Damaged Neocortical Perineuronal Nets Due to Experimental Focal Cerebral Ischemia in Mice, Rats and Sheep

As part of the extracellular matrix (ECM), perineuronal nets (PNs) are polyanionic, chondroitin sulfate proteoglycan (CSPG)-rich coatings of certain neurons, known to be affected in various neural diseases. Although these structures are considered as important parts of the neurovascular unit (NVU), their role during evolution of acute ischemic stroke and subsequent tissue damage is poorly understood and only a few preclinical studies analyzed PNs after acute ischemic stroke. By employing three models of experimental focal cerebral ischemia, this study was focused on histopathological alterations of PNs and concomitant vascular, glial and neuronal changes according to the NVU concept. We analyzed brain tissues obtained 1 day after ischemia onset from: (a) mice after filament-based permanent middle cerebral artery occlusion (pMCAO); (b) rats subjected to thromboembolic MACO; and (c) sheep at 14 days after electrosurgically induced focal cerebral ischemia. Multiple fluorescence labeling was applied to explore simultaneous alterations of NVU and ECM. Serial mouse sections labeled with the net marker Wisteria floribunda agglutinin (WFA) displayed largely decomposed and nearly erased PNs in infarcted neocortical areas that were demarcated by up-regulated immunoreactivity for vascular collagen IV (Coll IV). Subsequent semi-quantitative analyses in mice confirmed significantly decreased WFA-staining along the ischemic border zone and a relative decrease in the directly ischemia-affected neocortex. Triple fluorescence labeling throughout the three animal models revealed up-regulated Coll IV and decomposed PNs accompanied by activated astroglia and altered immunoreactivity for parvalbumin, a calcium-binding protein in fast-firing GABAergic neurons which are predominantly surrounded by neocortical PNs. Furthermore, ischemic neocortical areas in rodents simultaneously displayed less intense staining of WFA, aggrecan, the net components neurocan, versican and the cartilage link protein (CRTL) as well as markers in net-bearing neurons such as the potassium channel subunit Kv3.1b and neuronal nuclei (NeuN). In summary, theconsistent observations based on three different stroke models confirmed that PNs are highly sensitive constituents of the NVU along with impaired associated GABAergic neurons. These results suggest that PNs could be promising targets of future stroke treatment, and further studies should address their reorganization and plasticity in both stabilizing the acute stroke as well as supportive effects during the chronic phase of stroke

Improved Anticancer Activities of a New Pentafluorothio-Substituted Vorinostat-Type Histone Deacetylase Inhibitor

The development of new anticancer drugs is necessary in order deal with the disease and with the drawbacks of currently applied drugs. Epigenetic dysregulations are a central hallmark of cancerogenesis and histone deacetylases (HDACs) emerged as promising anticancer targets. HDAC inhibitors are promising epigenetic anticancer drugs and new HDAC inhibitors are sought for in order to obtain potent drug candidates. The new HDAC inhibitor SF5-SAHA was synthesized and analyzed for its anticancer properties. The new compound SF5-SAHA showed strong inhibition of tumor cell growth with IC50 values similar to or lower than that of the clinically applied reference compound vorinostat/SAHA (suberoylanilide hydroxamic acid). Target specific HDAC inhibition was demonstrated by Western blot analyses. Unspecific cytotoxic effects were not observed in LDH-release measurements. Pro-apoptotic formation of reactive oxygen species (ROS) and caspase-3 activity induction in prostate carcinoma and hepatocellular carcinoma cell lines DU145 and Hep-G2 seem to be further aspects of the mode of action. Antiangiogenic activity of SF5-SAHA was observed on chorioallantoic membranes of fertilized chicken eggs (CAM assay). The presence of the pentafluorothio-substituent of SF5-SAHA increased the antiproliferative effects in both solid tumor and leukemia/lymphoma cell models when compared with its parent compound vorinostat. Based on this preliminary study, SF5-SAHA has the prerequisites to be further developed as a new HDAC inhibitory anticancer drug candidate

Coalescent angiogenesis—evidence for a novel concept of vascular network maturation

Angiogenesis describes the formation of new blood vessels from pre-existing vascular structures. While the most studied mode of angiogenesis is vascular sprouting, specific conditions or organs favor intussusception, i.e., the division or splitting of an existing vessel, as preferential mode of new vessel formation. In the present study, sustained (33-h) intravital microscopy of the vasculature in the chick chorioallantoic membrane (CAM) led to the hypothesis of a novel non-sprouting mode for vessel generation, which we termed "coalescent angiogenesis." In this process, preferential flow pathways evolve from isotropic capillary meshes enclosing tissue islands. These preferential flow pathways progressively enlarge by coalescence of capillaries and elimination of internal tissue pillars, in a process that is the reverse of intussusception. Concomitantly, less perfused segments regress. In this way, an initially mesh-like capillary network is remodeled into a tree structure, while conserving vascular wall components and maintaining blood flow. Coalescent angiogenesis, thus, describes the remodeling of an initial, hemodynamically inefficient mesh structure, into a hierarchical tree structure that provides efficient convective transport, allowing for the rapid expansion of the vasculature with maintained blood supply and function during development

Impaired Neurofilament Integrity and Neuronal Morphology in Different Models of Focal Cerebral Ischemia and Human Stroke Tissue

As part of the neuronal cytoskeleton, neurofilaments are involved in maintaining cellular integrity. In the setting of ischemic stroke, the affection of the neurofilament network is considered to mediate the transition towards long-lasting tissue damage. Although peripheral levels of distinct neurofilament subunits are shown to correlate with the clinically observed severity of cerebral ischemia, neurofilaments have so far not been considered for neuroprotective approaches. Therefore, the present study systematically addresses ischemia-induced alterations of the neurofilament light (NF-L), medium (NF-M), and heavy (NF-H) subunits as well as of α-internexin (INA). For this purpose, we applied a multi-parametric approach including immunofluorescence labeling, western blotting, qRT-PCR and electron microscopy. Analyses comprised ischemia-affected tissue from three stroke models of middle cerebral artery occlusion (MCAO), including approaches of filament-based MCAO in mice, thromboembolic MCAO in rats, and electrosurgical MCAO in sheep, as well as human autoptic stroke tissue. As indicated by altered immunosignals, impairment of neurofilament subunits was consistently observed throughout the applied stroke models and in human tissue. Thereby, altered NF-L immunoreactivity was also found to reach penumbral areas, while protein analysis revealed consistent reductions for NF-L and INA in the ischemia-affected neocortex in mice. At the mRNA level, the ischemic neocortex and striatum exhibited reduced expressions of NF-L- and NF-H-associated genes, whereas an upregulation for Ina appeared in the striatum. Further, multiple fluorescence labeling of neurofilament proteins revealed spheroid and bead-like structural alterations in human and rodent tissue, correlating with a cellular edema and lost cytoskeletal order at the ultrastructural level. Thus, the consistent ischemia-induced affection of neurofilament subunits in animals and human tissue, as well as the involvement of potentially salvageable tissue qualify neurofilaments as promising targets for neuroprotective strategies. During ischemia formation, such approaches may focus on the maintenance of neurofilament integrity, and appear applicable as co-treatment to modern recanalizing strategies

Increased Immunosignals of Collagen IV and Fibronectin Indicate Ischemic Consequences for the Neurovascular Matrix Adhesion Zone in Various Animal Models and Human Stroke Tissue

Ischemic stroke causes cellular alterations in the "neurovascular unit" (NVU) comprising neurons, glia, and the vasculature, and affects the blood-brain barrier (BBB) with adjacent extracellular matrix (ECM). Limited data are available for the zone between the NVU and ECM that has not yet considered for neuroprotective approaches. This study describes ischemia-induced alterations for two main components of the neurovascular matrix adhesion zone (NMZ), i.e., collagen IV as basement membrane constituent and fibronectin as crucial part of the ECM, in conjunction with traditional NVU elements. For spatio-temporal characterization of these structures, multiple immunofluorescence labeling was applied to tissues affected by focal cerebral ischemia using a filament-based model in mice (4, 24, and 72 h of ischemia), a thromboembolic model in rats (24 h of ischemia), a coagulation-based model in sheep (2 weeks of ischemia), and human autoptic stroke tissue (3 weeks of ischemia). An increased fibronectin immunofluorescence signal demarcated ischemia-affected areas in mice, along with an increased collagen IV signal and BBB impairment indicated by serum albumin extravasation. Quantifications revealed a region-specific pattern with highest collagen IV and fibronectin intensities in most severely affected neocortical areas, followed by a gradual decline toward the border zone and non-affected regions. Comparing 4 and 24 h of ischemia, the subcortical fibronectin signal increased significantly over time, whereas neocortical areas displayed only a gradual increase. Qualitative analyses confirmed increased fibronectin and collagen IV signals in ischemic areas from all tissues and time points investigated. While the increased collagen IV signal was restricted to vessels, fibronectin appeared diffusely arranged in the parenchyma with focal accumulations associated to the vasculature. Integrin alpha(5) appeared enriched in the vicinity of fibronectin and vascular elements, while most of the non-vascular NVU elements showed complementary staining patterns referring to fibronectin. This spatio-temporal characterization of ischemia-related alterations of collagen IV and fibronectin in various stroke models and human autoptic tissue shows that ischemic consequences are not limited to traditional NVU components and the ECM, but also involve the NMZ. Future research should explore more components and the pathophysiological properties of the NMZ as a possible target for novel neuroprotective approaches

Impaired Neurofilament Integrity and Neuronal Morphology in Different Models of Focal Cerebral Ischemia and Human Stroke Tissue

As part of the neuronal cytoskeleton, neurofilaments are involved in maintaining cellular integrity. In the setting of ischemic stroke, the affection of the neurofilament network is considered to mediate the transition towards long-lasting tissue damage. Although peripheral levels of distinct neurofilament subunits are shown to correlate with the clinically observed severity of cerebral ischemia, neurofilaments have so far not been considered for neuroprotective approaches. Therefore, the present study systematically addresses ischemia-induced alterations of the neurofilament light (NF-L), medium (NF-M), and heavy (NF-H) subunits as well as of u-internexin (INA). For this purpose, we applied a multi-parametric approach including immunofluorescence labeling, western blotting, qRT-PCR and electron microscopy. Analyses comprised ischemia-affected tissue from three stroke models of middle cerebral artery occlusion (MCAO), including approaches of filament-based MCAO in mice, thromboembolic MCAO in rats, and electrosurgical MCAO in sheep, as well as human autoptic stroke tissue. As indicated by altered immunosignals, impairment of neurofilament subunits was consistently observed throughout the applied stroke models and in human tissue. Thereby, altered NF-L immunoreactivity was also found to reach penumbral areas, while protein analysis revealed consistent reductions for NF-L and INA in the ischemia-affected neocortex in mice. At the mRNA level, the ischemic neocortex and striatum exhibited reduced expressions of NF-L- and NF-H-associated genes, whereas an upregulation for Ina appeared in the striatum. Further, multiple fluorescence labeling of neurofilament proteins revealed spheroid and bead-like structural alterations in human and rodent tissue, correlating with a cellular edema and lost cytoskeletal order at the ultrastructural level. Thus, the consistent ischemia-induced affection of neurofilament subunits in animals and human tissue, as well as the involvement of potentially salvageable tissue qualify neurofilaments as promising targets for neuroprotective strategies. During ischemia formation, such approaches may focus on the maintenance of neurofilament integrity, and appear applicable as co-treatment to modern recanalizing strategies

Novel compounds with antiangiogenic and antineoplastic potency for growth control of testicular germ cell tumors

Der testikuläre Keimzelltumor ist die häufigste maligne Tumorerkrankung junger Männer im Alter zwischen 25 und 45 Jahren. Durch die stete Verbesserung der chemotherapeutischen Behandlungsmöglichkeiten gilt der testikuläre Keimzelltumor heute als Paradebeispiel einer heilbaren Tumorerkrankung. In etwa 20% der Fälle kommt es jedoch zu einer Entwicklung einer Cisplatinresistenz, welche mit einer schlechten Prognose einhergeht und den Einsatz höher dosierter Chemotherapien erfordert. Gerade für diese, meistens sehr junge Patientengruppe, ist die Entwicklung neuartiger Therapieansätze daher von großer Bedeutung. In der vorliegenden Arbeit wurden mittels einer computerbasierten Methode völlig neue und bislang unbekannte Substanzen (HP-2, HP-14) identifiziert und deren Wirksamkeit in vitro und in vivo überprüft. Es konnte gezeigt werden, dass HP-2 und HP-14 nicht nur die Proliferation von Endothelzellen, sondern auch von VEGFR-2 exprimierenden testikulären Keimzelltumorzellen dosisabhängig um bis zu 90% inhibieren, während sie das Wachstum von VEGFR-2-negativen Zelllinien nicht beeinflussen. Anhand verschiedener Angiogenesemodelle ließ sich zeigen, dass HP-2 und HP-14 in der Lage sind, angiogeneserelevante Endothelzellfunktionen zu inhibieren. So wurden sowohl die VEGF-vermittelte Zellmigration als auch die Ausbildung kapillarähnlicher Strukturen von Endothelzellen durch die Substanzen deutlich reduziert. Zusätzliche in vivo Überprüfungen im Chorioallantoismembranmodell bestätigten das antiangiogenetische Potential und die gute Verträglichkeit der neuen Substanzen. Zusätzlich konnte gezeigt werden, dass HP-2 und HP-14 auch direkt eine wachstumsinhibitorische Wirkung auf Tumorzellen ausüben. Der Einfluss von HP-2 und HP-14 auf zellbiologisch wichtige Prozesse, wie z.B. den Zellzyklus, bei Tumorzellen wurde mittels Durchflusszytometrie untersucht. Es konnte gezeigt werden, dass HP-2 und HP-14 zu einem S-Phasen Arrest der TKZT Zellen führen. Im Western Blot bestätigte sich, dass Proteine, die beim Zellzyklus eine wichtige Rolle spielen, wie z.B. p21 und p27 verändert reguliert waren und zu einem S-Phasen Arrest der testikulären Keimzelltumorzellen führten. Die Resistenz gegenüber cisplatinbasierter Chemotherapie geht mit einer schlechten Prognose in testikulären Keimzelltumoren einher. Daher wurde großer Wert darauf gelegt, die Effektivität der beiden neuen HP-Substanzen auch bei cisplatinresistenten testikulären Keimzelltumoren zu evaluieren. Beide Substanzen inhibierten auch hier dosisabhängig das Wachstum cisplatinresistenter TKZT Zellen. In Kombinationsexperimenten mit Cisplatin erzielten HP-2 und HP-14 additive wachstumsinhibitorische Effekte in testikulären Keimtumorzellen unabhängig von der Cisplatinresistenz. Unter Verwendung von testikulären Keimzelltumorzellen, die auf die Chorioallantoismembran befruchteter Hühnereier appliziert wurden, konnte das antineoplastische Potenzial zumindest von HP-14 auch in vivo aufgezeigt werden. Genexpressionsanalysen ergaben, dass die Behandlung von HP-14 und Cisplatin, allein oder in Kombination, zu einer Änderung des Genexpressionsmusters von Genen führt, die den zellulären Funktionen der Erkennung und Reparatur von DNA Schäden, aber auch des Wachstums, zugeordnet werden. Insgesamt kann daher festgehalten werden, dass mit der Identifikation der HP-Substanzen interessante Kandidaten für die zielgerichtete Therapie von Hodenkrebs, besonders für jene, die resistent gegenüber der konventionellen cisplatinbasierten Chemotherapie sind, gefunden wurden. Die Antitumoraktivität von HP-14 konnte bereits durch in vivo Überprüfungen bestätigt werden. Weitere Untersuchungen sind nötig, um eine abschließende Bewertung der hier vorgestellten Möglichkeit zur Behandlung von Hodenkrebs vorzunehmen. Die erhobenen Daten bilden dafür eine entsprechende Basis.Testicular cancer is one of the most common malignancies in young men between the ages of 25 and 45. Due to the improvement of chemotherapeutic treatment options, today the testicular germ cell cancer (TGCT) represents a curable disease. However, 20% of the patients with cisplatin-resistant or -refractory disease have an unfavorable prognosis which requires the use of a high dose chemotherapy. Therefore, it is very important to identify new treatment options in order to develop therapies with minimal side effects to improve the quality of life of these mostly young patients. In the present study, previously unknown compounds with putative antiangiogenic and antiproliferative properties (HP-2 and HP-14) were identified by an in silico screening approach and their efficiency was verified in vitro and in vivo. It has been shown that HP-2 and HP-14 reduced the growth of endothelial cells and VEGFR-2 expressing testicular germ cell tumor cells by up to 90%, while they failed to suppress the growth of VEGFR-2 lacking tumor cells. It was demonstrated that HP-2 and HP-14 reduced endothelial cell functions relevant to angiogenesis, as they inhibited the VEGF induced endothelial cell migration. Additional in vivo evaluations by using the chick chorioallantoic membrane model confirmed the antiangiogenic potency and good tolerability of the novel substances. HP-2 and HP-14 also have direct inhibitory effects on growth of testicular germ cell tumor cells. The antiproliferative mode of action of the novel compounds was analyzed using flow cytometry and western blot analyzes. Treatment of TGCT cells with HP-compounds raised the expression of the cell cycle-inhibiting molecule p21, which resulted in an S-phase arrest of the cell cycle. Resistance to cisplatin-based chemotherapy is associated with poor prognosis in testicular cancer. In this respect, the efficiency of the HP-2 and HP-14 in platinum resistant TGCT tumors was also evaluated. Both compounds inhibited the growth of cisplatin-resistant TGCT cells in a dose- dependent manner. In combination experiments with cisplatin, HP-14 revealed additive growth inhibitory effects in TGCT cells irrespective of the level of cisplatin resistance. Using TGCT cells inoculated on the chorioallantoic membrane of fertilized chicken eggs, the antineoplastic potency of HP-14 was also demonstrated in vivo. Gene expression profiling revealed changes in the expression pattern of genes related to cellular functions such as DNA damage detection and repair, as well as in cellular growth after treatment with both cisplatin and HP-14, alone or in combination. In summary, this study shows that the identified novel compound HP-14 effectively inhibits the growth of cisplatin-resistant TGCT cells and suppresses tumor growth and tumor angiogenesis. Thus, HP-14 may be qualified as a new candidate for targeted treatment of testicular cancer, particularly those showing resistance to the conventionally applied platinum-based chemotherapies

Synthetic Small Molecule Modulators of Hsp70 and Hsp40 Chaperones as Promising Anticancer Agents

A class of chaperones dubbed heat shock protein 70 (Hsp70) possesses high relevance in cancer diseases due to its cooperative activity with the well-established anticancer target Hsp90. However, Hsp70 is closely connected with a smaller heat shock protein, Hsp40, forming a formidable Hsp70-Hsp40 axis in various cancers, which serves as a suitable target for anticancer drug design. This review summarizes the current state and the recent developments in the field of (semi-)synthetic small molecule inhibitors directed against Hsp70 and Hsp40. The medicinal chemistry and anticancer potential of pertinent inhibitors are discussed. Since Hsp90 inhibitors have entered clinical trials but have exhibited severe adverse effects and drug resistance formation, potent Hsp70 and Hsp40 inhibitors may play a significant role in overcoming the drawbacks of Hsp90 inhibitors and other approved anticancer drugs

Synthetic Small Molecule Modulators of Hsp70 and Hsp40 Chaperones as Promising Anticancer Agents

A class of chaperones dubbed heat shock protein 70 (Hsp70) possesses high relevance in cancer diseases due to its cooperative activity with the well-established anticancer target Hsp90. However, Hsp70 is closely connected with a smaller heat shock protein, Hsp40, forming a formidable Hsp70-Hsp40 axis in various cancers, which serves as a suitable target for anticancer drug design. This review summarizes the current state and the recent developments in the field of (semi-)synthetic small molecule inhibitors directed against Hsp70 and Hsp40. The medicinal chemistry and anticancer potential of pertinent inhibitors are discussed. Since Hsp90 inhibitors have entered clinical trials but have exhibited severe adverse effects and drug resistance formation, potent Hsp70 and Hsp40 inhibitors may play a significant role in overcoming the drawbacks of Hsp90 inhibitors and other approved anticancer drugs