Positional Relations of the Cervical Vagus Nerve Revisited.

OBJECTIVES: The cervical part of the vagus nerve (CVN) has become an important target for stimulation therapy to treat epilepsy and psychiatric conditions. For this purpose, the CVN is visualized in the carotid sheath, assuming it to be localized dorsomedially between the carotid artery (CA) and the internal jugular vein (JV). The aim of our morphological study was therefore to revisit the CVN relationships to the CA and JV, hypothesizing it to have common variations to this classical textbook anatomy. MATERIALS AND METHODS: Positional relations of the CVN, CA and JV were investigated in the carotid sheath of 35 cadavers at the C3 to C6 level. Positional relations of the CVN, CA and JV were documented on the basis of a 3 × 3 chart. RESULTS: Eighteen different arrangements of the CVN, CA and JV were observed. The typical topographic relationship of the CVN dorsomedially between the CA and JV was only found in 42% of all cases. The CVN was located dorsally or (dorso-)laterally to the CA in 80% and dorsally or (dorso-)medially of the JV in 96% of all cases. CONCLUSIONS: Classical textbook anatomy of the CVN is only present in a minority of cases. Positional variations in contrast to textbook anatomy are considerably more frequent than previously described, which might be a hypothetical morphological explanation for the lack of efficacy or side effects of CVN stimulation. Furthermore, the position of the CVN relative to the internal jugular vein is more consistent than to the CA.

Structure, Function and Insertion of the Human Vocal Folds.

We here summarize the structures of the laryngeal vocal fold as well as its insertion structures at the anterior commissure and at the area of the vocal process and place these findings within the context of biomechanical, functional, and clinical implications.

Different Patterns of Cartilage Mineralization Analyzed by Comparison of Human, Porcine, and Bovine Laryngeal Cartilages.

Laryngeal cartilages undergo a slow ossification process during aging, making them an excellent model for studying cartilage mineralization and ossification processes. Pig laryngeal cartilages are similar to their human counterparts in shape and size, also undergo mineralization, facilitating the study of cartilage mineralization. We investigated the processes of cartilage mineralization and ossification and compared these with the known processes in growth plates. Thyroid cartilages from glutaraldehyde-perfused male minipigs and from domestic pigs were used for X-ray, light microscopic, and transmission electron microscopic analyses. We applied different fixation and postfixation solutions to preserve cell shape, proteoglycans, and membranes. In contrast to the ossifying human thyroid cartilage, predominantly cartilage mineralization was observed in minipig and domestic pig thyroid cartilages. The same subset of chondrocytes responsible for growth plate mineralization is also present in thyroid cartilage mineralization. Besides mineralization mediated by matrix vesicles, a second pattern of cartilage mineralization was observed in thyroid cartilage only. Here, the formation and growth of crystals were closely related to collagen fibrils, which served as guide rails for the expansion of mineralization. It is hypothesized that the second pattern of cartilage mineralization may be similar to a maturation of mineralized cartilage after initial matrix vesicles-mediated cartilage mineralization.

The influence of biomechanical parameters on the expression of VEGF and endostatin in the bone and joint system.

Many degenerative processes in the skeletal system are induced by mechanical overload. Osteoarthritis and spontaneous tendon ruptures are two examples of mechanically influenced diseases. Incubator-housed compression apparatuses and cyclic strain chambers are adequate models to investigate the cellular processes. Recent studies have shown that growth factors are involved in the transduction pathways of mechanical overload leading to tissue degradation. Vascular endothelial growth factor (VEGF) is a dimerized, 45 kDa peptide that normally attracts endothelial cells in wound healing. VEGF can be detected in the superficial zone of the tibial plateau in osteoarthritic (OA) patients with degenerative changes but not in healthy articular cartilage. Blood vessels are only rarely observed in OA cartilage suggesting that there are other roles for VEGF in cartilage. VEGF is also detectable in ruptured but not in normal tendons. The mechanically induced expression of VEGF in avascular tissues like articular cartilage or fibrocartilage of contact areas from gliding tendons initiates degenerative processes. Chondrocytes from OA cartilage also express the VEGF receptor 2. In vitro assays have shown that VEGF binds the VEGFR-2 leading to a phosphorylation of MAP kinases (ERK1/2) with subsequent transcription factor accumulation (activator protein 1 = AP-1). One of the antagonists of VEGF is endostatin. Endostatin, a fragment of collagen type XVIII, is expressed in avascular tissues and has the potency to decrease VEGF induced effects (ERK1/2 phosphorylation). The increase in matrix metalloproteinase (MMP) production and the decrease in tissue inhibitor metalloproteinase (TIMP) synthesis is a result of the signal transduction cascade activation. MMPs participate in the degradation processes of osteoarthritis whereas TIMPs are inhibitors of the MMPs. Taken together mechanically induced VEGF is involved in the destruction and endostatin in the maintenance of avascular tissues of the bone and joint system.

Locally applied angiogenic factors--a new therapeutic tool for meniscal repair.

Tears in the peripheral part of the menisci have a better healing potential than tears in the central part, because the central two-thirds of the menisci are avascular. The avascular status of the meniscus is maintained by the expression of antiangiogenic factors such as endostatin. The distribution of endostatin in the menisci correlates with the degree of vascularization. Endostatin immunostaining is strong in the avascular zone and reduced in the vascularized outer one-third. Endostatin interacts with signal transduction of the vascular endothelial growth factor (VEGF) by reducing VEGF-induced kinase (Erk1/2) phosphorylation. VEGF plays an important role in angiogenesis in fetal menisci and it is down-regulated in the adult meniscus. We hypothesized that healing of meniscal tears in the avascular zone can be promoted by the local application of the angiogenic factor VEGF. To evaluate this hypothesis a tear was created in the avascular zone of the medial meniscus in 18 merino sheep. The tear was then repaired with an uncoated suture (group 1), a suture coated with PDLLA (group 2), and by a suture coated with PDLLA/VEGF (group 3). After 6 weeks we observed increased factor VIII immunostaining in the VEGF-treated group. However, in this treatment group (VEGF/PDLLA) no meniscus healed. In the uncoated suture group and in the PDLLA-coated suture group partial healing was observed in three animals and complete healing in three animals, respectively. Factor VIII expression is normally restricted to vascular endothelial cells. In this study, however, single endothelial cells could be detected in the menisci of the VEGF/PDLLA group. This finding suggests that the application of VEGF might have stimulated proliferation of vascular endothelial cells but the application of VEGF was not successful in stimulating the more complex process of vasculogenesis. Further immunohistochemical examinations of the specimen have shown that in the VEGF/PDLLA group there is strong immunostaining against matrix metalloproteinase 13 (MMP-13). In vitro studies have shown that VEGF can stimulate chondrocytes to proliferate but also to express MMP-13 via HIF1-alpha induction. Since meniscal fibrochondrocytes express the VEGF receptor 2 (KDR) the induction of MMP expression might be another factor which inhibits healing despite increased angiogenesis. In conclusion, the local application of VEGF via PDLLA-coated sutures does not promote meniscal healing. A single growth factor might not always be a promising tool for the promotion of tissue repair. Further studies have to find out if growth factor combinations (VEGF and angiopoitin) might be more effective in stimulating vasculogenesis during meniscal healing.

Expression and regulation of antimicrobial peptides in articular joints.

Articular joint infection is a surprisingly rare event considering the frequency of joint arthrocentesis and other invasive procedures applied to limb joints. This observation led us to the hypothesis that a local "chemical shield" in the form of antimicrobial proteins provides synovial membrane and articular cartilage with resistance to infection. We subsequently began a systematic analysis of in vitro and in vivo antimicrobially active proteins in healthy articular joints and in disease states such as pyogenic arthritis, rheumatoid arthritis, and osteoarthritis. An anatomical approach with systematic characterization combined with antimicrobial testing revealed expression and production of human antibiotic peptides and proteins. In this review, we focus on the most prominent antimicrobial proteins in articular joints, which we have identified as lysozyme, lactoferrin, secretory phospholipase A2, RNase 7, CAP37, the cathelicidin LL37, and especially the human beta-defensin-2 and -3 (HBD-2/-3). Activation pathways and possible antimicrobial functions are discussed and the involvement in non-antimicrobial processes such as tissue remodelling is also considered.

Human vagus nerve branching in the cervical region.

BACKGROUND: Vagus nerve stimulation is increasingly applied to treat epilepsy, psychiatric conditions and potentially chronic heart failure. After implanting vagus nerve electrodes to the cervical vagus nerve, side effects such as voice alterations and dyspnea or missing therapeutic effects are observed at different frequencies. Cervical vagus nerve branching might partly be responsible for these effects. However, vagus nerve branching has not yet been described in the context of vagus nerve stimulation. MATERIALS AND METHODS: Branching of the cervical vagus nerve was investigated macroscopically in 35 body donors (66 cervical sides) in the carotid sheath. After X-ray imaging for determining the vertebral levels of cervical vagus nerve branching, samples were removed to confirm histologically the nerve and to calculate cervical vagus nerve diameters and cross-sections. RESULTS: Cervical vagus nerve branching was observed in 29% of all cases (26% unilaterally, 3% bilaterally) and proven histologically in all cases. Right-sided branching (22%) was more common than left-sided branching (12%) and occurred on the level of the fourth and fifth vertebra on the left and on the level of the second to fifth vertebra on the right side. Vagus nerves without branching were significantly larger than vagus nerves with branches, concerning their diameters (4.79 mm vs. 3.78 mm) and cross-sections (7.24 mm2 vs. 5.28 mm2). DISCUSSION: Cervical vagus nerve branching is considerably more frequent than described previously. The side-dependent differences of vagus nerve branching may be linked to the asymmetric effects of the vagus nerve. Cervical vagus nerve branching should be taken into account when identifying main trunk of the vagus nerve for implanting electrodes to minimize potential side effects or lacking therapeutic benefits of vagus nerve stimulation.

Endostatin/collagen XVIII--an inhibitor of angiogenesis--is expressed in cartilage and fibrocartilage.

UNLABELLED: Aim of the study was to get a deeper insight in the mechanisms regulating avascularity of cartilaginious tissues. In the center of our interest was the expression of the anti-angiogenic fragment of collagen XVIII and its potency to inhibit angiogenesis. We observed a strong endostatin/collagen XVIII production in articular and fibrocartilage and an inhibitory potency concerning the VEGF-signalling pathway. INTRODUCTION: Cartilaginous tissue is mainly avascular and shows a limited intrinsic capacity for healing. Aim of this study was to investigate the expression of the antiangiogenic peptide endostatin/collagen XVIII in cartilage and fibrocartilage. RESULTS: In fetal epiphyseal cartilage of humans high endostatin/collagen XVIII levels could be detected by ELISA whereas significantly lower levels were found in articular cartilage of adults. In the fibrocartilaginous tissue of the menisci, there was no significant difference in the endostatin/collagen XVIII concentrations between samples of fetuses and adults. But in the menisci of adults, endostatin/collagen XVIII concentrations were higher in the internal avascular two thirds of the meniscus whereas in the fetal menisci higher endostatin/collagen XVIII concentrations were found in the external third. Endostatin/collagen XVIII immunostaining of rat articular cartilage shows that endostatin/collagen XVIII downregulation starts soon after birth. In fetal cartilage and fibrocartilage of rats and humans, endostatin/collagen XVIII could be immunostained in the extracellular matrix and in the pericellular matrix of endothelial cells, fibrochondrocytes and chondrocytes. In adult cells, weak endostatin/collagen XVIII immunostaining was restricted to the pericellular matrix of fibrochondrocytes and chondrocytes. The detection of endostatin/collagen XVIII could be verified by in situ hybridization. Chondrocytes in vitro released measurable amounts of endostatin/collagen XVIII into culture supernatants. Stimulation of chondrocytes with EGF, as an example of a growth factor, or dexamethasone had no influence on endostatin/collagen XVIII expression. Endostatin inhibited VEGF-induced phosphorylation of MAPK in chondrocytes. CONCLUSIONS: The spatial and temporal expression of endostatin/collagen XVIII in cartilaginous tissue and its potency regarding inactivation of VEGF signalling suggests that this antiangiogenic factor is important not only for the development but also for the maintenance of avascular zones in cartilage and fibrocartilage. EXPERIMENTAL PROCEDURES: We analyzed the spatial and temporal expression of endostatin/collagen XVIII--an endogenous angiogenesis inhibiting factor--in cartilage and fibrocartilage of humans and rats by immunohistochemical and biochemical (ELISA) methods and by in situ hybridization. To elucidate possible factors responsible for the induction or suppression of endostatin/collagen XVIII in cartilaginous tissues, chondrocytes (cell line C28/I2) were exposed to EGF and dexamethason. To study the possible interaction of endostatin/collagen XVIII with angiogenic factors, the immortalized human chondrocytes (C28/I2) have been incubated with VEGF and the phosphorylation of the MAPK Erk 1/2 (extracellular-regulated kinases), a known signal transduction pathway for VEGF has been determined under the influence of endostatin.

VEGF expression in adult permanent thyroid cartilage: implications for lack of cartilage ossification.

Vascular endothelial growth factor (VEGF) has been shown to play an important role during endochondral bone formation in hypertrophic cartilage remodeling, ossification, and angiogenesis, but it is not expressed in normal adult articular cartilage. Thyroid cartilage undergoes only partial ossification beginning at the age of about 20. Because it never completely ossifies, we investigated a possible role of VEGF and its receptors (VEGFRs) as well as the angiogenetic inhibitor endostatin in this permanent cartilage. In analysis of cartilage samples from all specimens evaluated, VEGF121 and VEGF165 were identified as the only VEGF splice forms expressed. In addition to VEGF, VEGFR-2 (kinase domain region/fetal liver kinase 1), but not VEGFR-1 (fms-like tyrosine kinase 1), was detectable by RT-PCR in cartilage. However, VEGFR-2 expression was only detectable up to the age of 19 years. Deposition of VEGF and VEGFR was confirmed by immunohistochemistry. VEGF concentrations measured by ELISA in thyroid cartilage increased with age in males but decreased in females. Endostatin concentrations measured by ELISA in thyroid cartilage were three times lower than in articular cartilage and showed no change with age, either in females or males. VEGF was immunostained within the intra- and pericellular matrices of some but not all chondrocytes. Thus, apart from its production in hypertrophic chondrocytes of growth plates, VEGF is also produced in single chondrocytes of thyroid cartilage. The data allow us to speculate that thyroid cartilage persists in an embryological state until it has reached its final size. After reaching its final size at the end of the second decade, VEGFR-2 is downregulated and ossification starts in the posterior part of the thyroid cartilage, proceeding ventrally. Both proteins, VEGF121 and VEGF165, should contribute to this process. VEGF concentration is high and changes in an age-related and sex-specific manner. Therefore, we postulate that VEGF is at least one of the key factors that is important for the lifelong ossification in thyroid cartilage.

The role of vascular endothelial growth factor in glucocorticoid-induced bone loss: evaluation in a minipig model.

Vascular endothelial growth factor (VEGF) has been recently shown to play an important role during endochondral bone formation in hypertrophic cartilage remodeling, ossification, and angiogenesis. To our knowledge there are no previous studies investigating the role of VEGF in osteoporosis. We hypothesized that VEGF expression in bone would be reduced under glucocorticoid (GC) treatment and tested this in a minipig model. As part of a larger study, 17 primiparous sows (Göttingen minipig) were allocated to two experimental groups when they were 15 months old: a control group (n = 9) and a group receiving GC treatment for 15 months (n = 8). All animals were fed a semisynthetic diet until they were sacrificed. The GC group received prednisolone orally at a daily dose of 1 mg/kg body wt for 8 weeks and thereafter 0.5 mg/kg body wt. VEGF levels in lumbar vertebrae were measured by enzyme-linked immunosorbent assay (ELISA) and verified by Western blot analysis. VEGF and its receptors (VEGFR) were localized by immunohistochemistry. Expression of VEGF-mRNA was analyzed by reverse transcription-polymerase chain reaction. VEGF protein was quantified in supernatants of cultivated osteoblasts by ELISA. Spinal bone mineral density was assessed in vivo by quantitative computed tomography. Expression of cyclooxygenase-2 (COX-2) protein was investigated by immunohistochemistry. High VEGF concentrations were measured in normal lumbar vertebrae whereas VEGF concentrations were 60% lower (P < 0.0001) in GC-treated minipigs. VEGF levels were associated (r = 0.7) with rates of spinal trabecular bone loss, which differed significantly (P < 0.0013) between controls (-0.47 +/- 2.2% SEM) and GC-treated minipigs (-12.8 +/- 2.3% SEM). Osteoblasts were immunopositive for VEGF. VEGF receptors VEGFR-2 (KDR, flk-1) and VEGFR-1 (flt-1) could be immunostained on osteoclasts and osteoblasts. VEGF-mRNA and protein were detectable in the lumbar vertebrae of all animals. The expression of COX-2 protein was decreased in GC-treated animals. VEGF is produced in osteoblasts and its concentration is decreased in GC-treated animals as well as in osteoblasts exposed to GC. Since reductions in VEGF concentrations correlate with parallel measurement of bone mineral density in GC-treated minipigs we hypothesize that VEGF may be an important modulating factor for bone remodeling, specifically in GC-induced osteoporosis. GC inhibit COX-2 and hence prostaglandin E2 (PGE2) production. Since PGE2 is able to increase VEGF synthesis, this may be the link between GC and VEGF decrease.

The cavernous body of the human efferent tear ducts contributes to regulation of tear outflow.

PURPOSE: To test the hypothesis that the surrounding vascular plexus of the lacrimal sac and the nasolacrimal duct contributes to the regulation of tear outflow. METHODS: Experiments in 30 probands aged between 15 and 37 years were performed in both nasolacrimal systems of each subject by observing with an endoscope the transit time of an applied tear drop containing fluorescein dye until its entry into the inferior meatus of the nose. Four different experiments were performed to determine the median transit time under normal conditions and the influence on transit time of a decongestant drug, a foreign body on the ocular surface, and a decongestant drug applied together with a foreign body on the ocular surface. Comparisons were made between the right and left nasolacrimal system, in males and females, eyeglass wearers and non-eyeglass wearers, and the different experiments and the results statistically analyzed. RESULTS: The tear transit time was independent of side (right or left), gender, or eyeglass wear. It showed great individual variability. Application of a decongestant drug or placement of a foreign body on the ocular surface both prolonged the dye transit time significantly. Application of a decongestant drug simultaneously with placement of a foreign body shortened the dye transit time significantly compared with the effect of the decongestant drug alone but revealed no significant difference compared with application of a foreign body alone. CONCLUSIONS: The cavernous body of the lacrimal sac and nasolacrimal duct plays an important role in the physiology of tear outflow regulation. It is subject to autonomic control and is integrated into a complex neuronal reflex feedback mechanism starting with the dense innervation of the cornea. Moreover, its function can be pharmacologically influenced.

Animal model for the absorption of lipophilic substances from tear fluid by the epithelium of the nasolacrimal ducts.

PURPOSE: To compare the nasolacrimal tissues of several species to see how closely they resemble the human and to measure nasolacrimal absorption of a substance, to show that an absorption pathway exists for substances placed in the external eye, other than directly through the cornea or conjunctiva. METHODS: The nasolacrimal systems of six different vertebrates were investigated by light microscopy to find a species with a nasolacrimal system comparable to that of humans, for use in absorption experiments. In addition to primates, rabbits were revealed by histology to have a lacrimal system closely comparable to that of humans. The rabbit lacrimal system had a stratified epithelium consisting of two layers. Subepithelially, the lamina propria was composed of two strata: loose connective tissue containing elastic fibers and lymphatic cells and a rich venous plexus comparable to a cavernous body. Rabbits were therefore chosen for the absorption experiments. (3)H-cortisol was dropped into the eyes of female rabbits. After 21, 43, or 146 minutes, the rabbits were killed, the blood collected, and the nasolacrimal systems prepared and embedded for histologic examination. Serum was obtained from the clotted blood, and radioactivity was counted. Autoradiographs of sections of rabbit nasolacrimal duct were also prepared. RESULTS: Uptake of radioactivity into the serum was high and increased with time. After 21 minutes, maximum incorporation of the applied radioactivity into the blood the level was 7.1%; after 43 minutes, 12.4%; and after 146 minutes, 15.5%. Transport of radioactivity was visualized in autoradiographs of rabbit nasolacrimal systems. CONCLUSIONS: (3)H-cortisol is incorporated from the nasolacrimal ducts into the blood of rabbits. The comparable morphology of rabbits and humans suggests that absorption of cortisol would also take place in humans. Future investigations of the nasolacrimal passage are needed to understand whether absorption of normal tear fluid components in the nasolacrimal ducts is a physiological function that also plays a role in pathologic conditions such as dry eye. The similarities between rabbit and human nasolacrimal ducts support the use of the rabbit for such studies.

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). In fetal tendons high endostatin levels could be quantified by ELISA whereas low levels were found in adult tissue. In fetal tendons endostatin could also be immunostained in endothelial cells but mainly in fibroblasts. In adult Achilles tendons endostatin immunostaining was restricted to endothelial cells. In the tibialis posterior tendon--as an example for "wrap around"--endostatin immunostaining remained positive in the fibrocartilage adjacent to the medial malleolus. Fibrochondrocytes of the type II collagen positive fibrocartilage were intensively stained with the endostatin antibody. Factor VIII immunostaining showed that this region was largely avascular. Monolayer cultures of tendon cells released measurable amounts of endostatin into their culture supernatants. Application of intermittent hydrostatic pressure increased endostatin expression significantly. The conditioned media of tendon fibroblasts cultivated under intermittent hydrostatic pressure inhibited the proliferation of HUVEC in a dose dependent way. The spatial expression of endostatin in adult gliding tendons suggests that mechanical factors are involved in the regulation of this anti-angiogenic factor. In accordance, tendon cells exposed to intermittent hydrostatic pressure inhibit endothelial cell proliferation via humoral factors and produce endostatin. These findings support the view that the development and maintenance of avascular zones in tendons might be caused by a mechanically induced upregulation of anti-angiogenic factors.

Angiogenesis in fetal tendon development: spatial and temporal expression of the angiogenic peptide vascular endothelial cell growth factor.

In traction tendons, whose line of action corresponds to that of the muscle, few blood vessels are uniformly distributed within the tendon tissue. In gliding tendons, which change their direction of pull, an avascular zone is normally found in the region where the tendon wraps around the pulley. This avascular fibrocartilaginous gliding zone is predisposed for degenerative changes and spontaneous rupture. Since factors regulating angiogenesis in tendons are largely unknown, we analyzed the expression of the vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 (flt-1) and VEGFR-2 (KDR) in human fetal and adult tendon tissue by immunohistochemical, biochemical, and molecular biology methods. In order to elucidate whether mechanical stress might influence VEGF expression in tendon tissue we loaded primary cultures of rat tenocytes with intermittent hydrostatic pressure in a special cell culture chamber (amplitude: 0.2 Mpa, frequency: 0.1 Hz, time period: 5 h/day) and measured VEGF expression using ELISA. In fetal tendons high VEGF levels could be quantified by ELISA, whereas negligible ones were found in adult tissue. VEGF could be immunostained in tenocytes and endothelial cells. In the tibialis posterior tendon - as an example for a gliding tendon - VEGF immunostaining decreased in the gliding zone adjacent to the bony hypomochlion between week 20 and week 24 after gestation. This region remained largely avascular during the fetal period. In the peritendineum and in regions proximally and distally of the gliding zone immunostaining for VEGF was positive and factor VIII-positive microvessels could be detected. In these vessels, the VEGFR-1 (flt-1) and the VGEFR-2 could also be visualized. Reverse transcription-polymerase chain reaction (RT-PCR) confirmed the results regarding VEGF expression and showed further that the splice variants VEGF(121) and VEGF(165) are expressed exclusively during angiogenesis in fetal tendons. Monolayer cultures of tendon cells released measurable amounts of VEGF. Application of intermittent hydrostatic pressure decreased VEGF expression significantly. Thus, the angiogenic peptide VEGF is present in human fetal tendons, which are exposed to traction, but not in the avascular zone of gliding tendons, which are predominantly exposed to compressive and shearing forces. These findings support the view that the development of avascular zones in tendons might be caused by a mechanically induced downreglation of VEGF expression.

The human false vocal folds -- an analysis of antimicrobial defense mechanisms.

Inflammatory processes often lead to pathologic changes in the area of the larynx. A moistening function of the false vocal folds has been described frequently. Up to now we have little knowledge of the role of the false vocal folds in protection against pathogenic agents. The present study analyzes the structures of the false vocal folds in their relations to antimicrobial defense mechanisms. Investigations were performed on false vocal folds of larynges from 34 cadavers using histologic, histochemical and immunohistochemical methods. Seromucous glands, together with epithelial and goblet cells of the folds, synthesize a complex mucus layer. In all of the investigated samples this layer contains carbohydrates including N-acetyl-glucosamine, N-acetyl-galactosamine, galactose, mannose, fucose, and sialic acids. Furthermore, antimicrobial peptides like lactoferrin, lysozyme, alpha and beta defensins are also found in these structures. IgA, produced by plasma cells in the false vocal folds, is frequently integrated in the secretory product. Synthesized mucins, antimicrobial peptides and immunoglobulins form a specialized protective substance that is secreted mainly at the true vocal folds. Here the layer functions to lubricate the true vocal folds, resulting in positive functional consequences during vocal production. Moreover, together with immunocompetent cells, the protective layer seems to play a major role in antigen defense and prevents invasion of pathogenic agents.

Programmable cells of monocytic origin (PCMO): a source of peripheral blood stem cells that generate collagen type II-producing chondrocytes.

The focus of this study was a new adult pluripotent cell derived from human peripheral blood monocytes identified as a "programmable cell of monocytic origin" (PCMO). In contrast to bone marrow-derived stem cells, these cells can be harvested from peripheral venous blood without aspiration of the bone marrow and have multilineage potential comparable to that of mesenchymal stem cells (MSC). The aim of this study was to evaluate the potential of PCMOs to differentiate into collagen type II-producing chondrocytes using various extrinsic cues (TGFbeta-1, IGF-1, BMP-2, and BMP-7). Collagen type I and II proteins were localized using immunohistochemistry and quantified by enzyme-linked immunosorbent assays (ELISA). The shape of the differentiating PCMOs was monitored with electron microscopy. Collagen type I and II messenger RNA expression was analyzed using real-time reverse transcriptase-polymerase chain reaction (RT PCR) and regular RT PCR. Immunohistochemistry revealed a strong accumulation of collagen type II after a 6-week incubation period with BMP-2, BMP-7, TGF-beta, IGF-I, and TGF-beta, and IGF-1. Collagen type I was only mildly induced by the applied stimulants. Electron microscopy findings showed a shift from a monocyte-like structure to a chondrocyte-like structure after 2 weeks of stimulation. Stimulation of PCMOs with BMP-2, BMP-7, TGF-beta, IGF-I, and TGF-beta, and IGF-1 induced a chondrogenic differentiation with continuous expression of collagen type II mRNA and protein over several weeks time. Collagen type I and II expression in undifferentiated PCMOs or in control cells incubated without any stimulant was not detected. PCMOs have the potential to differentiate into collagen type II synthesizing chondrocytes. The ability to reprogram and differentiate PCMOs from peripheral blood into sizable quantities might enable their clinical application in cartilage repair after mechanical injury or in cases of osteoarthritis.

Human beta-defensin 3 mediates tissue remodeling processes in articular cartilage by increasing levels of metalloproteinases and reducing levels of their endogenous inhibitors.

OBJECTIVE: Beta-defensins are broad-spectrum antimicrobial peptides (APs) that are components of innate immunity. Recent investigations showed the induction of beta-defensins in synovial membranes of osteoarthritic (OA) joints and suggested that they have functions other than the ability to kill microbes. As a result of these findings, we undertook this study to investigate the production of human beta-defensin 3 (HBD-3) in OA cartilage and to determine its influence on chondrocyte function. METHODS: Healthy and OA cartilage were assessed for HBD-3 expression by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. HBD-3 expression in C28/I2 chondrocytes after administration of tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1) was determined by real-time RT-PCR and immunodot blot. Enzyme-linked immunosorbent assay experiments were used to study the effects of HBD-3 in cultured articular chondrocytes and in healthy and OA cartilage discs. Immunohistochemical analyses were performed to study the expression of mouse beta-defensins (MBDs) in OA cartilage of STR/Ort mice. RESULTS: HBD-3 was induced in OA cartilage without bacterial challenge. Cytokines involved in the pathogenesis of OA, namely, TNFalpha and IL-1, were strong inducers of HBD-3 in cultured chondrocytes. Application of the recombinant HBD-3 protein to cultured chondrocytes and cartilage discs resulted in increased production of cartilage-degrading matrix metalloproteinases and in down-regulation of their endogenous regulators, tissue inhibitors of metalloproteinases 1 and 2. Furthermore, STR/Ort mice, which are genetically predisposed to develop OA-like lesions in the knee joint, demonstrated an increased expression of MBDs 3 and 4 in cartilage compared with that in healthy animals. CONCLUSION: These findings widen our knowledge of the functional spectrum of APs and demonstrate that HBD-3 is a multifunctional AP with the ability to link host defense mechanisms and inflammation with tissue-remodeling processes in articular cartilage. Moreover, our data suggest that HBD-3 is an additional factor in the pathogenesis of OA.

Quantitative assessment of blood vessels of the human Achilles tendon: an immunohistochemical cadaver study.

BACKGROUND: The pathogenesis of Achilles tendon rupture remains unclear, but vascular patterns may play an important role. Hypoxia is considered to be an important factor in the aetiology of tendon degeneration. METHODS: Statements from the literature regarding the vascularization of the Achilles tendon are controversial. We determined the vascular density of the Achilles tendon using a new method involving antibodies against laminin, a component of the basement membrane. RESULTS: The blood supply of the Achilles tendon mainly arose from the anterior paratenon of the tendon from which vessels run into the tendon. The proximal part of the tendon was supplied by a recurrent branch of the posterior tibial artery, while the distal part of the tendon was vascularized by the rete arteriosum calcaneare, supplied by the fibular and posterior tibial arteries. An avascular area could be seen close to the insertion of the tendon to the calcaneus. Three regions with different vascular density could be determined in the Achilles tendon. The distal part of the Achilles tendon had a vascular density of 56.6 vessels/cm(2). In the middle part of the tendon, the vascular density was much lower: 28.2 vessels/cm(2). The proximal part of the tendon had a vascular density of 73.4 vessels/cm(2). CONCLUSION: The reduced vascularization in the tendon waist may be a predisposing factor for degeneration and spontaneous rupture of the human Achilles tendon.