Hiroki Takata

Bone Marrow Angiotensin AT1 Receptor Regulates Differentiation of Monocyte Lineage Progenitors From Hematopoietic Stem Cells

Background—The angiotensin II (Ang II) type 1 (AT1) receptor is expressed in bone marrow (BM) cells, whereas it remains poorly defined how Ang II regulates differentiation/proliferation of monocyte-lineage cells to exert proatherogenic actions. Methods and Results—We generated BM chimeric apoE−/− mice repopulated with AT1-deficient (Agtr1−/−) or wild-type (Agtr1+/+) BM cells. The atherosclerotic development was significantly reduced in apoE−/−/BM-Agtr1−/− mice compared with apoE−/−/BM-Agtr1+/+ mice, accompanied by decreased numbers of BM granulocyte/macrophage progenitors (GMP:c-Kit+Sca-1−Lin−CD34+CD16/32+) and peripheral blood monocytes. Macrophage-colony-stimulating factor (M-CSF)–induced differentiation from hematopoietic stem cells (HSCs:c-Kit+Sca-1+Lin−) to promonocytes (CD11bhighLy-6Glow) was markedly reduced in HSCs from Agtr1−/− mice. The expression of M-CSF receptor c-Fms was decreased in HSCs/promonocytes from Agtr1−/− mice, accompanied by a marked inhibition in M-CSF–induced phosphorylation of PKC-&dgr; and JAK2. c-Fms expression in HSCs/promonocytes was mainly regulated by TNF-&agr; derived from BM CD45−CD34− stromal cells, and Ang II specifically regulated the TNF-&agr; synthesis and release from BM stromal cells. Conclusions—Ang II regulates the expression of c-Fms in HSCs and monocyte-lineage cells through BM stromal cell–derived TNF-&agr; to promote M-CSF–induced differentiation/proliferation of monocyte-lineage cells and contributes to the proatherogenic action.

Periaortic adipose tissue-specific activation of the renin-angiotensin system contributes to atherosclerosis development in uninephrectomized apoE / mice

Chronic kidney disease (CKD) is an independent risk factor for the development of cardiovascular disease. The perivascular adipose tissue is closely implicated in the development of atherosclerosis; however, the contribution to CKD-associated atherogenesis remains undefined. Eight-week-old apoE-deficient mice were uninephrectomized and fed a high-cholesterol diet starting at 12 wk of age. The atherosclerotic lesion area in the thoracic aorta was comparable in 16-wk-old uninephrectomized (UNX) mice and sham control mice; however, the lesion area was markedly exaggerated in 20-wk-old UNX mice compared with the control (54%, P < 0.05). While the accumulation of monocytes/macrophages and the mRNA expression levels of inflammatory cytokines/chemokines in the thoracic periaortic adipose tissue (PAT) did not differ between the two groups, angiotensinogen (AGT) mRNA expression and the angiotensin II (ANG II) concentration in the PAT were significantly higher in 16-wk-old UNX mice than in the control (1.9- and 1.5-fold increases vs. control, respectively; P < 0.05). ANG II concentrations in both the plasma and epididymal white adipose tissue (WAT) were comparable between the two groups, suggesting that PAT-specific activation of the renin-angiotensin system (RAS) is primarily involved in CKD-associated atherogenesis. The homeostasis model assessment-insulin resistance (HOMA-IR) index and plasma insulin level after glucose loading were significantly elevated in 16-wk-old UNX mice. In vitro stimulation of preadipocytes with insulin exaggerated the AGT mRNA expression along with increased mRNA expression of PPARγ. These findings suggest that PAT-specific RAS activation probably primarily contributes in accelerating atherosclerotic development in UNX mice and could thus represent a therapeutic target for preventing CKD-associated atherogenesis.

Bone Marrow AT1 Augments Neointima Formation by Promoting Mobilization of Smooth Muscle Progenitors via Platelet-Derived SDF-1α

Objectives—Bone marrow (BM)-derived endothelial progenitor cells (EPCs) and vascular smooth muscle progenitor cells (VPCs) contribute to neointima formation, whereas the angiotensin II (Ang II) type 1 receptor (AT1)-mediated action on BM-derived progenitors remains undefined. Methods and Results—A wire-induced vascular injury was performed in the femoral artery of BM-chimeric mice whose BM was repopulated with AT1-deficient (BM-Agtr1−/−) or wild-type (BM-Agtr1+/+) cells. Neointima formation was profoundly reduced by 38% in BM-Agtr1−/− mice. Although the number of circulating EPCs (Sca-1+Flk-1+) and extent of reendothelialization did not differ between the 2 groups, the numbers of both circulating VPCs (c-Kit−Sca-1+Lin−) and tissue VPCs (Sca-1+CD31−) incorporated into neointima were markedly decreased in BM-Agtr1−/− mice. The accumulation of aggregated platelets and their content of stromal cell–derived factor-1&agr; (SDF-1&agr;) were significantly reduced in BM-Agtr1−/− mice, accompanied by a decrease in the serum level of SDF-1&agr;. Thrombin-induced platelets aggregation was dose-dependently inhibited (45% at 0.1 IU/mL, P<0.05) in Agtr1−/− platelets compared with Agtr1+/+ platelets, accompanied by the reduced expression and release of SDF-1&agr;. Conclusions—The BM-AT1 receptor promotes neointima formation by regulating the mobilization and homing of BM-derived VPCs in a platelet-derived SDF-1&agr;–dependent manner without affecting EPC-mediated reendothelialization.

Vascular angiotensin II type 2 receptor attenuates atherosclerosis via a kinin/NO-dependent mechanism

Introduction: The angiotensin II (Ang II) type 1 receptor exerts pro-atherogenic action by augmenting oxidative stress, whereas the Ang II type 2 receptor (AT2)-mediated effect on atherosclerosis remains controversial. Materials and methods: AT2 transgenic (AT2-Tg) mice, which overexpress AT2 in their vascular smooth muscle cells, were crossed with apoE-deficient (apoE-/-) mice to generate AT2 transgenic apoE-/- mice (AT2-Tg/apoE-/-). Results: A subpressor dose of Ang II infusion exaggerated atherosclerosis development in apoE-/- mice, which was markedly suppressed in AT2-Tg/apoE-/- mice. Inhibitors of nitric oxide (NO) synthase (L-NAME) or bradykinin type 2 receptor completely abolished AT2-mediated anti-atherogenic actions. The vascular cell adhesion molecule-1 expression levels and degree of monocyte/macrophage accumulation in the intima were also considerably reduced in AT2-Tg/apoE-/- mice; these phenomena were completely reversed by L-NAME treatment. Ang II infusion significantly enhanced the accumulation of dihydroethidium-positive mononuclear cells in the intima and mRNA expression levels of Nox2, a phagocytic cell-type NADPH oxidase subunit in apoE-/- mice, which was completely inhibited in AT2-Tg/apoE-/- mice. Conclusions: Vascular AT2 stimulation exerts anti-atherogenic actions in an endothelial kinin/NO-dependent manner, and its anti-oxidative effect is likely to be exerted by inhibiting the accumulation of superoxide-producing mononuclear leukocytes.

Loxoprofen Sodium, a Non-Selective NSAID, Reduces Atherosclerosis in Mice by Reducing Inflammation

Recently, it is suggested that the use of nonsteroidal anti-inflammatory drugs (NSAID) may contribute to the occurrence of cardiovascular events, while the formation of atherosclerotic lesions is related to inflammation. Loxoprofen sodium, a non-selective NSAID, becomes active after metabolism in the body and inhibits the activation of cyclooxygenase. We fed apoE−/− mice a western diet from 8 to 16 weeks of age and administered loxoprofen sodium. We measured atherosclerotic lesions at the aortic root. We examined serum levels of cholesterol and triglycerides with HPLC, platelet aggregation, and urinary prostaglandin metabolites with enzyme immune assay. Atherosclerotic lesion formation was reduced to 63.5% and 41.5% as compared to the control in male and female apoE−/− mice treated with loxoprofen sodium respectively. Urinary metabolites of prostaglandin E2, F1α, and thromboxane B2, and platelet aggregation were decreased in mice treated with loxoprofen sodium. Serum levels of cholesterol and triglycerides were not changed. We conclude that loxoprofen sodium reduced the formation of early to intermediate atherosclerotic lesions at the proximal aorta in mice mediated by an anti-inflammatory effect.

Bone Marrow Angiotensin AT2 Receptor Deficiency Aggravates Atherosclerosis Development by Eliminating Macrophage Liver X Receptor-mediated Anti-atherogenic Actions

Background: Bone marrow (BM) Angiotensin II (Ang II) type 1 (AT1) receptor plays a crucial role in atherosclerosis development; however, the effect of BM Ang II type 2 (AT2) receptor on atherogenesis remains undefined. Methods and results: We generated BM chimera apoE-deficient (apoE−/−) mice whose BM cells were repopulated with AT2-deficient (Agtr2−/−) or wild-type (Agtr2+/+) cells. After 2 months of a high-cholesterol diet, the atherosclerotic lesion area was significantly increased in the apoE−/−/BM-Agtr2−/− mice compared with the apoE−/−/BM-Agtr2+/+ mice (51%, P < 0.05), accompanied by an augmented accumulation of lesion macrophages. Although phenotypic polarization in BM-derived macrophages and lipopolysaccharide-induced expression of proinflammatory cytokines in thioglycollate-induced peritoneal macrophages (TGPMs) were not affected by AT2-deficiency, mRNA and protein expression levels of macrophage liver X receptor β (LXRβ) were significantly decreased in Agtr2−/− TGPMs compared with Agtr2+/+ TGPMs. Anti-inflammatory effects of LXR agonist (GW3965) were markedly inhibited in Agtr2−/− TGPMs. Furthermore, the expression levels of ATP-binding cassette transporter ABCA1 and CCR7 were much lower in Agtr2−/− TGPMs than Agtr2+/+ TGPMs, accompanied by a significantly reduced cholesterol efflux. Conclusions: Our findings demonstrate that BM-AT2 deficiency aggravates atherosclerosis, at least in part, by eliminating the anti-atherogenic properties of macrophages elicited by LXRβ activation.