The mechanisms of inflammation in gout and pseudogout (CPP-induced arthritis).

Recent advances have stimulated new interest in the area of crystal arthritis, as microcrystals can be considered to be endogenous "danger signals" and are potent stimulators of immune as well as non-immune cells. The best known microcrystals include urate (MSU), and calcium pyrophosphate (CPP) crystals, associated with gout and pseudogout, respectively. Acute inflammation is the hallmark of the acute tissue reaction to crystals in both gout and pseudogout. The mechanisms leading to joint inflammation in these diseases involve first crystal formation and subsequent coating with serum proteins. Crystals can then interact with plasma cell membrane, either directly or via membrane receptors, leading to NLRP3 activation, proteolytic cleavage and maturation of pro-interleukin-1ß (pro-IL1ß) and secretion of mature IL1ß. Once released, this cytokine orchestrates a series of events leading to endothelial cell activation and neutrophil recruitment. Ultimately, gout resolution involves several mechanisms including monocyte differentiation into macrophage, clearance of apoptotic neutrophils by macrophages, production of Transforming Growth Factor (TGF-ß) and modification of protein coating on the crystal surface. This review will examine these different steps.

Implication of dipeptidylpeptidase IV activity in human bronchial inflammation and in bronchoconstriction evaluated in anesthetized rabbits.

BACKGROUND: Decreased dipeptidylpeptidase IV (DPPIV) activity within the human nasal mucosa has previously been shown to contribute to the severity of chronic inflammatory rhinosinusitis. OBJECTIVE: To investigate and correlate the role of DPPIV activity with regard to bronchial inflammation. METHODS: DPPIV/CD26 activity/concentration was investigated in the bronchial tissue of human subjects suffering from chronic bronchial inflammation. In addition, the effect of a recombinant Aspergillus fumigatus DPPIV (fuDPPIV) was investigated on histamine-induced bronchoconstriction in anesthetized rabbits. RESULTS AND CONCLUSIONS: DPPIV/CD26 was present in submucosal seromucous glands, in leukocytes and to a very low degree in endothelial cells of human bronchi. DPPIV activity was correlated with tissue CD26 content measured by immunoassay. As previously reported for the nasal mucosa, DPPIV/CD26 activity was inversely correlated with the degree of airway inflammation. Systemic pretreatment with recombinant fuDPPIV markedly reduced the increase in histamine-induced airway resistance in rabbits. In conclusion, DPPIV activity modulates lower airway tone by degrading unknown peptidic substrates released by histamine in response to an allergen. Contrasting with our observations in the nose, this modulation is apparently not mediated via a neurokinin (NK1) receptor.

Arthritis is linked to local and systemic activation of coagulation and fibrinolysis pathways.

BACKGROUND: Activation of coagulation and fibrinolysis play a role in the pathophysiology of experimental arthritis. OBJECTIVE: To determine the extent of activation of the coagulation and fibrinolytic pathways in different joint diseases in humans and to ascertain the factors that may influence fibrin deposition within the joint. METHODS: Plasma from normal subjects (controls, n= 21) and plasma and synovial fluid samples from patients with rheumatoid arthritis (RA; n = 64), osteoarthritis (OA; n = 29), spondyloarthropathy (SpA; n = 22) and crystal arthritis (CA; n = 25) were analyzed for the levels of TF (tissue factor) and tissue factor pathway inhibitor (TFPI) activities, thrombin-antithrombin III (TAT) complexes, and F1 + 2 (thrombin fragment), fibrin d-dimer and thrombin-activated fibrinolysis inhibitor (TAFI) antigenic levels. The measurements were analyzed by pairwise correlation with each other as well as with standard parameters of inflammation [C-reactive protein (CRP), joint leukocyte count]. Inter-group comparisons were performed to look for disease-specific differences. RESULTS: Compared with healthy controls, patients with joint diseases had higher levels of TAT, F1 + 2 and d-dimers in their plasma. In the synovial fluid, TF activity, TAT, d-dimers, and TAFI were significantly higher in inflammatory arthritides than in OA. The levels were highest in RA patients. In the plasma, TF activity was correlated with TAT and d-dimer levels with CRP, TFPI, and TAT. In the synovial fluid, TF activity correlated with plasma CRP levels, synovial fluid leukocyte count, and synovial TAT and TAFI levels. In addition, synovial d-dimers correlated with CRP, and synovial TAFI levels were correlated with synovial F1 + 2 and TAT. CONCLUSIONS: Activation of the coagulation and fibrinolytic cascades in the joint and in the circulation is evident in both inflammatory and degenerative joint diseases. Within the joint, inflammatory mechanisms leading to TF-mediated activation of the coagulation pathway and subsequent fibrin deposition is the most likely explanation for the observed findings. In the plasma, the link between inflammation (CRP increase) and TF activation is weak, and a non-TF-mediated mechanism of coagulation activation could explain these findings. RA is characterized by significantly higher levels of TAT in the synovial fluid and plasma than other arthritides. Although fibrinolytic activity is linked to inflammation, the increased amounts of TAFI in the joint, particularly in RA, may explain why fibrin formation is so prominent in this condition compared with other joint diseases.

PPARgamma expression in normal human placenta, hydatidiform mole and choriocarcinoma.

Peroxisome proliferator-activated receptor (PPAR) gamma belongs to a subclass of nuclear hormone receptors that execute their transcriptional functions as heterodimers with the retinoid X receptors (RXR). PPARgamma plays a pivotal role in cellular differentiation. This study investigated PPARgamma protein expression in normal human placentas, hydatidiform moles and choriocarcinoma, using immunohistochemical and Western blot analyses. In first trimester normal placenta, PPARgamma was mainly localized in the nuclei of the villous cytotrophoblastic cells, whereas at term it was mainly localized in the nuclei of the syncytiotrophoblast. Extravillous cytotrophoblast of cell islands and cell columns also showed nuclear PPARgamma immunostaining. A striking result was the altered expression patterns of PPARgamma in pathological tissues; PPARgamma showed a reduced immunostaining in the trophoblastic diseases. In hydatidiform moles, PPARgamma was mainly localized in the nuclei of the trophoblastic collections of the pathological villi and in the extravillous trophoblastic cells, whereas in the choriocarcinoma, only a few trophoblastic cells showed weak PPARgamma nuclear immunostaining. These findings suggest an involvement of PPARgamma in trophoblast differentiation during normal placental development. The down-regulation of PPARgamma expression in the gestational trophoblastic diseases analysed in this study provides a new insight into the progression of these pathologies.

Plasminogen activator inhibitor type-1 deficiency attenuates murine antigen-induced arthritis.

OBJECTIVE: To examine the role of plasminogen activator inhibitor type-1 (PAI-1), the major fibrinolytic inhibitor, in vivo during murine antigen-induced arthritis (AIA). METHODS: AIA was induced in PAI-1-deficient mice and control wild-type mice. Arthritis severity was evaluated by technetium 99m (99mTc) uptake in the knee joints and by histological scoring. Intra-articular fibrin deposition was examined by immunohistochemistry and synovial fibrinolysis quantitated by tissue D-dimer measurements and zymograms. RESULTS: Joint inflammation, quantitated by 99mTc uptake, was significantly reduced in PAI-1(-/-) mice on day 7 after arthritis onset (P<0.01). Likewise, synovial inflammation, evaluated by histological scoring, was significantly decreased in PAI-1-deficient mice on day 10 after arthritis onset (P<0.001). Articular cartilage damage was significantly decreased in PAI-1(-/-) mice, as shown by histological grading of safranin-O staining on day 10 after arthritis onset (P<0.005). Significantly decreased synovial accumulation of fibrin was observed by day 10 in arthritic joints of PAI-1(-/-) mice (P<0.005). Accordingly, the synovial tissue content of D-dimers, the specific fibrin degradation products generated by plasmin, were increased in PAI-1(-/-) mice (P<0.02). Finally, as expected, PA activity was increased in synovial tissues from PAI-1(-/-) mice, as shown by zymographic analysis. CONCLUSIONS: These results indicate that deficiency of PAI-1 results in increased synovial fibrinolysis, leading to reduced fibrin accumulation in arthritic joints and reduced severity of AIA.

Plasma apolipoprotein(a) co-deposits with fibrin in inflammatory arthritic joints.

Extravascular coagulation and diminished fibrinolysis are processes that contribute to the pathology of both inflammatory arthritis and atherosclerosis. We hypothesized that, given its homology with plasminogen, apolipoprotein (apo) (a), the distinctive glycoprotein of the atherogenic lipoprotein (Lp) (a), may be equally implicated in inflammatory arthritis. We detected the presence of apo(a) as part of Lp(a) in human arthritic synovial fluid. The abundance of apo(a) in synovial fluid rose in proportion to plasma apo(a) levels and was higher in inflammatory arthritides than in osteoarthritis. In addition, apo(a) immunoreactive material, but not apo(a) transcripts, was detected in inflammatory arthritic synovial tissues. These data indicated that synovial fluid apo(a) originates from circulating Lp(a) and that diffusion of Lp(a) through synovial tissue is facilitated in inflammatory types of arthritis. In synovial tissues, apo(a) co-localized with fibrin. These observations could be reproduced in a model of antigen-induced arthritis, using transgenic mice expressing human Lp(a). Although in this mouse model the presence of apo(a) did not change the severity of arthritis, the co-localization of apo(a) with fibrin in synovial tissue suggests that, in humans, apo(a) may modulate locally the fibrinolytic activity and may thus contribute to the persistence of intra-articular fibrin in inflammatory arthritis.

Accelerated nephrotoxic nephritis is exacerbated in C1q-deficient mice.

C1q deficiency strongly predisposes to the development of systemic lupus erythematosus in humans and mice. We used the model of accelerated nephrotoxic nephritis in C1q-deficient mice to explore the mechanisms behind these associations. C1q-deficient mice developed severe glomerular thrombosis within 4 days of induction of disease, whereas wild-type mice developed mild injury. These findings suggest that C1q protects from immune-mediated glomerular injury. This exacerbated thrombosis was also seen in mice triply deficient in C1q, factor B, and C2, excluding a major pathogenic role for the alternative pathway of complement in this phenomenon. However, these mice did not develop elevated creatinine levels. No exacerbation of accelerated nephrotoxic nephritis was observed in mice doubly deficient in factor B and C2, suggesting a protective role for C1q against renal inflammation that is proximal to C2 activation. There were increased murine IgG deposits, neutrophil numbers, and apoptotic cells in the glomeruli of C1q-deficient mice compared with wild-type mice. Renal expression of genes encoding procoagulant proteins was also enhanced in C1q-deficient mice. The increased IgG deposits and apoptotic cells in the glomeruli of C1q-deficient mice suggest that the exacerbation of disease may be due to a defect in the clearance of immune complexes and/or apoptotic cells from their kidneys.

Amelioration of collagen-induced arthritis by thrombin inhibition.

The deleterious role of fibrin deposition in arthritic joints prompted us to explore the effect of the thrombin inhibition on the course of collagen-induced arthritis (CIA) in the mouse. CIA was induced in male DBA/1J mice using native chicken type II collagen. The thrombin inhibitor polyethyleneglycol-hirudin (PEG-hirudin) was given for 16 days, starting 20 days after the first immunization (preventive treatment) or at the onset of clinical signs of arthritis (curative treatment). All the mice treated with PEG-hirudin had a significantly prolonged clotting time compared with control mice. PEG-hirudin, administered in a preventive way, led to significantly reduced incidence and severity of CIA during most of the treatment period, as assessed by clinical scoring. Accordingly, histological features showed a significant diminution of synovial hyperplasia in PEG-hirudin-treated mice compared with untreated mice. There was also a significant downmodulation of the synovial proinflammatory IL-1beta and IL-12p35 cytokine mRNAs in treated mice. Intra-articular fibrin, evaluated by immunohistochemistry, was significantly reduced in treated mice compared with control mice and correlated with both clinical and histological scorings. Most importantly, once arthritis was established, PEG-hirudin also showed a curative effect. In conclusion, PEG-hirudin can both prevent the onset of CIA in a dose-dependent manner and ameliorate established arthritis, suggesting that thrombin inhibition may offer a new therapeutic approach in arthritis.

Enhanced expression of genes involved in coagulation and fibrinolysis in murine arthritis.

STATEMENT OF FINDINGS: We have analyzed the pattern of procoagulant and fibrinolytic gene expression in affected joints during the course of arthritis in two murine models. In both models, we found an increased expression of tissue factor, tissue factor pathway inhibitor, urokinase plasminogen activator, and plasminogen activator inhibitor 1, as well as thrombin receptor. The observed pattern of gene expression tended to favor procoagulant activity, and this pattern was confirmed by functional assays. These alterations would account for persistence of fibrin within the inflamed joint, as is seen in rheumatoid arthritis.

Effect of thrombin inhibition on synovial inflammation in antigen induced arthritis.

OBJECTIVE: To determine the effect of the thrombin inhibitor, hirudin, on the pathogenesis of murine antigen induced arthritis (AIA). METHODS: AIA was induced by intra-articular injection of methylated bovine serum albumin in the knee joints of previously immunised mice. Hirudin (injected subcutaneously 3 x 200 microg/mouse/day) was given over 13 days, starting three days before arthritis onset, and its anticoagulant effect monitored by clotting times. Arthritis severity was evaluated by technetium-99m ((99m)Tc) uptake in the knee joints and by histological scoring. In addition, intra-articular fibrin deposition was examined by immunohistochemistry, and synovial cytokine mRNA expression measured by RNase protection. RESULTS: Joint inflammation, measured by (99m)Tc uptake, was significantly reduced in hirudin treated mice at days 7 and 10 after arthritis onset. Histologically, synovial thickness was markedly decreased in hirudin treated mice compared with untreated ones. By contrast, no difference in articular cartilage proteoglycan content was found between both groups. Intra-articular fibrin deposition and synovial interleukin 1beta mRNA levels, were slightly reduced ( approximately 20%) in arthritic joints from hirudin treated mice compared with untreated ones at day 10 of AIA. CONCLUSION: Hirudin reduces joint inflammation associated with AIA by fibrin-dependent and independent mechanisms.

Effect of interleukin 17 on proteoglycan degradation in murine knee joints.

OBJECTIVE: To evaluate the effect of murine interleukin 17 (IL17) on cartilage catabolism and joint inflammation by direct intra-articular injection of the cytokine into murine knee joints. METHODS: Knees of normal C57 Bl mice were injected once or repeatedly with recombinant IL17 or IL1beta. Inflammation was estimated by technetium-99m pertechnetate ((99)Tc) uptake and histological scoring of tissue sections. Proteoglycan depletion was evaluated by histological scoring of safranin O stained sections. Effects on proteoglycan synthesis were studied by (35)SO(4) incorporation. RESULTS: A single intra-articular injection of IL17 (10 ng/knee) produced effects very similar to those of IL1beta (10 ng/knee). No inflammation was detected at six or 24 hours by (99)Tc uptake. However, safranin O staining showed depletion of proteoglycan at 48 hours. Repeated injections of IL17 induced joint inflammation and cartilage proteoglycan depletion as shown by histological scoring. Unlike IL1beta, proteoglycan depletion induced by IL17 seemed to be the result of increased degradation only, as no suppression of (35)SO(4) incorporation was seen. CONCLUSION: These findings confirm, in vivo, the catabolic effects of IL17 on cartilage. IL17 is thus the first T cell cytokine showing a direct catabolic effect on cartilage in addition to stimulatory effects on macrophages and synoviocytes, making it a potentially important cytokine in the pathogenesis of arthritis.

Exacerbation of antigen-induced arthritis in urokinase-deficient mice.

In rheumatoid arthritis, synovial expression of urokinase (uPA) activity is greatly increased (Busso, N., V. Péclat, A. So, and A. -P. Sappino. 1997. Ann. Rheum. Dis. 56:550- 557). We report the same effect in murine antigen-induced arthritis. uPA-mediated plasminogen activation in arthritic joints may have deleterious effects via degradation of cartilage and bone matrix proteins as well as beneficial effects via fibrin degradation. We evaluated these contrasting effects in vivo by analyzing the phenotype of uPA-deficient (uPA-/-) and control mice during antigen-induced arthritis. Joint inflammation was comparable in both groups up to day 3 and subsequently declined in control mice, remaining significantly elevated in uPA-/- mice on days 10 and 30 after arthritis onset. Likewise, synovial thickness was markedly increased in uPA-deficient mice persisting for up to 2 mo, whereas it subsided in control animals. Bone erosion was exacerbated in uPA-/- mice on day 30. By contrast, no difference in articular cartilage proteoglycan content was found between both groups. Significantly increased accumulation of fibrin was observed by day 30 in arthritic joints of uPA-/- mice. We hypothesized that synovial fibrin deposition plays a role in joint inflammation. Accordingly, defibrinogenation of uPA-/- mice by ancrod significantly decreased the sustained joint inflammation. All the above observations were reproducible in plasminogen-deficient (Pln-/-) mice. In conclusion, synovial fibrin deposition plays a role as a nonimmunological mechanism which sustains chronic arthritis.

Plasminogen activation in synovial tissues: differences between normal, osteoarthritis, and rheumatoid arthritis joints.

OBJECTIVE: To analyse the functional activity of the plasminogen activators urokinase (uPA) and tissue type plasminogen activator (tPA) in human synovial membrane, and to compare the pattern of expression between normal, osteoarthritic, and rheumatoid synovium. The molecular mechanisms underlying differences in PA activities between normal and pathological synovial tissues have been further examined. METHODS: Synovial membranes from seven normal (N) subjects, 14 osteoarthritis (OA), and 10 rheumatoid arthritis (RA) patients were analysed for plasminogen activator activity by conventional zymography and in situ zymography on tissue sections. The tissue distribution of uPA, tPA, uPA receptor (uPAR), and plasminogen activator inhibitor type-1 (PAI-1) was studied by immunohistochemistry. uPA, tPA, uPAR, and PAI-1 mRNA values and mRNA distribution were assessed by northern blot and in situ hybridisations respectively. RESULTS: All normal and most OA synovial tissues expressed predominantly tPA catalysed proteolytic activity mainly associated to the synovial vasculature. In some OA, tPA activity was expressed together with variable amounts of uPA mediated activity. By contrast, most RA synovial tissues exhibited considerably increased uPA activity over the proliferative lining areas, while tPA activity was reduced when compared with N and OA synovial tissues. This increase in uPA activity was associated with increased levels of uPA antigen and its corresponding mRNA, which were localised over the synovial proliferative lining areas. In addition, in RA tissues, expression of the specific uPA receptor (uPAR) and of the plasminogen activator inhibitor-type 1

Urokinase and type I plasminogen activator inhibitor production by normal human hepatocytes: modulation by inflammatory agents.

We examined the effects of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor-alpha and transforming growth factor-beta) on the plasminogen activator system (urokinase, tissue-type plasminogen activator, type 1 plasminogen activator inhibitor) in primary cultures of human hepatocytes. We show that interleukin-1 beta and tumor necrosis factor-alpha increase urokinase-type plasminogen activator production, reinforcing the concept that increased urokinase production is associated with inflammatory processes. By contrast, the same agents (i.e., interleukin-1 beta and tumor necrosis factor-alpha) do not stimulate plasminogen activator inhibitor type 1 production. This latter observation rules out hepatocytes as a major cellular source of plasmatic plasminogen activator inhibitor type 1 during acute-phase-related responses. Among the inflammatory agents used, transforming growth factor-beta was found to be the most effective modulator of both urokinase-type plasminogen activator and plasminogen activator inhibitor type 1, inducing severalfold increases of activity of urokinase-type plasminogen activator, antigen and the corresponding mRNA and increasing plasminogen activator inhibitor type 1 antigen and mRNA levels. Urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 modulation by transforming growth factor-beta may play a critical role in hepatic pathophysiology.

Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells.

A human epithelial cell line, WISH, and a mouse cell line, LB6-uPAR, transfected with the human urokinase receptor (uPAR), both expressed high affinity uPAR but undetectable levels of urokinase (uPA). In two independent assays, binding of exogenous pro-uPA produced an up to threefold enhancement of migration. The migration was time and concentration dependent and did not involve extracellular proteolysis. This biologic response suggested that uPAR can trigger an intracellular signal. Since this receptor is a glycosyl-phosphatidylinositol-linked protein, we postulated that it must do so by interacting with other proteins, among which, by analogy to other systems, would be a kinase. To test this hypothesis, we carried out a solid phase capture of uPAR from WISH cell lysates using either antibodies against uPAR or pro-uPA adsorbed to plastic wells, followed by in vitro phosphorylation of the immobilized proteins. SDS-PAGE and autoradiography revealed two phosphorylated protein bands of 47 and 55 kD. Both proteins were phosphorylated on serine residues. Partial sequence of the two proteins showed a 100% homology to cytokeratin 18 (CK18) and 8 (CK8), respectively. A similar pattern of phosphorylation was obtained with lysates from A459 cells, a lung carcinoma, but not HL60, LB6-uPAR or HEp3 cell lysates, suggesting that the identified multiprotein uPAR-complex may be specific for simple epithelia. Moreover, immunocapture with antibody to another glycosyl-phosphatidylinositol-linked protein, CD55, which is highly expressed in WISH cells, was ineffective. The kinase was tentatively identified as protein kinase C, because it was inhibited by an analogue of staurosporine more specific for PKC and not by a PKA or tyrosine kinase inhibitors. The kinase was tentatively identified as PKC epsilon because of its resistance to PMA down-modulation, independence of Ca2+ for activity, and reaction with a specific anti-PKC epsilon antibody in Western blots. Cell fractionation into cytosolic and particulate fractions revealed that all four proteins, the kinase, uPAR, CK18, and CK8, were present in the particulate fraction. In vivo, CK8, and to a lesser degree CK18, were found to be phosphorylated on serine residues. Occupation of uPAR elicited a time-dependent increase in the phosphorylation intensity of CK8, a cell shape change and a redistribution of the cytokeratin filaments. These results strongly suggest that uPAR serves not only as an anchor for uPA but participates in a signal transduction pathway resulting in a pronounced biological response.

Tratamento com fertilizacao in vitro em retroejaculacao / Retroejaculation and treatment with in vitro fertilization - case report

Os autores descrevem o tratamento fertilizacao in vitro realizado com sucesso em um caso de infertilidade masculina por ejaculacao retrograda. Apos o preparo da bexiga urinaria, o meio HTF-Human TubalFluid foi injetado na bexiga, e a seguir solicitou-se ao paciente se masturbar e ter miccao natural em recipiente apropriado. A amostra coletada foi preparada, obtendo-se um semen com boa concentracao e motilidade. Com a estimulacao da ovulacao feita com o protocolo longo, utilizando GnRHa + hMG + hCG e aspiracao tranvaginal son controle ecografico obtiveram-se seis oocitos, dos quais quatro fertilizaram e foram transferidos ao utero. O suporte da fase lutea foi feito com progesterona oleosa e estradiol transdermico. No 14 dia pos-transferencia foi confirmada a gestacao pela dosagem de B-hCG e no 30 dia foi verificada a presenca de saco gestacional unico e topico com batimentos cardiacos presentes.

Down-regulation of retinoic acid receptor activity associated with decreased alpha and gamma isoforms expression in F9 embryonal carcinoma cells differentiated by retinoic acid.

F9 embryonal carcinoma cells differentiate in response to retinoic acid (RA). To investigate the regulation of RA receptors (RARs) expression during this process, cDNA probes specific for the major RAR isoforms were used. In contrast to the level of RAR beta 2 mRNA which was high in cells treated 5 days with RA and below detection in untreated cells, as previously described, the steady state levels of RAR alpha 1, alpha 2, gamma 1, and gamma 2 mRNAs were markedly decreased in the RA-differentiated cells as compared to untreated cells. The down-regulation of the RA-responsive system in differentiated cells was also evident in gel shift assays as a marked decrease in binding capacity to a retinoid acid response element (beta 2RARE), as well as in chloramphenicol acetyltransferase (CAT) assays as a sixfold decrease in RA-mediated transacting activity via this element. The down-regulation of RAR DNA-binding and transacting activity coincided with the burst in tissue plasminogen activator secretion and thus, occurred at the hinge between early and late differentiation. The down-regulation of RA responsiveness may constitute an important event in the transition between early and late differentiation stage in F9 cells.

Interaction of vasculotropin/vascular endothelial cell growth factor with human umbilical vein endothelial cells: binding, internalization, degradation, and biological effects.

Vasculotropin/vascular endothelial cell growth factor (VAS/VEGF) is a newly purified growth factor with a unique specificity for vascular endothelial cells. We have investigated the interactions of VAS/VEGF with human umbilical vein endothelial cells (HUVE cells). 125I-VAS/VEGF was found to HUVE cells in a saturable manner with a half-maximum binding at 2.8 ng/ml. Scatchard analysis did show two classes of high-affinity binding sites. The first class displayed a dissociation constant of 9 pM with 500 sites/cell. The dissociation constant and the number of binding sites of the second binding class were variable for different HUVE cell cultures (KD = 179 +/- 101 pM, 5,850 +/- 2,950 sites/cell). Half-maximal inhibition of 125I-VAS/VEGF occurred with a threefold excess of unlabeled ligand. Basic fibroblast growth factor (bFGF) and heparin did not compete with 125I-VAS/VEGF binding. In contrast, suramin and protamin sulfate completely displaced 125I-VAS/VEGF binding from HUVE cells. VAS/VEGF was shown to be internalized in HUVE cells. Maximum internalization (55% of total cell-associated radioactivity) was observed after 30 min. 125I-VAS/VEGF was completely degraded 2-3 hr after binding. At 3 hr, the trichloroacetic acid (TCA)-soluble radioactivity accumulated in the medium was 60% of the total radioactivity released by HUVE cells. No degradation fragment of 125I-VAS/VEGF was observed. Chloroquine completely inhibited degradation. VAS/VEGF was able to induce angiogenesis in vitro in HUVE cells. However, it did not significantly modulate urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), and tissue factor (TF). Prostacyclin production was only stimulated at very high VAS/VEGF concentrations. Taken together, these results indicate that VAS/VEGF might be a potent inducer of neovascularization resulting from a direct interaction with endothelial cells. The angiogenic activity seems to be independent of the plasminogen activator or inhibitor system.