Pericyte-derived extracellular vesicle-mimetic nanovesicles ameliorate erectile dysfunction via lipocalin 2 in diabetic mice

Abstract

Diabetes mellitus is one of the main causes of erectile dysfunction (ED). Men with diabetic ED do not respond well to oral phosphodiesterase-5 inhibitors owing to neurovascular dysfunction. Pericyte-derived extracellular vesicle-mimetic nanovesicles (PC-NVs) are known to promote nerve regeneration in a mouse model of cavernous nerve injury. Here, we report that administration of PC-NVs effectively promoted penile angiogenesis and neural regeneration under diabetic conditions, thereby improving erectile function. Specifically, PC-NVs induced endothelial proliferation and migration and reduced cell apoptosis under diabetic conditions. In addition, PC-NVs induced neural regeneration in STZ-induced diabetic mice in dorsal root ganglion and major pelvic ganglion explants in vivo and ex vivo under high-glucose conditions. We found that lipocalin 2 (Lcn2) is a new target of PC-NVs in this process, demonstrating that PC-NVs exert their angiogenic and nerve-regeneration effects by activating MAP kinase and PI3K/Akt and suppressing P53 signaling pathway in an Lcn2-dependent manner. Our findings provide new conclusive evidence that PC-NVs can promote neurovascular regeneration and recovery of erectile function under diabetic conditions via an Lcn2-dependent mechanism. Thus, local administration of PC-NVs may be a promising treatment strategy for the treatment of diabetic ED.

Keywords: Diabetes mellitus; Erectile dysfunction; Lipocalin2; Nanovesicles; Neurovascular regeneration; Pericyte.

Figure 1

Preparation and characterization of MCP-derived PC-NVs. (A) Schematic overview of PC-NVs extraction procedure. (B) Representative TEM phase images for detecting isolated PC-NVs. Scale bar = 100 nm. (C) Enrichment diameter distribution of PC-NVs, measured by dynamic light scattering. (D) Representative Western blot analysis of EV markers from three independent experiments that yielded similar results. GM130: negative PV marker; TSG101, Alix100 and CD81: positive EV markers. HBS, HEPES-buffered saline.

Figure 2

PC-NVs improve erectile function in diabetic mice. (A) Representative ICP responses for age-matched control (C) and diabetic mice 2 weeks after intracavernous injection of HBS (H) or PC-NVs (0.5, 1, and 5 μg/20 μl) on days -3 and 0. Stimulus interval indicated by a solid bar. (B and C) Staining for endothelial cells (PECAM-1; red), smooth muscle cells (α-SMA; green), and pericytes (NG2; green) of cavernous from age-matched control (C) and diabetic mice 2 weeks after intracavernous injection of HBS (H) or PC-NVs (5 μg/20 μl) on days -3 and 0. Scale bar = 100 μm. (D and E) Ratios of mean maximal ICP and total ICP (area under the curve) to MSBP calculated for each group and presented as means ± SEM (n = 5). (F-H) Quantitative analysis of cavernous endothelial cell, smooth muscle cell and pericyte content, quantified by Image J and presented as means ± SEM (n = 7; *P < 0.05; ^#P < 0.001). DM, diabetes mellitus; HBS, HEPES-buffered saline; STZ, streptozotocin.

Figure 3

PC-NVs enhance angiogenesis by increasing proliferation and decreasing apoptosis of MCECs under HG conditions. (A) Tube-formation assay. MCECs were treated with PC-NVs (1 µg/mL) or HBS under NG or HG conditions for 3 days. Representative images were acquired at 18 hours. Scale bar = 100 µm. (B) Ex vivo mouse aortic ring microvessel-outgrowth assay. Aorta rings were treated with PC-NVs (1 µg/mL) or HBS under NG or HG conditions for 5 days. Representative images of sprouting microvessels were acquired at 5 days. Scale bar = 100 µm. (C) Migration assay. MCECs were treated with PC-NVs (1 µg/mL) or HBS under NG or HG conditions for 3 days. Representative images of migrated cells were acquired at 24 hours. Scale bar = 100 µm. (D) Master junctions, quantified using Image J and presented as means ± SEM (n= 5). (E) Intensity of microvessel sprouting area from aortic rings, quantified using Image J and presented as means ± SEM (n = 9). Relative ratio of the NG group was defined as 1. (F) Migrated cells, quantified using Image J and presented as means ± SEM (n = 5). (G and H) Immunofluorescence staining of MCECs with anti-BrdU antibody (red) (G) and TUNEL assay (H) in cells treated with PC-NVs (1 µg/mL) or HBS under NG or HG conditions for 3 days. Nuclei were labeled with DAPI. Scale bars = 50 μm. (I and J) Number of BrdU-positive (I) or TUNEL-positive (J) endothelial cells per high-power field (HPF). Results are presented as means ± SEM (n = 5; *P < 0.05; ^#P < 0.001). DAPI, 4,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling.

Figure 4

PC-NVs induce neural regeneration under diabetic or HG conditions. (A) nNOS (red) and NF (green) staining in cavernous tissue from age-matched control (C) and diabetic mice 2 weeks after intracavernous injection of HBS or PC-NVs (5 μg/20 μl) on days -3 and 0. Nuclei were labeled with DAPI. Scale bar = 25μm. (B and C) Quantitative analysis of nNOS- and NF-positive axonal areas by ImageJ, presented as means ± SEM (n = 6). (D and E) βIII-tubulin staining in mouse MPG (red) and DRG (green) tissue treated with PC-NVs (1 µg/mL) or HBS under NG or HG conditions for 5 days. Scale bar = 100 μm. (F and G) βIII-tubulin-immunopositive neurite length in MPG or DRG tissue, quantified using Image J and presented as means ± SEM (n = 7). (H) Representative Western blots for neurotrophic factors (NGF, NT3, and BDNF) in cavernous tissue from age-matched control (C) and diabetic mice 2 weeks after intracavernous injection of HBS or PC-NVs (5 μg/20 μl) on days -3 and 0. (I-K) Band intensity values of each neurotropic factor normalized to the density of β-actin, quantified using Image J and presented as means ± SEM (n = 6; *P < 0.05; ^#P < 0.001). The relative ratio in the control group was defined as 1. DM, diabetes mellitus; HBS, HEPES-buffered saline.

Figure 5

Lcn2 as a novel target of PC-NV-triggered neurovascular regeneration under diabetic conditions. (A and B) Analysis of RNA-sequencing data from MCECs treated with PC-NVs (1 µg/mL) or HBS under HG conditions for 3 days. (A) DEGs between HG+HBS and HG+PC-NVs, shown by scatter plot. (B). Seven genes met criteria for significant DEGs (fold change > 4 and log2 > 6); Lcn2 (red) identified as a target for PC-NV-induced neurovascular regeneration. (C and D) Lcn2 Relative expression in MCECs treated with PC-NVs (1 µg/mL) or HBS under HG conditions in MCP lysates (C, top) and PC-NVs (C, bottom), detected by Western blotting. (D) Band intensity of Lcn2 normalized to that of β-actin, quantified using Image J and presented as means ± SEM (n = 5). (E) Representative ICP responses for age-matched control (C) and diabetic mice obtained 2 weeks after intracavernous injection of HBS (H), PC-NVs (shCon) (5 μg/20 μl), or PC-NVs (Lcn2-KD) (5 μg/20 μl) on days -3 and 0. The stimulus interval indicated by a solid bar. (F) Tube-formation assay in MCECs treated with HBS, PC-NVs (shCon), or PC-NVs (Lcn2-KD) under NG or HG conditions for 3 days. (G) Ex vivo mouse aortic ring microvessel-outgrowth assay. Aorta rings were treated with HBS, PC-NVs (shCon), or PC-NVs (Lcn2-KD) under NG or HG conditions for 5 days. (H and I) βIII-tubulin staining in mouse MPG (H) and DRG (I) tissue, treated with HBS, PC-NVs (shCon), or PC-NVs (Lcn2-KD) under NG or HG conditions for 5 days. (J and K) Ratios of mean maximal ICP and total ICP (area under the curve) to MSBP, calculated for each group and presented as means ± SEM (n = 5). (L) Master junctions, quantified using Image J and presented as means ± SEM (n = 9). (M) Intensity of microvessel sprouting area from aortic rings, quantified using Image J and presented as means ± SEM (n = 5). (N and O) βIII-tubulin-immunopositive neurite length in MPG or DRG tissue, quantified using Image J and presented as means ± SEM (n = 4; ^#P < 0.001). DM, diabetes mellitus; HBS, HEPES-buffered saline.

Figure 6

Identification of the PC-NVs/Lcn2 signaling pathway responsible for promoting neurovascular regeneration. (A) Heatmap of log2-normalized intensities of DEGs from age-matched controls (C) and diabetic mice, determined 2 weeks after intracavernous injections of HBS, shCon-PC-NVs (5 μg/20 μl) or Lcn2-KD-PC-NVs (5 μg/20 μl) on days -3 and 0. Color density expressed fold-change magnitude in the indicated multiples. Red, up-regulated; blue, down-regulated. (B) Protein-protein interaction network of DEGs. The line thickness corresponds to the interaction strength between protein (C) Representative Western blots for MAPK, PI3K, Akt, and Trp53 in cavernous tissue from age-matched control (C) and diabetic mice, 2 weeks after intracavernous injections of HBS, shCon-PC-NVs (5 μg/20 μl) or Lcn2-KD-PC-NVs (5 μg/20 μl) on days -3 and 0. (D-G) Band intensities of each protein normalized to the density of β-actin, quantified using Image J and presented as means ± SEM (n = 4; *P < 0.05; ^#P < 0.001). DM, diabetes mellitus; HBS, HEPES-buffered saline.