Temporal Proteomic Remodeling of Cerebral Arteries in Male Hypertensive Rats

Figures

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  Figure 1.. Identification of DEPs (differentially expressed proteins) in aged hypertensive rats. Volcano plots comparing protein abundance in cerebral arteries from 30- (A) and 40-week-old (B) spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) controls. Log2-fold change difference and unadjusted P values are presented in a volcano plot. Red=significantly upregulated and blue=significantly downregulated in SHR compared with WKY. C, Venn diagram representing the number of shared DEPs from each comparison. D and E, Pathway enrichment analysis in STRING of DEPs from the 2 time point comparisons using the Reactome database.37 Top 10 most significant pathways are presented with −log10-transformed false-discovery rate (FDR).

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  Figure 2.. Aged hypertensive rats have reduced SRF (serum response factor) expression and a proteomic signature similar to SRF knockout mice. A, Immunofluorescence staining of SRF (orange), αSMA (α-smooth muscle actin; green), and PECAM (platelet endothelial cell adhesion molecule)-1 (CD31; red) in cerebral arteries from Wistar-Kyoto (WKYs) control rats and spontaneously hypertensive rats (SHRs) at 30 and 40 weeks of age. Images were acquired with a ×20 lens. Scale bar, 20 µm. B, Arithmetic mean fluorescent intensity of SRF in the 4 groups (WKY 30w=light blue; WKY 40w=dark blue; SHR 30w=light orange; and SHR 40w=dark orange; n=4 biological replicates in each group with 2–4 technical replicates). Mean±SEM is presented, the 2-way ANOVA test and the Fisher LSD post hoc test (uncorrected). *P<0.05 and **P<0.01. C, Venn diagram representing the number of overlapping DEPs (differentially expressed proteins) from the 2 time points and an SRF knockout artery database from a previous publication.11 D, Fold change differences of shared DEPs from the 2 SHR vs WKY comparisons, respectively. Core SRF–associated genes are highlighted in orange. E, Pathway enrichment analysis in STRING of shared DEPs from…

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  Figure 3.. Time-dependent regulation of M-phase association proteins. A, STRING network of proteins with significant association to M-phase. Protein-protein-interaction (PPI) cluster is visualized in green. Outer red rings are proteins (nodes) with the highest number of interactions (degrees) in the network. B, Bar chart depicting the number of proteins in the network with 0 to 4, 5 to 9, and 10 to 14 degrees. C, Pathway enrichment analysis in STRING of PPI cluster using the gene ontology biological processes database. The top 10 most significant pathways are presented with −log10-transformed false-discovery rate (FDR). D, Dot plot depicting log fold change differences of significantly regulated proteins associated with the M-phase pathway at the 30-week comparison of spontaneously hypertensive rats (SHRs) vs Wistar-Kyoto (WKY) control. The respective fold change difference from the 40-week comparison is included. E and F, Dot plot depicting subanalysis of upregulated or downregulated protein clusters in the M-phase pathway (Student t test: **P<0.01 and *P<0.05).

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  Figure 4.. Time-dependent regulation of Rho GTPase association proteins. A, STRING network of proteins with significant association to the Rho GTPase signaling pathway. Protein-protein-interaction (PPI) cluster is visualized in blue (first neighbors of Cdc42 [cell division cycle 42]) and green (second neighbors of Cdc42). The outer red ring depicts the protein with the highest number of interactions (degrees) in the network. B, Number of proteins with 0 to 4, 5 to 9, 10 to 14, 15 to 19, 20 to 24, 25 to 29, and 30 to 34 degrees. C, Pathway enrichment analysis in STRING of Cdc42-PPI cluster (both first and second neighbors) using the gene ontology biological processes database. The top 10 most significant pathways are presented with −log10-transformed false-discovery rate (FDR). D, Log fold change differences of significantly regulated proteins associated with the Rho GTPase signaling pathway at the 40-week comparison of spontaneously hypertensive rats (SHRs) vs Wistar-Kyoto (WKY) control. The respective fold change difference from the 30-week comparison is included. E and F, Dot plot depicting subanalysis of upregulated or downregulated protein clusters in the Rho GTPase signaling pathway…

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  Figure 5.. Vascular remodeling of the basilar artery from 30- and 40-week-old hypertensive rats. A, Exemplary Sirius red–stained cerebral arteries from 30- and 40-week-old Wistar-Kyoto (WKY) control or spontaneously hypertensive rats (SHRs). B, Media thickness (i), circumference (ii), media-to-circumference (M:C; iii) ratio, and mean RBG (red, blue, green) converted value in the different groups (iv). C, Immunohistochemistry analysis of (αSMA [α-smooth muscle actin] in orange and nuclei (Hoechst) in white) in cerebral arteries from WKY controls and SHRs (n=4 biological replicates per group). D, Average nuclear diameter (feret) of the cell in the media layer of the artery. E, Cell number-to-wall area ratio (i) and total nuclear area-to-wall area ratio (ii). Images were acquired with a ×20 lens. Scale bar, 100 µm. Mean±SEM is presented, the 2-way ANOVA test and the Fisher LSD post hoc test (uncorrected). ns indicates nonsignificant. P>0.05, *P<0.05, and **P<0.01. WKY blue, SHR=orange. Row interactions from 2-way ANOVA test; #P<0.05 and ##P<0.01.

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  Figure 6.. Dysregulation of ECM (extracellular matrix) proteins. Principal component analysis plots based on ECM-associated proteins28,29 in spontaneously hypertensive rats (SHRs) vs Wistar-Kyoto (WKY) control at 30 weeks of age (A) or 40 weeks of age (B). C, i, Scatter plot depicting fold change difference of all ECM-associated proteins. ****P<0.0001 in the Student t test comparison. ii, Venn diagram comparing differentially expressed ECM–associated proteins at each time point (P<0.05). iii, The Fisher exact test comparing ECM protein overlap with the ECM database. The line represents P=0.05. D, Scatter plots of shared (i) and unique (ii and iii) differentially expressed ECM–associated proteins (=27) from 30- and 40-week comparisons, respectively. Red=upregulated, blue=downregulated, and gray line=simple linear regression curve. E, Trajectory of differentially expressed ECM–associated proteins as in D, highlighting proteins changed in the same direction in blue and opposite direction in orange.