Opposing USP19 splice variants in TGF-ß signaling and TGF-ß-induced epithelial-mesenchymal transition of breast cancer cells.

Ubiquitin-specific protease (USP)19 is a deubiquitinating enzyme that regulates the stability and function of multiple proteins, thereby controlling various biological responses. The alternative splicing of USP19 results in the expression of two major encoded variants that are localized to the endoplasmic reticulum (ER) (USP19-ER) and cytoplasm (USP19-CY). The importance of alternative splicing for the function of USP19 remains unclear. Here, we demonstrated that USP19-CY promotes TGF-ß signaling by directly interacting with TGF-ß type I receptor (TßRI) and protecting it from degradation at the plasma membrane. In contrast, USP19-ER binds to and sequesters TßRI in the ER. By decreasing cell surface TßRI levels, USP19-ER inhibits TGF-ß/SMAD signaling in a deubiquitination-independent manner. Moreover, USP19-ER inhibits TGF-ß-induced epithelial-mesenchymal transition (EMT), whereas USP19-CY enhances EMT, as well as the migration and extravasation of breast cancer cells. Furthermore, USP19-CY expression is correlated with poor prognosis and is higher in breast cancer tissues than in adjacent normal tissues. Notably, the splicing modulator herboxidiene inhibits USP19-CY, increases USP19-ER expression and suppresses breast cancer cell migration. Targeting USP19 splicing or its deubiquitinating activity may have potential therapeutic effects on breast cancer.

New calcification model for intact murine aortic valves.

Calcific aortic valve disease (CAVD) is a common progressive disease of the aortic valves, for which no medical treatment exists and surgery represents currently the only therapeutic solution. The development of novel pharmacological treatments for CAVD has been hampered by the lack of suitable test-systems, which require the preservation of the complex valve structure in a mechanically and biochemical controllable system. Therefore, we aimed at establishing a model which allows the study of calcification in intact mouse aortic valves by using the Miniature Tissue Culture System (MTCS), an ex vivo flow model for whole mouse hearts. Aortic valves of wild-type mice were cultured in the MTCS and exposed to osteogenic medium (OSM, containing ascorbic acid, ß-glycerophosphate and dexamethasone) or inorganic phosphates (PI). Osteogenic calcification occurred in the aortic valve leaflets that were cultured ex vivo in the presence of PI, but not of OSM. In vitro cultured mouse and human valvular interstitial cells calcified in both OSM and PI conditions, revealing in vitro-ex vivo differences. Furthermore, endochondral differentiation occurred in the aortic root of ex vivo cultured mouse hearts near the hinge of the aortic valve in both PI and OSM conditions. Dexamethasone was found to induce endochondral differentiation in the aortic root, but to inhibit calcification and the expression of osteogenic markers in the aortic leaflet, partly explaining the absence of calcification in the aortic valve cultured with OSM. The osteogenic calcifications in the aortic leaflet and the endochondral differentiation in the aortic root resemble calcifications found in human CAVD. In conclusion, we have established an ex vivo calcification model for intact wild-type murine aortic valves in which the initiation and progression of aortic valve calcification can be studied. The in vitro-ex vivo differences found in our studies underline the importance of ex vivo models to facilitate pre-clinical translational studies.

The roadmap of WT1 protein expression in the human fetal heart.

The transcription factor Wilms' Tumor-1 (WT1) is essential for cardiac development. Deletion of Wt1 in mice results in disturbed epicardial and myocardial formation and lack of cardiac vasculature, causing embryonic lethality. Little is known about the role of WT1 in the human fetal heart. Therefore, as a first step, we analyzed the expression pattern of WT1 protein during human cardiac development from week 4 till week 20. WT1 expression was apparent in epicardial, endothelial and endocardial cells in a spatiotemporal manner. The expression of WT1 follows a pattern starting at the epicardium and extending towards the lumen of the heart, with differences in timing and expression levels between the atria and ventricles. The expression of WT1 in cardiac arterial endothelial cells reduces in time, whereas WT1 expression in the endothelial cells of cardiac veins and capillaries remains present at all stages studied. This study provides for the first time a detailed description of the expression of WT1 protein during human cardiac development, which indicates an important role for WT1 also in human cardiogenesis.

Tbx5 is required for avian and Mammalian epicardial formation and coronary vasculogenesis.

RATIONALE: Holt-Oram syndrome is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene. Overexpression of Tbx5 in the chick proepicardial organ impaired coronary blood vessel formation. However, the potential activity of Tbx5 in the epicardium itself, and the role of Tbx5 in mammalian coronary vasculogenesis, remains largely unknown. OBJECTIVE: To evaluate the consequences of altered Tbx5 gene dosage during proepicardial organ and epicardial development in the embryonic chick and mouse. METHODS AND RESULTS: Retroviral-mediated knockdown or upregulation of Tbx5 expression in the embryonic chick proepicardial organ and proepicardial-specific deletion of Tbx5 in the embryonic mouse (Tbx5(epi-/)) impaired normal proepicardial organ cell development, inhibited epicardial and coronary blood vessel formation, and altered developmental gene expression. The generation of epicardial-derived cells and their migration into the myocardium were impaired between embryonic day (E) 13.5 to 15.5 in mutant hearts because of delayed epicardial attachment to the myocardium and subepicardial accumulation of epicardial-derived cells. This caused defective coronary vasculogenesis associated with impaired vascular smooth muscle cell recruitment and reduced invasion of cardiac fibroblasts and endothelial cells into myocardium. In contrast to wild-type hearts that exhibited an elaborate ventricular vascular network, Tbx5(epi-/-) hearts displayed a marked decrease in vascular density that was associated with myocardial hypoxia as exemplified by hypoxia inducible factor-1α upregulation and increased binding of hypoxyprobe-1. Tbx5(epi-/-) mice with such myocardial hypoxia exhibited reduced exercise capacity when compared with wild-type mice. CONCLUSIONS: Our findings support a conserved Tbx5 dose-dependent requirement for both proepicardial and epicardial progenitor cell development in chick and in mouse coronary vascular formation.

Cardiac endothelial cells express Wilms' tumor-1: Wt1 expression in the developing, adult and infarcted heart.

Myocardial infarction is the leading cause of death worldwide. Due to their limited regenerative capacity lost cardiomyocytes are replaced by a non-contractile fibrotic scar tissue. The epicardial layer of the heart provides cardiac progenitor cells during development. Because this layer regains embryonic characteristics in the adult heart after cardiac injury, it could serve as a promising source for resident cardiac progenitor cells. Wilms' tumor-1 (Wt1) is associated with the activation and reactivation of the epicardium and therefore potentially important for the differentiation and regenerative capacity of the epicardium. To gain more insight into the regulation of Wt1 we examined the spatiotemporal expression pattern of Wt1 during murine development and after cardiac injury. Interestingly, we found that Wt1 is expressed in the majority of the cardiac endothelial cells within the myocardial ventricular layer of the developing heart from E12.5 onwards. In the adult heart only a subset of coronary endothelial cells remains positive for Wt1. After myocardial infarction Wt1 is temporally upregulated in the endothelial cells of the infarcted area and the border zone of the heart. In vitro experiments show that endothelial Wt1 expression can be induced by hypoxia. We show that Wt1 is associated with endothelial cell proliferation: Wt1 expression is higher in proliferating endothelial cells, Wt1 knockdown inhibits the proliferation of endothelial cells, and Wt1 regulates CyclinD1 expression. Finally, endothelial cells lacking Wt1 are not capable to establish a proper vascular network in vitro. Together, these results suggest a possible role for Wt1 in cardiac vessel formation in development and disease.

The epicardium as modulator of the cardiac autonomic response during early development.

The cardiac autonomic nervous system (cANS) modulates heart rate, contraction force and conduction velocity. The embryonic chicken heart already responds to epinephrine prior to establishment of the cANS. The aim of this study was to define the regions of the heart that might participate in modulating the early autonomic response to epinephrine. Immunofluorescence analysis reveals expression of neural markers tubulin beta-3 chain and neural cell adhesion molecule in the epicardium during early development. In addition, expression of the ß2 adrenergic receptor, the receptor for epinephrine, was found in the epicardium. Ex-ovo micro-electrode recordings in hearts with inhibition of epicardial outgrowth showed a significantly reduced response of the heart rate to epinephrine compared to control hearts. This study suggests a role for the epicardium as autonomic modulator during early cardiac development.

Extraembryonic endoderm cells as a model of endoderm development.

In recent years the multipotent extraembryonic endoderm (XEN) stem cells have been the center of much attention. In vivo, XEN cells contribute to the formation of the extraembryonic endoderm, visceral and parietal endoderm and later on, the yolk sac. Recent data have shown that the distinction between embryonic and extraembryonic endoderm is not as strict as previously thought due to the integration, and not the displacement, of the visceral endoderm into the definitive embryonic endoderm. Therefore, cells from the extraembryonic endoderm also contribute to definitive endoderm. Many research groups focused on unraveling the potential and ability of XEN cells to both support differentiation and/or differentiate into endoderm-like tissues as an alternative to embryonic stem (ES) cells. Moreover, the conversion of ES to XEN cells, shown recently without genetic manipulations, uncovers significant and novel molecular mechanisms involved in extraembryonic endoderm and definitive endoderm development. XEN cell lines provide a unique model for an early mammalian lineage that complements the established ES and trophoblast stem cell lines. Through the study of essential genes and signaling requirements for XEN cells in vitro, insights will be gained about the developmental program of the extraembryonic and embryonic endodermal lineage in vivo. This review will provide an overview on the current literature focusing on XEN cells as a model for primitive endoderm and possibly definitive endoderm as well as the potential of using these cells for therapeutic applications.

TGFß and BMP signaling in cardiac cushion formation: lessons from mice and chicken.

Cardiac cushion formation is crucial for both valvular and septal development. Disruption in this process can lead to valvular and septal malformations, which constitute the largest part of congenital heart defects. One of the signaling pathways that is important for cushion formation is the TGFß superfamily. The involvement of TGFß and BMP signaling pathways in cardiac cushion formation has been intensively studied using chicken in vitro explant assays and in genetically modified mice. In this review, we will summarize and discuss the role of TGFß and BMP signaling components in cardiac cushion formation.

An in vivo map of bone morphogenetic protein 2 post-transcriptional repression in the heart.

The Bmp2 3'untranslated region (UTR) sequence bears a sequence conserved between mammals and fishes that can post-transcriptionally activate or repress protein synthesis. We developed a map of embryonic cells in the mouse where this potent Bmp2 regulatory sequence functions by using a lacZ reporter transgene with a 3'UTR bearing two loxP sites flanking the ultra-conserved sequence. Cre-recombinase-mediated deletion of the ultra-conserved sequence caused strong ectopic expression in proepicardium, epicardium and epicardium-derived cells (EPDC) and in tissues with known epicardial contributions (coronary vessels and valves). Transient transfections of reporters in the epicardial/mesothelial cell (EMC) line confirmed this repression. Ectopic expression of the recombined transgene also occurred in the aorta, outlet septum, posterior cardiac plexus, cardiac and extracardiac nerves and neural ganglia. Bmp2 is dynamically regulated in the developing heart. 3'UTR-mediated mechanisms that restrain BMP2 synthesis may be relevant to congenital heart and vasculature malformations and to adult diseases involving aberrant BMP2 synthesis.

An autonomous BMP2 regulatory element in mesenchymal cells.

BMP2 is a morphogen that controls mesenchymal cell differentiation and behavior. For example, BMP2 concentration controls the differentiation of mesenchymal precursors into myocytes, adipocytes, chondrocytes, and osteoblasts. Sequences within the 3'untranslated region (UTR) of the Bmp2 mRNA mediate a post-transcriptional block of protein synthesis. Interaction of cell and developmental stage-specific trans-regulatory factors with the 3'UTR is a nimble and versatile mechanism for modulating this potent morphogen in different cell types. We show here, that an ultra-conserved sequence in the 3'UTR functions independently of promoter, coding region, and 3'UTR context in primary and immortalized tissue culture cells and in transgenic mice. Our findings indicate that the ultra-conserved sequence is an autonomously functioning post-transcriptional element that may be used to modulate the level of BMP2 and other proteins while retaining tissue specific regulatory elements.

Superimposed Tissue Formation in Human Aortic Valve Disease: Differences between Regurgitant and Stenotic Valves.

The formation of superimposed tissue (SIT), a layer on top of the original valve leaflet, has been described in patients with mitral regurgitation as a major contributor to valve thickening and possibly as a result of increased mechanical stresses. However, little is known whether SIT formation also occurs in aortic valve disease. We therefore performed histological analyses to assess SIT formation in aortic valve leaflets (n = 31) from patients with aortic stenosis (n = 17) or aortic regurgitation due to aortic dilatation (n = 14). SIT was observed in both stenotic and regurgitant aortic valves, both on the ventricular and aortic sides, but with significant differences in distribution and composition. Regurgitant aortic valves showed more SIT formation in the free edge, leading to a thicker leaflet at that level, while stenotic aortic valves showed relatively more SIT formation on the aortic side of the body part of the leaflet. SIT appeared to be a highly active area, as determined by large populations of myofibroblasts, with varied extracellular matrix composition (higher collagen content in stenotic valves). Further, the identification of the SIT revealed the presence of foldings of the free edge in the diseased aortic valves. Insights into SIT regulation may further help in understanding the pathophysiology of aortic valve disease and potentially lead to the development of new therapeutic treatments.

Characterization of Degenerative Mitral Valve Disease: Differences between Fibroelastic Deficiency and Barlow's Disease.

Degenerative mitral valve disease causing mitral valve prolapse is the most common cause of primary mitral regurgitation, with two distinct phenotypes generally recognized with some major differences, i.e., fibroelastic deficiency (FED) and Barlow's disease. The aim of this review was to describe the main histological, clinical and echocardiographic features of patients with FED and Barlow's disease, highlighting the differences in diagnosis, risk stratification and patient management, but also the still significant gaps in understanding the exact pathophysiology of these two phenotypes.

Oncofetal Protein CRIPTO Is Involved in Wound Healing and Fibrogenesis in the Regenerating Liver and Is Associated with the Initial Stages of Cardiac Fibrosis.

Oncofetal protein, CRIPTO, is silenced during homeostatic postnatal life and often re-expressed in different neoplastic processes, such as hepatocellular carcinoma. Given the reactivation of CRIPTO in pathological conditions reported in various adult tissues, the aim of this study was to explore whether CRIPTO is expressed during liver fibrogenesis and whether this is related to the disease severity and pathogenesis of fibrogenesis. Furthermore, we aimed to identify the impact of CRIPTO expression on fibrogenesis in organs with high versus low regenerative capacity, represented by murine liver fibrogenesis and adult murine heart fibrogenesis. Circulating CRIPTO levels were measured in plasma samples of patients with cirrhosis registered at the waitlist for liver transplantation (LT) and 1 year after LT. The expression of CRIPTO and fibrotic markers (αSMA, collagen type I) was determined in human liver tissues of patients with cirrhosis (on a basis of viral hepatitis or alcoholic disease), in cardiac tissue samples of patients with end-stage heart failure, and in mice with experimental liver and heart fibrosis using immuno-histochemical stainings and qPCR. Mouse models with experimental chronic liver fibrosis, induced with multiple shots of carbon tetrachloride (CCl4) and acute liver fibrosis (one shot of CCl4), were evaluated for CRIPTO expression and fibrotic markers. CRIPTO was overexpressed in vivo (Adenoviral delivery) or functionally sequestered by ALK4Fc ligand trap in the acute liver fibrosis mouse model. Murine heart tissues were evaluated for CRIPTO and fibrotic markers in three models of heart injury following myocardial infarction, pressure overload, and ex vivo induced fibrosis. Patients with end-stage liver cirrhosis showed elevated CRIPTO levels in plasma, which decreased 1 year after LT. Cripto expression was observed in fibrotic tissues of patients with end-stage liver cirrhosis and in patients with heart failure. The expression of CRIPTO in the liver was found specifically in the hepatocytes and was positively correlated with the Model for End-stage Liver Disease (MELD) score for end-stage liver disease. CRIPTO expression in the samples of cardiac fibrosis was limited and mostly observed in the interstitial cells. In the chronic and acute mouse models of liver fibrosis, CRIPTO-positive cells were observed in damaged liver areas around the central vein, which preceded the expression of αSMA-positive stellate cells, i.e., mediators of fibrosis. In the chronic mouse models, the fibrosis and CRIPTO expression were still present after 11 weeks, whereas in the acute model the liver regenerated and the fibrosis and CRIPTO expression resolved. In vivo overexpression of CRIPTO in this model led to an increase in fibrotic markers, while blockage of CRIPTO secreted function inhibited the extent of fibrotic areas and marker expression (αSMA, Collagen type I and III) and induced higher proliferation of residual healthy hepatocytes. CRIPTO expression was also upregulated in several mouse models of cardiac fibrosis. During myocardial infarction CRIPTO is upregulated initially in cardiac interstitial cells, followed by expression in αSMA-positive myofibroblasts throughout the infarct area. After the scar formation, CRIPTO expression decreased concomitantly with the αSMA expression. Temporal expression of CRIPTO in αSMA-positive myofibroblasts was also observed surrounding the coronary arteries in the pressure overload model of cardiac fibrosis. Furthermore, CRIPTO expression was upregulated in interstitial myofibroblasts in hearts cultured in an ex vivo model for cardiac fibrosis. Our results are indicative for a functional role of CRIPTO in the induction of fibrogenesis as well as a potential target in the antifibrotic treatments and stimulation of tissue regeneration.

Stress-induced remodelling of the mitral valve: a model for leaflet thickening and superimposed tissue formation in mitral valve disease.

AIMS: In mitral valve prolapse (MVP), leaflet thickening has recently been suggested to be due, in addition to a myxomatous degeneration, to the presence of a superimposed tissue (SIT), defined as an additional fibrous layer on top of the original leaflet. The mechanisms of SIT formation are currently unknown. We hypothesized that SIT formation would result from excessive leaflet stress and we used a unique ex vivo model to assess the correlation between leaflet remodelling and the type and location of mechanical stress and to elucidate the mechanisms underlying SIT formation. METHODS AND RESULTS: Human diseased mitral valves (MVs; n = 21) were histologically analysed for SIT formation and original leaflet thickening. The SIT comprised of various compositions of extracellular matrix and could reach more than 50% of total leaflet thickness. Original leaflet and SIT thickness did not show significant correlation (r = -0.27, P = 0.23), suggesting different regulatory mechanisms. To study the role of the mechanical environment on MV remodelling, mouse MV were cultured in their natural position in the heart and subjected to various haemodynamic conditions representing specific phases of the cardiac cycle and the MVP configuration. SIT formation was induced in the ex vivo model, mostly present on the atrial side, and clearly dependent on the duration, type, and extent of mechanical stress. Specific stainings and lineage tracing experiments showed that SIT comprises of macrophages and myofibroblasts and is associated with the activation of the transforming growth factor-beta and bone morphogenetic protein signalling pathways. Migration of valvular interstitial cells and macrophages through breakages of the endothelial cell lining contributed to SIT formation. CONCLUSIONS: Mechanical stresses induce specific cellular and molecular changes in the MV that result in SIT formation. These observations provide the first insights in the mechanism of SIT formation and represent an initial step to identify potential novel and early treatment for MVP.

Immunofluorescent Visualization of BMP Signaling Activation on Paraffin-Embedded Tissue Sections.

Immunohistochemistry allows the detection of the presence, localization, and activation of proteins in biological tissues by using the ability of antibodies to bind to specific antigens. Cellular signaling can be visualized using antibodies raised against phosphorylated proteins. Phosphorylated Smad1, Smad5, and Smad9 are the activated signaling molecules of the BMP pathway that transfer BMP signals from the cell surface to the nucleus. Here we describe the detection of phospho-Smad1/5/9 on paraformaldehyde-fixed and paraffin-embedded tissue sections by immunofluorescence.

In vivo and in vitro Approaches Reveal Novel Insight Into the Ability of Epicardium-Derived Cells to Create Their Own Extracellular Environment.

Human epicardium-derived cells (hEPDCs) transplanted in the NOD-SCID mouse heart after myocardial infarction (MI) are known to improve cardiac function, most likely orchestrated by paracrine mechanisms that limit adverse remodeling. It is not yet known, however, if hEPDCs contribute to preservation of cardiac function via the secretion of matrix proteins and/or matrix proteases to reduce scar formation. This study describes the ability of hEPDCs to produce human collagen type I after transplantation into the infarct border zone, thereby creating their own extracellular environment. As the in vivo environment is too complex to investigate the mechanisms involved, we use an in vitro set-up, mimicking biophysical and biochemical cues from the myocardial tissue to unravel hEPDC-induced matrix remodeling. The in vivo contribution of hEPDCs to the cardiac extracellular matrix (ECM) was assessed in a historical dataset of the NOD-SCID murine model of experimentally induced MI and cell transplantation. Analysis showed that within 48 h after transplantation, hEPDCs produce human collagen type I. The build-up of the human collagen microenvironment was reversed within 6 weeks. To understand the hEPDCs response to the pathologic cardiac microenvironment, we studied the influence of cyclic straining and/or transforming growth beta (TGFß) signaling in vitro. We revealed that 48 h of cyclic straining induced collagen type I production via the TGFß/ALK5 signaling pathway. The in vitro approach enables further unraveling of the hEPDCs ability to secrete matrix proteins and matrix proteases and the potential to create and remodel the cardiac matrix in response to injury.

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System.

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

Regional differences in WT-1 and Tcf21 expression during ventricular development: implications for myocardial compaction.

BACKGROUND: Morphological and functional differences of the right and left ventricle are apparent in the adult human heart. A differential contribution of cardiac fibroblasts and smooth muscle cells (populations of epicardium-derived cells) to each ventricle may account for part of the morphological-functional disparity. Here we studied the relation between epicardial derivatives and the development of compact ventricular myocardium. RESULTS: Wildtype and Wt1CreERT2/+ reporter mice were used to study WT-1 expressing cells, and Tcf21lacZ/+ reporter mice and PDGFRα-/-;Tcf21LacZ/+ mice to study the formation of the cardiac fibroblast population. After covering the heart, intramyocardial WT-1+ cells were first observed at the inner curvature, the right ventricular postero-lateral wall and left ventricular apical wall. Later, WT-1+ cells were present in the walls of both ventricles, but significantly more pronounced in the left ventricle. Tcf21-LacZ + cells followed the same distribution pattern as WT-1+ cells but at later stages, indicating a timing difference between these cell populations. Within the right ventricle, WT-1+ and Tcf21-lacZ+ cell distribution was more pronounced in the posterior inlet part. A gradual increase in myocardial wall thickness was observed early in the left ventricle and at later stages in the right ventricle. PDGFRα-/-;Tcf21LacZ/+ mice showed deficient epicardium, diminished number of Tcf21-LacZ + cells and reduced ventricular compaction. CONCLUSIONS: During normal heart development, spatio-temporal differences in contribution of WT-1 and Tcf21-LacZ + cells to right versus left ventricular myocardium occur parallel to myocardial thickening. These findings may relate to lateralized differences in ventricular (patho)morphology in humans.

Mutations in a TGF-ß ligand, TGFB3, cause syndromic aortic aneurysms and dissections.

BACKGROUND: Aneurysms affecting the aorta are a common condition associated with high mortality as a result of aortic dissection or rupture. Investigations of the pathogenic mechanisms involved in syndromic types of thoracic aortic aneurysms, such as Marfan and Loeys-Dietz syndromes, have revealed an important contribution of disturbed transforming growth factor (TGF)-ß signaling. OBJECTIVES: This study sought to discover a novel gene causing syndromic aortic aneurysms in order to unravel the underlying pathogenesis. METHODS: We combined genome-wide linkage analysis, exome sequencing, and candidate gene Sanger sequencing in a total of 470 index cases with thoracic aortic aneurysms. Extensive cardiological examination, including physical examination, electrocardiography, and transthoracic echocardiography was performed. In adults, imaging of the entire aorta using computed tomography or magnetic resonance imaging was done. RESULTS: Here, we report on 43 patients from 11 families with syndromic presentations of aortic aneurysms caused by TGFB3 mutations. We demonstrate that TGFB3 mutations are associated with significant cardiovascular involvement, including thoracic/abdominal aortic aneurysm and dissection, and mitral valve disease. Other systemic features overlap clinically with Loeys-Dietz, Shprintzen-Goldberg, and Marfan syndromes, including cleft palate, bifid uvula, skeletal overgrowth, cervical spine instability and clubfoot deformity. In line with previous observations in aortic wall tissues of patients with mutations in effectors of TGF-ß signaling (TGFBR1/2, SMAD3, and TGFB2), we confirm a paradoxical up-regulation of both canonical and noncanonical TGF-ß signaling in association with up-regulation of the expression of TGF-ß ligands. CONCLUSIONS: Our findings emphasize the broad clinical variability associated with TGFB3 mutations and highlight the importance of early recognition of the disease because of high cardiovascular risk.

Recruitment of intra- and extracardiac cells into the myocardial lineage during mouse development.

The tubular heart differentiates from the bilateral cardiac fields in the splanchnic mesoderm. The expression of smooth muscle proteins has been shown to accompany the early phases of cardiac muscle formation. In this study we show that during elongation of the arterial pole of the mouse linear heart tube, alpha-smooth muscle actin (alpha-Sma) expression extends in the area that has been shown to become recruited into the myocardial lineage, but does not yet express myocardial markers. These data suggest that alpha-Sma identifies mesodermal cells that during subsequent development will be recruited into the myocardial lineage. Myocardium formation is not only observed at the arterial pole, but also at the venous pole and in the intracardiac mesenchyme. This results in the formation of the caval and pulmonary myocardium, the smooth-walled atrial myocardium, the myocardial atrioventricular septum, and the myocardial outlet septum. To determine whether recruitment into the myocardial lineage also takes place in these regions, the spatiotemporal pattern of expression of alpha-Sma and of the myocardial markers sarcoplasmatic reticulum calcium ATPase (Serca2a), alpha-myosin heavy chain (Mhc), and beta-Mhc were examined. We show that prior to the expression of myocardial markers, alpha-Sma is expressed in these regions, which suggests that these mesodermal cells become recruited into the cardiac lineage after formation of the linear heart tube.