Supplemental Digital Content is available in the text. Atherosclerosis is an age-related disease characterized by systemic oxidative stress and low-grade inflammation. The role of telomerase and telomere length in atherogenesis remains contentious. Short telomeres of peripheral leukocytes are predictive for coronary artery disease. Conversely, attenuated telomerase has been demonstrated to be protective for atherosclerosis. Hence, a potential causative role of telomerase in atherogenesis is critically debated.+ cells and significantly reduced CD4+ T-cell proliferation. The latter was telomerase dependent because oxidative stress had no effect on the proliferation of primary lymphocytes isolated from telomerase knockout mice. In contrast, myeloid cell proliferation was unaffected by oxidative stress nor reliant on telomerase. Telomerase reverse transcriptase deficiency had no effect on regulatory T-cell (Treg) numbers in vivo or suppressive function ex vivo. Adoptive transfer of telomerase reverse transcriptase–/– Tregs into Rag2–/– ApoE–/– (recombination activating gene 2/apolipoprotein E) double knockout mice demonstrated that telomerase function was not required for the ability of Tregs to protect against atherosclerosis. However, telomere length was critical for Treg function.In this study, we used multiple mouse models to investigate the regulation of telomerase under oxidative stress as well as its impact on atherogenesis in vitro and in vivo. Using primary lymphocytes and myeloid cell cultures, we demonstrate that cultivation under hyperoxic conditions induced oxidative stress resulting in chronic activation of CD4reg function, provided that telomere length is not critically short. We suggest that oxidative stress may contribute to atherosclerosis via suppression of telomerase and acceleration of telomere attrition in Tregs.Telomerase contributes to lymphocyte proliferation but plays no major role in T We and others have shown previously that the majority of T-cells in an atherosclerotic lesion are CD4+ T-helper type 1 cells that produce interferon-γ12 and deficiency in either interferon-γ or the transcription factor T-bet (required for T-helper type 1 differentiation) attenuates the progression of atherosclerosis in cholesterol-fed low-density lipoprotein receptor null mice (Ldlr−/−). Conversely, reduction or dysfunction of the regulatory T-cell population (Treg) leads to increased atherosclerosis.14 Furthermore, adoptive transfer of the Treg subpopulation into hypercholesterolemic mice reduces lesion development.5 Recently, it has also been demonstrated that prolonged hypercholesterolemia impairs Treg cells but not effector T-cell accumulation in atherosclerotic lesions and reversal of hypercholesterolemia can prevent loss of lesional Treg cells.12 The ratio of regulatory to effector T-cells is, therefore, critical in determining the outcome of atherosclerosis.Atherosclerosis is an age-related systemic disease characterized by systemic oxidative stress and low-grade chronic inflammation, which is driven both by innate and adaptive immune responses. In particular, distinct subpopulations of T-cells have opposing roles in the development of atherosclerotic lesions,1247See accompanying editorial on page reg cells do not arise solely from thymic generation but can also be induced by rapid turnover from the memory T-cell pool.15 Once generated, they are susceptible to apoptosis and have limited replicative potential, which is directly related to telomere length.16 Telomeres consist of tandem TTAGGG DNA repeats at the ends of chromosomes as well as the shelterin complex. They function to maintain chromosomal integrity during cell division and protect against chromosomal instability. In cells without telomerase activity, telomeres shorten with every cell division because of the end replication problem,17 as well as because of increased oxidative stress.18 Telomere shortening can be compensated or slowed down by concomitant activity of telomerase, a ribonucleoprotein composed of an RNA subunit (telomerase RNA component [TERC]), containing the template for telomere repeat addition, and a telomerase reverse transcriptase (TERT) subunit. Accordingly, late-generation telomerase knockout mice (TERT−/− or TERC−/−) display an age-related phenotype.21Human T22 while telomere dysfunction in late-generation TERC−/− mice reduces the number and function of T- and B- lymphocytes.24 The end replication problem is not the only mechanism that can contribute to telomere shortening resulting eventually in cellular senescence. Telomeres have been shown to be favored targets for DNA damage.26 Because oxidative stress plays a major role in chronic inflammatory diseases, it has been suggested that telomere damage may be involved in their pathophysiology. However, the exact mechanisms driving telomere damage and shortening under conditions of chronic oxidative stress are still not fully understood.Regarding its specific role in T-lymphocytes, telomerase levels control the lifespan of T lymphocytes33 There remains a paucity of data on the role of telomerase and its regulation in atherosclerosis. The increased prevalence of atherosclerosis in older people is associated with a decline in the function of lymphoid progenitors and mature T-cells,34 and shorter telomeres have been observed in leukocytes of human patients with advanced coronary atherosclerosis.35 However, it is unclear as to whether accelerated telomere shortening or inherited telomere length are the most important influences in the development of atherosclerosis.37Telomere length in leukocytes has been investigated in a variety of clinical studies involving patients with either existing or developing coronary heart disease (CHD).31 This hypothesis, however, is in direct contradiction to the recent demonstration of Poch et al38 who have shown that a mouse model that lacks telomerase activity and displays significantly shorter telomeres is in fact protected from atherosclerosis,38 as well as the observations that aged rabbits develop less atheroma under a high-cholesterol diet compared with younger animals.39 Several human studies have also failed to identify any association between telomere length and atherosclerosis40 or between telomere length and mortality in subjects over the age of 65 years.43 However, it has been suggested that studies showing no correlation have examined older populations37 while until the age of 60 years there is a correlation between telomere length and mortality which includes cardiovascular deaths.44 Adding further complexity to the interpretation of the human studies is the fact that factors, including race/ethnicity and sex, all influence outcome.37It has been previously suggested that telomere shortening or dysfunction in telomerase activity in leukocytes, including T-cells, may be causative in the progression of this disease.+ T-lymphocytes but not CD11b+ myeloid cell expansion. Lack of Tert in cells with sufficiently long telomeres within a population of Treg T-lymphocytes is not detrimental to their suppressive function. In contrast, short telomeres diminished Treg number and function.In this study, we aimed to address some of the controversy surrounding the role of telomerase activity and telomere length with regards to T-cell activation and proliferation. Moreover, we aimed to address the functional requirement of telomerase for regulatory T-cell–mediated atheroprotection. We report here that oxidative stress suppresses telomerase activity and attenuates proliferation of CD4online-only Data Supplement.The data that support the findings of this study are available from the corresponding author on reasonable request. Details of the major resources and detailed methods can be found in the 45 (Jax strain B6.129S-Tert tm1Yjc/J), and TERC knockout, generated by Blasco et al46 (Jax strain B6.Cg-Terc tm1Rdp/J), animals were purchased from Jackson Laboratory, Maine. Generation and initial phenotypic characterization of the mTert-GFP (green fluorescent protein) mice have been published previously.51 The mTert-GFP mouse2 contains a reporter cassette in which the expression of the gene for GFP is under the control of a 4.4-kb fragment of the promoter of murine Tert. As such GFP expression in this model represents mTert promoter activity as an indicator of TERT transcription. Rag2−/− ApoE−/− (recombination activating gene 2/apolipoprotein E) double knockout mice and CD28−/− mice were originally obtained from Charles River. All mice were held under the UK Home office animal licenses PPL 60/3864 or PO11C464C. Details for each line used to obtain the data for each figure are included in Table I in the online-only Data Supplement.Animal work was authorized and approved by the Cambridge and Newcastle University Ethics review boards. All animal procedures were performed conforming to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Both male and female mice were used in all studies. TERT knockout, generated by Chiang et al47 Assessment of CD4+ cell purity is demonstrated in Figure I in the online-only Data Supplement. Splenocytes were cultured in a 24-well plate (2×106 cells/2 mL per well). MACSibead mouse T-cell, CD3 and CD28 antibody coated, expansion beads (Miltenyi 130-093-627) were added to medium as described.47 TA-65 activator (TA65) is a telomerase activator purified from Astragalus membranaceous52 and provided by TA-Science Inc . BIBR 1532 (Tocris Bioscience), a telomerase inhibitor,53 was dissolved in dimethyl sulfoxide and used as the indicated concentration.Cells were isolated and cultured as described previously.55 Cells were labelled with 10-μM dihydroethdium (Molecular Probes) as described56 or 5-μM Mitosox Red (Molecular Probes).Dihydroethidium and Mitosox are established methods to measure superoxide levels.−/− splenocytes and the immortal fibroblast cell line 3T3 were used as negative and positive controls .Telomeric Repeat Amplification Protocol kit (Roche) was performed as per the manufacturer’s instructions. TERTreg Detection Kit  as per manufactures instructions. In our hands, ≥98% of CD4+ T-cells can be identified as T-cells by CD3+ staining .After isolation, splenocytes were labeled using the T−/− ApoE−/− mice were transplanted with 107 splenocytes from CD28−/− mice and either PBS or 106 CD4+ CD25+ regulatory T-cells from either Tert−/− mice or wild-type (WT) littermates. Mice were fed an atherogenic Western diet  for 7 weeks. Atherosclerosis was quantified in the aortic root as described previously.57Rag2U test was used to compare groups of 2, and 2-way ANOVA with Bonferroni post hoc analysis was used to compare groups of ≥3. Statistical significance was set at P<0.05.After a test for normality, statistical analysis was performed as appropriate and indicated in the legend of each figure. Data are presented as mean±SEM or as dot for individual experiments with a line representing the median. A Mann–Whitney 59 In this study, we used hyperoxic culture conditions to study the effect of chronic oxidative stress on lymphocyte growth kinetics and T-cell activation.Increased oxidative stress and acute inflammation as well as increased T-cell activity are key characteristics of atherosclerosis. We have previously established hyperoxia as a model of chronic mild oxidative stress.+ T-cells were cultured for 10 days at either physiological oxygen levels  or hyperoxia (40% oxygen). Assessment of cell purity is shown in Figure I in the online-only Data Supplement. When cultured under hyperoxic conditions, both the total splenocyte population and CD4+ T-cells displayed a significantly higher level of dihydroethidium staining intensity indicating increased mitochondrial superoxide levels .Splenocytes or CD4+ cells cultured under hyperoxic conditions had an elevated interferon γ mRNA expression relative to splenoctyes cultured at physoxia . Having demonstrated that hyperoxia and oxidative stress are associated with aspects of T-cell activation, we were interested to examine the effect of oxidative stress on splenocyte proliferation. Splenocytes cultured at physoxia demonstrated a 30-fold increase in cell number over 28 days. In contrast, splenocytes cultured in either 20% or 40% oxygen saturation showed a dose-dependent attenuation of proliferation by 15% or 50%, respectively, compared with physoxia . To reflect the physiological environment, the total splenocyte population was cocultured in the presence of T-cell–specific activation under conditions of physoxia or hyperoxia. After 14 days, total cell numbers of the individual subpopulations were quantified by flow cytometry to extrapolate rates of proliferation. CD4+ T-cells displayed the greatest proliferative response with 100-fold expansion for 14 days under 3% oxygen  attenuated by hyperoxia-induced oxidative stress. However, the CD11b+ were not experimentally stimulated to proliferate under these experimental conditions.Given the heterogeneity of cell populations residing in the spleen, flow cytometry was used to study the effect of oxidative stress on the proliferation of individual splenocyte subpopulations Figure D–1F. Gat60 and that telomerase facilitates statin-induced T-cell proliferation.47 The latter led us to hypothesize that the oxidative stress–induced attenuation of T-cell proliferation in this study might be mediated via the suppression of telomerase activity. To test this hypothesis, we quantified telomerase activity in WT splenocytes cultured at either physoxia or hyperoxia. Hyperoxia resulted in a significant reduction of telomerase activity, and all detectable telomerase activity was suppressed after 2 weeks of culture  overexpressing fibroblasts, thereby preventing telomere maintenance and halting cell proliferation−/−), the catalytic subunit of telomerase.61 Although these mice lack functional telomerase activity, they do not have significantly shorter telomeres than WT animals.47 No significant difference in the rates of proliferation in Tert−/− splenocytes cultured under physoxic or hyperoxic conditions were observed  TERT knockout mice, which lack expression of telomerase reverse transcriptase  and cultured under the different oxygen conditions . In contrast, hyperoxia significantly (P<0.0001) attenuated mTert-GFP expression in splenocytes at all time points investigated . mTert-GFP expression mimicked the proliferative trends of each cell subpopulation in the different oxygen conditions shown in Figure Having demonstrated that oxidative stress suppresses telomerase activity, we next aimed to ascertain whether this reduction in telomerase was at a transcriptional level of s Figure A. Under reg cells modulate the development of atherosclerosis in mice. Using adoptive transfer studies, we demonstrated that naturally arising Treg cells are potent inhibitors of atherosclerosis in several different mouse models.5 We next examined whether telomerase-deficient Tregs retain the potential to suppress atherosclerosis in vivo. Surprisingly, Tert−/− mice contained the same number of both CD4+ T-cells and Treg cells, including the CD4+CD25+FoxP3+ population, as WT mice . To evaluate the functional properties of telomerase-deficient T-cells, we purified Treg cells and CD4+CD25– effector T-cells and assessed their proliferative potential ex vivo . Treg cells were suppressive of effector T-cells proliferation, regardless of Tert expression  TERC−/− mice had shorter telomeres compared with WT mice from the same strain .62 We compared the percentage of CD4+ T-cells and Tregs in the spleen of both these TERC−/− and WT mice  leads to an increased risk of CHD.65 Although in our animal studies absence of telomerase had no effect on Treg maintenance, it is important to point out that because of the long telomeres in laboratory mouse strains, this may not reflect the human biology of telomeres. Indeed, late-generation TERC knockout mice with shorter telomeres are considered more representative of the human situation.Multiple studies have confirmed the association of short leukocyte telomere length and the incidence of CHD in humans.66 Although the proatherogenic function of T-helper type 1 CD4+ as well as CD8+ T-cells is well established, it is also emerging that Treg cells are critical in offsetting the detrimental effects of both adaptive and innate immune responses. What remains unclear is how the plaque environmental milieu influences the net effect of these 2 antagonistic responses and how the molecular mechanisms controlling the survival, proliferation, and suppressive function of the Treg population contribute to disease progression. We propose that within the atherosclerotic plaque, chronic oxidative stress conditions may lead to suppression of telomerase and acceleration of telomere attrition in Tregs. This in turn may contribute to premature senescence of Treg cells and therefore progression of atherosclerosis.It is known that the adaptive immune system plays a pivotal role in the development and progression of atherosclerosis.67 Accumulated DNA damage in peripheral blood cells not only occurs in patients with coronary artery disease and acute myocardial infarction68 but is also associated with outcome.69 Moreover, high-cholesterol diet and atherosclerosis have been associated with increased DNA damage in peripheral lymphocytes in rabbits.70 Mitochondrial localization of the TERT protein has been demonstrated to regulate levels of mitochondrial derived reactive oxygen species and TERT can translocate to the mitochondria reducing mitochondrial derived reactive oxygen species production in the process, which is independent of its canonical role in the nucleus.60 It had been shown in different cell types that lack of TERT specifically in the mitochondria is detrimental to mitochondrial function and morphology.71 However, such a mitochondrial role has to date not been described in immune cells. We and others have previously demonstrated that TERT can to bind to mitochondrial DNA which also protects cells from DNA damage and oxidative stress.72 An antioxidant role of TERT has also recently been described by Beyer et al53 who have demonstrated that loss of telomerase activity in cells of healthy human vessels results in a switch from nitrous oxide to a proinflammatory hydrogen peroxide, which mediates vascular dilation. Conversely, restoration of telomerase activity in arterioles from humans with coronary artery disease reverts the mediator of flow-induced dilation from hydrogen peroxide to nitrous oxide.53 The data in our current study demonstrate that oxidative stress attenuates TERT expression at a transcriptional level in lymphocytes. Such reduction in telomerase influences both telomerase nuclear activity and would also diminish some noncanonical functions of telomerase and thereby resistance to oxidative stress further contributing to disease. Indeed, we have demonstrated that oxidative stress and attenuated TERT expression are both associated with an increased production of mitochondrial derived superoxide. We are currently investigating the impact of telomerase activation by TA-65 on canonical (telomere length) and noncanonical  pathways in patients with myocardial infarction . Noncanonical roles for telomerase may possibly also explain the difference in phenotype between the TERT−/− and TERC−/− mice in addition to differences in telomere length that are already differentially regulated at the level of heterozygotes in both knockout models.73 The TERC−/− mice might still benefit from noncanonical roles of telomerase because of the presence of TERT.Oxidative stress within the atherosclerosis microenvironment may have other detrimental effects that could contribute to disease progression. Oxidative stress can directly damage both genomic DNA, in the absence of telomere shortening, and mitochondrial DNA, which is linked to vascular senescence and atherosclerosis.76 Furthermore, nonimmune cells have been shown to have accelerated telomere attrition and develop premature senescence when cultured under hyperoxic conditions.47 This could represent one mechanism that would explain why patients with short telomeres in lymphocytes are predisposed to CHD, a hypothesis supported by our recent work demonstrating that immunosenescence can be an independent predictor of disease outcome in older people, possibly via accelerated atherosclerosis.77Correlative evidence from human population studies collectively suggests an association of short telomeres in lymphocytes with conditions of increased oxidative stress, including smoking, obesity, and CHD.38 may be explained by global immunosenescence and a dysfunctional immune system, where the lack of myeloid cell proliferation is responsible for reduced plaque progression. Of note, our experiments demonstrate that in contrast to T-cells, proliferation of myeloid cells was indeed not influenced by oxidative stress. Therefore, the plaque environment seems to modulate the local immune response in a manner conducive for chronic inflammation. A dichotomous protective versus pathological role of telomerase as a result of tissue specificity has already been described (reviewed in 78). Telomerase activation has been demonstrated to reduce reactive oxygen species production and thereby inflammation in the endothelium whereas increased telomerase activity within the vascular smooth muscle layer can result in abnormal proliferation and vascular remodeling in hypertensive rats.78The only study showing an atheroprotective effect of global telomerase deficiency as well as shortened telomeres in the ApoE mouse model79 remains a potentially powerful therapeutic intervention.Finally, even in light of recent studies demonstrating that critically short telomeres can be protective for atherosclerosis, our current data suggest that restoring telomerase activity in T-cells, for example, through small molecule activators such as TA-65,We acknowledge the Newcastle University Flow Cytometry Core Facility for assistance with the generation of Flow Cytometry data.www.BHF.org.uk) as well as the Newcastle Healthcare Charity . N.M. Al Zhrany was funded by a stipend from the Government of Saudi Arabia.This study was supported, in part, by British Heart Foundation Project Grants PG/15/85/31744 and PG/12/47/29681 (None.


Though it is highly conserved, the physiological function of TMEM184A remains unknown. The objective of this study was to investigate the expression and effects on vascular regeneration of TMEM184A using the adult zebrafish regenerating caudal fin as an in vivo model. Here, we show that Tmem184a is expressed in vascular endothelial cells (ECs) of mature and regenerating zebrafish fins. Transient morpholino (MO)-mediated knockdown of Tmem184a using two validated MOs results in tangled regenerating vessels that do not grow outward and limit normal overall fin regeneration. A significant increase in EC proliferation is observed. Consistent with in vitro work with tissue culture vascular cells, heparin has the opposite effect and decreases EC proliferation which also hinders overall fin regeneration. Collectively, our study suggests that Tmem184a is a novel regulator of angiogenesis.Transmembrane protein 184A (TMEM184A) was recently identified as the heparin receptor in vascular cells. Heparin binds specifically to TMEM184A and induces anti-proliferative signaling  Heparan sulfates (HS) are long chains of polysaccharides covalently attached to a class of proteins called proteoglycans (HSPGs). Though HSPGs are expressed ubiquitously, their functions are tissue specific and depend on certain properties of the glycosaminoglycan chains  and the core protein. These chains have binding sites for several different types of proteins, and these interactions are a critical part of the regulation of many biological processes. One notable example is angiogenesis, the development of new vasculature from existing vessels  interacts normally with TMEM184A. We hypothesized that TMEM184A functions specifically in the vasculature in vivo to modulate vascular cell proliferation and other behaviors. To begin to elucidate the in vivo function of TMEM184A, we employed the adult zebrafish regenerating caudal fin as a model for angiogenesis/wound healing.Transmembrane protein 184A (TMEM184A) was recently identified as the heparin receptor in vascular ECs and smooth muscle cells (VSMCs)  which expresses EGFP specifically in ECs. Upon amputation of the caudal fin, a plexus is formed and new vessels develop over several days. The new ECs do not arise from progenitor stem cells but rather sprout from the existing vessels  results in excess EC proliferation and disorganized neovascularization with hindered outgrowth. Heparin injection into regenerating vasculature decreases EC proliferation and affects overall fin regeneration. These results are remarkably consistent with our previous in vitro work. Together, these findings suggest that Tmem184a is a modulator of angiogenesis. Our study emphasizes the regenerating zebrafish fin as a strong method to study vascular regeneration and highlights the existence of a novel player in the mechanism of angiogenesis.Here we report the Danio rerio) C32 strain described previously  approved the protocols performed in the manuscript . Lehigh University's Animal Welfare Assurance Number is A-3877-01. All experiments were performed to minimize pain and discomfort. The zebrafish (Tg(Fli1:EGFP) transgenic line  fish was injected for microscopy assays . The entire fin of wild-type C32 fish was injected with MO or a specific treatment when preparing fin lysates for western blotting. All fins were harvested 1 day post injection, or 4 dpa.The 2O. Two non-overlapping MOs targeting Tmem184a were used: one that targeted the translation start site, ATG MO, and one targeting the splice site between pre-mRNA exons 2 and 3, SS MO. A mismatched standard control (Con MO) was also used to control for off target effects. The ATG MO was tagged with lissamine rhodamine, and the SS MO and Con MO were tagged with fluorescein. Sequences of MOs were as follows: ATG MO 5′CTGAGAGTAGTTTCATTCATCCTGA3′; SS MO 5′AAACAGGCACACTCACTGAATGGGC3′; Con MO 5′CCTCTTACCTCAGTTACAATTTATA3′. Microinjection and electroporation procedures were performed as described previously using a Norishinge IM 300 microinjector and visualized under Nikon SMZ 800 with a 1x objective  were purchased from GeneTools, LLC  and dissolved in sterile ddHInitial regenerating vascular phenotypes in Con and ATG MO-injected fins were assessed with confocal microscopy. The regenerating portion of each fin was harvested and fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (1x PBS) for either overnight at 4°C or for 4 h at room temperature. Fins were dehydrated in 100% methanol and stored in −20°C until further use. Fins were rehydrated from methanol to 1x PBS through a series of washes. Fins were mounted into glycerol on a microscope slide and covered with a coverslip. Fins were imaged for EGFP and lissamine-rhodamine with a 10x objective of a Zeiss LSM 880 confocal microscope at room temperature. Higher resolution tiled z-stacks were taken to image the entire fin and to allow us to zoom in to analyze vasculature around the third and fourth fin rays of each side.Tg(Fli1:EGFP) fish were anesthetized and 100 μg/mL unfractionated heparin from porcine skin (Sigma), 1% phenol red in 1x PBS, or 100 μg/mL chondroitin sulfate (Sigma) was injected into the third and fourth fin rays of the dorsal regenerating caudal fin. The uninjected ventral fin rays served as an internal control. After 24 h (4 dpa), fins were imaged using a Nikon Eclipse 80i microscope with a 4x objective and a Nikon Eclipse E1000M microscope with 1x or 4x objectives. Brightfield and EGFP fluorescence (FITC filter) images were obtained for both injected and uninjected sides of each fin. The fins were then harvested as the MO-injected fins and processed for vascular phenotype, total and vasculature regenerate length, and cell proliferation analyses.At 3 dpa, RRID:SCR_003070) as previously reported .Brightfield and regenerating vasculature  images obtained with a Nikon Eclipse E1000M microscope and NIS elements software were exported as TIFs. Regenerating vasculature images were superimposed over the corresponding brightfield image. The transparency of the vasculature image was set to 50–75%, and the brightness and contrast were changed so individual fin rays became clear and easily measurable. Total and vascular regeneration length from the amputation plane of the third fin ray of each side (uninjected vs. injected) of each fin was measured using NIH ImageJ . To analyze only H3P-positive cells in Fli1-positive ECs above the amputation plane, H3P was pseudocolored to red for all fins, and EGFP was pseudocolored to green in SS MO-injected fins. Z stacks/images were projected in 3D using the Show Volume View function and alpha blending was applied. Individual H3P/Fli1-positive nuclei were confirmed by zooming in and rotating in several directions and counted without software.Cell proliferation was detected by immunostaining for histone-3-phosphate (H3P) as described previously  ontogenic and regenerating fins were sectioned as previously described  fins. Sections were incubated with commercial primary antibodies against GFP  and TMEM184A (TMEM184A-INT). We observed remarkably specific expression of TMEM184A in ECs of both ontogenic and regenerating sections  in whole regenerating fins and quantified H3P in Fli1-positive ECs Figure . To obsein vivo. We injected heparin into the third and fourth fin rays of one side of 3 dpa fins and harvested at 4 dpa for phenotype analysis and the H3P assay. Chondroitin sulfate was used as a control for heparin due to its similar size and sulfation and does not mimic heparin effects. Phenol red alone  was used as a negative control. There was a decrease in vascular regeneration in heparin-injected fins compared to controls  line, visualization of the vasculature with microscopy is simple , EC adherens junction proteins such as VE-cadherin, co-receptor Neuropilin-1, and certain HSPGs  and promote tissue outgrowth. At the molecular level, there are several explanations for why these effects on proliferation would limit outgrowth including an imbalance of tip and stalk cells, effects on EC migration, displacement of HS chains by Tmem184a and/or heparin, and interactions with other heparin/HS binding proteins. During regeneration, ECs compete for the tip cell position which leads the sprouting vessel. HS chains are involved in tip/stalk cell maintenance through creating gradients of growth factors and chemokines to direct migrating ECs. Interactions between HS and Tmem184a required for maintaining such a gradient may have been disrupted in the Tmem184a knockdown fins. It is also possible that heparin is interacting through more than just Tmem184a. Migration of proliferating ECs has been shown to be controlled in response to VEGFR signaling, and altered Vegfr2 signaling in the Tmem184a MO and/or heparin-injected fins could have caused decreased migration. The regenerating caudal fin has limited supporting vascular cells , and the literature suggests that these supporting cells are found in larger arterial vessels rather than the distal tips of newly sprouting vessels  Tmem184a plays in the vasculature will be critical for advancing our knowledge of mechanisms responsible for pathological angiogenesis and may also provide the opportunity for evaluating this novel receptor as a molecular target for angiogenic therapies.In conclusion, our data are the first to report an SF and KR carried out experimental work. SF, MI, and LL planned the study and wrote the manuscript. All authors analyzed data, reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In vitro studies using VSMC were performed to assess the functional relevance of one of the validated genes. We identified 1624 and 135 differentially expressed genes within carotid atherosclerosis samples of symptomatic compared to asymptomatic patients using the E-MEXP-2257 and GSE21545 datasets, respectively . Only 7 differentially expressed genes or 0.4%  were consistent between the datasets. We validated the differential expression of ARSB which was upregulated 1.15-fold (P = 0.029) in atherosclerosis from symptomatic patients. In vitro incubation of VSMCs with the ARSB inhibitor L-ascorbic acid resulted in marked upregulation of SIRT1 and AMPK. This study suggests that ARSB may represent a novel target to limit carotid embolization.The aim of this study was to identify genes for which the expression within carotid atherosclerosis was reproducibly associated with the symptoms of cerebral embolization. Two publically available microarray datasets E-MEXP-2257 and GSE21545 were analysed using GeneSpring 11.5. The two datasets utilized a total of 22 and 126 carotid atherosclerosis samples, obtained from patients with and without symptoms of cerebral embolization, respectively. To assess whether the findings were reproducible we analysed carotid atherosclerosis samples from another 8 patients with and 7 patients without symptoms of cerebral embolization using real-time PCR.  Carotid atherosclerosis is estimated to be responsible for ~20% of all ischemic strokes5. Atherosclerotic plaque rupture and cerebral embolization is believed to be the mechanism by which carotid atherosclerosis leads to cerebral symptoms, such as transient ischemic attack and stroke1. However, the identification of the so-called “vulnerable plaque” has been elusive. The first whole-genome gene expression study of stroke was published a decade ago6. The authors at that time profiled peripheral blood mononuclear cells of stroke patients and compared them with those of healthy donors6. Almost 200 differentially expressed genes were identified; however, those assessed had limited diagnostic value with estimated specificity and sensitivity less than 80%6. A number of previous studies have examined differential gene expression in symptomatic carotid atherosclerosis; however, findings have not been consistent across the studies15. All these results question the value of differential gene expression in representing true molecular determinants of stroke. The current study re-examined previously published microarray datasets of carotid artery atherosclerosis to gain further insight. An attempt has been made to identify consistent and reproducible differentially expressed genes using publically available microarray datasets that utilized carotid endarterectomy samples from symptomatic and asymptomatic patients.The prevalence of carotid artery stenosis is approximately 4% to 8% in adults aged 50 to 79 years13. Suitable datasets were required to be publically available, to assess whole-genome gene expression within carotid plaque biopsies from patients both with and without symptoms of cerebral embolization, and to use commercially available microarrays of the same chip platform. The publically available raw array data were downloaded from Gene Expression Omnibus (GEO) provided by the National Center for Biotechnology Information (NCBI) or ArrayExpress provided by the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) public repositories.Two human microarray datasets were included in this study to establish a consensus set of differentially expressed genes in carotid atherosclerosis associated with the recent symptoms of cerebral embolization16. Briefly, the raw data matrix downloaded from GEO or ArrayExpress public repositories was uploaded into GeneSpring GX 11.5 (Agilent Technologies Pty Ltd) and the standard normalization procedures recommended for the Affymetrix GeneChip arrays was followed. Expression values were normalized using percentile shift normalization with default settings. These included normalization to 75th percentile. The expression profile of carotid atherosclerosis samples obtained from participants with symptoms of cerebral embolization was compared to those without symptoms. Since all samples represented advanced atherosclerotic tissue, only small differences in gene expression were expected between patients with and without symptoms of cerebral embolization. Previous evidence suggested that small changes in gene expression are able to induce significant phenotypic differences17. In line with this, genes with ≥1.15-absolute fold differential expression between groups based on P-value < 0.05 by non-parametric Mann–Whitney U-test with no mathematical correction for multiple testing were considered to be differentially expressed.In order to identify differentially expressed genes in carotid atherosclerotic tissue between symptomatic and asymptomatic patients, we analysed each original microarray dataset as described previously1. Quantitative real-time reverse transcription PCR (qRT-PCR) assay was performed to assess the relative expression of arylsulphatase B (ARSB) as this was consistently differential expressed in the array analyses. The relative expression of ARSB in each sample was calculated by using the Concentration-Ct-standard curve method and normalized using the average expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene for each sample using the Rotor-Gene Q operating software version 2.0.24 (Qiagen). GAPDH was chosen as the “housekeeping” gene since analyses showed its expression to be similar in carotid biopsies from symptomatic and asymptomatic patients. The QuantiTect SYBR Green one-step RT-PCR Kit (Qiagen) was used according to the manufacturer’s instructions with 20 ng of total RNA as template. All reactions were independently repeated in duplicate to assess the repeatability of the results and the mean of the two values for each sample was used for analyses. The QuantiTect Primer Assays QT00026684 and QT00079247 (Qiagen) were used for the ARSB and GAPDH assessments, respectively. Mann–Whitney U test was performed to identify differences in expression levels of ARSB between carotid atherosclerosis biopsies of patients with and without symptoms of cerebral embolization. Data were presented as box-and-whisker plots with median and interquartile range with maximum and minimum data points (whiskers) for relative expression. Statistical significance was defined at the conventional 5% level. All computations were performed using the Stata/MP 13.1 statistical software . Ethical approval was granted from The Townsville Hospital (TTH) and Health Services Committees, written informed consent was obtained from each participant, and the protocols conformed to the ethical guidelines of the Declaration of Helsinki.Using total RNA obtained from atherosclerosis removed from the proximal internal carotid (PIC) arteries of 8 patients with and 7 patients without symptoms of cerebral embolization, we assessed the validity of microarray findings . Total RNA was extracted from PIC biopsies stored in RNAlater (Sigma-Aldrich) at −80 °C using RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. Symptomatic patients presented with focal neurological symptoms related to their anterior cerebral circulation such as transient ischemic attack (TIA) or stroke within 6 weeks of surgery; asymptomatic patients presented with no history of neurological symptomsSIRT1) and protein kinase AMP-activated catalytic subunit alpha 1  under inflammatory conditions in vitro. These two ARSB-associated bioactive molecules were specifically selected to test their individual and combined ability to upregulate the SIRT1/AMPK metabolic pathway that exerts strong anti-inflammatory, anti-atherogenic, and plaque-stabilizing effects19. We used human vascular smooth muscle cells  that were plated at a seeding density of 1 × 105 cells/well and maintained at 37 °C, 5% CO2, in Dulbecco’s Modified Eagle Medium  containing 10% fetal bovine serum . Cells were growth arrested at 90% confluency by incubation in DMEM + 0.1% FBS overnight (18 hours). Control cultures (n = 6) were exposed to DMEM + 10% FBS comprising 10% v/v conditioned media generated from human monocytic THP-1 cells exposed to 10 µg/ml endotoxin  over a period of 24 hours. Experimental cultures were exposed to the same pro-inflammatory media but supplemented with either L-ascorbic acid  or chondroitin sulphate sodium salt , or a combination of both (n = 6 cultures). All cells were harvested after a 24-hour experimental period and subjected to RNA extraction followed by qRT-PCR using the Qiagen’s QuantiTect Primer Assay QT00009436 (AMPK) and QT00051261 (SIRT1) as outlined above.We investigated the effects of chondroitin sulphate, a natural substrate for ARSB, and L-ascorbic acid, an ARSB inhibitor, on the expression of sirtuin 1 , and ~27% of participants (6/22) had a positive history of smoking , and ~49% of participants (62/126) had a positive history of smoking  within carotid plaques of symptomatic compared to asymptomatic patients in E-MEXP-2257 and GSE21545, respectively  gene was the only gene consistently upregulated in symptomatic compared to asymptomatic patients identified in both E-MEXP-2257  and GSE21545  datasets (Table F3) gene and the ubiquitin like modifier activating enzyme 6 (UBA6) gene were found to be upregulated in symptomatic compared to asymptomatic patients in the E-MEXP-2257 dataset but downregulated in symptomatic compared to asymptomatic patients in the GSE21545 dataset (Table GAS6) gene, the uncharacterized LOC730101 (LOC730101), and the secretory leukocyte peptidase inhibitor (SLPI) gene were identified to be downregulated in symptomatic compared to asymptomatic patients in both datasets (Table GPR135) gene was found to be downregulated in symptomatic compared to asymptomatic patients in the E-MEXP-2257 dataset but upregulated in symptomatic compared to asymptomatic patients in the GSE21545 dataset  and L-ascorbic acid (ARSB inhibitor) on important anti-atherogenic pathways represented by the MPK Fig.  and SIRTRT1 Fig. . The incRT1 Fig.  but not MPK Fig. . ImportaMPK Fig.  and addiRT1 Fig. . These f13. By focusing on genes that were simultaneously differentially expressed in the datasets analysed, we were able to discover and validate the upregulation of ARSB in carotid plaques of symptomatic compared to asymptomatic patients, not acknowledged in the original microarray studies. It is important to note that the protein product of this gene is involved in degradative processes of sulphated proteoglycans, the major component of virtually all extracellular matrices (ECMs)20. Furthermore, previous data suggest an inverse association between enzymatic activity of ARSB and the stability of sulphated proteoglycans within the ECM21. In line with this, Koledgie et al. found that plaque rupture sites contain very little proteoglycan content relative to stable lesions22, consistent with a degradative process.We analysed publically available microarray datasets from previous gene expression studies that utilised carotid plaque biopsies from patients with and without symptoms of cerebral embolizationARSB within the carotid plaque biopsies of symptomatic patients identified in both microarray datasets was relatively small, we were able to confirm these findings by qRT-PCR using carotid plaque biopsies obtained from another group of symptomatic and asymptomatic patients. This led us to hypothesize that upregulation of ARSB may represent an important pathological mechanism associated with symptoms of cerebral embolization, consistent with previous findings that even small changes in gene expression can induce major phenotypic differences17. The ARSB enzyme catalyses de-sulphation of ubiquitous glycosaminoglycans such as chondroitin sulphate23. Published evidence suggests that plasma concentration of under-sulphated chondroitin is elevated in patients with carotid artery disease24, while sulphated chondroitin has been long known to exhibit anti-atherogenic properties in rodents, primates, and humans28. Several historical studies from the 1960s and 1970s report reduced incidence of coronary events and cardiovascular mortality in atherosclerotic subjects treated with chondroitin sulphate30. Recent data suggests that anti-atherogenic actions of chondroitin sulphate may occur through interfering with the pro-inflammatory activation of monocytes and endothelial cells by tumor necrosis factor (TNF) alpha31, a cytokine thought to be crucially involved in the pathogenesis of atherosclerotic plaque32. Although the authors did not elucidate the exact mechanism of action of chondroitin sulphate31, it is possible that upregulation of anti-inflammatory microRNAs, the negative regulators of gene expression, could play a role35. Previous studies suggest an inhibitory effect of chondroitin sulphates on gene expression through modification of microRNAs36. Importantly, the ARSB enzyme is inhibited by L-ascorbic acid37. Due to the lack of a well-developed animal model of carotid atherosclerosis associated with cerebral embolization, we further investigated the effect of L-ascorbic acid and chondroitin sulphate on important anti-atherogenic pathways in vitro. We found that chondroitin sulphate and L-ascorbic acid administered together induced a remarkable upregulation in the expression of SIRT1 and AMPK genes in VSMCs exposed to inflammatory conditions in vitro. These findings suggest that chondroitin sulphate formulated with L-ascorbic acid may serve as a potent activator of the SIRT1/AMPK pathway. This may hold promise as a novel therapeutic approach for carotid atherosclerosis since the SIRT1/AMPK pathway is key to a number of vasculoprotective processes. In particular, SIRT1 is the nicotinamide adenosine dinucleotide (NAD)-dependent deacetylase that has been associated with inhibition of the proatherogenic VSMC foam cell formation possibly through the suppressing of the nuclear factor kappa B (NF-κB) signalling pathway38. AMPK is the main energy-sensing kinase in all eukaryotic cells and has been implicated in stabilizing atherosclerotic plaques through the inhibition of the mammalian target of rapamycin (mTOR) signalling pathway39. The downregulation of genes for secretory leukocyte peptidase inhibitor (SLPI), uncharacterized LOC730101, and growth arrest specific 6 (GAS6) in carotid atherosclerosis associated with the symptoms of cerebral embolization in both datasets included in this study was also demonstrated. The role of SLPI and LOC730101 in human carotid atherosclerosis is largely unknown. Some evidence suggests that more severe atherosclerosis in humans is associated with an increase in GAS6 expression40, while similar expression of GAS6 in human carotid arteries with and without atherosclerosis has been reported41. Further investigation of the role of these three genes in carotid atherosclerosis is needed.Although the upregulation of The limitations of this study include the relatively small number of patients included in the original microarray datasets. In particular, although both datasets collectively included 148 carotid atherosclerosis tissue samples, the total number of 38 ischemic events was relatively small and findings need to be substantiated by larger studies. We also observed very limited overlap and consistency between the genes differentially expressed in the two datasets suggesting the heterogeneous nature of the patients investigated. Further genome-wide gene expression studies involving histologically standardized sets of patients are needed. In view of these limitations we sought to validate important microarrays finding using another set of carotid artery biopsies obtained from patients with and without symptoms of cerebral embolization. The assessment of independent samples helps to minimize the possibility that selection biases adversely affected the generalizability of the findings. We did, however, only assess mRNA not protein levels due to limited availability of carotid artery biopsies. Finally, the exact mechanism by which chondroitin sulphate combined with L-ascorbic acid upregulated the SIRT1/AMPK pathway as well as the therapeutic doses of these two bioactive molecules remains to be elucidated.In conclusion, a decade after the first microarrays for stroke, its molecular determinants are still poorly understood. The outcome of this study highlight a potential role for arylsulfatase B in promoting atherosclerosis-related stroke and warrants its further investigation as a therapeutic target that could be of potential clinical benefit.Supplemental Table ISupplemental Table II


To evaluate tacrolimus as therapeutic option for diabetic nephropathy (DN) based on molecular profile and network-based molecular model comparisons.We generated molecular models representing pathophysiological mechanisms of DN and tacrolimus mechanism of action (MoA) based on literature derived data and transcriptomics datasets. Shared enriched molecular pathways were identified based on both model datasets. A newly generated transcriptomics dataset studying the effect of tacrolimus on mesangial cells in vitro was added to identify mechanisms in DN pathophysiology. We searched for features in interference between the DN molecular model and the tacrolimus MoA molecular model already holding annotation evidence as diagnostic or prognostic biomarker in the context of DN.Thirty nine molecular features were shared between the DN molecular model, holding 252 molecular features and the tacrolimus MoA molecular model, holding 209 molecular features, with six additional molecular features affected by tacrolimus in mesangial cells. Significantly affected molecular pathways by both molecular model sets included cytokine-cytokine receptor interactions, adherens junctions, TGF-beta signaling, MAPK signaling, and calcium signaling. Molecular features involved in inflammation and immune response contributing to DN progression were significantly downregulated by tacrolimus , interleukin 4, or interleukin 10). On the other hand, pro-fibrotic stimuli being detrimental to renal function were induced by tacrolimus like the transforming growth factor beta 1 (TGFB1), endothelin 1 (EDN1), or type IV collagen alpha 1 (COL4A1).Patients with DN and elevated TNF levels might benefit from tacrolimus treatment regarding maintaining GFR and reducing inflammation. TGFB1 and EDN1 are proposed as monitoring markers to assess degree of renal damage. Next to this stratification approach, the use of drug combinations consisting of tacrolimus in addition to ACE inhibitors, angiotensin receptor blockers, TGFB1- or EDN1-receptor antagonists might warrant further studies. Tacrolimus is a powerful immunosuppressive drug belonging to the group of calcineurin inhibitors (CNIs), which was introduced in clinical use to tackle organ rejection in solid organ transplantation . TacroliTacrolimus is used for the treatment of lupus nephritis and also a clinical trial in the context of IgA glomerulonephritis has recently been conducted NCT0122445]6]. [6]. 5][6. [6]. 5]In this study we investigated the effects of tacrolimus on pathophysiological mechanisms of diabetic nephropathy on the level of in-silico constructed mechanism of action and disease pathophysiology molecular models respectively. We furthermore searched for molecular markers showing the potential for identifying the cohort of diabetic nephropathy patients benefitting the most from the immunosuppressive properties of tacrolimus.Molecular features affected by tacrolimus were extracted from scientific literature taking into consideration genes linked via NCBI gene2pubmed associations to publications which were annotated with the MeSH term “tacrolimus”. In addition deregulated transcripts were extracted from two published transcriptomics studies from Kern and colleagues as well as Maluf and colleagues focusing on the effect of tacrolimus on renal tissue, in particular on renal fibroblasts and post-kidney transplants, respectively 12]. Tra. Tra12].Construction of a diabetic nephropathy molecular model followed the same procedure as construction of the tacrolimus MoA model using as input features associated via gene2pubmed to publications holding “diabetic nephropathies” as major MeSH term.A molecular pathway enrichment analysis was conducted for the two constructed molecular models using the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 . Results2 in air. Cells were seeded onto six-well cell culture plates (Greiner Bio one) and were used for experiments at the fifth passage. Confluent cells were cultured in serum free media and FK 506 (Abcam) dissolved in DMSO (Sigma) in a final concentration of 1 μM for 24h. The control group was treated in serum free media and DMSO only and a total of four independent experiments were conducted. Trizol® and chloroform reagent was used to extract RNA (Invitrogen). The RNA pellet was dissolved in 10 μl sterile water and stored at –80°C.Primary human mesangial cells  were cultured in MCDB-131 media (Gibco-invitrogen), supplemented with 10% FBS, 100U penicillin/ml 100ug/ml streptomycin and 2mmol L-Glutamine  at 37°C in humid atmosphere with 5%COhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84908).Total RNA from human mesangial cells was reverse transcribed, labelled and hybridised to Affymetrix Human Gene (HuGene) 2.0 ST Array (Affymetrix) according to the manufacturer’s protocol. The oligo R package was used for pre-processing and normalization of expression data. The statistical analysis of microarrays (SAM) method available in the TIGR MultiExperimentViewer was used in order to identify differentially regulated transcripts between tacrolimus treated and untreated cells 18]. Tra. Tra18].The two constructed molecular models, namely the one representing tacrolimus MoA and the one reflecting diabetic nephropathy pathophysiology on a molecular network level were compared to each other with respect to molecular feature overlap as previously described . DeregulBiomarker evidence for diabetic nephropathy diagnosis and prognosis was gathered from scientific literature for molecular features in the diabetic nephropathies molecular model being affected by tacrolimus. Therefore molecular features were extracted from publications annotated with the major MeSH term “diabetic nephropathies” as well as the MeSH terms “biological markers” OR “genetic markers” neglecting profiling studies by excluding publications annotated with one of the following MeSH terms: “gene expression studies”, “microarray analysis”, “proteomics”, “metabolomics”, or “genome-wide association study”. Publications however had to be either of publication type “clinical trial” or annotated with “humans” or “disease models, animal”. Prognostic markers were extracted from publications further holding the MeSH term “prognosis”, whereas diagnostic markers were extracted from publications annotated either with “diagnosis”, “diagnosis, differential”, “early diagnosis”, or “diabetic nephropathies/diagnosis”. Next to considering molecular features assigned via gene2pubmed to the respective marker papers, we mined for molecular features using their respective gene names and considered perfect matches as hits.calcium signaling with 28 molecular features out of the 252 being assigned to this pathway . A set of signaling cascades were among the list of enriched molecular pathways like e.g. Adipocytokine signaling , MAPK signaling , TGF-beta signaling , or PPAR signaling . Next to these signaling cascades, molecular pathways linked to inflammation and immune response were enriched like for example cytokine-cytokine receptor interactions , Toll-like receptor signaling , or chemokine signaling . In addition the renin-angiotensin system  as well as the complement and coagulation system  were enriched with molecular features from the constructed DN molecular model.583 molecular features could be extracted from scientific literature being associated with diabetic nephropathy. The constructed molecular model for diabetic nephropathies in total held 252 molecular features in 15 process units ranging in size from 3 to 58 molecular features. The top enriched molecular pathway of the 17 identified molecular pathways was The tacrolimus MoA molecular model held 209 molecular features assigned to 19 process units ranging in size from 3 to 38 molecular features. From the 785 unique protein coding genes forming the input for model construction, 582 protein-coding genes originated from the two transcriptomics studies with 217 genes coming from literature mining for tacrolimus-associated molecular features. Five of the enriched molecular pathways in DN were also significantly enriched based on the set of molecular features in the tacrolimus MoA molecular model, namely cytokine-cytokine receptor interactions, adherens junctions, TGF-beta signaling, MAPK signaling, as well as calcium signaling .The use of heterogeneous data sets for constructing the tacrolimus MoA molecular model could be seen very critically but can also be seen from a positive angle. With input data coming from fibroblasts as well as from post-transplant kidneys it is not possible to reconstruct cell-specific molecular pathways, but on the other hand it allows assessing the effect of tacrolimus on kidney as a whole. Following this line we furthermore assessed the effect of tacrolimus on mesangial cells using gene expression profiling.21534 transcripts remained after preprocessing and normalization steps which were used for hierarchical cluster analysis in order to see whether there was an effect of tacrolimus on mesangial cells in culture. 1—Pearson correlation and average linkage were used as distance measure and linkage rule. In the cluster dendrogram, a clear separation between the tacrolimus-treated and the untreated control samples was detectable although absolute differences in gene expression between samples were only minor. Nevertheless 222 differentially regulated transcripts were identified using the SAM with a false discovery rate of lower than 5%. 154 transcripts were upregulated after tacrolimus treatment with 68 being downregulated. The deregulated transcripts could be mapped to 204 unique protein coding genes on the level of Ensembl Gene IDs with 141 upregulated and 63 downregulated genes.39 of the 252 individual molecular features of the DN molecular model were also part of the tacrolimus MoA molecular model thus being affected by tacrolimus. In addition six further molecular features of the DN molecular model could be identified as deregulated after tacrolimus treatment in mesangial cells .Three molecular features had annotation evidence as marker for diabetic nephropathy, with TNF being reported as diagnostic marker for DN and TGFB1 and EDN1 showing evidence as prognostic marker for DN.A set of molecular features involved in inflammation and immune response like the tumor necrosis factor alpha (TNF), interleukins 4 and 10 , or the heat shock protein family A (Hsp70) member 1B (HSPA1B) were all downregulated by tacrolimus.Inflammation is recognized as a major factor in diabetic nephropathy 21], sub, sub21],TNF was previously reported as biomarker for diagnosing diabetic nephropathy and also proposed as potential drug target in order to slow disease progression . Next toThe downside of tacrolimus on renal tissue becomes evident when looking at upregulated molecular features in the DN molecular model . EspeciaAnother pro-fibrotic molecule, namely endothelin 1 (EDN1), was upregulated by tacrolimus in mesangial cells, thus reinforcing the finding that tacrolimus rather activates than inhibits TGF-beta signaling .Based on these findings further studies are warranted stratifying DN patients based on TNF levels in order to identify patients being prone for treatment with low-dose tacrolimus 44]. TGF. TGF44].A further strategy might be to use drugs that block detrimental effects induced by tacrolimus like e.g. blocking TGF-beta signaling cascade with TGFB1 antibodies or using endothelin 1 receptor antagonists which are currently in clinical testing in the context of diabetic nephropathy. Such strategies were already proposed as alternative to conventional strategies of calcineurin inhibitor avoidance, withdrawal, or minimization in the context of renal transplantation . The endInflammatory mechanisms, and in particular the tumor necrosis factor signaling cascade, contributing to progression of diabetic nephropathy might be counterbalanced by tacrolimus treatment. Using TNF as predictive marker and TGFB1 and EDN1 as monitoring markers might be a valuable strategy finding the subset of diabetic nephropathy patients benefitting the most from tacrolimus treatment.


Biventricular pacing is an important modality to improve left ventricular (LV) synchronization and long-term function. However, the biological effects of this treatment are far from being elucidated and existing animal models are limited and demanding. Recently, we introduced an implanted device for double-site epicardial pacing in rats and echocardiographically demonstrated favorable effects of LV and biventricular (LV-based) pacing modes typically observed in humans. Here, this new animal model was further characterized. Electrodes were implanted either on the right atria (RA) and right ventricle (RV) or on the RV and LV. Following recovery, rats were either used for invasive hemodynamic measurements  or exposed to sustained RV vs. biventricular tachypacing for 3 days. RV pacing compromised, while LV-based pacing modes markedly enhanced cardiac performance. Changes in LV performance were associated with prominent compensatory changes in arterial resistance. Sustained RV tachypacing increased the electrocardiogram QTc interval by 7.9 ± 3.1 ms , dispersed refractoriness between the right and left pacing sites and induced important molecular changes mainly in the early-activated septal tissue. These effects were not observed during biventricular tachypacing (n = 6). Our results demonstrate that the rat is an attractive new model to study the biological consequences of LV dyssynchrony and resynchronization. Compelling evidence indicates that right ventricular (RV) pacing can be detrimental to left ventricular (LV) function. The sequence of electrical activation during RV pacing may largely resemble the activation pattern of left bundle branch block (LBBB)4, and can result in dyssynchronous LV contraction6. In accordance, RV pacing increases the risk for heart failure (HF) even in patients without pre-existing LV dysfunction10.The electromechanical and molecular consequences of various ventricular pacing modes have become a subject of intense interest in recent years due to profound clinical implications11. BiV pacing has been demonstrated to improve contractile performance in patients with dyssynchrony and to improve long-term survival in selected patients2. Although the clinical effectiveness of CRT is remarkable, there are multiple questions concerning dyssynchrony and CRT that remain ill-defined at present. For example, patient vulnerability to HF progression as a result of iatrogenic RV pacing is variable and optimal management of this problem is controversial13. In addition, it is estimated that around 30% of selected patients based on current indications do not benefit from CRT. Clinical data indicate that improvement in dyssynchrony is a prerequisite for obtaining beneficial effects of CRT, but the extent of such improvement may correlates poorly with clinical outcomes14. Although detailed evaluation including assessment of RV dyssynchrony may supplement LV dyssynchrony information and improve outcome prediction15, responsiveness to CRT also appears to involve factors beyond mechano-energetic considerations3. Recent studies exploring the cellular and molecular impacts of CRT in the failing heart indicate a highly complicated picture of cellular and molecular sequence of events, which is still far from being elucidated and which might be the key for better treatment adjustment and prediction of outcome19. Thus, reliable models that support extensive research of the complex biology of dyssynchrony and resynchronization are of paramount importance for obtaining new clinical insights in this field.Cardiac resynchronization therapy (CRT) by means of biventricular (BiV) pacing was developed as a strategy to overcome the deleterious effects of electromechanical dyssynchrony in the failing heart and represents a major advance in HF therapy21. In addition, the ability to induce clinically relevant manipulations such as hypertension, diabetes as well as molecular and genetic modifications, are quite limited in large animal models. While rodents have many such advantages and are already extensively used in HF research22, technical difficulties in electrode implantation have largely prevented proper validation of the effects of various pacing modes in rodents. Two murine studies utilizing single site pacing of RV and M-mode echocardiography have managed to clearly demonstrate dyssynchrony between the septum and the LV lateral wall24. However, single site ventricular pacing is methodologically problematic since it requires comparison to the spontaneous rhythm, which is different from the pacing state not only in ventricular electromechanics but also in beating rate and atrio-ventricular synchrony. Recently, using double site bipolar electrode implantation in combination with electrical recordings and speckle-tracking echocardiography, we managed to characterize for the first time the effects of various ventricular pacing modes on LV electromechanical synchrony in rats25. We found that RV pacing induces marked LV dyssynchrony compared to right atrial (RA) pacing or sinus rhythm. In contrast, LV pacing and, to a greater extent BiV pacing, diminished LV dyssynchrony25. These findings support the notion that rodent cardiac pacing mimics important electromechanical features seen in humans. However, the actual effects of different pacing modes on LV performance and on the myocardial properties of the rodent heart are still unknown. Therefore, in the present study we used pressure-volume (PV) loop recordings to characterize the effects of different ventricular pacing modes on cardiac performance. Next, we compared myocardial properties following application of sustained RV or BiV tachypacing for 72 hours in conscious freely moving rats. Our findings indicate similarities in the effects of various pacing modes to humans, but also demonstrate remarkable effects on arterial elastance, which were not described in humans to a similar extent. In addition, we found that sustained RV tachypacing induces prominent electrical and biochemical changes in myocardial properties compared to BiV pacing, including the activation of JNK in the early-activated myocardium, which was not previously identified in this setting. Overall, our data mark this model as an attractive new tool to study the complex pathophysiology of ventricular dyssynchrony and resynchronization.Current animal studies dealing with dyssynchrony and resynchronization almost exclusively utilize large animals (mainly dogs). Although insights coming from such models are extremely valuable, their highly demanding nature restricts usage for proof-of-principle studies and widespread experimentation at multiple laboratories25. Thus, it is imperative to characterize in details the hemodynamic effects of RV pacing as compared to normally conducted, synchronous contraction. To enable such analysis we implanted pacing electrodes on both the RA and the RV and utilized them to induce either overdrive atrial (A) pacing (synchronous) or sequential atrial and RV (A + RV) pacing (dyssynchronous) with similar beating rate and atrioventricular (AV) coupling. Using conventional ECG analysis we adjusted the AV interval during A + RV pacing to achieve complete RV pacing activation sequence . Moreover, in humans a reduction of Ea during RV pacing might be associated with pacing-induced mitral regurgitation33. Thus, the elevated Ea we observed during RV pacing implies absence of RV pacing-induced mitral regurgitation in our model. Indeed, we did not detect mitral retrograde flow waves during any of the overdrive pacing modes (n = 5). In addition, we did not notice acute changes in left atrial diameter between the different ventricular pacing modes .An additional important finding was a prominent increase of Ea during A + RV pacing and a further increase during RV-only pacing Table . Since p25. A typical example of obtained electrocardiograms is shown in Fig. Following the above analysis of RV pacing-induced dyssynchrony relative to synchronous pacing, we next used implanted RV and LV pacing electrodes to compare the hemodynamic effects of various ventricular pacing modes. ECG recordings confirmed the previously described effects on QRS duration and morphologyP-V loop recordings indicated profound differences in LV hemodynamic performance, when LV and BiV pacing were compared with RV pacing Fig. . LV systIn an attempt to further clarify the mechanisms leading to the marked differences in LV performance between RV pacing and LV-based pacing modes, inferior vena-cava occlusion experiments were performed. Under our standard experimental conditions without mechanical ventilation, these attempts were challenging and largely failed to induce meaningful curves that could be properly analyzed. Supplemental Fig. Following detailed analysis of the acute consequences of various pacing modes, we next utilized our model to examine for the first time, the effects of sustained RV tachypacing vs. BiV tachypacing in freely moving rats. At baseline, there were no differences between the RV-paced and BiV-paced groups in any of the measured electrophysiological parameters including spontaneous corrected QT interval (QTc-NSR), paced QT interval during RV site pacing and LV site pacing, epicardial activation time during pacing of both sites and VERP during pacing of both sites.RV tachypacing for 72 hours significantly prolonged the QTc-NSR relative to baseline, a phenomenon which was not detected following BiV tachypacing Fig. . AnalysiTo further elucidate the electrophysiological details, we analyzed epicardial activation time and VERP during right (Rt.) site pacing and Lt. site pacing. Epicardial activation was not affected by the different pacing modes Fig. . However23. Analysis of phosphorylated ERK, P38 or AKT did not reveal differential effects between the animals exposed to the different pacing modes  and late-activated  parts of the mid-LV were analyzed focusing specifically on molecules and signals that were previously implicated in the pathophysiology of electromechanical dyssynchronyLevels of Cx43 were also evaluated following RV vs. BiV pacing. While expression appeared to be globally increased in the tachypaced animals relative to shams, it did not consistently vary between the different pacing modes  we found that A + RV pacing did not influence measures of preload  of the rat heart. Moreover, considering the unchanged epicardial activation time in both groups, the observed differences in paced-QT during LV site pacing also support prolongation of APD in the RV free wall. Although our current analysis cannot determine the VERP in the early-activated septal part of the LV, we may speculate that it is also prolonged and contributes to the global increase in non-paced QT following RV tachypacing. Regional changes of VERP in the RV paced rats are somewhat reminiscent to published data in the dog model of LBBB where such changes were related to regional differences in load as well as to the extent of myocardial stretch applied at different time points during the action potential23. In contrast to Bilchick et al.23, here we could directly compare between RV-paced tissue and BiV-paced tissue, under similar beating rate and similar AV synchrony, a mission which is impossible using single site pacing methodology. The most important and somewhat unexpected finding here was a differential effect of RV tachypacing on JNK signaling, in contrast with P38, ERK and AKT previously implicated in the myocardial response to dyssynchrony in the dog heart3. It will be important to further explore whether these discrepancies reflect species dependent differences, or alternatively, it might reflect temporal variations due to the relatively short period of pacing we used here in comparison with the 3–6 weeks period applied in the dog model3. In any case, considering the fact that JNK is a stress-activated protein kinase with clear importance in the myocardium54, this new finding and its implications on the pathophysiology of myocardial dyssynchrony should be further explored in details. It is for example known that JNK is a major regulator of cardiac KChIP2 gene expression, and activation of JNK can diminish the transient outward K+ current (Ito) by transcriptional down-regulation of KChIP255. Whether such mechanism may contribute to differential changes in APD in the RV-paced rat heart is an important possibility that might be explored based on the current findings.Our biochemical results comparing myocardial tissue from the septal wall and the lateral wall of the LV, also support differential effects of RV pacing on the early-activated and late-activated regions as previously suggested in the mouse heart3. Moreover, by using rats it should be rather straightforward to combine our model with other insults leading to myocardial dysfunction such as hypertension, diabetes mellitus, high output failure and more. Further echocardiography regional dyssynchrony assessment in parallel with ventricular arrhythmias substrate evaluation56, is another important future direction. In summary, our current findings support the notion that the instrumented rat model is an important new advance in this field of research and may be used to shed light on fundamental effects of dyssynchrony and resynchronization on the tissue level.Other observed changes in the RV-paced rats including CAMKII activation and increased expression of osteopontin can reflect the profound effects of this dyssynchronous mode of tachypacing on the rat myocardium as opposed to BiV tachypacing. Thus, we may expect that with further prolongation of the tachypacing period profound differences in cardiac function would be noted between animals exposed to RV tachypacing and those exposed to BiV tachypacing. This may lay the foundation for a rat model, which is rather similar to the dog model of dyssynchronous heart failure induced by tachypacing in combination with LBBBThe study was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. All the experiments reported in this study were approved by the institutional ethics committee of Ben-Gurion University of the Negev, Israel . Adult male Sprague-Dawley rats were obtained from Harlan Laboratories . The animals were kept under standardized conditions throughout the study, according to home office guidelines: 12:12 light:dark cycles at 20–24 °C and 30–70% relative humidity. Animals were free-fed autoclaved rodent chow and had free access to reverse osmosis filtered water. At the end of the experiments animals were sacrificed by extraction of the heart under deep Nembutal-induced anesthesia.57. Briefly, the electrical connections to the epicardial surface were done using miniature-bipolar hook electrodes that were designed by our group for various in-vivo electrophysiological applications in rodents58. Each electrode contains a distal head with two sharp tungsten pins that are curved and isolated by insulted coating up to their tips. By means of small lateral thoracotomy the electrode can be fixed on the selected epicardial surface without the need for additional suturing. The implantable device is composed of an 8 pin ‘female’ connector that is attached by highly flexible insulted electrical wires  to a set of two miniature-bipolar hook electrodes. Additional electrodes for peripheral pseudo-ECG measurements are also implanted subcutaneously. Electron beam radiation is applied for sterilization of the device before its use. For device implantation rats (250–320 g) were anesthetized  and mechanically ventilated. The animals were placed on a warmed heating pad and under sterile conditions the two miniature-bipolar hook electrodes were implanted on the desired sites. Following chest closure the 8-pin ‘female’ connector was exteriorized through the skin of the back. Postoperative recovery and analgesia were done as described previously25. Buprenorphine (0.1 mg/Kg) was given by subcutaneous injection during immediate recovery and every 24 hours during the first 3 days. In addition, Dipyrone (400 mg/500 ml) was added to the drinking water during the first 3 days. The animals were monitored on a daily basis for signs of stress or inappropriate weight loss, according to guidance from the Ben-Gurion University veterinary services  #A5060-01, and fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)).The implantable device and the surgical technique that were utilized to permanently locate two bipolar-electrodes in predetermined epicardial locations were previously described in detail25. 2. Comparison between the effect of sustained RV tachypacing and BiV tachypacing (600 BPM) applied for 72 hours (n = 14). In section #1 hemodynamic measurements were initially done in animals implanted with right atria (RA) and RV electrodes (group A) to fully characterize the effect of RV pacing with and without optimal atrio-ventricular (A-V) coupling (n = 8). Next, electrodes were implanted on the RV and LV (group B) to directly compare between the different ventricular pacing modes using conventional P-V loop recordings (n = 8), inferior vena-cava occlusion studies  and echocardiographic evaluation directed towards mitral regurgitation and atrial dimensions assessment during the different pacing modes (n = 5). In part #2 animals were divided into three groups: shams, which were connected to the system without pacing (n = 2), sustained RV pacing (n = 6) and sustained BiV pacing (n = 6).Overall 39 successfully implanted rats were evaluated in this work. The study contained two principle parts: 1. Acute hemodynamic measurements during various pacing modes (n = 23). In this section each animal served as its own control and the effects of various pacing modes were compared in each animal as previously done for strain analysis2 through a face mask. Body temperature was rectally monitored and maintained at 37 ± 0.5 °C by a circulating water warming pad. Electrical stimulation (2 ms square pulses) was applied using isolation units  at a double diastolic threshold. A Labview 7.1 based program previously developed in our laboratory57 was used to control electrical stimulation as well as ECG recordings  through an A/D converter  as previously described25.Rats were evaluated 6–8 days after implantation of the pacing device. Animals were anesthetized by 3% isoflurane in an induction chamber followed by constant administration of 2% isoflurane with supplemental O61. Based on previous echocardiographic measurements from our laboratory under isoflurane anesthesia we estimated stroke volume of 0.9 μl/g of body weight for each animal. While this estimation might be somewhat biased from the absolute values, our analysis focused on the comparison of the effects of different pacing modes in each animal. Thus, the relative changes between the pacing modes were the main focus of our study. Following equilibration, pressure–volume curves were acquired during various pacing modes using continuous override pacing at a 150 ms cycle length (CL). The P-V loop acquisition protocol was as follows: Following optimal positioning of the catheter in the LV, it was fixed in place and LV P-V loops (1–2 minutes each) were sequentially recorded in sinus rhythm followed by override pacing at the different pacing sites. In group A the modes of the pacing were: (1) RA pacing alone; (2) sequential RA-RV pacing at the longest AV interval producing a right ventricular activation sequence without fusion as confirmed by QRS morphology , ejection fraction (EF), stroke work (SW), the maximal slope of the systolic pressure increment (dP/dtmax), the diastolic pressure decrement (dP/dtmin), and the time constant of isovolumic relaxation (Tau). At the end of all P-V loop recordings animals were sacrificed and correct positioning of the epicardial electrodes was confirmed by post-mortem operation. For description of inferior vena-cava occlusion attempts under RV vs. BiV pacing see supplemental methods.In each animal the right common carotid artery was exposed and a 1.9-Fr pressure-volume (P-V) catheter  was inserted into the right common carotid artery and passed in a retrograde fashion through the aortic valve into the LV to monitor pressure and volume as well as admittance magnitude and phase angle signals in the LV. Data were acquired using a ADI LabChart acquisition and analysis software which was also used for offline analysis. Signals were acquired at a sampling rate of 1,000 Hz using an Advantage PV system . The steps involved in the setup and use of the Advantage system, i.e., catheter calibration and measures of myocardial conductivity and permittivity were done as previously described in the literatureogy Fig. ; (3) RV 25 with a Vivid 7 echocardiography system  and 10S probe. Following optimal positioning of the transducer in an apical four chamber view, it was fixed in place by the operator, while clips were sequentially recorded during override pacing (150 ms Cycle length) under the different pacing modes . Presence or absence of mitral regurgitant jet was assessed with 2D color Doppler and continuous-wave Doppler. Maximal atrial diameter in the apical four chamber view was also evaluated during the different pacing modes.Analysis was done under isoflurane sedation (1%) as previously described57. Pacing was done using optically isolated units . Pseudo ECG and epicardial signals were recorded using conventional amplifier . The animals were allowed to adapt for 24 h in the cage. On the 7th postoperative day, baseline pseudo ECG recordings were recorded and measurements of ventricular effective refractory period (VERP) were evaluated through each pacing electrode (RV and LV). VERP was determined at twice-diastolic threshold as the longest S1-S2 interval that failed to generate a response. The programed stimulation protocol included ten S1 stimuli at 100-ms cycle length, followed by S2 decremented in 2-ms intervals. QT and epicardial activation time measurements were done as previously described25. QTc was calculated using normalized Bazett’s equation adapted for rats: QTcn − B = QT/(RR/f)1/2, where f is the normalization factor according to the basal RR duration (150 ms for rats)62.On the 6th postoperative day animals were placed in dedicated pacing and recording cages where the back connector was linked by an elastic cable to the pacing and recording apparatus as previously describedAfter baseline measurements, continuous RV pacing (n = 6) or BiV pacing (n = 6) was initiated at a 100 ms CL. Confirmation of effective pacing was continuously monitored through online pseudo ECG recordings. For the BiV pacing group we routinely confirmed effective pacing through each of the pacing channels (RV and LV) at least twice daily. At the end of 72 hours of pacing all electrophysiological parameters were re-evaluated, before the animals were sacrificed.63. Membranes were incubated with primary antibodies including: rabbit anti-phospho-ERK 1/2 ; rabbit anti-phospho-p38 ; rabbit anti-phospho-JNK ; mouse anti-total-JNK , rabbit anti-phospho-CaMKII ; rabbit anti-total-CaMKII , rabbit anti-phospho-Akt Ser473 ; mouse anti-osteopontin ; rabbit anti-Connexin 43 . GAPDH was evaluated using rabbit anti-GAPDH  and served as loading control. Filters were extensively washed in TBS-T and then were incubated with horseradish peroxidase-conjugated secondary antibody. Secondary antibody was a species-specific horseradish peroxidase-conjugated goat anti-rabbit  or goat anti-mouse . Bands were visualized by using the ECL system  and were quantified with computerized densitometry and ImageJ (version 1.24) software. Densitometric values were reported after normalization for gel-loading differences and relative to the average value obtained from protein extracts of sham-operated control rats. For description of real-time QT-PCR analysis and histological analysis of connexin43 see supplemental methods.All animals exposed to 72 hours of RV pacing (n = 6), BiV pacing (n = 6) or sham-operation with no pacing (n = 2) were sacrificed and tissue samples from the septum (early activated) and the free wall (late activated) of the LV were snap frozen in liquid nitrogen. Protein lysates were obtained in RIPA buffer containing protease and phosphatase inhibitors and prepared for immunoblot analysis as previously describedP < 0.05. Unless otherwise stated p-values are displayed graphically as follows: *p < 0.05, **p < 0.01, ns = not significant.Values are expressed as means ± SE. In part #1 comparison between the different pacing modes was done utilizing 1-way ANOVA of repeated measures and Tukey multiple comparisons post-test. Inferior vena-cava occlusion analysis data was compared using Wilcoxon rank test. In part #2 comparisons were done using paired or unpaired t-test as required. For n of less than 6 or parameters that are not normally distributed non-parametric testing was performed. The criterion for significance was set at Supplementary data


BRCA1 mutated MANEC of his ileum. The tumor presented a susceptibility to a combined chemotherapy and the PARP1-inhibitor olaparib.Adenocarcinomas or combined adeno-neuroendocrine carcinomas (MANEC) of small bowel usually have a dismal prognosis with limited systemic therapy options. This is the first description of a patient showing a germline-related BRCA1 mutation in different tumor locations. There was no known personal history of a previous malignant tumor. Nevertheless we identified his BRCA1 mutation as germline-related. A systemic treatment was started including Gemcitabine followed by selective internal radiotherapy (SIRT) to treat liver metastases and in the further course Capecitabine but this treatment finally failed after 9 months and all liver metastases showed progression. The treatment failure was the reason to induce an individualized therapeutic approach using combined chemotherapy of carboplatin, paclitaxel and the Poly (ADP-ribose) polymerase- (PARP)-inhibitor olaparib analogous to the treatment protocol of Oza et al.A 74-year old male patient presented with a metastasized MANEC of his ileum. Due to clinical symptoms his ileum-tumor and the single brain metastasis were removed. We verified the same pathogenic (class 5) All liver metastases demonstrated with significant tumor regression after 3 months and could be removed. In his most current follow up from December 2017 (25 months after his primary diagnosis) the patient is in a very good general condition without evidence for further metastases.BRCA1 mutation in small bowel adeno-neuroendocrine carcinoma (MANEC). Our findings offer a personalized treatment option. The germline background was unexpected in a 74-year old man with no previously known tumor burden. We should be aware of the familiar background in tumors of older patients as well.We present first evidence of a therapy susceptible germline-related  Small bowel carcinomas are rare and account only for 0.5% of all cancers. The 5 year overall survival rates for patients with small-bowel adenocarcinoma is 34.9%. To best of our knowledge no valid prognostic or survival data exist to the mixed adeno-neuroendocrine carcinoma (MANEC)-subtype of the small bowel. There is a strong need for a more effective and personalized systemic treatment option in carcinoma of the small bowel. Most recently Schrock, A et al.  describeBRCA1 and BRCA2 and others  [The most important pathway in DNA double strand break (DNA-dbs.) repair is homologous recombination (HR). The HR pathway is dependent on different proteins including , RAD52) .BRCA1/2 deficient cells show a high degree of chromosomal instability which increases the risk of malignant transformation [BRCA1/2 (gBRCA-mut) were described in carcinoma of the ovary in 5–18% and in some other solid malignant tumors including breast carcinoma, pancreatic carcinoma and less frequent in prostate carcinoma. It became apparent that BRCA mutations also occur in sporadic carcinomas and these tumors respond to Poly (ADP-ribose) polymerase- (PARP) inhibition. The full significance of somatic BRCA mutations as a biomarker for the prediction of therapeutic response to PARP inhibition is not entirely clear but ovarian cancer patients with a somatic BRCA mutation are equally likely to benefit from treatment with PARP inhibitors as those with a germline mutation of BRCA. Some authors suggest to perform somatic BRCA testing in routine clinical diagnostics of all advanced tumors, which are potentially amenable to PARP inhibitors [Effective DNA repair is essential to maintain DNA integrity – so ormation , 5. Germhibitors , 6–11.http://arup.utah.edu/database/BRCA/orClinVardatabase:http://www.ncbi.nlm.nih.gov/clinvar/ or Universal mutation database BRCA share: http://www.umd.be/BRCA1/).Each BRCA variant was classified according to the established IARC classification. This classification ranges from class 1 variants  via class 2 , class 3 (uncertain), class 4 (likely pathogenic) to class 5 variants (definitively pathogenic) according to different databases (compare: BRCA mutation database: Tumor areas were marked on H&E-stained tissue slides by a pathologist (AQ) and DNA was extracted from corresponding unstained 10 μm thick slides by manual macrodissection. After proteinase K treatment, the DNA was automatically purified using the Maxwell 16 FFPE Tissue LEV DNA Purification Kit  on the Maxwell 16 Instrument (Promega) following the manufacturers’ protocol. The DNA concentration was measured using a real-time qPCR-based method.BRCA1 and BRCA2 mutation and were further analyzed using a panel of 12 other genes resulting altogether in a total of 452 amplicons. Additionally, in these tumor manifestations microsatellite status was checked using five different mononucleotide markers: BAT25, BAT26, NR-21, NR-22 and NR-27 [By parallel sequencing, all tumor manifestations were analyzed for http://www.broadinstitute.org/igv/, Cambridge; USA). A 5% cutoff for variant calls was used and results were only interpreted if the coverage was > 200×.Isolated DNA (10 ng each) was amplified with the Human BRCA1 and BRCA2 GeneRead DNAseq Targeted Panel V2  or with a customized GeneRead DNAseq Alignment and annotation was done using a modified version of a previously described method . ResultiThe primary MANEC of the ileum showed two different tumor components: One gland-forming adenocarcinoma-component including PAS-positive mucins in the tumor cell cytoplasm or gland lumina as well as a solid tumor forming neuroendocrine carcinoma-component showing a strong and diffuse immunohistochemical positivity for synatophysin and to a lesser content chromogranin A as well Fig. . The tumClinicalTrials.gov Identifier: NCT02807181). Because of anemia and poor general condition the administration of cisplatin was withheld. In the further course of treatment an additional tumor site in the ileum was detected and progression with cerebral metastasis occurred. The ileum tumor and one brain metastasis were removed. The ileum tumor was classified as a mixed adeno-neuroendocrine carcinoma (MANEC). Tumor manifestations in the liver and in the brain could be characterized as metastases from the adenocarcinoma component  12/2015–03/2016) followed by selective internal radiotherapy (SIRT) analogous to the SIRCCA study protocol . We verified the same BRCA1 mutation (c.3700_3704delGTAAA p.V1234Qfs*8) with a very high allele frequency up to 96% in all locations using ultra deep sequencing in the formalin-fixed paraffin embedded material . The MANEC was microsatellite stable. The mutational hot spots of all other analyzed genes  were wild type.In the further course we patient was treated with capecitabine  05–09/2016). This treatment finally failed 4 months later and all liver metastases showed progression. The treatment failure was the reason to analyze the tumor-DNA for molecular alterations using two panels of 14 genes including BRCA 1 and 2, administered intravenously on day 1) and carboplatin (4 mg/mL per min) in total five cycles for the following 4 months.According to the encouraging experiences using combined platin-based chemotherapy and PARP-inhibitors in BRCA-mutated ovarian carcinomas the molecular results have been the trigger for the personalized therapeutic approach using combined chemotherapy of carboplatin, paclitaxel and the PARP1-inhibitor olaparib analogous to the treatment protocol of Oza et al.  followinAll tumor manifestations demonstrated with a significant tumor regression in the MINT analysis and Ga-68-Dotatate PET-CT after 3 months of treatment  of the small bowel. There was no known personal history of a previous malignant tumor. Nevertheless, the high allele frequency of the BRCA1 mutation in all tumor manifestations suggested a germline background. Consequently, we tested histologically tumor-free ileum mucosa and were able to detect the same BRCA1 mutation with an allele frequency of 48%. This finding clearly identifies the mutation as germline and a deletion of the wild type BRCA1 allele in the tumor. The homogeneous distribution of BRCA1 mutated tumor cells in the primary tumor and in the different metastasis analyzed here suggests a clonal origin and a causative role of this second hit. Even though a gBRCA1 mutation could be a marker for prostate cancer we did not find any indications for prostate cancer [To the best of our knowledge this is the first description of pathogenic and treatable germline-related e cancer .BRCA1 mutation in our treated patient, because there was no known personal history of a prior malignant tumor in the 74 year-old man. The germline background of the BRCA1 mutated small bowel carcinoma (like in ovarian adenocarcinoma) is especially promising for a successful combination therapy using platinum-based chemotherapy and a PARP inhibitor (e.g. Olaparib). It has to be questioned whether the somatic pathogenic BRCA mutation itself is the main predictor of an effective PARP inhibition [According to treatment protocols established in BRCA mutated ovarian carcinomas we successfully applied combined chemotherapy of carboplatin, paclitaxel and the PARP1-inhibitor olaparib. Initially, we assumed a somatic background of the hibition .BRCA1/2 somatic mutations or the promoter methylation of BRCA some other alterations are potential markers of an effective PARP inhibition  [Beyond ylation) .gBRCA mutated ovarian carcinoma are sensitive to therapies using immune-checkpoint inhibition most likely due to its higher mutational load compared to wild type ovarian carcinoma [BRCA mutated MANEC also benefit from immune-checkpoint inhibition as an additional future treatment option.Additionally, first evidence came up that arcinoma . It remaBRCA1 mutation in a metastasized mixed adeno-neuroendocrine carcinoma (MANEC) of small bowel.Our findings are remarkable in many ways: Here we provide first evidence of a therapy susceptible The germline background of our patient was unexpected because he is a 74-year old man with no previously known tumor burden. Currently, there is increasing evidence that the first germline associated tumor manifestation  may occur beyond the sixth decade of living which means that we should be aware of the familiar background in tumors of older patients as well .