Tissue Inhibitor Of Metalloproteinase-1 mRNA Expression Research Articles

Impact of tissue inhibitor of metalloproteinases-3 genetic variants on clinicopathological characteristics of urothelial cell carcinoma.

To investigate the distribution of single nucleotide polymorphism (SNP) of tissue inhibitor of metalloproteinases-3 (TIMP-3) in patients with/without urothelial cell carcinoma (UCC), three loci of TIMP-3 SNPs (rs9862 C/T, rs9619311 T/C, rs11547635 C/T) were genotyped via TaqMan allelic discrimination for 424 UCC patients and 848 non-UCC participants. Furthermore, the TIMP-3 mRNA expression and its correlation with clinical characters of urothelial bladder carcinoma was analyzed using The Cancer Genome Atlas database (TCGA). The distribution of all 3 studied SNPs of TIMP-3 was insignificantly different between the UCC and non-UCC groups. However, significantly lower tumor T status was found in TIMP-3 SNP rs9862 CT + TT variant than the wild type (OR: 0.515, 95% CI: 0.289-0.917, P = 0.023). Moreover, the muscle invasive tumor type was significantly correlated to the TIMP-3 SNP rs9619311 TC + CC variant in the non-smoker subgroup (OR: 2.149, 95% CI: 1.143-4.039, P = 0.016). With the TIMP-3 expression data provided in TCGA, significantly higher TIMP-3 mRNA expression was observed in UCC with high tumor stage (P < 0.0001), high tumor T status (P < 0.0001) and high lymph node status (P = 0.0005). In conclusions, TIMP-3 SNP rs9862 variant is associated with lower tumor T status of UCC while TIMP-3 SNP rs9619311 variant is correlated to muscle invasive UCC development in non-smoker.

Changes of heparin-binding protein in severe burn patients during shock stage and its effects on human umbilical vein endothelial cells and neutrophils

Changes of heparin-binding protein in severe burn patients during shock stage and its effects on human umbilical vein endothelial cells and neutrophils

Anti-aging Effect of Mixed Extract from Medicinal Herbs

Purpose: This study was conducted to confirm the applicability of a mixed herbal extract (MHE) as an anti-aging cosmetic ingredient by investigating its skin anti-aging activities in vitro and in vivo.Methods: In this study, we prepared MHE using an ultrasonic extraction containing Forsythiae fructus, Tribuli fructus, Solomon’s seal, Siberian ginseng, Ponciri fructus and Ginseng. We investigated the anti-aging effect of the MHE for skin in dermal fibroblasts. The anti-aging activity was determined by the type I collagen synthesis levels. Matrix metalloproteinase-1 (MMP1) and tissue inhibitor of metalloproteinases 1 (TIMP1) mRNA levels were measured by qRT-PCR. MMP1 protein levels were evaluated by blotting analysis. Clinical tests of skin moisture, elasticity, texture, and wrinkles were performed using cosmetics containing 1% MHE.Results: The MHE induced the upregulation of pro-collagen type I synthesis and TIMP1 mRNA expression. The MHE led to the downregulation of MMP1 mRNA levels and protein levels. Furthermore, after skin application of cosmetics containing 1% MHE, skin hydration, elasticity, texture, and crow’s feet were improved 4 weeks after the treatment.Conclusion: MHE has an anti-aging effect by promoting collagen synthesis and suppressing MMP1 gene expression in vitro, and it has a skin improvement effect in vivo. Therefore, the MHE was shown to have value as a functional cosmetic ingredient.

Phenotypic and Functional Transformation in Smooth Muscle Cells Derived from a Superficial Thrombophlebitis-affected Vein Wall

Superficial thrombophlebitis (ST) is a frequent pathology, but its exact incidence remains to be determined. This study tested the hypothesis whether relationships exist among smooth muscle cells (SMCs) derived from ST, varicose great saphenous veins (VGSVs), and normal great saphenous veins (GSVs). Forty-one samples of ST, VGSVs, and GSVs were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, and senescence in SMCs from the three vein walls were compared by various methods. Bax, Bcl-2, caspase-3, matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 messenger RNA (mRNA) and protein expressions were detected by fluorescence quantitative PCR and Western blot. An obvious decrease in cytoskeletal filaments was observed in thrombophlebitic vascular smooth muscle cells (TVSMCs). The quantity of proliferation, migration, adhesion, and senescence in TVSMCs was significantly higher than in varicose vascular smooth muscle cells and normal vascular smooth muscle cells (NVSMCs) (all P < 0.05). Bax and caspase-3 mRNA and protein expression were decreased, while Bcl-2 mRNA and protein expression were increased in the TVSMCs compared with the varicose vascular smooth muscle cells and the NVSMCs (all P < 0.05). MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA and protein expression were significantly increased in the TVSMCs compared with the VVGSVs and the NVSMCs (all P < 0.05). SMCs derived from ST are more dedifferentiated and demonstrate increased cell proliferation, migration, adhesion, and senescence, as well as obviously decreased cytoskeletal filaments. These results suggest that the phenotypic and functional differences could be related to the presence of atrophic and hypertrophic vein segments during the disease course among SMCs derived from ST, VGSVs, and GSVs.

The pathomechanism of human myxomatous valvular degeneration at the mechanical and cellular level.

The purpose of this study was to explore the pathomechanism of human myxomatous valve degeneration by investigating changes in the phenotype of valvular cells, the metabolism of the extracellular matrix and their mechanical properties. Mitral valve specimens were harvested from patients who had undergone valve replacement, and divided into two groups: patients with a myxomatous mitral valve and a control group. Histological investigation showed that the morphology of the extracellular matrix was looser and less coordinated in myxomatous valves than in controls. α-SMA (α-smooth muscle actin) and Vimentin were positive and DNA (deoxyribonucleic acid) assay of leaflets and expression of SMemb (embryonic smooth muscle myosin heavy chain), MMP-13 (matrix Metalloproteinases-13), MMP-1 mRNA (messenger Ribonucleic Acid) of the myxomatous valves were increased while the hydroxyproline content, expression of TIMP-1 (tissue inhibitor of metalloproteinase-1) mRNA and mechanical properties were decreased compared with controls. Compared to the quiescent interstitial cells in non-myxomatous valves, interstitial cells in myxomatous valves exhibit myofibroblast activation and express excessive levels of matrix metalloproteinases. The balance between MMP/TIMP was disrupted. We conclude that overactivation of VICs (Valvular interstitial cells) and the imbalance of MMP/TIMP could be important features of the pathomechanism of myxomatous mitral valve degeneration.

Leonurus sibiricus root extracts decrease airway remodeling markers expression in fibroblasts.

Bronchial asthma is believed to be provoked by the interaction between airway inflammation and remodeling. Airway remodeling is a complex and poorly understood process, and controlling it appears key for halting the progression of asthma and other obstructive lung diseases. Plants synthesize a number of valuable compounds as constitutive products and as secondary metabolites, many of which have curative properties. The aim of this study was to evaluate the anti-remodeling properties of extracts from transformed and transgenic Leonurus sibiricus roots with transformed L. sibiricus roots extract with transcriptional factor AtPAP1 overexpression (AtPAP1). Two fibroblast cell lines, Wistar Institute-38 (WI-38) and human fetal lung fibroblast (HFL1), were incubated with extracts from transformed L. sibiricus roots (TR) and roots with transcriptional factor AtPAP1 over-expression (AtPAP1 TR). Additionally, remodeling conditions were induced in the cultures with rhinovirus 16 (HRV16). The expressions of metalloproteinase 9 (MMP)-9, tissue inhibitor of metalloproteinases 1 (TIMP-1), arginase I and transforming growth factor (TGF)-β were determined by quantitative polymerase chain reaction (qPCR) and immunoblotting methods. AtPAP1 TR decreased arginase I and MMP-9 expression with no effect on TIMP-1 or TGF-β mRNA expression. This extract also inhibited HRV16-induced expression of arginase I, MMP-9 and TGF-β in both cell lines (P<0·05) Our study shows for the first time to our knowledge, that transformed AtPAP1 TR extract from L. sibiricus root may affect the remodeling process. Its effect can be attributed an increased amount of phenolic acids such as:chlorogenic acid, caffeic acid or ferulic acid anddemonstrates the value of biotechnology in medicinal research.

Neuroprotective effect of neuregulin-1β on spinal cord ischemia reperfusion injury

Objective: This study was designed to see if neuregulin-1β (NRG-1β) plays a protective role in spinal cord ischemia and reperfusion injury (SCII). Design: Animal research. Setting: China. Participants: NA. Interventions: Forty-eight SD rats were randomly divided into control group (n = 16), SCII model group (n = 16) and NRG-1β-treated group (n = 16). In control group, the abdominal aorta was isolated but not clipped. The rats in NRG-1β-treated group were treated with 10μg/kg NRG-1β during developing SCII model. Outcome Measures: Neurological scores were evaluated. At 3, 6, 12 and 24 h after the reperfusion, rats were killed. Pathological changes of spinal cord were assessed with HE staining, and immunohistochemical staining of matrix metalloproteinases-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). MMP-9 and TIMP-1 mRNA levels were assessed using real-time PCR. Results: NRG-1β reduced the damage of SCII in the rats. The expression of MMP-9 protein and mRNA in NRG-1β treatment group was significantly lower than the model group (P < 0.05) at 6 h, 12 h and 24 h after the perfusion. The expression of TIMP-1 protein and mRNA in the treatment group was significantly higher than the model group at 12 h and 24 h after the perfusion. Conclusion: NRG-1β reduced the reperfusion damage in rat model of SCII, in which process MMP-9 and TIMP-1 were probably involved.

Effect of medicated serum of Curcumae Radix extract on mRNA expression of TIMP-1, MMPs-13 and aI-collagen of HSC-T6 cell

To study the effect of medicated serum of Curcumae Radix (Yujin) on the mRNA expression of Tissue inhibitor of metalloproteinase-1 (TIMP-1), Matrix metalloproteinases-13 (MMPs-13) and aI-collagen of Hepatic stellate cell-T6 (HSC-T6) cell. Twenty SD rats were randomly divided into 4 groups: high dose of Yujin group (16.2gkg−1), medium dose of Yujin group (8.1gkg−1), low dose of Yujin group (4.05gkg−1) and blank control group (with the same volume of 0.9% saline). Yujin extract or 0.9% saline were administered daily by gavage to rats for 4d, after once administration of full-day dose for 1h on the fourth day, ether anaesthesia was given, and blood was taken from abdominal aortic in asepsis condition for preparation of medicated serum. HSC-T6 cells were divided into 5 groups: high dose medicated serum of Yujin group, medium dose medicated serum of Yujin group, low dose medicated serum of Yujin group, medicated serum of blank control group and negative control group (added with the same volume of PBS instead of rat serum), after 48h of simultaneous acting on HSC-T6 cells in all groups by the medicated serum with a concentration of 10%, the mRNA expression level of TIMP-1, MMPs-13 and aI-collagen was analyzed with RT-PCR. Compared with the negative control group, the mRNA expression level of TIMP-1, MMPs-13 and aI-collagen in all experimental groups increased significantly. Compared with the control group, the mRNA expression of aI-collagen and TIMP-1 was obviously inhibited in all medicated serum of Yujin groups (P<0.01), meanwhile, the mRNA expression level of MMPs-13 was effectively improved (P<0.05). The medicated serum of Yujin had an effect on the production and degradation of Extracellular matrix (ECM) of HSC-T6 cell.

Correlation between the mRNA expression of tissue inhibitor of metalloproteinase-1 and apparent diffusion coefficient on diffusion-weighted imaging in rats' liver fibrosis

To explore the correlation between the apparent diffusion coefficient (ADC) and mRNA expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in different stages of liver fibrosis in rats. Methods: A model of liver fibrosis in rats was established by intraperitoneal injection of high-fat diet combined with porcine serum. After drug administration for 4 weeks, 48 rats served as a model group and 12 rats served as a control group, then they underwent diffusion weighted imaging (DWI) scanning. The value of ADC was calculated at b value=800 s/mm2. The rats were sacrificed and carried out pathologic examination after DWI scanning immediately. The mRNA expression of TIMP-1 was detected by real time-polymerase chain reaction (RT-PCR). The rats of hepatic fibrosis were also divided into a S0 group (n=4), a S1 group (n=11), a S2 group (n=12), a S3 group (n=10), and a S4 group (n=9) according to their pathological stage. The value of ADC and the expression of TIMP-1 mRNA among the different stage groups of liver fibrosis were compared, and the correlation between ADC and the TIMP-1 mRNA were analyzed. Results: The ADC value and the TIMP-1 mRNA expression were significantly different between the control group and the liver fibrosis group (F=46.54 and 53.87, P<0.05). There were significant differences in the value of ADC between every two groups (all P<0.05), except the control group vs the S1 group, the S1 group vs the S2 group, and the S2 group vs the S3 group (all P>0.05). For the comparison of TIMP-1 mRNA, there was no significant difference between the S1 group and the S2 group, the S3 group and the S4 group (both P>0.05). There were significant differences among the rest of the groups (all P<0.05). Rank correlation analysis showed that there was a negative correlation between the ADC value and the TIMP-1 mRNA expression (r=-0.76, P<0.01). Conclusion: When the value of ADC decreases in the progress of rats' liver fibrosis, the mRNA expression of TIMP-1 increases gradually, and there is a negative correlation between them.

Tissue inhibitor of metalloproteinases 3 promoter methylation and mRNA expression in cardia carcinoma and its relationship with prognosis

Objective To explore the relationship of the methylation levels of tissue inhibitor of metalloproteinases 3(TIMP-3) gene promoter CpG islands with the invasiveness and prognosis in cardia carcinoma. Methods The tumors tissues were collected from 65 patients with cardia carcinoma. The methylation levels of the promoter of TIMP-3 gene were detected by methylation-specific PCR(MSP), the mRNA expression levels of TIMP-3 gene were detected by RT-PCR. The relationship of TIMP-3 gene ectopic methylation with invasiveness and prognosis of the cardia carcinoma patients was analyzed. Results The positive expression rates of TIMP-3 mRNA in gastric cancer and normal gastric tissues were 53.8 % (35/65) and 96.9 % (63/65), respectively. The difference was not statistically significant(Fisher exact test, P=0.912). The positive rate of TIMP-3 mRNA was negatively correlated with the depth of tumor invasion and lymph node metastasis [mucosa and muscular vs. serosa and mucosa outside of the serosa and muscula: 83.3 % (10/12) vs. 45.3 % (24/53); with lymph node metastasis vs. without lymph node metastasis: 73.9 % (17/23) vs. 40.5 % (17/42)] (both P<0.05). There was a negative correlation between TIMP-3 gene promoter methylation and TIMP-3 mRNA expression (r=-0.276, P=0.026). The size of tumor and TIMP-3 gene promoter methylation were both the independent influencial factors of prognosis in cardia carcinoma(both P<0.05). Conclusion The methylation of promoter region in CpG islands plays an important role in the invasion and metastasis of cardia carcinoma, and it can be used as an independent predictor of biological behavior and prognosis. Key words: Stomach neoplasms; Tissue inhibitor of metalloproteinase 3; Methylation; RNA, messenger; Prognosis

Tissue Inhibitor of Metalloproteinase-1 Is Confined to Tumor-Associated Myofibroblasts and Is Increased With Progression in Gastric Adenocarcinoma.

The tissue inhibitor of metalloproteinase-1 (TIMP-1) inhibits the extracellular matrix-degrading activity of several matrix metalloproteinases, thereby regulating cancer cell invasion and metastasis. Studies describing the expression pattern and cellular localization of TIMP-1 in gastric cancer are, however, highly discordant. We addressed these inconsistencies by performing immunohistochemistry and in situ hybridization analyses in a set of 49 gastric cancer lesions to reexamine the TIMP-1 localization. In addition, we correlated these findings to clinicopathological parameters. We show that strong expression of TIMP-1 protein and mRNA was observed in a subpopulation of stromal fibroblast-like cells at the periphery of the cancer lesions. In a few cases, a small fraction of cancer cells showed weak expression of TIMP-1 protein and mRNA. The stromal TIMP-1-expressing cells were mainly tumor-associated myofibroblasts. In the normal-appearing mucosa, scattered TIMP-1 protein was only found in chromogranin A positive cells. TIMP-1-positive myofibroblasts at the invasive front of the tumors were more frequently seen in intestinal than in diffuse histological subtype cases (p=0.009). A significant trend to a higher number of cases showing TIMP-1 staining in myofibroblasts with increasing tumor, node, metastasis (TNM) stage was also revealed (p=0.041). In conclusion, tumor-associated myofibroblasts are the main source of increased TIMP-1 expression in gastric cancer.

The Clinical Utility of TIMP3 Expression in ACTH-Secreting Pituitary Tumor.

In recent years, the tissue inhibitor of metalloproteinase-3 (TIMP3) plays a pivotal role in tumorigenesis, while the role of TIMP3 in adrenocorticotrophic hormone (ACTH)-secreting pituitary adenomas remains unclear. In this study, 86 sporadic pituitary tumor specimens, including ACTH (40), GH (18), PRL-secreting (8), and non-functioining (20) and non-tumorous pituitary samples (n = 10) were available, and then, the mRNA and protein expression of TIMP3 was quantified by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), western blotting, and immunohistochemistry, respectively. Our findings showed that TIMP3 expression was significantly correlated with Ki-67 expression and the invasiveness of pituitary adenomas. TIMP3 mRNA and protein expression were reduced in ACTH-secreting pituitary adenomas and the other three types of pituitary adenomas compared to adjacent non-tumorous pituitary tissues (all p < .01). On the other hand, the expression of TIMP3 was negatively correlated with tumor size and Ki-67 in ACTH-secreting pituitary adenomas. TIMP3 mRNA expression was significantly lower in invasive pituitary adenomas than that in noninvasive ones (1.92-fold, p < .05). TIMP3 protein levels were also significantly lower in the majority of invasive adenomas (1.41-fold, p < .05) Furthermore, TIMP3 mRNA and protein expression were significantly lower in pituitary giant adenomas than those in microadenomas (2.58-fold, p < .05). In conclusion, the expression of TIMP3 is low in pituitary adenomas including ACTH-secreting pituitary adenomas and negatively associated with tumor aggressiveness. TIMPs may play a potential role in the progression of ACTH-secreting pituitary adenomas and be useful as a biomarker of invasiveness.

Antiadhesive and anti-inflammatory effects of pirfenidone in postoperative intra-abdominal adhesion in an experimental rat model

BackgroundPirfenidone (PF) is a potent antifibrotic and anti-inflammatory agent. We investigated the protective effect of PF against postoperative intra-abdominal adhesions. Material and methodsThirty male Sprague–Dawley rats were divided into three groups (n = 10 in each group). In group 1 (control), adhesion induction was performed by cecal abrasion, and no treatment was administered. In group 2 (vehicle), for 2 wk after adhesion induction, 0.4%-carboxymethylcellulose was administered by gavage. In group 3 (PF treatment), for 2 wk after adhesion induction, 500-mg/kg/d PF was administered by gavage. On the 15th postoperative day, the animals were killed, and cecal and peritoneal tissues were excised. The adhesions were graded macroscopically. The protein concentrations and mRNA expression levels of the following genes were measured in the tissues: matrix metallopeptidase-9 (MMP-9); tissue inhibitor of metalloproteinase-1 (TIMP-1); tumor necrosis factor-alpha (TNF-α); and transforming growth factor-beta 1 (TGF-β1). The tissue samples were also evaluated histopathologically. ResultsMacroscopic and histopathologic evaluation showed that PF-reduced adhesion and inflammation (P < 0.001, P = 0.004, respectively). Pretreatment with PF-reduced TIMP-1, TNF-α, and TGF-β1 protein concentrations (P < 0.001, P < 0.001, and P < 0.001, respectively) and mRNA expression levels (P = 0.030, P = 0.005, and P = 0.016, respectively) and increased MMP-9 protein concentrations (P < 0.001) and mRNA expression (P = 0.021). ConclusionsThe findings of this study suggest that PF can be used as a protective agent to prevent the development of peritoneal adhesions and inflammation during the postoperative period.

Influence of Expression Plasmid of Connective Tissue Growth Factor and Tissue Inhibitor of Metalloproteinase-1 shRNA on Hepatic Precancerous Fibrosis in Rats.

In this study, influence caused by expression plasmids of connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1) short hairpin RNA (shRNA) on mRNA expression of CTGF,TIMP-1,procol-α1 and PCIII in hepatic tissue with hepatic fibrosis, a precancerous condition, in rats is analyzed. To screen and construct shRNA expression plasimid which effectively interferes RNA targets of CTGF and TIMP-1 in rats. 50 cleaning Wistar male rats are allocated randomly at 5 different groups after precancerous fibrosis models and then injection of shRNA expression plasimids. Plasmid psiRNA-GFP-Com (CTGF and TIMP-1 included), psiRNA-GFP-CTGF, psiRNA-GFP-TIMP-1 and psiRNA- DUO-GFPzeo of blank plasmid are injected at group A, B, C and D, respectively, and as model control group that none plasimid is injected at group E. In 2 weeks after last injection, to hepatic tissue at different groups, protein expression of CTGF, TIMP-1, procol-α1and PC III is tested by immunohistochemical method and,mRNA expression of CTGF,TIMP-1,procol-α1 and PCIII is measured by real-time PCR. One-way ANOVA is used to comparison between-groups. Compared with model group, there is no obvious difference of mRNA expression among CTGF,TIMP-1,procol-α1,PC III and of protein expression among CTGF, TIMP-1, procol-α1, PC III in hepatic tissue at group injected with blank plasmid. Expression quantity of mRNA of CTGF, TIMP-1, procol-α1 and PCIII at group A, B and C decreases, protein expression of CTGF, TIMP-1, procol-α1, PC III in hepatic tissue is lower, where the inhibition of combination RNA interference group (group A) on procol-α1 mRNA transcription and procol-α1 protein expression is superior to that of single interference group (group B and C) (P<0.01 or P<0.05). RNA interference on CTGF and/or TIMP-1 is obviously a inhibiting factor for mRNA and protein expression of CTGF, TIMP-1, procol-α1 and PCIII. Combination RNA interference on genes of CTGF and TIMP-1 is superior to that of single RNA interference, and this could be a contribution for prevention of precancerous condition.

Expression and regulative function of tissue inhibitor of metalloproteinase 3 in the goat ovary and its role in cultured granulosa cells

Tissue inhibitor of metalloproteinase 3 (TIMP3) played a key role in female reproduction. However, its expression and function in goat are still unclear. In the present study, the full-length cDNA of goat TIMP3 was cloned from adult goat ovary; meanwhile, we demonstrated that putative TIMP3 protein shared a highly conserved amino acid sequence with known mammalian homologs. Real-time PCR results showed that TIMP3 was widely expressed in the tissues of adult goat. In the ovary, increasing expression of TIMP3 mRNA was discovered during the growth process of follicle and corpus luteum. Immunohistochemistry results suggested that TIMP3 protein existed in oocytes of all types of follicles, corpus luteum and granulosa and theca cells of primary, secondary, and antral but not primordial follicles. In vitro, human chorionic gonadotropin (hCG) stimulated the expression of TIMP3 in goat granulosa cells. hCG-induced TIMP3 mRNA expression was reduced by the inhibitors of protein kinase A, protein kinase C, MAPK kinase, or p38 kinase. Functionally, over-expression of TIMP3 significantly increased apoptosis and decreased the viability of cultured granulosa cells. Knockdown of TIMP3 could decrease hCG-induced progesterone secretion and the mRNA abundance of key steroidogenic enzymes (StAR, p450scc and HSD3B) as well as ECM proteins (DCN and FN). These findings provided evidence that the hCG induced expression of TIMP3 may play an important role in regulating goat granulosa cell survival and steroidogenesis.

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.

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

Losartan attenuates paraquat-induced pulmonary fibrosis in rats.

Paraquat (PQ) is one of the most widely used herbicides in the world and can cause pulmonary fibrosis in the cases with intoxication. Losartan, an angiotensin II type 1 receptor antagonist, has beneficial effects on the treatment of fibrosis. The aim of this study was to examine the effect of losartan on pulmonary fibrosis in PQ-intoxicated rats. Adult male Sprague Dawley rats (n = 32, 180-220 g) were randomly assigned to four groups: (i) control group; (ii) PQ group; (iii) PQ + losartan 7d group; and (iv) PQ + losartan 14d group. Losartan treatment (intragastrically (i.g.), 10 mg/kg) was performed for 7 and 14 days after a single i.g. dose of 40 mg/kg PQ. All rats were killed on the 16th day, and hematoxylin-eosin and Masson's trichrome staining were used to examine lung injury and fibrosis. The levels of hydroxyproline and transforming growth factor β1 (TGF-β1), matrix metallopeptidase 9 (Mmp9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) messenger RNA (mRNA) expression and relative expression levels of collagen type I and III were also detected. PQ caused a significant increase in hydroxyproline content, mRNA expression of TGF-β1, Mmp9, and TIMP-1, and relative expression levels of collagen type I and III ( p < 0.05), while losartan significantly decreased the amount of hydroxyproline and downregulated TGF-β1, Mmp9, and TIMP-1 mRNA and collagen type I and III expressions ( p < 0.05). Histological examination of PQ-treated rats showed lung injury and widespread inflammatory cell infiltration in the alveolar space and pulmonary fibrosis, while losartan could markedly reduce such damage and prevent pulmonary fibrosis. The results of this study indicated that losartan could reduce lung damage and prevent pulmonary fibrosis induced by PQ.

Effects of captlpril on tissue inhibitorof metalloproteinase-1 in rat hepatic stellate cells

Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) and captlpril on tissue inhibitorof metalloproteinase-1 (TIMP-1) mRNA expression in culured hepatic stellate cells (HSCs) in vivo.Methods HSC-T6 rat hepatic stellate cell line was chosen as the study model of the activated HSCs.The cultured HSCs were randomized into control group,Ang Ⅱ group (1 × 10-5,1 × 10-7 mol/L) and AngⅡ + captlpril group (1 × 10-5,1 × 10-7 mol/L).HSCs were harvested to measure the TIMP-1 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR).Results TIMP-1 gene expression levels in different groups of Ang Ⅱ (1 × 10-5 mol/L,1 × 10-7 mol/L) were 1.145 ±0.219 and 0.860±0.115 respectively,and those in control group was 0.523 ± 0.056.TIMP-1 expression levels in different groups of Ang Ⅱ ± captlpril (1 × 10-5 mol/L,1 × 10-7 mol/L) were 0.740 ± 0.089 and 0.590 ± 0.073 respectively,and those in Ang Ⅱ group were 0.850 ± 0.107 and 0.620 ± 0.103 respectively.Conclusion Angiotensin Ⅱ can increase the type Ⅰ collage mRNA expression of HSCs,which can be inhibited by captlpril. Key words: Angiotensin Ⅱ ; Captlpril; Hepatic stellate cells; Tissue inhibitorof metalloproteinase-1

Preventive effects of punicalagin against ultraviolet B-induced damage to human HaCaT keratinocytes

Objective To investigate the mechanisms underlying the protection by punicalagin against ultraviolet B (UVB)-induced damage to keratinocytes.Methods Cultured human HaCaT keratinocytes were divided into several groups:blank control group receiving no treatment,punicalagin groups treated with various concentrations of punicalagin,UVB group irradiated with UVB at 30 mJ/cm2,combination groups pretreated with different concentrations of punicalagin followed by UVB radiation at 30 mJ/cm2.The concentrations of punicalagin were 5,10,20,40 and 80 μmol/L in the cell proliferation assay,10,20 and 40 μmol/L in the other assays.After additional culture for different durations,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferation of HaCaT cells,Hoechst and propidium iodide (PI) staining as well as flow cytometry to detect the apoptosis in cells,reverse transcription-PCR to quantify the mRNA expressions of matrix metalloproteinase-1 (MMP1) and tissue inhibitor of metalloproteinase-1 (TIMP1) in HaCaT cells,Western blot to determine the phosphorylation levels of the mitogen-activated protein kinase (MAPK) pathway-related proteins including P38,JNK and ERK.Statistical analysis was carried out by t test,one-way analysis of variance,and Dunnett's t-test.Results As the MTT assay showed,punicalagin at 10-40 μmol/L showed stronger pre-protective effects against UVB-induced damage to HaCaT cells compared with punicalagin at the other concentrations.The number of cells highly positive for both Hoechst and PI staining was larger in the UVB group than that in the blank control group,but smaller in the combination groups than in the UVB group.The percentage of apoptotic cells increased significantly in the UVB group compared with the blank control group (9.82％ ± 0.11％ vs.1.24％ ± 0.91％,P ＜ 0.01),but decreased significantly in the three combination groups (punicalagin (10,20 and 40 μmol/L) + UVB) compared with the UVB group (6.38％ ± 0.14％,5.24％ ± 0.17％ and 3.77％ ± 0.11％ vs.9.82％ ± 0.11％,all P＜ 0.01).Theexpression of MMP1 mRNA was significantly higher,but that of TIMP1 mRNA was significantly lower in the UVB group than in the blank control group (both P ＜ 0.01),whereas no statistically significant difference was observed in the expression of MMP1 or TIMP1 mRNA between the punicalagin groups and blank control group (all P ＞ 0.05).The pretreatment with punicalagin significantly reduced the expression level of MMP1 mRNA (P ＜ 0.01),but elevated that of TIMP1 mRNA (P ＜ 0.01) in the combination groups compared with the UVB group.As Western blot showed,the phosphorylation levels of P38,JNK and ERK were markedly increased in the UVB group (all P ＜0.01),but experienced no significant changes in the punicalagin groups (all P ＞ 0.05) compared with the blank control group,and decreased to different degrees in the combination groups compared with the UVB group (all P ＜0.01).Conclusion Punicalagin has a pre-protective effect on UVB-induced damage to HaCaT cells. Key words: Keratinocytes; Ultraviolet rays; Matrix metalloproteinase 1; Extracellular signal-regulated MAP kinases; Punicalagin; Matrix metalloproteinase inhibitory factor