TGF-beta1 antisense therapy modulates expression of matrix metalloproteinases in keloid-derived fibroblasts

Transforming growth factor-beta1 (TGF-beta1) has been identified as an important regulator of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta1. The purpose of this study was to analyze the effect of TGF-beta1 targeting on the expression of matrix metalloproteinases (MMPs) in fibroblasts cultured from earlobe keloids. The expression of MMP-2 and -9 in tissue samples from keloids was investigated by immunohistochemistry. The effect of TGF-beta1 targeting using antisense oligonucleotides on the expression of MMPs in keloid-derived fibroblasts was analysed by ELISA and multiplex RT-PCR. Immunohistochemical studies demonstrated an increased expression of MMP protein in tissue samples from keloids compared to normal human skin. Antisense TGF-beta1 oligonucleotide treatment significantly downregulated MMP-9 secretion in vitro. In conclusion, TGF-beta1 antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in keloids.     

TGF-beta antisense oligonucleotides reduce mRNA expression of matrix metalloproteinases in cultured wound-healing-related cells

The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix. The 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. We investigated the effect of TGF-beta antisense oligonucleotides on the mRNA expression of matrix metalloproteinases in cultured human keratinocytes, fibroblasts and endothelial cells using multiplex RT-PCR. The treatment of keratinocytes and fibroblasts with TGF-beta antisense oligonucleotides resulted in a significant decrease of expression of mRNA of MMP-1 and MMP-9 compared to controls. Accordingly, a decreased expression of MMP-1 mRNA in endothelial cells was detectable. Other MMPs were not affected. Affecting all dermal wound-healing-related cell types, TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in chronic wounds. Pharmaceutical intervention in this area ultimately may help clinicians to proactively intervene in an effort to prevent normal wounds from becoming chronic.     

反义寡核苷酸抑制神经母细胞瘤LA-N-5细胞血管内皮生长因子mRNA表达及其生物效应的研究

目的研究血管内皮生长因子(VEGF)反义寡核苷酸(ASODN)转染对神经母细胞瘤LA-N-5细胞VEGF mRNA表达的影响以及对肿瘤细胞增殖、分化的影响。方法用LipofectamineTM2000介导的VEGF ASODN和错义寡核苷酸(MSODN)转染LA-N-5细胞,半定量RT-PCR检测各组细胞VEGF165和VEGF121 mRNA转染前后不同时间表达的变化;MTT法测定转染后各组细胞的生长曲线及抑制率。结果半定量RT-PCR检测结果显示,在转染后72 h VEGF165和VEGF121 mRNA的表达:ASODN组为0.346±0.029和0.227±0.036,ASODN+LipofectamineTM2000组为0.275±0.035和0.165±0.017。ASODN组和ASODN+LipofectamineTM2000组均显著抑制VEGF mRNA的表达,ASODN+LipofectamineTM2000组抑制作用较ASODN组更强(P<0.05);转染后ASODN组和ASODN+LipofectamineTM2000组细胞增殖显著受抑,在48 h时抑制率最高,分别为(39.92±2.7...     

Increased intrathecal levels of the angiogenic factors VEGF and TGF-beta in Alzheimer's disease and vascular dementia.

The aim of the present study was to investigate, in patients with Alzheimer's disease (AD), and vascular dementia (VAD), patterns of local release of vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-beta), two cytokines having a pivotal role in hypoxia-induced angiogenesis. The intrathecal levels of these molecules were related to the clinical severity of these diseases and to the intrathecal levels of beta-amyloid protein.Significantly increased cerebrospinal fluid (CSF) levels of both VEGF and TGF-beta were observed in 20 patients with AD and in 26 patients with VAD compared to healthy controls. Interestingly, there was significant correlation between the CSF levels of TGF-beta and VEGF in all the individuals studied.Our study demonstrates, both in patients with AD and in patients with VAD, an intrathecal production of VEGF, a cytokine which plays a pivotal role in angiogenesis. These results suggest that vascular factors might not only play a role in the pathogenesis of VAD but also in the pathogenesis of AD. In addition, we show in AD and VAD an intrathecal production of TGF-beta, a cytokine exerting on one hand anti-inflammatory and angiogenic properties, but on the other promoting amyloidogenesis.     

MIF反义寡核苷酸对巨噬细胞表达MIF的影响

目的：研究巨噬细胞移动抑制因子（MIF）反义寡核苷酸对体外巨噬细胞表达MIF的影响。 方法： 设计特异性MIF寡核苷酸，其序列为:反义: 5’-TACGGATACAAGTAGCAC-3’; 正义: 5’－ATGCCTATGTTCATCGTG-3’;错义: 5’－CTCTCAGACTCGATCTGT-3’。通过脂质体包裹后将其分别转染至小鼠巨噬细胞，观察MIF反义寡核苷酸对脂多糖（LPS）刺激巨噬细胞表达MIF的影响。 结果： LPS刺激后6 h，巨噬细胞表达MIF mRNA和合成分泌的MIF量增加，9-12 h达高峰。MIF反义寡核苷酸转染巨噬细胞后，可见LPS刺激的MIF mRNA和MIF的表达均明显少于LPS刺激组、LPS刺激＋转染正义寡核苷酸组和 LPS刺激＋转染错义寡核苷酸组（P<0.05），与非LPS刺激组无显著差异。 结论： LPS可刺激小鼠巨噬细胞合成和表达MIF。MIF反义寡核苷酸可显著抑制巨噬细胞MIF的过度表达。     

人反义VEGF基因对肾癌细胞VEGF表达的影响

目的：构建反义VEGF基因真核表达载体，研究其对肾癌细胞VEGF表达的影响。 方法： 克隆人VEGF基因，将反义VEGF基因定向克隆于质粒pcDNA3.1(-)真核表达载体，酶切鉴定；转染人肾癌细胞，G418筛选阳性克隆。RT-PCR方法检测VEGFmRNA的表达，免疫组化法检测VEGF基因的蛋白表达，MTT法测细胞生长，流式细胞术检测细胞周期。 结果： 成功构建反义VEGF基因真核表达载体；反义 VEGF组VEGFmRNA的表达受到抑制，明显低于空载体组和对照组VEGFmRNA的表达,空载体组VEGFmRNA的表达没有受到影响；反义 VEGF组的VEGF蛋白表达明显低于空载体组和对照组，空载体组和对照组VEGF蛋白的表达无显著差异；反义 VEGF组细胞生长减慢，G1期细胞比例增加，S期细胞比例减少。 结论： 人反义VEGF基因可明显降低肾癌细胞VEGF基因在转录和翻译水平的表达，抑制肾癌细胞生长，为肾癌基因治疗提供一定的实验依据。     

WT1 expression in angiogenic tumours of the skin

AIMS: To determine the expression of WT1 in endothelial proliferations and tumours. Endothelial cells are derived from angioblasts which differentiate into bone marrow stem cells (BMSC). BMSC are characterized by the constitutive expression of the WT1 gene and we have postulated that its expression may be maintained during the differentiation of angioblasts to endothelial cells. METHODS AND RESULTS: The expression of WT1 was studied in human umbilical vein-derived (HUVEC) and brain microvascular endothelial cells (HBME) as well as in a Kaposi sarcoma (KS) cell line in vitro. Forty-two human skin biopsy samples of endothelial proliferations and tumours were analysed for the protein expression of WT1 using the monoclonal antibodies for wt-WT1 (6F-H2) and its 17AA+ variant (2C12). WT1 expression was detectable in HUVEC and KS cells and all WT1 splice variants examined (17AA+/- KTS+/-) were detectable in KS cells, while the 17AA+/- and KTS- variants were present in HUVEC. Immunohistochemical analysis of the 42 human skin biopsy samples revealed cytoplasmic WT1 expression using wild-type specific antibody (6FH2) in microvessels, which is maintained during neoangiogenesis (inflammation, haemorrhage, peritumoral angiogenesis). Around one-third of haemangiomas (3/10) and non-HIV-Kaposi sarcomas (7/18) expressed the WT1 protein in the cytoplasm of tumour cells compared with its frequent expression in angiosarcomas (7/8) using the same antibody (6FH2). The nuclear 17AA+ isoform of WT1 was detectable at protein level in a small proportion of KS cases exclusively (3/7). CONCLUSION: Our data suggest that WT1 protein expression is maintained during angiogenesis and malignant transformation of endothelial cells and can be considered as a new endothelial marker.     

Increased expression of pro-inflammatory cytokines and metalloproteinase-1 by TGF-beta1 in synovial fibroblasts from rheumatoid arthritis and normal individuals

Transforming growth factor (TGF)-beta1 is expressed abundantly in the rheumatoid synovium. In this study, the inflammatory effect of TGF-beta1 in rheumatoid arthritis (RA) was investigated using cultured fibroblast-like synoviocytes (FLS) from RA and osteoarthritis (OA) patients, as well as non-arthritic individuals. mRNA expressions of IL-1beta, tumour necrosis factor (TNF)-alpha, IL-8, macrophage inflammatory protein (MIP)-1alpha and metalloproteinase (MMP)-1 were increased in RA and OA FLS by TGF-beta1 treatment, but not in non-arthritic FLS. Enhanced protein expression of IL-1beta, IL-8 and MMP-1 was also observed in RA FLS. Moreover, TGF-beta1 showed a synergistic effect in increasing protein expression of IL-1beta and matrix metalloproteinase (MMP)-1 with TNFalpha and IL-1beta, respectively. Biological activity of IL-1 determined by mouse thymocyte proliferation assay was also enhanced by 50% in response to TGF-beta1 in the culture supernatant of RA FLS. DNA binding activities of nuclear factor (NF)-kappaB and activator protein (AP)-1 were shown to increase by TGF-beta1 as well. These results suggest that TGF-beta1 contributes for the progression of inflammation and joint destruction in RA, and this effect is specific for the arthritic synovial fibroblasts.     

Abnormal gene expression in skin fibroblasts from a Hutchinson-Gilford patient.

We had the opportunity to investigate a new case of Hutchinson-Gilford progeria, a rare disease commonly regarded as a model in the study of aging. Two strains of fibroblasts (strains 1 and 2) were derived from two pieces of a skin biopsy. These two populations multiplied as normal cells at low population doubling level but senesced rapidly and stopped proliferating after 14 or 15 population doubling levels. Interestingly, an unusual pattern of growth in clusters was observed for strain 1. The level of collagen and noncollagen protein synthesis of both strains of affected fibroblasts was similar to that of normal fibroblasts as determined by [3H]proline incorporation measurement and was similarly affected by varying serum concentrations. The pattern of the main types of newly synthesized collagen polypeptides analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was similar in normal and progeria cells. The steady-state level of mRNAs coding for macromolecules of the extracellular matrix did not provide any differences between affected and control fibroblasts except for a strong increase of elastin and of alpha 1 and alpha 2 type IV procollagen mRNA mainly in strain 1 and less marked in strain 2. Interestingly, senescent progeria fibroblasts exhibited a reduced level of all the tested mRNAs, whereas collagen type IV and elastin mRNAs remained elevated. As suggested by immunofluorescence and immunoblotting studies, the increased amount of type IV mRNAs was paralleled by an enhanced production of type IV collagen by fibroblasts in vitro. Histologic examination of the skin revealed a superabundant network of abnormal elastic fibers in the reticular dermis and a thickening of basement membranes. The relationship between these alterations and aging in progeria is discussed.     

Extracellular matrix modulates expression of cell-surface proteoglycan genes in fibroblasts

Cell-surface proteoglycans are involved in many functions, including interactions with components of the extracellular microenvironment. They also act as coreceptors that bind and modify the actions of various growth factors, cytokines, and the extracellular matrix (ECM). This study investigated the regulation by the ECM of the expression of cell-surface proteoglycans (CD44, syndecan-1-4, betaglycan, glypican-1). We examined the changes in the expression levels of cell-surface proteoglycan genes in intact tendon, monolayer culture, and under various culture conditions. There was a significant increase in the expression of CD44 and syndecan-4 mRNAs during cell isolation from the tendon. With the switch to a 3D culture environment, there was a significant increase in the expression of CD44 at each passage point relative to its expression in 2D at those passage points. Syndecan-4 mRNA also increased steadily at each passage point in 3D culture environment. This influence on cell surface proteoglycans gene expression may indicate that collagen gel culture mimics in vivo tendon environment. This study provides further insight into the regulation of cell-surface proteoglycans in ligament and tendon fibroblasts by the ECM and 3D culture conditions.     

不同作用靶点的bFGF反义脱氧寡核苷酸对胶质瘤SWO-38细胞效应的观察

目的:比较3条针对不同位点的碱性成纤维细胞生长因子(bFGF)反义脱氧寡核苷酸抑制神经胶质瘤SWO-38细胞bFGF表达的效应。方法:用脂质体介导bFGF反义脱氧寡核苷酸的转染入SWO-38细胞, MTT及流式细胞术检测细胞增殖及凋亡的改变, Western-blot的方法检测bFGF蛋白水平的改变。结果:3条脱氧寡核苷酸链的细胞增殖抑制率分别为49%, 33%, 51%, 促进细胞凋亡率分别为35%、27%、18%, 对bFGF蛋白的表达降低分别为63%、42%、11%。结论:对细胞增殖活力、凋亡率、bFGF蛋白水平均有明显效应的脱氧寡核苷酸序列才是理想的反义物质。     

Differences in the expression of glycosaminoglycans in human fibroblasts derived from gingival overgrowths is related to TGF-beta up-regulation

Glycosaminoglycans (GAGs) play important roles in cell behavior and have the ability to bind and modulate cytokines. Using primary cultured fibroblasts from hereditary gingival fibromatosis (HGF), normal gingiva (NG), and NG treated with cyclosporin-A (NGc) we show changes in the expression and structural characteristics of GAGs as well as in the expression of enzymes involved in their biosynthesis and degradation. In addition, we show the over-expression of TGF-β1 and TGF-β type II receptor in HGF and NGc. There is an increase in the GAGs retained in the cellular fraction, and the fine structure of galactosaminoglycans show a decrease in α-l-iduronic acid content in HGF and NGc. Elevated extracellular levels of low molecular weight hyaluronan (HA) are found in HGF due to increase in the expression of HA synthase 3 and hyaluronidases 1 and 2. The results bring new insights to the accumulation of extracellular matrix related to TGF-β over-expression.     

Simvastatin inhibits growth factor expression and modulates profibrogenic markers in lung fibroblasts

Simvastatin is best known for its antilipidemic action and use in cardiovascular disease due to its inhibition of 3-hydroxy-3-methylglutaryl CoenzymeA (HMG CoA) reductase, a key enzyme in the cholesterol synthesis pathway. Inhibition of biological precursors in this pathway also enables pleiotrophic immunomodulatory and anti-inflammatory capabilities, including modulation of growth factor expression. Connective tissue growth factor (CTGF) and persistent myofibroblast formation are major determinants of the aggressive fibrotic disease, idiopathic pulmonary fibrosis (IPF). In this study we used human lung fibroblasts derived from healthy and IPF lungs to examine Simvastatin effects on CTGF gene and protein expression, analyzed by RT-PCR and ELISA, respectively. Simvastatin significantly inhibited (P < 0.05) CTGF gene and protein expression, overriding the induction by transforming growth factor-beta1, a known potent inducer of CTGF. Such Simvastatin suppressor action on growth factor interaction was reflected functionally on recognized phenotypes of fibrosis. alpha-smooth muscle actin expression was downregulated and collagen gel contraction reduced by 4.94- and 7.58-fold in IMR90 and HIPF lung fibroblasts, respectively, when preconditioned with 10 microM Simvastatin compared with transforming growth factor-beta1 treatment alone after 24 h. Our data suggest that Simvastatin can modify critical determinants of the profibrogenic machinery responsible for the aggressive clinical profile of IPF, and potentially prevents adverse lung parenchymal remodeling associated with persistent myofibroblast formation.     

Replicative potential of individual cell hybrids derived from young and old donor human skin fibroblasts

Neonatal, adult, and aged donor human skin fibroblast cell cultures have been characterized for the population doubling potential and in vitro-in vivo relationship. Hybrid cells derived from individual whole-cell fusions of replicating GRC 387 (aged) and CSC 303 (neonatal) cells demonstrated that an intermediate mode of replication between that of the two parental cell lines occurred; therefore the longevity of the aged fibroblast cells was enhanced. The GRC 387 cells have an extended post-replicative phase-out period in comparison with the CSC 303 cells, and the experimental hybrids demonstrated a 20–25 % increase of this period over that of the GRC 387 cells.     

Stage-dependent expression of an angiogenic agent and vascular organization in experimental skin tumor development

Increased angiogenesis and expression of antibodies to vascular endothelial growth factor (VEGF), an angiogenic agent, have been shown in the tumor development of many tissues. Areas of skin expressing VEGF and total volume of vessels expressing laminin in the wall were measured in chemical carcinogen-exposed mice using CAS-200 morphometry apparatus having a sensitivity exceeding 99% and reproducibility exceeding 99%. The area of VEGF expression was increased in carcinogen-exposed skin, dysplasia and in well-differentiated squamous cell carcinomas, but decreased in squamous cell carcinomas with decreased degree of differentiation. The vessel volume increased prior to the formation of tumors in carcinogen-exposed skin as well as in highly malignant neoplasms. In well-differentiated squamous cell carcinomas with an expansive growth pattern, the vessels were parallel to the basal membrane, in moderately differentiated tumors the vessels were in the direction of tumor invasion, and in poorly differentiated tumors, active angiogenesis consisted of numerous, enlarged vessels within the tumor. This study showed increased VEGF expression and number of vessels occurring in early stages of skin tumor development, pointing to a role of angiogenesis in chemical risk assessment and in cancer prevention. Altered vessel structure and vessel arrangement were distinct in later stages of tumor growth and in malignant neoplasms, pointing to the utility of detailed vessel analysis in neoplasm characterization.     

乳糖化多聚赖氨酸导向反义寡核苷酸抗乙型肝炎病毒的作用

目的 研究乳糖化多聚赖氨酸（Gal-PLL）导向反义寡核苷酸对乙型肝炎病毒（HBV）基因表达的特异性抑制作用。方法 根据聚合酶链反应（PCR）产物直接测序结果,在HBV U5样序列区合成了一段16聚硫代磷酸的反义寡核苷酸,并将其与肝靶向配体GaL-PLL连接,在2.2.15细胞比较,观察了它们对HBV基因表达的影响,结果 通过测序确定了2.2.15细胞中的HBV DNA属于HBV表面抗原ayw1亚型,并将此用于反义寡核苷酸的序列设计;经荧光组化分析表明,Gal-PLL对大鼠肝组织有选择性亲和力;Gal-PLL与DNA形成复合物的最佳摩尔比为2：1,在相同实验条件下,硫代磷酸反义寡核苷酸对HBsAg和HBeAg的抑制率分别为70％和58％,而Gal-PLL硫代磷酸反义寡核苷酸对HBsAg和HBeAg的抑制率分别为96％和82％,同时培养上清液和细胞中HBV DNA的含量明显下降,无关序列寡核酸无明显效果,寡核苷酸对细胞未产生毒性,结论 肝靶向配体和反义寡核酸的复合物可以通过无唾液酸糖蛋白受体靶入2.2.15细胞内,并对HBV基因表达和复制产生特异性抑制作用。     

Modulation of collagen synthesis and its gene expression in human skin fibroblasts by tocotrienol-rich fraction

Introduction

Skin aging may occur as a result of increased free radicals in the body. Vitamin E, the major chain-breaking antioxidant, prevents propagation of oxidative stress, especially in biological membranes. In this study, the molecular mechanism of tocotrienol-rich fraction (TRF) in preventing oxidative stress-induced skin aging was evaluated by determining the rate of total collagen synthesis and its gene expression in human skin fibroblasts.

Material and methods

Primary culture of human skin fibroblasts was derived from circumcision foreskin of 9 to 12 year-old boys. Fibroblast cells were divided into 5 different treatment groups: untreated control, hydrogen peroxide (H₂O₂)-induced oxidative stress (20 µM H₂O₂ exposure for 2 weeks), TRF treatment, and pre- and post-treatment of TRF to H₂O₂-induced oxidative stress.

Results

Our results showed that H₂O₂-induced oxidative stress decreased the rate of total collagen synthesis and down-regulated COL I and COL III in skin fibroblasts. Pre-treatment of TRF protected against H₂O₂-induced oxidative stress as shown by increase in total collagen synthesis and up-regulation of COL I and COL III (p<0.05) genes. However, similar protective effects against H₂O₂-induced oxidative stress were not observed in the post-treated fibroblasts.

Conclusions

Tocotrienol-rich fraction protects against H₂O₂-induced oxidative stress in human skin fibroblast culture by modulating the expression of COL I and COL III genes with concomitant increase in the rate of total collagen synthesis. These findings may indicate TRF protection against oxidative stress-induced skin aging.