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. 2023 May 26;9(21):eade7280.
doi: 10.1126/sciadv.ade7280. Epub 2023 May 26.

Nitric oxide synthase and reduced arterial tone contribute to arteriovenous malformation

Lawrence Huang  1 , Feng Cheng  1 , Xuetao Zhang  1 , Jacek Zielonka  2 , Matthew A Nystoriak  3 , Weiwei Xiang  1 , Kunal Raygor  1 , Shaoxun Wang  1 , Aditya Lakshmanan  1 , Weiya Jiang  1 , Sai Yuan  1 , Kevin S Hou  1 , Jiayi Zhang  1 , Xitao Wang  1 , Arsalan U Syed  3 , Matea Juric  2 , Takamune Takahashi  4 , Manuel F Navedo  3 , Rong A Wang  1
Affiliations

Nitric oxide synthase and reduced arterial tone contribute to arteriovenous malformation

Lawrence Huang et al. Sci Adv. .

Abstract

Mechanisms underlying arteriovenous malformations (AVMs) are poorly understood. Using mice with endothelial cell (EC) expression of constitutively active Notch4 (Notch4*EC), we show decreased arteriolar tone in vivo during brain AVM initiation. Reduced vascular tone is a primary effect of Notch4*EC, as isolated pial arteries from asymptomatic mice exhibited reduced pressure-induced arterial tone ex vivo. The nitric oxide (NO) synthase (NOS) inhibitor NG-nitro-l-arginine (L-NNA) corrected vascular tone defects in both assays. L-NNA treatment or endothelial NOS (eNOS) gene deletion, either globally or specifically in ECs, attenuated AVM initiation, assessed by decreased AVM diameter and delayed time to moribund. Administering nitroxide antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl also attenuated AVM initiation. Increased NOS-dependent production of hydrogen peroxide, but not NO, superoxide, or peroxynitrite was detected in isolated Notch4*EC brain vessels during AVM initiation. Our data suggest that eNOS is involved in Notch4*EC-mediated AVM formation by up-regulating hydrogen peroxide and reducing vascular tone, thereby permitting AVM initiation and progression.

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Figures

Fig. 1.
Fig. 1.. Impaired in vivo cerebral vascular tone in mice expressing Notch4* in ECs.
(A) Representative images of middle cerebral and anterior cerebral arteries and branches in live postnatal day 16 (P16) Tie2-tTA and Tie2-tTA;TRE-Notch4* mice visualized through an open cranial window. Scale bars, 150 μm. aCSF, artificial cerebrospinal fluid. (B) Arterial and arteriolar tone in the absence (−) and presence (+) of NG-nitro-l-arginine (L-NNA) treatment (30 mg/kg) (percentage change from maximum passive diameter). (In the absence of L-NNA, artery: Tie2-tTA, n = 24 mice; Tie2-tTA;TRE-Notch4*, n = 27 mice; P = 0.1385, t test; arteriole: Tie2-tTA, n = 27 mice; Tie2-tTA;TRE-Notch4*, n = 29 mice; P = 0.001, t test.) (In the presence of L-NNA, artery: Tie2-tTA, n = 10 mice; Tie2-tTA;TRE-Notch4*, n = 10 mice; P = 0.0936, t test; arteriole: Tie2-tTA, n = 10 mice; Tie2-tTA;TRE-Notch4*, n = 10 mice; P = 0.913, t test.) (C) Arteriolar tone by edge detection from line profiles (Tie2-tTA, n = 17 mice; Tie2-tTA;TRE-Notch4*, n = 19 mice; P = 0.02, t test).
Fig. 2.
Fig. 2.. Impaired ex vivo cerebral vascular tone in mice expressing Notch4* in ECs.
(A) Representative diameter recordings from pressurized Tie2-tTA and Tie2-tTA;TRE-Notch4* cerebral arteries over a range of intravascular pressures. Solid and dashed lines indicate active and passive diameters, respectively. (B) Arterial tone (percentage change from maximum passive diameter) measured in isolated cerebral arteries over a range of intravascular pressures. (n = 8 arteries from seven mice per group; Tie2-tTA versus Tie2-tTA;TRE-Notch4* at 20 mmHg, P = 0.4783; 40 mmHg, P = 0.0667; 60 mmHg, P = 0.0121; 80 and 100 mmHg, P < 0.0001, t test). (C) Representative diameter recordings from pressurized (60 mmHg) cerebral arteries in the absence and presence of 50 μM L-NNA. The maximum passive diameter was recorded at the end of the experiment in Ca2+-free perfusate containing nifedipine (nifed; 1 μM) and forskolin (fsk; 0.5 μμ). (D) L-NNA–induced constriction (percentage change in diameter normalized to maximum passive diameter for vessels shown in (D). (n = 5 arteries from four mice per group; P = 0.0431, t test.) (E) Arterial tone at 60 mmHg of intravascular pressure in the absence (−) and presence (+) of 50 μM L-NNA (n = 5 arteries from four mice per group; Tie2-tTA versus Tie2-tTA;TRE-Notch4* in the absence of L-NNA, P = 0.0085; in the presence of L-NNA, P = 0.3763, t test). Error bars, SEM.
Fig. 3.
Fig. 3.. L-NNA decreased AVM diameter in brains of mice expressing Notch4* in ECs.
(A) Lectin-perfused vasculature on P24 surface cerebral cortex of Tie2-tTA and Tie2-tTA;TRE-Notch4* mice treated with saline or L-NNA (a, artery; v, vein; dashed lines, example AV connections; arrowheads, example points of measurement for AV connection diameter; scale bars, 100 μm). (B) Arteriovenous (AV) connection diameter (Tie2-tTA + saline, n = 7 mice; Tie2-tTA + L-NNA, n = 9 mice; Tie2-tTA;TRE-Notch4* + saline, n = 6 mice; Tie2-tTA;TRE-Notch4* + L-NNA, n = 6 mice; P = 0.0002, t test). (C) Fraction of AV connections with a diameter of ≥30 μm over total AV connections quantified [same n numbers as in (B); P < 0.0001, t test]. (D) Lectin-perfused vasculature on P12 surface cerebral cortex of Tie2-tTA and Tie2-tTA;TRE-Notch4* mice treated with saline or L-NNA (dashed lines, example AV connections; arrowheads, example points of measurement for AV connection diameter; scale bars, 100 μm). (E) AV connection diameter [Tie2-tTA + saline, n = 6 mice; Tie2-tTA + L-NNA, n = 7 mice; Tie2-tTA;TRE-Notch4* + saline, n = 4 mice; Tie2-tTA;TRE-Notch4* + L-NNA, n = 5 mice; Tie2-tTA;TRE-Notch4* + L-NNA versus Tie2-tTA;TRE-Notch4* + saline, Padj < 0.0001; Tie2-tTA;TRE-Notch4* + L-NNA versus Tie2-tTA + saline, Padj = 0.2631; Tie2-tTA;TRE-Notch4* + L-NNA versus Tie2-tTA + L-NNA, Padj = 0.3582; Tukey’s multiple comparisons test following a two-way analysis of variance (ANOVA) to analyze the effects of Notch4*EC and L-NNA]. (F) Fraction of AV connections with a diameter of ≥12.5 μm over total AV connections quantified [same n numbers as in (E); Tie2-tTA;TRE-Notch4* + L-NNA versus Tie2-tTA;TRE-Notch4* + saline, Padj < 0.0001; Tie2-tTA;TRE-Notch4* + L-NNA versus Tie2-tTA + saline, Padj = 0.5605; Tie2-tTA;TRE-Notch4* + L-NNA versus Tie2-tTA + L-NNA, Padj = 0.5336; Tukey’s multiple comparisons test following a two-way ANOVA to analyze the effects of Notch4*EC and L-NNA]. Error bars, SEM.
Fig. 4.
Fig. 4.. L-NNA treatment delayed illness progression in mice expressing Notch4* in ECs.
(A) Kaplan-Meier analysis of Tie2-tTA;TRE-Notch4* mice treated with saline (n = 34 mice) or L-NNA (n = 18 mice), with monitoring starting on P12. Blue indicates median moribund age (P < 0.0001, log-rank/Mantel-Cox test). (B) Heart-to-body weight ratio at P24 (Tie2-tTA + saline, n = 3 mice; Tie2-tTA + L-NNA, n = 3 mice; Tie2-tTA;TRE-Notch4* + saline, n = 7 mice; Tie2-tTA;TRE-Notch4* + L-NNA, n = 4 mice; P = 0.002, t test.). Error bars, SEM.
Fig. 5.
Fig. 5.. eNOS deletion decreased AVM diameter in brains of mice expressing Notch4* in ECs.
(A) Vasculature on P24 surface cerebral cortex of Tie2-tTA and Tie2-tTA;TRE-Notch4* mice without (eNOS+/+) and with (eNOS−/−) endothelial nitric oxide synthase (eNOS) deletion. (B) AV connection diameter (Tie2-tTA;TRE-Notch4*;eNOS+/+, n = 9 mice; Tie2-tTA;TRE-Notch4*;eNOS−/−, n = 12 mice; P = 0.0114, t test). (C) Fraction of AV connections with a diameter of ≥30 μm over total AV connections quantified [same n numbers as in (B); P = 0.0044, t test]. For (B) and (C), n = 3 mice per group for Tie2-tTA;eNOS+/+ and Tie2-tTA;eNOS−/−. (D) Kaplan-Meier analysis of Tie2-tTA;TRE-Notch4* mice without (eNOS+/+) and with (eNOS−/−) eNOS deletion (Tie2-tTA;TRE-Notch4*;eNOS+/+, n = 17 mice; Tie2-tTA;TRE-Notch4*;eNOS−/−, n = 24 mice; P = 0.0024, log-rank/Mantel-Cox test). (E) Heart-to-body weight ratio at P24 (Tie2-tTA;TRE-Notch4*;eNOS+/+, n = 8 mice; Tie2-tTA;TRE-Notch4*;eNOS−/−, n = 10 mice; P = 0.0062, t test; Tie2-tTA;eNOS+/+, n = 4 mice; Tie2-tTA;eNOS−/−, n = 5 mice). (F) Vasculature on P24 surface cerebral cortex of Tie2-tTA and Tie2-tTA;TRE-Notch4* mice without and with endothelial-specific deletion of eNOS. (G) AV connection diameter [Tie2-tTA;TRE-Notch4*;eNOSf/f, n = 6 mice; Tie2-tTA;TRE-Notch4*;Cdh5(PAC)-CreERT2;eNOSf/f, n = 7 mice; P = 0.0004, t test; n = 6 mice per group for Tie2-tTA;eNOSf/f and Tie2-tTA;Cdh5(PAC)-CreERT2;eNOSf/f]. (H) Kaplan-Meier analysis of Tie2-tTA;TRE-Notch4* mice without and with endothelial-specific eNOS deletion [Tie2-tTA;TRE-Notch4*;eNOSf/f, n = 6 mice; Tie2-tTA;TRE-Notch4*;Cdh5(PAC)-CreERT2;eNOSf/f, n = 9 mice; P = 0.017, log-rank/Mantel-Cox test]. (I) Heart-to-body weight ratio at P24 [Tie2-tTA;TRE-Notch4*; Cdh5(PAC)-CreERT2;eNOSf/f, n = 6 mice; Tie2-tTA;TRE-Notch4*;eNOSf/f, n = 5 mice; P = 0.0245, t test; Tie2-tTA;eNOSf/f, n = 6 mice; Tie2-tTA; Cdh5(PAC)-CreERT2;eNOSf/f, n = 6 mice]. Dashed lines, example AV connections; arrowheads, example points of measurement for AV connection diameter; scale bars, 100 μm. For Kaplan-Meier analyses, monitoring started on P12, and blue indicates median moribund age. Error bars, SEM.
Fig. 6.
Fig. 6.. Endothelial Notch4* expression did not result in a significant change in cGMP production in aortae, DAF staining in cerebral vasculature, or nitrite/nitrate levels in isolated cerebral vessels.
(A) Acetylcholine (ACh)–stimulated cyclic guanosine monophosphate (cGMP) production in aorta (P12: Tie2-tTA, n = 8 mice; Tie2-tTA;TRE-Notch4*, n = 8 mice; P = 0.8248, t test; 8 weeks: Tie2-tTA, n = 7 mice; Tie2-tTA;TRE-Notch4*, n = 8 mice; P = 0.3754, t test). (B) 4-Amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) staining and lectin-perfused vasculature on P12 surface cerebral cortex of Tie2-tTA and Tie2-tTA;TRE-Notch4*, with or without L-NNA treatment. Scale bars, 100 μm. (C) Quantification of L-NNA sensitive DAF-FM fluorescence intensity by arterial diameter (A.U., arbitrary units; Tie2-tTA, n = 6 mice; Tie2-tTA;TRE-Notch4*, n = 6 mice; > 60 μm, P = 0.389; 35 to 55 μm, P = 0.4138; <30 μm, P = 0.5951, t test.) (D) Nitrite/nitrate level in isolated brain blood vessels at P12 (Tie2-tTA, n = 8 mice; Tie2-tA;TRE-Notch4*, n = 9 mice; P = 0.7562, t test) and P16 (n = 6 for both groups; P = 0.3785, t test). Error bars, SEM.
Fig. 7.
Fig. 7.. Tempol treatment delayed illness progression and decreased brain AVM diameter in moribund mice expressing Notch4* in ECs.
(A) Kaplan-Meier analysis of Tie2-tTA;TRE-Notch4* mice treated with vehicle (n = 55 mice) or Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; n = 26 mice), with monitoring starting on P12. Blue indicates median moribund age (P < 0.0001, log-rank/Mantel-Cox test). (B) Lectin-perfused vasculature on surface cerebral cortex of moribund Tie2-tTA;TRE-Notch4* mice and age-matched Tie2-tTA mice treated with vehicle or Tempol (dashed lines, example AV connections; arrowheads, example points of measurement for AV connection diameter; scale bars, 100 μm). (C) AV connection diameter (Tie2-tTA + vehicle, n = 6 mice; Tie2-tTA + Tempol, n = 7 mice; Tie2-tTA;TRE-Notch4* + vehicle, n = 10 mice; Tie2-tTA;TRE-Notch4* + Tempol, n = 10 mice; P = 0.0043, t test). (D) Fraction of AV connections with a diameter of ≥12.5 μm over total AV connections quantified [same n numbers as in (C); P = 0.0012, t test]. (E) Age at which Tie2-tTA;TRE-Notch4* mice were moribund. Error bars, SEM.
Fig. 8.
Fig. 8.. Increased hydroperoxide and NT but not peroxynitrite levels in isolated cerebral vessels of mice expressing Notch4* in ECs.
(A) Hydrogen peroxide product o-MitoPhOH in isolated brain blood vessels at P16 [Tie2-tTA + vehicle, n = 13 mice; Tie2-tTA + NG-nitro arginine-methyl ester (L-NAME), n = 10 mice; Tie2-tA;TRE-Notch4* + vehicle, n = 13 mice; Tie2-tA;TRE-Notch4* + L-NAME, n = 11 mice; Tie2-tTA + vehicle versus Tie2-tTA;TRE-Notch4* + vehicle, Padj = 0.0122; Tie2-tTA + vehicle versus Tie2-tTA + L-NAME, Padj = 0.3899; Tie2-tTA;TRE-Notch4* + vehicle versus Tie2-tTA;TRE-Notch4* + L-NAME, Padj = 0.0116; Tukey’s multiple comparisons test following a two-way ANOVA to analyze the effects of Notch4*EC and L-NAME]. (B) Peroxynitrite-specific product cyclo-o-MitoPh in isolated brain blood vessels at P16. Cyclo-o-MitoPh was below the detection limit for Tie2-tTA and Tie2-tTA;TRE-Notch4* blood vessels. Tie2-tTA blood vessels treated with SIN-1 served as a positive control for peroxynitrite. (C) Expression of 3-nitrotyrosine (3-NT) in brain blood vessels at P16. Scale bars, 100 μm. (D) Fluorescence intensity of 3-NT normalized to that of CD31 (Tie2-tTA, n = 6 mice; Tie2-tA;TRE-Notch4*, n = 8 mice; artery, P = 0.0305; vein, P = 0.0051; capillary, P = 0.0091, t test). Error bars, SEM.
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