Biomedical subjects

A Ghahary

Publications and source records attributed to A Ghahary.

At least 37 records · Page 2Linked to original sources

PubMed1999

Lack of class I major histocompatibility antigens on trophoblast of periimplantation blastocysts and term placenta in the pig.

In this study, the pattern of expression of class I major histocompatibility (MHC) antigens and mRNA on periimplantation blastocysts and term placental tissue was determined for the pig. Class I MHC antigens could not be detected immunohistochemically either on extra-embryonic membranes or on the embryonic portion of Day 14, 16, 22, and 25 blastocysts. Nor could class I MHC antigens be detected on the outer trophoblast epithelium and inner endodermal surface of the chorioallantoic membrane or on the outer and inner surfaces of the amnion at term. However, MHC class I antigens were detected on the vascular mesoderm found in both the chorion and amnion at term, and in Day 25 extra-embryonic membranes. Uterine endometrial cells and tissues and maternal peripheral blood leukocytes stained strongly for class I MHC antigens. There was a large difference in the intensity of class I MHC mRNA signal, detected by Northern blot analysis, in embryo/fetus-derived tissues compared to that in maternal tissues. The embryos appeared to express even less class I MHC mRNA than did the extra-embryonic membranes. In addition, in situ hybridization of Day 16 blastocysts indicated class I MHC mRNA to be ubiquitously expressed at low levels in embryos and extra-embryonic tissues compared to uterine endometrial tissue controls. Taken together, these results indicate that class I MHC antigens are either not expressed on the surface of the extra-embryonic/fetal membranes during gestation in the pig or are expressed at very low levels, and that specific mRNA is expressed at correspondingly low levels.

Allantois↗

PubMed1999

Antisense preproendothelin-oligoDNA therapy for vasospasm in a canine model of subarachnoid hemorrhage.

OBJECT: The purpose of this study is twofold: 1) to test antisense genetic techniques used in the prevention of cerebral vasospasm in a canine model of subarachnoid hemorrhage (SAH), targeting the endothelin-1 (ET-1) gene; and 2) to determine if fibrinolysis of subarachnoid clot with recombinant tissue plasminogen activator (rtPA) could enhance the effect of antisense treatment. METHODS: A total of 39 dogs were studied in this experiment. Placebo (six animals), rtPA (six animals), antisense preproET-1 oligodeoxynucleotide (ASOD; five animals), or rtPA plus ASOD (combined treatment; six animals) was injected into the cisterna magna 30 minutes after a second SAH was induced on the 2nd day of the experiment. The animals were observed until Day 7, when they underwent follow-up angiography and then were killed; their basilar arteries were removed for analysis. Control animals included in this study (two animals in each group) received placebo, rtPA, ASOD, or rtPA plus ASOD without induction of SAH, or rtPA with mismatched (nonsense) preproET-1 oligodeoxynucleotide following SAH. Six additional dogs were analyzed earlier following SAH. Dogs that received placebo developed severe vasospasm (51+/-8% of baseline caliber). Administration of ASOD alone resulted in a mild reduction in vasospasm (64+/-13% of baseline caliber) and rtPA alone resulted in a moderate reduction in vasospasm (81+/-5% of baseline caliber); however, the combined therapy of rtPA plus ASOD almost completely prevented vasospasm (95+/-6%, of baseline caliber), which was significantly different from all other groups (p < 0.05). Morphological analysis of the basilar arteries yielded results similar to angiography with respect to vasospasm severity. The ASOD treatment combined with rtPA resulted in reduced ET-1 expression, as demonstrated by immunohistochemical staining of the arteries, and reduced preproET-1 levels on Day 4, as measured by reverse transcription-polymerase chain reaction. Nonsense DNA sequences had no effect on the vessels. CONCLUSIONS: Antisense preproET-1 oligodeoxynucleotide treatment, when combined with clot lysis caused by rtPA, reduced vasospasm in the canine model of SAH, and this effect appeared to be related to reduced ET-1 synthesis. The results of this experiment support a causative role for ET-1 early in the course of vasospasm development in dogs. The apparent additive therapeutic effects of antisense and fibrinolytic treatments could be due to clot lysis, which allows better delivery of oligodeoxynucleotides to arteries within the subarachnoid space.

Animals↗

PubMed1998

Induction of transforming growth factor beta 1 by insulin-like growth factor-1 in dermal fibroblasts.

Transforming growth factor beta1 (TGF-beta1) belongs to a family of multifunctional modulatory proteins involved in cell growth, differentiation, development, and wound healing. Although the biological activities of TGF-beta1 have been extensively studied, its regulation remains obscure. Here we report the effects of insulin-like growth factor-1 (IGF-1) on the expression of TGF-beta1 by dermal fibroblasts and suggest a possible mechanism. An enzyme-linked immunosorbent assay (ELISA) specific for TGF-beta revealed a greater than twofold increase (12.3 +/- 1.6 vs. 4.8 +/- 0.8 pg/10(4) cells, n = 7, P < 0.05) in the protein in conditioned medium obtained from IGF-1-treated cells compared to that from untreated controls. Similar results were obtained by the mink lung epithelial cell growth inhibition assay. The results of Northern analysis revealed a dose-dependent increase in TGF-beta1 mRNA in response to IGF-1 treatment. Using the optimum concentration of IGF-1 (100 ng/ml), a greater than twofold increase (25.43 +/- 5.7 vs. 12.13 +/- 4.5, P < 0.05) in TGF-beta1 mRNA was observed. This effect persisted for at least 48 h after IGF-1 was removed from the culture medium. Nuclear run-on assay showed that this stimulation was due, at least in part, to an increase in the rate of transcription of the TGF-beta1 gene. Treatment of human dermal fibroblasts with IGF-1 caused a substantial increase in c-fos and c-jun mRNA expression within 30 and 60 min, respectively. In contrast to c-jun mRNA which was constitutively expressed by dermal fibroblasts, the expression of c-fos mRNA was transient and only detectable between 15 and 60 min. Greater than 58% of the increase in TGF-beta1 caused by IGF-1 could be blocked by the addition of anti-TGF-beta1 neutralizing antibody to the culture medium, suggesting that autoinduction of TGF-beta1 may be involved. An increase in IGF-1-induced TGF-beta1 should be important in many different physiological processes such as cellular proliferation, differentiation, and wound healing. These findings also suggest that induction of TGF-beta1 mRNA and protein by IGF-1 may be a mechanism by which this cytokine is regulated in physiological and/or pathological conditions.

Animals↗

PubMed1998

Expression and localization of insulin-like growth factor-1 in normal and post-burn hypertrophic scar tissue in human.

The migration of epithelial cells from dermal appendages toward the wound surface is essential for re-epithelialization of partial thickness burn injuries. This study provides evidence that these cells in vivo synthesize a mitogenic and fibrogenic factor, insulin-like growth factor-1 (IGF-1), which may promote the development of the post-burn fibroproliferative disorder, hypertrophic scarring (HSc). An evaluation of 7 post-burn hypertrophic scars, 7 normal skin samples obtained from the same patients and 4 mature scars revealed that IGF-1 expressing cells from the disrupted sweat glands tend to reform small sweat glands of 4-10 cells/gland in post-burn HSc. The number of these cells increases with time and the glands become larger in mature scar. Other epithelial cells such as those found in sebaceous glands and basal and suprabasal keratinocytes, also express IGF-1 protein and mRNA as detected by Northern and RT-PCR analysis of RNA obtained from whole skin and separated epidermis and dermis. However, cultured keratinocytes did not express mRNA for IGF-1. Histological comparisons between normal and HSc sections show no mature sebaceous glands in dermal fibrotic tissues but the number of IGF-1 producing cells including infiltrated immune cells was markedly higher in the dermis of hypertrophic scar tissues relative to that of the normal control. In these tissues, but not in normal dermis, IGF-1 protein was found associated with the extracellular matrix. By in situ hybridization, IGF-1 mRNA was localized to both epithelial and infiltrated immune cells. Collectively, these findings suggest that in normal skin, fibroblasts have little or no access to diffusible IGF-1 expressed by epithelial cells of the epidermis, sweat and sebaceous glands; while following dermal injury when these structures are disrupted, IGF-1 may contribute to the development of fibrosis through its fibrogenic and mitogenic functions. Reformation of sweat glands during the later stages of healing may, therefore, limit this accessibility, and lead to scar maturation.

Adolescent↗

PubMed1998

Genetically modified dermal keratinocytes express high levels of transforming growth factor-beta1.

In an attempt to genetically modify cultured keratinocytes with transforming growth factor-beta1 (TGF-beta1), which has been proven to be one of the most important cytokines involved in wound healing, two constructs were made. One, designated pG3Z:K14-TGF-beta1, is a plasmid in which the expression of TGF-beta1 is driven by the keratin 14 promoter. The other, designated pLin-TGF-beta1, is a retroviral vector in which the retroviral 5' long-terminal repeat promoter drives expression. In both constructs, the deletion of a small fragment of the noncoding region of the TGF-beta1 gene was made to differentiate the transcript from that for endogenously expressed TGF-beta1. Different types of cells were transfected with the pG3Z:K14-TGF-beta1 construct using the calcium phosphate method. The pLin-TGF-beta1 construct was propagated in a retroviral packaging cell line and conditioned medium that contained high titers of the virus was used to transduce keratinocytes or other types of cells grown in standard culture. Northern analysis, used to evaluate the expression of TGF-beta1 mRNA in the pG3Z:K14-TGF-beta1 transfected keratinocyte C1-177 cell line, showed a smaller TGF-beta1 transcript compared with that endogenously expressed by dermal fibroblasts. The level of TGF-beta1 protein evaluated by enzyme-linked immunosorbent assay was significantly higher in medium conditioned by either the K14-TGF-beta1 transfected or the pLin-TGF-beta1 transduced keratinocytes, compared with that obtained from control cells; however, the level of TGF-beta1 protein was unchanged in cultures of pG3Z:K14-TGF-beta1 transfected nonkeratinocyte cells such as fetal and adult fibroblasts. Using the mink lung epithelial cell growth inhibition assay, we found an increase in TGF-beta1 activity in conditioned medium from the pG3Z:K14-TGF-beta1 transfected cells. To evaluate possible paracrine effects of the keratinocyte derived TGF-beta1, a coculture system was established with pLin-TGF-beta1 transduced keratinocytes grown in the upper chamber and dermal fibroblasts in the lower chamber. The results showed that TGF-beta1 released from keratinocytes diffused to the lower chamber where it stimulated collagen production by dermal fibroblasts. In summary, we demonstrate here that primary cultured keratinocytes can be genetically modified to express high levels of TGF-beta1 and suggest that this offers a potential approach for the therapy of dermal lesions such as nonhealing wounds.

Cells, Cultured↗

PubMed1998

Fibroblasts from post-burn hypertrophic scar tissue synthesize less decorin than normal dermal fibroblasts.

1. Fibroblast cultures were established from biopsies of hypertrophic scar and normal dermis taken from nine patients recovering from second- and third-degree burns. The capacity of these fibroblasts to synthesize the small proteoglycan decorin was assessed by quantitative Western blot analysis of conditioned medium collected from confluent cultures. Levels of mRNA for decorin were assessed by quantitative Northern analysis. Since transforming growth factor-beta 1 is implicated in various fibrotic conditions, including post-burn hypertrophic scar, its effect on decorin synthesis by these paired fibroblast cell strains was assessed. 2. Production of decorin was lower in all cell strains of hypertrophic scar fibroblasts tested, compared with normal dermal fibroblasts cultured from the same patients (mean 49 +/- 23%; P < 0.001, n = 9). Levels of mRNA for decorin were also lower (mean 59 +/- 28%; P < 0.02, n = 7) but those for biglycan and versican were not significantly different. Four pairs of cell strains were examined at more than one passage and the differences in decorin protein were found to be phenotypically persistent. Treatment of confluent cultures with transforming growth factor-beta 1 for 3 days caused a reduction in both decorin protein and mRNA in all six strains of hypertrophic scar fibroblasts tested and in five of six strains of normal dermal fibroblasts. An increase in the length of the dermatan sulphate chain on decorin, a previously reported characteristic of this glycosaminoglycan in hypertrophic scar, was seen in all but two of the strains treated with transforming growth factor-beta 1. The depression of decorin synthesis by transforming growth factor-beta 1 was reversed on removal of the agent and passaging the fibroblasts. 3. The reduced capacity of fibroblasts in hypertrophic scar tissue to synthesize decorin may have implications for the development of the condition since this small proteoglycan is involved in tissue organization and may also play a role in modulating the activity in vivo of fibrogenic cytokines such as transforming growth factor-beta 1.

Adolescent↗

PubMed1998

IFN-alpha2b suppresses the fibrogenic effects of insulin-like growth factor-1 in dermal fibroblasts.

The interferon (IFN) proteins, including IFN-alpha2b have been used as antifibrogenic factors to modulate the expression of extracellular matrix (ECM) proteins associated with fibroproliferative disorders in skin. This study was conducted to determine if IFN-alpha2b can counteract the fibrogenic effects of insulin-like growth factor-1 (IGF-1), which is present in large quantity in fibrotic dermis. Human dermal fibroblasts were established in culture and treated with either vehicle (control), 2000 U/ml IFN-alpha2b alone, 100 ng/ml IGF-1 alone, or both IFN-alpha2b and IGF-1. The results showed that treatment with IFN-alpha2b inhibited the proliferation of dermal fibroblasts, reduced the steady-state levels of type I procollagen mRNA in the cells, and reduced the production of collagen as measured by hydroxyproline in conditioned medium. However, this treatment also increased levels of collagenase mRNA in the cells and collagenase activity in the medium. Cells treated with IGF-1 showed increased proliferation and collagen production and decreased collagenase. Cells treated with both IFN-alpha2b and IGF-1 exhibited a 44% reduction in hydroxyproline production (p < 0.05) and a 363% increase in collagenase activity over cells treated with IGF-1 alone (p < 0.01). These results indicate that when IGF-1 and IFN-alpha2b are used individually, they function as fibrogenic and antifibrogenic factors for dermal fibroblasts, respectively, and that fibrogenic effects of IGF-1 on cell proliferation, collagen, and collagenase expression can be counteracted by IFN-alpha2b. These findings support the potential use of IFN-alpha2b as a therapeutic agent for treatment of fibroproliferative disorders, such as postburn hypertrophic scarring.

Cell Division↗

PubMed1998

Transforming growth factor-beta in thermally injured patients with hypertrophic scars: effects of interferon alpha-2b.

Hypertrophic scarring is a common dermal fibroproliferative disorder that leads to poor quality wound healing, prolongs rehabilitation, and increases morbidity following major thermal and other injuries to the deep dermis. Local and systemic transforming growth factor (TGF)-beta has been implicated as a fibrogenic cytokine in the pathogenesis of many fibrotic disorders, whereas interferon (IFN) alpha-2b may improve the pathologic features of dermal fibrosis directly or by antagonizing the effects of TGF-beta and histamine. Nine patients with severe hypertrophic scarring were evaluated for 8 weeks before treatment with subcutaneous recombinant IFN alpha-2b, 2 x 10(6) IU three times per week for 24 weeks. Clinical assessment was performed using standardized photography, a burn scar assessment tool, and serial scar volume measurements. Monthly measurements of serum TGF-beta and plasma Ntau-methylhistamine were made prior to, during, and after IFN alpha-2b therapy and compared with 27 age-matched controls. Serial biopsies of the hypertrophic scars and normal skin were performed for evaluation of mast cell numbers. Significant improvement in scar assessment occurred in 7 of 9 patients, and 3 of 9 demonstrated significant reductions in scar volume with interferon therapy beyond that occurring during the 8-week control period. For the entire group, mean rates of improvement were significantly better during interferon therapy with no recurrence following treatment. Before interferon therapy, serum TGF-beta was significantly higher in the burn patients with hypertrophic scarring than in a control population (123.04 +/- 36.48 vs. 56.85 +/- 8.38 ng/ml, p < 0.05). Within 3 months of IFN alpha-2b therapy, serum TGF-beta levels fell significantly and remained within the normal range during therapy and after interferon therapy was stopped. Plasma Ntau-methylhistamine levels were also significantly elevated in the hypertrophic scar patients as compared with age and sex-matched controls (153.6 +/- 92.07 vs. 48.3 +/- 28.9 pg/ml, p < 0.05), and significant reductions were achieved with interferon therapy and maintained after interferon was discontinued. Paired biopsies of hypertrophic scarring and normal tissue demonstrated increased numbers of mast cells in hypertrophic scars compared with normal uninjured skin from the same patients (2.65 +/- 1.63 vs. 1.04 +/- 0.62 cells/high power field, p < 0.001); however, no significant change in mast cell content of the hypertrophic scars accompanied interferon therapy. Patients with severe hypertrophic scarring demonstrate increased levels of serum TGF-beta and plasma Ntau-methylhistamine following thermal injury. A significant clinical improvement in scar quality and volume occurred during IFN alpha-2b therapy, which was associated with normalization of serum TGF-beta and plasma Ntau-methylhistamine levels. A double-blind, placebo-controlled trial will be required to further assess the usefulness of subcutaneous treatment with IFN alpha-2b for the treatment of hypertrophic scarring.

Adult↗

PubMed1997

Determination of plasma Ntau-methylhistamine in vivo by isotope dilution using benchtop gas chromatography-mass spectrometry.

A practical sensitive and specific method for determination of the stable metabolite of histamine, Ntau-methylhistamine, in human plasma using benchtop gas chromatography-stable isotope dilution mass spectrometry has been developed. Ntau-Methylhistamine, a principal metabolite of histamine in humans, was extracted and purified from human plasma using a two-step procedure with Sep-Pak silica cartridges. Quantitation of Ntau-methylhistamine was made possible by the synthesis of Ntau-[2H3]methylhistamine used as an internal standard. Derivatization with pentafluoropropionyl anhydride of extracts of human plasma yielded the bis-pentafluoropropionyl derivative of Ntau-methylhistamine for measurement using selected ion monitoring of the m/z 417/420 ion pair after electron impact on a benchtop gas chromatography-mass spectrometry (GC-MS). By improvements in the plasma extraction technique, inclusion of a synthetic internal standard and the development of a sensitive and stable derivative of the histamine metabolite, Ntau-methylhistamine was found to be significantly elevated in the plasma of patients with the dermal fibroproliferative disorder, hypertrophic scarring as compared to age-matched normal volunteers (98.5+/-29.5 pg/ml, n=9, versus 43.3+/-16.5 pg/ml, n=8, p<0.05). As such, this method affords a sensitive, specific and practical approach to measurement of histamine metabolites in plasma and other biological fluids.

Burns↗

PubMed1997

Cytoskeleton regulates expression of genes for transforming growth factor-beta 1 and extracellular matrix proteins in dermal fibroblasts.

Cytoskeleton not only controls cell morphology but also regulates cell growth, migration, differentiation, and gene expression, events which are fundamental to embryogenesis, carcinogenesis, and wound healing. We have recently reported that reorganization of cytoskeleton induces expression of mRNA for transforming growth factor-beta 1 (TGF-beta 1), collagenase, and tissue inhibitor of metalloproteinase-I (TIMP-I) in dermal fibroblasts. In this report we have examined the role of gene transcription in this induction. As judged by nuclear run-on assay, trypsin, EGTA (ethylene glycol-bis (beta-aminoethyl ether) N, N, N', N', tetra-acetic acid), or cytochalasin C (Chs) increased the rate of transcription of the TGF-beta 1 gene by 2.0, 2.7, and 1.6 fold, respectively, and of the collagenase gene by 5.3, 6.2, and 3.3 fold. The rate of transcription of the TIMP-I gene was increased by trypsin (4.3 fold) or EGTA (3.8 fold) but unaffected by Chs. Cytochalasin induced an increase in the rate of transcription of procollagen I (alpha 1), procollagen I (alpha 2), and fibronectin genes by 1.4, 1.5, and 1.9 fold respectively, while trypsinization or EGTA treatment had no or little effects on these gene. Since transcription of the TGF-beta 1 gene is believed to be largely governed by the activating protein 1 (AP1) complex, we also examined the expression of mRNA for c-fos and c-jun protoon-coproteins. Trypsinization induced rapid (within 30 min) and transient expression of c-fos mRNA. A 2.4 fold increase in c-jun mRNA was apparent after 4 hr and persisted for at least 24 hr. Actinomycin D (Act D) suppressed the induction of TGF-beta 1 mRNA by Chs but had less effect on the TGF-beta 1 mRNA in trypsinized cells which had been replated for 4 hr, suggesting that the half life of TGF-beta 1 mRNA is reduced in cells with a disassembled cytoskeleton. Simultaneous treatment with Chs and cycloheximide (Cxm) resulted in a superinduction of TGF-beta 1 mRNA by 88 +/- 23% (n = 4, P < 0.05), which was abrogated by preexposure to Act D. In contrast, the induction of collagenase mRNA by Chs was totally blocked by Cxm, indicating that the Cxm-mediated superinduction is selective and that protein synthesis is required for induction of this mRNA. Our results suggest that the activities of genes for proteins involved in the structure (Type I collagen and fibronectin), turnover (collagenase and TIMP-1) and regulation (TGF-beta 1) of extracellular matrix (ECM), are all governed at least in part by the status of the cytoskeleton. Since the cytoskeleton is reorganized during cell division, migration, and differentiation, these results may have implications for the regulation of ECM during such processes as embryogenesis, carcinogenesis, and wound healing.

Cells, Cultured↗

PubMed1997

Hypertrophic scars, keloids, and contractures. The cellular and molecular basis for therapy.

Keloids, hypertrophic scars, and contractures are a result of aberrations of the normal wound healing process. An understanding of the cellular and molecular events that are implicated in the development of these fibroproliferative disorders will allow for optimization of wound healing. In turn, treatment choices can be based on the most current scientific information available.

Adult↗

PubMed1997

Liposome-associated interferon-alpha-2b functions as an anti-fibrogenic factor for human dermal fibroblasts.

This study was conducted to determine whether interferon-alpha-2b (IFN-alpha-2b) can be encapsulated in liposomes without compromising its anti-fibrogenic effects on human dermal fibroblasts. The rationale for this approach is that systemic administration of IFN-alpha-2b by injection for treatment of dermal fibrosis is uncomfortable, requires a large quantity of the cytokine, and cannot be easily used in children. Liposomes are potentially useful as vehicles for the topical delivery of drugs if they can be encapsulated without loss of biologic activity. Empty sonicated vesicles composed of dioleoyl-phosphatidylcholine:dioleoyl-phosphatidylglycerol at a molar ratio of 7:3 were mixed with various concentrations of IFN-alpha-2b and then dried and rehydrated. An enzyme-linked immunosorbent assay (ELISA) was used to determine the efficiency of encapsulation and the stability of the preparation under experimental conditions. Greater than 80% of added IFN-alpha-2b became associated with the liposomes and remained encapsulated for up to 5 d at 4 degrees C. The rate of release increased markedly at 37 degrees C. Liposome-encapsulated IFN-alpha-2b (2000 units per ml) significantly reduced the proliferation of dermal fibroblasts (60 +/- 8.8 vs. 100 +/- 8, mean +/- SEM, p < 0.05, n = 8) and the levels of mRNA for type I (41.5 +/- 8.7% vs 100 +/- 18, p < 0.05, n = 4) and type III (68 +/- 8.4% vs 100 +/- 4.9%, p < 0.05, n = 3) procollagen, as analyzed on northern blots. This was consistent with the reduction found in collagen in conditioned medium from treated fibroblasts. In contrast, treatment increased levels of mRNA for collagenase (241 +/- 42% vs 100 +/- 3.4, p < 0.05, n = 3) and collagenase activity (289 +/- 5.8% vs 100 +/- 10.9%, p < 0.05, n = 9) in conditioned medium. This last effect was probably not due to a reduction in TIMP-1 (tissue inhibitor of metalloproteinase-1) because levels of mRNA for this inhibitor were not lower in treated cells. The efficacy of liposome-associated IFN-alpha-2b in vitro supports the concept of the topical use of this anti-fibrogenic agent for treatment of fibroproliferative disorders.

Cell Division↗

PubMed1997

Nitric oxide synthase expression and nitric oxide production are reduced in hypertrophic scar tissue and fibroblasts.

We have recently demonstrated that human dermal fibroblasts express both constitutive and inducible nitric oxide synthases (NOS) and produce nitric oxide (NO). In this study, NOS expression and NO production were compared in human fibroblasts derived from hypertrophic scar (HSc) and site-matched normal dermis. NOS expression and NO production in HSc fibroblasts are significantly reduced when compared with normal fibroblasts. Without stimulation, the HSc fibroblasts produced 7.37 +/- 1.17 nmol and the normal fibroblasts produced 11.15 +/- 0.79 nmol/10(6) cells/96 h (paired t test, p < 0.01, n = 6) NO as determined by the Griess reaction. After stimulation with 200 units interferon-gamma per ml and 40 microg lipopolysaccharide per ml, both HSc and normal fibroblasts produced significantly higher NO, indicating that the HSc fibroblasts retain the capacity to express inducible NOS (iNOS). The Ca2+-dependent NOS (cNOS) activity in the cytosol of HSc fibroblasts (1.43 pmol/min/g of protein) was significantly lower than that in normal fibroblasts (2.60 pmol/min/g of protein), as determined by citrulline assay (p < 0.01, n = 4). The mRNAs for endothelial cNOS and iNOS in both HSc and normal fibroblasts were detectable by RT-PCR. Flow cytometry confirmed that the untreated HSc fibroblasts expressed less endothelial cNOS protein than untreated normal fibroblasts. Because NO markedly inhibits cell proliferation, our results suggest that after thermal injury, fibroblasts in the scar tissue may undergo phenotypic alteration leading to reduced endothelial cNOS expression. The low levels of endogenous NO in HSc fibroblasts might be responsible for the cellularity of post-burn HSc, a characteristic feature of this fibrotic condition.

Adolescent↗

PubMed1997

Immunohistochemical localization of transforming growth factor beta and tumor necrosis factor alpha in the lungs of fibrosis-prone and "non-fibrosing" mice during the latent period and early phase after irradiation.

To evaluate the possibility that TGF-beta and TNF-alpha are involved in fibrosis induced in mouse lung by irradiation, the proportion of cells immunoreactive for each was compared in two strains of mice. C3HeB/FeJ mice develop only classical pneumonitis during the early phase, whereas C57L/J mice develop small, tightly packed areas of inflammation which undergo fibrosis during the latent period, and exhibit progressive fibrosis of large regions of intense inflammation during the early phase. Very few cells were immunoreactive for an antibody to the latency-associated peptide (LAP) of TGF-beta during the latent period in C3HeB/FeJ mice, and no cells were positive during the early phase. In contrast, between 0.7 and 10% of cells were positive in C57L/J mice in lesions without fibrosis and in lesions in the early stages of fibrosis. Fibroblasts positive for LAP were seen only in lesions containing fibrosis. A similar pattern of immunoreactivity was seen in C57L/J mice using an antibody which recognizes active TGF-beta, with the exception that positive fibroblasts were observed within areas of inflammation without fibrosis. Thus the association of active TGF-beta with fibroblasts might be a characteristic of the initiation of fibrosis in this model. TNF-alpha was detected in macrophages in all classes of lesions, and minor differences between the strains did not appear to be biologically meaningful.

Animals↗

PubMed1996

Chemical characterization and quantification of proteoglycans in human post-burn hypertrophic and mature scars.

1. Samples of normal skin from four patients, post-burn hypertrophic scar from five patients and post-burn mature scar from six patients were analysed for hydroxyproline, water and uronic acid and extracted with guanidinium chloride to yield the proteoglycan pool. A large chondroitin sulphate proteoglycan and biglycan were purified from one hypertrophic scar biopsy and decorin from a normal skin biopsy, by ion-exchange chromatography, gel-filtration and hydrophobic interaction chromatography. These purified proteoglycans were used in an inhibition ELISA assay to estimate the quantities of each in the tissue samples. 2. Samples of post-burn hypertrophic scar had on average 30% less hydroxyproline, 12% more water and 2.4 times as much uronic acid as normal skin. These differences were all statistically significant, whereas the small differences between mature scars and normal skin were not. The content of decorin in hypertrophic scars was only 25% of that in normal skin whereas the large chondroitin sulphate proteoglycan and biglycan were each about 6-fold higher. The mature scars had slightly elevated levels of large chondroitin sulphate proteoglycan and biglycan and a reduced content of decorin compared with normal skin but these differences were not statistically significant. 3. The results suggest that aberrant proteoglycan metabolism is a significant factor contributing to the altered physical properties of hypertrophic scars and that maturation of post-burn scars is dependent on a return of the relative proportions and concentrations of proteoglycans to those characteristic of normal dermis.

Burns↗

PubMed1996

Human dermal fibroblasts produce nitric oxide and express both constitutive and inducible nitric oxide synthase isoforms.

Nitric oxide (NO) is produced by a variety of human and animal cells and is involved in a broad array of physiological and pathophysiological processes. It can cause vasodilation, serve as a neurotransmitter, and have anti-neoplastic, anti-microbial, and anti-proliferative effects. In this study, we have demonstrated that fibroblasts derived from human skin spontaneously produce NO and that this production can be enhanced by stimulating the cells with interferon-gamma and lipopolysaccharide. The production of NO by human dermal fibroblasts can be blocked by NG-monomethyl-L-arginine (L-NMMA). The inhibitory effect of L-NMMA on NO production was restored by addition of L-arginine but not D-arginine. By measuring the rate of conversion of [14C]L-arginine to [14C]L-citrulline, we show that unstimulated cells expressed only Ca2+-dependent NO synthase (NOS) activity (1.36 +/- 0.57 pmol/mg/min; n = 4) whereas stimulated cells expressed both Ca2+-dependent (2.60 +/- 0.54 pmol/mg/min; n = 4) and -independent (1.59 +/- 0.14 pmol/mg/min; n = 4) NOS activities. With reverse transcription polymerase chain reaction (RT-PCR), the 422-bp RT-PCR product for human endothelial constitutive NOS and the 462-bp RT-PCR product for human hepatocyte inducible NOS were detected in proportion to the amount of mRNA-related RT-cDNA added to the reaction mixture. Further evidence by immunocytochemistry demonstrated that human dermal fibroblasts express both constitutive and inducible NOS proteins. These data collectively suggest that in addition to macrophages and other inflammatory cells, nitric oxide production by dermal fibroblasts could be important during the inflammatory stages of wound healing and possibly also in the later stages of proliferation and tissue remodeling after skin injury in humans.

Animals↗

PubMed1996

Collagenase production is lower in post-burn hypertrophic scar fibroblasts than in normal fibroblasts and is reduced by insulin-like growth factor-1.

We recently demonstrated that the accumulation of extracellular matrix in post-burn hypertrophic scarring (HSc) tissues is, in part, caused by an overexpression of mRNA for fibronectin, type I, and type III procollagen. Here, we report that five different fibroblast cell strains derived from HSc tissues are deficient in collagenase activity relative to paired fibroblasts from normal skin of the same patients. Quantitative analysis demonstrated significantly lower (52.5 +/- 16.8% vs 100 +/- 8.3%; n = 9; p < 0.05) collagenase activity in conditioned medium from HSc fibroblasts relative to that obtained from the control. The expression of collagenase mRNA was also significantly depressed (51 +/- 7% vs 100 +/- 11%; n = 5; p < 0.05) in four of five strains of HSc fibroblasts examined. The level of mRNA for collagenase in both HSc and normal fibroblasts increased with serial passage, but at any given passage number, the expression of this transcript was lower in HSc fibroblasts. Insulin-like growth factor 1 (IGF-1), which is present at the site of HSc in high quantity, reduced collagenase mRNA but increased type I collagen mRNA expression in a time-dependent manner. The collagenase activity in conditioned medium derived from IGF-1-treated normal dermal fibroblasts was reduced (23.1 +/- 7.81% vs 100 +/- 6.6%; n = 7; p < 0.05). A significant reduction in collagenase mRNA and activity was also found in HSc fibroblasts following IGF-1 treatment. These findings suggest that IGF-1-induced suppression of collagenase mRNA and activity may be a mechanism by which IGF-1 promotes the development of post-burn HSc.

Adolescent↗

PubMed1995

Enhanced expression of mRNA for insulin-like growth factor-1 in post-burn hypertrophic scar tissue and its fibrogenic role by dermal fibroblasts.

Hypertrophic scarring (HSc) which frequently develops in patients following severe thermal injury is characterized by accumulation of extracellular matrix (ECM) proteins including type I and type III collagen. In this study, we examined the presence and quantity of IGF-1 mRNA transcripts in post-burn HSc. The results of dot blot experiments showed a 77.5% (100 +/- 8.15 vs 177.5 +/- 19, p < 0.01) increase in expression of IGF-1 IIIRNA in HSc tissue relative to normal dermis obtained from the same patients. A Northern blot analysis confirmed the specificity of the IGF-1 cDNA. This cDNA visualized four different transcripts with apparent sizes of 7.0, 3.9, 1.8 and 1.0 kb, similar to those previously reported. The possible fibrogenic role of IGF-1 was examined by analyzing the effect of this growth factor on the expression of mRNA for the pro alpha 1(I) chain of type I procollagen and the pro alpha 1(III) chain of type III procollagen in dermal fibroblasts. IGF-1 increased the expression of these transcripts as early as 6 h and the effect was maximal at 24 h. Quantitative analysis by densitometry showed a 149 and 166% increase in pro alpha 1(I) and pro alpha 1(III) mRNA after 24 h of IGF-1 treatment, respectively. This effect seems to be specific as the abundance of mRNA for the pro alpha 2(I) chain of type I procollagen or TIMP-II was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗