Generation and Comparative Analysis of an Itga8-CreER T2 Mouse with Preferential Activity in Vascular Smooth Muscle Cells.

All current smooth muscle cell (SMC) Cre mice similarly recombine floxed alleles in vascular and visceral SMCs. Here, we present an Itga8-CreER T2 knock-in mouse and compare its activity with a Myh11-CreER T2 mouse. Both Cre drivers demonstrate equivalent recombination in vascular SMCs. However, Myh11-CreER T2 mice, but not Itga8-CreER T2 mice, display high activity in visceral SMC-containing tissues such as intestine, show early tamoxifen-independent activity, and produce high levels of CreERT2 protein. Whereas Myh11-CreER T2 -mediated knockout of serum response factor (Srf) causes a lethal intestinal phenotype precluding analysis of the vasculature, loss of Srf with Itga8-CreER T2 (Srf Itga8 ) yields viable mice with no evidence of intestinal pathology. Male and female Srf Itga8 mice exhibit vascular contractile incompetence, and angiotensin II causes elevated blood pressure in wild type, but not Srf Itga8 , male mice. These findings establish the Itga8-CreER T2 mouse as an alternative to existing SMC Cre mice for unfettered phenotyping of vascular SMCs following selective gene loss.

SENCR stabilizes vascular endothelial cell adherens junctions through interaction with CKAP4.

SENCR is a human-specific, vascular cell-enriched long-noncoding RNA (lncRNA) that regulates vascular smooth muscle cell and endothelial cell (EC) phenotypes. The underlying mechanisms of action of SENCR in these and other cell types is unknown. Here, levels of SENCR RNA are shown to be elevated in several differentiated human EC lineages subjected to laminar shear stress. Increases in SENCR RNA are also observed in the laminar shear stress region of the adult aorta of humanized SENCR-expressing mice, but not in disturbed shear stress regions. SENCR loss-of-function studies disclose perturbations in EC membrane integrity resulting in increased EC permeability. Biotinylated RNA pull-down and mass spectrometry establish an abundant SENCR-binding protein, cytoskeletal-associated protein 4 (CKAP4); this ribonucleoprotein complex was further confirmed in an RNA immunoprecipitation experiment using an antibody to CKAP4. Structure-function studies demonstrate a noncanonical RNA-binding domain in CKAP4 that binds SENCR Upon SENCR knockdown, increasing levels of CKAP4 protein are detected in the EC surface fraction. Furthermore, an interaction between CKAP4 and CDH5 is enhanced in SENCR-depleted EC. This heightened association appears to destabilize the CDH5/CTNND1 complex and augment CDH5 internalization, resulting in impaired adherens junctions. These findings support SENCR as a flow-responsive lncRNA that promotes EC adherens junction integrity through physical association with CKAP4, thereby stabilizing cell membrane-bound CDH5.

CRISPR-Cas9-Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin-Brief Report.

Objective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research.

Serum Response Factor Is Essential for Maintenance of Podocyte Structure and Function.

Podocytes contain an intricate actin cytoskeleton that is essential for the specialized function of this cell type in renal filtration. Serum response factor (SRF) is a master transcription factor for the actin cytoskeleton, but the in vivo expression and function of SRF in podocytes are unknown. We found that SRF protein colocalizes with podocyte markers in human and mouse kidneys. Compared with littermate controls, mice in which the Srf gene was conditionally inactivated with NPHS2-Cre exhibited early postnatal proteinuria, hypoalbuminemia, and azotemia. Histologic changes in the mutant mice included glomerular capillary dilation and mild glomerulosclerosis, with reduced expression of multiple canonical podocyte markers. We also noted tubular dilation, cell proliferation, and protein casts as well as reactive changes in mesangial cells and interstitial inflammation. Ultrastructure analysis disclosed foot process effacement with loss of slit diaphragms. To ascertain the importance of SRF cofactors in podocyte function, we disabled the myocardin-related transcription factor A and B genes. Although loss of either SRF cofactor alone had no observable effect in the kidney, deficiency of both recapitulated the Srf-null phenotype. These results establish a vital role for SRF and two SRF cofactors in the maintenance of podocyte structure and function.

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal-contractile coupling.

CRISPR-Cas9 genome editing of a single regulatory element nearly abolishes target gene expression in mice--brief report.

OBJECTIVE: To ascertain the importance of a single regulatory element in the control of Cnn1 expression using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) genome editing. APPROACH AND RESULTS: The CRISPR/Cas9 system was used to produce 3 of 18 founder mice carrying point mutations in an intronic CArG box of the smooth muscle cell-restricted Cnn1 gene. Each founder was bred for germline transmission of the mutant CArG box and littermate interbreeding to generate homozygous mutant (Cnn1(ΔCArG/ΔCArG)) mice. Quantitative reverse transcription polymerase chain reaction, Western blotting, and confocal immunofluorescence microscopy showed dramatic reductions in Cnn1 mRNA and CNN1 protein expression in Cnn1(ΔCArG/ΔCArG) mice with no change in other smooth muscle cell-restricted genes and little evidence of off-target edits elsewhere in the genome. In vivo chromatin immunoprecipitation assay revealed a sharp decrease in binding of serum response factor to the mutant CArG box. Loss of CNN1 expression was coincident with an increase in Ki-67 positive cells in the normal vessel wall. CONCLUSIONS: CRISPR/Cas9 genome editing of a single CArG box nearly abolishes Cnn1 expression in vivo and evokes increases in smooth muscle cell DNA synthesis. This facile genome editing system paves the way for a new generation of studies designed to test the importance of individual regulatory elements in living animals, including regulatory variants in conserved sequence blocks linked to human disease.

G-protein-coupled receptor-2-interacting protein-1 is required for endothelial cell directional migration and tumor angiogenesis via cortactin-dependent lamellipodia formation.

OBJECTIVE: Recent evidence suggests G-protein-coupled receptor-2-interacting protein-1 (GIT1) overexpression in several human metastatic tumors, including breast, lung, and prostate. Tumor metastasis is associated with an increase in angiogenesis. We have showed previously that GIT1 is required for postnatal angiogenesis during lung development. However, the functional role of GIT1 in pathological angiogenesis during tumor growth is unknown. APPROACH AND RESULTS: In the present study, we show inhibition of angiogenesis in matrigel implants as well as reduced tumor angiogenesis and melanoma tumor growth in GIT1-knockout mice. We demonstrate that this is a result of impaired directional migration of GIT1-depleted endothelial cells toward a vascular endothelial growth factor gradient. Cortactin-mediated lamellipodia formation in the leading edge is critical for directional migration. We observed a significant reduction in cortactin localization and lamellipodia formation in the leading edge of GIT1-depleted endothelial cells. We specifically identified that the Spa homology domain (aa 250-420) of GIT1 is required for GIT1-cortactin complex localization to the leading edge. The mechanisms involved extracellular signal-regulated kinases 1 and 2-mediated Cortactin-S405 phosphorylation and activation of Rac1/Cdc42. Finally, using gain of function studies, we show that a constitutively active mutant of cortactin restored directional migration of GIT1-depleted cells. CONCLUSION: Our data demonstrated that a GIT1-cortactin association through GIT1-Spa homology domain is required for cortactin localization to the leading edge and is essential for endothelial cell directional migration and tumor angiogenesis.