Hypothyroidism increases angiotensinogen gene expression associated with vascular smooth muscle cells cholesterol metabolism dysfunction and aorta remodeling in Psammomys obesus

Abstract

It has been previously shown that clinical cardiovascular manifestations can be caused by mild changes in thyroid function. However, the implication of angiotensinogen (Agt) and vascular smooth muscle cells (VSMCs) dysfunction in the pathophysiology of cardiovascular manifestations in hypothyroidism have not yet been investigated. We induced experimental hypothyroidism in Psammomys obesus by administering carbimazole for five months. At the end of the experiment, the animals were sacrificed and histopathological analysis was performed using Masson's trichrome staining of the aorta and thyroid gland. The expression of the Agt gene and the genes implicated in cholesterol metabolism regulation in the liver and VSMCs was determined by qRT-PCR. Histological observations revealed profound remodeling of the aorta structure in animals with hypothyroidism. In addition, Agt gene expression in the liver was significantly increased. In vitro study, showed that VSMCs from hypothyroid animals overexpressed 3-hydroxy-3-methylglutaryl coenzyme A reductase (Hmgcr) and Acyl CoA:cholesterol acyltransferase (Acat) 1, with failure to increase the efflux pathway genes (ATP-binding cassette subfamily G member (Abcg) 1 and 4). These results suggest that hypothyroidism leads to vascular alterations, including structural remodeling, VSMCs cholesterol metabolism dysfunction, and their switch to a synthetic phenotype, together with hepatic Agt gene overexpression.

Figure 1

Several follicles of varying sizes and shapes form the thyroid gland of control Psammomys obesus, sand rat. A single layer of follicular cells delimited the colloid (C) surrounding the follicle (A). Dramatic changes in the morphology of the thyroid gland in Psammomys obesus rendered hypothyroid by carbimazole. Follicular hypertrophy and hyperplasia, total absence of colloid, and a significant vascular invasion, with enlargement of these vessels (B).

Figure 2

Relative amount of mRNA (ng/µg) normalized to the 18S mRNA level of the genes involved in cholesterol metabolism (Srebp2, Hmgcr, Lrp, Abcg1, Abcg4) and the Agt gene in the liver of the control and hypothyroid Psammomys obesus (n = 5 − 6 for each group). The values are presented as means ± SEMs. *p < 0.05 hypothyroid versus control group.

Figure 3

(A) Analysis of proliferation (n = 6) and nuclear axis length (n = 100) of control and hypothyroid Psammomys obesus VSMCs in secondary culture. (B) SMCs of control and hypothyroid Psammomys obesus in secondary culture fixed in Bouin’s aqueous and stained with May-Grünwald Giemsa. Scale bar, 50 µm. (C) VSMCs mRNA expression (ng/µg) of Srebp2, Hmgcr, Lrp, Abcg4, and Acat1 of control and hypothyroid group (n = 5 − 6 for each group). *Hypothyroid versus control VSMCs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

mRNA levels (ng/μg) of genes involved in cholesterol metabolism in VSMCs of the control and hypothyroid group in the absence (– T₃) and presence of T₃ (+ T₃) in the culture medium for 48 h and 96 h. *p < 0.05, **p < 0.01, ***p < 0.001 versus the corresponding values in the absence of T₃.

Figure 5

Cholesterol content in VSMCs of the control and hypothyroid Psammomys obesus, cultured without (Control) or with T₃ (+ T₃), cyclodextrin-cholesterol complexes (+ CCC), and CCC combined with T₃ (+ T₃ + CCC). **p < 0.01, ****p < 0.0001 versus control.

Figure 6

mRNA levels (ng/µg) of Srebp2, Hmgcr, Lrp, Abcg1, Abcg4, and Acat1 in VSMCs from control and hypothyroid Psammomys obesus gerbils following addition of T₃ (+ T₃), CCC (+ CCC), and CCC combined with T₃ (+ T₃ + CCC) in the culture medium. The values are presented as means ± SEMs. *versus Control group. *p < 0.05. Hypothyroidism alters the cellular and connective structures of the aortic wall.

Figure 7

The aortic vascular wall of the control animals was composed of an intima (I) consisting of a single layer of endothelial cells (E.C) with flattened nuclei. The media (M) was formed by smooth muscle cells (SMCs), separated by elastic lamellae (E.L), which appeared very wavy. The media was separated from the intima and adventitia by the internal (I.E.L) and external elastic lamella, respectively (Asterisk black denoted aortic lumen) (A,D) Endothelial cells are hypertrophied and separated from the I.E.L by a much-thickened subendothelial space containing collagens (black arrow). Thrombus formation (green arrow) and interrupted I.E.L (red arrow) were also observed with infiltration by SMCs in the first interlamellar space, characterized by a synthetic phenotype (yellow arrow) (B) Hypothyroidism also caused disorganization of the media, which was manifested by the thinning, ruptures, and fragmentation of E.L (orange arrow) with a decrease in their degree of undulation (C). In certain regions of the aortic wall, we observed an increased accumulation of collagens (E).