| BackgroundCoronary artery disease (CAD) seriously threatens human life and becomes one of the most important diseases that affect longevity and survival quality of aging. Hypercholesterolemia (HC) -induced endothelial dysfunction is the first step of atherogenesis. The mechanisms involving in the pathological changes of endothelial dysfunction include: changes in expression and bioactivity of nitric oxide synthase (NOS), changes in activation of NOS-related cofactors, increasing in nitric oxide (NO) production but decreasing in NO bioavailability, et al. Impairment of NO bioavailability and NO/cGMP/cGK signaling pathway is thought to be one of the most important mechanisms.NO is synthesized from L-arginine by NOS, including eNOS, nNOS and iNOS. The concentration of endothelial-derived NO is very low under physiological condition, about 0.1100nM. It induces vasorelaxation of vascular smooth muscle through activating cGMP/cGK pathway. Hypercholesterolemia-induced inflammation activates iNOS and increases NO production. NO reacts with superoxide amion (˙O2-) to form ONOOˉwhich possess high cytotoxicity. Inhibition of the key point of hypercholesterolemia is thought to preserve NO/cGMP/cGK signaling pathway and reverse endothelial dysfunction.Myeloperoxidase(MPO) is an oxidase located in azurophil granule of monocytes and neutrophilic granulocyte. When inflammation occurs, it was released out of cell where it caused tissue injury nearby through reacting with H2O2 and generating hypochloric acid. Finally, it plays a key role in artherosclerosis. It has been shown that MPO accumulated in basal lamina under the vascular endothelium during hypercholesterolemia. So it could be supposed that MPO leads to endothelial dysfunction through sinking NO. In addition, MPO can regulate˙O2ˉgeneration and consumption. So it is proposed that MPO maybe cause endothelial dysfunction through reducing the NO bioavailability which caused by the increasing ONOOˉproduction. Therefore, it is possible that MPO is one of the key factors involved in vascular endothelial dysfunction. However, more evidences are also needed to be further provided about it.Increasing studies have reported that Peroxisome proliferator-activated receptorγ(PPARγ) plays an important role in anti-inflammation and improves the vascular endothelial function. But the exact mechanisms are still unclear. MPO is one of the major inflammatory factors. It has been reported that PPARγagonists regulated the expression of MPO gene in different manners according to different cell types. Therefore, we proposed whether the PPARγagonists could ameliorate the vascular endothelial function and attenuate the artherosclerosis.Objective1. To testify the previous results that vascular NO/cGMP/cGK pathway impairment during hypercholesterolemia could lead to endothelial dysfunction;2. To determine whether MPO mediates hypercholesterolemia-induced endothelial dysfunction through impairing vascular NO/cGMP/cGK pathway;3. To observe whether PPARγagonists attenuate hypercholesterolemia-induced endothelial dysfunction, and whether it is related to MPO regulation.PART ONE MPO mediates endothelial dysfunction in rats with hypercholesterolemia1 Methods1.1 Animals and GroupsMale healthy Wistar rats (n=32) were randomly divided into four groups:①Control group (n=8): Fed with normal diet for 8 weeks;②Hypercholesterolemia group(n=8): Fed with high-cholesterol diet for 8 weeks;③Hypercholesterolemia + Dapsone (DDS) group (Inhibitor of MPO) (n=8): Fed with high-cholesterol diet for 8 weeks, combined with DDS via intraperitoneal injection in the last 6 days;④Hypercholesterolemia + DMSO group (n=8):Fed with high-cholesterol diet for 8 weeks, combined with DMSO via intraperitoneal injection in the last 6 days. 1.2 Index DetectionCHO, TG and LDL-CHO were quantitatively analyzed by relative reagent kits. Vascular endothelial function was determined by comparing vasorelaxation to ACh (acetylcholine), an endothelium-dependent vasodilator, and SNP, an endothelium-independent vasodilator in vascular rings in vitro. The vascular MPO activity was detected by MPO activity assay Kit. The total content of NOx was detected according to the nitrate/nitrite colorimetric assay. The level of cGMP was measured by RIA Kit.2 Results2.1 Establishment of diet-induced hypercholesterolemic modelAfter 8 weeks, the content of serum lipid which is profiled such as serum Total cholesterol (CHO), Triglyeride (TG),Low density lipoprotein cholesterol (LDL-CHO) in the HC group increased significantly compared with that of before administration of high-cholesterol diet and the ordinary diet (Table1, Table2).2.2 Hypercholesterolemia induce endothelial dysfunctionACh-induced vasorelaxation in the HC group is more potent (log EC50 [mol/L]: -6.61±0.11, 50.51%±1.00%) than that in the ordinary diet control group (log EC50 [mol/L]: -7.29±0.06, maximal response: 97.88%±3.89%) (P<0.01, Figure1a, 1b, 2a, 2b). SNP-induced vasorelaxation were unchanged between the two groups above (Figure1a, 1b, 3).2.3 Hypercholesterolemia impaired vascular NO/cGMP/cGK signaling pathway2.3.1 NO decreased in the vessel of Hypercholesterolemic ratsTo testify the signaling pathway involved in hypercholesterolemia-induced endothelial dysfunction, we detected the NOx content in vessel. Compared with the normal control group, NOx content decreased significantly in the HC group (P<0.01, Figure4).2.3.2 cGMP decreased in vessel of Hypercholesterolemic ratsVascular NO bioactivity was determined by cGMP content. Compared with the normal control group, vascular cGMP content decreased significantly in the HC group (P<0.01, Figure5). These results above indicate that hypercholesterolemia reduces NO bioactivity and impairs the vascular NO/cGMP/cGK signaling pathway which results in endothelial dysfunction.2.4 Vascular MPO activity increased in Hypercholesterolemic ratsWe detected the changes of MPO activity. Compared with the normal control group, MPO activity increased significantly in HC group (P<0.01, Figure6).2.5 Negative correlation between vascular endothelia function and MPO activityWe analyzed the correlation between vascular endothelial function and MPO activity. It shows that the logEC50 of ACh-induced vasorelaxation was positively correlated to the MPO activity (r=0.624, P<0.01, Figure7). There was also a negative correlation between the maximum relaxation response to ACh and vascular MPO activity (r=-0.739, P<0.01, Figure 8).2.6 Negative correlation between vascular NOx/cGMP and MPO activityTo determine the direct relation between MPO and hypercholesterolemia-induced NO/cGMP/cGK signaling pathway impairment, correlation between vascular NOx/cGMP content and MPO activity were analysised. We found both vascular NOx and cGMP negatively correlated to MPO activity respectively (r=-0.768, -0.955 P<0.01, Figure9, 10). These results suggest that hypercholesterolemia-induced NO/cGMP/cGK signaling pathway impairment is associated with vascular endothelia dysfunction.2.7 DDS attenuated vascular MPO activity in hypercholesterolemiaTo estimate the role of MPO in hypercholesterolemia-induced vascular endothelial dysfunction, we use the MPO inhibitor DDS. Compared with the ordinary control group, the MPO activity in HC group significantly increased (P<0.01). DDS obviously reduced the MPO activity in hypercholesterolemic model (P<0.01), while DMSO had no significant effects on it (Figure 11).2.8 DDS preserved hypercholesterolemia-induced endothelial dysfunctionCompared with HC group, the logEC50 decreased significantly in DDS group. Meanwhile, ACh induced maximum relaxation increased obviously (P<0.01, Figure12b, 14a). Interestingly, the maximum relaxation response to ACh in DMSO group is much higher than that in HC group (P<0.01, Figure12b). However, logEC50 between these two groups were comparable (Figure 12a). There were no differences in 10-10mol/L-10-6 mol/L SNP-induced vasorelaxation among all these groups (Figure 13, 14b).2.9 DDS ameliorated hypercholesterolemia-induced NO/cGMP/cGK signaling pathway impairment2.9.1 DDS increased vascular NOx content in hypercholesterolemic ratsVascular NOx content was detected to determine the signaling pathway involving in MPO mediated hypercholesterolemia-induced endothelial dysfunction. DDS enhanced the production of NO in hypercholesterolemic rats (P<0.01), while DMSO treatment had no significant effects (Figure 15).2.9.2 DDS increased vascular cGMP content in hypercholesterolemic ratsDDS increased the vascular cGMP content in hypercholesterolemic rats (P<0.01), while DMSO has no obvious effects (Figure16).These data demonstrated that MPO participated in hypercholesterolemia-induced impairment of vascular NO bioactivity. The results above indicate that the vascular MPO activity increased in the hypercholesterolemic rats, and then impaired NO/cGMP/cGK signaling pathway, which finally resulted in endothelial dysfunction.PART TWO Pioglitazone protects endothelial function in hypercholesterolemic rats by depressing vascular MPO activity1 Methods1.1 Animals and GroupsMale healthy Wistar rats (n=40) were randomly divided into five groups:①Control group (n=8): Fed with normal diet for eight weeks;②Hypercholesterolemic group(n=8): Fed with high-cholesterol diet for 8 weeks and gave oil as solvent by intragastric administration;③Hypercholesterolemia + PIO group (n=8):Fed with high-cholesterol diet for 8 weeks, combined with PIO via intragastric administration in the last 4 weeks;④Hypercholesterolemia + DDS (the inhibitor of MPO) group (n=8) : Fed with high-cholesterol diet for 8 weeks, combined with DDS via intraperitoneal injection in the last 6 days;⑤Hypercholesterolemia + PIO + DDS group (n=8):Fed with high-cholesterol diet for 8 weeks, combined with PIO via intragastric administration in the last 4 weeks and DDS via intraperitoneal injection in the last 6 days.1.2 Index DetectionSee part1.2 Results 2.1 Establishment of diet-induced hypercholesterolemic modelThere were no significant differences in serum lipid in all groups before the experiment. After 4 weeks, the content of serum lipid profiles such as serum total cholesterol (CHO), Triglyeride (TG),Low density lipoprotein cholesterol (LDL-CHO) is much higher in the HC group than that of before high-cholesterol feeding and than that of the control group (Table3). PIO significantly reduced the high level of serum lipid in hypercholesterolemia rats (Table4, Figure17, 18).2.2 PIO preserved hypercholesterolemia-induced endothelial dysfunctionCompared with the HC group, PIO markedly reduced the LogEC50 (P<0.01, Figuer19a) and increased the maximum relaxation (P<0.01, Figuer19b, 21a). There were no significant differences in ACh-induced vasorelaxation between PIO and DDC groups. The ACh-induced vasorelaxation in PIO combined with DDS group also has no significant difference from PIO group (Figure19a, 19b, 21a). Addition of SNP resulted in comparable relaxations in rings from all groups (Figure20, 21b).2.3 PIO reduced MPO activity of hypercholesterolemic vesselPIO obviously reduced the MPO activity in hypercholesterolemic model (P<0.01), comparable to that of DDS group (P<0.01, Figure22).2.4 Negative correlation between vascular endothelial function and MPO activity after PIO treatmentThe LogEC50 of ACh-induced vasorelaxation were positively correlated to vascular MPO activity (P<0.01, Figure23). There was a negative correlation between the maximum relaxation response to ACh and vascular MPO activity (P<0.01, Figure 24). The reducing of MPO activity and attenuating of endothelial function indicate that PIO protects hypercholesterolemia-induced endothelial dysfunction through inhibiting vascular MPO activity.2.5 PIO ameliorated hypercholesterolemia-induced impairment of NO/cGMP/cGK signaling pathway2.5.1 PIO increased vascular NO production in hypercholesterolemic rats Vascular NOx content was detected to determine the role of MPO in the protection of PIO on hypercholesterolemia-induced endothelial dysfunction. Treatment with PIO enhanced the production of NO in hypercholesterolemic rats (P<0.05). There were no marked differences between PIO and DDC groups(Figure 25).2.5.2 PIO increased vascular cGMP content in hypercholesterolemic rats PIO increased the vascular cGMP content in hypercholesterolemic rats (P<0.01). There were no significant differences between PIO and DDC groups (Figure 26).These results indicate that PIO may preserve hypercholesterolemia-induced the impairment of NO/cGMP/cGK signaling pathway through inhibiting vascular MPO activity.2.6 Negative correlation between vascular NOx/cGMP content and MPO activity after PIO treatmentTo determine the direct relation between MPO and the protective effect of PIO on hypercholesterolemia-induced impairment of NO/cGMP/cGK signaling pathway, correlation between vascular NOx/cGMP content and MPO activity were analysised after PIO treament. Vascular NOx and cGMP content negatively correlated to MPO activity respectively (P<0.01, Figure27, 28).These results demonstrate that PIO protects hypercholesterolemia-induced endothelial dysfunction by inhibiting vascular MPO activity and preserving the integrity of NO/cGMP/cGK signaling pathway.Conclusion(1) MPO participates in hypercholesterolemia-induced endothelial dysfunction by decreasing the production and/or bioactivity of vascular NO and impairing NO/cGMP/cGK signaling pathway;(2) PPARγagonists PIO reverse hypercholesterolemia-induced endothelial dysfunction by inhibiting vascular MPO activity. |
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