Study On The Vascular Endothelium Permeability Mediated By VEGF And Its Molecular Mechanism In Pathogenesis Of HFRS

Hantaanvirus have the potential to cause two different types of diseases inhuman: hemorrhagic fever with renal syndrome (HFRS) and hantaviruspulmonary syndrome (HPS). HFRS is manifested clinically by the triad of fever,hemorrhage, and renal failur. Enhanced vascular permeability and plasma leakageare the central event in the pathogenesis and hallmark of HFRS. The illness has acomplex course with five regonized phases: febrile, hypotensive/shock, oliguric,diuretic, and convalescent. The key of the discussion that follows is the severeform of HFRS that is usually associated with Hantaan virus. Increasedendothelial permeability is a common pathogenic feature for HFRS.As we know, VEGF(vascular endothelial growth factor) is reported50,000times more potent than histamine in inducing vascular permeability. Hantaanvirusprimarily infect endothelial cells and enhance the permeability of endothelialcells, and it maybe is associated with VEGF. However, the pathogenesis ofvascular leakage haven’t been clarificated yet. It is a systemic response,exceptefor VEGF, the role of additional immune cell, chemokine, or cytokine responsesin hantavirus-directed endothelial cell permeability cannot be excluded.Such asTNF-α、IFN-γ、ang-1and cell-cell junction proteins and so on.Therefor, some experiments to measure VEGF, sVEGFR and TNF-α, IFN-γand Ang-1(angiopoietin1) in different phases of the patients with hemorrhagicfever with renal syndrome and to detect the permeability induced by VEGF inHTNV-infected HUVECs and VEGFR2knockdown cells were performed. Theresults are as follows. The measurement of the plasma cytokines in patients with HFRSTo detect the plasma of patients of HFRS the sera were collected from68HFRS patients in different phases and20healthy controls. The cytokines levelsin the serum were determined by using sandwich enzyme-linked immunosorbentassay (ELISA) kits.The plasma VEGF levels in patients with HFRS were increased to337.6±238.0pg/mL in febrile phases, reached the highest level of556.0±185.2pg/mL during hypotensive/shock phase, sharply decreased to259.0±152.8pg/mLin oliguric phase, and then maintained at normal levels of223.1±110.6pg/mLand246.5±202.0pg/mL during both diuretic and convalescent phases, comparedwith212.4±116.8pg/ml in healthy controls. The patterns of VEGF changes duringdifferent phases were further confirmed in20individual patients with HFRS bydynamical analysis, showing a similar increased levels of561.1±194.3pg/mL inhypotensive/shock phase and similar various levels between212.5～397.5pg/mLat other phases.These results suggest that the increased VEGF may play an important role invascular permeability at hypotensive/shock phase of the patients with HFRS, andsVEGFR2, ANG-1, TNF-α and IFN-γ may play synergetic or antagonistic effectsfor VEGF-mediated micro-vascular permeability.The detection of the permeability induced by VEGF and HTNVinfection on HUVECs and VEGFR2knockdown cellsThe two kind of cells, HUVECs and VEGFR2knockdown cells, wereseeded respectively onto vitronectin-coated24-Transwell chanmbers at a celldensity of2×105cells and grown in EBM-2basal medium with10%of fetal calfserum. The cells were infected with pathogenic HTNV(strain76-118) at amultiplicity of infection(MOI)of0.5. After3days postinfection, cells werestarved overnight with EBM-2basal medium without FBS.To detected the permeability of the cells mentioned above theFITC-dextran was added to the upper chamber of monolayers in the presence orabsence of VEGF. The FITC-dextran leaked into the lower chamber was measured in a fluorescence using Tecan Genios Pro Microplate Reader (490-nmexcitation,530-nm emission), and fluorescence intensity measurements wereexpressed directly over the basal permeability of monolayers.Pathogenic hantaan viruses can not enhance endothelial cell permeabilityalone. But Hantaan virus enhence sensibility of HUVECs and VEGFR2knockdown HUVECs in response to VEGF. On the other hand, Hantaanvirus-directed endothelial cell permeability can be inhibited by Ang-1.The relationship between VEGF and cell-cell junction protein is accociatedwith permeability of endothelial cellsEndothelial paracellular permeability is regulated by the apicalintercellular junctional complex referred to as the apical junctional complex. Themajor constituents of this complex are tight junctions (TJs) and subjacentadherens junctions(AJs). Claudin-1is a crucial regulator of TJ permeability inendothelial cell while VE-cadherin is another one of AJ. We focus on selectedmethods used to investigate the influence of their interaction with VEGF, HV, andother cytokines.The permeability of vascular endothelial cells cultured in vitro wasdetermined by measuring transepithelial electrical resistance (TER) over the celllayer, using a Millicell-ERS-2volt-ohmmeter (Millipore, Billerica, MA). Valueswere obtained by subtraction of a background value and by multiplication by thearea of the filter. The TER of both infected and mock-infected cell layers wasmeasured daily to evaluate integrity of TJ and AJ.There was a sharply decrease of TER in the initial3hours, then itdecreased smoothly. It was demonstrated that cell-cell junction was destructedafter stimulated by VEGF, especially in the timepoint of3hours.Then wedetected mRNA levels and protein levels of claudin-1and VE-cadherin byrealtime-PCR and western blot. It was revealed that VEGF down-regulated theexpression of claudin-1and VE-cadherin both in mRNA levels and protein levels.To prove the results above we measure claudin-1and VE-cadherin again in the flow cytometry and indirect immunofluorescence and got the similar results.Conclusion: These results suggest that the increased VEGF athypotensive/shock phase of the patients with HFRS may play an important role invascular permeability. Meanwhile, HTNV infected endothelial cells enhanced thesensibility to VEGF and the effect is inhibited by Ang-1. VEGF downregulateexpression of claudin-1and VE-cadherin which enhance the permeability ofendothelial cells from paracellular pathway. The primary results mentioned abovedemonstrate that the VEGF induced hypermeability of vascular endothelial cellsboth in HFRS patients and also in endothelial cells cultured in vitro. We hope itwould be helpful to elucidate the pathogenesis of HFRS and new drugsdesignment.