Published online before print January 3, 2008, doi: 10.1161/CIRCRESAHA.107.160861
© 2008 American Heart Association, Inc.
Integrative Physiology |
Non–Cell-Autonomous Roles for the Planar Cell Polarity Gene Vangl2 in Development of the Coronary Circulation
From the Institute of Human Genetics (H.M.P., V.H., J.D.P., B.C., D.J.H.), Newcastle University, Newcastle upon Tyne, UK; Medical Research Council Mammalian Genetics Unit (J.N.M.), Harwell, Oxon, UK; and Department of Molecular Genetics (K.K.), Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima, Japan.
Correspondence to Deborah J. Henderson, Institute of Human Genetics, Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ, United Kingdom. E-mail d.j.henderson{at}ncl.ac.uk
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Abstract |
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Establishment of cellular polarity is essential for the development of many tissues. In this study, we describe defects in the formation of the coronary vasculature in the loop-tail (Lp) mutant in which the planar cell polarity (PCP) gene, Vangl2, is disrupted. Although Vangl2 is expressed exclusively in the myocardial cells of the developing heart, the coronary vessels do not develop an intact smooth muscle layer, and there are enlarged, ectopic vessels on the surface of the heart. Reduced fibronectin deposition in the subepicardial space is associated with limited migration of epicardially derived cells (EPDCs) into the ventricular myocardium and likely contributes to these defects. Analysis of cardiomyocytes shows that the actin cytoskeleton is disrupted and the cytoarchitecture of the ventricular myocardium is abnormal in Lp/Lp hearts. Moreover, activation of RhoA/Rho kinase signaling is disrupted in these cells. Conditional inhibition of myocardial Rho kinase activity disrupts the organization of the cardiomyocytes and formation of the coronary vessels to produce the same spectrum of defects as seen in Lp. These data suggest that Vangl2 and Rho kinase act cell autonomously in the myocardium to regulate the organization of cardiomyocytes but also have non–cell-autonomous effects on the formation of the coronary vasculature.
Key Words: Vangl2 • planar cell polarity • cardiac development • myocardium • coronary vasculature
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Introduction |
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The coronary arteries that channel oxygen rich blood throughout the ventricular myocardium are formed from cells that originally derive from a region of the splanchnic mesoderm known as the proepicardium (reviewed in1–3). These pluripotential proepicardial cells translocate to the surface of the heart and cover the entirety of the ventricles and the atria with a continuous, polarized monolayer: the epicardium.4 The epicardial cells undergo a rapid epithelial-to-mesenchymal transformation (EMT) and migrate first into the subepicardial extracellular mesenchyme. From there, a proportion migrate into the adjacent myocardium to give rise to a variety of mesenchymal cell types: fibroblasts that form the fibrous skeleton of the heart, supporting the valve structures and contributing to the propagation of the heart beat; endothelial cells that line the coronary arteries; and smooth muscle cells that stabilize and strengthen the coronary vasculature.5–7 Once in contact with the myocardium, the epicardial cells release trophic factors such as retinoic acid, erythropoietin, fibroblast growth factors, and Wnts,8,9 which are essential for the growth and development of the compact myocardium. Without this stimulation, the ventricular myocardium remains thin and the embryonic heart fails.
The noncanonical Wnt planar cell polarity (PCP) signaling pathway plays essential roles in the establishment of tissue polarity throughout the developing vertebrate embryo.10,11 It signals via the small GTPases RhoA and Rac, which ultimately modulate the actin cytoskeleton to bring about changes in cell adhesion, polarity and movement. Rho kinase (ROCK) acts downstream of RhoA in this pathway to mediate many of these effects (reviewed elsewhere10). Normal cardiac development is dependent on PCP signaling12–15 and both the loop-tail (Lp) mouse mutant,12,13 which has a mutation in the Vangl2 gene, and the Dishevelled 2 (Dvl2) knockout mouse15 have defects in the alignment of the ventricular chambers with the great vessels (double outlet right ventricle). In addition, abnormal polarization of cardiomyocytes in Lp mutants prevent their migration into the developing outflow tract cushions (myocardialization) and disrupts the formation of the muscular subpulmonary infundibulum of the right ventricle.13 In this study, we describe abnormalities in the formation of the coronary vasculature in Lp, resulting from non–cell-autonomous effects on migration and/or differentiation of transformed epicardial cells.
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Materials and Methods |
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Embryo Preparation and Analysis
Lp mice were bred and genotyped as described previously.12 No heart malformations were observed in stage matched Lp/+ embryos and these were used with +/+ embryos as controls. The ROCK-CAT dominant-negative (DN) mice16 (BRC no. 01294) were provided by RIKEN BioResource Center (Tsukuba, Japan). Mlc2v-cre mice17 were obtained from Dr P Riley (University College London, UK). Animals were maintained according to the Animals (Scientific Procedures) Act 1986, United Kingdom.
Immunohistochemistry
Immunoperoxidase staining was performed on paraformaldehyde fixed, paraffin-embedded sections,13 carefully matched for stage and position within the heart. Immunofluorescence and rhodamine/phalloidin (Sigma) staining was performed on cryosections using laser confocal microscopy (Zeiss 510 meta LSCM). The Vangl2 antibody was produced and used as previously.18 The retinaldehyde dehydrogenase (RALDH)2 antibody was a gift from Dr P McCaffery (University of Massachusetts Medical School, Waltham). 
-Smooth muscle actin (clone 1A4) and -actinin were from Sigma Chemical Co; WT1 was from Cell Signaling; ROCK1, ROCK2, and fibronectin were from Santa Cruz Biotechnology; platelet endothelial cell-adhesion molecule (PECAM) (CD31) was from Pharmingen; N-cadherin was from BD Transduction; connexin 43 was from Chemicon; EphB4 was from R&D Laboratories; myosin heavy chain (MHC) IIB was from Covance; and phospho-myosin phosphatase (MYPT)696 was from Upstate. Zinc fixative (BD Pharmingen) was used before PECAM staining of paraffin-embedded sections. Whole-mount antibody staining was performed as described previously.19 Each experiment was repeated a minimum of 3 times on an average of six embryos per genotype and included appropriate controls.
Analysis of Migrating Epicardially Derived Cells
Epicardially derived cell (EPDC) counts were performed within 12 carefully matched 145-µm2 squares of the ventricular wall of embryonic day (E)14.5 Lp/+ (n=5) and Lp/Lp (n=5) fetuses. Thus, for each sample, EPDCs migrated from a region of epicardium 145 µm in length. Only Lp/Lp with normal myocardial wall thickness were included in this analysis. In addition to the total number of WT1-positive migrating cells within a sampled area, the numbers found within the outer 70 µm and inner 70 µm of the ventricular myocardium were noted separately. A total of 1022 Lp/+ and 840 Lp/Lp WT1-labeled cells were sampled in each case. Statistical significance was determined using the 
2 test.
cDNA Probes and In Situ Hybridization
Slide in situ hybridization using digoxigenin-labeled Vangl2 riboprobe was performed as previously.13
Epicardial Transformation Assays
Hearts from E12.5 mouse embryos were dissected and the ventricles placed epicardial-side down on 1.5 mg/mL collagen type I gels (Vitrogen 100; Collagen Esthetics).5,9 Gels were incubated for 13 days before labeling with anti–-smooth muscle actin (SMA) (Sigma clone 1A4), anti-
SMA (ICN), or phalloidin and analysis using confocal microscopy.
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Results |
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Lp/Lp Fetuses Display Abnormal Coronary Development
During prior studies in Lp mice,12,13 it was noted that the left main ventral coronary artery, which is normally present on the surface of the left ventricle in control hearts by the 15th day of gestation (E15.5), was absent in the majority of Lp/Lp fetuses. Instead, blood pools were seen over the surface of the ventricles (Figure 1A and 1B). By E16.5, enlarged, tortuous vessels were apparent on the ventricular surface (Figure 1D, compare with 1C). These defects suggested an abnormality in the formation or remodeling of the coronary vessels in Lp/Lp mutants.
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We used an anti-PECAM (CD31) antibody to label the endothelial cells lining the coronary vessels. At E15.5, a vascular plexus was observed forming at the interventricular region and at the base of the great arteries. This appeared more disorganized in Lp/Lp hearts (Figure 1E, 1F, 1H, and 1I), particularly at the base of the great arteries, where the coronary arteries connect with the aortic root (arrows in Figure 1E and 1H). However, histological analysis showed that these vessels always connected to the aortic root and never with the pulmonary trunk (data not shown). To clarify whether coronary veins, as well as arteries, are affected in Lp/Lp hearts, we used an antibody raised against EphB4, a specific marker of veins. Whole-mount EphB4 staining of isolated Lp/Lp and control hearts at E15.5 showed disorganization of vessels at the coronary plexus, similar to that observed following PECAM staining (Figure I in the online data supplement, available at http://circres.ahajournals.org; and Figure 1F and 1I). By E17.5, PECAM labeling demonstrated remodeling such that the vessels had become buried within the compact myocardium in control hearts (Figure 1K). In contrast, enlarged vessels, filled with blood cells and correlating with the ectopic blood vessels seen in isolated hearts (Figure 1D), could be seen over the surface of the ventricles in Lp/Lp fetuses (Figure 1N). Sectioning revealed these vessels lay within the subepicardial region (Figure 1L, 1M, 1O, and 1P). Although there were increased numbers of sinusoids within the Lp/Lp ventricular myocardium (data not shown), there was no evidence of hemorrhaging into the subepicardial region from the lumen of the heart.
Formation of the Coronary Blood Vessel Wall Is Impaired in Lp/Lp
Coronary endothelial tubes are stabilized and strengthened by the addition of vascular smooth muscle cells (SMCs) derived from the epicardium,1–3 following a base to apex gradient. Thus at E14.5, although the endothelial lining of the coronary vessels is readily visualized, there was no evidence of SMC recruitment (data not shown). However by E15.5, cells around the endothelial tubes within the ventricular wall expressed SMA (Figure 2A and 2C) and by E18.5, a continuous layer of SMA-positive cells was seen (Figure 2E and 2G). In contrast, SMA labeling was not found surrounding the endothelial tubes in matched regions of the myocardial wall of Lp/Lp fetuses at E15.5. Moreover, SMA labeling was abnormal in Lp/Lp at E18.5; the vessels were irregular in shape and the vessel wall was of variable thickness with patchy SMA staining (Figure 2F and 2H). Vessels correlating with those observed following PECAM staining could be seen on the surface of the heart without any evidence of SMA labeling (Figure 2F). Thus, there appears to be two main defects in the coronary vasculature in Lp/Lp hearts: vessels within the myocardial wall fail to develop an intact SMC layer and extensive ectopic vessels form in the subepicardium, on the surface of the heart.
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Vangl2 Is Expressed in the Myocardium but Not in Coronary Progenitors
In situ hybridization and immunohistochemistry showed that Vangl2 transcripts and protein were found throughout the ventricular myocardium at E9.5 to E11.5 (Figure 3A, 3E, and 3G and data not shown). By E12.5, both were still expressed in the myocardium, localizing to the compacting myocardium of the ventricular wall and the trabeculations of the left ventricle (Figure 3B, 3F, and 3H). Although at E14.5 Vangl2 mRNA expression levels were declining, transcripts and protein were still found in these areas (Figure 3D), and the time course of ventricular Vangl2 expression was similar to that of the outflow tract myocardium.13 Throughout the time period investigated, high levels of Vangl2 expression were observed in the epithelia of the gut and developing lungs (Figure 3A, 3B, and 3D), as described previously.20 Neither Vangl2 mRNA or protein was detected in the epicardium or in the coronary vessels at any stage examined (arrowheads in Figure 3C, 3F, and 3H and data not shown).
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Altered Fibronectin Deposition in the Subepicardial Space in Lp/Lp Embryos
Because the formation of an intact epicardium is a prerequisite for the development of the coronary circulation and the thickening and compaction of the ventricular myocardium,21–24 we followed the addition of the epicardium to the heart. Using antibodies raised against Wilms tumor 1 (WT1) on sectioned E9.5 and E10.0 embryos, we observed an apparently normal proepicardium in +/+, Lp/+, and Lp/Lp embryos (arrows in Figure 4A and 4B). Furthermore, using RALDH2 as well as WT1 antibodies, an intact epicardium covering the entire ventricular myocardium was observed in +/+, Lp/+ and Lp/Lp (Figure 4A through 4H). However, by E13.5, epicardial blisters, filled with what appeared to be red blood cells, were a common finding in Lp/Lp embryos (Figure 4G and 4H). These blisters correlated with the ectopic coronary vessels found in the subepicardial space at later stages of gestation (compare with Figure 1O). In addition, the myocardial wall was thinned in many of the Lp/Lp fetuses (Figure 4K and 4L).
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As abnormalities in PCP signaling have been associated with defects in fibronectin deposition in other systems,25,26 we looked at fibronectin localization in ventricles from Lp/Lp embryos and fetuses and their wild-type littermates. At E10.5, as the epicardium was forming, fibronectin was apparent on the myocardial surface (Figure 4I and 4J). However, from E11.5 through to E14.5, fibronectin deposition was markedly reduced in the subepicardial space of Lp/Lp hearts when compared with their wild-type littermates, and this region was markedly thinned (arrows in Figure 4K and 4L and data not shown).
Migration of EPDCs Into the Myocardium Is Impaired in Lp
Following the application of the epicardium, a proportion of its cells undergo EMT and migrate firstly into the subepicardial space and then into the myocardium.1–3 WT1 is a sensitive and specific marker for these epicardially derived cells (EPDCs) (Figure 5A through 5D).7 Close analysis of the cells within the epicardial monolayer showed that these cells were more flattened in the Lp/Lp compared with their control littermates (Figure 5C and 5D). Expression of the gap junction protein connexin 43 confirmed this observation, localizing to a multicellular layer in the control fetuses, compared with a single, flattened monolayer in the Lp/Lp (Figure 5E and 5F).
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We also used WT1 labeling to evaluate the behavior of EPDCs as they migrated into the myocardium at E13.5 and E14.5 (Figure 5A through 5D and data not shown). Although there was no significant difference between the numbers of EPDCs leaving the epicardium in Lp/Lp fetuses when compared with control littermates, when we analyzed their penetration into the myocardium, only 12% (101/840 cells; interembryonic variation, 2% to 23%) of the cells progressed into the inner, luminal region of the ventricular wall, with the remaining 88% found in the outer region. Many of these EPDCs were found in the subepicardium, just below the epicardial monolayer (Figure 5D). This contrasted with 32% of EPDCs that penetrated into the inner region in the Lp/+ fetuses (324/1022; interembryonic variation, 19% to 39%; P>0.001; Figure 5A through 5D). Notably, the WT1-positive EPDCs within the ventricular myocardium from +/+ and Lp/+ embryos had ovoid shaped nuclei, suggesting polarized cells and migration (Figure 5C). In contrast, the WT1-positive nuclei in Lp/Lp fetuses lacked obvious polarity (Figure 5D).
Lp/Lp EPDCs Form Normal SMCs in Explant Culture
Because Vangl2 is not expressed in the epicardium or its derivatives, yet SMC recruitment to the vessels is abnormal, we suspected a non–cell-autonomous role for Vangl2 within the myocardium. To test this idea, we used a collagen gel explant system to determine whether Lp/Lp epicardial cells undergo normal EMT, migrate efficiently into collagen, and form SMCs5,9 when isolated from the Lp/Lp myocardial environment. Following explantation of ventricular segments, EPDCs migrated into the collagen gels, and after 13 days in culture, cells with the morphology of vascular SMCs that expressed SMA and SMA were observed in both Lp/Lp and wild-type explants (Figure 6A through 6F). Both Lp/+ and Lp/Lp cells exhibited characteristic actin stress fibers (Figure 6G and 6H), as has been described previously.27 Indeed, cultures from the +/+, Lp/+, and Lp/Lp fetuses were indistinguishable. These data support a non–cell-autonomous role for Vangl2 in the development of the coronary circulation.
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Cytoskeletal Disorganization in Lp/Lp Myocardium
We sought to determine the effects of loss of Vangl2 function on the development of the myocardium. Antibodies to N-cadherin were used to outline the cells as this is found throughout the cardiomyocyte plasma membrane at this time. Interestingly, although polarization of cardiomyocytes is not established until late in gestation,28 Lp/Lp cardiomyocytes appeared more rounded than their +/+ and Lp/+ littermates, even at this early stage (Figure 7A and 7B). -Actinin labeling was indistinguishable between Lp/Lp and control littermates showing that intact sarcomeres were formed, although again, in some regions the cardiomyocytes appeared disorganized (Figure 7C and 7D). Because the downstream components of the Vangl2-mediated PCP pathway are known to modulate the actin cytoskeleton,10,11 we also used rhodamine/phalloidin labeling to identify the filamentous actin cytoskeleton of the myocardial cells. This confirmed the disorganization of the ventricular wall in Lp/Lp embryos, showing that the Lp/Lp cells were rounded and irregular in size and shape (Figure 7E and 7F). Moreover, the phalloidin staining was more punctate in Lp/Lp fetuses than in control littermates, suggesting that the actin cytoskeleton of the cardiomyocytes is disrupted. Because Vangl2 and the PCP pathway regulate cytoskeletal organization via RhoA/ROCK signaling, we used phosphorylation-specific antibodies to key components to determine whether normal activation of the pathway had occurred. Localization of phospho-MYPT696, a key target of ROCK signaling, was disrupted in Lp/Lp myocardium at E13.5, appearing more punctuate, rather than being distributed evenly throughout the cortical region of the cardiomyocytes. This was particularly noticeable in the myocardium adjacent to the subepicardial space (arrows in Figure 7G and 7H). MHC IIB, which lies further downstream in the pathway, also localizes to the cortical region of cardiomyocytes; in this case, expression was reduced in Lp/Lp hearts (Figure 7I and 7J). In addition, although MHC IIB was apically localized in the epicardium of Lp/+ hearts, it was found in both the apical and basal compartment of epicardial cells in Lp/Lp hearts (arrows in Figure 7I and 7J).
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ROCK Also Acts Non–Cell-Autonomously to Regulate Coronary Vessel Formation
These data suggest that RhoA/ROCK activation is disrupted in Lp/Lp hearts and that this may be responsible for the cytoskeletal abnormalities and myocardial disorganization observed. We therefore examined the expression of ROCK1 and ROCK2 in Lp hearts from E10.5 onwards (Figure 8A and 8B and data not shown). Both ROCK1 and ROCK2 were expressed in the epicardium and myocardium; thus, it is possible that ROCK1 and/or ROCK2 could be acting in either of these tissues to affect coronary vessel development. We were able to specifically determine whether ROCK function within the myocardium (ie, in the same tissue as Vangl2) is necessary for normal coronary artery formation using ROCK-CAT DN mice.16 When intercrossed with Mlc2v-cre mice,17 the DN construct is expressed via Cre-loxP recombination and disrupts ROCK function solely in the myocardium (Figure 8C). Analysis of ROCK-CAT DN;Mlc2v-cre fetuses (n=15) revealed the same spectrum of anomalies in coronary vessel formation as seen in Lp/Lp hearts. Hematoxylin/eosin staining of sectioned hearts revealed discontinuities in the epicardium and epicardial blisters, similar to those observed in Lp/Lp hearts (Figure 8E and 8F). Moreover, disorganized vessel-like structures were seen at the ventricular surface (Figure 8E and 8H), in some cases coalescing to form large holes (Figure 8I). Staining of E15.5 hearts for SMA revealed reduced staining of vascular SMCs within the myocardial wall (Figure 8G through 8I). RhoA-CAT DN16; Mlc2v-cre fetuses showed similar defects to the ROCK-CAT DN;Mlc2v-cre (supplemental Figure II and data not shown).
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Rhodamine/phalloidin staining of the ROCK-CAT DN;Mlc2v-cre hearts confirmed that the actin cytoskeleton was disrupted specifically in the myocardium of the ventricle, with staining appearing more punctate than in control tissue (Figure 8J and 8K). Moreover, this cytoskeletal disruption was associated with abnormalities in the organization of the cardiomyocytes within the wall of the ventricle, closely resembling that seen in Lp/Lp fetuses (compare 8K with 7D). The expected pattern of apically located phalloidin staining was observed in gut epithelium in ROCK-CAT DN;Mlc2v-cre fetuses, showing that the cytoskeleton was disrupted specifically in the ventricular cardiomyocytes (Figure 8L). Notably, in contrast to the Lp/Lp fetuses, fibronectin deposition appeared normal in the subepicardial space in ROCK-CAT DN;Mlc2v-cre fetuses (supplemental Figure III), suggesting that the cytoskeletal disruption and disorganization of the cardiomyocytes was sufficient to cause the coronary vessels defects seen in these mice.
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Discussion |
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These studies suggest a non–cell-autonomous role for Vangl2-PCP signaling in coronary artery formation. The coronary vessels in Lp/Lp hearts fail to develop a normal SMC layer and enlarged ectopic vessels are found in the subepicardium, on the surface of the heart. Loss of functional Vangl2 results in reduced deposition of fibronectin in the subepicardial space, which may limit normal migration of EPDCs into the myocardium. In addition activation of RhoA/ROCK signaling in the myocardium is disrupted, which causes disorganization within the ventricular wall via effects on the cytoskeleton. Although the precise mechanism by which PCP signaling regulates cytoskeletal organization remains unclear, mislocalization of activated MYPT is likely to have major implications for polarization and organization of ventricular cardiomyocytes. These data suggest that PCP signaling may be important at a number of levels for development of the mature ventricular myocardium and coronary vessels.
Proper polarization and organization of the cardiomyocytes occurs late in gestation28 and is essential for the mechanical and electrical coupling of the myocardium. The abnormalities in myocardial cellular architecture that we observe in Lp/Lp and ROCK DN;Mlc2v-cre fetuses may reflect abnormalities in this process. As well as a requirement for Vangl2 in polarization of cardiomyocytes during myocardialization of the proximal outflow cushions,13 our data suggest a more general role for Vangl2 in cardiomyocyte polarity. This correlates with the defects in organization of the ventricular cardiomyocytes that we recently described for another PCP gene, Scrib,29 suggesting that these genes may act together to regulate cardiomyocyte polarization. Reciprocal signaling between the myocardium and epicardium is known to be essential for normal maturation of the ventricular wall.1 Disrupted polarity and/or disorganization of the myocardium may prevent normal signaling, perhaps because of mislocalization of signaling components within the myocardium. Disrupted signaling from the myocardium could affect the epicardium (as suggested by the abnormal phenotype and mislocalization of MHC IIB in the epicardial monolayer in Lp/Lp), which may then affect subsequent signaling back to the myocardium. It has also been shown (reviewed elsewhere1) that the EPDCs within the myocardium are required for the thickening of the wall. Limited migration of EPDCs in Lp hearts, resulting in deficiency of trophic factors, extracellular matrix molecules and extracellular matrix–modulating molecules that they release, could be related to the thinning and the disorganization of the ventricular myocardium. A detailed analysis of the establishment of cardiomyocyte polarity in Lp, and its relationship with EPDC colonization of the ventricular wall and signaling between these distinct cell types, will be the subject of future studies.
In addition to the defects in organization of the ventricular myocardium, a deficiency in the deposition of extracellular fibronectin in the subepicardial space was observed in Lp/Lp hearts. Although the subepicardial extracellular matrix is deposited by both myocardial and epicardial cells, fibronectin is deposited by the (Vangl2-expressing) myocardium and can be observed before the appearance of the epicardium.30 Because fibronectin is permissive for cell migration, its reduction in Lp/Lp embryos after the formation of an intact epicardium may also affect the efficiency of migration of EPDCs into the myocardium. Fibronectin matrices have been shown to be deficient at tissue boundaries in Xenopus embryos in which Vangl2 is disrupted.25,26 These defects were associated with defects in the polarized cell movements that occur during the process of convergence and extension. This therefore resembles the Lp/Lp heart, where fibronectin deposition is reduced at the epicardial–myocardial boundary and cell migration is impaired.
The majority of PECAM-stained coronary vessels are found in the subepicardial space in Lp/Lp, on the surface of the heart. Although coronary vessels are normally found in this position in many organisms, including humans, the main coronary vessels are usually found embedded within the ventricular wall in mice. Although the precise mechanism by which the endocardial vessels become surrounded by myocardium in rodents remain unclear, it probably relates to both the active migration of EPDCs into the ventricular wall and to the continued growth and compaction of the ventricular myocardium, processes that are disrupted to a variable degree in Lp. Indeed it seems likely that the presence of the majority of coronary vessels within the subepicardium of Lp/Lp hearts results from a combination of these abnormalities. Similarly, the failure of the Lp/Lp coronary vessels to mature and become enveloped in a continuous SMC layer may also have a complicated etiology. Reduced numbers of EPDCs in the inner region of the myocardial wall may be a contributory factor, but because vascular SMC recruitment is flow dependent, abnormal cardiac function secondary to myocardial thinning may also play a role.
As well as playing important roles in the myocardium, as we show here, ROCK has also been suggested to play cell autonomous roles in formation of the coronary vessels via the epicardium.27,31 These studies have shown that ROCK is essential for both EMT and SMC differentiation of epicardial cells, highlighting the importance of interactions between the 2 cell types in formation of the coronary vasculature and showing that there are multiple steps. Abnormalities in any of these factors can result in defects in the coronary circulation.
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Acknowledgments |
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Sources of Funding
This project was funded by the British Heart Foundation (BS/05/003 and PG/05/041). We also thank Friends for Life for funds for technical support for the project.
Disclosures
None.
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Footnotes |
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Original received February 19, 2007; resubmission received July 30, 2007; revised resubmission received December 19, 2007; accepted December 21, 2007.
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References |
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