Stimulation of cyclic GMP production via AT2 and B2 receptors in the pressure-overloaded aorta after banding

Abdominal aortic banding induces upregulation of the angiotensin II (Ang II) type-2 (AT2) receptor, thereby decreasing the contractile response to Ang II in the thoracic aorta of the rat. The aim of this study was to use a mouse model to clarify the mechanisms by which the banding elicits upregulation of the aortic AT2 receptor and the subsequent attenuation of Ang II responsiveness. Concomitantly with the elevation in blood pressure and plasma renin concentration after banding, AT2-receptor mRNA levels in the thoracic aorta rapidly increased in mice within 4 days. Upregulation of the AT2 receptor, as well as blood pressure elevation after banding, was abolished by losartan administration. The contractile response to Ang II was depressed in aortic rings of banding mice but not of sham mice, and was restored by either the AT2-receptor antagonist PD123319 or the bradykinin B2-receptor antagonist icatibant. cGMP content in the thoracic aorta of banding mice was 9-fold greater than that of sham mice, and the elevation was reduced to sham levels 1 hour after intravenous injection of PD123319 or icatibant. When aortic rings were incubated with Ang II, cGMP content increased in banding rings but not in sham rings; the pretreatment with PD123319 or icatibant inhibited Ang II-induced cGMP production. These results suggest that aortic banding induces upregulation of the AT2 receptor through increased circulating Ang II via the AT1 receptor, thereby activating a vasodilatory pathway in vessels through the AT2 receptor via the kinin/cGMP system.     

Angiotensin II stimulates endothelial NO synthase phosphorylation in thoracic aorta of mice with abdominal aortic banding via type 2 receptor

Abdominal aortic banding in mice induces upregulation of angiotensin II (Ang II) type 2 (AT2) receptors in the pressure-overloaded thoracic aorta. To clarify mechanisms underlying the vascular AT2 receptor-dependent NO production, we measured aortic levels of endothelial NO synthase (eNOS), eNOS phosphorylated at Ser633 and Ser1177, protein kinase B (Akt), and Akt phosphorylated at Ser473 in thoracic aortas of mice after banding. Total eNOS, both forms of phosphorylated eNOS, Akt, and phosphorylated Akt levels, as well as cGMP contents, were significantly increased 4 days after banding. The administration of PD123319 (an AT2 receptor antagonist) or icatibant (a bradykinin B2 receptor antagonist) abolished the banding-induced upregulation of both forms of phosphorylated eNOS, as well as elevation of cGMP, but did not affect the upregulation of eNOS, Akt, and phosphorylated Akt. In the in vitro experiments using aortic rings prepared from banded mice, Ang II produced significant increases in both forms of phosphorylated eNOS, as well as cGMP, and these effects were blocked by PD123319 and icatibant. Ang II-induced eNOS phosphorylation and cGMP elevation in aortic rings were inhibited by protein kinase A (PKA) inhibitors H89 and KT5720 but not by phosphatidylinositol 3-kinase inhibitors wortmannin and LY24002. The contractile response to Ang II was attenuated in aortic rings from banded mice via AT2 receptor, and this attenuation was blocked by PKA inhibitors. These results suggest that the activation of AT2 receptor by Ang II induces phosphorylation of eNOS at Ser633 and Ser1177 via a PKA-mediated signaling pathway, resulting in sustained activation of eNOS.     

Blood pressure is the major driving force for plaque formation in aortic-constricted ApoE-/- mice

OBJECTIVE: Using an aortic constriction model in mice, we studied whether the increase in pressure or the activation of the renin-angiotensin system (RAS) and its main receptors is the main driving force for plaque progression. METHODS: Male ApoE mice underwent sham surgery or placement of a suprarenal silver clip around the aorta (AoC). Half the group was treated with the selective AT1 receptor antagonist losartan (30 mg/kg per day) for 4 weeks. RESULTS: Anesthetized mean arterial pressure (MAP) was increased in AoC mice compared to sham (106 +/- 3 versus 90 +/- 1 mmHg, P < 0.001). Losartan reduced MAP in sham mice (78 +/- 2 mmHg, P < 0.01) but not in AoC (AoC losartan 104 +/- 2 mmHg). Plasma renin concentration (PRC) was increased in AoC mice compared to sham [1.6 +/- 0.3 versus 0.8 +/- 0.2 milliGoldblatt units (mGU)/ml, P < 0.001]. Losartan treatment augmented this difference (18.7 +/- 3.7 versus 4.6 +/- 1.7 mGU/ml, P < 0.01). AT2 receptor mRNA expression was increased 5.8-fold by aortic constriction in thoracic aorta (P < 0.05) and the major site for expression of the AT2 receptor protein was within the plaques. The plaque area was increased in AoC mice compared to sham (0.61 +/- 0.09 versus 0.07 +/- 0.01%, P < 0.001); however, losartan did not alter plaque area. CONCLUSIONS: Our data do not support a role for the AT1 receptor in the progression of atherosclerosis in this model, since blockade with losartan did not alter plaque distribution. Furthermore, we found no support for the counteraction of atherogenesis by increased activity of the RAS acting on the AT2 receptor. Our data suggest that increased pressure is the main driving force for atherosclerosis in this model.     

Stimulation of the AT2 receptor reduced atherogenesis in ApoE(-/-)/AT1A(-/-) double knock out mice

AT1 receptor blockers (ARB) and in part ACE inhibitors (ACI) potentially exert beneficial effects on atherogenesis independent of AT1 receptor inhibition. These pleiotropic effects might be related to angiotensin II mediated activation of the AT2 receptor. To analyze this hypothesis we investigated the development of atherosclerosis and the role of ACIs and ARBs in apolipoprotein E-deficient (ApoE(-/-)) mice and in ApoE/AT1A receptor double knockout mice (ApoE(-/-)/AT1A(-/-)). ApoE(-/-) mice and ApoE(-/-)/AT1A(-/-) mice were fed cholesterol-rich diet for 7 weeks. Vascular oxidative stress, endothelial dysfunction, and atherosclerotic lesion formation were evident in ApoE(-/-) mice, but were markedly reduced in ApoE(-/-)/AT1A(-/-) mice. Concomitant treatment of ApoE(-/-)/AT1A(-/-) mice with either telmisartan or ramipril had no additional effect on blood pressure, vascular oxidative stress, AT2 receptor expression, and endothelial function. Remarkably, atherosclerotic lesion formation was increased in ramipril treated ApoE(-/-)/AT1A(-/-) mice compared to untreated ApoE(-/-)/AT1A(-/-) mice whereas pharmacological AT1 receptor inhibition with telmisartan had no additional effect on atherogenesis. Moreover, chronic AT2 receptor inhibition with PD123,319 significantly increased plaque development in ApoE(-/-)/AT1A(-/-) mice. In additional experiments, direct AT2 receptor stimulation reduced atherogenesis in ApoE(-/-)/AT1A(-/-) mice. Taken together, our data demonstrate a relevant antiatherosclerotic role of the AT2 receptor in atherosclerotic mice and provide novel insight in RAS-physiology.     

Repeated exposure to stressors do not accelerate atherosclerosis in ApoE-/- mice

Psychosocial stress is suggested to play a significant role in development of cardiovascular disease. To evaluate the effects of repeated exposure to stress on atherosclerosis in atherosclerosis-prone ApoE(-/-) mice we used five different stressors. We further sought to determine whether stress combined with high salt diet induces dysfunctional neurohormonal regulation and impaired salt excretion, thus amplifying the atherogenic potential of salt. The five stressors were evaluated in male C57BL/6 mice and ApoE(-/-) mice (studies I and II) and then used in female ApoE(-/-) mice to study their effect on atherosclerosis (study III). The mice in study III received standard or high salt diet (8%) alone or in combination with stress for 12 weeks. Urine and plasma were collected for corticosterone and lipid analysis, respectively. Acute blood pressure (BP) and heart rate (HR) responses to stress were measured using telemetry. Plaque burden was assessed in the thoracic aorta and aortic root. Plaque morphology was investigated regarding macrophages and collagen content. Urinary corticosterone chronically increased in stressed mice (P<0.05 control vs. stress, P<0.05 control salt vs. stress salt). BP and HR increased acutely during all stressors (P<0.05). Body weight gain decreased significantly in the stress group (P<0.05 vs. control). However, stress did not alter plasma lipid levels, plaque area or plaque morphology. Increased BP and HR suggest an acute stress-related response in ApoE(-/-) mice. Furthermore, stress chronically decreased body weight gain and increased urinary corticosterone levels. Notably, despite an apparent stress effect, stress affected neither atherogenesis nor plaque morphology.     

Angiotensin type 1 receptor blocker reduces intimal neovascularization and plaque growth in apolipoprotein E-deficient mice

The interactions between the renin-angiotensin system and neovascularization in atherosclerotic plaque development are unclear. We investigated the effects of angiotensin II type 1 receptor antagonism in the pathogenesis of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice with a special focus on plaque neovascularization. ApoE(-/-) mice fed a high-fat diet were randomly assigned to 1 of 2 groups and administered vehicle or olmesartan for 12 weeks. Quantification of plaque areas at the aortic root and in the thoracic and abdominal aorta revealed that, in all 3 of the regions, olmesartan reduced intimal neovessel density and the mRNA levels of toll-like receptor (TLR) 2 and TLR4. Olmesartan increased the levels of collagen and elastin, reduced the level of macrophages in the aortic root, and reduced the mRNA and the activity of matrix metalloproteinase (MMP) 2 in aortic roots and thoracic aortas. Aortic ring assay revealed that olmesartan-treated ApoE(-/-) mice had a markedly lower angiogenic response than that of untreated ApoE(-/-) mice. Bone marrow-derived endothelial progenitor cell-like c-Kit(+) cells from olmesartan-treated ApoE(-/-) mice showed marked impairment of cellular functions and lower expression of TLR2/TLR4 and MMP-2 compared with those of untreated controls. MMP-2 deficiency reduced intimal neovessel density and atherosclerotic lesion formation. Olmesartan and small-interfering RNA targeting TLR2 reduced the levels of TLR2, and MMP-2 mRNA induced angiotensin II in cultured endothelial cells. Angiotensin II type 1 receptor antagonism appears to inhibit intimal neovascularization in ApoE(-/-) mice, partly by reducing TLR2/TLR4-mediated inflammatory action and MMP activation, thus decreasing atherosclerotic plaque growth and increasing plaque instability.     

Induction of angiotensin II subtype 2 receptor-mediated blood pressure regulation in synthetic diet-fed rats

OBJECTIVE: Chronic feeding of a purified synthetic diet induces renin-angiotensin system-dependent moderate high blood pressure in normal Sprague-Dawley rats. The present study was designed to characterize the angiotensin II (Ang II) receptor type 2 (AT2)-specific mechanism of blood pressure regulation in these rats. METHODS: The effect of the AT2 receptor antagonist PD123319 (PD) on blood pressure was examined in vivo in synthetic diet-fed rats. Ang II-dependent contraction of aortic rings prepared from the synthetic diet-fed rats was also investigated. RESULTS: After 8 weeks of feeding the synthetic diet, the mean arterial pressure (MAP) was significantly elevated above levels measured in control rats (117 +/- 2 versus 102 +/- 3 mmHg, P < 0.05). Intravenous administration of PD to conscious hypertensive rats elicited an immediate dose-dependent increase in MAP that was sustained for approximately 7.4 min with 3 mg/kg PD. The angiotensin converting enzyme inhibitor captopril, but not the Ang II type 1 receptor blocker losartan, significantly attenuated the effect of PD on blood pressure. PD did not increase the plasma level of catecholamines. The PD-dependent blood pressure increase was not observed in normotensive control rats. Aortic ring assays revealed that functional activation of the AT2 receptor occurs only in the hypertensive rats, and this AT2 response is abolished by indomethacin (5 micromol/l) but not by Nomega-nitro-L-arginine methyl ester (100 Fmol/l). CONCLUSION: These results clearly demonstrate that AT2 receptor-mediated blood pressure regulation is functional in this experimental model of hypertension. Furthermore, cyclooxygenase metabolites might be the key mediators for the AT2 receptor-mediated blood pressure-lowering action.     

Angiotensin type 2 receptor-mediated phosphorylation of eNOS in the aortas of mice with 2-kidney, 1-clip hypertension

To evaluate the role of vascular angiotensin II (Ang II) type 2 (AT2) receptor in renovascular hypertension, we investigated expressions of AT2 receptor and endothelial nitric oxide synthase (eNOS) in thoracic aortas of mice with 2-kidney, 1-clip (2K1C) hypertension. The mRNA levels of AT2 receptor in aortas, but not those of AT1 and bradykinin B2 receptors, increased 14 days but not 42 days after clipping. The contractile response to Ang II (>0.1 micromol/L) was attenuated in aortic rings excised 14 days after clipping and was restored to that of rings from sham mice by antagonists of AT2 receptor (PD123319) and B2 receptor (icatibant). The aortic levels of total eNOS, phosphorylated eNOS at Ser1177 (p-eNOS), total Akt, and phosphorylated Akt at Ser473 (p-Akt) were increased in 2K1C mice on day 14, whereas only eNOS levels were increased on day 42. The aortic cGMP levels were 20-fold greater in 2K1C mice on day 14 compared with sham mice. Administration of nicardipine for 4 days before the excision of aortas 14 days after clipping not only reduced blood pressure but also decreased the aortic levels of eNOS, p-eNOS, Akt, p-Akt, and cGMP to sham levels, whereas the administration of PD123319 or icatibant to 2K1C mice decreased p-eNOS and cGMP to sham levels without affecting blood pressure and the levels of eNOS, Akt and p-Akt. These results suggest that vascular NO production is enhanced by increased eNOS phosphorylation via the activation of AT2 receptors in the course of 2K1C hypertension.     

Role of bradykinin, nitric oxide, and angiotensin II type 2 receptor in imidapril-induced angiogenesis

The angiotensin II (Ang II)-Ang II type 1 receptor pathway is proangiogenic, whereas studies showed that some angiotensin-converting enzyme inhibitors also stimulate angiogenesis in the setting of tissue ischemia, leaving a controversy of Ang II-mediated angiogenesis. We investigated whether an angiotensin-converting enzyme inhibitor imidapril-induced angiogenesis might be mediated via the tissue bradykinin pathway. To rule out the conventional effects of Ang II on angiogenesis, we used Ang II type 1a receptor knockout (AT1aKO) mice. We examined the effects of the angiotensin-converting enzyme inhibitor imidapril on angiogenesis in a hindlimb ischemia model using AT1aKO mice. After induction of hindlimb ischemia, AT1aKO mice were treated with or without imidapril (1.0 or 0.1 mg/kg per day for 21 days). Angiogenesis was quantified by laser Doppler blood flowmetry and capillary density. Angiogenesis was reduced in AT1aKO mice compared with wild-type mice. Imidapril with either low or high doses enhanced angiogenesis in AT1aKO mice (P<0.01). Ang II type 2 receptor antagonist (PD123319; 30 mg/kg per day) and B1 receptor antagonist (DesArg9-[Leu8]-bradykinin; 50 nmol/kg per day) suppressed the imidapril-induced angiogenesis in AT1aKO mice to an extent even lower than that of nontreated AT1aKO mice. B2 receptor antagonist (Hoechst 140; 100 microg/kg/d) and NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester; 20 mg/kg per day) moderately attenuated the imidapril-mediated angiogenesis. RT-PCR revealed that vascular endothelial growth factor receptor 2 mRNA was reduced with PD123319, DesArg9-[Leu8]-bradykinin, or Hoechst 140, and vascular endothelial growth factor mRNA abundance was suppressed with PD123319 or DesArg9-[Leu8]-bradykinin. In conclusion, imidapril elicited angiogenesis in the setting of tissue ischemia in AT1aKO mice. This angiogenic effect might involve the Ang II-Ang II type 2 receptor pathway in addition to the bradykinin-B1 and bradykinin-B2 receptor/NO-dependent pathways.     

Losartan reduces the vasorelaxant effect of atrial natriuretic peptide on basal tone in aorta

The Ca2+ -and receptor-dependencies of the basal tone seen in angiotensin II (Ang II)-conditioned rabbit thoracic aortic rings were investigated. Ca2+ -free Krebs significantly and partially reversibly reduced basal tone in aortic rings that had recovered from an earlier challenge with Ang II; rings not previously exposed to Ang II were unaffected. The effect of Ca2+ -free Krebs was similar to the reduction in basal tone evoked by atrial natriuretic peptide (ANP), but was smaller than that seen with exposure to Ca2+ -free Krebs+EGTA+sodium nitroprusside (SNP). Pretreating rings with Ca2+ free Krebs blocked the vasorelaxant effects of ANP and Ca2+ -free Krebs+EGTA+SNP. Losartan, an AT1 receptor antagonist, significantly attenuated ANP-induced relaxation, but did not otherwise alter basal tension in either unstimulated or Ang II-conditioned rings. The AT2 receptor antagonist, PD 123319, had no effect. These data suggest that transient exposure to Ang II induces prolonged, AT1-dependent increases in intracellular free Ca2+ which are antagonized by ANP.     

Role of angiotensin receptor subtypes in mesenteric vascular proliferation and hypertrophy.

The aim of this study was to explore the regulation of angiotensin receptors after chronic infusion with angiotensin II (Ang II) and to clarify the relative roles of the angiotensin type 1 (AT(1)) and type 2 (AT(2)) receptors in the mediation of Ang II-induced mesenteric vascular hypertrophy. In male Sprague-Dawley rats, Ang II infusion at a dose of 58.3 ng/min by subcutaneous osmotic minipumps for 14 days led to increased mesenteric weight and wall:lumen ratio of the vessels and proliferation of smooth muscle cells. These vascular changes were attenuated by either valsartan, an AT(1) receptor antagonist, at a dose of 30 mg. kg(-1). d(-1) by gavage, or PD123319, an AT(2) receptor antagonist, at a dose of 830 ng/min by intraperitoneally implanted osmotic minipumps. Ang II infusion was associated with hypertension, which was prevented by valsartan, but not PD123319. (125)I-Sar(1), Ile(8) Ang II binding to mesenteric vasculature was increased after Ang II infusion. Valsartan treatment was associated with reduced Ang II binding to both receptor subtypes, whereas PD123319 was associated with reduced Ang II binding to only the AT(2) receptor subtype. These findings suggest that the trophic and proliferative effects of Ang II on the mesenteric vasculature are mediated by both AT(1) and AT(2) receptors.     

Angiotensin III stimulates aldosterone secretion from adrenal gland partially via angiotensin II type 2 receptor but not angiotensin II type 1 receptor

Angiotensin II (Ang II) and Ang III stimulate aldosterone secretion by adrenal glomerulosa, but the angiotensin receptor subtypes involved and the effects of Ang IV and Ang (1-7) are not clear. In vitro, different angiotensins were added to rat adrenal glomerulosa, and aldosterone concentration in the medium was measured. Ang II-induced aldosterone release was blocked (30.3 ± 7.1%) by an Ang II type 2 receptor (AT2R) antagonist, PD123319. Candesartan, an Ang II type 1 receptor (AT1R) antagonist, also blocked Ang II-induced aldosterone release (42.9 ± 4.8%). Coadministration of candesartan and PD123319 almost abolished the Ang II-induced aldosterone release. A selective AT2R agonist, CGP42112, was used to confirm the effects of AT2R. CGP42112 increased aldosterone secretion, which was almost completely inhibited by PD123319. In addition to Ang II, Ang III also induced aldosterone release, which was not blocked by candesartan. However, PD123319 blocked 22.4 ± 10.5% of the Ang III-induced aldosterone secretion. Ang IV and Ang (1-7) did not induce adrenal aldosterone secretion. In vivo, both Ang II and Ang III infusion increased plasma aldosterone concentration, but only Ang II elevated blood pressure. Ang IV and Ang (1-7) infusion did not affect blood pressure or aldosterone concentration. In conclusion, this report showed for the first time that AT2R partially mediates Ang III-induced aldosterone release, but not AT1R. Also, over 60% of Ang III-induced aldosterone release may be independent of both AT1R and AT2R. Ang III and AT2R signaling may have a role in the pathophysiology of aldosterone breakthrough.     

Regulation of interleukin-8 expression by HMG-CoA reductase inhibitors in human vascular smooth muscle cells

Interleukin-8 (IL-8) is a potent chemotactic factor that has been implicated in atherogenesis. HMG-CoA reductase inhibitors (statins) may reduce the cardiovascular risk and vulnerability of atherosclerotic plaque through nonlipid mechanisms such as inhibition of cytokine expression. In this study, we investigated the effects of statins on IL-8 synthesis in human vascular smooth muscle cells (VSMCs). Addition of angiotensin II (Ang II) increased IL-8 production in VSMCs in a time (0-24 h)- and dose (10(-8)-10(-6) mol/l)-dependent manner with increased IL-8 mRNA accumulation. The Ang II type 1 receptor (AT1R) antagonist candesartan, but not the Ang II type 2 receptor (AT2R) antagonist PD123319, significantly blocked Ang II-induced IL-8 production. Addition of fluvastatin decreased the basal and Ang II-induced IL-8 production in VSMCs in a dose (10(-8)-10(-5) mol/l)-dependent manner with a decrease in IL-8 mRNA accumulation. The effect of fluvastatin on IL-8 production was completely reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate, but not in the presence of squalene or farnesyl-pyrophosphate. Lipophilic cerivastatin also significantly decreased IL-8 production, while hydrophilic pravastatin showed no effect on IL-8 levels. In conclusion, we demonstrated for the first time that Ang II increased IL-8 production and fluvastatin decreased the basal and Ang II-induced IL-8 production in human VSMCs. These findings suggested that Ang II may exacerbate atherosclerosis through induction of IL-8 in VSMCs, while statins may exert therapeutic effects by modulating IL-8 synthesis in patients with atherosclerotic disease.     

Effects of valsartan on angiotensin II-induced migration of human coronary artery smooth muscle cells.

The migration as well as proliferation of coronary artery medial smooth muscle cells (SMC) into the intima is proposed to be an important process of intimal thickening in coronary atherosclerosis. In the current study, we examined the effects of the angiotensin type 1 receptor antagonist valsartan on angiotensin II (Ang II)-induced migration of cultured human coronary artery SMC using Boyden's chamber methods. Ang II significantly stimulated human coronary artery SMC migration in a concentration-dependent manner between 10(-6) and 10(-8) mol/l when cells of passage 4 to 6 were used. However, the migration response to Ang II was moderately decreased in cells of passage 10 to 12, and was markedly decreased in cells of passage 15 to 17, compared to that of passage 4 to 6. Ang II-induced migration was blocked by the Ang II type 1 (AT1) receptor antagonist valsartan in a concentration-dependent manner. By contrast, the Ang II type 2 (AT2) receptor antagonist PD 123319 did not affect Ang II-induced migration. Ang II modestly increased the cell number of human coronary artery SMC after a 24-h incubation. This increase in cell numbers was also clearly blocked by valsartan, but not by PD 123319. These results indicate that Ang II stimulates migration as well as proliferation via AT1 receptors in human coronary artery SMC when cells of passage 4 to 6 are used. Valsartan may prevent the progression of coronary atherosclerosis through an inhibition of Ang II-induced migration and proliferation in these cells, although in vivo evidence is lacking.     

Atheroprotection via cannabinoid receptor-2 is mediated by circulating and vascular cells in vivo

Low-dose oral tetrahydrocannabinol (THC) reduces progression of atherosclerosis in mice. THC activates central cannabinoid-1 receptors (CB1) with subsequent psychoactive effects as well as peripheral cannabinoid-2 receptors (CB2). In order to dissect the underlying mechanisms, we performed experiments under selective CB2 stimulation as well as after genetic disruption of the CB2 receptor. Atherosclerosis prone apolipoprotein E-deficient mice were crossed with cannabinoid receptor-2 deficient mice to obtain ApoE −/− CB2 −/− double knockout mice. After 8 weeks of a high-cholesterol diet, immunohistochemical stainings of the aortic root revealed that vascular leukocyte infiltration in atherosclerotic plaques was accelerated in ApoE −/− CB2 −/− mice compared with ApoE −/− mice. This was accompanied by increased release of reactive oxygen species as measured using L012-enhanced chemiluminescence, and by decreased endothelial function as assessed in isolated aortic rings in organ chamber experiments. ApoE −/− mice treated with the selective CB2 agonist JWH 133 during a high-cholesterol diet showed decreased atherosclerotic lesion formation, improved endothelial function and reduced levels of reactive oxygen species. To assess whether CB2 expression in circulating cells influences atherosclerosis, irradiated ApoE −/− mice were repopulated with bone marrow-derived cells from ApoE −/− and ApoE −/− CB2 −/− mice and were fed a high-cholesterol diet for 8 weeks. CB2 deficiency in bone marrow-derived cells increased leukocyte infiltration into the vessel wall, but had no impact on plaque formation. Cell culture experiments revealed that CB2 activation diminishes ROS generation in vascular cells. Selective CB2 receptor stimulation modulates atherogenesis via impact on both circulating proinflammatory and vascular cells.     

Angiotensin II atherogenicity in apolipoprotein E deficient mice is associated with increased cellular cholesterol biosynthesis.

Angiotensin II (Ang II) was shown to be an important risk factor for accelerated atherosclerosis. Inhibition of Ang II action on the arterial wall by blocking its production with angiotensin converting enzyme (ACE) inhibitors, or by blocking binding to its receptors on cells with antagonists was shown to attenuate atherogenesis in animal model of atherosclerosis. We questioned whether Ang II atherogenicity is related to a stimulatory effect of Ang II on macrophage cholesterol biosynthesis. Angiotensin II injected intraperitoneally once a day (0.1 ml of 10(-7) M per mouse) for a period of 30 days, to the apolipoprotein E deficient mice increased the atherosclerotic lesion area by 95% (P < 0.01 vs. control), compared to placebo-injected mice, with no significant effect on blood pressure or on plasma cholesterol levels. On using mouse peritoneal macrophages (MPMs) that were harvested after intraperitoneally injection of Ang II, an increased rate of cellular cholesterol biosynthesis (measured as incorporation of [3H]acetate into cholesterol) by up to 90% (P < 0.01 vs. control) was observed. In mice treated with the ACE inhibitor, Fosinopril (25 mg/kg per day) a reduction in their MPM's cholesterol synthesis by up to 70% (P < 0.01 vs. control) was obtained. In vitro studies in human monocyte-derived macrophages (HMDM), in MPMs from control BALB/c mice, and in J-774 A.1 macrophage-like cell line demonstrated up to 44, 34 and 30% stimulation of macrophage cholesterol biosynthesis, respectively, following cell incubation with 10(-7) M Ang II for 18 h at 37 degrees C. The stimulatory effect of Ang II on macrophage cholesterol biosynthesis could be related to its interaction with the macrophage AT1 receptor, as Losartan (10(-5) M), an AT1 blocker, but not PD 123319 (10(-5) M), an AT2 blocker, prevented the stimulatory effect on macrophage cholesterol synthesis. Furthermore, in cells that lack the AT1 receptor (RAW macrophages), Ang II did not increase cellular cholesterol synthesis. Ang II increased macrophage 3-hydroxy-3-methyl glutaryl CoA (HMG CoA) reductase mRNA levels in a dose dependent manner in J-774 A.1 macrophages and in MPM. Losartan, the AT1 receptor antagonist clearly attenuated this mRNA induction. We thus conclude that Ang II stimulation of macrophage cholesterol biosynthesis is related to its interaction with the AT1 receptor, followed by stimulation of macrophage HMG CoA reductase gene expression, which leads to increased cellular cholesterol biosynthesis, and can possibly result in macrophage cholesterol accumulation and foam cell formation.     

Bone marrow-derived monocyte chemoattractant protein-1 receptor CCR2 is critical in angiotensin II-induced acceleration of atherosclerosis and aneurysm formation in hypercholesterolemic mice

Angiotensin II (Ang II) is implicated in atherogenesis by activating inflammatory responses in arterial wall cells. Ang II accelerates the atherosclerotic process in hyperlipidemic apoE-/- mice by recruiting and activating monocytes. Monocyte chemoattractant protein-1 (MCP-1) controls monocyte-mediated inflammation through its receptor, CCR2. The roles of leukocyte-derived CCR2 in the Ang II-induced acceleration of the atherosclerotic process, however, are not known. We hypothesized that deficiency of leukocyte-derived CCR2 suppresses Ang II-induced atherosclerosis. METHODS AND RESULTS: A bone marrow transplantation technique (BMT) was used to develop apoE-/- mice with and without deficiency of CCR2 in leukocytes (BMT-apoE-/-CCR2+/+ and BMT-apoE-/-CCR2-/- mice). Compared with BMT-apoE-/-CCR2+/+ mice, Ang II-induced increases in atherosclerosis plaque size and abdominal aortic aneurysm formation were suppressed in BMT-apoE-/-CCR2-/- mice. This suppression was associated with a marked decrease in monocyte-mediated inflammation and inflammatory cytokine expression. CONCLUSIONS: Leukocyte-derived CCR2 is critical in Ang II-induced atherosclerosis and abdominal aneurysm formation. The present data suggest that vascular inflammation mediated by CCR2 in leukocytes is a reasonable target of therapy for treatment of atherosclerosis.     

Regulation of collagen synthesis in mouse skin fibroblasts by distinct angiotensin II receptor subtypes

We examined the possibility of whether angiotensin (Ang) II type 1 (AT1) and type 2 (AT2) receptor stimulation differentially regulates collagen production in mouse skin fibroblasts. Both AT1 and AT2 receptors were expressed in neonatal skin fibroblasts prepared from wild-type mice to a similar degree, and the AT1a receptor was exclusively expressed as opposed to the AT1b receptor. In wild-type fibroblasts, Ang II increased collagen synthesis accompanied by an increase in expression of tissue inhibitor of metalloproteinase (TIMP)-1, and these increases were inhibited by valsartan, an AT1 receptor blocker, but augmented by PD123319, an AT2 receptor antagonist. Ang II decreased basal and IGF-I-induced collagen production and inhibited TIMP-1 expression in neonatal skin fibroblasts prepared from AT1a knockout (KO) mice. These Ang II-mediated inhibitory effects on collagen production and TIMP-1 expression observed in AT1a KO fibroblasts were attenuated by the addition of PD123319 or a tyrosine phosphatase inhibitor, sodium orthovanadate, but not affected by a serine/threonine phosphatase inhibitor, okadaic acid. Moreover, we demonstrated that transfection of a catalytically inactive, dominant negative SHP-1 (Src homology 2-containing protein-tyrosine phosphatase-1) mutant inhibited the Ang II-mediated inhibitory effect on both collagen synthesis and TIMP-1 expression in AT1a KO fibroblasts. These results suggest that AT1a receptor stimulation increases collagen production in skin fibroblasts at least in part due to the inhibition of collagen degradation via the increase in TIMP-1 expression, whereas AT2 receptor stimulation exerts inhibitory effects on TIMP-1 expression, which is mediated at least partially by the activation of SHP-1, thereby possibly inhibiting collagen production.     

Angiotensin II stimulates DNA and protein synthesis in vascular smooth muscle cells from human arteries: role of extracellular signal-regulated kinases.

OBJECTIVE: This study investigates the growth effects and associated signaling pathways of angiotensin II (Ang II) in human vascular smooth muscle cells. METHODS: Cultured vascular smooth muscle cells derived from resistance arteries (< 300 microm diameter) from subcutaneous gluteal biopsies of healthy subjects (n = 6) and human aortic vascular smooth muscle cells were used. Cells were studied between passages 3 and 6. Both 3H-thymidine and 3H-leucine incorporation were measured as indices of vascular smooth muscle cell hyperplasia (DNA synthesis) and cell hypertrophy (protein synthesis), respectively. Growth effects of Ang II (10(-12) - 10(-6) mol/l), in the absence and presence of 10(-5) mol/l losartan (AT1 antagonist) and PD123319 (AT2 antagonist), were determined. Ang II-induced effects were compared to those of endothelin-1. To determine whether extracellular signal-regulated kinase (ERK)-dependent pathways play a role in Ang II-mediated growth, cells were pretreated with the selective ERK kinase (MEK) inhibitor, PD98059 (10(-5) mol/l). ERK activation was determined by Western blot in the absence and presence of PD98059. RESULTS: Ang II dose-dependently increased 3H-thymidine incorporation in cells from aorta (Emax = 276 +/- 10.4% of control) and resistance arteries (Emax = 284 +/- 5.1% of control). Ang II also stimulated 3H-leucine incorporation in cells from aorta (Emax = 162 +/- 11.6 of control) and resistance arteries (Emax 175 +/- 10% of control). Unlike Ang II, endothelin-1 failed to significantly alter cellular growth, except at high concentrations (> 10(-7) mol/l), where it had a weak stimulatory effect Losartan, but not PD123319, blocked Ang II-stimulated growth responses. Ang II significantly increased phosphorylation of ERK-1 and ERK-2, with maximum responses obtained at 5 min. PD98059 inhibited Ang II-stimulated ERK activity and abrogated agonist-induced DNA and protein synthesis. Losartan, but not PD123319 inhibited Ang II-induced phosphorylation of ERK-1 and ERK-2. CONCLUSIONS: Ang II stimulates both hyperplasia and hypertrophy in vascular smooth muscle cells from human arteries. These growth effects are mediated via Ang II receptors of the AT1 subtype that are linked to ERK-dependent signaling pathways.