Kazuhiko Takaya

Vascular Endothelial Growth Factor (VEGF) Expression in Human Coronary Atherosclerotic Lesions Possible Pathophysiological Significance of VEGF in Progression of Atherosclerosis

BACKGROUND Vascular endothelial growth factor (VEGF) is an important angiogenic factor reported to induce migration and proliferation of endothelial cells, enhance vascular permeability, and modulate thrombogenicity. VEGF expression in cultured cells (smooth muscle cells, macrophages, endothelial cells) is controlled by growth factors and cytokines. Hence, the question arises of whether VEGF could play a role in atherogenesis. METHODS AND RESULTS Frozen sections from 38 coronary artery segments were studied. The specimens were characterized as normal with diffuse intimal thickening, early atherosclerosis with hypercellularity, and advanced atherosclerosis (atheromatous plaques, fibrous plaques, and totally occlusive lesions). VEGF expression as well as the expression of 2 VEGF receptors, flt-1 and Flk-1, were studied with immunohistochemical techniques in these samples at the different stages of human coronary atherosclerosis progression. The expression of VEGF mRNA was also studied with reverse transcription-polymerase chain reaction. Normal arterial segments showed no substantial VEGF expression. Hypercellular and atheromatous lesions showed distinct VEGF positivity of activated endothelial cells, macrophages, and partially differentiated smooth muscle cells. VEGF positivity was also detected in endothelial cells of intraplaque microvessels within advanced lesions. In totally occlusive lesions with extensive neovascularization, intense immunostaining for VEGF was observed in accumulated macrophages and endothelial cells of the microvessels. Furthermore, VEGF mRNA expression was detected in atherosclerotic coronary segments but not in normal coronary segments. The immunostainings for flt-1 and Flk-1 were detected in aggregating macrophages in atherosclerotic lesions and also in endothelial cells of the microvessels in totally occlusive lesions. CONCLUSIONS These results demonstrate distinct expression of VEGF and its receptors (flt-1 and Flk-1) in atherosclerotic lesions in human coronary arteries. Considering the multipotent actions of VEGF documented experimentally in vivo and in vitro, our findings suggest that VEGF may have some role in the progression of human coronary atherosclerosis, as well as in recanalization processes in obstructive coronary diseases.

Kidney produces a novel acylated peptide, ghrelin

Ghrelin is a novel growth hormone‐releasing peptide with a unique acylated structure. Here we reveal that prepro‐ghrelin gene is expressed in the mouse kidney and glomerulus. We also show by reverse‐phase high performance liquid chromatography coupled with radioimmunoassay that the mouse kidney does produce ghrelin. The ghrelin immunoreactivity in the mouse kidney is 6.79±0.48 fmol/mg (n=5), which is much more abundant than that in the mouse plasma of 0.339±0.029 fmol/μl (n=6). Furthermore, prepro‐ghrelin gene is expressed in cultured rat mesangial cells, fibroblast‐like NRK‐49F cells and mouse podocytes, but not in rat epithelial cell‐like NRK‐52E cells. Ghrelin receptor gene is also expressed in the rat kidney. These findings demonstrate that the kidney, glomerulus and renal cells express prepro‐ghrelin gene and ghrelin is produced locally in the kidney, and suggest the endocrine and/or paracrine roles of ghrelin in the kidney.

Plasma Ghrelin and Desacyl Ghrelin Concentrations in Renal Failure

Ghrelin is a novel hormone that possesses growth hormone (GH)-releasing, cardiovascular, and metabolic activities. Ghrelin is a unique acylated polypeptide, and the naked peptide, desacyl ghrelin, does not have the activity. This study examines plasma ghrelin concentrations in 41 patients with mild to severe renal diseases. Two kinds of radioimmunoassays were used: amino-terminal immunoreactivity represents ghrelin alone (N-IR), and carboxyl-terminal immunoreactivity corresponds to the sum of both ghrelin and desacyl ghrelin (C-IR). In all subjects, the plasma N-IR was much smaller than the C-IR, indicating that desacyl ghrelin predominates over ghrelin in the circulation. The plasma C-IR, but not N-IR, was significantly correlated with the serum creatinine level and was increased 2.8-fold in patients with end-stage renal disease compared with those in patients with normal renal function. The plasma GH concentration was significantly correlated with the plasma N-IR and the C-IR, as well as with the serum creatinine level. Bilateral nephrectomy in mice caused marked increase in the plasma C-IR without significant changes in the local C-IR and ghrelin mRNA level in the stomach, which is the main site of ghrelin production. These findings suggest that circulating ghrelin concentrations play a role in the regulation of blood GH concentrations and that the kidney is an important site for clearance and/or degradation of desacyl ghrelin. Furthermore, elevation of blood GH levels in renal failure seems to be caused by a mechanism other than alteration in the circulating ghrelin concentration.

Vascular Endothelial Growth Factor Suppresses C-Type Natriuretic Peptide Secretion

Angiogenesis plays a pivotal role not only in wound healing and tumor progression but also in diabetic angiopathy, arteriosclerosis, and collateral formation of obstructive vascular diseases. Vascular endothelial growth factor (VEGF) is now thought to be an endothelium-specific and potent angiogenic factor. We previously demonstrated that C-type natriuretic peptide (CNP), originally isolated from porcine brain, is produced by endothelial cells and proposed that CNP can exert control over vascular tone and growth as a local vascular regulator. In the present study, we examined the effect of VEGF on CNP secretion from endothelial cells using the specific radioimmunoassay for CNP we developed. VEGF (1 to 100 ng/mL) dose-dependently suppressed CNP secretion from cultured bovine endothelial cells, and 100 ng/mL VEGF suppressed endothelial CNP secretion to 28% of control levels (31.7 +/- 5.5 versus 8.9 +/- 0.8 fmol/mL, vehicle versus VEGF). VEGF also suppressed CNP mRNA expression in endothelial cells 9 hours after administration. In contrast, basic fibroblast growth factor (20 ng/mL), an endothelium-nonspecific angiogenic factor, significantly stimulated CNP secretion by 290%. These results indicate that VEGF can regulate vascular tone and growth in the process of angiogenesis through suppression of endothelial secretion of CNP, which is an endothelium-derived vasorelaxing and growth-inhibitory peptide.

Expression of the RET proto-oncogene in normal human tissues, pheochromocytomas, and other tumors of neural crest origin

Abstract In order to evaluate the roles of the RET proto-oncogene in normal human tissues and tumors derived from the neural crest cells, we examined the expression of RET in a variety of adult human tissues, pheochromocytomas, medullary thyroid carcinomas (MTCs), ganglioneuromas, and a neurinoma. Northern blot analysis demonstrated that RET is normally expressed in the adrenal medulla and cerebellum among adult human tissues. RET transcripts were detected in all of 11 sporadic and one familial pheochromocytomas. The levels of RET mRNA were higher in 8 of 12 pheochromocytomas than in the normal adrenal medulla, indicating that RET is overexpressed in the majority of sporadic pheochromocytomas. There was a considerable difference in the level of RET expression in each pheochromocytoma ranging from 0.2 to 10 times the transcripts found in the normal adrenal medulla. The sizes of the transcripts of 7.0, 6.0, 4.5, and 3.9 kb were the same as those found in the adrenal medulla, suggesting no rearrangements of the RET gene in pheochromocytomas. Southern blot analysis revealed neither amplification nor gross structural changes in the RET gene. RET was also expressed at high levels in four MTCs examined, whereas its transcripts were detected at low abundance in only one of three ganglioneuromas. RET was not expressed in a neurinoma. These results suggest that RET may play some roles in a limited range of adult human tissues, and that its high levels of expression may have relevance to development or growth of pheochromocytomas and MTCs.

Molecular cloning of hamster brain and atrial natriuretic peptide cDNAs. Cardiomyopathic hamsters are useful models for brain and atrial natriuretic peptides.

Brain and atrial natriuretic peptides (BNP and ANP) are cardiac hormones with diuretic, natriuretic, and vasodilatory activities. Cardiomyopathic hamsters are widely used animal models of heart failure. Due to the structural divergence of BNP among species, examination on pathophysiological roles of BNP using cardiomyopathic hamsters is so far impossible. We therefore isolated hamster BNP and ANP cDNAs, and investigated synthesis and secretion of these peptides in normal and cardiomyopathic hamsters. The COOH-terminal 32-residue peptide of cloned hamster preproBNP with 122 amino acids, preceded by a single arginine residue, supposedly represents hamster BNP showing < 50% homology to rat BNP. Alpha-hamster ANP, 28-residue peptide, is identical to alpha-rat ANP. In hamsters, BNP and ANP occur mainly in the ventricle and the atrium, respectively. The 32-wk-old hypertrophic cardiomyopathic BIO14.6 strain exhibited ventricular hypertrophy. The 32-wk-old dilated cardiomyopathic BIO53.58 strain remained at the stage without apparent heart failure. In BIO14.6 and BIO53.58 strains at this age, ventricular BNP and ANP gene expressions are augmented, and the plasma BNP concentration is elevated to 136 and 108 fmol/ml, respectively, three times greater than the elevated plasma ANP concentration, which well mimics changes of the plasma BNP and ANP concentrations in human heart failure. Cardiomyopathic hamsters, therefore, are useful models to investigate the implication of BNP in human cardiovascular diseases.

Identification of Cis-elements of the Human Endothelin-A Receptor Gene and Inhibition of the Gene Expression by the Decoy Strategy

Previously, we succeeded in molecular cloning of the cDNA and the gene for human endothelin-A receptor (ET-AR). In the present study, we define cis-elements in the 5′-flanking region of the ET-AR gene. Deletion analyses were performed in A7r5 cells, rat vascular smooth muscle cell line, and Chinese hamster ovary cells using ET-AR promoter-luciferase gene constructs including 5 kilobases of the 5′-flanking region. These analyses demonstrated the existence of one negative regulatory element (−2.0 kilobases to −857 bases) and two positive regulatory elements (−137 to −53 and −53 to +251). Gel mobility shift assay revealed a nuclear protein binding to the region (−104 to −78) (R1). DNase I footprinting analysis showed a footprint spanning from −91 to −83 whose sequence is CCCCACCTT (ETA-P1). When a plasmid including R1 fragments (R1 decoy) was co-transfected into A7r5 cells with ET-AR (−137 to +251)-luciferase gene construct, it significantly reduced the luciferase activity in a dose-dependent manner. Moreover, R1 decoy down-regulated the endogenous ET-AR mRNA in A7r5 cells by a maximum of 75%. Thus, we identified cis-elements that regulate basal transcriptional activity of the ET-AR gene and proved the feasibility to suppress the expression of the ET-AR gene by the DNA decoy strategy using the positive regulatory element we identified.

Cis elements for transcriptional regulation of the human endothelin-A receptor gene

Summary: To elucidate the transcriptional regulation of the endothelin-A receptor (ET-AR) gene, we defined the cis elements in the 5‘-flanking region by functional analysis. Chimeric gene constructs were prepared using serial deletion mutants of a 5-kilobase (kb) fragment of the human ET-AR 5’-flanking region and the luciferase gene and were transfected into a rat vascular smooth-muscle cell line, A7r5. Deletion of the region between −2.0 kb and - 857 bases caused a twofold increase in luciferase activity, and deletion from - 137 bases to −53 bases reduced the activity by 50%. These results indicate the existence of a negative regulatory element and a positive regulatory element, respectively. The gel shift assay revealed the existence of nuclear proteins in A7r5 cells that bind to the region containing the positive regulatory element. Thus, we identified cis regulatory elements and nuclear factors that regulate the expression of the ET-AR gene.

Current topics in pheochromocytoma

Pheochromocytoma is the tumor that produces catecholamines and originates from chromaffin cells, which are differentiated from sympathoadrenal progenitor cells of neural crest under the influence of glucocorticoids. Genetic abnormalities of familial pheochromocytomas have elucidated oncogenic genetic bases of the tumor, including gene abnormalities of the RET proto-oncogene in multiple endocrine neoplasia type 2, VHL gene in von Hippel Lindaus disease or the NF1 gene in neurofibromatosis. Co-localization of various substances with catecholamines in the tumor, including neuropeptide Y, opioid peptides or adrenomedulOFF peptide elevating cAMP production, is recognized. The significance of these substances in modulating clinical features of pheochromocytomas is not fully understood.

AUGMENTED EXPRESSION OF THE ENDOTHELIN-A RECEPTOR GENE IN CULTURED MESANGIAL CELLS FROM STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS

1. To elucidate the role of endothelin (ET) receptor in hypertension, we studied the expression of the ET‐A receptor (ET‐ AR) gene and the ET‐B receptor (ET‐BR) gene in cultured mesangial cells isolated from stroke‐prone spontaneously hypertensive rats (SHRSP) and Wistar‐Kyoto (WKY) rats.