Urinary nerve growth factor (NGF) levels are decreased in aged female patients with overactive bladder syndrome (OAB), in type 1 diabetic mice and in aging rats. ProNGF in the same populations is unchanged. This imbalance was linked to a high activity of the proteolytic enzyme metalloproteinase-9 (MMP-9) that degrade NGF into peptides. On the other hand, the inhibitor of receptor p75NTR, THX-B, restored normal levels of NGF in urine from mice with type 1 diabetes. We here examine in vitro the effect of THX-B on the activity of MMP-9 in bladder cells and its consequences on the levels of secreted NGF by urothelial and smooth muscle cells.

Primary culture of urothelial and smooth muscle cells (SMCs) were grown from mouse bladder. Expression of NGF and MMP-9 were assessed by RT-qPCR, by immunohistochemistry and by immunoblotting. Levels of microRNAs were measured by RT-qPCR. NGF and proNGF secretion were evaluated using ELISA kits and MMP-9 activity by enzymatic assays.

NGF and MMP-9 mRNAs were expressed in both cell types at similar level. Microscopy confirmed the presence of both proteins in the cytoplasm of cells. Urothelial cells and SMCs culture supernatant contained significant amounts of NGF and proNGF. On the other hand, MMP-9 protein content was 7 times higher in SMCs than in urothelial cells, which was confirmed by high levels of miR-491-5p in the latter. However, secretion of active MMP-9 in the medium was 40 times higher in urothelial cells medium. Incubation with THX-B (5 µg/mL) for 24 hours abolished the synthesis and secretion of MMP-9 and doubled the concentration of NGF secreted in the medium of urothelial cells. ProNGF secretion levels in the same medium were not affected. On the other hand, THX-B had little effects on SMCs both at the level of proNGF, NGF and MMP-9. Interestingly, when growing urothelial cells on a membrane delineating a lower and upper compartment, most of the secretion of NGF from urothelial cells occurred at the apical portion of the cell layer, the basal medium receiving much less NGF. THX-B incubated for 24 hours increased the release of NGF only in the basal compartment. Finally, the effect of THX-B on NGF secretion appears to be linked to p75NTR-linked cyclic AMP pathway.

The bladder is a major source of NGF and proNGF. Our data suggest that the secretion of NGF and proNGF from urothelial and smooth muscle cells in vivo occur both into urine and into the space between muscle and urothelium. THX-B, which successfully restored normal contractile activity in a type 1 diabetic rodent model, has a direct effect on bladder cells, particularly by increasing secretion of NGF toward the basal environment where the smooth muscle may be its primary target.

Urothelial cells in vitro secrete most of MMP-9. As well, these cells are the primary target for THX-B, decreasing expression and secretion of MMP-9 and subsequently enhancing secretion of NGF. These results are in accordance with our previous publications on OAB patients and diabetes type 1 in rodents and suggest that p75 antagonism could be a therapeutic tool to improve OAB by targeting primarily the urothelium.

Mossa AH, Galan A, Cammisotto PG, Velasquez Flores M, Shamout S, Barcelona P, Saragovi HU, Campeau L. Antagonism of proNGF or its receptor p75NTR reverses remodelling and improves bladder function in a mouse model of diabetic voiding dysfunction. Diabetologia. 2020 Sep;63(9):1932-1946. doi: 10.1007/s00125-020-05222-4.
Mossa AH, Cammisotto PG, Shamout S, Campeau L. Imbalance of nerve growth factor metabolism in aging women with overactive bladder syndrome. World J Urol. 2020 Sep 1. doi: 10.1007/s00345-020-03422-6.