Single cell transcriptional landscape of mouse cavernous tissues in normal and diabetic conditions.

(A) Schematic workflow of this study. Cavernous tissues from sixteen-week-old male mice were used for single cell RNA sequencing (n=5 for each group). DM, diabetes mellitus. (B) Visualization of single cell data from mouse cavernous tissues using UMAP. Each cell type is indicated by a different color. FCP, fibrochondrocyte progenitors; FC, fibrochondrocytes; Chond, chondrocytes; FB, fibroblasts; Myo-FB, myofibroblasts; LEC, lymphatic endothelial cells; VEC, vascular endothelial cells; SMC, smooth muscle cells. (C) Heatmap of known cell type marker genes used for annotation. (D) UMAP projection of four fibroblast clusters and expression of marker genes of four fibroblast subsets. Egr1 for Egr1 high FB; Cxcl12 for Cxcl12 high FB; Mgp for Reticular FB; Rps25 and Rps17 for Ribosomal gene high FB. (E) Dot plot showing the expression of top five marker genes of each fibroblast subset. (F) DEGs between diabetic and normal conditions in Fibroblasts. The top 10 (based on log-fold change) DEGs are indicated with gene names, and genes identified as having high or low expression in diabetes in previous studies are indicated with gene names in red or blue. DEGs with adjusted p- value > 0.05 were indicated in gray. (G) Gene ontology analysis of the DEGs higher in normal compared to diabetes in Fibroblasts. (H) Gene ontology analysis of the DEGs higher in diabetes compared to normal in Fibroblasts.

Identification of Lbh as a marker of pericytes.

(A) Expression of well-known marker genes of SMC and Pericyte (Cnn1 for SMC, Rgs5 and Pdgfrb for Pericyte). (B) Significantly enriched gene sets associated with function of SMC and Pericyte. Gene sets with positive normalized enrichment score (NES) are enriched in Pericyte, and negative values are enriched in SMC. (C) Violin plots showing expression of Lbh in each cell type. (D) LBH (red)/a-SMA (green) and LBH (red)/CD31(green) staining in MCPs, aorta SMC, bladder, aorta, and kidney vascular tissues. Nuclei were labeled with DAPI (blue). Scale bars, 25 µm (MCPs and aorta SMC), 50 µm (bladder top panel and kidney vascular) and 100 µm (bladder bottom panel). (E) LBH (red)/a-SMA (green) and LBH (red)/PDGFRβ (green) staining in mouse cavernosum, dorsal artery, dorsal vein tissues. Nuclei were labeled with DAPI (blue). Scale bars, 100 µm (cavernosum), 25 µm (dorsal artery) and 50 µm (dorsal vein). Arrows indicate the LBH expressed pericytes. MCPs, mouse cavernous pericytes; SMC, smooth muscle cell; DAPI, 4,6-diamidino-2-phenylindole. ***P < 0.001 by DESeq2 test.

Cell-cell interactions between pericytes and other cell types in normal and diabetes.

(A) CellphoneDB dot plots showing angiogenesis-associated ligand-receptor interactions from pericytes to other cell types in normal and diabetic samples. P-values are indicated as circle sizes. The means of the average expression level of the interaction are indicated by color. (B) CellphoneDB dot plots showing angiogenesis- associated ligand-receptor interactions from other cell types to pericytes in normal and diabetic samples. P- values are indicated as circle sizes. The means of the average expression level of the interaction are indicated by color. (C) The inferred VEGF signaling pathway network in normal and diabetes using CellChat. The width of line represents the communication probability. The color of line matches the sender of the signal. (D) GSEA plots showing the gene sets downregulated in diabetic pericytes compared to normal pericytes. (E) Heatmap showing the regulon activities of angiogenesis-related transcription factors in pericytes and SMCs in normal and diabetes. Scaled values of regulon activity scores are displayed on the heatmap. **P < 0.01; ***P < 0.001 by generalized linear model. (F) Proteome profiler mouse angiogenesis array analysis of mouse penis tissues from age-matched control and diabetic mice. The relative expression of each protein was determined by comparing the respective plots to the positive control (reference spot). The frame dot line indicates changed proteins between control and diabetic mice. Expression of the indicated proteins was quantified by assessing the intensity of the dot using Image J. DM, diabetes mellitus.

LBH improves erectile function under diabetic conditions through induction of neurovascular regeneration.

(A and B) Representative images of immunofluorescence staining of LBH (green)/CD140b (red) in cavernosum tissues and LBH (red) in MCPs under normal and diabetic conditions (in vivo and in vitro). Nuclei were labeled with DAPI (blue). Scale bar, 100 µm. (C and D) Quantification of LBH or CD140b expression in in vivo and in vitro by using Image J, and results are presented as means ± SEM (n = 4). (E) Representative western blots for LBH of MCPs under NG and HG conditions, and mouse penis tissues from age-matched control and diabetic mice. (F) Normalized band intensity ratio of LBH to β-actin was quantified using Image J, and results are presented as means ± SEM (n = 4). (G) Representative intracavernous pressure (ICP) responses for the age-matched control and diabetic mice stimulated at 2 weeks after intracavernous injections with PBS, lentiviruses ORF control particles (NC), and ORF clone of mouse Lbh (LBH O/E) (20 µL for PBS, 5×104 IFU/mouse for lentiviral particles). The stimulus interval is indicated by a solid bar. (H) Ratios of mean maximal ICP and total ICP (area under the curve) versus MSBP were calculated for each group, and the results are presented as means ± SEM (n = 5). (I and J) Cavernous CD31 (endothelial cell, red), NG2 (pericyte, green), neurofilament (red), and nNOS (green) staining in cavernous tissues from age-matched control (C) and diabetic mice stimulated at 2 weeks after intracavernous injections with lentiviruses ORF control particles (NC) and ORF clone of mouse Lbh (LBH O/E). Scale bars, 100 µm (left), 25 µm (right). (K-N) Quantitative analysis of cavernous endothelial cell, pericyte, and neuronal cell content were quantified by Image J, and results are presented as means ± SEM (n = 4). The relative ratio in the NG or control group was defined as 1. **P < 0.01; ***P < 0.001. DM, diabetes mellitus; PBS, phosphate-buffered saline; MCPs, mouse cavernous pericytes; NG, normal glucose; HG, high glucose; DAPI, 4,6-diamidino-2-phenylindole; MSBP, mean systolic blood pressure; ns, not significant.

LBH as a marker of pericyte in human corpus cavernosum.

(A) Visualization of single cell data from human corpus cavernosum using UMAP. Each cell type is indicated by a different color. EC, endothelial cells; FB, Fibroblasts; SMC, Smooth muscle cell; MFB, Myofibroblast; SWC, Schwann cell; MAC, Macrophage; T, T cell. (B) Expression of marker genes of SMC (ACTA2 and CNN1) and marker genes of Pericyte (RGS5 and LBH). (C) Biological processes identified through gene ontology analysis of clusters annotated as Pericyte, SMC, and MFB. (D) LBH (green) staining in cavernous tissues from two patients with diabetic erectile dysfunction and two patients with congenital penile curvature who had normal erectile function during reconstructive penile surgery. Scale bar, 100 µm. (E) LBH (red) staining in primary cultured human cavernous pericytes under NG and HG conditions for 3 days. (F and G) LBH immunopositive areas were quantified by Image J, and results are presented as means ± SEM (n = 4). Nuclei were labeled with DAPI (blue). The relative ratio in the control or NG group was defined as 1. ***P < 0.001. NG, normal glucose; HG, high glucose; DM, diabetes mellitus; DAPI, 4,6-diamidino-2-phenylindole.

LBH-interacting protein identification in mouse cavernous pericytes.

(A) LBH was immunoprecipitated (IP) from whole-MCPs lysates, resolved on SDS-PAGE gels, and stained with Coomassie blue solution. Gel bands indicated by red frame dot line were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. (B) Co-IP of LBH, CRYAB and Vimentin from whole-MCPs lysates followed by immunoblot analysis to detect CRYAB, Vimentin and LBH. (C and D) Representative images of immunofluorescence staining of LBH (green)/CRYAB (red) and LBH (green)/VIM (red) in normal penis tissues and MCPs. Nuclei were labeled with DAPI (blue). Scale bar, 100 µm (top), 25 µm (bottom). (E) Protein-protein interaction network of LBH, CRYAB, VIM, and 1st and 2nd interactors of LBH. Lines connecting molecules show interaction sources in color. (F) Biological pathways involving molecules in PPI network identified by gene ontology analysis. (G) Significantly enriched gene sets associated with angiogenesis and nerve system in normal pericytes compared to diabetic pericytes in single cell data. (H) Representative western blots for CRYAB and VIM of MCPs under NG and HG conditions (left), and mouse penis tissues from age-matched control and diabetic mice (right). (I) Normalized band intensity ratio of CRYAB and VIM to β-actin was quantified using Image J, and results are presented as means ± SEM (n = 4). The relative ratio in the NG or control group was defined as 1. **P < 0.01; ***P < 0.001. MCPs, mouse cavernous pericytes; NG, normal glucose; HG, high glucose; DM, diabetes mellitus; DAPI, 4,6-diamidino-2-phenylindole.