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 (2-3-4-5). 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

Percutaneous balloon angioplasty (PTA) technique

This is a pilot study of six hemodialysis patients with arteriovenous grafts (AVGs), who suffered from persistent restenosis and were recruited to undergo vitamin D exposure treatment after successful PTA. Patients gave informed consent and the study was approved by the local ethical committee and was also in adherence with the Declaration of Helsinki. In short, post-angioplasty area created by balloon inflation clamp just at the proximal site to restenosis was a target lesion where 0.5 μg of calcitriol (Kyowa Hakko Kirin Co., Ltd, Tokyo, Japan) was administered directly through a side-hole catheter. We used iodine-contrast mixture when administered calcitriol to visualize perfect direct vitamin D exposure for 2 minutes. Percent patency (%patency), defined as angiographic visualization of a patent lesion, 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. It was then compared with the prior %patency, calculated 3 months post-conventional PTA well before the calcitriol intervention, since all six patients had needed repeated-PTA within 3 months due to severe recurrent VA restenosis.

In vitro study

Human VSMCs were purchased from Takara Bio Inc. (Tokyo, Japan) and were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and penicillin-streptomycin solution (culture medium) in a humidified incubator maintained at 37°C, with 5% CO₂. Cells were maintained at 70%-80% confluence by passaging as needed. Cells at passage <10 were used for the present study. VSMCs were cultured with the culture medium in six-well plates until they were 70%-80% confluent and were then cultured under the designated concentrations (0, 0.1, 1, 10, 100, 1000 nM) of calcitriol. To mimic clinical vitamin D intervention after PTA, we designed calcitriol-treated arms of two different exposure times (15 minutes and 24 hours).

Cell death detection ELISA

To investigate apoptosis induced by enzastaurin treatment, the Cell Death Detection ELISA (CDDE) plus (Roche, Mannheim, Germany), a sandwich enzyme immunoassay for detection of nucleosomes released during apoptotic process, was performed. Cells (5 x 10³ per well) were disseminated in 96-well microtiter plates (Sarstedt) in 200 µl of growth medium. After an overnight attachment period, cells were treated with 1, 2, and 10 µM of enzastaurin for 24 hours at 37°C. After accomplishing the ELISA procedure, as recommended by manufacturer, release of nucleosomes was determined by measuring the absorption spectrophotometrically at 405 nm. Accumulation of the detached mononucleosomes and oligonucleosomes into the cytoplasm was calculated as enrichment factor (absorbance of sample/absorbance of corresponding negative control). Assay was performed in duplicate and repeated twice.

Cell proliferation assay

The cell proliferation assay was performed by measuring 5-Bromo-2’-deoxy-uridine (BrdU) incorporation. To determine cell proliferation, human VSMCs were plated in 96-well plates and allowed to attach for 24 hours. Cells were then loaded with BrdU in the last 4 hours of treatment. BrdU incorporation was quantified by an immunofluorescence assay kit (Roche Applied Science, Mannheim, Germany), following manufacturer’s instructions. Three fields were chosen randomly from various sections to ensure objectivity of sampling. The total 100 cells from each field were counted, and BrdU-positive cell and the ratio of BrdU-positive cell per 100 cells were calculated. Assay was performed in triplicate and repeated thrice.

Immunohistochemical analyses

The tissue specimens of vascular stenotic anastomosis were obtained from a hemodialysis patient who underwent VA surgery, and fixed in 10% buffered formalin and embedded in paraffin for routine processing. Serial sections of 3-5 µm thickness were obtained from the paraffin embedded block. We stained them for hematoxylin and eosin (HE) and Elastica-van Gieson (EVG). For immunohistochemistry (IHC), adjacent sections were stained for vitamin D receptor (VDR, 1:10000 dilution; Abcam Inc., Cambridge, UK) as described elsewhere (12) and incubated with horseradish-conjugated secondary antibody (Nichirei Corporation, Tokyo, Japan).

Statistical analyses

Results are expressed as the mean RSD, unless otherwise noted. Paired or unpaired t-test was employed to assess the statistical differences. P-values <0.05 were considered significant.

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).

Fig. 1 - Fig. 2 -

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).

Fig. 3 -

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.

Fig. 4 -Fig. 5 -

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).

Fig. 6 -

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.

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

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).

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).

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).

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).

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).

• 1. Rotmans JI Pasterkamp G Verhagen HJ Pattynama PM Blankestijn PJ Stroes ES Hemodialysis access graft failure: time to revisit an unmet clinical need? J Nephrol 2005 18 1 9 20
• 2. Mattana J Effiong C Kapasi A Singhal PC Leukocyte-polytetrafluoroethylene interaction enhances proliferation of vascular smooth muscle cells via tumor necrosis factor-alpha secretion. Kidney Int 1997 52 6 1478 1485
• 3. Ezzahiri R Lemson MS Kitslaar PJ Leunissen KM Tordoir JH Haemodialysis vascular access and fistula surveillance methods in The Netherlands. Nephrol Dial Transplant 1999 14 9 2110 2115
• 4. Cinat ME Hopkins J Wilson SE A prospective evaluation of PTFE graft patency and surveillance techniques in hemodialysis access. Ann Vasc Surg 1999 13 2 191 198
• 5. Tordoir JH Hofstra L Leunissen KM Kitslaar PJ Early experience with stretch polytetrafluoroethylene grafts for haemodialysis access surgery: results of a prospective randomised study. Eur J Vasc Endovasc Surg 1995 9 3 305 309
• 6. Roy-Chaudhury P Sukhatme VP Cheung AK Hemodialysis vascular access dysfunction: a cellular and molecular viewpoint. J Am Soc Nephrol 2006 17 4 1112 1127
• 7. Nikkari ST Clowes AW Restenosis after vascular reconstruction. Ann Med 1994 26 2 95 100
• 8. Allaire E Clowes AW Endothelial cell injury in cardiovascular surgery: the intimal hyperplastic response. Ann Thorac Surg 1997 63 2 582 591
• 9. Aoshima Y Mizobuchi M Ogata H et al. Vitamin D receptor activators inhibit vascular smooth muscle cell mineralization induced by phosphate and TNF-α. Nephrol Dial Transplant 2012 27 5 1800 1806
• 10. Wong MS Leisegang MS Kruse C et al. Vitamin D promotes vascular regeneration. Circulation 2014 130 12 976 986
• 11. Valcheva P Cardus A Panizo S et al. Lack of vitamin D receptor causes stress-induced premature senescence in vascular smooth muscle cells through enhanced local angiotensin-II signals. Atherosclerosis 2014 235 2 247 255
• 12. Isidro RA Cruz ML Isidro AA et al. Immunohistochemical expression of SP-NK-1R-EGFR pathway and VDR in colonic inflammation and neoplasia. World J Gastroenterol 2015 21 6 1749 1758
• 13. Manson JE Mayne ST Clinton SK Vitamin D and prevention of cancer—ready for prime time? N Engl J Med 2011 364 15 1385 1387
• 14. Wasse H Huang R Long Q et al. Very high-dose cholecalciferol and arteriovenous fistula maturation in ESRD: a randomized, double-blind, placebo-controlled pilot study. J Vasc Access 2014 15 2 88 94
• 15. Katsanos K Karnabatidis D Kitrou P Spiliopoulos S Christeas N Siablis D Paclitaxel-coated balloon angioplasty vs. plain balloon dilation for the treatment of failing dialysis access: 6-month interim results from a prospective randomized controlled trial. J Endovasc Ther 2012 19 2 263 272
• 16. Portugaller RH Kalmar PI Deutschmann H The eternal tale of dialysis access vessels and restenosis: are drug-eluting balloons the solution? J Vasc Access 2014 15 6 439 447
• 17. Mary A Hénaut L Boudot C et al. Calcitriol prevents in vitro vascular smooth muscle cell mineralization by regulating calcium-sensing receptor expression. Endocrinology 2015 156 6 1965 1974
• 18. Kassi E Adamopoulos C Basdra EK Papavassiliou AG Role of vitamin D in atherosclerosis. Circulation 2013 128 23 2517 2531
• 19. Rebsamen MC Sun J Norman AW Liao JK 1alpha,25-dihydroxyvitamin D3 induces vascular smooth muscle cell migration via activation of phosphatidylinositol 3-kinase. Circ Res 2002 91 1 17 24
• 20. Tukaj C Trzonkowski P Pikuła M Hallmann A Tukaj S Increased migratory properties of aortal smooth muscle cells exposed to calcitriol in culture. J Steroid Biochem Mol Biol 2010 121 1-2 208 211
• 21. Raymond MA Désormeaux A Labelle A et al. Endothelial stress induces the release of vitamin D-binding protein, a novel growth factor. Biochem Biophys Res Commun 2005 338 3 1374 1382
• 22. Mary A Hénaut L Boudot C et al. Calcitriol prevents in vitro vascular smooth muscle cell mineralization by regulating calcium-sensing receptor expression. Endocrinology 2015 156 6 1965 1974