Elevated TGFβ signaling contributes to cerebral small vessel disease in mouse models of Gould syndrome

Abstract

Cerebral small vessel disease (CSVD) is a leading cause of stroke and vascular cognitive impairment and dementia. Studying monogenic CSVD can reveal pathways that are dysregulated in common sporadic forms of the disease and may represent therapeutic targets. Mutations in collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause highly penetrant CSVD as part of a multisystem disorder referred to as Gould syndrome. COL4A1 and COL4A2 form heterotrimers [a1α1α2(IV)] that are fundamental constituents of basement membranes. However, their functions are poorly understood and the mechanism(s) by which COL4A1 and COL4A2 mutations cause CSVD are unknown. We used histological, molecular, genetic, pharmacological, and in vivo imaging approaches to characterize central nervous system (CNS) vascular pathologies in Col4a1 mutant mouse models of monogenic CSVD to provide insight into underlying pathogenic mechanisms. We describe developmental CNS angiogenesis abnormalities characterized by impaired retinal vascular outgrowth and patterning, increased numbers of mural cells with abnormal morphologies, altered contractile protein expression in vascular smooth muscle cells (VSMCs) and age-related loss of arteriolar VSMCs in Col4a1 mutant mice. Importantly, we identified elevated TGFβ signaling as a pathogenic consequence of Col4a1 mutations and show that genetically suppressing TGFβ signaling ameliorated CNS vascular pathologies, including partial rescue of retinal vascular patterning defects, prevention of VSMC loss, and significant reduction of intracerebral hemorrhages in Col4a1 mutant mice aged up to 8 months. This study identifies a novel biological role for collagen α1α1α2(IV) as a regulator of TGFβ signaling and demonstrates that elevated TGFβ signaling contributes to CNS vascular pathologies caused by Col4a1 mutations. Our findings suggest that pharmacologically suppressing TGFβ signaling could reduce the severity of CSVD, and potentially other manifestations associated with Gould syndrome and have important translational implications that could extend to idiopathic forms of CSVD.

Keywords: Basement membrane; CSVD; Gould syndrome; TGFβ; Type IV collagen.

Figure 1.. 4PBA partially ameliorates developmental retinal vascular defects in Col4a1 mutant mice.

(A) P7 retinas immunolabeled for CD31 and (B) quantification of vascular outgrowth showing significantly impaired retinal angiogenesis in Col4a1^+/G1344D mice but not their 4PBA treated counterparts when compared to their respective controls. From left to right, n= 15, 17, 16, and 10 retinas. (C-F) P7 radial arterioles immunolabeled for CD31 (green) and NG2 (magenta) and quantification showing increased numbers of arteriolar mural cells (white arrows in C and E) in Col4a1^+/G1344D (C-D) and Col4a1^+/G394V (E-F) mice that are reduced by 4PBA. n= 12 mice per group. Data are presented as mean±SD, *p<0.05; **p<0.01; ****p<0.0001, two-way ANOVA. Scale bars: 500μm (A), and 100μm (C and E). NT, no treatment.

Figure 2.. Col4a1 mutant mice have abnormal retinal vascular patterning.

(A) Retinal radial arterioles and (B) whole retinas immunolabeled for calponin and (C) quantification showing increased intensity of arteriolar calponin labeling, (D) peripheral extension of calponin labeling along radial arterioles and (E) increased number of calponin⁺ primary arterioles (white arrows in B) in 1MO Col4a1^+/G1344D mice that are not prevented by 4PBA treatment. From left to right, n= 10, 12, 6, and 9 retinas. (F) Retinal radial arterioles immunolabeled for αSMA and (G) retinas immunolabeled for CD31 and αSMA and (H) quantification showing increased arteriolar αSMA labeling intensity, (I) increased numbers of primary arterioles and (J) reduced complexity of precapillary arteriolar arborization in 1MO Col4a1^+/G1344D mice. 4PBA reduced αSMA labeling intensity in Col4a1^+/+ and Col4a1^+/G1344D retinas and improved precapillary arteriolar arborization in Col4a1^+/G1344D retinas. From left to right, n= 10, 9, 6, and 9 retinas. Data are presented as mean±SD, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, two-way ANOVA. Scale bars: 100μm (A and B) and 50μm (F and G).

Figure 3.. Col4a1 mutant mice have elevated TGFβ signaling that is reduced by 4PBA.

(A) qPCR analyses showing increased expression of TGFβ target genes in P7 Col4a1^+/G1344D brains. n= 8 brains per group. (B) Representative images and (C) quantification of in vivo bioluminescence in P7 mice carrying the SBE-luciferase reporter gene showing elevated TGFβ signaling in Col4a1^+/G1344D mice that is reduced by 4PBA. From left to right n= 12, 8, 6, and 8 mice. (D) qPCR analyses showing increased expression of TGFβ target genes in 1MO Col4a1^+/G1344D brains that is generally reduced by 4PBA. From left to right, n= 6, 7, 5, and 6 brains. Data are presented as mean±SD, *p<0.05; **p<0.01; ****p<0.0001, two-way ANOVA.

Figure 4.. Genetically reducing TGFβ signaling partially ameliorates developmental retinal vascular defects in Col4a1 mutant mice.

(A) qPCR analyses of P7 and (B) P30 brains showing that Tgfb1 heterozygosity decreased TGFβ target gene expression in brains from Col4a1^+/+ and Col4a1^+/G1344D mice. From left to right, n= 6, 5, 9, and 4 mice (A), and n= 5, 7, 5, and 6 mice (B). (C) P7 retinas immunolabeled for CD31 and (D) quantification of retinal vascular outgrowth showing that Tgfb1 heterozygosity does not prevent retinal angiogenesis defects in Col4a1^+/G1344D mice. From left to right n= 14, 8, 10, and 8 retinas. (E) P7 retinal arterioles immunolabeled for CD31 and NG2 and (F) quantification showing that Tgfb1 heterozygosity normalizes the number of arteriolar mural cells (white arrows in C) in Col4a1^+/G1344D retinas. n= 13 retinas per group. Data are presented as mean±SD, **p<0.01; ****p<0.0001, two-way ANOVA. Scale bars: 500μm (C) and 100μm (E).

Figure 5.. Genetically reducing TGFβ signaling partially restores vascular patterning in Col4a1 mutant retinas.

(A) Retinal radial arterioles and (B) whole retina immunolabeled for calponin and (C-E) quantification showing that Tgfb1 heterozygosity does not change the intensity (C) or pattern of calponin labeling (D-E) in 1MO Col4a1^+/G1344D mice. White arrows in B indicate calponin⁺ primary arterioles. From left to right, n= 8, 11, 12, and 10 retinas. (F) Radial arterioles immunolabeled for αSMA and (G) radial arterioles immunolabeled for CD31 and αSMA and (H-J) quantification showing that Tgfb1 heterozygosity improves precapillary arteriolar arborization (J) but not arteriolar αSMA labeling intensity (H) or number of primary arterioles (I) in 1MO Col4a1^+/G1344D mice. From left to right, n= 12, 13, 12, and 11 retinas (H) and n= 12, 13, 12, and 15 retinas (I-J). Data are presented as mean±SD, *p<0.05; **p<0.01, two-way ANOVA. Scale bars: 100μm (A, B, F) and 50μm (G).

Figure 6.. Tgfb1 heterozygosity reduces ICH severity in Col4a1 mutant mice.

(A-F) Prussian blue-stained brain sections and quantification of brain hemosiderin showing that Tgfb1 heterozygosity reduces ICH severity in P7 (A-B) and 1MO (C-D) Col4a1^+/G1344D mice but not in exercised 3MO Col4a1^+/G1344D mice (E-F). From left to right, n= 12, 12, 11, and 11 mice (B), n= 12, 15, 10, and 12 mice (D), and n=12, 11, 12, 13 mice (F). Data are presented as mean±SD, *p<0.05; **p<0.01; ***p<0.001, two-way ANOVA.

Figure 7.. Tgfb1 heterozygosity prevents age-related vascular defects in Col4a1 mutant mice.

(A) Prussian blue-stained brain sections from P7, 1MO and 8MO Col4a1^+/G1344D mice and quantification of hemosiderin showing an age-dependent shift in ICH distribution. C, cortical; and SC, subcortical. n= 12 mice per group. (B) 1MO, 3MO and 8MO radial arterioles immunolabeled for αSMA and quantification showing (C) a progressive loss of VSMC coverage and (D) increased arteriolar diameter in Col4a1^+/G1344D mice. Notably, Tgfb1 heterozygosity prevented VSMC loss in Col4a1^+/G1344D mice and reduced arteriolar diameter in both Col4a1^+/+ and Col4a1^+/G1344D mice at 1MO. n=10 mice per group. (E) Prussian blue-stained brain sections from 8MO mice and (F) quantification of hemosiderin showing that ICH severity in Col4a1^+/G1344D is significantly reduced by Tgfb1 heterozygosity. From left to right, n= 9, 12, 11, and 10 mice. Data are presented as mean±SD, *p<0.05; **p<0.01, ***p<0.001; ****p<0.0001, two-way ANOVA. Scale bar: 100μm.