Single cell transcriptome analysis of cavernous tissues reveals the key roles of pericytes in diabetic erectile dysfunction

Reviewer #1 (Public Review):

In this study, the researchers aimed to investigate the cellular landscape and cell-cell interactions in cavernous tissues under diabetic conditions, specifically focusing on erectile dysfunction (ED). They employed single-cell RNA sequencing to analyze gene expression patterns in various cell types within the cavernous tissues of diabetic individuals. The researchers identified decreased expression of genes associated with collagen or extracellular matrix organization and angiogenesis in several cell types, including fibroblasts, chondrocytes, myofibroblasts, valve-related lymphatic endothelial cells, and pericytes. They also discovered a newly identified marker, LBH, that distinguishes pericytes from smooth muscle cells in mouse and human cavernous tissues. Furthermore, the study revealed that pericytes play a role in angiogenesis, adhesion, and migration by communicating with other cell types within the corpus cavernosum. However, these interactions were found to be significantly reduced under diabetic conditions. The study also investigated the role of LBH and its interactions with other proteins (CRYAB and VIM) in maintaining pericyte function and highlighted their potential involvement in regulating neurovascular regeneration. Overall, the manuscript is well-written and the study provides novel insights into the pathogenesis of ED in patients with diabetes and identifies potential therapeutic targets for further investigation.

Reviewer #2 (Public Review):

Summary: In this manuscript, the authors performed single cell RNA-sequencing of cells from the penises of healthy and diabetes mellitus model (STZ injection-based) mice, identified *Lbh* as a marker of penis pericytes, and report that penis-specific overexpression of *Lbh* is sufficient to rescue erectile function in diabetic animals. In public human single cell RNA-sea datasets, the authors report that *LBH* is similarly specific to pericytes and down regulated in diabetic patients. Additionally, the authors report discovery of CRYAB and VIM1 as protein interacting partners with LBH.

The authors contributions are of interest to the erectile dysfunction community and their *Lbh* overexpression experiments are especially interesting and well-conducted. However, claims in the manuscript regarding the specificity of *Lbh* as a pericyte marker, the mechanism by which *Lbh* overexpression rescues erectile function, cell-cell interactions impaired by diabetes, and protein-interaction partners require qualification or further evidence to justify.

Major claims and evidence:

1. Marker gene specificity and quantification: One of the authors' major contributions is the identification of *Lbh* as a marker of pericytes in their data. The authors present qualitative evidence for this marker gene relationship, but it is unclear from the data presented if *Lbh* is truly a specific marker gene for the pericyte lineage (either based on gene expression or IF presented in Fig. 2D, E). Prior results (see Tabula Muris Consortium, 2018) suggest that *Lbh* is widely expressed in non-pericyte cell types, so the claims presented in the manuscript may be overly broad. Even if *Lbh* is not a globally specific marker, the authors' subsequent intervention experiments argue that it is still an important gene worth studying.
2. Cell-cell communication and regulon activity changes in the diabetic penis: The authors present cell-cell communication analysis and TF regulon analysis in Fig 3 and report differential activities in healthy and DM mice. These results are certainly interesting, however, no statistical analyses are performed to justify claimed changes in the disease state and no validations are performed. It is therefore challenging to interpret these results, and the relevant claims do not seem well supported.
3. Rescue of ED by Lbh overexpression: This is a striking and very interesting result that warrants attention. By simple overexpression of the pericyte marker gene Lbh, the authors report rescue of erectile function in diabetic animals. While mechanistic details are lacking, the phenomenon appears to have a large effect size and the experiments appear sophisticated and well conducted. If anything, the authors appear to underplay the magnitude of this result.
4. Mechanistic claims for rescue of ED by Lbh overexpression: The authors claim that cell type-specific effects on MPCs are responsible for the rescue of erectile function induced by Lbh overexpression. This causal claim is unsupported by the data, which only show that Lbh overexpression influences MPC performance. In vivo, it's likely that Lbh is being over expressed by diverse cell types, any of which could be the causal driver of ED rescue. In fact, the authors report rescue of cell type abundance in endothelial cells and neuronal cells. Therefore, it cannot be concluded that MPC effects alone or in principal are responsible for ED rescue.
5. Protein interaction data: The authors claim that CRYAB and VIM1 are novel interacting partners of LBH. However, the evidence presented (2 blots in Fig. 6A,B) lack the relevant controls. It is possible that CRYAB and VIM1 are cross-reactive with the anti-LBH antibody or were not washed out completely. The abundance of bands on the Coomassie stain in Fig. 6A suggests that either event is plausible. Therefore, the evidence presented is insufficient to support the claim that CRYAB and VIM1 are protein interacting partners of LBH.

**Impact**: These data will trigger interest in Lbh as a target gene within the erectile dysfunction community.

Reviewer #3 (Public Review):

Bae et al. described the key roles of pericytes in cavernous tissues in diabetic erectile dysfunction using both mouse and human single-cell transcriptomic analysis. Erectile dysfunction (ED) is caused by dysfunction of the cavernous tissue and affects a significant proportion of men aged 40-70. The most common treatment for ED is phosphodiesterase 5 inhibitors; however, these are less effective in patients with diabetic ED. Therefore, there is an unmet need for a better understanding of the cavernous microenvironment, cell-cell communications in patients with diabetic ED, and the development of new therapeutic treatments to improve the quality of life.

Pericytes are mesenchymal-derived mural cells that directly interact with capillary endothelial cells (ECs). They play a vital role in the pathogenesis of erectile function as their interactions with ECs are essential for penile erection. Loss of pericytes has been associated with diabetic retinopathy, cancer, and Alzheimer's disease and has been investigated in relation to the permeability of cavernous blood vessels and neurovascular regeneration in the authors' previous studies. This manuscript explores the mechanisms underlying the effect of diabetes on pericyte dysfunction in ED. Additionally, the cellular landscape of cavernous tissues and cell type-specific transcriptional changes were carefully examined using both mouse and human single-cell RNA sequencing in diabetic ED. The novelty of this work lies in the identification of a newly identified pericyte (PC)-specific marker, LBH, in mouse and human cavernous tissues, which distinguishes pericytes from smooth muscle cells. LBH not only serves as a cavernous pericyte marker, but its expression level is also reduced in diabetic conditions. The LBH-interacting proteins (Cryab and Vim) were further identified in mouse cavernous pericytes, indicating that these signaling interactions are critical for maintaining normal pericyte function. Overall, this study demonstrates the novel marker of pericytes and highlights the critical role of pericytes in diabetic ED.