Urotensin II receptor deficiency ameliorates ligation-induced carotid intimal hyperplasia partially through the RhoA-YAP1 pathway.

Intimal hyperplasia (IH) is a common pathological feature of vascular proliferative diseases, such as atherosclerosis and restenosis after angioplasty. Urotensin II (UII) and its receptor (UTR) are widely expressed in cardiovascular tissues. However, it remains unclear whether the UII/UTR system is involved in IH. Right unilateral common carotid artery ligation was performed and maintained for 21 days to induce IH in UTR knockout (UTR-/-) and wild-type (WT) mice. Histological analysis revealed that compared with WT mice, UTR-deficient mice exhibited a decreased neointimal area, angiostenosis and intima-media ratio. Immunostaining revealed fewer smooth muscle cells (SMCs), endothelial cells and macrophages in the lesions of UTR-/- mice than in those of WT mice. Protein interaction analysis suggested that the UTR may affect cell proliferation by regulating YAP and its downstream target genes. In vitro experiments revealed that UII can promote the proliferation and migration of SMCs, and western blotting also revealed that UII increased the protein expression of RhoA, CTGF, Cyclin D1 and PCNA and downregulated p-YAP protein expression, while these effects could be partly reversed by urantide. To evaluate the translational value of UTRs in IH management, WT mice were also treated with two doses of urantide, a UTR antagonist, to confirm the benefit of UTR blockade in IH progression. A high dose of urantide (600 µg/kg/day), rather than a low dose (60 µg/kg/day), successfully improved ligation-induced IH compared with that in mice receiving vehicle. The results of the present study suggested that the UII/UTR system may regulate IH partly through the RhoA-YAP signaling pathway.

Diabetic status aggravates atherosclerosis in aged ApoE-/- mice.

Streptozocin (STZ) aggravates diabetic atherosclerosis in aged ApoE-/- mice. (A). Study design: ApoE-/- mice were given STZ (50 mg/kg/day) or vehicle by intraperitoneal injection for five days consecutively to induce diabetes. (B). Body weight and blood glucose levels were measured in mice on the baseline, 16th, and 32nd weeks. (C). Representative oil red O staining of en face aorta and quantifications of the lesional area of the whole aortic tree (D). Representative micrographs stained by H&E and Oil red O (left), and quantifications of microscopic atherosclerotic area. All data are expressed as mean ± SEM; All data were tested for normality and equal variance. For analysis of body weight and blood glucose, two-way ANOVA analysis was used (B). For normally or approximately normally distributed data, a Student's t-test was performed (C, D). n = 6 mice/group. *p<0.05 and **p<0.01 vs vehicle group.

Genetic deficiency of protein inhibitor of activated STAT3 suppresses experimental abdominal aortic aneurysms.

Aim: Signal transducer and activator of transcription (STAT) signaling is critical for the pathogenesis of abdominal aortic aneurysms (AAAs). Though protein inhibitor of activated STAT3 (PIAS3) negatively modulates STAT3 activity, but its role in AAA disease remains undefined. Method: AAAs were induced in PIAS3 deficient (PIAS3-/-) and wild type (PIAS3+/+) male mice via transient intra-aortic elastase infusion. AAAs were assessed by in situ measurements of infrarenal aortic external diameters prior to (day 0) and 14 days after elastase infusion. Characteristic aneurysmal pathologies were evaluated by histopathology. Results: Fourteen days following elastase infusion, aneurysmal aortic diameter was reduced by an approximately 50% in PIAS3-/- as compared to PIAS3+/+ mice. On histological analyses, PIAS3-/- mice showed less medial elastin degradation (media score: 2.5) and smooth muscle cell loss (media score: 3.0) than those in PIAS3+/+ mice (media score: 4 for both elastin and SMC destruction). Aortic wall leukocyte accumulation including macrophages, CD4+ T cells, CD8+ T cells and B cells as well as mural neovessel formation were significantly reduced in PIAS3-/- as compared to PIAS3+/+ mice. Additionally, PIAS3 deficiency also downregulated the expression levels of matrix metalloproteinases 2 and 9 by 61% and 70%, respectively, in aneurysmal lesion. Conclusion: PIAS3 deficiency ameliorated experimental AAAs in conjunction with reduced medial elastin degradation and smooth muscle cell depletion, mural leukocyte accumulation and angiogenesis.

Urotensin II Enhances Advanced Aortic Atherosclerosis Formation and Delays Plaque Regression in Hyperlipidemic Rabbits.

Accumulated evidence shows that elevated urotensin II (UII) levels are associated with cardiovascular diseases. However, the role of UII in the initiation, progression, and regression of atherosclerosis remains to be verified. Different stages of atherosclerosis were induced in rabbits by a 0.3% high cholesterol diet (HCD) feeding, and either UII (5.4 µg/kg/h) or saline was chronically infused via osmotic mini-pumps. UII promoted atherosclerotic fatty streak formation in ovariectomized female rabbits (34% increase in gross lesion and 93% increase in microscopic lesion), and in male rabbits (39% increase in gross lesion). UII infusion significantly increased the plaque size of the carotid and subclavian arteries (69% increase over the control). In addition, UII infusion significantly enhanced the development of coronary lesions by increasing plaque size and lumen stenosis. Histopathological analysis revealed that aortic lesions in the UII group were characterized by increasing lesional macrophages, lipid deposition, and intra-plaque neovessel formation. UII infusion also significantly delayed the regression of atherosclerosis in rabbits by increasing the intra-plaque macrophage ratio. Furthermore, UII treatment led to a significant increase in NOX2 and HIF-1α/VEGF-A expression accompanied by increased reactive oxygen species levels in cultured macrophages. Tubule formation assays showed that UII exerted a pro-angiogenic effect in cultured endothelial cell lines and this effect was partly inhibited by urantide, a UII receptor antagonist. These findings suggest that UII can accelerate aortic and coronary plaque formation and enhance aortic plaque vulnerability, but delay the regression of atherosclerosis. The role of UII on angiogenesis in the lesion may be involved in complex plaque development.

Kunming mouse strain is less susceptible to elastase-induced abdominal aortic aneurysms.

BACKGROUND: Porcine pancreatic elastase (PPE) is successfully used to induce abdominal aortic aneurysm (AAA) in mice. However, differences between mouse strains in susceptibility to PPE induction have been reported. Kunming mouse is one of the most frequently used strains in China but whether it is suitable for induction of AAA by PPE application remains unclear. METHODS: PPE infusion (1.5 units/ml) in temporary controlled aorta was performed to induce AAAs in both C57BL/6J and Kunming mice. Phosphate-buffered saline (PBS) application was used as vehicle control. The aorta diameters of all mice were measured at days 0 and 14 after surgery to evaluate the AAA formation. RESULTS: After 14 days of PPE or PBS infusion, all mice were sacrificed and aorta tissues were collected for histological staining analysis. At the 14th day after infusion, PPE successfully induced aortic dilation in Kunming mice and typical AAA in C57BL/6J mice. The aorta diameter increased by 0.23 mm in Kunming mice after PPE infusion, while it was 0.72 mm in the C57BL/6J strain. PPE induced mild elastin degradation, smooth muscle cell (SMC) depletion and mural leucocyte infiltration in Kunming mice, but in PPE-sensitive C57BL/6J mice, it induced total loss of SMCs, elastin disappearance and diffused infiltrated leucocytes in aortic aneurysmal segments. The effects of PPE in inducing angiogenesis and upregulating matrix metalloproteinase 2 and 9 expression in Kunming mice were also weaker than that in C57BL/6J mice. CONCLUSION: At the reported dose of PPE, Kunming mouse is not as susceptible to AAA formation as C57BL/6J mice. The failure of PPE to induce AAA formation in Kunming mice may be associated to its inability to boost a strong inflammatory response.

Dapagliflozin Ameliorates the Formation and Progression of Experimental Abdominal Aortic Aneurysms by Reducing Aortic Inflammation in Mice.

BACKGROUND: Dapagliflozin, a sodium glucose transporter protein-2 (SGLT-2) inhibitor, reduces the risk for cardiovascular diseases. However, the influence of dapagliflozin on nondissecting abdominal aortic aneurysms (AAAs) remains unclear. METHODS: AAAs were created in male C57BL/6 mice via intra-aortic porcine pancreatic elastase (PPE) infusion. Mice were daily treated with dapagliflozin (1 or 5 mg/kg body weight) or an equal volume of vehicle through oral gavage beginning one day prior to PPE infusion for 14 days. To investigate its translational value, dapagliflozin or vehicle was also administered to mice with existing AAAs in another cohort. Aortic diameters were measured prior to (day 0 for baseline) and 14 days after PPE infusion. After sacrifice, mice aortae were collected, and following histological analyses were performed. RESULTS: Dapagliflozin treatment significantly reduced aneurysmal aortic expansion following PPE infusion as compared to vehicle treatment especially at 5 mg/kg body weight (approximately 21% and 33% decreases in 1 and 5 mg/kg treatment groups, respectively). The dose-dependent attenuation of AAAs by dapagliflozin was also confirmed on histological analyses. Dapagliflozin remarkably reduced aortic accumulation of macrophages, CD4+ T cells, and B cells particularly following dapagliflozin treatment at 5 mg/kg. Dapagliflozin treatment also markedly attenuated medial SMC loss. Though the difference was not significant, dapagliflozin treatment tended to attenuate CD8+ T cells and elastin degradation. Dapagliflozin treatment at 5 mg/kg caused a 53% reduction in neovessel density. Furthermore, dapagliflozin treatment mitigated further progress of existing AAAs. CONCLUSION: Dapagliflozin treatment ameliorated PPE-induced AAAs by inhibiting aortic leukocytes infiltration and angiogenesis.

Temporal and Quantitative Analysis of Aortic Immunopathologies in Elastase-Induced Mouse Abdominal Aortic Aneurysms.

OBJECTIVE: Elastase-induced abdominal aortic aneurysm (AAA) model is widely used for aneurysmal pathogenesis and translational research. However, temporal alternations in aneurysmal histologies remain unknown. This study is aimed at analyzing temporal immunopathologies of aneurysmal aorta following experimental AAA induction. METHODS: Male C57BL/6J mice at the age of 10-14 weeks received intra-aortic infusion of elastase to induce AAAs. Aortic diameters at the baseline and indicated days after AAA induction were measured, and aortae were collected for histopathological analysis. RESULTS: Aorta diameters increased from 0.52 mm at the baseline levels to 0.99 mm, 1.34 mm, and 1.41 mm at days 7, 14, and 28, respectively, corresponding 90%, 158%, and 171% increases over the baseline level. Average aortic diameters did not differ between days 14 and 28. Severe elastin degradation and smooth muscle cell depletion were found at days 14 and 28 as compared to the baseline and day 7. No difference in the scores of medial elastin and SMC destruction was noted between days 14 and 28. Consistent results were found for leukocyte accumulation, neoangiogenesis, and matrix metalloproteinase expression. Twenty-eight days after AAA induction, all aneurysmal pathologies showed an attenuated trend, although most histopathological parameters did no differ between days 14 and 28. CONCLUSION: Our data suggest that almost aneurysmal immunohistopathologies reach maximal 14 days following AAA induction. Analysis of day 14 histologies is sufficient for AAA pathogenesis and translational studies in elastase-induced mouse experimental AAAs.