Effect of Rosuvastatin on Cholesterol Efflux Capacity and Endothelial Function in Type 2 Diabetes Mellitus and Dyslipidemia

Abstract

Background: Quality and quantity of high-density lipoprotein cholesterol (HDL-C) may be associated with cardiovascular risk. We investigated the effect of rosuvastatin on cholesterol efflux (CE) for HDL function and vascular health.

Methods and Results: We enrolled 30 dyslipidemic patients with type 2 diabetes mellitus and 20 healthy subjects as controls. Vascular health was assessed on flow-medicated dilation (FMD), nitroglycerin-induced dilatation of the brachial artery and carotid artery intima-media thickness (cIMT). These parameters were compared between patients and controls, and between baseline and at 12 weeks of treatment with rosuvastatin 20 mg. Age and body mass index were 49.8±11.3 years and 25.8±3.7 kg/m² in the patients, and 28.8±3.2 years and 22.4±2.4 kg/m² in the controls, respectively. The biomarkers related to lipid and glucose metabolism and lipoprotein (a), high-sensitivity C-reactive protein, and cIMT were significantly higher, and CE and FMD were significantly lower in the patients than in the controls. In the patients, rosuvastatin 20 mg decreased low-density lipoprotein cholesterol by 54.1% and increased HDL-C by 4.8%. The CE increased significantly after rosuvastatin treatment (12.26±2.72% vs. 14.05±4.14%). FMD also increased, and lipoprotein (a) and cIMT decreased significantly and were associated with changes of CE.

Conclusions: Rosuvastatin-induced changes in HDL function are significantly associated with cardiovascular benefit.

Reducing low-density lipoprotein cholesterol (LDL-C) is the primary target in the management of dyslipidemia in patients with a high risk of cardiovascular disease (CVD). Patients who have achieved LDL-C below the currently recommended targets, however, still experience cardiovascular events.¹ This may result, in part, from high triglycerides and low high-density lipoprotein cholesterol (HDL-C), which is associated with insulin resistance and recognized as atherogenic dyslipidemia.

Editorial p 1251

Low HDL-C and high triglycerides is common and is recognized as an independent risk factor for cardiovascular morbidity and mortality.² There is solid evidence that plasma HDL-C has a protective role in CVD.^3,4 Increasing HDL-C alone to reduce CVD risk, however, has not yet been confirmed in large randomized clinical trials, and whether HDL function is more important than HDL concentration in reducing the risk of CVD remains controversial.⁵ Nonetheless, to reduce further the risk of coronary artery disease, raising HDL-C and lowering triglycerides may be the secondary therapeutic target for patients who achieve LDL-C below the currently recommended target but are still at high risk. Previously, a subgroup analysis of the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study suggested that the addition of fenofibrate to statins in patients with high triglycerides and low HDL-C led to favorable results for cardiovascular morbidity.⁶ Recently, an ACCORD follow-up study provided evidence that fenofibrate effectively reduced CVD in participants with high triglycerides (>204 mg/dL) and low HDL-C (<34 mg/dL) (HR, 0.73; 95% CI: 0.56–0.95) after an additional 5 years of follow-up (total, 9.7 years).⁷

An intervention that increases HDL-C, however, is not necessarily accompanied by an enhancement of HDL function. There is an argument suggesting that HDL-C level per se does not represent the functionality of the HDL system.⁸ Several studies in humans have shown that raising HDL-C using cholesteryl ester transfer protein inhibitors or niacin fails to improve any clinical cardiovascular outcomes.^9–11 Thus, increasing the concentration of HDL-C alone might be ineffective, indicating that qualitative changes in HDL-C are required to result in clinical benefit. It has been increasingly recognized that functional properties of HDL rather than the level of HDL-C are likely to be important for the cardioprotective effects of HDL-C.^12,13

Statin treatment has proven a cardiovascular benefit in subjects with type 2 diabetes mellitus (T2DM).¹⁴ There are limited studies, however, on HDL function after statin therapy. Furthermore, previous clinical studies were inconsistent.^15–17 HDL function assessed in vitro did not change significantly after 4 weeks of treatment with rosuvastatin.¹⁵ In contrast, cholesterol efflux (CE) increased in response to pitavastatin treatment alone or to a combination of simvastatin and bezafibrate.^16,17 Recently, 1 study showed that CE did not change after 12 months of treatment with rosuvastatin, but CE was associated with incident CVD in individuals on statin therapy.¹⁸ Therefore, we investigated the effect of rosuvastatin, a potent statin, on HDL function using CE, vascular health using a Doppler system, and atheromatous status in carotid arteries.

Methods

Patients and Design

We recruited 30 patients with T2DM and dyslipidemia from the diabetes clinics at Seoul National University Bundang Hospital (SNUBH) and 20 healthy participants from the wider community as controls. Inclusion criteria for the patient group were as follows: (1) T2DM with HbA1c ≥6.0%; (2) age ≥20 years; (3) LDL-C ≥100 mg/dL; and (4) HDL-C <40 mg/dL in men and <50 mg/dL in women with one or more risk factors: body mass index (BMI) ≥25 kg/m², triglyceride ≥150 mg/dL, hypertension, current smoker, or family history of coronary artery disease.

Exclusion criteria included type 1 DM, statin within the previous 12 weeks, liver function abnormalities (aspartate or alanine aminotransferase [AST or ALT] >3-fold above the upper reference range), or contraindication to statins.

For the healthy control group, individuals who were aged between 20 and 35 years and had normal glucose and lipid profiles without cardiovascular risk were selected.

First, we compared baseline biomarkers and vascular health between the patients and the healthy control group. Various biomarkers related to lipid and glucose metabolism, and high-sensitivity C-reactive protein (hsCRP) for inflammation and lipoprotein (a) were measured. CE for HDL function, flow-mediated dilation (FMD) for vascular endothelial function, and carotid intima-media thickness (cIMT) were assessed.

Second, in the patient group, we compared CE at baseline with that after 1 week and 12 weeks of treatment with rosuvastatin 20 mg daily to assess the short- and long-term effects, respectively. The biomarkers were compared between baseline and after 12 weeks of treatment with rosuvastatin.

All subjects provided written informed consent to participate before inclusion in this study. This study was approved by the Institutional Review Board of SNUBH (IRB no. B-1403-241-008) and complied with the principles of the Declaration of Helsinki. This study was registered at ClinicalTrials.gov: NCT02185963.

Anthropometry and Biochemistry

Height and body weight were measured by standard methods with the participants in light clothing. BMI was calculated as body weight (kg) divided by the square of the height (m). After 12 h of overnight fasting, venous blood samples were taken for biochemistry assays at baseline and after rosuvastatin treatment. Serum total cholesterol, triglycerides, HDL-C and LDL-C were measured using a Hitachi 747 Clinical Chemistry Analyzer (Hitachi, Tokyo, Japan). Non-HDL-C was calculated as total cholesterol-HDL-C. Serum apolipoprotein A-I (apoA-I) and apolipoprotein B (apoB) were measured using a Beckmann Coulter AU5822 (Brea, CA, USA). Lipoprotein (a) was measured using particle enhanced immunoturbidimetry on a Hitachi 717 analyzer (Tina-quant, Roche, Switzerland).

Plasma glucose concentration was measured using the glucose oxidase method (747 Clinical Chemistry Analyzer; Hitachi). HbA1c was measured using a Bio-Rad Variant II Turbo HPLC Analyzer (Bio-Rad, Hercules, CA, USA) in the National Glycohemoglobin Standardization Program (NGSP) level II certified laboratory at SNUBH. Fasting insulin was measured on radioimmunoassay (Linco, St. Louis, MO, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) and β-cell function (HOMA-β) were calculated.¹⁹ AST/ALT and creatinine were measured using an Architect Ci8200 analyzer (Abbott Laboratories, Abbott Park, IL, USA). Urinary albumin was measured using turbidimetry (A&T 502X, A&T, Tokyo, Japan), and urinary creatinine was measured using the Jaffe method (Hitachi 7170, Hitachi). The ratio of urinary albumin to creatinine concentration (mg/g) was used for albuminuria.

Vascular Function

Vascular health was assessed with the following 3 methods: (1) FMD (endothelium dependent); (2) nitroglycerin-induced dilation (NID) (endothelium independent); and (3) cIMT.

FMD and NID were measured in the brachial artery according to the guidelines of the International Brachial Artery Reactivity Task Force.²⁰ An optimal brachial artery image was obtained above the antecubital fossa at rest. Thereafter, blood pressure cuff was inflated to 250 mmHg for 5 min to induce hyperemia. A longitudinal image of the artery was measured at approximately 60 s after deflation. FMD was defined as the percent change in brachial artery diameter within 1 min after ischemia compared with baseline.

After a 15-min resting interval, 0.6 mg nitroglycerin as an exogenous nitric oxide (NO) donor was administered sublingually, and vascular relaxation was measured at 3 min for NID, which was defined as the percent change within 3 min of nitroglycerin treatment. Reproducibility of the mean difference was 1.9% in FMD and 2.2% in NID, respectively, similar to a previous study.²¹

For measurement of cIMT, B-mode ultrasound (Philips iU22, San Jose, CA, USA) was carried out using a linear array 5–12-MHz scan head. Far-wall cIMT measurements were performed with longitudinal 2-D images in the end-diastolic phase of the cardiac cycle using the QLAB instrument (Philips). In addition, maximum IMT or plaque thickness was measured at the distal common carotid artery, bulb, and proximal internal carotid artery areas on both sides. cIMT was measured by a single sonographer who was a registered vascular technologist, was certified by the American Registry for Diagnostic Medical Sonography, and had 10 years of experience in carotid sonography. All measurements were obtained without prior clinical information about the subjects. The intraclass correlation coefficient was 0.93, and the median for differences between the pairs of measurements was 2.7%.

CE Capacity

The CE capacity was measured using THP-1 cells as previously described.^17,22 After collecting venous blood, plasma samples were stored at −80℃ until assay and were prepared by centrifugation at 1,811 g for 15 min at 4℃. We used apoB-depleted plasma samples that were prepared by adding 40 μL polyethylene glycol solution in 200 mmol/L glycine at pH 10 to 100 μL plasma. The samples were then centrifuged at 7,960 g at 4℃ for 15 min, and the supernatants were transferred to new tubes. The THP-1 human monocytes were differentiated into macrophages by adding 100 nmol/L phorbol myristate acetate. Subsequently, macrophages were loaded with 50 μg/mL acetyl LDL, 3 μmol/L T0901317 (dual LXR/FXR agonist), and 2 μCi/mL [³H] cholesterol for 24 h followed by equilibration in medium containing 2% bovine serum albumin. Then, 4 μg of apoB-depleted plasma from individual patients, diluted in medium, was added. After 3 h, the supernatant medium was removed, and radioactivity within the medium was determined by liquid scintillation counting. Then cells were lyzed in 0.5 mol/L NaOH, and the radioactivity remaining within the cells was measured by liquid scintillation counting. CE was expressed as the percentage of counts measured from the medium in relation to the total counts measured from the medium and cell lysate.

Statistical Analysis

Data are given as mean±SD. Baseline characteristics between the patient and control groups were compared using Student’s t-test or Mann-Whitney U-test. Paired t-test or Wilcoxon signed-rank test was used to compare various factors, including HDL function, before and after statin treatment. Pearson correlation analysis was conducted to evaluate the association between vascular function and biomarkers. Partial correlation analysis was also conducted after adjusting for age and changes in BMI, LDL-C, and HbA1c. Multivariable linear regression analysis was used to assess any association between changes in CE and those in FMD or cIMT after adjusting for relevant factors. P<0.05 was considered to be significant. Statistical analysis was performed using SPSS Statistics for Windows (version 20.0, IBM, Armonk, NY, USA).

Results

Major Parameters

Clinical and biochemical characteristics and HDL function in the 30 dyslipidemic patients with T2DM (mean age, 49.83±11.33 years) and the 20 healthy controls (mean age, 28.80±3.16 years) are listed in Table 1. Mean BMI in the patients and healthy controls was 25.77±3.65 and 22.42±2.44 kg/m², respectively. Among the T2DM patients, 7 were managed with lifestyle modification alone, 8 with metformin alone, and 15 with metformin plus other hypoglycemic agents.

Table 1. Baseline Characteristics

	Control (n=20)	T2DM+dyslipidemia (n=30)	P-value
Anthropometric parameters
Male	10 (50)	15 (50)	–
Median duration of DM (years)	–	6.20±6.14	–
Age (years)	28.80±3.16	49.83±11.33	<0.01
Height (cm)	168.41±10.54	165.28±9.79	0.296
Weight (kg)	63.89±11.11	70.82±13.84	0.057
BMI (kg/m2)	22.42±2.44	25.77±3.65	<0.01
Lipid profile
Total cholesterol (mg/dL)	174.25±27.11	235.13±35.90	<0.01
Triglycerides (mg/dL)	95.70±48.25	218.73±83.93	<0.01
HDL-C (mg/dL)	58.20±13.13	43.07±5.59	<0.01
LDL-C (mg/dL)	96.20±19.21	150.13±27.24	<0.01
ApoA-1 (mg/dL)	146.20±21.61	124.48±14.28	<0.01
ApoB (mg/dL)	82.85±17.25	133.00±19.11	<0.01
Non-HDL-C (mg/dL)	116.05±27.49	192.07±33.46	<0.01
Lipoprotein (a) (mg/dL)	12.95±12.02	27.30±22.66	<0.01
Glucose homeostasis
Fasting plasma glucose (mg/dL)	91.95±7.56	153.33±56.53	<0.01
HbA1c (%)	5.20±.21	8.01±1.93	<0.01
Fasting plasma insulin (μIU/L)	8.16±2.96	10.86±4.60	0.015
HOMA-IR	1.87±0.74	4.03±1.93	<0.01
HOMA-β	104.32±37.06	54.31±24.85	<0.01
Others
AST (IU/mL)	25.90±22.19	31.47±18.17	0.357
ALT (IU/mL)	19.40±11.87	40.83±29.11	<0.01
Creatinine (mg/mL)	0.78±0.15	0.83±0.19	0.247
Urine microalbumin/Cr ratio (mg/g)	8.67±11.21	211.06±955.49	<0.01
hsCRP (mg/dL)	0.07±0.06	0.38±1.14	<0.01
CE (%)	14.84±4.86	12.39±2.77	0.034
Vascular function
FMD (%)	12.45±3.10	10.09±3.25	0.020
NID (%)	19.77±6.19	15.10±5.80	0.011
Maximum cIMT
R common carotid area (mm)	0.51±0.09	0.87±0.46	<0.01
R bulb area (mm)	0.51±0.11	1.15±0.63	<0.01
R internal carotid area (mm)	0.43±0.10	0.83±0.58	<0.01
L common carotid area (mm)	0.50±0.10	0.90±0.45	<0.01
L bulb area (mm)	0.52±0.16	1.26±0.62	<0.01
L internal carotid area (mm)	0.43±0.12	0.91±0.47	<0.01

Data given as n (%) or mean±SD. ALT, alanine aminotransferase; ApoA-1, apolipoprotein A-1; ApoB, apolipoprotein B; AST, aspartate aminotransferase; BMI, body mass index; CE, cholesterol efflux; cIMT, carotid intima-media thickness; DM, diabetes mellitus; FMD, flow-mediated dilation; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, homeostasis model assessment of insulin resistance; HOMA-β, homeostasis model assessment of β-cell function; hsCRP, high-sensitivity C-reactive protein; L, left; LDL-C, low-density lipoprotein cholesterol; NID, nitroglycerin-induced dilation; R, right; T2DM, type 2 diabetes mellitus.

The patients had significantly lower HDL-C and apoA-I and higher total cholesterol, triglycerides, LDL-C, apoB, and lipoprotein (a). As expected, the patients had higher glucose and insulin concentrations and HbA1c than the controls. As a result, HOMA-IR was greater and HOMA-β was lower in the patients than in the controls. Furthermore, albuminuria was more prevalent and hsCRP was significantly higher in the patients than in the controls.

Baseline CE was lower in patients than in controls (12.39±2.77% vs. 14.84±4.86%, P=0.034). In the vascular health evaluation, both FMD and NID were significantly lower in the patient group than in the control group (all P<0.05). Maximum cIMT was higher in the patients than in the controls (all P<0.01).

Change in Parameters After Rosuvastatin Treatment

Among the 30 patients, 29 completed 12 weeks of treatment with rosuvastatin. As shown in Table 2, BMI, glycemic indices, HOMA-IR, and HOMA-β did not change after 12 weeks of treatment with rosuvastatin. Regarding lipid profiles, total cholesterol, triglycerides, LDL-C, apoB, and lipoprotein (a) decreased significantly, while HDL-C increased significantly in response to rosuvastatin treatment. hsCRP concentration also significantly decreased after rosuvastatin treatment.

Table 2. Change in Parameters After 12-Week 20-mg Daily Rosuvastatin

	T2DM+dyslipidemia (n=29)		P-value
	Baseline	At 12 weeks
Anthropometric parameters
BMI (kg/m2)	25.95±3.58	26.09±3.70	0.303
Lipid profiles
Total cholesterol (mg/dL)	234.66±36.43	134.90±24.70	<0.01
Triglycerides (mg/dL)	210.24±71.10	144.17±64.35	<0.01
HDL-C (mg/dL)	42.83±5.53	44.90±7.34	0.029
LDL-C (mg/dL)	150.52±27.64	69.03±20.91	<0.01
ApoA-1 (mg/dL)	123.39±13.25	130.33±17.80	0.032
ApoB (mg/dL)	133.14±19.45	73.33±15.84	<0.01
Non-HDL-C (mg/dL)	191.83±34.03	90.00±23.52	<0.01
Lipoprotein (a) (mg/dL)	28.07±22.65	22.69±19.80	0.007
Glucose homeostasis
Fasting plasma glucose (mg/dL)	154.28±57.29	138.93±38.80	0.116
HbA1c (%)	8.04±1.95	7.80±1.57	0.487
Fasting plasma insulin (μIU/L)	10.98±4.64	11.78±5.41	0.309
HOMA-IR	4.08±1.94	4.06±2.14	0.939
HOMA-β	54.69±25.20	67.50±41.26	0.124
Others
AST (IU/mL)	31.90±18.33	33.83±20.34	0.617
ALT (IU/mL)	41.66±29.27	40.48±25.39	0.798
Creatinine (mg/mL)	0.84±0.19	0.82±0.16	0.344
Urine microalbumin/Cr ratio (mg/g)	217.96±971.64	170.46±709.12	0.259
hsCRP (mg/dL)	0.39±1.16	0.10±0.08	<0.01
CE (%)	12.26±2.72	14.05±4.14	0.010
Vascular function
FMD (%)	10.15±3.29	11.29±3.50	0.043
NID (%)	15.27±5.83	15.53±4.87	0.768
Maximum cIMT
R common carotid area (mm)	0.85±0.46	0.84±0.43	0.316
R bulb area (mm)	1.16±0.64	1.09±0.63	0.025
R internal carotid area (mm)	0.82±0.59	0.78±0.53	0.031
L common carotid area (mm)	0.90±0.45	0.88±0.43	0.252
L bulb area (mm)	1.27±0.63	1.16±0.53	0.018
L internal carotid area (mm)	0.91±0.48	0.85±0.41	0.029

Data given as mean±SD. Abbreviations as in Table 1.

In the present study, CE was measured after 1 and 12 weeks of treatment with rosuvastatin to evaluate the short- and long-term effects, respectively. CE did not change significantly from baseline after 1 week, but it increased significantly by 14.6% (from 12.26±2.72% to 14.05±4.14%) compared with baseline after 12 weeks of treatment (Table 2; Figure 1).

Figure 1.

Change in cholesterol efflux after treatment with rosuvastatin (20 mg daily) for 1 and 12 weeks. P-values calculated using paired t-test.

Regarding the evaluation of endothelial function, FMD increased by 11.2% (from 10.15±3.29% to 11.29±3.50%) after 12 weeks of treatment, but NID did not change. Maximum cIMT at the right and left bulb and internal carotid areas in both carotid arteries decreased significantly from baseline (Table 2).

The correlations between changes in CE and changes in FMD, lipoprotein (a), and maximum cIMT at the right and left bulb areas were analyzed to investigate whether changes in CE were directly associated with vascular health. The changes in CE correlated positively with those of FMD and negatively with those of lipoprotein (a) and maximum cIMT at both the right and left bulb areas after adjusting for age and changes in BMI, LDL-C, and HbA1c (Figure 2).

Figure 2.

Change in cholesterol efflux vs. change in (A) flow-mediated dilation (FMD); (B) lipoprotein (a), and (C,D) carotid intima-media thickness (cIMT) at the (C) right and (D) left bulb areas after adjusting for age and changes in body mass index, low-density lipoprotein cholesterol, and hemoglobin A1c.

Change in CE

The results of multivariable linear regression analysis are listed in Table 3. We evaluated whether changes in CE were independently associated with vascular health status measures such as FMD or cIMT. Relevant factors such as age, BMI, HbA1c, and changes in LDL-C, lipoprotein (a), and hsCRP were included in the models. We found that change in CE was significant regarding the association with that in FMD and in cIMT at bulb areas on both sides (Table 3). Change in LDL-C was also significantly associated with that in cIMT at both sides.

Table 3. Multivariate Indicators of ΔFMD and ΔcIMT

	ΔFMD		ΔcIMT
			Right bulb area		Left bulb area
	Standardized β	P-value	Standardized β	P-value	Standardized β	P-value
Age (years)	0.168	0.396	−0.339	0.066	−0.183	0.374
BMI (kg/m2)	0.003	0.986	0.051	0.752	0.009	0.960
HbA1c (%)	0.198	0.329	0.008	0.964	0.186	0.781
ΔLDL-C (mg/dL)	−0.208	0.298	0.487	0.010	0.418	0.026
ΔLipoprotein (a) (mg/dL)	−0.021	0.925	0.104	0.542	0.173	0.450
ΔhsCRP (mg/dL)	−0.160	0.397	0.153	0.552	0.201	0.310
ΔCE (%)	0.646	0.007	−0.569	0.007	−0.637	0.009

Δ, change; HbA1c, glycated hemoglobin. Other abbreviations as in Table 1.

Discussion

In the present study, we confirmed that HDL function as assessed by CE was lower and other vascular health conditions were inferior in patients with T2DM and dyslipidemia than in healthy individuals. Treatment with rosuvastatin 20 mg for 12 weeks increased HDL function as estimated by CE and improved lipid profiles. Circulating levels of biomarkers related to vascular health, such as hsCRP, lipoprotein (a), and cIMT, were significantly higher in the patients than in the healthy controls, and decreased after rosuvastatin treatment.

Treatment with statins is well documented to reduce cardiovascular events and to be associated with an LDL-lowering effect.^23,24 The present study has confirmed the lipid-modulating properties of rosuvastatin: decreases in LDL-C, apoB, triglycerides, and non-HDL-C. A residual risk for CVD, however, has still been reported after intensive lipid-lowering therapy.¹ In this circumstance, HDL-C has attracted attention because it has several well-documented functions to protect against CVD.²⁵ HDL also inhibit vascular inflammation and oxidative modification of LDL. Thus, HDL-C exerts a beneficial role in vascular health, particularly in endothelial function.^25,26

Recently, HDL function is recognized as an important target for lipid modulating strategies.^25,27–31 CE is an established method for evaluating HDL function^28,31 and the ability to promote CE from macrophages in the artery wall is considered to be a main cardioprotective function of HDL.^27,29 Individuals with CVD are well known to have impaired CE.^28,31 Data on the CE of T2DM patients compared with normal controls, however, are scarce. One study found that the CE of HDL in patients with a long duration of DM (>10 years) was lower than that in non-diabetic controls.³² Similarly, in the present study, baseline CE capacity of patients was impaired compared with that of healthy controls.

In the present study, the CE capacity of HDL increased after 12 weeks of treatment with rosuvastatin, along with improvement of lipid profiles. Treatment with certain types of statins may improve qualitative aspects of HDL such as the CE capacity and quantitative aspects of lipid profiles. Several studies have investigated HDL function after statin treatment, but inconsistent results have been reported: some studies showed increased CE,^16,17 some showed no effects,^30,31 and others showed decreased CE.¹⁵ These studies used various types of statins, and the patients had various phenotypes, such as dyslipidemia or DM. Duration of treatment varied from 4 to 16 weeks. In contrast, we used 20 mg rosuvastatin, a potent statin, which might have a favorable effect on HDL function, particularly in patients who have both DM and dyslipidemia.

HDL is known to play an important role in endothelial function by increasing the production of NO.³³ HDL isolated from healthy individuals promotes the synthesis of NO in endothelial cells, which contributes to endothelial repair.³⁴ These endothelial-vasoprotective effects of HDL are reported to be impaired in individuals with T2DM.³⁵ We compared endothelial-vasoprotective effects of HDL between middle-aged DM patients and young healthy controls. As expected, we found decreased endothelial-vascular function of HDL in T2DM patients, impaired vascular relaxation, and thicker cIMT. We previously investigated cIMT and other vascular functions in a community-based elderly cohort.³⁶ cIMT in the present middle-aged patients with T2DM and dyslipidemia was higher than that in elderly people aged >65 years (0.87–0.90 mm in common carotid artery areas vs. 0.75–0.81 mm in distal common carotid artery areas, respectively; P=0.058). Although we did not measure cIMT in age-matched controls in the present study, we could confirm that cIMT in middle-aged patients with DM was similar to or a little higher than that in the elderly subjects aged >65 years in the previous study.³⁶

Treatment with rosuvastatin 20 mg for 12 weeks improved vascular endothelial function and decreased cIMT at the right and left bulb areas; these results were consistent with previous studies.^37–39 In addition, 2 studies have investigated cIMT changes after moderate doses of rosuvastatin in East Asian patients with dyslipdemia.^40,41 They found that 5–10 mg rosuvastatin slowed the progression of cIMT significantly at 12 months and at 2 years compared with 10–20 mg pravastatin treatment.^40,41

Inflammatory biomarkers such as hsCRP, tumor necrosis factor-α, interleukin-6, and adhesion molecules are reported to predict future cardiovascular events.⁴² HDL has the ability to suppress the production of these inflammatory cytokines and inhibit the expression of endothelial cell adhesion molecules.^38,43 Morgantini et al reported that the anti-inflammatory properties of HDL were impaired in patients with T2DM.⁴⁴ Consistent with these findings, the present study showed that inflammatory markers such as hsCRP and lipoprotein (a) were higher in patients than in healthy controls. Lipoprotein (a) has proatherogenic and prothrombotic effects.⁴⁵ Lipoprotein (a) promotes the expression of pro-inflammatory cytokines and induces endothelial dysfunction.⁴⁶ Elevation of lipoprotein (a) and low-grade inflammation commonly coexist, and both play deleterious roles on endothelial integrity, at both the functional and structural levels.⁴⁶ In the present study, lipoprotein (a) decreased significantly after 12 weeks of treatment with rosuvastatin. Moreover, change in CE was associated with change in FMD, lipoprotein (a), and cIMT after adjusting for age and change in BMI, LDL-C, and HbA1c (Figure 2). These changes support the favorable effect of rosuvastatin on the qualitative aspect of HDL-C and its association with vascular health beyond an LDL-lowering effect.

We do not know whether the improvements in vascular health status after rosuvastatin treatment could have been caused by reduction in LDL-C or by other pleiotropic effects of the drug. The present finding that the increase in CE induced by rosuvastatin was independently associated with an increase in FMD and a decrease in cIMT after adjusting for relevant factors suggests that the alteration in CE induced by rosuvastatin is an independent predictor of vascular health.

The present study did have several limitations. First, the subjects consisted of only a small sample of relatively healthy patients with T2DM and dyslipidemia. Second, in the patient group, various anti-diabetic treatments were used, including lifestyle modification alone, metformin, or metformin in combination with sulfonylurea or a dipeptidyl peptidase-4 inhibitor; this may have affected HDL function and/or vascular health. Nevertheless, these treatments were maintained throughout the study period.

Several studies have reported that proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors decrease lipoprotein (a).^47,48 A recent study reported that PCSK9 inhibited CE via downregulation of gene expression for the ATP-binding cassette transporter B1, and that co-incubation with an anti-PCSK9 antibody prevented this effect.⁴⁹ This suggests that PCSK9 inhibitors might have a positive effect on CE. If another group had been treated with PCSK9 inhibitors in the present study, the results could have been illuminating, but unfortunately those agents were not available in South Korea at the time of the present study.

The present study did have several advantages. We assessed vascular health status and HDL function using various methods, including CE, vascular endothelial-dependent or -independent function, cIMT, and various biomarkers. We examined CE to confirm short- (1 week) and long-term (12 weeks) effects of rosuvastatin.

Conclusions

Rosuvastatin 20 mg increased HDL function, improved vascular endothelial function and atherogenesis in carotid arteries, and favorably changed biomarkers related to lipid metabolism and atherosclerosis in patients who had both DM and dyslipidemia. This suggests that, in addition to its LDL-lowering effects, the improvement in HDL function after rosuvastatin treatment may contribute to its beneficial effect in cardiovascular morbidity and mortality.

Acknowledgments

This work was supported by research grants from the Korea Diabetes Association (06-2014-108) and Astra Zeneca (2014). The funding agencies had no role in the study design, data collection or analysis, decision to publish or preparation of the manuscript. The sole responsibility for the content of this paper lies with the authors.

Disclosures

The authors declare no conflicts of interest.

Author Contributions

K.Y.J., K.M.K. and S.L. researched data and contributed to the experimental design and discussion. K.Y.J., K.M.K., S.K.H., H.M.Y., T.J.O., S.H.C., K.S.P. and H.C.J. researched data and contributed to the discussion. K.Y.J., K.M.K. and S.L. drafted the manuscript. All authors edited and revised the manuscript and approved the final version. S.L. is responsible for the integrity of the work as a whole.

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