Direct GPCR-EGFR interaction enables synergistic membrane-to-nucleus information transfer.

We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or -mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.

A Thromboxane A2 Receptor-Driven COX-2-Dependent Feedback Loop That Affects Endothelial Homeostasis and Angiogenesis.

BACKGROUND: TP (thromboxane A2 receptor) plays an eminent role in the pathophysiology of endothelial dysfunction and cardiovascular disease. Moreover, its expression is reported to increase in the intimal layer of blood vessels of cardiovascular high-risk individuals. Yet it is unknown, whether TP upregulation per se has the potential to affect the homeostasis of the vascular endothelium. METHODS: We combined global transcriptome analysis, lipid mediator profiling, functional cell analyses, and in vivo angiogenesis assays to study the effects of endothelial TP overexpression or knockdown/knockout on the angiogenic capacity of endothelial cells in vitro and in vivo. RESULTS: Here we report that endothelial TP expression induces COX-2 (cyclooxygenase-2) in a Gi/o- and Gq/11-dependent manner, thereby promoting its own activation via the auto/paracrine release of TP agonists, such as PGH2 (prostaglandin H2) or prostaglandin F2 but not TxA2 (thromboxane A2). TP overexpression induces endothelial cell tension and aberrant cell morphology, affects focal adhesion dynamics, and inhibits the angiogenic capacity of human endothelial cells in vitro and in vivo, whereas TP knockdown or endothelial-specific TP knockout exerts opposing effects. Consequently, this TP-dependent feedback loop is disrupted by pharmacological TP or COX-2 inhibition and by genetic reconstitution of PGH2-metabolizing prostacyclin synthase even in the absence of functional prostacyclin receptor expression. CONCLUSIONS: Our work uncovers a TP-driven COX-2-dependent feedback loop and important effector mechanisms that directly link TP upregulation to angiostatic TP signaling in endothelial cells. By these previously unrecognized mechanisms, pathological endothelial upregulation of the TP could directly foster endothelial dysfunction, microvascular rarefaction, and systemic hypertension even in the absence of exogenous sources of TP agonists.

Aspirin sensitivity of PIK3CA-mutated Colorectal Cancer: potential mechanisms revisited.

PIK3CA mutations are amongst the most prevalent somatic mutations in cancer and are associated with resistance to first-line treatment along with low survival rates in a variety of malignancies. There is evidence that patients carrying PIK3CA mutations may benefit from treatment with acetylsalicylic acid, commonly known as aspirin, particularly in the setting of colorectal cancer. In this regard, it has been clarified that Class IA Phosphatidylinositol 3-kinases (PI3K), whose catalytic subunit p110α is encoded by the PIK3CA gene, are involved in signal transduction that regulates cell cycle, cell growth, and metabolism and, if disturbed, induces carcinogenic effects. Although PI3K is associated with pro-inflammatory cyclooxygenase-2 (COX-2) expression and signaling, and COX-2 is among the best-studied targets of aspirin, the mechanisms behind this clinically relevant phenomenon are still unclear. Indeed, there is further evidence that the protective, anti-carcinogenic effect of aspirin in this setting may be mediated in a COX-independent manner. However, until now the understanding of aspirin's prostaglandin-independent mode of action is poor. This review will provide an overview of the current literature on this topic and aims to analyze possible mechanisms and targets behind the aspirin sensitivity of PIK3CA-mutated cancers.

The Role of ABCG2 in the Pathogenesis of Primary Hyperuricemia and Gout-An Update.

Urate homeostasis in humans is a complex and highly heritable process that involves i.e., metabolic urate biosynthesis, renal urate reabsorption, as well as renal and extrarenal urate excretion. Importantly, disturbances in urate excretion are a common cause of hyperuricemia and gout. The majority of urate is eliminated by glomerular filtration in the kidney followed by an, as yet, not fully elucidated interplay of multiple transporters involved in the reabsorption or excretion of urate in the succeeding segments of the nephron. In this context, genome-wide association studies and subsequent functional analyses have identified the ATP-binding cassette (ABC) transporter ABCG2 as an important urate transporter and have highlighted the role of single nucleotide polymorphisms (SNPs) in the pathogenesis of reduced cellular urate efflux, hyperuricemia, and early-onset gout. Recent publications also suggest that ABCG2 is particularly involved in intestinal urate elimination and thus may represent an interesting new target for pharmacotherapeutic intervention in hyperuricemia and gout. In this review, we specifically address the involvement of ABCG2 in renal and extrarenal urate elimination. In addition, we will shed light on newly identified polymorphisms in ABCG2 associated with early-onset gout.

Impact of DICER1 and DROSHA on the Angiogenic Capacity of Human Endothelial Cells.

RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.

Generation of Cardiomyocytes From Vascular Adventitia-Resident Stem Cells.

RATIONALE: Regeneration of lost cardiomyocytes is a fundamental unresolved problem leading to heart failure. Despite several strategies developed from intensive studies performed in the past decades, endogenous regeneration of heart tissue is still limited and presents a big challenge that needs to be overcome to serve as a successful therapeutic option for myocardial infarction. OBJECTIVE: One of the essential prerequisites for cardiac regeneration is the identification of endogenous cardiomyocyte progenitors and their niche that can be targeted by new therapeutic approaches. In this context, we hypothesized that the vascular wall, which was shown to harbor different types of stem and progenitor cells, might serve as a source for cardiac progenitors. METHODS AND RESULTS: We describe generation of spontaneously beating mouse aortic wall-derived cardiomyocytes without any genetic manipulation. Using aortic wall-derived cells (AoCs) of WT (wild type), αMHC (α-myosin heavy chain), and Flk1 (fetal liver kinase 1)-reporter mice and magnetic bead-associated cell sorting sorting of Flk1+ AoCs from GFP (green fluorescent protein) mice, we identified Flk1+CD (cluster of differentiation) 34+Sca-1 (stem cell antigen-1)-CD44- AoCs as the population that gives rise to aortic wall-derived cardiomyocytes. This AoC subpopulation delivered also endothelial cells and macrophages with a particular accumulation within the aortic wall-derived cardiomyocyte containing colonies. In vivo, cardiomyocyte differentiation capacity was studied by implantation of fluorescently labeled AoCs into chick embryonic heart. These cells acquired cardiomyocyte-like phenotype as shown by αSRA (α-sarcomeric actinin) expression. Furthermore, coronary adventitial Flk1+ and CD34+ cells proliferated, migrated into the myocardium after mouse myocardial infarction, and expressed Isl-1+ (insulin gene enhancer protein-1) indicative of cardiovascular progenitor potential. CONCLUSIONS: Our data suggest Flk1+CD34+ vascular adventitia-resident stem cells, including those of coronary adventitia, as a novel endogenous source for generating cardiomyocytes. This process is essentially supported by endothelial cells and macrophages. In summary, the therapeutic manipulation of coronary adventitia-resident cardiac stem and their supportive cells may open new avenues for promoting cardiac regeneration and repair after myocardial infarction and for preventing heart failure.

Precursor-Directed Biosynthesis and Fluorescence Labeling of Clickable Microcystins.

Microcystins, cyclic nonribosomal heptapeptides, are the most well-known cyanobacterial toxins. They are exceptionally well studied, but open questions remain concerning their physiological role for the producing microorganism or their suitability as lead compounds for anticancer drug development. One means to study specialized metabolites in more detail is the introduction of functional groups that make a compound amenable for bioorthogonal, so-called click reactions. Although it was reported that microcystins cannot be derivatized by precursor-directed biosynthesis, we successfully used this approach to prepare clickable microcystins. Supplementing different azide- or terminal alkyne containing amino acid analogues into the cultivation medium of microcystin-producing cyanobacteria strains, we found that these strains differ strongly in their substrate acceptance. Exploiting this flexibility, we generated more than 40 different clickable microcystins. We conjugated one of these derivatives with a fluorogenic dye and showed that neither incorporation of the unnatural amino acid analogue nor attachment of the fluorescent label significantly affects the cytotoxicity against cell lines expressing the human organic anion transporting polypeptides 1B1 or 1B3. Using time-lapse microscopy, we observed that the fluorescent microcystin is rapidly taken up into eukaryotic cells expressing these transporters.

Necrotizing fasciitis caused by the treatment of chronic non-specific back pain.

BACKGROUND: Chronic back pain is a multifactorial disease that occurs particularly in adults and has many negative effects on the quality of daily life. Therapeutic strategies are often multimodal and designed for a long-term therapy period. In some cases, one option is joint infiltration or intrathecal injection with local anaesthetics. An adverse effect of this intervention may be necrotic fasciitis, a disease with high mortality and few therapeutic options. CASE PRESENTATION: This case shows a 53-year-old female patient who developed necrotic fasciitis after infiltrations of the sacroiliac joint and after epidural-sacral and intrathecal injections. CONCLUSION: Thanks to early and aggressive surgical intervention, antibiotic treatment and hyperbaric oxygenation, she survived this serious complication and was able to return to life.

ABC Transport Proteins in Cardiovascular Disease-A Brief Summary.

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters may play an important role in the pathogenesis of atherosclerotic vascular diseases due to their involvement in cholesterol homeostasis, blood pressure regulation, endothelial function, vascular inflammation, as well as platelet production and aggregation. In this regard, ABC transporters, such as ABCA1, ABCG5 and ABCG8, were initially found to be responsible for genetically-inherited syndromes like Tangier diseases and sitosterolemia. These findings led to the understanding of those transporter's function in cellular cholesterol efflux and thereby also linked them to atherosclerosis and cardiovascular diseases (CVD). Subsequently, further ABC transporters, i.e., ABCG1, ABCG4, ABCB6, ABCC1, ABCC6 or ABCC9, have been shown to directly or indirectly affect cellular cholesterol efflux, the inflammatory response in macrophages, megakaryocyte proliferation and thrombus formation, as well as vascular function and blood pressure, and may thereby contribute to the pathogenesis of CVD and its complications. Furthermore, ABC transporters, such as ABCB1, ABCC2 or ABCG2, may affect the safety and efficacy of several drug classes currently in use for CVD treatment. This review will give a brief overview of ABC transporters involved in the process of atherogenesis and CVD pathology. It also aims to briefly summarize the role of ABC transporters in the pharmacokinetics and disposition of drugs frequently used to treat CVD and CVD-related complications.

Multipotent mesenchymal stromal cells promote tumor growth in distinct colorectal cancer cells by a ß1-integrin-dependent mechanism.

Tumor-stroma interactions play an essential role in the biology of colorectal carcinoma (CRC). Multipotent mesenchymal stromal cells (MSC) may represent a pivotal part of the stroma in CRC, but little is known about the specific interaction of MSC with CRC cells derived from tumors with different mutational background. In previous studies we observed that MSC promote the xenograft growth of the CRC cell-line DLD1. In the present study, we aimed to analyze the mechanisms of MSC-promoted tumor growth using various in vitro and in vivo experimental models and CRC cells of different mutational status. MSC specifically interacted with distinct CRC cells and supported tumor seeding in xenografts. The MSC-CRC interaction facilitated three-dimensional spheroid formation in CRC cells with dysfunctional E-cadherin system. Stable knock-downs revealed that the MSC-facilitated spheroid formation depended on ß1-integrin in CRC cells. Specifically in α-catenin-deficient CRC cells this ß1-integrin-dependent interaction resulted in a MSC-mediated promotion of early tumor growth in vivo. Collagen I and other extracellular matrix compounds were pivotal for the functional MSC-CRC interaction. In conclusion, our data demonstrate a differential interaction of MSC with CRC cells of different mutational background. Our study is the first to show that MSC specifically compared to normal fibroblasts impact early xenograft growth of distinct α-catenin deficient CRC cells possibly through secretion of extracellular matrix. This mechanism could serve as a future target for therapy and metastasis prevention.

[Multimodal pain management in a patient with atypical cervicogenic headache]. / Patient mit einer atypischen Form zervikogener Kopfschmerzen.

A 45-year-old patient presented with an eight-year history of persistent unilateral headache associated with recurrent episodes of ipsilateral conjunctival injections, eyelid edema and ptosis. Prior ineffective pharmacological treatment strategies included tramadol, non-steroidal anti-inflammatory drugs and triptans. Palpation of right suboccipital trigger points revealed tenderness in the area of the greater occipital nerve and reinforced the symptoms. The diagnosis of cervicogenic headache was confirmed by symptom resolution following right greater occipital nerve blockade. A multimodal treatment strategy (physical therapy, nerve blockade, pharmacological treatment) was chosen and an emphasis was put on optimizing pharmacological pain relief using the opioid analgesic tapentadol and the tricyclic antidepressant amitriptyline as an adjuvant analgesic. Importantly, the patient reported a substantial and consistent pain reduction and considerable quality of life improvement during implementation of the treatment regimen.

Homoarginine levels are regulated by L-arginine:glycine amidinotransferase and affect stroke outcome: results from human and murine studies.

BACKGROUND: Endogenous arginine homologues, including homoarginine, have been identified as novel biomarkers for cardiovascular disease and outcomes. Our studies of human cohorts and a confirmatory murine model associated the arginine homologue homoarginine and its metabolism with stroke pathology and outcome. METHODS AND RESULTS: Increasing homoarginine levels were independently associated with a reduction in all-cause mortality in patients with ischemic stroke (7.4 years of follow-up; hazard ratio for 1-SD homoarginine, 0.79 [95% confidence interval, 0.64-0.96]; P=0.019; n=389). Homoarginine was also independently associated with the National Institutes of Health Stroke Scale+age score and 30-day mortality after ischemic stroke (P<0.05; n=137). A genome-wide association study revealed that plasma homoarginine was strongly associated with single nucleotide polymorphisms in the L-arginine:glycine amidinotransferase (AGAT) gene (P<2.1 × 10(-8); n=2806), and increased AGAT expression in a cell model was associated with increased homoarginine. Next, we used 2 genetic murine models to investigate the link between plasma homoarginine and outcome after experimental ischemic stroke: (1) an AGAT deletion (AGAT(-/-)) and (2) a guanidinoacetate N-methyltransferase deletion (GAMT(-/-)) causing AGAT upregulation. As suggested by the genome-wide association study, homoarginine was absent in AGAT(-/-) mice and increased in GAMT(-/-) mice. Cerebral damage and neurological deficits in experimental stroke were increased in AGAT(-/-) mice and attenuated by homoarginine supplementation, whereas infarct size in GAMT(-/-) mice was decreased compared with controls. CONCLUSIONS: Low homoarginine appears to be related to poor outcome after ischemic stroke. Further validation in future trials may lead to therapeutic adjustments of homoarginine metabolism that alleviate stroke and other vascular disorders.

Myeloperoxidase deficiency ameliorates progression of chronic kidney disease in mice.

Myeloperoxidase (MPO) is an enzyme expressed in neutrophils and monocytes/macrophages. Beside its well-defined role in innate immune defence, it may also be responsible for tissue damage. To identify the role of MPO in the progression of chronic kidney disease (CKD), we investigated CKD in a model of renal ablation in MPO knockout and wild-type mice. CKD was induced by 5/6 nephrectomy. Mice were followed for 10 wk to evaluate the impact of MPO deficiency on renal morbidity. Renal ablation induced CKD in wild-type mice with increased plasma levels of MPO compared with controls. No difference was found between MPO-deficient and wild-type mice regarding albuminuria 1 wk after renal ablation, indicating similar acute responses to renal ablation. Over the next 10 wk, however, MPO-deficient mice developed significantly less albuminuria and glomerular injury than wild-type mice. This was accompanied by a significantly lower renal mRNA expression of the fibrosis marker genes plasminogen activator inhibitor-I, collagen type III, and collagen type IV as well as matrix metalloproteinase-2 and matrix metalloproteinase-9. MPO-deficient mice also developed less renal inflammation after renal ablation, as indicated by a lower infiltration of CD3-positive T cells and F4/80-positive monocytes/macrophages compared with wild-type mice. In vitro chemotaxis of monocyte/macrophages isolated from MPO-deficient mice was impaired compared with wild-type mice. No significant differences were observed for mortality and blood pressure after renal ablation. In conclusion, these results demonstrate that MPO deficiency ameliorates renal injury in the renal ablation model of CKD in mice.

Impact of genetic variability in the ABCG2 gene on ABCG2 expression, function, and interaction with AT1 receptor antagonist telmisartan.

The ATP-binding cassette transporter ABCG2 plays a prominent role in cardiovascular and cancer pathophysiology, is involved in the pathogenesis of gout, and affects pharmacokinetics of numerous drugs. Telmisartan, a widely used AT1 receptor antagonist, inhibits the transport capacity of ABCG2 and may cause drug-drug interactions, especially in individuals carrying polymorphism that facilitate the telmisartan-ABCG2 interaction. Thus, the aim of this study was to identify ABCG2 polymorphisms and somatic mutations with relevance for the telmisartan-ABCG2 interaction. For this purpose, a cellular system for the conditional expression of ABCG2 was established. ABCG2 variants were generated via site-directed mutagenesis. Interaction of telmisartan with these ABCG2 variants was investigated in HEK293-Tet-On cells using the pheophorbide A efflux assay. Moreover, expression of ABCG2 variants was studied in these cells. Importantly, protein levels of the Q141K and F489L variant were significantly reduced, a phenomenon that was partly reversed by pharmacological proteasome inhibition. Moreover, basal pheophorbide A efflux capacity of S248P, F431L, and F489L variants was significantly impaired. Interestingly, inhibition of ABCG2-mediated pheophorbide A transport by telmisartan was almost abolished in cells expressing the R482G variant, whereas it was largely increased in cells expressing the F489L variant. We conclude that the arginine residue at position 482 of the ABCG2 molecule is of major importance for the interaction of telmisartan with this ABC transporter. Furthermore, individuals carrying the F489L polymorphism may be at increased risk of developing adverse drug reactions in multi-drug regimens involving ABCG2 substrates and telmisartan.

Deletion of vascular thromboxane A2 receptors and its impact on angiotensin II-induced hypertension and atherosclerotic lesion formation in the aorta of Ldlr-deficient mice.

The thromboxane A2 receptor (TP) has been shown to play a role in angiotensin II (Ang II)-mediated hypertension and pathological vascular remodeling. To assess the impact of vascular TP on Ang II-induced hypertension, atherogenesis, and pathological aortic alterations, i.e. aneurysms, we analysed Western-type diet-fed and Ang II-infused TPVSMC KO/Ldlr KO, TPEC KO/Ldlr KO mice and their respective wild-type littermates (TPWT/Ldlr KO). These analyses showed that neither EC- nor VSMC-specific deletion of the TP significantly affected basal or Ang II-induced blood pressure or aortic atherosclerotic lesion area. In contrast, VSMC-specific TP deletion abolished and EC-specific TP deletion surprisingly reduced the ex vivo reactivity of aortic rings to the TP agonist U-46619, whereas VSMC-specific TP knockout also diminished the ex vivo response of aortic rings to Ang II. Furthermore, despite similar systemic blood pressure, there was a trend towards less atherogenesis in the aortic arch and a trend towards fewer pathological aortic alterations in Ang II-treated female TPVSMC KO/Ldlr KO mice. Survival was impaired in male mice after Ang II infusion and tended to be higher in TPVSMC KO/Ldlr KO mice than in TPWT/Ldlr KO littermates. Thus, our data may suggest a deleterious role of the TP expressed in VSMC in the pathogenesis of Ang II-induced aortic atherosclerosis in female mice, and a surprising role of the endothelial TP in TP-mediated aortic contraction. However, future studies are needed to substantiate and further elucidate the role of the vascular TP in the pathogenesis of Ang II-induced hypertension, aortic atherosclerosis and aneurysm formation.

Ceacam1 deletion causes vascular alterations in large vessels.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance and endothelial survival. However, its role in the morphology of macrovessels remains unknown. Mice lacking Ceacam1 (Cc1-/-) exhibit hyperinsulinemia, which causes insulin resistance and fatty liver. With increasing evidence of an association among hyperinsulinemia, fatty liver disease, and atherosclerosis, we investigated whether Cc1-/- exhibited vascular lesions in atherogenic-prone aortae. Histological analysis revealed impaired endothelial integrity with restricted fat deposition and aortic plaque-like lesions in Cc1-/- aortae, likely owing to their limited lipidemia. Immunohistochemical analysis indicated macrophage deposition, and in vitro studies showed increased leukocyte adhesion to aortic wall, mediated in part by elevation in vascular cell adhesion molecule 1 levels. Basal aortic eNOS protein and NO content were reduced, in parallel with reduced Akt/eNOS and Akt/Foxo1 phosphorylation. Ligand-induced vasorelaxation was compromised in aortic rings. Increased NADPH oxidase activity and plasma 8-isoprostane levels revealed oxidative stress and lipid peroxidation in Cc1-/- aortae. siRNA-mediated CEACAM1 knockdown in bovine aortic endothelial cells adversely affected insulin's stimulation of IRS-1/PI 3-kinase/Akt/eNOS activation by increasing IRS-1 binding to SHP2 phosphatase. This demonstrates that CEACAM1 regulates both endothelial cell autonomous and nonautonomous mechanisms involved in vascular morphology and NO production in aortae. Systemic factors such as hyperinsulinemia could contribute to the pathogenesis of these vascular abnormalities. Cc1-/- mice provide a first in vivo demonstration of distinct CEACAM1-dependent hepatic insulin clearance linking hepatic to macrovascular abnormalities.

Isoprostanes and neuroprostanes: total synthesis, biological activity and biomarkers of oxidative stress in humans.

Isoprostanes (IsoPs) and neuroprostanes (NeuroPs) are formed in vivo by a free radical non-enzymatic mechanism involving peroxidation of arachidonic acid (AA, C20:4 n-6) and docosahexaenoic acid (DHA, C22:6 n-3) respectively. This review summarises our research in the total synthesis of these lipid metabolites, as well as their biological activities and their utility as biomarkers of oxidative stress in humans.

The role of EGFR in vascular AT1R signaling: From cellular mechanisms to systemic relevance.

The epidermal growth factor receptor (EGFR) belongs to the ErbB-family of receptor tyrosine kinases that are of importance in oncology. During the last years, substantial evidence accumulated for a crucial role of EGFR concerning the action of the angiotensin II type 1 receptor (AT1R) in blood vessels, resulting form AT1R-induced EGFR transactivation. This transactivation occurs through the release of membrane-anchored EGFR-ligands, cytosolic tyrosine kinases, heterocomplex formation or enhanced ligand expression. AT1R-EGFR crosstalk amplifies the signaling response and enhances the biological effects of angiotensin II. Downstream signaling cascades include ERK1/2 and p38 MAPK, PLCγ and STAT. AT1R-induced EGFR activation contributes to vascular remodeling and hypertrophy via e.g. smooth muscle cell proliferation, migration and extracellular matrix production. EGFR transactivation results in increased vessel wall thickness and reduced vascular compliance. AT1R and EGFR signaling pathways are also implicated the induction of vascular inflammation. Again, EGFR transactivation exacerbates the effects, leading to endothelial dysfunction that contributes to vascular inflammation, dysfunction and remodeling. Dysregulation of the AT1R-EGFR axis has been implicated in the pathogenesis of various cardiovascular diseases and inhibition or prevention of EGFR signaling can attenuate part of the detrimental impact of enhanced renin-angiotensin-system (RAAS) activity, highlighting the importance of EGFR for the adverse consequences of AT1R activation. In summary, EGFR plays a critical role in vascular AT1R action, enhancing signaling, promoting remodeling, contributing to inflammation, and participating in the pathogenesis of cardiovascular diseases. Understanding the interplay between AT1R and EGFR will foster the development of effective therapeutic strategies of RAAS-induced disorders.

Transcriptional impact of EGFR activation in human female vascular smooth muscle cells.

Vascular smooth muscle cells (VSMC) are critical for the vascular tone, but they can also drive the development of vascular diseases when they lose their contractile phenotype and de-differentiate. Previous studies showed that the epidermal growth factor receptor (EGFR) of VSMC is critical for vascular health, but most of the underlying mechanisms by which VSMC-EGFR controls vascular fate have remained unknown. We combined RNA-sequencing and bioinformatics analysis to characterize the effect of EGFR-activation on the transcriptome of human primary VSMC (from different female donors) and to identify potentially affected cellular processes. Our results indicate that the activation of human VSMC-EGFR is sufficient to trigger a phenotypical switch toward a proliferative and inflammatory phenotype. The extent of this effect is nonetheless partly donor-dependent. Our hypothesis-generating study thus provides a first insight into mechanisms that could partly explain variable susceptibilities to vascular diseases in between individuals.

EGFR activation differentially affects the inflammatory profiles of female human aortic and coronary artery endothelial cells.

Endothelial cells (EC) are key players in vascular function, homeostasis and inflammation. EC show substantial heterogeneity due to inter-individual variability (e.g. sex-differences) and intra-individual differences as they originate from different organs or vessels. This variability may lead to different responsiveness to external stimuli. Here we compared the responsiveness of female human primary EC from the aorta (HAoEC) and coronary arteries (HCAEC) to Epidermal Growth Factor Receptor (EGFR) activation. EGFR is an important signal integration hub for vascular active substances with physiological and pathophysiological relevance. Our transcriptomic analysis suggested that EGFR activation differentially affects the inflammatory profiles of HAoEC and HCAEC, particularly by inducing a HCAEC-driven leukocyte attraction but a downregulation of adhesion molecule and chemoattractant expression in HAoEC. Experimental assessments of selected inflammation markers were performed to validate these predictions and the results confirmed a dual role of EGFR in these cells: its activation initiated an anti-inflammatory response in HAoEC but a pro-inflammatory one in HCAEC. Our study highlights that, although they are both arterial EC, female HAoEC and HCAEC are distinguishable with regard to the role of EGFR and its involvement in inflammation regulation, what may be relevant for vascular maintenance but also the pathogenesis of endothelial dysfunction.