Mechanism underlying follicular hyperproliferation and oncogenesis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a skin disorder that causes chronic painful inflammation and hyperproliferation, often with the comorbidity of invasive keratoacanthoma (KA). Our research, employing high-resolution immunofluorescence and data science approaches together with confirmatory molecular analysis, has identified that the 5'-cap-dependent protein translation regulatory complex eIF4F is a key factor in the development of HS and is responsible for regulating follicular hyperproliferation. Specifically, eIF4F translational targets, Cyclin D1 and c-MYC, orchestrate the development of HS-associated KA. Although eIF4F and p-eIF4E are contiguous throughout HS lesions, Cyclin D1 and c-MYC have unique spatial localization and functions. The keratin-filled crater of KA is formed by nuclear c-MYC-induced differentiation of epithelial cells, whereas the co-localization of c-MYC and Cyclin D1 provides oncogenic transformation by activating RAS, PI3K, and ERK pathways. In sum, we have revealed a novel mechanism underlying HS pathogenesis of follicular hyperproliferation and the development of HS-associated invasive KA.

Conformationally Defined Rexinoids for the Prevention of Inflammation and Nonmelanoma Skin Cancers.

Compound 1 is a potent rexinoid that is highly effective in cancer chemoprevention but elevates serum triglycerides. In an effort to separate the lipid toxicity from the anticancer activity of 1, we synthesized four new analogs of rexinoid 1, of which three rexinoids did not elevate serum triglycerides. Rexinoids 3 and 4 are twice as potent as rexinoid 1 in binding to Retinoid X receptor (RXR). All-trans retinoic acid (ATRA) plays a key role in maintaining skin homeostasis, and rexinoids 3-6 are highly effective in upregulating the genes responsible for the biosynthesis of ATRA. Inflammation plays a key role in skin cancer, and rexinoids 3 and 4 are highly effective in diminishing LPS-induced inflammation. Rexinoids 3 and 4 are highly effective in preventing UVB-induced nonmelanoma skin cancer (NMSC) without displaying any overt toxicities. Biophysical studies of rexinoids 3 and 5 bound to hRXRα-ligand binding domain (LBD) reveal important conformational and dynamical differences in the ligand binding domain.

Epigenetic regulation in the pathogenesis of non-melanoma skin cancer.

Understanding of cancer with the help of ever-expanding cutting edge technological tools and bioinformatics is revolutionizing modern cancer research by broadening the space of discovery window of various genomic and epigenomic processes. Genomics data integrated with multi-omics layering have advanced cancer research. Uncovering such layers of genetic mutations/modifications, epigenetic regulation and their role in the complex pathophysiology of cancer progression could lead to novel therapeutic interventions. Although a plethora of literature is available in public domain defining the role of various tumor driver gene mutations, understanding of epigenetic regulation of cancer is still emerging. This review focuses on epigenetic regulation association with the pathogenesis of non-melanoma skin cancer (NMSC). NMSC has higher prevalence in Caucasian populations compared to other races. Due to lack of proper reporting to cancer registries, the incidence rates for NMSC worldwide cannot be accurately estimated. However, this is the most common neoplasm in humans, and millions of new cases per year are reported in the United States alone. In organ transplant recipients, the incidence of NMSC particularly of squamous cell carcinoma (SCC) is very high and these SCCs frequently become metastatic and lethal. Understanding of solar ultraviolet (UV) light-induced damage and impaired DNA repair process leading to DNA mutations and nuclear instability provide an insight into the pathogenesis of metastatic neoplasm. This review discusses the recent advances in the field of epigenetics of NMSCs. Particularly, the role of DNA methylation, histone hyperacetylation and non-coding RNA such as long-chain noncoding (lnc) RNAs, circular RNAs and miRNA in the disease progression are summarized.

In-silico Studies and Wet-Lab Validation of Camptothecin Derivatives for Anti-Cancer Activity Against Liver (HepG2) and Lung (A549) Cancer Cell Lines.

BACKGROUND: In the present study, we have explored the utility of QSAR modelling, in silico ADMET, docking, chemical semi-synthesis, and in vitro evaluation studies for the identification of active camptothecin (CPT) derivatives against cancer-targeting human liver (HepG2) and lung (A549) cancer cell lines. METHODS: Two QSAR models were developed as screenings tools using the multiple linear regression (MLR) method followed by ADMET and docking studies. The regression coefficient (r2) and cross-validation regression coefficients (rCV2T) of the QSAR model for the HepG2 cell line was 0.95 and 0.90, respectively, and for the A549 cell line, it was 0.93 and 0.81, respectively. RESULTS: In silico studies show that CPT derivatives (CPT-1 and CPT-6) possess drug-like properties. Docking performed on DNA Topoisomerase-I showed significant binding affinity. Finally, predicted active derivatives were chemically semi synthesized, spectroscopically characterized, and evaluated in-vitro for cytotoxic/anticancer activity against HepG2 and A549 cell lines. CONCLUSION: The experimental results are consistent with the predicted results. These findings may be of immense importance in the anticancer drug development from an inexpensive and widely available natural product, camptothecin.

Patched1 haploinsufficiency severely impacts intermediary metabolism in the skin of Ptch1+/-/ODC transgenic mice.

The study of dominantly heritable cancers has provided insights about tumor development. Gorlin syndrome (GS) is an autosomal dominant disorder wherein affected individuals develop multiple basal cell carcinomas (BCCs) of the skin. We developed a murine model of Ptch1 haploinsufficiency on an ornithine decarboxylase (ODC) transgenic background (Ptch1+/-/ODCt/C57BL/6) that is more sensitive to BCCs growth as compared with Ptch1+/+/ODCt/C57BL/6 littermates. Ptch1+/-/ODCt/C57BL/6 mice show an altered metabolic landscape in the phenotypically normal skin, including restricted glucose availability, restricted ribose/deoxyribose flow and NADPH production, an accumulation of α-ketoglutarate, aconitate, and citrate that is associated with reversal of the tricarboxylic acid cycle, coupled with increased ketogenic/lipogenic activity via acetyl-CoA, 3-hydroybutyrate, and cholesterol metabolites. Also apparent was an increased content/acetylation of amino-acids, glutamine and glutamate, in particular. Accordingly, metabolic alterations due to a single copy loss of Ptch1 in Ptch1+/-/ODCt/C57BL/6 heterozygous mice may provide insights about the cancer prone phenotype of BCCs in GS patients, including biomarkers/targets for early intervention.

MicroRNAs as potential biomarkers to predict the risk of urinary retention following intradetrusor onabotulinumtoxin-A injection.

AIMS: MicroRNAs (miRs) control post-transcriptional gene expression, and this is relevant in understanding better chronic diseases and treatment outcomes. The role of miRs in the pathology and treatment outcomes of overactive bladder (OAB) is unknown. In this study, we assessed the differential expression of miRs in OAB patients responding with either normal or elevated post-void residual volumes (PVRs) ≥200 mL following intradetrusor injection of onabotulinumtoxin-A (onaBoNT-A). METHODS: Female OAB patients refractory to OAB drugs were consented for this study. Cystoscopic-guided punch bladder biopsy was obtained at the time of injection of onaBoNT-A 100 units. The expression of 13 miR species, selected for their known effect on neurotrophin expression and smooth muscle function, was measured. PVRs and urine nerve growth factor (NGF) levels were measured at baseline and at the follow-up visit. RESULTS: Fourteen patients with mean age of 66 years were consented. Of these patients, nine maintained PVRs <200 mL after onaBoNT-A injection to comprise the low PVR group. The other five patients with PVRs ≥200 mL comprised the high PVR group. The expression of miR221 and miR125b was upregulated by 11- and 2-fold, respectively, in patients who responded with low PVRs after onaBoNT-A (P < 0.05). Urine NGF levels at baseline were not different between the two groups. CONCLUSIONS: This study suggests that deficiency in the pretreatment expression of miR221 and miR125b may predispose OAB patients to high PVRs following intradetrusor onaBoNT-A. Additional studies are needed to better understand the role of miRs in OAB.

BDNF overexpression in the bladder induces neuronal changes to mediate bladder overactivity.

Elevated levels of brain-derived neurotrophic factor (BDNF) in urine of overactive bladder (OAB) patients support the association of BDNF with OAB symptoms, but the causality is not known. Here, we investigated the functionality of BDNF overexpression in rat bladder following bladder wall transfection of either BDNF or luciferase (luciferase) transgenes (10 µg). One week after transfection, BDNF overexpression in bladder tissue and elevation of urine BDNF levels were observed together with increased transcript of BDNF, its cognate receptors (TrkB and p75NTR), and downstream PLCγ isoforms in bladder. BDNF overexpression can induce the bladder overactivity (BO) phenotype which is demonstrated by the increased voiding pressure and reduced intercontractile interval during transurethral open cystometry under urethane anesthesia. A role for BDNF-mediated enhancement of prejunctional cholinergic transmission in BO is supported by the significant increase in the atropine- and neostigmine-sensitive component of nerve-evoked contractions and upregulation of choline acetyltransferase, vesicular acetylcholine transporter, and transporter Oct2 and -α1 receptors. In addition, higher expression of transient receptor channels (TRPV1 and TRPA1) and pannexin-1 channels in conjunction with elevation of ATP and neurotrophins in bladder and also in L6/S1 dorsal root ganglia together support a role for sensitized afferent nerve terminals in BO. Overall, genomic changes in efferent and afferent neurons of bladder induced by the overexpression of BDNF per se establish a mechanistic link between elevated BDNF levels in urine and dysfunctional voiding observed in animal models and in OAB patients.

Molecular correlates in urine for the obesity and prostatic inflammation of BPH/LUTS patients.

PURPOSE: Benign prostatic hyperplasia (BPH) is strongly associated with obesity and prostatic tissue inflammation, but the molecular underpinning of this relationship is not known. Here, we examined the association between urine levels of chemokines/adipokines with histological markers of prostate inflammation, obesity, and lower urinary tract symptoms LUTS in BPH patients. METHODS: Frozen urine specimens from 207 BPH/LUTS patients enrolled in Nashville Men's Health Study were sent for blinded analysis of 11 analytes, namely sIL-1RA, CXC chemokines (CXCL-1, CXCL-8, CXCL-10), CC chemokines (CCL2, CCL3, CCL5), PDGF-BB, interleukins IL-6, IL-17, and sCD40L using Luminex™ xMAP® technology. After adjusting for age and medication use, the urine levels of analytes were correlated with the scales of obesity, prostate inflammation grade, extent, and markers of lymphocytic infiltration (CD3 and CD20) using linear regression. RESULTS: sIL-1RA levels were significantly raised with higher BMI, waist circumference and waist-hip ratio in BPH patients after correction for multiple testing (P = 0.02). Men with greater overall extent of inflammatory infiltrates and maximal CD3 infiltration were marginally associated with CXCL-10 (P = 0.054) and CCL5 (P = 0.054), respectively. CCL3 in 15 patients with moderate to severe grade inflammation was marginally associated with maximal CD20 infiltration (P = 0.09), whereas CCL3 was undetectable in men with mild prostate tissue inflammation. There was marginal association of sCD40L with AUA-SI scores (P = 0.07). CONCLUSIONS: Strong association of sIL-1RA in urine with greater body size supports it as a major molecular correlate of obesity in the urine of BPH patients. Increased urine levels of CXCL-10, CCL5, and CCL3 were marginally associated with the scores for prostate tissue inflammation and lymphocytic infiltration. Overall, elevated urinary chemokines support that BPH is a metabolic disorder and suggest a molecular link between BPH/LUTS and prostatic inflammation.

Autophagy Activation Alleviates Amyloid-ß-Induced Oxidative Stress, Apoptosis and Neurotoxicity in Human Neuroblastoma SH-SY5Y Cells.

Autophagy is an evolutionary conserved catabolic process that ensures continuous removal of damaged cell organelles and long-lived protein aggregates to maintain cellular homeostasis. Although autophagy has been implicated in amyloid-ß (Aß) production and deposition, its role in pathogenesis of Alzheimer's disease remains elusive. Thus, the present study was undertaken to assess the cytoprotective and neuroprotective potential of autophagy on Aß-induced oxidative stress, apoptosis and neurotoxicity in human neuroblastoma SH-SY5Y cells. The treatment of Aß1-42 impaired the cell growth and redox balance, and induced apoptosis and neurotoxicity in SH-SY5Y cells. Next, the treatment of rapamycin (RAP) significantly elevated the expression of autophagy markers such as microtubule-associated protein-1 light chain-3 (LC3), sequestosome-1/p62, Beclin-1, and unc-51-like kinase-1 (ULK1) in SH-SY5Y cells. RAP-induced activation of autophagy notably alleviated the Aß1-42-induced impairment of redox balance by decreasing the levels of pro-oxidants such as reactive oxygen species, lipid peroxidation and Ca2+ influx, and concurrently increasing the levels of antioxidant enzymes such as superoxide dismutase and catalase. The RAP-induced autophagy also ameliorated Aß1-42-induced loss of mitochondrial membrane potential and apoptosis. Additionally, the activated autophagy provided significant neuroprotection against Aß1-42-induced neurotoxicity by elevating the expression of neuronal markers such as synapsin-I, PSD95, NCAM, and CREB. However, 3-methyladenine treatment significantly exacerbated the neurotoxic effects of Aß1-42. Taken together, our study demonstrated that the activation of autophagy provided possible neuroprotection against Aß-induced cytotoxicity, oxidative stress, apoptosis, and neurotoxicity in SH-SY5Y neuronal cells.

Effect of Intravesical Liposome-Based Nerve Growth Factor Antisense Therapy on Bladder Overactivity and Nociception in a Rat Model of Cystitis Induced by Hydrogen Peroxide.

The aim of this study was to evaluate whether liposome-based local suppression of nerve growth factor (NGF) in the bladder has effects on bladder hypersensitivity in a rat cystitis model induced by intravesical instillation of hydrogen peroxide (HP). HP (1.5%) was intravesically administered to adult female Sprague-Dawley rats. Liposomes complexed with NGF antisense oligonucleotide (OND) labeled with TYE563 fluorescent tag were intravesically instilled on day 2. Red fluorescence from the TYE 563 tag was observed with fluorescent microscopy on day 3. Four separate groups of rats were used in the following experiments: (a) sham-liposome group, (b) sham-OND group, (c) cystitis-liposome group, and (d) cystitis-OND group. Saline or 1.5% HP was intravesically administered on day 0. Empty liposomes or liposomes-antisense OND were instilled into the bladder on day 2. The following experiments were conducted to evaluate the effect of NGF antisense treatment on day 7: (a) continuous cystometry was performed in an awake condition; (b) pain behavior induced by instillation of resiniferatoxin into the bladder, including licking behavior (lower abdominal licking) and freezing behavior (motionless head-turning toward lower abdomen), was observed; (c) immunohistochemical staining of the bladder and L6 DRG for NGF was performed; (d) the expression of several genes in the bladder was analyzed by reverse transcription polymerase chain reaction (RT-PCR); and (e) after Fast Blue was injected into the bladder wall, Fast Blue-positive or -negative cells in DRG neurons were separately collected by using a laser-capture microdissection method 7 days later. RT-PCR was performed to evaluate gene expressions in captured neuronal cells. The expression of TYE563 was identified only in the urothelial layer. In cystometric investigation, intercontraction intervals (ICI) were significantly (p = 0.001) shorter in the cystitis-liposome group in comparison to the sham-liposome group. ICI was significantly (p = 0.007) longer in the cystitis-OND group compared to the cystitis-liposome group. Comparisons of the sham-liposome and the sham-OND groups showed no significant difference in ICI (p = 0.56). Licking events did not significantly differ among the four groups. In contrast, the cystitis-liposome group showed significantly more freezing events than the sham-liposome group did (p = 0.002). A significant reduction in the number of freezing events was observed in the cystitis-OND group compared to the cystitis-liposome group (p = 0.04). Immunofluorescence staining demonstrated that NGF expression in the mucosa (p = 0.02) and L6 DRG (p = 0.01) was significantly higher in the cystitis-liposome group than it was in the sham-liposome group. The expression of NGF was significantly lower in the mucosa (p = 0.002) and L6 DRG (p = 0.01) in the cystitis-OND group compared to the cystitis-liposome group. RT-PCR showed that the expression of NGF and TRPV1 mRNA in the mucosa was significantly higher in the cystitis-liposome group than it was in the sham-liposome group (p = 0.001 and 0.03, respectively). On the other hand, these gene expressions were significantly lower in the cystitis-OND group than they were in the cystitis-liposome group (p = 0.007 and 0.02, respectively). The cystitis-liposome group showed significantly higher expression of TRPA1, P2X3, and BDNF mRNA in labeled bladder afferent neurons than the sham-liposome group did (p = 0.03, 0.01, and 0.001, respectively). These gene expressions were significantly lower in the cystitis-OND group compared to the cystitis-liposome group (p = 0.04, 0.006, and 0.03, respectively). The study indicated that intravesical application of liposome-NGF antisense OND significantly improved bladder hypersensitivity induced by chemical cystitis in rats. Intravesical treatment with liposome-OND conjugates could be a novel local therapy of hypersensitive bladder disorders such as bladder pain syndrome/interstitial cystitis.

Intravesical liposome therapy for interstitial cystitis.

Over the past two decades, there has been lot of interest in the use of liposomes as lipid-based biocompatible carriers for drugs administered by the intravesical route. The lipidic bilayer structure of liposomes facilitates their adherence to the apical membrane surface of luminal cells in the bladder, and their vesicular shape allows them to co-opt the endocytosis machinery for bladder uptake after instillation. Liposomes have been shown to enhance the penetration of both water-soluble and insoluble drugs, toxins, and oligonucleotides across the bladder epithelium. Empty liposomes composed entirely of the endogenous phospholipid, sphingomyelin, could counter mucosal inflammation and promote wound healing in patients suffering from interstitial cystitis. Recent clinical studies have tested multilamellar liposomes composed entirely of sphingomyelin as a novel intravesical therapy for interstitial cystitis. In addition, liposomes have been used as a delivery platform for the instillation of botulinum toxin in overactive bladder patients. The present review discusses the properties of liposomes that are important for their intrinsic therapeutic effect, summarizes the recently completed clinical studies with intravesical liposomes and covers the latest developments in this field.

Neuroprotection Through Rapamycin-Induced Activation of Autophagy and PI3K/Akt1/mTOR/CREB Signaling Against Amyloid-ß-Induced Oxidative Stress, Synaptic/Neurotransmission Dysfunction, and Neurodegeneration in Adult Rats.

Autophagy is a catabolic process involved in the continuous removal of toxic protein aggregates and cellular organelles to maintain the homeostasis and functional integrity of cells. The mechanistic understanding of autophagy mediated neuroprotection during the development of neurodegenerative disorders remains elusive. Here, we investigated the potential role of rapamycin-induced activation of autophagy and PI3K/Akt1/mTOR/CREB pathway(s) in the neuroprotection of amyloid-beta (Aß1-42)-insulted hippocampal neurons in rat model of Alzheimer's disease (AD) like phenotypes. A single intra-hippocampal injection of Aß1-42 impaired redox balance and markedly induced synaptic dysfunction, neurotransmission dysfunction, and cognitive deficit, and suppressed pro-survival signaling in the adult rats. Rapamycin administration caused a significant reduction of mTOR complex 1 phosphorylation at Ser2481 and a significant increase in levels of autophagy markers such as microtubule-associated protein-1 light chain-3 (LC3), beclin-1, sequestosome-1/p62, unc-51-like kinase 1 (ULK1). In addition, rapamycin induced the activation of autophagy that further activated p-PI3K, p-Akt1 (Ser473), and p-CREB (Ser183) expression in Aß1-42-treated rats. The activated autophagy markedly reversed Aß1-42-induced impaired redox homeostasis by decreasing the levels of prooxidants-ROS generation, intracellular Ca2+ flux and LPO, and increasing the levels of antioxidants-SOD, catalase, and GSH. The activated autophagy also provided significant neuroprotection against Aß1-42-induced synaptic dysfunction by increasing the expression of synapsin-I, synaptophysin, and PSD95; and neurotransmission dysfunction by increasing the levels of CHRM2, DAD2 receptor, NMDA receptor, and AMPA receptor; and ultimately improved cognitive ability in rats. Wortmannin administration significantly reduced the expression of autophagy markers, p-PI3K, p-Akt1, and p-CREB, as well as the autophagy mediated neuroprotective effect. Our study demonstrate that autophagy can be an integrated part of pro-survival (PI3K/Akt1/mTOR/CREB) signaling and autophagic activation restores the oxidative defense mechanism(s), neurodegenerative damages, and maintains the integrity of synapse and neurotransmission in rat model of AD.

Investigational drugs for bladder pain syndrome (BPS) / interstitial cystitis (IC).

INTRODUCTION: Bladder pain syndrome (BPS)/interstitial cystitis (IC) is associated with sensory lower urinary tract symptoms. Unfortunately, many of the existing oral treatments are ineffective in most patients of BPS/IC, which is the motivation for developing new drugs and therapeutic approaches. This review covers the latest drugs that have been investigated in BPS/IC patients. Intravesical treatments offer the opportunity to directly target the painful bladder with less systemic side effects. AREAS COVERED: In this review, the authors analyze the existing literature supporting the treatment of BPS/IC with conventional drugs including heparin, hyaluronic acid, chondroitin sulfate, and dimethylsulfoxide (DMSO). Furthermore, investigational drugs such as tanezumab and adalimumab, capable of sequestering nerve growth factor (NGF), and Tumor necrosis factor-α (TNF- α) are discussed. Investigational treatments such as liposomes, botulinum toxin (BTX), liposomal BTX, PD-0299685 (a Ca(2+) channel ɑ2δ ligand), continuous intravesical lidocaine, and AQX-1125 (a novel SHIP1 activating compound) are also covered. EXPERT OPINION: New investigational drugs offer promising improvements in clinical outcomes for BPS/IC patients; however, BPS/IC is a chronic pain disorder that is very vulnerable to a strong placebo effect. In addition, BPS/IC is a heterogeneous disorder that can be classified into several phenotypes. Since different phenotypes of BPS/IC respond differently to systemic and intravesical treatments, the authors believe that new drugs developed for BPS/IC are more likely to meet their predetermined clinical endpoints if the inclusion/exclusion criterion is tailored to specific phenotype of BPS/IC patients.

Constitutive expression Of NGF And P75(NTR) affected by bladder distension and NGF antisense treatment.

AIMS: It is known that bladder exposure to noxious stimuli elicits nerve growth factor (NGF) expression with region wise differences. Here, we investigated the effect of bladder distension (cystometry) and bladder wall injection of NGF antisense oligonucleotide (ODN) together as well as separately on spontaneous (constitutive) expression of NGF and its cognate p75 neurotrophin receptor (p75(NTR)). METHOD: Under isoflurane anesthesia, either 15µg of protamine sulfate (vehicle) alone or complexed with 1.5µg of NGF antisense or scrambled ODN was injected (10µL) at 4 sites in bladder wall of 24 adult female Sprague-Dawley rats and 6 rats were left untreated (n=30). Under urethane anesthesia, cystometry (CMG) was performed in treated and control rats. Fluorescent ODN and NGF/p75(NTR) expression was localized in harvested tissue. KEY FINDINGS: Complexation of ODN with protamine was essential for the retention of ODN in bladder tissue as the uncomplexed ODN was untraceable after injection. Bladder distension from CMG raised the expression of NGF and p75(NTR) relative to CMG naïve rats. The groups treated with vehicle, scrambled and antisense ODN were indistinct with regard to CMG parameters, but the intense immunoreactivity of NGF and p75(NTR) seen in the vehicle and scrambled ODN groups was reduced following treatment with NGF antisense. SIGNIFICANCE: The constitutive expression of NGF and p75(NTR) is responsive to bladder distension and administration of NGF antisense. Complexation with protamine reduces the clearance of ODN and demonstrates the potential of ODN nanoparticles as an option for reducing the inducible NGF expression in OAB patients following intradetrusor injection.

Liposome Based Intravesical Therapy Targeting Nerve Growth Factor Ameliorates Bladder Hypersensitivity in Rats with Experimental Colitis.

PURPOSE: Pelvic organ cross sensitization is considered to contribute to overlapping symptoms in chronic pelvic pain syndrome. Nerve growth factor over expression in the bladder is reportedly involved in the symptom development of bladder pain syndrome/interstitial cystitis. We examined whether a reduction of over expressed nerve growth factor in the bladder by intravesical treatment with liposome and oligonucleotide conjugates would ameliorate bladder hypersensitivity in a rat colitis model. MATERIALS AND METHODS: Adult female rats were divided into 1) a control group, 2) a colitis-oligonucleotide group with intracolonic TNBS (2,4,6-trinitrobenzene sulfonic acid) enema and intravesical liposome-oligonucleotide treatments, 2) a colitis-saline group with intracolonic TNBS and intravesical saline treatments, 4) a sham oligonucleotide group with intravesical liposome-oligonucleotide treatment without colitis and 5) a sham-saline group with intravesical saline treatment without colitis. Liposomes conjugated with nerve growth factor antisense oligonucleotide or saline solution were instilled in the bladder and 24 hours later colitis was induced by TNBS enema. Effects of nerve growth factor antisense treatment were evaluated by pain behavior, cystometry, molecular analyses and immunohistochemistry 10 days after TNBS treatment. RESULTS: In colitis-oligonucleotide rats nerve growth factor antisense treatment ameliorated pain behavior and decreased a reduction in the intercontraction interval in response to acetic acid stimulation as well as nerve growth factor expression in the bladder mucosa. All were enhanced in colitis-saline rats compared to sham rats. CONCLUSIONS: Nerve growth factor over expression in the bladder mucosa and bladder hypersensitivity induced after colitis were decreased by intravesical application of liposome-oligonucleotide targeting nerve growth factor. This suggests that local antinerve growth factor therapy could be effective treatment of bladder symptoms in chronic pelvic pain syndrome.

Spontaneous Recovery of Reflex Voiding Following Spinal Cord Injury Mediated by Anti-inflammatory and Neuroprotective Factors.

OBJECTIVE: To investigate the time-dependent changes in expression of cytokines that characterizes the spontaneous recovery of reflex voiding after spinal cord injury (SCI). SCI is known to reorganize the neural circuitry of micturition reflex after injury. METHODS: Under isoflurane anesthesia, spinal cord of 18 adult female Sprague-Dawley rats was completely transected at the Th9-10 level. Awake cystometry was performed at each time point on controls and 6 SCI animals, and bladder was then harvested for analysis of 29 proteins Millipore kit or enzyme-linked immunosorbent assay. Prophylactic dose of ampicillin 100 mg/kg was administered periodically to all SCI animals. RESULTS: Spontaneous recovery of voiding after SCI at 12 weeks was evident from increased intercontractile interval and voiding efficiency during cystometry. Expression of proinflammatory interleukins ([IL] IL-1α and IL-1ß, IL-2, IL-5, IL-6, IL-18, tumor necrosis factor alpha [TNF-α]) and CXC chemokines (CXCL1, CXCL2, CXCL10), CX3CL1, and CCL2 showed significant elevation at 4 and at 8 weeks with slight decrease at 12 weeks. In contrast, expression of anti-inflammatory IL-10 and neuroprotective factors, CXCL-5, and leptin, was elevated at 8 and at 12 weeks (P < .05). In contrast, expression of CCL3, CCL5, and growth factors (vascular endothelial growth factor, nerve growth factor, epidermal growth factor, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor) did not show any significant temporal change after SCI. CONCLUSION: Spontaneous recovery of reflex voiding at 12 weeks was marked by increased endogenous expression of anti-inflammatory cytokine IL-10 and neuroprotective factors, CXCL-5, and leptin, which suggests that pharmacological suppression of inflammation, can hasten the emergence of reflex voiding after SCI.

Advances in intravesical therapy for urinary tract disorders.

INTRODUCTION: Intravesical therapy is a valuable option in the clinical management of urinary tract disorders such as interstitial cystitis/ painful bladder syndrome (IC/PBS) and refractory overactive bladder. This review will cover the latest advances in this field using polymer and liposomes as delivery platform for drugs, protein and nucleic acids. AREAS COVERED: This review summarizes the significance of intravesical therapy for lower urinary tract disorders. The recent advancement of liposomes as a drug delivery platform for botulinum toxin, tacrolimus and small interfering RNA is discussed. The importance of polymers forming indwelling devices and hydrogels are also discussed, where all preparations improved efficacy parameters in rodent models. Clinical experience of treating IC/PBS with indwelling devices and liposomes are summarized and preclinical evidence about the downregulation of target gene expression in rodent bladder with liposomes complexed with siRNA is also reviewed. EXPERT OPINION: There have been several advances in the field of intravesical therapy for improving clinical outcomes. One of the most promising research avenues is the repurposing of drugs, given previously by other routes of administration, such as tacrolimus. Intravesical therapy also opens up novel therapeutic targets with improved efficacy and safety for underactive bladder.

Urine chemokines indicate pathogenic association of obesity with BPH/LUTS.

OBJECTIVES: High prevalence of lower urinary tract symptoms (LUTS) consistent with benign prostate hyperplasia (BPH) is associated with obesity and prostatic inflammation. Here, we investigated whether chemokines associated with obesity and prostatic inflammation can be measured in normally voided urine of BPH/LUTS patients to demonstrate the mechanistic association between obesity and BPH/LUTS. METHODS: Frozen urine specimens of BPH/LUTS patients enrolled in the Nashville Men's Health Study were sent for blinded analysis to University of Pittsburgh. Thirty patients were blocked by their AUA-SI (>7 or ≤7) and prostatic enlargement (<40, 40-60, >60 cc). Clinical parameters including age, prostate size, and medications were derived from chart review. CXC chemokines (CXCL-1, CXCL-8, and CXCL-10), CC chemokines (CCL2 and CCL3), and sIL-1ra were measured in thawed urine using Luminex™ xMAP(®) technology and ELISA for NGF. RESULTS: Urinary CCL2 levels were several fold higher compared with the other six proteins, of which CCL3 was detectable in less than one-fourth of patients. Urine levels of sIL-1ra and CXCL-8 were significantly associated with increasing BMI and waist circumference in BPH patients. CXCL-8 showed a marginal association with overall AUA-SI scores, as well as obstructive (p = 0.08) symptom subscores. Prostate volume was inversely and marginally associated with urinary CXCL-10 (p = 0.09). CONCLUSIONS: Urine levels of CXCL-8, CXCL-10, and sIL-1ra were associated with varying degrees with LUTS severity, prostate size, and obesity, respectively. These findings in urine are consistent with past studies of chemokine levels from expressed prostatic secretions and demonstrate the potential of noninvasively measured chemokine in urine to objectively classify BPH/LUTS patients.

Inflammasomes are important mediators of prostatic inflammation associated with BPH.

BACKGROUND: There is mounting evidence to support the role of inflammation in benign prostate hyperplasia (BPH), and a recent study reported expression of inflammasome derived cytokine IL-18 in prostate biopsy of BPH patients. Here we examined the expression of inflammasome-derived cytokines and activation of nucleotide-binding oligomerization domain-like receptor with pyrin domain protein 1 (NLRP) 1 inflammasome in a rat model of prostatic inflammation relevant to BPH. METHODS: Prostatic inflammation was experimentally induced in three-month-old male Sprague-Dawley rats by intraprostatic injection (50 µL) of either 5 % formalin or saline (sham) into the ventral lobes of prostate. 7 days later, prostate and bladder tissue was harvested for analysis of inflammasome by Western blot, immunohistochemistry and downstream cytokine production by Milliplex. RESULTS: Expression of interleukins, CXC and CC chemokines were elevated 2-15 fold in formalin injected prostate relative to sham. Significant expression of NLRP1 inflammasome components and caspase-1 in prostate were associated with significant elevation of pro and cleaved forms of IL-1ß (25.50 ± 1.16 vs 3.05 ± 0.65 pg/mg of protein) and IL-18 (1646.15 ± 182.61 vs 304.67 ± 103.95 pg/mg of protein). Relative to prostate tissue, the cytokine expression in bladder tissue was much lower and did not involve inflammasome activation. CONCLUSIONS: Significant upregulation of NLRP1, caspase-1 and downstream cytokines (IL-18 and IL-1ß) suggests that a NLRP1 inflammasome is assembled and activated in prostate tissue of this rat model . Recapitulation of findings from human BPH specimens suggests that the inflammasome may perpetuate the inflammatory state associated with BPH. Further clarification of these pathways may offer innovative therapeutic targets for BPH-related inflammation.

4-Hydroxy-trans-2-nonenal (4-HNE) induces neuronal SH-SY5Y cell death via hampering ATP binding at kinase domain of Akt1.

Inhibition mechanism(s) of protein kinase B/Akt1 and its consequences on related cell signaling were investigated in human neuroblastoma SH-SY5Y cells exposed to 4-hydroxy-trans-2-nonenal (4-HNE), one of the most reactive aldehyde by-products of lipid peroxidation. In silico data indicate that 4-HNE interacts with kinase domain of Akt1 with the total docking score of 6.0577 and also forms H-bond to Glu234 residue similar to highly potent Akt1 inhibitor imidazopiperidine analog 8b, in which the protonated imidazole nitrogen involves in two hydrogen bonds between Glu234 and Asp292. The strong hydrogen bonding with Glu234 and hydrophobic interactions with several residues, namely Leu156, Gly157, Val164, Ala177, Tyr229, Ala230, Met281 and Thr291, at the vicinity which is normally occupied by the ribose of ATP, appear to be the main causes of Akt1 inhibition and lead to the significant conformational change on this region of protein. Results of mutational docking prove that Glu234 plays a major role in 4-HNE-mediated Akt1 inhibition. In silico data on Akt inhibition were further validated by observing the down-regulated levels of phosphorylated (Thr308/Ser493) Akt1 as well as the altered levels of the downstream targets of pAkt, namely downregulated levels of pGSK3ß (Ser9), ß-catenin, Bcl2 and upregulated levels of pro-apoptotic markers, namely Bad, Bax, P(53) and caspase-9/3. The cellular fate of such pAkt inhibition was evidenced by increased reactive oxygen species, degraded nuclei, transferase dUTP nick end labeling positive cells and upregulated levels of pJNK1/2. We identified that 4-HNE-mediated Akt1 inhibition was due to the competitive inhibition of ATP by 4-HNE at the kinase domain of ATP binding sites.