Kum Whang

Inhibition of hypoxia inducible factor-1α (HIF-1α) decreases vascular endothelial growth factor (VEGF) secretion and tumor growth in malignant gliomas

SummaryIntroductionHypoxia inducible factor-1α (HIF-1α) regulates vascular endothelial growth factor (VEGF), the presumed principal mediator of angiogenesis in malignant gliomas, under normal physiologic conditions. We examined the effect of HIF-1α on VEGF secretion, tumor growth, and angiogenesis in malignant gliomas.MethodsWe examined 175 human gliomas for expression of HIF-1α and its downstream-regulated proteins. HIF-1α expression and VEGF secretion in glioma cell lines under normoxia and hypoxia were examined using␣ELISA and Western blot. Malignant glioma cell lines were transfected with dominant-negative HIF-1α (DN-HIF-1α) expression vector or siRNA constructs against the HIF-1α gene. Growth studies were conducted on cells with the highest VEGF/HIF-1α inhibition isolated from stable transfected cell lines. MIB-1-labeling index and microvascular density (MVD) measurements were performed on the in vivo tumors.ResultsHIF-1 expression correlates with malignant glioma phenotype and was not confined to perinecrotic, pseudopalisading cells. VEGF and HIF-1 expression was high in glioma cell lines even under normoxia, and increased after exposure to hypoxia or growth factor stimulation. Cells transfected with DN-HIF-1α or HIF-1α siRNA demonstrated decreased HIF-1α and VEGF secretion. In vivo but not in vitro growth decreased in response to VEGF and HIF-1 inhibition. HIF-1 siRNA studies showed decreased VEGF secretion and in vitro and in vivo growth of glioma cell lines. MVD was unchanged but MIB-1 proliferation index decreased for both types of HIF-1 inhibition.ConclusionsVEGF and HIF-1α are elevated in malignant gliomas. HIF-1α inhibition results in VEGF secretion inhibition. HIF-1α expression affects glioma tumor growth, suggesting clinical applications for malignant glioma treatment.

Silencing of Hypoxia Inducible Factor-1α by RNA Interference Attenuates Human Glioma Cell Growth In vivo

Purpose: Higher-grade gliomas are distinguished by increased vascular endothelial cell proliferation and peritumoral edema. These are thought to be instigated by vascular endothelial growth factor, which, in turn, is regulated by cellular oxygen tension. Hypoxia inducible factor-1α (HIF-1α) is a main responder to intracellular hypoxia and is overexpressed in many human cancers, including gliomas. Experimental Design: We investigated the role of HIF-1α in glioma growth in vivo using RNA interference (RNAi) in U251, U87, and U373 glioma cells. Results: We found that RNAi can be used to significantly attenuate glioma growth by reducing HIF-1α levels constitutively using short hairpin RNAs and transiently using small interfering RNAs (siRNA). HIF-1α levels on average were reduced 55% in normoxia and 71% in hypoxia. Vascular endothelial growth factor and GLUT-1 levels were reduced 81% and 71%, respectively, in the stable HIF-1α–reduced clones. These clones showed significant growth attenuation (up to 73%) compared with negative controls when grown in vivo in mouse flanks. Cellular proliferation was also reduced significantly, as determined by MIB-1 staining. Treating gliomas grown in mouse flank transiently with siRNA against HIF-1α by intratumoral injection resulted in a significant reduction of HIF-1α activity. This activity was followed using a hypoxia-responsive luciferase construct that enabled hypoxia imaging in vivo. Tumor volume in these siRNA injection experiments was reduced by 50% over the negative controls. Conclusions: These results indicate that transient RNAi directed against HIF-1α can effectively curb glioma growth in vivo.

A comparison of the cell lines used in meningioma research.

BACKGROUND Immortal cell lines and cell lines derived from operative specimens transplanted into animal models are used in meningioma research. We address 2 criticisms of the mouse xenograft flank tumor model: Why are tumor induction rates derived from operative specimens low and inconsistent? Are flank tumors meningiomas? METHODS Meningioma cell cultures were processed for Giemsa-band karyotyping and flow cytometry. Mouse flank tumors induced subcutaneously were analyzed microscopically, immunohistochemically, and ultrastructurally. Giemsa-band studies identified meningiomas with simple karyotype (< or =1 chromosomal abnormality) or complex karyotype (multiple chromosomal abnormalities). RESULTS Cell cultures with complex karyotypes (IOMM-Lee, CH-157 MN, 2 operative specimens) grew rapidly in vitro and induced tumors in 49 (98%) of 50 animals. Meningioma cell cultures with simple karyotypes grew slowly in vitro and showed small, nongrowing tumors in mouse flanks (10/10). Meningioma flank tumors were vimentin-positive with ultrastructural features consistent with meningiomas. Cell cultures with complex karyotypes grew faster in cell culture and consistently induced flank tumors, unlike meningiomas with simple karyotypes. CONCLUSIONS Meningioma cell lines transplanted into flanks of nude mice exhibit microscopic, immunohistochemical, and ultrastructural features of meningiomas. The ease of monitoring tumor growth in the subcutaneous mouse flank model is its primary advantage, although we recognize an intracranial location is more biologically desirable.

Dynamic CT Perfusion Imaging for the Detection of Crossed Cerebellar Diaschisis in Acute Ischemic Stroke

Objective Although the detection of crossed cerebellar diaschisis (CCD) by means of different imaging modalities is well described, little is known about its diagnosis by computed tomography perfusion (CTP) imaging. We investigated the detection rate of CCD by CTP imaging and the factors related to CCD on CTP images in patients with acute ischemic stroke. Materials and Methods CT perfusion maps of cerebral blood volume (CBV), cerebral blood flow (CBF), mean transit time (MTT), and time-to-peak (TTP) obtained from 81 consecutive patients affected by an acute ischemic stroke were retrospectively reviewed. Whole-brain perfusion maps were obtained with a multichannel CT scanner using the toggling-table technique. The criteria for CCD was a unilateral supratentorial ischemic lesion and an accompanying decrease in perfusion of the contralateral cerebellar hemisphere on the basis of CTP maps by visual inspection without a set threshold. Maps were quantitatively analyzed in CCD positive cases. Results The criteria for CCD were fulfilled in 25 of the 81 cases (31%). Detection rates per CTP map were as follows: MTT (31%) > TTP (21%) > CBF (9%) > CBV (6%). Supratentorial ischemic volume, degree of perfusion reduction, and infratentorial asymmetry index correlated strongly (R, 0.555-0.870) and significantly (p < 0.05) with each other in CCD-positive cases. Conclusion It is possible to detect CCD on all four of the CTP-based maps. Of these maps, MTT is most sensitive in detecting CCD. Our data indicate that CTP imaging is a valid tool for the diagnosis of CCD in patients affected by an acute hemispheric stroke.

Neuregulin 1 up-regulates the expression of nicotinic acetylcholine receptors through the ErbB2/ErbB3-PI3K-MAPK signaling cascade in adult autonomic ganglion neurons

We investigated effects of Neuregulin 1 (NRG1) on the expression of nicotinic acetylcholine receptor (nAChR) in major pelvic ganglion (MPG) from adult rat. MPG neurons were found to express transcripts for type I and III NRG1s as well as α and β‐type epidermal growth factor (EGF)‐like domains. Of the four ErbB receptor isoforms, ErbB1, ErbB2, and ErbB3 were expressed in MPG neurons. Treating MPG with NRG1β significantly increased the transcript and protein level of the nAChR α3 and β4 subunits. Consistent with these molecular data, nicotinic currents (IACh) were significantly up‐regulated in NRG1β‐treated sympathetic and parasympathetic MPG neurons. In contrast, the type III NRG1 and the α form of the NRG1 failed to alter the IACh. Inhibition of the ErbB2 tyrosine kinase completely abolished the effects of NRG1β on the IACh. Stimulation of the ErbB receptors by NRG1β activated the phosphatidylinositol‐3‐kinase (PI3K) and mitogen‐activated protein kinase (MAPK). Immunoblot analysis revealed that PI3K‐mediated activation of Akt preceded Erk1/2 activation in NRG1β‐treated MPG neurons. Furthermore, specific PI3K inhibitors abrogated the phosphorylation of Erk1/2, while inhibition of MEK did not prevent the phosphorylation of Akt. Taken together, these findings suggest that NRG1 up‐regulates nAChR expression via the ErbB2/ErbB3‐PI3K‐MAPK signaling cascade and may be involved in maintaining the ACh‐mediated synaptic transmission in adult autonomic ganglia.

Accuracy of the detection of infratentorial stroke lesions using perfusion CT: an experimenter-blinded study

IntroductionAlthough perfusion CT (PCT) for the detection of supratentorial stroke is well established, there is a dearth of evidence of its effectiveness in the detection of infratentorial stroke. Hence, this study compared sensitivity, specificity, and accuracy of PCT maps between infratentorial and supratentorial stroke lesions.MethodsOne hundred patients with acute stroke who had successfully undergone near whole-brain PCT with the toggling table technique and follow-up MRI were included. Wilcoxon Mann–Whitney test was performed at P < 0.01.ResultsThere was no significant statistical difference in the accuracy (91.79% vs. 93.23% in regional cerebral blood volume; 92.26% vs. 95.31% in regional cerebral blood flow; 89.17% vs. 92.71% in mean transit time; 89.76% vs. 92.19% in time to peak; P > 0.01 in all PCT maps) between supratentorial and infratentorial stroke. Also, there was no remarkable difference in both sensitivity and specificity of PCT maps.ConclusionThis was the first study to investigate the accuracy of PCT with the toggling table technique in detection of infratentorial stroke lesions. Clinically, PCT is highly reliable and accurate in detecting infratentorial stroke lesions.

Expression profiles of high voltage-activated calcium channels in sympathetic and parasympathetic pelvic ganglion neurons innervating the urogenital system.

Among the autonomic ganglia, major pelvic ganglia (MPG) innervating the urogenital system are unique because both sympathetic and parasympathetic neurons are colocalized within one ganglion capsule. Sympathetic MPG neurons are discriminated from parasympathetic ones by expression of low voltage-activated Ca2+ channels that primarily arise from T-type α1H isoform and contribute to the generation of low-threshold spikes. Until now, however, expression profiles of high voltage-activated (HVA) Ca2+ channels in these two populations of MPG neurons remain unknown. Thus, in the present study, we dissected out HVA Ca2+ channels using pharmacological and molecular biological tools. Reverse transcription-polymerase chain reaction analysis showed that MPG neurons contained transcripts encoding all of the known HVA Ca2+ channel isoforms (α1B, α1C, α1D and α1E), with the exception of α1A. Western blot analysis and pharmacology with ω-agatoxin IVA (1 μM) confirmed that MPG neurons lack the α1A Ca2+ channels. Unexpectedly, the expression profile of HVA Ca2+ channel isoforms was identical in the sympathetic and parasympathetic neurons of the MPG. Of the total Ca2+ currents, ω-conotoxin GVIA-sensitive N-type (α1B) currents constituted 57 ± 5% (n = 9) and 60 ± 3% (n = 6), respectively; nimodipine-sensitive L-type (α1C and α1D) currents made up 17 ± 4% and 14 ± 2%, respectively; and nimodipine-resistant and ω-conotoxin GVIA-resistant R-type currents were 25 ± 3% and 22 ± 2%, respectively. The R-type Ca2+ currents were sensitive to NiCl2 (IC50 = 22 ± 0.1 μM) but not to SNX-482, which was able to potently (IC50 = 76 ± 0.4 nM) block the recombinant α1E/β2a/α2δ Ca2+ currents expressed in human embryonic kidney 293 cells. Taken together, our data suggest that sympathetic and parasympathetic MPG neurons share a similar but unique profile of HVA Ca2+ channel isoforms.

Differential changes of calcium binding proteins in the rat striatum after kainic acid-induced seizure

It has been suggested that calcium binding proteins protect against Ca2+ overload, thus rendering neurons more resistant against excitotoxicity. The influence of kainic acid, which induces status epilepticus, on the expressions of calbindin D28k, parvalbumin and calretinin was examined in the rat striatum by immunohistochemistry and microdensitometry. At 1, 3 and 6 days after kainic acid-induced seizure, the number of calretinin-positive neurons in the striatum was significantly lower than in control rats. However, no significant difference was observed in the number of calbindin D28k- and parvalbumin-positive neurons in control and seizure rats. At 1, 3 and 6 days after seizure the optical densities of calretinin- and parvalbumin-positive neurons in the striatum were significantly lower than in control rats. Our finding concerning the selective loss of calretinin-positive neurons in seizure groups suggests that calcium binding proteins in the striatum have differential vulnerabilities to kainic acid-induced seizure.

Gamma Knife surgery for low-flow cavernous sinus dural arteriovenous fistulas.

OBJECT The purpose of this study was to assess the efficacy of Gamma Knife surgery (GKS) for treating cavernous sinus dural arteriovenous fistulas (CSDAVFs). METHODS Of the 4123 GKSs performed between May 1992 and March 2009, 890 procedures were undertaken to treat vascular lesions. In 24 cases, the vascular lesion that was treated was a dural arteriovenous fistula, and in 6 of these cases, the lesion involved the cavernous sinus. One of these 6 cases was lost to follow-up, leaving the other 5 cases (4 women and 1 man) to comprise the subjects of this study. All 5 patients had more than 1 ocular symptom, such as ptosis, chemosis, proptosis, and extraocular movement palsy. In all patients, CSDAVF was confirmed by conventional angiography. Three patients were treated by GKS alone and 2 patients were treated by GKS combined with transarterial embolization. The median follow-up period after GKS in these 5 cases was 30 months (range 9-59 months). RESULTS All patients experienced clinical improvement, and their improvement in ocular symptoms was noticed at a mean of 17.6 weeks after GKS (range 4-24 weeks). Two patients received embolization prior to GKS but did not display improvement in ocular symptoms. An average of 20 weeks (range 12-24 weeks) was needed for complete improvement in clinical symptoms. There were no treatment-related complications during the follow-up period. CONCLUSIONS Gamma Knife surgery should be considered as a primary, combined, or additional treatment option for CSDAVF in selected cases, such as when the lesion is a low-flow shunt without cortical venous drainage. For those selected cases, GKS alone may suffice as the primary treatment method when combined with close monitoring of ocular symptoms and intraocular pressure.

Middle Meningeal Artery Embolization in Recurrent Chronic Subdural Hematoma Combined with Arachnoid Cyst.

Chronic subdural hematoma (CSDH) is a collection of old blood and its breakdown products between the surface of the brain parenchyma and the outermost layer called the dura. The most common treatment option for primary CSDH is burr-hole trephination; however, the treatment method for recurrent CSDH is still widely debated. An arachnoid cyst (AC) is a sac filled with cerebrospinal fluid located between the brain or spinal cord and the arachnoid membrane, which is one of the three meninges covering the brain or spinal cord. Although it is rare, the cyst is associated with CSDH in juveniles, and the recurrence rate of CSDH increases in such cases. Much of the literature has supported the preventive role of middle meningeal artery (MMA) embolization in recurrent CSDH. We report a 13-year-old male patient with recurrent CSDH and AC where the early intervention of MMA embolization was proven effective in preventing the further recurrence of CSDH.