Association of cyclophilin A level and pulse pressure in predicting recurrence of cerebral infarction

Abstract

Cyclophilin A (CypA), secreted from vascular smooth muscle cells and inflammatory cells in response to oxidative stress, promotes vascular atherosclerosis and development of carotid stenosis. Increased concentration of plasma CypA in acute cerebral infarction was demonstrated clinically. The primary aim of this study was to investigate the prognostic impact between CypA level and outcome in patients with acute ischemic stroke. Admission serum CypA concentrations were detected in 66 acute cerebral infarction patients and in 52 healthy individuals. Inflammatory biomarkers, including high‐sensitivity C‐reactive protein, adhesion molecules, interleukins, and matrix‐metalloproteases, were also assessed. We also examined the relationship between plasma biomarkers, blood pressure (BP), pulse pressure, the carotid artery velocity, the prognostic assessment with modified Rankin scale, and stroke recurrence. Plasma CypA concentration was higher on the first day of hospitalization in the high BP stroke group than in normal BP stroke group, which was statistically significant, which was observed even in the third month and sixth month follow‐up outpatient periods. For stroke recurrence prediction, there was an important association between the higher (>60) pulse pressure on the seventh day of hospitalization and CypA level on the third month and sixth month follow‐up outpatient periods. Our study revealed higher circulating serum levels of CypA in the hypertensive stroke group than in the non‐hypertensive stroke group. We expect that elevated plasma CypA level and raised pulse pressure during hospitalization to become valuable biomarkers in predicting stroke recurrence in the sixth month assessment of acute cerebral infarction.

INTRODUCTION

Cerebral infarction appears to recur highly in the first year after onset of ischemic stroke, despite appropriate secondary prevention measures and therapy. Most of the neurological recovery of stroke survivors occurs in the first 6 months, yet improvement may be observed up until 18 months after stroke. Making a prognostic outcome prediction immediately after a cerebral stroke is difficult for neurological physicians. Such a prediction depends on many factors, including reperfusion therapies at different stages in stroke management, initial clinical assessment scores, underlying cardiovascular diseases, and stroke subtype.

Up to 50% of strokes may be attributable to hypertension, and there is controversy about which component of blood pressure (BP) contributes to the outcome after stroke. Previous meta-analysis studies have demonstrated an association between increase in pulse pressure (PP) and mortality from myocardial infarction (MI) and risk prediction of coronary heart disease (CHD). Increasingly more evidence indicates that PP is also linked to incident stroke and its occurrence risk, although there are some controversial reports on stroke outcome. In addition to BP, previous studies have included many investigations of independent predictors of cerebrovascular diseases recurrence in recent decades. Each biomarker has its own advantages on some aspect of vascular biochemistry. However, there have been some limitations or controversial results in the application of these biomarkers in terms of clinical utility.

Cyclophilin A (CypA), a ubiquitously expressed intracellular protein, is secreted by monocytes, macrophages, and vascular smooth muscle cells (VSMCs) in response to inflammatory and oxidative stimuli. CypA performs its extracellular functions in promoting the development of atherosclerosis, facilitating the proliferation and migration of VSMCs, activating proinflammatory pathways in endothelial cells, being a chemoattractant, and augmenting the production of reactive oxygen species (ROS). CypA can be overexpressed in various illnesses, such as sepsis, autoimmune diseases, rheumatoid arthritis, cancer, cerebrovascular disease, and cardiovascular diseases. Of note, circulating plasma CypA level shows prognostic effect in some diseases, such as chronic obstructive pulmonary disease, CHD, acute MI, congestive heart failure, and hemorrhagic stroke. Some interesting findings concerning plasma CypA in hypertension and carotid stenosis of cerebral stroke have also been published in previous studies. Such data suggest that CypA might be a potential biomarker for reflecting the severity and assessing prognosis after acute inflammation. Therefore, adding objective paraclinical parameters to precise outcome prediction techniques remains a challenge.

The primary aim of the present study was to investigate the relationship between prognostic outcome after stroke and inflammatory biomarkers, including adhesion molecules, interleukins, matrix-metalloproteases (MMP) and plasma CypA.

MATERIALS AND METHODS

Study design

This study was conducted according to the tenets of the recent revision of the Declaration of Helsinki. Informed written consent for the collection of samples and subsequent analysis was obtained from all patients or their relatives. The study was approved by the Independent Ethics Committee of the Changhua Christian Hospital (number: 071216).

Patients

The study group comprised patients who were treated for acute stroke at the Department of Neurology, Changhua Christian Hospital, during the period January 2010 to January 2012. Demographic data and risk factor profiles of patients and controls are shown in Table .

Clinical characteristics and biochemical variables

	Normal BP Group (first day)	High BP Group (first day)	Normal BP Group (seventh day)	High BP Group (seventh day)
	(n = 22)	(n = 44)	(n = 22)	(n = 44)	P value (first day)	P value (seventh day)
Demographic data
Age (years)	60 ± 11	62 ± 7			.399
Body mass index (kg/m2)	25.79 ± 4.37	25.25 ± 4.10			.664
Smoking index (packyear)	37.20 ± 37.78	35.94 ± 18.34			.917
Intima-media thickness (mm)	0.90 ± 0.14	0.88 ± 0.15			.533
Systolic blood pressure (mmHg)	131 ± 9	162 ± 17	147 ± 9	155 ± 20	.001	.296
Diastolic blood pressure (mmHg)t	79 ± 7	94 ± 15	92 ± 9	92 ± 12	.001	1.000
Pulse pressure	51.8 ± 10.42	68.23 ± 20.19	25.08 ± 28.63	43.49 ± 31.13	.001*,#	.045*,#
>Second stroke	1.12 ± 0.33	1.15 ± 0.36	1.41 ± 0.51	1.35 ± 0.48	.778	.665
Biomarkers
Plasma Cyclophilin A (ng/mL)	79.24 ± 49.98	120.6 ± 62.91	79.24 ± 49.98	120.5 ± 63.67	.019	.020
Plasma CyP A after 3 month (ng/mL)	80.14 ± 14.52	138.4 ± 48.08	80.14 ± 14.52	131.4 ± 37.91	.049	.038
Plasma CyP A after 6 month (ng/mL)	67.16 ± 17.03	145.8 ± 62.16	67.16 ± 17.04	140.4 ± 60.32	.001	.010 ,#
ICA PSV	56.74 ± 17.84	55.43 ± 18.20	56.74 ± 17.85	54.78 ± 17.99	.828	.743
CCA PSV	72.80 ± 23.75	66.84 ± 15.40	72.80 ± 23.75	66.81 ± 15.61	.428	.318
Modified Rankin scale (3 months)	2 ± 1	2 ± 1	2 ± 1	2 ± 1	.603	.603
Modified Rankin scale (6 months)	2 ± 1	1 ± 1	2 ± 1	1 ± 1	.039	.013

Inclusion criteria

We defined a clinically definite stroke as new clinical symptoms or signs of a focal disturbance of cerebral function of a vascular origin lasting more than 24 hours. The ischemic stroke is diagnosed by brain computed tomography (CT scan) or magnetic resonance image scan (MRI scan). All patients with acute ischemic stroke for whom peripheral blood samples had been taken within 6 hours to 1 week after stroke occurrence were eligible for inclusion.

Exclusion criteria

Patients with infection within the recent week, other neurological diseases (eg, head trauma, intracranial or subarachnoid hemorrhage, epidural or subdural hemorrhage, aneurysm, transient ischemic attack, or neurological deficits due to neoplasm) were excluded. Subjects with culture-confirmed infection after stroke were also excluded.

Methods

Clinicians recorded data at the time of assessment using a standardized structured pro forma and, in patients who consented, we drew blood for measurement of inflammatory markers. BP, PP, and other vital signs were collected on the first day and seventh day of hospitalization, and third and sixth month follow-up outpatient periods. Baseline serum creatinine, creatine phosphokinase, hemoglobin, and hemoglobinA1c levels were routinely measured after stroke. Baseline plasma lipid and glucose levels were measured after an 8-hour fast. Plasma was isolated from heparin anticoagulated blood samples and stored at −80°C until analyzed.

Stroke neurologists reviewed the clinical features and assessed the neurological outcome of each patient and clinical progress in the coming day and seventh day of hospitalization and the following third and sixth month follow-up outpatient periods.

Measurement of plasma CypA, CD147, MMP-2, and MMP-9 concentrations

Plasma CD147 levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit purchased from R&D System (Minneapolis, Minnesota). Plasma CypA levels were measured using an ELISA kit from Wuhan USCN Science (Wuhan, China). The plasma levels of total MMP-2 and total MMP-9 were measured using an ELISA kit purchased from R&D System. All the assays were performed according to manufacturer instructions.

Ultrasound assessment of carotid artery

We measured the thickness of carotid intima-media thickness (CIMT) and plaques present in the carotid wall by underwent high-resolution duplex Doppler ultrasound. CIMT is measured between the thickness of tunica intima and tunica media, the innermost two layers of the wall of the carotid vessel wall. Carotid plaques represent a focal thickening of the intima-media >1 mm, protruding into the lumen of the carotid artery with acoustic shadowing, which is at least twice as thick as the IMT on either side. The degree of stenosis was evaluated according to the European Carotid Surgery Trial criteria. Heterogeneous hypoechoic plaques with zones of varying echogenicity and surface irregularities or ulcerations and those with a severe degree of stenosis (≥70%) are considered “complex” plaques that place patients at major risk for stroke.

Assessment of stroke severity and outcome

On admission, stroke severity influencing the neurological status was assessed using the modified Rankin scale (mRS) method. Subsequent functional outcome was assessed using the mRS at third and sixth month follow-up outpatient periods, derived either directly from evaluation of face-to face interview or discussion with caretakers or relatives. Disease recurrence, mortality, as well as the therapeutic follow-up, were recorded at the second and third clinical visits. All patients were assessed at 1 year after disease occurrence for clinical evaluation by mRS (favorable (mRS = 1-2), poor (mRS = 3-6)). The endpoint was death within 6 months and the time of recurrence. The cause of death was recorded. We had collected the cumulative data about the frequency of recurrent cerebral infarction since 2010.

Statistical analysis

Comparison of categorical variables was analyzed using the X² test. The paired t test and one-way analysis of variance (ANOVA) were used to exam the differences between the mean values in the non-hypertension and hypertension groups with controls for body mass index (BMI), smoking index, and lipid profiles. Multiple linear regressions were applied to assess the importance of various factors contributing to hypertension. A partial correlation was applied in the comparison between BP and the plasma level of CypA with controls for BMI, smoking index, and lipid profile. The Student's t-test was used to assess the differences and compare the correlation between stroke and control subjects in plasma CypA concentration. Multivariable model included age, sex, baseline mRS as a continuous variable, hypertension, dyslipidemia, diabetes mellitus, atrial fibrillation, smoking, and body mass index. Further multiple regression analyses were performed using the same model, but by replacing CypA concentration with high-sensitivity C-reactive protein (hs-CRP) concentration as one of the covariates. This was done to investigate the association between these variables. Receiver Operator Characteristic (ROC) curves were constructed to assess the predictive accuracy of CypA for carotid medial thickness and plaque by calculating the Area Under the Curve (AUC). Correlations between CypA concentrations and CIMT and plaque were analyzed using Spearman's rho coefficient. Significance was assigned at 2-sided P value < .05. All statistical analyses were performed using the statistical software package SPSS (Version 17.0; Chicago, IL, USA).

RESULTS

Baseline patient characteristics

The demographic and biochemical characteristics of the enrolled patient and the control groups are shown in Table . Stroke subtype of those enrolled patients is lacunar or small infarction due to small-vessel occlusion. We divided the stroke subjects into 2 groups based on their blood pressure on the first day of hospitalization, 22 (33%) with normal-BP (BP < 140/90 mmHg) and 44 (67%) with high-BP (BP > 140/90 mmHg). High-BP patients were more than 100 times (OR, 109; 95% CI) more likely to develop a stroke than normal-BP subjects. There was no relationship of statistical significance between BP on the first and seventh day of hospitalization and age, smoking, BMI, stroke recurrence, mRS assessment, CIMT, and flow velocity in systolic and diastolic phase of internal and common carotid arteries in both groups (Table ).

Correlation between blood pressure and plasma CypA concentration

On the first-day of hospitalization, plasma CypA concentration was higher in the high-BP group than in the normal-BP group with statistically significant (P = 0.019), even in the third month (P = 0.049) and sixth month (P = 0.001) follow-up outpatient periods.

Correlation between pulse pressure and plasma CypA concentration

On the first and seventh day of hospitalization, the measured pulse pressure was higher in the high-BP group (68.23 ± 20.19 and 43.49 ± 31.13) than in the normal-BP group (51.8 ± 10.42 and 25.08 ± 28.63) (Table ). We set 60 as a cutoff number for PP. On the seventh day of hospitalization, plasma CypA level was higher in the patients with higher PP (>60) with statistical significance, even in the third month (P = .001) and sixth month (P = .004) follow-up outpatient periods. In Table , there was no significant relationship between plasma CypA concentration and CIMT (P = .654/.339) and total plaque (P = .202/.205) of the carotid artery, regardless of the value of PP on the first and seventh day of hospitalization.

Association between pulse pressure (PP) on first and seventh day hospitalization and biochemical variables

	Low-PP (first day)	High-PP (first day)	Low-PP (seventh day)	High-PP (seventh day)	First day/seventh day
(n = 27)	(n = 23)	(n = 27)	(n = 23)	P-value
Plasma CyP A (first)	122.2 ± 60.08	127.0 ± 55.74	122.2 ± 59.57	136.3 ± 57.19	.777/.488
Plasma CyP A (3 months)	111.1 ± 45.89	143.2 ± 50.23	105.3 ± 31.56	178.9 ± 4.27	.189/.001
Plasma CyP A (6 months)	105.8 ± 52.38	151.2 ± 65.23	98.44 ± 35.91	195.7 ± 73.61	.132/.004
Plasma MMP-2 (1 day)	272.0 ± 49.10	276.4 ± 90.01	272.0 ± 51.70	285.3 ± 119.0	.827/.592
Plasma MMP-2 (6 months)	303.7 ± 43.81	314.2 ± 85.42	302.8 ± 50.03	317.8 ± 96.37	.651/.564
Plasma MMP-9 (1 day)	71.76 ± 87.10	50.72 ± 37.68	58.98 ± 64.24	68.04 ± 90.26	.288/.711
Plasma MMP-9 (6 months)	45.78 ± 87.10	63.83 ± 54.04	52.99 ± 40.96	61.94 ± 43.14	.224/.577
CIMT (mm)	0.89 ± 0.13	0.92 ± 0.18	0.90 ± 0.14	0.95 ± 0.19	.654/.339
Total plaque index	4 ± 4	6 ± 5	5 ± 5	7 ± 4	.202/.205

Correlation between pulse pressure and recurrent cerebral infarction

There was a positive correlation between recurrent infarction and significantly higher systolic BP and PP on the first (P = .019*, P = .044*) and seventh day (P = .042*, P = .004*) of hospitalization. In Table , it revealed the probability of recurrent cerebral infarction even in the normal-BP group, however. Regression analysis showed that the higher PP on the seventh day of hospitalization plays an important role in predicting recurrent infarction (>2 strokes). There was no significant relationship between recurrent infarction and carotid stenosis, systolic and diastolic flow velocity of the internal and common carotid artery.

Association between stroke recurrence and biochemical variables

	0-First stroke	≥Second stroke
	(n = 42)	(n = 8)	P value
Systolic blood pressure (mmHg) (first)	150 ± 20	171 ± 25	.019
Diastolic blood pressure (mmHg) (first)	90 ± 15	97 ± 18	.310
Systolic blood pressure (mmHg) (7 days)	151 ± 15	171 ± 30	.044
Diastolic blood pressure (mmHg) (7 days)	93 ± 12	91 ± 14	.845
Pulse pressure (first)	60.10 ± 14.36	74.57 ± 28.37	.042
Pulse pressure (7 days)	58.68 ± 9.75	79.25 ± 23.68	.004
ICA PSV	53.80 ± 19.10	57.18 ± 16.46	.523
CCA PSV	69.08 ± 17.67	67.69 ± 17.98	.789
ICA/CCA PSV	0.29 ± 0.01	0.33 ± 0.108	.124
Stenosis (%)	17.75 ± 19.88	21.09 ± 20.29	.535

DISCUSSION

Post-stroke inflammation is known to deteriorate cerebral damage at the acute phase of a stroke. More importantly, reactive oxygen species and some inflammatory cytokines lead to cell death and maintain inflammation, may result in brain swelling and penumbra expansion in the infarction area and affect the prognosis of stroke. The result of our study demonstrated higher plasma level of CypA in stroke patients from the first day of the emergency room visit and even lasted for 6 months, especially in patients with higher PP. Besides, those patients with higher PP over 60 mmHg during the 7 days of hospitalization after acute infarction have a higher chance of having a recurrent stroke in the following 6 months. We made an inference and proposed that higher CypA plasma level and elevated PP will exert an important mutual influence on the prognosis and the recurrent stroke of the patients with acute cerebral infarction through this study.

More than 50% of patients with cerebral stroke had a prior history of hypertension. Elevated BP presents in more than 75% of acute stroke stages and is associated with poor consequence. High systolic blood pressure (SBP) is associated with an increased risk of early stroke recurrence. Uncontrolled BP could affect the outcome after cerebral stroke with a J-shaped/U-shaped relationship. Stroke subtype of those enrolled patients is lacunar or small infarction due to small-vessel occlusion. A few patients with massive infarction got infection somewhere later, therefore they were not enrolled in the study. We could not measure the accurate size of infarction in the brain image without the appropriate computer software. In this study, most of the stroke patients had elevated BP, including systolic and diastolic, with statistical significance. However, there was no relationship between the BP on the first and seventh day of hospitalization and the stroke recurrence, mRS assessment, and CIMT in both groups. Hypertension may be the most initial factor in stroke occurrence, but many other confounding factors could induce subsequent atherosclerosis formation.

Hypertension contributes to vascular endothelial dysfunction and inflammation, followed by smooth muscle cell proliferation and vascular wall stiffening. The higher level of biomarkers and pro-inflammatory cytokines, such as hs-CRP, intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1 (VCAM-1), could be predictors of incident hypertension and even related to PP.

As previous studies have indicated, plasma CypA might be a hypertension-related biomarker in vivo among the various vascular cytokines and exhibit a critical role in the early pathogenesis of essential hypertension. In this present study, plasma CypA concentration was higher in the high-BP stroke patients with statistical significance on the first day of hospitalization, and even in the third month and sixth month follow-up outpatient periods.

The pulse pressure, the difference between the systolic and diastolic BP (SBP and DBP), represents the continuously pulsatile component of BP and is an indirect marker of arterial stiffness. Persistently elevated PP greater than 40 mmHg is responsible for exerting mechanical stress on the arterial wall, inducing endothelial dysfunction, enhancing adhesion of inflammatory cells to endothelium, contributing to the development of increased CIMT and stiffness, and subsequent systemic vascular injury and carotid atherosclerosis. Each 10 mmHg increment in PP was associated with a 0.008 mm increase in CIMT. In the present study, the measured PP of the first and seventh day of hospitalization was above the normal range in both groups, though it was higher in the high-BP patients. Even though they had good control of BP in high-BP group, their PP was still higher than the normal range. As demonstrated in the Framingham Heart Study of the general population, the combined use of static (ie, SBP or mean arterial pressure [MAP]) and dynamic (ie, PP) methods have shown a linear relationship with cardiovascular risk. High PP in the acute stroke period is known to have a J-shaped association with increased risk of stroke recurrence in acute infarction stroke. Some studies have demonstrated that high PP may increase the risk of stroke recurrence with poor outcome, although some controversy exists with small sample sizes, short follow-up duration, or no adjustments parameters. Accumulating evidence has indicated that PP was superior to other BP parameters, including SBP, DBP, and MAP, in predicting the prognosis of serious cardiovascular and cerebrovascular morbidity and mortality. A recent study from the Taiwan stroke registry showed a U-shape relationship between PP at admission and 3-month functional outcome after ischemic stroke. Lots of studies of antihypertensive drug therapy on changes in PP vs changes in CIMT indicate slight or inconsistent reductions of wall thickness which are substantially less than that obtained with lipid-lowering agents. Our study revealed that focus on close monitoring with good control of PP rather than BP may be an important step in regulating post-stroke hemodynamics and preventing the recurrence of ischemic stroke.

Increased PP was associated with higher circulating levels of N-terminal pro-brain natriuretic peptide (NT-pro-BNP), mid-regional pro-atrial natriuretic peptide (MR-proANP), and MMP-2. Apart from PP, other biomarkers have their own advantages in the mechanisms of vascular biochemistry, such as monocyte chemoattractant protein 1 (MCP-1) and ICAM-1 in vascular plaques progression, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in plaque destabilization, circulating soluble CD40 ligand and MMP in vascular endothelial inflammation, and osteopontin (OPN) as a marker in vascular calcification. ROS was the key to stimulate CypA secretion from endothelial cells and VSMCs, suggesting the existence of extracellular CypA and ROS in the pathogenesis of vascular remodeling. Increased ROS formation in hypertension leads to arterial atherosclerosis with increased CIMT and stenosis. Extracellular CypA overexpression in VSMCs showed increased CIMT in animal studies. Recent studies suggest that CypA may have an important role in the development and progression of atherosclerotic plaque and promotion of vulnerable lesions and induce subsequent plaque rupture. There is strong evidence that CypA is expressed in the atherosclerotic plaque of patients with MI. Elevated plasma CypA concentrations were positively correlated with stenosis severity and CIMT. Higher CIMT value is not only a surrogate marker of stroke beyond the classical risk factors but a stronger independent predictor for cerebrovascular event recurrence. Elevated CypA level has also been proven to be significantly related to cerebral ischemic stroke in hypertensive patients. We set 60 as a cutoff number of PP in this study. There was statistical significance between the CypA level on the third and sixth month follow-up outpatient periods and higher than 60 of PP on the seventh day of hospitalization but not in PP lower than 60. No matter if the pulse pressure was higher or lower than 60, no significant association was revealed between PP in the first day of hospitalization and CypA concentration, CIMT and total plaques of the carotid artery. A PP level higher than 60 in the seventh day of hospitalization had a positive relationship with CypA levels in the third and sixth month follow-up outpatient periods, but without connection with CIMT and total plaques of the carotid artery, and mRS assessment, although a few normotensive patients also had a chance of stroke recurrence.

As predictors of cerebral stroke outcome, inflammatory markers for early neurological worsening and poor outcome after stroke. S100β for delayed infarct expansion, brain natriuretic peptide (BNP) for functional outcome after stroke, and brain-derived neurotrophic factor (BDNF) polymorphism for outcome after traumatic brain injury, have been demonstrated in many previous studies' analyses. Higher PP also has been connected to a positive relationship in the risk of stroke occurrence in meta-analysis studies and a significant influence in deteriorating the long-term prognosis of elderly stroke patients. We found that stroke patients with higher CypA concentrations and elevated PP over 60 mmHg in the seventh day of hospitalization after acute infarction have a higher chance of recurrent cerebral stroke (P = .004), and are positively associated. This means that failure to adequately control PP after acute cerebral infarction would lead to deterioration of the inflammatory status within the arterial environment and make the post-stroke patients having a higher chance of recurrent stroke. In conclusion, we made an inference that higher CypA plasma level and elevated PP will exert an important mutual influence on the prognosis and the recurrent stroke of the patients with acute cerebral infarction. They may significantly improve the predictive value and reflect the 6-month recurrence rate of cerebral infarction from the current study.

There were some limitations in the present study. First, the number of participants in the study with acute ischemic stroke is small, which restricted our exploration and analysis of the correlation between risk factors and CypA concentration. Nonetheless, the study revealed significantly higher plasma levels of CypA in stroke patients than in non-stroke subjects. Second, only mild stroke patients, as assessed by the mRS, were enrolled in this study. This may have created some bias within this investigation. Third, due to fewer numbers of inflammatory biomarkers studied, it was difficult to test inflammatory biomarkers systematically. A broader study with sufficient sample size is needed for investigation in the future.

CONCLUSION

In addition to the positive correlation of plasma CypA with hypertension and CIMT demonstrated previously, we made an inference and proposed that higher CypA plasma level and elevated PP will exert an important mutual influence on the prognosis and the recurrent stroke of the patients with acute cerebral infarction through this study. They might have a significant part in predicting the 6-month outcome after acute ischemic stroke. More research is needed to focus on CypA temporal profiles and the consequences thereof for the predictive model with a longer follow-up period. The exploration of CypA in the pathophysiology of ischemic stroke may be applied to a broad spectrum of vascular diseases to determine a cause-effect relationship.

ACKNOWLEDGMENTS

The authors thank the patients who participated the study.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

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