Targeting the ERK Signaling Pathway to Halt Adventitia Remodeling of Ascending Thoracic Aortic Aneurysm

Figures

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  Figure 1. Histological Analysis of Human Adventitia Samples (A) H&E and (B) Van Gieson's stainings. Scale bar = 50 μm. White arrows point to vasa vasorum (VV). Images are representative of n = 1 sample of each group. (C-E) Bar graphs of morphometric measurements for (C) VV density, (D) VV lumen area, (E) VV perimeter size, and (F) VV wall thickness. N = 4 controls and 6 ascending thoracic aortic aneurysms (AsTAA). In (C, D, and F), data are presented as individual values and means ± SEM. In (E), curves show the frequency distribution of the VV perimeter size from all samples in the 2 groups. Analysis by unpaired Mann-Whitney test. ∗∗P < 0.01. AsTAA = ascending thoracic aortic aneurysm; H&E = hematoxylin and eosin.

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  Figure 2. In situ Characterization of Adventitial Pericyte-Like Cells (APCs) in Human Aorta Adventitia Using Immunohistochemistry (A) Vasa vasorum (VV)-associated APCs are characterized by expression of platelet-derived growth factor receptor beta (PDGFRβ) and lack of CD31. (B and C) Immunofluorescence images showing examples of VV surrounded by PDGFRβ+ APCs (green). In (B), CD31 labels the VV lumen (white), although in (C), alpha-smooth muscle actin (α-SMA) identifies vascular smooth muscle cells (VSMCs) within the VV tunica media (red). (D) Immunofluorescence images showing coverage of VV with PDGFRβ+ APCs (green, indicated by white arrows) in the 2 groups. α-SMA (red) labels VSMCs. 4’,6-diamidino-2-phenylindole (DAPI) (blue) labels the nuclei. Scale bar = 50 μm. (E) Bar graphs showing a significant decline in APC coverage in ascending thoracic aortic aneurysm (AsTAA) samples compared with controls. N = 4 control and 6 AsTAA. Data are presented as individual values and means ± SEM. Analysis by unpaired Mann-Whitney U test. (F) Curves showing the frequency distribution of the percentage of APC coverage of VV from all samples in the 2 groups. (G) Correlation study between the APC coverage…

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  Figure 3. Antigenic Characterization of Isolated Adventitial Pericyte-Like Cells (APCs) (A and B) Immunofluorescence images showing antigenic profile of (A) Control-adventitial pericyte-like cells, and (B) ascending thoracic aortic aneurysm (AsTAA)-APCs at passage 4 of culture. Both groups show strong positivity for pericyte markers neural glial antigen 2 (NG2) and Platelet-derived growth factor receptor beta (PDGFRβ), and negativity for tested endothelial cells, vascular smooth muscle cells, and fibroblast markers. N = 5. Images are representative of 1 sample from each group. 4’,6-diamidino-2-phenylindole (DAPI) labels nuclei in blue. Scale bar = 50 μm. (C) Bar graphs showing the marker quantification. Data are presented as individual values and means ± SEM. N = 5. Expression of each marker between the 2 groups was compared using an unpaired Mann-Whitney U test (all not significant).

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  Figure 4. Transcriptomic Analysis of Control and Ascending Thoracic Aortic Aneurysm Adventitial Pericyte-Like Cells (AsTAA APCs) (A) A whole-genome RNA sequencing study was conducted on control and ascending thoracic aortic aneurysm (AsTAA)-derived adventitial pericyte-like cells (APCs) (n = 3/group). (B) Principal component analysis. (C) Hierarchical clustering of the top 10K transcripts. (D) Mean average plot showing that 19 transcripts were significantly upregulated and 31 transcripts downregulated in AsTAA-APCs compared with control cells. Differentially expressed genes (DEGs) were defined as those with a false discovery rate (FDR) < 0.05 and a fold change >2. (E) Heatmap showing all protein-coding DEGs, ranked from highest to lowest fold change. (F and G) Lollipop plots showing enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways based on DEGs. Upregulated pathways are shown in (F), and downregulated pathways are shown in (G). On the y-axis are the Gene Ontology (GO) terms. The x-axis indicates the statistical significance (–Log(10)FDR). The circle size indicates the number of genes involved in each pathway, whereas the lollipop color indicates the enrichment score. IL =…

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  Figure 5. Ascending Thoracic Aortic Aneurysm Adventitial Pericyte-Like Cells (AsTAA-APCs) Show Impaired Functional and Molecular Properties (A) Growth curve showing hyperproliferative status of ascending thoracic aortic aneurysm adventitial pericyte-like cells (AsTAA-APCs) compared with controls. N = 5. Analysis by 2-way analysis of variance. (B) Wound closure migration assay comparing nonstimulated and platelet-derived growth factor BB (PDGFBB)-stimulated cells. Images were snapped after 8 and 10 hours and compared with the initial time zero. Bar graphs report the percentage of wound closure. N = 4. (C) Secreted angiogenic factors in adventitial pericyte-like cell (APC)-conditioned media analyzed by ELISA. N = 6. (D) Angiogenic tube formation assay of APCs cocultured with human aortic ECs (AoECs). Images are representative of 1 sample from each group. N = 4 APCs, n = 1 AoECs. (E) Gelatinase activity fluorescence assay. Results are presented as U/mg. N = 4. (F) Expression of MMP2, MMP9 and tissue inhibitor of metalloproteinases (TIMP-1) transcripts as assessed by quantitative reverse transcription polymerase chain reaction. N = 6. (G) Transwell invasion assay testing the APC capacity to…

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  Figure 6. P-ERK Levels in Ascending Thoracic Aortic Aneurysm Adventitial Pericyte-Like Cells (AsTAA-APCs) With or Without PD0325901 Treatment (A) Western blot analysis showing that AsTAA-APCs express higher levels of P-ERK compared with control APCs. N = 4. Data are presented as individual values and means ± SEM. Analysis by unpaired Mann-Whitney U test. (B) PD0325901 (MEK inhibitor - MEKi) in a dose of 250 nM successfully inhibited ERK phosphorylation/activation in vitro. N = 4 AsTAA-APCs. (C) Immunofluorescence images showing MEKi treatment inhibited P-ERK (green) nuclear translocation. 4’,6-diamidino-2-phenylindole (DAPI) labels nuclei in blue. N = 2 AsTAA-APCs. Images are representative of 1 sample. In (B and C), APCs were incubated for 48 hours with a growth factor (GF)-free and serum-free medium to switch off molecular signaling, followed by incubation with either the MEKi or its vehicle for 1h, finally followed by stimulation with a GF mix to induce ERK phosphorylation. (D) Viability assay in AsTAA-APCs using Calcein-AM (green, viable cells) and ethidium homodimer III (EthD-III - red, dead cells). N = 3. Images are representative of 1 sample. (E) Brightfield optical images showing…

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  Figure 7. In vitro PD0325901 Treatment Restored Ascending Thoracic Aortic Aneurysm Adventitial Pericyte-Like Cell (AsTAA-APC) Functional and Molecular Properties Comparison of AsTAA-APCs treated for 10 days with either PD0325901 (MEKi) or vehicle. (A) Analysis of proliferating cells using EdU (yellow) and Ki67 (green). DAPI labels nuclei in blue. Images are representative of 1 sample from each group. Scale bar = 50 μm. Bar graphs reporting the percentage of cells positive for EdU and Ki67. N = 5. (B) Secreted angiogenic factors in AsTAA-APC conditioned media measured by enzyme-linked immunosorbent assay (. N = 4. (C and D) Angiogenic tube formation assay with MEKi/Vehicle-treated AsTAA-APCs cocultured with human aortic ECs (AoECs). Images are representative of 1 sample from each group. The bar graph reports the cumulative length of angiogenic networks per imaging field. N = 4 AsTAA-APCs, n = 1 AoECs. (E) Wound closure migration assay comparing nonstimulated and platelet-derived growth factor BB (PDGFBB)-stimulated cells. Images were snapped after 8 and 10 hours and compared with time zero. Bar graphs report the percentage of the wounds closed. N = 3. (F) Immunofluorescence images and bar…

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  Figure 8. Transcriptomic Analysis of Ascending Thoracic Aortic Aneurysm Adventitial Pericyte-Like Cells (AsTAA-APCs) Conditioned With the MEKi or Vehicle (A) A whole-genome RNA-Sequencing study was conducted on AsTAA-APCs conditioned for 10 days with mitogen-activated protein kinase kinase inhibitor (MEKi) (PD0325901) or its vehicle, dimethyl sulfoxide (DMSO) (n = 3). (B) Principal component analysis. (C) Hierarchical clustering of the top 10K transcripts. (D) Mean average plot showing 728 transcripts significantly upregulated and 673 downregulated in MEKi-conditioned AsTAA-APCs compared with vehicle-treated cells. Differentially expressed genes (DEGs) were defined as those with a false discovery rate (FDR) < 0.05 and a fold change >2. (E-H) Lollipop plots showing enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) and biological process pathways based on DEGs. Upregulated pathways are shown in (E and F), and downregulated pathways are shown in (G and H). On the y-axis are the Gene Ontology (GO) terms. The x-axis indicates the statistical significance (–Log(10)FDR). The circle size indicates the number of genes involved in each pathway, whereas the lollipop color indicates the…

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  Figure 9. Oral PD0325901 Treatment for 14 Days Halted Thoracic Aortic Aneurysm (TAA) Progression in a Murine Model (A) Cartoon showing the experimental protocol. Mice were started on oral PD0325901 (10 mg/kg/d) or vehicle (dimethyl sulfoxide, DMSO) embedded in jelly 72 hours post-Angiotensin II (ANG-II) minipump implantation and followed up for 14 days. Control mice, included as a reference, did not receive any treatment. (B) Western blot showing protein expression for total extracellular signal-regulated kinase 1/2 (ERK1/2) and P-ERK in aorta lysates. N = 3 per group. (C) Images acquired using a stereo microscope (Leica) showing the mouse aortas after harvesting. (D) Images of baseline and final echocardiography showing the ascending aorta and proximal arch. Images representative of 1 sample from each group. (E and F) Inner edge-to-edge diameters were presented as a fold change of the measurements at the final time point against basal timepoints. (G) Pulse wave Doppler images of baseline and final echocardiography representative of 1 sample per group. (H) Pulse wave velocity (PWV) was calculated using the equation (d-d0)/(T2-T1), where (d-d0) = distance, T1 = start time, and T2 = end…

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  Figure 10. PD0325901 Treatment Inhibits Angiotensin-II (ANG-II)-Induced Aortic Remodeling (A) Hematoxylin and eosin (H&E) images and bar graphs showing aortic medial area. (B) Van Gieson (EVG) elastin staining images and bar graphs showing elastin medial content. (C) Azan Mallory images and bar graphs showing medial collagen content. (D) Immunohistochemistry (IHC) staining showing active MMP9 expression (brown, 3,3′-diaminobenzidine). Bar graphs report the expression of active MMP9 limited to the tunica media. (E) IHC images and bar graphs showing expression of α-Smooth muscle actin (α-SMA - red fluorescence) in medial vascular smooth muscle cells. (F) IHC images and bar graphs showing recruited CD45+ cells (green). Arrows point to positive cells. (G) Fluorescence images and bar graphs showing apoptotic adventitial cells (Tunel+, in green). Arrows point to positive nuclei. (H) IHC images and bar graphs showing pericytes (platelet-derived growth factor receptor beta [PDGFRβ]+ cells) in the adventitia. In all fluorescent images, 4’,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei (blue). All images are representative of 1 sample from each group. Scale bar = 50 μm. Data are presented…

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  Figure 11. Schematic Illustration Summarizing the Key Findings From Both the Human Patient and Mouse Studies The cartoon illustrates the potential adventitial pericyte-like cells (APC)-driven mechanism in ascending thoracic aortic aneurysm (TAA) pathogenesis (left) and the beneficial effects of MEK inhibition therapy (MEKi - PD0325901) in rescuing APC function, preventing aortic wall remodeling, and halting TAA progression (right). Although further investigation is needed to confirm the role of APCs in TAA pathogenesis and progression in vivo, our in vitro findings strongly suggest that APCs are key players in TAA.