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Biomedical subjects

G S Schultz

Publications and source records attributed to G S Schultz.

At least 19 recordsLinked to original sources
PubMed2003

Connective tissue growth factor in tear film of the horse: detection, identification and origin.

BACKGROUND: Healing of corneal ulcers in horses is often associated with profound corneal stromal fibrosis and scar formation resulting in visual impairment. Connective tissue growth factor (CTGF) is a fibrogenic cytokine involved in wound healing and scarring. The purpose of this study was to determine whether CTGF was present in the tear fluid of normal horse eyes and the eyes of horses with corneal ulcers in order to evaluate the role of CTGF in corneal wound healing and corneal scar formation. METHODS: Tear fluid samples were collected from 65 eyes of 44 horses; 32 samples from normal eyes, 21 samples from eyes with corneal ulceration, and 12 samples from the unaffected contralateral eyes of horses with ulcers. CTGF levels in the tears were determined by enzyme immunoassay using goat IgG against human CTGF. Antigenetic similarity of human and horse CTGF was established in a bio-equivalence assay. The identity of horse CTGF was confirmed by western blot. Lacrimal and nictitating membrane glands were investigated by immunohistochemistry in the attempt to clarify the origin of tear fluid CTGF. RESULTS: CTGF was detected in tear film of 23 normal unaffected eyes (72%) and 8 normal contralateral eyes (67%), with the mean CTGF levels (+/- SEM) being 51.5+/-19.2 and 13.4+/-3.9 ng/ml respectively. CTGF was found in 8 eyes with corneal ulcers (38%) with the mean CTGF concentration of 26.3+/-14.8 ng/ml. Western blot identified the protein detected as CTGF. The identification of CTGF in lacrimal glands suggests a major role of these glands in the presence of CTGF in tears. CONCLUSIONS: CTGF is present in horse tear fluid and derives, at least partly, from the lacrimal gland. Equine CTGF has strong antigenic similarity with human CTGF. Corneal disease leads to a decrease of CTGF concentrations in tears. The possible role of CTGF in the healing process of ocular surface requires further investigation.

Animals↗
PubMed2003

Novel antisense oligonucleotides targeting TGF-beta inhibit in vivo scarring and improve surgical outcome.

The scarring response is an important factor in many diseases throughout the body. In addition, it is a major problem in influencing results of surgery. In the eye, for example, post-operative scarring can determine the outcome of surgery. This is particularly the case in the blinding disease glaucoma, where several anti-scarring regimens are currently used to improve glaucoma surgery results, but are of limited use clinically because of severe complications. We have recently identified transforming growth factor-beta (TGF-beta) as a target for post-operative anti-scarring therapy in glaucoma, and now report the first study of novel second-generation antisense phosphorothioate oligonucleotides against TGF-beta in vivo. Single applications of a TGF-beta OGN at the time of surgery in two different animal models closely related to the surgical procedure performed in glaucoma patients, significantly reduced post-operative scarring (P<0.05) and improved surgical outcome. Our findings suggest that TGF-beta antisense oligonucleotides have potential as a new therapy for reducing post-surgical scarring. Its long-lasting effects after only a single administration at the time of surgery make it particularly attractive clinically. Furthermore, although we have shown this agent to be useful in the eye, it could have widespread applications anywhere in the body where the wound-healing response requires modulation.

Amino Acid Sequence↗
PubMed2001

Pterygia pathogenesis: corneal invasion by matrix metalloproteinase expressing altered limbal epithelial basal cells.

OBJECTIVE: To assess the potential role of matrix metalloproteinases (MMPs) in the pathogenesis of pterygia by comparing the immunolocalization patterns of MMPs in altered limbal basal stem cells, activated fibroblasts, and areas of elastotic degeneration adjacent to the pterygia. METHODS: Nine primary and 1 recurrent pterygia along with normal superior limbal-conjunctival tissue and cornea were immunostained with mouse monoclonal antibodies specific for MMP-1, MMP-2, MMP-3, MMP-9, membrane type 1 (MT1)-MMP (MMP-14), and membrane type 2-MMP (MMP-15). RESULTS: Normal conjunctival, limbal, and corneal cells lacked significant immunostaining except for cell surface MT1-MMP. In contrast, altered limbal basal epithelial cells of the 9 primary and 1 recurrent pterygia immunostained for all 6 MMPs. Activated and altered fibroblasts associated with the pterygia immunostained primarily for MMP-1. In contrast, stromal areas of elastotic degeneration (pingueculae) showed variable immunostaining of MMPs. CONCLUSIONS: Altered limbal basal epithelial cells (pterygium cells) immunostained for multiple types of MMPs in contrast to normal conjunctival, limbal, and corneal cells. The pterygium cells invading over Bowman's layer produce elevated MMP-1, MMP-2, and MMP-9 expression, which probably are the main MMPs responsible for the dissolution of Bowman's layer. Pterygium cells may also cause activation of fibroblasts at the head of the pterygium, leading to the initial cleavage of fibrillar collagen in Bowman's layer by the production of MMP-1. Altered fibroblasts in areas of elastotic degeneration (pingueculae) trailing behind the pterygium constitute a second type of tumor, which is noninvasive. CLINICAL RELEVANCE: These data of altered MMP expression support the concept that altered basal limbal epithelial cells play a key role in the formation and migration of a pterygium.

Antibodies, Monoclonal↗
PubMed2001

Effect of healing on the expression of transforming growth factor beta(s) and their receptors in chronic venous leg ulcers.

The transforming growth factor betas are of major importance in the wound repair process; however, no studies to date have investigated the role of the transforming growth factor beta receptors in chronic venous leg ulcers or what effect healing has on these proteins. To determine whether the transforming growth factor beta peptides and their receptors are expressed in chronic venous wounds, we used immunofluorescent analysis and quantitative competitive reverse transcription polymerase chain reaction to identify the protein and mRNA expression, respectively. Biopsy samples from wounds and normal skin were collected from 12 patients with chronic venous leg ulcers and three patients undergoing reconstructive surgery, respectively. Additionally four of the chronic venous leg ulcer patients were re-biopsied between 2 and 8 wk after the first biopsy when the wounds had entered the healing phase. The tissue excised from the ulcers included the surrounding intact skin, the ulcer edge, and the ulcer base. Immunofluorescent staining for transforming growth factors beta1, beta2, and beta3 was observed within the epidermis of the skin surrounding the chronic venous ulcers and in fibroblasts and inflammatory cells of the dermis, although this staining was not as strong as that seen in normal unwounded skin. Very little staining could be seen within the ulcers for any of the ligands, however. In contrast the transforming growth factor beta type I receptor was observed throughout the ulcers and the normal unwounded skin biopsies, particularly in the basal epidermal cells. No immunofluorescence for the type II transforming growth factor beta receptor was observed in any of the ulcer biopsies investigated, although it was observed throughout the epidermis and in fibroblasts and inflammatory cells in the surrounding skin. Quantitative, competitive reverse transcription polymerase chain reaction was used to analyze mRNA expression for transforming growth factor beta1 and the type II receptor in the nonhealing ulcers and normal unwounded skin biopsies. These studies revealed that transforming growth factor beta1 and transforming growth factor beta receptor II mRNA was expressed in all the chronic nonhealing ulcers albeit at very low levels for the type II receptor. In marked contrast to the staining observed in nonhealing chronic ulcers, positive immunostaining was observed for the transforming growth factor betas and both the type I and type II receptors in healing ulcers. These results suggest that the absence of a viable receptor complex for the transforming growth factor betas in nonhealing chronic venous ulcers may contribute to wound chronicity.

Aged↗
PubMed2001

Amniotic fluid concentrations of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 during pregnancy and labor.

OBJECTIVE: This study was undertaken to determine whether obstetric factors affect the concentrations of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 in the amniotic fluid. STUDY DESIGN: We prospectively collected amniotic fluid samples from 109 women at various stages of pregnancy and labor and determined matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 concentrations by means of enzyme-linked immunosorbent sandwich assay systems. With multiple regression analysis we evaluated relationships between amniotic fluid matrix metalloproteinase 9 concentration and tissue inhibitor of metalloproteinase 1 concentration and the following factors: gestational age, presence of labor, cervical dilatation, membrane status, presence of clinical chorioamnionitis, and microbial colonization of the amniotic fluid. RESULTS: The detectable presence of amniotic fluid matrix metalloproteinase 9 was independently associated with intra-amniotic infection, labor, cervical dilatation, and spontaneous rupture of membranes. Chorioamnionitis and amniotic fluid matrix metalloproteinase 9 concentrations were correlated with tissue inhibitor of metalloproteinase 1 levels. CONCLUSIONS: Intra-amniotic infection, advanced labor, and rupture of membranes before the onset of labor were independently associated with the presence of matrix metalloproteinase 9 in the amniotic fluid. Both pathologic and physiologic processes appear to produce shifts in the balance between degradation and synthesis of the extracellular matrix.

Amniotic Fluid↗
PubMed2000

TGF-beta1, -beta2, and -beta3 in vitro: biphasic effects on Tenon's fibroblast contraction, proliferation, and migration.

PURPOSE: To compare the effects of the three human transforming growth factor-beta (TGF-beta) isoforms and different concentrations of TGF-beta on human Tenon's capsule fibroblasts (HTF), with a view to delineating the role of this growth factor in the subconjunctival scarring response after glaucoma filtration surgery. METHODS: Application of recombinant human TGF-beta1, -beta2, and -beta3 (range 0-10(-8) M) was assessed using several assays of HTF function: fibroblast-mediated collagen contraction, proliferation, and migration. RESULTS: All three isoforms of TGF-beta behaved in a similar manner in vitro. They each stimulated HTF-mediated collagen contraction, proliferation, and migration with a characteristic concentration-dependent response, with peak activities at 10(-9), 10(-12), and 10(-9) M, respectively, that were significantly different from control (P<0.05). At concentrations above and below peak activities, HTF activity was reduced, demonstrating biphasic effects of TGF-beta. CONCLUSIONS: TGF-beta1, -beta2, and -beta3 have similar actions in vitro; this is demonstrated by their effects on several HTF-mediated functions. TGF-beta induces a response in HTF that is concentration-dependent, with different functions being maximally stimulated at different concentrations. This biphasic response highlights the significance of the concentration profile of TGF-beta at the wound site. These findings are important in filtration surgery, where constant changes in the local environment occur due to the passage of aqueous and the wound healing process. The varying levels of TGF-beta in the aqueous and subconjunctival tissues may thus significantly modify the conjunctival scarring response.

Cell Division↗
PubMed2000

Causes and effects of the chronic inflammation in venous leg ulcers.

The pathogenesis of venous leg ulcers is multifactorial. In this review article new physiological, molecular and cellular abnormalities in venous ulcers related to the chronic inflammation are presented and discussed. Venous hypertension causes disturbed microcirculation and pathological changes of the capillaries, which eventually locks the condition in a self-amplifying, detrimental cascade with persistent elevated levels and activities of pro-inflammatory cytokines and proteases preventing progress into a healing phase. As a consequence fibroblasts senescence and become less responsive to growth factors the older the ulcers become. Current data imply there is no deficiency but rather an unfavorable distribution of growth factors in venous ulcers. An imbalance in proteolytic enzymes and their endogenous inhibitors is a common finding in chronic venous leg ulcers. Variation in disease severity and concomitant ailments in this heterogeneous patient group may explain the contradictory results in the literature. Thus, to advance the areas of research further, longitudinal studies involving larger number of patients are required to identify the major pathogenic factors.

Chronic Disease↗
PubMed2000

Enhanced short-term plasmid transfection of filtration surgery tissues.

PURPOSE: To quantify and localize plasmid transfection of filtration surgery tissues using two delivery techniques. METHODS: Full-thickness filtering procedures were performed on eyes of New Zealand albino rabbits. In 10 eyes, naked plasmid DNA in saline was either injected beneath Tenon's capsule at the filtration site or absorbed into a collagen shield that was then placed external to the sclerostomy and under the Tenon's capsule. Forty-eight hours after surgery, levels of the reporter gene, chloramphenicol acetyltransferase (CAT) were measured in samples of ocular tissues. In two additional eyes, the ss-galactosidase (ss-GAL:) reporter gene expression was localized histologically. RESULTS: Injection of plasmid DNA in saline vehicle into the filtration bleb produced readily detectable CAT activity in bleb tissue (conjunctiva, Tenon's capsule, and sclera) whereas CAT activity was nearly undetectable in samples of the cornea, iris-ciliary body, and tissues located opposite the bleb site. Delivery of the plasmid DNA into the bleb through a collagen shield increased CAT activity 30-fold over injection of plasmid in saline (2711 +/- 567 mU/mg versus 92 +/- 38 mU/mg). ss-Gal activity was imaged only in the region of the bleb, and microscopic examination showed ss-Gal activity localized to Tenon's capsule fibroblasts, with minimal ss-Gal activity observed in inflammatory cells or scleral fibroblasts. CONCLUSIONS: Transfection of filtration tissues is enhanced by absorption of naked DNA into a collagen shield. Furthermore, transfection is localized to the fibroblasts and inflammatory cells of the filtration bleb site. Gene therapy using naked plasmid DNA and a simple collagen shield delivery vehicle may be useful for regulating wound healing after glaucoma surgery.

Animals↗
PubMed1999

Keratinocyte collagenase-1 expression requires an epidermal growth factor receptor autocrine mechanism.

In response to cutaneous injury, expression of collagenase-1 is induced in keratinocytes via alpha2beta1 contact with native type I collagen, and enzyme activity is essential for cell migration over this substratum. However, the cellular mechanism(s) mediating integrin signaling remain poorly understood. We demonstrate here that treatment of keratinocytes cultured on type I collagen with epidermal growth factor receptor (EGFR) blocking antibodies or a specific receptor antagonist inhibited cell migration across type I collagen and the matrix-directed stimulation of collagenase-1 production. Additionally, stimulation of collagenase-1 expression by hepatocyte growth factor, transforming growth factor-beta1, and interferon-gamma was blocked by EGFR inhibitors, suggesting a required EGFR autocrine signaling step for enzyme expression. Collagenase-1 mRNA was not detectable in keratinocytes isolated immediately from normal skin, but increased progressively following 2 h of contact with collagen. In contrast, EGFR mRNA was expressed at high steady-state levels in keratinocytes isolated immediately from intact skin but was absent following 2 h cell contact with collagen, suggesting down-regulation following receptor activation. Indeed, tyrosine phosphorylation of the EGFR was evident as early as 10 min following cell contact with collagen. Treatment of keratinocytes cultured on collagen with EGFR antagonist or heparin-binding (HB)-EGF neutralizing antibodies dramatically inhibited the sustained expression (6-24 h) of collagenase-1 mRNA, whereas initial induction by collagen alone (2 h) was unaffected. Finally, expression of collagenase-1 in ex vivo wounded skin and re-epithelialization of partial thickness porcine burn wounds was blocked following treatment with EGFR inhibitors. These results demonstrate that keratinocyte contact with type I collagen is sufficient to induce collagenase-1 expression, whereas sustained enzyme production requires autocrine EGFR activation by HB-EGF as an obligatory intermediate step, thereby maintaining collagenase-1-dependent migration during the re-epithelialization of epidermal wounds.

Animals↗
PubMed1999

Amniotic fluid matrix metalloproteinase-9 levels in women with preterm labor and suspected intra-amniotic infection.

OBJECTIVE: To determine the accuracy of amniotic fluid (AF) matrix metalloproteinase-9 measurements for diagnosing intra-amniotic infection in women with preterm labor. METHODS: We performed amniocenteses in 44 women between 22 and 35 weeks' gestation who presented to our center with preterm labor and clinical suspicion of intra-amniotic infection. Each sample was analyzed by glucose measurement, Gram stain, and culture for aerobes, anaerobes, and mycoplasmas. We tested the AF for matrix metalloproteinase-9 using gelatin zymography and a commercial enzyme-linked immunosorbent assay (ELISA) system. We calculated accuracy and confidence intervals (CIs) for AF matrix metalloproteinase-9, glucose, and Gram stain for diagnosing intra-amniotic infection, using culture as the criterion standard. RESULTS: All patients who had matrix metalloproteinase-9 detectable by ELISA also demonstrated matrix metalloproteinase-9 by zymography. Six cases of intra-amniotic infection were confirmed by culture (prevalence 14%). The performance statistics of AF matrix metalloproteinase-9 for diagnosing intra-amniotic infection were: sensitivity 83% (95% CI 53, 99), specificity 95% (95% CI 88, 99), positive predictive value 71% (95% CI 37, 99), and negative predictive value 97% (95% CI 92, 99). Two women had false-positive results; one had gram-negative rods on the AF Gram stain and developed clinical signs and symptoms of chorioamnionitis several hours after amniocentesis and the other had a purulent vaginal discharge and an AF glucose level less than 15 mg/dL. Both delivered within 24 hours of amniocentesis. CONCLUSION: Measuring matrix metalloproteinase-9 in the AF appeared to be reliable for diagnosing intra-amniotic infection. An elevated matrix metalloproteinase-9 concentration in the AF at a preterm gestational age may portend imminent delivery regardless of microbiologic confirmation of intra-amniotic infection.

Adult↗
PubMed1999

Transforming and epidermal growth factors in degenerated intervertebral discs.

We studied the presence of anabolic growth factors in human herniated intervertebral discs (IVD) using a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Messenger RNA (mRNA) was isolated from the nucleus pulposus using oligo (dT)25 superparamagnetic beads and probing with gene-specific primers in RT-PCR. mRNA coding for TGF-alpha (3/10), EGF (0/10), TGF-beta1 (0/10) and TGF-beta3 (2/10) or the EGF receptor (EGF-R; 0/10) and TGF-beta type-II receptor (0/10) was found only occasionally. Beta-actin was always present and positive sample controls confirmed the validity of the RT-PCR assay. These RT-PCR findings were confirmed using immunohistochemical staining of EGF and TFG-beta, whereas TGF-alpha protein was always found associated with discocytes. We conclude that the nucleus pulposus of the herniated IVD is vulnerable to proteolytic degradation and depletion of proteoglycans due to the lack and/or low production of anabolic growth factors/receptors which could increase the local synthesis of the extracellular matrix.

Actins↗
PubMed1998

Analysis of protease activity in human otitis media.

Chronic otitis media is a common problem associated with a nonintact tympanic membrane frequently involving Staphylococcus aureus and Pseudomonas aeruginosa. The virulence of Pseudomonas bacteria is related to the production of two matrix metalloproteinases, elastase and alkaline protease. Serine proteases, such as neutrophil elastase, are produced by the host inflammatory response. These proteases are thought to contribute to tissue destruction and assist bacterial invasion during infection. This preliminary study was done to identify protease activity in otorrhea samples from patients with otitis media and a nonintact tympanic membrane and to examine the ability of selective protease inhibitors to decrease protease activity. Ilomostat (galardin) is a synthetic, specific inhibitor of matrix metalloproteinases including P. aeruginosa elastase and alkaline protease, whereas alpha1-antitrypsin inhibits serine proteases including neutrophil elastase. Samples were collected and cultured from 20 patients with otorrhea resulting from tympanic membrane perforations or pressure-equalization tubes. A protease assay that used azocasein as the substrate was used to quantify protease activity, with and without addition of selective protease inhibitors. Cultures revealed P. aeruginosa alone in 7 samples, P. aeruginosa plus other organisms in 10, and S. aureus alone in 3. Protease activity was detected in 15 (75%) of the samples. A statistically significant (p < 0.05) decrease in protease activity was seen with the addition of alpha1-antitrypsin or Ilomostat plus alpha1-antitrypsin, but not with Ilomostat alone. Analyzing the 10 samples with the highest protease activity, a statistically significant decrease in activity was seen with Ilomostat or alpha1-antitrypsin alone and with both Ilomostat and alpha1-antitrypsin together. Bacteriologic type, source of sample, age and gender of the subject, and duration of infection were not significantly related to protease activity. This is the first study to quantify protease activity and inhibition by selective protease inhibitors in human otorrhea. Protease inhibitors effectively decrease protease activity in most cases and in addition to standard antibiotic therapy might prove beneficial in the treatment of otitis media with a nonintact tympanic membrane. This study supports future clinical investigations into the role of proteases and inhibition of protease activity in the treatment of otitis media.

Anti-Bacterial Agents↗
PubMed1998

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.

Adult↗
PubMed1998

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.

Adrenergic beta-Agonists↗
PubMed1997

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.

Acute Disease↗
PubMed1997

Inhibition of transforming growth factor alpha stimulation of human squamous cell carcinoma of the head and neck with anti-TGF-alpha antibodies and tyrphostin.

BACKGROUND: Transforming growth factor alpha (TGF-alpha) and its receptor (EGF-R) may regulate normal and malignant epithelial cell growth by an autocrine mechanism. We investigated the role of TGF-alpha in regulating head and neck SCC tumor growth. METHODS: TGF-alpha and EGF-R levels were measured in 7 SCC cell lines and 14 SCC biopsies by RIA, Scatchard, and Western analysis. TGF-alpha autocrine stimulation of DNA synthesis in SCC cell lines was assessed by incubation with TGF-alpha neutralizing antibodies and tyrphostin AG 1478, a selective and potent inhibitor of EGF-R kinase. RESULTS: All SCC cell lines synthesized TGF-alpha and expressed elevated EGF-R levels compared to normal keratinocytes. Twelve of the 14 SCC biopsies contained TGF-alpha protein and 8 had specific EGF-R. Exogenous TGF-alpha or EGF significantly increased DNA synthesis in 4 of 5 SCC cell lines. TGF-alpha neutralizing antibodies or tyrphostin AG 1478 reduced DNA synthesis in the two SCC cell lines (FaDu and SCC9) tested. CONCLUSIONS: These results indicate that SCC cell lines and tumors usually synthesize TGF-alpha, have elevated levels of EGF-R, and are mitogenically stimulated by a TGF-alpha autocrine system. Selective inhibition of the TGF-alpha system by EGF-R kinase inhibitors or TGF-alpha neutralizing antibodies may be useful strategies for treating SCC that overexpress TGF-alpha and its receptor.

Autocrine Communication↗
PubMed1997

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.

Acute Disease↗