UV调节角质形成细胞VEGFRs表达和激活及其功能和机制研究
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摘要
背景：
在正常生理或病理状态下,血管新生都是重要的生物学过程,主要由生长因子和其相应的酪氨酸激酶受体(receptor tyrosine kinases, RTKs)控制,其中,最主要的就是血管内皮生长因子(vascular endothelial growth factor, VEGF)家族及其受体(VEGF receptors, VEGFRs,包括VEGFR-1, VEGFR-2, VEGFR-3及辅助受体Neuropilins,即NRP-1和NRP-2)。
目前的研究发现,VEGF家族包括VEGF-A, PIGF (placenta growth factor,胎盘生长因子),VEGF-B, VEGF-C, VEGF-D, VEGF-E和svVEGF (snake venom VEGF,蛇毒VEGF). VEGF-A,即一般意义上的VEGF,以往称为血管通透性因子(vascular permeability factor, VPF),存在于多种细胞和组织中,与机体内的生理和病理活动密切相关,如肿瘤的血管新生和血管形成。VEGF在基因转录过程中由于不同的剪接方式形成几种分子量不同的VEGF同种异构体(VEGF isoforms),在人类主要为VEGF111、VEGF121、VEGF145、VEGF165、VEGF189、VEGF206等。
表皮角质形成细胞是VEGF的主要分泌细胞之一,在某些炎症性皮肤病如银屑病、特应性皮炎等情况下,皮肤角质形成细胞分泌VEGF明显增加,通过旁分泌作用促进真皮层微血管增生。角质形成细胞主要分泌VEGF111、VEGF121、 VEGF165和VEGF189等,其中以VEGF165效应最强。VEGF通过与其受体相结合发挥生物学效应,这些受体分为跨膜酪氨酸激酶受体和非酪氨酸激酶受体(即辅助受体),前者包括VEGFR-1, VEGFR-2, VEGFR-3,后者则包括NRP-1和NRP-2,而VEGFR-2则被认为是介导VEGF促血管新生效应的主要受体。近年来,越来越多的证据表明,VEGFRs家族不仅仅表达在血管内皮细胞上,也表达于包括上皮细胞在内的一些非血管内皮细胞上。而且,VEGF/VEGFRs已经不再局限于血管新生和／或血管通透性的增加,该信号途径还参与了其他多种功能,包括诱导肿瘤的转移、参与炎症反应、保护神经和肝脏、参与骨髓干细胞的迁移等。因此,有必要对VEGF/VEGFRs进行更深入的研究。
银屑病(Psoriasis)是一种由多基因遗传决定的、多环境因素刺激诱导的免疫异常性慢性炎症性增生性皮肤病,其特征包括表皮角质形成细胞增生过度和异常分化,淋巴细胞(主要为T细胞)炎性浸润以及真皮血管改变如血管新生、扩张、迂曲等。之前,我们研究了VEGFRs在HaCaT细胞株、正常人表皮和银屑病非皮损区、邻近皮损区和皮损区角质形成细胞中的表达情况,并研究了VEGF通过VEGFR-2对HaCaT细胞和表皮角质形成细胞增殖、迁移和黏附的作用以及VEGFRs在银屑病不同发展阶段的可能作用。结果表明：VEGFR-1和VEGFR-2在正常表皮基底层和棘层有显著表达,颗粒层表达较少,角质层无表达；而VEGFR-3, NRP-1和NRP-2均匀地表达于除角质层以外的其它表皮各层。在银屑病患者皮肤中,VEGFR-1和VEGFR-2强烈表达于银屑病非皮损区和邻近皮损区除角质层以外的其它各层角质形成细胞、银屑病皮损区表皮全层角质形成细胞,包括角质层角化不全细胞。在银屑病非皮损区、邻近皮损区VEGFR-3的分布与正常表皮中的分布一致,而银屑病皮损区则在包含角质层角化不全细胞的表皮各层均表达VEGFR-3; VEGFR-1、VEGFR-2和VEGFR-3mRNA表达水平从正常表皮到银屑病非皮损区、邻近皮损区和皮损区逐渐增加,皮损区表皮角质形成细胞VEGFRs mRNA表达水平最高；VEGFRs和NRPs在角质形成细胞内的分布形式亦有所差异：VEGFRs沿着细胞间连接呈膜性分布,而NRPs在细胞膜和细胞浆中均有分布。此外,VEGF可促进角质形成细胞的增殖和迁移而降低角质形成细胞的黏附功能。抗VEGFR-2抗体预处理能显著抑制VEGF诱导的对角质形成细胞的以上作用。
日光中的紫外线(ultraviolet, UV)与皮肤息息相关。皮肤中的角质形成细胞和黑素细胞是UV照射的重要靶位,急性UV辐射可增强皮肤角质形成细胞VEGF、白介素-8(IL-8)、碱性成纤维细胞因子(bFGF)等细胞因子的表达和释放,致使真皮血管扩张和增生,产生红斑反应；而慢性UV辐射可导致皮肤老化,产生皮角、日光性角化病等癌前期病变甚至皮肤癌。然而,临床上采用窄谱中波紫外线(narrowband UVB, NB-UVB)台疗银屑病等免疫炎症性皮肤病效果显著,其机制涉及减少局部T细胞的浸润、激活调节性T细胞等多个方面,此外,UV照射后皮损处细胞因子、细胞表面抗原和受体表达的改变也使得炎症介质的效应作用抑制和减弱。鉴于UVB辐射可增强皮肤角质形成细胞VEGF的表达的肯定性,结合我们课题组之前的发现,我们思考,为何NB-UVB光疗不但没有加重银屑病患者皮损的红斑反应和鳞屑性表皮增生,却反而起到了一个较好的治疗作用?会不会因为UV同时通过调节角质形成细胞VEGFRs的表达而改变了VEGF/VEGFRs信号通路在银屑病等炎症性皮肤病中的作用?如果UV能调节角质形成细胞VEGFRs的表达,那么,它又是通过什么途径达到该调节作用的呢?带着这样的疑问,我们设计和进行了本研究。如有新的发现,我们不但重复和肯定了我们课题组之前的工作,还将在国际上首次报道UV对角质形成细胞VEGFRs表达的影响及其相关的可能机制和作用,以及通过改变VEGF/VEGFRs信号通路而产生的对银屑病等炎症性皮肤疾病的治疗作用,为UV治疗这类皮肤病提供了更多新的依据。
研究目的：
利用UV(包括UVA和UVB)辐射正常人皮肤角质形成细胞或者正常人皮肤,以及采用NB-UVB治疗银屑病患者皮损,研究UV辐射前后上述细胞或者表皮VEGF和VEGFRs的表达变化情况,明确UV对角质形成细胞VEGF和VEGFRs表达以及VEGFRs磷酸化的影响；探索UV调节角质形成细胞VEGFRs表达和磷酸化的可能信号机制；进一步明确VEGFRs在正常人和银屑病表皮角质形成细胞中的功能,并为UV治疗银屑病等免疫性炎症性皮肤病提供新的理论依据。
研究方法：
第一部分：首先,分离和培养正常人表皮角质形成细胞,采用不同剂量(高、中、低)的UVB,分不同时间段对正常人皮肤角质形成细胞进行辐射,然后,以MTT法测定各组细胞增殖活性；流式细胞仪测定各组细胞的凋亡率；提取总RNA,采用逆转录-聚合酶链反应(Reverse transcript polymerase chain reaction, RT-PCR)法半定量测定VEGF和VEGFRs的mRNA含量变化及其趋势；提取总蛋白,采用Western Blot法半定量测定VEGF、VEGFRs和P-VEGFRs的蛋白质含量变化及其趋势；以间接免疫荧光法检测UVB辐射前后VEGF、VEGFRs和P-VEGFRs在正常角质形成细胞上的表达和定位；采用Western Blot法筛选出UVB影响角质形成细胞VEGFRs表达和磷酸化的可能信号机制。其次,入组一定数量的正常健康人,确定其各自的最小红斑量(minimal erythema dose, MED),采用低(1MED)、高(3MEDs)两种剂量的UVB辐射正常人臀部皮肤,24h后切取三处皮肤(未照光、低剂量、高剂量)进行活检,组织在OCT包埋后,做成冰冻切片,行间接免疫荧光,原位观察UVB辐射前后VEGF、VEGFRs和P-VEGFRs在正常人表皮中的表达和定位情况。
第二部分：将UVB光源换成UVA,其他处理方法基本同上。
第三部分：首先,按照一定的入选标准,收集多个同一银屑病患者NB-UVB光疗前、中、后皮损处皮肤；其次,作为比较,招募多个银屑病患者单独外用0.05%卤米松乳膏作治疗处理,并切取治疗前、中、后的皮损处皮肤。所有标本OCT包埋后做成6μm冰冻切片,用间接免疫荧光标记表皮中的VEGF、VEGFRs和P-VEGFRs,原位分析它们在两种治疗前、中、后表皮中的表达和分布情况。
研究结果：
一、VEGFR-2信号途径被中等剂量UVB激活后可促进正常人角质形成细胞的存活：
1、中等剂量的UVB辐射可促进培养的正常人角质形成细胞VEGF和VEGFR-1、VEGFR-2、NRP-1mRNA和蛋白的表达；
2、中等剂量的UVB辐射可促进正常人皮肤表皮VEGF和VEGFR-1、 VEGFR-2、NRP-1的表达；
3、中等剂量UVB辐射所致的VEGFR-1、VEGFR-2、NRP-1的表达上调与VEGF的表达无关,而主要由UVB辐射后导致角质形成细胞发生缺氧和氧化应激所致；
4、中等剂量UVB辐射可促进培养的正常人角质形成细胞和正常人表皮所表达的VEGFR-1和VEGFR-2发生磷酸化,即可以激活VEGFR-1和VEGFR-2;
5、中等剂量UVB辐射所致的VEGFR-1和VEGFR-2的激活亦不依赖于角质形成细胞自分泌产生的VEGF,而与蛋白激酶C (PKC)以及Sarc家族激酶(SFKs)的激活有关；
6、高剂量的UVB辐射能促进培养的正常人角质形成细胞和正常人皮肤表皮VEGF的表达,却未能明显促进VEGFR-1、VEGFR-2、NRP-1的表达和磷酸化；采用VEGF中和抗体处理高剂量UVB辐射后的细胞,可一定程度上恢复UVB诱导的VEGFR-1、VEGFR-2、NRP-1的表达增高及其磷酸化的发生；
7、中等剂量UVB辐射角质形成细胞所产生的VEGF对细胞具有一定的保护作用,而高剂量UVB辐射角质形成细胞所产生的大量VEGF则表现为一种炎症介质,对细胞具有明显的损伤作用；
8、UVB辐射可剂量依赖性促进角质形成细胞的凋亡,当细胞被VEGFR-2中和抗体处理后,能明显加重中等剂量UVB辐射所致的细胞凋亡,减少细胞的存活；当VEGFR-1被其中和抗体处理后,则未显著显示出上述作用,表明VEGFR-2信号途径的激活对UVB辐射后的角质形成细胞具有促生存作用；
9、采用VEGFR-2中和抗体处理可抑制中等剂量UVB辐射所致的角质形成细胞ERK1/2和Akt信号途径的激活,表明UVB诱导的VEGFR-2信号途径的激活可通过进一步激活ERK1/2和Akt来促进细胞存活。
二、VEGFR-1和VEGFR-2信号途径被UVA激活后均可促进正常人角质形成细胞的存活：
1、在一定剂量范围内,UVA辐射呈剂量依赖性促进培养的正常人角质形成细胞VEGFR-1、VEGFR-2、NRP-1mRNA和蛋白的表达；
2、在一定剂量范围内,UVA辐射可呈剂量依赖性增强正常人皮肤表皮VEGFR-1、vegfr-2、NRP-1的表达；
3、无论是对培养的正常人角质形成细胞,还是在正常人皮肤表皮,UVA辐射均不能诱导VEGF的表达增强；
4、UVA辐射可促进培养的正常人角质形成细胞和正常人表皮所表达的VEGFR-1和VEGFR-2发生磷酸化,即可以激活VEGFR-1和VEGFR-2;
5、UVA辐射所致的VEGFR-1和VEGFR-2的激活亦与蛋白激酶C (PKC)以及Sarc家族激酶(SFKs)的激活有关；
6、UVA辐射可剂量依赖性促进角质形成细胞的凋亡,当细胞被VEGFR-1和/或VEGFR-2中和抗体处理后,均能明显加重UVA辐射所致的细胞凋亡,减少细胞的存活,表明VEGFR-1和VEGFR-2信号途径的激活对UVA辐射后的角质形成细胞均具有促生存作用；
7、采用VEGFR-1和/或VEGFR-2中和抗体处理可抑制UVA辐射所致的角质形成细胞ERK1/2和/或Akt信号途径的激活,表明UVA诱导的VEGFR-1和VEGFR-2的激活可通过进一步激活ERK1/2和/或Akt来促进细胞存活。
三、银屑病皮损中高表达的VEGFRs可被NB-UVB和外用0.05%卤米松乳膏两种疗法下调
1、银屑病皮损中高表达的VEGFRs可被NB-UVB光疗显著下调,皮损基底部角质形成细胞的VEGFRs的表达首先出现下降,随着UVB剂量的逐渐增加,VEGFRs的表达位置也逐渐往上迁移,至疗程结束时,表皮全层均不再过表达VEGFRs;同时,P-VEGFR的表达可在随着VEGFRs往上迁移的部位而加强,但其在疗程结束后完全消失。
2、银屑病皮损中高表达的VEGFRs亦可被外用0.05%卤米松乳膏显著下调,但下调方式不同于NB-UVB,表现为弥漫性、逐渐均匀地下降。然而,至疗程结束时,虽然皮损得到明显改善,但VEGFRs的表达仍明显高于正常皮肤。P-VEGFR的表达与VEGFRs相似。
3、银屑瘸皮损中VEGFRs的表达高低与患者病情,即PASI评分呈正相关。
结论：
1、正常人角质形成细胞VEGFRs的表达可被一定剂量范围内的UVA和UVB上调,该调节不依赖于UVB所致的VEGF的自分泌,而依赖于UV诱导的细胞缺氧和氧化应激；
2、UV可不依赖于VEGF直接激活角质形成细胞VEGFRs,这与UV所致的PKC途径的激活有关；
3、UV辐射后VEGFRs的激活可促进角质形成细胞的存活,该作用虽为一种保护机制,但在长期、慢性UV辐射下,为皮肤光老化和光源性皮肤肿瘤的发生提供了潜在可能；
4、UVB可显著下调银屑病角质形成细胞VEGFRs的表达,该调节方式不同于倍他米松乳膏；
5、银屑病皮损处表皮VEGFRs表达异常可能不是银屑病的启动因素,但这种继发性改变可能间接参与了表皮增生和角化不全的发生,而UV可通过有效调节VEGFRs的表达和功能而影响角质形成细胞的生物学行为,表明了VEGFRs在角质形成细胞生理和病理过程中的作用。
Background:
Angiogenesis is a complex process by which new blood vessels arise from the pre-existing vasculature under both physical and pathological conditions. Angiogenesis is mostly controlled by some growth factors and their receptor tyrosine kinases, among which the most important are vascular endothelial growth factor (VEGF) and their receptors, VEGF receptors (VEGFRs), including VEGFR-1, VEGFR-2, VEGFR-3and their co-receptors, neuropilins (including NRP-1and NRP-2).
The VEGF family includes VEGF-A, P1GF (placenta growth factor), VEGF-B, VEGF-C, VEGF-D, VEGF-E and svVEGF (snake venom VEGF). The generally referred VEGF is VEGF-A (also called vascular permeability factor, VPA), which is associated with physical activities and pathological process in many kinds of cells and tissues of the body, such as new blood vessel formation in tumors. To date, seven isoforms of human VEGF have been identified, which range in length from111to206amino acid residues (VEGF111,121,145,165,183,189,206).
Human epidermal keratinocytes are a major source of VEGF, which normally express VEGF121, VEGF165and VEGF189, of which VEGF165has the strongest effect. The effect of VEGF is mediated by binding with its receptors, including transmembrane, tyrosine kinase VEGFR-1, VEGFR-2, VEGFR-3and non-tyrosine kinase neuropilins (including NRP-1and NRP-2). VEGFR-2has a more crucial role in angiogenesis than VEGFR-1and VEGFR-3. Recently, mounting evidence demonstrated that VEGFRs was not only expressed in endothelial cells, but also in some non-endothelial cells, including epithelial cells and tumor cells, and the VEGF/VEGFR signaling mediated effects are so various that they are no longer limited in enhancing angiogenesis and vascular permeability. So, further works need to be done to investigate the functional significance of VEGFRs.
Psoriasis is a common, chronic, inflammatory, immune aberrant and proliferative skin disease that is determined by polygene and induced by multiple environmental factors. The characteristics of psoriasis include hyperplasia and altered differentiation of epidermal keratinocytes (acanthosis), infiltration of lymphocytes (mainly T cells) into the dermis and epidermis, and dilation and growth of capillary vessels in the dermis. Angiogenesis plays especially important roles in the pathogenesis of psoriasis. We previously examined the expression of VEGFRs in HaCaT cells, normal human epidermal keratinocytes, psoriatic nonlesional, perilesional and lesional keratinocytes and epidermis. The distribution pattern of these receptors varied in epidermis:VEGFRs show a membranous staining pattern along the intercellular junctions in the basal and suprabasal layers of the epidermis except for the horny cell layer. The staining pattern of NRPs is differed from that for VEGFRs. NRPs are stained in the membrane and in the cytoplasm of keratinocytes in the whole epidermis except for the stratum corneum. Specifically, VEGFR-1and VEGFR-2strongly labeled keratinocytes in stratum basal and spinosum adjacent to basal, while all layers of the epidermis except horny cell layer demonstrated a uniform expression pattern of VEGFR-3, NRP-1, and NRP-2. Moreover, NRP-1and NRP-2show diffuse intense fluorescence compared with VEGFRs. In psoriatic epidermis, VEGFR-1and VEGFR-2strongly labeled nonlesional, perilesional keratinocytes in all layers of the epidermis except the stratum corneum, and lesional keratinocytes in all viable layers, including the parakeratotic stratum corneum. A uniform expression pattern of VEGFR-3was detected in psoriatic epidermis except for stratum corneum. Interestingly, parakeratotic keratinocytes in lesional psoriatic stratum corneum also showed intense fluorescence for VEGFRs, suggesting VEGFRs might play important roles in different stages of psoriasis. We also showed that VEGF affected cell proliferation, migration, and adhesion of HaCaT cells and normal human keratinocytes via VEGFR-2signaling pathway.
Ultraviolet (UV) is one of the most important components in the sunlight, and is closely related to human skin. Keratinocytes and melanocytes are important target cells for UV in the skin, an acute UV exposure can enhance the expression and release of cytokines like VEGF、interleukin-8(IL-8) and basic fibroblast growth factor (bFGF) from keratinocytes, resulting in skin erythema caused by blood vessel dilation and angiogenesis in the dermis; whereas repeated and chronic UV exposure can lead to skin photoaging, including development of pre-cancerous lesions like cutaneous horn, actinic keratosis or even skin cancers. In the meantime, narrowband UVB (NB-UVB) phototherapy has been used clinically to treat immuo-inflammatory dermatoses including psoriasis with good effectiveness. The underlying mechanisms involves depleting regional infiltration of T cells, activating Reg T cells, and importantly, the changed expression of cytokines, cellular surface antigens and receptors in the UV-irradiated lesions can also inhibit the inflammation mediators-induced effects. As UVB can enhance the expression of VEGF, we asked that, integrating our previous findings, why NB-UVB would not aggravate lesional erythema and epidermal hyperplasia in psoriasis, but achieved a good therapeutic result? Would UV modulate VEGF/VEGFRs signaling pathway in psoriatic epidermis? If the hypothesis is true, how does it work? With such a list of questions, we designed and performed this study. If new findings could be discovered, we would demonstrate for the first time that UV affects the expression of VEGFRs and its underlying mechanisms and roles in normal keratinocytes. In addition, we would provide more evidence relating to UV phototherapy, if it is useful for treating psoriasis by interfering with VEGF/VEGFRs signaling in psoriatic epidermis.
Objective:
1. By using normal human keratinocytes and normal human epidermis, we investigated whether UV (including UVA and UVB) could regulate the expression of VEGF and VEGFRs, whether UV could activate VEGFRs. To further clarify the underlying mechanisms and possible roles of VEGFRs in normal human keratinocytes.
2. By using psoriatic human epidermis, we investigated whether NB-UVB phototherapy could regulate the expression of VEGF and VEGFRs, to further demonstrate the roles of VEGFRs in psoriatic epidermis, and to provide more evidence for the mechanism of UVB phototherapy.
Methods:
1. First, normal human keratinocytes were seperated from adolescent foreskin and cultured with serum free medium. By applying different doses of UVB (high, moderate, and low), keratinocytes were irradiated and harvested at various times after irradiation. Then, cellular viability was determined by MTT assay; apoptosis rate was determined by using flow cytometry; Total RNA was extracted and VEGF/VEGFRs mRNAs were determined by reverse transcript polymerase chain reaction (RT-PCR); Total protein was extracted and VEGF, VEGFRs and phospho-VEGFRs proteins were determined by Western Blot; Indirect immunofluorescence was used to detect the expression of VEGF, VEGFRs, and phospho-VEGFRs in keratinocytes before and after UVB irradiation; Western Blot was used to illucidate the mechanisms and possible pathways for the UVB-induced regulation and activation of VEGFRs. In addition, to recruit a few healthy individuals, their minimal erythema dose (MED) to UVB was dertermined respectively, then, two doses of UVB (1MED,3MEDs) was adopted to irradiate the healthy skin in the buttocks.24h after irradiation, biopsies from three sites (sham-irradiated,1MED,3MEDs) were taken, embedded in OCT and snap frozen, and Indirect immunofluorescence was performed to detect in situ expression of VEGF, VEGFRs, and phospho-VEGFRs in normal human epidermis before and after UVB irradiation.
2. Second, replace the light source of UVB with UVA, other methods were similar to above.
3. Third, according to enrollment criteria, to collect a few psoriasis patients, and take biopsies from their skin lesions before, under, and after NB-UVB phototherapy; For comparison, another few psoriasis patients were enrolled with treatment of topical Halomethasone monohydrate0.05%cream, and biopsies were taken from their skin lesions before, under, and after treatment. All specimens were embedded in OCT and snap frozen, cut into6μm slides, and Indirect immunofluorescence was performed to detect in situ expression of VEGF, VEGFRs, and phospho-VEGFRs in psoriatic human epidermis before, under, and after the two treatments.
Results:
1. Activation of VEGFR-2signaling in response to moderate dose of ultraviolet B promotes survival of normal human keratinocytes:
a) Moderate dose of UVB enhanced the mRNA and protein expression of VEGF, VEGFR-1, VEGFR-2, and NRP-1in normal human keratinocytes;
b) Moderate dose of UVB enhanced the expression of VEGF, VEGFR-1, VEGFR-2, and NRP-1in normal human epidermis;
c) Moderate dose UVB-induced upregulation of VEGFR-1, VEGFR-2, and NRP-1was independent of autocrine VEGF in keratinocytes, but mainly caused by UVB-induced hypoxia and oxidative stress.
d) Moderate dose UVB promoted tyrosine phosphorylation of VEGFR-1and VEGFR-2in normal human keratinocytes and epidermis, that is, VEGFR-1and VEGFR-2were activated by UVB;
e) Moderate dose UVB-induced tyrosine phosphorylation of VEGFR-1and VEGFR-2was again independent of autocrine VEGF, but mainly caused by UVB-induced activation of protein kinase C (PKC) and Sarc family kinases (SFKs);
f) High dose of UVB upregulated the expression of VEGF in normal human keratinocytes and epidermis, but it failed to affect significant expression and activation of VEGFRs. Treatment of VEGF neutralizing antibody after high dose of UVB could restore UVB-induced expression and activation of VEGFRs in some degree;
g) Moderate dose UVB-induced VEGF plays a protective role for keratinocytes, whereas high dose of UVB-induced, over-released VEGF acted as a proinflammatory factor, which caused obvious damage to keratinocytes;
h) UVB dose-dependently induced cell apoptosis. Incubation of VEGFR-2but not VEGFR-1neutralizing antibody significantly accelerated cell apoptosis induced by moderate dose of UVB, and reduced cell survival, suggesting that activation of VEGFR-2signaling pathway played a pro-survival role for keratinocytes;
i) Incubation of VEGFR-2neutralizing antibody significantly inhibited activation of ERK1/2and Akt induced by moderate dose of UVB, suggesting UVB-induced activation of VEGFR-2signaling promotes cell survival via ERK1/2and PI3K/Akt pathway.
2. Activation of both VEGFR-1and VEGFR-2signaling by UVA promotes survival of normal human keratinocytes:
a) UVA dose-dependentlyupregulated the mRNA and protein expression of VEGFR-1, VEGFR-2, and NRP-1in normal human keratinocytes;
b) UVA dose-dependently upregulated the expression of VEGFR-1, VEGFR-2, and NRP-1in normal human epidermis;
c) UVA could not induce the expression of VEGF in normal human keratinocytes or epidermis;
d) UVA promoted tyrosine phosphorylation of VEGFR-1and VEGFR-2in normal human keratinocytes and epidermis, that is, VEGFR-1and VEGFR-2were activated by UVA;
e) UVA-induced tyrosine phosphorylation of VEGFR-1and VEGFR-2was also mainly caused by UVA-induced activation of PKC and SFKs;
f) UVA dose-dependently induced cell apoptosis. Incubation of both VEGFR-1and VEGFR-2neutralizing antibody significantly accelerated cell apoptosis induced by UVA, and reduced cell survival, suggesting that activation of VEGFR-1and VEGFR-2signaling pathways played a pro-survival role for keratinocytes in response to UVA;
i) Incubation of VEGFR-1and VEGFR-2neutralizing antibody significantly inhibited activation of ERK1/2and Akt induced by UVA, suggesting UVA-induced activation of VEGFR-1and VEGFR-2signaling both promotes cell survival via ERK1/2and PI3K/Akt pathway.
3. Over-expressed VEGFRs in psoriatic epidermis were normalized by UVB phototherapy in a way different from that by halomethasone
a) The over-expressed VEGFRs in the whole psoriatic epidermis was significantly down-regulated both during and after phototherapy, and interestingly, their expression decreased in a down to top advancement way, that is, VEGF receptors over-expressed in the basal layers were much more sensitive to UVB than that in upper layers, and the location of VEGF receptors shafted to the upper epidermis, even to the stratum corneum after NB-UVB treatment. In addition, VEGFRs were activated in psoriatic epidermis, but their activation was enhanced with their upward movement during NB-UVB treatment, and became extinct after whole therapy.
b) The overall result from treatment by halomethasone was similar to that by UVB phototherapy. However, the process was quite different, in which VEGF, VEGFRs and phospho-VEGFRs decreased in a gradual, homogeneous manner. Although the skin lesions were greatly improved, the expression of VEGFRs was still higher than normal skin, the expression of phospho-VEGFR was similar to VEGFRs, suggesting VEGF and its receptors are not target molecules of halomethasone, and their expression automatically declined due to improvement of the skin lesions by treatment of the drug.
c) The expression of VEGFRs in psoriatic epidermis is positively correlated with patients' PASI scoring.
Conclusions:
1. The expression of VEGFRs in keratinocytes can be upregulated by UVA and UVB irradiation, which is independent of UVB-induced autocrine release of VEGF, but partly dependent of UV-induced cell hypoxia and oxidative stress;
2. UV activates VEGFRs in keratinocytes independent of VEGF, but via activation of PKC signaling pathway;
3. Activation of VEGFRs by UV promotes survival of normal human keratinocytes. Although it acts an protective effect for keratinocytes, it provides potential opportunities for skin photoaging, malignant transformation and development of skin caners under long-term, chronic UV exposure.
4. UVB phototherapy significantly downregulates the expression of VEGFRs in psoriatic epidermis, in a way which was different from that by treatment of halomethasone0.05%cream;
5. The over-expressed VEGFRs in psoriatic epidermis may not be initial factors in psoriasis pathogenesis, however, this secondary pathological change of VEGFRs may be involved in the development of epidermal hyperplasia and parakeratosis. As UV can affect the biological behavior of keratinocytes through modulating the expression and function of VEGFRs, we demonstrate the important roles of VEGFRs in physiological and pathological conditions of epidermal keratinocytes.
在正常生理或病理状态下,血管新生都是重要的生物学过程,主要由生长因子和其相应的酪氨酸激酶受体(receptor tyrosine kinases, RTKs)控制,其中,最主要的就是血管内皮生长因子(vascular endothelial growth factor, VEGF)家族及其受体(VEGF receptors, VEGFRs,包括VEGFR-1, VEGFR-2, VEGFR-3及辅助受体Neuropilins,即NRP-1和NRP-2)。
目前的研究发现,VEGF家族包括VEGF-A, PIGF (placenta growth factor,胎盘生长因子),VEGF-B, VEGF-C, VEGF-D, VEGF-E和svVEGF (snake venom VEGF,蛇毒VEGF). VEGF-A,即一般意义上的VEGF,以往称为血管通透性因子(vascular permeability factor, VPF),存在于多种细胞和组织中,与机体内的生理和病理活动密切相关,如肿瘤的血管新生和血管形成。VEGF在基因转录过程中由于不同的剪接方式形成几种分子量不同的VEGF同种异构体(VEGF isoforms),在人类主要为VEGF111、VEGF121、VEGF145、VEGF165、VEGF189、VEGF206等。
表皮角质形成细胞是VEGF的主要分泌细胞之一,在某些炎症性皮肤病如银屑病、特应性皮炎等情况下,皮肤角质形成细胞分泌VEGF明显增加,通过旁分泌作用促进真皮层微血管增生。角质形成细胞主要分泌VEGF111、VEGF121、 VEGF165和VEGF189等,其中以VEGF165效应最强。VEGF通过与其受体相结合发挥生物学效应,这些受体分为跨膜酪氨酸激酶受体和非酪氨酸激酶受体(即辅助受体),前者包括VEGFR-1, VEGFR-2, VEGFR-3,后者则包括NRP-1和NRP-2,而VEGFR-2则被认为是介导VEGF促血管新生效应的主要受体。近年来,越来越多的证据表明,VEGFRs家族不仅仅表达在血管内皮细胞上,也表达于包括上皮细胞在内的一些非血管内皮细胞上。而且,VEGF/VEGFRs已经不再局限于血管新生和／或血管通透性的增加,该信号途径还参与了其他多种功能,包括诱导肿瘤的转移、参与炎症反应、保护神经和肝脏、参与骨髓干细胞的迁移等。因此,有必要对VEGF/VEGFRs进行更深入的研究。
银屑病(Psoriasis)是一种由多基因遗传决定的、多环境因素刺激诱导的免疫异常性慢性炎症性增生性皮肤病,其特征包括表皮角质形成细胞增生过度和异常分化,淋巴细胞(主要为T细胞)炎性浸润以及真皮血管改变如血管新生、扩张、迂曲等。之前,我们研究了VEGFRs在HaCaT细胞株、正常人表皮和银屑病非皮损区、邻近皮损区和皮损区角质形成细胞中的表达情况,并研究了VEGF通过VEGFR-2对HaCaT细胞和表皮角质形成细胞增殖、迁移和黏附的作用以及VEGFRs在银屑病不同发展阶段的可能作用。结果表明：VEGFR-1和VEGFR-2在正常表皮基底层和棘层有显著表达,颗粒层表达较少,角质层无表达；而VEGFR-3, NRP-1和NRP-2均匀地表达于除角质层以外的其它表皮各层。在银屑病患者皮肤中,VEGFR-1和VEGFR-2强烈表达于银屑病非皮损区和邻近皮损区除角质层以外的其它各层角质形成细胞、银屑病皮损区表皮全层角质形成细胞,包括角质层角化不全细胞。在银屑病非皮损区、邻近皮损区VEGFR-3的分布与正常表皮中的分布一致,而银屑病皮损区则在包含角质层角化不全细胞的表皮各层均表达VEGFR-3; VEGFR-1、VEGFR-2和VEGFR-3mRNA表达水平从正常表皮到银屑病非皮损区、邻近皮损区和皮损区逐渐增加,皮损区表皮角质形成细胞VEGFRs mRNA表达水平最高；VEGFRs和NRPs在角质形成细胞内的分布形式亦有所差异：VEGFRs沿着细胞间连接呈膜性分布,而NRPs在细胞膜和细胞浆中均有分布。此外,VEGF可促进角质形成细胞的增殖和迁移而降低角质形成细胞的黏附功能。抗VEGFR-2抗体预处理能显著抑制VEGF诱导的对角质形成细胞的以上作用。
日光中的紫外线(ultraviolet, UV)与皮肤息息相关。皮肤中的角质形成细胞和黑素细胞是UV照射的重要靶位,急性UV辐射可增强皮肤角质形成细胞VEGF、白介素-8(IL-8)、碱性成纤维细胞因子(bFGF)等细胞因子的表达和释放,致使真皮血管扩张和增生,产生红斑反应；而慢性UV辐射可导致皮肤老化,产生皮角、日光性角化病等癌前期病变甚至皮肤癌。然而,临床上采用窄谱中波紫外线(narrowband UVB, NB-UVB)台疗银屑病等免疫炎症性皮肤病效果显著,其机制涉及减少局部T细胞的浸润、激活调节性T细胞等多个方面,此外,UV照射后皮损处细胞因子、细胞表面抗原和受体表达的改变也使得炎症介质的效应作用抑制和减弱。鉴于UVB辐射可增强皮肤角质形成细胞VEGF的表达的肯定性,结合我们课题组之前的发现,我们思考,为何NB-UVB光疗不但没有加重银屑病患者皮损的红斑反应和鳞屑性表皮增生,却反而起到了一个较好的治疗作用?会不会因为UV同时通过调节角质形成细胞VEGFRs的表达而改变了VEGF/VEGFRs信号通路在银屑病等炎症性皮肤病中的作用?如果UV能调节角质形成细胞VEGFRs的表达,那么,它又是通过什么途径达到该调节作用的呢?带着这样的疑问,我们设计和进行了本研究。如有新的发现,我们不但重复和肯定了我们课题组之前的工作,还将在国际上首次报道UV对角质形成细胞VEGFRs表达的影响及其相关的可能机制和作用,以及通过改变VEGF/VEGFRs信号通路而产生的对银屑病等炎症性皮肤疾病的治疗作用,为UV治疗这类皮肤病提供了更多新的依据。
研究目的：
利用UV(包括UVA和UVB)辐射正常人皮肤角质形成细胞或者正常人皮肤,以及采用NB-UVB治疗银屑病患者皮损,研究UV辐射前后上述细胞或者表皮VEGF和VEGFRs的表达变化情况,明确UV对角质形成细胞VEGF和VEGFRs表达以及VEGFRs磷酸化的影响；探索UV调节角质形成细胞VEGFRs表达和磷酸化的可能信号机制；进一步明确VEGFRs在正常人和银屑病表皮角质形成细胞中的功能,并为UV治疗银屑病等免疫性炎症性皮肤病提供新的理论依据。
研究方法：
第一部分：首先,分离和培养正常人表皮角质形成细胞,采用不同剂量(高、中、低)的UVB,分不同时间段对正常人皮肤角质形成细胞进行辐射,然后,以MTT法测定各组细胞增殖活性；流式细胞仪测定各组细胞的凋亡率；提取总RNA,采用逆转录-聚合酶链反应(Reverse transcript polymerase chain reaction, RT-PCR)法半定量测定VEGF和VEGFRs的mRNA含量变化及其趋势；提取总蛋白,采用Western Blot法半定量测定VEGF、VEGFRs和P-VEGFRs的蛋白质含量变化及其趋势；以间接免疫荧光法检测UVB辐射前后VEGF、VEGFRs和P-VEGFRs在正常角质形成细胞上的表达和定位；采用Western Blot法筛选出UVB影响角质形成细胞VEGFRs表达和磷酸化的可能信号机制。其次,入组一定数量的正常健康人,确定其各自的最小红斑量(minimal erythema dose, MED),采用低(1MED)、高(3MEDs)两种剂量的UVB辐射正常人臀部皮肤,24h后切取三处皮肤(未照光、低剂量、高剂量)进行活检,组织在OCT包埋后,做成冰冻切片,行间接免疫荧光,原位观察UVB辐射前后VEGF、VEGFRs和P-VEGFRs在正常人表皮中的表达和定位情况。
第二部分：将UVB光源换成UVA,其他处理方法基本同上。
第三部分：首先,按照一定的入选标准,收集多个同一银屑病患者NB-UVB光疗前、中、后皮损处皮肤；其次,作为比较,招募多个银屑病患者单独外用0.05%卤米松乳膏作治疗处理,并切取治疗前、中、后的皮损处皮肤。所有标本OCT包埋后做成6μm冰冻切片,用间接免疫荧光标记表皮中的VEGF、VEGFRs和P-VEGFRs,原位分析它们在两种治疗前、中、后表皮中的表达和分布情况。
研究结果：
一、VEGFR-2信号途径被中等剂量UVB激活后可促进正常人角质形成细胞的存活：
1、中等剂量的UVB辐射可促进培养的正常人角质形成细胞VEGF和VEGFR-1、VEGFR-2、NRP-1mRNA和蛋白的表达；
2、中等剂量的UVB辐射可促进正常人皮肤表皮VEGF和VEGFR-1、 VEGFR-2、NRP-1的表达；
3、中等剂量UVB辐射所致的VEGFR-1、VEGFR-2、NRP-1的表达上调与VEGF的表达无关,而主要由UVB辐射后导致角质形成细胞发生缺氧和氧化应激所致；
4、中等剂量UVB辐射可促进培养的正常人角质形成细胞和正常人表皮所表达的VEGFR-1和VEGFR-2发生磷酸化,即可以激活VEGFR-1和VEGFR-2;
5、中等剂量UVB辐射所致的VEGFR-1和VEGFR-2的激活亦不依赖于角质形成细胞自分泌产生的VEGF,而与蛋白激酶C (PKC)以及Sarc家族激酶(SFKs)的激活有关；
6、高剂量的UVB辐射能促进培养的正常人角质形成细胞和正常人皮肤表皮VEGF的表达,却未能明显促进VEGFR-1、VEGFR-2、NRP-1的表达和磷酸化；采用VEGF中和抗体处理高剂量UVB辐射后的细胞,可一定程度上恢复UVB诱导的VEGFR-1、VEGFR-2、NRP-1的表达增高及其磷酸化的发生；
7、中等剂量UVB辐射角质形成细胞所产生的VEGF对细胞具有一定的保护作用,而高剂量UVB辐射角质形成细胞所产生的大量VEGF则表现为一种炎症介质,对细胞具有明显的损伤作用；
8、UVB辐射可剂量依赖性促进角质形成细胞的凋亡,当细胞被VEGFR-2中和抗体处理后,能明显加重中等剂量UVB辐射所致的细胞凋亡,减少细胞的存活；当VEGFR-1被其中和抗体处理后,则未显著显示出上述作用,表明VEGFR-2信号途径的激活对UVB辐射后的角质形成细胞具有促生存作用；
9、采用VEGFR-2中和抗体处理可抑制中等剂量UVB辐射所致的角质形成细胞ERK1/2和Akt信号途径的激活,表明UVB诱导的VEGFR-2信号途径的激活可通过进一步激活ERK1/2和Akt来促进细胞存活。
二、VEGFR-1和VEGFR-2信号途径被UVA激活后均可促进正常人角质形成细胞的存活：
1、在一定剂量范围内,UVA辐射呈剂量依赖性促进培养的正常人角质形成细胞VEGFR-1、VEGFR-2、NRP-1mRNA和蛋白的表达；
2、在一定剂量范围内,UVA辐射可呈剂量依赖性增强正常人皮肤表皮VEGFR-1、vegfr-2、NRP-1的表达；
3、无论是对培养的正常人角质形成细胞,还是在正常人皮肤表皮,UVA辐射均不能诱导VEGF的表达增强；
4、UVA辐射可促进培养的正常人角质形成细胞和正常人表皮所表达的VEGFR-1和VEGFR-2发生磷酸化,即可以激活VEGFR-1和VEGFR-2;
5、UVA辐射所致的VEGFR-1和VEGFR-2的激活亦与蛋白激酶C (PKC)以及Sarc家族激酶(SFKs)的激活有关；
6、UVA辐射可剂量依赖性促进角质形成细胞的凋亡,当细胞被VEGFR-1和/或VEGFR-2中和抗体处理后,均能明显加重UVA辐射所致的细胞凋亡,减少细胞的存活,表明VEGFR-1和VEGFR-2信号途径的激活对UVA辐射后的角质形成细胞均具有促生存作用；
7、采用VEGFR-1和/或VEGFR-2中和抗体处理可抑制UVA辐射所致的角质形成细胞ERK1/2和/或Akt信号途径的激活,表明UVA诱导的VEGFR-1和VEGFR-2的激活可通过进一步激活ERK1/2和/或Akt来促进细胞存活。
三、银屑病皮损中高表达的VEGFRs可被NB-UVB和外用0.05%卤米松乳膏两种疗法下调
1、银屑病皮损中高表达的VEGFRs可被NB-UVB光疗显著下调,皮损基底部角质形成细胞的VEGFRs的表达首先出现下降,随着UVB剂量的逐渐增加,VEGFRs的表达位置也逐渐往上迁移,至疗程结束时,表皮全层均不再过表达VEGFRs;同时,P-VEGFR的表达可在随着VEGFRs往上迁移的部位而加强,但其在疗程结束后完全消失。
2、银屑病皮损中高表达的VEGFRs亦可被外用0.05%卤米松乳膏显著下调,但下调方式不同于NB-UVB,表现为弥漫性、逐渐均匀地下降。然而,至疗程结束时,虽然皮损得到明显改善,但VEGFRs的表达仍明显高于正常皮肤。P-VEGFR的表达与VEGFRs相似。
3、银屑瘸皮损中VEGFRs的表达高低与患者病情,即PASI评分呈正相关。
结论：
1、正常人角质形成细胞VEGFRs的表达可被一定剂量范围内的UVA和UVB上调,该调节不依赖于UVB所致的VEGF的自分泌,而依赖于UV诱导的细胞缺氧和氧化应激；
2、UV可不依赖于VEGF直接激活角质形成细胞VEGFRs,这与UV所致的PKC途径的激活有关；
3、UV辐射后VEGFRs的激活可促进角质形成细胞的存活,该作用虽为一种保护机制,但在长期、慢性UV辐射下,为皮肤光老化和光源性皮肤肿瘤的发生提供了潜在可能；
4、UVB可显著下调银屑病角质形成细胞VEGFRs的表达,该调节方式不同于倍他米松乳膏；
5、银屑病皮损处表皮VEGFRs表达异常可能不是银屑病的启动因素,但这种继发性改变可能间接参与了表皮增生和角化不全的发生,而UV可通过有效调节VEGFRs的表达和功能而影响角质形成细胞的生物学行为,表明了VEGFRs在角质形成细胞生理和病理过程中的作用。
Background:
Angiogenesis is a complex process by which new blood vessels arise from the pre-existing vasculature under both physical and pathological conditions. Angiogenesis is mostly controlled by some growth factors and their receptor tyrosine kinases, among which the most important are vascular endothelial growth factor (VEGF) and their receptors, VEGF receptors (VEGFRs), including VEGFR-1, VEGFR-2, VEGFR-3and their co-receptors, neuropilins (including NRP-1and NRP-2).
The VEGF family includes VEGF-A, P1GF (placenta growth factor), VEGF-B, VEGF-C, VEGF-D, VEGF-E and svVEGF (snake venom VEGF). The generally referred VEGF is VEGF-A (also called vascular permeability factor, VPA), which is associated with physical activities and pathological process in many kinds of cells and tissues of the body, such as new blood vessel formation in tumors. To date, seven isoforms of human VEGF have been identified, which range in length from111to206amino acid residues (VEGF111,121,145,165,183,189,206).
Human epidermal keratinocytes are a major source of VEGF, which normally express VEGF121, VEGF165and VEGF189, of which VEGF165has the strongest effect. The effect of VEGF is mediated by binding with its receptors, including transmembrane, tyrosine kinase VEGFR-1, VEGFR-2, VEGFR-3and non-tyrosine kinase neuropilins (including NRP-1and NRP-2). VEGFR-2has a more crucial role in angiogenesis than VEGFR-1and VEGFR-3. Recently, mounting evidence demonstrated that VEGFRs was not only expressed in endothelial cells, but also in some non-endothelial cells, including epithelial cells and tumor cells, and the VEGF/VEGFR signaling mediated effects are so various that they are no longer limited in enhancing angiogenesis and vascular permeability. So, further works need to be done to investigate the functional significance of VEGFRs.
Psoriasis is a common, chronic, inflammatory, immune aberrant and proliferative skin disease that is determined by polygene and induced by multiple environmental factors. The characteristics of psoriasis include hyperplasia and altered differentiation of epidermal keratinocytes (acanthosis), infiltration of lymphocytes (mainly T cells) into the dermis and epidermis, and dilation and growth of capillary vessels in the dermis. Angiogenesis plays especially important roles in the pathogenesis of psoriasis. We previously examined the expression of VEGFRs in HaCaT cells, normal human epidermal keratinocytes, psoriatic nonlesional, perilesional and lesional keratinocytes and epidermis. The distribution pattern of these receptors varied in epidermis:VEGFRs show a membranous staining pattern along the intercellular junctions in the basal and suprabasal layers of the epidermis except for the horny cell layer. The staining pattern of NRPs is differed from that for VEGFRs. NRPs are stained in the membrane and in the cytoplasm of keratinocytes in the whole epidermis except for the stratum corneum. Specifically, VEGFR-1and VEGFR-2strongly labeled keratinocytes in stratum basal and spinosum adjacent to basal, while all layers of the epidermis except horny cell layer demonstrated a uniform expression pattern of VEGFR-3, NRP-1, and NRP-2. Moreover, NRP-1and NRP-2show diffuse intense fluorescence compared with VEGFRs. In psoriatic epidermis, VEGFR-1and VEGFR-2strongly labeled nonlesional, perilesional keratinocytes in all layers of the epidermis except the stratum corneum, and lesional keratinocytes in all viable layers, including the parakeratotic stratum corneum. A uniform expression pattern of VEGFR-3was detected in psoriatic epidermis except for stratum corneum. Interestingly, parakeratotic keratinocytes in lesional psoriatic stratum corneum also showed intense fluorescence for VEGFRs, suggesting VEGFRs might play important roles in different stages of psoriasis. We also showed that VEGF affected cell proliferation, migration, and adhesion of HaCaT cells and normal human keratinocytes via VEGFR-2signaling pathway.
Ultraviolet (UV) is one of the most important components in the sunlight, and is closely related to human skin. Keratinocytes and melanocytes are important target cells for UV in the skin, an acute UV exposure can enhance the expression and release of cytokines like VEGF、interleukin-8(IL-8) and basic fibroblast growth factor (bFGF) from keratinocytes, resulting in skin erythema caused by blood vessel dilation and angiogenesis in the dermis; whereas repeated and chronic UV exposure can lead to skin photoaging, including development of pre-cancerous lesions like cutaneous horn, actinic keratosis or even skin cancers. In the meantime, narrowband UVB (NB-UVB) phototherapy has been used clinically to treat immuo-inflammatory dermatoses including psoriasis with good effectiveness. The underlying mechanisms involves depleting regional infiltration of T cells, activating Reg T cells, and importantly, the changed expression of cytokines, cellular surface antigens and receptors in the UV-irradiated lesions can also inhibit the inflammation mediators-induced effects. As UVB can enhance the expression of VEGF, we asked that, integrating our previous findings, why NB-UVB would not aggravate lesional erythema and epidermal hyperplasia in psoriasis, but achieved a good therapeutic result? Would UV modulate VEGF/VEGFRs signaling pathway in psoriatic epidermis? If the hypothesis is true, how does it work? With such a list of questions, we designed and performed this study. If new findings could be discovered, we would demonstrate for the first time that UV affects the expression of VEGFRs and its underlying mechanisms and roles in normal keratinocytes. In addition, we would provide more evidence relating to UV phototherapy, if it is useful for treating psoriasis by interfering with VEGF/VEGFRs signaling in psoriatic epidermis.
Objective:
1. By using normal human keratinocytes and normal human epidermis, we investigated whether UV (including UVA and UVB) could regulate the expression of VEGF and VEGFRs, whether UV could activate VEGFRs. To further clarify the underlying mechanisms and possible roles of VEGFRs in normal human keratinocytes.
2. By using psoriatic human epidermis, we investigated whether NB-UVB phototherapy could regulate the expression of VEGF and VEGFRs, to further demonstrate the roles of VEGFRs in psoriatic epidermis, and to provide more evidence for the mechanism of UVB phototherapy.
Methods:
1. First, normal human keratinocytes were seperated from adolescent foreskin and cultured with serum free medium. By applying different doses of UVB (high, moderate, and low), keratinocytes were irradiated and harvested at various times after irradiation. Then, cellular viability was determined by MTT assay; apoptosis rate was determined by using flow cytometry; Total RNA was extracted and VEGF/VEGFRs mRNAs were determined by reverse transcript polymerase chain reaction (RT-PCR); Total protein was extracted and VEGF, VEGFRs and phospho-VEGFRs proteins were determined by Western Blot; Indirect immunofluorescence was used to detect the expression of VEGF, VEGFRs, and phospho-VEGFRs in keratinocytes before and after UVB irradiation; Western Blot was used to illucidate the mechanisms and possible pathways for the UVB-induced regulation and activation of VEGFRs. In addition, to recruit a few healthy individuals, their minimal erythema dose (MED) to UVB was dertermined respectively, then, two doses of UVB (1MED,3MEDs) was adopted to irradiate the healthy skin in the buttocks.24h after irradiation, biopsies from three sites (sham-irradiated,1MED,3MEDs) were taken, embedded in OCT and snap frozen, and Indirect immunofluorescence was performed to detect in situ expression of VEGF, VEGFRs, and phospho-VEGFRs in normal human epidermis before and after UVB irradiation.
2. Second, replace the light source of UVB with UVA, other methods were similar to above.
3. Third, according to enrollment criteria, to collect a few psoriasis patients, and take biopsies from their skin lesions before, under, and after NB-UVB phototherapy; For comparison, another few psoriasis patients were enrolled with treatment of topical Halomethasone monohydrate0.05%cream, and biopsies were taken from their skin lesions before, under, and after treatment. All specimens were embedded in OCT and snap frozen, cut into6μm slides, and Indirect immunofluorescence was performed to detect in situ expression of VEGF, VEGFRs, and phospho-VEGFRs in psoriatic human epidermis before, under, and after the two treatments.
Results:
1. Activation of VEGFR-2signaling in response to moderate dose of ultraviolet B promotes survival of normal human keratinocytes:
a) Moderate dose of UVB enhanced the mRNA and protein expression of VEGF, VEGFR-1, VEGFR-2, and NRP-1in normal human keratinocytes;
b) Moderate dose of UVB enhanced the expression of VEGF, VEGFR-1, VEGFR-2, and NRP-1in normal human epidermis;
c) Moderate dose UVB-induced upregulation of VEGFR-1, VEGFR-2, and NRP-1was independent of autocrine VEGF in keratinocytes, but mainly caused by UVB-induced hypoxia and oxidative stress.
d) Moderate dose UVB promoted tyrosine phosphorylation of VEGFR-1and VEGFR-2in normal human keratinocytes and epidermis, that is, VEGFR-1and VEGFR-2were activated by UVB;
e) Moderate dose UVB-induced tyrosine phosphorylation of VEGFR-1and VEGFR-2was again independent of autocrine VEGF, but mainly caused by UVB-induced activation of protein kinase C (PKC) and Sarc family kinases (SFKs);
f) High dose of UVB upregulated the expression of VEGF in normal human keratinocytes and epidermis, but it failed to affect significant expression and activation of VEGFRs. Treatment of VEGF neutralizing antibody after high dose of UVB could restore UVB-induced expression and activation of VEGFRs in some degree;
g) Moderate dose UVB-induced VEGF plays a protective role for keratinocytes, whereas high dose of UVB-induced, over-released VEGF acted as a proinflammatory factor, which caused obvious damage to keratinocytes;
h) UVB dose-dependently induced cell apoptosis. Incubation of VEGFR-2but not VEGFR-1neutralizing antibody significantly accelerated cell apoptosis induced by moderate dose of UVB, and reduced cell survival, suggesting that activation of VEGFR-2signaling pathway played a pro-survival role for keratinocytes;
i) Incubation of VEGFR-2neutralizing antibody significantly inhibited activation of ERK1/2and Akt induced by moderate dose of UVB, suggesting UVB-induced activation of VEGFR-2signaling promotes cell survival via ERK1/2and PI3K/Akt pathway.
2. Activation of both VEGFR-1and VEGFR-2signaling by UVA promotes survival of normal human keratinocytes:
a) UVA dose-dependentlyupregulated the mRNA and protein expression of VEGFR-1, VEGFR-2, and NRP-1in normal human keratinocytes;
b) UVA dose-dependently upregulated the expression of VEGFR-1, VEGFR-2, and NRP-1in normal human epidermis;
c) UVA could not induce the expression of VEGF in normal human keratinocytes or epidermis;
d) UVA promoted tyrosine phosphorylation of VEGFR-1and VEGFR-2in normal human keratinocytes and epidermis, that is, VEGFR-1and VEGFR-2were activated by UVA;
e) UVA-induced tyrosine phosphorylation of VEGFR-1and VEGFR-2was also mainly caused by UVA-induced activation of PKC and SFKs;
f) UVA dose-dependently induced cell apoptosis. Incubation of both VEGFR-1and VEGFR-2neutralizing antibody significantly accelerated cell apoptosis induced by UVA, and reduced cell survival, suggesting that activation of VEGFR-1and VEGFR-2signaling pathways played a pro-survival role for keratinocytes in response to UVA;
i) Incubation of VEGFR-1and VEGFR-2neutralizing antibody significantly inhibited activation of ERK1/2and Akt induced by UVA, suggesting UVA-induced activation of VEGFR-1and VEGFR-2signaling both promotes cell survival via ERK1/2and PI3K/Akt pathway.
3. Over-expressed VEGFRs in psoriatic epidermis were normalized by UVB phototherapy in a way different from that by halomethasone
a) The over-expressed VEGFRs in the whole psoriatic epidermis was significantly down-regulated both during and after phototherapy, and interestingly, their expression decreased in a down to top advancement way, that is, VEGF receptors over-expressed in the basal layers were much more sensitive to UVB than that in upper layers, and the location of VEGF receptors shafted to the upper epidermis, even to the stratum corneum after NB-UVB treatment. In addition, VEGFRs were activated in psoriatic epidermis, but their activation was enhanced with their upward movement during NB-UVB treatment, and became extinct after whole therapy.
b) The overall result from treatment by halomethasone was similar to that by UVB phototherapy. However, the process was quite different, in which VEGF, VEGFRs and phospho-VEGFRs decreased in a gradual, homogeneous manner. Although the skin lesions were greatly improved, the expression of VEGFRs was still higher than normal skin, the expression of phospho-VEGFR was similar to VEGFRs, suggesting VEGF and its receptors are not target molecules of halomethasone, and their expression automatically declined due to improvement of the skin lesions by treatment of the drug.
c) The expression of VEGFRs in psoriatic epidermis is positively correlated with patients' PASI scoring.
Conclusions:
1. The expression of VEGFRs in keratinocytes can be upregulated by UVA and UVB irradiation, which is independent of UVB-induced autocrine release of VEGF, but partly dependent of UV-induced cell hypoxia and oxidative stress;
2. UV activates VEGFRs in keratinocytes independent of VEGF, but via activation of PKC signaling pathway;
3. Activation of VEGFRs by UV promotes survival of normal human keratinocytes. Although it acts an protective effect for keratinocytes, it provides potential opportunities for skin photoaging, malignant transformation and development of skin caners under long-term, chronic UV exposure.
4. UVB phototherapy significantly downregulates the expression of VEGFRs in psoriatic epidermis, in a way which was different from that by treatment of halomethasone0.05%cream;
5. The over-expressed VEGFRs in psoriatic epidermis may not be initial factors in psoriasis pathogenesis, however, this secondary pathological change of VEGFRs may be involved in the development of epidermal hyperplasia and parakeratosis. As UV can affect the biological behavior of keratinocytes through modulating the expression and function of VEGFRs, we demonstrate the important roles of VEGFRs in physiological and pathological conditions of epidermal keratinocytes.
引文
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