Distribution of osteoprotegerin in unruptured intracranial aneurysms in humans: association with aneurysm wall protective remodeling.

OBJECTIVE: Aneurysm wall inflammation is associated with lesion instability in unruptured intracranial aneurysms (UIAs). However, most UIAs remain unruptured during lifelong follow-ups because of simultaneous protective remodeling against the inflammatory response. The protective effects of osteoprotegerin (OPG) in intracranial and abdominal aortic aneurysms have been suggested using rodent models; however, the role of this protein in UIAs in humans remains unclear. Herein, the authors examined the relationship between OPG expression and aneurysm wall integrity in intraoperatively resected UIAs by using immunohistochemical and immunofluorescence staining. METHODS: Sixteen UIA wall tissue specimens resected between 2017 and 2022 were analyzed. Aneurysm growth was defined as an enlargement > 1 mm or an obvious morphological change over the course of more than 6 months. Three high-power fields were randomly selected from areas expressing high and low levels of OPG within the same aneurysm. To clarify the role of OPG in the human aneurysm wall, the authors compared averaged values for the following pathological features between the 2 OPG expression groups: aneurysm wall thickness, collagen, macrophages, smooth muscle cells, and transforming growth factor beta 1 (TGF-ß1). Immunohistochemical staining within the entire tissue area was also analyzed to determine the relationships between OPG expression and different aneurysm growth patterns. Pathological findings were compared between high and low OPG expression levels using the Wilcoxon signed-rank test. RESULTS: The heterogeneous expression of OPG was detected in the walls of UIAs. Lesions expressing high OPG levels had thicker aneurysm walls (327 vs 180 µm, p = 0.002) and higher expression levels of TGF-ß1 (8.5% vs 5.4%, p = 0.002) than those expressing low OPG levels. The expression of TGF-ß1 was colocalized with that of OPG mainly in the tunica media. Furthermore, lesions expressing high OPG levels had larger α-SMA+ areas (25% vs 13%, p = 0.002). Aneurysm growth was observed in 6 of 9 UIAs with available data: whole sac expansion in 4 and secondary aneurysm formation in 2. Among the 6 UIAs with aneurysm growth, OPG expression was relatively higher in the UIAs with an internal elastic lamina than in those without (17% vs 6.9%). CONCLUSIONS: Aneurysm wall integrity was associated with OPG expression in the aneurysm wall. Collectively, the study results indicated that OPG is associated with protective remodeling, which may contribute to the retention of aneurysm wall structures.

Angiotensin II Type 1 Receptor Blocker Prevents Abdominal Aortic Aneurysm Progression in Osteoprotegerin-Deficient Mice via Upregulation of Angiotensin (1-7).

Background Angiotensin II type 1 receptor blockers (ARBs) have been shown to limit the growth of abdominal aortic aneurysm (AAA), but their efficacy is controversial. This study aimed to investigate the molecular mechanism underlying the protective effect of ARBs against AAA progression. Methods and Results Olmesartan, an ARB, was administered to wild-type and osteoprotegerin-knockout (Opg-KO) mice starting 2 weeks before direct application of CaCl2 to aortas to induce AAA. The protective effect of olmesartan against AAA in wild-type and Opg-KO mice was compared at 6 weeks after AAA induction. Olmesartan prevented AAA progression in Opg-KO mice, including excessive aortic dilatation and collapse of tunica media, but not in wild-type mice. Deficiency of the Opg gene is known to cause excessive activation of the tumor necrosis factor-related apoptosis-inducing ligand-induced c-Jun N-terminal kinase/matrix metalloproteinase 9 pathway, resulting in prolonged AAA progression. Olmesartan attenuated the upregulation of phosphorylated c-Jun N-terminal kinase and matrix metalloproteinase 9 expression in the aortic wall of Opg-KO mice. In cultured vascular smooth muscle cells, tumor necrosis factor-related apoptosis-inducing ligand-induced c-Jun N-terminal kinase phosphorylation and matrix metalloproteinase 9 expression were inhibited by angiotensin (1-7), the circulating levels of which are increased by ARBs. Furthermore, administering an angiotensin (1-7) antagonist to Opg-KO mice diminished the protective effect of olmesartan against AAA progression. Conclusions Olmesartan prevented AAA progression in Opg-KO mice by upregulating angiotensin (1-7), suggesting that angiotensin (1-7) may be a key factor that mediates the protective effect of ARBs.

Smad4 regulates the nuclear translocation of Nkx2-5 in cardiac differentiation.

Bmp plays an important role in cardiomyocyte differentiation, but the function of Smad4 in Bmp signaling remains elusive. Here, we show that disruption of the Smad4 gene in cardiac progenitors expressing Sfrp5 led to embryonic lethality with hypoplastic heart formation. Although the expression of Nkx2-5 is regulated by Bmp signaling, expression of Nkx2-5 was weakly detected in the mutant heart. However, the nuclear translocation of Nkx2-5 was impaired. Expression of CK2 or PP1, which could alter the phosphorylation status of the NLS of Nkx2-5, was not affected, but Nkx2-5 was found to bind to Smad4 by co-immunoprecipitation experiments. Introduction of Smad4 into cells derived from Smad4 conditional knockout embryonic hearts restored the nuclear localization of Nkx2-5, and exogenous Nkx2-5 failed to translocate into the nucleus of Smad4-depleted fibroblasts. These results suggest that Smad4 plays an essential role in cardiomyocyte differentiation by controlling not only transcription but also the nuclear localization of Nkx2-5.

Disruption of Osteoprotegerin has complex effects on medial destruction and adventitial fibrosis during mouse abdominal aortic aneurysm formation.

Aortic aneurysm refers to dilatation of the aorta due to loss of elasticity and degenerative weakening of its wall. A preventive role for osteoprotegerin (Opg) in the development of abdominal aortic aneurysm has been reported in the CaCl2-induced aneurysm model, whereas Opg was found to promote suprarenal aortic aneurysm in the AngII-induced ApoE knockout mouse aneurysm model. To determine whether there is a common underlying mechanism to explain the impact of Opg deficiency on the vascular structure of the two aneurysm models, we analyzed suprarenal aortic tissue of 6-month-old ApoE-/-Opg-/- mice after AngII infusion for 28 days. Less aortic dissection and aortic lumen dilatation, more adventitial thickening, and higher expression of collagen I and Trail were observed in ApoE-/-Opg-/- mice relative to ApoE-/-Opg+/+ mice. An accumulation of α-smooth muscle actin and vimentin double-positive myofibroblasts was noted in the thickened adventitia of ApoE-/-Opg-/- mice. Our results suggest that fibrotic remodeling of the aorta induced by myofibroblast accumulation might be an important pathological event which tends to limit AngII-induced aortic dilatation in ApoE -/-Opg-/- mice.

Changes in lipid metabolism and capillary density of the skeletal muscle following low-intensity exercise training in a rat model of obesity with hyperinsulinemia.

Although exercise is effective in improving obesity and hyperinsulinemia, the exact influence of exercise on the capillary density of skeletal muscles remains unknown. The aim of this study was to investigate the effects of low-intensity exercise training on metabolism in obesity with hyperinsulinemia, focusing specifically on the capillary density within the skeletal muscle. Otsuka Long-Evans Tokushima fatty (OLETF) rats were used as animal models of obesity with hyperinsulinemia, whereas Long-Evans Tokushima Otsuka (LETO) rats served as controls (no obesity, no hyperinsulinemia). The animals were randomly assigned to either non-exercise or exercise groups (treadmill running for 60 min/day, for 4 weeks). The exercise groups were further divided into subgroups according to training mode: single bout (60 min, daily) vs. multiple bout (three bouts of 20 min, daily). Fasting insulin levels were significantly higher in OLETF than in LETO rats. Among OLETF rats, there were no significant differences in fasting glucose levels between the exercise and the non-exercise groups, but the fasting insulin levels were significantly lower in the exercise group. Body weight and triacylglycerol levels in the liver were significantly higher in OLETF than in LETO rats; however, among OLETF rats, these levels were significantly lower in the exercise than in the non-exercise group. The capillary-to-fiber ratio of the soleus muscle was significantly higher in OLETF than in LETO rats; however, among OLETF rats, the ratio was lower in the exercise group than in the non-exercise group. No significant differences in any of the studied parameters were noted between the single-bout and multiple-bout exercise training modes among either OLETF or LETO rats. These results suggest that low-intensity exercise training improves insulin sensitivity and fatty liver. Additionally, the fact that attenuation of excessive capillarization in the skeletal muscle of OLETF rats was accompanied by improvement in increased body weight.