Levels of endothelial cell stimulating angiogenesis factor and vascular endothelial growth factor are elevated in psoriasis

Neovascularization appears to play an early and important part in the evolution of psoriatic plaques. We studied the distribution and production of two known angiogenesis factors, endothelial cell stimulating angiogenesis factor (ESAF) and vascular endothelial growth factor (VEGF), in the skin of patients with chronic plaque psoriasis and normal control subjects. Our results showed that tissue levels of ESAF and VEGF were significantly elevated in involved as compared with normal control skin (P = 0.006 and P < 0. 0001, respectively). Tissue levels of ESAF and VEGF were also raised in involved skin as compared with uninvolved skin in patients with psoriasis (P = 0.001 and P < 0.0001, respectively). Tissue levels of ESAF and VEGF in plaques of psoriasis correlated closely with the clinical severity of psoriasis (r = 0.6 and r = 0.9, respectively). Serum levels of ESAF and VEGF were significantly raised in patients with psoriasis as compared with control subjects (P = 0.001 and P = 0.02, respectively). In vitro culture studies revealed that ESAF is produced by both keratinocytes and fibroblasts in approximately equal quantities in normal skin, whereas VEGF is secreted predominately by keratinocytes. A similar pattern is seen in both involved and uninvolved skin of patients with psoriasis. However, there is increased secretion of both factors in keratinocytes and fibroblasts from involved and uninvolved skin as compared with normal control skin (P < 0.001). The increased levels and secretion in plaques of psoriasis of two molecules, ESAF and VEGF, known to promote new blood vessel formation, suggest a pathogenetic role for them in this disease.     

Ultraviolet B-induced skin angiogenesis is associated with a switch in the balance of vascular endothelial growth factor and thrombospondin-1 expression

We have previously shown that targeted overexpression of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in the epidermis prevented chronic ultraviolet B (UVB)-induced angiogenesis and cutaneous photodamage in mice, suggesting that angiogenesis plays a critical role in the mediation of UVB effects on the skin. Nevertheless, the molecular regulation of angiogenesis factors and inhibitors by acute UVB irradiation still remains to be elucidated. We performed quantitative analyses of cutaneous vascularity and of vascular endothelial growth factor (VEGF) and TSP-1 expression after acute UVB irradiation of mouse skin. Skin vascularity in the upper dermis was greatly increased until day 8 after a single dose of UVB irradiation (200 mJ per cm2) and associated with upregulation of VEGF mRNA expression, with downregulation of TSP-1 mRNA, and with protein expression in the hyperplastic epidermis. After 13 days, skin vascularity was normalized with downregulation of VEGF mRNA expression and upregulation of TSP-1 mRNA expression to the levels observed in non-UVB-irradiated skin. In contrast, the angiogenic UVB response was prolonged in TSP-1-deficient mice. We found a pronounced induction of the TSP-1 receptor CD36 in CD31-positive vessels on day 8 after UVB irradiation, associated with vascular endothelial cell apoptosis. These results demonstrate that acute UVB irradiation leads to a shift toward a proangiogenic environment and they suggest that the balance between VEGF and TSP-1 plays a critical role in the control of angiogenesis and vascular regression induced by acute UVB irradiation.     

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     

Increased expression of MAP2 inhibits melanoma cell proliferation,invasion and tumor growth in vitro and in vivo

Please cite this paper as: Increased expression of MAP2 inhibits melanoma cell proliferation, invasion and tumor growth in vitro and in vivo. Experimental Dermatology 2010; 19 : 958–964. Abstract: Malignant melanoma (MM) is characterized by aggressive metastasis and high mortality rate. Microtubule‐associated proteins 2 (MAP2) is expressed abundantly in majority of melanocytic nevi and primary melanomas, but absent in metastatic melanomas. To determine whether MAP2 correlates with tumor progression of MM, we investigated the effects of MAP2 inhibition on the biological behaviour of metastatic melanoma in vitro and in vivo. Our results demonstrated that adenovirus‐mediated MAP2 induced apoptotic cell death and cell cycle arrest in metastatic human and mouse melanoma cell lines in vitro, and substantially inhibited the growth of melanomas in nude mice in vivo. In addition, intracellular expression of MAP2 was found to induce the morphologic alteration, suppress the migration and invasion and affect the assembly, stabilization and bundling of microtubules in melanoma cells. This is the first study that MAP2 expression significantly inhibits the growth of MM in vivo. Our results suggest that MAP2 may serve as a promising molecular target for therapy and chemoprevention of MM in humans.     

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.     

Expression of basic fibroblast growth factor,vascular endothelial growth factor,and thrombospondin-1 related to microvessel density in nonaggressive and aggressive basal cell carcinomas

The aggressive forms of basal cell carcinoma (BCC) are biologically different in a number of features from their nonaggressive counterparts. The expression of basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF), and Thrombospondin-1 (TSP-1) was examined in primary culture samples of nonaggressive and aggressive BCCs. We studied the relationship between neoangiogenesis by counting microvessels, tumor aggressiveness, and the expression of three different angiogenic factors. bFGF mRNA was detectable in all BCC samples, but there was no significant difference in the levels of expression between the two types. VEGF mRNA was also detectable in all BCC samples. VEGF expression in the aggressive type was approximately 3.5-fold higher than in the nonaggressive type. TSP-1 expression was variable and not related to the type of BCC. The mean value of microvessel density (MVD) was significantly higher for the aggressive type than for the nonaggressive type. There was a significant correlation between VEGF levels and MVD. No significant relationships were found between bFGF, TSP-1 mRNA expression, and MVD. In conclusion, the results of present study suggest that VEGF expression and angiogenesis might play important roles in the progression to aggressive BCC.     

Expression and localization of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 in human epidermal appendages: a comparison study by immunofluorescence

Background.  Vascular endothelial growth factor (VEGF) promotes angiogenesis and plays important roles in neovascularization and development of tissues. VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and are originally considered specific to endothelial cells. We have previously shown that keratinocytes from human normal skin express VEGFRs. This poses the question of whether these receptors are also expressed by epidermal appendages, as epidermal appendages are lined with epithelial cells.
Objective.  To investigate the expression of VEGFR-2 compare with VEGF in epidermal appendages, including hair follicles, eccrine sweat glands and sebaceous glands.
Methods.  Monoclonal antibodies to VEGF and VEGFR-2 were used for immunohistochemical examination of cryostat-cut sections of normal human skin specimens from 11 donors undergoing cosmetic surgery.
Results.  Immunoreactivities for VEGF and VEGFR-2 principally showed parallel intense expression in anagen hair follicle (including outer root sheat, inner root sheath, dermal papillae epidermal matrix), sebaceous glands (ductal and secretory portions) and eccrine sweat glands (ductal and secretory portions), respectively. In particular, abundant expression of VEGF was found in the follicular basement membrane zone surrounding the bulb matrix and in the ductal and secretory portions of eccrine sweat glands.
Conclusion.  A potential VEGF/VEGFR-2 autocrine pathway may be defined by the coexpression of VEGF and VEGFR-2 in human skin epidermal appendages.     

Knockdown of Skp2 by siRNA inhibits melanoma cell growth in vitro and in vivo

BACKGROUND: Low levels of p27Kip1 expression are associated with poor prognosis in various malignancies including malignant melanoma. Recently, it has been reported that S phase kinase-interacting protein 2 (Skp2), the specific ubiquitin ligase subunit that targets p27Kip1 for degradation, was overexpressed and was inversely related to p27Kip1 levels in malignant melanoma with poor prognosis. OBJECTIVE: We investigated whether small interfering RNA (siRNA)-mediated gene silencing of Skp2 can be employed in order to inhibit p27Kip1 down-regulation and suppress melanoma cell growth as a consequence in vitro and in vivo. METHODS: We constructed a plasmid vector, which synthesizes siRNAs to determine the effects of decreasing the high constitutive levels of Skp2 protein in melanoma cells. Western blot and real-time RT-PCR were performed to examine the decreases of Skp2 protein and mRNA in vitro. Furthermore, melanoma cells were injected into the back of nude mice subcutaneously to examine the suppression of tumorigenicity targeting Skp2 gene silencing in vivo. RESULTS: Skp2 protein was decreased and the p27Kip1 protein was accumulated in Skp2 siRNA transfected melanoma cells. Skp2 siRNA inhibited the cell growth of melanoma cells in vitro. Moreover, Skp2 siRNA also suppressed tumor proliferation in vivo. CONCLUSION: Our results suggest that siRNA-mediated gene silencing of Skp2 can be a potent tool of cancer gene therapy for suppression of p27Kip1 degradation in malignant melanoma.     

Photochemotherapy inhibits angiogenesis and induces apoptosis of endothelial cells in vitro

BACKGROUND: Photochemotherapy has long been used in the treatment of psoriasis; however, its mechanism has not been completely elucidated. Psoriasis is now regarded as an angiogenesis-related disease. Recent studies indicated that the inhibition of angiogenesis by photochemotherapy could be an underlying mechanism. It was found that photochemotherapy can downregulate the expression of angiogenic factors in keratinocytes. However, the direct effect of photochemotherapy on endothelial cells has not been studied. METHODS: In this study, we determined the effect of photochemotherapy on the proliferation of human microvascular endothelial cells through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and cell cycle analysis. The migration assay and in vitro tube formation assay were used to investigate the migration properties and tube formation ability of human microvascular endothelial cells after psoralen plus UVA (PUVA) treatment. The apoptosis of endothelial cells elicited by photochemotherapy was also analyzed with fluorescence-activated cell sorting analysis (FACS). RESULTS: UVA (0.8-5.0 J/cm(2)) irradiation with the presence of 8-methoxypsoralen (8-MOP) (300 ng/ml) resulted in a dose-dependent reduction in the cell viabilities of endothelial cells. FACS data showed an accumulation of cells in G0/G1 phase of cell cycle and apoptotic features of cell death after UVA irradiation with psoralen. The migration properties and tube formation ability of endothelial cells were dramatically inhibited by photochemotherapy. CONCLUSION: Our results showed that photochemotherapy inhibits angiogenesis and induces apoptosis of human microvascular endothelial cells in vitro, which may be a possible mechanism of photochemotherapy in the treatment of psoriasis.     

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     

重组人色素上皮衍生因子对HaCaT细胞体外增殖和白细胞介素6、8及血管内皮生长因子表达的影响

目的 探讨重组人色素上皮衍生因子（rhPEDF）对HaCaT细胞体外增殖和白细胞介素6（IL-6）、IL-8及血管内皮生长因子（VEGF）表达的影响。 方法 HaCaT细胞分4组进行不同处理,分别为100 μg/L rhPEDF组、50 μg/L rhPEDF组、25 μg/L rhPEDF组及对照组（只加RPMI 1640培养液）。采用CCK8法检测不同浓度rhPEDF作用于HaCaT细胞24、48、72 h后对细胞体外增殖的影响。RT-PCR和Western印迹法检测rhPEDF对HaCaT细胞IL-6、IL-8、VEGF mRNA及其蛋白表达的影响。统计分析方法采用两因素方差分析、单因素方差分析、SNK-q检验以及Pearson相关分析。 结果 25、50、100 μg/L的rhPEDF作用24、48、72 h后对HaCaT细胞体外增殖均有不同程度的抑制作用。随时间延长和rhPEDF浓度的增加,rhPEDF对HaCaT细胞体外增殖的抑制作用均逐渐增强（F = 1115、329.9,均P < 0.001）。与对照组相比,不同浓度（100、50、25 μg/L）rhPEDF作用于HaCaT细胞48 h后,VEGF、IL-6、IL-8 mRNA及蛋白的表达均下降,差异均有统计学意义（P < 0.05）。随rhPEDF浓度的增加,各浓度rhPEDF组VEGF mRNA、IL-6和IL-8蛋白表达均出现下调,与对照组比较,差异均有统计学意义（P < 0.05）;100 μg/L rhPEDF组的IL-6、IL-8 mRNA表达明显低于25 μg/L rhPEDF组（P < 0.05）;100 μg/L rhPEDF组VEGF蛋白表达分别低于50、25 μg/L rhPEDF组（P < 0.05）,而50与25 μg/L rhPEDF组间差异无统计学意义（P > 0.05）。 结论 rhPEDF可抑制HaCaT细胞体外增殖,并可在mRNA及蛋白水平下调IL-6、IL-8、VEGF的表达。     

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.     

环氧合酶-2和血管内皮生长因子mRNA在皮肤鳞状细胞癌组织中的表达

目的 探讨环氧合酶-2(COX-2)、血管内皮生长因子(VEGF)mRNA在皮肤鳞状细胞癌(SCC)和正常人皮肤组织中的表达以及它们之间相互关系。方法 应用逆转录-聚合酶链反应(RT-PCR)方法,检测24例SCC和10例正常人皮肤组织中COX-2和VEGF mRNA的表达。结果 RT-PCR结果显示,在正常人皮肤组织中COX-2和VEGF mRNA呈较弱表达或无表达,吸光度平均值分别为(0.01±0.01)和(0.02±0.02);有79.2%(19/24)SCC组织中COX-2 mRNA表达水平增高,平均值为(0.56±0.48),与正常皮肤组织比较差异有统计学意义(P<0.05);VEGF mRNA在SCC组织中全部高表达(24/ 24,100%),平均值为(0.66±0.35),与正常人皮肤组织比较差异有统计学意义(P<0.05)。经相关性分析两者之间的表达呈明显正相关(r=0.86)。结论 COX-2可能与SCC血管形成有关,且其作用可能通过上调VEGF通道来发挥作用。     

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。     

人肝细胞生长因子对体外培养的黑素细胞生物学活性的影响

目的 探讨人肝细胞生长因子（rhHGF）对体外培养正常人黑素细胞生物学活性的影响。方法 分别用不同浓度的rhHGF（0.1，1，10，20，60，80，100 μg/L）作用于体外培养的正常人黑素细胞，采用噻唑蓝（MTT）法检测细胞增殖及酪氨酸酶活性，用倒置显微镜对比观察不同时期rhHGF（60 μg/L）处理组黑素细胞形态的改变，并用流式细胞仪分析黑素细胞周期及凋亡情况。结果 0.1 ~ 100 μg/L rhHGF有促进黑素细胞增殖作用，在20，60，80 μg/L浓度时促增殖比较显著（P ＜ 0.01），rhHGF各浓度组均增加黑素细胞酪氨酸酶活性（P ＜ 0.01或P ＜ 0.05）；浓度为60 μg/L的rhHGF能使黑素细胞数目增多、胞体增大、树突增多，并有促进黑素细胞由G0/G1期进入S期及抑制细胞凋亡的作用。结论 rhHGF可增强体外培养的人黑素细胞的生物学活性。     

Insulin-like growth factor-II regulates the expression of vascular endothelial growth factor by the human keratinocyte cell line HaCaT

Psoriasis is a chronic, relapsing skin disease characterized by enhanced angiogenesis. The pathogenetic process resulting in hypervascularity remains to be further investigated. It has been reported that a potent angiogenic factor, vascular endothelial growth factor (VEGF) is overexpressed in psoriatic epidermis and that the level of insulin-like growth factor II (IGF-II) is significantly elevated in the tissue fluid and serum of the psoriatic lesion. We considered the possibility that IGF-II might function as a paracrine inducer of VEGF. Here, we demonstrated that exposure of HaCaT keratinocytes to IGF-II induced both mRNA and protein expression of VEGF through the MAP kinase (extracellular signal-regulated kinase (ERK2) pathway. Particularly, we determined that phosphorylation of ERK2 but not p38 and JNK1/2 was activated by IGF-II in a time-dependent manner. Additionally, we found that IGF-II treatment induced the expression of MDM2 through the MAP kinase pathway. Moreover, the increase of MDM2 resulted in decreased levels of p53 followed by increased expression of HIF-1alpha and VEGF. Taken together, these results suggest that IGF-II enhances the expression of VEGF in HaCaT cells by increasing HIF-1alpha levels.     

Tranilast inhibits the cell growth of normal human keratinocytes in vitro

Tranilast is used clinically as a drug for hypertrophic scars or keloids. Recently, the roles of keratinocytes in the pathogenesis of those conditions have been noted. Therefore, we first examined the effect of tranilast on the cell growth of normal human keratinocytes. A cell growth assay demonstrated that the cell number significantly decreased during 48?h cultures with the addition of tranilast (5–400?μM) compared with a control (tranilast 0) in a dose-dependent manner. Morphologically, cell spreading was decreased and the cell body was elongated with higher concentrations (200–400?μM) of tranilast, and the cell area decreased significantly. The effect was not due to cytotoxicity. The inhibition of cell growth and the changes in cell morphology by the treatment of 100?μM tranilast reversed after the removal of the tranilast. Immunohistochemical staining revealed that F-actin and vinculin expression with tranilast-treated keratinocytes decreased significantly in a dose-dependent manner (100–400?μM). In addition, cell cycle examination showed that 400?μM of tranilast caused G0/G1 arrest with the keratinocytes. From these data we concluded that tranilast inhibited the growth of normal human keratinocytes, and one of its mechanisms may involve decreasing cell spreading by inhibition of F-actin fiber and focal contact formation with the cells.     

沙利度胺对HaCaT细胞分泌血管内皮细胞生长因子及肿瘤坏死因子α的影响

目的 探讨沙利度胺对HaCaT细胞增殖及血管内皮细胞生长因子（VEGF）和肿瘤坏死因子α（TNF-α）表达的影响。方法 体外培养的HaCaT细胞分为阴性对照组及不同浓度沙利度胺实验组;采用水溶性四氮唑法（WST-1）检测沙利度胺对HaCaT细胞增殖的影响,实时定量PCR法检测HaCaT细胞VEGF及TNF-α mRNA的表达水平,ELISA法检测HaCaT细胞VEGF及TNF-α蛋白的表达水平。采用单因素方差分析进行统计分析。结果 沙利度胺浓度在0.01 ～ 100 nmol/L之间时,可抑制VEGF mRNA（0.439 ～ 0.634,P ＜ 0.01）与蛋白（0.587 ～ 0.923,P ＜ 0.05）的表达;浓度在0.1 ～ 100 nmol/L之间时,可抑制TNF-α mRNA（0.493 ～ 0.587,P ＜ 0.05）与蛋白（0.408 ～ 0.617,P ＜ 0.01）的表达。结论 沙利度胺在一定浓度范围可抑制角质形成细胞增殖及减少VEGF及TNF-α表达。 【关键词】 角蛋白细胞; 沙立度胺; 血管内皮生长因子类; 肿瘤坏死因子α