Striated Myogenesis and Peristalsis in the Fetal Murine Esophagus Occur without Cell Migration or Interstitial Cells of Cajal

Esophageal striated myogenesis progresses differently from appendicular myogenesis, but the mechanism underlying this process is incompletely understood. Early theories of transdifferentiation of smooth muscle into striated muscle are not supported by transgenic fate-mapping experiments; however, the origin of esophageal striated muscle remains unknown. To better define the process of striated myogenesis, we examined myogenesis in murine fetal cultured esophageal whole-organ explants. Embryonic day 14.5 (E14.5) esophagi maintained a functional contractile phenotype for up to 7 days in culture. Striated myogenesis, as evidenced by myogenin expression, proceeded in a craniocaudal direction along the length of the esophagus. Esophageal length did not change during this process. Complete, but not partial, mechanical disruption of the rostral esophagus inhibited myogenesis distally. Addition of fibroblast growth factor-2 (FGF-2) to the culture media failed to inhibit striated myogenesis, but attenuated smooth muscle actin expression and reduced peristaltic activity. Inhibition of c-kit failed to inhibit peristalsis. These results suggest that striated myogenic precursors are resident along the entire length of the esophagus by day 14.5 and do not migrate along the esophagus after E14.5. Induction of myogenesis craniocaudally appears to require physical continuity of the esophagus and is not inhibited by FGF-2. Finally, peristalsis in E14.5 esophagi appears not to be regulated by interstitial cells of Cajal

Smooth muscle Notch1 mediates neointimal formation after vascular injury

Notch1 regulates binary cell fate determination and is critical for angiogenesis and cardiovascular development. However, the pathophysiological role of Notch1 in the postnatal period is not known. We hypothesize that Notch1 signaling in vascular smooth muscle cells (SMCs) may contribute to neointimal formation after vascular injury. METHODS AND RESULTS: We performed carotid artery ligation in wild-type, control (SMC-specific Cre recombinase transgenic [smCre-Tg]), general Notch1 heterozygous deficient (N1+/-), SMC-specific Notch1 heterozygous deficient (smN1+/-), and general Notch3 homozygous deficient (N3-/-) mice. Compared with wild-type or control mice, N1+/- and smN1+/- mice showed a 70% decrease in neointimal formation after carotid artery ligation. However, neointimal formation was similar between wild-type and N3-/- mice. Indeed, SMCs derived from explanted aortas of either N1(+/-)- or smN1+/- mice showed decreased chemotaxis and proliferation and increased apoptosis compared with control or N3-/- mice. This correlated with decreased staining of proliferating cell nuclear antigen-positive cells and increased staining of cleaved caspase-3 in the intima of N1(+/-)- or smN1+/- mice. In SMCs derived from CHF1/Hey2-/- mice, activation of Notch signaling did not lead to increased SMC proliferation or migration. CONCLUSIONS: These findings indicate that Notch1, rather than Notch3, mediates SMC proliferation and neointimal formation after vascular injury through CHF1/Hey2 and suggest that therapies that target Notch1/CHF1/Hey2 in SMCs may be beneficial in preventing vascular proliferative diseases

Regulation of Bronchomotor Tone in Conscious Calves

peer reviewedaudience: researcher, professional, studentThe purpose of this study was to investigate the effects of some alpha and beta sympathomimetic and sympatholytic drugs on respiratory impedance in healthy conscious calves. Ten Friesian calves were investigated in this study. The forced oscillation technique was used to measure the resistance (Rrs) and the reactance (Xrs) of the respiratory system at frequencies ranging from 4 to 26 Hz. Isoprenaline (1 microgram/kg i.v.), propranolol (3 micrograms/kg i.v.), noradrenaline (2 micrograms/kg i.v.), xylazine (20 micrograms/kg i.v.) and yohimbine (0.25 mg/kg i.v.) were were administered. Isoprenaline induced a significant decrease of Rrs. An increase of Rrs after administration of propranolol was observed but without any change of the frequency dependence of Rrs. A small increase in the resonant frequency was also recorded. A decrease of Rrs was recorded after yohimbine injection. Noradrenaline and xylazine administration increased the resistances and the resonant frequency and induced a negative frequency dependence of Rrs. These results suggest that (1) the major effects of beta adrenergic drugs are on the central airways, (2) the alpha adrenergic system may play a role on the regulation of bronchomotor tone in calves, (3) the effects of alpha adrenergic drugs are on both central and peripheral airways and (4) the forced oscillation technique allows the differentiation of calibre changes occurring in small and large airways

Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle.

Current approaches to monitor and quantify cell division in live cells, and reliably distinguish between acytokinesis and endoreduplication, are limited and complicate determination of stem cell pool identities. Here we overcome these limitations by generating an in vivo reporter system using the scaffolding protein anillin fused to enhanced green fluorescent protein, to provide high spatiotemporal resolution of mitotic phase. This approach visualizes cytokinesis and midbody formation as hallmarks of expansion of stem and somatic cells, and enables distinction from cell cycle variations. High-resolution microscopy in embryonic heart and brain tissues of enhanced green fluorescent protein-anillin transgenic mice allows live monitoring of cell division and quantitation of cell cycle kinetics. Analysis of cell division in hearts post injury shows that border zone cardiomyocytes in the infarct respond with increasing ploidy, but not cell division. Thus, the enhanced green fluorescent protein-anillin system enables monitoring and measurement of cell division in vivo and markedly simplifies in vitro analysis in fixed cells

Investigation of the effects of histamine inhalation on the tracheobronchial tree of calves by the forced oscillation technique.

Effects of histamine inhalation were investigated with two different techniques in nine conscious, healthy calves. The oesophageal balloon technique was used to measure the dynamic respiratory compliance (Cdyn) and the pulmonary resistance (RL). The reactance (Xrs) and the resistance (Rrs) of the respiratory system were measured at high frequencies by the forced oscillation technique. These parameters were recorded before and after histamine inhalation. Three histamine dihydrochloride solution concentrations were used (16 mg/ml; 32 mg/ml; 64 mg/ml). Histamine inhalation induced a decrease in Cdyn and Xrs an increase in Rrs, RL and of the resonant frequency and a negative frequency dependence of Rrs. Some of these changes were satisfactorily correlated with the histamine solution concentrations. The resonant frequency was well correlated with Cdyn. It was concluded that histamine inhalation induces a decrease of the calibre of small and large airways and a non-homogeneous behaviour of the pulmonary ventilation in awake calves. The forced oscillation technique can be used to perform an inhalation provocation test in unsedated animals

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca2+ release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca2+ channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca2+ entry through VGCCs triggers RyR-mediated Ca2+ release via a Ca2+-induced Ca2+ release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca2+ release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP3Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca2+]i. The initial abrupt sub-PM [Ca2+]i upstroke was all but abolished by block of VGCCs (by 5 μM nicardipine), depletion of intracellular Ca2+ stores (with 10 μM cyclopiazonic acid) or inhibition of IP3Rs (by 2 μM xestospongin C or 30 μM 2-APB), but was not affected by block of RyRs (by 50–100 μM tetracaine or 100 μM ryanodine). Inhibition of either IP3Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation–contraction coupling in this phasic visceral smooth muscle occurs by Ca2+ entry through VGCCs which evokes an initial IP3R-mediated Ca2+ release activated via a CICR mechanism