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.