Increased α₁-adrenergic contraction in small mesenteric arteries from cavin-1-deficient mice.

<p>A) Original traces showing contraction to 60 mM KCl and to cumulative addition of cirazoline in one representative wild type (WT; black) and knock out (KO; grey) mouse. Concentration-response curves for the α₁-adrenergic agonist cirazoline (C, control conditions; D, +300 μM of the NOS-inhibitor L-NAME; E, in the presence of 20 μM ODQ; F, in the presence of 10 μM ryanodine; G, in the presence of 20 μM dynasore) in small mesenteric arteries shows increased α₁-adrenergic contractility in the absence of cavin-1. As a reference, contraction to cirazoline in WT and KO is replicated in grey in panel D, E, F and G. The statistical difference between WT-drug and KO-drug is indicated by the p-value in the graphs. Panel B shows integrated force in response to depolarization (60 mM KCl).</p

Cavin-1-reporter staining of systemic blood vessels.

<p>A) The aorta was dissected, fixed briefly in paraformaldehyde, and stained using x-gal. Bottom shows an intact aorta and top shows an opened aorta from the luminal side. B) Small mesenteric arteries with the superior mesenteric artery on the left. C) The vascular supply of the cerebral hemispheres. D) X-gal stained arteries and a vein in the prostate was paraffin embedded, sectioned, and counterstained using hematoxylin-eosin.</p

Elevated arginase1 expression and increased arginase inhibitor-induced relaxation of Cavin-1^−/− arteries.

<p>A and B) Western blotting for arginase 1 in small mesenteric arteries (SMA) A) and in aorta B) from wild type (WT) and cavin-1 knock out (KO) mice. C) SMA were mounted in a wire-myograph and pre-contraction with 40 mM KCl. Relaxation to increasing concentrations of NOHA, an arginase inhibitor, was then assessed.</p

Cavin-1 deficient mice exhibit reduced vasomotion.

<p>Panel A) shows original traces from two wild type (WT; black) and two knock out (KO; grey) arteries, mounted in a wire-myograph, after prolonged (1 h) incubation with cirazoline (0.1 μM). Note the absence of rhythmic force oscillations (vasomotion) in KO. Panel B) shows the fraction of vessels exhibiting vasomotion in WT and KO in the presence and absence of the gap junction blocker 18-α-glycyrrhetinic acid (18-α-GA). Panel C) shows expression of connexin 40 and 43 in the aorta and D) shows that 18-α-GA does not reduce the difference between WT and KO in α₁-adrenergic contraction.</p

Reduced expression of caveolae-associated proteins in cavin-1^−/− mice.

<p>A) 20 μg of protein from small mesenteric artery lysates was subjected to western blotting for cavins and caveolins. GAPDH or HSP90 were used as loading controls. B) Electron micrographs showing membrane sections from wild type (WT) and cavin-1-deficient (KO) small mesenteric arteries. Top panels show endothelial cells (EC) and bottom panels show smooth muscle cells (SMC). Middle panel in B shows quantification of caveolae per μm membrane in endothelial cells (EC) and in smooth muscle cells (SMC) from WT and KO animals, respectively. Right panel shows size distribution of caveolae in endothelium compared to smooth muscle in WT arteries.</p

Unchanged mean arterial blood pressure in cavin-1-deficient mice.

<p>Mean arterial blood pressure was measured in the carotid artery of anaesthetized mice in control conditions (A) and after infusion of the nitric oxide synthase inhibitor L-NAME (B). Panel C shows heart rate in control conditions.</p

DataSheet1_Transcription factor GATA6 promotes migration of human coronary artery smooth muscle cells in vitro.PDF

Vascular smooth muscle cell plasticity plays a pivotal role in the pathophysiology of vascular diseases. Despite compelling evidence demonstrating the importance of transcription factor GATA6 in vascular smooth muscle, the functional role of GATA6 remains poorly understood. The aim of this study was to elucidate the role of GATA6 on cell migration and to gain insight into GATA6-sensitive genes in smooth muscle. We found that overexpression of GATA6 promotes migration of human coronary artery smooth muscle cells in vitro, and that silencing of GATA6 in smooth muscle cells resulted in reduced cellular motility. Furthermore, a complete microarray screen of GATA6-sensitive gene transcription resulted in 739 upregulated and 248 downregulated genes. Pathways enrichment analysis showed involvement of transforming growth factor beta (TGF-β) signaling which was validated by measuring mRNA expression level of several members. Furthermore, master regulators prediction based on microarray data revealed several members of (mitogen activated protein kinase) MAPK pathway as a master regulators, reflecting involvement of MAPK pathway also. Our findings provide further insights into the functional role of GATA6 in vascular smooth muscle and suggest that targeting GATA6 may constitute as a new approach to inhibit vascular smooth muscle migration.</p

MiR-29 depletion by genetic deletion of Dicer in smooth muscle increases detrusor stiffness in Ca²⁺-free conditions.

<p>Real-time quantitative polymerase chain reaction (<i>n</i>=5-7) for miR-29b (A), miR-29c (B) and elastin (Eln, C) in control (Ctrl) and Dicer knockout (KO) mouse bladders. Electron micrographs from control (D) and Dicer KO (E) detrusor. SMC: smooth muscle cell; N: neural varicosity. White arrowheads point to elastin fibrils. Scale bars represent 1 µm. Quantitative morphometry of collagen fibril diameters (F) and basal membrane thickness (G). (H) Western blots for Eln, Gapdh and Cav1 in detrusors from control and smooth muscle-specific Dicer knockout (KO) mice. Summarized data on Eln expression in control and Dicer KO bladders (I, <i>n</i>=12). (J) Passive circumference-stress curves in Dicer KO and control bladder strips (<i>n</i>=12). (K) Passive circumference-stress relations in control and elastase-treated rat detrusor strips (n=4).</p

Repression of miR-29 after outlet obstruction is associated with increased levels of miR-29 target proteins.

<p>(A) Western blots for eight miR-29 targets in sham-operated control bladders and at 6 weeks of obstruction. Gapdh and Cav1 were used as loading controls (<i>n</i>=6 throughout). (B) Relative fold change at 6 weeks of obstruction (vs. sham) of miR-29 target messenger RNAs (mRNA; black circles) and proteins (white circles). The fold change of mRNA compared to the fold change of the protein was significantly different throughout, except for Eln and Mybl2. MRNA expression is from the microarrays (<i>n</i>=6−8), and protein expression is from the western blots in A. Protein expression was normalized first to Gapdh and then to the mean for the sham-operated controls. (C) Immunofluorescence staining for collagen IV (col4a1, green) in sham-operated control and 6-wk obstructed detrusor. Scale bars represent 100 µm. The fluorescent pear-shaped profile in the center of the 6 weeks specimen represents the outline of a muscle bundle. Individual cell profiles are visible within the bundles. Nuclei are stained blue and the outer bladder surface is facing upward in both micrographs. </p

Rat bladder outlet obstruction leads to SMAD and extracellular-signal-regulated kinase (ERK) phosphorylation and to increased transcript levels of established transforming growth factor-β (TGF-β) targets.

<p>Phosphorylation of SMAD proteins (A) SMAD2/3 and (B) SMAD1/5/8 was measured by western blotting using phosphorylation-sensitive antibodies at various times following outlet obstruction. Gapdh was used as loading and normalization control. Protein expression was normalized first to Gapdh and then to the mean for the sham-operated controls. (C) Transcript levels for three established TGF-β targets are shown at 6 weeks of obstruction. The fold changes were large so the log₂ expression is shown on the y-axis. Data is from the mRNA microarray described in further detail in the text. (D) Time-course of ERK1/2 activation. All samples: n=6-8. * <i>p</i> < 0.05 versus sham. *** <i>p</i> < 0.001 versus sham.</p