Innovative in vivo rat model for global cerebral hypoxia: a new approach to investigate therapeutic and preventive drugs.

Introduction: Severe acute global cerebral hypoxia can lead to significant disability in humans. Although different animal models have been described to study hypoxia, there is no endogenous model that considers hypoxia and its effect on the brain as an independent factor. Thus, we developed a minimally invasive rat model, which is based on the non-depolarizing muscle blocking agent rocuronium in anesthetized animals. This drug causes respiratory insufficiency by paralysis of the striated muscles. Methods: In this study, 14 rats underwent 12 min of hypoxemia with an oxygen saturation of approximately 60% measured by pulse oximetry; thereafter, animals obtained sugammadex to antagonize rocuronium immediately. Results: Compared to controls (14 rats, anesthesia only), hypoxic animals demonstrated significant morphological alterations in the hippocampus (cell decrease in the CA 1 region) and the cerebellum (Purkinje cell decrease), as well as significant changes in hypoxia markers in blood (Hif2α, Il1ß, Tgf1ß, Tnfα, S100b, cspg2, neuron-specific enolase), hippocampus (Il1ß, Tnfα, S100b, cspg2, NSE), and cerebellum (Hif1α, Tnfα, S100b, cspg2, NSE). Effects were more pronounced in females than in males. Discussion: Consequently, this model is suitable to induce hypoxemia with consecutive global cerebral hypoxia. As significant morphological and biochemical changes were proven, it can be used to investigate therapeutic and preventive drugs for global cerebral hypoxia.

Targeting actin inhibits repair of doxorubicin-induced DNA damage: a novel therapeutic approach for combination therapy.

Severe side effects often restrict clinical application of the widely used chemotherapeutic drug doxorubicin. In order to decrease required substance concentrations, new concepts for successful combination therapy are needed. Since doxorubicin causes DNA damage, combination with compounds that modulate DNA repair could be a promising strategy. Very recently, a role of nuclear actin for DNA damage repair has been proposed, making actin a potential target for cancer therapy in combination with DNA-damaging therapeutics. This is of special interest, since actin-binding compounds have not yet found their way into clinics. We find that low-dose combination treatment of doxorubicin with the actin polymerizer chondramide B (ChB) synergistically inhibits tumor growth in vivo. On the cellular level we demonstrate that actin binders inhibit distinctive double strand break (DSB) repair pathways. Actin manipulation impairs the recruitment of replication factor A (RPA) to the site of damage, a process crucial for homologous recombination. In addition, actin binders reduce autophosphorylation of DNA-dependent protein kinase (DNA-PK) during nonhomologous end joining. Our findings substantiate a direct involvement of actin in nuclear DSB repair pathways, and propose actin as a therapeutic target for combination therapy with DNA-damaging agents such as doxorubicin.

Endothelial autophagic flux hampers atherosclerotic lesion development.

Blood flowing in arteries generates shear forces at the surface of the vascular endothelium that control its anti-atherogenic properties. However, due to the architecture of the vascular tree, these shear forces are heterogeneous and atherosclerotic plaques develop preferentially in areas where shear is low or disturbed. Here we review our recent study showing that elevated shear forces stimulate endothelial autophagic flux and that inactivating the endothelial macroautophagy/autophagy pathway promotes a proinflammatory, prosenescent and proapoptotic cell phenotype despite the presence of atheroprotective shear forces. Specific deficiency in endothelial autophagy in a murine model of atherosclerosis stimulates the development of atherosclerotic lesions exclusively in areas of the vasculature that are normally resistant to atherosclerosis. Our findings demonstrate that adequate endothelial autophagic flux limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence and inflammation.

Autophagy is required for endothelial cell alignment and atheroprotection under physiological blood flow.

It has been known for some time that atherosclerotic lesions preferentially develop in areas exposed to low SS and are characterized by a proinflammatory, apoptotic, and senescent endothelial phenotype. Conversely, areas exposed to high SS are protected from plaque development, but the mechanisms have remained elusive. Autophagy is a protective mechanism that allows recycling of defective organelles and proteins to maintain cellular homeostasis. We aimed to understand the role of endothelial autophagy in the atheroprotective effect of high SS. Atheroprotective high SS stimulated endothelial autophagic flux in human and murine arteries. On the contrary, endothelial cells exposed to atheroprone low SS were characterized by inefficient autophagy as a result of mammalian target of rapamycin (mTOR) activation, AMPKα inhibition, and blockade of the autophagic flux. In hypercholesterolemic mice, deficiency in endothelial autophagy increased plaque burden only in the atheroresistant areas exposed to high SS; plaque size was unchanged in atheroprone areas, in which endothelial autophagy flux is already blocked. In cultured cells and in transgenic mice, deficiency in endothelial autophagy was characterized by defects in endothelial alignment with flow direction, a hallmark of endothelial cell health. This effect was associated with an increase in endothelial apoptosis and senescence in high-SS regions. Deficiency in endothelial autophagy also increased TNF-α-induced inflammation under high-SS conditions and decreased expression of the antiinflammatory factor KLF-2. Altogether, these results show that adequate endothelial autophagic flux under high SS limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence, and inflammation.

Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice.

Deep venous thrombosis (DVT) is one of the most common cardiovascular diseases, but its pathophysiology remains incompletely understood. Although sterile inflammation has recently been shown to boost coagulation during DVT, the underlying molecular mechanisms are not fully resolved, which could potentially identify new anti-inflammatory approaches to prophylaxis and therapy of DVT. Using a mouse model of venous thrombosis induced by flow reduction in the vena cava inferior, we identified blood-derived high-mobility group box 1 protein (HMGB1), a prototypical mediator of sterile inflammation, to be a master regulator of the prothrombotic cascade involving platelets and myeloid leukocytes fostering occlusive DVT formation. Transfer of platelets into Hmgb1-/- chimeras showed that this cell type is the major source of HMGB1, exposing reduced HMGB1 on their surface upon activation thereby enhancing the recruitment of monocytes. Activated leukocytes in turn support oxidation of HMGB1 unleashing its prothrombotic activity and promoting platelet aggregation. This potentiates the amount of HMGB1 and further nurtures the accumulation and activation of monocytes through receptor for advanced glycation end products (RAGE) and Toll-like receptor 2, leading to local delivery of monocyte-derived tissue factor and cytokines. Moreover, disulfide HMGB1 facilitates formation of prothrombotic neutrophil extracellular traps (NETs) mediated by RAGE, exposing additional HMGB1 on their extracellular DNA strands. Eventually, a vicious circle of coagulation and inflammation is set in motion leading to obstructive DVT formation. Therefore, platelet-derived disulfide HMGB1 is a central mediator of the sterile inflammatory process in venous thrombosis and could be an attractive target for an anti-inflammatory approach for DVT prophylaxis.

In vitro and in vivo characterization of the actin polymerizing compound chondramide as an angiogenic inhibitor.

AIMS: Inhibiting angiogenesis is a major approach in tumour therapy. To combat angiogenesis, the tubulin cytoskeleton has emerged as an interesting target in many pre- and clinical studies. Contrarily, the actin cytoskeleton has been largely neglected as a potential drug target in angiogenesis. However, due to the development of drug resistances, new therapeutic strategies are always needed in tumour treatment. Therefore, the therapeutic potential of actin-binding small molecules is of particular interest. METHODS AND RESULTS: We investigate the impact of chondramide (Ch), an actin polymerizing myxobacterial compound, on angiogenesis and underlying signalling. Chondramide treatment not only reduces the migration of endothelial cells but also the maturation of endothelial tube networks on matrigel. These observations can partly be explained by a disintegration of stress fibres due to aggregation and subsequent accumulation of actin in cellular structures known as 'aggresomes'. Chondramide treatment impairs the maturation of focal adhesions and reduces the amount of active ß1 integrin at the cell surface. Accordingly, signalling events downstream of focal adhesions are reduced. Thus, we observed that the activity of Src and downstream factors Rho-GTPases Rac1 and Rho is reduced upon Ch treatment. In vivo, Ch was well tolerated in mice and vascularization of a tumour xenograft as well as of the developing retina was significantly reduced. CONCLUSION: Chondramide diminishes angiogenesis via two ways: (i) the disintegration of stress fibres and (ii) the reduction of promigratory signals. Our findings highlight Ch as a novel class of therapeutic lead compound with anti-angiogenic potential.