TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin.     

(-)Epigallocatechin gallate hampers collagen destruction and collagenase activation in ultraviolet-B-irradiated human dermal fibroblasts: involvement of mitogen-activated protein kinase.

Ultraviolet (UV) irradiation leads to distinct changes in skin connective tissues by degradation of collagen, which is a major structural component in the extracellular matrix most likely mediated by matrix metalloproteinases (MMP), collagenases. These changes in collagenous skin tissues have been suggested to be causes of the skin wrinkling observed in premature aging of the skin. This study mimicked the action of environmental ultraviolet on skin and investigated whether (-)epigallocatechin gallate (EGCG), a bioactive catechin component of green tea, mechanistically inhibited activation of MMP-1, MMP-8, and MMP-13 and destruction of collagen in UV-B irradiated human dermal fibroblasts by modulating cellular signaling pathways. Cell viability was moderately decreased by > or = 30% in human dermal fibroblasts treated with 100 mJ/cm2 UV-B, accompanying a substantial generation of reactive oxygen species evidenced by DCF staining. Western blot analysis and immunocytochemical staining revealed that EGCG markedly suppressed collagen degradation enhanced in UV-B-exposed human dermal fibroblast. Pre-treatment of fibroblasts with EGCG also inhibited UV-B-induced production of collagenases, MMP-1, MMP-8 and MMP-13, in a dose-dependent manner. In addition, EGCG rapidly and substantially hampered UV-B irradiation-induced activation of ASK-1 and phosphorylation of MAPK, JNK, p38 MAPK, and ERK1/2, in dermal fibroblasts. These results demonstrate that EGCG has abilities to hamper UV-B-induced collagenolytic MMP production via interfering with the MAPK-responsive pathways. Therefore, EGCG may be a potential agent for the prevention and treatment of skin photoaging.     

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.     

The unexpected anabolic phenotype and extended longevity of skin fibroblasts after chronic glucocorticoid excess

Intense stress can challenge tissue homeostasis and accelerate the ageing process. However, several lines of evidence indicate that repeated mild stresses can have beneficial and even life-prolonging effects. Hypersecretion of glucocorticoids (GC) represents the major hormonal response to stress. Besides its life-sustaining role, GC excess, usually due to several side-effects that promote a "catabolic" phenotype, can be detrimental for several tissues. Cushing's syndrome patients are characterized by chronic endogenous GC excess and consequently at the time of diagnosis they have an atrophic elderly-like skin. Interestingly, when Cushing's syndrome fibroblasts were removed from the high-GC milieu in vivo and cultured in vitro under standard conditions they express an "anabolic" phenotype, i.e. they restore their ability for collagen synthesis, they secrete reduced levels of metalloproteases (MMP-1 and MMP-2) and have an increased proliferative capacity and contractility. Furthermore, these cells exhibit a significant extension of their proliferative lifespan, while they respond better to exogenous stress by producing significantly higher levels of heat-shock protein-70 (HSP70). These results imply that long-term hypercortisolism in vivo can have beneficial consequences on fibroblast physiology in vitro.     

A potent,selective inhibitor of matrix metalloproteinase-3 for the topical treatment of chronic dermal ulcers

The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix (required for cell migration) and growth factors and their receptors. The overexpression of MMP-3 (stromelysin-1) and MMP-13 (collagenase-3) is associated with nonhealing wounds, whereas active MMPs-1, -2, -9, and -14 are required for normal wound healing to occur. We describe the synthesis and enzyme inhibition profile of (3R)-3-[([(1S)-2,2-dimethyl-1-(([(1S)-2-methoxy-1-phenylethyl]amino)carbonyl)propyl]amino)carbonyl]-6-(3-methyl-4-phenylphenyl)hexanoic acid (UK-370,106, 7), which is a potent inhibitor of MMP-3 (IC(50) = 23 nM) with >1200-fold weaker potency vs MMP-1, -2, -9, and -14. MMP-13, which may also contribute to the pathology of chronic wounds, was inhibited about 100-fold less potently by compound 7. Compound 7 potently inhibited cleavage of [(3)H]-fibronectin by MMP-3 (IC(50) = 320 nM) but not cleavage of [(3)H]-gelatin by either MMP-2 or -9 (up to 100 microM). Compound 7 had little effect, at MMP-3 selective concentrations, on keratinocyte migration over a collagen matrix in vitro, which is a model of the re-epithelialization process. Following iv (rat) or topical administration to dermal wounds (rabbit), compound 7 was cleared rapidly (t(1/2) = 23 min) from plasma, but slowly (t(1/2) approximately 3 days) from dermal tissue. In a model of chronic dermal ulcers, topical administration of compound 7 for 6 days substantially inhibited MMP-3 ex vivo. These data suggest compound 7 is sufficiently potent to inhibit MMP-3-mediated matrix degradation while leaving unaffected cellular migration mediated by MMPs 1, 2, and 9. These properties make compound 7 a suitable candidate for progression to clinical trials in human chronic dermal wounds, such as venous ulcers.     

Matrix metalloproteinase-9 expression and release from skin fibroblasts interacting with keratinocytes: Upregulation in response to sulphur mustard

Matrix metalloproteinases (MMPs), especially MMP-9 and MMP-2, degrade various proteins of the extracellular matrix, including collagen type IV the major component of basement membranes which also separate the epidermis from the dermis. Although previous work indicates the contribution of MMPs and their inhibitors (TIMPs) to the pathophysiology of skin lesions induced by the toxic chemical warefare agent sulphur mustard (SM), little is known about the underlying molecular and cellular mechanisms. In this study we demonstrate in a 3D-skin model that topical application of SM significantly upregulated basal MMP-9 mRNA expression and release from the cells as shown by qRT-PCR and zymography, whereas that of MMP-2, membrane-type 1 (MT1)-MMP, TIMP-1 and TIMP-2 remained almost unaffected by SM. Further studies in neonatal human dermal fibroblasts (NHDF) and HaCaT keratinocytes revealed that MMP-9 was not secreted from these cells, neither with or without exposure to SM. However, when NHDF and HaCaT were cocultivated, MMP-9 was expressed and released from the cell mixture, suggesting that interaction between both cell types is essential for MMP-9 production. Moreover, SM-treatment of NHDF/HaCaT cocultures further upregulated MMP-9 biosynthesis and secretion, which was consistent with our findings obtained in the 3D-skin model. Addition of conditioned medium derived from SM-exposed HaCaT cells to NHDF was able to stimulate MMP-9 secretion and also increased the migratory potential of NHDF as shown in a scratch-wound healing assay and a fluorescent cell invasion assay. In contrast, culture supernatants of SM-treated NHDF had not such an effect on HaCaT cells. Taken together, our findings provide first evidence that SM exposure of skin stimulates keratinocytes to release soluble factors which in turn induce enhanced MMP-9 secretion and invasiveness of fibroblasts in vitro. This provides a potential mechanism probably contributing to SM-evoked tissue injury in vivo.     

Inhibition of plasmin-mediated prostromelysin-1 activation by interaction of long chain unsaturated fatty acids with kringle 5

C18 unsaturated fatty acids were here found to inhibit proMMP (matrix metalloproteinase)-3 activation by plasmin. This effect was suppressed by lysine ligand competitors, indicating that it was mediated by binding to kringle domains. Surface plasmon resonance analysis demonstrated that oleic acid interacted to a similar extent with plasmin and kringle 5 (KD values of 3.4 x 10(-8) and 5.9 x 10(-8)M) while interaction with kringles 1-2-3 was 10-fold lower. Furthermore, oleic acid stimulated the amidolytic activity of plasmin and mini-plasmin, but not micro-plasmin. Oleic acid also enhanced u-PA (urokinase-type plasminogen activator)-mediated plasminogen activation over 50-fold. Taken together, these data indicate that inhibition of plasmin-induced proMMP-3 activation by unsaturated fatty acids was mediated through their preferential binding to kringle 5. The influence of elaidic acid on the plasmin/MMP-3/MMP-1 proteolytic cascade was assessed ex vivo. Exogenous addition of plasmin to dermal fibroblasts or supplementation of gingival fibroblast culture medium with plasminogen triggered this cascade. In both instances, elaidic acid totally abolished proMMP-3 and proMMP-1 activation. Additionally, a significant decrease in lattice retraction and collagen degradation in a range similar to that obtained with Batimastat was observed when human gingival fibroblasts were cultured in plasminogen-containing type I collagen gels, indicative of the dual influence of unsaturated fatty acids on MMP activation and activity. In conclusion, unsaturated fatty acids or molecules with similar structures could be attractive target for the development of natural pharmacological inhibitors directed against plasmin and/or MMPs in different pathological contexts such, skin UV irradiation, vascular diseases and tumour growth and invasion.     

TGF-beta antisense oligonucleotides modulate expression of matrix metalloproteinases in isolated fibroblasts from radiated skin

BACKGROUND: Transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. The purpose of this study was to analyze the effect of TGF-beta targeting on the expression of matrix metalloproteinases (MMPs) in fibroblasts isolated from radiation-induced chronic dermal wounds. MATERIALS AND METHODS: The expression of MMPs in tissue samples from radiation-induced chronic dermal wounds was investigated by immunohistochemistry and microarray technique. The effect of TGF-beta targeting using antisense oligonucleotides on the expression of MMPs in isolated fibroblasts was analysed by ELISA and multiplex RT-PCR. RESULTS: Immunohistochemical investigation and microarray analysis demonstrated an increased expression of MMP protein and mRNA in tissue samples from radiation-induced chronic dermal wounds compared to normal human skin. Antisense TGF-beta oligonucleotide treatment significantly down-regulated MMP secretion in vitro. CONCLUSION: TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in radiation-induced chronic wounds.     

In vitro evaluation of chloroaluminum phthalocyanine nanoemulsion and low-level laser therapy on human skin dermal equivalents and bone marrow mesenchymal stem cells

Nanotechnology and tissue engineering are promising scientific fields in the development of advanced materials useful to human health. This article describes the preparation of a nanocarrier for the controlled release of a photosensitizer compound associated with low-level light therapy for skin wound healing treatment and applicable to other skin diseases. A biological model was used as an in vitro skin equivalent based on a three-dimensional culture of fibroblasts and mesenchymal stem cells and denominated by dermal equivalent (DE). Results show that it is possible to use the photomodulation process to control the wound healing in a scratching process and to induce the biomolecules release, both of which are related with the inflammatory wound healing process. In the studies, the MMP-2 and MMP-9 expression from zymography analyses were evaluated. All results showed a dependence on enzymatic activity relating to lowlevel laser applications which indicates a potential application in wound healing processes based on phototherapy and nanotechnology.     

The mechanism involved in the inhibitory action of tranilast on collagen biosynthesis of keloid fibroblasts.

Tranilast, an anti-allergic drug inhibiting the release of substances such as histamine and prostaglandins from mast cells, was previously reported to suppress collagen synthesis of fibroblasts derived from keloid tissues. However, the inhibitory mechanism on collagen synthesis is unknown. We studied its inhibitory mechanism on collagen synthesis by culturing fibroblasts from keloid and hypertrophic scar tissues of humans. Collagen synthesis of fibroblasts from keloid and hypertrophic scar tissue is greater than that from healthy human skin. Tranilast (3-100 microM) did not inhibit prolyl hydroxylase (the rate-limiting enzyme in collagen synthesis) activity. Tranilast (3-300 microM) suppressed the collagen synthesis of fibroblasts from keloid and hypertrophic scar tissue but not healthy skin fibroblasts. Tranilast (30-300 microM) inhibited the release of transforming growth factor (TGF)-beta 1 from keloid fibroblasts, which enhances the collagen synthesis of keloid fibroblasts. Anti-TGF-beta 1 antibody (50 microliter/ml) inhibited the collagen synthesis, although diphenhydramine (10 microM) and indomethacin (10 microM) did not show any inhibition. These results suggest that tranilast inhibits collagen synthesis of fibroblasts from keloid and hypertrophic scar tissue through suppressing the release of TGF-beta 1 from the fibroblasts themselves.     

The effect of tripeptide-copper complex on human hair growth in vitro

The tripeptide-copper complex, described as a growth factor for various kinds of differentiated cells, stimulates the proliferation of dermal fibroblasts and elevates the production of vascular endothelial growth factor, but decreased the secretion of transforming growth factor-beta1 by dermal fibroblasts. Dermal papilla cells (DPCs) are specialized fibroblasts, which are important in the morphogenesis and growth of hair follicles. In the present study, the effects of L-alanyl-L-histidyl-L-lysine-Cu2+ (AHK-Cu) on human hair growth ex vivo and cultured dermal papilla cells were evaluated. AHK-Cu (10(-12) - 10(-9) M) stimulated the elongation of human hair follicles ex vivo and the proliferation of DPCs in vitro. Annexin V-fluorescein isothiocyanate/propidium iodide labeling and flow cytometric analysis showed that 10(-9) M AHK-Cu reduced the number of apoptotic DPCs, but this decrease was not statistically significant. The ratio of Bcl-2/Bax was elevated, and the levels of the cleaved forms of caspase-3 and PARP were reduced by treatment with 10(-9) M AHK-Cu. The present study proposed that AHK-Cu promotes the growth of human hair follicles, and this stimulatory effect may occur due to stimulation of the proliferation and the preclusion of the apoptosis of DPCs.     

皮肤恶性黑色素瘤患者瘤组织中MMP-9、Nm23-H1和MTA1蛋白表达水平及临床意义

目的 探讨皮肤恶性黑色素瘤(CMM)组织中基质金属蛋白酶(MMP)-9、Nm23-H1和肿瘤转移相关基因蛋白1(MTA-1)表达水平及其临床意义.方法 收集2013年10月-2015年12月收治的CMM 64例存档的蜡块标本作为CMM组,选取同期外科切取的28例皮肤交界痣标本及18例正常皮肤分别作为皮肤交界痣组与正常对照组.检测3组MMP-9、Nm23-H1和MTA-1蛋白的表达水平.结果 MMP-9、MTA-1蛋白在CMM组中阳性表达率最高,在皮肤交界痣组及正常对照组中阳性表达率逐渐降低(P ＜0.05);CMM组Nm23-H1蛋白阳性率低于正常对照组及皮肤交界痣组(P＜0.05);MMP-9、MTA-1蛋白在CMM组中阳性表达率随Clark分级的增高逐渐升高,有淋巴结转移者阳性表达率高于无淋巴结转移者(P＜0.01);Nm23-H1在CMM组中阳性表达率随Clark分级的增高逐渐降低,有淋巴结转移者阳性表达率低于无淋巴结转移者(P＜0.01).结论 MMP-9、Nm23-H1和MTA1的表达水平在一定程度反映了CMM的病情,联合检测MMP-9、Nm23-H1和MTA1表达水平可为临床评估患者病情提供准确资料.     

Identification of benzene metabolites in dermal fibroblasts as nonphenolic: regulation of cell viability, apoptosis, lipid peroxidation and expression of matrix metalloproteinase 1 and elastin by benzene metabolites

The skin is exposed to benzene and its derivatives, prevalent environmental chemicals. They may impair the structural integrity of the skin by increased expression of matrix metalloproteinase 1 (MMP-1; degrades structural collagen) and elastin, synthesized primarily by the dermal fibroblasts. We examined the metabolism of benzene in dermal fibroblasts and identified the benzene metabolites as toluene, benzaldehyde, aniline and benzoic acid. These metabolites were not toxic to the cells with regard to cell viability, apoptosis and lipid peroxidation, unlike the phenolic benzene metabolites (hydroquinone, t-butyl hydroquinone and phenol) or hydrogen peroxide. Toluene and phenol, which compose cigarette smoke, and benzaldehyde stimulated MMP-1 and/or elastin expression. In summary, the dermal fibroblasts metabolize benzene to nonphenolic metabolites that are less toxic to the cellular components than the phenolic benzene derivatives. Toluene, benzaldehyde and phenol can directly cause facial wrinkling and impaired structural integrity by upregulating MMP-1 and/or elastin.     

Immunohistochemical analysis of radiation-induced non-healing dermal wounds of the head and neck

Persistent, poorly healing wounds are a significant clinical problem in patients who have had previous irradiation. The pathology of chronic dermal ulcers is characterised by excessive proteolytic activity which degrades the extracellular matrix (required for cell migration) and growth factors and their receptors. Interestingly, the molecular basis of radiation-induced dermal wounds is poorly understood. The aim of this study was to investigate, by immunohistochemistry, the expression of the endothelial marker vWF, of angiogenic bFGF, VEGF and IL-8, of collagenases MMP-2 and MMP-9 and their inhibitors TIMP-1 and TIMP-2, in tissue samples from radiation-induced chronic dermal wounds and healthy control skin. Performing immunohistochemical detection of microvessels, an equivalent density of microvessels was observed within tissue samples from normal healthy skin and from radiation-induced non-healing cutaneous wounds. Investigation of angiogenic bFGF and VEGF demonstrated a decreased expression of both factors in the radiation-induced dermal wounds. The expression of angiogenic IL-8 was weak in both the healthy skin samples and the radiation-induced wounds. In addition, an increased expression of collagenases MMP-2 and MMP-9 protein within the radiation-induced wounds was demonstrated. While the expression of TIMP-1 showed no difference of expression between normal control skin and tissue samples from radiation-induced wounds, TIMP-2 expression was slightly increased compared to healthy controls. Our data suggest that radiation-induced dermal injuries often fail to heal because of decreased angiogenesis and persistently high concentrations of MMPs with an imbalance of their tissue inhibitors. The basic mechanisms of wound healing in radiation-induced dermal wounds at the molecular level need to be understood further for the development of innovative treatment strategies.     

丹皮酚对TNF-α诱导真皮成纤维细胞MMP-9 mRNA及细胞因子表达的影响

目的通过研究丹皮酚对TNF-α诱导的真皮成纤维细胞MMP-9 mRNA及细胞因子表达的影响,探讨丹皮酚抗皮肤炎症的可能机制。方法采用RT-PCR法考察正常人真皮成纤维细胞中MMP-9 mRNA表达情况以及丹皮酚对TNF-α诱导的真皮成纤维细胞MMP-9 mRNA表达的影响。采用ELISA法测定丹皮酚对TNF-α诱导的真皮成纤维细胞表达IL-1β、IL-6及IL-8的影响。结果MMP-9在正常真皮成纤维细胞上表达极弱,TNF-α可以诱导MMP-9 mRNA的表达,丹皮酚可以抑制TNF-α诱导引起的MMP-9 mRNA表达的上调。成纤维细胞可分泌少量的IL-1β、IL-6及IL-8,TNF-α可以明显增加成纤维细胞IL-1β和IL-8的分泌量,丹皮酚可以抑制TNF-α诱导的IL-1β和IL-8的产生,但对IL-6的分泌无影响。结论丹皮酚可以抑制TNF-α诱导真皮成纤维细胞引起的MMP-9 mRNA及炎症因子IL-1β和IL-8表达水平的上调。     

Transforming growth factor-beta 1-induced collagen production in cultures of cardiac fibroblasts is the result of the appearance of myofibroblasts

Transforming growth factor-beta 1 (TGF-beta 1), which appears in high concentrations in fibrotic cardiac tissue, is a potent inductor of tissue collagen deposition and of the differentiation of fibroblasts to myofibroblasts. It is accepted that TGF-beta 1 is a potent stimulator of collagen secretion by fibroblasts. The aim of the present study was to determine which type of cells, fibroblasts and/or myofibroblasts are stimulated, in terms of collagen production, by TGF-beta 1. Therefore, using cultures of second-passage rat cardiac fibroblasts, we investigated the dose- (0.003-15 ng/ml) and time-dependence (2-48 h) of the TGF-beta 1-induced effects on collagen production and on the appearance of myofibroblasts, as estimated by the presence of alpha-smooth muscle actin (alpha-SMA; a marker of myofibroblasts). The reversibility of the TGF-beta 1-stimulated effects was also studied. The dose- and time-dependent stimulation of collagen production was closely associated with the induction of alpha-SMA. TGF-beta 1 did not change the cell phenotype or increase collagen production in rat cardiac fibroblasts cultures after a long incubation (24-28 h) at low concentrations (< 1 ng/ml), or after a short incubation (2-4 h) at high concentrations (1-15 ng/ml). However, after a long incubation at high concentrations, TGF-beta 1 changed the cell phenotype and increased collagen production in these cultures through the differentiation of fibroblasts to myofibroblasts. A maximal increase of collagen production (two-fold, p < 0.001) was observed after incubation of fibroblasts with 15 ng/ml TGF-beta 1 for 48 h. Under these conditions, alpha-SMA was increased by 3.5-fold (p < 0.001) and second-passage cultures of fibroblasts and their offspring in the next passage consisted mainly of myofibroblasts. The stimulation of collagen by 15 ng/ml TGF-beta 1 for 48 h was irreversible. In fact, additional incubation of these second-passage TGF-beta 1-stimulated cultures without TGF-beta 1 for 2 days did not decrease the high activity of collagen production. Moreover, the third-passage offspring of these TGF-beta 1-stimulated fibroblasts cultured without TGF-beta 1 also showed a higher production of collagen compared with control fibroblasts. Furthermore, the increased collagen production in the third-passage fibroblast offspring of the second-passage TGF-beta 1-stimulated fibroblasts could not be further stimulated by TGF-beta 1. Thus, the activity of collagen production in TGF-beta 1-stimulated cultures and in their next passage offspring is not sensitive to TGF-beta 1. Our data suggest that TGF-beta 1-stimulated collagen production in cultures of adult rat cardiac ventricular fibroblasts cannot be explained by a direct stimulation of collagen production, either in fibroblasts or in myofibroblasts. Instead, TGF-beta 1 induces differentiation of fibroblasts to myofibroblasts, the latter having a higher activity for collagen production than the former.     

IL-18BPa:Fc cooperates with immunosuppressive drugs in human whole blood

The proinflammatory cytokine interleukin (IL)-18 appears to be involved in the pathogenesis of diseases associated with immunoactivation and inflammation. Consequently, blockage of IL-18 bioactivity by use of IL-18 binding protein (IL-18 BP) is likely a promising therapeutic concept. In the present study, we investigated immunomodulatory activities of IL-18 BPa:Fc in human whole blood cultures. We report that IL-18 BPa:Fc (200 ng/mL) significantly inhibited lipopolysaccharide (LPS, 10 ng/mL)/IL-12 (5 ng/mL)-induced release of interferon-gamma (IFNgamma) and matrix metalloproteinase-9 (MMP-9) from whole blood cultures of healthy donors. Notably, IL-18 BPa:Fc (200 ng/mL) further reinforced dexamethasone (5 nM)- or mycophenolic acid (2 microM)-mediated reduction of LPS/IL-12-induced IFNgamma production by an additional 50.5 or 49.9%, respectively. To investigate effects of IL-18 BP:Fc in the context of autoimmune diseases, experiments were performed with whole blood obtained from patients with systemic lupus erythematosus or Wegener's granulomatosis undergoing immunosuppressive therapy. After ex vivo stimulation with LPS (10 ng/mL), production of IFNgamma and MMP-9 was determined. Both mediators likely contribute to renal inflammation frequently seen in these diseases. In accord with the aforementioned data, LPS (10 ng/mL)-induced IFNgamma was significantly reduced by coincubation with IL-18 BPa:Fc at 200 ng/mL. IL-18 BPa:Fc also inhibited production of MMP-9. The present data demonstrate that IL-18 BPa:Fc has the potential to amplify anti-inflammatory actions of immunosuppressive drugs, and thus may prove to be a valuable novel pharmacological component in the treatment of human autoimmune diseases.     

Dexpanthenol Modulates Gene Expression in Skin Wound Healing in vivo

Topical application of dexpanthenol is widely used in clinical practice for the improvement of wound healing. Previous in vitro experiments identified a stimulatory effect of pantothenate on migration, proliferation and gene regulation in cultured human dermal fibroblasts. To correlate these in vitro findings with the more complex in vivo situation of wound healing, a clinical trial was performed in which the dexpanthenol-induced gene expression profile in punch biopsies of previously injured and dexpanthenol-treated skin in comparison to placebo-treated skin was analyzed at the molecular level by Affymetrix? GeneChip analysis. Upregulation of IL-6, IL-1β, CYP1B1, CXCL1, CCL18 and KAP 4-2 gene expression and downregulation of psorasin mRNA and protein expression were identified in samples treated topically with dexpanthenol. This in vivo study might provide new insight into the molecular mechanisms responsible for the effect of dexpanthenol in wound healing and shows strong correlations to previous in vitro data using cultured dermal fibroblasts.