III. Results
3.6. Endothelial dysfunction and hyperhomocysteinemia in PD
Forty PD patients treated with levodopa, 33 PD patients treated with levodopa/entacapone, 22 untreated PD patients, and 30 controls were enrolled, and FMD was compared between groups.
The demographic characteristics of the groups are summarized in Table 11. No significant differences in age, sex, medication use, smoking, or BMI were found among groups. Among patients with PD, disease duration was significantly lower in the untreated group, and the daily levodopa dose was significantly higher in the levodopa treatment group.
FMD was significantly lower in PD patients with levodopa (6.0 ± 1.8%) compared to the levodopa/entacapone treatment group (7.2 ± 1.4%, p=0.03), the untreated group (7.8 ± 1.2%, p<0.05) and controls (8.5 ± 2.9%, p<0.05).
FMD in the levodopa/entacapone treatment group was intermediate between the levodopa group and the untreated group or controls (Figure 8). Mean time to maximal diameter in FMD was not significantly different between groups (48.8 ± 8.9 sec in the levodopa treatment group, 49.7 ± 9.0 sec in the levodopa/entacapone treatment group, 48.2 ± 9.2 sec in the untreated group and 49.2 ± 9.4 sec in controls). The level of homocysteine was significantly higher in the levodopa treatment group compared to the other groups. Among the FMD tertile subgroups, homocysteine level was significantly lower in the highest tertile compared to the lowest (Figure 8). Correlation analysis showed that the level of homocysteine was negatively correlated with FMD (r=-0.271, p=0.002;
Figure 9). This association remained significant after adjusting for age (r=-0.398, p=0.001) because age was partially associated with homocysteine levels (r=0.176, p=0.077). In addition, the level of homocysteine showed a positive correlation with levodopa dose (r=0.369, p<0.001) and a negative correlation with vitamin B12 level (r=-0.198, p=0.045).
In multivariate logistic regression model adjusting for age, sex, duration of levodopa treatment, UPDRS III, hypertension, diabetes mellitus, dyslipidemia, and BMI, the uppermost homocysteine quartiles (odds ratio, 5.70; 95% confidence interval, 1.60-20.27; p=0.007) and age (odds ratio, 1.09;
95% confidence interval, 1.02-1.17; p=0.009) were independent predictors of the lowest tertile of FMD (Table 12).
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Table 11. Demographic characteristics of patients with Parkinson’s disease and controls
Characteristic PD-L (n=40) PD-L/E (n=33) PD-N (n=22) Controls (n=30) p
Entacapone therapy duration (mo) 18.75 (6.9)
Levodopa (mg/day) 400.1 (123.8) 343.2 (127.3) 0.04
Entacapone (mg/day) 0 (0) 600.0 (0.0) NS
Levodopa equivalent dose 483.5 (139.2) 454.7 (196.0) NS
Body mass index (kg/m2) 23.8 (2.2) 23.9 (2.2) 23.5 (2.1) 24.3 (1.7) NS Values are expressed as mean (SD) or numbers (percentage). PD-L, Parkinson’s disease with levodopa treatment; PD-L/E,
Parkinson’s disease with levodopa/entacapone treatment; PD-N, untreated Parkinson’s disease. aCompared to PD-N at p<0.05.
bCompared to controls at p<0.05. cCompared to PD-L/E, PD-N or controls at p<0.05. NS, not significant.
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Figure 4. Flow-mediated dilation in patients with PD and controls. Box-and-whisker plot revealed that flow-mediated dilation was significantly decreased in PD patients with levodopa treatment compared with those with levodopa/entacapone treatment (p=0.03), no treatment (p<0.05), and controls (p<0.05). The black horizontal line in each box represents the median, the boxes represent the interquartile range, and the whiskers represent the minimum and maximum. *p=0.03, **p<0.05, ***p<0.05.
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Figure 5. Relationship between flow-mediated dilation and homocysteine
Correlation analysis showed that the level of homocysteine was significantly negatively correlated with flow-mediated dilation.
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Table 12. Multivariate-adjusted odds ratios of variables associated with the lowest tertile of FMD.
Univariate OR Multivariate OR (95% CI) p
Age (y) 1.081 1.09 (1.02-1.17) 0.011
UPDRS III 1.021 1.01 (0.95-1.06) NS
Hypertension 1.34 1.29 (0.46-3.62) NS
Diabetes 1.45 1.79 (0.39-8.07) NS
Dyslipidemia 1.69 2.25 (0.51-10.06) NS
BMI (kg/m2) 0.86 0.87 (0.68-1.11) NS
Homocysteine quartiles
1st (< 10.9) Reference Reference
2nd (10.9 – 12.7) 1.14 0.93 (0.18-4.5) 0.928
3rd (12.7 – 14.5) 2.65 2.69 (0.62-11.56) 0.186
4th (> 14.5) 5.86 6.33 (1.61-26.65) 0.012
OR = odds ratio; CI = confidence intervals; UPDRS=unified Parkinson’s disease rating scale; BMI=body mass index; NS = not significant. Odds ratios were adjusted for age, sex, duration of levodopa medication, UPDRS III, HTN, DM, dyslipidemia, and BMI.
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3.7. Effect of levodopa on endothelial function in PD: serial follow-up study
This study included 36 patients with de novo PD. Homocysteine levels and brachial artery FMD were investigated before and after 10-15 months of levodopa (LD; n=18) or dopamine agonist (DA; n=18) treatment. Homocysteine increased (11.52 ± 0.45 to 14.33 ± 0.68, p < 0.05) during levodopa treatment, but not in response to DA (10.59 ± 0.38 to 11.38 ± 0.67, p=0.184). FMD decreased after levodopa treatment (8.60 ± 0.46 to 7.210 ± 0.402, p=0.002) but there were no significant changes after DA treatment (8.33 ± 0.38 to 8.226 ± 0.330, p=0.266; Table 12) Correlation analysis revealed that the changes in homocysteine levels were negatively correlated with changes in FMD (r=-0.309, p=0.067).
This indicates that 1 year of levodopa treatment may adversely affect endothelial function in de novo PD.
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Table 13. Change of FMD and level of homocysteine after levodopa and dopamine agonist treatment Levodopa (n= 18) Dopamine agonist (n=18)
Characteristic baseline follow up baseline follow up
Age (years) 69.7 (4.3) 65.3 (7.0) a
Sex (male) 7 (38.8%) 37 (49%)
Disease duration (months) 14.3 (5.6) 16.2 (6.7)
Motor UPDRS 17.5 (3.8) 15.1 (2.5) 16.7 (3.0) 15.7 (2.1)
Levodopa daily dose 0 231.9 (61.7) 0 202.8 (51.7)
FMD (%) 8.6 (0.4) 7.2 (0.4) 8.3 (0.4) a 8.2 (0.3)
Homocysteine 11.5 (0.45) 14.3 (0.6) 10.5 (0.3)a 11.4 (0.7)
Values are expressed as mean (SD) or numbers (with percentages). PD, Parkinson’s disease, UPDRS, Unified Parkinson’s Disease Rating Scale, a Significant difference between baseline and follow-up at p < 0.05.
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3.8. Effect of endothelial dysfunction on cognition in PD
A total of 91 dementia-free PD patients were enrolled. FMD was positively correlated with verbal memory (delayed and recognition) and visual recognition memory, as well as some frontal domain scores (Figure 6). However, given the large number of comparisons (17 cognition tests * 1 FMD parameter = 17), the results did not survive multiple comparison corrections (a = 0.002 [(0.05/
(17)]).
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Figure 6. Correlations between FMD and cognition in PD
R=0.28
P=0.007 R=0.25
P=0.01
R=0.23 P=0.03
R=0.21 P=0.04
R=0.21 P=0.06 R=0.21 P=0.05
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3.9. Effect of subthalamic deep brain stimulation on FMD and HRV
Twenty patients with advanced PD who underwent subthalamic (STN) DBS surgery were enrolled.
HRV and FMD were investigated before and 1 year after surgery. FMD improved (6.01 ± 1.51 to 6.79
± 1.57, p=0.04), but there were no significant changes in HRV parameters (Table 13; Figure 7). While the level of homocysteine slightly decreased (13.9 ± 4.1 to 13.1 ± 3.1, p=0.041), there was no significant correlation between changes in homocysteine levels and alterations in FMD (r=-0.209, p=0.087).
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Table 14. Change of FMD and level of homocysteine after DBS surgery in PD Patients undergoing STN-DBS (n= 20 )
Characteristic baseline follow up
Age (years) 64.5 (8.0)
Sex (male) 12 (60%)
Disease duration (months) 123.5 (64.1)
Motor UPDRS (Off) 46.6 (13.2) 25.1 (11.3)
Levodopa equivalent daily dose 681 (133) 331.9 (61.7)
FMD (%) 6.01 (1.5) 6.79 (1.57)*
Homocysteine 13.9 (4.1) 13.1 (3.1)
Values are expressed as mean (SD) or numbers (with percentages). PD, Parkinson’s disease, UPDRS, Unified Parkinson’s Disease Rating Scale. a Significant difference between before and after DBS at p < 0.05.
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Figure 7. The difference of FMD and HRV parameter before and after DBS
SDNN, standard deviation of the normal-to-normal RR interval; RMSSD, root mean square difference between successive RR intervals; TP, total spectral power; LF, low frequency; HF, high frequency. * Significant difference before and after DBS at P < 0.05.
* NS
NS NS
NS NS
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IV. Discussion
4.1. Cardiovascular autonomic system in PD
PD and ET are the most common movement disorders in the elderly. Both ET patients and the elderly can display subtle parkinsonian signs [54]. Furthermore, a recent study demonstrated that one in five patients with ET have a tremor at rest [55]. In patients with PD, postural tremors occur as frequently as tremors at rest, and may be the presenting symptom [56]. Accordingly, differentiating between ET and PD is often challenging [4,5]. To improve the early diagnosis of ET and PD, positron emission tomography (PET), cardiac MIBG scanning, and olfaction studies have been proposed [57,58].
However, simple markers are also needed to allow the accurate identification of PD during the early stages. Our results showed that almost all parameters of HRV were significantly lower in patients with PD compared to those with ET, whereas differences between the ET and control groups were not significant. This suggests that HRV may be helpful in differentiating PD from ET at the early stage of PD. Although PD and ET differ in their pathogenesis, their clinical features are similar.
Patients with TDPD usually respond poorly to levodopa treatment, and their prognosis is favorable.
In addition, some patients with definite signs of PD have normal 18F-dopamine PET scans, and those with isolated resting or action tremors have consistently abnormal striatal dopamine transporter (DAT) uptake [59]. As such, early TDPD is commonly misdiagnosed as ET, emphasizing the need for an additional simple diagnostic tool capable of distinguishing between the two conditions. The distinction is important in determining prognosis, treatment planning, and identifying eligible patients for clinical studies.
TP and SDNN represent overall cardiac autonomic dysfunction, and RMSSD represents parasympathetic function. While LF predominantly represents the sympathetic nervous system, the HF component is generally a marker of the vagal nervous system. In our study, most HRV parameters were significantly lower in the PD group compared to the ET and control groups. Among HRV components, overall cardiac autonomic dysfunction (TP, SDNN) and cardiac sympathetic dysfunction (LF) was more prominent than parasympathetic dysfunction (HF, RMSSD). Our findings suggest that cardiac sympathetic dysfunction is prominent, but cardiac parasympathetic dysfunction is also present in early PD. This is consistent with previous studies reporting prominent peripheral sympathetic denervation combined with significant central autonomic dysfunction in the brainstem including the
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dorsal vagal nucleus in PD. However, an understanding of the pathological role played by HRV is needed to validate its usefulness as an early non-invasive imaging marker in PD.
Dementia is one of the most disabling non-motor symptoms that occurs in PD. Our findings show that decreased cardiac MIBG uptake increases the risk for the development of dementia.
Several studies have indicated that cognition, olfaction, visual hallucination and RBD are associated with decreased cardiac MIBG uptake in PD [25-27]. By contrast, striatal dopamine depletion is not well correlated with non-motor symptoms [60,61] but rather is correlated with motor severity and predicts the motor progression [62]. This suggests that the cardiac MIBG uptake might provide information regarding the presence of an extranigral alpha-synuclein pathology. Our results are in agreement with those of a previous Japanese follow up study, which showed a significant difference in the motor progression and the occurrence of dementia between the two groups that were divided according to delayed H/M ratio [63].
The clinical significance of HRV on cognition remains unclear. Some cross-sectional studies found that HRV in patients with MCI was similar to that of healthy controls [64], but others showed that HRV was slightly impaired compared to controls when subjects were standing [65,66]. Our findings demonstrate that in early PD, visuospatial and frontal function are correlated with some HRV parameters. There are several explanations for these observed associations. First, low HRV as a reflection of autonomic dysfunction might directly underlie cognitive impairment by causing dysregulation of cerebral perfusion [67,68]. Furthermore, it is possible that low HRV might reflect established cerebral lesions and neurodegenerative processes present in the brain [69]. Secondly, that low HRV is associated with increased blood pressure variability, which is associated with cognitive decline and structural brain changes [70]. Executive function is mainly controlled by the prefrontal cortex of the brain. Reduced HRV is associated with hypoactivity of the prefrontal cortex, which is likely to affect executive function [71]. Furthermore, the frontal cortex is able to adjust HRV via subcortical structures such as the amygdala. This cortico-subcortical inhibitory circuit represents the structural connection between neuropsychological processes (such as cognitive function) and physiologic processes (such as HRV). Abnormalities in the cortico-subcortical circuit can be reflected in HRV. Future brain imaging studies could provide new insight into the biology of these associations [72].
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4.2. Vascular endothelial system in PD
Levodopa treatment is the gold standard therapy in PD patients. However, this therapy increases serum levels of homocysteine, due to its metabolism via catechol-O-methyltransferase [73]. Several reports have shown that hyperhomocysteinemia might be associated with increased prevalence of coronary artery disease, carotid intima media thickness, peripheral neuropathy, and cognitive impairment in patients with PD [74-77], although there have been conflicting results concerning the risk of stroke in PD [78].
It is generally accepted that endothelial dysfunction is part of the early pathogenesis of atherosclerosis [46]. Along with traditional vascular risk factors, hyperhomocysteinemia is known to decrease FMD [79]. In this study, FMD was significantly lower in the levodopa treatment group compared to those in the levodopa/entacapone treatment group and controls. In addition, homocysteine level was negatively correlated with FMD, and was an independent predictor of the lowest tertile, indicating that endothelial dysfunction as assessed by the FMD is associated with chronic levodopa treatment in PD patients.
In our prospective follow-up study in de novo PD, FMD decreased after 1 year of levodopa treatment.
However, there was no correlation between the changes in homocysteine levels and FMD. In addition, STN DBS surgery could decrease levodopa dosage by almost 50%, and improve motor and endothelial function but had no direct effect on cardiac autonomic function.
Decreased FMD has been found in patients with cerebrovascular risk factors [80] and has been shown to have prognostic significance in the development of cardiovascular events [81]. In addition to traditional vascular risk factors, hyperhomocysteinemia is known to decrease FMD [79]. However, it is unclear whether hyperhomocysteinemia is a direct cause of decreased FMD or a marker of atherosclerosis. Several studies demonstrated that acute changes in homocysteine by methionine loading can cause decreased FMD in healthy elderly patients [82]. In this regard, the results of the present study provide evidence of a possible association between FMD and homocysteine levels in patients with PD. In line with our findings, Yong et al. demonstrated that changes in homocysteine levels after treatment with levodopa affect cerebral hemodynamics, such as pulsatility index. This may reflect systemic vascular resistance and increased vascular stiffness associated with endothelial dysfunction [83]. Similarly, in patients with ischemic stroke, hyperhomocysteinemia was independently associated with an increased pulsatility index in the cerebral arteries [84]. In addition,
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several reports have demonstrated that homocysteine-lowering therapy (using folic acid or vitamin B) can improve endothelial dysfunction as assessed by FMD [85] and reduce progression of carotid intima media thickness (a marker of early atherosclerosis) in asymptomatic subjects with hyperhomocysteinemia [86]. However, our prospective study shows that changes in homocysteine levels after levodopa treatment or DBS surgery did not correlate with FMD changes. It is possible levodopa may generate more oxidative stress, which may affect endothelial function [87].
Several recent studies report evidence of vascular involvement in PD, supporting our findings. For example, CSF biomarkers of angiogenesis are increased in PD, and are associated with gait impairments, increased blood brain barrier permeability, white matter lesions and cerebral microbleeds, indicating that abnormal angiogenesis may be present in PD pathogenesis and contribute to dopa-resistant symptoms [88]. Other lines of evidence include a human pathological study of PD cases showing endothelial degeneration and preservation of basement membrane, leading to an increase in string vessel formation in PD. String vessels have no function in circulation, suggesting cerebral hypoperfusion may contribute to the neuronal degeneration characteristic of PD [45].
V. Conclusion
Neurodegeneration does not occur in isolation but is believed to result from various chronic insults such as neurotoxic material, mitochondrial dysfunction, and accumulation of abnormal proteins that interact over time leading to selective neuronal loss.
Cardiac autonomic dysfunction occurs early in Lewy body disorders, including PD, DLB, and RBD, reflecting the early accumulation of Lewy bodies in cardiac sympathetic postganglionic nerves. HRV was significantly lower in early PD and DLB, and higher HRV was associated with better cognitive function. In addition, a reduction in cardiac MIBG uptake was associated with subsequent risk of dementia, suggesting reduced uptake may reflect wider extension of alpha-synuclein pathology in PD Traditional vascular risk factors and brain white matter changes contribute to the neurodegenerative process in PD. Vascular endothelial dysfunction occurs during early stages of atherosclerosis, and plays an important role in selective permeability of molecules entering the brain from the bloodstream.
FMD was significantly lower in PD patients, which was associated with levodopa treatment. STN-DBS could reduce levodopa dosage and improve FMD with significant motor improvement. The
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modification of these parameters represents a promising therapeutic approach to treat PD.
Figure 8. Proposed mechanism of the relationship between cardiac autonomic and vascular endothelial dysfunction in PD.
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