The Synergistic Protection Of Pericyte On Vascular Reactivity And Vascular Permeability After Sepsis And Its Mechanism

Sepsis is a clinically common critical disease with a high incidence and mortality,which is estimated to affect at least 3 million patients in our country every year,potentially leading to 1 million deaths.The vascular hyporeactivity and leakage are the important pathological and physiological processes during the occurrence and progression of sepsis,and they play important roles in occurrence of multiple organ dysfunction.About 30%-40%of ICU patient death is associated with the vascular dysfunction.At present,a series of treatment measures have been proposed based on the mechanisms of vascular hyporeactivity and vascular leakage after sepsis,but these measures are often contradictory in treating vascular hyporeactivity and vascular leakage,for example,when vasoconstrictors are used to increase vascular reactivity,they can result in the endothelial cytoskeleton contraction and aggravate vascular leakage.Nevertheless,there is no good treatment to solve this issue.Pericyte?PC?is a pluripotent cell,with contraction function,which can regulate the irrigation flow and permeability of microcirculation.PC is encapsulated within the basement membrane?BM?and ensheathe the capillary endothelium throughout the body.Recent studies indicated that PC participated in the pathogenesis of inflammation,immune dysfunction and abnormal angiogenesis.The dysfunction of PC is related to the pathological progress of several diseases,including Parkinson's,Alzheimer's diseases,diabetic retinopathy diseases,myocardial infarction and pulmonary fibrosis diseases.However,it is unknown whether PC could synergetically regulate vascular vasomotor and endothelial barrier function after sepsis.Therefore,using cecal ligation and puncture?CLP?and lipopolysaccharide?LPS,10mg/kg body weight?-induced sepsis models in rats and transgenic mice,and rat mesenteric vascular endothelial cells?VECs?and vascular smooth muscle cells?VSMCs?,we investigated the roles of PC in vascular reactivity and vascular permeability after sepsis and the underlying mechanisms in the present study.Main research contents:Part I:The relationship between the changes of PC amount and structure and vascular reactivity and permeability after sepsisTo determine the roles of PC amount in regulation of vascular reactivity and permeability after sepsis,CLP and intravenously injection of LPS were used to establish the septic rats and GFP-PDGFR-?transgenic mice.The changes of PC amount and structure in mesenteric microvessels were observed at 6,12 and 24h after sepsis,and the changes of the vascular reactivity of mesenteric arterioles to NE and Ach and the permeability of the mesenteric venules were monitored at 6,12 and 24h after sepsis.Part II:The synergistic effects of exogenous PCs infusion on improving the vascular hyporeactivity and leakage after septic ratsTo determine the synergistically protective effects of pericytes on the vascular vasomotor and endothelial barrier functions in septic rats,the location and amount of PC in mesenteric microvessels,the vascular reactivity of mesenteric arterioles and the permeability of the mesenteric venules were measured after PCs infusion in septic rats.Part III:The rescue effect of PCs infusion on vascular vasomotor and endothelial barrier function in PC-deficient ratsTo further determine the synergistically protective effects of pericytes on the vascular vasomotor and endothelial barrier function in septic rats,we established the PC-deficient rat model by CP-673451?a PDGFR inhibitor?.The relationship between the decrease of PCs in rat mesentery and the vascular reactivity and vascular permeability,and the effects of PCs infusion on the vascular reactivity and vascular permeability were observed.Part?:The mechanisms of synergistic effects of pericytes on vascular reactivity and permeability1.The direct effect of PCs on the contractile function of VSMCs and the barrier function of VECsThe direct contact between PCs and VSMCs and VECs,and its effect on the contractile response of VSMCs and barrier function of VECs were observed with LPS-stimulated VSMCs and VECs.2.Effects of PC-derived microvesicles?PCMV?on vascular reactivity and permeabilityThe number of MVs secreted by PCs and the absorption of PCMV in VSMCs and VECs were observed.Effects of PCMV on vascular reactivity and permeability in vivo and in vitro were observed.The types of growth factors and microRNAs in PCMV,and its effect on the contractile response of VSMCs and barrier function of VECs were observed.Results:Part I:The relationship between the changes of PC amount and structure and vascular reactivity and permeability after sepsis1.The amount of PC?PDGFR-?and NG-2?markers in the mesenteric microvessels was significantly decreased in CLP and LPS-induced septic rats,and the desquamation of PC in mesenteric microvessels after sepsis was significantly detected.The constriction and relaxing response of mesenteric arterioles,the blood flow velocity,and the expression of p-MLC₂₀0 in SMA were significantly decreased after sepsis.The permeability of the mesenteric venules was increased,the expression of ZO-1 and VE-cadherin in SMV was decreased,the integrity of tight junctions disrupted.It was suggested that the desquamation of PC was closely related to the changes of vascular reactivity and permeability in septic rats.2.As the same as septic rats,PC significantly fell off in the fluorescent labeled-pericyte mice with CLP and LPS-induced sepsis.The constriction and relaxing response of mesenteric arterioles was significantly decreased,and the vascular permeability was significantly increased.The results further demonstrated that the desquamation of PC was closely related to the changes of vascular reactivity and permeability in septic mice.Part II:The synergistic effects of exogenous PCs infusion on improving the vascular hyporeactivity and leakage after septic ratsExogenous PCs could colonize on the mesenteric microvessels,and most of them were colonized at 24h.PCs infusion could improve the 72-hour survival rate of septic rats,improve the vascular reactivity and the blood flow velocity and increase the expression of p-MLC₂₀0 in SMA.Also,PCs infusion could decrease the vascular leakage,improve the integrity of tight junctions and increase the expression of ZO-1 and VE-cadherin in SMV in septic rats,poly?i:C?pre-stimulated PC had a better effect.The results suggested that PCs had the synergistically protective effects on the vascular reactivity and permeability after sepsis.Part III:The rescue effect of PCs infusion on vascular vasomotor and endothelial barrier function in PC-deficient ratsCP-673451 may knock down the PC in the mesenteric microvessels and established the PC-deficient rats.PCs infusion could rescue the PC loss-induced damage of vascular vasomotor and endothelial barrier function.Sepsis could aggravate the vascular hyporeactivity and leakage in PC-deficient rats,and PCs infusion could synergistically improve them,poly?i:C?pre-stimulated PC had a better effect.The results suggested that PCs infusion could restore the vascular reactivity and permeability in PC-deficient rats with or without sepsis.Part?:The mechanisms of synergistic effects of pericytes on vascular reactivity and permeability1.The direct effect of PCs on the contractile function of VSMCs and the barrier function of VECsPCs could directly contact with VSMCs and VECs to form a connection,which could directly regulate the contractile response of VSMCs and barrier function of VECs.2.Effects of PC-derived microvesicles?PCMV?on vascular reactivity and permeabilityPC could produce microvesicles to synergetically regulate and protect the vascular vasomotor and endothelial barrier function.Mechanisms study indicated that PCMV could carry Ang-1,miR-145 and miR-132 to play effects.These results suggested that PCMV could play the synergistically protective role in the vascular reactivity and permeability via carring Ang-1,miR-145 and miR-132 after sepsis.Conclusion1.PC loss and structure destruction played an important role in the vascular hyporeactivity and leakage after sepsis.2.Exogenous PCs infusion could synergistically improve the vascular hyporeactivity and leakage after sepsis.3.PCs infusion could rescue the vascular vasomotor and endothelial barrier function in PC-deficient rats with or without sepsis.4.The synergistic effects of pericytes on vascular reactivity and permeability include the direct and paracrine mechanism.The direct mechanism might be that PCs directly contacts with VSMCs and VECs to form tight and gap junctions,and forms the physical coverage.The paracrine mechanism is that PCs secrete MV containing many active substances,such as Ang-1,miR-145 and miR-132,to play effects.Ang-1 could directly act on VSMCs and VECs to play effects.miR-145 could mainly act on VSMCs to improve the contractile response of VSMCs via inhibiting the expression of Sphk2 and S1PR1 and increasing the expression of S1PR2 and p-MLC₂₀;miR-132 could mainly act on VECs to improve the barrier function of VECs via inhibiting the expression of Sphk2 and S1PR2 and increasing the expression of S1PR1,ZO-1 and VE-cadherin.