Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states

Elevated transgelin reduces function of endothelial colony forming cells from gestational diabetic pregnancies

poster abstractFetal exposure to maternal diabetes predisposes children to future complications including hypertension and cardiovascular disease. A key mechanism by which these complications are thought to occur and persist is through the functional impairment of vascular progenitor cells, including endothelial colony forming cells (ECFCs). Previously, we showed that ECFCs exposed to gestational diabetes exhibit functional deficits, such as impaired vessel formation, but also differential gene expression compared to uncomplicated controls. One gene that was confirmed to be significantly upregulated in ECFCS from diabetic pregnancies was transgelin, an actin-binding smooth muscle protein. However, the functional consequences of increased transgelin in ECFCs are unknown. Therefore, to determine if transgelin is sufficient and required to induce dysfunction of ECFCs from diabetic pregnancies, transgelin protein levels were manipulated using genetic methods. Specifically, lentiviral overexpression and siRNA knockdown techniques were used in ECFCs from control and diabetic pregnancies respectively. Network formation assays and trans-well migration assays were performed to assess whether alteration of transgelin levels impact ECFC vasculogenesis and migration. Decreasing transgelin expression in diabetes-exposed ECFCs increased network formation (n=15, p<0.05) and cell migration (n=12, p<0.05). Conversely, overexpression of transgelin in ECFCs from uncomplicated pregnancies decreased network formation (n=12, p<0.05). Additional studies are underway to further elucidate intracellular signaling altered as a result of increased transgelin expression in diabetes-exposed ECFCs. Delineating the mechanisms underlying ECFC functional deficits will aid in the understanding of how and why chronic vascular complications persist in children born to mothers with diabetes

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

Epigenetic regulation in neonatal ECFCs following intrauterine exposure to gestational diabetes

poster abstractGestational diabetes (GDM) complicates up to 10% of pregnancies. In addition to acute risks, the children of diabetic mothers have an increased risk of obesity, diabetes, and hypertension, starting in childhood. While the causes of this increased risk are unknown, previous studies in our lab have identified functional deficits in endothelial colony forming cells (ECFCs) isolated from the cord blood of GDM pregnancies. This study focused on identifying genes that have altered epigenetic modifications that result in abnormal mRNA and protein expression in ECFCs from the cord blood GDM pregnancies. The objective of this study was to identify mRNA expression and DNA methylation alterations in ECFCs that may help identify the causes of ECFC dysfunction following intrauterine exposure to GDM. ECFCs were obtained from control and GDM pregnancies. DNA, RNA, and protein samples were isolated in parallel from ECFCs. RNA microarray analysis using the Affymetrix Human 1.0 Gene Array was used to identify gene expression alterations in GDM ECFCs compared to control ECFCs. Genome-wide DNA methylation was assessed using an Infinium 450K Methylation Array for DNA samples at >450,000 CpG sites. Correlation analysis was performed to identify possible sites that have altered CpG methylation and RNA expression. RNA expression results were validated using qRT-PCR and western blotting. Bisulfite sequencing of genomic DNA from the ECFCs was performed to identify additional sites with altered methylation for regions not included in the DNA methylation array. Of the 28,000 genetic loci tested, 596 mRNAs were altered between control and GDM ECFCs (p<0.01). More stringent criteria identified 38 genes for further investigation by limiting analysis to genes that exhibited increased or decreased expression by at least 50%, with a p<0.01. PLAC8 was identified as being increased 5-fold by microarray analysis, a result which was confirmed in two cohorts by qRT-PCR and western blotting. Analysis of the methylation array and bisulfite sequencing results revealed 3 regions surrounding the transcriptional start site of PLAC8 gene whose CpG methylation negatively correlate with RNA expression in samples from control and GDM ECFCs. In contrast, a CpG island is fully unmethylated in both control and GDM ECFCs. The discovery of CpG sites whose methylation correlates with PLAC8 mRNA expression in ECFCs is consistent with the hypothesis that intrauterine exposure to GDM results in epigenetic changes. Analysis of methylation at this site could be used as a biomarker for children of mothers with GDM who may be at risk for disease later in life. Using bisulfite pyrosequencing, we are currently developing assays to quickly determine if methylation of the PLAC8 putative promoter region is altered in cord blood mononuclear cells obtained from GDM or healthy control pregnancies. We are also investigating the role of methylation in regulating PLAC8 RNA expression, determining if there is altered histone modifications and transcription factor binding in these regions, and examining other genes that may comprise a molecular signature of ECFC dysfunction

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

Fanconi anemia genes are highly expressed in primitive CD34(+ )hematopoietic cells

BACKGROUND: Fanconi anemia (FA) is a complex recessive genetic disease characterized by progressive bone marrow failure (BM) and a predisposition to cancer. We have previously shown using the Fancc mouse model that the progressive BM failure results from a hematopoietic stem cell defect suggesting that function of the FA genes may reside in primitive hematopoietic stem cells. METHODS: Since genes involved in stem cell differentiation and/or maintenance are usually regulated at the transcription level, we used a semiquantitative RT-PCR method to evaluate FA gene transcript levels in purified hematopoietic stem cells. RESULTS: We show that most FA genes are highly expressed in primitive CD34-positive and negative cells compared to lower levels in more differentiated cells. However, in CD34(- )stem cells the Fancc gene was found to be expressed at low levels while Fancg was undetectable in this population. Furthermore, Fancg expression is significantly decreased in Fancc -/- stem cells as compared to wild-type cells while the cancer susceptibility genes Brca1 and Fancd1/Brac2 are upregulated in Fancc-/- hematopoietic cells. CONCLUSIONS: These results suggest that FA genes are regulated at the mRNA level, that increased Fancc expression in LTS-CD34(+ )cells correlates with a role at the CD34(+ )differentiation stage and that lack of Fancc affects the expression of other FA gene, more specifically Fancg and Fancd1/Brca2, through an unknown mechanism

Versatile Coordination of Cyclopentadienyl-Arene Ligands and Its Role in Titanium-Catalyzed Ethylene Trimerization

Cationic titanium(IV) complexes with ansa-(η5-cyclopentadienyl,η6-arene) ligands were synthesized and characterized by X-ray crystallography. The strength of the metal-arene interaction in these systems was studied by variable-temperature NMR spectroscopy. Complexes with a C1 bridge between the cyclopentadienyl and arene moieties feature hemilabile coordination behavior of the ligand and consequently are active ethylene trimerization catalysts. Reaction of the titanium(IV) dimethyl cations with CO results in conversion to the analogous cationic titanium(II) dicarbonyl species. Metal-to-ligand backdonation in these formally low-valent complexes gives rise to a strongly bonded, partially reduced arene moiety. In contrast to the η6-arene coordination mode observed for titanium, the more electron-rich vanadium(V) cations [cyclopentadienyl-arene]V(NiPr2)(NC6H4-4-Me)+ feature η1-arene binding, as determined by a crystallographic study. The three different metal-arene coordination modes that we experimentally observed model intermediates in the cycle for titanium-catalyzed ethylene trimerization. The nature of the metal-arene interaction in these systems was studied by DFT calculations.

The Xpc gene markedly affects cell survival in mouse bone marrow

The XPC protein (encoded by the xeroderma pigmentosum Xpc gene) is a key DNA damage recognition factor that is required for global genomic nucleotide excision repair (G-NER). In contrast to transcription-coupled nucleotide excision repair (TC-NER), XPC and G-NER have been reported to contribute only modestly to cell survival after DNA damage. Previous studies were conducted using fibroblasts of human or mouse origin. Since the advent of Xpc−/− mice, no study has focused on the bone marrow of these mice. We used carboplatin to induce DNA damage in Xpc−/− and strain-matched wild-type mice. Using several independent methods, Xpc−/− bone marrow was ∼10-fold more sensitive to carboplatin than the wild type. Importantly, 12/20 Xpc−/− mice died while 0/20 wild-type mice died. We conclude that G-NER, and XPC specifically, can contribute substantially to cell survival. The data are important in the context of cancer chemotherapy, where Xpc gene status and G-NER may be determinants of response to DNA-damaging agents including carboplatin. Additionally, altered cell cycles and altered DNA damage signalling may contribute to the cell survival end point

Inappropriate use of the title 'chiropractor' and term 'chiropractic manipulation' in the peer-reviewed biomedical literature

BACKGROUND: The misuse of the title 'chiropractor' and term 'chiropractic manipulation', in relation to injury associated with cervical spine manipulation, have previously been reported in the peer-reviewed literature. The objectives of this study were to - 1) Prospectively monitor the peer-reviewed literature for papers reporting an association between chiropractic, or chiropractic manipulation, and injury; 2) Contact lead authors of papers that report such an association in order to determine the basis upon which the title 'chiropractor' and/or term 'chiropractic manipulation' was used; 3) Document the outcome of submission of letters to the editors of journals wherein the title 'chiropractor', and/or term 'chiropractic manipulation', had been misused and resulted in the over-reporting of chiropractic induced injury. METHODS: One electronic database (PubMed) was monitored prospectively, via monthly PubMed searches, during a 12 month period (June 2003 to May 2004). Once relevant papers were located, they were reviewed. If the qualifications and/or profession of the care provider/s were not apparent, an attempt was made to confirm them via direct e-mail communication with the principal researcher of each respective paper. A letter was then sent to the editor of each involved journal. RESULTS: A total of twenty four different cases, spread across six separate publications, were located via the monthly PubMed searches. All twenty four cases took place in one of two European countries. The six publications consisted of four case reports, each containing one patient, one case series, involving twenty relevant cases, and a secondary report that pertained to one of the four case reports. In each of the six publications the authors suggest the care provider was a chiropractor and that each patient received chiropractic manipulation of the cervical spine prior to developing symptoms suggestive of traumatic injury. In two of the four case reports contact with the principal researcher revealed that the care provider was not a chiropractor, as defined by the World Federation of Chiropractic. The authors of the other two case reports did not respond to my communications. In the case series, which involved twenty relevant cases, the principal researcher conceded that the term chiropractor had been inappropriately used and that his case series did not relate to chiropractors who had undergone appropriate formal training. The author of the secondary report, a British Medical Journal editor, conceded that he had misused the title chiropractor. Letters to editors were accepted and published by all four journals to which they were sent. To date one of the four journals has published a correction. CONCLUSION: The results of this year-long prospective review suggests that the words 'chiropractor' and 'chiropractic manipulation' are often used inappropriately by European biomedical researchers when reporting apparent associations between cervical spine manipulation and symptoms suggestive of traumatic injury. Furthermore, in those cases reported here, the spurious use of terminology seems to have passed through the peer-review process without correction. Additionally, these findings provide further preliminary evidence, beyond that already provided by Terrett, that the inappropriate use of the title 'chiropractor' and term 'chiropractic manipulation' may be a significant source of over-reporting of the link between the care provided by chiropractors and injury. Finally, editors of peer-reviewed journals were amenable to publishing 'letters to editors', and to a lesser extent 'corrections', when authors had inappropriately used the title 'chiropractor' and/or term 'chiropractic manipulation'

Current understanding of the relationship between cervical manipulation and stroke: what does it mean for the chiropractic profession?

The understanding of the relationship between cervical manipulative therapy (CMT) and vertebral artery dissection and stroke (VADS) has evolved considerably over the years. In the beginning the relationship was seen as simple cause-effect, in which CMT was seen to cause VADS in certain susceptible individuals. This was perceived as extremely rare by chiropractic physicians, but as far more common by neurologists and others. Recent evidence has clarified the relationship considerably, and suggests that the relationship is not causal, but that patients with VADS often have initial symptoms which cause them to seek care from a chiropractic physician and have a stroke some time after, independent of the chiropractic visit