Interstitial cystitis/Bladder Pain Syndrome (IC/BPS) is a chronic inflammatory disease characterized by visceral pain and voiding symptoms. IC/BPS is still an unsolved enigma with ineffective ...diagnosis criteria and treatment. A main limitation in IC/BPS understanding is the lack of appropriate preclinical model. Cyclophosphamide (CYP) is commonly used as an experimental model for IC/BPS in rodent. However, the proposed models are very aggressive, contrasting with what occurs in clinic, and often associated with severe toxicity and high mortality rate. In addition, visceral pain, the hallmark symptom of IC/BPS, has been validated in only few of them. In this study, we developed a chronic model of CYP-induced IC/BPS in female rat. In our protocol, no severe weight loss occurred and the survival rate was 100%. In accordance to human pathology, chronic CYP-injected rats developed severe painful behavior whereas only sparse inflammation was observed. Inflammatory response was characterized by bladder edema and focal urothelial damage but absence of massive infiltrate. This chronic model showed persistent symptoms indicative for a central sensitization mechanism. We further demonstrate that CYP-induced chronic visceral pain was significantly reduced by curative treatment with clinically relevant compounds (gabapentin, ibuprofen, and Ialuril ®). We therefore developed and validated a rat model of chronic cystitis that shares strong similarity with human non-ulcerative IC/BPS features without overtly affecting the animal health. This model will thus provide mechanistic insights of the disease and help to evaluate therapeutic agents for IC/BPS.
Pulmonary arterial hypertension (PAH) is a devastating cardiopulmonary disorder with various origins. All forms of PAH share a common pulmonary arteriopathy characterised by vasoconstriction, ...remodelling of the pre-capillary pulmonary vessel wall, and in situ thrombosis. Although the pathogenesis of PAH is recognised as a complex and multifactorial process, there is growing evidence that potassium channels dysfunction in pulmonary artery smooth muscle cells is a hallmark of PAH. Besides regulating many physiological functions, reduced potassium channels expression and/or activity have significant effects on PAH establishment and progression. This review describes the molecular mechanisms and physiological consequences of potassium channel modulation. Special emphasis is placed on KCNA5 (Kv1.5) and KCNK3 (TASK1), which are considered to play a central role in determining pulmonary vascular tone and may represent attractive therapeutic targets in the treatment of PAH.
For more than a decade, the understanding of RNA interference (RNAi) has been a growing field of interest. Micro-RNAs (miRNAs) are small regulatory RNAs that play an important role in disease ...development and progression and therefore represent a potential new class of therapeutic targets. However, delivery of RNAi-based oligonucleotides is one of the most challenging hurdles to RNAi-based drug development. Electropermeabilization (EP) is recognized as a successful non-viral method to transfer nucleic acids into living cells both in vitro and in vivo. EP is the direct application of electric pulses to cells or tissues that transiently permeabilize plasma membranes, allowing the efficient delivery of exogenous molecules. The present review focused on the mechanism of RNAi-based oligonucleotides electrotransfer, from cellular uptake to intracellular distribution. Biophysical theories on oligonucleotide electrotransfer will be also presented. The advantages and few drawbacks of EP-mediated delivery will also be discussed.
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Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with limited therapeutic options. Although exposed to stressful conditions, pulmonary artery (PA) smooth muscle cells (PASMCs) ...exhibit a "cancer-like" pro-proliferative and anti-apoptotic phenotype. HDAC6 is a cytoplasmic histone deacetylase regulating multiple pro-survival mechanisms and overexpressed in response to stress in cancer cells. Due to the similarities between cancer and PAH, we hypothesized that HDAC6 expression is increased in PAH-PASMCs to face stress allowing them to survive and proliferate, thus contributing to vascular remodeling in PAH. We found that HDAC6 is significantly up-regulated in lungs, distal PAs, and isolated PASMCs from PAH patients and animal models. Inhibition of HDAC6 reduced PAH-PASMC proliferation and resistance to apoptosis in vitro sparing control cells. Mechanistically, we demonstrated that HDAC6 maintains Ku70 in a hypoacetylated state, blocking the translocation of Bax to mitochondria and preventing apoptosis. In vivo, pharmacological inhibition of HDAC6 improved established PAH in two experimental models and can be safely given in combination with currently approved PAH therapies. Moreover, Hdac6 deficient mice were partially protected against chronic hypoxia-induced pulmonary hypertension. Our study shows for the first time that HDAC6 is implicated in PAH development and represents a new promising target to improve PAH.
Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders, where epithelial defects drive, at least in part, some of the pathology. We reconstituted human intestinal epithelial organ, by ...using three-dimension culture of human colon organoids. Our aim was to characterize morphological and functional phenotypes of control (non-IBD) organoids, compared to inflamed organoids from IBD patients. The results generated describe the epithelial defects associated with IBD in primary organoid cultures, and evaluate the use of this model for pharmacological testing of anti-inflammatory approaches. Human colonic tissues were obtained from either surgical resections or biopsies, all harvested in non-inflammatory zones. Crypts were isolated from controls (non-IBD) and IBD patients and were cultured up to 12-days. Morphological (size, budding formation, polarization, luminal content), cell composition (proliferation, differentiation, immaturity markers expression), and functional (chemokine and tight junction protein expression) parameters were measured by immunohistochemistry, RT-qPCR or western-blot. The effects of inflammatory cocktail or anti-inflammatory treatments were studied in controls and IBD organoid cultures respectively. Organoid cultures from controls or IBD patients had the same cell composition after 10 to 12-days of culture, but IBD organoid cultures showed an inflammatory phenotype with decreased size and budding capacity, increased cell death, luminal debris, and inverted polarization. Tight junction proteins were also significantly decreased in IBD organoid cultures. Inflammatory cytokine cocktail reproduced this inflammatory phenotype in non-IBD organoids. Clinically used treatments (5-ASA, glucocorticoids, anti-TNF) reduced some, but not all parameters. Inflammatory phenotype is associated with IBD epithelium, and can be studied in organoid cultures. This model constitutes a reliable human pre-clinical model to investigate new strategies targeting epithelial repair.
Background and Purpose
Thrombin is massively released upon tissue damage associated with bleeding or chronic inflammation. The effects of this thrombin on tissue regrowth and repair has been scarcely ...addressed and only in cancer cell lines. Hence, the purpose of the present study was to determine thrombin's pharmacological effects on human intestinal epithelium growth, proliferation and apoptosis, using three‐dimensional cultures of human colon organoids.
Experimental Approach
Crypts were isolated from human colonic resections and cultured for 6 days, forming human colon organoids. Cultured organoids were exposed to 10 and 50 mU·mL−1 of thrombin, in the presence or not of protease‐activated receptor (PAR) antagonists. Organoid morphology, metabolism, proliferation and apoptosis were followed.
Key Results
Thrombin favoured organoid maturation leading to a decreased number of immature cystic structures and a concomitant increased number of larger structures releasing cell debris and apoptotic cells. The size of budding structures, metabolic activity and proliferation were significantly reduced in organoid cultures exposed to thrombin, while apoptosis was dramatically increased. Both PAR1 and PAR4 antagonists inhibited apoptosis regardless of thrombin doses. Thrombin‐induced inhibition of proliferation and metabolic activity were reversed by PAR4 antagonist for thrombin's lowest dose and by PAR1 antagonist for thrombin's highest dose.
Conclusions and Implications
Overall, our data suggest that the presence of thrombin in the vicinity of human colon epithelial cells favours their maturation at the expense of their regenerative capacities. Our data point to thrombin and its two receptors PAR1 and PAR4 as potential molecular targets for epithelial repair therapies.
The Focal adhesion kinase (FAK) is a ubiquitous cytoplasmic tyrosine‐kinase promoting tumor progression and metastasis processes by acting in cancer cells and their tumor microenvironment partners. ...FAK overexpression in primary colon tumors and their metastasis is associated to poor colorectal cancer (CRC) patients’ outcome. Eight FAK mRNA alternative splice variants have been described and contribute to additional level of FAK activity regulation, some of them corresponding to overactivated FAK isoforms. To date, FAK mRNA alternative splice variants expression and implication in CRC processes remain unknown. Here, using different human CRC cells lines displaying differential invasive capacities in an in vivo murine model recapitulating the different steps of CRC development from primary tumors to liver and lung metastasis, we identified three out of the eight mRNA variants (namely FAK0, FAK28 and FAK6) differentially expressed along the CRC process and the tumor sites. Our results highlight an association between FAK0 and FAK6 expressions and the metastatic potential of the most aggressive cell lines HT29 and HCT116, suggesting that FAK0 and FAK6 could represent aggressiveness markers in CRC. Our findings also suggest a more specific role for FAK28 in the interactions between the tumors cells and their microenvironment. In conclusion, targeting FAK0, the common form of FAK, might not be a good strategy based on the numerous roles of this kinase in physiological processes. In contrast, FAK6 or FAK28 splice variants, or their corresponding protein isoforms, may putatively represent future therapeutic target candidates in the development of CRC primary tumors and metastasis.
What's new?
Overexpression of the focal adhesion kinase (FAK) is associated with poor outcome in patients with colorectal cancer but the role of the eight splice variants of FAK remains unknown. Here the authors correlated FAK splice variant expression in colorectal tumor cell lines with invasiveness in mouse models. FAK0 and FAK6 splice variant expression was associated with higher aggressiveness and metastatic potential, underscoring that distinct FAK splice variants may represent new targets in the development of drugs against colorectal cancer and associated metastasis.
Initiation of adaptive mucosal immunity occurs in organized mucosal lymphoid tissues such as Peyer's patches of the small intestine. Mucosal lymphoid follicles are covered by a specialized ...follicle-associated epithelium (FAE) that contains M cells, which mediate uptake and transepithelial transport of luminal Ags. FAE cells also produce chemokines that attract Ag-presenting dendritic cells (DCs). TLRs link innate and adaptive immunity, but their possible role in regulating FAE functions is unknown. We show that TLR2 is expressed in both FAE and villus epithelium, but TLR2 activation by peptidoglycan or Pam(3)Cys injected into the intestinal lumen of mice resulted in receptor redistribution in the FAE only. TLR2 activation enhanced transepithelial transport of microparticles by M cells in a dose-dependent manner. Furthermore, TLR2 activation induced the matrix metalloproteinase-dependent migration of subepithelial DCs into the FAE, but not into villus epithelium of wild-type and TLR4-deficient mice. These responses were not observed in TLR2-deficient mice. Thus, the FAE of Peyer's patches responds to TLR2 ligands in a manner that is distinct from the villus epithelium. Intraluminal LPS, a TLR4 ligand, also enhanced microparticle uptake by the FAE and induced DC migration into the FAE, suggesting that other TLRs may modulate FAE functions. We conclude that TLR-mediated signals regulate the gatekeeping functions of the FAE to promote Ag capture by DCs in organized mucosal lymphoid tissues.
Melanoma is a very aggressive skin tumor that can be cured when diagnosed and treated in its early stages. However, at the time of identification, the tumor is frequently in a metastatic stage. ...Intensive research is currently ongoing to improve the efficacy of the immune system in eliminating cancer cells. One approach is to boost the activation of cytotoxic T cells by IL-12 cytokine that plays a central role in the activation of the immune system. In parallel, physical methods such as electropermeabilization-based treatments are currently under investigation and show promising results.
In this study, we set electrical parameters to induce a partial-irreversible electropermeabilization (pIRE) of melanoma to induce a sufficient cell death and potential release of tumor antigens able to activate immune cells. This protocol mimics the situation where irreversible electropermeabilization is not fully completed. Then, a peritumoral plasmid IL-12 electrotransfer was combined with pIRE treatment. Evaluation of the tumor growth and survival was performed in mouse strains having a different immunological background (C57Bl/6 (WT), nude and C57Bl6 (TLR9-/-)).
pIRE treatment induced apoptotic cell death and a temporary tumor growth delay in all mouse strains. In C57Bl/6 mice, we showed that peritumoral plasmid IL-12 electrotransfer combined with tumor pIRE treatment induced tumor regression correlating with a local secretion of IL-12 and IFN-γ. This combined treatment induced a growth delay of distant tumors and prevented the emergence of a second tumor in 50% of immunocompetent mice.
The combination of pIL-12 GET and pIRE not only enhanced survival but could bring a curative effect in wild type mice. This two-step treatment, named Immune-Gene Electro-Therapy (IGET), led to a systemic activation of the adaptive immune system and the development of an anti-tumor immune memory.
•CernitinTM inhibit enzyme COX-2 and reduce the production of prostaglands which are known to cause pain during inflammation.•Simultaneously, CernitinTMtreatment may lead to recycling the AA to the ...cell membrane resulting in reduced AA release.•CernitinTM treatment inhibits conversion of testosterone to DHT, through inhibition of 5α-reductase enzyme.•Thus, decreasing AR/DHT interaction which ultimately leads to decrease proliferation and reduces PSA level.
Cernitin™ pollen extracts (brand name Cernilton®) alleviates symptoms related to common lower uro-genital tract disorders in men. The underlying mechanisms are ill-defined but the inflammatory pathway could be one of them. In a previous in vitro study it was shown that Cernitin™ induce a regulatory effect on inflammatory parameters.
In this study, male Sprague Dawley rats were used to validate the effects of Cernitin™ in chronic prostatitis and benign prostatic hyperplasia. Pain was assessed by von Frey assay.
Cernitin™ exhibited significant pain relief in the induced prostatitis rat model and was associated with a significant decrease in the intraprostatic level of COX-2 and MCP-1 in the prostatic tissue homogenates. In a parallel study, Cernitin™ treatment led to a significant decrease in prostate weight in rats with testosterone induced BPH. Concurrently, a significant decrease in the percentage of proliferation marker, Ki-67, and androgen receptor expressing cells was observed. Similarly, a low level of cytoplasmic 5α-reductase expression was observed in Cernitin™- and finasteride-treated animals.
The current in vivo experiments support the use of Cernitin™ as an anti-inflammatory and symptom reducing agent that could, in part, explain the impact of Cernitin™ on the management of chronic pelvic pain in men.
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