabetes mellitus

Background The imbalance between matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) may play an important role in impaired healing of diabetic foot ulcer. Aim of the work This study was designed to investigate the association of MMP-9 and TIMP-1 levels in diabetic foot ulcer among Egyptian patients with type 2 diabetes mellitus attending the outpatient clinic of National institute of diabetes and endocrinology. Patients and methods This study was conducted on a total number of 90 participants comprising three groups: group I included 35 type 2 diabetic patients without foot ulcer. Group II included 35 type 2 diabetic patients having various degrees of foot ulcer complications. Group III included 20 apparently healthy participants (as controls). In addition to the routine clinical and laboratory investigations, MMP-9 and TIMP-1 were measured. All patients were randomly taken from the outpatient clinic of National institute of diabetes and endocrinology. Results The study results showed that the higher levels of plasma MMP-9 and the lower levels of TIMP-1 were associated with patients with diabetic foot ulcer. Conclusion The measurement of plasma MMP-9 and TIMP-1 in type 2 diabetic patients may help us to know the patients at most risk to develop diabetic foot ulcer.

Aldosterone Induces Tissue Inhibitor of Metalloproteinases-1 Expression and Further Contributes to Collagen Accumulation: From Clinical to Bench Studies.

Aldosterone induces myocardial fibrosis. Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a key factor of myocardial fibrosis. This study tested the hypothesis that aldosterone induces TIMP-1 expression and contributes to the fibrotic process. We prospectively enrolled 54 patients with primary aldosteronism, and measured plasma TIMP-1 and echocardiographic parameters. In the cell study, we investigated the possible molecular mechanism by which aldosterone induces TIMP-1 secretion and the effects on collagen accumulation. In the animal study, we measured serum TIMP-1 levels, cardiac TIMP-1 levels, and cardiac structure in an aldosterone infusion mouse model using implantation of aldosterone pellets. In patients with primary aldosteronism, plasma TIMP-1 was correlated with 24-hour urinary aldosterone, left ventricular mass, and impairment of left ventricular diastolic function. In human cardiac fibroblasts, TIMP-1 protein and mRNA expressions were significantly increased by aldosterone through the glucocorticoid receptor/PI3K/Akt/nuclear factor-κB pathway. TIMP-1 small-interfering RNA significantly reduced aldosterone-induced collagen accumulation, and aldosterone did not alter the levels of collagen1a1 or matrix metalloproteinase-1 mRNA. The aldosterone-induced TIMP-1 expression was inversely related to matrix metalloproteinase-1 activity. Furthermore, in the animal model, the serum and cardiac levels of TIMP-1 were significantly elevated in the mice that received aldosterone infusion. This elevation was blocked by RU-486 but not by eplerenone, suggesting that the effect was through glucocorticoid receptors. In a long-term aldosterone infusion model, serum TIMP-1 was associated with serum aldosterone level, cardiac structure, and fibrosis. In conclusion, aldosterone induced TIMP-1 expression in vivo and in vitro. This increased TIMP-1 expression resulted in enhanced collagen accumulation via the suppression of matrix metalloproteinase-1 activity.

Crucial Factors of the Inflammatory Microenvironment (IL-1 beta/TNF-alpha/TIMP-1) Promote Maintenance of the Malignant Hemopoietic Clone of Myelofibrosis By Stimulating the in Vitro Survival/Proliferation/Migration of Circulating CD34+ stem/Progenitor Cells

Introduction. Myelofibrosis (MF), an acquired clonal disorder of the hematopoietic stem/progenitor cell (HSPC) with a dysregulation in JAK/STAT signalling (mutations in JAK2, MPL and Calreticulin (CALR) genes), is characterized by a state of chronic inflammation. It is argued that the up-regulated production of proinflammatory cytokines by both HSPCs and the surrounding stromal cells generates a microenvironment that selects for the malignant clone. Only recently, it has been hypothesized that the sustained inflammatory microenvironment of MF can alter crucial biological processes, leading to genomic instability and cancer progression. Here we tested the in vitro functional effects of pivotal players of the inflammatory microenvironment (the extracellular ATP nucleotide and selected cytokines, such as Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α or the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)) on the HSPCs from MF patients.Methods: Circulating CD34+/CD34+ CD38- cells from MF patients (JAK2V617F (17 cases) and CALR (9 cases) mutations) or cord blood (CB; 8 samples) were phenotypically and functionally characterized after in vitro incubation with or without ATP (1000 μM), IL-1β (10 ng/mL), TNF-α (10 ng/mL) or TIMP-1 (100 ng/mL) (alone or in combination). Cells were then analyzed for survival/apoptosis (Annexin-V/Propidium Iodide staining), phenotype (evaluation of CD63 (TIMP-1 receptor), CXCR4 and CD38 expression), cell cycle and clonogenic capacity. Migration was assessed first towards a CXCL12 gradient in the presence or absence of the pro-inflammatory factors. In parallel experiments, CD34+ cells from MF patients were co-cultured with normal mesenchymal stromal cells (MSCs) in the presence or absence of the pro-inflammatory cytokines and then evaluated for their ability to migrate towards a CXCL12 gradient. Plasma TIMP-1, TNF-α, IL-1β and CXCL12 were measured by ELISA assay.Results: The plasma levels of TIMP-1, TNF-α, IL-1β, CXCL12 and the number of circulating CD34+, CD34+ CD38-, CD34+ CD63+, CD34+ CD184+ cells were increased in MF patients. According to mutational status, the CD34+ CD63+ cells were higher in the CALR+ patients.The survival of MF CD34+ cells was strongly stimulated by in vitro incubation with TNF-α or IL-1β as compared with the CB-derived CD34+ cells or untreated cells. By multiple cytokine combinations, IL-1β/TIMP-1, IL-1β /ATP or IL-1β /TNF-α treatments significantly promote the survival of MF CD34+ cells as compared with the normal counterparts or the untreated cells. Various combinations with IL-1β were also effective in stimulating survival of CD34+CD38- cells.IL-1β/TIMP-1 and IL-1β/TNF-α/TIMP-1, but not factors alone, significantly increased the CFU-C growth of MF patients as compared with the CB-derived counterparts and the untreated cells. Moreover, comparing CALR+ vs JAK2V617F+ patients, the colony formation of JAK2V617F+ patients was mainly promoted by the IL-1β/TNF-α treatment.Along with clonogenic capacity stimulation, exposure of CD34+ cells from MF patients to IL-1β/TNF-α/TIMP-1 significantly increases the S-phase cells, suggesting that these pro-inflammatory factors stimulated cell-cycle progression in dormant CD34+ MF cells.Migration of CD34+ cells from MF was significantly increased in CXCL12 treated cells. In addition, exposure of MF CD34+ cells to IL-1β/TNF-α, IL-1β/TIMP-1 or IL-1β/TNF-α/TIMP-1 significantly promotes cell migration in comparison with the CB-derived counterparts or SDF-1 alone. MF migrated cells in the presence of IL-1β/TNF-α significantly upregulate CD63 expression. Intriguingly, colony formation of MF migrated CD34+ cells in the presence of IL-1β/TNF-α or IL-1β/TNF-α/TIMP-1 was potently increased. Finally, co-culture systems with normal MSCs in the presence of pro-inflammatory factors revealed that MF CD34+ cells display increased migration ability toward CXCL12 gradient.Conclusions: Altogether our findings suggest that in MF the inflammatory niche plays a key role in the maintenance of the malignant clone. Thus, the interplay between the pro-inflammatory cytokines promote and select the HSPCs with higher proliferative activity, clonogenic potential and migration capability. Targeting these microenvironmental interactions may be a clinically relevant approach.D.F. and D.S. equally contributed DisclosuresMartinelli:Pfizer: Consultancy; Ariad: Consultancy; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy; AMGEN: Consultancy; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy.

Elevated circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) levels are associated with neuroendocrine differentiation in castration resistant prostate cancer.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a 28.5 kDa secreted glycoprotein that inhibits matrix metalloproteinase (MMP) activity. Our group has previously shown that elevated plasma TIMP-1 levels predict poor survival in metastatic castration-resistant prostate cancer (CRPC) patients; however, the underlying source and impact of elevated circulating TIMP-1 protein is unknown. In this study, we used qRT-PCR, ELISA and immunohistochemistry to evaluate TIMP-1 expression in androgen-sensitive and resistant prostate cancer (PC) cell lines, tumor tissues and patient sera, and to correlate TIMP-1 levels to expression of chromogranin A (CGA), an established marker of neuroendocrine differentiation (NED). We also explored the relationship between TIMP-1 overexpression and induction of NED by overexpressing TIMP-1 in androgen-sensitive LNCaP cells, as well as by inducing NED of LNCaP cells with IL-6. Patients with CRPC have significantly higher serum TIMP-1 levels compared to patients with hormone-sensitive disease. Although circulating TIMP-1 levels were increased, peripheral blood cells were not the source of elevation. Instead, elevated TIMP-1 expression was associated with higher expression of CGA in both blood and metastatic tumor tissue. We further show that androgen receptor (AR) and PSA non-expressing prostate cancer cell lines known to display NED phenotypes such as PC-3, PC-3M, and DU145 cells, expressed high levels of TIMP-1, in contrast to AR (+) and PSA (+) adenocarcinoma cell lines such as LNCaP, VCaP, and LAPC-4, which had barely detectable levels of TIMP-1. In addition, ectopic overexpression of TIMP-1 in LNCaP cells did not induce NED. However, TIMP-1 mRNA expression was elevated >10-fold during IL-6-induced NED of LNCaP cells, suggesting that TIMP-1 overexpression accompanies, but is not the driving force for NED. Finally, we show that conditioned media from androgen-resistant PC-3, PC-3M, and DU145 cells induced TIMP-1 mRNA expression in primary prostate stromal fibroblasts in an ERK and NF-κB dependent manner. We provide in vitro and clinical evidence to support the association between NED and elevated circulating TIMP-1 expression in CRPC. Our observation supports further evaluation of TIMP-1 as a tissue and serum biomarker for NED in CRPC.

A randomized pilot study on single-port versus conventional laparoscopic rectal surgery: effects on postoperative pain and the stress response to surgery.

Potential benefits of single-port laparoscopic surgery may include improved cosmetic results, less postoperative pain, surgical trauma and faster recovery. Results of randomized prospective studies with a focus on single-port rectal surgery have not yet been presented. The aim of the present study was to compare single-port and conventional laparoscopic surgery for rectal cancer in terms of short-term outcomes including postoperative pain and trauma-induced changes in certain bioactive substances. Patients with non-metastasized rectal cancer were prospectively randomized to single-port (n = 20) or conventional laparoscopic rectal surgery (n = 20). Postoperative pain was assessed at rest, at coughing and during mobilization, with a numeric pain ranking score and was recorded at 6 h after the operation and subsequently every morning daily for 4 days. Levels of C-reactive protein (CRP), interleukin-6 (IL-6) and tissue inhibitor of metalloproteinases-1 (TIMP-1) were determined. Blood samples were collected preoperatively (baseline), and 6, 24, 48, 72 and 96 h after skin incision. Pain scores were significantly reduced in the single-port group on postoperative days 2, 3 and 4 during coughing and mobilization. In addition, the patients in the single-port group suffered significantly less pain at rest at 6 h after surgery and on postoperative days 1, 3 and 4. The levels of the three markers increased significantly after surgery. The increase was similar between groups for plasma IL-6 and TIMP-1 at all time points, while the CRP levels were significantly lower in the single-port group at 6 (p < 0.001) and 24 h (p < 0.05) after skin incision. Abdominal incisions lengths were significantly shorter in the single-port group (p = 0.001). There was no significant difference between groups in operating time and blood loss, morbidity or mortality rate. The short-term oncological outcome in the two groups was similar. Single-port rectal surgery may reduce postoperative pain. Although CRP levels were lower at some time points, results of the present randomized, pilot study suggest that the trauma-induced inflammatory response of single-port operations may be similar to the trauma-induced inflammatory response of conventional laparoscopic surgery.

Abstract 1098: TIMP-1: A potential biomarker of neuroendocrine differentiation in metastatic castration-resistant prostate cancer (mCRPC)

Abstract BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a 28.5 kDa secreted glycoprotein that inhibits matrix metalloproteinase (MMP) activity. Recent studies have demonstrated that TIMP-1 promotes tumor growth in an MMP-independent manner. We have previously shown that elevated plasma TIMP-1 predicts worse survival in mCRPC patients; however, the mechanism underpinning this association is unknown. Normal and malignant neuroendocrine (NE) cells, from various organs, express TIMP-1 as measured by immunohistochemistry (IHC). We hypothesized that elevated blood levels of TIMP-1 in mCRPC patients results from androgen deprivation therapy-induced NE differentiation. METHODS: In this study, we used qPCR, ELISA and IHC to correlate the expression of TIMP-1 and NE markers such as chromogranin A (CGA) in 7 prostate cancer cell lines, 37 patient serum samples and prostate cancer (adenocarcinoma and NE cancer) tissue microarrays. RESULTS: We first examined TIMP-1 and NE marker (NSE and PTHLH) mRNA expression in 7 prostate cancer cell lines. Our results show that PC-3, PC-3M, DU145 cells express high levels of TIMP-1 as well as NE markers. On the other hand, LNCAP, VCAP and LAPC-4 cells had barely detectable levels of TIMP-1 and much lower expression of NE markers. We then measured serum TIMP-1 and CGA levels from CRPC patient samples by ELISA. Patients with castration resistant disease had significantly higher serum TIMP-1 levels (mean value of 373.5 ng/ml) than patients with hormone sensitive disease (mean value of 304.8 ng/ml) (p=0.01), suggesting that castration resistance in prostate cancer patients is associated with increased TIMP-1 production. Interestingly, when we divided patients using a cutoff of 100 ng/ml (based on cutoffs reported in published literature), serum TIMP-1 was significantly higher in the high CGA group (p=0.018), indicating an association between TIMP-1 production and existing NE markers. We also compared PSA values between low and high CGA groups and found that the high CGA group was associated with lower PSA levels (p=0.029). Next, we explored TIMP-1 expression in NE tumor tissues. We performed TIMP-1 and CGA IHC staining in archival liver neuroendocrine and thyroid cancers and found that TIMP-1 expression was associated with CGA expression. In prostate cancer specimens, normal prostate epithelial cells expressed high levels of TIMP-1, but the expression was lost in adenocarcinomas. In contrast, prostatic neuroendocrine cancers expressed high levels of TIMP-1 as well as the NE marker CGA. CONCLUSIONS: These observations support the further evaluation of TIMP-1 as a tissue and serum biomarker for NE differentiation in CRPC. Note: This abstract was not presented at the meeting. Citation Format: Yixuan Gong, Uma Chippada-Venkata, Xudong Dai, Matthew D. Galsky, Jiaoti Huang. TIMP-1: A potential biomarker of neuroendocrine differentiation in metastatic castration-resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1098. doi:10.1158/1538-7445.AM2014-1098

Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is an endogenous inhibitor of matrix metalloproteinases (MMPs) with reported tumor promoting, as well as inhibitory, effects. These paradoxical properties are presumably mediated by different biological functions, MMP-dependent as well as -independent, and probably related to TIMP-1 levels of protein expression, post-translational modifications, and cellular localization. TIMP-1 is an N-glycosylated protein that folds into two functional domains, a C- and an N-terminal domain, with six disulfide bonds. Furthermore, TIMP-1 is processed in the N-terminal sequence. These three biochemical properties make TIMP-1 difficult to produce in conventional bacterial, insect, or yeast expression systems. We describe here a HEK293 cell-based strategy for production and purification of secreted and N-glycosylated recombinant his6-tagged human TIMP-1 (his6-rTIMP-1), which resulted in large amounts of highly purified and bioactive protein. Matrix-assisted laser desorption ionization mass spectrometry confirmed the N- and C-termini of his6-rTIMP-1, and N-glycosylation profiling showed a match to the N-glycosylation of human plasma TIMP-1. The his6-rTIMP-1 was bioactive as shown by its proper inhibitory effect on MMP-2 activity, and its stimulatory effect on cell growth when added to the growth medium of four different breast cancer cell lines. This study provides an easy set-up for large scale production and purification of bioactive, tagged recombinant human TIMP-1, which structurally and functionally is similar to endogenous human TIMP-1, while using an expression system that is adaptable to most biochemical and biomedical laboratories including those that do not perform protein purifications routinely.

Positron Emission Tomography/Computed Tomography and Biomarkers for Early Treatment Response Evaluation in Metastatic Colon Cancer

Treatment options for metastatic colon cancer (mCC) are widening. We prospectively evaluated serial 2-deoxy-2-[18F]fluoro-d-glucose positron-emission tomography/computed tomography (PET/CT) and measurements of tissue inhibitor of metalloproteinases-1 (TIMP-1), carcinoembryonic antigen (CEA), and liberated domain I of urokinase plasminogen activator receptor (uPAR(I)) for early assessment of treatment response in mCC patients. Thirty-three mCC patients scheduled for first-line chemotherapy with capecitabine and oxaliplatin (CAPOX) and bevacizumab participated; 27 were evaluated by PET/CT before treatment, after one and four treatment series. Morphological and metabolic response was independently assessed according to Response Evaluation Criteria in Solid Tumors and European Organization for Research and Treatment of Cancer PET criteria. Plasma TIMP-1, plasma uPAR(I), and serum CEA were determined. Metabolic response after one treatment course predicted the ability of CAPOX and bevacizumab to induce morphological response after four treatment series with a sensitivity of 80%, specificity of 69%, and odds ratio of 13.9 (95% confidence interval [CI] 1.9; 182). Early metabolically stable or progressive disease was associated with shorter progression-free survival (hazard ratio [HR] = 3.2 [CI 1.3; 7.8]). Biomarker levels at early evaluation were associated with shorter OS (TIMP-1 per unit increase on a log-2-transformed ng/mL scale: HR = 2.6 [CI 1.4; 4.9]; uPAR(I) per 25 fmol/mL increase: HR = 1.5 [CI 1.1; 2.1]). This monocentric study demonstrated predictive value of early metabolic PET response and prognostic value of TIMP-1 and uPAR(I) levels in mCC treated with CAPOX and bevacizumab. Results support investigation of PET/CT, TIMP-1, and uPAR(I) guided early treatment adaptation in mCC.

Plasma Matrix Metalloproteinase-9 Response to Downhill Running in Humans

Matrix metalloproteinase-9 is a proteolytic enzyme capable of degrading proteins of the muscle extracellular matrix. Systemic levels of MMP-9 or its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), have the potential to serve as blood markers of exercise-induced muscle damage. The purpose of this study was to determine if an eccentrically-dominated task, downhill running (DHR), produces changes in plasma MMP-9 or TIMP-1 and examine the relationship between MMP-9/TIMP-1 levels and indirect indicators of muscle damage. Subjects were sedentary (SED, n=12) or had a history of concentrically-biased training (CON, n=9). MMP-9 and TIMP-1 were measured before (Pre-Ex), immediately after (Post-Ex), and 1-, 2-, 4-, and 7-days post-DHR (-10°), and compared to discomfort ratings, creatine kinase activity and strength loss. At 1-day Post-Ex, discomfort increased (5.6 ± 7.8 to 45.5 ± 19.9 mm; 0-100 mm scale), strength decreased (-6.9 ± 1.6%) and CK increased (162.9 ± 177.2%). MMP-9 was modestly but significantly increased at Post-Ex in both CONC and SED (32.7 ± 33.6%) and at 4-days in SED (66.9 ± 88.1%), Individual responses were variable, however. There were no correlations between MMPs and discomfort ratings, plasma CK or strength. While plasma MMP-9 changes may be detectable in the systemic circulation after DHR, they are small and do not correspond to other markers of damage.

The circulating level of MMP-9 and its ratio to TIMP-1 as a predictor of severity in patients with community-acquired pneumonia

BackgroundHere, we have examined the plasma levels of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1), the MMP-9/TIMP-1 molar ratio, the TIMP-1 single nucleotide polymorphisms (SNPs) 372C/T and the susceptibility to community-acquired pneumonia (CAP). MethodsAn enzyme-linked immunosorbent assay (ELISA) was used to measure plasma MMP-9 and TIMP-1 concentrations in 60 patients with CAP and 60 healthy individuals. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to detect the TIMP-1 SNPs 372C/T. ResultsThe plasma MMP-9, TIMP-1 levels and the MMP-9/TIMP-1 molar ratio were significantly increased in patients with CAP compared to normal controls. The MMP-9/TIMP-1 molar ratio decreased significantly in patients with CAP after treatment. Furthermore, the plasma TIMP-1 concentration was positively correlated with the pneumonia severity index (PSI), the CURB score, the results of the acute physiology and chronic health evaluation II (APACHE II) and the length of the hospital stay. No significant difference was found in the genotype distribution of TIMP-1 372C/T between patients with CAP and normal controls. ConclusionsWe hypothesize that MMP-9 levels and the MMP-9/TIMP-1 molar ratio play a role in the development of CAP and are related to the severity of CAP. Based on our data, we suggest that incorporating plasma MMP-9 levels and the MMP-9/TIMP-1 molar ratio into a clinical evaluation will aid in CAP diagnosis.

Plasma TIMP-1 in patients with metastatic colorectal cancer treated with first-line oxaliplatin-based therapy with or without cetuximab: Results from the Nordic VII study.

e14710 Background: Plasma Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) is a biomarker in patients with metastatic colorectal cancer (mCRC). TIMP-1 inhibits matrix metalloproteinases and has tumor promoting functions. High levels of TIMP-1 associate with reduced sensitivity to irinotecan-based but not oxaliplatin-based treatment in patients with mCRC. Based on our prior studies, we here tested the hypothesis that high pretreatment plasma TIMP-1 correlate with short overall survival (OS) and short progression-free survival (PFS) but not response rate (RR) in patients with mCRC included in the NORDIC VII Study in which patients received oxaliplatin-based therapy. Methods: In the NORDIC VII Study patients were randomized to: A) Nordic FLOX; B) Nordic FLOX + cetuximab or C) Nordic FLOX intermittently + cetuximab continuously. Plasma samples for TIMP-1 analysis were available from 426 patients. Plasma TIMP-1 was determined using the MAC15 antibody in-house validated kinetic ELISA. Results: The three study populations were comparable and not different from the total intention to treat population. The median pretreatment plasma TIMP-1 was 269 ng/mL (58-1318 ng/mL). KRAS mutated in 149 patients (35%). There was no interaction between treatment arms and pretreatment plasma TIMP-1 either for PFS (p = 0.16) or OS (p = 0.64). High pretreatment plasma TIMP-1 was associated with shorter PFS (HR=1.22, 1.07-1.39, p=0.003) and shorter OS (HR=1.55, 1.36-1.80, p&lt;0.0001). RR did not correlate significantly to pretreatment plasma TIMP-1 (OR=1.16, 0.91-1.49, p=0.22). Multivariate analysis (TIMP-1, Köhne index, KRAS and BRAF status, CRP, CEA, age, gender) demonstrated that high pretreatment plasma TIMP-1 was not an independent biomarker of PFS (HR=1.06, 0.88-1.28, p=0.52) or OS (HR=1.19, 0.96-1.47, p=0.11). Conclusions: Plasma TIMP-1 is a prognostic biomarker in patients with mCRC treated with 1st line oxaliplatin-based therapy +/- cetuximab. A predictive effect of plasma TIMP-1 could only be estimated for cetuximab treatment, and no significant treatment by marker interaction was observed.

Abstract 5068: Differential regulation of tissue inhibitor of metalloproteinase (TIMP-1) expression in primary stromal fibroblast cells by androgen sensitive and resistant prostate cancer cells.

Abstract Background: The tissue inhibitor of metalloproteinase 1 (TIMP-1) regulates matrix metalloproteinases (MMPs) activities and is involved in cancer invasion and metastasis. TIMP-1 level is significantly elevated in prostate cancer patient blood and the elevated plasma TIMP-1 level predicts decreased survival of metastatic castration-resistant prostate cancer (mCRPC) patients. Methods and findings: In this study, we examined serum TIMP-1 levels in 114 prostate cancer patient samples by ELISA and found mCRPC patients have significantly higher serum TIMP-1 (mean value of 373.5 ng/ml) than patients with recurrent hormone-sensitive disease (mean value of 304.8 ng/ml) (p&amp;lt;0.001). Then we examined TIMP-1 expression in androgen-sensitive (AS) and androgen resistant (AR) prostate cancer cell lines (LNCaP, VCaP, LAPC-4, 22RV1, DU145, PC-3 and PC-3M) and primary prostate stromal fibroblast cells (HPrF) by realtime PCR and ELISA. Our results showed that AS and AR cells regulate TIMP-1 expression differently. AS cells barely expressed any TIMP-1, whereas AR prostate cancer cells not only expressed moderate amount of TIMP-1, but also induced stromal fibroblast cells to secrete more TIMP-1. But overall, fibroblast cells secreted at least 10 times more TIMP-1 than AR prostate cancer cells, indicating that stromal fibroblasts are the major source of TIMP-1 in prostate cancer. To identify the soluble factors secreted by AR cells to induce TIMP-1 expression in HPrF, we analyzed cytokine and growth factor profiles of AR and AS cell condition medium (CM) by RayBio cytokine and growth factor arrays and found that multiple cytokines and growth factors were upregulated in AR cell line condition medium including IL-6, IL-10, GM-SCF, GRO alpha and etc. More investigation is underway to verify the roles of the cytokines and growth factors in TIMP-1 regulation in prostate cancer. In addition, using pharmaceutical inhibitors, we showed that AZD6244 (a MEK inhibitor) and BAY11-7082 (an NF-kB inhibitor) completely blocked prostate cancer cell line CM-induced TIMP-1 expression in stromal fibroblasts. Conclusion: Our studies have demonstrated that androgen resistance is associated with TIMP-1 overproduction in prostate cancer and suggested that we can potentially inhibit TIMP-1 overexpression in prostate cancer patients with MEK and NF-kB inhibitors. Citation Format: Yixuan Gong, Lan He, Matthew Galsky, William Oh. Differential regulation of tissue inhibitor of metalloproteinase (TIMP-1) expression in primary stromal fibroblast cells by androgen sensitive and resistant prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5068. doi:10.1158/1538-7445.AM2013-5068 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Tissue Inhibitor of Metalloproteinases-1 as a Biological Marker in Colorectal Cancer: Influence of Smoking on Plasma Levels?

At present plasma tissue inhibitor of metalloproteinases-1 (TIMP-1) is undergoing validation as a biological marker in colorectal cancer (CRC). The clinical implementation of plasma TIMP-1 in prognosis, prediction, screening and monitoring CRC requires robust information as to the influence of preanalytical factors, including inter- and intrapersonal biological variations. The aim of the present study was to evaluate the possible effects of smoking on the level of TIMP-1 in plasma from healthy subjects. Forty-six never-smokers and 48 daily smokers participated in the 13-week study. Smokers were randomized into 3 groups of 16 subjects each: one group continued to smoke, a second group refrained from smoking and used a transdermal nicotine patch, and a third group refrained from smoking and used placebo patches. Plasma TIMP-1 levels were determined using ELISA. No significant differences in TIMP-1 levels could be shown between the smoking group, the 2 different abstinent smoking groups and the never-smoking group. Smoking does not appear to have any influence on TIMP-1 levels in plasma collected from healthy subjects.