Col4a1 mutation in mice causes defects in vascular function and low blood pressure associated with reduced red blood cell volume

Abstract

Collagen type IV is the major structural component of the basement membrane and COL4A1 mutations cause adult small vessel disease, familial porencephaly and hereditary angiopathy with nephropathy aneurysm and cramps (HANAC) syndrome. Here, we show that animals with a Col4a1 missense mutation (Col4a1(+/Raw)) display focal detachment of the endothelium from the media and age-dependent defects in vascular function including a reduced response to nor-epinephrine. Age-dependent hypersensitivity to acetylcholine is abolished by inhibition of nitric oxide synthase (NOS) activity, indicating that Col4a1 mutations affect vasorelaxation mediated by endothelium-derived nitric oxide (NO). These defects are associated with a reduction in basal NOS activity and the development of heightened NO sensitivity of the smooth muscle. The vascular function defects are physiologically relevant as they maintain in part the hypotension in mutant animals, which is primarily associated with a reduced red blood cell volume due to a reduction in red blood cell number, rather than defects in kidney function. To understand the molecular mechanism underlying these vascular defects, we examined the deposition of collagen type IV in the basement membrane, and found it to be defective. Interestingly, this mutation also leads to activation of the unfolded protein response. In summary, our results indicate that mutations in COL4A1 result in a complex vascular phenotype encompassing defects in maintenance of vascular tone, endothelial cell function and blood pressure regulation.

Figure 1

Vascular phenotypes of Col4a1^+/Raw mice. (A,B) Mild and more severe cerebral haemorrhaging in Col4a1^+/Raw pups (arrow). (C) Predicted (50%) and observed (19.8%) percentage of Col4a1^+/Raw animals (31/156) (black) at weaning (χ² = 56.6, one degree of freedom, p<0.0001). (D) Lack of gross histological phenotype in mutant aortae (E) Normal total collagen and elastin (F) deposition in mutant animals. (Data shown for 6 month old animals, see supplemental data for 3 and 9 month old animals). (G) Focal endothelial detachment in aortae from Col4a1^+/Raw animals (arrow). (H) Higher magnification of EM analysis. Size 1μm (g,h), 10μm (d-f).

Figure 2

Assessment of vascular function. (A) Mutant animals (black bars) have reduced amounts of contraction (mN) in K-PSS (p=0.0012 two way Anova, n=6 for 3 and 6 month old animals, n=10 for 11-15 month old). (B) Reduced contraction to Nor-Epinephrine in 6 month old mutant animals (n=6) (two way Anova p<0.001, 6 months). (C) 11-15 Month old mutant animals display increased vasodilation to low concentrations of ACh (two way Anova, n=10, p<0.0001). Incubation of vessels in LNAME (labelled +L-NAME) before administration of ACh abolishes vasodilation. (D) Vasodilation to SNP in 11-15 month old mutant animals shows increased relaxation (two way Anova, n=10, p<0.0001). (E) Vasodilation to Iso-Prenaline in 11-15 month old animals (n=6). WT (open circles), Col4a1^+/Raw (filled circles). Error bars indicate standard error of measurement. * p< 0.05, ** p< 0.01, *** p<0.001 post hoc test

Figure 3

Reduced basal NOS function in Col4a1^+/Raw. (A) Contraction obtained by administration of L-NAME as measure of amount of NO in 11-15 month animals expressed as percentage of contraction obtained with KCl. (n= 14 WT, n=11 Col4a1^+/Raw, t-test p <0.05). (B) qRT-PCR for PRKG1 (cGMP dependant protein kinase I). Expressed as relative units to 18S RNA levels. (n=6) (C). eNOS mRNA levels in 12 month old WT (white) and Col4a1^+/Raw (black) (n=6) 3 animals as determined by qRT-PCR relative to 18S RNA levels (p = 0.17 t-test). (D) eNOS, and sGC protein expression levels in aortae of 11-15 month old animals. Predominant band of coomassie gel is given as loading control (entire gel is provided in Supplemental Fig. 3). Error bars indicate standard error of measurement. * p< 0.05 t-test.

Figure 4

Collagen type IV and blood pressure regulation. (A) Systolic blood pressure of 4-6 months old animals. (Col4a1^+/Raw 99 mm Hg (n=6), WT 124 mm Hg (n=5), p<0.001 t-test) (B) Systolic blood pressure of 14 month old animals. (Col4a1^+/Raw 85.1mm Hg, WT 108.1mm Hg, n=6, p< 0.001, t-test). (C) Mean arterial blood pressure of 40 day old animals (Col4a1^+/Raw 70 mm Hg (n=5), WT 91 mm Hg (n=7), p<0.01, t-test). (D) Systolic blood pressure of 14 month old animals before and during treatment with L-NAME. Measurements were collected at day 0, day 7 and day 14 of treatment. (WT, n=9, Col4a1^+/Raw n=7) * p< 0.05, ** p< 0.01, *** p<0.001 t- test

Figure 5

Low blood pressure is associated with reduced blood volume. (A) Sodium excretion in 40 day old animals (Col4a1^+/Raw 0.05 μmol/min (n=5), WT 0.2 μmol/min (n=7), p<0.05 t-test) (B) Plasma aldosterone levels in 40 days old animals. (Col4a1^+/Raw 1044 nmol/L (n=5), WT 476 nmol/L (n=7), p<0.01 t-test) (C) Blood volume in animals (Col4a1^+/Raw 88.9 μl/g body weight (n=10), WT 120 μl/g body weight (n=13), p<0.01 t-test). (D) Plasma volume of animals. (E) Reduced hematocrit in mutant animals (Col4a1^+/Raw 38% (n=15), WT 45% (n=19), p<0.001 t-test). (F) Reduced red blood cell number in Col4a1^+/Raw animals. (Col4a1^+/Raw 8.55 × 10⁶ RBC/μl (n=10), WT 9.50 × 10⁶ RBC/μl (n=12), p<0.01 t-test). Error bars indicate standard error of measurement. * p< 0.05, ** p< 0.01, *** p<0.001 t- test

Figure 6

Col4a1^+/Raw mutation results in UPR activation. (A) Interrupted staining for Col4a2 in vascular BM of descending aortae from mutant animals in contrast to continuous laminin (LM) staining. (arrow indicate presence of Col4a2 in BM. Arrowhead indicate absence of Col4a2 as indicated by green staining in merged image) Size 20 μm. Detail (white square) is provided below panel A. (B) Increased Bip mRNA expression levels in mutant animals. Expressed as relative units to 18S RNA levels (p< 0.05 t-test, n=9).