Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Chemokine (C-C motif) Ligand 2 Expressing Adventitial Fibroblast Expansion During Loeys-Dietz Syndrome Aortic Aneurysm Formation

doi: 10.1161/ATVBAHA.124.322069

Figure Lengend Snippet: A) Long-axis echocardiographic measurements of Tgfbr2 G357W/+ and WT aortic root and ascending aorta at 8 and 24 weeks ( Tgfbr2 G357W/+ : n=12; WT: n=10; at each time point) Box and whisker plots display 95% CI (box), mean (line) and range (whiskers). ***P<0.001 (2-way ANOVA test). B) Representative echocardiographic images demonstrating Tgfbr2 G357W/+ aortic root dilation. C) Elastin Von Gieson (EVG) staining of Tgfbr2 G357W/+ and WT aortic root/ascending aorta segments at 8 and 24 weeks, highlighting extensive elastin fragmentation in Tgfbr2 G357W/+ aneurysmal aortic wall (60x magnification; scale bar: 25μm). D) Trichrome staining of Tgfbr2 G357W/+ and WT aortic root/ascending aorta segments at 8 and 24 weeks. Blue: collagen; red: smooth muscle (60x magnification; scale bar: 25 μm). E) Elastin fragmentation quantification in Tgfbr2 G357W/+ and WT aortic root/ascending aorta at 8 and 24 weeks. Column plot displays mean with SD error bars (Kruskal-Wallis test).

Article Snippet: Tgfbr2 G357W/+ mice (Strain #: 024634) were gifted to us from Dr. Dietz and the colony augmented with mice purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Whisker Assay, Staining

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Chemokine (C-C motif) Ligand 2 Expressing Adventitial Fibroblast Expansion During Loeys-Dietz Syndrome Aortic Aneurysm Formation

doi: 10.1161/ATVBAHA.124.322069

Figure Lengend Snippet: A) Uniform manifold approximation and projection (UMAP) dimensional reduction of >9000 cells from n = 8 Tgfbr2 G357W/+ and n = 8 WT 24-week aortic root/ascending aortas, clustering into 9 unique cell types (Left) . UMAP grouped by genotype shows overlapping clustering of both genotypes (blue: Tgfbr2 G357W/+ ; red: WT) except for enrichment of Tgfbr2 G357W/+ inflammatory fibroblasts and macrophages (Right) . B) Dot plot for top cluster specific genes. Color of dot denotes relative gene expression (blue low; red high) and dot size represents percentage of cells expressing the gene. C) Feature plot representing the location of cluster specific genes: Myh11 – Smooth muscle cells (SMCs), Fibroblasts – Pi16 ; C3 – Inflammatory fibroblasts; Tbx20 – Valve interstitial cells (VICs); Pecam1 – Endothelial cells (EC); Cd68 – macrophages; Ccl21a – lymphatic endothelium; Cd3d – T cells; Upk3b – paracardial cells.

Article Snippet: Tgfbr2 G357W/+ mice (Strain #: 024634) were gifted to us from Dr. Dietz and the colony augmented with mice purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Gene Expression, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Chemokine (C-C motif) Ligand 2 Expressing Adventitial Fibroblast Expansion During Loeys-Dietz Syndrome Aortic Aneurysm Formation

doi: 10.1161/ATVBAHA.124.322069

Figure Lengend Snippet: A) Uniform manifold approximation and projection (UMAP) dimensional reduction of 24-week Tgfbr2 G357W/+ and WT aortic root/ascending aortas. Red circle identifies SMCs on UMAP clustering ( Left ) that were reclustered and split by genotype ( Right ). B) Violin plot comparison of SMC contractile genes between Tgfbr2 G357W/+ and WT SMCs. C) Representative violin plot of select genes increased in Tgfbr2 G357W/+ SMCs compared to WT (***P<0.001). D) Representative violin plot of select genes decreased in Tgfbr2 G357W/+ SMCs compared to WT (***P<0.001). E) Cell-cell communication prediction of outgoing and incoming interaction strength in WT (Top) and Tgfbr2 G357W/+ (Bottom) by cell cluster. F ) TGFβ signaling network between cell clusters split by genotype. Line width represents signaling strength. G) Violin plot of gene expression of Tgfbr1 ( Top ) and Tgfbr2 ( Bottom ) in each cell cluster (blue - Tgfbr2 G357W/+ ; red – WT). SMCs – smooth muscle cells, UMAP - uniform manifold approximation and projection.

Article Snippet: Tgfbr2 G357W/+ mice (Strain #: 024634) were gifted to us from Dr. Dietz and the colony augmented with mice purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Comparison, Gene Expression

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Chemokine (C-C motif) Ligand 2 Expressing Adventitial Fibroblast Expansion During Loeys-Dietz Syndrome Aortic Aneurysm Formation

doi: 10.1161/ATVBAHA.124.322069

Figure Lengend Snippet: A) UMAP clustering of 24wk Tgfbr2 G357W/+ , Fbn1 C1041G/+ , and respective WT control aortic samples with low resolution clustering clusters into 5 major cell types. B) Selection and reclustering of only SMCs clusters into 3 SMC subtypes – contractile SMCs (yellow), Tnnt2+ SMCs (blue), and modSMCs (purple). C) Feature plots of canonical gene markers for each SMC subtype Myh11 – contractile SMCs, Tnnt2 – Tnnt2 + SMCs, Tnfrsf11b - modSMCs. D) Feature plot of cumulative gene score from previously identified top 100 modSMC genes in 24-week Fbn1 C1041G/+ samples. Score intensity only observed in Fbn1 C1041G/+ sample. E) Venn diagram comparing SMC differentially expressed genes between Tgfbr2 G357W/+ and 129S6/SvEvTac WT control compared to differentially expressed genes between Fbn1 C1041G/+ and C57BL/6 WT control. F) Venn diagram comparing SMC differentially expressed genes between Tgfbr2 G357W/+ and 129S6/SvEvTac WT control compared to differentially expressed genes between Fbn1 C1041G/+ and C57BL/6 WT control and top 100 previously identified 24-week modSMC markers.

Article Snippet: Tgfbr2 G357W/+ mice (Strain #: 024634) were gifted to us from Dr. Dietz and the colony augmented with mice purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Control, Selection

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Chemokine (C-C motif) Ligand 2 Expressing Adventitial Fibroblast Expansion During Loeys-Dietz Syndrome Aortic Aneurysm Formation

doi: 10.1161/ATVBAHA.124.322069

Figure Lengend Snippet: A) Uniform manifold approximation and projection (UMAP) dimensional reduction of 24-week Tgfbr2 G357W/+ and WT aortic root/ascending aortas. Red circle identifies fibroblasts on UMAP clustering ( Left ). Reclustered fibroblasts result in 2 distinct fibroblast clusters: Resident and Inflammatory fibroblast ( Right ). B) Top KEGG mouse Gene Ontology pathways for each fibroblast cluster independent of genotype. C) Heat map of top differentially expressed genes between Resident fibroblast and Inflammatory fibroblast clusters. D) 24-week Fibroblast cluster UMAP plot colored by genotype (blue - Tgfbr2 G357W/+ ; red – WT). The top pole of the fibroblasts is specific to only Tgfbr2 G357W/+ samples. E) Feature plot of Cytokine-cytokine receptor interaction KEEG GO gene score. Highest scores associate with Tgfbr2 G357W/+ inflammatory fibroblasts. F) Violin plots of multiple collagen subtypes and inflammatory markers enriched in 24-week Tgfbr2 G357W/+ vs. WT mice (***P<0.001). G) RNA in situ hybridization staining for Ccl2 and fibroblast marker Pi16 co-localized to the aortic adventitia and enriched in 24-week Tgfbr2 G357W/+ compared to WT (60x magnification; scale bar: 50μm). H) UMAP plot of 8wk Tgfbr2 G357W/+ and WT aortic samples showing all expected cell clusters. I) Subselection, reclustering, and grouping by genotype shows similar distribution of Tgfbr2 G357W/+ and WT fibroblasts in UMAP space. F) Violin plots of multiple collagen subtypes and inflammatory markers enriched in 8-week Tgfbr2 G357W/+ vs. WT. UMAP - uniform manifold approximation and projection, GO – gene ontology

Article Snippet: Tgfbr2 G357W/+ mice (Strain #: 024634) were gifted to us from Dr. Dietz and the colony augmented with mice purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: RNA In Situ Hybridization, Staining, Marker

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Chemokine (C-C motif) Ligand 2 Expressing Adventitial Fibroblast Expansion During Loeys-Dietz Syndrome Aortic Aneurysm Formation

doi: 10.1161/ATVBAHA.124.322069

Figure Lengend Snippet: A) Cd68 immunofluorescence staining of Tgfbr2 G357W/+ and WT aortic root. Red – Cd68, blue – Hoechst nuclear stain; green – elastin autofluorescence. Top : 10x magnification; scale bar: 100μm. Bottom : 20x magnification; scale bar: 50μm. B) UMAP projection of macrophages after reclustering into 3 clusters: MΦ1–3. C) Feature plots of cluster defining genes. MΦ1/Resident macrophages: F13a1, Lyve1, and Gas6 ; MΦ2/M1 macrophages: Cd74, H2-Aa, Cd40 ; MΦ3/M2 macrophages Arg1, Ccl24, Mrc1 . D) UMAP projection split by genotype identified similar resident clusters but substantially increased MΦ2 and MΦ3 clusters in Tgfbr2 G357W/+ . E) Bar plot by percentage and absolute cell count for each macrophage cluster by genotype. F) Feature plot of MΦ1/Resident ( F13a1 ), MΦ2/M1 ( Ccr2 ), and MΦ3/M2 ( Arg1 ) macrophage defining genes split by genotype. Very few MΦ2/M1 and MΦ3/M2 macrophages exist within the WT samples. UMAP - uniform manifold approximation and projection.

Article Snippet: Tgfbr2 G357W/+ mice (Strain #: 024634) were gifted to us from Dr. Dietz and the colony augmented with mice purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Immunofluorescence, Staining, Cell Characterization

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Chemokine (C-C motif) Ligand 2 Expressing Adventitial Fibroblast Expansion During Loeys-Dietz Syndrome Aortic Aneurysm Formation

doi: 10.1161/ATVBAHA.124.322069

Figure Lengend Snippet: A) Violin plot of highly differentially expressed inflammatory signaling genes CCL2 , IL6 , and CXCL2 specific to LDS fibroblasts, as seen also in Tgfbr2 G357W/+ inflammatory fibroblasts. B) RNA in situ hybridization for CCL2 enhanced in LDS aortic root adventitia in comparison to donor aortic root (scale bar: 100μm). C) Immunohistochemistry staining for MCP1 protein (encoded by CCL2 ) in LDS and donor aortic root samples also shows increased staining in the LDS adventitia vs. donor (scale bar: 200μm). D) Immunohistochemistry staining for macrophages (CD68) in human LDS and donor aortic root samples. Macrophages are more highly concentrated in the LDS adventitia than that of donor (scale bar: 200μm). ***P<0.001

Article Snippet: Tgfbr2 G357W/+ mice (Strain #: 024634) were gifted to us from Dr. Dietz and the colony augmented with mice purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: RNA In Situ Hybridization, Comparison, Immunohistochemistry, Staining

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Chemokine (C-C motif) Ligand 2 Expressing Adventitial Fibroblast Expansion During Loeys-Dietz Syndrome Aortic Aneurysm Formation

doi: 10.1161/ATVBAHA.124.322069

Figure Lengend Snippet: A) Violin plots of contractile genes ( MYH11, ACTA2, CNN1 ), synthetic and proteolytic genes ( FN1, COL1A2, MMP2 ), and TGFβ related genes ( TGFB1, TGFBR1, TGFBR2 ) comparing SMCs and modSMCs. B) Biological Process Gene Ontology terms for LDS modSMCs compared to donor modSMCs. C) Violin plots for select modSMC genes comparing donor and LDS gene expression. ***P<0.001

Article Snippet: Tgfbr2 G357W/+ mice (Strain #: 024634) were gifted to us from Dr. Dietz and the colony augmented with mice purchased from the Jackson Laboratory (Bar Harbor, ME).

Techniques: Gene Expression