H3K9ac of TGFßRI in human umbilical cord: a potential biomarker for evaluating cartilage differentiation and susceptibility to osteoarthritis via a two-step strategy.

BACKGROUND: Epidemiological investigation and our previous reports indicated that osteoarthritis had a fetal origin and was closely associated with intrauterine growth retardation (IUGR). Human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) could be programmable to "remember" early-life stimuli. Here, we aimed to explore an early-warning biomarker of fetal-originated adult osteoarthritis in the WJ-MSCs. METHODS: Firstly, two kinds of WJ-MSCs were applied to evaluate their chondrogenic potential in vitro through inducing chondrogenic differentiation as the first step of our strategy, one from newborns with IUGR and the other from normal newborns but treated with excessive cortisol during differentiation to simulate the excessive maternal glucocorticoid in the IUGR newborns. As for the second step of the strategy, the differentiated WJ-MSCs were treated with interleukin 1ß (IL-1ß) to mimic the susceptibility to osteoarthritis. Then, the expression and histone acetylation levels of transforming growth factor ß (TGFß) signaling pathway and the expression of histone deacetylases (HDACs) were quantified, with or without cortisol receptor inhibitor RU486, or HDAC4 inhibitor LMK235. Secondly, the histone acetylation and expression levels of TGFßRI were further detected in rat cartilage and human umbilical cord from IUGR individuals. RESULTS: Glycosaminoglycan content and the expression levels of chondrogenic genes were decreased in the WJ-MSCs from IUGR, and the expression levels of chondrogenic genes were further reduced after IL-1ß treatment, while the expression levels of catabolic factors were increased. Then, serum cortisol level from IUGR individuals was found increased, and similar changes were observed in normal WJ-MSCs treated with excessive cortisol. Moreover, the decreased histone 3 lysine 9 acetylation (H3K9ac) level of TGFßRI and its expression were observed in IUGR-derived WJ-MSCs and normal WJ-MSCs treated with excessive cortisol, which could be abolished by RU486 and LMK235. At last, the decreased H3K9ac level of TGFßRI and its expression were further confirmed in the cartilage of IUGR rat offspring and human umbilical cords from IUGR newborn. CONCLUSIONS: WJ-MSCs from IUGR individuals displayed a poor capacity of chondrogenic differentiation and an increased susceptibility to osteoarthritis-like phenotype, which was attributed to the decreased H3K9ac level of TGFßRI and its expression induced by high cortisol through GR/HDAC4. The H3K9ac of TGFßRI in human umbilical cord could be a potential early-warning biomarker for predicting neonatal cartilage dysplasia and osteoarthritis susceptibility.

Lentivirus-delivered ACE siRNA rescues the impaired peak bone mass accumulation caused by prenatal dexamethasone exposure in male offspring rats.

Angiotensin I converting enzyme (ACE) is a major component of the renin-angiotensin system (RAS). Our previous study demonstrated that activated bone RAS was associated with low peak bone mass induced by prenatal dexamethasone exposure (PDE) in male offspring rats. However, we did not determine whether the inhibition of ACE expression could rescue PDE-induced low peak bone mass. In the present study, we treated pregnant Wistar rats with dexamethasone (0.2 mg/kg.d) on gestational days 9-20 and obtained eight weeks old male offspring rats. Some of the offspring rats from the PDE group were injected lentivirus delivered-ACE siRNA (LV-ACE siRNA) through the intra-bone marrow for 4 weeks. We found that the intra-bone marrow injection of LV-ACE siRNA rescued the impaired peak bone mass accumulation caused by PDE in male offspring rats. Moreover, LV-ACE siRNA ameliorated PDE-induced inhibition of osteogenesis and alleviated PDE-induced RAS activation in the bone tissues in vivo. Our in vitro findings further confirmed that LV-ACE siRNA reversed the suppressed osteogenic differentiation caused by dexamethasone, which can be attributed to alleviated RAS activation. In conclusion, LV-ACE siRNA rescued impaired peak bone mass accumulation caused by PDE through alleviation of local bone RAS activation in male offspring rats.

The low-expression programming of 11ß-HSD2 mediates osteoporosis susceptibility induced by prenatal caffeine exposure in male offspring rats.

BACKGROUND AND PURPOSE: Prenatal caffeine exposure (PCE) can cause developmental toxicity of long bones in offspring, but the long-term effects and the underlying mechanism have not been fully clarified. Here, we investigated the effects of PCE peak bone mass accumulation and osteoporosis susceptibility in offspring and its intrauterine programming mechanism. EXPERIMENTAL APPROACH: Pregnant Wistar rats were administrated intragastrically with saline or caffeine (120 mg·kg-1 ·day-1 ) on gestational days 9-20. The serum and bone samples were collected from the fetal and postnatal offspring for bone mass, genes expression and corticosterone analysis. Then, rat bone marrow mesenchymal stem cells (BMSCs) were treated with corticosterone in vitro to confirm the molecular mechanism. KEY RESULTS: PCE caused fetal bone dysplasia in male and female offspring. In adulthood, PCE reduced peak bone mass and increased osteoporosis susceptibility in male offspring but not in females. Meanwhile, PCE only decreased the H3K9ac and expression levels of 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) before and after birth in the male offspring but not in the females. Moreover, the high level of corticosterone induced by PCE down-regulated the H3K9ac and expression levels of 11ß-HSD2 through promoting glucocorticoid receptor (GR; NR3C1) into the nucleus of bone marrow mesenchymal stem cells (BMSCs) and recruiting histone deacetylase 11 (HDAC11) binding to 11ß-HSD2 promoter region, which further enhanced the effect of corticosterone on suppressing osteogenic function of BMSCs. CONCLUSION AND IMPLICATIONS: PCE caused osteoporosis susceptibility in male adult offspring, which attributed to the low-functional programming of 11ß-HSD2 induced by corticosterone via GR/HDAC11 signalling.

Articular damages in multi-generational female offspring due to prenatal caffeine exposure correlates with H3K9 deacetylation of TGFß signaling pathway.

Adverse environment during pregnancy could lead to maternal glucocorticoid overexposure in utero, and then induce the intrauterine growth retardation (IUGR) and the programmed change in cartilage development. The transforming growth factor ß (TGFß) signaling pathway plays a crucial role in the process of chondrogenesis, cartilage growth, development, maturation, and phenotype maintenance. Our previous results had shown that prenatal caffeine exposure (PCE) could result in the damaged articular cartilage in offspring rats. However, whether this change could transmit to multiple generations was still unknown. In this study, pregnant Wistar rats received either saline or caffeine (120 mg/kg, i.g.) once daily from gestational day 9-20 (GD9-20). The female offspring mated with normal male rats to generate the following generations. We obtained the articular cartilages in subsequent F1 to F3 female offspring. The H3K9 acetylation and expression of the TGFß signaling pathway were detected; the content of the cartilage matrix was detected. The results showed that PCE reduced the H3K9 acetylation and the expression of the TGFß signaling pathway, then reduced the extracellular matrix in F1, F2, and F3 generations. in vitro, corticosterone could induce the H3K9 deacetylation of the TGFß signaling pathway, thus inhibiting the expression of the TGFß signaling pathway and extracellular matrix. The overall results revealed that PCE induced a multi-generational damaged articular cartilage in female offspring rats, which was partially related to the maternal high glucocorticoid-induced H3K9 hypoacetylation of TGFß signaling pathway.

GR/HDAC2/TGFßR1 pathway contributes to prenatal caffeine induced-osteoarthritis susceptibility in male adult offspring rats.

Prenatal caffeine exposure (PCE) induces developmental toxicity of multi-organ and susceptibility to multi-disease in offspring. However, the effects of PCE on osteoarthritis susceptibility in adult offspring and its intrauterine programming mechanism remain to be further investigated. Here, we found that PCE induced susceptibility to osteoarthritis in male adult offspring rats, which was related to the inhibited function of cartilage matrix synthesis from fetuses to adults. Meanwhile, PCE consistently downregulated the H3K9ac and expression levels of transforming growth factor ß receptor 1 (TGFßR1), and then blocked TGFß signaling pathway, which contributed to the suppressed cartilage matrix synthesis. Moreover, the high level of corticosterone caused by PCE reduced the H3K9ac level on TGFßR1 promoter region through acting on glucocorticoids receptor (GR) and recruiting histone deacetylase 2 (HDAC2) into the nucleus of fetal chondrocytes. Taken together, PCE induced osteoarthritis susceptibility in male adult offspring rats, which was attributed to the low-functional programming of TGFßR1 induced by corticosterone via GR/HDAC2 signaling.

Subchondral bone dysplasia partly participates in prenatal dexamethasone induced-osteoarthritis susceptibility in female offspring rats.

Prenatal dexamethasone exposure (PDE) induces developmental toxicities of multi-organs and susceptibility to multi-diseases in offspring. However, the effects of PDE on osteoarthritis susceptibility in adult offspring and its mechanism have not been reported. In the present study, we treated pregnant Wistar rats with dexamethasone (0.2 mg/kg) daily on gestational days (GD) 9-20. Some pregnant rats were sacrificed on GD20, and the rest were delivered to obtain the postnatal offspring. The adult female offspring rats were performed with ovariectomy or sham operation during postnatal weeks 22-28. We found that PDE led to osteoarthritis phenotypes in articular cartilage and an increase in modified Mankin's score, but reduced the cartilage thickness in female adult offspring rats, which were more evident after ovariectomy. Moreover, PDE reduced the bone mass of subchondral bone in female adult offspring, which was aggravated by ovariectomy. The correlation analysis results indicated that the osteoarthritic phenotype and cartilage thickness were closely associated with the decreased bone mass of subchondral bone induced by PDE. Further, PDE retarded the development of primary and secondary ossification centers, then led to subchondral bone dysplasia, which could be partly mediated by the inhibited osteogenic function before and after birth. Collectively, the subchondral bone dysplasia partly participated in osteoarthritis susceptibility induced by PDE in female offspring rats.

Subchondral bone dysplasia mediates susceptibility to osteoarthritis in female adult offspring rats induced by prenatal caffeine exposure.

Our previous studies confirmed that prenatal caffeine exposure (PCE) could induce susceptibility to osteoarthritis in adult offspring rats due to poor chondrocyte differentiation, but its mechanism remains to be further investigated. This study aimed to explore whether subchondral bone dysplasia mediates susceptibility to osteoarthritis in adult offspring rats induced by PCE. Pregnant Wistar rats were treated with caffeine (120 mg/kg.d) or saline from gestational day (GD) 9 to 20. The female offspring were euthanized to collect femurs at GD20, postnatal week (PW) 6, and PW28 (non-ovariectomy and ovariectomy groups) to detect osteoarthritis-like phenotype, subchondral bone mass, ossification center development, and other evidence. The results showed that PCE increased the Mankin score of pathological articular cartilage, but decreased articular cartilage thickness and subchondral bone mass, which were more obvious after ovariectomy. Meanwhile, the correlation analysis results demonstrated that the Mankin score of articular cartilage was significantly negatively correlated with subchondral bone mass, and the thickness of articular cartilage was significantly positively correlated with subchondral bone mass. Further, the length and area of the primary and secondary ossification centers, the number of osteoblasts, and the related genes' expression of osteogenic differentiation (e.g., Runx2, BSP, ALP, and OCN) were all significantly decreased in the PCE group before and after birth. Taken together, PCE induced susceptibility to osteoarthritis in adult female offspring, which was likely related to the subchondral bone dysplasia and reduction of subchondral bone mass production due to developmental disorder of primary and secondary ossification centers caused by osteoblast differentiation disability before and after birth.

Nicotine exposure during pregnancy programs osteopenia in male offspring rats via α4ß2-nAChR-p300-ACE pathway.

Prenatal nicotine exposure (PNE) induces developmental toxicity in offspring. However, the long-term harmful effects on bone development and the intrauterine programming mechanism attributed to PNE remain unclear. In the present research, pregnant Wistar rats were injected subcutaneously with nicotine (2 mg/kg/d) to obtain and analyze bone samples from the fetal and adult offspring. Bone marrow mesenchymal stem cells (BMSCs) were treated with nicotine during osteogenic differentiation to clarify the related molecular mechanisms. The results indicated that PNE led to bone dysplasia in the fetuses and reduced bone mass in the adult offspring, which was mediated by the sustained activation of the local bone renin angiotensin system (RAS) and suppressed osteogenic differentiation before and after birth. In vitro, nicotine suppressed BMSCs' osteogenic function through promoting angiotensin-converting enzyme (ACE) expression and activating RAS. Furthermore, nicotine induced histone acetylase p300 into the nuclei of the BMSCs by acting on the α4ß2-nicotinic acetylcholine receptor (α4ß2-nAChR), leading to the increased histone 3 lysine 9 acetylation level of ACE and RAS activation. Taken together, the sustained activation of local bone RAS mediated prenatal nicotine-induced osteopenia in adult offspring via the α4ß2-nAChR-p300-ACE pathway.-Xiao, H., Wen, Y., Pan, Z., Shangguan, Y., Magdalou, J., Wang, H., Chen, L. Nicotine exposure during pregnancy programs osteopenia in male offspring rats via α4ß2-nAChR-p300-ACE pathway.

Prenatal caffeine exposure induces liver developmental dysfunction in offspring rats.

We previously showed that prenatal caffeine exposure (PCE) induces intrauterine growth retardation (IUGR) and high susceptibility to nonalcoholic fatty liver disease in offspring rats, and the underlying mechanisms are associated with fetal overexposure to maternal glucocorticoids. Herein, we aimed to verify whether PCE disrupts liver development before and after birth and explore its possible programming mechanism. In vivo, reduced fetal weights and increased IUGR rates were accompanied by fetal liver developmental dysfunction in PCE rats. Increased fetal serum corticosterone and decreased insulin-like growth factor 1 (IGF1) levels were observed. Both male and female fetal livers exhibited increased glucocorticoid function-related gene (GR/C/EBPα) expression and inhibited IGF1 signaling pathway (IGF1/IGF1R/Akt2) expression. At PW6, the levels of serum corticosterone and glucocorticoid function-related genes in PCE offspring livers were decreased, while serum IGF1 and liver IGF1 signaling pathway expression were increased, accompanied by obvious catch-up growth and enhanced liver function. Furthermore, in PCE adult offspring under chronic stress, serum corticosterone and liver GR/C/EBPα expression levels were elevated, while the serum IGF1 and liver IGF1 signaling pathway levels were decreased. In vitro, cortisol (not caffeine) upregulated GR and C/EBPα expression and downregulated IGF1R expression. The IGF1R expression downregulated by cortisol was partially reversed by GR or C/EBPα knockdown. In conclusion, PCE induced liver developmental dysfunction in fetal rats and catch-up growth in IUGR offspring. The mechanisms may be closely associated with GR/C/EBPα upregulation and IGF1/IGF1R signaling pathway downregulation in the fetal liver, caused by intrauterine programming of the liver glucocorticoid-IGF1 axis induced by glucocorticoid overexposure.

Glucocorticoid programming mechanism for hypercholesterolemia in prenatal ethanol-exposed adult offspring rats.

Our previous studies showed that prenatal ethanol exposure (PEE) elevated blood total cholesterol (TCH) level in adult offspring rats. This study was aimed at elucidating the intrauterine programming mechanism of hypercholesterolemia in adult rats induced by PEE. Pregnant Wistar rats were intragastrically administered ethanol (4 mg/kg∙d) from gestational day (GD) 9 to 20. The offspring rats were euthanized at GD20 and postnatal week 24. Results showed that PEE decreased serum TCH and HDL-C levels (female and male) as well as LDL-C level (female only) in fetal rats but increased serum TCH level and the TCH/HDL-C and LDL-C/HDL-C ratios in adult rats. Furthermore, PEE elevated serum corticosterone levels but inhibited hepatic insulin-like growth factor 1 (IGF1) signaling pathway, cholesterol synthesis and output in fetal rats. The conversed changes were observed in adult rats. Moreover, histone acetylation (H3K9ac and H3K14ac) and expression of hepatic reverse cholesterol transport (RCT) related genes, scavenger receptor BI and low-density lipoprotein receptor were decreased before and after birth by PEE. In HepG2 cells, cortisol negatively regulated the IGF1 signaling pathway and cholesterol metabolic genes, but this inhibition of the cholesterol metabolic genes could be reversed by glucocorticoid receptor antagonist RU486, whereas exogenous IGF1 treatment only reversed the downregulation of RCT genes by cortisol. We confirmed a "two programming" mechanism for PEE-induced hypercholesterolemia in adult rats. The "first programming" was a glucocorticoid (GC)-induced persistent reduction of RCT genes by epigenetic modifications, and the "second programming" was the negative regulation of cholesterol synthesis and output by the GC-IGF1 axis.

Two intrauterine programming mechanisms of adult hypercholesterolemia induced by prenatal nicotine exposure in male offspring rats.

Epidemiologic studies showed that low birth weight is associated with high cholesterol and an increased risk of cardiovascular diseases in adulthood. This study aimed to elucidate the intrauterine programming mechanisms of adult hypercholesterolemia. The results showed that prenatal nicotine exposure (PNE) caused intrauterine growth retardation and hypercholesterolemia in male adult offspring rats. Hepatic cholesterol synthesis and output were deceased in utero but increased in adults; hepatic reverse cholesterol transport (RCT) persistently deceased before and after birth. Meanwhile, PNE elevated serum corticosterone level and decreased hepatic IGF1 pathway activity in male fetuses, whereas converse changes were observed in male adults. The chronic stress model and cortisol-treated HepG2 cells verified that excessive glucocorticoid (GC)-induced GC-IGF1 axis programming enhanced hepatic cholesterol synthesis and output. In addition, PNE decreased the expression of specific protein 1 and P300 enrichment and H3K27 acetylation at the promoter region of genes responsible for RCT both in fetal and adult, male livers and reduced expression of those genes, similar alterations were also confirmed in cortisol-treated HepG2 cells, suggesting that excessive GC-related programming induced continuous RCT reduction by epigenetic modification. Taken together, the "2-programming" approach discussed above may ultimately contribute to the development of hypercholesterolemia in male adult offspring.-Zhou, J., Zhu, C., Luo, H., Shen, L., Gong, J., Wu, Y., Magdalou, J., Chen, L., Guo, Y., Wang, H. Two intrauterine programming mechanisms of adult hypercholesterolemia induced by prenatal nicotine exposure in male offspring rats.

Activation of local bone RAS by maternal excessive glucocorticoid participated in the fetal programing of adult osteopenia induced by prenatal caffeine exposure.

This study was aimed to investigate whether and how prenatal caffeine exposure (PCE) could induce osteopenia in the adult offspring. Pregnant rats were treated with prenatal caffeine 12 mg/100 g body weight per day from pregnant day 9 to 20, while rat bone marrow mesenchymal stem cells (BMSCs) were treated with exogenous corticosterone during osteogenic induction. Shorter femur and primary ossification center was observed in the PCE offspring, as well as less bone trabecular and poor biomechanical intensity. Local gene expression of glucocorticoid receptor (GR) and angiotensin converting enzyme (ACE), as well as angiotensin 2 content, was found to be stimulated, while the expression of bone gamma-carboxyglutamate protein (BGLAP), alkaline phosphatase (ALP) and bone sialoprotein (BSP) was found to be suppressed, with hypomethylation of ACE promoter. Corticosterone (1250 nM) suppressed osteogenic differentiation of BMSCs and gene expression of BGLAP, ALP and BSP, which was attenuated by enalapril, while it stimulated ACE mRNA expression and induced hypomethylation of ACE promoter, which was attenuated by mifepristone. It indicated that PCE caused bone growth retardation and adult osteopenia in offspring, which might be triggered by the activation of local RAS induced by excessive maternal glucocorticoid, while the hypomethylation of ACE gene might be the key point of the sustained activation of the local RAS.

Synergistic effects of prenatal nicotine exposure and post-weaning high-fat diet on hypercholesterolaemia in rat offspring of different sexes.

Hypercholesterolaemia is considered a disease with intrauterine origin. Recently, we reported that prenatal nicotine exposure (PNE) induced an abnormal level of total cholesterol in rat offspring before and after birth. However, there were little data about sex differences in serum cholesterol level in PNE offspring. In addition, many previous studies reported that blood cholesterol is associated with daily diet. This study was designed to analyse the interaction among PNE, high-fat diet (HFD) and sex on cholesterol metabolism in the rat. Pregnant Wistar rats were administered 2 mg/kg nicotine subcutaneously from gestational day (GD) 11 until parturition. After weaning, pups were fed with normal diet or HFD till 24 weeks, and then, serum cholesterol phenotypes and hepatic cholesterol metabolism-related genes were tested. Results showed that PNE manifested a distinct programming effect on cholesterol phenotype and cholesterol metabolism-related genes. HFD aggregated PNE-induced hypercholesterolaemia in adult offspring and exacerbated liver cholesterol metabolism dysfunction in PNE offspring. There was no sex difference in serum cholesterol level, but there were interactions among PNE, HFD and sex on cholesterol metabolic genes in adult offspring, which indicates that cholesterol metabolism in female offspring is more likely to be affected by PNE and HFD. In conclusion, HFD exacerbated PNE-induced hypercholesterolaemia, and sex differences existed in liver cholesterol metabolic genes in PNE- or HFD-treated offspring.

Glucocorticoid-activation system mediated glucocorticoid-insulin-like growth factor 1 (GC-IGF1) axis programming alteration of adrenal dysfunction induced by prenatal caffeine exposure.

Glucocorticoids play a major factor in fetal maturation and fate decision after birth. We have previously demonstrated that prenatal caffeine exposure (PCE) resulted in adrenal dysplasia. However, its molecular mechanism has not been clarified. In the present study, a rat model of intrauterine growth retardation (IUGR) was established by PCE, and offspring were sacrificed. Moreover, NCI-H295 A cells were used to confirm glucocorticoid-related molecular mechanism. Results showed that PCE fetal weight decreased, and the IUGR rate increased, while serum corticosterone levels increased but insulin-like growth factor 1 (IGF1) levels decreased. Fetal adrenals exhibited an activated glucocorticoid-activation system, and the downregulated expression of IGF1 signal pathway and steroidal synthetases. For adult rats, there was no significant change in the glucocorticoid-activation system in the PCE group, the IGF1 signal pathway showed increased trend, and the expression levels of adrenal steroidal synthetases were close to normal. The data in vitro showed that the cortisol of 1200 nM can inhibit the expression of adrenocortical cell steroidal synthetases and IGF1 signal pathway when compared with the control. Meanwhile, the glucocorticoid-activation system was activated while GR inhibitor mifepristone can reverse the effect of cortisol. Furthermore, cortisol can also promote GR into the nucleus after its activation. Based on these findings, we speculated that high concentrations of glucocorticoid in utero led to GR in the nucleus through its activation and then inhibited the IGF1 signaling pathway by activating the glucocorticoid-activation system, which could further downregulate steroid synthesis.

Intrauterine Programming of Glucocorticoid-Insulin-Like Growth Factor-1 Axis-Mediated Developmental Origin of Osteoporosis Susceptibility in Female Offspring Rats with Prenatal Caffeine Exposure.

Epidemiologic investigations suggest that excessive intake of caffeine during pregnancy is one of the risk factors for osteoporosis in adult offspring. However, the phenomena and mechanisms have remained obscure. This study found that prenatal caffeine exposure (PCE) leads to persistent bone dysplasia in gestational day 20 and postnatal week 12 offspring rats and increases the susceptibility to osteoporosis in postnatal week 28 offspring rats. In the embryonic period, PCE increases the concentration of serum corticosterone and inhibits the expression of insulin-like growth factor-1 (IGF1) and osteogenic differentiation genes. After birth, the recovery of IGF1 expression in PCE offspring is unable to completely compensate osteogenic function, and chronic stress can lead to a further decrease in IGF1 expression. In vitro experiments found that corticosterone instead of caffeine restrains mineralized nodule formation and osteoblast differentiation by inhibiting IGF1 expression. The corticosterone inhibits H3K9 and H3K14 histone acetylation of IGF1 in osteoblasts through glucocorticoid receptor and CCAAT and enhancer binding protein α, respectively. In conclusion, glucocorticoid instead of caffeine inhibits bone IGF1 expression via glucocorticoid receptor and CCAAT and enhancer binding protein α and mediates the PCE-induced bone dysplasia and bone mass reduction in offspring fetal rats, which may contribute to osteoporosis susceptibility in adulthood.

Prenatal caffeine exprosure increases adult female offspring rat's susceptibility to osteoarthritis via low-functional programming of cartilage IGF-1 with histone acetylation.

Our previous in vivo studies showed that prenatal caffeine exposure (PCE) could restrain the development of chondrogenesis, which may delay fetal articular cartilage development and increase susceptibility to osteoarthritis in adults. So, the goal of the current study is to clarify theincreasing susceptibility to adult osteoarthritis in caffeine-exposed female offspring and its'mechanism. Pregnant rats were treated with 120 mg/kg·d caffeine or equal volumes of saline from gestational day (GD) 9 to 20. knee joints were collected from GD20 female fetuses and 18-week old female offspring which was treated with strenuous running for 6 weeks (55 min/day at 20 m/min) load to induce osteoarthritis. Knee joints from GD20 fetuses and adult offspring were collected for histochemistry and immunohistochemistry. Next, chondrocytes were isolated from 1-day-old newborn rats and in vitro studies were conducted where the cells in primary culture were exposed to 1, 10, and 100 µM caffeine and 250, 500, and 1,250 nM corticosterone. Insulin-like growth factor 1 (IGF-1) signal pathway genes' expression levels in fetal chondrocytes were studied, and IGF-1 histone acetylation was detected in vitro. Immunohistochemical results showed low expression levels of IGF-1 signaling genes (IGF-1, IRS-1, AKT, and COL2A1) both in fetal and adult cartilage with PCE. For adult offspring, histological results and Mankin score revealed increased cartilage destruction and accelerated osteoarthritis progression in PCE group with strenuous running exercise. Analysis in vitro revealed that caffeine and corticosterone impeded the expression of IGF-1 signaling pathway aggrecan and COL2A1 genes, but only corticosterone decreased H3K9 and H3K27 acetylation in the IGF-1 promoter region. In concluson, PCE low functional programmed cartilage IGF-1 by histone acetylation modification via overexposure to corticosterone and delayed articular cartilage development from fetus to adults. Then, the delayed cartilage development increased susceptibility to osteoarthritis in offsprings.

Influencing factors, underlying mechanism and interactions affecting hypercholesterolemia in adult offspring with caffeine exposure during pregnancy.

Epidemiological surveys suggest that adult hypercholesterolemia has an intrauterine origin and exhibits gender differences. Our previous study demonstrated that adult rats with intrauterine growth retardation (IUGR) offspring rats induced by prenatal caffeine exposure (PCE) had a higher serum total cholesterol (TCH) level. In this study, we aimed to analyze the influencing factors, underlying mechanism and interactions affecting hypercholesterolemia in adult offspring with caffeine exposure during pregnancy. Pregnant rats were administered caffeine (120 mg/kg d) from gestational day 11 until delivery. Offspring rats fed a normal diet or a high-fat diet (HFD) were euthanized at postnatal week 24, and blood and liver samples were collected. The results showed that PCE could increase the serum levels of TCH and low-density lipoprotein-cholesterol (LDL-C), and the hepatic expression of HMG CoA reductase (HMGCR) and apolipoprotein B (ApoB), but decreased the high-density lipoprotein-cholesterol (HDL-C) level and the hepatic expression of scavenger receptor B1 (SR-B1) and LDL receptor (LDLR). Furthermore, PCE, HFD and gender interact with each other to influence the serum cholesterol phenotype and expression of hepatic cholesterol metabolic genes. These results suggest that the hypercholesterolemia in adult offspring rats induced by PCE mainly resulted from enhanced synthesis and the weakened reverse transport of cholesterol in the liver, furthermore HFD could aggravate this effect, which is caused by hepatic cholesterol metabolic disorders. Moreover, cholesterol metabolism in female rats was more sensitive to neuroendocrine changes and HFD than that in males. This study confirmed the influencing factors (such as a HFD and female gender) of hypercholesterolemia in IUGR offspring providing theoretical and experimental bases for the effective prevention of fetal-originated hypercholesterolemia.

Intrauterine programming mechanism for hypercholesterolemia in prenatal caffeine-exposed female adult rat offspring.

Clinical and animal studies have indicated that hypercholesterolemia and its associated diseases have intrauterine developmental origins. Our previous studies showed that prenatal caffeine exposure (PCE) led to fetal overexposure to maternal glucocorticoids (GCs) and increased serum total cholesterol levels in adult rat offspring. This study further confirms the intrauterine programming of PCE-induced hypercholesterolemia in female adult rat offspring. Pregnant Wistar rats were intragastrically administered caffeine (30, 60, and 120 mg/kg/d) from gestational day (GD)9 to 20. Female rat offspring were euthanized at GD20 and postnatal wk 12; several adult rat offspring were additionally subjected to ice-water swimming stimulation to induce chronic stress prior to death. The effects of GCs on cholesterol metabolism and epigenetic regulation were verified using the L02 cell line. The results showed that PCE induced hypercholesterolemia in adult offspring, which manifested as significantly higher levels of serum total cholesterol and LDL cholesterol (LDL-C) as well as higher ratios of LDL-C/HDL cholesterol. We further found that the cholesterol levels were increased in fetal livers but were decreased in fetal blood, accompanied by increased maternal blood cholesterol levels and reduced placental cholesterol transport. Furthermore, analysis of PCE offspring in the uterus and in a postnatal basal/chronic stress state and the results of in vitro experiments showed that hepatic cholesterol metabolism underwent GC-dependent changes and was associated with cholesterol synthase via abnormalities in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) histone acetylation. We concluded that, to compensate for intrauterine placentally derived decreases in fetal blood cholesterol levels, high intrauterine GC levels activated fetal hepatic CCAAT enhancer binding protein α signaling and down-regulated Sirtuin1 expression, which mediated the high levels of histone acetylation ( via H3K9ac and H3K14ac) and expression of HMGCR. This GC-dependent cholesterol metabolism programming effect was sustained through adulthood, leading to the occurrence of hypercholesterolemia.-Xu, D., Luo, H. W., Hu, W., Hu, S. W., Yuan, C., Wang, G. H., Zhang, L., Yu, H., Magdalou, J., Chen, L. B., Wang, H. Intrauterine programming mechanism for hypercholesterolemia in prenatal caffeine-exposed female adult rat offspring.

Role of TGFß Signaling in Maternal Ethanol-Induced Fetal Articular Cartilage Dysplasia and Adult Onset of Osteoarthritis in Male Rats.

Based on our previous findings that prenatal ethanol exposure in offspring increased susceptibility to adult osteoarthritis, this study aimed to further investigate the direct toxicity of ethanol on fetal articular cartilage development. Rat bone marrow-derived stroma cells were capsulated in alginate beads, incubated in a chondrogenic differentiation medium, and cultured for 4 weeks with ethanol treatment at concentrations of 0, 4, 20, and 100 mM. Pregnant rats were treated with ethanol (4 g/kg/day) from gestational days (GDs) 9 to 20. At GD20 and postnatal weeks 2, 6, and 12, 8 male offspring were sacrificed, and 8 male offspring rats of 8-weeks old in each group were treated with or without intraarticular injection of papain for 4 weeks to verify the susceptibility of adult osteoarthritis. Ethanol treatment resulted in poor differentiation of bone marrow-derived stroma cells to chondrocytes and suppressed the expression of the transforming growth factor-ß (TGFß)-smad2/3-Sox9 signaling pathway. In animal experiments, the shape of articular cartilage in the ethanol treatment group was more disordered than that of the control group, the matrix was not deep, and the cartilage was thin, which showed poor cartilage development. The TGFß signaling pathway in the ethanol treatment group was persistently low at all time points. After intraarticular injection of papain, histological analyses, and the Mankin score revealed increased cartilage destruction in the ethanol treatment group. Ethanol caused articular cartilage dysplasia that was programmed in adulthood via a low-functional TGFß signaling pathway, and the tolerance of this articular cartilage to external stimuli was significantly decreased.