Myocardial tissue caveolae.

Caveolae and their coat proteins, caveolins (Cav), are cave-like invaginations found in the plasma membrane of a variety of cells. These unique vesicles and their coat proteins, Cavs, have diverse effects on endothelial function, nitric oxide synthesis regulation, signal transduction, cholesterol metabolism, and apoptosis. Animal studies in Cav knockout mice demonstrate the vital role of these structural proteins on endothelial and vascular function. Genetic studies have proposed that beside neoplasia, Cavs may play a role in the development of atherosclerosis, cardiomyopathy, long QT syndrome, pulmonary fibrosis, and muscular dystrophy. The role of Cav expression in atherosclerotic disease is poorly understood and remains controversial. Interestingly, there is emerging evidence between low Cav-1 levels and the vulnerable plaque, which could potentially identify Cav-1 as a novel plaque biomarker. Cavs, through intricate biochemical pathways involving endothelial nitric oxide synthase and mitogen-activated protein kinase, are known to affect the cardiovascular system at multiple levels. In the present review, we aim to highlight the nature and types of caveolae, caveolar signaling mechanisms and regulation, and the pathophysiology of Cavs as it pertains to the cardiovascular system. Ongoing research is needed to clarify the diagnostic and prognostic role of these novel proteins and to determine how the effects of Cavs can translate into clinical medicine.

Caveolin-1 deletion exacerbates cardiac interstitial fibrosis by promoting M2 macrophage activation in mice after myocardial infarction.

Adverse remodeling following myocardial infarction (MI) leading to heart failure is driven by an imbalanced resolution of inflammation. The macrophage cell is an important control of post-MI inflammation, as macrophage subtypes secrete mediators to either promote inflammation and extend injury (M1 phenotype) or suppress inflammation and promote scar formation (M2 phenotype). We have previously shown that the absence of caveolin-1 (Cav1), a membrane scaffolding protein, is associated with adverse cardiac remodeling in mice, but the mechanisms responsible remain to be elucidated. We explore here the role of Cav1 in the activation of macrophages using wild type C57BL6/J (WT) and Cav1(tm1Mls/J) (Cav1(-/-)) mice. By echocardiography, cardiac function was comparable between WT and Cav1(-/-) mice at 3days post-MI. In the absence of Cav1, there were a surprisingly higher percentage of M2 macrophages (arginase-1 positive) detected in the infarcted zone. Conversely, restoring Cav1 function after MI in WT mice by adding back the Cav1 scaffolding domain reduced the M2 activation profile. Further, adoptive transfer of Cav1 null macrophages into WT mice on d3 post-MI exacerbated adverse cardiac remodeling at d14 post-MI. In vitro studies revealed that Cav1 null macrophages had a more pronounced M2 profile activation in response to IL-4 stimulation. In conclusion, Cav1 deletion promotes an array of maladaptive repair processes after MI, including increased TGF-ß signaling, increased M2 macrophage infiltration and dysregulation of the M1/M2 balance. Our data also suggest that cardiac remodeling can be improved by therapeutic intervention regulating Cav1 function during the inflammatory response phase.

Imbalanced expression of Vcan mRNA splice form proteins alters heart morphology and cellular protein profiles.

The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart's pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican's expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan ((tm1Zim)), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan ((tm1Zim)) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.

Versican G1 domain and V3 isoform overexpression results in increased chondrogenesis in the developing chick limb in ovo.

Previous work has shown that versican proteoglycan is highly expressed in the extracellular matrix of precartilage limb mesenchyme. Although much of versican's role in chondrogenesis has been attributed to its glycosaminoglycan complement, N- and C-terminal G1 and G3 domains of versican have been shown to possess distinct functions when expressed ectopically. This study was undertaken to test the hypothesis that overexpression of the versican G1 domain and short V3 isoform, comprised of only G1 and G3, in the chick wing in ovo would result in increased chondrogenesis, suggesting function for discrete versican domains in limb skeletal development. Recombinant adenoviruses encoding G1 and V3 proteins were microinjected into proximal HH19-25 chick wing buds which resulted in significant enlargement of humeral primordia at HH35. Enhanced cartilage deposition appeared due to increased chondrogenic aggregation as a result of recombinant G1 or V3 overexpression, further implicating versican in early stages of limb development.

Fibulin-1 is required for morphogenesis of neural crest-derived structures.

Here we report that mouse embryos homozygous for a gene trap insertion in the fibulin-1 (Fbln1) gene are deficient in Fbln1 and exhibit cardiac ventricular wall thinning and ventricular septal defects with double outlet right ventricle or overriding aorta. Fbln1 nulls also display anomalies of aortic arch arteries, hypoplasia of the thymus and thyroid, underdeveloped skull bones, malformations of cranial nerves and hemorrhagic blood vessels in the head and neck. The spectrum of malformations is consistent with Fbln1 influencing neural crest cell (NCC)-dependent development of these tissues. This is supported by evidence that Fbln1 expression is associated with streams of cranial NCCs migrating adjacent to rhombomeres 2-7 and that Fbln1-deficient embryos display patterning anomalies of NCCs forming cranial nerves IX and X, which derive from rhombomeres 6 and 7. Additionally, Fbln1-deficient embryos show increased apoptosis in areas populated by NCCs derived from rhombomeres 4, 6 and 7. Based on these findings, it is concluded that Fbln1 is required for the directed migration and survival of cranial NCCs contributing to the development of pharyngeal glands, craniofacial skeleton, cranial nerves, aortic arch arteries, cardiac outflow tract and cephalic blood vessels.

Characterization of the cardiac phenotype in neonatal Ts65Dn mice.

The Ts65Dn mouse is the most-studied of murine models for Down syndrome. Homology between the triplicated murine genes and those on human chromosome 21 correlates with shared anomalies of Ts65Dn mice and Down syndrome patients, including congenital heart defects. Lethality is associated with inheritance of the T65Dn chromosome, and anomalies such as right aortic arch with Kommerell's diverticulum and interrupted aortic arch were found in trisomic neonates. The incidence of gross vascular abnormalities was 17% in the trisomic population. Histological analyses revealed interventricular septal defects and broad foramen ovale, while immunohistochemistry showed abnormal muscle composition in the cardiac valves of trisomic neonates. These findings confirm that the gene imbalance present in Ts65Dn disrupts crucial pathways during cardiac development. The candidate genes for congenital heart defects that are among the 104 triplicated genes in Ts65Dn mice are, therefore, implicated in the dysregulation of normal cardiogenic pathways in this model.

Cartilage link protein 1 (Crtl1), an extracellular matrix component playing an important role in heart development.

To expand our insight into cardiac development, a comparative DNA microarray analysis was performed using tissues from the atrioventricular junction (AVJ) and ventricular chambers of mouse hearts at embryonic day (ED) 10.5-11.0. This comparison revealed differential expression of approximately 200 genes, including cartilage link protein 1 (Crtl1). Crtl1 stabilizes the interaction between hyaluronan (HA) and versican, two extracellular matrix components essential for cardiac development. Immunohistochemical studies showed that, initially, Crtl1, versican, and HA are co-expressed in the endocardial lining of the heart, and in the endocardially derived mesenchyme of the AVJ and outflow tract (OFT). At later stages, this co-expression becomes restricted to discrete populations of endocardially derived mesenchyme. Histological analysis of the Crtl1-deficient mouse revealed a spectrum of cardiac malformations, including AV septal and myocardial defects, while expression studies showed a significant reduction in versican levels. Subsequent analysis of the hdf mouse, which carries an insertional mutation in the versican gene (CSPG2), demonstrated that haploinsufficient versican mice display septal defects resembling those seen in Crtl1(-/-) embryos, suggesting that reduced versican expression may contribute to a subset of the cardiac abnormalities observed in the Crtl1(-/-) mouse. Combined, these findings establish an important role for Crtl1 in heart development.

Periostin regulates collagen fibrillogenesis and the biomechanical properties of connective tissues.

Periostin is predominantly expressed in collagen-rich fibrous connective tissues that are subjected to constant mechanical stresses including: heart valves, tendons, perichondrium, cornea, and the periodontal ligament (PDL). Based on these data we hypothesize that periostin can regulate collagen I fibrillogenesis and thereby affect the biomechanical properties of connective tissues. Immunoprecipitation and immunogold transmission electron microscopy experiments demonstrate that periostin is capable of directly interacting with collagen I. To analyze the potential role of periostin in collagen I fibrillogenesis, gene targeted mice were generated. Transmission electron microscopy and morphometric analyses demonstrated reduced collagen fibril diameters in skin dermis of periostin knockout mice, an indication of aberrant collagen I fibrillogenesis. In addition, differential scanning calorimetry (DSC) demonstrated a lower collagen denaturing temperature in periostin knockout mice, reflecting a reduced level of collagen cross-linking. Functional biomechanical properties of periostin null skin specimens and atrioventricular (AV) valve explant experiments provided direct evidence of the role that periostin plays in regulating the viscoelastic properties of connective tissues. Collectively, these data demonstrate for the first time that periostin can regulate collagen I fibrillogenesis and thereby serves as an important mediator of the biomechanical properties of fibrous connective tissues.

Versican proteolysis mediates myocardial regression during outflow tract development.

An important phase of cardiac outflow tract (OFT) formation is the remodeling of the distal region of the common outlet in which the myocardial sleeve is replaced by with smooth muscle. Here we demonstrate that expression of the proteoglycan versican is reduced before the loss of myocardium from the distal cardiac outlet concomitant with an increase in production of the N-terminal cleavage fragment of versican. To test whether versican proteolysis plays a role in OFT remodeling, we determined the effects of adenoviral-mediated expression of a versican isoform devoid of known matrix metalloproteinase cleavage sites (V3) and an N-terminal fragment of versican (G1). V3 expression promoted an increase in thickness of the proximal OFT myocardial layer independent of proliferation. In contrast, the G1 domain caused thinning and interruptions of the OFT myocardium. These in vivo findings were consistent with findings using cultured primary cardiomyocytes showing that the V3 promoted myocardial cell-cell association while the G1 domain caused a loss of myocardial cell-cell association. Taken together, we conclude that intact versican and G1-containing versican cleavage products have opposing effects on myocardial cells and that versican proteolysis may facilitate the loss of distal myocardium during OFT remodeling.

Periostin promotes atrioventricular mesenchyme matrix invasion and remodeling mediated by integrin signaling through Rho/PI 3-kinase.

Recent evidence suggests that extracellular matrix components may play a signaling role in embryonic valve development. We have previously identified the spatiotemporal expression patterns of periostin in developing valves, but its function during this process is largely unknown. To evaluate the functional role periostin plays during valvulogenesis, two separate three-dimensional culture assay systems, which model chick atrioventricular cushion development, were employed. These assays demonstrated that cushion mesenchymal cells adhered and spread on purified periostin in a dose-responsive manner, similar to collagen I and fibronectin via alpha(v)beta(3) and beta(1) integrin pairs. Periostin overexpression resulted in enhanced mesenchyme invasion through 3D collagen gels and increased matrix compaction. This invasion was dependent on alpha(v)beta(3) more than beta(1) integrin signaling, and was mediated differentially by Rho kinase and PI 3-kinase. Both matrix invasion and compaction were associated with a colocalization of periostin and beta(1) integrin expression to migratory cell phenotype in both surface and deep cells. The Rho/PI 3-kinase pathway also differentially mediated matrix compaction. Both Rho and PI 3-kinase were involved in normal cushion mesenchyme matrix compaction, but only PI 3-kinase was required for the enhanced matrix compaction due to periostin. Taken together, these results highlight periostin as a mediator of matrix remodeling by cushion mesenchyme towards a mature valve structure.

Proteolytic cleavage of versican during cardiac cushion morphogenesis.

The proteoglycan versican is essential to the formation of endocardial cushion mesenchyme by epithelial-mesenchymal transformation (EMT). A potentially important factor in the regulation of versican activity during cushion EMT is proteolysis by ADAMTS metalloproteinases. Using antibodies to the DPEAAE neoepitope created by ADAMTS proteolysis of versican, we detected the amino terminal 70-kDa versican cleavage fragment in cardiac cushions. Initially (i.e., 9.5 days post coitum [dpc]), the fragment is associated with endocardial cells undergoing EMT and with newly derived mesenchymal cells. ADAMTS-1 and its cofactor fibulin-1 were also associated with these cells. As cushions become increasingly populated with mesenchymal cells (10.5-12.5 dpc), the fragment remains asymmetrically distributed compared with the pattern of total versican. Highest levels of the fragment are present in regions immediately subjacent to the endocardium characterized as having densely packed, rounded cells, lacking cellular protrusions. With further development (i.e., 12.5-14.5 dpc), the pattern of fragment distribution within cushions broadens to include the ECM surrounding loosely packed mesenchymal cells in the cushion core. Together, the findings reveal that versican proteolysis leading to the production of the 70-kDa fragment is integral to the formation and differentiation of endocardial cushion mesenchyme.

Hot hearts in the Sonoran Desert: the 11th Weinstein Cardiovascular Development Conference in Tucson.

The 11th Annual Weinstein Cardiovascular Development Conference was held May 19-22, 2005 at the Westward Look Resort and Conference Center in Tucson, Arizona. The Westward Look was the site of the 6th Weinstein Meeting in 1999, and this year, 330 basic research scientists and research clinicians returned to Tucson for 3 days of meetings, Mariachis, and margaritas. The meeting was hosted by the cardiovascular research group at the University of Arizona and offered flavors of the desert southwest that included record temperatures, the Skopopelli conference logo modified from the Kokopelli of Native American mythology (Fig. 1), and liberal use of a cattle prod to encourage speaker timeliness.

GATA factors lie upstream of Nkx 2.5 in the transcriptional regulatory cascade that effects cardiogenesis.

Members of the GATA-4, -5, and -6 subfamily of transcription factors are co-expressed with the homeoprotein Nkx 2.5 in the precardiac mesoderm during the earliest stages of its specification and are known to be important determinants of cardiac gene expression. Ample evidence suggests that GATA factors and Nkx 2.5 cross-regulate each other's expression; however, the temporal order of the expression of these transcription factors in vivo remains unresolved, and thus precise definition of the role of the products of the genes they transcribe in early development has been difficult to assess. We employed P19 CL6 mouse embryonic carcinoma cells as a model to investigate this problem, because these cells, like embryonic stem cells, can be induced to differentiate along multiple lineages. Here we demonstrate that when P19 CL6 cells are induced to differentiate to a cardiogenic lineage, the expression of GATA-4 and GATA-6 is up-regulated prior to the transcriptional activation of Nkx 2.5. Moreover, over-expression of GATA-4 or -6 at the time of Nkx 2.5 induction results in a significant up-regulation of endogenous Nkx 2.5 transcription. Finally, it is known that a Nkx-dependent enhancer is necessary for GATA-6 expression within cardiomyocytes of the developing mouse embryo. We demonstrate that within undifferentiated P19 CL6 cells, GATA-6 expression is subject to active repression by a novel upstream element that possesses binding sites for factors involved in transcriptional repression that are conserved between mammalian species.

Detection of betaig-H3, a TGFbeta induced gene, during cardiac development and its complementary pattern with periostin.

Regulation of normal cardiac development involves numerous transcription factors, cytoskeletal proteins, signaling molecules, and extracellular matrix proteins. These key molecular components act in concert to induce morphological changes essential for the proper development of a functional four-chambered heart. Growth factors such as BMPs and TGFbeta's play a role in migration, proliferation and differentiation during cardiac development and are important regulators of the extracellular matrix (ECM). Genes responsive to these morphogens are likely to play an equally significant role during cardiac development. Therefore, we sought to clone the chicken TGFbeta induced gene betaig-H3 and evaluate its spatio-temporal expression during heart morphogenesis. Our studies show by Northern analysis, whole mount and section in situ hybridization experiments that betaig-H3 is expressed primarily in the mesenchyme of the atrioventricular and outflow tract cushions and later in the right and left atrioventricular valve leaflets and supporting valve structures. The mRNA expression domains of betaig-H3 show a complementary pattern compared to that of its highly homologous relative, periostin.

Limb chondrogenesis is compromised in the versican deficient hdf mouse.

It has been suggested that the matrix proteoglycan, versican, may perform a functional role during early events of limb skeletogenesis largely by virtue of its spatiotemporal expression pattern in precartilage mesenchymal aggregations. The versican-deficient hdf transgenic mouse has provided the first model to explore the implications of a null mature versican on limb chondrogenesis. Due to lethality of hdf homozygous embryos prior to limb cartilage differentiation, high-density micromass cultures were employed to compare the chondrogenic capacity of hdf mutant limb mesenchyme to that of wild-type. In homozygous hdf mesenchyme, aggregation was severely compromised and neither cartilage-characteristic Type II collagen nor alcian blue positive foci were detected during a 6-day period of culture. Three-dimensional culture of hdf mutant mesenchyme, however, showed that in a permissive environment mutant cells also expressed Type II collagen. Results strongly suggest that mature versican proteoglycan is essential for precartilage aggregation and subsequent cartilage differentiation.

Normal distribution of melanocytes in the mouse heart.

We report the consistent distribution of a population of pigmented trp-1-positive cells in several important septal and valvular structures of the normal mouse (C57BL/6) heart. The pigmented cell population was first apparent by E16.5 p.c. in the right atrial wall and extended into the atrium along the interatrial septum. By E17.5, these cells were found along the apical membranous interventricular septum near or below the surface of the endocardium. The most striking distribution of dark pigmented cells was found in the tricuspid and mitral valvular leaflets and chordae tendineae. The normal distribution of pigmented cells in the valvuloseptal apparatus of C57BL/6 adult heart suggests that a premelanocytic lineage may participate in the earlier morphogenesis of the valve leaflets and chordae tendineae. The origin of the premelanocyte lineage is currently unknown. The most likely candidate populations include the neural crest and the epicardially derived cells. The only cell type in the heart previously shown to form melanocytes is the neural crest. The presence of neural crest cells, but not melanocytes, in some of the regions we describe has been reported by others. However, previous reports have not shown a contribution of melanocytes or neural crest derivatives to the atrioventricular valve leaflets or chordae tendineae in mouse hearts. If these cells are of neural crest origin, it would suggest a possibly greater contribution and persistence of neural crest cells to the valvuloseptal apparatus than has been previously understood.

Immunolocalization of chick periostin protein in the developing heart.

The process that cardiac cushions undergo to form the mature septa and valves of the adult heart is poorly understood. Periostin is an extracellular molecule that is expressed during cushion mesenchyme formation and throughout valvulogenesis. Once thought to be an osteoblast-specific factor, studies have shown this molecule is antiosteogenic. We have produced an antibody to chicken periostin and examined periostin's localization in the developing avian heart. This antibody recognized proteins from chick heart lysates around 90 kD molecular weight as predicted from the chick periostin mRNA and other periostin orthologs. Periostin immunolocalization was first evident as fibrous strands in the cushion mesenchyme. At HH25, periostin was detected on the basal surface of the trabecular endothelium and also on the endocardial epithelium of the atrioventricular cushion. We hypothesize that periostin may function in the organization of extracellular matrix molecules, providing cues necessary for attachment and spreading during the epithelial-to-mesenchymal transitions of the endocardial epithelium. Enhanced secretion of periostin in the region of delamination may directly or indirectly promote change in the myocardium that precedes or mediates delamination of the leaflet. At later stages of development (HH34-38), periostin was seen predominantly in the fibrous regions of the heart, such as the left atrioventricular valve (LAV), annulus, cardiac skeleton, and adventitia. We propose that periostin is induced by sheer stress and may be an essential molecular component for structures of the heart that undergo mechanical stress or tension during the cardiac cycle.

Versican expression during skeletal/joint morphogenesis and patterning of muscle and nerve in the embryonic mouse limb.

Versican, an extracellular matrix proteoglycan, has been implicated in limb development and is expressed in precartilage mesenchymal condensations. However, studies have lacked precise spatial and temporal investigation of versican localization during skeletogenesis and its relationship to patterning of muscle and nerve during mammalian limb development. The transgenic mouse line hdf (heart defect), which bears a lacZ reporter construct disrupting Cspg2 encoding versican, allowed ready detection of hdf transgene expression through histochemical analysis. Hdf transgene expression in whole mount heterozygous embryos and localization of versican relative to cartilage, muscle, and nerve tissues in paraffin-embedded limb sections of wild-type embryos from 10.5-14 days postcoitum were evaluated by lacZ histochemistry, immunohistochemistry, and in situ hybridization. Versican was localized within precartilage condensations and nascent cartilages with expression diminishing during maturation of the cartilage model at later time points. Interestingly, versican remained highly expressed in developing synovial joint interzones, suggesting potential function for versican in joint morphogenesis. Isolated myoblasts, incipient skeletal muscle masses, and neurites were not present in areas of strong versican expression within the developing limb. Versican-expressing tissues may reserve space for the future limb skeleton and developing joints and may aid in patterning of muscle and nerve by deterring muscle migration and innervation into these regions.

Identification and detection of the periostin gene in cardiac development.

Periostin, a member of the fasciclin gene family, acts as a cell adhesion molecule through binding to cell surface integrins. Periostin expression has previously been shown to increase substantially following transforming growth factor beta (TGF-beta) and bone morphogenetic protein stimulation. As these molecules are indispensable for cardiac development, we sought to clone the chicken ortholog of periostin and evaluate its spatiotemporal expression pattern during heart morphogenesis. We show by Northern analysis, whole mount and section in situ hybridization experiments that periostin is predominantly expressed in the developing endothelium of the ventricular trabeculae as well as in the endothelium and mesenchyme of the outflow tract and atrioventricular endocardial cushions. Cardiac expression continues into fetal development where periostin is seen predominantly in the valve leaflets and supporting chordae tendinae.