Discussion
To the best of our knowledge, this is the first study evaluating the
effect of endothelial cell BH4 deficiency and the underlying mechanisms
on maternal vascular adaptations in uterine arteries during pregnancy.
The key findings are 1) Selective deficiency in maternal endothelial
cell Gch1 /BH4 biosynthesis during pregnancy leads to vascular
dysfunction due to loss of NOS-derived vasodilators in both in aortas
and uterine arteries from Gch1fl/fl Tie2cre
mice; 2) In uterine arteries the vascular impairment is incompletely
compensated for by an increase in EDHF-mediated vasodilation mediated by
an increase in intermediate and large-conductance
Ca2+-activated K+ channels. 3) Oral
supplementation of BH4 and 5MTHF, but not BH4 alone, preserves vascular
endothelial cell vasodilator function, and thus prevent progressive
pregnancy-induced hypertension in mice with endothelial cell BH4
deficiency. Taken together, these findings identify a novel role for
endothelial cell Gch1 and BH4 biosynthesis in vascular
adaptations to pregnancy.
Pregnancy is associated with a 10-fold increase in uterine artery blood
flow. In order to accommodate this without increases in systemic blood
pressure the uterine artery undergoes significant changes with enhanced
vasodilation and reduced constriction, leading to reduced vascular
resistance and increased blood flow (Cooke
& Davidge, 2003; Nelson, Steinsland,
Johnson, Suresh, Gifford & Ehardt, 1995). Nitric oxide is a key
mediator of this adaptive response. Plasma NO levels are higher in
viable than non-viable pregnancies and is inversely correlated uterine
artery pulsatility index (Battaglia,
Morotti, Montaguti, Mariacci, Facchinetti & Pilu, 2022) and loss of
NOS3 (eNOS) associated with uterine artery dysfunction, reduced placenta
nutrient transport and fetal growth restriction
(Kusinski et al., 2012). In the current
study, we have shown that loss of maternal endothelial specific BH4 is
alone sufficient to causes maladaptive uterine artery remodeling with
enhanced constrictor and reduced dilator response in both aorta and
uterine artery. The enhanced constrictor response observed in
endothelial cell Gch1 knockout mice is likely mediated in part by
reduced tonic production of NOS derived vasodilators as the difference
between genotypes were abolished in the presence of the NOS inhibitor
L-NAME. This is in keeping with clinical studies where acute
administration of L-NAME greatly reduced forearm blood flow in pregnant
women compared to non-pregnant controls
(Anumba, Robson, Boys & Ford, 1999;
Williams, Vallance, Neild, Spencer &
Imms, 1997). In addition, uterine arteries from pregnant endothelial
cell Gch1 knockout mice had increase passive stiffness. Previous,
studies in aorta have shown that increase vascular stiffness is
associated with enhance agonist mediated contractile response
(Zhang, Li, Gao, Wang, Cheng & Wang,
2022) and thus it is possible that this mechanism may also contribute
to the increase contractile response observed in our current study.
Interestingly, prior to pregnancy compensatory changes in NOS mediated
dilation were sufficient to maintain normal vasoconstrictor and dilator
responses in endothelia cell Gch1 knockout mice. We have
previously shown in aorta that in the absence of endothelial cell BH4,
eNOS become uncoupled and produces H2O2instead of NO, which acts as an endothelium-dependent vasodilator
partially compensating for the loss of eNOS derived NO
(Chuaiphichai et al., 2017;
Chuaiphichai et al., 2014). However,
during pregnancy this compensatory mechanism is lost with NOS mediated
vasodilation making minimal contribution to uterine artery vasodilation
in endothelial cell Gch1 knockout mice. Pregnancy is a state of
mild oxidative stress (Morris et al.,
1998; Palm, Axelsson, Wernroth & Basu,
2009; Toescu, Nuttall, Martin, Kendall &
Dunne, 2002). Increase expression of antioxidant defenses have
previously been observed in pregnancy
(Jenkins, Wilson, Roberts, Miller,
McKillop & Walker, 2000). Interestingly, catalase levels have been
found to be increased in pre-eclampsia as compared to normal pregnancy
(Gohil, Patel & Gupta, 2011). It is
possible that in this altered redox environment, NOS derived
H2O2 is no longer an effective
vasodilator in endothelial cell Gch1 knockout mice.
We show that pregnancy is associated with an increase in EDHF mediated
vasodilation in uterine arteries. This is consistent with other studies
which have shown enhanced EDHF mediated dilation in pregnancy
(Luksha, Nisell & Kublickiene, 2004;
Zhu et al., 2013). However, EDHF
upregulation in the absence of endothelial cell BH4, was not sufficient
to compensate fully for the loss of NOS mediated vasodilation in the
uterine artery. In uterine arteries from both wild type andGch1fl/flTie2cre mice both large and
intermediate conductance Ca2+-activated
K+ channels were responsible for the majority of the
EDHF mediated dilation. This is in keeping with previous studies which
have shown an increased expression and activity of BKCachannels (Hu et al., 2017) and
SKCa channels (Zhu et al.,
2013) in uterine arteries of pregnant sheep. Further interrogation of
EDHF response showed in wild type mice SKCa channels
made up approximately 40% of the EDHF response, however, in endothelial
cell Gch1 knockout mice this component was markedly reduced to
only 20%. Recent studies have shown metabolic regulation of
SKCa channel in coronary endothelial cells with reduced
expression observed endothelial cells from diabetic arteries
(Liu et al., 2020). Pregnancy represents
a significantly altered metabolic state, further studies interrogating
if similar mechanisms are driving reduced SKCa channel
activity in our currently study will be key to address this.
Reduced vascular BH4 is a hallmark of multiple cardiovascular conditions
(Hink et al., 2001;
Landmesser et al., 2003;
Li et al., 2006;
Mollnau et al., 2003). Oxidative stress
causes oxidation of BH4 to BH2 and B, which are incapable of acting as a
cofactor for NOS leading to uncoupled NOS. Markers of oxidative stress
are present in the placenta and maternal circulation of patients with
pre-eclampsia (Raijmakers, Dechend &
Poston, 2004) and we have previously shown reduced BH4 in placental
extravascular vesicles from women with hypertensive pregnancies
(Chuaiphichai et al., 2021). Yet in a
landmark clinical trial the antioxidant vitamins C and E failed to
prevent the development of pre-eclampsia in high risk pregnancies
(Poston, Briley, Seed, Kelly & Shennan,
2006), and simple strategies attempting to restore or augment NO with
NO donors in pre-eclampsia have been disappointing
(Meher & Duley, 2007). One possible
explanation for these results is the failure to specifically target eNOS
uncoupling and consequent altered NO/ROS signalling. Thus, augmenting
BH4 levels may be a rational therapeutic strategies to treat vascular
complication in pregnancy. However, in this study we found that oral BH4
supplementation alone was not sufficient to restore vascular function in
uterine arteries and thus prevent progressive hypertension inGch1fl/fl Tie2cre mice. We have previously shown
that oral supplementation of BH4 is not a consistent approach to
increase vascular BH4 levels, either in mice or in patients
(Chuaiphichai et al., 2021;
Cunnington et al., 2012a;
Cunnington et al., 2012b), due to
oxidation of BH4 to BH2 and B. Interestingly, vascular supplementation
of BH4 can be achieved by combining the BH4 with the
5-methyltetrahydrofoalte. The enzyme dihydrofolate reductase (DHFR)
reduces dihydrofolate to the fully reduced folate, tetrahydrofolate, and
can also reduce oxidized BH2 to regenerate BH4. We have previously
demonstrated that supplementation of BH4 with 5-MTHF, restored BH4
levels in pregnancy (Chuaiphichai et al.,
2021). In this study we show for the first time that that the
combination of BH4 + 5-MTHF is sufficient to restore vascular function
in Gch1fl/fl Tie2cre mice. 5-MTHF has been
shown to be an effective treatment to augment vascular BH4 levels in
patients (Antoniades et al., 2006),
exemplifying the early translational potential of this approach –
particularly since folates are already approved for use by pregnant
women.
Taken together, this study demonstrated that deficiency in maternal
endothelial cell BH4 biosynthesis leads to systemic vascular dysfunction
and progressive pregnancy-induced hypertension, which could be reversed
by supplementation with BH4 and 5-MTHF. Thus, targeting endothelial cellGch1 and BH4 biosynthesis by supplementation with BH4 and 5-MTHF
may provide a novel therapeutic target for the prevention and treatment
of pregnancy-related hypertension such as pre-eclampsia.