Vitamin D intervention does not improve vascular regeneration in diet-induced obese male mice with peripheral ischemia
Vitamin D promotes or inhibits neovascularization in a disease context-dependent manner
- Supplementation of high palmitate medium with vitamin D, niacin or both vitamins increased EC tube formation, which relies primarily on cell migration, and also maintained tube stability over time.
- Both vitamins increased stress response and anti-inflammatory gene expression, but only vitamin D improved recovery of hind limb use following ischemic injury.
Despite widespread vitamin supplement use for the promotion of vascular health, it is not clear whether this approach is beneficial
- Recent meta-analyses [1] and primary end point analyses from the VITamin D and OmegA-3 TriaL (VITAL) indicate no general cardiovascular benefit associated with consumption of the most popular supplements, including vitamin D
- However, vitamin D supplementation has been associated with improved endothelial function in subgroup analyses of individuals with diabetes
- The effects of vitamin D intervention on peripheral ischemia in the setting of obesity and hyperlipidemia, and the potential vascular regenerative mechanisms involved are unclear
Endothelial cell culture
- Primary human adult dermal microvascular endothelial cells from a single donor were maintained in Medium 199 (Life Technologies, Carlsbad, CA, USA) supplemented with EGM-2MV SingleQuots (Lonza).
- For vitamin treatments, medium was supplemented with niacin (Sigma Aldrich, Oakville, ON, Canada) solubilized in cell culture grade water and/or the active form of vitamin D (1,25-dihydroxyvitamin D3; Sigma Aldrich.)
- Initially, we compared the effects of vehicle treatments (water and DMSO) under both BSA and palmitate conditions to confirm that there were no effects.
Tube formation and stability
- Cells were seeded onto a thin layer of growth-factor-replete Matrigel basement membrane matrix (Corning, Bedford, MA, USA) in a 96-well plate.
- Resulting tube networks were imaged by light microscopy at 18 h to assess maximum tube formation and at 24 and 42 h to assuage tube stability using an Olympus IX71 inverted microscope.
Global gene expression
- Cells were treated for 16 h with control medium (BSA) or media containing palmitate (0.5 mM) supplemented with either niacin (10 μM) or vitamin D (10 nM).
- RNA was prepared as previously described.
- Microarray file data were imported into Partek Genomics Suite v6.6 using the RMA algorithm. Statistically significant differences were determined by ANOVA (P<.05).
qRT-PCR
- Differential expression of genes of interest from microarray analyses was independently confirmed by qRT–PCR using TaqMan assays for CXCL8, DDIT3, IL1A, HMOX1, SMOX, BMX, MANF, BAMBI, CEBPB, CDK1, MCM5, CYP24A1 and MIR126
Endothelial cell proliferation
- To determine population doubling rates, cells were seeded at 2500 cells/cm2 and incubated with control medium (BSA) or media containing palmitate (0.5 mM) supplemented with niacin (10 μM) or vitamin D (10 nM) for 16 h.
Mouse model of diet-induced obesity and hyperlipidemia
- Five-week-old male 129S6/SvEvTac mice (Taconic, Germantown, NY, USA) were fed a Western diet containing 42% of calories from animal fat ad libitum for a total of 17 weeks.
- Immediately prior to sacrifice, mice were fasted for 6 h and weighed, and blood glucose was determined by handheld glucometer (Bayer, Mississauga, ON, Canada). All remaining parameters were determined postmortem.
Hind limb ischemia and vitamin intervention
- Mice underwent right hind limb surgery with femoral and saphenous artery ligation followed by complete excision of the femoral artery.
- Given the compromised mobility of mice following ischemic injury, we administered vitamin supplements by injection to ensure that the entire daily dose was received in the event of decreased food consumption
- To ensure complete solubilization of each treatment, the vehicle for niacin was sterile water (5:4:1, volumetric ratio)
- Each mouse received sequential, individual injections of each solution on opposing sides of the peritoneal cavity
Gait analyses
- Functional recovery of the ischemic hind limb was assessed on days 3, 9, and 15 postsurgery by gait analyses using a Catwalk system (Noldus, Leesburg, VA, USA)
- Mice were recorded as they traversed a glass walkway, and the duration of contact for each paw was recorded to generate digital gait maps
- Mean paw contact times were used to calculate hind limb use ratios of injured (right) to uninjured (left) limbs
- A ratio of <1.0 indicates decreased use of the injured limb
- Complete recovery of limb use is indicated by a hindlimb use ratio of 1.0
Histology:
- Serial cross sections (5 μm) were cut at three equally spaced locations spanning the length of the muscle.
- Sections were stained with hematoxylin and eosin and analyzed using ImageJ
- Total myofiber and adipose tissue areas per section were calculated using a customized, automated ImageJ protocol
- Ratios of regenerating to nonregenerating myofibers per mouse were calculated
- Adipose areas were normalized to total section areas and reported as percentages
- Muscle inflammation was determined by immunostaining for the mouse pan-macrophage marker, F4/80 (Invitrogen, Thermo Fisher Scientific, Mississauga, ON, Canada).
- Fibrosis was diagnosed by Masson's Trichrome staining
- total macrophage and collagen areas were quantified using a customized ImageJ procedure
Statistical analyses
- All statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA), except for microarray analyses.
Niacin and vitamin D improve endothelial cell angiogenic function in high palmitate.
- Media supplemented with niacin (10 μM), vitamin D (10 nM) or the combination of niacIN plus vitamin D significantly increased tube formation at 18 h in the presence of lipotoxic conditions (Fig. 1A, B).
- All three treatments significantly increased the tube stability over the 24 h following maximum tube formation, suggesting no additive or synergistic effects.
Niacin and vitamin D have distinct effects on endothelial cell gene expression in high palmitate
- Palmitate induced expression of many cell stress response genes, including those involved in ER stress (DDIT3, CXCL8), oxidative stress (HMOX1) and inflammation (IL1A).
- The top 10 GO categories and KEGG pathways were predominantly related to cellular responses to stress.
- Treatment did not alter the expression of niacin or vitamin D receptors GPR109A and VDR, nor was general cell viability affected as measured by MTT assays.
Niacin, but not vitamin D, improves recovery of hind limb function in obese mice with metabolic syndrome
- We used Western-diet-fed 129S6 mice with surgically induced hind limb ischemia.
- All mice exhibited characteristics of metabolic syndrome as indicated by increased adiposity, hyperlipidemia, and hepatic steatosis compared to our previous observations of age-matched lean control mice (129S6).
- Over the 2-week treatment period, hind limb contact time ratios were significantly increased only in niacin-treated mice (Fig. 4A, B).
Vitamin D does not promote vascular or myofiber regeneration of tibialis anterior muscles.
- Quantification revealed no significant differences in percentages of regenerating, nonregenerating, or necrotic myofibers compared to controls given vitamin D alone or in combination with niacin (Fig. 5D).
Vitamin D does not decrease inflammation or fibrosis of tibialis anterior muscles
- Sustained inflammation and increased interstitial fibrosis are associated with impaired myofiber regeneration [18].
- We quantified macrophage content and collagen deposition in each mouse and determined a fibrosis location score for each mouse to provide relative quantification of collagen.
- Treatment with vitamin D alone or in combination with niacin, did not significantly impact the location of collagen deposition, whereas mice treated with the same amount of vitamin D showed consistent decreases in inflammation and fibrosis.
Discussion Although observational studies have suggested an inverse relationship between vitamin D intake and PVD risk in human populations, there are no data yet available from large randomized, placebo-controlled vitamin D primary prevention or intervention trials.
- Recent reports indicate that vitamin D administration prior to injury can promote vascular regeneration in lean mouse models of peripheral ischemia and carotid artery damage, with and without diabetes [7], [22].
- Similarly, vitamin D limits malignant tumor progression in lean mice through antiangiogenic effects on tumor vasculature [23], [24]
- The timing of vitamin D treatment determines its therapeutic effect on angiogenesis and neovascularization, and may be related to the inhibition of cell cycle gene expression.
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