Comparison of Expression and Proliferative Effect of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its Receptors on Human Astrocytoma Cell Lines

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide considered to be a potent regulator of astrocytes. It has been reported that PACAP also affects astrocytoma cell properties, but the proliferative effects of this peptide in previous reports were inconsistent. The purpose of this study was to search for correlations between malignant potential, PACAP/PACAP receptor expression, and the proliferative potential of four astrocytoma cell lines (KNS-81, KINGS-1, SF-126, and YH-13). Immunohistochemical observations were performed using astrocyte lineage markers with a view to establishing malignant potential, which is inversely correlated to differentiation status in astrocytoma cells. YH-13 showed the most undifferentiated astrocyte-like status, and was immunopositive to a cancer stem cell marker, CD44. These observations suggest that YH-13 is the most malignant of the astrocytoma cell lines tested. Moreover, the strongest PAC1-R immunoreactivity was observed in YH-13 cells. Using real-time PCR analysis, no significant differences among cell lines were detected with respect to PACAP mRNA, but PAC1-R and VPAC1-R mRNA levels were significantly increased in YH-13 cells compared with the other cell lines. Furthermore, when cell lines were treated with PACAP (10^?11 M) for 3 days, the YH-13 cell line, but not of the other cell lines, exhibited a significantly increased cell number. These results suggest that PACAP receptor expression is correlated with the malignant and proliferative potential of astrocytoma cell lines.     

Accumulation of beta-amyloid in the brain microvessels accompanies increased hyperphosphorylated tau proteins following microsphere embolism in aged rats

To define mechanisms underlying neurovascular injury following brain embolism-induced neurodegeneration, we investigated temporal and spatial pathological changes in brain microvessels up to 12 weeks after microsphere embolism (ME) induction in aged male rats. Mild ME upregulated endothelial nitric oxide synthase (eNOS) and protein tyrosine nitration in brain microvessels. Strong beta-amyloid immunoreactivity coincident with increased eNOS immunoreactivity was observed in microvessels. Immunoblotting of purified brain microvessels revealed that beta-amyloid accumulation significantly increased 1 week after ME induction and remained elevated for 12 weeks. Importantly, beta-amyloid accumulation in brain parenchyma was also observed in areas surrounding injured microvessels at 12 weeks. Levels of Alzheimer's-related hyperphosphorylated tau proteins also concomitantly increased in neurons surrounding regions of beta-amyloid accumulation 12 weeks after ME induction, as did glycogen synthase kinase (GSK3beta) (Tyr-216) phosphorylation. Taken together, ME-induced aberrant eNOS expression and subsequent protein tyrosine nitration in microvessels preceded beta-amyloid accumulation both in microvessels and brain parenchyma, leading to hyperphosphorylation of neuronal tau proteins through GSK3beta activation.     

Spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ZSET1446/ST101) treatment rescues olfactory bulbectomy-induced memory impairment by activating Ca2+/calmodulin kinase II and protein kinase C in mouse hippocampus

Olfactory bulbectomy (OBX) in mice elicits impaired memory and cognitive functions. Here, we found that chronic oral administration of spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ZSET1446/ST101) (0.1-1 mg/kg/day), a novel cognitive enhancer, significantly improved memory deficits as assessed by Y-maze and novel object recognition tasks in OBX mice. Immunostaining of cholinergic neurons in the medial septum by using an anti-choline acetyltransferase antibody indicated that chronic ZSET1446 treatment did not rescue cholinergic neurons. However, chronic treatment significantly restored OBX-induced decreases both in calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) phosphorylation without improving decreased extracellular signal-regulated kinase phosphorylation in the hippocampal CA1 region. Consistent with enhanced CaMKII and PKC phosphorylation, ZSET1446 treatment improved glutamate receptor 1 (Ser-831) phosphorylation in the hippocampal CA1 region. ZSET1446 treatment also significantly rescued impaired long-term potentiation (LTP) in the hippocampal CA1 region of OBX mice. Taken together, the cognition-enhancing effect of ZSET1446 is probably mediated in part by stimulation of CaMKII and PKC activities, which in turn rescue impaired hippocampal LTP in OBX mice.     

Angiogenic effects of adrenomedullin in ischemia and tumor growth

Adrenomedullin (AM) is a novel vasodilating peptide involved in the regulation of circulatory homeostasis and implicated in the pathophysiology of cardiovascular disease. We tested the hypothesis that AM also possesses angiogenic properties. Using laser Doppler perfusion imaging, we found that AM stimulated recovery of blood flow to the affected limb in the mouse hind-limb ischemia model. AM exerted this effect in part by promoting expression of vascular endothelial growth factor (VEGF) in the ischemic limb, and immunostaining for CD31 showed the enhanced flow to reflect increased collateral capillary density. By enhancing tumor angiogenesis, AM also promoted the growth of subcutaneously transplanted sarcoma 180 tumor cells. However, heterozygotic AM knockout mice (AM+/-) showed significantly less blood flow recovery with less collateral capillary development and VEGF expression than their wild-type littermates. Similarly, mice treated with AM22-52, a competitive inhibitor of AM, showed reduced capillary development, and growth of sarcoma 180 tumors was inhibited in AM+/- and AM22-52-treated mice. Notably, administration of VEGF or AM rescued blood flow recovery and capillary formation in AM+/- and AM22-52-treated mice. In cocultures of endothelial cells and fibroblasts, AM enhanced VEGF-induced capillary formation, whereas in cultures of endothelial cells AM enhanced VEGF-induced Akt activation. These results show that AM possesses novel angiogenic properties mediated by its ability to enhance VEGF expression and Akt activity. This may make AM a useful therapeutic tool for relieving ischemia; conversely, inhibitors of AM could be useful for clinical management of tumor growth.     

Pediatric liver failure with massive sinusoidal infiltration of histiocytes

Histiocytic neoplasms, such as Langerhans cell histiocytosis (LCH) and disseminated juvenile xanthogranuloma (JXG), can involve the liver and sometimes cause liver failure. We aimed to classify non-LCH histiocytic proliferating disorders that do not exhibit typical disseminated JXG histology. We examined four pediatric patients who presented with liver failure and splenomegaly. Two patients with liver cirrhosis without cholestasis underwent liver transplantation (LT). The other two patients presented with giant cell hepatitis causing neonatal/infantile acute liver failure (ALF). The infantile ALF patient also underwent LT. Liver dysfunction developed after LT in all three transplant cases and the grafts exhibited massive sinusoidal infiltration of histiocytes with hemophagocytosis, similar to the native liver. The neonatal ALF patient was treated with an LCH-type chemotherapy regimen, and is alive and well at 18 months. Infiltrating histiocytes were positive for CD68 and CD163, and negative for CD1a, CD207, and S-100 protein. The BRAF V600E mutation was not present. Liver histological findings were not consistent with conventional disseminated JXG or LCH, although the histological findings in other organs overlapped those of well-known histiocytic neoplasms. The histological and immunohistochemical findings of infiltrating histiocytes suggest that these four cases constituted a disseminated JXG-like systemic disease.