Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells That Contribute to the Fibrous Cap

Abstract

Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including "vascular disease," "disorder of artery," and "occlusion of artery," as well as disease-related cellular functions including "cellular movement" and "cellular growth and proliferation." In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE-/- and Ldlr-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall.

Fig 1. TCF21 Vascular Disease Network built with differential gene expression data from siTCF21 RNA-Seq studies in HCASMC.

Interaction of the network nodes identified through enrichment of differentially expressed genes in functionally annotated categories was visualized with Cytoscape. Node color was mapped to log fold change with green representing genes that are downregulated along with TCF21 and red representing genes that are upregulated, node size was mapped to absolute expression value in control cells, and font size to enrichment Q-value. Edges contain arrows to indicate the direction of interaction and are colored to distinguish types of interactions. Green edges represent functional interaction (protein-protein binding, protein modification, molecular cleavage, phosphorylation, and protein-DNA interactions); magenta edges represent gene expression (expression and transcription) relationships; red edges represent activation; and blue edges inhibition.

Fig 2. TCF21 regulates basic cellular functions in vascular smooth muscle cells in vitro.

A) HCASMC transduced with TCF21 overexpressing lentivirus (pWPI-TCF21) or empty lentivirus (pWPI empty) were labeled with the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU), which was visualized with a fluorescent azide, allowing identification and quantification of proliferating cells with immunofluorescence microscopy, reported here as a percent with baseline being all DAPI positive cells. HCASMC showed an increase in the percentage of TCF21 overexpressing cells compared to DAPI stained cells (33.1% ± 2.3 control vs. 51% ± 4.1 overexpressing cells, P<0.001). B) HCASMC transduced with knockdown lentiviruses showed a decreased percentage of dividing cells (43.4% ± 4.4 control vs. 30.8% ± 2.4 knockdown cells, P<0.01). C) siTCF21 produced a decrease in apoptosis in HCASMC as measured by a caspase activity assay (41,824±1872 vs. 18,837±1302, P<0.0001). D) TCF21 regulation of HCASMC migration was evaluated with a gap closure assay. TCF21 overexpressing cells transduced with pWPI-TCF21 lentivirus covered a significantly larger surface area after 12 hours of study compared to cells transduced with the empty pWPI vector (27.8 ± 2.7 vs. 15.8 ± 1.16, P<0.001). E) HCASMC treated with siTCF21 compared to siCTRL showed significantly increased expression of ACTA2, TAGLN, and MYH11 SMC marker genes (P<0.05/P<0.01/P<0.05 respectively). F) The ACTA2 locus as visualized on the University of California Santa Cruz genome browser. Data provided here reveals evidence for a likely enhancer region in the first intron, as indicated by DNase hypersensitivity measured in human aortic SMC, and histone modification data showing enrichment of H3K27Ac and H3K4me1 at this same site, as well as clustering of a number of transcription factor binding sites. G) Chromatin immunoprecipitation for TCF21 binding to the enhancer region of the ACTA2 locus by ChIP-qPCR (P<0.05). H) Dual luciferase assays in rat aortic smooth muscle cells with a reporter construct containing the human ACTA2 promoter and first intron (SMA-luc). A TCF21 expression construct was transfected with human TCF3 (E12), TCF3 (E47), TCF12, and Twist1 murine expression vectors, showing specific suppression of transcription of the SMA-luc reporter (1.0 ± 0.01 vs. 0.1 ± 0.009, P<0.01 for TCF21 alone). I) Similar dual luciferase assays using a 3 E-box containing minimal promoter construct (E-luc) based on the nucleotide sequence of the first intron (n = 3, 3 replicates), again showing TCF21 mediated suppression of transcription (1.0 ± 0.29 vs. 0.21 ± 0.02, P<0.01 for TCF21 alone).

Fig 3. Tcf21 expressing cells are found in atherosclerotic lesions.

Tcf21 ^lacZ/+, ApoE ^-/- mice were fed HFD from 4 weeks of age for 4, 8, 12, or 20 weeks, proximal aortic tissues were harvested, and Tcf21 gene expression evaluated by Xgal staining to visualize β-galactosidase activity, and sections were counterstained with nuclear fast red to visualize disease lesion architecture. For each timepoint, boxes in low-power images at left indicate regions examined at high power in panels to the right. At 4 weeks of HFD there was no expression in the lesions although clusters of lacZ expressing cells identified in the media in regions below the disease lesions (arrows). At the 8-week timepoint cells with β-galactosidase activity were seen extending from the media to the luminal surface of the lesion (black arrows) in the vicinity of the forming fibrous cap (purple arrows). By 12 weeks of HFD there was extensive labeling of cells in lesions, with the appearance of β-galactosidase positive cells in the vicinity of the fibrous cap (purple arrows). Also, there was extensive staining of cells in areas of disrupted medial structure (white arrows). After 20 weeks of HFD, Tcf21-expressing cells had decreased in the lesions but formed a narrow band of cells associated with the fibrous cap (purple arrows).

Fig 4. Tcf21 expressing cells associate with the fibrous cap.

Xgal stained lesions in Tcf21 ^lacZ/+, ApoE ^-/- mice fed HFD for 20 weeks were evaluated by immunohistochemistry for expression of fibrous cap markers. Tcf21 reporter expressing cells (blue indicator) were identified in the media and adventitia (black arrows, middle panels), and in association with the fibrous cap (blue arrows, right panels). β-galactosidase negative cells in the luminal aspect of the fibrous cap stained positive for Tagln, as well as growth factor receptors Tgfbr2 and Pdgfrb (red indicator). In addition, in the region of the fibrous cap, cells were identified that showed staining for Tcf21 expression as well as Tagln and growth factor receptors (purple arrows).

Fig 5. Tcf21 expressing cells in ApoE ^-/- lesions give rise to smooth muscle cells in the fibrous cap.

Tcf21 ^iCre/+, ApoE ^-/- mice were administered tamoxifen to activate expression of an inducible MerCreMer construct knocked into the Tcf21 locus [31]. Cre mediated recombination of a tomato (tdT) reporter at the constitutively expressed ROSA26 locus allowed lineage tracing of Tcf21 expressing cells. Animals received tamoxifen at 6–8 weeks of HFD and tissues were harvested at 12 weeks of diet (A-D), or received tamoxifen 4–6 weeks of age prior to HFD and tissues harvested at 16 weeks (E). A) Intramyocardial coronary artery showing Tcf21 expression (tdT) in adventitial cells surrounding the Tagln positive medial SMC. B) In an early lesion without a defined fibrous cap, tdT positive cells were seen throughout the lesion and in the media (pink arrow), with Tagln staining (green arrows) being restricted to cells in the media. C) In the Tagln and tdT merged imaging (right panel), lesion Tagln positive cells are identified in the fibrous cap (green arrows) and some of these cells co-stain for tdT fluorescence (yellow arrows), indicating that they previously expressed Tcf21. Tagln expressing cells in the media (green arrows, Tagln only imaging) do not colocalize with tdT staining in this location. D) Tagln positive cells in the fibrous cap were also positive for tdT fluorescence, suggesting that the identified SMC had expressed Tcf21. Also, co-staining of tdT and Tagln is noted in the media (yellow color). E) Animals received tamoxifen at 4 weeks of age prior to HFD and tissues were harvested at 12 weeks of diet. In an early lesion without a defined fibrous cap, tdT positive cells are seen throughout the adventitia, in the media and in the plaque. In, intima; M, media; Ad, adventitia; Lu, lumen.

Fig 6. Tcf21 lineage traced cells in Ldlr ^-/- lesions give rise to smooth muscle cells in the fibrous cap.

Tcf21 ^iCre/+, Ldlr ^-/- mice were administered tamoxifen to activate expression of an inducible MerCreMer construct knocked into the Tcf21 locus to produce recombination and thus expression of a floxed STOP tandem dimer tomato (tdT) reporter gene. Animals received tamoxifen chow at 3–5 weeks of age and were subsequently fed HFD for 16–20 weeks before sacrifice. A) tdT positive cells are seen throughout the lesion and in the adventitia with Tagln staining cells identified in the media and fibrous cap. B) A number of tdT lineage traced cells in the fibrous cap express the SMC marker Acta2 as shown by colocalization of fluorescent markers (yellow arrows). tdT positive cells within the lesion did not express Tagln, as shown by red color (red arrow). C) Some cells in the adventitia stain for periostin expression and colocalize with tdT staining in this location (yellow arrows). Staining for periostin and colocalization with Tcf21 lineage cells was also observed in the fibrous cap and subcap (yellow arrows). D) Extensive colocalization was noted for Pdgfra antibody staining and tdT fluorescence in the adventitia and fibrous cap. In, intima; M, media; Ad, adventitia; FC, fibrous cap, Lu, lumen.

Fig 7. Rate of cell division in vascular lesions is related to Tcf21 expression.

A) EdU staining was performed in Tcf21 ^iCre/+, ROSA ^tdT/+, Ldlr ^-/- mice treated with tamoxifen and EdU and placed on HFD for 9, 12, or 15 weeks prior to tissue collection. Co-localization of Tcf21 tdT fluorescence (red) and EdU fluorescent staining (green) allowed identification of the sdividing cells that were expressing Tcf21 (yellow arrows, image from 12 week timepoint). B) The ratio of Tcf21+EdU+ cells compared to total Tcf21+ cells was significantly increased at 12 weeks of diet, consistent with a proliferative response in Tcf21+ cells. Comparison of percentage differences between 0 and 9 weeks, and from 9 to 12 weeks approached significance, P = 0.06; there was not a statistical difference between 12 and 15 weeks, P = 0.11. C, D) Rates of cell division in vascular lesions were compared between Tcf21 ^lacZ/+, ApoE ^-/- animals and ApoE ^-/- animals on HFD for 20 wks by quantifying the relative number of dividing cells identified by EdU fluorescence compared to the total number of cells identified by DAPI fluorescence. EdU fluorescence (green) was merged with red pseudocolored DAPI fluorescence, yellow arrows indicate yellow nuclei that are positive for both EdU and DAPI fluorescence. E) A statistically significant decrease in this percentage in Tcf21 ^lacZ/+, ApoE ^-/- animals compared to ApoE ^-/- animals suggested a correlation between Tcf21 expression and rate of cell division in the vascular lesions. In, intima; M, media; Ad, adventitia; FC, fibrous cap, Lu, lumen.

Fig 8. Differential TCF21 gene expression in fibrous cap of stable vs. ruptured atherosclerotic plaque.

A) Microdissection laser capture was employed to harvest tissue from endarterectomy samples from vessels with stable plaque or evidence of plaque rupture. An example of a stable plaque and a ruptured plaque with necrotic core (NC) and related luminal thrombus (T) are shown. These representative sections show the types of plaque regions that were harvested for RNA isolation. B) qPCR quantitation of TCF21 mRNA levels in harvested tissue. TCF21 expression was significantly lower in fibrous cap tissue from the ruptured plaque.