Hyperglycemia Attenuates Aortic Aneurysm By Modulating The Expression Of Cathepsin S And Cystatin C In Mice

ObjectivesEpidemiological evidence suggests an inverse relationship between diabetes mellitus (DM) and abdominal aortic aneurysms (AAA); however, the mechanisms remain unknown. AAA formation involves extensive medial matrix protein proteolysis, smooth muscle cells loss, angiogenesis, and inflammatory cell accumulation, leading to progressive dilatation and eventual rupture. Previous studies have found that Cathepsin S contributed to all of these pathological events, thereby promoting Ang Ⅱ-induced AAA formation in Apoe-/-mice. Several studies have documented that high glucose levels influenced the expression and activity of cathepsin S, however, there is a lack of direct evidence for the effect of glucose on cathepsin S gene expression in AAA. We induced AAA in hyperglycemic mice to investigate the effect of hyperglycemia on the expression of cathepsin S and Cystatin C in experimental aortic aneurysm development.Methods1. Experimental modelsSixteen apolipoprotein E-deficient (ApoE-/-) C57BL/6 mice were divided into AAA group (n= 8) and DM+AAA group (n= 8). Hyperglycemia was induced by consecutive intraperitoneal injection of streptozotocin (STZ:50 mg/kg) dissolved in citrate buffer for 5 days. Control mice were also subjected to the same procedure without STZ. Serum glucose levels and body weight were monitored for 3 or more weeks after STZ injection. Hyperglycemia was confirmed by measuring casual blood glucose levels exceeding 300 mg/dl prior to AAA induction and also at the time of sacrifice.2. Induction of DM and AAAInduction of AAAs began 3 weeks after STZ or buffer injection, generating either hyperglycemic (DM+AAA, n= 8) or normoglycemic (AAA, n= 8) AAA mice. Angiotensin II (Ang II:1000 ng/kg/min) was administered subcutaneously by Alzet osmotic minipumps (model 2004) continuously for 28 days. Mice were euthanized with intentional chloral hydrate overdose after 28 days of Ang II infusion. Blood was then collected by cardiac puncture and the aortic tissues were harvested.3. Aortic diameter measurementMaximum aortic diameter was measured on the mice before AAA induction and prior to sacrifice via transabdominal ultrasound imaging at 40 MHz. All the examiners were blinded to the study group assignment.4. Enzyme-linked immunosorbent assay (ELISA)Blood samples were centrifuged at 5000 rpm for 10 minutes at 4℃ to isolate the plasma. The plasma levels of cystatin C were measured using mouse cystatin C ELISA kits according to manufacturer’s protocol.5. Quantitative real-time polymerase chain reaction (qRT-PCR)Total RNA was extracted from aortic samples using TRIzol reagent. Equal amounts (1μg) of RNA were reverse transcribed using M-MLV RTase cDNA Synthesis Kit. Real-time PCR was performed with SYBR-Green PCR Master Mix using an ABI 7900HT Fast Real-Time PCR system. β-actin was chosen as an internal control. A comparative Ct method (2-△△CT) was used to analyze the relative gene expression.6. Western Blotting analysisAortic tissues were extracted in a lysis buffer in the presence of protease inhibitor cocktail. Protein samples were quantified with the Bio-Rad Dc protein assay kit. Equal amounts of protein extracts (30μg/lane) were loaded on a 12% SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with anti-cystatin C antibody (1:10,000) and anti-cathepsin S antibody (1:100). The immunoblotting for housekeeping protein β-actin was performed to assure equal protein loading. Detection and quantification were performed with the LI-COR Odyssey Infrared imaging system.7. Histology and ImmunohistochemistryThe samples of aneurysms were fixed in 4% paraformaldehyde overnight and then embedded in paraffin and cut into 5μm sections. According to the SP method, endogenous peroxidase activity was blocked by using 3% hydrogen peroxide for 15 min. The sections were incubated with anti-cystatin C antibody (1:500) or anti-cathepsin S antibody (1:10) at 4℃ overnight, then with secondary HRP-conjugated antibody for 30 min at room temperature. The stained specimens were exposed to the 3,3-diam-inobenzidine (DAB) and counterstained with hematoxylin. Primary antibodies were replaced with PBS in the negative controls. The number of positive cells was counted by 2 independent observers.8. Analysis of elastin fragmentation and apoptotic cellsWeigert staining was carried out to analyze the elastin fragmentation according to the manufacturer’s instructions. Aortic wall elastin fragmentation was graded based on the degree of elastin filament breaks.Results1. Hyperglycemia reduces experimental AAA diameter.Although there were no significant differences in aortic diameters between the 2 groups before perfusion (0.70 ± 0.04mm vs 0.68±0.04mm, P> 0.05). However, DM+AAA group had significantly decreased aortic diameters compared to AAA group after perfusion (1.13 ± 0.05mm vs 1.35±0.09mm, P< 0.05).2. Hyperglycemia increases cystatin C expression and reduces aortic cathepsin S expression.Cystatin C expression was analyzed both systemically and within the aortic wall. Plasma cystatin C levels were significantly increased in DM+AAA group compared to AAA group (1347.19+120.25pg/mL vs 1060.17±54.28pg/mL, P<0.05). Using qRT-PCR and western blotting, we found that cystatin C mRNA and protein levels were higher in DM+AAA group than in AAA group. Additionally, the expression of cathepsin S mRNA and protein decreased in the DM+AAA group compared to the AAA group (P< 0.05). Immunohistochemical analysis showed that AAA in DM+AAA group expressed higher levels of cystatin C, but lower levels of cathepsin L than the AAA group (P<0.05).3. Hyperglycemia reduces elastin degradation and cell apoptosis in AAA lesionsWeigert staining demonstrated significantly reduced elastin fragmentation in AAA lesions from DM+AAA group compared with those from AAA group (2.00+ 0.27 vs 2.88±0.23, P< 0.05).ConclusionsHyperglycemia increased cystatin C expression and decreased cathepsin S expression, resulting in reduced AAA diameter and medial elastin degradation in experimental aneurysm. These results highlight the roles of cathepsin S and cystatin C in AAA pathophysiology, and suggest a candidate mechanism for hyperglycemic inhibition of AAA. Novel therapeutic strategies can therefore be developed using selective and reversible cathepsin inhibitors to reduce the morbidity and mortality of AAA.