Cushion Tissue Research Articles

Loss of myocardial Hey2/Hrt2 function disrupts rightward shift of atrioventricular cushion tissue and causes tricuspid atresia.

Endocardial cushion tissue is primordia of the valves and septa of the adult heart, and its malformation causes various congenital heart diseases (CHDs). Tricuspid atresia (TA) is defined as congenital absence or agenesis of the tricuspid valve caused by endocardial cushion defects. However, little is known about what type of endocardial cushion defect causes TA. Using three-dimensional volume rendering image analysis, we demonstrated morphological changes of endocardial cushion tissue in developing Hey2/Hrt2 KO mouse embryos that showed malformation of the tricuspid valve, which resembled human TA at neonatal period. In control embryos, atrioventricular (AV) endocardial cushion tissues showed rightward shift to form a tricuspid valve. However, the rightward shift of endocardial cushion tissue was disrupted in Hey2/Hrt2 KO embryos, leading to the misalignment of AV cushions. We also found that muscular tissue filled up the space between the right atrium and ventricle, resulting in the absence of the tricuspid valve. Moreover, analysis using tissue-specific conditional KO mice showed that HEY2/HRT2-expressing myocardium may physically regulate the AV shift. Disruption of rightward cushion movement is an initial cue of TA phenotype, and myocardial HEY2/HRT2 is necessary for the regulation of proper alignment of AV endocardial cushion tissue.

Morphology, adipocyte size, and fatty acid analysis of dairy cattle digital cushions, and the effect of body condition score and age

The digital cushion is an essential part of maintaining a healthy foot, working to dissipate foot strike and body weight forces and lameness from claw horn disruption lesions. Despite the importance of the digital cushion, little is known about the basic anatomy, adipocyte morphology, and fatty acid composition in relation to age, limb position, and body condition score. In total, 60 claws (from 17 cows) were selected and collected from a herd, ensuring that body condition score data and computed micro-tomography were known for each animal. Digital cushion tissue underwent histological staining combined with stereology, systematic random sampling, and cell morphology analysis, in addition to lipid extraction followed by fatty acid analysis. The results describe digital cushion architecture and adipocyte sizes. Adipocyte size was similar across all 4 claws (distal left lateral and medial and distal right lateral and medial) and across the ages (aged 2-7 yr); however, animals with body condition score of 3.00 or more at slaughter had a significantly increased cell size in comparison to those with a score of less than 2.50. Of 37 fatty acid methyl esters identified, 5 differed between either the body condition score or different age groups. C10:0 capric acid, C14:0 myristic acid, C15:0 pentadecanoic acid, and C20:0 arachidic acid percentages were all lesser in lower body condition score cows, whereas C22:1n-9 erucic acid measurements were lesser in younger cows. Saturated fatty acid, monounsaturated fatty acid, and polyunsaturated fatty acid percentages were not altered in the different claws, ages, or body condition score groups. Triglyceride quantities did not differ for claw position or age but had decreased quantities in lower body condition score animals. Digital cushion anatomy, cellular morphology, and fatty acid composition have been described in general and also in animals with differing ages, body condition scores, and in the differing claws. Understanding fat deposition, mobilization, and composition are essential in not only understanding the roles that the digital cushion plays but also in preventing disorders and maintaining cattle health and welfare.

Msx1 upregulates p27 expression to control cellular proliferation during valvuloseptal endocardial cushion formation in the chick embryonic heart.

Cushion tissues, the primordia of valves and septa of the adult heart, are formed in the atrioventricular (AV) and outflow tract (OFT) regions of the embryonic heart. The cushion tissues are generated by the endothelial-mesenchymal transition (EMT), involving many soluble factors, extracellular matrix, and transcription factors. Moreover, neural crest-derived mesenchymal cells also migrate into the OFT cushion. The transcription factor Msx1 is known to be expressed in the endothelial and mesenchymal cells during cushion tissue formation. However, its exact role in EMT during cushion tissue formation is still unknown. In this study, we investigated the expression patterns of Msx1 mRNA and protein during chick heart development. Msx1 mRNA was localized in endothelial cells of the AV region at Stage 14, and its protein was first detected at Stage 15. Thereafter, Msx1 mRNA and protein were observed in the endothelial and mesenchymal cells of the OFT and AV regions. in vitro assays showed that ectopic Msx1 expression in endothelial cells induced p27, a cell-cycle inhibitor, expression and inhibited fibroblast growth factor 4 (FGF4)-induced cell proliferation. Although the FGF signal reduced the EMT-inducing activities of transforming growth factor β (TGFβ), ectopic Msx1 expression in endothelial cells enhanced TGFβ signaling-induced αSMA, an EMT marker, expression. These results suggest that Msx1 may support the transformation of endothelial cells due to a TGFβ signal in EMT during cushion tissue formation.

Knowing Real Cardiac Anatomy Enhances Understanding of Cardiac Development

New ideas about cardiac chamber formation, interatrial septum formation and development of the outflow region can be better understood by knowing real cardiac anatomy. The definitive chambers of the heart can be regionalized into components that provide a basis for understanding their formation during prenatal embryonic development. Each atrium has a venous inlet, a body, an appendage and a vestibule, while each ventricle consists of an inlet, an apical trabeculated portion and an outlet. During development several precursors combine to form the components of definitive chambers. Recent studies have challenged the classical version of interatrial septum formation, particularly the presence of a secondary atrial septum (septum secundum), and the closure of the primary atrial foramen (ostium primum). These studies suggest that the primary atrial septum (septum primum) merges with a fold of myocardium from the roof of the right atrium rather than with a second septum. This fold can be demonstrated in the dissection laboratory. Closure of the primary atrial foramen is accomplished by fusion of the ingrowing vestibular spine fusing with the mesenchymal cap of the primary septum and the merged AV endocardial cushions. This forms the anterior inferior atrial buttress that might be considered a secondary atrial septum. Finally, formation of the outflow region can be better understood using a tripartite explanation rather that the traditional bipartite version. During cardiac looping, the outflow region elongates and develops a bend in the middle. This flexure can define the outflow region into proximal, intermediate and distal segments. From these segments the outflow regions of the ventricles, the aortic valves and the intrapericardial roots of the great vessels are formed. The septum dividing the proximal segment is formed exclusively from endocardial cushion tissue, while the septum that partitions the intermediate and distal outflow regions is derived from cushion tissue and neural crest ectomesenchyme. Traditionally the aorticopulmonary septum was described as partitioning the entire outflow region. More recent examination of outflow tract development suggests that the aorticopulmonary septum only partitions the distal portion of the outflow region and the aortic sac. The intrapericardial and extrapericardial roots of the great vessels form from the division of the distal outflow region and the aortic sac.

Role of Periostin in Cardiac Valve Development.

Although periostin plays a significant role in adult cardiac remodeling diseases, the focus of this review is on periostin as a valvulogenic gene. Periostin is expressed throughout valvular development, initially being expressed in endocardial endothelial cells that have been activated to transform into prevalvular mesenchyme termed "cushion tissues" that sustain expression of periostin throughout their morphogenesis into mature (compacted) valve leaflets. The phenotype of periostin null indicates that periostin is not required for endocardial transformation nor the proliferation of its mesenchymal progeny but rather promotes cellular behaviors that promote migration, survival (anti-apoptotic), differentiation into fibroblastic lineages, collagen secretion and postnatal remodeling/maturation. These morphogenetic activities are promoted or coordinated by periostin signaling through integrin receptors activating downstream kinases in cushion cells that activate hyaluronan synthetase II (Akt/PI3K), collagen synthesis (Erk/MapK) and changes in cytoskeletal organization (Pak1) which regulate postnatal remodeling of cells and associated collagenous matrix into a trilaminar (zonal) histoarchitecture. Pak1 binding to filamin A is proposed as one mechanism by which periostin supports remodeling. The failure to properly remodel cushions sets up a trajectory of degenerative (myxomatous-like) changes that over time reduce biomechanical properties and increase chances for prolapse, regurgitation or calcification of the leaflets. Included in the review are considerations of lineage diversity and the role of periostin as a determinant of mesenchymal cell fate.

Abstract 16895: Lrp1, an Endocytic Vesicle Trafficking Protein, is Associated With Congenital Heart Diseases - Through a "non-Autonomous" Action

Backgrounds: Congenital heart diseases affect nearly 1% of neonates per year in the United States and are the leading cause of neonatal death. Despite a strong indication of genetic contribution to congenital heart diseases, the developmental processes and genetic components are poorly understood. From the large-scale forward genetic screen using inbred mice chemically mutagenized with ethyl-nitrosourea (ENU), a novel finding of endocytic trafficking gene mutations, including Lrp1, were recovered as causing congenital heart disease phenotypes. Endocytosis plays roles in modulating cell signaling by regulating the trafficking of the internalized endocytic vesicles to different endosomal compartments, some destined for fusion with lysosomes and degradation while others are recycled to the surface by recycling endosomes. Mutations in these genes causes a spectrum of congenital heart disease comprising of outflow tract abnormalities and alignment defects. Methods and Results: The LRP1 [line 1554(MGI 96828)] mutant mouse line recovered from a forward genetic screen results from a missense (C4232R) mutation in the region encoding the epidermal growth factor (EGF) repeat domain of LRP1. The mutants exhibit a spectrum of septation and outflow tract defects including endocardial cushion defects, double outlet of right ventricle with normal related great arteries as well as DORV with D-malposition of great arteries (Taussig-Bing anomalies). Mouse embryonic fibroblasts (MEF) from LRP1 m/m mutants demonstrated decreased cell-surface expression and increased retention in the endosomes. Migration assay of MEF demonstrated decreased migration of LRP1 m/m compared with control with normal Golgi orientation. The decreasing migration LRP1 m/m also observed in the endocardial cushion tissue from E9.5-10.5 embryos. Different cell lineages crucial to cardiac development with tissue-specific genetic manipulations were used to define the roles of the LRP1 pathway in these three fields of cells during outflow tract development and septation. Cardiac neural crest cell (Wnt1-Cre) knockout of LRP1 recapitulate LRP1 m/m cardiac phenotypes as well as Nkx2.5/Tie2-cre double knockout of LRP1. RNA expression profile suggests Notch pathway and integrin pathway are associated with LRP1 during cardiac development. Conclusions: We provide the first evidence of endocytic vesicle trafficking pathway associated with the development of congenital heart diseases with malalignment/septation and outflow tract defects

Double- Chambered Left Ventricle and Abnormal Papillary Muscle Formation

Papillary muscles develop separately from mitral valve leaflet and chordae, they origin from myocardial ridge of the anterior wall and to the posterior wall of the left ventricle (LV). While chordae and mitral valve leaflets origin from a cushion tissue. The myocardial ridge gradually loosens from the ventricular wall and meanwhile the cushion tissue transforms into leaflet and chordae1. Abnormal papillary muscle formation may present a muscle bridge and divide the LV into two chambers. The most rare form is the superior inferior LV subdivision which our cases represent.

Expression of the Tgfβ2 Gene During Chick Embryogenesis

We performed a comprehensive analysis of the expression of transforming growth factor (TGF) β2 during chick embryogenesis from stage 6 to 30 (Hamburger and Hamilton, J Morphol 1951;88:49-92) using in situ hybridization. During cardiogenesis, Tgfβ2 was expressed in the endothelial/mesenchymal cells of the valvulo-septal endocardial cushion tissue and in the epicardium until the end of embryogenesis. During the formation of major arteries, Tgfβ2 was localized in smooth muscle progenitors but not in the vascular endothelium. During limb development, Tgfβ2 was expressed in the mesenchymal cells in the presumptive limb regions at stage 16, and thereafter it was localized in the skeletal muscle progenitors. In addition, strong Tgfβ2 expression was seen in the mesenchymal cells in the pharyngeal arches. Tgfβ2 mRNA was also detected in other mesoderm-derived tissues, such as the developing bone and pleura. During ectoderm development, Tgfβ2 was expressed in the floor plate of the neural tube, lens, optic nerve, and otic vesicle. In addition, Tgfβ2 was expressed in the developing gut epithelium. Our results suggest that TGFβ2 plays an important role not only in epithelial-mesenchymal interactions but also in cell differentiation and migration and cell death during chick embryogenesis. We also found that chick and mouse Tgfβ2 RNA show very similar patterns of expression during embryogenesis. Chick embryos can serve as a useful model to increase our understanding in the roles of TGFβ2 in cell-cell interactions, cell differentiation, and proliferation during organogenesis.

Two-photon microscopy-guided femtosecond-laser photoablation of avian cardiogenesis: noninvasive creation of localized heart defects

Embryonic heart formation is driven by complex feedback between genetic and hemodynamic stimuli. Clinical congenital heart defects (CHD), however, often manifest as localized microtissue malformations with no underlying genetic mutation, suggesting that altered hemodynamics during embryonic development may play a role. An investigation of this relationship has been impaired by a lack of experimental tools that can create locally targeted cardiac perturbations. Here we have developed noninvasive optical techniques that can modulate avian cardiogenesis to dissect relationships between alterations in mechanical signaling and CHD. We used two-photon excited fluorescence microscopy to monitor cushion and ventricular dynamics and femtosecond pulsed laser photoablation to target micrometer-sized volumes inside the beating chick hearts. We selectively photoablated a small (∼100 μm radius) region of the superior atrioventricular (AV) cushion in Hamburger-Hamilton 24 chick embryos. We quantified via ultrasound that the disruption causes AV regurgitation, which resulted in a venous pooling of blood and severe arterial constriction. At 48 h postablation, quantitative X-ray microcomputed tomography imaging demonstrated stunted ventricular growth and pronounced left atrial dilation. A histological analysis demonstrated that the laser ablation produced defects localized to the superior AV cushion: a small quasispherical region of cushion tissue was completely obliterated, and the area adjacent to the myocardial wall was less cellularized. Both cushions and myocardium were significantly smaller than sham-operated controls. Our results highlight that two-photon excited fluorescence coupled with femtosecond pulsed laser photoablation should be considered a powerful tool for studying hemodynamic signaling in cardiac morphogenesis through the creation of localized microscale defects that may mimic clinical CHD.

THE GLUTATHIONE ACCUMULATION ON MIXED BUD TISSUES DURING THE DORMANCY OF JAPANESE PEAR

Glutathione (GSH) can exert effects in the regulation of several growth and developmental events. Considering that glutathione can play an important function during the bud dormancy, the glutathione pool on Japanese pear (Pyrus pyryfolia) bud tissues of plants exposed to different environmental conditions through the winter season was analyzed. Mixed buds of plants kept under natural conditions of cold accumulation (NC) presented higher GSH/GSSG ratio and a significant increase in the total glutathione content in concomitance with the endodormancy overcoming. A correspondingly greater glutathione reductase (GR) activity, enzyme which reduces GSSG back to GSH, was also found in the bud tissues of plants under NC. Bud cushion and shoot tissues presented a less significant increase in the total glutathione content and GR activity. Cold deprived (CD) plants did not show bud break and a considerable decline on the glutathione content, through the reduction of GR activity, was observed in buds. From these results, it can be assumed that the chilling exposure has an important influence on the GSH accumulation and on keeping high GSH/GSSG ratio in bud tissues at breaking time. This trait can be considered as a part of the complex dormancy overcoming process.

Lipid content and fatty acid composition of the digital cushion of bulls offered different amounts of linseed1

Previous research suggests that the digital cushion, a shock-absorbing structure in the claw, plays an important role in protecting cattle from lameness. This study aimed to assess the degree to which nutritional factors influence the composition of the digital cushion. This involved quantifying lipid content and fatty acid composition differences in digital cushion tissue from cattle offered diets with different amounts of linseed. Forty-six bulls were allocated to 1 of 4 treatments, which were applied for an average of 140 +/- 27 d during the finishing period. The treatments consisted of a linseed supplement offered once daily on top of the basal diet (grass silage:concentrate) at 0, 400, 800, or 1,200 g of supplement/animal per day. For each treatment, the concentrate offered was adjusted to ensure that total estimated ME intake was constant across treatments. Target BW at slaughter was 540 kg. Legs were collected in 3 batches after 120, 147 and 185 d on experiment. Six samples of the digital cushion were dissected from the right lateral hind claw of each animal. Lipids were extracted and expressed as a proportion of fresh tissue, and fatty acid composition of the digital cushion was determined by gas chromatography. Data were analyzed by ANOVA, with diet, location within the digital cushion, and their interactions as fixed effects and fat content (grams per 100 g of tissue) as a covariate. Linear or quadratic contrasts were examined. The lipid content of digital cushion tissue differed between sampling locations (P < 0.001) but did not vary by treatment. There were quadratic responses to increasing linseed supplementation for several fatty acids. Although the overall proportion of C18:3n-3 (the most abundant fatty acid in linseed) did not differ (P < 0.33) by treatment, there was a quadratic influence of diet on total PUFA concentration (P = 0.003) and a linear increase in C18:3n-3 as a proportion of total PUFA (P = 0.01) in the digital cushion. This work demonstrates that dietary fatty acid composition influences the concentration of fatty acids incorporated in the digital cushion of cattle. Based on the large number of quadratic responses among the fatty acids, it appears there is a threshold amount of fatty acid incorporation in the digital cushion.

Computational modeling of epithelial–mesenchymal transformations

An epithelial–mesenchymal transformation (EMT) involves alterations in cell–cell and cell–matrix adhesion, the detachment of epithelial cells from their neighbors, the degradation of the basal lamina and acquisition of mesenchymal phenotype. Here we present Monte Carlo simulations for a specific EMT in early heart development: the formation of cardiac cushions. Cell rearrangements are described in accordance with Steinberg's differential adhesion hypothesis, which states that cells possess a type-dependent adhesion apparatus and are sufficiently motile to give rise to the tissue conformation with the largest number of strong bonds. We also implement epithelial and mesenchymal cell proliferation, cell type change and extracellular matrix production by mesenchymal cells. Our results show that an EMT is promoted more efficiently by an increase in cell–substrate adhesion than by a decrease in cell–cell adhesion. In addition to cushion tissue formation, the model also accounts for the phenomena of matrix invasion and mesenchymal condensation. We conclude that in order to maintain epithelial integrity during EMT the number of epithelial cells must increase at a controlled rate. Our model predictions are in qualitative agreement with available experimental data.

Lycopodiopsis derbyi Renault from the Corumbataí Formation in the state of São Paulo (Guadalupian of Paraná Basin, Southern Brazil): New data from compressed silicified stems

Lycopodiopsis derbyi Renault was analyzed on the basis of compressed silicified stems from four Guadalupian outcrops of the Paraná Basin (Corumbataí Formation) in the State of São Paulo, Southern Brazil. Dichotomous stems have been recorded, and three different branch regions related to apoxogenesis are described. The most proximal region has larger, clearly rhomboidal leaf cushions, with protruding upper edges; the intermediate transitional region also has rhombic leaf cushions, but they are smaller and less elongated than the lower in the same axis; finally, the most distal region reveals only incipient cushions, with inconspicuous infrafoliar bladders; interspersed microphylls were still attached. A well preserved branch representative of this most distal region was sectioned; it has a siphonostelic cylinder similar to that previously described for L. derbyi. The cortex, however, shows new traits, such as a short portion of elongated cells between the periderm and the external cortex (or leaf cushion tissue). The stems were apparently silicified prior to their final burial but were probably not transported for long distances. Their final burial may have taken place during storm events, which were common during the deposition of the Corumbataí Formation. These stems are commonly deformed due to compression, mainly because the internal cortical portions rapidly decayed prior to silicification due to their thin-walled tissue, and are therefore not preserved. The common alkalinity of a shallow marine environment such as that in which the Corumbataí Formation was deposited, should mobilize the silica and favors petrifaction. Based on the new data, an emended diagnosis is proposed and a modification of the identification key published by Thomas and Meyen in 1984 for Upper Paleozoic Lycopsida is suggested.

Periostin promotes a fibroblastic lineage pathway in atrioventricular valve progenitor cells.

Differentiation of prevalvular mesenchyme into valve fibroblasts is an integral step towards the development of functionally mature cardiac valves. Although clinically relevant, little is known regarding the molecular and cellular mechanisms by which this process proceeds. Genes that are regulated in a spatio-temporal pattern during valve remodeling are candidates for affecting this differentiation process. Based on its expression pattern, we have focused our studies on the role of the matricellular gene, periostin, in regulating the differentiation of cushion mesenchymal cells into valve fibroblasts. Herein, we demonstrate that periostin expression is coincident with and regulates type I collagen protein production, a major component of mature valve tissue. Adenoviral-mediated knock-down of periostin in atrioventricular mesenchyme resulted in a decrease in collagen I protein expression and aberrant induction of myocyte markers indicating an alteration in AV mesenchyme differentiation. In vitro analyses using a novel "cardiotube" assay further demonstrated that expression of periostin regulates lineage commitment of valve precursor cells. In these cells, expression of periostin and collagen I are regulated, in part, by TGFbeta-3. We further demonstrate that TGFbeta-3, through a periostin/collagen pathway, enhances the viscoelastic properties of AV cushion tissue surface tension and plays a crucial role in regulating valve remodeling. Thus, data presented here demonstrate that periostin, a TGFbeta-3 responsive gene, functions as a crucial mediator of chick AV valve maturation via promoting mesenchymal-to-fibroblast differentiation while blocking differentiation of alternative cell types (myocytes).

Roles of TGFβ and BMP during valvulo–septal endocardial cushion formation

The primordia of valves and the atrioventricular septum arise from endocardial cushion tissue that is formed in the outflow tract (OFT) and in the atrioventricular (AV) regions during cardiogenesis. Abnormal development of the endocardial cushion results in various congenital heart diseases. Endocardial epithelial-mesenchymal transformation (EMT) is a critical process in cushion tissue formation and is regulated by many factors, such as growth factors, intercellular signaling molecules, transcription factors, and extracellular matrices. A signal that is produced by the myocardium of the AV and OFT regions and transferred to the adjacent endocardium across the extracellular matrix mediates EMT. Studies in vitro and genetic analyses have shown that transforming growth factor beta and bone morphogenetic protein play central roles in the regulation of EMT during cushion tissue formation.

Cardiac cushions modulate action potential phenotype during heart development

The extracellular matrix plays an important role in cardiac function. Its role in the generation and modulation of electrical activity in the early stages of heart development has not been studied extensively. Our study demonstrates that the extracellular matrix in cardiac cushions can alter the action potential phenotype by direct contact with cardiomyocytes from different regions of the heart. We also demonstrate that fibronectin, an important and abundant component of the cardiac extracellular matrix, partially mimics the effects of the cushion tissue in altering the changes in action potential. Fibronectin increases I(Ca) (2+) and acutely increases cytosolic calcium. These findings suggest that the composition of the cardiac extracellular matrix during development plays an important role in defining patterns of electrical activity in the developing heart.

A combination of right ventricular hypertrabeculation/noncompaction and Ebstein's anomaly

A 35-year-old man admitted with iterative extremities edema for three months and mild chest complaint associated with activity without chest distress for five days. He underwent an initial 2DE that did not confirm HVM/NVM and 3DE that strengthened the diagnosis of HVM/NVM. The limitations of 2DE may lead to misinterpretation of prominent trabeculations as “false tendons,” and may also lead to underestimation of the severity of HVM/NVM. In contrast to 2DE, 3DE provides for pyramid-shaped datasets that encompass the entire right ventricular. Specifically, right ventricular can be sectioned in userselected planes and an unlimited number and angles of such planes can be used. Intracavitary echodensities that are suspicious for trabeculations can be tracked in multiple directions from base to apex. To our knowledge, this is the first report on only right ventricular HVM/NVM associated with Ebstein's malformation. The leaflets of the tricuspid valve develop from endocardial cushion tissue and the myocardium. The tricuspid valve leaflets become freely movable after delamination of the tension apparatus within the myocardium. Although the coexistence of right ventricular HVM/NVM and Ebstein's malformation could be a coincidence, I believe that both defects could be caused by a developmental arrest of the right ventricular myocardium. That is to say, the genetic change that is responsible for Ebstein's anomaly also plays a role in the differentiation of the right myocardium.

Latent Transforming Growth Factor-β-Binding Protein-4 Regulates Transforming Growth Factor-β1 Bioavailability for Activation by Fibrogenic Lung Fibroblasts in Response to Bleomycin

Recent evidence suggests that subsets of lung fibroblasts differentially contribute to fibrogenic progression. We have previously shown that a subset of rat lung fibroblasts with fibrogenic characteristics [Thy-1 (-) fibroblasts] responds to stimuli (bleomycin, interleukin-4, etc) with increased latent transforming growth factor (TGF)-beta activation, whereas non-fibrogenic Thy-1-expressing [Thy-1 (+)] fibroblasts do not. Activation of latent TGF-beta1 by interstitial lung fibroblasts is critical for fibrogenic responses. To better understand the susceptibility of fibrogenic fibroblasts to the stimulation of TGF-beta activation, we examined the role of latent TGF-beta-binding proteins (LTBPs), key regulators of TGF-beta bioavailability and activation, in TGF-beta1 activation by these fibroblasts. Treatment of fibroblasts with bleomycin up-regulated LTBP-4 mRNA, protein, and soluble LTBP-4-bound large latent TGF-beta1 complexes in Thy-1 (-) fibroblasts to significantly higher levels than in Thy-1 (+) fibroblasts. Bleomycin-induced TGF-beta1 activation required LTBP-4, since lung fibroblasts deficient in LTBP-4 did not activate TGF-beta1. Expression of LTBP-4 restored TGF-beta1 activation in response to bleomycin, but expression either of LTBP-4 lacking the TGF-beta-binding site or only the TGF-beta-binding domain did not. Bleomycin treatment of mice increased LTBP-4 expression in the lung. Thy-1 knockout mice had increased levels of both LTBP-4 expression and TGF-beta activation, as well as enhanced Smad3 phosphorylation compared with wild-type mice. Together, these data identify a critical role for LTBP-4 in the regulation of latent TGF-beta1 activation in bleomycin-induced lung fibrosis.

ROCK1 Expression is Regulated by TGFβ3 and ALK2 During Valvuloseptal Endocardial Cushion Formation

During early heart development at the looped heart stage, endothelial cells in the outflow tract and atrioventricular (AV) regions transform into mesenchyme to generate endocardial cushion tissue. This endocardial epithelial-mesenchymal transition (EMT) is regulated by several regulatory pathways, including the transforming growth factor-beta (TGFbeta), bone morphogenetic protein (BMP), and Rho-ROCK pathways. Here, we investigated the spatiotemporal expression pattern of ROCK1 mRNA during EMT in chick and examined whether TGFbeta or BMP could induce the expression of ROCK1. At the onset of EMT, ROCK1 expression was up-regulated in endothelial/mesenchymal cells. A three-dimensional collagen gel assay was used to examine the mechanisms regulating the expression of ROCK1. In AV endocardium co-cultured with associated myocardium, ROCK1 expression was inhibited by either anti-TGFbeta3 antibody, anti-ALK2 antibody or noggin, but not SB431542 (ALK5 inhibitor). In cultured preactivated AV endocardium, TGFbeta3 protein induced the expression of ROCK1, but BMP did not. AV endothelial cells that were cultured in medium supplemented with TGFbeta3 plus anti-ALK2 antibody failed to express ROCK1. These results suggest that the expression of ROCK1 is up-regulated at the onset of EMT and that signaling mediated by TGFbeta3/ALK2 together with BMP is involved in the expression of ROCK1.

Accessory Atrioventricular Pathways

By definition, accessory atrioventricular pathways are aberrant muscle bundles that connect the atrium to a ventricle outside of the regular atrioventricular conduction system. Clinically, they may manifest as substrates for ventricular preexcitation. The first accessory pathway in a patient who suffered from Wolff-Parkinson-White syndrome was described in 1943 by Wood, Wolferth, and Geckler.1 Shortly after, Ohnell created a reconstruction of an accessory pathway that very elegantly showed the close proximity of the pathway to the fibrous attachment of the mitral valve and its relationship with the sulcus coronarius (Figure, A).2 Subsequent histological studies have demonstrated unequivocally that these pathways are the anatomic substrates for the classical Wolff-Parkinson-White variety of preexcitation. Figure. A, Ohnell’s depiction of an accessory bundle in the left atrioventricular groove.2 Reprinted with permission from Blackwell Publishing. B, Histological section stained with Masson’s trichrome (fibrous tissue in green and myocardium in red) to show an accessory left-sided pathway (arrow) skirting the mitral annulus. C, Histological section showing a broad right-sided accessory pathway (arrow) that is formed by a myocardial pouch extending from the right atrial appendage to the ventricle and ultimately traced to a small right ventricular vein. This pathway is some distance from the fibrous annulus of the tricuspid valve. *Valvar annulus. Article p 1508 Accessory atrioventricular pathways are found most often in the parietal atrioventricular junctional areas, including the paraseptal areas. They breach the insulation provided by the fibrofatty tissues of the atrioventricular groove (sulcus tissue) and the hingelines (fibrous annulus) of the valves. They are rarely found in the area of fibrous continuity between the aortic and mitral valves because in this area, there is usually a wide gap between the atrial myocardium and ventricular myocardium to accommodate the aortic outflow tract. On the left parietal side, the accessory pathways tend …