Systematic variation of RhoA activity reveals an inhibitory impact of active RhoA on the homeostasis and angiogenic capacity of human endothelial cells
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ABSTRACT:
PROVIDER: PRJNA757958 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15647570_1.fastq.gz | Fastqsanger.gz | |||
| SRR15647570_2.fastq.gz | Fastqsanger.gz | |||
| SRR15647571_1.fastq.gz | Fastqsanger.gz | |||
| SRR15647571_2.fastq.gz | Fastqsanger.gz | |||
| SRR15647572_1.fastq.gz | Fastqsanger.gz |
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Project description:The small GTPase RhoA regulates a variety of cellular processes, including cell motility, proliferation, survival and permeability. In addition, there are reports suggesting that the RhoA-ROCK axis plays a role in VEGF-mediated angiogenesis, whereas other work has shown opposite effects. To elucidate this conflicting data, we examined HUVEC and HCAEC after stable overexpression (lentiviral transduction) of constitutively active (G14V/Q63L), dominant-negative (T19N), or wild-type RhoA using a variety of in vitro angiogenesis assays (proliferation, migration, tube formation, angiogenic sprouting, endothelial cell viability) and a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. We observed that expression of active as well as wild-type RhoA but not expression of dominant-negative RhoA significantly increased endothelial cell death as well as inhibited endothelial cell proliferation, migration, tube formation and angiogenic sprouting of endothelial cells in vitro and reduced HUVEC-related angiogenesis in vivo. Inhibition of RhoA by C3 transferase antagonized inhibitory RhoA effects and strongly enhanced VEGF-induced angiogenic sprouting in control-treated cells. In contrast, inhibition of RhoA effectors ROCK1/2 and LIMK1/2 had no significant effect on RhoA-related effects, but again increased angiogenic sprouting and migration of control-treated cells. In line with these data, VEGF did not activate RhoA in HUVEC as measured by a FRET-based biosensor. Furthermore, global transcriptome and subsequent bioinformatic gene ontology (GO) enrichment analyses revealed that constitutively active RhoA induces a differentially expressed gene pattern that is enriched for GO biological process terms such as mitotic nuclear division, cell proliferation, cell motility and cell adhesion and includes a significant decrease in VEGFR-2 and NOS3 expression. Thus, our data demonstrate that increased RhoA activity has the potential to trigger endothelial dysfunction and anti-angiogenic effects independently of its well-characterized downstream effectors ROCK and LIMK.
2022-07-06 | GSE182806 | GEO
Project description:NCoR1 limits angiogenic capacity by altering Notch signaling
| PRJNA996109 | ENA
Project description:NCoR1 limits angiogenic capacity by altering Notch signaling [RNA]
| PRJNA996110 | ENA
Project description:NCoR1 limits angiogenic capacity by altering Notch signaling [ATAC-seq]
| PRJNA1014001 | ENA
Project description:Background The small GTPase RhoA (Ras homolog gene family, member A) regulates a variety of cellular processes, including cell motility, proliferation, survival, and permeability. In addition, there are reports indicating that RhoA-ROCK (rho associated coiled-coil containing protein kinase) activation is essential for VEGF (vascular endothelial growth factor)-mediated angiogenesis, whereas other work suggests VEGF-antagonistic effects of the RhoA-ROCK axis. Methods and Results To elucidate this issue, we examined human umbilical vein endothelial cells and human coronary artery endothelial cells after stable overexpression (lentiviral transduction) of constitutively active (G14V/Q63L), dominant-negative (T19N), or wild-type RhoA using a series of in vitro angiogenesis assays (proliferation, migration, tube formation, angiogenic sprouting, endothelial cell viability) and a human umbilical vein endothelial cells xenograft assay in immune-incompetent NOD scid gamma mice in vivo. Here, we report that expression of active and wild-type RhoA but not dominant-negative RhoA significantly inhibited endothelial cell proliferation, migration, tube formation, and angiogenic sprouting in vitro. Moreover, active RhoA increased endothelial cell death in vitro and decreased human umbilical vein endothelial cell-related angiogenesis in vivo. Inhibition of RhoA by C3 transferase antagonized the inhibitory effects of RhoA and strongly enhanced VEGF-induced angiogenic sprouting in control-treated cells. In contrast, inhibition of RhoA effectors ROCK1/2 and LIMK1/2 (LIM domain kinase 1/2) did not significantly affect RhoA-related effects, but increased angiogenic sprouting and migration of control-treated cells. In agreement with these data, VEGF did not activate RhoA in human umbilical vein endothelial cells as measured by a Förster resonance energy transfer-based biosensor. Furthermore, global transcriptome and subsequent bioinformatic gene ontology enrichment analyses revealed that constitutively active RhoA induced a differentially expressed gene pattern that was enriched for gene ontology biological process terms associated with mitotic nuclear division, cell proliferation, cell motility, and cell adhesion, which included a significant decrease in VEGFR-2 (vascular endothelial growth factor receptor 2) and NOS3 (nitric oxide synthase 3) expression. Conclusions Our data demonstrate that increased RhoA activity has the potential to trigger endothelial dysfunction and antiangiogenic effects independently of its well-characterized downstream effectors ROCK and LIMK.
| S-EPMC9238636 | biostudies-literature
Project description:Corepressors negatively regulate gene expression by chromatin compaction. Targeted regulation of gene expression could provide a means to control endothelial cell phenotype. We hypothesize that by targeting corepressor proteins, endothelial angiogenic function can be improved. To study this, the expression and function of nuclear corepressors in human umbilical vein endothelial cells (HUVEC) and in murine organ culture was studied. RNA-seq revealed that nuclear receptor corepressor 1 (NCoR1), Silencing Mediator of Retinoid and Thyroid hormone receptors (SMRT) and repressor element-1 silencing transcription factor (REST) are the highest expressed corepressors in HUVECs. Knockout and knockdown strategies demonstrated that the depletion of NCoR1 increased the angiogenic capacity of endothelial cells, whereas depletion of SMRT or REST did not. Interestingly, the effect was VEGF signaling independent. NCoR1 depletion significantly upregulated angiogenesis-associated genes, especially tip cell genes, including ESM1, DLL4 and NOTCH4, as observed by RNA- and ATAC-seq. Confrontation assays comparing cells with and without NCoR1-deficiency revealed that loss of NCoR1 promotes a tip-cell position during spheroid sprouting. Moreover, a proximity ligation assay identified NCoR1 as a direct binding partner of the Notch-signaling-related transcription factor RBPJk. Luciferase assays showed that siRNA-mediated knockdown of NCoR1 promotes RBPJk activity. Furthermore, NCoR1 downregulation prompts upregulation of several elements in the Notch signaling cascade. Downregulation of NOTCH4, but not NOTCH1, prevented the positive effect of NCoR1 knockdown on spheroid outgrowth. Collectively, these data indicate that decreasing NCOR1 expression is an attractive approach to promote angiogenic function. This SuperSeries is composed of the SubSeries listed below.
2024-02-12 | GSE237654 | GEO
Project description:Corepressors negatively regulate gene expression by chromatin compaction. Targeted regulation of gene expression could provide a means to control endothelial cell phenotype. We hypothesize that by targeting corepressor proteins, endothelial angiogenic function can be improved. To study this, the expression and function of nuclear corepressors in human umbilical vein endothelial cells (HUVEC) and in murine organ culture was studied. RNA-seq revealed that nuclear receptor corepressor 1 (NCoR1), Silencing Mediator of Retinoid and Thyroid hormone receptors (SMRT) and repressor element-1 silencing transcription factor (REST) are the highest expressed corepressors in HUVECs. Knockout and knockdown strategies demonstrated that the depletion of NCoR1 increased the angiogenic capacity of endothelial cells, whereas depletion of SMRT or REST did not. Interestingly, the effect was VEGF signaling independent. NCoR1 depletion significantly upregulated angiogenesis-associated genes, especially tip cell genes, including ESM1, DLL4 and NOTCH4, as observed by RNA- and ATAC-seq. Confrontation assays comparing cells with and without NCoR1-deficiency revealed that loss of NCoR1 promotes a tip-cell position during spheroid sprouting. Moreover, a proximity ligation assay identified NCoR1 as a direct binding partner of the Notch-signaling-related transcription factor RBPJk. Luciferase assays showed that siRNA-mediated knockdown of NCoR1 promotes RBPJk activity. Furthermore, NCoR1 downregulation prompts upregulation of several elements in the Notch signaling cascade. Downregulation of NOTCH4, but not NOTCH1, prevented the positive effect of NCoR1 knockdown on spheroid outgrowth. Collectively, these data indicate that decreasing NCOR1 expression is an attractive approach to promote angiogenic function.
2024-02-12 | GSE237653 | GEO
Project description:Corepressors negatively regulate gene expression by chromatin compaction. Targeted regulation of gene expression could provide a means to control endothelial cell phenotype. We hypothesize that by targeting corepressor proteins, endothelial angiogenic function can be improved. To study this, the expression and function of nuclear corepressors in human umbilical vein endothelial cells (HUVEC) and in murine organ culture was studied. RNA-seq revealed that nuclear receptor corepressor 1 (NCoR1), Silencing Mediator of Retinoid and Thyroid hormone receptors (SMRT) and repressor element-1 silencing transcription factor (REST) are the highest expressed corepressors in HUVECs. Knockout and knockdown strategies demonstrated that the depletion of NCoR1 increased the angiogenic capacity of endothelial cells, whereas depletion of SMRT or REST did not. Interestingly, the effect was VEGF signaling independent. NCoR1 depletion significantly upregulated angiogenesis-associated genes, especially tip cell genes, including ESM1, DLL4 and NOTCH4, as observed by RNA- and ATAC-seq. Confrontation assays comparing cells with and without NCoR1-deficiency revealed that loss of NCoR1 promotes a tip-cell position during spheroid sprouting. Moreover, a proximity ligation assay identified NCoR1 as a direct binding partner of the Notch-signaling-related transcription factor RBPJk. Luciferase assays showed that siRNA-mediated knockdown of NCoR1 promotes RBPJk activity. Furthermore, NCoR1 downregulation prompts upregulation of several elements in the Notch signaling cascade. Downregulation of NOTCH4, but not NOTCH1, prevented the positive effect of NCoR1 knockdown on spheroid outgrowth. Collectively, these data indicate that decreasing NCOR1 expression is an attractive approach to promote angiogenic function.
2024-02-12 | GSE242671 | GEO
Project description:Intercellular transfer of miR-200c-3p impairs the angiogenic capacity of cardiac endothelial cells
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Project description:Dynamic interactions between RhoA and Rac1, members of the Rho small GTPase family, play a vital role in the control of cell migration. Using predictive mathematical modelling and experimental validation in MDA-MB-231 mesenchymal breast cancer cells, we show that Rac1 and RhoA interactions via the PAK-family kinases can produce bistable, switch-like responses to a graded PAK inhibition. Using a small chemical inhibitor of PAK we confirm the model conjecture demonstrating that cellular RhoA and Rac activation levels respond in a bistable manner to PAK inhibition where for a given inhibition level these levels are high or low depending on the history of the system. Consequently, we show that downstream signalling, actin dynamics and cell migration also behave in a bistable fashion, displaying abrupt switches and hysteresis in response to PAK inhibition. In summary, our results demonstrate that PAK is a critical component in the Rac1-RhoA inhibitory crosstalk that mediates bistable GTPase activity and cell migration switches.
2017-03-29 | MSV000080751 | MassIVE