Novel 5-aminopyrazoles endowed with anti-angiogenetic properties: Design, synthesis and biological evaluation.

The promising anti-angiogenetic properties of previously synthesized pyrazolyl ureas provided the rationale for the synthesis of novel 5-aminopyrazoles 2-5, differently decorated on the pyrazole nucleus. All the derivatives were tested by MTT assays and proved to be non-cytotoxic against eight different tumor cell lines and normal fibroblasts. An EdU proliferation assay was carried out on human foreskin fibroblasts and VEGF stimulated human umbilical vein endothelial cells which confirmed the absence of cytotoxicity of the compounds on human cells up to 20 µM concentration. To evaluate the influence of the newly synthesized pyrazoles on MAPK and PI3K signaling pathways, the phosphorylation of ERK1/2 and Akt was analyzed by Western blots from HFF and HUVEC cell lysates stimulated with growth factors in the presence or absence of the compounds. Pyrazoles 3b and 3c showed a significant inhibition of Akt phosphorylation in both tested cell lines with lower phosphorylation levels than the reference compound GeGe-3 in HUVEC. Furthermore, derivatives 2 and 3 appeared to strongly affect the migration of HFF cells in a wound healing assay, confirming their potential ability to interfere with the angiogenesis process. The new pyrazole library extends the structure-activity relationships of the previously isolated compounds and highlights the attractiveness of this chemical class for pathological cell migration and angiogenesis.

Synthesis, functional proteomics and biological evaluation of new 5-pyrazolyl ureas as potential anti-angiogenic compounds.

Based on biological results of previous synthesized pyrazolyl ureas able to interfere with angiogenesis process, we planned and synthesized the new benzyl-urea derivatives 2-4; some of them showed an interesting anti-proliferative profile and particularly 4e potently inhibited HUVEC proliferation. To shed light on the mechanism of action of 4e, its interactome has been deeply inspected to identify the most prominent protein partners, mainly taking into account kinome and phosphatome, through drug affinity responsive target stability experiments, followed by targeted limited proteolysis analysis. From these studies, PP1γ emerged as the most reliable 4e potential target in HUVEC. Molecular docking simulations on PP1γ were carried out to predict 4e binding mode. To assess its potential anti-angiogenic effect, 4e was tested in vitro to verify interference on kinase and phosphate activities. Overall, our results evidenced for 4e an interesting anti-angiogenic action, probably due to its action at intracellular level on PP1γ signalling pathways.

New Series of Pyrazoles and Imidazo-Pyrazoles Targeting Different Cancer and Inflammation Pathways.

(1) Background: different previously synthesized pyrazoles and imidazo-pyrazoles showed interesting anti-angiogenic action, being able to interfere with ERK1/2, AKT and p38MAPK phosphorylation in different manners and with different potency; (2) Methods: here, a new small compound library, endowed with the same differently decorated chemical scaffolds, has been synthetized to obtain new agents able to inhibit different pathways involved in inflammation, cancer and human platelet aggregation. (3) Results: most of the new synthesized derivatives resulted able to block ROS production, platelet aggregation and p38MAPK phosphorylation both in platelets and Human Umbilical Vein Endothelial cells (HUVEC). This paves the way for the development of new agents with anti-angiogenic activity.

Reduction of Neglected Displaced Fractures of the Distal Femoral Physis.

BACKGROUND: We performed open osteoclasis, soft-tissue release, and fracture fragment reduction and fixation to treat 10 cases of neglected physeal fractures of the distal aspect of the femur with severe deformity. To our knowledge, no specific surgical procedure for this problem has been reported in the literature. DESCRIPTION: The procedure is typically performed through an extensile anterolateral approach. With use of an osteotome, the typically abundant fracture callus is disrupted and partially removed to recreate the original fracture line. Through periosteal dissection, an extensive musculoperiosteal detachment and release is achieved to facilitate fracture reduction while protecting the physis from further injury. ALTERNATIVES: Knee rehabilitation in closed, nondisplaced or minimally displaced fractures1.Open callus osteoclasis in combination with a Z-shaped quadriceps tenoplasty, reduction, and plaster cast immobilization2.Open subperiosteal osteoclasis, reduction, and tibial traction3.Open callus osteoclasis, reduction, and condylar plating4.Sequestrectomy with preservation of a periosteal sleeve to treat osteomyelitis complicating an open fracture1.Transfemoral amputation to treat gas gangrene or vascular injury following severe open injury1,5-7. RATIONALE: This procedure was developed in remote medical facilities where patients are often first seen >21 days after the original injury. By that time, closed reduction or standard open reduction and internal fixation techniques are no longer possible. After 6 months of fracture age, the procedure is inefficient. EXPECTED OUTCOMES: This procedure allows correction of limb malalignment and shortening while preserving the growth plate1. IMPORTANT TIPS: In some cases, hypertrophic fracture callus might be mistaken for the femoral diaphysis.An extensive musculoperiosteal release will facilitate reduction of the fracture fragments.The adequacy of reduction must be assessed in all 3 planes intraoperatively.The adequacy of reduction must be assessed in all 3 planes intraoperatively.The adequacy of reduction must be assessed in all 3 planes intraoperatively.

[Total hip replacements in a country with limited resources: evaluation of medium-term results]. / Arthroplasties totales de hanche dans un pays a ressources limitees : evaluation des resultats a moyen terme.

INTRODUCTION: Total hip arthroplasty (THA) has been regularly performed in the public hospital in Bobo-Dioulasso since 2010. The objective of thisstudywas to assess the medium-termresults of total hip arthroplastyat the Bobo-Dioulasso UniversityHospital. Dioulasso. PATIENTS AND METHODS: This was a non-randomized single-center retrospectivestudydesigned in accordance with the recommendations of the Declaration of Helsinki. Wereviewed the medical files and evaluated 29 patients, i.e. 33 hipsoperated for THA at the Bobo-Dioulasso UniversityHospitalfromJanuary 1st, 2010 to December 31, 2015. RESULTS: ONATF was the indication for ATH in 78.79% of cases (n = 28). THA wascementedin 43% of cases. Complications wereposterior dislocations (n = 4), infection (n = 1) and loosening (n = 2). The meanfollow-up was 78.3 months. At the last follow-up, the mean PMA was 15.13 and the mean Harris score was 84.36. The inclination of the cupswasbetween 45 ° and 50 °. The femoral stems werecenteredin 94% of cases (n = 32). There was one case of peri-prosthetic ossification and one case of acetabularrim. CONCLUSION: Total hip arthroplasty has become a common and well-codified practice in orthopedicsurgerywithreliable and veryencouragingresults.

INTRODUCTION: L'arthroplastie totale de la hanche (ATH) est régulièrement pratiquée dans l'hôpital public à Bobo-Dioulasso depuis 2010. L'objectif de cette étude était d'évaluer les résultats à moyen terme des arthroplasties totales de hanches au CHU de Bobo-Dioulasso. PATIENTS ET MÉTHODES: Il s'agissait d'une étude rétrospective monocentrique non randomisée rédigée conformément aux recommandations de la déclaration d'Helsinki. Nous avons revu les dossiers médicaux et évaluer 29 patients soit 33 hanches opérées pour PTH au CHU de Bobo-Dioulasso du 1er Janvier 2010 au 31 Décembre 2015. RÉSULTATS: L'ONATF était l'indication de l'ATH dans 78,79% des cas (n=28). Les PTH étaient cimentées (n=14) ou non. Les complications étaient les luxations postérieures (n=4), l'infection (n=1) et le descellement (n=2). Le recul moyen était de 78,3 mois. Au dernier recul, le PMA moyen était de 15,13 et le score de Harris moyen de 84,36. L'inclinaison des cupules était comprise entre 45° et 50°. Les tiges fémorales étaient centrées 94% des cas (n=32). Il a été noté un cas d'ossification péri prothétique et un cas de liséré acétabulaire. CONCLUSION: L'arthroplastie totale de hanche est devenue une pratique courante et bien codifiée en chirurgie orthopédique avec des résultats fiables et très encourageants.

Induced Pluripotent Stem Cells to Understand Mucopolysaccharidosis. I: Demonstration of a Migration Defect in Neural Precursors.

Background: Mucopolysaccharidosis type I-Hurler (MPS1-H) is a severe genetic lysosomal storage disorder due to loss-of-function mutations in the IDUA gene. The subsequent complete deficiency of alpha l-iduronidase enzyme is directly responsible of a progressive accumulation of glycosaminoglycans (GAG) in lysosomes which affects the functions of many tissues. Consequently, MPS1 is characterized by systemic symptoms (multiorgan dysfunction) including respiratory and cardiac dysfunctions, skeletal abnormalities and early fatal neurodegeneration. Methods: To understand mechanisms underlying MPS1 neuropathology, we generated induced pluripotent stem cells (iPSC) from a MPS1-H patient with loss-of-function mutations in both IDUA alleles. To avoid variability due to different genetic background of iPSC, we established an isogenic control iPSC line by rescuing IDUA expression by a lentivectoral approach. Results: Marked differences between MPS1-H and IDUA-corrected isogenic controls were observed upon neural differentiation. A scratch assay revealed a strong migration defect of MPS1-H cells. Also, there was a massive impact of IDUA deficiency on gene expression (340 genes with an FDR <0.05). Conclusions: Our results demonstrate a hitherto unknown connection between lysosomal degradation, gene expression and neural motility, which might account at least in part for the phenotype of MPS1-H patients.

Characterization of Proangiogenic Monocytes from Blood in Patients with Chronic Ischemic Diabetic Foot Ulcers and Controls.

Many persons with diabetes mellitus have limb ischemia, which is a major clinical problem. A subset of human monocytes that expresses TIE-2 may enhance neovascularization. We performed 179 phlebotomies on 142 patients (or donors), including 61 patients/donors without diabetes or ischemia (controls), 39 diabetic nonischemic patients (controls), and 42 diabetic patients with severe limb ischemia requiring amputation. We compared these groups for the presence of TIE-2-positive proangiogenic monocytes. The proportion of proangiogenic monocytes in the venous blood (on hospital admission) was significantly increased in diabetic patients without ischemia (9.22% ± 1.19%), compared to controls (6.53% ± 0.58%) or ischemic diabetic patients (5.44% ± 0.56%) (P < 0.05). In this pilot evaluation, we succeeded in extracting potential proangiogenic TIE-2 monocytes from the blood of diabetic patients without ischemia, but less in patients with ischemia. The implications for therapeutic neoangiogenesis require further studies.

Management of neglected distal femur epiphyseal fracture-separation.

INTRODUCTION: Neglected epiphyseal fracture-separations of the distal femur are rare. Still reported in developing countries, they lead to therapeutic issues. The objective of the study is to describe their characteristics and to propose treatment options. MATERIALS AND METHODS: Ten years of ongoing study was held in our orthopedics department. All patients with a neglected epiphyseal fracture-separations of the distal femur after a knee trauma were included in the study. Pre-operative and post-operative data were collected and analyzed. RESULTS: A total of 13 cases of neglected traumatic epiphyseal fracture-separations of the distal femur were found among 8616 in-patients of the department. It was mainly boys (9M/4F) around 16 years that were received 14 weeks after knee trauma. Most of the injuries were an AO-type 33-E/2.1 (Simple Salter-Harris' type II) (n=12) distal femur malunion (n=10). Associated complications were cutaneous opening (n=7), superficial infection (n=4), deep infection (n=4). Fractures were management surgically (n=12) by an open osteoclasis procedure (n=9), debridement (n=7) and a thigh amputation (n=1). The outcome was better if an open osteoclasis procedure was early performed in closed distal femur mal-union with a complementary rehabilitation program. CONCLUSION: Specialized trauma care facilities must be increased, and trauma education programs must be undertaken to avoid neglected epiphyseal fracture-separations of the distal femur in developing countries.

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro.

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

The tyrosine-kinase inhibitor sunitinib targets vascular endothelial (VE)-cadherin: a marker of response to antitumoural treatment in metastatic renal cell carcinoma.

BACKGROUND: Vascular endothelial (VE)-cadherin is an endothelial cell-specific protein responsible for endothelium integrity. Its adhesive properties are regulated by post-translational processing, such as tyrosine phosphorylation at site Y685 in its cytoplasmic domain, and cleavage of its extracellular domain (sVE). In hormone-refractory metastatic breast cancer, we recently demonstrated that sVE levels correlate to poor survival. In the present study, we determine whether kidney cancer therapies had an effect on VE-cadherin structural modifications and their clinical interest to monitor patient outcome. METHODS: The effects of kidney cancer biotherapies were tested on an endothelial monolayer model mimicking the endothelium lining blood vessels and on a homotypic and heterotypic 3D cell model mimicking tumour growth. sVE was quantified by ELISA in renal cell carcinoma patients initiating sunitinib (48 patients) or bevacizumab (83 patients) in the first-line metastatic setting (SUVEGIL and TORAVA trials). RESULTS: Human VE-cadherin is a direct target for sunitinib which inhibits its VEGF-induced phosphorylation and cleavage on endothelial monolayer and endothelial cell migration in the 3D model. The tumour cell environment modulates VE-cadherin functions through MMPs and VEGF. We demonstrate the presence of soluble VE-cadherin in the sera of mRCC patients (n = 131) which level at baseline, is higher than in a healthy donor group (n = 96). Analysis of sVE level after 4 weeks of treatment showed that a decrease in sVE level discriminates the responders vs. non-responders to sunitinib, but not bevacizumab. CONCLUSIONS: These data highlight the interest for the sVE bioassay in future follow-up of cancer patients treated with targeted therapies such as tyrosine-kinase inhibitors.

Angiogenic factor-driven inflammation promotes extravasation of human proangiogenic monocytes to tumours.

Recruitment of circulating monocytes is critical for tumour angiogenesis. However, how human monocyte subpopulations extravasate to tumours is unclear. Here we show mechanisms of extravasation of human CD14dimCD16+ patrolling and CD14+CD16+ intermediate proangiogenic monocytes (HPMo), using human tumour xenograft models and live imaging of transmigration. IFNγ promotes an increase of the chemokine CX3CL1 on vessel lumen, imposing continuous crawling to HPMo and making these monocytes insensitive to chemokines required for their extravasation. Expression of the angiogenic factor VEGF and the inflammatory cytokine TNF by tumour cells enables HPMo extravasation by inducing GATA3-mediated repression of CX3CL1 expression. Recruited HPMo boosts angiogenesis by secreting MMP9 leading to release of matrix-bound VEGF-A, which amplifies the entry of more HPMo into tumours. Uncovering the extravasation cascade of HPMo sets the stage for future tumour therapies.

Design, synthesis and biological evaluation of new pyrazolyl-ureas and imidazopyrazolecarboxamides able to interfere with MAPK and PI3K upstream signaling involved in the angiogenesis.

Taking into account the structure activity relationship information given by our previous studies, we designed and synthesized a small library of pyrazolylureas and imidazopyrazolecarboxamides fluorinated on urea moiety and differently decorated on pyrazole nucleus. All compounds were preliminary screened by Western blotting technique to evaluate their activity on MAPK and PI3K pathways by monitoring ERK1/2, p38MAPK and Akt phosphorylation, and also screened with a wound healing assay to assess their capacity in inhibiting endothelial cell migration, using human umbilical vein endothelial cells stimulated with VEGF. Pyrazoles and imidazopyrazoles did not show the same activity profile. SAR consideration showed that specific substituents and their position in pyrazole nucleus, as well as the type of substituent on the phenylurea moiety play a pivotal role in determining increase or decrease of kinases phosphorylation. On the other hand the loss of flexibility in imidazopyrazole derivatives is responsible for activity potentiation. Screening of the compound library for inhibition of endothelial cell migration, a function required for angiogenesis, showed significant activity for compound 3. This compound might interfere with cell migration by modulating the activity of different upstream target kinases. Therefore, compound 3 represents a potential inhibitor of angiogenesis. Furthermore, it may be used as a tool to identify unknown mediators of endothelial migration and thereby unveiling new therapeutic targets for controlling pathological angiogenesis in diseases such as cancers.

The pyrazolyl-urea GeGe3 inhibits tumor angiogenesis and reveals dystrophia myotonica protein kinase (DMPK)1 as a novel angiogenesis target.

The limitation of targeting VEGF/VEGFR2 signalling to stop angiogenesis in cancer therapy has been blamed on re-activation of alternative receptor tyrosine kinases by compensatory angiogenic factors. Targeting MAPK and PI3K signaling pathways in endothelial cells may be an alternative or complementary approach. Herein we aimed to evaluate the antitumor and antiangiogenic potential of a novel pyrazolyl-urea kinase inhibitor, GeGe3, and to identify its kinase targets. We found GeGe3 to inhibit the proliferation of HUVEC and endothelial tube formation. GeGe3 impaired inter-segmental angiogenesis during development of zebrafish embryos. In mice, GeGe3 blocked angiogenesis and tumor growth in transplanted subcutaneous Lewis Lung Carcinomas. Screening for GeGe3-targeted kinases revealed Aurora B, Aurora C, NEK10, polo-like kinase (PLK)2, PLK3, DMPK1 and CAMK1 as candidate targets. Biochemical analysis of these kinases showed DMPK1 regulation upon VEGF challenge. Investigation of the role of DMPK1 in endothelial cells revealed DMPK1 as a novel mediator of angiogenesis that controls the activation of MAPK signaling, proliferation and migration. GeGe3 alters angiogenesis by targeting DMPK in tumor endothelial cells and pericytes. The pyrazolyl-urea GeGe3, a novel blocker of MAPK and PI3K pathways, strongly inhibits physiological and tumor angiogenesis. We also report GeGe3-targeted kinase DMPK as a novel mediator of angiogenesis.

5'-Ectonucleotidase/CD73 expression on lymph-circulating lymphocytes and lymphatic endothelial cells offers new paths to explore barrier function.

5'-Nucleotidase/CD73 is a key enzyme in the regulation of purinergic signaling, hydrolyzing extracellular AMP to produce adenosine, which is critical in the blood vascular system and in immunosuppression. CD73 is expressed by both blood endothelial cells and lymphatic endothelial cells. Although the role of CD73 on blood endothelial cells in controlling vascular permeability and leukocyte trafficking has been studied, the role of lymphatic CD73 has thus far remained unknown. In this issue of European Journal of Immunology, Yegutkin et al. [Eur. J. Immunol. 2015. 45: 562-573] compare CD73 activity in the endothelia of lymphatics and blood vessels and investigate the CD73(+) lymphocyte subpopulations possibly involved in immunoregulation. This Commentary will discuss how the authors' work sheds light on the differential use of CD73 by these two cell populations to control endothelial permeability and sprouting.

Dynamic phosphorylation of VE-cadherin Y685 throughout mouse estrous cycle in ovary and uterus.

We previously reported that vascular endothelial growth factor induced vascular endothelial (VE)-cadherin tyrosine phosphorylation at Y685 in a Src-dependent manner in vitro. Here, we studied the occurrence of Y685 phosphorylation in vivo in the female reproductive tract because it is a unique model of physiological vascular remodeling dependent on vascular endothelial growth factor. We first developed and characterized an anti-phospho-specific antibody against the site Y685 of VE-cadherin to monitor VE-cadherin phosphorylation along the four phases of mouse estrous cycle, termed proestrus, estrus, metestrus, and diestrus. A dynamic profile of tyrosine phosphorylated proteins was observed in both uterus and ovary throughout mouse estrous cycle, including kinase Src, which was found highly active at the estrus phase. The extent of tyrosine phosphorylated VE-cadherin was low at proestrus but strongly increased at estrus and returned to baseline at metestrus and diestrus, suggesting a potent hormonal regulation of this specific process. Indeed, C57Bl/6 female mice treatment with pregnant mare serum gonadotropin and human chorionic gonadotropin confirmed a significant increase in phosphoY685-VE-cadherin compared with that in untreated mice. These results demonstrate that VE-cadherin tyrosine phosphorylation at Y685 is a physiological and hormonally regulated process in female reproductive organs. In addition, this process was concomitant with the early steps of vascular remodeling taking place at estrus stage, suggesting that phosphoY685-VE-cadherin is a biomarker of endothelial cell activation in vivo.

VE-cadherin Y685F knock-in mouse is sensitive to vascular permeability in recurrent angiogenic organs.

Covalent modifications such as tyrosine phosphorylation are associated with the breakdown of endothelial cell junctions and increased vascular permeability. We previously showed that vascular endothelial (VE)-cadherin was tyrosine phosphorylated in vivo in the mouse reproductive tract and that Y685 was a target site for Src in response to vascular endothelial growth factor in vitro. In the present study, we aimed to understand the implication of VE-cadherin phosphorylation at site Y685 in cyclic angiogenic organs. To achieve this aim, we generated a knock-in mouse carrying a tyrosine-to-phenylalanine point mutation of VE-cadherin Y685 (VE-Y685F). Although homozygous VE-Y685F mice were viable and fertile, the nulliparous knock-in female mice exhibited enlarged uteri with edema. This phenotype was observed in 30% of females between 4 to 14 mo old. Histological examination of longitudinal sections of the VE-Y685F uterus showed an extensive disorganization of myometrium and endometrium with highly edematous uterine glands, numerous areas with sparse cells, and increased accumulation of collagen fibers around blood vessels, indicating a fibrotic state. Analysis of cross section of ovaries showed the appearance of spontaneous cysts, which suggested increased vascular hyperpermeability. Electron microscopy analysis of capillaries in the ovary showed a slight but significant increase in the gap size between two adjacent endothelial cell membranes in the junctions of VE-Y685F mice (wild-type, 11.5 ± 0.3, n = 78; and VE-Y685F, 12.48 ± 0.3, n = 65; P = 0.045), as well as collagen fiber accumulation around capillaries. Miles assay revealed that either basal or vascular endothelial growth factor-stimulated permeability in the skin was increased in VE-Y685F mice. Since edema and fibrotic appearance have been identified as hallmarks of initial increased vascular permeability, we conclude that the site Y685 in VE-cadherin is involved in the physiological regulation of capillary permeability. Furthermore, this knock-in mouse model is of potential interest for further studies of diseases that are associated with abnormal vascular permeability.

Evidence for post-translational processing of vascular endothelial (VE)-cadherin in brain tumors: towards a candidate biomarker.

Vessel abnormalities are among the most important features in malignant glioma. Vascular endothelial (VE)-cadherin is of major importance for vascular integrity. Upon cytokine challenge, VE-cadherin structural modifications have been described including tyrosine phosphorylation and cleavage. The goal of this study was to examine whether these events occurred in human glioma vessels. We demonstrated that VE-cadherin is highly expressed in human glioma tissue and tyrosine phosphorylated at site Y(685), a site previously found phosphorylated upon VEGF challenge, via Src activation. In vitro experiments showed that VEGF-induced VE-cadherin phosphorylation, preceded the cleavage of its extracellular adhesive domain (sVE, 90 kDa). Interestingly, metalloproteases (MMPs) secreted by glioma cell lines were responsible for sVE release. Because VEGF and MMPs are important components of tumor microenvironment, we hypothesized that VE-cadherin proteolysis might occur in human brain tumors. Analysis of glioma patient sera prior treatment confirmed the presence of sVE in bloodstream. Furthermore, sVE levels studied in a cohort of 53 glioma patients were significantly predictive of the overall survival at three years (HR 0.13 [0.04; 0.40] p ≤ 0.001), irrespective to histopathological grade of tumors. Altogether, these results suggest that VE-cadherin structural modifications should be examined as candidate biomarkers of tumor vessel abnormalities, with promising applications in oncology.

Soluble VE-cadherin in rheumatoid arthritis patients correlates with disease activity: evidence for tumor necrosis factor α-induced VE-cadherin cleavage.

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that principally attacks synovial joints. However, accelerated atherosclerosis and increased cardiovascular morbidity and mortality are major clinical consequences of endothelial dysfunction in RA patients. Tumor necrosis factor α (TNFα) is the major mediator of inflammation in RA, related to vascular injury by targeting VE-cadherin, an endothelium-specific adhesion molecule of vital importance for endothelium integrity and angiogenesis. We undertook this study to examine the mechanisms regulating VE-cadherin processing by TNFα and their occurrence in RA. METHODS: Human umbilical vein endothelial cells were used in primary culture and treated with recombinant TNFα to study VE-cadherin cleavage. Cell lysates and conditioned media were analyzed by Western blotting for VE-cadherin cytoplasmic domain and extracellular domain (VE-90) generation, respectively. VE-90 was analyzed at baseline and at the 1-year followup in sera from 63 RA patients (from the Very Early Rheumatoid Arthritis cohort) with disease duration of <6 months. RESULTS: TNFα induced a time-dependent shedding of VE-90 in cell media. This effect was prevented by tyrosine kinase inhibitors (genistein and PP2) or by knocking down Src kinase. In contrast, tyrosine phosphatase blockade enhanced VE-cadherin cleavage, confirming the requirement of tyrosine phosphorylation processes. In addition, using the matrix metalloproteinase (MMP) activator APMA and the MMP inhibitor GM6001, we demonstrated that MMPs are involved in TNFα-induced VE-cadherin cleavage. Of major importance, VE-90 was detected in sera from the 63 RA patients and was positively correlated with the Disease Activity Score at baseline and after 1-year followup. CONCLUSION: These findings provide the first evidence of VE-cadherin proteolysis upon TNFα stimulation and suggest potential clinical relevance of soluble VE-cadherin in management of RA.