Journal: Nature Communications

Article Title: Molecular mechanisms of coronary artery disease risk at the PDGFD locus

doi: 10.1038/s41467-023-36518-9

Figure Lengend Snippet: a UCSC browser screenshot at 11q22.3 locus showing position of PDGFD gene and lncRNA AP002989.1 relative to the candidate SNP rs2019090, and b overlap of rs2019090 with ChIP-seq tracks for CAD risk transcription factors SMAD3 and TCF21. Also shown are ATAC-seq open chromatin and active enhancer histone modification H3K27ac ChIP-seq tracks in human coronary artery smooth muscle cells (HCASMC), as well as ENCODE layered H3K27ac for HUVEC (blue) and NHLF (purple) cells. Genomic coordinates refer to hg19 assembly. c Genomic sequence at rs2019090 for protective and disease alleles, with FOXC1/C2 motifs indicated. d Co-localization of coronary artery disease (CAD) GWAS association data on the x -axis and PDGFD eQTL association data (GTEx v8, aorta) on the y -axis. P -values represent original GWAS findings, not a statistical test to determine variant causality. e Position weight matrices for FOXC1 and FOXC2, as per the JASPAR database. f , g CRISPRi epigenetic silencing by transduction of dCas9KRAB and single guide RNAs targeted around rs2019090 in cell lot 59386145, a HCASMC primary culture with AA genotype. Expression of PDGFD , ** p = 0.0033, **** p < 0.0001 and lncRNA AP002989.1 were evaluated by quantitative RT-PCR. Data were normalized relative to control and expressed as mean ± s.e.m. with p -values obtained using ordinary one-way ANOVA with Dunnett’s multiple comparisons post-hoc test. Dots represent three technical replicates from three biologically independent samples. Source data are provided in the Source Data File.

Article Snippet: Gene expression was assessed using TaqMan qPCR probes (Thermo Fisher) for PDGFD (Hs00228671_m1), AP002989.1 (hs04980451_m1), FOXC1 (Hs00559473_s1), FOXC2 (Hs00270951_s1), PDGFRA (Hs00998018_m1), PDGFRB (Hs01019589_m1), CCL2 (Hs00234140_m1), and CCL7 (Hs00171147_m1) according to the manufacturer’s instructions on a ViiA7 Real-Time PCR system (Applied Biosystems, Foster City, CA).

Techniques: ChIP-sequencing, Modification, Sequencing, Variant Assay, Transduction, Expressing, Quantitative RT-PCR, Control

Journal: Nature Communications

Article Title: Molecular mechanisms of coronary artery disease risk at the PDGFD locus

doi: 10.1038/s41467-023-36518-9

Figure Lengend Snippet: Results of enhancer trap assay for a FOXC1 , * p = 0.0126 and b FOXC2 , * p = 0.0155, ** p = 0.0040, or empty control (pWPI) co-transfected with luciferase reporters with three copies of the 150 base pair region containing the A allele (Ax3) or T allele (Tx3) cloned into the minimal promoter-driven luciferase reporter vector pLUC-MCS (MCS). A7r5 rat vascular smooth muscle cells were used for these assays. Values represent mean ± s.e.m. of four biologically independent experiments expressed as fold change relative to pWPI-empty plasmid with p-values obtained with two-sided unpaired t-test. c Results of quantitative polymerase chain reaction (qPCR) analysis for PDGFD , n = 5 biologically independent knockdown samples, ** p = 0.0026, ** p = 0.0058, * p = 0.0123, and n = 4 biologically independent over-expression samples in 1508 cell lot and n = 3 for all other lots and conditions; *** p = 0.0004, *** p = 0.0002. Analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons post-hoc test or d AP002989.1 expression with knockdown (KD), n = 6 biologically independent knockdown samples in 1508 cell lot, or over-expression (OE) of FOXC1 , n = 3 biologically independent samples for all other conditions and cell lots, in HCASMC carrying different genotypes for rs2019090. Each dot represents a biologically independent sample. Data were normalized relative to controls and expressed as mean ± s.e.m with p -values using ordinary one-way ANOVA with Dunnett’s multiple comparisons post hoc test. e qPCR analysis for expression levels of PDGFD , ** p = 0.0028, f FOXC1 , ** p = 0.0024, g AP002989.1 , h PDGFRA , ** p = 0.0039, and i PDGFRB , ** p = 0.0068 with PDGFD knockdown (KD) in HCASMC. Each dot represents a biologically independent sample, n = 3. Data were expressed as mean ± s.e.m with p -values using a two-sided unpaired t-test. j qPCR analysis for expression levels of PDGFD , n = 6 for PD(Low) and PD(Mid), n = 4 for PD(High ) , *** p < 0.001, k FOXC1 , n = 6 for PD(Low) and PD(Mid), n = 4 for PD(High), ** p = 0.0055, * p = 0.0177 l AP002989.1 , n = 5 for PD(Low), n = 3 for PD(Mid), n = 2 for PD(High), m PDGFRA , n = 6 for PD(Low) and PD(High), n = 4 for PD(Mid), *** p = 0.0006, * p = 0.0189, *** p = 0.0004 and n PDGFRβ , n = 6 for PD(Low) and PD(Mid), n = 4 for PD(High), * p = 0.0244, ** p = 0.0035, * p = 0.0305 with PDGFD overexpression (OE) in HCASMC. Data grouped based on expression levels of PDGFD and expressed as mean ± s.e.m of biologically independent samples with p -values obtained from one-way ANOVA with Dunnett’s multiple comparisons post-hoc test. Each dot represents a biological replicate. Data represented as relative expression as control ratio (treatment of scramble siRNA (si-Ctl, KD control) or empty-pWPI (Ctl, OE control). Source data are provided in the Source Data File.

Article Snippet: Gene expression was assessed using TaqMan qPCR probes (Thermo Fisher) for PDGFD (Hs00228671_m1), AP002989.1 (hs04980451_m1), FOXC1 (Hs00559473_s1), FOXC2 (Hs00270951_s1), PDGFRA (Hs00998018_m1), PDGFRB (Hs01019589_m1), CCL2 (Hs00234140_m1), and CCL7 (Hs00171147_m1) according to the manufacturer’s instructions on a ViiA7 Real-Time PCR system (Applied Biosystems, Foster City, CA).

Techniques: TRAP Assay, Control, Transfection, Luciferase, Clone Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction, Knockdown, Over Expression, Expressing

Journal: Nature Communications

Article Title: Molecular mechanisms of coronary artery disease risk at the PDGFD locus

doi: 10.1038/s41467-023-36518-9

Figure Lengend Snippet: a Schematic of experimental design showing that dissected aortic tissues were harvested for single cell RNA sequencing (scRNAseq) and histology analyses from SMC-specific lineage tracing control (Ctl) and lineage tracing Pdgfd knockout (KO) mice. Eight-week-old mice, 2 Ctl and 3 KO captures (two mice per capture), were treated with tamoxifen twice at 3-day intervals and subsequently fed high fat diet for 16 weeks and then sacrificed. Tissues were digested to single cells, tdTomato positive (tdT+) fluorescence and negative (tdT-) cells collected and captured on the10x Chromium controller, libraries generated and sequenced. Created with BioRender.com. b Uniform manifold approximation and projection (UMAP) of scRNAseq results identified 13 different clusters at 2.6 clustering resolution, with respective biological cluster identities as defined by cluster marker genes. c UMAP displaying expression of indicated markers reflecting unique cluster identity: Cnn1 , SMC; Fn1 , FMC; Ibsp , CMC; Rgs5 , pericytes. d UMAP visualizing dimension reduction plots of Pdgfd and Pdgfrb expression. e UMAP images comparing feature expression of tdTomato positive cells from Ctl and KO mice. The dotted line is generated based on the Ctl image. Arrows indicate increase in SMC number and decrease in transition cell (CMC) number. f Bar plot presenting the average percentage of lineage traced cells and g non-lineage traced cells in Ctl and KO groups.

Article Snippet: Gene expression was assessed using TaqMan qPCR probes (Thermo Fisher) for PDGFD (Hs00228671_m1), AP002989.1 (hs04980451_m1), FOXC1 (Hs00559473_s1), FOXC2 (Hs00270951_s1), PDGFRA (Hs00998018_m1), PDGFRB (Hs01019589_m1), CCL2 (Hs00234140_m1), and CCL7 (Hs00171147_m1) according to the manufacturer’s instructions on a ViiA7 Real-Time PCR system (Applied Biosystems, Foster City, CA).

Techniques: RNA Sequencing, Control, Knock-Out, Fluorescence, Generated, Marker, Expressing

Journal: Nature Communications

Article Title: Molecular mechanisms of coronary artery disease risk at the PDGFD locus

doi: 10.1038/s41467-023-36518-9

Figure Lengend Snippet: a X-gal staining visualizing β-galactosidase activity (lacZ, blue precipitate) to determine the cellular location of Pdgfd expression in mouse model atherosclerosis. Aortic root sections were also stained with a generic nuclear marker nuclear fast red (NFR), immunohistochemistry for the Cd68 macrophage marker or Cnn1 marker for SMC identification. b Quantification of total vessel area, n = 25 control and 17 KO mouse sections. c Quantification of lesion, n = 26 control and 17 KO mouse sections, and d acellular areas in Ctl and KO groups expressed as a ratio of the total vessel area per section, n = 10 control and 10 KO mouse sections, p < 0.0001. e Representative images identifying expression of the tdTomato gene to visualize the SMC lineage traced cells in aortic sections. f Quantification of tdTomato positive ( tdT +) area relative to total vessel area, n = 18 control and 14 KO mouse sections, p < 0.0001. g Representative sections stained for Cnn1, a marker of the differentiated SMC. h Quantification of Cnn1 positive (Cnn1+) area at the media, n = 25 control and 17 KO mouse sections, p = 0.0061 and i compared to total cross-sectional area expressed as a ratio of the total vessel area per section, n = 25 control and 17 KO mouse sections, p = 0.0042. j Representative images of Cd68-stained aortic root area to quantify monocyte recruitment. k Quantification of Cd68 positive (Cd68+) area relative to the vessel area, n = 23 control and 17 KO mouse sections, p = 0.004. l Representative images of Col2a1 RNAscope of the aortic root in Ctl and KO mice. m Quantitative RNAscope of Col2a1 , n = 12 control and 12 KO mouse sections, p = 0.0052 and n Ibsp expression, n = 13 control and 13 KO mouse sections, p = 0.0074. o Representative images stained for calcium deposits with alizarin red S. p Quantification of calcium deposits, n = 9 control and 9 KO mouse sections, p = 0.0076. Each dot represents quantification from identical level sections from individual animals. Data expressed as mean ± s.e.m with p -values using a two-sided unpaired t-test.

Article Snippet: Gene expression was assessed using TaqMan qPCR probes (Thermo Fisher) for PDGFD (Hs00228671_m1), AP002989.1 (hs04980451_m1), FOXC1 (Hs00559473_s1), FOXC2 (Hs00270951_s1), PDGFRA (Hs00998018_m1), PDGFRB (Hs01019589_m1), CCL2 (Hs00234140_m1), and CCL7 (Hs00171147_m1) according to the manufacturer’s instructions on a ViiA7 Real-Time PCR system (Applied Biosystems, Foster City, CA).

Techniques: Staining, Activity Assay, Expressing, Marker, Immunohistochemistry, Control, RNAscope

Journal: Nature Communications

Article Title: Molecular mechanisms of coronary artery disease risk at the PDGFD locus

doi: 10.1038/s41467-023-36518-9

Figure Lengend Snippet: a Schematic of experimental design showing that SMC-specific lineage tracing wildtype mice were treated with tamoxifen at 8 weeks of age and tissues harvested after 8 and 16 weeks of high fat diet. Blocking Pdgfd antibody or isotype control antibody administration,10 mg/kg subcutaneously twice weekly, was initiated at 11 weeks and continued until animals were sacrificed after either 8 weeks exposure to the diet (5 weeks antibody) or 16 weeks diet (13 weeks antibody), and scRNAseq conducted at these timepoints. Created with BioRender.com. b Heatmap showing gene expression changes after 5 weeks of antibody treatment. The Fblst-1 cluster shows early downregulation of Pdgfd-regulated genes, and FMC and CMC cluster cells beginning to show evidence of upregulation of these genes as the SMC lineage cells are undergoing phenotypic transition in the developing lesion. Yellow color indicates differential downregulated genes, genes in red text reside in window of lead SNP ± 500 kilobases. c Heatmap showing decreases in Pdgfd regulated genes across different cell clusters in targeted animals compared to controls. Yellow color indicates differential downregulated genes, genes in red text reside in window of lead SNP ± 500 kilobases. d Bar plot presenting the average percentage of lineage traced cells and e non-lineage traced cells in Ctl antibody and Pdgfd blocking antibody groups.

Article Snippet: Gene expression was assessed using TaqMan qPCR probes (Thermo Fisher) for PDGFD (Hs00228671_m1), AP002989.1 (hs04980451_m1), FOXC1 (Hs00559473_s1), FOXC2 (Hs00270951_s1), PDGFRA (Hs00998018_m1), PDGFRB (Hs01019589_m1), CCL2 (Hs00234140_m1), and CCL7 (Hs00171147_m1) according to the manufacturer’s instructions on a ViiA7 Real-Time PCR system (Applied Biosystems, Foster City, CA).

Techniques: Blocking Assay, Control, Gene Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: Platelet-Derived Growth Factor-D Activates Complement System to Propagate Macrophage Polarization and Neovascularization

doi: 10.3389/fcell.2021.686886

Figure Lengend Snippet: RPE-specific overexpression of PDGF-D in vivo. (A) Schematic diagram illustrating the AAV vector carrying GFP (AAV-GFP) or human PDGF-D (AAV-PDGF-D) gene and RPE specific VDM2 promoter. (B) Real-time PCR results of relative mRNA expression of PDGF-D in retinae or RPE-choroid complex from mice injected with AAV-GFP or AAV-PDGF- D for 4 weeks. (C) Representative immunoblotting showing PDGF-D expression in mouse retinae or RPE-choroid complex. (D) Quantifications of PDGF-D protein levels in (C) . (E) Immunofluorescence images highlighting RPE-specific expression of GFP or PDGF-D in mouse RPE-choroid complex with 4 weeks AAV-GFP or AAV-PDGF-D injection, respectively. Scale bar: 50 μm. n = 5, **** p < 0.0001, ns: not significant.

Article Snippet: For analysis of flat-mounted retinas, 1 week after AAV-PDGF-D subretinal injection, SB290157 (MCE, cat: HY-101502A) was injected intraperitoneally (30 mg/kg body weight) every 2 days.

Techniques: Over Expression, In Vivo, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Injection, Western Blot, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Platelet-Derived Growth Factor-D Activates Complement System to Propagate Macrophage Polarization and Neovascularization

doi: 10.3389/fcell.2021.686886

Figure Lengend Snippet: RNA-seq and transcriptomic analysis of PDGF-D-induced complement pathway. (A) Volcano plot showing the differentially expressed genes (DEFs) from the RNA-seq data. (B) Biological function enrichment gene ontology (GO) analysis of DEGs showing the enriched biological processes in mouse PDGF-D-overexpressing RPE-choroids. (C,D) Heatmap of the DEGs associated to complement pathway (C) and chemokines and chemokine receptors (D) in mouse RPE-choroids injected with AAV-GFP or AAV-PDGF-D. (E,F) Immunoblot of C1q (E) and C3 (F) expression in RPE-choroid complex and retinae from mice with AAV-GFP or AAV-PDGF-D injection. (G) Immunofluorescence staining showing the IA/IE and C1q expression in mouse retinae and choroids with AAV-GFP or AAV-PDGF-D injection. (H) Quantification of IA/IE and C1q expression in (G) . (I) Functional network analysis of DEGs showing pathways related to activation of the complement system, immune cell migration and activation of immune responses in PDGF-D-overexpressing RPE-choroids. Scale bar: 50 μm. n = 5–6, *** p < 0.001.

Article Snippet: For analysis of flat-mounted retinas, 1 week after AAV-PDGF-D subretinal injection, SB290157 (MCE, cat: HY-101502A) was injected intraperitoneally (30 mg/kg body weight) every 2 days.

Techniques: RNA Sequencing, Injection, Western Blot, Expressing, Immunofluorescence, Staining, Functional Assay, Activation Assay, Migration

Journal: Frontiers in Cell and Developmental Biology

Article Title: Platelet-Derived Growth Factor-D Activates Complement System to Propagate Macrophage Polarization and Neovascularization

doi: 10.3389/fcell.2021.686886

Figure Lengend Snippet: PDGF-D induced macrophage polarizations. (A) Real-time PCR analysis of markers of M1 and M2 macrophage polarization regulated by PDGF-D in mouse retinal pigment epithelium. (B) Immunofluorescence staining for IBA1 + and CD16/32 + (M1 marker) cells in mouse retinae and choroids with AAV-GFP or AAV-PDGF-D injection. (C) Quantifications of IBA1 + and CD16/32 + macrophage densities in (B) . (D) Immunofluorescence staining of IBA1 + and CD206 + (M2 marker) cells in mouse retinae and choroids with AAV-GFP or AAV-PDGF-D injection. (E) Quantification of CD206 + macrophage cell density in retinae and choroids in (D) . Scale bar: 50 μm. n = 5, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: For analysis of flat-mounted retinas, 1 week after AAV-PDGF-D subretinal injection, SB290157 (MCE, cat: HY-101502A) was injected intraperitoneally (30 mg/kg body weight) every 2 days.

Techniques: Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Marker, Injection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Platelet-Derived Growth Factor-D Activates Complement System to Propagate Macrophage Polarization and Neovascularization

doi: 10.3389/fcell.2021.686886

Figure Lengend Snippet: PDGF-D overexpression increased vascular endothelial cell density and mural cell coverage in mouse retinae and choroids. (A) Real-time PCR results showing angiogenic genes upregulated by PDGF-D overexpression in RPE-choroid complex. (B) Immunofluorescence staining of CD31 + endothelial cells (EC) and α-SMA + smooth muscle cells (SMC) in mouse retinae and choroids injected with AAV-GFP or AAV-PDGF-D. (C) Quantifications of CD31 + cell density and SMC coverage in retinae and choroids in (B) . Scale bar: 50 μm. n = 5, * p < 0.05, ** p < 0.01, **** p < 0.0001, ns: not significant.

Article Snippet: For analysis of flat-mounted retinas, 1 week after AAV-PDGF-D subretinal injection, SB290157 (MCE, cat: HY-101502A) was injected intraperitoneally (30 mg/kg body weight) every 2 days.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Injection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Platelet-Derived Growth Factor-D Activates Complement System to Propagate Macrophage Polarization and Neovascularization

doi: 10.3389/fcell.2021.686886

Figure Lengend Snippet: PDGF-D promotes choroid sprouting and pathological choroidal neovascularization (CNV). (A) PDGF-D protein treatment increased mouse choroidal sprouting at different days. (B) Quantifications of choroidal sprouting areas in (A) . (C) RPE-choroid wholemount immunofluorescence staining of IB4 + neovessels and IBA1 + macrophages in laser-induced CNVs with AAV-GFP or AAV-PDGF-D overexpression. (D) Quantifications of neovascular areas and IBA1 + macrophages in (C) . (E) Immunofluorescence staining of CD31 + ECs and IBA1 + macrophages in laser-induced CNVs. (F) Quantifications of the CD31 + ECs and IBA1 + macrophages in e. Scale bar: 50 μm. n = 5, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant.

Article Snippet: For analysis of flat-mounted retinas, 1 week after AAV-PDGF-D subretinal injection, SB290157 (MCE, cat: HY-101502A) was injected intraperitoneally (30 mg/kg body weight) every 2 days.

Techniques: Immunofluorescence, Staining, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: Platelet-Derived Growth Factor-D Activates Complement System to Propagate Macrophage Polarization and Neovascularization

doi: 10.3389/fcell.2021.686886

Figure Lengend Snippet: Inhibiting complement C3 cascade by SB290157 alleviates PDGF-D-induced inflammation and pathological neovascularization. (A) SB290157 (20 μM) inhibited migration of mouse and human macrophages treated with conditioned medium from PDGF-D-treated HRPE cells (PDGF-D-CM) for 24 h. Conditioned medium from HRPE cells without PDGF-D treatment (CTL-CM) was used as a control. (B) Immunofluorescence staining of IBA1 + macrophages in PDGF-D-overexpressing RPE-choroids treated with or without SB290157. (C) Quantification of IBA1 + macrophages in retinae and choroids in (B) . (D) Immunofluorescence staining of CD31 + ECs, NG2 + pericytes and α-SMA + SMCs in PDGF-D-overexpressing RPE-choroids treated with or without SB290157. (E) Quantification of the CD31 + , SMA + , and NG2 + cells in retinae and choroids in (D) . (F) Real-time PCR results showing that SB290157 inhibited PDGF-D-induced upregulation of angiogenic genes. (G) RPE-choroid wholemount immunofluorescence staining showing SB290157 reduced IB4 + neovessels and IBA1 + macrophages in laser-induced CNVs with AAV-GFP or AAV-PDGF-D overexpression. (H) Quantification of IB4 + and IBA1 + cells in (G) . Scale bar: 50 μm. n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For analysis of flat-mounted retinas, 1 week after AAV-PDGF-D subretinal injection, SB290157 (MCE, cat: HY-101502A) was injected intraperitoneally (30 mg/kg body weight) every 2 days.

Techniques: Migration, Control, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Over Expression