Fig 2: Identification of the signaling pathway mediating the effects of MCP-EV-delivered Hebp1 in CNI-induced ED mice. (A) Heatmap of log2-normalized intensities of significantly differentially expressed genes in Signaling Explorer Antibody Arrays from sham-operated and CNI-induced ED mice that received two intracavernous injections (administered on days -3 and 0) of shHebp1 MCP-EVs (10 µg per mouse in 20 µl PBS), shCon MCP-EVs (10 µg per mouse in 20 µl PBS), or PBS only. Color density shows fold-changes, with different multiples indicated by different colors. Red, up-regulated; blue, down-regulated. (B) Proteins significantly regulated by MCP-EV-delivered Hebp1 in CNI-induced ED mice, identified by a PPI network of differentially expressed genes. The thickness of lines corresponds to the strength of the interaction between the proteins. Red box highlights claudin family genes. (C) Representative Western blots for claudin -1, -2, -3, and -11 in CC tissue from the indicated groups. (D) Ox-LDL (green) immunostaining in CC tissues from the indicated groups. Nuclei were labeled with the DNA dye DAPI (blue). Scale bar, 100 µm. (E-I) Expression of the indicated proteins, quantified by densitometric analysis of the corresponding protein bands, and Ox-LDL immunopositive areas, determined using an image analyzer. Results are presented as means ± SEM (n = 4; *P < 0.05, ***P < 0.001). The relative ratio of the sham operation group was defined as 1. ED, erectile dysfunction; MCPs, mouse cavernous pericytes; EVs, extracellular vesicles; PBS, phosphate-buffered saline; Ox-LDL, oxidized low-density lipoprotein; DAPI, 4,6-diamidino-2-phenylindole; ns, not significant.

Fig 3: Hebp1 expression is decreased in the penis of CNI-induced ED mice. (A-C) Representative (left) and high-magnification (right) images of double-immunofluorescence staining of normal mouse CC and DNB tissue using antibodies against Hebp1 (red), PECAM-1 (A; green), NG2 (B; green), and NF (C; green). Nuclei were labeled with the DNA dye DAPI (blue). Scale bars, 400 µm (left), 100 µm (right top and middle), and 25 µm (right bottom). (D) Representative (left) and high-magnification (right) images of Hebp1 (red) immunofluorescence staining in penis tissues from sham-operated and CNI-induced ED mice (7 days after CNI). (E-G) Quantitative analyses of Hebp1 expression in total penis tissue (E), the CC (F), and DNBs (G) were performed using an image analyzer. Results are presented as means ± SEM (n = 4; **P < 0.01, ***P < 0.001 vs. sham operation groups). The relative ratio of the sham operation group was defined as 1. CC, corpus cavernosum; DNBs, dorsal nerve bundles; ED, erectile dysfunction; CNI, cavernous nerve injury; NF, neurofilament; DAPI, 4,6-diamidino-2-phenylindole.

Fig 4: Hebp1 protein improves erectile function in CNI-induced ED mice. (A) Representative ICP responses for sham-operated and CNI-induced ED mice stimulated 1 week after two intracavernous injections (administered on days -3 and 0) of Hebp1 protein (1.0 and 5 µg per mouse). The stimulus interval is indicated by a solid bar. (B, C) Ratios of mean maximal ICP and total ICP (area under the curve) to MSBP were calculated for each group. Results are presented as means ± SEM (n = 5). (D, E) Immunofluorescence staining of CC and DNB tissues for PECAM-1 (D; green), NG2 (D; red), and NF (E; green) after ICP studies. Nuclei were labeled with the DNA dye DAPI (blue). Scale bars, 100 µm (D) and 25 µm (E). (F) NF (green) immunofluorescence staining in mouse MPGs exposed to LPS (2.5 µg/ml) and then treated with Hebp1 (5 µg/ml) or PBS for 5 days. Scale bar, 100 µm. (G-I) Quantitative analysis of CC endothelial cell (G, PECAM-1), pericyte (H, NG2) and neuronal cell (I, NF) contents using an image analyzer. Results are presented as means ± SEM (n = 5). (J) Neurite sprouting, quantified using an image analyzer. Results are presented as means ± SEM (n = 4; *P < 0.05, **P < 0.01, ***P < 0.001). The relative ratio of the sham operation or control group was defined as 1. ED, erectile dysfunction; CNI, cavernous nerve injury; NF, neurofilament; DAPI, 4,6-diamidino-2-phenylindole; MPG, major pelvic ganglion; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; MSBP, mean systolic blood pressure; DNB, dorsal nerve bundles; ns, not significant.

Fig 5: Schematic depiction of the proposed mechanism for MCP-EV-delivered Hebp1 in CNI-induced ED mice. EVs, extracellular vesicles; ECs, endothelial cells; Ox-LDL, oxidized low-density lipoprotein; CNI, cavernous nerve injury; ED, erectile dysfunction.