Residual carcinoma cells after chemoradiotherapy for esophageal squamous cell carcinoma patients: striving toward appropriate judgment of biopsy.

In esophageal squamous cell carcinoma (ESCC) patients who are treated with chemoradiotherapy (CRT), identification of the presence or absence of residual or recurrent carcinoma is usually pivotal in their clinical management. In addition, the extent of carcinoma invasion into the esophageal wall could determine the clinical outcome of these patients following CRT. Therefore, in this study, we evaluated the response to CRT both macroscopically and histologically in a consecutive series of 42 ESCC patients receiving neoadjuvant chemoradiotherapy following curative esophageal resection at Tohoku University Hospital between August 2011 and December 2012. The histological grading of tumor regression was as follows: grade 3, markedly effective (no viable residual tumor cells); grade 2, moderately effective (residual tumor cells in less than one-third of the tumor); grade 1, slightly effective (1b, residual tumor cells in one-third to two-thirds of the tumor; 1a, residual tumor cells in more than two-thirds of the tumor); and grade 0, ineffective. In this study, we selected grade 2 and 1b cases because they might show a complete response with definitive CRT. We evaluated the presence of any residual in situ lesions and tumor depth in detail. The grading of tumor regression in primary sites was as follows: grade 3 (7 cases), grade 2 (16 cases), grade 1b (13 cases), and grade 1a (6 cases). The concordance rate between macroscopic and histopathological evaluation on the depth of the tumor was 40% (17/42). Among 29 cases (grade 2 and grade 1b), intraepithelial lesions were not detected in 17 cases, and tumor nests were not detected in the lamina propria mucosae in 9 cases. The results of this study highlight the difficulties of detecting residual carcinoma cells using conventional endoscopic biopsy in patients who have received CRT. Therefore, when residual cancer is clinically suspected in patients who have received CRT, the biopsy specimen should be obtained from the deep layer of the esophagus whenever possible. Additionally, close follow-up is required using positron emission tomography/computed tomography, endoscopy, and other radiological evaluations.

Rat kidney thromboxane receptor: molecular cloning, signal transduction, and intrarenal expression localization.

Thromboxane (TX) plays important roles in control of renal hemodynamics and water and electrolyte metabolism, and is involved in the pathophysiology of many renal diseases. The aim of the present study is to isolate a rat kidney cDNA encoding functional TX receptor, and to reveal its intrarenal expression localization. A clone (rTXR2) was isolated from a rat kidney cDNA library by a homology screening approach. rTXR2 was shown to encode the amino acid sequence containing seven transmembrane spanning domains representing rat (r) TX receptor. The membrane from COS-7 cells transiently transfected with rTXR2 cDNA was shown to be specifically bound by a thromboxane receptor antagonist, SQ29548. Either in Xenopus oocyte expression or in transfected COS-7 cells, rTX receptor was shown to be linked with Ca2+ messenger system. TX receptor-mediated increase in cytosolic Ca2+ was also observed in cultured glomerular mesangial cells. In situ hybridization showed that rTX receptor mRNA was detected in renal glomeruli, smooth muscle cells in renal arterioles, and transitional cell epithelium of renal pelvis. Reverse transcription linked to PCR applied to microdissected nephron segments indicated the presence of rTX receptor mRNA exclusively in the glomerulus. In conclusion, we have cloned a functional rat kidney TX receptor, which is expressed specifically in renal glomerulus, arterial smooth muscle cells, and transitional cell epithelium of renal pelvis. The present study will provide important insights into the etiology and pathophysiology of renal diseases with relation to TX metabolism.

Hypoxia-induced endothelial apoptosis through nuclear factor-kappaB (NF-kappaB)-mediated bcl-2 suppression: in vivo evidence of the importance of NF-kappaB in endothelial cell regulation.

The transcription factor nuclear factor-kappaB (NF-kappaB) plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes involved in endothelial activation. Although recent reports have documented the contribution of NF-kappaB to apoptosis, it is still controversial. Especially, the role of NF-kappaB in endothelial apoptosis is largely unknown. Hypoxia significantly induced human aortic endothelial cell death and apoptosis in a time-dependent manner (P<0.01), accompanied by NF-kappaB activation. Decrease in total cell number and increase in apoptotic cells induced by hypoxia were significantly attenuated by NF-kappaB decoy, but not by scrambled decoy, oligodeoxynucleotides (ODNs) (P<0.01). Increase in DNA fragmentation induced by hypoxia was also significantly inhibited by NF-kappaB decoy ODNs as compared with scrambled decoy ODNs (P<0.01). Moreover, transfection of NF-kappaB decoy ODNs resulted in a significant decrease in caspase-3-like activity, which is a common pathway for apoptosis, compared with scrambled decoy ODNs. Importantly, transfection of NF-kappaB decoy ODNs significantly increased protein of bcl-2, an inhibitor of apoptosis, and did not alter bax, a promoter of apoptosis, thereby resulting in a significant increase in the ratio of bcl-2 to bax (P<0.01). bcl-2 mRNA was also decreased by hypoxia, whereas transfection of NF-kappaB decoy ODNs significantly attenuated decrease in bcl-2 mRNA. These results demonstrate that activation of NF-kappaB by hypoxia induced endothelial apoptosis in a bcl-2-dependent manner. The importance of NF-kappaB in endothelial apoptosis was confirmed by the observation that pyrrolidine dithiocarbamate, a potent NF-kappaB inhibitor, prevented endothelial apoptosis, caspase 3-like activity, and bcl-2 downregulation induced by hypoxia. To test this hypothesis in vivo, we transfected NF-kappaB decoy ODNs into rat intact carotid artery after reperfusion injury. Reperfusion injury was associated with a significant increase in endothelial apoptosis at 24 hours, whereas NF-kappaB decoy ODN treatment markedly decreased terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive endothelial cells at 24 hours after reperfusion (P<0.01). Here, using synthetic double-stranded DNA with high affinity for NF-kappaB as a decoy approach, we demonstrated that activation of NF-kappaB by hypoxia caused aortic endothelial cell death and apoptosis through the suppression of bcl-2. NF-kappaB-mediated endothelial apoptosis induced by hypoxia may be involved in the pathogenesis of endothelial dysfunction observed in cardiovascular ischemic diseases.

Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat diabetic hind limb ischemia model: molecular mechanisms of delayed angiogenesis in diabetes.

BACKGROUND: Because no study has documented the angiogenic properties of hepatocyte growth factor (HGF) in a diabetes model, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease in diabetes. METHODS AND RESULTS: Because intramuscular injection of luciferase plasmid by the hemagglutinating virus of Japan (HVJ)-liposome method had much higher efficiency than injection of naked plasmid, we used the HVJ-liposome method to transfect the human HGF gene into the rat diabetic hindlimb model. As expected, transfection of human HGF vector resulted in a significant increase in blood flow as assessed by laser Doppler imaging and capillary density, even in the diabetes model, accompanied by the detection of human HGF protein. Interestingly, the degree of natural recovery of blood flow was significantly greater in nondiabetic rats than in diabetic rats. Thus, in an in vitro culture system, we further studied the molecular mechanisms of how diabetes delayed angiogenesis. Importantly, high-D-glucose treatment of endothelial cells resulted in a significant decrease in matrix metalloproteinase (MMP)-1 protein and ets-1 expression in human aortic endothelial cells. Similarly, high D-glucose significantly decreased mRNA and protein of HGF in endothelial cells. Downregulation of MMP-1 and ets-1 by high D-glucose might be due to a significant decrease in HGF, because HGF stimulated MMP-1 production and activated ets-1. CONCLUSIONS: Overall, intramuscular injection of human HGF plasmid induced therapeutic angiogenesis in a rat diabetic ischemic hindlimb model as a potential therapy for peripheral arterial disease. The delay of angiogenesis in diabetes might be due to downregulation of MMP-1 and ets-1 through a decrease in HGF by high D-glucose.

Potential contribution of a novel antifibrotic factor, hepatocyte growth factor, to prevention of myocardial fibrosis by angiotensin II blockade in cardiomyopathic hamsters.

BACKGROUND: Because hepatocyte growth factor (HGF) prevented and/or regressed fibrosis in liver and pulmonary injury models, HGF may play an important role in the pathogenesis of fibrotic cardiovascular disease. Because angiotensin (Ang) II significantly decreased local HGF production, we performed (1) in vitro experiments using fibroblasts and (2) administration of an ACE inhibitor (temocapril) and an Ang II type 1 receptor antagonist (CS-866) to cardiomyopathic hamsters. METHODS AND RESULTS: In human fibroblasts, HGF significantly increased the production of matrix metalloprotease-1 (MMP-1) and urokinase plasminogen activator, whereas HGF also significantly attenuated the reduction of MMP-1 activity induced by Ang II. In contrast, HGF significantly decreased transforming growth factor (TGF)-beta mRNA stimulated by Ang II, whereas HGF also decreased basal TGF-beta protein level without affecting growth. Similarly, in rat cardiac fibroblasts, HGF inhibited the expression and production of TGF-beta, whereas HGF upregulated its specific receptor, c-met. Conversely, in vivo experiments revealed that administration of temocapril and CS-866 to cardiomyopathic hamsters resulted in a significant decrease in fibrotic area and increase in cardiac HGF concentration and mRNA (P<0.01), whereas cardiac concentration and mRNA of HGF were significantly decreased in cardiomyopathic hamsters. In contrast, mRNA expression of collagen III was markedly decreased by treatment with temocapril and CS-866. CONCLUSIONS: Here, we demonstrated that Ang II blockade prevented myocardial fibrosis in the cardiomyopathic hamster, accompanied by a significant increase in cardiac HGF. Overall, increase in local HGF expression may participate in the prevention of myocardial injury by Ang II blockade through its antifibrotic action.

Transfection of antisense p53 tumor suppressor gene oligodeoxynucleotides into rat carotid artery results in abnormal growth of vascular smooth muscle cells.

BACKGROUND: Although loss of activity of an antioncogene, the p53 tumor suppressor gene product, has been postulated in the pathogenesis of human restenosis, little is known about the role of p53 in the regulation of vascular smooth muscle cell (VSMC) growth. In this study, to clarify the role of p53 in the pathogenesis of restenosis, we examined transfection of antisense p53 oligodeoxynucleotides (ODN) into VSMC in vitro and rat carotid artery in vivo. METHODS AND RESULTS: The specificity of antisense p53 ODN was confirmed by a significant decrease in p53 protein. Transfection of antisense p53 ODN into VSMC resulted in a significant increase in DNA synthesis and cell number as compared with sense and scrambled ODN (P<0.01). Importantly, transfection of antisense p53 ODN into rat intact carotid artery resulted in a significant increase in the ratio of neointima to medial area at 2 and 4 weeks after transfection, accompanied by a significant decrease in p53 protein (P<0.01). Moreover, cotransfection of wild-type p53 plasmid completely abolished neointimal formation induced by antisense p53 ODN. The sustained effect of a single antisense ODN administration was confirmed by the kinetics of ODN in the vessel wall with the use of FITC-labeled ODN. CONCLUSIONS: Overall, the present study demonstrated that loss of p53 by antisense p53 ODN resulted in an abnormal VSMC growth in vitro and in vivo. These results demonstrated the potential contribution of p53 to the pathogenesis of restenosis.

Phosphorylation of p38 mitogen-activated protein kinase downstream of bax-caspase-3 pathway leads to cell death induced by high D-glucose in human endothelial cells.

Because high D-glucose significantly stimulates endothelial cell death, we examined the molecular mechanisms of high D-glucose-induced endothelial apoptosis. Treatment of human aortic endothelial cells with high D-glucose (25 mmol/l), but not mannitol and L-glucose, resulted in a significant decrease in cell number and a significant increase in apoptotic cells as compared with a physiological concentration (5 mmol/l). Interestingly, high D-glucose treatment significantly increased bax protein, accompanied by translocation of bax protein from cytosol to mitochondria-enriched heavy membrane fraction. In contrast, the expression and distribution of bcl-2 protein were not altered by high D-glucose. In addition, the activity of caspase-3 proteases was increased after exposure to high glucose, whereas caspase inhibitors prevented endothelial cell death induced by high D-glucose. On the other hand, p38 mitogen-activated protein kinase (MAPK) was markedly phosphorylated and showed sustained phosphorylation after stimulation. A specific inhibitor of p38 MAPK, SB 203580, and the overexpression of kinase-inactive p38 MAPK significantly attenuated cell death induced by high D-glucose in human aortic endothelial cells, whereas at 6 h after high D-glucose treatment, SB 203580 and overexpression of kinase-inactive p38 MAPK did not attenuate caspase-3 activation induced by high D-glucose. Importantly, caspase inhibitors significantly attenuated the sustained phosphorylation of p38 MAPK induced by high D-glucose. Thus, we finally focused the MAPK kinase (MEK) kinase 1 (MEKK1) to further examine the cross-talk between p38 MAPK and the bax-caspase proteases pathway. High D-glucose treatment induced MEKK1 cleavage, whereas caspase inhibitors significantly attenuated the cleavage. Importantly, kinase-inactive MEKK1 also blocked the phosphorylation of p38 MAPK induced by high D-glucose. Here, we demonstrated that high D-glucose induced apoptosis in human endothelial cells through activation of the bax-caspase proteases pathway and through phosphorylation of p38 MAPK mediated by MEKK1. Phosphorylation of p38 MAPK downstream of the bax-caspase pathway may play a pivotal role in endothelial apoptosis mediated by high D-glucose.

Beneficial effect of intracoronary verapamil on microvascular and myocardial salvage in patients with acute myocardial infarction.

OBJECTIVES: We assessed the acute effect of intracoronary injection of verapamil on microvascular function after primary percutaneous translumanal coronary angioplasty (PTCA) for acute myocardial infarction (AMI) with myocardial contrast echocardiography (MCE) in relation to functional outcomes. BACKGROUND: Recent clinical studies have documented the potential of verapamil for possible increase in coronary blood flow after primary PTCA. METHODS: Forty patients with a first AMI were randomly assigned to the verapamil group (n = 20) or the control group (n = 20). In the verapamil group, verapamil (0.5 mg) was injected into the infarct-related artery shortly after PTCA, followed by the oral administration. We performed MCE with an intracoronary injection of sonicated microbubbles before and after verapamil. To assess microvascular integrity, we determined the baseline-subtracted peak intensity in the risk area and the ratio of the no reflow zone plus the low reflow zone to the risk area (low reflow ratio). We determined the average wall motion score (dyskinesia/akinesia = 3; normal = 0) in the risk area on the day of AMI and a mean of 24 days later. RESULTS: The low reflow zone was observed shortly after PTCA in 14 verapamil group patients, and the low reflow ratio decreased after verapamil (0.39 +/- 0.23 vs. 0.29 +/- 0.17 [mean +/- SD], p < 0.05). Peak intensity significantly (p < 0.05) increased from 6 +/- 5 to 12 +/- 6 after verapamil. The reduction in wall motion score from the acute (day -1) to the late stage (day -24) was significantly greater in the verapamil group than in the control group (0.7 +/- 0.8 vs. 0.2 +/- 1.3, respectively, p < 0.05). CONCLUSIONS: Intracoronary administration of verapamil after primary PTCA can attenuate microvascular dysfunction and thereby augment myocardial blood flow in patients with AMI, leading to better functional outcome than with PTCA alone.

Intravenous nicorandil can preserve microvascular integrity and myocardial viability in patients with reperfused anterior wall myocardial infarction.

OBJECTIVES: We assessed whether the intravenous administration of nicorandil, an adenosine triphosphate (ATP)-sensitive K+ channel opener, exerts beneficial effect on microvascular function and functional and clinical outcomes in patients with acute myocardial infarction (AMI). BACKGROUND: Experimental studies documented that ATP-sensitive K+ channel opener exerts cardioprotection after prolonged ischemia. METHODS: We randomly divided 81 patients with a first anterior AMI into two groups, nicorandil (n = 40) and control groups (n = 41). All patients received successful coronary angioplasty within 12 h after the symptom onset and underwent myocardial contrast echcardiography (MCE) with the intracoronary injection of sonicated microbubbles. In the nicorandil group, we injected 4 mg of nicorandil followed by the infusion at 6 mg/h for 24 h and by oral nicorandil (15 mg/day). RESULTS: The improvement in regional left ventricular function, wall motion score and regional wall motion was significantly better in the nicorandil group then in the control group. Intractable congestive heart failure, malignant ventricular arrhythmia and pericardial effusion were more frequently found in the control group than in the nicorandil group (15% vs. 37%, 5% vs. 20% and 8% vs. 37%, p < 0.05, respectively). The frequency of sizable MCE no reflow phenomenon was significantly lower in the nicorandil group than in the control group (15% vs. 33%, p < 0.05). CONCLUSIONS: Intravenous nicorandil in conjunction with coronary angioplasty is associated with better functional and clinical outcomes compared to angioplasty alone in patients with an anterior AMI. Myocardial contrast echocardiography findings imply that an improvement in microvascular function with nicorandil may be attributable to this better outcome.

Additive effects of nicorandil on coronary blood flow during continuous administration of nitroglycerin.

OBJECTIVES: We examined whether patients with ischemic heart disease (IHD) should be treated with nicorandil, an adenosine triphosphate-sensitive potassium channel opener, in addition to the regular use of nitrates. BACKGROUND: It has been reported that nicorandil possibly has additive effects on nitroglycerin (NTG) treatment for angina, but the mechanism is not clear. METHODS: We directly measured anterograde coronary blood flow (CBF) with a Doppler guide wire to examine the effects of intravenous administration of NTG (0.3 mg) and nicorandil (6 mg) during continuous administration of NTG at a sufficient dose (25 microg/min) in subjects with normal and stenotic coronary arteries. RESULTS: Additional systemic administration of NTG decreased anterograde CBF (normal -19.7%; stenotic -21.2%). In contrast, nicorandil increased anterograde CBF in both normal (54.6%) and stenotic (89.6%) coronary arteries, without the coronary steal phenomenon. There was a tendency toward nicorandil-dilated diameters in the patients with stenotic arteries (p = 0.06). There were no effects of additional administration on pulmonary artery wedge pressure. There was no difference in changes in heart rate and mean aortic blood pressure between NTG and nicorandil therapy. CONCLUSIONS: These results suggest that in patients treated with nitrates, additional administration of nicorandil is more useful, in terms of increasing CBF, than additional administration of nitrates. Adjunctive use of nicorandil with nitrates may provide the further benefit of myocardial protection and may improve the prognosis of patients with IHD.

Transcriptional suppression of type 1 angiotensin II receptor gene expression by peroxisome proliferator-activated receptor-gamma in vascular smooth muscle cells.

Angiotensin (A) II plays a critical role in vascular remodeling, and its action is mediated by type 1 AII receptor (AT1R). Recently, 15-deoxy-(Delta)(12,14)-prostaglandin J(2) and thiazolidinediones have been shown to be ligands for peroxisome proliferator-activated receptor (PPAR)-gamma and activate PPAR-gamma. In the present work, we have studied the effect of PPAR-gamma on AT1R expression in rat vascular smooth muscle cells (VSMCs). We observed that: 1) endogenous AT1R expression was significantly decreased by PPAR-gamma ligands both at messenger RNA and protein levels, whereas AT1R messenger RNA stability was not affected; 2) AII-induced increase of (3)H-thymidine incorporation into VSMCs was inhibited by PPAR-gamma ligands; 3) rat AT1R gene promoter activity was significantly suppressed by PPAR-gamma ligands, and PPAR-gamma overexpression further suppressed the promoter activity; 4) transcriptional analyses using AT1R gene promoter mutants revealed that a GC-box-related sequence within the -58/-34 region of the AT1R gene promoter was responsible for the suppression; 5) Sp1 overexpression stimulated AT1R gene transcription via the GC-box-related sequence, which was inhibited by additional PPAR-gamma overexpression; 6) electrophoretic mobility shift assay suggested that Sp1 could bind to the GC-box-related sequence whereas PPAR-gamma could not; 7) antibody supershift experiments using VSMC nuclear extracts revealed that protein-DNA complexes formed on the GC-box-related sequence, which were decreased by PPAR-gamma coincubation, were mostly composed of Sp1; and 8) glutathione S-transferase pull-down assay revealed a direct interaction between PPAR-gamma and Sp1. Taken together, it is suggested that activated PPAR-gamma suppresses AT1R gene at a transcriptional level by inhibiting Sp1 via a protein-protein interaction. PPAR-gamma ligands, thus, may inhibit AII-induced cell growth and hypertrophy in VSMCs by AT1R expression suppression and possibly be beneficial for treatment of diabetic patients with hypertension and atherosclerosis.

Cyclic AMP inhibited proliferation of human aortic vascular smooth muscle cells, accompanied by induction of p53 and p21.

Although cAMP is an important second messenger that plays a pivotal role in the regulation of platelet aggregation and dilatation of blood vessels, little is known about the action of cAMP on the growth of vascular smooth muscle cells (VSMCs). Thus, we initially studied the effects of cAMP accumulation by using various cAMP stimulants, including a phosphodiesterase type 3 inhibitor (cilostazol) on human aortic VSMC growth. Accumulation of cAMP inhibited the platelet-derived growth factor (PDGF)-stimulated VSMC growth in a dose-dependent manner (P<0.01), whereas PDGF significantly stimulated the growth of human VSMCs. Thus, we focused on the role of cell cycle regulatory genes, especially on a negative regulator, an anti-oncogene, p53. The protein of p53 was potentiated by cilostazol as well as forskolin and 8-bromo-cAMP, whereas PDGF decreased p53 expression. Upregulation of p53 protein by cAMP was further confirmed by the observation that the decrease in p21, a p53-inducible protein, by PDGF was significantly attenuated by cilostazol in a dose-dependent manner (P<0.01). These results revealed that accumulation of cAMP inhibited VSMC proliferation, which was at least in part due to an increase in p53-p21 expression. Because p53 and p21 have been reported to induce apoptosis, we examined apoptotic cells for cAMP accumulation. Incubation of VSMCs with cilostazol resulted in a significant increase in apoptotic cells in a dose-dependent manner compared with vehicle treatment as assessed by nuclear chromatic morphology (P<0.01); forskolin also stimulated apoptotic cells. Consistent with nuclear staining, DNA fragmentation in VSMCs treated with forskolin as well as 8-bromo-cAMP and cilostazol was significantly increased compared with DNA fragmentation in VSMCs treated with vehicle, whereas PDGF significantly decreased the rate of DNA fragmentation (P<0.01). Overall, these results demonstrated that cAMP inhibited the proliferation of human aortic VSMCs, accompanied by p53-p21-mediated apoptosis. Analogues of cAMP that have direct inhibitory effects on VSMC proliferation can be considered as potential antiproliferative drugs against VSMC growth.

Association of a mutation in thiazide-sensitive Na-Cl cotransporter with familial Gitelman's syndrome.

Gitelman's syndrome is a variant of Bartter's syndrome, characterized by hypokalemia, hypomagnesemia, hypocalciuria, and hypovolemia. We have observed familial cases of Gitelman's syndrome, and a possible mutation in thiazide-sensitive Na-Cl cotransporter was investigated in this kindred. The proband was a 47-yr-old Japanese female, and her mother was also affected. Her parents and maternal grandparents are consanguineous. By using PCR-amplification and direct sequencing, we identified a novel non-conservative missense mutation at 623 amino acid position, which substitutes proline for leucine (L623P), and also creates an Nci I restriction site in the exon 15. The mutation was not detected in normal healthy subjects (n = 102). Nci I digestion of PCR-amplified exon 15 DNA fragments from individuals in the family indicated the autosomal recessive inheritance of the disorder. In conclusion, the L623P mutation in the thiazide-sensitive Na-Cl cotransporter gene is suggested to impair the transporter activity, and to underlie this familial Gitelman's syndrome; Gitelman's syndrome observed in this kindred has been inherited in an autosomal recessive fashion.

Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as cytokine supplement therapy.

Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells and acts as a survival factor against endothelial cell death. Therefore we hypothesized that a decrease in local vascular HGF might be related to the pathogenesis of peripheral arterial disease. We initially evaluated vascular HGF concentration in the vessels of patients with arteriosclerosis obliterans. Consistent with in vitro findings that hypoxia downregulated vascular HGF production, vascular HGF concentration in the diseased segments of vessels from patients with arteriosclerosis obliterans was significantly decreased as compared with disease-free segments from the same patients (P<0.05), accompanied by a marked reduction in HGF mRNA. On the other hand, a novel therapeutic strategy for ischemic diseases that uses angiogenic growth factors to expedite and/or augment collateral artery development has recently been proposed. Thus in view of the decreased endogenous vascular HGF, rhHGF (500 micrograms/animal) was intra-arterially administered through the internal iliac artery of rabbits in which the femoral artery was excised to induce unilateral hind limb ischemia, to evaluate the angiogenic activity of HGF, which could potentially have a beneficial effect in hypoxia. Administration of rhHGF twice on days 10 and 12 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemic model as assessed by angiography (P<0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-treated animals. In addition, we examined the feasibility of intravenous administration of rhHGF in a moderate ischemia model. Importantly, intravenous administration of rhHGF also resulted in a significant increase in angiographic score as compared with vehicle (P<0.01). Overall, a decrease in vascular HGF might be related to the pathogenesis of peripheral arterial disease. In the presence of decreased endogenous HGF, administration of rhHGF induced therapeutic angiogenesis in the rabbit ischemic hind limb model, as potential cytokine supplement therapy for peripheral arterial disease.

PDI and glutathione-mediated reduction of the glutathionylated variant of human lysozyme.

A mutant human lysozyme, designated as C77A-a, in which glutathione is bound to Cys95, has been shown to mimic an intermediate in the formation of a disulfide bond during folding of human (h)-lysozyme. Protein disulfide isomerase (PDI), which is believed to catalyze disulfide bond formation and associated protein folding in the endoplasmic reticulum, attacked the glutathionylated h-lysozyme C77A-a to dissociate the glutathione molecule. Structural analyses showed that the protein is folded and that the structure around the disulfide bond, buried in a hydrophobic core, between the protein and the bound glutathione is fairly rigid. Thioredoxin, which has higher reducing activity of protein disulfides than PDI, catalyzed the reduction with lower efficiency. These results strongly suggest that PDI can catalyze the disulfide formation in intermediates with compact structure like the native states in the later step of in vivo protein folding.

Anti-apoptotic action of hepatocyte growth factor through mitogen-activated protein kinase on human aortic endothelial cells.

OBJECTIVE: To investigate the molecular mechanisms of the anti-apoptotic action of hepatocyte growth factor (HGF), a novel angiogenic growth factor that may have a pivotal role in the regulation of endothelial cells, on human aortic endothelial cells. METHODS: An index of cell number and death was determined using a water-soluble tetrazolium salt dye assay, DNA fragmentation enzyme-linked immunosorbent assay, and non-confocal fluorescence microscopy of nuclear staining with Hoechst 33258 and propidium iodide. Extracellular-signal-regulated protein kinase (ERK) and the p38 mitogen-activated protein kinase (p38MAPK) were analysed by Western blotting using a phospho-specific antibody. RESULTS: Treatment of quiescent endothelial cells with HGF resulted in significant dose-dependent increases in cell numbers and decreases in lactate dehydrogenase (LDH) release. Moreover, HGF significantly attenuated endothelial cell death induced by culture in serum-free conditions. We therefore focused on the signal transduction system, and in particular on ERK and p38MAPK. ERK was markedly phosphorylated by HGF. The contribution of ERK to cell growth was supported by the observation that addition of PD98059, a specific inhibitor of MAPK kinase, significantly attenuated the increase in endothelial cell numbers induced by HGF, in a dose-dependent manner. Similarly, PD98059 also attenuated the decrease in LDH release and DNA fragmentation by HGF under serum-free conditions. Interestingly, ERK was re-phosphorylated at 12 h after stimulation. Re-phosphorylation of ERK was the result of induction of endogenous HGF by exogenously added HGF, as addition of neutralizing anti-HGF antibody to the conditioned medium attenuated re-phosphorylation of ERK at 12 h. In contrast, although p38MAPK was also phosphorylated by HGF, SB203580, a specific inhibitor of p38MAPK, failed to change the endothelial cell growth induced by HGF. CONCLUSION: We have demonstrated that the anti-apoptotic action of HGF against endothelial cell death was mainly through phosphorylation of ERK on human endothelial cells.

Potential of microvascular reperfusion with adjunctive pharmacological intervention: its impact on myocardial perfusion and functional outcomes in patients with acute myocardial infarction.

One of the major limitations of reperfusion therapy in acute myocardial infarction (AMI) is the presentation of no-reflow phenomenon. In 25 to 30% of patients with AMI. myocardial blood flow is occasionally profoundly reduced, even after coronary recanalisation, because of microvascular dysfunction so-called no-reflow phenomenon. Patients with this phenomenon are regarded as a high risk group among patients with reperfused AMI. Clinical studies using myocardial contrast echocardiography have demonstrated that intracoronary injection of calcium antagonists or potassium channel agonists in conjunction with coronary reperfusion can augment myocardial blood flow and that this was associated with better functional and clinical outcomes than with percutaneous transluminal coronary angioplasty alone. Thus, it is possible to prevent reperfusion injury and improve cardiac function using a adjunctive pharmacological intervention, either intravenously or by infusion directly into the coronary artery.

1H and 15N NMR study of human lysozyme.

The 15N signal assignment of human lysozyme was carried out by using 1H-1H and 1H-15N two dimensional experiments. To solve the severe overlap problem of the NH signals, uniform labeling of the protein with 15N was introduced. The uniformly 15N labeled protein was prepared using a high-expression system of Saccharomyces cerevisiae. From the analyses of 1H and 15N NMR spectra, all of the backbone 15N signals of the molecule were assigned to each specific residue in the amino acid sequence. Recently published proton signal assignments [Redfield & Dobson (1990) Biochemistry, 29, 7201-7214] were confirmed by these complementary data. In addition, assignments were extended to side chain 15NH2 groups of asparagine and glutamine. Elements of secondary structure were deduced from the pattern of sequential and medium-range NOE connectivities. Two beta-sheets and four alpha-helices could be identified in the protein, which were in good agreement with those determined by X-ray crystallography. The interaction between human lysozyme and its inhibitor N-acetyl-chitotriose was investigated by 15N-1H HMQC spectra. Most of the 15N-NH cross-peaks in the spectra were separated well enough to be followed during the titration experiment. Residues whose NH proton signals decrease in intensity upon complex formation, are located mainly around subsites B, C, and D. Local conformational changes were observed around the fourth helix adjacent to the cleft of human lysozyme.

Functional analysis and chromosomal gene assignment of rat kidney prostaglandin EP3 receptor.

We have reported two isoformes of rat prostaglandin EP3 receptor with their different carboxyl-terminal tails (rEP3A and rEP3B receptors), which are derived by alternative RNA splicing, and both receptors have been shown to be localized to renal distal tubules. In the present study, we characterized the signal transduction system of rat kidney EP3 receptors either in a renal cell line mimicking renal distal tubule cells, TKC2, or in COS-7 cells by functional expression of these receptors. We also examined the chromosomal localization of the EP3 receptor gene by fluorescence in situ hybridization (FISH). In TKC2 cells, vasopressin (AVP, 10(-7) M), prostaglandin (PG) E2 (10(-7) M), or forskolin (10(-8) M) markedly stimulated cyclic AMP formation. Overexpression of the rEP3A receptor significantly attenuated the AVP-, PGE2- or forskolin-induced cyclic AMP formation, whereas there was no change with rEP3B receptor expression. On the other hand, in COS-7 cells transfected with rEP3A receptor cDNA, PGE2 (10(-7) M) did not affect cytosolic free calcium concentration ([Ca2+]i), whereas transfection of rEP3B receptor cDNA evoked PGE2-induced increases in [Ca2+]i. Moreover, we have revealed that the rEP3 receptor gene is localized to rat chromosome 2q44-45. In conclusion, rEP3A or rEP3B receptor is suggested as a mediator of the natriuretic/diuretic action of PGE2 in renal distal tubules via a decrease in cyclic AMP formation or an increase in [Ca2+]i, respectively. Information of the gene assignment of rat EP3 receptor to rat chromosome 2q44-45 is useful for further analysis of the role of EP3 receptor in genetically hypertensive rat models.

A family with von Hippel-Lindau disease revealed by pheochromocytoma.

Von Hippel-Lindau (VHL) disease is an inherited neoplastic disease characterized by a predisposition to develop retinal angiomas, central nervous system hemangioblastomas, renal cell carcinomas, pancreatic cysts and pheochromocytomas. Recently, we encountered three members of the same family who each had both VHL disease and pheochromocytoma. As in all three patients we suspected pheochromocytoma, the diagnosis of VHL disease should be considered. The possible presence of VHL disease was initially investigated in all three patients based on the presence of pheochromocytoma. A mutational analysis of the VHL gene revealed the presence of a missense mutation, consisting of a G to A transversion, at nucleotide 713 in all three patients. This germline point mutation in the VHL gene is often detected in type 2 VHL disease with pheochromocytoma. Genetic analysis seems to be useful for early detection of VHL disease, even when the formal criteria for diagnosis of this disease are lacking.