Fasudil alleviates the vascular endothelial dysfunction and several phenotypes of Fabry disease

Abstract

Fabry disease (FD), a lysosomal storage disorder, is caused by defective α-galactosidase (GLA) activity, which results in the accumulation of globotriaosylceramide (Gb3) in endothelial cells and leads to life-threatening complications such as left ventricular hypertrophy (LVH), renal failure, and stroke. Enzyme replacement therapy (ERT) results in Gb3 clearance; however, because of a short half-life in the body and the high immunogenicity of FD patients, ERT has a limited therapeutic effect, particularly in patients with late-onset disease or progressive complications. Because vascular endothelial cells (VECs) derived from FD-induced pluripotent stem cells display increased thrombospondin-1 (TSP1) expression and enhanced SMAD2 signaling, we screened for chemical compounds that could downregulate TSP1 and SMAD2 signaling. Fasudil reduced the levels of p-SMAD2 and TSP1 in FD-VECs and increased the expression of angiogenic factors. Furthermore, fasudil downregulated the endothelial-to-mesenchymal transition (EndMT) and mitochondrial function of FD-VECs. Oral administration of fasudil to FD mice alleviated several FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat insensitivity. Our findings demonstrate that fasudil is a novel candidate for FD therapy.

Keywords: Fabry disease; drug screening; fasudil; iPSCs; vascular endothelial cells.

Graphical abstract

Figure 1

Screening of hit compounds from a clinical chemical library using FD-VECs (A) Schematic overview of the drug screening procedure using FD-VECs that were differentiated from FD-iPSCs. The abilities of 2,107 preclinical or clinical chemical compounds to improve the endothelial functionality of FD-VECs were examined. In the first screen, compounds inducing cytotoxicity and/or aberrant morphological changes of FD-VECs were excluded. The effects of the compounds on the ability of FD-VECs to form tube-like structures were evaluated in the second and third screens. (B) Scatterplot showing the total tube lengths of FD-VECs treated with DMSO (negative control), the TGF-β inhibitor SB431542 (SB; positive control, 5 μM), and the first screened clinical compounds (5 μM). Two independent experiments were conducted. FD, FD1-VECs. (C) The relative total tube lengths of FD-VECs treated with DMSO (negative control), the TGF-β inhibitor SB431542 (SB; positive control), or the second screened clinical compounds at different concentrations (0.5, 1, or 5 μM). Data are represented as mean ± SEM (n = 3); ∗p < 0.05 and ∗∗∗p < 0.001 (Student’s t test). 1, D-4476; 2, fasudil; 3, T0070907; 4, N-(4-butoxyphenyl)acetamide; 5, lomerizine; 6, aconitine; 7, tolfenamic acid; 8, quinine-ethyl-carbonate; 9, corticosterone; 10, nialamide; 11, amikacin; 12, bromocriptine; 13, GR-159897; 14, PK 11195; 15, eprazinone. (D) Western blot analysis of p-SMAD2, SMAD2, TSP1, KDR, eNOS, and GAPDH in WT-VECs, untreated FD-VECs, and FD-VECs treated with the indicated compounds. Data are represented as mean ± SEM (n = 5); ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (Student’s t test). WT, WT-VECs; FD, FD1-VECs; E, eprazinone; L, lomerizine; TA, tolfenamic acid; F, fasudil; D, D-4476; T, T0070907.

Figure 2

The effect of fasudil on the angiogenesis of FD-VECs (A) Tube-like structure formation by WT-VECs, untreated (FD) or fasudil-treated FD-VECs, and gene-corrected FD-VECs (FD[c]). Data are represented as mean ± SEM (n = 3; p < 0.05 (Student’s t test). WT, WT-VECs; FD, FD1-VECs; FD(c), gene-corrected FD1-VECs. Scale bar: 200 μm. (B) Western blot analysis of p-SMAD2, SMAD2, TSP1, KDR, eNOS, and GAPDH in the cells described in (A). Data are represented as mean ± SEM (n = 7); ∗p < 0.05 and ∗∗∗p < 0.001 (Student’s t test). (C) Immunostaining of TSP1 and KDR in the cells described in (A). Scale bar: 50 μm.

Figure 3

Fasudil downregulates the EndMT of FD-VECs (A) Western blot analysis of EndMT-associated factors (CD31, COL1A1, ACTA2, SNAI1, and TWIST) in WT-VECs, FD-VECs, and gene-corrected FD-VECS (FD[c]). GAPDH expression was used as a loading control. Data are represented as mean ± SEM (n = 4); ∗p < 0.05 and ∗∗∗p < 0.001 (Student’s t test). WT, WT-VECs; FD, FD1-VECs; FD(c), gene-corrected FD1-VECs. (B) Western blot analysis of EndMT-associated factors in WT-VECs, untreated (FD) or fasudil-treated FD-VECs, and gene-corrected FD-VECs (FD[c]). Data are represented as mean ± SEM (n = 4); ∗∗p < 0.01 (Student’s t test). (C) Immunostaining of EndMT-associated factors in the cells described in (B). Scale bar: 50 μm.

Figure 4

Fasudil alleviates impaired metabolic processes in FD-VECs (A) The transcriptional levels of ROS-related genes (GSTM1, NCF2, and PPARGC1A) in WT-VECs, untreated (FD) or fasudil-treated FD-VECs, and gene-corrected FD-VECs (FD[c]). Data are represented as mean ± SEM (n = 4); ∗p < 0.05 and ∗∗p < 0.01 (Student’s t test). WT, WT-VECs; FD, FD1-VECs; FD(c), gene-corrected FD1-VECs. (B) Representative images showing the fluorescence intensity of ROS in the cells described in (A). Data in the graph are represented as mean ± SEM (n = 4); ∗p < 0.05 and ∗∗p < 0.01 (Student’s t test). Scale bar: 50 μm. (C) Extracellular flux analysis of the oxygen consumption rate (OCR) in the cells described in (A). Data are represented as mean ± SEM (n = 6). O, oligomycin; F, FCCP; R&A, rotenone and antimycin A.

Figure 5

Fasudil alleviates FD phenotypes in FD mice after oral administration (A) Schematic overview of the treatment of FD mice (Gla^−/−/TSP1^Tg) with fasudil (10 or 30 mg/kg/day for 6 months). As a control, FD mice were administered PBS. (B) Echocardiography analysis of WT mice (n = 6) and FD mice treated with PBS (n = 4) or fasudil at 10 or 30 mg/kg (n = 9). The left ventricular mass/body weight ratio, ejection fraction, fractional shortening, and cardiac output were measured. Data are represented as mean ± SEM; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (Student’s t test). (C) Analysis of sweat secretion by WT mice and FD mice treated with or without fasudil. Data are represented as mean ± SEM (n = 3); ∗p < 0.05 (Student’s t test). (D) Heat tolerance analysis of WT mice and FD mice treated with or without fasudil. Increased latency of paw withdrawal indicates hyposensitivity to thermal pain. Data are represented as mean ± SEM (n = 5); ∗p < 0.05 (Student’s t test).

Figure 6

Fasudil alleviates fibrosis and inflammation in the renal tissues of FD mice (A) Schematic overview of the treatment of FD mice (Gla^−/−/TSP1^Tg) with fasudil (30 mg/kg/day for 6 months). As a control, FD mice were administered PBS. (B) Immunohistochemical analysis of CD31 and ACTA2 in renal tissues from WT mice (n = 3) and FD mice (Gla^−/−/TSP1^Tg) administered PBS (n = 3) or fasudil (n = 4). Data in the graph are represented as mean ± SEM; ∗∗p < 0.01 (Student’s t test). Scale bar: 100 μm. (C) Immunohistochemical analysis of COL1A1 in renal tissues from WT mice (n = 3) and FD mice (Gla^−/−/TSP1^Tg) administered PBS (n = 3) or fasudil (n = 4). Data in the graph are represented as mean ± SEM; ∗p < 0.05 (Student’s t test). Scale bar: 100 μm. (D) Immunohistochemical analysis of inflammation-associated markers (LCN2 and F4/80) in renal tissues from WT mice (n = 3) and FD mice (Gla^−/−/TSP1^Tg) administered PBS (n = 3) or fasudil (n = 4). Data in the graphs are represented as mean ± SEM; ∗p < 0.05 and ∗∗p < 0.01 (Student’s t test). Scale bar: 100 μm. (E) Western blot analysis of fibrosis-related markers (ACTA2 and COL1A1), inflammation markers (LCN2 and F4/80), p-SMAD2, SMAD2, and GAPDH in kidney lysates from FD mice (Gla^−/−/TSP1^Tg) treated with or without fasudil. Data are represented as mean ± SEM (n = 3); ∗p < 0.05 and ∗∗p < 0.01 (Student’s t test). (F) A schematic illustration for the underlying mechanism of fasudil in FD models.