Christian J. Hoffmann

Regulation of Foxo‐1 and the angiopoietin‐2/Tie2 system by shear stress

Transcription factor Foxo‐1 can be inactivated via Akt‐mediated phosphorylation. Since shear stress activates Akt, we determined whether Foxo‐1 and the Foxo‐1‐dependent, angiogenesis‐related Ang‐2/Tie2‐system are influenced by shear stress in endothelial cells. Expression of Foxo‐1 and its target genes p27Kip1 and Ang‐2 was decreased under shear stress (6 dyn/cm2, 24 h), nuclear exclusion of Foxo‐1 by phosphorylation increased. eNOS and Tie2 were upregulated. No effects on Ang‐1 expression were detected. In conclusion, Foxo‐1 and Ang‐2/Tie2 are part of the molecular response to shear stress, which may regulate angiogenesis.

Up‐regulation of the peroxiredoxin‐6 related metabolism of reactive oxygen species in skeletal muscle of mice lacking neuronal nitric oxide synthase

Although neuronal nitric oxide synthase (nNOS) plays a substantial role in skeletal muscle physiology, nNOS‐knockout mice manifest an only mild phenotypic malfunction in this tissue. To identify proteins that might be involved in adaptive responses in skeletal muscle of knockout mice lacking nNOS, 2D‐PAGE with silver‐staining and subsequent tandem mass spectrometry (LC‐MS/MS) was performed using extracts of extensor digitorum longus muscle (EDL) derived from nNOS‐knockout mice in comparison to C57Bl/6 control mice. Six proteins were significantly (P≤ 0.05) more highly expressed in EDL of nNOS‐knockout mice than in that of C57 control mice, all of which are involved in the metabolism of reactive oxygen species (ROS). These included prohibitin (2.0‐fold increase), peroxiredoxin‐3 (1.9‐fold increase), Cu2+/Zn2+‐dependent superoxide dismutase (SOD; 1.9‐fold increase), heat shock protein β‐1 (HSP25; 1.7‐fold increase) and nucleoside diphosphate kinase B (2.6‐fold increase). A significantly higher expression (4.1‐fold increase) and a pI shift from 6.5 to 5.9 of peroxiredoxin‐6 in the EDL of nNOS‐knockout mice were confirmed by quantitative immunoblotting. The concentrations of the mRNA encoding five of these proteins (the exception being prohibitin) were likewise significantly (P≤ 0.05) higher in the EDL of nNOS‐knockout mice. A higher intrinsic hydrogen peroxidase activity (P≤ 0.05) was demonstrated in EDL of nNOS‐knockout mice than C57 control mice, which was related to the presence of peroxiredoxin‐6. The treatment of mice with the chemical NOS inhibitor l‐NAME for3 days induced a significant 3.4‐fold up‐regulation of peroxiredoxin‐6 in the EDL of C57 control mice (P≤ 0.05), but did not alter its expression in EDL of nNOS‐knockout mice. ESR spectrometry demonstrated the levels of superoxide to be 2.5‐times higher (P≤ 0.05) in EDL of nNOS‐knockout mice than in C57 control mice while an in vitro assay based on the emission of 2,7‐dichlorofluorescein fluorescence disclosed the concentration of ROS to be similar in both strains of mice. We suggest that the up‐regulation of proteins that are implicated in the metabolism of ROS, particularly of peroxiredoxin‐6, within skeletal muscles of nNOS‐knockout mice functionally compensates for the absence of nNOS in scavenging of superoxide.

Expression of ADAMTS1 in endothelial cells is induced by shear stress and suppressed in sprouting capillaries.

ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3‐kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP‐1. The anti‐angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70‐kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress‐treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress‐treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non‐perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress‐dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis. J. Cell. Physiol. 226: 350–361, 2011.

Vascular Signal Transducer and Activator of Transcription-3 Promotes Angiogenesis and Neuroplasticity Long-Term After Stroke

Center for Stroke Research Berlin, Charite-Universitatsmedizin Berlin, Germany; Dept of Neurology, Charite-Universitatsmedizin Berlin, Germany; Max-Delbruck Center for Molecular Medicine, Berlin, Germany; Institute of Clinical Neurobiology, University Hospital, University of Wurzburg, Germany; Cluster of Excellence NeuroCure, Charite-Universitatsmedizin Berlin, Germany; German Center for Neurodegenerative Diseases (DZNE), Partner Site, Berlin, Germany; German Center for Cardiovascular Diseases (DZHK), Partner Site, Berlin, GermanyBackground— Poststroke angiogenesis contributes to long-term recovery after stroke. Signal transducer and activator of transcription-3 (Stat3) is a key regulator for various inflammatory signals and angiogenesis. It was the aim of this study to determine its function in poststroke outcome. Methods and Results— We generated a tamoxifen-inducible and endothelial-specific Stat3 knockout mouse model by crossbreeding Stat3floxed/KO and Tie2-CreERT2 mice. Cerebral ischemia was induced by 30 minutes of middle cerebral artery occlusion. We demonstrated that endothelial Stat3 ablation did not alter lesion size 2 days after ischemia but did worsen functional outcome at 14 days and increase lesion size at 28 days. At this late time point vascular Stat3 expression and phosphorylation were still increased in wild-type mice. Gene array analysis of a CD31-enriched cell population of the neurovascular niche showed that endothelial Stat3 ablation led to a shift toward an antiangiogenic and axon growth-inhibiting micromilieu after stroke, with an increased expression of Adamts9. Remodeling and glycosylation of the extracellular matrix and microglia proliferation were increased, whereas angiogenesis was reduced. Conclusions— Endothelial Stat3 regulates angiogenesis, axon growth, and extracellular matrix remodeling and is essential for long-term recovery after stroke. It might serve as a potent target for stroke treatment after the acute phase by fostering angiogenesis and neuroregeneration.

Vascular Stat3 Promotes Angiogenesis and Neuroplasticity Long-Term After Stroke

Center for Stroke Research Berlin, Charite-Universitatsmedizin Berlin, Germany; Dept of Neurology, Charite-Universitatsmedizin Berlin, Germany; Max-Delbruck Center for Molecular Medicine, Berlin, Germany; Institute of Clinical Neurobiology, University Hospital, University of Wurzburg, Germany; Cluster of Excellence NeuroCure, Charite-Universitatsmedizin Berlin, Germany; German Center for Neurodegenerative Diseases (DZNE), Partner Site, Berlin, Germany; German Center for Cardiovascular Diseases (DZHK), Partner Site, Berlin, GermanyBackground— Poststroke angiogenesis contributes to long-term recovery after stroke. Signal transducer and activator of transcription-3 (Stat3) is a key regulator for various inflammatory signals and angiogenesis. It was the aim of this study to determine its function in poststroke outcome. Methods and Results— We generated a tamoxifen-inducible and endothelial-specific Stat3 knockout mouse model by crossbreeding Stat3floxed/KO and Tie2-CreERT2 mice. Cerebral ischemia was induced by 30 minutes of middle cerebral artery occlusion. We demonstrated that endothelial Stat3 ablation did not alter lesion size 2 days after ischemia but did worsen functional outcome at 14 days and increase lesion size at 28 days. At this late time point vascular Stat3 expression and phosphorylation were still increased in wild-type mice. Gene array analysis of a CD31-enriched cell population of the neurovascular niche showed that endothelial Stat3 ablation led to a shift toward an antiangiogenic and axon growth-inhibiting micromilieu after stroke, with an increased expression of Adamts9. Remodeling and glycosylation of the extracellular matrix and microglia proliferation were increased, whereas angiogenesis was reduced. Conclusions— Endothelial Stat3 regulates angiogenesis, axon growth, and extracellular matrix remodeling and is essential for long-term recovery after stroke. It might serve as a potent target for stroke treatment after the acute phase by fostering angiogenesis and neuroregeneration.

Suppression of zinc finger protein 580 by high oxLDL/LDL-ratios is followed by enhanced expression of endothelial IL-8.

OBJECTIVE The Interleukin 8 (IL-8) response of endothelial cells to lipoproteins has well known implications for the development and progression of atherosclerosis. In this study we sought for the role of zinc finger protein 580 (ZNF580) in the endothelial IL-8 response to lipoproteins. METHODS In human umbilical vein endothelial cells (HUVEC) ZNF580 and IL-8 levels were examined by real-time-RT-PCR, immunoblotting and immunostaining or ELISA, respectively. RESULTS ZNF580 is located in the nucleus and regulated by LDL and HDL depending on the oxLDL/LDL-ratio but not by TNFα. IL-8 expression profiles are inversely influenced by the oxLDL/LDL-ratio, both in vitro and in vivo. Knock down of ZNF580 enhances the expression and release of IL-8 and increases monocyte arrest under flow conditions in vitro. CONCLUSIONS ZNF580 is a novel factor in the lipoprotein-dependent regulation of IL-8 and monocyte arrest. Therefore it may be a new potential target for intervention in atherosclerosis.

Passive Entrapment of Tumor Cells Determines Metastatic Dissemination to Spinal Bone and Other Osseous Tissues.

During the metastatic process tumor cells circulate in the blood stream and are carried to various organs. In order to spread to different organs tumor cell—endothelial cell interactions are crucial for extravasation mechanisms. It remains unclear if tumor cell dissemination to the spinal bone occurs by passive entrapment of circulating tumor cells or by active cellular mechanisms mediated by cell surface molecules or secreted factors. We investigated the seeding of three different tumor cell lines (melanoma, lung and prostate carcinoma) to the microvasculature of different organs. Their dissemination was compared to biologically passive microbeads. The spine and other organs were resected three hours after intraarterial injection of tumor cells or microbeads. Ex vivo homogenization and fluorescence analysis allowed quantification of tumor cells or microbeads in different organs. Interestingly, tumor cell distribution to the spinal bone was comparable to dissemination of microbeads independent of the tumor cell type (melanoma: 5.646% ± 7.614%, lung: 6.007% ± 1.785%, prostate: 3.469% ± 0.602%, 7 μm beads: 9.884% ± 7.379%, 16 μm beads: 7.23% ± 1.488%). Tumor cell seeding differed significantly between tumor cells and microbeads in all soft tissue organs. Moreover, there were significant differences between the different tumor cell lines in their dissemination behaviour to soft tissue organs only. These findings demonstrate that metastatic dissemination of tumor cells to spinal bone and other osseous organs is mediated by passive entrapment of tumor cells similar to passive plugging of microvasculature observed after intraarterial microbeads injection.