Rebecca K. Studer

Role for Protein Kinase C in the Mediation of Increased Fibronectin Accumulation by Mesangial Cells Grown in High-Glucose Medium

The fibronectin content of RMC cultures grown for 8–14 days in medium containing 30 mM (540 mg/dl) D-glucose was increased 30–60% relative to that of control cells cultured in 10 mM (180 mg/dl) glucose. Addition of equiosmolar concentrations (20 mM, 360 mg/dl) of L-glucose, 3-O-methylglucose, or mannitol to 10 mM glucose media did not alter fibronectin accumulation compared with values observed in 10 mM glucose alone. The basal phosphorylation of the 80,000-Mr MARCKS protein, which is a substrate for PKC, and the phosphorylation induced by acute (15-min) exposure of cells to 15% FCS or to 0.1 μM (50 ng/ml) PDBu were all increased in cells grown in 30 mM compared with 10 mM glucose. By contrast, phosphorylation of the 80,000-Mr protein in response to a maximal concentration of PDBu (1 μM, 500 ng/ml) was not different in cells grown in 30 mM compared with 10 mM glucose. The acute increases in phosphorylation of the 80,000-Mr protein were blocked by the PKC inhibitor calphostin C. Chronic (7-day) exposure of mesangial cells grown in 10 mM glucose to 0.1 μM of the PKC agonist PDBu also resulted in a sustained 40% increase in 80,000-Mr phosphorylation and a 20–30% increase in fibronectin accumulation. As assessed by [35S]methionine incorporation, mesangial cell fibronectin synthesis was increased by exposure to PDBu, a finding consistent with earlier observations with 30 mM glucose. The increase in 80,000-Mr proteimesangial cells grown in either 0.1 μM PDBu or 30 mM glucose was abolished in cells whose PKC activity had been downregulated by exposure to PMA. Culture of mesangial cells with the thromboxane-endoperoxide analogue U-46619 in 30 mM, but not 10 mM, glucose increased fibronectin accumulation compared withvalues obtained in 30 mM glucose alone. These results demonstrate a sustained activation of PKC in mesangial cells cultured in a high concentration of glucose and support a role for PKC activation in the mediation of enhanced fibronectin accumulation induced by glucose. Because nonmetabolizable glucose analogues and mannitol were ineffective, metabolism of glucose is implicated in expression of these actions. The results also suggest that a high ambient glucose concentration may interact with thromboxane and/or prostaglandin endoperoxide in the activation of PKC and the enhancement of fibronectin accumulation by mesangial cells.

Nitric Oxide Inhibition of Transforming Growth Factor-β and Collagen Synthesis in Mesangial Cells

Culture of mesangial cells (MCs) in 5.6 vs. 30.0 mmol/1 glucose for 3 weeks induced a sustained increase in protein kinase C (PKC) activity, transforming growth factor (TGF)-β1 mRNA, bioactive TGF-β, and collagen synthesis. Nitric oxide (NO), generated exogenously by the NO donor S-nitroso-N-acetyl, D,L-penicillamine (SNAP) or endogenously after the exposure of MC to interleukin-1β (IL-1β), suppressed bioactive TGF-β in MCs cultured in 5.6 or 30.0 mmol/1 glucose and suppressed or abolished increases in TGF-β1 mRNA and collagen synthesis induced by high concentrations of glucose or phorbol 12,13-dibutyrate without altering values obtained with normal glucose concentrations. SNAP had a transient suppressive effect on PKC activity, which may explain at least in part some of the actions of SNAP. The selective inhibitor of PKC, bisindolylmaleimide (GFX), mimicked NO action. The ability of SNAP and IL-lβ to suppress TGF-β and collagen synthesis was not mediated by cGMP, since the cGMP analog, 8-Br-PET-cGMP, did not mimic NO action and an antagonist of cGMP-dependent protein kinase, Rp-8-pCPT-cGMPs, did not prevent the inhibitory actions of SNAP. N-ω-L-arginine methyl ester (NMMA) increased TGF-β in glomerular capillary endothelial cells (GCECs) and stimulated collagen synthesis by MC in a co-culture with GCECs. Captopril inhibited TGF-β and collagen synthesis and increased cGMP in co-cultures of GCECs and MCs. These effects of captopril were abolished by NMMA, implying mediation by NO. Thus, endogenous NO produced by GCECs may modulate TGF-β production by both GCECs and MCs and act to suppress matrix protein synthesis by MCs.

Antioxidant Inhibition of Protein Kinase C-Signaled Increases in Transforming Growth Factor-Beta in Mesangial Cells

Protein kinase C (PKC)-signaled increases in transforming growth factor beta (TGF beta) have been implicated in the stimulation of matrix protein synthesis induced by high concentrations of glucose, thromboxane, angiotension II (AII), and other stimuli in cultured glomerular mesangial cells. In the present study, the effects of several antioxidants on mesangial cell responses to high glucose, thromboxane, and AII were examined. alpha-Tocopherol blocked increases in PKC, TGF beta bioactivity, collagen, and/or fibronectin synthesis induced in mesangial cells by high glucose, the thromboxane analog U46619, and AII. By contrast, alpha-tocopherol did not alter increases in matrix protein synthesis in mesangial cells in response to exogenous TGF beta, a cytokine that does not activate PKC in mesangial cells and whose actions to stimulate matrix protein synthesis in these cells are not blocked by PKC inhibition or downregulation. Taurine and N-acetylcystein similarly inhibited activation of PKC and increases in TGF beta in response to high glucose, U46619, and AII. alpha-Tocopherol but not taurine or N-acetylcysteine partially blocked increases in PKC activity in mesangial cells in response to the diacylglycerol (DAG) analog, phorbol dibutyrate (PDBu). Thus, alpha-tocopherol may have direct effects on interaction of the PKC system of mesangial cells with DAG that are not shared by N-acetylcysteine or taurine. Increases in TGF beta have been implicated in the pathogenesis of glomerulosclerosis in diabetes and other nephropathies. The capacity of antioxidants to block increases in TGF beta in mesangial cells in response to high glucose, thromboxane, and All suggests their potential therapeutic utility to attenuate glomerulosclerosis.

Coculture of bone marrow mesenchymal stem cells and nucleus pulposus cells modulate gene expression profile without cell fusion.

Study Design. Changes in gene expression profile and cell fusion of mesenchymal stem cells (MSC) and nucleus pulposus cells (NPC) after coculture were analyzed. Objective. To investigate the mechanisms of the interaction between NPC and MSC such us differentiation, stimulatory effect, and cell fusion. Summary of Background Data. Introduction of exogenous cells to supplement and replenish intervertebral disc cell population offers a potential approach to treat intervertebral disc degeneration (IDD). Recent evidences showed that intradiscal injection of MSC effectively alter the course of IDD in vivo, and the regenerative potential may result from up-regulated extracellular matrix protein synthesis mediated by MSC and NPC interaction. Methods. Using a double labeling cell system and flow activated cell sorting, we quantitatively analyzed changes in the gene expression profile of human male MSC and female NPC after coculture in a 3-dimensional system that allows short distance paracrine interactions typical of the nucleus pulposus. Furthermore, we analyzed for cell fusion in the cell interaction by fluorescence in situ hybridization (FISH) for X and Y chromosomes, using a 3-dimensional culture system to allow cell-to-cell interactions conducive to cell fusion. Results. Two weeks of coculture cell interaction in a 3-dimensional environment induces a change in MSCs towards a more chondrogenic gene expression profile indicating MSC differentiation, and NPC gene expression changes in matrix and chondrogenic genes demonstrating only a modest trophic effect of MSC on NPC. Moreover, FISH analysis demonstrated that cell fusion is not responsible for MSC plasticity in the interaction with NPCs. Conclusion. This study clarifies the mechanism of MSCs and NPCs interaction in a 3-dimensional environment, excluding cell fusion. These data support the use of undifferentiated MSC for stem cell therapy for IDD treatment.

p38 MAPK Inhibition Modulates Rabbit Nucleus Pulposus Cell Response to IL-1

Analysis of disc gene expression implicated IL‐1 in the development of intervertebral disc degeneration (IDD) in a rabbit stab model. The purpose of these studies is to determine the role of p38 Mitogen Activated Protein Kinase (p38 MAPK) signaling in nucleus pulposus cell response to IL‐1, and to compare rabbit nucleus pulposus (rNP) cell responses to IL‐1 activation with those in a stab model of disc degeneration. NP cells maintained in alginate bead culture were exposed to IL‐1, with or without p38 MAPK inhibition. RNA was isolated for reverse transcription polymerase chain reaction (RT‐PCR) analysis of gene expression, conditioned media analyzed for accumulation of nitric oxide (NO) and prostaglandin E‐2 (PGE‐2), and proteoglycan synthesis measured after 10 days. IL‐1 upregulation of mRNA for cycloxygenase‐2 (COX‐2), matrix metalloproteinase‐3 (MMP‐3), IL‐1, and IL‐6, was blunted by p38 inhibition while downregulation of matrix proteins (collagen I, collagen II, aggrecan) and insulin‐like‐growth‐factor I (IFG‐1) was also reversed. mRNA for tissue inhibitor of matrixmetalloproteinase‐1 (TIMP‐1) was modestly increased by IL‐1, while those for Transforming Growth Factor‐β (TGF‐β) SOX‐9, and versican remained unchanged. Blocking p38 MAPK reduced IL‐1 induced NO and PGE‐2 accumulation and partially restored proteoglycan synthesis. p38 MAPK inhibition in control cells increased mRNA for matrix proteins (aggrecan, collagen II, versican, collagen I) and anabolic factors (IGF‐1, TGF, and SOX‐9) from 50% to 120%, decreased basal PGE‐2 accumulation, but had no effect on message for TIMP‐1, MMP‐3, or COX‐2. Inhibition of p38 MAPK in cytokine‐activated disc cells blunts gene expression and production of factors associated with inflammation, pain, and disc matrix catabolism while reversing IL‐1 downregulation of matrix protein gene expression and proteoglycan synthesis. The results support the hypothesis that IL‐1 could be responsible for many of the mRNA changes seen in rabbit NP in the stab model of disc degeneration, and uphold the concept that development of molecular techniques to block p38 MAPK could provide a therapeutic approach to slow the course of intervertebral disc degeneration.

p38 MAPK inhibition in nucleus pulposus cells: a potential target for treating intervertebral disc degeneration.

Study Design. Human nucleus pulposus cells were cultured in alginate beads and activated with IL-1&bgr; or TNF-&agr;, with and without inhibition of p38 mitogen activated protein kinase (p38 MAPK) activity. Cell production of factors modulating the anabolic/catabolic balance of the disc was determined. Objective. To determine the role of signaling through p38 MAPK in nucleus pulposus cells response to inflammatory cytokines and whether it might be a valid target for the development of molecular therapies for disc degeneration. Summary of Background Data. Multiple factors contribute to intervertebral disc degeneration (IDD), and development of effective therapies depends on understanding the underlying cellular pathophysiology. Interleukin-1&bgr; and tumor necrosis factor-&agr; are implicated in the development of IDD, and p38 MAPK is part of cytokine and mechanical stress signal pathways in other cells. These studies determine whether inhibiting p38 MAPK can decrease factors that negatively affect the metabolic balance and viability of nucleus pulposus cells. Materials and Methods. Degenerated intervertebral disc tissue was obtained from patients undergoing elective surgical procedures. Nucleus pulposus cells in alginate bead culture were exposed to IL-1 or TNF-&agr;, with or without p38 MAPK inhibition, and conditioned media analyzed for accumulation of nitric oxide (NO), prostaglandin E2 (PGE2), IL-6, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) through 10 days. Results. Inhibition of p38 MAPK decreased PGE2 in conditioned medium of control, unstimulated cells while not affecting TIMP-1 accumulation. Blocking cytokine activation of p38 MAPK reduced IL-1 and TNF-&agr; induced PGE2 and IL-6 accumulation. p38 MAPK inhibition increased the ratio of TIMP-1 to MMP-3 in conditioned medium of cells activated by IL-1 or TNF-&agr;. Conclusion. Inhibition of p38 MAPK in cytokine-activated disc cells blunts production of factors associated with inflammation, pain, and disc matrix catabolism. The data support further analysis of these effects on the anabolic/catabolic balance of nucleus pulposus cells and suggest that molecular techniques blocking this signal could provide a therapeutic approach to slow the course of intervertebral disc degeneration.

Characterization of intervertebral disc aging: longitudinal analysis of a rabbit model by magnetic resonance imaging, histology, and gene expression.

Study Design. A cohort of young, healthy New Zealand White rabbits was followed longitudinally with serial magnetic resonance imaging (MRI) analysis and terminal analysis of histologic changes and gene expression. Objective. To examine the changes observed during normal aging in the intervertebral disc. Summary of Background Data. Although there is a correlation between aging and the onset of intervertebral disc degeneration (IDD), evidence suggests that distinct pathways are involved in these processes. Our group has characterized a reproducible rabbit model of IDD by MRI, radiograph, histology, and mRNA expression. However, no similar analysis has been performed longitudinally for intervertebral disc aging to allow comparison of these 2 important processes. Methods. Four skeletally mature female NZW rabbits were housed for 122 weeks, and lumbar spine MRIs were characterized serially. Histologic and quantitative gene expression analysis of the nucleus pulposus of these aging animals was performed, and compared with adult and young rabbits. Results. Mean MRI index decreased by <25% through 120 weeks. The histologic analysis showed changes in cell composition, with abundant notochordal cells in the young, chondrocyte-like cells and notochordal cells in the adult, and clusters of hypertrophic chondrocytes in the aging discs. The PCR analysis of the nucleus pulposus showed that gene expression of collagen decreased, whereas that for proteoglycans increased with aging. BMP-2, TIMP-1, and SOX-9 expression was significantly lower in the young compared with adult discs and TGF-&bgr;1 demonstrated lower gene expression in young and aging animals. Conclusion. Although dramatic cellular changes were observed, age-related MRI changes occurred in this rabbit model of normal aging at a much slower rate than in a previous injury model of degeneration. In addition, the gene expression analysis of the nucleus pulposus demonstrated remarkable differences between aging and injury induced degeneration. These results suggest that aging and injury contribute uniquely to the process of IDD.

Clinical diagnosis of potentially treatable early articular cartilage degeneration using optical coherence tomography

A series of bench to operating room studies was conducted to determine whether it is feasible to use optical coherence tomography (OCT) clinically to diagnose potentially reversible early cartilage degeneration. A human cadaver study was performed to confirm the reproducibility of OCT imaging and grading based on identification of changes to cartilage OCT form birefringence using a polarized OCT system approved for clinical use. Segregation of grossly normal appearing human articular cartilage into two groups based on the presence or absence of OCT form birefringence showed that cartilage without OCT form birefringence had reduced ability to increase proteoglycan synthetic activity in response to the anabolic growth factor IGF-1. The bench data further show that IGF-1 insensitivity in cartilage without OCT form birefringence was reversible. To show clinical feasibility, OCT was then used arthroscopically in 19 human subjects. Clinical results confirmed that differences to OCT form birefringence observed in ex vivo study were detectable during arthroscopic surgery. More prevalent loss of cartilage OCT form birefringence was observed in cartilage of human subjects in groups more likely to have cartilage degeneration. This series of integrated bench to bedside studies demonstrates translational feasibility to use OCT for clinical studies on whether human cartilage degeneration can be diagnosed early enough for intervention that may delay or prevent the onset of osteoarthritis.

Human Nucleus Pulposus Cells React to IL-6 : Independent Actions and Amplification of Response to IL-1 and TNF-α

Study Design. Human nucleus pulposus cells were activated with IL-6 plus IL-6 soluble receptor (sR) in the presence or absence of IL-1&bgr; or TNF-&agr;. Cell production of factors modulating the anabolic/catabolic balance of the disc and proteoglycan synthesis were determined. Objective. To evaluate NP cell response to exogenous IL-6, and how IL-6 modulates IL-1 and TNF-&agr; actions in these cells. Summary of Background Data. Interleukin-6 (IL-6) is produced by cervical and lumbar herniated discs and is associated with neurological symptoms of intervertebral disc degeneration. It upregulates catabolic gene expression and downregulates matrix protein gene expression in chondrocytes. However, no studies have evaluated the effects of IL-6 on disc nucleus pulposus (NP) cells. Methods. NP cells from degenerated human discs were expanded in monolayer, maintained in alginate bead culture, and activated with IL-6 plus IL-6 soluble receptor (sR), in the presence or absence of IL-1&bgr; or TNF-&agr;. Conditioned media was collected and analyzed for nitrite, PGE-2, TIMP-1, MMP-3, VEGF, and IL-8. Proteoglycan synthesis was assayed as 35S-sulfate incorporation normalized to DNA content and relative gene expression measured by rtPCR. Results. IL-6 + sR decreased collagen and aggrecan message, proteoglycan synthesis, and exacerbated the downregulation of proteoglycan synthesis effected by IL-1. PGE-2 synthesis was increased by IL-6 + sR, as was the induction of COX-2 mRNA. IL-6 + sR also enhanced IL-1 and TNF-&agr; stimulated synthesis of PGE-2. IL-6 + sR induced MMP-3 approximately twofold and increased gene expression and synthesis in cells exposed to IL-1 and TNF-&agr;. MMP-13 induction by TNF-&agr; was also potentiated by IL-6 + sR. IL-6 + sR induced IL-6 gene expression and increased that stimulated by TNF-&agr; fourfold. Conclusion. The results suggest maneuvers to diminish IL-6 production in the disc could provide some protection against the adverse effects of IL-1 and TNF-&agr;, thus, helping preserve disc composition, structure, and function.

Differentiation of intervertebral notochordal cells through live automated cell imaging system in vitro

Study Design. We demonstrated the differentiation of notochordal cells by direct observation using a live automated cell imaging system. We also hypothesized that notochordal cells have characteristics of chondrocyte-like cells. Objective. To determine characteristics of notochordal cells by matrix protein expression and their differentiation using a live automated cell imager. Summary of Background Data. Although notochordal cells are critical to homeostasis of intervertebral disc, their fate has not been extensively studied and there is little evidence of notochordal cells as progenitors. Methods. Notochordal cells purified from rabbit nucleus pulposus were isolated after serial filtration. Notochordal cells in 3-dimensional culture were compared to chondrocyte-like cells by 35S sulfate incorporation into proteoglycan and reverse transcription polymerase chain reaction for gene expression(collagen II and aggrecan). Notochordal cells in 2-D culture were used for immunocytochemical staining (collagen II, aggrecan, and SOX9) and time-lapsed cell tracking study. Results. Notochordal cells were capable of proteoglycan production at a rate comparable to chondrocyte-like cells (108% ± 22.6% to chondrocyte-like cells) and expressed collagen II, aggrecan, and SOX9. In time-lapsed cell tracking analysis, notochordal cells were slower in population doubling time than chondrocyte-like cells and differentiated into 3 morphologically distinct cell types: vacuolated cells (area: 2392 ± 507.1 &mgr;m2, velocity: 0.09 ± 0.01 &mgr;m/min); giant cells (area: 12678 ± 1637.0 &mgr;m2, velocity: 0.08 ± 0.01 &mgr;m/min) which grew rapidly without cell division; polygonal cells (area: 3053 ± 751.2 &mgr;m2, 0.14 ± 0.01 &mgr;m/min) morphologically similar to typical differentiation type of chondrocyte-like cells (area: 2671 ± 235.6 &mgr;m2, 0.19 ± 0.01 &mgr;m/min). Rarely, notochordal cells formed clusters analogous to that observed in vivo. Conclusion. These studies demonstrate a chondrocyte phenotype of notochordal cells and are the first direct evidence of notochordal cell differentiation, suggesting that they may act as progenitor cells, which has the potential to lead to their use in novel approaches to regeneration of degenerative intervertebral disc.