Haplodeficiency of Klotho Gene Causes Arterial Stiffening via Upregulation of Scleraxis Expression and Induction of Autophagy - PubMed (original) (raw)

Haplodeficiency of Klotho Gene Causes Arterial Stiffening via Upregulation of Scleraxis Expression and Induction of Autophagy

Kai Chen et al. Hypertension. 2015 Nov.

Abstract

The prevalence of arterial stiffness increases with age, whereas the level of the aging-suppressor protein klotho decreases with age. The objective of this study is to assess whether haplodeficiency of klotho gene causes arterial stiffness and to investigate the underlying mechanism. Pulse wave velocity, a direct measure of arterial stiffness, was increased significantly in klotho heterozygous (klotho(+/-)) mice versus their age-matched wild-type (WT) littermates, suggesting that haplodeficiency of klotho causes arterial stiffening. Notably, plasma aldosterone levels were elevated significantly in klotho(+/-) mice. Treatment with eplerenone (6 mg/kg per day IP), an aldosterone receptor blocker, abolished klotho deficiency-induced arterial stiffening in klotho(+/-) mice. Klotho deficiency was associated with increased collagen and decreased elastin contents in the media of aortas. In addition, arterial matrix metalloproteinase-2, matrix metalloproteinase-9, and transforming growth factor-β1 expression and myofibroblast differentiation were increased in klotho(+/-) mice. These klotho deficiency-related changes can be blocked by eplerenone. Protein expression of scleraxis, a transcription factor for collagen synthesis, and LC3-II/LC3-I, an index of autophagy, were upregulated in aortas of klotho(+/-) mice, which can be abolished by eplerenone. In cultured mouse aortic smooth muscle cells, aldosterone increased collagen-1 expression that can be completely eliminated by small interfering RNA knockdown of scleraxis. Interestingly, aldosterone decreased elastin levels in smooth muscle cells, which can be abolished by small interfering RNA knockdown of Beclin-1, an autophagy-related gene. In conclusion, this study demonstrated for the first time that klotho deficiency-induced arterial stiffening may involve aldosterone-mediated upregulation of scleraxis and induction of autophagy, which led to increased collagen-1 expression and decreased elastin levels, respectively.

Keywords: Becn1 protein, mouse; Scx protein, mouse; autophagy; collagen; elastin; myofibroblasts; vascular stiffness.

© 2015 American Heart Association, Inc.

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Figures

Figure 1

Figure 1

Haplodeficiency of Klotho gene (klotho+/−) increased arterial pulse wave velocity (PWV) and serum aldosterone levels. (A) PWV was measured in klotho+/− and age-mated WT mice by 10-MHz Doppler probes (n=14). (B) Serum aldosterone levels were measured by ELISA (n=6). Data are expressed as mean±SE and analyzed by a one-way ANOVA. **p<0.01 vs. WT group.

Figure 2

Figure 2

Blockade of aldosterone receptors abolished the increase of PWV in klotho+/− mice. PWV was measured after treatment with eplerenone for 3 weeks. Data are expressed as mean±SE and analyzed by two-way ANOVA. n=7. *p<0.05 vs. WT group; #p<0.05 vs. klotho+/−-vehicle group.

Figure 3

Figure 3

Haplodeficiency of klotho gene increased collagen expression but decreased elastin levels in aortas which can be abolished by blockade of aldosterone receptors. (A) Immunohistochemical analysis of collagen-1 (blue) and elastin (brown). (B) Western blot analysis of collagen-1 and elastin. Data are expressed as mean±SE and analyzed by a two-way ANOVA. n=5. *p<0.05, **p<0.01 vs. WT group; #p<0.05, ##p<0.01 vs. klotho+/−-vehicle group. Scale bar = 20 μm.

Figure 4

Figure 4

Haplodeficiency of klotho gene increased arterial MMP2, MMP9 and TGFβ1 expression which can be eliminated by blockade of aldosterone receptors. (A) Immunohistochemical staining results of MMP2 and MMP9. (B) Western blot analysis of MMP2 and MMP9 expression. (C) Western blot analysis of TGFβ1 expression. Data are expressed as mean±SE and analyzed by two-way ANOVA. n=5, *p<0.05, **p<0.01 vs WT group; #p<0.05, ##p<0.01 vs klotho+/−-vehicle group. Scale bar = 20 μm

Figure 5

Figure 5

Klotho+/− increased aortic myofibroblasts differentiation which can be abolished by blockade of aldosterone receptors. Myofibroblast differentiation was evaluated by α-SMA positive cells using immunohistochemical staining. The semi-quantitative data are expressed as mean±SE and analyzed by two-way ANOVA. n=5, **p<0.01 vs WT group; #p<0.05 vs klotho+/−-vehicle group. Scale bar = 20 μm.

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