Thomas Krahn

Cardioprotection by Ecto-5'-Nucleotidase (CD73) and A2B Adenosine Receptors

Background— Ecto-5′-nucleotidase (CD73)–dependent adenosine generation has been implicated in tissue protection during acute injury. Once generated, adenosine can activate cell-surface adenosine receptors (A1AR, A2AAR, A2BAR, A3AR). In the present study, we define the contribution of adenosine to cardioprotection by ischemic preconditioning. Methods and Results— On the basis of observations of CD73 induction by ischemic preconditioning, we found that inhibition or targeted gene deletion of cd73 abolished infarct size-limiting effects. Moreover, 5′-nucleotidase treatment reconstituted cd73−/− mice and attenuated infarct sizes in wild-type mice. Transcriptional profiling of adenosine receptors suggested a contribution of A2BAR because it was selectively induced by ischemic preconditioning. Specifically, in situ ischemic preconditioning conferred cardioprotection in A1AR−/−, A2AAR−/−, or A3AR−/− mice but not in A2BAR−/− mice or in wild-type mice after inhibition of the A2BAR. Moreover, A2BAR agonist treatment significantly reduced infarct sizes after ischemia. Conclusions— Taken together, pharmacological and genetic evidence demonstrate the importance of CD73-dependent adenosine generation and signaling through A2BAR for cardioprotection by ischemic preconditioning and suggests 5′-nucleotidase or A2BAR agonists as therapy for myocardial ischemia.

Circulating tumour cells escape from EpCAM-based detection due to epithelial-to-mesenchymal transition

BackgroundCirculating tumour cells (CTCs) have shown prognostic relevance in metastatic breast, prostate, colon and pancreatic cancer. For further development of CTCs as a biomarker, we compared the performance of different protocols for CTC detection in murine breast cancer xenograft models (MDA-MB-231, MDA-MB-468 and KPL-4). Blood samples were taken from tumour bearing animals (20 to 200 mm2) and analysed for CTCs using 1. an epithelial marker based enrichment method (AdnaTest), 2. an antibody independent technique, targeting human gene transcripts (qualitative PCR), and 3. an antibody-independent approach, targeting human DNA-sequences (quantitative PCR). Further, gene expression changes associated with epithelial-to-mesenchymal transition (EMT) were determined with an EMT-specific PCR assay.MethodsWe used the commercially available Adna Test, RT-PCR on human housekeeping genes and a PCR on AluJ sequences to detect CTCs in xenografts models. Phenotypic changes in CTCs were tested with the commercially available “Human Epithelial to Mesenchymal Transition RT-Profiler PCR Array”.ResultsAlthough the AdnaTest detects as few as 1 tumour cell in 1 ml of mouse blood spiking experiments, no CTCs were detectable with this approach in vivo despite visible metastasis formation. The presence of CTCs could, however, be demonstrated by PCR targeting human transcripts or DNA-sequences - without epithelial pre-enrichment. The failure of CTC detection by the AdnaTest resulted from downregulation of EpCAM, whereas mesenchymal markers like Twist and EGFR were upregulated on CTCs. Such a change in the expression profile during metastatic spread of tumour cells has already been reported and was linked to a biological program termed epithelial-mesenchymal transition (EMT).ConclusionsThe use of EpCAM-based enrichment techniques leads to the failure to detect CTC populations that have undergone EMT. Our findings may explain clinical results where low CTC numbers have been reported even in patients with late metastatic cancers. These results are a starting point for the identification of new markers for detection or capture of CTCs, including the mesenchymal-like subpopulations.

Plasma biomarkers of myocardial fibrosis and remodeling in terminal heart failure patients supported by mechanical circulatory support devices.

BACKGROUND In this study we analyzed putative biomarkers for myocardial remodeling in plasma from 55 endstage heart failure patients with the need for mechanical circulatory support (MCS). We compared our data to 40 healthy controls and examined if MCS by either ventricular assist devices or total artificial hearts has an impact on plasma concentrations of remodeling biomarkers. METHODS & RESULTS Plasma biomarkers were analysed pre and 30 days post implantation of a MCS device using commercially available enzyme linked immunosorbent assays (ELISA). We observed that the plasma concentrations of remodeling biomarkers: tissue inhibitor of metalloproteinase 1 (TIMP1), tenascin C (TNC), galectin 3 (LGALS3), osteopontin (OPN) and of neurohumoral biomarker brain natriuretic peptide (BNP), are significantly elevated in patients with terminal heart failure compared to healthy controls. We did not find elevated plasma concentrations for matrix metalloproteinase 2 (MMP2) and procollagen I C-terminal peptide (PCIP). However, only BNP plasma levels were reduced by MCS, whereas the concentrations of remodeling biomarkers remained elevated or even increased further 30 days after MCS. LGALS3 plasma concentrations at device implantation were significantly higher in patients who did not survive MCS due to multi organ failure (MOF). CONCLUSIONS Our findings indicate that mechanical unloading in endstage heart failure is not reflected by a rapid reduction of remodeling plasma biomarkers.

Efomycine M, a new specific inhibitor of selectin, impairs leukocyte adhesion and alleviates cutaneous inflammation

Specific interference with molecular mechanisms guiding tissue localization of leukocytes may be of great utility for selective immunosuppressive therapies. We have discovered and characterized efomycines, a new family of selective small-molecule inhibitors of selectin functions. Members of this family significantly inhibited leukocyte adhesion in vitro. Efomycine M, which was nontoxic and showed the most selective inhibitory effects on selectin-mediated leukocyte-endothelial adhesion in vitro, significantly diminished rolling in mouse ear venules in vivo as seen by intravital microscopy. In addition, efomycine M alleviated cutaneous inflammation in two complementary mouse models of psoriasis, one of the most common chronic inflammatory skin disorders. Molecular modeling demonstrated a spatial conformation of efomycines mimicking naturally occurring selectin ligands. Efomycine M might be efficacious in the treatment of human inflammatory disorders through a similar mechanism.

Diagnostic leukapheresis enables reliable detection of circulating tumor cells of nonmetastatic cancer patients

Significance The infrequent detection of circulating tumor cells (CTCs) has hindered their clinical implication and their potential use in the sense of a “liquid biopsy” for cancer diagnosis and therapy. Hypothesizing that the limited blood volume commonly used for CTC analysis (1–10 mL) accounts for variable detection rates, we used leukapheresis to screen large blood volumes for CTCs. This enabled a more reliable detection of CTCs at high frequency even in nonmetastatic cancer patients. Thus, diagnostic leukapheresis may facilitate the routine clinical use of CTCs as biomarkers for personalized medicine. Combined with technologies for single-cell molecular genetics or cell biology, it may significantly improve prediction of therapy response and monitoring, especially in early systemic cancer. Circulating tumor cells (CTCs) are promising biomarkers for diagnosis and therapy in systemic cancer. However, their infrequent and unreliable detection, especially in nonmetastatic cancer, currently impedes the clinical use of CTCs. Because leukapheresis (LA) targets peripheral blood mononuclear cells, which have a similar density to CTCs, and usually involves processing the whole circulating blood, we tested whether LA could substantially increase CTC detection in operable cancer patients. Therefore, we screened LA products generated from up to 25 L of blood per patient in two independent studies, and found that CTCs can be detected in more than 90% of nonmetastatic breast cancer patients. Interestingly, complete white blood cell sampling enabled determining an upper level for total CTC numbers of about 100,000 cells (median, 7,500 CTCs) per patient and identified a correlation of CTC numbers with anatomic disease spread. We further show that diagnostic leukapheresis can be easily combined with the US Food and Drug Administration-approved CellSearch system for standardized enumeration of CTCs. Direct comparison with 7.5 mL of blood revealed a significantly higher CTC frequency in matched LA samples. Finally, genomic single-cell profiling disclosed highly aberrant CTCs as therapy-escaping variants in breast cancer. In conclusion, LA is a clinically safe method that enabled a reliable detection of CTCs at high frequency even in nonmetastatic cancer patients, and might facilitate the routine clinical use of CTCs as in the sense of a liquid biopsy. Combined with technologies for single-cell molecular genetics or cell biology, it may significantly improve prediction of therapy response and monitoring of early systemic cancer.

Elevated plasma levels of TNF-alpha and interleukin-6 in patients with diastolic dysfunction and glucose metabolism disorders.

BackgroundDiabetes mellitus (DM) has reached epidemic proportions and is an important risk factor for heart failure (HF). Left ventricular diastolic dysfunction (LVDD) is recognized as the earliest manifestation of DM-induced LV dysfunction, but its pathophysiology remains incompletely understood. We sought to evaluate the relationship between proinflammatory cytokine levels (TNF-alpha, IL-6) and tissue Doppler derived indices of LVDD in patients with stable coronary artery disease.MethodsWe enrolled 41 consecutive patients (mean age 65+/-10 years) submitted for coronary angiography. Echocardiographic assessment was performed in all patients. Pulsed tissue Doppler imaging was performed at the mitral annulus and was characterized by the diastolic early relaxation velocity Em. Conventional transmitral flow was measured with pw-doppler. Early (E) transmitral flow velocity was measured. LVDD was defined as E/Em ratio ≥ 15, E/Em 8-14 was classified as borderline. Plasma levels of TNF-alpha and IL-6 were determined in all patients. A standardized oral glucose tolerance test was performed in subjects without diabetes.ResultsPatients with E/Em ratio ≥ 15, classified as LVDD and those with E/Em ratio 8-14 (classified as borderline) had significantly higher IL-6 (P = 0,001), TNF-alpha (P < 0,001) and NT-pro- BNP (P = 0,001) plasma levels compared to those with normal diastolic function. TNF-alpha and IL-6 levels remains significantly elevated after adjustment for sex, age, left ventricular ejection function, body mass index, coronary heart disease, smoking, hypertension and diabetes mellitus with linear regression analysis. Furthermore, in subjects LVDD or borderline LV diastolic function, 75% had diabetes or IGT, respectively. When subjects without diabetes were excluded, both IL-6 (P = 0,006) and TNF-alpha (P = 0,002) remained significantly elevated in subjects with E/Em ratio ≥ 15.ConclusionThis study reveals that increased plasma levels of IL-6 and TNF-alpha were associated with LVDD. These findings suggest a link between low-grade inflammation and the presence of LVDD. An active proinflammatory process may be of importance in the pathogenesis of diastolic dysfunction.

Enumeration and Molecular Characterization of Tumor Cells in Lung Cancer Patients Using a Novel In Vivo Device for Capturing Circulating Tumor Cells

Purpose: The use of circulating tumor cells (CTC) as “liquid biopsy” is limited by the very low yield of CTCs available for subsequent analyses. Most in vitro approaches rely on small sample volumes (5–10 mL). Experimental Design: Here, we used a novel approach, the GILUPI CellCollector, which enables an in vivo isolation of CTCs from peripheral blood. In total, 50 lung cancer patients were screened in two subsequent device applications before and after therapy (n = 185 applications). Results: By in vivo isolation, 58% (108/185) of the patients were positive for ≥1 CTC (median, 5 CTCs; range, 1–56 cells) as compared with 27% (23/84; range, 1–300 cells) using the FDA-cleared CellSearch system. Furthermore, we could show that treatment response during therapy was associated with significant decreases in CTC counts (P = 0.001). By dPCR, mutations in the KRAS and EGFR genes relevant for treatment decisions could be detected in CTCs captured by in vivo isolation and confirmed in the primary tumors of the same patients. Conclusions: In vivo isolation of CTCs overcomes blood volume limitations of other approaches, which might help to implement CTC-based “liquid biopsies” into clinical decision making. Clin Cancer Res; 22(9); 2197–206. ©2015 AACR.

Metabolic syndrome with or without diabetes contributes to left ventricular diastolic dysfunction

Objective Left ventricular diastolic dysfunction (LVDD) is considered a precursor of diabetic cardiomyopathy, while the metabolic syndrome (MetS) is associated with an increased risk of cardiovascular morbidity and mortality. This study aimed to evaluate the association between LVDD, MetS and glucose metabolism disturbances classifi ed by oral glucose tolerance testing (oGTT). Methods and results The presence of LVDD was evaluated in 166 subjects with normal ejection fraction, 43 (26%) of whom had type 2 diabetes at inclusion. In subjects without diabetes, an oGTT was performed. The MetS was diagnosed as indentifi ed by the NCEPIII-criteria, while LVDD was verifi ed and graded according to the current guidelines. MetS was diagnosed in 97 (59%) patients, 44% of whom had known diabetes. The prevalence of LVDD was 68% in subjects with MetS vs. 19% in patients without MetS, respectively (P < 0.001). A severe form of LVDD was observed in 34% and 15% of patients with and without MetS, respectively (P= 0.001), whereupon the prevalence of mild and severe diastolic dysfunction increased with the number of MetS criteria (P= 0.001). In the MetS group, early diastolic tissue relaxation velocity (E´) was signifi cantly reduced (6.9 ± 1.8 cm/s vs. 7.7 ± 2.1 cm/s; P= 0.009) and the E/E´ ratio was signifi cantly higher (10.5 ± 3.9 vs. 9.1 ± 3.0 cm/s, P= 0.015) as compared to the group without MetS (n = 69). Conclusion MetS was associated with a higher prevalence and severity of LVDD, whereupon coexisting diabetes aggravates these fi nding. Patients displaying MetS with concomitant LVDD might represent a target population in which appropriate medical care for early heart failure prevention should be initiated

Absence of adenosine-mediated aortic relaxation in A2A adenosine receptor knockout mice

Adenosine mediates vascular responses through four receptor subtypes: A(1), A(2A), A(2B), and A(3). The role of A(2A) receptors in aortic vascular tone was investigated using A(2A) adenosine receptor (AR) knockout (A(2A)KO) and corresponding wild-type (A(2A)WT) mice. Isolated aortic rings from A(2A)WT and A(2A)KO mice were precontracted with phenylephrine (10(-7) M), and concentration responses for adenosine analogs and selective agonists/antagonists were obtained. Nonselective adenosine analog (NECA; EC(50) = 6.78 microM) and CGS-21680 (A(2A)AR selective agonist; EC(50) = 0.013 microM) produced concentration-dependent relaxation (maximum of 25% and 28% relaxation at 10(-5) M NECA and CGS-21680, respectively) in A(2A)WT aorta. In A(2A)KO aorta, NECA (EC(50) = 0.075 microM) induced concentration-dependent contraction (maximum contraction of 47% at 10(-6) M; P < 0.05 compared with A(2A)WT), whereas CGS-21680 produced no response. SCH-58261 (10(-6) M; A(2A)AR selective antagonist) abolished both NECA- and CGS-21680-mediated vasorelaxation in A(2A)WT (P < 0.05), whereas no change was observed in A(2A)KO. When DPCPX (10(-5) M; A(1) selective antagonist) was used in NECA concentration response, greater vasorelaxation was observed in A(2A)WT (50% vs. 25% in controls at 10(-5) M; P < 0.05), whereas lower contraction was seen in A(2A)KO tissues (5% vs. 47% in controls at 10(-6) M; P < 0.05). Aortic endothelial function, determined by response to acetylcholine, was significantly higher in WT compared with KO (66% vs. 51%; P < 0.05). BAY 60-6583 (A(2B) selective agonist) produced similar relaxation in both KO and WT tissues. In conclusion, A(2A)AR KO mice had significantly lower aortic relaxation and endothelial function, suggesting that the A(2A)AR plays an important role in vasorelaxation, probably through an endothelium-dependent mechanism.

Cardiovascular autonomic neuropathy contributes to left ventricular diastolic dysfunction in subjects with Type 2 diabetes and impaired glucose tolerance undergoing coronary angiography.

Diabet. Med. 28, 311–318 (2011)