Simultaneous spawning by female stream goby Rhinogobius sp. and the association with brood cannibalism by nesting males.

A laboratory experiment was conducted by varying the undersurface area of nesting substratum and the number of females in an experimental tank to elucidate the determinants of the mating pattern in the stream goby, Rhinogobius sp. cross-band type. Males with larger nests tended to attract two or more females to their nest in a tank. Moreover, males spawned simultaneously with multiple females and entire brood cannibalism by males was rarely observed under a female-biased sex ratio. When males spawned with a single female with low fecundity, however, entire brood cannibalism occurred at a high frequency, suggesting that a male guarding a nest with fewer eggs consumes the brood. Therefore, spawning behaviour of females that leads to a large egg mass would decrease the risk of entire brood cannibalism. In this species, simultaneous spawning by multiple females in a nest serves as a female counter-measure against entire brood cannibalism. These results suggest that a conflict of interest between the sexes through brood cannibalism is a major determinant of simultaneous spawning.

Interleukin-10 gene transfer to peritoneal mesothelial cells suppresses peritoneal dissemination of gastric cancer cells due to a persistently high concentration in the peritoneal cavity.

Interleukin (IL)-10 has potent biological properties including an inhibitory action on the proliferation and metastasis of various cancer cells. However, it is difficult to maintain a high concentration of this cytokine as it has a short half life. In this study, we evaluated whether peritoneal mesothelial cells (PMCs) could be suitable for maintaining a high concentration of IL-10 using adenoviral gene transfer. We also evaluated the therapeutic effects of an intraperitoneal injection with adenoviral vector containing mouse IL-10 gene (Ad-mIL-10) using a mouse peritoneal dissemination model of MKN45 gastric cancer cells. We demonstrated that in vitro transfection efficiency of a recombinant adenovirus containing the bacterial beta-galactosidase gene (Ad-LacZ) was approximately 10-fold higher for primarily isolated PMCs than MKN45. The entire peritoneum was transfected until 3 weeks after an intraperitoneal Ad-LacZ injection. Ad-mIL-10 treatment increased intraperitoneal IL-10 levels until 3 weeks after treatment, and then significantly inhibited peritoneal cancer growth by inhibiting angiogenesis. This treatment also improved cachexia and prolonged mice survival. We thus concluded that IL-10 gene transfer in PMCs could be a new strategy for the prevention of peritoneal dissemination of gastric cancer due to the resulting persistently high IL-10 concentration in the peritoneal cavity.

Microbubble destruction with ultrasound augments neovascularisation by bone marrow cell transplantation in rat hind limb ischaemia.

OBJECTIVE: To examine the effects of microbubble destruction with ultrasound (MB) combined with bone marrow derived mononuclear cell transplantation (BMT) into ischaemic tissues in rat hind limb ischaemia. METHODS AND RESULTS: Unilateral hind limb ischaemia was surgically induced in Lewis rats. At postoperative day 7, rats were randomly divided into three groups: a vehicle treated group, an ultrasound treated group, and an MB treated group. MB treatment increased vascular endothelial growth factor mRNA as assessed by real time polymerase chain reaction (3.0-fold, p < 0.05). At four weeks, the MB group had increases in laser Doppler blood flow index (LDBFI; 1.2-fold, p < 0.05), angiographically detectable collateral vessels (angiographic score: 1.4-fold, p < 0.01), and capillary to muscle fibre ratio (1.4-fold, p < 0.01) in ischaemic limbs compared with the vehicle treated group. No differences were seen between the vehicle and ultrasound treated groups. Secondly, rats were allocated to vehicle treatment, BMT (5 x 10(6) cells/rat), or a combination of MB and BMT (MB+BMT) at seven days after hind limb ischaemia. BMT treatment significantly increased LDBFI, angiographic score, and capillary to muscle fibre ratio compared with vehicle treatment. Interestingly, MB+BMT treatment produced significantly greater LDBFI (1.2-fold, p < 0.01), angiographic score (1.5-fold, p < 0.01), and capillary to muscle fibre ratio (1.5-fold, p < 0.05) than BMT treatment alone. CONCLUSIONS: MB may be a useful technique to enhance BMT induced neovascularisation.

Dominant-negative c-Jun inhibits rat cardiac hypertrophy induced by angiotensin II and hypertension.

Cardiac activator protein-1 (AP-1), composed of c-Jun, is significantly activated by hypertension or angiotensin II (AngII). This study was undertaken to elucidate whether c-Jun could be the potential target for treatment of cardiac hypertrophy. We constructed recombinant adenovirus carrying dominant-negative mutant of c-Jun (Ad.DN-c-Jun). Using catheter-based technique of adenoviral gene transfer, we achieved global myocardial transduction of DN-c-Jun in rats, to specifically inhibit cardiac AP-1. (1) AngII (200 ng/kg/min) infusion in rats caused cardiac hypertrophy, increased cardiac p70S6 kinase activity by 1.3-fold (P<0.05) and enhanced the gene expression of cardiac hypertrophic markers. Ad.DN-c-Jun, which was transferred to the heart 2 days before AngII infusion, prevented cardiac hypertrophy (P<0.01), decreased p70S6 kinase phosphorylation (P<0.05), and suppressed cardiac gene expression of brain natriuretic peptide, collagen I, III, and IV, monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) (P<0.01). (2) In genetically hypertensive rats with cardiac hypertrophy, cardiac gene transfer of Ad.DN-c-Jun, without affecting hypertension, regressed cardiac hypertrophy (P<0.05), and suppressed p70S6 kinase phosphorylation by 20% (P<0.05) and suppressed the enhanced expression of collagen I, III, and IV, MCP-1 and PAI-1. These results provided the first evidence that in vivo blockade of cardiac c-Jun inhibits pathologic cardiac hypertrophy.

Angiotensin converting enzyme inhibitor prevents left ventricular remodelling after myocardial infarction in angiotensin II type 1 receptor knockout mice.

BACKGROUND: It is well known that angiotensin converting enzyme (ACE) inhibitors and angiotensin II type 1 (AT1) receptor blockers (ARBs) prevent left ventricular (LV) remodelling after myocardial infarction (MI). However, it is still not clear whether inhibition of the AT1 receptor is enough to prevent LV remodelling after MI. OBJECTIVE: To elucidate the effects of ACE inhibitors that are not mediated by the AT1 receptor on LV remodelling, MI was experimentally induced in wild-type (WT-MI) mice and AT1 receptor knockout (KO-MI) mice. METHODS: Mice were divided into six groups: WT-control, KO-control, WT-MI, KO-MI, WT-MI treated with an ACE inhibitor, and KO-MI treated with an ACE inhibitor. Four weeks after MI, cardiac function was assessed by Doppler echocardiography and non-infarcted myocardial mRNA expression by northern blot analysis. RESULTS: Cardiac function decreased significantly in the MI groups compared with the sham operated groups. Additionally, in the MI groups end diastolic dimension, E wave velocity, the ratio of peak velocity of E wave to A wave, deceleration rate of E wave, and mRNA expression of atrial natriuretic peptide, brain natriuretic peptide, and collagens I and III increased significantly compared with the sham groups. LV remodelling after MI was prevented in KO-MI mice compared with WT-MI mice. ACE inhibitor administration significantly attenuated progressive LV remodelling in both WT and KO-MI groups. CONCLUSION: ACE inhibitors can prevent the LV remodelling process that accompanies cardiac dysfunction after MI, even in AT1 KO mice. These findings suggest that ACE inhibitors prevent LV remodelling after MI by mechanisms other than inhibition of angiotensin AT1 receptor mediated effects.

Effects of eplerenone on transcriptional factors and mRNA expression related to cardiac remodelling after myocardial infarction.

OBJECTIVE: To examine the effects of eplerenone, a selective aldosterone blocker, on cardiac function after myocardial infarction (MI) and myocardial remodelling related transcriptional factors and mRNA expression in non-infarcted myocardium. METHODS: MI was induced by ligation of the coronary artery in Wistar rats. Rats were randomly assigned to a vehicle treated group or an eplerenone treated group (100 mg/kg/day). RESULTS: At four weeks after MI, left ventricular (LV) end diastolic pressure, LV weight, and LV end diastolic dimension were increased in MI rats. Eplerenone significantly reduced the increase in LV end diastolic pressure, LV weight, and LV end diastolic dimension. In the MI rats the decreased ejection fraction indicated systolic dysfunction and the increased E wave to A wave ratio and E deceleration rate indicated diastolic dysfunction. Eplerenone significantly attenuated this systolic and diastolic dysfunction. Myocardial interstitial fibrosis, transcriptional activities of activator protein 1 and nuclear factor kappaB, and mRNA expression of monocyte chemoattractant protein 1, plasminogen activator inhibitor 1, atrial natriuretic peptide, brain natriuretic peptide, and collagen types I and III were significantly increased at four weeks after MI. Eplerenone significantly attenuated interstitial fibrosis and suppressed transcriptional activity and mRNA expression of these genes. CONCLUSIONS: When administered after MI, eplerenone prevents cardiac remodelling accompanied by systolic and diastolic dysfunction and inhibits abnormal myocardial transcriptional activities and gene expression.

Correlation of MAP kinases with COX-2 induction differs between MKN45 and HT29 cells.

BACKGROUND: Mitogen-activated protein (MAP) kinases, including extracellular signal-regulated kinases (ERK),c-Jun NH2-terminal kinases (JNK) and p38 MAP kinase (p38 MAPK) are important intermediates of the signal-transduction pathway from the cell surface to the nucleus. Expression of cyclooxygenase (COX)-2, associated with proliferation, apoptosis or both of gastrointestinal cancer cells, is mediated through MAP kinase families. However, the correlation between respective MAP kinase signals and COX-2 in the proliferation of gastric and colon cancer cells has not been well elucidated. AIM: We examined the effect of selective inhibitors of MAP kinases and COX-2 on serum-induced proliferation of gastric (MKN45) and colon (HT29) cancer cells. METHODS: After 24-h serum starvation, cancer cells were stimulated with 2% serum and COX-2 inhibitors (NS398 10 micromol/L, or etodolac 100 micromol/L) or 1 h after preincubation with inhibitors for ERK (PD98059 20 micromol/L) or p38 MAPK (SB203580 10 micromol/L). Phosphorylated MAP kinases and COX-2 protein were evaluated by Western blotting, and the proliferation of cancer cells was estimated by 3H-thymidine incorporation. Transcription factors nuclear factor-kappaB and CREB were assayed by an electorophoretic mobility shift assay. RESULTS: Serum increased the proliferation of MKN45 and HT29 cells by 280% and 200%, respectively, compared with the control levels (100%). In both cancer cells, phosphorylated MAP kinases were increased within 30 min after stimulation. PD98059 and SB203580 inhibited the serum-induced proliferation of MKN45 by 21% and 51% and of HT29 by 81% and 69%, respectively. NS398 and etodolac inhibited the proliferation of HT29 by 21% and 41%, respectively, but not that of MKN45. PD98059 and SB203580 also suppressed serum-induced expression of COX-2 protein in HT29 cells. In addition to the activation of MAP kinases and COX-2, activities of nuclear factor-kappaB and CREB were also increased during HT29 cell proliferation. CONCLUSIONS: These results suggest that the correlation of MAP kinases with COX-2 induction for cell proliferation differs between MKN45 and HT29 cells.

Dominant-negative mutant of c-Jun gene transfer: a novel therapeutic strategy for colorectal cancer.

Activator protein-1 (AP-1), a transcription factor, is activated through many oncogenic signals. However, its biological role in colorectal cancer has not been fully elucidated. To investigate the role of AP-1 in colorectal cancer, we constructed an adenovirus-expressing TAM67, a dominant-negative mutant of c-Jun lacking the transactivation domain of wild c-Jun (DN-c-Jun), to inhibit endogenous AP-1. AP-1 DNA-binding activity was increased in colon cancer cells (HT-29 cells) by serum stimulation, followed by an increase in both [(3)H]thymidine incorporation and cell number. Transfection of Ad-DN-c-Jun to HT-29 cells significantly inhibited serum-induced cell proliferation in vitro. As shown by flow cytometric analysis, DN-c-Jun significantly inhibited entrance into S phase after serum stimulation, thereby leading to G(1) arrest. In vivo transfection of Ad-DN-c-Jun into xenografted HT-29 cell tumors in nude mice significantly decreased tumor volume on day 21 after treatment. A change was associated with decrease in Ki-67 labeling index. These observations together showed that AP-1 is a critical modulator for proliferation and cell cycle of HT-29 cells. We obtained the first evidence that DN-c-Jun gene transfer exerted a significant antitumor effect on colon cancer both in vitro and in vivo. DN-c-Jun gene transfer may be a new candidate for treatment of colorectal cancer.

Activation of mitogen-activated protein kinases in the non-ischemic myocardium of an acute myocardial infarction in rats.

As one of the signal transduction pathways related to myocardial remodeling, mitogen-activated protein kinases (MAPKs) possibly play an important role in ischemic heart disease, but it is still unknown whether myocardial MAPKs are activated in the non-ischemic region of an acute myocardial infarction (AMI). Therefore, the present study investigated the myocardial activity of extracellular signal-regulated kinases (ERKs), c-Jun NH2 terminal kinases (JNKs) and p38MAPK during the acute phase of an infarction of the rat heart, and measured the geometrical ventricular changes by echocardiography. All MAPKs were significantly activated in the ischemic myocardium (IM), non-ischemic septal wall (SW), and right ventricular wall (RV). Furthermore, the activation patterns of MAPKs differed in each region. The activation of p44ERK, JNKs and p38MAPK in the IM occurred rapidly after myocardial ischemia, followed by those in the SW and RV. The activator protein-1 DNA binding activities of the IM, SW and RV increased significantly at I day after coronary ligation. Echocardiography showed increased SW motion and RV dilatation. In conclusion, this is the first in vivo evidence that myocardial MAPKs are activated in the non-ischemic region of an AMI. Echocardiographic results suggest that acceleration of workload and/or stretch may partially induce the activation of MAPKs.

Significance of chymase-dependent angiotensin II-forming pathway in the development of vascular proliferation.

BACKGROUND: Vascular tissues of humans and dogs contain chymase as an angiotensin II-forming enzyme. In this study, we investigated whether chymase-dependent angiotensin II formation plays a crucial role in the development of vascular proliferation in dog grafted veins. METHODS AND RESULTS: The right external jugular vein of dogs was grafted to the ipsilateral carotid artery. As a control group, the right external jugular veins in dogs that had not received grafts were used. In the chymase inhibitor-treated group, the vein was infiltrated with 10 micromol/L Suc-Val-Pro-Phe(P)(OPh)(2) and was grafted to the carotid artery. In the placebo-treated group, ACE activity in the grafted veins was significantly lower than that in the control veins up to 7 days after the operation, whereas chymase activity was increased significantly. After 7 days, the mRNA levels of collagen I, collagen III, and fibronectin, all of which are induced by an increase of angiotensin II action, were significantly increased in the grafted veins, and the intima-media ratio of the grafted veins was also increased. In the chymase inhibitor-treated group, the chymase activity in the grafted veins 7 days after the operation was suppressed to 12.1%. The elevated mRNA levels of fibronectin, collagen I, and collagen III in the grafted veins were significantly suppressed by treatment with the chymase inhibitor, and the intima-media ratio was also decreased significantly. CONCLUSIONS: We demonstrate for the first time that chymase-dependent angiotensin II formation plays an important role in the development of vascular proliferation in the grafted veins.

Gene transfer of dominant-negative mutants of extracellular signal-regulated kinase and c-Jun NH2-terminal kinase prevents neointimal formation in balloon-injured rat artery.

We previously reported that extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), belonging to mitogen-activated protein kinases, are rapidly activated in balloon-injured artery. Therefore, we examined the role of these kinase activations in neointimal formation by using an in vivo gene transfer technique. We made the dominant-negative mutants of ERK (DN-ERK) and JNK (DN-JNK) to specifically inhibit endogenous ERK and JNK activation, respectively. Before balloon injury, these mutants were transfected into rat carotid artery using the hemagglutinating virus of Japan liposome method. In vivo transfection of DN-ERK and DN-JNK significantly suppressed the activation of ERK and JNK, respectively, after balloon injury, confirming successful expression of the transfected genes. Neointimal formation at 14 and 28 days after injury was prevented by gene transfer of DN-ERK or DN-JNK. Furthermore, bromodeoxyuridine labeling index and total cell-counting analysis at 7 days showed that either DN-ERK or DN-JNK remarkably suppressed smooth muscle cell (SMC) proliferation in both the intima and the media after injury. Gene transfer of wild-type ERK (W-ERK) or JNK (W-JNK) significantly enhanced neointimal hyperplasia at 14 days after injury. Furthermore, DN-ERK and DN-JNK significantly suppressed serum-induced SMC proliferation in vitro. We obtained the first evidence that in vivo gene transfer of DN-ERK or DN-JNK prevented neointimal formation in balloon-injured artery by inhibiting SMC proliferation. Thus, ERK and JNK activation triggers SMC proliferation, leading to neointimal formation. These kinases may be the new therapeutic targets for prevention of vascular diseases.

Expression of gene for EIIIA- and EIIIB- fibronectin, fetal types of fibronectin, during gastric ulcer healing in rats.

Fibronectin (FN) is important for wound healing via cell proliferation, adhesion, differentiation, and matrix formation. Fetal types of FN mRNA, which include the region of EIIIA or EIIIB, are well expressed during embryonic development and wound healing. This study was done to investigate the mRNA expression of full-length FN, EIIIA- and EIIIB-FN, and the localization of FN in the gastric tissues during ulcer healing with northern blot analysis and immunohistochemical technique. Gastric ulcers in rats were produced by acetic acid. EIIIA- and EIIIB-FN mRNA were not detected in normal gastric tissues, but were expressed in the ulcerated tissues throughout the healing phase. However, on day 60 (in the scarred phase), the EIIIA- and EIIIB-FN mRNA had disappeared. The levels of full-length FN mRNA were increased from day 3 to 32 compared with the control levels, and decreased to the control levels on day 60. Full-length FN was predominantly localized at the mesenchyme around the infiltrating inflammatory cells in the granulation tissues and the basement membranes of the nonproliferating epithelial cells, which were regenerated at the ulcer margin. Thus, fetal gene transcripts of FN suggest the important role of fetal FN in gastric ulcer healing, mainly via the migration of mesenchymal and epithelial cells.

Fibrillar collagen specifically regulates human vascular smooth muscle cell genes involved in cellular responses and the pericellular matrix environment.

Proliferation and alpha(v)beta(3) integrin-dependent migration of vascular smooth muscle cells are suppressed on polymerized type I collagen. To identify genes specifically regulated in human smooth muscle cells by polymerized collagen, we used the suppressive subtraction hybridization technique. Compared with smooth muscle cells cultured on monomer collagen, polymerized collagen suppresses the following: (1) a number of other extracellular matrix proteins, including fibronectin, thrombospondin-1, tenascin-C, and cysteine-rich protein 61; (2) actin binding proteins including alpha-actinin; (3) signaling molecules; (4) protein synthesis-associated proteins; and (5) genes with unknown functions. Some of the identified genes, including cysteine-rich protein 61, show unique kinetics of mRNA regulation by monomer or polymerized collagen distinct from growth factors, suggesting extracellular matrix-specific gene modulation. Moreover, in vivo balloon catheter-mediated injury to the rat carotid artery induces many of the genes that are suppressed by polymerized collagen. Protein levels of thrombospondin-1 and fibronectin are also suppressed by polymerized collagen. Thrombospondin-1-mediated smooth muscle cell migration on vitronectin is significantly inhibited after culture on polymerized collagen for 24 hours, which is associated with decreased alpha-actinin accumulation at focal adhesions. Thus, polymerized type I collagen dynamically regulates gene expression, pericellular accumulation of extracellular matrix molecules, and the response to a given matrix molecule.

Angiotensin blockade inhibits increased JNKs, AP-1 and NF- kappa B DNA-binding activities in myocardial infarcted rats.

Inhibition of the renin-angiotensin system has been shown to prevent left ventricular remodeling after myocardial infarction. However, the effect of angiotensin on the signal transduction pathway of left ventricular remodeling after myocardial infarction is as yet unknown. The purpose of this study was to measure myocardial MAPKs and AP-1, NF- kappa B, and Sp-1 DNA-binding activities after myocardial infarction. Moreover, we evaluated the effects of angiotensin converting enzyme (ACE) inhibitor and angiotensin receptor blocker (ARB) on signal transduction pathway. Myocardial infarction was produced by ligation of the coronary artery in Wistar rats. Temocapril (ACE inhibitor) (3 and 30 mg/kg/day) and candesartan cilexitil (ARB) (1 and 10 mg/kg/day) were orally administered once a day. After ligation of the left descending coronary artery, JNKs (p46JNK and p55JNK) increased to 2.0- (P<0.01) and 2.8-fold (P<0.01) at 7 days, respectively. ERKs (p44ERK and p42ERK) and p38 activities did not increase significantly. AP-1 and NF- kappa B binding activities increased at 5 days, reached their peak 2.2- and 2.0-fold at 7 days. Sp-1 did not change. ACE inhibitor and ARB inhibited JNKs, NF- kappa B and AP-1 activities. Increased JNKs, AP-1, NF- kappa B, and Sp-1 DNA-binding activities were suppressed by both drugs in the infarcted region. Doppler-echocardiography showed that ACE inhibitor and ARB prevented the dilatation of left ventricular cavity at 14 days and improved diastolic filling pattern. JNKs, AP-1 and NF- kappa B activation in myocardial infarcted rats could be responsible for left ventricular remodeling after myocardial infarction and angiotensin may be related to the activation of these signals.

Quantitative assessment of DNA microarrays--comparison with Northern blot analyses.

DNA microarray is a powerful technology that provides the expression profile of thousands of genes. However, less attention has been paid to its quantitative aspect. In this study, we constructed a small-scale DNA microarray that contains 84 genes and characterized its quantitative aspect. Analyses with this microarray showed that 17 genes were induced, whereas 8 genes were suppressed at least twofold during the differentiation of mouse embryonic stem cells. When repeated with the same combination of fluorescent dyes for probe labeling, the microarray produced consistent data (correlation coefficient = 0.991). In contrast, data were less consistent when repeated with the reverse combination of dyes (correlation coefficient = 0.945). The effect of dye combination was particularly evident in several genes. Total RNA (15 microg) and poly(A) RNA (0.5 microg) showed comparable sensitivity and produced essentially identical data (correlation coefficient = 0.983). The sensitivity of the DNA microarrays was slightly inferior to that of Northern blot analyses. In most genes, data obtained with the two methods were consistent. However, in 4 of 46 genes compared, DNA microarrays failed to detect the expression changes that were revealed by Northern blot. These data demonstrated that DNA microarrays provide quantitative data comparable to Northern blot in general, but a few issues must be considered when analyzing data.

Effects of combination of ACE inhibitor and angiotensin receptor blocker on cardiac remodeling, cardiac function, and survival in rat heart failure.

BACKGROUND: The mechanism and treatment of diastolic heart failure are poorly understood. We compared the effects of an ACE inhibitor, an angiotensin receptor blocker (ARB), and their combination on diastolic heart failure in Dahl salt-sensitive (DS) rats. METHODS AND RESULTS: DS rats fed an 8% NaCl diet from 7 weeks of age were treated with benazepril 10 mg/kg alone, valsartan 30 mg/kg alone, or combined benazepril and valsartan at 5 and 15 mg/kg, respectively, or at 1 and 3 mg/kg, respectively. At 16 weeks of age, DS rats exhibited prominent concentric left ventricular (LV) hypertrophy and diastolic dysfunction with preserved systolic function, as estimated by echocardiography. Despite comparable hypotensive effects among all drug treatments, the combination of benazepril 5 mg/kg and valsartan 15 mg/kg improved diastolic dysfunction and survival in DS rats more effectively than ACE inhibitor or ARB alone. Furthermore, the increase in LV endothelin-1 levels and hydroxyproline contents in DS rats was significantly suppressed only by combined benazepril and valsartan, and LV atrial natriuretic peptide mRNA upregulation in DS rats was suppressed to a greater extent by the combination therapy than monotherapy. CONCLUSIONS: The combination of ACE inhibitor and ARB, independently of the hypotensive effect, improved LV phenotypic change and increased LV endothelin-1 production and collagen accumulation, diastolic dysfunction, and survival in a rat heart failure model more effectively than either agent alone, thereby providing solid experimental evidence that the combination of these 2 agents is more beneficial than monotherapy for treatment of heart failure.

Dominant negative c-jun gene transfer inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia in rats.

We previously reported that activator protein-1 (AP-1), containing c-Jun, is rapidly activated in balloon-injured artery. Therefore, we examined the role of c-Jun in vascular smooth muscle cell (SMC) proliferation, by using in vitro and in vivo gene transfer techniques. (1) Serum (2%) stimulation significantly increased AP-1 DNA binding activity in aortic SMCs, followed by the increase in both 3H-thymidine incorporation and cell number. Aortic SMCs were infected with recombinant adenovirus containing TAM67, a dominant negative c-Jun lacking transactivation domain of wild c-Jun (Ad-DN-c-Jun), to specifically inhibit AP-1. Ad-DN-c-Jun significantly inhibited serum-induced SMC proliferation, by inhibiting the entrance of SMC into S phase. (2) The effect of DN-c-Jun was examined on balloon injury-induced intimal hyperplasia in rats. Before balloon injury, DN-c-Jun was transfected into rat carotid artery using the hemagglutinating virus of Japan-liposome method. In vivo transfection of DN-c-Jun significantly inhibited vascular SMC proliferation in the intima and the media and subsequently prevented intimal thickening at 14 days after balloon injury. We obtained the first evidence that DN-c-Jun gene transfer prevented vascular SMC proliferation in vitro and in vivo, and c-Jun was involved in balloon injury-induced intimal hyperplasia. Thus, AP-1 seems to be the new therapeutic target for treatment of vascular diseases.

In vivo activation of rat aortic platelet-derived growth factor and epidermal growth factor receptors by angiotensin II and hypertension.

It is unclear whether the previous in vitro evidence of a link between angiotensin II (Ang II) and growth factor receptors can apply to the in vivo situation. In this study, we examined vascular platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptor activation in stroke-prone spontaneously hypertensive rats (SHRSP) and the role of Ang II. Tyrosyl phosphorylation of the growth factor receptors was determined by Western blot analysis coupled with immunoprecipitation. Tyrosyl phosphorylation of the aortic PDGF beta-receptor, but not the EGF receptor, was chronically increased in SHRSP with hypertension, compared with normotensive rats, being accompanied by increased extracellular signal-regulated kinase (ERK) activity. Treatment of SHRSP with ACE inhibitors (perindopril or enalapril) significantly reduced aortic PDGF beta-receptor tyrosyl phosphorylation and ERK activity, whereas treatment with hydralazine failed to reduce these activities. Therefore, these aortic changes in SHRSP were mediated by Ang II in response to vascular ACE. Ang II was infused into rats to examine the effects on aortic growth factor receptors. Chronic Ang II infusion, via the angiotensin type 1 receptor, significantly increased activation of the aortic PDGF beta-receptor but not the EGF receptor. Thus, the aortic PDGF beta-receptor, activated by ACE-mediated Ang II, seems to be responsible for vascular remodeling in hypertensive rats.

Important role of angiotensin II-mediated c-Jun NH(2)-terminal kinase activation in cardiac hypertrophy in hypertensive rats.

In vitro studies on the role of the mitogen-activated protein (MAP) kinase family (extracellular signal-regulated kinase [ERK], c-Jun NH(2)-terminal kinase [JNK], and p38) in cardiac hypertrophic response have produced confusing and contradictory results. We examined the in vivo role of the angiotensin II type 1 (AT(1)) receptor in cardiac MAP kinase activities during both the onset and development of cardiac hypertrophy in stroke-prone spontaneously hypertensive rats (SHRSP). In both the acute and chronic phases of cardiac hypertrophy in SHRSP, cardiac JNK activities were significantly increased compared with those in normotensive rats, whereas there was no prominent increase in cardiac ERK or p38 activities in SHRSP. Losartan, an AT(1) receptor antagonist, prevented the onset of cardiac hypertrophy and regressed the progression of cardiac hypertrophy in SHRSP, being accompanied by the reduction of JNK activity and activator protein-1 (AP-1) activity in SHRSP. However, in spite of the normalization of blood pressure, hydralazine did not prevent or regress cardiac hypertrophy and did not decrease JNK or AP-1 activity in SHRSP. Inversely, hydralazine significantly increased the cardiac ERK activity in SHRSP by enhancing its phosphorylation. In conclusion, we have obtained the first evidence that the AT(1) receptor is involved in the enhanced cardiac JNK activity in both the onset and development of cardiac hypertrophy of hypertensive rats. We propose that JNK is involved in AT(1) receptor-mediated cardiac hypertrophy in vivo, in part mediated by the activation of AP-1.