皮肤基底细胞癌组织中血管内皮细胞生长因子受体-2的表达

目的 研究血管内皮生长因子受体-2(VEGFR-2)在皮肤基底细胞癌(BCC)组织中的表达情况.方法 提取BCC组织的mRNA,以RT-PCR法检测标本中VEGFR-2mRNA的表达.同时应用蛋白质印迹法检测VEGFR-2蛋白的表达,免疫荧光法检测VEGFR-2在BCC组织中的定位.结果 BCC组织标本mRNA和蛋白水平中均可以检测到VEGFR-2的表达,并较正常人对照有显著增加.VEGFR-2在BCC细胞主要表现为膜性分布.结论 皮肤BCC组织细胞能够高异常表达VEGFR-2,可能是导致BCC细胞肿瘤性增殖的原因之一.     

Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages

To localize epidermal growth factor (EGF) receptors in normal human epidermis and other skin structures, two different light microscopic methods were used. EGF binding [( 125I]EGF/R) to the extracellular portion of the EGF receptor was studied by incubating intact skin samples with [125I]EGF, sectioning the tissues, and performing autoradiography. Immunoreactive EGF receptor molecules (IR-EGF/R) were localized with a mono-specific anti-EGF receptor antibody using a 2-step indirect immunocytochemical method (horseradish peroxidase) and detergent permeabilized tissues. This latter method measured the total pool of EGF receptors: occupied and/or internalized forms, precursor forms, and partially degraded forms of the EGF receptor that retain immunoreactivity. Both the [125I]EGF/R and IR-EGF/R localization studies indicated that EGF receptors were present in basal epidermal keratinocytes, sebocytes, outer root sheath cells in hair follicles, smooth muscle cells of arrector pili muscles, and dermal arteries. The highest levels of [125I]EGF/R and IR-EGF/R were found in the dermal ducts of eccrine sweat glands. The distribution of both [125I]EGF/R and IR-EGF/R was not consistent with the concept that EGF exclusively is involved in cellular division and proliferation in normal human epidermis and its appendages, i.e., EGF receptors were also found in tissues that do not undergo rapid proliferation. The present study indicates that EGF may have a more complex regulatory role in the skin than was previously thought.     

Expression of the soluble vascular endothelial growth factor receptor-1 in cutaneous melanoma: role in tumour progression

Background Vascular endothelial growth factor (VEGF)‐A, placenta growth factor (PlGF) and their corresponding membrane receptors are involved in autocrine and paracrine regulation of melanoma growth and metastasis. Besides the membrane receptors, a soluble form of the VEGF receptor (VEGFR)‐1 (sVEGFR‐1) has been identified, that behaves both as a decoy receptor, sequestering VEGF‐A and PlGF, and as an extracellular matrix (ECM) molecule, promoting endothelial cell adhesion and migration through the interaction with α5β1 integrin. Objectives To analyse whether sVEGFR‐1 plays a role during melanoma progression. Methods sVEGFR‐1 expression was evaluated in a panel of 36 melanoma cell lines and 11 primary human melanocyte cultures by quantitative real‐time polymerase chain reaction analysis and in specimens of primary or metastatic melanoma lesions from 23 patients by immunohistochemical analysis. Results sVEGFR‐1 expression was highly upregulated in melanoma cell lines with respect to human melanocytes. Interestingly, cell lines obtained from cutaneous metastases showed a significant reduction of sVEGFR‐1 expression, as compared with cell lines derived from primary tumours. These results were confirmed by immunohistochemical analysis of sections from primary skin melanomas and the corresponding cutaneous metastases, suggesting that modulation of sVEGFR‐1 expression influences ECM invasion by melanoma cells and metastasis localization. Moreover, we provide evidence that adhesion of melanoma cells to sVEGFR‐1 is favoured by the activation of a VEGF‐A/VEGFR‐2 autocrine loop. Conclusions Our data strongly suggest that sVEGFR‐1 plays a role in melanoma progression and that low sVEGFR‐1/VEGF‐A and sVEGFR‐1/transmembrane VEGFR‐1 ratios might predict a poor outcome in patients with melanoma.     

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.     

Expression of vascular endothelial growth factor in a human hemangiosarcoma cell line (ISO-HAS)

Abstract Vascular endothelial growth factor (VEGF), in addition to being a specific mitogen of endothelial cells in vitro, is also known to induce angiogenesis in vivo. These functions suggest that VEGF may play an important role in the growth of hemangiosarcomas. Previous studies have demonstrated the expression of VEGF and its receptors, flt-1 or KDR/flk-1, in hemangiosarcomas by immunohistochemical staining and in situ hybridization. In the present study, however, we demonstrated that tumor cells of the hemangiosarcoma cell line ISO-HAS express mRNA of VEGF and its two receptors, flt-1 and KDR, and secrete VEGF protein. VEGF mRNA expression and protein secretion were found to be enhanced by phorbol 12-myristate 13-acetate. In addition, we demonstrated that ISO-HAS cells respond to recombinant human VEGF₁₆₅ with a dose-dependent up-regulation of cell proliferation and growth. These results suggest that the VEGF-VEGF receptor system plays a role in proliferation and growth of hemangiosarcoma cells. Received: 7 September 2000 / Revised: 2 December 2000 / Accepted: 3 March 2001     

Role of vascular endothelial growth factor in keloids: a clinicopathologic study

Background  Despite their benign nature, keloids are usually associated with considerable cosmetic effects and may lead to functional problems. Recently, it has been reported that vascular endothelial growth factor (VEGF), a potent angiogenic factor, is overexpressed in keloid tissue and may have a potential role in its evolution.
Methods  Twenty patients with keloids were included in this study and classified into two groups according to the treatment received: intralesional triamcinolone acetonide 20 mg/mL (group 1) and cryotherapy spray technique (group 2). Treatment was continued until clearance or for a maximum of six sessions, and the follow-up period was 1 year. Skin biopsies were taken from patients before and after treatment to evaluate keloid pathology and from patients and 10 healthy controls to detect the immunohistochemical expression of VEGF.
Results  Histopathologic examination revealed a remarkable resolution of the nodular arrangement of collagen after therapy, particularly in group 1. A statistically significant difference in VEGF expression was found between patients before therapy and controls, and between patients before and after therapy in each group. There was no significant difference in the treatment outcome between intralesional steroids and cryotherapy. No significant correlation was observed between the clinical variables of keloids and both VEGF expression and clinical response to therapy.
Conclusion  VEGF seems to play an important role in the pathogenesis of keloids and may be a useful guide in the evaluation of keloid therapeutics. Modulation of its production may provide a valuable treatment for keloids.     

血管内皮生长因子受体家族在HaCaT细胞中的表达

目的 探讨血管内皮生长因子受体(VEGFR)家族在角质形成细胞系HaCaT细胞的表达。方法 RT-PCR法检测HaCaT细胞中VEGFR家族中VEGFR-1、VEGFR-2、VEGFR-3及neuropilin (NRP)-1、NRP-2的mRNA表达,蛋白免疫印迹法检测VEGFR家族蛋白质的表达,同时免疫荧光法定位VEGFR家族在HaCaT细胞中的表达情况。结果 RT-PCR发现VEGFR-1、VEGFR-2、VEGFR-3及NRP-1、NRP-2 mRNA在HaCaT细胞中均有表达;蛋白免疫印迹发现VEGFR-1、VEGFR-2、VEGFR-3蛋白质相对分子质量均为180 000,NRP-1、NRP-2为140 000。HaCaT细胞膜及细胞质内检测到较强的VEGFR家族荧光信号,以细胞膜表达为强。结论 在mRNA及蛋白质水平,HaCaT细胞均表达VEGFR-1、VEGFR-2、VEGFR-3及NRP-1、NRP-2。     

血管内皮生长因子和转化生长因子β1在尖锐湿疣组织中的表达

目的:探讨血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)和转化生长因子β1(transforminggrowthfactorbeta1,TGFβ1)在尖锐湿疣(condylomaacuminatum,CA)组织中的表达及其可能作用。方法:观察并定量计数27例CA病变组织的血管数,应用逆转录(RT)-PCR和免疫组化方法检测VEGF和TGFβ1的表达情况,同时以17例正常包皮组织作为对照。结果:CA组织的血管数比正常包皮组织明显增多(t=5.059,P<0.01);VEGF和TGFβ1的mRNA在CA的表达水平分别为0.880±0.054、1.136±0.145;而二者在正常包皮组织内表达水平分别为0.448±0.095、0.784±0.085(P<0.05);VEGF和TGFβ1在CA组织除角质层外的表皮全层细胞均有强表达,正常包皮组织中为弱表达,且主要位于基底层细胞;VEGF和TGFβ1的表达呈显著正相关(r=0.792,P<0.001)。结论:CA组织细胞可产生并分泌较多的TGFβ1和VEGF,两者可能与CA组织中的血管生成有关。     

N-Acetylcysteine downregulates vascular endothelial growth factor production by human keratinocytes in vitro

Abstract The present study was designed to evaluate the action of various antioxidants including N-acetylcysteine (NAC) and the flavonoids resveratrol and quercetin on the production of VEGF by human keratinocytes (HKC). NAC, resveratrol, and quercetin dose-dependently suppressed the incorporation of ³H-thymidine into HKC. Values of median inhibitory concentration for NAC, resveratrol, and quercetin were 10 mM, 55 μM, and 15 μM, respectively (P < 0.01). RT-PCR demonstrated VEGF 121 and VEGF 206 expression in all HKC samples. HKC showed baseline expression and a progressive gradual time-dependent increase in VEGF secretion (510 ± 75 pg/ml at 24 h), and EGF (2.5–100 ng/ml) enhanced the secretion of VEGF in a dose-dependent fashion. HKC were incubated with NAC (2.5–20 mM) for 2 h prior to the addition of EGF (5 ng/ml) or PMA (10 ng/ml), and a significant decrease (P < 0.01) was found after 24 h of incubation with 2.5 mM NAC. However, neither resveratrol nor quercetin reduced the synthesis of this cytokine. In summary we conclude that NAC and the flavonoid antioxidants resveratrol and quercetin inhibit HKC proliferation regardless of the stage of differentiation and that NAC significantly inhibits VEGF secretion in basal and EGF- or PMA-treated HKC. Received: 28 February 2000 / Revised: 21 July 2000 / Accepted: 11 October 2000     

Inhibition of vascular endothelial growth factor expression in keloid fibroblasts by vector-mediated vascular endothelial growth factor shRNA: a therapeutic potential strategy for keloid

Vascular endothelial growth factor (VEGF) plays important roles in the regulation of angiogenesis and inflammation in both pathological and physiological wound repair. Several strategies have been developed for keloid therapy; however, a universally effective treatment has not been explored to date. In this study, three potential short interfering RNA (siRNA) sequences for the VEGF gene were cloned into expression plasmids and transfected into keloid fibroblasts. PGC-VEGF shRNA 2 (short hairpin RNA 2) plasmid significantly inhibited VEGF expression determined by real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and fibroblasts growth was also significantly by (methyl thiazolyl tetrazolium) MTT assay and apoptosis detection, whereas the control transfection showed no obviously effects. Plasminogen activator inhibitor-1 (PAI-1) expression in pGC-VEGF shRNA2 group was also obviously downregulated when compared to the pGC-VEGF shRNA negative control and mock group. These results suggest that modulation of VEGF production by vector-based RNAi in keloid fibroblasts may be a therapeutic potential strategy for keloid. Guo-You Zhang and Cheng-Gang Yi contributed equally to this work.     

Expression and localization of tissue kallikrein mRNAs in human epidermis and appendages

Tissue kallikreins are a group of serine proteases that are found in many organs and biologic fluids. Tissue kallikrein genes (KLKs) are found on chromosome 19q13.3-4 as a gene cluster encoding 15 different serine proteases. In skin, two tissue kallikrein proteins, hK5 and hK7, are expressed in the stratum corneum and are known to be involved in desquamation of corneocytes. The possible involvement of other kallikrein proteins has not been clarified, however, nor has the significance of each member in the serine protease activity of skin been delineated. In the study described here, we examined expression and localization of KLK mRNA in normal human skin by means of RT-PCR and in situ hybridization. Quantitative RT-PCR analysis showed abundant expression of KLK1 and KLK11 mRNA, moderate expression of KLK4, KLK5, KLK6, KLK7, and KLK13 mRNA, and low expression of KLK8 mRNA in normal human skin. For KLK4, KLK8, and KLK13 mRNA, splice variants were identified to be their major mRNA species. Two variants for KLK13 mRNA were novel. The amount of the serine protease inhibitor Kazal-type 5 (SPINK5) mRNA was comparable to KLK1 and KLK11 mRNA. In situ hybridization revealed intense expression of all KLK mRNA studied except KLK12 mRNA in the stratum granulosum of normal epidermis, where SPINK5 mRNA coexisted. Excluding KLK13 mRNA, they are also expressed in hair sheath, eccrine sweat glands, and sebaceous glands. Coexpression of various KLK and SPINK5 mRNA suggests that their proteins are the candidates to balance and maintain serine protease activities in both the skin and appendages.     

Expression of endothelial nitric oxide synthase and vascular endothelial growth factor in human malignant melanoma and their relation to angiogenesis

BACKGROUND: Angiogenesis is the major and key factor for growth and invasion of tumours, including malignant melanoma (MM), but the factors that contribute to tumour angiogenesis are still unclear. OBJECTIVE: To study expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) in human MM and their relation to angiogenesis. To investigate the correlation between eNOS and VEGF and the role of nitric oxide (NO) generated by eNOS in the process of mediating angiogenesis by VEGF. METHODS: Tissue sections from 31 patients with MM were examined using immunohistochemistry and morphological quantitative analysis for protein expression of eNOS and VEGF. Microvessel density (MVD) was counted in endothelial cells in immunostained by anti-FVIII:RAg antibody. RESULTS: Positive eNOS and VEGF immunostaining were observed in 77.4% and 83.9% of MM lesions, respectively, whereas pigmented naevi never expressed eNOS and VEGF. A positive correlation between eNOS and VEGF in MM was observed. Expression of eNOS and VEGF was positively correlated with MVD expression in MM, and MVD expression in MM was stronger than in pigmented naevi. Expression of eNOS and VEGF was not correlated with lymph node metastasis. CONCLUSIONS. On the basis of the current data showing that malignant melanocytic tumours displayed strong VEGF and eNOS expression, whereas benign melanocytic proliferations showed no immunoreactivity for VEGF and eNOS, such expression may be used as a discriminating factor to distinguish malignant melanoma from pigmented naevi. Expression of eNOS and VEGF may contribute to angiogenesis of MM, eNOS probably plays an important role in mediating VEGF-induced angiogenesis.     

血管内皮细胞生长因子在斑秃皮损中的表达

血管内皮细胞生长因子（vasculaqr endothelial cell growth factor,VEGF)是一种特殊作用于血管内皮细胞的丝裂原，在体内能诱导血管形成。为探讨VEGF为斑秃的关系，应用免疫组化的方法检测了VEGF在30例斑秃皮损中的表达和分布，以16例正常人头皮为对照。结果表明斑秃皮损中VEGF的表达明显低于正常对照。提示VEGF的表达下调可能与斑秃的发病有关。     

Immunohistochemical localization by monoclonal antibody of human epidermal growth factor in mixed tumours of the skin

Immunohistochemical distribution of human epidermal growth factor (hEGF) was described in 17 cases of mixed tumour of the skin with monoclonal antibody. In normal sweat glands, epithelial cells in the secretory portion and in the transitional area between secretory portion and duct showed prominent staining for hEGF. In the salivary pleomorphic adenoma type of mixed tumour of the skin, luminal tumour cells of tubular and duct-like structures gave a very characteristic hEGF staining reaction. The tumour cells showing strong staining for hEGF were scattered throughout the solid foci in this type of mixed tumour. Tubular epithelial cells in the clear cell adenoma type also displayed a positive hEGF reaction. And apocrine mixed tumours strong staining for hEGF occurred on the apical side of tubular and ductal tumour cells. In view of the immunohistochemical staining patterns for hEGF, the histologic origin of mixed tumours of the skin is suggested to be cells in the secretory portion and those in the transitional portion between secretory portion and duct of the sweat gland.     

Expression of vascular endothelial growth factor (VEGF) in various compartments of the human hair follicle

Abstract Hair follicle vascularization appears to be closely related to the processes involved in hair cycle regulation, in which growth factors, cytokines and other bioactive molecules are involved. In particular, vascular endothelial growth factor (VEGF), essential for angiogenesis and vascular permeability, may be responsible for maintaining proper vasculature around the hair follicle during the anagen growth phase. The aim of our study was to compare the in vitro angiogenic capacity, i.e. the steady-state expression of the VEGF gene, of different cultured cell types derived from normal human hair follicles, corresponding to different follicular compartments. Human dermal papilla cells (DPC), fibrous sheath fibroblasts, dermal fibroblasts, and follicular and interfollicular keratinocytes were cultured and studied in vitro for VEGF expression at the mRNA level using RT-PCR, and for VEGF protein synthesis by radioimmunoprecipitation and immunocytochemistry. In vivo examination for VEGF expression in human terminal hair follicles was performed using immunohistochemical methods. In the present report the expression of four different VEGF molecular isoforms, differing in their angiogenic capacity, are described in different cultured follicular cell types for the first time. Cultured follicular cells strongly expressed mRNA of four VEGF molecular species identified as the 121-, 145-, 165- and 189-amino acid splice variants, the most prominent being the 121-amino acid molecule. DPC, and also other mesenchymal cells such as fibrous sheath fibroblasts and dermal fibroblasts, in vivo and in vitro strongly expressed VEGF mRNA and synthesized a 46-kDa VEGF protein, whereas follicular and interfollicular keratinocytes in vitro expressed lower levels of VEGF mRNA and proteins than mesenchymal cells. As the highest expression of VEGF was found in DPC, we suggest that DPC are mainly responsible for angiogenic processes possibly related to the human hair cycle. Received: 14 January 1998 / Received after revision: 16 July 1998 / Accepted: 30 July 1998     

血管内皮生长因子和血管生成素在斑秃皮损中的表达

目的:探讨血管内皮生长因子(VEGF)和血管生成素在斑秃患者皮损处的表达情况.方法:取新鲜斑秃患者皮损和正常人头皮行冰冻切片,采用免疫组化的方法检测VEGF和血管生成素蛋白的分布和表达.结果:人毛囊的毛乳头、真皮鞘,皮脂腺等处均表达VEGF和血管生成素蛋白.斑秃患者此两种因子表达明显低于正常对照(P<0.05),且表达强度与斑秃严重程度呈负相关.结论:VEGF和血管生成素表达下调可能与斑秃的发病有关.