Bodo Kurz

Mechanical Overload Induces VEGF in Cartilage Discs via Hypoxia-Inducible Factor

VEGF (vascular endothelial growth factor) is not only one of the most important angiogenesis factors, but is involved also in inflammatory processes. Recent studies have shown that VEGF as well as its receptor VEGFR-2 are expressed on osteoarthritic chondrocytes, but not on normal adult chondrocytes. Since mechanical overload is one of the causative factors for osteoarthritis, we studied its effect on VEGF expression on bovine cartilage disks that were compressed once with a strain of 50% and a strain rate of 1/second. Under these conditions, control disks (without pressure) were completely negative for VEGF expression as evidenced by immunocytochemical stainings as well as by enzyme-linked immunosorbent assay (ELISA) measurements. In contrast, 4 days after mechanical overload, the cartilage disks were positive in both detection methods. In addition, after mechanical overload chondrocytes were strongly immunopositive for hypoxia-inducible factor-1alpha (HIF-1alpha), the limiting protein of the dimeric transcription factor HIF-1 that is known to induce VEGF expression. Furthermore, the matrix metalloproteases MMP-1, MMP-3, and MMP-13, could be easily detected in pressure-treated disks by immunohistochemistry whereas staining in controls was low or undetectable. The tissue inhibitors of metalloproteinases (TIMP-1 and -2) could be detected in controls but not in samples treated with mechanical overload. To prove that increased MMP or decreased TIMP expression could be a result of the autocrine action of VEGF on chondrocytes, we repeated the experiments in the presence of a specific inhibitor for the kinase activity of the VEGFR-2. This inhibitor was effective to reduce mechanically induced MMP-1, -3, and -13 immunostaining and to restore TIMP expression. Taking together, these findings indicate that VEGF is induced in chondrocytes by mechanical overload and mediates destructive processes in osteoarthritis as an autocrine factor.

Combination of reduced oxygen tension and intermittent hydrostatic pressure: a useful tool in articular cartilage tissue engineering

Cartilage cells are normally studied under atmospheric pressure conditions and without loading. However, since cartilage exists in a condition of reduced oxygen and intermittent hydrostatic pressure we hypothesized lower partial oxygen pressures (PO2) and different intermittent hydrostatic pressures (IHP) would increase articular chondrocyte proliferation and matrix production and to stabilize chondrocyte phenotype in vitro. Monolayers of adult bovine articular chondrocytes were cultured under 5% or 21% PO2 in combination with IHP (0.2 MPa amplitude, frequencies 5/5s = 0.1 Hz, 30/2 or 2/30 min on/off loading). We measured proliferation (3H-thymidine incorporation) and collagen secretion (protein-binding assay, collagen type II-ELISA and immunocytochemical staining of pericellular collagen types I, II and IX). Reduced PO2 stimulated proliferation and collagen type II and IX secretion of chondrocytes in comparison to 21% PO2. Additionally, collagen type I expression was delayed by low PO2, indicating a stabilization of the cell phenotype. IHP 5/5s and 30/2 min inhibited proliferation but increased collagen secretion (pericellular collagen type IX was decreased). IHP 30/2 min delayed first expression of collagen type I. In contrast, IHP 2/30 min increased proliferation, but lowered collagen expression. All stimulating or inhibiting effects of PO2 and IHP were additive and vice versa. Reduced PO2 and different settings of IHP increased proliferation, collagen secretion, and phenotype stability of chondrocytes. The oxygen- and IHP-induced effects were additive, suggesting that a combination of these parameters might be a useful tool in cartilage tissue engineering.

β-Adrenoceptor-mediated effects in rat cultured thymic epithelial cells

Sympathetic nerves were visualized in sections from rat thymus by immunostaining of tyrosine hydroxylase, the rate‐limiting enzyme of catecholamine biosynthesis, and by glyoxylic acid‐induced fluorescence of catecholamines. Catecholaminergic nerve fibres were detected in close connection to thymic epithelial cells which therefore might be preferred target cells. To evaluate this, rat immunocytochemically defined, cultured thymic epithelial cells were investigated for adrenoceptors and adrenergic effects. In rat cultured thymic epithelial cells mRNA for β1‐ and β2‐adrenoceptors was detected by reverse transcription‐polymerase chain reaction by use of sequence‐specific primers. Specific, saturable binding to the cultivated cells was observed with the β‐adrenoceptor agonist CGP 12177. Adrenaline, noradrenaline or the β‐adrenoceptor agonist, isoprenaline, increased intracellular adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) levels in cultivated thymic epithelial cells dose‐dependently about 25 fold. The pharmacological properties revealed that this response was mediated by receptors of the β1‐ and the β2‐subtypes. The selective β3‐adrenoceptor agonist BRL 37344 had no effect on cyclic AMP levels. The increase in cyclic AMP was downregulated by preincubation with glucocorticoids like dexamethasone or cortisol which also changed the relative importance of β1‐/β2‐adrenoceptors to the response. Incubation with isoprenaline or the adenylate cyclase activator forskolin decreased basal and serum‐stimulated proliferation of thymic epithelial cells. However, adrenergic stimulation of thymic epithelial cells did not induce interleukin 1 production. Since thymic epithelial cells create a microenvironment which influences the maturation and differentiation of thymocytes to T‐lymphocytes, their observed capacity to respond to catecholamines provides novel evidence for the suggestion that adrenergic stimulation may interfere with the regulation of immune functions.

Reactive oxygen species induce expression of vascular endothelial growth factor in chondrocytes and human articular cartilage explants

Vascular endothelial growth factor (VEGF) promotes cartilage-degrading pathways, and there is evidence for the involvement of reactive oxygen species (ROS) in cartilage degeneration. However, a relationship between ROS and VEGF has not been reported. Here, we investigate whether the expression of VEGF is modulated by ROS.Aspirates of synovial fluid from patients with osteoarthritis (OA) were examined for intra-articular VEGF using ELISA. Immortalized C28/I2 chondrocytes and human knee cartilage explants were exposed to phorbol myristate acetate (PMA; 0–20 μg/ml), which is a ROS inducer, or 3-morpholino-sydnonimine hydrochloride (SIN-1; 0–20 μM), which is a ROS donor. The levels of VEGF protein and nitric oxide (NO) production were determined in the medium supernatant, using ELISA and Griess reagent, respectively. Gene expression of VEGF-121 and VEGF-165 was determined by splice variant RT-PCR. Expression of VEGF and VEGF receptors (VEGFR-1 and VEGFR-2) was quantified by real-time RT-PCR.Synovial fluid from OA patients revealed markedly elevated levels of VEGF. Common RT-PCR revealed that the splice variants were present in both immortalized chondrocytes and cartilage discs. In immortalized chondrocytes, stimulation with PMA or SIN-1 caused increases in the levels of VEGF, VEGFR-1 and VEGFR-2 mRNA expression. Cartilage explants produced similar results, but VEGFR-1 was only detectable after stimulation with SIN-1. Stimulation with PMA or SIN-1 resulted in a dose-dependent upregulation of the VEGF protein (as determined using ELISA) and an increase in the level of NO in the medium.Our findings indicate ROS-mediated induction of VEGF and VEGF receptors in chondrocytes and cartilage explants. These results demonstrate a relationship between ROS and VEGF as multiplex mediators in articular cartilage degeneration.

Mechanical factors influence the expression of endostatin––an inhibitor of angiogenesis––in tendons

Avascular zones of tendons are predisposed for degenerative changes and spontaneous rupture. Therefore, we analyzed the expression of the endogenous angiogenesis inhibiting factor endostatin in human fetal and adult tendons by immunohistochemical and biochemical methods. Moreover, to elucidate factors involved in the regulation of vascularity, we exposed primary cultures of rat tendon cells to intermittent hydrostatic pressure (0.2 MPa, 0.1 Hz for 24 h), and measured the endostatin content by ELISA and the effect of the conditioned medium to the proliferation of human umbilical vein endothelial cells (HUVEC).

Immunohistochemical detection of estrogen receptor α in articular chondrocytes from cows, pigs and humans: in situ and in vitro results

Clinical observations suggest that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis, but only little is known about the influence of these hormones on articular cartilage cells. The effect of estradiol is mediated by estrogen receptors alpha and beta. The goal of the present study was to search for estrogen receptor alpha in articular tissue from cows, pigs and humans by immunohistochemistry to form a basis for in vitro studies. In addition, we also tried to detect estrogen receptor alpha in cultivated articular chondrocytes from cows and bulls under certain culture conditions. Estrogen receptor alpha is detected by the use of antibody 13H2 in articular chondrocytes from cows, bulls, pigs and humans. Chondrocytes are physiologically exposed to reduced oxygen tension. In isolated articular chondrocytes from cows and bulls incubated either with 21% O2 or with 5% O2 positive cells were also found. These positive results therefore encourage testing the influence of estradiol on cultivated articular cartilage cells in these species under different culture conditions.

Catecholamines and lipopolysaccharide synergistically induce the release of interleukin-6 from thymic epithelial cells

The thymus as the major site of T-cell development is exposed to circulating hormones as well as to neurotransmitters released from peripheral nerves. We investigated the influence of catecholamines on the synthesis of interleukin-1 (IL-1) and IL-6 by cultured rat thymic epithelial cells. Basal or lipopolysaccharide (LPS)-stimulated production of IL-1 was not affected by catecholamines. Release of IL-6 was stimulated only scarcely by catecholamines or tumor necrosis factor-alpha (TNF-alpha) and moderately by LPS alone. However, co-stimulation with adrenaline, noradrenaline, or the beta-adrenoceptor agonist isoproterenol (isoprenaline) had an additive (TNF-alpha) or synergistic (LPS) effect on IL-6 release. The synergistic effect was dose-dependent on catecholamine or LPS concentrations. It was mediated by beta-adrenoceptors that are linked to elevation of intracellular cAMP levels, since it was promoted by beta-adrenoceptor agonists and could be blocked by beta-adrenoceptor antagonists. Co-incubation of LPS with agents directly raising cAMP-levels like forskolin or dibutyryl cAMP yielded even stronger IL-6 induction. After co-stimulation IL-6 mRNA was first detected after 3-4 h and a constant increase of IL-6 bioactivity in the culture supernatant was measured for up to 48 h. Since IL-6 is an important factor for thymocyte differentiation and proliferation, the findings demonstrate an influence of neuronal or hormonal catecholamines on the thymic microenvironment that is created by thymic epithelial cells.

Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and low oxygen partial pressure

ZusammenfassungIm Bereich des sog. „tissue engineering“ wird derzeit an einem künstlichen Knorpelersatz gearbeitet, der zur Deckung von Defekten im Gelenkknorpel verwendet werden soll. Meist werden dabei verschiedene künstliche Matrices in vitro mit autologen Chondrozyten besiedelt und auf ihre Verwendbarkeit als Implantat (Resorbierbarkeit, Antigenität, Toxizität und Integration in bestehendes Gewebe) hin untersucht. Oft ist bei diesen Untersuchungen die zur Verfügung stehende autologe Knorpelzellmenge zu gering, um größere Defekte behandeln oder verschiedene Matrices miteinander vergleichen zu können. Daher ist eine In-vitro-Vermehrung der Chondrozyten notwendig, bei der die Zellen jedoch gleichzeitig dedifferenzieren und fibroblastenartig werden. Deshalb ist es von Interesse, Parameter zu untersuchen, die eine Redifferenzierung induzieren. Das Ziel dieser Untersuchung war es, den Einfluss von intermittierendem hydrostatischem Druck und niedrigem Sauerstoffpartialdruck auf die Redifferenzierung von Rindergelenkknorpelzellen im Monolayer und in dreidimensionaler (3-D-)Alginatkultur zu testen. Der Redifferenzierungsprozess wurde durch immunzytochemischen Nachweis von Typ-II-Kollagen überprüft. Die Vitalität der Zellen wurde mit Hilfe des Trypan-Blau-Ausschlusstests bestimmt. Die Chondrozyten wurden durch 2wöchige Kultur in Plastikflaschen bei 20 % O2 dedifferenziert. Danach wurden sie als Monolayer oder 3-D-Alginatkultur subkultiviert und für 3 Wochen 3 unterschiedlichen Stimuli zum Zwecke der Redifferenzierung ausgesetzt: 1.) 20 % O2 (= 20,26 kPa Po2) + 5 % CO2 + 75 % N2; 2.) 5 % O2 (= 5,07 kPa Po2) + 5 % CO2 + 90 % N2; 3.) 5 % O2 (= 5,07 kPa Po2) + 5 % CO2 + 90 % N2 + 8 h intermittierende hydrostatische Druckbelastung (Frequenz: 30 min Belastung bei 3 bar absolut und 2 min Entlastung bei 1 bar absolut) pro Tag. Im Monolayer konnte nach 3 Wochen für keine der 3 Kulturbedingungen immunzytochemisch neusynthetisiertes Typ-II-Kollagen nachgewiesen werden. Daher scheint eine Redifferenzierung der dedifferenzierten Chondrozyten im Monolayer unter den untersuchten Bedingungen nicht möglich zu sein. In der 3-D-Alginatkultur wurde bei den bei 20 % O2 gehaltenen Chondrozyten kein immunzytochemisch detektierbares Typ-II-Kollagen gefunden. Bei 5 % O2 war eine starke Typ-II-Produktion in der gesamten Kugel zu verzeichnen. Im Vergleich zu diesen zeigten Chondrozyten aus Kugeln, die zusätzlich noch mit intermittierendem hydrostatischem Druck behandelt wurden, eine geringere Typ-II-Kollagenproduktion (sowohl im Bezug auf die Menge der Immunfärbung als auch auf die Anzahl der Typ-II-produzierenden Zellen) und eine Häufung der positiven Zellen im Randbereich der Kugel. Die Vitalität der Zellen in den Alginatkugeln lag nach 3 Wochen bei ca. 90 %. Unsere Untersuchungen haben gezeigt, dass der Sauerstoffpartialdruck ein wichtiger Parameter in der Kultivierung von Gelenkknorpelzellen darstellt. Ein reduzierter Sauerstoffpartialdruck förderte oder induzierte die Redifferenzierung von dedifferenzierten Chondrozyten in Alginatkultur.SummaryOne of the goals in the field of tissue engineering is the development of artificial cartilage for the treatment of cartilage defects. Therefore autologous chondrocytes are seeded on different artificial matrices to test their possible use as implants (resorption, antigenicity, toxicity and their integration in the tissue). One of the main problems in these experiments is that usually the amount of available chondrocytes is too low for treating large-scale defects or for comparing different matrices. An in-vitro-multiplication of the cells is needed which causes the chondrocytes to dedifferentiate and become fibroblast-like. Therefore parameters which induce a redifferentiation are of great interest. The objective of this study was to determine the influence of intermittent hydrostatic pressure and low oxygen partial pressure on the redifferentiation of dedifferentiated bovine articular chondrocytes in monolayer and three-dimensional alginate bead culture. The redifferentiation process was monitored by immunocytochemical detection of newly synthesized collagen type II. The viability of the cells was determined by the trypanblue exclusion test. The chondrocytes were dedifferentiated by a two week culture in plastic flasks with an oxygen level of 20 %. After this they were subcultured in monolayer or three-dimensional alginate culture and subjected to three different stimuli for three weeks in order to redifferentiate: 1.) 20 % O2 (= 20,26 kPa Po2) + 5 % CO2 + 75 % N2; 2.) 5 % O2 (= 5,07 kPa Po2) + 5 % CO2 + 90 % N2; 3.) 5 % O2 (= 5,07 kPa Po2) + 5 % CO2 + 90 % N2 + 8 h/d of intermittent hydrostatic pressure (frequency: 3 bar absolute for 30 min and 1 bar absolute for 2 min). In the monolayer there was no detectable collagen type II found by immunocytochemistry under either of the three culture conditions. Therefore a redifferentiation of dedifferentiated chondrocytes was not possible in monolayer cultures with the tested parameters. In the three-dimensional alginate culture there was no immunocytochemical staining of collagen type II found in the beads cultured with 20 % oxygen. With 5 % oxygen we found a strong collagen type II-production by chondrocytes throughout the whole bead. The intermittent hydrostatic pressure combined with 5 % oxygen lead to a decreased collagen type II-production compared to cells subjected to 5 % oxygen only. Also chondrocytes closer to the edge of these beads were more often immunopositive and seemed to produce more immunoreactive collagen type II. The viability of the chondrocytes in the alginate culture was close to 90 % after three weeks. Our experiments showed that oxygen partial pressure is an important parameter in the cultivation of articular chondrocytes. Reduced partial oxygen pressure promoted or induced the redifferentiation of dedifferentiated chondrocytes in alginate culture.

Vulnerability of the superficial zone of immature articular cartilage to compressive injury

OBJECTIVE The zonal composition and functioning of adult articular cartilage causes depth-dependent responses to compressive injury. In immature cartilage, shear and compressive moduli as well as collagen and sulfated glycosaminoglycan (sGAG) content also vary with depth. However, there is little understanding of the depth-dependent damage caused by injury. Since injury to immature knee joints most often causes articular cartilage lesions, this study was undertaken to characterize the zonal dependence of biomechanical, biochemical, and matrix-associated changes caused by compressive injury. METHODS Disks from the superficial and deeper zones of bovine calves were biomechanically characterized. Injury to the disks was achieved by applying a final strain of 50% compression at 100%/second, followed by biomechanical recharacterization. Tissue compaction upon injury as well as sGAG density, sGAG loss, and biosynthesis were measured. Collagen fiber orientation and matrix damage were assessed using histology, diffraction-enhanced x-ray imaging, and texture analysis. RESULTS Injured superficial zone disks showed surface disruption, tissue compaction by 20.3 ± 4.3% (mean ± SEM), and immediate biomechanical impairment that was revealed by a mean ± SEM decrease in dynamic stiffness to 7.1 ± 3.3% of the value before injury and equilibrium moduli that were below the level of detection. Tissue areas that appeared intact on histology showed clear textural alterations. Injured deeper zone disks showed collagen crimping but remained undamaged and biomechanically intact. Superficial zone disks did not lose sGAG immediately after injury, but lost 17.8 ± 1.4% of sGAG after 48 hours; deeper zone disks lost only 2.8 ± 0.3% of sGAG content. Biomechanical impairment was associated primarily with structural damage. CONCLUSION The soft superficial zone of immature cartilage is vulnerable to compressive injury, causing superficial matrix disruption, extensive compaction, and textural alteration, which results in immediate loss of biomechanical function. In conjunction with delayed superficial sGAG loss, these changes may predispose the articular surface to further softening and tissue damage, thus increasing the risk of development of secondary osteoarthritis.

Effect of Transmitters and Co-Transmitters of the Sympathetic Nervous System on Interleukin-6 Synthesis in Thymic Epithelial Cells

In the thymus, sympathetic nerves run in septa in close connection to subcapsular/perivascular thymic epithelial cells (TEC). Since TEC are supposed to create a microenvironment of cytokines necessary for the development of thymocytes to T cells, we investigated the influence of sympathetic transmitters and co-transmitters on interleukin-6 (IL-6) synthesis in cultivated rat TEC that express markers of perivascular/subcapsular TEC. Noradrenaline and ATP stimulated IL-6 production in the culture supernatants 14- and 23-fold over basal values after 24 h. Co-stimulation with noradrenaline and ATP yielded an additive effect. Synthesis of IL-6 was concentration-dependent upon ATP and appeared to be mediated by P2 purinoceptors. During 24 h stimulation with 1 mM ATP, two thirds of the ligand was degraded mainly to ADP, production of AMP and adenosine was minor or negligible. Thus, in TEC, transmitters and co-transmitters of the sympathetic nervous system have a co-stimulatory effect on synthesis of IL-6 that is an important factor for thymocyte differentiation and proliferation.