Abstract
Purpose
Neointimal hyperplasia (NH) causes vascular access (VA) stenosis, which results in serious under-dialytic morbidity in hemodialysis patients. We sought to assess whether a vitamin D intervention to NH lesions leads to better VA patency and examined clinical and in vitro studies.
Methods
A pilot clinical study of six hemodialysis patients was conducted to elucidate whether 0.5 μg calcitriol injection to stenotic lesion after balloon angioplasty (PTA) maintains better vessel patency until the next follow-up angiography. Localized vitamin D exposure was utilized by delivering and fixing calcitriol intensively at the stenotic lesion through a side-hole catheter with balloon clamping. We also performed vascular smooth muscle cell (VSMC) culture to detect both apoptosis (cell death detection assay) and cell viability (5-Bromo-2′-deoxy-uridine incorporation), and explored the efficacy of vitamin D to inhibit VSMC proliferation. Additionally, immunohistochemistry (IHC) was conducted to examine vitamin D receptor (VDR) expression at NH lesion, obtained from VA surgery.
Results
Percent patency, the proportion between stenotic and non-stenotic vessel diameters, increased significantly (p = 0.03) after directly catheter-delivered 0.5 μg calcitriol administration. In vitro VSMC studies, 0.1 nM calcitriol significantly (p<0.05) enhanced apoptosis and cell-cycle inhibition for two different calcitriol exposure times (15 minutes and 24 hours). IHC staining revealed that VDR-positive hyperplastic cells were observed at NH lesion.
Conclusions
Intensive vitamin D exposure at NH lesion has an ability to inhibit further VSMC proliferation, and presumably leads to greater patency rate for recurrent VA stenosis. Further studies are needed to clarify whether its unique property is exhibited through VDR-mediated mechanism.
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
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 × 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).
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 (
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).
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.
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 (
Cell proliferation assay. Calcitriol exposure at 0.1 nM for 15 minutes clearly inhibited vascular smooth cell proliferation (
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).
Histological features of neointimal hyperplasia (NH) of vessel stenosis, obtained from vascular access surgery. Representative cross-sections of the hyperplastic vessel wall (
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.
Footnotes
Acknowledgement
The authors thank Division of Nephrology, Nagoya Daini Red Cross Hospital for kindly advising immunohistochemistry imaging.
Financial support: This research was supported by Grants-in-Aid from ROD21.
Conflict of interest: The authors have no conflicts of interest.