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Aim: NADPH oxidases are important sources of reactive oxygen species (ROS). Several Nox homologues are present together in the vascular system but whether they exhibit crosstalk at the activity level is unknown. To address this, vessel function of knockout mice for the cytosolic Nox organizer proteins p47phox, NoxO1 and a p47phox-NoxO1-double knockout were studied under normal condition and during streptozotocin-induced diabetes.
Results: In the mouse aorta, mRNA expression for NoxO1 was predominant in smooth muscle and endothelial cells, whereas p47phox was markedly expressed in adventitial cells comprising leukocytes and tissue resident macrophages. Knockout of either NoxO1 or p47phox resulted in lower basal blood pressure. Deletion of any of the two subunits also prevented diabetes-induced vascular dysfunction. mRNA expression analysis by MACE (Massive Analysis of cDNA ends) identified substantial gene expression differences between the mouse lines and in response to diabetes. Deletion of p47phox induced inflammatory activation with increased markers of myeloid cells and cytokine and chemokine induction. In contrast, deletion of NoxO1 resulted in an attenuated interferon gamma signature and reduced expression of genes related to antigen presentation. This aspect was also reflected by a reduced number of circulating lymphocytes in NoxO1-/- mice.
Innovation and conclusion: ROS production stimulated by NoxO1 and p47phox limit endothelium-dependent relaxation and maintain blood pressure in mice. However, NoxO1 and p47phox cannot substitute each other despite their similar effect on vascular function. Deletion of NoxO1 induced an anti-inflammatory phenotype, whereas p47phox deletion rather elicited a hyper-inflammatory response.
Anaphylactic shock is a severe allergic reaction involving multiple organs including the bronchial and cardiovascular system. Most anaphylactic mediators, like platelet-activating factor (PAF), histamine, and others, act through G protein – coupled receptors, which are linked to the heterotrimeric G proteins Gq /G 11 , G12/G13 , and Gi . The role of downstream signaling pathways activated by anaphylactic mediators in defi ned organs during anaphylactic reactions is largely unknown. Using genetic mouse models that allow for the conditional abrogation of G q /G 11 - and G 12 /G 13 -mediated signaling pathways by inducible Cre/loxP-mediated mutagenesis in endothelial cells (ECs), we show that Gq /G11 -mediated signaling in ECs is required for the opening of the endothelial barrier and the stimulation of nitric oxide formation by various infl ammatory mediators as well as by local anaphylaxis. The systemic effects of anaphylactic mediators like histamine and PAF, but not of bacterial lipopolysaccharide (LPS), are blunted in mice with endothelial G alpha q/G alpha 11 deficiency. Mice with endothelium-specific G alpha q /G alpha 11 deficiency, but not with G alpha 12/G alpha 13 deficiency, are protected against the fatal consequences of passive and active systemic anaphylaxis. This identifies endothelial Gq/G11 -mediated signaling as a critical mediator of fatal systemic anaphylaxis and, hence, as a potential new target to prevent or treat anaphylactic reactions.
Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant.
Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair.
Objective: To determine the role of the AMPKα2 subunit in vascular repair.
Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2-/- versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice.
Conclusions: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia.
Background: In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFKappaB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO). Methodology/Principal Findings: Overexpression of a dominant negative AMPKalpha2 mutant in tumor necrosis factor-alpha-stimulated human endothelial cells resulted in increased NFKappaB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKalpha2-/- mice the interleukin (IL)-1beta induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFKappaB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKalpha2-/- mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IKappaB and p65, indicating a link between AMPK and the IKappaB kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPKalpha2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKalpha2+/+ versus AMPKalpha2-/- mice. Conclusions: These results demonstrate that the IKK is a direct substrate of AMPKalpha2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFKappaB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK.
BACKGROUND: In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks.
RESULTS: We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired.
CONCLUSIONS: Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.
Cytochrome P450 (CYP) signalling pathway has been shown to play a vital role in the vasoreactivity of wild type mouse ophthalmic artery. In this study, we determined the expression, vascular responses and potential mechanisms of the CYP-derived arachidonic acid metabolites. The expression of murine CYP (Cyp2c44) and soluble epoxide hydrolase (sEH) in the wild type ophthalmic artery was determined with immunofluorescence, which showed predominant expression of Cyp2c44 in the vascular smooth muscle cells (VSMC), while sEH was found mainly in the endothelium of the wild type ophthalmic artery. Artery of Cyp2c44−/− and sEH−/− mice were used as negative controls. Targeted mass spectrometry-based lipidomics analysis of endogenous epoxide and diols of the wild type artery detected only 14, 15-EET. Vasorelaxant responses of isolated vessels in response to selective pharmacological blockers and agonist were analysed ex vivo. Direct antagonism of epoxyeicosatrienoic acids (EETs) with a selective inhibitor caused partial vasodilation, suggesting that EETs may behave as vasoconstrictors. Exogenous administration of synthetic EET regioisomers significantly constricted the vessels in a concentration-dependent manner, with the strongest responses elicited by 11, 12- and 14, 15-EETs. Our results provide the first experimental evidence that Cyp2c44-derived EETs in the VSMC mediate vasoconstriction of the ophthalmic artery.
AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPKα1 and α2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPKα−/−) or endothelial-specific deletion (AMPKαΔEC) of the AMPKα subunits. In control and AMPKα1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPKα1 or α2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPKα1 subunit attenuated phenylephrine-mediated contraction in an eNOS- and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPKα1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPKα1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.
Myeloid-specific deletion of the AMPK2 subunit alters monocyte protein expression and atherogenesis
(2019)
The AMP-activated protein kinase (AMPK) is an energy sensing kinase that is activated by a drop in cellular ATP levels. Although several studies have addressed the role of the AMPKα1 subunit in monocytes and macrophages, little is known about the α2 subunit. The aim of this study was to assess the consequences of AMPKα2 deletion on protein expression in monocytes/macrophages, as well as on atherogenesis. A proteomics approach was applied to bone marrow derived monocytes from wild-type mice versus mice specifically lacking AMPKα2 in myeloid cells (AMPKα2∆MC mice). This revealed differentially expressed proteins, including methyltransferases. Indeed, AMPKα2 deletion in macrophages increased the ratio of S-adenosyl methionine to S-adenosyl homocysteine and increased global DNA cytosine methylation. Also, methylation of the vascular endothelial growth factor and matrix metalloproteinase-9 (MMP9) genes was increased in macrophages from AMPKα2∆MC mice, and correlated with their decreased expression. To link these findings with an in vivo phenotype, AMPKα2∆MC mice were crossed onto the ApoE-/- background and fed a western diet. ApoExAMPKα2∆MC mice developed smaller atherosclerotic plaques than their ApoExα2fl/fl littermates, that contained fewer macrophages and less MMP9 than plaques from ApoExα2fl/fl littermates. These results indicate that the AMPKα2 subunit in myeloid cells influences DNA methylation and thus protein expression and contributes to the development of atherosclerotic plaques.
Cytochrome P450-derived epoxyeicosatrienoic acids (EETs) stimulate endothelial cell proliferation and angiogenesis. In this study, we investigated the involvement of the forkhead box, class O (FOXO) family of transcription factors and their downstream target p27Kip1 in EET-induced endothelial cell proliferation. Incubation of human umbilical vein endothelial cells with 11,12-EET induced a time- and dose-dependent decrease in p27Kip1 protein expression, whereas p21Cip1 was not significantly affected. This effect on p27Kip1 protein was associated with decreased mRNA levels as well as p27Kip1 promoter activity. 11,12-EET also stimulated the time-dependent phosphorylation of Akt and of the forkhead factors FOXO1 and FOXO3a, effects prevented by the phosphatidylinositol 3-kinase inhibitor LY 294002. Transfection of endothelial cells with either a dominant-negative or an “Akt-resistant”/constitutively active FOXO3a mutant reversed the 11,12-EET-induced down-regulation of p27Kip1, whereas transfection of a constitutive active Akt decreased p27Kip1 expression independently of the presence or absence of 11,12-EET. To determine whether these effects are involved in EET-induced proliferation, endothelial cells were transfected with the 11,12-EET-generating epoxygenase CYP2C9. Transfection of CYP2C9 elicited endothelial cell proliferation and this effect was inhibited in cells co-transfected with CYP2C9 and either a dominant-negative Akt or constitutively active FOXO3a. Reducing FOXO expression using RNA interference, on the other hand, attenuated p27Kip1 expression and stimulated endothelial cell proliferation. These results indicate that EET-induced endothelial cell proliferation is associated with the phosphatidylinositol 3-kinase/Akt-dependent phosphorylation and inactivation of FOXO factors and the subsequent decrease in expression of the cyclin-dependent kinase inhibitor p27Kip1.
Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) are important modulators of endothelial cell homeostasis. We investigated the signaling pathway linking the activation of CYP 2C9 to enhanced endothelial cell proliferation. Overexpression of CYP 2C9 in cultured human endothelial cells markedly increased proliferation. This effect was paralleled by an up-regulation of the G1 phase regulatory protein, cyclin D1. The specific CYP 2C9 inhibitor, sulfaphenazole, prevented both the enhanced cell proliferation and up-regulation of cyclin D1. CYP 2C9 overexpression also decreased the activity of the c-Jun N-terminal kinase (JNK). Coexpression of wild type JNK with CYP 2C9 attenuated the CYP 2C9-induced increase in cyclin D1 expression and abolished the CYP 2C9-induced proliferation response. In contrast, cotransfecting dominant negative JNK with CYP 2C9 restored the CYP 2C9-mediated up-regulation of cyclin D1 and proliferation. The inactivation of JNK is linked to its dephosphorylation by dual specificity mitogen-activated protein (MAP) kinase phosphatases (MKPs). Overexpression of CYP 2C9 significantly increased the expression of MKP-1, as did incubation with 11,12-EET. These data demonstrate that the mitogenic effect of CYP 2C9 is due to the generation of EETs, which promote the MKP-1-mediated dephosphorylation and inactivation of JNK, effects ultimately culminating in the expression of cyclin D1 and endothelial cell proliferation.
Inhibition of the soluble epoxide hydrolase (sEH) has beneficial effects on vascular inflammation and hypertension indicating that the enzyme may be a promising target for drug development. As the enzymatic core of the hydrolase domain of the human sEH contains two tyrosine residues (Tyr383 and Tyr466) that are theoretically crucial for enzymatic activity, we addressed the hypothesis that the activity of the sEH may be affected by nitrosative stress. Epoxide hydrolase activity was detected in human and murine endothelial cells as well in HEK293 cells and could be inhibited by either authentic peroxynitrite (ONOO−) or the ONOO− generator 3-morpholino-sydnonimine (SIN-1). Protection of the enzymatic core with 1-adamantyl-3-cyclohexylurea in vitro decreased sensitivity to SIN-1. Both ONOO− and SIN-1 elicited the tyrosine nitration of the sEH protein and mass spectrometry analysis of tryptic fragments revealed nitration on several tyrosine residues including Tyr383 and Tyr466. Mutation of the latter residues to phenylalanine was sufficient to abrogate epoxide hydrolase activity. In vivo, streptozotocin-induced diabetes resulted in the tyrosine nitration of the sEH in murine lungs and a significant decrease in its activity. Taken together, these data indicate that the activity of the sEH can be regulated by the tyrosine nitration of the protein. Moreover, nitrosative stress would be expected to potentiate the physiological actions of arachidonic acid epoxides by preventing their metabolism to the corresponding diols.
Background
Cytochrome-P450 (CYP450) epoxygenases metabolise arachidonic acid (AA) into four different biologically active epoxyeicosatrienoic acid (EET) regioisomers. Three of the EETs (i.e., 8,9-, 11,12- and 14,15-EET) are rapidly hydrolysed by the enzyme soluble epoxide hydrolase (sEH). Here, we investigated the role of sEH in nociceptive processing during peripheral inflammation.
Results
In dorsal root ganglia (DRG), we found that sEH is expressed in medium and large diameter neurofilament 200-positive neurons. Isolated DRG-neurons from sEH-/- mice showed higher EET and lower DHET levels. Upon AA stimulation, the largest changes in EET levels occurred in culture media, indicating both that cell associated EET concentrations quickly reach saturation and EET-hydrolyzing activity mostly effects extracellular EET signaling. In vivo, DRGs from sEH-deficient mice exhibited elevated 8,9-, 11,12- and 14,15-EET-levels. Interestingly, EET levels did not increase at the site of zymosan-induced inflammation. Cellular imaging experiments revealed direct calcium flux responses to 8,9-EET in a subpopulation of nociceptors. In addition, 8,9-EET sensitized AITC-induced calcium increases in DRG neurons and AITC-induced calcitonin gene related peptide (CGRP) release from sciatic nerve axons, indicating that 8,9-EET sensitizes TRPA1-expressing neurons, which are known to contribute to mechanical hyperalgesia. Supporting this, sEH-/- mice showed increased nociceptive responses to mechanical stimulation during zymosan-induced inflammation and 8,9-EET injection reduced mechanical thresholds in naive mice.
Conclusion
Our results show that the sEH can regulate mechanical hyperalgesia during inflammation by inactivating 8,9-EET, which sensitizes TRPA1-expressing nociceptors. Therefore we suggest that influencing the CYP450 pathway, which is actually highly considered to treat cardiovascular diseases, may cause pain side effects.
Cystathionine γ lyase (CSE) is the major source of hydrogen sulfide-derived species (H2Sn) in endothelial cells and plays an important role in protecting against atherosclerosis. Here we investigated the molecular mechanisms underlying the regulation of CSE expression in endothelial cells by fluid shear stress/flow. Fluid shear stress decreased CSE expression in human and murine endothelial cells and was negatively correlated with the transcription factor Krüppel-like factor (KLF) 2. CSE was identified as a direct target of the KLF2-regulated microRNA, miR-27b and high expression of CSE in native human plaque-derived endothelial cells, was also inversely correlated with KLF2 and miR-27b levels. One consequence of decreased CSE expression was the loss of Prx6 sulfhydration (on Cys47), which resulted in Prx6 hyperoxidation, decamerization and inhibition, as well as a concomitant increase in endothelial cell reactive oxygen species and lipid membrane peroxidation. H2Sn supplementation in vitro was able to reverse the redox state of Prx6. Statin therapy, which is known to activate KLF2, also decreased CSE expression but increased CSE activity by preventing its phosphorylation on Ser377. As a result, the sulfhydration of Prx6 was partially restored in samples from plaque containing arteries from statin-treated donors. Taken together, the regulation of CSE expression by shear stress/disturbed flow is dependent on KLF2 and miR-27b. Moreover, in murine and human arteries CSE acts to maintain endothelial redox balance at least partly by targeting Prx6 to prevent its decamerization and inhibition of its peroxidase activity.
Proline-rich tyrosine kinase 2 (PYK2) can be activated by angiotensin II (Ang II) and reactive oxygen species. We report that in endothelial cells, Ang II enhances the tyrosine phosphorylation of endothelial NO synthase (eNOS) in an AT1-, H2O2-, and PYK2-dependent manner. Low concentrations (1–100 µmol/liter) of H2O2 stimulated the phosphorylation of eNOS Tyr657 without affecting that of Ser1177, and attenuated basal and agonist-induced NO production. In isolated mouse aortae, 30 µmol/liter H2O2 induced phosphorylation of eNOS on Tyr657 and impaired acetylcholine-induced relaxation. Endothelial overexpression of a dominant-negative PYK2 mutant protected against H2O2-induced endothelial dysfunction. Correspondingly, carotid arteries from eNOS–/– mice overexpressing the nonphosphorylatable eNOS Y657F mutant were also protected against H2O2. In vivo, 3 wk of treatment with Ang II considerably increased levels of Tyr657-phosphorylated eNOS in the aortae of wild-type but not Nox2y/– mice, and this was again associated with a clear impairment in endothelium-dependent vasodilatation in the wild-type but not in the Nox2y/– mice. Collectively, endothelial PYK2 activation by Ang II and H2O2 causes the phosphorylation of eNOS on Tyr657, attenuating NO production and endothelium-dependent vasodilatation. This mechanism may contribute to the endothelial dysfunction observed in cardiovascular diseases associated with increased activity of the renin–angiotensin system and elevated redox stress.
Renal cell carcinoma alters endothelial receptor expression responsible for leukocyte adhesion
(2016)
Renal cell carcinoma (RCC) escapes immune recognition. To elaborate the escape strategy the influence of RCC cells on endothelial receptor expression and endothelial leukocyte adhesion was evaluated. Human umbilical vein endothelial cells (HUVEC) were co-cultured with the RCC cell line, Caki-1, with and without tumor necrosis factor (TNF)-alpha. Intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial (E)-selectin, standard and variants (V) of CD44 were then analysed in HUVEC, using flow cytometry and Western blot analysis. To determine which components are responsible for HUVEC-Caki-1 interaction causing receptor alteration, Caki-1 membrane fragments versus cell culture supernatant were applied to HUVECS. Adhesion of peripheral blood lymphocytes (PBL) and polymorphonuclear neutrophils (PMN) to endothelium was evaluated by co-culture adhesion assays. Relevance of endothelial receptor expression for adhesion to endothelium was determined by receptor blockage. Co-culture of RCC and HUVECs resulted in a significant increase in endothelial ICAM-1, VCAM-1, E-selectin, CD44 V3 and V7 expression. Previous stimulation of HUVECs with TNF-alpha and co-cultivation with Caki-1 resulted in further elevation of endothelial CD44 V3 and V7 expression, whereas ICAM-1, VCAM-1 and E-selectin expression were significantly diminished. Since Caki-1 membrane fragments also caused these alterations, but cell culture supernatant did not, cell-cell contact may be responsible for this process. Blocking ICAM-1, VCAM-1, E-selectin or CD44 with respective antibodies led to a significant decrease in PBL and PMN adhesion to endothelium. Thus, exposing HUVEC to Caki-1 results in significant alteration of endothelial receptor expression and subsequent endothelial attachment of PBL and PMN.
In this meeting report, particularly addressing the topic of protection of the cardiovascular system from ischemia/reperfusion injury, highlights are presented that relate to conditioning strategies of the heart with respect to molecular mechanisms and outcome in patients’ cohorts, the influence of co-morbidities and medications, as well as the contribution of innate immune reactions in cardioprotection. Moreover, developmental or systems biology approaches bear great potential in systematically uncovering unexpected components involved in ischemia–reperfusion injury or heart regeneration. Based on the characterization of particular platelet integrins, mitochondrial redox-linked proteins, or lipid-diol compounds in cardiovascular diseases, their targeting by newly developed theranostics and technologies opens new avenues for diagnosis and therapy of myocardial infarction to improve the patients’ outcome.
Recent advances in basic cardiovascular research as well as their translation into the clinical situation were the focus at the last "New Frontiers in Cardiovascular Research meeting". Major topics included the characterization of new targets and procedures in cardioprotection, deciphering new players and inflammatory mechanisms in ischemic heart disease as well as uncovering microRNAs and other biomarkers as versatile and possibly causal factors in cardiovascular pathogenesis. Although a number of pathological situations such as ischemia-reperfusion injury or atherosclerosis can be simulated and manipulated in diverse animal models, also to challenge new drugs for intervention, patient studies are the ultimate litmus test to obtain unequivocal information about the validity of biomedical concepts and their application in the clinics. Thus, the open and bidirectional exchange between bench and bedside is crucial to advance the field of ischemic heart disease with a particular emphasis of understanding long-lasting approaches in cardioprotection.
The interaction of macrophages with apoptotic cells is required for efficient resolution of inflammation. While apoptotic cell removal prevents inflammation due to secondary necrosis, it also alters the macrophage phenotype to hinder further inflammatory reactions. The interaction between apoptotic cells and macrophages is often studied by chemical or biological induction of apoptosis, which may introduce artifacts by affecting the macrophages as well and/or triggering unrelated signaling pathways. Here, we set up a pure cell death system in which NIH 3T3 cells expressing dimerizable Caspase-8 were co-cultured with peritoneal macrophages in a transwell system. Phenotype changes in macrophages induced by apoptotic cells were evaluated by RNA sequencing, which revealed an unexpectedly dominant impact on macrophage proliferation. This was confirmed in functional assays with primary peritoneal macrophages and IC-21 macrophages. Moreover, inhibition of apoptosis during Zymosan-induced peritonitis in mice decreased mRNA levels of cell cycle mediators in peritoneal macrophages. Proliferation of macrophages in response to apoptotic cells may be important to increase macrophage numbers in order to allow efficient clearance and resolution of inflammation.