The lazaroid tirilazad is a new inhibitor of direct and indirect UVA-induced lipid peroxidation in human dermal fibroblasts

Lipid peroxidation caused by oxidative stress within the tissue leads to destruction and dysfunction of cellular membranes. Human dermal fibroblasts in the skin are subject to constant photooxidative stress caused mainly by deeply penetrating UVA irradiation. Therefore, the membrane damage caused by this photooxidative stress may be a major promoter of photoaging and photocarcinogenic processes initiated and promoted by long-term UVA exposure of the skin. The oxidative destruction is counterbalanced by a complex network of enzymatic and nonenzymatic antioxidants creating the skins line of defence against UVA-induced reactive oxygen species. The lazaroid tirilazad represents a new synthetic group of antioxidants with structural molecular similarity to glucocorticosteroids. We investigated the antioxidative capacity of tirilazad by determining its effects on the levels of malondialdehyde (MDA), as a marker of lipid peroxidation, induced directly or indirectly by UVA in human dermal fibroblasts. In a time- and dose-dependent kinetic, we demonstrated that fibroblasts incubated with tirilazad are well protected against subsequent UVA irradiation and show no increase in MDA levels similar to the unirradiated controls. This was also observed when lipid peroxidation was caused chemically by incubation of human dermal fibroblasts with 200 M Fe³⁺-citrate and 1 mM ascorbyl phosphate as a model of indirect UVA-induced skin damage. Lysates of fibroblasts treated this way showed a tenfold increase in MDA levels, whereas preincubation with tirilazad resulted in a significantly lower increase in MDA levels. Furthermore, in a comparison with the well-established radical scavenger Trolox, an -tocopherol analogue, tirilazad offered better protection to the membranes. Our results demonstrate for the first time that the lazaroid tirilazad is an effective inhibitor of direct and indirect UVA-induced increases in MDA as a marker of lipid peroxidation in human dermal fibroblasts.     

Adaptive cellular protection against UVA-1-induced lipid peroxidation in human dermal fibroblasts shows donor-to-donor variability and is glutathione dependent

Photo-oxidative stress and subsequent lipid peroxidation (LPO) is one of the major mechanisms of UVA-related skin pathology. The skin's protection system against photo-oxidative stress involves low molecular scavengers as well as highly specialised antioxidant enzymes like glutathione peroxidase (GPX). Against repetitive UVA-1 exposures in vitro it is partly adaptive, as recent studies have shown exemplarily for antioxidant enzymes. We now investigated in vitro by repetitively irradiating human dermal fibroblasts with UVA-1 whether this adaptive response might reflect itself in reduced cellular membrane damage, that is, LPO. Our experiments show that the degree of cellular protection against LPO and the adaptive potential of the cells against a repetitive UVA-1 exposure varies from donor-to-donor and depends highly on glutathione.     

Signaling events during induction of plasminogen activator inhibitor-1 expression by sphingosylphosphorylcholine in cultured human dermal fibroblasts

Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid metabolite that can enhance wound healing. In a search for effectors downstream of SPC in the wound-healing process, we found that the expression of the gene for plasminogen activator inhibitor-1 (PAI-1) was significantly affected. ELISA and western blot analyses showed that SPC markedly induced PAI-1 production in human dermal fibroblasts cultured in vitro. Inhibition by pre-treatment with pertussis toxin (PTx), but not by tyrphostin A47 (a receptor tyrosine kinase inhibitor), indicated that PTx-sensitive G proteins were involved in SPC-induced PAI-1 expression. SPC elicited a rapid and transient increase in intracellular calcium levels ([Ca2+]i), measured using laser scanning confocal microscopy, which was partly mediated through PTx-sensitive G proteins. Pre-treatment with thapsigargin, but not with EGTA, abolished SPC-induced PAI-1 expression, indicating the importance of Ca2+ release from internal stores. Phorbol-12-myristate-13-acetate (PMA) induced the expression of PAI-1, and pre-treatment with Ro 31-8220 (a PKC inhibitor) markedly suppressed SPC-induced PAI-1 expression. SPC-induced PAI-1 expression was also significantly suppressed by PD98059 (a specific MAPK kinase 1/2 inhibitor). Consistent with this result, SPC stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase (ERK). Together, these results suggest that SPC induces PAI-1 production through a G protein-coupled calcium increase and downstream kinase signaling events in cultured human dermal fibroblasts.     

溶菌酶对体外培养的人成纤维细胞基质金属蛋白酶-1和12及赖氨酸氧化酶基因表达的影响

目的 探讨溶菌酶对体外培养的人成纤维细胞基质金属蛋白酶-1和12及赖氨酸氧化酶基因表达的影响。方法 采用酶消化法进行体外人皮肤成纤维细胞原代培养,然后将不同浓度的溶菌酶（0,1 × 10-8,1 × 10-7 mol/L）加入体外培养的人皮肤成纤维细胞中,待药物作用后,提取总RNA,通过逆转录反应获得cDNA并进行体外扩增,对扩增产物进行琼脂糖凝胶电泳,根据条带的平均光密度A值的比值来判断人成纤维细胞中基质金属蛋白酶（MMP）-1、MMP-12及赖氨酸氧化酶（LOX）mRNA的表达水平。结果 对体外培养的人成纤维细胞进行溶菌酶干预,经β肌动蛋白内参校正后,RT-PCR示对照组、低剂量组、高剂量组MMP-1、MMP-12 mRNA表达水平三组间差异具有统计学意义（F值分别为6.98和4.44,P值均 < 0.05）。SNK-q检验显示,低剂量组与对照组、高剂量组比较,差异均无统计学意义（P > 0.05）,高剂量组与对照组相比,差异有统计学意义（P < 0.05）。LOX mRNA表达水平三组间差异具有统计学意义（F = 5.24,P < 0.05）,SNK-q检验显示,低剂量组、高剂量组与对照组相比,差异具有统计学意义（P < 0.05）,低剂量组与高剂量组相比,差异无统计学意义（P > 0.05）。结论 溶菌酶可以下调MMP-1和MMP-12及上调LOX基因的转录水平。     

Investigation of keratinocyte regulation of collagen I synthesis by dermal fibroblasts in a simple in vitro model

BACKGROUND: Hypertrophic scarring and skin graft contracture are major causes of morbidity after burn injuries. A prominent feature is excessive fibroplasia with accumulation of increased fibrillar collagen relative to normal scar tissue. The application of split-thickness skin grafts or cultured epithelial autografts to burn wounds is known to reduce scarring and contraction. OBJECTIVES: To investigate further how the keratinocyte influences underlying fibroblast behaviour by examining the influence of keratinocytes on fibroblast collagen synthesis, using a new assay for collagen synthesis never previously applied to skin cell biology. METHODS: We investigated the influence of the keratinocyte on fibroblast synthesis of type I collagen using an immunoassay for the aminoterminal propeptide of type I collagen (P1NP) in conditioned medium from monocultures and cocultures of keratinocytes and fibroblasts over 14 days. The importance of the physical presence of the keratinocyte was investigated by comparing cocultures of keratinocytes and fibroblasts against fibroblast monocultures with keratinocyte-conditioned medium. Pharmacological agents known to promote fibroblast proliferation [basic fibroblast growth factor (bFGF)], keratinocyte proliferation [insulin-like growth factor (IGF)-1], modify scarring in vivo[tumour necrosis factor (TNF)-alpha] or modify collagen biochemistry [putrescine, estrone, estradiol and beta-aminopropionitrile (beta-APN)] were then investigated for their effect on collagen synthesis in fibroblasts and in keratinocyte/fibroblast cocultures. RESULTS: Keratinocytes in coculture with fibroblasts, and keratinocyte-conditioned medium, both reduced fibroblast P1NP synthesis. Of the pharmacological agents investigated, bFGF, IGF-1, TNF-alpha and beta-APN all increased collagen synthesis both in monocultures of fibroblasts and in cocultures of keratinocytes and fibroblasts. CONCLUSIONS: Fibroblast collagen synthesis appears to be downregulated by keratinocyte-derived cytokines. Fibroblast growth factors and proinflammatory cytokines appear to be able partially to overcome this downregulation and to increase collagen synthesis.     

Hypoxia-induced increase of matrix metalloproteinase-1 synthesis is not restored by reoxygenation in a three-dimensional culture of human dermal fibroblasts

BACKGROUND: Delayed wound healing is multi-factorial. Although ischemic change is considered to be crucial, little is known about the effects of hypoxia or reoxygenation on the connective tissue metabolism by human dermal fibroblasts. OBJECTIVE: The aim of this study is to determine whether or not hypoxia (2% O(2)) or reoxygenation (20% O(2)) affects mRNA expression and production of matrix metalloproteinase-1 (MMP-1), type I collagen, tissue inhibitors of metalloproteinase-1 (TIMP-1), and transforming growth factor-beta1 (TGF-beta1) by human dermal fibroblasts in a three-dimensional culture. METHODS: We introduced the three-dimensional culture of human dermal fibroblasts with experimental wound. After wounding, cells were incubated under hypoxic (2%) or normoxic (20%) condition, and harvested at 24, 36, 48, and 72 h (n=8). In the reoxygenation study (n=4), cells were first exposed to a hypoxic condition for 72 h and further incubated under a normoxic condition for 72 h. RESULTS: The relative ratio (hypoxia/normoxia) of MMP-1 mRNA expressions were significantly elevated at 36 and 48 h compared with those at 12 h (P<0.05). The relative ratio of proMMP-1 was also significantly increased at 48 and 72 h compared with that at 12 h (P<0.001 and P<0.05, respectively). There were no significant changes in mRNA and protein levels of type I collagen, TGF-beta1, and TIMP-1. In a reoxygenic condition, 72 h reoxygenation after 72 h hypoxia, the hypoxia-induced alterations of MMP-1 and carboxyterminal propeptide of type I procollagen (PIP) were not restored. CONCLUSION: Our results indicate that hypoxia may be responsible for delayed wound healing by inducing an increase of MMP-1 synthesis.     

Baicalein inhibits matrix metalloproteinase 1 expression via activation of TRPV1‐Ca‐ERK pathway in ultraviolet B–irradiated human dermal fibroblasts

Increased matrix metalloproteinase 1 (MMP‐1) expression is a feature of photo‐aged skin. We investigated the effects of baicalein and sulphoraphane on ultraviolet B (UVB) irradiation–induced MMP‐1 expression and apoptosis using human dermal fibroblasts. UVB irradiation not only increased MMP‐1 expression, but also caused apoptosis. Both baicalein and sulphoraphane protected cells from UVB irradiation–induced apoptosis, but only baicalein inhibited MMP‐1 expression. UVB irradiation activated 12‐lipoxygenase, and its product, 12‐hydroxyeicosatetraenoic acid, activated TRPV1 channels. The resulting UVB irradiation–induced Ca²⁺ increase was blocked by the 12‐lipoxygenase inhibitor baicalein and the TRPV1 blocker capsazepine, but not by the Nrf2 inducer sulphoraphane. UVB irradiation also increased ROS generation and decreased Nrf2 protein levels. UVB irradiation–induced MMP‐1 expression was blocked by the Ca²⁺ chelator BAPTA, by capsazepine and by TRPV1 silencing. However, induction was unaffected by the antioxidant N‐acetylcysteine. ERK phosphorylation and JNK phosphorylation were induced by UVB irradiation, but only ERK phosphorylation was Ca²⁺ sensitive. Increased MMP‐1 expression was blocked by PD98059, but not by SP600125. Thus, increased MMP‐1 expression is mediated by increased cytosolic Ca²⁺ and ERK phosphorylation. UVB irradiation–induced ROS generation is also Ca²⁺ sensitive, and UVB irradiation–induced apoptosis is caused by increased ROS. Thus, baicalein, by blocking the UVB irradiation–induced cytosolic Ca²⁺ increase, protects cells from UVB irradiation–induced MMP‐1 expression and apoptosis. In contrast, sulphoraphane, by decreasing cellular ROS, protects cells from only UVB‐induced apoptosis. Thus, targeting 12‐lipoxygenase may provide a therapeutic approach to improving the health of photo‐aged human skin.     

Cytokine regulation of proliferation and ICAM-1 expression of human dermal microvascular endothelial cells in vitro.

The effects of recombinant human interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF) on the cell proliferation and the expression of intercellular adhesion molecule-1 (ICAM-1) were assessed in cultured human dermal microvascular endothelial cells (HDMEC). IL-1 alpha and IL-1 beta stimulated the proliferation of HDMEC in a dose-dependent manner, whereas in control experiments using human umbilical vein endothelial cells (HUVEC), IL-1 alpha and IL-1 beta did not stimulate HUVEC growth. Also GM-CSF stimulated the proliferation of HDMEC, whereas IL-6 did not affect endothelial cell growth in vitro. Treatment with IL-1 alpha, IL-1 beta, and TNF markedly increased the expression of ICAM-1 on HDMEC in a time- and dose-dependent manner, in contrast to IL-6 and GM-CSF. By pre-embedding immunoelectron microscopy, membrane-bound expression of ICAM-1 was visualized with pronounced labeling in areas of microvillous cell protrusions. The TNF-induced expression of ICAM-1 on HDMEC was blocked by co-incubation with a neutralizing antibody against TNF, but not with neutralizing antibodies against IL-1 alpha, IL-1 beta, or IL-6. In addition, co-incubation of HDMEC with TNF and the retinoid compound acitretin, dexamethasone, or indomethacin did not abrogate the TNF-induced ICAM-1 expression. These results disclose IL-1 as a major, multifunctional endothelial cell-targeted cytokine and further confirm the concept that pro-inflammatory cytokines exert differential regulatory effects on dermal microvascular endothelial cell proliferation and expression of cell-adhesion molecules.     

Improvement of the dermal epidermal junction in human reconstructed skin by a new c-xylopyranoside derivative

Skin aging entails drastic changes in the extracellular dermal matrix (ECM) and dermal-epidermal junction (DEJ). These biological alterations are reflected in the clinical signs of aged skin. A new C-xylopyranoside derivative, C-beta-D-xylopyranoside-2-hydroxy-propane (C-Xyloside) has been shown to induce neo-synthesis of matrix proteins such as glycosaminoglycans and heparan sulfate proteoglycans. The aim of this study was to assess the effects of C-Xyloside on markers of the dermal epidermal junction. Basement membrane components, collagen IV, collagen VII and laminin 5 as well as sub-epidermal dermal markers, pro-collagen I and fibrillin 1 were analysed using immunohistochemistry in a reconstructed skin model, including a dermal equivalent populated with living fibroblasts. Levels of mRNA of collagen VII alpha1 and collagen IV alpha1 were evaluated in dermal fibroblasts using RT-PCR. The results showed that C-Xyloside significantly induced a higher deposition of basement membrane and DEJ proteins in the reconstructed skin model and increased collagen VII gene expression. These findings indicate that, in addition to stimulating glycosaminoglycan and heparan sulfate proteoglycan expression, C-Xyloside improves the morphogenesis of the whole DEJ, and strongly suggests beneficial effects in aged skin from restoring DEJ integrity.     

Redox regulation of cellular stress response by ferulic acid ethyl ester in human dermal fibroblasts: role of vitagenes

Skin is one of the main targets for reactive oxygen species; thus, reactive oxygen species-induced damage and protein and lipid modifications occur, and skin can undergo a wide array of diseases, from photosensitivity to cancer. In this study, human dermal fibroblasts exposed to hydrogen peroxide (0-1000 micromol/L) exhibited a marked increase in both protein carbonyls and 4-hydroxy-2-nonenal, which are indices of protein and lipid oxidation, respectively. An amount of 25 micromol/L ferulic acid ethyl ester, a well-known nutritional antioxidant, significantly counteracted both protein and lipid oxidation and reduced the loss in cell viability elicited by 500 micromol/L of hydrogen peroxide. A common way for cells to react to oxidative stress is up-regulation of vitagenes. To the vitagene family belong the heat shock proteins heme oxygenase-1 and heat shock protein-70, which are involved in the cellular defense against oxidative stress by different mechanisms. The administration of 25 micromol/L ferulic acid ethyl ester significantly decreased hydrogen peroxide-induced protein and lipid oxidation. Dermal fibroblasts exposed to 25 micromol/L ferulic acid ethyl ester in the presence of 500 micromol/L hydrogen peroxide showed an increased level of both heme oxygenase-1 and heat shock protein-70 compared with dermal fibroblasts treated with hydrogen peroxide alone. These findings provide evidence for the protective role of vitagenes in free radical-induced skin damage and highlight the potential protective use of nutritional antioxidants, such as ferulic acid and its derivatives.     

Insufficient expression of the melanocortin‐1 receptor by human dermal fibroblasts contributes to excess collagen synthesis in keloid scars

Activation of the α‐melanocyte‐stimulating hormone (αMSH)/melanocortin‐1 receptor (MC1R) signalling pathway exerts antagonistic actions on cutaneous inflammatory and fibrogenic responses in addition to promoting pigment production. Herein, the expression of MC1R by keloid‐derived fibroblasts and keloid scar tissue was investigated using a range of techniques. MC1R mRNA expression levels in five different keloid fibroblast cell lines were significantly reduced to less than half compared with five normal fibroblast cell lines (P < 0.05). Immunohistological analysis of tissue samples indicated that MCR1 immunoreactivity in both epidermal and dermal compartments of five keloid tissue samples was dramatically decreased compared with normal skin (P < 0.05). Insufficient expression of MC1R on human dermal fibroblasts might abolish the αMSH‐mediated suppression of collagen production and myofibroblast transformation elicited by the profibrotic cytokine‐transforming growth factor‐β1. Restoration of reduced MC1R by dermal fibroblasts may lead to novel scar‐reducing therapeutic approaches for treating this refractory fibrotic disease.     

Caveolin-1 is a negative regulator of MMP-1 gene expression in human dermal fibroblasts via inhibition of Erk1/2/Ets1 signaling pathway

Background

Caveolar raft domains, also termed caveolae, are flask shaped invaginations that require the expression of the structural protein caveolin-1 (cav-1). Matrix metalloproteinase 1 (MMP-1) is a collagenase capable of degrading insoluble triple helical collagens. Deregulation of MMP-1 contributes to various pathological processes, including tissue fibrosis and impaired wound healing.

Objective

In this study we investigated the role of cav-1 in MMP-1 gene regulation in human dermal fibroblasts.

Methods

Fibroblasts were isolated from healthy subjects. Western blot was used to analyze protein levels and quantitative real time RT-PCR was used to measure mRNA expression. Cells were transiently transfected with siRNA oligos against acid sphingomyelinase (ASMase) and cav-1, or transduced with adenoviruses overexpressing ASMase and cav-1. The specific pharmacological inhibitors UO126 and SP600125 were used to block Erk1/2 and JNK activity.

Results

This study shows that siRNA-mediated depletion of ASMase or cav-1, results in upregulation of MMP-1 gene expression. Similarly, MMP-1 expression was decreased after overexpresssion of cav-1 via an adenoviral vector. Depletion of cav-1 had no effect on JNK phosphorylation, while it resulted in an increase in Erk1/2 and Ets1 phosphorylation levels. Furthermore, in cav-1 depleted cells treated with the Erk inhibitor UO126, there was no increase in the levels of phospho-Erk1/2, phospho-Ets1, and MMP-1, suggesting that cav-1 mediated effects on MMP-1 and phospho-Ets1 are Erk1/2 dependent.

Conclusions

In conclusion, this study has revealed an important role for cav-1 as a negative regulator of MMP-1 gene expression via inhibition of Erk1/2/Ets1 signaling. Cav-1 could potentially be a therapeutic target in diseases with deregulated extracellular matrix (ECM) turnover.     

Correlation between lipoprotein(a) and lipid peroxidation in psoriasis: role of the enzyme paraoxonase-1

Background Psoriasis is a chronic, inflammatory skin disease associated with abnormal plasma lipid metabolism and with a high frequency of cardiovascular events. Modifications of plasma lipids and an increase in the levels of biochemical markers of lipid peroxidation have been reported in subjects with psoriasis, suggesting a relationship between psoriasis, lipoproteins and oxidative damage. Objectives To investigate further the relationship between lipoproteins and oxidative stress in psoriasis. Method The levels of plasma lipids, lipoprotein(a) [Lp(a)] and markers of lipid peroxidation were evaluated in subjects with psoriasis (n = 23) and in controls (n = 25). In the same subjects, the activity of paraoxonase‐1 (PON1), an antioxidant and an anti‐inflammatory enzyme associated with high‐density lipoproteins, was investigated. Results The results showed higher levels of Lp(a) in the serum of patients with psoriasis compared with controls (P < 0·001). Higher levels of lipid hydroperoxides (P < 0·001) and lower PON1 activity were observed in the serum of patients compared with healthy subjects, confirming that psoriasis is associated with oxidative stress. The imbalance between oxidative stress and antioxidant enzymes, and the increase of Lp(a) serum levels was related to the extent and severity of psoriasis. Finally, our results demonstrated that Lp(a) levels were positively correlated with markers of lipid peroxidation and negatively related to PON1 activity, suggesting that subjects with higher levels of Lp(a) are more exposed to oxidative damage. Conclusions Our results provide further evidence that oxidative stress and impairment of the antioxidant system in the plasma of patients may play a role in pathogenesis and progression of psoriasis and related complications.     

The up-regulation of angiotensin II receptor type 1 and connective tissue growth factor are involved in high-glucose-induced fibronectin production by cultured human dermal fibroblasts

BACKGROUND: The expansion of extracellular matrix in diabetes occur in many tissues, including skin, but the underlying mechanisms are poorly understood. We were interested to study whether the activation of angiotensin II/receptor type 1 pathway with the consequent involvement of CTGF may be the possible cause of high-glucose-induced matrix abnormalities in cultured dermal fibroblasts. OBJECTIVE: We aimed to investigate the effect of high glucose on the generation of angiotensin II and the expression of angiotensin II receptors, and on the expression of CTGF mediating the fibronectin production in cultured human dermal fibroblasts. METHODS: Cell culturing, ELISA, semi-quantitative RT-PCR, Western blotting. RESULTS: High glucose treatment of cultured dermal fibroblasts led to: (1) the angiotensin II receptor type 1 was up-regulated at the level of mRNA and protein, unlike the receptor type 2; (2) the generation of angiotensin II and the mRNA expression of all components of the local renin-angiotensin system were not altered; (3) the mRNA and protein expression of CTGF was up-regulated, and this effect was cancelled by losartan; (4) the fibronectin production was increased, also was cancelled by losartan, while an anti-CTGF-neutralizing antibody only partly reduced it; (5) TGFbeta1 expression, the secretion of total and active TGFbeta1 were not changed; (6) the hyperosmotic action of high glucose had no effect. CONCLUSION: The up-regulation of angiotensin II receptor type 1 and the consequent increase of CTGF expression, independently of TGFbeta1, participate in high-glucose-induced fibronectin production in cultured human dermal fibroblasts.     

NADPH oxidase is a novel target of delphinidin for the inhibition of UVB‐induced MMP‐1 expression in human dermal fibroblasts

We investigated the reported antiphotoaging effects of the major anthocyanidin delphidin and sought to identify its specific molecular target during UVB‐induced MMP‐1 expression. Delphinidin treatment significantly inhibited UVB‐induced MMP‐1 expression in primary cultured human dermal fibroblasts (HDF), an effect associated with the suppression of MKK4‐JNK1/2, MKK3/6‐p38 and MEK‐ERK1/2 phosphorylation. Further investigation revealed that delphinidin significantly inhibited UVB‐induced ROS production and NOX activity. Interestingly, the inhibitory effect of delphinidin on UVB‐induced NOX activity was stronger than that of apocynin, a pharmaceutical NOX inhibitor. Fractioned cell analysis results using a Western blot assay showed that this effect occurred through the inhibition of UVB‐induced P47^phox (a NOX subunit) translocation from the cytosol to the membrane. Pull down assays demonstrated that delphinidin binds directly to P47^phox in vitro. Collectively, our results suggest that delphinidin targets NOX, resulting in the suppression of UVB‐induced MMP‐1 expression in human dermal fibroblasts.