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D R Inman

Publications and source records attributed to D R Inman.

At least 19 recordsLinked to original sources
PubMed1995

Modulation of adhesive properties of DEAE-dextran with laminin.

Human squamous epithelial cells produce lower amounts of laminin and fibronectin when cultured on DEAE-dextran than when cultured on gelatin-coated polystyrene (Biotechnol. Bioeng., 33:1235). The epithelial cells also spread much more slowly on DEAE-dextran than they do on gelatin-coated polystyrene. To determine if the low level of matrix production by cells grown on DEAE-dextran contributed to the slowness of cell spreading on this substrate, microcarriers made from DEAE-dextran were treated with exogenous laminin (10 micrograms/cm2 of surface area) and then examined for ability to support cell adhesion. Squamous epithelial cells spread as rapidly on the laminin-treated DEAE-dextran as they did on gelatin-coated polystyrene (much more rapidly than on untreated DEAE-dextran). This indicates 1) that laminin can bind to DEAE-dextran in a fashion that is biologically usable by anchorage-dependent cells, and 2) that when laminin is bound to DEAE-dextran, the failure of squamous epithelial cells to rapidly spread is overcome. These data support the hypothesis that failure of the cells to synthesize an intact extracellular matrix on DEAE-dextran is responsible, at least in part, for the slowness with which cells spread on this substrate.

Adhesives↗
PubMed1995

Human skin in organ culture. Elaboration of proteolytic enzymes in the presence and absence of exogenous growth factors.

Proteinase levels were assessed in organ culture fluids from human neonatal foreskin maintained under growth factor-free conditions and in the presence of a combination of growth factors (ie, epidermal growth factor, insulin, hydrocortisone, pituitary extract, and all-trans-retinoic acid). Analysis of culture fluids by gelatin zymography revealed the presence of 92-kd and 72-kd gelatinases. There was a greater amount of 92-kd gelatinase activity in the presence of growth factors whereas the levels of 72-kd gelatinase were similar in growth factor-free and growth factor-containing media. Experiments with keratinocytes and fibroblasts in monolayer culture and with isolated dermal tissue in organ culture indicated that the epithelial component was responsible for most of the 92-kd gelatinase activity whereas fibroblasts were primarily responsible for the 72-kd gelatinase activity. Activation with aminophenyl mercuric acetate, requirement for divalent cations, inhibition with EDTA, and insensitivity to inhibition with phenylmethyl sulfonyl fluoride indicated that both gelatinases were metalloproteinases. In additional studies, culture fluids were examined for the presence of plasminogen activator activity. This was detected in culture fluids from tissues maintained under both conditions but was increased in the growth factor-containing medium. The increased amount seen in the growth factor-containing medium appeared to be due almost entirely to a single factor, ie, all-trans-retinoic acid. In monolayer culture, both keratinocytes and fibroblasts produced plasminogen activator; the level was higher in keratinocyte culture fluids than in culture fluids from fibroblasts.

Cells, Cultured↗
PubMed1994

all-trans-retinoic acid preserves viability of fibroblasts and keratinocytes in full-thickness human skin and fibroblasts in isolated dermis in organ culture.

Human dermal fibroblast and human epidermal keratinocyte survival was examined under various conditions in organ culture. Using cell recovery from organ-cultured tissue as the criterion, it was observed that no keratinocytes and few fibroblasts survived incubation for 10-12 days in serum-free basal medium containing a low level (0.15 mM) of extracellular Ca2+. Increasing the extracellular Ca2+ concentration to 1.4 mM or treating the tissue with 3 microM retinoic acid (RA) under low Ca2+ conditions resulted in increased keratinocyte and fibroblast survival; the two treatments together were more effective than either treatment alone. The same treatments preserved fibroblast survival when pieces of isolated dermal tissue were incubated in organ culture and also supported fibroblast survival in monolayer culture. These findings indicate that recovery of keratinocytes and fibroblasts from skin after maintenance in organ culture provides a simple but definitive measure of the viability of the major cellular elements present in the tissue. These findings suggest that RA treatment enhances survival of both fibroblasts and keratinocytes and that these effects of RA can be seen at physiological Ca2+ concentrations as well as at suboptimal levels of extracellular Ca2+. Finally, these results indicate that the dermis is a direct target of RA.

Adult↗
PubMed1994

All-trans retinoic acid (RA) stimulates events in organ-cultured human skin that underlie repair. Adult skin from sun-protected and sun-exposed sites responds in an identical manner to RA while neonatal foreskin responds differently.

Adult human skin from a sun-protected site (hip) and from a sun-exposed site (forearm) was maintained in organ culture for 12 d in the presence of a serum-free, growth factor-free basal medium. Cultures were incubated under conditions optimized for keratinocyte growth (i.e., in 0.15 mM extracellular Ca2+) or for fibroblast growth (i.e., in 1.4 mM extracellular Ca2+). Treatment with all-trans retinoic acid (RA) induced histological changes in the organ-cultured skin under both conditions which were similar to the changes seen in intact skin after topical application. These included expansion of the viable portion of the epidermis and activation of cells in the dermis. In sun-damaged skin samples, which were characterized by destruction of normal connective tissue elements and presence of thick, dark-staining elastotic fibers, a zone of healthy connective tissue could be seen immediately below the dermo-epidermal junction. This zone was more prominent in RA-treated organ cultures than in matched controls. Associated with these histological changes was an increase in overall protein and extracellular matrix synthesis. In concomitant studies, it was found that RA treatment enhanced survival and proliferation of adult keratinocytes and adult dermal fibroblasts under both low- and high-Ca2+ conditions. In all of these assays, responses of sun-protected and sun-exposed skin were identical. In contrast, responses of neonatal foreskin to RA were similar to those of adult skin in the presence of low-Ca2+ culture medium, but under conditions of high extracellular Ca2+ RA provided little or no additional stimulus. Together these studies suggest that the ability of RA to enhance repair of sun-damaged skin (documented in previous studies) may reflect its ability to influence the behavior of skin in a manner that is age dependent but independent of sun-exposure status.

Adult↗
PubMed1994

Expression of serine proteinases and metalloproteinases in organ-cultured human skin. Altered levels in the presence of retinoic acid and possible relationship to retinoid-induced loss of epidermal cohesion.

Neonatal human foreskin obtained at circumcision was cut into 2 x 2-mm pieces and placed in organ culture. Culture medium consisted of a serum-free, growth factor-free basal medium containing either 0.15 mmol/L Ca2+ or 1.4 mmol/L Ca2+. Some cultures were left as control, whereas others were treated with 3 mumol/L all-trans retinoic acid (RA). In the presence of RA, epidermal cohesion was disrupted and the upper layers separated from the viable epidermis beneath. This effect was observed under both low Ca2+ and high Ca2+ conditions. At 2-day intervals, culture fluids were collected and analyzed for serine and metalloproteinase activities. Serine proteinase activity was detected in the culture fluids and virtually all of the detected activity was dependent on the presence of plasminogen. Activity was elevated in the RA-treated tissues and this was due to increased amounts of both urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA). Elastase and cathepsin G were not detected in either control or RA-treated cultures. Increased plasminogen activator levels were also detected in RA-treated keratinocytes and fibroblasts in monolayer culture. Significant amounts of t-PA (though not u-PA) were found in fibroblast culture fluids, whereas both t-PA and u-PA were detected in culture fluids from keratinocytes. Metalloproteinase activity was also detected in the culture fluids of control and RA-treated tissues but in contrast to plasminogen activator, metalloproteinase activity decreased in the presence of RA. Casein and gelatin zymographic studies indicated the presence of both 92- and 72-kd gelatinases and stromelysin-1 and suggested that the decreased activity was primarily due to reduction in the 92- and 72-kd gelatinases. When serine proteinase inhibitors (aprotinin and soybean trypsin inhibitor) were included in the culture medium throughout the incubation period, epidermal discohesion was reduced. A metalloproteinase inhibitor, tissue inhibitor of metalloproteinase-2, did not have this effect. Taken together, these data show that a number of proteolytic enzymes are produced during organ culture of human skin. They suggest that these proteases may influence the structural integrity of the tissue.

Cells, Cultured↗
PubMed1993

Use of recombinant and synthetic peptides as attachment factors for cells on microcarriers.

Polystyrene culture dishes and polystyrene microcarriers were coated with Pronectin-F and poly-L-lysine (polylysine), either alone or in combination. Pronectin-F is a recombinant peptide containing repeats of the RGD cell-attachment sequence from fibronectin. Polylysine is a polymer of L-lysine. Pronectin-F supported attachment of Madin-Darby Canine Kidney (MDCK) cells at concentrations as low as 0.025 micrograms/cm2 of surface area. The cells rapidly spread after attachment. Polylysine at concentrations of 0.05-0.5 micrograms/cm2 also supported cell attachment but cells did not rapidly spread after attachment to this substrate. Higher concentrations of polylysine could not be used because of toxicity. When the two peptides were used in conjunction, MDCK cells attached and spread at lower peptide concentrations than they did when either substrate was used alone. These findings suggest that recombinant Pronectin-F alone or in conjunction with a cationic polymer could be a useful replacement for materials such as gelatin or collagen which are currently used as microcarrier surfaces.

Amino Acid Sequence↗
PubMed1993

Retinoid toxicity for fibroblasts and epithelial cells is separable from growth promoting activity.

Three different retinoids with widely varying capacity to stimulate skin repair in vivo and stimulate fibroblast and epithelial cell growth in vitro were examined for capacity to lyse the same cells at high concentrations. These included all-trans retinoic acid (RA), tetrahydro tetramethyl napthalenyl benzoic acid (TTNPB), and its biologically inactive structural analogue, meta-carboxy TTNPB. Despite their differing capacities to stimulate skin repair and cell growth, all of the agents were cytotoxic for fibroblasts and epithelial cells over the same range of concentrations (0.6-3 x 10(-5) M). Cytotoxicity for both fibroblasts and epithelial cells was blocked by addition of phosphatidylcholine to the cells along with the retinoid. In the presence of high concentrations of RA (0.75-3 x 10(-5) M) and phosphatidylcholine, proliferation was observed. The proliferative response was greater under these conditions than in the presence of an optimal concentration of RA (0.75-3 x 10(-6) M) without phosphatidylcholine. These data suggest that toxicity of retinoids can be separated, at least partially, from their growth-promoting activities.

Cell Division↗
PubMed1993

Effects of sodium lauryl sulfate on human skin in organ culture: comparison with all-trans-retinoic acid and epidermal growth factor.

Human skin organ cultures were established from 2-mm punch biopsies and incubated under serum-free conditions in basal medium containing either 0.15 or 1.4 mM extracellular Ca2+. Organ cultures were treated with concentrations of sodium lauryl sulfate (SLS) that had previously been shown to support growth of human epidermal keratinocytes and human dermal fibroblasts in monolayer culture. Epidermal growth factor (EGF), alone and in combination with insulin and bovine pituitary extract, fetal bovine serum and all-trans retinoic acid (RA) were also examined for comparative purposes. The addition of SLS to culture medium containing low extracellular Ca2+ had no effect on the viability or histological appearance of the organ-cultured skin. Complete degeneration of the tissue occurred in the presence of SLS just as it did under control conditions. When SLS was added to culture medium containing high extracellular Ca2+, the basal layer of keratinocytes was much thinner than under control conditions. When EGF or EGF in combination with insulin and pituitary extract were utilized in place of SLS, identical results were obtained. That is, there was no preservation of the basal epithelial layer in the presence of low-Ca2+ culture medium and in the presence of high-Ca2+ culture medium, the basal layer was thinner than in control tissue. Virtually identical results were also obtained in medium containing 10% fetal bovine serum. In contrast, when RA was included in low-Ca2+ culture medium, the basal epithelium was maintained in a viable, histologically healthy condition. However, normal epithelial differentiation did not occur and the upper layers of the epidermis separated from the basal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion↗
PubMed1993

Effects of all-trans-retinoic acid on melanocyte adhesion and motility.

Human epidermal melanocytes were treated with all-trans-retinoic acid (RA) and examined for adhesion to bovine serum albumin-, fibronectin- and laminin-coated culture dishes. Control and treated cells were also examined for motility into micropore filters coated with the same proteins. Treatment of the cells with 3 x 10(-6) M RA for 3-4 days resulted in inhibition of attachment to all three substrates. Decreased attachment was observed within 1.5 h. Inhibition of attachment was not due to toxicity because differences between control and treated cells disappeared by 18 h, when most of the cells (approximately 75%) were attached and spread on all three substrates. The same treatment that inhibited adhesion also reduced migration into the interstices of micropore filters coated with the same three proteins. In additional experiments, human and mouse melanoma cell lines were examined in place of normal melanocytes. RA treatment also blocked adhesion and motility of these cells. The malignant melanoma cells were less sensitive to RA than normal melanocytes in the adhesion assay but were equally sensitive in the motility assay. The ability of RA to inhibit melanocyte adhesion and motility as well as melanocyte growth could explain, in part, the capacity of retinoids to modulate melanocyte function in hyperpigmented skin lesions.

Animals↗
PubMed1993

All-trans retinoic acid and extracellular Ca2+ differentially influence extracellular matrix production by human skin in organ culture.

Two-mm full-thickness punch biopsies of human skin were placed in organ culture in a serum-free, growth factor-free basal medium. Under conditions of low extracellular Ca2+ (0.15 mmol/L), the tissue quickly degenerated. However, degeneration was prevented when the extracellular Ca2+ concentration was increased to 1.4 mmol/L. The tissue remained histologically normal in appearance and biochemically active for up to 12 days. The addition of 3 mumol/L all-trans retinoic acid (RA) to the low-Ca2+ culture medium also prevented tissue degeneration. However, in contrast to what was seen in the presence of 1.4 mmol/L Ca2+, epidermal differentiation did not occur normally in the presence of RA. Rather, the upper layers of the epidermis routinely separated from the underlying basal cells. Fibronectin production by the organ cultured skin was examined. Biosynthetic labeling/immunoprecipitation studies demonstrated that incubation of the tissue in basal medium containing 1.4 mmol/L Ca2+ resulted in a high level of fibronectin production relative to the amount produced in basal medium containing 0.15 mmol/L Ca2+. In contrast, the addition of 3 mumol/L RA to the low Ca2+ basal medium did not stimulate fibronectin production. Similar results were observed in enzyme-linked immunosorbent assays where the addition of Ca2+ to a final concentration of 1.4 mmol/L stimulated fibronectin and thrombospondin production whereas RA (3 mumol/L) did not. Although RA by itself failed to stimulate extracellular matrix production, the addition of 3 mumol/L RA to basal medium containing 1.4 mmol/L Ca2+ led to a further increase in fibronectin production over that seen in the presence of 1.4 mmol/L Ca2+ alone. Taken together, these data indicate that although either 1.4 mmol/L Ca2+ or 3 mumol/L RA facilitates survival of organ-cultured skin in basal medium, they have very different effects on extracellular matrix production. This supports the view, based on histological appearance, that the two treatments work through different mechanisms. The data further support the suggestion that the two treatments may have additive or even synergistic effects.

Calcium↗
PubMed1992

Modulation of growth in normal and malignant melanocytic cells by all-trans retinoic acid.

Human epidermal melanocytes were examined for proliferation under various conditions in the presence or absence of all-trans retinoic acid (RA). Under conditions which supported proliferation, RA at concentrations of 0.25-1.0 microgram/ml inhibited cell growth but was not cytotoxic. When melanocytes were cultured under conditions which by themselves did not support growth, RA did not overcome the growth limitation. Treatment of melanocytes with RA altered their morphological appearance. Alterations included retraction of dendritic processes, increased flattening, and a slight darkening of the cytoplasm in some of the cells. However, when examined biochemically, there was no significant change in the amount of malanin per cell or in tyrosinase activity. RA also inhibited proliferation of six different malignant melanoma lines. Inhibition was observed over the same RA concentrations and over the same time course in the melanoma cells as was seen in melanocytes. Inhibition of melanocyte and melanoma cell proliferation was slowly reversed following removal of RA from the culture medium. These results indicate that RA can inhibit proliferation of melanocytic cells.

Animals↗
PubMed1992

Modulation of Ca2+ levels in keratinocytes by all-trans retinoic acid.

Human epidermal keratinocytes, that have been growth-arrested by removal of epidermal growth factor from the culture medium, are stimulated to proliferate by all-trans retinoic acid (RA). The same treatment inhibits the onset of differentiated features and reduces cell-substrate adhesion. In the present study we show that the same treatment results in a decrease in total cell-associated Ca2+ as measured by changes in the amount of 45Ca2+ bound to cells at equilibrium following RA treatment and by a decrease in intracellular free Ca2+ levels as measured with the Ca(2+)-sensitive dye, Indo-1. The alterations in Ca2+ levels were evident within an hour after RA treatment, were in the range of 30-35% and occurred over the same RA concentration range that stimulated proliferation (i.e., 0.25-1.0 micrograms/ml). When the extracellular Ca2+ concentration was elevated from the normal level of 0.15-1.4 mM, intracellular free Ca2+ increased by a factor of 2 while total cell-associated Ca2+ increased approximately 6-fold. Even under conditions of high extracellular Ca2+, RA was able to reduce cell-associated and intracellular free Ca2+. These data indicate that RA has the capacity to lower Ca2+ levels in keratinocytes concomitantly with its effects on biological behavior.

Calcium↗
PubMed1991

Production of fibronectin by human tumor cells and interaction with exogenous fibronectin: comparison of cell lines obtained from colon adenocarcinomas and squamous carcinomas of the upper aerodigestive tract.

Cell lines derived from 13 different human colon adenocarcinomas were examined for production of fibronectin by ELISA and for cell-surface expression of fibronectin by indirect immunofluorescence. Two squamous epithelial cell lines obtained from tumors of the upper aerodigestive tract were used as controls. None of the 13 colon carcinoma lines produced detectable amounts of fibronectin or showed detectable cell-surface staining with anti-fibronectin. The 2 squamous epithelial cell lines, in contrast, produced large amounts of fibronectin which could be detected in the culture medium and bound to the substratum. The squamous carcinoma cells also stained brightly when examined in the viable state by immunofluorescence with anti-fibronectin. In addition to being studied for fibronectin production, each cell line was also examined for the ability to interact with exogenous fibronectin in an adhesion assay. None of the colon carcinoma cells were adherent to fibronectin-coated culture dishes while the 2 squamous carcinoma cells rapidly attached and spread on this substratum. These data suggest that cell lines derived from adenocarcinomas of the colon are deficient in production of fibronectin and in their ability to interact with exogenous fibronectin. In their degree of deficiency, the colon carcinoma cells are significantly different from several different types of human tumor cell. The failure of the colon carcinoma cells to synthesize detectable amounts of fibronectin endogenously or to interact with exogenous fibronectin may explain, in part, the low degree of adhesive interaction which these cells have for their substratum. This, in turn, may influence the in vitro and in vivo properties of colon carcinoma cells.

Adenocarcinoma↗
PubMed1991

Inhibition of epithelial cell adhesion by retinoic acid. Relationship to reduced extracellular matrix production and alterations in Ca2+ levels.

Human squamous epithelial cells maintained in growth factor-deficient medium were examined for sensitivity to all-trans retinoic acid (retinoic acid). Under conditions of low external Ca2+ (0.15 mmol/l [millimolar]), or high external Ca2+ (1.4 mmol/l), retinoic acid stimulated proliferation. Concomitantly, cell-substrate adhesion was decreased. Enzyme-linked immunosorbent assays were used to assess production of two extracellular matrix components, ie, fibronectin and thrombospondin. In the presence of retinoic acid, production of both was decreased. Because both fibronectin and thrombospondin serve as epithelial cell adhesion factors, the decreased production of these moieties could contribute to reduced adhesion. Using 45Ca2+ to measure total cell-associated Ca2+ and the Ca2(+)-sensitive dye Indo-1 to measure intracellular free Ca2+, it was found that concentrations of retinoic acid that altered cell-substrate adhesion in the squamous epithelial cells had no effect on total, cell-associated Ca2+, but reduced intracellular free Ca2+ by 50% to 60%. Because Ca2+ is a regulator of adhesion, the ability of retinoic acid to modulate Ca2+ levels in the squamous epithelial cells may explain, in part, how retinoic acid influences their adhesiveness.

Calcium↗
PubMed1989

Modulation of fibronectin production in normal human melanocytes and malignant melanoma cells by interferon-gamma and tumor necrosis factor-alpha.

Normal cultured human epidermal melanocytes and melanoma cells derived from three different malignant melanomas were examined for synthesis of extracellular matrix components before and after treatment for one day with interferon-gamma, tumor necrosis factor-alpha, or both. Treatment of the cells with either cytokine individually had minimal effects on fibronectin levels. Treatment of the cells with the two agents in combination greatly stimulated fibronectin production as indicated by biosynthetic labeling and immunoprecipitation and by enzyme-linked immunosorbent assay. Synthesis of laminin was decreased slightly by the same treatment whereas thrombospondin production was stimulated slightly. The same treatment reduced melanocyte viability slightly but significantly inhibited proliferation and altered the morphology of the melanocytes. The treated cells became flattened and polygonal in shape while the untreated cells exhibited a bipolar shape with one or more long dendritic processes. These morphologic changes were not seen in cultures treated with interferon-gamma or tumor necrosis factor-alpha individually. The effects of interferon-gamma and tumor necrosis factor-alpha on fibronectin production by epidermal melanocytes are in contrast to the effects of the same treatments on fibronectin production by epidermal keratinocytes where fibronectin production is decreased but are similar to the effects of transforming growth factor-beta on a number of other cell types in which increased synthesis of fibronectin occurs and is associated with decreased growth.

Cells, Cultured↗