Vitamin D is a regulator of endothelial nitric oxide synthase and arterial stiffness in mice

Abstract

The vitamin D hormone 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] is essential for the preservation of serum calcium and phosphate levels but may also be important for the regulation of cardiovascular function. Epidemiological data in humans have shown that vitamin D insufficiency is associated with hypertension, left ventricular hypertrophy, increased arterial stiffness, and endothelial dysfunction in normal subjects and in patients with chronic kidney disease and type 2 diabetes. However, the pathophysiological mechanisms underlying these associations remain largely unexplained. In this study, we aimed to decipher the mechanisms by which 1,25(OH)2D3 may regulate systemic vascular tone and cardiac function, using mice carrying a mutant, functionally inactive vitamin D receptor (VDR). To normalize calcium homeostasis in VDR mutant mice, we fed the mice lifelong with the so-called rescue diet enriched with calcium, phosphate, and lactose. Here, we report that VDR mutant mice are characterized by lower bioavailability of the vasodilator nitric oxide (NO) due to reduced expression of the key NO synthesizing enzyme, endothelial NO synthase, leading to endothelial dysfunction, increased arterial stiffness, increased aortic impedance, structural remodeling of the aorta, and impaired systolic and diastolic heart function at later ages, independent of changes in the renin-angiotensin system. We further demonstrate that 1,25(OH)2D3 is a direct transcriptional regulator of endothelial NO synthase. Our data demonstrate the importance of intact VDR signaling in the preservation of vascular function and may provide a mechanistic explanation for epidemiological data in humans showing that vitamin D insufficiency is associated with hypertension and endothelial dysfunction.

Figures

Figure 1.

Mean arterial pressure and the RAAS are unchanged in VDR-deficient mice fed the rescue diet. A, Ratio of heart weight (HW) to body weight (BW) in WT and VDRΔ/Δ mice. B, Representative histological heart sections demonstrating cardiac hypertrophy in VDRΔ/Δ mice. Hematoxylin and eosin staining after perfusion fixation of 9-month-old mice. C, Mean arterial pressure (MAP) in the ascending aorta. D, Kidney renin mRNA levels were elevated in 9-month-old VDRΔ/Δ mice fed the normal diet (ND, left) but not in VDRΔ/Δ mice fed the rescue diet (RD, right) enriched in calcium, phosphate, and lactose. E and F, Serum (E) and urinary (F) aldosterone levels were elevated in 9-month-old VDRΔ/Δ mice fed the normal diet but not those fed the rescue diet as measured by ELISA. No differences between the groups were observed in renin mRNA or aldosterone levels at 3 months of age. G, Plasma renin activity (PRA) was similar in 3- and 9-month-old WT and VDRΔ/Δ mice fed the rescue diet. Values in A and C to G are means ± SEM of 4 to 8 mice per group. *, P < .05 comparing VDRΔ/Δ with WT mice fed the same diet.

Figure 2.

VDR deficiency alters vascular and cardiac parameters in aged mice. Aortic and cardiac pressure analysis by catheterization of the ascending aorta and left ventricle. Values are means ± SEM of 4 to 8 mice per group. *, P < .05 comparing 9-month-old VDRΔ/Δ with WT mice. PP, pulse pressure; LVEF, LV ejection fraction measured by echocardiography; Max dP/dt, the first derivative of the developed pressure; EDP, end-diastolic LV pressure; Tau, isovolumetric relaxation time.

Figure 3.

Aortic functional changes due to VDR deficiency. A, Parameters of arterial stiffening are increased in 9-month-old VDRΔ/Δ mice. Ascending aortic impedance (left) was evaluated as the ratio of aortic pressure to luminal average blood velocity (n = 4–8 each). Augmentation index (right) was calculated as a ratio of the pressure at the augmentation point and the pulse pressure (n = 4–8 each). B, Representative Doppler echograms of the aortic flow velocities recorded in WT mice (top panels) and VDRΔ/Δ mice (bottom panels). Values are means ± SEM. *, P < .05 comparing 9-month-old VDRΔ/Δ with WT mice.

Figure 4.

Aortic structural changes in VDR-deficient mice. A, Changes in the collagen and elastin protein content in the thoracic aorta of VDRΔ/Δ mice. Bars are collagen/elastin content expressed as a percentage of that of WT mice at the respective age (n = 6–9 each). B, Representative histological thoracic aorta sections stained with hematoxylin and eosin (left) and elastic lamellae stained by resorcin-fuchsin (middle and right) in 9-month-old WT and VDRΔ/Δ mice. Right panels are higher magnification views of the insets depicted in middle panels. C, Scanning electron micrograph of elastin residues in the thoracic aorta of 9-month-old WT and VDRΔ/Δ mice. Values are means ± SEM. *, P < .05 comparing 9-month-old VDRΔ/Δ with WT mice.

Figure 5.

VDR controls NO production and NOS3 gene expression in vivo. A, NO production assessed by measuring nitrite and nitrate levels in ultrafiltrated serum (n = 4–8 each), and urine (n = 6 each). B, Reduced mRNA expression of NOS3 in the aorta of VDRΔ/Δ mice (n = 5–8 each). C, Reduced aortic protein expression of NOS3 in VDRΔ/Δ mice (n = 4–6 each). Values are means ± SEM. *, P < .05; **, P < .01 comparing VDRΔ/Δ with WT mice.

Figure 6.

VDR and NOS3 expression in aortic endothelial and smooth muscle cells. A, LacZ reporter gene expression visualized by X-Gal staining to detect localization of the VDR. Intense staining is present in the endothelial and medial layer of the thoracic aorta (blue) in the VDRΔ/Δ mouse, whereas staining is completely absent in the WT mouse. The parathyroid gland was used as a positive control for VDR expression. Right panels are higher magnification views of the insets depicted in the left panels. B, mRNA expression of VDR and NOS3 in the endothelial and smooth muscle layer harvested by laser capture microdissection from cryosections of the thoracic aorta from 9-month-old WT and VDRΔ/Δ mice and normalized to the total aorta preparation. Values are means ± SEM. *, P < .05 comparing VDRΔ/Δ with WT mice (4–5 samples per group).

Figure 7.

Vitamin D is a direct transcriptional regulator of the NOS3 gene. A, In vitro exposure to 1,25(OH)2D3 and 25(OH)D3 stimulated expression of NOS3 mRNA in the aorta. Rings of the thoracic aorta isolated from 3-month-old WT and VDRΔ/Δ mice were incubated with 10−8 M 1,25(OH)2D3, 10−7 M 25(OH)D3, or vehicle (Veh, ethanol) for 24 hours in organ culture (n = 3 each). B, 1,25(OH)2D3 increases NOS3 gene transcription in HEK-293 cells transiently transfected with the pCMV6 plasmid construct containing human VDR (hVDR) and the PCC2FOS plasmid construct containing the full-length human NOS3 GFP-tagged gene, including the promoter region (hNOS3). GFP expression (4–6 samples per group) was determined 24 hours after stimulation with 10−10, 10−9, 10−8, or 10−7 M 1,25(OH)2D3 or vehicle (ethanol). Values are means ± SEM. *, P < .05 comparing 1,25(OH)2D3 vs vehicle (in A) or 1,25(OH)2D3 vs vehicle (double-transfected HEK-293 cells) and vs NOS3 single-transfected HEK-293 cells (vehicle and 1,25(OH)2D3 treatment) (in B).

Figure 8.

VDR-driven mechanism regulating endothelial NO production by stimulation of NOS3 transcription. After binding to its ligand 1,25(OH)2D3, the activated VDR stimulates NOS3 transcription, resulting in increased production of NO, which in turn causes vascular smooth muscle cell relaxation, thereby reducing arterial stiffness and cardiac afterload.