The FASEB Journal express article 10.1096/fj.03-0197fje. Published online January 20, 2004.
Endoglin regulates nitric oxide-dependent vasodilatation Mirjana Jerkic,* Juan V. Rivas-Elena,* Marta Prieto,* Rosalia Carrón,* Francisco Sanz-Rodríguez,† Fernando Pérez-Barriocanal,* Alicia Rodríguez-Barbero,* Carmelo Bernabéu,† and J. M. López-Novoa* *Instituto “Reina Sofía” de Investigación Nefrológica, Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, Campus Miguel de Unamuno, 37007 Salamanca; and †Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain Corresponding author: J. M. López-Novoa, Departamento de Fisiología y Farmacología, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain. E-mail:
[email protected] ABSTRACT Endoglin is a membrane glycoprotein that plays an important role in cardiovascular development and angiogenesis. We examined the role of endoglin in the control of vascular tone by measuring nitric oxide (NO)-dependent vasodilation in haploinsufficient mice (Eng+/−) and their Eng+/+ littermates. The vasodilatory effect of acetylcholine, bradykinin, and sodium nitroprusside was assessed in anesthetized mice; in isolated, perfused hindlimbs; and in aortic rings. The substantial hypotensive and vasodilatory response induced by acetylcholine and bradykinin in Eng+/+ was markedly reduced in Eng+/− mice. Both kinds of animals had similar responses to sodium nitroprusside, suggesting that the deficient vasodilatory effect is not due to a NO response impairment. Urinary and plasma concentrations of nitrites, a NO metabolite, were lower in Eng+/− than in Eng+/+ mice. The levels of endothelial nitric oxide synthase (eNOS) in kidneys and femoral arteries were about half in Eng+/− than in Eng+/+ mice and were also reduced in primary cultures of aortic endothelial cells from Eng+/− compared with those from Eng+/+ mice. Furthermore, overexpression or suppression of endoglin in cultured cells induced a marked increase or decrease in the protein levels of eNOS, respectively. Thus, our results in vivo and in vitro demonstrate a relationship between endoglin and NO-dependent vasodilation mediated by the regulation of eNOS expression. Key words: endoglin haploinsufficiency ● vascular tone ● eNOS expression
H
ereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, is an autosomal dominant vascular dysplasia that affects 1:10,000 individuals. This disorder is associated with epistaxis and telangiectasias in the majority of patients and with pulmonary and cerebral arteriovenous malformations (PAVM and CAVM, respectively) in 15-20% of cases (1-3). These abnormalities are caused primarily by the dilatation of postcapillary venules, which eventually connect directly to arterioles without intervening capillaries (4). Direct shunting of blood through CAVMs may lead to ischemic and/or hemorrhagic infarctions (3).
HHT is a heterogeneous disorder in terms of its clinical manifestations. This is explained in part at the molecular level by the involvement of at least two different loci. The candidate gene for HHT1 was mapped to chromosome 9q33–34 and was identified as endoglin (5). Endoglin (CD105) is a homodimeric membrane glycoprotein that, in association with transforming growth factor (TGF)-β receptors, binds TGF-β1, TGF-β3, activin, BMP-2, and BMP-7 (6). Endoglin is constitutively expressed on endothelial cells of capillaries, veins, and arteries (7, 8) and in contractile cells such as vascular smooth muscle cells (9) and mesangial cells (10). Endoglin gene mutation in HHT-1 is associated to endoglin haploinsufficiency and not to the production of dominant negative forms of endoglin (11). As endoglin haploinsufficiency leads to the vascular disorder, this implies that endoglin levels are critical in maintaining vascular homeostasis. Several authors have generated endoglin null mice and have shown that the embryos die at midgestation of vascular and cardiovascular defects, demonstrating the critical role of endoglin in vascular development (12–15). The vascular endothelium secretes an array of vasoactive substances including nitric oxide (NO), prostacyclin (PGI2), endothelium-derived hyperpolarizing factor (EDHF), and endothelin (ET) to name but a few. NO is synthesized in the vascular endothelium by the endothelial nitric oxide synthase (eNOS) and is a very potent smooth muscle relaxant, PGI2 is also a vascular muscle relaxant, while ET-1 is a very potent vasoconstrictor. Interestingly, the expression of eNOS and ET-1 has been shown to be increased by TGF-β1 in wound repair. As endoglin is a regulatory component of the TGF-β1 receptor system, expressed on all types of endothelial cells and capable of modulating specific responses to this multipotent growth factor, it is, therefore, possible that reduced endoglin expression may result in disruption of the delicate balance in the secretion of endothelium-derived vasodilators and vasoconstrictors, thus predisposing the organism to local dilation of blood vessels. However, to date nothing is known about the possible role of endoglin in vascular tone. Thus, the main goal of our study has been to explore whether endoglin is involved in vascular tone control. Specifically, we have assessed NO-mediated vasodilation in endoglin haploinsufficient mice, as well as the possible alterations in NOS isoforms expression. MATERIALS AND METHODS Experimental animals Eng+/− mice were generated by homologous recombination using embryonic stem cells of 129/Ola (129) origin as described previously by Bourdeau et al. (12, 15) and by backcrossing onto the C57BL/6 (B6) background. B6-Eng+/− mice of N4 were kindly given by Dr. Michelle Letarte, (Hospital for Sick Children, Toronto, Canada) and backcrossed again in our facilities with C57BL/6. N5 and N6 generations of B6-Eng+/− mice were used in the present studies, as well as their B6-Eng+/+ littermate controls. These Eng+/− mice do not show clinical signs of HHT, in contrast with those of 129/Ola background, in which signs of HHT were evident (15). Genotypes were determined by PCR analysis of tail DNA isolated using two sets of primers amplifying a 470-bp nucleotide product in the mutated allele and a 300 bp nucleotide product in the wild-type allele, respectively (12, 16). Mice were kept in ventilated rooms, in a germ-free facility, under controlled conditions of light, temperature, humidity, food, and water (Unidad de Experimentación Animal, Edificio
Departamental, University of Salamanca, Spain) and fed on a standard chow for laboratory mice (Panlab, Madrid, Spain) and water ad libitum. Studies were performed in male animals of 4–6 months of age, weighing between 20 and 25 g. Studies were designed in a paired form with the Eng+/− animals and their Eng+/+ control littermates with normal endoglin expression. In all the procedures, animals were treated in accordance with the international and national institutions guidelines for the care and use of laboratory animals: Conseil de l´Europe (published in the Official Daily N. L358/1-358/6, 18th December 1986), Spanish Government (published in Boletín Oficial del Estado N. 67, pp. 8509–8512, 18th March 1988, and Boletín Oficial del Estado N. 256, pp. 31349–31362, 28th October 1990), and the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health (NIH publication no. 85–23). Blood pressure measurements Mice were anaesthetized with sodium pentobarbital, 40 mg/kg body wt., body temperature was maintained at 37°C, and tracheotomy was performed. The right carotid artery was cannulated with PE-10 tubing connected to PE-50 tubing and to a pressure transducer for continuous measurement of arterial blood pressure. Arterial pressure was recorded in a digital data recorder (MacLab/4e, AD Instruments) and analyzed using Chart v 3.4 (an application program). The left jugular vein was catheterized for the administration of vasoactive substances. Supplemental doses of anesthesia (sodium pentobarbital: 10 mg/kg body wt.) were administrated as required. We assessed the changes in mean arterial pressure (MAP) of Eng+/+ and Eng+/− mice in response to the vasodilators acetylcholine (Ach), bradykinin, and sodium nitroprusside as well as vasoconstrictor substances (angiotensin II, L-NAME). Substances were given as a bolus, and time intervals between injections were as long as necessary for allowing blood pressure to return to the basal level. Acetylcholine (Ach) was given in three doses: 0.4, 0.6, and 0.8 µg/kg body wt. Bradykinin was given in four doses: 0.8, 1.2, 1.6, and 2.4 µg/kg body wt. Angiotensin II was given in three doses: 0.15, 0.2, and 0.3 µg/kg body wt. Sodium nitroprusside was given at a dose of 0.5 mg/kg body wt. L-NAME was given in a single dose of 50 mg/kg body wt., as the effect was very long lasting. Some animals received first acetylcholine or bradykinin and then L-NAME. Other animals received first L-NAME and, when the pressure was stabilized, acetylcholine or bradykinin. Animals showing any sign of hemorrhage and animals with a systolic blood pressure
