Gene expression analysis of embryonic stem cells expressing VE-cadherin (CD144) during endothelial differentiation

Background: Endothelial differentiation occurs during normal vascular development in the developing embryo. This process is recapitulated in the adult when endothelial progenitor cells are generated in the bone marrow and can contribute to vascular repair or angiogenesis at sites of vascular injury or ischemia. The molecular mechanisms of endothelial differentiation remain incompletely understood. Novel approaches are needed to identify the factors that regulate endothelial differentiation. Methods: Mouse embryonic stem (ES) cells were used to further define the molecular mechanisms of endothelial differentiation. By flow cytometry a population of VEGF-R2 positive cells was identified as early as 2.5 days after differentiation of ES cells, and a subset of VEGF-R2+ cells, that were CD41 positive at 3.5 days. A separate population of VEGF-R2+ stem cells expressing the endothelial-specific marker CD144 (VE-cadherin) was also identified at this same time point. Channels lined by VE-cadherin positive cells developed within the embryoid bodies (EBs) formed by differentiating ES cells. VE-cadherin and CD41 expressing cells differentiate in close proximity to each other within the EBs, supporting the concept of a common origin for cells of hematopoietic and endothelial lineages. Results: Microarray analysis of \u3e45,000 transcripts was performed on RNA obtained from cells expressing VEGF-R2+, CD41+, and CD144+ and VEGF-R2-, CD41-, and CD144-. All microarray experiments were performed in duplicate using RNA obtained from independent experiments, for each subset of cells. Expression profiling confirmed the role of several genes involved in hematopoiesis, and identified several putative genes involved in endothelial differentiation. Conclusion: The isolation of CD144+ cells during ES cell differentiation from embryoid bodies provides an excellent model system and method for identifying genes that are expressed during endothelial differentiation and that are distinct from hematopoiesis

ERG is required for the differentiation of embryonic stem cells along the endothelial lineage

<p>Abstract</p> <p>Background</p> <p>The molecular mechanisms that govern stem cell differentiation along the endothelial lineage remain largely unknown. Ets related gene (ERG) has recently been shown to participate in the transcriptional regulation of a number of endothelial specific genes including VE-cadherin (CD144), endoglin, and von Willebrand's Factor (vWF). The specific role of the ETS factor ERG during endothelial differentiation has not been evaluated.</p> <p>Results</p> <p>ERG expression and function were evaluated during the differentiation of embryonic stem cells into embryoid bodies (EB). The results of our study demonstrate that ERG is first expressed in a subpopulation of vascular endothelial growth factor receptor 2 (VEGF-R2) expressing cells that also express VE-cadherin. During ES cell differentiation, ERG expression remains restricted to cells of the endothelial lineage that eventually coalesce into primitive vascular structures within embryoid bodies. ERG also exhibits an endothelial cell (EC)-restricted pattern during embryogenesis. To further define the role of ERG during ES cell differentiation, we used a knockdown strategy to inhibit ERG expression. Delivery of three independent shRNA led to 70-85% reductions in ERG expression during ES cell differentiation compared to no change with control shRNA. ERG knockdown was associated with a marked reduction in the number of ECs, the expression of EC-restricted genes, and the formation of vascular structures.</p> <p>Conclusion</p> <p>The ETS factor ERG appears to be a critical regulator of EC differentiation.</p

Gene expression analysis of embryonic stem cells expressing VE-cadherin (CD144) during endothelial differentiation

<p>Abstract</p> <p>Background</p> <p>Endothelial differentiation occurs during normal vascular development in the developing embryo. This process is recapitulated in the adult when endothelial progenitor cells are generated in the bone marrow and can contribute to vascular repair or angiogenesis at sites of vascular injury or ischemia. The molecular mechanisms of endothelial differentiation remain incompletely understood. Novel approaches are needed to identify the factors that regulate endothelial differentiation.</p> <p>Methods</p> <p>Mouse embryonic stem (ES) cells were used to further define the molecular mechanisms of endothelial differentiation. By flow cytometry a population of VEGF-R2 positive cells was identified as early as 2.5 days after differentiation of ES cells, and a subset of VEGF-R2⁺cells, that were CD41 positive at 3.5 days. A separate population of VEGF-R2⁺stem cells expressing the endothelial-specific marker CD144 (VE-cadherin) was also identified at this same time point. Channels lined by VE-cadherin positive cells developed within the embryoid bodies (EBs) formed by differentiating ES cells. VE-cadherin and CD41 expressing cells differentiate in close proximity to each other within the EBs, supporting the concept of a common origin for cells of hematopoietic and endothelial lineages.</p> <p>Results</p> <p>Microarray analysis of >45,000 transcripts was performed on RNA obtained from cells expressing VEGF-R2⁺, CD41⁺, and CD144⁺and VEGF-R2^-, CD41^-, and CD144^-. All microarray experiments were performed in duplicate using RNA obtained from independent experiments, for each subset of cells. Expression profiling confirmed the role of several genes involved in hematopoiesis, and identified several putative genes involved in endothelial differentiation.</p> <p>Conclusion</p> <p>The isolation of CD144⁺cells during ES cell differentiation from embryoid bodies provides an excellent model system and method for identifying genes that are expressed during endothelial differentiation and that are distinct from hematopoiesis.</p

Gene expression analysis of embryonic stem cells expressing VE-cadherin (CD144) during endothelial differentiation

Background: Endothelial differentiation occurs during normal vascular development in the developing embryo. This process is recapitulated in the adult when endothelial progenitor cells are generated in the bone marrow and can contribute to vascular repair or angiogenesis at sites of vascular injury or ischemia. The molecular mechanisms of endothelial differentiation remain incompletely understood. Novel approaches are needed to identify the factors that regulate endothelial differentiation. Methods: Mouse embryonic stem (ES) cells were used to further define the molecular mechanisms of endothelial differentiation. By flow cytometry a population of VEGF-R2 positive cells was identified as early as 2.5 days after differentiation of ES cells, and a subset of VEGF-R2+ cells, that were CD41 positive at 3.5 days. A separate population of VEGF-R2+ stem cells expressing the endothelial-specific marker CD144 (VE-cadherin) was also identified at this same time point. Channels lined by VE-cadherin positive cells developed within the embryoid bodies (EBs) formed by differentiating ES cells. VE-cadherin and CD41 expressing cells differentiate in close proximity to each other within the EBs, supporting the concept of a common origin for cells of hematopoietic and endothelial lineages. Results: Microarray analysis of \u3e45,000 transcripts was performed on RNA obtained from cells expressing VEGF-R2+, CD41+, and CD144+ and VEGF-R2-, CD41-, and CD144-. All microarray experiments were performed in duplicate using RNA obtained from independent experiments, for each subset of cells. Expression profiling confirmed the role of several genes involved in hematopoiesis, and identified several putative genes involved in endothelial differentiation. Conclusion: The isolation of CD144+ cells during ES cell differentiation from embryoid bodies provides an excellent model system and method for identifying genes that are expressed during endothelial differentiation and that are distinct from hematopoiesis

Nesprin-1 and actin contribute to nuclear and cytoskeletal defects in lamin A/C-deficient cardiomyopathy

Lamin A/C mutations are the most common cause of familial dilated cardiomyopathy (DCM) but the pathogenetic mechanisms are incompletely understood. Nesprins are spectrin repeat-containing proteins that interact with lamin A/C and are components of the linker-of-nucleoskeleton-and-cytoskeleton (LINC) complex that connects the nuclear envelope to the actin cytoskeleton. Our aim was to determine whether changes in nesprin-1 and actin might contribute to DCM in homozygous Lmna knockout (Lmna-/-) mice. Here we find that Lmna-/- cardiomyocytes have altered nuclear envelope morphology, disorganization of nesprin-1 and heterogeneity in the distribution of nuclear and cytoskeletal actin. Functional interactions of nesprin-1 with nuclear G-actin and with the cytoskeletal γ-actin, α-cardiac actin and α-smooth muscle actin (α-SMA) isoforms were shown by immunoprecipitation and Western blotting. At 4-6weeks of age, Lmna-/- mice had normal levels of γ-actin and α-cardiac actin, but α-SMA expression was increased by 50%. In contrast to the predominant vascular distribution of α-SMA in WT ventricular sections, α-SMA had a diffuse staining pattern in Lmna-/- sections. Osmotic swelling studies showed enhanced radial swelling in Lmna-/- cardiomyocytes indicative of cytoskeletal instability. The distensibility of Lmna-/- cardiomyocytes with osmotic stress was reduced by addition of α-SMA-specific fusion peptide. Our findings support a model in which uncoupling of the nucleus and cytoskeleton associated with disruption of the LINC complex promotes mechanical instability and defective force transmission in cardiomyocytes. Changes in the distribution and expression patterns of nuclear and cytoskeletal actin suggest that diverse transcriptional and structural defects may also contribute to DCM in Lmna-/- mice

Irreversible triggers for hypertrophic cardiomyopathy are established in the early postnatal period

Background: Hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere protein genes, and left ventricular hypertrophy (LVH) develops as an adaptive response to sarcomere dysfunction. It remains unclear whether persistent expression of the mutant gene is required for LVH or whether early gene expression acts as an immutable inductive trigger. Objectives: The aim of this study was to use a regulatable murine model of HCM to study the reversibility of pathological LVH. Methods: The authors generated a double-transgenic mouse model, tTA x αMHC R403Q , in which expression of the HCM-causing Arg403Gln mutation in the α-myosin heavy chain (MHC) gene is inhibited by doxycycline administration. Cardiac structure and function were evaluated in groups of mice that received doxycycline for varying periods from 0 to 40 weeks of age. Results: Untreated tTA x αMHC R403Q mice showed increased left ventricular (LV) mass, contractile dysfunction, myofibrillar disarray, and fibrosis. In contrast, mice treated with doxycycline from conception to 6 weeks had markedly less LVH and fibrosis at 40 weeks. Transgene inhibition from 6 weeks reduced fibrosis but did not prevent LVH or functional changes. There were no differences in LV parameters at 40 weeks between mice with transgene inhibition from 20 weeks and mice with continuous transgene expression. Conclusions: These findings highlight the critical role of the early postnatal period in HCM pathogenesis and suggest that mutant sarcomeres manifest irreversible cardiomyocyte defects that induce LVH. In HCM, mutation-silencing therapies are likely to be ineffective for hypertrophy regression and would have to be administered very early in life to prevent hypertrophy development