Bernhard Tillmann

The role of vasculature and angiogenesis for the pathogenesis of degenerative tendons disease.

More than 100 years ago Wilhelm Roux (1895) introduced the term “functional adaptation to anatomy and physiology”. Compared with other organ systems the functional adaptation processes are best identifiable in the locomotor system, like for example in the two types of tendons: traction and gliding tendons. Traction tendons are tendons where the direction of pull is in line with the direction of the muscle (e.g. Achilles tendon). Gliding tendons (e.g. tibialis posterior tendon) change direction by turning around a bony or fibrous hypomochlion. In this region the tendon is subjected to intermittent compressive and shear forces and the extracellular matrix consists of avascular fibrocartilage. Avascularity is considered to be a key factor for the etiology of degenerative tendon disease. The repair capability after repetitive microtrauma is strongly compromised in avascular tissue of gliding tendons. Reduced vascularity is not a specific feature of gliding tendons; several studies have shown that the number and size of blood vessels are largely shortened in the waist of the Achilles tendon. However, histological biopsies from degenerated Achilles tendons and Doppler flow examinations revealed a high blood vessel density in patients with degenerative tendon disease.

The splice variants VEGF121 and VEGF189 of the angiogenic peptide vascular endothelial growth factor are expressed in osteoarthritic cartilage

OBJECTIVE Vascular endothelial growth factor (VEGF) has recently 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 cartilage. Since genes expressed during development often reappear in the disease state, we investigated whether VEGF and its receptors (VEGFRs) are expressed in osteoarthritic (OA) cartilage. METHODS VEGF production in OA cartilage from the tibial plateau was measured by enzyme-linked immunosorbent assay. Deposition of VEGF and VEGFR was determined by immunohistochemistry. Expression of messenger RNA for the different VEGF splice forms and for VEGFR was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS Increased VEGF concentrations were measured in OA cartilage from the tibial plateau, while VEGF was almost undetectable in normal cartilage but could be immunostained within the intracellular and pericellular matrices of OA chondrocytes. In analyses of cartilage samples from all 10 OA patients evaluated, VEGF121 and VEGF189 were identified as the only VEGF splice forms expressed. RT-PCR and immunohistochemistry for VEGF in normal hyaline cartilage yielded negative findings. In addition to VEGF, VEGFR-2 (kinase domain region/fetal liver kinase 1), but not VEGFR-1 (fms-like tyrosine kinase 1), could be detected by RT-PCR in OA cartilage and immunostained on OA chondrocytes. CONCLUSION Apart from its production in hypertrophic chondrocytes, VEGF is also produced in chondrocytes of OA cartilage. While the splice variant VEGF189 binds to extracellular matrix proteoglycans, VEGF121 is diffused freely. Both proteins should contribute to the inflammatory process by autocrine/paracrine stimulation of chondrocytes, chemotaxis of macrophages, and promotion of angiogenesis.

Vascular endothelial growth factor (VEGF) induces matrix metalloproteinase expression in immortalized chondrocytes

VEGF (vascular endothelial growth factor), an important angiogenesis factor, appears also to be involved in inflammatory processes. Recent studies have shown that VEGF and its receptors (VEGFR) are expressed on osteoarthritic, but not on normal adult, chondrocytes. To elucidate possible functions of VEGF in osteoarthritic cartilage, the effects of VEGF were studied on immortalized human chondrocytes. Activated matrix metalloproteinase (MMP)‐1, MMP‐3, MMP‐13, tissue inhibitor of metalloproteinases (TIMP)‐1, TIMP‐2, interleukin (IL)‐1β, IL‐6, and tumour necrosis factor‐α (TNF‐α) were measured in culture supernatants by enzyme‐linked immunosorbent assays, nitric oxide with the Griess reagent, and cell proliferation by [3H]thymidine incorporation. VEGFR‐2 mRNA was quantified by real‐time reverse transcription‐polymerase chain reaction and the protein was identified by immuno‐gold electron microscopy. Intracellular signal transduction effects were determined by western blots and electrophoretic mobility shift assays. The chondrocyte cell lines C28/I2, C20/A4, and T/C28a2/a4 expressed functionally active VEGFR‐2. VEGF stimulation induced receptor phosphorylation, activation of the mitogen‐activated protein kinases ERK 1/2, and long‐lasting activation of the transcription factor AP‐1 (activator protein‐1). VEGF increased secreted MMP‐1, MMP‐3, and especially MMP‐13, which could be effectively reduced by an inhibitor of VEGFR‐2 kinase activity. Interestingly, VEGF diminished the expression of TIMP‐1 and especially TIMP‐2. Under hypoxic conditions, as occur in cartilage, the reduction in TIMP levels was even greater. Furthermore, VEGF induced IL‐1β, IL‐6, TNF‐α, and nitric oxide expression to a small extent and stimulated the proliferation of immortalized chondrocytes. These findings indicate that VEGF is an autocrine stimulator of immortalized chondrocytes that mediates mainly destructive processes in osteoarthritis. Copyright

STRUCTURE AND VASCULARIZATION OF THE CRUCIATE LIGAMENTS OF THE HUMAN KNEE JOINT

The structure and vascularization of the human anterior and posterior cruciate ligament were investigated by light microscopy, transmission electron microscopy, injection techniques and by immunohistochemistry. The major part of the anterior and posterior cruciate ligament is composed of bundles of type I collagen. Type III collagen-positive fibrils separate the bundles. The major cell type is the elongated fibroblast, lying solitarily between the parallel collagen fibrils. The histologic structure of the cruciate ligaments is not homogeneous. In both ligaments there is a zone where the tissue resembles fibrocartilage. In the anterior cruciate ligament the fibrocartilaginous zone is located 5–10 mm proximal of the tibial ligament insertion in the anterior portion of the ligament. In the posterior cruciate ligament the fibrocartilage is located in the central part of the middle third. Within those zones the cells are arranged in columns and the cell shape is round to ovoid. Transmission electron microscopy reveals typical features of chondrocytes. The chondrocytes are surrounded by a felt-like pericellular matrix, a high content of cellular organelles and short processes on the cell surface. The pericellular collagen is positive for type II collagen. The major blood supply of the cruciate ligaments arises from the middle geniculate artery. The distal part of both cruciate ligaments is vascularized by branches of the lateral and medial inferior geniculate artery. Both ligaments are surrounded by a synovial fold where the terminal branches of the middle and inferior arteries form a periligamentous network. From the synovial sheath blood vessels penetrate the ligament in a horizontal direction and anastomose with a longitudinally orientated intraligamentous vascular network. The density of blood vessels within the ligaments is not homogeneous. In the anterior cruciate ligament an avascular zone is located within the fibrocartilage of the anterior part where the ligament faces the anterior rim of the intercondylar fossa. The fibrocartilaginous zone of the middle third of the posterior cruciate ligament is also avascular. According to Pauwel’s theory of the ”causal histogenesis” (1960) the stimulus for the development of fibrocartilage within dense connective tissue is shearing and compressive stress. In the anterior cruciate ligament this biomechanical situation may occur when the ligament impinges on the anterior rim of the intercondylar fossa when the knee is fully extended. Compressive and shearing stress in the center of the middle third of the posterior cruciate ligament may result from twisting of the fiber bundles.

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.

Structure and vascularization of the acetabular labrum with regard to the pathogenesis and healing of labral lesions

IntroductionThe aim of this study is to examine the structure and vascularization of the acetabular labrum with regard to the pathogenesis and healing of labral tears.Materials and methodsThe labral tissue was characterized immunohistochemically and under light microscopy; the collagen fibril texture was demonstrated by scanning electron microscopy after temporary staggered maceration of the tissue; and the vascularization of the acetabular labrum was studied immunohistochemically using antibodies against laminin.ResultsThe peripheral aspect of the acetabular labrum consists of dense connective tissue. The internal layer consists of type II collagen-positive fibrocartilage. Scanning electron microscopy revealed three distinct layers in the acetabular labrum: (1) the articular surface was covered by a meshwork of thin fibrils; (2) beneath the superficial network, there is a layer of lamella-like collagen fibrils; (3) the majority of the collagen fibrils are oriented in a circular manner. Blood vessels enter the labrum from the adjacent joint capsule. The distribution of vessels within the labrum is not homogenous. Blood vessels can be detected only in the peripheral one-third of the labrum. The internal part is avascular.ConclusionThe result of this study demonstrates that the structure of the acetabular labrum is highly significant for the direction of traumatic and dysplastic labral lesions. The biomechanical analysis of the structure suggests that the labrum is stressed by compressive load. Therefore, excision or removal of the labrum may alter physiological functions such as enhancing joint stability and load distribution. The vascular pattern identified should encourage surgeons to develop repair strategies of peripheral labral tears to maintain its functions in the hip.

Curving and looping of the internal carotid artery in relation to the pharynx: frequency, embryology and clinical implications

Variations of the course of the internal carotid artery in the parapharyngeal space and their frequency were studied in order to determine possible risks for acute haemorrhage during pharyngeal surgery and traumatic events, as well as their possible relevance to cerebrovascular disease. The course of the internal carotid artery showed no curvature in 191 cases, but in 74 cases it had a medial, lateral or ventrocaudal curve, and 17 preparations showed kinking (12) or coiling (5) out of a total of 265 dissected carotid sheaths and 17 corrosion vascular casts. In 6 cases of kinking and 2 of coiling, the internal carotid artery was located in direct contact with the tonsillar fossa. No significant sex differences were found. Variations of the internal carotid artery leading to direct contact with the pharyngeal wall are likely to be of great clinical relevance in view of the large number of routine procedures performed. Whereas coiling is ascribed to embryological causes, curving is related to ageing and kinking is thought to be exacerbated by arteriosclerosis or fibromuscular dysplasia with advancing age and may therefore be of significance in relation to the occurrence of cerebrovascular symptoms.

The angiogenic peptide vascular endothelial growth factor is expressed in foetal and ruptured tendons

Abstract The Achilles tendon is one of the most common sites of injury and rupture. Evidence suggests that local vascularisation is involved in this aetiology. We investigated the expression of one important angiogenic factor, the vascular endothelial growth factor (VEGF), in normal and pathologic human Achilles tendons using immunohistochemical, biochemical, molecular and cell biology methods. VEGF could be immunostained in tenocytes of ruptured and foetal Achilles tendons, but not in normal adult ones. In microvessels, the VEGF receptor VEGFR-1 (flt-1) could be visualised as well. High VEGF levels were measured in homogenates from ruptured adult, lower ones in foetal and negligible concentrations in normal adult Achilles tendons using enzyme-linked immunoassay (ELISA) and Western blot experiments. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that the splice variants VEGF121 and VEGF165 are exclusively expressed. In tenocytes cultivated from newborn rat Achilles tendons, hypoxia or epidermal growth factor (EGF) raised VEGF production moderately whereas their combination resulted in a strong, synergistic induction. These results prove the presence of an angiogenic peptide and vascularisation in ruptured and foetal tendons and support the view that microtrauma or degeneration in the Achilles tendon precedes its rupture.

Blood supply of the tibialis anterior tendon.

Abstract Injection techniques and immunohistochemical methods (antibodies against laminin) were performed to uncover the vascular pattern of the human tibialis anterior tendon with regard to spontaneous rupture of this tendon. Proximally, the blood supply of the tibialis anterior tendon mainly arises from the anterior tibial artery. Distally, the tendon is supplied by branches of the medial tarsal artery. Blood vessels enter the peritenon via vinculae from the posterior side. From the peritenon, the blood vessels penetrate the tendon and anastomose with a longitudinally orientated intratendinous network. Compared with the surrounding peritenon, the number of vessels in the tendon substance is greatly reduced. The distribution of blood vessels within the anterior tibial tendon is not homogenous. The posterior part of the tendon has a complete vascular network that extends from the musculotendinous junction to the insertion at the bone. In the anterior half of the tendon, there is an avascular zone between 45 and 67 mm in length. The location of the avascular zone correlates well with the location of the most frequent site of spontaneous rupture of the tibialis anterior tendon reported in the literature. Hypovascularity has to be considered as an etiological cofactor for spontaneous rupture of the tibialis anterior tendon.

Blood supply of the peroneal tendons: Injection and immunohistochemical studies of cadaver tendons

We studied the vascular pattern of human peroneal tendons with injection techniques and immunohistochemically by using antibodies against laminin. The main blood supply arises from the peroneal artery. The distal part of the peroneus longus tendon is supplied by branches of the medial tarsal artery. Blood vessels enter the peritenon of both tendons via a mesotenon from the posterior aspect. From the peritenon, the blood vessels penetrate the peroneus brevis and peroneus longus tendons and anastomose with a longitudinally-oriented intratendinous network. The amount of vessels in the tendon substance is consistently less than in the surrounding peritenon. The distribution of blood vessels in the peroneal tendons is not homogeneous. In the region where the peroneus brevis tendon passes through the fibular groove, the longitudinally-oriented intratendinous vascular network is interrupted and the tendon is almost avascular. In this region, the tendon is squeezed between the peroneus longus tendon and the bony slide bearing of the lateral malleolus. The peroneus longus tendon has two avascular zones. In the region where the peroneus longus tendon curves around the lateral malleolus and the peroneal trochlea of the calcaneus, the anterior part of the tendon which is directed towards the pulley is avascular. A second avascular zone is located more distally in the region where the tendon changes direction and wraps around the cuboid.