Biomedical subjects

A Moriguchi

Publications and source records attributed to A Moriguchi.

At least 37 records · Page 2Linked to original sources

PubMed1999

Pancreatic Schwannoma: report of a case.

We present herein a case of solitary schwannoma of the pancreas and also review 26 previously reported cases from the English and Japanese literature. Primary schwannoma of the pancreas is a rare tumor. A 50-year-old female was discovered to have a large mass in the upper abdomen on ultrasonography. An examination by computed tomography (CT) scan, magnetic resonance imaging (MRI), and ultrasonography revealed a solid and cystic tumor in the left upper quadrant of the abdomen. A distal pancreatectomy with a splenectomy was performed to remove this tumor. A microscopic examination identified the tumor to be situated in the pancreas while it was composed of cells that originated from Schwann cells. Only 26 cases of pancreatic schwannoma have previously been reported in the English and Japanese literature. We describe in detail the characteristic findings based on image analyses, including CT scan, MRI, ultrasonography, and angiography, in these 26 cases.

Diagnostic Imaging↗

PubMed1999

Effect of propranolol on central neurotransmitter release in Wistar rats analysed by brain microdialysis.

1. The effect of propranolol on amino acid neurotransmitter release in the rostral ventrolateral medulla (RVLM) was examined in urethane-anaesthetized male Wistar rats. 2. Amino acids released in the RVLM in response to intravenous administration of propranolol (0.4 mg/kg per min; n = 6) or nitroglycerin (0.02 mg/kg per min; n = 5) were determined by the brain microdialysis method. 3. Amino acids in dialysates were analysed by high-performance liquid chromatography and were quantified by ultraviolet absorbance. 4. Administration of both intravenous propranolol and nitroglycerin significantly decreased arterial blood pressure. Heart rate was decreased only by propranolol. 5. The reduction in arterial blood pressure produced by intravenous propranolol was accompanied by a decrease in the release of the excitatory amino acid glutamate in the RVLM. 6. The reduction in arterial blood pressure following intravenous nitroglycerin was not accompanied by a release of glutamate. 7. There were no significant changes in the levels of other amino acids (glycine, taurine, GABA) following either propranolol or nitroglycerin. 8. The decrease in glutamate release in the RVLM may account, in part, for the central depressor mechanism of propranolol.

Adrenergic beta-Antagonists↗

PubMed1999

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.

Aged↗

PubMed1999

Inhibition of the p53 tumor suppressor gene results in growth of human aortic vascular smooth muscle cells. Potential role of p53 in regulation of vascular smooth muscle cell growth.

Loss of activity of the p53 tumor suppressor gene product has been postulated in the pathogenesis of human restenosis. Although the antioncogenes p53 and retinoblastoma (Rb) susceptibility gene have been reported to play a pivotal role in cell cycle progression in various cells, the role of p53 and Rb in the growth of human vascular smooth muscle cells (VSMC) has not yet been clarified. We used antisense strategy against p53 and Rb genes by the viral envelope-liposomal method. Transfection of antisense p53 oligodeoxynucleotides (ODN) alone resulted in an increase in DNA synthesis compared with control (P<0.01). Similarly, transfection of antisense Rb ODN alone resulted in a higher DNA synthesis rate than control (P<0.01). Moreover, increase in VSMC number was only induced by transfection of antisense p53 ODN alone or cotransfection of p53/Rb ODN (P<0.01), whereas a single transfection of antisense Rb ODN had little effect on cell number. Therefore, we hypothesized that this discrepancy is due to the induction of apoptosis mediated by p53. Interestingly, apoptotic cells were markedly increased in VSMC transfected with antisense Rb ODN alone, accompanied by the induction of p53 protein. The number of apoptotic cells was attenuated by cotransfection of antisense p53 ODN (P<0.01). We finally examined the molecular mechanisms of apoptosis induced by the absence of Rb. In VSMC transfected with antisense Rb ODN, bax, a promoter of apoptosis, was significantly increased in VSMC transfected with antisense Rb ODN (P<0.01), whereas bcl-2 and Fas did not play a pivotal role in the induction of apoptosis. Overall, these data first demonstrated that the antioncogenes p53 and Rb negatively regulated the cell cycle in VSMC, suggesting that the modulation of their activity may mediate VSMC growth such as that in restenosis and atherosclerosis. The presence of p53 plays a pivotal role in the regulation of apoptosis in human VSMC growth, probably through the bax pathway. These results provide evidence that p53 is a functional link between cell growth and apoptosis in VSMC.

Analysis of Variance↗

PubMed1999

Prevention of renal damage by angiotensin II blockade, accompanied by increased renal hepatocyte growth factor in experimental hypertensive rats.

Hepatocyte growth factor (HGF) is a unique growth factor that has many protective functions against renal damage. Our previous study demonstrated that HGF stimulated the growth of endothelial and epithelial cells without the replication of mesangial cells. Moreover, angiotensin (Ang) II significantly decreased local HGF production in mesangial cells. Therefore, we examined the effects of Ang II blockade on renal HGF expression and renal damage in experimental hypertensive rats. An angiotensin-converting enzyme inhibitor (cilazapril; 10 mg. kg(-1). d(-1)), an Ang II type 1 receptor antagonist (E-4177; 30 mg. kg(-1). d(-1)), hydralazine (8 mg. kg(-1). d(-1)), and vehicle were administered to 16-week-old stroke-prone spontaneously hypertensive rats (SHR-SP) for 3 weeks. Renal damage was evaluated with a computer analysis system, and renal HGF mRNA was measured by Northern blot analysis. Blood pressure of SHR-SP was significantly decreased by all drug treatments compared with vehicle. Moreover, cilazapril, E-4177, and hydralazine significantly decreased the thickening and necrosis of blood vessels compared with vehicle. Similarly, degeneration and necrosis of glomeruli were also markedly improved by cilazapril and E-4177 (P<0.01). We next examined the effects of Ang II blockade on renal HGF expression in SHR-SP. Renal HGF mRNA was markedly decreased in SHR-SP compared with Wistar-Kyoto rats, although Ang II blockade by cilazapril and E-4177 but not hydralazine significantly increased renal HGF mRNA in SHR-SP. Ang II blockade significantly increased renal HGF (a protective growth factor for tubular epithelial cells); thus, we examined tubular histological appearance. Degeneration and necrosis of tubules were significantly improved by cilazapril and E-4177 treatment (P<0.01). In addition, cell infiltration into the glomeruli and hemorrhage were also significantly reduced in SHR-SP treated with cilazapril or E-4177. The present data demonstrated the prevention of renal damage by Ang II blockade in SHR-SP, which was accompanied by a significant increase in renal HGF mRNA. Given the strong mitogenic activity and antiapoptotic actions of HGF on endothelial and epithelial cells, we believe that increased local HGF production by the blockade of Ang II may improve renal function in hypertension.

Angiotensin II↗

PubMed1999

Activation of the brain angiotensin system by in vivo human angiotensin-converting enzyme gene transfer in rats.

The possibility of the brain-specific expression of a component of the renin-angiotensin system was evaluated in the present study. We used the hemagglutinating virus of Japan-liposome complex to transfect human angiotensin-converting enzyme (ACE) cDNA, driven by the cytomegalovirus enhancer and beta-actin promoter, into the lateral cerebroventricle of male Sprague-Dawley rats. We evaluated the time course of hemodynamics, the tissue levels of angiotensin (Ang) II and vasopressin, and ACE activity. Intracerebroventricular transfection of the human ACE gene increased both blood pressure and heart rate. Transfected rats exhibited higher concentrations of brain Ang II and increased brain ACE activity. This activation of the brain angiotensin system was accompanied by increased vasopressin production. The increases in blood pressure and heart rate were abolished by intracerebroventricular administration of an ACE inhibitor or Ang II type 1 receptor antagonist. The expression of the transgene was widely distributed in the periventricular cell layer, the cortex, the hypothalamic nuclei, and the brain stem. Expression in the neuronal cells persisted for up to 14 days. Thus, this hemagglutinating virus of Japan-liposome method is a highly efficient system for gene delivery and is extremely useful for functional gene transfection. This novel hypertensive model may enable characterization of the functions of the renin-angiotensin system in the brain and determination of its role in the pathogenesis of hypertension.

Angiotensin II↗

PubMed1999

Hepatocyte growth factor (HGF) as a potential index of severity of hypertension.

Hepatocyte Growth Factor (HGF) is a mesenchyme-derived pleiotropic factor that regulates cell growth, cell motility, and morphogenesis of various cells, and is thus considered a humoral mediator of epithelial-mesenchymal interactions. We previously identified HGF as a novel member of the family of endothelium-specific growth factors. Moreover, the presence of a local HGF system (HGF and its specific receptor, c-met) has been demonstrated in vascular cells both in vitro and in vivo. HGF might contribute to the protection and/or repair of vascular endothelial cells injured by high blood pressure. If so, serum HGF level might be elevated in response to endothelial cell damage. To test this hypothesis, we measured serum levels of HGF in hypertensive and normotensive patients. Serum HGF concentration in hypertensive patients without any complications was significantly higher than that in normal subjects. Interestingly, serum HGF concentration in hypertensive patients with complications was significantly higher than that in either hypertensive patients without complications or normotensive subjects. Of importance, hypertensive patients treated with antihypertensive drugs showed the same level of serum HGF concentration as normotensive subjects. In contrast, serum HGF concentration in diabetic patients without hypertension was significantly lower than that in normal subjects, whereas serum HGF concentration in diabetic patients with hypertension was significantly higher than that in normal subjects. Moreover, serum HGF concentration in diabetic patients with hypertensive complications was even higher than that in diabetics without complications. This review discusses the possibility that HGF may be considered as a new index of the severity of hypertension.

Hepatocyte Growth Factor↗

PubMed1998

Stimulatory effect of lipoprotein (a) on proliferation of human mesangial cells: role of lipoprotein (a) in renal disease.

As lipoprotein (a) [Lp(a)] abnormalities would accelerate glomerular injury, we studied the effect of Lp (a) on proliferation of cultured human mesangial cells (MC). Transfection of human apo (a) gene into human hepatoma cells, HepG2 cells, producing human apo B, resulted in the formation of Lp (a), while no Lp (a) was detected in control cells. In contrast, free apo (a) was detected in the medium of apo (a)-transfected MC. Incubation of cultured medium of HepG2 cells transfected with apo (a) gene with MC resulted in a significant increase in cell number compared to control (P<0.01). In contrast, little effect of transfection of apo (a) gene directly into MC on growth of MC was observed. Of importance, addition of LDL into the medium of MC transfected with apo (a) vector resulted in a significant increase in number of MC compared to control, whereas LDL did not show any effects on MC growth. As active TGF-beta was not detected in the medium of MC, and addition of neutralizing anti-TGF-beta antibody did not alter growth of MC, Lp (a) stimulated growth of MC via the independent mechanisms from the inhibition of TGF-beta activation.

Apolipoproteins A↗

PubMed1998

Synthesis and structure-activity relationships of 7-(2-aminoalkyl)morpholinoquinolones as anti-Helicobacter pylori agents.

A series of the titled compounds was synthesized and tested for anti-Helicobacter pylori activities. We discovered Y-34867 having the most potent activity against Helicobacter pylori among the quinolones tested along with high photostability. Furthermore, Y-34867 showed an excellent therapeutic effect in the experimental Helicobacter pylori infected Mongolian gerbil model.

4-Quinolones↗

PubMed1998

A novel vascular modulator, hepatocyte growth factor (HGF), as a potential index of the severity of hypertension.

HGF (hepatocyte growth factor), a member of endothelium-specific growth factors, might contribute to protection and/or repair of vascular endothelial cells injured by high blood pressure (BP). If so, serum HGF level might be elevated in response to endothelial cell damage. To test this hypothesis, we measured serum levels of HGF in hypertensive and normotensive patients. Serum HGF concentration in hypertensive patients without any complication was significantly higher than normal subjects (p < 0.001). Serum HGF concentration showed a significant positive correlation with BP (p < 0.01). Interestingly, serum HGF concentration in hypertensive patients with complications was significantly higher than that in hypertensive patients without complication and normotensive subjects (p < 0.01). Of importance, hypertensive patients treated with antihypertensive drugs showed the same level of serum HGF concentration as normotensive subjects (p < 0.001). The present study demonstrated that serum concentration of HGF is significantly elevated dependent on the severity of hypertension, suggesting that HGF may be a new index of the severity of hypertension.

Antihypertensive Agents↗

PubMed1998

Adrenal, kidney, and heart angiotensins in female murine Ren-2 transfected hypertensive rats.

We analyzed by high-performance liquid chromatography and radioimmunoassay angiotensin I (Ang I), Ang II, Ang-(1-7), and metabolites in the adrenal, kidney and heart of normotensive female Sprague-Dawley (SD) and transgenic hypertensive [TGR(mRen-2)27] rats carrying the murine Ren-2d renin gene. The monogenetic model of hypertensive rats had significant increases in adrenal Ang II; whereas in the kidney Ang II was unchanged, but Ang I and Ang-(1-7) were significantly lower. Cardiac Ang I, Ang II, and Ang-(2-10) were significantly reduced in transgenic rats, while Ang-(2-7) was increased. In SD and transgenic rats kidney and adrenal angiotensins increased primarily during estrus or proestrus. In female transgenic rats the increased adrenal Ang II and the sustained renal Ang II may contribute to the established phase of hypertension.

Adrenal Glands↗

PubMed1998

Central amino acids mediate cardiovascular response to angiotensin II in the rat.

To elucidate the role of the rostral ventrolateral medulla (RVLM) in cardiovascular control through the release of central amino acid neurotransmitters, experiments were performed in Sprague-Dawley (normotensive) rats and spontaneously hypertensive rats (SHR) anesthetized with urethane by using microdialysis sampling from the RVLM for determination of amino acid neurotransmitters. The baseline release of the excitatory amino acid neurotransmitter, glutamate (GLU) from the RVLM in SHR was higher and those of the inhibitory amino acid neurotransmitters, glycine (GLY), taurine (TAU), and gamma-aminobutyric acid (GABA), were lower than in normotensive rats. Microinjection of angiotensin II (ANG II) into the RVLM caused a dose-dependent increase in mean arterial pressure (MAP) and heart rate (HR), accompanied by increased release of GLU in the RVLM. In contrast, microinjection of the ANG II type 1 receptor (AT1) antagonist CV 11974 into the RVLM reduced MAP and HR, accompanied by increased release of GLY, TAU and GABA. These changes in MAP and HR after administration of ANG II or AT1 antagonist were partially blocked by the use of the corresponding antagonist of each amino acid neurotransmitter. Furthermore, these effects were more prominently seen in SHR than in normotensive rats. These results suggest that the release of amino acid neurotransmitters mediate the cardiovascular effects of the angiotensin system in the RVLM, which may be involved in the generation of hypertension in SHR.

Adrenergic alpha-Agonists↗

PubMed1998

Potential role of hepatocyte growth factor in the maintenance of renal structure: anti-apoptotic action of HGF on epithelial cells.

BACKGROUND: Mesangial cells (MC) are known to secrete various vasoactive substances that may control endothelial and epithelial cell growth. Therefore, the cell-cell interactions among these cells may be important in the control of renal function. However, the exact mechanisms of maintaining the cell-cell interactions are not yet understood. We have focused on the role of hepatocyte growth factor (HGF) in the regulation of cell-cell interactions, since HGF has many protective functions in the kidney. To investigate the role of HGF in renal injury, we examined (1) the effects of HGF on epithelial injury induced by serum deprivation, and (2) the role of local HGF production in the maintenance of renal structure. METHODS: Apoptotic changes in epithelial cells were assessed by nuclear morphology and DNA fragmentation assay. Transfection of human HGF vector into epithelial cells was performed by a highly efficient viral-liposome method. The effects of secreted HGF on the growth of renal cells were examined using a co-culture system. RESULTS: The addition of recombinant HGF (rHGF) stimulated the growth of rat and porcine epithelial cells. Moreover, the decrease in number of epithelial cells by serum deprivation was significantly attenuated by rHGF. Interestingly, apoptotic changes in epithelial cells induced by serum deprivation were also significantly attenuated by rHGF (P < 0.01). As a model of gene therapy, the effects of overexpression of human HGF gene in epithelial cells on apoptosis induced by serum deprivation were examined. Transfection of human HGF vector into epithelial cells also attenuated epithelial cell death induced by serum deprivation through the inhibition of apoptosis, accompanied by increased HGF production (P < 0.01). In addition, HGF also prevented endothelial injury induced by tumor necrosis factor-alpha and dexamethasone. Given the presence of a local HGF system, we measured local HGF secreted from renal cells. Immunoreactive HGF was observed in the conditioned medium of MC, but not epithelial cells, while the specific receptor of HGF, c-met, was expressed in epithelial cells. Of importance, co-culture of MC with epithelial cells resulted in a significant increase in number of epithelial cells, which was significantly abolished by neutralizing anti-HGF antibody. CONCLUSIONS: Overall, these results demonstrate that local production of HGF in MC may maintain the growth of epithelial and endothelial cells through its anti-apoptotic action.

Animals↗

PubMed1998

Actions of hepatocyte growth factor as a local modulator in the kidney: potential role in pathogenesis of renal disease.

Endothelial cells are known to secrete various vasoactive substances that may control mesangial cell growth, whereas mesangial cells also secrete various growth factors and cytokines that may regulate endothelial cells. Therefore, it is apparent that cell-cell interactions among renal cells are important in the control of renal function. Indeed, co-culture of endothelial cells with mesangial cells resulted in a significant decrease in mesangial cell growth. However, the exact mechanisms of maintaining the cell-cell interactions are not yet understood. We have focused on the role of hepatocyte growth factor (HGF) in the regulation of cell-cell interactions, since HGF has been reported to have many organ protective functions in the kidney. Our present data demonstrated that addition of recombinant HGF (rHGF) stimulated DNA synthesis and growth of endothelial cells in a dose-dependent manner. However, there was no stimulatory effect of rHGF on the growth of mesangial cells in the time and dose-dependent manner. Therefore, we examined the presence of the local renal HGF system and its potential role in renal disease. The presence of secreted local HGF was observed in the conditioned medium from endothelial and mesangial cells. The presence of HGF mRNA was also detected in endothelial and mesangial cells. Interestingly, the specific receptor of HGF, c-met, was also expressed in both cells. Next, regulation of local HGF secretion was studied under stimulation with rHGF, angiotensin (Ang) II, and transforming growth factor (TGF)-beta. Of importance, rHGF significantly stimulated local HGF secretion from mesangial cells by positive feedback. In contrast, TGF-beta significantly decreased HGF secretion in mesangial cells and endothelial cells. Angiotensin II also significantly decreased local HGF production in mesangial cells in a dose-dependent manner. Finally, the effect of local HGF production from mesangial cells was studied using a co-culture system. Co-culture of mesangial cells with endothelial cells resulted in a significant increase in number of endothelial cells, which was abolished by co-incubation with neutralizing anti-HGF antibody. Overall, these results demonstrated the local production of HGF, which has stimulatory effects on growth of endothelial cells, but not mesangial cells. Local HGF secretion from mesangial cells may maintain the growth of endothelial cells. Negative regulation of local HGF production by Ang II and TGF-beta may play an important role in the pathogenesis of renal disease. Taken together, dysfunction of cell-cell regulation in the kidney due to decreased local HGF production may be an initial trigger for the development of renal disease such as glomerulonephritis.

Angiotensin II↗

PubMed1998

Hepatocyte growth factor as a potential index of complication in diabetes mellitus.

OBJECTIVE: Diabetes mellitus (DM), characterized by the premature development of microvascular and macrovascular disease, shows a loss of vasodilatory properties of resistance vessels. However, the mechanisms of endothelial dysfunction in diabetes have not yet been clarified. Hepatocyte growth factor (HGF) in vascular cells was down-regulated by high glucose levels, potentially accelerating the endothelial dysfunction in DM. In this study, the serum HGF level was measured to investigate further the role of HGF in DM. METHODS: Tissue and circulating HGF levels were measured in the KKAy mouse, a rodent model of non-insulin-dependent diabetes mellitus (NIDDM), and lean C57 BL control mice. Then, serum HGF concentrations were measured in NIDDM patients without liver, kidney or lung damage. For the study of serum HGF concentration, 30 normotensive and age-matched 58 DM patients were studied. The 58 DM patients were divided into 26 patients without hypertension and 32 patients with hypertension [22 patients without hypertensive complications (WHO I) and 10 patients with hypertensive complications (WHO II + III)]. RESULTS: The serum HGF concentration in KKAy mice was significantly lower than that in control mice (P < 0.01), at 14 weeks of age when they exhibit features of diabetes. Similarly, tissue HGF concentrations in the heart and kidney were decreased significantly in KKAy mice compared with control C57 BL mice (P< 0.05). The serum HGF concentration showed a significant negative correlation with hemoglobin (Hb) A(Ic) concentration (P< 0.01, r= -0.41). Since the serum HGF concentration is a potential index of the severity of hypertension, the serum levels of HGF in DM patients without and with hypertension were examined. The serum HGF concentration in DM patients without hypertension was significantly lower than that in normal subjects (P< 0.05), whereas that in DM patients with hypertension was significantly higher than that in normal subjects (P < 0.01). Moreover, the serum HGF concentration in DM patients with hypertensive complications was further higher than that in others (P < 0.01). CONCLUSION: The present data showed that serum, cardiac and renal HGF concentrations in KKAy mice were significantly decreased compared with control mice. Therefore, a decrease in local HGF may be a trigger of endothelial dysfunction in DM. Clinical data also demonstrated a significant negative correlation between serum HGF and HbA(Ic) concentrations in diabetic patients without complications. In contrast, the serum concentration of HGF was significantly elevated depending on the severity of hypertension. These results suggest that HGF may be a new index of complications such as hypertension in DM.

Animals↗

PubMed1998

Inhibition of growth of human vascular smooth muscle cells by overexpression of p21 gene through induction of apoptosis.

The senescent cell-derived inhibitor (sdi)-1 protein (p21 product) has been identified as a downstream mediator of the tumor suppressor p53 in the regulation of cell cycle progression through a G1 phase checkpoint. Given the importance of cell cycle inhibition for the treatment of restenosis, in this study we focused on the function of p21 gene in inhibiting proliferation of vascular smooth muscle cells (VSMC). To test the hypothesis, we transfected human p21 gene into human aortic VSMC using hemagglutinating virus of Japan-liposome-mediated transfer. Initially, we examined the successful transfection of human p21 gene into VSMC. p21 protein was increased in VSMC transfected with p21 vector as compared with control vector. Accompanied by increased p21 protein, transfection of p21 vector resulted in a significant decrease in number of VSMC induced by 2% serum (P<.01). Although p21 has been reported to play an important role in the regulation of apoptosis in some cells, apoptosis mediated by p21 is still controversial. Therefore, we hypothesized that overexpression of p21 mediates apoptosis in human VSMC, in addition to the blockade of cell cycle progression. First, we assessed the concordance between morphologic analysis and apoptosis as determined by nuclear staining with Hoechst 33342. Cells transfected with p21 gene exhibited the characteristic features of cell shrinkage, membrane blebbing, and rounding that are typical of apoptotic death. Of greater interest, a significant increase in apoptotic cells was observed in VSMC transfected with p21 vector as compared with control vector (P<.01). These results were confirmed by the measurement of DNA fragmentation. Consistent with nuclear staining, DNA fragmentation in VSMC transfected with human p21 gene was significantly increased as compared with that in VSMC transfected with control vector (P<.05). To study the molecular mechanisms of apoptosis mediated by overexpression of p21 gene, the protein levels of bax, a promoter of apoptosis, and bcl-2, an inhibitor of apoptosis, were also measured by Western blotting. Overexpression of p21 gene significantly increased protein of bax (P<.05), whereas transfection of p21 gene did not alter bcl-2 protein. Importantly, the ratio of bax to bcl-2 was significantly increased in VSMC transfected with human p21 vector as compared with control vector (P<.05). Overall, these results demonstrated that inhibition of VSMC growth by overexpression of human p21 gene was accompanied by induction of apoptosis through an inappropriate increase in bax protein. These results suggest that regulation of cell cycle by p21 may be closely linked to programmed cell death/apoptosis in human VSMC.

Aorta↗

PubMed1998

Conditioned medium from HepG2 cells transfected with human apolipoprotein(a) gene stimulates growth of human vascular smooth muscle cells: effects of overexpression of human apolipoprotein(a) gene.

Lipoprotein(a) [Lp(a)] is well known to stimulate growth of vascular smooth muscle cells (VSMCs), resulting in atherosclerosis. Its mechanism is postulated to be decreased in active transforming growth factor (TGF)-beta. However, the exact mechanisms and cellular processing from apolipoprotein(a) [apo(a)] to Lp(a) have not yet been clarified because no cultured cells producing apo(a) are available. Therefore, it is necessary to establish apo(a)-producing cells to study the role of apo(a). We evaluated the effects of overexpression of human apo(a) gene on human aortic VSMC growth. First, we tested whether transfection of apo(a) gene into human hepatoma cells, HepG2 cells, producing human apoB resulted in the formation of Lp(a). Transfection of apo(a) gene into HepG2 cells resulted in detectable levels of Lp(a) in the medium, as assessed by ELISA and Western blot, whereas no Lp(a) was detected in the medium of HepG2 cells transfected with control vector and untransfected HepG2 cells. Expression of apo(a) mRNA was also confirmed by reverse transcription-polymerase chain reaction. In contrast, Western blotting showed a single band detected by specific anti-apo(a) antibody, but not anti-apoB antibody, in the medium of apo(a)-transfected VSMCs. These results demonstrate that Lp(a) can be formed from apo(a) on HepG2 cells, whereas transfection of apo(a) gene into VSMCs resulted in the production of apo(a) alone but not Lp(a). Next, we examined the biological effects of overexpression of apo(a) gene on growth of VSMCs and endothelial cells. Incubation of cultured medium of HepG2 cells transfected with apo(a) gene with human VSMCs or endothelial cells resulted in a significant increase in cell number compared with the conditioned medium of HepG2 cells transfected with control vector. In contrast, transfection of apo(a) gene directly into VSMCs caused no significant effect on VSMC growth. Therefore, we measured TGF-beta concentration in the conditioned medium of VSMCs. However, using ELISA, only latent but not active TGF-beta was detected in the medium of VSMCs. Moreover, addition of neutralizing anti-TGF-beta antibody did not alter VSMC growth. These results suggest that Lp(a) could stimulate growth of VSMCs via the independent mechanisms from the inhibition of TGF-beta activation. Overall, these data demonstrate that overexpression of apo(a) gene in cells producing apoB results in formation of Lp(a), resulting in a mitogenic action on human endothelial cells and VSMCs. These results provide new information to understand the mechanisms of the mitogenic action of Lp(a) and suggest the role of Lp(a) in the pathogenesis of atherosclerosis.

Apolipoproteins↗

PubMed1998

Negative regulation of local hepatocyte growth factor expression by angiotensin II and transforming growth factor-beta in blood vessels: potential role of HGF in cardiovascular disease.

Because hepatocyte growth factor (HGF) is a member of the endothelium-specific growth factors, we hypothesized that HGF may play a role in cardiovascular disease. Therefore we first examined the role of local HGF production in endothelial cell (EC) growth. Addition of anti-HGF antibody to EC resulted in a significant decrease in EC number. Moreover, coculture of vascular smooth muscle cells (VSMC) with EC resulted in an increase in EC number that was completely inhibited by anti-HGF antibody, suggesting that HGF secreted from EC and VSMC regulates EC growth in an autocrine-paracrine manner. Interestingly, transforming growth factor (TGF)-ss significantly decreased HGF secretion from EC, whereas interleukin 6 stimulated immunoreactive HGF secretion. In human VSMC, TGF-ss and angiotensin II suppressed local HGF production in a dose-dependent manner. Interestingly, anti-TGF-beta antibody resulted in significant but not complete inhibition of the decrease in local HGF production. To further study the regulation of local HGF production, we used a coculture system. Coculture of VSMC with EC resulted in a significant decrease in local HGF secretion. The decrease in local HGF production by coculture was significantly attenuated by anti-TGF-beta antibody, suggesting that inhibition of local HGF production in the coculture system was due to TGF-beta activation. Moreover, a further decrease in local HGF production in the coculture system by angiotensin II was also observed. Finally, we studied the role of angiotensin II in the regulation of the local HGF system in vivo by using a balloon injury rat model. Of importance, local HGF production was significantly decreased in balloon-injured arteries compared with intact vessels, accompanied by a reduction of HGF mRNA. An angiotensin-converting enzyme inhibitor (cilazapril) or an angiotensin II type 1 receptor antagonist (E-4177) significantly stimulated local vascular HGF production associated with the inhibition of neointimal formation after balloon injury compared with vehicle. In contrast, hydralazine did not alter local HGF production or neointimal formation despite decreasing blood pressure to a similar level as that in rats treated with cilazapril or E-4177. Overall, local HGF secretion from vascular cells was negatively regulated by TGF-beta and angiotensin II. The present study also demonstrated that blockade of angiotensin II significantly inhibited neointimal formation, accompanied by a significant increase in local vascular HGF production in vivo in the balloon injury model. Given the strong mitogenic activity of HGF on endothelial cells, increased local HGF production by blockade of angiotensin II may enhance reendothelialization after balloon injury. Downregulation of the local vascular HGF system by TGF-beta and vascular angiotensin may play an important role in the pathogenesis of cardiovascular diseases.

Angiotensin II↗