The urothelial barrier in interstitial cystitis/bladder pain syndrome: its form and function, an overview of preclinical models.

PURPOSE OF REVIEW: Investigating bladder pain syndrome/interstitial cystitis (IC/BPS) preclinically is challenging. Various research models have been used to mimic the urothelial barrier closely and replicate the disease. The aim of this review is to discuss preclinical research related to the urothelial barrier in context of IC/BPS. RECENT FINDINGS: In vivo models mimic IC/BPS mainly with toxic substances in the urine, with protaminesulfate and proteoglycan deglycolysation resembling a temporary impaired barrier as seen in IC/BPS. This temporary increased permeability has also been found in vitro models. Glycosaminoglycan replenishment therapy has been described, in vivo and in vitro, to protect and enhance recover properties of the urothelium. The roles of immune and neurogenic factors in the pathogenesis of IC/BPS remains relatively understudied. SUMMARY: Preclinical studies provide opportunities to identify the involvement of specific pathologic pathways in IC/BPS. For further research is warranted to elucidate the primary or secondary role of permeability, together with inflammatory and neurogenic causes of the disease.

MRI as a Tool to Assess Interstitial Cystitis Associated Bladder and Brain Pathologies.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, often incapacitating condition characterized by pain seeming to originate in the bladder in conjunction with lower urinary tract symptoms of frequency and urgency, and consists of a wide range of clinical phenotypes with diverse etiologies. There are currently no diagnostic tests for IC/BPS. Magnetic resonance imaging (MRI) is a relatively new tool to assess IC/BPS. There are several methodologies that can be applied to assess either bladder wall or brain-associated alterations in tissue morphology and/or pain. IC/BPS is commonly associated with bladder wall hyperpermeability (BWH), particularly in severe cases. Our group developed a contrast-enhanced magnetic resonance imaging (CE-MRI) approach to assess BWH in preclinical models for IC/BPS, as well as for a pilot study for IC/BPS patients. We have also used the CE-MRI approach to assess possible therapies to alleviate the BWH in preclinical models for IC/BPS, which will hopefully pave the way for future clinical trials. In addition, we have used molecular-targeted MRI (mt-MRI) to quantitatively assess BWH biomarkers. Biomarkers, such as claudin-2, may be important to assess and determine the severity of BWH, as well as to assess therapeutic efficacy. Others have also used other MRI approaches to assess the bladder wall structural alterations with diffusion-weighted imaging (DWI), by measuring changes in the apparent diffusion coefficient (ADC), diffusion tensor imaging (DTI), as well as using functional MRI (fMRI) to assess pain and morphological MRI or DWI to assess anatomical or structural changes in the brains of patients with IC/BPS. It would be beneficial if MRI-based diagnostic tests could be routinely used for these patients and possibly used to assess potential therapeutics.

Innovative Approaches in the Battle Against Cancer Recurrence: Novel Strategies to Combat Dormant Disseminated Tumor Cells.

Cancer recurrence remains a great fear for many cancer survivors following their initial, apparently successful, therapy. Despite significant improvement in the overall survival of many types of cancer, metastasis accounts for ~90% of all cancer mortality. There is a growing understanding that future therapeutic practices must accommodate this unmet medical need in preventing metastatic recurrence. Accumulating evidence supports dormant disseminated tumor cells (DTCs) as a source of cancer recurrence and recognizes the need for novel strategies to target these tumor cells. This review presents strategies to target dormant quiescent DTCs that reside at secondary sites. These strategies aim to prevent recurrence by maintaining dormant DTCs at bay, or eradicating them. Various approaches are presented, including: reinforcing the niche where dormant DTCs reside in order to keep dormant DTCs at bay; promoting cell intrinsic mechanisms to induce dormancy; preventing the engagement of dormant DTCs with their supportive niche in order to prevent their reactivation; targeting cell-intrinsic mechanisms mediating long-term survival of dormant DTCs; sensitizing dormant DTCs to chemotherapy treatments; and, inhibiting the immune evasion of dormant DTCs, leading to their demise. Various therapeutic approaches, some of which utilize drugs that are already approved, or have been tested in clinical trials and may be considered for repurposing, will be discussed. In addition, clinical evidence for the presence of dormant DTCs will be reviewed, along with potential prognostic biomarkers to enable the identification and stratification of patients who are at high risk of recurrence, and who could benefit from novel dormant DTCs targeting therapies. Finally, we will address the shortcomings of current trial designs for determining activity against dormant DTCs and provide novel approaches.

SuperGAG biopolymers for treatment of excessive bladder permeability.

Few therapeutic options exist for treatment of IC/BPS. A novel high MW GAG biopolymer ("SuperGAG") was synthesized by controlled oligomerization of CS, purified by TFF and characterized by SEC-MALLS and 1H-NMR spectroscopy. The modified GAG biopolymer was tested in an OVX female rat model in which bladder permeability was induced by a 10-minute intravesicular treatment with dilute (1 mg/ml) protamine sulfate and measured by classical Ussing Chamber TEER measurements following treatment with SuperGAG, chondroitin sulfate, or saline. The effect on abrogating the abdominal pain response was assessed using von Frey filaments. The SuperGAG biopolymer was then investigated in a second, genetically modified mouse model (URO-MCP1) that increasingly is accepted as a model for IC/BPS. Permeability was induced with a brief exposure to a sub-noxious dose of LPS and was quantified using contrast-enhanced MRI (CE-MRI). The SuperGAG biopolymer restored impermeability to normal levels in the OVX rat model as measured by TEER in the Ussing chamber and reduced the abdominal pain response arising from induced permeability. Evaluation in the URO-MCP1 mouse model also showed restoration of bladder impermeability and showed the utility of CE-MRI imaging for evaluating the efficacy of agents to restore bladder impermeability. We conclude novel high MW SuperGAG biopolymers are effective in restoring urothelial impermeability and reducing pain produced by loss of the GAG layer on the urothelium. SuperGAG biopolymers could offer a novel and effective new therapy for IC/BPS, particularly if combined with MRI to assess the efficacy of the therapy.

In vivo and ex vivo assessment of bladder hyper-permeability and using molecular targeted magnetic resonance imaging to detect claudin-2 in a mouse model for interstitial cystitis.

OBJECTIVES: To determine if the URO-MCP-1 mouse model for bladder IC/BPS is associated with in vivo bladder hyper-permeability, as measured by contrast-enhanced MRI (CE-MRI), and assess whether molecular-targeted MRI (mt-MRI) can visualize in vivo claudin-2 expression as a result of bladder hyper-permeability. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, painful condition of the bladder that affects primarily women. It is known that permeability plays a substantial role in IC/BPS. Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. Claudin-2 is a molecular marker that is associated with increased hyperpermeability in the urothelium. MATERIALS AND METHODS: CE-MRI was used to measure bladder hyper-permeability in the URO-MCP-1 mice. A claudin-2-specific mt-MRI probe was used to assess in vivo levels of claudin-2. The mt-MRI probe consists of an antibody against claudin-2 conjugated to albumin that had Gd-DTPA (gadolinium diethylenetriamine pentaacetate) and biotin attached. Verification of the presence of the mt-MRI probe was done by targeting the biotin moiety for the probe with streptavidin-horse radish peroxidase (SA-HRP). Trans-epithelial electrical resistance (TEER) was also used to assess bladder permeability. RESULTS: The URO-MCP-1 mouse model for IC/BPS was found to have a significant increase in bladder permeability, following liposaccharide (LPS) exposure, compared to saline-treated controls. mt-MRI- and histologically-detectable levels of the claudin-2 probe were found to increase with LPS -induced bladder urothelial hyper-permeability in the URO-MCP-1 IC mouse model. Levels of protein expression for claudin-2 were confirmed with immunohistochemistry and immunofluorescence imaging. Claudin-2 was also found to highly co-localize with zonula occlidens-1 (ZO-1), a tight junction protein. CONCLUSION: The combination of CE-MRI and TEER approaches were able to demonstrate hyper-permeability, a known feature associated with some IC/BPS patients, in the LPS-exposed URO-MCP-1 mouse model. This MRI approach could be clinically translated to establish which IC/BPS patients have bladder hyper-permeability and help determine therapeutic options. In addition, the in vivo molecular-targeted imaging approach can provide invaluable information to enhance our understanding associated with bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing further therapeutic strategies.

Assessing bladder hyper-permeability biomarkers in vivo using molecularly-targeted MRI.

The objective was to investigate if some of the key molecular players associated with bladder hyper-permeability in interstitial cystitis/bladder pain syndrome (IC/BPS) could be visualized with molecularly-targeted magnetic resonance imaging (mt-MRI) in vivo. IC/BPS is a chronic, painful condition of the bladder that affects primarily women. It has been demonstrated over the past several decades that permeability plays a substantial role in IC/BPS. There are several key molecular markers that have been associated with permeability, including glycolsaminoglycan (GAG), biglycan, chondroitin sulfate, decorin, E-cadherin, keratin 20, uroplakin, vascular endothelial growth factor receptor 1 (VEGF-R1), claudin-2 and zonula occludens-1 (ZO-1). We used in vivo molecularly-targeted MRI (mt-MRI) to assess specific urothelial biomarkers (decorin, VEGF-R1, and claudin-2) associated with bladder hyper-permeability in a protamine sulfate (PS)-induced rat model. The mt-MRI probes consisted of an antibody against either VEGF-R1, decorin or claudin-2 conjugated to albumin that had also Gd-DTPA (gadolinium diethylene triamine penta acetic acid) and biotin attached. mt-MRI- and histologically-detectable levels of decorin and VEGF-R1 were both found to decrease following PS-induced bladder urothelial hyper-permeability, whereas claudin-2, was found to increase in the rat PS model. Verification of the presence of the mt-MRI probes were done by targeting the biotin moiety for each respective probe with streptavidin-hose radish peroxidase (HRP). Levels of protein expression for VEGF-R1, decorin and claudin-2 were confirmed with immunohistochemistry. In vivo molecularly-targeted MRI (mt-MRI) was found to successfully detect alterations in the expression of decorin, VEGFR1 and claudin-2 in a PS-induced rat bladder permeability model. This in vivo molecularly-targeted imaging approach has the potential to provide invaluable information to enhance our understanding of bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing novel therapeutic strategies.

Singlet oxygen-activatable Paclitaxel prodrugs via intermolecular activation for combined PDT and chemotherapy.

Systemic side effects and high hydrophobicity are major disadvantages of paclitaxel (PTX), one of the most popular anticancer drugs. Here, we present singlet oxygen (SO)-activatable and mitochondria-targeted PTX prodrugs to overcome these problems and boost the cytotoxic effect of photodynamic therapy (PDT). Three PTX prodrugs were prepared by conjugating PTX with various cationic groups. Hydrophobicity was determined in LogD7.4 value. Mitochondrial localization was confirmed by fluorescence confocal microscopy and uptake of mitochondria-specific fluorescence probe. Dark- and photo-toxicity were measured in AY-27 cells with MTT assay. All three prodrugs showed better hydrophilicity than PTX and improved phototoxicity when combined with protoporphyrin IX (PpIX) PDT. In conclusion, SO-activatable and higher hydrophilic PTX prodrugs were successfully prepared. This approach could be used to improve the antitumor efficacy of PDT without the systemic side effects of PTX.

Reduced urothelial regeneration in rat bladders augmented with permeable porcine small intestinal submucosa assessed by magnetic resonance imaging.

Augmentation enterocystoplasty remains the gold standard surgical bladder reconstruction procedure to increase the capacity and compliance of dysfunctional bladders. Since the use of the patient's intestine has severe risks of complications, alternative biodegradable matrices have been explored. Porcine small intestinal submucosa (SIS) has gained immense interests in bladder reconstruction due to its favorable properties. However, trials have shown inconsistent regeneration with SIS, attributed to the heterogeneity in microstructures and mechanical properties. We hypothesize that uneven SIS permeability to urine is a factor responsible for the inconsistency. We measured permeability to urine in situ using a contrast enhanced-magnetic resonance imaging (MRI), and evaluated urothelium regeneration using immunohistochemical staining of urothelial cell markers in SIS-augmented rat bladders. Results showed significant differences in permeability among SIS-augmented rat bladders. Commercial SIS scaffolds were then categorized into nonleaky and leaky groups based on MRI results. Hematoxylin and eosin staining showed higher numbers of inflammatory cells in leaky SIS on day 14 relative to nonleaky SIS. In addition, trichrome staining showed major changes in the distribution of collagen on day 28 between SIS-augmented bladder groups. Furthermore, expressions of urothelium-associated markers (cytokeratins AE1/AE3, claudin 4, and uroplakin III) were completed in bladders augmented with nonleaky SIS, whereas limited urothelial differentiation was noticed in leaky SIS-augmented bladders at post-augmentative day 14. These results show that scaffold permeability to urine may be responsible for variations in regenerative capacity of porcine SIS. Applications of MRI technique will be helpful to understand a relationship between biomaterial property and regenerative capacity. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1778-1787, 2018.

Phosphatidylserine targeted single-walled carbon nanotubes for photothermal ablation of bladder cancer.

Bladder cancer has a 60%-70% recurrence rate most likely due to any residual tumour left behind after a transurethral resection (TUR). Failure to completely resect the cancer can lead to recurrence and progression into higher grade tumours with metastatic potential. We present here a novel therapy to treat superficial tumours with the potential to decrease recurrence. The therapy is a heat-based approach in which bladder tumour specific single-walled carbon nanotubes (SWCNTs) are delivered intravesically at a very low dose (0.1 mg SWCNT per kg body weight) followed 24 h later by a short 30 s treatment with a 360° near-infrared light that heats only the bound nanotubes. The energy density of the treatment was 50 J cm-2, and the power density that this treatment corresponds to is 1.7 W cm-2, which is relatively low. Nanotubes are specifically targeted to the tumour via the interaction of annexin V (AV) and phosphatidylserine, which is normally internalised on healthy tissue but externalised on tumours and the tumour vasculature. SWCNTs are conjugated to AV, which binds specifically to bladder cancer cells as confirmed in vitro and in vivo. Due to this specific localisation, NIR light can be used to heat the tumour while conserving the healthy bladder wall. In a short-term efficacy study in mice with orthotopic MB49 murine bladder tumours treated with the SWCNT-AV conjugate and NIR light, no tumours were visible on the bladder wall 24 h after NIR light treatment, and there was no damage to the bladder. In a separate survival study in mice with the same type of orthotopic tumours, there was a 50% cure rate at 116 days when the study was ended. At 116 days, no treatment toxicity was observed, and no nanotubes were detected in the clearance organs or bladder.

Efficient activation of a visible light-activatable CA4 prodrug through intermolecular photo-unclick chemistry in mitochondria.

Photo-unclick chemistry mediates visible and near IR-controlled drug release via a singlet oxygen (SO)-cleavable linker. Due to the limited diffusion distance of SO in biological systems, a photosensitizer and the SO-cleavable linker have been conjugated in one molecule or mixed in nano-drug delivery systems. In this communication, we demonstrate a new strategy to activate prodrugs with photo-unclick chemistry in an intermolecular fashion using an SO-cleavable CA4 prodrug and a mitochondria-specific photosensitizer, protoporphyrin IX, formed from prodrug hexyl-5-aminolevulinate.

Targeting dormant micrometastases: rationale, evidence to date and clinical implications.

In spite of decades of research, cancer survival has increased only modestly. This is because most research is based on models of primary tumors. Slow recognition has begun that disseminated, dormant cancer cells (micrometastatic cells) that are generally resistant to chemotherapy are the culprits in recurrence, and until these are targeted effectively we can expect only slow progress in increasing overall survival from cancer. This paper reviews efforts to understand the mechanisms by which cancer cells can become dormant, and thereby identify potential targets and drugs either on the market or in clinical trials that purport to prevent metastasis. This review targets the most recent literature because several excellent reviews have covered the literature from more than two years ago. The paper also describes recent work in the authors' laboratories to develop a screening-based approach that does not require understanding of mechanisms of action or the molecular target. Success of this approach shows that targeting micrometastatic cells is definitely feasible.

In the absence of overt urothelial damage, chondroitinase ABC digestion of the GAG layer increases bladder permeability in ovariectomized female rats.

Loss of integrity of the protective impermeability barrier in the urothelium has been identified as significant in bladder dysfunction. In this study, we tested the theory that the luminal layer of glycosaminoglycans (GAG) serves as an important component of barrier function. The peptide polycation protamine sulfate (PS), 1 mg/ml, was instilled intravesically for 10 min into rat bladders. Chondroitinase ABC (ChABC), 63 IU/ml, was instilled into an additional six rats for 30 min to digest the GAG layer. Unmanipulated controls and sham-injected controls were also performed. After 24 h, the rats were euthanized, the bladders were removed, and permeability was assessed in the Ussing chamber and by diffusion of FITC-labeled dextran (4 kDa) to measure macromolecular permeability. The status of tight junctions was assessed by immunofluorescence and electron microscopy. In control and sham treated rat bladders, the transepithelial electrical resistance were means of 2.5 ± 1.1 vs. 2.6 ± 1.1 vs 1.2 ± 0.5 and 1.01 ± 0.7 kΩ·cm(2) in the PS-treated and ChABC-treated rat bladders (P = 0.0016 and P = 0.0039, respectively). Similar differences were seen in dextran permeability. Histopathology showed a mild inflammation following PS treatment, but the ChABC-treated bladders were indistinguishable from controls. Tight junctions generally remained intact. ChABC digestion alone induced bladder permeability, confirming the importance of the GAG layer to bladder barrier function and supports that loss of the GAG layer seen in bladder biopsies of interstitial cystitis patients could be a significant factor producing symptoms for at least some interstitial cystitis/painful bladder syndrome patients.

A Feasibility Study to Determine Whether Clinical Contrast Enhanced Magnetic Resonance Imaging can Detect Increased Bladder Permeability in Patients with Interstitial Cystitis.

PURPOSE: Interstitial cystitis/bladder pain syndrome is a bladder pain disorder associated with voiding symptomatology and other systemic chronic pain disorders. Currently diagnosing interstitial cystitis/bladder pain syndrome is complicated as patients present with a wide range of symptoms, physical examination findings and clinical test responses. One hypothesis is that interstitial cystitis symptoms arise from increased bladder permeability to urine solutes. This study establishes the feasibility of using contrast enhanced magnetic resonance imaging to quantify bladder permeability in patients with interstitial cystitis. MATERIALS AND METHODS: Permeability alterations in bladder urothelium were assessed by intravesical administration of the magnetic resonance imaging contrast agent Gd-DTPA (Gd-diethylenetriaminepentaacetic acid) in a small cohort of patients. Magnetic resonance imaging signal intensity in patient and control bladders was compared regionally and for entire bladders. RESULTS: Quantitative assessment of magnetic resonance imaging signal intensity indicated a significant increase in signal intensity in anterior bladder regions compared to posterior regions in patients with interstitial cystitis (p <0.01) and significant increases in signal intensity in anterior bladder regions (p <0.001). Kurtosis (shape of probability distribution) and skewness (measure of probability distribution asymmetry) were associated with contrast enhancement in total bladders in patients with interstitial cystitis vs controls (p <0.05). Regarding symptomatology interstitial cystitis cases differed significantly from controls on the SF-36®, PUF (Pelvic Pain and Urgency/Frequency) and ICPI (Interstitial Cystitis Problem Index) questionnaires with no overlap in the score range in each group. ICSI (Interstitial Cystitis Symptom Index) differed significantly but with a slight overlap in the range of scores. CONCLUSIONS: Data suggest that contrast enhanced magnetic resonance imaging provides an objective, quantifiable measurement of bladder permeability that could be used to stratify bladder pain patients and monitor therapy.

Impaired Expression of Prostaglandin E2 (PGE2) Synthesis and Degradation Enzymes during Differentiation of Immortalized Urothelial Cells from Patients with Interstitial Cystitis/Painful Bladder Syndrome.

PURPOSE: The differentiated superficial cells of the urothelium restrict urine flow into the bladder wall. We have demonstrated that urothelial cells isolated from bladders of patients with interstitial cystitis/painful bladder syndrome (IC/PBS) fail to release PGE2 in response to tryptase. This study examines the expression of PGE2 synthesis and degradation enzymes in urothelial cells during differentiation. MATERIALS AND METHODS: We measured immunoprotein expression of cyclooxygenase-2 (COX-2), prostaglandin E2 synthase (PGES) and 15-hydroxyprostaglandin dehydrogenase (PGDH) in human urothelial cells and in immortalized urothelial cells isolated from the bladders of IC/PBS patients or normal subjects during stratification and differentiation produced by increased calcium and fetal bovine serum (Ca/FBS) in the culture medium for 1, 3 and 7 days. RESULTS: PGES immunoprotein expression increased during differentiation in normal and IC/PBS urothelial cells. COX-2 expression also increased in cells from normal patients following differentiation. Remarkably, no COX-2 expression was detectable in urothelial cells isolated from 3 out of 4 IC/PBS patients. PGDH immunoprotein expression decreased in normal cells after 1 and 3 days of Ca/FBS addition, but returned to normal after 7 days. PGDH expression was unchanged during differentiation at 1 and 3 days, but was more than 2-fold higher at 7 days compared to day 0 in the IC/PBS cells. Urothelial cells isolated from IC/PBS patients demonstrated no PGE2 release in response to tryptase under any of the experimental conditions studied. CONCLUSIONS: Taken together, our results indicate that PGE2 release is compromised during stratification and differentiation in IC/PBS urothelium and may contribute to impaired barrier function.

Identification of novel drugs to target dormant micrometastases.

BACKGROUND: Cancer-specific survival has changed remarkably little over the past half century, mainly because metastases that are occult at diagnosis and generally resistant to chemotherapy subsequently develop months, years or even decades following definitive therapy. Targeting the dormant micrometastases responsible for these delayed or occult metastases would represent a major new tool in cancer patient management. Our hypothesis is that these metastases develop from micrometastatic cells that are suppressed by normal extracellular matrix (ECM). METHODS: A new screening method was developed that compared the effect of drugs on the proliferation of cells grown on a normal ECM gel (small intestine submucosa, SISgel) to cells grown on plastic cell culture plates. The desired endpoint was that cells on SISgel were more sensitive than the same cells grown as monolayers. Known cancer chemotherapeutic agents show the opposite pattern. RESULTS: Screening 13,000 compounds identified two leads with low toxicity in mice and EC50 values in the range of 3-30 µM, depending on the cell line, and another two leads that were too toxic to mice to be useful. In a novel flank xenograft method of suppressed/dormant cells co-injected with SISgel into the flank, the lead compounds significantly eliminated the suppressed cells, whereas conventional chemotherapeutics were ineffective. Using a 4T1 triple negative breast cancer model, modified for physiological metastatic progression, as predicted, both lead compounds reduced the number of large micrometastases/macrometastases in the lung. One of the compounds also targeted cancer stem cells (CSC) isolated from the parental line. The CSC also retained their stemness on SISgel. Mechanistic studies showed a mild, late apoptotic response and depending on the compound, a mild arrest either at S or G2/M in the cell cycle. CONCLUSIONS: In summary we describe a novel, first in class set of compounds that target micrometastatic cells and prevent their reactivation to form recurrent tumors/macrometastases.

Abnormalities in Expression of Structural, Barrier and Differentiation Related Proteins, and Chondroitin Sulfate in Feline and Human Interstitial Cystitis.

PURPOSE: We analyzed the urothelium of cats diagnosed with feline interstitial cystitis to determine whether abnormalities in protein expression patterns could be detected and whether the expression pattern was similar to that in patients with human interstitial cystitis/bladder pain syndrome. The proteins analyzed are involved in cell adhesion and barrier function, comprise the glycosaminoglycan layer or are differentiation markers. MATERIALS AND METHODS: Formalin fixed biopsies from 8 cats with feline interstitial cystitis and from 7 healthy control cats were labeled by immunohistochemistry and scored with a modified version of a system previously used for human samples. Cluster analysis was performed to investigate relationships between markers and samples. RESULTS: Of the feline interstitial cystitis bladders 89% showed abnormal protein expression and chondroitin sulfate patterns while only 27% of normal tissues showed slight abnormalities. Abnormalities were found in most feline interstitial cystitis samples, including biglycan in 87.5%, chondroitin sulfate, decorin, E-cadherin and keratin-20 in 100%, uroplakin in 50% and ZO-1 in 87.5%. In feline interstitial cystitis bladders about 75% of chondroitin sulfate, biglycan and decorin samples demonstrated absent luminal staining or no staining. Cluster analysis revealed that feline interstitial cystitis and normal samples could be clearly separated into 2 groups, showing that the urothelium of cats with feline interstitial cystitis is altered from normal urothelium. CONCLUSIONS: Feline interstitial cystitis produces changes in luminal glycosaminoglycan and several proteins similar to that in patients, suggesting some commonality in mechanism. Results support the use of feline interstitial cystitis as a model of human interstitial cystitis.

Contrast enhanced magnetic resonance imaging as a diagnostic tool to assess bladder permeability and associated colon cross talk: preclinical studies in a rat model.

PURPOSE: Interstitial cystitis/painful bladder syndrome is a devastating disease associated with multiple symptoms. It is usually diagnosed based on pain, urgency and frequency in the absence of other known causes. To our knowledge there is no diagnostic test to date. MATERIALS AND METHODS: In a model of rats intravesically exposed to protamine sulfate we performed in vivo diagnostic contrast enhanced magnetic resonance imaging with intravesical administration of Gd-diethylenetriamine pentaacetic acid contrast medium via a catheter to visualize increased bladder urothelium permeability. Gd-diethylenetriamine pentaacetic acid was administered intravenously to visualize secondary tissue effects in the colon. RESULTS: Bladder urothelium and colon mucosa were assessed 24 hours after bladder protamine sulfate exposure. Enhanced contrast magnetic resonance imaging established bladder urothelium leakage of Gd-diethylenetriamine pentaacetic acid according to the change in magnetic resonance imaging signal intensity in rats exposed to protamine sulfate vs controls (mean ± SD 399.7% ± 68.7% vs 39.2% ± 12.2%, p < 0.0001) as well as colon related uptake of contrast agent (mean 65.2% ± 17.1% vs 20.8% ± 9.8%, p < 0.01) after bladder protamine sulfate exposure. The kinetics of Gd-diethylenetriamine pentaacetic acid uptake and excretion were also assessed during 20 minutes of bladder and 30 minutes of colon exposure with increased signal intensity at 7 and 12 minutes, respectively. CONCLUSIONS: These preliminary studies indicate that contrast enhanced magnetic resonance imaging can be used to monitor primary bladder urothelium loss of permeability and secondary enhanced contrast medium in the colon mucosa. It can be considered a potential clinical diagnostic method for interstitial cystitis/painful bladder syndrome that involves loss of the permeability barrier. It can also be used to assess visceral organ cross talk.

Temporal expression of hyaluronic acid and hyaluronic acid receptors in a porcine small intestinal submucosa-augmented rat bladder regeneration model.

INTRODUCTION: Hyaluronic acid (HA), a non-sulfated glycosaminoglycan, is an essential component of the extracellular matrix (ECM). Since HA is involved in many phases of wound healing and may play a key role in tissue repair and regeneration, this study was intended to understand temporal and spatial expression of HA and HA receptors (HARs) during the course of bladder regeneration in rats. MATERIALS AND METHODS: Sprague-Dawley rats were subjected to partial cystectomy followed by augmentation with porcine small intestinal submucosal (SIS) prepared from distal sections of the small intestine. SIS-augmented bladders were harvested between postoperative days 2 and 56. RESULTS: Bladder regeneration proceeded without complications. All augmented bladders had complete urothelial lining and smooth muscle bundles by day 56 post-augmentation. Temporal and spatial distributions of HA and HARs were studied by immunohistochemistry in regenerating bladders. The strongest HA immunoreactivity was observed in the ECM on postoperative days 28 and 56. Cluster of differentiation 44 (CD44) immunoreactivity was detected in the cytoplasm of urothelial cells on day 56; and LYVE-1 immunoreactivity was exclusively limited to lymphatic vessels on days 28 and 56. CONCLUSIONS: We demonstrated that HA was synthesized throughout the course of bladder wound healing and regeneration; and HA deposition coincided with urothelial differentiation. Expression of CD44 and LYVE-1 followed the same temporal pattern as HA deposition. Therapeutic modalities through local delivery of exogenous HA to improve the outcome of SIS-mediated bladder regeneration might need to be coordinated with HAR expression in order to achieve maximal regenerative responses as opposed to fibrosis.

Increased bladder permeability in interstitial cystitis/painful bladder syndrome.

The definition of interstitial cystitis (IC) has evolved over the years from being a well-defined entity characterized by diagnostic lesion (Hunner's ulcer) in the urothelium to a clinical diagnosis by exclusion (painful bladder syndrome (PBS). Although the etiology is unknown, a central theme has been an association with increased permeability of the bladder. This article reviews the evidence for increased permeability being important to the symptoms of IC/PBS and in treating the disorder. Recent work showing cross-communication among visceral organs is also reviewed to provide a basis for understanding IC/PBS as a systemic disorder of a complex, interconnected system consisting of the bladder, bowel and other organs, nerves, cytokine-responding cells and the nervous system.

Development and characterization of a preclinical model of breast cancer lung micrometastatic to macrometastatic progression.

Most cancer patients die with metastatic disease, thus, good models that recapitulate the natural process of metastasis including a dormancy period with micrometastatic cells would be beneficial in developing treatment strategies. Herein we report a model of natural metastasis that balances time to complete experiments with a reasonable dormancy period, which can be used to better study metastatic progression. The basis for the model is a 4T1 triple negative syngeneic breast cancer model without resection of the primary tumor. A cell titration from 500 to 15,000 GFP tagged 4T1 cells implanted into fat pad number four of immune proficient eight week female BALB/cJ mice optimized speed of the model while possessing metastatic processes including dormancy and beginning of reactivation. The frequency of primary tumors was less than 50% in animals implanted with 500-1500 cells. Although implantation with over 10,000 cells resulted in 100% primary tumor development, the tumors and macrometastases formed were highly aggressive, lacked dormancy, and offered no opportunity for treatment. Implantation of 7,500 cells resulted in >90% tumor take by 10 days; in 30-60 micrometastases in the lung (with many animals also having 2-30 brain micrometastases) two weeks post-implantation, with the first small macrometastases present at five weeks; many animals displaying macrometastases at five weeks and animals becoming moribund by six weeks post-implantation. Using the optimum of 7,500 cells the efficacy of a chemotherapeutic agent for breast cancer, doxorubicin, given at its maximal tolerated dose (MTD; 1 mg/kg weekly) was tested for an effect on metastasis. Doxorubicin treatment significantly reduced primary tumor growth and lung micrometastases but the number of macrometastases at experiment end was not significantly affected. This model should prove useful for development of drugs to target metastasis and to study the biology of metastasis.