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Asian Journal of Andrology logoLink to Asian Journal of Andrology
. 2026 Mar 17;28(5):516–525. doi: 10.4103/aja202569

Phenotypic modulation of corpus cavernosum smooth muscle cells in type 2 diabetic rats is mediated by regulated exosomes secretion via neutral sphingomyelinase-2

Feng-Zhi Chen 1,2,*, Xin-Tao Zhang 1,2,*, Qin-Yu Zeng 3, Bing-Xin Lu 4, Li Wang 5, Hong-Bing Mei 1,2,6,✉, An-Yang Wei 4,✉
PMCID: PMC13623336  PMID: 41846426

Abstract

Phenotypic modulation of corpus cavernosum smooth muscle cells (CCSMCs) is closely related to the occurrence of erectile dysfunction (ED). However, the effect of exosomes (EXOs) on the phenotypic modulation of CCSMCs with diabetes mellitus (DM)-related ED is not fully understood. Using a rat model of type 2 DM, we found an important role for phenotypic modulation of CCSMCs in DM-related ED. EXO secretion by CCSMCs is also driven by pathological changes to the corpus cavernosum in diabetic rats. We used CCSMCs subjected to platelet-derived growth factor-BB as an in vitro model of phenotypic modulation and isolated EXOs from the supernatants of cultured CCSMCs by ultracentrifugation. The results suggest that phenotypic modulation enhances the capacity of CCSMCs to secrete EXOs and that inhibiting EXO secretion could restore the contractile phenotype of CCSMCs in vitro. Notably, a further mechanistic study revealed that regulated EXO secretion could change the phenotype of CCSMCs via the neutral sphingomyelinase-2 (nSMase2) pathway. Taken together, our results indicate that the modulation of EXO biogenesis and secretion may be a novel therapeutic approach to improve type 2 DM-induced ED by maintaining the contractile phenotype of CCSMCs.

Keywords: corpus cavernosum smooth muscle cells, exosomes, neutral sphingomyelinase-2, phenotypic modulation

INTRODUCTION

Men with erectile dysfunction (ED) have a reduced quality of life. Initiating and maintaining an erection involves neurological, hormonal, and vascular mechanisms.1,2 The corpus cavernosum is very similar to the vascular system and is thus considered a specialized vascular bed. Corpus cavernosum smooth muscle cells (CCSMCs) form the basic structure of the erectile process of the penis, playing a critical role in hemodynamic changes during penile erectile activity.3 These cells are influenced by various factors, and any factor causing damage to the CCSMCs may lead to ED.4,5

The phenotypic modulation of CCSMCs can alter the normal structure and function of the corpus cavernosum, which is closely related to the occurrence of ED.6,7 CCSMCs are divided into two types, the contractile and synthetic phenotypes, similar to vascular smooth muscle cells (VSMCs). CCSMCs exhibit remarkable plasticity and change their phenotype based on local environmental variations, such as diabetes mellitus (DM), hypoxic conditions, and cavernous nerve damage. Switching their phenotype from a contractile state to a synthetic state is also called the phenotypic modulation of CCSMC. Phenotypic modulation in CCSMCs can reduce the contraction and relaxation capacity of cells, increase the synthesis of extracellular matrix, and even cause cavernous fibrosis and hemodynamic changes of the penis. Thus, phenotypic modulation of CCSMC plays a crucial role in the development of ED. Our previous study demonstrated that in rats with type 1 DM-associated ED, CCSMCs not only underwent apoptosis and fibrosis but also underwent phenotypic modulation.8

Exosomes (EXOs), secreted by cells such as smooth muscle cells and mesenchymal stem cells, have recently received increased attention. EXOs are defined as the lipid bilayer nanovesicles of 30–200 nm in diameter. Multivesicular bodies directly fuse with the plasma membrane, releasing intraluminal vesicles into the extracellular environment as EXOs containing numerous microRNAs, mRNAs, and proteins.9 EXO shuttling has been identified as a novel avenue for cell-to-cell communication by delivering complex cargos of signaling molecules to recipient cells. Currently, accumulating evidence suggests that EXOs may have a wide range of biological activities, including cell survival, proliferation and differentiation, immunomodulation, and tissue regeneration.10,11,12,13 However, whether EXOs contribute to the phenotypic modulation of CCSMCs in DM-related ED remains to be clarified.

Neutral sphingomyelinase-2 (nSMase2) is a member of the nSMase family, which is a key enzyme in EXO secretion and functions in various cell types. Specifically, the activity of nSMase2 plays a significant role in EXO biogenesis, cargo selection, and the fate of multivesicular bodies. Consequently, it directly impacts the bioactivity of EXOs.14,15 nSMase2 is activated in vascular cells by tumor necrosis factor-alpha,16 shear stress,17 and oxidative stress18 and may contribute to endothelium activation,19 inflammation,20 and vascular calcification.21 However, as we have known so far, few studies have yet explored the role played by nSMase2 in CCSMC phenotypic modulation.

In this study, we explored the effect of regulated EXO secretion on phenotypic modulation of CCSMCs with type 2 DM-related ED. Performing in vitro experiments to determine the impacts of platelet-derived growth factor-BB (PDGFBB), a known synthetic phenotype stimulator, on EXO secretion and phenotypic modulation of CCSMC. We aimed to provide insights into the role of regulated EXO secretion in phenotypic modulation of CCSMC with type 2 DM-related ED and elucidate the underlying mechanisms.

MATERIALS AND METHODS

Animal subjects and treatments

Six-week-old male Sprague–Dawley rats were obtained from the Laboratory Animal Center of Southern Medical University (Guangzhou, China). Their breeding and treatment protocols were approved by the Ethics Committee of Nanfang Hospital, Southern Medical University (Guangzhou, China; Approval No. IACUC-LAC-20220916-005).

Following our established protocol,22 rats underwent a one-week acclimatization before being fed a high-fat diet (D12451: 24% fat, 41% carbohydrate, 24% protein; total calorific value: 4.73 kcal g−1; Stoyer Center of Experimental Animal Holding, Guangzhou, China) for four weeks. They then received two intraperitoneal injections of streptozocin (STZ; 30 mg kg−1, Sigma-Aldrich, St. Louis, MO, USA), one week apart. One week after the second STZ injection, random blood glucose levels were measured with a glucose meter (Roche, Mannheim, Germany) by tail tip snipping. Blood glucose readings above 16.7 mmol l−1 indicated the presence of DM. The weights of the rats were measured every two weeks, starting on the first day of the high-fat diet. An intraperitoneal insulin tolerance test was conducted eight weeks after STZ injection to determine insulin resistance. Concurrently, fasting serum glucose and lipid profiles were analyzed using a biochemical reagent kit (Nanjing Jiancheng Chemical Industrial Co., Ltd., Nanjing, China), while insulin levels were measured by an enzyme-linked immunosorbent assay (Mercodia Co., Uppsala, Sweden). The erectile function of rats with DM was assessed ten weeks after the STZ injection. Additionally, 10 age-matched rats without DM and subjected to sham surgery were used as the normal control (NC) group.

Erectile function assessment in vivo

Erectile function was evaluated using our established method.22 Anesthesia was induced before the right major pelvic ganglion was dissected and exposed. Then, a bipolar stainless-steel hook electrode, connected to the signal generator (MP150; BIOPAC Systems Inc., Goleta, CA, USA), was placed on the right cavernous nerves to stimulate penile erection. The electrical stimulation pattern was set to 5 V, 10 Hz, a 5-ms pulse width, and a 50-s duration. Concurrently, a 23-gauge needle with heparin was inserted into the proximal right corpus cavernosum to measure the intracavernous pressure (ICP). Subsequently, a 20-gauge cannula was inserted into the left carotid artery to monitor the systemic mean arterial pressure (MAP). The ratio of ICP to MAP (ICP/MAP) was recorded for each rat.

Histological examination

Masson’s trichrome staining and immunohistochemistry were performed according to established protocols and previous reports. The antibodies included anti-alpha-smooth muscle actin (anti-α-SMA; 1:100; Abcam, Cambridge, UK), anti-smoothelin (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-myosin heavy chain 11 (anti-Myh11; 1:50; Santa Cruz Biotechnology), anti-osteopontin (anti-OPN; 1:100; Abcam), and anti-collagen I (1:250; Proteintech, Wuhan, China). Digital images of Masson’s trichrome staining and immunohistochemistry were acquired with an Olympus microscope (Olympus Corporation, Tokyo, Japan). The percentage of positive areas and the smooth muscle-to-collagen ratio were calculated using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Double fluorescence staining was performed according to established protocols. The sections were blocked for 1 h, then incubated overnight with the mixture of primary antibodies–anti-CD63 (1:50; Santa Cruz Biotechnology) and anti-α-SMA (1:100; Proteintech); or the mixture of primary antibodies–anti-nSMase2 (1:50; Santa Cruz Biotechnology) and anti-α-SMA (1:100; Proteintech). Subsequently, the sections were immersed in fluorescent secondary antibodies (1:50 each; goat anti-mouse or goat anti-rabbit; Invitrogen, Carlsbad, CA, USA) for 1 h. Digital images were captured using a fluorescence microscope (Olympus Corporation).

Cell culture and analysis

CCSMCs were isolated and cultured using the explant method as previously established.8,23 Rat penile cavernous tissue explants (1–2 mm3) were placed into culture flasks at a spacing of 0.5 cm. Then, 2.5 ml of prepared Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Gaithersburg, MD, USA; containing 10% fetal bovine serum, 100 U ml−1 penicillin, and 100 U ml−1 streptomycin) was added to the culture flasks, and the flasks were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. On the third day, CCSMCs began to migrate from the explants. As PDGFBB is a well-established stimulator for inducing VSMC phenotypic modulation, we utilized it to promote in vitro phenotypic modulation of CCSMCs, as previously reported.23 Then, CCSMCs were pretreated with different concentrations of GW4869 (0, 0.1 µmol l−1, 1 µmol l−1, and 10 µmol l−1; Sigma-Aldrich) for 12 h, followed by the stimulation with PDGFBB (20 ng ml−1; Peprotech, Rocky Hill, CA, USA) for 48 h, while the NC group received no treatment. Cells were then harvested for further studies. All the following cell experiments were performed in triplicate.

Isolation and characterization of CCSMC-derived EXOs

As described in our previously published protocol,22 on reaching 80% cell confluence, the conventional culture medium was substituted with a serum-free medium, and CCSMCs were cultured for another 48 h. Subsequently, the medium was collected, and EXOs were isolated using a multistep centrifugation protocol (300g for 10 min, 2000g for 20 min, 10 000g for 20 min, and 110 000g for 70 min; Beckman Coulter Optima L-90K ultracentrifuge; Beckman Coulter, Fullerton, CA, USA). The EXO pellets were resuspended in phosphate-buffered saline (PBS). Western blot was used to determine the CD63, CD81, and calnexin protein levels, and transmission electron microscopy (H7500 TEM; Hitachi, Tokyo, Japan) was used to determine the morphology and ultrastructure of the EXOs.

Nanoparticle trafficking analysis

Assessments of the absolute concentration and size distribution of the EXOs were conducted using NanoSight NS300 (Malvern Instruments Ltd., Malvern, UK). Nanoparticle tracking analysis (NTA) automatically tracks and measures sizes particles based on Brownian motion and diffusion coefficients. After isolation, EXOs were diluted in 1 ml of filtered PBS. Control medium and filtered PBS were used as controls. The detection threshold was similar for all the samples.

Immunofluorescent staining of CCSMCs

Cells were fixed in 4.0% paraformaldehyde in PBS for 10 min at room temperature, followed by a 5-min wash with PBS before permeabilization with 0.25% Triton X-100 (MP Biomedicals, Irvine, CA, USA) in PBS for 10 min. The cells were then incubated in blocking buffer (containing 10% goat serum) for 30 min at room temperature prior to incubation with anti-nSMase2 (1:50; Santa Cruz Biotechnology) overnight at 4°C. After multiple washes with PBS, the coverslips were incubated with fluorescent secondary antibodies (Invitrogen) for 30 min at 37°C. For the negative control group, only the secondary antibodies were added, without primary antibodies. Finally, the coverslips containing the CCSMCs were examined by fluorescence microscopy (Olympus Corporation).

Cell counting kit-8 (CCK-8) cell proliferative assay

Cell viability was determined by measuring the metabolism of a CCK-8 assay (Dojindo, Kumamoto, Japan). Briefly, after the cells were incubated with the different treatments, 10 µl of sterile CCK-8 solution was added to each well for 2 h at 37°C. The optical density (OD) values were measured with a microplate reader (SpectraMax M5; Molecular Devices, Sunnyvale, CA, USA) at 450 nm.

Scratch wound-healing assay

Cells were wounded with a 200 µl pipette tip and incubated in serum-free medium. Images of the leading edge of the scratch were captured by light microscopy (Olympus Corporation) at specified time points. The scratch closure area was calculated as follows: migration area = (A0 − An), where A0 represents the initial wound area and An represents the remaining wound area at the metering point. The migration area of CCSMCs was quantified using ImageJ software (Analyze-Measure plugins; National Institutes of Health, Bethesda, MD, USA).

Quantitative real time polymerase chain reaction (qRT-PCR) analysis

Total RNA was isolated from CCSMCs and used for qRT-PCR analyses. Primer sequences were as follows: β-actin, 5′-GATCAAGATCATTGCTCCTCCTG-3′ (sense), 5′-AGGGTG TAAAACGCAGCTCA-3′ (anti-sense); α-SMA, 5′-TTCAATGT CCCTGCCATGTA-3′ (sense), 5′-CATCTCCAGAGCCAGCACA-3′ (anti-sense); smoothelin, 5′-TCAAGCAGATGTTGCTGGAC-3′ (sense), 5′-ACAGAAAGCCATCCATCAC-3′ (anti-sense); Myh11, 5′-GATGTGGTGCAGAAAGCTCA-3′ (sense), 5′-TGAGA ATCCATCGGAAAAGG-3′ (anti-sense); OPN, 5′-CCAGCCAAG GACCAACTACA-3′ (sense), 5′-AGTGTTTGCTGTAATGCGCC-3′ (anti-sense); and collagen I, 5′-ATCAGCCCAAACCCCAAGGAGA-3′ (sense), 5′-CGCAGGAAGGTCAGCTGGATAG-3′ (anti-sense).

Western blot analysis

Cells and tissue were harvested and lysed for preparation, and then, the total protein concentration was measured by a bicinchoninic acid (BCA) assay kit. After heat denaturation, equal amounts of protein extracts were run on a 10% or 12% tris–glycine gradient gel and then used for western blot analysis. The antibodies included anti-α-SMA (1:3000; Proteintech), anti-smoothelin (1:500; Santa Cruz Biotechnology), anti-Myh11 (1:500; Santa Cruz Biotechnology), anti-OPN (1:1000; Abcam), anti-collagen I (1:1000; Proteintech), and anti-nSMase2 (1:500; Santa Cruz Biotechnology). The band intensity was quantified using ImageJ software (National Institutes of Health) with β-actin as a control.

Statistical analyses

Data were expressed as mean ± standard deviation (s.d.). Comparisons between two or more groups were performed using the mean of the unpaired Student’s t-test or analysis of variance (ANOVA), respectively, followed by the least significance difference (LSD) test for multiple comparisons or the Dunnett T3 test for samples with unequal variances. P < 0.05 was considered statistically significant.

RESULTS

Body weight and serum concentration

The body weights of rats after STZ injection in the DM group decreased slightly but remained stable (Figure 1a). The insulin tolerance test showed that diabetic rats lacked a response to insulin compared with the NC rats (all P < 0.05; Figure 1b). The levels of serum glucose, triglycerides, and total cholesterol significantly increased, but the level of serum insulin significantly decreased in diabetic rats by the eighth week after STZ injection (all P < 0.05; Supplementary Figure 1 (57KB, tif) ).

Figure 1.

Figure 1

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Body weight, insulin challenge test, and erectile function evaluation of the rats (n = 10 animals per group). (a) Body weight was measured every two weeks. (b) Insulin challenge test carried out 8 weeks following the first STZ injection. (c) Representative recordings of ICP in response to electrostimulation of the cavernous nerve and MAP in NC and DM groups (voltage: 5 V). (d) A bar graph of the ratio of the voltage-dependent ICP to MAP. *P < 0.05. ICP: intracavernous pressure; MAP: mean arterial blood pressure; NC: normal control rats; DM: rats with diabetes mellitus; STZ: streptozocin.

Erectile function assessment in vivo and histological changes

Representative traces of the ICP from the electrical cavernous nerve stimulation are shown in Figure 2a. The ICP/MAP ratio was significantly lower in diabetic rats than that in NC rats (P < 0.05; Figure 1c and 1d). The smooth muscle and collagen contents were evaluated by Masson’s trichrome staining. The smooth muscle-to-collagen ratio in diabetic rats decreased compared with that in NC rats (P < 0.05; Supplementary Figure 2 (161.7KB, tif) ).

Figure 2.

Figure 2

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Phenotypic modulation of CCSMCs occurred in type 2 diabetic rats. (a) Representative immunohistochemical staining images of phenotype-associated proteins (α-SMA, smoothelin, Myh11, and OPN) and collagen I in penile tissues in each group. (b) The bar graph represents the relative density of phenotype-associated proteins and collagen I in penile tissues in each group. (c) Western blot analyses of phenotype-associated proteins and collagen I expression levels in penile tissues in each group. (d) The bar graph represents the relative quantification of phenotype-associated proteins and collagen I compared with that of β-actin in penile tissues in each group. *P < 0.05. CCSMCs: corpus cavernosum smooth muscle cells; NC: normal control rats; DM: rats with diabetes mellitus; α-SMA: alpha-smooth muscle actin; Myh11: myosin heavy chain 11; OPN: osteopontin; protein+ area: the area of protein positive staining.

Phenotypic modulation of CCSMCs in rats with type 2 DM

To investigate phenotypic modulation in the CCSMCs of diabetic rats, we examined the expression levels of phenotype-associated proteins and collagen I by western blot and immunohistochemical staining. The results of the immunohistochemical experiment showed that, in comparison to the NC group, the DM group exhibited a significant reduction in the protein levels of α-SMA, smoothelin, and Myh11. Conversely, there was a notable increase in the levels of OPN and collagen I in the DM group (all P < 0.05; Figure 2a and 2b). Western blot analysis also showed that the expression levels of α-SMA, smoothelin, and Myh11 were significantly downregulated in diabetic rats, while the expression levels of OPN and collagen I were significantly upregulated (all P < 0.05; Figure 2c and 2d). These results collectively indicate that phenotypic modulation occurs in the CCSMCs of diabetic rats, leading to enhanced collagen synthesis by these cells.

EXOs are detectable in the corpus cavernosum tissue of rats with type 2 DM

Immunofluorescence staining of the corpus cavernosum showed that the exosomal marker CD63 was present extensively in the penile tissue of diabetic rats. Moreover, double fluorescence staining showed significant CD63 expression in the CCSMCs of the corpus cavernosum tissue (Figure 3). These results show the presence of EXOs in the CCSMCs of rats with type 2 DM.

Figure 3.

Figure 3

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EXOs were observed in CCSMCs of the penile tissue of type 2 diabetic rats. Representative double fluorescence staining images of CD63 and α-SMA in corpus cavernosum tissue of type 2 diabetic rats. EXOs: exosomes; CCSMCs: corpus cavernosum smooth muscle cells; α-SMA: alpha-smooth muscle actin.

Effect of phenotypic modulation on EXO secretion in CCSMCs

We first investigated whether EXOs could be secreted by CCSMCs in vitro. Transmission electron microscopy showed that EXOs isolated from CCSMCs had a typical cup-shaped morphology and were less than 200 nm in diameter (Figure 4a). The western blot assay suggested that CCSMC-derived EXOs were enriched with exosomal markers such as CD63 and CD81 (Figure 4b). NTA, immunofluorescence, and western blot were then performed to explore the effects of phenotypic modulation on EXO secretion in CCSMCs. NTA analysis suggested that treatment of CCSMCs with PDGFBB resulted in a significant increase in EXO secretion (mean ± s.d.: 1.1×109 ± 0.2×109 vs 4.5×108 ± 0.9×108; Figure 4c). Given the role of the nSMase2 pathway in EXO biogenesis, we examined the expression of nSMase2 in CCSMCs as an indicator of their ability to secrete EXOs. CCSMCs treated with PDGFBB showed a significantly increased expression level of nSMase2 compared with the NC group (P < 0.05; Figure 4d–4f), verified by immunofluorescence and western blot. In addition, double fluorescence staining of corpus cavernosum tissue showed that the expression level of nSMase2 was considerably increased in the CCSMCs of diabetic rats compared with those of the NC group, despite the presence of fewer CCSMCs (Figure 5). These results indicate that phenotypic modulation enhances the capacity of CCSMCs to secrete EXOs both in vitro and in vivo.

Figure 4.

Figure 4

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Phenotypic modulation enhances the capacity of CCSMCs to secrete EXOs in vitro. (a) Representative transmission electron micrographs of CCSMCs-derived EXOs with a cup-shaped morphology. (b) Western blot results indicating positive expression for the CD63 and CD81 proteins in EXOs derived from CCSMCs but negative expression for calnexin, a protein found in the endoplasmic reticulum and present in CCSMCs lysates. (c) Nanoparticle tracking analysis of the concentration of EXOs in supernatant of CCSMCs. (d) CCSMCs were prepared for the detection of nSMase2 expression using immunofluorescence staining. (e) Western blot analyses measuring nSMase2 expression levels in CCSMCs. (f) The bar graph represents the relative quantification of nSMase2 compared with that of β-actin. Cell experiments performed: n = 3. *P < 0.05. CCSMCs: corpus cavernosum smooth muscle cells; EXOs: exosomes; nSMase2: neutral sphingomyelinase-2; NC: CCSMCs with no treatment; PDGFBB: CCSMCs treated with platelet-derived growth factor-BB; GW4869: CCSMCs treated with GW4869; PDGFBB+GW4869: CCSMCs were pretreated with GW4869, followed by stimulation with platelet-derived growth factor-BB; NS: not significant.

Figure 5.

Figure 5

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Increased expression levels of nSMase2 were detected in CCSMCs undergoing phenotypic modulation in type 2 diabetic rats. Representative double fluorescence staining images of α-SMA (red) and nSMase2 (green) in corpus cavernosum tissue of type 2 diabetic rats. NC: normal control rats; DM: rats with diabetes mellitus; CCSMCs: corpus cavernosum smooth muscle cells; α-SMA: alpha-smooth muscle actin; nSMase2: neutral sphingomyelinase-2.

EXO secretion regulates the CCSMC phenotype, mediated by nSMase2

To examine the role of EXOs in phenotypic modulation of CCSMCs, we inhibited EXO release using an nSMase2 inhibitor, GW4869, in the presence of PDGFBB. It showed that 10 µmol l−1 GW4869 efficiently inhibited the expression of nSMase2 in CCSMCs. A CCK-8 assay showed that PDGFBB significantly promotes the proliferation of CCSMCs (both P < 0.05; Figure 6a), while inhibition of nSMase2 (10 µmol l−1) in CCSMCs reduced the cell proliferation induced by PDGFBB (both P < 0.05; Figure 6a). In the cell scratch wound-healing assay, PDGFBB promoted CCSMC migration, and this migratory response was impaired by nSMase2 inhibition (P < 0.05; Figure 6b and 6c). These experiments demonstrated that, in CCSMCs, PDGFBB significantly downregulated all examined smooth muscle cell-specific genes associated with the contractile phenotype (including α-SMA, smoothelin, and Myh11), while upregulating the synthetic phenotype marker gene OPN. Inhibition of nSMase2 during PDGFBB treatment led to significant upregulation of contractile phenotype biomarker genes and downregulation of OPN at both the protein and mRNA levels (all P < 0.05; Figure 6d and 6e and Supplementary Figure 3 (93.3KB, tif) ).

Figure 6.

Figure 6

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Exosomes secretion could regulate phenotype of CCSMCs mediated by nSMase2. (a) Proliferation activity of CCSMCs evaluated by Cell Counting Kit-8 cell proliferative assay. (b) Migration activity of CCSMCs evaluated by scratch wound-healing assay. (c) Statistical analysis of the area of wound healing between each group. (d) Phenotype-associated genes and collagen I were detected by quantitative reverse transcriptase-polymerase chain reaction. (e) Western blot analyses measuring the expression levels of phenotype-associated proteins and collagen I in CCSMCs in each group. Cell experiments performed: n = 3. *P < 0.05. OD: optical density; PDGFBB: CCSMCs treated with platelet-derived growth factor-BB; GW4869: CCSMCs treated with GW4869; PDGFBB+GW4869: CCSMCs were pretreated with GW4869, followed by stimulation with platelet-derived growth factor-BB; CCSMCs: corpus cavernosum smooth muscle cells; nSMase2: neutral sphingomyelinase-2; NC: CCSMCs with no treatment; α-SMA: alpha-smooth muscle actin; Myh11: myosin heavy chain 11; OPN: osteopontin.

DISCUSSION

Our previous studies23,24 showed that, in rats with type 1 DM or bilateral cavernous nerve injuries, there is a notable decline in the expression of contractile smooth muscle cell markers coupled with an increase in the expression of synthetic smooth muscle cell markers. These molecular shifts suggest a phenotypic transition of CCSMCs from a contractile to a synthetic state. This process is often linked to reduced cellular contractility and impaired tissue function. In addition, the phenotypic modulation of CCSMCs might play a key role in the development of ED. In this study, we established a type 2 diabetic rat model using a combination of a high-fat diet and multiple injections of low-dose STZ. Similar to the rat models of type 1 DM and bilateral cavernous nerve injury, reduced expression of contractile phenotypic proteins (such as α-SMA, smoothelin, and Myh11) and increased expression of a synthetic element (OPN) were detected in the penile corpus cavernosum tissue of rats with type 2 DM. Collagen deposition is a characteristic of VSMC phenotypic modulation, and so, we simultaneously detected the expression of collagen I and found that the expression of collagen I was significantly increased in rats with type 2 DM compared with controls. These results further support the idea that erectile function is maintained through the contractile phenotype of CCSMCs, while the synthetic phenotype may play a role in the development of diabetes-related ED.

The contractile and synthetic phenotypes of smooth muscle cells represent opposite ends of a continuum. The transition from a contractile to a synthetic phenotype in response to local stimuli is linked to the development of various cardiovascular conditions, including diabetic vascular complications, atherosclerosis, hypertension, and transplant arteriopathy.25 Similar to other kinds of smooth muscle cells, CCSMCs can transition from a contractile state to a synthetic state based on local environmental cues as a result of the bidirectional differentiation capacity.8 In theory, this phenotypic modulation results in the downregulation of contractile phenotype markers, such as α-SMA, smoothelin, and Myh11, and the upregulation of synthetic phenotype markers like OPN, accompanied by collagen deposition. CCSMCs with the synthetic phenotype possess a stronger proliferative capacity but exhibit poorer contractility. Moreover, they may have a greater ability to synthesize and secrete bioactive substances.

EXOs, acting as novel intercellular communication messengers, can function in a paracrine and autocrine manner by delivering biologically active proteins, lipids, and RNA species.26,27 Thus, we aimed to determine the correlation between EXO secretion and CCSMC phenotypic modulation and whether regulated EXO secretion could change the cellular phenotype. CCSMC-derived EXOs were enriched with exosomal markers, such as CD63 and CD81. Moreover, double fluorescence staining showed significant expression of CD63 in the cavernosal smooth muscle cells of rats with type 2 DM. These results indicate that CCSMCs can secrete EXOs, alongside other cells such as adipose-derived stem cells,22 macrophages,28 and many kinds of cancer cells.29,30 In addition, we treated CCSMCs with PDGFBB, a known stimulator to induce VSMC phenotypic modulation because of its mitogenic potential.31 Our results showed that PDGFBB treatment causes CCSMCs to change their phenotype from contractile to synthetic, accompanied by elevated EXO secretion. Thus, phenotypic modulation of CCSMCs is linked to elevated EXO secretion, although it is not clear what the link is between phenotypic modulation and EXO secretion. Phenotypic modulation is driven by cytoskeleton remodeling and single mutations in contractile proteins such as actin α2 smooth aorta muscle (ACTA2) or Myh11.32,33 Additionally, actin cytoskeletal proteins are regulated by Rab27A and Rab27B, which are crucial for the secretion of EXOs by managing the trafficking of multivesicular bodies.34 Consequently, the cytoskeletal alterations associated with CCSMC phenotypic transformation may facilitate the trafficking of multivesicular bodies and the secretion of EXOs.

nSMase is a member of the sphingomyelinase family, which catalyzes the cleavage of the phosphodiester bond in sphingomyelin to form ceramide and phosphocholine.14 Three nSMases, including nSMase1, nSMase2, and nSMase3, have been identified.35 Different nSMases are thought to function in different cellular compartments and support different cellular functions. Among them, nSMase2 is the most studied protein isoform and has been implicated in multiple cellular processes, such as cell growth arrest, apoptosis, and inflammatory signaling, as well as in pathologies like cancer metastasis, Alzheimer’s disease, and other organ system disorders.14,36,37 EXO biogenesis and trafficking are very complex, involving many cellular components. The endosomal sorting complex, required for transport of components, and the nSMase pathway are responsible for orchestrating protein sorting and packaging to multivesicular bodies. Protein post-translational modifications, such as ubiquitination or glycosylation, may also be implicated in protein targeting.38,39 The processes that follow the docking of multivesicular bodies to the plasma membrane are regulated by the Rab family of small guanosine triphosphatases (GTPases).40 These various and complex mechanisms of EXO synthesis may not be strictly delineated and may instead have shared components and various levels of interdependency.41 Given the critical role of the nSMase2 pathway in EXO biogenesis and secretion15,42 and the correlation between phenotypic modulation and EXO secretion, we tested the effect of phenotypic modulation on nSMase2 expression. Our data, combined with previous reports, indicate that nSMase2 plays an important role in EXO secretion and that factors which promote CCSMC phenotypic transformation may also promote EXO secretion by increasing the expression of nSMase2 in CCSMCs.

GW4869, recognized as an nSMase2 inhibitor in both in vitro and in vivo studies, was used to evaluate the role of nSMase2 in cellular responses and physiological functions.43,44 Then, in the presence of PDGFBB, we inhibited EXO release from CCSMCs using GW4869. The results showed that 10 µmol l−1 GW4869 efficiently inhibited the expression of nSMase2 in CCSMCs, which is consistent with previous studies.45,46 We then explored the effects and potential mechanisms of regulated EXO secretion on CCSMC phenotypic modulation. Inhibition of nSMase2 reduced the proliferation and migration of CCSMCs induced by PDGFBB. Furthermore, nSMase2 inhibition restored the phenotypic modulation of CCSMCs induced by PDGFBB treatment. These results suggest that regulated EXO secretion can change the phenotype of CCSMCs via the nSMase2 pathway.

There are several limitations to our current study. First, we lack in vivo trials to verify the reversal of the phenotypic modulation of CCSMCs in diabetic rats by GW4869, improving erectile function. However, we have shown that phenotypic modulation of CCSMCs is linked to increased EXO secretion and nSMase2 expression and that GW4869 can restore the contractile phenotype of CCSMCs in vitro. Intraperitoneal injection of GW4869 into mice reduces inflammation and atherosclerosis,20 and in vivo experiments have shown that GW4869 is mainly absorbed by endothelial cells and macrophages rather than smooth muscle cells after intraperitoneal injection. This raises questions about the in vivo effect of GW4869 on the phenotypic modulation of CCSMCs. The cellular uptake of GW4869 by CCSMCs may be very limited, and even if GW4869 did reverse the phenotypic transformation of CCSMCs, this may be achieved by regulation of EXO secretion from endothelial cells and macrophages rather than by directly acting on CCSMCs. Second, it is still a challenge to fully understand the cargoes and roles of the EXOs secreted by CCSMCs, and this has not been included in this study. The effects of EXOs secreted by various cells under different conditions can differ because of diversity in their cargo composition.47 Adipose-derived stem cell-derived exosomes (ADSC-EXOs), mesenchymal stem cell-derived exosomes (MSC-EXOs), and CCSMC-EXOs differ in their dynamics and functions and have all been investigated for their potential to improve ED in rats.22,48,49 As reported by Song et al.,49 EXOs secreted by CCSMCs under normal conditions ameliorate DM-induced ED by inhibiting fibrosis and modulating the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway. EXOs derived from CCSMCs cultured under diabetic conditions may have different cargoes and thus do not improve ED. Based on our hypothesis, these EXOs are taken up by adjacent CCSMCs in a paracrine manner, thereby promoting the phenotypic modulation of these adjacent CCSMCs. This process also triggers further EXO secretion by these CCSMCs, forming a vicious cycle. These EXOs could be taken up by neighboring endothelial cells, leading to endothelial dysfunction. These may be the mechanisms underlying diabetic ED.

In our study, we have demonstrated the key role of phenotypic modulation of CCSMCs in a rat model of type 2 DM. There is an inverse relationship between the expression of contractile phenotype markers and the secretion of EXOs. Additionally, regulation of EXO secretion can alter the phenotype of CCSMCs, likely through mechanisms involving the nSMase2-dependent pathway.

CONCLUSIONS

Phenotypic modulation of CCSMCs is involved in the pathogenesis of diabetic ED. Regulated EXO secretion can change the phenotype of CCSMCs via the nSMase2 pathway. Thus, the regulation of EXO biogenesis and secretion may be a novel therapeutic approach to improve ED induced by type 2 DM by maintaining the contractile phenotype of CCSMCs.

AUTHOR CONTRIBUTIONS

FZC, AYW, and HBM took responsibility for the integrity of the data and accuracy of the data analysis. FZC, XTZ, and HBM developed the review concept and design of the review. FZC, XTZ, QYZ, and AYW drafted the manuscript. XTZ, HBM, BXL, LW, and AYW supervised the entire project. All authors contributed to acquisition, analysis, and/or interpretation of data, and read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

Supplementary Figure 1

Serum biochemistry of the rat. (a) Levels of glucose in rat serum biochemistry evaluation. (b) Levels of insulin in rat serum determined by enzyme-linked immunosorbent assay. (c) Levels of triglycerides and total cholesterol in rat serum biochemistry evaluation. *P < 0.05. NC: normal control rats; DM: rats with diabetes mellitus; TG: triglycerides; TC: total cholesterol; n: 10 animals per group.

AJA-28-516_Suppl1.tif (57KB, tif)
Supplementary Figure 2

Masson trichrome in penile tissues. (a) Smooth muscle (red) and collagen (blue) tissues stained by masson trichrome in each group. (b) The bar graph denotes the ratio between smooth muscle and collagen content. The ratio of smooth muscle to collagen was defined as the percentage of smooth muscle area in a given field. *P < 0.05. NC: normal control rats; DM: rats with diabetes mellitus.

AJA-28-516_Suppl2.tif (161.7KB, tif)
Supplementary Figure 3

Western blot analyses measuring the expression levels of phenotype-associated proteins and collagen I normalized to the β-actin level in CCSMCs in each group. (a) The bar graph represents the relative quantification of α-SMA compared with that of β-actin. (b) The bar graph represents the relative quantification of smoothelin compared with that of β-actin. (c) The bar graph represents the relative quantification of Myh11 compared with that of β-actin. (d) The bar graph represents the relative quantification of OPN compared with that of β-actin. (e) The bar graph represents the relative quantification of collagen I compared with that of β-actin. Cell experiments performed: n = 3. *P < 0.05. PDGFBB: CCSMCs treated with platelet-derived growth factor-BB; GW4869: CCSMCs treated with GW4869; PDGFBB+ GW4869: CCSMCs were pretreated with GW4869, followed by stimulation with platelet-derived growth factor-BB; CCSMCs: corpus cavernosum smooth muscle cells; NC: CCSMCs with no treatment; α-SMA: alpha-smooth muscle actin; Myh11: Myosin heavy chain 11; OPN: Osteopontin.

AJA-28-516_Suppl3.tif (93.3KB, tif)

ACKNOWLEDGMENTS

This research was supported by the Shenzhen Science and Technology Innovation Program (No. JCYJ20210324102802007), the Sanming Project of Medicine in Shenzhen (No. SZSM202111007), and the Shenzhen Key Medical Discipline Construction Fund (No. SZXK020).

Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure 1

Serum biochemistry of the rat. (a) Levels of glucose in rat serum biochemistry evaluation. (b) Levels of insulin in rat serum determined by enzyme-linked immunosorbent assay. (c) Levels of triglycerides and total cholesterol in rat serum biochemistry evaluation. *P < 0.05. NC: normal control rats; DM: rats with diabetes mellitus; TG: triglycerides; TC: total cholesterol; n: 10 animals per group.

AJA-28-516_Suppl1.tif (57KB, tif)
Supplementary Figure 2

Masson trichrome in penile tissues. (a) Smooth muscle (red) and collagen (blue) tissues stained by masson trichrome in each group. (b) The bar graph denotes the ratio between smooth muscle and collagen content. The ratio of smooth muscle to collagen was defined as the percentage of smooth muscle area in a given field. *P < 0.05. NC: normal control rats; DM: rats with diabetes mellitus.

AJA-28-516_Suppl2.tif (161.7KB, tif)
Supplementary Figure 3

Western blot analyses measuring the expression levels of phenotype-associated proteins and collagen I normalized to the β-actin level in CCSMCs in each group. (a) The bar graph represents the relative quantification of α-SMA compared with that of β-actin. (b) The bar graph represents the relative quantification of smoothelin compared with that of β-actin. (c) The bar graph represents the relative quantification of Myh11 compared with that of β-actin. (d) The bar graph represents the relative quantification of OPN compared with that of β-actin. (e) The bar graph represents the relative quantification of collagen I compared with that of β-actin. Cell experiments performed: n = 3. *P < 0.05. PDGFBB: CCSMCs treated with platelet-derived growth factor-BB; GW4869: CCSMCs treated with GW4869; PDGFBB+ GW4869: CCSMCs were pretreated with GW4869, followed by stimulation with platelet-derived growth factor-BB; CCSMCs: corpus cavernosum smooth muscle cells; NC: CCSMCs with no treatment; α-SMA: alpha-smooth muscle actin; Myh11: Myosin heavy chain 11; OPN: Osteopontin.

AJA-28-516_Suppl3.tif (93.3KB, tif)

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