Abstract Benign Prostatic Hyperplasia (BPH) is a highly prevalent health issue exhibited by ~50% of men by age 50, and 75% by age 80 which may be associated with bladder outlet obstruction (BOO) and lower urinary tract symptoms (LUTS). First line treatments include α1-adrenoceptors antagonists (e.g., tamsulosin) to decrease smooth muscle tone in the prostate and bladder neck and 5α-reductase inhibitors (e.g., finasteride) to block conversion of testosterone to more potent dihydrotestosterone for reducing prostate volume. However, these agents are often ineffective in preventing disease progression to urinary retention which may necessitate surgical intervention in many patients. Multiple lines of evidence support that apart from aging, the pathogenesis of LUTS comprises multiple modifiable factors including compromised prostatic perfusion, oxidative stress mediated inflammation, fibrosis, and neuroplasticity. Clinical evidence argues against “one size fits all” therapy and highlights the preference of treatment options with lower risk of sexual side effects which is underscored by the FDA approval of phosphodiesterase-5 inhibitor (PDE5I), tadalafil, and its recent (2021) combination with finasteride (i.e., Entadfi) as a single pill for BPH/LUTS. Since some patients develop refractoriness to tadalafil, there is a compelling reason to advance our understanding of nitric oxide (NO•)-cGMP signaling in the pathophysiology of BPH/LUTS. The canonical target for NO• is soluble guanylate cyclase (sGC) that catalyzes the production of cGMP. The responsiveness of sGC is dependent upon cytochrome B5 reductase-3 (CYB5R3) that maintains the sGC heme in the NO• sensitive reduced state (Fe2+). We recently reported that aged (≥24 mo) male mice without hormonal manipulation recapitulate the BOO phenotype of high bladder pressure/low urine flow compared to low pressure/high flow of adult mice (9 mo), and that daily oral treatment with the sGC activator, cinaciguat (10 mg/kg/2 wks) reversed prostate hyperplasia and BOO in these aged mice. We propose that the aged mouse is a suitable model for testing our novel hypothesis that BPH/LUTS arises in the milieu of age- related oxidative stress potentiating the neurodegeneration of NO• producing (nitrergic) nerves and inflammation-induced inactivation of CYB5R3 to decrease cGMP-PKG signaling. The vicious feed forward cycle of BPH/LUTS can be circumvented by cinaciguat, a heme mimetic that can stimulate cGMP production when sGC is oxidized and the heme displaced, while its combination with a PDE5I can check PDE5 upregulation due to prolonged elevation of cGMP. This is supported by our selective knockout (KO) of CYB5R3 in the prostate/bladder, with both models to be compared with prostatic tissue from aged men with BPH. Our aims are: 1) Probe the role of the NO•-sGC-cGMP signaling pathway in the pathogenesis of BPH/BOO/LUTS in aged mice with cinaciguat treatment; 2) Study the impact of tissue specific deletion of CYB5R3 in t...