Biomedical subjects

J Varani

Publications and source records attributed to J Varani.

At least 37 records · Page 2Linked to original sources

PubMed1999

Endothelial cell determinants of susceptibility to neutrophil-mediated killing.

Vascular endothelial cell injury plays an important role in the pathogenesis of inflammatory-mediated tissue injury. In the current study, we assessed injury in primary cultures of endothelial cells obtained from different sites within the same species, comparing rat dermal microvascular and rat lung microvascular endothelial cells. Dermal microvascular-derived endothelial cells were more sensitive to killing by PMA (phorbol myristate acetate)-activated human neutrophils than were endothelial cells derived from lung microvasculature. Lung endothelial cells stimulated with interferon-gamma plus lipopolysaccharide (IFNgamma + LPS) generated high levels of nitric oxide (*NO), while dermal endothelial cells stimulated with IFNgamma + LPS generated significantly lower levels of *NO. Under conditions of *NO generation (IFNgamma + LPS stimulation), or in the presence of the *NO donor, S-nitroso-N-acetyl penicillamine (SNAP), endothelial cell killing by PMA-activated neutrophils was reduced. Lung endothelial cells stimulated with PMA generated less superoxide (02*-) than dermal endothelial cells. Under conditions of *NO generation (IFNgamma + LPS stimulation), or in the presence of SNAP, O2*- release from endothelial cells was reduced. Endothelial cell-derived *NO appeared to play a significant role in attenuating the neutrophil-mediated killing. The differences in the ability of endothelial cells to generate *NO and 02*- underlies, at least in part, the differences in susceptibility of these cells to injury by activated neutrophils.

Animals↗

PubMed1999

Can we learn from the pathogenetic strategies of group A hemolytic streptococci how tissues are injured and organs fail in post-infectious and inflammatory sequelae?

The purpose of this review-hypothesis is to discuss the literature which had proposed the concept that the mechanisms by which infectious and inflammatory processes induce cell and tissue injury, in vivo, might paradoxically involve a deleterious synergistic 'cross-talk', among microbial- and host-derived pro-inflammatory agonists. This argument is based on studies of the mechanisms of tissue damage caused by catalase-negative group A hemolytic streptococci and also on a large body of evidence describing synergistic interactions among a multiplicity of agonists leading to cell and tissue damage in inflammatory and infectious processes. A very rapid cell damage (necrosis), accompanied by the release of large amounts of arachidonic acid and metabolites, could be induced when subtoxic amounts of oxidants (superoxide, oxidants generated by xanthine-xanthine oxidase, HOCl, NO), synergized with subtoxic amounts of a large series of membrane-perforating agents (streptococcal and other bacterial-derived hemolysins, phospholipases A2 and C, lysophosphatides, cationic proteins, fatty acids, xenobiotics, the attack complex of complement and certain cytokines). Subtoxic amounts of proteinases (elastase, cathepsin G, plasmin, trypsin) very dramatically further enhanced cell damage induced by combinations between oxidants and the membrane perforators. Thus, irrespective of the source of agonists, whether derived from microorganisms or from the hosts, a triad comprised of an oxidant, a membrane perforator, and a proteinase constitutes a potent cytolytic cocktail the activity of which may be further enhanced by certain cytokines. The role played by non-biodegradable microbial cell wall components (lipopolysaccharide, lipoteichoic acid, peptidoglycan) released following polycation- and antibiotic-induced bacteriolysis in the activation of macrophages to release oxidants, cytolytic cytokines and NO is also discussed in relation to the pathophysiology of granulomatous inflammation and sepsis. The recent failures to prevent septic shock by the administration of only single antagonists is disconcerting. It suggests, however, that since tissue damage in post-infectious syndromes is caused by synergistic interactions among a multiplicity of agents, only cocktails of appropriate antagonists, if administered at the early phase of infection and to patients at high risk, might prevent the development of post-infectious syndromes.

Animals↗

PubMed1999

Role of matrix metalloproteinases in models of macrophage-dependent acute lung injury. Evidence for alveolar macrophage as source of proteinases.

Matrix metalloproteinases (MMPs) have been implicated in the tissue injury seen in neutrophil-dependent models of acute lung injury. However, the role of MMPs in macrophage-dependent models of lung injury is unknown. To address this issue, the macrophage-dependent immunoglobulin A immune complex-induced lung injury model and the macrophage-dependent portion of the lipopolysaccharide-induced acute lung injury model in the rat were assessed for MMP involvement and for the source of these activities. In both models, injury was inhibited by the recombinant human tissue inhibitor of metalloproteinases-2. Bronchoalveolar lavage fluids (BALFs) from injured animals in both models showed increased levels of MMPs. Characterization of MMP production by isolated lung fibroblasts, endothelial cells, type II epithelial cells, and alveolar macrophages revealed that only the macrophage had the same spectrum of MMP activity as seen in the BALF. Further, isolated alveolar macrophages from injured lungs showed evidence of in vivo activation with the release of the same spectrum of MMP activities. Together these studies show that MMPs are produced during macrophage-dependent lung injury, that these MMPs play a role in the development of the lung injury, and that the alveolar macrophage is the likely source of these MMPs.

Animals↗

PubMed1999

Characterization of matrix metalloproteinases produced by rat alveolar macrophages.

Evidence presented in the accompanying article (Gibbs, D. F., T. P. Shanley, R. L. Warner, H. S. Murphy, J. Varani, and K. J. Johnson. 1999. Role of matrix metalloproteinases in models of macrophage-dependent acute lung injury: evidence for alveolar macrophage as source of proteinases. Am. J. Respir. Cell Mol. Biol. 20:1145-1154) implicates alveolar macrophage matrix metalloproteinases (MMPs) in two models of acute lung inflammation in the rat. As a prerequisite to understanding which specific MMPs might be involved in the injury and how they might function, it was necessary to know the spectrum of enzymes present. To this end, alveolar macrophages were obtained from normal rat lungs by bronchoalveolar lavage, placed in culture with and without various agonists, and assessed by a variety of techniques for MMPs. The identification process involved characterization by gelatin, beta-casein, and kappa-elastin zymography, with confirmation of identity by Western blot/immunoprecipitation. Message levels of detected MMPs were assessed by Northern blot. Rat alveolar macrophages were found to produce a low constitutive level of MMP-2 (72-kD gelatinase A) that was only modestly upregulated following stimulation with phorbol myristate acetate, bacterial lipopolysaccharide, or immunoglobulin A-containing immune complexes. Although control cells were found to produce little or no MMP-9 (92-kD gelatinase B) or MMP-12 (metalloelastase), both enzymes were markedly upregulated upon stimulation. In the same stimulated macrophages there was little activity against type I collagen (associated with MMP-13 [collagenase-3] on the basis of Western blotting), no activity suggestive of stromelysin or matrilysin, and no measurable secretion of the serine proteinases, elastase and cathepsin G. These data demonstrate the ability of rat alveolar macrophages to elaborate certain MMPs under proinflammatory conditions, consistent with their possible involvement in the progression of acute inflammation.

Animals↗

PubMed1999

Characteristics of nonmalignant and malignant human prostate in organ culture.

Prostate tissue was obtained from 52 radical prostatectomies immediately upon surgery. From each specimen, a small piece of tissue was fixed in 10% buffered formalin and used for histology, cytokeratin staining, staining with the antibodies to the proliferation-associated antigen (Ki-67), and histochemical evaluation of the epithelial-stromal basement membrane. A second piece was used for the isolation of epithelial cells and stromal cells in monolayer culture. The remainder of each specimen was cut into cubes (approximately 1 mm on a side) and incubated in organ culture for up to 20 days. At the end of the incubation period, tissue was fixed in 10% buffered formalin and examined as described above with zero-time tissue. These studies showed that normal epithelial and stromal elements survived in organ culture in the presence of a serum-free medium containing a mixture of growth factors (epidermal growth factor, insulin, pituitary extract, and dihydrotestosterone). In many of the tissues examined at 4 days, individual glands resembled those seen immediately after surgery, with a single layer of basal epithelial cells and a layer of secretory cells above. By Day 8, the secretory epithelium was lost in many places and basal cells proliferated to fill in the lumens of the glands. All of the nonmalignant glands were reactive with the anti-cytokeratin antibody (K903), and there was a large increase in the number of cells staining for Ki-67 as compared with zero-time tissue. Staining with the Periodic Acid Schiff (PAS) and PAS-methenamine silver (PASME) reagents revealed an intact basement membrane around virtually all of the epithelial structures. The basement membrane appeared to be thickened in some areas. In places where a gland was cut during the processing of the tissue, epithelial cells migrated out of the gland and covered the cut surface of the tissue piece. There was no detectable basement membrane separating the epithelium from the stroma at these sites. Whereas nonmalignant epithelial cells were preserved in the growth factor- and dihydrotestosterone-supplemented culture medium, most of the malignant cells rapidly lysed under the same conditions. However, when phorbol myristate acetate was included in the culture medium, many of the tumor cells remained viable. This was seen with the more well-differentiated tumors as well as with tumors that were highly anaplastic. All of the tumor cells were nonreactive with anti-cytokeratin antibody, and only a few cells stained for Ki-67. The basement membrane surrounding malignant cells was thin and, in places, appeared to be discontinuous. Where malignant glands were cut in the processing of the tissue, cells did not migrate out over the cut surface. In summary, this study identifies culture conditions for the successful maintenance of human prostate tissue for several days in organ culture. Histological/histochemical features that distinguish nonmalignant and malignant tissue are present in this model.

Cell Division↗

PubMed1998

Heterogeneity of vascular endothelial cells: differences in susceptibility to neutrophil-mediated injury.

Vascular endothelial cells are key participants in the development of inflammatory-mediated tissue injury. Not all tissues have the same response to inflammation and the role of vascular endothelial cells in this response is unclear. The objective of this study was to compare cultured primary endothelial cells derived from a variety of microvascular and macrovascular sources within the same species, rat. We developed and compared primary cultures of endothelial cells from dermis, kidney and lung microvasculature, pulmonary artery and vein, renal artery and vein, and aorta. Endothelial cells were similar in their growth characteristics, ability to take up DiI-Ac-LDL, and expression of ICAM-1, regardless of their site of origin. In contrast, susceptibility to injury by PMA-activated neutrophils differed markedly among cells from different tissues as well as between microvascular and macrovascular endothelial cells from the same tissue. This study demonstrated that while endothelial cells maintain some fundamental characteristics regardless of their origin, clear site-specific differences are evident in the sensitivity of endothelial cells to injury mediated by activated neutrophils.

Animals↗

PubMed1998

Elaboration of matrix metalloproteinase inhibitors by human skin in organ culture and by skin cells in monolayer culture: relationship to invasion.

Functional and immunochemical approaches were used to assess matrix metalloproteinase (MMP) inhibitors, e.g., tissue inhibitor of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2), in organ cultures of normal human skin maintained under growth factor free conditions or in medium supplemented with a combination of growth factors including epidermal growth factor, insulin, and pituitary extract. It has previously been shown that under growth factor free conditions, normal skin structure and function are maintained for several days, while in the presence of these exogenous growth factors, the epithelial cells invade the stroma [Invasion and Metastasis 1993;13:225-233]. TIMP-1 was detected in equivalent amounts in organ culture fluids under both conditions. TIMP-2 was not detected under either condition. Normal epidermal keratinocytes, normal dermal fibroblasts, and three different epithelial tumor cell lines were also examined for MMP inhibitor expression. Keratinocytes and fibroblasts produced high levels of both TIMP-1 and TIMP-2, but in neither cell type was there a significant difference between growth factor free and growth factor containing conditions. In contrast, the three epithelial tumor cell lines produced low to undetectable levels of both TIMP-1 and TIMP-2. These data suggest that acquisition of local invasive capacity is not dependent on a reduction in MMP inhibitor expression. A reduction in MMP inhibitors may accompany the transition from invasive to metastatic tumors.

Blotting, Western↗

PubMed1998

Human psoriatic skin in organ culture: comparison with normal skin exposed to exogenous growth factors and effects of an antibody to the EGF receptor.

Organ cultures were established from psoriatic lesional skin of 24 different individuals and maintained for 8 days under serum-free, growth-factor-free conditions. Nonlesional skin from 14 of the same individuals and normal skin from another 12 individuals were also maintained in organ culture. At the end of the incubation period, the tissues were fixed in formalin and examined histologically. Lesional skin continued to express features of psoriatic plaque, which included irregularly shaped epithelial cells arranged in a disorganized fashion, and elongation of the rete ridges with a thickening in their lower portion. Abnormal epidermal differentiation and separation of the upper epidermal layers from the lower layers was also a consistent feature. In contrast, nonlesional skin from psoriatic patients exhibited a histological appearance which resembled that of site-matched normal skin. When normal skin was exposed to a growth-factor-enriched culture medium during the 8-day incubation period, it exhibited a histological appearance similar to that of psoriatic skin. In addition to abnormal histological features, the psoriatic skin in organ culture released higher amounts of matrix metalloproteinase-9 (MMP-9; 92-kD gelatinase B/type IV collagenase) into the culture fluid than either nonlesional skin or normal skin. Organ cultures of psoriatic lesional skin from 6 individuals were maintained for 8 days in the presence of an antibody to the human epidermal growth-factor (EGF) receptor. The abnormal histological features of the psoriatic tissue were partially ameliorated in the presence of the antibody. These data suggest that growth factors which act through the EGF receptor help to maintain the psoriatic phenotype in organ culture. They also suggest that organ culture may provide a useful tool with which to elucidate the pathophysiological mechanisms of altered keratinocyte proliferation and differentiation in psoriasis.

Antibodies, Monoclonal↗

PubMed1998

Preservation of human skin structure and function in organ culture.

Human keratinocytes can be maintained in monolayer culture under serum-free conditions for an extended period of time. Under low Ca2+ conditions (e.g., 0.05-0.15 mM), an undifferentiated state is maintained and the cells proliferate optimally. When the Ca2+ concentration is raised to approximately 1.0 mM, differentiation occurs and growth shows. Human dermal fibroblasts can also be maintained in monolayer culture under serum-free conditions, but in contrast to keratinocytes, a physiological level of extracellular Ca2+ (above approximately 1.0 mM) is required. A variety of growth factors stimulate proliferation of both cell types but do not replace the Ca2+ requirement of the fibroblast population. All-trans retinoic acid also promotes proliferation of both cell types and, most interestingly, replaces the requirement-for a physiological level of Ca2+ in the fibroblast cultures. Human skin can be maintained in organ culture for an extended period of time under serum-free conditions. Conditions optimized for fibroblast proliferation (either physiological Ca2+ or all-trans retinoic acid) are required. In the presence of culture conditions optimized for the epithelial cell component, both the epidermis and dermis rapidly lyse. These data suggest that the fibroblast is the critical component in maintaining homeostasis of skin, and that maintenance of the epidermis as well as the dermis depends on the viability and functioning of these cells.

Aging↗

PubMed1998

Molecular mechanisms of intrinsic skin aging and retinoid-induced repair and reversal.

Past studies have shown that topical treatment of sun-exposed skin with all-trans retinoic acid improves the clinical and histologic appearance of the skin. This is associated with a reduction in matrix metalloproteinase elaboration and with expression of a newly synthesized collagenous matrix. Whether retinoid therapy might have a similar impact on the appearance of intrinsically aged skin is not known. This study, using human skin in organ culture and epidermal keratinocytes and fibroblasts in monolayer culture, show that retinoic acid stimulates growth of both keratinocytes and fibroblasts and stimulates extracellular matrix production by the fibroblasts. Adult skin from sun-exposed and sun-protected sites responds equally well to retinoic acid, whereas neonatal skin is much less responsive under the same conditions. The implications of this are (i) that retinoids may be able to repair intrinsically aged skin as well as photoaged skin, and (ii) that retinoids modulate human skin cell function in a manner that is age-related, and not simply a response to photodamage.

Adult↗

PubMed1997

Pathophysiology of premature skin aging induced by ultraviolet light.

BACKGROUND: Long-term exposure to ultraviolet irradiation from sunlight causes premature skin aging (photoaging), characterized in part by wrinkles, altered pigmentation, and loss of skin tone. Photoaged skin displays prominent alterations in the collagenous extracellular matrix of connective tissue. We investigated the role of matrix-degrading metalloproteinases, a family of proteolytic enzymes, as mediators of collagen damage in photoaging. METHODS: We studied 59 whites (33 men and 26 women, ranging in age from 21 to 58 years) with light-to-moderate skin pigmentation, none of whom had current or prior skin disease. Only some of the participants were included in each of the studies. We irradiated their buttock skin with fluorescent ultraviolet lights under standard conditions and obtained skin samples from irradiated and nonirradiated areas by keratome or punch biopsy. In some studies, tretinoin and its vehicle were applied to skin under occlusion 48 hours before ultraviolet irradiation. The expression of matrix metalloproteinases was determined by in situ hybridization, immunohistology, and in situ zymography. Irradiation-induced degradation of skin collagen was measured by radioimmunoassay of soluble cross-linked telopeptides. The protein level of tissue inhibitor of matrix metalloproteinases type 1 was determined by Western blot analysis. RESULTS: A single exposure to ultraviolet irradiation increased the expression of three matrix metalloproteinases -- collagenase, a 92-kd gelatinase, and stromelysin -- in skin connective tissue and outer skin layers, as compared with nonirradiated skin. The degradation of endogenous type I collagen fibrils was increased by 58 percent in irradiated skin, as compared with nonirradiated skin. Collagenase and gelatinase activity remained maximally elevated (4.4 and 2.3 times, respectively) for seven days with four exposures to ultraviolet irradiation, delivered at two-day intervals, as compared with base-line levels. Pretreatment of skin with tretinoin (all-trans-retinoic acid) inhibited the induction of matrix metalloproteinase proteins and activity (by 70 to 80 percent) in both connective tissue and outer layers of irradiated skin. Ultraviolet irradiation also induced tissue inhibitor of matrix metalloproteinases-1, which regulates the enzyme. Induction of the inhibitor was not affected by tretinoin. CONCLUSIONS: Multiple exposures to ultraviolet irradiation lead to sustained elevations of matrix metalloproteinases that degrade skin collagen and may contribute to photoaging. Treatment with topical tretinoin inhibits irradiation-induced matrix metalloproteinases but not their endogenous inhibitor.

Administration, Cutaneous↗

PubMed1997

Human squamous carcinoma cell invasion in organ-cultured skin.

In a previous study we showed that normal human epidermal keratinocytes were capable of invading the dermis of organ cultured skin when the tissue was treated with an exogenous source of epithelial growth factors (Fligiel and Varani (1993) Invasion Metastasis, 13, 225-233). Here we examined human squamous carcinoma cells from three different tumors for ability to invade the dermis in the same model. Invasion occurred in 40-80% of the tissues, depending on the tumor line, and was observed with equal frequency under both growth factor-free conditions and in the presence of exogenous growth factors. These data demonstrate that malignant human epithelial cells have the capacity to invade the dermis of organ-cultured skin. Unlike normal keratinocytes, there appears to be no exogenous growth factor requirement for invasion by the malignant cells.

Carcinoma, Squamous Cell↗

PubMed1997

Diethyldithiocarbamate and nitric oxide synergize with oxidants and with membrane-damaging agents to injure mammalian cells.

The effect of diethyldithiocarbamate (DDC) and sodium nitroprusside (SNP) on the killing of endothelial cells and on the release of arachidonate by mixtures of oxidants and membrane-damaging agents was studied in a tissue culture model employing bovine aortic endothelial cells labeled either with 51Chromium or 3arachidonic acid. While exposure to low, subtoxic concentrations of oxidants (reagent H2O2, glucose-oxidase generated peroxide, xanthine xanthine oxidase, AAPH-generated peroxyl radical, menadione-generated oxidants) did not result either in cell death or in the loss of membrane-associated arachidonic acid, the addition of subtoxic amounts of a variety of membrane-damaging agents (streptolysin S, PLA2, histone, taurocholate, wheatgerm agglutinin) resulted in a synergistic cell death. However, no significant amounts of arachidonate were released unless proteinases were also present. The addition to these reaction mixtures of subtoxic amounts of DDC (an SOD inhibitor and a copper chelator) not only very markedly enhanced cell death but also resulted in the release of large amounts of arachidonate (in the complete absence of added proteinases). Furthermore, the inclusion in DDC-containing reaction mixtures of subtoxic amounts of SNP, a generator of NO, further enhanced, in a synergistic manner, both cell killing and the release of arachidonate. Cell killing and the release of arachidonate induced by the DDC and SNP-containing mixtures of agonists were strongly inhibited by catalase, glutathione, N-acetyl cysteine, vitamin A, and by a nonpenetrating PLA2 inhibitor as well as by tetracyclines. A partial inhibition of cell killing was also obtained by 1,10-phenanthroline and by antimycin. It is suggested that DDC might amplify cell damage by forming intracellular, loosely-bound complexes with copper and probably also by depleting antioxidant thiols. It is also suggested that "cocktails" containing oxidants, membrane-damaging agents, DDC, and SNP might be beneficial for killing of tumor cells in vivo and for the assessment of the toxicity of xenobiotics in vitro.

Adolescent↗

PubMed1996

Molecular basis of sun-induced premature skin ageing and retinoid antagonism.

Damage to skin collagen and elastin (extracellular matrix) is the hallmark of long-term exposure to solar ultraviolet irradiation, and is believed to be responsible for the wrinkled appearance of sun-exposed skin. We report here that matrix-degrading metalloproteinase messenger RNAs, proteins and activities are induced in human skin in vivo within hours of exposure to ultraviolet-B irradiation (UVB). Induction of metalloproteinase proteins and activities occurred at UVB doses well below those that cause skin reddening. Within minutes, low-dose UVB upregulated the transcription factors AP-1 and NF-kappa B, which are known to be stimulators of metalloproteinase genes. All-trans retinoic acid, which transrepresses AP-1 (ref. 8), applied before irradiation with UVB, substantially reduced AP-1 and metalloproteinase induction. We propose that elevated metalloproteinases, resulting from activation of AP-1 and NF-kappa B by low-dose solar irradiation, degrade collagen and elastin in skin. Such damage, if imperfectly repaired, would result in solar scars, which through accumulation from a lifetime of repeated low-dose sunlight exposure could cause premature skin ageing (photoageing).

Adult↗

PubMed1996

Deferoxamine interferes with adhesive functions of activated human neutrophils.

Deferoxamine is a potent chelator of ferric iron. Past studies have shown that deferoxamine interferes with acute inflammatory tissue injury in a number of animal models. In cell culture, it inhibits neutrophil-medicated killing of endothelial cells. Both the animal model and cell culture data are thought to reflect the capacity of deferoxamine to interfere with the superoxide anion- and and ferric iron-dependent reduction of hydrogen peroxide to the hydroxyl radical (Fenton Reaction). The present study describes a second mechanism by which deferoxamine may interfere with the acute inflammatory response. Here it is shown that deferoxamine has the capacity to inhibit neutrophil adhesion to lung epithelial cells and vascular endothelial cells. Adhesion of phorbol ester-stimulated neutrophils to both cell types is reduced by 70-80%. The inhibitory effects are reversible and are overcome when ferric iron is present along with deferoxamine in a 2:1 molar ratio. Concentrations of deferoxamine that prevent neutrophil adhesion also prevent neutrophil-mediated killing of the same target cells. In contrast, deferoxamine does not significantly inhibit activation-induced up-regulation of neutrophil surface adhesion structures (CD11b/CD18) and does not prevent binding of a monoclonal antibody that recognizes beta 2 integrins in the high-affinity state. Release of proteolytic enzymes from activated cells is also not significantly inhibited by deferoxamine. Taken together, these data indicate that deferoxamine modulates neutrophil adhesive functions associated with the activated state. The ability of deferoxamine to interfere with neutrophil binding to target cells may contribute to its anti-inflammatory activity.

Adult↗

PubMed1996

Perfluorocarbon protects lung epithelial cells from neutrophil-mediated injury in an in vitro model of liquid ventilation therapy.

Liquid ventilation with perfluorocarbon has been effective in improving gas exchange and pulmonary function in the setting of acute respiratory failure. How improvement is brought about remains unknown. In the present study, we examined perfluorocarbon for effects on neutrophil function and for effects on neutrophil-epithelial cell interactions that could underlie its in vivo activity. Exposure of neutrophils in vitro to perfluorocarbon followed by washing did not interfere with their ability to generate oxidants or release proteolytic enzymes upon subsequent stimulation. Likewise, such treatment did not interfere with subsequent adhesion of the neutrophils to monolayers of epithelial cells or with neutrophil-induced injury to these cells. In contrast, when perfluorocarbon was added to neutrophils and epithelial cells together and the neutrophils then stimulated, it reduced their adhesive interaction with the target cells and concomitantly reduced target cell injury. In companion studies, cells were obtained by bronchial lavage of perfluorocarbon-treated patients with acute respiratory distress syndrome (ARDS) and analyzed for oxidant production. Oxidant-generating capacity by the cells obtained from the bronchial lavage fluid was similar to that of peripheral blood neutrophils. These data are consistent with the suggestion that perfluorocarbon protects cells in vitro from neutrophil-mediated injury, at least in part, by acting as a mechanical barrier. These findings are consistent with a potential mechanism of action in vivo.

Bronchoalveolar Lavage Fluid↗

PubMed1996

All-trans retinoic acid reduces membrane fluidity of human dermal fibroblasts. Assessment by fluorescence redistribution after photobleaching.

All-trans retinoic acid (RA) preserves human dermal fibroblast viability and stimulates proliferation in vitro. These effects are mediated, at least in part, by reducing the extracellular Ca2+ requirement. The same concentrations of RA that reduce the extracellular Ca2+ requirement also interrupt movement of Ca 2+ across the fibroblast plasma membrane. Based on these observations, we have examined the effects of RA on membrane properties that could influence Ca2+ movement. Fibroblasts were labeled with 1-acyl-2-(N-4- nitrobenzo-2-oxa-1,3 diazole)-amino-caproyl phosphatidyl-choline (a fluorescent phospholipid analogue) and examined for fluorescence redistribution after photobleaching (FRAP) with a pulse of intense light as a measure of membrane fluidity. Using this approach, we observed that membrane fluidity was higher when the cells were incubated in medium containing a low (sub-optimal) level of extracellular Ca2+ (0.15 mmol/L) than in a medium containing an optimal concentration (1.4 mmol/L). Treatment of the cells with 3 micromol/L RA reduced membrane fluidity of the cells under both high- and low-Ca2+ conditions. These findings demonstrate that RA has a direct effect on the plasma membrane of human dermal fibroblasts. This provides a possible mechanism for the previously identified inhibition of Ca2+ movement across the membrane of the same cells and for the previously identified protective effects against lysis under low-Ca2+ conditions.

Calcium↗

PubMed1996

Growth factor-induced epidermal invasion of the dermis in human skin organ culture: dermal invasion correlated with epithelial cell motility.

We have developed a model of human squamous epithelial cell invasion in human skin organ culture. Epidermal invasion of the dermis occurs in this model when the tissue is exposed to an exogenous source of epithelial cell growth factor. In the present study we sought to determine to what extent growth factor-induced invasion correlates with the ability of the growth factor to induce epithelial cell motility. Histological examination of tissue treated with epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1) or keratinocyte growth factor (KGF) showed that only HGF and EGF were inducers of invasion while KGF- and IGF-1-treated tissues were histologically similar to untreated controls. In parallel studies, HGF and EGF were found to be potent stimulators of epidermal keratinocyte motility while IGF-1 was less effective and KGF was even less so. None of the growth factors stimulated dermal fibroblast motility while HGF and to a lesser extent IGF-1 (but not EGF or KGF) stimulated motility of dermal vascular endothelial cells. Thus, there is a strong correlation between growth factor capacity to induce epidermal keratinocytes to invade the underlying dermal tissue and to induce motility in the invasive population of cells.

Adult↗