Investigation Of Cyanate And Its Carbamylated Products On Endothelial Injury

Aims Part 1: To investigate cyanate and its carbamylated products' effect on endothelial angiogenesis function.Part 2: To investigate the carbamylation level of high-density lipoprotein(HDL)in end-stage renal disease patients(ESRD)and the effects of carbamylated-HDL(C-HDL)on endothelial repair properties.Methods Part 1:(1)The mice were divided into two groups: the control group(n = 10)and cyanate group(n = 10).After 16 weeks of administration,unilateral hindlimb ischemia was surgically performed.Hindlimb blood perfusion was measured with laser Doppler perfusion imaging(LDPI)at day 0,7,21 after surgery.Gastrocnemius tissue from the ischemic hindlimb was harvested 21 days after surgery and stained with monoclonal CD31 antibodies.(2)Human arterial endothelial cells(HAECs)were cultured and treated with cyanate or carbamylated medium to test the migration,tube formation and proliferation abilities.Western blot was used to test the potential signal transduction pathway.(3)117 patients undergoing coronary angiography at Shanghai Ruijin Hospital with stable angina and CTO of at least one major epicardial coronary artery were enrolled in this study.Patients were then classified into either the poor coronary collateralization group(Rentrop score 0-1)or good coronary collateralization group(Rentrop score 2-3). The concentration of homocitrulline(Hcit)-marker of cyanate exposure was detected in the serum of the patients from both groups.Logistic regression analysis was used to detect if increasing Hcit levels were an independent predictor of poor collateral growth after adjusting for potential confounders.Part 2:(1)HDL was isolated from healthy controls(n = 22)and patients with ESRD(n = 30),besides,the carbamylation level of HDL was detected.(2)HAECs were treated with C-HDL or native HDL to assess their migration,proliferation and angiogenesis properties.HDL-associated paraoxonase1(PON1)activity was also determined by spectrophotometry assay.Western blot was used to test the potential signal transduction pathway associated with the inhibitory effects of C-HDL in endothelial cells.Results Part 1:(1)A reduction in blood perfusion recovery at day 21 was observed in the ischemic tissue of cyanate-treated mice.Likewise,there were fewer capillaries in the ischemic hindlimb tissue of cyanate-exposed mice.(2)Our in vitro study showed that cyanate,together with its carbamylated products,inhibited the migration,proliferation,and tube formation abilities of endothelial cells.Further research revealed that cyanate regulated angiogenesis partly by interrupting the VEGFR2/PI3K/Akt pathway.(3)Hcit levels were increased in patients with poor coronary collateralization(n = 58)compared with those with high collateralization(n = 59).In addition,Logistic regression analysis showed that elevated Hcit concentration was a strong and independent predictor of poor coronary collateral growth in patients with stable agina.Part 2:(1)Compared with healthy controls,the carbamylation level of HDL in ESRD patients was increased.(2)In vitro,C-HDL significantly inhibited migration,angiogenesis and proliferation in endothelial cells.Mechanistic studies revealed that HDL-associated PON1 activity negatively correlated with carbamylation level of HDL in ESRD patients.In addition,C-HDL suppressed the activation of VEGFR2 and SR-BI signaling pathways in endothelial cells.Conclusions Part 1: Impaired angiogenesis induced by cyanate might contribute to poor coronary collateral growth in patients with stable angina and chonic total occulsion.Part 2: This study identified a significantly increased carbamylation level of HDL in ESRD.Furthermore,C-HDL inhibited endothelial cell repair function.