Zebrafish Tie-2 shares a redundant role with Tie-1 in heart development and regulates vessel integrity

Tie-2 is a member of the receptor tyrosine kinase family and is required for vascular remodeling and maintenance of mammalian vessel integrity. A number of mutations in the human TIE2 gene have been identified in patients suffering from cutaneomucosal venous malformations and ventricular septal defects. How exactly Tie-2 signaling pathways play different roles in both vascular development and vascular stability is unknown. We have generated a zebrafish line carrying a stop mutation in the kinase domain of the Tie-2 receptor. Mutant embryos lack Tie-2 protein, but do not display any defect in heart and vessel development. Simultaneous loss of Tie-1 and Tie-2, however, leads to a cardiac phenotype. Our study shows that Tie-1 and Tie-2 are not required for early heart development, yet they have redundant roles for the maintenance of endocardial-myocardial connection in later stages. Tie-2 and its ligand Angiopoietin-1 have also been reported to play an important role in vessel stability. We used atorvastatin and simvastatin, drugs that cause bleeding in wild-type zebrafish larvae, to challenge vessel stability in tie-2 mutants. Interestingly, recent clinical studies have reported hemorrhagic stroke as a side effect of atorvastatin treatment. Exposure of embryos to statins revealed that tie-2 mutants are significantly protected from statin-induced bleeding. Furthermore, tie-2 mutants became less resistant to bleeding after VE-cadherin knockdown. Taken together, these data show that atorvastatin affects vessel stability through Tie-2, and that VE-cadherin and Tie-2 act in concert to allow vessel remodeling while playing a role in vessel stability. Our study introduces an additional vertebrate model to study in vivo the function of Tie-2 in development and disease

Novel Role for Mast Cells in Omental Tissue Remodeling and Cell Recruitment in Experimental Peritoneal Dialysis

Because of its dynamic structure, the omentum plays a key role in the immunity of the peritoneal cavity by orchestrating peritoneal cell recruitment. Because mast cells accumulate in the omentum upon experimental peritoneal dialysis (PD) and may produce angiogenic/profibrotic factors, it was hypothesized that mast cells mediate omental tissue remodeling during PD. Daily treatment with conventional PD fluid (PDF) for 5 wk resulted in a strong omental remodeling response, characterized by an approximately 10-fold increase in mast cell density (P < 0.01), an approximately 20-fold increase in vessel density (P < 0.02), an approximately 20-fold increase in the number of milky spots (P < 0.01), and a four-fold increase in submesothelial matrix thickness (P < 0.0003) in wild-type rats. In contrast, all PDF-induced omental changes were significantly reduced in mast cell–deficient Ws/Ws rats or in wild-type rats that were treated orally with a mast cell stabilizer cromoglycate. A time-course experiment showed mast cell accumulation immediately before the formation of blood vessels and milky spots. Functionally, PDF evoked a peritoneal cell influx, which was significantly reduced in Ws/Ws rats (P < 0.04) and in wild-type rats that were treated with cromoglycate (P < 0.03). Cromoglycate treatment also completely prevented PDF-induced omental adhesions to the catheter tip (P � 0.0002). Mesothelial damage, angiogenesis, and fibrosis of mesentery and parietal peritoneum as well as glucose absorption rate and ultrafiltration capacity proved to be mast cell independent. Data strongly support the hypothesis that mast cells mediate PDF-induced omental tissue remodeling and, subsequently, peritoneal cell influx and adhesion formation, providing therapeutic possibilities of modulating omental function

Mesothelial cell transplantation in models of acute inflammation and chronic peritoneal dialysis

OBJECTIVES: Mesothelial cell (MC) injury caused by continuous exposure to unphysiological peritoneal dialysis (PD) fluid and by episodes of peritonitis can eventually lead to peritoneal adhesions and peritoneal fibrosis. In the present study, we evaluated the possibility of using autologous genetically modified MCs for transplantation after the induction of peritoneal injury by acute inflammatory mediators or chronic instillation of PD fluid. METHODS: Rats were injected intraperitoneally either once with N-formyl-methionyl-leucyl-phenylalanine (fMLP), or thioglycollate, or PD fluid [i.e., Dianeal (Baxter Healthcare, Deerfield, Illinois, USA) or Physioneal (Baxter, Nivelles, Belgium)], or chronically (up to 8 weeks) with Dianeal. From 2 to 48 hours later, animals were injected with syngeneic MCs genetically modified to express the LacZ reporter gene. Rats were sacrificed 2 days later and expression of beta-galactosidase (beta-Gal) was visualized by X-Gal staining of excised tissues. Quantification of the percent area of beta-Gal-positive MCs on part of the parietal peritoneum was performed using computerized image analysis. RESULTS: The highest numbers of repopulated genetically modified MCs were observed 8 hours after a single thioglycollate injection, approximately 0.66% of a representative 2-cm2 area selected for study (corresponding to approximately 10% of the peritoneal surface). The number of genetically modified MCs found to repopulate the peritoneal surface following short-term injury varied with inflammatory mediator (thioglycollate > PD fluid > fMLP) and duration of exposure. No obvious differences were observed between the two PD fluids tested. Reimplantation of syngeneic genetically modified MCs was also observed after chronic instillation of PD fluid. CONCLUSIONS: These data demonstrate that transplanted genetically modified MCs repopulate the denuded areas on the peritoneal surface that were caused by acute or chronic inflammation. This technique opens possibilities of MC transplantation and gene therapy in order to prevent complications relevant to the continuous ambulatory PD setting

Apparent successful mesothelial cell transplantation hampered by peritoneal activation

Apparent successful mesothelial cell transplantation hampered by peritoneal activation.BackgroundMesothelial cell transplantation has been suggested to improve mesothelial repair after surgery, recurrent peritonitis and peritoneal dialysis.MethodsIn this study we evaluated mesothelial cell transplantation during the resolution phase of experimentally thioglycollate-induced peritonitis in rats. To this end 4 × 106 DiO-labeled autologous mesothelial cells were transplanted 1 week after peritonitis induction. Peritoneal inflammation and permeability characteristics were evaluated after another week.ResultsMesothelial cell transplantation after peritonitis resulted in incorporation of these cells in the parietal mesothelial lining, leading to an acute transient submesothelial thickening which was not seen in transplanted animals without prior peritonitis induction. Long-term functioning of these repopulated mesothelial cells leaded to peritoneal activation as evidenced by a ∼twofold increase in peritoneal lymphocytes (P < 0.01) and omental mast cell counts (P < 0.05), accompanied by the induction of inflammation markers monocyte chemoattractant protein-1 (MCP-1) (P < 0.01) and hyaluronan (P < 0.01) in the transplanted peritonitis group, but not in rats with peritonitis without mesothelial cell transplantation or in control rats without mesothelial cell transplantation (all four parameters P < 0.01). In addition, trapping of transplanted mesothelial cells in the milky spots of omental tissue and lymphatic stomata of the diaphragm both in control and thioglycollate rats seems to increase microvascular permeability, reflected by apparent increased diffusion rates of small solutes and proteins.ConclusionAltogether, our data underscore the importance of controlling peritoneal (patho)physiology and function in mesothelial transplantation protocols

Comparative Ultrastructural and Stereological Analyses of Unruptured and Ruptured Saccular Intracranial Aneurysms

Insight into processes leading to rupture of intracranial aneurysms (IAs) may identify biomarkers for rupture or lead to management strategies reducing the risk of rupture. We characterized and quantified (ultra)structural differences between unruptured and ruptured aneurysmal walls. Six unruptured and 6 ruptured IA fundi were resected after microsurgical clipping and analyzed by correlative light microscopy for quantitative analysis (proportion of the vessel wall area) and transmission electron microscopy for qualitative ultrastructural analysis. Quantitative analysis revealed extensive internal elastic lamina (IEL) thickening in ruptured IA (36.3% ± 15%), while thin and fragmented IEL were common in unruptured IA (5.6% ± 7.1%). Macrophages were increased in ruptured IA (28.3 ± 24%) versus unruptured IA (2.7% ± 5.5%), as were leukocytes (12.85% ± 10% vs 0%). Vasa vasorum in ruptured but not in unruptured IA contained vast numbers of inflammatory cells and extravasation of these cells into the vessel wall. In conclusion, detection of thickened IEL, leaky vasa vasorum, and heavy inflammation as seen in ruptured IA in comparison to unruptured IA may identify aneurysms at risk of rupture, and management strategies preventing development of vasa vasorum or inflammation may reduce the risk of aneurysmal rupture