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Dirk M Hermann

Publications and source records attributed to Dirk M Hermann.

At least 19 recordsLinked to original sources
PubMed2024

Proteomic Characterization of 1000 Human and Murine Neutrophils Freshly Isolated From Blood and Sites of Sterile Inflammation.

Neutrophils are indispensable for defense against pathogens. Injured tissue-infiltrated neutrophils can establish a niche of chronic inflammation and promote degeneration. Studies investigated transcriptome of single-infiltrated neutrophils which could misinterpret molecular states of these post mitotic cells. However, neutrophil proteome characterization has been challenging due to low harvests from affected tissues. Here, we present a workflow to obtain proteome of 1000 murine and human tissue-infiltrated neutrophils. We generated spectral libraries containing ∼6200 mouse and ∼5300 human proteins from circulating neutrophils. 4800 mouse and 3400 human proteins were recovered from 1000 cells with 102-108 copies/cell. Neutrophils from stroke-affected mouse brains adapted to the glucose-deprived environment with increased mitochondrial activity and ROS-production, while cells invading inflamed human oral cavities increased phagocytosis and granule release. We provide an extensive protein repository for resting human and mouse neutrophils, identify proteins lost in low input samples, thus enabling the proteomic characterization of limited tissue-infiltrated neutrophils.

Proteomics↗
PubMed2006

Human vascular endothelial growth factor protects axotomized retinal ganglion cells in vivo by activating ERK-1/2 and Akt pathways.

Based on its trophic effects on neurons and vascular cells, vascular endothelial growth factor (VEGF) is a promising candidate for the treatment of neurodegenerative diseases. To evaluate the therapeutic potential of VEGF, we here examined effects of this growth factor on the degeneration of axotomized retinal ganglion cells (RGCs), which, as CNS-derived neurons, offer themselves in an excellent way to study neuroprotection in vivo. Making use of a transgenic mouse line that constitutively expresses human VEGF under a neuron-specific enolase promoter, we show that (1) the VEGF-transgenic retina overexpresses human VEGF, (2) RGCs carry the VEGF receptor-2, and (3) vascular networks in normal and axotomized VEGF-transgenic (tg) retinas do not differ from control animals. After axotomy, RGCs of VEGF-tg mice were protected against delayed degeneration, as compared with wild-type littermates. Western blots revealed increased phosphorylated ERK-1/2 and Akt and reduced phosphorylated p38 and activated caspase-3 levels in axotomized VEGF-transgenic retinas. Intravitreous injections of pharmacological ERK-1/2 (PD98059) or Akt (LY294002) inhibitors showed that VEGF exerts neuroprotection by dual activation of ERK-1/2 and Akt pathways. In view that axotomy-induced RGC death occurs slowly and considering that RGCs are CNS-derived neurons, we predict the clinical implementation of VEGF in neurodegenerative diseases of both brain and retina.

Animals↗
PubMed2006

Increased balloon-induced inflammation, proliferation, and neointima formation in apolipoprotein E (ApoE) knockout mice.

BACKGROUND AND PURPOSE: The pathophysiology of vascular lesions after balloon angioplasty remains poorly understood. A major limitation of most experimental studies in this regard is that injury was assessed in healthy arteries. Our aim was to study the effects of hypercholesterolemia in a mouse vascular injury model that mimics human balloon angioplasty. METHODS: Carotid balloon distension was performed in wild-type (WT) mice on a normal diet (ND), in apolipoprotein E-deficient (ApoE-/-) mice on ND and in ApoE-/- mice fed a high cholesterol diet (CD). RESULTS: Medial cell death (TUNEL) was elevated in all mice at 1 hour and 1 day after angioplasty without differences between the groups. We found enhanced intimal inflammation (%CD45-positive cells) and vascular cell adhesion molecule-1 expression at 7 days (P < 0.05; n > or = 4) as well as increased proliferation rates (BrdU-index) in ApoE-/- CD at 7 and 28 days postinjury (P < 0.05; n > or = 5). Four weeks after injury, these events led to enhanced neointima in ApoE-/- CD compared with WT ND mice (intima/media, P < 0.001; n > or = 8). The amount of lesion formation paralleled the incremental increase in total plasma cholesterol in WT ND, ApoE-/- ND and ApoE-/- CD (P < 0.01). CONCLUSIONS: Carotid balloon distension injury in ApoE-/- mice on CD induced enhanced inflammation and proliferation leading to increased neointima. Further applications of this microballoon catheter in genetically modified mice will provide opportunities to elucidate molecular mechanisms of vascular lesion formation in a model that reflects clinical balloon angioplasty. This know-how may pave the way to catheter-based interventions of human microvessels in the peripheral or cerebral circulation.

Angioplasty, Balloon↗
PubMed2006

TAT fusion proteins against ischemic stroke: current status and future perspectives.

The delivery of proteins across the blood-brain barrier is severely limited by the proteins' size and biochemical properties. Thus, a large number of peptides have been characterized in recent years that efficaciously prevent neuronal death in vitro, but which may not be applied in vivo, since they are unable to cross cell membrane barriers. In the 1990ies, it had been shown that the HIV TAT protein is able to cross cell membranes even when coupled with larger peptides. Subsequent studies with fusion proteins of the 11-amino acid protein transduction domain of HIV-TAT revealed that TAT fusion proteins may successfully be used for therapeutic purposes in vivo, even when systemically applied. Indeed, intravenous delivery of TAT proteins linked with anti-apoptotic (Bcl-XL) and neurotrophic (GDNF) factors resulted in a rapid and highly efficacious transduction of the brain tissue. When administered after focal cerebral ischemia, intravenous TAT-Bcl-XL and TAT-GDNF significantly reduced brain injury, both when applied after severe and mild ischemic insults. These data provided the first in vivo evidence of the efficacy of fusion proteins in the ischemic brain, thus raising new hopes that neuroprotection is feasible after stroke. Yet, molecular biological studies have pointed out more recently that there are also limitations of the TAT protein strategy, which still need to be addressed. The development of clinically-applicable treatments for ischemic stroke based on fusion protein technologies deserves concerted efforts in the future.

Apoptosis↗
PubMed2006

The phosphatidylinositol-3 kinase/Akt pathway mediates VEGF's neuroprotective activity and induces blood brain barrier permeability after focal cerebral ischemia.

Based on its trophic influence on neurons and vascular cells, vascular endothelial growth factor (VEGF) is a promising candidate for stroke treatment. VEGF's survival-promoting effects are purchased at the expense of an increased blood brain barrier permeability, which potentially compromises tissue survival. The mechanisms via which VEGF protects the brain against ischemia remained unknown. We examined signaling pathways underlying VEGF's neuroprotective activity in our transgenic mouse line, which expresses human VEGF165 under a neuron-specific enolase (NSE) promoter. We show that VEGF receptor-2 (Flk-1) is expressed on ischemic neurons and astrocytes and is activated by VEGF. Following 90-min episodes of middle cerebral artery occlusion, VEGF increased phosphorylated (but not total) Akt and ERK-1/-2 and reduced phosphorylated mitogen activated protein kinase/p38 and c-Jun NH2-terminal kinase (JNK)-1/-2 levels, at the same time decreasing inducible NO synthase expression in ischemic neurons. Inhibition of Akt with Wortmannin reversed VEGF's neuroprotective properties, diminished brain swelling, and restored the vascular permeability induced by VEGF to below levels in WT animals. The aggravation of brain injury by Wortmannin was associated with the restitution of p38, but not of JNK-1/-2, ERK-1/-2, or inducible NOS (iNOS). Our data demonstrate that VEGF mediates both neuroprotection and blood brain barrier permeability via the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Based on our observation that VEGF neuroprotection and vascular leakage depend on PI3K/Akt, which is putatively regulated by VEGF receptor-2, we predict that it may not easily be possible to make use of VEGF's neuroprotective function without accepting its unfavorable consequence, the increased vascular permeability.

Androstadienes↗
PubMed2006

Inhibition of multidrug resistance transporter-1 facilitates neuroprotective therapies after focal cerebral ischemia.

The blood-brain barrier possesses active transporters carrying brain-permeable xenobiotics back into the blood against concentration gradients. We demonstrate that multidrug resistance transporter (Mdr)-1 is upregulated on capillary endothelium after focal cerebral ischemia; moreover, Mdr-1 deactivation by pharmacological inhibition or genetic knockout preferably enhances the accumulation and efficacy of two neuroprotectants known as Mdr-1 substrates in the ischemic brain. We predict that Mdr-1 inhibition may greatly facilitate neuroprotective therapies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Role of drug efflux carriers in the healthy and diseased brain.

The blood-brain barrier is a natural diffusion barrier, which expresses active carriers extruding drugs on their way to the brain back into the blood against concentration gradients. Whereas these so-called adenosine triphosphate-binding cassette (ABC) transporters prevent the brain entry of toxic compounds under physiological conditions, they complicate pharmacotherapies in neurological disease. Recent observations in animal models of ischemic stroke, drug-resistant epilepsy, and brain cancer showed that the prototype of ABC transporters, ABCB1, is upregulated on brain injury, deactivation of this carrier considerably enhancing the accumulation of neuroprotective, antiepileptic, and chemotherapeutic compounds. These studies provide the proof of concept that the efficacy of brain-targeting drugs may significantly be improved when drug efflux is blocked. Under clinical conditions, efforts currently are made to enhance drug accumulation by selecting new compounds that do not bind to efflux carriers or deactivating ABC transporters by targeted downregulation or pharmacological inhibition. We predict that strategies aiming at circumventing drug efflux may greatly facilitate progress in neurological therapies.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

Sleep EEG changes after middle cerebral artery infarcts in mice: different effects of striatal and cortical lesions.

STUDY OBJECTIVES: Hemispheric stroke in humans is associated with sleep-wake disturbances and sleep electroencephalogram (EEG) changes. The correlation between these changes and stroke extent remains unclear. In the absence of experimental data, we assessed sleep EEG changes after focal cerebral ischemia of different extensions in mice. DESIGN: Following electrode implantation and baseline sleep-wake EEG recordings, mice were submitted to sham surgery (control group), 30 minutes of intraluminal middle cerebral artery (MCA) occlusion (striatal stroke), or distal MCA electrocoagulation (cortical stroke). One and 12 days after stroke, sleep-wake EEG recordings were repeated. The EEG recorded from the healthy hemisphere was analyzed visually and automatically (fast Fourier analysis) according to established criteria. MEASUREMENTS AND RESULTS: Striatal stroke induced an increase in non-rapid eye movement (NREM) sleep and a reduction of rapid eye movement sleep. These changes were detectable both during the light and the dark phase at day 1 and persisted until day 12 after stroke. Cortical stroke induced a less-marked increase in NREM sleep, which was present only at day 1 and during the dark phase. In cortical stroke, the increase in NREM sleep was associated in the wake EEG power spectra, with an increase in the theta and a reduction in the beta activity. CONCLUSION: Cortical and striatal stroke lead to different sleep-wake EEG changes in mice, which probably reflect variable effects on sleep-promoting and wakefulness-maintaining neuronal networks.

Animals↗
PubMed2005

Blood pressure evolution after acute ischemic stroke in patients with and without sleep apnea.

BACKGROUND AND PURPOSE: Sleep apnea (SA) is an independent risk factor for arterial hypertension and is present in 50% to 70% of patients with ischemic stroke. The effects of SA on blood pressure (BP) and stroke outcome in the acute stroke phase are essentially unknown. METHODS: We studied 41 consecutive patients admitted within 96 hours after stroke onset. Stroke severity on admission (National Institutes of Health Stroke Scale [NIHSS]) and stroke outcome at discharge (modified Rankin Disability Scale [mRS]) were assessed. Nocturnal breathing was assessed with an ambulatory device the first night after admission. SA was defined by an apnea-hypopnea-index (AHI) > or =10/hour, and moderate-severe SA (MSSA) was defined by an AHI >30/hour. BP monitoring was performed during the first 36 hours after admission. A nondipping status (NDS) was defined by a ratio >0.9 of mean systolic BP during nights 1 to 2/mean systolic BP during day 2. RESULTS: SA was found in 28 (68%) and MSSA in 11 (27%) of 41 patients. A correlation was found between AHI and both NIHSS (r=0.331; P=0.035) and mRS (r=0.341; P=0.031). Patients with MSSA had higher systolic and diastolic BP values during night 1 (P=0.003), day 2 (P=0.004), and night 2 (P=0.03). NDS was found in 26 (63%) patients. Nondippers had a similar AHI but higher NIHSS (P=0.004) and mRS (P=0.005) than dippers. AHI and NDS were confirmed to be independent predictors for both stroke severity and stroke outcome in a multiple stepwise linear regression model. CONCLUSIONS: SA severity is associated with high 24-hour BP values but only weakly with stroke severity and outcome. Conversely, NDS is linked with a more severe stroke and a poorer evolution but not with SA severity. These data suggest different, although overlapping, pathophysiological and clinical implications of circadian and nocturnal BP values in acute stroke.

Adult↗
PubMed2005

Brain-derived erythropoietin protects from focal cerebral ischemia by dual activation of ERK-1/-2 and Akt pathways.

Apart from its hematopoietic function, erythropoietin (Epo) exerts neuroprotective functions in brain hypoxia and ischemia. To examine the mechanisms mediating Epo's neuroprotective activity in vivo, we made use of our transgenic mouse line tg21 that constitutively expresses human Epo in brain without inducing excessive erythrocytosis. We show that human Epo is expressed in tg21 brains and that cortical and striatal neurons carry the Epo receptor. After middle cerebral artery occlusion, human Epo potently protected brains of tg21 mice against ischemic injury, both when severe (90 min) and mild (30 min) ischemia was imposed. Histochemical studies revealed that Epo induced an activation of JAK-2, ERK-1/-2, and Akt pathways in the ischemic brain. This activation was associated with elevated Bcl-XL and decreased NO synthase-1 and -2 levels in neurons. Intracerebroventricular injections of selective inhibitors of ERK-1/-2 (PD98059) or Akt (wortmannin) pathways revealed that both ERK-1/-2 and Akt were required for Epo's neuroprotective function, antagonization of either pathway completely abolishing tissue protection. On the other hand, ERK-1/-2 and Akt blockade did not reverse the neuronal NO synthase-1/-2 inhibition, indicating that Epo down-regulates these NO synthases in an ERK-1/-2 and Akt independent manner. On the basis of our data, the dual activation of ERK-1/-2 and Akt is crucial for Epo's neuroprotective activity.

Androstadienes↗
PubMed2005

Aggravation of ischemic brain injury by prion protein deficiency: role of ERK-1/-2 and STAT-1.

The cellular isoform of prion protein, PrPc, may confer neuroprotection in the brain, according to recent studies. To elucidate the role of PrPc in stroke pathology, we subjected PrPc-knockout (Prnp(0/0)), wild-type and PrPc-transgenic (tga20) mice to 30 min of intraluminal middle cerebral artery occlusion, followed by 3, 24 or 72 h reperfusion, and examined how PrPc levels influence brain injury and cell signaling. In immunohistochemical experiments and Western blots, we show that PrPc expression is absent in the brains of Prnp(0/0) mice, detectable in wild-type controls and approximately 4.0-fold elevated in tga20 mice. We provide evidence that PrPc deficiency increases infarct size by approximately 200%, while transgenic PrPc restores tissue viability, albeit not above levels in wild-type animals. To elucidate the mechanisms underlying Prnp(0/0)-induced injury, we performed Western blots, which revealed increased activities of ERK-1/-2, STAT-1 and caspase-3 in ischemic brains of Prnp(0/0)mice. Our data suggest a role of cytosolic signaling pathways in Prnp(0/0)-induced cell death.

Animals↗
PubMed2005

TAT-GDNF in neurodegeneration and ischemic stroke.

The delivery of proteins across the blood-brain barrier is severely limited by their size and biochemical properties. Numerous peptides have been characterized in recent years that prevent neuronal death in vitro, but cannot be used therapeutically, since they do not cross cell membrane barriers. It has been shown in the 1990s that the HIV TAT protein is able to cross cell membranes even when coupled with larger peptides. It appears, therefore, that TAT fusion proteins may enter the brain, even when used systemically. Indeed, the systemic delivery of a TAT protein linked with glial-derived neurotrophic factor (GDNF) successfully transduced central nervous system (CNS) neurons in mice. When administered after optic nerve transection and focal cerebral ischemia, TAT-GDNF protected retinal ganglion cells and brain neurons from cell death, elevated tissue Bcl-XL levels and attenuated the activity of the executioner caspase-3. These findings demonstrate the in vivo efficacy of fusion proteins in clinically relevant disease models, raising hopes that neuroprotection may become eventually feasible in human patients.

Animals↗
PubMed2005

Signal transduction pathways involved in melatonin-induced neuroprotection after focal cerebral ischemia in mice.

Because of its favorable action profile in humans, melatonin is a particularly interesting candidate as a neuroprotectant in acute ischemic stroke. Until now, the signaling mechanisms mediating melatonin's neuroprotective actions remained essentially uninvestigated. Herein, we examined the effects of melatonin, administered either orally for 9 wk as a stroke prophylactic (4 mg/kg/day) or intraperitoneally immediately after reperfusion onset (4 mg/kg), on the activation of signal transduction pathways in mice submitted to 90 min of intraluminal middle cerebral artery occlusion, followed by 24 hr of reperfusion. In these studies, melatonin significantly reduced ischemic infarct size by approximately 30-35%, as compared with animals receiving diluent (sham) treatment, independent of whether the indole was administered prior to or after ischemia. Under both conditions, animals receiving melatonin exhibited elevated phosphorylated Akt levels in their brains, as determined by Western blots. Additionally, phosphorylation levels of mitogen-activated protein kinase/extracellular-regulated kinase (ERK)-1/-2 and Jun kinase (JNK)-1/-2 were increased following prophylactic, but not acute, melatonin treatment. Our data suggest a role of phosphatidyl inositol-3 kinase/Akt signaling in acute melatonin-induced neuroprotection, while ERK-1/-2 and/or JNK-1/-2 rather appear to be involved in melatonin's long-term effects.

Animals↗
PubMed2005

Tissue-plasminogen activator-induced ischemic brain injury is reversed by melatonin: role of iNOS and Akt.

In vivo studies showed that tissue-plasminogen activator (t-PA) may aggravate neuronal injury after focal cerebral ischemia. We hypothesized that t-PA impairs survival-promoting cell signaling in the ischemic brain, which may be reversed by a neuroprotectant, i.e. melatonin. We examined the effects of t-PA (10 mg/kg, i.v.), administered alone or in combination with melatonin (4 mg/kg, i.p.), on ischemic injury, inducible nitric oxide synthase (iNOS) expression as well as Akt, Bcl-X(L) and caspase-3 signaling following 90 min of intraluminal middle cerebral artery (MCA) occlusion in mice. t-PA, delivered immediately after reperfusion onset, increased infarct volume at 24 hr after MCA occlusion, in accordance with previous findings. Melatonin reduced infarct size when administered alone and reversed the t-PA-induced brain injury. Immunohistochemical studies showed that t-PA treatment was associated with an accumulation of iNOS positive cells in ischemic brain areas, which was abolished after co-delivery of melatonin. Western blots revealed that t-PA decreased phosphorylated Akt levels, but did not influence Bcl-X(L) expression and caspase-3 activity in ischemic brain lysates. Co-treatment with melatonin restored phosphorylated Akt levels, increased Bcl-X(L) expression and reduced caspase-3 activity. We provide evidence that t-PA-induced brain injury is accompanied by an activation of iNOS and inhibition of phosphatidylinositol-3 kinase/Akt. That melatonin reversed these signaling changes and the t-PA-induced brain injury makes this indole attractive as an add-on treatment with thrombolytics.

Animals↗
PubMed2005

Tissue plasminogen activator-induced ischemic injury is reversed by NMDA antagonist MK-801 in vivo.

In vitro studies suggested that tissue plasminogen activator (t-PA) may aggravate ischemic injury by enhancing N-methyl-D-aspartate (NMDA) receptor signalling. It remained unclear whether NMDA signalling is also relevant for t-PA toxicity in vivo. We herein examined effects of intravenous t-PA (10 mg/kg), administered alone or in combination with the NMDA antagonist MK-801 (0.2 mg/kg), following 90 min of middle cerebral artery occlusion in mice. In our study, MK-801 alone, administered intraperitoneally, neither affected infarct volume nor brain swelling at 24 h after reperfusion. t-PA significantly increased infarct size, in accordance with previous findings. t-PA-induced ischemic injury was completely abolished and brain swelling markedly reduced when t-PA-treated animals received additional MK-801 injections. To elucidate how t-PA influences brain damage, we examined actions of t-PA on the expression of NO synthases by immunohistochemistry, showing that t-PA does not influence neuronal NO synthase, but increases inducible NO synthase in ischemic areas. The effect of t-PA on inducible NO synthase levels was completely reversed after cotreatment with MK-801. Our study provides in vivo evidence in a model of focal cerebral ischemia that t-PA-induced brain injury involves an NMDA receptor-dependent mechanism.

Analysis of Variance↗
PubMed2004

Aggravation of focal cerebral ischemia by tissue plasminogen activator is reversed by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor but does not depend on endothelial NO synthase.

BACKGROUND AND PURPOSE: It has repeatedly been reported that the thrombolytic tissue plasminogen activator (tPA) may aggravate ischemic injury after stroke. The underlying mechanisms, however, remain unknown. We hypothesized that tPA induces an inhibition of endothelial NO synthase (eNOS) after focal ischemia that is responsible for ischemic damage and may be restored by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. METHODS: We examined the effects of tPA, administered either alone or in combination with rosuvastatin, on ischemic injury, on eNOS expression, and cell signaling after 90 minutes of intraluminal middle cerebral artery occlusion. RESULTS: In wild-type mice, tPA delivered immediately after ischemia significantly increased infarct volume 24 hours after reperfusion. Coadministration of rosuvastatin completely reversed the tPA-induced brain damage. Western blots of ischemic brain lysates showed that tPA markedly diminished eNOS levels, increased extracellular regulated kinase (ERK)-2, and decreased MAP kinase/p38 activity. Cotreatment with rosuvastatin prevented the decrease in eNOS, reduced ERK-1/-2 and normalized p38 levels. To elucidate the role of eNOS in tPA-induced ischemic injury, we also evaluated tPA effects in eNOS knockout mice. In eNOS knockout animals, tPA again significantly increased infarct size after transient focal ischemia. CONCLUSIONS: In a mouse model of focal cerebral ischemia, tPA induces eNOS inhibition, ERK-2 activation, and p38 inhibition, possibly as part of a more complex signaling response exacerbating brain injury. 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition reverses the effects of tPA by a mechanism independent of eNOS.

Animals↗
PubMed2004

Erythropoietin protects from axotomy-induced degeneration of retinal ganglion cells by activating ERK-1/-2.

Apart from its hematopoietic function, erythropoietin (Epo) exerts neuroprotective activity upon reduced oxygenation or ischemia of brain, retina, and spinal cord. To examine whether Epo has an impact on the retrograde degeneration of retinal ganglion cells (RGCs) following optic nerve transection in vivo, we made use of our transgenic mouse line tg21 that constitutively expresses human Epo preferentially in neuronal cells without inducing polycythemia. We show that the tg21 retina expresses human Epo and that RGCs in this mouse line carry the Epo receptor. Upon axotomy, the RGCs of Epo transgenic tg21 mice were protected against degeneration, as compared with wild-type control animals. Western blot analysis revealed decreased phosphorylation levels of STAT-5 and reduced expression of Bcl-XL in RGCs of axotomized tg21 animals, suggesting that the corresponding pathways are not crucial for Epo's neuroprotective activity. Increased phosphorylation levels of ERK-1/-2 and Akt, as well as decreased caspase-3 activity, however, were observed in injured tg21 retinae. Injection of selective inhibitors of ERK-1/-2 (PD98059) or Akt (Wortmannin) pathways into the vitreous space revealed that transgenic Epo protected the RGCs by a pathway involving ERK-1/-2 but not Akt. In view that axotomy-induced degeneration of RGC occurs slowly, and considering the earlier data on the safety and efficacy of Epo in human stroke patients, we predict the clinical implementation of recombinant human Epo not only in patients with acute ischemic stroke, but also with more delayed degenerative neurological diseases.

Animals↗