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T R Billiar

Publications and source records attributed to T R Billiar.

At least 55 records · Page 3Linked to original sources
PubMed2000

Cysteine 230 modulates tumor necrosis factor-related apoptosis-inducing ligand activity.

Biologically active tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein is known to form a homotrimer in solution. Unexpectedly, the recombinant active human TRAIL protein purified from bacteria produced two bands (a Mr 21,000 monomer derived from the disruption of the trimer in SDS gels and a Mr 42,000 dimer) on nonreducing SDS gels. The treatment of this TRAIL protein with DTT, a reducing agent, abolished formation of the Mr 42,000 band, suggesting that the Mr 42,000 band was the result of intermolecular disulfide bridge formation. Inspection of the amino acid sequence of human TRAIL protein identified a unique cysteine residue at position 230, and subsequent site-directed mutagenesis revealed that this amino acid residue is responsible for the appearance of the Mr 42,000 dimer. The binding analysis using the TRAIL protein and a TRAIL receptor (death receptor 5) revealed that both the dimer and the trimer bind to death receptor 5 with similar affinity. Interestingly, mutation of cysteine 230 to glycine completely abolished the apoptotic activity of TRAIL protein. The disruption of the dimer in the mixture of TRAIL dimer and trimer increased the apoptotic activity slightly, suggesting that the dimer has less apoptotic activity than the trimer. Therefore, our data indicate that cysteine 230 is not only required for TRAIL function but also modulates the apoptotic activity of TRAIL by forming an intermolecular disulfide bridge.

Apoptosis↗
PubMed2000

Attenuation of myocardial ischemia/reperfusion injury by superinduction of inducible nitric oxide synthase.

BACKGROUND: Nitric oxide (NO) has been implicated as a mediator in myocardial ischemia/reperfusion (I/R) injury, but its functional properties have been conflicting. We investigated whether NO has a protective role against I/R injury. METHODS AND RESULTS: Using endothelial NO synthase knockout (eNOS KO) mice, inducible NOS KO mice, the NO donor S-nitroso-N-acetylpenicillamine (SNAP), and the NOS inhibitor N-iminoethyl-L-ornithine (L-NIO), we performed studies of isolated perfused hearts subjected to 30 minutes of global ischemia followed by reperfusion. After 60 minutes of reperfusion, nitrite levels in the coronary effluent in the SNAP and eNOS KO groups were significantly elevated compared with other groups. Immunoblot and immunohistochemistry showed that iNOS was markedly induced in the eNOS KO hearts. Under spontaneous beating conditions during reperfusion, increased NO activity was correlated with a prevention of the hyperdynamic contractile response and enhanced myocardial protection, as evidenced by a reduction in myocardial injury and infarct size. During prolonged reperfusion, SNAP-treated hearts were able to preserve contractile functions for 180 minutes, whereas L-NIO-treated hearts showed a sustained deterioration in contractility. CONCLUSIONS: NO protects against I/R injury by preventing the hyperdynamic response of isolated perfused hearts during early reperfusion. In the eNOS KO hearts, a paradoxical increase in NO production was seen, accompanied by a superinduction of iNOS, possibly due to an adaptive mechanism.

Animals↗
PubMed2000

Characterization of rat CD14 promoter and its regulation by transcription factors AP1 and Sp family proteins in hepatocytes.

CD14, a 55kDa glycoprotein, serves as a lipopolysaccharide (LPS) recognition molecule. CD14 is a monocyte differentiation antigen expressed by myeloid-derived cells, or other cells such as hepatocytes, as either a membrane-bound protein or a soluble serum protein. Increasing evidence indicates that soluble CD14 in plasma is an acute-phase protein derived, among other sources, from liver cells. Although information is available on the cellular expression of CD14, little is known about the cis- and trans-acting factors that regulate basal CD14 transcription in liver cells. We show here that liver cells have a relatively high basal CD14 transcription rate as determined by nuclear run-on assay. We cloned and sequenced an 883bp 5'-flanking region of the rat CD14 gene and demonstrated functional promoter activity in liver cells. Sequence analysis revealed that, like in the human and mouse CD14 genes, multiple Sp1 and AP1 binding elements exist in rat CD14. Site-directed mutagenesis and transient transfection assays demonstrated that an Sp1 element located at -836 and an AP1 element located at -270 are required for basal promoter activity in liver cells. Electrophoretic mobility shift assays indicate that both Sp1 and Sp3 nuclear factors interact with the -836 Sp1 element, while the AP1-related proteins Fra-2 and JunD bind to the AP1 motif. These data provide novel insights into the regulation of basal CD14 expression in liver cells.

3T3 Cells↗
PubMed2000

Cyclic nucleotides suppress tumor necrosis factor alpha-mediated apoptosis by inhibiting caspase activation and cytochrome c release in primary hepatocytes via a mechanism independent of Akt activation.

Cyclic nucleotides have been previously shown to modulate cell death processes in many cell types; however, the mechanisms by which cyclic nucleotides regulate apoptosis are unclear. In this study, we demonstrated that cAMP as well as cGMP analogs suppressed tumor necrosis factor alpha (TNFalpha) plus actinomycin D (ActD)-induced apoptosis in a dose-dependent manner in cultured primary hepatocytes. Furthermore, forskolin, which increases intracellular cAMP levels, also effectively suppressed TNFalpha+ActD-induced apoptosis. Activation of multiple caspases was suppressed in cells exposed to TNFalpha+ActD in the presence of cAMP or cGMP analogs. TNFalpha+ActD-induced cytochrome c release from mitochondria was also inhibited by cAMP or cGMP, reinforcing our conclusion that cyclic nucleotides interfere with the early signaling events of TNFalpha-mediated apoptosis. We evaluated the possibility that cAMP and cGMP inhibit apoptosis by activating the serine/threonine kinase Akt, which is known to promote cell survival. Both cAMP- and cGMP-elevating agents led to marked increases in Akt activation that was inhibited by the phosphatidylinositol 3'-kinase inhibitors, LY294002 and wortmannin. However, complete inhibition of cyclic nucleotide-induced Akt activation had little effect on cyclic nucleotide-mediated cell survival, indicating the existence of other survival pathways. Interestingly, the specific inhibitor of protein kinase A (PKA), KT5720, blocked cGMP-mediated protection but only partially prevented the anti-apoptotic effect of cAMP, indicating that both PKA-dependent and -independent mechanisms are involved in cAMP-mediated suppression of apoptosis signaling. Our data suggest that multiple survival signaling pathways coexist in cells and that cyclic nucleotides delay apoptosis by interfering with apoptosis signaling by both PKA-dependent and -independent mechanisms.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Cellular non-heme iron content is a determinant of nitric oxide-mediated apoptosis, necrosis, and caspase inhibition.

In this report, we tested the hypothesis that cellular content of non-heme iron determined whether cytotoxic levels of nitric oxide (NO) resulted in apoptosis versus necrosis. The consequences of NO exposure on cell viability were tested in RAW264.7 cells (a cell type with low non-heme iron levels) and hepatocytes (cells with high non-heme iron content). Whereas micromolar concentrations of the NO donor S-nitroso-N-acetyl-DL-penicillamine induced apoptosis in RAW264.7 cells, millimolar concentrations were required to induce necrosis in hepatocytes. Caspase-3 activation and cytochrome c release were evident in RAW264.7 cells, but only cytochrome c release was detectable in hepatocytes following high dose S-nitroso-N-acetyl-DL-penicillamine exposure. Pretreating RAW264.7 cells with FeSO(4) increased intracellular non-heme iron to levels similar to those measured in hepatocytes and delayed NO-induced cell death, which then occurred in the absence of caspase-3 activation. Iron loading was also associated with the formation of intracellular dinitrosyl-iron complexes (DNIC) upon NO exposure. Cytosolic preparations containing DNIC as well as pure preparations of DNIC suppressed caspase activity. These data suggest that non-heme iron content is a key factor in determining the consequence of NO on cell viability by regulating the chemical fate of NO.

Animals↗
PubMed2000

Endothelial nitric oxide synthase protects aortic allografts from the development of transplant arteriosclerosis.

BACKGROUND: Inducible nitric oxide synthase (iNOS) is up-regulated in rejecting allografts and is protective against allograft arteriosclerosis; it suppresses neointimal smooth muscle cell accumulation and inhibits adhesion of platelets and leukocytes to the endothelium. However, the functional importance of endothelial NOS (eNOS) in the rejecting allografts remains unclear. METHODS: We examined the effects of selective eNOS deficiency in aortic allografts in a murine chronic rejection model using grafts from eNOS knockout (KO) mice (C57BL/6 background; H2b) and normal C3H (H2K) as recipients. Grafts from wild-type C57BL/6 mice served as controls. Grafts from iNOS KO mice served as a second group of controls where the contribution from iNOS was eliminated but eNOS was preserved. Aortic grafts were harvested and analyzed at days 10-14, 18-22, and 26-30 after transplantation. RESULTS: Endothelial NOS-deficient grafts showed significantly increased intima/media ratios at days 26-30 compared to controls. Immunostaining demonstrated that in eNOS KO grafts, eNOS was not detectable whereas iNOS was expressed prominently in infiltrating recipient mononuclear cells. In control grafts, eNOS expression was preserved in the endothelium even by day 30, and associated with a decrease in intimal thickening. We further demonstrated that early overexpression of iNOS by ex vivo gene transfer completely prevented the development of arteriosclerosis associated with eNOS deficiency. CONCLUSIONS: We found that eNOS plays a protective role in allografts, and that in eNOS-deficient allografts, early overexpression of iNOS is capable of preventing the development of allograft arteriosclerosis. In allografts with dysfunctional vascular endothelium and impaired eNOS activity as a result of ischemia or native arteriosclerotic disease, iNOS gene therapy may serve to improve their long-term survival and function.

Animals↗
PubMed2000

Optimizing cardiovascular gene therapy: increased vascular gene transfer with modified adenoviral vectors.

BACKGROUND: Adenovirus is widely used as a vector for gene transfer to the vasculature. However, the efficiency of these vectors can be limited by ineffective viral-target cell interactions. Viral attachment, which largely determines adenoviral tropism, is mediated through binding of the adenoviral fiber coat protein to the Coxsackievirus and adenovirus receptor, while internalization follows binding of the adenoviral RGD motif to alpha(v)-integrin receptors. Modifications of the fiber coat protein sequence have been successful for targeting the adenovirus to more prevalent receptors in the vasculature, including heparan sulfate-containing receptors and alpha(v)-integrin receptors. HYPOTHESIS: Modified adenoviral vectors targeted to receptors more prevalent in the vasculature result in an increased transfer efficiency of the virus in vitro and in vivo even in the presence of clinically relevant doses of heparin. DESIGN: We tested 2 modified E1- and E3-deleted Ad5 type adenoviral vectors containing the beta-galactosidase gene. AdZ.F(pK7) contains multiple positively charged lysines in the fiber coat protein that target the adenovirus to heparan sulfate receptors, while AdZ.F(RGD) contains an RGD integrin-binding sequence in the fiber coat protein that allows binding to alpha(v)-integrin receptors. The gene transfer efficiency of these modified viruses was compared in rat aortic smooth muscle cells in vitro and in an in vivo porcine model of balloon-induced arterial injury. Because of the use of heparin during most vascular surgical procedures and the concern that heparin might interfere with the binding of AdZ.F(pK7) to heparan sulfate receptors, the effect of heparin on the in vitro and in vivo transfer efficiency of these 2 modified adenoviruses was evaluated. RESULTS: In vitro infection of rat aortic smooth muscle cells with AdZ.F(pK7) and AdZ.F(RGD) resulted in significantly higher levels of beta-galactosidase expression compared with the unmodified adenovirus (mean +/- SEM, 1766.3 +/- 89.1 and 44.8 +/- 3.4 vs 10.1 +/- 0.7 mU per milligram of protein; P<.001). Following heparin administration, the gene transfer efficiency achieved with AdZ.F(pK7) diminished slightly in a concentration-dependent manner. However, the transfer efficiency was still greater than with the unmodified virus (mean +/- SEM, 1342.3 +/- 101.8 vs 4.8 +/- 0.4 mU per milligram of protein; P<.001). In vivo, following injury to the pig iliac artery with a 4F Fogarty balloon catheter, we found that AdZ.F(pK7) transduced the artery approximately 35-fold more efficiently than AdZ.F and 3-fold more efficiently than AdZ.F(RGD) following the administration of intravenous heparin, 100 U/kg body weight, and heparinized saline irrigation. CONCLUSIONS: Modifications of the adenovirus that lead to receptor targeting resulted in significantly improved gene transfer efficiencies. These improvements in transfer efficiencies observed with the modified vectors decreased slightly in the presence of heparin. However, AdZ.F(pK7) was still superior to AdZ.F(RGD) and AdZ.F despite heparin administration. These data demonstrate that modifications of adenoviral vectors that enhance binding to heparan sulfate receptors significantly improve gene transfer efficiency even in the presence of heparin and suggest an approach to optimize gene transfer into blood vessels.

Adenoviridae↗
PubMed2000

cAMP induces CD14 expression in murine macrophages via increased transcription.

CD14, a glycoprotein that binds bacterial lipopolysaccharide, plays a critical role in the inflammatory response to infection by gram-negative bacteria. Studies were undertaken to determine whether cyclic adenosine monophosphate (cAMP) regulates CD14 expression in macrophages. Incubation of RAW 264.7 cells with 8-Br-cAMP resulted in a significant increase in steady-state CD14 mRNA levels. The increase in mRNA levels was also associated with both cell-associated and soluble CD14 protein. H89 completely blocked the 8-Br-cAMP-induced CD14 mRNA up-regulation. There was no change in CD 14 mRNA half-life in the presence of 8-Br-cAMP. The CD14 gene transcription rate was increased about twofold after exposure to 8-Br-cAMP. cAMP-dependent increases in CD14 mRNA were also observed in rat peritoneal macrophages, demonstrating that this is an authentic response of mature macrophages. This study provides evidence that cAMP and protein kinase A are important regulators of CD14 expression in macrophages.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Nitric oxide induces angiogenesis and upregulates alpha(v)beta(3) integrin expression on endothelial cells.

Nitric oxide (NO) has been implicated as a mediator of angiogenesis. However, its precise role in angiogenesis and its mechanism of action have not been established. We performed in vivo and in vitro angiogenesis assays using NO donor S-nitroso-N-acetylpenicillamine (SNAP) and NO synthase inhibitor N-iminoethyl-l-ornithine (L-NIO). SNAP significantly increased and L-NIO significantly suppressed capillary ingrowth into subcutaneously implanted Matrigel plugs in mice. For the in vitro angiogenesis assay, human umbilical vein endothelial cells (HUVECs) (4 x 10(4) cells/well) were treated with placebo, SNAP (100 microM), or L-NIO (100 microM) and cultured on Matrigel for 18 h. The typical capillary networks formed on Matrigel by HUVECs as a result of cell migration and differentiation were quantified by computer-assisted image analysis as a measure of angiogenesis. Treatment of HUVECs with SNAP significantly increased the capillary network area compared with control, 8701 +/- 693 vs 6258 +/- 622 area units (P < 0.05), whereas L-NIO significantly decreased the capillary area (4540 +/- 342, P < 0.05). Furthermore, we have shown with a blocking monoclonal antibody that formation of capillary networks on Matrigel is mediated by the functional expression of the alpha(v)beta(3) integrin, which plays a role in facilitating endothelial cell adhesion to basement membrane matrix and endothelial cell migration. After an 18-h culture, flow cytometry revealed that SNAP significantly upregulated and L-NIO significantly downregulated in a concentration-dependent manner alpha(v)beta(3) integrin expression on endothelial cells. In conclusion, NO induces angiogenesis in vivo and in vitro by promoting endothelial cell migration and differentiation into capillaries. One possible mechanism might involve the upregulation of alpha(v)beta(3) integrin on endothelial cells, a critical mediator of cell-matrix adhesion and migration.

Animals↗
PubMed2000

Role of inducible nitric oxide synthase in postoperative intestinal smooth muscle dysfunction in rodents.

BACKGROUND & AIMS: We have shown that intestinal manipulation leads to a significant inhibition of circular muscle contraction. We hypothesized that the inflammatory mediator inducible nitric oxide (NO) plays a role in surgically induced ileus. METHODS: Rats and inducible NO synthase (iNOS) knockout and wild-type mice underwent a simple intestinal manipulation. Reverse-transcription polymerase chain reaction and immunohistochemistry were used to detect and localize iNOS expression. Nitrite and NO production were measured in muscularis cultures. Spontaneous and bethanechol-stimulated jejunal circular muscle contractions were measured in an organ bath. RESULTS: Intestinal manipulation resulted in significant iNOS messenger RNA induction in mucosa and muscularis. Immunohistochemistry localized iNOS in phagocytes within the muscularis. Nitrite and NO production increased 59.8-fold 24 hours after manipulation. L-n(6)-(1-iminoethyl) lysine (L-NIL) inhibited this response. In control rats, selective iNOS inhibition did not increase spontaneous muscle activity, but after manipulation L-NIL significantly improved spontaneous activity. iNOS knockout mice showed a significant 81% decrease in neutrophil infiltration into the muscularis after intestinal manipulation compared with wild-types. Contractile activity was normal in knockout mice after intestinal manipulation. CONCLUSIONS: These results show that leukocyte-derived inducible NO inhibits gastrointestinal motility after manipulation and plays an essential role in the initiation of intestinal inflammation.

Animals↗
PubMed2000

The role of nitric oxide in apoptosis.

Nitric oxide (NO) has been shown to play important roles in modulating cell viability. Both proapoptotic and antiapoptotic functions of NO have been reported. This article discusses our current understanding on the mechanisms responsible for the dual actions of NO in regulating apoptosis and propose that one common mechanism for NO-mediated inhibition of apoptosis is the inhibition of caspases via nitrosylation.

Animals↗
PubMed2000

Nitric oxide prevents p21 degradation with the ubiquitin-proteasome pathway in vascular smooth muscle cells.

PURPOSE: We have shown that gene transfer of the inducible nitric oxide synthase (iNOS) gene to injured arteries inhibits the development of intimal hyperplasia. One mechanism by which nitric oxide (NO) may inhibit this process is through the upregulation of the cyclin-dependent kinase inhibitor p21, which induces a G0/G1 cell cycle arrest, leading to an inhibition of vascular smooth muscle cell (VSMC) proliferation. Because NO induced such a dramatic upregulation of p21 and because p21 is a universal inhibitor of the cell cycle, this study aimed to determine how NO upregulates p21 protein expression in VSMCs. METHODS: p21 messenger RNA (mRNA) levels in rat aortic smooth muscle cells (RASMCs) were determined by Northern blot analysis after treatment with S-nitroso-N-acetylpenicillamine (SNAP) or after adenoviral iNOS gene transfer. p21 protein levels in RASMCs in similar conditions were determined by Western blot analysis. Levels of ubiquinated p21 in these same treatment groups were assessed by immunoprecipitation of p21 from RASMCs, followed by western blot analysis for ubiquitin. Protein tyrosine and protein serine/threonine phosphatase activity after treatment with SNAP, plus or minus the phosphatase inhibitors calyculin A or cantharidin, were measured with (32)P-labeled myelin basic protein as a substrate. RESULTS: NO exposure by the NO-donor SNAP or iNOS gene transfer induced a dose- and time-dependent increase in p21 protein expression in RASMCs. p21 mRNA levels were significantly increased after SNAP treatment only at the 6-hour point, but were not increased at 24 hours. In contrast, protein levels were increased from 6 to 24 hours, and transcriptional inhibitors did not inhibit this increase in protein synthesis. The increase in p21 protein expression induced by NO was associated with less of the ubiquinated form of p21 at both early and late points. Furthermore, NO induced an increase in both protein tyrosine and protein serine/threonine phosphatase activity. Inhibition of these phosphatases with calyculin A or cantharidin prevented the upregulation of p21 protein expression by NO. CONCLUSION: These data indicate that one mechanism by which NO upregulates p21 protein expression is through the prevention of p21 protein degradation by the ubiquitin-proteasome pathway in association with increased protein tyrosine and serine/threonine phosphatase activity.

Adenosine Triphosphatases↗
PubMed2000

Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to induce apoptosis in various tumor cells but not in nontransformed, normal cells. Preclinical studies in mice and nonhuman primates have shown that administration of TRAIL can induce apoptosis in human tumors, but that no cytotoxicity to normal organs or tissues is found. The susceptibility of tumor cells to TRAIL and an apparent lack of activity in normal cells has lead to a proposal to use TRAIL in cancer therapy. Here, we assessed the sensitivity of hepatocytes from rat, mouse, rhesus monkey and human livers to TRAIL-induced apoptosis. TRAIL induced apoptosis in normal human hepatocytes in culture but not in hepatocytes isolated from the other species. Human hepatocytes showed characteristic features of apoptosis, including cytoplasmic shrinkage, the activation of caspases and DNA fragmentation. Apoptosis and cell death in human hepatocytes was massive and rapid, occurring in more than 60% of the cells exposed to TRAIL within 10 hours. These results indicate that there are species differences in sensitivity to TRAIL, and that substantial liver toxicity might result if TRAIL were used in human cancer therapy.

Animals↗
PubMed2000

Nitric oxide prevents tumor necrosis factor alpha-induced rat hepatocyte apoptosis by the interruption of mitochondrial apoptotic signaling through S-nitrosylation of caspase-8.

Mitochondrial cytochrome c release plays a critical role in apoptotic signal cascade after the activation of cell surface death receptors. We investigated the role played by nitric oxide (NO) in mitochondrial apoptotic signaling in tumor necrosis factor alpha (TNF-alpha) plus actinomycin D (TNF-alpha/ActD)-induced apoptosis. NO produced either by S-nitroso-N-acetyl-DL-penicillamine (SNAP) or inducible NO synthase (iNOS) prevented TNF-alpha/ActD-induced apoptosis in hepatocytes and also inhibited both caspase-8-like (IETDase) and caspase-3-like protease (DEVDase) activity as well as mitochondrial cytochrome c release. Recombinant human (rh) caspase-8 induced the cleavage of the cytochrome c-effluxing factor Bid and cytochrome c release from purified mitochondria in the reconstitution system with Bid(+/+) cytosol, but not with Bid(-/-) cytosol. The addition of SNAP and the caspase-8 inhibitor Ac-IETD-fmk inhibited caspase-8-dependent Bid cleavage and cytochrome c release. The inhibitory effect of NO on caspase-8 was reversed by dithiothreitol (DTT). Furthermore, rh-caspase-8 was found to be modified by S-nitrosylation with 1.7 moles of NO bound per mole of enzyme. Treatment of hepatocytes with interleukin 1beta (IL-1beta) plus interferon gamma (IFN-gamma), which induced iNOS expression and NO production, suppressed TNF-alpha/ActD-induced Bid cleavage and mitochondrial cytochrome c release. The NOS inhibitor N(G)-monomethyl-L-arginine (NMA) inhibited the protective effects of IL-1beta and IFN-gamma. The liver-specific NO donor V-PYRRO/NO also inhibited in vivo elevation of IETDase activity, Bid cleavage, and mitochondrial cytochrome c release in the livers of rats injected with TNF-alpha plus D-galactosamine. Our results indicate that one mechanism by which NO protects hepatocytes from TNF-alpha/ActD-induced apoptosis is via the interruption of mitochondrial apoptotic signaling through S-nitrosylation of caspase-8.

Animals↗
PubMed2000

Inducible nitric oxide synthase (iNOS) expression upregulates p21 and inhibits vascular smooth muscle cell proliferation through p42/44 mitogen-activated protein kinase activation and independent of p53 and cyclic guanosine monophosphate.

OBJECTIVE: Overexpression of the inducible nitric oxide synthase (iNOS) gene inhibits neointimal hyperplasia after arterial injury. The purpose of this study was to examine the mechanism by which nitric oxide (NO) inhibits vascular smooth muscle cell (VSMC) proliferation, specifically focusing on signaling pathways known to be activated by NO, including cyclic guanosine monophosphate (cGMP), p53, and p42/44 mitogen-activated protein kinase (MAPK). METHODS AND RESULTS: VSMCs that were subjected to iNOS gene transfer demonstrated a reduction in proliferation (80%) that was associated with a marked increase in p21 expression. The antiproliferative and p21 stimulatory effects of NO were not suppressed by the soluble guanylate cyclase inhibitor ODQ, implicating cGMP-independent signaling. The role of p53 in NO-mediated upregulation of p21 and inhibition of proliferation was evaluated using p53 -/- VSMCs. A similar reduction in cellular proliferation and upregulation of p21 expression were achieved with iNOS gene transfer as well as treatment with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP), demonstrating the p53-independent nature of these NO-mediated pathways. The transfer of the iNOS gene activated the p42/44 MAPK, and inhibition of this MAPK pathway with PD98059 partially blocked the antiproliferative effects of NO and completely inhibited the p21 stimulatory effects of NO. For confirmation that iNOS overexpression upregulated p21 in vivo, injured rat carotid arteries were infected with an adenoviral vector carrying the iNOS gene and demonstrated a marked upregulation of p21 expression at three days. However, the ability of NO to inhibit VSMC proliferation does not solely depend on p21 upregulation since the NO-donor SNAP-inhibited VSMC proliferation in p21 -/- VSMCs. CONCLUSION: Nitric oxide inhibits VSMC proliferation in association with the upregulation of p21; both occur independent of p53 and cGMP while being partially mediated through the p42/44 MAPK signaling cascade. This represents one potential mechanism by which NO inhibits VSMC proliferation.

Animals↗
PubMed2000

Apoptosis in sepsis.

Sepsis demonstrates a marked dysregulation of the immune system in its ability to fight infection. Previous models have focused on the mechanisms which upregulate and sustain the heightened immune response without addressing the role of down-regulation effectors. Attention has been drawn to these down-regulating mechanisms and their precise role in the pathophysiology of sepsis. Apoptosis is an evolutionarily conserved, energy-dependent mode of cell death requiring the initiation and regulation of complex genetic programs. It is the body's main method of getting rid of cells which are in excess, damaged, or no longer needed in a controlled manner. The role of this cellular phenomenon in physiology and pathophysiology has been the subject of intense scrutiny over the last decade. Much work has demonstrated that dysregulation of apoptosis does occur in immune and nonimmune cells in in vitro and in vivo models of sepsis. The difficulty has been in tying the phenomenology of apoptosis into the pathophysiology of sepsis. Further work is needed to make these connections to elucidate rational approaches for clinical applications of immunomodulation in sepsis.

Animals↗
PubMed2000

Induced nitric oxide inhibits IL-6-induced stat3 activation and type II acute phase mRNA expression.

Inducible nitric oxide synthase (iNOS) can be coexpressed with acute phase reactants in hepatocytes; however, it is unknown if NO can regulate the acute phase response. We tested the hypothesis that iNOS-derived nitric oxide (NO) attenuates the acute phase response by inhibiting IL-6-enhanced Stat3 DNA-binding activity and type II acute phase mRNA expression. iNOS was overexpressed in cultured rat hepatocytes via transduction with a replication defective adenovirus containing cDNA for human iNOS (AdiNOS), and Stat3 DNA-binding activity was determined by electrophoretic mobility shift assay (EMSA). EMSAs demonstrated that AdiNOS inhibits IL-6-induced Stat3 activation and that this inhibition is reversible in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMA). The induction of beta-fibrinogen mRNA by IL-6, a Stat3 dependent process, is attenuated in AdiNOS-transduced cells and partially reversed by L-NMA. Thus, iNOS overexpression suppresses IL-6-induced Stat3 activation and type II acute phase mRNA expression in cultured hepatocytes. This suppression may represent a mechanism by which NO down-regulates the acute phase response.

Acute-Phase Reaction↗