Celiprolol Activates eNOS Through the PI3K-Akt Pathway and Inhibits VCAM-1 Via NF-κB Induced by Oxidative Stress
Vascular cell adhesion molecule-1 (VCAM-1) and reactive oxygen species play critical roles in early atherogenesis, and nitric oxide (NO) is an important regulator of the cardiovascular system. Although celiprolol, a specific β1-antagonist with weak β2-agonistic action, stimulates endothelial nitric oxide synthase (eNOS) production, the mechanisms remain to be determined. Because it was recently reported that phosphatidylinositol 3-kinase (PI3K) and its downstream effector Akt are implicated in the activation of eNOS and that regulation of VCAM-1 expression is mediated via nuclear factor-κB (NF-κB), we hypothesized that celiprolol activates phosphorylation of eNOS through the PI3K-Akt signaling pathway; that celiprolol modulates VCAM-1 expression, which is associated with inhibiting NF-κB phosphorylation; and that celiprolol suppresses NAD(P)H oxidase p22phox, p47phox, gp91phox, and nox1 expression in the left ventricle of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. eNOS and Akt phosphorylation upregulated by celiprolol alone were suppressed by treatment with celiprolol plus wortmannin. Increased expression of VCAM-1, p22phox, p47phox, gp91phox, nox1, activated p65 NF-κB, c-Src, p44/p42 extracellular signal-regulated kinases, and their downstream effector p90 ribosomal S6 kinase phosphorylation in DOCA rats was inhibited by celiprolol. Celiprolol administration resulted in a significant improvement in cardiovascular remodeling and suppression of transforming growth factor-β1 gene expression. In conclusion, celiprolol suppresses VCAM-1 expression because of inhibition of oxidative stress, NF-κB, and signal transduction, while increasing eNOS via stimulation of the PI3K-Akt signaling pathway and improving cardiovascular remodeling.
- receptors, adrenergic, β
- adrenergic receptor blockers
- nitric oxide
- oxidative stress
- cell adhesion molecules
Leukocyte adhesion to the endothelium and infiltration into tissue have been found to contribute to the tissue damage and impairment of vascular perfusion in a broad array of systemic diseases, including atherosclerosis and hypertension.1 Localized accumulation of leukocytes is mediated by the endothelial expression of specific adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1, or platelet-endothelial cell adhesion molecule-1.2 VCAM-1 is an early marker of endothelial activation and dysfunction, leukocyte infiltration, and vascular remodeling, and a recent study demonstrated that VCAM-1 plays a key role in early atherogenesis.3 Endothelium-derived relaxing factor, nitric oxide (NO), is an important component of vascular homeostasis.4 Recently, some investigators5,6 have shown that the serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-OH kinase (PI3K), phosphorylates human endothelial NO synthase (eNOS) on serine 1177 in response to varied stimuli, such as growth factors and shear stress. Furthermore, expression of adhesion molecules, including VCAM-1, can be regulated by NO, and increased levels of NO are associated with decreased leukocyte adhesion molecule expression.7 The mechanisms by which NO modulates expression of VCAM-1 are unclear. However, regulation of VCAM-1 expression occurs at the transcriptional level and is in part mediated through the transcription factor nuclear factor-κB (NF-κB).8 Activated NF-κB is involved in the expression of several proinflammatory genes and is present in endothelial cells in the early lesions of atherosclerosis.9 In addition, several lines of evidence indicate that reactive oxygen species (ROS) are implicated in the activation of NF-κB. Enhanced production of ROS, most notably superoxide and hydrogen peroxide (H2O2), contributes to the dysregulation of physiologic processes, which leads to structural and functional alterations in hypertension.10 Angiotensin II (Ang II) stimulates the generation of ROS in smooth muscle cells (SMCs) by activation of NAD(P)H oxidases and might also be implicated in ROS production in endothelial cells.11
The phagocyte NAD(P)H oxidase is composed of a membrane-associated 22-kDa α-subunit (p22phox) and a 91-kDa β-subunit (gp91phox), with cytosolic components composed of p47phox, p67phox, and p40phox.12 Moreover, 2 gp91phox homologues, nox1 and nox4, are expressed at much higher levels than is gp91phox in SMCs, and nox1 is responsible for increased superoxide production, serum-induced mitogenesis, and activation of redox-sensitive signaling in vitro.13 Recently, some investigators and we have shown that in spontaneously hypertensive rats, NAD(P)H oxidase p22phox14 and p47phox15 expression was upregulated in the vasculature, which might contribute to endothelial dysfunction and vascular hypertrophy.
β-Adrenoceptor antagonists (β-blockers) are widely used as effective antihypertensive agents, and we have previously shown that celiprolol, a specific β1-antagonist with weak β2-agonist action, stimulates eNOS mRNA and protein expression and improves cardiovascular remodeling in hypertensive rats.16 Kakoki et al17 reported that celiprolol augmented endothelium vasodilation in rat aortas and that this action was attenuated by treatment with NAN-190, a 5-hydroxytryptamine1A (5-HT1A) antagonist. Noda et al18 also reported that celiprolol showed vasodilatory action by way of an NO-cGMP system in porcine coronary arteries by acting on other β-adrenoceptor sites in the endothelium. Thus, celiprolol might be expected to improve endothelial function. Clinically, NOS activity was increased and generation of superoxide anion was decreased by treatment with celiprolol in hypertensive patients.19 However, the signal mechanism and effects on VCAM-1 expression by which celiprolol stimulates eNOS phosphorylation are unclear. We hypothesized that in the left ventricle (LV) of deoxycorticosterone acetate (DOCA)-salt hypertensive rats, celiprolol (1) activates phosphorylation of eNOS through the PI3K-Akt signaling pathway; (2) modulates VCAM-1 expression, which is associated with inhibition of NF-κB phosphorylation; and (3) reduces production of ROS by suppressing NAD(P)H oxidase subunit p22phox, p47phox, gp91phox, and nox1 expression.
All procedures were performed in accordance with our institutional guidelines for animal research and with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Animal Models and Experimental Design
Twenty-eight 6-week-old, male, normotensive Wistar rats (Oriental Bioservice, Kanto Inc, Ibaraki, Japan) were used, and DOCA-salt hypertension was induced in 21 rats as described previously.16 Rats received weekly subcutaneous injections of DOCA (30 mg/kg) after right nephrectomy and were given 1% saline as drinking water (DOCA-V, n=7). Eight of the 14 remaining DOCA-salt rats were treated with celiprolol (Nippon Shinyaku Co, Ltd) in their drinking water for 5 weeks, and a fresh drug solution was prepared daily (DOCA-Ce, n=8). The drug was given at an average dose of 10 mg · kg−1 · d−1 (subpressor dose), which was adjusted weekly to the drinking habits of the animals.16 The remaining 6 DOCA-salt rats were treated with celiprolol plus wortmannin, a PI3K inhibitor (DOCA-Ce+Wo, n=6). An osmotic minipump (model 2ML4, Alzet Corp) containing wortmannin (1 mg · kg−1 · d−1) dissolved in 8% dimethyl sulfoxide was implanted. Age-matched, sham-operated rats (ShC, n=7) served as a control group.
All procedures used for mRNA extraction, cDNA synthesis, polymerase chain reaction (PCR), and quantification of PCR products were described in detail in our previous report.20 Levels of VCAM-1; eNOS; NAD(P)H oxidase p22phox, p47phox, gp91phox, and nox1; atrial natriuretic peptide (ANP); β-myosin heavy chain (β-MHC); type I collagen, transforming growth factor (TGF)-β1; and glyceraldehyde 3-phosphate dehydrogenase mRNA were measured as described previously.15,21
Western Blot Analysis
VCAM-1; eNOS; and NAD(P)H oxidase p22phox, p47phox, and gp91phox proteins were measured as described previously.16,22
Activity of eNOS, Akt, p65NF-κB, p60c-Src, ERK1/2, and p90RSK
eNOS, Akt, p65NF-κB, p60c-Src, p44/p42 extracellular signal-regulated kinase (ERK1/2), and p90 ribosomal S6 kinase (p90RSK) phosphorylation was measured as described in detail previously.23
Measurement of H2O2
H2O2 production in LV homogenates was measured with the 2′,7′-dichlorodihydrofluorescein-diacetate method, as described previously.15
Determination of NADPH Oxidase Activity
NADPH oxidase activity in the LV was assessed by the measurement of superoxide-enhanced lucigenin chemiluminescence, as described previously.24
Immunohistochemistry of eNOS
A total of 24 2-μm paraffin sections were processed for the avidin-biotin-horseradish peroxidase complex technique, as previously mentioned.22
Histologic examination was performed as described in detail previously.22,23
Effects of Propranolol, Another β-Blocker
To elucidate whether celiprolol is a special β-blocker with potency to increase eNOS production and improve cardiovascular remodeling, we performed a supplementary experiment to evaluate the effects of another β-blocker, propranolol, on eNOS mRNA and protein expression and cardiovascular remodeling in DOCA-salt hypertensive rats. Five DOCA-salt rats were treated with propranolol (DOCA-Pro, n=5) in their drinking water for 5 weeks. The drug was given at an average dose of 10 mg · kg−1 · d−1 (subdepressor dose).
All values are expressed as mean±SEM. Mean values were compared among the 3 or 4 groups by ANOVA and the Bonferroni post hoc test for multiple comparisons. A value of P<0.05 was considered statistically significant.
Physiologic Profiles After 5-Week Treatment With Celiprolol in DOCA-Salt Rats
Body weight (BW), systolic blood pressure (SBP), LV weight (LVW) to BW ratio, and heart rate in the 4 groups are presented in the Table. There were no differences in BW among the 4 groups. In contrast, DOCA-salt rats had a higher LVW/BW compared with ShC rats. Long-term celiprolol therapy in DOCA-salt rats significantly decreased LVW/BW. However, long-term therapy with celiprolol plus wortmannin in DOCA-salt rats significantly increased LVW/BW compared with celiprolol therapy alone. DOCA-salt rats had markedly higher SBP by the tail-cuff method than did ShC rats. Long-term celiprolol or celiprolol plus wortmannin therapy did not significantly affect SBP. There were no significant differences in heart rate among the 4 groups.
The morphological appearance of coronary arteries in the 4 groups are shown in Figures 1E through 1H and listed in the Table. The wall-to-lumen ratio was significantly increased in DOCA-salt rats compared with that in ShC animals. Long-term celiprolol treatment caused significant amelioration of this ratio. However, long-term celiprolol plus wortmannin therapy significantly increased the wall-to-lumen ratio. Perivascular fibrosis was significantly greater in DOCA-salt rats than in ShC rats. Long-term celiprolol treatment also caused significant improvement in perivascular fibrosis, but long-term therapy with celiprolol plus wortmannin significantly increased fibrosis.
eNOS and Akt Phosphorylation and eNOS Expression
Phosphorylation of eNOS and Akt in the LV was significantly lower in DOCA-salt rats than in ShC animals (Figures 2A and 2B). Long-term celiprolol administration to DOCA-salt rats significantly increased eNOS and Akt phosphorylation. However, long-term celiprolol plus wortmannin therapy reduced eNOS and Akt phosphorylation compared with celiprolol therapy alone. Moreover, eNOS mRNA and protein levels in the LV were significantly decreased in DOCA-salt rats compared with ShC rats (Figures 2C and 2D). Long-term celiprolol treatment in DOCA-salt rats significantly increased eNOS mRNA and protein levels. However, long-term celiprolol plus wortmannin therapy reduced eNOS mRNA and protein expression compared with celiprolol therapy alone.
Immunoreactivity for eNOS
There was a tendency for decreased immunoreactivity to eNOS in DOCA-salt rats compared with ShC rats. Immunoreactivity for eNOS was obviously enhanced in the small coronary artery by long-term celiprolol treatment. However, long-term celiprolol plus wortmannin therapy reduced immunoreactivity for eNOS compared with celiprolol therapy alone (Figures 1A through 1D).
VCAM-1 mRNA and protein levels in the LV were significantly higher in DOCA-salt rats than in ShC animals (Figures 3A and 3B). Long-term celiprolol therapy in DOCA-salt rats significantly decreased VCAM-1 mRNA and protein levels.
NAD(P)H Oxidase Subunits
NAD(P)H oxidase subunit p22phox, p47phox, and gp91phox mRNA and protein levels in the LV were significantly higher in DOCA-salt rats than in ShC rats. Long-term celiprolol therapy in DOCA-salt rats significantly decreased p22phox, p47phox, and gp91phox mRNA and protein levels. Gene expression of nox1 mRNA was also significantly increased in DOCA-salt rats compared with ShC rats. Long-term celiprolol treatment of DOCA-salt rats reduced nox1 mRNA levels (Figures 4A through 4D and 5A through 5C⇓).
H2O2 production in the LV was significantly increased in DOCA-salt compared with that in ShC rats (127.4±4.3 vs 81.9±2.8 fluorescence intensity units/mg LVW; P<0.01; Figure 5D). Long-term celiprolol treatment of DOCA-salt rats significantly reduced H2O2 production (113.7±3.7 fluorescence intensity units/mg LVW vs DOCA-V; P<0.05).
NADPH Oxidase Activity
NADPH oxidase activity in the LV was significantly higher in DOCA-salt than in ShC rats (196.7±10.4% vs 100±4.3%; P<0.01; Figure 5D). Long-term celiprolol treatment of DOCA-salt rats significantly reduced NADPH oxidase activity (143.9±3.8% vs DOCA-V; P<0.01).
p65NF-κB, p60c-Src, ERK1/2, and p90RSK Phosphorylation
Phosphorylation of p65NF-κB, p60c-Src, ERK1/2, and p90RSK in the LV was significantly higher in DOCA-salt than in ShC rats. Long-term celiprolol therapy of DOCA-salt rats significantly decreased the phosphorylation of p65NF-κB, p60c-Src, ERK1/2, and p90RSK (Figures 6A through 6D).
Gene Expression of ANP, β-MHC, Type I Collagen, and TGF-β1
Expression of fetal-type genes, such as those for ANP and β-MHC, and gene expression of type I collagen and TGF-β1 mRNA in the 3 groups are shown in Figures 7A through 7D. Gene expression of ANP, β-MHC, type I collagen, and TGF-β1 in the LV was significantly higher in DOCA-salt than in ShC rats. Long-term celiprolol treatment of DOCA-salt rats reduced the expression of ANP, β-MHC, type I collagen, and TGF-β1 mRNA.
Effect of Propranolol on eNOS Expression and Cardiovascular Remodeling
After long-term propranolol administration for 5 weeks, the SBP in DOCA-V and DOCA-Pro rats was similar and significantly higher than in ShC rats (DOCA-Pro, 186±6 mm Hg vs ShC; P<0.01). Long-term propranolol therapy in DOCA-salt rats did not decrease LVW/BW (DOCA-Pro, 2.83±0.09 mg/g vs DOCA-V; P=NS). The levels of eNOS mRNA (0.15±0.02 vs DOCA-V; P=NS) and protein (43±6% vs DOCA-V; P=NS) in the LV were not changed by administration of propranolol compared with DOCA-V. In addition, the wall-to-lumen ratio (0.23±0.02 vs DOCA-V; P=NS) and perivascular fibrosis (0.65±0.05 vs DOCA-V; P=NS) were not improved by propranolol therapy. These results suggest that celiprolol might have a potency to increase eNOS production and ameliorate cardiovascular remodeling, independent of BP, compared with propranolol.
The present study investigated the effect of a subdepressor dose of celiprolol on eNOS phosphorylation, mRNA and protein expression, VCAM-1 mRNA and protein expression, oxidative stress, and cardiovascular remodeling in the LV of DOCA-salt hypertensive rats.
First, we demonstrated that celiprolol increases the expression of eNOS mRNA and protein and activates phosphorylation of eNOS through the PI3K-Akt signaling pathway. eNOS catalyzes the synthesis of NO in blood vessels, which plays a critical role in maintaining BP homeostasis and vascular integrity.25 The vasodilating action of celiprolol is considered to be endothelium dependent in part. However, the precise activating molecular mechanisms are not fully clarified. Kakoki et al17 showed that celiprolol might act on the 5-HT1A receptor because the vasodilating action of celiprolol was attenuated by treatment with a 5-HT1A receptor antagonist in the rat aorta. Moreover, Noda et al18 showed that celiprolol acts at another β-adrenoceptor site in the endothelium in porcine coronary arteries. We hypothesized that celiprolol activates phosphorylation of eNOS through the PI3K-Akt signaling pathway. We confirmed this hypothesis by using wortmannin, a PI3K inhibitor, and showed that celiprolol activates eNOS phosphorylation through the PI3K-Akt pathway. On activation, Akt phosphorylates eNOS, the major site in rats being serine 1177.26 Phosphorylation of eNOS at serine 1177 is thought to enhance the Ca2+-calmodulin sensitivity of eNOS, which permits greater levels of NO generation in the presence of lesser increases in intracellular Ca2+ content. Apparently, activation of eNOS, which is independent of the increase in intracellular Ca2+ content and has been ascribed to fluid shear stress and pulsatile flow in multiple studies, appears to rely on the PI3K-Akt-eNOS phosphorylation sequence.27 Current evidence strongly suggests that in the case of celiprolol, PI3K acts through the serine/threonine kinase Akt to respond in regulating the phosphorylation and activation of eNOS. However, it is not known exactly why celiprolol activates the PI3K-Akt pathway. To elucidate this point, further research will be necessary.
Second, we demonstrated that celiprolol inhibits VCAM-1 expression and that this was associated with inhibition of p65 NF-κB phosphorylation. VCAM-1 plays a critical role in adhesion and recruitment of mononuclear leukocytes during times of chronic inflammation, and its expression has been linked to the early phase of atherosclerosis in animal models.28 Expression of adhesion molecules, including VCAM-1, can be regulated by NO, and increased levels of NO are associated with decreased leukocyte adhesion molecule expression.7 Indeed, exogenous NO donors might downregulate cytokine-induced VCAM-1 expression in cultured endothelial cells in vitro.7 However, the mechanisms by which NO modulates expression of VCAM-1 have not been fully delineated. Regulation of VCAM-1 expression occurs at the transcriptional level and is in part mediated via the transcription factor NF-κB.8 Indeed, the cytokine-induced transcriptional enhancer in the VCAM-1 promoter requires the combined interactions of NF-κB with other nuclear activators, such as activator protein-1.29 Inhibition of VCAM-1 expression by celiprolol might in part be due to enhanced eNOS activity and inhibition of p65 NF-κB phosphorylation.
Third, we demonstrated that celiprolol inhibits NAD(P)H oxidase p22phox, p47phox, gp91phox, and nox1 expression and also inhibits oxidative stress-mediated signal transduction pathways, such as c-Src and ERK1/2 and its downstream effector p90RSK. In addition, production of H2O2 in the LV was also reduced by treatment with celiprolol. Thus, celiprolol might reduce the production of ROS by suppressing NAD(P)H oxidase expression. Clinically, celiprolol was reported to reduce the production of superoxide anion in essential hypertensive patients.19 This mechanism might be related to the inhibition of NAD(P)H oxidase subunit expression. The DOCA-salt hypertensive rat is well known for its suppressed plasma renin and angiotensin levels. However, we have recently demonstrated that angiotensin-converting enzyme and Ang II type 1 receptor mRNA expression is upregulated in the LV of DOCA-salt hypertensive rats.30 Thus, the local tissue renin-angiotensin system might be augmented in this low-renin hypertensive rat model and might play a key role in the regulation of growth processes. Ang II stimulates vascular NAD(P)H oxidase to produce superoxide, which not only inactivates NO and impairs vasomotor function but also contributes to atherogenesis by the activation of VCAM-1.31 In addition, Tummala et al32 showed that Ang II stimulates VCAM-1 expression via activation of oxidative signaling pathways involving the redox-sensitive transcription factor NF-κB and the upregulation of genes downstream from NF-κB, including VCAM-1. Moreover, Li et al33 demonstrated that carotid and femoral arterial VCAM-1 expression and superoxide levels were increased in DOCA-salt hypertensive rats. These results suggest that Ang II might play a role in stimulating superoxide and raise the possibility that the increased superoxide in DOCA-salt hypertension might contribute to VCAM-1 expression.
In the current study, to elucidate whether celiprolol might have the potency to increase cardiac eNOS release and affect cardiovascular remodeling without altering BP, we evaluated the effects of the nonselective β-blocker, propranolol, on eNOS expression and cardiovascular remodeling with the same protocols. As a consequence, however, a subdepressor dose of propranolol failed to improve cardiovascular remodeling and the production of eNOS. In addition, Asanuma et al34 recently demonstrated that because a subdepressor dose of propranolol increased neither coronary blood flow nor cardiac NO production, celiprolol mediates coronary vasodilation and improves myocardial ischemia through NO-dependent mechanisms without a change in BP. These findings suggest that the available data support the notion that celiprolol might possess cardioreparative properties by way of cardiac NO production beyond its hemodynamic effect on BP.
Celiprolol increases eNOS by stimulation of the PI3K-Akt signaling pathway. Celiprolol suppresses VCAM-1 expression, which is associated with the suppression of NF-κB activation. The production of ROS was also decreased by suppression of NAD(P)H oxidase subunit expression. These actions lead to an improvement in cardiovascular remodeling. Celiprolol might have significant therapeutic potential in the treatment of atherosclerotic hypertension.
This work was supported in part by scientific research grant-in-aid 14570691 from the Japan Society for the Promotion of Science, a research grant from the Seki Minato Foundation, and a Dokkyo University School of Medicine investigator-initiated research grant. We thank Kazumi Akimoto, PhD, for technical assistance with the reverse transcription-PCR; Noriko Suzuki for preparing and staining tissue sections for histologic investigation; and Yasuko Mamada and Mika Nomura for technical assistance.
- Received November 20, 2002.
- Revision received December 19, 2002.
- Accepted September 11, 2003.
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