Age-dependent changes in intervertebral disc cell mitochondria and bioenergetics.

Robust cellular bioenergetics is vital in the energy-demanding process of maintaining matrix homeostasis in the intervertebral disc. Age-related decline in disc cellular bioenergetics is hypothesised to contribute to the matrix homeostatic perturbation observed in intervertebral disc degeneration. The present study aimed to measure how ageing impacted disc cell mitochondria and bioenergetics. Age-related changes measured included matrix content and cellularity in disc tissue, as well as matrix synthesis, cell proliferation and senescence markers in cell cultures derived from annulus fibrosus (AF) and nucleus pulposus (NP) isolated from the discs of young (6-9 months) and older (36-50 months) New Zealand White rabbits. Cellular bioenergetic parameters were measured using a Seahorse XFe96 Analyzer, in addition to quantitating mitochondrial morphological changes and membrane potential. Ageing reduced mitochondrial number and membrane potential in both cell types. Also, it significantly reduced glycolytic capacity, mitochondrial reserve capacity, maximum aerobic capacity and non-glucose-dependent respiration in NP. Moreover, NP cells exhibited age-related decline in matrix synthesis and reduced cellularity in older tissues. Despite a lack of changes in mitochondrial respiration with age, AF cells showed an increase in glycolysis and altered matrix production. While previous studies report age-related matrix degenerative changes in disc cells, the present study revealed, for the first time, that ageing affected mitochondrial number and function, particularly in NP cells. Consequently, age-related bioenergetic changes may contribute to the functional alterations in aged NP cells that underlie disc degeneration.

Predictors of pressure ulcer incidence following traumatic spinal cord injury: a secondary analysis of a prospective longitudinal study.

STUDY DESIGN: Secondary analysis of data from a prospective cohort study. OBJECTIVES: The objective of this study was to identify the medical and demographic factors associated with the development of pressure ulcers during acute-care hospitalization and inpatient rehabilitation following acute spinal cord injury. SETTING: The study was carried out at acute hospitalization, inpatient rehabilitation and outpatient rehabilitation sites at a university medical center in the United States. METHODS: Adults with acute traumatic spinal cord injury (n=104) were recruited within 24-72 h of admission to the hospital. Pressure ulcer incidence was recorded. RESULTS: Thirty-nine participants out of 104 (37.5%) developed at least one pressure ulcer during acute-care hospitalization and inpatient rehabilitation. Univariate logistic regression analyses revealed significant association of pressure ulcer incidence for those with pneumonia and mechanical ventilation (P=0.01) and higher injury severity (ASIA A) (P=0.01). Multiple logistic regression showed that the odds of formation of a first pressure ulcer in participants with ASIA A was 4.5 times greater than that for participants with ASIA B, CI (1-20.65), P=0.05, and 4.6 times greater than that for participants with ASIA C, CI (1.3-16.63), P=0.01. CONCLUSION: Among individuals with acute traumatic SCI, those with high-injury severity were at an increased risk to develop pressure ulcers. Pneumonia was noted to be associated with the formation of pressure ulcers.

Effects of secreted factors in culture medium of annulus fibrosus cells on microvascular endothelial cells: elucidating the possible pathomechanisms of matrix degradation and nerve in-growth in disc degeneration.

OBJECTIVE: To test whether the interaction between annulus fibrosus cells (AFCs) and endothelial cells (ECs) disrupts matrix homeostasis and stimulates production of innervation mediators. METHODS: Human microvascular ECs were cultured in the conditioned media of AF cell culture derived from degenerated human surgical specimen. Matrix-metalloproteinases (MMPs) and platelet-derived growth factor (PDGF) of ECs of this culture were analyzed by qRT-PCR, Western, and immunofluorescence. Vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and nerve growth factor (NGF) in the media of this cell culture were assayed by ELISA. To determine the effects of ECs on AFCs, qRT-PCR was performed to determine mRNA levels of collagen I, II and aggrecan in AFCs cultured in EC conditioned media. RESULTS: Compared to ECs cultured in naïve media, ECs exposed to AFC conditioned media expressed higher mRNA and protein levels of key biomarkers of invasive EC phenotype, MMP-2 (2×), MMP-13 (4×), and PDGF-B (1.5-2×), and NGF (24.9 ± 15.2 pg/mL vs 0 in naïve media). Treatment of AF cells with EC culture conditioned media decreased collagen type II expression two fold. Considerable quantities of pro-angiogenic factors IL-8 (396.7 ± 302.0 pg/mL) and VEGF (756.2 ± 375.9 pg/mL) were also detected in the conditioned media of untreated AF cell culture. DISCUSSION: AFCs from degenerated discs secreted factors which stimulated EC production of factors known to induce matrix degradation, angiogenesis, and innervation. IL-8 and VEGF maybe the secreted factors from AFCs which mediate a pro-angiogenic stimulus often implicated in the development of disc degeneration.

Viral co-infection, autoimmunity, and CSF HIV antibody profiles in HIV central nervous system escape.

Antiretroviral therapy (ART) suppresses plasma and cerebrospinal fluid (CSF) HIV replication. Neurosymptomatic (NS) CSF escape is a rare exception in which CNS HIV replication occurs in the setting of neurologic impairment. The origins of NS escape are not fully understood. We performed a case-control study of asymptomatic (AS) escape and NS escape subjects with HIV-negative subjects as controls in which we investigated differential immunoreactivity to self-antigens in the CSF of NS escape by employing neuroanatomic CSF immunostaining and massively multiplexed self-antigen serology (PhIP-Seq). Additionally, we utilized pan-viral serology (VirScan) to deeply profile the CSF anti-viral antibody response and metagenomic next-generation sequencing (mNGS) for pathogen detection. We detected Epstein-Barr virus (EBV) DNA more frequently in the CSF of NS escape subjects than in AS escape subjects. Based on immunostaining and PhIP-Seq, there was evidence for increased immunoreactivity against self-antigens in NS escape CSF. Finally, VirScan revealed several immunodominant epitopes that map to the HIV envelope and gag proteins in the CSF of AS and NS escape subjects. Whether these additional inflammatory markers are byproducts of an HIV-driven process or whether they independently contribute to the neuropathogenesis of NS escape will require further study.

Spine degeneration in a murine model of chronic human tobacco smokers.

OBJECTIVE: To investigate the mechanisms by which chronic tobacco smoking promotes intervertebral disc degeneration (IDD) and vertebral degeneration in mice. METHODS: Three month old C57BL/6 mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 6 months) to model long-term smoking in humans. Total disc proteoglycan (PG) content [1,9-dimethylmethylene blue (DMMB) assay], aggrecan proteolysis (immunobloting analysis), and cellular senescence (p16INK4a immunohistochemistry) were analyzed. PG and collagen syntheses ((35)S-sulfate and (3)H-proline incorporation, respectively) were measured using disc organotypic culture. Vertebral osteoporosity was measured by micro-computed tomography. RESULTS: Disc PG content of smoke-exposed mice was 63% of unexposed control, while new PG and collagen syntheses were 59% and 41% of those of untreated mice, respectively. Exposure to tobacco smoke dramatically increased metalloproteinase-mediated proteolysis of disc aggrecan within its interglobular domain (IGD). Cellular senescence was elevated two-fold in discs of smoke-exposed mice. Smoke exposure increased vertebral endplate porosity, which closely correlates with IDD in humans. CONCLUSIONS: These findings further support tobacco smoke as a contributor to spinal degeneration. Furthermore, the data provide a novel mechanistic insight, indicating that smoking-induced IDD is a result of both reduced PG synthesis and increased degradation of a key disc extracellular matrix protein, aggrecan. Cleavage of aggrecan IGD is extremely detrimental as this results in the loss of the entire glycosaminoglycan-attachment region of aggrecan, which is vital for attracting water necessary to counteract compressive forces. Our results suggest identification and inhibition of specific metalloproteinases responsible for smoke-induced aggrecanolysis as a potential therapeutic strategy to treat IDD.

p38 MAPK inhibition selectively mitigates inflammatory mediators and VEGF production in AF cells co-cultured with activated macrophage-like THP-1 cells.

OBJECTIVES: Recent data have suggested that macrophages are involved in the pathogenesis of discogenic back pain and enhance the secretion of inflammatory mediators in co-cultured annulus fibrosus (AF) cells. The purpose of these studies is to determine the role of p38 mitogen-activated protein kinase (p38 MAPK) signaling in the interactions between macrophage and AF cells. METHODS: Human AF cells were co-cultured with phorbol myristate acetate-stimulated macrophage-like THP-1 cells with and without p38 MAPK inhibition. Conditioned media from co-cultured cells were assayed for interleukin (IL)-6, IL-8, prostaglandin E2 (PGE2), PGF2alpha, and vascular endothelial growth factor (VEGF). Naïve and macrophage-exposed AF cell responses to 10ng/ml tumor necrosis factor-alpha (TNF-alpha) were compared using the same outcome measures. RESULTS: IL-6, IL-8, PGE2, PGF2alpha, and VEGF were secreted in greater quantities by cells maintained in co-culture compared to macrophages or AF cells cultured alone. SB202190 blunted IL-6, PGE2, and PGF2alpha production in a dose-dependent manner in co-culture. However, it did not suppress IL-8 and VEGF production. TNF-alpha-stimulated AF cell inflammatory mediators were up-regulated by macrophage exposure. SB202190 successfully suppressed IL-6, IL-8, PGE2, and PGF2alpha secretion in macrophage-exposed AF cells in response to TNF-alpha. CONCLUSIONS: Annular injury can result in macrophage infiltration, and this can cause enhanced inflammatory mediator and VEGF production by AF cells. The p38 MAPK pathway signals are responsible for much of IL-6 and PG secretion from AF cells with macrophage-like cells, suggesting that blockade of this signal may serve as a therapeutic approach to discogenic pain.

Enhancing effect of staurosporine on NO production in rat peritoneal macrophages via a protein kinase C-independent mechanism.

Staurosporine (3-100 nM), frequently used as a protein kinase C (PKC) inhibitor, increased accumulation of nitrite in the culture medium of rat peritoneal macrophages up to 6 times above the control level. Moreover, when used in combination with the stable analogue of cyclic AMP, dibutyrylcyclic AMP (db cyclic AMP; 0.1 mM), and/or a cytokine, tumour necrosis factor-alpha (TNF alpha; 100 u ml-1), staurosporine synergistically potentiated, up to 30 times, nitrite accumulation. On the other hand, the other PKC inhibitors, calphostin C and H-7 (10 nM-10 microM) were not effective under the same conditions. The staurosporine-induced nitrite accumulation, in both the presence and the absence of TNF alpha and/or db cyclic AMP was effectively inhibited by the protein synthesis inhibitor, cycloheximide, or by the nitric oxide (NO) synthesis inhibitor, NG-monomethyl-L-arginine (L-NMMA). Thus our data suggest that staurosporine may enhance NO production in macrophages via intracellular mechanisms unrelated to the PKC inhibition.

Inhibition of swine microglial cell phagocytosis of Cryptococcus neoformans by femtomolar concentrations of morphine.

Microglia are important immune effector cells within the brain. The phagocytosis of nonopsonized Cryptococcus neoformans by swine microglia was used as an in vitro model for studies on cellular mechanisms of opiate-mediated immunomodulation in the brain. Morphine inhibited potently (IC50 approximately 10(-16) M) the phagocytosis of C. neoformans by primary cultures of neonatal pig microglia. The mu opioid agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) also suppressed phagocytosis but with a much lower potency than morphine (IC50 approximately 10(-8) M). The inhibitory effects of morphine and DAMGO were blocked by equimolar concentrations of naloxone and by the selective mu opiate receptor antagonist beta-funaltrexamine. Pertussis toxin but not cholera toxin reversed the inhibitory effects of both morphine and DAMGO. Our data suggest that morphine inhibits phagocytosis of C. neoformans by swine microglia via a mechanism involving mu opiate receptors coupled to a pertussis toxin-sensitive Gi/G(o) protein signaling pathway.

cAMP analogues and cholera toxin stimulate the accumulation of nitrite in rat peritoneal macrophage cultures.

Rat peritoneal macrophages incubated with the two stable analogues of cAMP, dibutyryl-cAMP and 8-bromo-cAMP, as well as with cholera toxin, released nitrite in a dose-dependent manner. Cholera toxin and dibutyryl-cAMP enhanced nitrite release induced by bacterial lipopolysaccharide. The stimulatory effects of all these substances were inhibited by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine, dexamethasone and the protein synthesis inhibitor, cycloheximide. Our data indicate that the activation of cAMP-dependent pathway(s) can induce nitric oxide synthesis in rat peritoneal macrophages even in the absence of immunological stimuli, such as exogenous cytokines or lipopolysaccharide, although the exact mechanism of this phenomenon remains to be established.

Influence of SIN-1 and sodium nitroprusside (NANP) on ox-LDL metabolism in macrophages.

Effects of NO-donors (3-morpholinosydnonimine-SIN-1 and sodium nitroprusside NaNP) on the accumulation and degradation of oxidized LDL (ox-LDL) by macrophages were studied. Ox-LDL, but not native-LDL (n-LDL) suppressed the LPS-stimulated biosynthesis of NO by macrophages. SIN-1 at low concentrations < 100 microM was without any effect while SIN-1 at high concentration (300 microM) and NaNP (30-300 microM) stimulated the accumulation and degradation of ox-LDL by macrophages. The pretreatment of macrophages with NG-monomethyl-L-arginine (L-NMMA, 3 microM) for 24 hours had the same stimulatory effect. The inhibition of endogenous formation of NO, by L-NMMA profoundly changed the pattern of action of NO-donors on ox-LDL catabolism by macrophages; the stimulatory action of SIN-1 was transformed to the inhibitory action on the accumulation and degradation of ox-LDL whereas NaNP lost its stimulatory action entirely. Our interpretation of this unexpected interactions between SIN-1, NaNP and L-NMMA is as follows. Endogenous NO in macrophages inhibits the accumulation of ox-LDL and therefore, the stimulatory effect of L-NMMA has been overcome by exogenous NO from SIN-1. However, NO at high concentrations promotes lipid accumulation in macrophages and thereby, in the absence of L-NMMA, SIN-1 at high concentrations and NaNP produced a paradoxical stimulatory effect in macrophages. NaNP is not a proper NO-donor and its mode of action differed from that of SIN-1. In conclusion, NO at low physiological concentrations keeps scavenger receptors of macrophages downregulated and hence endogenous NO may show anti-atherogenic properties.

[Menisci and synovial folds of the metacarpophalangeal joint of the thumb]. / Menisci und Synovialfalten des Daumengrundgelenkes.

The inner surface of 25 thumb metacarpophalangeal joints were investigated and the arrangement and structure of different folds protruding into the joint cavity at the level of the joint cleft studied. At the ulnar and radial sides, compact wedgeshaped folds are found consisting of collagenous fibres, which connect with the fibrous layer of the joint capsule. The circularly arranged fibrous tissue is covered by a thin layer of cartilaginous cells. From a structural and functional point of view they are comparable to the menisci of the knee joint. In contrast, a different kind of tissue is found at the palmar and dorsal circumference of the joint cleft: typical synovial folds, consisting of loose connective tissue and small fat lobules. These are suggested not to fulfill a particular mechanical function but to act as a malleable spacer which is able to conform to the requirements of joint mobility.

Application of a semiautomated contour segmentation tool to identify the intervertebral nucleus pulposus in MR images.

BACKGROUND AND PURPOSE: Accurate identification of the NP in MR images is crucial to properly and objectively assess the intervertebral disk. Therefore, computerized segmentation of the NP in T2WI is necessary to produce repeatable and accurate results with minimal user input. MATERIAL AND METHODS: A semiautomated CS method was developed to identify the NP in T2WI on the basis of intensity differences compared with the AF. The method was validated by segmenting computer-generated images with a known ROI. The method was tested by using 63 MR images of rabbit lumbar disks, which were segmented to detect disk degeneration. An ICC was used to assess the repeatability of this method compared with manual segmentation. RESULTS: The error in the detected area of the rabbit NP by using CS was -3.49% ± 4.4% (mean ± SD) compared with 22.36% ± 5.55% by using manual segmentation. Moreover, the method was capable of detecting disk degeneration in a known rabbit puncture model of disk degeneration. Finally, this method had an ICC of 0.97 and 0.99 in regard to segmenting the area and calculating the MR imaging index of the NP, deeming it highly repeatable. CONCLUSIONS: The CS method is a semiautomated computer method able to segment the NP of the rabbit disk and detect disk degeneration. In addition, it could assist in clinical detection, assessment, and monitoring of early degeneration in human disks.

Ouabain enhances the lipopolysaccharide-induced nitric oxide production by rat peritoneal macrophages.

Incubation of rat peritoneal macrophages with the bacterial product lipopolysaccharide (LPS) caused significant increases in the production of nitrite, a stable endproduct of nitric oxide (NO). Addition of the digitalis-derived glycoside ouabain, which alone is without effect, significantly enhanced the stimulating effects of LPS. The Na+ ionophore monensin was without any effect, while the Ca2+ ionophore A23187, like ouabain, enhanced the effect of LPS. Thus ouabain may enhance the LPS-stimulated NO production in rat peritoneal macrophages via still unknown mechanisms in which Ca2+ ions are most likely involved.

[Alloplastic replacement of the anterior cruciate ligament. Comparative studies of ruptured Dacron, Trevira and Ligapro ligaments]. / Alloplastischer Ersatz des vorderen Kreuzbandes. Vergleichende Untersuchungen an rupturierten Dacron-, Trevira- und Ligaprobändern.

Histological investigations were performed on 24 polyethylene terephthalate ligaments (eight Dacron, eleven Trevira and five Ligapro) which had served as substitute for the human anterior cruciate ligament. Because of rupture or loosening they had been explanted six to 48 months after operation. Only in one Trevira ligament a longitudinal direction of collagenous lamellae could be seen, all other ligaments had only few collagenous fibers, mainly orientated in circular direction. In the fibrous tissue of all three kinds of ligaments there were signs of inflammation. Foreign body giant cells have immigrated--they mainly enclose the graft fibres in Dacron and Ligapro ligaments. In Trevira and Ligapro ligaments the foreign body giant cells phagocyte particles of graft fibers after increasing implantation times. The inflammatory reaction to graft fibers and their particles may also damage host tissue. Regarding these results we conclude that the Polyethylene terephthalate ligaments are not qualified as substitutes for the anterior cruciate ligament.

Induction of DNA synthesis by microtubule depolymerization is mediated by actin filaments.

Colcemid (0.4 microgram/ml) induced DNA synthesis in quiescent chick embryo fibroblasts, and increased the amount of F-actin. Cytochalasin D, which disorganized actin filaments, inhibited the stimulation of DNA synthesis by colcemid. These results suggest that actin filaments are involved in the sequence of events leading from the depolymerization of microtubules to the initiation of DNA synthesis. They also demonstrate that depolymerization of microtubules can influence the amount of F-actin in the cell.

Distinction between signaling mechanisms in lipid rafts vs. caveolae.

The relative importance of lipid rafts vs. specialized rafts termed caveolae to influence signal transduction is not known. Here we show that in cells lacking caveolae, the dually acylated protein, endothelial nitric oxide synthase (eNOS), localizes to cholesterol-rich lipid raft domains of the plasma membrane. In these cells, expression of caveolin-1 (cav-1) stimulates caveolae biogenesis, promotes the interaction of cav-1 with eNOS, and the inhibition of NO release from cells. Interestingly, in cells where cav-1 does not drive caveolae assembly, despite equal levels of cav-1 and eNOS and localization of both proteins to raft domains of the plasmalemma, the physical interaction of eNOS with cav-1 is dramatically less resulting in less inhibition of NO release. Thus, cav-1 concentrated in caveolae, not in rafts, is in closer proximity to eNOS and is necessary for negative regulation of eNOS function, thereby providing the first clear example of spatial regulation of signaling in this organelle that is distinct from raft domains.

Toward identification of acid/base catalysts in the active site of enolase: comparison of the properties of K345A, E168Q, and E211Q variants.

High-resolution crystallographic data show that Glu 168 and Glu 211 lie on opposite surfaces of the active site from Lys 345. Two different proposals for general base catalysis have emerged from these structural studies. In one scheme, the carboxylate side chains of Glu 168 and Glu 211 are proposed to ionize a trapped water molecule and the OH- serves as the base [Lebioda, L., & Stec, B. (1991) Biochemistry 30, 2817-2822]. In the other proposal, the epsilon-amino group of Lys 345 functions in general base catalysis [Wedekind, J. E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994) Biochemistry 33, 9333-9342]. Genes encoding site specific mutations of these active site residues of yeast enolase, K345A, E168Q, and E211Q, have been prepared. The respective protein products of the wild type and mutant genes were expressed in Escherichia coli and isolated in homogeneous form. All three mutant proteins possess severely depressed activities in the overall reaction- < 1 part in 10(5) of wild type activity. Properties of the three mutant proteins in partial reactions were examined to define more clearly the roles of these residues in the catalytic cycle. The K345A variant fails to catalyze the exchange of the C-2 proton of 2-phospho-D-glycerate with deuterium in D2O, whereas both the E211Q and E168Q mutant proteins are functional in this partial reaction. For E211Q and E168Q enolases, exchange is essentially complete prior to appearance of product, and this observation provides further support for an intermediate in the normal reaction. K345A enolase is inactive in the ionization of tartronate semialdehyde phosphate (TSP), whereas both E168Q and E211Q proteins alter the tautomeric state or catalyze ionization of bound TSP. Wild type enolase catalyzes hydrolysis of (Z)-3-chloro-2-phosphoenolpyruvate by addition of OH- and elimination of Cl- at C-3. This reaction mimics the addition of OH- to C-3 of phosphoenolpyruvate in the reverse reaction with the normal product. All three mutant proteins are depressed in their abilities to carry out this reaction. In single-turnover assays, the activities vary in the order K345A > E168Q >> E211Q. These results suggest that Lys 345 functions as the base in the ionization of 2-PGA and that Glu 211 participates in the second step of the reaction.

Nature of the transition state of the protein-tyrosine phosphatase-catalyzed reaction.

The dephosphorylation of p-nitrophenyl phosphate by Yersinia protein-tyrosine phosphatase (PTPase) and by the rat PTP1 has been examined by measurement of heavy-atom isotope effects at the nonbridge oxygen atoms [18(V/K)nonbridge], at the bridging oxygen atom [18(V/K)bridge], and the nitrogen atom in the leaving group 15(V/K). The effects were measured using an isotope ratio mass spectrometer by the competitive method and thus are effects on V/K. The results for the Yersinia PTPase and rat PTP1, respectively, are 1.0142 +/- 0.0004 and 1.0152 +/- 0.0006 for 18(V/K)bridge; 0.9981 +/- 0.0015 and 0.9998 +/- 0.0013 for 18(V/K)nonbridge; and 1.0001 +/- 0.0002 and 0.9999 +/- 0.0003 for 15(V/K). The magnitudes of the isotope effects are similar to the intrinsic values measured in solution, indicating that the chemical step is rate-limiting for V/K. The transition state for phosphorylation of the enzyme is dissociative in character, as is the case in solution. Binding of the substrate is rapid and reversible, as is the binding-induced conformational change which brings the catalytic general acid into the active site. Cleavage of the P-O bond and proton transfer from the general acid Asp to the leaving group are both far advanced in the transition state, and there is no development of negative charge on the departing leaving group. Experiments with several general acid mutants give values for 18(V/K)bridge of around 1.0280, 15(V/K) of about 1.002, and 18(V/K)nonbridge effects of from 1.0007 to 1.0022. These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion but suggest more nucleophilic participation than in the solution reaction.

Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt.

Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding beta-galactosidase (beta-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with beta-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of beta-gal-infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.

Trafficking of endothelial nitric-oxide synthase in living cells. Quantitative evidence supporting the role of palmitoylation as a kinetic trapping mechanism limiting membrane diffusion.

To examine endothelial nitric-oxide synthase (eNOS) trafficking in living endothelial cells, the eNOS-deficient endothelial cell line ECV304 was stably transfected with an eNOS-green fluorescent protein (GFP) fusion construct and characterized by functional, biochemical, and microscopic analysis. eNOS-GFP was colocalized with Golgi and plasma membrane markers and produced NO in response to agonist challenge. Localization in the plasma membrane was dependent on the palmitoylation state, since the palmitoylation mutant of eNOS (C15S/C26S eNOS-GFP) was excluded from the plasma membrane and was concentrated in a diffuse perinuclear pattern. Fluorescence recovery after photobleaching (FRAP) revealed eNOS-GFP in the perinuclear region moving 3 times faster than the plasmalemmal pool, suggesting that protein-lipid or protein-protein interactions are different in these two cellular domains. FRAP of the palmitoylation mutant was two times faster than that of wild-type eNOS-GFP, indicating that palmitoylation was influencing the rate of trafficking. Interestingly, FRAP of C15S/C26S eNOS-GFP but not wild-type eNOS-GFP fit a model of protein diffusion in a lipid bilayer. These data suggest that the regulation of eNOS trafficking within the plasma membrane and Golgi are probably different mechanisms and not due to simple diffusion of the protein in a lipid bilayer.