| Background:Atherosclerotic abdominal aortic aneurysm (AAA) is a life-threatening aortic disease in the elderly. Among the numerous pathophysiologic mechanisms, inflammation plays pivotal role.26S proteasome, the central proteolytic unit of the the ubiquitin - proteasome system (UPS), plays a critic role in the regulation of inflammation. β 5 is one of three functional constitutive subunits of proteasome, mediating the chymotrypsin-like activity. By the action of the intended proinflammatory cytokines, the β 5 subunits can be transferred into β 5i, composing the immunoproteasome and mediating the enhanced chymotrypsin-like activity. Recently, we have discovered the enhancement of P 5 and β 5i function in human and mice AAA tissues. Bortezomib (BTZ), a specific proteasome inhibitor, has been reported to be able to attenuate the AAA formation in angiotensin Ⅱ (Ang Ⅱ)-induced ApoE-/- mice model, while the precise molecular mechanism remain elusive. Moreover, whether the immune subunit β 5i participate in the pathological process of AAA formation remains unknown.Objective:We intend to reveal the underlying mechanisms of how proteasome and the immune subunit β 5i regulate the local inflammatory response during AAA formation.Materials and methods:We collected aortic tissue from AAA patients and control. We also induced the AAA in ApoE-/- mice by Ang Ⅱ infusion (1,000 ng/min/kg). Drug treatment was carried out by intraperitoneal injection of low dose BTZ (50μg/kg,2 times per week) and subcutaneous injection of PR-957 (10mg/kg, every one day). Here, the mice were divided into four groups:Sham, BTZ/PR-957, Ang Ⅱ, and Ang Ⅱ + BTZ/PR-957. The chymotrypsin-like proteasome peptidase activities were measured by activity assay kit. The expression level of β5i was analyzed by Western Blot analysis. The gross anatomy morphology was pictured in each group. Aortic structural remodeling was observed by H&E staining, Masson staining, Gomori’s aldehyde fuchsin staining and immunohistochemical staining. The inflammatory cells in the aorta were observed by flow cytometry analysis as well as immunohistochemical staining. To decide the subtypes of CD4+T lymphocytes, the expression levels specific cytokines were detected by qPCR analysis. The apoptotic state of the aortic vascular smooth muscle cells (VSMC) were determined by TUNEL staining. And the activities of matrix metalloproteinase (MMPs) were determined by zymography.Results:1.Low dose BTZ treatment markedly prevented AAA formation.2.Low dose BTZ treatment significantly attenuated the inflammation response by reducing the CD3+ T lymphocytes infiltration.3.The mRNA expression of intercellular cell adhesion molecule-1 (ICAM-1) was upregulate in Ang Ⅱ group, while downregulated in Ang Ⅱ+BTZ group. Furthermore, NF-κB signaling pathway was activated during AAA formation and was inhibited by BTZ.4.The protein expression of (35i expression was elevated in AAA tissue both from AAA patients and murine AAA model.5.PR-957 injection significantly reduced Ang Ⅱ-induced AAA formation in mice (the incidence, severity and diameter) by effectively inhibiting the expression of (35i.6.The inflammatory responses, the vascular remodeling and degradation of elastic fibers within the vessel wall induced by Ang Ⅱ-infusion were all inhibited by PR-957 treatment in ApoE-/- mice.7. The infiltration of CD3+T lymphocytes and its subsequently differention into CD4+Helper T cell (Th) and CD8+ Cytotoxic T cell (Tc) in AAA tissue, were all significantly inhibited by PR-957 treatment.8. The mRNA expression of Th17-related cytokines and transcription factors were upregulated by Ang Ⅱ infusion in ApoE-/- mice and downregulated by PR-957 treatment. On the contrary, the mRNA expression of Treg-related cytokines and transcription factors were downregulated by Ang II infusion in ApoE-/- mice while upregulated by PR-957 treatment.9. The apoptosis of VSMC in the AAA tissue was significantly increased while suppressed by PR-957 treatment. Simultaneously, the increased activities of MMPs in AAA tissue were also suppressed by PR-957 treatment.Conclusion:In this study, we proved that proteasome activation plays a critical role in AAA formation in vivo partially by regulating T lymphocytes infiltration through regulating the activation of NF-κB signaling pathway. Moreover, we proved that the expression of immunoproteasome subunit β5i was elevated in AAA tissue and promoted the Ang Ⅱ-induced AAA formation in ApoE-/- mice by increasing the infiltration and differentiation of T lymphocytes within the abdominal aorta. |
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