The Potential Role of Urinary Microbiota in Bladder Carcinogenesis: A Systematic Review.

PURPOSE: The quantitative objective of the current systematic review was to identify the potential role of urinary microbiota in bladder cancer (BC) carcinogenesis, invasiveness, progression, and metastasis. MATERIALS AND METHODS: The proposed systematic review was conducted in accordance with critical review according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement, and the Joanna Briggs Institute (JBI) methodology for systematic reviews. The search strategy aimed to find both published and unpublished studies up to the January 2024. A JBI appraisal checklist was used to assess possible biases. RESULTS: This systematic review was centered on 27 studies comprising 926 BC patients. Overall, 412 control individuals were compared with BC patients. The most common sampling method was midstream urine collection. Regarding microbial alpha diversity, there was no statistically significant difference between cancerous and healthy samples (n = 8), recurrent and not recurrent (n = 1), responders versus non-responders(n = 1), tumor grades (n = 1), and collection methods (n = 1). However, five studies reported higher diversity in controls, and five other studies reported, conversely, high levels of alpha diversity in BC patients or recurrent cases. Furthermore, a responder (RE) to treatment and a non-muscle invasive bladder cancer (NMIBC) groups demonstrated significant difference with non-responder (NR) and muscle invasive bladder cancer (MIBC), respectively. In terms of beta-diversity, nine studies reported significant diversity between BC patients and controls, one article demonstrated difference between recurrent and not recurrent patients, a study reported significant difference in RE and NR groups whereas another showed opposite, and others (n = 4) did not find any difference between BC, controls, MIBC and NMIBC patients, or between tumor grades. One study reported a difference between the collection method and beta-diversity in males and another reported the difference in females. CONCLUSION: The included studies demonstrate that the composition of urinary microbiota is altered in patients with BC. However, the differentially enriched genera in the urine of these patients vary between studies, and there is too much heterogeneity across studies to make any reliable and valid conclusions. Furthermore, well-designed research is necessary to assess the role of microbiota in the carcinogenesis and progression of BC.

Clofarabine Has a Superior Therapeutic Window as compared to Gemcitabine in Preclinical Bladder Cancer Models.

Current standard-of-care systemic therapy options for locally advanced and metastatic bladder cancer (BC), which are predominantly based on cisplatin-gemcitabine combinations, are limited by significant treatment failure rates and frailty-based patient ineligibility. We previously addressed the urgent clinical need for better-tolerated BC therapeutic strategies using a drug screening approach, which identified outstanding antineoplastic activity of clofarabine in preclinical models of BC. To further assess clofarabine as a potential BC therapy component, we conducted head-to-head comparisons of responses to clofarabine versus gemcitabine in preclinical in vitro and in vivo models of BC, complemented by in silico analyses. In vitro data suggest a distinct correlation between the two antimetabolites, with higher cytotoxicity of gemcitabine, especially against several nonmalignant cell types, including keratinocytes and endothelial cells. Accordingly, tolerance of clofarabine (oral or intraperitoneal application) was distinctly better than for gemcitabine (intraperitoneal) in patient-derived xenograft models of BC. Clofarabine also exhibited distinctly superior anticancer efficacy, even at dosing regimens optimized for gemcitabine. Neither complete remission nor cure, both of which were observed with clofarabine, were achieved with any tolerable gemcitabine regimen. Taken together, our findings demonstrate that clofarabine has a better therapeutic window than gemcitabine, further emphasizing its potential as a candidate for drug repurposing in BC. PATIENT SUMMARY: We compared the anticancer activity of clofarabine, a drug used for treatment of leukemia but not bladder cancer, and gemcitabine, a drug currently used for chemotherapy against bladder cancer. Using cell cultures and mouse models, we found that clofarabine was better tolerated and more efficacious than gemcitabine, and even cured implanted tumors in mouse models. Our results suggest that clofarabine, alone or in combination schemes, might be superior to gemcitabine for the treatment of bladder cancer.

The microbiome of kidney stones and urine of patients with nephrolithiasis.

The incidence of nephrolithiasis is rising worldwide. Although it is a multifactorial disease, lifestyle plays a major role in its etiology. Another considerable factor could be an aberrant microbiome. In our observational single-center study, we aimed to investigate the composition of bacteria in kidney stones and urine focusing on patients with features of metabolic syndrome. Catheterized urine and kidney stones were collected prospectively from 100 consecutive patients undergoing endoscopic nephrolithotomy between 2020 and 2021 at our clinic. Microbiome composition was analyzed via 16S rRNA gene amplicon sequencing. Detection of bacteria was successful in 24% of the analyzed kidney stones. These patients had a prolonged length of stay compared to patients without verifiable bacteria in their stones (2.9 vs 1.5 days). Patients with features of metabolic syndrome were characterized by kidney stones colonized with classical gastrointestinal bacteria and displayed a significant enrichment of Enterococcaceae and Enterobacteriaceae. Stones of patients without features of metabolic syndrome characterized by Ureaplasma and Staphylococcaceae. Patients with bacteria in their kidney stones exhibit a longer length of stay, possibly due to more complex care. Patients presenting with features of metabolic syndrome displayed a distinct stone microbiome compared to metabolically fit patients. Understanding the role of bacteria in stone formation could enable targeted therapy, prevention of post-operative complications and new therapeutic strategies.

Evaluation of APOBEC3 expression as prognostic marker in squamous cell carcinoma of the penis.

Squamous cell carcinoma of the penis (PSC) is a rare disease with limited information on the molecular events leading to malignant transformation. In a third of PSC cases, presence of human papilloma virus (HPV) is found. The APOBEC3 family of proteins is known to play a significant role in defense against HPV infection, but their role in PSC is largely unknown. In this study, we aim to assess mRNA expression levels of APOBEC3 family members in HPV+ and HPV- PSC to get insight into their association with clinicopathological features and to evaluate their prognostic impact. Expression levels of six APOBEC3 family members in tissue from 50 patients with PSC were determined by RT-PCR and correlated with clinical and histopathological features. Lower expression of APOBEC3A, APOBEC3B, and APOBEC3C was observed in advanced PSC stages. Except for APOBEC3D, HPV+ samples showed higher expression of APOBEC3s compared to HPV- samples. In univariate analyses, APOBEC3A and APOBEC3C expression tended to be associated with disease-free survival and APOBEC3A expression with overall survival; however, multivariable analyses failed to confirm these associations with outcome. More extensive external validation and functional laboratory studies are needed to evaluate further their role in PSC development and progression.

Molecular biomarkers to help select neoadjuvant systemic therapy for urothelial carcinoma of the bladder.

PURPOSE OF REVIEW: In this review, we aimed to summarize the available evidence on pretreatment molecular biomarkers that may help to predict oncologic and pathologic outcomes in patients treated with neoadjuvant systemic therapy (NAST) for urothelial carcinoma of the bladder (UCB). RECENT FINDINGS: Several readily available and easily measurable blood-based biomarkers (e.g., neutrophil to lymphocyte or platelet-lymphocyte ratios) seems to help improve the selection of UCB patients who are most likely to benefit from NAST. Recent evidence suggests liquid biopsy including circulating tumor DNA (ctDNA) to be a promising tool to guide the administration of NAST in UCB patients. Pretreatment molecular and genetic characterization of transurethral resection of the bladder tumor samples may also help understand the tumor biology as luminal and basal tumor subtypes seems to be more responsive to NAST, while claudin-low and luminal-infiltrated tumor subtypes are less. In the context of neoadjuvant immunotherapy, programmed death-ligand 1 (PD-L1) status and ctDNA remain the only biomarker with possible value as the clinical utility of tumor mutational burden remains controversial/poor. SUMMARY: Biomarker approach is a necessary step to usher the age of precision/personalized medicine for muscle-invasive UCB with the overarching good to prevent both over- and under-therapy. The present review may offer a robust framework to compare and assess current and future molecular biomarkers for the selection of NAST in muscle-invasive UCB.

Molecular and Pharmacological Bladder Cancer Therapy Screening: Discovery of Clofarabine as a Highly Active Compound.

BACKGROUND: The heterogeneity of bladder cancers (BCs) is a major challenge for the development of novel therapies. However, given the high rates of recurrence and/or treatment failure, the identification of effective therapeutic strategies is an urgent clinical need. OBJECTIVE: We aimed to establish a model system for drug identification/repurposing in order to identify novel therapies for the treatment of BC. DESIGN, SETTING, AND PARTICIPANTS: A collection of commercially available BC cell lines (n = 32) was comprehensively characterized. A panel of 23 cell lines, representing a broad spectrum of BC, was selected to perform a high-throughput drug screen. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Positive hits were defined as compounds giving >50% inhibition in at least one BC cell line. RESULTS AND LIMITATIONS: Amongst >1700 tested chemical compounds, a total of 471 substances exhibited antineoplastic effects. Clofarabine, an antimetabolite drug used as third-line treatment for childhood acute lymphoblastic leukaemia, was amongst the limited number of drugs with inhibitory effects on cell lines of all intrinsic subtypes. We, thus, reassessed the substance and confirmed its inhibitory effects on commercially available cell lines and patient-derived cell cultures representing various disease stages, intrinsic subtypes, and histologic variants. To verify these effects in vivo, a patient-derived cell xenograft model for urothelial carcinoma (UC) was used. Well-tolerated doses of clofarabine induced complete remission in all treated animals (n = 12) suffering from both early- and late-stage disease. We further took advantage of another patient-derived cell xenograft model originating from the rare disease entity sarcomatoid carcinoma (SaC). Similarly to UC xenograft mice, clofarabine induced subcomplete to complete tumour remissions in all treated animals (n = 8). CONCLUSIONS: The potent effects of clofarabine in vitro and in vivo suggest that our findings may be of high clinical relevance. Clinical trials are needed to assess the value of clofarabine in improving BC patient care. PATIENT SUMMARY: We used commercially available cell lines for the identification of novel drugs for the treatment of bladder cancer. We confirmed the effects of one of these drugs, clofarabine, in patient-derived cell lines and two different mouse models, thereby demonstrating a potential clinical relevance of this substance in bladder cancer treatment.

Expression Analysis and Mutational Status of Histone Methyltransferase KMT2D at Different Upper Tract Urothelial Carcinoma Locations.

The gene coding for histone methyltransferase KMT2D is found among the top mutated genes in upper tract urothelial carcinoma (UTUC); however, there is a lack of data regarding its association with clinicopathologic features as well as survival outcomes. Therefore, we aimed to investigate KMT2D expression, mutation patterns, and their utility as prognostic biomarkers in patients with UTUC. A single-center study was conducted on tumor specimens from 51 patients treated with radical nephroureterectomy (RNU). Analysis of KMT2D protein expression was performed using immunohistochemistry (IHC). Customized next-generation sequencing (NGS) was used to assess alterations in KMT2D exons. Cox regression was used to assess the relationship of KMT2D protein expression and mutational status with survival outcomes. KMT2D expression was increased in patients with a previous history of bladder cancer (25% vs. 0%, p = 0.02). The NGS analysis of KMT2D exons in 27 UTUC tumors revealed a significant association between pathogenic KMT2D variants and tumor location (p = 0.02). Pathogenic KMT2D variants were predominantly found in patients with non-pelvic or multifocal tumors (60% vs. 14%), while the majority of patients with a pelvic tumor location (81% vs. 20%) did not harbor pathogenic KMT2D alterations. Both IHC and NGS analyses of KMT2D failed to detect a statistically significant association between KMT2D protein or KMT2D gene alteration status and clinical variables such as stage/grade of the disease or survival outcomes (all p > 0.05). KMT2D alterations and protein expression were associated with UTUC features such as multifocality, ureteral location, and previous bladder cancer. While KMT2D protein expression and KMT2D mutational status do not seem to have prognostic value in UTUC, they appear to add information to improve clinical decision-making regarding the type of therapy.

The microbiome in urinary tract infections in children - an update.

PURPOSE OF REVIEW: Urinary tract infection (UTI) is one of the most common pediatric infections worldwide. Recently introduced 16S rRNA sequencing allows detailed identification of bacteria involved in UTI on a species-based level. The urogenital microbiome in children is scarcely investigated, with underlying conditions differing from adults. Improvement in diagnostic and therapeutic approaches can help to minimize unnecessary antibiotic treatments, thereby protecting the physiological microbiome. RECENT FINDINGS: Healthy bladders of children display a distinct microbiome than those of adults. UTI is characterized by changes in bacterial composition, with a high prevalence of Enterobacterales. There is a correlation between bacterial species and the pH of the urine, so a characteristic age-related pathogen pattern can be found due to the acidic urine in infants and more alkaline urine in older children. Recently, new methods were proposed to overcome the suboptimal diagnostic performance of urine cultures and urine dipstick test. This allows precise treatment decisions and helps to prevent chronification of UTI, related voiding dysfunctions and renal scaring, systemic abiosis, and the development of antibiotic resistance. SUMMARY: Uropathogens involved in UTIs in children should be identified with precision to allow targeted therapeutic decisions. This can also help preventing the destruction of the microbiome homeostasis, which could result in a life-long dysbiosis. New treatment approaches and recolonization with probiotics are necessary due to increasing intrinsic antibiotic resistance of bacteria.

Discovery of Molecular DNA Methylation-Based Biomarkers through Genome-Wide Analysis of Response Patterns to BCG for Bladder Cancer.

BACKGROUND: Bacillus Calmette-Guérin (BCG) immunotherapy, the standard adjuvant intravesical therapy for some intermediate and most high-risk non-muscle invasive bladder cancers (NMIBCs), suffers from a heterogenous response rate. Molecular markers to help guide responses are scarce and currently not used in the clinical setting. METHODS: To identify novel biomarkers and pathways involved in response to BCG immunotherapy, we performed a genome-wide DNA methylation analysis of NMIBCs before BCG therapy. Genome-wide DNA methylation profiles of DNA isolated from tumors of 26 BCG responders and 27 failures were obtained using the Infinium MethylationEPIC BeadChip. RESULTS: Distinct DNA methylation patterns were found by genome-wide analysis in the two groups. Differentially methylated CpG sites were predominantly located in gene promoters and gene bodies associated with bacterial invasion of epithelial cells, chemokine signaling, endocytosis, and focal adhesion. In total, 40 genomic regions with a significant difference in methylation between responders and failures were detected. The differential methylation state of six of these regions, localized in the promoters of the genes GPR158, KLF8, C12orf42, WDR44, FLT1, and CHST11, were internally validated by bisulfite-sequencing. GPR158 promoter hypermethylation was the best predictor of BCG failure with an AUC of 0.809 (p-value < 0.001). CONCLUSIONS: Tumors from BCG responders and BCG failures harbor distinct DNA methylation profiles. Differentially methylated DNA regions were detected in genes related to pathways involved in bacterial invasion of cells or focal adhesion. We identified candidate DNA methylation biomarkers that may help to predict patient prognosis after external validation in larger, well-designed cohorts.

Secretion of severe acute respiratory syndrome coronavirus 2 in urine.

PURPOSE OF REVIEW: Despite the plethora of publications discussing the severe respiratory coronavirus 2 (SARS-CoV-2), evidence of viral secretion in urine is sparse. RECENT FINDINGS: We could identify 34 publications including a total of 2172 patients. Among those, 549 patients were tested for SARS-CoV-2 secretion in urine, which was detected in only 38 patients (6.9%). Within the seven studies displaying positive results, the majority of positive patients (86.8%) was from not yet peer-reviewed studies including weak data and heterogeneous techniques for sample testing. Furthermore, none of the studies available in the literature addressed the virulence of detected viral RNA in urine. SUMMARY: Overall, only seven studies were able to detect SARS-CoV-2 secretion in urine, all of them with a considerably low rate of positivity. However, these studies were of rather low quality considering their methodology. Despite this, as SARS-CoV-2 has been detected in urine, it is of importance to discuss safety and urinary hygiene protocols. Until further research provides valid data on viral shedding and virulence in urine, potential risk of transmission through urine cannot be ruled out. Therefore, safety and hygiene measures need to be discussed.

Poly(ADP-ribose) polymerase inhibitors in prostate and urothelial cancer.

PURPOSE OF REVIEW: The aim of this article is to give an overview of poly(ADP-ribose) polymerase inhibitors (PARPis) trials in prostate cancer and to discuss emerging approaches with potential future clinical implementation in both prostate and urothelial cancer. RECENT FINDINGS: PARPis are a class of drugs that can be applied for the treatment of homologous recombination repair (HRR)-deficient tumors. Tumors are potentially sensitive to PARPi harbor mutations in genes relevant for DNA damage repair, such as BRCA1/2 or ATM, which are present to a significant degree in metastatic prostate and urothelial cancer patients. Several PARPis have been successfully tested in clinical trials for HRR-deficient metastatic castration-resistant prostate cancer (mCRPC), and olaparib and rucaparib have recently received breakthrough approval in BRCA1/2 mutated mCRPC. Combination treatment of PARPis with androgen-receptor inhibitors or with checkpoint inhibitors and earlier frontline applications are currently being evaluated, and clinical trials enrolling bladder cancer (BCa) patients with HRR deficiency have recently been initiated. SUMMARY: Approximately 10% of mCRPC patients and 34% of metastatic BCa patients have tumors with HRR deficiency and may benefit from PARPi treatment. Correct identification of these patients as well as determining the most adequate time point for drug administration will be key to successful clinical implementation.

PD1/PD-L1 therapy in metastatic renal cell carcinoma.

PURPOSE OF REVIEW: The aim of the article to summarize recent changes of treatment options in metastatic renal cell carcinoma (mRCC) with a special emphasis on immune checkpoint inhibition. RECENT FINDINGS: The introduction of checkpoint inhibitor (CPI) therapy has led to a paradigm change in advanced renal cell carcinoma (RCC). Dual immune checkpoint inhibition or the combination of CPI and tyrosine kinase inhibitors (TKIs) was shown to improve survival when compared with the former standard of care sunitinib. Moreover, these novel strategies were shown to enable unprecedented rates of complete and durable responses, particularly with dual checkpoint inhibition. Although the treatment landscape has rapidly evolved, it remains unknown which combination is the best for the individual patient. Pivotal trials have used sunitinib as a comparator but no head to head comparisons have been conducted between novel agents so far. Moreover, no predictive biomarker has been identified yet to bring the best treatment to the individual patient. SUMMARY: The aim of this review is to summarize the findings of CPI-based trials conducted in RCC and to discuss the future of mRCC treatment.

The prognostic impact of tumour NSD2 expression in advanced prostate cancer.

Purpose: To assess the prognostic significance of the nuclear receptor binding SET protein 2 (NSD2), a co-activator of the NFkB-pathway, on tumour progression in patients with advanced prostate cancer (PCa).Methods: We retrospectively assessed NSD2 expression in 53 patients with metastatic and castration-resistant PCa. Immunohistochemical staining for NSD2 was carried out on specimen obtained from palliative resection of the prostate. Univariable and multivariable analyses were performed to assess the association between NSD2 expression and PCa progression.Results: Of the 53 patients, 41 had castration-resistant PCa and 48 men had metastases at time of tissue acquisition. NSD2 expression was increased in tumour specimen from 42 patients (79.2%). In univariable Cox regression analyses, NSD2 expression was associated with PSA progression, progression on imaging and overall survival (p = 0.04, respectively). In multivariable analyses, NSD2 expression did not retain its association with these endpoints.Conclusions: NSD2 expression is abnormal in almost 80% of patients with advanced PCa. Expression levels of this epigenetic regulator are easily detected by immunohistochemistry while this biomarker exhibited prognostic value for PCa progression and death in univariable analysis. Further studies on NSD2 involvement in PCa proliferation, progression, metastasis and resistance mechanisms are needed.

Epigenetic alterations of testicular germ cell tumours.

PURPOSE OF REVIEW: Testicular germ cell tumours (TGCTs) exhibit, in contrast to other cancer types, a relatively low mutational burden. However, numerous epigenetic alterations have been shown to impact TGCT. In this review, we summarize the most relevant findings of the past 2 years. RECENT FINDINGS: Recent studies focused on the functions of microRNAs and the impact of aberrant DNA methylation. Moreover, several epigenetic drugs with antineoplastic effects in TGCTs were identified. SUMMARY: Aberrant DNA methylation and differentially expressed microRNAs have an important effect on TGCT pathogenesis. Moreover, differential DNA methylation patterns were found to be specific for different TGCT subtypes. Various microRNAs, such as miR-371a-3p, were found to be highly sensitive and specific biomarkers for TGCT. The epigenetic drugs guadecitabine, animacroxam, and JQ1 showed promising effects on TGCT in preclinical in-vivo and in-vitro studies.

Prevalence and Prognostic Value of the Polymorphic Variant 1245A>C of HSD3B1 in Castration-resistant Prostate Cancer.

INTRODUCTION: The purpose of this study was to investigate the prevalence and prognostic value of the polymorphic variant (1245A>C), a single nucleotide polymorphism (SNP) of the HSD3B1 gene, in the tumors of patients with castration-resistant prostate cancer (CRPC). MATERIALS AND METHODS: We retrospectively evaluated 44 patients with CRPC who underwent palliative transurethral resection of the prostate. Genomic DNA was extracted from formalin-fixed and paraffin-embedded material, and 1245A>C SNP of the HSD3B1 gene was analyzed via Sanger sequencing. Cox regression analysis was used to assess the prognostic value of the respective SNP with time to progression as well as cancer-specific and overall survival in the subgroup of patients receiving second systemic treatment. RESULTS: The SNP was present in 20 patients (51.2%) who received second line systemic treatment additionally to androgen deprivation, of which 16 (80%) patients were heterozygous and 4 (20%) were homozygous. Correlation analysis revealed no association of the SNP with any clinical characteristics at initiation of second-line systemic treatment. Moreover, the presence of the variant (1245A>C) of HSD3B1 was not associated with any survival endpoint. CONCLUSIONS: The variant allele 1245C of the HSD3B1 gene is present in approximately one-half of patients with CRPC; however, it is not associated with oncologic outcomes. These findings, however, need to be interpreted with caution as the sample size is small. Further research on biomarkers is needed to help tailor clinical decision making in prostate cancer, especially in the increasingly complex therapeutic landscape of CRPC.

Hepatocyte specific expression of an oncogenic variant of ß-catenin results in lethal metabolic dysfunction in mice.

BACKGROUND: Wnt/ß-catenin signaling plays a crucial role in embryogenesis, tissue homeostasis, metabolism and malignant transformation of different organs including the liver. Continuous ß-catenin signaling due to somatic mutations in exon 3 of the Ctnnb1 gene is associated with different liver diseases including cancer and cholestasis. RESULTS: Expression of a degradation resistant form of ß-catenin in hepatocytes resulted in 100% mortality within 31 days after birth. Ctnnb1CAhep mice were characterized by reduced body weight, significantly enlarged livers with hepatocellular fat accumulation around central veins and increased hepatic triglyceride content. Proteomics analysis using whole liver tissue revealed significant deregulation of proteins involved in fat, glucose and mitochondrial energy metabolism, which was also reflected in morphological anomalies of hepatocellular mitochondria. Key enzymes involved in transport and synthesis of fatty acids and cholesterol were significantly deregulated in livers of Ctnnb1CAhep mice. Furthermore, carbohydrate metabolism was substantially disturbed in mutant mice. CONCLUSION: Continuous ß-catenin signaling in hepatocytes results in premature death due to severe disturbances of liver associated metabolic pathways and mitochondrial dysfunction. METHODS: To investigate the influence of permanent ß-catenin signaling on liver biology we analyzed mice with hepatocyte specific expression of a dominant stable form of ß-catenin (Ctnnb1CAhep ) and their WT littermates by serum biochemistry, histology, electron microscopy, mRNA profiling and proteomic analysis of the liver.

Boosting the hypoxic response in myeloid cells accelerates resolution of fibrosis and regeneration of the liver in mice.

We have recently shown that targeting Vascular Endothelial Growth Factor (VEGF) specifically in scar-infiltrating myeloid cells prevented remodeling of the sinusoidal vasculature and abrogated the resolution of murine liver fibrosis, thereby unmasking an unanticipated link between angiogenesis and resolution of fibrosis. In a gain of function approach, we wanted to test the impact of VEGF overexpression in myeloid cells on fibrolysis. We observe that genetic inactivation of the von Hippel Lindau protein (VHL), a negative regulator of Hypoxia-inducible factors (HIF) in myeloid cells, leads to increased VEGF expression and most importantly, accelerated matrix degradation and reduced myofibroblast numbers after CCl4 challenge. This is associated with enhanced expression of MMP-2 and -14 as well as lower expression of TIMP-2 in liver endothelial cells. In addition, we report increased expression of MMP-13 in scar-associated macrophages as well as improved liver regeneration upon ablation of VHL in myeloid cells. Finally, therapeutic infusion of macrophages nulli-zygous for VHL or treated with the pharmacologic hydroxylase inhibitor and HIF-inducer Dimethyloxalylglycine (DMOG) accelerates resolution of fibrosis. Hence, boosting the HIF-VEGF signaling axis in macrophages represents a promising therapeutic avenue for the treatment of liver fibrosis.

Hepatocyte specific expression of an oncogenic variant of ß-catenin results in cholestatic liver disease.

BACKGROUND: The Wnt/ß-catenin signaling pathway plays a crucial role in embryonic development, tissue homeostasis, wound healing and malignant transformation in different organs including the liver. The consequences of continuous ß-catenin signaling in hepatocytes remain elusive. RESULTS: Livers of Ctnnb1CA hep mice were characterized by disturbed liver architecture, proliferating cholangiocytes and biliary type of fibrosis. Serum ALT and bile acid levels were significantly increased in Ctnnb1CA hep mice. The primary bile acid synthesis enzyme Cyp7a1 was increased whereas Cyp27 and Cyp8b1 were reduced in Ctnnb1CA hep mice. Expression of compensatory bile acid transporters including Abcb1, Abcb4, Abcc2 and Abcc4 were significantly increased in Ctnnb1CA hep mice while Ntcp was reduced. Accompanying changes of bile acid transporters favoring excretion of bile acids were observed in intestine and kidneys of Ctnnb1CA hep mice. Additionally, disturbed bile acid regulation through the FXR-FGF15-FGFR4 pathway was observed in mice with activated ß-catenin. MATERIALS AND METHODS: Mice with a loxP-flanked exon 3 of the Ctnnb1 gene were crossed to Albumin-Cre mice to obtain mice with hepatocyte-specific expression of a dominant stable form of ß-catenin (Ctnnb1CA hep mice). Ctnnb1CA hep mice were analyzed by histology, serum biochemistry and mRNA profiling. CONCLUSIONS: Expression of a dominant stable form of ß-catenin in hepatocytes results in severe cholestasis and biliary type fibrosis.

Inhibition of intestinal bile acid absorption improves cholestatic liver and bile duct injury in a mouse model of sclerosing cholangitis.

BACKGROUND AND AIMS: Approximately 95% of bile acids (BAs) excreted into bile are reabsorbed in the gut and circulate back to the liver for further biliary secretion. Therefore, pharmacological inhibition of the ileal apical sodium-dependent BA transporter (ASBT/SLC10A2) may protect against BA-mediated cholestatic liver and bile duct injury. METHODS: Eight week old Mdr2(-/-) (Abcb4(-/-)) mice (model of cholestatic liver injury and sclerosing cholangitis) received either a diet supplemented with A4250 (0.01% w/w) - a highly potent and selective ASBT inhibitor - or a chow diet. Liver injury was assessed biochemically and histologically after 4weeks of A4250 treatment. Expression profiles of genes involved in BA homeostasis, inflammation and fibrosis were assessed via RT-PCR from liver and ileum homogenates. Intestinal inflammation was assessed by RNA expression profiling and immunohistochemistry. Bile flow and composition, as well as biliary and fecal BA profiles were analyzed after 1week of ASBT inhibitor feeding. RESULTS: A4250 improved sclerosing cholangitis in Mdr2(-/-) mice and significantly reduced serum alanine aminotransferase, alkaline phosphatase and BAs levels, hepatic expression of pro-inflammatory (Tnf-α, Vcam1, Mcp-1) and pro-fibrogenic (Col1a1, Col1a2) genes and bile duct proliferation (mRNA and immunohistochemistry for cytokeratin 19 (CK19)). Furthermore, A4250 significantly reduced bile flow and biliary BA output, which correlated with reduced Bsep transcription, while Ntcp and Cyp7a1 were induced. Importantly A4250 significantly reduced biliary BA secretion but preserved HCO3(-) and biliary phospholipid secretion resulting in an increased HCO3(-)/BA and PL/BA ratio. In addition, A4250 profoundly increased fecal BA excretion without causing diarrhea and altered BA pool composition, resulting in diminished concentrations of primary BAs tauro-ß-muricholic acid and taurocholic acid. CONCLUSIONS: Pharmacological ASBT inhibition attenuates cholestatic liver and bile duct injury by reducing biliary BA concentrations in mice.

Resolution of liver fibrosis requires myeloid cell-driven sinusoidal angiogenesis.

UNLABELLED: Angiogenesis is a key feature of liver fibrosis. Although sinusoidal remodeling is believed to contribute to fibrogenesis, the impact of sinusoidal angiogenesis on the resolution of liver fibrosis remains undefined. Myeloid cells, particularly macrophages, constantly infiltrate the fibrotic liver and can profoundly contribute to remodeling of liver sinusoids. We observe that the development of fibrosis is associated with decreased hepatic vascular endothelial growth factor (VEGF) expression as well as sinusoidal rarefication of the fibrotic scar. In contrast, the resolution of fibrosis is characterized by a rise in hepatic VEGF levels and revascularization of the fibrotic tissue. Genetic ablation of VEGF in myeloid cells or pharmacological inhibition of VEGF receptor 2 signaling prevents this angiogenic response and the resolution of liver fibrosis. We observe increased expression of matrix metalloproteases as well as decreased expression of tissue inhibitor of metalloproteases confined to sinusoidal endothelial cells in response to myeloid cell VEGF. Remarkably, reintroduction of myeloid cell-derived VEGF upon recovery restores collagenolytic acitivity and the resolution of fibrosis. CONCLUSION: We identify myeloid cell-derived VEGF as a critical regulator of extracellular matrix degradation by liver endothelial cells, thereby unmasking an unanticipated link between angiogenesis and the resolution of fibrosis.