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B, in Mononuclear Cells of Normal Subjects: Evidence of an Antiinflammatory Action
Division of Endocrinology, Diabetes and Metabolism, State University of New York at Buffalo, and Kaleida Health, Buffalo, New York 14209
Address all correspondence and requests for reprints to: Paresh Dandona, M.D., Ph.D., F.R.C.P., F.A.C.P., F.A.C.C., Diabetes-Endocrinology Center of Western New York, 3 Gates Circle, Buffalo, New York 14209. E-mail: pdandona{at}kaleidahealth.org.
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Abstract |
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B (NF-B) in mononuclear cells. Four groups of eight normal subjects were given 1) 160 mg daily of valsartan, 2) 80 mg daily of simvastatin, 3) 40 mg quinapril, or 4) no treatment. Fasting blood samples were obtained before treatment and at d 1, 8, and 14 (7 d after the cessation of the drug). After valsartan, ROS generation by polymorphonuclear cells and mononuclear cells fell significantly by more than 40% (P < 0.01). NF-B binding activity and the expression of total cellular p65, a protein component of NF-B, fell significantly (P < 0.01). The expression of inhibitor B (IB) increased significantly (P < 0.05). Plasma C-reactive protein (CRP) concentration fell significantly (P < 0.01). All indices, except IB, reverted toward baseline, 7 d after the cessation of the drug. IB persisted in an elevated state. Neither quinapril nor simvastatin given for 7 d produced a suppression of ROS generation, intranuclear NF-B, p65, or CRP, and these two agents did not alter cellular IB either. The untreated controls also did not demonstrate a change in their ROS generation or NF-B binding activity or plasma CRP concentration. We conclude that valsartan at a modest dose exerts a profound and rapid ROS and inflammation-suppressive effect that may be relevant to its potential beneficial effects in atherosclerosis, diabetes, and congestive cardiac failure. In contrast, quinapril and simvastatin produced no similar effect over the period of 1 wk. Our observations may also have implications to clinical situations in which a rapid antiinflammatory effect is required. |
Introduction |
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B (IB) and its ubiquitination in vitro (1, 2, 3). This allows cytosolic nuclear factor B (NF-B) to translocate to the nucleus to induce an increase in the transcription of proinflammatory cytokines, adhesion molecules, and enzymes generating ROS (4, 5, 6). Thus, apart from being a major vasopressor effector of the renin-angiotensin system, angiotensin II is probably also a modulator of ROS generation and of inflammation. Its proinflammatory action is probably of relevance to atherosclerosis (7). It is noteworthy that angiotensin converting enzyme (ACE) inhibitors (8, 9) and angiotensin receptor blockers (ARBs) improve cardiovascular outcomes in patients with coronary heart disease and diabetes mellitus (8, 9).
Thus far, there is no information regarding the effect of either ARB or ACE inhibitors on either ROS generation or other mediators of inflammation in vivo. It is, therefore, important to investigate whether such agents are antiinflammatory and/or ROS suppressive. Recent work has shown that the key proinflammatory transcription factor, NF-B, is activated in proinflammatory states including atherosclerosis. NF-B regulates the transcription of genes for proinflammatory cytokines (e.g. TNF
and IL-6), adhesion molecules (e.g. intercellular adhesion molecule-1 and vascular cell adhesion molecule-1), chemokines (e.g. monocyte chemoattractant protein-1), and protein subunits of ROS-generating enzymes [e.g. reduced form of nicotinamide adenine dinucleotide phosphate oxidase (p47phox)] (4, 6, 10, 11). IB is a protein that binds to NF-B and prevents its translocation into the nucleus and, thus, prevents the transcription of proinflammatory genes. We have recently used mononuclear cells (MNCs) prepared from blood samples to investigate the pro- or antiinflammatory actions of various agents and drugs including hydrocortisone (12), troglitazone (13, 14), and insulin (15). Monocytes and T cells are known to initiate the endothelial inflammation leading to atherosclerosis (16). The rapid antiinflammatory and ROS-suppressive effect of hydrocortisone is observed in 4–6 h. Using the same model, we have shown that carvedilol and nadolol suppress ROS generation by leukocytes and oxidative stress, as observed in lipid peroxidation or amino acid oxidation after 1 wk of administration of modest doses of these drugs. HMG-CoA reductase inhibitors (statins) and ACE inhibitors improve cardiovascular outcomes, and both have been shown to lower plasma C-reactive protein (CRP) concentrations (17, 18, 19). This indicates that they have an antiinflammatory effect.
In view of the above, we have now investigated the effect of valsartan, an ARB, on ROS generation by leukocytes [polymorphonuclear cell (PMN) and MNC], NF-B, IB, and plasma CRP concentrations, key indices of inflammation, after a short period of therapy in normal subjects. We also compared the action of quinapril (an ACE inhibitor) and simvastatin with that of valsartan over the same period of time.
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Subjects and Methods |
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Four sets of eight subjects [mean age, 32.4 ± 5.6 yr (range, 26–42); mean body mass index, 24.6 ± 3.0 kg/m2 (range, 19.5–28)] were included in the study. All subjects came to the Research Center at the Diabetes-Endocrinology Center of Western New York at 0800–0900 h in the fasting state. A blood sample was obtained, and they were started on either valsartan (160 mg daily) or quinapril (40 mg daily) or simvastatin (80 mg daily) for 7 d. A second fasting blood sample was obtained on d 8, 24 h after the last dose of the drug. A third blood sample was obtained 7 d after the cessation of the drug. The fourth group was not given any drug but had fasting samples obtained on d 1, 8, and 14, as in the three drug-taking groups. All subjects participating in the study signed informed consent. The study protocol was approved by the Human Research Committee of the State University of New York at Buffalo based at Millard Fillmore Hospital. Compliance regarding drug intake was assured by administering eight tablets of the drugs to each subject and by determining that each subject had one remaining tablet at the end.
ROS generation assay
Respiratory burst activity of PMN or MNC was measured by detection of superoxide radical via chemiluminescence, as described previously (20, 21). Briefly, blood samples were collected in Na-EDTA as an anticoagulant. Three and a half milliliters of anticoagulated blood sample were carefully layered over 3.5 ml PMN medium (Robbins Scientific Corp., Sunnyvale, CA). Samples were centrifuged, and at the end of the centrifugation, two bands separated at the top of the red blood cell pellet. The top band consisted of MNC, whereas the bottom band consisted of PMN. The bands were harvested and repeatedly washed with Hanks’ balanced salt solution. This method provides yields greater than 95% pure PMN and MNC suspensions. Five hundred microliters of MNC or PMN (2 x 105 cells) were delivered into a Chronolog Lumi-Aggregometer cuvette. Fifteen microliters of 10 mM Luminol were then added, followed by 1.0 µl of 10 mM fMLP. Chemiluminescence was recorded for 15 min.
NF-B EMSA
DNA-binding protein extracts were prepared from MNC by the method described by Andrews and Faller (22). Total protein concentrations were determined using bicinchoninic acid protein assay (Pierce, Rockland, IL). EMSA was performed using a NF-B activity and binding protein detection kit (Life Technologies, Long Island, NY). The double-stranded oligonucleotide containing the consensus sequence for the NF-B binding site was radiolabeled with 
-P32 by T4 kinase. Then, 5 µg of the nuclear extract were mixed with the 5x incubation buffer [50 mM Tris (pH 7.5), 500 mM NaCl, 5 mM DTT, 5 mM EDTA, 20% glycerol, and 0.4 mg/ml sonicated salmon sperm], and the mixture was preincubated at 4 C for 15 min. Labeled oligonucleotide (60,000 cpm) was added, and the mixture was incubated at room temperature for 20 min. Samples were then applied to wells of 6% nondenaturing polyacrylamide gel. The gel was dried under vacuum and exposed to x-ray film. Densitometry was performed using molecular analyst software (Bio-Rad, Hercules, CA).
Total IB and p65 (Rel A) Western blotting
IB and p65 Western blotting were performed as described previously (12, 13).
Plasma CRP measurement
Plasma CRP was assayed with an ELISA kit from Diagnostic Systems Laboratories Inc. (Webster, TX).
Statistical analysis
Statistical analysis was performed using SigmaStat software (Jandel Scientific, San Rafael, CA). All data on ROS generation and CRP were normalized to a baseline of 100% in view of the interindividual variability and are expressed accordingly as percentage of the basal. A paired t test was used to compare all the indices measured in this study. Results are expressed as mean ± SD.
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Results |
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ROS generation by PMN fell from 175 ± 96 mV (100%) to 53 ± 18.6% of the basal on the eighth day, 24 h after the last dose of valsartan (P < 0.001). It returned to the baseline 7 d after cessation of the drug (95 ± 23.4%). ROS generation by MNC fell from 404 ± 242 mV (100%) to 56.3 ± 11.5% of the basal on the eighth day, 24 h after the last dose of valsartan (P < 0.001). It returned to the baseline 7 d after cessation of the drug (99 ± 25.2%). In contrast, quinapril and simvastatin were not able to suppress ROS generation by PMN and MNC (Fig. 1
). ROS generation did not alter in the control group either.
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B binding activity and total NF-B (Rel A: p65) in MNC homogenates
Valsartan intake caused a significant decrease in intranuclear NF-B binding activity levels (P < 0.01) (Fig. 2A). This decrease was statistically significant on the eighth day. NF-B binding activity returned to the basal level after the cessation of valsartan intake. NF-B gel retardation assay confirmed that it was a p50 and p65 heterodimer (Fig. 2B). p65 (Rel A) protein expression fell significantly as well after valsartan intake (P < 0.05) (Fig. 2C). Thus, both the total NF-B as protein as well as its activity in terms of DNA binding fell significantly. Neither quinapril nor simvastatin or the control group altered either p65 expression or NFB binding activity.
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B expression in MNC homogenates
IB expression in MNC homogenates increased significantly after valsartan intake (P < 0.05) (Fig. 3), at wk 1; it remained elevated at wk 2. IB expression did not alter after simvastatin or quinapril or in control subjects.
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Plasma CRP concentration fell significantly from a basal level of 127.9 ± 154.6 ng/ml (100%) to 67.4 ± 22.2% of the basal at wk 1 and to 122.3 ± 122.0% of the basal level at wk 2 (P < 0.05). Plasma CRP concentration did not alter in the quinapril or simvastatin or the control group (Fig. 4).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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B, as reflected in its binding to the consensus sequence polynucleotide and the diminished levels of p65 (Rel A), the protein that is a part of the NF-B heterodimer. IB expression increased whereas plasma CRP concentration fell. Therefore, valsartan has a comprehensive antiinflammatory effect at the cellular and molecular level, as well as in the plasma. It is of interest that quinapril, an ACE inhibitor, did not suppress any of the mediators of inflammation investigated in this study. Because angiotensin II is proinflammatory, it is surprising that this should be so. However, it is possible that the reduction in plasma angiotensin II concentrations after the administration of quinapril takes longer than the blockade of the angiotensin II receptor after valsartan. Whether valsartan shares this property with other ARBs is worthy of additional investigation. Similarly, an investigation of quinapril after longer administration would be of interest. It should be noted that ramipril (10 mg) caused a reduction in CRP in the Heart Outcomes Prevention Evaluation (HOPE) study quite apart from reducing the frequency of cardiovascular events. Simvastatin at a high dose (80 mg daily) also did not produce a significant effect during this period.
This antiinflammatory effect of valsartan would potentially inhibit the transcription of NF-B-modulated proinflammatory cytokines and adhesion molecules and enzymes responsible for ROS generation, like the reduced form of nicotinamide adenine dinucleotide phosphate oxidase. It is of interest that IB expression remained elevated 1 wk after the withdrawal of the drug, whereas NF-B reverted to levels greater than the baseline. The mechanism underlying this discrepancy is not clear. It is possible that the persistent increase in IB at wk 2 is a reflection of the increase in NF-B, because the latter is one of the regulators of the expression of IB gene. It is noteworthy that CRP fell significantly by over 30% in 1 wk; the magnitude of this fall is greater than that reported recently by following pravastatin therapy (14%) for 6 months (23). It is also of interest that simvastatin and quinapril (at high doses) did not induce a reduction in CRP, although both reduce CRP after a long duration of administration.
The reduction in ROS generation would potentially reduce oxidative damage of lipids, amino acids, and proteins. Oxidative damage of lipids leads to lipid peroxidation, which is crucial in the formation of foam cells from the monocyte-macrophage, which takes up oxidized low-density lipoprotein by the scavenger receptor (7, 24).
These data provide a mechanism that may explain the beneficial effects of valsartan and other ARBs in long-term outcome studies in patients with atherosclerosis, those with congestive cardiac failure, and those with renal complications of diabetes. These drugs block the action of angiotensin II at the AT1 receptor level. Angiotensin II is known to promote oxidative stress and to be proinflammatory. Leukocytes are known to express angiotensin II receptors (25). Thus, valsartan, which blocks angiotensin II, may be expected to inhibit inflammation and oxidative stress and, thus, the progression of atherosclerosis. The blockade of the AT1 receptor by valsartan may allow the activation of the AT2 receptor by angiotensin II (26). This may, in turn, facilitate nitric oxide generation (27) that may contribute to an antiinflammatory effect. These observations also have implications for the action of ACE inhibitors because they reduce the concentration and bioavailability of angiotensin II in plasma. However, ACE inhibitors probably take longer to exert this effect.
The recent Losartan Intervention for Endpoint Reduction in Hypertension (LIFE) study demonstrating a significant fall in cardiovascular mortality and morbidity in both diabetic and nondiabetic populations after intake of another angiotensin receptor blocker, losartan, is consistent with the mechanistic data presented here (28). Furthermore, it is also of interest that our short-term studies with ß-blockers (nadolol) and a combined - and ß-blocker (carvedilol) (21, 29) demonstrated a significant suppression of ROS generation by leukocytes but did not alter plasma CRP concentrations.
The comprehensive antiinflammatory effect of valsartan is similar to that we have described for insulin and the insulin sensitizers of the thiazolidinedione class (13, 14, 15, 30). The antiinflammatory effects of these drugs are of relevance to potential prevention of atherosclerosis as well as the restoration of insulin sensitivity in insulin resistant states. Thus, losartan has been shown to prevent both cardiovascular complications and type 2 diabetes (28).
The fact that these rapid and marked antiinflammatory changes have been observed in normal subjects within the period of 1 wk of the administration of a modest dose of valsartan suggest 1) a potent antiinflammatory effect of this drug and 2) a tonic proinflammatory effect of angiotensin II in normal subjects. It is likely that these effects would be more impressive in patients with inflammatory changes given higher doses of the drug for longer periods. The ability of valsartan to induce a rapid antiinflammatory effect like that of thiazolidenediones may also allow it to be used in clinical situations in which we require a rapid antiinflammatory effect. The absence of an effect by simvastatin or quinapril at relatively high doses in our study also suggests that the antiinflammatory effect of these drugs is relatively slow to develop.
In conclusion, valsartan inhibits ROS generation by PMN and MNC and, thus, reduces the oxidative load. It also suppresses NF-B while inducing IB and suppressing plasma CRP concentration. These actions are consistent with an antiinflammatory and potential anti-atherogenic effect of valsartan and possibly other ARBs in the long term. Our data are also the first to distinguish between ARBs, on the one hand, and ACE inhibitors and statins in terms of the rapidity of onset.
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Footnotes |
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B, inhibitor B; MNC, mononuclear cell; NF-B, nuclear factor B; PMN, polymorphonuclear cell; ROS, reactive oxygen species. Received November 21, 2002.
Accepted May 21, 2003.
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References |
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B activation induced by intracellular oxidative stress. Arterioscler Thromb Vasc Biol 20:645–651B: ten years after. Cell 87:13–20[CrossRef][Medline]
B and IB proteins: new discoveries and insights. Annu Rev Immunol 14:649–683[CrossRef][Medline]
B in the immune system. Annu Rev Immunol 12:141–179[Medline]
B: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336:1066–1071B and Rel: participants in a multiform transcriptional regulatory system. Int Rev Cytol 143:1–62[Medline]
B expression and diminished nuclear NF-B in human mononuclear cells following hydrocortisone injection. J Clin Endocrinol Metab 84:3386–3389B and stimulation of inhibitor B by troglitazone: evidence for an anti-inflammatory effect and a potential antiatherosclerotic effect in the obese. J Clin Endocrinol Metab 86:1306–1312B suppressive and inhibitor-B stimulatory effects of troglitazone in obese patients with type 2 diabetes: evidence of an antiinflammatory action? J Clin Endocrinol Metab 86:3250–3256B and stimulates IB in mononuclear cells in obese subjects: evidence for an anti-inflammatory effect? J Clin Endocrinol Metab 86:3257–3265This article has been cited by other articles:
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