Rongqin Ren

BMPER, a novel endothelial cell precursor-derived protein, antagonizes bone morphogenetic protein signaling and endothelial cell differentiation

ABSTRACT The development of endothelial cell precursors is essential for vasculogenesis. We screened for differentially expressed transcripts in endothelial cell precursors in developing mouse embryoid bodies. We cloned a complete cDNA encoding a protein that contains an amino-terminal signal peptide, five cysteine-rich domains, a von Willebrand D domain, and a trypsin inhibitor domain. We termed this protein BMPER (bone morphogenetic protein [BMP]-binding endothelial cell precursor-derived regulator). BMPER is specifically expressed in flk-1-positive cells and parallels the time course of flk-1 induction in these cells. In situ hybridization in mouse embryos demonstrates dorsal midline staining and staining of the aorto-gonadal-mesonephric region, which is known to host vascular precursor cells. BMPER is a secreted protein that directly interacts with BMP2, BMP4, and BMP6 and antagonizes BMP4-dependent Smad5 activation. In Xenopus embryos, ventral injection of BMPER mRNA results in axis duplication and downregulation of the expression of Xvent-1 (downstream target of Smad signaling). In an embryoid body differentiation assay, BMP4-dependent differentiation of endothelial cells in embryoid bodies is also antagonized by BMPER. Taken together, our data indicate that BMPER is a novel BMP-binding protein that is expressed by endothelial cell precursors, has BMP-antagonizing activity, and may play a role in endothelial cell differentiation by modulating local BMP activity.

A concentration-dependent endocytic trap and sink mechanism converts Bmper from an activator to an inhibitor of Bmp signaling.

Bmper, which is orthologous to Drosophila melanogaster crossveinless 2, is a secreted factor that regulates Bmp activity in a tissue- and stage-dependent manner. Both pro- and anti-Bmp activities have been postulated for Bmper, although the molecular mechanisms through which Bmper affects Bmp signaling are unclear. In this paper, we demonstrate that as molar concentrations of Bmper exceed Bmp4, Bmper dynamically switches from an activator to an inhibitor of Bmp4 signaling. Inhibition of Bmp4 through a novel endocytic trap-and-sink mechanism leads to the efficient degradation of Bmper and Bmp4 by the lysosome. Bmper-mediated internalization of Bmp4 reduces the duration and magnitude of Bmp4-dependent Smad signaling. We also determined that Noggin and Gremlin, but not Chordin, trigger endocytosis of Bmps. This endocytic transport pathway expands the extracellular roles of selective Bmp modulators to include intracellular regulation. This dosage-dependent molecular switch resolves discordances among studies that examine how Bmper regulates Bmp activity and has broad implications for Bmp signal regulation by secreted mediators.

Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

Endothelial cell migration is an important step during angiogenesis, and its dysregulation contributes to aberrant neovascularization. The bone morphogenetic proteins (BMPs) are potent stimulators of cell migration and angiogenesis. Using microarray analyses, we find that myosin-X (Myo10) is a BMP target gene. In endothelial cells, BMP6-induced Myo10 localizes in filopodia, and BMP-dependent filopodial assembly decreases when Myo10 expression is reduced. Likewise, cellular alignment and directional migration induced by BMP6 are Myo10 dependent. Surprisingly, we find that Myo10 and BMP6 receptor ALK6 colocalize in a BMP6-dependent fashion. ALK6 translocates into filopodia after BMP6 stimulation, and both ALK6 and Myo10 possess intrafilopodial motility. Additionally, Myo10 is required for BMP6-dependent Smad activation, indicating that in addition to its function in filopodial assembly, Myo10 also participates in a requisite amplification loop for BMP signaling. Our data indicate that Myo10 is required to guide endothelial migration toward BMP6 gradients via the regulation of filopodial function and amplification of BMP signals.

Gene Expression Profile Signatures Indicate a Role for Wnt Signaling in Endothelial Commitment From Embryonic Stem Cells

We have used global gene expression analysis to establish a comprehensive list of candidate genes in the developing vasculature during embryonic (ES) cell differentiation in vitro. A large set of genes, including growth factors, cell surface molecules, transcriptional factors, and members of several signal transduction pathways that are known to be involved in vasculogenesis or angiogenesis, were found to have expression patterns as expected. Some unknown or functionally uncharacterized genes were differentially regulated in flk1+ cells compared with flk1− cells, suggesting possible roles for these genes in vascular commitment. Particularly, multiple components of the Wnt signaling pathway were differentially regulated in flk1+ cells, including Wnt proteins, their receptors, downstream transcriptional factors, and other components belonging to this pathway. Activation of the Wnt signal was able to expand vascular progenitor populations whereas suppression of Wnt activity reduced flk1+ populations. Suppression of Wnt signaling also inhibited the formation of matured vascular capillary-like structures during late stages of embryoid body differentiation. These data indicate a requisite and ongoing role for Wnt activity during vascular development, and the gene expression profiles identify candidate components of this pathway that participate in vascular cell differentiation.

Dual Role for Glucocorticoids in Cardiomyocyte Hypertrophy and Apoptosis

Glucocorticoids and their synthetic derivatives are known to alter cardiac function in vivo; however, the nature of these effects and whether glucocorticoids act directly on cardiomyocytes are poorly understood. To explore the role of glucocorticoid signaling in the heart, we used rat embryonic H9C2 cardiomyocytes and primary cardiomyocytes as model systems. Dexamethasone (100 nm) treatment of cardiomyocytes caused a significant increase in cell size and up-regulated the expression of cardiac hypertrophic markers, including atrial natriuretic factor, β-myosin heavy chain, and skeletal muscle α-actin. In contrast, serum deprivation and TNFα exposure triggered cardiomyocyte apoptosis, and these apoptotic effects were inhibited by dexamethasone. Both the hypertrophic and anti-apoptotic actions of glucocorticoids were abolished by the glucocorticoid receptor (GR) antagonist RU486 and by short hairpin RNA-mediated GR depletion. Blocking the activity of the mineralocorticoid receptor had no effect on these glucocorticoid-dependent cardiomyocyte responses. Aldosterone (1 μm) activation of GR also promoted cardiomyocyte hypertrophy and cell survival. To elucidate the mechanism of the dual glucocorticoid actions, a genome-wide microarray was performed on H9C2 cardiomyocytes treated with vehicle or dexamethasone in the absence or presence of serum. Serum dramatically influenced the transcriptome regulated by GR, revealing potential glucocorticoid signaling mediators in both cardiomyocyte hypertrophy and apoptosis. These studies reveal a direct and dynamic role for glucocorticoids and GR signaling in the modulation of cardiomyocyte function.

Essential role of stress hormone signaling in cardiomyocytes for the prevention of heart disease

Significance Stress is increasingly associated with heart disease. Glucocorticoids are primary stress hormones, yet their direct role in the heart is poorly understood. Mice lacking the glucocorticoid receptor specifically in cardiomyocytes die prematurely from heart failure. The deficiency in glucocorticoid signaling leads to the aberrant regulation of a large cohort of genes strongly associated with both cardiovascular and inflammatory disease processes. These findings reveal an obligate role for cardiomyocyte glucocorticoid receptors in maintaining normal heart function and define a paradigm for stress in cardiovascular disease. Heart failure is a leading cause of death in humans, and stress is increasingly associated with adverse cardiac outcomes. Glucocorticoids are primary stress hormones, but their direct role in cardiovascular health and disease is poorly understood. To determine the in vivo function of glucocorticoid signaling in the heart, we generated mice with cardiomyocyte-specific deletion of the glucocorticoid receptor (GR). These mice are born at the expected Mendelian ratio, but die prematurely from spontaneous cardiovascular disease. By 3 mo of age, mice deficient in cardiomyocyte GR display a marked reduction in left ventricular systolic function, as evidenced by decreases in ejection fraction and fractional shortening. Heart weight and left ventricular mass are elevated, and histology revealed cardiac hypertrophy without fibrosis. Removal of endogenous glucocorticoids and mineralocorticoids neither augmented nor lessened the hypertrophic response. Global gene expression analysis of knockout hearts before pathology onset revealed aberrant regulation of a large cohort of genes associated with cardiovascular disease as well as unique disease genes associated with inflammatory processes. Genes important for maintaining cardiac contractility, repressing cardiac hypertrophy, promoting cardiomyocyte survival, and inhibiting inflammation had decreased expression in the GR-deficient hearts. These findings demonstrate that a deficiency in cardiomyocyte glucocorticoid signaling leads to spontaneous cardiac hypertrophy, heart failure, and death, revealing an obligate role for GR in maintaining normal cardiovascular function. Moreover, our findings suggest that selective activation of cardiomyocyte GR may represent an approach for the prevention of heart disease.

Bone Morphogenetic Protein Endothelial Cell Precursor–Derived Regulator Regulates Retinal Angiogenesis In Vivo in a Mouse Model of Oxygen-Induced Retinopathy

Objective—Bone morphogenetic proteins (BMPs) are potently proangiogenic; however, the mechanisms underlying the regulation of vessel development by BMPs are not fully understood. To assess the significance of BMP endothelial cell precursor-derived regulator (BMPER) in blood vessel formation in vivo, we investigated its role in retinal angiogenesis. Methods and Results—In a model of oxygen-induced retinopathy, Bmper mRNA expression and protein levels are downregulated, correlating with the initiation of Sma and Mad related protein phosphorylation in endothelial cells. Moreover, Bmper haploinsufficiency results in an increased rate of retinal revascularization, with retinas from Bmper+/− mice displaying increased numbers of branching points and angiogenic sprouts at the leading edge of the newly formed vasculature. Furthermore, although Bmper haploinsufficiency does not alter Bmp expression, it does lead to an increase in BMP signaling, as evidenced by increased phosphorylated Sma and Mad related protein levels in endothelial cells and increased expression of known BMP target genes. Conclusion—These observations provide compelling evidence that BMPER is important in the regulation of BMP signaling and revascularization in the hypoxic retina. These bring forth the possibility of novel therapeutic approaches for pathological angiogenesis based on manipulation of BMP signaling.

BMPER regulates cardiomyocyte size and vessel density in vivo

BACKGROUND BMPER, an orthologue of Drosophila melanogaster Crossveinless-2, is a secreted factor that regulates bone morphogenetic protein activity in endothelial cell precursors and during early cardiomyocyte differentiation. Although previously described in the heart, the role of BMPER in cardiac development and function remain unknown. METHODS BMPER-deficient hearts were phenotyped histologically and functionally using echocardiography and Doppler analysis. Since BMPER -/- mice die perinatally, adult BMPER +/- mice were challenged to pressure-overload-induced cardiac hypertrophy and hindlimb ischemia to determine changes in angiogenesis and regulation of cardiomyocyte size. RESULTS We identify for the first time the cardiac phenotype associated with BMPER haploinsufficiency. BMPER messenger RNA and protein are present in the heart during cardiac development through at least E14.5 but is lost by E18.5. BMPER +/- ventricles are thinner and less compact than sibling wild-type hearts. In the adult, BMPER +/- hearts present with decreased anterior and posterior wall thickness, decreased cardiomyocyte size and an increase in cardiac vessel density. Despite these changes, BMPER +/- mice respond to pressure-overload-induced cardiac hypertrophy challenge largely to the same extent as wild-type mice. CONCLUSION BMPER appears to play a role in regulating both vessel density and cardiac development in vivo; however, BMPER haploinsufficiency does not result in marked effects on cardiac function or adaptation to pressure overload hypertrophy.

The Molecular Pathophysiology, Differential Diagnosis, and Treatment of MPO Deficiency

Myeloperoxidase (MPO) deficiency is a common immune system variant affecting ~1:2000 people in the United States. MPO is partially responsible for the antimicrobial properties of both neutrophils and monocytes as a critical component in creating hypochlorous acid in phagosomes. While >95% of people with MPO are asymptomatic, a small percentage of people with MPO deficiency present with severe, often recurrent, microbial infections necessitating a clear understanding of its diagnosis and underlying pathophysiology. Primary “hereditary” MPO deficiency can be due to any number of recently recognized MPO mutations. Secondary or “acquired” causes, on the other hand, are also common due to a variety of exposures and concomitant diseases. The differentiation of primary and secondary causes can generally be made by assessing the clinical history and defining patterns of MPO expression. Generally, simple laboratory tests and/or clinical presentation and history can differentiate the underlying cause. No specific therapy is indicated in patients with MPO deficiency beyond microbe-specific therapy for their infections, along with strict control of blood glucose in diabetic patients. *Corresponding author: Monte S. Willis, McAllister Heart Institute, University of North Carolina at Chapel Hill, 2340B Medical Biomolecular Research Building, 103 Mason Farm Road, Chapel Hill, NC 27599-7525, USA, Tel: (919) 843-1938; Fax: (919) 843-4585; E-mail: monte_willis@med.unc.edu Received December 13, 2011; Accepted March 28, 2012; Published March 29, 2012 Citation: Ren R, Fedoriw Y, Willis MS (2012) The Molecular Pathophysiology, Differential Diagnosis, and Treatment of MPO Deficiency. J Clinic Experiment Pathol 2:109. doi:10.4172/2161-0681.1000109 Copyright:

Glucocorticoid signaling in cardiac disease.

Abstract As major mediators of stress regulation, glucocorticoids have an essential role in maintaining cardiovascular homeostasis under both physiological and pathological conditions. The release of glucocorticoids into the peripheral circulation is adjusted by the hypothalamic-pituitary-adrenal axis in response to various pathological challenges such as sepsis, starvation, and psychological stress. Clinically, dysregulation of the glucocorticoid-mediated signaling as a result of either excess ligand or receptor hypersensitivity is connected with the progression of unfavorable cardiovascular events such as cardiac hypertrophy, atherosclerosis, and coronary artery disease. The direct effects of glucocorticoids on cardiac tissues are mediated by two steroid receptors, the glucocorticoid receptor and mineralocorticoid receptor, which are both expressed by cardiomyocytes. Although each receptor has some shared responses to glucocorticoids, each receptor also has unique effects on cardiac functions. Elucidating the selective actions of each receptor is critical for determining the proper pharmaceutical targets in cardiovascular diseases.