Prenatal inflammation-induced NF-κB dyshomeostasis contributes to renin-angiotensin system over-activity resulting in prenatally programmed hypertension in offspring

Studies involving the use of prenatally programmed hypertension have been shown to potentially contribute to prevention of essential hypertension (EH). Our previous research has demonstrated that prenatal inflammatory stimulation leads to offspring’s aortic dysfunction and hypertension in pregnant Sprague-Dawley rats challenged with lipopolysaccharide (LPS). The present study found that prenatal LPS exposure led to NF-κB dyshomeostasis from fetus to adult, which was characterized by PI3K-Akt activation mediated degradation of IκBα protein and impaired NF-κB self-negative feedback loop mediated less newly synthesis of IκBα mRNA in thoracic aortas (gestational day 20, postnatal week 7 and 16). Prenatal or postnatal exposure of the IκBα degradation inhibitor, pyrollidine dithiocarbamate, effectively blocked NF-κB activation, endothelium dysfunction, and renin-angiotensin system (RAS) over-activity in thoracic aortas, resulting in reduced blood pressure in offspring that received prenatal exposure to LPS. Surprisingly, NF-κB dyshomeostasis and RAS over-activity were only found in thoracic aortas but not in superior mesenteric arteries. Collectively, our data demonstrate that the early life NF-κB dyshomeostasis induced by prenatal inflammatory exposure plays an essential role in the development of EH through triggering RAS over-activity. We conclude that early life NF-κB dyshomeostasis is a key predictor of EH, and thus, NF-κB inhibition represents an effective interventional strategy for EH prevention.

Scientific RepoRts | 6:21692 | DOI: 10.1038/srep21692 we observed aortic dysfunction with unknown mechanisms 18 . NF-κ B signal is the main downstream pathway after the inflammation challenge. To determine the role of NF-κ B signal in prenatal inflammatory-induced conduit artery dysfunction, we firstly assessed the mRNA and protein levels of NF-κ B family components in the thoracic aorta of prenatal LPS-stimulated offspring at gestational day (GD) 20 or postnatal week 7. By real-time RT-PCR analysis, we observed that prenatal LPS stimulation significantly decreased IκBα but increased IκBβ mRNA expression in thoracic aortas of offspring at both GD 20 (Fig. 2a) and postnatal week 7 (Fig. 2b), as compared to control animals. In addition, prenatal LPS exposure significantly decreased the mRNA expression levels of IKKβ and IKKγ in offspring's thoracic aortas at GD 20 (Fig. 2a); however, there was no significant change at postnatal week 7 (Fig. 2b). Immunoblotting further validated the decreased Iκ Bα , increased phosphorylated (p)-p65 and total p65 protein levels in thoracic aortas of prenatal LPS-stimulated offspring at postnatal week 7, as compared with those in the control group (Fig. 2c). To find more direct evidences of NF-κ B activation in the thoracic aorta of prenatal inflammation-induced offspring, we evaluated the NF-κ B p65 DNA binding activity using a non-radioactive universal EZ-TFA Transcription Factor Assay kit. This kit combines the principle of the electrophoretic mobility shift assay (EMSA) with the 96-well based enzyme-linked immunoabsorbent assay (ELISA) instead of the conventional EMSA. Our data showed that p65 DNA binding activity in thoracic aortas of prenatal LPS-stimulated offspring was significantly higher than that in the control offspring (Fig. 2d). We also observed a significantly higher level of tumor necrosis factor-α (TNF-α ), a downstream target of NF-κ B, in thoracic aortas of prenatal LPS-stimulated offspring at the age of 7 weeks (Fig. 2e). These data indicate a predisposition of early life NF-κ B activation, characterized by the low expression of Iκ Bα at both the mRNA and the protein levels, in the conduit artery of prenatal inflammation-induced offspring.
Interestingly, the mRNA expression of IKKs, which implicates in Iκ Bα phosphorylation and degradation, did not significantly change at postnatal week 7, indicating that mechanisms upstream of IKK or other signal pathways contributed to the down-regulation of Iκ Bα . Previous findings reported that PI3K-Akt activation leads to IκBα degradation through IKK-dependent 33 and -independent pathway 34,35 . Our current finding of increased phosphorylated IκBα in thoracic aortas of offspring from prenatal exposure to LPS, promoted us firstly to explore whether there exists an activation of PI3K-Akt in thoracic aortas of offspring from prenatal exposure to LPS. As expected, phosphorylated Akt ser473 and its downstream target phosphorylated GSK-3β ser9 were significantly increased in thoracic aortas of offspring that received prenatal exposure to LPS at the age of 7 weeks (Fig 3a). To  Thoracic aortic p65, IκBα, IκBβ, IKKα, IKKβ and IKKγ mRNA expression were determined by real-time RT-PCR at gestational day 20 (a) and postnatal week 7 (b) (n = 6 offspring in each group). (c) Iκ Bα , Iκ Bβ , phosphorylated (p)-p65 and total p65 protein expression were determined by immunoblotting in thoracic aortas of offspring at postnatal week 7. β -actin was taken as internal control. Representative plots of 2 from 8 offspring in each group (left panel) and statistical data of relative densitometry, normalized by β -actin (right panel), were shown. (d) NF-κ B p65 DNA binding activity in thoracic aortas of 7-week-old offspring were determined by a NF-κ B EZ-TFA transcription factor assay colorimetric kit. Positive, competitive, and negative controls were carried out using TNF-α -treated Hela whole cell extract, unlabeled competitor oligonucleotide containing the identical consensus sequence as the capture probe in other samples, and without any capture probe, respectively (n = 6 offspring in each group). (e) Expression levels of TNF-α and IL-6 in the thoracic aortas of 7-week-old offspring were measured by ELISA (n = 8 offspring in each group). Con indicates prenatal vehicle control; LPS, prenatal LPS stimulation. Error bars represent S.D. *p < 0.05 and **p < 0.01, LPS versus Con.
Scientific RepoRts | 6:21692 | DOI: 10.1038/srep21692 further address the mechanism by which IκBα mRNA expression was downregulated, we determined the binding ability of NF-κ B (p65) to the promoter of IκBα (NFKBIA) because previous reports found that NF-κ B can bind to multiple sites within the IκBα promoter thereby promoting its transcription in the recovery stage as a negative feedback loop after NF-κ B activation 36 . Our chromatin immunoprecipitation (ChIP) assays found that decreased binding of p65 appeared on all three potential binding sites of IκBα promoter (− 275/− 266 bp, − 181/− 172 bp and − 31/− 21 bp) in thoracic aortas of offspring that received prenatal exposure to LPS (Fig. 3b). This tendency was also the same in offspring from prenatal exposure to LPS at the age of 16 weeks (Data not shown).
To further resolve whether the early life NF-κ B activation in fetal thoracic aorta was triggered by the direct damage from LPS or by the indirect damage caused by maternal derived pro-inflammatory cytokines exposure, (a) Expression of Akt, GSK-3β , phosphorylated (p)-Akt ser473 and p-GSK-3β ser9 at the protein level was determined by immunoblotting in thoracic aortas of offspring at postnatal week 7. β -actin was taken as internal control. Representative plots of 2 from 8 offspring in each group (left panel) and statistical data of relative densitometry, normalized by β -actin (right panel), were shown. (b) Schematic structure of the IκBα (Nfkbia) promoter with putative NF-κ B-binding sites (top panel), and ChIP analysis of NF-κ B (p65) binding to the IκBα promoter in thoracic aortas at the age of 7 weeks old (bottom panel) (n = 5 offspring in each group). Con indicates prenatal vehicle control; LPS, prenatal LPS stimulation. Error bars represent S.D. *p < 0.05 and **p < 0.01, LPS versus Con.
we assessed the protein or mRNA levels of tumor-necrosis factor-α (TNF-α ) and interleukin-6 (IL-6) in maternal serum, placenta, as well as the embryo at different time points after the last i.p. injection of LPS. We found that the expression levels of TNF-α and IL-6 in maternal serum and placenta were immediately increased 2 hours (2 h) after last time LPS administration (Fig. 4a,b). However, only IL-6 mRNA expression in the fetus was significantly increased after 48 h of LPS administration (Fig. 4c). The delayed IL-6 mRNA elevation in fetus, in comparison to its rapid and robust increases in maternal serum and placenta, suggests that the damage on embryonic development might be caused indirectly by maternal derived pro-inflammatory cytokines exposure, rather than directly from in utero LPS.
Collectively, our data demonstrate that both PI3K-Akt-mediated degradation of Iκ Bα and impaired NF-κ B self-negative feedback loop on newly Iκ Bα re-synthesis implicates in Iκ Bα down-regulation and NF-κ B over-activation in conduit arteries, leading to hypertension in the offspring exposed to an inflammatory stimulus during the prenatal period.
Prenatal or postnatal administration of the NF-κB inhibitor, PDTC, prevents elevated blood pressure in inflammation-induced PPH rats. To further explore the physiological role of NF-κ B activation on the development of hypertension in offspring of prenatal inflammatory stimulation, we used the inhibitor of Iκ Bα degradation, PDTC, in vivo to block NF-κ B activation simultaneously with LPS stimulation or postnatally in adulthood. Treatment with PDTC started at postnatal week 7 and evaluated the NF-κ B activity and blood pressure at postnatal week 16. Our results identified a decrease in Iκ Bα , with reported increases in phosphorylated p65 (p-p65) and total p65 protein levels in thoracic aortas of prenatal LPS-stimulated offspring at the age of 16 weeks (Fig. 5a, LPS group and Fig. 5b, LPS + Ve group). Conversely, elevated Iκ Bα, decreased p-p65 and total p65 protein expression in thoracic aortas after PDTC administration at both prenatal (Fig. 5a, L + P group) and postnatal stage (Fig. 5b, LPS + PDTC group). Our data suggests that NF-κ B activation in the conduit artery of prenatal LPS-stimulated offspring is a lifelong persistent event. In addition, our data also suggests that PDTC efficiently inactivates NF-κ B signaling in the offspring's thoracic aortas. Our published research in this area had shown that prenatal LPS stimulation leads to development of offspring's hypertension with no gender differences 15 . In the current study, we were interested in exploring whether persistent NF-κ B activation was associated with the development of hypertension in both male and female offspring. We found that prenatal PDTC administration significantly abolished offspring's mean systolic artery pressure (MSAP) elevation induced by prenatal LPS stimulation (L + P group), compared with that in offspring of prenatal LPS stimulation alone (LPS group) (LPS, 129 ± 1 mmHg versus L + P, 115 ± 2 mmHg; 8 male and 8 female offspring for each group, p < 0.05) (Fig. 5c). Our results also demonstrated at postnatal 16 weeks old, daily PDTC administration inhibited offspring's MSAP elevation induced by prenatal LPS exposure (LPS + Ve, 124 ± 1 mmHg versus LPS + PDTC, 109 ± 3 mmHg; 8 male and 8 female offspring for each group, p < 0.05) (Fig. 5d). However, postnatal PDTC treatment had no effect on MSAP in control offspring (data not shown). These results indicate that early life and persistent NF-κ B activation is highly associated with the offspring's gradual elevation in blood pressure.

Prenatal PDTC administration alleviates RAS over-activity and improves endothelial dysfunction in thoracic aortas of inflammation-induced PPH rats.
To explore the mechanisms by which PDTC inhibited the offspring's blood pressure elevation, we first determined the local RAS activity in thoracic aortas since it is known to contribute to the chronic process of secondary structural damages during hypertension development 25 . The protein levels of RAS family members, such as Ang II, ACE, ACE2, AT1R and AT2R, were determined by immunostaining or immunoblotting. We found that prenatal LPS stimulation increased protein levels of ACE, AT1R and Ang II, but had no effect on ACE2 and AT2R in thoracic aortas ( Fig. 6a,b, LPS group). The increase in ACE and Ang II protein levels by prenatal LPS exposure was obviously abolished by prenatal PDTC treatment ( Fig. 6a,b, L + P group). However, prenatal PDTC treatment did not significantly influence the protein expression levels of ACE2, AT1R and AT2R (Fig. 6a, L + P group).
Furthermore, we also assessed the protein levels of p-eNOS ser1176 and total eNOS, known markers of vascular endothelium function 37 , by immunostaining in thoracic aortas. The p-eNOS ser1176 level was significantly decreased in thoracic aortas of prenatal LPS-stimulated offspring (Fig. 6c,d, LPS group); however, this decrease in p-eNOS ser1176 expression was also abolished by prenatal PDTC administration (Fig. 6c,d, L + P group). Although the eNOS protein level was unchanged in both L + P group and LPS group (Fig. 6c,d), the ratio of p-eNOS ser1176 to total eNOS expression was decreased in thoracic aortas of prenatal LPS-stimulated offspring (LPS group) whereas it was increased by prenatal PDTC administration (L + P group) (Fig. 6e).

Postnatal long-term PDTC administration prevents offspring from RAS over-activity and endothelial dysfunction at thoracic aortas of inflammation-induced PPH rats.
In this segment of our research, we wanted to determine the protective effect of PDTC on offspring's thoracic aortas in the model of postnatal PDTC administration. We assessed the offspring's RAS activity by measuring the expression levels of relevant proteins (Ang II, ACE, ACE2, AT1R and AT2R ) in thoracic aortas of offspring at the age of 16 weeks after 9 weeks of daily PDTC administration that started at postnatal week 7. The protein levels of ACE and AT1R were increased in thoracic aortas of prenatal LPS-stimulated offspring (LPS + Ve group) compared to those in control (Con + Ve group). Postnatal PDTC treatment abolished the changes of ACE but not AT1R protein expression in thoracic aortas of offspring with prenatal LPS exposure (LPS + PDTC group). There was no significant difference of ACE2 and AT2R expression among these groups (Fig. 7a). We found higher Ang II expression in thoracic aortas of prenatal LPS-stimulated offspring (LPS + Ve group) compared to controls (Con + Ve group). Interestingly, this elevated expression of Ang II was attenuated by postnatal long-term PDTC administration (LPS + PDTC group) (Fig. 7b). On the other hand, the expression of p-eNOS ser1176 protein was decreased in thoracic aortas of prenatal LPS-stimulated offspring (LPS + Ve group) compared with that of controls (Con + Ve group), suggesting that prenatal LPS exposure led to the suppressed level of p-eNOS ser1176 protein at an older age. Interestingly, this decrease in p-eNOS ser1176 protein expression mediated by prenatal LPS exposure was efficiently rescued by the PDTC treatment (LPS + PDTC group). The ratio of p-eNOS ser1176 to total eNOS showed a similar trend among these groups since there was no change in the expression of total eNOS protein under these conditions (Fig. 7c). Representative blot (top panel) and statistical data of relative densitometry (bottom panel), normalized by β -actin, were shown (n = 6 offspring in each group). (c,d) Mean systolic artery pressure (MSAP) was measured by standard tail-cuff method in 16-week-old conscious offspring from prenatal PDTC administration simultaneously with LPS stimulation (c) or post-birth PDTC daily administration from 7 to 16 weeks old (d) (n = 8 male and 8 female offspring in each group). Con indicates prenatal vehicle control; LPS, prenatal LPS stimulation; L + P, prenatal LPS plus PDTC administration; Con + Ve, offspring from Con group accepted saline daily from 7 to 16 weeks old as vehicle control; LPS + Ve, offspring from LPS group accepted saline daily from 7 to 16 weeks old as model control; LPS + PDTC, offspring from LPS group accepted PDTC daily from 7 to 16 weeks old. Error bar represents S.D. *p < 0.05 or **p < 0.01, LPS versus Con or LPS + Ve versus Con + Ve, respectively; # p < 0.05 or ## p < 0.01, L + P versus LPS or LPS + PDTC versus LPS + Ve, respectively.   Our previous report have demonstrated that prenatal PDTC treated control offspring only showed transient increased Ang II expression in kidney 1 day after birth, but soon reversed to the same level in adult 16 and the postnatal PDTC treatment did not have any obvious effects on RAS activity in control offspring (data not shown). Thus, our data supports a newly defined network that early life NF-κ B activation is responsible for RAS over-activity and endothelium dysfunction in conduit artery of PPH rats.

Prenatal LPS stimulation shows no effect on RAS mRNA transcript in thoracic aortas of inflammation-induced PPH rats at early life.
To further determine the expression changes of RAS components in offspring's thoracic aortas after prenatal LPS stimulation, we measured the transcript levels of AT1R, AT2R, and ACE in offspring's thoracic aortas at GD 20 or postnatal week 7 by real-time RT-PCR, respectively. Interestingly, there was no significant change in the mRNA expression of RAS components in thoracic aortas of prenatal LPS-stimulated offspring at both GD 20 and postnatal week 7 (Fig. 8a,b). The upregulation in RAS activity was delayed in comparison to NF-κ B activation in thoracic aortas of prenatal LPS-stimulated offspring. This suggested that early life and persistent NF-κ B activation is a critical factor for the elevation in RAS activity in the local vasculature of inflammation-induced PPH rats, although other risk factors may also exist.
Prenatal LPS exposure leads to resistance artery lesion but not NF-κB activation nor RAS over-activity in inflammation-induced PPH rats at the postnatal age of 16 weeks. Because damage of resistance artery structure plays a critical role in the progression of EH, we focused our efforts on detecting the pathological changes of superior mesenteric arteries in prenatal LPS-stimulated offspring at the age of 16 weeks. As expected, we observed obvious enlargement of the media muscular layer existed in superior mesenteric arteries of prenatal LPS-stimulated offspring (LPS group) at 16 weeks of age. In addition, we also detected that some endothelial layers were detached or even desquamated from the basement membrane in superior mesenteric arteries of prenatal LPS-stimulated offspring (LPS group). In contrast, prenatal PDTC administration prevented the enlargement of the media muscular layer and lesion of endothelial layer in superior mesenteric arteries that were caused by prenatal LPS exposure (Fig. 9a).
We previously reported inconsistent changes of ACE expression in conduit arteries and resistance arteries in spontaneous hypertensive rats 26 . As such, it suggests a tissue specific role of RAS activity during the development of hypertension. Henceforth, this prompted us to determine NF-κ B and RAS activity in resistance arteries after prenatal LPS exposure by detecting the protein expression of p-p65, p65, and the targeted RAS family of proteins. We did not observe any significant change in any of these targeted proteins (Fig. 9b,c). The consistent changes between NF-κ B and RAS activity in conduit and resistance arteries, taken together with the finding that RAS activity upregulation was delayed in comparison to NF-κ B activation in conduit arteries (Fig. 8), suggest that NF-κ B upregulating RAS activity in conduit artery is critical to aortic dysfunction and hypertension development.

Discussion
The present study demonstrated that prenatal inflammatory exposure caused conduit but not resistance artery Iκ Bα /Iκ Bβ imbalance, leading to early life NF-κ B activation. This dyshomeostasis evoked progressive dysfunction of RAS in adult offspring's conduit artery, and subsequently aggravated blood pressure resulting in the development of hypertension. The major findings of this study is that prenatal LPS exposure caused a NF-κ B activation, characterized by decreased Iκ Bα level through both PI3K-Akt-mediated degradation of Iκ Bα and less newly synthesized IκBα mRNA by impaired NF-κ B self-negative feedback loop, from fetus to adult. As such, this indirect damage on the fetus is mainly caused by the fetal exposure of maternal-derived pro-inflammatory cytokines. In addition, we have also found that prenatal or postnatal application of specific Iκ Bα degradation inhibitor PDTC prevented the offspring's blood pressure elevation and endothelium dysfunction. Furthermore, we showed that prenatal LPS exposure led to higher levels of ACE and Ang II in aortic tissue, which could be abolished by both prenatal and postnatal PDTC application. Finally, we found that prenatal LPS exposure resulted in resistance artery lesion but not NF-κ B activation or RAS over-activity in offspring at the age of 16 weeks. These findings support our hypothesis that early life NF-κ B dyshomeostasis in conduit artery, induced by prenatal inflammatory stimulation, is predictive of EH development in adult.
Inflammation plays an important role in vascular dysfunction and hypertension 38 . Recent research has focused on the effect of these inflammatory mediators in specific regard to their effects on the conduit artery; however, their effects during the progression of hypertension are largely unknown. The current study focuses on the effects of prenatal inflammatory stimuli on the development of hypertension. We identified that prenatal exposure to inflammatory stimuli causes higher levels of pro-inflammatory factors and NF-κ B activation in the fetus (48 h after the last LPS administration) as well as in thoracic aortas of 7-week-old offspring. We also found that NF-κ B p65 DNA binding activity and expression of pro-inflammatory factor TNF-α were significantly higher in the thoracic aorta of prenatal inflammation stimulated offspring at the age of 7 weeks. As persistent NF-κ B activation is a nexus for inflammation response and tissue damage 39 , a deep understanding on how distinct pathways activate or inhibit NF-κ B activity may be indispensable for us to search and identify the potential targets for treatment or prevention of certain human diseases. NF-κ B activation is reported to exist in kidney 40,41 and heart 42 of older spontaneous hypertensive rats with unknown mechanisms. In the present study, using a classic immune-inflammation stimulator LPS during the second trimester of fetal development, we found that persistent NF-κ B activation can be programed during the fetal development by inflammatory challenge, and blocking NF-κ B activation by PDTC could prevent hypertension development in offspring of prenatal inflammatory stimulation. Thus, we believe that early life and persistent NF-κ B activation programed by prenatal inflammatory stimuli is a critical factor for inflammation ignition and maintenance in the early stage of hypertension development.
NF-κ B and Iκ B proteins play as an integrated network in regulating NF-κ B activation. At rest state, Iκ Bα maintains NF-κ B in an inactive form in the cytoplasm by blocking the nuclear localization signals of NF-κ B proteins, while Iκ Bβ can sequester p65 in the cytoplasm by masking NF-κ B nuclear localization sequences 43,44 . Upon stimulation, IKKs phosphorylate the classical Iκ B proteins Iκ Bα , Iκ Bβ and Iκ Bε at specific serine residues, which lead to their proteasomal degradation, and nuclear translocation of free NF-κ B. However, NF-κ B-dependent transcription of Iκ Bα gene leads to rapid re-synthesis of the Iκ Bα protein, and inhibition of NF-κ B-dependent transcription at nucleus 45 . At the same time, newly synthesized Iκ Bβ shows a hypo-phosphorylated form, which mainly stays in the nucleus, binds DNA with p65 and c-Rel. This DNA-bound NF-κ B/Iκ Bβ complexes are resistant to Iκ Bα , and can prolong pro-inflammatory gene expression 46,47 . In the current study, we found that both mRNA and protein levels of Iκ Bα were diminished in aortic tissue from fetus to adult after prenatal inflammatory exposure, whereas no significant changes existed in Iκ Bβ expression. Mechanically, we found no expression changes of IKKs, but increased PI3K-Akt activation mediated degradation of Iκ Bα protein and impaired NF-κ B self-negative feedback loop on newly Iκ Bα synthesis implicated in Iκ Bα down-regulation in conduit arteries of prenatal inflammation-stimulated offspring. This indicates a specific gene re-imprinting of Iκ Bα after prenatal inflammatory stimulation is responsible for impaired NF-κ B clearance capacity and activation.
The RAS is described as a cascade of biochemical reactions, whose activity is essential for cardiovascular homeostasis 48 . The increased Ang II level leads to the expression of IL-6, MCP-1 and TNF-α in monocytes in vascular tissue, which contributes to the vascular inflammation leading to vascular damage 49 . Our previous studies demonstrated that systemic levels of Ang II 16 , NO and endothelin 1 15 remained unchanged but the levels of renal renin, Ang II expression 16 and thoracic aortas' Ang II, AT1R 30 were significantly increased in prenatal inflammation-induced PPH rats. The present study found that ACE over-expression was responsible for increased Ang II expression in thoracic aortas of prenatal inflammation stimulated offspring. Interestingly, increased Ang II, AT1R, ACE expression and NF-κ B activation didn't exist in the resistance artery of prenatal LPS-stimulated offspring, which is consistent with previous finding in spontaneous hypertensive rats 26 . This similarity with spontaneous hypertensive rats further supported our idea that prenatal inflammatory stimulation programed hypertension could also be good model to study EH in the future.
Increased tissue Ang II favors the expression of regulatory, structural, and cytokine genes through the activation of NF-κ B signal pathway in EH, which plays important roles in long-term control of blood pressure, vascular remodeling, cardiac hypertrophy and inflammation 50,51 . Our current study found that NF-κ B activation, occurring in thoracic aortic tissue in both fetus and adult, took place prior to the expression changes of RAS components. Increased ACE and Ang II expression were reversed by both prenatal and postnatal PDTC administration. However, we could not find any direct evidence of increased NF-κ B binding to ACE promoter in thoracic aortas of offspring from prenatal exposure to LPS (data not show). Our study demonstrates that this delayed RAS over-activity is indirectly caused by NF-κ B activation through the pro-inflammatory nexus preceding hypertension development in PPH rats. Intriguingly, prenatal or postnatal PDTC application predominantly reversed ACE but showed no effect on AT1R receptor expression. This might be the reason that PDTC blocked the reported positive feedback loop between NF-κ B and RAS (NF-κ B → ACE → Ang II → NF-κ B) 52 . Together with the finding that the consistent changes between NF-κ B and RAS activity only in conduit but not in resistance arteries, we demonstrate that prenatal PDTC treatment can block the initial factor of NF-κ B activation. We also demonstrate that postnatal PDTC treatment can break the RAS positive feedback, both of which could prevent the development of prenatal programed hypertension.
Endothelial cells release endothelial-relaxing factors and therefore have a critical role in vasodilation, inhibition of platelet aggregation and monocyte invasion. Endothelium dysfunction is associated with a variety of diseases including atherosclerosis, diabetes mellitus, coronary artery disease, hypertension and hypercholesterolemia 53 . In the current study, we found decreased eNOS phosphorylation level in conduit artery of PPH rats at 16 weeks old, which was restored by PDTC administration at fetus or after birth. The activation of NF-κ B → ACE → Ang II → Reactive oxygen species signal pathway plays a major role in the pathogenesis of endothelium dysfunction 54 . We previously found no significant vasodilation reactivity at the age of 12 weeks, which indicated the proper endothelium function at that time point 18 . This suggests that the endothelium dysfunction might be a secondary event of NF-κ B → ACE → Ang II → Reactive oxygen species → NF-κ B positive feedback signal pathway during the development of hypertension in prenatal inflammation-induced offspring.
In summary, the present study has established a new and direct link between programmed NF-κ B dyshomeostasis, early life NF-κ B activation and RAS over-activity in conduit artery in prenatal inflammation-induced PPH rats. Following prenatal inflammatory stimulation, early life and adult lifelong NF-κ B activation ignites the NF-κ B → ACE → Ang II → NF-κ B positive feedback loop in conduit artery, which in turn predisposes to aortic dysfunction and development of prenatal inflammation programmed hypertension. Since prenatal inflammatory exposure is an important unresolved public health problem, the present findings provide a new notion to reduce the incidence of adult hypertension at an early life stage. As NF-κ B dyshomeostasis might be predictive of PPH, it would be beneficial to identify safe agents to efficiently modulate the NF-κ B signaling pathway for prevention or treatment of EH starting at the neonatal stage.

Animals.
Nulliparous time-dated pregnant SD rats were purchased from the Animal Centre of Third Military Medical University (Chongqing, China). All animals took standard laboratory rat chow and tap water ad artitrium. Rats were housed individually in a room at constant temperature (24 °C) and under a 12-h light-dark cycle until parturition. Pups were raised with a lactating mother until 4 weeks of age, at which time they were weaned to cages containing four pups for each 15 . This study was conducted in accordance with the principles outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures and protocols were approved by the local animal ethics committee at Third Military Medical University.
Study I: Prenatal PDTC study. Time-dated pregnant (GD 8) SD rats (250 g to 300 g) were randomly divided into three groups (n = 8 in each group) as described previously 16 . The pregnant rats in these groups were intraperitoneally (i.p) administered with saline (Control group), LPS 0.79 mg/kg (LPS group), or LPS 0.79 mg/kg plus PDTC 0.609 mmol/kg (L + P group), respectively. LPS was given on GD 8, 10 and 12, whereas PDTC was given daily from GD 8 to 14. The pregnant rats in the LPS group were given saline injection on GD 9, 11, 13 and 14, and pregnant rats in the control group were given saline daily from GD 8 to14. After delivery, the size of the litter was reduced to 8 for each mother by random selection to avoid the effect of nutritional disproportion.
Study II: Postnatal PDTC study. For postnatal PDTC administration, pups from the aforementioned LPS group in Study I were randomly separated into two groups and daily i.p administered with PDTC (0.609 mmol/kg, LPS + PDTC group) or with vehicle saline (LPS + Ve group) from postnatal week 7 to week 16. Aforementioned control group offspring in Study I received saline daily starting at postnatal week 7 were taken as vehicle control (Con + Ve).
Collection of serum and tissues. Blood samples from pregnant rats were obtained via heart puncture 2 h after the last LPS i.p injection. After clotting for a half hour and then centrifuging at 3000 rpm for 20 min, the serum was collected and stored at − 80 °C for later use.
Placental and embryonic tissues were collected and then stored at RNA later solution (Tiangen Biotech, Beijing, China) at − 80 °C from pregnant rats that were anaesthetized at indicated time points after the last LPS i.p injection.

Real-time RT PCR.
Real-time RT-PCR was performed as previously described 16 . Briefly, the thoracic aortic p65, IκBα, IκBβ, IKKα, IKKβ, IKKγ, TNF-α and IL-6 mRNA expressions were assessed by real-time RT-PCR when the offspring were at gestational day 20 or postnatal week 7, respectively. Total RNA was extracted from thoracic aortas using Trizol (Roche Molecular Biochemicals, Mannheim, Germany) and total RNA (1 μ g) was then reverse-transcribed into cDNA using a First Stand cDNA Synthesis Kit (Toyobo, Osaka, Japan). Primer sequences for real-time RT-PCR were listed in Table 1. Each real-time PCR reaction was carried out in a total volume of 20 μ l with Quanti Tect SYBR Green PCR Master Mix (MJ Research, Waltham, MA, USA) according to the following conditions: 2 min at 95 °C, 40 cycles at 95 °C for 10 s, 60 °C for 15 s, 72 °C for 20 s, using ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA).
Relative mRNA expression was calculated by normalizing the relative gene expression to the control group after normalized the cycle threshold value by the internal control β-actin.
For Ang II immunofluorescence, goat anti-rabbit IgG H&L (FITC) antibodies (Abcam) were used for an additional 60 minute incubation after the primary antibody incubation and washing. Sections were then incubated with 1 mg/ml DAPI for 30 min, washed three times with PBS, and mounted into Vectashield ® mounting medium (Vector Laboratories, Burlingame, CA). The coverslips were visualized under a Leica confocal laser scanning microscope (Leica).
The histological structure of superior mesenteric artery was analyzed by standard H&E staining after sections were dewaxed and rehydrated.
The investigators were blinded for acquiring the images.