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

Tomita, N; Koike, H; Yamasaki, K; Kunugiza, Y; Yamamoto, K; Ogihara, T; Morishita, R Author Information

Although injury of epithelial cells has been reported to be responsible for renal disease such as acute renal failure, its molecular mechanisms are largely unknown. As hypoxia has been postulated as the initial trigger of epithelial injury, we studied the molecular mechanisms of apoptosis induced by hypoxia in human renal epithelial cells. Severe hypoxia caused epithelial cell death, accompanied by a significant increase in LDH release (p<0.01). In addition, hypoxic treatment of epithelial cells resulted in a significant increase in apoptotic cells as assessed by cell morphology (p<0.01). The apoptotic change in epithelial cells under hypoxic condition was also confirmed by a significant increase in caspase-3-like activity and release of cytochrome c (p<0.01). The decrease in epithelial cell number was completely abolished by addition of a wide-spectrum caspase inhibitor, Z-VAD, rather than Z-DEVD, a specific caspase-3 inhibitor (p<0.01). Thus, we further studied the molecular mechanisms of apoptosis induced by hypoxia. Anti-apoptotic factors, Bcl-2 and Bcl-xL, were significantly decreased in epithelial cells under a hypoxic condition as assessed by Western blotting (p<0.01). In contrast, hypoxia did not alter their location. Of particular importance, translocation of a proapoptotic factor, Bax, from the cytoplasm to the mitochondrial membrane was observed in response to hypoxia, whereas total Bax protein was not changed by hypoxia. Overall, this study demonstrated that hypoxia caused epithelial cell death induced by caspase-3-like activity-dependent apoptosis. The pro-apoptotic mechanisms of hypoxia in epithelial cells largely depend on a significant decrease in Bcl-2 and Bcl-xL. In addition, the present results demonstrate that translocation of Bax from the cytosol to the mitochondrial membrane occurred under hypoxia, thereby leading to pathological tissue destruction.

BACKGROUND:Although lipoprotein(a) (Lp[a]) is a risk factor for atherosclerosis, no study has documented the effects of Lp(a) on angiogenesis. In this study, we examined collateral formation in peripheral arterial disease (PAD) model in Lp(a) transgenic mice. In addition, we examined the feasibility of gene therapy by using an angiogenic growth factor, hepatocyte growth factor (HGF), to treat PAD in the presence of high Lp(a).METHODS AND RESULTS:In Lp(a) transgenic mice, the degree of natural recovery of blood flow after operation was significantly lower than that in nontransgenic mice. Of importance, there was a significant negative correlation between serum Lp(a) concentration and the degree of natural recovery of blood flow (P<0.05). In addition, Lp(a) significantly stimulated the growth of vascular smooth muscle, accompanied by the phosphorylation of ERK. These data demonstrated the association of impairment of collateral formation with serum Lp(a) concentration. Thus, we examined the feasibility of therapeutic angiogenesis by using HGF, with the goal of progression to human gene therapy. Intramuscular injection of HGF plasmid resulted in a significant increase in blood flow even in Lp(a) transgenic mice, accompanied by the detection of human HGF protein. A significant increase in capillary density also was detected in Lp(a) transgenic mice transfected with human HGF compared with control (P<0.01).CONCLUSIONS:Overall, a high serum Lp(a) concentration impaired collateral formation. Although the delay of angiogenesis in high serum Lp(a) might diminish angiogenesis, intramuscular injection of HGF plasmid induced therapeutic angiogenesis in the Lp(a) transgenic ischemic hindlimb mouse model as potential therapy for PAD.

BACKGROUND:Kidney targeted gene transfer has been attempted by many researchers over the last 10 years; however, unfortunately, no reliable technique for gene transfer to the kidney has been established. At experimental level several in vivo gene transfer methods have been reported. METHODS:We were the first to report successful in vivo gene transfer into the kidney using the HVJ-liposome method. Since then, this method has been modified to achieve highly efficient gene transfer. In this study, we have developed a renal glomerulus-specific gene transfer method using HVJ-liposomes with anti-Thy 1 antibody, OX-7. RESULTS:Following systemic delivery of fluoroisothiocyanate (FITC)-labeled oligodeoxynucleotides (ODN) by HVJ-liposomes coupled with OX-7, we observed fluorescence in renal glomeruli from 2 h post-administration. To examine the efficacy of this delivery system, NF-kappaB or scrambled (SD) decoy ODN was administered by HVJ-liposomes coupled with OX-7 into a crescent glomerulonephritis, anti-glomerular basement membrane (GBM) model. Animals given SD decoy ODN developed severe glomerulonephritis by day 7 with heavy albuminuria, glomerular crescent formation and up-regulated renal expression of IL-1beta and ICAM-1. In contrast, NF-kappaB decoy ODN treatment substantially inhibited the disease with a reduction in alubuminuria, histological damage and the renal expression of inflammatory cytokines. CONCLUSIONS:This study has demonstrated that systemic delivery of HVJ-liposomes coupled with OX-7 results in efficient ODN transfer in rat glomeruli. NF-kappaB, but not SD decoy ODN administered systemically via HVJ-liposomes complexed with OX-7 showed clear therapeutic potential for glomerulonephritis. This novel ODN transfer method combined with decoy strategy has the potential to lead to the establishment of a new therapeutic approach to glomerular diseases.

A missense mutation (N1411) in Presenilin-2 (PS-2) gene is associated with early-onset familial Alzheimer's disease. In this study, SK-N-SH human neuroblastoma cells were transfected with wild-type and mutant PS-2 gene to examine presenilin-2 effects on apoptosis. Serum deprivation resulted in enhanced apoptosis in mutant PS-2 comparing with wild-type PS-2. Similarly, mutant PS-2 induced lactate dehydrogenase release to greater extent than wild-type PS-2. Time course experiment demonstrated that the increase in caspase-3-like activity was more pronounced and accelerated in mutant PS-2, compared to wild-type PS-2. While a significant decrease in bcl-2, an anti-apoptotic molecule, occurred in the cells overexpressing mutant PS-2, no significant change was observed in bax, a pro-apoptotic molecule, as compared with the cells overexpressing wild-type PS-2. Our study demonstrated that mutant PS-2 induces apoptosis accompanied by increased caspase-3-like activity and decreased bcl-2 expression in neuronal cells after serum-deprivation.

Injury of endothelial cells has been postulated to be an initial trigger of the progression of atherosclerosis in patients with diabetes. Previously, we demonstrated high D-glucose induced endothelial apoptosis through the bax-caspase pathway and the potential contribution of hepatocyte growth factor (HGF) to the pathogenesis of endothelial dysfunction. In this study, we analyzed the molecular mechanisms of the protective actions of HGF against endothelial cell death under high D-glucose conditions. High concentrations of D-glucose resulted in a significant increase in apoptosis and necrosis. In contrast, HGF attenuated high D-glucose-induced apoptosis and necrosis (P < 0.01). High D-glucose significantly increased bax protein, but not bcl-2, and activated caspase 3-like and 9, whereas HGF significantly increased bcl-2 expression without affecting bax level and attenuated the increase in caspase 3 and 9 activity. Interestingly, high D-glucose resulted in translocation of bax protein from cytosol to the mitochondrial membrane, whereas HGF inhibited the bax translocation. Importantly, this bax translocation was also completely blocked by overexpressed bcl-2. These findings suggest that HGF can activate bcl-2 expression and inhibit translocation of bax protein upstream of the mitochondria, thereby leading to the inhibition of caspase 3 and 9 activation. HGF may be an important factor in the maintenance of endothelial function.

Injury of endothelial cells has been assumed to be an initial trigger of the development of atherosclerosis. In this study, we investigated the molecular mechanisms of endothelial cell death induced by hypoxia, which leads to oxidative stress. To study the relation between hypoxia-induced cell death and activation of nuclear factor-κB (NF-κB) in a hypoxic state, we evaluated the effect of 2 antioxidant drugs, probucol and pyrrolidine dithiocarbamate (PDTC), on human endothelial apoptosis. Although hypoxic treatment of human aortic endothelial cells resulted in a significant decrease in cell number and a significant increase in apoptotic cells compared with that of cells under normoxia ( P <0.01), treatment with probucol (50 μmol/L) or PDTC (100 μmol/L) significantly attenuated the decrease in cell number ( P <0.01) and was accompanied by inhibition of NF-κB activation. Furthermore, downregulation of bcl-2 caused by hypoxia was inhibited by these drugs. We further investigated the translocation of bax protein from the cytoplasm to the mitochondrial heavy fraction membrane, as translocation of bax protein is considered to be a determinant of apoptosis. Interestingly, we found that antioxidant treatment inhibited the translocation of bax protein caused by hypoxia. Moreover, upregulation of p53, a proapoptotic molecule, was observed in hypoxia, whereas treatment with probucol attenuated the expression of p53 accompanied by suppression of NF-κB activation. These data suggest functional links between p53 and endothelial apoptosis through the activation of NF-κB. Overall, the current study demonstrated that oxidative stress induced apoptosis in human aortic endothelial cells through the downregulation of bcl-2, translocation of bax, and upregulation of p53, probably through NF-κB activation. Oxidative stress may play an important role in endothelial apoptosis mediated by hypoxia, through the activation of NF-κB.

Abstract—Injury of endothelial cells has been assumed,to be an initial trigger of the development,of atherosclerosis. In this study, we investigated the molecular mechanisms of endothelial cell death induced by hypoxia, which leads to oxidative stress. To study the relation between,hypoxia-induced cell death and activation of nuclear factor- kB (NF-kB) in a hypoxic state, we evaluated the effect of 2 antioxidant drugs, probucol and pyrrolidine dithiocarbamate (PDTC), on human,endothelial apoptosis. Although hypoxic treatment of human,aortic endothelial cells resulted in a significant decrease in cell number,and a significant increase in apoptotic cells compared,with that of cells under normoxia (P,0.01), treatment with probucol (50 mmol/L) or PDTC (100 mmol/L) significantly attenuated the decrease in cell number (P,0.01) and was accompanied by inhibition of NF-kB activation. Furthermore, downregulation of bcl-2 caused by hypoxia was inhibited by these drugs. We further investigated the translocation of bax protein from the cytoplasm to the mitochondrial heavy fraction membrane, as translocation of bax protein is considered to be a determinant of apoptosis. Interestingly, we found that antioxidant treatment inhibited the translocation of bax protein caused by hypoxia. Moreover, upregulation of p53, a proapoptotic molecule, was observed in hypoxia, whereas treatment with probucol attenuated the expression of p53 accompanied,by suppression of NF-kB activation. These data suggest functional links between p53 and endothelial apoptosis through the activation of NF-kB. Overall, the current study demonstrated,that oxidative stress induced apoptosis in human,aortic endothelial cells through the downregulation of bcl-2, translocation of bax, and upregulation of p53, probably through NF-kB activation. Oxidative stress may play an important role in endothelial apoptosis mediated by hypoxia, through the activation of NF-kB. (Hypertension. 2001; 38:48-55.) Key Words: hypoxia,n apoptosis n endothelium-derived factors n bax E

P83 Background: Hepatocyte growth factor (HGF), an angiogenic growth factor, may play a pivotal role in the regulation of endothelial cells (EC), since HGF demonstrated the mitogenic and anti-apoptotic actions in endothelial cells. However, it is still unclear how HGF demonstrated those actions. Therefore, in this study, we have focused on the signal transduction systems. Methods: Human aortic EC death was induced by serum-deprived condition. Cell growth was assessed by WST assay, and thymidine incorporation and cell death was measured by LDH release and caspase 3 activity. Phosphorylation of ERK, STAT and Akt was measured by western blotting as assessed by the specific phospho-specific antibody. Results: Treatment of EC with recombinant HGF (rHGF) resulted in a significant increase in cell growth (WST: control; 0.267±0.02, rHGF 100ng/ml; 0.385±0.003, p≤0.01) and also attenuated the cell death (LDH release: control; 0.94±0.04, rHGF 100ng/ml; 0.43±0.024, p≤0.01). ERK and STAT3 (Ser727) was phosphorylated by rHGF (100 ng/ml) as assessed by the specific phospho-specific antibody, while PD98059, the inhibitor of MEK, attenuated endothelial cell growth (HGF; 0.385±0.003, HGF+PD 30uM; 0.203±0.003, p≤0.01) and the phosphorylation of STAT3 (Ser727). On the other hand, STAT3 (Tyr705), which was usually stimulated by JAK, was not phosphorylated by rHGF. In addition, Akt on the down stream of PI3 kinase was also phosphorylated by rHGF, while the specific inhibitor of PI3kinase, wortomanin and LY294002, markedly attenuated endothelial cell death assessed by LDH release and caspase3 activity. Conclusions: Overall, this study demonstrated that HGF stimulated cell growth through ERK cascade including STAT3(Ser727) activation, not JAK-STAT3(Tyr705) pathway, and inhibited endothelial cell death through PI3 kinase-Akt pathway. HGF may stimulate angiogenesis through both mitogenic and anti-cell death function in endothelial cells.

Angiogenic growth factors play important roles in angiogenic responses, such as vasculogenesis and angiogenesis in response to hypoxia. A novel angiogenic growth factor, hepatocyte growth factor (HGF), has been reported to inhibit endothelial cell death. However, its molecular mechanisms are largely unknown. Thus, we studied (1) the effects of HGF on hypoxia-induced endothelial apoptosis and (2) the molecular mechanisms of the antiapoptotic actions of HGF in endothelial cells. Severe hypoxia increased the cell death rate in human aortic endothelial cells, whereas HGF significantly attenuated cell death. In addition, hypoxic treatment resulted in a significant increase in apoptotic cells, whereas HGF could attenuate apoptosis, accompanied by attenuation of the increase in caspase-3-like activity (P<0.01). Of importance, HGF significantly increased Bcl-2, an inhibitor of apoptosis, in a dose-dependent manner under normoxic and hypoxic conditions (P<0.01), whereas hypoxic conditions resulted in a significant decrease in Bcl-2, In contrast, HGF failed to affect Bcl-xL, which is also well known as an inhibitor of apoptosis under both normoxic and hypoxic conditions, whereas Bcl-xL was significantly decreased in endothelial cells exposed to hypoxia (P<0.01). No significant change in Bax, a promoter of apoptosis, was also observed in endothelial cells under hypoxia, whereas HGF did not affect Bax. Overall, this study demonstrated that HGF prevented endothelial cell death induced by hypoxia through its antiapoptotic action. The antiapoptotic mechanisms of HGF in hypoxia-induced endothelial cell death largely depend on Bcl-2, but not Bcl-xL and Bax.

P197 Background Since the mechanisms of atherosclerosis or restenosis after angioplasty have been postulated to involve an increase in TGF-β, a selective decrease in TGF-βmay have therapeutic value. Thus, we employed the ribozyme a unique class of RNA molecules that not only store information but also process catalytic activity, to selectively inhibit TGF-βexpression. Methods & Results We constructed ribozyme oligonucleotides targeted to the sequence of TGF-βgene which shows 100 % homology among human, rat and mouse. The specificity of ribozyme against TGF-βgene was confirmed by selective inhibition of TGF-βmRNA in cultured human VSMC as well as balloon-injured blood vessels in vivo. In vitro study Transfection of TGF-βribozyme ON into human VSMC by cationic liposome significantly decreased mRNA expression and protein of TGF-βinduced by Ang II as compared to control In vivo study Transfection of FITC-labeled ribozyme ON resulted in fluorescence in the balloon-injured vessels at 1 day after transfection. The fluorescence was localized primarily in cell nuclei , and persisted for up to 2 weeks after transfection.And then,transfection of TGF-βribozyme ON into rat cartid balloon injry model significantly decreased mRNA expression of TGF-βinduced by balloon injry ,and have significant inhibitory effects on neointimal formation after vascular injury(P<0.01), whereas DNA-based control oligonucleotides and mismatched ribozyme oligonucleotides did not have any inhibitory effect on neointimal formation. Inhibition of neointimal formation was accompanied by 1) a reduction in collagen synthesis and mRNA expression of collagen I and III, and 2) a significant decrease in DNA synthesis as assessed by PCNA staining. Conclusion Overall, this study provides the first evidence that selective blockade of TGF-βby ribozyme resulted in inhibition of neointimal formation, accompanied by a reduction in collagen synthesis and DNA synthesis in a rat model. We anticipate that ribozyme ON pharmacokinetics will facilitate the potential clinical utility of the ribozyme strategy.

replication of dead myocytes by genetically modified cardiac myocytes, and

Background —Because the mechanisms of atherosclerosis or restenosis after angioplasty have been postulated to involve an increase in transforming growth factor (TGF)-β, a selective decrease in TGF-β may have therapeutic value. Thus, we used the ribozyme strategy to actively cleave the targeted gene to selectively inhibit TGF-β expression. Methods and Results —We constructed ribozyme oligonucleotides (ONs) targeted to the sequence of the TGF-β gene that shows 100% homology among the human, rat, and mouse species. The specificity of ribozyme against TGF-β gene was confirmed by selective inhibition of TGF-β mRNA in cultured vascular smooth muscle cells as well as balloon-injured blood vessels in vivo. Importantly, the marked decrease in TGF-β resulted in significant inhibition of neointimal formation after vascular injury in a rat carotid artery model ( P <0.01), whereas DNA-based control ONs and mismatched ribozyme ONs did not have any inhibitory effect on neointimal formation. Inhibition of neointimal formation was accompanied by (1) a reduction in collagen synthesis and mRNA expression of collagen I and III and (2) a significant decrease in DNA synthesis as assessed by proliferating cell nuclear antigen staining. Moreover, we modified ribozyme ONs containing phosphorothioate DNA and RNA targeted to the TGF-β gene. Of importance, modified ribozyme ONs showed a further reduction in TGF-β expression. Conclusions —Overall, this study provides the first evidence that selective blockade of TGF-β resulted in inhibition of neointimal formation, accompanied by a reduction in collagen synthesis and DNA synthesis in a rat model. We anticipate that modification of ribozyme ON pharmacokinetics will facilitate the potential clinical utility of the ribozyme strategy.

The transcription factor nuclear factor-kappa B (NF-kappa B) 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-kappa B to apoptosis, it is still controversial. Especially, the role of NF-kappa B 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-kappa B activation. Decrease in total cell number and increase in apoptotic cells induced by hypoxia were significantly attenuated by NF-kappa B decoy, but not by scrambled decoy, oligodeoxynucleotides (ODNs) (P<0.01). Increase in DNA fragmentation induced by hypoxia was also significantly inhibited by NF-kappa B decoy ODNs as compared with scrambled decoy ODNs (P<0.01). Moreover, transfection of NF-kappa B 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-kappa B 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-kappa B decoy ODNs significantly attenuated decrease in bcl-2 mRNA. These results demonstrate that activation of NF-kappa B by hypoxia induced endothelial apoptosis in a bcl-2-dependent manner. The importance of NF-kappa B in endothelial apoptosis was confirmed by the observation that pyrrolidine dithiocarbamate, a potent NF-kappa B inhibitor, prevented endothelial apoptosis, caspase 3-like activity, and bcl-2 downregulation induced by hypoxia. To test this hypothesis in vivo, we transfected NF-kappa B 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-kappa B 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-kappa B as a decoy approach, we demonstrated that activation of NF-kappa B by hypoxia caused aortic endothelial cell death and apoptosis through the suppression of bcl-2. NF-kappa B-mediated endothelial apoptosis induced by hypoxia may be involved in the pathogenesis of endothelial dysfunction observed in cardiovascular ischemic diseases.

Glomerular expression of cytokines, interleukin-1 (IL-1), and tumor necrosis factor-alpha (TNF-alpha), together with leukocytic infiltration, are prominent features in crescentic glomerulonephritis. Because these cytokines are targets for nuclear transcription factor-kappaB (NF-kappaB), the use of NF-kappaB decoy oligodeoxynucleotide (ODN) treatment was evaluated in an experimental disease model. Crescentic glomerulonephritis was induced in primed Wistar rats by injection of sheep antiglomerular basement membrane serum. Thirty minutes after injection, rats were anesthetized and the left kidney was perfused with NF-kappaB decoy ODN or scrambled ODN control mixed with a virus-liposome complex, and then killed 7 d later. Animals given the scrambled control ODN developed severe glomerulonephritis by day 7 with heavy proteinuria, glomerular crescents and interstitial lesions, marked leukocytic infiltration, and upregulated renal expression of cytokines (IL-1 and TNF-alpha) and adhesion molecules (intercellular adhesion molecule-1). In contrast, NF-kappaB decoy ODN treatment substantially inhibited the disease with a 50% reduction in proteinuria, a threefold reduction in histologic damage, a 50% reduction in leukocytic infiltration, and a 50 to 80% reduction in the renal expression of cytokines and leukocyte adhesion molecules. In conclusion, this study has demonstrated that NF-kappaB plays a key role in cytokine-mediated renal injury and that NF-kappaB decoy ODN treatment has clear therapeutic potential in rapidly progressive glomerulonephritis.