Remodeling of the vascular tunica media is essential for development of collateral vessels in the canine heart

Previous studies have shown that neointima formation and adventitial remodeling play an important role in the enlargement of collateral vessels (CVs) during coronary arteriogenesis in the dog heart. In this study, we investigated the importance of remodeling of the tunica media in the same model. Basal membrane (BM), contractile and cytoskeletal components of smooth muscle cells (SMCs) were studied in growth of coronary CVs induced by chronic occlusion of the left circumflex (LCX) coronary artery by routine histology, electron microscopy (EM), and immunoconfocal microscopy using antibodies against α-smooth actin (α-SM actin), calponin, desmin, and laminin. In addition, matrix metalloproteinase-2 (MMP-2) and tissue inhibitor-1 of matrix metalloproteinase (TIMP-1) were investigated. The data showed that (1) in normal small arteries (NVs) laminin formed a network in which SMCs were encaged;α-SM actin, calponin and desmin were evenly expressed in SMCs; (2) in early (2 weeks) growing CVs the laminin network was disrupted, desmin was significantly reduced in SMCs, but α-SM actin and calponin still highly expressed; (3) in actively (6 weeks) growing CVs laminin was still weak in the tunica media (TM), but without network-like structure. Desmin was further reduced in SMCs of TM, whereas α-SM actin and calponin showed little changes, although they were significantly decreased in intimal SMCs; (4) in mature CVs, the network-like structure was re-formed, and α-SM actin, calponin, and desmin were all similar to that in normal vessels; (5) histology for BM confirmed laminin staining; (6) EM revealed that in NVs the SMCs contained abundant contractile filaments and were surrounded by a layer of BM whereas in growing CVs, BM structure was not observed, but the SMCs in the media still contained many myofilaments; (7) MMP-2 was highly expressed in the media of early growing vessels, but decreased in TM of actively growing vessels where TIMP-1 expression was high. In conclusion, our data revealed features of TM of growing CVs. Disruption and degradation of BM facilitate SMC proliferation, and together with reduction of desmin and fragmentation of the internal elastic lamina enable the vascular wall to expand and enlarge when blood pressure and shear stress increase. MMP2 may be an important player in regulating SMC phenotype, proliferation, migration and maintaining integrity of the vascular wall through governing proteolysis during arteriogenesis. (Mol Cell Biochem 264: 201–210, 2004)     

Remodeling of lung interstitium but not resistance vessels in canine pacing-induced heart failure.

We previously showed that pacing-induced heart failure in dogs results in an enhancement of pulmonary vascular reactivity. In the present study we hypothesized that enhanced matrix deposition and structural remodeling of lung resistance microvessels would underlie these functional changes. Using biochemical measures, we found no difference in the normalized lung content of hyaluronan, uronic acid, and collagen between control dogs and dogs paced for 1 mo, although lung dry weight and noncollagen protein content increased significantly in the paced group (P < 0.05). From separate Formalin-fixed lung lobes, 5-microm frozen sections were prepared and stained with Masson's trichrome, and vascular structure was evaluated using standard morphometric techniques. When perivascular fluid cuffs were excluded from the measure of wall thickness, collagen and media volume fractions in any size range did not differ between paced and control groups. Similarly, in the paced group, medial thickness in <400-microm arterial or venular microvessels did not vary significantly from that in the controls. In contrast, the relationship of interstitial fluid pressure to lung water was significantly shifted to the right in the paced group, such that normal tissue pressures were observed, despite the increased water content. We conclude that although 1 mo of pacing-induced heart failure results in altered interstitial function, the attendant pulmonary hypertension and/or hormonal responses are insufficient to induce medial hypertrophy or other remodeling of the extra-alveolar microvasculature.     

NgBR is essential for endothelial cell glycosylation and vascular development

NgBR is a transmembrane protein identified as a Nogo‐B‐interacting protein and recently has been shown to be a subunit required for cis‐prenyltransferase (cisPTase) activity. To investigate the integrated role of NgBR in vascular development, we have characterized endothelial‐specific NgBR knockout embryos. Here, we show that endothelial‐specific NgBR knockout results in embryonic lethality due to vascular development defects in yolk sac and embryo proper. Loss of NgBR in endothelial cells reduces proliferation and promotes apoptosis of the cells largely through defects in the glycosylation of key endothelial proteins including VEGFR2, VE‐cadherin, and CD31, and defective glycosylation can be rescued by treatment with the end product of cisPTase activity, dolichol phosphate. Moreover, NgBR functions in endothelial cells during embryogenesis are Nogo‐B independent. These data uniquely show the importance of NgBR and protein glycosylation during vascular development.     

Na,K-ATPase is essential for embryonic heart development in the zebrafish

Na,K-ATPase is an essential gene maintaining electrochemical gradients across the plasma membrane. Although previous studies have intensively focused on the role of Na,K-ATPase in regulating cardiac function in the adults, little is known about the requirement for Na,K-ATPase during embryonic heart development. Here, we report the identification of a zebrafish mutant, heart and mind, which exhibits multiple cardiac defects, including the primitive heart tube extension abnormality, aberrant cardiomyocyte differentiation, and reduced heart rate and contractility. Molecular cloning reveals that the heart and mind lesion resides in the alpha1B1 isoform of Na,K-ATPase. Blocking Na,K-ATPase alpha1B1 activity by pharmacological means or by morpholino antisense oligonucleotides phenocopies the patterning and functional defects of heart and mind mutant hearts, suggesting crucial roles for Na,K-ATPase alpha1B1 in embryonic zebrafish hearts. In addition to alpha1B1, the Na,K-ATPase alpha2 isoform is required for embryonic cardiac patterning. Although the alpha1B1 and alpha2 isoforms share high degrees of similarities in their coding sequences, they have distinct roles in patterning zebrafish hearts. The phenotypes of heart and mind mutants can be rescued by supplementing alpha1B1, but not alpha2, mRNA to the mutant embryos, demonstrating that alpha1B1 and alpha2 are not functionally equivalent. Furthermore, instead of interfering with primitive heart tube formation or cardiac chamber differentiation, blocking the translation of Na,K-ATPase alpha2 isoform leads to cardiac laterality defects.     

Recycling of methylthioadenosine is essential for normal vascular development and reproduction in Arabidopsis

5'-Methylthioadenosine (MTA) is the common by-product of polyamine (PA), nicotianamine (NA), and ethylene biosynthesis in Arabidopsis (Arabidopsis thaliana). The methylthiol moiety of MTA is salvaged by 5'-methylthioadenosine nucleosidase (MTN) in a reaction producing methylthioribose (MTR) and adenine. The MTN double mutant, mtn1-1mtn2-1, retains approximately 14% of the MTN enzyme activity present in the wild type and displays a pleiotropic phenotype that includes altered vasculature and impaired fertility. These abnormal traits were associated with increased MTA levels, altered PA profiles, and reduced NA content. Exogenous feeding of PAs partially recovered fertility, whereas NA supplementation improved fertility and also reversed interveinal chlorosis. The analysis of PA synthase crystal structures containing bound MTA suggests that the corresponding enzyme activities are sensitive to available MTA. Mutant plants that expressed either MTN or human methylthioadenosine phosphorylase (which metabolizes MTA without producing MTR) appeared wild type, proving that the abnormal traits of the mutant are due to MTA accumulation rather than reduced MTR. Based on our results, we propose that the key targets affected by increased MTA content are thermospermine synthase activity and spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A.     

Remodeling is at the heart of chromatin

Chromatin modifications are integral elements of chromosome structure and its function and the vasculature depends on tissue-specific genome regulation for its development. A general concept for the de-regulation of chromatin modifications in cardiac and vascular disease is also emerging. The recognition that metabolic memory contributes to disease persistence highlights the benefit of early and aggressive treatment. As for the importance of memory, we do know that good metabolic control delays the onset of long-term diabetic complications. There are striking parallels between the timing of disease and the development of complications. Landmark multicenter clinical trials on diabetes patients have popularized the concept that glucose is also a demonstrable determinant for the development of complications, indicating the prolonged benefit of intensive therapy and the lasting damage of conventional therapy. Each cell type experiences thousands of modifications to the epigenome in response to environmental changes it is exposed to. Therefore, history is neither lost nor forgotten and previous experiences and exposure may form future memories. There is now a strong resurgence in research trying to understand gene-environment interactions and to determine what commits specific vascular cell types to specific memories. Recent insights show that cardiac gene expression is distinguished by specific chromatin remodeling events and histone modifications that are associated with heart disease.     

FOG-2, a cofactor for GATA transcription factors, is essential for heart morphogenesis and development of coronary vessels from epicardium

We disrupted the FOG-2 gene in mice to define its requirement in vivo. FOG-2(-/-) embryos die at midgestation with a cardiac defect characterized by a thin ventricular myocardium, common atrioventricular canal, and the tetralogy of Fallot malformation. Remarkably, coronary vasculature is absent in FOG-2(-/-) hearts. Despite formation of an intact epicardial layer and expression of epicardium-specific genes, markers of cardiac vessel development (ICAM-2 and FLK-1) are not detected, indicative of failure to activate their expression and/or to initiate the epithelial to mesenchymal transformation of epicardial cells. Transgenic reexpression of FOG-2 in cardiomyocytes rescues the FOG-2(-/-) vascular phenotype, demonstrating that FOG-2 function in myocardium is required and sufficient for coronary vessel development. Our findings provide the molecular inroad into the induction of coronary vasculature by myocardium in the developing heart.     

ADMP2 is essential for primitive blood and heart development in Xenopus

We describe here the cloning of a new member of the TGF-beta family with similarity to the anti-dorsalizing morphogenetic proteins (ADMPs). This new gene, ADMP2, is expressed in a broad band of mesendoderm cells that appear to include the progenitors of the endoderm and the ventral mesoderm. Antisense morpholino oligonucleotide knockdown of ADMP2 results in near-complete disruption of primitive blood and heart development, while the development of other mesoderm derivatives, including pronephros, muscle and lateral plate is not disrupted. Moreover, the development of the primitive blood in ADMP2 knockdown embryos cannot be rescued by BMP. These results suggests that ADMP2 plays an early role in specifying presumptive ventral mesoderm in the leading edge mesoderm, and that ADMP2 activity may be necessary to respond to BMP signaling in the context of ventral mesoderm induction.     

Mab21l2 is essential for embryonic heart and liver development

During mouse embryogenesis, proper formation of the heart and liver is especially important and is crucial for embryonic viability. In this study, we showed that Mab21l2 was expressed in the trabecular and compact myocardium, and that deletion of Mab21l2 resulted in a reduction of the trabecular myocardium and thinning of the compact myocardium. Mab21l2-deficient embryonic hearts had decreased expression of genes that regulate cell proliferation and apoptosis of cardiomyocytes. These results show that Mab21l2 functions during heart development by regulating the expression of such genes. Mab21l2 was also expressed in the septum transversum mesenchyme (STM). Epicardial progenitor cells are localized to the anterior surface of the STM (proepicardium), and proepicardial cells migrate onto the surface of the heart and form the epicardium, which plays an important role in heart development. The rest of the STM is essential for the growth and survival of hepatoblasts, which are bipotential progenitors for hepatocytes and cholangiocytes. Proepicardial cells in Mab21l2-deficient embryos had defects in cell proliferation, which led to a small proepicardium, in which α4 integrin expression, which is essential for the migration of proepicardial cells, was down-regulated, suggesting that defects occurred in its migration. In Mab21l2-deficient embryos, epicardial formation was defective, suggesting that Mab21l2 plays important roles in epicardial formation through the regulation of the cell proliferation of proepicardial cells and the migratory process of proepicardial cells. Mab21l2-deficient embryos also exhibited hypoplasia of the STM surrounding hepatoblasts and decreased hepatoblast proliferation with a resultant loss of defective morphogenesis of the liver. These findings demonstrate that Mab21l2 plays a crucial role in both heart and liver development through STM formation.     

RBFOX2 is required for establishing RNA regulatory networks essential for heart development

Human genetic studies identified a strong association between loss of function mutations in RBFOX2 and hypoplastic left heart syndrome (HLHS). There are currently no Rbfox2 mouse models that recapitulate HLHS. Therefore, it is still unknown how RBFOX2 as an RNA binding protein contributes to heart development. To address this, we conditionally deleted Rbfox2 in embryonic mouse hearts and found profound defects in cardiac chamber and yolk sac vasculature formation. Importantly, our Rbfox2 conditional knockout mouse model recapitulated several molecular and phenotypic features of HLHS. To determine the molecular drivers of these cardiac defects, we performed RNA-sequencing in Rbfox2 mutant hearts and identified dysregulated alternative splicing (AS) networks that affect cell adhesion to extracellular matrix (ECM) mediated by Rho GTPases. We identified two Rho GTPase cycling genes as targets of RBFOX2. Modulating AS of these two genes using antisense oligos led to cell cycle and cell-ECM adhesion defects. Consistently, Rbfox2 mutant hearts displayed cell cycle defects and inability to undergo endocardial-mesenchymal transition, processes dependent on cell-ECM adhesion and that are seen in HLHS. Overall, our work not only revealed that loss of Rbfox2 leads to heart development defects resembling HLHS, but also identified RBFOX2-regulated AS networks that influence cell-ECM communication vital for heart development.     

Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leads to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor^flox/flox mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.     

GATA6 is essential for embryonic development of the liver but dispensable for early heart formation

Several lines of evidence suggest that GATA6 has an integral role in controlling development of the mammalian liver. Unfortunately, this proposal has been impossible to address directly because mouse embryos lacking GATA6 die during gastrulation. Here we show that the early embryonic deficiency associated with GATA6-knockout mice can be overcome by providing GATA6-null embryos with a wild-type extraembryonic endoderm with the use of tetraploid embryo complementation. Analysis of rescued Gata6-/- embryos revealed that, although hepatic specification occurs normally, the specified cells fail to differentiate and the liver bud does not expand. Although GATA6 is expressed in multiple tissues that impact development of the liver, including the heart, septum transversum mesenchyme, and vasculature, all are relatively unaffected by loss of GATA6, which is consistent with a cell-autonomous requirement for GATA6 during hepatogenesis. We also demonstrate that a closely related GATA factor, GATA4, is expressed transiently in the prehepatic endoderm during hepatic specification and then lost during expansion of the hepatic primordium. Our data support the proposal that GATA4 and GATA6 are functionally redundant during hepatic specification but that GATA6 alone is available for liver bud growth and commitment of the endoderm to a hepatic cell fate.