Towards gene therapy of postoperative adhesions: fiber and transcriptional modifications enhance adenovirus targeting towards human adhesion cells.

Postoperative abdominal/pelvic peritoneal adhesions are a major source of morbidity (bowel obstruction, infertility, ectopic gestation as well as chronic pelvic pain) in women. In this study, we screened various transduction and transcription modifications of adenovirus (Ad) to identify those that support maximal Ad-mediated gene delivery to human adhesion fibroblasts, which in turn would enhance the efficacy of this novel treatment/preventative strategy for postoperative adhesions. We transduced primary cultures of human peritoneal adhesion fibroblasts with fiber-modified Ad vectors Ad5-RGD-luc, Ad5-Sigma-luc, Ad5/3-luc and Ad5-CAV2-luc as well as transcriptional targeting viruses Ad5-survivin-luc, Ad5-heparanase-luc, Ad5-mesothelin (MSLN)-CRAd-luc and Ad5-secretory leukoprotease inhibitor (SLPI)-luc, and compared their activity to wild-type Ad5-luc. At 48 h, luciferase activity was measured and normalized to the total protein content in the cells. Among the fiber-modified Ad vectors, Ad5-Sigma-luc and among the transcriptional targeting modified Ad vectors, Ad5-MSLN-CRAd-luc showed significantly increased expression levels of luciferase activity at 5, 10 and 50 plaque forming units/cell in adhesion fibroblast cells compared with wild-type Ad5-luc (p < 0.05). Specific modifications of Ad improve their gene delivery efficiency towards human peritoneal adhesion fibroblasts. Developing a safe localized method to prevent/treat postoperative adhesion formation would have a major impact on women health.

Uterine fibroids are characterized by an impaired antioxidant cellular system: potential role of hypoxia in the pathophysiology of uterine fibroids.

PURPOSE: Fibroids are the most common smooth muscle overgrowth in women. This study determined the expression and the effect of hypoxia on two potent antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) on human fibroid cells. METHODS: Immortalized human leiomyoma (fibroid) and myometrial cells were subjected to hypoxia (2 % O2, 24 h). Total RNA and cell homogenate were obtained from control and treated cells; CAT and SOD mRNA and activity levels were determined by real-time RT-PCR and ELISA, respectively. RESULTS: Fibroid cells have significantly lower antioxidant enzymes, SOD and CAT mRNA and activity levels than normal myometrial cells (p < 0.05). Hypoxia treatment significantly increased SOD activity in myometrial cells while significantly decreasing CAT activity in fibroid cells (p < 0.05). There was no significant difference in CAT mRNA levels or activity in response to hypoxia in myometrial cells. Also, there was no significant difference in SOD mRNA levels in response to hypoxia in myometrial cells. CONCLUSION: This is the first report to show that uterine fibroids are characterized by an impaired antioxidant cellular enzymatic system. More importantly, our results indicate a role for hypoxia in the modulation of the balance of those enzymes in fibroid and myometrial cells. Collectively, these results shed light on the pathophysiology of fibroids thereby providing potential targets for novel fibroid treatment.

A simple and rapid purification of kallikrein from rat submandibular gland.

Rat submandibular kallikrein was isolated in an 87% yield by a very quick and simple procedure involving hydrophobic interaction chromatography. Furthermore, that purification method was superior to both aprotinin-affinity chromatography and immunoaffinity chromatography for the purification of rat submandibular kallikrein. The kallikrein purified by hydrophobic interaction chromatography consisted of a number of isoenzymes. The major component of Mr 38,000 seen on SDS-gel electrophoresis was found to be the glycosylated kallikrein, whereas the minor component of Mr 26,000 represented the non-glycosylated enzyme.

T-cell receptor gene rearrangement in canine mycosis fungoides: further support for a canine model of cutaneous T-cell lymphoma.

Canine cutaneous T-cell lymphoma (CTCL) is a morphologic and immunophenotypic simulant of human mycosis fungoides (MF) characterized by an infiltrate of atypical, hyperconvoluted, epidermotropic T cells. To further support our hypothesis that canine MF is a useful model for the study of human CTCL, we have used Southern blotting to search for clonal T-cell proliferations in canine MF. Cellular DNA was extracted from normal dog buffy coat cells (n = 8), lesional canine MF skin (n = 8), canine MF buffy coat cells (n = 7), normal dog skin (n = 3), and normal human buffy coat cells (n = 5), digested with a panel of restriction enzymes and Southern blotted onto nylon membranes. All cases of canine MF were also immunophenotyped with anti-canine monoclonal antibodies to CD4, CD8, CD18, CD45RA, canine class II, T-cell activation antigens, and pan-B-cell antigens. Normal dogs gave reproducible digestion patterns in blood and skin, which differed from the human germline patterns when probed with a human T-cell receptor (TCR), beta chain constant region (C beta) cDNA. Common germline bands between the species included the 3.5-kb Eco RI, 3.4-kb Bam HI, 5.4-kb Sac I. These results confirmed that the TCR-beta gene is evolutionarily conserved between dog and man. Immunostaining revealed that 3/7 cases were CD4+ canine CTCL and 4/7 were CD8+ canine CTCL. Rearranged bands, deletion of germline bands, as well as minor alterations in electrophoretic mobility were observed in lesional DNA from seven of eight cases of canine MF, with at least two restriction digests in each case. Dog rearrangements were best detected with Bgl II, Eco RI, Eco RV, and Sac I, whereas deletions were detected with Bgl II, Sac I, Eco RV, and Bam HI. These studies demonstrate the presence of clonal TCR rearrangement in canine MF, further supporting the similarity of this tumor to human MF and its role as an animal model of CTCL.

Submandibular enzymatic vasoconstrictor messenger RNA in rat kidney.

Recently, we reported the isolation and identification of a potent vasoconstrictor enzyme from the rat submandibular gland, a member of the rat kallikrein gene family, which we named submandibular enzymatic vasoconstrictor (SEV). We studied whether messenger RNA (mRNA) for SEV is present in the kidney and isolated glomeruli, using the polymerase chain reaction assay with primers specific to the entire rat kallikrein family that would amplify a 430-bp fragment from their mRNA. As a probe we used a phosphorus-32-labeled oligonucleotide specific for SEV mRNA. A fragment of the predicted size was obtained on Southern blot for amplified renal RNA; however, no signal was obtained with glomerular RNA. To further confirm the presence of SEV mRNA in the kidney, polymerase chain reaction was repeated using primers specific to SEV mRNA that would amplify a 372-bp fragment from SEV mRNA alone. Again, a fragment of the predicted size was obtained on Southern blot after amplification of renal RNA but not RNA from the glomeruli. Southern blot of polymerase chain reaction-amplified RNA with primers that amplified the entire kallikrein gene family, using kallikrein complementary DNA that recognizes all members of the kallikrein gene family as a probe, revealed a 430-bp fragment for both renal and glomerular RNA, indicating that glomeruli contain mRNA for a member or members of the kallikrein family other than SEV. When the Southern blots were hybridized with a 32P-labeled oligonucleotide probe specific for glandular kallikrein, a fragment of the predicted size was obtained from amplified renal RNA but not glomerular RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuronal beta-amyloid precursor protein gene expression: regulation by aurintricarboxylic acid.

beta-Amyloid precursor protein (beta-APP) and its derivative, amyloid beta-protein (beta-A4), may cause death of differentiated neurons and aurintricarboxylic acid (ATA), a metabolic inhibitor, improves neuronal survival. Therefore, we studied the effect of ATA on neuronal beta-APP gene expression. ATA decreased beta-APP mRNA levels by increasing its degradation, without changing the rate of transcription. ATA decreased both steady state and interleukin-1 (IL1)-induced increase in beta-APP mRNA levels. These effects of ATA were associated with rounding of cells suggestive of decreased cell adhesion or neurite retraction that was completely reversible when ATA was removed. However, beta-APP mRNA levels continued to remain suppressed in neurons that were actively regrowing neurites following discontinuation of ATA. In studies carried out upto 24 h, ATA did not damage cells as determined by Trypan blue exclusion, lactate dehydrogenase (LDH)-release and transmission electron microscopy. The findings suggest that constitutive or steady state levels of beta-APP mRNA may not be essential for the survival and growth of neurons and that ATA suppresses beta-APP expression without causing cell damage. These observations may be a basis for studying whether ATA or a related compound could beneficially regulate beta-APP levels in vivo.

Molecular characterization of fibroblasts isolated from human peritoneum and adhesions.

OBJECTIVE: To determine the response of adhesion and peritoneal fibroblasts to hypoxia. DESIGN: Prospective experimental study. SETTING: University medical center. PATIENT(S): Primary cultures of fibroblasts established from the peritoneal and adhesion tissues of the same patients (n = 2) to minimize genetic variations. INTERVENTION(S): Hypoxia treatment of the primary cultured fibroblast. MAIN OUTCOME MEASURE(S): Analyze the expression of extracellular matrix (ECM) components, metalloproteinases and their tissue inhibitors, growth factors, and cytokines in adhesion and peritoneal fibroblasts under normal and hypoxic conditions by reverse transcriptase/polymerase chain reaction analysis. RESULT(S): Compared to peritoneal fibroblasts, adhesion fibroblasts had a significant increase in the basal mRNA levels for collagen I, fibronectin, MMP-1, TIMP-1, TGF-beta 1, TGF-beta 2, and IL-10. Hypoxia resulted in a further increase in collagen 1, fibronectin, TIMP-1, TGF-beta 1, TGF-beta 2, IL-10, and IFN-gamma mRNA levels in both peritoneal and adhesion fibroblasts. The increase was more profound in adhesion fibroblasts. CONCLUSION(S): Hypoxia induces molecular changes in both peritoneal and adhesion fibroblasts, creating a milieu that favors adhesion development. The effect of hypoxia was more profound on adhesion fibroblasts.

Analysis of p53 gene mutations in keloids using polymerase chain reaction-based single-strand conformational polymorphism and DNA sequencing.

BACKGROUND: Keloids are the result of a dysregulated wound healing process. They are characterized by the formation of excess scar tissue that proliferates beyond the boundaries of the original wound. Somatic mutations of p53 have been implicated as causal events in up to 50% of all human malignancies. In addition, p53 has been shown to play an important role in controlling cell proliferation and apoptosis. We hypothesize that mutations in p53 can lead to a hyperproliferative state that can result in keloid formation. OBJECTIVE: To detect p53 DNA mutations in tissues and cultured fibroblasts from skin lesions of 7 patients with keloids. DESIGN: The polymerase chain reaction followed by single-strand conformational polymorphism analysis and direct DNA sequencing were used to detect p53 gene mutations. SETTING: The Department of Dermatology, Henry Ford Hospital, Detroit, Mich. PATIENTS: Seven patients with keloids seen for routine surgical excision of their lesions. Normal DNA specimens were obtained from buccal smears and healthy skin samples from these patients. RESULTS: Mutations in the p53 were identified in all patients by polymerase chain reaction followed by single-strand conformational polymorphism analysis and subsequently confirmed by DNA sequencing. A mutation in exon 5 resulting in amino acid substitution was found in 1 of the patients in keloid tissue and cultured keloid fibroblasts (codon 156, CGC-->CCC, arginine-->proline). Frameshift mutations in exons 5 and 6 caused by the insertion or deletion of a nucleotide at different positions were found in 6 patients with keloids in both keloid tissues and cultured fibroblasts. Mutations in exon 4 resulting in amino acid substitution were found in all patients in both keloid tissues and cultured fibroblasts (all in codon 72, CGC-->CCC, arginine-->proline). No p53 mutations were detected in buccal smears or cultured fibroblasts from healthy skin samples of any of the patients. CONCLUSIONS: Focal mutations in p53 may increase cell proliferation and decrease cell death in the dysregulated growth patterns that have been clinically documented. An understanding of the pattern of all growth dysregulation related to keloids may lead to new therapeutic strategies.

Prospective, single-blind, randomized, controlled study to assess the efficacy of the 585-nm flashlamp-pumped pulsed-dye laser and silicone gel sheeting in hypertrophic scar treatment.

OBJECTIVE: To determine the efficacy of the 585-nm flashlamp-pumped pulsed-dye laser and silicone gel sheeting in the treatment of hypertrophic scars in lighter- and darker-skinned patients. DESIGN: Prospective, single-blind, randomized, internally controlled, comparison investigation. SETTING: Large academic dermatology department. PATIENTS: Twenty patients with hypertrophic scars (19 completed the laser treatments and 18 completed the silicone gel sheeting treatments). MAIN OUTCOME MEASURES: Clinical measurements included hypertrophic scar blood flow, elasticity, and volume. Patients' subjective complaints of pruritus, pain, and burning were also monitored. Histological assessment of fibrosis, number of telangiectasias, and number of mast cells was performed. Statistically significant improvements in clinical measurements and patients' subjective complaints determined treatment success. RESULTS: Mean scar duration was 32 months (range, 4 months to 20 years). There was an overall reduction in blood flow, volume, and pruritus over time (P = .001, .02, and .005, respectively). However, no differences were detected among treatment and control groups. There was no reduction in pain or burning (0-40 weeks), elasticity (8-40 weeks), or fibrosis (0-40 weeks, n = 5 biopsies) in the treated or control sections of the scars. Unlike in a previous study, the number of mast cells in the scars was similar to the number of mast cells in healthy skin. CONCLUSION: Clinical results demonstrate that the improvements in scar sections treated with silicone gel sheeting and pulsed-dye laser were no different than in control sections.

Effect of hypoxia on stimulatory effect of TGF-beta 1 on MMP-2 and MMP-9 activities in mouse fibroblasts.

OBJECTIVE: Because chronic low-grade hypoxia has been implicated in the pathogenesis of fibrosis and postoperative adhesion formation, we hypothesized that hypoxia may modulate the effect of transforming growth factor-beta 1 (TGF-beta 1) on matrix metalloproteinase (MMP)-2 and MMP-9 at both transcriptional and translational levels. METHODS: Mouse fibroblasts were placed in a hypoxic environment with or without 1 ng/mL TGF-beta 1 for varying periods of time. Zymography was performed on cell supernatants collected after each treatment. Gelatinolytic bands corresponding to MMP-2 and MMP-9 were quantiated by densitometry. Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was also performed for MMP-2 and MMP-9 on total RNA extracted from cells after each treatment. Analysis of PCR-amplified products was performed by 2% agarose gel followed by ethidium bromide staining of DNA bands. Scanning densometry was used to determine the ratio of intensity of each band relative to beta-actin. RESULTS: Hypoxia resulted in a 64% decrease in MMP-9 activity and 80% decrease in MMP-9 mRNA level but did not affect MMP-2 mRNA level or activity. TGF-beta 1 treatment resulted in 180% and 50% increases in MMP-2 and MMP-9 activities, respectively. Increases of 37.5% and 40% in MMP-2 and MMP-9 mRNA levels, respectively, were seen. However, under hypoxic conditions, TGF-beta1 resulted in a 160% increase and 45% decrease in MMP-2 and MMP-9 activities and a 37.5% increase and 71% decrease in MMP-2 and MMP-9 mRNA levels, respectively. CONCLUSION: Hypoxia suppresses the stimulatory effect of TGF-beta1 on the activity of MMP-9 but not MMP-2. This may suggest an important role for MMP-9 under hypoxic conditions in the pathogenesis of tissue fibrosis and postoperative adhesion formation.

Effects of prostaglandin E(2) on proliferation and apoptosis of epithelial ovarian cancer cells.

OBJECTIVE: There is strong evidence indicating that prostaglandins (PG) and their synthesizing enzyme cyclooxygenase-2 (COX-2) play an important role in tumorigenesis. The purposes of the present study were to determine the pattern of expression of COX-2 and the effect of PG treatment on proliferation and apoptosis in epithelial ovarian cancer cells. METHODS: Two epithelial ovarian cancer cell lines, MDAH-2774 and SKOV3, were grown in flasks to confluence. Cells were then treated with exogenous dimethyl prostaglandin E(2) (dmPGE(2)) at increasing concentrations of 0-10 microg/mL. Total RNA was extracted from cells at different treatment doses and subjected to reverse transcriptase-polymerase chain reaction for the semiquantitative analysis of COX-2, Bcl-2, and bax expression. Flow cytometry was performed to assess effect of treatment on the cell cycle. The TUNEL assay was used to assess apoptosis. RESULTS: We found that COX-2 was constitutively expressed in the MDAH-2774 and SKOV3 epithelial ovarian cancer cells as determined by detection of a 304-bp amplified fragment using specific primers for the COX-2 gene. Treatment of both cell lines with dmPGE(2) resulted in dose-dependently higher expression of COX-2, Bcl-2, and bax mRNA compared with untreated cells. These changes were associated with an increase in the proliferative fraction and with a simultaneous reduction in apoptosis. CONCLUSIONS: Prostaglandin E(2) stimulated proliferation and reduced apoptosis in epithelial ovarian cancer cells. These effects were associated with overexpression of COX-2 and an increase in the ratio of Bcl-2:bax mRNA.

Regulation of inducible nitric oxide synthase in post-operative adhesions.

BACKGROUND: The deficiency of the inducible nitric oxide synthase (iNOS) substrate, L-arginine (L-Arg), the co-factor tetrahydrobiopterin (H4B) or molecular oxygen may lead to lower NO levels, which enhances the development of adhesion phenotype. METHODS: We utilized high-performance liquid chromatography (HPLC) and immunoprecipitation with nitrotyrosine antibody to determine the levels of H4B, citrulline and protein nitration in fibroblasts established from normal peritoneal and adhesion tissues. RESULTS: The level of H4B was dramatically attenuated in adhesion fibroblasts. The immunoprecipitation with nitrotyrosine antibody revealed higher protein nitration in adhesion compared with normal fibroblasts. There were higher accumulations of citrulline in adhesion fibroblasts as compared with normal fibroblasts. In addition, peritoneal fibroblasts treated with 2% oxygen for 24 h and implanted back into the peritoneal cavity of the rats exhibited marked increase in severity of adhesion as well as extensive distribution involving many sites and organs. CONCLUSIONS: Control of the catalytic activity of iNOS in adhesion fibroblasts may be because of subsaturating amounts of L-Arg and H4B which allow iNOS to generate a combination of reactive oxygen species in addition to NO, thereby influencing NO bioavailability and function.

Quantitation of interferon gamma mRNA levels in psoralen/UVA-treated HUT-78 cells by competitive PCR.

We have developed a method to accurately quantitate IFN gamma mRNA in HUT-78 cells before and after PUVA treatment by the competitive RT/PCR technique, which could be utilized to accurately quantitate any mRNA species of interest. Total RNA was isolated from HUT-78 cells before and after PUVA treatment. A synthetic IFN gamma mRNA was made to contain a 54 bp deletion in the middle of IFN gamma cDNA gene and used as an internal standard. 0.5 microgram of target RNA was co-reverse transcribed and co-amplified with increasing concentrations of synthetic IFN gamma RNA using the same primers. The products of the synthetic RNA were separated from that of the target RNA by gel electrophoresis. This allowed determination of the amount of target IFN gamma mRNA to be quantitated by extrapolating against a standard curve. PUVA treatment of HUT-78 cells resulted in an increase in IFN gamma mRNA level from 32 to 80 pg/microgram of total RNA, suggesting that PUVA induces transcription of T-helper 1 cytokines as part of its mechanism of action.

Borrelia burgdorferi DNA is undetectable by polymerase chain reaction in skin lesions of morphea, scleroderma, or lichen sclerosus et atrophicus of patients from North America.

BACKGROUND: Borrelia burgdorferi has been linked to the pathogenesis of morphea and lichen sclerosus et atrophicus (LSA). However, considerable controversy still exists as to the actual role, if any, that this spirochete plays in the development of these diseases. Antibody titer determinations have been inconclusive and polymerase chain reaction (PCR) studies have yielded conflicting results. OBJECTIVE: We sought to show whether PCR analysis detected B. burgdorferi in archival tissue specimens from the involved skin of 20 North American patients with morphea, 10 patients with LSA, and four patients with scleroderma. METHODS: We used two different sets of PCR primers for the B. burgdorferi flagellin gene, one specific for European strains of B. burgdorferi, and another common to both European and American strains. A subset of these samples were further amplified with nested PCR primers. RESULTS: None of the samples showed PCR products with either primer sets, whereas purified B. burgdorferi DNA and lesional erythema chronicum migrans tissues, which were used as positive controls, yielded easily detectable products with all primer sets. CONCLUSION: These data suggest that B. burgdorferi infection plays no role in the development of morphea, LSA, or scleroderma in North American patients; these findings further support the recent observations that B. burgdorferi strain variability is associated with differential spectra of disease in North America compared with that found in various parts of Europe.

T-cell cytokine network in cutaneous lupus erythematosus.

BACKGROUND: A variety of immunologic abnormalities have been described in systemic and experimental lupus erythematosus (LE). Several T-cell defects, especially in helper T (Th) cell cytokines, have been reported. OBJECTIVE: Our purpose was to identify the Th cytokine profile in cutaneous LE. METHOD: Total RNA was extracted from punch biopsy specimens from 19 patients with cutaneous LE (nine, discoid LE; two, subacute cutaneous LE; and eight, systemic LE) and from four healthy control subjects. RNA was reverse transcribed into complementary DNA and amplified with polymerase chain reaction (PCR) primers specific for interleukin-2 (IL-2), IL-4, IL-5, IL-10, interferon gamma (IFN-gamma), and beta actin. PCR products were detected by agarose gel electrophoresis and Southern blot with 32P-labeled, nested probes. RESULTS: Sixteen of 19 cutaneous LE specimens lacked IL-2, all were negative for IL-4, and 10 of 19 had detectable IL-10, whereas IFN-gamma and IL-5 messenger RNAs were present in the majority of LE specimens. IFN-gamma and IL-10 mRNAs were found in all normal skin controls, whereas IL-2, IL-4, and IL-5 mRNAs were undetectable. Functional IFN-gamma protein was evidenced by intercellular adhesion molecule-1 and HLA-DR staining of keratinocytes in nine of nine LE specimens but not in normal skin. The pattern of cytokine mRNAs, intercellular adhesion molecule-1, and/or HLA-DR expression in cutaneous LE specimens did not vary with different subtypes of LE, antinuclear antibody titer, or the magnitude of inflammation. CONCLUSION: The presence of IL-5 mRNA in cutaneous LE specimens suggests that Th type 2 cells combine with local IFN-gamma production to augment disease and may be related to the pathophysiology of cutaneous LE.

Characterization of kallikrein isolated from rat submandibular glands by a simple and rapid purification procedure.

Numerous biochemical properties (e.g. Mr, carbohydrate content, pI) were determined for kallikrein isolated from rat submandibular glands by a simple, rapid purification procedure. The kinetic behaviour of the enzyme towards various inhibitors and synthetic substrates was investigated. The effects of different salts and detergents on the esterolytic activity of the rat tissue kallikrein were recorded.

Cytokine mRNA changes during the treatment of hypertrophic scars with silicone and nonsilicone gel dressings.

BACKGROUND: Treatment of hypertrophic scars can be difficult for both patients and physicians. Silicone-containing gel dressings have been reported to be an effective alternative treatment for hypertrophic scars, yet the mechanism of action of these dressings is unknown. OBJECTIVE: To determine whether silicone is an essential factor in the treatment of hypertrophic scars and investigate the effects of occlusive dressing therapy on the expression of key wound healing mediators. METHODS: A pilot paired comparison, nonrandomized study was conducted comparing a silicone gel sheeting (Silastic [SGS]) with a hydrogel dressing (ClearSite). The effects of the dressings were compared side by side in the treatment of 15 hypertrophic scars at both the clinical and molecular levels through the use of reverse transcriptase/polymerase chain reaction to evaluate effects on the expression of interleukin 8 (IL-8), basic fibroblast growth factor (bFGF), granulocyte-macrophage colony-stimulating factor (GMCSF), epidermal growth factor (EGF), transforming growth factor beta (TGF-beta), and fibronectin. RESULTS: Comparable clinical improvement of the hypertrophic scars was obtained with both dressings. Treatment of hypertrophic scars resulted in increased mean levels of IL-8, bFGF, and GMCSF mRNA; while mean TGF beta and fibronectin mRNAs decreased after treatment with both dressings. Comparison between the two dressings revealed significant changes in IL-8 and fibronectin mRNA levels after treatment with ClearSite, while only fibronectin changes were significant after treatment with SGS with respect to normal skin. Only ClearSite induced significant changes in IL-8 and bFGF levels when untreated scars were compared with posttreatment lesions, suggesting that the hydrogel augments collagenolysis via promotion of inflammation. CONCLUSIONS: This study demonstrates that silicone is not a necessary component of occlusive dressings in the treatment of hypertrophic scars. The pathogenesis of hypertrophic scars is further elucidated by demonstrating that there is molecular evidence for extensive connective tissue remodeling occurring during occlusive dressing therapy.

Kallikrein messenger RNA in rat arteries and veins.

Glandular kallikrein (EC 3.4.21.8) belongs to a subgroup of serine proteases coded by a multigene family. A kininogenase resembling glandular kallikrein has been identified in vascular tissue; however, it is not clear whether it is synthesized by vascular tissue or taken up from plasma. To determine the potential for kallikrein synthesis in vascular tissues, we tested whether messenger RNA (mRNA) for glandular kallikrein is present in rat arteries and veins. Poly(A+) RNA was isolated from pools of arteries or veins (n = 3, 30 rats each). Poly(A+) RNA from the kidney and liver was used as a positive and negative control, respectively. As a probe, we used rat pancreatic kallikrein 32P-labeled complementary DNA, which recognizes mRNA of the entire rat kallikrein family. Slot-blot analysis indicated that kallikrein mRNA was present in mRNA from the arteries, veins, and kidney but not from the liver. Poly(A+) RNA from arteries and veins contained approximately 1% as much kallikrein mRNA as that from the kidney. To confirm the slot-blot results and determine whether the mRNA for true glandular kallikrein was present in vascular tissue, we employed a polymerase chain reaction assay, first using primers specific for the entire kallikrein family (which amplify a 430-bp fragment) and then using primers specific for true glandular kallikrein mRNA (which amplify a 370-bp fragment). After the polymerase chain reaction assay, both arteries and veins showed fragments of these sizes when tested with rat kallikrein complementary DNA probe, thus confirming the presence of glandular kallikrein mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of type I and III collagen expression in human peritoneal mesothelial cells in response to hypoxia and transforming growth factor-beta1.

Overexpression and accumulation of extracellular matrix is central to peritoneal adhesion formation following surgically induced tissue trauma. Transforming growth factor-beta1 and hypoxia have been implicated in tissue fibrosis and postoperative adhesion formation. To extend this observation we examined whether transforming growth factor-beta1 and/or hypoxia regulate the expression of type I and III collagen in human peritoneal mesothelial cells. Cultured human mesothelial cells were maintained under hypoxia (2% oxygen), or treated with transforming growth factor-beta1 (1 ng/ml) or a combination of hypoxia and transforming growth factor-beta1. Total cellular RNA from treated and untreated cells were collected and subjected to multiplex reverse transcription/polymerase chain reaction to quantitate collagen I and III mRNA levels in response to these treatments. The results indicate that 6 hours of hypoxia increased collagen III mRNA by 7.2 fold which was further increased to 9.4 fold following transforming growth factor-beta1 treatment; in contrast collagen I mRNA decreased by 0.42 fold which was further decreased by 0.3 fold following transforming growth factor-beta1 treatment. Transforming growth factor-beta1 treatment under normal conditions resulted in an 8.4-fold increase and a 0.3-fold decrease in collagen III and I mRNA levels, respectively. Hypoxia treatment also resulted in a 1.9-fold increase in transforming growth factor-beta1 mRNA level compared with control. The ratio of type III/I collagen was increased in response to transforming growth factor-beta1 treatment under hypoxic condition. In conclusion, the data suggest that hypoxia may modulate extracellular matrix production by human mesothelial cells via a transforming growth factor-beta1 dependent mechanism.