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Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis.

Diaz MF, Li N, Lee HJ, Adamo L, Evans SM, Willey HE, Arora N, Torisawa YS, Vickers DA, Morris SA, Naveiras O, Murthy SK, Ingber DE, Daley GQ, García-Cardeña G, Wenzel PL - J. Exp. Med. (2015)

Bottom Line: Furthermore, Ncx1 heartbeat mutants, as well as static cultures of AGM, exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB).Similar to flow-exposed cultures, transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution.These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030 Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030 Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030.

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Developmental pathways are modulated by WSS. (A) Gene expression was determined by Illumina WG-6 BeadChips in E10.5 AGM-derived cells after 6 or 36 h of culture with WSS or static conditions. The Venn diagram displays overlap of genes differentially expressed between static and WSS at 6 and 36 h (n = 3 independent experiments at each time point; unpaired Student’s t test: P < 0.01; twofold threshold). Genes listed in red are up-regulated with application of WSS, whereas genes in blue are down-regulated. (B) Ingenuity pathway analysis reveals roles for WSS in several canonical pathways. (C) Functional themes are identified by network visualization of gene sets enriched by WSS at 6 and 36 h (p-value cutoff 0.05, FDR 0.8, overlap coefficient cutoff 0.5). Each node represents between 10 and 600 genes, where distance between nodes and thickness of edge lines reflects overlap of genes. Direction of enrichment is depicted for 6 h as color of inner node and for 36 h as color of node border (up, red; down, blue).
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fig5: Developmental pathways are modulated by WSS. (A) Gene expression was determined by Illumina WG-6 BeadChips in E10.5 AGM-derived cells after 6 or 36 h of culture with WSS or static conditions. The Venn diagram displays overlap of genes differentially expressed between static and WSS at 6 and 36 h (n = 3 independent experiments at each time point; unpaired Student’s t test: P < 0.01; twofold threshold). Genes listed in red are up-regulated with application of WSS, whereas genes in blue are down-regulated. (B) Ingenuity pathway analysis reveals roles for WSS in several canonical pathways. (C) Functional themes are identified by network visualization of gene sets enriched by WSS at 6 and 36 h (p-value cutoff 0.05, FDR 0.8, overlap coefficient cutoff 0.5). Each node represents between 10 and 600 genes, where distance between nodes and thickness of edge lines reflects overlap of genes. Direction of enrichment is depicted for 6 h as color of inner node and for 36 h as color of node border (up, red; down, blue).

Mentions: We and others previously identified NO as a critical regulator of hematopoietic progenitor expansion in response to WSS and blood flow (Adamo et al., 2009; North et al., 2009; Wang et al., 2011). To more precisely define the signaling mechanisms triggered by WSS, we conducted global gene expression profiling of ex vivo cultures of AGM. Notch, Wnt, and eicosanoid signaling have previously been found in proteomic analyses to respond rapidly to fluid shear stress in aortic endothelial cells (Wang et al., 2007). Informed by these studies and our own serial measurements of gene subsets, we selected an early time point (6 h) and one demonstrated previously to enhance hematopoietic activity (36 h) to perform global gene expression analysis (Adamo et al., 2009). In brief, WSS was applied to dissociated E10.5 AGM cultured within microfluidic channels as described previously (Li et al., 2014), replicates were lysed, and RNA was processed for analysis by Illumina Mouse WG-6 v2.0 Expression BeadChips (45,200 transcripts). Unsupervised hierarchical clustering of unfiltered genes positioned 6- and 36-h cultures in two distinct groups, followed by segregation of static and WSS cultures (not depicted). Differential gene expression analysis (P < 0.01, twofold threshold) revealed significant change in 1,435 unique transcripts at 6 h, 347 at 36 h, and 109 common to both time points (Fig. 5 A and Dataset S1). By Ingenuity pathway analysis, these genes were found to encode enzymes and transcription regulators required for signaling through several overlapping pathways, including G protein–coupled receptors (GPCRs), calcium transport, nuclear factor of activated T cells (NFAT), phospholipase C, and PI3K (Btk, Chp1, Nfatc3, and Plcb3; Fig. 5 B). Components of the NFκB signaling pathway were also enriched, including the TRAF family member associated NFκB activator (Tank) and IKBε regulatory subunit (Nfkbie). Several other kinases, enzymes, and transcription factors were found to contribute to signaling through CREB, such as the α regulatory subunit of cAMP-dependent protein kinase (Prkar1a), G protein–binding protein (Gnb1l), Creb5, and CREB-binding protein (Crebbp). Wnt/β-catenin regulators and ligands were also enriched (Akt3, Dkk1, Wnt1, and Wnt7b). Functional enrichment analysis of unfiltered genes by GSEA (Mootha et al., 2003; Subramanian et al., 2005) was used to construct a global network wherein overlapping gene sets clustered together (Merico et al., 2010) and connections between pathways could be visualized at 6 and 36 h in parallel (Fig. 5 C). Wnt signaling functionally clustered with the adaptive immune system and cell cycle, whereas Notch, TGF-β, and EGFR/CREB/MAPK clustered as distinct groups. Notably, several functional groups that emerged from network analysis are known regulators or effectors of prostaglandin production and signaling, including calcium, MAPK, CREB, Wnt, NFκB, and biological oxidation involving prostaglandin-endoperoxide synthase function of COX1 and COX2 (Ptgs1 and Ptgs2; Fig. 5 C and Dataset S2; Tsatsanis et al., 2006; Goessling et al., 2011).


Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis.

Diaz MF, Li N, Lee HJ, Adamo L, Evans SM, Willey HE, Arora N, Torisawa YS, Vickers DA, Morris SA, Naveiras O, Murthy SK, Ingber DE, Daley GQ, García-Cardeña G, Wenzel PL - J. Exp. Med. (2015)

Developmental pathways are modulated by WSS. (A) Gene expression was determined by Illumina WG-6 BeadChips in E10.5 AGM-derived cells after 6 or 36 h of culture with WSS or static conditions. The Venn diagram displays overlap of genes differentially expressed between static and WSS at 6 and 36 h (n = 3 independent experiments at each time point; unpaired Student’s t test: P < 0.01; twofold threshold). Genes listed in red are up-regulated with application of WSS, whereas genes in blue are down-regulated. (B) Ingenuity pathway analysis reveals roles for WSS in several canonical pathways. (C) Functional themes are identified by network visualization of gene sets enriched by WSS at 6 and 36 h (p-value cutoff 0.05, FDR 0.8, overlap coefficient cutoff 0.5). Each node represents between 10 and 600 genes, where distance between nodes and thickness of edge lines reflects overlap of genes. Direction of enrichment is depicted for 6 h as color of inner node and for 36 h as color of node border (up, red; down, blue).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: Developmental pathways are modulated by WSS. (A) Gene expression was determined by Illumina WG-6 BeadChips in E10.5 AGM-derived cells after 6 or 36 h of culture with WSS or static conditions. The Venn diagram displays overlap of genes differentially expressed between static and WSS at 6 and 36 h (n = 3 independent experiments at each time point; unpaired Student’s t test: P < 0.01; twofold threshold). Genes listed in red are up-regulated with application of WSS, whereas genes in blue are down-regulated. (B) Ingenuity pathway analysis reveals roles for WSS in several canonical pathways. (C) Functional themes are identified by network visualization of gene sets enriched by WSS at 6 and 36 h (p-value cutoff 0.05, FDR 0.8, overlap coefficient cutoff 0.5). Each node represents between 10 and 600 genes, where distance between nodes and thickness of edge lines reflects overlap of genes. Direction of enrichment is depicted for 6 h as color of inner node and for 36 h as color of node border (up, red; down, blue).
Mentions: We and others previously identified NO as a critical regulator of hematopoietic progenitor expansion in response to WSS and blood flow (Adamo et al., 2009; North et al., 2009; Wang et al., 2011). To more precisely define the signaling mechanisms triggered by WSS, we conducted global gene expression profiling of ex vivo cultures of AGM. Notch, Wnt, and eicosanoid signaling have previously been found in proteomic analyses to respond rapidly to fluid shear stress in aortic endothelial cells (Wang et al., 2007). Informed by these studies and our own serial measurements of gene subsets, we selected an early time point (6 h) and one demonstrated previously to enhance hematopoietic activity (36 h) to perform global gene expression analysis (Adamo et al., 2009). In brief, WSS was applied to dissociated E10.5 AGM cultured within microfluidic channels as described previously (Li et al., 2014), replicates were lysed, and RNA was processed for analysis by Illumina Mouse WG-6 v2.0 Expression BeadChips (45,200 transcripts). Unsupervised hierarchical clustering of unfiltered genes positioned 6- and 36-h cultures in two distinct groups, followed by segregation of static and WSS cultures (not depicted). Differential gene expression analysis (P < 0.01, twofold threshold) revealed significant change in 1,435 unique transcripts at 6 h, 347 at 36 h, and 109 common to both time points (Fig. 5 A and Dataset S1). By Ingenuity pathway analysis, these genes were found to encode enzymes and transcription regulators required for signaling through several overlapping pathways, including G protein–coupled receptors (GPCRs), calcium transport, nuclear factor of activated T cells (NFAT), phospholipase C, and PI3K (Btk, Chp1, Nfatc3, and Plcb3; Fig. 5 B). Components of the NFκB signaling pathway were also enriched, including the TRAF family member associated NFκB activator (Tank) and IKBε regulatory subunit (Nfkbie). Several other kinases, enzymes, and transcription factors were found to contribute to signaling through CREB, such as the α regulatory subunit of cAMP-dependent protein kinase (Prkar1a), G protein–binding protein (Gnb1l), Creb5, and CREB-binding protein (Crebbp). Wnt/β-catenin regulators and ligands were also enriched (Akt3, Dkk1, Wnt1, and Wnt7b). Functional enrichment analysis of unfiltered genes by GSEA (Mootha et al., 2003; Subramanian et al., 2005) was used to construct a global network wherein overlapping gene sets clustered together (Merico et al., 2010) and connections between pathways could be visualized at 6 and 36 h in parallel (Fig. 5 C). Wnt signaling functionally clustered with the adaptive immune system and cell cycle, whereas Notch, TGF-β, and EGFR/CREB/MAPK clustered as distinct groups. Notably, several functional groups that emerged from network analysis are known regulators or effectors of prostaglandin production and signaling, including calcium, MAPK, CREB, Wnt, NFκB, and biological oxidation involving prostaglandin-endoperoxide synthase function of COX1 and COX2 (Ptgs1 and Ptgs2; Fig. 5 C and Dataset S2; Tsatsanis et al., 2006; Goessling et al., 2011).

Bottom Line: Furthermore, Ncx1 heartbeat mutants, as well as static cultures of AGM, exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB).Similar to flow-exposed cultures, transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution.These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030 Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030 Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030.

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