Sexual dimorphism of miRNA signatures in feto-placental endothelial cells is associated with altered barrier function and actin organization.

Endothelial function and the risk for endothelial dysfunction differ between males and females. Besides the action of estrogen, sex chromosome gene expression and programming effects also provoke this sexual dimorphism. MicroRNAs (miRNAs) have emerged as regulators of endothelial cell function and dysfunction. We here hypothesized distinct miRNA expression patterns in male versus female human endothelial cells that contribute to the functional differences. We used our well-established model of fetal endothelial cells isolated from placenta (fpEC) and analyzed sexual dimorphic miRNA expression and potentially affected biological functions. Next-generation miRNA sequencing of fpEC isolated after pregnancies with male and female neonates identified sex-dependent miRNA expression patterns. Potential biological pathways regulated by the altered set of miRNAs were determined using mirPath and mirSystem softwares, and suggested differences in barrier function and actin organization. The identified pathways were further investigated by monolayer impedance measurements (ECIS) and analysis of F-actin organization (Phalloidin). Nine miRNAs were differentially expressed in fpEC of male versus female neonates. Functional pathways most significantly regulated by these miRNAs included 'Adherens junction', 'ECM receptor interaction' and 'Focal adhesion'. These pathways control monolayer barrier function and may be paralleled by altered cytoskeletal organization. In fact, monolayer impedance was higher in fpEC of male progeny, and F-actin staining revealed more pronounced peripheral stress fibers in male versus female fpEC. Our data highlight that endothelial cell function differs between males and females already in utero, and that altered miRNAs are associated with sex dependent differences in barrier function and actin organization.

Descemet's Membrane Modulation of Posterior Corneal Fibrosis.

Purpose: 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. Methods: 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. Results: 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. Conclusions: 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.

The Impact of Photorefractive Keratectomy and Mitomycin C on Corneal Nerves and Their Regeneration.

PURPOSE: To determine how photorefractive keratectomy (PRK) and mitomycin C (MMC) affect corneal nerves and their regeneration over time after surgery. METHODS: Twenty-eight New Zealand rabbits had corneal epithelial scraping with (n = 3) and without (n = 3) MMC 0.02% or -9.00 diopter PRK with (n = 6) and without (n = 16) MMC 0.02%. Corneas were removed after death and corneal nerve morphology was evaluated using acetylcholinesterase immunohistochemistry and beta-III tubulin staining after 1 day for all groups, after 1 month for PRK with and without MMC, and 2, 3, and 6 months after PRK without MMC. Image-Pro software (Media Cybernetics, Rockville, MD) was used to quantitate the area of nerve loss after the procedures and, consequently, regeneration of the nerves over time. Opposite eyes were used as controls. RESULTS: Epithelial scraping with MMC treatment did not show a statistically significant difference in nerve loss compared to epithelial scraping without MMC (P = .40). PRK with MMC was significantly different from PRK without MMC at 1 day after surgery (P = .0009) but not different at 1 month after surgery (P = .90). In the PRK without MMC group, nerves regenerated at 2 months (P < .0001) but did not return to the normal preoperative level of innervation until 3 months after surgery (P = .05). However, the morphology of the regenerating nerves was abnormal-with more tortuosity and aberrant innervation compared to the preoperative controls-even at 6 months after surgery. CONCLUSIONS: PRK negatively impacts the corneal nerves, but they are partially regenerated by 3 months after surgery in rabbits. Nerve loss after PRK extended peripherally to the excimer laser ablated zone, indicating that there was retrograde degeneration of nerves after PRK. MMC had a small additive toxic effect on the corneal nerves when combined with PRK that was only significant prior to 1 month after surgery. [J Refract Surg. 2018;34(12):790-798.].

IL-1 and TGF-ß Modulation of Epithelial Basement Membrane Components Perlecan and Nidogen Production by Corneal Stromal Cells.

Purpose: 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. Methods: Marker-verified rabbit keratocytes, corneal fibroblasts, myofibroblasts were stimulated with TGF-ß1, IL-1α, IL-1ß, TGF-ß3, 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). Results: IL-1α or -1ß significantly upregulated perlecan mRNA expression in keratocytes. TGF-ß1 or -ß3 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-ß1 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. Conclusions: IL-1 and TGF-ß1 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.

Posterior stromal cell apoptosis triggered by mechanical endothelial injury and basement membrane component nidogen-1 production in the cornea.

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.

Fibrocyte migration, differentiation and apoptosis during the corneal wound healing response to injury.

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 TGFß requisite for myofibroblast survival.

Epithelial basement membrane injury and regeneration modulates corneal fibrosis after pseudomonas corneal ulcers in rabbits.

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 (TGFß) 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 TGFß to maintain viability. The endothelium and Descemet's membrane may serve a similar function modulating TGFß penetration into the posterior stroma-with the source of TGFß likely being the aqueous humor.

Endocrine and metabolic adaptations to pregnancy; impact of obesity.

Adaptations of maternal endocrine and metabolic homeostasis are central to successful pregnancy. They insure that an adequate and continuous supply of metabolic fuels is available for the growing fetus. Healthy pregnancy is classically described as a mild diabetogenic state with significant adjustments in both insulin production and sensitivity. The placenta contributes to the endocrine adaptations to pregnancy through the synthesis of various hormones which may impact insulin action. Obesity has the highest prevalence among metabolic disease in pregnancy. This article summarizes the literature addressing the endocrine and metabolic adaptations implemented during normal pregnancy. Mechanisms of regulation are further examined in the context of maternal obesity.

Identification of early transcriptome signatures in placenta exposed to insulin and obesity.

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.

Obesity-induced down-regulation of the mitochondrial translocator protein (TSPO) impairs placental steroid production.

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/m(2)) and 90 lean (body mass index 19-25 kg/m(2)) 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.

Hyperinsulinemia stimulates angiogenesis of human fetoplacental endothelial cells: a possible role of insulin in placental hypervascularization in diabetes mellitus.

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-ß3 (+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.

Variable promoter methylation contributes to differential expression of key genes in human placenta-derived venous and arterial endothelial cells.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Differential response of arterial and venous endothelial cells to extracellular matrix is modulated by oxygen.

Binding of endothelial cell (EC) integrins to extracellular-matrix (ECM) components is one of the key events to trigger intracellular signaling that will ultimately result in proper vascular development. Even within one tissue, the endothelial phenotype differs between arteries and veins. Here, we tested the hypothesis that anchorage dependent processes, such as proliferation, viability, survival and actin organization of venous (VEC) and arterial EC (AEC) differently depend on ECM proteins. Moreover,because of different oxygen tension in AEC and VEC, we tested oxygen as a co-modulator of ECM effects. Primary human placental VEC and AEC were grown in collagens I and IV, fibronectin, laminin, gelatin and uncoated plates and exposed to 12 and 21% oxygen. Our main findings revealed that VEC are more sensitive than AEC to changes in the ECM composition. Proliferation and survival of VEC, in contrast to AEC, were profoundly increased by the presence of collagen I and fibronectin when compared with gelatin or uncoated plates. These effects were reversed by inhibition of focal adhesion kinase (Fak) and modulated by oxygen. VEC were more susceptible to the oxygen dependent ECM effects than AEC. However, no differential ECM effect on actin organization was observed between the two cell types. These data provide first evidence that AEC and VEC from the same vascular loop respond differently to ECM and oxygen in a Fak-dependent manner.