Bone morphogenetic protein-2 can mediate myocardial regulation of atrioventricular cushion mesenchymal cell formation in mice

Transformation of endocardial endothelial cells into invasive mesenchyme is a critical antecedent of cardiac cushion tissue formation. The message for bone morphogenetic protein (BMP)-2 is known to be expressed in myocardial cells in a manner consistent with the segmental pattern of cushion formation [Development 109(1990) 833]. In the present work, we localized BMP-2 protein in atrioventricular (AV) myocardium in mice at embryonic day (ED) 8.5 (12 somite stage) before the onset of AV mesenchymal cell formation at ED 9.5. BMP-2 protein expression was absent from ventricular myocardium throughout the stages examined. After cellularization of the AV cushion at ED 10.5, myocardial BMP-2 protein expression was diminished in AV myocardium, whereas cushion mesenchymal cells started expressing BMP protein. Expression of BMP-2 in cushion mesenchyme persisted during later stages of development, ED 13.5-16, during valuvulogenesis. Intense expression of BMP-2 persisted in the valve tissue in adult mice. Based on the expression pattern, we performed a series of experiments to test the hypothesis that BMP-2 mediates myocardial regulation of cardiac cushion tissue formation in mice. When BMP-2 protein was added to the 16-18 somite stage (ED 9.25) AV endocardial endothelium in culture, cushion mesenchymal cells were formed in the absence of AV myocardium, which invaded into collagen gels and expressed the mesenchymal marker, smooth muscle (SM) alpha-actin; whereas the endothelial marker, PECAM-1, was lost from the invaded cells. In contrast, when noggin, a specific antagonist to BMPs, was applied together with BMP-2 to the culture medium, AV endothelial cells remained as an epithelial monolayer with little expression of SM alpha-actin, and expression of PECAM-1 was retained in the endocardial cells. When noggin was added to AV endothelial cells cocultured with associated myocardium, it blocked endothelial transformation to mesenchyme. AV endothelium treated with BMP-2 expressed elevated levels of TGFbeta-2 in the absence of myocardium, as observed in the endothelium cocultured with myocardium. BMP-2-supported elevation of TGFbeta-2 expression in endocardial cells was abolished by noggin treatment. These data indicated that BMP signaling is required in and BMP-2 is sufficient for myocardial segmental regulation of AV endocardial cushion mesenchymal cell formation in mice.     

Myocardial specificity for initiating endothelial-mesenchymal cell transition in embryonic chick heart correlates with a particulate distribution of fibronectin

The early chick heart tube consists of myocardium and endothelium separated by a myocardially derived basement membrane (MBM). As development proceeds, the endothelium undergoes a transition into mesenchyme in a regionally specific manner; only the atrioventricular (AV) and outflow tract, but not the ventricular endothelium, is transformed into mesenchyme, the progenitor of heart septa and valves. Recent experiments have shown that an EDTA extract of MBM can initiate AV endothelium to form mesenchyme in an in vitro collagen gel culture system. Two-dimensional gel electrophoresis of AV region EDTA extracts showed potentially three isoelectric forms of fibronectin (Fn), while extracts from ventricle contained only two forms. The purpose of the present study was to further investigate the significance of these regional differences by testing of specific myocardial regions (AV vs ventricle) for their ability to induce endothelium to form mesenchyme in vitro, and to immunohistochemically determine if a regionally specific distribution of Fn exists in the MBM that can be correlated with previous electrophoretic data. Embryonic heart regions cultured on three-dimensional collagen gels showed that AV endothelium could only form mesenchyme if cocultured with AV myocardium. Coculture with ventricular myocardial explants did not initiate differentiation of AV endothelium. In contrast, ventricular endothelial cells did not form mesenchyme when cocultured with AV or ventricle myocardium. Immunohistochemical localization of Fn revealed three distinct morphological patterns of distribution in the AV-MBM, i.e., an intense lamina densa staining, diffuse staining in fibrils, and as particles. The Fn localized in particles (0.1 to 0.5 micron in diameter) appeared as a gradient of decreasing concentration extending from the myocardium toward the endothelium. In contrast, no particulate Fn staining was observed in the ventricular region. EDTA extraction selectively depleted the particulate form of Fn. Previous work has shown that this extract, which contains several lower Mr proteins in addition to Fn, is biologically active in initiating mesenchyme formation from AV endothelium in vitro. These results show that a regionally specific interaction of the myocardium with the endothelium is required to initiate the formation of prevalvular mesenchyme. This interaction may be mediated by a multicomponent complex involving Fn and other proteins which appear as a regionally distinct particulate only in areas of endothelial differentiation.     

Initial expression of type I procollagen in chick cardiac mesenchyme is dependent upon myocardial stimulation

Formation of the atrioventricular (AV) mesenchyme is a critical step in early heart development. Endothelial cells are activated and transformed into a mesenchymal population that invades the cell-free myocardial basement membrane. This process can be duplicated in collagen gel culture, where it has been established that myocardium or its secretory products activate the endothelium. The purpose of the present study was to determine when these activated endothelial and/or mesenchymal cells start producing type I collagen in situ. These results were compared to those obtained from a culture model of mesenchyme formation. The production of type I collagen was monitored using a monoclonal antibody (M38) that recognizes the carboxy-terminal propeptide of human type I procollagen. The initial expression of the latter within activated AV endothelial and mesenchymal cells in ovo was 48 hr following activation. Prior to this time, only the myocardium was reactive with M38. AV explants of early hearts on collagen gels revealed staining of activated endothelial and mesenchymal cells with M38 after 48 hr in coculture with myocardial tissue. Explants that were prevented from activating (myocardium removed) never expressed the M38 antigen. Similarly, AV endothelial monolayers grown in the presence of myocardial conditioned medium activated and expressed type I collagen after 48 hr in culture, whereas those grown in standard medium did not. These results establish the initial expression of type I collagen within activated AV endothelium and mesenchyme. In addition, the data suggest that the expression of type I collagen within the AV mesenchyme may be dependent on extrinsic influences that induce the AV endothelium to transform into mesenchyme.     

Formation of myocardium after the initial development of the linear heart tube.

Well after formation of the primary linear heart tube, the mesenchymal cardiac septa become largely myocardial, and myocardial sleeves are formed along the caval and pulmonary veins. This second wave of myocardium formation can be envisioned to be the result of recruitment of cardiomyocytes by differentiation from flanking mesenchyme and/or by migration from existing myocardium (myocardialization). As a first step to elucidate the underlying mechanism, we studied in chicken heart development the formation of myocardial cells within intra- and extracardiac mesenchymal structures. We show that the second wave of myocardium formation proceeds in a caudal-to-cranial gradient in vivo. At the venous pole, loosely arranged networks of cardiomyocytes are observed in the dorsal mesocardium from H/H19 onward, in the atrioventricular cushion region from H/H26 onward, and in the proximal outflow tract (conus) from H/H29 onward. The process is completed at H/H stage 43. Subsequently, we determined the potential of the different cardiac compartments to form myocardial networks in a 3D in vitro culture assay. This analysis showed that the competency to form myocardial networks in vitro is a characteristic of the myocardium that is flanked by intra- or extracardiac mesenchyme, i.e., the inflow tract, atrioventricular canal, and outflow tract. These cardiac compartments can be induced to form myocardial networks by a temporally released or secreted signal that is similar throughout the entire heart. Atrial and ventricular compartments are not competent and do not produce the inducer. Moreover, cardiac cushion mesenchyme was found to be able to (trans-)differentiate into cardiomyocytes in the in vitro culture assay. The combined observations suggest that a common mechanism and molecular regulatory pathway underlies the recruitment of mesodermal cells into the cardiogenic lineage during this second wave of myocardium formation through the entire heart.     

Fibroblast growth factor (FGF)-4 can induce proliferation of cardiac cushion mesenchymal cells during early valve leaflet formation

While much has been learned about how endothelial cells transform to mesenchyme during cardiac cushion formation, there remain fundamental questions about the developmental fate of cushions. In the present work, we focus on the growth and development of cushion mesenchyme. We hypothesize that proliferative expansion and distal elongation of cushion mesenchyme mediated by growth factors are the basis of early valve leaflet formation. As a first step to test this hypothesis, we have localized fibroblast growth factor (FGF)-4 protein in cushion mesenchymal cells at the onset of prevalve leaflet formation in chick embryos (Hamburger and Hamilton stage 20-25). Ligand distribution was correlated with FGF receptor (FGFR) expression. In situ hybridization data indicated that FGFR3 mRNA was confined to the endocardial rim of the atrioventricular (AV) cushion pads, whereas FGFR2 was expressed exclusively in cushion mesenchymal cells. FGFR1 expression was detected in both endocardium and cushion mesenchyme as well as in myocardium. To determine whether the FGF pathways play regulatory roles in cushion mesenchymal cell proliferation and elongation into prevalvular structure, FGF-4 protein was added to the cushion mesenchymal cells explanted from stage 24-25 chick embryos. A significant increase in proliferative ability was strongly suggested in FGF-4-treated mesenchymal cells as judged by the incorporation of 5'-bromodeoxyuridine (BrdU). To determine whether cushion cells responded similarly in vivo, a replication-defective retrovirus encoding FGF-4 with the reporter, bacterial beta-galactosidase was microinjected into stage 18 chick cardiac cushion mesenchyme along the inner curvature where AV and outflow cushions converge. As compared with vector controls, overexpression of FGF-4 clearly induced expansion of cushion mesenchyme toward the lumen. To further test the proliferative effect of FGF-4 in cardiac cushion expansion in vivo (ovo), FGF-4 protein was microinjected into stage 18 chick inner curvature. An assay for BrdU incorporation indicated a significant increase in proliferative ability in FGF-4 microinjected cardiac cushion mesenchyme as compared with BSA-microinjected controls. Together, these results suggest a role of FGF-4 for cardiac valve leaflet formation through proliferative expansion of cushion mesenchyme.     

Properties of xanthosine 5'-monophosphate-amidotransferase from Escherichia coli.

Xanthosine 5′-phosphate (XMP)-amidotransferase catalyzes the formation of guanosine 5′-phosphate (GMP) by aminating XMP with either the amide group of glutamine (amidotransferase) or ammonia (aminase). The glutamine-supported activity of the purified enzyme from Escherichia coli has been studied, and its properties have been compared with those of other amidotransferases. The following results have been obtained. (i) The glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), irreversibly inhibits the amidotransferase activity. A maximal rate of inhibition by DON is achieved in the presence of XMP, ATP, and Mg²⁺ with a pseudo-first-order rate constant of 0.276 min^?1. (ii) The total number of sulfhydryl groups is approximately 22 per dimer (126,000 M_r). In the absence of substrates, about 8 sulfhydryl groups per dimer are titratable with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), and in the presence of XMP, ATP, and Mg²⁺ an additional 6 cysteine residues per dimer become exposed. When the amidotransferase activity is inactivated by DON, only 8 sulfhydryl groups are titratable. DTNB, p-chloromercuribenzoate, and bromopyruvate all selectively inactivate the amidotransferase activity. These results are consistent with the hypothesis that cysteine residues which are exposed by the substrates are involved in the amidotransferase activity. (iii) The purified XMP amidotransferase contains a glutaminase activity which can be measured in the absence of GMP formation. The glutaminase activity requires XMP, Mg²⁺, and either psicofuranine, an analog of adenosine, or inorganic pyrophosphate (PP_i) and is inhibited by p-chloromercuribenzoate and DON. Maximal stimulation is observed with 100 μm psicofuranine or PP_i, and there is no further stimulation in the presence of both effectors. The apparent K_m is 31 μm with PP_i and 13 μm with psicofuranine; the V for glutamine hydrolysis is about 60% of the rate of the amidotransferase activity. The cooperative interactions between the binding of PP_i and psicofuranine have been confirmed. In the presence of 2.5 μm psicofuranine the K_m for PP_i is reduced 20-fold, but the maximal velocity is unchanged. Similarly, the apparent K_m for psicofuranine is reduced by low concentrations (10 μm) of PP_i. The “uncoupling” of the hydrolysis of glutamine from the amination of XMP is the basis for the reported inhibitory effects of psicofuranine and PP_i on the amidotransferase activity. (iv) Tris buffer selectively inhibits the XMP-amidotransferase activity by inhibiting the glutaminase activity. This inhibition is time dependent and reversible and may explain the previous reports on the inability of this enzyme to use glutamine as a substrate.     

Induction of an epithelial-mesenchymal transition by an in vivo adheron-like complex

The embryonic vertebrate heart consists of two epithelia: the myocardium and endothelium, separated by the myocardial basement membrane (MBM). The myocardium has been shown to induce endothelial transformation into prevalvular mesenchyme in a temporally and site restricted manner. Previously, we hypothesized that the myocardial-endothelial interaction is mediated in vivo by aggregates of 30-nm particles in the MBM which can be removed by EDTA extraction. These MBM extracts contain fibronectin and other lower Mr proteins and can initiate an epithelial-mesenchymal transition in the AV (atrioventricular canal) endothelium of embryonic chick heart in collagen gel culture. These and other data suggested that the 30-nm multicomponent particles are similar, structurally and compositionally, to multimolecular complexes, termed adherons, secreted by L6 muscle cells in culture. The purpose of this study was to (1) test whether the removal of the 30-nm particles from MBM extracts of embryonic chick hearts would remove the in vitro biological activity and (2) determine if the fractionated MBM extracts can cause AV endothelial cells to follow the same differentiation pathway observed in vivo by monitoring immunohistochemically the cell surface expression of N-CAM. Results showed that centrifugation of extract at 100,000g for 1 hr produced a supernatant fraction that was unable to initiate mesenchyme formation from AV endothelium. However, the resuspended pellet fraction did initiate differentiation of endothelium into mesenchyme. Conditioned medium from L6 skeletal muscle cultures could not substitute for the EDTA extract of embryonic heart. Endothelial cells undergoing the transition to form mesenchyme, both in vivo and in vitro, showed a concomitant decrease in N-CAM staining. This suggested that the pellet-induced formation of migrating cells in the collagen gels is not the result a novel in vitro phenomenon.     

The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye

During the development of the anterior segment of the eye, neural crest mesenchyme cells migrate between the lens and the corneal epithelium. These cells contribute to the structures lining the anterior chamber: the corneal endothelium and stroma, iris stroma, and trabecular meshwork. In the present study, removal of the lens or replacement of the lens with a cellulose bead led to the formation a disorganized aggregate of mesenchymal cells beneath the corneal epithelium. No recognizable corneal endothelium, corneal stroma, iris stroma, or anterior chamber was found in these eyes. When the lens was replaced immediately after removal, a disorganized mass of mesenchymal cells again formed beneath the corneal epithelium. However, 2 days after surgery, the corneal endothelium and the anterior chamber formed adjacent to the lens. When the lens was removed and replaced such that only a portion of its anterior epithelial cells faced the cornea, mesenchyme cells adjacent to the lens epithelium differentiated into corneal endothelium. Mesenchyme cells adjacent to lens fibers did not form an endothelial layer. The cell adhesion molecule, N-cadherin, is expressed by corneal endothelial cells. When the lens was removed the mesenchyme cells that accumulated beneath the corneal epithelium did not express N-cadherin. Replacement of the lens immediately after removal led to the formation of an endothelial layer that expressed N-cadherin. Implantation of lens epithelia from older embryos showed that the lens epithelium maintained the ability to support the expression of N-cadherin and the formation of the corneal endothelium until E15. This ability was lost by E18. These studies provide evidence that N-cadherin expression and the formation of the corneal endothelium are regulated by signals from the lens. N-cadherin may be important for the mesenchymal-to-epithelial transformation that accompanies the formation of the corneal endothelium.     

Extracellular matrix from embryonic myocardium elicits an early morphogenetic event in cardiac endothelial differentiation

A critical step in early cardiac morphogenesis can be faithfully duplicated in culture using a hydrated collagen substratum, and thereby serves as a useful model system for studying the molecular mechanisms of cell differentiation. Results from previous work suggested that the myocardium in the atrioventricular canal (AV) region of the developing chick heart secretes extracellular proteins into its associated basement membrane, which may function to promote an epithelial-mesenchymal transition of endothelium to form prevalvular fibroblasts (E. L. Krug, R. B. Runyan, and R. R. Markwald, 1985, Dev. Biol. 112, 414-426; C. H. Mjaatvedt, R. C. Lepera, and R. R. Markwald, 1987, Dev. Biol., in press). In the present study we show that an EDTA-soluble extract of embryonic chick hearts can substitute for the presence of myocardium, the presumptive stimulator tissue, in initiating mesenchyme formation from AV endothelium in culture. Ventricular endothelium was unresponsive to this material in keeping with observed in situ behavior. AV endothelial cells did not survive beyond 4-5 days when cultured in the absence of either the EDTA-soluble heart extract, myocardial conditioned medium, or the myocardium itself. Antibody prepared against a particulate fraction of the EDTA-solubilized heart extract immunohistochemically localized this material to the myocardial basement membrane. In addition, conditioned medium from embryonic myocardial cultures effectively induced mesenchyme formation. Neither a variety of growth factors nor a sarcoma basement membrane preparation were effective in promoting mesenchyme formation indicating a selectivity of the responding embryonic AV endothelial cells to myocardial basement membrane. These observations reflect a truly inductive phenomenon as there was an absolute dependence on the presence of the stimulating substance/tissue and retention, in culture, of both the temporal and regional characteristics observed in situ. This is in contrast to the results of others investigating the cytodifferentiation of committed cells whose phenotypic expression can be either accelerated or diminished but not obligatorily regulated by a specific agent, thus making the interpretation of data difficult, if not irrelevant, to the study of differentiation. The results of this study provide direct experimental support for the hypothesis that extracellular matrix can indeed serve as a direct stimulator or "secondary inducer" of cytodifferentiation.     

Heart-valve mesenchyme formation is dependent on hyaluronan-augmented activation of ErbB2-ErbB3 receptors

Heart septation and valve malformations constitute the most common anatomical birth defects. These structures arise from the endocardial cushions within the atrioventricular canal (AVC) through dynamic interactions between cushion cells and the extracellular matrix (termed cardiac jelly). Transformation of endothelial cells to mesenchymal cells is essential for the proper development of the AVC and subsequent septation and valve formation. Atrioventricular septal defects can result from incomplete endocardial cushion morphogenesis. We show that hyaluronan-deficient AVC explants from Has2(-/-) embryos, which normally lack mesenchyme formation, are rescued by heregulin treatment, which restores phosphorylation of ErbB2 and ErbB3. These events were blocked using a soluble ErbB3 molecule, as well as with an inhibitor of ErbB2, herstatin. We show further that ErbB3 is activated during hyaluronan treatment of Has2(-/-) explants. These data provide a link between extracellular matrix-hyaluronan and ErbB receptor activation during development of early heart-valve and septal mesenchyme.     

Heterogeneity of chemical composition of lipid droplets in endothelial inflammation and apoptosis

Lipid droplets (LDs) play regulatory role in various cells but their significance in endothelial pathophysiology is still not well understood. Here, we studied LDs in in situ endothelial cells (ECs) in isolated blood vessels stimulated with pro-inflammatory or pro-apoptotic stimuli using Raman and fluorescence imaging. Endothelial inflammation induced by murine TNF-α (mTNF-α) was featured by overexpression of ICAM-1, vWF, increased production of PGI₂, and was associated with the formation of low number of LDs. However in the presence of atglistatin, the inhibitor of triacyclglycerols hydrolysis, the number of LDs significantly increased. In contrast, in endothelium stimulated by human TNF-α (hTNF-α) or FasL, apart from endothelial inflammation, displayed also apoptosis as evidenced by high annexin expression and significant LDs formation. Raman imaging confirmed that LDs were localized in endothelium and revealed significant heterogeneity in biochemical composition of endothelial LDs that dependent on endothelial stimuli. Repertoire of LDs included LDs rich in highly unsaturated lipids, assigned to the inflammation, as well as LDs featured by more saturated lipids linked to apoptosis, where Raman signals indicating content of cholesterol and phospholipids were higher for endothelial apoptosis in comparison to endothelial inflammation. The heterogeneity in chemical composition of LDs suggested more complex pathophysiological role of endothelial LDs then previously appreciated.     

Nitric oxide signaling during myocardial angiogenesis

Ischemic heart disease develops as a consequence of coronary atherosclerotic lesion formation. Coronary collateral vessels and microvascular angiogenesis develop as an adaptive response to myocardial ischemia, which ameliorates the function of the damaged heart. Angiogenesis, the formation of new blood vessels from pre-existing vascular bed, is of paramount importance in the maintenance of vascular integrity both in the repair process of damaged tissue and in the formation of collateral vessels in response to tissue ischemia. Angiogenesis is modulated by a multitude of cytokines/chemokines and growth factors. In this regard, angiogenesis cannot be viewed as a single process. It is likely that different mediators are involved in different phases of angiogenesis. Vascular endothelial cells (ECs) produce nitric oxide (NO), an endothelium-derived labile molecule, which maintains vascular homeostasis and thereby prevents vascular atherosclerotic changes. In patients with ischemic heart disease, the release of endothelium-derived NO is decreased, which plays an important role in the atherosclerotic disease progression. In recent years, endothelium-derived NO has been shown to modulate angiogenesis in vitro and in vivo. In this review, we summarize recent progress in the field of the NO-mediated regulation of postnatal angiogenesis, particularly in response to myocardial ischemia. (Mol Cell Biochem 264: 25–34, 2004)     

Structural analyses on the matrical organization of glycosaminoglycans in developing endocardial cushions

Extracellular glycosaminoglycans (GAG) were examined in embryonic rat valvular primordia (cushion tissue) to determine if there are specific, in situ, intermolecular associations of GAG and if the passage of migrating cushion cells alters matrical organization. Precursor incorporation studies and colloidal iron staining controlled by acidified methylation, pH, and polysaccharidase digestion indicated that both hyaluronate (HA) and chondroitin sulfate (CHS) were secreted into the premigratory matrix with the predominant GAG being HA. Premigratory matrix was revealed by scanning electron microscopy after routine fixation as a microfibrillar stroma; addition of cetylpyridinium chloride (CPCL) to the fixative resulted in the retention of an additional matrical component superimposed upon the microfibrillar stroma. TEM analysis of the CPCL-dependent matrix revealed that it was composed of intertwined 3-nm filaments, electron-dense, amorphous material, and 30-nm granules. Collagen-like microfibrils were associated primarily with the filamentous component of the CPCL-dependent matrix. Ultracytochemical results obtained with dialyzed iron binding regulated by pH and polysaccharidase and protease digestion suggests that the 3-nm filaments contain HA and the granules contain both CHS and protein. Commensurate with cushion cell formation and migration, X-ray dispersive analysis and polyanionic histochemical criteria indicated increased deposition of CHS in the postmigratory matrix (i.e., matrix transversed by cells). Ultrastructurally, the CPCL-dependent components of the postmigratory matrix became progressively restructured within the wedge of migrating cells. In contrast to premigratory matrix, fewer 3-nm filaments were evident, while 30-nm granules heavily studded the collagen-like microfibrils. Physical fixation controls confirmed the variations between pre- and postmigratory matrices. These results suggest that modification in the matrix organization of embryonic heart GAG may be correlated with the migration of cushion tissue mesenchyme.     

Avian corneal innervation: Inhibition of nerve ring formation by 6-diazo-5-oxo-l-norleucine

Normal innervation of embryonic avian cornea is achieved in two distinct phases. During phase I, nerves extend from the ventrotemporal region both dorsally and ventrally around the cornea, but not into it, ultimately encircling the 10th-day cornea. Phase II commences as nerves extend radially from the ring into the corneal stroma and from there into the epithelium. The effect of the glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), on this normal sequence of events has been examined. In ovo administration of 5 μg DON on the 5th day of development inhibits the incorporation of [³⁵S]sulfate in sulfated glycosaminoglycans in both the cornea and control tissues and inhibits the completion of phase I. Phase II of corneal innervation appears to be affected only indirectly and extension of nerves into the cornea does occur. However, the number of nerves entering the DON-treated cornea is dramatically reduced. Administration of DON on the 7th or 9th days of development does not affect corneal innervation, but does demonstrate a clear effect on [³⁵S]sulfate incorporation in sulfated glycosaminoglycans by the cornea and control tissues. These data suggest that nerve ring completion is not a prerequisite for extension of nerves into the cornea and suggest an integral role for glycosaminoglycans in facilitating phase I, but not phase II, of corneal innervation.     

Wnt signaling in heart valve development and osteogenic gene induction

Wnt signaling mediated by β-catenin has been implicated in early endocardial cushion development, but its roles in later stages of heart valve maturation and homeostasis have not been identified. Multiple Wnt ligands and pathway genes are differentially expressed during heart valve development. At E12.5, Wnt2 is expressed in cushion mesenchyme, whereas Wnt4 and Wnt9b are predominant in overlying endothelial cells. At E17.5, both Wnt3a and Wnt7b are expressed in the remodeling atrioventricular (AV) and semilunar valves. In addition, the TOPGAL Wnt reporter transgene is active throughout the developing AV and semilunar valves at E16.5, with more localized expression in the stratified valve leaflets after birth. In chicken embryo aortic valves, genes characteristic of osteogenic cell lineages including periostin, osteonectin, and Id2 are expressed specifically in the collagen-rich fibrosa layer at E14. Treatment of E14 aortic valve interstitial cells (VICs) in culture with osteogenic media results in increased expression of multiple genes associated with bone formation. Treatment of VIC with Wnt3a leads to nuclear localization of β-catenin and induction of periostin and matrix gla protein but does not induce genes associated with later stages of osteogenesis. Together, these studies provide evidence for Wnt signaling as a regulator of endocardial cushion maturation as well as valve leaflet stratification, homeostasis, and pathogenesis.     

Synthesis and evaluation of polymeric continuous bed (monolithic) reversed-phase gradient stationary phases for capillary liquid chromatography and capillary electrochromatography

There is a demand of novel high resolution separation media for separation of complex mixtures, particularly biological samples. One of the most flexible techniques for development of new separation media currently is synthesis of the continuous bed (monolithic) stationary phases. In this study the capillary format gradient stationary phases were formed using continuous bed (monolith) polymerization in situ. Different reversed-phase stationary phase gradients were tailored and their resolution using capillary liquid chromatography and capillary electrochromatography at isocratic mobile phase conditions was evaluated. It is demonstrated, that efficiency and resolution of the gradient stationary phases can be substantially increased comparing to the common (isotropic) stationary phases. The proposed formation approach of the gradient stationary phase is reproducible and compatible with the capillary format or microchip format separations. It can be easily automated for the separation optimizations or mass production of the capillary columns or chips.