Expression And Function Of Vascular Endothelial Growth Factor Receptors In Normal Human Skin And Psoriatic Epidermal Keratinocytes

[BACKGROUND]Psoriasis is a common chronic,inflammatory,immune aberrant and proliferative disease of the skin that is determined by polygene and induced by multiple environmental factors.The characters of psoriasis include hyperplasia and altered differentiation of epidermis(acanthosis),infiltration of leukocytes into both the dermis and epidermis,and dilation and growth of blood vessels.Especially, angiogenesis play important roles in the pathogenesis of psoriasis. Angiogenesis is the complex process by which new blood vessels arise from the pre-existing vasculature under physical or pathological conditions.Angiogenesis is controlled by some growth factors and their receptor tyrosine kinases.Among them, the most important is vascular endothelial growth factors and their receptors,VEGF receptors(VEGFRs),which include VEGFR-1,VEGFR-2,VEGFR-3 and their co-receptors,neuropilins(including NRP-1 and NRP-2).To date,the VEGF family include VEGF-A,P1GF(placenta growth factor), VEGF-B,VEGF-C,VEGF-D,VEGF-E and svVEGF(snake venom VEGF).The general referred VEGF is VEGF-A(vascular permeability factor,VPA),which is associated with cellular physical and pathological activities in many tissues and cells.To date,six isoforms of human VEGF have been identified,which range in length from 121 to 206 amino acid residues(VEGF_{121,145,165,183,189,206}).Human skin epidermal keratinocytes is one major sources of VEGF and which normally express VEGF₁₂₁,VEGF₁₆₅and VEGF₁₈₉and specifically,VEGF₁₆₅is the strongest.The effect of VEGF is mediated by its receptors,including transmembrane VEGFR-1,VEGFR-2,VEGFR-3 and neuropilins,VEGFR-2 has a more crucial effect in angiogenesis.Recently,more and more investigations had found that VEGFRs was expressed not only in endothelial cells,but in some non-endothelial cells,and the effect of VEGF/VEGFR was not limited in enhancing angiogenesis and vascular permeability.The function of VEGF/VEGFR signal is multiple.Further investigations should be done to elucidate it.[OBJECTIVES]To investigate the expression of VEGFR in normal human skin and psoriatic nonlesional,perilesional and lesional skin,the effect of VEGF on proliferation, migration and adhesion of epidermal keratinocytes,the functional significance of VEGFRs in different stages of psoriasis and the regulating effect of calcium and VEGF on the expression of VEGFRs in psoriasis.[METHODS]In the first part:Total RNA and protein was extracted from the primary cultured epidermal keratinocytes.The mRNA and protein level of VEGFRs and NRPs was determined by reverse transcript polymerase chain reaction(RT-PCR)and Western-blot,separately.We further localized these receptors by immunofiuorescence (IF)staining in the epidermis of surgical skin specimens.The effect of VEGF on proliferation,migration and adhesion was observed by an exogenous VEGF₁₆₅.And further,the epidermal keratinocytes were preincubated with a neutralized anti-VEGFR-2 antibody to elucidate the importance of VEGFR-2 in the bioactivities of epidermal keratinocytes. In the second part:Skin samples from 17 patients with chronic plaque psoriasis and 11 normal controls were included.The expression of VEGFRs in psoriatic keratinocytes at mRNA and protein levels was determined by RT-PCR,real time PCR and Western blot analysis.Localization of the VEGFRs in skin lesions was determined by immunofluorescent method.Since keratinocyte proliferation and differentiation rely on calcium concentrations,and VEGF is overexpressed in psoriatic epidermis,we further investigated the roles of calcium and VEGF in regulating the expression of VEGFRs.[RESULTS]â… .Expression of VEGFRs and NRPs in normal human skin:1.VEGFR-1,VEGFR-2,VEGFR-3 and NRP-1,NRP-2 mRNA is expressed in normal human epidermal keratinocytes.2.The protein level of VEGFR-1,VEGFR-2,VEGFR-3 and NRP-1,NRP-2 is expressed in normal human epidermal keratinocytes.3.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-1 and VEGFR-2 strongly 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-1 and NRP-2 show diffuse intense fluorescence as compared with VEGFRs.â…¡.The association between VEGFR-2 and bioactivities of epidermal keratinocytes1.VEGF₁₆₅increased the proliferation of keratinocytes in a dose-dependent manner and this induction reached plateau at a concentration of 10 ng/ml. VEGFR-2 neutralizing antibody could partially block this induction; 2.The motility of keratinocytes was greatly affected by elevated concentration of VEGF₁₆₅in a dose-dependent manner,which was reduced by VEGFR-2 neutralizing antibody.3.VEGF reduced adhesion ofkeratinocytes,which could be completely reversed by VEGFR-2 neutralizing antibody.â…¢.Expression of VEGFRs in psoriasis1.VEGFR-1 and VEGFR-2 strongly 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-3 was detected in both normal and psoriatic epidermis except for stratum corneum.Interestingly, parakeratotic keratinocytes in lesional psoriatic stratum comeum also showed intense fluorescence for VEGFRs2.mRNA and protein levels of VEGFRs were overexpressed in psoriatic keratinocytes as compared to normal keratinocytes.â…£.The effects of calcium and VEGFon expression of VEGFRs in nonlesional psoriatic keratinocytes1.Calcium enhanced expression of VEGF₁₂₁,VEGF₁₆₅and VEGF₁₈₉in a dose-dependent manner and maximally at 1.0mM;2.In a dose-dependent manner,calcium regulated the expression of VEGFRs in a parabolic pattern:calcium maximally enhanced the expression of VEGFRs mRNA at a concentration of 1.0mM;3.VEGF₁₆₅at 10 ng/ml markedly increases expression of VEGFR-1,VEGFR-2 and VEGFR-3 in nonlesional psoriatic keratinocytes;4.Exposure of nonlesional psoriatic keratinocytes to bevacizumab at concentration of 1.0 mg/ml moderately to mostly reduced the amount of VEGF-induced VEGFR-1,VEGFR-2 and VEGFR-3 protein synthesis. However,no relevant inhibition of overexpression of VEGFRs was seen when nonlesional psoriatic keratinocytes were treated with 1.0 mM calcium addition with 1.0 mg/ml bevacizumab.[CONCLUSIONS]â… .VEGFR-1,VEGFR-2,VEGFR-3 and their co-receptors,NRP-1 and NRP-2 are expressed in normal human epidermal keratinocytes in vitro at mRNA and protein levels;â…¡.VEGFRs and NRPs are expressed in normal human epidermis except for the corny layer.VEGFR-1 and VEGFR-2 are localized primarily in basal cell layer and adjacent spinosum stratum,while VEGFR-3,NRP-1 and NRP-2 are uniformly expressed in the epidermis;â…¢.VEGF enhances the proliferation and migration,while inhibits the adhesion of epidermal keratinocytes partly through VEGFR-2;â…£.VEGFR-1,VEGFR-2 and VEGFR-3 are overexpressed by psoriatic lesional keratinocytes,compared with normal epidermal keratinocytes,at mRNA and protein levels;â…¤.The distribution pattern of VEGFR-1,VEGFR-2 and VEGFR-3 may be associated with proliferation,migration and differentiation of psoriatic epidermal keratinocytes;â…¥.VEGF and calcium may be two factors enhancing expression of VEGFR-1, VEGFR-2 and VEGFR-3 in psoriasis;â…¦.Calcium enhances expression of VEGFR-1,VEGFR-2 and VEGFR-3 independent of VEGF.