Biomedical subjects

A Ghahary

Publications and source records attributed to A Ghahary.

At least 55 records · Page 3Linked to original sources

PubMed1995

Immunolocalization of TGF-beta 1 in human hypertrophic scar and normal dermal tissues.

Following severe thermal injury and other injuries to the deep dermis of the skin, patients frequently develop hypertrophic scarring (HSc) which is characterized by an over-abundance of extracellular matrix (ECM) proteins including collagen. Our previous work revealed a synchronous elevation in expression of mRNA for transforming growth factor (TGF)-beta 1, type I and type III procollagen in human HSc tissue, suggesting a possible role of locally synthesized TGF-beta 1 in matrix production. In this study the immunoreactive sites of TGF-beta 1 protein in hypertrophic and normal dermal (ND) tissue obtained from the same patients have been determined by an immunoperoxidase staining system. We used two TGF-beta 1-specific rabbit polyclonal antibodies known as anti-LC and anti-CC which were prepared to different synthetic preparations of a peptide corresponding to the first 1-30 amino acids of the amino-terminus of mature TGF-beta 1. These antibodies have affinity for two distinct epitopes of TGF-beta 1. The anti-LC antibody localized TGF-beta 1 to non-proliferating/differentiated epidermal cells, suprabasal keratinocytes in both normal and HSc tissues. The intensity of this staining was significantly higher (150 +/- 26 sq. micron vs 77 +/- 7 sq. micron, n = 5, P < 0.05) in normal tissues compared to HSc tissues. When the anti-CC antibody was used as the primary antibody, intense staining of focal regions was observed in the dermis of HSc tissue, but not in ND tissue obtained from the same patient. These foci contained collagen which was nodular, distributed in whorl-like arrangements and highly enriched with microvessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1995

Regulation of collagen synthesis and mRNA expression in normal and hypertrophic scar fibroblasts in vitro by interferon-gamma.

Development of hypertrophic scarring (HTS) is a major problem for patients who survive extensive thermal injuries. HTS and other fibroproliferative disorders are associated with excessive accumulation of collagen and other extracellular matrix proteins. Recent in vitro and in vivo studies have demonstrated that proteins of the interferon family have an inhibitory effect on collagen production by fibroblasts in some fibroproliferative disorders. This study investigated the effects of interferon-gamma (IFN-gamma) on cell proliferation, collagen production, and expression of types I and III procollagen mRNA in human postburn HTS fibroblasts. Paired fibroblast cultures were established from explants of biopsies obtained from HTS and normal skin (matched for location and skin tension) in five patients recovering from thermal injuries. Thus, normal dermis from each patient was used as a paired control. Administration of IFN-gamma (1000 U/ml) to proliferating fibroblast cultures for 5 days resulted in 51% reduction (P < 0.05) in HTS cell proliferation. Using hydroxyproline as an index for collagen production, a 34% reduction (P < 0.05) in collagen synthesis was observed in HTS fibroblast culture media after treatment with IFN-gamma (1000 u/ml) for 48 hr. Northern blot analysis demonstrated 55 and 36% reductions (P < 0.05 for each) in type I and type III procollagen mRNA levels, respectively, after treatment for 12 hr with IFN-gamma (1000 u/ml). The effect of IFN-gamma on each of these parameters was at least as pronounced in HTS fibroblasts as their normal controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1995

Alteration in cell morphology triggers transforming growth factor-beta 1, collagenase, and tissue inhibitor of metalloproteinases-I expression in normal and hypertrophic scar fibroblasts.

Using immunocytochemistry and Northern blot analysis, we investigated the role of cell morphology and reorganization of the cytoskeleton in the expression of transforming growth factor-beta 1 (TGF-beta 1) in human dermal fibroblasts. Disruption of the cytoskeleton was induced by three different agents--trypsin, ethyl-eneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or cytochalasin--and was confirmed by staining with rhodamine-labeled phalloidin. Immunocytochemical staining with antibodies specific for TGF-beta 1 revealed a cell-shape-related induction of TGF-beta 1. Northern blot analysis of total RNA showed a significant increase in the expression of TGF-beta 1 mRNA as early as 4 h and peaking at 12 h after disruption of the cytoskeleton. Quantitative analysis of TGF-beta 1 mRNA expression at 4 h after treatment with trypsin, EGTA, or cytochalasin C showed increases of 2.6-, 3.3-, and 2.6-fold, respectively. Disruption of the cytoskeleton by trypsin, EGTA, or cytochalasin C increased mRNA for collagenase by 3.8-fold, 2.3-fold, or 2.5-fold, respectively. The expression of mRNA for tissue inhibitor of metalloproteinases I (TIMP-I) also showed a 3.2-fold increase by trypsin, a 3.6-fold increase by EGTA, and a 2.5-fold increase by cytochalasin C. Cell-shape-related induction of TGF-beta 1, collagenase, and TIMP-I genes appears to be selective, as the levels of mRNA for fibronectin and type I procollagen were not significantly altered. These data suggest that gene expression of TGF-beta 1, collagenase, and TIMP-I is governed by the status of the cytoskeleton microfilament organization, which may be a mechanism of gene regulation during cell division, migration, and differentiation, events fundamental to wound healing.

Actins↗

PubMed1995

Immunohistochemical localization of the proteoglycans decorin, biglycan and versican and transforming growth factor-beta in human post-burn hypertrophic and mature scars.

The distributions of the small proteoglycans, decorin and biglycan and the large proteoglycan, versican, in normal skin and post-burn hypertrophic and mature scars, were compared using monoclonal and polyclonal antibodies to the core proteins. Biglycan and versican were virtually undetectable in normal dermis but readily seen in hypertrophic scars. Staining for decorin was strong throughout the dermis in normal skin but restricted to the deep dermis and a narrow zone under the epidermis in hypertrophic scar--areas which did not stain for versican. Decorin was absent or reduced in the nodules in these specimens. In mature post-burn scars, staining for all three proteoglycans demonstrated an intensity that was intermediate between that in normal dermis and that in the nodules of the hypertrophic scars. Transforming growth factor-beta was present in the nodules of hypertrophic scars but the deep dermis of these specimens stained most intensely for this cytokine which was also found in the dermis of mature scars but was not detectable in normal dermis. The apparent co-distribution of decorin and transforming growth factor-beta suggests that this proteoglycan may play an active role in the resolution of the scars. Changes in proteoglycan type and distribution could possibly account, at least in part, for the derangement of collagen and the altered physical properties of hypertrophic scar tissue.

Adult↗

PubMed1995

The effect of interferon alpha 2b on the expression of cytoskeletal proteins in an in vitro model of wound contraction.

Wound contraction is an essential component of wound healing. However, the development of scar contractures in tissues and organs disrupts normal organ integrity and produces functional deformities. Although interferons alpha and gamma inhibit extracellular matrix protein production by fibroblasts, their effects on cytoskeletal protein mediated-wound contraction are as yet unclear. The fibroblast-populated collagen lattice is an in vitro assay that simulates wound contraction. When matched pairs of human hypertrophic scar and normal dermal fibroblast cultures established from patients recovering from a thermal injury were used, interferon-alpha 2b exposure before lattice formation was found to significantly inhibit contraction in a treatment time-dependent manner (p < 0.05). Fibroblasts generated contractile forces that were triphasic and serum sensitive (p < 0.01). Comparison of hypertrophic scar and normal dermal fibroblasts revealed no significant differences in ability to induce lattice contraction. Northern blot analysis of mRNAs for the intracellular contractile proteins revealed that interferon-alpha 2b significantly down-regulated mRNA levels of the actin isoforms beta and gamma (50% to 60%) but had no significant effect on alpha-tubulin, vimentin, and alpha-actinin. Fibroblast-populated collagen lattices were stained with rhodamine-labeled phalloidin to reveal filamentous actin proteins. Marked morphologic alterations of the stress fibers were associated with reductions in lattice contraction after interferon-alpha 2b treatment. Thus interferon-alpha 2b's inhibition of wound contraction in vitro is associated with reductions in mRNA for beta and gamma actin and distinct morphologic alterations in fibroblast stress fiber morphology.

Adolescent↗

PubMed1995

Effect of retinoic acid on expression of transforming growth factor-beta by retinal pigment epithelial cells in culture.

OBJECTIVE: To study the effect of retinoic acid on the expression of transforming growth factor-beta (TGF-beta) by human retinal pigment epithelial (RPE) cells in culture. MAIN OUTCOME MEASURES: Expression of TGF-beta 1 by RPE cells after 24 hours in culture at the messenger RNA level (Northern analysis) and the protein level (enzyme-linked immunosorbent assay [ELISA]). RESULTS: On the basis of cell morphology, growth characteristics and the results of immunohistochemical studies, we concluded that the cells used in these experiments were RPE in origin. Immunohistochemical studies confirmed TGF-beta 1 expression by the RPE cells in culture. Densitometry showed that retinoic acid reduced the level of TGF-beta 1 mRNA expression by 41% compared with control samples. ELISA showed that retinoic acid inhibited TGF-beta expression when compared with the baseline level of TGF-beta in media from RPE cells in culture. CONCLUSIONS: These findings may have implications for the pharmacotherapy of proliferative vitreoretinopathy.

Blotting, Northern↗

PubMed1994

Differential effects of thermal injury on circulating insulin-like growth factor binding proteins in burn patients.

The results of this report provide evidence that insulin-like growth factor-1 binding proteins (IGFBPs) in human sera are differentially regulated as a result of severe burn injury. Using the ligand binding technique, 125I-IGF-1 visualizes 5 different protein bands corresponding to those previously reported for IGFBP-1 to 4 with apparent sizes of 23-42 kd in serum samples prepared from severely burned patients and healthy individuals. The level of IGFBP-3 was significantly decreased within 3-5 days of injury and remained depressed for up to 20 days post injury. The average level of this binding protein reached its lowest value within 3-5 days of the injury (3.8 +/- 1.48% relative to day 0-1 value, n = 4, p < 0.01). Serum samples from 3 of 4 patients showed no recovery within 20 days post injury and the level of IGFBP-3 remained significantly depressed (p < 0.01). In contrast, the levels of IGFBP-2 and IGFBP-4 increased 2 and 3 fold in the same serum samples within 3-5 days of the burn injury, respectively. This increase returns to normal (day 0-1 value) within 7-10 days for IGFBP-2, but the level of IGFBP-4 remained elevated 4 fold relative to the day 0-1 (p < 0.01). However, the abundance of IGFBP-1 in these serum samples was not significantly altered by the burn injury. By controlling for protein loading, these apparent alterations of IGFBPs in the sera of burned patients were not due to hemodilution. Similarly, significant reductions in IGFBP-3 were not likely due to IGFBP-3 specific protease activity in the sera of burn patients since incubation of sera from burn patients and normal individuals at 37 degrees C did not alter the pattern of IGFBPs in sera obtained from normal individuals. Of interest, the level of IGF-1 protein in these samples was also markedly reduced following severe burn injury similar to IGFBP-3. The results of this study suggest that a marked reduction of serum IGF-1 seen in burn patients is associated with a significant reduction of IGFBP-3, a major IGF-1 binding protein in human serum.

Adult↗

PubMed1994

Expression of mRNA for transforming growth factor-beta 1 is reduced in hypertrophic scar and normal dermal fibroblasts following serial passage in vitro.

We have recently demonstrated a synchronous elevation in the expression of mRNA for type I, type III procollagen and transforming growth factor-beta 1 in post-burn hypertrophic scar tissue compared to normal skin obtained from the same patient. In an attempt to examine whether these effects are phenotypically persistent, we compared the expression of mRNA for transforming growth factor-beta 1 in cultured fibroblasts from post-burn hypertrophic scar and normal dermis established from the same patients at passages 3, 5, 7, and 10. When a cDNA probe specific for transforming growth factor-beta 1 was used, a transcript with an apparent size of 2.5 kb was detected in both hypertrophic and normal fibroblasts at various passages. This cDNA also hybridized with a transcript with an apparent size of 4.9 kb. The abundance of the 2.5-kb transcript was decreased to 64% and 33% in hypertrophic scar and normal fibroblasts respectively, between passage 3 and 7, whereas the intensity of the 4.9-kb message was not significantly altered. This change seems to be selective because no significant alteration in the expression of mRNA for the heat-shock-like protein (Nb29) was observed. These findings suggest that the effect of passage on the expression of transforming growth factor-beta 1 in hypertrophic scar tissue fibroblasts is more pronounced than in normal cells derived from the same patient.

Adolescent↗

PubMed1994

Insulin-like growth factor-1 in the arterial wall after exposure to periarterial blood.

Intimal proliferation is thought to be initiated by the migration and proliferation of smooth muscle cells after endothelial damage. These changes may be induced, in part, by mitogenic growth factors such as insulin-like growth factor-1 (IGF-1). This study was designed to investigate the role of locally synthesized IGF-1 and its receptor in the arterial wall in response to the exposure to periarterial blood. Rat femoral arteries were exposed to periarterial blood for various time periods (control, 1, 3, 7, 14, and 21 d). Total ribonucleic acid was extracted from the arteries of 10 to 15 animals, and the expression of IGF-1 messenger ribonucleic acid in treated and untreated arteries was analyzed using dot blot analysis. To identify and localize IGF-1 receptors on the arterial walls, an in situ ligand binding of IGF-1 to the arterial sections was utilized using [125I]IGF-1 as a tracer. Our results revealed that luminal narrowing was maximum at 7 days posttreatment. Intimal proliferation occurred at 14 and 21 days. The results of dot blot analysis showed that the expression of IGF-1 messenger ribonucleic acid was increased four-fold by Day 3 and remained elevated up to 7 days, then gradually decreased. In situ [125I]IGF-1 binding to the normal rat femoral artery localized IGF-1 receptors to the arterial wall. There was a marked increase in the number of receptors at 3 and 7 days after treatment with periarterial blood. These results suggest that locally synthesized IGF-1 and its receptor may function in an autocrine and/or paracrine loop as part of the response of the arterial wall to periarterial blood, resulting in intimal proliferation.

Animals↗

PubMed1993

Expression and regulation of insulin-like growth factor binding protein-1 in the rat uterus throughout estrous cycle.

In previous studies, we have demonstrated that IGF-1 and IGF-1 receptor are expressed in rat uterus and that the expression is up-regulated by estrogen. The present study examines the expression and regulation of IGFBP-1 and IGFBP-3 in rat uterus throughout the estrous cycle. The stage of the estrous cycle in 16 mature female rats was determined by microscopic examination of daily prepared vaginal smears. Rat uteri were then used for RNA extraction. The results of the Northern blot analysis demonstrate that uterine cells express both IGFBP-1 and IGFBP-3 mRNA throughout the estrous cycle. When autoradiograms were quantitated by a densitometry, a significant reduction in expression of IGFBP-1 mRNA was found in uteri at stages of proestrous and estrous relative to that in diestrus. Although the level of IGFBP-3 mRNA varied in uteri throughout estrous cycle but this variation was not statistically significant. The lowest expression of IGFBP-1 (8.5% relative to diestrus, p < 0.05, n = 4) and IGFBP-3 (71% relative to diestrus) was found in the uteri prepared from rats at the stage of proestrus, while the highest expression of IGFBP-1 and IGFBP-3 was observed in the uteri obtained from rats at the stage of diestrus and metestrus, respectively. Using anti-rabbit IGFBP-1 antibody raised against an oligo-synthetic IGFBP-1 peptide, immunohistochemical staining demonstrates the presence of IGFBP-1 in the luminal and stromal glandular epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Gas chromatography-mass spectrometry determination of 18O2 in 18O-labelled 4-hydroxyproline for measurement of collagen synthesis and intracellular degradation.

The use of gas chromatography-mass spectrometry (GC-MS) and 18O2, a stable isotope which is incorporated into collagen during the post-translational conversion of proline to hydroxyproline, offers the potential advantages of high levels of sensitivity and specificity as compared to other techniques for measuring rates of collagen synthesis and degradation in vitro and vivo. Trifluoracetylation and methanol esterification of hydroxyproline yields two derivatives of hydroxyproline: N,O-trifluoroacetyl methyl 4-hydroxy-L-proline (N,O-TFA-Hyp) and N-trifluoroacetyl methyl 4-hydroxy-L-proline (N-TFA-Hyp). In the past, N-TFA-Hyp, which yields the 16O/18O-containing m/z 182/184 ion pair [M-COOH3]+ when analyzed by electron impact ionization GC-MS, has been proposed for analysis of 18O-enriched collagen. Although N,O-TFA-Hyp can be converted to N-TFA-Hyp by solvolysis, we find that this leads to degradation of the chromatography in GC-MS and demonstrate here that this extra chemical step is unnecessary if the m/z 278/280 ion pair (representing the [M-COOCH3]+. fragment) is measured by selected ion monitoring. By labelling fibroblasts in culture with 18O2, a sample of isotope-enriched collagen was obtained which was used to calibrate the GC-MS over the range 0.5-49% atom percent enrichment (APE). The greater sensitivity of 18O2 versus [15N]proline for labelling newly synthesized collagen was demonstrated by the finding of a ten-fold higher enrichment in the former isotope when administered to cell cultures at the same precursor APE. Thus, the approach described herein permits the determination of total hydroxyproline and APE on the same sample avoiding additional processing steps while maintaining the quality of chromatography and the sensitivity of detection. Measurement of absolute rates of both collagen synthesis and intracellular degradation of newly synthesized collagen in cell cultures is thus possible. Preliminary results comparing collagen metabolism in pairs of fibroblasts from hypertrophic scars and normal skin in post-burn patients are presented.

Cicatrix↗

PubMed1993

Regulation of collagen synthesis and messenger RNA levels in normal and hypertrophic scar fibroblasts in vitro by interferon alfa-2b.

Hypertrophic scars, which commonly occur after thermal and traumatic injury of the skin, are a fibroproliferative disorder of the dermal matrix wherein components of the inflammatory process, including the fibrotic growth factor, transforming growth factor-beta, appear to activate dormant fibroblasts leading to cellular proliferation and excessive matrix synthesis. To investigate the potential beneficial role and mechanism of interferon alfa-2b in controlling excessive collagen production in hypertrophic scar, we measured dose response, time of onset, and duration of action in hypertrophic scar fibroblasts in vitro and compared them with those of site-matched normal fibroblasts obtained from four patients after thermal injury. Interferon alfa-2b reduced collagen protein synthesis and type I messenger RNA levels in both hypertrophic scar and normal fibroblasts after treatment, but these changes were apparent only after approximately 72 hours. Significant reductions in collagen synthesis occurred in four pairs of normal and hypertrophic scar fibroblasts (p < 0.05), accompanied by significant reductions in type I (p < 0.05) but not type III procollagen messenger RNA. Hypertrophic scar fibroblasts recovered completely from the effects of interferon alfa-2b on procollagen type I messenger RNA within 48 hours of cessation of treatment in contrast to normal skin fibroblasts, in which the reduction in type I procollagen messenger RNA by interferon alfa-2b persisted beyond 72 hours after treatment. These data suggest that interferon alfa-2b reduces collagen synthesis in both normal and hypertrophic fibroblasts but the hypertrophic fibroblast may remain less sensitive to its effects.

Journal Article↗

PubMed1993

Expression of fibronectin messenger RNA in hypertrophic and normal dermal tissues and in vitro regulation by interferon alfa-2b.

After severe thermal injury, hypertrophic scarring which is associated with accumulation of extracellular matrix proteins including fibronectin, frequently develops. We have recently demonstrated that interferon alfa-2b significantly reduces the level of type 1 procollagen messenger RNA expressed by both hypertrophic and normal dermal fibroblasts. In this report, we provide evidence that this cytokine also significantly decreases the expression of fibronectin messenger RNA in human hypertrophic scar and normal dermal fibroblasts. Four dermal fibroblast cell strains were established in cell culture from four human postburn hypertrophic scar tissues with the use of normal dermal fibroblasts from the same patients as controls. These cells were then treated with 2000 U/ml interferon alfa-2b in culture medium at various times. The results of Northern analysis of interferon-treated dermal fibroblasts indicate that this cytokine reduced the expression of fibronectin messenger RNA as early as 12 hours after treatment and reached its lowest level (24% relative to untreated fibroblasts) after 96 hours. When the expression of fibronectin messenger RNA was quantified by densitometry for each individual paired cell strain, a differential response to interferon treatment was found among cell strains. The level of fibronectin messenger RNA expression decreased from 17.2% to 69% in hypertrophic scar fibroblasts and 47% to 83.7% in normal fibroblasts relative to that of untreated control values. Although this decrease was less pronounced in normal fibroblasts than in hypertrophic scar fibroblasts, this reduction was significant in both interferon alfa-2b treated hypertrophic scar fibroblasts (6.39 +/- 0.71 versus 2.88 +/- 0.9, n = 4, p < 0.05) and normal cells compared with untreated controls (5.47 +/- 0.89 versus 3.64 +/- 0.99, n = 4, p < 0.05) as assessed with Student's paired t test. Rehybridization of the RNA blot prepared from interferon alfa-2b treated and untreated hypertrophic scar fibroblasts with a complementary DNA for the tissue inhibitor of metalloproteinase type 2 gelatinase inhibitor showed no significant changes in abundance of this transcript. This result suggests that this cytokine selectively suppresses the expression of fibronectin messenger RNA and that this reduction is not due to RNA loading. A dot blot analysis of total RNA extracted from these tissues was carried out to compare the expression of fibronectin messenger RNA between human hypertrophic scar tissues and normal dermis obtained from the same patients. The blot was initially hybridized with fibronectin complementary DNA and subsequently with a complementary DNA for the tissue inhibitor of metalloproteinase type 2 to correct for RNA loading. When the ratio of fibronectin to tissue inhibitor or metalloproteinase type 2 messenger RNA expression for each hypertrophic scar tissue was compared with its normal control, this ratio was fourfold higher in human hypertrophic scar tissues relative to normal controls. In contrast, the expression of this message in cultured hypertrophic scar fibroblasts was not significantly different from that in normal fibroblasts. The results of this study suggest that hypertrophic scarring developing after thermal injury is associated with an overexpression of fibronectin messenger RNA, and interferon alfa-2b may be of therapeutic value to down-regulate the expression of this transcript.

Journal Article↗

PubMed1993

Procollagen types I and III and transforming growth factor-beta gene expression in the arterial wall after exposure to periarterial blood.

The stiffening and thickening of the arterial wall after subarachnoid hemorrhage may reflect increased connective tissue. The purpose of this study was to examine the nature of collagen synthesis in response to periarterial blood. Rat femoral arteries were exposed to periarterial blood for varying lengths of time (control, 1, 3, 7, and 14 d). Dot-blot analysis of total ribonucleic acid extracted from the arteries (n = 10 to 15 animals each) demonstrated that the expression of procollagen Types I and III messenger ribonucleic acid increased at 7 (threefold) and 14 days. The expression of transforming growth factor-beta (TGF-beta), an important regulator of collagen synthesis, was markedly increased by 3 days (threefold), followed by a gradual decline. There were marked differences in procollagen Types I and III and TGF-beta gene expression between arteries exposed to blood and sham-operated arteries for a period of 7 days (n = 25 animals). Northern blot analysis of total ribonucleic acid extracted from cultured vascular smooth muscle cells showed that the treatment with a higher concentration of serum for 48 hours increased the expression of procollagen Types I and III and TGF-beta, whereas exposure to oxyhemoglobin did not. After exposure to periarterial blood, arterial walls show increased synthesis of procollagen Types I and III, perhaps a response to the increased secretion of TGF-beta, which in turn could be the result of exposure to serum factors.

Animals↗

PubMed1993

Enhanced expression of mRNA for transforming growth factor-beta, type I and type III procollagen in human post-burn hypertrophic scar tissues.

To explore the possible role of locally synthesized transforming growth factor beta-1 (TGF-beta 1) and procollagen gene expression in postburn hypertrophic scars, we have compared mRNA levels for type I and type III procollagen and TGF-beta 1 in human hypertrophic scar tissue with normal dermis obtained from the same patients as a control. Northern blot analysis of total RNA extracted from hypertrophic scar tissue and normal skin demonstrated two transcripts for the pro-alpha 1(I) chain (5.8 kb and 4.8 kb) and for the pro-alpha 1(III) chain (5.4 kb and 4.8 kb) and one transcript (4.9 kb) for TGF-beta 1. Quantitative analysis of dot blot autoradiograms of mRNA from three samples of hypertrophic scar tissue and normal skin showed average increases of 102% (p < 0.05) for pro-alpha 1(I), 91% (p < 0.06) for pro-alpha 1(III), and 61% (p < 0.05) for TGF-beta 1. Three additional hypertrophic scar samples were quantitatively analyzed on Northern blots and showed increases of 246%, 102%, and 250% of the specific messages for pro-alpha 1(I), pro-alpha 1(III), and TGF-beta 1 relative to a normal skin control. Two transcripts (4.9 kb and 2.5 kb) for TGF-beta 1 were identified in cultured fibroblasts. In contrast to the results from tissue, the level of these transcripts in fibroblasts cultured from hypertrophic scar tissue and normal skin were not significantly different, suggesting that the synthesis of this growth factor is stimulated in tissue by a presently unknown mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1992

Effects of partial hepatectomy on hepatic insulinlike growth factor binding protein-1 expression.

Insulinlike growth factor binding proteins modulate the action of the insulinlike growth factors in various bioassays and may regulate the bioavailability of the insulinlike growth factors in vivo. Because the insulinlike growth factors may influence hepatic regeneration, we have examined the effect of partial hepatectomy on serum insulinlike growth factor binding proteins and on the abundance of insulinlike growth factor binding protein-1 messenger RNA in the liver. All rats were fasted before and after partial hepatectomy or sham operation to avoid the confounding effects of difference in food intake. Using a conventional protocol, 70% of the liver was removed, and groups of four or five rats were killed at different intervals after partial hepatectomy. Sham-operated rats served as controls. Pooled sera from each group of rats were analyzed by ligand blotting with 125I-insulinlike growth factor-I. Liver RNA from individual rats was analyzed by slot-blot and Northern-blot hybridization. A small decrease in the 39- to 42-kD insulinlike growth factor binding protein was apparent in sera from both the sham-operated and partial hepatectomized rats. In contrast, a dramatic increase (fivefold) in the 29-kD serum insulinlike growth factor binding protein (insulinlike growth factor binding protein-1) was apparent only in the partial hepatectomized rats. Hepatic insulinlike growth factor binding protein-1 messenger RNA abundance was significantly increased (1.99 +/- 0.18-fold; p less than 0.05) at 1 hr, reached a peak of 2.32 +/- 0.22-fold (p less than 0.01) at 3 hr after partial hepatectomy and returned to basal levels over the subsequent 6 to 12 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Rat glioma cell lines C6 and 9L synthesize type 1 collagen in vitro.

Glial cell lines (C6, a glioma and 9L, a gliosarcoma) grown in vitro produce type 1 collagen which is detectable in the extracellular matrix by immunocytochemistry. Northern blot analysis using a cDNA specific for the proalpha2 (I) chain of procollagen indicates the presence of a single transcript with an apparent size of 4.8 kb in the C6 cell line, whereas two transcripts with apparent sizes of 5.8 and 4.8 kb are visualized in the 9L cells. The stimulatory effect of ascorbic acid on collagen production is detectable by a 20-27% increase in the concentration of hydroxyproline in the culture medium from the two glioma cell lines. Therefore these glioma cell lines provide a valuable model system for comparative investigations on the regulation of type 1 collagen synthesis by nonmesenchymal cells of neuroepithelial origin.

Animals↗

PubMed1991

Insulin-like growth factor-I expression is not increased in the retina of diabetic BB/W-rats.

A combination of immunocytochemistry, in situ hybridization and ligand binding were used to investigate the localization of IGF-I and its receptor in the retina of diabetic and non-diabetic BB/W-rats. Immunocytochemical localization revealed the presence of IGF-I in retinal pigment epithelium, ganglion cells, Muller cell processes and in microvessels. In most sites immunoreactivity was increased in the diabetic retina compared to that of non-diabetic BB/W-rats. In microvessels, however, immunoreactivity was decreased in diabetes. In situ hybridization using an antisense IGF-I riboprobe provided evidence of IGF-I synthesis in all retinal layers with a similar grain density in diabetic and non-diabetic rats. Autoradiographic localization of IGF-I receptors, using [125I]-IGF-I binding, demonstrated a diffuse localization in all retinal layers, with an increase in diabetic animals. These findings suggest that IGF-I synthesis is not altered in the diabetic retina, and that the increased immunoreactivity of IGF-I detectable in the various layers of the retina from diabetic rats may be due to an increased uptake of blood-derived IGF-I suggested by increased receptor density in diabetic rats.

Animals↗