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).