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Inflammation has a key role in the progression of the abdominal aortic aneurysm (AAA). The vitamin D receptor (VDR) is a widely expressed nuclear receptor with an expression pattern that comprises most leukocytes, endothelial cells as well as vascular smooth muscle cells.1, 2 In vitro studies show that activation of the VDR has strong immune-modulatory (anti-inflammatory) effects, and in the context of atherosclerotic disease it has been proposed that activation of the VDR may quench vascular inflammation. Although the validity of this concept has been well established in animal models, clinical studies consistently fail to show a benefit of VDR activation.3, 4 The basis for this discrepancy is unclear. Possible explanations include differences in inflammatory responses between preclinical disease models and the human situation.5, 6 Moreover, it has been pointed out that observations from situations of true vitamin D deficiency cannot simply be extrapolated to conditions characterized by a (sub) normal vitamin D status.7 Considering the apparent gap between preclinical expectations and outcomes of clinical interventions, we decided to explore the anti-inflammatory potential of VDR activation on the human vasculature.

The pathology of AAA is characterized by a broad and intense inflammation that comprises almost all aspects of the native and adaptive immune response.8, 9 In some patients, open surgical repair is indicated, thereby providing access to tissue. As such, this procedure provides a unique opportunity to test the anti-inflammatory potential of pharmaceutical interventions.

In the present study, we investigated the anti-inflammatory potential of a 2–4-week treatment with the potent VDR agonist, paricalcitol, in patients scheduled for elective, open AAA repair. Results from this study show that the anti-inflammatory potential of the VDR agonist paricalcitol in patients with subnormal vitamin D status is restricted to a selective effect on the calcineurin/NFAT axis, an observation that is supported by the ability of paricalcitol to effectively suppress lymphocyte proliferation in the mixed lymphocyte reaction (MLR).

MATERIALS AND METHODS

Patient Populations

This open proof‐of‐concept study was approved by the Medical Ethical Committee of the Leiden University Medical Center. Written informed consent was obtained from all patients. Patients scheduled for open AAA repair were eligible for the study. Decision for open repair was based on anatomical (eg, neck and elongation), and patients’ characteristics (eg, age) and preferences. Patients with impaired liver dysfunction (ALAT greater than three times upper limit of the reference values, hypercalcemia and/or hyperphosphatemia, patients on digoxin as well as patients with inflammatory disease or (suspected) so‐called inflammatory aortic aneurysms, were excluded from participation in the study. The study was started in November 2008 and the final patient was included in November 2010.

Patients received paricalcitol 1 μg once a day in the 2–4 weeks preceding their planned elective open repair. The final dose was taken in the evening before the surgery. Control AAA wall samples were obtained from the LUMC biobank, these samples were matched for sex, age, maximum AAA diameter, and statin use. AAA wall tissue was taken from the anterior–lateral aneurysm wall at the level of the maximal diameter of the aneurysm. All wall samples (viz., samples both study samples and biobank samples) were collected immediately after opening of the aneurysm sac. Adhering thrombus was carefully removed and wall samples were immediately halved. One-half was snap‐frozen in CO2‐cooled isopentane or liquid nitrogen and stored at −80 °C until use for mRNA (RT‐PCR) and protein (ELISA) analysis. The other half was fixed in formaldehyde (24 h), decalcified (Kristensens solution, 120 h), and paraffin embedded for histological analysis. All analyses were performed in an investigator‐blind manner.

Assessment of baseline vitamin D status was not foreseen in the study protocol. In the light of the study findings, we considered information on the vitamin D status of AAA patients in retrospect relevant. To that end, we measured vitamin D levels in the available plasma samples from AAA patients who participated in the PHAST study.10 Selected samples were all from patients from the same study center as the patients from whom aneurysm wall samples were available.

Immunohistochemistry

Slides were incubated overnight with antibodies against myeloperoxidase (MPO; rabbit polyclonal, 1:4000 dilution, DakoCytomation, Heverlee, Belgium), CD3 (polyclonal rabbit, 1:400 dilution, Abcam, Cambridge, UK), CD4 (clone 4B12, 1:200 dilution, DakoCytomation), CD8 (clone C8/144B, 1:200 dilution, DakoCytomation), CD20 (clone L26, 1:1000 dilution, DakoCytomation), CD68 (clone KP6, 1:1200, DakoCytomation), and CD138 (clone B‐B4, 1:1000 dilution, Serotec, Oxford, UK).9

For each section, six representative medium power fields (three photographs for ‘medial’ and three for the ‘adventitial’ layer) were photographed at a × 20 magnifier, and the number of positive cells counted.

Semi-quantitative mRNA Analysis

Total RNA extraction was performed using RNAzol (Campro Scientific, Veenendaal, The Netherlands) and glass beads.9 Copy DNA was prepared using kit #A3500 (Promega, Leiden, The Netherlands) and quantitative real‐time PCR (Taqman system) analysis was performed for human interleukin (IL)‐1β, IL-2, IL-4, IL‐6, IL‐8, IL-10, tumor necrosis factor‐α, interferon‐β, MCP‐1, perforin, B-lymphocyte-induced maturation protein-1 (BLIMP-1), matrix metalloproteinase (MMP)-2, -3, -9, and 12, and the cathepsins K, L, and S, on the ABI‐7500 Fast system (Life Biosciences, Nieuwerkerk aan den IJssel, The Netherlands) using established primer/probe sets (Assays on Demand, Life Biosciences) and Taqman Gene Expression Master Mix (Life Biosciences). Analyses were performed according to the manufacturer’s instructions. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) expression was used as a reference and for normalization.

Protein Analysis

Aortic wall tissues were pulverized in liquid nitrogen and homogenized in two volumes lysis buffer (10 mM Tris (pH 7.0), 0.1 mM CaCl2, 0.1 M NaCl, 0.25% (v/v) Triton X‐100). Samples were subsequently centrifuged at 10 000 g for 15 min at 4 °C, and the supernatant protein extract was snap‐frozen in liquid nitrogen and stored at –80 °C until analysis. Protein content in thawed protein extracts homogenates was determined with a BCA protein assay kit (Pierce, Rockford, IL, USA). Cytokine/chemokine protein levels in these homogenates were measured by separate ELISAs for IL‐6, IL‐8 (PeliKane compact kit, Sanquin, Amsterdam, The Netherlands), and MCP‐1 (Quantikine kit, R&D Systems, Abingdon, UK).

Relative tissue MMP2 (pro- and activated form) content was assessed by western blot using anti-MMP2 (sc-10736, Santa Cruz Biotechnology, Dallas, TX, USA) and GAPDH (sc-25778, Santa Cruz Biotechnology) for normalization. Blots were visualized using the supersignal West femto substrate kit (Life Technologies, Bleiswijk, The Netherlands) and chemiluminiscense visualized and quantified on a Chemidoc Touch Imaging system (Bio-Rad Laboratories, Veenendaal, The Netherlands).

T-cell Proliferation

MLRs were set-up with 50 μl of 1 × 10E6 donor PBMCs with 10E5 irradiated (30 Gy/3000 rad) HLA-mismatched stimulator cells (antigen-specific stimulus), or donor PBMCs were stimulated with the mitogen phytohemagglutinin (non-antigen-specific stimulus) in triplicate in 96-well round-bottomed plates (Greiner Bio-one) in the presence of different concentrations paricalcitol or one of the comparators (tacrolimus or mycophenolic acid). Proliferation was measured on day 5 by the incorporation of 3H-thymidine, which was added during the last 16 h of culture. The results were expressed as the median counts per minute for each triplicate culture.

Statistical Analysis

Statistical analyses were performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). For the comparisons, a P‐value <0.05 was considered statistically significant. Differences between the groups were evaluated by ANOVA (normally distributed data) or the Kruskal–Wallis test in case of non‐normally distributed continuous data. As all the observations in the paper fit within a theoretical framework, no correction for multiple testing was performed.

RESULTS

Patient Population

Baseline characteristics of the pre‐operative paricalcitol intervention group (n=11) and matched control group (n=11) used in the molecular analysis are shown in Table 1. Paricalcitol was well tolerated and there were no drop-outs.

Table 1 Baseline patient characteristics of the paricalcitol intervention study
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Plasma levels showed that all patients in the parallel AAA cohort are vitamin D insufficient by current standards11 (median level 43 (30–62 (IQR) nmol/l)).

Paricalcitol Intervention

AAAs are characterized by a broad cellular inflammatory component consisting of macrophages, neutrophils, and T and B cells.8, 9 Immunohistochemical analysis showed that paricalcitol reduced the aortic wall T-cell (CD3+) and T-helper cell (CD4+) content (P<0.024 and P<0.032 respectively), but did not influence the relative abundance of the other inflammatory cell types (ie, monocytes/macrophages (CD68+), neutrophils (MPO+), cytotoxic T cells (CD8+), B cells (CD20+), and plasma cells (CD138+)) (Figure 1) or distribution over the media and adventitia (Figure 2).

Figure 1
figure 1

Effect of paricalcitol on aneurysm wall leukocyte content. Semi-quantitative analysis of aortic wall T cells (CD3), T-helper cells (CD4), and cytotoxic T cells (CD8), B cells (CD20), monocyte/macrophage (CD68), plasma cells (CD138), and neutrophil (myeloperoxidase (MPO)) content. Cell counts are based on reflect the number of positive cells per six medium power fields. Cell content is expressed as the number of cells per mm2. Non-treated controls (white bars); paricalcitol-treated patients (gray bars). Boxplots indicate the interquartile ranges with the median. The vertical lines represent the range. *P<0.03.

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Figure 2
figure 2

Immunohistological staining for CD3 and CD4 in AAA wall samples from control and paricalcitol-treated individuals. AAA, abdominal aortic aneurysm.

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Evaluation of an effect of paricalcitol treatment on the mRNA levels of inflammatory mediators showed a selective reduction in the Th1/Th2 cytokines IL-2, 4, and 10. No effect was seen on the mRNA and protein levels of general proinflammatory cytokines (Table 2; Figure 3). Paricalcitol did not influence the expression of the B-cell-associated marker BLIMP-1 or the cytotoxic T-cell/NK cell marker perforin. On the protease level, paricalcitol reduced the expression of the cathepsins K and L, and increased the expression of MMP2 (Table 2). No effect was found on the expression of other MMPs. The change in MMP2 mRNA expression was not followed by an increase aneurysm wall protein content. On the contrary, VDR activation reduced both pro- and activated MMP2 content (relative expression: 1.27 (0.50) vs 0.74 (0.24) (proMMP2) and 0.31 (0.13) vs 0.18 (0.04) (activated MMP2), P<0.04).

Table 2 Relative mRNA expression of selected inflammatory mediators, proteases, cytokines, and cell activation markers (log transcript level relative to GAPDH (GAPDH=0))
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Figure 3
figure 3

Similar aneurysm wall protein interleukin-6, interleukin-8, and monocyte chemo-attractant protein-1 content in non-treated controls (white bars) and paricalcitol-treated patients (gray bars). Boxplots indicate the interquartile ranges with the median. The vertical lines represent the range. No differences were found between the two groups (ANOVA). IL, interleukin.

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An apparent selective effect on CD4+ T-helper cells, T-helper cell-associated cytokines, and a selective effect on cathepsin K and L expression imply an effect of paricalcitol on the calcineurin/NFAT signaling pathway. We explored such an effect12 in a MLR. Figure 4 shows that the effect size of paricalcitol-mediated T-cell proliferation was almost equal to that of tacrolimus. Cytotoxicity assays showed that this effect is not explained by excess cell death (results not shown).

Figure 4
figure 4

Relative effects of tacrolimus, paricalcitol, and mycophenolic acid (MPA) on the specific and non-specific T-cell response in a mixed lymphocyte reaction (control condition=100%). *P<0.04. PHA, phytohemagglutinin.

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DISCUSSION

This clinical study shows that a brief 2–4-week intervention with the VDR agonist paricalcitol exerts clear effects on aspects of vascular inflammation in the AAA. The selective effect on T-helper cells, the proteases cathepsin K and L, and the results of the mixed lymphocyte response are consistent with a selective effect on the calcineurin/NFAT axis.

Reports on the anti-inflammatory potential of VDR agonists are multiple and diverse. Reported mechanisms include, among others quenching of NFκB13 and MAPK14, 15 activation, as well as interference with macrophage activation16 or the Ang-2-Tie-2-MLC kinase cascade.17 Using the AAA as a clinical example of comprehensive vascular inflammation,8, 9 we show that the effects of VDR activation through the vitamin D analog paricalcitol are restricted, and include an effect on the aneurysm wall T-helper (CD4+) cell content and its associated cytokines, as well as an effect on the expression of the cysteine proteases cathepsins K and L. The ~75% reductions in both T-helper cell content and in the T-helper cell-related cytokines IL-2, 4, and 10 suggest that the reduction in cytokine expression primarily reflects a reduction of aortic wall cell content rather than an effect on cell activation. For logistic reasons, assessment of vitamin D status was not included in the study protocol. As of the remarkable findings, we considered information on the vitamin D status of AAA patients relevant. To address this point, we assessed vitamin D plasma levels in plasma samples from a group of AAA patients in this trial’s study center who participated in the PHAST study. All these patients were vitamin D insufficient, viz., none of the AAA patients tested was vitamin D deficient or sufficient.11

The observed selective effects on T-cell activation and the (contrasting) effect on the cysteine proteases cathepsin K and L converge at the level of the calcineurin/NFAT axis, such an effect that has been clearly demonstrated for vitamin D in in vitro studies.18, 19 A NFAT-1c responsive element has been described for the cathepsin L promoter region,20 as such the reduced cathepsin L expression may reflect reduced promoter activity. An alternative (but non-exclusive) explanation is that the reduced cathepsin L expression mirrors a reduction in CD4 content. Increased cathepsin K expression seemingly contrasts with the findings for cathepsin L. Yet, cathepsin K expression is in part regulated by NFAT-2,21 a factor that in contrast to NFAT-1c requires phosphorylation rather than dephosphorylation (NFAT-1c) for nuclear transfer. Hence, it has been pointed out that calcineurin inhibitors promote NFAT-2 activity.22 No effect was found of VDR agonist on RANKL expression,23 (results not shown). Opposed associations have been reported for a link between MMP2 expression and the calcineurin axis, as such it is unclear whether the increased MMP2 expression links to an effect on this axis or, alternatively, reflects a separate effect of VDR activation or a statistical type I error.24, 25 This latter possibility is supported by reduced MMP2 protein content in paricalcitol-treated patients.

In two previous studies, we have shown that brief 2–4-week pre-operative interventions with doxycycline and the ACE inhibitor ramipril profoundly reduced aortic wall inflammation through, respectively, the effects on the AP-1 (doxycycline)26 and NFκB27 proinflammatory pathways. Similarly, it was found that statins dose dependently reduce NFκB-driven inflammation in the aneurysm wall.28 Lacking clear effects on other inflammatory pathways, VDR activation appears highly selectively influencing NFAT-mediated inflammation. Our observations not necessary exclude an effect of VDR activation on other pathways as reported in experimental studies. Yet, such effects may only be apparent when studying vitamin-D-deficient individuals, and are missed in the real for life situation with most patients having suboptimal (ie, with plasma levels beyond 20 nmol/l as found in AAA patients) or normal vitamin D levels.

The observed selective effect of VDR activation on NFAT-mediated inflammation implies a role for VDR agonists in pathologies with established benefit from NFAT inhibition inhibitors, either as an low-toxic, moderate potent mono therapy or alternatively as an add-on therapy, allowing tempering of calcineurin inhibitor dose, thereby potentially limiting the negative side effects of this class of compounds.

A critical point is whether the observed effects of VDR activation are beneficial in the context of AAA disease or vascular disease in general (atherosclerosis). Although cathepsin K29 and L30 have both been implicated in the process of aneurysm formation,31 an apparent dominant role in AAA progression is challenged by the observation that reductions in cathepsin K and L expression during, respectively, statin28 and ACE inhibitor therapy27 are not followed by an effect on aneurysms growth. It is unclear whether and if the effects of VDR activation on T-helper cell content will influence AAA disease. A role for T-helper cells in the context of AAA disease remains elusive32 with reported observations from animal studies being inconclusive,33, 34 and clinically accelerated aneurysm progression during intense immune suppression.35

Along these lines, the potential benefit of VDR activation in the context of atherosclerotic disease remains unclear with clear benefits in preclinical studies, and consistent epidemiological association between vitamin D (sub) deficiency and manifest atherosclerotic disease,2 but very limited evidence for a benefit of vitamin D supplementation on manifestations of atherosclerotic disease.3, 4

This interventional study has a number of limitations. The study is not placebo controlled and small. Yet given the progressive decline in open AAA repair procedures, interventional studies such as this become more and more difficult to perform. We have chosen for an individual matching procedure of cases and control material from our tissue bank; this approach reduces clinical variation in the study and removes potential strong confounders, and is very suited to observe subtle differences in pathways. However, the matched design does not circumvent biases as in the double-blinded randomized trials. The consistent data from the different platforms used in this study make a type I error extremely unlikely. A larger sample size would obviously have resulted in smaller confidence intervals. We cannot exclude that minor effects on other inflammatory pathways are missed due to a type II statistical error. Yet, if such effects exist, they presumably compare weakly with the effect exerted on the NFAT pathways, and with pleiotropic effects on NFκB and AP-1 signaling exerted by, respectively, statins28 and ACE inhibitors,27 and doxycycline.26 A further limitation of the study is that evaluation of baseline vitamin D status was not included in the study protocol. We therefore assessed vitamin D levels in plasma from AAA patients participating PHAST study who were included in same center as the patients in this study.10 Without any exception, all patients in this AAA group had insufficient vitamin D plasma levels, as such it is unclear whether and how the observations from this study translate to vitamin-D-deficient and -sufficient individuals.

In conclusion, the anti-inflammatory potential of VDR activation in vitamin-D-insufficient individuals is highly selective and appears mediated by an effect on the NFAT/calcineurin axis.