The Potential Effect Of Arginase I On Athersclerosis

BackgroudInflammation plays an important role in the generation and development of atherosclerosis. Inflammation is often related to the development of atherosclerosis, severity and its consequences. The secretion of inflammatory factors can trigger chemotaxis and migration of monocytes and macrophage, and further to aggravate inflammatory reaction. Smooth muscle cells (SMCs) locate in the middle layer of vascular wall and keep "quiescent" under normal physiological conditions. However, these kinds of cells often acquire a state of dedifferentiation and exhibit the character of "synthetic" phenotype under atherosclerotic conditions, contributing to vascular pathological changes by synthesizing and secreting certain inflammatory factors.It has been demonstrated that arginase Ⅰ have the ability to promote SMCs proliferation. However, its role in inflammation has remains unknown. Both arinase and nitric oxide synthetase (NOS) catalyze L-arginine, which can be catatlyzed by induced nitric oxide synthetase (iNOS) to produce excess nitric oxide (NO) in cytoplasm. Under inflammatory conditions, the iNOS can produce excessive nitric oxide (NO), which can produce proinflammatory effects of ONOO-by reaction with intracellular superoxide anion (O2-). Then, it may inhibit the inflammation to decrease the catalyzing substrate of iNOS.Based on the previous studies, we make the following assumptions:1) Arginase Ⅰ has the ability to inhibit inflammation;2) Arginase Ⅰ inhibits the inflammation by decreasing catalyzing substrate of pro-inflammatory iNOS.Based on the above ideas, we design the experiments in vivo and in vitro to validate the hypothesize in the present study. We aim to investigate the role of Arg Ⅰ in inflammaiton of SMCs and try to look for new opportunities and therapeutic targets in SMC associated inflammatory disease.Material and Methods1. Cell Culture and Inflammatory Cell Model: Human aorta smooth muscular cells (hASMCs) were cultured and lipopolysaccharide (LPS) was used to induce inflammatory reaction in hASMCs in our experiments.2. ARG Ⅰ Interference in hASMCs: Transient transfection with Lipofectamine2000was performed in our experiments.3. Animal Model and ARG Ⅰ Interference: New Zealand White rabbits were subjected to balloon injury of the abdominal aorta and fed with high fat diet to establish atherosclerotic plaque model. At the end of12weeks, lentivirus vectors carried ARG Ⅰ or si-ARG Ⅰ was locally delivered to plaques as our laboratory described before. The rabbits were continued to fed with high fat food for4weeks, then were anesthetized and killed for further studies.4. ELISAAssay: To determine the extent of cellular inflammatory reaction by detecting released TNFa in cell culture media supernatant.5. Real-time RT-PCR, Western-blot Analysis and Enzyme Activity Measurement: To determine the effect of LPS on the leve of gene, on protein content or on catalyzing activities of Arg Ⅰ and iNOS, Real-time RT-PCR, Western-blot and enzyme activity measurement were performed, respectively. 6. Monocyte Chemotaxis and Migration: Co-culture system of THP1and hASMCs was established in Transwells to determine the effect of Arg Ⅰ on hASMCs.7. Immunocytochemistry and Immunohistochemistry: To indicate the locations of the Arg Ⅰ and iNOS, subunit of NF-κB, components of atherosclerotic plaque and inflammatory factors in it, immunocytochemistry and immunohistochemistry were performed, respectively.8. FCM and Laser Confocal Microscopy: To determine the content of intracellular ONOO-, ROS and O2.-, flowcytometry and laser confocal microscopy were chosen in our experiments.Results1. Induction of LPS on produce of TNFα in hASMCs is time-dose dependent LPS (>10ng/ml) induced TNFa release in hASMCs, and TNFα content increased as LPS stimulating dose increased or stimulating time extended. LPS caused quick release of TNFα, but the rate of quantity increasing of TNFα reduced after24hours, and the rate decreased more obviously after48hours.2. LPS increases TNFα production via iNOS activity in hASMCs LPS induced TNFa release in hASMCs, but this course was suppressed in the presence of iNOS inhibitors (AG or1400W), and the release of TNFα was abolished by about70%to80%, proving that LPS increased TNFa production via iNOS activity in hASMCs.3. LPS upregulates expression of Arg Ⅰ and iNOS simultaneously Expression of Arg Ⅰ and iNOS increased with the stimulus of LPS in both gene and protein level, but the increasing velocity of Arg Ⅰ lagged behind iNOS. iNOS mRNA level achieved peak after8hours, whereas Arg Ⅰ mRNA level achieved peak after12hours, and then both of their amplification declined; on protein level, iNOS continued to increase with LPS stimulus, whereas Arg Ⅰ began to decline48hours after stimulus.4. Substrate competition between Arg Ⅰ and iNOS Accordant with above tendency, the catalytic activity of the two enzymes increased with LPS stimulus. But different with the continuous increase of iNOS activity, Arg Ⅰ activity apparently declined after48hours. Moreover, iNOS activity slightly increased as Arg Ⅰ activity began to decline. After pretreating cells with L-arginine which is the common substrate of the two enzymes, both of their catalytic activities increased after12hours and continued to increase till72hours after LPS stimulus.5. Arg Ⅰ suppresses TNFα production and monocyte chemotaxis and migration Arg Ⅰ apparently suppressed TNFα release induced by LPS in hASMCs, and Arg Ⅰ inhibition or interference augmented TNFα release. Monocyte chemotaxis and migration to hASMCs apparently increased after hASMCs being induced by LPS, whereas Arg Ⅰ upregulation decreased Monocyte chemotaxis and migration and Arg Ⅰ downregulation increased the chemotaxis and migration.6. Arg Ⅰ inhibits NO release without decreasing iNOS expression Immunohistochemistry showed that Arg Ⅰ and iNOS were largely colocalized in hASMCs. NO release was decreased with Arg Ⅰ elevation but increased with Arg Ⅰ inhibition or interference; whereas Arg Ⅰ elevation had no effect on iNOS expression, indicating Arg Ⅰ suppresses NO release through substrate competition other than decreasing iNOS expression.7. Arg Ⅰ decreases ROS,O2.-, and ONOO-generation in hASMCs LPS-induced generation of ROS, O2.-, and ONOO-was attenuated by intra-hASMCs Arg Ⅰ elevation, and the increase of O2.-andONOO-generation was also attenuated by iNOS inhibition, without apparent affect on ROS generation.8. Arg Ⅰ inhibits TNFα produce via NF-kB activation LPS induced NF-kB activation, and the latter caused TNFα generation in cells. Arg Ⅰ decreased TNFα produce via suppressing NF-kB activation.9. Arg Ⅰ attenuates atherosclerotic plaque inflammation Arg Ⅰ transfection caused decrease of TNFα and iNOS expression and lessen of macrophages in plaque, and Arg Ⅰ inhibition acted conversely. The area ratio between TNFa and SMCs showed that Arg Ⅰ attenuated the ratio apparently. Conclusion1. Arginase Ⅰ has the ability to inhibit inflammation in SMCs.2. Arginase Ⅰ can inhibit inflammation in atherosclerotic plaque.3. Arginase Ⅰ inhibit inflammation by competiting common catalyzing substrate with the pro-inflammation factor iNOS and then reduce the production of toxic ONOO-generation. BackgroundAtherosclerotic plaque rupture is the main cause of myocardial infarction, cerebral thrombosis and other cardiovascular or cerebral vascular acute events. Inflammatory cell infiltration and secondary inflammatory response triggered within the atherosclerotic plaque play important roles in plaque rupture. And inflammation exists through the whole process of the atherosclerosis:for occurrence, development, plaque rupture, thrombosis and even leads to death. Therefore, it can stabilize the vulnerable atherosclerotic plaque and then prevent cardiovascular or cerebral vascular acute events to suppress the inflammation effectively.Vascular smooth muscle cells (VSMCs) act as "double-edge sword" in the generation and development of atherosclerosis. In the early stage of atherosclerosis, the proliferation of SMCs may contribute to the formation of plaque; And in the middle stage of atherosclerosis, the formation of foam cells derived from SMCs can promote the development of plaque; While in the late stage of atherosclerosis, the proliferation of SMCs may contribute to the thickness of fibrous cap and then increase the stability of the atherosclerotic plaque. It has been documented that Arg Ⅰ can promote SMCs proliferation. And our previous studies also have demonstrated the inflammation inhibited ability of Arg Ⅰ. However, the role of Arg Ⅰ in the stability of atherosclerotic plaque remains unknown.Based on previous studies, we make the following hypothesis:1) Over expression of Arg Ⅰ can increase the stability of atherosclerotic plaque in the advanced stage of atherosclerosis;2) Arg Ⅰ promote the stability of atherosclerotic plaque through suppressing inflammation of macrophages and promoting SMCs proliferation.Based on the above ideas, we design the experiments both in vivo and in vitro to validate the hypothesis in the present studies. Our studies try to demonstrate the role of Arg Ⅰ in the stability of atherosclerotic plaque and look for new opportunities and therapeutic target for vulnerable plaque stability and cardiovascular acute events prevention.Meterials and Methods1. Animal Model: Male New Zealand White rabbits were fed with high fat diet (1%cholesterol) and subjected to balloon injury to establish atherosclerosis model.2. Cell Culture: Human mononuclear cells derived macrophage (MDM) and human aorta smooth muscle cells were cultured.3. Arginase Ⅰ Intervention: Lentiviral vector and arginase inhibitors were both used in our experiments.4. Sirius Red Staining and Oil Red O Staining: The methods were used to indicate macroscopic structure, collagen content and lipid content of the plaque.5. Immunohistochemistry and Immunocytochemistry: Immunohistochemistry was used to detect the content of smooth muscle cells and macrophages, the expression of TNFα and IL-6as well as the positive rate of proliferating cell nuclear antigen (PCNA) in the plaque; immunocytochemistry was used to indicating the location and expression of aginase Ⅰ and iNOS in macrophages.6. Monocyte Chemotaxis and Migration: Co-culture system of human monocyte and human MDM were used in our experiments. 7. Western Blot and Enzyme Activity Measurement: The methods were used to detect the expression and catalyzing activities of Arg Ⅰ and iNOS.8. Flow Cytometry: To detect the intracellular production of ROS and O2.-,FCM were used.9. ELISA: ELISA were used to detect the content of TNF α and IL-6in serum and in cell culture supernatant.10. EDU Incorporation: EDU incorporation was used to evaluate the proliferation of hASMCs.Results1. Arginase Ⅰ affects morphosis and components of atherosclerotic plaque: Plaques transfected with arginase Ⅰ had more compact texture and less necrosis cores than plaques transfected with LacZ and treated with physiological saline; collagen content and quantity of smooth muscle cells increased(both p<0.01,VS.LacZ transfection), whereas quantity of macrophages and lipid cores significantly decreased((both p<0.01,VS.LacZ transfection); plaque vulnerability index significantly decreased(both p<0.01, VS.LacZ transfection).2. Arginase Ⅰ suppresses inflammation in plaque: Concentration of TNF and IL-6in plaques significantly decreased by arginase Ⅰ transfection (both p<0.01, VS. LacZ transfection), whereas the concentration of the above cytokines in the serum did not significantly alter (p>0.05, VS. LacZ transfection); expression of IL-1β wasn't significantly affected by arginase Ⅰ transfection; expression of arginase Ⅰ and activity of arginase were significantly increased (both p<0.01, VS. LacZ transfection) by arginase Ⅰ transfection, while expression of arginase Ⅱ was depressed significantly(p<0.05, VS. LacZ transfection).3. Arginase Ⅰ suppresses inflammation induced by LPS in macrophages: Production of TNFa and IL-6induced by LPS was depressed by arginase I upregulation in human macrophages and mice macrophages (All p<0.01, VS. LPS alone group); while the release of the above cytokines was increased by arginase Ⅰ downregulation or inhibition(All p<0.01, VS. LPS alone group),and inhibition acted more obviously than downregulation. Further studies validated that LPS induced monocytes chemotaxis and migration to macrophages, and upregulation of arginase Ⅰ in macrophages aggravated monocytes chemotaxis and migration (Both p<0.01, VS.LPS alone group),while arginase Ⅰ downregulation or inhibition suppressed monocytes chemotaxis and migration (All p<0.01, VS. LPS alone group)5. Arginase Ⅰ exerts effect of suppressing inflammation via substrate competition with iNOS: Induction of LPS on protein expression and enzyme activity of arginase and iNOS appeared to be time dependent; addition of L-arginine increased the expression of TNF and IL-6induced by LPS in macrophages and inverted the suppression of arginase Ⅰ on the two cytokines; whereas iNOS inhibitor suppressed the release of the two cytokines induced by LPS. Arginase Ⅰ transfection increased intracellular arginase activity, but decreased iNOS activity, and the two enzymes colocalized largely in cells. But regulation of Arginase Ⅰ didn't change the protein expression of iNOS. The results suggested that Arginase Ⅰ exerts effect of suppressing inflammation via substrate competition with iNOS rather than reducing its expression.6. Arginase Ⅰ decreases O2.-, ROS and ONOO-generation in macrophages: Arginase Ⅰ elevation attenuated production of O2.-, ROS and ONOO-induced by LPS, whereas arginase Ⅰ suppression or inhibition acted conversely; iNOS inhibition augmented production of O2.-andONOO-induced by LPS, but had little effect on ROS.7. Arginase Ⅰ contributes to proliferation of smooth muscle cells: Quantity of smooth muscle cells in plaque transfected with arginase Ⅰ was significantly increased (p<0.01, VS. LacZ transfection) compared with plaque transfected with LacZ or treated with physiological saline; arginase Ⅰ elevation promoted proliferation of smooth muscle cells in plaque and cultured human aorta smooth muscle cells, while arginase Ⅰ depression or inhibition acted conversely.Conclusion1. Arginase Ⅰ contributes to atherosclerotic plaque stability;2. Arginase Ⅰ has effect of suppressing inflammation;3. Arginase Ⅰ exerts the effect of suppressing inflammation through substrate competition with the pro-inflammation factor iNOS and reducing the production of ONOO-by decreasing the catalysate NO of iNOS;4. Arginase Ⅰ contributes to the proliferation of human aorta smooth muscle cells;5. Arginase Ⅰ exerts the effect of stabilizing atherosclerotic plaque through suppressing inflammation and promoting proliferation of smooth muscle cells.