Daisuke Matsuda

Pyripyropene A, an Acyl–Coenzyme A:Cholesterol Acyltransferase 2–Selective Inhibitor, Attenuates Hypercholesterolemia and Atherosclerosis in Murine Models of Hyperlipidemia

Objective—Pyripyropene A (PPPA) of fungal origin is the first compound that has been found to strongly and selectively inhibit acyl–coenzyme A:cholesterol acyltransferase 2 (ACAT2) isozyme activity in vitro. The purpose of the present study was to investigate in vivo efficacy of the ACAT2-selective inhibitor in atherosclerosis. Methods and Results—PPPA treatment (10 to 100 mg/kg) caused 30.5±4.7% to 55.8±3.3% inhibition of the cholesterol absorption from the mouse intestine. When PPPA (10 to 50 mg/kg per day) was orally administered to apolipoprotein E–knockout mice for 12 weeks, the levels of plasma cholesterol, very-low-density lipoprotein (VLDL), and low-density lipoprotein (LDL) and hepatic cholesterol content were lowered. Furthermore, the ratio of cholesteryl oleate (exclusively synthesized in hepatic ACAT2) to cholesteryl linoleate in VLDL- and LDL-derived cholesteryl ester decreased, indicating that hepatic ACAT2 activity was inhibited by PPPA. PPPA-treated mice had reduced atherogenic lesion areas that were lowered by 26.2±3.7% to 46±3.8% in the aortae and by 18.9±3.6% to 37.6±6.0% in the hearts. Conclusion—Our findings indicate that ACAT2-selective inhibition in the intestine and the liver can be effective against atherosclerosis and that PPPA appears to be a potential antiatherogenic lead compound. This study is the first demonstration of the in vivo efficacy of PPPA, an ACAT2-selective inhibitor, in atherosclerosis. PPPA-treated atherogenic mice showed a decrease in intestinal cholesterol absorption and cholesterol and cholesteryl oleate levels in both LDL and VLDL, resulting in protection of atherosclerosis development.

Genomic expression and single-nucleotide polymorphism profiling discriminates chromophobe renal cell carcinoma and oncocytoma

BackgroundChromophobe renal cell carcinoma (chRCC) and renal oncocytoma are two distinct but closely related entities with strong morphologic and genetic similarities. While chRCC is a malignant tumor, oncocytoma is usually regarded as a benign entity. The overlapping characteristics are best explained by a common cellular origin, and the biologic differences between chRCC and oncocytoma are therefore of considerable interest in terms of carcinogenesis, diagnosis and clinical management. Previous studies have been relatively limited in terms of examining the differences between oncocytoma and chromophobe RCC.MethodsGene expression profiling using the Affymetrix HGU133Plus2 platform was applied on chRCC (n = 15) and oncocytoma specimens (n = 15). Supervised analysis was applied to identify a discriminatory gene signature, as well as differentially expressed genes. High throughput single-nucleotide polymorphism (SNP) genotyping was performed on independent samples (n = 14) using Affymetrix GeneChip Mapping 100 K arrays to assess correlation between expression and gene copy number. Immunohistochemical validation was performed in an independent set of tumors.ResultsA novel 14 probe-set signature was developed to classify the tumors internally with 93% accuracy, and this was successfully validated on an external data-set with 94% accuracy. Pathway analysis highlighted clinically relevant dysregulated pathways of c-erbB2 and mammalian target of rapamycin (mTOR) signaling in chRCC, but no significant differences in p-AKT or extracellular HER2 expression was identified on immunohistochemistry. Loss of chromosome 1p, reflected in both cytogenetic and expression analysis, is common to both entities, implying this may be an early event in histogenesis. Multiple regional areas of cytogenetic alterations and corresponding expression biases differentiating the two entities were identified. Parafibromin, aquaporin 6, and synaptogyrin 3 were novel immunohistochemical markers effectively discriminating the two pathologic entities.ConclusionsGene expression profiles, high-throughput SNP genotyping, and pathway analysis effectively distinguish chRCC from oncocytoma. We have generated a novel transcript predictor that is able to discriminate between the two entities accurately, and which has been validated both in an internal and an independent data-set, implying generalizability. A cytogenetic alteration, loss of chromosome 1p, common to renal oncocytoma and chRCC has been identified, providing the opportunities for identifying novel tumor suppressor genes and we have identified a series of immunohistochemical markers that are clinically useful in discriminating chRCC and oncocytoma.

Somatic Pairing of Chromosome 19 in Renal Oncocytoma Is Associated with Deregulated ELGN2-Mediated Oxygen-Sensing Response

Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase ELGN2, a key protein that regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of ELGN2 in renal oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of renal oncocytoma.

Identification of copy number alterations and its association with pathological features in clear cell and papillary RCC.

We report and characterize the copy number alterations (CNAs) in 35 clear cell and 12 papillary renal cell carcinomas (RCC) using Affymetrix 100K SNP arrays. Novel gain and loss regions are identified in both subtypes. In addition, statistically significant CNA are detected and associated with the pathological features: VHL mutation status, tumor grades, and sarcomatoid component in clear cell RCC and in types 1 and 2 of papillary RCC. Florescence in situ hybridization confirmed the copy number gain in the transforming growth factor, beta-induced gene (TGFBI), which is a possible oncogene for clear cell RCC.

Synthesis and structure-activity relationship of pyripyropene A derivatives as potent and selective acyl-CoA:cholesterol acyltransferase 2 (ACAT2) inhibitors: part 1.

In an effort to develop potent and selective inhibitors toward ACAT2, structure-activity relationship studies were carried out using derivatives based on pyripyropene A (PPPA, 1). We have successfully developed novel PPPA derivatives with a 7-O-substituted benzoyl substituent that significantly exhibit more potent ACAT2 inhibitory activity and higher ACAT2 isozyme selectivity than 1.

Selective Inhibition of Acyl-CoA:cholesterol Acyltransferase 2 Isozyme by Flavasperone and Sterigmatocystin from Aspergillus Species

Five known fungal metabolites, aurasperone A, aurasperone D, averufanin, flavasperone and sterigmatocystin, were isolated from the culture broths of Aspergillus species as inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT) in the cell-based assay using ACAT1- and ACAT2-expressing CHO cells. These compounds share a similar polycyclic skeleton. Among them, flavasperone and sterigmatocystin, having an angular skeleton, showed selective inhibition toward ACAT2 isozyme, while the others having a linear one had no selectivity in inhibition.

New verticilides, inhibitors of acyl-CoA:cholesterol acyltransferase, produced by Verticillium sp. FKI-2679

Verticillium sp. FKI-2679, a soil isolate, was found to produce inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT) in a cell-based assay using ACAT1- and ACAT2-expressing CHO cells. Three new compounds, verticilides A2, A3 and B1, were isolated along with a known compound, verticilide A1, from the fermentation broth of the fungus by solvent extraction, ODS column chromatography, silica gel column chromatography and preparative HPLC. Structure elucidation showed that these compounds were new cyclic depsipeptide. Verticilides A1, A2, A3 and B1 showed a degree of selectivity towards ACAT2, with IC50s 8.5–11-fold more potent than observed against ACAT1.

New isochaetochromin, an inhibitor of triacylglycerol synthesis in mammalian cells, produced by Penicillium sp. FKI-4942: I. Taxonomy, fermentation, isolation and biological properties

A new bis-naphtho-γ-pyrone isomer named isochaetochromin A1 was isolated along with known isochaetochromins B1 and B2 from the culture broth of Penicillium sp. FKI-4942 by solvent extraction, silica gel column chromatography and HPLC. Among them, isochaetochromin B1 showed the most potent inhibitory activity of triacylglycerol synthesis with an IC50 value of 5.6 μM, followed by isochaetochromins B2 (IC50, 11 μM) and A1 (33 μM).

Dinapinones, novel inhibitors of triacylglycerol synthesis in mammalian cells, produced by Penicillium pinophilum FKI-3864

Dinapinone A, a novel biaryl dihydronaphthopyranone, was isolated from the culture broth of Penicillium pinophilum FKI-3864 by solvent extraction, silica gel and ODS column chromatography and HPLC. Dinapinone A showed very potent inhibition of triacylglycerol (TG) synthesis in intact Chinese hamster ovary K1 (CHO-K1) cells with an IC50 value of 0.097 μM. Dinapinone A was found to be a mixture of stereoisomers, resulting in its separation into dinapinones A1 and A2 by HPLC using a C30 reverse-phase column. Dinapinone A1 did not inhibit TG synthesis in CHO-K1 cells even at 12 μM, and dinapinone A2 showed less potent inhibition (IC50; 0.65 μM) than dinapinone A; however, a mixture of isolated dinapinones A1 and A2 (a 1:1 ratio) recovered the potent TG inhibitory activity (IC50; 0.054 μM). A similar effect of dinapinone on TG synthesis in intact Raji cells was also observed.

Cavernous hemangioma of the adrenal gland.

Cavernous hemangioma is a rare benign tumor, which is histologically represented by cystic spaces lined with endothelial cells. The first report of adrenal cavernous hemangioma was published in 1955, and only approximately 50 cases have been reported to date. Here we present a case of cavernous hemangioma of the adrenal gland , which is treated by laparoscopic resection. A 51-year-old man was referred to our institute after an ulcer was found on his head. Because the ulcer could not be ruled out from metastatic disease, we carried out chest/abdominal computed tomography (CT). Although chest CT showed no evidence of lung mass or mediasinum lymph node swelling, abdominal CT revealed the circumscribed and enhanced mass located at the upper pole of the left kidney (4.5 ¥ 4.0 ¥ 3.5 cm) (Fig. 1a). Magnetic resonance imaging showed a hypo-intense signal mass on T1-weighted images and a heterogeneously hyper-intense signal mass on T2-weighted images. All of the adrenal-related hormones, laboratory examinations, and plasma levels of tumor markers in the patient were within normal limits. Because malignant diseases could not be excluded clinically, the patient underwent laparoscopic resection of the left adrenal gland and the resected tumor measured 4.0 ¥ 4.0 ¥ 3.5 cm and weighed 67 g. In a section of the gross specimen, a cystic tumor with septa and blood clots were observed (Fig. 1b). Pathological examinations of the left adrenal gland revealed blood-filled lacunae, hematoma, and endothelial lining, which was confirmed by hematoxylin and eosin staining (Fig. 1c) and immunoperoxidase factor VIII staining (Fig. 1d). The tumor was histopathologically diagnosed as cavernous hemangioma. Four years after surgical removal of the tumor, no recurrence was evidenced by follow-up CT and physical examinations. Adrenal hemangiomas are still etiologically ambiguous. Histopathologically, hemangiomas are mainly classified into two types: (i) cavernous hemangioma, which is described as an enlarged mass of blood-filled sinusoidal channels that have eroded and displaced normal tissues; and (ii) capillary hemangioma, which is described as a small tuft of sub-mucosal capillaries arranged in radiating loops or lobules. Because adrenal hemangiomas lack documentation of specific features, preoperative diagnosis is still frequently difficult and patients are often misdiagnosed of other adrenal tumors. It is most important that the cavernous hemangioma should be included in the differential diagnosis of adrenal neoplasm, although cavernous hemangioma of the adrenal gland is rare. In the present case, since any specific findings such as calcification and peripheral enhancement were not found , the patient could not be diagnosed as having cavernous hemangioma at the time of discovery. Although cavernous hemangioma requires conservative treatment by periodical follow-up if the tumor is asymptomatic and small, surgical resection must be required for excluding malignant disease, removing pressure-related symptoms, and precluding spontaneous hemorrhage. Laparoscopic resection of adrenal tumors has become the standard procedure lately, and this procedure must be required for adrenal hemangioma as well. The present case was successfully treated by laparoscopic resection without any complications.