human coronary artery smooth muscle cells pasmc  (Lonza)

Journal: bioRxiv

Article Title: A Molecular Pathway for Arterial-Specific Association of Vascular Smooth Muscle Cells

doi: 10.1101/2019.12.27.889782

Figure Lengend Snippet: A,B, Schematic diagrams illustrating the experimental design for using the mrc1a promoter to drive ectopic mosaic expression of cxcl12b in veins. A, A Tol2(mrc1a:cxcl12b-2a-mCherry) DNA construct co-translationally expressing cxcl12b and mCherry under the control of the mrc1a promoter is injected into Tg(tagln:eGFP) transgenic zebrafish embryos at the 1 cell stage. B, At 4 dpf tol2(mrc1a:cxcl12b-2a-mCherry) -injected zebrafish larvae are analyzed for vSMC (eGFP) association at sites of mCherry (i.e. cxcl12b ) expression in the dorsal aorta and cardinal vein. C,D , Representative confocal images of the mid-trunk of 4 dpf Tg(tagln:eGFP) transgenic larvae injected with either control Tol2(mrc1a) “empty vector” (C) or Tol2(mrc1a:cxcl12b-2a-mCherry) (D). eGFP-expressing vSMCs are shown in green, cxcl12b-2a-mCherry expression in dorsal aorta (DA) or cardinal vein (CV) endothelium is shown in magenta. E , Quantification of eGFP-positive vSMC associated with the dorsal aorta (DA) or cardinal vein (CV) in 4 dpf Tg(tagln:eGFP) transgenic zebrafish injected with either control Tol2(mrc1a) “empty vector” (black columns) or Tol2(mrc1a:cxcl12b-2a-mCherry) (green columns), showing strongly increased association of vSMCs with the cardinal vein. F , Schematic diagrams showing potential models for direct (left) versus indirect (right) mechanisms for promoting arterial recruitment of vSMC via CXCL12. G , Schematic diagram illustrating the 3D pulmonary artery smooth muscle cell (PASMC) motility assay. CXCL12, PDGFB, or nothing (control) is placed within the collagen gel to determine if PASMCs migrate towards these potential chemoattractants. H, Representative lateral images of 3D collagen gels showing PASMCs within the collagen matrix for each gel condition. I , Quantification of the relative number of PASMCs invading the collagen gel. The control is set to 100% and the CXCL12 and PDGFB conditions normalized to this level of invasion. Scale bars = 75 µm (panels C,D), 200 µm (panel H). Box plots are graphed showing the median versus the first and third quartiles of the data (the middle, top, and bottom lines of the box respectively). The whiskers demonstrate the spread of data within 1.5x above and below the interquartile range. All data points are shown as individual dots, with outliers shown above or below the whiskers. P-values are indicated above statistically significant datasets.

Article Snippet: Human coronary artery smooth muscle cells (PASMC, Lonza) were cultured in 10% FBS in Advanced DMEM base media (Gibco) on 1mg/ml gelatin coated tissue culture flasks.

Techniques: Expressing, Construct, Control, Injection, Transgenic Assay, Plasmid Preparation, Motility Assay

Journal: bioRxiv

Article Title: A Molecular Pathway for Arterial-Specific Association of Vascular Smooth Muscle Cells

doi: 10.1101/2019.12.27.889782

Figure Lengend Snippet: A,B, PDGFB transcript (A) and protein (B) in HUVEC cells cultured in vitro in a confluent cell monolayer for up to 8 hours with (“+CXCL12”) or without (“CTRL”) added recombinant CXCL12. Relative PDGFB transcript levels (A) and protein levels (B) were measured by qPCR and Western blot, respectively, showing an upregulation of both PDGFB transcript and PDGFB protein levels in response to stimulation by CXCL12. C-E , PDGFB transcript (C) and protein levels (D,E) in HUVEC cells cultured in vitro in a confluent cell monolayer and treated with either control, CXCR4, or CXCL12 siRNAs. Relative PDGFB transcript (C) and protein (E,F) levels were measured by qPCR and Western blot, respectively, showing suppression of both PDGFB transcript and protein in response to either CXCR4 or CXCL12 knockdown. Values in A, C, and E are averaged from three individual experiments and expressed as a percentage of control. Error bars ± s.d. (A,C). F, Confocal images of immunohistochemically stained transverse sections through the dorsal aorta of E12.5 Cxcr4+/- heterozygous sibling (F) and Cxcr4-/- mutant (G) mice, probed for platelet derived growth factor B (PDGFB; green) and for smooth muscle 22 alpha (SM22, aka transgelin) for vascular smooth muscle cells (vSMC, red). G, Quantification of relative PDGFB protein expression in Cxcr4+/- heterozygous embryos versus Cxcr4-/- homozygous mutant embryos. Values are expressed as a percentage of heterozygous control and averaged from five individual mice per condition. H , Schematic diagram of a zebrafish larva with the red box highlighting the area imaged in panels I and J. I,J, Whole mount in situ hybridization of the mid-trunk of 2.5 dpf zebrafish injected with control (I) or cxcl12b (J) RNA, showing upregulation of pdgfb transcript in response to exogenous cxcl12b . Red and blue brackets in panel J indicate the dorsal aorta and cardinal vein, respectively. K, Western blot of whole embryo protein lysate from 2.5 dpf zebrafish injected with either control (left) or cxcl12b (right) RNA, probed for pdgfb (top) or alpha tubulin (bottom), showing upregulation of pdgfb protein levels in response to exogenous cxcl12b . Images are representative of data from three individual experiments. M , Schematic diagram illustrating the proposed model for endothelial-autonomous chemokine signaling driving increased endothelial PDGFB ligand production, thereby indirectly promoting vSMC acquisition by arteries. Putative upstream regulators of CXCL12 and CXCR4 are noted in red. Scale bars = 50 µm (panel F). Box plots are graphed showing the median versus the first and third quartiles of the data (the middle, top, and bottom lines of the box respectively). The whiskers demonstrate the spread of data within 1.5x above and below the interquartile range. All data points are shown as individual dots, with outliers shown above or below the whiskers. P-values are indicated above statistically significant datasets.

Article Snippet: Human coronary artery smooth muscle cells (PASMC, Lonza) were cultured in 10% FBS in Advanced DMEM base media (Gibco) on 1mg/ml gelatin coated tissue culture flasks.

Techniques: Cell Culture, In Vitro, Recombinant, Western Blot, Control, Knockdown, Staining, Mutagenesis, Derivative Assay, Expressing, In Situ Hybridization, Injection