Intrinsic Gata4 expression sensitizes the aortic root to dilation in a Loeys-Dietz syndrome mouse model

Abstract

Loeys-Dietz syndrome (LDS) is an aneurysm disorder caused by mutations that decrease transforming growth factor-β (TGF-β) signaling. Although aneurysms develop throughout the arterial tree, the aortic root is a site of heightened risk. To identify molecular determinants of this vulnerability, we investigated the heterogeneity of vascular smooth muscle cells (VSMCs) in the aorta of Tgfbr1 ^M318R/+ LDS mice by single cell and spatial transcriptomics. Reduced expression of components of the extracellular matrix-receptor apparatus and upregulation of stress and inflammatory pathways were observed in all LDS VSMCs. However, regardless of genotype, a subset of Gata4-expressing VSMCs predominantly located in the aortic root intrinsically displayed a less differentiated, proinflammatory profile. A similar population was also identified among aortic VSMCs in a human scRNAseq dataset. Postnatal VSMC-specific Gata4 deletion reduced aortic root dilation in LDS mice, suggesting that this factor sensitizes the aortic root to the effects of impaired TGF-β signaling.

Figure 1.. Downregulation of transcripts associated with extracellular matrix-receptor interactions and upregulation of stress and inflammation pathways in Tgfbr1^M318R/+ LDS VSMCs.

(A) Uniform manifold approximation and projection (UMAP) of aortic cells from control (Tgfbr1^+/+) and LDS (Tgfbr1^M318R/+) mice. (B) Dot plot of cluster defining transcripts used to identify endothelial cells, leukocytes, fibroblasts, and VSMCs. Color of the dot represents a scaled average expression while the size indicates the percentage of cells in which the transcript was detected. (C) ClueGO gene enrichment analysis network of transcripts dysregulated in LDS VSMCs relative to controls. Each node represents a term/pathway or individual genes associated with that term. The color of the node corresponds to the ClueGO group to which each node belongs. The size of the node indicates significance of the enrichment calculated by the ClueGO algorithm. (D) ClueGO network in which terms differentially enriched among transcripts downregulated in LDS VSMCs are highlighted in blue, while those enriched among transcripts upregulated in LDS VSMCs are highlighted in red. (E) Dot plot showing expression of a selection of transcripts significantly dysregulated in LDS VSMCs. (F,G) EnrichR gene over-representation analysis for the ENCODE and ChEA Consensus transcription factors (TF) databases showing the top three most significant terms associated with transcripts that are downregulated (F) or upregulated (G) in LDS VSMCs.

Figure 2.. MERFISH reveals spatially heterogeneous transcriptional profiles in LDS VSMCs.

MERFISH images of the proximal aorta of LDS (A) and control (B) mice, scale bar is 1 mm. The first panel displays all detected transcripts across the aortic tissue, with key anatomic landmarks indicated. Subsequent panels depict the colocalization of Myh11 and transcripts of interest. Insets note regions of the ascending aorta and aortic root that are presented at higher magnification.

Figure 3.. Transcriptionally and spatially-defined VSMC subclusters with distinct responses to LDS-causing mutations can be identified in both murine and human aortas.

(A) UMAP of VSMCs from control (Tgfbr1^+/+) and LDS (Tgfbr1^M318R/+) mice shown split by genotype. (B) Dot plot showing enrichment of cluster-defining transcripts in VSMC1 and VSMC2. For a given transcript, the color of the dot represents a scaled average expression while the size indicates the percentage of cells in which it was detected. (C) RNA in situ hybridization showing the expression of Gata4 along the length of the murine aorta in a 16-week old control animal. (D) UMAP of control and LDS VSMCs from human patients and dot plot of cluster defining markers in this dataset split by aortic region (Pedroza et al., 2023). (E,F) UMAP overlayed with weights for CoGAPS patterns 4 and 5, in mouse and human scRNAseq datasets. (G,H) Violin plots showing the distribution of pattern 4 and 5 weights in VSMC subclusters from mouse and human scRNAseq datasets. P-values refer to Wilcoxon test. (I) EnrichR gene over-representation analysis for the ENCODE and ChEA Consensus TF databases showing the top four most significant terms associated with transcripts that define CoGAPs Patterns 4 and 5. (J) ClueGO network of terms differentially enriched in mouse and human LDS VSMC2 relative to VSMC1. Terms highlighted in blue are enriched in VSMC1, while those highlighted in red are enriched in VSMC2.

Figure 4.. Gata4 mRNA and protein are upregulated in the aortic root of LDS mice.

(A) Representative images of RNA in situ hybridization for Gata4 in the aortic root and ascending aorta of control and LDS (Tgfbr1^M318R/+) mice. Insets identify the location shown at higher magnification in the subsequent panel. Scale bars 50 and 200 microns, respectively. (B) Representative images of immunofluorescence for GATA4in the aortic root and ascending aorta of control and LDS mice. Insets identify the location shown at higher magnification in the subsequent panel. Scale bars 50 and 200 microns, respectively. (C) Immunoblot for Gata4 expression relative to ß-actin in aortic root lysates of control (n=3) and LDS mice (n=3), and related quantification of immunoblot, P-value refers to two-tailed Student’s t-test.

Figure 5.. Gata4 protein is upregulated in LDS aortic root of Gata4^Ctrl and effectively ablated in Gata4^SMcKO mice.

Representative images of immunofluorescence for GATA4 at 16 weeks of age. Three independent biological replicates are shown per genotype abbreviated as follows Control (Tgfbr1^+/+) and LDS (Tgfbr1^M318R/+) with (Gata4^SMcKO) or without (Gata4^Ctrl) smooth muscle specific deletion of Gata4 Insets identify location shown at higher magnification in subsequent panels. Images were acquired at 20x magnification. Scale bars 50 and 200 microns, respectively.

Figure 6.. Smooth muscle-specific deletion of Gata4 (Gata4^SMcKO) reduces aortic root size and growth and improves aortic root media architecture in LDS mice.

(A) Aortic root diameter of Ctrl (Tgfbr1^+/+) and LDS (Tgfbr1^M318R/+) with (Gata4^SMcKO) or without (Gata4^Ctrl) smooth muscle specific deletion of Gata4 as measured by echocardiography at 8 and 16 weeks of age and aortic root growth from 8-16 weeks. P-values refer to Brown-Forsythe AN0VA. (B) Representative VVG-stained aortic root sections from three independent biological replicates per genotype. Insets identify area shown at higher magnification in the subsequent panel. Scale bars 50 and 200 microns, respectively.

Figure 7.. Smooth muscle-specific deletion of Gata4 results in reduced expression of Agtr1a.

Representative images of RNA in situ hybridization for Agtr1a in the aortic root of mice at 16 weeks of age. Three independent biological replicates are shown per genotype abbreviated as follows Control (Tgfbr1^+/+) and LDS (Tgfbr1^M318R/+) with (Gata4^SMcKO) or without (Gata4^Ctrl) smooth muscle specific deletion of Gata4. Insets identify location shown at higher magnification in subsequent panels. Images were acquired at 20x magnification. Scale bars 50 and 200 microns, respectively.

Figure 8.. Smooth muscle-specific deletion of Gata4 results in reduced expression of Cebpb.

Representative images of RNA in situ hybridization for Cebpb in the aortic root of mice of indicated genotype at 16 weeks of age. Three independent biological replicates are shown per genotype abbreviated as follows Control (Tgfbr1^+/+) and LDS (Tgfbr1^M318R/+) with (Gata4^SMcKO) or without (Gata4^Ctrl) smooth muscle specific deletion of Gata4. Insets identify location shown at higher magnification in subsequent panels. Images were acquired at 20x magnification. Scale bars 50 and 200 microns, respectively.