Vascular tube formation on matrix metalloproteinase-1-damaged collagen.

Connective tissue damage and angiogenesis are both important features of tumour growth and invasion. Here, we show that endothelial cells maintained on a three-dimensional lattice of intact polymerised collagen formed a monolayer of cells with a cobblestone morphology. When the collagen was exposed to organ culture fluid from human basal cell tumours of the skin (containing a high level of active matrix metalloproteinase-1 (MMP-1)), degradation of the collagen matrix occurred. The major degradation products were the $3 over 4$- and $1 over 4$-sized fragments known to result from the action of MMP-1 on type I collagen. When endothelial cells were maintained on the partially degraded collagen, the cells organised into a network of vascular tubes. Pretreatment of the organ culture fluid with either tissue inhibitor of metalloproteinase-1 (TIMP-1) or neutralising antibody to MMP-1 prevented degradation of the collagen lattice and concomitantly inhibited endothelial cell organisation into the vascular network. Purified (activated) MMP-1 duplicated the effects of skin organ culture fluid, but other enzymes including MMP-9 (gelatinase B), elastase or trypsin failed to produce measurable fragments from intact collagen and also failed to promote vascular tube formation. Together, these studies suggest that damage to the collagenous matrix is itself an important inducer of new vessel formation.

Vitamins as hormones.

Vitamins A and D are the first group of substances that have been reported to exhibit properties of skin hormones, such as organized metabolism, activation, inactivation, and elimination in specialized cells of the tissue, exertion of biological activity, and release in the circulation. Vitamin A and its two important metabolites, retinaldehyde and retinoic acids, are fat-soluble unsaturated isoprenoids necessary for growth, differentiation and maintenance of epithelial tissues, and also for reproduction. In a reversible process, vitamin A is oxidized IN VIVO to give retinaldehyde, which is important for vision. The dramatic effects of vitamin A analogues on embryogenesis have been studied by animal experiments; the clinical malformation pattern in humans is known. Retinoic acids are major oxidative metabolites of vitamin A and can substitute for it in vitamin A-deficient animals in growth promotion and epithelial differentiation. Natural vitamin A metabolites are vitamins, because vitamin A is not synthesized in the body and must be derived from carotenoids in the diet. On the other hand, retinoids are also hormones - with intracrine activity - because retinol is transformed in the cells into molecules that bind to and activate specific nuclear receptors, exhibit their function, and are subsequently inactivated. The mechanisms of action of natural vitamin A metabolites on human skin are based on the time- and dose-dependent influence of morphogenesis, epithelial cell proliferation and differentiation, epithelial and mesenchymal synthetic performance, immune modulation, stimulation of angiogenesis and inhibition of carcinogenesis. As drugs, vitamin A and its natural metabolites have been approved for the topical and systemic treatment of mild to moderate and severe, recalcitrant acne, photoaging and biologic skin aging, acute promyelocytic leukaemia and Kaposi's sarcoma. On the other hand, the critical importance of the skin for the human body's vitamin D endocrine system is documented by the fact that the skin is both the site of vitamin D (3)- and 1,25-dihydroxyvitamin D (3) [1, 25(OH) (2)D (3)]-synthesis and a target organ for 1,25(OH) (2)D (3). 1,25(OH) (2)D (3) is not only essential for mineral homeostasis and bone integrity, but also for numerous further physiologic functions including regulation of growth and differentiation in a broad variety of normal and malignant tissues, including cells derived from prostate, breast and bone. In keratinocytes and other cell types, 1,25(OH) (2)D (3) regulates growth and differentiation. Consequently, vitamin D analogues have been introduced for the treatment of the hyperproliferative skin disease psoriasis. Other newly detected functions of vitamin D analogues include profound effects on the immune system as well as protection against cancer and other diseases, including autoimmune and infectious diseases, in various tissues. Current investigation of the biological effects of vitamin D analogues are likely to lead to new therapeutic applications that, besides cancer prevention, may include the prevention and treatment of infectious as well as of inflammatory skin diseases. This review summarizes existing knowledge on vitamins A and D, the major vitamin-hormones of the skin.

Upregulation of calcium-sensing receptor and mitogen-activated protein kinase signalling in the regulation of growth and differentiation in colon carcinoma.

In the present study, we demonstrate that Ca2+-induced growth inhibition and induction of differentiation in a line of human colon carcinoma cells (CBS) is dependent on mitogen-activated protein (MAP) kinase signaling and is associated with upregulation of extracellular calcium-sensing receptor (CaSR) expression. When CBS cells were grown in Ca2+-free medium and then switched to medium supplemented with 1.4 mM Ca2+, proliferation was reduced and morphologic features of differentiation were expressed. E-cadherin, which was minimally expressed in nonsupplemented medium, was rapidly induced in response to Ca2+ stimulation. Sustained activation of the extracellular signal-regulated kinase (ERK) occurred in Ca2+-supplemented medium. When an inhibitor of ERK activation (10 microM U0126) was included in the Ca2+-supplemented culture medium, ERK-activation did not occur. Concomitantly, E-cadherin was not induced, cell proliferation remained high and differentiation was not observed. The same level of Ca2+ supplementation that induced MAP kinase activation also stimulated CaSR upregulation in CBS cells. A clonal isolate of the CBS line that did not upregulate CaSR expression in response to extracellular Ca2+ was isolated from the parent cells. This isolate failed to produce E-cadherin or undergo growth inhibition/induction of differentiation when exposed to Ca2+ in the culture medium. However, ERK-activation occurred as efficiently in this isolate as in parent CBS cells or in a cloned isolate that underwent growth reduction and differentiation in response to Ca2+ stimulation. Together, these data indicate that CaSR upregulation and MAP kinase signalling are both intermediates in the control of colon carcinoma cell growth and differentiation. They appear to function, at least in part, independently of one another.

Antagonism of epidermal growth factor receptor tyrosine kinase ameliorates the psoriatic phenotype in organ-cultured skin.

Psoriatic plaque skin incubated for eight days in organ culture in the presence of a potent epidermal growth factor (EGF) receptor tyrosine kinase (RTK) antagonist reverted to a more normal histological appearance, while untreated psoriatic plaque skin retained histological features associated with the psoriatic phenotype. In concomitant studies it was shown that the EGF-RTK antagonist had no significant effect on histological features of non-psoriatic skin and no effect on dermal function, i.e. elaboration of both type I procollagen and matrix metalloproteinase-1 (MMP-1; interstitial collagenase). When human epidermal keratinocytes were treated with the EGF-RTK antagonist in monolayer culture, growth inhibition was seen (ED(50) = approximately 0.06 microM). When dermal fibroblasts were exposed to the EGF-RTK antagonist in monolayer culture, proliferation, MMP-1 and type I procollagen production were essentially unaffected at concentrations which interfered with keratinocyte growth (up to 1 microM). The capacity of the EGF-RTK antagonist to modulate the histological features of psoriatic skin in organ culture under conditions in which normal skin architecture and dermal function are largely unaffected suggests a potential for anti-psoriatic therapy.

Keratinocyte stimulation of matrix metalloproteinase-1 production and proliferation in fibroblasts: regulation through mitogen-activated protein kinase signalling events.

Incubation of human dermal fibroblasts in keratinocyte-conditioned culture medium led to a 5.7-fold increase in the level of matrix metalloproteinase-1. Virtually all of the matrix metalloproteinase-1 - inducing activity could be related to agonists acting through members of the epidermal growth factor receptor family or to agonists acting through the interleukin-1 receptor. The same keratinocyte-conditioned medium also induced a modest increase in fibroblast proliferation (approximately 1.8-fold). Growth-stimulating activity could be attributed to epidermal growth factor receptor (but not interleukin-1 receptor) function. In fibroblasts exposed to keratinocyte-conditioned medium, mitogen-activated protein kinase signalling through both the extracellular signal-related kinase pathway and p38 pathway occurred. When recombinant epidermal growth factor or recombinant interleukin-1beta were used as a control, they induced mitogen-activated protein kinase signalling consistent with the combined effects of epidermal growth factor receptor - specific and interleukin-1 receptor - specific agonists in keratinocyte-conditioned medium. Recombinant epidermal growth factor stimulated both matrix metalloproteinase-1 induction and proliferation while recombinant interleukin-1beta stimulated matrix metalloproteinase-1 elaboration but not fibroblast growth. An inhibitor of extracellular signal-related kinase pathway signalling (U0126) blocked induction of matrix metalloproteinase-1 production induced by keratinocyte-conditioned medium (as well as by epidermal growth factor or interleukin-1beta), and also inhibited proliferation. A p38 signalling inhibitor (SB203580) blocked matrix metalloproteinase-1 elaboration induced by keratinocyte-conditioned medium or interleukin-1beta, but did not inhibit matrix metalloproteinase-1 elaboration or cell growth induced by epidermal growth factor. These data indicate that keratinocyte-fibroblast interactions are mediated by multiple stimulating agents acting on specific receptors to induce signalling through different mitogen-activated protein kinase pathways leading to altered expression of key biological functions.

Induction of matrix metalloproteinase-1 (MMP-1) during epidermal invasion of the stroma in human skin organ culture: keratinocyte stimulation of fibroblast MMP-1 production.

Organ cultures of human skin were incubated for 8 days under growth factor-free conditions or exposed to 10 ng ml(-1) of human recombinant epidermal growth factor (EGF) during the incubation period. Normal histological features were preserved in the absence of growth factor, while epithelial cells underwent a proliferative response and invaded the underlying stroma in the presence of exogenous EGF. The same concentrations of EGF that induced stromal invasion also resulted in up-regulation of matrix metalloproteinase-9 (MMP-9; 92-kD gelatinase B) in organ culture and keratinocyte monolayer culture, and expression of MMP-1 (interstitial collagenase) in organ culture and fibroblast monolayer culture. When skin organ cultures were exposed to a potent, irreversible EGF-receptor tyrosine kinase (EGF-RTK) antagonist along with EGF, abnormal histological features were reversed, and MMP-9 production was suppressed. In contrast, EGF-RKT antagonism had only a modest inhibitory effect on MMP-1 production. Culture fluid from keratinocytes grown in monolayer culture stimulated fibroblast proliferation and MMP-1 elaboration. Treatment of fibroblasts with the same EGF-RTK antagonist inhibited keratinocyte-induced fibroblast proliferation but had only a modest inhibitory effect (approximately 20% inhibition) on MMP-1 production. In contrast, treatment of dermal fibroblasts with Interleukin-1 Receptor Antagonist had no effect on keratinocyte-induced fibroblast growth but strongly inhibited MMP-1 production (greater than 70% inhibition). These data indicate that stromal invasion by epithelial cells in EGF-treated skin is associated with events occurring in both the epidermis and dermis. The direct effect of the exogenous growth factor appears to be primarily on the epidermis. Dermal events reflect, at least in part, a response to factors elaborated in the epidermis.

Anti-CD11a ameliorates disease in the human psoriatic skin-SCID mouse transplant model: comparison of antibody to CD11a with Cyclosporin A and clobetasol propionate.

The present study assesses the applicability of human skin-SCID (severe combined immunodeficiency) mouse chimeras in testing antipsoriatic therapeutics. Three agents were examined: (1) a monoclonal antibody to the alpha subunit of leukocyte function associated antigen-1 integrin (CD11a); (2) Cyclosporin A; and (3) clobetasol propionate (Temovate), a potent topical corticosteroid used clinically in the treatment of psoriasis. Skin transplanted to SCID mice from normal human volunteers or from psoriatic lesional skin was allowed to heal for 3 to 5 weeks before application of test reagents. During this period, psoriatic skin, which was 3.8-fold thicker than the corresponding normal skin before transplantation, maintained its phenotype (ie, increased epidermal thickness, rete ridges with blunted ends, and intralesional presence of T lymphocytes). Transplanted normal human skin, however, underwent a hyperplastic response during this period, resulting in a 2.4-fold increase in epidermal thickness. After the healing period, animals transplanted with normal or psoriatic skin were treated for 14 days by daily intraperitoneal injection of either Cyclosporin A or a monoclonal antibody to human CD11a, or by topical application of clobetasol propionate. At the end of the treatment period, the mice were killed and the tissue evaluated morphometrically for changes in epidermal thickness and immunohistologically for the presence of T lymphocytes. Both Cyclosporin A and anti-CD11a reduced the epidermal thickness of transplanted psoriatic skin, whereas neither reagent significantly reduced the thickness of transplanted normal skin. T lymphocytes were detected in the skin from treated animals; there did not seem to be any reduction in the number of T lymphocytes. Clobetasol propionate reduced the epidermal thickness of both normal and psoriatic skin. These data indicate that, in this model, therapies directed against pathophysiologic mechanisms that contribute to psoriasis can be distinguished from treatments that block epidermal hyperplasia occurring as a consequence of xenografting. Our observations provide evidence for the activity of anti-CD11a in an animal model of human psoriasis.

The role of metalloelastase in immune complex-induced acute lung injury.

Matrix metalloproteases (MMPs) are a group of zinc-dependent endopeptidases that can degrade every component of the extracellular matrix. Under normal circumstances, the levels of MMPs are tightly regulated at both transcriptional and posttranscriptional levels. However, they are up-regulated in pathological states such as inflammation. Previous investigations have suggested that MMP-12 (metalloelastase) may be an important mediator in the pathogenesis of chronic lung injury. In this study we investigated the role of metalloelastase in the pathogenesis of acute lung injury using mice containing a targeted disruption of the metalloelastase gene. Neutrophil influx into the alveolar space in metalloelastase-deficient animals was reduced to approximately 50% of that observed in parent strain mice following the induction of injury by immune complexes. In addition, lung permeability in metalloelastase-deficient mice was approximately 50% of that of injured parent strain animals with normal levels of metalloelastase and this was correlated with histological evidence of less lung injury in the metalloelastase-deficient animals. Collectively, the data suggest that metalloelastase is necessary for the full development of acute alveolitis in this model of lung injury. Further, the data suggest that reduced injury in metalloelastase-deficient mice is due in part to decreased neutrophil influx into the alveolar space.

Role of stromelysin 1 and gelatinase B in experimental acute lung injury.

Matrix metalloproteinases (MMPs) are upregulated locally in sites of inflammation, including the lung. Several MMP activities are upregulated in acute lung injury models but the exact role that these MMPs play in the development of the lung injury is unclear due to the absence of specific inhibitors. To determine the involvement of individual MMPs in the development of lung injury, mice genetically deficient in gelatinase B (MMP-9) and stromelysin 1 (MMP-3) were acutely injured with immunoglobulin G immune complexes and the intensity of the lung injury was compared with genetically identical wild-type (WT) mice with normal MMP activities. In the WT mice there was upregulation of gelatinase B and stromelysin 1 in the injured lungs which, as expected, was absent in the genetically deficient gelatinase B- and stromelysin 1-deficient mice, respectively. In the deficient mice there was little in the way of compensatory upregulation of other MMPs. The gelatinase B- and the stromelysin 1-deficient mice had less severe lung injury than did the WT controls, suggesting that both MMPs are involved in the pathogenesis of the lung injury. Further, the mechanism of their involvement in the lung injury appears to be different, with the stromelysin 1-deficient mice having a reduction in the numbers of neutrophils recruited into the lung whereas the gelatinase B-deficient mice had the same numbers of lung neutrophils as did the injured WT controls. These studies indicate, first, that both gelatinase B and stromelysin 1 are involved in the development of experimental acute lung injury, and second, that the mechanisms by which these individual MMPs function appear to differ.

Matrix metalloproteinases (MMPs) in fresh human prostate tumour tissue and organ-cultured prostate tissue: levels of collagenolytic and gelatinolytic MMPs are low, variable and different in fresh tissue versus organ-cultured tissue.

Prostate tissue was obtained from 22 radical prostatectomies (performed for clinical management of prostate carcinoma) immediately after surgery. A small piece of tissue was fixed immediately in formalin and used for routine histology while a second piece was frozen in OCT and used for immuno-histochemistry. Another small piece was used for isolation of epithelial and stromal cells. The remainder of the tissue was cut into 2 x 2 mm pieces and incubated in organ culture for 8 days. In organ culture, non-malignant, basal epithelial cells underwent a proliferative response. This was accompanied by de-differentiation of glandular structures and by migration of epithelial cells across the surface of the tissue. Erosion of the basement membrane could also be seen in places, but was not widespread. Invasion of epithelial cells into the adjacent stroma was not evident. Production of matrix metalloproteinases (MMPs) with gelatinolytic activity or collagenolytic activity was assessed in organ culture and compared to expression patterns in fresh tissue. MMP-1 (interstitial collagenase) and MMP-9 (92-kDa gelatinase B) were undetectable or low in fresh tissue specimens. Both enzymes were detected in organ culture and both increased over time. Even after 6 days, however, there was only a low level of gelatin-hydrolytic activity and no measurable collagen-hydrolytic activity. In past studies we used organ cultures of normal skin and malignant skin tumours (basal cell carcinomas) to help elucidate the role of collagenolytic and gelatinolytic MMPs in epithelial cell invasion (Varani et al, 2000). Compared to MMP levels observed in skin, levels of these enzymes in prostate are low. The low level of collagenolytic and gelatinolytic MMPs in fresh prostate tissue and in organ-cultured prostate tissue may help explain why there is little tissue destruction in many primary prostate tumours and why the majority of such tumours remain confined to the prostate for extended periods.

Inhibition of type I procollagen synthesis by damaged collagen in photoaged skin and by collagenase-degraded collagen in vitro.

Type I and type III procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced procollagen synthesis. In the present study, we investigated type I procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I procollagen gene expression by in situ hybridization and for type I procollagen protein by immunostaining. Both mRNA and protein were reduced ( approximately 65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I procollagen production was because of inherently reduced capacity of skin fibroblasts in severely photodamaged forearm skin to synthesize procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I procollagen synthesis. For these studies, fibroblasts from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of fibroblasts were isolated from the two skin sites. Fibroblasts from the two sites had similar growth capacities and produced virtually identical amounts of type I procollagen protein. These findings indicate that the lack of type I procollagen synthesis in sun-damaged skin is not because of irreversible damage to fibroblast collagen-synthetic capacity. It follows, therefore, that factors within the severely photodamaged skin may act in some manner to inhibit procollagen production by cells that are inherently capable of doing so. Interactions between fibroblasts and the collagenous extracellular matrix regulate type I procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. Fibroblasts are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is approximately 3.6-fold greater in photodamaged skin than in sun-protected skin, and some fibroblasts are surrounded by debris. To model this situation, skin fibroblasts were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal fibroblasts, whereas intact collagen did not. Fibroblasts cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) fibroblasts from photoaged and sun-protected skin are similar in their capacities for growth and type I procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I procollagen synthesis.

Cyclic stretching of mesangial cells up-regulates intercellular adhesion molecule-1 and leukocyte adherence: a possible new mechanism for glomerulosclerosis.

Intraglomerular hypertension is a primary causal factor in the progressive glomerulosclerosis that characterizes diabetic nephropathy or severe renal ablation. However, inflammation of the glomerular mesangium also participates in at least the early phase of these diseases. In glomerulonephritis, where inflammation is thought to be the predominant causal factor, intraglomerular hypertension is also often present. Mesangial cells (MCs) are critical in orchestrating key functions of the glomerulus including extracellular matrix metabolism, cytokine production, and interaction with leukocytes. Because MCs are subject to increased stretching when intraglomerular hypertension is present, and in glomerulonephritis MC/leukocyte interactions seem to be mediated primarily via the up-regulation of intercellular adhesion molecule-1 (ICAM-1), we examine the possibility that cyclic stretching is a stimulus for increased MC ICAM-1 activity. We demonstrate that the normal low levels of MC ICAM-1 mRNA and protein are dramatically up-regulated by even short intervals of cyclic stretch. This effect is dose- and time-dependent, and requires little amplitude and a brief period of elongation for significant induction. Stretch-induced MC ICAM-1 also leads to a marked elevation in phagocytic leukocyte adherence. This stimulated adherence is equal or greater than that induced by the inflammatory cytokine tumor necrosis factor-alpha, whereas an additive effect occurs when both are applied in combination. Our results indicate that stretch-induced ICAM-1 may provide a direct link between hypertension and inflammation in the progression of injury and glomerulosclerosis in diabetes, renal ablation, and other forms of glomerulonephritis.

Heparin-binding epidermal-growth-factor-like growth factor activation of keratinocyte ErbB receptors Mediates epidermal hyperplasia, a prominent side-effect of retinoid therapy.

Sun-protected human skin was maintained in organ culture and treated with all-trans retinoic acid in the presence or absence of reversible or irreversible pharmacologic antagonists of c-erbB receptor tyrosine kinase activity. In the absence of these inhibitors, all-trans retinoic acid induced epidermal hyperplasia comparable to that induced in intact skin by all-trans retinol or all-trans retinoic acid itself. There was a strong correlation between inhibition of epidermal hyperplasia in organ culture and inhibition of epidermal-growth-factor-dependent keratinocyte growth in monolayer culture. In additional studies it was shown that all-trans retinoic acid could overcome the known inhibitory effects of calcium on expression of HB-EGF-like growth factor mRNA in organ-cultured skin. Further, it was shown that an antibody to HB-EGF-like growth factor inhibited retinoid-stimulated epidermal hyperplasia in organ culture and reduced proliferation in cultured keratinocytes. In contrast, the c-erbB receptor tyrosine kinase antagonists and the neutralizing HB-EGF-like growth factor antibody were ineffective in inhibiting all-trans-retinoic-acid-dependent survival and proliferation of human dermal fibroblasts. Taken together, these data indicate (i) that retinoid-induced epidermal hyperplasia in human skin proceeds through c-erbB, and (ii) that HB-EGF-like growth factor is one of the c-erbB ligands mediating this effect.

Decreased extracellular-signal-regulated kinase and increased stress-activated MAP kinase activities in aged human skin in vivo.

The ability of human skin to rejuvenate itself diminishes with the passage of time, resulting in increased fragility. This increased fragility reflects both reduced growth of skin cells and loss of collagenous connective tissue. Oxidative damage plays a central role in cellular aging. Cellular responses to growth signals and oxidative stress are mediated, in part, by growth-factor-activated and stress-activated MAP kinases. We report here that the extracellular-signal-regulated MAP kinase pathway is reduced and the stress-activated MAP kinase pathway is increased in old, compared with young, human skin in vivo. Extracellular-signal-regulated kinase activity was 45% lower in old skin (mean age 84.3 y) relative to young skin (mean age 23.8 y). This lower extracellular- signal-regulated kinase activity resulted from reduced activation, since total extracellular-signal-regulated kinase protein levels did not differ between young and old skin, whereas phosphorylated (i.e., activated) extracellular-signal-regulated kinase protein was reduced 60% in old skin. Cyclin D2, which is regulated by extracellular-signal-regulated kinase and functions to promote cell cycle progression, was reduced 50% in old skin compared with young skin. In contrast, stress-activated MAP kinase activity was elevated 3.4-fold in old skin compared with young skin. This increased activity resulted from enhanced activation, since total stress-activated MAP kinase protein levels were similar in old and young skin. Transcription factor c-Jun, which is activated by stress-activated MAP kinases and promotes expression of connective-tissue-degrading matrix metalloproteinases, was elevated 2-fold in old skin compared with young skin. Treatment of old skin with vitamin A (retinol) for 7 d stimulated extracellular-signal-regulated kinase activity, consistent with its demonstrated ability to stimulate cell growth in old human skin. Taken together, these data indicate that alterations in MAP kinase activities play a key role in the pathophysiology of human skin aging.

Time-dependent inhibition of immune complex-induced lung injury by catalase: relationship to alterations in macrophage and neutrophil matrix metalloproteinase elaboration.

Rats were subjected to acute lung injury by the intra-alveolar formation of IgG immune complexes of bovine serum albumin (BSA) and anti-BSA. In this model of injury, complement activation occurs and large numbers of neutrophils invade the interstitium and alveolar space. In the present study, animals were treated with intratracheal catalase concomitantly with anti-BSA or after a lag period of 5-120 min. Catalase treatment at time-zero or at 5 min post injury failed to prevent lung injury as indicated by permeability change, histological features, and neutrophil influx. However, treatment after a delay of 15-30 min (but not 120 min) afforded substantial protection. Consistent with past findings [19], lung injury was accompanied by an accumulation of matrix metalloproteinase 9 (MMP-9) in bronchoalveolar lavage (BAL) fluid. There was a strong correlation between inhibition of injury and reduction in MMP-9 levels. In vitro studies conducted in parallel revealed that unstimulated alveolar macrophages did not produce measurable MMP-9, while there was a large induction following exposure to the same immune complexes that initiated injury in vivo. MMP-2 was also slightly upregulated under the same conditions. Concomitant treatment with catalase greatly inhibited MMP-9 production by macrophages in response to immune complexes, but this treatment had little effect on basal production of either MMP-9 or MMP-2 by macrophage. The same concentration of catalase that suppressed MMP-9 elaboration also inhibited the production of tumor necrosis factor alpha. In contrast, when neutrophils were treated with catalase and then exposed to immune complexes, the antioxidant failed to prevent the release of either MMP-2 or MMP-9. Taken together, these findings demonstrate that antioxidant treatment interferes with elaboration of MMPs by alveolar macrophages. Protection against lung injury is correlated with reduction in MMP levels in the BAL fluid.

Troglitazone improves psoriasis and normalizes models of proliferative skin disease: ligands for peroxisome proliferator-activated receptor-gamma inhibit keratinocyte proliferation.

BACKGROUND: Psoriasis is often treated with agents that activate nuclear hormone receptors for glucocorticoids, retinoids, and vitamin D. The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a related nuclear hormone receptor that can be activated by its ligands, including the thiazolidinediones. OBJECTIVE: To assess whether treatment with troglitazone, a currently available thiazolidinedione used to treat diabetes mellitus, has an effect on psoriasis in normoglycemic patients and whether ligands for PPARgamma have an effect on models of psoriasis. DESIGN: Open-label administration of troglitazone in patients with psoriasis and evaluation of drug actions in cellular, organ, and transplant models of psoriasis. SETTING: University and community hospital outpatient departments and university laboratories. PATIENTS: Patients with chronic, stable plaque psoriasis and control subjects. Five patients with psoriasis received troglitazone (none withdrew); 10 different untreated patients and 10 controls provided tissue samples. INTERVENTIONS: Oral troglitazone therapy at various dosages in patients with psoriasis; also, use of troglitazone, ciglitazone, and 15-deoxy-delta-12,14-prostaglandinJ2 in psoriasis models. MAIN OUTCOME MEASURES: Investigator-determined clinical results in patients and cell counts and histological evidence in models. RESULTS: All patients' psoriasis improved substantially during troglitazone therapy. Peroxisome proliferator-activated receptor-gamma was expressed in human keratinocytes; ligands for PPARgamma inhibited the proliferation of normal and psoriatic human keratinocytes in culture. Troglitazone treatment normalized the histological features of psoriatic skin in organ culture and reduced the epidermal hyperplasia of psoriasis in the severe combined immunodeficient mouse and human skin transplant model of psoriasis (P<.05 compared with untreated controls). CONCLUSIONS: Peroxisome proliferator-activated receptor-gamma might be a useful intracellular target for the treatment of psoriasis; further study is needed to assess the clinical value of ligands for PPARgamma, including troglitazone.

Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin.

Tissue from 54 histologically-identified basal cell carcinomas of the skin was obtained at surgery and assayed using a combination of functional and immunochemical procedures for matrix metalloproteinases (MMPs) with collagenolytic activity and for MMPs with gelatinolytic activity. Collagenolytic enzymes included MMP-1 (interstitial collagenase), MMP-8 (neutrophil collagenase) and MMP-13 (collagenase-3). Gelatinolytic enzymes included MMP-2 (72-kDa gelatinase A/type IV collagenase) and MMP-9 (92-kDa gelatinase B/type IV collagenase). Inhibitors of MMP activity including tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2) were also assessed. All three collagenases and both gelatinases were detected immunochemically. MMP-1 appeared to be responsible for most of the functional collagenolytic activity while gelatinolytic activity reflected both MMP-2 and MMP-9. MMP inhibitor activity was also present, and appeared, based on immunochemical procedures, to reflect the presence of TIMP-1 but not TIMP-2. As a group, tumours identified as having aggressive-growth histologic patterns were not distinguishable from basal cell carcinomas with less aggressive-growth histologic patterns. In normal skin, the same MMPs were detected by immunochemical means. However, only low to undetectable levels of collagenolytic and gelatinolytic activities were present. In contrast, MMP inhibitor activity was comparable to that seen in tumour tissue. In previous studies we have shown that exposure of normal skin to epidermal growth factor in organ culture induces MMP up-regulation and activation. This treatment concomitantly induces stromal invasion by the epithelium (Varani et al (1995) Am J Pathol 146: 210-217; Zeigler et al (1996b) Invasion Metastasis 16: 11-18). Taken together with these previous data, the present findings allow us to conclude that the same profile of MMP/MMP inhibitors that is associated with stromal invasion in the organ culture model is expressed endogenously in basal cell carcinomas of skin.

Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin.

Damage to human skin due to ultraviolet light from the sun (photoaging) and damage occurring as a consequence of the passage of time (chronologic or natural aging) are considered to be distinct entities. Photoaging is caused in part by damage to skin connective tissue by increased elaboration of collagen-degrading matrix metalloproteinases, and by reduced collagen synthesis. As matrix metalloproteinase levels are known to rise in fibroblasts as a function of age, and as oxidant stress is believed to underlie changes associated with both photoaging and natural aging, we determined whether natural skin aging, like photoaging, gives rise to increased matrix metalloproteinases and reduced collagen synthesis. In addition, we determined whether topical vitamin A (retinol) could stimulate new collagen deposition in sun-protected aged skin, as it does in photoaged skin. Sun-protected skin samples were obtained from 72 individuals in four age groups: 18-29 y, 30-59 y, 60-79 y, and 80+ y. Histologic and cellular markers of connective tissue abnormalities were significantly elevated in the 60-79 y and 80+ y groups, compared with the two younger age groups. Increased matrix metalloproteinase levels and decreased collagen synthesis/expression were associated with this connective tissue damage. In a separate group of 53 individuals (80+ y of age), topical application of 1% vitamin A for 7 d increased fibroblast growth and collagen synthesis, and concomitantly reduced the levels of matrix-degrading matrix metalloproteinases. Our findings indicate that naturally aged, sun-protected skin and photoaged skin share important molecular features including connective tissue damage, elevated matrix metalloproteinase levels, and reduced collagen production. In addition, vitamin A treatment reduces matrix metalloproteinase expression and stimulates collagen synthesis in naturally aged, sun-protected skin, as it does in photoaged skin.

Hemolysis of human red blood cells induced by the combination of diethyldithiocarbamate (DDC) and divalent metals: modulation by anaerobiosis, certain antioxidants and oxidants.

The objective of the present communication is to describe the role played by combinations between diethydithiocarbamate (DDC) and divalent metals in hemolysis of human RBC. RBC which had been treated with DDC (10-50 microM) were moderately hemolyzed (about 50%) upon the addition of subtoxic amounts of Cu2+ (50 microM). However, a much stronger and a faster hemolysis occurred either if mixtures of RBC-DDC were immediately treated either by Co2+ (50 microM) or by a premixture of Cu2+ and Co2+ (Cu:Co) (50 microM). While Fe2+ and Ni2+, at 50 microM, initiated 30-50% hemolysis when combined with DDC (50 microM), on a molar basis, Cd2+ was at least 50 fold more efficient than any of the other metals in the initiation of hemolysis by DDC. On the other hand, neither Mn2+ nor Zn2+, had any hemolysis-initiating effects. Co2+ was the only metal which totally blocked hemolysis if added to DDC prior to the addition of the other metals. Hemolysis by mixtures of DDC + (Cu:Co) was strongly inhibited by anaerobiosis (flushing with nitrogen gas), by the reducing agents glutathione, N-acetyl cysteine, mercaptosuccinate, ascorbate, TEMPO, and alpha-tocopherol, by the PLA2 inhibitorbromophenacylbromide (BrPACBr), by tetracycline as well as by phosphatidyl choline, cholesterol and by trypan blue. However, TEMPO, BrPACBr and PC were the only agents which inhibited hemolysis induced by DDC: Cd2+ complexes. On the other hand, none of the classical scavengers of reactive oxygen species (ROS) employed e.g dimethylthiourea, catalase, histidine, mannitol, sodium benzoate, nor the metal chelators desferal and phenanthroline, had any appreciable inhibitory effects on hemolysis induced by DDC + (Cu:Co). DDC oxidized by H2O2 lost its capacity to act in concert either with Cu2+ or with Cd2+ to hemolyze RBC. While either heating RBC to temperatures greater than 37 degrees C or exposure of the cells to glucose-oxidase-generated peroxide diminished their susceptibility to hemolysis, exposure to the peroxyl radical from AAPH, enhanced hemolysis by DDC + (Cu:Co). The cyclovoltammetry patterns of DDC were drastically changed either by Cu2+, Co2+ or by Cd2+ suggesting a strong interaction of the metals with DDC. Also, while the absorbance spectrum of DDC at 280 nm was decreased by 50% either by Co2+, Cd2+ or by H2O2, a 90% reduction in absorbance occurred if DDC + H2O2 mixtures were treated either by Cu2+ or by Co2+, but not by Cd2+. Taken together, it is suggested that DDC-metal chelates can induce hemolysis by affecting the stability and the integrity of the RBC membrane, and possibly also of the cytoskeleton and the role played by reducing agents as inhibitors might be related to their ability to deplete oxygen which is also supported by the inhibitory effects of anaeobiosis.

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.