VEGF在类胰蛋白酶引起皮下毛细血管通透性增加中的作用研究

VEGF在类胰蛋白酶引起皮下毛细血管通透性增加中的作用研究 VEGF在类胰蛋白酶引起皮下毛细血管通透性增加中的 作用研究 VEGF is involved in the increase of dermal microvessel permeability induced by tryptase Bai Qianming Wang Xinhong Xu Yali Yin Lianhua Li Xiaobo 5 10 15 20 25 30 35 40 45 Fudan University Shanghai Medical College ShangHai 200032 Abstract Mast cells abundantly reside near the dermal vessels and release tryptase in the condition of cutaneous hypersensitivity and allergy which is meanifested by rapid edema due to increased vascular permeability However the mechanism of mast cell tryptase promoting permeability remains to be defined In this study we investigated the effect of tryptasehuman mast cells HMC-1 supernatant on the permeability of human dermal microvascular endothelial cells HDMECs and studied whether vascular endothelial growth factor VEGF is involved in this effect or not HMC-1 cells released tryptase pro-degranulating agent a23187 dose-dependently and HMC-1 cells density-dependently Both tryptase and HMC-1 supernatant can promote permeability of HDMECs dramatically dose-dependently which was resisted by tryptase inhibitor APC366 a specific tryptase inhibitor and partially reversed by a VEGF neutralizing antibody Tryptase added to HDMECs caused a significant increase of mRNA and protein levels of VEGF VEGF receptors Flt-1 and Flk-1 by Real-time RT-PCR assay and Western-blot respectively Our results suggest that mast cells tryptase can up-regulate the expression of VEGF and VEGF receptors The VEGF neutralizing antibody can block the dermal microvessel hypermeability induced by tryptase Thus VEGF is involved in the increase of tryptse-induced dermal microvessel hypermeability and edema Keywords tryptase mast cells VEGF endothelial cells permeability skin 0 Introduction Mast cells are the key cells of cutaneous hypersensitivity reactions and allergic inflammatory responses such as contact dermatitiseczema and nettle rash In the skin mast cells reside mostly in the dermis associated with blood vessels and appendages[1] Mast cells activation results in the prompt release of granule-stored mediators such as chymase proteoglycans histamine cytokines and so on The predominant mediator is tryptase which is a kind of serine protease with tetrameric conformation-tryptase and β-tryptase are most abundantly expressed in and secreted by mast cells Human mature β-tryptase is stored in the mast cells granules and released upon activation while α-tryptase is apparently processed only to the proenzyme stage and is constituively secreted along with pro-tryptase In healthy individuals only-tryptase can be detected whereas -trypatase is undectable However significant elevations of circulating β-tryptase levels were observed in patients with allergic diseases[23] Cellular response to tryptase is mediated mainly through protease-activated receptor-2 PAR-2 that belongs to the G-protein-coupled receptors PAR-2 is stimulated by proteolytic cleavage of their extracellular domain unmasking a new N-terminus acting as a tethered ligand[4] PAR-2 is localized on keratinocytes especially in the granular layer endothelial cells hair follicles myoepithelial cells of sweat glands and dermal dendritic-like cells PAR-2 is also highly expressed in keratinocytes and endothelial cells of inflamed skin[5] Edema caused by increased vascular permeability is a major manifestation of allergic responses in skin[6] Tryptase may contribute to vascular permeability by the direct or indirect generation of bradykinin from kininogens[7] Intradermal injection of tryptase can induce the immediate cutaneous reaction manifested with increase of dermal microvascular permeability This effect can be inhibited by synthetic tryptase inhibitor APC366 or a combination of the Foundations Specialized Research Fund for the Doctoral Program of higher education 200802461083 2009 CMA-LOREAL China Skin Hair Grant funds Brief author introduction柏乾明1983-男博士研究生研究方向动脉粥样 硬化 Correspondance author 李晓波 1980-女讲师研究方向动脉粥样硬化 E-mail xblifcom -1- histamine H1 and H2 antagonists suggesting that the tryptase response is mediated by histamine[8] Kawabata et al reported that PAR-2 activating peptide PAR-2AP causes enhancement of vascular permeability and edema in rat hind paws[9] But Steinhoff et al have shown that the vascular action of PAR-2AP in rat hind paws is due to the release of sensory peptides such as 50 55 60 65 70 75 80 85 calcitonin gene-related peptide and substance P from sensory nerve endings[10] Itoh et al reported the elevation of intracellular Ca2 and activation of PKC after PAR-2 stimulation might trigger the formation of actin stress fibers and disappearance of tight junctional proteins such as VE-cadherin which leads to barrier dysfunction[11] So far the mechanisms of enhancement of vascular permeability caused by tryptase are still not clear and need further study Vascular endothelial growth factor VEGF also known as vascular permeability factor or VPF is a potent agent that causes hyperpermeability of blood vessels[12] In patients with delayed hypersensitivity the amount of VEGF produced in lesional scales was approximately 25 times higher than that in normal stratum corneum[13] In patients with allergic contact dermatitis the mRNAs encoding VPFVEGF two VPFVEGF vascular endothelial cell receptors flt-1 and flk-1 were all strikingly over-expressed in dermal microvascular cells[14] Microvessel permeability can be increased by mast cell tryptase VEGF is an important agent mediating microvessel permeability Interestingly tryptase VEGF and VEGF receptors all abundantly reside in the dermal allergic and hypersensitive reactions Based on the above reports we hypothesize that tryptase may increase the dermal microvascular permeability as well as edema through regulating the expression of VEGF and VEGF receptors Then we set up the model of hyperpermeability in cultured HDMECs Human dermal microvascular endothelial cells using purified human β-tryptase or tryptase released by human mast cell line HMC-1 The effects of tryptase and tryptase inhibitor APC366 on VEGF and VEGF receptor flt-1 flk-1 expression were investigated We used a VEGF neutralizing antibody to inhibit VEGF and then observed the permeability of HDMECs monolayers 1 Materials and Methods 11 Reagents Culture media reagents and SuperScript TMIII First–Strand Synthesis System for RT-PCR were purchased from Invitrogen Carlsbad CA USA SV Total RNA Isolation Kit was from Promega Madison WI USA SYBR Green real-time PCR Master Mix was from Toyobo Company Osaka JP Primary antibody against von Willebrand factor vWF CD34 vascular endothelial growth factor VEGF fms-like tyrosine kinase Flt-1 kinase insert domain containing receptor Flk-1 and Glyceraldehydes-3-phosphate dehydrogenase GAPDH were purchased from SantaCruz Biotechnology Inc Santa Cruz CA Anti-human VEGF Antibody for inhibiting VEGF was obtained from RD Systems Minneapolis MN USA SuperSignal West Pico Chemiluminescent Substrate was obtained from Pierce Biotechnology Inc Rockford IL USA -trypatase was kindly provided by Dr Shunlin Ren Division of Gastroenterology Virginia Commonwealth University RichmondVA USA All other reagents were from Sigma-Aldrich Chemical Co St Louis MO unless otherwise mentioned 12 Isolation culture and identification of human dermal microvascular endothelial cells HDMECs The method of HDMECs isolation and culture was set up based on literatures published previously[15-18] Briefly human neonatal foreskins were cut into small pieces and digested by 05 mgml Dispase dissolved in sodium acetate at 37?C for 1 h After removal of the epidermis the -2- 90 95 100 105 110 115 120 125 dermal fragments were treated with 1 collagenase I at 37?C for 1 h The microvascular segments were passed through a 100-μm nylon mesh cell strainer collected and purified by Percoll gradient centrifugation The fraction with a density 1048 gml which was rich in microvascular fragments was removed and applied to gelatin-precoated tissue-culture dishes and cultured in Dulbeccos modified Eagles medium DMEM 1000 mgL glucose supplemented with 10 mM HEPES 10 mM L-glutamine 15 Uml heparin 1 μgml hydrocortisone acetate 325 μgml glutathione 005 mM dibutyryl cyclic AMP 5 μgml insulin 5 μgml transferin 5 μM 2-mercaptoethanol 100 Uml penicillin 100 μg ml streptomycin and 20 fatal bovine serum The HDMECs were identified on the basis of morphological characteristics immunofluorescent staining of Von Willebrand factor vWF and CD34 The expressions of CD34 and vWF in HDMECs were also quantified with flow cytometry All experiments used HDMECs at passages 2-4 13 Culture of human mast cell line HMC-1 The human mast cell line HMC-1 was kindly obtained from Second Military Medical University Shanghai China The cells were cultured in 75 cm2 flasks in Iscoves modified Dulbeccos medium IMDM supplemented with 10 fetal bovine serum FBS 100 IUml penicillin and 100 μgml streptomycin in humidified air with 5 CO2 at 37?C Collected HMC-1 cells were activated and degranulated in the addition of pro-degranulating agent a23187 The HMC-1 supernatant containing tryptase is collected centrifuged filtered and then used as conditioned medium henceforth referred as HMC-1 supernatant in the following experiments The activity of tryptase released from HMC-1 was quantified by monitoring hydrolysis of tosyl-L-Gly-Pro-Lysp-nitroanilide t6140 using a standard spectrophotometric assay at 405-nm wavelength As described below optimal stimulation and release were achieved by incubating HMC-1 cells 1x107ml for 2 h with a23187 1 μgml at 37?C 14 Measurement of vascular permeability in cultured HDMECs As described on previous literature[19] HDMECs were grown to confluent monolayers on gelatin-coated membranes in double-chamber tissue culture plates Transwell membrane 04 μM pore size Corning Costar After 2 d chambers were examined microscopically for integrity and uniformity of endothelial monolayers The confluent monolayers were incubated with APC366 250 μgml anti-VEGF antibody 01 μgml followed by activation by either tryptase or HMC-1 supernatant for 18 h as described At the end of the incubation period FITC-conjugated dextran 1 mgml Mr 42000 Sigma-Aldrich was added to the upper chambers and fluorescence in the lower chamber was measured 1 h later with a fluorescence reader Experiments were performed in triplicate and repeated multiple times 15 Quantitative real-time RT-PCR Total RNA was extracted according to the suppliers instructions Two micrograms of total RNA were reversely transcribed and amplified The relative mRNA level was measured by real-time PCR using SYBR-Green Real-time PCR Master Mix Toyobo Osaka Japan with SYBR-Green I Specific primer pairs for VEGF Flt-1 Flk-1 and GAPDH were listed in Table 1 130 135 -3- Table 1 Primer pairs used to amplify PCR products Gene Sequence 5-3 Product size GeneBank No VEGF Forward CAACATCACCATGCAGATTATGC 132bp NM_001033756 Reverse CCCACAGGGATTTTCTTGTCTT Flt-1 Forward TGGCTGCGACTCTCTTCTG 118bp NM_002019 Reverse CAAAGGAACTTCATCTGGGTCC Flk-1 Forward GGCCCAATAATCAGAGTGGCA 104bp NM_002253 Reverse TGTCATTTCCGATCACTTTTGGA GAPDH Forward CATGAGAAGTATGACAACAGCCT 113bp NM_002046 Reverse AGTCCTTCCACGATACCAAAGT 16 Western-blot Total cell lysates of HDMECs were extracted on ice with 1 NP40 05 sodium 140 145 150 155 160 deoxycholate and 01 SDS in PBS with proteinase inhibitor cocktail Sigma 30 μg of total protein were separated on 10 SDS-PAGE gels and transferred onto a polyvinylidene difluoride membrane using a Bio-Rad Mini-Blot transfer apparatus as described previously[20] Membranes were blocked in PBS containing 5 nonfat dried milk for 1 h The membranes were incubated in primary antibodies against VEGF 1500 dilution Flt-1 1200 dilution Flk-1 1200 dilution and GAPDH 11000 dilution for overnight at 4?C After washing the membranes were then incubated in a 14000 dilution of a secondary antibody goat anti-rabbit IgG-HRP conjugate Bio-Rad at room temperature for 1 h in TBS washing buffer containing 05 Tween 20 After washing protein bands were visualized using Western Lightning Chemiluminescences Reagent and developed on Bio Light Film Kodak The bands densities were quantified by densitometry using software of GIS Bio-Tanon Shanghai China VEGF Flt-1 and Flk-1 protein levels were normalized to the GAPDH levels All Western-blot experiments were repeated at least three times with separate cells preparation 17 Statistical analysis Data were presented as mean?SEM Statistical significance was assessed by one-way ANOVA and discrepancies between groups were considered statistically significant at P,005 2 Results 21 Culture and identification of human dermal microvascular endothelial cells HDMECs All HDMECs gave typical confluent cobblestone appearance Fig 1A and had positive reactions to the antibodies against vWF Fig 1B and CD34 Fig 1C Negative control without first antibody exhibited no staining Fig 1D The expressions of vWF and CD34 were also quantified with flow cytometry Exceed 90 cells were positive for vWF and CD34 which suggested the purity of the primary cells exceeded 90 -4- 165 170 175 180 Figure 1 Primary culture and purity check Morphology of passage 2 human dermal microvascular endothelial cells HDMECs was shown in A To confirm the purity of HDMECs we used von Willebrands factor B and CD34 C immunostaining which are specific for endothelial cells Negative control without first antibody staining is displayed in D X 200 22 Measurement of the tryptase activity of HMC-1 supernatant To affirm the existence of tryptase in the conditioned medium we incubated the HMC-1 supernatant made with HMC-1 suspension in the presence and absence of pro-degranulating agent a23187 1 μgml with substrate t6140 N-Tosylglycyl-L-prolyl-L-lysine 4-nitroanilide acetate salt 8 mmolL for 10 minutes in the reaction buffer 40 mM HEPES012 M NaClpH 74 OD value of the reactions were detected by spectrophotometer at 405nm each 30 seconds The control is set with only the reaction buffer As shown in Fig 2A the change of OD405 formation of t6140-derived product digested by tryptase was linear for at least 10 minutes Then 5 minutes was chosen to be the reaction time Tryptase was released in the HMC-1 supernatant which is increased dramatically by pro-degranulating agent a23187 Fig 2B A23187 stimulated HMC-1 cells to release tryptase dose-dependently Fig 2C On the other way tryptase was released from HMC-1 cells by 1 μgml a23187 in density-dependent manner Fig 2D In the following experiments HMC-1 supernatant was prepared using 1x107 HMC-1 cellsml treated with 1 μgml a23187 -5- 185 190 Figure 2 Effect of tryptaseHMC-1 supernatant on the permeability of HDMECs As described in the Methods the permeability of HDMECs after indicated treatment was detected by measuring fluorescence in the lower chamber at 490nm after incubation with FITC-dextran for 1 h in the upper chamber The changes of OD490 in the lower chamber after 1h of incubation were calculated for displaying the permeability of HDMECs A Effect of tryptase and APC366tryptase at different concentrations on the permeability of HDMECs The heparin control was also shown B Effect of HMC-1 supernatant and APC366HMC-1 supernatant at different concentrations on the permeability of HDMECs C Effect of anti-VEGF antibody on the increase of permeability stimulated by tryptase D Effect of anti-VEGF antibody on the increase of permeability stimulated by HMC-1 supernatant P 005 compared to the group of non-addition P 005 compared to the group only treated with tryptase P 005 compared to the group only treated with HMC-1 supernatant 195 23 Effect of tryptaseHMC-1 supernatant on the permeability of HDMECs As described in the method the amount of FITC-dextran in the lower chamber leaked from the HDMECs layer was detected to scale the permeability of HDMECs The permeability of HDMECs with different treatments was quantified by the percentage of OD490 change The 200 205 210 confluent monolayers were treated with tryptase or HMC-1 supernatant for 18 h in the presence and absence of APC366 a selective inhibitor of tryptase 250 μgml pretreatment As shown in Fig3A tryptase promoted permeability of HDMECs dramatically in a dose-dependent manner which was resisted by APC366 Because-tryptase was added into HDMECs accompanied by heparin as stabilizer heparin control was also studied In result the heparin added into the cells had no effect on the permeability Fig 3B showed that HMC-1 supernatant enhanced the permeability of HDMECs dose-dependently which was resisted by APC366 To investigate whether VEGF is involved in the hypermeability anti-VEGF antibody 01 μgml was preincubated on HDMECs to block VEGF The data was normalized to groups treated with normal goat IgG As a result inhibition of VEGF significantly attenuated tryptase-induced permeability Fig 3C but only modestly attenuated HMC-1 supernatant-induced permeability Fig 3D -6- 215 220 225 230 Figure 3 Effect of tryptaseHMC-1 supernatant on the permeability of HDMECs As described in the Methods the permeability of HDMECs after indicated treatment was detected by measuring fluorescence in the lower chamber at 490nm after incubation with FITC-dextran for 1 h in the upper chamber The changes of OD490 in the lower chamber after 1h of incubation were calculated for displaying the permeability of HDMECs A Effect of tryptase and APC366tryptase at different concentrations on the permeability of HDMECs The heparin control was also shown B Effect of HMC-1 supernatant and APC366HMC-1 supernatant at different concentrations on the permeability of HDMECs C Effect of anti-VEGF antibodySU5416 on the increase of permeability stimulated by tryptase D Effect of anti-VEGF antibody on the increase of permeability stimulated by HMC-1 supernatant P 005 compared to the group of non-addition P 005 compared to the group only treated with tryptase P 005 compared to the group only treated with HMC-1 supernatant 24 Effect of tryptase and APC366tryptase on the VEGF Flt-1 and Flk-1 protein levels in HDMECs To study the mechanism of resistance of tryptase-induced hypermeability by anti-VEGF antibody the protein levels of VEGF Flt-1 and Flk-1 in HDMECs of indicated treatments were analyzed by Western-blot Different concentrations of tryptase were added into HDMECs for 18 h in the absence and presence of APC366 The heparin control was also analyzed As a result increases in VEGF Fig 4A Flt-1 Fig 4B and Flk-1 Fig 4C protein levels following the addition of tryptase to the cells in culture were concentration dependent APC366 resisted the upregulation of VEGF Fig 4A Flt-1 Fig 4B and Flk-1 Fig 4C by tryptase The heparin added into the cells had no effect on these protein expressions -7- 235 240 245 250 255 Figure 4 Effect of tryptase and APC366tryptase on the VEGF Flt-1 and Flk-1 protein levels in HDMECs Different concentrations of tryptase 0 1 5 and 10 nmolL indicated as Lane 1 2 3 and 4 respectively were added into HDMECs for 18 h in the absence and presence of APC366 The VEGF A Flt-1 B and Flk-1 C protein levels were detected by Western-blot and normalized to GAPDH The heparin control was also analyzed P 005 compared to the group of non-addition P 005 compared to the group without APC366 at the same concentration of tryptase or HMC-1 supernatant 25 Effect of tryptase on the VEGF Flt-1 and Flk-1 mRNA levels in HDMECs To determine whether varieties of VEGF Flt-1 and Flk-1exposed to tryptase are mediated by varieties of their RNA expression we studied the effect of tryptase on VEGF Flt-1 and Flk-1 mRNA expression in HDMECs by Real-time RT-PCR GAPDH was determined in parallel and used as an internal standard Different concentrations of tryptase were added into HDMECs for 6 h The expression levels were normalized to heparin control As figure 5 shown tryptase up-regulated VEGF Flt-1 and Flk-1 mRNA levels significantly Figure 5 Effect of tryptase on the VEGF Flt-1 and Flk-1 mRNA levels in HDMECs Different concentrations of tryptase 0 1 and 10 nmolL were added into HDMECs for 6 h Real time RT-PCR analysis of VEGF A Flt-1 B and Flk-1 C mRNA levels are shown in Figure 5 GAPDH was determined in parallel and used as an internal standard The heparin control was also analyzed The expression levels were normalized to heparin control P 005 compared to the group of heparin control -8- 3 Discussion Our study reveals that tryptase can upregulate the expression of VEGF and VEGF receptors VEGF inhibitor can partly block the dermal microvascular hyperpermeability induced by tryptase 260 265 270 275 280 285 290 295 300 By the reason of the primary HDMECs cultures approximate the physiologic cell features in vivo more than cloned or transformed cell lines we isolated and cultured primary HDMECs from newborn human foreskins The HDMEC monolayer has cobblestone morphology compatible with endothelial cells and stained positive with anti-vWF The microvascular characteristic of these endothelial cells was confirmed by the presence of CD34 which is the distinguishing feature of microvascular endothelial cells[21] Mast cell tryptase is one of the crucial molecules released by mast cells that are ubiquitous in the body and critical for allergic reactions in skin The subunits of the β-tryptase tetramer are held together by hydrophobic and polar interactions between subunits[22] Heparin stabilizes tryptase in its physiologically active tetrameric conformation[23] β-Tryptase used in our experiments was stabilized by heparin glycosaminoglycan at ,12 molar ratio Identical concentrations of heparin were added to cells as a control In our study APC366 N- 1-Hydroxy-2-naphthoyl - L-arginyl-L-prolinamide a selective inhibitor of mast cell tryptase was used as an antagonist of tryptase to confirm the specific effect of tryptase Edema is a prominent manifestation of allergic reactions in skin which is caused by enhanced vascular permeability Greatly expanded populations of dermal mast cells gather in cutaneous sites when inflammation Tryptase released by mast cells play an important role in vascular permeability increase Our data also confirmed either purified tryptase or HMC-1-released tryptase stimulated permeability of HDMECs To investigate whether tryptase-induced hyperpermeability is mediated by VEGF we blocked the effect of VEGF using anti-VEGF antibody In result tryptase stimulated permeability of HDMECs dramatically in a dose-dependent manner which was resisted by APC366 and reversed by anti-VEGF partially Hereby VEGF is at least partially responsible to the enhancement of permeability induced by tryptase We also did parallel experiments using mast cells conditioned media containing tryptase Human mast cell line HMC-1 containing mostly β-tryptase and traces of chymase and other mediators is a very useful tool for in vitro studies on a large number of human mast cells of the mucosal phenotype Tryptase activity assay demonstrated that tryptase was released in the HMC-1 supernatant By pretreating the HMC-1 supernatant with APC366 we observed a marked decrease in the permeability Thus tryptase in the HMC-1 supernatant participated in the hyperpermeability of HDMECs Our results also suggested VEGF is involved in the enhancement of hyperpermeability induced by HMC-1 cells Based on our data APC366 dramatically inhibited the increase of permeability induced by either exogenous tryptase or supernatants from mast cells Inhibition of VEGF significantly attenuated tryptase-induced permeability but only modestly attenuated mast cell-induced permeability Fig 3D One of the possibility is the components of HMC-1 supernatant are complicated including chymase histamine heparin TNF-α and SCF etc with the exclusion of tryptase These mediators may interfere with the effect of anti-VEGF antibody The mechanisms of enhancement of vascular permeability caused by tryptase were studied in literature too As mentioned in the introduction tryptase may contribute to vascular permeability by variety of mechanisms The present study focuses on the role of VEGF in the tryptase-induced hyperpermeability The VEGF family includes VEGF-A placenta growth factor PlGF VEGF-B VEGF-C and VEGF-D The original member of the VEGF family VEGF-A also known as -9- vascular permeability factor was characterized as a potent inducer of vascular permeability[24-27] In the present work VEGF-A is represented as VEGF Anti-human VEGF antibody was used to block VEGF-A VEGF-A binds to the 2 types III receptor tyrosine kinases VEGF receptor-1 305 310 315 320 325 330 335 340 345 350 355 VEGFR-1 namely Flt-1 and VEGFR-2 namely KDR or Flk-1 which are primarily expressed by vascular endothelial cells Previous studies have revealed that VEGF expressions by epidermal keratinocytes and endothelial expression of VEGF receptors are up regulated in cutanous inflammation[28] Our study suggested tryptase might promote permeability of HDMECs by increasing the expression of VEGF and VEGF receptors Flt-1 and Flk-1 in the endothelial cells However the specific mechanism by which tryptase stimulates expression of VEGF and VEGF receptors should be elucidated in the further study 4 Conclusion In conclusion mast cells tryptase may promote the dermal microvessel permeability which can be reversed by VEGF inhibitor VEGF is involved in the increase of dermal microvessel hyperpermeability and edema induced by tryptase Such knowledge may lead to novel means of controlling allergic reaction in skin References [1] Garbuzenko E Nagler A Pickholtz D Gillery P Reich R Maquart FX Levi-Schaffer F Human mast cells stimulate fibroblast proliferation collagen synthesis and lattice contraction a direct role for mast cells in skin fibrosis[J] Clin Exp Allergy 2002 32 2 237-246 [2] Fukuoka Y Schwartz LB Human beta-tryptase detection and characterization of the active monomer and prevention of tetramer reconstitution by protease inhibitors[J] Biochemistry 2004 43 33 10757-10764 [3] Fukuoka Y Schwartz LB The B12 anti-tryptase monoclonal antibody disrupts the tetrameric structure of heparin-stabilized beta-tryptase to form monomers that are inactive at neutral pH and active at acidic pH[J] J Immunol 2006 176 5 3165-3172 [4] Kanke T Takizawa T Kabeya M Kawabata A Physiology and pathophysiology of proteinase-activated receptors PARs PAR-2 as a potential therapeutic target[J] J Pharmacol Sci 2005 97 1 38-42 [5] Steinhoff M Corvera CU Thoma MS Kong W McAlpine BE Caughey GH Ansel JC Bunnett NW Proteinase-activated receptor-2 in human skin tissue distribution and activation of keratinocytes by mast cell tryptase[J] Exp Dermatol 1999 8 4 282-294 [6] Bazzoni G Dejana E Endothelial cell-to-cell junctions molecular organization and role in vascular homeostasis[J] Physiol Rev 2004 84 3 869-901 [7] Imamura T Dubin A Moore W Tanaka R Travis J Induction of vascular permeability enhancement by human tryptase dependence on activation of prekallikrein and direct release of bradykinin from kininogens[J] Lab Invest 1996 74 5 861-870 [8] Molinari JF Moore WR Clark J Tanaka R Butterfield JH Abraham WM Role of tryptase in immediate cutaneous responses in allergic sheep[J] J Appl Physiol 1995 79 6 1966-1970 [9] Kawabata A Kuroda R Minami T Kataoka K Taneda M Increased vascular permeability by a specific agonist of protease-activated receptor-2 in rat hindpaw[J] Br J Pharmacol 1998 125 3 419-422 [10] Steinhoff M Vergnolle N Young SH Tognetto M Amadesi S Ennes HS Trevisani M Hollenberg MD Wallace JL Caughey GH Mitchell SE Williams LM Geppetti P Mayer EA Bunnett NW Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism[J] Nat Med 2000 6 2 151-158 [11] Itoh Y Sendo T Oishi R Physiology and pathophysiology of proteinase-activated receptors PARs role of tryptasePAR-2 in vascular endothelial barrier function[J] J Pharmacol Sci 2005 97 1 14-19 [12] Bates DO Harper SJ Regulation of vascular permeability by vascular endothelial growth factors[J] Vascul Pharmacol 2002 39 4-5 225-237 [13] Brown LF Olbricht SM Berse B Jackman RW Matsueda G Tognazzi KA Manseau EJ Dvorak HF Van de WL Overexpression of vascular permeability factor VPFVEGF and its endothelial cell receptors in delayed hypersensitivity skin reactions[J] J Immunol 1995 154 6 2801-2807 [14] Zhang Y Matsuo H Morita E Increased production of vascular endothelial growth factor in the lesions of atopic dermatitis[J] Arch Dermatol Res 2006 297 9 425-429 [15] Davison PM Bensch K Karasek MA Isolation and growth of endothelial cells from the microvessels of the newborn human foreskin in cell culture[J] J Invest Dermatol 1980 75 4 316-321 [16] Davison PM Karasek MA Human dermal microvascular endothelial cells in vitro effect of cyclic AMP on cellular morphology and proliferation rate[J] J Cell Physiol 1981 106 2 253-258 [17] Marks RM Czerniecki M Penny R Human dermal microvascular endothelial cells an improved method for tissue culture and a description of some singular properties in culture[J] In Vitro Cell Dev Biol 1985 - 10 - 360 365 370 375 380 385 390 395 400 21 11 627-635 [18] Gupta K Ramakrishnan S Browne PV Solovey A Hebbel RP A novel technique for culture of human dermal microvascular endothelial cells under either serum-free or serum-supplemented conditions isolation by panning and stimulation with vascular endothelial growth factor[J] Exp Cell Res 1997 230 2 244-251 [19] Orlova VV Economopoulou M Lupu F Santoso S Chavakis T Junctional adhesion molecule-C regulates vascular endothelial permeability by modulating VE-cadherin-mediated cell-cell contacts[J] J Exp Med 2006 203 12 2703-2714 [20] Li X Zhou T Zhi X Zhao F Yin L Zhou P Effect of hypoxiareoxygenation on CD73 ecto-5-nucleotidase in mouse microvessel endothelial cell lines[J] Microvasc Res 2006 72 1-2 48-53 [21] Wang XH Chen SF Jin HM Hu RM Differential analyses of angiogenesis and expression of growth factors in micro- and macrovascular endothelial cells of type 2 diabetic rats[J] Life Sci 2009 84 7-8 240-249 [22] Ren S Lawson AE Carr M Baumgarten CM Schwartz LB Human tryptase fibrinogenolysis is optimal at acidic pH and generates anticoagulant fragments in the presence of the anti-tryptase monoclonal antibody B12[J] J Immunol 1997 159 7 3540-3548 [23] Blair RJ Meng H Marchese MJ Ren S Schwartz LB Tonnesen MG Gruber BL Human mast cells stimulate vascular tube formation Tryptase is a novel potent angiogenic factor[J] J Clin Invest 1997 99 11 2691-2700 [24] Bates DO Harper SJ Regulation of vascular permeability by vascular endothelial growth factors[J] Vascul Pharmacol 2002 39 4-5 225-237 [25] Dvorak HF Discovery of vascular permeability factor VPF [J] Exp Cell Res 2006 312 5 522-526 [26] Dvorak HF Brown LF Detmar M Dvorak AM Vascular permeability factorvascular endothelial growth factor microvascular hyperpermeability and angiogenesis[J] Am J Pathol 1995 146 5 1029-1039 [27] Ribatti D The crucial role of vascular permeability factorvascular endothelial growth factor in angiogenesis a historical review[J] Br J Haematol 2005 128 3 303-309 [28] Brown LF Olbricht SM Berse B Jackman RW Matsueda G Tognazzi KA Manseau EJ Dvorak HF Van de WL Overexpression of vascular permeability factor VPFVEGF and its endothelial cell receptors in delayed hypersensitivity skin reactions[J] J Immunol 1995 154 6 2801-2807 VEGF 在类胰蛋白酶引起皮下毛细血管通透性增加中 的作用研究 柏乾明王新红许雅丽殷莲华李晓波 复旦大学上海医学院生理与病理生理学系上海 200032 摘要目的 本文研究 VEGF 在类胰蛋白酶引起皮肤微血管内皮细胞通透性增加中的作用 方法和结果成功分离培养人真皮微血管内皮细胞HDMEC纯化的类胰蛋白酶和人肥 大细胞系 HMC-1 脱颗粒产生的上清液均可浓度依赖性增加 HDMEC 通透性该效应可被特异性 的类胰蛋白酶抑制剂 APC366 所抑制而 VEGF 中和抗体可部分抑制该效应实时 RT-PCR 和 Western-blot 检测发现类胰蛋白酶可上调 VEGF 及其受体 flt-1 和 KDRmRNA 和蛋白水平 结论 类胰蛋白酶可以上调皮肤微血管内皮细胞 VEGF 及其受体表达VEGF 抑制剂一定程度 上可以缓解类胰蛋白酶引起的血管内皮细胞通透性增加类胰蛋白酶很可能通过调节 VEGF 及其受体的表达来引起皮下血管内皮通透性增加皮下水肿发生 关键词病理学类胰蛋白酶肥大细胞VEGF内皮细胞皮肤 中图分类号R3632 - 11 -