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Hypertension Plus Diabetes Mimics the Cardiomyopathy Induced by Nitric Oxide Inhibition in Rats
Hypertension Plus Diabetes Mimics the Cardiomyopathy Induced by Nitric Oxide Inhibition in Rats* Rita C. Sampaio, BS; Jose E. Tanus-Santos, MD, PhD; Silvia E. S. F. C. Melo, PharmD; Stephen Hyslop, PhD; Kleber G. Franchini, MD, PhD; Iara M. Luca, PhD; and Heitor Moreno, Jr, MD, PhD
Study objectives: We compared the myocardial lesions caused by the long-term inhibition of nitric oxide (NO) biosynthesis with those associated with renovascular hypertension (two-kidney, one-clip model [2K-1C]) and superimposed streptozotocin-induced diabetes mellitus (DM). Design: Prospective trial. Setting: University laboratory. Interventions: Male Wistar rats were classified into the following groups: (1) a control group; (2) the L-NAME group (treatment with the NO synthase inhibitor N-nitro-L-arginine methyl ester [LNAME], 75 mol per rat per day, orally); (3) the 2K-1C group (renovascular hypertension); (4) the DM group (treatment with streptozotocin, 60 mg/kg via intraperitoneal route); and (5) the 2K-1C plus DM group (renovascular hypertension and streptozotocin-induced DM). Arterial BP was measured by a tail-cuff method for 3 weeks, after which histologic and stereological analysis of the heart was done and cardiac NO synthase type 3 (NOS3) levels were assessed by Western blotting. The circulating levels of nitrates/nitrites and thromboxane B2 (TXB2, the stable metabolite of thromboxane A2) were also measured. Results: In DM and 2K-1C rats, the myocardial lesions consisted mainly of recent myocardial infarcts, which were more severe in the 2K-1C plus DM group. In L-NAME–treated rats, multiple foci of reparative fibrosis and fresh myocardial necrosis resembled the severe lesions found in the 2K-1C plus DM group. Although NOS3 protein expression increased (19 to 44%; p < 0.05) in all treated groups, serum nitrate/nitrite levels decreased only in the L-NAME group and the 2K-1C plus DM group. These two groups also showed a more pronounced increase in TXB2 concentrations. Conclusions: These results indicate that the association of hypertension and DM mimics the alterations induced by L-NAME in rats, which suggests a role for NO in the pathophysiology of hypertensive-diabetic cardiomyopathy. (CHEST 2002; 122:1412–1420) Key words: diabetes mellitus; heart; hypertension; myocardial diseases; N-nitro-L-arginine methyl ester; nitric oxide Abbreviations: ANOVA ⫽ analysis of variance; BSA ⫽ bovine serum albumin; BW ⫽ body weight; DM ⫽ diabetes mellitus; HW ⫽ heart weight; HWI ⫽ heart weight index; IP ⫽ intraperitoneal; 2K-1C ⫽ two-kidney, one-clip model; L-NAME ⫽ N-nitro-L-arginine-methyl ester; LVW ⫽ left ventricular weight; LVWI ⫽ left ventricular weight index; NO ⫽ nitric oxide; NOS3 ⫽ nitric oxide synthase type 3; RVW ⫽ right ventricular weight; RVWI ⫽ right ventricular weight index; TCP ⫽ tail-cuff pressure; TXA2 ⫽ thromboxane A2; TXB2 ⫽ thromboxane B2
clinical and morphologic features of hyperT hetensive-diabetic cardiomyopathy were described
⬎ 2 decades ago.1 Although there is still controversy regarding the existence of “hypertensive”2 or “diabetic” cardiomyopathy, epidemiologic data have confirmed an increased likelihood of developing heart *From the Departments of Pharmacology (Drs. Sampaio, TanusSantos, Melo, Hyslop, and Moreno) and Medicine (Dr. Franchini), Faculty of Medical Sciences, and Department of Histology (Dr. Luca), Institute of Biology, State University of Campinas, Campinas, Brazil. Manuscript received October 16, 2001; revision accepted February 28, 2002. 1412
failure when hypertension and diabetes mellitus (DM) are combined.3 Since the incidence of DM is increasing rapidly, a significant rise in the incidence of accelerated heart failure secondary to diabetic cardiomyopathy is anticipated, especially when hyDr. Moreno was partly supported by Conselho Nacional de Pesquisa e Desenvolvimento. Drs. Tanus-Santos and Sampaio were supported by Fundac¸a˜o de Auxı´lio a` Pesquisa do Estado de Sa˜o Paulo grant No. 97/11236-0. Correspondence to: Jose E. Tanus-Santos, MD, PhD, University of Sao Paulo, Faculty of Medicine of Ribeirao Preto, Dept of Pharmacology, Av Bandeirantes, 3900, Ribeirao Preto, SP, Brazil 14049-900; e-mail: [email protected] Laboratory and Animal Investigations
pertension is a concomitant synergistic factor. Moreover, there is an increased prevalence of hypertension in diabetic patients and vice versa, suggesting that both conditions commonly coexist.4 The relationship among hypertension, diabetes, and endothelial function is complex. Since hypertensive-diabetic rats showed more severe coronary microvascular abnormalities,5 hypertension was suggested to exacerbate diabetic cardiomyopathy by further impairing the blood supply to the myocardium.6 In both disorders, the decreased production or bioavailability of nitric oxide (NO) causes endothelial dysfunction7 that may involve coronary vessels. A dysregulation of cardiac myocyte NO synthase type 3 (NOS3) was suggested to be involved in hypertension-induced cardiac alterations,8 and an increased expression of a dysfunctional NOS3 has been reported in an experimental model of diabetes,9 thus indicating that the NO system has a crucial role in both conditions. We have previously described hypertensive cardiomyopathy secondary to endothelial dysfunction following administration of the NO synthase inhibitor N-nitro-L-arginine-methyl ester (LNAME).10 In the present study, we compared the myocardial alterations following the chronic inhibition of NO biosynthesis with those observed in hypertensivediabetic rats. We also evaluated the responses to DM and renovascular hypertension alone (two-kidney, one-clip model [2K-1C]). We hypothesized that similar mechanisms could be activated in hypertensivediabetic and L-NAME–induced cardiomyopathy. To examine the role of NO in the development of hypertensive-diabetic cardiomyopathy, the cardiac levels of NOS3 protein were evaluated by Western blotting and serum nitrate and nitrite concentrations were measured. Since thromboxane A2 (TXA2), an eicosanoid produced by activated platelets and endothelium, is frequently involved in the pathophysiology of cardiovascular diseases,11 the plasma levels of thromboxane B2 (TXB2), the stable metabolite of TXA2, were also quantified.
Materials and Methods Experimental Groups Male Wistar rats (150 to 200 g at the start of the study) were provided by Central Animal Services of the university, and were classified as follows: (1) a control group (n ⫽ 14), sham-operated rats that received tap water alone; (2) the L-NAME group (n ⫽ 31), rats that received L-NAME in drinking water; (3) the 2K-1C group (n ⫽ 26), rats with a silver clip placed around one renal artery; (4) the DM group (n ⫽ 27), rats that received a single intraperitoneal (IP) dose of streptozotocin, 60 mg/kg; and (5) the 2K-1C plus DM group (n ⫽ 26), rats that received a single www.chestjournal.org
dose of streptozotocin and had one renal artery clipped. The animals were killed after 3 weeks. This investigation conformed to the National Research Council guidelines. Induction of Goldblatt II Hypertension Rats were anesthetized with sodium pentobarbital, 40 mg/kg IP, a left flank incision was done to allow a silver clip (0.2-mm inner diameter) to be placed around the renal artery.12 In sham-operated control rats, the clip was removed immediately after being fixed around the vessel and the kidney was repositioned in the abdomen. Long-term Treatment With L-NAME L-NAME was dissolved in the drinking water at a concentration of 1.2 mM to give a daily intake of approximately 75 mol per rat per day.10 The average daily intake of water and food did not differ significantly among the five groups. Streptozotocin Administration Diabetes was induced with an IP injection of streptozotocin, 60 mg/kg, dissolved in citrate buffer. BP Measurements Arterial BP was measured twice a week by a tail-cuff method,13 and the mean of these two determinations was considered to be the mean for that week. The same procedure was applied to weight gain. Heart Weight Indexes After 3 weeks of treatment, the rats were killed with a lethal dose of sodium pentobarbital, and the heart was removed, washed with saline solution, and then fixed in 10% formalin for 24 h. Subsequently, the atria were removed and the ventricles weighed to obtain the heart weight (HW). The left ventricular weight (LVW) and right ventricular weight (RVW) were determined after separating the ventricles. The HW index (HWI) was calculated by dividing the HW by the body weight (BW). Correspondingly, the LVW index (LVWI) and the RVW index (RVWI) were calculated by dividing the LVW and RVW by the BW. Histologic Analysis The left and right ventricles were cut into five equidistant rings perpendicular to the long axis of the heart. All rings were embedded in paraffin, and 5-m sections were stained with hematoxylin-eosin. For each rat, one section of each of the five ventricular rings was studied by light microscopy. Cardiomyocyte size was determined by measuring the cell diameters by using an optical microscope system supplied with a graduated eyepiece micrometer and a 100 ⫻ objective (1,000 ⫻ magnification). Fifteen cells randomly selected from the subepicardial, midmyocardial, and subendocardial regions were measured in each animal of the different experimental groups. The investigator responsible was unaware of the corresponding groups for slides examined. Stereological Procedures Myocardial lesions were evaluated quantitatively using a stereological method that allowed the blind analysis of lesions CHEST / 122 / 4 / OCTOBER, 2002
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resulting from recent infarcts, ie, focal lesions showing the onset or progress of death in cardiac myocytes, as well as the presence of fibrosis in scar tissue. In this method,14,15 the counting grid consisted of a combination of points and parallel lines (in this case, a 10 ⫻ objective with a graticule containing 100 points and 10 parallel lines). Each point corresponded to the center of a polygon of known area. The distance between the lines served as the point of reference for calibrating the system and also for determining the area of the lesion. For sample areas with a square format, the area (distance squared) was given by the formula: A ⫽ ⌫共Ap兲 ⫻ Pt where A was the area of the test point, ⌫ (Ap) was the sum of areas of the test points used, and Pt was the total number of test points. Western Blotting of Myocardial NOS3 For Western blotting, the thoracic cavity was opened and the heart rapidly removed. The left ventricle was minced coarsely and immediately homogenized in approximately 10 volumes of solubilization buffer (1% Triton-X 100, 100 mM Tris-HCl, pH 7.4, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM ethylenediamine tetra-acetic acid, 2 mM phenylmethylsulfonyl fluoride, and 0.1 mg of aprotinin per milliliter) at 4°C using a polytron PTA 20S generator (model PT 10/35; Brinkman Instruments; Westbury, NY) operated at maximum speed for 30 s. The extracts were centrifuged at 10,000g at 4°C for 30 min to remove insoluble material. The resulting supernatant was used for the assay. Protein concentrations were determined with a dye-binding assay using Bio-Rad reagent (Bio-Rad Laboratories; Richmond, CA) and bovine serum albumin (BSA) as the standard. Equal amounts of total protein were for all samples diluted with Laemmli sample buffer containing 100 mM dithiothreitol and heated in a boiling water bath for 4 min, after which they were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (8% polyacrylamide gels) in a miniature gel apparatus (Mini-Protean; Bio-Rad Laboratories). Electrotransfer of proteins from the gel to the nitrocellulose membrane was done for 90 min at 120 V (constant) in a miniature transfer apparatus (Mini-Protean). Nonspecific protein binding to the nitrocellulose membrane was reduced by preincubating the filter overnight at 4°C in blocking buffer (5% defatted dry milk, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20). The nitrocellulose membrane blot was incubated with anti-NOS3 antibodies (Santa Cruz Biotechnology; Santa Cruz, CA) diluted in 10 mL of blocking buffer (3% BSA instead of defatted dry milk) overnight at 4°C, and then washed for 60 min in blocking buffer without milk or BSA. The blots were subsequently incubated with 2 Ci of [125I] protein A, 30 Ci/g, in 10 mL of blocking buffer for 2 h at room temperature, and then washed again for 30 min as described above. [125I] Protein A bound to the antibody was detected by autoradiography using preflashed Kodak XAR film (Eastman Kodak; Rochester, NY) stored at ⫺ 80° for 24 h. Band intensities were quantified by optical densitometry of the developed autoradiographs. Plasma Measurements Thromboxane B2: Venous blood samples were collected from the tail vein into tubes containing ethylenediamine tetra-acetic acid at baseline and 3 weeks after treatment. The plasma was separated by centrifugation and stored at 20°C until assayed. Plasma samples were extracted using C18 reverse-phase cartridges (Waters Corporation; Milford, MA), and the TXB2 levels 1414
were determined with a commercial enzyme immunoassay (Cayman Chemical; Ann Arbor, MI). The detection limit of this assay was 13.3 pg/mL. Nitrates and Nitrites: The circulating levels of nitrates/nitrites were used to estimate NO production.16 Blood samples from a caudal vein were collected into heparinized tubes at baseline and 3 weeks after treatment. The plasma was separated by centrifugation and stored at 20°C until assayed. Plasma nitrates/nitrites levels were determined spectrophotometrically using a commercial assay kit (Cayman Chemical). Briefly, plasma samples were filtered at 4°C by centrifugation through filters with a molecular weight cutoff of 10,000 (Centricon; Millipore; Milford, MA). After the addition of nitrate reductase to convert nitrates to nitrites, Griess reagent was added to the mixture and the nitrite concentrations were determined by measuring the resulting absorbance at 550 nm in a SpectraMax 340 multiwell plate reader (Molecular Devices; San Diego, CA). The nitrite concentrations of the samples were calculated by reference to a standard curve of nitrite run in parallel. The detection limit of this assay was 2.5 M. Insulin: Insulin levels were determined at baseline and 3 weeks after treatment using a commercial enzyme immunoassay (Rat insulin enzyme immunoassay kit; SPI-BIO; Massy, France). Creatinine: Renal function was assessed by measuring creatinine levels by the Jaffe method at baseline and 3 weeks after treatment. Statistical Analysis Results are expressed as mean ⫾ SEM. Analysis of variance (ANOVA) for repeated measurements was used to assess the differences in BW and tail-cuff pressure (TCP). One-way ANOVA was used to compare HW, LVW, RVW, HWI, LVWI, and RVWI. When ANOVA results were significant, the Duncan test was used to determine the differences among groups. In all cases, p ⬍ 0.05 was considered to be significant. All calculations were done using SigmaStat software (SPSS Science; Chicago, IL).
Results BW and TCP Table 1 and Figure 1 show the BW and TCP, respectively. The two groups of rats treated with streptozotocin had a lower BW than the control group (p ⬍ 0.05) after 3 weeks. TCP increased equally in the L-NAME, 2K-1C, and 2K-1C plus DM groups (p ⬍ 0.05). TCP increased in the DM group after 3 weeks (p ⬍ 0.05). Cardiac Weight and Cardiac Weight Indexes Table 1 shows the HW, RVW, and LVW, and their respective weight indexes. There were no significant differences in these parameters in the control, L-NAME, and DM groups. In contrast, HW, HWI, LVW, and LVWI increased after 3 weeks of treatment (p ⬍ 0.05) in the 2K-1C and the 2K-1C plus DM groups when compared to the control group. Histologic and Stereological Analysis There were no evident histologic lesions in the hearts of control rats. The heart of L-NAME–treated Laboratory and Animal Investigations
Table 1—Characteristics of Study Patients in the Experimental Groups After 3 Weeks of Study* Parameters
Control (n ⫽ 16)
L-NAME (n ⫽ 31)
2K-1C (n ⫽ 21)
DM (n ⫽ 27)
2K-1C Plus DM (n ⫽ 20)
BW, g HW, mg HWI, mg/g LVW, mg LVWI, mg/g RVW, mg RVWI, mg/g
262 ⫾ 19 700 ⫾ 54 2.6 ⫾ 0.3 550 ⫾ 70 2.1 ⫾ 0.3 150 ⫾ 28 0.6 ⫾ 0.1
240 ⫾ 36 640 ⫾ 98 2.7 ⫾ 0.2 510 ⫾ 82 2.1 ⫾ 0.2 130 ⫾ 21 0.5 ⫾ 0.1
250 ⫾ 26 870 ⫾ 61† 3.5 ⫾ 0.6† 710 ⫾ 54† 2.9 ⫾ 0.2† 160 ⫾ 15 0.6 ⫾ 0.1
205 ⫾ 40† 570 ⫾ 97 2.8 ⫾ 0.3 430 ⫾ 80 2.1 ⫾ 0.2 140 ⫾ 25 0.6 ⫾ 0.1
198 ⫾ 33† 750 ⫾ 47† 3.8 ⫾ 0.8† 625 ⫾ 72† 3.2 ⫾ 0.2† 125 ⫾ 20 0.7 ⫾ 0.1
*Data are presented as mean ⫾ SEM. †p ⬍ 0.05 vs control.
rats showed unambiguous foci of reparative fibrosis consistent with organized myocytolytic necrosis. In addition, recent myocardial necrosis with granulation tissue showing blood vessels and many fibroblasts was also seen in these rats (Fig 2). Furthermore, some of these rats showed interstitial and perivascular cardiac fibrosis. The rats in the other three experimental groups had myocardial lesions consisting mainly of recent myocardial infarcts with dark coagulated myocytes, contraction bands, and dead myocytes lacking nuclei. Although similar lesions were observed in these three groups, the myocardial lesions in the DM group were greater than in the 2K-1C group. Even more widespread lesions were present in the 2K-1C plus DM group compared to the 2K-1C group and the DM group (Fig 3). Figure 4 provides a quantitative comparison of the myocardial lesions in the different groups studied. In agreement with the qualitative histologic analysis, stereological analysis showed larger myocardial le-
Figure 1. TCP during the 3 weeks of study. E ⫽ control group (n ⫽ 16); F ⫽ L-NAME group (n ⫽ 31); f ⫽ 2K-1C group (n ⫽ 21); 䡺 ⫽ DM group (n ⫽ 27); ƒ ⫽ 2K-1C plus DM group (n ⫽ 20). The points represent the mean ⫾ SEM. *p ⬍ 0.05 vs basal for L-NAME, 2K-1C, and 2K-1C plus DM groups. #p ⬍ 0.05 for all groups at week 3 vs control group. www.chestjournal.org
sions in the DM group and the 2K-1C plus DM group compared to the 2K-1C group (p ⬍ 0.05). The diameters of myocytes in the DM group were similar to those in control rats in the three myocardial regions (Table 2). In contrast, the diameters of midmyocardial and subepicardial myocytes were greater in the 2K-1C group and the 2K-1C plus DM group compared to the control group (p ⬍ 0.05 for both). A small increase in the diameter of midmyocardial myocytes was observed in the L-NAME group (p ⬍ 0.05). Western Blotting of Myocardial NOS3 Figure 5 shows a representative Western blot of NOS3 in the hearts of rats from the different groups, as well as the average ⫾ SEM values (n ⫽ 4) of densitometric readings obtained with anti-NOS3 antibody. Increased levels of NOS3 protein were seen in the 2K-1C, DM, and 2K-1C plus DM groups (19 to 23% higher than in the control group; p ⬍ 0.05). Treatment with L-NAME produced a more pronounced increase in the NOS3 protein levels (44% higher than in the control group; p ⬍ 0.05).
Figure 2. L-NAME: subendocardial infarct showing a wellestablished granulation tissue with blood vessels, many fibroblasts, and little collagenization (Massons trichrome, original ⫻ 128). CHEST / 122 / 4 / OCTOBER, 2002
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DM group and the 2K-1C plus DM group (p ⬍ 0.05 for both).
Discussion
Figure 3. 2K-1C plus DM: recent myocardial infarct with dark coagulated myocytes lacking nuclei (hematoxylin-eosin, original ⫻ 128).
Plasma Measurements: Nitrates and Nitrites: The circulating levels of nitrates/nitrites were unaltered in the control, 2K-1C, and DM groups, but decreased in the LNAME and 2K-1C plus DM groups (p ⬍ 0.05 for both; Fig 6, top, A). TXB2: TXB2 concentrations increased after 3 weeks in the four experimental groups (p ⬍ 0.05), but not in the control group (Fig 6, bottom, B). This increase was more marked in the L-NAME and 2K-1C plus DM groups. Insulin, Glucose, and Creatinine: Table 3 shows the plasma insulin, glucose, and creatinine levels at baseline and after 3 weeks. There were no significant changes in the creatinine levels in all of the experimental groups (Table 3). Marked increases in glucose and decreases in insulin levels occurred in the
Figure 4. Total area of myocardial lesions (micrometers squared) after 3 weeks. Columns represent the mean ⫾ SEM. *p ⬍ 0.05 vs 2K-1C group. 1416
In the present study, the association of hypertension and DM resulted in more severe cardiomyopathy than would be anticipated with each condition. The severity of the hypertensive-diabetic cardiomyopathy resembled that seen after the inhibition of NO synthesis, thus suggesting a role for NO in both cardiomyopathies. Although the histologic changes in the heart were qualitatively alike in the 2K-1C, DM, and 2K-1C plus DM groups, quantitative analysis indicated larger lesions in the last group. These findings are comparable to those found at autopsy in a large group of patients with either hypertension, DM, or both conditions.17 In this case, the hearts of patients with concomitant hypertension and DM showed more severe and generalized fibrosis than those of patients with hypertension or DM alone.17 The histologic analysis in the present study suggested that the severity of L-NAME–induced myocardial lesions was analogous to that in rats with DM and superimposed hypertension (the 2K-1C plus DM group). This suggestion is supported by the observation that, although the total area of myocardial lesions produced by L-NAME differed from that in the 2K-1C plus DM group, the multiple foci of reparative fibrosis and fresh myocardial necrosis seen after treatment with L-NAME resembled the more severe and extensive lesions present in the 2K-1C plus DM group. The multiple foci of reparative fibrosis seen after treatment with L-NAME were similar to those described in previous studies,10,18 and represented remotely infarcted myocardium. Thus, long-term inhibition of NO synthesis may cause an early activation of the pathophysiologic mechanisms underlying the more recent cardiac lesions found in the 2K-1C plus DM group. An increase in BP enhances the mechanical stress on cardiomyocytes and is an important stimulus for cardiac hypertrophy.19 The similar rises in BP levels in the 2K-1C, 2K-1C plus DM, and L-NAME groups contrasted with the different grades of hypertrophic responses in these experimental groups, and suggested that mechanisms other than a pressure overload are also activated.10 Indeed, hypertrophy has been suggested to be related to humoral factors such as plasma renin activity or angiotensin II levels, but not to the mechanical overload.20 The larger HW, HWI, LVW, and LVWI were associated with a significantly greater myocyte diameter in the midmyocardium and subepicardium in Laboratory and Animal Investigations
Table 2—Myocyte Diameter in the Left Ventricle Subendocardium, Midmyocardium, and Subepicardium of the Experimental Groups* Regions
Control (n ⫽ 6)
L-NAME (n ⫽ 6)
2K-1C (n ⫽ 6)
DM (n ⫽ 6)
2K-1C Plus DM (n ⫽ 6)
Subendocardium, m Midmyocardium, m Subepicardium, m
12.8 ⫾ 0.8 13.0 ⫾ 0.2 10.3 ⫾ 0.8†
13.4 ⫾ 0.2 14.8 ⫾ 0.8† 12.2 ⫾ 0.5
13.7 ⫾ 0.4 18.3 ⫾ 1.6†‡ 13.2 ⫾ 0.4†
12.0 ⫾ 0.8 13.5 ⫾ 0.5 10.8 ⫾ 0.7‡
13.7 ⫾ 0.5 18.2 ⫾ 1.3†‡ 13.4 ⫾ 0.7†
*Data are presented as mean ⫾ SEM. †p ⬍ 0.05 vs control. ‡p ⬍ 0.05 vs the other two regions.
the 2K-1C group and the 2K-1C plus DM group. However, no remarkable increases were observed in the cardiac indexes and myocyte diameter of LNAME–treated rats. These findings confirmed previous studies demonstrating that ventricular hypertrophy and increased cardiomyocyte diameter occur in 2K-1C–treated rats or after aortic stenosis, but not after treatment with L-NAME.10,20 –22 This lack of a hypertrophic response after treatment with L-NAME may be explained by a limitation imposed on ventricular growth attributed to a more pronounced loss of cardiomyocytes that undergo apoptosis after inhibition of NO biosynthesis.23 Interestingly, one study24 has demonstrated that different doses of L-NAME, which produced the same increase in systolic BP, resulted in fibrosis that was proportional to the dose of L-NAME administered. Thus, a more powerful inhibition of NO biosynthesis may cause more severe myocardial lesions in rats treated with increasing doses of L-NAME, or when DM is superimposed on hypertension. Rats treated with streptozotocin, 60 mg/kg, gained weight and showed hypoinsulinemia and hyperglyce-
Figure 5. Representative Western blot of the NOS3 levels in the hearts of rats from control group (CT), L-NAME group, DM group, 2K-1C group, and 2K-1C plus DM group (densitometric readings [mean ⫾ SEM], n ⫽ 4 blots per group). *p ⬍ 0.05 vs control group; **p ⬍ 0.01 vs control group. IB ⫽ immunoblot. www.chestjournal.org
mia as reported previously with a slightly lower dose of streptozotocin (55 mg/kg).6 The increase in the BP of diabetic rats agrees with previous findings showing a trend for arterial pressure to increase when glucose
Figure 6. Top, A: basal and week 3 serum nitrate/nitrite levels. Bottom, B: basal and week 3 serum TXB2 in control group (open columns), L-NAME group (filled columns), 2K-1C group (horizontally striped columns), DM group (vertically striped columns), and 2K-1C plus DM group (stippled columns). Columns represent the mean ⫾ SEM. *p ⬍ 0.05 vs basal. CHEST / 122 / 4 / OCTOBER, 2002
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Table 3—Plasma Creatinine, Glucose, and Insulin Concentrations After 3 Weeks in the Groups Studied* Parameters Creatinine, mg/dL Basal Week 3 Glucose, mg/dL Basal Week 3 Insulin, ng/mL Basal Week 3
Control (n ⫽ 16)
L-NAME (n ⫽ 31)
2K-1C (n ⫽ 21)
DM (n ⫽ 27)
2K-1C Plus DM (n ⫽ 20)
0.4 ⫾ 0.1 0.3 ⫾ 0.1
0.4 ⫾ 0.1 0.3 ⫾ 0.1
0.4 ⫾ 0.1 0.3 ⫾ 0.1
0.3 ⫾ 0.1 0.4 ⫾ 0.1
0.4 ⫾ 0.1 0.4 ⫾ 0.1
114 ⫾ 15 105 ⫾ 10
112 ⫾ 11 84 ⫾ 11
111 ⫾ 14 104 ⫾ 13
105 ⫾ 20 361 ⫾ 115†‡
150 ⫾ 24 427 ⫾ 107†‡
1.0 ⫾ 0.1 0.6 ⫾ 0.1
0.9 ⫾ 0.1 0.8 ⫾ 0.1
0.9 ⫾ 0.2 0.8 ⫾ 0.1
0.9 ⫾ 0.1 0.1 ⫾ 0.1†‡
0.8 ⫾ 0.1 0.0 ⫾ 0.0†‡
*Data are presented as mean ⫾ SEM. †p ⬍ 0.05 vs control. ‡p ⬍ 0.05 vs basal.
levels are not controlled.25 Importantly, levels of NOS3 protein were increased in diabetic rat hearts, maybe as a result of enhanced oxidative stress caused by hyperglycemia.26 Although similar increases (19 to 23%) in the levels of NOS3 protein were also observed in the 2K-1C group and the 2K-1C plus DM group, treatment with L-NAME produced the greatest increase in NOS3 protein (44% higher than the control group). Curiously, the increased expression of NOS3 did not produce concomitant changes in the plasma nitrate/nitrite levels in any of the all groups. The plasma nitrate/nitrite concentrations decreased only in the L-NAME and 2K-1C plus DM groups, which showed the most severe myocardial lesions. Although the precise relationship between serum nitrate/nitrite levels and NO production is still unclear,27 these results suggest an important decrease in NO bioavailability16 in both groups. Indeed, diabetic rats show impaired acetylcholineinduced aortic relaxation and nitrate/nitrite production attributed to an abnormal metabolism of NO.28 A reduced bioavailability of NO caused by a lack of substrates or cofactors may lead to the uncoupling of NOS3 or increased NO degradation by reactive oxygen species.9,29 Complex mechanisms regulate NOS3 at the transcriptional and posttranscriptional levels.30 Although NOS3 is expressed predominantly in coronary and endocardial endothelial cells, and to a lesser extent in cardiac myocytes,31 the regulation of the expression of this enzyme in the heart is not well studied, especially in pathophysiologic conditions. Increased expression of NOS3 was reported in the heart after banding of the aorta,32 or after the development of hypertension in spontaneously hypertensive rats.33 The increased NOS3 levels seen in the 2K-1C group and the L-NAME group did not agree with previous studies showing decreased levels of NOS3 messenger RNA in 2K-1C–treated rats,34 or after 6 weeks of 1418
treatment with L-NAME, 60 mg/kg/d.35 Differences in rat strain, and in the dose and duration of treatment with L-NAME, could explain the discrepancies between these studies. These same reasons could account for the extent and severity of the myocardial lesions seen here compared to previous studies. A hypercoagulable or prothrombotic state, partially due to platelet abnormalities, has been described in hypertension36 and DM,37 and may involve changes in the bioavailability of NO. This is mainly because endogenous NO, besides controlling vascular tone, also regulates leukocyte adhesion and platelet aggregation. Thus, the more severe myocardial lesions and greater increases in TXB2 levels seen in L-NAME–treated and 2K-1C plus DM-treated rats may be partially explained by a more extensive reduction in the availability of NO in both groups. This greater reduction in NO availability may have increased platelet activation and released more TXA2 than in the other experimental groups. This suggestion is in line with studies demonstrating that hyperglycemia per se, besides inducing platelet hyperactivity,37 can also chemically inactivate NO,38 thereby potentiating the decreases of NO bioavailability seen in hypertension.7 In further support of this hypothesis, inhibitors of NO biosynthesis have been shown to enhance platelet aggregation.39 In addition, increased TXA2 production, indicative of platelet activation, has been found in acute coronary disease.40 Together, these findings point to a lack of NO as a key factor in the diabetic-hypertensive patients. There is one limitation in the present study. The reduced NO availability we have shown in diabetichypertensive cardiomyopathy could be further confirmed by examining the effects of a treatment that increases the availability of NO. Such a treatment would probably result in a normalization or attenuation of alterations in diabetic-hypertensive cardioLaboratory and Animal Investigations
myopathy. Since this issue has not been previously addressed, further studies exploring this possibility are warranted. The experimental data presented here are supported by a recently published clinical study41 comparing hypertensive diabetic, hypertensive, and healthy subjects. Interestingly, hypertensive diabetic patients had the highest plasma levels of von Willebrand factor and soluble P-selectin, which reflect a prothrombotic state associated with low NO availability.41 Therefore, a major clinical implication of our findings is that improving NO availability might serve as an important element in the therapy of diabetic-hypertensive patients. Indeed, a decreased capacity to enhance NO production in treated diabetic nephropathy has been suggested to accelerate the renal disease.42 In conclusion, our data support the hypothesis that the cardiac abnormalities found in hypertension are similar to found in other pathophysiologic conditions such as DM,2 probably because of the activation of related underlying mechanisms. Moreover, the combination of hypertension and DM enhanced the severity of these abnormalities to such an extent that the effect resembled dose-dependent L-NAME– induced cardiomyopathy. These findings suggest a role for the NO system in the pathophysiology of diabetic-hypertensive cardiomyopathy. References 1 Factor SM, Minase T, Sonnenblick EH. Clinical and morphological features of human hypertensive-diabetic cardiomyopathy. Am Heart J 1980; 99:446 – 458 2 Lip GY, Felmeden DC, Li-Saw-Hee FL, et al. Hypertensive heart disease: a complex syndrome or a hypertensive “cardiomyopathy”? Eur Heart J 2000; 21:1653–1665 3 Grossman E, Messerli FH. Diabetic and hypertensive heart disease. Ann Intern Med 1996; 125:304 –310 4 Sowers JR, Epstein M, Frohlich ED. Diabetes, hypertension, and cardiovascular disease: an update. Hypertension 2001; 37:1053–1059 5 Factor SM, Minase T, Cho S, et al. Coronary microvascular abnormalities in the hypertensive-diabetic rat: a primary cause of cardiomyopathy? Am J Pathol 1984; 116:9 –20 6 Mathis DR, Liu SS, Rodrigues BB, et al. Effect of hypertension on the development of diabetic cardiomyopathy. Can J Physiol Pharmacol 2000; 78:791–798 7 Kojda G, Harrison D. Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure. Cardiovasc Res 1999; 43:562–571 8 Bayraktutan U, Yang ZK, Shah AM. Selective dysregulation of nitric oxide synthase type 3 in cardiac myocytes but not coronary microvascular endothelial cells of spontaneously hypertensive rat. Cardiovasc Res 1998; 38:719 –726 9 Hink U, Li H, Mollnau H, et al. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res 2001; 88:E14 –E22 10 Moreno H Jr, Metze K, Bento AC, et al. Chronic nitric oxide inhibition as a model of hypertensive heart muscle disease. Basic Res Cardiol 1996; 91:248 –255 www.chestjournal.org
11 Humphrey PP, Hallet P, Hornby EJ, et al. Pathophysiological actions of thromboxane A2 and their pharmacological antagonism by thromboxane receptor blockade with GR32191. Circulation 1990; 81:I42–I52, I59 –I60 12 Goldblatt HLJ, Hanzal RF, Summerville WW. Studies on experimental hypertension: the production of persistent elevation of systolic blood pressure by means of renal ischemia. J Exp Med 1934; 59:347–379 13 Zatz R. A low cost tail-cuff method for the estimation of mean arterial pressure in conscious rats. Lab Anim Sci 1990; 40:198 –201 14 Weibel E. Stereological methods: practical methods for biological morphometry (vol 1). New York, NY: Academic Press, 1979 15 Coelho-Filho OR, De Luca IM, Tanus-Santos JE, et al. Pravastatin reduces myocardial lesions induced by acute inhibition of nitric oxide biosynthesis in normocholesterolemic rats. Int J Cardiol 2001; 79:215–221 16 Jungersten L, Edlund A, Petersson AS, et al. Plasma nitrate as an index of nitric oxide formation in man: analyses of kinetics and confounding factors. Clin Physiol 1996; 16:369 –379 17 van Hoeven KH, Factor SM. A comparison of the pathological spectrum of hypertensive, diabetic, and hypertensivediabetic heart disease. Circulation 1990; 82:848 – 855 18 Moreno H Jr, Piovesan Nathan L, Pereira Costa SK, et al. Enalapril does not prevent the myocardial ischemia caused by the chronic inhibition of nitric oxide synthesis. Eur J Pharmacol 1995; 287:93–96 19 Hefti MA, Harder BA, Eppenberger HM, et al. Signaling pathways in cardiac myocyte hypertrophy. J Mol Cell Cardiol 1997; 29:2873–2892 20 Matsubara BB, Matsubara LS, Zornoff LA, et al. Left ventricular adaptation to chronic pressure overload induced by inhibition of nitric oxide synthase in rats. Basic Res Cardiol 1998; 93:173–181 21 Bartunek J, Weinberg EO, Tajima M, et al. Chronic N(G)nitro-L-arginine methyl ester-induced hypertension: novel molecular adaptation to systolic load in absence of hypertrophy. Circulation 2000; 101:423– 429 22 Arnal JF, el Amrani AI, Chatellier G, et al. Cardiac weight in hypertension induced by nitric oxide synthase blockade. Hypertension 1993; 22:380 –387 23 Pessanha MG, Mandarim-de-Lacerda CA. Influence of the chronic nitric oxide synthesis inhibition on cardiomyocytes number. Virchows Arch 2000; 437:667– 674 24 Pechanova O, Bernatova I, Pelouch V, et al. L-NAMEinduced protein remodeling and fibrosis in the rat heart. Physiol Res 1999; 48:353–362 25 Fitzgerald SM, Brands MW. Nitric oxide may be required to prevent hypertension at the onset of diabetes. Am J Physiol Endocrinol Metab 2000; 279:E762–E768 26 Stockklauser-Farber K, Ballhausen T, Laufer A, et al. Influence of diabetes on cardiac nitric oxide synthase expression and activity. Biochim Biophys Acta 2000; 1535:10 –20 27 Moshage H. Nitric oxide determinations: much ado about NO.-thing? Clin Chem 1997; 43:553–556 28 Kobayashi T, Kamata K. Effect of chronic insulin treatment on NO production and endothelium-dependent relaxation in aortae from established STZ-induced diabetic rats. Atherosclerosis 2001; 155:313–320 29 Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 2000; 87:840 – 844 30 Govers R, Rabelink TJ. Cellular regulation of endothelial nitric oxide synthase. Am J Physiol Renal Physiol 2001; 280:F193–F206 31 Shah AM, MacCarthy PA. Paracrine and autocrine effects of CHEST / 122 / 4 / OCTOBER, 2002
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32 33 34 35
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nitric oxide on myocardial function. Pharmacol Ther 2000; 86:49 – 86 Barton CH, Ni Z, Vaziri ND. Effect of severe aortic banding above the renal arteries on nitric oxide synthase isotype expression. Kidney Int 2001; 59:654 – 661 Vaziri ND, Ni Z, Oveisi F, et al. Effect of antioxidant therapy on blood pressure and NO synthase expression in hypertensive rats. Hypertension 2000; 36:957–964 Kobayashi N, Kobayashi K, Hara K, et al. Benidipine stimulates nitric oxide synthase and improves coronary circulation in hypertensive rats. Am J Hypertens 1999; 12:483– 491 Kobayashi N, Hara K, Watanabe S, et al. Effect of imidapril on myocardial remodeling in L-NAME-induced hypertensive rats is associated with gene expression of NOS and ACE mRNA. Am J Hypertens 2000; 13:199 –207 Lip GY, Li-Saw-Hee FL. Does hypertension confer a hypercoagulable state? J Hypertens 1998; 16:913–916 Trovati M, Anfossi G. Insulin, insulin resistance and platelet function: similarities with insulin effects on cultured vascular smooth muscle cells. Diabetologia 1998; 41:609 – 622
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38 Brodsky SV, Morrishow AM, Dharia N, et al. Glucose scavenging of nitric oxide. Am J Physiol Renal Physiol 2001; 280:F480 –F486 39 Chen LY, Mehta JL. Variable effects of L-arginine analogs on L-arginine-nitric oxide pathway in human neutrophils and platelets may relate to different nitric oxide synthase isoforms. J Pharmacol Exp Ther 1996; 276:253–257 40 Hirsh PD, Hillis LD, Campbell WB, et al. Release of prostaglandins and thromboxane into the coronary circulation in patients with ischemic heart disease. N Engl J Med 1981; 304:685– 691 41 Ouvina SM, La Greca RD, Zanaro NL, et al. Endothelial dysfunction, nitric oxide and platelet activation in hypertensive and diabetic type II patients. Thromb Res 2001; 102: 107–114 42 Earle KA, Mehrotra S, Dalton RN, et al. Defective nitric oxide production and functional renal reserve in patients with type 2 diabetes who have microalbuminuria of African and Asian compared with white origin. J Am Soc Nephrol 2001; 12:2125–2130
Laboratory and Animal Investigations
Study objectives: We compared the myocardial lesions caused by the long-term inhibition of nitric oxide (NO) biosynthesis with those associated with renovascular hypertension (two-kidney, one-clip model [2K-1C]) and superimposed streptozotocin-induced diabetes mellitus (DM). Design: Prospective trial. Setting: University laboratory. Interventions: Male Wistar rats were classified into the following groups: (1) a control group; (2) the L-NAME group (treatment with the NO synthase inhibitor N-nitro-L-arginine methyl ester [LNAME], 75 mol per rat per day, orally); (3) the 2K-1C group (renovascular hypertension); (4) the DM group (treatment with streptozotocin, 60 mg/kg via intraperitoneal route); and (5) the 2K-1C plus DM group (renovascular hypertension and streptozotocin-induced DM). Arterial BP was measured by a tail-cuff method for 3 weeks, after which histologic and stereological analysis of the heart was done and cardiac NO synthase type 3 (NOS3) levels were assessed by Western blotting. The circulating levels of nitrates/nitrites and thromboxane B2 (TXB2, the stable metabolite of thromboxane A2) were also measured. Results: In DM and 2K-1C rats, the myocardial lesions consisted mainly of recent myocardial infarcts, which were more severe in the 2K-1C plus DM group. In L-NAME–treated rats, multiple foci of reparative fibrosis and fresh myocardial necrosis resembled the severe lesions found in the 2K-1C plus DM group. Although NOS3 protein expression increased (19 to 44%; p < 0.05) in all treated groups, serum nitrate/nitrite levels decreased only in the L-NAME group and the 2K-1C plus DM group. These two groups also showed a more pronounced increase in TXB2 concentrations. Conclusions: These results indicate that the association of hypertension and DM mimics the alterations induced by L-NAME in rats, which suggests a role for NO in the pathophysiology of hypertensive-diabetic cardiomyopathy. (CHEST 2002; 122:1412–1420) Key words: diabetes mellitus; heart; hypertension; myocardial diseases; N-nitro-L-arginine methyl ester; nitric oxide Abbreviations: ANOVA ⫽ analysis of variance; BSA ⫽ bovine serum albumin; BW ⫽ body weight; DM ⫽ diabetes mellitus; HW ⫽ heart weight; HWI ⫽ heart weight index; IP ⫽ intraperitoneal; 2K-1C ⫽ two-kidney, one-clip model; L-NAME ⫽ N-nitro-L-arginine-methyl ester; LVW ⫽ left ventricular weight; LVWI ⫽ left ventricular weight index; NO ⫽ nitric oxide; NOS3 ⫽ nitric oxide synthase type 3; RVW ⫽ right ventricular weight; RVWI ⫽ right ventricular weight index; TCP ⫽ tail-cuff pressure; TXA2 ⫽ thromboxane A2; TXB2 ⫽ thromboxane B2
clinical and morphologic features of hyperT hetensive-diabetic cardiomyopathy were described
⬎ 2 decades ago.1 Although there is still controversy regarding the existence of “hypertensive”2 or “diabetic” cardiomyopathy, epidemiologic data have confirmed an increased likelihood of developing heart *From the Departments of Pharmacology (Drs. Sampaio, TanusSantos, Melo, Hyslop, and Moreno) and Medicine (Dr. Franchini), Faculty of Medical Sciences, and Department of Histology (Dr. Luca), Institute of Biology, State University of Campinas, Campinas, Brazil. Manuscript received October 16, 2001; revision accepted February 28, 2002. 1412
failure when hypertension and diabetes mellitus (DM) are combined.3 Since the incidence of DM is increasing rapidly, a significant rise in the incidence of accelerated heart failure secondary to diabetic cardiomyopathy is anticipated, especially when hyDr. Moreno was partly supported by Conselho Nacional de Pesquisa e Desenvolvimento. Drs. Tanus-Santos and Sampaio were supported by Fundac¸a˜o de Auxı´lio a` Pesquisa do Estado de Sa˜o Paulo grant No. 97/11236-0. Correspondence to: Jose E. Tanus-Santos, MD, PhD, University of Sao Paulo, Faculty of Medicine of Ribeirao Preto, Dept of Pharmacology, Av Bandeirantes, 3900, Ribeirao Preto, SP, Brazil 14049-900; e-mail: [email protected] Laboratory and Animal Investigations
pertension is a concomitant synergistic factor. Moreover, there is an increased prevalence of hypertension in diabetic patients and vice versa, suggesting that both conditions commonly coexist.4 The relationship among hypertension, diabetes, and endothelial function is complex. Since hypertensive-diabetic rats showed more severe coronary microvascular abnormalities,5 hypertension was suggested to exacerbate diabetic cardiomyopathy by further impairing the blood supply to the myocardium.6 In both disorders, the decreased production or bioavailability of nitric oxide (NO) causes endothelial dysfunction7 that may involve coronary vessels. A dysregulation of cardiac myocyte NO synthase type 3 (NOS3) was suggested to be involved in hypertension-induced cardiac alterations,8 and an increased expression of a dysfunctional NOS3 has been reported in an experimental model of diabetes,9 thus indicating that the NO system has a crucial role in both conditions. We have previously described hypertensive cardiomyopathy secondary to endothelial dysfunction following administration of the NO synthase inhibitor N-nitro-L-arginine-methyl ester (LNAME).10 In the present study, we compared the myocardial alterations following the chronic inhibition of NO biosynthesis with those observed in hypertensivediabetic rats. We also evaluated the responses to DM and renovascular hypertension alone (two-kidney, one-clip model [2K-1C]). We hypothesized that similar mechanisms could be activated in hypertensivediabetic and L-NAME–induced cardiomyopathy. To examine the role of NO in the development of hypertensive-diabetic cardiomyopathy, the cardiac levels of NOS3 protein were evaluated by Western blotting and serum nitrate and nitrite concentrations were measured. Since thromboxane A2 (TXA2), an eicosanoid produced by activated platelets and endothelium, is frequently involved in the pathophysiology of cardiovascular diseases,11 the plasma levels of thromboxane B2 (TXB2), the stable metabolite of TXA2, were also quantified.
Materials and Methods Experimental Groups Male Wistar rats (150 to 200 g at the start of the study) were provided by Central Animal Services of the university, and were classified as follows: (1) a control group (n ⫽ 14), sham-operated rats that received tap water alone; (2) the L-NAME group (n ⫽ 31), rats that received L-NAME in drinking water; (3) the 2K-1C group (n ⫽ 26), rats with a silver clip placed around one renal artery; (4) the DM group (n ⫽ 27), rats that received a single intraperitoneal (IP) dose of streptozotocin, 60 mg/kg; and (5) the 2K-1C plus DM group (n ⫽ 26), rats that received a single www.chestjournal.org
dose of streptozotocin and had one renal artery clipped. The animals were killed after 3 weeks. This investigation conformed to the National Research Council guidelines. Induction of Goldblatt II Hypertension Rats were anesthetized with sodium pentobarbital, 40 mg/kg IP, a left flank incision was done to allow a silver clip (0.2-mm inner diameter) to be placed around the renal artery.12 In sham-operated control rats, the clip was removed immediately after being fixed around the vessel and the kidney was repositioned in the abdomen. Long-term Treatment With L-NAME L-NAME was dissolved in the drinking water at a concentration of 1.2 mM to give a daily intake of approximately 75 mol per rat per day.10 The average daily intake of water and food did not differ significantly among the five groups. Streptozotocin Administration Diabetes was induced with an IP injection of streptozotocin, 60 mg/kg, dissolved in citrate buffer. BP Measurements Arterial BP was measured twice a week by a tail-cuff method,13 and the mean of these two determinations was considered to be the mean for that week. The same procedure was applied to weight gain. Heart Weight Indexes After 3 weeks of treatment, the rats were killed with a lethal dose of sodium pentobarbital, and the heart was removed, washed with saline solution, and then fixed in 10% formalin for 24 h. Subsequently, the atria were removed and the ventricles weighed to obtain the heart weight (HW). The left ventricular weight (LVW) and right ventricular weight (RVW) were determined after separating the ventricles. The HW index (HWI) was calculated by dividing the HW by the body weight (BW). Correspondingly, the LVW index (LVWI) and the RVW index (RVWI) were calculated by dividing the LVW and RVW by the BW. Histologic Analysis The left and right ventricles were cut into five equidistant rings perpendicular to the long axis of the heart. All rings were embedded in paraffin, and 5-m sections were stained with hematoxylin-eosin. For each rat, one section of each of the five ventricular rings was studied by light microscopy. Cardiomyocyte size was determined by measuring the cell diameters by using an optical microscope system supplied with a graduated eyepiece micrometer and a 100 ⫻ objective (1,000 ⫻ magnification). Fifteen cells randomly selected from the subepicardial, midmyocardial, and subendocardial regions were measured in each animal of the different experimental groups. The investigator responsible was unaware of the corresponding groups for slides examined. Stereological Procedures Myocardial lesions were evaluated quantitatively using a stereological method that allowed the blind analysis of lesions CHEST / 122 / 4 / OCTOBER, 2002
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resulting from recent infarcts, ie, focal lesions showing the onset or progress of death in cardiac myocytes, as well as the presence of fibrosis in scar tissue. In this method,14,15 the counting grid consisted of a combination of points and parallel lines (in this case, a 10 ⫻ objective with a graticule containing 100 points and 10 parallel lines). Each point corresponded to the center of a polygon of known area. The distance between the lines served as the point of reference for calibrating the system and also for determining the area of the lesion. For sample areas with a square format, the area (distance squared) was given by the formula: A ⫽ ⌫共Ap兲 ⫻ Pt where A was the area of the test point, ⌫ (Ap) was the sum of areas of the test points used, and Pt was the total number of test points. Western Blotting of Myocardial NOS3 For Western blotting, the thoracic cavity was opened and the heart rapidly removed. The left ventricle was minced coarsely and immediately homogenized in approximately 10 volumes of solubilization buffer (1% Triton-X 100, 100 mM Tris-HCl, pH 7.4, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM ethylenediamine tetra-acetic acid, 2 mM phenylmethylsulfonyl fluoride, and 0.1 mg of aprotinin per milliliter) at 4°C using a polytron PTA 20S generator (model PT 10/35; Brinkman Instruments; Westbury, NY) operated at maximum speed for 30 s. The extracts were centrifuged at 10,000g at 4°C for 30 min to remove insoluble material. The resulting supernatant was used for the assay. Protein concentrations were determined with a dye-binding assay using Bio-Rad reagent (Bio-Rad Laboratories; Richmond, CA) and bovine serum albumin (BSA) as the standard. Equal amounts of total protein were for all samples diluted with Laemmli sample buffer containing 100 mM dithiothreitol and heated in a boiling water bath for 4 min, after which they were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (8% polyacrylamide gels) in a miniature gel apparatus (Mini-Protean; Bio-Rad Laboratories). Electrotransfer of proteins from the gel to the nitrocellulose membrane was done for 90 min at 120 V (constant) in a miniature transfer apparatus (Mini-Protean). Nonspecific protein binding to the nitrocellulose membrane was reduced by preincubating the filter overnight at 4°C in blocking buffer (5% defatted dry milk, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20). The nitrocellulose membrane blot was incubated with anti-NOS3 antibodies (Santa Cruz Biotechnology; Santa Cruz, CA) diluted in 10 mL of blocking buffer (3% BSA instead of defatted dry milk) overnight at 4°C, and then washed for 60 min in blocking buffer without milk or BSA. The blots were subsequently incubated with 2 Ci of [125I] protein A, 30 Ci/g, in 10 mL of blocking buffer for 2 h at room temperature, and then washed again for 30 min as described above. [125I] Protein A bound to the antibody was detected by autoradiography using preflashed Kodak XAR film (Eastman Kodak; Rochester, NY) stored at ⫺ 80° for 24 h. Band intensities were quantified by optical densitometry of the developed autoradiographs. Plasma Measurements Thromboxane B2: Venous blood samples were collected from the tail vein into tubes containing ethylenediamine tetra-acetic acid at baseline and 3 weeks after treatment. The plasma was separated by centrifugation and stored at 20°C until assayed. Plasma samples were extracted using C18 reverse-phase cartridges (Waters Corporation; Milford, MA), and the TXB2 levels 1414
were determined with a commercial enzyme immunoassay (Cayman Chemical; Ann Arbor, MI). The detection limit of this assay was 13.3 pg/mL. Nitrates and Nitrites: The circulating levels of nitrates/nitrites were used to estimate NO production.16 Blood samples from a caudal vein were collected into heparinized tubes at baseline and 3 weeks after treatment. The plasma was separated by centrifugation and stored at 20°C until assayed. Plasma nitrates/nitrites levels were determined spectrophotometrically using a commercial assay kit (Cayman Chemical). Briefly, plasma samples were filtered at 4°C by centrifugation through filters with a molecular weight cutoff of 10,000 (Centricon; Millipore; Milford, MA). After the addition of nitrate reductase to convert nitrates to nitrites, Griess reagent was added to the mixture and the nitrite concentrations were determined by measuring the resulting absorbance at 550 nm in a SpectraMax 340 multiwell plate reader (Molecular Devices; San Diego, CA). The nitrite concentrations of the samples were calculated by reference to a standard curve of nitrite run in parallel. The detection limit of this assay was 2.5 M. Insulin: Insulin levels were determined at baseline and 3 weeks after treatment using a commercial enzyme immunoassay (Rat insulin enzyme immunoassay kit; SPI-BIO; Massy, France). Creatinine: Renal function was assessed by measuring creatinine levels by the Jaffe method at baseline and 3 weeks after treatment. Statistical Analysis Results are expressed as mean ⫾ SEM. Analysis of variance (ANOVA) for repeated measurements was used to assess the differences in BW and tail-cuff pressure (TCP). One-way ANOVA was used to compare HW, LVW, RVW, HWI, LVWI, and RVWI. When ANOVA results were significant, the Duncan test was used to determine the differences among groups. In all cases, p ⬍ 0.05 was considered to be significant. All calculations were done using SigmaStat software (SPSS Science; Chicago, IL).
Results BW and TCP Table 1 and Figure 1 show the BW and TCP, respectively. The two groups of rats treated with streptozotocin had a lower BW than the control group (p ⬍ 0.05) after 3 weeks. TCP increased equally in the L-NAME, 2K-1C, and 2K-1C plus DM groups (p ⬍ 0.05). TCP increased in the DM group after 3 weeks (p ⬍ 0.05). Cardiac Weight and Cardiac Weight Indexes Table 1 shows the HW, RVW, and LVW, and their respective weight indexes. There were no significant differences in these parameters in the control, L-NAME, and DM groups. In contrast, HW, HWI, LVW, and LVWI increased after 3 weeks of treatment (p ⬍ 0.05) in the 2K-1C and the 2K-1C plus DM groups when compared to the control group. Histologic and Stereological Analysis There were no evident histologic lesions in the hearts of control rats. The heart of L-NAME–treated Laboratory and Animal Investigations
Table 1—Characteristics of Study Patients in the Experimental Groups After 3 Weeks of Study* Parameters
Control (n ⫽ 16)
L-NAME (n ⫽ 31)
2K-1C (n ⫽ 21)
DM (n ⫽ 27)
2K-1C Plus DM (n ⫽ 20)
BW, g HW, mg HWI, mg/g LVW, mg LVWI, mg/g RVW, mg RVWI, mg/g
262 ⫾ 19 700 ⫾ 54 2.6 ⫾ 0.3 550 ⫾ 70 2.1 ⫾ 0.3 150 ⫾ 28 0.6 ⫾ 0.1
240 ⫾ 36 640 ⫾ 98 2.7 ⫾ 0.2 510 ⫾ 82 2.1 ⫾ 0.2 130 ⫾ 21 0.5 ⫾ 0.1
250 ⫾ 26 870 ⫾ 61† 3.5 ⫾ 0.6† 710 ⫾ 54† 2.9 ⫾ 0.2† 160 ⫾ 15 0.6 ⫾ 0.1
205 ⫾ 40† 570 ⫾ 97 2.8 ⫾ 0.3 430 ⫾ 80 2.1 ⫾ 0.2 140 ⫾ 25 0.6 ⫾ 0.1
198 ⫾ 33† 750 ⫾ 47† 3.8 ⫾ 0.8† 625 ⫾ 72† 3.2 ⫾ 0.2† 125 ⫾ 20 0.7 ⫾ 0.1
*Data are presented as mean ⫾ SEM. †p ⬍ 0.05 vs control.
rats showed unambiguous foci of reparative fibrosis consistent with organized myocytolytic necrosis. In addition, recent myocardial necrosis with granulation tissue showing blood vessels and many fibroblasts was also seen in these rats (Fig 2). Furthermore, some of these rats showed interstitial and perivascular cardiac fibrosis. The rats in the other three experimental groups had myocardial lesions consisting mainly of recent myocardial infarcts with dark coagulated myocytes, contraction bands, and dead myocytes lacking nuclei. Although similar lesions were observed in these three groups, the myocardial lesions in the DM group were greater than in the 2K-1C group. Even more widespread lesions were present in the 2K-1C plus DM group compared to the 2K-1C group and the DM group (Fig 3). Figure 4 provides a quantitative comparison of the myocardial lesions in the different groups studied. In agreement with the qualitative histologic analysis, stereological analysis showed larger myocardial le-
Figure 1. TCP during the 3 weeks of study. E ⫽ control group (n ⫽ 16); F ⫽ L-NAME group (n ⫽ 31); f ⫽ 2K-1C group (n ⫽ 21); 䡺 ⫽ DM group (n ⫽ 27); ƒ ⫽ 2K-1C plus DM group (n ⫽ 20). The points represent the mean ⫾ SEM. *p ⬍ 0.05 vs basal for L-NAME, 2K-1C, and 2K-1C plus DM groups. #p ⬍ 0.05 for all groups at week 3 vs control group. www.chestjournal.org
sions in the DM group and the 2K-1C plus DM group compared to the 2K-1C group (p ⬍ 0.05). The diameters of myocytes in the DM group were similar to those in control rats in the three myocardial regions (Table 2). In contrast, the diameters of midmyocardial and subepicardial myocytes were greater in the 2K-1C group and the 2K-1C plus DM group compared to the control group (p ⬍ 0.05 for both). A small increase in the diameter of midmyocardial myocytes was observed in the L-NAME group (p ⬍ 0.05). Western Blotting of Myocardial NOS3 Figure 5 shows a representative Western blot of NOS3 in the hearts of rats from the different groups, as well as the average ⫾ SEM values (n ⫽ 4) of densitometric readings obtained with anti-NOS3 antibody. Increased levels of NOS3 protein were seen in the 2K-1C, DM, and 2K-1C plus DM groups (19 to 23% higher than in the control group; p ⬍ 0.05). Treatment with L-NAME produced a more pronounced increase in the NOS3 protein levels (44% higher than in the control group; p ⬍ 0.05).
Figure 2. L-NAME: subendocardial infarct showing a wellestablished granulation tissue with blood vessels, many fibroblasts, and little collagenization (Massons trichrome, original ⫻ 128). CHEST / 122 / 4 / OCTOBER, 2002
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DM group and the 2K-1C plus DM group (p ⬍ 0.05 for both).
Discussion
Figure 3. 2K-1C plus DM: recent myocardial infarct with dark coagulated myocytes lacking nuclei (hematoxylin-eosin, original ⫻ 128).
Plasma Measurements: Nitrates and Nitrites: The circulating levels of nitrates/nitrites were unaltered in the control, 2K-1C, and DM groups, but decreased in the LNAME and 2K-1C plus DM groups (p ⬍ 0.05 for both; Fig 6, top, A). TXB2: TXB2 concentrations increased after 3 weeks in the four experimental groups (p ⬍ 0.05), but not in the control group (Fig 6, bottom, B). This increase was more marked in the L-NAME and 2K-1C plus DM groups. Insulin, Glucose, and Creatinine: Table 3 shows the plasma insulin, glucose, and creatinine levels at baseline and after 3 weeks. There were no significant changes in the creatinine levels in all of the experimental groups (Table 3). Marked increases in glucose and decreases in insulin levels occurred in the
Figure 4. Total area of myocardial lesions (micrometers squared) after 3 weeks. Columns represent the mean ⫾ SEM. *p ⬍ 0.05 vs 2K-1C group. 1416
In the present study, the association of hypertension and DM resulted in more severe cardiomyopathy than would be anticipated with each condition. The severity of the hypertensive-diabetic cardiomyopathy resembled that seen after the inhibition of NO synthesis, thus suggesting a role for NO in both cardiomyopathies. Although the histologic changes in the heart were qualitatively alike in the 2K-1C, DM, and 2K-1C plus DM groups, quantitative analysis indicated larger lesions in the last group. These findings are comparable to those found at autopsy in a large group of patients with either hypertension, DM, or both conditions.17 In this case, the hearts of patients with concomitant hypertension and DM showed more severe and generalized fibrosis than those of patients with hypertension or DM alone.17 The histologic analysis in the present study suggested that the severity of L-NAME–induced myocardial lesions was analogous to that in rats with DM and superimposed hypertension (the 2K-1C plus DM group). This suggestion is supported by the observation that, although the total area of myocardial lesions produced by L-NAME differed from that in the 2K-1C plus DM group, the multiple foci of reparative fibrosis and fresh myocardial necrosis seen after treatment with L-NAME resembled the more severe and extensive lesions present in the 2K-1C plus DM group. The multiple foci of reparative fibrosis seen after treatment with L-NAME were similar to those described in previous studies,10,18 and represented remotely infarcted myocardium. Thus, long-term inhibition of NO synthesis may cause an early activation of the pathophysiologic mechanisms underlying the more recent cardiac lesions found in the 2K-1C plus DM group. An increase in BP enhances the mechanical stress on cardiomyocytes and is an important stimulus for cardiac hypertrophy.19 The similar rises in BP levels in the 2K-1C, 2K-1C plus DM, and L-NAME groups contrasted with the different grades of hypertrophic responses in these experimental groups, and suggested that mechanisms other than a pressure overload are also activated.10 Indeed, hypertrophy has been suggested to be related to humoral factors such as plasma renin activity or angiotensin II levels, but not to the mechanical overload.20 The larger HW, HWI, LVW, and LVWI were associated with a significantly greater myocyte diameter in the midmyocardium and subepicardium in Laboratory and Animal Investigations
Table 2—Myocyte Diameter in the Left Ventricle Subendocardium, Midmyocardium, and Subepicardium of the Experimental Groups* Regions
Control (n ⫽ 6)
L-NAME (n ⫽ 6)
2K-1C (n ⫽ 6)
DM (n ⫽ 6)
2K-1C Plus DM (n ⫽ 6)
Subendocardium, m Midmyocardium, m Subepicardium, m
12.8 ⫾ 0.8 13.0 ⫾ 0.2 10.3 ⫾ 0.8†
13.4 ⫾ 0.2 14.8 ⫾ 0.8† 12.2 ⫾ 0.5
13.7 ⫾ 0.4 18.3 ⫾ 1.6†‡ 13.2 ⫾ 0.4†
12.0 ⫾ 0.8 13.5 ⫾ 0.5 10.8 ⫾ 0.7‡
13.7 ⫾ 0.5 18.2 ⫾ 1.3†‡ 13.4 ⫾ 0.7†
*Data are presented as mean ⫾ SEM. †p ⬍ 0.05 vs control. ‡p ⬍ 0.05 vs the other two regions.
the 2K-1C group and the 2K-1C plus DM group. However, no remarkable increases were observed in the cardiac indexes and myocyte diameter of LNAME–treated rats. These findings confirmed previous studies demonstrating that ventricular hypertrophy and increased cardiomyocyte diameter occur in 2K-1C–treated rats or after aortic stenosis, but not after treatment with L-NAME.10,20 –22 This lack of a hypertrophic response after treatment with L-NAME may be explained by a limitation imposed on ventricular growth attributed to a more pronounced loss of cardiomyocytes that undergo apoptosis after inhibition of NO biosynthesis.23 Interestingly, one study24 has demonstrated that different doses of L-NAME, which produced the same increase in systolic BP, resulted in fibrosis that was proportional to the dose of L-NAME administered. Thus, a more powerful inhibition of NO biosynthesis may cause more severe myocardial lesions in rats treated with increasing doses of L-NAME, or when DM is superimposed on hypertension. Rats treated with streptozotocin, 60 mg/kg, gained weight and showed hypoinsulinemia and hyperglyce-
Figure 5. Representative Western blot of the NOS3 levels in the hearts of rats from control group (CT), L-NAME group, DM group, 2K-1C group, and 2K-1C plus DM group (densitometric readings [mean ⫾ SEM], n ⫽ 4 blots per group). *p ⬍ 0.05 vs control group; **p ⬍ 0.01 vs control group. IB ⫽ immunoblot. www.chestjournal.org
mia as reported previously with a slightly lower dose of streptozotocin (55 mg/kg).6 The increase in the BP of diabetic rats agrees with previous findings showing a trend for arterial pressure to increase when glucose
Figure 6. Top, A: basal and week 3 serum nitrate/nitrite levels. Bottom, B: basal and week 3 serum TXB2 in control group (open columns), L-NAME group (filled columns), 2K-1C group (horizontally striped columns), DM group (vertically striped columns), and 2K-1C plus DM group (stippled columns). Columns represent the mean ⫾ SEM. *p ⬍ 0.05 vs basal. CHEST / 122 / 4 / OCTOBER, 2002
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Table 3—Plasma Creatinine, Glucose, and Insulin Concentrations After 3 Weeks in the Groups Studied* Parameters Creatinine, mg/dL Basal Week 3 Glucose, mg/dL Basal Week 3 Insulin, ng/mL Basal Week 3
Control (n ⫽ 16)
L-NAME (n ⫽ 31)
2K-1C (n ⫽ 21)
DM (n ⫽ 27)
2K-1C Plus DM (n ⫽ 20)
0.4 ⫾ 0.1 0.3 ⫾ 0.1
0.4 ⫾ 0.1 0.3 ⫾ 0.1
0.4 ⫾ 0.1 0.3 ⫾ 0.1
0.3 ⫾ 0.1 0.4 ⫾ 0.1
0.4 ⫾ 0.1 0.4 ⫾ 0.1
114 ⫾ 15 105 ⫾ 10
112 ⫾ 11 84 ⫾ 11
111 ⫾ 14 104 ⫾ 13
105 ⫾ 20 361 ⫾ 115†‡
150 ⫾ 24 427 ⫾ 107†‡
1.0 ⫾ 0.1 0.6 ⫾ 0.1
0.9 ⫾ 0.1 0.8 ⫾ 0.1
0.9 ⫾ 0.2 0.8 ⫾ 0.1
0.9 ⫾ 0.1 0.1 ⫾ 0.1†‡
0.8 ⫾ 0.1 0.0 ⫾ 0.0†‡
*Data are presented as mean ⫾ SEM. †p ⬍ 0.05 vs control. ‡p ⬍ 0.05 vs basal.
levels are not controlled.25 Importantly, levels of NOS3 protein were increased in diabetic rat hearts, maybe as a result of enhanced oxidative stress caused by hyperglycemia.26 Although similar increases (19 to 23%) in the levels of NOS3 protein were also observed in the 2K-1C group and the 2K-1C plus DM group, treatment with L-NAME produced the greatest increase in NOS3 protein (44% higher than the control group). Curiously, the increased expression of NOS3 did not produce concomitant changes in the plasma nitrate/nitrite levels in any of the all groups. The plasma nitrate/nitrite concentrations decreased only in the L-NAME and 2K-1C plus DM groups, which showed the most severe myocardial lesions. Although the precise relationship between serum nitrate/nitrite levels and NO production is still unclear,27 these results suggest an important decrease in NO bioavailability16 in both groups. Indeed, diabetic rats show impaired acetylcholineinduced aortic relaxation and nitrate/nitrite production attributed to an abnormal metabolism of NO.28 A reduced bioavailability of NO caused by a lack of substrates or cofactors may lead to the uncoupling of NOS3 or increased NO degradation by reactive oxygen species.9,29 Complex mechanisms regulate NOS3 at the transcriptional and posttranscriptional levels.30 Although NOS3 is expressed predominantly in coronary and endocardial endothelial cells, and to a lesser extent in cardiac myocytes,31 the regulation of the expression of this enzyme in the heart is not well studied, especially in pathophysiologic conditions. Increased expression of NOS3 was reported in the heart after banding of the aorta,32 or after the development of hypertension in spontaneously hypertensive rats.33 The increased NOS3 levels seen in the 2K-1C group and the L-NAME group did not agree with previous studies showing decreased levels of NOS3 messenger RNA in 2K-1C–treated rats,34 or after 6 weeks of 1418
treatment with L-NAME, 60 mg/kg/d.35 Differences in rat strain, and in the dose and duration of treatment with L-NAME, could explain the discrepancies between these studies. These same reasons could account for the extent and severity of the myocardial lesions seen here compared to previous studies. A hypercoagulable or prothrombotic state, partially due to platelet abnormalities, has been described in hypertension36 and DM,37 and may involve changes in the bioavailability of NO. This is mainly because endogenous NO, besides controlling vascular tone, also regulates leukocyte adhesion and platelet aggregation. Thus, the more severe myocardial lesions and greater increases in TXB2 levels seen in L-NAME–treated and 2K-1C plus DM-treated rats may be partially explained by a more extensive reduction in the availability of NO in both groups. This greater reduction in NO availability may have increased platelet activation and released more TXA2 than in the other experimental groups. This suggestion is in line with studies demonstrating that hyperglycemia per se, besides inducing platelet hyperactivity,37 can also chemically inactivate NO,38 thereby potentiating the decreases of NO bioavailability seen in hypertension.7 In further support of this hypothesis, inhibitors of NO biosynthesis have been shown to enhance platelet aggregation.39 In addition, increased TXA2 production, indicative of platelet activation, has been found in acute coronary disease.40 Together, these findings point to a lack of NO as a key factor in the diabetic-hypertensive patients. There is one limitation in the present study. The reduced NO availability we have shown in diabetichypertensive cardiomyopathy could be further confirmed by examining the effects of a treatment that increases the availability of NO. Such a treatment would probably result in a normalization or attenuation of alterations in diabetic-hypertensive cardioLaboratory and Animal Investigations
myopathy. Since this issue has not been previously addressed, further studies exploring this possibility are warranted. The experimental data presented here are supported by a recently published clinical study41 comparing hypertensive diabetic, hypertensive, and healthy subjects. Interestingly, hypertensive diabetic patients had the highest plasma levels of von Willebrand factor and soluble P-selectin, which reflect a prothrombotic state associated with low NO availability.41 Therefore, a major clinical implication of our findings is that improving NO availability might serve as an important element in the therapy of diabetic-hypertensive patients. Indeed, a decreased capacity to enhance NO production in treated diabetic nephropathy has been suggested to accelerate the renal disease.42 In conclusion, our data support the hypothesis that the cardiac abnormalities found in hypertension are similar to found in other pathophysiologic conditions such as DM,2 probably because of the activation of related underlying mechanisms. Moreover, the combination of hypertension and DM enhanced the severity of these abnormalities to such an extent that the effect resembled dose-dependent L-NAME– induced cardiomyopathy. These findings suggest a role for the NO system in the pathophysiology of diabetic-hypertensive cardiomyopathy. References 1 Factor SM, Minase T, Sonnenblick EH. Clinical and morphological features of human hypertensive-diabetic cardiomyopathy. Am Heart J 1980; 99:446 – 458 2 Lip GY, Felmeden DC, Li-Saw-Hee FL, et al. Hypertensive heart disease: a complex syndrome or a hypertensive “cardiomyopathy”? Eur Heart J 2000; 21:1653–1665 3 Grossman E, Messerli FH. Diabetic and hypertensive heart disease. Ann Intern Med 1996; 125:304 –310 4 Sowers JR, Epstein M, Frohlich ED. Diabetes, hypertension, and cardiovascular disease: an update. Hypertension 2001; 37:1053–1059 5 Factor SM, Minase T, Cho S, et al. Coronary microvascular abnormalities in the hypertensive-diabetic rat: a primary cause of cardiomyopathy? Am J Pathol 1984; 116:9 –20 6 Mathis DR, Liu SS, Rodrigues BB, et al. Effect of hypertension on the development of diabetic cardiomyopathy. Can J Physiol Pharmacol 2000; 78:791–798 7 Kojda G, Harrison D. Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure. Cardiovasc Res 1999; 43:562–571 8 Bayraktutan U, Yang ZK, Shah AM. Selective dysregulation of nitric oxide synthase type 3 in cardiac myocytes but not coronary microvascular endothelial cells of spontaneously hypertensive rat. Cardiovasc Res 1998; 38:719 –726 9 Hink U, Li H, Mollnau H, et al. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res 2001; 88:E14 –E22 10 Moreno H Jr, Metze K, Bento AC, et al. Chronic nitric oxide inhibition as a model of hypertensive heart muscle disease. Basic Res Cardiol 1996; 91:248 –255 www.chestjournal.org
11 Humphrey PP, Hallet P, Hornby EJ, et al. Pathophysiological actions of thromboxane A2 and their pharmacological antagonism by thromboxane receptor blockade with GR32191. Circulation 1990; 81:I42–I52, I59 –I60 12 Goldblatt HLJ, Hanzal RF, Summerville WW. Studies on experimental hypertension: the production of persistent elevation of systolic blood pressure by means of renal ischemia. J Exp Med 1934; 59:347–379 13 Zatz R. A low cost tail-cuff method for the estimation of mean arterial pressure in conscious rats. Lab Anim Sci 1990; 40:198 –201 14 Weibel E. Stereological methods: practical methods for biological morphometry (vol 1). New York, NY: Academic Press, 1979 15 Coelho-Filho OR, De Luca IM, Tanus-Santos JE, et al. Pravastatin reduces myocardial lesions induced by acute inhibition of nitric oxide biosynthesis in normocholesterolemic rats. Int J Cardiol 2001; 79:215–221 16 Jungersten L, Edlund A, Petersson AS, et al. Plasma nitrate as an index of nitric oxide formation in man: analyses of kinetics and confounding factors. Clin Physiol 1996; 16:369 –379 17 van Hoeven KH, Factor SM. A comparison of the pathological spectrum of hypertensive, diabetic, and hypertensivediabetic heart disease. Circulation 1990; 82:848 – 855 18 Moreno H Jr, Piovesan Nathan L, Pereira Costa SK, et al. Enalapril does not prevent the myocardial ischemia caused by the chronic inhibition of nitric oxide synthesis. Eur J Pharmacol 1995; 287:93–96 19 Hefti MA, Harder BA, Eppenberger HM, et al. Signaling pathways in cardiac myocyte hypertrophy. J Mol Cell Cardiol 1997; 29:2873–2892 20 Matsubara BB, Matsubara LS, Zornoff LA, et al. Left ventricular adaptation to chronic pressure overload induced by inhibition of nitric oxide synthase in rats. Basic Res Cardiol 1998; 93:173–181 21 Bartunek J, Weinberg EO, Tajima M, et al. Chronic N(G)nitro-L-arginine methyl ester-induced hypertension: novel molecular adaptation to systolic load in absence of hypertrophy. Circulation 2000; 101:423– 429 22 Arnal JF, el Amrani AI, Chatellier G, et al. Cardiac weight in hypertension induced by nitric oxide synthase blockade. Hypertension 1993; 22:380 –387 23 Pessanha MG, Mandarim-de-Lacerda CA. Influence of the chronic nitric oxide synthesis inhibition on cardiomyocytes number. Virchows Arch 2000; 437:667– 674 24 Pechanova O, Bernatova I, Pelouch V, et al. L-NAMEinduced protein remodeling and fibrosis in the rat heart. Physiol Res 1999; 48:353–362 25 Fitzgerald SM, Brands MW. Nitric oxide may be required to prevent hypertension at the onset of diabetes. Am J Physiol Endocrinol Metab 2000; 279:E762–E768 26 Stockklauser-Farber K, Ballhausen T, Laufer A, et al. Influence of diabetes on cardiac nitric oxide synthase expression and activity. Biochim Biophys Acta 2000; 1535:10 –20 27 Moshage H. Nitric oxide determinations: much ado about NO.-thing? Clin Chem 1997; 43:553–556 28 Kobayashi T, Kamata K. Effect of chronic insulin treatment on NO production and endothelium-dependent relaxation in aortae from established STZ-induced diabetic rats. Atherosclerosis 2001; 155:313–320 29 Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 2000; 87:840 – 844 30 Govers R, Rabelink TJ. Cellular regulation of endothelial nitric oxide synthase. Am J Physiol Renal Physiol 2001; 280:F193–F206 31 Shah AM, MacCarthy PA. Paracrine and autocrine effects of CHEST / 122 / 4 / OCTOBER, 2002
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nitric oxide on myocardial function. Pharmacol Ther 2000; 86:49 – 86 Barton CH, Ni Z, Vaziri ND. Effect of severe aortic banding above the renal arteries on nitric oxide synthase isotype expression. Kidney Int 2001; 59:654 – 661 Vaziri ND, Ni Z, Oveisi F, et al. Effect of antioxidant therapy on blood pressure and NO synthase expression in hypertensive rats. Hypertension 2000; 36:957–964 Kobayashi N, Kobayashi K, Hara K, et al. Benidipine stimulates nitric oxide synthase and improves coronary circulation in hypertensive rats. Am J Hypertens 1999; 12:483– 491 Kobayashi N, Hara K, Watanabe S, et al. Effect of imidapril on myocardial remodeling in L-NAME-induced hypertensive rats is associated with gene expression of NOS and ACE mRNA. Am J Hypertens 2000; 13:199 –207 Lip GY, Li-Saw-Hee FL. Does hypertension confer a hypercoagulable state? J Hypertens 1998; 16:913–916 Trovati M, Anfossi G. Insulin, insulin resistance and platelet function: similarities with insulin effects on cultured vascular smooth muscle cells. Diabetologia 1998; 41:609 – 622
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38 Brodsky SV, Morrishow AM, Dharia N, et al. Glucose scavenging of nitric oxide. Am J Physiol Renal Physiol 2001; 280:F480 –F486 39 Chen LY, Mehta JL. Variable effects of L-arginine analogs on L-arginine-nitric oxide pathway in human neutrophils and platelets may relate to different nitric oxide synthase isoforms. J Pharmacol Exp Ther 1996; 276:253–257 40 Hirsh PD, Hillis LD, Campbell WB, et al. Release of prostaglandins and thromboxane into the coronary circulation in patients with ischemic heart disease. N Engl J Med 1981; 304:685– 691 41 Ouvina SM, La Greca RD, Zanaro NL, et al. Endothelial dysfunction, nitric oxide and platelet activation in hypertensive and diabetic type II patients. Thromb Res 2001; 102: 107–114 42 Earle KA, Mehrotra S, Dalton RN, et al. Defective nitric oxide production and functional renal reserve in patients with type 2 diabetes who have microalbuminuria of African and Asian compared with white origin. J Am Soc Nephrol 2001; 12:2125–2130
Laboratory and Animal Investigations