Pericyte-derived heme-binding protein 1 promotes angiogenesis and improves erectile function in diabetic mice

Abstract

Purpose: To comprehensively evaluate the effect on angiogenesis of heme-binding protein 1 (Hebp1) in the treatment of diabetes-induced erectile dysfunction.

Materials and methods: Mouse corpus cavernosum endothelial cells and pericytes were used for in vitro study. Four groups of mice were used: control nondiabetic mice and streptozotocin-induced diabetic mice receiving two intracavernous injections of phosphate-buffered saline, Hebp1 (1 µg), or Hebp1 (5 µg). The function of Hebp1 in diabetic conditions was evaluated by tube formation assay, aorta ring assay, migration assay, intracavernous pressure, immunofluorescence staining, and Western blot experiments.

Results: We report that Hebp1 is more highly expressed in mouse corpus cavernosum pericytes and can effectively promote endothelial cell angiogenesis under high-glucose conditions. Following exogenous administration of Hebp1 protein, we found that elevated Hebp1 levels can improve the erectile function of diabetic mice, which is achieved by reducing reactive oxygen species levels and activating the PI3K/AKT/eNOS signaling pathway.

Conclusions: Our findings demonstrate that Hebp1 can promote angiogenesis and improve erectile function under diabetic conditions.

Keywords: Diabetes mellitus; Erectile dysfunction; Heme-binding proteins; Pericytes.

Fig. 1. Decreased heme-binding protein 1 (Hebp1) expression under high glucose (HG) conditions. (A) Representative western blots for Hebp1 in mouse corpus cavernosum endothelial cells (MCECs) and mouse corpus cavernosum pericytes (MCPs). (B) Normalized band intensity values (n=4) were quantified by use of ImageJ software (n=4). ^***p<0.001 vs. MCEC group. The relative ratio of the MCEC group was arbitrarily set to 1. (C) Immunofluorescent staining of MCPs with antibodies against Hebp1 (green) and PDGFRβ (pericyte marker). Nuclei were labeled with the DNA dye DAPI. Scale bar indicates 100 µm. (D) The Hebp1-immunopositive area in PDGFRβ-expressing cells was quantified by use of ImageJ software (n=4). (E) Representative western blots for Hebp1 in MCPs exposed to normal glucose (NG) and HG conditions. (F) Normalized band intensity values were quantified by ImageJ software (n=4). ^***p<0.001 vs. NG group. The relative ratio of the NG group was arbitrarily set to 1. IF, immunofluorescent; WB, Western blots.

Fig. 2. Pericyte-derived heme-binding protein 1 (Hebp1) induces angiogenesis under high glucose (HG) conditions. (A-C) Tube formation assays (A), aortic ring assays (B), and migration assays (C) of cells treated with phosphate-buffered saline (PBS) or Hebp1 (200 ng/mL) under normal glucose (NG) and HG conditions. ×40 magnification. (D) Number of migrated endothelial cells was quantified by using ImageJ software (n=4). ^***p<0.001 vs. HG group. (E) Representative western blots for Hebp1 in mouse corpus cavernosum pericytes (MCPs) infected with shCon or shHebp1 lentivirus at three doses (1 µL, 1×10⁴ TU; 5 µL, 5×10⁴ TU; 10 µL, 1×10⁵ TU/mL culture medium). (F) Tube formation assay of mouse corpus cavernosum endothelial cells (MCECs) exposed to MCP culture media at indicated conditions. ×40 magnification. (G) The number of master junctions (n=4, left) and intensity of area of microvessels sprouting from aortic rings were quantified by use of ImageJ software (n=4, right). ^***p<0.001 vs. HG group. (H) Normalized band intensity values were quantified by use of ImageJ software (n=4). ^**p<0.01 vs. shCon group. (I) Number of master junctions was quantified by use of ImageJ software (n=4). ^***p<0.001 vs. complement medium group, ^**p<0.01 vs. MCPs-shCon cultured medium group. The relative ratio of the NG, shCon, or complement medium group was arbitrarily set to 1. shCon, small hairpin RNA scramble control; shHebp1, shRNA heme-binding protein 1.

Fig. 3. Heme-binding protein 1 (Hebp1) improves erectile function under diabetic conditions in mice with streptozotocin (STZ)-induced diabetes. (A) Representative intracavernous pressure (ICP) responses for age-matched control mice and mice with STZ-induced diabetes stimulated at 2 weeks after intracavernous injection of phosphate-buffered saline (PBS) (P; 20 µL) or Hebp1 (1 µg/20 µL and 5 µg/20 µL). The stimulus interval is indicated by a solid bar (1 minute). (B, C) Ratios of mean maximal ICP (B) and total ICP (C) (area under the curve) to mean systemic blood pressure (MSBP) were calculated for each group (n=5, ^**p<0.01, ^***p<0.001). DM, diabetes mellitus; ns, not significant.

Fig. 4. Heme-binding protein 1 (Hebp1) increases corpus cavernosum endothelial cell, pericyte, and neuronal cell content. (A, B) Corpus cavernosum pericyte (A, NG2, red), endothelial cell (A, PECAM-1, green), and nerve (B, βIII-tubulin, red) staining in corpus cavernosum tissue from age-matched control mice and mice with streptozotocin (STZ)-induced diabetes stimulated at 2 weeks after intracavernous injection of PBS (20 µL) or Hebp1 (1 µg/20 µL and 5 µg/20 µL). Nuclei were labeled with DAPI (blue). Scale bars, 100 µm (A) or 25 µm (B). (C-E) Quantification of the NG2, PECAM-1, and βIII-tubulin immunopositive areas in corpus cavernosum tissue using ImageJ software. ^*p<0.05, ^**p<0.01, ^***p<0.001. The relative ratio of the control group was arbitrarily set to 1. DM, diabetes mellitus; ns, not significant.

Fig. 5. Heme-binding protein 1 (Hebp1) decreases corpus cavernosum reactive oxygen species production in mice with streptozotocin (STZ)-induced diabetes. (A, B) In situ detection of superoxide anion (A, hydroethidine, red) and nitrotyrosine (B, red) production in corpus cavernosum endothelial cells (A and B, green) from age-matched control mice and mice with STZ-induced diabetes stimulated at 2 weeks after intracavernous injection of PBS (20 µL) or Hebp1 (1 µg/20 µL and 5 µg/20 µL). Nuclei were labeled with DAPI (blue). Scale bars, 100 µm. (C, D) The ethidium bromide fluorescence-immunopositive cavernosum area and nitrotyrosine-immunopositive cavernosum area were quantified by use of ImageJ software (n=4). ^***p<0.001. The relative ratio of the control group was arbitrarily set to 1. DM, diabetes mellitus; ns, not significant.

Fig. 6. Heme-binding protein 1 (Hebp1) increases PI3K/AKT/eNOS signaling in mice with streptozotocin (STZ)-induced diabetes. (A) Representative western blots for p-PI3K/PI3K, p-AKT/AKT, and p-eNOS/eNOS in corpus cavernosum tissue from age-matched control mice and mice with STZ-induced diabetes stimulated at 2 weeks after intracavernous injection of PBS (20 µL) or Hebp1 (1 µg/20 µL and 5 µg/20 µL). (B-D) Normalized band intensity values were quantified by use of ImageJ software (n=4). ^*p<0.05, ^**p<0.01, ^***p<0.001. The relative ratio of the control group was arbitrarily set to 1. DM, diabetes mellitus; ns, not significant.