Objective The purpose of this study was to investigate the effects of insulin on human placental transcriptome and biological processes in first-trimester pregnancy. Study Design Maternal plasma and ...placenta villous tissue were obtained at the time of voluntary termination of pregnancy (7-12 weeks) from 17 lean (body mass index, 20.9 ± 1.5 kg/m2 ) and 18 obese (body mass index, 33.5 ± 2.6 kg/m2 ) women. Trophoblast cells were immediately isolated for in vitro treatment with insulin or vehicle. Patterns of global gene expression were analyzed using genome microarray profiling after hybridization to Human Gene 1.1 ST and real time reverse transcription–polymerase chain reaction. Results The global trophoblast transcriptome was qualitatively separated in insulin-treated vs untreated trophoblasts of lean women. The number of insulin-sensitive genes detected in the trophoblasts of lean women was 2875 ( P < .001). Maternal obesity reduced the number of insulin-sensitive genes recovered by 30-fold. Insulin significantly impaired several gene networks regulating cell cycle and cholesterol homeostasis but did not modify pathways related to glucose transport. Obesity associated with high insulin and insulin resistance, but not maternal hyperinsulinemia alone, impaired the global gene profiling of early gestation placenta, highlighting mitochondrial dysfunction and decreased energy metabolism. Conclusion We report for the first time that human trophoblast cells are highly sensitive to insulin regulation in early gestation. Maternal obesity associated with insulin resistance programs the placental transcriptome toward refractoriness to insulin with potential adverse consequences for placental structure and function.
Context:
Low concentrations of estradiol and progesterone are hallmarks of adverse pregnancy outcomes as is maternal obesity. During pregnancy, placental cholesterol is the sole source of sex ...steroids. Cholesterol trafficking is the limiting step in sex steroid biosynthesis and is mainly mediated by the translocator protein (TSPO), present in the mitochondrial outer membrane.
Objective:
The objective of the study was to investigate the effects of maternal obesity in placental sex steroid biosynthesis and TSPO regulation.
Design/Participants:
One hundred forty-four obese (body mass index 30–35 kg/m2) and 90 lean (body mass index 19–25 kg/m2) pregnant women (OP and LP, respectively) recruited at scheduled term cesarean delivery. Placenta and maternal blood were collected.
Setting:
This study was conducted at MetroHealth Medical Center (Cleveland, Ohio).
Main Outcome Measures:
Maternal metabolic components (fasting glucose, insulin, leptin, estradiol, progesterone, and total cholesterol) and placental weight were measured. Placenta (mitochondria and membranes separated) and cord blood cholesterol values were verified. The expression and regulation of TSPO and mitochondrial function were analyzed.
Results:
Plasma estradiol and progesterone concentrations were significantly lower (P < .04) in OP as compared with LP women. Maternal and cord plasma cholesterol were not different between groups. Placental citrate synthase activity and mitochondrial DNA, markers of mitochondrial density, were unchanged, but the mitochondrial cholesterol concentrations were 40% lower in the placenta of OP. TSPO gene and protein expressions were decreased 2-fold in the placenta of OP. In vitro trophoblast activation of the innate immune pathways with lipopolysaccharide and long-chain saturated fatty acids reduced TSPO expression by 2- to 3-fold (P < .05).
Conclusion:
These data indicate that obesity in pregnancy impairs mitochondrial steroidogenic function through the negative regulation of mitochondrial TSPO.
The purpose of this study was to investigate whether myofibroblast-related fibrosis (scarring) after microbial keratitis was modulated by the epithelial basement membrane (EBM) injury and ...regeneration. Rabbits were infected with Pseudomonas aeruginosa after epithelial scrape injury and the resultant severe keratitis was treated with topical tobramycin. Corneas were analyzed from one to four months after keratitis with slit lamp photos, immunohistochemistry for alpha-smooth muscle actin (α-SMA) and monocyte lineage marker CD11b, and transmission electron microscopy. At one month after keratitis, corneas had no detectible EBM lamina lucida or lamina densa, and the central stroma was packed with myofibroblasts that in some eyes extended to the posterior corneal surface with damage to Descemet's membrane and the endothelium. At one month, a nest of stromal cells in the midst of the SMA + myofibroblasts in the stroma that were CD11b+ may be fibrocyte precursors to myofibroblasts. At two to four months after keratitis, the EBM fully-regenerated and myofibroblasts disappeared from the anterior 60–90% of the stroma of all corneas, except for one four-month post-keratitis cornea where anterior myofibroblasts were still present in one localized pocket in the cornea. The organization of the stromal extracellular matrix also became less disorganized from two to four months after keratitis but remained abnormal compared to controls at the last time point. Myofibroblasts persisted in the posterior 10%–20% of posterior stroma even at four months after keratitis in the central cornea where Descemet's membrane and the endothelium were damaged. This study suggests that the EBM has a critical role in modulating myofibroblast development and fibrosis after keratitis—similar to the role of EBM in fibrosis after photorefractive keratectomy. Damage to EBM likely allows epithelium-derived transforming growth factor beta (TGFβ) to penetrate the stroma and drive development and persistence of myofibroblasts. Eventual repair of EBM leads to myofibroblast apoptosis when the cells are deprived of requisite TGFβ to maintain viability. The endothelium and Descemet's membrane may serve a similar function modulating TGFβ penetration into the posterior stroma—with the source of TGFβ likely being the aqueous humor.
•Myofibroblast-mediated fibrosis causes scarring after microbial keratitis.•Defective epithelial basement membrane underlies myofibroblast development.•Epithelial basement membrane regeneration triggers myofibroblast disappearance.
The endothelial compartment, comprising arterial, venous and lymphatic cell types, is established prenatally in association with rapid phenotypic and functional changes. The molecular mechanisms ...underpinning this process in utero have yet to be fully elucidated. The aim of this study was to investigate the potential for DNA methylation to act as a driver of the specific gene expression profiles of arterial and venous endothelial cells.
Placenta-derived venous and arterial endothelial cells were collected at birth prior to culturing. DNA methylation was measured at >450,000 CpG sites in parallel with expression measurements taken from 25,000 annotated genes. A consistent set of genomic loci was found to show coordinate differential methylation between the arterial and venous cell types. This included many loci previously not investigated in relation to endothelial function. An inverse relationship was observed between gene expression and promoter methylation levels for a limited subset of genes implicated in endothelial function, including NOS3, encoding endothelial Nitric Oxide Synthase.
Endothelial cells derived from the placental vasculature at birth contain widespread methylation of key regulatory genes. These are candidates involved in the specification of different endothelial cell types and represent potential target genes for environmentally mediated epigenetic disruption in utero in association with cardiovascular disease risk later in life.
The aim of this study was to determine whether bone marrow-derived fibrocytes migrate into the cornea after stromal scar-producing injury and differentiate into alpha-smooth muscle actin ...(αSMA) + myofibroblasts. Chimeric mice expressing green fluorescent protein (GFP) bone marrow cells had fibrosis (haze)-generating irregular phototherapeutic keratectomy (PTK). Multiplex immunohistochemistry (IHC) for GFP and fibrocyte markers (CD34, CD45, and vimentin) was used to detect fibrocyte infiltration into the corneal stroma and the development of GFP+ αSMA+ myofibroblasts. IHC for activated caspase-3, GFP and CD45 was used to detect fibrocyte and other hematopoietic cells undergoing apoptosis. Moderate haze developed in PTK-treated mouse corneas at 14 days after surgery and worsened, and persisted, at 21 days after surgery. GFP+ CD34+ CD45+ fibrocytes, likely in addition to other CD34+ and/or CD45+ hematopoietic and stem/progenitor cells, infiltrated the cornea and were present in the stroma in high numbers by one day after PTK. The fibrocytes and other bone marrow-derived cells progressively decreased at four days and seven days after surgery. At four days after PTK, 5% of the GFP+ cells expressed activated caspase-3. At 14 days after PTK, more than 50% of GFP+ CD45+ cells were also αSMA+ myofibroblasts. At 21 days after PTK, few GFP+ αSMA+ cells persisted in the stroma and more than 95% of those remaining expressed activated caspase-3, indicating they were undergoing apoptosis. GFP+ CD45+ SMA+ cells that developed from 4 to 21 days after irregular PTK were likely developed from fibrocytes. After irregular PTK in the strain of C57BL/6—C57/BL/6-Tg(UBC-GFP)30Scha/J chimeric mice, however, more than 95% of fibrocytes and other hematopoietic cells underwent apoptosis prior to the development of mature αSMA+ myofibroblasts. Most GFP+ CD45+ αSMA+ myofibroblasts that did develop subsequently underwent apoptosis—likely due to epithelial basement membrane regeneration and deprivation of epithelium-derived TGFβ requisite for myofibroblast survival.
•Bone marrow-derived fibrocytes migrated to the cornea as early as 1 day after injury.•Fibrocytes began to differentiate into αSMA+ myofibroblasts.•Fibrocyte-derived myofibroblasts develop in parallel to keratocyte-derived myofibroblasts after injury.•Myofibroblast precursors undergo apoptosis if the epithelial basement membrane regenerates.
Parents of an infant may be particularly vulnerable to peritraumatic distress (e.g., psychological distress experienced during or immediately following a traumatic event) associated with events such ...as the COVID-19 pandemic. Since peritraumatic distress could affect both their psychological well-being and their couple relationship functioning, it is essential to measure and document these symptoms within parents. The COVID-19 Peritraumatic Distress Index (CPDI; Qiu et al., 2020) was the first validated instrument to measure COVID-19 peritraumatic distress, but it has not yet been validated in French. This study aimed to assess the psychometric properties of the French-Canadian version of the CPDI (F-CPDI) in a sample of 492 parents (58% of mothers) of an infant in Quebec Province (Canada). The factor structure, internal consistency, and convergent validity of the instrument were tested. Results indicate that the F-CPDI has good internal consistency and supports the four-factor structure proposed by the authors of the original instrument. Results of correlation analyses indicated that peritraumatic distress was related to increased psychological distress, postpartum depression, and lower life satisfaction. Results indicate satisfactory psychometric qualities for the F-CPDI, providing researchers and mental health professionals access to a COVID-19 peritraumatic distress measure. This questionnaire can be used to assess peritraumatic distress in parents of an infant during a pandemic period, which is a first step towards offering adapted intervention strategies.
Les parents d'un nouveau-né peuvent être particulièrement vulnérables à la détresse péritraumatique (c'est-à-dire la détresse psychologique vécue pendant ou immédiatement après un événement traumatique) associée à des événements tels que la pandémie de COVID-19. Puisque la détresse péritraumatique pourrait affecter à la fois leur bien-être psychologique et le fonctionnement de leur relation de couple, il est essentiel de mesurer et de documenter ces symptômes chez les parents. L'indice de détresse péritraumatique reliée à la COVID-19 (IDPC; Qiu et al., 2020) a été le premier instrument validé pour mesurer la détresse péritraumatique reliée à la COVID-19, mais il n'a pas encore été validé en français. Cette étude visait à évaluer les propriétés psychométriques de la version franco-canadienne du CPDI (l'IDPC en français) auprès d'un échantillon de 492 parents (58 % de mères) d'un nouveau-né dans la province de Québec (Canada). La structure factorielle, la cohérence interne et la validité convergente de l'instrument ont été testées. Les résultats indiquent que l'IDPC a une bonne cohérence interne et soutient la structure à quatre facteurs proposée par les auteurs de l'instrument original. Les résultats des analyses de corrélation indiquent que la détresse péritraumatique est liée à une détresse psychologique accrue, à la dépression post-partum et à une satisfaction de vie moindre. Les résultats indiquent des qualités psychométriques satisfaisantes pour l'IDPC, permettant aux chercheurs et aux professionnels de la santé mentale d'avoir accès à un indice de détresse péritraumatique reliée à la COVID-19. Ce questionnaire peut être utilisé pour évaluer la détresse péritraumatique des parents d'un nouveau-né en période de pandémie, ce qui constitue une première étape pour proposer des stratégies d'intervention adaptées.
Public Significance Statement
The COVID-19 pandemic is associated with an increase in mental health problems, including peritraumatic distress (Qiu et al., 2020). This study tested the validity of the French-Canadian COVID-19 Peritraumatic Distress Index (F-CPDI; Qiu et al., 2020), the first validated instrument to measure COVID-19 peritraumatic distress. Results revealed satisfactory psychometric qualities for the F-CPDI and prevalence rate of peritraumatic distress reaching 20.5% in Quebec parents of an infant.
The purpose of this study was to evaluate the effect of removal of Descemet's basement membrane and endothelium compared with removal of the endothelium alone on posterior corneal fibrosis.
Twelve ...New Zealand White rabbits were included in the study. Six eyes had removal of the Descemet's membrane-endothelial complex over the central 8 mm of the cornea. Six eyes had endothelial removal with an olive-tipped cannula over the central 8 mm of the cornea. All corneas developed stromal edema. Corneas in both groups were cryofixed in optimum cutting temperature (OCT) formula at 1 month after surgery. Immunohistochemistry (IHC) was performed for α-smooth muscle actin (SMA), keratocan, CD45, nidogen-1, vimentin, and Ki-67, and a TUNEL assay was performed to detect apoptosis.
Six of six corneas that had Descemet's membrane-endothelial removal developed posterior stromal fibrosis populated with SMA+ myofibroblasts, whereas zero of six corneas that had endothelial removal alone developed fibrosis or SMA+ myofibroblasts (P < 0.01). Myofibroblasts in the fibrotic zone of corneas that had Descemet's membrane-endothelial removal were undergoing both mitosis and apoptosis at 1 month after surgery. A zone between keratocan+ keratocytes and SMA+ myofibroblasts contained keratocan-SMA-vimentin+ cells that were likely CD45- corneal fibroblasts and CD45+ fibrocytes.
Descemet's basement membrane has an important role in modulating posterior corneal fibrosis after injury that is analogous to the role of the epithelial basement membrane in modulating anterior corneal fibrosis after injury. Fibrotic areas had myofibroblasts undergoing mitosis and apoptosis, indicating that fibrosis is in dynamic flux.
Context:
The insulin/IGF system regulates fetal and placental growth and development. In a pregnancy complicated by maternal diabetes, placentas are hypervascularized and fetal insulin levels are ...elevated. In the fetal circulation, insulin can act on the placenta through insulin receptors present on the fetoplacental endothelial cells.
Objective:
We hypothesized that insulin exerts proangiogenic effects on the fetoplacental endothelial cells, thereby contributing to the placental hypervascularization in diabetes.
Design:
The effect of insulin on angiogenesis and proliferation of human fetoplacental endothelial cells was investigated by a 2-dimensional network formation assay, staining for actin fibers, automatic cell counting, and cell cycle analysis. The signaling pathways involved were identified using antibodies against activated signaling proteins and pharmacological inhibitors.
Results:
Insulin enhanced network formation by 23% (P < .05%) and caused actin reorganization. Insulin stimulated (P < .05) phosphorylation of insulin receptor (+320%), and insulin receptor substrate-1 (+140%), Akt (+177%), glycogen-synthase kinase-β3 (+70%), and endothelial nitric oxide synthase (eNOS; +100%) increased nitric oxide production and activated Ras-related C3 botulinum toxin substrate 1 (Rac1). Insulin did not induce ERK1/2 phosphorylation or proliferation. Inhibition of phosphatidylinositol 3-kinase, eNOS, and Rac1 signaling abolished the effects on network formation.
Conclusions:
Elevated fetal insulin levels may contribute to the placental hypervascularization in diabetes via the phosphatidylinositol 3-kinase/Akt/eNOS pathway and involve Rac1. However, insulin does not stimulate proliferation and may need to cooperate with other growth factors.
To determine whether (1) the in vitro expression of epithelial basement membrane components nidogen-1, nidogen-2, and perlecan by keratocytes, corneal fibroblasts, and myofibroblasts is modulated by ...cytokines/growth factors, and (2) perlecan protein is produced by stromal cells after photorefractive keratectomy.
Marker-verified rabbit keratocytes, corneal fibroblasts, myofibroblasts were stimulated with TGF-β1, IL-1α, IL-1β, TGF-β3, platelet-derived growth factor (PDGF)-AA, or PDGF-AB. Real-time quantitative RT-PCR was used to detect expression of nidogen-1, nidogen-2, and perlecan mRNAs. Western blotting evaluated changes in protein expression. Immunohistochemistry was performed on rabbit corneas for perlecan, alpha-smooth muscle actin, keratocan, vimentin, and CD45 at time points from 1 day to 1 month after photorefractive keratectomy (PRK).
IL-1α or -1β significantly upregulated perlecan mRNA expression in keratocytes. TGF-β1 or -β3 markedly downregulated nidogen-1 or -2 mRNA expression in keratocytes. None of these cytokines had significant effects on nidogen-1, -2, or perlecan mRNA expression in corneal fibroblasts or myofibroblasts. IL-1α significantly upregulated, while TGF-β1 significantly downregulated, perlecan protein expression in keratocytes. Perlecan protein expression was upregulated in anterior stromal cells at 1 and 2 days after -4.5 or -9 diopters (D) PRK, but the subepithelial localization of perlecan became disrupted at 7 days and later time points in -9-D PRK corneas when myofibroblasts populated the anterior stroma.
IL-1 and TGF-β1 have opposing effects on perlecan and nidogen expression by keratocytes in vitro. Proximate participation of keratocytes is likely needed to regenerate normal epithelial basement membrane after corneal injury.
This study was performed to determine whether cells in the posterior stroma undergo apoptosis in response to endothelial cell injury and to determine whether basement membrane component nidogen-1 was ...present in the cornea. New Zealand White rabbits had an olive tip cannula inserted into the anterior chamber to mechanically injure corneal endothelial cells over an 8 mm diameter area of central cornea with minimal injury to Descemet's membrane. At 1 h (6 rabbits) and 4 h (6 rabbits) after injury, three corneas at each time point were cryopreserved in OCT for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemistry (IHC) for vimentin and nidogen-1, and three corneas at each time point were fixed for transmission electron microscopy (TEM). Uninjured corneas were controls. Stromal cells over approximately the posterior 25% of the stroma overlying to the site of corneal endothelial injury underwent apoptosis detected by the TUNEL assay. Many of these apoptotic cells were vimentin+, suggesting they were likely keratocytes or corneal fibroblasts. Stromal cells peripheral to the site of endothelial injury and more anterior stromal cells overlying the site of endothelial injury did not undergo apoptosis. Stromal cell death was confirmed to be apoptosis by TEM. No apoptosis of stromal cells was detected in control, uninjured corneas. Nidogen-1 was detected in the stroma of unwounded corneas, with higher nidogen-1 in the posterior stroma than the anterior stroma. After endothelial scrape injury, concentrations of nidogen-1 appeared to be in the extracellular matrix of the posterior stroma and, possibly, within apoptotic bodies of stromal cells. Thus, posterior stromal cells, likely including keratocytes, undergo apoptosis in response to corneal endothelial injury, analogous to anterior keratocytes undergoing apoptosis in response to epithelial injury.
•Overlying keratocytes undergo apoptosis following mechanical injury to corneal endothelial cells in rabbits.•The epithelial basement membrane and Descemet's basement membrane have high concentrations of nidogen-1 in rabbits.•There is higher nidogen-1 in keratocytes and extracellular matrix in the posterior stroma compared to anterior stroma.
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