Keloids demonstrate high-level epidermal expression of vascular endothelial growth factor

BACKGROUND: Keloids are a major cause of morbidity, and arise after operation, injury, or cutaneous infection. Clinically, keloids differ from hypertrophic scars in that they grow beyond the original borders of the injury. Keloids occur most commonly for patients of African and Asian descent, and treatment options are multiple, indicating that there is no entirely satisfactory treatment for keloids. Angiogenesis inhibition has been shown to be effective in treatment of malignancy in both animal models and human beings. OBJECTIVE: We sought to determine whether keloids produce the potent angiogenic factor vascular endothelial growth factor (VEGF). METHODS: We performed in situ hybridization for VEGF on keloid tissue and normal skin. RESULTS: Our study demonstrated abundant production of VEGF in keloids and, surprisingly, the major source of VEGF was the overlying epidermis. CONCLUSIONS: Our results suggest that the overlying epidermis is the major source of keloid angiogenesis. These findings demonstrate that keloids are angiogenic lesions. Topical antiangiogenic therapy, directed at either down-regulating epidermal VEGF or inhibiting keratinocyte-derived VEGF activity on its endothelial receptors, may be useful in the treatment of keloids.     

瘢痕疙瘩成纤维细胞胶原合成的实验研究

目的 探讨瘢痕疙瘩中胶原过度聚集的原因.方法 从正常皮肤与活跃增生瘢痕疙瘩标本分别培养真皮成纤维细胞,采用³H-脯氨酸掺入法分别检测单层培养及胶原凝胶三维培养体系中成纤维细胞的胶原合成量.用斑点杂交法检测单层培养细胞的人前.α1(Ⅰ)型胶原mRNA水平.结果 瘢痕疙瘩成纤维细胞Ⅰ型胶原mRNA水平升高,其胶原合成量也显著高于正常真皮成纤维细胞(P<0.01).结论 在增生活跃瘢痕疙瘩中,成纤维细胞胶原合成功能处于活化状态,胶原合成增加是导致胶原过度积聚的重要原因.     

Collagen gene expression in keloids: analysis of collagen metabolism and type I, III, IV, and V procollagen mRNAs in keloid tissue and keloid fibroblast cultures

Regulation of collagen gene expression was studied in keloids and fibroblast cultures established from keloid biopsies from 9 patients. The collagen concentration in keloid tissue was not different from that in normal skin. The activities of 2 enzymes catalyzing intracellular collagen biosynthesis, prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT) were significantly elevated in the keloids, the mean increase in the former enzyme being 5-fold and in the latter 3-fold with respect to the controls. The mean procollagen production rate in the keloid fibroblasts was at the control level, with only 1 keloid cell line showing a procollagen synthesis rate higher than the mean value + 2 SD of the controls. The mean PH and GGT activities of the keloid fibroblasts were not elevated, but PH activity in 2 cell lines and GGT activity in 1 cell line were higher than the mean + 2 SD for the controls. Cellular type I, III, IV, and V procollagen mRNAs were measured by slot blot hybridization using specific human cDNA clones for the various collagen types. The amounts of type I, III, and V procollagen mRNAs corresponded to the ratios in which these collagen types are produced by fibroblasts. No synthesis of type IV procollagen mRNA by keloid fibroblasts was observed. The total amount of type I and III procollagen mRNAs correlated significantly (p less than 0.01) with the procollagen synthesis rate measured after radioactive labeling of the cells in the keloid and control fibroblasts, indicating that collagen production in these cells is mainly controlled by regulating the final steady state levels of collagen mRNA. The results suggest that fibroblasts isolated from keloids often synthesize normal amounts of collagen.     

Gene expression profiling reveals alteration of caspase 6 and 14 transcripts in normal skin of keloid-prone patients

Excessive scar formation in keloids points to altered tissue modeling and repair mechanisms. Dysregulation of cytokine and apoptotic cascades and their downstream signaling pathways might have a role in keloid development. Total RNA was isolated from biopsied keloidal tissue and adjacent normal skin of black patients, white patient’s scars, and normal skin of black and white patients, with normal wound healing. Apoptosis, cytokine and NFkB pathway microarrays were used to study and compare gene expression levels. Real-time PCR was used to verify microarray results in original samples and a separate, validation-set of samples. Significant differences were observed in the expression levels of members of caspase, cytokines and MAP kinase pathways, between the normal skin of keloid-prone and normal skin of keloid-resistant patients. Specifically, expression of caspase 6, and caspase 14 genes were different between normal skin of keloid-prone individuals and normal skin of keloid-resistant patients. Our results suggest that normal skin of keloid-prone individuals constitutively expresses a distinct gene profile which might contribute to their susceptibility to develop keloids.     

Co-ordinate induction of collagen type I and biglycan expression in keloids

Proteoglycans are macromolecules displaying structural roles as well as regulatory functions in the maintenance of the extracellular matrix. Biglycan/PG-I and decorin/PG-II are two small proteoglycans that are structurally related but differ considerably in their localization in vivo and behaviour in vitro. Decorin and, to a minor extent, biglycan, can be located at the surface of type I collagen fibrils and have been shown to influence collagen fibrillogenesis. However, the physiological role of biglycan in the dermis is not known. Biopsies obtained from keloids were bisected and processed for total RNA extraction and immunohistochemistry. Northern blot analysis of total RNA obtained from keloids with high growth tendency in vivo showed a marked induction of biglycan and collagen α1(I) mRNA expression in comparison with total RNA obtained from normal skin or keloids with little growth tendency. In contrast, decorin mRNA expression remained largely unaltered. Studying these biopsies by immunohistochemistry, decorin expression in the dermis was unaltered comparing normal and keloid tissue, whereas a markedly increased staining for biglycan was observed in the keloid tissue, which was most pronounced in the nodular formations, and was a characteristie feature of keloids. The altered expression of biglycan in keloid tissue might be involved in the abnormal regulation of extracellular matrix deposition either through the binding of growth factors or by influencing the three-dimensional organization of collagen fibres or associated molecules.     

瘢痕疙瘩相关基因的生物信息学研究

目的用基因芯片及生物信息学的方法确定瘢痕疙瘩致病的相关基因,探讨瘢痕疙瘩发生的分子机理。方法利用含有人约22000个基因的长寡核苷酸芯片,检测5例具有遗传家族史的瘢痕疙瘩与正常皮肤成纤维细胞的基因差异性的表达情况,采用非监督聚类的等阶聚类方法(Hierarchicalclustering)发现统计学意义上差异表达的基因,GO(geneontology)确定这些基因所属的功能群体,探讨其在瘢痕疙瘩发生发展中的重要作用。结果生物信息学分析表明,在5例瘢痕疙瘩成纤维细胞中表达趋势一致的上调基因有11个,下调基因有34个,其中与胶原代谢相关的差异表达基因数量最多。结论与正常皮肤成纤维细胞相比,瘢痕疙瘩发生过程中有多个基因的表达发生了明显地改变,其中代谢相关基因表达的改变可能在瘢痕疙瘩发生发展中发挥重要作用。     

结缔组织生长因子在瘢痕疙瘩中表达的研究

目的探讨结缔组织生长因子(CTGF)在瘢痕疙瘩发病中的作用。方法应用半定量逆转录聚合酶链反应技术检测30例瘢痕疙瘩患者皮损及对应邻近未受累皮肤中CTGFmRNA的表达,并以15例正常人皮肤组织作为对照。同时应用SP免疫组化技术对5例瘢痕疙瘩组织和5例正常人皮肤标本进行了检测。结果CTGFmRNA在瘢痕疙瘩及其边缘正常皮肤中的表达均明显高于正常人对照,差异有显著性(P<0.01)。瘢痕疙瘩CTGF的表达高低与病程无相关性(P>0.05)。免疫组化研究证实CTGF在瘢痕疙瘩中呈强表达,而在正常人皮肤中无表达。在瘢痕疙瘩组织边缘,CTGF表达呈现由强至弱的过渡现象。结论CTGF在瘢痕疙瘩中持续高度表达,提示其与瘢痕疙瘩的慢性纤维化有关。CTGF可能成为临床治疗瘢痕疙瘩的一个有力靶位。     

瘢痕疙瘩组织中ICAM-1、VEGF、c-fos表达的免疫组化检测

采取免疫组化SP方法检测了细胞间粘附分子 1(ICAM 1)、血管内皮细胞生长因子 (VEGF)、以及原癌基因 (c fos)在瘢痕疙瘩、普通瘢痕、正常皮肤组织中的表达。结果表明ICAM 1、VEGF、c fos在瘢痕疙瘩中表达皆增强。ICAM 1主要表达在真皮浅层血管、浸润的炎细胞、成纤维细胞 ;VEGF、c fos主要表达在表皮、真皮血管、皮肤附属器 ;部分瘢痕疙瘩标本成纤维细胞c fos染色阳性 ,提示瘢痕疙瘩组织中血管内皮细胞处于一种激活状态 ,瘢痕疙瘩组织血管内皮细胞和成纤维细胞增殖异常。     

成纤维细胞活化蛋白在瘢痕疙瘩组织中的表达及意义

 目的:了解成纤维细胞活化蛋白（FAP）在瘢痕疙瘩组织中的表达情况，探讨FAP在瘢痕疙瘩发病机制中的作用。方法:采用免疫组化染色技术检测30例瘢痕疙瘩组织（病例组）和20例正常皮肤组织（对照组）中FAP的表达强度，并比较两组间及瘢痕疙瘩不同临床分级之间FAP表达阳性率的差异。结果:病例组瘢痕疙瘩组织中FAP在成纤维细胞和血管内皮细胞内表达，阳性率为73.33％；对照组正常皮肤组织中未见FAP表达，两组间FAP表达阳性率比较,差异有统计学意义（  X²=26.19,P=0.001）；瘢痕疙瘩临床分级中,轻度与重度之间及中度与重度之间比较，FAP表达阳性率差异均有统计学意义（P值分别为0.002、0.006）。结论:瘢痕疙瘩组织中FAP表达阳性率明显高于正常皮肤组织；瘢痕疙瘩临床分级越严重，FAP表达阳性率越高；FAP可能参与瘢痕疙瘩的发病机制，针对FAP的干预可能有助于瘢痕疙瘩的治疗。     

瘢痕疙瘩组织中一氧化氮含量测定

目的 检测瘢痕疙瘩组织中一氧化氮（ＮＯ）的含量,探讨ＮＯ在瘢痕组织增生中的作用。方法 活检标本,奶疙瘩组、正常瘢痕组与正常皮肤组,每组８例。组织匀浆,用Ｇｒｉｅｓｓ试剂检测上清液中的ＮＯ２／ＮＯ３含量,以代表组织中ＮＯ的含量。结果 瘢痕疙瘩组织中ＮＯ含量显著高于正常瘢痕与正常皮肤组织中ＮＯ的含量,Ｐ值皆小于０．０１。正常瘢痕组与正常皮肤组之间差异无显著性。结论 ＮＯ可能在瘢痕疙瘩形成中起一定作用。     

瘢痕疙瘩组织中内皮素－1及碱性成纤维细胞生长因子mRNA的表达

目的 了解内皮素-1和碱性成纤维细胞生长因子 (bFGF)在瘢痕疙瘩组织的表达情况。 方法 组织活检标本分为 3组,瘢痕疙瘩、萎缩性瘢痕和正常皮肤。用地高辛标记的 cDNA探针,采取冰冻切片原位杂交的方法检测内皮素-1和 bFGF mRNA的表达。结果 瘢痕疙瘩真皮组织内内皮素-1 mRNA的表达明显强于萎缩性瘢痕和对照组。阳性染色主要位于真皮浅层血管和部分真皮成纤维细胞。瘢痕疙瘩组 6/8例真皮成纤维细胞内皮素-1 mRNA阳性,且呈弥漫性分布;萎缩性瘢痕和正常皮肤真皮成纤维细胞内皮素-1 mRNA无阳性着色。结论 内皮素-1和 bFGF mRNA在瘢痕疙瘩组织中的过度表达提示内皮素-1和 bFGF可能在瘢痕疙瘩的形成中起一定作用。     

Elevated prolidase activity in keloids: correlation with type I collagen turnover

BACKGROUND: Keloid pathogenesis involves an altered balance of extracellular matrix metabolism, mainly accumulation of type I collagen. This could be due to excessive synthesis or decreased degradation of matrix, or a combination of both processes. Prolidase, an imidodipeptide-cleaving cytosolic enzyme, plays an important role in the collagen catabolic process by recycling proline for collagen synthesis. Collagen accumulation in keloids is due to an imbalance in the steady state of collagen turnover. OBJECTIVES: To investigate prolidase activity and its role in the steady state of collagen turnover between normal skin and keloid tissue and their derived fibroblasts. METHODS: Ten sets of keloid and normal skin tissues and their derived fibroblasts were employed. Measurements were made of tissue prolidase activity, free proline level, and concentrations of the collagen synthesis product aminoterminal propeptide of type I procollagen (PINP) and the collagen degradative product carboxyterminal telopeptide of type I collagen (ICTP). Also, synthesis of collagens type I and III and matrix metalloproteinases 1 and 2 was investigated using Western blot analysis. RESULTS: Keloid tissues had a significant increase in prolidase activity, up to fourfold that in normal skin. The elevated prolidase activity was accompanied by an increase in tissue PINP and ICTP concentrations in keloid; in addition, the collagen turnover index (PINP/ICTP) was higher in keloids. CONCLUSIONS: The combination of elevated prolidase activity and associated higher collagen synthesis to degradation ratio in keloids suggests a possible metabolic process for the excessive accumulation of type I collagen in keloids.     

Activation of collagen gene expression in keloids: co-localization of type I and VI collagen and transforming growth factor-beta 1 mRNA

Untreated, clinically active keloids were examined as model system to study the spatial expression of extracellular matrix and transforming growth factor-beta 1 (TGF-beta 1) genes in fibrotic skin diseases. In situ hybridizations localized active expression of type I and VI collagen genes to the areas containing an abundance of fibroblasts and apparently representing the expanding border of the lesions. Within this zone, microvascular endothelial cells also expressed the type I collagen genes, as evaluated by simultaneous use of in situ hybridization for collagen gene expression and immunolocalization for factor VIII-related antigen, a marker for endothelial cell differentiation. Slot-blot hybridizations of RNA isolated from this zone suggested that the expression of type I and IV collagen genes was selectively enhanced, as compared to type III collagen gene expression. TGF-beta 1 protein and mRNA were also detected in areas active in type I and type VI collagen gene expression, indicating that TGF-beta 1 gene is transcribed and the corresponding protein is deposited in areas of elevated collagen gene expression, including microvascular endothelial cells. We conclude that the initial step in the development of fibrotic reaction in keloids involves the expression of the TGF-beta 1 gene by the neovascular endothelial cells, thus activating the adjacent fibroblasts to express markedly elevated levels of TGF-beta 1, as well as type I and VI collagen genes.     

Differences in collagen production between normal and keloid-derived fibroblasts in serum-media co-culture with keloid-derived keratinocytes

Keloids are characterized by the deposition of excessive extracellular-matrix collagen by abnormal fibroblasts in response to cutaneous injury. Studies to date have largely concentrated on the role of the keloid fibroblast in the pathogenesis of this lesion. Recent studies have highlighted the important concept of epithelial-mesenchymal interactions in normal skin biology. Extrapolating this to keloids in two recent serum-free in vitro studies, we demonstrated increased growth and proliferation, as well as induction of keloid-like collagen secretory characteristics in normal fibroblasts co-cultured with keloid-derived keratinocytes. Most fibroblast culture work to date has been performed in nutrient and growth factor-rich serum media. To investigate how a serum co-culture system might influence epithelial-mesenchymal interactions, [3H] proline incorporation was examined in normal and keloid fibroblasts co-cultured in serum with keratinocytes derived either from normal skin or keloid tissue. Results showed increased [3H] proline incorporation when normal fibroblasts were co-cultured with keloid keratinocytes, which was significantly increased when keloid fibroblasts were co-cultured with keloid keratinocytes. Taken with previous results, this study demonstrates a good correlation between both serum and serum-free co-culture systems, and supports the significance of epithelial-mesenchymal interactions in keloid pathogenesis.     

瘢痕旁和瘢痕下扩张器埋植治疗17例胸部瘢痕疙瘩

目的 探讨瘢痕旁和瘢痕下扩张器埋植治疗前胸部大面积瘢痕疙瘩的疗效。方法 从2006年3月至2009年6月,17例前胸部大面积瘢痕疙瘩患者共接受21个扩张器埋植。瘢痕面积最大15.7 cm × 5.5 cm,最小4.5 cm × 3.0 cm。其中瘢痕旁埋植12个,瘢痕下埋植9个。瘢痕旁埋植扩张器容量70 ~ 400 ml,瘢痕下埋植80 ~ 500 ml。经6 ~ 8周注水扩张后,行瘢痕疙瘩切除、扩张器取出和扩张皮瓣转移术,同时给予术中即时皮内注射复方倍他米松注射液、术后浅表电子束照射联合治疗,随访12 ~ 50个月。结果 除1个扩张器瘢痕下埋植后感染导致提前取出手术失败外,余20个扩张器均顺利完成整个治疗过程。主要并发症为扩张器外露4个,其中瘢痕旁1个,瘢痕下埋植3个,但未影响二期手术。扩张不满意2个,其中瘢痕旁和瘢痕下各1个。除2例复发外,余15例自觉症状均明显缓解,效果满意。2例复发患者均为扩张不满意,缝合时切口张力较大、且术后延期拆线者。结论 瘢痕旁和瘢痕下扩张器埋植为治疗前胸部大面积瘢痕疙瘩的较为理想的选择方法。切口缝合的张力是决定瘢痕疙瘩术后是否复发的关键。     

Gene profiling of keloid fibroblasts shows altered expression in multiple fibrosis-associated pathways

Keloids are benign tumors of the dermis that form during a protracted wound healing process. Susceptibility to keloid formation occurs predominantly in people of African and Asian descent. The key alteration(s) responsible for keloid formation has not been identified and there is no satisfactory treatment for this disorder. The altered regulatory mechanism is limited to dermal wound healing, although several diseases characterized by an exaggerated response to injury are prevalent in individuals of African ancestry. We have observed a complex pattern of phenotypic differences in keloid fibroblasts grown in standard culture medium or induced by hydrocortisone (HC). In this study Affymetrix-based microarray was performed on RNA obtained from fibroblasts cultured from normal scars and keloids grown in the absence and presence of HC. We observed differential regulation of approximately 500 genes of the 38,000 represented on the Affymetrix chip. Of particular interest was increased expression of several IGF-binding and IGF-binding-related proteins and decreased expression of a subset of Wnt pathway inhibitors and multiple IL-1-inducible genes. Increased expression of connective tissue growth factor and insulin-like growth factor binding protein-3 was observed in keloid fibroblasts only in the presence of HC. These findings support a role for multiple fibrosis-related pathways in the pathogenesis of keloids.     

肥大细胞在瘢痕疙瘩发病机理中作用的研究

为探讨肥大细胞在瘢痕疙瘩发病机理中的作用，用透射电镜观察活跃增生瘢痕疙瘩的超微结构。用组胺分别处理体外培养正常皮肤与瘢痕疙瘩成张纤维细胞，采用^3H-脯氨酸掺入、胃蛋白酶消化法检测其胶原合成量。结果：瘢痕疙瘩中有大量含有高度发达粗面内质网的成纤维细胞，还可见有脱颗粒的肥大细胞。瘢痕疙瘩成纤维细胞在胶原合成量已有增加的基础上，经一定浓度组胺作用，其胶原合成量仍有显著增加。结果示肥大细胞可有助于瘢痕疙瘩成纤维细胞胶原合成功能的活化及其活化表型的维持。