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Proteomic-based detection of a protein cluster dysregulated during cardiovascular development identifies biomarkers of congenital heart defects.

Nath AK, Krauthammer M, Li P, Davidov E, Butler LC, Copel J, Katajamaa M, Oresic M, Buhimschi I, Buhimschi C, Snyder M, Madri JA - PLoS ONE (2009)

Bottom Line: Additionally, a successful application of a cluster of protein biomarkers (WNT16, ST14 and Pcsk1) as a prenatal screen for CHDs was confirmed in a study of human amniotic fluid (AF) samples from women carrying normal fetuses and those with CHDs.The novel finding that WNT16, ST14 and Pcsk1 protein levels increase in fetuses with CHDs suggests that these proteins may play a role in the etiology of human CHDs.The information gained through this bed-side to bench translational approach contributes to a more complete understanding of the protein pathways dysregulated during cardiovascular development and provides novel avenues for diagnostic and therapeutic interventions, beneficial to fetuses at risk for CHDs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America. anjali.nath@aya.yale.edu

ABSTRACT

Background: Cardiovascular development is vital for embryonic survival and growth. Early gestation embryo loss or malformation has been linked to yolk sac vasculopathy and congenital heart defects (CHDs). However, the molecular pathways that underlie these structural defects in humans remain largely unknown hindering the development of molecular-based diagnostic tools and novel therapies.

Methodology/principal findings: Murine embryos were exposed to high glucose, a condition known to induce cardiovascular defects in both animal models and humans. We further employed a mass spectrometry-based proteomics approach to identify proteins differentially expressed in embryos with defects from those with normal cardiovascular development. The proteins detected by mass spectrometry (WNT16, ST14, Pcsk1, Jumonji, Morca2a, TRPC5, and others) were validated by Western blotting and immunoflorescent staining of the yolk sac and heart. The proteins within the proteomic dataset clustered to adhesion/migration, differentiation, transport, and insulin signaling pathways. A functional role for several proteins (WNT16, ADAM15 and NOGO-A/B) was demonstrated in an ex vivo model of heart development. Additionally, a successful application of a cluster of protein biomarkers (WNT16, ST14 and Pcsk1) as a prenatal screen for CHDs was confirmed in a study of human amniotic fluid (AF) samples from women carrying normal fetuses and those with CHDs.

Conclusions/significance: The novel finding that WNT16, ST14 and Pcsk1 protein levels increase in fetuses with CHDs suggests that these proteins may play a role in the etiology of human CHDs. The information gained through this bed-side to bench translational approach contributes to a more complete understanding of the protein pathways dysregulated during cardiovascular development and provides novel avenues for diagnostic and therapeutic interventions, beneficial to fetuses at risk for CHDs.

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Related in: MedlinePlus

Experimental Model: Applying Proteomic Profiling to a Hyperglycemia Induced Model of Cardiovascular Defects.Embryos cultured ex vivo exhibit normal organogenesis and vascular development (A, normal embryo inside a functional yolk sac containing circulating red blood cells). Embryos treated with 20 mM D-glucose display malformation of the embryo and vasculature (B, abnormal embryo and pooling of blood at one pole). PECAM staining of control yolk sacs reveals a mature, remodeled vasculature (C) while hyperglycemic yolk sacs appear immature and mal-developed (D). Normally by E9.5, EMT has begun within the cardiac endocardial cushions (E). Treatment with hyperglycemia results in an acellular endocardial cushion (F). E–F: α-Smooth muscle cell actin (green) labeling of the myocardium (Myo) and mesenchymal cells (Mes); PECAM (red) staining of the endocardium (En); CJ (Cardiac Jelly). Volcano plots (G–I)) depicting each peptide peak detected by LC-MS as a blue dot and plotting the log2 ratio (of treatment to control) against −log10 p value (G, HG vs Control; H, HG+rVEGF vs Control; I, HG+ nitric oxide donor vs Control). The red dots represent the 143 peptide peaks that were dysregulated by hyperglycemia but returned to control levels by both rVEGF (H) and nitric oxide donor (I). The red horizontal lines are drawn at p = 0.05 and the red vertical lines are drawn at log2 ratio = 1 and −1. Scale bars = 100 µm. (A–B is reprinted from Am J Pathol 2001, 158:1199-206 with permission from the American Society for Investigative Pathology.)
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pone-0004221-g001: Experimental Model: Applying Proteomic Profiling to a Hyperglycemia Induced Model of Cardiovascular Defects.Embryos cultured ex vivo exhibit normal organogenesis and vascular development (A, normal embryo inside a functional yolk sac containing circulating red blood cells). Embryos treated with 20 mM D-glucose display malformation of the embryo and vasculature (B, abnormal embryo and pooling of blood at one pole). PECAM staining of control yolk sacs reveals a mature, remodeled vasculature (C) while hyperglycemic yolk sacs appear immature and mal-developed (D). Normally by E9.5, EMT has begun within the cardiac endocardial cushions (E). Treatment with hyperglycemia results in an acellular endocardial cushion (F). E–F: α-Smooth muscle cell actin (green) labeling of the myocardium (Myo) and mesenchymal cells (Mes); PECAM (red) staining of the endocardium (En); CJ (Cardiac Jelly). Volcano plots (G–I)) depicting each peptide peak detected by LC-MS as a blue dot and plotting the log2 ratio (of treatment to control) against −log10 p value (G, HG vs Control; H, HG+rVEGF vs Control; I, HG+ nitric oxide donor vs Control). The red dots represent the 143 peptide peaks that were dysregulated by hyperglycemia but returned to control levels by both rVEGF (H) and nitric oxide donor (I). The red horizontal lines are drawn at p = 0.05 and the red vertical lines are drawn at log2 ratio = 1 and −1. Scale bars = 100 µm. (A–B is reprinted from Am J Pathol 2001, 158:1199-206 with permission from the American Society for Investigative Pathology.)

Mentions: Under normoglycemic conditions, control embryos exhibit normal organogenesis and vascular development of the yolk sac (Fig. 1A, whole embryo inside a functional yolk sac containing circulating red blood cells). Hyperglycemic embryos display malformation of the embryo proper and yolk sac vasculature (Fig. 1B, abnormal embryo with a nonfunctional vitelline circulation, evidenced by pooling of blood at one pole of the yolk sac). Whole mount staining of control yolk sacs for the endothelial marker PECAM-1 confirms the formation of a mature, remodeled vasculature with a hierarchy of arborizing large and small vessels (Fig. 1C) while hyperglycemic yolk sacs appear arrested at the primary capillary plexus stage of vascular development (Fig. 1D). By E9.5, endocardial cells (cardiac endothelial cells) of the endocardial cushions are actively undergoing EMT and invading the cardiac jelly in control embryos (Fig. 1E). Hyperglycemia results in an acellular endocardial cushion due to inhibition of EMT, which prematurely arrests cushion morphogenesis (Fig. 1F).


Proteomic-based detection of a protein cluster dysregulated during cardiovascular development identifies biomarkers of congenital heart defects.

Nath AK, Krauthammer M, Li P, Davidov E, Butler LC, Copel J, Katajamaa M, Oresic M, Buhimschi I, Buhimschi C, Snyder M, Madri JA - PLoS ONE (2009)

Experimental Model: Applying Proteomic Profiling to a Hyperglycemia Induced Model of Cardiovascular Defects.Embryos cultured ex vivo exhibit normal organogenesis and vascular development (A, normal embryo inside a functional yolk sac containing circulating red blood cells). Embryos treated with 20 mM D-glucose display malformation of the embryo and vasculature (B, abnormal embryo and pooling of blood at one pole). PECAM staining of control yolk sacs reveals a mature, remodeled vasculature (C) while hyperglycemic yolk sacs appear immature and mal-developed (D). Normally by E9.5, EMT has begun within the cardiac endocardial cushions (E). Treatment with hyperglycemia results in an acellular endocardial cushion (F). E–F: α-Smooth muscle cell actin (green) labeling of the myocardium (Myo) and mesenchymal cells (Mes); PECAM (red) staining of the endocardium (En); CJ (Cardiac Jelly). Volcano plots (G–I)) depicting each peptide peak detected by LC-MS as a blue dot and plotting the log2 ratio (of treatment to control) against −log10 p value (G, HG vs Control; H, HG+rVEGF vs Control; I, HG+ nitric oxide donor vs Control). The red dots represent the 143 peptide peaks that were dysregulated by hyperglycemia but returned to control levels by both rVEGF (H) and nitric oxide donor (I). The red horizontal lines are drawn at p = 0.05 and the red vertical lines are drawn at log2 ratio = 1 and −1. Scale bars = 100 µm. (A–B is reprinted from Am J Pathol 2001, 158:1199-206 with permission from the American Society for Investigative Pathology.)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2626248&req=5

pone-0004221-g001: Experimental Model: Applying Proteomic Profiling to a Hyperglycemia Induced Model of Cardiovascular Defects.Embryos cultured ex vivo exhibit normal organogenesis and vascular development (A, normal embryo inside a functional yolk sac containing circulating red blood cells). Embryos treated with 20 mM D-glucose display malformation of the embryo and vasculature (B, abnormal embryo and pooling of blood at one pole). PECAM staining of control yolk sacs reveals a mature, remodeled vasculature (C) while hyperglycemic yolk sacs appear immature and mal-developed (D). Normally by E9.5, EMT has begun within the cardiac endocardial cushions (E). Treatment with hyperglycemia results in an acellular endocardial cushion (F). E–F: α-Smooth muscle cell actin (green) labeling of the myocardium (Myo) and mesenchymal cells (Mes); PECAM (red) staining of the endocardium (En); CJ (Cardiac Jelly). Volcano plots (G–I)) depicting each peptide peak detected by LC-MS as a blue dot and plotting the log2 ratio (of treatment to control) against −log10 p value (G, HG vs Control; H, HG+rVEGF vs Control; I, HG+ nitric oxide donor vs Control). The red dots represent the 143 peptide peaks that were dysregulated by hyperglycemia but returned to control levels by both rVEGF (H) and nitric oxide donor (I). The red horizontal lines are drawn at p = 0.05 and the red vertical lines are drawn at log2 ratio = 1 and −1. Scale bars = 100 µm. (A–B is reprinted from Am J Pathol 2001, 158:1199-206 with permission from the American Society for Investigative Pathology.)
Mentions: Under normoglycemic conditions, control embryos exhibit normal organogenesis and vascular development of the yolk sac (Fig. 1A, whole embryo inside a functional yolk sac containing circulating red blood cells). Hyperglycemic embryos display malformation of the embryo proper and yolk sac vasculature (Fig. 1B, abnormal embryo with a nonfunctional vitelline circulation, evidenced by pooling of blood at one pole of the yolk sac). Whole mount staining of control yolk sacs for the endothelial marker PECAM-1 confirms the formation of a mature, remodeled vasculature with a hierarchy of arborizing large and small vessels (Fig. 1C) while hyperglycemic yolk sacs appear arrested at the primary capillary plexus stage of vascular development (Fig. 1D). By E9.5, endocardial cells (cardiac endothelial cells) of the endocardial cushions are actively undergoing EMT and invading the cardiac jelly in control embryos (Fig. 1E). Hyperglycemia results in an acellular endocardial cushion due to inhibition of EMT, which prematurely arrests cushion morphogenesis (Fig. 1F).

Bottom Line: Additionally, a successful application of a cluster of protein biomarkers (WNT16, ST14 and Pcsk1) as a prenatal screen for CHDs was confirmed in a study of human amniotic fluid (AF) samples from women carrying normal fetuses and those with CHDs.The novel finding that WNT16, ST14 and Pcsk1 protein levels increase in fetuses with CHDs suggests that these proteins may play a role in the etiology of human CHDs.The information gained through this bed-side to bench translational approach contributes to a more complete understanding of the protein pathways dysregulated during cardiovascular development and provides novel avenues for diagnostic and therapeutic interventions, beneficial to fetuses at risk for CHDs.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America. anjali.nath@aya.yale.edu

ABSTRACT

Background: Cardiovascular development is vital for embryonic survival and growth. Early gestation embryo loss or malformation has been linked to yolk sac vasculopathy and congenital heart defects (CHDs). However, the molecular pathways that underlie these structural defects in humans remain largely unknown hindering the development of molecular-based diagnostic tools and novel therapies.

Methodology/principal findings: Murine embryos were exposed to high glucose, a condition known to induce cardiovascular defects in both animal models and humans. We further employed a mass spectrometry-based proteomics approach to identify proteins differentially expressed in embryos with defects from those with normal cardiovascular development. The proteins detected by mass spectrometry (WNT16, ST14, Pcsk1, Jumonji, Morca2a, TRPC5, and others) were validated by Western blotting and immunoflorescent staining of the yolk sac and heart. The proteins within the proteomic dataset clustered to adhesion/migration, differentiation, transport, and insulin signaling pathways. A functional role for several proteins (WNT16, ADAM15 and NOGO-A/B) was demonstrated in an ex vivo model of heart development. Additionally, a successful application of a cluster of protein biomarkers (WNT16, ST14 and Pcsk1) as a prenatal screen for CHDs was confirmed in a study of human amniotic fluid (AF) samples from women carrying normal fetuses and those with CHDs.

Conclusions/significance: The novel finding that WNT16, ST14 and Pcsk1 protein levels increase in fetuses with CHDs suggests that these proteins may play a role in the etiology of human CHDs. The information gained through this bed-side to bench translational approach contributes to a more complete understanding of the protein pathways dysregulated during cardiovascular development and provides novel avenues for diagnostic and therapeutic interventions, beneficial to fetuses at risk for CHDs.

Show MeSH
Related in: MedlinePlus