The Role Of Adipocytes In Angiogenesis And Endothelial Dysfunction In Peritoneal Dialysis

Background Long-term peritoneal dialysis?PD?patients are at the high risk of cardiovascular death and withdrawing from PD due to peritoneal ultrafiltration failure,which are related to endothelial dysfunction.Vascular endothelial growth factor?VEGF?is an important cytokine which participates in endothelial dysfunction and angiogenesis.Furthermore,it shows an obvious upregulation of intercellular adhesion molecule-1?ICAM-1?and endothelin-1?ET-1?when endothelial dysfunction occurs.However,the exact mechanisms of angiogenesis and endothelial dysfunction in long-term PD patients still remained unknown.Studies have proved an increase of visceral adipose tissue in PD patients,which is the main source of adipocytokines.However,whether adipocytokines are associated with cardiovascular disease?CVD?or peritoneal ultrafiltration failure is unclear.Objective Explore the effect of adipocytes on angiogenesis and endothelial dysfunction in PD.Methods Part one,in vitro study.3T3-L1 preadipocytes were differentiated into adipocytes.Three groups of adipocytes were used here,1.Control group,cells cultured in regular medium;2.High glucose?HG?group,which used medium containing 139mmol/l glucose;3.High mannitol?HM?group,which share the same osmotic pressure with high glucose group.24h later,supernatant was collected and the concentration of VEGF was tested by enzyme linked immune sorbent assay?ELISA?.Mouse microvascular endothelial cells?ECs?were cultured and divided into 6 groups,1.Control group;2.High glucose?HG?group;3.High mannitol?HM?group;4.Con-adipocyte group:ECs cultured with the supernatant of adipocytes treated with regular medium;5.HG-adipocyte group:ECs cultured with the supernatant of adipocytes treated with high glucose;6.HM-adipocyte group:ECs cultured with the supernatant of adipocytes treated with high mannitol.12h later,assays for tube formation on Matrigel and migration through Transwell plate were tested in different EC groups.VEGF neutralizing antibody was added or not into the supernatant of adipocytes before the assays.24h later,ICAM-1 and ET-1 expression in ECs were tested by Western blot,and SB203580,a blocker of p38 mitogen activated protein kinase?MAPK?was used to explore the function of p38 MAPK in ICAM-1 and ET-1 expression in ECs.Part two,in vivo study.1×10¹² PFU/ml adeno associated virus?AAV?containing the gene encoding Preadipocyte factor-1?Pref-1?and control AAV were constructed.Male C57BL/6 mice?4 week?were divided into4 groups,1.Normal saline?NS?group:mice intraperitoneal injected with 100?l PBS at 2-week old and received intraperitoneal injected with 1.5ml NS per day since4-week old;2.Peritoneal dialysis group?PD?:mice intraperitoneal injected with100?l PBS at 2-week old and received intraperitoneal injected with 1.5ml peritoneal dialysis fluid?PDF?per day since 4-week old;3.PD+AAV-Control group:mice intraperitoneal injected with 100?l control AAV at 2-week old and received intraperitoneal injection of 1.5ml PDF per day since 4-week old;4.PD+AAV-Pref-1group:mice intraperitoneal injected with 100?l AAV containing Pref-1 at 2-week old and received intraperitoneal injection of 1.5ml PDF per day since 4-week old.30days later,visceral adipose tissues in mesenterium,omentum,posterior peritoneum and epididymis fat pad were collected and weighed.Peritoneal vessel density was detected by CD31 immunohistochemical staining.Evans blue was used to test vascular permeability of peritoneal membrane.ELISA was used to detect the expression of VEGF and ICAM-1 in mice serum.Results In vitro,more than 90%3T3-L1 preadipocytes were differentiated into adipocytes.Compared with Control group?1572.50±83.89pg/ml?and HM?1803.21±25.94pg/ml?group,VEGF produced by adipocytes in HG group was significantly increased?2088.36±107.18pg/ml??P<0.05?.High glucose inhibited tube formation of ECs?6.3±1.2/HP?compared with Control group?12.3±0.6/HP??P<0.01?.However,tube formation of ECs in HG-adipocyte group increased significantly?11.3±1.5/HP??P<0.01?,which was not obvious in HM-adipocyte group?8.7±0.6/HP??P<0.05?.The tube formation of ECs reduced after neutralized antibody of VEGF been added into HG-adipocyte supernatant?6.3±2.1/HP??P<0.05?.Migration of ECs increased in HG group?2.3±1.2/HP?compared with Control group?6.8±1.7/HP??P<0.05?.Furthermore,the migration of ECs in HG-adipocyte group was more obvious?36.8±2.1/HP??P<0.05?.The number of migrated cells reduced after neutralization of VEGF in HG-adipocyte supernatant?23.0±3.2/HP??P<0.05?.ICAM-1 of ECs cultured in HG-adipocytes group increased significantly?1.53±0.06?compared with HG group?1.30±0.10?and Con-adipocyte group?1.13±0.15??P<0.05?.Pretreatment of ECs with SB203580 resulted in a reduction of ICAM-1 in HG-adipocyte group?0.93±0.12 vs.1.19±0.03??P<0.05?.Similarly,ECs cultured in HG-adipocytes group produced more ET-1?2.24±0.27?than HG group?1.49±0.14?and Con-adipocyte group?1.45±0.25??P<0.05?,which had no relationship with p38MAPK?P>0.05?.In vivo,visceral adipose tissue in mice with PD increased?0.69±0.03g?compared with the mice injected with NS?0.23±0.08g??P<0.05?.Pref-1 significantly inhibited the accumulation of visceral adipose tissue in mice?0.13±0.03g??P<0.05?.Peritoneal angiogenesis was more obvious in PD mice than NS group?11.00±2.00/HP vs.3.67±1.15/HP??P<0.05?,which could be partially inhibited by AAV-Pref-1 injection?8.00±1.00/HP??P<0.05?.In the meanwhile,AAV-Pref-1 injection also reduced peritoneal permeability which was increased by PD treatment?0.12±0.06 vs.0.29±0.03??P<0.05?.Moreover,the expression of VEGF and ICAM-1 decreased in serum of PD mice with AAV-Pref-1 injection compared with others?VEGF,62.36±11.57 pg/ml;ICAM-1,38.84±8.06pg/ml??P<0.05?.Conclusion Adipocytes activated by high glucose in peritoneal dialysis fluid play an important role in endothelial dysfunction,potentiating peritoneal angiogenesis and CVD in PD patients,and inhibition of adipogenesis may be helpful in peritoneal protection and CVD prevention,maintaining the persistence and effectiveness of PD treatment.