TIMP-1 Immunoreactivity Research Articles

Melatonin pretreatment modulates anti-inflammatory, antioxidant, YKL-40, and matrix metalloproteinases in endotoxemic rat lung tissue.

We aimed to investigate the effects of melatonin administered before and during endotoxemia on the lung tissue of rats, cytokine, YKL-40, matrix metalloproteinase (MMP) and inhibitor levels, oxidative stress parameters, and energy balance. Sepsis was induced with lipopolysaccharide (LPS), the cell wall molecule of gram negative bacteria. Rats were divided into four groups, Control, LPS (Escherichia coli O127:B8, 20 mg/kg), melatonin (10 mg/kg), and melatonin+LPS (M+LPS). After injections, lung tissues samples were taken for experimental analyses. YKL-40, thiobarbituric acid reactive substances (TBARS), glutathione reductase (GR), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) enzymes levels were measured, high-energy components were analyzed; tumor necrosis factor-alpha (TNF-α), MMP-2, YKL-40, MMP-9, myeloperoxidase (MPO), tissue inhibitors of matrix metalloproteinase (TIMP)-1, and interleukin (IL)-10 immunoreactivities were investigated. In LPS group, YKL-40, creatine phosphate (both, p < 0.05), SOD, GR, adenosine mono-phophate (AMP), adenosine tri-phosphate (ATP) (for all, p < 0.01) were significantly decreased, while TBARS and adenosine di-phosphate (ADP) levels were increased (p < 0.01, p < 0.05; respectively) compared to other groups. MMP-2 and -9, TIMP-1, TNF-α, IL-10, and MPO immunoreactivity were investigated in LPS group. On the contrary, in M+LPS group, MMP-9, TIMP-1 immunoreactivities were not found and IL-10 and MMP-2 immunoreactivities were found with little involvement. In M+LPS group, YKL-40, GR, AMP, ATP, creatine phosphate (for all, p < 0.05), and SOD (p < 0.01) levels were significantly increased and TBARS levels were decreased (p < 0.05). In our study, we suggest that melatonin exerts a protective and curative effect by reducing the matrix metalloproteinase levels responsible for tissue damage balance, stimulating the release of antioxidant enzymes, regulating cytokines and energy balance during endotoxemia.

Dietary Supplementation with Omega-3 Polyunsaturated Fatty Acids Modulate Matrix Metalloproteinase Immunoreactivity in a Mouse Model of Pre-abdominal Aortic Aneurysm

Two-day infusion of angiotensin II to apolipoprotein E-deficient (ApoE(-/-)) mice provides a model of pre-abdominal aortic aneurysm. Long chain omega-3 polyunsaturated fatty acids (n-3 PUFAs) have anti-inflammatory effects. This study examined the effect of an eight-week low or high n-3 PUFA diet in ApoE(-/-) mice on matrix metalloproteinase (MMP) expression and elastin degradation. ApoE(-/-) mice were fed a low or high n-3 PUFA diet for eight weeks prior to two-day infusion with angiotensin II. The omega-3 index, MMP-2, MMP-9, TIMP-1, and TGF-β1 immunoreactivity, and elastin fragmentation were measured. The omega-3 index with the low and high n-3 PUFA diet was 3.78% and 13.03%, respectively. MMP-9 immunoreactive stain intensity was lower in mice fed the high, compared to the low n-3 PUFA diet in endothelial cells (suprarenal aorta), and inflammatory cells (suprarenal and infrarenal aorta). Inflammatory cells had higher TIMP-1 and TGF-β1 stain intensity in mice fed the high, compared to the low n-3 PUFA diet (suprarenal aorta). MMP-2 immunoreactivity was unaffected by diet. A non-significant trend for reduced elastin fragmentation was observed in mice fed the high n-3 PUFA diet. Dietary supplementation with n-3 PUFAs may have protective anti-inflammatory effects mediated through modulation of MMPs and TIMPs.

Residents' journal review

Residents' journal review

Effects of low-intensity laser therapy on periodontal tissue remodeling during relapse and retention of orthodontically moved teeth

This study was designed to investigate the effects of low-intensity laser therapy (LILT) on periodontal ligament (PDL) remodeling during relapse and retention after the completion of orthodontic movement. The maxillary central incisors (n = 104) of the 52 rats were randomly divided into five groups according to the treatment modality: baseline control group without any intervention (n = 8); relapse group without retainer after tooth movement (n = 24); retention group with fixed retainer after tooth movement (n = 24); lased relapse group without retainer after tooth movement and LILT (n = 24); lased retention group with retainer after tooth movement and LILT (n = 24). LILT was daily performed using a gallium-aluminum-arsenide diode laser in a biostimulation mode: wavelength of 780 nm, continuous waves at 70 mW output power, a preset low intensity of 1.75 W/cm(2) in contact mode, resulting in energy dose of 5 J/cm(2) per irradiation for 3 s. The animals were euthanized on days 1, 3, and 7 after removal of the orthodontic appliance. Real-time RT-PCR was performed for quantitative analysis of matrix metalloproteinases mRNA expression. Immunoreactivities of collagen and tissue inhibitor of metalloproteinase were observed on the compression and tension sides. LILT significantly facilitated the expression of five tested MMP mRNAs in both relapse and retention groups. TIMP-1 immunoreactivity was inhibited by LILT in both groups, whereas Col-I immunoreactivity was increased by LILT only in the retention group. These results indicate that LILT would act differently on the stability after orthodontic treatment according to additional retainer wearing or not. LILT when combined with a retainer on the moved teeth may shorten the retention period by accelerating periodontal remodeling in the new tooth position, whereas, LILT on the moved teeth left without any retainer would rather increase the rate of relapse after treatment.

P1-06-03: Predictive Value of HER2, Topoisomerase-II (Topo-II) and Tissue Inhibitor of Metalloproteinases (TIMP-1) for Efficacy of Taxane-Based Chemotherapy in Intermediate Risk Breast Cancer – Results from the EC-Doc Trial.

Abstract Background: Despite extensive research, there is still no consensus on optimal predictors for use of taxane-based chemotherapy (cht) in early breast cancer. Some studies have revealed HER2 as a significant predictive marker for efficacy of taxanes and anthracyclines. TIMP-1 and Topo-II are reported to be predictive for anthracycline efficacy. In our previous reports, both Ki-67≥20% and central G3 status emerged as significant predictors for taxane benefit. We have now compared HER2 and Topo-II (as protein expression and gene amplification) and TIMP-1 immunoreactivity as well as factor combinations (HT (HER2/TIMP-1) and 2T (Topo-II/TIMP-1) regarding their predictive value for benefit from taxane-based cht. Methods: The EC-Doc trial randomized 1950 patients with 1–3 positive LN to 6x CEF/CMF vs. 4xEC-4xDoc. Significantly better DFS and OS favoring EC-Doc have been previously reported (Nitz et al., SABCS 2008). Protein expression and gene amplification data as well central histology/grade were available for 772 patients. Survival analysis was performed using Cox proportional hazards and Kaplan-Meier statistics. Analysis of HER2 survival impact status was prospectively planned. Results: The entire and the investigated study populations did not differ regarding baseline characteristics. After median follow up of 64 months, both DFS (5y 90% vs. 80%, p=0.006) and OS (5y 95% vs. 92%, p=0.022) rates significantly favored EC-Doc vs. CEF in this cohort as well. HER2 over-expression (3+ and/or FISH≥2.0) was reported in 158 tumors (20%), Topo-II aberration (deletion or amplification) was reported in 78 (49.4%) HER2+ and in 83 (13.6%) HER2−negative tumors; 496 tumors were classified as TIMP-1 immunoreactive (65.2%). None of these factors were significantly prognostic for EFS in this collective. Regarding DFS, EC-Doc was strongly superior to FEC in HER2+ tumors (HR=0.29, 95%CI: 0.12−0.7, p=0.006) but not in HER2− tumors (p=0.18). In Topo-II aberrated tumors, the benefit of EC-Doc was remarkably strong (HR=0.28, 95% CI: 0.11−0.69, p=0.006), whereas the benefit was not significant in Topo-II normal tumors (p=0.16), which comprise more than ¾ of the total. In contrast, Topo-II protein overexpression (&amp;gt;10%) was not associated with a stronger benefit in either subgroup. The superiority of EC-Doc to FEC was significant in the larger group of TIMP-1 immunoreactive tumors (HR=0.57, p=0.025) but not in TIMP-1 negative tumors (p=0.14), similar behavior was seen in “HT” and “2T” subgroups (significance with HR about 0.5 in the “+” subgroups). In a multivariate model for DFS including age, tumor size, Ki-67, central grade, HR, HER2, TOPO_II aberration, TIMP-1 status, therapy and interactions of all these factors with therapy arm, the only significant therapy interaction was that of (high) Ki-67 (HR=0.76, 95% CI: 0.59−0.98, p=0.03); significant main effects in this model were age, central grade, and Ki-67. Conclusions: These data suggest predictive significance for Topo-II aberration, TIMP immunoreactivity and HER2 over-expression as well as a multivariate predictive significance of high Ki-67 for enhanced benefit of taxane-based cht. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-03.

Elevated plasma levels of tissue inhibitors of metalloproteinase-1 and their overexpression in muscle in human and mouse muscular dystrophy

To investigate the role of tissue inhibitors of metalloproteinases (TIMPs) in muscular dystrophy, we examined the expression of TIMP-1 using plasma and biopsied muscle from patients with various muscular dystrophies by ELISA, immunohistochemistry, and Western blot analysis. TIMP-1 immunolocalization was also studied in mouse models of muscular dystrophy. Plasma TIMP-1 was elevated and correlated with TGF-β1 in Duchenne muscular dystrophy (DMD) and congenital muscular dystrophy (CMD), but not in Becker muscular dystrophy. In dystrophic human muscles, TIMP-1 was immunopositive in the regenerating and non-regenerating muscle fibers, and interstitial cells that consist of activated fibroblasts and macrophages. TIMP-1 immunoreactivity was also closely associated with TGF-β1. Western blot analysis showed elevated TIMP-1 protein in muscles in DMD. The semiquantitative analysis of TIMP-1 staining intensity and tissue fibrosis showed that TIMP-1 immunoreactivity is closely associated with the extent of tissue fibrosis in human and mouse dystrophic muscles. In conclusion, the present study implied that the TGF-β1–TIMP-1 pathway is activated in dystrophic muscles and the overexpression of TIMP-1 may result in increased deposition of extracellular matrix leading to tissue fibrosis.

HER2,TOP2A, and TIMP-1 and Responsiveness to Adjuvant Anthracycline-Containing Chemotherapy in High-Risk Breast Cancer Patients

PURPOSE To evaluate whether the combination of HER2 with TIMP-1 (HT) or TOP2A with TIMP-1 (2T) more accurately identifies patients who benefit from cyclophosphamide, epirubicin, and fluorouracil (CEF) compared with cyclophosphamide, methotrexate, and fluorouracil (CMF) than these markers do when analyzed individually. PATIENTS AND METHODS The Danish Breast Cancer Cooperative Group (DBCG) 89D trial randomly assigned 980 high-risk Danish breast cancer patients to CMF or CEF. Archival tumor tissue was analyzed TIMP-1, and HER2-negative and TIMP-1 immunoreactive tumors were classified as HT nonresponsive and otherwise HT responsive. Similarly, the 2T panel was constructed by combining TOP2A and TIMP-1; tumors with normal TOP2A status and TIMP-1 immunoreactivity were classified as 2T-nonresponsive and otherwise 2T-responsive. Results In total, 623 tumors were available for analysis, of which 154 lacked TIMP-1 immunoreactivity, 188 were HER2 positive, and 139 had a TOP2A aberration. HT status was a statistically significant predictor of benefit from CEF compared with CMF (P(interaction) = .036 for invasive disease-free survival [IDFS] and .047 for overall survival [OS]). The 269 (43%) patients with a 2T-responsive profile had a significant reduction in IDFS events (adjusted hazard ratio, 0.48; 95% CI, 0.34 to 0.69; P < .001) and OS events (adjusted hazard ratio, 0.54; 95% CI, 0.38 to 0.77; P < .001). 2T status was a highly significant predictor of benefit from CEF compared with CMF (P(interaction) < .0001 for IDFS and .004 for OS). CONCLUSION The 2T profile is a more accurate predictor of incremental benefit from anthracycline-containing chemotherapy than HER2, TIMP-1, or TOP2A individually, and compared with these, 2T classifies a larger proportion of patients as sensitive to anthracyclines.

Immunohistochemical Study of Matrix Metalloproteinase 9 and Tissue Inhibitor of Matrix Metalloproteinase 1 in Benign and Malignant Breast Tissue – Strong Expression in Intraductal Carcinomas of the Breast

Objective: Matrix metalloproteinases (MMPs) are involved in carcinogenesis due to their tissue remodeling capability, and there is convincing evidence linking gelatinase B (MMP-9) with malignant cell invasion. Tissue inhibitor 1 of MMP (TIMP-1) is a strong inhibitor of MMP-9 but has also tumor-enhancing effects. Only few data exist on MMP-9 or TIMP-1 expression in tissue samples of different breast histology. Methods: MMP-9 and TIMP-1 immunoreactivity was examined in a wide range of breast tissue samples differing in histology from usual ductal hyperplasia (UDH) to fully developed ductal breast carcinoma. Immunohistochemical expression of MMP-9 was studied in 178 samples: 31 UDH samples, 29 atypical ductal hyperplasia (ADH) samples, 28 ductal carcinoma in situ (DCIS) samples and 90 ductal invasive carcinoma samples (30 samples of malignancy grades I, II and III, respectively). TIMP-1 expression was also analyzed in 178 breast tissue samples: 41 UDH, 21 ADH and 34 DCIS lesions, and 82 invasive ductal breast carcinomas (25 in grade I, 30 in grade II and 27 in grade III). Results: A significantly distinctive pattern of MMP-9 protein expression was shown in DCIS samples, where 85.7% of the cases showed moderate or strong positivity and negative staining was rare (p = 0.021). Negative or weakly positive MMP-9 staining was the most prominent finding in UDH (71%), ADH (69%) as well as in invasive carcinoma samples (64.4%). Various degrees of TIMP-1 expression were seen in 86.5% of all cases. DCIS and invasive carcinoma samples revealed similar immunostaining: at least some positivity was seen in 91.1% of the DCIS samples and 91.5% of infiltrative carcinomas. Thus, TIMP-1 negativity (22.2%) was significantly associated with hyperplastic lesions (p = 0.026). Conclusions: These results suggest that MMP-9 and TIMP-1 overexpression are early markers of breast carcinogenesis preceding tumor invasion. Apparently, DCIS carries the risk to evolve into a malignant phenotype according to these markers. The clinical importance of these findings is discussed.

Matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2 expression is not regulated by norgestimate or norelgestromin

Steroid hormones regulate endometrial expression of matrix metalloproteinases (MMPs) and their inhibitors. Synthetic progestins are widely used in oral contraceptives and for hormone replacement therapy. To assess whether the synthetic progestins norgestimate and its derivative norelgestromin (17-deacetylnorgestimate) modulate the expression of MMPs, Ishikawa endometrial cancer cells were separately treated with 17β-estradiol, 17α-hydroxyprogesterone, norgestimate and norelgestromin. Culture supernatants were assayed for MMPs 2, 3 and 9, and for tissue inhibitors of MMPs (TIMP-1 and TIMP-2) by enzyme-linked immunosorbent assays (ELISAs). No marked modulation of MMP-2 and TIMP-2 expression was observed upon incubation of the cells with the synthetic progestins. By ELISA, neither MMP-3 or MMP-9 nor TIMP-1 immunoreactivity was detected. Interestingly, TIMP-2 expression was downregulated by 17β-estradiol and 17α-hydroxyprogesterone.

The Relationship Between Expression of Matrix Metalloproteinases(MMPs)-2, 9 and Tissue Inhibitors of Metalloproteinase (TIMP)-1, 2 and Survival Time in Resected Non-Small Cell Lung Cancer

Background : Matrix metalloproteinases(MMPs) are a large family of proteolytic enzymes, which are involved in the degradation of many different components of the extracellular matrix. There is increasing evidence indicating that individual MMPs have important roles in tumor invasion by inactivating the MMPs. In this study, the correlation between MMPs and TIMPs expression, and the clinical outcome was investigated. Materials and Methods : Immunohistochemical staining of MMP-2, 9 and TIMP-1,2 were performed on paraffin-embedded tumor sections from 74 resected primary non-small cell lung cancers. Results : In 74 patients, MMP-2, MMP-9, TIMP-1, and TIMP-2 immunoreactivity was demonstrated in 24(34%), 19(26%) and 32(43%) of the paraffin-embedded tumors, respectively. The median survival of the MMP-2 positive cases was significantly shorter than that of the negative cases(20 vs 34 months). The median survival of the TIMP-2 positive cases was also was significantly longer than that of the negative cases (34 vs 18 months). The MMP-2, and MMP-9 expression level had a positively correlation with a more advanced stage and lymph node metastasis. There was inverse correlation between TIMP-2 expression and tumor invasion. The median survival of the MMP-2 negative/TIMP-2 positive cases was higher than that of the other cases. Conclusion : These results suggest that tumor invasion and lymph node metastasis are closely related to MMP-2 and MMP-9 expression. There was an inverse correlation between TIMP-2 and MMP-9 expression, and tumor invasion.

In vitro and in vivo expression of interstitial collagenase/MMP-1 by human mast cells.

Degradation of the extracellular matrix occurs under physiological and pathological conditions, thought to be principally mediated by a family of neutral proteolytic enzymes termed the matrix metalloproteinases (MMPs). The present study was initiated to determine whether mast cells have the ability to produce these proteases in diseased and normal human tissue. Immunohistochemistry and in situ hybridization was performed to localize interstitial collagenase protein and mRNA transcripts in diseased human tissue. The human mast cell line HMC-1 was cultured under serum free conditions, stimulated with phorbol mystrate acetate (PMA) and supernatants analyzed by Western blotting and zymography to determine the profile of secreted MMPs. The dog mast cell line BR, known to secrete gelatinolytic enzymes, was used in parallel studies. Total RNA was extracted and analyzed by RT-PCR for the expression of tissue inhibitors of MMP (TIMPs). Collagenase-1 protein and mRNA were expressed by tryptase and chymase positive human mast cells in all tissue analyzed. This proteinase wa also detected in the cytoplasm and conditioned media of HMC-1 cells. PMA induced gelatinolytic activity in both mast cell lines examined. TIMP-1 immunoreactivity was detected and TIMP-1, and-2 (but not TIMP-3) mRNA transcripts were amplified from HMC-1 cells. This is the first demonstration of the expression of collagenase-1 by human mast cells in both inflamed and normal tissues, and by a human mast cell line. MMPs secreted by these cells could contribute to the extensive matrix lysis characteristic of diseases such as rheumatoid arthritis and inflammatory ocular disorders. Alternatively collagenase-1 production by mast cells may play a critical role in cell invasion and migration into sites of inflammation.

Balance in asthma between matrix metalloproteinases and their inhibitors

Balance in asthma between matrix metalloproteinases and their inhibitors

Expression of Matrix Metalloproteinases and Tissue Inhibitor of Matrix Metalloproteinases in Non-Small-Cell Lung Cancer

Expression of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 was studied in non-small-cell lung cancer (NSCLC). Activity of MMP-2 and MMP-9 by gelatin zymography and expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNAs were examined in 11 lung cancer cell lines which included six small-cell lung cancer (SCLC) cell lines. Localization of MMP-2, MMP-9, TIMP-1, and TIMP-2 was examined by immunohistochemistry in 43 resected NSCLC (22 adenocarcinomas, 17 squamous cell carcinomas, 4 large cell carcinomas) using specific anti-human monoclonal antibodies. Expression of MMP-2 mRNA was detected in 5 (100%), MMP-9 in 1 (20%), TIMP-1 in 4 (80%), and TIMP-2 in 5 (100%) of 5 NSCLC cell lines examined. MMP-2 gelatinolytic activity also was detected in all five NSCLC cell lines, whereas MMP-9 activity was detected in only one cell line. In 43 patients, MMP-2, MMP-9, TIMP-1, and TIMP-2 immunoreactivity was demonstrated in 19 (44%), 9 (21%), 15 (35%), and 29 (67%) excised tumors, respectively. All stromal fibroblasts in tumor samples stained positive for MMP-2. There was a correlation between TIMP-2 immunoreactivity and disease stage (42% stage I versus 88% stages II, III, and IV) (p = 0.0024). Both cancer cell lines and NSCLC tumor samples frequently expressed MMP-2, MMP-9, TIMP-1, and TIMP-2; MMP-2 in particular was highly expressed in malignant cells and surrounding fibroblasts. These findings suggest that MMP-2 plays a more important role in invasion of NSCLC than MMP-9 and that TIMP-2 may have clinical relevance in NSCLC.

Role of tissue inhibitors of metalloproteinases (TIMPs) in colorectal carcinoma.

Increased production of matrix metalloproteinases (MMPs) has been associated with increases in invasive and metastatic potential in many types of human carcinoma. Tissue inhibitors of metalloproteinase (TIMP)-1 inhibits most interstitial collagenases and MMP-9. TIMP-2 binds specifically and noncovalently to the pro-form of MMP-2 and inhibits its enzyme activity. In this study, we examined TIMP-1 and TIMP-2 expressions in relation to clinicopathological variables in colorectal carcinoma with in situ hybridization and immunohistochemistry. TIMP-1 and TIMP-2 expressions were localized overwhelmingly to pericancer stromal cells, while malignant and normal mucosal cells were weak or negative. Strong stromal TIMP-1 immunoreactivity correlated with Dukes' stage (p=0.022), status of lymph node metastasis (p=0.044) and poor survival (p= 0.005). The degree of immunohistochemical staining of TIMP-2 did not correlate with all clinicopathological variables. The correlation between enhanced TIMP-1 expression and advanced stage and poor survival suggest a growth promoting activity of TIMP-1 in colorectal carcinoma.

Effect of immunization against the amino-terminal peptide (alpha N) of the alpha 43-subunit of inhibin on follicular atresia and expression of tissue inhibitor of matrix metalloproteinase (TIMP-1) in ovarian follicles of sheep.

Ewes actively immunized against alpha N, the N-terminal peptide of inhibin alpha 43 precursor, have lowered fertility associated with ovulation failure, restricted tissue remodelling and reduced matrix metalloproteinase-2 activity in the follicular fluid at the time of expected ovulation. This could be due to altered ratios of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase (TIMP-1), or to the onset of atresia in antral follicles destined to ovulate. The objectives of the present study were to investigate the effects of immunization against alpha N on the localization of TIMP-1 in ovine follicles, and on follicular growth and atresia in the follicular phase. Ewes were either immunized against alpha N or remained as controls and the ovaries were removed before (0, n = 4) and at 12 h (n = 4) and 24 h (n = 4) after hCG administration in a synchronized follicular phase, 48 h after removal of intravaginal pessaries. Observations were made on a single section taken through the largest follicle present in the ovaries of each ewe. There were no healthy antral follicles > 1 mm in immunized ovaries (0/29) compared with controls (16/31) (P < 0.001), whereas the proportion of healthy antral follicles < 1 mm was the same in each group (9/19 versus 5/12). TIMP-1 immunoactivity was localized in large luteal cells, smooth muscle and endothelial cells, and in all antral follicles, including oocytes. At the time of hCG administration, no TIMP-1 immunoreactivity was detected in the apical region of the follicular wall of large follicles (> 6 mm) compared with the rest of the follicle wall, but staining appeared in the apical granulosa layer 24 h later. In newly formed corpora lutea, TIMP-1 expression was found along the invaginating vascular layer. There was no effect of immunization on the patterns of TIMP-1 immunoreactivity, suggesting that changes in TIMP-1 are not involved in the effects of alpha N. These data are consistent with a paracrine role for alpha N in the selection and atresia of antral follicles, and for TIMP-1 in tissue reorganization and steroidogenesis at the time of ovulation.

Immunolocalization of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human subconjunctival tissues

Purpose. To evaluate the roles of fibroblast proteins in the remodeling of the subconjunctival connective tissue, we immunohistochemically assessed the expression of matrix metalloproteinases (MMP)−1 and −2, and the tissue inhibitors of matrix metalloproteinases (TIMP)−1 and −2 in cultured human subconjunctival fibroblasts and in normal and healing human subconjunctival connective tissue.Methods. Cultured fibroblasts derived from human subconjunctival connective tissue and surgical specimens of normal and healing conjunctiva were immunostained with monoclonal antibodies directed against human MMPs and TIMPs and examined by light and electron microscopy.Results. In the cultured fibroblasts, MMP-1 and TIMP-1 antibodies stained the cytoplasm in a fine granular pattern, suggesting localization of those proteins in the endoplasmic reticulum (ER) and Golgi apparatus. Antibodies to MMP-2 and TIMP-2 reacted with fibroblast cytoplasm in a granular pattern. Electron microscopy of those fibroblasts revealed MMP-1 and TIMP-1 immunoreactivity in the ER cisternae or on the membrane of the ER. In surgical samples, MMP-1 and TIMP-1 were immunohistochemically detected in healing subconjunctival tissue, but not in conjunctival epithelium or normal subconjunctival tissue.Conclusions. MMPs and TIMPs may be involved in remodeling of subconjunctival connective tissue and in fibroblast population after surgical interventions. These proteins may play a crucial role in the post-operative fibrotic process occurring during scar formation in subconjunctival tissue. Curr. Eye Res. 17: 445–451, 1998.This paper was presented in abstract form by Dr. Kawashima at the Annual Meeting of The Association for Research in Vision and Ophthalmology, May 13, 1997, Fort Lauderdale, FL, USA.

Immunolocalization of tissue inhibitor of metalloproteinases-1 within ovine periovulatory follicular and luteal tissues.

In previous studies, tissue inhibitor of metalloproteinases (TIMP)-1 mRNA increased in follicular tissue after the preovulatory gonadotropin surge and was expressed in luteal tissue. However, the localization of TIMP-1 protein within ovine periovulatory follicular and luteal tissues is unknown. The objectives of the present study were to 1) localize TIMP-1 within follicles collected before and after a preovulatory gonadotropin surge and within Day 3 and Day 10 corpora lutea (CL), 2) determine whether TIMP-1 was colocalized to Day 10 luteal cells with oxytocin or TIMP-2, and 3) determine whether TIMP-1 was present within secretory granules of large luteal cells. Ovaries were removed from ewes before (presurge; n = 4) or 12-14 h after (postsurge; n = 5) an LHRH-induced gonadotropin surge (36 h following PGF2 alpha-induced luteolysis; objective 1). Additionally, ovaries containing CL were collected on Days 3 (n = 5; objective 1) and 10 (n = 4, 3, and 2 for objectives 1, 2, and 3, respectively). TIMP-1 immunoreactivity was observed within the granulosa cells of postsurge but not presurge follicles. On Days 3 and 10, TIMP-1 was localized within luteal tissue in a cell-specific manner. On Day 10, many of the cells that were immunopositive for TIMP-1 were judged to be large luteal cells on the basis of morphology (diameter > 22 microns; round nucleus) and colocalization with oxytocin and TIMP-2. Electron microscopy demonstrated that TIMP-1 was localized to secretory granules undergoing exocytosis from Day 10 large luteal cells. These data indicate that TIMP-1 is produced by granulosa cells following a gonadotropin surge and is packaged in secretory granules by large steroidogenic cells of the ovine CL.