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.

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.

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.

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.