Study On Molecular Mechanisms Of Vascular Unstability In Venous Malformations And Preparation Of Pingyangmycin Hydrogel

Part 1. Evaluation of vascular stability in venous malformations Objective: Vascular unstability plays an important role in the development of numerous diseases(e.g. vascular anomalies, inflammation of the retina caused by diabetes and tumors),and is closely associated with the treatment and prognosis of patients with tumors. This part aims to study the dilated venous channels and disorganized vascular smooth muscle cell layer, and systematically evaluate the status of blood vessels in venous malformations.Methods: The expression levels of vascular endothelial cells marker CD34 and perivascular cells marker a-SMA were analyzed to evaluate the perivascular cell coverage by double-labeling immunohistochemistry and double-labeling immunofluorescence in human normal skin tissues and venous malformations. The vascular ultrastructure was detected in normal skin tissues and venous malformations by transmission electron microscopy. Vascular adhesion molecules were analyzed in normal skin tissues and venous malformations by immunohistochemistry and real-time quantitative PCR.Results: Double-labeling immunohistochemistry data showed that the vessels were composed of tightly adjacent endothelial cells and perivascular cells in normal skin tissues. However, venous malformations were characterized by enlarged venous channels and decreased perivascular cell coverage. Meanwhile, the changes of ultrastructures(e.g. incomplete or absent basement membrane, poor junctions or tortuous surface between adjacent endothelial cells, and poor or no contact between adjacent perivascular cells) were found in venous malformations. Moreover, the protein and gene levels of adhesion molecules(VE-cadherin and N-cadherin) were significantly down-regulated in venous malformations compared to those in normal skin tissues.Conclusions: The decreased perivascular cell coverage, abnormal ultrastructures and down-regulated adhesion molecules were involved in venous malformations, whichprovided additional and strong evidence revealing the unstable blood vessels in venous malformations.Part 2. The association of balancing effect between Tie2 and TGF-P with the unstable vessels in venous malformations Objective; Venous malformations were the most common slow-flow vascular malformations characterized by irregular venous channels, luminal thrombi and phleboliths. Thus, it is of great importance to explore underling mechanisms of unstable vessels in venous malformations. Previous studies have reported that constitutive activation of Tie2 resulting from the mutation of coding gene TEK were involved in the development of venous malformations. This part aims to study the biological effects of TEK mutation, the expression level of TGF-P,and the association of above changes with unstable vessels in venous malformations.Methods: The expression and phosphorylation levels of Tie2 were detected by immunohistochemistry in normal skin tissues and venous malfonnations. Meanwhile,the correlation of perivascular cell coverage with phosphorylation level of Tie2 was analyzed by serial sections, double-labeling immunohistochemistry and immunofluorescence. After that, the protein and gene expression level of TGF-P was detected by immunohistochemistry and real-time quantitative PCR and analyzed for the correlation with the expression and phosphorylation levels of Tie2 by double-labeling immunofluorescence in normal skin tissues and venous malformations. Finally, the in vtro studies by western blot and real-time quantitative PCR were carried out for exploring the underlying mechanisms.Results; The immunohistochemistry and immunofluorescence data showed that the expression and phosphorylation levels of Tie2 were significantly up-regulated and negatively correlated with the perivascular cell cover^e, while the expression ofTGF-P was significantly down-regulated and positively correlated with the perivascular cell coverage in venous malformations compared to normal skin tissues.Meanwhile, negative correlation between Tie2 and TGF-P were detected. The in vitro studies showed the expression and phosphorylation levels of Tie2 were significantly up-regulated, while TGF-P was down-regulated in endothelial cells of human venous malformations compared to human umbilical vein endothelial cells. Moreover, Ang-1(agonist of Tie2) could down-regulate the expression of TGF-P, while recombinant TGF-(3 could suppress the expression and phosphorylation levels of Tie2 in human umbilical vein endothelial cells.Conclusions: The possible balancing effect between Tie2 and TGF-P in endothelial cells was closely associated with the unstable vessels in venous malformations, which may be helpful to provide novel strategies for the treatment of vascular unstability-related diseases(e.g. vascular malformations and tumor angiogenesis).Part 3. TGF-p/CCN2 signaling pathway: a novel potent target for treatment of venous malformations Objective: Previous studies have revealed the decreased extracellular matrix deposition in the blood vessels of venous malformations. This part aims to evaluate the expression level of matricellular proteins CCN family regulating the production of extracellular matrix, the role of TGF-p/CCN2,and the association of above changes with unstable vessels in venous malformations.Methods: The expression and phosphorylation levels of Tie2, the expression of matricellular proteins CCN1-CCN6 and TGF-P as well as the phosphorylation of PDGFRp were detected by immunohistochemistry in normal skin tissues and venous malformations, and analyzed for correlation. Meanwhile, the concurrent expression of TGF-p/CCN2 was evaluated in venous malformations by double-labeling immunofluorescence and real-time quantitative PCR. The correlation of CCN2expression with perivascular cell coverage was analyzed by serial sections,double-labeling immunohistochemistry and immunofluorescence. The in vtro studies by western blot and real-time quantitative PCR were carried out for exploring the underlying mechanisms.Results; The immunohistochemistry data showed that CCN2 was strongly expressed in the fibroblast-like cells, vascular endothelial cells and perivascular cells,but significantly down-regulated in venous malformation. Spearman rank correlation test and clustering analysis revealed the positive correlation of CCN2 and TGF-p.Meanwhile, the expression level of CCN2 was positively correlated with the perivascular cell coverage in venous malformations. In addition,the concurrent down-regulation of CCN2 and TGF-P were found. The in vitro studies showed that exogenous TGF-P or CCN2 could up-regulate the expression of CCN2 and extracellular matrix components in human umbilical vein endothelial cells. Also, the sclerosant Bleomycin A5 could up-regulate the expression level of TGF-p/CCN2 and extracellular matrix. Most importantly, the up-regulation of TGF-p/CCN2 and a-SMA was detected in the patients treated with Bleomycin A5.Conclusions: TGF-p/CCN2 signaling pathway in endothelial cells was significantly down-regulated in venous malformations, which may be associated with the unstable vessels resulting from the decreased extracellular matrix deposition and abnormal perivascular cell coverage in venous malformations. TGF-(3/CCN2 signaling pathway may be a novel potent target for treatment of venous malformations.Part 4. Pingyangmycin/PEG-PCL-PEG hydrogel designed for the treatment of vascular malformations Objective: Vascular malformations affect the appearance and infiltrate normal tissues.Sclerotherapy is a preferable option for these diseases. This part aims to prepare a new drug delivery system, Pingyangmycin hydrochloride-loaded PEG-PCL-PEG hydrogel(PYM/PECE hydrogel), and improve the therapeutic effects of intravascular sclerotherapy and embolotherapy.Methods: The biodegradable and temperature sensitive PYM/PECE hydrogel was successfully synthesized. The effect of PYM/PECE hydrogel on cell growth curves and changes of cell morphology and activities measured by MTT assay were carried out in human umbilical vein endothelial cell line(HUVEC). The control-release effect of PYM/PECE hydrogel was evaluated both in vitro and in vivo. The therapeutic effects of sclerotherapy and embolotherapy were evaluated by macroscopic examinations and Hematoxylin-Eosin(H-E) staining of cross-section of rabbits' central auricular arteries. The gel degradation behavior and toxicity test were evaluated in healthy rats and rabbits.Results: The hydrogel system was a free-flowing sol at room temperature, forms a non-flowing gel at physiological temperature, and could sustain about 14 days in situ.The in vitro and in vivo results showed that the control-release effect of PYM/PECE hydrogel was obvious. The MTT results show that PYM/PECE hydrogel could decrease the cell-viability of HUVEC cells in a time-dependent manner more mildly than PYM-injections. The in vivo studies demonstrated 14 days after PYM/PECE hydrogel was injected, occlusion of auricular arterial lumen were detected at the injected site. Furthermore, no toxic responses of the hydrogel system were observed.Conclusions; Our data indicate that the PYM/PECE hydrogel could be a safe candidate for in situ gel-forming controlled drug delivery system. It showed excellent potential as an alternative to sclerotherapy and interventional embolization materials.