High‐dose atorvastatin improves vascular endothelial function in patients with leukoaraiosis

Abstract Objective Leukoaraiosis (LA), as an age‐related white matter degeneration, is mainly caused by chronic ischemia. Our study aims to explore the efficacy of different doses of atorvastatin (ATV) in the vascular endothelial function in patients with LA. Methods Our study enrolled 402 LA patients who were then randomly included as control or treated with ATV (10 mg), ATV (20 mg), or ATV (30 mg). The total cholesterol (TC), triglycerides (TG), high‐density lipoprotein cholesterol (HDL‐C), and low‐density lipoprotein cholesterol (LDL‐C) were detected by enzyme colorimetric assay. The high‐sensitivity C‐reactive protein (hs‐CRP) level, reactive hyperemia index (RHI), endothelin‐1 (ET‐1) content, and nitric oxide (NO) level were tested by latex agglutination test, peripheral arterial tonometry technology, radioimmunoassay, and nitrate reductase assay, respectively. Results After 8 weeks of ATV treatment, the levels of TC, LDL‐C, and HS‐CRP decreased significantly, and the trends were demonstrated in a more significant way with the increases of dose of ATV. The treatment with ATV at different doses elevated NO level and RHI and declined ET‐1 content. Gastrointestinal reaction, muscular pain, and increased aminopherase were observed after treatment with the ATV at different doses with more obvious symptoms detected accompanied by the increase of the dose. The RHI was in negative correlation with the ET‐1 and HS‐CRP while in positive correlation with NO. Conclusion Our study demonstrates that ATV can significantly improve the vascular endothelial function in LA patients with a dose‐dependent effect.

characterized by declined nitric oxide (NO) bioavailability, was present as the first stage during the development of coronary artery disease. 7 Vascular endothelial function, decreased with aging, is closely related to an elevated risk of cardiovascular disease, and particularly aerobic exercise, lifestyle modification as well as dietary adjustment had a favorable effect on vascular aging, 8 while the assessment of the vascular endothelial function lacks consistency. 9 Interestingly, atorvastatin (ATV) may enhance endothelial function for relatively moderate nicotine-dependent smokers. 10 Atorvastatin, as a synthetic inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A, presents with a long plasma half-life and lipidlowering ability and is commonly performed to reduce cholesterol levels. 11 As an HMG-CoA reductase inhibitor, ATV is often used in the treatment of hypercholesterolemia. 12 In addition, it is widely used for the treatment of dyslipidemias and exhibits protective effects against seizures, and a study demonstrated its effect on oxidative stress markers and certain neurotransmitter and on animal models of anxiety, seizures, and depression. 13 A high-fat diet could lead to endothelial dysfunction related to inflammation, and ATV might be capable of counter-regulating it. 14 Another study explored the role of local and systemic ATV application on periodontium by using immunohistochemical and histomorphometric analyses and demonstrated its beneficial effects on periodontium after the induction of experimental periodontitis, indicating that ATV can also be treated as a therapeutic and protective marker for periodontal disease. 15 A previous study investigated the effect of ATV therapy on disease activity, inflammation, arterial stiffness, and endothelial dysfunction in patients suffering from rheumatoid arthritis, and the results indicated that ATV therapy in patients with rheumatoid arthritis inhibited disease activity and vascular risk factors promoting the atheromatous lesion. 16 Thus, this study aims to explore the role of ATV in the vascular endothelial function in patients with LA, so as to provide a therapeutic method for LA treatment.

| Ethical statement
The present study was performed with the approval of the Ethics Committee of Jiaxing Maternity and Child Health Care Hospital and all subjects signed written consents.

| MRI and grading standards
Head MRI using 3.0 T superconducting MRI system (German with LA when symmetrical structure, unclear patchy fusion or nonfusion area, relative low density of T1WI signal (relative low density of CT signal), and high density of T2WI signal and T2FLAIR were observed in periventricular white matter of two lateral ventricles.
Foci exhibited low-density signal in MRI and long T1 and T2 signals.
All patients were divided into four levels according to the Aharon-Peretz standard, 18 and the concrete grading was presented as follows: LA-1, low-density area was observed on the frontal or occipital horn of lateral ventricle; LA-2, low-density area was observed on both the frontal and occipital horn of lateral ventricle; LA-3, successive low-density area was observed lateral ventricle with foci; LA-4, foci were observed around the cella lateralis and corona radiate area.  (Table 1), and there were no significant differences in parameters such as heart rate, blood pressure, and blood lipid (all P > .05). Patients in the ATV groups, aside from the regular treatment, were administered oral doses of different AVT concentrations (Lipitor, Pfizer Inc, 10, 20, and 40 mg, respectively) once every night for 8 weeks whereas the patients in the control group only received regular treatment. The diet and lifestyle of all subjects were maintained in the same routine during experiments.

| Grouping and treatment regimens
Before and after 8 weeks of treatment, venous blood samples were extracted on an empty stomach for detection of blood fat, levels of high-sensitivity C-reactive protein (HS-CRP), endothelin-1 (ET-1), and nitric oxide (NO). In addition, endothelial peripheral arterial tonometry (Endo-PAT) was used to detect and observe vascular endothelial function.

| Enzyme colorimetric assay
OLYMPUS AU400 automatic biochemical analyzer was employed.
The total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) reagents and standards were obtained by Olympus Optical Co., Ltd. and ApoA and ApoB reagents and standards by Jiuqiang Bio Co., Ltd. All patients in the control and ATV groups were tested, with no drinking or high-fat diet before detection. The venous blood samples extracted on an empty stomach were reserved in biochemical coagulation tubes; the serum was separated at 4000 r/min and detected within 2 hours. The TC, TG, HDL-C, and LDL-C were detected by enzyme colorimetric assay. All aforementioned indexes were detected within 24 hours of onset of LA and 8 weeks after ATV treatment on an empty stomach.

| Detection of HS-CRP
Heparin anticoagulant (3 mL) was extracted from a vein on an empty stomach from patients in the control, ATV (10 mg), ATV (20 mg), and ATV (40 mg) groups and was centrifuged for 5 minutes (4000 r/min) to separate blood plasma, after which it was then reserved in a refrigerator at 38°C for further use. HS-CRP in blood plasma was detected by latex agglutination test (BN100; Baldor Electric Company).

| Reactive hyperemia index (RHI) detection
The Endo-PAT technology (AUDEN) was employed for RHI detection. The principle was based on the biosensor system, in combination with the peripheral arterial tension measurement (PAT) for detecting the change in fingertip pulse volume (blood flow). All  TA B L E 1 Comparisons of clinical data of subjects among the control and ATV (10 mg), ATV (20 mg), and ATV (30 mg) groups patients while lying flat wore pneumatic cuff in the right arms and a probe containing sensor and pneumatic appliance was attached on the forefinger. After inflation, the blood flow signals were transferred to the computer, and then, detection was started when the signals were stable. The base signal was initially collected for 5 minutes, followed by inflation for 5 minutes and data collection, then exhaustion immediately, and recording for 5 minutes to end the detection. Finally, the RHI of blood vessel was measured by software.
The RHI was regarded as reference, and smaller RHI was indicative of more severe vascular endothelial dysfunction.

| Radioimmunoassay
Twelve hours after the patients were on empty stomach, 2 mL of elbow venous blood was extracted in a recumbent position and then transferred to a tube with 30 µL of 10% ethylene diamine tetraacetic acid (EDTA) and 40 µL of trasylol, mixed well at 40°C, centrifuged for 10 minutes (3000 r/min) to separate blood plasma for preservation at −70°C. Before detection, the samples were thawed at a room temperature, centrifuged for 5 minutes (3000 r/min) at 4°C. The radioimmunoassay was conducted for serum detection. The reference value of ET-1 was 50.82 ± 7.58 pg/mL.

| Nitrate reductase assay (NRA)
Nitrate reductase assay detected the NO level in the blood.

| Follow-up
Follow-up was conducted by outpatient reexamination and telephone contact for recording the adverse reactions, such as gastrointestinal reaction, muscular pain, and increased aminopherase.
Eight weeks after the treatment follow-up was stopped, a total of 402 cases were followed up, with a rate of 100%.

| Statistical analysis
The SPSS 19.0 software (IBM Corp.) was used for data analysis. The categorical data were presented in the form of number or percentage, and the chi-square test was highlighted for comparisons among multiple groups. The measurement data were detected using normality test and homogeneity of variance test. One-way analysis of variance (ANOVA) was conducted for comparisons among multiple groups, mean ± SD for data in normal distribution, t test for comparisons before and after ATV treatment, and Pearson correlation analysis for correlation analysis, with P < .05 signifying significant difference.

| The clinical data of enrolled subjects
According to the different doses of ATV, all patients were allocated into the control (201 cases), ATV (10 mg, 67 cases), ATV (20 mg, 67 cases), and ATV (40 mg, 67 cases) groups. There were no remarkable differences in terms of age, gender, disease history, heart rate, blood pressure, and blood fat before ATV treatment (all P > .05), as shown in Table 1.

| ATV treatment reduces the levels of TC and LDL-C in LA patients in a dose-dependent manner
Enzyme colorimetric assay was conducted to investigate whether

| ATV treatment decreases the HS-CRP level in LA patients in a dose-dependent manner
In order to determine the HS-CRP level in the control and ATV group. In the control group, the HS-CRP level dropped from 11.38 to 11.24, showing a relative low decreasing trend than that in the ATV groups ( Figure 3). It was concluded that ATV treatment could decrease the HS-CRP level in LA patients in a dose-dependent manner.

| ATV treatment elevates the levels of RHI, ET-1, and NO in LA patients
Subsequently, PAT technology, radioimmunoassay, and NRA were performed for determination of levels of RHI, ET-1, and NO, respectively. As shown in Figure 4, there were no significant differences It was suggested that ATV treatment could elevate the levels of RHI, ET-1, and NO in LA patients, but this effect was independent of its dose.

| RHI is negatively correlated with HS-CRP and ET-1 whereas exhibiting a positive correlation with NO level
Pearson correlation analysis ( Figure 5) was conducted to detect the correlation of RHI with HS-CRP, ET-1, and NO, which exhibited that the RHI was negatively correlated with HS-CRP and ET-1 (r = −.680 and −.678, both P < .05), whereas exhibiting a positive correlation with NO level (r = .647, P < .05).

| Treatment with ATV (40 mg) contributes to the most severe adverse reactions
The adverse reactions experienced by all patients in the four groups were recorded. The records highlighted no adverse reaction in the control group, while four cases (6.0%) of gastrointestinal reaction, five cases (7.5%) of muscle pain, and three cases (4.5%) of transaminase elevation were observed in the ATV (10 mg) group; five cases

| D ISCUSS I ON
Leukoaraiosis, or white matter changes, is in close association with increased age, history of stroke, hypertension, and diabetes mellitus. 19 It has been worldwide regarded as a part of the normal aging process, even though it is strongly associated with dementia or other disabilities, and its pathogenesis still has not been thoroughly acknowledged. 20  Initially, we found that the levels of TC, LDL-C, and HS-CRP decreased significantly in the ATV groups compared with the control group after 8-week of ATV treatment, indicating that a proper dose of ATV could promote vascular endothelial function. Serum cholesterol, either total or lipoprotein fractions, is in closely association with human both mid-and late-life depression. 21 A cohort study was conducted for determining the relative risk and incidence for myocardial infarction, and it verified that TC was a significant risk factor for myocardial infarction. 22 LDL-C level is strongly associated with sustained viral response for chronic hepatitis C infection, and it was concluded that LDL-C burden might identify those patients with pegIFN/RBV therapy. 23 LDL-C is also an well-acknowledged  27 Remarkably, ATV-induced increment in apelin was independently associated with changes in TC and LDL-cholesterol. 28 A study demonstrated that the ATV treatment had a close relation with remarkable reduction in target-to-background ratio (TBR) both in femoral aorta and in ascending artery, and TBR in both arteries associated with reductions in LDL-C, MDA-LDL-C, and HS-CRP. 29 ATV was able to decrease LDL-C and HS-CRP in patients of European or South Asian origin. 30 Most importantly, one study aimed at investigating the efficacy of ATV on levels of TC and HS-CRP in atrial fibrillation (AF) patients in Asia suggested that ATV was significantly effective in lowering serum levels of HS-CRP as well as TC so as to inhibit cardiovascular events. 31 HS-CRP, factor VII, and enhanced monocyte cytokine release were abnormalities, which could be attenuated by both ATV treatments. 32 A combination of ATV with Berberine reduces levels of TC, TG, LDL-C, and also oxidative stress and inflammation in rat models. 33 Altogether, we concluded that decreased levels of TC, LDL-C, and HS-CRP in the ATV groups after 8- week of ATV treatment promoted the vascular endothelial function.  34 One study demonstrated that ET-1 to some extent contributed to diminishing the endothelium-dependent vasodilatation among older men. 35 Moreover, NO and ET-1 are natural parts in vascular function, and the imbalance between these two markers is a characteristic of endothelial dysfunction and is significant in vascular disease progression. 36 The expression and effects of ET-1 along with its receptors are significantly altered during the development of cardiovascular disease and the increased production of ET-1 mediates many pathophysiological events contributing to the development of atherosclerosis and vascular complications in diabetes mellitus. 37 RHI was calculated using RH-PAT and was defined as the ratio of the digital pulse volume which could attenuate the severity in the patients with restenosis. 38 Significantly, one study offered evidence that ATV was of significant importance in reducing ET-1 level, which highlighted its antivasospastic effect on chronic vasospasm. 39 In addition, another study found that compared to non-ATV group, ET-1 expression decreased in human umbilical vein endothelial cells of the ATV group but NO level increased, signifying that ATV could inhibit oxidative stress as well as endothelial damage. 40 A prior study proved that liver ischemia-reperfusion injury leads to a dramatic increase in the apoptosis and necrosis of hepatocytes, aminopherase activity, and production of pro-inflammatory cytokines. 41 Our findings were in line with the aforementioned studies; thus, we conclude that ATV treatment promoted vascular endothelial function in patients with LA.
To conclude, our study demonstrates that different doses of

ACK N OWLED G M ENTS
We would like to give our sincere appreciation to the reviewers for their helpful comments on this article.