| Background: Aoritc dissection(AD) is the most devastating complication of thoracic aortic disease. The estimated annual incidence of AD is approximately 2-5 per 100 000 individuals, and the prevalence appears to be increasing dependent of the aging population. The most feared clinical consequence of AD progression is acute aortic rupture leading to death, which occurs in about 1-2% per hour within the initial 24 hours and almost half by 1 week after clinical symptoms onset for acute type A dissection patients who do not receive treatment. Although the advent of thoracic endovascular aortic repair has altered the management approach for aortic pathologies with lower risk of mortality and morbidity, especially for elder patients with greater comorbidities, many patients died before hospital admission or undergoing the endovascular therapy. Therefore, the mechanisms of AD need to be elucidated to further improve the prognosis. Increasing pathologic researches have shown that inflammatory mechanisms are involved in the process of vascular remodeling, which plays an important role in the development and progress of AD. Glucocorticoids have been widely used in clinical practice by reason of the powerful and effective anti-inflammatory properties. The randomized, double-blinded, placebo-controlled clinical trial concluded that preoperative methylprednisolone attenuated the inflammatory response with a faster recovery after endovascular repair for abdominal aortic aneurysms. To date, however, the relationship between glucocorticoid and AD was still unclear.Objective The objective of the study was to explore the potential effects of glucocorticoid on the development, progress and prognosis of AD, and to further elucidate the cellular and molecular mechanisms that involved in.Methods: In clinical study, the study protocol complied with the declaration of Helsinki, and was approved by the Ethical Committee of our hospital. The human samples were collected in our hospital, and signed informed consent forms for using the samples in the experiments were obtained from all subjects. The blood samples of AD patients, non-ruptured aortic aneurysm(n AA) patients and healthy volunteers were collected and detected for the serum cortisol and plasma adrenocorticotropic hormone(ACTH) by radioimmunoassay. At the same period of time, aortic specimens were obtained from AD and n AA patients, who underwent open surgery for aortic graft. Additionally, healthy aortic specimens were obtained from eight body donation volunteers. Immunohistochemistry was used to detect the level of glucocorticoid receptor(GCR). To evaluate the potential influence factors of serum cortisol, the clinical characteristics of gender, age, height, weight, systolic blood pressure, diastolic blood pressure, blood glucose, triglyceride, and tear number were collected to perform the multivariable linear regression in AD group. In animal study, the Institutional Animal Care and Use Committee of our university approved all the experiments. Six-month old male wide-type C57BL/6 mice were purchased from the animal centre of our university and randomly allocated to one of the three treatment groups according to the computer-generated randomization sequence stratified by weight: the first group received Angiotensin II infusion followed by intraperitoneal administration of corticosterone and served as the intervention group, the second group received Angiotension II infusion followed by intraperitoneal administration of phosphate buffer solution(PBS) and served as the model group, and the third group received saline infusion followed by intraperitoneal administration of PBS and served as the control group. To eliminate the interference of endogenous glucocorticoids, bilateral adrenalectomy was performed in all mice. The Kaplan-Meier curve was used to calculate the cumulative proportions of freedom from death. Blood pressure was measured using the tail-cuff method before and after micro-pump implantation every week. Ketamine-anesthetized mice were perfused with PBS via the left ventricle to remove blood from tissue; then the entire aorta from ascending aorta to iliac artery was excised in one piece and placed in sterile PBS. After the periadventitial fat was removed, the aortic specimen was photographed for measuring the external diameter with the help of image-pro plus software. After hematoxylin eosin staining of mice aorta, the media thickness of aorta was measured using image-pro plus software. The collagen volume fraction was assessed after Masson’s trichrome staining. The levels of GCR and macrophage were detected by immunohistochemistry. In cellular and molecular study, human aortic Smooth muscle cell(HA-SMC) and macrophage were cultured in dedicated conditional medium and intervened by GC. Supernatant cytokines of matrix metalloprotein-2(MMP-2), tissue inhibitor of metalloproteinase-2(TIMP-2) and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immuno sorbent assay. The migration and phenotype switch of HA-SMC were detected by scratch wound assay and cellular immunofluorescence, respectively. Co-culture experiments were performed in 24-well transwell membrane. The Image Streamχ imaging flow cytometry and cellular immunofluorescence detected the apoptosis and migration of HA-SMCs in coculture system, respectively. The multiplexed protein microarray and neutralizing antibody were used to screen the potential vital cytokines in the interaction between HA-SMC and macrophage. The Path Scan stress and apoptosis signaling antibody array kit based upon the sandwich immunoassay principle was used to determine the signaling molecules in coculture system, which was verified by western blot analysis.Results: Between October 2012 and December 2013, 82 patients diagnosed with AD, 68 patients with n AA and 76 healthy volunteers were prospectively registered in the study. Diagnosis of AD and n AA were confirmed by computed tomography angiography. The serum cortisol concentrations in AD patients, n AA patients, and healthy volunteers were 173.98±23.44 ng/m L, 136.60±21.83 ng/m L, and 129.00±27.56 ng/m L, respectively. The differences between AD and n AA patients(P<0.001), or healthy volunteers(P<0.001) were significant. Similarly, the concentrations of plasma ACTH were 26.76±7.41 pg/m L, 24.75±8.21 pg/m L, and 25.64±7.38 pg/m L for AD patients, n AA patients, and healthy volunteers, respectively. No significant differences were observed among them. The aortic specimens were obtained from eight AD, eight n AA patients and eight body donation volunteers. The percentages of GCR-positive staining were 14.25±1.31% in AD group, 13.39±1.42% in n AA group, and 12.55±2.12% in healthy group. No statistical difference was observed among them. We found the tear number was a potential influence factor for serum cortisol(b=0.920, P=0.029). In animal study, a total of 80 mice were randomly allocated into intervention group(n=32), model group(n=32), and control group(n=16). Nine mice(one in intervention group, five in model group, and three in control group) died before micro-osmotic pumps implantation, and 71 mice were included and analyzed in the experiment. Although no differences of cumulative proportions of freedom from death were found between model and intervention(P=0.139, Log-rank test), or control groups(P=0.107), the exogenous corticosterone intervention prolonged the survival time of mice. The blood pressure level in model group was significantly higher than that in control group at 7-, 14-, 21-, 28-day, and lower than that in intervention group at 7-, 14-, 21-day time point. The external diameter of ascending aorta in model group(1458.49±257.96 μm) was significantly higher than that in control(1094.61±154.57 μm, P<0.001) or intervention groups(1279.17±204.24 μm, P=0.005). The differences of external diameter of aortic arch and descending aorta between model and control groups were pronounced(aortic arch: 1449.48±290.34 μm vs. 986.38±151.16 μm, P<0.001; descending aorta: 1343.47±238.48 μm vs. 980.36±98.48 μm, P<0.001). The incidence of AD in model group was significantly higher than that in intervention group(29.6% vs. 6.5%, P=0.034), or control group(29.6% vs. 0%, P=0.037), which was confirmed by hematoxylin eosin staining. We found the media thickness in model group was significantly higher than that in control group(89.6±14.7 μm vs. 68.4±11.6 μm, P<0.001). No difference was observed between intervention and model groups(89.8±13.5 μm vs. 89.6±14.7 μm, P=0.937). The difference of collagen volume fraction was significant between model and control(24.2±6.0% vs. 31.7±6.3%, P=0.001) or intervention groups(24.2±6.0% vs. 30.1±8.2%, P=0.003). No significant difference of positive-GCR staining was observed between model group and intervention(9.3±3.0% vs. 8.5±2.1%, P=0.235) or control group(9.3±3.0% vs. 7.6±1.1%, P=0.115). But the positive-macrophage staining in model group was higher than that in control group or intervention group(9.3±3.5% vs. 2.2±1.0% or 7.4±3.2%, P<0.001 or 0.041). In celluar and molecular study, we found that glucocorticoids inhibited the MMP-2 secretion. However, no regulation role that glucocorticoids played on TIMP-2 secretion was observed in our experiment. In addition, high concentration of glucocorticoids inhibited the TNF-α secretion, and low concentration of glucocorticoids promoted the TNF-α secretion. Our results also showed that glucocorticoids significantly inhibited the HA-SMCs migration, and promoted the differentiation of HA-SMCs from synthetic to contractile phenotype. In coculture system, we found that co-culture of HA-SMCs with macrophages caused a decrease in apoptosis and glucocorticoids enhanced the inhibiting effect, but no regulatory role in migration was observed. The multiplexed protein microarray analysis found the interleukin-6(IL-6) or soluble tumor necrosis factor receptor II(TNF-s RII) might play a vital role in the cellular interaction. Therefore, the monoclonal mouse anti human IL-6 or TNF-s RII antibody was used to neutralize the cytokines. We found the inhibition effect of HA-SMC apoptosis induced by co-culture was eliminated by TNF-s RII antibody, not by the IL-6 antibody. We identified that the protein levels of bad, HSP27, p38 MAPK and total Ik Ba were significant differences between the groups by stress and apoptosis related signaling antibody array. Further, western blot analysis was used to verify the changes. We found neutralizing antibody of TNF-s RII and glucocorticoids affected phosphorylation of p38 and HSP27.Conclusions: Glucocorticoids play a protective role in vascular remodeling and lower the incidence of AD. Increased glucocorticoids decreased the margination, extravasation, and local activation of macrophages, resulting in the inhibition of MMP-2 secretion to protect the extracellular matrix from degradation. It inhibited the migration, the phenotype switch from contractile to synthetic, and the apoptosis of HA-SMCs induced by TNF-α to maintain the homeostasis of aortic wall. In addition, glucocorticoids suppressed the TNF-α secretion and increased the uncombined TNF-s RII level, which played a vital role in cellular interactions and inhibited the activation of p38 MAPK-HSP27 pathway. These findings suggest that glucocorticoids or TNF-s RII may be used as a novel predictor for the development of AD in the future. |
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