Modulation Of VEGFRs By UV And Their Functional Significance In Keratinocytes

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 halomethasonea) 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.