Martin Andrassy

A mechanism converting psychosocial stress into mononuclear cell activation

Little is known about the mechanisms converting psychosocial stress into cellular dysfunction. Various genes, up-regulated in atherosclerosis but also by psychosocial stress, are controlled by the transcription factor nuclear factor κB (NF-κB). Therefore, NF-κB is a good candidate to convert psychosocial stress into cellular activation. Volunteers were subjected to a brief laboratory stress test and NF-κB activity was determined in peripheral blood mononuclear cells (PBMC), as a window into the body and because PBMC play a role in diseases such as atherosclerosis. In 17 of 19 volunteers, NF-κB was rapidly induced during stress exposure, in parallel with elevated levels of catecholamines and cortisol, and returned to basal levels within 60 min. To model this response, mice transgenic for a strictly NF-κB-controlled β-globin transgene were stressed by immobilization. Immobilization resulted in increased β-globin expression, which could be reduced in the presence of the α1-adrenergic inhibitor prazosin. To define the role of adrenergic stimulation in the up-regulation of NF-κB, THP-1 cells were induced with physiological amounts of catecholamines for 10 min. Only noradrenaline resulted in a dose- and time-dependent induction of NF-κB and NF-κB-dependent gene expression, which depended on pertussis-toxin-sensitive G protein-mediated phosphophatidylinositol 3-kinase, Ras/Raf, and mitogen-activated protein kinase activation. Induction was reduced by α1- and β-adrenergic inhibitors. Thus, noradrenaline-dependent adrenergic stimulation results in activation of NF-κB in vitro and in vivo. Activation of NF-κB represents a downstream effector for the neuroendocrine response to stressful psychosocial events and links changes in the activity of the neuroendocrine axis to the cellular response.

High-Mobility Group Box-1 in Ischemia-Reperfusion Injury of the Heart

Background— High-mobility group box-1 (HMGB1) is a nuclear factor released by necrotic cells and by activated immune cells. HMGB1 signals via members of the toll-like receptor family and the receptor for advanced glycation end products (RAGE). Although HMGB1 has been implicated in ischemia/reperfusion (I/R) injury of the liver and lung, its role in I/R injury of the heart remains unclear. Methods and Results— Here, we demonstrate that HMGB1 acts as an early mediator of inflammation and organ damage in I/R injury of the heart. HMGB1 levels were already elevated 30 minutes after hypoxia in vitro and in ischemic injury of the heart in vivo. Treatment of mice with recombinant HMGB1 worsened I/R injury, whereas treatment with HMGB1 box A significantly reduced infarct size and markers of tissue damage. In addition, HMGB1 inhibition with recombinant HMGB1 box A suggested an involvement of the mitogen-activated protein kinases jun N-terminal kinase and extracellular signal-regulated kinase 1/2, as well as the nuclear transcription factor nuclear factor-&kgr;B in I/R injury. Interestingly, infarct size and markers of tissue damage were not affected by administration of recombinant HMGB1 or HMGB1 antagonists in RAGE−/− mice, which demonstrated significantly reduced damage in reperfused hearts compared with wild-type mice. Coincubation studies using recombinant HMGB1 in vitro induced an inflammatory response in isolated macrophages from wild-type mice but not in macrophages from RAGE−/− mice. Conclusions— HMGB1 plays a major role in the early event of I/R injury by binding to RAGE, resulting in the activation of proinflammatory pathways and enhanced myocardial injury. Therefore, blockage of HMGB1 might represent a novel therapeutic strategy in I/R injury.

Loss of pain perception in diabetes is dependent on a receptor of the immunoglobulin superfamily

Molecular events that result in loss of pain perception are poorly understood in diabetic neuropathy. Our results show that the receptor for advanced glycation end products (RAGE), a receptor associated with sustained NF-kappaB activation in the diabetic microenvironment, has a central role in sensory neuronal dysfunction. In sural nerve biopsies, ligands of RAGE, the receptor itself, activated NF-kappaBp65, and IL-6 colocalized in the microvasculature of patients with diabetic neuropathy. Activation of NF-kappaB and NF-kappaB-dependent gene expression was upregulated in peripheral nerves of diabetic mice, induced by advanced glycation end products, and prevented by RAGE blockade. NF-kappaB activation was blunted in RAGE-null (RAGE(-/-)) mice compared with robust enhancement in strain-matched controls, even 6 months after diabetes induction. Loss of pain perception, indicative of long-standing diabetic neuropathy, was reversed in WT mice treated with soluble RAGE. Most importantly, loss of pain perception was largely prevented in RAGE(-/-) mice, although they were not protected from diabetes-induced loss of PGP9.5-positive plantar nerve fibers. These data demonstrate, for the first time to our knowledge, that the RAGE-NF-kappaB axis operates in diabetic neuropathy, by mediating functional sensory deficits, and that its inhibition may provide new therapeutic approaches.

Critical role of RAGE and HMGB1 in inflammatory heart disease

Significance Myocardial inflammation leads in many cases to cardiomyopathy and contributes to progressive heart failure. The exact pathological mechanism of disease induction and progression in the setting of heart failure is unknown. High-mobility group box 1 (HMGB1), an evolutionarily abundant and highly conserved protein, promotes cardiac inflammation, and in turn immunity, as a damage-associated molecular pattern. HMGB1 stimulates immunity, at least in part, through interaction with its principal binding partner RAGE (receptor for advanced glycation end products). Here we show that HMGB1 and RAGE appear to be important components in cardiac troponin I-induced experimental autoimmune myocarditis as well as in patients with myocarditis. Both molecules represent potential drug targets and show significant potential in heart failure treatment. Autoimmune response to cardiac troponin I (TnI) induces inflammation and fibrosis in the myocardium. High-mobility group box 1 (HMGB1) is a multifunctional protein that exerts proinflammatory activity by mainly binding to receptor for advanced glycation end products (RAGE). The involvement of the HMGB1–RAGE axis in the pathogenesis of inflammatory cardiomyopathy is yet not fully understood. Using the well-established model of TnI-induced experimental autoimmune myocarditis (EAM), we demonstrated that both local and systemic HMGB1 protein expression was elevated in wild-type (wt) mice after TnI immunization. Additionally, pharmacological inhibition of HMGB1 using glycyrrhizin or anti-HMGB1 antibody reduced inflammation in hearts of TnI-immunized wt mice. Furthermore, RAGE knockout (RAGE-ko) mice immunized with TnI showed no structural or physiological signs of cardiac impairment. Moreover, cardiac overexpression of HMGB1 using adeno-associated virus (AAV) vectors induced inflammation in the hearts of both wt and RAGE-ko mice. Finally, patients with myocarditis displayed increased local and systemic HMGB1 and soluble RAGE (sRAGE) expression. Together, our study highlights that HMGB1 and its main receptor, RAGE, appear to be crucial factors in the pathogenesis of TnI-induced EAM, because inhibition of HMGB1 and ablation of RAGE suppressed inflammation in the heart. Moreover, the proinflammatory effect of HMGB1 is not necessarily dependent on RAGE only. Other receptors of HMGB1 such as Toll-like receptors (TLRs) may also be involved in disease pathogenesis. These findings could be confirmed by the clinical relevance of HMGB1 and sRAGE. Therefore, blockage of one of these molecules might represent a novel therapeutic strategy in the treatment of autoimmune myocarditis and inflammatory cardiomyopathy.

PKCβ modulates ischemia-reperfusion injury in the heart

Protein kinase C-betaII (PKCbetaII) is an important modulator of cellular stress responses. To test the hypothesis that PKCbetaII modulates the response to myocardial ischemia-reperfusion (I/R) injury, we subjected mice to occlusion and reperfusion of the left anterior descending coronary artery. Homozygous PKCbeta-null (PKCbeta(-/-)) and wild-type mice fed the PKCbeta inhibitor ruboxistaurin displayed significantly decreased infarct size and enhanced recovery of left ventricular (LV) function and reduced markers of cellular necrosis and serum creatine phosphokinase and lactate dehydrogenase levels compared with wild-type or vehicle-treated animals after 30 min of ischemia followed by 48 h of reperfusion. Our studies revealed that membrane translocation of PKCbetaII in LV tissue was sustained after I/R and that gene deletion or pharmacological blockade of PKCbeta protected ischemic myocardium. Homozygous deletion of PKCbeta significantly diminished phosphorylation of c-Jun NH(2)-terminal mitogen-activated protein kinase and expression of activated caspase-3 in LV tissue of mice subjected to I/R. These data implicate PKCbeta in I/R-mediated myocardial injury, at least in part via phosphorylation of JNK, and suggest that blockade of PKCbeta may represent a potent strategy to protect the vulnerable myocardium.

HMGB1 Is Associated with Atherosclerotic Plaque Composition and Burden in Patients with Stable Coronary Artery Disease

Objectives The role of inflammation in atherosclerosis is widely appreciated. High mobility group box 1 (HMGB1), an injury-associated molecular pattern molecule acting as a mediator of inflammation, has recently been implicated in the development of atherosclerosis. In this study, we sought to investigate the association of plasma HMGB1 with coronary plaque composition in patients with suspected or known coronary artery disease (CAD). Design HMGB1, high sensitive troponin T (hsTnT) and high sensitive C-reactive protein (hsCRP) were determined in 152 consecutive patients with suspected or known stable CAD who underwent clinically indicated 256-slice coronary computed tomography angiography (CCTA). Using CCTA, we assessed 1) coronary calcification, 2) non-calcified plaque burden and 3) the presence of vascular remodeling in areas of non-calcified plaques. Results Using univariate analysis, hsCRP, hsTnT and HMGB1 as well as age, and atherogenic risk factors were associated with non-calcified plaque burden (r = 0.21, p = 0.009; r = 0.48, p<0.001 and r = 0.34, p<0.001, respectively). By multivariate analysis, hsTnT and HMGB1 remained independent predictors of the non-calcified plaque burden (r = 0.48, p<0.01 and r = 0.34, p<0.001, respectively), whereas a non-significant trend was noticed for hs-CRP (r = 0.21, p = 0.07). By combining hsTnT and HMGB1, a high positive predictive value for the presence of non-calcified and remodeled plaque (96% and 77%, respectively) was noted in patients within the upper tertiles for both biomarkers, which surpassed the positive predictive value of each marker separately. Conclusions In addition to hs-TnT, a well-established cardiovascular risk marker, HMGB1 is independently associated with non-calcified plaque burden in patients with stable CAD, while the predictive value of hs-CRP is lower. Complementary value was observed for hs-TnT and HMGB1 for the prediction of complex coronary plaque.

Deletion of bone marrow‐derived receptor for advanced glycation end products inhibits atherosclerotic plaque progression

Eur J Clin Invest 2011; 41 (11): 1164–1171

Central Role of PKCβ in Neointimal Expansion Triggered by Acute Arterial Injury

We tested the hypothesis that PKC&bgr; contributes to vascular smooth muscle cell (SMC) migration and proliferation; processes central to the pathogenesis of restenosis consequent to vascular injury. Homozygous PKC&bgr; null (−/−) mice or wild-type mice fed the PKC&bgr; inhibitor, ruboxistaurin, displayed significantly decreased neointimal expansion in response to acute femoral artery endothelial denudation injury compared with controls. In vivo and in vitro analyses demonstrated that PKC&bgr;II is critically linked to SMC activation, at least in part via regulation of ERK1/2 MAP kinase and early growth response-1. These data highlight novel roles for PKC&bgr; in the SMC response to acute arterial injury and suggest that blockade of PKC&bgr; may represent a therapeutic strategy to limit restenosis.

AGE-modified albumin containing infusion solutions boosts septicaemia and inflammation in experimental peritonitis

HSA preparations for i.v. use are administered in critically ill patients. Although increasing intravascular osmotic pressure seems to be a pathophysiologically orientated treatment, clinical trials do not indicate a benefit for mortality in HSA–treated patients. Instead, there is evidence for inflammatory reactions upon infusion of different HSA batches. A neglected issue concerning the safety and quality of these therapeutics is processing–related post–transcriptional protein modifications, such as AGEs. We therefore tested the hypothesis that commercially available infusion solutions contain AGEs and studied whether these protein modifications influence outcome and inflammation in a murine model of sepsis induced by CLP. Screening of different HSA and Ig preparations in this study revealed an up to approximate tenfold difference in the amount of AGE modifications. Application of clinically relevant concentrations of CML–modified HSA in CLP led to increased inflammation and enhanced mortality in wild–type mice but not in mice lacking the RAGE. Lethality was paralleled by increased activation of the proinflammatory transcription factor NF–κB, NF–κB–dependent gene expression, and infiltration of inflammatory cells in the peritoneal cavity. This study implies that infusion solutions containing a high load of the AGE–modified protein have the potential to activate RAGE/NF–κB–mediated inflammatory reactions, causing increased mortality in experimental peritonitis.

Short-Term Akt Activation in Cardiac Muscle Cells Improves Contractile Function in Failing Hearts

Akt is a serine/threonine protein kinase that is activated by a variety of growth factors or cytokines in a phosphatidylinositol 3-kinase-dependent manner. By using a conditional transgenic system in which Akt signaling can be turned on or off in the adult heart, we previously showed that short-term Akt activation induces a physiological form of cardiac hypertrophy with enhanced coronary angiogenesis and maintained contractility. Here we tested the hypothesis that induction of physiological hypertrophy by short-term Akt activation might improve contractile function in failing hearts. When Akt signaling transiently was activated in murine hearts with impaired contractility, induced by pressure overload or doxorubicin treatment, contractile dysfunction was attenuated in both cases. Importantly, improvement of contractility was observed before the development of cardiac hypertrophy, indicating that Akt activation improves contractile function independently of its growth-promoting effects. To gain mechanistic insights into Akt-mediated positive inotropic effects, transcriptional profiles in the heart were determined in a pressure overload-induced heart failure model. Biological network analysis of differentially expressed transcripts revealed significant alterations in the expression of genes associated with cell death, and these alterations were reversed by short-term Akt activation. Thus, short-term Akt activation improves contractile function in failing hearts. This beneficial effect of Akt on contractility is hypertrophy-independent and may be mediated in part by inhibition of cell death associated with heart failure.