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. 2011 Aug 1;589(Pt 19):4777–4786. doi: 10.1113/jphysiol.2011.214726

Vitamin D insufficiency is associated with impaired vascular endothelial and smooth muscle function and hypertension in young rats

Marianne Tare 1, Sarah J Emmett 1, Harold A Coleman 1, Con Skordilis 1, Darryl W Eyles 2, Ruth Morley 3, Helena C Parkington 1
PMCID: PMC3213423  PMID: 21807617

Non-technical summary

Adverse environments during early life are linked with an increased risk of cardiovascular disease. There is an alarming increase in the prevalence of vitamin D (VitD) deficiency in women of reproductive age. We show that male and female rat offspring that were exposed to VitD deficiency in the womb and early life have high blood pressure. The arteries from VitD deficient offspring have an impaired ability to relax due to deficiencies in the production of two important factors, nitric oxide and endothelium-derived hyperpolarizing factor. VitD deficient female offspring have an additional impairment in the nitric oxide signalling pathway in the arterial muscle. The findings of this study are particularly relevant for women intending to become pregnant. Ensuring VitD sufficiency before and during pregnancy in women will reduce the burden of cardiovascular disease risk in their offspring.

Abstract

Abstract

Increasing evidence links vitamin D deficiency and cardiovascular dysfunction in human adults. There is a worldwide increase in the prevalence of vitamin D deficiency in women of reproductive age, particularly dark-skinned and/or veiled women and their infants. We used a rat model to determine the functional impact of vitamin D deficiency during intra uterine and early life on resistance artery reactivity and blood pressure in the offspring as young adults. Rat dams were maintained on vitamin D deficient or replete chow before and during pregnancy and lactation. The offspring were maintained on the same chow until studied at 7–8 weeks of age. Conscious blood pressure was measured. Endothelial and smooth muscle function were tested in mesenteric arteries on a pressure myograph. Vitamin D deficient male and female offspring had a 10-fold lower serum 25-hydroxyvitamin D (P < 0.0001) and markedly elevated blood pressures (11–20 mmHg, P < 0.001) and heart rates (21–40 beats min−1, P < 0.02) than control fed offspring. Serum calcium was unchanged. Mesenteric artery myogenic tone was doubled in vitamin D deficiency. Endothelium-derived nitric oxide-evoked dilation was halved in arteries from vitamin D deficient males and dioestrous females. Dilation attributed to endothelium-derived hyperpolarizing factor was all but abolished in vitamin D deficient oestrous females. Nitroprusside-evoked dilation was unaltered in arteries from males, but was markedly reduced in vessels of vitamin D deplete females. In conclusion, early life vitamin D deficiency is associated with endothelial vasodilator dysfunction, and this is likely to contribute to the accompanying elevation in blood pressure and an increased cardiovascular disease risk.

Introduction

The prevalence of vitamin D (VitD) deficiency is increasing in Western societies and is re-emerging as a public health issue (Holick, 2007; Lips, 2010). VitD insufficiency is thought to occur in approximately 50% of individuals in Western societies (Lips, 2010). Globally, around 1 billion people are estimated to be VitD insufficient or VitD deficient (Holick, 2011). Although, VitD deficiency has been associated with disorders of calcium/phosphorous handling and bone metabolism (e.g. osteomalacia and rickets), it is becoming increasingly evident that VitD has myriad effects on physiological function due, in large part, to the presence of VitD receptors in most tissues. VitD deficiency has been linked with autoimmune (Ponsonby et al. 2002), neurological (McGrath, 2001; Eyles et al. 2003), and cardiovascular diseases (Zittermann, 2006; Nemerovski et al. 2009; Valdivielso et al. 2009). There is an apparent inverse relationship between cardiovascular disease and UV exposure in adult humans (Rostand, 1997; Krause et al. 1998). There is an increased incidence of high blood pressures in dark-skinned individuals living at high latitudes (Boucher, 1998; Shaw & Pal, 2002), with increasing latitude (Rostand, 1997), in those who cover their skin when outdoors (Holick, 2007), or in winter months (Sherman et al. 1990; Carnevale et al. 2001; McGrath et al. 2001). The inverse association between VitD levels and blood pressure in humans (Kristal-Boneh et al. 1997; Pfeifer et al. 2001) appears to be independent of serum Ca2+ or parathyroid hormone (Kristal-Boneh et al. 1997). In the Framingham cohort, the incidence of a cardiovascular event in a 6 year period was doubled in individuals with low VitD levels (Wang et al. 2008). VitD supplementation increases flow-mediated dilation (Sugden et al. 2008; Harris et al. 2011) and reduces blood pressure in diabetic individuals (Sugden et al. 2008).

VitD deficiency in pregnant women is increasing in prevalence. VitD deficiency and even frank rickets in infants and young children is seen worldwide (Grover & Morley, 2001; Nozza & Rodda, 2001; Prentice, 2008). Offspring of women who are VitD deficient during gestation have lower levels of VitD at birth (Brooke et al. 1980), and dark skinned babies are at increased risk of VitD deficiency postnatally, since breast milk is a poor provider of VitD (Basile et al. 2006). A wealth of evidence from epidemiological and experimental studies shows that suboptimal conditions during early life can result in an increased risk of cardiovascular disease in adulthood (Gluckman et al. 2008). Offspring of VitD deficient rats are hypertensive, have enhanced aortic constriction (Weishaar & Simpson, 1987), cardiac hypertrophy and increased nephron number (Maka et al. 2008; Gezmish et al. 2010).

Blood pressure is elevated in mice lacking the gene encoding the VitD receptor (VDR−/−) (Li et al. 2002). In spontaneously hypertensive rats (SHR), VitD supplementation lowers blood pressure (Lucas et al. 1986; Borges et al. 1999). Mesenteric arteries of adult rats made VitD deficient had enhanced contractile responses (Bian et al. 1996), and exposure of aortic rings from SHR to VitD in vitro reduced the amplitude of the contractile response to some agents (Wong et al. 2008). Endothelium-dependent vasodilator function provides a significant counterbalance to vasoconstrictor influences in order to maintain normal blood pressure and tissue blood flow. Endothelial dysfunction is a prominent feature of cardiovascular disease (Félétou & Vanhoutte, 2006). Vascular endothelial function is vulnerable to the effects of early life environments (Poston, 2007), and the effect of early life VitD insufficiency on endothelial function has never been investigated. The aim of the present study was to test the hypothesis that early life VitD insufficiency leads to endothelial dysfunction which facilitates constriction in resistance blood vessels and contributes to elevated blood pressure. We used a rat model of VitD insufficiency in utero and early life, in view of the high prevalence of deficiency in pregnant women and their children. We studied vascular function and blood pressure in young adult male and female rats. The VitD levels achieved in this model approach those seen in dark-skinned pregnant women (Grover & Morley, 2001) and in children with rickets (Nozza & Rodda, 2001).

Methods

Ethical approval

All procedures were approved by the Physiology Animal Ethics Committee, Monash University and this study was conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. The experiments comply with the policies and regulations of The Journal of Physiology given by Drummond (2009).

Animals

Four-week-old female Sprague–Dawley rats received either a standard semi-purified chow (containing 1000 IU VitD, cholecalciferol; 4.5 g Ca2+ kg−1, AIN93G) or chow with no added VitD (Specialty Feeds, Glen Forrest, WA, Australia). Six weeks later, all were mated with VitD replete males. The females (14 per group) were maintained on VitD replete or deplete diets throughout pregnancy and lactation. Four days after birth each litter was reduced to 10 pups. Pups were weaned at 4 weeks of age. Offspring were maintained on the same chow as their mothers until experimentation (7–8 weeks of age). All rats were housed under incandescent light, which is free of UV irradiation in the VitD action spectrum (290–315 nm), with a 12 h light–dark cycle. For females, a vaginal smear was examined, the uterus was weighed, and the presence of follicles or corpora lutea was established to determine stage of the oestrous cycle.

Arterial pressure was measured in freely moving, conscious rats via a catheter inserted into the ventral tail artery under isofluorane anaesthesia (a 10 min procedure) (Parkington et al. 2004). The blood pressure values recorded during the final 30 min, 2–3 h after recovery from anaesthetic, were analysed. At the end of recording, blood was collected via the recording catheter and the animal anaesthetized with isoflurane and decapitated. Serum was stored at −70°C for determination of 25-hydroxy-VitD in accredited laboratories at the Royal Children's Hospital, Melbourne, using a radioimmunoassay kit (Immunodiagnostic Systems, Boldon, UK). The coefficient of variation for this laboratory is 10.2 and 10.1% at 30 and 100 nmol l−1, respectively. Serum Ca2+ levels were determined in the same laboratory.

Experimental protocols

The mesenteric arterial tree was isolated and placed in physiological saline solution (PSS) containing (mm): NaCl 120; KCl 5; NaHCO3 25; glucose 11; KH2PO4 1; MgSO4 1.2; CaCl2 2.5; gassed with 5% CO2 and 95% O2. A segment of a third order branch (∼250 μm outside diameter at 57 mmHg) was mounted on a pressure myograph (Living Systems Instrumentation, Burlington, VT, USA) as previously described (Parkington et al. 2004). Pressure within the segment was set at 57 ± 3 mmHg with no lumenal flow, and the outside was constantly superfused (Parkington et al. 2004). Diameter was recorded via Diamtrak software (Diamtrak, Flinders University, SA, Australia). Data were stored digitally for analysis using Axoscope software (Axon Instruments, Union City, CA, USA).

At the beginning of each experiment, smooth muscle constriction was tested with 10 μm phenylephrine. At the end of the experiment the arteries were exposed to PSS in which 100 mm NaCl had been replaced with KCl (HiK PSS). To study vasorelaxation, the segments were submaximally constricted (to 60–70% of the constriction evoked by HiK PSS) with arginine vasopressin (AVP; 1 pm to 5 nm) (Fig. 6B). If the level of myogenic tone was altered in the presence of endothelial blockers, the concentration of AVP used was adjusted accordingly to achieve the same level of preconstriction as outlined above. Then, endothelial vasodilator function was tested using a 2 min application of acetylcholine (ACh) to stimulate the endothelium. Increasing concentrations of ACh were applied discretely, with complete recovery of diameter to pre-dilation levels before the next concentration was applied. NO production was blocked using Nω-nitro-l-arginine methylester (l-NAME, 100 μm, 30 min) and prostanoid synthesis was blocked using indomethacin (1 μm, 30 min). Finally, the segment was again constricted and endothelium-independent relaxation was tested using sodium nitroprusside (SNP, 10 μm).

Figure 6. Influence of VitD deficiency on endothelium-independent vasodilation and vasoconstriction in mesenteric arteries.

Figure 6

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A, dilation evoked by sodium nitroprusside (SNP 10−5m) in PSS containing l-NAME plus indomethacin (males, n = 10; dioestrous females, n = 6; oestrous females n = 8). *Difference from control fed; †difference between females and males. B, diameter changes evoked by 100 mm K+ (HiK); and by 10 μm phenylephrine (PE). n = 5 in each group. Level of preconstriction in the presence of arginine vasopressin (AVP) (n = 4–6 per group).

Data analysis

Comparison between control and deficient groups was by Student's t test. Relaxations elicited by ACh (a) in control PSS, (b) in l-NAME and (c) in l-NAME plus indomethacin were integrated (area-under-curve) to take into account both the amplitude and duration of the responses (Tare et al. 2000), as the responses were shorter in arteries from VitD deplete rats. Sigmoid concentration–relaxation curves were constructed using Prism software (GraphPad Software Inc., La Jolla, CA, USA). Integrated relaxations were subtracted to estimate the component of the response attributable to NO (a–b), prostanoid (b–c), and EDHF (c alone) within each segment and results from all rats in each group were pooled (Tare et al. 2000). Constrictions to HiK and phenylephrine, basal tone and relaxation evoked by SNP were normalized to maximum diameter, obtained by exposure for 30 min at the end of the experiment in PSS containing zero Ca2+ and 3 mm EGTA. Two-way ANOVA was used to test the effects of VitD depletion in male and females, with post hoc Bonferroni testing as appropriate. Results were expressed as means ± SEM, except where indicated, and n = the number of offspring from separate mothers. A probability of P < 0.05 was accepted as statistically significant.

Results

VitD deprivation

Serum levels

Serum 25-hydroxy VitD levels were markedly lower in rats fed VitD deficient chow, while serum Ca2+ levels were not different (Table 1).

Table 1.

Effects of VitD deprivation in utero and into young adulthood on serum 25-hydroxyvitamin D and Ca2+, body mass, mean arterial pressure (MAP) and heart rate in 7- to 8-week-old rats

Males Females
Control fed Vit D deplete P Control fed Vit D deplete P
Serum 25OH-D (nmol l-1) 120 ± 7 13 ± 1 <0.001 138 ± 14 10 ± 1 <0.001
Serum Ca2+ (mmol l−1) 2.29 ± 0.06 2.19 ± 0.10 0.7 2.36 ± 0.05 2.29 ± 0.07 0.3
MAP (mmHg) 104 ± 1 115 ± 2 0.001 102 ± 2 122 ± 2 <0.001
Heart rate (beats min−1) 378 ± 6 418 ± 8 0.002 394 ± 5 415 ± 5 0.02
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For males, n = 13 for control fed and n = 12 for VitD deplete; for females, n = 8 and 13 for control fed and VitD deplete, respectively. Values are given as means ± SEM. Vit D, vitamin D; MAP, mean arterial pressure; 25OH-D, 25-hydroxyvitamin D.

Body weights

Body weight at day 1 was not different between VitD deficient and control-fed pups (Fig. 1A and B). Growth rates of the offspring were equivalent before weaning. After weaning the VitD deficient groups tended to have lower body weights and this was significant in the males (P = 0.003).

Figure 1. Growth of male and female offspring.

Figure 1

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Growth of Control and VitD deplete male (n = 14/group) and female (n = 14/group) offspring. *Difference from control fed.

Mean blood pressure and heart rate

In both males and females, mean arterial blood pressure and heart rate were significantly higher in VitD deplete compared with control-fed rats (Table 1).

Vascular function

Spontaneous tone

Following pressurization of the vessels, spontaneous tone development (myogenic tone) was greater in arteries of VitD deplete males and dioestrous females (22% of maximal diameter) compared with vessels from control fed rats (8–10%) (P < 0.0001) (Fig. 2). Arteries of oestrous females developed greater tone (33%) (P < 0.0001), and this was significantly increased to 60% in vessels from VitD deplete animals (Fig. 2).

Figure 2. VitD insufficiency on myogenic tone.

Figure 2

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Effects of VitD and sex on basal tone development in isolated small mesenteric arteries of males (n = 7–12) and of females in dioestrus (n = 7) and in oestrus (n = 6). *Difference from control fed; †difference from dioestrous females and males.

Endothelium-dependent vasodilation

Arteries were preconstricted to between 60 and 70% of the HiK evoked constriction using AVP (Fig. 6B). Stimulation of the endothelium with ACh evoked concentration-dependent dilation of segments pre-constricted with AVP. Despite the fact that each application of ACh was for 2 min, the duration of endothelium-dependent vasodilation was strikingly shorter in arteries from VitD deplete rats (Fig. 3).

Figure 3. Influence of VitD insufficiency on vasodilation.

Figure 3

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Examples of raw traces showing vasodilation evoked by endothelial stimulation with ACh 10 μm in mesenteric arteries from Control (upper trace) and VitD deplete (lower trace) rats.

In control PSS, maximum integrated endothelium-dependent vasodilations were significantly reduced in arteries from VitD deplete males and dioestrous females compared with vessels from their control fed counterparts (P < 0.0001, ANOVA) (Fig. 4). In arteries from oestrous females, maximum relaxation was smaller than in the other two groups (P < 0.0001) and was similar between control-fed and VitD deplete females (P = 0.18) (Fig. 4).

Figure 4. Effect of VitD deficiency on endothelium-dependent vasodilation.

Figure 4

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Integrated endothelium-dependent vasodilation in arteries from control fed (left panels) and VitD deplete rats (right panels), in control solution and in the presence of NO synthase blockade (l-NAME), and additional block of prostanoid synthesis (l-NAME + indomethacin, Indo). Number of animals: n = 6–7 for each group of control fed and n = 8–11 for VitD deplete rats. †Significant difference between VitD replete and VitD deplete in control PSS (ANOVA). *Individual point differences in control PSS versus in l-NAME.

Maximum integrated dilation in all tissues was reduced by l-NAME (P < 0.0001) (Fig. 4), with little additional effect of indomethacin. In tissues from VitD deplete male and dioestrous female rats, ACh-induced vasodilations were reduced by l-NAME. In arteries from oestrous females l-NAME all but abolished relaxation (Fig. 4).

Contributions of NO, prostanoid and EDHF to endothelium-dependent vasodilation

VitD depletion halved the NO component of endothelium-dependent dilation in mesenteric arteries of males and dioestrous females (P < 0.0001) (Fig. 5). The EDHF contribution was preserved in arteries from VitD deplete males (P = 0.2) and dioestrous females (P = 0.8). In contrast, in oestrous females the EDHF component was all but abolished in arteries of VitD deplete animals (P < 0.0001), with preservation of the NO component (P = 0.5) (Fig. 5). The small dilator prostanoid response was reduced in arteries from VitD deficient male rats (Fig. 5).

Figure 5. Contribution of NO, EDHF and prostanoids to endothelium-dependent vasodilation.

Figure 5

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Responses in individual tissues were subtracted to reveal the absolute contribution of NO, EDHF and prostanoids to endothelium-dependent responses. Dilation, positive values; constriction, negative values. †Significance difference between VitD replete and VitD deplete (ANOVA; *individual point differences, same n as for Fig. 4).

Smooth muscle responsiveness

Endothelium-independent dilation

SNP (10 μm) evoked equivalent dilations in arteries of all control fed rats, irrespective of sex (P = 0.03 by ANOVA) (Fig. 6A). VitD depletion had no effect on SNP-evoked vasodilation in arteries from males (n = 10). However, vasodilations in vessels of VitD deplete dioestrous (n = 6) and oestrous (n = 8) females were significantly reduced compared with responses in arteries of VitD deplete males (P = 0.004 and 0.0002, respectively) (Fig. 6A). Vasodilations in arteries of VitD deplete oestrous females were approximately halved compared with their control fed counterparts (P = 0.02).

Constriction

Exposure for 1 min to HiK PSS (that is in which 100 mm Na+ had been replaced by K+) or to normal PSS containing 10 μm phenylephrine reduced external diameter to an equivalent extent (Fig. 6B, n = 5 in all groups) and was similar irrespective of sex or VitD status.

Rate of constriction/relaxation

The 10–90% rise times for relaxations to SNP (+VitD 11 ± 2 s, n = 9 and −VitD 20 ± 5 s, n = 11, P = 0.1) and for contractions to HiK PSS (+VitD 4 ± 1 s, n = 6 and −VitD 8 ± 2 s, n = 8, P = 0.1) were not different in arteries from VitD replete versus VitD deplete rats. This suggested that there was no physical impediment to contraction or relaxation as a result of VitD status.

Vessel diameter

Maximum passive diameter (in Ca2+ free, EGTA-containing PSS) was not affected by VitD deprivation (P = 0.3).

Discussion

The present study provides the first report of the deleterious effects of early life VitD insufficiency on endothelium-dependent vasodilator function in resistance arteries. This was associated with an elevation in blood pressure. Several other novel observations to emerge from our study include the development of significantly greater basal tone in mesenteric arteries of VitD deplete rats and a blunted responsiveness to SNP in arteries from females. There was a striking influence of sex and/or endogenous sex steroids on the endothelium-derived vasodilator targeted by VitD insufficiency. Thus, while NO-mediated vasodilation was preferentially reduced in arteries from males and dioestrous females, the EDHF component of vasodilation was all but abolished in tissues from females in oestrus.

These results add to the growing body of evidence implicating VitD deficiency in adverse cardiovascular outcomes in humans (Kristal-Boneh et al. 1997; Krause et al. 1998; Valdivielso et al. 2009) and the ability of VitD to ameliorate the high blood pressure in SHR rats (Borges et al. 1999) and increase flow-mediated vasodilation, an indicator of endothelial vasodilator function, in adult humans (Harris et al. 2011; Jablonski et al. 2011). Blood pressure was elevated by 11 (males) and 20 mmHg (females) in our study, similar to the 20 mmHg reported previously for VitD deplete male rats (Weishaar & Simpson, 1987) and VitD receptor knockout mice (Li et al. 2002). In humans, for each 10–20 mmHg increase in blood pressure there is a 2-fold risk of cardiovascular disease (Chobanian et al. 2003).

The effects of VitD deficiency we report cannot be explained by low serum Ca2+, since levels were similar in deficient and control groups. Our observation that both blood pressure and heart rate were elevated in VitD insufficiency suggests the possibility that VitD may also have an effect on the cardiovascular control centres in the brain. Studies in rats demonstrate that maternal VitD insufficiency can adversely affect brain development in the fetus (Brown et al. 2003; Eyles et al. 2003). Effects of VitD insufficiency on cardiovascular centres in the brain have not been studied.

Blood pressure is elevated in mice lacking the VitD receptor, by a mechanism implicating renin, and is independent of Ca2+ or parathyroid hormone (Li et al. 2002). Renin increases angiotensin II levels, predisposing to the development of hypertension and oxidative stress (Ortiz et al. 2001), and has action on baroreceptor pathways (Lohmeier et al. 2002) and structure in blood vessels. In the present study, arterial diameter was not affected by VitD insufficiency and histological examination of 10 μm sections of vessels failed to disclose detectable morphological differences in mesenteric resistance arteries between treatment groups (data not shown). Furthermore, an earlier study by Weishaar and colleagues (1990) found that plasma renin activity was unaltered in VitD deficient, normocalcaemic rats.

The endothelium achieves vasorelaxation by a variety of mechanisms, with NO an important endothelium-dependent vasodilator, especially in larger vessels, and the contribution of EDHF becomes increasingly important as vessel size decreases (Félétou & Vanhoutte, 2006). Early life insults increase cardiovascular disease risk and this may be mediated in part by endothelial vasodilator dysfunction (Lamireau et al. 2002; Payne et al. 2003; Taylor et al. 2004; Franco et al. 2006; Poston, 2007; Torrens et al. 2009). NO and EDHF contributed approximately equally to overall endothelium-dependent vasodilation in pressurized small mesenteric arteries from our males, similar to previous observations (McCulloch & Randall, 1998), and dioestrous females. Reduction in EDHF contributes, at least in part, to the endothelial dysfunction in mesenteric arteries in SHR, due to alterations in the nature or density of the K+ channels involved in the hyperpolarization. VitD supplementation of SHR normalizes blood pressure and restores K+ channels in these animals (Borges et al. 1999). It is interesting that Borges and colleagues used female rats in their study of the effects of VitD in SHR, although the stage of the oestrous cycle was not documented. In the present study, EDHF-mediated vasodilation was preserved in mesenteric arteries of males and dioestrous females deprived of VitD since early life but was all but abolished in vessels of females in oestrus. VitD deficiency resulted in a halving of the contribution of NO to endothelium-dependent vasorelaxation, leaving EDHF intact in our males and dioestrous females. Endothelial NOS is upregulated by oestrogen (Chambliss & Shaul, 2002) and it may be that this circumvents, in part, the reduction in the NO component of endothelium-dependent vasodilation in VitD deficient oestrous females. The contribution of EDHF to endothelium-dependent vasodilation is approximately halved in mesenteric arteries of oestrous females compared with dioestrous females or males, suggesting that the elevated oestrogen, and thus NO production, may have a suppressive effect on EDHF (Bauersachs et al. 1996). The virtual absence of EDHF responses in tissues from VitD deficient oestrous females could reflect a convergence of the effects of oestrogen and VitD deficiency, perhaps at the level of genome transcription, a major site of action of both VitD and oestrogen. Sex differences in endothelium-dependent dilation in mesenteric artery have been reported in normotensive rats (McCulloch & Randall, 1998). Additionally, adverse prenatal conditions, e.g. maternal hypertension or dietary fat, have been found to have disparate effects on cardiovascular function based on sex (Denton et al. 2003; Khan et al. 2003). In our study, blood pressure (BP) was significantly elevated in both males (by 11 mmHg) and to an even greater extent in females (20 mmHg), and these rats were only 7–8 weeks of age. Interestingly, although numbers are small following separation of the female BP data according to the stage of the oestrous cycle (oestrus versus dioestrus), VitD deficiency appeared to have a greater effect during oestrus than in dioestrus.

Endothelium-independent relaxation to SNP was reduced in VitD deplete female rats and may explain, in part, the reduction in the NO component of endothelium-dependent vasorelaxation in these animals. Offspring of calorific or protein restricted dams may also exhibit similar reductions in responsiveness to SNP (Lamireau et al. 2002; Brawley et al. 2003). Of these studies, one was associated with a reduction in smooth muscle guanylyl cyclase but no change in endothelial NO synthase (Lamireau et al. 2002). The impaired endothelium-dependent vasodilation in vessels with intact SNP responsiveness in males indicates a reduction in NO production or bioavailability, consistent with increased oxidative stress (see above). In arteries from oestrous females, although SNP responsiveness was decreased, endothelium-dependent relaxation attributed to NO was unchanged reflecting the possibility of an increase in NO production in these vessels, perhaps reflecting the stimulatory effect of oestrogen on eNOS (see above).

The mesenteric vessels studied here developed modest myogenic tone, confirming previous observations in this tissue (Davis & Hill, 1999). Although not extensively studied, it appears that early life environments may alter myogenic tone development in a sex- and region-dependent manner (Hemmings et al. 2005; Xiao et al. 2009). Tone was significantly increased in tissues obtained from all VitD deplete rats (Fig. 1). Basal release of NO from the endothelium can influence vascular tone (Davis & Hill, 1999), and blockade of NO production with l-NAME increased tone by approximately 15% in arteries from males and dioestrous females and by around 30% in oestrous vessels (data not shown). However, the extent of tone development following blockade of NOS was equivalent in vessels from VitD replete and deplete males and females, suggesting that the deleterious effect of VitD deficiency on tone is unlikely to be via basal NO production. In SHR, the enhanced myogenic tone in mesenteric arteries is ameliorated with VitD supplementation (Borges et al. 1999), a factor which may contribute to the normalisation of BP in these treated rats.

There was no evidence for an effect of VitD insufficiency on the ability of the smooth muscle of the mesenteric artery to contract, in response to either HiK PSS depolarization or receptor activation with phenylephrine or AVP. In a previous study of VitD depletion in utero, sensitivity of aortic rings to noradrenaline was increased. These rat offspring were hypocalcaemic and the vascular hyper-responsiveness was reversed by normalization of serum Ca2+ (Weishaar & Simpson, 1987). In our rats, serum Ca2+ levels were preserved.

This study provides a clear demonstration that VitD insufficiency in early life has serious repercussions on vascular function. These findings are particularly relevant for pregnant women and their children. Serum levels of 25-hydroxy VitD in our VitD deplete rat dams and pups are similar to those found in dark-skinned and veiled pregnant women and in children with rickets. There is evidence that potentially modifiable factors operating around the time of conception, during pregnancy or early childhood can ‘programme’ later health (Nathanielsz et al. 2007; Poston, 2007; Gluckman et al. 2008; Nijland et al. 2008). VitD may be an important factor in this regard (McGrath, 2001), since its active form has many critical physiological functions, including regulating genomic stability (Chatterjee, 2001; Sutton & MacDonald, 2003). VitD supplementation is widely available, cheap and safe and provides an alternative to sunshine exposure when lifestyle choices preclude adequately increased skin exposure to sunlight.

Acknowledgments

The authors thank Donna West for preparing the animals, Michelle Kett for assistance with recording conscious BP, and Peter Verras and Ronda Greaves for assay of VitD and serum Ca2+ determinations. This work was supported by the Ramaciotti Foundation (Perpetual Trustees) (H.C.P.), NHMRC (H.C.P., M.T., H.A.C., D.W.E. and C.S.) and VicHealth (Victorian Health Promotion Foundation) (R.M.).

Glossary

Abbreviations

AVP

arginine vasopressin

BP

blood pressure

EDHF

endothelium-derived hyperpolarizing factor

NO

nitric oxide

NOS

nitric oxide synthase

SHR

spontaneously hypertensive rats

SNP

sodium nitroprusside

VitD

vitamin D

VDR

vitamin D receptor; PSS, physiological saline solution; HiK PSS, PSS with isosmolar replacement of Na+ with K+

Author contributions

M.T., S.J.E., H.C.P. experimental procedures. C.S. research assistance, H.A.C. analysis, D.W.E. provision of animals, R.M. clinical input, M.T., H.C.P., H.A.C., R.M. study design. All authors approved earlier versions of the manuscript. Functional studies done in Dept Physiology, Monash University, VitD & calcium assayed at Royal Chikdren's Hospital, Melbourne.

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