The Mechanism Of Cathepsin L's Role On Coronary Collaterals Circulation

PartⅠThe relationship of Cathepsin L and the coronary collateral formation in patients with coronary artery diseaseOBJECTIVE:To determine whether plasma Cat L can be used as a biomarker to predict coronary collateral formation in patients with CHD.METHODS:218 participants with CHD (age 67±11 years old) were included in this study. Patients underwent coronary angiography and categorized as either Poor or Rich collaterals based on the Rentrop scores. Plasma Cat L, pro-angiogenic factor placenta growth factor (PLGF), anti-angiogenic factors cystatin C and endostatin were measured. All statistic analyses were performed using SPSS statistical software v10.0.RESULTS:1.Our data demonstrate for the first time that elevated plasma Cat L, along with its pro-angiogenic factor PLGF, are independently associated with enhanced coronary artery collateral formation in CHD patients (P=0.034 and P=0.003 respectively).2.Among diabetes subgroup, patients with rich collateral have higher Cathepsin L level than those with poor collaterals. Among non-diabetes subgroup, patients with rich collaterals have higher PLGF level. Those may be due to the increasing inflammatory reactions in diabetes patients.3. Subgroup analysis shows that the correlation between Cathepsin L and coronary collaterals exists in patients with diabetes and acute coronary syndrome as well (p<0.05).4.According to the ROC analysis, the cutoff value of plasma Cathepsin L level is higher than 22.43 nM,which has the ability to find more patients with rich collaterals(sensibility is 42.6%, specificity is 79.3%).CONCLUSIONS:Plasma Cat L level maybe act as a useful biomarker to predict coronary collateral formation in CHD patients. Part II The impact of Cathepsin L on biological characteristics of HUVECOBJECTIVE:To investigate the influence of Cathepsin L on related HUVEC characteristics such as proliferation, apoptosis and invasion.METHODS:HUVECs were treated with different concentration of ectogenic nature Cathepsin L protein (10 nM and 30 nM),as well as with special selective cathepsin L inhibitor (Z-FF-FMK,10 uM) for 24 hours culture.The cell proliferation ability of HUVECs which were treated by above conditions were further confirmed by CCK8. The cell apoptosis was detected by AnnexinV/PI.The invasion ability were measured by Transwell plates precoated with typeⅠcollagen.RESULTS:1.It appeared that small dose of Cathepsin L protein perhaps inhibit the cell proliferation while large dose of Cathepsin L enhance the cell proliferation. After been inhibited by special selective inhibitor Z-FF-FMK,the cell proliferation increased. However,the proliferation rate didn't reach the statistical significance.2. The cell apoptosis was obviously increased by ectogenic Cathepsin L protein in a dose-dependent way while z-FF-FMK has the ability to protect the cell from early apoptosis,3.HUVEC migrated or infiltrated across the Transwell membrane more than the control group after stimulated by Cathepsin L protein while fewer than the control cells after stimulated by z-FF-FMK.CONCLUSIONS:The Cathepsin L protein enhanced the invasion ability of endothelial cell during angiogenesis. and stimulated cell apoptosis obviously which suggested other property apart from pro-angiogenesis. Part III The regulation of Cathepsin L on HUVEC angiogenesis functions under high glucose/palmitate burdenOBJECTIVE:To investigate the involvement of Cathepsin L in cell proliferation, cell apoptosis and invasion stimulated by high glucose/palmitate and to investigate the influence of high glucose/palmitate on Cathepsin L of HUVEC.METHODS:Cells were incubated in M199 supplemented with different concentration of glucose/palmitate or ordinary media as well as Cathepsin L protein, special selective Cathepsin L inhibitor (Z-FF-FMK) for 24h. The proliferation ability of HUVECs was further confirmed by CCK8, the cell apoptosis was detected by AnnexinV/PI, the invasion ability by transwell plates. Then after incubation of HUVEC with different concentration of glucose/palmitate, we analyzed the mRNA levels by realtime-PCR and protein expression by western blot. We also determined the enzyme activity by specific selective fluorogenic substrate.RESULTS:1. Not only the cell proliferation but also the cell migration/invasion ability were reduced after exposure to high glucose/palmitate stimulation. At the same time, cell apoptosis were increased by high glucose/palmitate burden.2. Large dose Cathepsin L protein still could promote cell migration/invasion even under the high glucose/palmitate burden. If inhibited by z-FF-FMK,cell migration/invasion decreased strikingly.3. The cell apoptosis was obviously increased by large dose Cathepsin L protein and decreased by z-FF-FMK even after being treated by high glucose/palmitate stimulation.4. High glucose/palmitate did not change the expression of gene and protein of Cathepsin L obviously but reduced enzyme acticity strikingly, That suggests a potential post-translational modification mechanism to regulate Cathepsin L.CONCLUSIONS:Cathepsin L protein enhanced endothelial cell invasion even under conditions of high glucose/palmitate level, which suggested the pro-angiogenesis effects of Cathepsin L.The down-regulation on Cathepsin L enzyme activity by high glucose/palmitate may suggest that the poor coronary collaterals formation in patients with diabetes is maybe related to impaired Cathepsin L function.