Status of research on vitamin D supplementation in treating or preventing liver fibrosis (2013)

Fibrosis represents a disease state with excess deposition of extracellular matrix components such as collagen and fibronectin in a given tissue 1-3. In fact, fibrosis can occur in every parenchymal organ, including the liver, kidney, lung, heart, or pancreas, contributing to abnormal function of each affected organ 1. Indeed, it has been estimated that fibrotic disorders account for up to 45% of deaths in developed countries [4 and references within]. In the liver, long-term heavy alcohol consumption 5, 6 and chronic infection with hepatitis viruses 7-10 are major risk factors for the activation and transformation of hepatic stellate cells into myofibroblasts, which produce collagen and alpha-smooth muscle actin, eventually leading to liver fibrosis 2, 3. Recent results have also indicated that hepatic fibrosis can take place following exposure to fast foods containing high fats and high fructose corn syrup 11. In these cases, the rates of collagen synthesis are usually far greater than that of collagen degradation by proteolytic enzymes such as metalloproteinases (MMPs) 1-3. The aetiological factors and underlying mechanisms of fibrosis are diverse and complex. For example, hepatic stellate cells (HSC) are activated by increased oxidative/nitrative stress-mediated injury of epithelial and endothelial cells with dysregulation of innate and adaptive immune cells. Hepatocyte apoptosis and endoplasmic reticulum (ER) stress, caused by accumulation of unfolded proteins, are likely to promote fibrosis observed in experimental models of nonalcoholic steatohepatitis 11 as well as patients 7-10. In addition, profibrotic cytokines such as transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor (PDGF) are potent stimulators of collagen biosynthesis 1-3. On the other hand, some synthetic drugs such as peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone or rosiglitazone can decrease collagen contents in experimental models and are being evaluated for approval of human usage 12. The beneficial and adverse effects of many other agents including the modulators of cannabinoid receptors have also been tested in experimental models and being evaluated in human patients with hepatic fibrosis 1, 12. Unfortunately, not a single agent has been approved by the Food and Drug Administration in the USA for treating fibrotic conditions to date 1, 4, 12. It has been well-established that vitamin D is critically important in regulating the intestinal absorption of calcium and phosphate, bone mineralization and maintenance of kidney function. In addition, the Endocrine Society 13 recently reported a scientific statement on the roles of vitamin D in nonskeletal functions including metabolic syndrome, cardiovascular system, skin, muscle, placenta and maternal health, and prevention of certain types of cancer. In these systems, vitamin D has a number of important effects, including in the modulation of innate or adaptive immune cells, blood pressure via regulating the renin-angiotensin pathway, cellular growth and differentiation, and many more [13 and references within]. However, this scientific statement did not summarize the effects of vitamin D supplementation on liver fibrosis, suggesting a need for studying vitamin D supplementation in patients with hepatic fibrosis. Active vitamin D (1,25-dihydroxyvitamin D or 1,25-(OH)2D3, calcitriol) is produced from 7-dehydrocholesterol by sunlight and dietary vitamin D via sequential hydroxylation reactions catalysed by specific cytochromes P450 in the liver (many P450 isoforms involved in 25-hydroxylation) and kidney (CYP27B1 for 1α-hydroxylation) respectively 7, 13, 14. Because of different sites of production and its target tissues, vitamin D is now considered a pleiotropic hormone. Its biological activity is mediated through interaction with its receptor, vitamin D receptor (VDR), which is ubiquitously expressed in many tissues including bone, intestine, kidney, and liver 13, 15. Upon binding of 1,25-(OH)2D3 to nuclear VDR, it becomes hyperphosphorylated and then interacts with the its binding partner retinoid receptor-X (RXR) to form an active heterodimer complex, which specifically binds to unique DNA sequence vitamin D response element (VDRE) in the promoter regions of its down-stream target genes. The vitamin D-VDR-VDRE cascade can both up- and down-regulate its target genes. For instance, osteocalcin, osteopontin and vitamin D-24-hydroxylase (CYP24) represent genes that are positively regulated by the vitamin D-VDR-RXR complex. In contrast, type I collagen, bone sialoprotein, parathyroid hormone (PTH) and PTH-related peptide are negatively regulated by the vitamin D-VDR-RXR heterodimeric complex [15 and references within]. It is well-established that plasma levels of 25-hydroxyvitamin D, which has a longer half-life than 1,25-(OH)2D3 and is thus considered a reliable biomarker for cellular vitamin D status 14, are significantly reduced (serum levels less than 30 ng/ml) in various types of chronic liver disease (CLD) 6-10 including alcoholic individuals with fibrosis/cirrhosis. Lower levels of serum 25-hydroxyvitamin D were also observed in NAFLD patients (metabolic syndrome with hepatic steatosis, necroinflammation and fibrosis) compared to those in control individuals 10. In fact, an inverse relationship between the level of 25-hydroxyvitamin D and the degree of hepatic cirrhosis has been described [14 and references within]. Vitamin D deficiency in cirrhotic patients is also strongly correlated with decreased bone density associated with chronic hepatic dysfunction 14. Although the exact mechanism remains unknown, vitamin D deficiency in CLD including cirrhosis patients likely results from a combination of multiple factors including decreased rates of vitamin D synthesis because of less sunlight exposure, decreased intestinal absorption and interruption of the enterohepatic circulation of vitamin D 14. Furthermore, vitamin D deficiency is known to be associated with clinical complications in cirrhosis patients such as bone disorders, cardiovascular problems, and muscle weakness 13, 14. Decreased levels of VDR and CYP27 have also been observed in patients with NAFLD 10, leading presumably to worsening of their liver disease. In contrast, a recent report summarizing the 4-year longitudinal study revealed that serum vitamin D levels do not correlate with the progression of HCV-associated fibrosis (n = 129 HCV cases vs 129 age-matched controls) 16, calling into question the role of vitamin D in CLD. In this issue, Mezey and colleagues 17 demonstrated that biologically active 1,25-(OH)2D3 (calcitriol) exposure actually: (1) slightly, but significantly decreased TGFβ1 level, (2) increased VDR expression, (3) suppressed TGFβ1-mediated type I collagen production (on both mRNA and protein levels) and the amounts of secreted collagen, (4) restored TGFβ1-mediated alterations of profibrotic markers (e.g. decreased MMP-9 and elevated TIMP-1 expression), (5) demonstrated VDRE interacting transcription factor proteins by UV-crosslink of nuclear protein-DNA complex, and (6) identified the VDR binding sites on the promoter of type I collagen in stellate cells. However, the rates of collagen degradation were similar regardless of different treatments. By conducting promoter assays, electrophoretic mobility shift assays (EMSA), and chromatin immunoprecipitation assays (CHIP), the authors demonstrated that VDR binds to the proximal Sp1.1 site (at −174 bp) as well as a newly identified distal site (at −2.3 kb region) on the type I collagen gene promoter. This study 17 not only confirmed the suppressive effect of vitamin D on collagen expression as recently reported by another laboratory 18 but also identified a new VDR binding site in the collagen gene promoter. In the latter study 18, calcitriol exposure inhibited proliferation of primary hepatic stellate cells and markedly suppressed the expression of collagen while it restored other profibrotic markers. Calcitriol administration (5 μg/kg/dose, ip twice a week for 10 weeks) also significantly prevented thioacetamide-induced liver fibrosis and extracellular matrix deposition in rats. More importantly, these results [17, 18 and references within] are consistent with many reports on vitamin D-dependent decreased expression of collagen and other fibrotic markers in other experimental models. Together, these data should serve as the basis for future translational research on vitamin D supplementation in treating or preventing the progression of hepatic fibrosis in humans. Despite many clinical and pre-clinical trials with vitamin D supplementation for treatment of diseases such as the metabolic syndrome, osteoporosis and fractures from falls 13, 14, very few clinical studies have been conducted to evaluate the beneficial effects and adverse toxicities of vitamin D supplementation on hepatic fibrosis. One recent study revealed that supplementation of a single oral megadose (300 000 IU) of vitamin D3 to alcoholic cirrhosis individuals, who were previously vitamin D-deficient, effectively raised the plasma 25-hydroxyvitamin D levels for up to 90 days 6. Although the number of cirrhotic patients was rather small (n = 4–20), administration of vitamin D3 (cholecalciferol) revealed more effective than vitamin D2 (ergocalciferol) in raising the plasma 25-hydroxyvitamin D levels determined at 7, 30 and 90 days. This result was likely related to higher levels of plasma vitamin D binding protein (DBP) observed in patients with vitamin D3 administration than those with vitamin D2 6. Another study 9 showed that the majority (~79%) of CLD of various aetiologies (n = 158 including 65 cirrhosis patients) have mildly or severely deficient levels of 25-hydroxyvitamin D. Many patients with cirrhosis showed severely decreased vitamin D levels. While these data do not establish a cause and effect relationship between vitamin D and its potential suppressive fibrogenic effect, other data shed light on this issue. For example, oral daily supplementation with a standard dose of vitamin D3 (50 μg/day) or vitamin D2 (1000 IU/day) not only elevated 25-hydroxyvitamin D levels with a median period of 4 months but also improved the severity of CLD, although surrogate markers (e.g. bilirubin) were used in this study 9. In another study 19, oral vitamin D3 supplementation to HCV patients (2000 IU/day for 12 weeks prior to starting antiviral therapy, n = 20) significantly increased the serum levels of 25-hydroxyvitamin D at 12 weeks and improved host responses (e.g. HCV RNA virus clearance) compared with the corresponding controls (n = 30 for non-supplemented control patients) [19 and references within]. The relatively high dose of vitamin D3 did not appear to cause any serious adverse toxicities beyond those observed with the antiviral agents used to treat HCV patients in two genotypes (genotypes 2-3 and 1), but significantly improved the fibrosis score and viral clearance 19. Taken together, these data 6, 7, 13, 14, 17-19 suggest a need for further laboratory and clinical study to evaluate the optimal dosage ranges, route of administration, duration, and beneficial and adverse effects of vitamin D supplementation on serum 25-hydroxyvitamin D levels and the progression of liver fibrosis. Furthermore, research should be directed to study the outcomes and effective doses of exogenously administered vitamin D and its noncalcemic analogues such as 20S-hydroxyvitamin D3, 17,20S-(OH)2pD3 (17,20S-dihydroxypregnacalciferol) and 17,20R-(OH)2pD3 [20 and references within]. The mechanisms by which the cellular levels of vitamin D, plasma DBP and nuclear VDR are decreased in the experimental models of CLD (and fibrosis in other tissues) should be also investigated in the future. The basic mechanistic studies will contribute to better understanding of the roles of vitamin D, DBP and VDR in the pathological progression of CLD and future translational applications in clinics. Despite the many reports about the beneficial effects of vitamin D on preventing fibrosis in experimental models, clinical investigation on the effect of vitamin D supplementation in treating human fibrosis remains lacking. In fact, a recent PubMed search with keywords ‘vitamin D’, ‘fibrosis’ and ‘clinical trial’ revealed 26 reports, although many of these studies do not directly deal with hepatic fibrosis. On the basis of the abundant safety information available for vitamin D and analogues 6, 7, 13, 14, 19, we speculate that large-scale multi-centre clinical evaluation of the effect of vitamin D supplementation on liver fibrosis (especially in early fibrosis) are in order. Indeed, this sort of data may further inform the potential for vitamin D or its less-toxic analogues in treating fibrosis in other tissues as well.