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While it is experimentally supported that impaired myocardial vascularization contributes to a mismatch between myocardial oxygen demand and supply, a mechanistic basis for disruption of coordinated tissue growth and angiogenesis in heart failure remains poorly understood. Silencing strategies that impair microRNA biogenesis have firmly implicated microRNAs in the regulation of angiogenesis, and individual microRNAs prove to be crucial in developmental or tumor angiogenesis. A high-throughput functional screening for the analysis of a whole-genome microRNA silencing library with regard to their phenotypic effect on endothelial cell proliferation as a key parameter, revealed several anti- and pro-proliferative microRNAs. Among those was miR-216a, a pro-angiogenic microRNA which is enriched in cardiac microvascular endothelial cells and reduced in expression under cardiac stress conditions. miR-216a null mice display dramatic cardiac phenotypes related to impaired myocardial vascularization and unbalanced autophagy and inflammation, supporting a model where microRNA regulation of microvascularization impacts the cardiac response to stress.

Acetylsalicylic acid (ASA) is widely used in secondary prevention of cardiovascular (CV) disease, mainly because of its antithrombotic effects. Here, we investigated whether ASA can prevent the progression of vessel wall remodelling, atherosclerosis, and CV complications in apolipoprotein E deficient (ApoE−/−) mice, a model of stable atherosclerosis, and in ApoE−/− mice with a mutation in the fibrillin-1 gene (Fbn1C1039G+/−), which is a model of elastic fibre fragmentation, accompanied by exacerbated unstable atherosclerosis. Female ApoE−/− and ApoE−/−Fbn1C1039G+/− mice were fed a Western diet (WD). At 10 weeks of WD, the mice were randomly divided into four groups, receiving either ASA 5 mg/kg/day in the drinking water (ApoE−/− (n = 14), ApoE−/−Fbn1C1039G+/− (n = 19)) or plain drinking water (ApoE−/− (n = 15), ApoE−/−Fbn1C1039G+/− (n = 21)) for 15 weeks. ApoE−/−Fbn1C1039G+/− mice showed an increased neutrophil–lymphocyte ratio (NLR) compared to ApoE−/− mice, and this effect was normalised by ASA. In the proximal ascending aorta wall, ASA-treated ApoE−/−Fbn1C1039G+/− mice showed less p-SMAD2/3 positive nuclei, a lower collagen percentage and an increased elastin/collagen ratio, consistent with the values measured in ApoE−/− mice. ASA did not affect plaque progression, incidence of myocardial infarction and survival of ApoE−/−Fbn1C1039G+/− mice, but systolic blood pressure, cardiac fibrosis and hypertrophy were reduced. In conclusion, ASA normalises the NLR, passive wall stiffness and cardiac remodelling in ApoE−/−Fbn1C1039G+/− mice to levels observed in ApoE−/− mice, indicating additional therapeutic benefits of ASA beyond its classical use.

Context.— The benefit of programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) as a method to select patients who may benefit from programmed death receptor-1 (PD-1)/PD-L1 immunotherapies remains uncertain in many tumor indications. Objectives.— To compare the commercially available, approved PD-L1 IHC assays (22C3, 28-8, SP142, SP263), specifically identifying the changes in staining output created by altering the detection method. Design.— This pilot study investigates the respective PD-L1 kit assay staining patterns and related scoring of tumor cells and immune cells on lung carcinoma and melanoma. Furthermore, the influence of the detection method (platform and related reagents) on PD-L1 antibody performance is studied. Results.— The SP142 kit reveals more immune cell staining but less tumor cell staining than the other PD-L1 kits. Alternatively, the 22C3 and 28-8 kits show good tumor cell sensitivity, but less pronounced immune cell staining, even in tonsil. Tumor cell staining by the SP263 kit is comparable to that of 22C3 and 28-8 kits, while immune cell staining is better. Strikingly, the selection of the detection method has a major impact on the sensitivity of the assay for PD-L1 detection per cell type. Switching the detection method of the kits could largely circumvent the observed staining differences. Conclusions.— The diverse sensitivities caused by the choice of the detection method should be taken into consideration when selecting PD-L1 kits or developing PD-L1 IHC laboratory-developed tests. When using alternative kits or laboratory-developed tests, it is strongly recommended to reestablish their clinical utility per therapeutic agent or compare them with the original kit.

Aims: Tumour cell and/or immune cell programmed cell death ligand 1 (PD-L1) expression is considered as a potential biomarker for anti-PD1 and anti-PD-L1 immunotherapy. Currently, different PD-L1 assays are used. This study aims to compare the staining patterns of two PD-L1 antibody clones in melanoma metastases and correlate them with PD-L1 mRNA expression. Methods and results: The immunohistochemistry assays were optimized and validated independently on a Ventana Benchmark Ultra (Ventana Medical Systems Inc., Tucson, AZ, USA) (E1L3N) and XT (SP142), using the same detection system. In total, 46 melanoma metastases were stained with both validated immunohistochemistry assays. Stained slides were digitized for qualitative and semi-quantitative evaluation; done by pathologist and semi-automated software analysis. A subset of 21 melanoma metastases was selected for quantification of the PDL1 mRNA expression. Accuracy and precision criteria were met for both assays. PD-L1 protein and mRNA expression showed remarkably good Spearman's coefficients of 0.90 (E1L3N) and 0.87 (SP142). Despite the remarkable correlation between both PD-L1 assays in expression patterns and quantification values (q > 0.90), E1L3N showed significantly more tumour cell staining than SP142. Conclusions: E1L3N and SP142 IHC assays were optimized and validated successfully and independently for sensitive and accurate PD-L1 detection. Concordance was best for immune cell scoring, while E1L3N tended to detect more tumour cells. Determination of the clinically relevant cut-off values for immune cell versus tumour cell detection requires further research.

Key points Cyclic stretch is known to alter intracellular pathways involved in vessel tone regulation. We developed a novel set-up that allows straightforward characterization of the biomechanical properties of the mouse aorta while stretched at a physiological heart rate (600 beats min–1). Active vessel tone was shown to have surprisingly large effects on isobaric stiffness. The effect of structural vessel wall alterations was confirmed using a genetic mouse model. This set-up will contribute to a better understanding of how active vessel wall components and mechanical stimuli such as stretch frequency and amplitude regulate aortic mechanics. Abstract Cyclic stretch is a major contributor to vascular function. However, isolated mouse aortas are frequently studied at low stretch frequency or even in isometric conditions. Pacing experiments in rodents and humans show that arterial compliance is stretch frequency dependent. The Rodent Oscillatory Tension Set-up to study Arterial Compliance is an in-house developed organ bath set-up that clamps aortic segments to imposed preloads at physiological rates up to 600 beats min–1. The technique enables us to derive pressure–diameter loops and assess biomechanical properties of the segment. To validate the applicability of this set-up we aimed to confirm the effects of distension pressure and vascular smooth muscle tone on arterial stiffness. At physiological stretch frequency (10 Hz), the Peterson modulus (EP; 293 (10) mmHg) for wild-type mouse aorta increased 22% upon a rise in pressure from 80–120 mmHg to 100–140 mmHg, while, at normal pressure, EP increased 80% upon maximal contraction of the vascular smooth muscle cells. We further validated the method using a mouse model with a mutation in the fibrillin-1 gene and an endothelial nitric oxide synthase knock-out model. Both models are known to have increased arterial stiffness, and this was confirmed using the set-up. To our knowledge, this is the first set-up that facilitates the study of biomechanical properties of mouse aortic segments at physiological stretch frequency and pressure. We believe that this set-up can contribute to a better understanding of how cyclic stretch frequency, amplitude and active vessel wall components influence arterial stiffening.

Apoptosis of macrophages and vascular smooth muscle cells (VSMCs) in advanced atherosclerotic plaques contributes to plaque progression and instability. Caspase-3, a key executioner protease in the apoptotic pathway, has been identified in human and mouse atherosclerotic plaques but its role in atherogenesis is not fully explored. We therefore investigated the impact of caspase-3 deletion on atherosclerosis by crossbreeding caspase-3 knockout (Casp3−/−) mice with apolipoprotein E knockout (ApoE−/−) mice. Bone marrow-derived macrophages and VSMCs isolated from Casp3−/−ApoE−/−mice were resistant to apoptosis but showed increased susceptibility to necrosis. However, caspase-3 deficiency did not sensitize cells to undergo RIP1-dependent necroptosis. To study the effect on atherosclerotic plaque development, Casp3+/+ApoE−/−and Casp3−/−ApoE−/−mice were fed a western-type diet for 16 weeks. Though total plasma cholesterol, triglycerides, and LDL cholesterol levels were not altered, both the plaque size and percentage necrosis were significantly increased in the aortic root of Casp3−/−ApoE−/−mice as compared to Casp3+/+ApoE−/−mice. Macrophage content was significantly decreased in plaques of Casp3−/−ApoE−/−mice as compared to controls, while collagen content and VSMC content were not changed. To conclude, deletion of caspase-3 promotes plaque growth and plaque necrosis in ApoE−/−mice, indicating that this antiapoptotic strategy is unfavorable to improve atherosclerotic plaque stability.

Autophagy is a subcellular process that plays an important role in the degradation of proteins and damaged organelles such as mitochondria (a process termed "mitophagy") via lysosomes. It is crucial for regulating protein and mitochondrial quality control and maintaining cellular homeostasis, whereas dysregulation of autophagy has been implicated in a wide range of diseases including atherosclerosis. Recent evidence has shown that the autophagic process becomes dysfunctional during the progression of atherosclerosis, regardless of whether there are many autophagy-stimulating factors (e.g., reactive oxygen species, oxidized lipids, and cytokines) present within the atherosclerotic plaque. This review highlights the recent insights into the causes and consequences of defective autophagy in atherosclerosis, with a special focus on the role of autophagy and mitophagy in plaque macrophages, vascular smooth muscle cells (VSMCs), and endothelial cells (ECs). It has been shown that defective autophagy can promote apoptosis in macrophages but that it accelerates premature senescence in VSMCs. In the ECs, defective autophagy promotes both apoptosis and senescence. We will discuss the discrepancy between these three cell types in their response to autophagy deficiency and underline the cell type-dependent role of autophagy, which may have important implications for the efficacy of autophagy-targeted treatments for atherosclerosis. ispartof: OXIDATIVE MEDICINE AND CELLULAR LONGEVITY vol:2018 ispartof: location:United States status: published

Cardiovascular ageing is a key determinant of life expectancy. Cellular senescence, a state of irreversible cell cycle arrest, is an important contributor to ageing due to the accumulation of damaged cells. Targeting cellular senescence could prevent age-related cardiovascular diseases. In this study, we investigated the effects of neuregulin-1 (NRG-1), an epidermal growth factor with cardioprotective and anti-atherosclerotic effects, on cellular senescence., info:eu-repo/semantics/published

Autophagy is triggered in vascular smooth muscle cells (VSMCs) of diseased arterial vessels. However, the role of VSMC autophagy in cardiovascular disease is poorly understood. Therefore, we investigated the effect of defective autophagy on VSMC survival and phenotype and its significance in the development of postinjury neointima formation and atherosclerosis. Tissue-specific deletion of the essential autophagy gene Atg7 in murine VSMCs (atg7−/− VSMCs) caused accumulation of SQSTM1/p62 and accelerated the development of stress-induced premature senescence as shown by cellular and nuclear hypertrophy, CDKN2A-RB-mediated G1 proliferative arrest and senescence-associated GLB1 activity. Transfection of SQSTM1-encoding plasmid DNA in Atg7+/+ VSMCs induced similar features, suggesting that accumulation of SQSTM1 promotes VSMC senescence. Interestingly, atg7−/− VSMCs were resistant to oxidative stress-induced cell death as compared to controls. This effect was attributed to nuclear translocation of the transcription factor NFE2L2 resulting in upregulation of several antioxidative enzymes. In vivo, defective VSMC autophagy led to upregulation of MMP9, TGFB and CXCL12 and promoted postinjury neointima formation and diet-induced atherogenesis. Lesions of VSMC-specific atg7 knockout mice were characterized by increased total collagen deposition, nuclear hypertrophy, CDKN2A upregulation, RB hypophosphorylation, and GLB1 activity, all features typical of cellular senescence. To conclude, autophagy is crucial for VSMC function, phenotype, and survival. Defective autophagy in VSMCs accelerates senescence and promotes ligation-induced neointima formation and diet-induced atherogenesis, implying that autophagy inhibition as therapeutic strategy in the treatment of neointimal stenosis and atherosclerosis would be unfavorable. Conversely, stimulation of autophagy could be a valuable new strategy in the treatment of arterial disease.

Apolipoprotein E deficient (ApoE(-/-)) mice with abeterozygous mutation in the flbrillin-1 gene (Fbn1(C1039G+/-) show spontaneous atherosclerotic plaque ruptures, disturbances in cerebral flow and sudden death when fed a Western-type diet (WD). The present study focused on motor coordination and spatial learning of ApoE(-/-) Fbn1(C1039G+/-) mice on WD for 20 weeks (n = 21). ApoE(-/-) mice on WD (n = 24) and ApoE(-/-) Fbn1(C1039G+/-) mice on normal diet (ND, n = 21) served as controls. Starting from 10 weeks of diet, coordination was assessed every two weeks by the following tests: gait analysis, stationary beam, wire suspension and accelerating rotarod. The Morris water maze test was performed after 13 weeks of diet to study spatial learning. At the end of the experiment (20 weeks of WD), the mice were sacrificed and the brachiocephalic artery and brain were isolated. From 12 weeks onward, gait analysis of ApoE(-/-) Fbn1(C1039G+/-) mice on WD revealed a progressive increase in track width as compared to ApoE(-/-) mice on WD and ApoE(-/-) Fbn1(C1039G+/-) mice on ND (at 20 weeks: 29.8 +/- 0.6 mm vs. 25.8 +/- 0.4 mm and 26.0 +/- 0.5 mm). Moreover, the stationary beam test showed a decrease in motor coordination of ApoE(-/-) Fbn1(C1039G+/-) mice on WD at 18 and 20 weeks. The wire suspension test and accelerating rotarod could not detect signs of motor impairment. Spatial learning was also not affected. Histological analysis of the brachiocephalic artery showed larger and more stenotic plaques in ApoE(-/-) Fbn1(C1039G+/-) mice on WD. Furthermore, the parietal cortex of ApoE(-/-) Fbn1(C1039G+/-) mice on WD showed pyknotic nuclei as a sign of hypoxia and the percentage of pyknosis correlated with track width. In conclusion, gait analysis may be an efficient method for analyzing hypoxic brain damage in the ApoE(-/-) nFb1(C1039G+/-) mouse model. This test could be of value to assess the effect of potential anti-atherosclerotic therapies in mice. (c) 2014 Elsevier Inc. All rights reserved.

Although cancer vaccination has yielded promising results in patients, the objective response rates are low. The right choice of adjuvant might improve the efficacy. Here, we review the biological rationale, as well as the preclinical and clinical results of polyinosinic:polycytidylic acid and its derivative poly-ICLC as cancer vaccine adjuvants. These synthetic immunological danger signals enhanced vaccine-induced anti-tumor immune responses and contributed to tumor elimination in animal tumor models and patients. Supported by these results, poly-ICLC-containing cancer vaccines are currently extensively studied in the ongoing trials, making it highly plausible that poly-ICLC will be part of the future approved cancer immunotherapies.

Autophagy is a reparative, life-sustaining process by which cytoplasmic components are sequestered in double-membrane vesicles and degraded on fusion with lysosomal compartments. Growing evidence reveals that basal autophagy is an essential in vivo process mediating proper vascular function. Moreover, autophagy is stimulated by many stress-related stimuli in the arterial wall to protect endothelial cells and smooth muscle cells against cell death and the initiation of vascular disease, in particular atherosclerosis. Basal autophagy is atheroprotective during early atherosclerosis but becomes dysfunctional in advanced atherosclerotic plaques. Little is known about autophagy in other vascular disorders, such as aneurysm formation, arterial aging, vascular stiffness, and chronic venous disease, even though autophagy is often impaired. This finding highlights the need for pharmacological interventions with compounds that stimulate the prosurvival effects of autophagy in the vasculature. A large number of animal studies and clinical trials have indicated that oral or stent-based delivery of the autophagy inducer rapamycin or derivatives thereof, collectively known as rapalogs, effectively inhibit the basic mechanisms that control growth and destabilization of atherosclerotic plaques. Other autophagy-inducing drugs, such as spermidine or add-on therapy with widely used antiatherogenic compounds, including statins and metformin, are potentially useful to prevent vascular disease with minimal adverse effects.

Background and Aims. Inflammatory mediators that cross-talk in different metabolically active organs are thought to play a crucial role in the pathogenesis of Nonalcoholic Steatohepatitis (NASH). This study was aimed at investigating the CD4+RORγt+ T-helper cells and their counterpart, the CD4+CD25+FOXP3+ regulatory T cells in the liver, subcutaneous adipose tissue (SAT), and abdominal adipose tissue (AAT) in a high fat diet (HFD) mouse model.Methods. C57BL6 mice were fed a HFD or a normal diet (ND). Liver enzymes, metabolic parameters, and liver histology were assessed. The expression of CD4+RORγt+ cells and regulatory T cells in different organs (blood, liver, AAT, and SAT) were analyzed by flow cytometry. Cytokine and adipokine tissue expression were studied by RT-PCR.Results. Mice fed a HFD developed NASH and metabolic alterations compared to normal diet. CD4+RORγt++ cells were significantly increased in the liver and the AAT while an increase of regulatory T cells was observed in the SAT of mice fed HFD compared to ND. Inflammatory cytokines were also upregulated.Conclusions. CD4+RORγt++ cells and regulatory T cells are altered in NASH with a site-specific pattern and correlate with the severity of the disease. These site-specific differences are associated with increased cytokine expression.

The neuregulin-1 (NRG-1)/receptor tyrosine-protein kinase erbB (ErbB) system is an endothelium-controlled paracrine system modulating cardiac performance and adaptation. Recent studies have indicated that NRG-1 has antifibrotic effects in the left ventricle, which were explained by direct actions on cardiac fibroblasts. However, the NRG-1/ErbB system also regulates the function of macrophages. In this study, we hypothesized that the antifibrotic effect of NRG-1 in the heart is at least partially mediated through inhibitory effects on macrophages. We also hypothesized that the antifibrotic effect of NRG-1 may be active in other organs, such as the skin and lung. First, in a mouse model of angiotensin II (ANG II)-induced myocardial hypertrophy and fibrosis, NRG-1 treatment (20 µg·kg−1·day−1 ip) significantly attenuated myocardial hypertrophy and fibrosis and improved passive ventricular stiffness (4 wk). Interestingly, 1 wk after exposure to ANG II, NRG-1 already attenuated myocardial macrophage infiltration and cytokine expression. Furthermore, mice with myeloid-specific deletion of the ErbB4 gene ( ErbB4F/FLysM-Cre+/−) showed an intensified myocardial fibrotic response to ANG II. Consistently, NRG-1 activated the ErbB4 receptor in isolated macrophages, inhibited phosphatidylinositide 3-kinase/Akt and STAT3 signaling pathways, and reduced the release of inflammatory cytokines. Further experiments showed that the antifibrotic and anti-inflammatory effects of NRG-1 were reproducible in mouse models of bleomycin-induced dermal and pulmonary fibrosis. Overall, this study demonstrates that the antifibrotic effect of NRG-1 in the heart is linked to anti-inflammatory activity NRG-1/ErbB4 signaling in macrophages. Second, this study shows that NRG-1 has antifibrotic and anti-inflammatory effects in organs other than the heart, such as the skin and lung. NEW & NOTEWORTHY Our study contributes to the understanding of the antifibrotic effect of neuregulin-1 during myocardial remodeling. Here, we show that the antifibrotic effect of neuregulin-1 is at least partially mediated through anti-inflammatory activity, linked to receptor tyrosine-protein kinase erbB-4 activation in macrophages. Furthermore, we show that this effect is also present outside the heart. Listen to this article’s corresponding podcast at http://ajpheart.podbean.com/e/nrg-1-inhibits-macrophage-activation-during-tissue-fibrosis/ .

Our study underscores the importance of elastin fragmentation in the vessel wall as an accelerator of atherosclerosis with enhanced inflammation and increased neovascularization, thereby promoting the development of unstable plaques that eventually may rupture. The present mouse model offers the opportunity to further investigate the role of key factors involved in plaque destabilization and potential targets for therapeutic interventions., Aims There is a need for animal models of plaque rupture. We previously reported that elastin fragmentation, due to a mutation (C1039G+/−) in the fibrillin-1 (Fbn1) gene, promotes atherogenesis and a highly unstable plaque phenotype in apolipoprotein E deficient (ApoE−/−) mice on a Western-type diet (WD). Here, we investigated whether plaque rupture occurred in ApoE−/−Fbn1C1039G+/− mice and was associated with myocardial infarction, stroke, and sudden death. Methods and results Female ApoE−/−Fbn1C1039G+/− and ApoE−/− mice were fed a WD for up to 35 weeks. Compared to ApoE−/− mice, plaques of ApoE−/−Fbn1C1039G+/− mice showed a threefold increase in necrotic core size, augmented T-cell infiltration, a decreased collagen I content (70 ± 10%), extensive neovascularization, intraplaque haemorrhage, and a significant increase in matrix metalloproteinase-2, -9, -12, and -13 expression or activity. Plaque rupture was observed in 70% of ascending aortas and in 50% of brachiocephalic arteries of ApoE−/−Fbn1C1039G+/− mice. In ApoE−/− mice, plaque rupture was not seen in ascending aortas and only in 10% of brachiocephalic arteries. Seventy percent of ApoE−/−Fbn1C1039G+/− mice died suddenly, whereas all ApoE−/− mice survived. ApoE−/−Fbn1C1039G+/− mice showed coronary plaques and myocardial infarction (75% of mice). Furthermore, they displayed head tilt, disorientation, and motor disturbances (66% of cases), disturbed cerebral blood flow (73% of cases; MR angiograms) and brain hypoxia (64% of cases), indicative of stroke. Conclusions Elastin fragmentation plays a key role in plaque destabilization and rupture. ApoE−/−Fbn1C1039G+/− mice represent a unique model of acute plaque rupture with human-like complications.

Cyclic stretch is known to alter intracellular pathways involved in vessel tone regulation. We developed a novel set-up that allows straightforward characterization of the biomechanical properties of the mouse aorta while stretched at a physiological heart rate (600 beats minCyclic stretch is a major contributor to vascular function. However, isolated mouse aortas are frequently studied at low stretch frequency or even in isometric conditions. Pacing experiments in rodents and humans show that arterial compliance is stretch frequency dependent. The Rodent Oscillatory Tension Set-up to study Arterial Compliance is an in-house developed organ bath set-up that clamps aortic segments to imposed preloads at physiological rates up to 600 beats min

Atherosclerosis remains the leading cause of death and disability in our Western society. To investigate whether the dynamics of leukocyte (sub)populations could be predictive for plaque inflammation during atherosclerosis, we analyzed innate and adaptive immune cell distributions in blood, plaques, and lymphoid tissue reservoirs in apolipoprotein E-deficient (ApoE−/−) mice and in blood and plaques from patients undergoing endarterectomy. Firstly, there was predominance of the CD11b+conventional dendritic cell (cDC) subset in the plaque. Secondly, a strong inverse correlation was observed between CD11b+cDC or natural killer T (NKT) cells in blood and markers of inflammation in the plaque (including CD3, T-bet, CCR5, and CCR7). This indicates that circulating CD11b+cDC and NKT cells show great potential to reflect the inflammatory status in the atherosclerotic plaque. Our results suggest that distinct changes in inflammatory cell dynamics may carry biomarker potential reflecting atherosclerotic lesion progression. This not only is crucial for a better understanding of the immunopathogenesis but also bares therapeutic potential, since immune cell-based therapies are emerging as a promising novel strategy in the battle against atherosclerosis and its associated comorbidities. The cDC-NKT cell interaction in atherosclerosis serves as a good candidate for future investigations.

Arterial hypertension (AHT) affects the voltage dependency of L-type Ca(2+) channels in cardiomyocytes. We analyzed the effect of angiotensin II (AngII)-induced AHT on L-type Ca(2+) channel-mediated isometric contractions in conduit arteries. AHT was induced in C57Bl6 mice with AngII-filled osmotic mini-pumps (4 weeks). Normotensive mice treated with saline-filled osmotic mini-pumps were used for comparison. Voltage-dependent contractions mediated by L-type Ca(2+) channels were studied in vaso-reactive studies in vitro in isolated aortic and femoral arteries by using extracellular K(+) concentration-response (KDR) experiments. In aortic segments, AngII-induced AHT significantly sensitized isometric contractions induced by elevated extracellular K(+) and depolarization. This sensitization was partly prevented by normalizing blood pressure with hydralazine, suggesting that it was caused by AHT rather than by direct AngII effects on aortic smooth muscle cells. The EC50 for extracellular K(+) obtained in vitro correlated significantly with the rise in arterial blood pressure induced by AngII in vivo. The AHT-induced sensitization persisted when aortic segments were exposed to levcromakalim or to inhibitors of basal nitric oxide release. Consistent with these observations, AngII-treatment also sensitized the vaso-relaxing effects of the L-type Ca(2+) channel blocker diltiazem during K(+)-induced contractions. Unlike aorta, AngII-treatment desensitized the isometric contractions to depolarization in femoral arteries pointing to vascular bed specific responses of arteries to hypertension. AHT affects the voltage-dependent L-type Ca(2+) channel-mediated contraction of conduit arteries. This effect may contribute to the decreased vascular compliance in AHT and explain the efficacy of Ca(2+) channel blockers to reduce vascular stiffness and central blood pressure in AHT.

The in vivo cutaneous nerve regeneration model using capsaicin is applied extensively to study the regenerative mechanisms and therapeutic efficacy of disease modifying mole- cules for small fiber neuropathy (SFN). Since mismatches between functional and morpho- logical nerve fiber recovery are described for this model, we aimed at determining the capability of the capsaicin model to truly mimic the morphological manifestations of SFN in diabetes. As nerve and blood vessel growth and regenerative capacities are defective in diabetes, we focused on studying the key regulator of these processes, the neuropilin-1 (NRP-1)/semaphorin pathway. This led us to the evaluation of NRP-1 receptor expression in epidermis and dermis of subjects presenting experimentally induced small fiber neuropa- thy, diabetic polyneuropathy and of diabetic subjects without clinical signs of small fiber neuropathy. The NRP-1 receptor was co-stained with CD31 vessel-marker using immuno- fluorescence and analyzed with Definiens1 technology. This study indicates that capsaicin application results in significant loss of epidermal NRP-1 receptor expression, whereas dia- betic subjects presenting small fiber neuropathy show full epidermal NRP-1 expression in contrast to the basal expression pattern seen in healthy controls. Capsaicin induced a decrease in dermal non-vascular NRP-1 receptor expression which did not appear in dia- betic polyneuropathy. We can conclude that the capsaicin model does not mimic diabetic neuropathy related changes for cutaneous NRP-1 receptor expression. In addition, our data suggest that NRP-1 might play an important role in epidermal nerve fiber loss and/or defec- tive regeneration and that NRP-1 receptor could change the epidermal environment to a nerve fiber repellant bed possibly through Sem3A in diabetes.

Bioavailability of nitric oxide (NO) is mostly studied in isolated blood vessels. We investigated changes in basal and receptor-stimulated endothelial NO synthase (eNOS) activity after isolation of wild-type and Marfan mouse aorta. Starting 1h after dissection, basal NO release was assessed at hourly intervals by its ability to suppress isometric contractions in aortic segments. Relaxation induced by acetylcholine or α(2)-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) was used to study stimulated NOS activity. One hour after dissection, phenylephrine- or prostaglandin F(2α)-induced force attained only 17 ± 4% or 31 ± 7% of maximum tension in the presence of N(Ω)-nitro-l-arginine-methylesther (l-NAME), and contractions increased to 63 ± 6% and 82 ± 11%, respectively at 5h. In contrast, acetylcholine or UK14304 relaxation curves changed minimally. l-NAME and eNOS-deficiency abolished basal NO production, unlike inhibitors of neuronal (N(Ω)-propyl-l-arginine) or inducible (1400W) NOS. Acetylcholine-induced relaxation was abolished by l-NAME, strongly suppressed by eNOS-deficiency and attenuated by N(Ω)-propyl-l-arginine. In a bioassay based on diethylamine NONOate concentration-response curves the suppression of contractile forces was interpolated into NO equivalents. This showed exponential decay of basal NO, which occurred three times faster in aortas from mice with Marfan syndrome, while acetylcholine-induced relaxation remained unaltered. Immunoblotting showed unchanged eNOS expression, or phosphorylation at Ser1177, Ser617 or Thr495 between 1h and 4h, but Akt phosphorylation declined gradually. The dramatic loss of basal NO release after tissue isolation shows that timing is crucial when studying NO responses. The preservation of receptor-induced relaxation implies differential regulation of basal and stimulated eNOS activity, and phosphoinositide-3-kinase/Akt signalling seems specifically associated with basal eNOS activity.

Evidence is accumulating that autophagy occurs in advanced atherosclerotic plaques. Although there is an almost relentless discovery of molecules that are involved in autophagy, studies of selective autophagy induction or inhibition using knockout mice are just now beginning to reveal its biological significance. Most likely, autophagy safeguards plaque cells against cellular distress, in particular oxidative injury, by degrading the damaged intracellular material. In this way, autophagy is protective and contributes to cellular recovery in an unfavorable environment. Pharmacological approaches have recently been developed to stabilize vulnerable, rupture-prone lesions through induction of autophagy. This approach has proven to be successful in short-term studies. However, how autophagy induction affects processes such as inflammation remains to be elucidated and is currently under investigation. This review highlights the possibilities for exploiting autophagy as a drug target for plaque stabilization.

Despite tremendous advances over the last 15 years in identifying vulnerable atherosclerotic plaques, the incidence of death and disability caused by such lesions still remains the number one health threat in developed countries. Therefore, new systemic or focal therapies aimed at decreasing the overall burden of disease, and a change to a more benign phenotype, are needed. Because cell death is a prominent feature of advanced atherosclerotic plaques with a major impact on plaque destabilization, an increasing number of compounds targeting the apoptotic or autophagic machinery in atherosclerosis are being explored, predominantly at the preclinical level. This review will provide an overview of these compounds, with a focus on both inhibition and stimulation of cell death, to prevent acute coronary syndromes and sudden cardiac death.

BACKGROUND AND PURPOSE Lithium chloride (LiCl) inhibits inositol monophosphatase (IMPase) at therapeutic concentrations. Given that LiCl induces death in cultured macrophages and that macrophages play an active role in atherosclerotic plaque destabilization, we investigated whether LiCl would induce selective macrophage death to stabilize the structure of the plaque. EXPERIMENTAL APPROACH The effect of LiCl was assessed on macrophages and smooth muscle cells (SMCs) in culture, in isolated atherosclerotic carotid arteries from rabbits and after local in vivo treatment via osmotic minipumps to rabbits with collared atherosclerotic carotid arteries. In addition, in vitro experiments were performed to elucidate the mechanism of LiCl-induced macrophage death. KEY RESULTS In vitro, whereas SMCs were highly resistant, LiCl induced macrophage death characterized by externalization of phosphatidylserine, caspase-3 cleavage and DNA fragmentation, all indicative of apoptosis. LiCl reduced inositol-1,4,5-trisphosphate levels in macrophages. Moreover, the IMPase inhibitor L-690 330 as well as IMPase gene silencing induced macrophage apoptosis. Both in vitro treatment of rabbit atherosclerotic carotid arteries with LiCl and local in vivo administration of LiCl to the plaques decreased plaque macrophages through apoptosis, as shown by terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL), without affecting SMCs. Vasomotor studies in vitro showed that LiCl did not affect the functionality of SMCs and endothelial cells. CONCLUSIONS AND IMPLICATIONS LiCl selectively decreased the macrophage load in rabbit atherosclerotic plaques via IMPase inhibition without affecting the viability or functionality of SMCs and endothelial cells. These data provide evidence for local administration of an IMPase inhibitor to stabilize atherosclerotic plaques.

Aim: Transglutaminase 2 (TG2) is important for the deposition and stability of the extracellular matrix via effects on cross-linking of matrix proteins and transforming growth factor β (TGFβ) activity. The purpose of this study was to investigate the effect of TG2 deficiency on the composi- tion of atherosclerotic plaques. Methods: Apolipoprotein E (ApoE)–/– mice were crossbred with TG2–/– mice to obtain ApoE–/–TG2–/– mice. ApoE–/– and ApoE–/–TG2–/– mice were fed a Western-type diet for 16 or 30 weeks to determine the effect of TG2 deficiency on early and advanced atherosclerosis, respectively. Results: Atherosclerotic plaques of ApoE–/–TG2–/– mice showed decreased cross-linking of matrix proteins, as well as decreased nuclear staining for phospho-Smad2/-Smad3, indicative of decreased TGFβ activity. Compared to ApoE–/– mice, plaque area was decreased by 45 and 48% in ApoE–/–TG2–/– mice after 16 and 30 weeks, respectively. Sirius red staining showed a significant decrease in collagen content in early and advanced atherosclerotic plaques of ApoE–/–TG2–/– mice. Furthermore, there was a significant increase in macrophages in advanced atherosclerotic plaques of ApoE–/–TG2–/– mice. Conclusion: TG2 deficiency resulted in a decreased collagen content and increased inflammation, which are features of a more unstable plaque.

Background and purpose: 7-Ketocholesterol, an oxysterol present in atherosclerotic lesions, induces smooth muscle cell (SMC) death, thereby destabilizing plaques. Statins protect patients from myocardial infarction, though they induce SMC apoptosis. We investigated whether statins and 7-ketocholesterol exerted additive cell death effects. Experimental approach: Cultured rabbit aorta SMCs (passage 2–6) were exposed to 7-ketocholesterol with or without fluvastatin, simvastatin or pravastatin. Uptake of neutral red (NR), monolayer protein, cleavage of the pan-caspase substrate Asp-Glu-Val-Asp-rhodamine110, cell morphology (light and electron microscopy) and processing of microtubule-associated protein 1 light chain 3 (LC3, immunoblot) were determined. Key results: NR uptake declined upon 18 h exposure to 25 μM 7-ketocholesterol (−41±3%, n=13), 100 μM fluvastatin (−59%) or 30–100 μM simvastatin (−28 to −74%). Oxysterol and high statin concentrations exerted additive effects, but lower concentrations (fluvastatin 10–30 μM, simvastatin 1–10 μM) partly reversed viability loss. 7-Ketocholesterol caused intense cytoplasmic vacuolization, processing of LC3-I to LC3-II, but little caspase activation (increase 29.5%). Fluvastatin (10–100 μM, 70–545% increase) and simvastatin (3–100 μM 43–322% increase) induced caspase activation without LC3 processing, but failed to activate caspases in 7-ketocholesterol-treated SMCs. Pravastatin up to 100 μM was always inactive. Conclusions and implications: 7-Ketocholesterol caused SMC death, mainly via autophagic vesicle formation with LC3 processing, whereas lipophilic statins evoked SMC apoptosis. Cell death following 7-ketocholesterol and low statin concentrations were not additive, presumably because the autophagic process interfered with statin-induced caspase activation. This further illustrates that drug effects in normal SMCs are not necessarily predictive for activities in atherosclerotic settings. British Journal of Pharmacology (2008) 154, 1236–1246; doi:10.1038/bjp.2008.181; published online 12 May 2008

The aim of the study was to determine the maximum tolerated dose (MTD), the dose limiting toxicity (DLT), and the pharmacokinetic profile (Pk) of bendamustine (BM) on a day 1 and 2 every 3 weeks schedule and to recommend a safe phase II dose for further testing. Patients with solid tumours beyond standard therapy were eligible. A 30-min intravenous infusion of BM was administered d1+d2 q 3 weeks. The starting dose was 120 mg m(-2) per day and dose increments of 20 mg m(-2) were used. Plasma and urine samples were analysed using validated high-performance liquid chromatography/fluorescence assays. Fifteen patients were enrolled. They received a median of two cycles (range 1-8). The MTD was reached at the fourth dose level. Thrombocytopaenia (grade 4) was dose limiting in two of three patients at 180 mg m(-2). One patient also experienced febrile neutropaenia. Lymphocytopaenia (grade 4) was present in every patient. Nonhaematologic toxicity including cardiac toxicity was not dose limiting with this schedule. Mean plasma Pk values of BM were tmax 35 min, t(1/2) 49.1 min, Vd 18.3 l m(-2), and clearance 265 ml min(-1) m(-2). The mean total amount of BM and its metabolites recovered in the first micturition was 8.3% (range 2.7-26%). The MTD of BM in the present dose schedule was 180 mg m(-2) on day 1+2. Thrombocytopaenia was dose limiting. The recommended dose for future phase II trials with this schedule is 160 mg m(-2) per day.

In the last decades, the search for mechanisms underlying progressive arterial stiffening and for interventions to avoid or reverse this process has gained much attention. In general, arterial stiffening displays regional variation and is, for example, during aging more prominent in elastic than in muscular arteries. We hypothesize that besides passive also active regulators of arterial compliance [i.e., endothelial and vascular smooth muscle cell (VSMC) function] differ between these arteries. Hence, it is conceivable that these vessel types will display different time frames of stiffening. To investigate this hypothesis segments of muscular arteries such as femoral and mesenteric arteries and elastic arteries such as the aorta and carotid artery were isolated from female C57BI6 mice (5-6 months of age, n = 8). Both microscopy and passive stretching of the segments in a myograph confirmed that passive mechanical properties (elastin, collagen) of elastic and muscular arteries were significantly different. Endothelial function, more specifically basal nitric oxide (NO) efficacy, and VSMC function, more specifically L-type voltage-gated Ca2+ channel (VGCC)-mediated contractions, were determined by alpha(1)-adrenoceptor stimulation with phenylephrine (PE) and by gradual depolarization with elevated extracellular K+ in the absence and presence of eNOS inhibition with L-NAME. PE mediated isometric contractions significantly increased after inhibition of NO release with L-NAME in elastic, but not in muscular vessel segments. This high basal eNOS activity in elastic vessels was also responsible for shifts of K+ concentration-contraction curves to higher external K+. VGCC-mediated contractions were similarly affected by depolarization with elevated K+ in muscular artery segments or in elastic artery segments in the absence of basal NO. However, K+-induced contractions were inhibited by the VGCC blocker diltiazem with significantly higher sensitivity in the muscular arteries, suggestive of different populations of VGCC isoforms in both vessel types. The results from the present study demonstrate that, besides passive arterial wall components, also active functional components contribute to the heterogeneity of arterial compliance along the vascular tree. This crucially facilitates the search for (patho) physiological mechanisms and potential therapeutic targets to treat or reverse large artery stiffening as occurring in aging-induced arterial stiffening.

Background: During abdominal sepsis, the inhibition of gastrointestinal (GI) motility together with mucosal barrier dysfunction will lead to increased bacterial translocation and maintenance of sepsis. The activation of the vagal anti-inflammatory pathway remains an appealing therapeutic strategy in sepsis. In this respect, selective alpha7 nicotinic acetylcholine receptor ([alpha]7nAChR) agonists have shown anti-inflammatory properties in several animal models of inflammation. Methods: Sepsis was induced in OF-1 mice by caecal ligation and puncture (CLP). GI transit was quantified, and cytokine levels were determined in serum and colon. Colonic permeability was assessed by means of Evans blue injection. We studied the effect of GTS-21, an [alpha]7nAChR agonist on the aforementioned parameters. Splenectomized animals as well as [alpha]7nAChR-knock-out animals (Chrna7-/-) were included to study the role of splenic macrophages and the [alpha]7nAChR during polymicrobial abdominal sepsis. Results: In septic animals, GTS-21 significantly ameliorated GI motility, lowered systemic and colonic levels of IL-6, decreased colonic permeability and decreased the number of positive cultures obtained from blood and mesenteric lymph nodes. Splenectomy prevented animals from developing sepsis-induced ileus. Chrna7-/- mice displayed a more severe septic phenotype, whereas GTS-21 remarkably was also beneficial in these animals. Conclusion: Our results show that peripheral targeting of the vagal anti-inflammatory pathway proves beneficial in an animal model of polymicrobial abdominal sepsis. A major role is allocated to splenic immune cells in the development of sepsis, as preventive splenectomy was protective for the development of sepsis. Data on the Chrna7-/- mice suggest that the beneficial effects mediated by GTS-21 on inflammation and motility might be related to activation of other receptors besides the [alpha]7nAChR.

Background Mobilization of the innate immune response to clear and metabolize necrotic and apoptotic cardiomyocytes is a prerequisite to heart repair after cardiac injury. Suboptimal kinetics of dying myocyte clearance leads to secondary necrosis, and in the case of the heart, increased potential for collateral loss of neighboring non-regenerative myocytes. Despite the importance of myocyte phagocytic clearance during heart repair, surprisingly little is known about its underlying cell and molecular biology. Objective To determine if phagocytic receptor MERTK is expressed in human hearts and to elucidate key sequential steps and phagocytosis efficiency of dying adult cardiomyocytes, by macrophages. Results In infarcted human hearts, expression profiles of the phagocytic receptor MER-tyrosine kinase (MERTK) mimicked that found in experimental ischemic mouse hearts. Electron micrographs of myocardium identified MERTK signal along macrophage phagocytic cups and Mertk −/− macrophages contained reduced digested myocyte debris after myocardial infarction. Ex vivo co-culture of primary macrophages and adult cardiomyocyte apoptotic bodies revealed reduced engulfment relative to resident cardiac fibroblasts. Inefficient clearance was not due to the larger size of myocyte apoptotic bodies, nor were other key steps preceding the formation of phagocytic synapses significantly affected; this included macrophage chemotaxis and direct binding of phagocytes to myocytes. Instead, suppressed phagocytosis was directly associated with myocyte-induced inactivation of MERTK, which was partially rescued by genetic deletion of a MERTK proteolytic susceptibility site. Conclusion Utilizing an ex vivo co-cultivation approach to model key cellular and molecular events found in vivo during infarction, cardiomyocyte phagocytosis was found to be inefficient, in part due to myocyte-induced shedding of macrophage MERTK. These findings warrant future studies to identify other cofactors of macrophage–cardiomyocyte cross-talk that contribute to cardiac pathophysiology.

Because cholesterol-independent effects of statins are difficult to determine in patients, we studied these pleiotropic effects in apolipoprotein E-deficient (ApoE−/−) mice with a mutation in the fibrillin-1 gene (Fbn1C1039G +/−). These mice develop exacerbated atherosclerosis and spontaneous plaque ruptures, accompanied by myocardial infarctions (MI) and sudden death. ApoE−/− Fbn1C1039G +/− mice were fed a Western diet (WD). At week 10 of WD, mice were divided in a control (WD), atorvastatin (10 mg/kg/day + WD) and cholesterol withdrawal group (cholW, normal chow). The latter was included to compare the effects of atorvastatin with dietary lipid lowering. Fifteen weeks later, the mice were sacrificed. CholW, but not atorvastatin, reduced plasma cholesterol. Survival increased from 50% to 90% both in cholW and atorvastatin treated mice. CholW as well as atorvastatin treatment increased plaque collagen and fibrous cap thickness, but they did not affect the amount of plaque macrophages and T cells. MMP-2 and MMP-9 activity was significantly lower and the expression of MMP-12, TNF-α and IL-1β was strongly reduced in both treatment groups. Blood monocytes and neutrophils returned to baseline levels (ApoE−/− mice before the onset of atherosclerosis). Importantly, atorvastatin but not cholW significantly reduced coronary stenosis (from 50 to 28%) and the occurrence of MI (from 43 to 10%). In conclusion, independent of cholesterol lowering, atorvastatin significantly reduced mortality, plaque vulnerability and inflammation to the same extent as cholW. In addition, atorvastatin but not cholW reduced coronary stenosis and the occurrence of MI. These data unequivocally illustrate the significance of the pleiotropic effects of atorvastatin in the prevention of cardiovascular morbidity and mortality.

Different immune cell types are present within atherosclerotic plaques. Dendritic cells (DC) are of special interest, since they are considered as the 'center of the immuniverse'. Identifying inflammatory DC subtypes within plaques is important for a better understanding of the lesion pathogenesis and pinpoints their contribution to the atherosclerotic process. We have developed a flow cytometry-based method to characterize and isolate different DC subsets (i.e. CD11b(+), Clec9A(+) and CD16(+) conventional (c)DC and CD123(+) plasmacytoid (p)DC) in human atherosclerotic plaques. We revealed a predominance of pro-inflammatory CD11b(+) DC in advanced human lesions, whereas atheroprotective Clec9A(+) DC were almost absent. CD123(+) pDC and CD16(+) DC were also detectable in plaques. Remarkably, plaques from distinct anatomical locations exhibited different cellular compositions: femoral plaques contained less CD11b(+) and Clec9A(+) DC than carotid plaques. Twice as many monocytes/macrophages were observed compared to DC. Moreover, relative amounts of T cells/B cells/NK cells were 6 times as high as DC numbers. For the first time, fluorescent activated cell sorting analysis of DC subsets in human plaques indicated a predominance of CD11b(+) cDC, in comparison with other DC subsets. Isolation of the different subsets will facilitate detailed functional analysis and may have significant implications for tailoring appropriate therapy.

L-type Ca2+ channel (VGCC) mediated Ca2+ influx in vascular smooth muscle cells (VSMC) contributes to the functional properties of large arteries in arterial stiffening and central blood pressure regulation. How this influx relates to steady-state contractions elicited by α1-adrenoreceptor stimulation and how it is modulated by small variations in resting membrane potential (Vm) of VSMC is not clear yet. Here, we show that α1-adrenoreceptor stimulation of aortic segments of C57Bl6 mice with phenylephrine (PE) causes phasic and tonic contractions. By studying the relationship between Ca2+ mobilisation and isometric tension, it was found that the phasic contraction was due to intracellular Ca2+ release and the tonic contraction determined by Ca2+ influx. The latter component involves both Ca2+ influx via VGCC and via non-selective cation channels (NSCC). Influx via VGCC occurs only within the window voltage range of the channel. Modulation of this window Ca2+ influx by small variations of the VSMC Vm causes substantial effects on the contractile performance of aortic segments. The relative contribution of VGCC and NSCC to the contraction by α1-adrenoceptor stimulation could be manipulated by increasing intracellular Ca2+ release from non-contractile sarcoplasmic reticulum Ca2+ stores. Results of this study point to a complex interactions between α1-adrenoceptor-mediated VSMC contractile performance and Ca2+ release form contractile or non-contractile Ca2+ stores with concomitant Ca2+ influx. Given the importance of VGCC and their blockers in arterial stiffening and hypertension, they further point toward an additional role of NSCC (and NSCC blockers) herein.

Vulnerable atherosclerotic plaques are prone to plaque rupture leading to acute cardiovascular syndromes and death. Elucidating the risk of plaque rupture is important to define better therapeutic or preventive strategies. In the present study, we investigated the effect of chronic intermittent mental stress on atherosclerotic plaque stability and cardiovascular mortality in apolipoprotein E-deficient (ApoE(-/-)) mice with a heterozygous mutation in the fibrillin-1 gene (Fbn1(C1039G+/)(-)). This mouse model displays exacerbated atherosclerosis with spontaneous plaque ruptures, myocardial infarction and sudden death, when fed a Western-type diet (WD). Female ApoE(-/-)Fbn1(C1039G+/-) mice were fed a WD for up to 25 weeks. After 10 weeks WD, mice were divided in a control (n = 27) and mental stress (n = 29) group. The chronic intermittent mental stress protocol consisted of 3 triggers: water avoidance, damp bedding and restraint stress, in a randomly assigned order lasting 6 h every weekday for 15 weeks. Chronic intermittent mental stress resulted in a significant increase in the amount of macrophages in atherosclerotic plaques of the proximal ascending aorta, whereas type I collagen and fibrous cap thickness were decreased. The coronary arteries of mental stress-treated mice showed larger plaques, more stenosis, and an increased degree of perivascular fibrosis. Moreover, myocardial infarctions occurred more frequently in the mental stress group. As compared to the control group, the survival of stressed ApoE(-/-)Fbn1(C1039G+/-) mice decreased from 67% to 52% at 25 weeks WD, presumably due to myocardial infarctions. In conclusion, chronic intermittent mental stress promotes plaque instability, myocardial infarctions, and mortality of ApoE(-/-)Fbn1(C1039G+/-) mice.

Dipeptidyl peptidase (DPP) II (E.C. 3.4.14.2) is an intracellular protease that releases, preferably at acidic pH, N-terminal dipeptides from oligopeptides with Pro or Ala in the penultimate position. The natural substrates and the physiological role of DPPII remain unclear. The aim of the present study was to investigate the involvement of DPPII activity in different forms of cell death (apoptosis, necrosis and autophagy) in human leukocytes. We determined specific DPP activities in leukocytes. Compared to other subpopulations of peripheral blood mononuclear cells (PBMC), we observed relatively high DPPII specific activity in monocytic cells, opening new perspectives for further investigation of the DPPII functions. A second intriguing finding was that DPPII specific activity increased during necrosis, whereas induction of apoptosis or autophagy did not affect any of the dipeptidyl peptidase activities. Finally, we showed that inhibition of DPPII (>90%) using the in vitro applicable, highly potent ( K i of 0.082 ± 0.048 nM) and selective DPPII inhibitor UAMC00039, did not induce any form of cell death in leukocytes. These data are of importance for a more precise interpretation of the in vitro and in vivo effects of other dipeptidyl peptidase inhibitors.

Efficient phagocytosis of cells undergoing apoptosis by macrophages is important to prevent immunological responses and development of chronic inflammatory disorders such as systemic lupus erythematosus, cystic fibrosis and atherosclerosis. To study phagocytosis of apoptotic cells (AC) by macrophages in tissue, we validated different apoptosis markers (DNA fragmentation, caspase-3 activation and cleavage of its substrate poly(ADP-ribose)polymerase-1) in combination with macrophage immunostaining. Human tonsils were used as a model because they show a high apoptosis frequency under physiological conditions as well as efficient phagocytosis of AC by macrophages. On the other hand, advanced human atherosclerotic plaques were examined since plaques show severely impaired phagocytosis of AC. Our results demonstrate that the presence of non-phagocytized terminal deoxynucleotidyl transferase end labelling (TUNEL)-positive AC represents a suitable marker of poor phagocytosis by macrophages in situ. Other markers for apoptosis, such as cleavage of caspase-3 or PARP-1, should not be used to assess phagocytosis efficiency, because activation of the caspase cascade and cleavage of their substrates can occur in AC when they have not yet been phagocytized by macrophages.

Previous studies demonstrated that macrophages are difficult to transfect. In the present study, we investigated whether J774A.1 macrophages can be efficiently transfected using nucleofector technology. Nucleofection of J774A.1 macrophages with mRNA resulted in transfection efficiencies up to 75% without cell death as compared to control pulsed macrophages. In contrast, introduction of DNA into J774A.1 cells caused apoptosis without expression of the gene of interest. Our results show that mRNA nucleofection is a new high-speed transfection method for macrophages.

Despite some progress in the field of gene transfer into hard-to-transfect cells, so far an efficient nonviral method for monocytes has not been available. A comparison of plasmid DNA with capped and polyadenylated mRNA for enhanced green fluorescent protein gene delivery into the commonly used monocytic cell lines U937 and THP-1 suggested that limited DNA trafficking may be the underlying cause of poor transfection results. As Nucleofector technology delivers DNA (or mRNA) straight into the nucleus, we obtained nucleofection efficiencies of up to 80% without significant cell toxicity. Moreover, as the DNA quickly reaches the nucleus, nucleofected cells were ready for analysis after only 2-6 h. The technique is suitable not only for monocytes but also for other hard-to-transfect cells.

3028 Background: Currently, programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) assays received approval in combination with an anti-PD-1 or anti-PD-L1 compounds. However, the FDA blueprint and other publications revealed differences in staining pattern between PD-L1 IHC assays. More precisely the SP142 assay detects less tumor cells (TC), but more immune cells (IC), while the 28-8 assay is more sensitive for TC and less appropriate for IC detection. SP263 stains TC and IC equally well. E1L3N IHC reveals IC and slightly more TC staining than SP142. This study investigates whether these staining discrepancies can be partly explained by specific epitope recognition. Methods: Linear epitope mapping was performed for PD-L1 antibody clones 28-8, E1L3N, SP142 and SP263. In brief, the PD-L1 sequence (Q9NZQ7, Uniprot) was split into 15 amino acid (AA) peptides with a peptide overlap of 14 AA . Each peptide was printed in duplicate on the PD-L1 microarray. The microarray was exposed to different concentration of the four PD-L1 antibodies. For the detection, sheep anti-rabbit IgG DyLight800 was used. Results: Epitope mapping for the E1L3N antibody revealed a linear epitope in the intracellular domain. The other clones showed weaker binding to multiple peptides. The 28.8 clone demonstrated binding to predominantly intracellular epitopes, while SP142 and SP263 clones showed binding to both intracellularly and extracellularly located epitopes. Blasting the epitope sequences of the PD-L1 antibodies did not disclose identity with another (non-PD-L1) human protein. Conclusions: Different binding characteristics were found for all four PD-L1 antibody clones in a linear epitope mapping experiment. This could lead to particular staining patterns depending on PD-L1 conformation (folding) or isoform expression. Two PD-L1 isoforms are known, with isoform 2 lacking AA 19-132 of the extracellular domain. Especially SP142 binding can be impacted in the case of dominant isoform 2 presence, since epitopes were observed in this spliced domain. Further investigation is needed into the potentially conformational epitopes of SP142, SP263 and 28.8 antibody clones as well as in the PD-L1 conformation and isoform expression in TC and IC.

α1-Adrenoceptor stimulation of mouse aorta causes intracellular Ca(2+) release from sarcoplasmic reticulum Ca(2+) stores via stimulation of inositoltriphosphate (IP3) receptors. It is hypothesized that this Ca(2+) release from the contractile and IP3-sensitive Ca(2+) store is under the continuous dynamic control of time-independent basal Ca(2+) influx via L-type voltage-gated Ca(2+) channels (LCC) residing in their window voltage range. Mouse aortic segments were α1-adrenoceptor stimulated with phenylephrine in the absence of external Ca(2+) (0Ca) to measure phasic isometric contractions. They gradually decreased with time in 0Ca, were inhibited with 2-aminoethoxydiphenyl borate, and declined with previous membrane potential hyperpolarization (levcromakalim) or with previous inhibition of LCC (diltiazem). Former basal stimulation of LCC with depolarization (15 mM K(+)) or with BAY K8644 increased the subsequent phasic contractions by phenylephrine in 0Ca. Although exogenous NO (diethylamine NONOate) reduced the phasic contractions by phenylephrine, stimulation of endothelial cells with acetylcholine in 0Ca failed to attenuate these phasic contractions. Finally, inhibition of the basal release of NO with N(Ω)-nitro-L-arginine methyl ester also attenuated the phasic contractions by phenylephrine. Results indicated that α1-adrenoceptor stimulation with phenylephrine causes phasic contractions, which are controlled by basal LCC and endothelial NO synthase activity. Endothelial NO release by acetylcholine was absent in 0Ca. Given the growing interest in the active regulation of arterial compliance, the dependence of contractile SR Ca(2+) store-refilling in basal conditions on the activity of LCC and basal eNOS may contribute to a more thorough understanding of physiological mechanisms leading to arterial stiffness.

Arterial stiffening is the root cause of a range of cardiovascular complications, including myocardial infarction, left ventricular hypertrophy, stroke, renal failure, dementia, and death, and a hallmark of the aging process. The most important in vivo parameter of arterial stiffness is pulse wave velocity (PWV). Clinically, PWV is determined noninvasively using applanation tonometry. Unlike the clinical value of arterial stiffness and PWV, techniques to determine PWV in mice are scarce. The only way to determine aortic PWV noninvasively in the mouse is by using ultrasound echo Doppler velocimetry. It is a fast, efficient, and accurate technique, but the required tools are expensive and technically complex. Here, we describe the development and validation of a novel technique to assess carotid–femoral PWV noninvasively in mice. This technique is based on applanation tonometry as used clinically. We were able to establish a reproducible reference value in wild-type mice (3.96±0.05 m/s) and to detect altered carotid–femoral PWV values in endothelial nitric oxide synthase knockout mice (4.66±0.05 m/s; P P

Underlying the aging process is a lifelong accumulation of molecular damage. When DNA damage is too extensive to be repaired or when the repairing cascades are impaired, e.g., during chronic oxidative stress associated with aging, apoptosis occurs. A very low, albeit elevated, rate of apoptosis can be an important factor in the pathogenesis of heart failure, making it a potential target for therapy. Necrosis is even more prominent in failing human hearts than apoptosis. Autophagy is an essential and protective pathway in the heart. However, during aging, the rate of protective autophagy declines. The progressive inhibition of autophagy in the aging heart is in part attributed to intralysosomal accumulation of lipofuscin. Cross-linked polymeric lipofuscin cannot be degraded by lysosomal hydrolases and leads to preferential allocation of lysosomal enzymes to lipofuscin-loaded lysosomes at the expense of active autolysosomes. Impaired autophagy further stimulates accumulation of damaged mitochondria, which are deficient in ATP production and which produce large amounts of reactive oxygen species (ROS). Moreover, oxidatively modified cytosolic proteins form large indigestible aggregates, enhancing lipofuscinogenesis and sensitizing cardiomyocytes to undergo apoptosis/necrosis, eventually leading to heart failure. Pharmacological inhibition of apoptosis and necrosis improves heart function and survival. Currently used drugs in heart failure therapy, such as ACE-inhibitors and β-adrenergic receptor blockers, prevent apoptosis. However, complete inhibition of apoptosis might have adverse effects. Furthermore, compounds that supplement the decreased levels of autophagy during aging, such as AMPK activators, mTOR inhibitors, and/or sirtuin activators, might be of great value in heart failure therapy by preventing apoptosis and necrosis and stimulating cardiomyocyte survival. However, the dosing should be carefully set in order to avoid excessive autophagic cell death.

Autoimmune diseases affect about one in 15 individuals in developed countries and are characterized by a breakdown in immune tolerance. Current therapeutic approaches against destructive immune responses in autoimmune diseases are based on non-specific agents systemically suppressing the function of many immune effector cells. This indiscriminate immunosuppression, however, often causes serious and sometimes life-threatening side effects. Therefore, the need for more specific treatments resulting in lower toxicity and longer-term solutions is high. Because of the established role of dendritic cells (DCs) in maintaining the balance between immunity and tolerance, tolerogenic (tol)DCs might be novel therapeutic targets to prevent undesirable (auto-)immune responses. The idea behind tolDC therapy is that it is a highly targeted, antigen-specific treatment that only affects the auto-reactive inflammatory response. The therapeutic potential of tolDCs has already been proven in experimental animal models of different autoimmune disorders as well as with in vitro experiments using ex vivo generated human tolDCs, thus the challenge remains in bringing tolDC therapy to the clinic, although first clinical trials have been conducted. In this review, we will extensively discuss the use of tolDCs for induction of antigen-specific tolerance in several autoimmune disease settings, from bench to bedside, including currently applied strategies to generate tolDCs as well as technical difficulties and challenges in the field.

KW-2149 is a new, semisynthetic, C-7-N-Substituted, mitomycin C analog showing antitumor activity both in vitro and in vivo, equal or superior to mitomycin C. In a phase I study, KW-2149 was administered as an i.v. bolus injection every 21 days and at a dose of 100 mg/m2 pulmonary toxicity was dose limiting. Animal studies have indicated since that KW-2149-induced pulmonary toxicity can be prevented by pretreatment with corticosteroids. This paper presents the results of a further phase I study of KW-2149 with corticosteroid pretreatment. Patients were treated with oral dexamethasone 8 mg every 12 h, starting 24 h before KW-2149 administration, for 5 days. KW-2149 was given as an i.v. bolus injection every 21 days. Seventeen patients were treated with a total of 48 courses. Six patients received 60 mg/m2 and 11 patients 75 mg/m2. Two courses were not evaluable for toxicity. Significant lung toxicity was observed in at least three patients treated with a dose of 75 mg/m2 KW-2149 and pulmonary toxicity was therefore considered the dose-limiting toxicity at 75 mg/m2. No other important side effects were noted. One partial response was observed in a patient with colorectal cancer. Pretreatment with dexamethasone failed to suppress KW-2149-induced lung toxicity.

After oral or parenteral administration of chemotherapeutic agents, these drugs are transported to the tissues by the blood in different fractions: plasma water, plasma proteins or cells. In this review, the role of the red blood cell in storage, transport and metabolism of different anti-cancer drugs is described.