Differential Effects of Calcitriol, FGF-23, and Klotho on Vascular Smooth Muscle Cell Calcification and Their Role in Medial Calcification.
Medial calcification, or Monckeberg's arteriosclerosis, is the pathological deposition of calcium-phosphate mineral along the elastic fibers in the middle layer of arteries and is associated with chronic kidney disease (CKD), diabetes, and ageing. It causes increased arterial stiffness and is correlated with an increased risk of total and cardiovascular mortality in type 2 diabetes patients  and CKD patients on hemodialysis . It is now widely believed to be an active process that involves four key events: 1) the trans-differentiation of Vascular Smooth Muscle Cells (VSMCs) into osteoblast-like cells, 2) the release of matrix vesicles, 3) the loss of calcification inhibitors, and 4) the degradation of the extracellular matrix . High concentrations of calcium and phosphate play a large role in the pathogenesis, as they are able to increase calcification in a concentration-dependent and synergistic manner [4-6].
Under normal conditions, proper mineral metabolism is maintained through the actions of the bone, kidney, and endocrine systems through the molecules vitamin D, parathyroid hormone (PTH), fibroblast growth factor 23 (FGF-23), and klotho. During CKD, patients display dysfunctional mineral metabolism, displaying decreased conversion of vitamin D to its active form 1,25-dihydroxyvitamin D (or calcitriol), increased expression of PTH (secondary hyperparathyroidism), and decreased expression of klotho. To treat secondary hyperparathyroidism, patients will often receive calcitriol supplementations. While research has shown that it does increase the survival rate of CKD patients , large doses of calcitriol can cause hypercalcemia and promote medial calcification. Less calcemic analogues, such as paricalcitol , have been created in order to avoid these side effects; however, research has been inconclusive whether calcitriol treatment increases or decreases medial calcification and whether it is a systemic or local reaction. Some studies have shown that calcitriol increases both in vitro and in vivo calcification [9-12], others have shown that it decreases calcifications [13-16], and the rest have shown mixed results [17-20]. Many of the recent studies that have shown positive results from calcitriol treatment believe it to be associated with an increase in klotho expression and subsequent reaction with FGF-23 [15, 16]. Because there are no clinically available treatments for medial calcification, understanding the mechanism behind calcitriol's effect as well as its interaction with FGF-23 and klotho could lead to the development of a potential therapy utilizing these molecules.
The purpose of this study was to: 1) observe the in vitro effects of 10, 100, and 1000 nM concentrations of calcitriol on VSMC calcification in the presence of normal and high phosphate, 2) examine the surface morphology and protein expression of VSMCs given 100 nM calcitriol in the presence of normal and high phosphate, and 3) observe the in vitro effects of combinations of calcitriol, FGF-23, and soluble klotho on VSMC calcification in the presence of high phosphate.
SUBJECTS AND METHODS
Calcitriol was obtained from Tocris Biosciences. Recombinant human klotho and FGF-23 were obtained from R&D Systems.
For western blot analysis, anti-alkaline phosphatase (ALP), anti-a-smooth muscle actin (aSMA), anti-smooth muscle myosin heavy chain (SM-MHC), and anti-klotho antibodies were obtained from Abcam. Anti-ERK 1 and anti-ERK 2 antibodies were obtained from Santa Cruz Biotechnology.
Human primary aortic VSMCs were purchased from ATCC. Cells were grown and maintained using Dulbecco's Modified Eagles Medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were incubated at 37[degrees]C with 5% C[O.sub.2], and media was changed every 2-3 days.
For the experiments, the HVSMCs were seeded in cell culture plates and allowed to grow to ~80% confluency. Upon reaching ~80% confluency (day 0), each group received the different treatments for 7 or 14 days, with the media being changed every 2-3 days. During this time, all media contained 10% charcoal-stripped FBS in place of regular FBS to remove residual vitamin D metabolites. For experiments, normal phosphate (NP) groups refers to groups receiving standard cell culture media, and high phosphate (HP) groups refers to groups receiving media supplemented with 3 mM inorganic phosphate in the form of dibasic sodium phosphate. Cells were at passage 8 for all experiments.
To examine concentration-dependent effects, three concentrations (10, 100, and 1000 nM) of calcitriol were added to both NP and HP media. For this experiment, cells were given the appropriate media for 14 days. For further examination of the effects of calcitriol supplementation, a concentration of 100 nM was chosen, as it was the lowest concentration that a change was observed in calcium deposition. For these experiments, cells were given the appropriate media for both 7 and 14 days. When examining the effects of the combinations of calcitriol, FGF-23, and soluble klotho, concentrations of 100 nM for calcitriol, 10 ng/mL for FGF-23, and 0.4 nM for soluble klotho were chosen and added to HP media. One group was grown in NP media with the vehicle for comparison. For this experiment, cells were given the appropriate media for 14 days.
Calcium Deposition Quantification
Media was removed from the cell cultures, and they were rinsed gently with 1x phosphate buffered saline (PBS). The cell layers were then decalcified by using 0.6 N HCl for 24 hours. HCl supernatants were collected, and the cell layers were then solubilized using 0.1 N NaOH/0.1% sodium dodecyl sulfate (SDS) solution.
Calcium concentration of HCl supernatant were determined with atomic absorption spectroscopy (AA). It was conducted using a Shimadzu AA-7000F (Shimadzu Corp) and Calcium Atomax Hollow Cathode Lamp (PerkinElmer) using a wavelength of 422.7 nM. Calcium content was normalized to intracellular protein content. Protein concentration of the NaOH/SDS solution was determined using the Pierce bicinchoninic acid (BCA) assay (Thermo Scientific) following manufacturer's instructions.
Visualization of Calcium Deposition
Visualization of the calcium deposition was done using scanning electron microscopy (SEM). Prior to seeding cells in the six well plates, Thermanox plastic coverslips were placed in wells with the treated side face-up. After receiving the appropriate media for 7 or 14 days, media was removed, and cells were fixed in 1/2 Kamovsky's fixative in 0.1 M sodium cacodylate buffer for up to two weeks. The samples were processed for SEM imaging by further fixation using 0.4% osmium tetroxide followed by serial dehydration, using increasing concentrations of ethanol and hexamethyldisilazane (HMDS). Lastly, samples were allowed to air dry overnight, mounted, and sputtered coated with 15 nm of platinum using an EMS 1150T ES sputter coater (Electron Microscopy Science). Samples were imaged using a Carl Zeiss EVO50VP Variable Pressure Scanning Electron Microscope (Zeiss).
Examination of Protein Expression
Protein was obtained by placing cells in lysis buffer (10 mM Hepes, 150 mM NaCl, 1.5 mM Mg[Cl.sub.2], 1 mM EDTA, 10 mM Na-pyrophosphate, 10 mM NaF, 0.1 mM Na-orthovanadate, 1% Na deoxycholate, 1% Triton x 100, and 0.1% SDS), scraped, sonicated on ice for 5 seconds, and centrifuged at 15000 RPM to remove large cellular debris. Protein concentration was determined using BCA assay and diluted such that 20 pg of protein was loaded in each well. Samples were loaded into the wells of 10% or 8% SDS-Page gel and ran at a constant voltage (120 V) for approximately 2 hours or until tracking blue dye reached the bottom. Once finished, gels were transferred to PDVF membrane overnight.
Membranes were blocked with 5% non-fat dry milk in 1x Tris-Buffered Saline with 0.05% Tween 20 (TBST) buffer for one hour at room temperature. Primary antibodies were added to 5% milk in 1x TBST buffer, solution was placed on membranes, and membranes were allowed to shake overnight at 4o C. Membranes were rinsed with 1x TBST. Secondary antibodies were added to 1% milk with 1x TBST buffer and placed on membranes for 1-2 hours at room temperature. ECL solution was added to each membrane, allowed to incubate 5 minutes at room temperature, and developed on film.
Data is presented as mean [+ or -] standard deviation, and error bars on graphs represent standard deviation. For all AA experiments, n = 6. For western blot and SEM, n = 3. When comparing two groups, student t-test was used ([alpha] = 0.05). When comparing multiple groups, one-way ANOVA with Fisher's Least Significant Difference (LSD) post-hoc analysis was used ([alpha] = 0.05).
Effects of Calcitriol Concentrations on VSMC Calcification
To examine any concentration-dependent effect of calcitriol, three concentrations of calcitriol (10, 100, and 1000 nM) were added to VSMCs in the presence of normal and high phosphate, and calcium deposition was quantified with AA. As seen in Figure 1 (left), in the NP groups, only 1000 nM calcitriol (94.29 [+ or -] 83.1 [micro]g/mg protein) was able to cause a significant increase in calcium content compared to the vehicle (18.29 [+ or -] 20.39 [micro]g/mg protein). In the HP groups, both 100 nM (3293.02 [+ or -] 1674.01 [micro]g/mg protein) and 1000 nM calcitriol (2608.60 [+ or -] 1182.96 [micro]g/mg protein) caused a significant increase in calcium content compared to the vehicle (1078.52 [+ or -] 325.92 [micro]g/mg protein), but no significant difference was found between them. It is worth noting the calcium content value of 1000 nM NP group was still ~10 times lower than the vehicle HP group.
In-Depth Analysis of Calcitriol Supplementation on VSMC Calcification
To further analyze the effects of calcitriol supplementation on VSMC calcification, one concentration of calcitriol was chosen (100 nM). The increase in calcification of VSMCs observed in the previous experiment was confirmed using AA and observed qualitatively using SEM. Protein expression (aSMA, SM-MHC, ALP, and Klotho) was examined using western blot analysis.
The calcium content of VSMCs can be seen in Figure 1 (right). After 7 days, the supplementation of 100 nM (26.71 [+ or -] 10.62 [micro]g/mg protein) caused a significant increase in calcification compared to the vehicle (4.08 [+ or -] 0.75 [micro]g/mg protein) in the NP groups, while there was no effect in the HP groups (100 nM calcitriol, 166.94 [+ or -] 17.88 [micro]g/mg protein; vehicle, 169.41 [+ or -] 54.12 [micro]g/mg protein). On the contrary, at 14 days, we observed a significant increase in calcium content with the addition of 100 nM calcitriol in the HP groups (100 nM calcitriol, 654.66 [+ or -] 299.92 [micro]g/mg protein; vehicle, 380.20 [+ or -] 49.62 [micro]g/mg protein) but not the NP groups (100 nM calcitriol, 61.90 [+ or -] 6.58 [micro]g/mg protein; vehicle, 56.17 [+ or -] 7.40 [micro]g/mg protein).
SEM images (at 1000x magnification) after 7 and 14 days can be seen in Figure 2. Comparing the images at day 7, there appears to be more small nodules in the HP groups compared to the NP groups. Although the AA data showed that there was increase in calcification with calcitriol supplementation in the day 7 NP groups, there does not appear to be distinct morphological differences between these two groups. The images after 14 days of treatment are similar to the 7 day images, in that the HP groups appear to have more nodule formations than the NP groups. Also, while AA data showed an increase in calcification with the supplementation of 100 nM calcitriol in the 14 day HP group, there again did not appear to be any morphological differences between the two groups.
The protein expression of [alpha]SMA, SM-MHC, ALP, and klotho were examined using western blot analysis. The relative protein expression can be found in Figure 3. At 7 and 14 days, there was a decrease in [alpha]SMA between the NP and HP groups. However, there did not appear to be any effect in [alpha]SMA expression caused by the addition of 100 nM in either group. SM-MHC expression followed a similar trend as [alpha]SMA but to a lesser degree. There also did not appear to be any effect of phosphate or calcitriol supplementation on ALP expression. Finally, there did not appear to be any change to the total klotho expression between any of the groups. Total ERK was used as a loading control.
Effects of Calcitriol, FGF-23, and Klotho Supplementation on VSMC Calcification
In order to observe the effects from the interaction of calcitriol, FGF-23, and klotho, various combinations were added to VSMCs in HP conditions, and calcium content was quantified using AA. As seen in Figure 4, the addition of FGF-23 and calcitriol (29.63 [+ or -] 4.81 [micro]g/mg protein) caused a significant decrease in calcium content compared to the vehicle (726.35 [+ or -] 537.60 [micro]g/mg protein) and was similar to the calcium content of VSMCs that did not receive phosphate supplementation (9.18 [+ or -] 7.67 [micro]g/mg protein). Surprisingly, the calcium content of the VSMCs that received the combination of calcitriol, FGF-23, and klotho was significantly higher than all the other groups (3406.51 [+ or -] 810.45 [micro]g/mg protein).
In order to determine if calcitriol treatment promotes VSMC calcification, many in vitro and in vivo studies have been conducted; however, the results have often been contradictory. When looking at the concentration-dependent effects of calcitriol on VSMC calcification, we observed that 100 and 1000 nM calcitriol supplementation was able to increase the calcification in the presence of high phosphate and only 1000 nM calcitriol supplementation was able to increase the calcification in the presence of normal phosphate. The first study to reveal detrimental effects of calcitriol supplementation on in vitro VSMC calcification, published in 1998 by Jono et al, showed that calcitriol supplementation (as low as 10 nM) on bovine VSMCs increased calcification in the presence of high phosphate . Another study revealed that calcitriol supplementation (100 and 300 nM) increased calcification in vitro rat VSMC grown with p glycerosphosphate. It is worth noting that they did not find a difference between the calcification caused by 100 and 300 nM , similar to our 100 and 1000 nM HP groups. It was recently showed that calcitriol supplementation increased calcification in mouse VSMC cultures in not only high phosphate conditions, but also in normal phosphate conditions . One study showed calcitriol decreased in vitro calcification; however, this study involved the addition of an inflammatory cytokine that accelerates calcification . This, however, could be due to calcitriol's role in immune system regulation , as calcitriol did not have an effect on calcification caused by high phosphate alone in this study . Taken altogether, it is clear that calcitriol alone does have a direct effect on the in vitro VSMC calcification.
Interestingly, the degree of calcification caused by 1000 nM calcitriol in normal phosphate conditions was ~10 times lower than the calcification caused by high phosphate alone. This could help explain the effects caused by FGF-23  and klotho knockout . FGF-23 null mice with a normal diet had accelerated mortality and increased calcification, calcitriol, serum calcium, and serum phosphate, but low-phosphate dietary restriction resulted in no arterial calcification, despite high serum calcitriol . Very similar results were found when observing klotho knockout mice. They displayed growth retardation, increased calcification, and increased serum calcium, phosphate, FGF-23, and calcitriol, but with normalization of serum phosphate levels, vascular calcification was abolished, despite the continued high serum calcium and calcitriol . These results suggest that while calcitriol can have an effect with normal phosphate, high phosphate is more important to the pathogenesis of medial calcification.
When we further examined the effects of 100 nM calcitriol supplementation on the VSMCs with AA, SEM, and western blot. At 7 days, 100 nM calcitriol supplementation did not increase calcification in the presence of high phosphate but did with normal phosphate. After 14 days, 100 nM calcitriol supplementation increased calcification in the presence of high phosphate but did not in the absence of high phosphate after 14 days. A time-dependent effect has not been previously noted; however, it is worth noting that calcitriol's effects on osteoblast differentiation is highly dependent on the stage of maturation of the cell . In fact, recent research has shown that calcitriol accelerates matrix vesicle formation in osteoblasts but only during the early phase of differentiation . Thus, it is possible that calcitriol has a similar stage-dependent effect on VSMCs, but more research needs to be conducted before any conclusion can be reached. Looking at the SEM images, nodule formations appear in large number in the calcification groups but not the control groups, suggesting that these are the mineral deposits. Comparing the vehicle groups to the 100 nM calcitriol groups, there do not appear to be any distinct morphological differences to suggest a cause for the increase in calcification caused by the calcitriol supplementation. To the best of our knowledge, we are the first group to observe calcifying VSMCs using SEM. When looking at protein expression, we observed a loss of [alpha]SMA and SM-MHC expression in the calcification groups compared to the control groups at 7 and 14 days, consistent with previous studies that show that VSMCs deposit mineral and undergo transition to an osteoblast-like phenotype with the addition of high phosphate . On the other hand, there did not appear to be a difference in [alpha]SMA and SM-MHC expression between the vehicle and 100 nM calcitriol groups at either 7 or 14 days. Han et al showed an increase in runx2 expression after the supplementation of 100 nM calcitriol . While we were unable to detect runx2 expression in our cells (data not shown), upregulation of runx2 is typically coupled with the loss of smooth muscle markers, so it is surprising that we did not see a difference in [alpha]SMA or SM-MHC. Our results actually suggest that the increase in calcification caused by calcitriol supplementation may be independent of the phenotypic change.
Lastly, we examined the effects of combinations of calcitriol, FGF-23, and soluble klotho. We saw a significant decrease in calcification with the combination of calcitriol and FGF-23, agreeing with the findings of Lim et al. In their study, this decrease is due to the increased expression of transmembrane klotho, as klotho siRNA was able to abolish this decrease in their study . It is believed that the increased expression of transmembrane klotho allows FGF-23 to react with its receptor and exert a beneficial effect. Soluble klotho alone is believed to be able to elicit a positive effect, as Hu et al showed that the addition of 0.4 nM soluble klotho decreased VSMC calcification in vitro . In their experiment, they added 2 mM inorganic phosphate, so it is possible that 0.4 nM was too low of a concentration to elicit a similar, beneficial effect on the VSMC calcification caused by 3 mM inorganic phosphate in our study. The most surprising result was that the addition of all three, calcitriol, FGF-23, and soluble klotho, caused an extreme increase in VSMC calcification. This is the first time this combination has been added to VSMCs in the presence of high phosphate in vitro. Research has shown that soluble klotho can still act as a coreceptor for FGF-23 signaling , although it is believed to not be as active as the membrane form. Because the FGF-23 + calcitriol group and the FGF-23 + klotho group do not experience an increase in calcification, it may be possible that the combination of calcitriol, FGF-23, and klotho allows for excess FGF-23 activity. Supporting this idea, Jimbo et al found that FGF-23 causes concentration-dependent increase of calcification in klotho-over-expressing VSMCs in the presence of high phosphate . While more research needs to be conducted to determine the mechanism behind this interaction, it is clear that calcitriol's effect on VSMC calcification is complicated and involves interactions with the other endocrine molecules, FGF-23 and klotho. This, in part, may help explain the contradictory results found both in vitro and in vivo research.
The authors would like to acknowledge Ms. Amanda Lawrence at I2AT at Mississippi State University for assistance with SEM preparation and imaging and Dr. James Stewart and Ms. Amber Kay at the Department of Biological Sciences at Mississippi State University for assistance with western blot analysis. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1125191. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
Conflict of Interest: KAB, None to declare; CLS, None to declare
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Kevin A Bennett, Rachel Hybart, Chartrisa LaShan Simpson
Department of Agricultural and Biological Engineering, Mississippi State University, Mississippi State, MS, United States
Corresponding Author: Chartrisa LaShan Simpson, email@example.com
Caption: Figure 1. (Left) Effects of three concentrations of calcitriol on VSMC calcification in the presence of normal and high phosphate after 14 days. * P < 0.05 compared to HP vehicle and # P < 0.05 compared to NP vehicle b y Fisher's LSD post hoc test. (Right) Effects of 100 nM calcitriol on VSMC calcification in the presence of normal and high phosphate after 7 and 14 days. * P < 0.05 compared to vehicle with same treatment by student t-test.
Caption: Figure 2. Representative SEM images (1000x magnification) of (A) NP vehicle, (B) HP vehicle, (C) NP 100 nM calcitriol, and (D) HP 100 nM calcitriol after 7 days of treatment, (E) NP vehicle, (F) HP vehicle, (G) NP 100 nM calcitriol, and (H) HP 100 nM calcitriol after 14 days of treatment. Scale bar is equal to 10 [micro]m.
Caption: Figure 3. Relative protein expression of [alpha]SMA, SM-MHC, ALP, and klotho. Total ERK was used as loading control.
Caption: Figure 4. Effects of combinations of 100 nM calcitriol, 10 ng/mL FGF- 23, and 0.4 nM soluble klotho on VSMC calcification in the presence of high phosphate after 14 days. * P < 0.05 compared to all groups and # P < 0.05 compared to HP vehicle by Fisher's LSD post hoc test.