Oxidative stress contributes to endothelial dysfunction in mouse models of hereditary hemorrhagic telangiectasia

Abstract

Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia caused by mutations in endoglin (ENG; HHT1) or activin receptor-like kinase (ALK1; HHT2) genes, coding for transforming growth factor-β (TGF-β) superfamily receptors. We demonstrated previously that endoglin and ALK1 interact with endothelial NO synthase (eNOS) and affect its activation. Endothelial cells deficient in endoglin or ALK1 proteins show eNOS uncoupling, reduced NO, and increased reactive oxygen species (ROS) production. In this study, we measured NO and H(2)O(2) levels in several organs of adult Eng and Alk1 heterozygous mice, to ascertain whether decreased NO and increased ROS production is a generalized manifestation of HHT. A significant reduction in NO and increase in ROS production were found in several organs, known to be affected in patients. ROS overproduction in mutant mice was attributed to eNOS, as it was L-NAME inhibitable. Mitochondrial ROS contribution, blocked by antimycin, was highest in liver while NADPH oxidase, inhibited by apocynin, was a major source of ROS in the other tissues. However, there was no difference in antimycin- and apocynin-inhibitable ROS production between mutant and control mice. Our results indicate that eNOS-derived ROS contributes to endothelial dysfunction and likely predisposes to disease manifestations in several organs of HHT patients.

Figure 1

Decreased NO production in tissues of Eng and Alk1 heterozygous mice. NO levels were measured using a microsensor in the presence or absence of the NOS inhibitor L-NAME, 1 mM. NO levels were significantly reduced in heart, lungs, and liver of (a) Eng ^+/− and (b) Alk1 ^+/− mice compared to their control littermates. Nitric oxide production was inhibited by L-NAME in all tissues of control mice but not in tissues of (a) Eng ^+/− or (b) Alk1 ^+/− mice. *P < 0.05, and ***P < 0.001 versus +/+ untreated mice; N = 6–8/group for Eng mice and 6–10/group for Alk1 mice.

Figure 2

ROS production is increased in tissues of mutant mice. H₂O₂ production was measured by Amplex red, to estimate tissue ROS production. H₂O₂ generation was increased in pulmonary, hepatic, and colonic tissues of (a) Eng ^+/− and (b) Alk1 ^+/− mice compared to those of corresponding wild-type mice. Cardiac ROS production was similar between mutant and control mice. *P < 0.05, **P < 0.01, and ***P < 0.001 versus +/+ mice; N = 10–15/group for Eng mice and 7–20/group for Alk1 mice.

Figure 3

ROS production is inhibited by L-NAME in tissues of mutant mice. H₂O₂ production was measured by Amplex red with and without the NOS inhibitor L-NAME. Results are expressed as a percentage of the respective control values (without inhibitors). L-NAME significantly inhibited H₂O₂ production in pulmonary, hepatic, and colonic tissues of (a) Eng ^+/− and (b) Alk1 ^+/− mice but had no effect on tissues of control mice. In cardiac tissue, L-NAME effect was not significantly different between mutant and control mice. *P < 0.05, **P < 0.01, and ^† P < 0.1 versus samples from corresponding control values; N = 6–13/group for both Eng and Alk1 mice.

Figure 4

Mitochondrial and NADPH-dependent ROS production does not differ between mutant and wild-type mice. Results are expressed as a percentage of the respective control values in the absence of inhibitor. (a) and (b) Antimycin, 50 μM, inhibited mitochondrial ROS production to the same level in tissues of both Eng ^+/−, Alk1 ^+/− and respective wild-type mice. N = 5–11/group for Eng mice and 5–15/group for Alk1 mice, except for liver samples from Alk1 ^+/+ mice, where N = 4. (c) and (d) Apocynin, 100 μM, inhibited NADPH-oxidase dependent H₂O₂ production in tissues of both mutant and wild-type mice. N = 5–8/group for both Eng and Alk1 mice, except for heart samples from both groups of mice, where N = 4. *P < 0.05, **P < 0.01, ***P < 0.001, and ^† P < 0.1 versus corresponding control values (samples without inhibitor).

Figure 5

Model of oxidative stress in mutant mice leading to vascular endothelial damage. In Eng ^+/− and Alk1 ^+/− mice, altered eNOS activation renders the enzyme refractory to regulation by TGF-β/BMP signaling and represents a critical event leading to excessive oxidative stress. Uncoupled eNOS produces low amounts of NO and high levels of oxygen radicals (•O₂ ⁻). The NOS inhibitor, L-NAME, inhibits ROS production in tissues of mutant mice. Superoxide dismutase (SOD) and the SOD mimetic compound Tempol convert •O₂ ⁻ into less harmful hydrogen peroxide (H₂O₂). A large portion of ROS is produced by mitochondria (Antimycin-inhibitable) and NADPH oxidases (Apocynin-inhibitable) however that percentage does not differ between mutant and control mice. Low NO cellular level may also inhibit mitochondrial K_ATP channel (mitoK_ATP) opening, trigger permeability transition pores (PTP) opening, and further increase the oxidative stress caused by mitochondrial ROS release.