Rodrigues, Stephen Fernandes MSc; dosSantos, Rosangela Aparecida PhD; de Oliveira, Maria Aparecida PhD; Rastelli, Viviani Milan PhD; Nucci, Gilberto de PhD; Tostes, Rita de Cássia PhD; Nigro, Dorothy PhD; Carvalho, Maria Helena PhD; Fortes, Zuleica B PhD
Cardiovascular disease (CVD) has become the main cause of death worldwide,1,2 and hypertension is one of the most important modifiable risk factors for CVD.3,4
Epidemiological surveys and interventional studies have revealed a higher prevalence of hypertension in the older than in younger age groups, attaining 50 to 75% in certain countries.5,6 This is complicated by the fact that the elderly are potentially more susceptible to other diseases like inflammatory diseases. Consequently, the combination of nonsteroidal antiinflammatory drugs (NSAIDs) and antihypertensive agents is more frequent in those people. It is estimated that as many as 20 million patients and 12% of the elderly in the United States take concurrent NSAIDs and antihypertensive medications.7,8
Association between treatment with NSAIDs (indomethacin, naproxen) and small increase in blood pressure (BP) levels has been demonstrated.9,10 NSAIDs may affect BP via alterations in sodium and water retention in kidneys, blockade of prostaglandin generation, mainly PGE2 and PGI2 (important vasodilators at renal vascular bed), and production of vasoconstricting factors, including endothelin-1 and P450-mediated metabolites of arachidonic acid.8 Moreover, it was found that NSAIDs affect BP most notably in hypertensive patients on antihypertensive medication.8 However, there are few studies on the influence of antihypertensive agents on the antiinflammatory activity of NSAIDs. We have demonstrated that drugs like enalapril and losartan, antihypertensive agents widely used, are able to reduce the antimigratory activity of diclofenac.11,12
Leukocyte extravasation is fundamental in the inflammatory response and can be divided into 3 steps: initial interaction of leukocytes with activated endothelium (rolling), leukocyte activation with firm adhesion to endothelial cells (adherence), and their extravasation into the surrounding tissues (migration).13
Some essential steps of the inflammatory process are regulated by the presence of calcium as the leukocyte paracellular migration.14 This process involves cell adhesion molecules (CAMs) that participate directly on it and are found both in leukocytes and endothelial cells. E-selectin, ICAM-1, and VCAM-1, among other adhesion molecules are involved in the interaction between leukocyte and vascular endothelial cells.13,15 Thus, the utilization of antihypertensive drugs, which act by blocking calcium channels, might affect the leukocyte transmigration.
Among the calcium blockers employed in clinical practice, amlodipine has been largely prescribed for the control of BP in the elderly because it has few side effects.16 Besides, it was demonstrated that amlodipine is able to inhibit the expression of CAMs like intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in endothelial cells in culture,17 and it reduces monocyte transmigration in vitro.18 However, to our knowledge, the effect of amlodipine on leukocyte migration has not yet been demonstrated in vivo and is therefore one of the objectives of the present study.
Parallel to inhibition of prostaglandins and thromboxanes, it was demonstrated that some NSAIDs like diclofenac are able to reduce the neutrophil rolling, adherence, and transmigration through endothelial layer by reducing the expression of CAMs (L-selectin, E-selectin, P-selectin, CD11/CD18, ICAM-1, and VCAM-1).11,12,19,20 Since amlodipine is an antihypertensive agent frequently employed to treat high BP and diclofenac is an antiinflammatory drug that is commonly used to treat skeletal/muscle disorders, we aim to study a possible interaction of amlodipine and diclofenac in SHR because both drugs influence the leukocyte transmigration and the expression of CAMs.
Male SHR (age, 14 weeks; body weight, 230 to 290 g) from our own breeding colony at the Institute were used. SHR were randomized into 4 groups that were age-matched and weight-matched. The groups consisted of SHR treated with vehicle (saline solution at 0.9%, PO), SHR treated with diclofenac potassium salt [Cataflan (Geigy, Sao Paulo, Brazil) at 1.0 mg·kg−1·d−1 for 15 d, PO], SHR treated with amlodipine [Norvasc (Pfizer, Sao Paulo, Brazil) at 10 mg·kg−1·d−1 for 15 d, PO], and SHR treated with diclofenac (1.0 mg·kg−1·d−1 for 15 d, PO) in association with amlodipine (10 mg·kg−1·d−1 for 15 d, PO). The selection of the dose of diclofenac was based on studies performed by the manufacturer21 and in our previous works.11,12 The doses chosen in this study have previously been shown to reduce leukocyte migration (diclofenac)11 and reduce BP levels in SHR (amlodipine 10 mg·kg−1).22 Although the t1/2 of diclofenac might be too short, we still could find the antiinflammatory effect 24 h after the last administration of once-a-day treatments. All animal procedures were in accordance with Ethics Committee of the Institute of Biomedical Sciences, University of Sao Paulo (under the protocol number 007/2004).
Body Weight Gain
Body weight gain was determined before the beginning of the treatments and weekly during the 15 d of treatment of SHR.
Arterial Blood Pressure
BP levels were determined before the beginning of the treatments and after 15 d of treatment of SHR. BP was measured in unanesthetized SHR by tail plethysmography using the Powerlab/4S system (ADInstruments Ltd., Castle Hill, Australia). SHR were placed in a warming chamber maintained at 40°C for about 10 min to allow pulses to be recorded. The cuff pressure was controlled, and the systolic pulses were detected. The average of 3 successive measurements was taken as the mean of systolic pressure values.
Direct Vital Microscopy of the Microcirculation
The experimental preparations used in this study were similar to that described previously.11,12,23 Male SHR were anesthetized with an IP injection of 45 mg·kg−1 sodium pentobarbital (Cristalia, Sao Paulo, Brazil), and the internal spermatic fascia of the wall of the scrotal chamber was exteriorized for microscopic examination in situ. This procedure does not require extensive surgical manipulation for the observation of the vascular network and provides a valuable mean for transilluminating a tissue for quantitative studies of the microcirculation. In addition, the preparation is not affected by respiratory movements of the rat, and its microcirculatory characteristics remain basically invariant throughout the course of the experiment. SHR were maintained on a special board thermostatically controlled at 37°C that included a transparent platform on which the tissue to be transilluminated was placed. The preparation was washed with warmed (37°C) Ringer-Locke solution, pH 7.4, containing 1% gelatin. The composition of the Ringer-Locke solution (in mmol/L) was 154.0 NaCl, 5.6 KCl, 2.0 CaCl2, 2 H2O, 6.0 NaHCO3 and 5.0 glucose (Merck S/A, Rio de Janeiro, Brazil). A transparent plastic film was arranged on the tissue to keep it moist during the stabilization time, which lasted 30 min. A television camera was incorporated into a trinocular microscope to facilitate observation of the enlarged image (×2500) on the video screen. Images were recorded on a video recorder with a ×40 longitudinal distance objective with 0.65 numerical aperture. Measurements of the vessel diameter were realized through an image-shearing monitor incorporated in the system. Vessels selected for study were postcapillary venules, and their diameters ranged from 14 to 18 μm (second-order venules).
In a series of experiments, leukocytes moving in the periphery of the axial stream in contact with the endothelium were considered to be “rollers”24 and their number was determined 30 min after surgery (stabilization time) and expressed as “rollers”/min without any stimulus except that induced by the “exposure trauma.” In these circumstances, only few adherent and migrated leukocytes are observed. The rolling leukocytes moved sufficiently slowly to be individually visible and were counted as they rolled past a 100-μm venule. The rats used in these experiments were not used for any other procedure.
TNFα-Induced Leukocyte Adhesion and Transmigration
In another series of experiments, the number of adherent (number per 100-μm length of venule) and migrated (number that accumulated in a 2500-μm2 standard area of connective tissue adjacent to a postcapillary venule) leukocytes was determined after 2 h from the injection of tumor necrosis factor α (TNFα 5 ng, 0.1 ml; R&D Systems, Minneapolis, MN, SC) into the scrotum. The scrotum was opened 1.5 h after the TNFα injection, and the internal spermatic fascia was prepared for intravital microscopy analysis, which occurred 0.5 h later. Three different fields were evaluated for each animal to avoid variability based on sampling. Data were then averaged for each animal. The rats used in these experiments were not used for any other procedure.
Venular Blood Flow Velocity, Venular Shear Rate, and Leukocyte Velocity
In another series of experiments, centerline velocity of red blood cells (VRBC) was measured using an optical Doppler velocimeter that was calibrated against a rotating glass disk coated with red blood cells. The measurements were made after the stabilization period (30 min after the induction of anesthesia at basal conditions). For microvessels smaller than 25 μm, VRBC = Vmean. Venular shear rates (γ) were calculated from the Newtonian definition: γ = 8 (Vmean/Dv) where Dv = venule diameter.25 Vessels selected for study were postcapillary venules of internal spermatic fascia, and their diameters ranged from 14 to 18 μm (second-order venules).
Blood samples for leukocyte count were collected from the tail of unanesthetized rats immediately before the real-time PCR protocol for the detection of ICAM-1 mRNA. SHRs were placed in a hold chamber, and their tails were warmed at 40°C for about 30 s to facilitate the blood collect. Blood was placed into tubes containing EDTA (Labor Vacuum, Sao Paulo, Brazil). Total leukocyte count was made after erythrocyte lyses with Cellmilise II solution (CELM, Sao Paulo, Brazil) in automatic counter (CELM CC-530, CELM, Sao Paulo, Brazil). Stained blood films were used for differential leukocyte counts.
Determination of Plasma Concentrations of Diclofenac and Amlodipine
Approximately 500 μL of blood were obtained from the orbital sinus before (blank samples) and after 15 min, 45 min, 6 h, and 24 h of the initial oral administration of diclofenac potassium salt (1 mg·kg−1) to SHR, SHR treated with amlodipine (10 mg·kg−1), and SHR treated with diclofenac (1 mg·kg−1) in association with amlodipine (10 mg·kg−1). The three first times were chosen to incorporate the peak plasmatic concentration of both drugs investigated, according to the literature26,27 and to previous tests performed by us. The 24-hour time was chosen because all the experimental procedures were run at this time interval. The blood was collected into heparinized tubes. The samples were subsequently centrifuged (1500 rev·min−1 for 10 min at 4°C), and plasma was stored at -20°C for further utilization. Plasma concentrations of diclofenac and amlodipine were analyzed by liquid chromatography-electrospray tandem mass spectrometry according to Oliveira et al28 and Padua et al.29 All frozen plasma samples were previously thawed at room temperature and centrifuged (3000 rev·min−1 for 5 min at 4°C) to precipitate solids. The analyte and internal standard [diclofenac potassium (Medley Indústria Farmacêutica, Sao Paulo, Brazil) and felodipine (Astra-Zeneca, Sao Paulo, Brazil)] were extracted from the plasma samples (150 μL) by liquid-liquid extraction using diethylether dichloromethane (60/40, vol/vol) and chromatographed on a C18 analytic column (Alltech, Prevail, C18; 3 μm; 100 × 4.6 mm). The mobile phase consisted of 2 solutions: (1) acetonitrile-water mixture (95/5, vol/vol) plus 5 mM ammonium formiate and 10 mM formic acid; (2) water plus 5 mM ammonium formiate and 10 mM formic acid. The method has a chromatographic total run time of 4.5 min. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer (Micromass, Manchester, UK) by multiple reaction monitoring. Acetonitrile (HPLCgrade), dichloromethane, and diethylether (analytic grade) were purchased from Mallinckrodt (St Louis, MO). Formic acid (analytic grade) was purchased from Merck (Rio de Janeiro, Brazil). Ultrapure water was obtained from an Elga UHQ ultrafiltration system (Elga, UK). The results represent plasma concentration curve of the drugs relative to the time and area under curve (AUC) values. The rats used in these experiments were not used for any other procedure.
Immunohistochemistry Analysis for Detection of ICAM-1, P-Selectin, and PECAM-1
Cross-sections of the unstimulated whole testis of SHR were prepared for the avidin-biotin/diaminobenzidine immunoperoxidase detection of ICAM-1, P-selectin, and PECAM-1 on the endothelium of venules of the internal spermatic fascia. Under anesthesia with pentobarbital sodium (45 mg·kg−1, IP), testis were excised through a longitudinal incision of the skin and dartos muscle in the midline over the ventral aspect of the scrotum. Specimens were then quick-frozen in hexane (Synth, Sao Paulo, Brazil). Sections of 8-μm thickness were cut on a Leica CM 1850 cryostat (Heidelberg, Germany) at -25oC and collected on glass slides previously coated with a thin layer of poly-L-lysine solution (Sigma, St. Louis, MO). Slides were incubated in acetone at −20°C for 10 min and stored in a freezer at −20°C for further utilization. Sections were washed once in 0.1 M PBS for 5 min and then incubated with biotin-conjugated monoclonal anti-rat ICAM-1 (CD54) antibody, clone 1A29, mouse IgG1 (1:10 in 0.1 M PBS plus Tween 20 0.3% solution; BD Pharmingen, San Diego, CA), biotin-conjugated monoclonal anti-rat PECAM-1 (CD31) antibody, clone TLD-3A12, mouse IgG1 (1:1000 in 0.1 M PBS plus Tween 20 0.3% solution; BD Pharmingen), or purified rabbit polyclonal anti-human P-selectin (CD62P) antibody (1:100 in 0.1 M PBS plus Tween 20 0.3% solution plus 5.0% rabbit serum; BD Pharmingen) overnight at 4°C. Slides were rinsed 3 times in 0.1 M PBS for 10 min each. Negative control sections were treated in the same manner as the experimental slides, except that the primary antibody was replaced by PBS 0.1 M. The slides incubated with anti-human polyclonal P-selectin antibody were afterwards incubated with biotin-conjugated secondary IgG anti-rabbit antibody (1:1000 in 0.1 M PBS plus Tween 20 0.3% solution; Vectastain ABC kit, Vector Laboratories, Burlingame, CA) for 1 h at room temperature and washed 3 times in 0.1 M PBS 0.1. Cross-sections were subsequently incubated with biotin-streptoavidin solution (Vectastain ABC kit, Vector Laboratories, Burlingame, CA) for 1 h at room temperature before 2 rinses in 0.1 M PBS (5 min each). Slides were incubated in diaminobenzidine solution (0.5 mg·mL−1 in 0.1 M PBS) in the presence of hydrogen peroxide (0.6 μL·mL−1 in 0.1 M PBS; Synth, Sao Paulo, Brazil) and afterwards washed twice in 0.1 M PBS and once in distillated water. Slides received Microscopy Entellan New mounting medium (Merck, Darmstadt, Germany) and were covered with cover glasses. Stain on the venular endothelial cells of the internal spermatic fascia was evaluated on an Axioskop 50 optical microscopy (Carls Zeiss, Gottingen, Germany) and analyzed by the software KS 300 3.0 (Zeiss, Germany). The results are expressed in arbitrary units.
Real-Time PCR for Detection of ICAM-1 mRNA
In another series of experiments, unstimulated spermatic fascias were dissected from pentobarbital sodium-anesthetized (45 mg·kg−1, IP) SHR treated with vehicle, diclofenac, amlodipine, or diclofenac plus amlodipine, frozen in liquid nitrogen, and stored at −70°C. Total cellular RNA was isolated from the spermatic fascia using TRizol Reagent (Invitrogen, San Diego, CA) according to the manufacturer's instruction. DNase I was employed to digest DNA to obtain RNA purification before RT reaction. Total RNA (2 μg) was used for first-strand cDNA synthesis [reverse transcriptase (RT)] using SuperScript II (Invitrogen), and RNaseOUT (Invitrogen) was also added to protect the RNA during this process. Amplification was performed by using of the following primers: 5′- CCT CTT GCG AAG ACG AGA AC - 3′ (forward) and 5′- ACT CGC TCT GGG AAC GAA TA - 3′ (reverse) (198 pb) for rat ICAM-1 and 5′- AAG ATT TGG CAC CAC ACT TTC TAC A - 3′ (forward) and 5′- CGG TGA GCA GCA CAG GGT - 3′ (reverse) (69 pb) for rat β-actin. The level of cDNA was analyzed by quantitative real-time PCR using Platinum SYBR green qPCR SuperMix UDG (Invitrogen). Real-time PCR reactions were performed, recorded, and analyzed by using Corbett Research system (Corbett Life Sciences, Sydney, Australia). PCR conditions used to amplify all genes were 2 min at 95°C and 40 cycles of 95°C for 15 s, 60°C for 1 min, and 72°C for 20 s. Specificity of the SYBR green assays was confirmed by melting-point analysis. Expression data were calculated from the cycle threshold (Ct) value using the ΔCt method for quantification.30 Gene expression of the β-actin mRNA was used for normalization. The results were expressed in fold increase.
Flow Cytometry for Detection of L-selectin and CD-18
To evaluate the effect of the different treatments on CD18 and L-selectin protein expressions, leukocytes from SHR were isolated from blood (6 mL) collected from the abdominal aorta under anesthesia (pentobarbital sodium 45 mg·kg−1, IP). Blood was initially placed into tubes containing EDTA (Labor Vacuum, Sao Paulo, Brazil). Erythrocyte lysis was subsequently performed using 0.13 M ammonium chloride solution (1:7, blood:ammonium chloride solution, vol/vol) for 5 min at 4°C, and a pellet of leukocytes was obtained after centrifugation (1200 rev·min−1, 5 min at 4°C) and discard of supernatant. Leukocytes were resuspended in 1 ml of HBSS (pH = 7.4) whose composition (in mmol/L) was 137.0 NaCl, 5.4 KCl, 0.7 Na2HPO4, 0.7 KH2PO4 e 5.5 glicose (Merck and Synth) plus 27.5 mM Tris base and 0.1% bovine serum albumine (BSA) (Sigma-Aldrich Co., St Louis, MO) and counted in Neubauer chamber. Samples were centrifuged (1200 rev·min−1, 10 min at 4°C) and resuspensed in HBSS plus Tris and BSA in a final concentration equal to 11.11 × 106 leukocytes/ml. Volume equal a 90 μL, equivalent to 1 × 106 cells, was incubated with or without primary antibody for 30 min at 4°C in the dark with antibody against CD18 (conjugated with 5.5 mg/L FITC; BD Pharmingen) or L-selectin (conjugated with 26 mg/L FITC, BD Pharmingen). As negative control, leukocytes were treated in the same manner as those obtained from experimental group rats, except that antibodies were not added. After incubation, cells were washed, resuspended in 500 μL of HBSS plus Tris and BSA, and the fluorescence was determined on an automated fluorescence-associated cell-separation system (FACS Calibur, BD). CellQuest software (BD) was used to quantify the results. Granulocytes were separated in a gate by their higher size and granulosity. Data were obtained from 5000 granulocytes. Results are expressed in intensity of fluorescence (arbitrary units ± SEM, n = 4 to 8).
Data are given as means ± SEM. One-way analysis of variance (ANOVA) followed by Tukey Multiple Comparison Test were used, except for the comparison of the plasma concentrations of the drugs when unpaired Student t test was used. The minimum acceptable level of significance was P at a value less than or equal to 0.05.
Body Weight Gain
The treatments tested did not interfere with the body weight gain in SHR (Table 1).
Arterial Blood Pressure
Diclofenac treatment did not change the BP levels of SHR, but amlodipine, combined or not with diclofenac, decreased the BP values compared to the vehicle-treated group (Table 1).
Direct Vital Microscopy of the Microcirculation
Venular diameters were not changed by any treatment tested in SHR (Table 2).
Diclofenac decreased the number of rollers in SHR, and its association with amlodipine did not interfere significantly with that reduction (Table 2). On the other hand, amlodipine did not change the leukocyte rolling compared to vehicle-treated SHR (Table 2). Both diclofenac and amlodipine reduced the number of adherent and migrated leukocytes of SHR after previous stimuli (2 hours) with TNFα; however, when administered combined, amlodipine decreased the effect of the diclofenac on the number of adherent and migrated leukocytes (Table 2).
Venular Blood Flow Velocity, Venular Shear Rate, and Leukocyte Velocity
Compared to vehicle-treatment, no tested treatment modified the venular blood flow velocity, venular shear rate, and leukocyte velocity of SHR (Figure 1).
There was no significant difference between the treatments with diclofenac, amlodipine, or diclofenac associated with amlodipine on the total or differential leukocyte countings in SHR (Figure 2).
Plasma Concentrations of Diclofenac and Amlodipine
The plasma concentration of diclofenac was not modified by amlodipine (Figure 3, A and B). Similarly, the plasma concentration of amlodipine was not changed by diclofenac (Figure 3, C and D).
Immunohistochemistry Analysis for Detection of ICAM-1, P-Selectin, and PECAM-1
Treatment of SHR with diclofenac and amlodipine, administered alone, decreased the protein expression of ICAM-1 in venular endothelial cells. However, when diclofenac and amlodipine were administered combined, the ICAM-1 expression was similar to the vehicle-treated group (Figures 4, A and B). No treatment altered the expression of P-selectin (Figure 5, A and B) or PECAM-1 (Figure 6, A and B) in venular endothelial cells of SHR.
Real-Time PCR of ICAM-1 mRNA
Treatment of SHR with diclofenac and amlodipine, administered alone, decreased the ICAM-1 mRNA in venular endothelial cells. However, when diclofenac and amlodipine were administered combined, the ICAM-1 expression was similar to that observed in SHR treated with the vehicle (Figure 7).
Flow Cytometry for Detection of L-selectin and CD-18
SHR treated with diclofenac, amlodipine, or the combination of both had no difference on the L-selectin (Figure 8) and CD-18 (Figure 9) expressions on granulocytes compared to vehicle-treated SHR.
In the present study, we demonstrated for the first time that amlodipine reduces in vivo leukocyte migration in SHR, interfering with ICAM-1 protein and gene expression. We also found that amlodipine reduces the effect of diclofenac on leukocyte transmigration in this model of hypertension. A pharmacokinetic drug interaction could explain our results; however, this possibility was discarded because no significant difference was found in plasma concentrations of the drugs given alone or in association.
NSAIDs have been widely used for treatment of several acute and chronic inflammatory diseases. They act mainly by blocking the action of the cyclooxygenase enzyme, and they consequently inhibit the synthesis of prostaglandins. Prostaglandins not only participate actively in the inflammatory process, but they also have important physiological roles such as in gastric protection.
Amlodipine is an antihypertensive drug that belongs to the L type-calcium channel blockers and is widely used for the control of high BP. When administered at the dose of 10 mg·kg−1·d−1, amlodipine reduced BP levels in SHR (approximately 30 mm Hg), similarly to that observed by Yokota et al.22
NSAIDs (for example, indomethacin and naproxen) are known to attenuate the effects of some antihypertensive agents.7,31 However, this is not a universal finding; diclofenac did not interfere with the BP-lowering effect of other antihypertensive drugs such as β-blockers,32 enalapril,11 and losartan.12 In fact, diclofenac did not interfere with either the BP of SHR or with the BP-lowering effect of amlodipine in the present study. This may be the consequence of the low dose of diclofenac used. In fact, Whelton33 demonstrated that diclofenac has a dose-dependent effect on BP; as we found, no effect would therefore be observed at low dose.
To investigate leukocyte endothelial interactions in the present study, we chose postcapillary venules in because they are considered to be the major site for leukocyte adhesion to the vascular wall in response to noxious stimuli.23 With intravital microscopy, leukocyte behavior can be dissected into the 3 main steps: rolling, adherence, and transmigration.
Rolling behavior can be studied without any stimulus except that induced by exposure trauma and was used in our study. Amlodipine did not interfere with rolling leukocytes nor alter the reduction of this parameter observed after diclofenac treatment.
Because a large number of mediators has been implicated in the leukocyte-endothelium interaction leading to adhesion and leukocyte transmigration during inflammation, the experimental strategy we employed to assess the contribution of specific mediators to this facet of the response involved exposure of venules to an exogenous irritant such as TNFα. This mediator is able to induce venular endothelial cells to synthesize CAMs like ICAM-1, facilitating the interaction endothelial cell-leukocyte.34 Furthermore, it is highly chemotactic for neutrophils.35 In this case, neutrophils are therefore most likely responsible for the leukocyte response seen in our study. We chose 2-hour time exposure of TNFα because it is the minimum time to observe adherence and transmigration. The reduction in leukocyte adherence and migration after the combination was not different from that observed with amlodipine alone, but it was of a lower magnitude than that observed with diclofenac; therefore, we may conclude that amlodipine reduced the effect of diclofenac but that diclofenac did not interfere with the effect of amlodipine.
Whether reduction of the BP levels caused by amlodipine participates in reducing leukocyte adherence and migration in vivo in SHR is unknown; however, Singhal et al18 observed that amlodipine reduces monocyte migration in vitro independently of its pressure-lowering effect. This observation supports the idea that effects other than BP reduction, such as antioxidant property, that are present in some antihypertensive drugs like amlodipine can contribute in a more important way to the antimigratory effect of that drug.
Besides the influence of CAMs on the leukocyte-endothelial interaction, the hemodynamic force represented by parameters like flow and resistance affects the transport of leukocytes to an injury site.36 To evaluate the possible interference of hemodynamic changes on the leukocyte behavior studied, we measured venular diameter and venular blood flow velocity to calculate the wall shear rate because the dependence of shear rate on leukocyte adhesion has been demonstrated in vivo37 and in vitro.38 Low shear rates promote leukocyte adherence to microvascular endothelium in postcapillary venules.37 Therefore, increase in shear rate might explain the decrease in leukocyte transmigration observed with the different treatments. However, no treatment tested increased the venular blood flow velocity and venular shear rate. Therefore, we can exclude interference of these parameters on the alterations observed.
An alteration on the number of circulating neutrophils caused by any treatment could explain why the administration of diclofenac and amlodipine reduced leukocyte adherence and transmigration. However, we did not detect a statistically significant difference in the number of circulating leukocytes after all treatments used. Thus, an influence of this parameter on the reduced migration could be excluded.
The mechanisms underlying leukocyte accumulation in a tissue depend on the interaction between the cells and the vascular endothelium. During the development of inflammatory responses, leukocytes roll along the lining endothelium of postcapillary venules and eventually become firmly attached to the vascular wall before migrating into tissues.13 Specific adhesion glycoproteins expressed on the surface of leukocytes and endothelial cells play an important role in the adhesion and migrated phenomena.39 It is known that both diclofenac19,20 and amlodipine17 are able to reduce the expression of several CAMs, thus exerting an antiinflammatory effect.
Members of the selectin family of CAMs, P-selectin expressed on endothelial cells and L-selectin or CD18 expressed on leukocytes, can mediate leukocyte rolling along the wall of the microvasculature.40 No drug used in our study modified P-selectin and L-selectin expressions in SHR. This may explain why amlodipine did not reduce the rolling leukocytes. The mechanism involved in the reduction in the number of rollers observed by us with diclofenac associated or not with amlodipine in SHR remains unknown; however, we hypothetize that it is the consequence of the diclofenac effect on the expression of other CAMs.
Reduction in CAMs on endothelial cells or on leukocytes could also be involved in the reduced leukocyte adherence and transmigration observed. In the present study we demonstrated that the administration of diclofenac or amlodipine reduced the ICAM-1 expression on endothelial cells of SHR. This finding might explain why both diclofenac and amlodipine reduced adherence and transmigration in SHR. In fact, reduction in ICAM-1 protein expression by diclofenac11,12,20 or amlodipine17 has been previously observed, but not the interference of each drug on each effect, reducing ICAM-1 expression in vivo as we demonstrated. Expression of PECAM-1 as well as of CD-18 seems not to be involved in the effect of the drugs alone or combined because no difference in the expression of these molecules was found after the treatments.
Reduction in ICAM-1 expression might be due to the reduced level of mRNA expression we found. This has been previously demonstrated by Sakai20 for diclofenac and by Yoshii et al41 for amlodipine when employed alone. We confirm these findings and extended the observation showing that the effect of diclofenac on ICAM-1 mRNA is reduced when it is combined with amlodipine.
The mechanism by which TNFα increases the number of adherent and migrated leukocytes might involve activation of the transcription factor NF-κB via NADPH activity-derived oxidant signaling, resulting in increased ICAM-1 expression.34,42,43. Thus, we hypothesize that diclofenac and amlodipine reduce the ICAM-1 mRNA; consequently, the protein expression of this molecule, either reducing the activity of NF-κB or NADPH oxidant signaling. In fact, it has been demonstrated that both diclofenac44 and amlodipine45,46 reduce, albeit slightly, the mRNA for NF-κB.
Antioxidant properties of these drugs could contribute to that effect. Yoshii et al41 demonstrated that amlodipine is able to reduce NADPH expression and activity and ICAM-1 expression in apolipoprotein E-deficient (ApoEKO) mice. Reduction in lipid peroxidation,47 free-radical-scavenging activity,48 and inhibition of superoxide generation in rat polimorphonuclear leukocytes49 have been described for diclofenac.
The most interesting finding of our study, the reduction of the antimigratory effect of the diclofenac when administered combined with amlodipine, may be explained by the blocking effect of amlodipine on the reduction of the ICAM-1 expression caused by diclofenac. The intimate mechanism involved in this interaction remains to be clarified.
Our data allow us to conclude that amlodipine reduces the leukocyte transmigration, interfering with ICAM-1 protein and gene expressions in SHR. Therefore, besides reducing BP levels, amlodipine may also be useful for modulation of the inflammatory process mainly characterized by leukocyte accumulation as atherosclerosis and myocardial and cerebral ischemia-reperfusion injuries.
When associated with diclofenac, amlodipine may hinder the full expression of the antiinflammatory effect of this NSAID. Thus, the use of this combination must be monitored closely in clinical practice, mainly in the elderly population, where amlodipine is widely used to reduce high BP levels. Due to the high incidence of musculoskeletal disorders, the use of antiinflammatory drugs like diclofenac is very common in this population, making the combination of both drugs not unusual.
We thank Antonio Garcia Soares Junior for good technical assistance.
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