Subclinical Thyrotoxicosis and Cardiovascular Risk: Assessment of Circulating Endothelial Progenitor Cells, Proangiogenic Cells, and Endothelial Function

Abstract

Background: Subclinical thyrotoxicosis (SCT) is defined by low or undetectable thyroid-stimulating hormones and normal thyroid hormones. The treatment of SCT is uncertain despite being associated with increased cardiovascular risk (CVR) and mortality. Circulating endothelial progenitor cells (cEPCs) and circulating angiogenic cells (CACs) have been found to be reduced in conditions with CVR. We aimed to evaluate whether endothelial function and cEPC and CAC counts were reduced in SCT and to study the in vitro effect of triiodothyronine (T3) on proangiogenic cell (PAC) function from young healthy controls.

Methods: cEPCs (quantified by flow cytometry, 20 SCT/20 controls), CACs following in vitro cultures (15 SCT/14 controls), paracrine function of CACs, endothelial function by flow-mediated dilation (FMD, 9 SCT/9 controls), and the effect of T3 on apoptosis and endothelial nitric oxide synthase (eNOS) expression in PACs were studied.

Results: p < 0.001, CD133⁺/VEGFR-2⁺ 0.4 (0.0-0.7) vs. 0.6 (0.0-4.6), p = 0.009, CD34⁺/VEGFR-2⁺ 0.3 (0.0-1.0) vs. 0.7 (0.1-4.9), p = 0.002; while CAC count was similar. SCT predicted a lower cEPC count after adjustment for conventional CVR factors. FMD was lower in SCT subjects versus controls (% mean ± SD, 2.7 ± 2.3 vs. 6.1 ± 2.3, p = 0.005). In vitro studies showed T3 increased early apoptosis and reduced eNOS expression in PACs.

Conclusions: In conclusion, SCT is associated with reduced cEPC count and FMD, confirming increased CVR in SCT. Future outcome trials are required to examine if treatment of this subclinical hyperactive state improves cardiovascular outcome.

Clinical trial registration: http://www.controlled-trials.com/isrctn/, identifier ISRCTN70334066.

Keywords: apoptosis; cardiovascular risk; circulating angiogenic cells; endothelial progenitor cells; proangiogenic cells; subclinical thyrotoxicosis.

Figure 1

Differential gene expression (analyzed by RT-qPCR) in macrophages (MФ), PACs, and human umbilical vein endothelial cells (HUVEC), all isolated from the same donor (n = 3). Gene expression of macrophage-specific markers (A) CD14 and (B) CD115. (C) Gene expression profile of endothelial cell surface marker CD31. (D) Gene expression of the hematopoietic stem cell surface marker CD34 in PACs. (E) CD144 (VE-cadherin) is expressed in endothelial cells but not in macrophages. (F) CD146 (melanoma cell adhesion molecule) cell adhesion molecule used as a marker for mature endothelial cell lineage. (G) Gene expression of endothelial cell-specific marker vWF, upregulated in mature endothelial cells but not in PACs and macrophages. (H) KDR vascular endothelial growth factor receptor 2 (VEGFR-2), which is highly expressed in endothelial cells HUVECs and PACs. HUVECs serve as a control cell type for comparison. All data were normalized to the reference gene TATA-box-binding protein (TBP). It appears that PACs are distinctively different from macrophages given their characteristic gene signature.

Figure 2

RT-PCR for TRα1 in PAC and HUVEC samples. All samples are shown positive for TRα1 mRNA expression. RT−, without reverse transcriptase. The size of DNA fragments was determined by comparison with a 100-bp DNA ladder molecular weight marker.

Figure 3

Graph showing apoptosis in proangiogenic cells (PACs) after staining with Annexin V and 7-AAD. Data are represented as mean ± SEM. ^* p < 0.05 versus normal (1× T3) and n = 11. +con, positive control, −con, negative control.

Figure 4

Effect of triiodothyronine (T3) on the expression of eNOS in PACs. Gene expression was quantified by real-time PCR and normalized to TBP (reference gene TATA-box-binding protein). Data are represented as mean ± SEM. ^* p < 0.05 versus normal (1× T3). +con, positive control; −con, negative control.