Mouse and human strategies identify PTPN14 as a modifier of angiogenesis and hereditary haemorrhagic telangiectasia

Abstract

Hereditary haemorrhagic telangiectasia (HHT) [corrected] is a vascular dysplasia syndrome caused by mutations in transforming growth factor-β/bone morphogenetic protein pathway genes, ENG and ACVRL1. HHT [corrected] shows considerable variation in clinical manifestations, suggesting environmental and/or genetic modifier effects. Strain-specific penetrance of the vascular phenotypes of Eng(+/-) and Tgfb1(-/-) mice provides further support for genetic modification of transforming growth factor-β pathway deficits. We previously identified variant genomic loci, including Tgfbm2, which suppress prenatal vascular lethality of Tgfb1(-/-) mice. Here we show that human polymorphic variants of PTPN14 within the orthologous TGFBM2 locus influence clinical severity of HHT, [corrected] as assessed by development of pulmonary arteriovenous malformation. We also show that PTPN14, ACVRL1 and EFNB2, encoding EphrinB2, show interdependent expression in primary arterial endothelial cells in vitro. This suggests an involvement of PTPN14 in angiogenesis and/or arteriovenous fate, acting via EphrinB2 and ACVRL1/activin receptor-like kinase 1. These findings contribute to a deeper understanding of the molecular pathology of HHT [corrected] in particular and to angiogenesis in general.

Figure 1. Fine mapping and functional validation of Tgfbm2 in congenic mice

(a) F1.C57/129.Tgfb1+/− mice were backcrossed >10 generations into inbred C57, at each generation selecting for both the Tgfb1^tm1N allele and D1Mit362¹²⁹, using genetic markers. When mice were >99.9% C57, except for a 26Mb 129 interval at the telomeric end of chromosome 1, littermates were used to generate C57. Tgfb1+/− congenics that were either homozygous C57 at (Tgfbm2^C57/C57) or homozygous 129 (Tgfbm2^129/129) at Tgfbm2. Tgfb1+/− intercrosses within the indicated mouse strains were assayed for viable Tgfb1−/− pups as a percentage of wild type neonates. Whereas Tgfb1−/− mice on a C57 (n>400) or congenic C57.Tgfbm2^C57/C57 (n=84) background always die in utero from vascular dysgenesis, Tgfbm2^129/129 (n=139) rescued C57.Tgfb1−/− mice from invariable prenatal lethality (p=0.02; χ2 test), almost to the extent observed on a pure 129 genetic background (n > 100, and see earlier publication). (b) Tgfb1+/− mice backcrossed > 20 generations to NIH selecting only for the Tgfb1^tm1N allele were unexpectedly found to harbor 129 genomic variants at Tgfbm2. These mice were segregated by Tgfbm2 genotype and assayed for frequency of viable Tgfb1−/− pups born on a NIH.Tgfbm2^129/129 background, compared to that on a NIH.Tgfbm2 ^NIH/NIH background. A similar comparative intercross analysis was undertaken on a panel of NIH.Tgfbm3 ^C57/C57 mice (lines 3, 4, 6 and 8; n > 100 mice for each genotype) that are sensitized to Tgfb1−/− prenatal lethality, showing a statistically significant association between pup viability and Tgfbm2^129/129 (p = 0.0002; logistic regression). All data in (b) are normalized relative to pup viability of the partner mouse strain homozygous for the NIH allele of Tgfbm2^NIH/NIH (light gray bars). Dark gray bar shows data for NIH mice heterozygous for Tgfbm2^129/NIH. Black bars show data for mice homozygous 129 for Tgfbm2^129/129.

Figure 2. Genomic structure of murine Tgfbm2 and human TGFBM2.

(a) The murine Tgfbm2 locus, indicating the position of the coselected 1Mb Tgfbm2¹²⁹ minimal interval (boxed region) spanning from exons 57–60 of Ush2a to exons 2–6 of Ptpn14 (Supplementary Data S1). Also indicated are 129S2Sv, C57 and “variant 129” haplotypes within this minimal Tgfbm2 interval, together with the positions of the peak genetic linkage from the original genetic mapping of this locus.(b) Human 1q41 region syntenic to Tgfbm2, indicating position of PTPN14 SNP rs2936018 and USH2A SNP rs700024.

Figure 3. SNPs within PTPN14 show genetic association with presence of PAVM in Dutch HHT patients

(a) 534 gene-centric SNPs tagging the five syntenic genes of TGFBM2 at 1q41,plus 72 TGFβ/BMP signalling “candidate genes”, were screened in 721 HHT patients and family members. Graph shows the distribution of −LOG₁₀ (p) values resulting from GC analysis of genetic association to PAVM in pooled HHT1 and HHT2. (b) The genomic map distribution of −LOG₁₀ (p) values at 1q41 for 76 gene-tagging SNPs across the TGFBM2 across. Black data points represent initial Dutch SNP screen; Red data points represent cumulative −LOG₁₀ (p) values for combined initial and extension studies of Dutch patient samples.

Figure 4. siPTPN14 stimulates in vitro angiogenesis

si-RNA KD of PTPN14 was optimized in HMEC-1 cells (a) and HUAECs (not shown) by Western analysis. (b, c) siRNA-treated HUAECs were cultured for 6 days in a 3D in vitro angiogenesis assay. (b) Phase contrast micrographs of siControl or siPTPN14-treated HUAEC at 6 days. Scale bar represents 150μM. (c) Vascular sprouts longer than one bead diameter (150μM), sprouts with and without lumens, and tip cells, were enumerated by morphology under phase contrast microsopy. Tip cell number is a surrogate for branching, and lumen formation a parameter of advanced angiogenesis. Graph shows quantification of the ratios of sprouts per bead, tip cells per bead and lumens per sprout, after treatment with siControl (black) or siPTPN14 (white). The analysis was performed blinded to sample identity. At least 25 beads in three different wells were analysed for each experiment. Two biological replicates were undertaken. Mean and SD are shown. * p<0.01, Students t-test.

Figure 5. ACVRL and EFNB2 show interdependent expression with PTPN14 in vitro

(a–f) HUAECs were treated with indicated siRNAs, and cultured for 2 days. Quantitative RT-PCR (a, c, e) and Western blot analysis (b, d, f) were then performed for EFNB2/EphrinB2 (a–d) and PTPN14 (e, f). (g) Cartoon summary of possible interactions implied by data in (a–f); PTPN14 down-regulation by ACVRL1 may be direct, or indirectly mediated via elevated EFNB2 levels. Data in panels a, c and e are representative of at least three independent biological replicates. Data in panels b, d and f, are representative of two independent biological replicates. Moreover, data in panels b, d and f, are from additional experiments, independent from those shown in a, c and e. Mean and SD are shown. *** p< 0.0001, ** p<0.001, * p<0.01, Students t-test.