There is growing evidence for a significant role of nitric oxide (NO•) signalling in prostate physiology and its dysregulation contributing benign prostatic hyperplasia (BPH). This premise is supported clinically by the efficacy of phosphodiesterase type-5 (PDE5) inhibitor treatment in reducing BPH-associated lower urinary tract symptoms (LUTS). However, the cellular targets and downstream mechanism for NO• signalling in the prostate are still unclear. Cytochrome B5 reductase type-3 (CYB5R3) is a flavoprotein that regulates soluble guanylate cyclase (sGC), the cognate target of NO• that generates cyclic GMP [1]. CYB5R3 is known to be downregulated in aging and pathology [2] and could be a source of NO•-signalling impairment. Thus, the goal of this study was to determine the consequences of prostatic CYB5R3 downregulation in a mouse model and correlate these with BPH phenotype in human prostates.

Generation of inducible β-actin CYB5R3 deficient mouse. A mouse with loxP sites flanking exon3 of the CYB5R3 gene [1] was crossed with a mouse expressing Cre recombinase linked to the tamoxifen-inducible β-actin promoter (Jackson labs, stock #004682). At 8-12 weeks, knockout (KO) and wildtype (WT) mice were injected with 4-hydroxytamoxifen into all eight prostate lobes and physiological recordings taken up to 24-weeks post-injection. All mouse strains were based on a C57Bl/6J background and maintained in a centralized husbandry facility on a 12-hour light/dark cycle (7 am – 7pm).

Metabolic cage assessments of adult and aged mice. Voiding behaviour analysis was performed using customized metabolic cages (Columbus Instruments Inc.) consisting of a climate-controlled cabinet on the same 12-hour light/dark cycle as their regular housing facility. Urine output, food and water consumption were recorded for up to 48 hours.

Clinical specimens. De-identified clinical specimens were obtained from the institutional biospecimens core. Formalin-fixed paraffin embedded (FFPE) prostate biopsies of men (50-70 years old) with and without BPH diagnosis were sectioned for histological examination. Samples from younger men (20-30 years) were obtained as controls. The FFPE specimens were verified by a clinical pathologist to be free of malignancies.
 
Histology and immunofluorescence. Mouse and human prostate FFPE sections were processed for chemical staining. For protein localization, sections were processed and probed with appropriate primary antibodies and visualized by fluorescence microscopy.

Data and statistical analysis. Data are expressed as mean ± standard deviation. Statistical analyses were performed in GraphPad Prism 10 software. Comparison of data sets were performed with one-way ANOVA followed by Tukey’s multiple comparison post-hoc test where the null hypothesis was rejected at p<0.05.

Histological examination of human prostate biopsies showed significantly decreased CYB5R3 expression in BPH diagnosed samples compared to younger and age-matched control samples (figure 1A). This correlated with increased inflammation and fibrosis (collagen and elastin) deposition in BPH samples compared to non-BPH tissues (figure 1B). Histological findings in humans were mirrored by CYB5R3 deficient mouse prostates (figure 2A and 2B). CYB5R3 deficient mice also showed increase in voiding frequency and voided volumes compared to WT littermates (figure 2C and 2D) without significant changes in flow rate or urine output/water intake ratios (figure 2E and 2F).

Natural aging is associated with decreased CYB5R3 expression in both humans and mice [2], Genetic deletion of prostate CYB5R3 in mice promotes inflammation and fibrosis reminiscent of human BPH phenotype and is accompanied by differences in voiding behaviour.

NO• is an important regulator of prostate homeostasis. These data indicate that aging related downregulation of CYB5R3 and in turn sGC is detrimental. Reestablishing physiological NO•-cGMP signalling in the prostate with sGC activators could be a promising therapy for BPH-LUTS [3].

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Zabbarova et al. Benign prostatic hyperplasia/obstruction ameliorated using a soluble guanylate cyclase activator. Journal of Pathology, 256(4), 442-454, 2022.