An Oligomeric Sulfated Hyaluronan and Silk-Elastinlike Polymer Combination Protects against Murine Radiation Induced Proctitis

Semisynthetic glycosaminoglycan ethers (SAGEs) are short, sulfated hyaluronans which combine the natural properties of hyaluronan with chemical sulfation. In a murine model, SAGEs provide protection against radiation induced proctitis (RIP), a side effect of lower abdominal radiotherapy for cancer. The anti-inflammatory effects of SAGE have been studied in inflammatory diseases at mucosal barrier sites; however, few mechanisms have been uncovered necessitating high throughput methods. SAGEs were combined with silk-elastinlike polymers (SELPs) to enhance rectal accumulation in mice. After high radiation exposure to the lower abdominal area, mice were followed for 3 days or until they met humane endpoints, before evaluation of behavioral pain responses and histological assessment of rectal inflammation. RNA sequencing was conducted on tissues from the 3-day cohort to determine molecular mechanisms of SAGE–SELP. After 3 days, mice receiving the SAGE–SELP combination yielded significantly lowered pain responses and amelioration of radiation-induced rectal inflammation. Mice receiving the drug–polymer combination survived 60% longer than other irradiated mice, with a fraction exhibiting long term survival. Sequencing reveals varied regulation of toll like receptors, antioxidant activities, T-cell signaling, and pathways associated with pain. This investigation elucidates several molecular mechanisms of SAGEs and exhibits promising measures for prevention of RIP

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

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

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

LL-37 instillation leads to local and systemic inflammatory changes and GM-0111 reduces the inflammatory effects of LL-37.

<p>The animals treated with LL-37 show decreased body weight gain (A), increased bladder weight (B), gross anatomical changes in the bladder (C), severity of inflammatory changes in histological observations (D), and the bladder concentration of IL-6 (E), PTX-3 (F), MPO activity (G) and blood level of SAP (H). *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001 compared to Normal or PBS pre-treatment/LL-37 treated control. Black horizontal bars are mean or median (necropsy and histology scores) values. BDL and the values in the parenthesis represent the number of samples that fell below the detection limit (dotted red line) of the ELISA out of the total samples tested (see Data Analysis in Materials and Methods section for details). Gray area represents the range of values found in normal animals.</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