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Vitamin D deficiency represents a pandemic social problem affecting over 1 billion of people worldwide, although its clinical relevance is still largely neglected (1).

Several studies have documented that the maintenance of adequate levels of vitamin D offers benefits in terms of survival, quality of life and for the maintenance of “house-keeping” homeostatic processes in different organs and tissues, including the cardiovascular system (2,3).

However, the exact threshold for the definition of the optimal levels of vitamin D required for the achievement of the cardioprotective effects is still debated. In fact, while a circulating 25(OH)D < 10 ng/ml is universally accepted as a severe deficiency, it is still unclear whether the cut-off for sufficiency is to be settled at 20 or 30 ng/ml (4).

In Chapter 2, we documented a progression of the risk of coronary artery disease with the lowering of vitamin D levels, although its prevalence and extent were significantly enhanced only for patients with levels < 10 ng/ml.

In addition, the present thesis documented, in Chapter 8, that, even among subjects with vitamin D < 10 ng/ml, yet the levels of circulating active hormone could be preserved, therefore pointing at the superiority of the direct evaluation of calcitriol levels rather than vitamin D precursors.

Indeed, the definition of the complex regulatory mechanisms responsible for the homeostasis of vitamin D levels still needs further clarification (5). In fact, both genetic factors and several clinical conditions, despite not conditioning the measured circulating 25(OH)D, can modulate its bioavailability and effects, therefore translating into cardiovascular consequences.

In Part 3 in fact, we showed that both genetic variants of vitamin D binding protein (VDBP) and of the 24-α hydroxylase responsible for vitamin D inactivation (CYP24A1), can be held responsible for the clinical manifestations of vitamin D deficiency, against comparable levels of vitamin D. In fact, carriers

of the G allele of rs7041 of VDBP; a variant associated with an enhanced binding of the vitamin to its transport-protein, displayed an increased thrombogenicity despite dual antiplatelet therapy (DAPT), only when this genetic status was associated with vitamin D deficiency.

Moreover, variability in the levels of the active hormonal form, calcitriol, could affect coronary calcifications and the inflammatory status. In Chapter 7, in fact, we showed that the wild-type genotype for rs2762939 of CYP24A1 were associated with coronary calcifications, potentially mediated by an increased inactivation of 1,25(OH)2D due to a higher enzymatic activity. In addition, in Chapter 8, we reported that activated vitamin D in ACS patients could be conditioned not only by the levels of its precursor, but also by other factors as hypertension and renal dysfunction. Indeed, the kidney represents the principal site of activation of vitamin D, and mainly at a tubular level, therefore explaining the reason for our positive association with the use of diuretics rather than with serum creatinine, instead representing a glomerular process. In addition, hypertension, by increasing the wall shear stress, could favor the induction of the pathway responsible for the paracrine production of calcitriol in the endothelium of the whole arterial system, thus increasing its circulating levels. On the contrary, an extremely focused vascular damage, as in case of an acute coronary syndrome, being dependent on the instabilization of a single atherosclerotic lesion, could also stimulate a local production of 1,25(OH)D, that could nevertheless result in too modest variations to be apparent at a systemic level. Such hypothesis, as much as the common pathogenesis of the spectrum of acute coronary events, could certainly explain our observation of similar levels of 1,25(OH)2D in different ACS types.

Therefore, present thesis directly points out at calcitriol as a marker of cardiovascular disease and a potential new target for a pharmacological approach to cardiovascular prevention.

In fact, 1,25(OH)2D, being linked to a pro-inflammatory and pro-thrombotic status and to vascular wall degeneration and remodeling, could represent an early predictor of cardiovascular risk, even in subjects without established coronary atherosclerosis.

In addition, the definition of the optimal levels of calcitriol for cardiovascular prevention could allow to optimize the therapy and to define which patients could mostly benefit from its supplementation, and especially for high-cardiovascular risk patients, as in the context of an ACS.

Indeed, the trials conducted so far on the topic are largely inadequate for answering to this issue, not being powered for the evaluation of cardiovascular endpoints and often failing in the restoration of adequate levels of vitamin D (6,7). The ongoing the VITamin D and OmegA-3 TriaL (VITAL) trial (8) will certainly shed more light on the topic, by enrolling 25,874 men and women across the U.S. taking daily dietary supplements of vitamin D3 or omega-3 fatty acids for the reduction of the risk of cancer, heart disease, and stroke.

Nevertheless, the majority of the studies were conducted with inactive 25(OH)D, then being subject to a differential activation and potentially not resulting in sufficient hormonal levels. In fact, the studies with direct calcitriol replacement have being conducted so far only in patients with renal failure, providing promising results, (9,10) that however, will certainly deserve confirmations in a more general population.

References

1. Holick MF. The vitamin D deficiency pandemic: Approaches for diagnosis, treatment and prevention. Rev Endocr Metab Disord. 2017;18(2):153-165

2. Gil Á, Plaza-Diaz J, Mesa MD. Vitamin D: Classic and Novel Actions. Ann Nutr Metab. 2018;72(2):87-95 3. Norman PE, Powell JT. Vitamin D and cardiovascular disease. Circ Res. 2014;114(2):379-9

4. Lugg ST, Howells PA, Thickett DR. Optimal Vitamin D Supplementation Levels for Cardiovascular Disease Protection. Dis Markers. 2015;2015:864370

5. Barry EL, Rees JR, Peacock JL, et al. Genetic Variants in CYP2R1, CYP24A1, and VDR Modify the Efficacy of Vitamin D3 Supplementation for Increasing Serum 25-Hydroxyvitamin D Levels in a Randomized Controlled Trial. The Journal of Clinical Endocrinology and Metabolism. 2014;99(10):E2133-E2137.

6. Theodoratou E, Tzoulaki I, Zgaga L, Ioannidis JP. Vitamin D and multiple health outcomes: umbrella review of systematic reviews and meta-analyses of observational studies and randomised trials. BMJ. 2014;348:2035

7. Rejnmark L, Bislev LS, Cashman KD, et al. Non-skeletal health effects of vitamin D supplementation: A systematic review on findings from meta-analyses summarizing trial data. Slominski AT, ed. PLoS ONE. 2017;12(7):e0180512..

8. Manson JE, Bassuk SS, Lee IM, Cook NR, Albert MA, Gordon D, Zaharris E, Macfadyen JG, Danielson E, Lin J, Zhang SM, Buring JE. The VITamin D and OmegA-3 TriaL (VITAL): rationale and design of a large randomized controlled trial of vitamin D and marine omega-3 fatty acid supplements for the primary prevention of cancer and cardiovascular disease. Contemp Clin Trials. 2012;33(1):159-71.

9. Verouti SN, Tsoupras AB, Alevizopoulou F, Demopoulos CA, Iatrou C. Paricalcitol effects on activities and metabolism of platelet activating factor and on inflammatory cytokines in hemodialysis patients. Int J Artif Organs. 2013;36(2):87-96 10. Shoben AB, Levin G, de Boer IH, et al. Variation in Oral Calcitriol Response in Patients With Stage 3-4 CKD. American Journal of Kidney Diseases. 2012;59(5):645-652.