A protective role for FGF-23 in local defence against disrupted arterial wall integrity? - PubMed (original) (raw)

A protective role for FGF-23 in local defence against disrupted arterial wall integrity?

Dongxing Zhu et al. Mol Cell Endocrinol. 2013.

Abstract

Increasing interest is focusing on the role of the FGF-23/Klotho axis in mediating vascular calcification. However, the underpinning mechanisms have yet to be fully elucidated. Murine VSMCs were cultured in calcifying medium for a 21 d period. FGF-23 mRNA expression was significantly up-regulated by 7d (1.63-fold; P<0.001), with a concomitant increase in protein expression. mRNA and protein expression of both FGFR1 and Klotho were confirmed. Increased FGF-23 and Klotho protein expression was also observed in the calcified media of Enpp1(-/-) mouse aortic tissue. Reduced calcium deposition was observed in calcifying VSMCs cultured with recombinant FGF-23 (10 ng/ml; 28.1% decrease; P<0.01). Calcifying VSMCs treated with PD173074, an inhibitor of FGFR1 and FGFR3, showed significantly increased calcification (50 nM; 87.8% increase; P<0.001). FGF-23 exposure induced phosphorylation of ERK1/2. Treatment with FGF-23 in combination with PD98059, an ERK1/2 inhibitor, significantly increased VSMC calcification (10 μM; 41.3% increase; P<0.01). Use of FGF-23 may represent a novel therapeutic strategy for inhibiting vascular calcification.

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Figures

Figure 1. In vitro calcification of murine aortic VSMCs cultured for 21 d under calcifying conditions

Quantification of (A) Alizarin red staining and (B) Alkaline phosphatase activity (mean moles pNPP hydrol/min/mg protein) (_n_=3). Fold change in (C) Alpl, (D) Runx2 and (E) PiT-1 RNA expression in VSMCs cultured with 2.5 mM βGP and 50 μg/ml ascorbic acid or control medium (_n_=4). VSMCs were treated with control (white bar) or βGP/AA containing (filled bar) medium. Results are presented as mean+/− S.E.M. **P<0.01; ***P<0.001 compared with day 0.

Figure 2. Up-regulation of FGF-23 expression during in vitro calcification of murine aortic VSMCs cultured for 21d with βGP and ascorbic acid

Fold change in the mRNA expression of (A) FGF-23, (B) Klotho and (C) FgfR1. (D) Recombinant mouse FGF-23 (rFGF-23) and Klotho (rKlotho) were used to verify specificity of commercial antibodies in VSMCs (E) FGF-23, Klotho and FGFR1 protein expression in corresponding VSMCs cultured with 2.5 mM βGP and 50 μg/ml ascorbic acid. VSMCs were treated with control (white bar) or βGP/AA containing (filled bar) medium. Results are presented as mean+/− S.E.M., _n_=4 *P<0.05; **P<0.01; ***P<0.001 compared with day 0.

Figure 3. Up-regulation of FGF-23 expression during in vitro calcification of murine aortic VSMCs cultured for 14d with 3mM Pi

(A) Calcium content was determined by quantification HCL leaching (μg/mg protein) (_n_=3), and visualised with alizarin red staining. Fold change in the mRNA expression of (B) Runx2, (C) Pit-1 (D) Alpl and (E) Fgf-23. VSMCs were treated with control (white bar) or βGP/AA containing (filled bar) medium. (F) Klotho and FGFR1 protein expression and (G) FgfR3 expression in corresponding VSMCs cultured with control [C] or 3mM Pi [P] medium (_n_=4). Results are presented as mean+/− S.E.M. *P<0.05; **P<0.01; ***P<0.001 compared with day 0.

Figure 4. Emergence of FGF-23 in the calcified aorta from the _Enpp1_−/− mouse in vivo

Medial aortic calcification was detected by alizarin red staining in (A) _Enpp1_−/− tissue compared to (B) Enpp1+/+ control. (C) Increased protein expression of FGF-23 was observed in the calcified media of _Enpp1_−/− aortic tissue (arrows). (D) No expression of FGF-23 was detected in the non-calcified media of the Enpp1+/+ control. Representative image of (E) _Enpp1_−/− compared to (F) Enpp1+/+ negative control tissue. Scare bars = 50μm.

Figure 5. Emergence of Klotho in the calcified aorta from the _Enpp1_−/− mouse in vivo

Medial aortic calcification was detected by alizarin red staining in (A) _Enpp1_−/− tissue compared to (B) Enpp1+/+ control. (C) Increased protein expression of Klotho was observed in the calcified media of _Enpp1_−/− aortic tissue. (D) Basal expression levels of Klotho was detected in the non-calcified media of the Enpp1+/+ control. Representative image of (E) _Enpp1_−/− compared to (F) Enpp1+/+ negative control tissue. (G) Fold change in the mRNA expression of FgfR1 in _Enpp1_−/− VSMCs compared to Enpp1+/+ control cells. Scare bars = 50μm.

Figure 6. Effect of FGF-23 treatment on the in vitro calcification of murine aortic VSMCs

VSMCs were incubated with FGF-23 (10–50ng/ml) in the presence of 3mM Pi for 9 days. (A) Calcium content was determined by quantification HCL leaching (μg/mg protein) (_n_=5), and visualised with alizarin red staining. Fold change in the mRNA expression of osteogenic markers (B) Ocn (C) Alpl and (D) Pit-1 (_n_=4). (E) Cell viability was assessed using the Alamar blue assay (OD570–OD620) (_n_=6). (F) Expression of cleaved caspase-3 protein as an indication of apoptosis. Results are presented as mean+/− S.E.M. *P<0.05; **P<0.01 compared with control.

Figure 7. Effect of FGFR1 inhibition on the in vitro calcification of murine aortic VSMCs

VSMCs were incubated with the FGFR1 inhibitor PD173074 (10–50nM) in the presence of 3mM Pi for 9 days. (A) Calcium content was determined by quantification HCL leaching (μg/mg protein) (_n_=5), and visualised with alizarin red staining. (B) Cell viability was assessed using the Alamar blue assay (OD570–OD620) (_n_=6). Results are presented as mean+/− S.E.M. ***P<0.001 compared with control.

Figure 8. FGF-23 induces Erk 1/2 activation in VSMCs

Effect of FGF-23 (10ng/ml) on the phosphorylation of Akt compared with total Akt, and Erk1/2 compared with total Erk1/2 shown by (A) western blot analysis and (B,C) densitometry quantification (_n_=3). (D) Effect of PD98059 (10μM) on calcium content (μg/mg protein) of VSMCs in the presence/absence of FGF-23 (10ng/ml) (_n_=5). Results are presented as mean+/− S.E.M. **P<0.01; *P<0.05 compared with a 0 exposure time (mins); b control and c FGF-23.

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A protective role for FGF-23 in local defence against disrupted arterial wall integrity? - PubMed (original) (raw)