Sigrid Nikol

Endogenous brain protection by granulocyte-colony stimulating factor after ischemic stroke.

Several lines of evidence have demonstrated beneficial effects of the hematopoietic factor G-CSF in experimental stroke. A conclusive demonstration of this effect in G-CSF deficient mice is, however, lacking. We therefore investigated the effect of G-CSF deficiency on infarct volumes, functional recovery, mRNA and protein expression of the matrix metalloproteinase 9 (MMP-9) after stroke. Furthermore we tested the efficacy of G-CSF substitution in G-CSF deficient animals to prevent the potential consequences of G-CSF deficiency. In the present study experimental stroke was induced in female non-treated wildtype (wt), G-CSF deficient mice and G-CSF substituted G-CSF deficient mice followed by assessment of infarct volumes, neurological outcome and sensorimotor function. In addition, immunohistochemistry and real-time PCR of the peri-ischemic area were performed. G-CSF deficient mice showed increased infarct volumes, whereas G-CSF substituted mice had a remarkable reduction in lesion size compared to wt mice. These findings are accompanied by an improvement in neurological and sensorimotor function. G-CSF deficiency resulted in an upregulation of MMP-9 in the direct peri-ischemic tissue. Treatment with G-CSF suppressed the upregulation of MMP-9. Taken together, G-CSF deficiency clearly resulted in enlarged infarct volumes, and worsened neurological outcome. G-CSF substitution abolished these negative effects, led to significant reduced lesion volumes, and improved neurological outcome. G-CSF mediated suppression of MMP-9 further demonstrates that endogenous G-CSF plays a significant role in brain protective mechanisms. We have shown for the first time that endogenous G-CSF is required for brain recovery mechanisms after stroke.

Up-regulation of nestin in the infarcted myocardium potentially indicates differentiation of resident cardiac stem cells into various lineages including cardiomyocytes.

To identify proteins involved in cardiac regeneration, a proteomics approach was applied. A total of 26 proteins, which displayed aberrant expression in mouse hearts infarcted through ligation of the left anterior descending coronary artery, were identified. These included the intermediate filament protein nestin, which was up‐regulated in the infarct border zone. Corresponding changes were observed for its mRNA. Nestin mRNA was also up‐regulated in hearts from 17 of 19 patients with end‐stage heart failure, including 4 with acute myocardial infarction in comparison with 8 donor hearts. Immunofluorescence confo‐cal laser scanning microscopy revealed that nestin is expressed, on the one hand, in small proportions of cardiomyocytes, endothelial cells, smooth muscle cells, neuronal cells, and fibroblasts. On the other hand, it was found to be coexpressed with the stem cell markers c‐kit, Sca‐1, Mdr‐1, and Abcg2 in small interstitial cells. In infarcted hearts from chimeric mice transplanted with bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice, less than 1% of nestin‐positive cells coexpressed EGFP, although EGFP‐positive cells were abundant in these. Consequently, enhanced expression of nestin in the injured myocardium might reflect spontaneous regenerative processes supposedly based on the differentiation of resident cardiac stem cells into diverse cardiac cell types. Scobioala, S., Klocke, R., Kuhlmann, M., Tian, W., Hasib, L., Milting, H., Koenig, S., Stelljes, M., El‐Banayosy, A., Tenderich, G., Michel, G., Breithardt, G., Nikol, S. Up‐regulation of nestin in the infarcted myocardium potentially indicates differentiation of resident cardiac stem cells into various lineages including cardiomyo‐cytes. FASEB J. 22, 1021–1031 (2008)

Effects of G-CSF treatment on neutrophil mobilization and neurological outcome after transient focal ischemia

Several recent studies demonstrated beneficial effects of G-CSF treatment (granulocyte colony-stimulating factor) in various CNS disease. Possible mechanisms underlying this activity are neuroprotection, anti-apoptosis, angiogenesis and anti-inflammation. Hence, we investigated the efficacy of G-CSF administration in experimental stroke by determining infarct volume and neurological score in wildtype, G-CSF-deficient and G-CSF-treated G-CSF-deficient mice. Besides, cerebral ischemia is followed by an upregulation of endothelial adhesion molecules which promote leukocyte recruitment to the injured area. In combination with G-CSF-induced leukocytosis, increased peripheral neutrophils could aggregate within microvasculature and additionally impair blood perfusion of the ischemic tissue. Therefore, we analyzed the neutrophil counts in both vessel and tissue compartment 2 and 5 days post-stroke by immunohistochemistry. Here we show that G-CSF deficiency leads to increased infarct volumes, whereas G-CSF substitution revokes detrimental effects by reducing lesion size and enhancing neurological outcome compared to untreated animals. Administration of G-CSF is accompanied by significant increase of circulating neutrophils 2 days post-ischemia but leukocytosis is restricted to the vessel compartment and has no deleterious effect on lesion formation and functional recovery. These observations are likely to be important for therapeutic targeting of G-CSF-mediated neuroprotection in stroke.

Paclitaxel Delivered to Adventitia Attenuates Neointima Formation without Compromising Re-Endothelialization after Angioplasty in a Porcine Restenosis Model

Purpose: To investigate the effect of paclitaxel delivered into the adventitia of pig femoral arteries on neointima formation and hyperplasia as well as re-endothelialization. Methods: Paclitaxel or vehicle was delivered into the adventitia of pig femoral arteries using a needle injection catheter following balloon overstretch. Arteries were then serially examined by angiography, Evans blue staining, morphometry, and immunohistochemistry for up to 12 weeks. Results: Local adventitial delivery of paclitaxel significantly attenuated neointima formation. The area of neointima (0.41±0.17 versus 2.75±0.81 mm2, p<0.01), the ratio of intima to media (0.12±0.05 versus 0.86±0.35, p<0.05), and the degree of stenosis (12.80%±3.13% versus 47.06%±7.25%, p<0.01) were significantly lower in the paclitaxel-treated group compared to controls. Furthermore, cell proliferation was significantly diminished following adventitial delivery of paclitaxel from day 3 to 21 compared to controls. Complete reendothelialization was observed 3 weeks after intervention in both groups of arteries treated with paclitaxel or vehicle alone. Conclusion: Paclitaxel delivered into the adventitia of pig femoral arteries effectively attenuates neointima formation after angioplasty without compromising re-endothelialization. Adventitial drug delivery may therefore be an alternative to drug-eluting stents for the prevention of restenosis.

Vascular Endothelial Growth Factor (VEGF165) and its Influence on Angiogenesis versus Arteriogenesis in Different Vascular Beds

PURPOSE To use local gene delivery to determine any district-specific influence of vascular endothelial growth factor (VEGF(165)) on angiogenesis and arteriogenesis in arteries of distinct developmental origin. METHODS Coronary and peripheral arteries were chronically occluded in 30 Pietrain pigs using a percutaneous approach and blinded stent-graft. DNA was delivered to the adventitia in dosages corresponding to 10% of the body weight-adapted amount used in clinical trials. The coronary arteries in 12 animals and the peripheral arteries in 12 animals were treated or used as controls (no occlusion or occlusion with transfection of the beta-galactosidase gene). Six additional animals were sacrificed at 1 or 3 weeks for expression analyses, while the other 24 animals were sacrificed at 5 months for expression analysis and histology. Angiography, polymerase chain reaction analyses, and immunohistochemistry were performed. RESULTS Expression of the VEGF gene was observed at 1 and 3 weeks following application, while transfected DNA was detected up to 5 months. New collaterals formed around occluded coronary arteries (2.63 +/- 0.69 fold, p<0.05 versus 1.24 +/- 0.40 fold for peripheral arteries), and angiographic arterial area increase was more pronounced in coronary (2.49 +/- 0.59 fold, p<0.05) than peripheral arteries (1.49 +/- 0.05 fold). There was no collateralization surrounding occluded peripheral arteries, but new arterial branches were seen (2.0 +/- 0.28, p<0.05 versus 1.07 +/- 0.31 for coronary). CONCLUSIONS The response to VEGF, whether it is predominantly angiogenesis or arteriogenesis, is dependent on the target vessel. These observed differences in the behavior of arteries may be related to their differing developmental origins, which may have important implications for future therapeutic strategies using VEGF in different vessels.

Activity of daily living is associated with circulating CD34+/KDR+ cells and granulocyte colony-stimulating factor levels in patients after myocardial infarction

The study aimed to investigate whether the extent of activities of daily living (ADL) of patients after myocardial infarction affect numbers of circulating CD34(+)/KDR(+) and CD45(+)/CD34(+) cells, which are supposed to protect structural and functional endothelial integrity. In a cross-sectional study, 34 male coronary artery disease patients with a history of myocardial infarction were assessed for times spent per week for specific physical ADL, including basic activities (instrumental ADL), leisure time activities, and sport activities, using a validated questionnaire. Individual specific activity times were multiplied with respective specific metabolic equivalent scores to obtain levels of specific activities. Numbers of circulating CD34(+)/KDR(+) and CD45(+)/CD34(+) cells were analyzed by flow cytometry. Furthermore, the colony-forming capacity of CD34(+) cells and the level of granulocyte colony-stimulating factor (G-CSF) in serum were measured. Analysis revealed that the extent of total activities and basic activities, as well as total activity time, were positively correlated with numbers of circulating CD34(+)/KDR(+) cells (r = 0.60, 0.56, and 0.55, P < 0.05). Higher levels of total activity were also associated with increased colony-forming capacity of CD34(+) cells (r = 0.54, P < 0.05) and with higher systemic levels of G-CSF (r = 0.44, P < 0.05). These findings indicate that even ADL-related activities of coronary artery disease patients after myocardial infarction exert stimulating effects on CD34(+)/KDR(+) cell mobilization, potentially mediated by increased G-CSF levels. This, in turn, potentially contributes to the beneficial effects of exercise on the diseased cardiovascular system.

First Report on an Inotropic Peptide Activating Tetrodotoxin-Sensitive, “Neuronal” Sodium Currents in the Heart

Background—New therapeutic approaches to improve cardiac contractility without severe risk would improve the management of acute heart failure. Increasing systolic sodium influx can increase cardiac contractility, but most sodium channel activators have proarrhythmic effects that limit their clinical use. Here, we report the cardiac effects of a novel positive inotropic peptide isolated from the toxin of the Black Judean scorpion that activates neuronal tetrodotoxin-sensitive sodium channels. Methods and Results—All venoms and peptides were isolated from Black Judean Scorpions (Buthotus Hottentotta) caught in the Judean Desert. The full scorpion venom increased left ventricular function in sedated mice in vivo, prolonged ventricular repolarization, and provoked ventricular arrhythmias. An inotropic peptide (BjIP) isolated from the full venom by chromatography increased cardiac contractility but did neither provoke ventricular arrhythmias nor prolong cardiac repolarization. BjIP increased intracellular calcium in ventricular cardiomyocytes and prolonged inactivation of the cardiac sodium current. Low concentrations of tetrodotoxin (200 nmol/L) abolished the effect of BjIP on calcium transients and sodium current. BjIP did not alter the function of Nav1.5, but selectively activated the brain-type sodium channels Nav1.6 or Nav1.3 in cellular electrophysiological recordings obtained from rodent thalamic slices. Nav1.3 (SCN3A) mRNA was detected in human and mouse heart tissue. Conclusions—Our pilot experiments suggest that selective activation of tetrodotoxin-sensitive neuronal sodium channels can safely increase cardiac contractility. As such, the peptide described here may become a lead compound for a new class of positive inotropic agents.

Recently Patented Applications of Homologous Cellular and Extracellular Agents as Therapeutics or Targets for the Prevention of Restenosis Post- Angioplasty

Currently available drug-eluting stents have been shown to reduce the prevalence of in-stent restenosis. However, their use is limited by their enormous cost and unwanted side effects associated with both drugs, sirolimus and paclitaxel, presently used to coat most of the stents clinically available. Due to their lack of selectivity with respect to targeted cell types these drugs do not only inhibit vascular smooth muscle cell proliferation underlying neointima formation, they also compromise endothelial repair increasing the risk for subacute thrombosis following implantation of drug-eluting stents. Accordingly, there is need for new cost-effective agents capable to inhibit restenosis without clinically relevant, unwanted side effects. In the present paper a selection of the most important patent applications published within the last 3 years and claiming the use of homologous cellular and extracellular agents as therapeutics or targets to prevent restenosis are reviewed. Such agents include c-Jun, the focal adhesion kinase (FAK) and its inhibitor FAK-related non-kinase (FRNK), estrogen receptors, variants of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) as well as some so far poorly characterized factors supposedly involved in the control of cell proliferation, inflammation and apoptosis. Such agents promise to be cost-effective and, in some cases, potentially devoid of unwanted side effects. Clinical long-term studies have yet to support such notions.

Proteomics: State of the Art and Its Relevance for Gene Therapy

The molecular mechanisms underlying most diseases, including those of the cardiovascular system, are widely unknown. Basically, pathological changes in the organism arise from protein alterations. Proteomics comprises a set of tools allowing the identification of protein alterations, i.e. changes of protein abundance and posttranslational modifications, associated with diseases. The linkage of information about such protein changes with functional alterations as revealed by physiological studies constitutes functional proteomics that enables the disclosure of disease mechanisms. Disease-linked protein alterations include those of suitable candidates for drug targets and disease biomarkers as well as therapeutic proteins/peptides. Since gene therapy depends on the function of a therapeutic protein encoded by a “therapeutic” gene, proteomic analyses provide the basis for the design and application of gene therapies. The storage and administration of experimental data obtained by the application of proteomic analyses is supported by speciesand tissue-specific protein databases and specific software. Publications in this field are reviewed in this chapter.

Stand der Gentherapie und der lokalen Medikamentenapplikation im kardiovaskulären Bereich

Die Identifizierung angiogener Wachstumsfaktoren ermoglichte die Entwicklung neuartiger Strategien zur Behandlung chronischer Gefasverschlusse. Die therapeutische Angiogenese spielt unter den gentherapeutischen Moglichkeiten im kardiovaskularen Bereich die derzeit groste Rolle. Sie stellt eine alternative Therapie fur Patienten mit einer fortgeschrittenen und therapierefraktaren ischamischen Herzerkrankung oder einer kritischen Beinischamie mit der Moglichkeit einer Kapillaraussprossung (Angiogenese) und einer Neubildung von Kollateralgefasen (Arteriogenese) dar. In-vitro- und experimentelle In-vivoUntersuchungen konnten die Effizienz der vermehrten Kollateralenbildung und der funktionellen Durchblutungsverbesserung bei der experimentellen myokardialen oder peripheren Ischamie sowohl fur rekombinante Gefaswachstumsfaktoren als auch fur gentherapeutische Strategien nachweisen. Die Vorteile gentherapeutischer Strategien sind die Minimierung systemischer Nebenwirkungen und die langsame und kontinuierliche Freisetzung des kodierten Faktors, was einen langer anhaltenden angiogenen Effekt erlaubt. In uberwiegend unkontrollierten klinischen Phase-I- und -Ha-Studien wurde gezeigt, dass Gefasbehandlungen mit Wachstumsfaktoren machbar, effizient und sicher sind. Die Ergebnisse placebokontrollierter und doppelblinder Studien mussen jedoch zur sicheren Beurteilung des therapeutischen Potenzials abgewartet werden. Die Weiterentwicklung der lokalen Medikamenten- und Genapplikation mittels spezieller Kathetersysteme wird in der Zukunft die Effizienz und Sicherheit der Gentherapie weiter verbessern konnen.