Low-Intensity Pulsed Ultrasound Counteracts Advanced Glycation End Products-Induced Corpus Cavernosal Endothelial Cell Dysfunction via Activating Mitophagy

Injury to corpus cavernosal endothelial cells (CCECs) is an important pathological basis of diabetes mellitus-induced erectile dysfunction (DMED), while low-intensity pulsed ultrasound (LIPUS) has been shown to improve erectile function in DMED. To further understand its therapeutic mechanism of action, in this study, we first demonstrated increased apoptosis and shedding in the CCECs of DMED patients, accompanied by significant mitochondrial injury by immunohistochemistry and electron microscopy of corpus cavernosum tissue. Next, we used advanced glycation end products (AGEs) to simulate the diabetic environment in vitro and found that AGES damaged mitochondria and inhibited angiogenesis in CCECs in a dose-dependent manner, while LIPUS treatment significantly reversed its effects. Mechanistic studies based on transcriptome sequencing showed that LIPUS significantly up-regulated LC3 and PARKIN protein levels in mitochondria, promoted mitophagy, and affected mitochondrial dynamics and reactive oxygen species (ROS) production. In addition, the protective effects of LIPUS were abrogated when mitophagy was inhibited by 3-methyladenine. In summary, LIPUS exerted potent inhibitory effects on AGES-induced CCEC failure via mitophagy, providing a theoretical basis for DMED treatment that encompasses the protection of endothelial structure and function.


Introduction
Diabetes mellitus-induced erectile dysfunction (DMED) is a common complication of diabetes that affects more than half of male diabetes patients, with a prevalence odd of approximately 3.5 times more than healthy men [1]. One of the most important cellular processes underlying pathological damage in diabetes is the increased formation of advanced glycation end products (AGEs). Proteins are modified into harmful AGEs with altered functions when their amino acid constituent groups interact with AGE precursors such as glyoxal, methylglyoxal, and deoxyglucosone [2].
Although the etiology of DMED is multifactorial, endothelial dysfunction is recognized as a key factor in the pathophysiology of the disease [3]. AGEs play an important role in diabetic vasculopathy, and the levels of AGEs are correlated with the severity of diabetic vascular complications [4]. The diabetic state perturbs vascular endothelial function by disrupting the generation and release of vasoactive substances and inhibiting endothelium-derived hyperpolarization [5,6]. Furthermore, AGEs damage vascular

Mitochondrial Dysfunction and Apoptosis Are Observed in the Corpus Cavernosal Endothelium of Dmed Patients
ED is induced by the detrimental actions of high glucose levels on endothelial cells [3]. To obtain an understanding of endothelial dysfunction in diabetes, we examined the corpus cavernosum endothelial cells (CCEC) of normal and DMED patients using scanning electron microscopy (SEM). In normal males, the ECs were neatly arranged with narrow gaps in between cells, resembling a tiled pattern. In contrast, the CC endothelium of DMED patients was recognizably swollen and disorganized, forming a papillary bulge on the surface. Parts of the endothelium had been lost, resulting in exposure of subcutaneous collagen fibers to the vascular lumen ( Figure 1A). We also used transmission electron microscopy (TEM) to examine subcellular structural changes in DMED ECs and found that the mitochondria showed obvious morphological changes, including increased size and decreased density ( Figure 1B). collagen fibers to the vascular lumen ( Figure 1A). We also used transmission electron microscopy (TEM) to examine subcellular structural changes in DMED ECs and found that the mitochondria showed obvious morphological changes, including increased size and decreased density ( Figure 1B). In addition, we used Von Willebrand Factor (VWF) to label ECs in order to investigate the apoptosis ratio between different cell types in DMED patients. VWF-positive DMED CCECs showed a significant increase in the percentage of γH2AX variant histone (γH2AX), a DNA damage double-stranded marker which could be detected during meiosis in germ cells or during apoptosis in non-germ cells, compared with those of normal CC (17.61% in DMED CC vs. 52.00% in normal CC) ( Figure 1C,D). In addition, although VWF-negative cells also showed increased levels of apoptosis in the DMED CC, the overall proportion of apoptotic VWF-negative cells was far lower than that of apoptotic EC cells (6.33% in VWF-negative cells and 16.00% in VWF-positive cells) ( Figure 1C,E). These In addition, we used Von Willebrand Factor (VWF) to label ECs in order to investigate the apoptosis ratio between different cell types in DMED patients. VWF-positive DMED CCECs showed a significant increase in the percentage of γH2AX variant histone (γH2AX), a DNA damage double-stranded marker which could be detected during meiosis in germ cells or during apoptosis in non-germ cells, compared with those of normal CC (17.61% in DMED CC vs. 52.00% in normal CC) ( Figure 1C,D). In addition, although VWF-negative cells also showed increased levels of apoptosis in the DMED CC, the overall proportion of apoptotic VWF-negative cells was far lower than that of apoptotic EC cells (6.33% in VWF-negative cells and 16.00% in VWF-positive cells) ( Figure 1C,E). These results indicated that although the diabetic state damaged all CC cells, ECs were more susceptible than other cells.

Ages Treatment Induces Apoptosis and Dysfunction of Human Ccecs In Vitro
Hyperglycemia caused by diabetes mainly exerts harmful effects by inducing the production of AGES. To further explore the effect of AGES on CCECs, we cultured CCECs with low (20 µg/mL, LAGES group) and high (100 µg/mL, HAGES group) concentrations of AGES. Examination by light microscopy revealed that AGES had dose-dependent effects on the growth rate, cell morphology, and colony characteristics of CCECs. After AGES treatment, cell morphology changed from oval to fibroblast-like, while the growth pattern of cell colonies altered from cobblestone-like to scattered ( Figure 2A); proliferation also decreased with high AGES concentration ( Figure 2B). Moreover, we found that AGES treatment induced apoptosis in CCECs: the addition of AGES at 20 µg/mL increased the early apoptosis ratio from 1.4% to 6.7% (p < 0.001) and increased the late apoptosis ratio from 5.4% to 19.5% (p < 0.001), while the increased AGES concentration (100 µg/mL) led to further increases in the level of apoptosis ( Figure 2C-E).  Examination of DMED CC tissue revealed localized endothelial shedding ( Figure 1A); repair of such injuries depends on the proliferation and migration of adjacent ECs. We found that AGES treatment also inhibited the migratory ability of CCECs through the cell scratch test (63.3% in NC vs. 54.5% in LAGES group vs. 43.0% in HAGES group, p < 0.0001) ( Figure 2F,G). Angiogenesis is another important function of CCECs required for the repair of damage to CC tissues. To test the effects of AGES on the angiogenic ability of CCECs, we performed assays to analyze both tube formation and vascular bud length. Firstly, analysis of tube formation by CCECs showed that total mesh area (positively correlated with tube formation) decreased from 175,209 in NC to 93,784 in LAGES group to 46,784 in HAGES group (p < 0.0001; Figure 2H,I). The second assay examined the spontaneous formation of vascular buds and the lengths of which represent the angiogenic ability of CCECs. We found a decrease in the ratio of vascular bud length to the radius of cell mass in AGES treatment groups (1.4 in NC vs. 1.2 in LAGES group vs. 1.0 in HAGES group, p = 0.0005) ( Figure 2J,K). Taken together, the above results indicated that AGES treatment not only disrupted CCEC function but also caused a loss of cell numbers.

AGES Treatment Perturbs the Transcriptome in Human CCECs
Although the EC dysfunction mediated by AGES has been studied [3], a comprehensive understanding at the molecular level is still lacking. Therefore, we explored the overall changes in the CCEC transcriptome induced by AGES treatment. Using set log 2 fold change > 2 and p < 0.05 as the threshold yielded 417 up-regulated genes and 528 down-regulated genes ( Figure 3A). Consistent with the TEM analysis of DMED CCECs, the transcriptomic changes revealed a significant enrichment of mitochondrial dysfunction and oxidative phosphorylation pathways, indicating that EC mitochondria were impaired in morphology and function. Disruption of energy metabolism and excessive ROS caused by mitochondrial dysfunction are important reasons for cell apoptosis and senescence, which was confirmed by an enrichment of the sirtuin signaling and antioxidant action of vitamin C pathways. Other pathways, such as the airway pathology in chronic obstructive pulmonary disease and immunogenic cell death signaling pathways, indicated abnormal inflammatory reactions in the diabetic state. In addition, endothelin-1 signaling and relaxin signaling were significantly activated and inhibited, respectively, suggesting that the endothelial regulation of smooth muscle contraction and relaxation was abnormal ( Figure 3B).
To further describe the changes in regulatory networks induced by AGES treatment, we used GENIE3 to predict altered networks of transcription factors and their target genes. A further comparison of differentially expressed genes revealed a significant up-regulation of some transcription factors at the central nodes of regulatory networks, such as ZNF580, TCF12, and RFX7, while others, such as HMGB2, MYC, and SMAD4, were significantly down-regulated ( Figure 3C). ZNF580, TCF12, HMGB2, and MYC have been shown to be closely related to angiogenesis in many studies [21][22][23][24]. In addition, Ingenuity Pathway Analysis (IPA) identified an enrichment of the inhibition of angiogenesis by TSP1 pathway in the AGES treatment group ( Figure 3B). Several angiogenesis-related genes showed significantly different expression patterns in the AGES-treated and untreated groups (NC) ( Figure 3D).

LIPUS Treatment Rescues AGES-Induced Impairment of Angiogenesis and Transcriptional Changes in CCECs
In animal models and clinical studies, LIPUS has been shown to improve erectile function and promote structural repair in the CC [16,25]. As AGES treatment significantly disrupted angiogenic function in CCECs, we assayed tube formation and vascular bud growth in CCECs treated with AGES and/or LIPUS to evaluate whether LIPUS stimulation could counteract the effects of AGES treatment. The results showed that LIPUS stimulation promoted CCEC angiogenesis in a dose-dependent manner, with an optimal therapeutic intensity of 100 mW/cm 2 ( Figure 4A-D).

LIPUS Treatment Rescues AGES-Induced Impairment of Angiogenesis and Transcriptional Changes in CCECs
In animal models and clinical studies, LIPUS has been shown to improve erectile function and promote structural repair in the CC [16,25]. As AGES treatment significantly disrupted angiogenic function in CCECs, we assayed tube formation and vascular bud growth in CCECs treated with AGES and/or LIPUS to evaluate whether LIPUS stimulation could counteract the effects of AGES treatment. The results showed that LIPUS stimulation promoted CCEC angiogenesis in a dose-dependent manner, with an optimal therapeutic intensity of 100 mW/cm 2 ( Figure 4A-D).  different doses of LIPUS stimulation under AGES treatment state. The total mesh area score is used to represent the level of tube formation in each group. n = 5 independent repeats. Data are shown as mean ± SD. ns, not significant; **, p < 0.01. (C,D) The radial length of CCEC mass vascular bud change after different doses of LIPUS stimulation with or without AGES treatment. n = 5 independent repeats. Data are shown as mean ± SD. ns, not significant; *, p < 0.05; **, p < 0.01. NC: negative control (0 µg/mL AGES); HAGES: high level of advanced glycation end products (100 µg/mL).
It is worth noting that LIPUS activates downstream signals through mechanical force conduction, while excessive energy may cause cell damage. We stimulated CCECs using LIPUS treatment at 100 mW/cm 2 and performed transcriptomic sequencing to evaluate the molecular effects in each pathway. LIPUS treatment yielded 583 up-regulated genes and 469 down-regulated genes ( Figure 5A). Further comparison among the three groups (untreated, AGES-treated, and AGES+LIPUS-treated) showed that the transcriptomic status of the AGES+LIPUS-treated group was similar to that of the untreated group ( Figure 5B). Furthermore, the selection of the top 2000 genes (by expression level) to perform principal component analysis (PCA) and generate 3D plots confirmed that LIPUS treatment promoted a more normal transcriptional state in AGES-treated CCECs ( Figure 5C). It is worth noting that LIPUS activates downstream signals through mechanical force conduction, while excessive energy may cause cell damage. We stimulated CCECs using LIPUS treatment at 100 mW/cm 2 and performed transcriptomic sequencing to evaluate the molecular effects in each pathway. LIPUS treatment yielded 583 up-regulated genes and 469 down-regulated genes ( Figure 5A). Further comparison among the three groups (untreated, AGES-treated, and AGES+LIPUS-treated) showed that the transcriptomic status of the AGES+LIPUS-treated group was similar to that of the untreated group ( Figure 5B). Furthermore, the selection of the top 2000 genes (by expression level) to perform principal component analysis (PCA) and generate 3D plots confirmed that LIPUS treatment promoted a more normal transcriptional state in AGES-treated CCECs ( Figure 5C). When performing the gene ennrichment analysis base on the differentially expressed genes (DEGs) between AGES-treated and AGES+LIPUS-treated groups, we found pathways related to cellular states such as "Cell aging", "Cell cycle", and "Apoptosis", and pathways related to mitochondria such as "mitochondrion organization", "apoptotic mi- When performing the gene ennrichment analysis base on the differentially expressed genes (DEGs) between AGES-treated and AGES+LIPUS-treated groups, we found path-ways related to cellular states such as "Cell aging", "Cell cycle", and "Apoptosis", and pathways related to mitochondria such as "mitochondrion organization", "apoptotic mitochondrial changes", and "Mitophagy-animal" were significantly enriched ( Figure 5D,E). This result indicated that the LIPUS treatment changed cellular and mitochondrial states via mitophagy or other mechanisms.

LIPUS Restores Mitochondrial Morphology and Reduces Mitochondrial ROS Production Induced by AGES in CCECs
Recent studies have demonstrated that mitophagy eliminates damaged and dysfunctional mitochondria, stabilizes mitochondrial structure and function, reduces mitochondrial ROS production, and maintains cell survival and growth [26,27]. To verify the pathway enrichment results of RNA sequencing, we used MitoSOX™ Red to label mitochondrial ROS and found that the AGES treatment increased the levels of mitochondrial ROS in a dose-dependent manner, while LIPUS stimulation rescued the effect of AGES treatment ( Figure 6A,B).

LIPUS Restores Mitochondrial Morphology and Reduces Mitochondrial ROS Production Induced by AGES in CCECs
Recent studies have demonstrated that mitophagy eliminates damaged and dysfunctional mitochondria, stabilizes mitochondrial structure and function, reduces mitochondrial ROS production, and maintains cell survival and growth [26,27]. To verify the pathway enrichment results of RNA sequencing, we used MitoSOX™ Red to label mitochondrial ROS and found that the AGES treatment increased the levels of mitochondrial ROS in a dose-dependent manner, while LIPUS stimulation rescued the effect of AGES treatment ( Figure 6A,B). Mitochondrial dynamics play an important role in diabetes establishment and progression as well as in associated conditions [28]. We observed that treatment with a high level of AGES (100 µg/mL) induced significant changes in the morphology of CCEC mitochondria, altering their state from fusion to fission, as evaluated by form factor Mitochondrial dynamics play an important role in diabetes establishment and progression as well as in associated conditions [28]. We observed that treatment with a high level of AGES (100 µg/mL) induced significant changes in the morphology of CCEC mitochondria, altering their state from fusion to fission, as evaluated by form factor (indicated the fusion level) (3.0 in untreated group, vs. 1.2 and AGES-treated group, p < 0.0001). However, LIPUS treatment partially reversed this change: in cells treated with 100 µg/mL AGES, stimulation by 100 or 200 mW/cm 2 LIPUS significantly increased the mitochondrial form factor from 1.2 in the AGES-treated group to 2.5 in the AGES+LIPUS-treated group (p = 0.0025) or 2.3 (p = 0.012), respectively ( Figure 6C,D).

LIPUS Activates Mitophagy in CCECs In Vitro
To validate the level of mitophagy, we performed ICC co-staining using Mito-Tracker ® Red and anti-LC3 antibody. LC3 is an autophagy marker involved in the formation of autophagosomes. Although AGES treatment increased the intensity of LC3 staining in the cytoplasm, staining was unchanged in mitochondrial regions. However, LIPUS treatment significantly increased the intensity of LC3 staining in both whole-cell cytoplasmic and mitochondrial regions, indicating activation of both autophagy and mitophagy ( Figure 7A,B). Furthermore, we examined the process of mitophagy by TEM. Under normal culture conditions, the structure of mitochondria in CCECs was intact and their internal morphology was clearly visible. However, while stimulation with AGES induced morphological changes, high levels of lysosomal-mitochondrial fusion were only observed in the LIPUS-stimulated group ( Figure 7C).

LIPUS Activates Mitophagy in CCECs In Vitro
To validate the level of mitophagy, we performed ICC co-staining using Mito-Tracker ® Red and anti-LC3 antibody. LC3 is an autophagy marker involved in the formation of autophagosomes. Although AGES treatment increased the intensity of LC3 staining in the cytoplasm, staining was unchanged in mitochondrial regions. However, LIPUS treatment significantly increased the intensity of LC3 staining in both whole-cell cytoplasmic and mitochondrial regions, indicating activation of both autophagy and mitophagy ( Figure 7A,B). Furthermore, we examined the process of mitophagy by TEM. Under normal culture conditions, the structure of mitochondria in CCECs was intact and their internal morphology was clearly visible. However, while stimulation with AGES induced morphological changes, high levels of lysosomal-mitochondrial fusion were only observed in the LIPUS-stimulated group ( Figure 7C).

Inhibition of Mitophagy Attenuates the Therapeutic Effects of LIPUS
Although we found that LIPUS promoted mitophagy in AGES-treated CCECs, the mechanism of action of LIPUS may be multi-faceted [29,30]. Therefore, we used 3-MA, a macroautophagy/mitophagy inhibitor, to verify the role of mitophagy in LIPUS-mediated protection against AGES-induced endothelial dysfunction. First, we used WB to verify the inhibitory efficiency of 3-MA, and the results showed that LIPUS significantly up-regulated the expression levels of autophagy (LC3 and p62) and mitophagy (PARKIN) proteins in mitochondria, while 3-MA treatment could inhibit this effect ( Figure 8A). We then used common endothelial cell markers (CD31) and CCEC-specific marker (KIT) to explore the effect of LIPUS-promoted mitophagy on the maintenance of endothelial cell status, and found that LIPUS restored the decreased CD31 expression induced by AGES, while 3-MA treatment also inhibited the effect of LIPUS ( Figure 8B).
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Inhibition of Mitophagy Attenuates the Therapeutic Effects of LIPUS
Although we found that LIPUS promoted mitophagy in AGES-treated CCECs, the mechanism of action of LIPUS may be multi-faceted [29,30]. Therefore, we used 3-MA, a macroautophagy/mitophagy inhibitor, to verify the role of mitophagy in LIPUS-mediated protection against AGES-induced endothelial dysfunction. First, we used WB to verify the inhibitory efficiency of 3-MA, and the results showed that LIPUS significantly up-regulated the expression levels of autophagy (LC3 and p62) and mitophagy (PARKIN) proteins in mitochondria, while 3-MA treatment could inhibit this effect ( Figure 8A). We then used common endothelial cell markers (CD31) and CCEC-specific marker (KIT) to explore the effect of LIPUS-promoted mitophagy on the maintenance of endothelial cell status, and found that LIPUS restored the decreased CD31 expression induced by AGES, while 3-MA treatment also inhibited the effect of LIPUS ( Figure 8B).  on the early apoptosis (B) and late apoptosis (C) of CCECs after LIPUS treatment. The existence of Annexin V (X-axis) and the nuclear staining of PI (Y-axis) by flow cytometry are shown. n = 5 independent repeats. Data are shown as mean ± SD. ns, not significant; *, p < 0.05. (F,G) Phalloidin staining shows typical changes of CCECs tube formation after 3-MA and/or LIPUS treatment. The total mesh area score is used to represent the level of tube formation in each group. n = 5 independent repeats. Data are shown as mean ± SD. ns, not significant; *, p < 0.05. AGES: advanced glycation end products; LIPUS: low-intensity pulsed ultrasound; 3-MA: 3-methyladenine.
When focused on cellular state and function CCECs, LIPUS reduced AGES-induced apoptosis in CCECs; this therapeutic effect was significantly inhibited by 3-MA treatment ( Figure 8C-E). Tube formation ability was similarly affected: combining LIPUS with 3-MA treatment resulted in a lower total mesh area than LIPUS treatment alone, and the results showed no significant difference when compared with the AGES-treated group ( Figure 8F,G).

Discussion
The incidence of diabetes and its complications is increasing year by year, and it is estimated that by 2025, there will be 300 million patients worldwide [31]. Diabetics are three times more likely to develop ED, a common complication of diabetes [32]. Although the pathogenesis of diabetic ED is multifactorial, endothelial dysfunction is considered to be an important pathological basis for this condition [33]. Insufficient production of nitric oxide in the ECs of diabetic patients inhibits the mechanism of cGMP-mediated smooth muscle relaxation and causes insufficient blood supply to the CC. Furthermore, in the diabetic state, apoptosis of ECs is increased, while the expression of cell connexins such as GJA1, CLDN5, and ZO-1 is decreased, leading to vascular leakage and failure to maintain erections [34]. In addition to impairing endothelial function, long-term diabetes can also cause extensive organic changes in the CC tissue, mainly manifested by morphological changes, decreased numbers of ECs, and increased levels of collagen [9].
Chronically elevated glucose levels have been reported to induce the formation of AGES, which are known to interfere with endothelial nitric oxide production by directly inhibiting the phosphorylation of endothelial nitric oxide synthase (eNOS) [35]. Previous studies have generally focused on the effect of AGES or high glucose on the endotheliumsmooth-muscle-erectile axis. However, EC function and survival also affect the stability of the CC structure, which may be the reason why many diabetic ED patients do not respond well to phosphodiesterase type 5 inhibitors (PDE5I) [36]. Bioinformatic analysis based on proteomics studies carried out on human umbilical vascular endothelial cells (HUVECs) revealed that the differentially regulated proteins were involved in various processes such as apoptosis and oxidative stress [37]. However, as our previous studies have shown that CCECs are more susceptible to diabetic damage than the vascular endothelium [9], studies based on HUVECs may not accurately reflect the diabetic conditions of CCECs and the cause of DMED. Therefore, we comprehensively evaluated the effects of AGES treatment on CCECs using RNA-seq and identified several highly enriched terms related to mitochondria. In addition, electron microscopy of CC tissue from patients as well as analysis of cultured CCECs showed apoptosis, shedding, and mitochondrial structural disorders of CCECs to be important features of DMED. Indeed, mitochondrial damage, increased ROS levels, and endothelial dysfunction induced by AGES stimulation are likely to be continuous changes caused by the same pathological process [38]. Therefore, when considering treatment for DMED, in addition to controlling blood glucose and AGES at the source, protecting and repairing the mitochondrial function of CCECs may have a more direct therapeutic effect.
Mitophagy is an autophagic response that specifically targets damaged, and hence, potentially cytotoxic, mitochondria. In recent years, mitophagy has been proven to play a protective role against the development of insulin resistance and diabetic conditions in cardiovascular, muscle, adipose, and kidney tissue [13,14,39]. Current studies indicate that LIPUS, which transmits mechanical energy, can safely and effectively treat ED, resulting from a variety of causes, and can reverse the pathological changes of the CC [16,40,41]. LIPUS also potently activates PINK1/Parkin-dependent mitophagy and plays a role in promoting β-cell resistance to diabetes-related hyperlipotoxicity [41]. Our study found that LIPUS promoted mitophagy and reduced mitochondrial ROS production as well as mitochondrial fission in a dose-dependent manner. In addition, LIPUS promoted CCEC angiogenesis, which is essential for the equilibrium between injury and endogenous regeneration capacity; disturbance of this balance in favor of the former results in the irreparable loss of the functional integrity of the endothelium and the exacerbation of vascular disease [42]. In addition, the mitophagy inhibitor 3-MA attenuated the protective effect of LIPUS in CCECs pre-treated with AGES, demonstrating that the effect of LIPUS was mitophagy-dependent. However, as a mechanism of cellular self-protection, the specific process by which LIPUS stimulates mitophagy requires further investigation.
However, there are some limitations to this study. For example, all CC tissue was derived from penile cancer patients. Although these patients had normal erectile function and tumor tissue was avoided during sampling, tumor compression or paracrine effects may still affect adjacent normal tissues, especially the morphology and function of CCEC. So, future tissue from healthy donors remains essential to confirm the conclusions of this study. Another limitation is that because DMED CC tissue is very precious and difficult to obtain, there were only two repeats in the Figure 1 part of our study. Although the conclusions of this part were verified by subsequent in vitro experiments and functional experiments, it is also necessary to further expand the sample size for verification.
In summary, we demonstrated that LIPUS restored AGES-induced CCEC damage by reducing apoptosis and promoting angiogenesis via protecting mitochondrial structure and function. LIPUS activated mitophagy by upregulating mitochondrial LC3, p62, and PARKIN, reduced levels of mitochondrial ROS, and blocked the AGES-induced changes in the mitochondrial state from fusion to fission. Therefore, our study provides theoretical support for LIPUS-based therapy and paves the way for further in vivo studies to explore improved treatment options for DMED.

Contact Information for Reagent and Resource Sharing
Requests for further information, resources, and reagents should be directed to and will be fulfilled by the Lead Contact or the first author, Yuzhuo Chen (chenyzh223@mail.sysu.edu.cn).

Experimental Models and Subject Details
The experiments performed in this study were approved by the Ethics Committee of The Fifth Hospital Affiliated Sun Yat-sen University (License No. 2022-L170-1). CC samples were obtained from the tumor margin of penile carcinoma resection, all patients reported good stimulated erections and early morning erections, under the age of 50. LIPUS Device (WBL-ED, ED-ZLT-F treatment head) was from Wanbeili Medical Device Co., LTD., Beijing, China. In the CCECs in vitro experiments, CCECs were treated with low (20 µg/mL, LAGES group) or high (100 µg/mL, HAGES group) concentrations of AGES with or without 10 mM 3-MA for 48 h [43][44][45], then, were stimulated with LIPUS three times, 5 min each time with a 1 min interval, according to previous studies [20,25].

Isolation and Culture of CCECs
Fresh CC tissue samples of approximately 1 × 1 cm size were first immersed in 15 mL PBS with vigorous shaking to remove residual blood cells. Then, tissues were cut into pieces of 1 × 1 mm size and enzymatically digested with 2.5 mg/mL collagenase type I (17104-019, gibco, US), 4 mg/mL collagenase type IV (17018029, gibco), 0.1 mg/mL neutral protease, and 2 mg/mL DNase I (#AMPD1, Sigma-Aldrich, US) at 37 • C for 20 min. Subsequently, the cell suspension was filtered through a 40 mm nylon mesh, and the cells were sorted by MACS with a Dead Cell Removal Kit (130-090-101, Miltenyi Biotec, Germany) to remove dead cells. Briefly, 10 6 cells were co-incubated with 20 µL MicroBeads at 4 • C for 30 min. After blocking and washing, magnetic separation with LS Columns (130-042-401; Miltenyi) and MidiMACS Separator (130-042-302; Miltenyi) was performed, retaining cells that were not adsorbed by the column. CCECs were cultured with EGM-2 medium and cell cloning began at approximately day 5 of culture, then fibroblast inhibitors were added for 7-10 days. When isolated colonies began to fuse with each other, the EC colonies were marked and digested using a clonal cylinder (C7983-50EA; Sigma-Aldrich), and the EC suspension could be further purified using CD31 magnetic beads (according to their purity) (130-091-935; Miltenyi).

Immunocytochemistry (ICC) and Immunohistochemical (IHC) Staining
The CCECs with the ability of adherence growth were cultured in the cell slide. After treatment, cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA). The cells were blocked with 5% bull serum albumin (BSA) for 1 h and incubated with relevant primary antibodies overnight at 4°C. These cells were further incubated with secondary antibodies for 2 h. Cell nuclei were labelled by Hoechst33342.
Sections were performed with rehydration, antigen retrieval, and blocking, and then incubated with appropriate primary antibodies at 4 • C overnight. Sections were further incubated with a secondary antibody for 2 h at room temperature. Nuclei were labelled with Hoechst33342 by incubating tissue sections for 15 min [46]. Images were captured with an OLYMPUS IX83 confocal microscope. Fluorescence intensity measurements were processed with Adobe Photoshop CC 2018 software v 23.0.2.

Endothelial Cell Functional Experiments
Cell migration: CCECs were seeded in a 6-well plate at a concentration of 1 × 10 6 per well. After two days, all cells completed contact with each other, and AGES and/or LIPUS treatment were performed according to the group. After 24 h, a 10 µL pipetting gun tip was used to make scratches and then the shed cells were removed. The results were observed 12 h later. The area ratio of cell migration regions and whole scratch regions in each group were recorded and counted.
Vascular bud length: CCECs were seeded in AggreWell (34411, STEMCELL, Canada) at a concentration of 3 × 10 5 per well; after 12 h, the cell mass was re-suspended and seeded on µ-Slide (81506, ibidi) with the pre-added 10 µL of Matrigel (256230, Corning, US) for 6 h. Next, cell supernatant was replaced with conditioned medium (Lonza, CC-3202, Switzerland) for 24 h and fixed. The ratio of cell mass diameter to the distance from the center of the cell mass to the farthest end of the vascular bud in each group was recorded and counted.

Cell Apoptosis Analysis
Annexin V-FITC apoptosis detection kit (A211-01, Vazyme Biotech, China) was used to measure cell apoptosis according to the manufacturer's protocol. In brief, CCECs were collected by EDTA-free trypsin, washed with ice-cold phosphate-buffered saline twice, and resuspended in 100 µL of binding buffer. Next, the cells were incubated with 5 µL of Annexin V-FITC and 5 µL of PI staining solution in the dark for 10 min, and then diluted with binding buffer to 500 µL. Cell apoptosis was subsequently analyzed by flow cytometry (Beckman Coulter, Inc., Brea, CA, USA); the fluorescence signals of Annexin V and PI conjugate were detected in fluorescence intensity channels B525-FITC and Y585-PE without a compensation set, respectively. The acquisition speed was 60 µL/min. The apoptotic rates were measured by FlowJo software, version 10.6.1 (FlowJo LCC, Becton Dickinson, Ashland, OR, USA).

RNA-Seq of CCECs In Vitro
The 1 × 10 5 CCECs cells were seeded in a six-well microplate. After 24 h, AGES at a final concentration of 100 µg/mL was added or not, and a standard dose of LIPUS stimulation was performed or not. The medium was changed every other day during culturing. After 2 days, all cells were harvested with TRIzol (15596026; Thermo Fisher) for mRNA sequencing. Library construction and sequencing were performed by Sinotech Genomics Co., Ltd. (Shanghai, China). Total RNA was isolated using RNeasy mini kit (74904; Qiagen). The poly(A)-containing mRNA molecules were purified using poly(T)-oligo-attached magnetic beads. Then, the mRNA underwent fragmentation, reverse transcription, purification, and enrichment. Clusters were generated by cBot with the library diluted to 10 pM, and then were sequenced on the Illumina NovaSeq 6000 (Illumina, USA). Paired-end sequence files (fastq) were mapped to the reference genome (GRh38-1.2.0) using HISAT2. Differential expression analysis for mRNA was performed using the R package edgeR. Differentially expressed RNAs with |log 2 (FC)| value > 1 and q value < 0.05, considered significantly modulated, were retained for the volcano plot and GO analysis.

Statistics and Reproducibility
Data in Figures 2D,F,H,J; 4B,D; 6D,E,F; and 7B,C,E were shown as mean ± SD. Data were first checked for normality with the D'Agostino-Pearson and Kolmogorov-Smirnov test, and statistical significance was calculated with the Kruskal-Wallis analysis and Dunn's test (for the result in Figures 2D,F,H,J, 4B,D, 6D,E,F and 7B,C,E unpaired, two-tailed), and the chi-square test ( Figure 1D,E) by GraphPad Prism 9.0.0 software. The confidence interval was 95%. Results were considered significant at p < 0.05. Statistical parameters are reported in the respective figures and figure legends. IHC and ICC staining for each disease type in this study were based on at least two biological repeats (five penile tumor patients with normal erectile function and two DMED patients). Scanning electron microscope and transmission electron microscope image ( Figure 1A,B) were based on two biological repeats.