Harnessing intestinal tryptophan catabolism to relieve atherosclerosis in mice.

Tryptophan (Trp) is an essential amino acid, whose metabolism is a key gatekeeper of intestinal homeostasis. Yet, its systemic effects, particularly on atherosclerosis, remain unknown. Here we show that high-fat diet (HFD) increases the activity of intestinal indoleamine 2, 3-dioxygenase 1 (IDO), which shifts Trp metabolism from the production of microbiota-derived indole metabolites towards kynurenine production. Under HFD, the specific deletion of IDO in intestinal epithelial cells leads to intestinal inflammation, impaired intestinal barrier, augmented lesional T lymphocytes and atherosclerosis. This is associated with an increase in serotonin production and a decrease in indole metabolites, thus hijacking Trp for the serotonin pathway. Inhibition of intestinal serotonin production or supplementation with indole derivatives alleviates plaque inflammation and atherosclerosis. In summary, we uncover a pivotal role of intestinal IDO in the fine-tuning of Trp metabolism with systemic effects on atherosclerosis, paving the way for new therapeutic strategies to relieve gut-associated inflammatory diseases.

Radial artery occlusion after cardiac catheterization and impact of medical treatment.

Background: The transradial artery approach is the preferred access for cardiac catheterization according to current guidelines. However, the most common complication is radial artery occlusion (RAO). Despite the rare indication for surgical reopening, the occluded radial artery is not available for further procedures or as a potential bypass graft. Still, treatment regimens for RAO are scarce. We now determined whether the addition of antithrombotic to antiplatelet therapy improves the rate of partial or complete regain of patency in RAO following transradial cardiac catheterization in a retrospective analysis. Patients and methods: In a two-center tertiary referral hospital retrospective analysis 4135 files of patients who had undergone transradial catheterization were screened for documented RAO. 141 patients were identified and 138 patients with complete information on the medical regimen and ultrasound examinations for a maximum of 3 months were included in the analysis, whereas 3 patients were excluded due to missing or incomplete follow-up information. Results: 3.3% of all patients that had undergone transradial catheterization featured an oligosymptomatic RAO, confirmed by color-coded duplex sonography. 21% of patients with additional anticoagulation regained full patency vs. 9% without additional anticoagulation (p = 0.07). 40% of patients with anticoagulation featured a partial or full regain of patency vs. 16% of patients without additional anticoagulation for a maximum of 3 months treatment (p = 0.006). No major bleedings were reported during the follow-up visits. Conclusions: RAO remains a rare complication of cardiac catheterization. The addition of antithrombotic therapy for 3 months appears to safely improve the partial or even full regain of radial patency in case of postinterventional RAO.

Thymic stromal lymphopoietin is a key cytokine for the immunomodulation of atherogenesis with Freund's adjuvant.

Adaptive immune responses regulate the development of atherosclerosis, with a detrimental effect of type 1 but a protective role of type 2 immune responses. Immunization of Apolipoprotein E-deficient (ApoE-/- ) mice with Freund's adjuvant inhibits the development of atherosclerosis. However, the underlying mechanisms are not fully understood. Thymic stromal lymphopoietin (TSLP) is an IL7-like cytokine with essential impact on type 2 immune responses (Th2). Thymic stromal lymphopoietin is strongly expressed in epithelial cells of the skin, but also in various immune cells following appropriate stimulation. In this study, we investigated whether TSLP may be crucial for the anti-atherogenic effect of Freund's adjuvant. Subcutaneous injection of complete Freund's adjuvant (CFA) rapidly led to the expression of TSLP and IL1ß at the site of injection. In male mice, CFA-induced TSLP occurred in immigrated monocytes-and not epithelial cells-and was dependent on NLRP3 inflammasome activation and IL1ß-signalling. In females, CFA-induced TSLP was independent of IL1ß and upon ovariectomy. CFA/OVA led to a more pronounced imbalance of the T cell response in TSLPR-/- mice, with increased INFγ/IL4 ratio compared with wild-type controls. To test whether TSLP contributes to the anti-atherogenic effects of Freund's adjuvant, we treated ApoE-/- and ApoE-/- /TSLPR-/- mice with either CFA/IFA or PBS. ApoE-/- mice showed less atherogenesis upon CFA/IFA compared with PBS injections. ApoE-/- /TSLPR-/- mice had no attenuation of atherogenesis upon CFA/IFA treatment. Freund's adjuvant executes significant immune-modulating effects via TSLP induction. TSLP-TSLPR signalling is critical for CFA/IFA-mediated attenuation of atherosclerosis.

Heart failure after pressure overload in autosomal-dominant desminopathies: Lessons from heterozygous DES-p.R349P knock-in mice.

BACKGROUND: Mutations in the human desmin gene (DES) cause autosomal-dominant and -recessive cardiomyopathies, leading to heart failure, arrhythmias, and AV blocks. We analyzed the effects of vascular pressure overload in a patient-mimicking p.R349P desmin knock-in mouse model that harbors the orthologue of the frequent human DES missense mutation p.R350P. METHODS AND RESULTS: Transverse aortic constriction (TAC) was performed on heterozygous (HET) DES-p.R349P mice and wild-type (WT) littermates. Echocardiography demonstrated reduced left ventricular ejection fraction in HET-TAC (WT-sham: 69.5 ± 2.9%, HET-sham: 64.5 ± 4.7%, WT-TAC: 63.5 ± 4.9%, HET-TAC: 55.7 ± 5.4%; p<0.01). Cardiac output was significantly reduced in HET-TAC (WT sham: 13088 ± 2385 µl/min, HET sham: 10391 ± 1349µl/min, WT-TAC: 8097 ± 1903µl/min, HET-TAC: 5793 ± 2517µl/min; p<0.01). Incidence and duration of AV blocks as well as the probability to induce ventricular tachycardias was highest in HET-TAC. We observed reduced mtDNA copy numbers in HET-TAC (WT-sham: 12546 ± 406, HET-sham: 13526 ± 781, WT-TAC: 11155 ± 3315, HET-TAC: 8649 ± 1582; p = 0.025), but no mtDNA deletions. The activity of respiratory chain complexes I and IV showed the greatest reductions in HET-TAC. CONCLUSION: Pressure overload in HET mice aggravated the clinical phenotype of cardiomyopathy and resulted in mitochondrial dysfunction. Preventive avoidance of pressure overload/arterial hypertension in desminopathy patients might represent a crucial therapeutic measure.

Rapid and automated risk stratification by determination of the aortic stiffness in healthy subjects and subjects with cardiovascular disease.

BACKGROUND: Aortic stiffness is an independent predictor of cardiovascular morbidity and mortality; thus, simple, rapid and preferably automated techniques are indispensable for pursuing a global risk stratification approach. We present an oscillometric technique for determination of the carotid-femoral pulse wave velocity (cfPWV), including the diagnostic accuracy, sensitivity and specificity, with emphasis on the training curve and procedural duration. METHODS: In a single-centre crossover study, we evaluated subjects free of known cardiovascular disease (CVD), subjects with CVD and a subgroup of subjects with peripheral artery disease (PAD) in terms of ankle-brachial index (ABI) and PWV measurements determined by oscillometry compared to tonometry. Pearson's correlation analysis was used to assess the relationship of the PWV measurements determined by both methods. Moreover, the time and cost of the examinations were compared. RESULTS: A total of 176 study subjects underwent assessments to obtain oscillometric and tonometric PWV measurements. The CVD-free subjects (n = 59) were younger (60.4±15.6 vs. 67.5±12.9 years, p = 0.003) than the subjects with CVD (n = 117). The PWV measurements showed significant correlations in CVD-free subjects (r = 0.797, p<0.001), in subjects with CVD (r = 0.817, p<0.001) and in the subgroup of subjects with PAD (r = 0.807, p<0.001). The examination duration was shorter for the oscillometric method than the tonometric method (4.4±0.5 vs. 9.2±0.8 min, p<0.001). CONCLUSION: Using a simple and rapid automated oscillometric method, we achieved good diagnostic accuracy for the determination of aortic stiffness through the PWV in both subjects with and without CVD. This method might be helpful in daily practice in terms of saving time and reducing procedural complexity for screening for cardiovascular morbidities and vascular damage in cases of atherosclerosis.

Brief report - Telomere length is a poor biomarker to predict 1-year mortality or cardiovascular comorbidity in patients with transcatheter aortic valve replacement.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) is a therapeutic option for patients with aortic valve stenosis at increased surgical risk. Telomeres are an established marker for cellular senescence and have served to evaluate cardiovascular diseases including severe aortic valve stenosis. In our study, we hypothesized that telomere length may be a predictor for outcome and associated with comorbidities in patients with TAVR. METHODS AND RESULTS: We analyzed leucocyte telomere length from 155 patients who underwent TAVR and correlated the results with 1-year mortality and severe comorbidities. The cohort was subdivided into 3 groups according to telomere length. Although a trend for a positive correlation of telomere length with a lower EuroSCORE could be found, telomere length was not associated with survival, aortic valve opening area or cardiovascular comorbidities (peripheral, coronary or cerebrovascular disease). Interestingly, long telomeres were significantly correlated to a reduced left ventricular ejection fraction (LVEF). CONCLUSION: In elderly patients with severe aortic valve stenosis, leucocyte telomere length did not predict post-procedural survival. The correlation between long telomere length and reduced LVEF in these patients deserves further attention.

Guidelines adherence or chronic total occlusion recanalization of the superficial femoral artery with a stentless approach: The next frontier?

In patients with intermittent claudication and long chronic total occlusions of the superficial femoral artery, a primary surgical bypass or stenting is recommended. However, due to the invasive nature, high complication rates and patient-related comorbidities, surgery is currently not the preferred method and full lesion stenting for long chronic total occlusions has the obvious consequences of permanent metallic implants. We report a case of a patient with a long chronic total occlusion of the superficial femoral artery with intermittent claudication. Endorsing an endovascular-first strategy, he was treated via an antegrade and retrograde approach with a complete recanalization and a stentless treatment with rotational atherectomy and drug-coated balloons. We believe this is a feasible endovascular strategy for the treatment of long chronic total occlusions of the superficial femoral artery for patients refusing open surgery. Further investigations are needed to evaluate long-term clinical outcomes of these novel techniques.

Analysis of Axin2 expression and function in murine models for pancreatic cancer.

BACKGROUND: The involvement of Wnt in carcinogenesis and progression of pancreatic cancer is currently intensely discussed. We evaluated activation of the Wnt signaling pathway by using a Wnt reporter mouse strain expressing ß-galactosidase under the control of the Axin2 promotor during pancreatitis induced formation of precancerous lesions. We also evaluated activation of Wnt signaling during interaction of pancreatic cancer with the tumor stroma. RESULTS: Activation of Wnt signaling was observed during acinar-to-ductal metaplasia after chronic as well as acute pancreatitis. Activation of Wnt signaling was also noticed during growth of pancreatic cancer in an orthotopic syngeneic pancreas cancer model. Activation of Wnt signaling was, however, not observed in carcinoma associated fibroblasts, but was detected in few cell clusters inside the tumor. Genetic ablation of Axin2 significantly reduced body weight without having a major impact on blood glucose concentration. However, ablation of Axin2 had no influence on the observed ß-galactosidase positive cell clusters or on tumor weight. CONCLUSION: These data demonstrate that the Wnt signaling pathway is activated during acinar-to-ductal metaplasia after injury to the pancreas. However these data do not support a major role of Wnt signaling or of Axin2 in carcinoma associated fibroblasts and tumor growth.

Intratumoral heterogeneity of the therapeutical response to gemcitabine and metformin.

Cancer heterogeneity and microenvironmental aspects within a tumor are considered key factors influencing resistance of carcinoma cells to distinct chemotherapeutical agents. We evaluated a high concentration of metformin in combination with gemcitabine on a syngeneic orthotopic mouse model using 6606PDA cells. We observed reduced tumor size and reduced cancer cell proliferation after three weeks of chemotherapy with either compound and noticed an additive effect between gemcitabine and metformin on tumor weight. Interestingly, distinct areas of the carcinoma responded differently to either compound. Metformin inhibited the proliferation of cancer cells close to the desmoplastic reaction, whereas gemcitabine inhibited the proliferation of cancer cells mainly 360-570 µm distant to the desmoplastic reaction. Indeed, co-culture of pancreatic stellate cells with 6606PDA, 7265PDA or MIA PaCa-2 cells increased gemcitabine resistance. Metformin resistance, however, was increased by high glucose concentration in the medium. Other factors such as hypoxia or the pH of the medium had no influence on gemcitabine or metformin induced inhibition of cancer cell proliferation. These data demonstrate a spatial heterogeneity in drug resistance within pancreatic adenocarcinomas and that microenvironmental aspects such as supply of glucose and the presence of pancreatic stellate cells regulate cancer cell sensitivity towards metformin or gemcitabine.

Characterization of novel carcinoma cell lines for the analysis of therapeutical strategies fighting pancreatic cancer.

BACKGROUND: Preclinical evaluations of chemotherapies depend on clinically relevant animal models for pancreatic cancer. The injection of syngeneic murine adenocarcinoma cells is one efficient option to generate carcinomas in mice with an intact immune system. However, this option is constrained by the paucity of appropriate cell lines. RESULTS: The murine pancreatic adenocarcinoma cell lines 6606PDA and 7265PDA were compared to the 6606l cell line isolated from a liver metastasis from mice suffering from pancreatic cancer. In tissue culture 6606PDA and 6606l proliferated faster than 7265PDA. 7265PDA cells were, however, significantly more sensitive to gemcitabine as assessed by BrdU-incorporation and trypan blue exclusion assays in vitro. Within 1 week after injection of either one of these three cell lines into the pancreas of C57BL/6J mice, carcinomas were observed by T2 weighted magnetic resonance imaging and histology. Three weeks after injecting 6606PDA or 6606l cells large carcinomas could be characterized, which were surrounded by extensive desmoplastic reaction. After injection of 7265PDA cells, however, remission of cancer was observed between the first and the third week. Compared to 6606PDA cell derived carcinomas a higher apparent diffusion coefficient was quantified by diffusion weighted magnetic resonance imaging in these tumors. This correlated with reduced cancer cell density observed on histological sections. CONCLUSION: All three cell lines can be used in vitro for testing combinatorial therapies with gemcitabine. The 6606PDA and 6606l cell lines but not the 7265PDA cell line can be used for evaluating distinct therapies in a syngeneic carcinoma model using C57BL/6J mice. Diffusion-weighted MRI proved to be an appropriate method to predict tumor remission.

Impact of diabetes type II and chronic inflammation on pancreatic cancer.

BACKGROUND: We explored if known risk factors for pancreatic cancer such as type II diabetes and chronic inflammation, influence the pathophysiology of an established primary tumor in the pancreas and if administration of metformin has an impact on tumor growth. METHODS: Pancreatic carcinomas were assessed in a syngeneic orthotopic pancreas adenocarcinoma model after injection of 6606PDA cells in the pancreas head of either B6.V-Lep(ob/ob) mice exhibiting a type II diabetes-like syndrome or normoglycemic mice. Chronic pancreatitis was then induced by repetitive administration of cerulein. Cell proliferation, cell death, inflammation and the expression of cancer stem cell markers within the carcinomas was evaluated by immunohistochemistry. In addition, the impact of the antidiabetic drug, metformin, on the pathophysiology of the tumor was assessed. RESULTS: Diabetic mice developed pancreatic ductal adenocarcinomas with significantly increased tumor weight when compared to normoglycemic littermates. Diabetes caused increased proliferation of cancer cells, but did not inhibit cancer cell necrosis or apoptosis. Diabetes also reduced the number of Aldh1 expressing cancer cells and moderately decreased the number of tumor infiltrating chloracetate esterase positive granulocytes. The administration of metformin reduced tumor weight as well as cancer cell proliferation. Chronic pancreatitis significantly diminished the pancreas weight and increased lipase activity in the blood, but only moderately increased tumor weight. CONCLUSION: We conclude that diabetes type II has a fundamental influence on pancreatic ductal adenocarcinoma by stimulating cancer cell proliferation, while metformin inhibits cancer cell proliferation. Chronic inflammation had only a minor effect on the pathophysiology of an established adenocarcinoma.

Diabetes increases pancreatic fibrosis during chronic inflammation.

Diabetes and fibrosis can be concurrent processes in several diseases such as cystic fibrosis or chronic pancreatitis. To evaluate whether diabetes can influence fibrosis and thus aggravate the pathological process, the progression of chronic pancreatitis was assessed in diabetic and non diabetic mice. For this purpose, insulin producing beta-cells in C57Bl/6J mice were selectively impaired by administration of streptozotocin. Chronic pancreatitis was then induced by repetitive administration of cerulein in normoglycaemic and hyperglycaemic mice. Diabetes caused enhanced collagen I deposition within three weeks of the onset of chronic pancreatitis and increased the proliferation of interstitial cells. This was accompanied by an increased number of interlobular fibroblasts, which expressed S100A4 (fibroblast-specific protein-1) and stimulation of α-smooth muscle actin expression of pancreatic stellate cells. In addition, the observed aggravation of chronic pancreatitis by diabetes also led to a significantly enhanced atrophy of the pancreas, increased infiltration of inflammatory chloracetate esterase positive cells and enhanced acinar cell death. We conclude that diabetes has a detrimental influence on the progression of chronic pancreatitis by aggravating fibrosis, inflammation and pancreatic atrophy.