| Objective:At present it is still unclear about the etiology of primary hypertension.The pathogenesis is still in exploring.Chronic inflammatory state in primary hypertension is a relatively new research propositions.Psgl-1 was discovered as P selectin ligand adhesion molecules.When the inflammatory response happened,Psgl-1 led to leukocytes rolling,then leukocytes captured by endothelial cells,induced adhesion and adhesion effect became strengthen,eventually mediated inflammatory response.Psgl-1 played an important role in the inflammatory response.The attention of targets for Psgl-1 in preventing cardiovascular disease increasing domestic and overseas.In our research,to determine the effect of psgl-1 on blood pressure regulation,we observed and compared blood pressure in male C57BL6/J and Psgl-1-/-mice after acute and chronic Angiotensin?treatment.Circulating levels of inflammatory regulation related factors were tested in Psgl-1 deficiency to find the underlying mechanism.To provide the basis for the potential therapeutic strategy of Psgl-1 in hypertension.Methods:8-10-week-old Male C57BL6/J and Psgl-1-/-mice were used in our research.1.The acute pressor response to Ang ? was measured in anesthetic mice by in vivo blood pressure recording via carotid artery catheterization.Cumulative Ang ?(0.2,2,20,200?g/kg)was administrated via jugular vein with 5 minutes interval.2.To determine whether the hematopoietic pool of Psgl-1 was responsible for the pressure regulation,C57BL6/J mice were irradiated and BMT was performed.Each recipient mouse was irradiated and injected with bone marrow cells from WT or Psgl-1-/-mice via the tail vein.6 weeks after BMT,the mice were used for blood pressure monitoring via carotid artery catherization.3.To test the potential therapeutic strategy of Psgl-1 blockade,the blocking antibody of Psgl-1 was injected to WT mice.For antibody injection experiments,a rat anti-mouse Psgl-1 antibody 4RA10 or isotype control rat IgG1 k(100?g in 200?l PBS)was injected into 8-week-old WT mice intraperitoneally overnight.The pressor responses to Ang ? were determined via blood pressure monitoring using catherization of carotid artery.4.After one week baseline BP recording,mice were anesthetized and subjected to subcutaneous implantation of osmotic minipump for testing the chronic pressor response to Ang ?.Control saline or Ang ? was continuously delivered at a concentration supporting and infusion rate of 500 ng/kg/min for two weeks.Blood pressure was measured in non-anesthetized mice by tail plethysmography using the BP-2000 Blood Pressure Analysis System every day for two weeks.Systemic infusion of Ang ?increased blood pressure measured by tail-cuff and carotid artery catheterization in both WT and Psgl-1-/-mice compared with saline-infused control mice.5.Plasma samples were collected via ventricular puncture at time of euthanasia.Plasma soluble P-selectin(sP-sel),soluble E-selectin(sE-sel),monocyte chemoattractant protein-1(MCP-1),chemokine(C-X-C motif)ligand 1(CXCL-1)and interleukin-17(IL-17)were measured with commercially available murine ELISA kits according to manufacturers' instructions.6.To study the effect of IL-17 on blood pressure,IL-17(1000ng in 200?l PBS)or PBS was injected to Psgl-1-/-mice.5 hours later,the pressor response to Ang ? was determined via blood monitoring using catherization of carotid artery.7.To examine the effect of angiotensin ? on IL-17 containing cells in WT and Psgl-1-/-mice,flow cytometry was performed.Cumulative dosages of Ang ? were intravenously administrated as in experiment of acute pressor response to Ang ?.60 minutes after last dose of Ang ?,the peripheral blood was collected for flow cytometry analysis.Cells were stained with corresponding fluorochrome-conjugated antibodies for leukocytes(Alexa Fluor 700 rat anti-mouse CD45),T cells(APC hamster anti-mouse CD3e),and IL-17 positive cells(PE rat anti-mouse IL-17A).8.We used HE staining for aorta and Sirius Red collagen staining for kidney.MCP-1,CXCL-1,ICAM-1,IL-6 and IL-17 in kidney,and Endothelin-1 and angiotensin ? receptor type lb in aorta were measured with real time PCR kit according to manufacturers' instructions.Results:1.The systolic and diastolic blood pressure elevations were recorded in response to different concentrations of Ang ?.In Psgl-1-/-mice,the concentration response of Ang? on systolic blood pressure was significantly reduced compared to WT mice.The response on diastolic blood pressure was also reduced in Psgl-1-/-mice.2.The pressor response to Ang ? was significantly reduced in WT mice receiving Psgl-1-/-bone marrow compared to WT mice from receiving WT bone marrow.3.The pressor responses to Ang? were significantly reduced in WT mice receiving Psgl-1 antibody compared to WT mice receiving control antibody.4.To determine effect of Psgl-1 deficiency on Ang?-mediated hypertension,the saline or Ang ? was chronically administrated via osmotic mini pumps into WT or Psgl-1-/-mice.Systolic blood pressure was similar between control WT and control Psgl-1-/-mice.However,Psgl-1 deficiency significantly reduced Ang ?-dependent increases in systolic blood pressure.5.Circulating levels of P-selectin,E-selectin,and interleukin-17 measured in WT and Psgl-1-/-mice after acute Ang ?administration is inhibited by Psgl-1 deficiency.6.Similarly,the levels of sP-sel,sE-sel,and IL-17 were significantly lower in WT mice receiving Psgl-1-/-BM or after Psgl-1 antibody treatment compared to corresponding control WT mice.7.The inhibition of Psgl-1 deficiency on Ang ?-induced pressure elevation was diminished in IL-17-treated Psgl-1-/-mice compared to PBS-treated Psgl-1-/-mice.8.There was no difference on number of leukocytes between WT and Psgl-1-/-mice.The number and percentage of neutrophils were significantly higher in Psgl-1-/-mice compared to WT mice.The percentage of lymphocytes was also higher in Psgl-1-/-mice compared to WT mice.No differences were detected between PBS-treated and Ang?-treated mice.9.Flow cytometry was performed after accumulative Ang? infusion,CD3+ T cells were significantly lower in Psgl-1-/-mice compared to WT mice either with or without Ang ?treatment.The Ang ? treatment had no effect on CD3+ cell distribution.The IL-17+ T cells were significantly lower in PBS-treated Psgl-1-/-mice compared to PBS-treated WT mice with or without Ang ? treatment.Following Ang ? infusion,IL-17+ T cells were significantly reduced compared to PBS-treated control mice.10.The plasma levels of P-selectin,E-selectin,CXCL-1 and MCP-1 were measured in WT and Psgl-1-/-mice after chronic Ang ? administration.The levels of P-selectin,E-selectin,CXCL-1 and MCP-1 were significantly increased in Ang ?-treated WT mice compared to control saline-treated WT mice.The Ang ?-induced P-selectin,E-selectin,CXCL-1 and MCP-1 elevations were inhibited in Psgl-1-/-mice after Ang ? treatment.11.The levels of collagen in kidney was significantly increased in Ang ?-treated WT mice compared to control saline-treated WT mice.The Ang ?-induced collagen in kidney elevation was inhibited in Psgl-1-/-mice after Ang ? treatment.12.The levels of CXCL-1,MCP-1,ICAM-1,IL-17 and IL-6 in kidney were also significantly increased in Ang ?-treated WT mice compared to control saline-treated WT mice.The Ang ?-induced CXCL-1,MCP-1,ICAM-1,IL-17 and IL-6 in kidney elevations were inhibited in Psgl-1-/-mice after Ang ? treatment.13.Also we found the levels of aorta media area and wall thickness were significantly increased in Ang ?-treated WT mice compared to control saline-treated WT mice.The Ang ?-induced aorta media area and wall thickness elevations were inhibited in Psgl-1-/-mice after Ang ? treatment.14.The levels of Endothelin-1 and angiotensin ? receptor type lb in aorta were significantly increased in Ang ?-treated WT mice compared to control saline-treated WT mice.But there were no differences between WT mice and Psgl-1-/-mice after chronic Ang ? treatment.Conclusion:In our study,we observed and compared blood pressure in male wild type and Psgl-1-/-mice after acute and chronic Angiotensin ? treatment.We found that Psgl-1 deficiency significantly reduced Ang ?-dependent increases in blood pressure.Blood-derived Psgl-1 plays more important role in protective effort Psgl-1 deficiency.Provided the evidence for the potential therapeutic strategy of Psgl-1 blockade.Circulating levels of P-selectin,E-selectin CXCL-1 and MCP-1 is inhibited by Psgl-1 deficiency.Ang ?-dependent pro-inflammatory is inhibited by Psgl-1 deficiency.IL-17 as a proinflammatory factor may participate in protective effort in blood pressure elevation after Ang ? treatment in Psgl-1 deficiency condition.Inflammatory infiltration and collagen deposition in kidney is inhibited by Psgl-1 deficiency after Ang ? treatment.The Ang ?-induced CXCL-1,MCP-1,ICAM-1,IL-17 and IL-6 in kidney elevations were inhibited in Psgl-1-/-mice after Ang ? treatment.The Ang ?-induced aorta media area and wall thickness elevations were inhibited in Psgl-1-/-mice after Ang ? treatment.Vascular remodeling is also inhibited by Psgl-1 deficiency after Ang ? treatment,but it has nothing with the levels of Endothelin-1 and angiotensin ? receptor type 1b in aorta.Our results provide the basis for the potential therapeutic strategy of Psgl-1 in hypertension. |
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