High Soluble Endoglin Levels Affect Aortic Vascular Function during Mice Aging

Abstract

Endoglin is a 180 kDa transmembrane glycoprotein that was demonstrated to be present in two different endoglin forms, namely membrane endoglin (Eng) and soluble endoglin (sEng). Increased sEng levels in the circulation have been detected in atherosclerosis, arterial hypertension, and type II diabetes mellitus. Moreover, sEng was shown to aggravate endothelial dysfunction when combined with a high-fat diet, suggesting it might be a risk factor for the development of endothelial dysfunction in combination with other risk factors. Therefore, this study hypothesized that high sEng levels exposure for 12 months combined with aging (an essential risk factor of atherosclerosis development) would aggravate vascular function in mouse aorta. Male transgenic mice with high levels of human sEng in plasma (Sol-Eng⁺) and their age-matched male transgenic littermates that do not develop high soluble endoglin (Control) on a chow diet were used. The aging process was initiated to contribute to endothelial dysfunction/atherosclerosis development, and it lasted 12 months. Wire myograph analysis showed impairment contractility in the Sol-Eng⁺ group when compared to the control group after KCl and PGF_2α administration. Endothelium-dependent responsiveness to Ach was not significantly different between these groups. Western blot analysis revealed significantly decreased protein expression of Eng, p-eNOS, and ID1 expression in the Sol-Eng⁺ group compared to the control group suggesting reduced Eng signaling. In conclusion, we demonstrated for the first time that long-term exposure to high levels of sEng during aging results in alteration of vasoconstriction properties of the aorta, reduced eNOS phosphorylation, decreased Eng expression, and altered Eng signaling. These findings suggest that sEng can be considered a risk factor for the development of vascular dysfunction during aging and a potential therapeutical target for pharmacological intervention.

Keywords: endoglin signaling; mice; soluble endoglin; vascular function.

Figure 1

Biochemical and ELISA analyses of plasma. Plasma concentration of total cholesterol (A), triglycerides (B), human sEng (C), mouse sEng (D), and weight (E) in Sol-Eng⁺ mice and control group. Representative microphotographs of hematoxylin-stained aortas from control group (F) and Sol-Eng⁺ (G) mice. Data are shown as a mean ± S.E.M., Mann–Whitney test, ** p ≤ 0.01. n = 6 mice per group.

Figure 2

Functional analysis of vascular reactivity. A maximal contraction to KCl (60 mM) (A), dose–response curve to cumulative addition of PGF_2α (B), dose–response curve to cumulative addition of Ach in pre-constricted aortic segments by PGF_2α (C), dose–response curve to cumulative addition of SNP in pre-constricted aortic segments by PGF_2α (D), dose–response curve to cumulative addition of Ach in pre-constricted aortic segments by PGF_2α in the presence of L-NAME (E). Data are shown as a mean ± S.E.M., Mann–Whitney test, * p < 0.05, ** p ≤ 0.01. n = 6 mice per group.

Figure 3

Expression of endothelial adhesion molecules in aortas of twelve-month-old male Sol-Eng⁺ and control mice. Expression of ICAM-1 (A) and VCAM-1 (B) in total protein extract from mice aortas. Densitometric quantification of immunoreactive bands (top panel: densitometric analysis, control = 100%) was recalculated to the GAPDH signal (bottom panel: representative immunoblots). Data are shown as a mean ± S.E.M, Mann–Whitney test. n = 5 mice per group.

Figure 4

Expression of membrane endoglin and selected molecules belonging to its signaling pathway in aortas of twelve-month-old male Sol-Eng⁺ and control mice. Expression of membrane endoglin (A), eNOS (B), p-eNOS (C), and ID1 (D) in total protein extracts from mice aortas. Densitometric quantification of immunoreactive bands (top panel: densitometric analysis, control = 100%) was recalculated to the GAPDH signal (bottom panel: representative immunoblots). Data are shown as mean ± S.E.M, Mann–Whitney test, * p ≤ 0.05, ** p ≤ 0.01. n = 5 mice per group.

Figure 5

Expression of markers related to smooth muscle cell reactivity. Expression of MLCK (A), p-MLCK (B), and αSMA (C) in total protein extract from mice aortas. Densitometric quantification of immunoreactive bands (top panel: densitometric analysis, control = 100%) was recalculated to the GAPDH signal (bottom panel: representative immunoblots). Data are shown as mean ± S.E.M, Mann–Whitney test. n = 5 mice per group.

Figure 6

Role of sEng with aging in vascular dysfunction. (A) In endothelial cells, sEng and aging reduce Eng expression (red arrow) and signaling resulting in reduced expression of eNOS (red arrow) and its reduced phosphorylation rate (red arrow). (B) Vasoconstriction process, localized in VSMC, consists in phosphorylation of myosin light chain by an activated form of myosin light chain kinase (MLCK). Binding to PGF_2α receptor or activation of K⁺ channel evoke changes in intracellular milieu, increasing calcium ion concentration. The presence of high levels of sEng and aging probably contribute to the alteration of vasoconstriction.