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Brain microangiopathy and macroangiopathy share common risk factors and biomarkers
Atherosclerosis 246 (2016) 71e77
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Brain microangiopathy and macroangiopathy share common risk factors and biomarkers Oh Young Bang a, *, Jong-Won Chung a, Sookyung Ryoo a, Gyeong Joon Moon b, Gyeong-Moon Kim a, Chin-Sang Chung a, Kwang Ho Lee a a b
Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Samsung Bioresearch Institute, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 October 2015 Received in revised form 12 December 2015 Accepted 25 December 2015 Available online 29 December 2015
Aims: Besides carotid or cardiac embolism, stroke can occur via microangiopathy (small arterial disease [SAD]) and macroangiopathy (intracranial atherosclerotic stroke [ICAS]) of the intracranial vasculature. There have been efforts to identify risk factors specific to microangiopathy and macroangiopathy, including vascular risk factors, and protein and genetic biomarkers. We hypothesized that despite the anatomic and pathophysiological differences between microvessels and macrovessels, microangiopathy and macroangiopathy share common risk factors during disease progression. Methods: Among 714 patients with acute infarctions within middle cerebral artery territory, 126 with SAD and 116 with ICAS were included in this study. Subclinical microangiopathy (degree of leukoaraiosis) and macroangiopathy (number of tandem stenosis) was graded in each patient. Inflammatory biomarkers (C-reactive protein, E-selectin, and LpPLA2), endothelial dysfunction (asymmetric dimethylarginine, urinary albumin-to-creatinine ratio, endostatin, and homocysteine), atherogenesis (lipoprotein(a), adiponectin, and resistin), and renal function (creatinine clearance and estimated glomerular filtration rate) were assessed. Results: Compared with the patients with isolated SAD, those with isolated ICAS were younger, were current smokers, and showed higher apoB levels (p < 0.05 in all cases). However, with the progression of subclinical microangiopathy, asymptomatic macroangiopathy worsened and vice versa. No significant differences in risk factors were observed between advanced SAD and ICAS. Decreased renal function was independently associated with progression of microangiopathy and macroangiopathy. Markers of endothelial dysfunction, but not the other markers, were significantly related to creatinine clearance level. Conclusions: Mild to moderate loss of renal function is strongly associated with both intracranial microangiopathy and macroangiopathy. Endothelial dysfunction may be associated with this relationship. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Intracranial atherosclerosis Small artery disease Risk factor Renal function Endothelial function
1. Introduction Besides carotid or cardiac embolism, stroke can occur via microangiopathy (small arterial disease [SAD]) and macroangiopathy (intracranial atherosclerotic stroke [ICAS]) of the intracranial vasculature. Both are common stroke subtypes worldwide.
* Corresponding author. Department of Neurology, Samsung Medical Center, Sungkyunkwan University, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea. E-mail address: [email protected] (O.Y. Bang). http://dx.doi.org/10.1016/j.atherosclerosis.2015.12.040 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
Numerous efforts have been made to identify the risk factors associated with intracranial microangiopathy and macroangiopathy [1]. Nevertheless, the apparent differences in risk factors between them are unclear. This could be caused by several reasons. First, the current stroke classification system may have limitations in defining intracranial microangiopathy and macroangiopathy. An autopsy study showed that intracranial atherosclerosis that was not severe was also responsible for parent territorial stroke [2]. Recently developed high-resolution magnetic resonance imaging (MRI) techniques can visualize intracranial wall plaque and provide information on the possible mechanisms of stroke [3]. Second, novel risk factors but not the conventional risk
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factors may differ between intracranial microangiopathy and macroangiopathy. Several recent studies investigated possible novel risk factors of intracranial macroangiopathy, which include metabolic syndrome and adipokines (adiponectin and resistin) [4e6], inflammatory markers [7,8], lipoprotein (a) [9,10], homocysteine [11], and endothelial dysfunction (asymmetric dimethylarginine [ADMA] and E-selectin) [6,7]. However, these biomarkers are also reported to be associated with cerebral microangiopathy [1,12]. We hypothesized that despite the anatomic and pathophysiological differences between microvessels and macrovessels, they share common risk factors because cerebral microangiopathy and macroangiopathy commonly coexist. Thus, we compared conventional and novel risk factors of stroke in patients with ICAS and SAD, in consideration of the degree of subclinical macroangiopathy (asymptomatic tandem stenosis) and microangiopathy (leukoaraiosis). 2. Patients and methods We prospectively recruited patients with acute symptomatic infarctions within the middle cerebral artery (MCA) territory admitted to a tertiary university stroke center between October 2008 and January 2012. We defined potential participants as patients who experienced focal or lateralizing symptoms within 7 days of symptom onset and showed relevant lesions on diffusionweighted imaging (DWI). Patients with potential sources of cardioaortic embolism, based on the modified Trial of Org 10172 in Acute Stroke Treatment (SSS-TOAST), extracranial atherosclerosis with significant (50%) stenosis in the relevant extracranial arteries, other stroke mechanisms (coagulopathy, vasculitis, moyamoya disease, internal carotid artery dissection, and others), or incomplete evaluations were excluded. Local institutional review boards approved this study. All the patients or patient's guardians provided informed consent with regard to participation in the study. During the study period, 1801 patients visited our center with acute ischemic stroke or TIA. Of 714 patients with acute MCA infarctions on DWI, 242 were included in this study. According to the lesion distribution on DWI and the presence of stenosis in the relevant MCA, we divided the patients into two groups: (1) the ICAS group, patients with infarcts within the MCA territory and relevant MCA stenosis, and (2) the SAD group, patients with deep infarctions and without stenotic lesions on the relevant MCA. We have reported that patients with intracranial atherosclerosis (especially branch occlusive disease [BOD]) are often misclassified as having microangiopathy or cryptogenic cause [13,14]. Thus, in this study, we regarded any degree of intracranial artery stenosis as macroangiopathy. 2.1. Workups We collected clinical information, including age, sex, and vascular risk factors. All patients underwent diagnostic testing that included routine blood tests, electrocardiography, at least 24 h of cardiac telemetry, and echocardiography. Our definitions of vascular risk factors were as follows: (1) Hypertension was deemed present when the patient had been undergoing treatment with antihypertensive agents or a blood pressure of either 160 mm Hg systolic or 90 mm Hg diastolic on at least 2 occasions after the acute phase of their ischemic stroke. (2) Diabetes mellitus was deemed present when the patient had been receiving medication for diabetes, had an elevated fasting glucose level 126 mg/dL or a 2-h plasma glucose level 200 mg/dL during their oral glucose tolerance test, or had a plasma glucose level 200 mg/dL along
with classic symptoms of hyperglycemia, hypoglycemic crisis, or hemoglobin A1c level >6.5%.8 (3) Dyslipidemia was determined to be present if the patient had been taking lipid-lowering agents or had a total cholesterol level >240 mg/dL, triglyceride level >200 mg/dL, or low-density lipoprotein cholesterol level >160 mg/ dL. ApoB and apoA1 levels were determined by using nephelometry (Behring Nephelometer, Marburg, Germany). (4) Coronary artery disease was regarded as being present when the patient had angina, myocardial infarction, or a history of coronary angioplasty or coronary bypass surgery. (5) Current smokers were those who regularly smoked at least 1 cigarette per day when admitted to the center. (6) Body mass index was measured within 3 days of admissions in most patients. In addition, creatinine clearance tests, i.e., creatinine clearance and estimated glomerular filtration rate (eGFR), were measured as markers of kidney function. Serum creatinine level was measured by using the Jaffe method with a Hitachi 7600-210 chemistry analyzer (Hitachi, Japan), and creatinine clearance was calculated according to the Cockcroft-Gault formula. eGFR was calculated by using the Modification of Diet in Renal Disease (MDRD) Study equation.
2.2. Novel biomarkers To further evaluate the possible mechanisms of subclinical microangiopathy and macroangiopathy, the following markers were determined in patients enrolled from August 2010 to January 2012 (n ¼ 113). Inflammatory biomarkers included high-sensitivity C-reactive protein [hs-CRP], lipoprotein-associated phospholipase A2 [LpPLA2] (a calcium-independent phospholipase-derived especially from macrophages), and E-selectin (also known as CD62E, a cell adhesion molecule expressed only on endothelial cells recruiting leukocytes to the site of injury). Biomarkers for endothelial dysfunction included ADMA (an endogenous inhibitor of nitric oxide synthase), urinary albumin-to-creatinine ratio (UACR), and homocysteine. The level of endostatin, an angiogenesis inhibitor that may suppress angiogenesis and collateral development, was measured. Atherogenic biomarkers included lipoprotein (a) (a lipideprotein complex with proatherogenic and prothrombotic properties) and adipokines (adiponectin and resistin, hormones secreted from adipose tissue). Amounts of protein in plasma were quantified by using the following enzyme-linked immunosorbent assay (ELISA) kits: human ADMA ELISA kit (Immundiagnostik, Cat No. K7828), human E-selectin ELISA kit (Biosensis, Cat No. BEK2089-2P), human adiponectin and resistin ELISA kits (AdipoGen, Cat Nos. AG-45A-0002PP-KI01 and AG-45A-0023PP-KI01, respectively), human Endostatin ELISA kit (Raybiotech, Cat No. ELHEndostatin-001), and human Lp-PL-A2 ELISA kit (EIAab, Cat No. E0867h). ELISAs were performed following the manufacturers' instructions. All ELISA absorbance readings were performed with reference to the standard curve. All samples were run in duplicate. ELISA plates were read by using the SpectraMax 340PC384 Microplate Reader and analyzed by using the SoftMax® Pro Data Analysis Software (Molecular Devices). Serum hs-CRP concentration was measured with the turbidimetric immunoassay by using an autochemistry analyzer (Hitachi 7600-210, Hitachi, Japan). The UACR was calculated. Plasma homocysteine levels were determined by using the method of Vester and Rasmussen [15]. Plasma lipoprotein(a) level was measured by using an immunoturbidimetric method (Roche Hitachi Modular P800, Japan). Blood samples were drawn after an overnight fast and centrifuged within 1 h after collection (mean ± SD, 3.25 ± 2.59 days after stroke onset) for blood biomarkers. Plasma was separated and frozen immediately at 70 C until analysis.
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2.3. Imaging analysis
3.1. Risk factors of microangiopathy and macroangiopathy
All participants underwent MRI on a 3-T system (Achieva; Philips Medical System, Best, the Netherlands), including DWI (repetition time, 2500 ms; echo time, 75 ms; matrix number, 128 128; 2 b values, 0 and 1000 s/mm2; slice thickness, 5 mm; interslice gap, 2 mm; 20 axial slices, and field of view, 240 mm), T2 fluidattenuated inversion recovery (FLAIR; using a fast-spin echo sequence with a repetition time/echo time of 11,000/125 ms, inversion time of 2800 ms, and a 320 252 matrix), gradient echo (repetition time, 645 ms; echo time, 16 ms; flip angle, 18 ; a 256 256 matrix; slice thickness, 5 mm; interslice gap, 2 mm; 20 axial slices; and field of view, 240 mm), and T1- and T2-weighted images. We also obtained three-dimensional, time-of-flight MR angiographic (MRA) of the intracranial arteries (repetition time, 25 ms; echo time, 3.5 ms; 80 slices of 0.45-mm thickness over contiguous sampling; flip angle, 20 ; a 880 450 matrix; and a field of view, 170 mm) and gadolinium-enhanced MRA of the extracranial arteries from all of the patients. We investigated MRI and MRA findings of subclinical macroangiopathy (asymptomatic tandem stenosis) and microangiopathy (leukoaraiosis). We measured degree of stenosis as in a previous study [16]. Besides relevant stenosis, we assessed any stenoocclusive lesions on nonrelevant intracranial and extracranial arteries and counted the number of steno-occlusive lesions on nonrelevant intracranial arteries as indicators of ICAS burden. In addition, we measured all of the patients' neuroimaging indicators of microangiopathy (leukoaraiosis). Leukoaraiosis was defined as a hyperintense white matter lesion on T2-FLAIR images lacking prominent hypointensity on T1-weighted images. We graded leukoaraiosis by using a modification of the method of Fazekas [17]. In this study, leukoaraiosis of Fazekas' grade 1 or 2 was defined as mild and 3 as severe.
The patient characteristics in these groups are shown in Table 1. Compared with the patients with isolated SAD, those with isolated ICAS were younger, had a higher prevalence of current smokers, and showed higher apoB levels (p < 0.05 in all of the cases). Among the biomarkers measured, only LpPLA2 differed between the groups, that is, higher in the ICAS group (1.67 ± 0.80) than in the SAD group (1.29 ± 0.57; p ¼ 0.019). However, the patients with ICAS had subclinical features of microangiopathy, and those with SAD often had tandem stenosis. Among the patients in the ICAS group, 77 had isolated ICAS (with or without mild leukoaraiosis), but 39 (32.5%) had severe leukoaraiosis. Similarly, among the patients in the SAD group, 108 had isolated SAD, but 18 (14.3%) had asymptomatic ICAS in nonrelevant vessels (Fig. 1A and B). With the progression of the subclinical features of microangiopathy (leukoaraiosis), asymptomatic macroangiopathy (tandem stenosis) worsened and vice versa (Fig. 1C and D). As a result, no significant differences in risk factors and biomarkers were observed between advanced SAD and ICAS (Table 1).
2.4. Statistical analysis We used commercially available software (STATA, version 13.1; Stata Corp, College Station, TX, USA) in our statistical analyses. We examined differences in discrete variables between the groups by using the c2, Fisher exact, or ManneWhitney test and examined differences in continuous variables by the KruskaleWallis test or the t test. Ordinal logistic regression analyses were further applied to investigate the independent clinical and laboratory factors associated with degree of leukoaraiosis and tandem stenosis. A multivariate logistic regression analysis was performed to verify the independent association between renal function and microangiopathy and macroangiopathy. Adjustment variables in the multivariable regression models were chosen among the potential outcome determinants that had significant clinical relevance or showed p values less than 0.20 in their associations with outcome in univariate analysis. Prior to adjustment variable selection variance inflation factor were calculated to check for possible multicollinearity. Bootstrap methods were applied to further validate multivariable analyses results. Creatinine clearance and eGFR was categorized into quartiles according to sample size to evaluate possible threshold effect. In all of the analyses p < 0.05 was considered statistically significant.
3. Results Of the 242 patients, 130 (53.7%) were male, and their average age was 64.7 ± 12.7 years. There were 126 patients (52.1%) in the SAD group and 116 (47.9%) in the ICAS group.
3.2. Loss of renal function as a common risk factor of microangiopathy and macroangiopathy Similar risk factors and biomarkers were involved in the progression of subclinical microangiopathy and macroangiopathy (Supplementary Tables I and II). As the degree of leukoaraiosis was increased, age and the prevalences of hypertension, diabetes, and current smoking habit were increased. Similarly, as the number of tandem stenosis was increased, these risk factors changed in the same manner. Both creatinine clearance and eGFR were decreased with the increase in both the degree of leukoaraiosis and the number of tandem stenosis in both the SAD and ICAS groups (Fig. 2). Multivariate logistic regression analysis was performed to evaluate the independent predictors of the progression of microangiopathy and macroangiopathy (Table 2). None of the adjusted variables included in the multivariable model showed increased risk for multicollinearity accessed by variance inflation factor (VIF < 3.0 for all variables). Decreased creatinine clearance was independently associated with both microangiopathy and macroangiopathy, although most patients showed mild to moderate loss of renal function. In 76.8% of the patients, creatinine clearance was higher than 60 (normal or stage 1 or 2 chronic kidney disease [CKD]). Other factors did not significantly add value, except age for microangiopathy and male sex, and increased hs-CRP level for macroangiopathy. A similar trend was observed when we used eGFR as a marker of renal impairment (Supplementary Table III and Supplementary Figure I). By bootstrap methods (1000 repeats), the significant association between creatinine clearance and leukoaraiosis and tandem stenosis remained significant. 3.3. Relationship between renal and endothelial functions Table 3 shows the correlations of creatinine clearance with markers of endothelial function, inflammation, and atherogenesis. Markers of endothelial dysfunction were significantly related to markers of renal dysfunction, creatinine clearance, and eGFR. On the contrary, most of the inflammatory or atherogenic markers were not related to any of the estimates of renal function. The path analysis was performed to simultaneously consider the direct, indirect, and total effects of renal dysfunction-related risk factors on cerebral micro- and macroangiopathy through renal dysfunction; endostatin was associated with decreased creatinine clearance, which was further associated with microangiopathy and
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Table 1 Comparison of risk factors between symptomatic microangiopathy and macroangiopathy.
Demographic Age (years) Male, n (%) Stroke risk factors Hypertension, n (%) Diabetes mellitus, n (%) Dyslipidemia, n (%) Coronary artery disease, n (%) Previous stroke or TIA, n (%) Current smoker, n (%) Metabolic syndrome, n (%) Body mass index (kg/m2) Laboratory parameters Fasting glucose (mg/dL) Total cholesterol (mg/dL) Triglyceride (mg/dL) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) Non-HDL cholesterol (mg/dL) Total cholesterol/HDL-C ratio LDL/HDL-C ratio ApoA1 (mg/dL) ApoB (mg/dL) C-reactive protein (mg/dL) Estimated GFR (mL/min) Creatinine clearance (mL/min)
SAD þ aSx ICAS
ICAS þ severe LA
(n ¼ 18)
(n ¼ 39)
0.044 0.169
74.1 ± 10.9 6 (33.3)*
71.1 ± 9.8 13 (33.3)y
0.304 1.000
43 (55.8) 21 (27.3) 33 (42.9) 3 (3.9) 11 (14.3) 26 (33.8) 35 (45.5) 24.0 ± 3.0
0.658 0.278 0.713 0.488 0.451 0.004 0.552 0.675
13 (72.2) 9 (50.0)* 4 (22.2) 3 (16.7) 4 (22.2) 0 (0) 11 (61.1) 25.3 ± 3.7
31 (79.5) 21 (53.8)y 11 (28.2) 2 (5.1) 7 (17.9) 73 (17.9) 26 (66.7) 24.7 ± 4.3
0.735 0.787 0.753 0.312 0.728 0.085 0.683 0.597
109.8 ± 33.3 186.9 ± 40.8 137.0 ± 75.5 46.6 ± 12.0 118.9 ± 37.1 140.3 ± 42.3 4.25 ± 1.42 2.74 ± 1.20 125.9 ± 25.7 103.3 ± 29.3 0.41 ± 0.81 97.0 ± 27.9 87.3 ± 31.4
0.820 0.833 0.466 0.884 0.978 0.968 0.490 0.672 0.690 0.047 0.152 0.327 0.927
129.9 ± 48.2* 187.7 ± 29.1 151.9 ± 100.7 44.8 ± 9.3 119.4 ± 20.9 142.8 ± 29.7 4.32 ± 0.98 2.76 ± 0.69 123.1 ± 17.1 96.9 ± 23.2 0.31 ± 0.47 78.8 ± 14.5* 66.0 ± 20.9*
123.6 ± 43.8y 189.7 ± 46.8 156.3 ± 128.6 49.1 ± 17.9 117.7 ± 30.7 140.6 ± 44.1 4.20 ± 1.38 2.63 ± 0.89 129.6 ± 33.8 96.6 ± 21.9 0.43 ± 0.72 86.8 ± 26.3y 70.8 ± 24.3y
0.627 0.865 0.901 0.243 0.832 0.847 0.729 0.567 0.643 0.980 0.531 0.232 0.475
SAD, isolated
ICAS, isolated
(n ¼ 108)
(n ¼ 77)
63.9 ± 12.5 69 (63.9)
60.1 ± 12.3 39 (50.6)
70 (64.8) 28 (25.9) 35 (32.4) 5 (4.6) 11 (10.2) 27 (25.0) 52 (48.1) 21.2 ± 3.2 108.7 ± 29.0 185.7 ± 38.5 148.2 ± 118.3 46.9 ± 11.8 118.8 ± 28.9 140.6 ± 33.4 4.21 ± 1.18 2.68 ± 0.88 123.9 ± 21.0 92.2 ± 22.7 0.27 ± 0.47 92.9 ± 28.4 86.8 ± 35.8
P value
P value
SAD, small arterial disease; ICAS, intracranial atherosclerotic stroke; LA, leukoaraiosis; aSx, asymptomatic; TIA, transient ischemic attack; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Apo, apolipoprotein; GFR, glomerular filtration rate. *p < 0.05, compared with isolated SAD. y p < 0.05, compared with isolated ICAS. ACR, albumin-to-creatinine ratio.
Fig. 1. Asymptomatic microangiopathy and macroangiopathy. (a) Asymptomatic microangiopathy (degree of leukoaraiosis by Fazekas' grade 0 vs. 1e2 vs. 3) and (b) macroangiopathy (number of any steno-occlusive lesions on non-relevant intracranial and extracranial arteries) among the SAD and ICAS patients. (c) Prevalence of asymptomatic ICAS among the SAD patients, according to severity of leukoaraiosis. (d) Prevalence of leukoaraiosis among the ICAS patients, according to concomitant asymptomatic ICAS.
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Fig. 2. Loss of renal function, as assessed based on creatinine clearance levels, with the increase in the burden of asymptomatic microangiopathy and macroangiopathy in the SAD and ICAS patients. (a) asymptomatic microangiopathy in SAD, (b) asymptomatic microangiopathy in ICAS, (c) asymptomatic macroangiopathy in SAD, and (d) asymptomatic macroangiopathy in ICAS.
Table 2 Multivariate testing for asymptomatic microangiopathy and macroangiopathy Leukoaraiosis Crude OR (95% CI) Age 1.10 Male 0.58 Hypertension 2.21 Diabetes mellitus 1.87 Dyslipidemia 0.73 Current smoker 0.35 Body mass index (kg/m2) 0.934 Fasting glucose (mg/dL) 1.002 Triglyceride (mg/dL) 1.001 HDL-cholesterol (mg/dL) 1.01 LDL-cholesterol (mg/dL) 0.998 C-reactive protein (mg/dL) 1.09 Creatinine clearance (mL/min) 1Q (61.0) 12.78 2Q (61.0e78.3) 7.08 3Q (78.4e99.7) 4.36 4Q (>99.7) 1
Tandem stenosis p Value
Adjusted OR (95% CI)
P value
Crude OR (95% CI)
(1.07e1.12) (0.36e0.92) (1.35e3.62) (1.12e3.12) (0.45e1.19) (0.20e0.62) (0.870e1.002) (0.995e1.008) (0.999e1.003) (0.99e1.03) (0.990e1.005) (0.94e1.26)
<0.001 0.022 0.002 0.017 0.201 <0.001 0.058 0.662 0.284 0.223 0.509 0.255
1.07 1.07 1.58 1.33
(1.04e1.11) (0.62e1.86) (0.92e2.73) (0.75e2.35)
<0.001 0.799 0.098 0.323
0.86 (0.44e1.66) 1.02 (0.93e1.11)
0.645 0.708
1.02 0.45 1.30 1.79 1.20 0.83 1.01 1.005 0.998 1.01 1.007 1.21
(6.01e27.19) (3.45e14.54) (2.18e8.72)
<0.001 <0.001 <0.001
2.93 (1.05e8.18) 2.13 (0.89e5.14) 2.18 (1.01e4.71) 1
0.040 0.090 0.048
(0.999e1.037) (0.28e0.72) (0.81e2.10) (1.09e2.93) (0.74e1.95) (0.49e1.43) (0.94e1.09) (0.999e1.012) (0.999e1.002) (0.99e1.03) (0.993e1.008) (1.03e1.42)
5.95 (1.49e3.84) 2.56 (1.32e4.98) 1.39 (0.72e2.70) 1
p Value 0.065 0.001 0.284 0.022 0.465 0.507 0.716 0.089 0.878 0.43 0.938 0.021
Adjusted OR (95% CI)
P value
0.99 0.50 1.005 1.47
(0.96e1.02) (0.30e0.83) (0.599e1.686) (0.74e2.94)
0.542 0.007 0.985 0.273
1.003 (0.994e1.012)
0.552
1.19 (1.01e1.40)
0.038
3.53 (1.23e5.89) 2.26 (1.14e4.50) 1.28 (0.62e2.49)
0.003 0.018 0.513
<0.001 <0.001 0.327 1
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Table 3 Relationship of renal function to markers of endothelial dysfunction, inflammation, and atherogenesis. Continuous variable
Endothelial dysfunction ADMA UACR Homocysteine Endostatin Inflammation hs-CRP LpPLA2 E-selectin Atherogenesis Lipoprotein(a) Adiponectin Resistin
Creatinine clearance
Estimated GFR
Correlation coefficient
P value
Correlation coefficient
P value
0.075 0.164 0.221 0.358
0.436 0.018 0.003 <0.001
0.270 0.164 0.367 0.389
0.004 0.018 <0.001 <0.001
0.064 0.208 0.052
0.322 0.031 0.647
0.085 0.139 0.084
0.185 0.155 0.461
0.120 0.262 0.162
0.065 0.006 0.093
0.013 0.079 0.172
0.839 0.417 0.077
The values are shown as Spearman correlation coefficient for continuous variables.
macroangiopathy (data not shown). 4. Discussion The main findings of this study are as follows: (a) cerebral microangiopathy and macroangiopathy often coexist, and with the progression of subclinical microangiopathy, asymptomatic macroangiopathy worsens and vice versa; (b) although several risk factors significantly differed between early-stage microangiopathy and macroangiopathy, no differences in risk factors were observed at advanced stages; (c) loss of renal function was independently associated with progression of both microangiopathy and macroangiopathy, which was a determinant of endothelial dysfunction. In the present study, one seventh of SAD patients had subclinical intracranial stenosis, while one third of ICAS patients had severe degree of leukoaraiosis. One recent carotid duplex study showed that carotid plaque is associated with lacunar infarcts and large white matter hyperintensity volume [18]. As a result, risk factor and biomarker profiles were similar between ICAS and SAD, but significantly different only between the patients with isolated (early stage) ICAS and those with SAD. The controversial results about the risk factors associated with intracranial microangiopathy and macroangiopathy may be caused by the difference in the proportion of patients with subclinical microangiopathy and macroangiopathy who were enrolled in the previous studies. CKD is a major global health problem. The worldwide prevalence of CKD is estimated to be higher than 7% in people aged 30 years or older [19], and much higher in stroke patients (20e30% in patients with acute ischemic stroke) [20,21]. The kidney and the brain share unique susceptibilities to vascular injury because the vasoregulation of the microvasculatures of the two organs is similar anatomically and functionally [21,22]. Renal impairment can be predictive of the presence and severity of cerebral small vessel disease [23]. In particular, CKD is an established risk factor of stroke and is also strongly associated with subclinical cerebrovascular abnormalities and cognitive impairment, partly because it shares several traditional and non-traditional risk factors. Meanwhile, uremia- and dialysis-related factors are sometimes associated with cerebrovascular diseases [21]. Moreover, our results and those of other studies suggest that renal function may act as the ‘Cinderella of cardiovascular risk profile’ [24,25]. Various risk factors such as oxidative stress, inflammation, and metabolic derangement were reported to be renal dysfunction-related risk factors of cardiovascular disease, and endothelial dysfunction may be the key contributing factor to cardiovascular disease [25e30]. In our study, no significant relationship was found between renal function and
inflammation. In patients with more-advanced renal impairment or coronary heart disease, inflammatory markers have been shown to be elevated [25,31]. However, in our cohort of mild to moderate loss of renal function and cerebral microangiopathies, inflammation may play a less important role. Therapeutic strategies for ICAS, including the use of antithrombotic interventions to prevent thromboembolism and restore blood flow, the use of statins to stabilize plaque, and identification and control of risk factors, have been regarded as different from those for SAD. On the contrary, the role of antithrombotics is limited [32], and control of risk factors is important in SAD. Our results suggest that therapeutic strategies targeting endothelial dysfunction may be important in both ICAS and SAD. Clinical trials have focused on antithrombotics in both ICAS (WASID [33] and SAMMPRIS [34]) and SAD (SPS3 [32]). Further clinical trials of treatments that restore endothelial function in patients with ICAS and SAD are warranted. The Chronic Renal Insufficiency Cohort (CRIC) study recently showed that lifestyle factors, such as nonsmoking, overweight (BMI of 25e30 kg/m2), and regular physical activity, were associated with reduced risk of CKD progression and also reduced risk of vascular events [35]. The strengths of this study include the consecutive recruitment of patients with comprehensive evaluation of novel and conventional risk factors. However, this study has several limitations. First, the data evaluated were from a unique population where intracranial atherosclerosis was prevalent. Accordingly, further investigations in different study populations are warranted. Second, this is a cross-sectional analysis of prospectively registered stroke patients. Prospective studies with longitudinal long-term follow-up in a larger cohort are needed to demonstrate serial changes in subclinical microangiopathy and macroangiopathy. Third, selected novel biomarkers were evaluated in the present study. Further studies are needed with more biomarkers. Lastly, subtypes of ICAS (BOD and non-BOD) [14] and SAD (red and white subtypes) [36] were not considered in the present study. Risk factor and biomarker profiles may differ between the subtype of ICAS and that of SAD. In conclusion, mild to moderate loss of renal function is strongly associated with both intracranial microangiopathy and macroangiopathy. Endothelial dysfunction may be related to this association. Strategies to reverse endothelial function have now been examined in a wide range of patients with vascular disease [37e39] and should be tested in patients with cerebral microangiopathy and macroangiopathy to prevent recurrent stroke and cognitive impairment. Further studies that examined biomarkers to find novel risk factors specific for microangiopathy and
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macroangiopathy deserve to be continued. A recent meta-analysis of genome-wide association studies showed that genetic risk factors are subtype-selective (macroangiopathy vs. microangiopathy vs. cardioembolism) [40]. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2015.12.040. Study funding This study was supported by the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI14C16240000). Conflicts of interest (financial or nonfinancial) None. Disclosure Authors report no disclosures. References [1] S.J. Kim, G.J. Moon, O.Y. Bang, Biomarkers for stroke, J. Stroke 15 (1) (2013) 27e37. [2] J. Gutierrez, M.S. Elkind, R. Virmani, et al., A pathological perspective on the natural history of cerebral atherosclerosis, Int. J. Stroke 10 (7) (2015) 1074e1080. [3] J.D. Bodle, E. Feldmann, R.H. Swartz, Z. Rumboldt, T. Brown, T.N. Turan, Highresolution magnetic resonance imaging: an emerging tool for evaluating intracranial arterial disease, Stroke 44 (1) (2013) 287e292. [4] O.Y. Bang, J.W. Kim, J.H. Lee, et al., Association of the metabolic syndrome with intracranial atherosclerotic stroke, Neurology 65 (2) (2005) 296e298. [5] O.Y. Bang, J.L. Saver, B. Ovbiagele, Y.J. Choi, S.R. Yoon, K.H. Lee, Adiponectin levels in patients with intracranial atherosclerosis, Neurology 68 (22) (2007) 1931e1937. [6] E. Lopez-Cancio, A. Galan, L. Dorado, et al., Biological signatures of asymptomatic extra- and intracranial atherosclerosis: the Barcelona-AsIA (Asymptomatic Intracranial Atherosclerosis) study, Stroke 43 (10) (2012) 2712e2719. [7] J.F. Arenillas, J. Alvarez-Sabin, C.A. Molina, et al., Progression of symptomatic intracranial large artery atherosclerosis is associated with a proinflammatory state and impaired fibrinolysis, Stroke 39 (5) (2008) 1456e1463. [8] A. Massot, D. Pelegri, A. Penalba, et al., Lipoprotein-associated phospholipase A2 testing usefulness among patients with symptomatic intracranial atherosclerotic disease, Atherosclerosis 218 (1) (2011) 181e187. [9] B.S. Kim, H.S. Jung, O.Y. Bang, C.S. Chung, K.H. Lee, G.M. Kim, Elevated serum lipoprotein(a) as a potential predictor for combined intracranial and extracranial artery stenosis in patients with ischemic stroke, Atherosclerosis 212 (2) (2010) 682e688. [10] J.F. Arenillas, C.A. Molina, P. Chacon, et al., High lipoprotein (a), diabetes, and the extent of symptomatic intracranial atherosclerosis, Neurology 63 (1) (2004) 27e32. [11] J.H. Yoo, C.S. Chung, S.S. Kang, Relation of plasma homocyst(e)ine to cerebral infarction and cerebral atherosclerosis, Stroke 29 (12) (1998) 2478e2483. [12] M. Fornage, Y.A. Chiang, E.S. O'Meara, et al., Biomarkers of inflammation and MRI-defined small vessel disease of the brain: the cardiovascular health study, Stroke 39 (7) (2008) 1952e1959. [13] O.Y. Bang, P.H. Lee, S.Y. Joo, J.S. Lee, I.S. Joo, K. Huh, Frequency and mechanisms of stroke recurrence after cryptogenic stroke, Ann. Neurol. 54 (2) (2003) 227e234. [14] S. Ryoo, J.H. Park, S.J. Kim, et al., Branch occlusive disease: clinical and magnetic resonance angiography findings, Neurology 78 (12) (2012) 888e896. [15] B. Vester, K. Rasmussen, High performance liquid chromatography method for rapid and accurate determination of homocysteine in plasma and serum, Eur.
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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Brain microangiopathy and macroangiopathy share common risk factors and biomarkers Oh Young Bang a, *, Jong-Won Chung a, Sookyung Ryoo a, Gyeong Joon Moon b, Gyeong-Moon Kim a, Chin-Sang Chung a, Kwang Ho Lee a a b
Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Samsung Bioresearch Institute, Seoul, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 October 2015 Received in revised form 12 December 2015 Accepted 25 December 2015 Available online 29 December 2015
Aims: Besides carotid or cardiac embolism, stroke can occur via microangiopathy (small arterial disease [SAD]) and macroangiopathy (intracranial atherosclerotic stroke [ICAS]) of the intracranial vasculature. There have been efforts to identify risk factors specific to microangiopathy and macroangiopathy, including vascular risk factors, and protein and genetic biomarkers. We hypothesized that despite the anatomic and pathophysiological differences between microvessels and macrovessels, microangiopathy and macroangiopathy share common risk factors during disease progression. Methods: Among 714 patients with acute infarctions within middle cerebral artery territory, 126 with SAD and 116 with ICAS were included in this study. Subclinical microangiopathy (degree of leukoaraiosis) and macroangiopathy (number of tandem stenosis) was graded in each patient. Inflammatory biomarkers (C-reactive protein, E-selectin, and LpPLA2), endothelial dysfunction (asymmetric dimethylarginine, urinary albumin-to-creatinine ratio, endostatin, and homocysteine), atherogenesis (lipoprotein(a), adiponectin, and resistin), and renal function (creatinine clearance and estimated glomerular filtration rate) were assessed. Results: Compared with the patients with isolated SAD, those with isolated ICAS were younger, were current smokers, and showed higher apoB levels (p < 0.05 in all cases). However, with the progression of subclinical microangiopathy, asymptomatic macroangiopathy worsened and vice versa. No significant differences in risk factors were observed between advanced SAD and ICAS. Decreased renal function was independently associated with progression of microangiopathy and macroangiopathy. Markers of endothelial dysfunction, but not the other markers, were significantly related to creatinine clearance level. Conclusions: Mild to moderate loss of renal function is strongly associated with both intracranial microangiopathy and macroangiopathy. Endothelial dysfunction may be associated with this relationship. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Intracranial atherosclerosis Small artery disease Risk factor Renal function Endothelial function
1. Introduction Besides carotid or cardiac embolism, stroke can occur via microangiopathy (small arterial disease [SAD]) and macroangiopathy (intracranial atherosclerotic stroke [ICAS]) of the intracranial vasculature. Both are common stroke subtypes worldwide.
* Corresponding author. Department of Neurology, Samsung Medical Center, Sungkyunkwan University, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea. E-mail address: [email protected] (O.Y. Bang). http://dx.doi.org/10.1016/j.atherosclerosis.2015.12.040 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
Numerous efforts have been made to identify the risk factors associated with intracranial microangiopathy and macroangiopathy [1]. Nevertheless, the apparent differences in risk factors between them are unclear. This could be caused by several reasons. First, the current stroke classification system may have limitations in defining intracranial microangiopathy and macroangiopathy. An autopsy study showed that intracranial atherosclerosis that was not severe was also responsible for parent territorial stroke [2]. Recently developed high-resolution magnetic resonance imaging (MRI) techniques can visualize intracranial wall plaque and provide information on the possible mechanisms of stroke [3]. Second, novel risk factors but not the conventional risk
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factors may differ between intracranial microangiopathy and macroangiopathy. Several recent studies investigated possible novel risk factors of intracranial macroangiopathy, which include metabolic syndrome and adipokines (adiponectin and resistin) [4e6], inflammatory markers [7,8], lipoprotein (a) [9,10], homocysteine [11], and endothelial dysfunction (asymmetric dimethylarginine [ADMA] and E-selectin) [6,7]. However, these biomarkers are also reported to be associated with cerebral microangiopathy [1,12]. We hypothesized that despite the anatomic and pathophysiological differences between microvessels and macrovessels, they share common risk factors because cerebral microangiopathy and macroangiopathy commonly coexist. Thus, we compared conventional and novel risk factors of stroke in patients with ICAS and SAD, in consideration of the degree of subclinical macroangiopathy (asymptomatic tandem stenosis) and microangiopathy (leukoaraiosis). 2. Patients and methods We prospectively recruited patients with acute symptomatic infarctions within the middle cerebral artery (MCA) territory admitted to a tertiary university stroke center between October 2008 and January 2012. We defined potential participants as patients who experienced focal or lateralizing symptoms within 7 days of symptom onset and showed relevant lesions on diffusionweighted imaging (DWI). Patients with potential sources of cardioaortic embolism, based on the modified Trial of Org 10172 in Acute Stroke Treatment (SSS-TOAST), extracranial atherosclerosis with significant (50%) stenosis in the relevant extracranial arteries, other stroke mechanisms (coagulopathy, vasculitis, moyamoya disease, internal carotid artery dissection, and others), or incomplete evaluations were excluded. Local institutional review boards approved this study. All the patients or patient's guardians provided informed consent with regard to participation in the study. During the study period, 1801 patients visited our center with acute ischemic stroke or TIA. Of 714 patients with acute MCA infarctions on DWI, 242 were included in this study. According to the lesion distribution on DWI and the presence of stenosis in the relevant MCA, we divided the patients into two groups: (1) the ICAS group, patients with infarcts within the MCA territory and relevant MCA stenosis, and (2) the SAD group, patients with deep infarctions and without stenotic lesions on the relevant MCA. We have reported that patients with intracranial atherosclerosis (especially branch occlusive disease [BOD]) are often misclassified as having microangiopathy or cryptogenic cause [13,14]. Thus, in this study, we regarded any degree of intracranial artery stenosis as macroangiopathy. 2.1. Workups We collected clinical information, including age, sex, and vascular risk factors. All patients underwent diagnostic testing that included routine blood tests, electrocardiography, at least 24 h of cardiac telemetry, and echocardiography. Our definitions of vascular risk factors were as follows: (1) Hypertension was deemed present when the patient had been undergoing treatment with antihypertensive agents or a blood pressure of either 160 mm Hg systolic or 90 mm Hg diastolic on at least 2 occasions after the acute phase of their ischemic stroke. (2) Diabetes mellitus was deemed present when the patient had been receiving medication for diabetes, had an elevated fasting glucose level 126 mg/dL or a 2-h plasma glucose level 200 mg/dL during their oral glucose tolerance test, or had a plasma glucose level 200 mg/dL along
with classic symptoms of hyperglycemia, hypoglycemic crisis, or hemoglobin A1c level >6.5%.8 (3) Dyslipidemia was determined to be present if the patient had been taking lipid-lowering agents or had a total cholesterol level >240 mg/dL, triglyceride level >200 mg/dL, or low-density lipoprotein cholesterol level >160 mg/ dL. ApoB and apoA1 levels were determined by using nephelometry (Behring Nephelometer, Marburg, Germany). (4) Coronary artery disease was regarded as being present when the patient had angina, myocardial infarction, or a history of coronary angioplasty or coronary bypass surgery. (5) Current smokers were those who regularly smoked at least 1 cigarette per day when admitted to the center. (6) Body mass index was measured within 3 days of admissions in most patients. In addition, creatinine clearance tests, i.e., creatinine clearance and estimated glomerular filtration rate (eGFR), were measured as markers of kidney function. Serum creatinine level was measured by using the Jaffe method with a Hitachi 7600-210 chemistry analyzer (Hitachi, Japan), and creatinine clearance was calculated according to the Cockcroft-Gault formula. eGFR was calculated by using the Modification of Diet in Renal Disease (MDRD) Study equation.
2.2. Novel biomarkers To further evaluate the possible mechanisms of subclinical microangiopathy and macroangiopathy, the following markers were determined in patients enrolled from August 2010 to January 2012 (n ¼ 113). Inflammatory biomarkers included high-sensitivity C-reactive protein [hs-CRP], lipoprotein-associated phospholipase A2 [LpPLA2] (a calcium-independent phospholipase-derived especially from macrophages), and E-selectin (also known as CD62E, a cell adhesion molecule expressed only on endothelial cells recruiting leukocytes to the site of injury). Biomarkers for endothelial dysfunction included ADMA (an endogenous inhibitor of nitric oxide synthase), urinary albumin-to-creatinine ratio (UACR), and homocysteine. The level of endostatin, an angiogenesis inhibitor that may suppress angiogenesis and collateral development, was measured. Atherogenic biomarkers included lipoprotein (a) (a lipideprotein complex with proatherogenic and prothrombotic properties) and adipokines (adiponectin and resistin, hormones secreted from adipose tissue). Amounts of protein in plasma were quantified by using the following enzyme-linked immunosorbent assay (ELISA) kits: human ADMA ELISA kit (Immundiagnostik, Cat No. K7828), human E-selectin ELISA kit (Biosensis, Cat No. BEK2089-2P), human adiponectin and resistin ELISA kits (AdipoGen, Cat Nos. AG-45A-0002PP-KI01 and AG-45A-0023PP-KI01, respectively), human Endostatin ELISA kit (Raybiotech, Cat No. ELHEndostatin-001), and human Lp-PL-A2 ELISA kit (EIAab, Cat No. E0867h). ELISAs were performed following the manufacturers' instructions. All ELISA absorbance readings were performed with reference to the standard curve. All samples were run in duplicate. ELISA plates were read by using the SpectraMax 340PC384 Microplate Reader and analyzed by using the SoftMax® Pro Data Analysis Software (Molecular Devices). Serum hs-CRP concentration was measured with the turbidimetric immunoassay by using an autochemistry analyzer (Hitachi 7600-210, Hitachi, Japan). The UACR was calculated. Plasma homocysteine levels were determined by using the method of Vester and Rasmussen [15]. Plasma lipoprotein(a) level was measured by using an immunoturbidimetric method (Roche Hitachi Modular P800, Japan). Blood samples were drawn after an overnight fast and centrifuged within 1 h after collection (mean ± SD, 3.25 ± 2.59 days after stroke onset) for blood biomarkers. Plasma was separated and frozen immediately at 70 C until analysis.
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2.3. Imaging analysis
3.1. Risk factors of microangiopathy and macroangiopathy
All participants underwent MRI on a 3-T system (Achieva; Philips Medical System, Best, the Netherlands), including DWI (repetition time, 2500 ms; echo time, 75 ms; matrix number, 128 128; 2 b values, 0 and 1000 s/mm2; slice thickness, 5 mm; interslice gap, 2 mm; 20 axial slices, and field of view, 240 mm), T2 fluidattenuated inversion recovery (FLAIR; using a fast-spin echo sequence with a repetition time/echo time of 11,000/125 ms, inversion time of 2800 ms, and a 320 252 matrix), gradient echo (repetition time, 645 ms; echo time, 16 ms; flip angle, 18 ; a 256 256 matrix; slice thickness, 5 mm; interslice gap, 2 mm; 20 axial slices; and field of view, 240 mm), and T1- and T2-weighted images. We also obtained three-dimensional, time-of-flight MR angiographic (MRA) of the intracranial arteries (repetition time, 25 ms; echo time, 3.5 ms; 80 slices of 0.45-mm thickness over contiguous sampling; flip angle, 20 ; a 880 450 matrix; and a field of view, 170 mm) and gadolinium-enhanced MRA of the extracranial arteries from all of the patients. We investigated MRI and MRA findings of subclinical macroangiopathy (asymptomatic tandem stenosis) and microangiopathy (leukoaraiosis). We measured degree of stenosis as in a previous study [16]. Besides relevant stenosis, we assessed any stenoocclusive lesions on nonrelevant intracranial and extracranial arteries and counted the number of steno-occlusive lesions on nonrelevant intracranial arteries as indicators of ICAS burden. In addition, we measured all of the patients' neuroimaging indicators of microangiopathy (leukoaraiosis). Leukoaraiosis was defined as a hyperintense white matter lesion on T2-FLAIR images lacking prominent hypointensity on T1-weighted images. We graded leukoaraiosis by using a modification of the method of Fazekas [17]. In this study, leukoaraiosis of Fazekas' grade 1 or 2 was defined as mild and 3 as severe.
The patient characteristics in these groups are shown in Table 1. Compared with the patients with isolated SAD, those with isolated ICAS were younger, had a higher prevalence of current smokers, and showed higher apoB levels (p < 0.05 in all of the cases). Among the biomarkers measured, only LpPLA2 differed between the groups, that is, higher in the ICAS group (1.67 ± 0.80) than in the SAD group (1.29 ± 0.57; p ¼ 0.019). However, the patients with ICAS had subclinical features of microangiopathy, and those with SAD often had tandem stenosis. Among the patients in the ICAS group, 77 had isolated ICAS (with or without mild leukoaraiosis), but 39 (32.5%) had severe leukoaraiosis. Similarly, among the patients in the SAD group, 108 had isolated SAD, but 18 (14.3%) had asymptomatic ICAS in nonrelevant vessels (Fig. 1A and B). With the progression of the subclinical features of microangiopathy (leukoaraiosis), asymptomatic macroangiopathy (tandem stenosis) worsened and vice versa (Fig. 1C and D). As a result, no significant differences in risk factors and biomarkers were observed between advanced SAD and ICAS (Table 1).
2.4. Statistical analysis We used commercially available software (STATA, version 13.1; Stata Corp, College Station, TX, USA) in our statistical analyses. We examined differences in discrete variables between the groups by using the c2, Fisher exact, or ManneWhitney test and examined differences in continuous variables by the KruskaleWallis test or the t test. Ordinal logistic regression analyses were further applied to investigate the independent clinical and laboratory factors associated with degree of leukoaraiosis and tandem stenosis. A multivariate logistic regression analysis was performed to verify the independent association between renal function and microangiopathy and macroangiopathy. Adjustment variables in the multivariable regression models were chosen among the potential outcome determinants that had significant clinical relevance or showed p values less than 0.20 in their associations with outcome in univariate analysis. Prior to adjustment variable selection variance inflation factor were calculated to check for possible multicollinearity. Bootstrap methods were applied to further validate multivariable analyses results. Creatinine clearance and eGFR was categorized into quartiles according to sample size to evaluate possible threshold effect. In all of the analyses p < 0.05 was considered statistically significant.
3. Results Of the 242 patients, 130 (53.7%) were male, and their average age was 64.7 ± 12.7 years. There were 126 patients (52.1%) in the SAD group and 116 (47.9%) in the ICAS group.
3.2. Loss of renal function as a common risk factor of microangiopathy and macroangiopathy Similar risk factors and biomarkers were involved in the progression of subclinical microangiopathy and macroangiopathy (Supplementary Tables I and II). As the degree of leukoaraiosis was increased, age and the prevalences of hypertension, diabetes, and current smoking habit were increased. Similarly, as the number of tandem stenosis was increased, these risk factors changed in the same manner. Both creatinine clearance and eGFR were decreased with the increase in both the degree of leukoaraiosis and the number of tandem stenosis in both the SAD and ICAS groups (Fig. 2). Multivariate logistic regression analysis was performed to evaluate the independent predictors of the progression of microangiopathy and macroangiopathy (Table 2). None of the adjusted variables included in the multivariable model showed increased risk for multicollinearity accessed by variance inflation factor (VIF < 3.0 for all variables). Decreased creatinine clearance was independently associated with both microangiopathy and macroangiopathy, although most patients showed mild to moderate loss of renal function. In 76.8% of the patients, creatinine clearance was higher than 60 (normal or stage 1 or 2 chronic kidney disease [CKD]). Other factors did not significantly add value, except age for microangiopathy and male sex, and increased hs-CRP level for macroangiopathy. A similar trend was observed when we used eGFR as a marker of renal impairment (Supplementary Table III and Supplementary Figure I). By bootstrap methods (1000 repeats), the significant association between creatinine clearance and leukoaraiosis and tandem stenosis remained significant. 3.3. Relationship between renal and endothelial functions Table 3 shows the correlations of creatinine clearance with markers of endothelial function, inflammation, and atherogenesis. Markers of endothelial dysfunction were significantly related to markers of renal dysfunction, creatinine clearance, and eGFR. On the contrary, most of the inflammatory or atherogenic markers were not related to any of the estimates of renal function. The path analysis was performed to simultaneously consider the direct, indirect, and total effects of renal dysfunction-related risk factors on cerebral micro- and macroangiopathy through renal dysfunction; endostatin was associated with decreased creatinine clearance, which was further associated with microangiopathy and
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Table 1 Comparison of risk factors between symptomatic microangiopathy and macroangiopathy.
Demographic Age (years) Male, n (%) Stroke risk factors Hypertension, n (%) Diabetes mellitus, n (%) Dyslipidemia, n (%) Coronary artery disease, n (%) Previous stroke or TIA, n (%) Current smoker, n (%) Metabolic syndrome, n (%) Body mass index (kg/m2) Laboratory parameters Fasting glucose (mg/dL) Total cholesterol (mg/dL) Triglyceride (mg/dL) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) Non-HDL cholesterol (mg/dL) Total cholesterol/HDL-C ratio LDL/HDL-C ratio ApoA1 (mg/dL) ApoB (mg/dL) C-reactive protein (mg/dL) Estimated GFR (mL/min) Creatinine clearance (mL/min)
SAD þ aSx ICAS
ICAS þ severe LA
(n ¼ 18)
(n ¼ 39)
0.044 0.169
74.1 ± 10.9 6 (33.3)*
71.1 ± 9.8 13 (33.3)y
0.304 1.000
43 (55.8) 21 (27.3) 33 (42.9) 3 (3.9) 11 (14.3) 26 (33.8) 35 (45.5) 24.0 ± 3.0
0.658 0.278 0.713 0.488 0.451 0.004 0.552 0.675
13 (72.2) 9 (50.0)* 4 (22.2) 3 (16.7) 4 (22.2) 0 (0) 11 (61.1) 25.3 ± 3.7
31 (79.5) 21 (53.8)y 11 (28.2) 2 (5.1) 7 (17.9) 73 (17.9) 26 (66.7) 24.7 ± 4.3
0.735 0.787 0.753 0.312 0.728 0.085 0.683 0.597
109.8 ± 33.3 186.9 ± 40.8 137.0 ± 75.5 46.6 ± 12.0 118.9 ± 37.1 140.3 ± 42.3 4.25 ± 1.42 2.74 ± 1.20 125.9 ± 25.7 103.3 ± 29.3 0.41 ± 0.81 97.0 ± 27.9 87.3 ± 31.4
0.820 0.833 0.466 0.884 0.978 0.968 0.490 0.672 0.690 0.047 0.152 0.327 0.927
129.9 ± 48.2* 187.7 ± 29.1 151.9 ± 100.7 44.8 ± 9.3 119.4 ± 20.9 142.8 ± 29.7 4.32 ± 0.98 2.76 ± 0.69 123.1 ± 17.1 96.9 ± 23.2 0.31 ± 0.47 78.8 ± 14.5* 66.0 ± 20.9*
123.6 ± 43.8y 189.7 ± 46.8 156.3 ± 128.6 49.1 ± 17.9 117.7 ± 30.7 140.6 ± 44.1 4.20 ± 1.38 2.63 ± 0.89 129.6 ± 33.8 96.6 ± 21.9 0.43 ± 0.72 86.8 ± 26.3y 70.8 ± 24.3y
0.627 0.865 0.901 0.243 0.832 0.847 0.729 0.567 0.643 0.980 0.531 0.232 0.475
SAD, isolated
ICAS, isolated
(n ¼ 108)
(n ¼ 77)
63.9 ± 12.5 69 (63.9)
60.1 ± 12.3 39 (50.6)
70 (64.8) 28 (25.9) 35 (32.4) 5 (4.6) 11 (10.2) 27 (25.0) 52 (48.1) 21.2 ± 3.2 108.7 ± 29.0 185.7 ± 38.5 148.2 ± 118.3 46.9 ± 11.8 118.8 ± 28.9 140.6 ± 33.4 4.21 ± 1.18 2.68 ± 0.88 123.9 ± 21.0 92.2 ± 22.7 0.27 ± 0.47 92.9 ± 28.4 86.8 ± 35.8
P value
P value
SAD, small arterial disease; ICAS, intracranial atherosclerotic stroke; LA, leukoaraiosis; aSx, asymptomatic; TIA, transient ischemic attack; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Apo, apolipoprotein; GFR, glomerular filtration rate. *p < 0.05, compared with isolated SAD. y p < 0.05, compared with isolated ICAS. ACR, albumin-to-creatinine ratio.
Fig. 1. Asymptomatic microangiopathy and macroangiopathy. (a) Asymptomatic microangiopathy (degree of leukoaraiosis by Fazekas' grade 0 vs. 1e2 vs. 3) and (b) macroangiopathy (number of any steno-occlusive lesions on non-relevant intracranial and extracranial arteries) among the SAD and ICAS patients. (c) Prevalence of asymptomatic ICAS among the SAD patients, according to severity of leukoaraiosis. (d) Prevalence of leukoaraiosis among the ICAS patients, according to concomitant asymptomatic ICAS.
O.Y. Bang et al. / Atherosclerosis 246 (2016) 71e77
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Fig. 2. Loss of renal function, as assessed based on creatinine clearance levels, with the increase in the burden of asymptomatic microangiopathy and macroangiopathy in the SAD and ICAS patients. (a) asymptomatic microangiopathy in SAD, (b) asymptomatic microangiopathy in ICAS, (c) asymptomatic macroangiopathy in SAD, and (d) asymptomatic macroangiopathy in ICAS.
Table 2 Multivariate testing for asymptomatic microangiopathy and macroangiopathy Leukoaraiosis Crude OR (95% CI) Age 1.10 Male 0.58 Hypertension 2.21 Diabetes mellitus 1.87 Dyslipidemia 0.73 Current smoker 0.35 Body mass index (kg/m2) 0.934 Fasting glucose (mg/dL) 1.002 Triglyceride (mg/dL) 1.001 HDL-cholesterol (mg/dL) 1.01 LDL-cholesterol (mg/dL) 0.998 C-reactive protein (mg/dL) 1.09 Creatinine clearance (mL/min) 1Q (61.0) 12.78 2Q (61.0e78.3) 7.08 3Q (78.4e99.7) 4.36 4Q (>99.7) 1
Tandem stenosis p Value
Adjusted OR (95% CI)
P value
Crude OR (95% CI)
(1.07e1.12) (0.36e0.92) (1.35e3.62) (1.12e3.12) (0.45e1.19) (0.20e0.62) (0.870e1.002) (0.995e1.008) (0.999e1.003) (0.99e1.03) (0.990e1.005) (0.94e1.26)
<0.001 0.022 0.002 0.017 0.201 <0.001 0.058 0.662 0.284 0.223 0.509 0.255
1.07 1.07 1.58 1.33
(1.04e1.11) (0.62e1.86) (0.92e2.73) (0.75e2.35)
<0.001 0.799 0.098 0.323
0.86 (0.44e1.66) 1.02 (0.93e1.11)
0.645 0.708
1.02 0.45 1.30 1.79 1.20 0.83 1.01 1.005 0.998 1.01 1.007 1.21
(6.01e27.19) (3.45e14.54) (2.18e8.72)
<0.001 <0.001 <0.001
2.93 (1.05e8.18) 2.13 (0.89e5.14) 2.18 (1.01e4.71) 1
0.040 0.090 0.048
(0.999e1.037) (0.28e0.72) (0.81e2.10) (1.09e2.93) (0.74e1.95) (0.49e1.43) (0.94e1.09) (0.999e1.012) (0.999e1.002) (0.99e1.03) (0.993e1.008) (1.03e1.42)
5.95 (1.49e3.84) 2.56 (1.32e4.98) 1.39 (0.72e2.70) 1
p Value 0.065 0.001 0.284 0.022 0.465 0.507 0.716 0.089 0.878 0.43 0.938 0.021
Adjusted OR (95% CI)
P value
0.99 0.50 1.005 1.47
(0.96e1.02) (0.30e0.83) (0.599e1.686) (0.74e2.94)
0.542 0.007 0.985 0.273
1.003 (0.994e1.012)
0.552
1.19 (1.01e1.40)
0.038
3.53 (1.23e5.89) 2.26 (1.14e4.50) 1.28 (0.62e2.49)
0.003 0.018 0.513
<0.001 <0.001 0.327 1
76
O.Y. Bang et al. / Atherosclerosis 246 (2016) 71e77
Table 3 Relationship of renal function to markers of endothelial dysfunction, inflammation, and atherogenesis. Continuous variable
Endothelial dysfunction ADMA UACR Homocysteine Endostatin Inflammation hs-CRP LpPLA2 E-selectin Atherogenesis Lipoprotein(a) Adiponectin Resistin
Creatinine clearance
Estimated GFR
Correlation coefficient
P value
Correlation coefficient
P value
0.075 0.164 0.221 0.358
0.436 0.018 0.003 <0.001
0.270 0.164 0.367 0.389
0.004 0.018 <0.001 <0.001
0.064 0.208 0.052
0.322 0.031 0.647
0.085 0.139 0.084
0.185 0.155 0.461
0.120 0.262 0.162
0.065 0.006 0.093
0.013 0.079 0.172
0.839 0.417 0.077
The values are shown as Spearman correlation coefficient for continuous variables.
macroangiopathy (data not shown). 4. Discussion The main findings of this study are as follows: (a) cerebral microangiopathy and macroangiopathy often coexist, and with the progression of subclinical microangiopathy, asymptomatic macroangiopathy worsens and vice versa; (b) although several risk factors significantly differed between early-stage microangiopathy and macroangiopathy, no differences in risk factors were observed at advanced stages; (c) loss of renal function was independently associated with progression of both microangiopathy and macroangiopathy, which was a determinant of endothelial dysfunction. In the present study, one seventh of SAD patients had subclinical intracranial stenosis, while one third of ICAS patients had severe degree of leukoaraiosis. One recent carotid duplex study showed that carotid plaque is associated with lacunar infarcts and large white matter hyperintensity volume [18]. As a result, risk factor and biomarker profiles were similar between ICAS and SAD, but significantly different only between the patients with isolated (early stage) ICAS and those with SAD. The controversial results about the risk factors associated with intracranial microangiopathy and macroangiopathy may be caused by the difference in the proportion of patients with subclinical microangiopathy and macroangiopathy who were enrolled in the previous studies. CKD is a major global health problem. The worldwide prevalence of CKD is estimated to be higher than 7% in people aged 30 years or older [19], and much higher in stroke patients (20e30% in patients with acute ischemic stroke) [20,21]. The kidney and the brain share unique susceptibilities to vascular injury because the vasoregulation of the microvasculatures of the two organs is similar anatomically and functionally [21,22]. Renal impairment can be predictive of the presence and severity of cerebral small vessel disease [23]. In particular, CKD is an established risk factor of stroke and is also strongly associated with subclinical cerebrovascular abnormalities and cognitive impairment, partly because it shares several traditional and non-traditional risk factors. Meanwhile, uremia- and dialysis-related factors are sometimes associated with cerebrovascular diseases [21]. Moreover, our results and those of other studies suggest that renal function may act as the ‘Cinderella of cardiovascular risk profile’ [24,25]. Various risk factors such as oxidative stress, inflammation, and metabolic derangement were reported to be renal dysfunction-related risk factors of cardiovascular disease, and endothelial dysfunction may be the key contributing factor to cardiovascular disease [25e30]. In our study, no significant relationship was found between renal function and
inflammation. In patients with more-advanced renal impairment or coronary heart disease, inflammatory markers have been shown to be elevated [25,31]. However, in our cohort of mild to moderate loss of renal function and cerebral microangiopathies, inflammation may play a less important role. Therapeutic strategies for ICAS, including the use of antithrombotic interventions to prevent thromboembolism and restore blood flow, the use of statins to stabilize plaque, and identification and control of risk factors, have been regarded as different from those for SAD. On the contrary, the role of antithrombotics is limited [32], and control of risk factors is important in SAD. Our results suggest that therapeutic strategies targeting endothelial dysfunction may be important in both ICAS and SAD. Clinical trials have focused on antithrombotics in both ICAS (WASID [33] and SAMMPRIS [34]) and SAD (SPS3 [32]). Further clinical trials of treatments that restore endothelial function in patients with ICAS and SAD are warranted. The Chronic Renal Insufficiency Cohort (CRIC) study recently showed that lifestyle factors, such as nonsmoking, overweight (BMI of 25e30 kg/m2), and regular physical activity, were associated with reduced risk of CKD progression and also reduced risk of vascular events [35]. The strengths of this study include the consecutive recruitment of patients with comprehensive evaluation of novel and conventional risk factors. However, this study has several limitations. First, the data evaluated were from a unique population where intracranial atherosclerosis was prevalent. Accordingly, further investigations in different study populations are warranted. Second, this is a cross-sectional analysis of prospectively registered stroke patients. Prospective studies with longitudinal long-term follow-up in a larger cohort are needed to demonstrate serial changes in subclinical microangiopathy and macroangiopathy. Third, selected novel biomarkers were evaluated in the present study. Further studies are needed with more biomarkers. Lastly, subtypes of ICAS (BOD and non-BOD) [14] and SAD (red and white subtypes) [36] were not considered in the present study. Risk factor and biomarker profiles may differ between the subtype of ICAS and that of SAD. In conclusion, mild to moderate loss of renal function is strongly associated with both intracranial microangiopathy and macroangiopathy. Endothelial dysfunction may be related to this association. Strategies to reverse endothelial function have now been examined in a wide range of patients with vascular disease [37e39] and should be tested in patients with cerebral microangiopathy and macroangiopathy to prevent recurrent stroke and cognitive impairment. Further studies that examined biomarkers to find novel risk factors specific for microangiopathy and
O.Y. Bang et al. / Atherosclerosis 246 (2016) 71e77
macroangiopathy deserve to be continued. A recent meta-analysis of genome-wide association studies showed that genetic risk factors are subtype-selective (macroangiopathy vs. microangiopathy vs. cardioembolism) [40]. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2015.12.040. Study funding This study was supported by the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI14C16240000). Conflicts of interest (financial or nonfinancial) None. Disclosure Authors report no disclosures. References [1] S.J. Kim, G.J. Moon, O.Y. Bang, Biomarkers for stroke, J. Stroke 15 (1) (2013) 27e37. [2] J. Gutierrez, M.S. Elkind, R. Virmani, et al., A pathological perspective on the natural history of cerebral atherosclerosis, Int. J. Stroke 10 (7) (2015) 1074e1080. [3] J.D. Bodle, E. Feldmann, R.H. Swartz, Z. Rumboldt, T. Brown, T.N. Turan, Highresolution magnetic resonance imaging: an emerging tool for evaluating intracranial arterial disease, Stroke 44 (1) (2013) 287e292. [4] O.Y. Bang, J.W. Kim, J.H. Lee, et al., Association of the metabolic syndrome with intracranial atherosclerotic stroke, Neurology 65 (2) (2005) 296e298. [5] O.Y. Bang, J.L. Saver, B. Ovbiagele, Y.J. Choi, S.R. Yoon, K.H. Lee, Adiponectin levels in patients with intracranial atherosclerosis, Neurology 68 (22) (2007) 1931e1937. [6] E. Lopez-Cancio, A. Galan, L. Dorado, et al., Biological signatures of asymptomatic extra- and intracranial atherosclerosis: the Barcelona-AsIA (Asymptomatic Intracranial Atherosclerosis) study, Stroke 43 (10) (2012) 2712e2719. [7] J.F. Arenillas, J. Alvarez-Sabin, C.A. Molina, et al., Progression of symptomatic intracranial large artery atherosclerosis is associated with a proinflammatory state and impaired fibrinolysis, Stroke 39 (5) (2008) 1456e1463. [8] A. Massot, D. Pelegri, A. Penalba, et al., Lipoprotein-associated phospholipase A2 testing usefulness among patients with symptomatic intracranial atherosclerotic disease, Atherosclerosis 218 (1) (2011) 181e187. [9] B.S. Kim, H.S. Jung, O.Y. Bang, C.S. Chung, K.H. Lee, G.M. Kim, Elevated serum lipoprotein(a) as a potential predictor for combined intracranial and extracranial artery stenosis in patients with ischemic stroke, Atherosclerosis 212 (2) (2010) 682e688. [10] J.F. Arenillas, C.A. Molina, P. Chacon, et al., High lipoprotein (a), diabetes, and the extent of symptomatic intracranial atherosclerosis, Neurology 63 (1) (2004) 27e32. [11] J.H. Yoo, C.S. Chung, S.S. Kang, Relation of plasma homocyst(e)ine to cerebral infarction and cerebral atherosclerosis, Stroke 29 (12) (1998) 2478e2483. [12] M. Fornage, Y.A. Chiang, E.S. O'Meara, et al., Biomarkers of inflammation and MRI-defined small vessel disease of the brain: the cardiovascular health study, Stroke 39 (7) (2008) 1952e1959. [13] O.Y. Bang, P.H. Lee, S.Y. Joo, J.S. Lee, I.S. Joo, K. Huh, Frequency and mechanisms of stroke recurrence after cryptogenic stroke, Ann. Neurol. 54 (2) (2003) 227e234. [14] S. Ryoo, J.H. Park, S.J. Kim, et al., Branch occlusive disease: clinical and magnetic resonance angiography findings, Neurology 78 (12) (2012) 888e896. [15] B. Vester, K. Rasmussen, High performance liquid chromatography method for rapid and accurate determination of homocysteine in plasma and serum, Eur.
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