In Aging, researchers have described how the well-known supplement fisetin may fight calcification of the blood vessels, seeing significant successes in both cellular and mouse models.
When calcium goes where it doesn’t belong
Calcification is not the same as ‘hardening’ of blood vessel walls (atherosclerosis), which occurs due to plaque deposits. Calcification occurs when phosphates in the blood cause calcium to precipitate, forming crystals; normally, regulatory processes prevent this from happening, but conditions such as chronic kidney disease [1] and systemic inflammation [2] can disrupt them, leading to stiff, dangerously narrow arteries.
Senescence of the smooth muscle cells of the vasculature (VSMCs) has been found to play a part. Exposing these cells to excessive phosphates, or excessive glucose, drives them senescent [3], and suppressing phosphate has been found to be beneficial in a rat model of kidney disease [4]. The p38/MAPK pathway also plays a significant role in this process, and previous work has found that activating it leads directly to additional calcification [5] and that inhibiting it prevents calcification [6].
As senolytics have been found to potentially alleviate this problem [7], these researchers took a close look at fisetin, which was not previously examined in vascular calcification, and its relationship to p38/MAPK.
Establishing a chain of causation
The researchers first took a population of human aortic cells and exposed them to both calcium and a phosphate donor. Under these conditions, as expected, the cells quickly began to express two calcification markers well above those of the control group. However, introducing even a single micromole of fisetin reduced both of these markers nearly to control-group levels, with increasing doses having no beneficial effects.
The fisetin was only effective when administered under the pro-calcium conditions; pre-treatment had no effect. Similarly, fisetin did not affect cells that were not exposed to pro-calcium conditions. However, in a cellular model of uremic conditions meant to reflect chronic kidney disease, fisetin reduced senescence- and calcification-related markers.
The researchers also investigated the role of p38/MAPK in these effects, focusing on four core RNA markers: the calcification markers BMP2, CBFA1, and ALPL along with the senescence marker CDKN1A.
They found that fisetin increases DUSP1, a negative regulator of the p38/MAPK pathway. Inhibiting this effect through another compound neutralized the effects of fisetin. Similarly, silencing or knocking down DUSP1 made calcification significantly worse and stopped fisetin from having any benefit. However, directly affecting p38 in these DUSP1-silenced cells was able to provide the same benefits as fisetin did in the unsilenced cells. Therefore, the causal chain is clear: fisetin affects DUSP1, which affects p38.
Effective on mouse models
The next experiment involved explanted mouse aortae, which were subjected to a pro-calcium environment. Fisetin reduced markers of both senescence and calcification, just as it had in the cellular experiments.
In living mice that were given cholecalciferol in order to induce calcification, fisetin had similar beneficial effects. While the anti-senescence and anti-calcification marker effects were not quite as strong as in the cellular and explant studies, there was still a very strong effect on actual calcification: the mice given both cholecalciferol and this supplement had arteries that looked much like those of the control group.
While these results are strongly positive, the researchers urge caution, as they did not have a model that perfectly recapitulates chronic kidney disease and its characteristic depletion of vitamin D. They note that while fisetin appears to be strongly effective against calcification itself, there may also be sex-dependent effects or pecularities that prevent it from having such benefits in actual people. Further work needs to be done to determine whether or not fisetin is effective in real-world situations involving calcification. However, fisetin is sold as a supplement, so it may be relatively inexpensive to conduct a clinical trial.
Literature
[1] Voelkl, J., Cejka, D., & Alesutan, I. (2019). An overview of the mechanisms in vascular calcification during chronic kidney disease. Current opinion in nephrology and hypertension, 28(4), 289-296.
[2] Voelkl, J., Egli-Spichtig, D., Alesutan, I., & Wagner, C. A. (2021). Inflammation: a putative link between phosphate metabolism and cardiovascular disease. Clinical Science, 135(1), 201-227.
[3] Zhang, M., Li, T., Tu, Z., Zhang, Y., Wang, X., Zang, D., … & Zhou, H. (2022). Both high glucose and phosphate overload promote senescence-associated calcification of vascular muscle cells. International Urology and Nephrology, 54(10), 2719-2731.
[4] Yamada, S., Tatsumoto, N., Tokumoto, M., Noguchi, H., Ooboshi, H., Kitazono, T., & Tsuruya, K. (2015). Phosphate binders prevent phosphate-induced cellular senescence of vascular smooth muscle cells and vascular calcification in a modified, adenine-based uremic rat model. Calcified Tissue International, 96, 347-358.
[5] Yang, Y., Sun, Y., Chen, J., Bradley, W. E., Dell’Italia, L. J., Wu, H., & Chen, Y. (2018). AKT-independent activation of p38 MAP kinase promotes vascular calcification. Redox biology, 16, 97-103.
[6] Kang, J. H., Toita, R., Asai, D., Yamaoka, T., & Murata, M. (2014). Reduction of inorganic phosphate-induced human smooth muscle cells calcification by inhibition of protein kinase A and p38 mitogen-activated protein kinase. Heart and vessels, 29, 718-722.
[7] Ceccherini, E., Gisone, I., Persiani, E., Ippolito, C., Falleni, A., Cecchettini, A., & Vozzi, F. (2024). Novel in vitro evidence on the beneficial effect of quercetin treatment in vascular calcification. Frontiers in Pharmacology, 15, 1330374.
