Biomedical subjects

Mark T Quinn

Publications and source records attributed to Mark T Quinn.

At least 37 records · Page 2Linked to original sources

PubMed2005

Effects of NADPH oxidase inhibitor in diabetic nephropathy.

BACKGROUND: We used apocynin to test the hypothesis that superoxide anion (O(-) (2)) from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase underlies the development of diabetic nephropathy in the rat. METHODS: Rats received apocynin (16 mg/kg/day) from 2 to 8 weeks after inducing diabetes mellitus (DM) with streptozotocin. RESULTS: DM increased excretion of hydrogen peroxide (H(2)O(2)), lipid peroxidation products (LPO), nitric oxide products (NOx), and protein. The kidneys of rats with DM had increased expression of p47phox and gp91phox and endothelial nitric oxide synthase (eNOS), and increased mesangial matrix with expression of fibronectin and collagen I. Apocynin prevented the increase in excretion of H(2)O(2), LPO, and protein in diabetic rats, increased renal NOx generation, and prevented the increased renal expression of gp91phox and the membrane fraction of p47phox, and reverted the mesangial matrix expansion. CONCLUSION: Activation of NADPH oxidase with translocation of p47phox to the membrane underlies the oxidative stress and limited NO generation, despite enhanced eNOS expression in a model of diabetic nephropathy. Apocynin prevents these changes and the associated proteinuria.

Acetophenones↗

PubMed2004

Oxidative stress in the infarcted heart: role of de novo angiotensin II production.

This study aimed to elucidate whether angiotensin (Ang) II generated de novo at the infarct site regulates the redox state of inflammatory cells participating in cardiac repair. On days 3-28 following ligation of the rat left coronary artery, we addressed at the infarct site: (a) the appearance and cellular origin of oxidative stress by monitoring the expression (mRNA and protein) of gp91phox (a subunit of superoxide producing NADPH oxidase) and the antioxidant superoxide dismutase (SOD), together with the presence of 3-nitrotyrosine (a marker of oxidative stress); (b) the presence of components requisite to Ang peptide generation; and (c) response to treatment with losartan (Los, 10mg/kg/day). We found at the infarct site, macrophage-derived oxidative stress appears during week 1 coincident with the appearance of components requisite to AngII generation and activity in these cells. Based on observed response to AT1 receptor antagonism with Los, we would suggest de novo AngII, in an autocrine manner, participates in the induction of oxidative stress while suppressing the expression of antioxidant defenses.

Angiotensin II↗

PubMed2004

Functional association of nox1 with p22phox in vascular smooth muscle cells.

The vascular NAD(P)H oxidases constitute important sources of ROS in the vessel wall and have been implicated in vascular disease. Vascular smooth muscle cells (VSMCs) from conduit arteries express two gp91phox homologs, Nox1 and Nox4, of which Nox1 is agonist-sensitive. Because p22phox has been shown to be functionally important in vascular cells stimulated with vasoactive hormones, the relationship of Nox1 and p22phox was investigated in VSMCs from rat and human aortas. Coimmunoprecipitation studies demonstrated that p22phox and hemagglutinin-tagged Nox1 associate in unstimulated VSMCs. These findings were confirmed by confocal microscopy, showing colocalization of the two proteins in their native states in the plasma membrane and submembrane areas of the cell. NADPH-driven superoxide production, as measured by electron spin resonance using 1-hydroxy-3-carboxypyrrolidine as a spin probe, is dependent on the coexpression of both subunits, suggesting the importance of the association for the functional integrity of the enzyme. These results indicate that in contrast to the neutrophil enzyme, VSMCs can use Nox1 rather than gp91phox as a catalytic center in the p22phox-based oxidase and that these two proteins are preassembled at or near the plasma membrane and submembrane vesicular structures in unstimulated cells.

Animals↗

PubMed2004

Identification of a novel tumor necrosis factor alpha-responsive region in the NCF2 promoter.

The phagocyte reduced nicotinamide adenine dinucleotide phosphate oxidase is a multiprotein enzyme that catalyzes the production of microbicidal oxidants. Although oxidase assembly involves association of several membrane and cytosolic oxidase proteins, one of the cytosolic cofactors, p67phox, appears to play a more prominent role in final activation of the enzyme complex. Based on the importance of p67phox, we investigated transcriptional regulation of the p67phox gene [neutrophil cytosolic factor 2 (NCF2)] and demonstrated previously that activator protein-1 (AP-1) was essential for basal transcriptional activity. As p67phox can be up-regulated by tumor necrosis factor alpha (TNF-alpha), which activates AP-1, we hypothesized that TNF-alpha might regulate NCF2transcription via AP-1. In support of this hypothesis, we show here that NCF2 promoter-reporter constructs are up-regulated by TNF-alpha but only when AP-1 factors were coexpressed. Consistent with this observation, we also demonstrate that NCF2 mRNA and p67phox protein are up-regulated by TNF-alpha in various myeloid cell lines as well as in human monocytes. It was surprising that mutagenesis of the AP-1 site in NCF2 promoter constructs did not eliminate TNF-alpha induction, suggesting additional elements were involved in this response and that AP-1 might play a more indirect role. Indeed, we used NCF2 promoter-deletion constructs to map a novel TNF-alpha-responsive region (TRR) located between -56 and -16 bp upstream of the translational start site and demonstrated its importance in vivo using transcription factor decoy analysis. Furthermore, DNase footprinting verified specific binding of factor(s) to the TRR with AP-1 binding indirectly to this region. Thus, we have identified a novel NCF2 promoter/enhancer domain, which is essential for TNF-alpha-induced up-regulation of p67phox.

Evaluation Studies as Topic↗

PubMed2004

Structure and regulation of the neutrophil respiratory burst oxidase: comparison with nonphagocyte oxidases.

Neutrophils play an essential role in the body's innate defense against pathogens and are one of the primary mediators of the inflammatory response. To defend the host, neutrophils use a wide range of microbicidal products, such as oxidants, microbicidal peptides, and lytic enzymes. The generation of microbicidal oxidants by neutrophils results from the activation of a multiprotein enzyme complex known as the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is responsible for transferring electrons from NADPH to O2, resulting in the formation of superoxide anion. During oxidase activation, cytosolic oxidase proteins translocate to the phagosome or plasma membrane, where they assemble around a central membrane-bound component known as flavocytochrome b. This process is highly regulated, involving phosphorylation, translocation, and multiple conformational changes. Originally, it was thought that the NADPH oxidase was restricted to phagocytes and used solely in host defense. However, recent studies indicate that similar NADPH oxidase systems are present in a wide variety of nonphagocytic cells. Although the nature of these nonphagocyte NADPH oxidases is still being defined, it is clear that they are functionally distinct from the phagocyte oxidases. It should be noted, however, that structural features of many nonphagocyte oxidase proteins do seem to be similar to those of their phagocyte counterparts. In this review, key structural and functional features of the neutrophil NADPH oxidase and its protein components are described, including a consideration of transcriptional and post-translational regulatory features. Furthermore, relevant details about structural and functional features of various nonphagocyte oxidase proteins will be included for comparison.

Animals↗

PubMed2003

Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells.

OBJECTIVE: Reactive oxygen species (ROS) that act as signaling molecules in vascular smooth muscle cells (VSMC) and contribute to growth, hypertrophy, and migration in atherogenesis are produced by multi-subunit NAD(P)H oxidases. Nox1 and Nox4, two homologues to the phagocytic NAD(P)H subunit gp91phox, both generate ROS in VSMC but differ in their response to growth factors. We hypothesize that the opposing functions of Nox1 and Nox4 are reflected in their differential subcellular locations. METHODS AND RESULTS: We used immunofluorescence to visualize the NAD(P)H subunits Nox1, Nox4, and p22phox in cultured rat and human VSMC. Optical sectioning using confocal microscopy showed that Nox1 is co-localized with caveolin in punctate patches on the surface and along the cellular margins, whereas Nox4 is co-localized with vinculin in focal adhesions. These immunocytochemical distributions are supported by membrane fractionation experiments. Interestingly, p22phox, a membrane subunit that interacts with the Nox proteins, is found in surface labeling and in focal adhesions in patterns similar to Nox1 and Nox4, respectively. CONCLUSIONS: The differential roles of Nox1 and Nox4 in VSMC may be correlated with their differential compartmentalization in specific signaling domains in the membrane and focal adhesions.

Animals↗

PubMed2003

AT1 blockade prevents glucose-induced cardiac dysfunction in ventricular myocytes: role of the AT1 receptor and NADPH oxidase.

Enhanced tissue angiotensin (Ang) II levels have been reported in diabetes and might lead to cardiac dysfunction through oxidative stress. This study examined the effect of blocking the Ang II type 1 (AT1) receptor on high glucose-induced cardiac contractile dysfunction. Rat ventricular myocytes were maintained in normal- (NG, 5.5 mmol/L) or high- (HG, 25.5 mmol/L) glucose medium for 24 hours. Mechanical and intracellular Ca2+ properties were assessed as peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening (+/-dL/dt), and intracellular Ca2+ decay (tau). HG myocytes exhibited normal PS; decreased +/-dL/dt; and prolonged TPS, TR90, and tau. Interestingly, the HG-induced abnormalities were prevented with the AT1 blocker L-158,809 (10 to 1000 nmol/L) but not the Janus kinase-2 (JAK2) inhibitor AG-490 (10 to 100 micromol/L). The only effect of AT1 blockade on NG myocytes was enhanced PS at 1000 nmol/L. AT1 antagonist-elicited cardiac protection against HG was nullified by the NADPH oxidase activator sodium dodecyl sulfate (80 micromol/L) and mimicked by the NADPH oxidase inhibitors diphenyleneiodonium (10 micromol/L) or apocynin (100 micromol/L). Western blot analysis confirmed that the protein abundance of NADPH oxidase subunit p47phox and the AT1 but not the AT2 receptor was enhanced in HG myocytes. In addition, the HG-induced increase of p47phox was prevented by L-158,809. Enhanced reactive oxygen species production observed in HG myocytes was prevented by AT1 blockade or NADPH oxidase inhibition. Collectively, our data suggest that local Ang II, acting via AT1 receptor-mediated NADPH oxidase activation, is involved in hyperglycemia-induced cardiomyocyte dysfunction, which might play a role in diabetic cardiomyopathy.

Acetophenones↗

PubMed2003

NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells.

Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, p22phox and gp91phox, as well as the Ca2+ concentration-responsive gp91phox homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for p22phox was not detectable, both gp91phox and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to p22phox or gp91phox did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death.

Antioxidants↗

PubMed2003

A dual role of the GTPase Rac in cardiac differentiation of stem cells.

The function of the GTPase Rac1, a molecular switch transducing intracellular signals from growth factors, in differentiation of a specific cell type during early embryogenesis has not been investigated. To address the question, we used embryonic stem (ES) cells differentiated into cardiomyocytes, a model that faithfully recapitulates early stages of cardiogenesis. Overexpression in ES cells of a constitutively active Rac (RacV12) but not of an active mutant (RacL61D38), which does not activate the NADPH oxydase generating ROS, prevented MEF2C expression and severely compromised cardiac cell differentiation. This resulted in poor expression of ventricular myosin light chain 2 (MLC2v) and its lack of insertion into sarcomeres. Thus ES-derived cardiomyocytes featured impaired myofibrillogenesis and contractility. Overexpression of MEF2C or addition of catalase in the culture medium rescued the phenotype of racV12 cells. In contrast, RacV12 specifically expressed in ES-derived ventricular cells improved the propensity of cardioblasts to differentiate into beating cardiomyocytes. This was attributed to both a facilitation of myofibrillogenesis and a prolongation in their proliferation. The dominant negative mutant RacN17 early or lately expressed in ES-derived cells prevented myofibrillogenesis and in turn beating of cardiomyocytes. We thus suggest a stage-dependent function of the GTPase during early embryogenesis.

Animals↗

PubMed2003

PB1 domain-mediated heterodimerization in NADPH oxidase and signaling complexes of atypical protein kinase C with Par6 and p62.

Maximal activation of NADPH oxidase requires formation of a complex between the p40(phox) and p67(phox) subunits via association of their PB1 domains. We have determined the crystal structure of the p40(phox)/p67(phox) PB1 heterodimer, which reveals that both domains have a beta grasp topology and that they bind in a front-to-back arrangement through conserved electrostatic interactions between an acidic OPCA motif on p40(phox) and basic residues in p67(phox). The structure enabled us to identify residues critical for heterodimerization among other members of the PB1 domain family, including the atypical protein kinase C zeta (PKC zeta) and its partners Par6 and p62 (ZIP, sequestosome). Both Par6 and p62 use their basic "back" to interact with the OPCA motif on the "front" of the PKC zeta. Besides heterodimeric interactions, some PB1 domains, like the p62 PB1, can make homotypic front-to-back arrays.

Amino Acid Sequence↗

PubMed2003

Inhibition of the neutrophil NADPH oxidase by adenosine is associated with increased movement of flavocytochrome b between subcellular fractions.

Adenosine is a potent inhibitor of reactive oxygen species (ROS) production by the NADPH oxidase in fMLF-stimulated neutrophils. Although much is known about the pharamacology and signal transduction of this effect, it is not known how adenosine affects assembly and localization of the NADPH oxidase components within the neutrophil. We report here that adenosine pretreatment of fMLF-stimulated neutrophils results in decreased plasma membrane/secretory granule content of the flavocytochrome b components (p22phox and gp91phox) of the NADPH oxidase, which correlates with inhibition of ROS production. Adenosine treatment did not affect upregulation of secretory and specific granule surface markers, confirming that degranulation was not impaired by adenosine. However, adenosine treatment did result in increased movement of cell-surface flavocytochrome b to heavy granule fractions in fMLF-stimulated neutrophils. These data suggest that adenosine-mediated effects on neutrophil ROS production are due, in part to endocytosis and/or redistribution of flavocytochrome b between various subcellular compartments.

Adenosine↗

PubMed2003

Superoxide production in the vasculature of lipopolysaccharide-treated rats and pigs.

Sepsis is associated with increased production of reactive oxygen species (ROS); however, the metabolic sources of increased ROS are not well understood. We hypothesized that the recently described nonphagocytic NAD(P)H oxidase system could be an important source of the ROS superoxide anion (O2-) during sepsis, and the interaction of O2- with nitric oxide (NO) may contribute to sepsis-induced vascular Injury. To evaluate this issue, we measured O2- production before and after treatment with lipopolysaccharide (LPS) in rats, who are Inducible NO synthase producers (NOSII) and in pigs, who do not produce NOSII. LPS increased O2- production in aorta from rats from 0.38 +/- 0.07 nmol/mg/10 min to 1.18 +/- 0.23 nmol/mg/10 min, (P = 0.001) in rats, and 0.63 +/- 0.05 nmol/mg/10 min to 1.5 +/- 1.6 nmol/mg/10 min (P = 0.001) in carotid arteries from pigs. Components of NAD(P)H oxidase, including p22(phox), gp91(phox), p47(phox), p67(phox), mRNA and p22(phox), and gp91(phox) proteins were present in rat aorta and aorta and carotid arteries from pigs. Expression mildly increased in rats, but not in pigs. In rats, NADH and NADPH greatly increased O2- production with no difference in untreated versus LPS-treated rats. The addition of L-NAME increased NADH-dependant O2- production from 75 +/- 3 nmol/O2-/mg/10 min to 113 +/- 7 nmoVO2-/mg/10 min in LPS-treated rats, but had no effect in untreated rats. In pigs, the NADH-stimulated O2- production was 43 +/- 8 nmol/mg/10 min before and 63 +/- 4.3 nmol/mg/10 min after LPS even without L-NAME (P < 0.05). In contrast to LPS-treated rats, L-NAME markedly decreased NADH-stimulated O2- production (63 +/- 4 nmol/mg/10 min to 33 +/- 5.6 nmol/mg/10 min, P < 0.01). Luminol-enhanced chemiluminescence was also Increased in porcine carotid arteries after LPS treatment, which is consistent with peroxynitrite formation. Our results indicate that components of NAD(P)H oxidase are present in vessels of pigs and rats and there is substantial NADH-dependent O2- production that is increased after LPS. However, the behavior of NAD(P)H oxidase in NOSII-producing and nonproducing species differs with a reduction of O2- by NO in rats and NO-dependent production in pigs.

Animals↗

PubMed2003

Inhibition of actin polymerization by peroxynitrite modulates neutrophil functional responses.

Peroxynitrite, a potent oxidant generated in inflammatory tissues, can nitrate tyrosine residues on a variety of proteins. Based on previous studies suggesting that actin might be a potential target for peroxynitrite-mediated nitration in neutrophils, we investigated the effects of peroxynitrite on actin function. We show here that peroxynitrite and the peroxynitrite generator (SIN-1) modified actin in a concentration-dependent manner, resulting in an inhibition of globular-actin polymerization and filamentous-actin depolymerization in vitro. The effects of peroxynitrite were inhibited by the pyrrolopyrimidine antioxidant PNU-101033E, which has been shown previously to specifically block peroxynitrite-mediated tyrosine nitration. Furthermore, spectrophotometric and immunoblot analysis of peroxynitrite-treated actin demonstrated a concentration-dependent increase in nitrotyrosine, which was also blocked by PNU-101033E. Activation of neutrophils in the presence of a nitric oxide donor (S-nitroso-N-acetylpenicillamine) resulted in nitration of exogenously added actin. Nitrated actin was also found in peroxynitrite-treated neutrophils, suggesting that actin may be an important intracellular target during inflammation. To investigate this issue, we analyzed the effect of peroxynitrite treatment on a number of actin-dependent neutrophil processes. Indeed, neutrophil actin polymerization, migration, phagocytosis, and respiratory burst activity were all inhibited by SIN-1 treatment in a concentration-dependent manner. Therefore, the ability of peroxynitrite to inhibit actin dynamics has a significant effect on actin-dependent, cellular processes in phagocytic cells and may modulate their host defense function.

Actins↗

PubMed2002

Novel role of gp91(phox)-containing NAD(P)H oxidase in vascular endothelial growth factor-induced signaling and angiogenesis.

Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell proliferation and migration, primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). Reactive oxygen species (ROS) derived from NAD(P)H oxidase are critically important in many aspects of vascular cell regulation, and both the small GTPase Rac1 and gp91(phox) are critical components of the endothelial NAD(P)H oxidase complex. A role of NAD(P)H oxidase in VEGF-induced angiogenesis, however, has not been defined. In the present study, electron spin resonance spectroscopy is utilized to demonstrate that VEGF stimulates O2*- production, which is inhibited by the NAD(P)H oxidase inhibitor, diphenylene iodonium, as well as by overexpression of dominant-negative Rac1 (N17Rac1) and transfection of gp91(phox) antisense oligonucleotides in human umbilical vein endothelial cells (ECs). Antioxidants, including N-acetylcysteine (NAC), various NAD(P)H oxidase inhibitors, and N17Rac1 significantly attenuate not only VEGF-induced KDR tyrosine phosphorylation but also proliferation and migration of ECs. Importantly, these effects of VEGF are dramatically inhibited in cells transfected with gp91(phox) antisense oligonucleotides. By contrast, ROS are not involved in mediating these effects of sphingosine 1-phosphate (S1P) on ECs. Sponge implant assays demonstrate that VEGF-, but not S1P-, induced angiogenesis is significantly reduced in wild-type mice treated with NAC and in gp91(phox-/-) mice, suggesting that ROS derived from gp91(phox)-containing NAD(P)H oxidase play an important role in angiogenesis in vivo. These studies indicate that VEGF-induced endothelial cell signaling and angiogenesis is tightly controlled by the reduction/oxidation environment at the level of VEGF receptor and provide novel insights into the NAD(P)H oxidase as a potential therapeutic target for angiogenesis-dependent diseases.

Animals↗

PubMed2002

Cytochrome b558-dependent NAD(P)H oxidase-phox units in smooth muscle and macrophages of atherosclerotic lesions.

OBJECTIVE: Despite studies implicating superoxide anion-producing oxidases in atherosclerosis, their characteristics, expression, and regulation in cells of lesions are poorly understood. We examined the following: (1) whether cytochrome b558-dependent NAD(P)H oxidase-phox peptides are expressed by intimal smooth muscle cells (iSMCs) and macrophages of human aortic atherosclerotic lesions and their regulation and (2) whether cytochrome b558-dependent NAD(P)H oxidase represents a major NAD(P)H oxidase in iSMCs. METHODS AND RESULTS: Using a combination of immunochemical and reverse transcription-polymerase chain reaction procedures, we demonstrate that p22(phox) and gp91(phox) (cytochrome b558) expression in normal intima was restricted to a quarter of the iSMCs. In fatty streaks, a similar fraction of iSMCs expressed cytochrome b558, whereas macrophages also expressed low levels of p47(phox) and p67(phox). In fibrofatty lesions, the majority of iSMCs expressed the cytochrome b558 subunits; p67(phox) was also detected. Macrophages and macrophage-derived foam cells expressed the 4 phox subunits that constitute superoxide-producing cytochrome b558-dependent NAD(P)H oxidase. These were upregulated by transforming growth factor-beta1 and interferon-gamma. Aortic lesions also expressed Thox1 and Nox4, and although their expression also increases with lesion severity, their expression is less frequent than that of gp91(phox). CONCLUSIONS: In human aortic fibrofatty lesions, a cytochrome b558-dependent NAD(P)H oxidase appears to be a major iSMC and macrophage oxidase whose expression is upregulated by cytokines.

Adolescent↗

PubMed2002

Dose-dependent regulation of NAD(P)H oxidase expression by angiotensin II in human endothelial cells: protective effect of angiotensin II type 1 receptor blockade in patients with coronary artery disease.

OBJECTIVE: Angiotensin II (Ang II)-mediated induction of vascular superoxide anion formation could contribute to the development of endothelial dysfunction, hypertension, and atherosclerosis. An NAD(P)H oxidase has been identified as a major endothelial source of superoxide anions. However, the molecular mechanism underlying the regulation of NAD(P)H oxidase activity in response to Ang II is not well understood. METHODS AND RESULTS: We investigated the dose-dependent regulation of superoxide anion formation and of NAD(P)H oxidase subunit expression in response to Ang II in human endothelial cells. Ang II regulates superoxide anion formation and the limiting subunit of endothelial NAD(P)H oxidase, gp91-phox, in a dose-dependent manner via Ang II type 1 (AT1) receptor-mediated induction and Ang II type 2 receptor-mediated partial inhibition at higher Ang II concentrations. Furthermore, AT1 receptor blocker therapy before coronary bypass surgery downregulates the gp91-phox expression in internal mammary artery biopsies of patients with coronary artery disease. CONCLUSIONS: Our data support a dose-dependent induction of proatherosclerotic oxidative stress in human endothelial cells in response to Ang II. The expression of NAD(P)H oxidase subunit gp91-phox is critical for endothelial superoxide anion formation. AT1 receptor blockade has an antiatherosclerotic and antioxidative potential by the reduction of oxidative stress in the vessel wall.

Angiotensin II↗

PubMed2002

Effect of fibrin sealant composition on human neutrophil chemotaxis.

The use of fibrin sealants offers one of the most physiologically compatible approaches to preventing postoperative adhesions. Although a number of fibrin sealant formulations have been developed, little is known about how the various components of these preparations affect the wound-healing process. Because one of the key steps in wound healing is the migration of phagocytic leukocytes, such as neutrophils, into the site of injury, we performed studies to characterize systematically the effects of various fibrin sealant components on neutrophil chemotaxis. Using a transwell chemotaxis assay, we found that increasing fibrin concentration resulted in an inhibition of the ability of the cells to migrate through the clots in a dose-dependent manner, and at fibrin clot concentrations >2.0 mg/mL chemotaxis was completely blocked. Factor XIII crosslinking of the clots also had a significant impact on neutrophil chemotaxis, and sealant preparations deficient in Factor XIII allowed neutrophil migration at much higher fibrin concentrations. The presence of various other fibrin sealant components such as plasminogen and fibrinolysis inhibitors (aprotinin and tranexamic acid) did not have any significant effects on the ability of neutrophils to migrate through fibrin clots as compared to control clots without these components. Overall, these studies show that the composition of fibrin sealant preparations can significantly affect neutrophil migration into the site of injury, which could possibly influence the wound healing process.

Chemotaxis, Leukocyte↗

PubMed2002

Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries: regulation by angiotensin II.

A major source of vascular smooth muscle cell (VSMC) superoxide is NAD(P)H oxidase. However, the molecular characteristics and regulation of this enzyme are unclear. We investigated whether VSMCs from human resistance arteries (HVSMCs) possess a functionally active, angiotensin II (Ang II)-regulated NAD(P)H oxidase that contains neutrophil oxidase subunits, including p22phox, gp91phox, p40phox, p47phox, and p67phox. mRNA expression of gp91phox homologues, nox1 and nox4, was also assessed in HVSMCs, human aortic smooth muscle cells, and rat VSMCs. HVSMCs were obtained from resistance arteries from gluteal biopsies of healthy subjects. gp91phox and nox4, but not nox1, were detected in HVSMCs. Nox1 and nox4, but not gp91phox, were expressed in human aortic smooth muscle cells and rat VSMCs. All NAD(P)H oxidase subunits were present in HVSMCs as detected by reverse transcriptase-polymerase chain reaction and immunoblotting. Ang II increased NAD(P)H oxidase subunit abundance. These effects were inhibited by cycloheximide. Acute Ang II stimulation (10 to 15 minutes) increased p47phox serine phosphorylation and induced p47phox and p67phox translocation. This was associated with NAD(P)H oxidase activation. In cells transfected with gp91phox antisense oligonucleotides, Ang II-mediated actions were abrogated. NADPH-induced superoxide generation was reduced by gp91ds-tat and apocynin, inhibitors of p47phox-gp91phox interactions. Our results suggest that HVSMCs possess a functionally active gp91phox-containing neutrophil-like NAD(P)H oxidase. Ang II regulates the enzyme by inducing phosphorylation of p47phox, translocation of cytosolic subunits, and de novo protein synthesis. These novel findings provide insight into the molecular regulation of NAD(P)H oxidase by Ang II in HVSMCs. Furthermore, we identify differences in gp91phox homologue expression in VSMCs from rats and human small and large arteries.

Adult↗