Study On Expression Of Hypoxia Inducible Factor-1α In HaCaT Cells Cultured Under Hypoxia And In Psoriatic Lesions Relationship With Angiogenesis-Related Protein

Psoriasis is a common chronic inflammatory disease of skin, Histological hallmarks of psoriatic skin include the infiltration of multiple immune cells, keratinocyte proliferation and increased dermal vascularity. Inseparable relationship Between the three, Angiogenesis play important roles in the pathogenesis of psoriasis. As a disease related to angiogenesis, Now psoriasis Research on the mechanism of angiogenesis has become a hot spot, In particular, some angiogenic factors have received increasing attention. Keratinocytes is a major source of pro-angiogenic, it can secrete vascular endothelial growth factor, transforming growth factor-α, fibroblast growth factors, tumor necrosis facto-α, inducible nitric oxide synthase, Angiopoietins,thymidine phosphorylase, interleukin-8. VEGF is found in one of the strongest and most specific direct factor in promoting angiogenesis, iNOS induced NO generation,and play the role of promoting angiogenesis.Microvascular is a place of exchange material skin tissue and circulating blood, Hyperblastosis or other factor surpass vascularity capacity,and leading to tissue hypoxia. Body feelings changes in oxygen concentration and make adaptive responses to hypoxia,to maintain tissue oxygen supply and to enhance the adaptability of cells. Hypoxia-inducible factor 1 is one of the most important factor hypoxia signal transduction, HIF-1 is a central regulator that induced gene expression and restore cell homeostasis when hypoxia. Under hypoxia, HIF-1 activates the expression of these genes by binding to a 50-base pair cis-acting HRE located in their enhancer and promoter regions, to adapt to changes in oxygen supply,that has important significance to pathophysiology.Some of the latest Studies display that hypoxia may be involved to the occurrence and development of psoriasis. Subcellular localization and transcriptional potency of the HIF-1αsubunits, Hypoxia blocks HIF-1αdegradation leading to accumulation, Induced the expression of target genes such as VEGF and iNOS. Therefore, We speculate that hypoxia may induce the expression of HIF-1αin psoriasis, Further to regulate some angiogenesis-related target gene expression, such as VEGF and iNOS,to play an important role in the process of development of psoriasis. We plan to confirmed by study of three parts as follows:The first to study relationship between HIF-1αand the expression of angiogenesis-related proteins in psoriatic lesions,the second to study relationship between HIF-1αand the expression of angiogenesis-related proteins on HaCaT cells cultured in hypoxia,the third to examine the effect of downregulated expression of HIF-1αon HaCaT cells cultured in hypoxia.these studies would further reveal HIF-1αplay a role of angiogenesis in psoriasis. Part I Relationship Study on Hypoxia Inducible Factor-1αand Angiogenesis -Related Protein Expression in Psoriatic LesionsObjective To investigate the expression of HIF-la,VEGF and iNOS protein in lesions of active psoriasis vulgaris and its correlations with angiogenesis.Methods The expression of HIF-1α,VEGF and iNOS proteins was detected in lesions and non- lesions of 32 cases of psoriasis vulgaris and the normal skin of 20 healthy control by immunohistochemical and western blotting methods, Special image analysis system SimplePCI was used for the quantitative analysis of expression of HIF-1α,VEGF and iNOS proteins, and CD34 marking vascular endothelial cell was used to measure the microvascular density (MVD).Results The average integral obsorbance of HIF-1α,VEGF and iNOS protein were 86.96±2.56,95.81±2.74 and 90.00±2.77 in 32 cases of psoriatic lesions, there waas a significantly difference compared with the control group. and expression of HIF-1αprotein (r=0.688,P<0.01),expression of VEGF protein(r=0.965,P<0.01) and expression of iNOS protein(r=0.899,P<0.01)had positive correlation with MVD in psoriasis respectively.It showed that VEGF and iNOS expression showed a positive association with HIF-1a expression in psoriatic lesions (r=0.721,P<0.01;r=0.873,P<0.01). The average integral obsorbance of HIF-la,VEGF and iNOS were 30.14±7.77,44.32±9.06, 48.76±10.08,MVD was 17.16±3.54 in 32 cases of psoriatic nonlesional skin which showing a no obvious difference in the expression of HIF-la,VEGF,iNOS,MVD with control group.Protein extracted from 20 healthy control and psoriasis skin samples of 32 patients and performed Western blot analysis, The results of Western blot had coincidence with the results of immunohistochemistry.Conclusion There has a overexpression of HIF-1αVEGF and iNOS in psoriatic lesional skin and they have a synergistic effect in new angiogenesis of the psoriasis.They may play an important role in the genesis and development of psoriasis.PartⅡStudy on expression of HIF-la and angiogenesis-related proteins in HaCaT Cells Cultured under Hypoxia ConditionsObjective To investigate the expression of HIF-la,VEGF and iNOS mRNA/protein in HaCaT Cells Cultured under Hypoxia conditions.Methods HaCaT cells was cultured under normoxic conditions and hypoxic conditions for 4h,8h,12h,24h respectively. The cell total RNA and whole cell protein were extracted. The expression level of HIF-1α,VEGF and iNOS mRNA/protein was measured by Real-time RT-PCR and western blotting repectively.Results There wasn't a significant difference for expression level of HIF-1αmRNA between the hypoxia group and normoxia group(p> 0.05), whereas, the expression level of HIF-1αprotein was obviously increased in HaCaT cells cultured under hypoxia conditions. The expression levels of VEGF and iNOS mRNA/protein were higher in HaCaT cells in hypoxia group than that in normoxic group (P<0.01)Conclusion (1) Hypoxia has no influence on the expression o HIF-1αmRNA,but could increase its protein expression level. (2) Hypoxia stimulation strongly upregulatesVEGF and iNOS mRNA/protein levels in HaCaT cells.PartⅢEfect of RNA Interference for HIF-la on Expression of Angiogenesis-related proteins in HaCaT cell under HypoxiaObjective To investigate the influence of RNA Interference targeting HIF-1αon expression of HIF-1α, VEGF and iNOS mRNA/ proteins in HaCaT cell under Hypoxia conditions.Methods Small interfering RNA(siRNA) targeting HIF-1αwas designed and synthesized,which was then transfected into HaCaT cells by using Lipofectamine 2000 reagent, at the same time the control group was set up. Total cellular RNA and whole cell protein was extracted from the treated HaCaT cells 24hrs after culture under under Hypoxia conditions. Real-time RT-PCR and Western blotting were used to detect the expression levels of VEGF and iNOS mRNA/proteins.Results The expression of HIF-la, VEGF and iNOS mRNA/ proteins in cultured HaCaT cells under hypoxia were significantly decreased in HIF-1α-siRNA transfected group compared with the control group (P<0.05).Conclusion Downregulation of HIF-1αexpression can inhibit expression of VEGF and iNOS mRNA/proteins in HaCaT cells under hypoxia. We speculated expression of VEGF and iNOS were regulated by HIF-1 in HaCaT cells under hypoxia.