Emily K. Blue

Gestational diabetes induces alterations in the function of neonatal endothelial colony-forming cells.

Background:Children born to mothers with gestational diabetes mellitus (GDM) experience increased risk of developing hypertension, type 2 diabetes mellitus, and obesity. Disrupted function of endothelial colony-forming cells (ECFCs) may contribute to this enhanced risk. The goal of this study was to determine whether cord blood ECFCs from GDM pregnancies exhibit altered functionality.Methods:ECFCs isolated from the cord blood of control and GDM pregnancies were assessed for proliferation, senescence, and Matrigel network formation. The requirement for p38MAPK in hyperglycemia-induced senescence was determined using inhibition and overexpression studies.Results:GDM-exposed ECFCs were more proliferative than control ECFCs. However, GDM-exposed ECFCs exhibited decreased network-forming ability in Matrigel. Aging of ECFCs by serial passaging led to increased senescence and reduced proliferation of GDM-exposed ECFCs. ECFCs from GDM pregnancies were resistant to hyperglycemia-induced senescence compared with those from controls. In response to hyperglycemia, control ECFCs activated p38MAPK, which was required for hyperglycemia-induced senescence. In contrast, GDM-exposed ECFCs showed no change in p38MAPK activation under equivalent conditions.Conclusion:Intrauterine exposure of ECFCs to GDM induces unique phenotypic alterations. The resistance of GDM-exposed ECFCs to hyperglycemia-induced senescence and decreased p38MAPK activation suggest that these progenitor cells have undergone changes that induce tolerance to a hyperglycemic environment.

Epigenetic Regulation of Placenta-Specific 8 Contributes to Altered Function of Endothelial Colony-Forming Cells Exposed to Intrauterine Gestational Diabetes Mellitus

Intrauterine exposure to gestational diabetes mellitus (GDM) is linked to development of hypertension, obesity, and type 2 diabetes in children. Our previous studies determined that endothelial colony-forming cells (ECFCs) from neonates exposed to GDM exhibit impaired function. The current goals were to identify aberrantly expressed genes that contribute to impaired function of GDM-exposed ECFCs and to evaluate for evidence of altered epigenetic regulation of gene expression. Genome-wide mRNA expression analysis was conducted on ECFCs from control and GDM pregnancies. Candidate genes were validated by quantitative RT-PCR and Western blotting. Bisulfite sequencing evaluated DNA methylation of placenta-specific 8 (PLAC8). Proliferation and senescence assays of ECFCs transfected with siRNA to knockdown PLAC8 were performed to determine functional impact. Thirty-eight genes were differentially expressed between control and GDM-exposed ECFCs. PLAC8 was highly expressed in GDM-exposed ECFCs, and PLAC8 expression correlated with maternal hyperglycemia. Methylation status of 17 CpG sites in PLAC8 negatively correlated with mRNA expression. Knockdown of PLAC8 in GDM-exposed ECFCs improved proliferation and senescence defects. This study provides strong evidence in neonatal endothelial progenitor cells that GDM exposure in utero leads to altered gene expression and DNA methylation, suggesting the possibility of altered epigenetic regulation.

Thymine DNA Glycosylase Represses Myocardin-induced Smooth Muscle Cell Differentiation by Competing with Serum Response Factor for Myocardin Binding

Myocardin is a serum response factor (SRF) co-activator that regulates transcription of many smooth muscle-specific genes and is essential for development of vascular smooth muscle. We used a yeast two-hybrid screen, with myocardin as bait in a search for factors that regulate myocardin transcriptional activity. From this screen, thymine DNA glycosylase (TDG) was identified as a myocardin-associated protein. TDG was originally identified as an enzyme involved in base excision repair of T:G mismatches caused by spontaneous deamination of methylated cytosines. However, TDG has also been shown to act as a transcriptional co-activator or co-repressor. The interaction between TDG and myocardin was confirmed in vitro by glutathione S-transferase pull down and in vivo by co-immunoprecipitation assays. We found that TDG abrogates myocardin induced expression of smooth muscle-specific genes and represses the trans-activation of the promoters of myocardin of these genes. Overexpression of TDG in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous TDG in SMCs increased smooth muscle-specific myosin heavy chain (SM MHC) and Telokin gene expression. Glutathione S-transferase pull-down assays demonstrated that TDG binds to a region of myocardin that includes the SRF binding domain. Furthermore, TDG was found to compete with SRF for binding to myocardin in vitro and in vivo, suggesting that TDG can inhibit expression of smooth muscle-specific genes, at least in part, through disrupting SRF/myocardin interactions. Finally, we demonstrated that the glycosylase activity of TDG is not required for its inhibitory effects on myocardin function. This study reveals a previously unsuspected role for the repair enzyme TDG as a repressor of smooth muscle differentiation via competing with SRF for binding to myocardin.

Post-translational regulation of the cellular levels of DAPK.

Death associated protein kinase (DAPK) is a large, multi-domain ser/thr kinase whose activities converge upon multiple signaling pathways that regulate autophagy, caspase-dependent cell death, cell adhesion and migration. The cellular levels of DAPK are post-translationally regulated by the combined activities of two degradation systems, including the ubiquitin proteasome and an extra-lysosomal proteolysis pathway. At least three distinct E3 ubiquitin ligases target DAPK, including mindbomb1, the chaperone dependent ligase, CHIP (carboxy terminus of Hsp70-interacting protein) and a cullin RING ligase complex, KLHL20-Cul3-RBX1. In addition, it appears that the cellular levels of DAPK are also regulated by an extra-lysosomal protease, cathepsin B. While protein quality control and recycling clearly benefit cells by removal of misfolded or toxic proteins and recycling of their components, the finding that multiple surveillance systems target DAPK suggests that these protein degradation systems also act to fine tune DAPK expression levels in response to specific signaling pathways.

Mesenchyme Homeobox 2 Enhances Migration of Endothelial Colony Forming Cells Exposed to Intrauterine Diabetes Mellitus.

Diabetes mellitus (DM) during pregnancy has long‐lasting implications for the fetus, including cardiovascular morbidity. Previously, we showed that endothelial colony forming cells (ECFCs) from DM human pregnancies have decreased vasculogenic potential. Here, we evaluate whether the molecular mechanism responsible for this phenotype involves the transcription factor, Mesenchyme Homeobox 2 (MEOX2). In human umbilical vein endothelial cells, MEOX2 upregulates cyclin‐dependent kinase inhibitor expression, resulting in increased senescence and decreased proliferation. We hypothesized that dysregulated MEOX2 expression in neonatal ECFCs from DM pregnancies decreases network formation through increased senescence and altered cell cycle progression. Our studies show that nuclear MEOX2 is increased in ECFCs from DM pregnancies. To determine if MEOX2 is sufficient and/or required to induce impaired network formation, MEOX2 was overexpressed and depleted in ECFCs from control and DM pregnancies, respectively. Surprisingly, MEOX2 overexpression in control ECFCs resulted in increased network formation, altered cell cycle progression, and increased senescence. In contrast, MEOX2 knockdown in ECFCs from DM pregnancies led to decreased network formation, while cell cycle progression and senescence were unaffected. Importantly, migration studies demonstrated that MEOX2 overexpression increased migration, while MEOX2 knockdown decreased migration. Taken together, these data suggest that altered migration may be mediating the impaired vasculogenesis of ECFCs from DM pregnancies. While initially believed to be maladaptive, these data suggest that MEOX2 may serve a protective role, enabling increased vessel formation despite exposure to a DM intrauterine environment. J. Cell. Physiol. 232: 1885–1892, 2017.

Kinetic analyses of vasculogenesis inform mechanistic studies

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess in vitro endothelial colony-forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts. A tissue cytometry approach was established to quantify the frequency and localization of dividing ECFCs. Additionally, Fiji TrackMate was used to quantify ECFC displacement and speed at the single-cell level during network formation. These novel approaches were then implemented to identify how intrauterine exposure to maternal diabetes mellitus (DM) impairs fetal ECFC vasculogenesis. Fetal ECFCs exposed to maternal DM form fewer initial network structures, which are not stable over time. Correlation analyses demonstrated that ECFC samples with greater division in branches form fewer closed network structures. Additionally, reductions in average ECFC movement over time decrease structural connectivity. Identification of these novel phenotypes utilizing the newly established methodologies provides evidence for the cellular mechanisms contributing to aberrant ECFC vasculogenesis.

Fetal hyperglycemia and a high-fat diet contribute to aberrant glucose tolerance and hematopoiesis in adult rats

Background:Children exposed to gestational diabetes mellitus (GDM) during pregnancy are at increased risk of obesity, diabetes, and hypertension. Our goal was to identify metabolic and hematopoietic alterations after intrauterine exposure to maternal hyperglycemia that may contribute to the pathogenesis of chronic morbidities.Methods:Streptozotocin treatment induced maternal hyperglycemia during the last third of gestation in rat dams. Offspring of control mothers (OCM) and diabetic mothers (ODM) were evaluated for weight, glucose tolerance, insulin tolerance, and hematopoiesis defects. The effects of aging were examined in normal and high-fat diet (HFD)-fed young (8-wk-old) and aged (11-mo-old) OCM and ODM rats.Results:Young adult ODM males on a normal diet, but not females, displayed improved glucose tolerance due to increased insulin levels. Aged ODM males and females gained more weight than OCM on a HFD and had worse glucose tolerance. Aged ODM males fed a HFD were also neutrophilic. Increases in bone marrow cellularity and myeloid progenitors preceded neutrophilia in ODM males fed a HFD.Conclusion:When combined with other risk factors like HFD and aging, changes in glucose metabolism and hematopoiesis may contribute to the increased risk of obesity, type 2 diabetes, and hypertension observed in children of GDM mothers.

Mycophenolic acid induces senescence of vascular precursor cells

Objective Endothelial dysfunction is central to the pathogenesis of many rheumatic diseases, typified by vascular inflammation and damage. Immunosuppressive drugs induce disease remission and lead to improved patient survival. However, there remains a higher incidence of cardiovascular disease in these patients even after adequate disease control. The purpose of this study was to determine the effect of mycophenolic acid (MPA), a commonly used immunosuppressive drug in rheumatology, on blood vessel or circulating endothelial colony forming cell number and function. Methods We tested whether mycophenolic acid exerts an inhibitory effect on proliferation, clonogenic potential and vasculogenic function of endothelial colony forming cell. We also studied potential mechanisms involved in the observed effects. Results Treatment with MPA decreased endothelial colony forming cell proliferation, clonogenic potential and vasculogenic function in a dose-dependent fashion. MPA increased senescence-associated β-galactosidase expression, p21 gene expression and p53 phosphorylation, indicative of activation of cellular senescence. Exogenous guanosine supplementation rescued diminished endothelial colony forming cell proliferation and indices of senescence, consistent with the known mechanism of action of MPA. Conclusion Our findings show that clinically relevant doses of MPA have potent anti-angiogenic and pro-senescent effects on vascular precursor cells in vitro, thus indicating that treatment with MPA can potentially affect vascular repair and regeneration. This warrants further studies in vivo to determine how MPA therapy contributes to vascular dysfunction and increased cardiovascular disease seen in patients with inflammatory rheumatic disease.

Transgelin induces dysfunction of fetal endothelial colony forming cells from gestational diabetic pregnancies

Fetal exposure to gestational diabetes mellitus (GDM) predisposes children to future health complications including hypertension and cardiovascular disease. A key mechanism by which these complications occur is through the functional impairment of vascular progenitor cells, including endothelial colony-forming cells (ECFCs). Previously, we showed that fetal ECFCs exposed to GDM have decreased vasculogenic potential and altered gene expression. In this study, we evaluate whether transgelin (TAGLN), which is increased in GDM-exposed ECFCs, contributes to vasculogenic dysfunction. TAGLN is an actin-binding protein involved in the regulation of cytoskeletal rearrangement. We hypothesized that increased TAGLN expression in GDM-exposed fetal ECFCs decreases network formation by impairing cytoskeletal rearrangement resulting in reduced cell migration. To determine if TAGLN is required and/or sufficient to impair ECFC network formation, TAGLN was reduced and overexpressed in ECFCs from GDM and uncomplicated pregnancies, respectively. Decreasing TAGLN expression in GDM-exposed ECFCs improved network formation and stability as well as increased migration. In contrast, overexpressing TAGLN in ECFCs from uncomplicated pregnancies decreased network formation, network stability, migration, and alignment to laminar flow. Overall, these data suggest that increased TAGLN likely contributes to the vasculogenic dysfunction observed in GDM-exposed ECFCs, as it impairs ECFC migration, cell alignment, and network formation. Identifying the molecular mechanisms underlying fetal ECFC dysfunction following GDM exposure is key to ascertain mechanistically the basis for cardiovascular disease predisposition later in life.