Exposure to stimulatory CpG oligonucleotides during gestation induces maternal hypertension and excess vasoconstriction in pregnant rats

Abstract

Bacterial infections increase risk for pregnancy complications, such as preeclampsia and preterm birth. Unmethylated CpG DNA sequences are present in bacterial DNA and have immunostimulatory effects. Maternal exposure to CpG DNA induces fetal demise and craniofacial malformations; however, the effects of CpG DNA on maternal cardiovascular health have not been examined. We tested the hypothesis that exposure to synthetic CpG oligonucleotides (ODNs) during gestation would increase blood pressure and cause vascular dysfunction in pregnant rats. Pregnant and nonpregnant female rats were treated with CpG ODN (ODN 2395) or saline (Veh) starting on gestational day 14or corresponding day for the nonpregnant groups. Exposure to CpG ODN increased systolic blood pressure in pregnant (Veh: 121 ± 2 mmHg vs. ODN 2395: 134 ± 2 mmHg,P< 0.05) but not in nonpregnant rats (Veh: 111 ± 2 mmHg vs. ODN 2395: 108 ± 5 mmHg,P> 0.05). Mesenteric resistance arteries from pregnant CpG ODN-treated rats had increased contractile responses to U46619 [thromboxane A2(TxA2) mimetic] compared with arteries from vehicle-treated rats [Emax(%KCl), Veh: 87 ± 4 vs. ODN 2395: 104 ± 4,P< 0.05]. Nitric oxide synthase (NOS) inhibition increased contractile responses to U46619, and CpG ODN treatment abolished this effect in arteries from pregnant ODN 2395-treated rats. CpG ODN potentiated the involvement of cyclooxygenase (COX) to U46619-induced contractions. In conclusion, exposure to CpG ODN during gestation induces maternal hypertension, augments resistance artery contraction, increases the involvement of COX-dependent mechanisms and reduces the contribution of NOS-dependent mechanisms to TxA2-induced contractions in mesenteric resistance arteries.

Keywords: Toll-like receptor 9; cyclooxygenase; hypertension; preeclampsia; vascular function.

Fig. 1.

Effects of in vivo treatment with oligodeoxynucleotide 2395 (ODN 2395) on blood pressure and spleen weights. On gestational day 19, systolic blood pressure (SBP; A) and spleen weights [%body weight (BW); B] of pregnant ODN 2395-treated rats were greater compared with vehicle-treated dams. Number of animals (n) are shown in parentheses. Values are means ± SE. *P < 0.05, vs. pregnant vehicle-treated rats; **P < 0.0001, vs. nonpregnant ODN 2395-treated rats.

Fig. 2.

Effects of in vivo treatment with ODN 2395 on maternal vasoconstriction. Systemic treatment with ODN 2395 increased contractile responses to U46619 in mesenteric arteries from pregnant but not from nonpregnant rats (A) but did not affect phenylephrine (PE)-induced contractions (B) and U46619-induced contractions (C) in aortic rings. NP, nonpregnant; V, vehicle-treated; P, pregnant; ODN, ODN 2395-treated. Values are means ± SE. *P < 0.05, pregnant vehicle-treated vs. pregnant ODN 2395-treated groups; #P < 0.05, pregnant vs. nonpregnant rats (main effect).

Fig. 3.

Effects of in vivo treatment with ODN 2395 on maternal vasodilation. Treatment with ODN 2395 did not affect relaxation responses to acetylcholine (ACh; A) and sodium nitroprusside (SNP; B). Values are means ± SE. *P < 0.05, nonpregnant vehicle-treated vs. pregnant vehicle-treated rats.

Fig. 4.

Effects of nonselective cyclooxygenase (COX) inhibition on U46619-induced contractions in arteries from rats treated with ODN 2395. Indomethacin (nonselective COX inhibitor) reduced U46619-induced contractions in mesenteric arteries from nonpregnant vehicle- and ODN 2395-treated rats (A) and from pregnant ODN 2395-treated rats but not in arteries from pregnant vehicle-treated animals (B). Values are means ± SE. *P < 0.05, arteries from vehicle-treated rats in the absence of inhibitor vs. arteries from vehicle-treated rats in the presence of indomethacin; #P < 0.05, arteries from ODN 2395-treated rats in the absence of inhibitor vs. arteries from ODN 2395-treated in the presence of indomethacin; $P < 0.05, arteries from pregnant vehicle-treated rats in the absence of inhibitor vs. arteries from pregnant ODN 2395-treated in the presence of indomethacin.

Fig. 5.

Effects of selective inhibition of COX-1 and COX-2 on U46619-induced contractions in arteries from rats treated with ODN 2395. SC560-induced inhibition of COX-1 attenuated contractile responses to U46619 in mesenteric resistance arteries from nonpregnant (A) and pregnant (B) ODN 2395-treated rats but not in vehicle-treated animals, whereas NS398-induced COX-2 inhibition reduced U46619-induced contractions in mesenteric arteries from both nonpregnant (C) and pregnant (D) vehicle-treated and ODN 2395-treated rats. Values are means ± SE. ^@P < 0.05, nonpregnant vehicle-treated group (no inhibitor) vs. nonpregnant ODN 2395-treated group in the presence of SC560 (A); #P < 0.05, ODN 2395-treated group (no inhibitor) vs. ODN 2395-treated group in the presence of inhibitor (A and B: SC560; C and D: NS398); $P < 0.05, pregnant vehicle-treated rats vs. pregnant ODN 2395-treated rats (no inhibitor; B and D); *P < 0.05, vehicle-treated group (no inhibitor) vs. vehicle-treated group in the presence of NS398 (C and D).

Fig. 6.

Effects of nitric oxide synthase (NOS) inhibition on U46619-induced contractions in arteries from rats treated with ODN 2395. N^G-nitro-l-arginine (l-NNA; nonselective NOS inhibitor) increased U46619-induced contractions in mesenteric arteries from nonpregnant vehicle- and ODN 2395-treated rats (A) and from pregnant vehicle-treated animals but did not have an effect on arteries from pregnant ODN 2395-treated rats (B). Values are means ± SE. *P < 0.05, arteries from vehicle-treated group (no inhibitor) vs. arteries from vehicle-treated group in the presence of l-NNA (A and B); #P < 0.05, arteries from nonpregnant ODN 2395-treated group (no inhibitor) vs. arteries from nonpregnant ODN 2395-treated group in the presence of l-NNA (A); $P < 0.05, arteries from pregnant vehicle-treated group vs. arteries from pregnant ODN 2395-treated group (no inhibitor; B); @P < 0.05, arteries from pregnant vehicle-treated group in the presence of l-NNA vs. arteries from pregnant ODN 2395-treated group in the presence of l-NNA (B).

Fig. 7.

Effect of in vivo treatment with ODN 2395 on protein expression. ODN 2395 treatment increased the expression of COX-2 (A and B) in mesenteric arteries from pregnant and nonpregnant rats and did not affect expression of COX-1 (C and D) and phosphorylation of endothelial nitric oxide synthase Ser1177 (eNOS^Ser1177; E and F) in arteries from any group. Number of animals (n) are shown in parentheses. Values are means ± SE. *P < 0.05, vehicle-treated group vs. ODN 2395-treated group.

Fig. 8.

Reactive oxygen species (ROS) production on arteries from rats treated with ODN 2395 or vehicle. ODN 2395 treatment did not affect dihydroethidium (DHE) fluorescent staining (×20 magnification) in mesenteric resistance arteries from nonpregnant rats (A) but increased fluorescent staining in arteries from pregnant dams (B). AU, arbitrary units. Number of animals (n) are shown in parentheses. Values are means ± SE. *P < 0.05, vehicle-treated group vs. ODN 2395-treated group.

Fig. 9.

Summary of findings and working hypothesis. Exposure to CpG ODN during gestation induces maternal hypertension and excessive constriction in maternal resistance arteries and alters the involvement of endothelium-derived factors to thromboxane A₂ (TxA₂)-induced contractions in mesenteric resistance vessels.