ABSTRACT
Background Vascular endothelial cell (EC) alignment in the direction of flow is an adaptive response that protects against aortic diseases, such as atherosclerosis. The Rho GTP ases are known to regulate this alignment. Herein, we analyze the effect of ARHGAP 18 on the regulation of EC alignment and examine the effect of ARHGAP 18 deficiency on the development of atherosclerosis in mice. Methods and Results We used in vitro analysis of ECs under flow conditions together with apolipoprotein E-/- Arhgap 18-/- double-mutant mice to study the function of ARHGAP 18 in a high-fat diet-induced model of atherosclerosis. Depletion of ARHGAP 18 inhibited the alignment of ECs in the direction of flow and promoted inflammatory phenotype, as evidenced by disrupted junctions and increased expression of nuclear factor-κB and intercellular adhesion molecule-1 and decreased endothelial nitric oxide synthase. Mice with double deletion in ARHGAP 18 and apolipoprotein E and fed a high-fat diet show early onset of atherosclerosis, with lesions developing in atheroprotective regions. Conclusions ARHGAP 18 is a protective gene that maintains EC alignments in the direction of flow. Deletion of ARHGAP 18 led to loss of EC ability to align and promoted atherosclerosis development.
Subject(s)
Aortic Diseases/genetics , Blood Flow Velocity/physiology , Endothelium, Vascular/metabolism , GTPase-Activating Proteins/genetics , Gene Expression Regulation , Plaque, Atherosclerotic/genetics , Animals , Aortic Diseases/metabolism , Aortic Diseases/pathology , Blotting, Western , Disease Models, Animal , Endothelium, Vascular/pathology , GTPase-Activating Proteins/biosynthesis , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Plaque, Atherosclerotic/metabolism , Plaque, Atherosclerotic/pathology , RNA/genetics , Signal TransductionABSTRACT
The formation of the vascular network requires a tightly controlled balance of pro-angiogenic and stabilizing signals. Perturbation of this balance can result in dysregulated blood vessel morphogenesis and drive pathologies including cancer. Here, we have identified a novel gene, ARHGAP18, as an endogenous negative regulator of angiogenesis, limiting pro-angiogenic signaling and promoting vascular stability. Loss of ARHGAP18 promotes EC hypersprouting during zebrafish and murine retinal vessel development and enhances tumor vascularization and growth. Endogenous ARHGAP18 acts specifically on RhoC and relocalizes to the angiogenic and destabilized EC junctions in a ROCK dependent manner, where it is important in reaffirming stable EC junctions and suppressing tip cell behavior, at least partially through regulation of tip cell genes, Dll4, Flk-1 and Flt-4. These findings highlight ARHGAP18 as a specific RhoGAP to fine tune vascular morphogenesis, limiting tip cell formation and promoting junctional integrity to stabilize the angiogenic architecture.
Subject(s)
GTPase-Activating Proteins/metabolism , Intercellular Junctions/metabolism , Melanoma, Experimental/blood supply , Neovascularization, Physiologic , rho GTP-Binding Proteins/metabolism , Animals , Cell Line, Tumor , Endothelial Cells/metabolism , GTPase-Activating Proteins/genetics , Human Umbilical Vein Endothelial Cells , Humans , Mice , Mice, Inbred C57BL , Retina/cytology , Retina/metabolism , Retina/pathology , Zebrafish/embryology , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
The regulation of the segment polarity gene wingless is essential for the correct patterning of the Drosophila ectoderm. We have previously shown that the asymmetric activation of wingless downstream of Hedghog-signaling depends on the T-box transcription factors, midline and H15. Hedgehog activates wingless anterior to the Hedgehog domain. midline/H15 are responsible in part for repressing wingless in cells posterior to the Hedgehog expressing cells. Here, we show that Midline binds the Groucho co-repressor directly via the engrailed homology-1 domain and requires an intact engrailed-homology-1 domain to repress wingless. In contrast, the regulation of Serrate, a second target of midline repression, is not dependent on the engrailed-homology-1 domain. Furthermore, we identify a midline responsive region of the wingless cis-regulatory region and show that Midline binds to sequences within this region. Mutating these sequences in transgenic reporter constructs results in ectopic reporter expression in the midline-expression domain, consistent with wingless being a direct target of Midline repression.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Repressor Proteins/metabolism , T-Box Domain Proteins/metabolism , Wnt1 Protein/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/genetics , Body Patterning/genetics , Body Patterning/physiology , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , DNA Primers/genetics , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental , Genes, Insect , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Jagged-1 Protein , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Protein Interaction Domains and Motifs , Repressor Proteins/chemistry , Repressor Proteins/genetics , Sequence Homology, Amino Acid , Serrate-Jagged Proteins , Signal Transduction , T-Box Domain Proteins/chemistry , T-Box Domain Proteins/genetics , Wnt1 Protein/chemistry , Wnt1 Protein/geneticsABSTRACT
Regional fates in the developing limbs of Drosophila melanogaster are controlled by selector gene transcription factors. Ventral fate in the fly leg is specified by the expression of the ligand Wingless. We present evidence that midline and H15, members of the Tbx20 class of T-box transcription factors, are key mediators of the Wingless signal in the formation of the ventral region of the fly leg. midline and H15 are restricted to identical ventral domains of expression through activation by Wingless and repression by the dorsal signal Decapentaplegic. midline and H15 function redundantly and cell autonomously in the formation of ventral-specific structures. Conversely, midline is sufficient to induce ventral fate. Finally, the induction of ectopic ventral fate by mid is compromised when Wingless signaling is attenuated, suggesting that Wingless acts both upstream and in parallel with midline/H15 to specify ventral fate. Based on these results, we propose that midline and H15 may be considered as the selector genes for ventral leg fate.
