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.

Implication of satellite cell behaviors in capillary growth via VEGF expression-independent mechanism in response to mechanical loading in HeyL-null mice.

Angiogenesis and muscle satellite cell (SC)-mediated myonuclear accretion are considered essential for the robust response of contraction-induced muscle hypertrophy. Moreover, both myonucleus and SCs are physically adjacent to capillaries and are the major sites for the expression of proangiogenic factors, such as VEGF, in the skeletal muscle. Thus, events involving the addition of new myonuclei via activation of SCs may play an important role in angiogenesis during muscle hypertrophy. However, the relevance among myonuclei number, capillary supply, and angiogenesis factor is not demonstrated. The Notch effector HeyL is specifically expressed in SCs in the skeletal muscle and is crucial for SC proliferation by inhibiting MyoD in overload-induced muscle hypertrophy. Here, we tested whether the addition of new myonuclei by SC in overloaded muscle is associated with angiogenic adaptation by reanalyzing skeletal muscle from HeyL-knockout (KO) mice, which show blunted responses of SC proliferation, myonucleus addition, and overload-induced muscle hypertrophy. Reanalysis confirmed blunted SC proliferation and myonuclear accretion in the plantaris muscle of HeyL-KO mice 9 wk after synergist ablation. Interestingly, the increase in capillary-to-fiber ratio observed in wild-type (WT) mice was impaired in HeyL-KO mice. In both WT and HeyL-KO mice, the expression of VEGFA and VEGFB was similarly increased in response to overload. In addition, the expression pattern of TSP-1, a negative regulator of angiogenesis, was also not changed between WT and HeyL-KO mice. Collectively, these results suggest that SCs activation-myonuclear accretion plays a crucial role in angiogenesis during overload-induced muscle hypertrophy via independent of angiogenesis regulators.

S-1-Propenylcysteine promotes IL-10-induced M2c macrophage polarization through prolonged activation of IL-10R/STAT3 signaling.

Atherosclerosis is a chronic inflammatory disease that may lead to the development of serious cardiovascular diseases. Aged garlic extract (AGE) has been reported to ameliorate atherosclerosis, although its mode of action remains unclear. We found that AGE increased the mRNA or protein levels of arginase1 (Arg1), interleukin-10 (IL-10), CD206 and hypoxia-inducible factor 2α (HIF2α) and decreased that of CD68, HIF1α and inducible nitric oxide synthase in the aorta and spleen of apolipoprotein E knockout mice. We also found that S-1-propenylcysteine (S1PC), a characteristic sulfur compound in AGE, increased the level of IL-10-induced Arg1 mRNA and the extent of M2c-like macrophage polarization in vitro. In addition, S1PC increased the population of M2c-like macrophages, resulting in suppressed the population of M1-like macrophages and decreased lipopolysaccharide-induced production of pro-inflammatory cytokines. These effects were accompanied by prolonged phosphorylation of the IL-10 receptor α (IL-10Rα) and signal transducer and activator of transcription 3 (STAT3) that inhibited the interaction between IL-10Rα and Src homology-2-containing inositol 5'-phosphatase 1 (SHIP1). In addition, administration of S1PC elevated the M2c/M1 macrophage ratio in senescence-accelerated mice. These findings suggest that S1PC may help improve atherosclerosis due to its anti-inflammatory effect to promote IL-10-induced M2c macrophage polarization.

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.

The SGLT2 Inhibitor Luseogliflozin Rapidly Normalizes Aortic mRNA Levels of Inflammation-Related but Not Lipid-Metabolism-Related Genes and Suppresses Atherosclerosis in Diabetic ApoE KO Mice.

Recent clinical studies have revealed the treatment of diabetic patients with sodium glucose co-transporter2 (SGLT2) inhibitors to reduce the incidence of cardiovascular events. Using nicotinamide and streptozotocin (NA/STZ) -treated ApoE KO mice, we investigated the effects of short-term (seven days) treatment with the SGLT2 inhibitor luseogliflozin on mRNA levels related to atherosclerosis in the aorta, as well as examining the long-term (six months) effects on atherosclerosis development. Eight-week-old ApoE KO mice were treated with NA/STZ to induce diabetes mellitus, and then divided into two groups, either untreated, or treated with luseogliflozin. Seven days after the initiation of luseogliflozin administration, atherosclerosis-related mRNA levels in the aorta were compared among four groups; i.e., wild type C57/BL6J, native ApoE KO, and NA/STZ-treated ApoE KO mice, with or without luseogliflozin. Short-term luseogliflozin treatment normalized the expression of inflammation-related genes such as F4/80, TNFα, IL-1ß, IL-6, ICAM-1, PECAM-1, MMP2 and MMP9 in the NA/STZ-treated ApoE KO mice, which showed marked elevations as compared with untreated ApoE KO mice. In contrast, lipid metabolism-related genes were generally unaffected by luseogliflozin treatment. Furthermore, after six-month treatment with luseogliflozin, in contrast to the severe and widely distributed atherosclerotic changes in the aortas of NA/STZ-treated ApoE KO mice, luseogliflozin treatment markedly attenuated the progression of atherosclerosis, without affecting serum lipid parameters such as high density lipoprotein, low density lipoprotein and triglyceride levels. Given that luseogliflozin normalized the aortic mRNA levels of inflammation-related, but not lipid-related, genes soon after the initiation of treatment, it is not unreasonable to speculate that the anti-atherosclerotic effect of this SGLT2 inhibitor emerges rapidly, possibly via the prevention of inflammation rather than of hyperlipidemia.

EPA Prevents the Development of Abdominal Aortic Aneurysms through Gpr-120/Ffar-4.

Abdominal aortic aneurysms (AAAs), which commonly occur among elderly individuals, are accompanied by a risk of rupture with a high mortality rate. Although eicosapentaenoic acid (EPA) has been reported to prevent AAA formation, the mechanism by which EPA works on vascular smooth muscle cells is unknown. This study aimed to investigate the mechanism by which orally-administered EPA prevents the formation of severe AAAs that develop in Osteoprotegerin (Opg) knockout (KO) mice. In the CaCl2-induced AAA model, EPA attenuated the enhanced progression of AAAs in Opg-KO mice, including the increase in aortic diameter with destruction of elastic fibers in the media. Immunohistochemical analyses showed that EPA reduced the phosphorylation of transforming growth factor beta-activated kinase-1/Map3k7 (Tak-1) and c-Jun NH2-terminal kinase (JNK), as well as the expression of Matrix metalloproteinase-9 (Mmp-9) in the media of the aorta. In smooth muscle cell cultures, rh-TRAIL-induced activation of the Tak-1-JNK pathway and increase in Mmp-9 expression were inhibited by EPA. Moreover, GW9508, a specific ligand for G-protein coupled receptor (Gpr)-120/Free fatty acid receptor (Ffar)-4, mimicked the effects of EPA. The effects of EPA were abrogated by knockdown of the Gpr-120/Ffar-4 receptor gene. Our data demonstrate that the Trail-Tak-1-JNK-Mmp-9 pathway is responsible for the enhancement of AAAs in Opg-KO mice, and that EPA inhibits the Tak-1-JNK pathway by activating Gpr-120/Ffar-4, which results in the attenuation of AAA development.

The Transcription Factor Hand1 Is Involved In Runx2-Ihh-Regulated Endochondral Ossification.

The developing long bone is a model of endochondral ossification that displays the morphological layers of chondrocytes toward the ossification center of the diaphysis. Indian hedgehog (Ihh), a member of the hedgehog family of secreted molecules, regulates chondrocyte proliferation and differentiation, as well as osteoblast differentiation, through the process of endochondral ossification. Here, we report that the basic helix-loop-helix transcription factor Hand1, which is expressed in the cartilage primordia, is involved in proper osteogenesis of the bone collar via its control of Ihh production. Genetic overexpression of Hand1 in the osteochondral progenitors resulted in prenatal hypoplastic or aplastic ossification in the diaphyses, mimicking an Ihh loss-of-function phenotype. Ihh expression was downregulated in femur epiphyses of Hand1-overexpressing mice. We also confirmed that Hand1 downregulated Ihh gene expression in vitro by inhibiting Runx2 transactivation of the Ihh proximal promoter. These results demonstrate that Hand1 in chondrocytes regulates endochondral ossification, at least in part through the Runx2-Ihh axis.

Osteoprotegerin Prevents Development of Abdominal Aortic Aneurysms.

Abdominal aortic aneurysms (AAAs), which commonly occur among elderly individuals, are accompanied by a risk of rupture and subsequent high mortality. Establishment of medical therapies for the prevention of AAAs requires further understanding of the molecular pathogenesis of this condition. This report details the possible involvement of Osteoprotegerin (OPG) in the prevention of AAAs through inhibition of Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In CaCl2-induced AAA models, both internal and external diameters were significantly increased with destruction of elastic fibers in the media in Opg knockout (KO) mice, as compared to wild-type mice. Moreover, up-regulation of TRAIL expression was observed in the media by immunohistochemical analyses. Using a culture system, both the TRAIL-induced expression of matrix metalloproteinase-9 in smooth muscle cells (SMCs) and the chemoattractive effect of TRAIL on SMCs were inhibited by OPG. These data suggest that Opg may play a preventive role in the development of AAA through its antagonistic effect on Trail.

Evidence of Notch-Hesr-Nrf2 Axis in Muscle Stem Cells, but Absence of Nrf2 Has No Effect on Their Quiescent and Undifferentiated State.

Nrf2 is a master regulator of oxidative stresses through the induction of anti-oxidative genes. Nrf2 plays roles in maintaining murine hematopoietic stem cells and fly intestinal stem cells. The canonical Notch signaling pathway is also crucial for maintaining several types of adult stem cells including muscle stem cells (satellite cells). Here, we show that Dll1 induced Nrf2 expression in myogenic cells. In addition, primary targets of Notch signaling, Hesr1 and Hesr3, were involved in the up-regulation of Nrf2 mRNA and expression of its target genes. In vitro, Nrf2 had anti-myogenic and anti-proliferative effects on primary myoblasts. In vivo, although Nrf2-knockout mice showed decreased expression of its target genes in muscle stem cells, adult muscle stem cells of Nrf2-knockout mice did not exhibit the phenotype. Taken together, in muscle stem cells, the Notch-Hesr-Nrf2 axis is a pathway potentially inducing anti-oxidative genes, but muscle stem cells either do not require Nrf2-mediated anti-oxidative gene expression or they have a complementary system compensating for the loss of Nrf2.

Induction of Timp1 in smooth muscle cells during development of abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is known to develop mainly by the increased diameter of aorta through metalloproteinases (MMPs). Although activities of MMPs are tightly regulated by the presence of tissue inhibitor of MMPs (TIMPs) and imbalances between MMPs and TIMPs may serve to fragility of arterial wall, little is known about TIMPs behavior in aneurysmal formation. Here, we utilized a murine experimental AAA model, and found that by immunohistochemical analysis, Timp1 as and Timp1 mRNA levels was also revealed in aortic tissue in AAA by RT-PCR. In cultured vascular smooth muscle cells (SMCs), Tumor Necrosis Factor (TNF)-alpha significantly activated both Mmp9 and Timp1 expression, and they were blocked by Jun kinase inhibitor (SP600125) in a dose-dependent manner. Interestingly, a proteasome inhibitor (MG132), which is known as an agent for inhibition of the nuclear factor-kappa B (NF-kappaB), significantly inhibited the TNF-alpha-induced expression of Timp1, whereas MG132, which also works as an activator of c-Jun/AP-1 pathway, strongly increased Mmp9. Taken together, inflammatory cytokines, including TNF-alpha, may simultaneously induce MMPs and TIMPs for the remodeling of the medial layer, leading to the increased diameter of the aorta, the aneurysm.

Ectopic retinoic acid signaling affects outflow tract cushion development through suppression of the myocardial Tbx2-Tgfß2 pathway.

The progress of molecular genetics has enabled us to identify the genes responsible for congenital heart malformations. However, recent studies suggest that congenital heart diseases are induced not only by mutations in certain genes, but also by abnormal maternal factors. A high concentration of maternal retinoic acid (RA), the active derivative of vitamin A, is well known as a teratogenic agent that can cause developmental defects. Our previous studies have shown that the maternal administration of RA to mice within a narrow developmental window induces outflow tract (OFT) septum defects, a condition that closely resembles human transposition of the great arteries (TGA), although the responsible factors and pathogenic mechanisms of the TGA induced by RA remain unknown. We herein demonstrate that the expression of Tbx2 in the OFT myocardium is responsive to RA, and its downregulation is associated with abnormal OFT development. We found that RA could directly downregulate the Tbx2 expression through a functional retinoic acid response element (RARE) in the Tbx2 promoter region, which is also required for the initiation of Tbx2 transcription during OFT development. Tgfb2 expression was also downregulated in the RA-treated OFT region and was upregulated by Tbx2 in a culture system. Moreover, defective epithelial-mesenchymal transition caused by the excess RA was rescued by the addition of Tgfß2 in an organ culture system. These data suggest that RA signaling participates in the Tbx2 transcriptional mechanism during OFT development and that the Tbx2-Tgfß2 cascade is one of the key pathways involved in inducing the TGA phenotype.

Mastermind-like 1 (MamL1) and mastermind-like 3 (MamL3) are essential for Notch signaling in vivo.

Mastermind (Mam) is one of the elements of Notch signaling, a system that plays a pivotal role in metazoan development. Mam proteins form transcriptionally activating complexes with the intracellular domains of Notch, which are generated in response to the ligand-receptor interaction, and CSL DNA-binding proteins. In mammals, three structurally divergent Mam isoforms (MamL1, MamL2 and MamL3) have been identified. There have also been indications that Mam interacts functionally with various other transcription factors, including the p53 tumor suppressor, ß-catenin and NF-κB. We have demonstrated previously that disruption of MamL1 causes partial deficiency of Notch signaling in vivo. However, MamL1-deficient mice did not recapitulate total loss of Notch signaling, suggesting that other members could compensate for the loss or that Notch signaling could proceed in the absence of Mam in certain contexts. Here, we report the generation of lines of mice null for MamL3. Although MamL3-null mice showed no apparent abnormalities, mice null for both MamL1 and MamL3 died during the early organogenic period with classic pan-Notch defects. Furthermore, expression of the lunatic fringe gene, which is strictly controlled by Notch signaling in the posterior presomitic mesoderm, was undetectable in this tissue of the double-null embryos. Neither of the single-null embryos exhibited any of these phenotypes. These various roles of the three Mam proteins could be due to their differential physical characteristics and/or their spatiotemporal distributions. These results indicate that engagement of Mam is essential for Notch signaling, and that the three Mam isoforms have distinct roles in vivo.

CCN3/NOV inhibits BMP-2-induced osteoblast differentiation by interacting with BMP and Notch signaling pathways.

We elucidate the role of CCN3/NOV, a member of the CCN family proteins, in osteoblast differentiation using MC3T3-E1 osteoblastic cells. Transduction with CCN3 adenovirus (AdCCN3) alone induced no apparent changes in the expression of osteoblast-related markers, whereas cotransduction with BMP-2 adenovirus (AdBMP-2) and AdCCN3 significantly inhibited the AdBMP-2-induced mRNA expression of Runx2, osterix, ALP, and osteocalcin. Immunoprecipitation-western analysis revealed that CCN3 associated with BMP-2. Compared to transduction with AdBMP-2 alone, cotransduction with AdBMP-2 and AdCCN3 attenuated the expression of phosphorylated Smad1/5/8 and the mRNA for Id1, Id2, and Id3. Transduction with AdCCN3 stimulated the expression of cleaved Notch1, the mRNA expression of Hes1 and Hey1/Hesr1, and the promoter activities of Hes1 and Hey1. The inhibitory effects of CCN3 on the expression of BMP-2-induced osteoblast-related markers were nullified in Hey1-deficient osteoblastic cells. These results indicate that CCN3 exerts inhibitory effects on BMP-2-induced osteoblast differentiation by its involvement of the BMP and Notch signaling pathways.

HESR1/CHF2 suppresses VEGFR2 transcription independent of binding to E-boxes.

The bHLH transcription factor HESR1 (CHF2) acts downstream of notch to regulate cardiovascular development and angiogenesis, at least in part through down-regulation of the VEGF receptor, VEGFR2. Surprisingly, we find that HESR1 interacts with the promoter in endothelial cells (EC) not through direct binding to the E-boxes, but through intermediary interactions with GC-box-binding proteins. The bHLH and orange domains of HESR1 are sufficient for repression in EC, likely through recruitment of co-repressors, however, the C-terminal YRPW motif is not required. The VEGFR2 promoter contains a functional initiator element but no TATA box, however, addition of a TATA sequence renders the promoter resistant to inhibition by HESR1. In agreement with this finding, the NrCAM, TK, and CMV promoters, which have TATA boxes, cannot be repressed. Thus, HESR1 represses VEGFR2 through interactions with SP-1-like factors and requires an Inr element in the absence of a TATA box. Our findings illuminate an important mechanism for notch/HESR1 regulation of VEGF-induced angiogenesis.

Targeted disruption of hesr2 results in atrioventricular valve anomalies that lead to heart dysfunction.

Genes involved in the Notch signaling pathway have been shown to be critical regulators of cardiovascular development. In vitro studies have revealed that the Notch signaling pathway directly regulates transcription of hairy and enhancer of split-related (hesr) genes, encoding basic helix-loop-helix transcription factors. To assess the functional role of hesr genes in cardiovascular development, we generated mice with a targeted disruption of the hesr2 gene and used echocardiography to analyze heart function of the mutant mice. In the early postnatal period, a majority of hesr2 homozygous mice die as a result of congestive heart failure accompanied by pronounced heart enlargement. Transthoracic echocardiography on 5-day-old homozygous mice revealed tricuspid and mitral valve regurgitation and a dilated left ventricular chamber with markedly diminished fractional shortening of the left ventricle. The hemodynamic anomalies were accompanied by morphological changes, such as dysplastic atrioventricular (AV) valves, a perimembranous ventricular septal defect, and a secundum atrial septal defect. AV valve regurgitations attributable to dysplasia of the AV valves were most likely responsible for the heart dysfunction in hesr2 homozygous mice. These observations indicate that the Notch signaling target hesr2 plays an important role in the formation and function of the AV valves. In addition, hesr2 activity may be important for proper development of cardiomyocytes, thereby assuring normal left ventricular contractility. Because of the unique spectrum of cardiac anomalies expressed by hesr2-null mice, they represent a useful model system for elucidating the genetic basis of heart dysfunction.