Vascular inflammation and associated leukocyte influx is a hallmark in the pathogenesis of atherosclerosis. In both animal models and human subjects, inhibiting monocyte recruitment is beneficial in preventing atherosclerosis and its clinical manifestations. The trafficking signals that recruit cells to areas of inflammation are provided by small secreted proteins called chemokines. Chemokines play a major role in the pathogenesis of inflammation, and redundancy among the chemokine signaling pathways means that blocking one pathway could result in another assuming its function. Therefore, we aim to block a cell’s response to a range of chemokine-induced directional migration signals. Slit2 treatment inhibits monocyte migration in vitro using transwell migration assays, and in vivo, using a murine peritonitis model of inflammatory cell influx. This inhibition is shown to be dose- and time- dependent. Furthermore, Slit2 inhibits monocyte adhesion to activated endothelial cell monolayers. These data may suggest a therapeutic role for Slit2 in atherosclerosis.

Vascular inflammation and associated leukocyte influx is a hallmark in the pathogenesis of atherosclerosis. In both animal models and human subjects, inhibiting monocyte recruitment is beneficial in preventing atherosclerosis and its clinical manifestations. The trafficking signals that recruit cells to areas of inflammation are provided by small secreted proteins called chemokines. Chemokines play a major role in the pathogenesis of inflammation, and redundancy among the chemokine signaling pathways means that blocking one pathway could result in another assuming its function. Therefore, we aim to block a cell’s response to a range of chemokine-induced directional migration signals. Slit2 treatment inhibits monocyte migration in vitro using transwell migration assays, and in vivo, using a murine peritonitis model of inflammatory cell influx. This inhibition is shown to be dose- and time- dependent. Furthermore, Slit2 inhibits monocyte adhesion to activated endothelial cell monolayers. These data may suggest a therapeutic role for Slit2 in atherosclerosis.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) is in the family of structural renal tract birth defects. CAKUT is the major cause of chronic kidney disease and renal failure in children and adults <40 years of age. ROBO2 is a receptor for the SLIT2 ligand. ROBO2/SLIT2 signaling has been shown to play important roles in neuronal migration and in early renal tract development. Our laboratory has recently identified ROBO2 as a novel CAKUT-causing gene. So far, total 26 ROBO2 mutations have been identified in patients with CAKUT. However, most of these mutations are missense amino acid substitutions and their functional significances are unclear, although they are predicted to be disease-causing by several bioinformatics prediction software. OBJECTIVE: To clarify uncertainties and confusions in the CAKUT field regarding the causality for ROBO2 missense mutations, we performed functional analysis of a ROBO2 missense mutation p.G114W (c.340G>T) that was identified in a CAKUT family. This p.G114W ROBO2 mutation is located in the first Ig domain of the ROBO2 extracellular region. The ROBO2 first Ig domain is the binding site for ligand SLIT2 that is required for ROBO2-SLIT2 signaling chemorepulsive activity in neuronal migration. We hypothesize that this p.G114W ROBO2 mutation would disrupt the SLIT2-ROBO2 binding and compromise its chemorepulsive activity in a sensitive functional neuronal migration assay. METHODS: Site directed mutagenesis was used to introduce c.340G>T point mutation into a ROBO2 cDNA fusion construct that contains the first Ig domain. Point mutation was verified using Sanger sequencing. Mutant ROBO2 cDNA and wildtype control constructs were purified using Qiagen Midiprep kit and transfected in HEK cells via calcium phosphate co-precipitation. The conditioned medium (CM) containing ROBO2 fusion proteins were analyzed by Western Blot. Neuronal migration assays were performed using postnatal anterior subventricular zone (SVZa) tissue explants that were isolated from postnatal day 1 to 5 (P1-5) Sprague-Dawley rat brain. RESULTS: By Sanger sequencing, we verified the c.340G>T point mutation in the ROBO2 cDNA fusion construct. By GFP fluorescence and Western blot analysis, we found abundant expressions of ROBO2 fusion protein in the conditioned medium of transfected HEK cells. In SVZa neuronal migration assays, we found that, when compared to the wild type fusion protein, the mutant ROBO2 fusion protein with the p.G114W amino acid substitution lost its function to block SLIT2-medicated inhibition of neuronal migration at both 50% conditioned medium and 100% conditioned medium concentrations. CONCLUSION: Our results show that ROBO2 p.G114W is a loss-of-function mutation disrupting normal SLIT2-ROBO2 chemorepulsive activity on SVZa neuronal migration, suggesting that the presence of this missense mutation compromises SLIT2-ROBO2 signaling and contributes to the development of CAKUT. / 2020-07-03T00:00:00Z

Il a été récemment montré que la vasorine (Vasn), d’abord décrite comme une protéine membranaire de type I qui neutralise TGFb, était exprimée par les ostéocytes et les ostéoblastes et impliquée dans le métabolisme du phosphate et du calcium. L’analyse MicroCT a montré que des souris Vasn -/- présentaient des signes de rachitisme avec des volumes osseux trabéculaires et corticaux fortement réduits. Ici, nous explorons pour la première fois l’expression et les fonctions de Vasn dans le territoire craniofacial. Nous démontrons que 1) Vasn est exprimée au sein de la dent en formation par les odontoblastes et les cémentoblastes et dans l’os alvéolaire par les ostéoblastes et les ostéocytes, 2) des souris Vasn -/- présentent un phénotype dentaire mixte associant des signes de rachitisme tels qu’une croissance réduite du squelette, une dentine minéralisée moins épaisse, des chambres pulpaires élargies et des signes supplémentaires évoquant une hypophosphatasie , principalement une formation perturbée de la racine avec des canaux dentaires élargis. Toutes ces données suggèrent un rôle important de Vasn dans la minéralisation de l’os et de la dent, probablement en régulant le dépôt de matrice et son organisation, plutôt que le processus de minéralisation lui-même. / Vasorin (Vasn), first described as a type I membrane protein which neutralizes TGFβ, has been recently shown to be expressed by osteocytes and osteoblasts and involved in the phosphate and calcium metabolism. MicroCT analysis have indicated that Vasn -/- mice displayed signs of rickets with dramatically reduced trabecular and cortical bone volumes. Here, we explored for the first time Vasn expression and function in the craniofacial area. We demonstrated that 1) Vasn is expressed in the forming tooth by odontoblasts and cementoblasts and in the alveolar bone by osteoblasts and osteocytes, 2) Vasn-/- mice present a mix cranial and dental phenotype associating signs of rickets such as, reduced skull growth, reduced thickness of mineralized dentin, and enlarged pulp chambers, and additional signs evokating hypophosphatasia, namely disturbed root formation with enlarged root canals. All these data suggest an important role of Vasn in bone and tooth mineralization, probably by regulating matrix deposition and organization rather than the mineralisation process itself.

Les métastases osseuses sont des complications fréquentes de nombreuses tumeurs solides et sontresponsables, sur le plan clinique, de fractures osseuses, d’hypercalcémie et de douleurs. A l’heure actuelle,il n’existe aucun traitement curatif ; la compréhension des mécanismes impliqués dans la formation et ledéveloppement des métastases osseuses est donc nécessaire afin d’envisager de nouvelles approchesthérapeutiques.Une analyse transcriptomique comparative entre une lignée humaine de cancer du sein, les MDA-MB-231, et leur sous-population ostéotropique, les B02, a montré une surexpression de composants de la voie designalisation Slit/Robo.Les récepteurs Robo et leurs ligands Slits, initialement identifiés comme facteurs clés du guidage axonal lorsdu développement, sont également impliqués dans la migration des cellules cancéreuses.Afin d’étudier le rôle de ces récepteurs Robo1 et 4 dans la dissémination métastatique à l’os, nousavons inhibé l’expression de ces gènes dans les cellules B02. Des expériences in vitro et in vivo montrentalors un rôle antagoniste de Robo1/4. En effet, l’inhibition de Robo1 augmente la croissance des tumeursprimaires, tandis que celle de Robo4 la diminue. Ces récepteurs régulent également différemment laformation de lésions ostéolytiques ainsi que la croissance tumorale de ces métastases. In vitro, l’absence deRobo1 augmente l’invasion, tandis que celle de Robo4 la diminue. Nous avons également montré quel’inhibition de Robo4 ralentit la colonisation de la moelle osseuse par les cellules B02 à des temps précoces.Enfin, l’étude d’une cohorte de patientes atteintes d’un cancer du sein montre une corrélation entrel’expression de Robo4 et la rechute métastatique osseuse.Cette étude montre l’implication de la voie Slit/Robo dans la dissémination métastatique à l’os, etsemble par conséquent constituer une approche thérapeutique judicieuse pour traiter ces métastases. / Bone metastases are a common complication of many solid tumors and are clinically responsible ofbone fractures, hypercalcemia, and pain. Currently, there is no curative treatment; understanding themechanisms involved in the formation and development of bone metastases is therefore necessary toconsider new therapeutic approaches.A comparative transcriptomic analysis between the human breast cancer cell line MDA-MB-231, andits osteotropic subpopulation, B02, has shown an up-regulation of Slit/Robo signaling pathway.Robo receptors and their ligands Slits were initially identified as key factors in axon guidance duringdevelopment. However, these proteins are also involved in the migration of cancer cells.To investigate the role of these receptors Robo1 and 4 in breast cancer bone metastasis, theexpression of these genes was inhibited in B02 cells. In vitro and in vivo experiments point out antagonisticrole of Robo1/4. Indeed, inhibition of Robo1 expression increases primary tumors growth, while Robo4invalidation reduces it. These receptors also differently regulate the formation of osteolytic lesions and extentof skeletal tumor burden. In vitro, Robo1 depletion induces an increased invasion, whereas Robo4 depletiondecreases it. We also showed that inhibition of Robo4 reduces survival and growth of B02 cells in the bonemarrow at early times of invasion. Finally, a cohort study of breast cancer patients shows a strong correlationbetween Robo4 expression and metastatic relapse in bone.This study shows the involvement of the Slit/Robo pathway in bone metastasis, and therefore seemsto be a judicious therapeutic approach to treat these metastases.

Glomerular derangement is the major feature of a diverse array of kidney disorders that lead to end-stage kidney disease (ESKD). Podocyte dysfunction is central to the underlying pathophysiology of many common glomerular diseases, including diabetic nephropathy (DN), focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), and genetic forms of nephrotic syndrome, and is associated with heavy proteinuria. Loss of podocyte foot process structure, or effacement, is the key feature of podocyte injury in these proteinuric glomerular disorders, also called "podocytopathies". However, the degree of effacement can vary: For instance, it is very disseminated (diffuse) in minimal change disease, but more variable (segmental) in focal segmental glomerulosclerosis. Recent work has shown that nephrin, ROBO2, and SLIT2 are proteins implicated in these podocyte foot process effacement and podocytopathy. We sought to evaluate changes in the expression of these proteins using immunofluorescence microscopy, and the degree of foot process effacement in podocytopathies using ultrastructural morphometry. In podocytopathies, we saw increased expression of ROBO2 and decreased expression of nephrin indicating that, upregulation of ROBO2 may lead to podocyte injury and podocyte injury may result in loss of nephrin. In addition, SLIT2, which binds ROBO2, was found in tubules and in glomeruli. A higher degree of geometric mean foot process width was vii observed in podocytopathies as compared to normal kidney. These findings can be used in the clinical setting to diagnose and monitor disease treatment. / 2024-02-07T00:00:00Z

Les métastases osseuses sont des complications fréquentes de nombreuses tumeurs solides et sontresponsables, sur le plan clinique, de fractures osseuses, d'hypercalcémie et de douleurs. A l'heure actuelle,il n'existe aucun traitement curatif ; la compréhension des mécanismes impliqués dans la formation et ledéveloppement des métastases osseuses est donc nécessaire afin d'envisager de nouvelles approchesthérapeutiques.Une analyse transcriptomique comparative entre une lignée humaine de cancer du sein, les MDA-MB-231, et leur sous-population ostéotropique, les B02, a montré une surexpression de composants de la voie designalisation Slit/Robo.Les récepteurs Robo et leurs ligands Slits, initialement identifiés comme facteurs clés du guidage axonal lorsdu développement, sont également impliqués dans la migration des cellules cancéreuses.Afin d'étudier le rôle de ces récepteurs Robo1 et 4 dans la dissémination métastatique à l'os, nousavons inhibé l'expression de ces gènes dans les cellules B02. Des expériences in vitro et in vivo montrentalors un rôle antagoniste de Robo1/4. En effet, l'inhibition de Robo1 augmente la croissance des tumeursprimaires, tandis que celle de Robo4 la diminue. Ces récepteurs régulent également différemment laformation de lésions ostéolytiques ainsi que la croissance tumorale de ces métastases. In vitro, l'absence deRobo1 augmente l'invasion, tandis que celle de Robo4 la diminue. Nous avons également montré quel'inhibition de Robo4 ralentit la colonisation de la moelle osseuse par les cellules B02 à des temps précoces.Enfin, l'étude d'une cohorte de patientes atteintes d'un cancer du sein montre une corrélation entrel'expression de Robo4 et la rechute métastatique osseuse.Cette étude montre l'implication de la voie Slit/Robo dans la dissémination métastatique à l'os, etsemble par conséquent constituer une approche thérapeutique judicieuse pour traiter ces métastases.

Lokale Invasion und Ausbreitung von Tumorzellen entlang von Nerven und Gefäßen limitieren den Erfolg kurativer Therapien von Patienten mit Pankreaskarzinom (PDAC). Der axon guidance Faktor Slit2 und seine Robo-Rezeptoren steuern die Navigation von Nerven und Gefäßen sowie die Motilität von Epithelzellen. Sie stellen somit attraktive Regulatoren der klinisch bedeutsamen Ausbreitungswege des PDAC dar. Zielsetzung der vorgelegten Arbeit war die Charakterisierung der Expression von Slit2 im PDAC und seiner Funktion für Tumorwachstum und -ausbreitung. Quantitative Analysen belegten eine deutliche Reduktion der Slit2 mRNA Expression in humanen PDAC Proben im Vergleich zu gesundem Gewebe. Zudem korrelierten Slit2 mRNA-Werte unterhalb des Medians mit einer höheren Inzidenz lymphatischer Metastasierung und einem gesteigerten Prozentsatz befallener Lymphknoten. Die Slit2-Rezeptoren Robo1 und 4 wiesen hingegen vergleichbare Immunreaktivität im Tumor und gesundem Gewebe auf, wobei eine differentielle Lokalisation in Epithelien, Nerven und Gefäßen zu beobachten war. Die Re-Expression von Slit2 in Slit2-defizienten Zelllinien führte zu einer Hemmung der gerichteten Migration und Invasion. Der Robo1-Rezeptor knockdown hingegen stimulierte die Motilität von Tumorzellen mit endogener Slit2 Expression. Slit2-konditioniertes Medium aus Tumorzellen hemmte die Lamellipodienbildung und die Migration von Endothelzellen. In orthotopen humanen Xenograft-Modellen und einem murinen, syngenen Tumormodell reduzierte die Re-Expression von Slit2 in PDAC Zellen Tumorwachstum, Invasion, Metastasierung und Angiogenese. Zudem verminderte die Induktion von Slit2 in PDAC Zellen deren gerichtete Migration entlang aussprießender Neuriten in einem ex vivo Model. Die vorliegenden Daten weisen Slit2 die Funktion eines Tumorsuppressors im duktalen Pankreaskarzinom zu. Ein Verlust der Slit2-Robo Aktivität könnte somit Metastasierung und neuronale Invasion fördern und einen aggressiveren Phänotyp begünstigen. / Early dissemination of pancreatic ductal adenocarcinoma (PDAC) via vascular routes and neural invasion limits curative therapy, suggesting a central role for the interaction of tumor cells with blood vessels and nerves in the tumor stroma. Slit2 and its Robo receptors constitute a system of guidance cues that function in axon guidance, angiogenesis and epithelial morphogenesis, respectively. Here, we studied the expression of Slit2 in PDAC and its function for tumor growth and dissemination. Slit2 mRNA expression was reduced in specimens of human PDAC as compared to non-transformed pancreas and low Slit2 mRNA expression correlated with a higher incidence and a higher extent of lymphatic metastasis. In contrast, the Slit2 receptors Robo1 and Robo4 were uniformly present in clinical samples of PDAC and healthy pancreas and displayed differential localization on epithelial tumor cells, nerves and tumor vasculature. Stable or inducible re-expression of Slit2 in Slit2-deficient PDAC cell lines inhibited directed migration and invasion. Conversely, Robo1-knockdown stimulated the motility of PDAC cells with endogenous Slit2 expression. Tumor cell derived Slit2, furthermore, suppressed lamellipodia formation and migration of primary endothelial cells. In vivo studies in orthotopic human xenograft and mouse syngeneic pancreatic cancer models revealed that re-expression of Slit2 in PDAC cells inhibited tumor growth, invasion, metastasis and angiogenesis. In addition, induction of Slit2 in PDAC cells impaired the unidirectional migration along outgrowing neurites in ex vivo co-cultures of tumor cells and dorsal root ganglia. These data provide evidence for a functional role of Slit2 as a tumor suppressor in human PDAC. A loss of Slit2-Robo activity as observed in human PDAC samples, might consequently promote metastasis and neural invasion and favors a more aggressive phenotype.