Peptide IRW upregulates ACE2 in spontaneously hypertensive rats via dopamine/D1R signaling pathway

Figures

• 

  Fig. 1. Construction of WGCNA modules and transcriptomic analysis of mesenteric arteries in response to IRW treatment. (A) Clustering dendrogram of genes, with dissimilarity based on topological, together with assigned module colors. (B) Scale-free topology fit used to select the soft-thresholding power. (C) Heatmap of the module-trait association. Each row corresponds to a module eigengene, column to a trait. Each cell contains the corresponding correlation (R) and p value. Colors were assigned randomly in the WGCNA analysis. (D) Gene Ontology and (E) KEGG enrichment of genes identified within the red module identified by WGCNA. (F) Chord diagram depicting the genes associated with the enriched KEGG pathways of interest. (G) PPI network of genes within the red module identified by WGCNA. Both node color and size represent their interactions with other proteins. ACE2, the target gene, shows a single direct interaction with DPP4, which is highlighted with a red circle. Proteins encoded by genes enriched in the KEGG pathway are highlighted with blue circles.

• 

  Fig. 2. Metabolomic profiling and WGCNA of mesenteric arteries following IRW treatment. (A) PCA and (B) PLS-DA score plots. (C) Volcano plot was constructed by plotting the fold change (IRW/Untreated) of each metabolite against p value. Fold change > 1.3, p < 0.05 (in red): significantly increased; fold change < 0.83, p < 0.05 (in blue): significantly decreased compared to Untreated. (D) Pathway enrichment analysis of differential metabolites. Each node represents one metabolic pathway; the node color and size represent its p value and pathway impact values, respectively. (E) Clustering dendrogram of metabolites, with dissimilarity based on topological, together with assigned module colors. (F) Scale-free topology fit used to select the soft-thresholding power. (G) Heatmap of the module-trait association. Each row corresponds to a module eigengene, and the column corresponds to the trait (ACE2 expression). Cells display the correlation coefficient (top) and the p-value (bottom, in parentheses). Positive and negative correlation coefficients indicate positive and negative correlations with ACE2 expression, respectively.

• 

  Fig. 3. Integrative network analysis and functional validation of the dopamine-D1R-Nr4a1 pathway regulating ACE2 expression. (A) IPA network of identified transcription factors. (B) IPA network highlighting molecules interacting with Nr4a1. Solid and dotted lines represent direct and indirect interactions, respectively. Colored lines indicate predicted effects: orange for activation, blue for inhibition, yellow for findings inconsistent with downstream molecule state, and pink for direct experimentally observed interactions without a defined activation or inhibition effects. (C) Schematic summary of the proposed signaling pathway. (D-I) Effects of dopamine treatment on ACE2, Nr4a1, and D1R expression in EA.hy926 cells, with or without co-treatment with the D1R antagonist SCH23390, or under Nr4a1 knockdown conditions. Cells were treated with dopamine (0.01–10 μM) for 2 h, followed by the detection of ACE2 and Nr4a1 mRNA expression (D) or their protein levels (E). Cells treated with dopamine (1 μM) and SCH23390 (10 μM) for 2 h were analyzed for mRNA (F) and protein levels (G). Nr4a1 knockdown was conducted using Nr4a1 siRNA, followed by dopamine treatment (1 μM) for 2 h, and detection of…

• 

  Fig. 4. Antihypertensive effect of IRW is mediated by D1R and associated with upregulation of the aortic ACE2-Ang (1–7) pathway. The D1R receptor antagonist SCH23390 (20 µg/kg body weight/h) or saline was chronically infused for 7 days before initiating IRW treatment. Co-treatment with SCH23390 and IRW continued for an additional 7 days, following the timeline shown in (A). During the study, (B) body weight, (C) mean arterial pressure, (D) systolic blood pressure, (E) diastolic blood pressure, and (F) heart rate were recorded over a 24-h period. Aortas collected at the endpoint were analyzed for protein levels of (G) ACE2, (H) Ang (1–7), and (I) D1R. Results are expressed as mean ± SEM (4 ∼ 6). *, **, *** and **** indicate p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively, compared with the untreated control.

• 

  Fig. 5. IRW influenced enzymes associated with its metabolites. (A) Networks involving two metabolites of IRW: kynurenine and dopamine. (B) Potential connections between kynurenine and dopamine. Aortas were collected at the endpoint to evaluate the concentrations of (C) PLP, a cofactor for (D) KYNU and (E) DDC. Results are expressed as mean ± SEM; n = 6. * indicates p < 0.05 compared with the untreated control.

• 

  Fig. 6. Mechanistic summary of IRW-induced ACE2 upregulation via the dopamine/D1R/Nr4a1 pathway, contributing to blood pressure reduction in SHRs.