International Journal of Cardiology 197 (2015) 200–202
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Letter to the Editor
Endothelial progenitor cells: Cardiovascular protection in Parkinson's disease?☆ Gianni Pezzoli a, Ferruccio Cavanna b, Erica Cassani a, Michela Barichella a, Giovanna Pinelli a, Laura Iorio a, Chiara Pusani a, Margherita Canesi a, Francesca Natuzzi a, Roberta Cazzola c, Benvenuto Cestaro c, Emanuele Cereda d,⁎ a
Parkinson Institute, Istituti Clinici di Perfezionamento, Milano, Italy Centro Analisi Monza (CAM), Monza-Brianza, Italy c Department of Biomedical and Clinical Sciences, “L. Sacco Hospital”, School of Clinical Nutrition, Faculty of Medicine and Surgery, University of Milano, Milano, Italy d Nutrition and Dietetics Service, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy b
a r t i c l e
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Article history: Received 1 June 2015 Accepted 19 June 2015 Available online 26 June 2015 Keywords: Parkinson disease Endothelial progenitor cells (EPCs) Cardiovascular risk
Parkinson's disease (PD) is characterized by reduced dopaminergic activity and previous animal-model studies have shown that the neurotransmitter dopamine modulates EPC function and mobilization from the bone marrow [1]. In a pilot study, we have observed that PD patients have significantly higher EPC levels than controls [2]. However, the strength of the evidence was hampered by the small sample size, the method used for EPC determination and the potential confounding effect of dopaminergic treatment. Furthermore, we did not take into account the role of important confounders, such as cardiovascular (CV) risk factors and related treatments. Conversely, another study has reported no difference in circulating EPCs between PD patients and controls [3]. Therefore, we investigated whether differences in counts of circulating EPCs occur between PD patients and controls, and, if so, whether such counts are modified by dopamine-replacement therapy. We studied: – de novo (drug naïve) PD patients (DPD) having at least a one-year follow-up;
☆ Source of support: This work was supported by the “Fondazione Grigioni per il Morbo di Parkinson”. ⁎ Corresponding author at: Fondazione IRCCS Policlinico San Matteo, Viale Golgi 19, 27100 Pavia, Italy. E-mail address:
[email protected] (E. Cereda).
http://dx.doi.org/10.1016/j.ijcard.2015.06.071 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
– PD patients treated with levodopa for more than 1 year (LTPD), not using catechol-O-methyl-transferase (COMT) inhibitors [3], matched (1:1) for age (± 1 year), gender and body mass index (BMI; ±1 kg/m2) to DPD patients; – healthy controls matched to DPD (1:1) and LTPD (1:1) patients for age (±1 year), gender and BMI (±1 kg/m2).
Patients were recruited consecutively among patients attending the Parkinson Institute out-patient clinic between January 2013 and June 2014. Healthy controls were recruited over the same period among the people living with patients seen at the Parkinson Institute. Exclusion criteria were: current or past smoking; hypertension; diabetes; CV disease; total cholesterol N 250 mg/dL, any-organ failure; history of cancer or any chronic inflammatory disease; autoimmune or blood disorders; current infection; use of lipid-lowering medications, NSAIDs or oral contraceptives in the previous 6 months; hormone replacement therapy; alcohol intake N10 g/day; body weight changes in the previous 6 months; use of any-type dietary supplements; previous neurosurgical procedure for PD. All PD patients were studied by brain MRI to exclude the presence of major vascular abnormalities. For the evaluation of EPCs we used a modified ISHAGE (International Society for Hematotherapy and Graft Engineering) protocol [4]. Accordingly, after immunostaining with fluorescent-conjugated antibodies against human CD34, CD133, kinase insert domain receptor (KDR) and CD45, samples were lysed and the absolute count of EPCs (CD34+/CD133+/KDR+/CD45+) was determined by flow cytometry (Navios™; Beckman Coulter) Additional blood samples were obtained for the evaluation of full blood count, blood glucose, serum creatinine, total cholesterol, HDL and LDL cholesterol and triglycerides. We assessed also dietary habits by means of a food frequency questionnaire. Particularly, as PD patients are generally advised to take advantage from the redistribution of protein intake throughout the day [5] and EPC counts are influenced by dietary protein content [6], we considered that the relevant covariate should be the number of high-protein dishes consumed weekly at the main meals.
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G. Pezzoli et al. / International Journal of Cardiology 197 (2015) 200–202 Table 1 Characteristics of the study population a by group. Characteristic
De-novo PD patients (N = 27)
Levodopa-treated PD patients (N = 27)
Control patients (N = 54)
P-value⁎
Age (years), mean (SD) Male gender, N (%) Disease duration (years), mean (SD) Levodopa dosage (mg/day), mean (SD) Levodopa dosage (mg/kg/day), mean (SD) BMI (kg/m2), mean (SD) White blood count (/103/μL), mean (SD) Glucose (mg/dL), mean (SD) Total cholesterol (mg/dL), mean (SD) HDL cholesterol (mg/dL), mean (SD) LDL cholesterol (mg/dL), mean (SD) Triglycerides (mg/dL), mean (SD) Serum creatinine (mg/dL), mean (SD) Number of high-protein dishes at main meals per week ≥8, N (%) EPC counts, cells/mL
61.4 (10.6) 16 (59) 1.1 (0.2) – – 28.6 (5.9) 6.41 (1.02) 94.3 (10.2) 196 (31)b 53 (10) 119 (39)b 124 (49) 0.86 (0.23) 22 (81) 4603 (2108)b
61.7 (9.6) 16 (59) 4.2 (2.4) 434 (191) 6.5 (3.6) 27.6 (4.7) 6.48 (1.68) 90.2 (12.9) 186 (32)b 52 (10) 112 (36)b 107 (47) 0.85 (0.19) 17 (63) 4410 (2603)b
61.5 (10.5) 32 (59) – – – 28.6 (5.0) 6.52 (1.25) 94.0 (9.5) 216 (33) 56 (12) 135 (32) 127 (45) 0.88 (0.22) 53 (98) 2338 (821)
0.994 1.000 b0.001 – – 0.691 0.939 0.257 0.001 0.249 0.013 0.183 0.822 b0.001 b0.001
Abbreviations: PD, Parkinson's disease; BMI, body mass index; HDL, high density lipoprotein; LDL, low density lipoprotein; AST, aspartate aminotransferase; ALT, alanine aminotransferase. ⁎ P-values according to one-way ANOVA (multiple-group comparison of continuous variables) or Student's t test (two-group comparison of continuous variables) or Fisher's exact test (comparison of categorical variables). a Sample size calculation was based on a previous pilot investigation [2]. We calculated that the recruitment of at least 19 patients for each group (PD and controls) would have a statistical power of 80% in showing an effect size of 0.9 with a two-tailed type-I error b5%. Considering the inclusion of two different groups of PD patients (de-novo and treated with levodopa) and the number of between-group comparisons, with an adjusted two-tailed type-I error b1.3%, we estimated that we would need to recruit 27 patients in each PD group and 54 controls. b Significantly different from controls by post-hoc comparison [Scheffe's test].
The study protocol was approved by the local ethics committee and conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as revised in 2013. Informed consent was obtained from all subjects. The general characteristics of the three study groups are shown in Table 1. Compared to PD patients, healthy controls had higher total and LDL cholesterol levels and protein intake. Conversely, EPC counts in both DPD and LTPD patients were significantly higher than in controls while no difference was found between the two groups of PD patients. In multivariate analysis, both DPD and LTPD patients remained characterized by higher circulating EPCs (Table 2). Finally, after adjusting for cholesterol levels and dietary protein intake, in LTPD increases in levodopa dose (mg/kg/day) were associated with higher EPC counts (for linear increase over tertiles of distribution of dose, β coefficient [standard error], 1123 cells/mL [521]; P = 0.043). Based on previous reports, higher EPC counts in PD patients were expected to partly depend on a favorable cardiometabolic profile, which is improved further by dopamine-replacement therapy [7]. It has also been reported that dopamine regulates mobilization of EPCs from the bone marrow in rodents, counts being higher in the presence of low dopamine levels and lower after dopamine treatment [1]. With this study, we have confirmed that PD patients have higher counts of EPCs than healthy people from the general population. We conducted multiple-adjusted analysis and included a highly selected study population to avoid the bias associated with many potential confounders (smoking and dietary habits, nutritional status, comorbidities, CV risk factors and related treatments) [6,8], although this may affect generalizability of the results. Furthermore, we included, for the first time, drug-
Table 2 Multivariate analysis of predictors of EPCs in the study population (variance in EPC counts explained by the model 46%). Predictors
Coefficient
Std. error
t-Value
P-value
Age (year) Male gender DPD LTPD Protein intakea Total cholesterol (mg/dL) White blood count (103/μL)
18 247 1236 1052 1814 12 515
15.0 369 521 518 489 5.9 266
1.23 0.67 2.37 2.03 3.71 2.03 1.94
0.221 0.505 0.020 0.045 b0.001 0.047 0.056
Abbreviations: DPD, de novo Parkinson's disease; LTPD, levodopa-treated Parkinson's disease. a Number of high-protein dishes at main meals per week ≥8.
naïve patients. Data showed that high EPC counts are independent of dopamine-replacement therapy and, on the other hand, that EPCs are directly correlated with daily levodopa dosage. Our results support the absence of a role for endothelial dysfunction in the pathophysiology of PD. In addition, levodopa replacement therapy is a safe and efficacious intervention without relevant implications on CV risk. PD is a disease model characterized by the progressive reduction in nigro-striatal dopaminergic activity occurring at both central and peripheral levels [9]. Higher EPCs in DPD patients are likely to depend mainly on this factor. Besides, higher levodopa doses may compensate additional dopaminergic activity loss in LTPD, reducing at the same time sympathetic activation and CV risk [7] rather than inducing sympathetic overactivity which has negative effect on EPC count [10]. However, the determinants of circulating EPC counts in PD deserve further investigation. Future studies should address also the functional properties of EPCs in PD as we limited their identification to cell surface markers and we did not demonstrate any colony forming ability. Finally, prospective studies controlling for relevant CV risk factors and confounders are required to clarify the role of EPCs as a marker of CV disease and risk in PD. The association between CVD and PD needs to be clarified, since the evidence still is inconsistent and recent studies have suggested that CV disease and mortality are more likely to be due to autonomic dysfunction [11] rather than atherosclerosis progression. Acknowledgments Study guarantor Dr Cereda had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Statement of authorship All the authors significantly contributed to the work and approved the manuscript for submission. Particularly, author contributions were as follows: Study concept and design: Pezzoli, Cavanna, Cassani, and Cereda. Acquisition of data: Cavanna, Cassani, Barichella, Pinelli, Iorio, Pusani, and Canesi. Analysis and interpretation of data: Pezzoli, Cavanna, Cereda, Cazzola, and Cestaro.
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Drafting of the manuscript: Cereda. Critical revision of the manuscript for important intellectual content: Pezzoli, Cavanna, Cassani, Cazzola, Cestaro, and Cereda. Statistical analysis: Cereda. Obtained funding: Pezzoli and Cavanna. Administrative, technical, or material support: Cavanna, Cassani, Barichella, and Natuzzi. Study supervision: Pezzoli and Barichella. Additional contributions The authors wish to thank Jennifer S Hartwig MD for assistance in editing the manuscript. Full financial disclosures of all authors for the past 2 years The authors also certify that over the last three years there were no affiliations with or involvement in any organization or entity with a direct financial interest in the subject matter or materials discussed herein. Emanuele Cereda has received consultancy honoraria and investigator grants from the “Fondazione Grigioni per il Morbo di Parkinson” and the Fondazione IRCCS Policlinico San Matteo and Nutricia Italia. References [1] D. Chakroborty, U.R. Chowdhury, C. Sarkar, R. Baral, P.S. Dasgupta, S. Basu, Dopamine regulates endothelial progenitor cell mobilization from mouse bone marrow in tumor vascularization, J. Clin. Invest. 118 (4) (2008) 1380–1389.
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