The Effects of Atorvastatin on Endothelial Function in Diabetic Patients and Subjects at Risk for Type 2 Diabetes

PANAYIOTIS A. ECONOMIDES, ANTONELLA CASELLI, ELIZABETH TIANI, LALITA KHAODHIAR, EDWARD S. HORTON, AND ARISTIDIS VEVES

Joslin Diabetes Center (P.A.E., E.T., E.S.H.) and Microcirculation Laboratory (A.C., L.K., A.V.), Beth Israel Deaconess Downloaded from https://academic.oup.com/jcem/article/89/2/740/2840787 by guest on 28 September 2021 Medical Center, Harvard Medical School, Boston Massachusetts 02215

We have investigated the effect of atorvastatin on the endo- risk group, there was a decrease in the C-reactive protein thelial function of patients with diabetes and subjects at risk [median (25–75 percentile), 0.12 mg/dl (0.07–0.27 mg/dl) at exit for type 2 diabetes in a 12-wk, prospective, randomized, visit vs. 0.24 mg/dl (0.07–0.35 mg/dl) at baseline; P < 0.05] and placebo-controlled, double-blind clinical trial. The flow- TNF␣ [median (25–75 percentile), 2.6 pg/ml (1.8–4.1 pg/ml) at mediated dilation (FMD; endothelium dependent) and nitro- exit visit vs. 4.4 pg/ml (3.6–6.0 pg/ml) at baseline; P < 0.05] in glycerin-induced dilation (endothelium independent) in the the atorvastatin-treated patients, whereas in the diabetes brachial artery and the vascular reactivity at the forearm skin group, a decrease in endothelin-1 (mean ؎ SD, 0.97 ؎ 0.29 pg/ml were measured. FMD improved in the atorvastatin-treated, at exit visit vs. 1.19 ؎ 0.42 pg/ml at baseline; P < 0.05) and at-risk subjects [median (25–75 percentile), 7.2% (2.9–9.6%) at plasminogen activator inhibitor-1 [median (25–75 percentile), exit visit vs. 6.6% (2.9–9.5%) at baseline; P < 0.05]. A similar 18 ng/ml (9–24 ng/ml) at exit visit vs. 27 ng/ml (7–41 ng/ml) at improvement of FMD was found in atorvastatin-treated dia- baseline; P < 0.05] were observed. We conclude that atorva- betic patients [median (25–75 percentile), 5.6 (3.9–7.9) at exit statin improves endothelial function and decreases levels of No changes were markers of endothelial activation and inflammation. (J Clin .[0.07 ؍ visit vs. 4.2 (3.2–7.2) at baseline; P observed in nitroglycerin-induced dilation and the microcir- Endocrinol Metab 89: 740–747, 2004) culation reactivity measurements in either group. In the at-

NDOTHELIAL FUNCTION IS abnormal in both the nitric oxide synthase expression and nitric oxide production, E macro- and microcirculation in subjects with type 1 or antiinflammatory action and effects on thrombosis, and fa- type 2 diabetes mellitus (1–3). Furthermore, endothelial func- vorable effects on plaque architecture and stability (9–11). tion is impaired in healthy subjects who are at risk of de- Thus, it is currently accepted that statins have pleiotropic veloping type 2 diabetes by virtue of having one or both properties that may contribute to the observed reduction in parents with type 2 diabetes, with or without impaired glu- cardiovascular disease (12). cose tolerance (3, 4). In addition, subjects at risk of devel- Direct action on the endothelial cell has emerged as one of oping diabetes have increased levels of inflammatory cyto- the most prominent mechanisms through which statins may kines and biochemical markers of endothelial dysfunction exert their beneficial effects (13, 14). Therefore, if this hy- suggesting that inflammation and endothelial dysfunction pothesis is correct, it would be expected that treatment with may be contributing factors to the development of diabetes statins should improve the impaired endothelial function (3, 5, 6). when it is impaired in conditions such as diabetes or the Statins have been shown to lower serum cholesterol levels prediabetic stage, even under conditions of normolipidemia. markedly and reduce cardiovascular morbidity and mortal- The main objective of this study was to study the effect of ity (7). Although it was initially thought that the reduction atorvastatin, one of the most powerful statins in providing in cardiovascular disease was solely related to their lipid- total cholesterol, LDL, and triglyceride reduction, on the lowering capacity, over the last few years, it has been rec- endothelial function of the micro- and macrocirculation. To ognized that statins may additionally act through mecha- this end, we have conducted a double-blind, randomized, nisms that are independent of low-density lipoprotein (LDL) placebo-controlled clinical trial that included subjects with cholesterol lowering to provide a 30% relative risk reduction impaired endothelial function divided into one group of of major coronary events (8). Several such mechanisms have healthy subjects at risk of developing diabetes and one group been proposed, including the up-regulation of endothelial of patients with type 1 or 2 diabetes.

Abbreviations: CRP, C-reactive protein; cv, coefficient of variation; Subjects and Methods FMD, flow-mediated dilation; HDL, high-density lipoprotein; LDL, low- density lipoprotein; PAI, plasma activator inhibitor; tPA, tissue plas- Subjects minogen activator. A total of 77 subjects were included in the study. Subjects were JCEM is published monthly by The Endocrine Society (http://www. enrolled if they were between the ages of 21 and 80 yr and at risk for type endo-society.org), the foremost professional society serving the en- 2 diabetes (either having a first-degree relative with type 2 diabetes and docrine community. normal glucose tolerance or impaired glucose tolerance defined as a 2-h

740 Economides et al. • Effect of Atorvastatin in Diabetes J Clin Endocrinol Metab, February 2004, 89(2):740–747 741 blood glucose value between 140–199 mg/dl during a 75-g oral glucose Endothelium-independent vasodilation in the macrocirculation was as- tolerance test) or had type 1 or type 2 diabetes. Diabetes was defined sessed by studying brachial artery diameter changes 5 min after the according to the recommendations of the American Diabetes Associa- administration of 400 ␮g of sublingual nitroglycerine (nitroglycerine- tion Expert Committee on the Classification and Diagnosis of Diabetes induced dilation). This test was performed 15 min after the reactive (15). hyperemia test and after obtaining a new baseline reading. To avoid confounding factors known to affect endothelial function After the baseline clinical and laboratory evaluations, participants in and/or glucose metabolism, the following exclusion criteria were ap- all three groups were randomized to either 20 mg atorvastatin treatment plied: treatment with lipid-lowering drugs during the previous 3 or corresponding placebo. The randomization procedure was carried months, cardiac arrhythmia, congestive heart failure, uncontrolled hy- out in a double-blind fashion, and the codes were kept masked until the pertension, recent stroke, chronic renal disease, macroalbuminuria (ex- end of the study. pressed as albumin to creatinine ratio Ͼ 300 ␮g/mg), severe dyslipi- Participants were asked to return for the exit visit after a 12-wk demia (triglycerides Ͼ 600 mg/dl or cholesterol Ͼ 300 mg/dl), or any treatment period. The diabetic patients were also asked to continue with other serious chronic disease requiring active treatment. Subjects were their same diabetes medications and dosages and were encouraged to also excluded if they were on any of the following medications: continue with their usual meal plan and physical activity level. In case glucocorticoids, antineoplastic agents, psychoactive agents, and problems with diabetes control were encountered, any modification to bronchodilators. the diabetes management was recorded. During the exit visit, blood tests Downloaded from https://academic.oup.com/jcem/article/89/2/740/2840787 by guest on 28 September 2021 The protocol was approved by the ethics committee or institutional for glycosylated hemoglobin, glucose, biochemical markers of inflam- review board at each center, and all participants gave written informed mation, and endothelial function were taken, and the vascular reactivity consent. Volunteers for the study were recruited through local adver- in the micro- and macrocirculation was measured. tisement at the Joslin Diabetes Center and the Beth Israel Deaconess Medical Center in Boston. Data analysis

Methods The Minitab statistical package (Minitab Inc., State College, PA) for personal computers was used for the statistical analysis. A two-tailed Volunteers attended the Joslin Diabetes Center Clinical Research comparison was assumed. The analyses were performed using a paired Center to undergo the clinical and laboratory evaluations. A general t test for parametrically distributed data and the Wilcoxon matched-pair physical examination was performed by a study physician. Subjects signed rank test for nonparametrically distributed data to compare were studied at all visits after an overnight fast. Participants were asked baseline data and changes in all variables at the end of the study within not to take their diabetes medications (sulfonylureas or metformin) for each group. The t test was used to compare the baseline characteristics 12 h before any of the studies, and those participants taking insulin were asked to omit the rapid-acting insulin the morning of each visit. TABLE 1. Baseline characteristics of the studied subjects Plasma glucose, total serum cholesterol, LDL cholesterol, high- density lipoprotein (HDL) cholesterol, triglycerides, liver function tests, At risk of type 2 Diabetic electrolytes, blood urea nitrogen, and creatinine were measured using diabetes patients the Synchron CX analyzer (Beckman/Coulter, Brea, CA). Routine uri- Total patients (no.) 37 40 nalysis was also performed. The glycosylated hemoglobin (normal Age (yr) 49 Ϯ 12 53 Ϯ 13 range, 4–6%) was determined in whole blood using ion-exchange HPLC Males (%) 20 (54%) 23 (58%) (Tosoh 2.2, Tokyo, Japan). Soluble intercellular adhesion molecule [co- With first-degree relative 25 (68%) — efficient of variation (cv), 4.4%], soluble vascular cell adhesion molecule (NGT) (no.) (cv, 5.0%), and endothelin-1 (cv, 4.4%) were measured in plasma by an With IGT (no.) 12 (32%) — ELISA method (R&D Systems, Minneapolis, MN). TNF␣ (cv, 4.7%) and Type 1/2 diabetes (no.) — 20/20 high-sensitivity C-reactive protein (CRP; cv, 5.1%) were measured by Diabetes duration (yr) — 8 Ϯ 12 chemiluminescent immunoassay. Von Willebrand factor (cv, 6.1%), BMI (kg/m2) 29.5 Ϯ 5.8 29.8 Ϯ 9.4 plasma activator inhibitor (PAI; cv, 6.0%) antigen, and tissue plasmin- Systolic BP (mm Hg) 126 Ϯ 17 127 Ϯ 13 ogen activator (tPA; cv, 4.5%) antigen were also measured by an ELISA Diastolic BP (mm Hg) 81 Ϯ 10 79 Ϯ 9 method (Diagnostica Stago, Parsippany, NJ). Fasting glucose (mg/dl)a 90 Ϯ 11 178 Ϯ 79 a Ϯ Ϯ HbA1c (%) 5.4 0.4 8.0 1.6 Vascular reactivity tests Total cholesterol (mg/dl) 206 Ϯ 40 205 Ϯ 42 LDL (mg/dl) 125 Ϯ 31 124 Ϯ 35 All eligible participants returned for a second visit to the Microcir- HDL (mg/dl) 59 Ϯ 19 60 Ϯ 14 culation Laboratory at the Beth Israel Deaconess Medical Center to Triglycerides (mg/dl) 118 Ϯ 78 102 Ϯ 75 undergo the vascular reactivity tests. All measurements were performed Urine albumin to creatinine 5.7 Ϯ 4.1 32.3 Ϯ 58.8 in the morning, while the subjects were in a fasting state. The investi- ratioa gators who performed the vascular reactivity measurements were Antihypertensive treatment 6 (16%) 16 (40%) blinded to the medical history of the subjects. These studies were carried ␤ blockers 1 0 out in a temperature-controlled room (24–26 C) and after a 30-min ACE inhibitors 2 11 acclimatization period. Diuretic 1 0 The vascular reactivity of the forearm skin microcirculation was eval- Calcium antagonists 1 0 uated by Laser Doppler perfusion imaging measurements before and Combination 1 5 after the iontophoresis of acetylcholine chloride (endothelium-depen- Diabetes treatment dent vasodilation) and sodium nitroprusside (endothelium-indepen- Diet — 1 (3%) dent vasodilation), as previously described (16). The reproducibility of Oral agents — 15 (37%) the technique has been previously reported by our group. The cv of the Insulin — 24 (60%) baseline measurement was 14.1%, and during maximal hyperemic re- Aspirin 2 (5%) 7 (18%) sponse after the iontophoresis, it was 13.7% (17). To assess the endothelium-dependent reactivity in the macrocircu- Results are presented as mean Ϯ SD or no. (%). Conversion factors lation, the flow-mediated dilation (FMD) of the brachial artery was to Systeme Internationale units are: glucose ϫ 0.05551; total choles- measured by using a high-resolution ultrasound with a 10.0-MHz linear terol, ϫ 0.0259; LDL, ϫ 0.0259; HDL, ϫ 0.0259; triglycerides, ϫ array transducer and an HDI Ultramark 9 System (Advanced Technol- 0.0113. ogy Laboratories, Bothel, WA). All measurements were in accordance NGT, Normal glucose tolerance; IGT, impaired glucose tolerance; with recently published guidelines (18). Reactive hyperemia is produced BMI, body mass index; BP, blood pressure; HbA1c, glycosylated he- by inflating a pneumatic tourniquet distally to the brachial artery to 50 moglobin; ACE, angiotensin-converting enzyme. mm Hg above the systolic pressure for 5 min and then deflating it. a P Ͻ 0.0001. 742 J Clin Endocrinol Metab, February 2004, 89(2):740–747 Economides et al. • Effect of Atorvastatin in Diabetes

TABLE 2. Baseline measurements of vascular reactivity and biochemical markers of endothelial function

At risk of type 2 diabetes Diabetic patients Resting brachial artery diameter (mm) 3.4 Ϯ 0.7 3.6 Ϯ 0.7 FMD (% of increase over baseline) 5.8 (4.0–9.3) 5.0 (3.2–7.8) Nitroglycerin-induced dilation (% of increase over baseline) 15.2 (11.4–20.9) 13.4 (10.1–17.1) Baseline blood flow before the iontophoresis of acetylcholine (V) 0.83 Ϯ 0.27 0.80 Ϯ 0.28 Acetylcholine-induced skin vasodilation (% of increase over baseline) 158 Ϯ 77 149 Ϯ 77 Sodium nitroprusside skin induced vasodilation (% of increase over baseline) 88 Ϯ 49 93 Ϯ 44 ICAM (ng/ml) 251 Ϯ 83 290 Ϯ 105 VCAM (ng/ml)a 614 Ϯ 228 789 Ϯ 218 Endothelin-1 (pg/ml)b 0.86 Ϯ 0.33 1.09 Ϯ 0.36 TNF␣ (pg/ml) 3.8 (2.3–5.6) 4.0 (2.3–8.2) High-sensitivity CRP (mg/dl) 0.22 (0.07–0.37) 0.37 (0.15–0.70) c Ϯ Ϯ von Willebrand Factor (%) 90 58 131 48 Downloaded from https://academic.oup.com/jcem/article/89/2/740/2840787 by guest on 28 September 2021 PAI-1 antigen (ng/ml) 22.7 (13.0–44.8) 17.6 (6.7–33) tPA antigen (ng/ml) 7.2 Ϯ 3.9 6.9 Ϯ 3.7

Data are presented as mean Ϯ SD or median (25–75 percentiles). ICAM, Intercellular adhesion molecule; VCAM, vascular cell adhesion molecule. a P Ͻ 0.01. b P Ͻ 0.05. c P Ͻ 0.001. between those receiving active treatment and those receiving placebo in Results in subjects at risk of developing diabetes (group 1) all groups. Correlation between variables was tested using both uni- variate and multivariate analyses (Pearson’s correlation and Spearman Atorvastatin-treated patients showed a significant reduc- correlation analysis for parametrically and nonparametrically distrib- tion in total cholesterol, LDL, and triglycerides, whereas no uted data and analysis and multiple stepwise regression analysis). The changes were observed in HDL (Table 3). No changes were Ϯ results are presented as mean sd and median (25–75 percentile). observed in weight, systolic and diastolic blood pressure, and fasting glucose. Finally, no changes were observed in Corrections for multiple comparisons any parameter in the placebo-treated subjects. We tested whether the number of significant results for a single group The main results on the endothelium-dependent and was consistent with chance or not. There are 14 separate comparisons -independentvasodilatoryresponsesinthemacro-andmicro- (see Tables 4 and 5) for each of the four groups (at risk of type 2 diabetes and diabetic patients, treated or placebo). With 14 significance tests and circulation are shown in Table 4. A significant improvement a nominal two-sided ␣ ϭ 0.05, one would expect 0.9 significant results in the FMD in the brachial artery diameter was found in the per group (0.7 ϭ 14 ϫ 0.05), and, if results for different variables are atorvastatin-treated patients after 3 months of treatment (P Ͻ independent, then the number of significant tests would follow a Poisson 0.05). In contrast, no significant changes in the brachial artery distribution. We observed no significant results in placebo-treated, at- risk patients and one significant result in placebo-treated diabetic sub- dilation were seen in response to nitroglycerin. No signifi- jects, both of which are consistent with chance. In contrast, in atorvas- cant changes were seen in the endothelium-dependent and tatin-treated patients in both groups we observed three significant -independent responses in the skin microcirculation. results, suggesting that these results are real findings. A reduction in CRP and TNF␣ levels was observed in the atorvastatin-treated patients (P Ͻ 0.05, Table 5). No changes Results were found in any of the biochemical endothelial measure- A total of 77 subjects were randomized into two groups; ments in the placebo-treated group. No correlations were 37 subjects at risk of developing diabetes were in the first found between the FMD changes and changes in total cho- group, and 40 diabetic patients were in the second group. lesterol and LDL, triglycerides, CRP, and TNF␣ (Figs. 1 and 2). Details about the demographic characteristics of the two study groups are shown in Table 1. There were no differences Results in diabetic patients (group 2) between the groups in age, gender, body mass index, blood pressure, and lipid levels. Results of the baseline measure- Atorvastatin-treated patients showed a significant reduc- ments of vascular reactivity and biochemical markers of en- tion in total cholesterol and LDL and a decrease in HDL dothelial function are given in Table 2. All vascular reactivity (Table 3). Of interest, the placebo group also had a small but measurements were similar in the two groups, but the vas- statistically significant reduction in total and LDL choles- cular cell adhesion molecule, endothelin-1, and von Wille- terol. No changes were observed in weight, systolic and brand Factor levels were higher in the diabetic group. diastolic blood pressure, and glycemic control in either of the Participants in both groups were randomized to either two groups. active or placebo treatment. There were no statistically sig- The FMD improved in the atorvastatin-treated patients nificant differences in any of the baseline characteristics be- but failed to reach statistical significance (P ϭ 0.07, Table 4). tween those individuals assigned to the active treatment and As with the placebo group, no changes were observed in the those receiving placebo in either group (Tables 3 and 4). nitroglycerine-induced dilation and microvascular reactivity Thirty subjects (81%) at risk of developing diabetes and 37 measurements. No changes in the vascular reactivity were diabetic patients (93%) completed the study. There were no observed in the placebo-treated group. differences between those who did not complete the study A significant reduction was observed in the endothelin-1 and those who did. and PAI-1 levels in the atorvastatin group (Table 5). In ad- Economides et al. • Effect of Atorvastatin in Diabetes J Clin Endocrinol Metab, February 2004, 89(2):740–747 743

TABLE 3. Results of changes in demographic characteristics in the two groups who completed a 3-month treatment period with atorvastatin or placebo

At risk type 2 diabetes Diabetic patients Active (n ϭ 15) Placebo (n ϭ 15) Active (n ϭ 19) Placebo (n ϭ 18) Age (yr) 48 Ϯ 13 49 Ϯ 11 51 Ϯ 14 55 Ϯ 11 Weight (kg) Baseline 85 Ϯ 15 81 Ϯ 21 88 Ϯ 24 87 Ϯ 20 Exit 85 Ϯ 16 81 Ϯ 20 88 Ϯ 24 87 Ϯ 20 P NS NS NS NS Systolic BP (mm Hg) Baseline 126 Ϯ 12 123 Ϯ 21 128 Ϯ 14 125 Ϯ 13 Exit 122 Ϯ 9 122 Ϯ 17 129 Ϯ 21 124 Ϯ 19 P NS NS NS NS Diastolic BP (mm Hg) Downloaded from https://academic.oup.com/jcem/article/89/2/740/2840787 by guest on 28 September 2021 Baseline 80 Ϯ 779Ϯ 10 80 Ϯ 978Ϯ 9 Exit 77 Ϯ 17 80 Ϯ 979Ϯ 11 78 Ϯ 11 P NS NS NS NS Fasting glucose (mg/dl) Baseline 92 Ϯ 12 89 Ϯ 10 163 Ϯ 72 182 Ϯ 79 Exit 93 Ϯ 12 90 Ϯ 10 183 Ϯ 138 193 Ϯ 110 P NS NS NS NS

HbA1c (%) Baseline 5.4 Ϯ 0.5 5.2 Ϯ 0.3 7.9 Ϯ 1.1 8.1 Ϯ 1.9 Exit 5.5 Ϯ 0.5 5.2 Ϯ 0.5 7.9 Ϯ 1.1 7.9 Ϯ 1.8 P NS NS NS NS Total cholesterol (mg/dl) Baseline 193 Ϯ 42 216 Ϯ 32 205 Ϯ 40 208 Ϯ 47 Exit 153 Ϯ 41 209 Ϯ 27 149 Ϯ 28 192 Ϯ 32 P Ͻ0.01 NS Ͻ0.0001 Ͻ0.01 LDL cholesterol (mg/dl) Baseline 115 Ϯ 31 129 Ϯ 28 124 Ϯ 36 125 Ϯ 37 Exit 81 Ϯ 36 125 Ϯ 21 73 Ϯ 22 113 Ϯ 30 P Ͻ0.01 NS Ͻ0.0001 Ͻ0.05 HDL cholesterol (mg/dl) Baseline 53 Ϯ 966Ϯ 24 63 Ϯ 14 59 Ϯ 14 Exit 55 Ϯ 11 61 Ϯ 19 58 Ϯ 15 59 Ϯ 16 P NS NS Ͻ0.05 NS TG (mg/dl) Baseline 132 Ϯ 96 116 Ϯ 71 101 Ϯ 78 106 Ϯ 79 Exit 91 Ϯ 45 129 Ϯ 77 105 Ϯ 104 106 Ϯ 72 P Ͻ0.05 NS NS NS

Data are presented as mean Ϯ SD. Conversion factors to Systeme Internationale units are: glucose, ϫ 0.05551; total cholesterol, ϫ 0.0259; ϫ ϫ ϫ LDL, 0.0259; HDL, 0.0259; triglycerides, 0.0113. NS, Not significant; BP, blood pressure; HbA1c, glycosylated hemoglobin; Tg, triglycerides. dition, a significant reduction was observed in the tPA in treatment with statins on endothelial function (21–24). How- both the atorvastatin and placebo groups. No correlations ever, two recent studies have reported a beneficial effect of were found between the FMD changes and changes in total statins on the endothelial function in type 2 diabetic patients. cholesterol, LDL, endothelin 1, PAI-1, and tPA. The first one used simvastatin 20–40 mg and had a target of LDL less than 80 mg, whereas the second study used a Discussion regimen similar to the present study, atorvastatin 20 mg daily In the present study, we have shown that treatment with (25, 26). One common characteristic of the above studies is 20 mg atorvastatin daily for a 3-month period resulted in that they included subjects with dyslipidemia. Atorvastatin significant improvement of endothelial function in the 40 mg once daily improved the flow-mediated vasodilation macrocirculation in healthy subjects at risk of developing in patients with type 1 diabetes (27). Cerivastatin was also type 2 diabetes. A similar but nonsignificant improvement shown to improve endothelial function in elderly diabetic was observed in diabetic patients. Atorvastatin also reduced patients within 3 d without affecting the lipid profiles (28). the CRP and TNF␣ levels in the subjects at risk of developing In addition to the reported effects on the endothelial function, diabetes and the endothelin-1 and PAI-1 levels in the diabetic treatment with atorvastatin decreased the carotid intima me- patients. dial thickness, a validated surrogate cardiovascular end Treatment with statins improved the endothelial function point, in patients with hyperlipidemia (29). in patients with coronary artery disease and in postmeno- In the present study, we found a positive effect of ator- pausal normocholesterolemic women (19, 20). Regarding the vastatin on the endothelial function of healthy subjects at risk effect of statins in diabetes, the data are conflicting, with the of developing type 2 diabetes, a population that is known to majority of the published studies suggesting no effect of have impaired endothelial function (3, 4). Their mean base- 744 J Clin Endocrinol Metab, February 2004, 89(2):740–747 Economides et al. • Effect of Atorvastatin in Diabetes

TABLE 4. Changes in endothelial function in the two groups who completed a 3-month period treatment with atorvastatin or placebo

At risk of type 2 diabetes Diabetic patients Active (n ϭ 15) Placebo (n ϭ 15) Active (n ϭ 19) Placebo (n ϭ 18) Brachial artery diameter (mm) Baseline 3.6 Ϯ 0.9 3.1 Ϯ 0.6 3.7 Ϯ 0.8 3.5 Ϯ 0.7 Exit 3.6 Ϯ 0.9 3.1 Ϯ 0.5 3.6 Ϯ 0.8 3.3 Ϯ 0.6 P NS NS NS NS FMD (% of increase over baseline) Baseline 6.6 (2.9–9.5) 7.1 (4.4–9.8) 4.2 (3.2–7.2) 5.8 (3.3–7.6) Exit 7.2 (2.9–9.6) 7.0 (4.2–9.1) 5.6 (3.9–7.9) 6.2 (3.6–8.8) P Ͻ0.05 NS 0.07 NS NID (% of increase over baseline) Baseline 14.1 (10.8–21.0) 15.2 (14.0–24.5) 12.5 (10.3–16.2) 16.3 (10.0–17.4) Exit 13.7 (11.6–19.3) 15.5 (12.1–23.2) 11.9 (10.7–15.8) 13.7 (10.6–17.5) Downloaded from https://academic.oup.com/jcem/article/89/2/740/2840787 by guest on 28 September 2021 P NS NS NS NS Baseline laser flux (V) Baseline 0.94 Ϯ 0.28 1.00 Ϯ 0.25 1.00 Ϯ 0.35 0.88 Ϯ 0.23 Exit 1.00 Ϯ 0.30 0.99 Ϯ 0.21 1.01 Ϯ 0.42 1.00 Ϯ 0.26 P NS NS NS NS Ach (% of increase over baseline) Baseline 133 Ϯ 55 159 Ϯ 67 146 Ϯ 86 163 Ϯ 67 Exit 121 Ϯ 60 152 Ϯ 75 157 Ϯ 84 130 Ϯ 78 P NS NS NS NS SNP (% of increase over baseline) Baseline 67 Ϯ 28 96 Ϯ 54 94 Ϯ 49 86 Ϯ 41 Exit 78 Ϯ 52 97 Ϯ 61 109 Ϯ 75 90 Ϯ 39 P NS NS NS NS

Data are presented as mean Ϯ SD or median (25–75 percentiles). NS, Not significant; NID, nitroglycerin-induced dilation; Ach, acetylcholine; SNP, sodium nitroprusside.

TABLE 5. Changes in inflammatory cytokines and biochemical markers of endothelial function

At risk of type 2 diabetes Diabetic patients Active (n ϭ 15) Placebo (n ϭ 15) Active (n ϭ 19) Placebo (n ϭ 18) High-sensitivity CRP (mg/ml) Baseline 0.24 (0.07–0.35) 0.20 (0.06–0.53) 0.30 (0.11–0.62) 0.41 (0.17–0.76) Exit 0.12 (0.07–0.27) 0.23 (0.05–0.43) 0.34 (0.07–0.60) 0.35 (0.13–0.56) P Ͻ0.05 NS NS NS SICAM (ng/ml) Baseline 251 Ϯ 55 251 Ϯ 106 294 Ϯ 105 286 Ϯ 109 Exit 257 Ϯ 55 275 Ϯ 68 289 Ϯ 95 278 Ϯ 98 P NS NS NS NS SVCAM (ng/ml) Baseline 602 Ϯ 214 625 Ϯ 248 732 Ϯ 168 849 Ϯ 252 Exit 602 Ϯ 211 595 Ϯ 224 721 Ϯ 238 808 Ϯ 200 P NS NS NS NS Endothelin (pg/ml) Baseline 0.82 Ϯ 0.25 0.89 Ϯ 0.40 1.19 Ϯ 0.42 1.02 Ϯ 0.29 Exit 0.81 Ϯ 0.24 0.83 Ϯ 0.29 0.97 Ϯ 0.29 0.98 Ϯ 0.30 P NS NS Ͻ0.05 NS TNF␣ (pg/ml) Baseline 4.4 (3.6–6.0) 2.8 (2.1–4.6) 3.8 (2.5–6.2) 5.8 (17–13.0) Exit 2.6 (1.8–4.1) 3.5 (1.8–6.2) 4.1 (1.8–6.6) 4.3 (2.6–7.0) P Ͻ0.05 NS NS NS PAI-1 (ng/ml) Baseline 29 (15–56) 21 (9–38) 27 (7–41) 11 (7–30) Exit 23 (11–41) 21 (14–50) 18 (9–24) 11 (9–33) P NS NS Ͻ0.05 NS tPA (ng/ml) Baseline 8.1 Ϯ 4.8 6.3 Ϯ 2.5 7.3 Ϯ 3.5 6.8 Ϯ 4.0 Exit 8.2 Ϯ 4.3 7.0 Ϯ 2.7 6.3 Ϯ 3.4 5.9 Ϯ 3.6 P NS NS Ͻ0.01 Ͻ0.05 vWF (%) Baseline 88 Ϯ 40 92 Ϯ 72 144 Ϯ 49 121 Ϯ 46 Exit 93 Ϯ 40 93 Ϯ 58 139 Ϯ 48 118 Ϯ 39 P NS NS NS NS

Data are presented as mean Ϯ SD or median (25–75 percentiles). NS, Not significant; SICAM, soluble intercellular adhesion molecule; SVCAM, soluble vascular cell adhesion molecule; vWF, von Willebrand factor. Economides et al. • Effect of Atorvastatin in Diabetes J Clin Endocrinol Metab, February 2004, 89(2):740–747 745 Downloaded from https://academic.oup.com/jcem/article/89/2/740/2840787 by guest on 28 September 2021

FIG. 1. Changes in FMD of the brachial artery (endothelium-dependent vasodilation) vs. changes in the LDL cholesterol level in healthy FIG. 2. Changes in high-sensitivity (hs) CRP vs. changes in the LDL subjects at risk for type 2 diabetes (A) and diabetic patients (B). No cholesterol level in healthy subjects at risk for type 2 diabetes (A) and association was found between changes in FMD and LDL cholesterol diabetic patients (B). As with FMD, no association was found between in both groups. D, Difference between exit and baseline visits. changes in hsCRP and LDL cholesterol in both groups. D, Difference between exit and baseline visits. line lipid levels were similar to those that are currently recommended as targets for successful treatment. In addition, pendent vasodilation in either studied group, which is a concentrations of total cholesterol and LDL attained in the finding that is in agreement with previous studies (30). Pre- atorvastatin-treated subjects were well below the currently vious studies from our unit and elsewhere have shown that recommended levels. Thus, to our knowledge, this is the first both these measurements are impaired in subjects at risk of study to show a positive effect of atorvastatin on the endo- developing diabetes and diabetic patients (31). Although the thelial function of subjects at risk of developing diabetes with reasons for this difference in the response to atorvastatin no dyslipidemia. treatment in the micro- and macrocirculation are not clear, it A similar but nonsignificant improvement was also found should be remembered that atherosclerosis is a process that in the endothelial function of the diabetic patients. The main is confined in the macrocirculation and is influenced by fac- reason for this failure is that, on the basis of preliminary tors such as hyperlipidemia and insulin resistance. In con- reports, we anticipated a considerably higher improvement trast, the microcirculation is mainly influenced by hypergly- of the endothelial function (19, 28). Therefore, we believe that cemia, and this is best seen by the fact that clinical this marginal failure to reach statistical significance is likely microvascular complications, such as retinopathy, neurop- to represent a type 2 statistical error and that the observed athy, and nephropathy, are not present in the prediabetic improvement is real. It should also be emphasized that, in stage. Further studies will be required to examine whether contrast to previous studies, dyslipidemia was not an inclu- other therapeutic interventions, such as treatment with sion criterion in the present study, and therefore, as with the angiotensin-converting enzyme inhibitors, may be better at-risk group, the baseline mean cholesterol levels were close candidates in reversing microvascular abnormalities. to the currently recommended therapeutic targets, whereas Previous studies have shown that statins can reduce CRP the atorvastatin-treated patients reached much lower levels. levels in nondiabetic subjects with dyslipidemia and diabetic Therefore, these results indicate that treatment with statins patients in a relatively short period of time (32–34). In the can be beneficial even in the presence of normal plasma total present study, a 50% reduction in CRP levels was observed and LDL cholesterol concentrations. in the at-risk group. In the same group, a significant reduc- Treatment with atorvastatin did not result in any changes tion was also observed in TNF␣ levels. Previous in vitro in the microcirculation endothelium-dependent and -inde- studies have suggested that statin treatment can reduce the 746 J Clin Endocrinol Metab, February 2004, 89(2):740–747 Economides et al. • Effect of Atorvastatin in Diabetes

TNF␣ production by human monocytes stimulated by oxi- of the FMD in the diabetic group might have been more dized LDL (35). This is the first study to indicate that treat- dramatic if the reduction in the HDL was not present. Further ment with a statin can reduce TNF␣ levels in humans. Fur- trials that will be focused on these issues will be required ther studies will be needed to further explore these findings. before definite conclusions are reached. Endothelin-1 levels were higher in the diabetic group and The present study has its limitations. The main limitation were reduced in the atorvastatin-treated patients. Previous in is that this is a small study that focused on showing a proof vitro studies have shown that statins reduce the expression of principle, and as such, it did not include a large cohort of of endothelin-1 in endothelial cells (36, 37). Our findings are subjects that could allow full statistical analysis. Thus, the consistent with these studies and are the first to observe such study was mainly powered to detect changes in the FMD en effect in humans. We believe that endothelin levels did not because there was no information available at that time re- change in the at-risk group because initial concentrations garding the effect of atorvastatin on biochemical markers of were not as high as in the diabetic patients, and therefore, a endothelial dysfunction. This is probably the main reason possible beneficial effect could not be seen. that discrepancies were found in the response of the various Downloaded from https://academic.oup.com/jcem/article/89/2/740/2840787 by guest on 28 September 2021 Atorvastatin has also been shown to reduce the expression endothelial markers (such as TNF␣, CRP, and PAI-1) in the of PAI-1 in human vascular smooth muscle and endothelial at-risk and diabetic patients who were treated with atorva- cells (38, 39). In the present study, atorvastatin treatment had statin. Further studies will be required to further explore the a beneficial effect in both groups, but a statistical significance findings of the present study before solid conclusions can be was reached only in the diabetic patients. The main reason reached. for not reaching significance in the at-risk group is related to Another limitation may be the fact that, in group 1, we the small number of tested subjects and the considerable included healthy subjects with parental history of type 2 variation in the PAI-1 levels. Finally, a reduction in the tPA diabetes and subjects with impaired glucose tolerance, was observed in both the actively treated and placebo-treated whereas in group 2, we included both type 1 and type 2 diabetic patients. The reduction in PAI-1 levels may be the diabetic patients. However, because previous studies have main factor in the atorvastatin-treated patients, but the rea- shown similar impairment in the endothelial function of sons in the placebo group are not clear. The fact that a small subjects with parental history of type 2 diabetes and subjects but significant reduction in total and LDL cholesterol levels with impaired glucose tolerance or type 1 and 2 diabetic was also observed in the diabetic placebo-treated patients patients, we believe that our inclusion criteria did not affect further indicates this possibility. the study outcomes (1–4). We believe that the most impor- Statins are currently thought to have pleiotropic effects tant factor was to have the at-risk and diabetic groups being that are independent of their lipid-lowering function (40). matched for age, gender, and lipidemia, and this was fully However, previous studies have shown an association be- achieved. tween the cholesterol changes and endothelial function. In conclusion, in the present study, we have shown that Thus, in one study, drastic reduction of LDL cholesterol by atorvastatin improves the endothelial function of the macro- apheresis, which resulted in a postapheresis mean LDL of 33 circulation and decreases levels of markers of endothelial mg/dl, was accompanied by a considerable improvement of activation in diabetic patients and in subjects at risk of de- the endothelial function while significant correlations where veloping type 2 diabetes. found between LDL levels and endothelial function (41). In addition, other studies have shown that isolated low HDL Acknowledgments levels are associated with endothelial dysfunction, which is completely restored to normal levels by a rapid increase in Received June 30, 2003. Accepted October 22, 2003. HDL (42). Low HDL levels are thought to cause reduced Address all correspondence and requests for reprints to: Aristidis nitric oxide bioavailability because of reduced endothelial Veves, M.D., Microcirculation Laboratory, Palmer 317, Beth Israel Dea- nitric oxide synthase expression and/or activity (43). coness Medical Center, West Campus, One Deaconess Road, Boston, In this study, no relationship was found between the Massachusetts 02215. E-mail: [email protected]. This work was supported by a clinical research grant from Pfizer Inc. achieved total and LDL cholesterol reduction and the (to A.V.) and, in part, by Grant RR 01032 to the Beth Israel Deaconess changes in the FMD or the biochemical markers of endothe- Medical Center General Clinical Research Center from the National lial dysfunction. In addition, a reduction was noted in the Institutes of Health, a William Randolph Hearst Fellowship provided by HDL cholesterol in the atorvastatin-treated diabetic patients the William Randolph Hearst Foundation, and a Mary K. Iacocca Fel- lowship provided by the Iacocca Foundation. despite an increase in the FMD. The reasons for the discrep- ancy between our study and the previous ones are not clear but may be related to the fact that we included patients References without severe dyslipidemia and that the achieved changes 1. Williams SB, Cusco JA, Roddy MA, Johnstone MT, Creager MA 1996 Im- in the HDL and LDL levels were as extreme as in the previous paired nitric oxide-mediated vasodilation in patients with non-insulin-dependent diabetes mellitus. J Am Coll Cardiol 27:567–574 studies. In addition, it should be emphasized that the main 2. 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