Molecular insights into treatment-resistant erectile dysfunction: staining patterns of human corpora cavernosa—a preliminary study

Introduction

Erectile dysfunction (ED) is a prevalent condition that affects millions of men globally and significantly impairs quality of life (1,2). Treatment options include lifestyle modifications, oral medications such as phosphodiesterase type 5 (PDE5) inhibitors, injected vasodilators, vacuum erection devices, and surgical interventions (3,4). Although PDE5 inhibitors have revolutionized the management of ED, they fail to produce effective results in 30–35% of patients and do not address the underlying pathophysiological mechanisms (1). This limitation has driven research into novel therapeutics targeting alternative molecular pathways, including centrally acting dopaminergic and melanocortin receptor agonists, and peripherally acting agents such as soluble guanylate cyclase stimulators and Rho-kinase inhibitors (2,5). Understanding molecular expression patterns in patients unresponsive to conventional therapies is critical for advancing targeted treatment strategies. Contemporary reviews also highlight growing interest in nutraceuticals for ED—many targeting endothelial-nitric oxide (NO) and oxidative stress pathways—though evidence quality remains variable (6).

The RhoA/Rho-kinase (ROCK) pathway is known to play a key role in maintaining penile flaccidity, and increased expression has been identified in the cavernous arteries of ED patients (7,8). Lysyl oxidase (LOX1), a protein associated with oxidative stress and fibrosis, has been shown to correlate with body mass index (BMI), and its inhibition has improved erectile function in animal models (9,10). CD31, a marker of vascular endothelium, serves as a useful indicator for quantifying microvascular density (11). Alpha-smooth muscle actin (α-SMA) is a well-documented contributor to structural changes in ED (12). The objective of this study was to evaluate the expression patterns of potential therapeutic targets—ROCK1/2, LOX1, CD31, and α-SMA—in corpus cavernosum tissue of men with treatment-resistant ED. We hypothesized that patients with treatment-resistant ED exhibit elevated expression of ROCK1/2 and α-SMA compared to other biomarkers. To test this hypothesis, we conducted an immunohistochemical (IHC) analysis of corpora cavernosa specimens from patients undergoing penile prosthesis placement. We present this article in accordance with the STROBE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-391/rc).

Methods

Formalin-fixed human corpora cavernosa specimens were obtained from 14 patients with ED undergoing penile prosthesis placement at Marshall University Medical Center (Huntington, WV, USA). Comorbid conditions such as diabetes mellitus, hypertension, hyperlipidemia, and coronary artery disease were defined according to standard clinical diagnostic criteria as documented in the medical record. Because all included specimens were obtained from patients undergoing penile prosthesis placement for treatment-refractory ED, the study lacked a non-ED control group. Tissue specimens were submitted to an external testing facility for histopathological preparation and IHC analysis. Due to the exploratory nature of the study, a formal power calculation was not performed. Staining and scoring were conducted by Comparative Biosciences, Inc., Sunnyvale, CA, using standardized chromogen-based horseradish peroxidase/3,3'-diaminobenzidine (HRP/DAB) techniques. Formalin-fixed paraffin-embedded (FFPE) tissue blocks were sectioned at approximately 5 µm thickness and stained to detect biomarkers, including Rho-associated protein kinase (ROCK1/ROCK2), CD31 (endothelial cell marker), α-SMA, and lysyl oxidase (LOX1). Specific primary antibodies were used as follows: CD31/PECAM-1, rabbit polyclonal (Sino Biological, Beijing, China; Cat# 50408, 1:1,000); OLR1/LOX1, rabbit polyclonal (Sino Biological, Cat# 10585-T26, 1:1,000); ROCK1, rabbit polyclonal (Sino Biological, Cat# 109567-T08, 1:200); and α-SMA, rabbit polyclonal (Invitrogen, Waltham, MA, USA; Cat# PA5-85070, 1:500). Detection used the ImmPRESS^® HRP Goat Anti-Rabbit IgG Polymer kit (Vector, Newark, CA, USA; MP-7451-50) with DAB chromogen (Cell Signaling, Danvers, MA, USA; 8059S). Antigen retrieval was performed in citrate buffer, pH 6.0 (Novus, Centennial, CO, USA; NB900-62075). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide (VWR^®, Radnor, PA, USA; BDH7690-1), and nonspecific binding was minimized using CAS blocking solution (Life Technologies, Carlsbad, CA, USA; 008120). Antibody specificity and staining conditions were optimized at the testing facility; staining was judged specific by comparison to control slides and concordance with known antigen distribution according to facility standard operating procedures (SOPs).

FFPE tissue sections underwent deparaffinization, rehydration, and antigen retrieval in heated citrate buffer (pH 6.0, 95 °C) for 20 minutes under low pressure. Following antigen retrieval, the sections were cooled at room temperature for 30 minutes, rinsed in deionized water, and washed twice with phosphate-buffered saline (PBS) for 1 minute each. Endogenous peroxidase activity was blocked by incubating the sections with 3% hydrogen peroxide (VWR^®) in PBS for 10 minutes, followed by two additional PBS washes (2 minutes each).

Protein blocking was performed using CAS blocking solution (Life Technologies™) for 60 minutes at room temperature, and the sections were incubated with the respective primary antibodies for 60 minutes at room temperature. After incubation, slides were washed twice with PBS (2 minutes each) and incubated with an HRP-conjugated goat anti-rabbit IgG polymer detection kit (ImmPRESS^®, Vector Laboratories^®, Newark, CA, USA) for 45 minutes at room temperature. Following three washes in PBS (2 minutes each), chromogen-based HRP/DAB staining was performed using the SignalStain^® DAB Substrate Kit (Cell Signaling Technology^®, Danvers, MA, USA), with development for 1–2 minutes. The slides were rinsed in PBS, counterstained with hematoxylin, and underwent dehydration before being cover-slipped.

The IHC protocol, adapted from the standardized chromogen-based HRP/DAB technique, included meticulous steps to ensure antigen retrieval, staining specificity, and signal development. The reagents used, such as the citrate buffer (Novus Biologicals^®, Centennial, CO, USA) and other solutions, were selected to optimize staining results.

The stained slides were evaluated by a single pathologist, blinded to patients’ clinical characteristics, using light microscopy to assess staining intensity and distribution. Staining intensity was graded on a multi-point scale: 0 (negative), ± (equivocal), +1 (weak/mild), +2 (moderate), +3 (strong), and +4 (intense). When staining intensity varied within a given sample, ranges (e.g., 2+/3+) were recorded to capture intra-sample heterogeneity. Given the small sample size and exploratory design, results were analyzed descriptively without inferential statistics to avoid underpowered subgroup comparisons. This detailed approach ensured reliable detection of the target biomarkers, critical for assessing the pathological features in human corpora cavernosa specimens.

Statistical analysis

Due to the exploratory nature of this pilot study and the limited sample size (n=14), formal hypothesis testing was not performed. Data were summarized using descriptive statistics, including means, standard deviations, and qualitative trends in biomarker expression. No inferential analyses were conducted to avoid underpowered subgroup comparisons.

Ethical consideration

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Marshall University Institutional Review Board (protocol No. 1301243-1). Written informed consent was obtained from all patients prior to tissue collection.

Results

Baseline characteristics were collected for all patients (Table 1). The study included 14 male patients with a mean age of 62±18 years. Comorbid conditions included diabetes mellitus (n=4), hypertension (n=7), hyperlipidemia (n=7), coronary artery disease (n=7), chronic obstructive pulmonary disease (COPD) (n=2), peripheral vascular disease (PD) (n=4), and hypogonadism (n=5).

Quantitative analysis of IHC staining

Staining intensity was scored using a multi-point scale (+1 to +4) for biomarkers ROCK1/2, α-SMA, CD31, and LOX1. The results demonstrate distinct expression patterns across patient subgroups.

ROCK1/2 expression

• Patients with hypertension (mean ROCK1/2 score: +3.0±0.5) and coronary artery disease (mean ROCK1/2 score: +3.1±0.4) showed higher ROCK1/2 expression compared to patients without these conditions (mean ROCK1/2 score: +2.0±0.5).
• Older patients (≥60 years) had elevated ROCK1/2 expression (mean ROCK1/2 score: +3.0) compared to younger patients (<60 years; mean ROCK1/2 score: +2.2).

α-SMA expression

Elevated α-SMA expression was noted in patients with hypertension (mean α-SMA score: +3.1±0.4) and longer ED duration (>10 years; mean α-SMA score: +3.0±0.3) compared to those without these factors (mean α-SMA score: +2.0±0.5).

CD31 expression

Smokers, both current (mean CD31 score: +1.8±0.5) and former (mean CD31 score: +1.5±0.5), exhibited increased CD31 expression compared to non-smokers (mean CD31 score: +0.8±0.5).

LOX1 expression

Patients with coronary artery disease exhibited elevated LOX1 expression (mean LOX1 score: +2.0±0.5) compared to those without coronary artery disease (mean LOX1 score: +1.0±0.3).

Comparative expression across biomarkers

Overall, ROCK1/2 and α-SMA expression levels were consistently higher across all tissue samples (mean scores: +2.8 and +2.7, respectively) compared to CD31 and LOX1 (mean scores: +1.2 and +1.5, respectively) (Table 2). Figure 1 provides an example of representative tissue staining. To further summarize these patterns, Figure 2 illustrates the mean staining intensities of ROCK1/2, α-SMA, CD31, and LOX1 across five patient subgroups—hypertension, coronary artery disease, advanced age, prolonged ED duration, and smoking status.

Figure 1 Immunohistochemical staining of corporal cavernosal tissue from patient EDA17, who had a history of coronary artery disease and hypogonadism with no smoking history. Staining scores: Rho-associated protein kinase (ROCK1/2) +3/+4, CD31 +2/+3, α-SMA +2, and LOX1 +2. Brown color represents positive staining. Scale bar: 100 μm. α-SMA, alpha smooth muscle actin; LOX1, lysyl oxidase.

Figure 2 Mean staining intensity of four biomarkers—Rho-associated protein kinase (ROCK1/2), CD31, α-SMA, and LOX1—across five patient conditions: hypertension, coronary artery disease, older age, long ED duration, and smokers. α-SMA, alpha smooth muscle actin; ED, erectile dysfunction; LOX1, lysyl oxidase.

Summary of key findings

• ROCK1/2 expression was elevated in patients with hypertension, coronary artery disease, and advanced age.
• α-SMA expression correlated with hypertension and prolonged ED duration.
• CD31 expression was most pronounced in smokers.
• LOX1 expression was elevated in coronary artery disease patients.

These results indicate distinct patterns of biomarker expression, providing insights into the potential roles of these proteins in the pathophysiology of ED and associated comorbid conditions.

Discussion

This study provides novel insights into the molecular landscape of treatment-resistant ED by highlighting the differential expression of key biomarkers in corpus cavernosum tissue. We observed increased expression of Rho-associated protein kinases (ROCK1/2) and α-SMA in patients with chronic ED, particularly those with comorbid hypertension, hyperlipidemia, and advanced age. Elevated CD31 expression was noted in patients with a history of smoking, while higher levels of LOX1 were associated with coronary artery disease. These findings underscore the complex interplay between vascular, fibrotic, and endothelial dysfunctions in the pathophysiology of treatment-resistant ED. Our tissue-level findings involve endothelial and contractile markers; notably, several nutraceutical approaches purport to modulate related NO/oxidative pathways, although clinical support remains limited (6).

The elevated expression of ROCK1/2 aligns with prior studies implicating the RhoA/ROCK pathway in maintaining penile flaccidity and contributing to ED (7,8). Chronic activation of this pathway, as evidenced by a mean staining intensity of +3.0 in hypertensive and coronary artery disease patients compared to +2.0 in patients without these conditions, likely exacerbates smooth muscle dysfunction and reinforces vascular comorbidities as critical contributors to treatment-resistant ED. These findings reinforce the potential of the ROCK pathway as a therapeutic target, particularly in patients unresponsive to PDE5 inhibitors (13).

Similarly, increased α-SMA expression, which can be observed in both smooth muscle cells and myofibroblasts, reflects smooth muscle and fibrotic activity in cavernosal tissue. While additional markers such as myosin, desmin, or vimentin would be required to distinguish these cell types more precisely, our findings align with previous research linking vascular remodeling and fibrosis to ED in hypertensive patients (12). The observed mean staining intensity of +3.1 in hypertensive patients, compared to +2.0 in non-hypertensive patients, is associated with a prominent role for smooth muscle fibrosis in tissue dysfunction and decreased compliance, which likely diminishes the efficacy of conventional treatments.

Elevated CD31 expression in smokers, both current (mean +1.8) and former (mean +1.5), compared to non-smokers (+0.8), suggests persistent endothelial alterations associated with tobacco exposure. While CD31 is a structural endothelial marker and does not directly assess endothelial function (e.g., eNOS phosphorylation), these findings are consistent with literature indicating long-term vascular changes in smokers. Future studies incorporating functional endothelial assays will be necessary to establish whether these structural differences translate into persistent endothelial dysfunction that contributes to treatment-resistant ED. This suggests that prolonged endothelial impairment may play a role in the refractory nature of ED among current and former smokers, highlighting the lasting vascular effects of tobacco use (14).

The association of LOX1 expression with coronary artery disease is further consistent with overlapping molecular pathways between ED and systemic vascular diseases. The mean LOX1 staining intensity of +2.0 in coronary artery disease patients, compared to +1.2 in those without, highlights its potential role in oxidative stress and endothelial dysfunction. While LOX1’s role in ED remains underexplored, its involvement in cavernosal fibrosis, as demonstrated in preclinical studies, suggests it could serve as a biomarker for cardiovascular risk assessment in ED patients. While LOX1’s role in ED remains underexplored, its involvement in cavernosal fibrosis, as demonstrated in preclinical studies, suggests it could serve as a biomarker for cardiovascular risk assessment in ED patients (10,15).

Notably, ROCK1/2 and α-SMA expression levels were higher across all tissue samples compared to CD31 and LOX1, suggesting that the ROCK pathway and fibrosis might play more prominent roles in treatment-resistant ED. These findings emphasize the heterogeneity of treatment-resistant ED, driven by diverse underlying pathologies. Personalized therapeutic approaches may be beneficial—for instance, targeting the ROCK pathway and fibrosis in patients with elevated ROCK1/2 and α-SMA expression or focusing on endothelial-specific interventions for those with higher CD31 or LOX1 expression.

Despite its novel findings, this study has limitations. The small cohort size (n=14) limits statistical power, particularly for subgroup analyses, and the results should be interpreted as exploratory hypothesis-generating findings rather than definitive conclusions. Furthermore, the semi-quantitative nature of IHC scoring introduces a degree of subjectivity. Additionally, due to the preliminary nature of the study, fluorescent analysis or quantitative techniques like Western blotting were not performed. All slides were graded by one experienced pathologist, blinded to patients’ clinical characteristics, which provided consistency but introduced subjectivity; inter-observer variability was not assessed. The absence of control group is also a notable limitation. The cross-sectional design precludes conclusions about the progression of molecular changes over time or their causal role in ED. Finally, because several patients presented with multiple comorbid conditions, overlap between groups may introduce confounding that could not be fully disentangled in this pilot study. Future studies should validate these findings in larger, more diverse cohorts and explore the prognostic value of these biomarkers in predicting disease progression or treatment response.

Despite these limitations, the findings from this preliminary analysis provide a valuable foundation for future research. Building upon these observations, future studies should aim to incorporate functional assessments, such as penile hemodynamics or vascular reactivity tests, which would provide deeper insights into the clinical relevance of these molecular alterations. Expanding the biomarker panel to include additional pathways involved in fibrosis and endothelial dysfunction could enhance our understanding of treatment-resistant ED and its relationship to systemic vascular diseases.

Conclusions

This study reinforces associations between the ROCK pathway, fibrosis, and endothelial alterations and the molecular pathophysiology of treatment-resistant ED. These findings emphasize the heterogeneity of treatment-resistant ED, suggesting that tailored therapeutic approaches may be beneficial; however, functional validation will be required before personalized therapies can be realized. Furthermore, the overlap between ED and systemic vascular diseases underscores the importance of comprehensive cardiovascular risk assessment and management in these patients.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-391/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-391/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-391/prf

Funding: This work was supported by the Harris Willits Chair in Urology Endowment Fund.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-391/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Marshall University Institutional Review Board (Protocol No. 1301243-1). Written informed consent was obtained from all patients prior to tissue collection.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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