The effects of ultraviolet A and reactive oxygen species on the mRNA expression of 72-kDa type IV collagenase and its tissue inhibitor in cultured human dermal fibroblasts

The effects of ultraviolet A (UVA) radiation and reactive oxygen species (ROS), generated with a xanthine and xanthine oxidase (XOD) system, on collagen enzymatic degradation involving the matrix metalloproteinase (MMP) and its tissue inhibitor of metalloproteinase (TIMP) were investigated using cultured human dermal fibroblasts. Total RNA was isolated and subjected to Northern blot analysis using cDNA clones for human interstitial collagenase (MMP-1), 72-kDa type IV collagenase (MMP-2) and TIMP-2. UVA irradiation resulted in an increase in MMP-1 mRNA up to 2.3-fold, but did not stimulate MMP-2 or TIMP-2 mRNA expression. In contrast, ROS induced by the xanthine and XOD system resulted in a dose-related increase in the level of MMP-2 mRNA up to 2.1-fold and a decrease in the level of TIMP-2 mRNA by 49% in the same fibroblasts. Catalase, used as scavenger, essentially prevented the ROS-induced alterations in MMP-2 and TIMP-2 mRNA levels. These results suggest that ROS produced in the dermis may contribute to biological changes in the connective tissue matrix observed in photoaging skin by accelerating the MMP-2-related matrix degradation system. Received: 6 September 1994     

苯甲酸雌二醇及UVA辐照对体外培养的人皮肤成纤维细胞胶原合成的影响

目的探讨苯甲酸雌二醇对体外培养的人成纤维细胞增殖、胶原合成及其对UVA抑制胶原合成的影响。方法在培养的人成纤维细胞中分别加入不同剂量的苯甲酸雌二醇,继续培养48h,用MTT法测定细胞的增殖情况,同时应用RT-PCR方法检测经不同剂量苯甲酸雌二醇及10J/cm²UVA处理后人成纤维细胞Ⅰ、Ⅲ型前胶原mRNA的表达情况。结果MTT法检测结果显示,苯甲酸雌二醇对体外培养的成纤维细胞增殖无明显促进作用。RT-PCR结果提示,苯甲酸雌二醇处理组两型前胶原的表达水平明显上调(P<0.05),而UVA照射后两型前胶原的表达显著下降,苯甲酸雌二醇可在一定水平对抗其作用(P<0.05)。结论苯甲酸雌二醇对体外培养的成纤维细胞Ⅰ、Ⅲ型前胶原的合成有一定的促进作用,并可对抗紫外线抑制胶原合成的作用。     

The ratio of glycosaminoglycans to collagen in the skin in malignant melanoma

Summary The interaction between glycosaminoglycans and collagen was determined in 24 subjects with malignant melanomas. The analysis indicates that the ratio of hexosamine to collagen was higher in the center of the tumor than in the outer areas. Our findings could result from dysregulation of the biosynthesis of glycosaminoglycanes.Dedicated in gratitude to Prof. J. Kühnau, MD, former director of the Institute of Physiological Chemistry, University of Hamburg on the occasion of his 80th birthday     

In vitro degradation of tropoelastin by reactive oxygen species

Abstract The effects of reactive oxygen species (ROS) on elastin molecules (tropoelastin) were studied in vitro. ROS generated by ultraviolet A and hematoporphyrin rapidly degraded tropoelastin within 5 min. Their degradative activity was inhibited by the addition of NaN₃. Treatment of tropoelastin with copper sulfate/ascorbic acid resulted in degradation of tropoelastin producing fragments of molecular weight 45, 30 and 10 kDa within 30 min. The degradation of tropoelastin was partially blocked by the addition of mannitol. ROS induced by the xanthine/xanthine oxidase system also degraded tropoelastin within 6 h. The degradation was blocked by catalase but not by superoxide dismutase (SOD). ROS generated by copper-ascorbate seems to be unique in that it cleaves relatively specific sites of the tropoelastin molecule. Thus ROS may play a degradative role in elastin metabolism which may cause the elastolytic changes or the deposition of fragmented elastic fibers in photoaged skin or age-related elastolytic disorders. Received: 15 January 1998     

Effects of histamine on collagen synthesis by cultured fibroblasts derived from guinea pig skin

Summary Fibroblast-like cells derived from guinea-pig skin were cultured for 3 h in the presence of various concentrations of histamine. The total protein synthesized was determined by the incorporation of radioactive proline, and the collagenous protein synthesized was measured by the incorporation of labeled hydroxyproline in the cell layer and medium. Synthesis of both total and collagenous protein increased in the presence of histamine in the concentration range of 10¹–10³ g/ml. The ratio of collagen to total protein synthesized also increased at these concentrations. However, in no case was an increase found when H₁ antagonist (chlorpheniramine) and H₂ antagonist (cimetidine) were added with the histamine. DNA synthesis was not affected by histamine at the concentrations used. These results suggest that histamine increases the synthesis of collagen by fibroblast-like cells through H₁ and H₂-receptors.     

活性氧簇在皮炎湿疹发病中的作用

活性氧簇是重要的氧的衍生物,氧化还原反应状态的平衡对维持机体的内环境起着重要的作用.皮肤是氧应激损害的主要靶器官,过多活性氧造成组织损伤,其主要通过影响核因子-κB、NO、蛋白激酶介导的磷酸化和基因异常表达发挥作用.多种炎症性皮肤疾病与活性氧簇相关,活性氧簇在皮炎、湿疹的发病中起着重要作用,抗氧化剂有望成为治疗这类疾病的新途径.     

Stimulation of collagen expression and glycosaminoglycan synthesis by midkine in human skin fibroblasts

Midkine is a retinoid-inducible regulator of cell differentiation and cell growth in developing embryonic tissues. The effects of midkine on the expression of the extracellular matrix components, collagens and glycosaminoglycans, in dermal fibroblasts were studied. Midkine did not alter fibroblast proliferation. Collagen synthesis was stimulated in a dose- and exposure time-dependent manner and the maximum twofold stimulation was achieved at a concentration of 100 ng/ml for 72 h treatment. Midkine increased the synthesis of type I and III collagens to the same extent. Glycosaminoglycan synthesis was also stimulated twofold, mainly acounted for by an increase in hyaluronan synthesis. Midkine enhanced mRNA levels of α ₁ (I), α ₁ (III), α ₁ (VI) collagens, transforming growth factor β1 and matrix metalloprotease-2 but did not alter the mRNA levels of elastin, MMP-1 and tissue inhibitor of metalloproteases-1. These results suggest that midkine is a potent stimulator of collagen and glycosaminoglycan synthesis and may prove useful in the therapy of delayed wound healing. Received: 9 October 1996     

Effects of glucocorticosteroids on cultured human skin fibroblasts

Summary As previously found, the glucocorticosteroid clobetasol-17-propionate inhibits cell proliferation during the early growth stage of normal baby foreskin fibroblasts and collagen synthesis in confluent cultures of these cells. The degree of inhibition of cell proliferation decreases with increasing cell density and, moreover, is transient.The anabolic steroids nandrolone and nandrolone-phenyl-propionate have similar effects on these cells. Likewise the magnitude of the inhibition is dose-dependent.When present together the two types of drug do not act in an additive manner. Even at low concentrations the anabolic steroids abolish the inhibitory effect of the glucocorticosteroid on cell proliferation. Furthermore, in this case only the inhibitory effect of the glucocorticosteroid on collagen synthesis is found and there is no further increase in this effect due to the presence of the anabolic steroids.Our results imply that the use of low concentrations of anabolic steroids combined with glucocorticosteroids in topical application to the skin may abolish some of the undesirable side effects of the glucocorticosteroids.     

金雀异黄素对体外培养的人皮肤成纤维细胞生物学活性和胶原合成的影响

目的 观察会雀异黄素对体外培养的正常人皮肤成纤维细胞生长和胶原合成的影响.方法 采用噻哗蓝(MTT)比色法检测不同浓度金雀异黄索对体外培养的人皮肤成纤维细胞存活力的影响,并绘制细胞的生长曲线;流式细胞仪检测细胞周期的变化;RT-PCR检测不同浓度金雀异黄素对成纤维细胞⒈型胶原mRNA表达的影响.结果 0.03125,0.0625,0.125,0.25,0.5,1 mg/L金雀异黄素作用24 h时,成纤维细胞增殖率分别为97.7%,113.8%,132.5%,116.4%,94.5%和83.3%.当金雀异黄素质量浓度大于0.5 mg/L,对成纤维细胞的增殖表现出抑制作用.浓度在0.0625～0.25 mg/L之间时,对成纤维细胞的增殖表现出促进作用.0.0625、0.125、0.25 mg/L金雀异黄素作用于成纤维细胞后,S期(41.15%±2.88%,61.89%±3.16%,48.18%±1.68%)和G2期(9.76%±3.99%,10.40%±0.54%,7.46%±2.47%)细胞明显高于对照组(S期为30.12%±0.60%,G2期为0.61%±0.16%),两组比较,差异均有统计学意义(P均＜0.05);而G1期细胞(49.08%±3.58%,30.04%±1.89%,44.36%±3.92%)明显低于对照组(69.27%±0.73%),两组比较,差异均有统计学意义(P均＜0.05);3个浓度组Ⅰ型胶原mRNA的表达(0.4814±0.0138,0.5767±0.0291,0.5675±0.0272)均高于对照组(0.4101±0.0236),两组比较,差异均有统计学意义(P均＜0.01).1 mg/L和0.5 mg/L的金雀异黄素作用成纤维细胞后,Ⅰ型胶原mRNA的表达(0.1662±0.0165,0.2017±0.0203)低于对照组,两组比较,差异均有统计学意义(P均＜0.01).结论 一定浓度的金雀异黄素可以促进正常人皮肤成纤维细胞的增殖和生长,并促进成纤维细胞Ⅰ型胶原mRNA的表达.     

申克孢子丝菌酵母相与白念珠菌诱导中性粒细胞活性氧簇的差异性表达

目的 研究人中性粒细胞吞噬申克孢子丝菌酵母相和白念珠菌后胞内活性氧簇（ROS）的实时表达水平及对两者杀菌能力的差异。 方法 应用密度梯度离心法分离人外周血中性粒细胞,并通过ROS探针（DCFH-DA）、流式细胞仪和激光共聚焦显微镜检测申克孢子丝菌酵母相临床株和白念珠菌标准株ATCC 90028作用后,中性粒细胞内ROS的实时表达水平及对上述2种真菌孢子的杀菌率。 结果 申克孢子丝菌酵母相临床株和白念珠菌标准株ATCC 90028作用中性粒细胞60 min后,申克孢子丝菌组中性粒细胞内ROS表达水平达到峰值（平均荧光强度159.67 ± 11.34）,并显著高于白念珠菌组（112.22 ± 9.66）,经LSD-t检验,P < 0.01;而作用120 ～ 180 min后,胞内ROS表达水平（120 min为89.01 ± 9.81;180 min为57.63 ± 8.46）迅速下降,显著低于白念珠菌组中ROS的同期表达水平（120 min时为110.25 ± 7.28;180 min时为109.98 ± 9.00,经LSD-t检验,120 min时P < 0.05,180 min时P < 0.01）。激光共聚焦显微镜观察到,ROS主要表达于吞噬上述真菌孢子的中性粒细胞中,并被募集到被吞噬的部分孢子表面。中性粒细胞对申克孢子丝菌180 min杀菌率（19.21% ± 3.68%）亦显著低于其对白念珠菌孢子的杀菌率（26.63% ± 4.97%）,经LSD-t检验,P < 0.01。 结论 中性粒细胞吞噬上述2种真菌孢子后,胞内ROS呈明显的差异性表达。与白念珠菌相比,中性粒细胞内ROS水平的迅速下降在一定程度上抑制了其对申克孢子丝菌的杀伤效能。 【关键词】 孢子丝菌属; 念珠菌,白色; 中性白细胞; 活性氧     

Generation of reactive oxygen species by Candida albicans in relation to morphogenesis

Candida albicans is able to generate significant amounts of reactive oxygen species (ROS). In this study, ROS generation by yeast and hyphal forms of the strain 3153 A was analyzed to determine whether ROS generation could be a major factor in the invasive behavior of germinative cells. Furthermore, the virulent strain CA6 and its avirulent and agerminative mutant VIR3 were compared. ROS were measured by lucigenin-enhanced chemiluminescence and a cytochrome c assay. During the blastoconidial phase of all strains moderate amounts of ROS were found at cell concentrations > 1 x 10(5)/ml. However, ROS generation appeared to be specifically inhibited at cell concentrations > 1 x 10(8)/ml, and this was found in both assays. As shown in comparative experiments, the medium used for measurement markedly affected the total amount of ROS. Hyphae of strain 3153 A generated a significantly higher amount of ROS than yeast cells and cells with germ tubes (P < 0.001). The strain CA6 showed significantly higher ROS generation than the VIR3 strain for both blastoconidiae and after 30 min of induction of hypha formation (P < 0.05). In conclusion, hypha formation, usually acknowledged as a major factor in Candida pathogenicity, was associated with markedly increased ROS formation. ROS generation was not closely linked to the ability to form hyphae, but was highest in germinative cells.     

Synthesis of glycosaminoglycans and collagen in skin fibroblasts cultured from a patient with lichen myxedematosus

Summary A patient is described with typical skin lesions of lichen myxedematosus and IgG-type lambda paraproteinemia. Fibroblasts cultured from the skin of the patient and from the skin of control persons were used to study glycosaminoglycan and collagen synthesis; the cultures were labelled with ³H-glucosamine and ³H-proline, respectively. Fibroblasts from the patient grew to a cell density which was lower than that of the control fibroblasts. The production of glycosaminoglycans was increased in lichen myxedematosus cultures, so that the ratio of hyaluronic acid to sulphated glucosaminoglycans was higher in the patient's cultures than in control cultures. Collagen production in the patient's cultures was about half of that in control cultures, whereas the ratio of type III to type I collagen was normal.     

枸杞多糖粗提物对紫外线诱导HaCaT细胞清除活性氧的影响

目的 探讨枸杞多糖粗提物对紫外线诱导的HaCaT细胞清除活性氧(ROS)的影响及可能机制.方法 将HaCaT细胞分为空白组、枸杞多糖组、UVA组、UVB组、UVA+枸杞多糖组、UVB+枸杞多糖组.用噻唑蓝法检测细胞增殖活性;分光光度计检测枸杞多糖粗提物对UVA和UVB吸收情况;用DCFH-DA荧光探针检测细胞内ROS水平;酶生化法测定胞质超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性及乳酸脱氢酶(LDH)漏出量.结果 0、100、200、300、400、500、600、1 500、2 000 mg/L枸杞多糖粗提物对HaCaT细胞增殖活性无明显影响.枸杞多糖粗提物对280 ～400 nm紫外线透光率较大;与空白组比较,UVA和UVB组LDH漏出量、ROS水平显著升高,胞内SOD及GSH-Px活力降低,差异均有统计学意义(P＜0.001或0.05).照射前加枸杞多糖粗提物(UVA+枸杞多糖组、UVB+枸杞多糖组)可明显升高细胞内SOD、GSH-Px活性,减少LDH释放,降低ROS水平,与UVA或UVB组比较,差异均有统计学意义(P＜0.05).结论 枸杞多糖粗提物不具有遮光剂的作用,但可有效清除ROS,降低LDH漏出率,抑制紫外线致HaCaT细胞光损伤,可能与其增强抗氧化酶活性有关.     

Ultraviolet light induces Stat3 activation in human keratinocytes and fibroblasts through reactive oxygen species and DNA damage

Please cite this paper as: Ultraviolet light induces Stat3 activation in human keratinocytes and fibroblasts through reactive oxygen species and DNA damage. Experimental Dermatology 2010; 19: 654–660. Abstract: Stat3 is activated by the outer stressors, such as ultraviolet (UV) exposure. In this study, we investigated the Stat3 response to UV irradiation in human epidermal keratinocytes and dermal fibroblasts. Results indicated that UVB and UVC differentially activate Stat3 in these cells. The UV‐induced Stat3 activation was mediated by both reactive oxygen species (ROS) and DNA damage, and the dominancy of ROS and DNA damage to activate Stat3 depended on the wavelength of UV. By using fibroblasts from a patient with xeroderma pigmentosum A (XP‐A) and those transfected with human XPA gene, we found that UVB activates Stat3 via both ROS and DNA damage, while UVC does so mainly via DNA damage. The present data suggest that Stat3 activation in UV‐exposed human skin is one of the initial events where DNA damage and ROS are involved.     

咪喹莫特对瘢痕疙瘩成纤维细胞增殖和胶原产生的影响

目的 探讨咪喹莫特对瘢痕疙瘩成纤维细胞增殖和胶原产生的影响.方法 从手术切除的瘢痕疙瘩组织中培养成纤维细胞,加入不同浓度的咪喹莫特作用后,观察细胞的形态学变化,MTT法检测细胞的活性.免疫组化和蛋白免疫印迹法检测咪喹莫特对瘢痕疙瘩成纤维细胞Ⅰ型、Ⅲ型前胶原产生的影响.结果 在10～100μg/mL范围内,咪喹莫特能显著抑制瘢痕疙瘩成纤维细胞的增殖,且存在剂量和时间依赖性;进一步检测到咪喹莫特作用后瘢痕疙瘩成纤维细胞Ⅰ型、Ⅲ型前胶原表达减弱.结论 咪喹莫特能有效抑制瘢痕疙瘩成纤维细胞的增殖和Ⅰ型、Ⅲ型前胶原的产生.     

Solar simulated irradiation modulates gene expression and activity of antioxidant enzymes in cultured human dermal fibroblasts

Exposure of skin to solar irradiation generates reactive oxygen species that damage DNA, membranes, mitochondria and proteins. To protect against such damage, skin cells have evolved antioxidant enzymes including glutathione peroxidase (GSH-Px), copper and zinc-dependent superoxide dismutase (SOD1), the mitochondrial manganese-dependent superoxide dismutase (SOD2), and catalase. This report examines the effect of a single low or moderate dose exposure to solar-simulating combined UVB and UVA irradiation on the gene expression and activities of these antioxidant enzymes in cultured normal human fibroblasts. We find that both doses initially decrease GSH-Px, SOD2 and catalase activities, but within 5 days after irradiation the activities of the enzymes return to pre-irradiation level (catalase) or are induced slightly (SOD1, GSH-Px) or substantially (SOD2) above the basal level. For SOD1, SOD2 and catalase, the higher dose also detectably modulates the mRNA level of these enzymes. Our results indicate that the effects of a single physiologic solar simulated irradiation dose persist for at least several days and suggest that skin cells prepare for subsequent exposure to damaging irradiation by upregulating this antioxidant defense system, in particular the mitochondrial SOD2. Our findings are consistent with the existence of a broad-based SOS-like response in irradiated human skin.     

Scleroderma fibroblasts: Some aspects of in vitro assessment of collagen synthesis

Summary Fibroblasts were cultured from skin biopsies of patients with systemic sclerosis in different stages of the disease. In vitro synthesis of collagen was checked after a pulse with tritiated proline. The ratio between type I and type III collagen was normal in all patients. Six of seven cultures derived from patients in the active state showed an increased synthesis of collagen relative to other proteins. Addition of serum (normal and diseased) to the culture medium did not stimulate synthesis of collagen in any culture with normal collagen synthesis.

---

Abbreviations ANA antinuclear antibodies - ENA extractable nuclear antibodies - SS systemic scleroderma - CRST calcinosis-Raynaud-sclerodactylia-telangiectasia - SD standard deviation Supported by the Deutsche Forschungsgemeinschaft (MU 378/10, Kr 558/3)