Lee, Wee Chee
(2020)
Effects of vitamin D on vascular function and oxidative stress in the microcirculation of diabetics.
PhD thesis, Universiti Sains Malaysia.
Abstract
Diabetes mellitus contributes to macro- and microvascular complications,
leading to adverse cardiovascular events. Vitamin D deficiency is associated with the
development of diabetes-related cardiovascular complications. This study was divided
into two parts: (i) animal study and (ii) human study. This animal study aims to
determine the effects of vitamin D deficiency on (a) microvascular endothelial and
smooth muscle functions in normal and diabetic rats; (b) the changes to endothelial
nitric oxide synthase (eNOS) protein expression and oxidative stress parameters in
mesenteric arterial tissue of normal and diabetic rats; (c) to study whether oral
calcitriol supplementation is able to ameliorate microvascular dysfunction in vitamin
D-deficient rats. This human study aims to evaluate the effects of vitamin D deficiency
on oxidative stress status in subcutaneous arteries of diabetic patients. Animal study:
(a) Male Sprague-Dawley (SD) rats were subdivided into three equal groups of 10 rats
each: (i) rats receiving 10-weeks of normal diet (Group NC), (ii) rats receiving 10-
weeks of vitamin D-deficient diet (Group ND) and (iii) rats receiving 10-weeks of
vitamin D-deficient diet with four weeks of oral calcitriol supplementation, starting
from week 7 (Groups NDS). (b) Streptozotocin-induced diabetic male SD rats were
subdivided into three equal groups of 10 rats each: (i) diabetic rats receiving 10-weeks
of normal diet (Group DC), (ii) diabetic rats receiving 10-weeks of vitamin D-deficient
diet (Group DD) and (iii) diabetic rats receiving 10-weeks of vitamin D-deficient diet
with four weeks of oral calcitriol supplementation, starting from week 7 of diabetes
induction (Groups DDS). At the end of 10 weeks, all rats were sacrificed. Rats’
mesenteric arteries were isolated and dissected to undergo vascular function studies
using wire myograph. Protein expression of eNOS in mesenteric arterial tissue was
determined using Western blot. Immunohistochemistry was used to detect the
presence and localization of eNOS in mesenteric arteries. Superoxide dismutase
(SOD) and malondialdehyde (MDA) levels in mesenteric arterial tissue; fasting blood
glucose (FBG), serum 25(OH)D and calcium levels in blood were also measured.
Human study: Diabetic patients were categorised into two groups based on serum
25(OH)D levels: (i) vitamin D non-deficient diabetic patients (Group DNP, n = 10)
and (ii) vitamin D-deficient diabetic patients (Group DDP, n = 13). The levels of SOD
and MDA in subcutaneous arterial tissue were measured. Results of animal study:
(a) Normal rats. Endothelium-dependent relaxation to acetylcholine (ACh) was
significantly attenuated in mesenteric arteries of vitamin D-deficient rats. Reduced
SOD levels and protein expression of eNOS were observed in vitamin D-deficient rats.
However, calcitriol supplementation showed no significant improvement in these
parameters. Endothelium-dependent contraction to calcium ionophore (CaI) was
augmented in vitamin D-deficient rats receiving calcitriol supplementation. Increased
calcium levels were also found in calcitriol-supplemented vitamin D-deficient rats. (b)
Diabetic rats. ACh-induced endothelium-dependent relaxation was significantly
impaired in mesenteric arteries of vitamin D-deficient diabetic rats. Reduced SOD
levels and protein expression of eNOS and enhanced MDA levels were found in
vitamin D-deficient diabetic rats. These impairments were successfully ameliorated by
calcitriol supplementation. Augmented CaI-induced endothelium-dependent
contraction and impaired sodium nitroprusside (SNP)-induced endotheliumindependent
relaxation occurred in vitamin D-deficient diabetic rats. However,
calcitriol supplementation failed to show improvement in these vascular responses
There were no significant differences in endothelium-independent relaxation to
salbutamol (SB) and contraction to phenylephrine (PE) as well as in general
parameters such as body weight changes and FBG levels between study groups in both
normal and diabetic rats. Results of human study: Markedly augmented MDA levels
were found in subcutaneous arterial tissues of vitamin D-deficient diabetic patients.
However, SOD levels in vitamin D-deficient diabetic patients showed the reduced
trend (p = 0.072) compared to vitamin D non-deficient diabetic patients. In conclusion,
this study demonstrated that vitamin D deficiency attenuates microvascular
endothelial function in both normal and diabetic rats. The impairment for endothelial
function was likely due to the diminished nitric oxide contribution, associated with
reduced eNOS protein expression and augmented oxidative stress. Vitamin D
deficiency in diabetic rats also impairs vascular smooth muscle function. The study
also showed that calcitriol supplementation to diabetic rats with vitamin D deficiency
improves endothelium-mediated vasodilation, by upregulating eNOS expression and
improving oxidative stress status. However, calcitriol supplementation to normal rats
with vitamin D deficiency induces hypercalcaemia, leading to augmented
endothelium-dependent contraction. Besides that, vitamin D deficiency in diabetic
patients as well showed augmented oxidative stress.
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