PCSK5 promotes angiogenesis and cardiac repair after myocardial infarction

Fig. 1. Plasma PCSK5 levels are increased in patients with acute MI and correlate with cardiac function. A The Venn diagram illustrated 104 genes that were commonly upregulated in both infarcted mice (GSE184986) and in infarcted patients (GSE132143). The top 7 secretory proteins were shown. B qPCR analysis detected the PCSK5 expression at the border zone of left ventricular (LV) in both controls and patients with myocardial infarction (MI), n = 5 per group. C ELISA measured plasma PCSK5 levels in controls (n = 30) and patients with AMI (n = 28). D Receiver operating characteristic curve was generated for plasma PCSK5 levels to identify AMI patients who exhibited an increased ejection fraction after a six-month follow-up (n = 28). E Western blot analysis measured the expressions of PCSK5 and GAPDH at the border zone of LV in sham mice and mice post-MI, n = 6 per group. F, G The expressions of PCSK5 and GAPDH were measured by Western blot (F) and qPCR (G) analyses at the border, infarct, and remote zone of hearts from sham and mice at 28 days post-MI, n = 6 per group. H Immunostaining at the border zone of LV indicated the expressions of PCSK5 and CD31 in both controls (n = 6) and MI…

Fig. 2. Endothelial-specific PCSK5 promotes cardiac repair following MI. A A schematic diagram was created in BioRender. Guo, J. (2026) https://BioRender.com/loszhp6. It depicted the MI operation in endothelial cells-deficient Pcsk5 (Pcsk5ECKO) mice and wild-type littermates (Pcsk5WT). MI was induced in 8–10 week-old male mice. Echocardiographic assessments were conducted at 0, 7, 14, and 28 days post-surgery. B qPCR analysis detected the Pcsk5 expression in ECs from Pcsk5WT and Pcsk5ECKO mice (n = 6 per group). C ELISA analysis detected the plasma PCSK5 levels in Pcsk5WT and Pcsk5ECKO mice, both sham-operated and at 28 days post-MI, n = 6 per group. D Echocardiography showed ejection fraction (EF%) and fractional shortening (FS%) of Pcsk5ECKO and Pcsk5WT mice at day 0, 7, 14, and 28 post-MI. n = 3 for Pcsk5WT + Sham and Pcsk5ECKO + Sham, n = 7 for Pcsk5WT + MI and Pcsk5ECKO + MI. E Masson staining elucidated the infarcted area from suture to apex in Pcsk5WT and Pcsk5ECKO mice at 28 days post-MI. n = 6 per group. Scale bar represents 5 mm. F TUNEL staining was performed at the border zone in Pcsk5WT and Pcsk5ECKO mice at 28 days post-MI. Scale bar represents 50 μm. n = 6 per group. G A…

Fig. 3. PCSK5 promotes angiogenesis following MI. A Uniform Manifold Approximation and Projection (UMAP) plots showed single-nucleus RNA sequencing (snRNA-seq) analysis conducted at the border zone of LV in three AAV-Pcsk5 and three AAV-Ctrl mice hearts at 7 days post-MI, cell types distinguished by color coding. B, C The heatmap presented the expression of differentially expressed genes (DEGs) in ECs ordered by samples (B), and representative Gene Ontology (GO) Biological processes terms were displayed in (C). D Heatmap showed selected transcription factor activities inferred with DoRothEA from gene expression data generated in ECs from AAV-Pcsk5 and AAV-Ctrl mice at 7 days post-MI. E UMAP plots showed snRNA-seq analyzed in ECs from AAV-Pcsk5 and AAV-Ctrl mice, with cell types distinguished by color coding and separated by sample at 7 days post-MI. F The histogram showed the proportion of EC subpopulation in AAV-Pcsk5 and AAV-Ctrl mice at 7 days post-MI. G Vlnplot analysis displayed ECs’ pseudotime in three AAV-Pcsk5 and three AAV-Ctrl mice hearts at 7 days post-MI. Boxplots show the first quartile, median, and third quartile, with the whiskers indicating ±1.5 times the inter-quartile…

Fig. 4. PCSK5 promotes the VEGFA signaling pathway in hearts post-MI. A CellChat analysis identified altered signaling pathways in the interactions among all cell types in AAV-Pcsk5 and AAV-Ctrl mice at 7 days post-MI. B Circle plots of significant (P value ≤ 0.05) cell-cell communication depicted differentially regulated VEGF pathways and interactions in heart tissues of AAV-Pcsk5 and AAV-Ctrl mice at 7 days post-MI. The line thickness indicated the interaction strength of signals from sending and receiving cells. Arrows indicated directionality. (C) Vlnplot analysis displayed the Vegfa expression in ECs from AAV-Pcsk5 and AAV-Ctrl mice at 7 days post-MI. D, E ELISA analysis measured the plasma VEGFA levels in Pcsk5ECKO and Pcsk5WT mice, as well as AAV-Pcsk5 and AAV-Ctrl mice at 28 days post-MI, n = 6 per group. F–H The protein expressions of ERK1/2, PCSK5, GAPDH, and the phosphorylation of ERK1/2 at the border zone of LV, were detected by Western blot analysis in AAV-Pcsk5 and AAV-Ctrl mice (F) or in AAV-EC-Pcsk5 and AAV-Ctrl mice (G) or in Pcsk5WT and Pcsk5ECKO mice (H) at 28 days post-MI, n = 6 per group. I, J The protein expressions of ERK1/2, PCSK5, and the phosphorylation of…

Fig. 5. PCSK5-mediated cleavage of VEGFA promotes the angiogenic effects. A–C Tube formation assays (A), EdU assays (B), and transwell assays (C) were employed to evaluate the tube formation, cell migration, and proliferation in HUVECs transfected with either Ad-Ctrl or Ad-PCSK5, followed by treatment with the VEGFA antibody Bevacizumab (20 ng/ml for 24 h), n = 4 biological replicates per group. Scale bar represents 50 μm. D The interaction mode and binding sites of PCSK5 and VEGFA were predicted by HDOCK analysis. E The Arg158 and Asn164 of PCSK5 were replaced with alanine (designated as PCSK5mut). 100 μg/mL recombinant human PCSK5 or PCSK5mut were incubated with 100 μg/mL human VEGFA peptide at 37 °C for 6 hs. Samples were detected by Western blot analysis (n = 3 independent experiments). F, G The cleaved VEGFA in cell supernatant (F) or the protein expressions of ERK1/2 and PCSK5, and the phosphorylation of ERK1/2 in cell lysate (G) were detected by Western blot analysis in HUVECs transfected with Lentivirus-Ctrl (Lenti-Ctrl), Lentivirus-PCSK5 (Lenti-PCSK5), or Lentivirus-PCSK5mut (Lenti-PCSK5mut). VEGFA cleavage (%) = the cleaved VEGFA/total VEGFA. n = 4 biological replicates per…

Fig. 6. VEGFA promotes PCSK5 transcription by ETS1. A Transcription factors binding to the PCSK5 promoter (±2000 bp region surrounding the transcription initiation site of PCSK5) were predicted by HOMER. A Venn diagram illustrated the differentially expressed transcription factors in MI patients (GSE132143) compared to controls. B HUVECs were transfected with Control or ETS1 lentivirus, as well as Control or ETS1 siRNAs. RT-qPCR analysis examined mRNA levels of PCSK5 and ETS1. n = 4 biological replicates. C, D Luciferase activity was detected in HEK293T cells transfected with luciferase reporter constructs containing various segments of the PCSK5 promoter (C) or reporter constructs containing PCSK5 promoter and ETS1 binding sites mutation truncations (D), followed by transfection with ETS1-FLAG, n = 6 biological replicates. E Snapshots of ChIP-seq tracks (GSE41166) demonstrated ETS1 enrichment at the promoter of PCSK5 in HUVECs treated with VEGFA (50 ng/ml). F, G The enrichment of ETS1 at the promoter of PCSK5 was validated by ChIP-qPCR analysis in HUVECs treated with VEGFA (100 ng/ml) for 0 or 24 h (F, n = 5 biological replicates) or in mouse cardiac ECs (G, n = 4 biological…

Fig. 7. Semaglutide promotes the angiogenic activity by upregulating PCSK5 expression in ECs. A Heatmap showed the expression profiles of genes regulated by semaglutide in HUVECs treated with semaglutide (100 nM) for 48 h and transfected with either Control siRNAs or PCSK5 siRNAs. B Representative Gene Ontology Biological Process (GO-BP) terms showed that semaglutide upregulated genes were associated with angiogenesis in HUVECs. C Gene Set Enrichment Analysis (GSEA) revealed significant positive enrichment of the angiogenesis gene set in HUVECs treated with semaglutide compared to HUVECs treated with PBS (Ctrl). D Correlation analyses examined the relationship between plasma levels of PCSK5 and GLP-1 in patients with AMI (n = 42). E HUVECs were treated with semaglutide (100 nM) for 48 h and transfected with Control siRNAs or ETS1 siRNAs. The protein levels of PCSK5, ETS1, and the phosphorylation of ETS1 were detected by Western blot analysis. Quantitative and statistical analyses were shown on the right and in Fig. S11D, n = 4 biological replicates. F Luciferase activity was detected in HEK293T cells transfected with luciferase reporter constructs containing PCSK5 promoter or ETS1…

Fig. 8. Semaglutide promotes angiogenesis by PCSK5. A A schematic diagram was created in BioRender. Guo, J. (2026) https://BioRender.com/fjpfytl. It depicted the MI operation in Pcsk5ECKO mice or Pcsk5WT littermates with semaglutide treatment. Pcsk5ECKO mice were treated with tamoxifen to conditionally knock out Pcsk5 in endothelial cells. Semaglutide (60 μg/kg) or vehicle was given to mice via subcutaneous injection daily after MI surgery. MI was surgically induced in 8–10 week-old male mice. Echocardiographic assessments were conducted at 0, 7, 14, and 28 days post-surgery. The heart tissues were harvested at 28 days post-MI. B Echocardiography showed EF% and FS% of Pcsk5ECKO and Pcsk5WT mice with or without semaglutide treatment at day 0, 7, 14, and 28 post-MI, n = 6 per group. C IB4 and α-SMA staining at the border zone of LV showed the capillary and arteriole densities in Pcsk5ECKO and wild-type mice with or without semaglutide treatment at 28 days post-MI. Scale bar represents 100 μm. n = 6 per group. D The schematic diagram was created in BioRender. Guo, J. (2026) https://BioRender.com/0w6zmr8. It illustrated that plasma PCSK5 levels were elevated in AMI patients compared to…