Prevention of Anti-microbial Peptide LL-37-Induced Apoptosis and ATP Release in the Urinary Bladder by a Modified Glycosaminoglycan

<div><p>Interstitial cystitis (IC), often referred to in combination with painful bladder syndrome, is a chronic inflammatory disease of the bladder. Current therapies primarily focus on replenishing urothelial glycosaminoglycan (GAG) layer using GAG analogs and managing pain with supportive therapies. However, the elusive etiology of IC and the lack of animal models to study the disease have been major hurdles developing more effective therapeutics. Previously, we showed an increased urinary concentration of antimicrobial peptide LL-37 in spina bifida patients and used LL-37 to develop a mouse model of cystitis that mimics important clinical findings of IC. Here we investigate (1) the molecular mechanism of LL-37 induced cystitis in cultured human urothelial cells and in mice, (2) the protective effects of GM-0111, a modified GAG, within the context of this mechanism, (3) the physiological and molecular markers that correlate with the severity of the inflammation, and (4) the protective effects of several GAGs using these biomarkers in our LL-37 induced cystitis model. We find that LL-37 quickly induces release of ATP and apoptosis in the urothelium. These changes can be inhibited by a chemically-modified GAG, GM-0111. Furthermore, we also find that GAG analogs provide varying degrees of protection against LL-37 challenge in mice. These findings suggest that GM-0111 and possibly GAG molecules prevent the development of cystitis by blocking the apoptosis and the concurrent release of ATP from the urothelium.</p></div

LL-37 induces ATP release from human urothelial cells (HUCs) (A) and GM-0111 reduces LL-37 induced ATP release in a dose-dependent manner (B).

<p>HUCs were challenged with LL-37 at various concentrations for 15 min and the culture supernatants were analyzed for ATP. To determine the inhibitory effects of GM-0111 against LL-37 induced ATP release, HUCs were treated with GM-0111 for 30 min prior to the 15 min LL-37 challenge (3 µM). Dotted red line indicates the basal concentration of ATP in the cell culture supernatant.</p

LL-37 induces inflammatory changes in the urinary bladder (A, B, C, and D: normal bladder; E, F, G, and H: 24 hr after 250 µM LL-37 treatment).

<p>Microscopic observations show that LL-37 causes edema (*), massive infiltration of leukocytes including PMNs and occasional hemorrhage (black arrow head) along with urothelial erosion (arrow). A and E are photostiched images of the entire bladder surface stained with H&E (lower panels are the enlarged views of the square regions). B and F are immunostained images for Ly6G, the surface antigen from PMNs (blue arrowheads). C and G are immunostained images for IL-6 showing strong immunoreactivities of IL-6 at the submucosal area in the LL-37 treated bladder. D and H are immunostained images for PTX-3 showing marked increase of PTX-3 positive cells in the LL-37 treated bladder. Lu: lumen, Mu: mucosa, ML: muscle layer.</p

Comparison of the emission wavelengths by a single fluorescent dye on in vivo 3-photon imaging of mouse brains

Multiphoton microscopy (MPM) is a powerful imaging technology for brain research. The imaging depth in MPM is partly determined by emission wavelength of fluorescent labels. It has been demonstrated that a longer emission wavelength is favorable for signal detection as imaging depth increases. However, there has been no comparison with near-infrared (NIR) emission. In order to quantitatively analyze the effect of emission wavelength on 3-photon imaging of mouse brains in vivo, we utilize the same excitation wavelength to excite a single fluorescent dye and simultaneously collect NIR and orange-red emission fluorescence at 828[Formula: see text]nm and 620[Formula: see text]nm, respectively. Both experimental and simulation results show that as the imaging depth increases, NIR emission decays less than orange-red fluorescent emission. These results show that it is preferable to shift the emission wavelength to NIR to enable more efficient signal collection deep in the brain

GM-0111 reduces LL-37 induced apoptosis in human urothelial cells (HUCs).

<p>HUCs pretreated with GM-0111 at 0, 0.1, 1.0, and 10 mg/mL for 30 min were challenged with LL-37 (25 µM) for 15 min at 37°C. Cells were stained with annexin V-FITC (shown in green) and 7-AAD (shown in red) to visualize (A) with microscopy (magnification 10<i>x</i>) and to quantify (B) the apoptotic cells with flow cytometry. **<i>p</i><0.01, ***<i>p</i><0.001 and NS, not significant (<i>p</i>>0.05) by Dunnet’s <i>t</i>-test.</p

LL-37 induces apoptotic cell death in human urothelial cells (HUCs).

<p>Cultured HUCs were treated with LL-37 for 15 min at 37°C. HUCs were then collected and labeled with annexin V-FITC (shown in green) and 7-AAD (shown in red). A, Fluorescence microscopy shows the increased number of apoptotic cells in LL-37 treated HUCs (magnification 10<i>x</i>). B, Flow cytometry analysis of HUCs shows that LL-37 induces apoptotic cell death at ≥ 10 µM or higher concentrations of LL-37. *<i>p</i><0.05 and ***<i>p</i><0.001 by Dunnet’s <i>t</i>-test.</p

LL-37 induces apoptotic cell death in the urinary bladder.

<p>The mucosal cells in the urinary bladder undergo apoptotic cell death and are removed quickly. TUNEL stain (top panels) and corresponding bright field (bottom panels) images of bladders from (A) normal animal, (B) normal bladder treated with TACS nuclease for positive control, (C) 1 hr after LL-37 (320 µM) challenge, and (D) 3 hr after LL-37 challenge. Red lines indicate the urothelial layers. Lu is the lumen of the bladder.</p

Multivariate analysis of various observations in LL-37 induced cystitis model and the inhibitory effects of GM-0111.

<p>Each intersecting graph shows the correlation between the two respective observational parameters. Graphs in the diagonal array represent the distribution of data from each parameter pooled from normal and LL-37/GM-0111 treated animals. Green lines indicate the correlation in each set of parameters and red dotted lines indicate the values following similar trends. Bold rectangular areas represent the correlation between physiological measurements and the concentrations of molecular markers. The values inside boxes are Spearman correlation coefficients (ρ).</p