l-arginine/NOS/NO signaling pathway plays a critical role in controlling variety of vascular diseases. However, whether NOS inhibition by L-NAME suppresses late embryonic development is undefined. The aim of this study is to determine whether NOS inhibition by L-NAME is critical for late embryonic rat hind limb development. The pregnant rat at E13.5 administrated L-NAME by consecutive intraperitoneal injection. The embryos been harvested from E16.5 to E 20.5. Hematoxylin and Eosin Staining, Immunofluorescence and Immunohistochemistry performed to determine hind limb Vasculogenesis, HUVEC culture, Adenoviral PFKFB3 infection, Real time PCR and western blot were performed to determine whether l-arginine/NOS/NO pathway controlling late embryonic hind limb development through PFKFB3 mediated angiogenetic pathway. NOS inhibition by L-NAME resulting in late embryonic hind limb developmental defects characterized by severe hemorrhage. The in vivo studies showed that NOS inhibition strongly suppressed hind limb angiogenetic remodeling by impairing differentiation of endothelial cells and smooth muscle cells, and extracellular matrix synthesis. For underlie mechanism, our studies indicated that L-NAME treatment dramatically suppresses PFKFB3 expression in hematopoietic progenitor cells, tubulogenetic endothelial cells and smooth muscle cells. Knockdown of PFKFB3 dramatically inhibits the expression of angiogenetic genes, as well as tubulogenesis and extracellular matrix related genes. Taken together, our data in this study demonstrated that l-arginine-eNOS-NO pathway is important for rat hind limb development during late embryonic stage. This could be both a useful animal model and a promising therapeutic treatment for defects of late embryonic developmental hind limbs.

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Objective: Smooth muscle cell (SMC) phenotypic switching is associated with development of a variety of occlusive vascular diseases. Paeonol has been reported to be involved in suppressing SMC proliferation. However, it is still unknown whether paeonol can regulate SMC phenotypic switching, and which eventually result in suppressing vasculogenesis. Methods: Murine left common carotid artery was injured by completely ligation, and paeonol was administrated by intraperitoneal injection. Hematoxylin and eosin (H&E) staining was performed to visualize vascular neointima formation. Rat aortic SMCs were used to determine whether paeonol suppresses cell proliferation and migration. And murine hind limb ischemia model was performed to confirm the function role of paeonol in suppressing vasculogenesis. Results: Complete ligation of murine common carotid artery successfully induced neointima formation. Paeonol treatment dramatically reduced the size of injury-induced neointima. Using rat aortic primary SMC, we identified that paeonol strongly suppressed cell proliferation, migration, and decreased extracellular matrix deposition. And paeonol treatment dramatically suppressed vasculogenesis after hind limb ischemia injury. Conclusion: Paeonol could regulate SMC phenotypic switching through inhibiting proliferation and migration of SMC, which results in inhibiting ischemia-induced vasculogenesis.

SM22α Smooth muscle protein 22α MHC Myosin heavy chain Vascular smooth muscle cells phenotypic switching contributes to development of variety vascular diseases, including post angioplasty restenosis, aneurysm, atherosclerosis and pulmonary hypertension 1,2. Maturated smooth muscle cell exhibits dramatically plasticity. The principal function of maturated smooth muscle cells is contraction regulating. However, smooth muscle cell can undergo phenotypic switching from differentiated stage to dedifferentiated stage, which characterized by reduced expression of smooth muscle specific genes, such as smooth muscle myosin heave chain, smooth muscle light chains, smooth muscle α-actin, SM22α, smooth muscle α-tropomyosin, smoothelin, h1-calponin, h-calponin, h-caldesmon, β-vinculin, metavinculin, telokin and desmin, whereas enhanced proliferation and migration 1,3. Emerging evidence indicated that Traditional Chinese Medicine is effective in treatment variety cardiovascular diseases. Salvia miltiorrhiza is a traditional Chinese medicine and widely used for treatment of cardiovascular diseases. Tanshinone II A is a key ingredient separated from salvia miltiorrhiza. Previously reports demonstrated that Tanshinone II A attenuates angiotensin II induced cardiac hypertrophy and cardiac fibroblast proliferation through MEK/ERK pathway 4,5. Tanshinone II A inhibits inflammatory response induced by myocardial infarction 6. Tanshinone II A suppresses cells growth on human hepatocellular carcinoma cells 7 , human gastric carcinoma cells. Tanshinone II A prevents apoptosis in PC12 induced by serum starvation 8. Tanshinone II A inhibits HCC cell invasion through suppressing the activity of MMPs 9. In treatment of vascular diseases, Tanshinone II A attenuates atherosclerosis calcification through suppressing oxidative stress 10. Tanshinone II A inhibits proliferation of pulmonary artery smooth muscle cells induced by hypoxia 11. Tanshinone II A induces pulmonary artery smooth muscle cell apoptosis through suppressing JAK1/STATS signaling pathway 12. And inhibits vascular smooth muscle migration through suppressing ERK1/2 MAPK signaling pathway 13. However, whether Tanshinone II A plays a critical role during pathological vascular remodeling is largely unknown. Our preliminary data shown that Tanshinone II A significant promotes KLF4 expression. This study aimed to test hypothesis that Tanshinone II A regulates pathological vascular remodeling through KLF4 mediated smooth muscle cell phenotypic switching. Material and methods Mouse common carotid artery complete ligation injury animal model. Completed ligation of mouse left common carotid artery induced vascular remodeling was performed as previously described 14. Briefly, Mouse were pretreated with Tanshinone II A (5 mg/kg) for 3 consecutive days and anesthetized with ketamine (80 mg/kg) and xylazine (5 mg/kg) by intraperitoneal injection 15. Exposed the left common carotid arteries and completely ligated at bifurcation with 6-0 silk. The right common carotid artery was going the same...

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