Vitamin D deficiency promotes intracranial aneurysm rupture

Abstract

Intracranial aneurysm rupture causes severe disability and high mortality. Epidemiological studies show a strong association between decreased vitamin D levels and an increase in aneurysm rupture. However, the causality and mechanism remain largely unknown. In this study, we tested whether vitamin D deficiency promotes aneurysm rupture and examined the underlying mechanism for the protective role of vitamin D against the development of aneurysm rupture utilizing a mouse model of intracranial aneurysm. Mice consuming a vitamin D-deficient diet had a higher rupture rate than mice with a regular diet. Vitamin D deficiency increased proinflammatory cytokines in the cerebral arteries. Concurrently, vitamin D receptor knockout mice had a higher rupture rate than the corresponding wild-type littermates. The vitamin D receptors on endothelial and vascular smooth muscle cells, but not on hematopoietic cells, mediated the effect of aneurysm rupture. Our results establish that vitamin D protects against the development of aneurysmal rupture through the vitamin D receptors on vascular endothelial and smooth muscle cells. Vitamin D supplementation may be a viable pharmacologic therapy for preventing aneurysm rupture.

Introduction

Intracranial aneurysm rupture results in subarachnoid hemorrhage with potentially devastating consequences. Mortality from aneurysmal rupture is up to 40%, with all-cause mortality at 6 months of approximately 60% and life-long dependency in 30% of survivors.^{1 –3} Small unruptured aneurysms are cautiously observed with serial imaging, while larger aneurysms undergo microsurgical clipping and endovascular embolization. ⁴ However, the adverse outcome rates for these invasive therapies can be significant, especially for large aneurysms or those in the posterior circulation. ⁵ Pharmacological prevention of rupture may forgo the risks of invasive treatment and is an attractive alternative.^{6 –10}

Clinical studies have shown a link between vitamin D deficiency and intracranial aneurysm rupture.^{11 –15} Exposure to sunlight is a major factor in maintaining normal serum vitamin D levels. Higher latitudes and winter months predispose populations to lower vitamin D levels.^16,17 Interestingly, multiple clinical studies have shown that decreased sunlight during winter months was correlated with an increase in aneurysmal rupture.^11,12,15 In recent years, the link between aneurysmal rupture and reduced vitamin D levels has been significantly strengthened from several clinical studies.^{13,18 –20} Despite these clinical studies showing an association between vitamin D deficiency and intracranial aneurysm rupture, a causal link has not been established, and the underlying biological mechanism remains unknown. Thus, in this study, using a mouse intracranial aneurysm model, we examined the causal link between vitamin D deficiency and aneurysm rupture and investigated the underlying mechanism by which vitamin D protects against the development of aneurysm rupture.

Materials and methods

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Mouse model of intracranial aneurysm

Experiments were conducted according to National Research Council’s Guide for the Care and Use of Laboratory Animals (Eighth Edition, 2011), and approved by the Institutional Animal Care and Use Committee of St. Joseph’s Hospital and Medical Center (Phoenix, AZ). The study was carried out adhering to ARRIVE guidelines, including blinding and randomization. ²¹ Details of our intracranial aneurysm mouse model were previously described.^{8,22 –26} We used C57BL/6J (The Jackson Laboratory, Bar Harbor, ME) and cell type-specific vitamin D receptor (VDR) knockout (KO) mice. ²⁷ We generated the endothelial cell (EC)-specific VDR KO (VDR^f/fTie2-Cre⁺), the vascular smooth muscle cell (VSMC)-specific VDR KO mice (VDR^f/fSM22-Cre⁺), the myeloid lineage cell (macrophages, monocytes, and granulocytes)-specific VDR KO mice (VDR^f/fLysM-Cre⁺), and their corresponding control littermates (VDR^f/f-Cre^–) using the Cre-lox breeding system. Intracranial aneurysms were induced by a combination of hypertension and a single-dose injection of elastase in the cerebrospinal fluid, as previously described (see Supplemental materials, Figure S1A).^{8,23 –26} We used the classical deoxycorticosterone acetate (DOCA)-salt hypertension method that involves a nephrectomy, followed by implantation of a subcutaneous DOCA pellet and switching to 1% sodium chloride drinking water one week later. ⁸ At this same point, a single dose of elastase (35.0 milli-units) was injected into the cerebrospinal fluid in the right basal cistern.^{8,22 –26} The same lot of elastase was used for all study groups. All mice were subjected to the same day/night light cycle, and no mice were exposed to natural light sources. 10–12-week-old male mice were used. Blood pressures were measured weekly after aneurysm induction using a tail cuff-based method.

Dietary supplement

The control diet contained 2000 IU/kg of vitamin D. Vitamin D-deficient diet (VDDD) was free of vitamin D (Research Diets Inc., New Brunswick, NJ). Mice were fed either the control diet or VDDD for 7 weeks before aneurysm induction. The diet duration was in accordance with the time range reported in the literature for both mice and rats.^{28 –30} Serum blood levels of vitamin D and calcium were measured at the time of aneurysm induction. 200 µL of blood was collected and placed in a K₃EDTA (tripotassium ethylenediaminetetraacetic acid) Minicollect tube (Greiner Bio-One International GmBH, Austria).

Evaluation of aneurysm formation and rupture

To determine whether an aneurysmal rupture occurred, two blinded observers performed neurological examinations daily, as previously established.^{22 –24} Mice were euthanized when they developed neurological symptoms associated with aneurysm rupture.^23,24,26,31 In this model, aneurysmal rupture has been reported to occur within three weeks of aneurysm induction (Figure S1A).^23,24 Therefore, asymptomatic mice were euthanized 21 days after aneurysm induction.^23,24,26,31 To visualize cerebral arteries and aneurysms, brain samples were perfused with phosphate-buffered saline, followed by a gelatin-containing 1% bromophenol blue dye. Two blinded observers assessed the intracranial arteries for the formation of aneurysms by examining the Circle of Willis and its major branches under a dissecting microscope (10X). Intracranial aneurysms were defined as localized outward bulging of the vascular wall in the Circle of Willis or its major primary branches.^8,22

Real-time PCR detection of cytokines

To assess the levels of inflammation-related cytokines in the cerebral arteries, we collected total RNA samples from cerebral arteries (Circle of Willis, including aneurysms) 5 days after aneurysm induction as previously described.^26,32 We measured mRNA expression levels of IL-1β (interleukin-1β), MCP-1 (monocyte chemoattractant protein-1), MMP-9 (matrix metallopeptidase 9), TNF-α (tumor necrosis factor-α), NF-κB (nuclear factor kappa B), TIMP-1/2 (tissue inhibitors of matrix metalloproteinases -1/2), RXR-α (retinoid X receptor alpha), iNOS (inducible nitric oxide synthase), VEGF (vascular endothelial cell growth factor) and TGF-β (transforming growth factor-β), and VDR.

RNA was extracted using the RNeasy Mini Kit (Qiagen, CA) and transcribed to cDNA using the QuantiTect reverse transcription kit (Qiagen). The mRNA expression levels were determined using SYBR Green technology (Applied Biosystems, CA). Quantitative values were obtained from the threshold cycle value (CT), and the data were analyzed by the 2^−ΔΔCT method. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression was quantified and used as an internal RNA control (See Supplemental materials for the primer sets used).

Experimental design and statistical analysis

To determine the role of VD deficiency in the formation and rupture of intracranial aneurysm, we employed two groups of animals treated with either control diet or VD deficient diet (Figure 1). To investigate the underlying mechanisms of VD deficiency on aneurysm formation/rupture, we used RT-PCR to determine mRNA levels of inflammatory cytokines in cerebral arteries in vitamin D-deficient diet and control diet-treated mice (Figure 2). To identify the cell type that mediates the effects of vitamin D against the development of aneurysm rupture, we compared the incidence of aneurysmal rupture in endothelial VD receptor (VDR) knockout (KO) mice (VDR^f/fTie2-Cre⁺, Figure 3), VSMC VDR KO mice (VDR^f/fSM22-Cre⁺, Figure 4), myeloid-lineage leukocyte VDR KO mice (VDR^f/fLysM-Cre⁺, Figure 5) with their corresponding control littermates (VDR^f/f-Cre⁻). The total and per group number of mice used in these studies is summarized in Supplemental materials Table S1. Inclusion and exclusion criteria for evaluating aneurysm rupture were predefined in a table encompassing all possible outcomes.

Figure 1.

Dietary vitamin D deficiency promotes aneurysmal rupture in a mouse model of intracranial aneurysm. (a and b) There was a significantly increased rupture rate in the vitamin D-deficient group compared to the control diet group. There was no difference in the incidence of aneurysms between the control diet and vitamin D deficient diet groups. (c) Symptom-free survival rate was lower in the vitamin D-deficient group. (d and e) Vitamin D deficient diet effectively decreased 25-hydroxy vitamin D in the serum but did not affect serum Ca²⁺ in the serum (n = 15 for both, P > 0.05) and (f) No difference in blood pressure between the two diet groups. ∗P < 0.05.

Figure 2.

Effect of dietary vitamin D deficiency on inflammation. Compared to the control diet group, in vitamin D-deficient mice, IL-1β, TNF-α, NF-κB, MMP-9, and MCP-1 in cerebral arteries were significantly higher, while TIMP-1 and TIMP-2 were significantly lower. VDR was also significantly higher. ∗P < 0.05. There was no difference between the two groups in mRNA levels of RXR-α, VEGF, TGF-β, and iNOS.

Figure 3.

Endothelial cell-specific knockout of vitamin D receptors increases the rupture rate of aneurysms. (a) No difference in the incidence of aneurysms between endothelial cell-specific vitamin D receptor knockout mice (VDR^f/fTie2-Cre⁺) and control mice (VDR^f/f). (b) Significantly increased rupture rate in VDR^f/fTie2-Cre⁺ mice. (c) Significantly lower symptom-free survival rate in VDR^f/fTie2-Cre⁺ mice and (d) No difference in blood pressure between the knockout group and control littermates. Fisher’s exact test was used to analyze the rupture rate of aneurysms. Log-rank (Mantel-Cox) test was used for the analysis of the survival rate. ∗P < 0.05.

Figure 4.

Smooth muscle cell-specific knockout of vitamin D receptors increases the rupture rate of aneurysms. (a) No difference in the incidence of aneurysms between smooth muscle cell-specific vitamin D receptor knockout mice (VDR^f/fSM22-Cre⁺) and control mice (VDR^f/f). (b) Significantly increased rupture rate in VDR^f/fSM22-Cre⁺ mice. (c) Significantly lower symptom-free survival rate in VDR^f/fSM22-Cre⁺ mice and (d) No difference in blood pressure between the knockout group and control littermates. Fisher’s exact test was used to analyze the rupture rate of aneurysms. Log-rank (Mantel-Cox) test was used for the analysis of the survival rate. ∗P < 0.05.

Figure 5.

Myeloid lineage cell-specific knockout of vitamin D receptors has no effect on the rupture rate of aneurysms. There was no difference in incidence rate (a), rupture rate of aneurysms (b), symptom-free survival rate (c) or blood pressures (d) between myeloid lineage cell-specific vitamin D receptor knockout mice (VDR^f/fLysM-Cre⁺) and control mice (VDR^f/f). Fisher’s exact test was used to analyze the rupture rate of aneurysms. Log-rank (Mantel-Cox) test was used for the analysis of the survival rate.

We used G*Power (Version 3.1.9.4) to calculate the sample size a priori based on the following criteria: For PCR experiments using Mann-Whitney analysis, α = 0.05, power of 0.8, and effect size of 1.8, the sample size was determined to be 5 for each group. For aneurysm rupture study using Fisher exact analysis, α = 0.05, power of 0.8, based on our previous experience with the model, assuming P1 = 0.8∼0.9, and P2 = 0.3∼0.4 the sample size was determined to be 10∼36 for each group. For the continuous data in Figure 2, the normality was examined by the Kolmogorov-Smirnov test in GraphPad Prism, and all data passed the exam.

Fisher’s exact test was used to analyze the incidences of aneurysm formation and rupture. For other continuous variables, we used the Mann-Whitney test. P-values <0.05 were considered statistically significant. Data are expressed as means ± SD.

Results

Vitamin D deficiency promoted aneurysmal rupture in a mouse model of intracranial aneurysm

As a first step, we assessed the effects of vitamin D deficiency on the formation and rupture of intracranial aneurysms. While there was no significant difference in the overall incidence of aneurysm formation between the groups fed with the control diet and the VDDD (85% vs. 94%; n = 13 vs. n = 17; Figure 1(a)), vitamin D deficiency significantly increased the aneurysm rupture rate (Figure 1(b); control diet vs. VDDD: 55% vs. 94%; P < 0.05). Accordingly, vitamin D-deficient mice had a significantly worse symptom-free survival rate than the control mice (control diet vs. VDDD: 54% vs. 12%, P < 0.05; Figure 1(c)). Vitamin D-deficient diet effectively reduced 25-hydroxy vitamin D concentration in the serum (control diet vs. vitamin D-deficient (VDDD): 36 ± 13 vs. 11 ± 2 ng/mL, n = 17 for both, P < 0.05, Figure 1(d)). However, the vitamin D-deficient diet did not have any effects on the serum Ca²⁺ concentration (control diet vs. vitamin D-deficient (VDDD): 4.7 ± 0.2 vs. 4.6 ± 0.2 ng/mL, n = 15 for both, P > 0.05, Figure 1(e)), excluding the possibility that the protective effect of vitamin D is through its effects on the serum Ca²⁺. There was no significant difference in blood pressure (Figure 1(f)) between the two groups at any time point.

Vitamin D deficiency increased proinflammatory cytokines in the cerebral arteries

To explore the mechanisms by which vitamin D deficiency promotes aneurysmal rupture, we used real-time RT-PCR to determine mRNA levels of inflammatory cytokines in cerebral arteries in vitamin D-deficient and control mice.

We found that mRNA levels of IL-1β, TNF-α, NF-κB, MMP-9, and MCP-1 in cerebral arteries were significantly higher in vitamin D-deficient mice than in the control group (IL-1 $\mathrm{\beta }$ : 1.0 ± 0.5 vs. 3.4 ± 1.7; TNF-α: 1.0 ± 0.5 vs. 1.6 ± 0.2; NF-κB: 1.0 ± 0.2 vs. 1.9 ± 0.4; MMP-9: 1.0 ± 0.2 vs. 2.2 ± 0.3; MCP-1: 1.0 ± 0.4 vs. 2.2 ± 0.9; n = 5∼6, all P < 0.05, Figure 2). In contrast, mRNA levels of TIMP-1 and TIMP-2 were significantly lower in vitamin D-deficient mice than in the control group (TIMP-1: 1.0 ± 0.3 vs. 0.5 ± 0.2; TIMP-2: 1.0 ± 0.6 vs. 0.5 ± 0.1; n = 5 for all, P < 0.05, Figure 2). Interestingly, vitamin D receptor levels were higher in vitamin D-deficient mice than in the control group (1.0 ± 0.7 vs. 2.0 ± 0.6; n = 6 for both, P < 0.05. Figure 2).

Vitamin D deficiency promotes aneurysmal rupture via the vitamin D receptors on the endothelial and vascular smooth muscle cells

To identify the cell type that mediates the protective effects of vitamin D against the development of aneurysm rupture, we utilized mice lacking vitamin D receptors in endothelial cells (EC), vascular smooth muscle cells (VSMC), and inflammatory cells. We compared the incidence of aneurysmal rupture among VDR^f/fTie2-Cre⁺ (endothelial VDR KO) mice, VDR^f/fSM22-Cre⁺ (VSMC VDR KO mice), VDR^f/fLysM-Cre⁺ (myeloid-lineage leukocyte VDR KO mice), and their corresponding control littermates (VDR^f/f-Cre⁻).

There were significant differences in the rupture rate and symptom-free survival rate between mice lacking vitamin D receptors in endothelial cells and control littermates (VDR^f/f vs. VDR^f/fTie2-Cre⁺: rupture rate: 40% vs. 92%; symptom-free survival rate: 60% vs. 8%; n = 10 vs. n = 14, both P < 0.05, Figure 3(a) to (c)). There were also significant differences in the rupture rate and symptom-free survival rate between mice lacking vitamin D receptors in vascular smooth muscle cells and control littermates (VDR^f/f vs. VDR^f/f SM22-Cre⁺: rupture rate: 36% vs. 86%; symptom-free survival rate: 64% vs. 14%, n = 12 vs. n = 15, both P < 0.05, Figure 4(a) to (c)). There was no significant difference in blood pressure between VDR^f/fTie2-Cre⁺ mice and control littermates (Figure 3(d)) or between VDR^f/fSM22-Cre⁺ mice and control littermates (Figure 4(d)).

Unlike VDR^f/fTie2-Cre⁺ or VDR^f/fSM22-Cre⁺ VDR KO mice, there was no difference in the rupture rate or symptom-free survival rate between mice lacking vitamin D receptors in leukocytes and control littermates (VDR^f/f vs. VDR^f/fLysM-Cre⁺: rupture rate: 55% vs. 54%; symptom-free survival rate: 45% vs. 41%, n = 13 vs. n = 15, both P = 0.83, Figure 5(a) to (c)). There was no significant difference in blood pressure between VDR^f/fLysM-Cre⁺ mice and control littermates (Figure 5(d)).

Discussion

Epidemiological and clinical studies have shown an association between decreased sunlight exposure, vitamin D deficiency, and intracranial aneurysm rupture.^{11 –15,18 –20} However, the underlying mechanism and the specific role of vitamin D in the rupture event is not well understood. In our experiments, vitamin D deficiency promoted aneurysm rupture while enhancing inflammation of cerebral arteries. Mechanisms independent of calcium seem to mediate these effects, given normal serum calcium levels in the vitamin D-deficient group. Vitamin D deficiency upregulated the proinflammatory cytokines IL-1β, TNF-α, NF-κB, MMP-9, and MCP-1, while downregulating the protective cytokines TIMP-1 and TIMP-2 in the cerebral arteries. Experiments using cell-type specific vitamin D receptor knockout mice found that vitamin D protects against the development of aneurysm rupture through its effects on vascular cells, specifically endothelial and vascular smooth muscle cells. Taken together, our findings indicate that vitamin D, affecting vascular vitamin D receptors, protects against the development of aneurysm rupture through the suppression of vascular inflammation.

Within the central nervous system vitamin D has significant protective effects. In traumatic brain injury, vitamin D supplementation has been shown to reduce neurocognitive deficits, edema, blood brain barrier disruption and promotes hippocampal neuronal survival. ³³ This neuroprotection is afforded through modulating microglial M2 polarization and neuroinflammation via the TLR4/MyD88/NF-κB pathway.^33,34 Furthermore, vitamin D was shown to be an anti-oxidant against D-Galactose induced Alzheimer’s in mice. ³⁵ Vitamin D receptors are expressed on both endothelial and vascular smooth muscle cells (VSMCs).^36,37 In endothelial cells, vitamin D suppresses NADPH oxidase, reduces expression of cyclooxygenase- 2, and increases production of nitric oxide by endothelial nitric oxide synthase. ³⁸ It, therefore, helps reduce levels of proinflammatory prostaglandins and oxidative stress to the vascular cells, which may prevent rupture.^38,39 Oxidative stress and inflammation upregulate matrix-degrading proteins and nuclear factor-κB (NF-κB). ⁴⁰ Activation of NF-κB induces the phenotypic changes of endothelial and vascular smooth muscle cells, resulting in excessive vascular remodeling and inflammation. ⁴⁰ VSMC transition to a proinflammatory phenotype has been shown to contribute to the pathophysiology of intracranial aneurysm. ⁴¹ The inhibition of NF-κB by vitamin D may prevent aneurysm rupture by blocking the phenotypic changes of vascular cells and reducing vascular inflammation. ³⁸

Activation of the VSMC vitamin D receptor has been shown to decrease the expression of TNF-α and increase the expression of NF-κB inhibitor, IκB-α. ⁴² The salient inflammatory role of TNF-α in intracranial aneurysm rupture has been shown through knockout and inhibition experiments. ⁴³ Vitamin D also regulates the expression of MMPs and affect the metabolism of collagen and elastin in VSMC. ³⁸ MMPs are responsible for degrading the extracellular matrix, inducing macrophage infiltration, and are upregulated in ruptured aneurysm walls. ⁴⁰ Knockout of tissue inhibitors of MMPs (TIMP-1 and TIMP-2) was shown to promote aneurysm progression, which is in agreement with our findings. ⁴⁴ These inflammatory and tissue remodeling cytokines may collectively mediate the protective effect of vitamin D against the development of aneurysm rupture.

Our results show that vitamin D supplementation may be a potential and practical strategy for reducing intracranial aneurysm rupture. Populations with decreased yearly exposure to sunlight or with low dietary intake of vitamin D may be the most to benefit from supplementation. However, vitamin D supplementation has not shown effect in peripheral arterial disease mitigation and certain populations such as those with kidney disease should proceed cautiously.^45,46 Consultation with a physician is mandatory.

There are several limitations in this study. First, the animal model employed in this study may not entirely replicate the full spectrum of biological events leading to aneurysm rupture, as the aneurysms were induced rather than occurring spontaneously. It is well-established that vascular inflammation plays a pivotal role in the pathophysiology of intracranial aneurysms in both humans and animals. Nevertheless, there could be significant disparities in the factors that trigger vascular inflammation between human aneurysms and this specific model. Despite these distinctions, the phenotypes of intracranial aneurysms in this model closely resemble those found in human intracranial aneurysms.^8,23 Most notably, this model exhibits the ultimate outcomes of aneurysmal rupture and associated neurological symptoms that are consistent with human aneurysms, indicating a similarity in the underlying biological processes.^8,47 Second, although we did not find a difference in serum calcium levels in our mice with vitamin D deficiency, we cannot completely exclude the role of vitamin D in local calcium metabolism. Under hemodynamic stresses, inflammation, and oxidative stress, VSMCs have been shown to switch their phenotype into osteogenic-like cells, which deposit a calcium-rich matrix.^{48 –50} Therefore, it may be possible that in a state of vitamin D deficiency, the proinflammatory state leads to osteogenic switching of VSMCs and a paradoxical process of microcalcification within the arterial wall. It is then during this microcalcification stage that the aneurysm wall may become vulnerable to rupture.

Third, as this was an initial investigation of the role of vitamin D in the pathophysiology of intracranial aneurysms, we used only male mice. We have shown that the incidence of aneurysm formation and rupture rates are higher in ovariectomized female mice than in male mice, indicating the protective effects of estrogen against the formation and rupture of intracranial aneurysms. ⁵¹ Especially given the interplay of vitamin D with sex differences, the difference in ruptures among male, non-ovariectomized female, and ovariectomized female mice should be carefully examined in future studies. Furthermore, the markers used for cell type (SM22, LysM, Tie2) are not specific to tissues solely within the intracranial compartment, therefore an unmeasured non-target effect may influence some of the results obtained.

Conclusion

We show that vitamin D deficiency promotes aneurysm rupture in a mouse aneurysm model. This occurs through a proinflammatory state generated at the level of vascular endothelial and smooth muscle cells. Vitamin D supplementation may be a practical treatment for the prevention of rupture in patients with intracranial aneurysms.

Supplemental Material

sj-pdf-1-jcb-10.1177_0271678X241226750 - Supplemental material for Vitamin D deficiency promotes intracranial aneurysm rupture

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X241226750 for Vitamin D deficiency promotes intracranial aneurysm rupture by Tetsuro Kimura, Redi Rahmani, Takeshi Miyamoto, Yoshinobu Kamio, Daisuke Kudo, Hiroki Sato, Taichi Ikedo, Jacob F Baranoski, Hiroki Uchikawa, Jinglu Ai, Michael T Lawton and Tomoki Hashimoto in Journal of Cerebral Blood Flow & Metabolism