Exploration Of Characteristics Of Traditional Chinese Medicine Syndromes In Hypertensive Disorders Of Pregnancy And Mechanism Of RGS2 Regulating Uterine Artery Myogenic Response

ObjectTo observe and explore characteristics of traditional Chinese medicine (TCM) syndromes in hypertensive disorders of pregnancy (HDP), furthermore, to provide a new therapy idea of TCM for HDP. To determine the mechanism of utroplacental ischemia related to vascular smooth muscle dysfunction, and explore the G protein signaling pathway involved in augmented myogenic tone of uterine artery. To determine the effect of intracellular Ca2+store and release on vasoconstriction and vasodilation.Methods1. Clinical studyWe recorded the demographic data in the questionnaire of hypertensive disorders of pregnancy and evaluated the clinical symptom of HDP patients who were in line with HDP diagnostic criteria with TCM symptoms score, and analyzed the law in distribution of TCM syndromes related with age, parity, blood pressure and content of urinary protein.2. Experimental study1) In vivoIn this study, three groups in nonpregnant mice including RGS2+/+、RGS2+/- and RGS2-/-. RGS2+/+ and RGS2-/-mice were used in pregnant study.① In nonpregnant virgin mice study, Ultrasound Doppler was used for the measurement of uterine artery blood flow velocity (PSV, EDV and MV) in RGS2-/-. RGS2+/+ and RGS2-/-mice. And based on these data, we calculated the impedance index (RI, PI, S/D) of uterine artery such as in three groups.② In pregnant study, we also observed the entire index (PSV, EDV, MV, RI, PI and S/D) on G10, G15 and G19 in RGS2-/-and RGS2+/+mice via Ultrasound Doppler.2) Ex vivo③ We increased the intraluminal pressure to detect the myogenic tone of uertine artery segments isolated from RGS2-/-. RGS2+/+ and RGS2-/-mice via video microscopy. And %myogenic tone, myogenic response sensitivity and resting myogenic tone were calculated from data recorded by Ionoptix.④ Acetylcholine (ACh) was facilitated for testing endothelium-dependent vasodilatory response in all three groups.⑤ Wall tension, mechanical properties and vascular structure of uterine arteries were recorded and analyzed via Ionoptix system.⑥ Gq/11 blocker, WU-07047 and Gi/b inhibitor, pertussis toxin (PTX) were applied to determining the effect on pressure-induced myogenic response in uterine arteries from different genotypes.⑤ We also observed the impact of Ryanodin (RyRs inhibitor), thapsigargin (SERCA blocker) and TPEN (internal stores Ca2+chelator) on uterine artery in three groups.⑥ We used fura-2 imaging for in vitro assessment of internal stores Ca2+release.Results1. Clinical study1) In 87 HDP patients, average age (30.4±5.32), average SBP (151.53±13.83) mmHg, average DBP (99.33±15.67) mmHg.30 patients belonged to gestational hypertension,51 belonged to preeclampsia (18 belonged to severe preeclampsia),0 belonged to eclampsia,1 belonged to chronic hypertension,5 belonged to chronic hypertension with superimposed preeclampsia2) In 87 HDP patients,23 patients (26.44%) belonged to insufficiency of the spleen with overabundance of dampness type,26 patients (29.89%) belonged to spleen deficiency and liver hyperactivity type,22 patients (25.29%) belonged to hyperactivity of liver fire due to yin deficiency type,16 patients (18.38%) belonged to edema due to the kidney insufficiency type.3) The distribution of TCM syndromes was related to age (P<0.01).4) The distribution of TCM syndromes was related to parity (P<0.05).5) The distribution of TCM syndromes was related to blood pressure (P<0.05).6) The distribution of TCM syndromes was not related to content of urinary protein(P> 0.05).2. Experimental study1) In vivo① In nonpregnant virgin mice study, PSV, LDV, and MV trended lower from RGS2+/+to Rgs2-/-mice. Moreover, all indices of uterine artery flow impedance, including PSV-to-LDV ratio, RI, and PI were significantly increased in both Rgs2+/-and Rgs2-/-relative to RGS2+/+mice.② In pregnant study, we found the impedance indexes of uterine artery (PI, RI) were significantly increased in RGS2-/-relative to RGS2+/+mice (P<0.01), and PSV, EDV were obviously lower than RGS2+/+mice(P<0.01).2) Ex vivo③ At 80 mmHg of intraluminal pressure, myogenic tone was significantly lower in RGS2+/+ relative to Rgs2+/- and Rgs2-/-, and remained elevated at all intraluminal pressures. RGS2 deficiency increased the sensitivity of uterine artery myogenic response. When compared to RGS2+/+, uterine arteries from Rgs2+/-and Rgs2-/-mice showed increased resting myogenic tone.② Stimulation of uterine arteries with increasing concentrations of acetylcholine caused similar vasodilatory responses in uterine arteries from RGS2+/+, Rgs2+/-, and Rgs2-/-mice.③ No significant difference of wall tension, mechanical property and vascular structure were observed from three genotypes.④ PTX had no effect on myogenic response in uterine arteries from RGS2+/+mice. In contrast, the same concentration of PTX decreased myogenic tone in uterine arteries from Rgs2+/-and Rgs2-/-mice. At 80 mmHg, PTX treatment reduced myogenic tone in both Rgs2+/-and Rgs2-/-to RGS2+/+control level. Treatment of uterine arteries with the novel G q inhibitor, WU-07047, reduced myogenic response to similar levels at all intraluminal pressures in all genotypes. The effect of WU-07047 on myogenic tone was more pronounced in arteries from Rgs2+/-and Rgs2-/-mice.⑤ In the presence of thapsigargin, myogenic response was almost completely abolished in both groups. In contrast, ryanodine treatment increased myogenic response in RGS2+/+arteries to Rgs2-/-level from 40 to 140 mmHg intraluminal pressures. In Rgs2-/-arteries, ryanodine significantly increased myogenic tone only at 40 mmHg, compared to Rgs2-/-control. In both groups, the effect of ryanodine on myogenic tone was almost completely abolished when co-incubated with TPEN to chelate Ca2+in internal stores.⑥ We found that total intracellular Ca2+concentration trended higher from RGS2-/-to RGS2+/+(P<0.01). The fluorescent signal triggered by Ca2+-free PSS in Rgs2-/-SMCs was almost completely abolished when the cells were pretreated with ryanodine(P<0.01). In the presence of ryanodine, total ionomycin-induced fura-2 fluorescent signal was enhanced in RGS2+/+(P<0.01) while it was attenuated in Rgs2-/-SMCs (P<0.01).Conclusion1. The type of TCM syndromes in hypertensive disorders of pregnancy included 4 types such as insufficiency of the spleen with overabundance of dampness type, spleen deficiency and liver hyperactivity type, hyperactivity of liver fire due to yin deficiency type, edema due to the kidney insufficiency type.2. The distribution of TCM syndromes was related to age, parity, blood pressure but content of urinary protein. It provided the evidence for setting up the clinic trial of TCM.3. This study shows that G protein regulation by RGS2 is critical to maintaining normal uterine artery diameter and blood flow.4. RGS2, a GTPase-activating protein for Gq/11 and Gi/o class G proteins, affects UABF and myogenic tone of uterine artery in nonpregnant mice via altering the intracellular Ca2+ release.5. RGS2 deficiency may increase the number of the intracellular Ca2+pool, and it may alter the function of RyRs to affect the myogenic response by regulating intrcellular Ca2+ release.6. RGS2 protein played a critical role in organ perfusion especially uterine perfusion. It may become a target protein to provide a new vision in diagnosis, therapy and prediction of utroplacental dysfunction linked to hypertensive disorder of pregnancy especially preeclampsia.