H. Scott Baldwin

The transcription factor NF-ATc is essential for cardiac valve formation

Nuclear factor of activated T cells (NF-AT) is the name of a family of four related transcription factors that may be needed for cytokine gene expression in activated lymphocytes. Here we report that mice with a targeted disruption of the NF-ATc gene show an unexpected and dramatic defect in cardiac morphogenesis, with selective absence of the aortic and pulmonary valves, leading to death in utero from congestive heart failure at days 13.5–17.5 of gestation. In contrast, tricuspid and mitral valve morphogenesis is normal. NF-ATc is the first transcription factor known to be expressed only in the endothelial cells of the heart. As in T cells, nuclear translocation of NF-ATc in cardiac endothelial cells is controlled by the calcium-regulated phosphatase calcineurin,: NF-ATc remains cytoplasmic in normal embryos cultured with cyclosporin A, an inhibitor of calcineurin. Abnormal development of the cardiac valves and septae is the most frequent form of birth defect, yet few molecular regulators of valve formation are known. Our results indicate that NF-ATc may play a critical role in signal-transduction processes required for normal cardiac valve formation.

Pancreatic Islet Production of Vascular Endothelial Growth Factor-A Is Essential for Islet Vascularization, Revascularization, and Function

To investigate molecular mechanisms controlling islet vascularization and revascularization after transplantation, we examined pancreatic expression of three families of angiogenic factors and their receptors in differentiating endocrine cells and adult islets. Using intravital lectin labeling, we demonstrated that development of islet microvasculature and establishment of islet blood flow occur concomitantly with islet morphogenesis. Our genetic data indicate that vascular endothelial growth factor (VEGF)-A is a major regulator of islet vascularization and revascularization of transplanted islets. In spite of normal pancreatic insulin content and β-cell mass, mice with β-cell–reduced VEGF-A expression had impaired glucose-stimulated insulin secretion. By vascular or diffusion delivery of β-cell secretagogues to islets, we showed that reduced insulin output is not a result of β-cell dysfunction but rather caused by vascular alterations in islets. Taken together, our data indicate that the microvasculature plays an integral role in islet function. Factors modulating VEGF-A expression may influence islet vascularity and, consequently, the amount of insulin delivered into the systemic circulation.

Endocardial Cells Form the Coronary Arteries by Angiogenesis through Myocardial-Endocardial VEGF Signaling

The origins and developmental mechanisms of coronary arteries are incompletely understood. We show here by fate mapping, clonal analysis, and immunohistochemistry that endocardial cells generate the endothelium of coronary arteries. Dye tracking, live imaging, and tissue transplantation also revealed that ventricular endocardial cells are not terminally differentiated; instead, they are angiogenic and form coronary endothelial networks. Myocardial Vegf-a or endocardial Vegfr-2 deletion inhibited coronary angiogenesis and arterial formation by ventricular endocardial cells. In contrast, lineage and knockout studies showed that endocardial cells make a small contribution to the coronary veins, the formation of which is independent of myocardial-to-endocardial Vegf signaling. Thus, contrary to the current view of a common source for the coronary vessels, our findings indicate that the coronary arteries and veins have distinct origins and are formed by different mechanisms. This information may help develop better cell therapies for coronary artery disease.

The N-myc downstream regulated gene (NDRG) family: diverse functions, multiple applications

The N‐myc downstream regulated gene (NDRG) family of proteins consists of 4 members, NDRG1–4, which are well conserved through evolution. The first member to be discovered and responsible for the family name was NDRG1, because its expression is repressed by the proto‐oncogenes MYCN and MYC. All family members are characterized by an α/β hydrolase‐fold motif; however, the precise molecular and cellular function of these family members has not been fully elucidated. Although the exact function of NDRG family members has not been clearly elucidated, emerging evidence suggests that mutations in these genes are associated with diverse neurological and electrophysiological syndromes. In addition, aberrant expression as well as tumor suppressor and oncogenic functions affecting key hallmarks of carcinogenesis such as cell proliferation, differentiation, migration, invasion, and stress response have been reported for several of the NDRG proteins. In this review, we summarize the current literature on the NDRG family members concerning their structure, origin, and tissue distribution. In addition, we review the current knowledge regarding the regulation and signaling of the NDRG family members in development and normal physiology. Finally, their role in disease and potential clinical applications (their role as detection or prognostic markers) are discussed.—Melotte, V., Qu, X., Ongenaert, M., van Criekinge, W., de BruÏne, A. P., Baldwin, H. S., van Engeland, M. The N‐myc downstream regulated gene (NDRG) family: diverse functions, multiple applications. FASEB J. 24,4153–4166 (2010). www.fasebj.org

Early embryonic vascular development.

Establishment of a functional vascular system is imperative for normal embryonic growth and development. Building on the excellent descriptive studies of endothelial cell position and behavior, it is now possible to begin to define the mechanisms directing endothelial cell differentiation, commitment, migration and organization into a tube. What are the factors that control differentiation of mesoderm into angioblasts and what is needed for these angioblasts to organize into a vascular bed? These processes involve the careful orchestration of a diverse group of molecules including components of the extracellular matrix, cell adhesion receptors, growth factors and their receptors. This review has discussed just a few of the components that have been implicated as potentially important in the establishment of the vascular system. However, our understanding of how vessels form in the embryo is really quite rudimentary. We are just beginning to understand the delicate balance of both angiogenic and angiostatic processes that result in the intricate patterning and diverse function that is characteristic of developing endothelial cells. We have not even discussed important factors such as the role of blood flow and apoptosis in maintaining and remodeling vascular beds within the embryo nor have we addressed the development of endothelial heterogeneity in the mature animal [reviewed in Ref. 98]. There are certain to be new molecules identified as well as the delineation of new roles for known molecules. In addition, advances in genetic manipulation and improvements in both in vitro and in vivo model systems should provide important new insights into this critical process of vascular ontogeny.

Bone Morphogenetic Protein 4 Promotes Pulmonary Vascular Remodeling in Hypoxic Pulmonary Hypertension

We show that 1 of the type II bone morphogenetic protein (BMP) receptor ligands, BMP4, is widely expressed in the adult mouse lung and is upregulated in hypoxia-induced pulmonary hypertension (PH). Furthermore, heterozygous null Bmp4lacZ/+ mice are protected from the development of hypoxia-induced PH, vascular smooth muscle cell proliferation, and vascular remodeling. This is associated with a reduction in hypoxia-induced Smad1/5/8 phosphorylation and Id1 expression in the pulmonary vasculature. In addition, pulmonary microvascular endothelial cells secrete BMP4 in response to hypoxia and promote proliferation and migration of vascular smooth muscle cells in a BMP4-dependent fashion. These findings indicate that BMP4 plays a dominant role in regulating BMP signaling in the hypoxic pulmonary vasculature and suggest that endothelium-derived BMP4 plays a direct, paracrine role in promoting smooth muscle proliferation and remodeling in hypoxic PH.

Noninvasive, In Utero Imaging of Mouse Embryonic Heart Development With 40-MHz Echocardiography

BACKGROUND The increasing number of transgenic and targeted mutant mice with embryonic cardiac defects has resulted in the need for noninvasive techniques to examine cardiac structure and function in early mouse embryos. We report the first use of a novel 40-MHz ultrasound imaging system in the study of mouse cardiac development in utero. METHODS AND RESULTS Transabdominal scans of mouse embryos staged between 8.5 and 13.5 days of gestation (E8.5 to E13.5) were obtained in anesthetized mice. Atrial and ventricular contractions could be discerned from E9.5, and changes in cardiac morphology were observed from E9.5 to E13.5. Hyperechoic streaming patterns delineated flow through the umbilical, vitelline, and other major blood vessels. Diastolic and systolic ventricular areas were determined by planimetry of the epicardial borders, and fractional area change was measured as an index of contractile function. Significant increases in ventricular size were documented at each stage between E10.5 and E13.5, and the ability to perform serial imaging studies over 3 days of embryonic development is described. Finally, the detection of vascular cell adhesion molecule 1 (VCAM-1) homozygous null mutant embryos demonstrates the first example of noninvasive, in utero analysis of cardiac structure and function in a targeted mouse mutant. CONCLUSIONS We used 40-MHz echocardiography to identify key elements of the early mouse embryonic cardiovascular system and for noninvasive dimensional analysis of developing cardiac ventricles. The ability to perform serial measurements and to detect mutant embryos with cardiac defects highlights the usefulness of the technique for investigating normal and abnormal cardiovascular development.

Regulation of the Murine Nfatc1 Gene by NFATc2

NFAT proteins play a key role in the inducible expression of cytokine genes in T lymphocytes. NFATc1 and NFATc2 are the predominant NFAT family members in the peripheral immune system. NFATc2 is found abundantly in the cytoplasm of resting T cells, whereasNfatc1 expression is induced during T cell activation. To investigate Nfatc1 regulation, we characterized the structure of the murine Nfatc1 gene and its 5′-flanking region. A 290-bp sequence proximal to the transcription start site is highly conserved between mouse and human and possesses both basal and inducible promoter activities. Multiple binding sites for transcription factors were identified within this region, including a consensus NFAT-binding site. This promoter segment was cyclosporin A-sensitive, and mutation of the NFAT site abrogated inducible promoter activity and inhibited formation of an inducible DNA·protein complex containing NFATc2 in primary T cells. Overexpression of NFATc2 increased inducibleNfatc1 promoter activity, whereas this inducibility was attenuated in NFATc2−/− splenocytes. This study suggests that pre-existing NFATc2 contributes to the subsequent induction ofNfatc1 during T cell activation.

Enhanced Sensitivity for High Spatial Resolution Lipid Analysis by Negative Ion Mode Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry

We have achieved enhanced lipid imaging to a ~10 μm spatial resolution using negative ion mode matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry, sublimation of 2,5-dihydroxybenzoic acid as the MALDI matrix, and a sample preparation protocol that uses aqueous washes. We report on the effect of treating tissue sections by washing with volatile buffers at different pHs prior to negative ion mode lipid imaging. The results show that washing with ammonium formate, pH 6.4, or ammonium acetate, pH 6.7, significantly increases signal intensity and number of analytes recorded from adult mouse brain tissue sections. Major lipid species measured were glycerophosphoinositols, glycerophosphates, glycerolphosphoglycerols, glycerophosphoethanolamines, glycerophospho-serines, sulfatides, and gangliosides. Ion images from adult mouse brain sections that compare washed and unwashed sections are presented and show up to 5-fold increases in ion intensity for washed tissue. The sample preparation protocol has been found to be applicable across numerous organ types and significantly expands the number of lipid species detectable by imaging mass spectrometry at high spatial resolution.

Tgfβ signaling is required for atrioventricular cushion mesenchyme remodeling during in vivo cardiac development

The transforming growth factorβ (Tgfβ) signaling pathway plays crucial roles in many biological processes. To understand the role(s) of Tgfβ signaling during cardiogenesis in vivo and to overcome the early lethality of Tgfbr2-/- embryos, we applied a Cre/loxp system to specifically inactivate Tgfbr2 in either the myocardium or the endothelium of mouse embryos. Our results show that Tgfbr2 in the myocardium is dispensable for cardiogenesis in most embryos. Contrary to the prediction from results of previous in vitro collagen gel assays, inactivation of Tgfbr2 in the endocardium does not prevent atrioventricular cushion mesenchyme formation, arguing against its essential role in epithelium-mesenchyme transformation in vivo. We further demonstrate that Tgfβ signaling is required for the proper remodeling of the atrioventricular canal and for cardiac looping, and that perturbation in Tgfβ signaling causes the double-inlet left ventricle (DILV) defect. Thus, our study provides a unique mouse genetic model for DILV, further characterization of which suggests a potential cellular mechanism for the defect.