Surgery for Aortic Prosthetic Valve Endocarditis in the Transcatheter Era.

Objectives: As surgical experience with infective endocarditis following transcatheter aortic valve replacement is scarce, this study compared the perioperative and short-term outcomes of patients suffering from endocarditis following surgical aortic valve replacement and transcatheter aortic valve replacement. Methods: Between January 2013 and December 2020, 468 consecutive patients were admitted to our center for surgery for IE. Among them, 98 were operated on for endocarditis following surgical aortic valve replacement and 22 for endocarditis following transcatheter aortic valve replacement. Results: The median EuroSCORE II (52.1 (40.6-62.0) v/s 45.4 (32.6-58.1), p = 0.207) and STS-PROM (1.8 (1.6-2.1) v/s 1.9 (1.4-2.2), p = 0.622) were comparable. Endocarditis following transcatheter aortic valve replacement accounted for 13.7% of the aortic prosthetic valve endocarditis between 2013 and 2015; this increased to 26.9% in the years 2019 and 2020.Concomitant procedures were performed in 35 patients (29.2%). The operative mortality was 26.5% in the endocarditis following surgical aortic valve replacement group and 9.1% in the endocarditis following transcatheter aortic valve replacement group (p = 0.098). Upon follow-up, survival at 6 months was found to be 98% in the group with endocarditis following surgical aortic valve replacement and 89% in the group with endocarditis following transcatheter aortic valve replacement (p = 0.081). Conclusions: Patients suffering from endocarditis following surgical aortic valve replacement and transcatheter aortic valve replacement present with comparable risk profiles and can be surgically treated with comparable results. Surgery as a curative option should not be rejected even in this intermediate-risk cohort.

Gastric Mucosal Devitalization (GMD): Using the Porcine Model to Develop a Novel Endoscopic Bariatric Approach.

BACKGROUND AND AIMS: As the pig model has similar gastrointestinal anatomy and physiology to humans, we used pigs to create a gastric mucosal devitalization (GMD) model in preparation for clinical translation of this technique as an endoscopic bariatric therapy (EBT). The aims of this study were to determine the ablation parameters and technique for a successful, safe, and feasible large surface area GMD that produces weight loss. METHODS: We performed GMD using argon plasma coagulation (APC) in 3 phases. Phase 1 assessed the ablation energy required to accomplish selective mucosal ablation using ex vivo pig stomachs (n = 2). Phase 2 assessed the optimal percentage of mucosal surface area to be treated and was performed on 10 pigs. Phase 3 assessed feasibility, efficacy, and safety with 8 pigs randomized into GMD (n = 4) or sham (SH, n = 4) and survived for 1 month. Body weights (GMD, n = 4, SH, n = 4) were measured daily in phase 3 for 1 month, and relative body weights were calculated and analyzed using one-tailed Student's t-test. Percent body fat was compared between GMD and SH at baseline and 1 month post-GMD. RESULTS: Phase 1 identified the optimal ablation parameters (120 W) that were then used in phase 2. Phase 2 revealed a trend that was suggestive that the optimal percent surface area to ablate was similar to that which is removed at laparoscopic sleeve gastrectomy. In phase 3, GMD was performed over 70% surface area of the greater curvature of the stomach in four pigs. GMD pigs had significantly lower relative body weight increase compared to SH at 1 month (1.375 ± 0.085 vs 1.575 ± 0.047, p = 0.0435). MRI showed a significantly lower body fat mass at 1 month in GMD pigs (5.9 ± 0.4% vs 12.7 ± 2.3%, p = 0.026) compared to SH. CONCLUSIONS: GMD resulted in decreased weight gain in the GMD group as evidenced by a lower relative body weight at 1 month. GMD in an animal model appears to show promise as a potential weight loss therapy.

Quantification of Multiple Bacteria in Calcified Structural Valvular Heart Disease.

Genome studies of heart valve tissue (HVT) in patients with structural valvular heart disease (sVHD) and acute infective endocarditis (aIE) showed polymicrobial infections. Subject of this study is the quantification of bacterial DNA in HVT of sVHD in comparison to aIE. It will be examined whether the bacterial DNA concentration can be used as surrogate marker to differentiate chronic and acute infections. DNA was isolated from HVT of 100 patients with sVHD and 23 microbiologically positively tested patients with aIE. Selected pathogens (Cutibacterium acnes, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, Clostridium difficile, and Klebsiella pneumoniae) were quantified using TaqMan-qPCR. Polymicrobial infiltration of HVT was investigated by immunohistologic methods. Of 100 sVHD patients, 94 tested positive for bacteria by 16S-rDNA and 72 by TaqMan-qPCR. In 29% of the sVHD cohort and in 70% of the aIE cohort, a coinfection with more than 2 bacteria was observed as indication of a polymicrobial infection. The most common pathogens in the sVHD patients were C. acnes (59%; 5-4074 pg/mL), E. faecalis (16%, 174-2781 pg/mL), and S. aureus (15%, 8-105 pg/mL). The DNA concentration of E. faecalis (P = 0.0285) and S. aureus (P = 0.0149) is significantly lower in the sVHD cohort than in the aIE cohort. sVHD is associated with bacterial infection and infiltration of the HVT in a majority of cases. TaqMan-qPCR is a valid instrument for the specific detection of bacteria in HVT and allows discrimination between sVHD and aIE for E. faecalis and S. aureus.

Bacterial infiltration in structural heart valve disease.

OBJECTIVES: The pathology of structural valvular heart disease (sVHD) ranges from basic diseases of rheumatologic origin to chronic degenerative remodeling processes after acute bacterial infections. Molecular genetic methods allow detection of the complete microbial spectrum in heart valve tissues independent of microbiological cultivation. In particular, whole-metagenome analysis is a sensitive and highly specific analytical method that allows a deeper insight into the pathogenicity of the diseases. In the present study we assessed the pathogen spectrum in heart valve tissue from 25 sVHD patients using molecular and microbiological methods. METHODS: Twenty-five sVHD patients were selected randomly from an observational cohort study (March 2016 to January 2017). The explanted native heart valves were examined using microbiological methods and immunohistological structural analysis. In addition, the bacterial metagenome of the heart valve tissue was determined using next-generation sequencing. RESULTS: The use of sonication as a pretreatment of valve tissue from 4 sVHD patients permitted successful detection of Clostridium difficile, Enterococcus faecalis, Staphylococcus saccharolyticus, and Staphylococcus haemolyticus using microbial cultivation. Histological staining revealed intramural localization. Metagenome analysis identified a higher rate of bacterial infiltration in 52% of cases. The pathogen spectrum included both gram-positive and gram-negative bacteria. CONCLUSIONS: Microbiological and molecular biological studies are necessary to detect the spectrum of bacteria in a calcified heart valve. Metagenome analysis is a valid method to gain new insight into the polymicrobial pathophysiology of sVHD. Our results suggest that an undetected proportion of sVHD might be triggered by chronic inflammation or influenced by secondary bacterial infiltration.

Gastric mucosal devitalization improves blood pressure, renin and cardiovascular lipid deposition in a rat model of obesity.

Background and study aims In lieu of the drawbacks of metabolic surgery, a method of mimicking resection of the gastric mucosa could be of value to those with obesity-related cardiovascular disease (CVD). Our study aims to investigate the effect of gastric mucosal devitalization (GMD) on blood pressure (BP) and cardiovascular lipid deposition in a rat model of obesity. Methods GMD of 70 % of the stomach was achieved by argon plasma coagulation. GMD was compared to sleeve gastrectomy (SG) and sham (SH) in a high-fat-diet-induced rat model of obesity (48 rats). At 8 weeks, we measured noninvasive BP, renin, vessel relaxation and ghrelin receptor regulation in the aorta. In addition, we quantified cardiac lipid deposition and lipid droplet deposition in cardiac muscle and aorta. Results GMD and SG were observed to have similar reductions in body weight, visceral adiposity, and serum lipid profile compared to SH rats. GMD resulted in a significant reduction in arterial BP compared to SH. Furthermore, there were significant reductions in plasma renin activity and percentage of phenylnephrine constriction to acetylcholine at the aortic ring in GMD rats compared to SH, providing insights into the mechanisms behind the reduced BP. Interestingly, the reduced BP occurred despite a reduction in endothelial ghrelin recteptor activation. Cardiac lipid content was significantly reduced in GMD rats. Lipid deposition, as illustrated by Nile Red stain, was reduced in cardiac muscle and the aorta. Conclusion GMD resulted in a significant improvement in BP, renin and cardiovascular lipid deposition. GMD deserves further attention as a method of treating obesity-related CVD.

Gastric mucosal devitalization is safe and effective in reducing body weight and visceral adiposity in a porcine model.

BACKGROUND AND AIMS: The early improvement in metabolic profile after sleeve gastrectomy (SG) indicates that the significant benefits of metabolic surgery are gastric in origin. We have previously demonstrated that devitalization of the gastric mucosa (without a reduction in gastric volume) in metabolically disturbed obese rats results in an improvement of obesity and its associated comorbidities. The aims of this study were to assess the technical feasibility, efficacy, and safety of gastric mucosal devitalization (GMD) in a large animal (porcine) model. METHODS: A 3-arm (GMD versus SG versus sham [SH]) prospective randomized controlled trial with an 8-week follow-up period was performed. The primary endpoint was relative weight loss. Secondary endpoints were absolute body weight, abdominal visceral adiposity, abdominal subcutaneous adiposity, organ lipid content, and serum ghrelin level. RESULTS: GMD resulted in a significant relative weight loss of 36% over SH at 8 weeks (P < .05). There was no significant difference in relative weight loss between GMD and SG at 4 weeks; however, SG resulted in a 29% superior relative weight loss at 8 weeks (P < .05). With regard to visceral adiposity, there was a significant benefit of GMD over SH at 8 weeks. Despite differences in relative weight loss, there was no significant difference in visceral adiposity between SG and GMD at 8 weeks. Significant improvements in GMD over SH were noted with regard to skeletal and heart muscle lipid content. GMD pigs at 8 weeks demonstrated regeneration of the gastric mucosa without ulceration or significant scarring. Despite mucosal regeneration, the abundance of serum ghrelin was significantly lower in the GMD cohort compared with the SG and SH cohorts. CONCLUSIONS: GMD was technically feasible and resulted in relative weight loss and an improvement in visceral adiposity. The benefits noted were out of proportion to what would be expected with weight loss alone.

Gastric mucosal devitalization reduces adiposity and improves lipid and glucose metabolism in obese rats.

BACKGROUND AND AIMS: The gastric mucosa is an endocrine organ that regulates satiation pathways by expression of orexigenic and anorexigenic hormones. Vertical sleeve gastrectomy (VSG) excludes gastric mucosa and reduces gastric volume. Our study aimed to investigate the independent effects of altering gastric mucosa on obesity and its related comorbidities. METHODS: Gastric mucosa devitalization (GMD) of 70% of the stomach was achieved by argon plasma coagulation in a high-fat diet rat model and was compared with VSG and sham surgery. In an 8-week follow-up study, we quantified body weight, visceral adiposity, insulin resistance index, cholesterol profiles, and free fatty acid profiles by enzyme-linked immunosorbent assay (ELISA). Following a 2-hour oral glucose tolerance test, the kinetics of ghrelin, glucagon-like peptide-1, peptide YY, and serum and liver bile acid levels were measured. Liver lipid content was quantified by ELISA. RESULTS: GMD resulted in significant reductions in body weight, visceral and subcutaneous adipose tissue, and hepatic steatosis as well as an improvement in lipid metabolism. GMD resulted in significant reductions in food intake and intestinal malabsorption of free fatty acids, both contributing to improved body composition and metabolic profile. Mechanistically, GMD resulted in a significant reduction in serum palmitate levels as well as an increase in serum and liver bile acid levels, known to alter glucose and lipid metabolism. Similar changes were noted when VSG rats were compared with sham surgery rats. CONCLUSIONS: Devitalization of gastric mucosa, independent of altering gastric volume, was able to reduce obesity-related comorbidities. The gastric mucosa may be a potential target for treating obesity and its associated comorbidities.

Tumor-associated energy homeostasis: hepatoblastoma and neuroblastoma affect glucose and lipid metabolism as well as ghrelin, GLP-1, and PYY in nude rats.

INTRODUCTION: The "metabolic competition" for nutrients between cancer cells and the patient has emerged as an important research area. For pediatric oncology, it remains unclear whether the neuroendokrine regulation of appetite by gastrointestinal hormones such as ghrelin "eat", GLP-1 (glucagon-like peptide, "do not eat"), and PYY (peptide tyrosine-tyrosine, "do not eat") is influenced by tumor growth. MATERIAL AND METHODS: In a prospective randomized study, human hepatoblastoma (HB) and neuroblastoma (NB) cells (3 × 10(6)) were transplanted into the abdominal wall of immune-incompetent (nu/nu) rats (ethic committee approval: TVV43/11). Sham-operated animals received cell culture medium only. Tumor growth was allowed for 8 weeks. Then, all the animals underwent a 2-hour oGTT (oral glucose tolerance test) and were assessed for serum levels of glucose, insulin, ghrelin, GLP-1, and PYY. Finally, all tumor masses and adipose tissues were excised and calculated. RESULTS: Total body weight (including tumor masses) differed for HB (329+31 g), but not for NB (358+22 g) compared with Sham (361+35 g). Subcutaneous adipose tissue was significantly decreased for both the tumor groups (HB=2.6 g, NB=2.1 g, and Sham=3.5 g). Only for NB, fasting glucose (3.4 + 0.6 mmol/L) and insulin (0.89+0.11 ng/mL) levels were significantly decreased compared with Sham (4.4+0.6 mmol/L; 1.19+0.36 ng/mL) only. During the oGTT (all data calculated as area under the curve, AUC) glucose levels were significantly increased for HB (104 ± 10) and NB (102 ± 13) compared with Sham (84 ± 3), but insulin levels remained similar for either group. Triglyceride levels were increased for HB (0.51 mmol/L) and especially NB (0.73 mmol/L) compared with Sham (0.34 mmol/L). Inflammatory parameters did not differ between the groups. Total ghrelin levels were significantly increased for NB (111 ± 10) and altered for HB (102 ± 15) compared with Sham (84 ± 8). Vice versa GLP-1 was statistically decreased in HB (92 ± 7) and NB (88 ± 12) compared with Sham (127 ± 13). Finally, PYY levels were nonsignificantly reduced for HB (117 ± 5) and NB (120 ± 4) compared with Sham (146 ± 12).

Establishing a reliable multiple reaction monitoring-based method for the quantification of obesity-associated comorbidities in serum and adipose tissue requires intensive clinical validation.

Multiple reaction monitoring (MRM)-based mass spectrometric quantification of peptides and their corresponding proteins has been successfully applied for biomarker validation in serum. The option of multiplexing offers the chance to analyze various proteins in parallel, which is especially important in obesity research. Here, biomarkers that reflect multiple comorbidities and allow monitoring of therapy outcomes are required. Besides the suitability of established MRM assays for serum protein quantification, it is also feasible for analysis of tissues secreting the markers of interest. Surprisingly, studies comparing MRM data sets with established methods are rare, and therefore the biological and clinical value of most analytes remains questionable. A MRM method using nano-UPLC-MS/MS for the quantification of obesity related surrogate markers for several comorbidities in serum, plasma, visceral and subcutaneous adipose tissue was established. Proteotypic peptides for complement C3, adiponectin, angiotensinogen, and plasma retinol binding protein (RBP4) were quantified using isotopic dilution analysis and compared to the standard ELISA method. MRM method variabilities were mainly below 10%. The comparison with other MS-based approaches showed a good correlation. However, large differences in absolute quantification for complement C3 and adiponectin were obtained compared to ELISA, while less marked differences were observed for angiotensinogen and RBP4. The verification of MRM in obesity was performed to discriminate first lean and obese phenotype and second to monitor excessive weight loss after gastric bypass surgery in a seven-month follow-up. The presented MRM assay was able to discriminate obese phenotype from lean and monitor weight loss related changes of surrogate markers. However, inclusion of additional biomarkers was necessary to interpret the MRM data on obesity phenotype properly. In summary, the development of disease-related MRMs should include a step of matching the MRM data with clinically approved standard methods and defining reference values in well-sized representative age, gender, and disease-matched cohorts.

Sleeve gastrectomy reduces xanthine oxidase and uric acid in a rat model of morbid obesity.

BACKGROUND: Serum uric acid (sUA) plays a major role in the development of morbidities associated with obesity, especially cardiovascular diseases. Within the purine pathway, xanthine oxidase (XOD) represents the key enzyme. The aim of this study was to investigate the dynamics of sUA and XOD following sleeve gastrectomy (SG) in a rat model of high-fat-diet (HFD) induced obesity. PATIENTS: Over a period of 11 weeks, 30 rats received a HFD, and 10 rats received a low fat diet (LFD). Thereafter, 10 randomly selected HFD rats and 10 LFD rats were sacrificed. The remaining 20 HFD rats were randomly assigned to either SG or sham operation (SH) and studied 14 days postoperatively. METHODS: The white adipose tissues (WAT) from visceral (intestinal and retroperitoneal) and inguinal (subcutaneous) depots were collected. sUA and urine UA (uUA) were measured by high performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Abundance and activity of XOD was investigated in the liver, colon, adipose tissue, and skeletal muscle by enzyme-linked immunosorbent assay (ELISA). RESULTS: HFD led to significant weight gain, elevated sUA levels, increased WAT and increase of XOD activity. Fourteen days postoperatively, SG rats showed a significant decrease of weight and adipose tissue, improved glucose metabolism, and changes of gut hormones. The sUA and uUA levels were significantly decreased following SG. Furthermore, XOD activity was significantly down-regulated in WAT. CONCLUSION: HFD induces elevated sUA levels by gain of WAT and increase of XOD activity. Following SG, the reduction of WAT as the major source of XOD and the lowering of XOD activity are the basis for the decrease of sUA.

Bariatric surgery in severely obese adolescents improves major comorbidities including hyperuricemia.

OBJECTIVE: Serum uric acid (sUA) is believed to contribute to the pathogenesis of metabolic comorbidities like hypertension, insulin-resistance (IR) and endothelial dysfunction (EDF) in obese children. The present pilot study investigated the association between sUA concentrations and loss of body weight following laparoscopic sleeve gastrectomy (LSG) or laparoscopic Roux-en-Y-gastric bypass (RYGB) in severely obese adolescents. MATERIALS/METHODS: 10 severely obese adolescents underwent either LSG (n=5) or RYGB (n=5). 17 normal weight, healthy, age- and gender-matched adolescents served as a normal weight peer group (NWPG). Pre- and 12 months postoperatively, sUA and relevant metabolic parameters (glucose homeostasis, transaminases, lipids) were compared. RESULTS: Preoperatively, sUA was significantly elevated in patients with severe obesity compared to NWPG. Twelve months after LSG and RYGB, a significant decrease in sUA, BMI, CVD risk factors, hepatic transaminases, and HOMA-IR was observed. Reduction in SDS-BMI significantly correlated with changes in sUA. CONCLUSIONS: sUA levels and metabolic comorbidities improved following bariatric surgery in severely obese adolescents. The impact of changes in sUA on long-term clinical complications of childhood obesity deserves further study.

The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro.

In allogenic and xenogenic transplantation, adequate immunosuppression plays a major role in graft survival, especially over the long term. The effect of immunosuppressive drugs on neural stem/progenitor cell fate has not been sufficiently explored. The focus of this study is to systematically investigate the effects of the following four different immunotherapeutic strategies on human neural progenitor cell survival/death, proliferation, metabolic activity, differentiation and migration in vitro: (1) cyclosporine A (CsA), a calcineurin inhibitor; (2) everolimus (RAD001), an mTOR-inhibitor; (3) mycophenolic acid (MPA, mycophenolate), an inhibitor of inosine monophosphate dehydrogenase and (4) prednisolone, a steroid. At the minimum effective concentration (MEC), we found a prominent decrease in hNPCs' proliferative capacity (BrdU incorporation), especially for CsA and MPA, and an alteration of the NAD(P)H-dependent metabolic activity. Cell death rate, neurogenesis, gliogenesis and cell migration remained mostly unaffected under these conditions for all four immunosuppressants, except for apoptotic cell death, which was significantly increased by MPA treatment.

Free fatty acid palmitate impairs the vitality and function of cultured human bladder smooth muscle cells.

BACKGROUND: Incidence of urinary tract infections is elevated in patients with diabetes mellitus. Those patients show increased levels of the saturated free fatty acid palmitate. As recently shown metabolic alterations induced by palmitate include production and secretion of the pro-inflammatory cytokine interleukine-6 (IL-6) in cultured human bladder smooth muscle cells (hBSMC). Here we studied the influence of palmitate on vital cell properties, for example, regulation of cell proliferation, mitochondrial enzyme activity and antioxidant capacity in hBSMC, and analyzed the involvement of major cytokine signaling pathways. METHODOLOGY/PRINCIPAL FINDINGS: HBSMC cultures were set up from bladder tissue of patients undergoing cystectomy and stimulated with palmitate. We analyzed cell proliferation, mitochondrial enzyme activity, and antioxidant capacity by ELISA and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-κB, JAK/STAT, MEK1, PI3K, and JNK in major cytokine signaling pathway regulation. We found: (i) palmitate decreased cell proliferation, increased mitochondrial enzyme activity and antioxidant capacity; (ii) direct inhibition of cytokine receptor by AG490 even more strongly suppressed cell proliferation in palmitate-stimulated cells, while counteracting palmitate-induced increase of antioxidant capacity; (iii) in contrast knockdown of the STAT3 inhibitor SOCS3 increased cell proliferation and antioxidant capacity; (iv) further downstream JAK/STAT3 signaling cascade the inhibition of PI3K or JNK enhanced palmitate induced suppression of cell proliferation; (v) increase of mitochondrial enzyme activity by palmitate was enhanced by inhibition of PI3K but counteracted by inhibition of MEK1. CONCLUSIONS/SIGNIFICANCE: Saturated free fatty acids (e.g., palmitate) cause massive alterations in vital cell functions of cultured hBSMC involving distinct major cytokine signaling pathways. Thereby, certain cytokines might counteract the palmitate-induced downregulation of cell proliferation and vitality. This could be an important link to clinical findings of increased risk of metabolic related bladder diseases such as overactive bladder (OAB) and bladder pain syndrome/interstitial cystitis (BPS/IC).

Combined proteomic and metabolomic profiling of serum reveals association of the complement system with obesity and identifies novel markers of body fat mass changes.

Obesity is associated with multiple adverse health effects and a high risk of developing metabolic and cardiovascular diseases. Therefore, there is a great need to identify circulating parameters that link changes in body fat mass with obesity. This study combines proteomic and metabolomic approaches to identify circulating molecules that discriminate healthy lean from healthy obese individuals in an exploratory study design. To correct for variations in physical activity, study participants performed a one hour exercise bout to exhaustion. Subsequently, circulating factors differing between lean and obese individuals, independent of physical activity, were identified. The DIGE approach yielded 126 differentially abundant spots representing 39 unique proteins. Differential abundance of proteins was confirmed by ELISA for antithrombin-III, clusterin, complement C3 and complement C3b, pigment epithelium-derived factor (PEDF), retinol binding protein 4 (RBP4), serum amyloid P (SAP), and vitamin-D binding protein (VDBP). Targeted serum metabolomics of 163 metabolites identified 12 metabolites significantly related to obesity. Among those, glycine (GLY), glutamine (GLN), and glycero-phosphatidylcholine 42:0 (PCaa 42:0) serum concentrations were higher, whereas PCaa 32:0, PCaa 32:1, and PCaa 40:5 were decreased in obese compared to lean individuals. The integrated bioinformatic evaluation of proteome and metabolome data yielded an improved group separation score of 2.65 in contrast to 2.02 and 2.16 for the single-type use of proteomic or metabolomics data, respectively. The identified circulating parameters were further investigated in an extended set of 30 volunteers and in the context of two intervention studies. Those included 14 obese patients who had undergone sleeve gastrectomy and 12 patients on a hypocaloric diet. For determining the long-term adaptation process the samples were taken six months after the treatment. In multivariate regression analyses, SAP, CLU, RBP4, PEDF, GLN, and C18:2 showed the strongest correlation to changes in body fat mass. The combined serum proteomic and metabolomic profiling reveals a link between the complement system and obesity and identifies both novel (C3b, CLU, VDBP, and all metabolites) and confirms previously discovered markers (PEDF, RBP4, C3, ATIII, and SAP) of body fat mass changes.

Cytokine effects on gap junction communication and connexin expression in human bladder smooth muscle cells and suburothelial myofibroblasts.

BACKGROUND: The last decade identified cytokines as one group of major local cell signaling molecules related to bladder dysfunction like interstitial cystitis (IC) and overactive bladder syndrome (OAB). Gap junctional intercellular communication (GJIC) is essential for the coordination of normal bladder function and has been found to be altered in bladder dysfunction. Connexin (Cx) 43 and Cx45 are the most important gap junction proteins in bladder smooth muscle cells (hBSMC) and suburothelial myofibroblasts (hsMF). Modulation of connexin expression by cytokines has been demonstrated in various tissues. Therefore, we investigate the effect of interleukin (IL) 4, IL6, IL10, tumor necrosis factor-alpha (TNFα) and transforming growth factor-beta1 (TGFß1) on GJIC, and Cx43 and Cx45 expression in cultured human bladder smooth muscle cells (hBSMC) and human suburothelial myofibroblasts (hsMF). METHODOLOGY/PRINCIPAL FINDINGS: HBSMC and hsMF cultures were set up from bladder tissue of patients undergoing cystectomy. In cytokine stimulated cultured hBSMC and hsMF GJIC was analyzed via Fluorescence Recovery after Photo-bleaching (FRAP). Cx43 and Cx45 expression was assessed by quantitative PCR and confocal immunofluorescence. Membrane protein fraction of Cx43 and Cx45 was quantified by Dot Blot. Upregulation of cell-cell-communication was found after IL6 stimulation in both cell types. In hBSMC IL4 and TGFß1 decreased both, GJIC and Cx43 protein expression, while TNFα did not alter communication in FRAP-experiments but increased Cx43 expression. GJ plaques size correlated with coupling efficacy measured, while Cx45 expression did not correlate with modulation of GJIC. CONCLUSIONS/SIGNIFICANCE: Our finding of specific cytokine effects on GJIC support the notion that cytokines play a pivotal role for pathophysiology of OAB and IC. Interestingly, the effects were independent from the classical definition of pro- and antiinflammatory cytokines. We conclude, that connexin regulation involves genomic and/or post-translational events, and that GJIC in hBSMC and hsMF depend of Cx43 rather than on Cx45.

Palmitate induced IL-6 and MCP-1 expression in human bladder smooth muscle cells provides a link between diabetes and urinary tract infections.

BACKGROUND: Urinary tract infections (UTI) are more frequent in type-2 diabetes mellitus patients than in subjects with normal glucose metabolism. The mechanisms underlying this higher prevalence of UTI are unknown. However, cytokine levels are altered in diabetic patients and may thus contribute to the development of UTI. Increased levels of free fatty acids (FFA), as observed in obese patients, can induce IL-6 production in various cell types. Therefore we studied the effects of the free fatty acid palmitate and bacterial lipopolysaccharide (LPS) on interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) expression and secretion in cultured human bladder smooth muscle cells (hBSMC). METHODOLOGY/PRINCIPAL FINDINGS: Biopsies were taken from patients undergoing cystectomy due to bladder cancer. Palmitate or LPS stimulated hBSMC were analysed for the production and secretion of the IL-6, gp80, gp80soluble, gp130, MCP-1, pSTAT3, SOCS3, NF-kappaB and SHP2 by quantitative PCR, ELISA, Western blotting, and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-kappaB and MEK1 in IL-6 and MCP-1 regulation. Palmitate upregulates IL-6 mRNA expression and secretion via NF-kappaB dependent pathways in a concentration- and time-dependent manner. MCP-1 was moderately upregulated by palmitate but was strongly upregulated by LPS involving NF-kappaB and MEK1 dependent pathways. Soluble IL-6 receptor (gp80soluble) was downregulated by palmitate and LPS, while membrane-bound gp80 was moderately upregulated. LPS increased SOCS3 and SHP2, whereas palmitate only induced SOCS3. Secondary finding: most of the IL-6 is secreted. CONCLUSIONS/SIGNIFICANCE: Bacterial infection (LPS) or metabolic alterations (palmitate) have distinct effects on IL-6 expression in hBSMC, (i) short term LPS induced autocrine JAK/STAT signaling and (ii) long-term endocrine regulation of IL-6 by palmitate. Induction of IL-6 in human bladder smooth muscle cells by fatty acids may represent a pathogenetic factor underlying the higher frequency and persistence of urinary tract infections in patients with metabolic diseases.