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.

Angiogenic and antifibrotic actions of hepatocyte growth factor improve cardiac dysfunction in porcine ischemic cardiomyopathy.

Impairment of cardiac function in ischemic cardiomyopathy has been postulated to be due to the decrease in blood flow and increase in collagen synthesis. Therefore, an approach to alter them directly by means of a growth factor may open up a new therapeutic concept in ischemic cardiomyopathy. From this viewpoint, hepatocyte growth factor (HGF) is a unique growth factor with angiogenic and antifibrotic effects. Thus, we examined the feasibility of gene therapy using HGF plasmid DNA for ischemic cardiomyopathy. Human HGF plasmid DNA at a dose of 0.4 or 4 mg was injected into ischemic myocardium of pigs induced by ameroid constrictor with the NOGA system. At 1 month after injection, the ischemic area was significantly reduced in the HGF group, accompanied by a significant increase in capillary density and regional myocardial perfusion in the ischemic area (P<0.01). In contrast, a significant decrease in fibrotic area was observed in the HGF group, associated with a significant decrease in collagen I, III and TGF-beta synthesis as compared to the control group (P<0.01). Consistently, cardiac function was significantly improved in the 4 mg HGF group as compared to the control group (P<0.05). Overall, the present in vivo experiments demonstrated that intramyocardial injection of human HGF plasmid DNA in ischemic cardiomyopathy resulted in a significant improvement in cardiac function through an increase in blood flow and decrease in fibrosis. These favorable outcomes suggest potential utility to treat patients with ischemic heart disease using HGF gene transfer. Currently, a phase I study using human HGF plasmid DNA is ongoing to test the validity of this concept.

Acceleration of wound healing by combined gene transfer of hepatocyte growth factor and prostacyclin synthase with Shima Jet.

Although skin diseases are one of the target diseases for gene therapy, there has been no practical gene transfer method. First, we examined gene transfer efficiency of the spring-powered jet injector, Shima Jet, which was originally developed as a non-needle jet injector of insulin. Local gene expression was about 100 times higher when the luciferase plasmid was transferred by the Shima Jet than by a needle. Gene transfer of beta-galactosidase revealed gene expression in the epidermis. Based on these results, we then examined the potential of gene therapy using the Shima Jet for wound healing. An increase of cellular proliferation of the epidermis and the number of microvessels in the granulation tissue was observed after hepatocyte growth factor (HGF) gene transfer. An increase in blood flow around the wound was observed after prostacyclin synthase (PGIS) gene transfer. Moreover, promotion on wound healing was observed in HGF gene transferred group, and further promotion was observed in combined gene transferred group as assessed by measuring wound area. These results indicate that co-transfer of HGF and PGIS genes by the Shima Jet could be an effective strategy to wound healing.

Development of efficient plasmid DNA transfer into adult rat central nervous system using microbubble-enhanced ultrasound.

Although gene therapy might become a promising approach for central nervous system diseases, the safety issue is a serious consideration in human gene therapy. To overcome this problem, we developed an efficient gene transfer method into the adult rat brain based on plasmid DNA using a microbubble-enhanced ultrasound method, since microbubble-enhanced ultrasound has shown promise for transfecting genes into other tissues such as blood vessels. Using the microbubble-enhanced ultrasound method, luciferase expression was increased approximately 10-fold as compared to injection of naked plasmid DNA alone. Interestingly, the site of gene expression was limited to the site of insonation with intracisternal injection, in contrast to previous studies using viruses. Expression of the reporter gene, Venus, was readily detected in the central nervous system. The transfected cells were mainly detected in meningeal cells with intracisternal injection, and in glial cells with intrastriatal injection. There was no obvious evidence of tissue damage by microbubble-enhanced ultrasound. Overall, the present study demonstrated the feasibility of efficient plasmid DNA transfer into the central nervous system, providing a new option for treating various diseases such as tumors.

Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle.

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.

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.

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.

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.

Differential effects among thiazolidinediones on the transcription of thromboxane receptor and angiotensin II type 1 receptor genes.

Peroxisome proliferator-activated receptor (PPAR)-gamma ligands thiazolidinediones (TZDs) have recently been reported to be anti-hypertensive and anti-atherosclerotic. We have previously shown that one of the TZDs troglitazone significantly suppressed the transcription of both thromboxane receptor (TXR) and angiotensin II type 1 receptor (AT1R) genes in vascular smooth muscle cells (VSMCs) by activating PPAR-gamma. In the present study, we compared the effects of troglitazone and other TZDs on the transcription of these genes. TXR and AT1R mRNAs in rat VSMCs were determined by semi-quantitative RT-PCR. Luciferase chimeric constructs containing either the 989-bp rat TXR gene promoter or the 1,969-bp rat AT1R gene promoter were transiently transfected into VSMCs. The cells were incubated with troglitazone, RS-1455 (a derivative of troglitazone which does not contain the hindered phenol resembling alpha-tocopherol), pioglitazone, or rosiglitazone for 12 h before harvesting. mRNA expression levels of TXR and AT1R were significantly decreased by troglitazone in contrast to rosiglitazone. TXR gene and AT1R gene transcription was significantly suppressed by troglitazone in a dose-dependent manner, while RS-1455 was less potent. Pioglitazone and rosiglitazone weakly suppressed the transcription of both genes in a manner almost similar to RS-1455. We have shown that troglitazone suppresses transcription of both the TXR and AT1R genes more potently than other TZDs. The structure of troglitazone and RS-1455 is identical except the hindered phenol, which is recently recognized to function as an antioxidant. Moreover, we have shown that the potency for activating PPAR-gamma is almost identical between troglitazone and RS-1455. We therefore speculate that the strong transcriptional suppression of the TXR and AT1R genes by troglitazone may be mediated in part by its antioxidant effect.

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.

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.

Renal tubule-specific transcription and chromosomal localization of rat thiazide-sensitive Na-Cl cotransporter gene.

The molecular mechanism underlying the renal expression localization of the thiazide-sensitive Na-Cl cotransporter (TSC) gene was studied. The TSC gene was localized to chromosome 19p12-14. In cultured cells, tissue-specific transcription activity of the 5'-flanking region of the rat rTSC gene (5'FL/rTSC) was demonstrated, and the major promoter region was located between position -580 and -141. To further examine the tissue-specific transcription, transgenic rats harboring the 5'FL/rTSC fused upstream of the LacZ gene were generated. Immunohistochemical analysis clearly showed that LacZ gene expression was co-localized to distal convoluted tubules (DCT) with TSC, indicating that the 5'FL/rTSC regulates the renal tubule-specific TSC expression. Because a transcription factor, HFH-3 (hepatocyte nuclear factor-3/folk head homologue-3), had also been localized to DCT, a possible role of the putative cis-acting element (HFH-3/rTSC, -400/-387 position) for HFH-3 binding in the tissue-specific transcription was examined. Deletion and mutation analyses suggested that transcription of the HFH-3/rTSC was actually responsive to HFH-3, and electrophoretic mobility shift assay showed a direct binding of in vitro synthesized HFH-3 to the HFH-3/rTSC. In conclusion, the rTSC gene is localized to rat chromosome 19p12--24. The transcription regulatory region of the TSC gene confers DCT-specific gene expression. DCT-specific transcription factor HFH-3 may be involved in the renal tubule-specific transcription of TSC gene.

Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat and rabbit hindlimb ischemia models: preclinical study for treatment of peripheral arterial disease.

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. Recently, a novel therapeutic strategy for ischemic diseases using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. We have previously reported that intra-arterial administration of recombinant HGF induced angiogenesis in a rabbit hindlimb ischemia model. In this study, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease rather than recombinant therapy, due to its disadvantages. Initially, we examined the transfection of 'naked' human HGF plasmid into a rat hindlimb ischemia model. Intramuscular injection of human HGF plasmid resulted in a significant increase in blood flow as assessed by laser Doppler imaging, accompanied by the detection of human HGF protein. A significant increase in capillary density was found in rats transfected with human HGF as compared with control vector, in a dose-dependent manner (P < 0.01). Importantly, at 5 weeks after transfection, the degree of angiogenesis induced by transfection of HGF plasmid was significantly greater than that caused by a single injection of recombinant HGF. As an approach to human gene therapy, we also employed a rabbit hindlimb ischemia model as a preclinical study. Naked HGF plasmid was intramuscularly injected in the ischemic hindlimb of rabbits, to evaluate its angiogenic activity. Intramuscular injection of HGF plasmid once on day 10 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemia 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-transfected animals. In addition, a significant increase in blood flow was assessed by a Doppler flow wire and the ratio in blood pressure of the ischemic limb to the normal limb was observed in rabbits transfected with HGF plasmid as compared with rabbits transfected with control vector (P < 0.01). Overall, intramuscular injection of naked human HGF plasmid induced therapeutic angiogenesis in rat and rabbit ischemic hindlimb models, as potential therapy for peripheral arterial disease.

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.

Mitogenic and antiapoptotic actions of hepatocyte growth factor through ERK, STAT3, and AKT in endothelial cells.

Hepatocyte growth factor (HGF), a member of the angiogenic growth factors, may play a pivotal role in the regulation of endothelial cells, inasmuch as HGF shows mitogenic and antiapoptotic actions in endothelial cells. Because the mechanism of these actions is still unclear, we examined the signal transduction system of HGF in human aortic endothelial cells. Treatment of endothelial cells with recombinant HGF (rHGF) resulted in a significant increase in DNA synthesis as assessed by thymidine incorporation. Importantly, phosphorylation of extracellular signal-related kinase (ERK) and Akt by rHGF was clearly observed. Thus, we further examined the effects of specific inhibitors of ERK or Akt on cell proliferation. Pretreatment with PD98059, a mitogen-activated protein kinase kinase inhibitor, significantly attenuated cell proliferation induced by rHGF, whereas inhibitors of phosphatidylinositol-3-OH kinase, wortmannin, and LY-294002, did not. Interestingly, treatment with rHGF significantly increased the phosphorylation of the signal transducers and activators of transcription (STAT)3 (Ser727), whereas PD98059 attenuated the phosphorylation of Ser727 induced by rHGF. In addition, treatment with rHGF significantly increased the promoter activity of c-fos, which includes the sis-inducible element and serum response element, whereas PD98059 completely attenuated the activation of the c-fos promoter induced by rHGF. In contrast, inhibition of Akt by wortmannin and LY-294002 failed to inhibit the phosphorylation of STAT3 and c-fos activation. On the other hand, treatment with rHGF attenuated the increase in LDH release and caspase-3 activity induced by tumor necrosis factor-alpha stimulation. In contrast to DNA synthesis, wortmannin and LY-294002 markedly attenuated the decrease in caspase-3 activity mediated by rHGF, whereas PD98059 did not. Overall, the present study demonstrated that HGF stimulated cell proliferation through the ERK-STAT3 (Ser727) pathway and had an antiapoptotic action through the phosphatidylinositol-3-OH kinase-Akt pathway in human aortic endothelial cells. These findings provide new perspectives in the role of HGF in cardiovascular disease.

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.

Suppression of rat thromboxane synthase gene transcription by peroxisome proliferator-activated receptor gamma in macrophages via an interaction with NRF2.

We have studied the transcription regulation of the rat thromboxane synthase (TXS) gene by peroxisome proliferator-activated receptor gamma (PPARgamma) in macrophages. The transcription activity of a cloned 5'-flanking region (1.6 kilobases) of the rat TXS gene (5'FL-TXS) was examined by luciferase reporter gene assay. TXS mRNA expression and the transcription activity of 5'FL-TXS were inhibited by PPARgamma ligands, 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)), and the thiazolidinedione troglitazone (TRO) in a dose-dependent manner. Overexpression of PPARgamma also significantly suppressed transcription, and further addition of PGJ(2) or TRO augmented the suppression. Deletion analysis showed that the element responsible for the PPARgamma effect is located in a region containing the nuclear factor E2 (NF-E2)/AP-1 site (-98/-88), which was indicated to be the major promoter of the TXS gene. By electrophoretic mobility shift assay using the NF-E2/AP-1 site and nuclear extracts from macrophages, we observed a specific protein-DNA complex formation, which was inhibited by a specific antibody against the transcription factor NRF2 (NF-E2-related factor 2). Moreover, the complex was decreased with PGJ(2), TRO, or in vitro translated PPARgamma. The transcription suppression by PPARgamma was confirmed using this truncated NRF2-binding element (-98/-88) by the reporter gene assay. Finally, a direct interaction between PPARgamma and NRF2 was confirmed by glutathione S-transferase pull-down assay. In conclusion, the NRF2-binding site (-98/-88) is the major promoter of 5'FL-TXS which can be suppressed by activated PPARgamma via a protein-protein interaction with NRF2 in macrophages.

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.

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.

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.