Bernhard Dorweiler

Adaptive suppression of the ATF4–CHOP branch of the unfolded protein response by toll-like receptor signalling

The endoplasmic reticulum (ER) unfolded protein response (UPR) restores equilibrium to the ER, but prolonged expression of the UPR effector CHOP (GADD153) is cytotoxic. We found that CHOP expression induced by ER stress was suppressed by prior engagement of toll-like receptor (TLR) 3 or 4 through a TRIF-dependent pathway. TLR engagement did not suppress phosphorylation of PERK or eIF-2α, which are upstream of CHOP, but phospho-eIF-2α failed to promote translation of the CHOP activator ATF4. In mice subjected to systemic ER stress, pretreatment with low dose lipopolysaccharide (LPS), a TLR4 ligand, suppressed CHOP expression and apoptosis in splenic macrophages, renal tubule cells and hepatocytes, and prevented renal dysfunction and hepatosteatosis. This protective effect of LPS did not occur in Trif−/− mice or in wild-type mice in which CHOP expression was genetically restored. Thus, TRIF-mediated signals from TLRs selectively attenuate translational activation of ATF4 and its downstream target gene CHOP. We speculate that this mechanism evolved to promote survival of TLR-expressing cells that experience prolonged levels of physiological ER stress in the course of the host response to invading pathogens.

Immunohistochemical Demonstration of Enzymatically Modified Human LDL and Its Colocalization With the Terminal Complement Complex in the Early Atherosclerotic Lesion

Treatment of low density lipoprotein (LDL) with degrading enzymes transforms the molecule to a moiety that is micromorphologically indistinguishable from lipoproteinaceous particles that are present in atherosclerotic plaques, and enzymatically modified LDL (E-LDL), but not oxidized LDL (ox-LDL), spontaneously activates the alternative complement pathway, as do lesion lipoprotein derivatives. Furthermore, because E-LDL is a potent inducer of macrophage foam cell formation, we propose that enzymatic degradation may be the key process that renders LDL atherogenic. In this article, we report the production of two murine monoclonal antibodies recognizing cryptic epitopes in human apolipoprotein B that become exposed after enzymatic attack on LDL. One antibody reacted with LDL after single treatment with trypsin, whereas recognition by the second antibody required combined treatment of LDL with trypsin and cholesterol esterase. In ELISAs, both antibodies reacted with E-LDL produced in vitro and with lesion complement activator derived from human atherosclerotic plaques, but they were unreactive with native LDL or ox-LDL. The antibodies stained E-LDL, but not native LDL or ox-LDL, that had been artificially injected into arterial vessel walls. With the use of these antibodies, we have demonstrated that early human atherosclerotic coronary lesions obtained at autopsy as well as lesions examined in freshly explanted hearts always contain extensive extracellular deposits of E-LDL. Terminal complement complexes, detected with a monoclonal antibody specific for a C5b-9 neoepitope, colocalized with E-LDL within the intima, which is compatible with the proposal that subendothelially deposited LDL is enzymatically transformed to a complement activator at the earliest stages in lesion development.

Extracellular Nampt Promotes Macrophage Survival via a Nonenzymatic Interleukin-6/STAT3 Signaling Mechanism *

Macrophages play key roles in obesity-associated pathophysiology, including inflammation, atherosclerosis, and cancer, and processes that affect the survival-death balance of macrophages may have an important impact on obesity-related diseases. Adipocytes and other cells secrete a protein called extracellular nicotinamide phosphoribosyltransferase (eNampt; also known as pre-B cell colony enhancing factor or visfatin), and plasma levels of eNampt increase in obesity. Herein we tested the hypothesis that eNampt could promote cell survival in macrophages subjected to endoplasmic reticulum (ER) stress, a process associated with obesity and obesity-associated diseases. We show that eNampt potently blocks macrophage apoptosis induced by a number of ER stressors. The mechanism involves a two-step sequential process: rapid induction of interleukin 6 (IL-6) secretion, followed by IL-6-mediated autocrine/paracrine activation of the prosurvival signal transducer STAT3. The ability of eNampt to trigger this IL-6/STAT3 cell survival pathway did not depend on the presence of the Nampt enzymatic substrate nicotinamide in the medium, could not be mimicked by the Nampt enzymatic product nicotinamide mononucleotide (NMN), was not blocked by the Nampt enzyme inhibitor FK866, and showed no correlation with enzyme activity in a series of site-directed mutant Nampt proteins. Thus, eNampt protects macrophages from ER stress-induced apoptosis by activating an IL-6/STAT3 signaling pathway via a nonenzymatic mechanism. These data suggest a novel action and mechanism of eNampt that could affect the balance of macrophage survival and death in the setting of obesity, which in turn could play important roles in obesity-associated diseases.

Atherogenic Properties of Enzymatically Degraded LDL: Selective Induction of MCP-1 and Cytotoxic Effects on Human Macrophages

The mechanisms underlying the selective accumulation of macrophages in early atherosclerotic lesions are poorly understood but are likely to be related to specific properties of altered low density lipoprotein (LDL) deposited in the subendothelium. Enzymatic, nonoxidative degradation of LDL converts the lipoprotein to a potentially atherogenic moiety, enzymatically altered LDL (E-LDL), which activates complement and is rapidly taken up by human macrophages via a scavenger receptor-dependent pathway. Immunohistological evidence indicates that E-LDL is present in an extracellular location in the early lesion. We report that E-LDL causes massive release of monocyte chemotactic protein 1 (MCP-1) from macrophages and that expression of interleukin 8 or RANTES remains unchanged. Release of MCP-1 was preceded by a rapid expression of MCP-1 mRNA, which was detectable after 15 minutes, reached maximum levels after 1 hour, and remained detectable for 12 hours after exposure to concentrations as low as 10 microg/mL E-LDL. MCP-1 mRNA induction and protein release by E-LDL exceeded that evoked by oxidized LDL. Release of MCP-1 was dependent on de novo protein synthesis and on the activity of tyrosine kinases. At higher concentrations, E-LDL, but not oxidized LDL, exerted toxic effects on macrophages that in part appeared to be due to apoptosis. The results show that E-LDL possesses major properties of an atherogenic lipoprotein.

An imbalance between specialized pro-resolving lipid mediators and pro-inflammatory leukotrienes promotes instability of atherosclerotic plaques

Chronic unresolved inflammation plays a causal role in the development of advanced atherosclerosis, but the mechanisms that prevent resolution in atherosclerosis remain unclear. Here, we use targeted mass spectrometry to identify specialized pro-resolving lipid mediators (SPM) in histologically-defined stable and vulnerable regions of human carotid atherosclerotic plaques. The levels of SPMs, particularly resolvin D1 (RvD1), and the ratio of SPMs to pro-inflammatory leukotriene B4 (LTB4), are significantly decreased in the vulnerable regions. SPMs are also decreased in advanced plaques of fat-fed Ldlr−/− mice. Administration of RvD1 to these mice during plaque progression restores the RvD1:LTB4 ratio to that of less advanced lesions and promotes plaque stability, including decreased lesional oxidative stress and necrosis, improved lesional efferocytosis, and thicker fibrous caps. These findings provide molecular support for the concept that defective inflammation resolution contributes to the formation of clinically dangerous plaques and offer a mechanistic rationale for SPM therapy to promote plaque stability.

Endovascular Aortic Arch Repair After Aortic Arch De-branching

PURPOSE Conventional surgical therapy of aortic arch aneurysms consists of aortic arch replacement requiring cardiopulmonary bypass and deep hypothermic circulatory arrest. This method is associated with significant morbidity and mortality, mainly due to neurologic complications and the sequelae of deep hypothermic circulatory arrest. Thus, it makes sense to work on developing less invasive surgical techniques. DESCRIPTION Surgical aortic arch de-branching is required before the supra-aortic vessels can be safely covered by an endovascular stent graft. We describe how the supra-aortic vessels can best be revascularized, followed by complete coverage of the aortic arch with endovascular stent grafts. EVALUATION We hereby present our case selection criteria, preoperative work-up, and surgical approach for aortic arch de-branching with supra-aortic revascularization, followed by complete coverage of the aortic arch by endovascular stent grafting. This techniques safeguards and pitfalls are described for a cohort of 26 patients. CONCLUSIONS Endovascular aortic arch repair after aortic arch de-branching has the potential to lower the morbidity and mortality rates in patients with aortic arch diseases.

Ischemia-Reperfusion Injury

The term ischemia-reperfusion injury describes the experimentally and clinically prevalent finding that tissue ischemia with inadequate oxygen supply followed by successful reperfusion initiates a wide and complex array of inflammatory responses that may both aggravate local injury as well as induce impairment of remote organ function. Conditions under which ischemia-reperfusion injury is encountered include the different forms of acute vascular occlusions (stroke, myocardial infarction, limb ischemia) with the respective reperfusion strategies (thrombolytic therapy, angioplasty, operative revascularization) but also routine surgical procedures (organ transplantation, free-tissue-transfer, cardiopulmonary bypass, vascular surgery) and major trauma/shock. Since the first recognition of ischemia-reperfusion injury during the 1970s, significant knowledge has accumulated and the purpose of this review is to present an overview over the current literature on the molecular and cellular basis of ischemia-reperfusion injury, to outline the clinical manifestations and to compile contemporary treatment and prevention strategies. Although the concept of reperfusion injury is still a matter of debate, it is corroborated by recent and ongoing clinical trials that demonstrated ischemic preconditioning, inhibition of sodium-hydrogen-exchange and administration of adenosine to be effective in attenuating ischemia-reperfusion injury.

Subendothelial infiltration of neutrophil granulocytes and liberation of matrix-destabilizing enzymes in an experimental model of human neo-intima

It was the objective of this study to examine the role of human neutrophil granulocytes (PMN) in an in-vitro model of human neo-intima developed for the study of atherosclerosis. Human granulocytes were subjected to a co-culture model of human endothelial and smooth muscle cells. Subendothelial lipid accumulation was achieved by addition of native LDL to the culture medium. Tissue samples were analyzed by immunohistochemistry and scanning/transmission electron microscopy, and culture supernatants were examined for the presence of interleukin-8 (IL-8), MCP-1, GRO-alpha, elastase and matrixmetalloproteinase-8 (MMP-8). Following addition of 2 mg/ml LDL, adherence, transmigration and infiltration depth of PMN was increased significantly when compared to controls. LDL challenging was paralleled by a time- and dose-dependent secretion of IL-8 from intimal smooth muscle cells. PMN infiltration was mediated by the IL-8-signalling pathway and accompanied by release of elastase and MMP-8 into the supernatant and induction of endothelial cell apoptosis. In conclusion, LDL-induced secretion of IL-8 by intimal smooth muscle cells provides a potential mechanism of PMN-recruitment into culprit lesions. The concomitant release of potent matrix-degrading enzymes and the induction of EC apoptosis may have implications for plaque destabilization and cardiovascular events.

Long-Term Follow-Up After Endovascular Treatment of Acute Aortic Emergencies

PurposeTo investigate the long-term outcome and efficacy of emergency treatment of acute aortic diseases with endovascular stent-grafts.MethodsFrom September 1995 to April 2007, 37 patients (21 men, 16 women; age 53.9 ± 19.2 years, range 18–85 years) with acute complications of diseases of the descending thoracic aorta were treated by endovascular stent-grafts: traumatic aortic ruptures (n = 9), aortobronchial fistulas due to penetrating ulcer or hematothorax (n = 6), acute type B dissections with aortic wall hematoma, penetration, or ischemia (n = 13), and symptomatic aneurysm of the thoracic aorta (n = 9) with pain, penetration, or rupture. Diagnosis was confirmed by contrast-enhanced CT. Multiplanar reformations were used for measurement of the landing zones of the stent-grafts. Stent-grafts were inserted via femoral or iliac cut-down. Two procedures required aortofemoral bypass grafting prior to stent-grafting due to extensive arteriosclerotic stenosis of the iliac arteries. In this case the bypass graft was used for introduction of the stent-graft.ResultsA total of 46 stent-grafts were implanted: Vanguard/Stentor (n = 4), Talent (n = 31), and Valiant (n = 11). Stent-graft extension was necessary in 7 cases. In 3 cases primary graft extension was done during the initial procedure (in 1 case due to distal migration of the graft during stent release, in 2 cases due to the total length of the aortic aneurysm). In 4 cases secondary graft extensions were performed—for new aortic ulcers at the proximal stent struts (after 5 days) and distal to the graft (after 8 months) and recurrent aortobronchial fistulas 5 months and 9 years after the initial procedure—resulting in a total of 41 endovascular procedures. The 30-day mortality rate was 8% (3 of 37) and the overall follow-up was 29.9 ± 36.6 months (range 0–139 months). All patients with traumatic ruptures demonstrated an immediate sealing of bleeding. Patients with aortobronchial fistulas also demonstrated a satisfactory follow-up despite the necessity for reintervention and graft extension in 3 of 6 cases (50%). Two patients with type B dissection died due to mesenteric ischemia despite sufficient mesenteric blood flow being restored (but too late). Two suffered from neurologic complications, 1 from paraplegia and 1 from cerebral ischemia (probably embolic), 1 from penetrating ulcer, and 1 from persistent ischemia of the kidney. Five of 9 (56%) patients with symptomatic thoracic aneurysm demonstrated endoleaks during follow-up and there was an increase in the aneurysm in 1.ConclusionEndovascular treatment is safe and effective for emergency treatment of life-threatening acute thoracic aortic syndromes. Results are encouraging, particularly for traumatic aortic ruptures. However, regular follow-up is mandatory, particularly in the other pathologies, to identify late complications of the stent-graft and to perform appropriate additional corrections as required.

MerTK receptor cleavage promotes plaque necrosis and defective resolution in atherosclerosis

Atherothrombotic vascular disease is often triggered by a distinct type of atherosclerotic lesion that displays features of impaired inflammation resolution, notably a necrotic core and thinning of a protective fibrous cap that overlies the core. A key cause of plaque necrosis is defective clearance of apoptotic cells, or efferocytosis, by lesional macrophages, but the mechanisms underlying defective efferocytosis and its possible links to impaired resolution in atherosclerosis are incompletely understood. Here, we provide evidence that proteolytic cleavage of the macrophage efferocytosis receptor c-Mer tyrosine kinase (MerTK) reduces efferocytosis and promotes plaque necrosis and defective resolution. In human carotid plaques, MerTK cleavage correlated with plaque necrosis and the presence of ischemic symptoms. Moreover, in fat-fed LDL receptor–deficient (Ldlr–/–) mice whose myeloid cells expressed a cleavage-resistant variant of MerTK, atherosclerotic lesions exhibited higher macrophage MerTK, lower levels of the cleavage product soluble Mer, improved efferocytosis, smaller necrotic cores, thicker fibrous caps, and increased ratio of proresolving versus proinflammatory lipid mediators. These findings provide a plausible molecular-cellular mechanism that contributes to defective efferocytosis, plaque necrosis, and impaired resolution during the progression of atherosclerosis.