Fisetin ameliorates vascular smooth muscle cell calcification via DUSP1-dependent p38 MAPK inhibition

Results

To investigate a possible impact of fisetin on VSMC calcification, a first series of experiments was performed in cultured primary human aortic VSMCs during control or pro-calcific conditions with addition of calcium and the phosphate donor β-glycerophosphate, in the absence and presence of increasing concentrations of fisetin (0 – 20 μM). As illustrated in Figure 1, calcification medium significantly up-regulated BMP2 and ALPL mRNA expression in VSMCs, effects significantly suppressed in the presence of 1 μM fisetin concentration. Lower fisetin concentrations did not significantly affect calcification medium-induced BMP2 and ALPL mRNA expression (Supplementary Figure 1).

Figure 1. Dose-dependent effects of fisetin on calcific marker expression in VSMCs during pro-calcific conditions. Relative mRNA expression (n=12) of BMP2 (A) and ALPL (B) in HAoSMCs treated for 48h with control (CTR) or calcification medium (Calc.) without and with the indicated concentrations of fisetin (Fis, 0 - 20 μM). **(p<0.01), ***(p<0.001) significant vs. control group; †(p<0.05), ††(p<0.01) significant vs. Calc.-treated group.

The calcification medium-induced increase of pro-calcific markers CBFA1, SP7, BGLAP and SPP1 mRNA expression (Figure 2A–2D), ALP activity (Figure 2E) as well as CBFA1 nuclear localization (Figure 2F) were all suppressed by 1 μM fisetin supplementation. Co-treatment with fisetin reduced expression of markers associated with senescence of VSMCs promoted by calcification medium, as shown by the mRNA expression of CDKN1A and GLB1 and further indicated by senescence-associated (SA)-β-galactosidase staining (Supplementary Figure 2). Treatment with fisetin alone did not consistently modify pro-calcific and senescence markers expression at 1 μM concentration, but high concentrations of fisetin (20 μM) had adverse effects in VSMCs (Supplementary Figure 3). Moreover, pre-treatment of VSMCs with 1 μM fisetin did not significantly modify calcification medium-induced pro-calcific marker expression (Supplementary Figure 4). More importantly, as illustrated by Osteosense fluorescence imaging and determination of calcium content (Figure 3), co-treatment with 1 μM fisetin significantly reduced calcification of VSMCs exposed to calcification medium. Taken together, fisetin inhibited pro-calcific signaling and calcification of VSMCs induced by mineral stress during high phosphate and calcium exposure.

Figure 2. Effects of fisetin on pro-calcific signaling in VSMCs during pro-calcific conditions. Relative mRNA expression (n=6) of CBFA1 (A), SP7 (B), BGLAP (C) and SPP1 (D) in HAoSMCs treated for 48h with control (CTR) or calcification medium (Calc.) without and with 1 μM fisetin (Fis). (E) Normalized ALP activity (n=6) in HAoSMCs treated for 7d with control (CTR) or calcification medium (Calc.) without and with 1 μM fisetin (Fis). *(p<0.05), **(p<0.01), ***(p<0.001) significant vs. control group; †(p<0.05), ††(p<0.01), †††(p<0.001) significant vs. Calc.-treated group. (F) CBFA1 (green) and nuclei (blue) shown by confocal imaging in HAoSMCs treated for 48h with control (CTR) or calcification medium (Calc.) without and with 1 μM fisetin (Fis). Scale bar: 20 μm.

Figure 3. Effects of fisetin on calcification of VSMCs during pro-calcific conditions. Calcification detected by Osteosense fluorescence imaging (A) and normalized calcium content (n=6, B) in HAoSMCs treated for 11d with control (CTR) or calcification medium (Calc.) without and with 1 μM fisetin (Fis). Calcified areas: white pseudocolor. **(p<0.01), ***(p<0.001) significant vs. control group; †††(p<0.001) significant vs. Calc.-treated group.

To elucidate the underlying mechanisms of the anti-calcific effects of fisetin in VSMCs, its potential to interfere with p38 MAPK activation was explored. As illustrated in Figure 4, calcification medium induced phosphorylation of p38 MAPK in VSMCs, effects significantly blunted in the presence of fisetin. Subsequently, the role of dual-specificity phosphatase 1 (DUSP1), a negative regulator of p38 MAPK activity was investigated. As shown in Figure 5, fisetin increased total DUSP1 protein abundance in VSMCs as well as the phosphorylation of DUSP1 at Ser³⁵⁹, a direct phosphorylation site for p44/42 MAPK that could inhibit DUSP1 degradation via the ubiquitination pathway [27]. Moreover, fisetin increased the abundance of phosphorylated and total p44/42 MAPK protein in VSMCs (Supplementary Figure 5). Pharmacological inhibition of p44/42 MAPK with LY3214996 blunted fisetin-induced phosphorylation of DUSP1 at Ser³⁵⁹ (Supplementary Figure 6).

Figure 4. Effects of fisetin on p38 MAPK phosphorylation in VSMCs during pro-calcific conditions. Representative Western blots and normalized phospho-p38 and total p38 MAPK protein abundance (n=5) in HAoSMCs treated for 30 min with control (CTR) or calcification medium (Calc.) without and with 1 μM fisetin (Fis). *(p<0.05) significant vs. control group; †(p<0.05) significant vs. Calc.-treated group.

Figure 5. Effects of fisetin on dual-specificity phosphatase 1 regulation in VSMCs. Representative Western blots and normalized phospho-DUSP1 and total DUSP1 protein abundance (n=6) in HAoSMCs treated for 30 min with control (CTR) or 1 μM fisetin (Fis). *(p<0.05) significant vs. control group.

In further experiments, the endogenous expression in VSMCs was suppressed by silencing of the DUSP1 gene using small interfering RNA (siRNA), during control and pro-calcific conditions with and without fisetin supplementation. As a result, transfection with DUSP1 siRNA significantly reduced DUSP1 expression in VSMCs as compared to negative control siRNA-transfected cells (Figure 6A). Calcification medium increased DUSP1 mRNA expression in VSMCs (Figure 6A and Supplementary Figure 7), which was not significantly affected by fisetin (Figure 6A). Knockdown of DUSP1 alone was sufficient to significantly up-regulate mRNA expression of pro-calcific markers in VSMCs (Figure 6B–6E). Furthermore, DUSP1 silencing abolished the protective effects of fisetin during pro-calcific conditions. In accordance, silencing of DUSP1 aggravated VSMC calcification induced by calcification medium and virtually abrogated the anti-calcific properties of fisetin (Figure 7).

Figure 6. Effects of dual-specificity phosphatase 1 knockdown on anti-calcifying properties of fisetin in VSMCs. Relative mRNA expression (n=10) of DUSP1 (A), BMP2 (B), CBFA1 (C), ALPL (D) and CDKN1A (E) in HAoSMCs transfected for 72h with negative control (Neg.si) or DUSP1 (DUSP1si) siRNA and treated for 48h with control (CTR) or calcification medium (Calc.) without and with 1 μM fisetin (Fis). **(p<0.01), ***(p<0.001) significant vs. Neg.si group; †(p<0.05), ††(p<0.01) significant vs. Neg.si+Calc.-treated group; ‡‡(p<0.01), ‡‡‡(p<0.001) significant between Neg.si+Calc.+Fis- and DUSP1si+Calc.+Fis-treated groups.

Figure 7. Effects of dual-specificity phosphatase 1 knockdown on the protective role of fisetin during VSMC calcification. Calcification detected by Osteosense fluorescence imaging (A) and normalized calcium content (n=7, B) in HAoSMCs transfected with negative control (Neg.si) or DUSP1 (DUSP1si) siRNA and treated for 11d with control (CTR) or calcification medium (Calc.) without and with 1 μM fisetin (Fis). Calcified areas: white pseudocolor. *(p<0.05), ***(p<0.001) significant vs. Neg.si group; †(p<0.05), ††(p<0.01) significant vs. Neg.si+Calc.-treated group; ‡‡‡(p<0.001) significant between Neg.si+Calc.+Fis- and DUSP1si+Calc.+Fis-treated groups.

Moreover, knockdown of DUSP1 was sufficient to increase phosphorylation of p38 MAPK in VSMCs (Figure 8A), but DUSP1 knockdown did not significantly affect SAPK/JNK or p44/42 MAPK phosphorylation (Supplementary Figure 8). Pharmacological inhibition of p38 MAPK with SB203580 significantly blunted the increased BMP2, CBFA1, ALPL and CDKN1A mRNA expression in DUSP1-silenced VSMCs (Figure 8C–8F). Treatment with the SB203580 inhibitor alone did not significantly modify DUSP1 or calcific marker mRNA expression in VSMCs (Supplementary Figure 9). Thus, knockdown of DUSP1 increased p38 MAPK-dependent pro-calcific signaling and aggravated calcification of VSMCs.

Figure 8. Role of p38 MAPK in dual-specificity phosphatase 1 knockdown-induced calcific marker expression in VSMCs. Representative Western blots and normalized phospho-p38 and total p38 MAPK protein abundance (n=4, A) in HAoSMCs transfected for 24h with negative control (Neg.si) or DUSP1 (DUSP1si) siRNA. Relative mRNA expression (n=8) of DUSP1 (B), BMP2 (C), CBFA1 (D), ALPL (E) and CDKN1A (F) in HAoSMCs transfected for 72h with negative control (Neg.si) or DUSP1 (DUSP1si) siRNA and treated without and with 10 μM p38 MAPK inhibitor SB203580 (SB). *(p<0.05), **(p<0.01), ***(p<0.001) significant vs. Neg.si group; †(p<0.05), ††(p<0.01), †††(p<0.001) significant vs. DUSP1si group.

Next experiments explored the effects of fisetin on pro-calcific marker expression in VSMCs during uremic conditions. Exposure of VSMCs to uremic serum from hemodialysis patients significantly increased BMP2, CBFA1, ALPL and CDKN1A mRNA expression as compared to VSMCs exposed to control serum from healthy volunteers (Figure 9). Treatment with fisetin significantly suppressed uremic serum-induced BMP2, CBFA1 and CDKN1A mRNA expression and tended to reduce ALPL mRNA expression, a difference, however, not reaching statistical significance (p=0.0551). Thus, fisetin treatment induced protective effects in VSMCs during uremic conditions.

Figure 9. Effects of fisetin on calcific marker expression in VSMCs during uremic conditions. Relative mRNA expression (n=10) of BMP2 (A), CBFA1 (B), ALPL (C) and CDKN1A (D) in HAoSMCs treated for 24h with 15% normal serum (NS) or uremic serum (US) without and with 1 μM fisetin (Fis). *(p<0.05), **(p<0.01), ***(p<0.001) significant vs. NS group; †(p<0.05) significant vs. respective serum alone group.

Additional experiments explored the effects of fisetin following angiotensin II stimulation. Again, fisetin ameliorated the expression of pro-calcific markers in VSMCs during angiotensin II treatment (Supplementary Figure 10).

To confirm the anti-calcific properties of fisetin, additional experiments were performed ex vivo in mouse aortic explants cultured during control or high phosphate conditions without and with co-treatment with fisetin. As shown in Figure 10, fisetin reduced calcification as well as the increased mRNA expression of Bmp2, Cbfa1, Alpl and Cdkn1a induced by phosphate exposure in mouse aortic explants. In addition, fisetin significantly reduced the abundance of phosphorylated p38 Mapk and tended to reduce total p38 Mapk protein abundance (p=0.0798) in mouse aortic explants during high phosphate conditions (Supplementary Figure 11).

Figure 10. Effects of fisetin ex vivo in calcifying mouse aortic explants. Alizarin Red staining (A) of mouse aortic arches cultured for 7d in medium supplemented with 1.6 mM phosphate (Pi) without and with 1 μM fisetin (Fis). Calcification: red staining; scale bar: 5 mm. Normalized calcium content (n=6, B) in mouse aortic explants cultured for 7d in medium supplemented with control (CTR) or 1.6 mM phosphate (Pi) without and with 1 μM fisetin (Fis). Relative mRNA expression (n=5) of Bmp2 (C), Cbfa1 (D), Alpl (E) and Cdkn1a (F) in mouse aortic explants cultured for 7d in medium supplemented with control (CTR) or 1.6 mM phosphate (Pi) without and with 1 μM fisetin (Fis). *(p<0.05), **(p<0.01), ***(p<0.001) significant vs. control group; †(p<0.05), ††(p<0.01), †††(p<0.001) significant vs. Pi-treated group.

Further experiments investigated the effects of fisetin in vivo in the cholecalciferol-induced VC mouse model. As shown by alizarin Red staining and quantification of calcium content, cholecalciferol overload induced aortic calcification in mice, effects significantly reduced by additional fisetin treatment (Figure 11A, 11B). Furthermore, fisetin suppressed cholecalciferol-induced Bmp2, Cbfa1, Alpl and Cdkn1a mRNA expression in the aortic tissues (Figure 11C–11F). High-dosed cholecalciferol significantly increased serum calcium and altered cystatin C, phosphate and fetuin A concentrations, while fisetin ameliorated most effects of cholecalciferol treatment (Supplementary Table 1).

Figure 11. Effects of fisetin in vivo during cholecalciferol overload. Alizarin Red staining (A) of aortas from mice receiving vehicle (CTR) or high-dosed cholecalciferol (vD) without and with fisetin (Fis). Calcification: red staining; scale bar: 5 mm. Normalized calcium content (n=6-9, B) in aortic tissue from mice receiving vehicle (CTR) or high-dosed cholecalciferol (vD) without and with fisetin (Fis). Relative mRNA expression (n=6-9) of Bmp2 (C), Cbfa1 (D), Alpl (E) and Cdkn1a (F) in aortic tissue from mice receiving vehicle (CTR) or high-dosed cholecalciferol (vD) without and with fisetin (Fis). *(p<0.05), **(p<0.01), ***(p<0.001) significant vs. control group; †(p<0.05), ††(p<0.01) significant vs. vD-treated group.

Discussion

This study discloses a powerful anti-calcific effect of fisetin in VSMCs during mineral stress, a condition typically observed in CKD [2]. Mechanistically, fisetin requires the phosphatase DUSP1 to inhibit p38 MAPK in order to mediate its protective effect on VSMC calcification (Figure 12).

Figure 12. Schematic illustration of mechanisms involved in the protective effects of fisetin during VSMC calcification. Mineral stress with disturbed phosphate and calcium homeostasis may trigger activation of pro-calcific signaling including phosphorylation and activation of p38 MAPK in VSMCs, which leads to a pro-calcific environment causing vascular calcification. Fisetin, a natural flavonol, induces the dual-specificity phosphatase 1 (DUSP1). Fisetin thereby inactivates p38 MAPK signaling through DUSP1 and inhibits further pro-calcific signaling and calcification of VSMCs.

Fisetin has previously been associated with protective effects in the vasculature and prevents neointimal hyperplasia [28], atherosclerosis [26], glucose-induced inflammation [29] and endothelial dysfunction [30] in animal models or cell culture. Especially the ability of fisetin to decrease vascular senescence has been well established [30, 31]. Also in the kidney, fisetin is associated with attenuation of fibrosis [25, 32]. The current observations now extend the protective effects of fisetin on the calcifying vasculature in vitro, ex vivo and in vivo. Although fisetin treatment altered calcium-phosphate homeostasis in cholecalciferol-treated mice and this might affect the calcification response, a direct effect on the vasculature is supported by aortic explant calcification model and cell culture experiments. A potential translational relevance of these observations for the CKD environment is underscored by the protective effects of fisetin in VSMCs exposed to uremic serum.

Mechanistically, the effects of fisetin appear to be mediated by inhibition of p38 MAPK during pro-calcific conditions. P38 MAPK appears to have a central role in pro-calcific signaling of VSMCs [9, 10, 33]. P38 MAPK inhibition has been shown to ameliorate VSMC calcification after phosphate exposure, while ERK1/2 MAPK or SAPK/JNK inhibition showed no effect [12]. The downstream effects of p38 MAPK activation in VC apparently are mediated through a mechanism involving RUNX2/CBFA1 [10]. P38 MAPK also exerts a critical role in the intracellular signaling pathways of inflammatory mediators, which are able to augment VC.

Accordingly, the pro-calcific effects of CRP [33], IL18 [34], ceramide [9] or angiotensin II [35] involve p38 MAPK.

P38 kinases react to a variety of cellular stress conditions and control a diverse array of cellular functions [11]. Phosphatases such as DUSP1 are induced by p38 MAPK and limit or terminate p38 MAPK signaling [11]. The current observations identify DUSP1 as a novel regulator of VSMC calcification. DUSP1 is expressed in VSMCs and plays an important role in maintaining the contractile phenotype by regulating p38 MAPK [36]. Fisetin reduces p38 MAPK phosphorylation in calcifying VSMCs apparently through DUSP1. Similarly, fisetin inhibits ubiquitination and proteasomal degradation of DUSP1 in osteoclasts and thereby limits p38 MAPK signaling [37]. In T-cells, angiotensin II increases proteasome activity and degrades DUSP1 [38]. Angiotensin II and aldosterone attenuate DUSP1 expression in VSMCs [30]. In accordance, we observed a reduced pro-calcific effect of angiotensin II in VSMCs treated with fisetin. Mechanistically, fisetin might activate ERK1/2 MAPK [39]. ERK1/2 could phosphorylate DUSP1 on Ser³⁵⁹, leading to its stabilization [27], but other ERK1/2-dependent phosphorylation sites could actually promote DUSP1 proteasomal degradation [40]. Therefore, the regulatory network of DUSP1 activation and its effect on MAPK appear intricate and multi-factorial [41]. The exact mechanisms how fisetin regulates DUSP1 are currently unknown.

Although the gene silencing experiments indicate a major role for DUSP1 in the anti-calcific effects of fisetin, the current experiments cannot rule out involvement of other pathways or mechanisms. Also, ubiquitination of NRF2 is negatively regulated by fisetin [42]. NRF2 is a powerful modulator of VC [43]. Other pathways might therefore contribute to the effects of fisetin in phosphate-treated VSMCs. In addition, the role of cellular senescence in the current observations should be interpreted with caution. P38 MAPK is important for the development of a senescence-associated secretory phenotype (SASP) [44] and the increased expression of senescence markers in calcifying VSMCs are ameliorated by fisetin. However, the role of DUSP1 in senescence appears complex [45, 46] and the primary effect of fisetin in this model may be mediated through other mechanisms than directly through senolytic properties [22]. In neuronal cells, DUSP1 is polyubiquitylated and increasingly degraded in response to glutamate-induced oxidative stress [47]. Thus, oxidative stress might shift the balance of DUSP1 expression and degradation. Increased oxidative stress is also a hallmark of phosphate-induced VC [2]. In turn, fisetin has been attributed with substantial antioxidant properties [23], which could therefore be involved in its protective effect during VC and subsequently alter expression of senescence markers.

Fisetin treatment has already been used in animal models of CKD and renal fibrosis [25, 32]. In addition, first clinical studies are being conducted on the beneficial effects of fisetin [48]. While the reduced renal function and uremic environment in CKD patients warrants caution, our observations suggest a potential benefit of fisetin treatment on VC. However, the current observations are limited by the model systems used. The artificial cell culture environment does not allow extrapolation to the in vivo situation, especially in terms of effective concentrations of fisetin. The cholecalciferol model rapidly induces VC, but differs from the situation in CKD, where active vitamin D levels are typically reduced. Therefore, CKD-specific or sex-dependent effects cannot be interpreted and further translational studies are required to delineate whether the putative benefits outweigh the risks of fisetin treatment in CKD.

In conclusion, this study shows a novel role of fisetin as powerful protective agent during phosphate-induced VSMC calcification. Mechanistically, this effect identifies a critical role of DUSP1 in the pro-calcific p38 MAPK signaling of VSMCs during calcifying conditions.

Author Contributions

IA and JV designed research and acquired funding; MR, SB, AS, MM, IJ and IA performed experiments; MR, SB, GB, IA and JV analyzed and interpreted data; IA and JV wrote the manuscript with comments and edits from all authors.

Acknowledgments

The authors gratefully acknowledge the technical assistance of A. Kurishev.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Ethical Statement and Consent

The blood collection was approved by the local ethics commission (approval number 1141/2019) and all volunteers provided informed consent. All animal experiments were approved by the local authorities (BMBWF Vienna, Austria), protocol number 2022-0.406.924.

Funding

This work was funded by the Austrian Science Fund (FWF) (10.55776/P34724) and the Faculty of Medicine of the Johannes Kepler University Linz through its internal funding programme Impetus Nr. I-04-23. Supported by Johannes Kepler Open Access Publishing Fund and the federal state Upper Austria.

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