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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 20, 2004 as doi:10.1096/fj.03-0197fje. |
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Instituto "Reina Sofía" de Investigación Nefrológica, Departamento de Fisiología & Farmacología, Universidad de Salamanca, Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain; and
# Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28040 Madrid, Spain
2Correspondence: Departamento de Fisiología y Farmacología, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain. E-mail: jmlnovoa{at}usal.es
SPECIFIC AIMS
Endoglin (CD105) is a homodimeric membrane glycoprotein that plays a major role in vascular development and function. Endoglin gene mutation in humans and the subsequent endoglin haploinsufficiency give rise to an autosomal dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia (HHT). However, so far nothing is known about the possible role of endoglin in vascular tone. Thus, the main goal of our study was to explore whether endoglin is involved in vascular tone control. Specifically, we have assessed NO-mediated vasodilation in endoglin haploinsufficient mice (Eng+/-) compared with their Eng+/+ littermates, as well as the possible alterations in nitric oxide synthase (NOS) isoforms expression.
PRINCIPAL FINDINGS
1. Endothelium-dependent hypotensive effects are reduced in Eng+/- mice
The hypotensive response to acetylcholine and bradykinin was higher in Eng+/+ than in Eng+/- mice (Fig. 1
). A bolus of the NOS inhibitor L-NAME induced a sustained hypertensive response in Eng+/+ animals but a lower response in Eng+/- animals. This was not due to the lack of a vasoconstrictor response in Eng+/- animals, as the infusion of angiotensin II had similar effects in both types of mice. Administration of an endothelium-independent NO donor, sodium nitroprusside, in animals treated with L-NAME to eliminate endogenous NO production induced a similar hypotensive response in Eng+/+ and Eng+/- animals. After nitric oxide inhibition by L-NAME infusion, the response to acetylcholine was similar in wild-type and endoglin-haploinsufficient animals.
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2. Endothelium-dependent vasodilation in isolated hind limbs and aortic rings is impaired in Eng+/- mice
Addition of acetylcholine to the perfusate liquid of isolated, perfused hind limbs of mice induced a marked and dose-dependent decrease in vascular resistance in Eng+/+ mice, whereas in Eng+/- mice the vasodilatory effect was markedly lower (Fig. 2
A). After inhibiting NO synthesis by adding L-NAME to the perfusion fluid, the vasodilatory effect of acetylcholine was markedly decreased in Eng+/+ but only slightly decreased in Eng+/- mice (Fig. 2B
); differences between both kinds of mice were not significant. Perfusion with L-NAME induced a higher increase in vascular resistance in Eng+/+ than in Eng+/- mice (Fig. 2C
). When the endothelium-independent NO donor sodium nitroprusside was given in the presence of L-NAME (to eliminate endogenous NO production), the vasodilator effect was similar in Eng+/+ and in Eng+/- mice (Fig. 2D
). When aortic rings from Eng+/+ and Eng+/- were first stimulated to 80–90% of maximum contraction with phenylephrine, subsequent relaxation in response to increasing concentrations of Ach was reduced in Eng+/- compared with Eng+/+ aortas. Relaxation induced by the NO donor sodium nitroprusside was similar for Eng+/+ and Eng+/- aortas. L-NAME markedly inhibited Ach-induced relaxation in Eng+/+ and Eng+/- aortas.
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3. eNOS expression and NO synthesis are decreased in Eng+/- mice
Levels of endothelial-type constitutive nitric oxide synthase (eNOS) in kidneys and femoral arteries of Eng+/- animals were found to be about half those in Eng+/+ mice, as determined by Western blot. No significant differences in iNOS expression in the kidneys or lungs from either group of animals were observed. Nitrite concentration in plasma and urine was reduced by 
50% in Eng+/- compared with Eng+/+ mice. Cultured mice aortic endothelial cells (MAEC) from Eng+/- had lower endoglin expression levels than those from Eng+/+ mice. Similarly, eNOS expression was reduced in MAEC from Eng+/- compared with those from Eng+/+ mice.
4. Decreased plasma and tissue TGF-ß1 levels in Eng+/- mice
Since eNOS expression has been shown to be regulated by TGF-ß and endoglin is a component of the TGF-ß receptor system, we postulated that the decreased eNOS expression in tissues from Eng+/- mice could be related to decreased TGF-ß1 levels. Plasma concentration of TGF-ß1 was lower in Eng+/- (10.9±1.9 ng/mL, n=7) than in Eng+/+ animals (14.3±2.9 ng/mL, n=7, P<0.05). mRNA levels of TGF-ß1 were lower in kidney and lung of Eng+/- mice compared with those of Eng+/+ animals.
5. Relationship between endoglin and eNOS expression
Further support for the endothelial cell involvement in eNOS down-regulation was obtained using RNA interference studies. Endoglin gene suppression in human endothelial cells results in a marked decrease of eNOS expression as determined by confocal microscopy analysis. To determine whether the relationship between endoglin and eNOS expression was endothelial cell specific or could be extended to other cell types, we assessed eNOS expression in a L6E9 myoblast cell line (characterized by the lack of endoglin expression) transfected with human endoglin. Endoglin-transfected L6E9 myoblasts showed a markedly higher eNOS expression than mock transfected cells as assessed by Western blot analysis.
CONCLUSIONS
Our results demonstrate a marked deficiency in NO-mediated vasodilation in endoglin-haploinsufficient mice, as well as a direct relationship between endoglin and eNOS expression in mice tissues, in primary cultures of endothelial cells and in transfectant myoblasts. Accordingly, it can be postulated that Eng+/- animals have an impaired NO-dependent vasodilatory response due to a decreased vascular eNOS expression. Heterozygous activin-like kinase-1 (Alk1) mutant mice (whose heterologous mutation in humans is associated with HHT type 2) are also hypertensive and show an impaired endothelium-dependent vascular response (personal communication from Dr. Paul Oh, University of Florida).
Several explanations could account for down-regulation of eNOS in endoglin-deficient mice. One explanation could be related to the fact that eNOS expression in endothelial cells is regulated by TGF-ß1. A second possibility derives from the fact that endothelial cells could change their phenotype and eNOS expression depending on the type of underlying extracellular matrix. Specifically, cultured human endothelial cells from the umbilical vein seeded on collagen I show decreased nitrite synthesis, NOS activity, eNOS protein content, and eNOS mRNA expression compared with cells grown on collagen IV. As endoglin seems to be involved in regulating cellular responses to TGF-ß1, including extracellular matrix synthesis, it may be that endoglin-deficient animals exhibit an alteration in the amount and/or type of ECM proteins in the vessel walls and that this alteration modifies the expression of eNOS by endothelial cells. This hypothesis is being assessed in our laboratory.
Thus, we can conclude that the deficiency in a single gene, endoglin, leads to a marked reduction in the effects of NO-dependent vasodilators, probably mediated by a marked decrease in eNOS expression. The possible relationship of these changes with the vascular abnormalities observed in patients with HHT1 and endoglin-deficient animals remains to be studied. Nonetheless, it can be speculated (Fig. 3
) that in physiological or pathological situations associated with increased blood volume and/or cardiac output or increased blood flow through an organ, such as in pregnancy, inflammation or infection, precapillary sphincters may not be able to dilate due to the eNOS deficiency. As a consequence, blood should cross the organs through "preferential channels," which act as arteriovenous shunts. If this situation is maintained for a relatively long period, the overall organization of the vessel network can be structurally modified. Nonperfused or hypoperfused capillaries would regress, probably by endothelial cell apoptosis, which results in capillary network destruction, giving rise to the formation of the arteriovenous malformations characteristic of HHT.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0197fje; 
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, n=8) and Eng+/– (•, n=8) mice. Asterisk represents significant difference with respect to basal value. Significant difference was observed with respect to Eng+/+ mice (#); P <0.05, 2-way ANOVA.
