Angiogenic T-Cells and Putative Endothelial Progenitor Cells in Hypertension-Related Cerebral Small Vessel Disease
Background and Purpose—Cerebral small vessel disease (CSVD) may be caused by endothelial dysfunction, whereas endothelial progenitor cells (EPC) may attenuate endothelial dysfunction. Their vitality is lower in CSVD. A subset of lymphocytes, angiogenic T-cells, is capable to stimulate EPC function. The purpose of our study was to explore the relation between CSVD manifestations, angiogenic T-cells, and EPC in hypertensive patients with CSVD.
Methods—We compared 32 essential hypertensive patients with CSVD (white matter lesions, asymptomatic lacunar infarcts, or microbleeds on 1.5-Tesla MRI) to 29 age-matched and sex-matched hypertensive controls. We counted angiogenic T-cells (CD3+/CD31+/CD184+) and putative EPC (CD31+/CD34+/CD45-/KDR+) by flow cytometry and determined EPC vitality by in vitro cluster formation.
Results—Putative EPC numbers were lower in hypertensive individuals with CSVD than in those without (10±7.103/mL versus 13±6.103/mL [median±interquartile range]; P=0.011). Angiogenic T-cell numbers were also lower in hypertensive individuals with CSVD than in those without (0.56±0.25.109/mL versus 0.78±0.50.109/mL; P=0.008). Higher angiogenic T-cell numbers independently related to absence of CSVD (odds ratio, 0.088; 95% confidence interval, 0.012–0.627).
Conclusions—Our data suggest that angiogenic T-cells and putative EPC independently relate to radiological CSVD manifestations in hypertensive patients.
Cerebral small vessel disease (CSVD) has several manifestations, such as white matter lesions, lacunar infarcts, and microbleeds. Blood–brain barrier dysfunction could play a key role in the pathogenesis of these abnormalities.1 Dysfunction of the blood–brain barrier may be attenuated by putative endothelial progenitor cells (EPC). Putative EPC are immature cells, which circulate in peripheral blood.2 They are involved in repair of endothelial damage2 and are possibly also involved in improving endothelial cell function.3 In CSVD, however, more severely affected patients have lower EPC vitality,4 whereas factors that regulate putative EPC in CSVD are largely unknown.2
Recent studies suggest that angiogenic T-cells (Tang) may regulate EPC function.5 Tang express platelet endothelial cell adhesion molecule (CD31) as well as the receptor for stromal-derived factor 1 (CD184).5 Furthermore, Tang promote the formation of new blood vessels and endothelial repair by stimulating the function of EPC.5
We hypothesized that CSVD patients have lower numbers of Tang, which may relate to lower EPC vitality. To test this hypothesis, we counted Tang and putative EPC and measured EPC functional properties in 2 groups of hypertensive patients, 1 with and 1 without CSVD in a case-control design.
Subjects and Methods
We included 32 hypertensive patients with CSVD and 29 hypertensive patients without CSVD from a longitudinal cohort study on hypertensive brain damage.6 We defined CSVD as the presence of 1 or more of the following MRI characteristics: (asymptomatic) lacunar infarcts; extensive white matter lesions; and brain microbleeds. For putative EPC quantification, we used flow cytometry identifying putative EPC as CD31+/CD34bright/CD45−/ KDR+.7 To determine the vitality of putative EPC, we used cell cultures with 2 different techniques2,3,8 and a telomerase assay to determine the degree of senescence in the cultured cells. To quantify Tang we used flow cytometry, defining Tang as CD3+/CD31+/CD184+. For a detailed description, please see Supplemental Methods (http://stroke.ahajournals.org).
Characteristics of the study population are provided online (Supplemental Table I). As a whole, risk factor profiles were similar between hypertensive patients with and without CSVD. Per definition, patients with CSVD had more extensive white matter lesions, more often had lacunar infarcts, and more often had microbleeds.
CSVD, EPC, and Circulating Tang
Data for T-cells and putative EPC in the patient groups with or without CSVD are shown in the Figure and the Table. Patients with CSVD had lower total T-cell counts, lower circulating Tang counts, as well as lower EPC counts. There was not a significant difference in ECFC or CFU-EC cluster counts or in EPC telomerase activity between the groups.
We performed binary logistic regression analyses to determine significant predictors for the presence of CSVD. After correction for total T-cell counts, as well as blood pressure measurements, only higher Tang numbers significantly related to the absence of CSVD (odds ratio, 0.088; 95% confidence interval, 0.012–0.627).
In the present study, we observed that hypertensive patients with CSVD had lower Tang and putative EPC numbers in their blood than hypertensive control patients without CSVD, independently of blood pressure levels. Therefore, we postulate that T-cells, next to EPC, may be involved in the pathogenesis of hypertension related CSVD.
We investigated whether Tang might be a regulating factor of EPC number and functionality in patients with CSVD. These cells form a subset of T-cells, which might play a role in vascular repair, because they stimulate putative EPC in their restorative capacities as evidenced in an in vitro study.5 Angiogenic T-cell numbers decrease with age.9 We found that numbers of Tang were lower in hypertensive patients with CSVD, independent of age. Furthermore, recent studies suggest that T-cells are involved in the pathogenesis of hypertension and, in particular, in hypertension-associated vascular damage.10,11 The relation between lower Tang and CSVD may specifically imply a role of Tang in CSVD. Still, the nature of this role remains elusive.
Our study has several limitations. First, our study design is cross-sectional and therefore the observed associations could also be an epiphenomenon of CSVD. Second, the definition of EPC remains controversial, hence the term putative EPC.2 However, for flow cytometry and for cultures we used established authoritative protocols. Third, although variables such as age, sex, and medication did not differ significantly between hypertensive patients with or without CSVD, we cannot exclude a mild effect of this difference because age and other risk factors inversely relate to EPC and Tang number.2,5 Furthermore, our study contains a relatively small number of patients, which could have led to statistical error. Therefore, future studies should be large enough to preclude such shortcoming. Notwithstanding these limitations, the strength of our study remains the novelty of our findings in a well-characterized group of hypertensive patients.
We found that T-cells and putative EPC relate to cerebral small vessel disease in hypertensive patients. In the process, Tang may play a role. These findings are novel and allow novel hypotheses regarding the pathophysiology of CSVD.
Sources of Funding
R.P.W.R. is funded by a research grant of the Netherlands Heart Foundation (2005B022). L.H.G.H. is funded by a research grant of the Novartis Foundation for Cardiovascular Excellence (003/07).
The online-only Data Supplement is available at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.111.632208/-/DC1.
- Received July 11, 2011.
- Accepted August 18, 2011.
- © 2012 American Heart Association, Inc.
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