Normalization of proliferation and tight junction formation in bladder epithelial cells from patients with interstitial cystitis/painful bladder syndrome by d-proline and d-pipecolic acid derivatives of antiproliferative factor

Abstract

Interstitial cystitis/painful bladder syndrome is a chronic bladder disorder with epithelial thinning or ulceration, pain, urinary frequency and urgency, for which there is no reliably effective therapy. We previously reported that interstitial cystitis/painful bladder syndrome bladder epithelial cells make a glycopeptide antiproliferative factor or 'APF' (Neu5Acα2-3Galβ1-3GalNAcα-O-TVPAAVVVA) that induces abnormalities in normal cells similar to those in interstitial cystitis/painful bladder syndrome cells in vitro, including decreased proliferation, decreased tight junction formation, and increased paracellular permeability. We screened inactive APF derivatives for their ability to block antiproliferative activity of asialylated-APF ('as-APF') in normal bladder cells and determined the ability of as-APF-blocking derivatives to normalize tight junction protein expression, paracellular permeability, and/or proliferation of interstitial cystitis/painful bladder syndrome cells. Only two of these derivatives [Galβ1-3GalNAcα-O-TV-(d-pipecolic acid)-AAVVVA and Galβ1-3GalNAcα-O-TV-(d-proline)-AAVVVA] blocked as-APF antiproliferative activity in normal cells (p < 0.001 for both). Both of these antagonists also 1) significantly increased mRNA expression of ZO-1, occludin, and claudins 1, 4, 8, and 12 in interstitial cystitis/painful bladder syndrome cells by qRT-PCR; 2) normalized interstitial cystitis/painful bladder syndrome epithelial cell tight junction protein expression and tight junction formation by confocal immunofluorescence microscopy; and 3) decreased paracellular permeability of (14) C-mannitol and (3) H-inulin between confluent interstitial cystitis/painful bladder syndrome epithelial cells on Transwell plates, suggesting that these potent APF antagonists may be useful for the development as interstitial cystitis/painful bladder syndrome therapies.

Figure 1. Structures of as-APF, d-proline as-APF, and d-pipecolic acid as-APF derivatives

Figure 2. Antiproliferative activity of as-APF, d-proline as-APF, and d-pipecolic acid as-APF derivatives in normal bladder epithelial cell explant cultures

Explanted normal bladder epithelial cells were treated with varying concentrations of as-APF (■), inactive control peptide (□), d-proline as-APF (▼), or d-pipecolic acid as-APF (○) for 48 hours prior to determination of ³H-thymidine incorporation. Assay was performed in triplicate twice; data are expressed as percent inhibition of thymidine incorporation compared to control cells incubated with medium alone +/- standard deviation.

Figure 3. Blocking of as-APF activity by d-proline as-APF and d-pipecolic acid as-APF in normal bladder epithelial cells

Explanted normal bladder epithelial cells were preincubated with varying concentrations of d-proline as-APF (■) or d-pipecolic acid as-APF (○) for 1.5 hours at 37°C, after which 125 nM as-APF was added to the medium; cell proliferation was assessed by ³H-thymidine incorporation 48 hours later. The assay was performed in triplicate twice; data are expressed as percent inhibition of thymidine incorporation compared to control cells incubated with medium alone +/- standard deviation.

Figure 4. Stimulation of IC/PBS cell proliferation by d-proline as-APF and d-pipecolic acid as-APF

Explanted cells from 3 IC/PBS donors were treated with varying concentrations of d-proline as-APF (■) or d-pipecolic acid as-APF (○); controls were incubated with culture medium alone (Δ). Cell proliferation was assessed by ³H-thymidine incorporation 48 hours later. The assay was performed in triplicate twice for each donor, and data points combined for all donors; data are expressed as counts per minute (CPM) +/- standard error of the mean.

Figure 5. RT-PCR analysis of tight junction protein mRNA expression in IC/PBS cells following treatment with d-proline as-APF or d-pipecolic acid as-APF

Explanted cells from IC/PBS donors were fed and treated with 2.5 μM d-proline as-APF (□), d-pipecolic acid as-APF ( ), inactive peptide ( ), or diluent control (■) twice weekly, and RNA was extracted at 9, 16, 23 and 30 days for qRT-PCR. By day 16, both APF derivatives were able to significantly (p < .05) stimulate tight junction protein expression in IC/PBS cells in vitro resulting in levels similar to those seen in bladder cells from age- and gender-matched normal donors ( ). PCR was performed in duplicate on three separate occasions for each sample; data are expressed as mean +/- standard error of the mean. (Data shown from experiment with the same IC/PBS cell donor treated simultaneously with either d-pipecolic acid as-APF or d-proline as-APF; d-proline as-APF was tested on cells from a total of 4 IC/PBS donors, with similar results)

Figure 6. Immunofluorescence confocal microscopy of IC/PBS cell explants treated with d-pipecolic acid as-APF, d-proline as-APF, or vehicle (PBS) alone for 9 days

Explanted cells from an IC/PBS donor were treated with 2.5 μM d-proline as-APF, d-pipecolic acid as-APF, or diluent (PBS) control twice weekly. On day 9, cells were fixed with acetone/ethanol and incubated with FITC-labeled anti-ZO-1 antibody, or primary antibodies against claudin or occludin proteins followed by FITC-labeled secondary antibodies. Images were obtained on a Zeiss LSM510 confocal laser scanning microscope. (Figure is representative of three separate experiments using cells from 3 IC/PBS donors)

Figure 7. Decreased paracellular permeability of IC/PBS cells by d-proline as-APF

Paracellular flux of [³H]-inulin (molecular weight: 5,200) or [¹⁴C]-mannitol (molecular weight: 184) was measured in IC/PBS cells cultured on Transwell membranes and treated with 2.5 μM d-proline as-APF (□), inactive peptide control ( ), diluent control (■), or calcium free medium ( ) for 16 days prior to incubation with tracer. Data are expressed as mean percent of the radioactivity applied to the apical medium that was recovered in the basal medium at 0.5 hr, 1 hr, 2 hrs, 4 hrs, 6 hrs +/- standard deviation. (Data shown from 4 experiments using cells from 4 different IC/PBS donors).

Figure 8. Decreased paracellular permeability of IC/PBS cells by d-pipecolic acid as-APF

Paracellular flux of [³H]-inulin (molecular weight: 5,200) or [¹⁴C]-mannitol (molecular weight: 184) was measured in IC/PBS cells cultured on Transwell membranes and treated with 2.5 μM d-pipecolic acid as-APF (□), inactive peptide control ( ), diluent control (■), or calcium free medium ( ) for 16 days prior to incubation with tracer. Data are expressed as mean percent of the radioactivity applied to the apical medium that was recovered in the basal medium at 0.5 hr, 1 hr, 2 hrs, 4 hrs, 6 hrs +/- standard deviation. (Data shown from 3 experiments using cells from 3 different IC/PBS donors).