Figure 1: Blood pressure and vasomotor function in aortas from
non-pregnant and pregnant wild-type andGch1fl/fl Tie2cre mice at E18.5 day of
gestation.
(A ) Systolic blood pressure prior to conception and at E18.5, a
significant increase in blood pressure was observed inGch1fl/fl Tie2cre mice both before conception
and at E18.5 compared with their wild type littermates
(*P<0.05 ; n=6 to 8 animals per group). (B )
Vascular function of in aorta from non-pregnant (NP) and pregnant (P)
mice at E18.5 day of gestation. Vasoconstrictions in response to
phenylephrine (PE) was significantly enhanced in aortas from pregnantGch1fl/fl Tie2cre mice compared to pregnant
wilt-type mice and non-pregnantGch1fl/fl Tie2cre mice
(*P<0.05 ; n=6 animals per group).
(C ) EC50 and
maximum contraction in response to PE. (D ) Absolute contraction
(mN) in response to KCL response (mN) in non-pregnant and pregnant mice
from both WT and Gch1fl/fl Tie2cre mice.
(E ) Vasoconstrictions in response to phenylephrine (PE) in the
presence of 100 µM L-NAME in aortas from non-pregnant (NP) and pregnant
(P) mice at E18.5 day of gestation of both genotypes. (F )
Endothelium-dependent vasodilatation in response to ACh was markedly
impaired in aortas from pregnantGch1fl/fl Tie2cre mice compared to pregnant WT
and non-pregnant Gch1fl/fl Tie2cre mice
(P<0.05 ; n=6 animals per group). (G )
EC50 and maximum relaxation in response to ACh.
(H ) Endothelium-dependent vasodilatations to ACh were totally
inhibited in all four groups in the presence of L-NAME. (I )
Endothelium-independent vasodilatations in response to the nitric oxide
donor, sodium nitroprusside (SNP) were significantly enhanced in both
pregnant wild-type and Gch1fl/fl Tie2cre mice
compared to non-pregnant wild-type andGch1fl/fl Tie2cre mice
(*P<0.05 , significant difference between non-pregnant
WT and pregnant WT, #P<0.05 , significant difference
between non-pregnant Gch1fl/fl Tie2cre vs
pregnant Gch1fl/fl Tie2cre mice; n=6 animals per
group).
Figure 2. Effect of endothelial cell BH4 deficiency on vascular
uterine artery function in pregnancy. Vascular function of isolated
uterine arteries (UA) from non-pregnant (NP) and pregnant (P) mice at
E18.5. (A ) Uterine artery diameters as determined by the
length-tension relationship at 100 mmHg. A significant increase in
diameter was observed with pregnancy in both groups
(*P<0.05 ; n=6 to 8 animals per group).
Vasoconstrictions in response to KCL response (45 mM) were significantly
increased in pregnant UA from both genotypes compared to non-pregnant
controls (*P<0.05 ; n=6 to 8 animals per group).
(B ) Cumulative dose response curved to the TxA2 mimetic, U46619
in non-pregnant and pregnant arteries from wild-type andGch1fl/fl Tie2cre mice. (C and D)EC50 and maximum contraction in response to U46619.
(E ) Vasoconstriction in response to U46619 in pregnant uterine
arteries in the presence or absence of non-selective NOS inhibitor, 100
µM L-NAME (*P<0.05 ; n=6 to 8 per group). (F )
Endothelium-dependent vasodilatation to acetylcholine (Ach) in uterine
arteries from non-pregnant and pregnant mice from both genotypes,
submaximally constricted with U46619. (G and H )
EC50 and maximum vasodilatation in response to ACh.
(I ) Endothelium-independent vasodilatations in response to the
nitric oxide donor, sodium nitroprusside (SNP) were significantly
enhanced in both pregnant wild-type andGch1fl/fl Tie2cre mice compared to non-pregnant
wild-type and Gch1fl/fl Tie2cre mice
(*P<0.05 ; n=6 to 8 animals per group).
Figure 3. Contribution of eNOS-derived vasodilators,
prostacyclin, and EDHF in non-pregnant and pregnant wild-type andGch1fl/fl Tie2cre uterine arteries at E18.5 day
of gestation . (A and B ) Endothelium-dependent
vasodilatations to acetylcholine (ACh) were determined in the presence
of the nitric oxide synthase inhibitor, L-NAME (100 μM) alone, or L-NAME
and the cyclooxygenase inhibitor, indomethacin (10 μM), or L-NAME,
indomethacin and EDHF blockers (apamin and charybdotoxin). (C )
Percentage contribution of NOS-derived vasodilators, prostacyclin and
EDHF-sensitive components (*P<0.05 ; n=6 to 8 animals
per group).
Figure 4. Contribution of SKca,
IKca and BKca channels in pregnant
wild-type and Gch1fl/fl Tie2cre uterine
arteries at E18.5 day of gestation. (A ) Endothelium-dependent
vasodilatations to acetylcholine (ACh) were determined in the presence
of the nitric oxide synthase inhibitor, L-NAME (100 μM) and the
cyclooxygenase inhibitor, indomethacin (10 μM), or L-NAME, indomethacin
and small-conductance Ca2+-activated
K+ channel (SKca) blocker, apamin (AP;
50 nM), or L-NAME, indomethacin, apamin and non-selectively intermediate
and large-conductance Ca2+-activated
K+ channels (IKca and
BKca , respectively) blocker, charybdotoxin (ChTx; 100
nM). (B ) Percentage contribution of apamin-sensitive component
(SKca), charybdotoxin-sensitive component
(IKca and BKca) in pregnant wild-type
and Gch1fl/fl Tie2cre uterine arteries at E18.5
day of gestation (*P<0.05 ; n=6 to 8 animals per group).
Figure 5. Supplementation of BH4 and 5MTHF but not BH4 alone
restores vascular function and prevents pregnancy-induced hypertension
in pregnant mice with endothelial cell BH4 deficiency. Non-pregnantGch1fl/fl Tie2cre andGch1fl/fl (Wild-type; WT) mice were
supplemented with BH4 (200 mg/kg/day) alone or BH4 with the fully
reduced folate, 5-methyltetrahydrofolate (5-MTHF; 15 mg/kg/day) or
control for 3 days before timed-matings, and throughout their subsequent
pregnancies. Blood pressure was determined before and throughout
pregnancy by non-invasive tail-cuff plethysmography. Vascular function
of isolated uterine arteries (UA) from pregnantGch1fl/fl Tie2cre and wild-type mice treated
with either BH4 alone or BH4+5MTHF or control was assessed by wire
myography at 18.5 day of gestation. (A and C ) Oral BH4
supplementation alone was not sufficient to prevent progressive
hypertension throughout pregnancy or (B and D ) restore
vascular dysfunction in uterine arteries fromGch1fl/fl Tie2cre mice
(*P<0.05 ; n=6 animals per group). (E andF ) The combination of 5-MTHF to BH4 oral supplementation was
sufficient to prevent progressive pregnancy-induced hypertension and
vascular dysfunction in UA from pregnantGch1fl/fl Tie2cre mice
(*P<0.05 ; n=6 animals per group).