Gene expression profiling of mouse cavernous endothelial cells for diagnostic targets in diabetes-induced erectile dysfunction

Abstract

Purpose: To investigate potential target genes associated with the diabetic condition in mouse cavernous endothelial cells (MCECs) for the treatment of diabetes-induced erectile dysfunction (ED).

Materials and methods: Mouse cavernous tissue was embedded into Matrigel, and sprouted cells were subcultivated for other studies. To mimic diabetic conditions, MCECs were exposed to normal-glucose (NG, 5 mmoL) or high-glucose (HG, 30 mmoL) conditions for 72 hours. An RNA-sequencing assay was performed to evaluate gene expression profiling, and RT-PCR was used to validate the sequencing data.

Results: We isolated MCECs exposed to the two glucose conditions. MCECs showed well-organized tubes and dynamic migration in the NG condition, whereas tube formation and migration were significantly decreased in the HG condition. RNA-sequencing analysis showed that MCECs had different gene profiles in the NG and HG conditions. Among the significantly changed genes, which we classified into 14 major gene categories, we identified that aging-related (9.22%) and angiogenesis-related (9.06%) genes were changed the most. Thirteen genes from the two gene categories showed consistent changes on the RNA-sequencing assay, and these findings were validated by RT-PCR.

Conclusions: Our gene expression profiling studies showed that Cyp1a1, Gclm, Igfbp5, Nqo1, Il6, Cxcl5, Olr1, Ctgf, Hbegf, Serpine1, Cyr61, Angptl4, and Loxl2 may play a critical role in diabetes-induced ED through aging and angiogenesis signaling. Additional research is necessary to help us understand the potential mechanisms by which these genes influence diabetes-induced ED.

Keywords: Diabetes mellitus; Erectile dysfunction; Gene expression; Penis; RNA sequencing.

Fig. 1. Isolation and characterization of primary cultured mouse cavernous endothelial cells (MCECs). (A) Representative phage images (screen magnification, ×40) of sprouted cells from mouse penis cavernous tissues at passage 0, and sprouted cells subcultured at passage 1. (B) Immunofluorescent staining of MCECs with antibodies against CD31 (an EC-positive marker), CD90 (a fibroblast marker), NG2 (a pericyte marker), and α-SMA (a smooth muscle cell marker). Nuclei were labeled with the DNA dye DAPI (4,6-diamidino-2-phenylindole). Scale bar indicates 100 µm.

Fig. 2. Decreased tube formation and migration in mouse cavernous endothelial cells (MCECs) exposed to the high-glucose (HG) condition. Phase images of MCECs (16 hours, screen magnification, ×40). MCECs were incubated in normal-glucose (NG) and HG conditions for 72 hours. (A) Tube formation assay was performed on Matrigel in 48-well dishes. Scale bar indicates 500 µm. (B) Migration assay in MCECs exposed to NG and HG conditions for same time with tube formation. After 24 hours, the images were taken by microscopy. Scale bar indicates 500 µm. (C) Number of tubes and (D) migrated cells per field. Each bar depicts the mean values (±standard error) from four separate experiments. ^**p<0.01 compared with the NG group.

Fig. 3. Analysis of two libraries of differentially expressed genes. (A) Total gene expression of mouse cavernous endothelial cells in normal-glucose (NG) and high-glucose (HG) conditions. (B) Differentially expressed genes in two libraries. (C) Differentially expressed gene analysis for significantly changed gene selection following the conditions set in advance. Two separate samples for each group were subject to analysis.

Fig. 4. The significantly altered genes of the RNA-sequencing data were allocated to gene ontology (GO) categories. (A) The percentage of total significantly changed genes allocated to the top 14 GO categories. (B) The percentage of genes in each category of total counted genes with detailed up-regulated and down-regulated gene numbers. The significantly altered genes were enriched in Aging and Angiogenesis GO categories, as showed with dotted frame.

Fig. 5. RT-PCR validation of differentially expressed genes in RNA sequencing. (A) Seven aging-related genes were evaluated in mouse cavernous endothelial cells (MCECs) exposed to normal-glucose (NG) and high-glucose (HG) conditions. (B) Six angiogenesis-related genes were evaluated in MCECs exposed to NG and HG conditions. (C, D) Each bar depicts the mean values (±standard error) from three separate experiments. ^*p<0.05 compared with the NG group.