Establishment and characterization of Fabry disease endothelial cells with an extended lifespan

Abstract

Fabry disease is an inborn error of glycosphingolipid catabolism resulting from a deficiency of lysosomal enzyme alpha-galactosidase A. The major clinical manifestations of the disease, such as stroke, cardiac dysfunction, and renal impairment, are thought to be caused by vasculopathy due to progressive accumulation of globotriaosylceramide in vascular endothelial cells. The pathogenesis of the vasculopathy has not been elucidated. Since in vitro studies using primary endothelial cells are hampered by the limited lifespan of these cells, the availability of cultured endothelial cells with an extended lifespan is critical for the study of the vasculopathy of Fabry disease. We therefore generated an endothelial cell line from a Fabry hemizygote by introduction of human telomerase reverse transcriptase gene. The cell line has markedly extended lifespan compared to parental primary cells. The cells stably express many key markers of endothelial cells such as von Willebrand factor, CD31, CD34, and endothelial nitric oxide synthase (eNOS) and retain functional characteristics such as uptake of acetylated low-density lipoprotein, responsiveness to angiogenic growth factors, up-regulation of eNOS production upon extracellular stimuli, and formation of tube-like structures on Matrigel basement membrane matrix. The cells show significantly reduced activity of alpha-galactosidase A compared with primary endothelial cells from normal individuals and accumulate globotriaosylceramide in lysosomes. This cell line will provide a useful in vitro model of Fabry disease and will facilitate systematic studies to investigate pathogenic mechanisms and explore new therapeutic approaches for Fabry disease.

Fig 1. hTERT-induced lifespan extension of Fabry ECs

(A) Expression level of hTERT was assessed by quantitative RT-PCR. (B) Growth rates of primary parental cells at passage 5 and hTERT-transduced cells at passage 26. hTERT-transduced cells proliferate actively with doubling time of 33.6 h, while primary cells essentially did not divide. (C) Population doubling (counted from passage 8) of hTERT transduced cells. (D and E) Microscopic photographs of SA β-gal cytochemistry of primary parental cells at passage 6 (D) and hTERT-transduced cells at passage 23 (E). Photos were taken at the same magnification. Scale bar: 250 μm

Fig 2. Characterization of IMFE1 cell line

(A) Morphology of IMFE1 cells under phase contrast microscope. (B–D) Immunofluorescence staining for vWF (B), CD31 (C) and CD34 (D). (E) eNOS expression analyzed by Western blot analysis. Left panel: Identification of eNOS expression in IMFE1 cell line. Primary human microvascular endothelial cells (HMVEC) and HeLa cells were used as positive and negative control respectively. Right panel: Persistent eNOS expression in IMFE1 cells at earlier and later passages (P). β-actin was used as loading control. (F) Uptake of DiI-Ac-LDL. Incorporated DiI (red) shows characteristic lysosomal-staining. Scale bar in (A): 100 μm

Fig 3. Responsiveness of IMFE1 cells to angiogenic growth factors and growth on Matrigel

(A) Growth response curves of IMFE1 cells 72hs after stimulation with bFGF and VEGF. Results are expressed as a ratio of the absorbance of the stimulated wells to the absorbance of the non-stimulated wells. Data are presented as mean ± SE (n = 6). (*P<0.0003, #P<0.002, stimulated wells compared with non-stimulated wells, two-tailed Student’s t test) (B) eNOS expression analyzed by Western blot. IMFE1 cells were incubated with medium containing 0–100 ng/ml VEGF or bFGF for 48 hr. β-Actin was used as loading control. (C) Tube formation assay. IMFE1 cells grown on Matrigel extracellular matrix formed tube-like structures that were similar to those formed by primary HMVEC.

Fig 4. Fabry phenotype of IMFE1 cell line

(A) α-Gal A activity of IMFE1 cells and primary ECs from four non-Fabry controls. The data are presented as mean ± SE (n ≥ 3). (B) Immunostaining of Gb3 in IMFE1 cells. Lysosomal-distribution of positive signal is apparent in some of the cells (arrows). Inset: Immunostaining of the cells after treatment with recombinant α-Gal A (0.4 IU/ml) for 48 hr.