EP3 is upregulated in cerebral small arteries of RHRsp, and deletion of EP3 does not affect blood pressure
COX-2/ PGE2 signaling is an important inflammatory pathway. Hypertension is strongly associated with the augmented expression of the COX-2/ PGE2/EP3 axis in both human and animal models[14, 15]. As such, we also observed significantly elevated expression of EP3 in the cerebral small arteries of RHRsp by immunofluorescence (Figure 1A), indicating that the PGE2/EP3 axis might be involved in the progression of CSVD induced by hypertension.
We then constructed the RHRsp model on EP3 knockout rats (EP3-/-) to determine whether EP3 receptor deficiency can attenuate the development of CSVD. First, we sought to determine whether the deletion of EP3 can affect the BP of the animals since hypertension is a crucial risk factor for CSVD and the PGE2 signaling is reported to have major effects on BP control through its receptors. We used two methods to monitor BP, including conscious BP measurement using the tail-cuff method and unconscious BP measurement using the invasive carotid artery catheterization. Results showed no significant difference in the baseline BP of either group (Figure 1B). Furthermore, we measured the BP of the rats after the 2k2c procedure. Both EP3-/- rats and the WT rats showed significantly increased BP compared to the sham-operated groups. However, there was still no difference between EP3-/- rats and the WT rats within the hypertensive group (Figure 1B), indicating that EP3 knockout did not impact both baseline BP and the induction of hypertension by the 2k2c procedure.
Deletion of EP3 attenuates the overexpression of ECM in the cerebral small arteries of RHRsp
Next, we tested whether disruption of EP3 modulates the progression of cerebral small artery remodeling in RHRsp. Two months after the 2k2c procedure, rats in the RHRsp group developed stable hypertension. By the end of 4-5months, the cerebral small arteries of RHRsp showed a significantly higher expression of ECM including fibronectin, laminin, collagen I, and collagen IV, compared with that observed in controls with normal BP. Intriguingly, EP3–/– RHRsp rats displayed a significant reduction in all four kinds of ECM, compared with that in WT RHRsp (Figure 2). These results indicated that despite no effect on hypertension, the deletion of EP3 still attenuated the overexpression of ECM in the cerebral small arteries of RHRsp.
Deletion of EP3 decreases the expression of ECM in BMVSMCs under AngII stimulation
As shown in above, our results confirmed that the deletion of EP3 successfully attenuated cerebral artery remodeling in RHRsp without alleviating high blood pressure. Hence, we assumed that EP3 may regulate the expression of ECM directly by affecting VSMCs. To further study the mechanism by which EP3 regulate VSMCs to express ECM in CSVD, BMVSMCs were extracted from both WT rats and EP3-/- rats. To mimic the elevated expression of ECM in vitro, we used ANGII to stimulate BMVSMCs, since both hypertensive humans and animals including RHRsp were reported to have an elevated level of ANGII[24, 25]. As shown in Figure 3, under the stimulation of ANGII, BMVSMCs exhibited significantly higher ECM expression including fibronectin, laminin, collagen I, and collagen IV. We found that the addition of the EP3 inhibitor L798106 could significantly alleviate this increased ECM expression induced by ANGII. Furthermore, the deletion of EP3 genes can significantly inhibit the baseline expression of ECM (without ANGII stimulation), and even under the stimulation of ANGII, a significantly higher expression of ECM was not observed in EP3-/- rat BMVSMCs. These results suggest that EP3 gene knockout can reverse the increased expression of ECM induced by ANGII in rat BMVSMCs.
Deletion of EP3 decreases the expression of ECM by suppressing TGF-β1 signaling in BMVSMCs under ANGII stimulation
TGF-β1 signaling has long been reported to play a role in regulating ECM expression. It has also been suggested that knockout of EP3 downregulates the expression of ECM in pulmonary arterioles by suppressing the TGF-β1/Smad pathway. In this study, we sought to determine whether EP3 can regulate the expression of ECM by altering the TGF-β1 signaling. As shown in Figure 4A-B, western blot analyses confirmed that the level of TGF-β1 and phosphorylation of Smad2/3 were elevated in rat BMVSMCs under ANGII stimulation. Inhibiting TGF-β1 signaling using LY364947 attenuated the high expression of ECM induced by ANGII in rat BMVSMCs (Figure 4A). Furthermore, the deletion of EP3 rescued ANGII-induced activation of TGF-β1 signaling, as evidenced by a decreased level of TGF-β1 and phosphorylation of Smad2/3(Figure 4B). These results indicated that the deletion of EP3 can decrease the expression of ECM by suppressing TGF-β1 signaling in BMVSMCs under ANGII stimulation.
Previous studies have shown that the EP3 receptor modulates multiple intracellular signaling pathways by coupling different types of heterotrimeric G proteins. As shown in Figure 4C, the downregulation of ECM by EP3 inhibition was abolished by pretreatment with pertussis toxin (PTX) in BMVSMCs, indicating that EP3 signaling regulated the expression of ECM via the PTX-sensitive G protein, Gi/o in BMVSMCs.
Deletion of EP3 attenuates decreased CBF in RHRsp
Cerebral small artery remodeling is the leading cause of decreased CBF in patients. In order to determine whether attenuated vascular remodeling caused by EP3 deletion can further improve CBF in RHRsp, we used PWI to monitor the global CBF of the animals. T2-weighted images and representative CBF images of the animals are shown in Figure 5. Consistent with previous studies on EP3-/- mice or mice treated with selective antagonists of EP3, our results showed no difference in CBF between normal WT rats and EP3-/- rats. As decreased CBF is seen in CSVD patients, our results showed that RHRsp demonstrated a lower global CBF, compared to the control rats. Intriguingly, EP3–/– RHRsp rats displayed a recovery of global CBF, compared with that in WT RHRsp. These results indicate that the deletion of EP3 can improve CBF in RHRsp, confirming our hypothesis that the deletion of EP3 can not only reverse vascular structure changes but also contribute to the functional recovery of blood supply in the brain.
Deletion of EP3 attenuates cognitive impairment in RHRsp
As seen above, the deletion of EP3 successfully attenuated vascular remodeling in RHRsp and improved the cerebral blood supply of the animals. We assume that these improvements may also help to attenuate the cognitive impairment in RHRsp. To this end, we used the Morris water maze test to evaluate the cognitive function of the animals. The Morris water maze test is designed to reflect the spatial learning and memory ability. During the hidden platform acquisition phase, on average, rats in all groups showed a progressive decrease in escape latency, indicating that subjects could learn the location of the platform. However, compared with the sham-operated group, RHRsp rats had a longer escape latency, and a statistical difference appeared on the third and fifth days. The deletion of EP3 (EP3-/-RHRsp) shortened the escape latency. However, there was no statistically significant difference (Figure 6). A no-platform probe test was conducted 24 h after the final day5 training trial. Figure 6 shows that the time spent in the target quadrant was decreased in RHRsp rats, compared with the sham-operated group. The deletion of EP3 (EP3–/– RHRsp) reversed this effect. The average number of times crossing the platform was higher in RHRsp than in sham-operated rats, and the deletion of EP3 (EP3–/– RHRsp) significantly improved the number. Taken together, these results show damaged spatial learning and memory ability in RHRsp, and the deletion of EP3 in RHRsp can rescue cognitive impairment. We cautiously speculate that the improvement of cognitive function in EP3–/–RHRsp could be partially attributed to the reduced vascular remodeling and increased CBF by the deletion of EP3.