Decline in estrogen (E2) after menopause possibly causes overactive bladder.  There is a report indicating systemic E2 application rather worsens bladder function [1].  E2 deficiency may also induce fibrosis that leads to bladder dysfunction [2], however, little has been known about relationship between fibrosis and platelete derived growth factor receptor (PDGFR) α+ cells in the bladder.  PDGF is essential to generate the extracellular matrix (ECM) [3].  This original work was aimed at investigating the effects of E2 on bladder function and assessing changes of bladder PDGFRα+ cells associated with inflammation and fibrosis.

C57BL/6 female mice (Charles River Laboratories) were used.  Bilateral ovariectomy (OVX) or sham surgery was performed at 7 weeks old and then E2 (3 μ/kg/day, administered every other day) was applied subcutaneously in OVX mice for two weeks before experiment.  Each protocol was done 1 (11 weeks old), 3 (19 weeks old) and 7 (35 weeks old) months after OVX (These were named 1, 3 and 7 month groups).  To perform in vivo cystometrogram (CMG), PE10 catheter was inserted one week before CMG.  Mice were restrained with specific holder under awake condition.  Saline was infused at 15 μl/hr after two hours acclimation of mice and voiding cycles were recorded for at least one hour after three reproducible micturition was confirmed.  Storage parameters were analyzed and data were expressed as mean ± SEM.  qPCR, western blotting (WB) was carried out to investigate the relationship between E2 deficiency/replacement and fibrosis marker, while immunohistochemistry (IHC) was done to check relationship between PDGFRα+ and estrogen receptor (ER) positive cells.  To further explore this, bladder was divided into urothelium/suburothelial layer (UT) and detrusor smooth muscle layer (SM) and then cultured with control media, E2 (10 ng/ml) or TNFα (100 ng/ml) for 7 days.  Cultured tissues were collected and frozen at -80°C to investigate fibrosis marker.

Bladder compliance was decreased in 1, 3 and 7 month OVX groups (20.3 ± 6.5, 10.2 ± 1.5 and 14.2 ± 2.7 μl/mmHg) compared to age-matched sham groups (35.1 ± 6.1, 24.6 ± 3.2 and 24.2 ± 4.5 μl/mmHg), while E2 recovered bladder compliance in 1 and 3 month (35.9 ± 4.9 and 22.2 ± 3.4 μl/mmHg) but not in 7 month group (13.3 ± 2.2 μl/mmHg).  Bladder capacity was reduced in 3 and 7 month OVX groups (155.7 ± 27.9 and 161.9 ± 23.7 μl) compared to sham (255.0 ± 16.7 and 256.3 ± 45.7 μl), but was not fully recovered by E2 (150.7 ± 16.8 μl and 197.7 ± 29.7 μl), whilst intercontraction interval (ICI) was also reduced in 3 and 7 month OVX groups (10.3 ± 1.8 and 10.7 ± 1.5 min) compared to age-matched sham groups (17.0 ± 1.1 and 17.0 ± 3.0 min) but was not rescued by E2 treatment (10.0 ± 1.1 and 13.1 ± 1.9 min).  Unexpectedly, E2 markedly increased the number of non-voiding contractions (NVCs) in 1, 3 and 7 month OVX+E2 groups in comparison with age-matched sham or OVX groups (Fig. 1).  E2 even induced an increase of NVCs in 1 month sham, demonstrating E2 failed to rescue OVX-induced bladder dysfunction and rather exacerbated the detrusor contraction.  Estrogen receptor (ERα) was dominantly immunoreactive against detrusor, however bladder interstitial cells were immunopositive upon ERβ.  Colocalizations of PDGFRα+ and ERβ positive interstitial cells were observed both in UT (Fig. 2) and SM.  Expression of PDGFRα and collagen 1a1 was upregulated in 3 month OVX group compared to sham, while E2 replacement further increased the expression of PDGFRα and fibrosis markers (collagen 1a1, interleukin (IL) 6, cadherin and TNFα) compared to age-matched sham or OVX groups.  In qPCR of cultured bladder tissue, TNFα dramatically upregulated the expression of Acta2, TIMP2, Tgfβ, Col1a1, Col1a2, cadherin and IL6 in UT but had marginal effects on SM.  Conversely, E2 increased the expression of fibrosis markers in SM but had little impact in UT.  In WB, signal intensity of ERβ was higher in 3 month OVX than age-matched sham group, while that was further upregulated in OVX+E2 group.

Combined effects of the upregulation of fibrosis markers in UT and SM may decrease bladder compliance and induce an increase in NVCs in in vivo CMG in systemic application of E2.  Coexpression of PDGFRα+ and ERβ immunoreactive interstitial cells demonstrates E2 might promote an increase in PDGFRα+ cells through ERβ which may play an important role in generating ECM.

Urothelium/suburothelial layer may be common site for the initiation of fibrosis.  Inflammation or E2 deficiency/replacement may cause fibrosis through the activation of PDGFRα+ cells.  These novel findings might point to a novel approach toward the treatment of bladder fibrosis.  We will further investigate the specific increase of these fibrosis markers as well as morphological changes after inducing inflammation using PDGFRα+/eGFP mice.

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