Differentially expressed miRNA
We identified a list of miRNA that were differentially expressed following the various manipulations. As mentioned previously, we focused on selected miRNAs that are known to be modified in a diabetic-like environment or that were highly changed after the addition of calcitriol. MiR-510 and miR-659 were over-expressed under diabetic-like conditions and decreased after calcitriol was added. MiR-126, miR-411, miR-20b, miR-15a and miR-181c were down-regulated under diabetic conditions and over-expressed after calcitriol was added.
MiR-510 was first identified in association with irritable bowel syndrome; its co-expression was involved in the regulation of 5-HT3 receptors in colonic enterocytes . Recently, Hezova et al.  demonstrated that miR-510 expression was elevated in regulatory T cells of diabetic patients. To the best of our knowledge, our findings are the first demonstration of miR-510 involvement in HUVEC exposed to a diabetic-like environment with significant changes induced by calcitriol. Manipulation of miR-510 may represent a beneficial effect of calcitriol if we consider the possible deleterious effect of increased miR-510 in DM. MiR-659 is not described in the database as being connected to a diabetic-like environment and appears to be novel in genomic control in the presence of diabetic-like conditions affected by vitamin D. Further investigation of the effect of the miRNA on their predicted target genes is warranted.
MiR-126 is highly enriched in endothelial cells and is involved in vascular integrity, angiogenesis and wound repair [25, 26]. Moreover, loss of plasma miR-126 is consistently associated with diabetes [27, 28]. Zampetaki et al.  found that high glucose concentrations (25 mmol/l equivalent to 500 mg/dl) significantly reduced miR-126 expression in endothelial apoptotic bodies and improved vascular growth factor (VEGF) signaling, leading to endothelial dysfunction [27, 29]. In patients with DM, low plasma levels of miR-126 could be clinically relevant and contribute to VEGF resistance and endothelial dysfunction . In this study, we also found in HUVEC a decreased expression of miR-126 exposed to a diabetic like environment and demonstrated that the addition of calcitriol elevated miR-126 expression, which can probably improve VEGF signaling and repair endothelial dysfunction.
Differential expression of miR-411 was reported in a miRNA profiling study of the hippocampus and the marginal division in a rat brain . In addition, miR-411 was reported to be down-regulated in an autosomal dominant muscle disorder (facioscapulohumeral muscular dystrophy) suggesting that reduction of miR-411 might have a positive effect on muscle regeneration and promote myoblast maturation . To our knowledge, no study has investigated the effect of diabetes and vitamin D on the function of miR-411 in endothelial cells. The relevance of its role in diabetes remains unknown and should be clarified in the future. The significant effect of vitamin D remains speculative at this stage.
Selected miRNAs and their target genes
MiR-15a, miR-20b and miR-181C were found to be down-regulated in a diabetic-like environment and up-regulated after the addition of calcitriol; they were chosen for further investigation at the level of their gene targets, which have been shown to be involved in the modulation of endothelial function. MiR-15a, a cell growth suppressor, was evaluated in human cancer cells and found to have a pro-apoptotic role by activating caspase 3/7, which reduces cell viability . In addition, miR-15a has been correlated with different pathophysiological events in the liver, which are also side-effects of anabolic steroids . MiR-15a was down-regulated in the plasma of diabetic patients  and in β-cells exposed to high glucose (33 mM equivalent to 600 mg/dl) for long periods . Mir-15a was also found to be up-regulated in endothelial cells and vascular smooth muscle cells after stimulation with KLF4, which indicate an option to suppress proliferative vascular disorders . MiR-20b was down-regulated in the plasma of diabetic patients  and similarly expressed in normal and diabetic dermatological tissue, but was significantly different from that of diabetic wounds during the course of healing . To our knowledge, no study has investigated the effect of diabetes and vitamin D on the function of miR-20b in endothelial cells.
Using pediatric cancer stem cells, Sanchez-Diaz et al.  showed that miR-181c regulate cell proliferation and the cell cycle, probably by affecting the Notch signaling pathway and the bone morphogenetic protein (BMP) pathway. Becker et al.  demonstrated that under the influence of anabolic steroids, miR-181c was down-regulated in bovine liver and might lead to uncontrolled proliferation in the liver.
To our knowledge, no study has investigated the effect of diabetes and vitamin D on the function of miR-181C in endothelial cells.
As mentioned above, these three miRNA that were down regulated in a diabetic environment were significantly up-regulated after addition of calcitriol. To the best of our knowledge, this is the first demonstration of the effect of calcitriol on the expression of these three miRNA under diabetic-like conditions in HUVEC.
In order to determine the potential genes involved in HUVEC exposed to a diabetic-like environment and calcitriol, we analyzed the predicted target genes of these 3 miRNA (miR-15a, miR-20b and miR-181c).
Seven predicted targets of the 3 deregulated miRNA were chosen for further analysis of the molecular pathways. In Figure 3, we observed that the target genes KLF9, KLF10, and KLF6 are directly and indirectly (via TXN, JUN and FOS) bound to TXNIP and to IL8 and correspond to immune, defense and metabolic biological processes. After calcitriol was added to the cells, we observed a down-regulation in KLF9, KLF10, KLF6, TXNIP and IL8 expression. These findings were negatively correlated with the miRNA expression patterns and therefore support their function.
Kruppel-like factors are members of the zinc finger family of transcription factors that regulate cellular differentiation and tissue development [38, 39]. KLF9 is a transcriptional regulator that is highly expressed in the rat brain, kidney, lung, and testis and regulates uterine endometrial cell proliferation, adhesion and differentiation [40, 41]. Panda et al.  showed that in Ishikawa cells (an endometrial adenocarcinoma cell line), miR-200c increased cell proliferation through KLF9 repression.
KLF10 plays a major role in mediating the effects of TGF-β through regulation of the Smad signaling pathway. It regulates gene transcription, inhibits cell proliferation, induces apoptosis and plays a role in activating the inflammatory response, including stress-induced inflammation, leading to increases in cardiovascular diseases, autoimmune abnormalities and DM . Yang et al.  showed that KLF10 induced endothelial cells to facilitate a possible pathway for TGF-β and regulated COX-1, which affects platelet aggregation. Using microarray gene expression, we demonstrated that in vascular smooth muscle cells, KLF10 was up-regulated under diabetic conditions and down-regulated after calcitriol was added . In this study, calcitriol had also a beneficial effect on KLF10 mRNA expression in HUVEC exposed to a diabetic environment.
KLF6 is one of the KLFs that are reportedly expressed in endothelial cells and is considered a damage-response factor [38, 39, 44]. KLF6 promotes tissue remodeling due to its ability to activate genes that are members of the TGF-β signaling pathway, which are implicated in vascular remodeling, tumor metastasis and apoptosis [39, 43]. Qi et al.  demonstrated that KLF6 was induced by high glucose concentrations (30 mmol/l equivalent to 600 mg/dl) in human kidney cells and binds to the TXNIP promoter region. In addition, in an in vivo study in diabetic rats, they showed that KLF6 and PPAR-γ (localized downstream of KLF6) play a key role in the regulation of TXNIP expression in the development of diabetes mellitus. The KLF target genes that were analyzed in this study and were found to be modified by a diabetic-like environment and calcitriol might provide unique information on the function of these transcription factors in endothelial cells and may be the basis of attractive research in the future in the field of DM in animal models and humans.
TXNIP is stimulated by high glucose concentrations. It is well-demonstrated that it promotes oxidative stress and apoptosis in several types of cells, including endothelial cells [15, 46, 47]. In this study, we showed that TXNIP mRNA expression in cultured HUVEC was up-regulated following exposure to a diabetic-like environment, while no significant changes was observed after the addition of calcitriol. IL8 is produced in several tissues, as well as in endothelial cells upon infection, inflammation, ischemia and trauma . We found that diabetic-like conditions stimulate the expression of this inflammation-related protein and that the addition of calcitriol had a beneficial effect on its expression.
Vitamin D deficiency and supplementation
It has been well established that vitamin D deficiency is correlated with high body fat and glucose levels and decreased insulin sensitivity. It is also an independent cardiovascular risk factor that predicts poor cardiovascular outcomes [49–51]. Grineva et al.  showed that in women at late reproductive age (mean age 46.1 ± 4.5) from the North‒West region of Russia, low 25(OH)D levels were associated with obesity, increased plasma glucose levels after OGTT and insulin resistance. A significant correlation between fasting insulin, 2 h OGTT glucose, insulin levels and low 25(OH)D levels was found in overweight and obese subpopulations. The authors suggested that vitamin D supplementation could be effective in the prevention of obesity and insulin resistance . The intervention between obesity, vitamin D and PTH is complex, suggesting possible effects at different levels. Alkharfy et al.  showed a beneficial effect of vitamin D supplementation in the metabolic profile of DM type 2 patients treated with insulin and oral hypoglycemic agents. The authors suggested that the effect of the medication could be mediated by an increase in HDL-cholesterol levels, particularly by vitamin D . Al-Daghri et al.  showed an improvement in LDL, total cholesterol and in homeostasis model assessment of β-cell function (HOMA-β) among a Saudi DM type 2 population receiving vitamin D3 supplementation for 18 months. This effect was more pronounced in women than in men. Vitamin D supplementation was suboptimal, which could explain the absence of significant increase in HDL-cholesterol levels and the increase in HOMA-insulin resistance index. Moreover the absence of relevant information concerning the anti-diabetic drugs and the diet composition of the patients could have limited the impact of the results .
Ongoing studies should provide an answer to the still-debated question, whether vitamin D supplementation can positively influence cardiovascular outcomes. Our study showed that vitamin D can influence the pro-atherogenic and inflammatory response seen in DM and therefore, might contribute to a preventive and therapeutic effect. Moreover, recent study showed that vitamin D may improve endothelial function in cardiovascular diseases and could possibly have a role in vascular protection .