Hiroyuki Kawahito

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.

Maternal High-Fat Diet Exaggerates Atherosclerosis in Adult Offspring by Augmenting Periaortic Adipose Tissue-Specific Proinflammatory Response

Objective— Maternal obesity elicits offspring’s metabolic disorders via developmental modifications of visceral adipose tissue; however, its effect on atherogenesis remains undefined. Perivascular adipose tissue has recently been implicated in vascular remodeling and vasoreactivity. We hypothesize that developmental modifications of perivascular adipose tissue by maternal high-fat diet (HFD) exposure promotes atherosclerosis in adult offspring. Approach and Results— Eight-week-old female apolipoprotein E-deficient mice were fed an HFD or normal diet (ND) during gestation and lactation. Offspring were fed a high-cholesterol diet from 8 weeks of age. Twenty-week-old male offspring of HFD-fed dams (O-HFD) showed a 2.1-fold increase in atherosclerotic lesion of the entire aorta compared with those of ND-fed dams (O-ND). Although mRNA expressions of interleukin-6, tumor necrosis factor, and monocyte chemotactic protein-1 and accumulation of macrophages in epididymal white adipose tissue were less in O-HFD than in O-ND, thoracic periaortic adipose tissue (tPAT) showed an exaggerated inflammatory response in O-HFD. Intra-abdominal transplantation of tPAT from 8-week-old O-HFD alongside the distal abdominal aorta exaggerated atherosclerosis development of the infrarenal aorta in recipient apolipoprotein E-deficient mice compared with tPAT from O-ND (210%, P<0.01). Although macrophage accumulation was rarely detected in tPAT of 8-week-old offspring, mRNA expression and protein levels of macrophage colony–stimulating factor were markedly elevated in O-HFD (2.3-fold, 3.3-fold, respectively, P<0.05), suggesting that increased macrophage colony–stimulating factor expression contributes to the augmented accumulation of macrophages, followed by the enhanced proinflammatory response. Conclusions— Our findings demonstrate that maternal HFD exaggerates atherosclerosis development in offspring by augmenting tPAT-specific inflammatory response proceeded by an increased expression of macrophage colony–stimulating factor.

Dipeptidyl peptidase-4 inhibitor sitagliptin improves pancreatic β-cell function in hypertensive diabetic patients treated with angiotensin receptor blockers

Introduction: Dipeptidyl peptidase (DPP)-4 inhibitors, a novel oral anti-diabetic agents, exert a protective effect on pancreatic β-cell function in patients with type 2 diabetic mellitus (T2DM). However, their beneficial effect in hypertensive T2DM patients treated with angiotensin receptor blockers (ARBs) has not been investigated. Methods: In this open-label multicenter randomized study, a total of 55 hypertensive T2DM patients treated with ARBs were randomly assigned to receive the DPP-4 inhibitor sitagliptin or sulfonylurea (SU). Results: After 24 weeks of treatment, a significant reduction in fasting blood glucose was only observed in the sitagliptin group, while HbA1c was significantly reduced in both groups. Homeostasis model assessment of insulin resistance was not significantly improved in either group. Indicators of pancreatic β-cell function, including proinsulin to insulin ratio and homeostasis model assessment of β-cell function, were significantly improved in the sitagliptin group, but not in the SU group. The beneficial effects of sitagliptin were observed in hypoglycemic drug naïve patients, but not in patients who had received SU monotherapy prior to the study. Conclusion: Treatment with the DPP-4 inhibitor sitagliptin might exert beneficial effects on pancreatic β-cell function in ARB-treated T2DM patients and its efficacy might be more pronounced in hypoglycemic drug naïve patients.

Transplantation of periaortic adipose tissue from angiotensin receptor blocker-treated mice markedly ameliorates atherosclerosis development in apoE–/– mice

Background: Perivascular adipose tissue is implicated in vasoreactivity; however, its effect on atherosclerosis remains undefined. Methods and results: We examined the effect of a high-cholesterol diet (HCD) on phenotypic alterations of the thoracic periaortic adipose tissue (tPAT) in apoE-deficient (apoE–/–) mice. Gene expression of the components of the renin angiotensin system and that of macrophage markers were significantly higher in apoE–/– mice fed an HCD than in those fed a chow diet (CD). These changes were absent both in angiotensin II (AngII) receptor blocker (ARB)-treated apoE–/– mice and in Ang II type 1 (AT1) receptor-deficient apoE–/– (Agtr1–/–/apoE–/–) mice. To evaluate their effect on atherosclerosis, we transplanted tPAT into apoE–/– mice alongside the distal abdominal aorta. Transplanted tPAT was harvested from apoE–/– and Agtr1–/–/apoE–/– mice fed a CD (tPAT-CD/apoE–/–, tPAT-CD/Agtr1–/–/apoE–/–), HCD (tPAT-HCD/apoE–/–, tPAT-HCD/Agtr1–/–/apoE–/–), or HCD in combination with ARB treatment (tPAT-HCD/ARB/apoE–/–). Four weeks after transplantation, a significantly increased oil red O-positive area was observed in the aorta of tPAT-HCD/apoE–/– mice than in tPAT-CD/apoE–/– mice. Such a change was absent in tPAT-HCD/ARB/apoE–/– and tPAT-HCD/Agtr1–/–/apoE–/– mice. Conclusions: Our findings demonstrated that AT1 receptor plays a crucial role in HCD-induced phenotypic alterations of tPAT, modulation of which could exert beneficial effects on atherosclerosis.

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.

Transplantation of brown adipose tissue inhibits atherosclerosis in apoE−/− mice: contribution of the activated FGF-21-adiponectin axis

Aims Brown adipose tissue (BAT) has been identified as an endocrine organ that maintains metabolic homeostasis; however, the effects on atherosclerosis remain undefined. Here, we investigated the effect of experimental BAT transplantation on atherosclerosis. Methods and results Interscapular BAT was dissected from 12-week-old wild-type mice and transplanted into the visceral cavity of 12-week-old apoE-/- mice. Oil-red O staining of whole aortas after 3 months of a high-cholesterol diet showed a significant decrease in atherosclerotic lesion area in BAT-transplanted mice by 20% compared with the sham control mice. A significant increase in oxygen consumption and energy expenditure, concomitant improvement of glucose tolerance, and lower triglyceride levels were observed in BAT-transplanted mice; however, serum cholesterol levels showed no difference between the two groups. Homologous transplantation of BAT from apoE-/- mice also showed a significant decrease in atherosclerotic lesion area by 28% compared with the sham control apoE-/- mice without affecting lipid levels, while epidydimal white adipose tissue transplantation did not affect atherosclerosis. In the combination of wild-type donor and apoE-/- recipient mice, both mRNA and protein levels of fibroblast growth factor 21 (FGF-21) were increased significantly in endogenous BAT in BAT-transplanted mice (180 and 38%, respectively, P < 0.05), accompanied by a higher concentration of circulating FGF-21 and noradrenaline (47 and 45%, respectively, P < 0.05). Concomitantly, serum adiponectin levels were elevated in BAT-transplanted mice (35%, P < 0.05), and showed an inverse correlation with atherosclerotic lesion area (r = 0.44, P < 0.05). Treatment with the nonselective β3-adrenergic receptor (AR) blocker completely abolished the anti-atherogenic effect of BAT transplantation and reduced concentrations of circulating FGF-21 and adiponectin to levels comparable with that of vehicle-treated BAT-transplanted mice. Conclusions Our findings demonstrate for the first time that anti-atherogenic action of BAT transplantation is BAT-specific and independent of lipid-lowering effect, accompanied by AR-mediated activation of the FGF-21-adiponectin axis.

Adrenergic receptor-mediated activation of FGF-21-adiponectin axis exerts atheroprotective effects in brown adipose tissue-transplanted apoE−/− mice

Brown adipose tissue (BAT) has been found as an endocrine organ that maintains metabolic homeostasis; however, the effects on atherosclerosis remain undefined. Here, we investigated the effect of experimental BAT transplantation on atherosclerosis. Interscapular BAT was dissected from wild-type mice and transplanted into the visceral cavity of 12-week-old apoE-/- mice. Oil-red O staining of whole aortas after 3 months of a high-cholesterol diet showed a significant decrease in atherosclerotic lesion area in BAT-transplanted mice by 32% compared with the sham control mice. Lipid profiles, except for serum triglyceride level, showed no difference between the 2 groups. BAT-transplanted mice showed higher concentrations of serum noradrenalin, fibroblast growth factor 21 (FGF-21), and adiponectin. Treatment with the β3-adrenergic receptor (AR) blocker completely abrogated the atheroprotective effects of BAT transplantation, with serum concentrations of FGF-21 and adiponectin being equivalent between the 2 groups. Homologous transplantation of BAT from apoE-/- mice also showed a significant decrease in atherosclerotic lesion area by 28% without affecting lipid profiles, while epidydimal white adipose tissue transplantation did not affect atherosclerosis. Serum and endogenous BAT concentrations of FGF-21 were significantly higher in BAT-transplanted mice than sham control mice. Concomitantly, serum adiponectin levels were elevated in BAT-transplanted mice and showed a significant inverse correlation with atherosclerotic lesion area. Our findings show for the first time that atheroprotective effect of BAT transplantation is BAT-specific and independent of lipid-lowering effect, accompanied by AR-mediated activation of the FGF-21-adiponectin axis.