Acid Sphingomyelinase/ceramide Modulates Functional And Structural Remodeling Of Carotid Arteries In Simulated Weightless Rats

During outer space flight, exposure to microgravity environment may induce adaptive changes in both function and structure of cardiovascular system. The disappearance of hydrostatic pressure will be followed by transmural pressure changes of the arterial system. Pressure of arteries in the upper body represents a status of “hypertension”, while the pressure of arteries in the lower body represents a state of “hypotension”. These changes can lead to a series of adaptive alterations in arterial system. Massive researches based on both human and animal models of microgravity or simulated microgravity have revealed hypertrophic changes and enhanced activity of local rennin-angiotensin system(L-RAS) in carotid arteries. Based on common conclusions of vascular physiology and pathology, these changes in structure and biological responses should come along with an obvious increase in vascular constriction. However, recent studies have demonstrated that the contractile responsiveness of carotid artery doesn’t change much or even has a tendency to reduce in simulated microgravity rats. The results show that there might be sophisticated mechanisms involved in regulative effects on carotid artery in simulated microgravity rats, which are still not quite clear.Acid sphingomyelinase(ASM) exists in almost all kinds of cell types in eukaryotes. It can cleave sphingolipids in the membrane into ceramide(Cer) and choline sphingomyelin, which have multiple functions in regulation. And it’s also a key factor in regulating cell death. Because ASM performs its functions mainly through ceramide, therefore it is always referred to as ASM/Cer. ASM/Cer exists in both vascular smooth muscle cells and endothelial cells. Diseases like atherosclerosis, hart failure and ischemia-reperfusion injury can lead to an up regulation of ASM/Cer level. Then it can cause the formation of atheromatous plaque, endothelial dysfunction, cardiac dysfunction and apoptosis of vascular smooth muscle cells via a series of mechanisms, in which local reactive oxygen species(ROS) may play a very important role.To find out whether ASM/Cer has changed after simulated microgravity exposure and how the change works in regulation of vascular function and structure in carotid artery of rats, we performed the following experiments:(1)4 week tail suspension hindlimb unloading was carried out to rats to simulate the effect of weightlessness. Western blot, immunohistochemistry and immunoinfluorescence staining were conducted to test the level of ASM/Cer in carotid artery.(2) Carotid arteries of suspended group(SUS) was incubated with C6-ceramide(C6-Cer) and carotid arteries of control group(CON) was incubated with C6-Cer or desipramine(Dpm). Then DHE fluorescent probe was used to evaluate the changes of superoxide anion(O2-·) level caused by incubation with C6-Cer and Dpm. Isometric force recording system was used to examine the changes of vascular function with or without incubation.(3)Western blot, immunohistochemistry and terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL) staining were used to test the apoptosis and proliferation of vascular smooth muscle cells in carotid artery. The effects of C6-Cer and Dpm incubation on apoptosis and proliferation level were also detected.The main findings of our study are as follows:(1)As is shown by western blot and immunohistochemistry results, 4 week tail suspension significantly decreased ASM expression in carotid artery. And immunoinfluorescence staining revealed a significant decrease of Cer content in SUS.(2)There was scarcely a change in vasoconstriction response to potassium chloride(KCl) and phenylephrine(PE) or vasodilation response to acetylcholine(ACh) and sodium nitroprusside(SNP) in carotid artery of SUS as well as O2-? level of carotid artery when compared with CON. C6-Cer incubation(10 μM) restored Cer content in SUS but didn’t change Cer content in CON. The level of O2-· and vasoconstriction response to KCl and PE of carotid artery in SUS was increased significantly after C6-Cer incubation, while no evident influence on the vasodilation response was observed. Increased vasoconstriction response to KCl and PE of carotid artery in SUS caused by C6-Cer incubation was erased by a contemporary incubation with superoxide dismutase(SOD, 50 U/L), a kind of O2-· scavenger. C6-Cer incubation had no influence on the level of O2-· and vasoconstriction or vasodilation response of carotid artery in CON. Dpm incubation decreased Cer content, the level of O2-· and vasoconstriction response of carotid artery in CON while had no effect on vasodilation response.(3)TUNEL staining and proliferating cell nuclear antigen(PCNA) test have shown that simulated microgravity promoted proliferation and restrained apoptosis of vascular smooth muscle cells of carotid artery. However, after C6-Cer incubation, which restored the content of Cer, the effect of simulated microgravity on vascular smooth muscle cells could be reversed. C6-Cer incubation had no effect on carotid artery of CON. Incubation with Dpm, which would decrease Cer content of carotidartery in CON, could promote proliferation and suppress apoptosis of vascular smooth cells.In conclusion, the results suggest that simulated weightlessness would decrease ASM/Cer content of carotid artery and through a reduced O2-· level to down regulate vasoconstriction response. The down regulated ASM/Cer may perform as a counterpart of other pro-vasoconstriction factors to maintain relative homeostasis of vascular function. Also, decreased ASM/Cer may up regulate proliferation and down regulate apoptosis. And that could be part of the mechanism which decreased ASM/Cer may participate in the process of vascular function and structure remodeling.