Diabetes and Early Development: Epigenetics, Biological Stress, and Aging.

Pregestational diabetes, either type 1 or type 2 diabetes, induces structural birth defects including neural tube defects and congenital heart defects in human fetuses. Rodent models of type 1 and type 2 diabetic embryopathy have been established and faithfully mimic human conditions. Hyperglycemia of maternal diabetes triggers oxidative stress in the developing neuroepithelium and the embryonic heart leading to the activation of proapoptotic kinases and excessive cell death. Oxidative stress also activates the unfolded protein response and endoplasmic reticulum stress. Hyperglycemia alters epigenetic landscapes by suppressing histone deacetylation, perturbing microRNA (miRNA) expression, and increasing DNA methylation. At cellular levels, besides the induction of cell apoptosis, hyperglycemia suppresses cell proliferation and induces premature senescence. Stress signaling elicited by maternal diabetes disrupts cellular organelle homeostasis leading to mitochondrial dysfunction, mitochondrial dynamic alteration, and autophagy impairment. Blocking oxidative stress, kinase activation, and cellular senescence ameliorates diabetic embryopathy. Deleting the mir200c gene or restoring mir322 expression abolishes maternal diabetes hyperglycemia-induced senescence and cellular stress, respectively. Both the autophagy activator trehalose and the senomorphic rapamycin can alleviate diabetic embryopathy. Thus, targeting cellular stress, miRNAs, senescence, or restoring autophagy or mitochondrial fusion is a promising approach to prevent poorly controlled maternal diabetes-induced structural birth defects. In this review, we summarize the causal events in diabetic embryopathy and propose preventions for this pathological condition. KEY POINTS: · Maternal diabetes induces structural birth defects.. · Kinase signaling and cellular organelle stress are critically involved in neural tube defects.. · Maternal diabetes increases DNA methylation and suppresses developmental gene expression.. · Cellular apoptosis and senescence are induced by maternal diabetes in the neuroepithelium.. · microRNAs disrupt mitochondrial fusion leading to congenital heart diseases in diabetic pregnancy..

Placental SARS-CoV-2 Infection and Its Implications for Increased Risk of Adverse Pregnancy Outcomes.

OBJECTIVE: Pregnant women are at increased risk of coronavirus disease 2019 (COVID-19). This could be explained through the prism of physiologic and immunologic changes in pregnancy. In addition, certain immunological reactions originate in the placenta in response to viral infections.This study aimed to investigate whether severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) can infect the human placenta and discuss its implications in the pathogenesis of adverse pregnancy outcomes. STUDY DESIGN: We conducted a retrospective cohort study in which we collected placental specimens from pregnant women who had a laboratory-confirmed SARS-CoV-2 infection. We performed RNA in situ hybridization assay on formalin-fixed paraffin-embedded tissues to establish the in vivo evidence for placental infectivity by this corona virus. In addition, we infected trophoblast isolated from uninfected term human placenta with SARS-CoV-2 variants to further provide in vitro evidence for such an infectivity. RESULTS: There was a total of 21 cases enrolled, which included 5 cases of spontaneous preterm birth (SPTB) and 2 intrauterine fetal demises (IUFDs). Positive staining of positive-sense strand of SARS-CoV-2 virions was detected in 15 placentas including 4 SPTB and both IUFDs. In vitro infection assay demonstrated that SARS-CoV-2 virions were highly capable of infecting both cytotrophoblast and syncytiotrophoblast. CONCLUSION: This study implies that placental SARS-CoV-2 infection may be associated with an increased risk of adverse obstetrical outcomes. KEY POINTS: · SARS-CoV-2 can effectively infect human placenta.. · Such infectivity is confirmed by in vitro experiments.. · Placental SARS-CoV-2 corelates with adverse obstetrical outcomes..

Small Molecule Activators of Mitochondrial Fusion Prevent Congenital Heart Defects Induced by Maternal Diabetes.

Most congenital heart defect (CHD) cases are attributed to nongenetic factors; however, the mechanisms underlying nongenetic factor-induced CHDs are elusive. Maternal diabetes is one of the nongenetic factors, and this study aimed to determine whether impaired mitochondrial fusion contributes to maternal diabetes-induced CHDs and if mitochondrial fusion activators, teriflunomide and echinacoside, could reduce CHD incidence in diabetic pregnancy. We demonstrated maternal diabetes-activated FoxO3a increases miR-140 and miR-195, which in turn represses Mfn1 and Mfn2, leading to mitochondrial fusion defects and CHDs. Two mitochondrial fusion activators are effective in preventing CHDs in diabetic pregnancy.

MicroRNA-322 overexpression reduces neural tube defects in diabetic pregnancies.

BACKGROUND: Hyperglycemia from pregestational diabetes mellitus induces neural tube defects in the developing fetus. Folate supplementation is the only effective way to prevent neural tube defects; however, some cases of neural tube defects are resistant to folate. Excess folate has been linked to higher maternal cancer risk and infant allergy. Therefore, additional interventions are needed. Understanding the mechanisms underlying maternal diabetes mellitus-induced neural tube defects can identify potential targets for preventing such defects. Despite not yet being in clinical use, growing evidence suggests that microRNAs are important intermediates in embryonic development and can serve as both biomarkers and drug targets for disease intervention. Our previous studies showed that maternal diabetes mellitus in vivo activates the inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) in the developing embryo and that a high glucose condition in vitro reduces microRNA-322 (miR-322) levels. IRE1α is an RNA endonuclease; however, it is unknown whether IRE1α targets and degrades miR-322 specifically or whether miR-322 degradation leads to neural tube defects via apoptosis. We hypothesize that IRE1α can inhibit miR-322 in maternal diabetes mellitus-induced neural tube defects and that restoring miR-322 expression in developing neuroepithelium ameliorates neural tube defects. OBJECTIVE: This study aimed to identify potential targets for preventing maternal diabetes mellitus-induced neural tube defects and to investigate the roles and relationship of a microRNA and an RNA endonuclease in mouse embryos exposed to maternal diabetes mellitus. STUDY DESIGN: To determine whether miR-322 reduction is necessary for neural tube defect formation in pregnancies complicated by diabetes mellitus, male mice carrying a transgene expressing miR-322 were mated with nondiabetic or diabetic wide-type female mice to generate embryos with or without miR-322 overexpression. At embryonic day 8.5 when the neural tube is not yet closed, embryos were harvested for the assessment of 3 miR-322 transcripts (primary, precursor, and mature miR-322), tumor necrosis factor receptor-associated factor 3 (TRAF3), and neuroepithelium cell survival. Neural tube defect incidences were determined in embryonic day 10.5 embryos when the neural tube should be closed if there is no neural tube defect formation. To identify which miR-322 transcript is affected by maternal diabetes mellitus and high glucose conditions, 3 miR-322 transcripts were assessed in embryos from dams with or without diabetes mellitus and in C17.2 mouse neural stem cells treated with different concentrations of glucose and at different time points. To determine whether the endonuclease IRE1α targets miR-322, small interfering RNA knockdown of IRE1α or overexpression of inositol-requiring transmembrane kinase/endoribonuclease 1α by DNA plasmid transfection was used to determine the effect of IRE1α deficiency or overexpression on miR-322 expression. RNA immunoprecipitation was performed to reveal the direct targets of inositol-requiring transmembrane kinase/endoribonuclease 1α. RESULTS: Maternal diabetes mellitus suppressed miR-322 expression in the developing neuroepithelium. Restoring miR-322 expression in the neuroepithelium blocked maternal diabetes mellitus-induced caspase-3 and caspase-8 cleavage and cell apoptosis, leading to a neural tube defect reduction. Reversal of maternal diabetes mellitus-inhibited miR-322 via transgenic overexpression prevented TRAF3 up-regulation in embryos exposed to maternal diabetes mellitus. Activated IRE1α acted as an endonuclease and degraded precursor miR-322, resulting in mature miR-322 reduction. CONCLUSION: This study supports the crucial role of the IRE1α-microRNA-TRAF3 circuit in the induction of neuroepithelial cell apoptosis and neural tube defect formation in pregnancies complicated by diabetes mellitus and identifies IRE1α and miR-322 as potential targets for preventing maternal diabetes mellitus-induced neural tube defects.

Obesity impacts placental function through activation of p-IRE1a-XBP1s signaling.

Maternal obesity is associated with a variety of obstetrical outcomes including stillbirth, preeclampsia, and gestational diabetes, and increases the risk of fetuses for congenital heart defects. Obesity during pregnancy represents a major contribution to metabolic dysregulation, which not only plays a key role in the pathogenesis of adverse outcome but also can potently induce endoplasmic reticulum (ER) stress. However, the mechanism associating such an obesogenic metabolic environment and adverse pregnancy outcomes has remained poorly understood. In this study, we aimed to determine whether the ER stress pathways (also named unfolded protein response (UPR)) were activated in the placenta by obesity. We collected placenta from the obese pregnancy (n = 12) and non-obese pregnancy (n = 12) following delivery by Caesarean-section at term. The specimens were assessed with immunocytochemistry staining and RT-QPCR. Our results revealed that in the obese placenta, p-IRE1α and XBP1s were significantly increased, CHOP and nine UPR chaperone genes were upregulated, including GRP95, PDIA6, Calnexin, p58IPK, SIL-1, EDEM, Herp, GRP58 and Calreticulin. However, Perk and BiP are not activated in the obese placenta. Our data suggest that upregulated p-IRE1α and XBP1s signaling, and UPR chaperone genes may play an important role in maternal obesity-induced placental pathology. In conclusion, this is the first report on ER stress and UPR activation in the placenta of maternal obesity. Our findings represent the first step in the understanding of one of the key ER signaling pathways, also referred to IRE1α-XBP1, in placental pathophysiology affected by obesity, which may be an important mechanism accounting for the observed higher maternal and perinatal risks.

SARS-CoV-2 infection induces activation of ferroptosis in human placenta.

Ferroptosis, a regulated non-apoptotic form of cell death, has been implicated in the response to varied types of infectious agents including virus. In this study, we sought to determine whether SARS-CoV-2 infection can induce activation of ferroptosis in the human placenta. We collected placentas from 23 pregnant females with laboratory-confirmed SARS-CoV-2 following delivery and then used RNA in situ hybridization assay for detection of viral positive-sense strand (PSS) to confirm that these placentas have been infected. We also used immunohistochemistry assay to assess expression levels of acyl-CoA synthetase long-chain family member 4 (ACSL4), an essential executioner of ferroptosis in the same specimens. Our results showed that ACSL4 expression was significantly increased in the group with positive positive-sense strand staining compared to their negative counterparts (p = 0.00022). Furthermore, we found that there was a positive trend for increased PSS staining along with increased ACSL4 expression. Our study supports that ferroptosis is activated in the response to SARS-CoV-2 infection in the human placenta, highlighting a molecular mechanism potentially linking this coronavirus infection and pathogenesis of adverse pregnancy outcomes.

Oxidative Stress Kinase Activation and Impaired Insulin Receptor Signaling Precede Overt Alzheimer's Disease Neuropathology.

BACKGROUND: The cascade of events that lead to Alzheimer's disease (AD) consists of several possible underlying signal transduction pathways. Apoptosis signal-regulating kinase 1 (ASK1) and insulin receptor (IR) signaling are implicated in AD. OBJECTIVE: We aimed to determine whether ASK1 activation and IR signaling impairment occurred prior to and during overt AD. METHODS: Immunostaining, immunoblotting, and quantitative PCR were used to assess the levels of ASK1 and IR signaling intermediates. Glucose uptake was determined in AD-patient derived inducible pluripotent stem cells (iPSCs). RESULTS: ASK1 signaling was activated in postmortem brain tissues acquired from APOE4 carriers, a causative heritable factor, and in brain tissues of AD subjects in comparison with those harboring the normal APOE3 variant, which was manifested with an increased phosphorylated ASK1 (p-ASK1) and reduced thioredoxin 1 (TRX1). ASK1 downstream signaling effectors were also significantly elevated in these APOE4 carriers and AD brain tissues. Increased insulin receptor substrate 1 (IRS1) phosphorylation at serine residues, and decreased p-AKT1, p-IRß, and GLUT3 expression were present in all APOE4 carriers and AD samples, suggesting impaired IR signaling leading to insulin resistance. ASK1 activation, IR signaling impairment, and GLUT3 reduction were also present in young AD transgenic mice prior to AD syndromes, AD mice at AD neuropathology onset, and AD iPSCs and their derived neurons prior to p-Tau aggregation. CONCLUSION: We conclude that the activation of oxidative stress-responsive kinases and reduced IR signaling precede and are persistent in AD pathogenesis. Our data further suggest possible crosstalk between ASK1 signaling and insulin resistance in AD etiology.

RNA Hypomethylation and Unchanged DNA Methylation Levels in the Cortex of ApoE4 Carriers and Alzheimer's Disease Subjects.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder, and ApoE4 variants are significant risk factors for AD. Epigenetic modifications are involved in AD pathology. However, it is unclear whether DNA/RNA methylation plays a role in AD pathology, and dysregulation of DNA/RNA methylation occurs in ApoE4 carriers. OBJECTIVE: The present study aimed to determine whether dysregulation of DNA/RNA methylation is present in the brains of ApoE4 carriers and AD patients. METHODS: In this study, postmortem brain tissues from carriers of ApoE4 and ApoE3, from AD and non- AD controls, were used in the analysis of DNA/RNA methylation, methyltransferases, and their demethylases. RESULTS: Immunofluorescence staining indicates that RNA methylation is suppressed in ApoE4 carriers. Further analysis shows that the expression of RNA methyltransferases and an RNA methylation reader is suppressed in ApoE4 carriers, whereas RNA demethylase expression is increased. RNA hypomethylation occurs in NeuN+ neurons in ApoE4 carriers and AD patients. Furthermore, in ApoE4 carriers, both DNA methyltransferases and demethylases are downregulated, and overall DNA methylation levels are unchanged. CONCLUSION: Our finding indicates that RNA methylation decreased in ApoE4 carriers before AD pathology and AD individuals. The expression of RNA methyltransferases and RNA methylation reader is inhibited, and RNA demethylase is upregulated in ApoE4 carriers, which leads to suppression of RNA methylation, and the suppression precedes the AD pathogenesis and persists through AD pathology.

Functional cargos of exosomes derived from Flk-1+ vascular progenitors enable neurulation and ameliorate embryonic anomalies in diabetic pregnancy.

Various types of progenitors initiate individual organ formation and their crosstalk orchestrates morphogenesis for the entire embryo. Here we show that progenitor exosomal communication across embryonic organs occurs in normal development and is altered in embryos of diabetic pregnancy. Endoderm fibroblast growth factor 2 (FGF2) stimulates mesoderm Flk-1+ vascular progenitors to produce exosomes containing the anti-stress protein Survivin. These exosomes act on neural stem cells of the neuroepithelium to facilitate neurulation by inhibiting cellular stress and apoptosis. Maternal diabetes causes Flk-1+ progenitor dysfunction by suppressing FGF2 through DNA hypermethylation. Restoring endoderm FGF2 prevents diabetes-induced survivin reduction in Flk-1+ progenitor exosomes. Transgenic Survivin expression in Flk-1+ progenitors or in utero delivery of survivin-enriched exosomes restores cellular homeostasis and prevents diabetes-induced neural tube defects (NTDs), whereas inhibiting exosome production induces NTDs. Thus, functional inter-organ communication via Flk-1 exosomes is vital for neurulation and its disruption leads to embryonic anomalies.

Maternal obesity-associated disruption of polarized lactate transporter MCT4 expression in human placenta.

Maternal obesity is associated with an increased risk of adverse pregnancy outcomes including stillbirth, and their etiology is thought to be related to placental and fetal hypoxia. In this study, we sought to investigate the levels of lactate in maternal and umbilical cord blood, a well characterized biomarker for hypoxia, and expression of plasma membrane lactate transporter MCT1 and MCT4 in the placental syncytiotrophoblast (STB), which are responsible for lactate uptake and extrusion, respectively, from pregnant women with a diagnosis of obesity following a Cesarean delivery at term. With use of approaches including immunofluorescence staining, Western blot, RT-qPCR and ELISA, our results revealed that in controls the expression of MCT1 was equally observed between basal (fetal-facing, BM) and microvillous (maternal-facing, MVM) membrane of the STB, whereas MCT4 was predominantly expressed in the MVM but barely detected in the BM. However, obese patients demonstrated significant decreased MCT4 abundance in the MVM coupled with concurrent elevated expression in the BM. We also found a linear trend toward decreasing MCT4 expression ratio of MVM to BM with increasing maternal pre-pregnancy BMI. Furthermore, our data showed that the lactate ratios of fetal cord arterial to maternal blood were remarkably reduced in obese samples compared to their normal counterparts. Collectively, these results suggest that the loss of polarization of lactate transporter MCT4 expression in placental STB leading to disruption of unidirectional lactate transport from the fetal to the maternal compartment may constitute part of mechanisms linking maternal obesity and pathogenesis of stillbirth.

A SARS-CoV-2 Delta Variant Case Manifesting as Extensive Placental Infection and Fetal Transmission.

INTRODUCTION: Studies indicate a very low rate of SARS-CoV-2 detection in the placenta or occasionally a low rate of vertical transmission in COVID-19 pregnancy. SARS-CoV-2 Delta variant has become a dominant strain over the world and possesses higher infectivity due to mutations in its spike receptor-binding motif. CASE PRESENTATION: To determine whether SARS-CoV-2 Delta variant has increased potential for placenta infection and vertical transmission, we analyzed SARS-CoV-2 infection in the placenta, umbilical cord, and fetal membrane from a case where an unvaccinated mother and her neonate were COVID-19 positive. A 35-year-old primigravida with COVID-19 underwent an emergent cesarean delivery due to placental abruption in the setting of premature rupture of membranes. The neonate tested positive for SARS-CoV-2 within the first 24 h, and then again on days of life 2, 6, 13, and 21. The placenta exhibited intervillositis, increased fibrin deposition, and syncytiotrophoblast necrosis. Sequencing of viral RNA from fixed placental tissue revealed SAR-CoV-2 B.1.167.2 (Delta) variant. Both spike protein and viral RNA were abundantly present in syncytiotrophoblasts, cytotrophoblasts, umbilical cord vascular endothelium, and fetal membranes. CONCLUSION: We report with strong probability the first SARS-CoV-2 Delta variant transplacental transmission. Placental cells exhibited extensive apoptosis, senescence, and ferroptosis after SARS-CoV-2 Delta infection.

SARS-CoV-2 invades cognitive centers of the brain and induces Alzheimer's-like neuropathology.

The neurotropism of SARS-CoV-2 and the phenotypes of infected neurons are still in debate. Long COVID manifests with "brain diseases" and the cause of these brain dysfunction is mysterious. Here, we analyze 34 age- and underlying disease-matched COVID-19 or non-COVID-19 human brains. SARS-CoV-2 RNA, nucleocapsid, and spike proteins are present in neurons of the cognitive centers of all COVID-19 patients, with its non-structural protein NSF2 detected in adult cases but not in the infant case, indicating viral replications in mature neurons. In adult COVID-19 patients without underlying neurodegeneration, SARS-CoV-2 infection triggers Aß and p-tau deposition, degenerating neurons, microglia activation, and increased cytokine, in some cases with Aß plaques and p-tau pretangles. The number of SARS-CoV-2 + cells is higher in patients with neurodegenerative diseases than in those without such conditions. SARS-CoV-2 further activates microglia and induces Aß and p-tau deposits in non-Alzheimer's neurodegenerative disease patients. SARS-CoV-2 infects mature neurons derived from inducible pluripotent stem cells from healthy and Alzheimer's disease (AD) individuals through its receptor ACE2 and facilitator neuropilin-1. SARS-CoV-2 triggers AD-like gene programs in healthy neurons and exacerbates AD neuropathology. An AD infectious etiology gene signature is identified through SARS-CoV-2 infection and silencing the top three downregulated genes in human primary neurons recapitulates the neurodegenerative phenotypes of SARS-CoV-2. Thus, our data suggest that SARS-CoV-2 invades the brain and activates an AD-like program.

Maternal obesity increases DNA methylation and decreases RNA methylation in the human placenta.

Maternal obesity is associated with increased risk of adverse pregnancy and birth outcomes. While increasing body of evidence supports that the etiology is related to fetal and placental hypoxia, molecular signaling changes in response to this pathophysiological condition in human placenta have remained elusive. Here by using varied approaches including immunocytochemistry staining, Western blot, RT-qPCR, and ELISA, we aimed to investigate the changes in epigenetic markers in placentas from obese pregnant women following delivery by Caesarean-section at term. Our results revealed that the levels of 5-methylcytosine (5mC), a methylated form commonly occurring in CpG dinucleotides and an important repressor of gene transcription in the genome, were significantly increased coupled with decreased activity of Ten-Eleven Translocation (TETs) enzymes that principally function by oxidizing 5mC in the obese placenta, consistent with hypoxia-induced genome-wide DNA hypermethylation observed in varied types of cells and tissues. N6-methyladenosine (m6A) represents the most abundant and conserved modification of gene transcripts, especially within mRNAs, which is stalled by m6A methyltransferases or "writers" including METTL-3/-14, WTAP, RBM15B, and KIAA1429. We further showed that obese placentas demonstrated significantly down-regulated levels of m6A along with reduced gene expression of WTAP, RBM15B, and KIAA1429. Our data support that maternal obesity-induced hypoxia may play an important role in triggering genome-wide DNA hypermethylation in the human placenta, and in turn leading to transcriptome-wide inhibition of RNA modifications. Our results further suggest that selectively modulating these pathways may facilitate development of novel therapeutic approaches for controlling and managing maternal obesity-associated adverse clinical outcomes.

Maternal diabetes induces senescence and neural tube defects sensitive to the senomorphic rapamycin.

Neural tube defects (NTDs) are the second most common structural birth defect. Senescence, a state of permanent cell cycle arrest, occurs only after neural tube closure. Maternal diabetes-induced NTDs are severe diabetic complications that lead to infant mortality or lifelong morbidity and may be linked to premature senescence. Here, we report that premature senescence occurs in the mouse neuroepithelium and disrupts neurulation, leading to NTDs in diabetic pregnancy. Premature senescence and NTDs were abolished by knockout of the transcription factor Foxo3a, the miR-200c gene, and the cell cycle inhibitors p21 and p27; transgenic expression of the dominant-negative FoxO3a mutant; or the senomorphic rapamycin. Double transgenic expression of p21 and p27 mimicked maternal diabetes in inducing premature neuroepithelium senescence and NTDs. These findings integrate transcription- and epigenome-regulated miRNAs and cell cycle regulators in premature neuroepithelium senescence and provide a mechanistic basis for targeting premature senescence and NTDs using senomorphics.

Restoring BMP4 expression in vascular endothelial progenitors ameliorates maternal diabetes-induced apoptosis and neural tube defects.

During mouse embryonic development, vasculogenesis initially occurs in the yolk sac, preceding neurulation. Our previous studies have demonstrated that maternal diabetes induces embryonic vasculopathy at early embryonic developmental stage by suppressing the expression of vascular growth factors including BMP4 (bone morphogenetic protein 4). This study aimed to determine whether restoring diabetes-inhibited BMP4 expression in Flk-1+ progenitors effectively prevented maternal diabetes-induced embryonic vasculopathy and NTDs. Transgenic (Tg) BMP4 expression in the vascular endothelial growth factor receptor 2 (Flk-1)-positive (Flk-1+) progenitors was achieved by crossing a Floxed BMP4 Tg mouse line with the Flk-1-Cre mouse line. Non-BMP4 Tg and BMP4 Tg embryos were harvested at E8.5 to assess the expression of BMP4, markers of endoplasmic reticulum stress, and expression of the Id genes, direct targets of BMP4; and the presence of cleaved caspase 3 and 8, apoptosis, and Smad signaling. BMP4 Tg overexpression neutralized its down-regulation by maternal diabetes in E8.5 embryos. Maternal diabetes-induced Flk-1+ progenitor apoptosis, impairment of blood island formation, and reduction of Flk-1+ progenitor number and blood vessel density, which were reversed by BMP4 Tg expression. BMP4 Tg expression in Flk-1+ progenitors blocked maternal diabetes-induced vasculopathy in early stage embryos (E7.5-E8.5) and consequently led to amelioration of maternal diabetes-induced neural tube defects (NTDs) at E10.5. BMP4 Tg expression inhibited maternal diabetes-induced endoplasmic reticulum stress and caspase cascade activation in the developing neuroepithelium, and reduced neuroepithelial cell apoptosis. BMP4 Tg expression re-activated Smad1/5/8 phosphorylation and reversed maternal diabetes-suppressed Smad4 expression. BMP4 Tg expression restored Id1 and Smad6 expression inhibited by maternal diabetes. In vitro, recombinant BMP4 protein blocked high glucose-induced Flk-1+ progenitor apoptosis and NTDs. These data demonstrate that BMP4 down-regulation in Flk-1+ progenitors are responsible for diabetes-induced yolk sac vasculopathy, and that restoring BMP4 expression prevents vasculopathy and rescues neuroepithelial cells from cellular organelle stress, leading to NTD reduction.

Deficiency of the oxidative stress-responsive kinase p70S6K1 restores autophagy and ameliorates neural tube defects in diabetic embryopathy.

BACKGROUND: Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium. OBJECTIVE: We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy. STUDY DESIGN: Female p70S6K1 heterogeneous knockout (p70S6K1+/-) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1+/-) mice to generate wild-type (WT), p70S6K1+/- and p70S6K1 knockout (p70S6K1-/-) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For in vitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions. RESULTS: Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes in vivo and high glucose in vitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation. CONCLUSION: The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy.

High Glucose Inhibits Neural Stem Cell Differentiation Through Oxidative Stress and Endoplasmic Reticulum Stress.

Maternal diabetes induces neural tube defects by suppressing neurogenesis in the developing neuroepithelium. Our recent study further revealed that high glucose inhibited embryonic stem cell differentiation into neural lineage cells. However, the mechanism whereby high glucose suppresses neural differentiation is unclear. To investigate whether high glucose-induced oxidative stress and endoplasmic reticulum (ER) stress lead to the inhibition of neural differentiation, the effect of high glucose on neural stem cell (the C17.2 cell line) differentiation was examined. Neural stem cells were cultured in normal glucose (5 mM) or high glucose (25 mM) differentiation medium for 3, 5, and 7 days. High glucose suppressed neural stem cell differentiation by significantly decreasing the expression of the neuron marker Tuj1 and the glial cell marker GFAP and the numbers of Tuj1+ and GFAP+ cells. The antioxidant enzyme superoxide dismutase mimetic Tempol reversed high glucose-decreased Tuj1 and GFAP expression and restored the numbers of neurons and glial cells differentiated from neural stem cells. Hydrogen peroxide treatment imitated the inhibitory effect of high glucose on neural stem cell differentiation. Both high glucose and hydrogen peroxide triggered ER stress, whereas Tempol blocked high glucose-induced ER stress. The ER stress inhibitor, 4-phenylbutyrate, abolished the inhibition of high glucose or hydrogen peroxide on neural stem cell differentiation. Thus, oxidative stress and its resultant ER stress mediate the inhibitory effect of high glucose on neural stem cell differentiation.

The current status and future of cardiac stem/progenitor cell therapy for congenital heart defects from diabetic pregnancy.

Pregestational maternal diabetes induces congenital heart defects (CHDs). Cardiac dysfunction after palliative surgical procedures contributes to the high mortality of CHD patients. Autologous or allogeneic stem cell therapies are effective for improving cardiac function in animal models and clinical trials. c-kit+ cardiac progenitor cells (CPCs), the most recognized CPCs, have the following basic properties of stem cells: self-renewal, multicellular clone formation, and differentiation into multiple cardiac lineages. However, there is ongoing debate regarding whether c-kit+ CPCs can give rise to sufficient cardiomyocytes. A new hypothesis to address the beneficial effect of c-kit+ CPCs is that these cells stimulate endogenous cardiac cells through a paracrine function in producing a robust secretome and exosomes. The values of other cardiac CPCs, including Sca1+ CPCs and cardiosphere-derived cells, are beginning to be revealed. These cells may be better choices than c-kit+ CPCs for generating cardiomyocytes. Adult mesenchymal stem cells are considered immune-incompetent and effective for improving cardiac function. Autologous CPC therapy may be limited by the observation that maternal diabetes adversely affects the biological function of embryonic stem cells and CPCs. Future studies should focus on determining the mechanistic action of these cells, identifying new CPC markers, selecting highly effective CPCs, and engineering cell-free products.

Magnetic Enhancement of Stem Cell-Targeted Delivery into the Brain Following MR-Guided Focused Ultrasound for Opening the Blood-Brain Barrier.

Focused ultrasound (FUS)-mediated blood-brain barrier disruption (BBBD) can enable even large therapeutics such as stem cells to enter the brain from the bloodstream. However, the efficiency is relatively low. Our previous study showed that human neural progenitor cells (hNPCs) loaded with superparamagnetic iron oxide nanoparticles (SPIONs) in culture were attracted by an external magnetic field. In vivo, enhanced brain retention was observed near a magnet mounted on the skull in a rat model of traumatic brain injury, where BBBD also occurs. The goal of the current study was to determine whether magnetic attraction of SPION-loaded hNPCs would also enhance their retention in the brain after FUS-mediated BBBD. A small animal magnetic resonance imaging (MRI)-guided FUS system operating at 1.5 MHz was used to treat rats (∼120 g) without tissue damage or hemorrhage. Evidence of successful BBBD was validated with both radiologic enhancement of gadolinium on postsonication TI MRI and whole brain section visualization of Evans blue dye. The procedure was then combined with the application of a powerful magnet to the head directly after intravenous injection of the hNPCs. Validation of cells within the brain was performed by staining with Perls' Prussian blue for iron and by immunohistochemistry with a human-specific antigen. By injecting equal numbers of iron oxide (SPIONs) and noniron oxide nanoparticles-loaded hNPCs, each labeled with a different fluorophore, we found significantly greater numbers of SPIONs-loaded cells retained in the brain at the site of BBBD as compared to noniron loaded cells. This result was most pronounced in regions of the brain closest to the skull (dorsal cortex) in proximity to the magnet surface. A more powerful magnet and a Halbach magnetic array resulted in more effective retention of SPION-labeled cells in even deeper brain regions such as the striatum and ventral cortex. There, up to 90% of hNPCs observed contained SPIONs compared to 60% to 70% with the less powerful magnet. Fewer cells were observed at 24 h posttreatment compared to 2 h (primarily in the dorsal cortex). These results demonstrate that magnetic attraction can substantially enhance the retention of stem cells after FUS-mediated BBBD. This procedure could provide a safer and less invasive approach for delivering stem cells to the brain, compared to direct intracranial injections, substantially reducing the risk of bleeding and infection.

Maternal diabetes and high glucose in vitro trigger Sca1+ cardiac progenitor cell apoptosis through FoxO3a.

Recent controversies surrounding the authenticity of c-kit+ cardiac progenitor cells significantly push back the advance in regenerative therapies for cardiovascular diseases. There is an urgent need for research in characterizing alternative types of cardiac progenitor cells. Towards this goal, in the present study, we determined the effect of maternal diabetes on Sca1+ cardiac progenitor cells. Maternal diabetes induced caspase 3-dependent apoptosis in Sca1+ cardiac progenitor cells derived from embryonic day 17.5 (E17.5). Similarly, high glucose in vitro but not the glucose osmotic control mannitol triggered Sca1+ cardiac progenitor cell apoptosis in a dose- and time-dependent manner. Both maternal diabetes and high glucose in vitro activated the pro-apoptotic transcription factor, Forkhead O 3a (FoxO3a) via dephosphorylation at threonine 32 (Thr-32) residue. foxo3a gene deletion abolished maternal diabetes-induced Sca1+ cardiac progenitor cell apoptosis. The dominant negative FoxO3a mutant without the transactivation domain from the C terminus blocked high glucose-induced Sca1+ cardiac progenitor cell apoptosis, whereas the constitutively active FoxO3a mutant with the three phosphorylation sites, Thr-32, Ser-253, and Ser-315, being replaced by alanine residues mimicked the pro-apoptotic effect of high glucose. Thus, maternal diabetes and high glucose in vitro may limit the regenerative potential of Sca1+ cardiac progenitor cells by inducing apoptosis through FoxO3a activation. These findings will serve as the guide in optimizing the autologous therapy using Sca1+ cardiac progenitor cells in cardiac defect babies born exposed to maternal diabetes.