Expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases in laryngeal and pharyngeal squamous cell carcinoma: A quantitative analysis.

Squamous cell carcinomas of the head and neck are known for their aggressive growth and propensity to metastasize. Invasion is facilitated by matrix metalloproteineases (MMPs). Tissue inhibitors of MMPs (TIMPs) negatively regulate MMP activity. MMP and TIMP expression in head and neck squamous cell carcinomas was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). qRT-PCR allows measurement of several mRNAs from as little as 4 microg of total cellular RNA. We measured MMP-1, MMP-2, MMP-9, and TIMP-1 expression in 8 specimens of primary tumors and adjacent normal tissue. MMP-1 was overexpressed in 6 of 8 tumors, and MMP-9 was overexpressed in 4 of 7 tumors. MMP-2 was expressed in 3 of 8 tumors and 3 of 8 normal samples. TIMP-1 was expressed in all specimens. This work demonstrates that qRT-PCR can be used to examine expression of specific mRNAs in clinical specimens. Therefore this method provides another tool for the molecular analysis of tumors.

Heterochromia after pediatric cataract surgery.

PURPOSE: Changes in iris color have been noted anecdotally after cataract surgery in infants, but they have not been studied systematically. The mechanism for these iris color changes has not previously been reported in the biomedical literature. METHODS: Photographs were taken of both eyes of 15 children and 11 rhesus monkeys who had undergone unilateral cataract surgery. Masked examiners reviewed the photographs and compared the iris color of the eyes that were operated on with the eyes that were not operated on. Between 4 and 6 weeks postoperatively, the level of prostaglandin F(2alpha) in the aqueous humor (n = 4) and vitreous humor (n = 2) was measured in both the operated and nonoperated eyes of 4 monkeys that had undergone a neonatal lensectomy during the first 5 days of life. RESULTS: Thirteen of 15 children had a darker iris color in the operated eye in relation to the nonoperated (control) eye. Four of 11 monkeys had a uniformly darker iris in the operated eye; the other 7 monkeys had regional darkening or patches of darker iris in the eye that was operated on. The prostaglandin F(2alpha) levels in neonatal monkeys were higher in the aqueous humor and in the vitreous humor of the operated eye in relation to the nonoperated eye. CONCLUSION: In some children, cataract surgery is associated with a darkening of the iris color in the operated eye. We speculate that this darkening results from an exuberant prostaglandin release stimulated by the cataract surgery and may occur through the same or a similar mechanism by which latanoprost causes the darkening of iris color.

Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells.

Adenosine, released in increased amounts by hypoxic tissues, is thought to be an angiogenic factor that links altered cellular metabolism caused by oxygen deprivation to compensatory angiogenesis. Adenosine interacts with 4 subtypes of G protein-coupled receptors, termed A(1), A(2A), A(2B), and A(3). We investigated whether adenosine causes proliferation of human retinal endothelial cells (HRECs) and synthesis of vascular endothelial growth factor (VEGF) and, if so, which adenosine receptor subtype mediates these effects. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), in a concentration-dependent manner, increased both VEGF mRNA and protein expression by HRECs, as well as proliferation. This proliferative effect of NECA was inhibited by the addition of anti-human VEGF antibody. NECA also increased insulin-like growth factor-I and basic fibroblast growth factor mRNA expression in a time-dependent manner and cAMP accumulation in these cells. In contrast, neither the A(1) agonist N(6)-cyclopentyladenosine nor the A(2A) agonist 2-p-(2-carboxyethyl) phenethylamino-NECA caused any of the above effects of NECA. The effects of NECA were not significantly attenuated by either the A(2A) antagonist SCH58261 or the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine. However, the nonselective adenosine receptor antagonist xanthine amine congener completely inhibited the effects of NECA. Addition of antisense oligonucleotide complementary to A(2B) adenosine receptor mRNA inhibited VEGF protein production by HRECs after NECA stimulation. Thus, the A(2B) adenosine receptor subtype appears to mediate the actions of adenosine to increase growth factor production, cAMP content, and cell proliferation of HRECs. Adenosine activates the A(2B) adenosine receptor in HRECs, which may lead to neovascularization by a mechanism involving increased angiogenic growth factor expression.

Expression of matrix metalloproteinases and tissue inhibitor of matrix metalloproteinases in mesothelial cells and their regulation by transforming growth factor-beta1.

Tissue injury and pelvic inflammation often results in peritoneal scar tissue formation. The objective of this study was to determine whether mesothelial cells which line the peritoneal cavity express matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), and if their expression is regulated by transforming growth factor-beta1, a key regulator of tissue fibrosis. For this purpose we used Met-5A cells, a cell line derived from human normal mesothelial cells, and for comparative analysis we used U-937 cells, a human monocytic/macrophage cell line. The cells were treated with transforming growth factor-beta1 (1 ng/ml) for various time periods and the levels of MMP and TIMP mRNA and protein expression were determined using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The results indicate that the mesothelial cells and macrophages express MMP-1 (collagenase-1), MMP-3 (stromelysin-1), TIMP-1 and TIMP-2 mRNA and protein at various levels, with significantly higher TIMPs than MMPs, and higher MMP-1 than MMP-3 (p < 0.001). The mesothelial cells express significantly less MMP-1, higher MMP- 3 and similar levels of TIMP mRNA compared to macrophages. In a time-dependent manner, treatment of the mesothelial cells with transforming growth factor-beta1 resulted in a significant decrease in the expression of MMP-1, while increasing the expression of TIMP-1 mRNA (p = 0.05). In contrast, MMP-3 and TIMP-2 expression was unaffected in mesothelial cells and in macrophages, compared to untreated controls. There was a significant increase in secreted MMP-1 and TIMP-2 by mesothelial cells following transforming growth factor-beta1 treatments in a time-dependent manner (p = 0.05 and p = 0.01), without affecting the secretion of these proteins by macrophages. A major portion of MMP-1 in the culture conditioned media of both cell types was found in complex with TIMP-1. The ratios of MMP-1/TIMPs production were significantly higher than MMP-3/TIMPs in mesothelial cells and macrophages, and progressively decreased following transforming growth factor-beta1 treatments (p < 0.05). In conclusion, these results indicate that mesothelial cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by transforming growth factor-beta1, a mechanism that in part may influence the outcome of peritoneal tissue repair and adhesion formation.

TGF-beta1 expression is reduced in hydrocephalic H-Tx rat brain.

Transforming growth factor-beta1 (TGF-beta1) is a cytokine with diverse biological effects. Overexpression of TGF-beta1 in mice has been shown to induce progressive hydrocephalus. We have used a quantitative RT-PCR method to analyze the TGF-beta1 expression in the brains of H-Tx rat, a model of congenital hydrocephalus. Our studies have shown that rather than increased expression, the 3- and 10-day hydrocephalic H-Tx rats have significantly lower TGF-beta1 levels than their normal siblings (p < 0.01). This difference became insignificant in the 21-day group. Besides, both hydrocephalic and normal H-Tx rats have significantly lower TGF-beta1 levels in all three age groups of 3-, 10- and 21-days than SD control rats (p < 0.01 in all three groups) although the difference tends to become less significant with development. We also tested the expression of another cytokine, the epidermal growth factor, and observed a similar reduction. This suggests that the TGF-beta1 expression change is not unique to the development of hydrocephalus in this rat model. Our hypothesis is that the TGF-beta1 expression decrease in the H-Tx rat is not the cause of the disease. Rather it might be the result of feedback inhibition by increase in the expression of the gene it regulates, including an extracellular matrix component. Effort is currently being made to test this hypothesis.

Messenger RNA levels for genes involved in extracellular matrix from human corneal scrapings before and after photorefractive keratectomy.

PURPOSE: To measure the levels of mRNA for genes important in cellular and extracellular matrix regulation in human corneal epithelium before and after photorefractive keratectomy (PRK) in order to explain myopic regression following surgery. METHODS: Scrapings from 26 normal corneas before a first photorefractive keratectomy were randomly pooled in two samples of 16 and 10 scraping, respectively, and compared to another 23 scrapings from corneas with myopic regression after a previous photorefractive keratectomy, also randomly pooled in another 2 samples of 16 and 7 scrapings each. The scrapings were analysed for seven different messenger RNAs involved in extracellular matrix using competition-based quantitative reverse-transcription polymerase chain reaction. RESULTS: Messenger RNAs for TGFalpha (Transforming growth factor-alpha), TGFbeta1 (Transforming growth factor-beta1), EGF-R (Epidermal growth factor-receptor) and TIMP1 (Tissue inhibitor metalloproteinase-1) were present in all samples. No mRNA for MMP9 (Metalloproteinase 9) or MMP2 (Metalloproteinase 2) were detected in any sample. Messenger RNA for collagen (alpha1) III was present in one sample following photorefractive keratectomy. CONCLUSIONS: The detection and measurement of levels of messenger RNA for selected growth factors, receptors, metalloproteinases and extracellular matrix proteins in ex vivo samples of human corneal epithelium is important and possible with a modified polymerase chain reaction technique. Messenger RNAs for Collagen III and for TGF-beta1 were elevated in one sample after photorefractive keratectomy.

Matrix metalloproteinase expression in human retinal microvascular cells.

The degree of hyperglycemia correlates with the development of diabetic retinopathy. We investigated the effect of glucose on the expression of matrix metalloproteinase (MMP)-2 and MMP-9 (72-kDa and 92-kDa type IV collagenases, respectively) by human retinal microvascular endothelial cells (HRECs). Cultured HRECs from nondiabetic and diabetic donors were exposed to 5 or 30 mmol/l glucose. Using gelatin zymography, conditioned medium (CM) from all cultures revealed a gelatinolytic band migrating at 65 kDa (representing the proform of MMP-2 that runs at 72 kDa under reducing conditions). This band was unchanged by glucose exposure or the disease state of the donors. CM from nondiabetic HREC cultures demonstrated an additional proteolytic activity migrating at 90 kDa when cells were exposed to 30 mmol/l glucose, but not when they were exposed to 5 mmol/l glucose. This same activity was seen in CM from HREC cultures of diabetic origin in the presence of both 5 and 30 mmol/l glucose. Western analysis confirmed the identity of the 65-kDa band as MMP-2. The anomalous activity at 90 kDa was identified as MMP-2 associated and co-migrating with a fibronectin fragment. Competition-based reverse transcription-polymerase chain reaction revealed that nondiabetic and diabetic HRECs expressed constitutively mRNA for MMP-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and fibronectin. After exposure to 5 or 30 mmol/l glucose, no changes were detected in mRNA levels in MMP-2 or MMP-9, their inhibitors TIMP-1 and TIMP-2, or fibronectin in either nondiabetic or diabetic HREC cultures. These results support the notion that modulation of MMP function by extracellular matrix components occurs in response to glucose and may be relevant to the development of diabetic retinopathy.

Changes in the expression of extracellular matrix (ECM) and matrix metalloproteinases (MMP) of proliferating rat parotid acinar cells.

Tissue morphogenesis, development, and maintenance of function are mediated by signals generated through the composition of the extracellular matrix. The regulation of the composition of matrix is determined by enzymes specific for their degradation, the matrix metalloproteinases. Chronic injections of the beta-adrenergic receptor agonist, isoproterenol, result in a non-neoplastic hypertrophy and hyperplasia of the rat parotid gland. The activity of matrix metalloproteinases, as measured by gelatin zymography and enzymatic digestion of Azocoll substrates by gland lysates, decreased significantly (P < 0.05) following 24 hrs of agonist treatment, and slowly recovered to control values by 6 days of treatment. Daily administration of the broad-spectrum matrix metalloproteinase inhibitor Galardin for 3 days in combination with isoproterenol resulted in enhanced gland hypertrophy compared with that produced by isoproterenol alone. Given alone, Galardin also caused hypertrophy. The relative abundance of mRNA for the extracellular matrix molecules, collagens I and III and fibronectin, declined rapidly following the initiation of beta-agonist treatment in vivo, while laminin B1 and B2 mRNA levels increased initially before declining below control levels. These changes in patterns of mRNA levels also were observed in the concentrations of glandular protein when Western dot blot analysis of collagens I and III and laminin, respectively, was used. The importance of laminin, in vivo, was demonstrated by coinjection of anti-laminin antibody along with isoproterenol, which resulted in the inhibition of beta-agonist-induced parotid gland hypertrophy and hyperplasia. These data suggest that modulation of the ECM is associated with isoproterenol-induced salivary gland hypertrophy and hyperplasia. It is likely that this modulation of the ECM takes place through transcriptional regulation of some ECM genes and regulation of matrix-degrading enzyme activity.

Insulin-like growth factor: receptor and binding proteins in human retinal endothelial cell cultures of diabetic and non-diabetic origin.

Human retinal endothelial cell (HREC) cultures of diabetic and non-diabetic origin were examined for the production of insulin-like growth factor I (IGF-I), IGF-I receptor and IGF-binding proteins (IGFBPs) using colloidal gold quantitative immunocytochemistry and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR). The levels of immunoreactivity for IGF-I receptor and for four IGFBPs (IGFBP-1, -2, -3 and -5) were significantly increased in diabetic HREC cultures. Moreover, diabetic HREC cultures showed significantly less immunoreactivity for IGF-I and for IGFBP-4 as compared to non-diabetic HREC cultures. Message levels for IGF-I decreased two-fold in diabetic HREC and correlated with protein levels. Message levels for IGFBP-1, -2 and -5 increased 1.5-, 1.7- and 1.6-fold, respectively, in diabetic HREC and correlated with protein levels. However, the protein levels for IGF-I receptor and IGFBP-3 and -4 did not correlate with mRNA levels. There were no differences in mRNA levels for IGF-I receptor and IGFBP-3 and -4 between diabetic and non-diabetic HREC cultures, suggesting a post-transcriptional regulation of IGF-I receptor and the two IGFBPs. The net effect, however, supports enhanced IGF-I action in HREC cultures of diabetic origin which is an important cellular event in diabetic retinopathy.

Human ageing impairs injury-induced in vivo expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 proteins and mRNA.

Proteolysis is an essential component of wound healing but, if uncontrolled, it may lead to degradation of the neo-matrix and a delay in wound repair. Despite numerous reports of impaired wound healing associated with increasing age, the control of proteolysis is completely unknown. Tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 inhibit the activity of matrix metalloproteinases and the pattern of regulation of these molecules determines in part the spatial and temporal regulation of proteolytic activity. This study reports on TIMP-1 and -2 protein localization using immunocytochemistry in healing wounds of healthy subjects of different ages from day 1 to 6 months post-wounding, and has quantified the mRNA levels for both inhibitors using reverse transcriptase-polymerase chain reaction (RT-PCR). TIMP-1 and TIMP-2 proteins are up-regulated from 24 h post-wounding, with a decrease in staining intensity by day 7 for TIMP-2 and by day 14 for TIMP-1. Steady-state mRNA levels for both TIMPs were significantly greater in normal young skin than in aged skin. In the young, there was a significant increase in mRNA expression for TIMP-1 and -2 by day 3 post-wounding, which decreased by day 14 and had returned to basal levels at day 21. In the wounds of the aged subjects, basal levels were observed for TIMP-1 and -2 at all time-points. These results suggest that intrinsic cutaneous ageing is associated with reduced levels of TIMP mRNA both in normal skin and during acute wound repair. These levels may be instrumental in dermal tissue breakdown in normal skin, retarded wound healing, and the predisposition of the elderly to chronic wound healing states.

Estrogen accelerates cutaneous wound healing associated with an increase in TGF-beta1 levels.

The cellular and molecular mechanisms underlying the effects of aging on human cutaneous wound healing are poorly understood, and the possible role of reproductive hormones in this process has never been investigated. We report that aging in healthy females was associated with a reduced rate of cutaneous wound healing, but an improved quality of scarring both microscopically and macroscopically, and with reduced levels of transforming growth factor-beta1 (TGF-beta1) immunostaining and steady-state mRNA in the wound. These age-related changes were reversed by the systemic administration of hormone replacement therapy (HRT). Moreover, ovariectomized young female rodents exhibited a marked delay in repair of acute incisional wounds, which was reversed by the topical application of estrogen. The cellular mechanism underlying these changes appears to involve an estrogen-induced increase in latent TGF-beta1 secretion by dermal fibroblasts. These results suggest that both the rate and quality of wound healing depend on reproductive hormone levels.

Expression of alpha-smooth muscle actin, TGF-beta 1 and TGF-beta type II receptor during connective tissue contraction.

Closure of rat mesenteric perforation is considered to occur by connective tissue contraction, a process that has been shown to be stimulated by transforming growth factor-beta 1. In the present study, we assessed the expression of alpha-smooth muscle actin during closure by quantitative-reverse transcription-polymerase chain reaction and in situ hybridization. The expression of transforming growth factor-beta 1 and transforming growth factor-beta type II receptor was also estimated in mesenteric membranes and free peritoneal cells after wounding. A larger expression of alpha-smooth muscle actin was seen around the wound edges compared to unwounded tissue. Both alpha-smooth muscle actin and transforming growth factor-beta type II receptor were expressed during Days 0, 3, 5, 7, and 10. The expression of alpha-smooth muscle actin on Day 5 was > 100 times higher than on Day 0. Transforming growth factor-beta 1 was expressed in both membranes and free peritoneal cells of unoperated control animals but down-regulated after wounding, a finding that has not been reported previously. It reappeared on Days 7 and 10 in free peritoneal cells but not in perforated membranes. The enhanced expression of alpha-smooth muscle actin and down-regulation of transforming growth factor-beta 1 expression after wounding appears to be important phenomena in tissue contraction and repair.

Single exposures to antiproliferatives: long-term effects on ocular fibroblast wound-healing behavior.

PURPOSE: To determine the long-term effects of single, 5-minute exposures to 5-fluorouracil (5FU) and mitomycin-C (MMC) on Tenon's capsule fibroblast migration, growth factor production, growth factor receptor expression, and extracellular matrix (ECM) production. METHODS: Monolayer cultures and the overlying growth medium of Tenon's capsule fibroblasts exposed to 5FU (0.25 to 25 mg/ml) or MMC (0.001 to 0.1 mg/ml) were harvested up to 48 days after treatment. The expression of growth factors and growth factor receptors, including transforming growth factor beta (TGFbeta), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF), and ECM molecules (collagen type I, collagen type III, and fibronectin) were quantitated at the mRNA and protein levels. The ability of fibroblasts exposed to 5FU and MMC to migrate to fetal calf serum was also investigated up to 48 days after treatment. RESULTS: Control cultures were found to produce the growth factors TGFbeta and bFGF but not EGF. Exposure to 5FU or MMC resulted in an initial significant increase (P < 0.05) in the production of TGFbeta and bFGF, with levels then decreasing toward those of controls. Cells exposed to 5FU or MMC exhibited an initial significant decrease (P < 0.05) in the number of TGFbeta, bFGF, and EGF growth factor receptors, with subsequent recovery toward control levels by day 48 after treatment. Both 5FU and MMC caused a significant reduction (P < 0.05) in collagen type I and fibronectin production compared to controls throughout the 48-day culture period. The production of collagen type III was initially elevated (P < 0.05) compared to controls after exposure to 5FU or MMC, production then decreasing toward control levels over the remainder of the 48-day culture period. The migration of cells exposed to 5FU or MMC was significantly reduced (P < 0.05) compared to controls up to 48 days after treatment; these cells exhibited a partial recovery of migratory ability throughout this period. CONCLUSIONS: Fibroblasts whose growth was arrested using single, short exposures to 5FU or MMC appear to be capable of performing several crucial aspects of wound healing, including the expression of growth factors and receptors and ECM molecules and the ability to migrate. These findings may help explain why in some patients treated with antiproliferatives, glaucoma filtration surgery fails because of scarring.

Extracellular-matrix gene expression during mouse submandibular gland development.

Early morphogenesis of mouse submandibular glands begins on late day 11 of fetal development when the epithelium begins to bud from the surrounding mandibular mesenchyme. Using total RNA collected from fetal BALB/c submandibular glands, steady-state levels of mRNA expression for extracellular matrix molecules were measured using quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR). By comparing the PCR amplification products of both the cellular mRNA and a synthetic template, pMATRIX, it was possible to measure the direct expression of collagens alpha2(I), alpha1(III), alpha1(IV), fibronectin, laminin B2, elastin and lysyl oxidase genes. There was an observed trend for an increasing concentration of collagen alpha2(I), collagen alpha1(III) and lysyl oxidase mRNA molecules per cell on day 16 of development. The relative abundance of elastin mRNA was detectable only on day 16. Fibronectin and laminin B2 were more constitutively present but had their highest copy number per cell on day 16. The presence of extracellular-matrix protein was confirmed by immunohistochemistry using day-16 fetal glands and adult glands. With the construction of the pMATRIX supertemplate and the advent of quantitative, competitive RT-PCR technology, it has been possible to measure small changes in the steady-state concentrations for extracellular-matrix mRNA during salivary gland development.

Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMPs) in normal skin and acute cutaneous wounds of healthy humans.

Despite the association of increasing age with chronic wound-healing disorders and an impaired rate of healing of acute cutaneous wounds, the role of matrix metalloproteinases (MMPs) is unknown. To determine the spatial and temporal patterns and activities of MMP-1, -2, -3 and -9, 132 healthy humans aged between 19 and 96 years underwent 4-mm punch biopsies followed by wound excision between day 1 and day 180 post-wounding. Wounds showed an age-related increase in MMP-2 and MMP-9 immunostaining from day 3; this was associated with degradation of gelatin as shown by zymograms and with increased proteinase activity as shown by azocoll assays. Distinct spatial localisations for each MMP were observed: MMP-2 was found in epidermal structures; MMP-9 was observed in inflammatory cells up to day 21; MMP-1 was localised to keratinocytes at the wound margin. Normal old skin showed pro-MMP-2 bands on zymography and increased MMP-2 immunostaining. These results indicate that: (1) intrinsic ageing is associated with the up-regulation of MMPs previously associated with chronic wound healing; (2) wound-tissue proteinases are essentially active up to day 21 postwounding; and (3) intrinsic ageing may predispose to tissue breakdown disorders because of MMP-2 up-regulation in normal skin.

Alpha-smooth muscle actin expression in rat and mouse mesenteric wounds after transforming growth factor-beta1 treatment.

Rat mesenteric perforations heal by contraction within 5 to 7 days, whereas mouse mesenteric perforations seldom close within 3 weeks unless stimulated by transforming growth factor-beta1. In this article, we quantified the expression of alpha-smooth muscle actin by quantitative-reverse transcription-polymerase chain reaction and the orientation of actin filaments at the wound margin by Fourier transformation image analysis after treatment with transforming growth factor-beta1. The expression of transforming growth factor-beta1 and its type II receptor was also assessed. Actin filaments were shown to increase with time at the wound margin in both species and the expression of alpha-smooth muscle actin mRNA increased simultaneously. Transforming growth factor-beta1 enhanced the alpha-smooth muscle actin expression four to five times in rats and three to four times in mice on day 5, but the number of copies expressed per cell was 15-fold higher in rats than in mice. Transforming growth factor-beta1 was down-regulated after wounding in free peritoneal cells of rats, but maintained until day 5 in transforming growth factor-beta1-treated mice. The main finding of this study was that untreated, normal rats expressed substantially more alpha-smooth muscle actin than mice. After treatment with transforming growth factor-beta1, this expression increased similarly in both species. It can be hypothesized that normal closure of mesenteric perforations requires a minimum level of actin expression. This level is not reached in normal mice, but is exceeded after stimulation. Perforations in the rat always close, because the alpha-smooth muscle actin expression is always above this level.

Differential expression of matrix metalloproteinases and their tissue inhibitors in leiomyomata: a mechanism for gonadotrophin releasing hormone agonist-induced tumour regression.

Tissue remodelling involving extracellular matrix (ECM) turnover plays a major role in leiomyoma growth and regression, regulated by the combined action of matrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs). We postulated that leiomyomata express MMP and TIMP mRNA and protein, and their expression is inversely regulated during tumour growth and gonadotrophin releasing hormone agonist (GnRHa)-induced regression. We therefore examined the expression of mRNA and protein for MMPs (interstitial collagenase, MMP-1; gelatinases, MMP-2 and MMP-9; and stromelysin, MMP-3) and TIMPs (TIMP-1 and TIMP-2) in leiomyoma and matched unaffected myometrium from GnRHa (lupron)-treated and untreated patients. Reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme analysis revealed that leiomyomata and myometrium expressed MMP-1, -2, -3 and -9, as well as TIMP-1 and -2 mRNA. Quantitative RT-PCR indicated that leiomyomata and myometrium during the secretory phase of the menstrual cycle expressed higher levels of MMP and TIMP mRNA compared to the proliferative phase (P < 0.05), with low to undetectable levels of MMP-1, -2 and -3 mRNA in the tumours. GnRHa therapy induced an overall reduction in MMP and TIMP mRNA expression in both leiomyomata and myometrium, but a significant decrease in TIMP-1, and an increase in MMP mRNA expression compared with untreated tumours (P < 0.05). Immunohistochemically, MMP-1, -2, -3 and -9 and TIMP-1 and -2 proteins were localized in leiomyomata and myometrial smooth muscle cells, arteriole wall and connective tissue fibroblasts, with an overall increase in MMP and a decrease in TIMP staining intensity in GnRHa-treated groups. The results suggest that MMP and TIMP expression in leiomyoma and myometrium are hormonally regulated, and that GnRHa-induced tumour regression is accompanied by an increase in MMP expression with a concomitant decrease in TIMP-1 expression, which may potentially provide an environment favouring ECM degradation.

Suppression of transforming growth factor-beta (TGF beta) and TGF beta receptor messenger ribonucleic acid and protein expression in leiomyomata in women receiving gonadotropin-releasing hormone agonist therapy.

The expression and cellular distribution of transforming growth factor-1 (TGF beta 1) through TGF beta 3 and TGF beta type I-III receptor messenger ribonucleic acid (mRNA) and protein were analyzed in leiomyomata from patients receiving GnRH agonist (GnRHa; leuprolide acetate) compared to those in untreated controls. Standard reverse transcription-PCR revealed that the unaffected myometrium and leiomyomata from leuprolide-treated and untreated patients express TGF beta 1-3 and TGF beta type I-III receptor mRNA. The myometrial and leiomyomata smooth muscle cells were the primary site of TGF beta 1-3 and TGF beta type I and II receptor mRNA and protein expression, as determined by in situ hybridization and immunohistochemical localization. These observations indicate that leiomyomata express a higher of level of TGF beta and TGF beta receptor mRNA and protein than unaffected myometrium during the secretory phase of the menstrual cycle, and women who received leuprolide acetate therapy had a substantially lower level of expression than untreated controls. Furthermore, competition-based quantitative reverse transcription-PCR using synthetic internal standards revealed that leiomyomata express a significantly higher number (copies per cell) of TGF beta type II receptor mRNA, followed by TGF beta 1, TGF beta type I receptor, TGF beta 2, and TGF beta 3 (P < 0.05). However, there was a significant decrease in the levels (copies per cell) of TGF beta 1, TGF beta 3, and TGF beta type I and type II receptor mRNA expression in leiomyomata from leuprolide-treated compared to untreated patients (P < 0.05). The data provide further evidence that leiomyomata express mRNA and protein for all components of the TGF beta system, and GnRHa therapy results in down-regulation of their expression. More specifically, these data suggest that TGF beta 1 and TGF beta 3 may play a more important role in leiomyomata growth than TGF beta 2, which leads us to propose that lowering TGF beta and receptor expression may have a direct effect on leiomyomata regression.

Biochemical analysis of acute and chronic wound environments.

The process of wound healing involves a complex interaction between numerous cell types, extracellular matrix molecules, and soluble mediators including growth factors and cytokines. This complex milieu is under active investigation for the purposes of beginning to understand how this environment regulates tissue repair. Quantitation of growth factors, cytokines, and matrix metalloproteinases within surgical wound fluids may help to elucidate this regulatory network, not only in noncomplicated wound healing but also in pathologic lesions such as chronic ulcers.

Horse complement protein C9: primary structure and cytotoxic activity.

Lack of hemolytic activity of horse serum is an inherent property of horse C9. To understand the molecular reasons for this deficiency we have cloned C9 cDNA from a horse liver cDNA library and have sequenced the cDNA yielding the complete coding sequence for horse C9. Purification of C9 from horse plasma and microsequencing established the N-terminus of the mature protein and verified that the correct horse C9 cDNA clone had been isolated. The deduced amino acid sequence corresponds to a mature protein of 526 amino acids that is 77% identical to human C9. It has the same domain structure as human C9 and contains 22 cysteines and four invariant tryptophans. The few differences include the N-terminus, which is an unblocked glycine in horse C9 but pyroglutamine in human C9, and three potential N-glycosylation sites compared to two in human C9. The N-terminal difference is unimportant since microsequencing of bovine C9, which is strongly hemolytic, established that it also has an unblocked glycine identical to horse C9. There are no obvious structural differences apparent that could resolve the differences in hemolytic potency between the two molecules. Aside from a few conservative replacements, both C9 sequences are identical between positions 250 and 360. This region includes the membrane interaction domain in C9 and the postulated transmembrane segment that is thought to constitute the wall of a putative transmembrane pore and, therefore, should be required for cytotoxicity. In agreement with this prediction we have observed that, in contrast to the marked decrease in hemolytic activity, horse C9 is very efficient in killing a variety of Gram-negative bacteria. These results demonstrate that horse C9 is a structurally competent molecule with efficient cytotoxic activity. Its inability to lyse erythrocytes may be related to the action of control proteins on target cell membranes.