An Efficacy of Intensive Vitamin D Delivery to Neointimal Hyperplasia in Recurrent Vascular Access Stenosis

Introduction

Vascular access (VA) failure, such as recurrent stenosis and thrombosis, constitutes a major cause of morbidity for patients on chronic hemodialysis for end-stage kidney disease (1).

Neointimal hyperplasia (NH) formation at the anastomosis site and/or the outflow veins, followed by in situ thrombosis, is a primary cause for recurrent VA failure, because NH is seen at the anastomosis involving an artery, a synthetic graft or the outflow veins (2–5). Vascular smooth muscle cell (VSMC) proliferation and migration into the luminal surface play a key role for NH lesion formation (6, 7), thus, an agent to disrupt the mechanism toward VSMC proliferation/migration could be a promising candidate to inhibit restenosis of VA, because post-angio-plastic tissue response drives further NH under the mechanism where damaged endothelial cells and VSMCs release growth factors such as basic fibroblast growth factor (bFGF) that simulate VSMC proliferation in the media (8).

Vitamin D exhibits its protective property for vascular calcification (9) and may be a promising option for vascular regeneration (10), partly via vitamin D receptor (VDR) (11). Although vitamin D and its receptor activators have been vigorously studied with respect to efficacy on VSMCs, it is still unclear whether vitamin D has an inhibitory property against VSMC-driven NH in VA vessel stenosis. We conducted both clinical and in vitro studies to investigate whether vitamin D, especially when administered at relatively higher concentrations for a short period of time, has an efficacy to suppress NH progression.

Methods

Results

AVG was affected at the axillary vein-graft anastomosis (Fig. 1). Conventional PTA was performed (Fig. 2A) and 0.5 μg calcitriol was then administered with radiocontrast via a side-hole catheter and 2-minute clamp by balloon inflation (Fig. 2B), resulting in high concentrations of vitamin D exposure at the stenotic lesion after PTA. Post-PTA angiography represented enlarged vessel (Fig. 2C) and favorable vessel diameter was maintained until the next follow-up angiography (Fig. 2D).

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Fig. 1

Contrast angiography showing a 74-year-old hemodialysis patient, undergoing a brachio-axillary AVG with marked stenosis at the graft-venous anastomosis.

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Fig. 2

Percutaneous balloon angioplasty (PTA) was performed (A) balloon inflation up to 14 atm. Two-minute balloon clamp technique was used and 0.5 μg calcitriol/contrast mixture was visualized at the stenotic lesion (B). Vessel enlargement was confirmed after successful PTA (C), and vessel patency was well maintained until the next follow-up study (D).

The %patency was well-maintained 3 months after the focus calcitriol strategy at NH lesions, compared with the one before the calcitriol intervention (p = 0.03, Fig. 3).

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Fig. 3

Percent patency (%patency), calculated as the proportion between stenotic and non-stenotic vessel diameters, was measured with the first follow-up angiography 3 months after local calcitriol injection, and compared with the prior %patency well before the calcitriol intervention. Vessel patency significantly increased after single dose of 0.5 μg calcitriol-treated PTA was performed intensively at stenotic vessels (p = 0.03).

Figure 4 clearly shows VSMC apoptosis was induced at certain concentrations of calcitriol (0.1-1 nM) for two different co-incubation times (15 minutes and 24 hours). Interestingly, 0.1 nM calcitriol consistently blocked VSMC proliferation for two different exposure times (15 minutes and 24 hours, Fig. 5), indicating that relatively short exposure of vitamin D may reduce VSMC proliferation possibly by enhancing apoptosis.

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Fig. 4

Cell death detection ELISA assay, a sandwich enzyme immunoassay for detection of nucleosomes, indicated that vascular smooth muscle cell apoptosis was induced extensively at 0.1 and 1.0 nM calcitriol treatment for 15 minutes (A, black bar) and 24 hours (B, grey bar).

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Fig. 5

Cell proliferation assay. Calcitriol exposure at 0.1 nM for 15 minutes clearly inhibited vascular smooth cell proliferation (A, black bar). Similar inhibitory effect was observed at 24-hour co-incubation of 0.1 nM and 1 nM calcitriol (B, grey bar).

Our histological findings manifested typical NH lesions of hypertrophic vessel wall obtained from VA surgery (Fig. 6A), and hyperplastic VSMCs were positively stained for EVG (Fig. 6B). Cross-sections of NH lesions were highly immune-reactive to VDR (Fig. 6C) and nuclear localization of VDR was observed at NH lesions (arrows, Fig. 6D).

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Fig. 6

Histological features of neointimal hyperplasia (NH) of vessel stenosis, obtained from vascular access surgery. Representative cross-sections of the hyperplastic vessel wall (A-D). NH lesion was identified (A) stained for hematoxylin and eosin (HE) (B) stained for Elastica-van Gieson (EVG). Scale bar, 500 μm in panels (A) and (B). NH lesion was abundantly stained for vitamin D receptor (VDR) in hyperplastic vessel wall (C). Nuclear staining (brown) for VDR was positively recognized at NH lesion (D, arrows). Scale bar, 100 μm in panel (C) and (D).

Discussion

The present study has first demonstrated that ‘intensive’ vitamin D treatment may inhibit NH formation of VA at frequently restenotic lesions, when a certain level of vitamin D is maintained at those lesions for a relatively shorter period of time. Our in vitro study supports possible mechanism, in which certain concentrations of vitamin D induce VSMC apoptosis, presumably inhibiting afferent vessel NH formation.

Vitamin D has also been presented as anti-proliferative property against various kinds of cancer cells (13). It is true that higher systemic administration of vitamin D increases serum 1,25-dihydroxylvitamin D levels, which may not eventually improve VA maturation in one clinical randomized controlled pilot study (14). Since NH in VA consists of VSMC proliferation at localized vessels, proper vitamin D delivery and exposure to stenotic lesions may effectively reduce recurrent vessel stenosis and improve the primary patency rate, without any adverse events, including hypercalcemia. Unfortunately, our data lacked serum calcium and phosphorous levels post-calcitriol intensive injection. Yet, it is of great interest that a single dose of 0.5 μg calcitriol, when ‘intensively’ administered, may or may not change bone mineral milieu in end-stage kidney disease.

Our results have shed some light into drug-coated balloon intervention, because several anti-proliferative agent-coating balloons render a promising option to frequent restenosis of VA (15, 16). It might be indicated that PTA balloon coated with anti-proliferative vitamin D receptor activator could interrupt cell growth of VSMC at NH lesion and eventually enhance VA patency.

Our study has limitations, such as a small pilot study and a relatively shorter observation period. Still, there is solid evidence that the majority of hemodialysis patients suffer both absolute vitamin D deficiency and VA failure.

VSMCs, considered key cells toward NH formation, are treated historically with calcitriol and other vitamin D receptor activators for 24 to 48 hours in vitro in conventional vitamin D studies, which have been relevant experiments for vitamin D effect on vascular calcification (9, 17). Specifically, 0.1-1 nM concentrations of vitamin D have been thought to play a crucial role for various effects (17). Those findings strengthen the rationale of our in vitro experiment design using 0.1-1.0 nM concentrations of calcitriol. Furthermore, 15-minute co-incubation of calcitriol, which was an extremely shorter exposure time than 24 hours, had similar anti-mitotic and pro-apoptotic effects on human VSMCs in our experiments. All of our experimental data potentially support our strategy for anti-NH formation, by applying intensively direct calcitriol exposure into VA stenotic lesion in clinical settings.

Calcitriol has been demonstrated to have diverse effects on VSMCs (18). At super-physiologically high doses, calcitriol induces VSMC migration through both the genomic and non-genomic pathways (19, 20). In contrast, calcitriol inhibits VSMC migration and proliferation by a mechanism that attenuates extracellular signal-regulated kinase 1/2 phosphorylation (21). Our in vitro data may support ‘biphasic’ effects of calcitriol, because SMCs treated with higher concentrations, enhance the inverse effects of calcitriol on both apoptosis and cell proliferation. Further studies are needed to elucidate this controversial issue from different approaches (22).

Prospective large-scale studies are encouraged to examine whether a targeted single dose of vitamin D, if administered intensively at NH lesions, improves the patency rate of VA by blocking VSMC proliferation, previously activated into the luminal surface of stenotic vessels. If that is the case, the crux of an argument is whether those vitamin D effects are VDR-mediated, and whether its unique property is recognized through genomic or non-genomic VDR-mediated mechanisms, simply because hyperplastic SMCs of NH lesions have immune-reactivity to VDR, according to our IHC results.

Conclusion

Localized vitamin D exposure at NH stenosis in VA failure has a potential to improve VA patency by the anti-proliferative mechanism through which vitamin D effectively enhance apoptosis to hyperplastic VSMCs.