Epigenetic regulation by hypoxia, N-acetylcysteine and hydrogen sulphide of the fetal vasculature in growth restricted offspring: A study in humans and chicken embryos.

Fetal growth restriction (FGR) is a common outcome in human suboptimal gestation and is related to prenatal origins of cardiovascular dysfunction in offspring. Despite this, therapy of human translational potential has not been identified. Using human umbilical and placental vessels and the chicken embryo model, we combined cellular, molecular, and functional studies to determine whether N-acetylcysteine (NAC) and hydrogen sulphide (H2S) protect cardiovascular function in growth-restricted unborn offspring. In human umbilical and placental arteries from control or FGR pregnancy and in vessels from near-term chicken embryos incubated under normoxic or hypoxic conditions, we determined the expression of the H2S gene CTH (i.e. cystathionine γ-lyase) (via quantitative PCR), the production of H2S (enzymatic activity), the DNA methylation profile (pyrosequencing) and vasodilator reactivity (wire myography) in the presence and absence of NAC treatment. The data show that FGR and hypoxia increased CTH expression in the embryonic/fetal vasculature in both species. NAC treatment increased aortic CTH expression and H2S production and enhanced third-order femoral artery dilator responses to the H2S donor sodium hydrosulphide in chicken embryos. NAC treatment also restored impaired endothelial relaxation in human third-to-fourth order chorionic arteries from FGR pregnancies and in third-order femoral arteries from hypoxic chicken embryos. This NAC-induced protection against endothelial dysfunction in hypoxic chicken embryos was mediated via nitric oxide independent mechanisms. Both developmental hypoxia and NAC promoted vascular changes in CTH DNA and NOS3 methylation patterns in chicken embryos. Combined, therefore, the data support that the effects of NAC and H2S offer a powerful mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy. KEY POINTS: Gestation complicated by chronic fetal hypoxia and fetal growth restriction (FGR) increases a prenatal origin of cardiovascular disease in offspring, increasing interest in antenatal therapy to prevent against a fetal origin of cardiovascular dysfunction. We investigated the effects between N-acetylcysteine (NAC) and hydrogen sulphide (H2S) in the vasculature in FGR human pregnancy and in chronically hypoxic chicken embryos. Combining cellular, molecular, epigenetic and functional studies, we show that the vascular expression and synthesis of H2S is enhanced in hypoxic and FGR unborn offspring in both species and this acts to protect their vasculature. Therefore, the NAC/H2S pathway offers a powerful therapeutic mechanism of human translational potential against fetal cardiovascular dysfunction in complicated pregnancy.

Premature Vascular Aging in Guinea Pigs Affected by Fetal Growth Restriction.

Cardiovascular risk associated with fetal growth restriction (FGR) could result from an early impaired vascular function. However, whether this effect results in premature vascular aging has not been addressed. We studied the ex vivo reactivity of carotid and femoral arteries in fetal (near term), adults (eight months-old) and aged (16 months-old) guinea pigs in normal (control) and FGR offspring. Additionally, an epigenetic marker of vascular aging (i.e., LINE-1 DNA methylation) was evaluated in human umbilical artery endothelial cells (HUAEC) from control and FGR subjects. Control guinea pig arteries showed an increased contractile response (KCl-induced) and a progressive impairment of NO-mediated relaxing responses as animals get older. FGR was associated with an initial preserved carotid artery reactivity as well as a later significant impairment in NO-mediated responses. Femoral arteries from FGR fetuses showed an increased contractility but a decreased relaxing response compared with control fetuses, and both responses were impaired in FGR-adults. Finally, FGR-HUAEC showed decreased LINE-1 DNA methylation compared with control-HUAEC. These data suggest that the aging of vascular function occurs by changes in NO-mediated responses, with limited alterations in contractile capacity. Further, these effects are accelerated and imposed at early stages of development in subjects exposed to a suboptimal intrauterine environment.

N-Acetylcysteine, a glutathione precursor, reverts vascular dysfunction and endothelial epigenetic programming in intrauterine growth restricted guinea pigs.

KEY POINTS: Intrauterine growth restriction (IUGR) is associated with vascular dysfunction, oxidative stress and signs of endothelial epigenetic programming of the umbilical vessels. There is no evidence that this epigenetic programming is occurring on systemic fetal arteries. In IUGR guinea pigs we studied the functional and epigenetic programming of endothelial nitric oxide synthase (eNOS) (Nos3 gene) in umbilical and systemic fetal arteries, addressing the role of oxidative stress in this process by maternal treatment with N-acetylcysteine (NAC) during the second half of gestation. The present study suggests that IUGR endothelial cells have common molecular markers of programming in umbilical and systemic arteries. Notably, maternal treatment with NAC restores fetal growth by increasing placental efficiency and reverting the functional and epigenetic programming of eNOS in arterial endothelium in IUGR guinea pigs. ABSTRACT: In humans, intrauterine growth restriction (IUGR) is associated with vascular dysfunction, oxidative stress and signs of endothelial programming in umbilical vessels. We aimed to determine the effects of maternal antioxidant treatment with N-acetylcysteine (NAC) on fetal endothelial function and endothelial nitric oxide synthase (eNOS) programming in IUGR guinea pigs. IUGR was induced by implanting ameroid constrictors on uterine arteries of pregnant guinea pigs at mid gestation, half of the sows receiving NAC in the drinking water (from day 34 until term). Fetal biometry and placental vascular resistance were followed by ultrasound throughout gestation. At term, umbilical arteries and fetal aortae were isolated to assess endothelial function by wire-myography. Primary cultures of endothelial cells (ECs) from fetal aorta, femoral and umbilical arteries were used to determine eNOS mRNA levels by quantitative PCR and analyse DNA methylation in the Nos3 promoter by pyrosequencing. Doppler ultrasound measurements showed that NAC reduced placental vascular resistance in IUGR (P < 0.05) and recovered fetal weight (P < 0.05), increasing fetal-to-placental ratio at term (∼40%) (P < 0.001). In IUGR, NAC treatment restored eNOS-dependent relaxation in aorta and umbilical arteries (P < 0.05), normalizing eNOS mRNA levels in EC fetal and umbilical arteries (P < 0.05). IUGR-derived ECs had a decreased DNA methylation (∼30%) at CpG -170 (from the transcription start site) and this epigenetic signature was absent in NAC-treated fetuses (P < 0.001). These data show that IUGR-ECs have common molecular markers of eNOS programming in umbilical and systemic arteries and this effect is prevented by maternal treatment with antioxidants.

Co-Infection by LF-89-Like and EM-90-Like Genogroups of Piscirickettsia Salmonis in Farmed Atlantic Salmon in Chile: Implications for Surveillance and Control of Piscirickettsiosis.

Piscirickettsiosis (SRS), caused by Piscirickettsia salmonis, is the main infectious disease that affects farmed Atlantic salmon in Chile. Currently, the official surveillance and control plan for SRS in Chile is based only on the detection of P. salmonis, but neither of its genogroups (LF-89-like and EM-90-like) are included. Surveillance at the genogroup level is essential not only for defining and evaluating the vaccination strategy against SRS, but it is also of utmost importance for early diagnosis, clinical prognosis in the field, treatment, and control of the disease. The objectives of this study were to characterize the spatio-temporal distribution of P. salmonis genogroups using genogroup-specific real-time probe-based polymerase chain reaction (qPCR) to discriminate between LF-89-like and EM-90-like within and between seawater farms, individual fish, and tissues/organs during early infection in Atlantic salmon under field conditions. The spatio-temporal distribution of LF-89-like and EM-90-like was shown to be highly variable within and between seawater farms. P. salmonis infection was also proven to be caused by both genogroups at farm, fish, and tissue levels. Our study demonstrated for the first time a complex co-infection by P. salmonis LF-89-like and EM-90-like in Atlantic salmon. Liver nodules (moderate and severe) were strongly associated with EM-90-like infection, but this phenotype was not detected by infection with LF-89-like or co-infection of both genogroups. The detection rate of P. salmonis LF-89-like increased significantly between 2017 and 2021 and was the most prevalent genogroup in Chilean salmon aquaculture during this period. Lastly, a novel strategy to identify P. salmonis genogroups based on novel genogroup-specific qPCR for LF-89-like and EM-90-like genogroups is suggested.

Transcriptional Profiling of Human Endothelial Cells Unveils PIEZO1 and Mechanosensitive Gene Regulation by Prooxidant and Inflammatory Inputs.

PIEZO1 is a mechanosensitive cation channel implicated in shear stress-mediated endothelial-dependent vasorelaxation. Since altered shear stress patterns induce a pro-inflammatory endothelial environment, we analyzed transcriptional profiles of human endothelial cells to determine the effect of altered shear stress patterns and subsequent prooxidant and inflammatory conditions on PIEZO1 and mechanosensitive-related genes (MRG). In silico analyses were validated in vitro by assessing PIEZO1 transcript levels in both the umbilical artery (HUAEC) and vein (HUVEC) endothelium. Transcriptional profiling showed that PIEZO1 and some MRG associated with the inflammatory response were upregulated in response to high (15 dyn/cm2) and extremely high shear stress (30 dyn/cm2) in HUVEC. Changes in PIEZO1 and inflammatory MRG were paralleled by p65 but not KLF or YAP1 transcription factors. Similarly, PIEZO1 transcript levels were upregulated by TNF-alpha (TNF-α) in diverse endothelial cell types, and pre-treatment with agents that prevent p65 translocation to the nucleus abolished PIEZO1 induction. ChIP-seq analysis revealed that p65 bonded to the PIEZO1 promoter region, an effect increased by the stimulation with TNF-α. Altogether this data showed that NF-kappa B activation via p65 signaling regulates PIEZO1 expression, providing a new molecular link for prooxidant and inflammatory responses and mechanosensitive pathways in the endothelium.

Specific arterio-venous transcriptomic and ncRNA-RNA interactions in human umbilical endothelial cells: A meta-analysis.

Whether arterial-venous differences of primary endothelial cells commonly used for vascular research are preserved in vitro remains under debate. To address this issue, a meta-analysis of Affymetrix transcriptomic data sets from human umbilical artery (HUAECs) and vein (HUVEC) endothelial cells was performed. The meta-analysis showed 2,742 transcripts differentially expressed (false discovery rate <0.05), of which 78% were downregulated in HUVECs. Comparisons with RNA-seq data sets showed high levels of agreement and correlation (p < 0.0001), identifying 84 arterial-venous identity markers. Functional analysis revealed enrichment of key vascular processes in HUAECs/HUVECs, including nitric oxide- (NO) and hypoxia-related genes, as well as differences in miRNA- and ncRNA-mRNA interaction profiles. A proof of concept of these findings in primary cells exposed to hypoxia in vitro and in vivo confirmed the arterial-venous differences in NO-related genes and miRNAs. Altogether, these data defined a cross-platform arterial-venous transcript profile for cultured HUAEC-HUVEC and support a preserved identity involving key vascular pathways post-transcriptionally regulated in vitro.

MiR-21-5p directly contributes to regulating eNOS expression in human artery endothelial cells under normoxia and hypoxia.

Clinical conditions associated with hypoxia and oxidative stress, such as fetal growth restriction (FGR), results in endothelial dysfunction. Previous reports show that changes in eNOS expression under these conditions are tightly controlled by DNA methylation and histone posttranslational modifications. However, the contribution of an orchestrating epigenetic mechanism, such as miRNAs, on the NO-related genes expression has not been addressed. We aimed to determine the levels of miRNAs highly expressed in normal endothelial cells (EC), miR-21 and miR-126, in FGR human umbilical artery EC (HUAEC), and their effects on hypoxia-dependent regulation of both, NO-related and oxidative stress-related genes. Results were validated by transcriptome analysis of HUAEC cultured under chronic low oxygen conditions. Cultured FGR-HUAEC showed decreased hsa-miR-21, DDAH1, SOD1, and NRF2, but increased miR-126, NOX4, and eNOS levels, compared with controls. MiR-21-5p levels in FGR were associated with increased hg-miR-21 gene promoter methylation, with no changes in hg-miR-126 gene promoter methylation. HUAEC exposed to hypoxia showed a transient increase in eNOS and DDAH11, paralleled by decrease miR-21-5p levels, but no changes in miR-126-3p and the other genes under study. Transcriptome profiling showed an inverse relationship among miR-21 and several transcripts targeted by miR-21 in HUAEC exposed to hypoxia, meanwhile miR-21-5p-mimic decreased eNOS and DDAH1 transcripts stability, blocking their induction by hypoxia. Consequently, FGR programs a hypoxia-related miRNA that contributes to the regulation of the NO pathway, involving a direct effect of miR-21-5p on eNOS transcript stability, not previously reported. Moreover, hypoxia downregulates miR-21-5p, contributing to increasing the expression of NO-related genes in arterial endothelial cells.

Adult vascular dysfunction in foetal growth-restricted guinea-pigs is associated with a neonate-adult switching in Nos3 DNA methylation.

AIM: Foetal growth restriction (FGR) is associated with endothelial dysfunction and cardiovascular diseases in adult subjects. Early vascular remodelling and epigenetic changes occurring on key endothelial genes might precede this altered vascular function. Further, it has been proposed that oxidative stress during development may determine some of these epigenetic modifications. To address this issue, we studied the in vivo and ex vivo vascular function and Nos3 promoter DNA methylation in arteries from eight-month-old guinea-pig born from control, FGR-treated and FGR-NAC-treated pregnancies. METHODS: Femoral and carotid arteries in vivo vascular function were determined by Doppler, whilst ex vivo vascular function and biomechanical properties were assessed by wire myography. Levels of eNOS mRNA and site-specific DNA methylation in Nos3 promoter in aorta endothelial cells (AEC) were determined by qPCR and pyrosequencing respectively. RESULTS: FGR adult showed an increased femoral vascular resistance (P < .05), stiffness (P < .05) and arterial remodelling (P < .01), along with an impaired NO-mediated relaxation (P < .001). These effects were prevented by maternal treatment with NAC. Endothelial-NOS mRNA levels were decreased in FGR adult compared with control and FGR-NAC (P < .05), associated with increased DNA methylation levels (P < .01). Comparison of Nos3 DNA methylation in AEC showed a differential methylation pattern between foetal and adult guinea-pigs (P < .05). CONCLUSION: Altogether, these data suggest that adult vascular dysfunction in the FGR does not result from early changes in Nos3 promoter DNA methylation, but from an altered vessel structure established during foetal development.