SRY Gene Transferred By Extracellular Vesicles Accelerates Atherosclerosis By Promotion Of Leukocyte Adherence To Endothelial Cells

Extracellular vesicles(EVs) are small membrane vesicles. EVs may often contain abundant functional transmembrane and cytosolic proteins, mRNAs,mi RNAs, and DNAs. EVs participate in important biological processes, such as surface-membrane trafficking and horizontal transfer of borne molecules among neighboring cells. Our previous study showed the existence of DNAs in EVs that could be transferred fromone cell to another by endocytosis or fusion. The transferred EV DNAs have the ability to influence the function of the recipient cells by increasing DNA-coding mRNA and protein levels. However, the role of the EV DNAs in the pathogenesis of disease is not clear. There is increasing evidence that EVs play a pivotal role in tumorigenesis, which can occur in adjacentand remote locations. There are several pieces of evidence that EVs, via intercellular communication, play an important pathological role in atherosclerosis by coordinating the actions of platelets and leucocytes adherent to endothelial cells 1. However, the role of EV DNAs in atherogenesis is unclear.The fundamental biological role of the Y chromosome is to impart male characteristics, but it is also linked to cardiovascular disease. For example, several studies have shown that SRY(sex determining region, Y), a gene in the Y chromosome, was responsible for increasing the angiotensin II and noradrenaline levels. In addition, polysomy of the Y chromosome(mostly 47, XYY karyotype) was related to increased risk of coronary artery disease(CAD) in men. We hypothesize that SRY DNAs in plasma EVs are involved in the pathogenesis of atherosclerosis by the transfer of SRY DNA to recipient cells and endothelial cells, and increased adherence of leukocytes to endothelial cells.1.1 Methods1.1.1 Increased SRY GCNs in plasma EVs or leukocytes from male patients with CADIn the first part, we observed the gene copy number(GCN), mRNA and protein expression of SRY in extracellular vesicles(EVs) and leukocytes from male coronary artery disease patients and the normal men by PCR, real-time PCR, DNA sequencing and immunoblotting.1.1.2 Transportable and functional SRY DNA in EVs from SRY-transfected HEK293 cells to receipt cellsIn second part, we observed the effect of EVs from SRY-HEK293 cells to the protein expression of CD11-a in THP-1 cells and ICAM-1 in HUVECs. Moreover, by EMSA, we measured the regulation of SRY protein to the promoter of CD11-a and ICAM-1 gene. The adhesion between the monocytes and endothelial cells was also measured after treatment of SRY-EVs.1.1.3 Effect of SRY DNA in EVs on the atherosclerosis in ApoE-/- miceIn this part, we observed aorta atherosclerotic plaques in vivo in the ApoE-/- mice after treatment of SRY-EVs. And the CD11-a and ICAM-1 protein expression in plasma from mice were measured by ELISA.1.2 Result1.2.1 We found, by PCR and gene sequencing, SRY DNA GCNs of SRY were higher in plasma EVs from male CAD patients than healthy malesubjects.And the GCNs in leukocytes from males were higher in CAD patients than healthy controls. Moreover, the mRNA and protein expressions of SRY in leukocytes were also higher in CAD patients than healthy controls. Immunoblotting showed that CD11-a protein expression was significantly increased in leukocytes from male patients with CAD compared with healthy male subjects1.2.2 PCR study showed cytomegalovirus(CMV)-SRY DNA in SRY-HEK293 cells, as well as in their EVs. The CD11-a protein expression was significantly increased in SRY-EV-treated THP-1 cells, and ICAM-1 protein expression was higher in SRY-EV-treated HUVECs than EV-treated control cells.We found increased DNA-binding ability of SRY protein to the promoters of ICAM-1 and CD11-a in SRY-EV-treated cells compared with vehicle and control EV groups by EMSA. Moreover, the presence of SRY-EVs increased the number of THP-1 cells that adhered to HUVECs.1.2.3 We found that the size of atherosclerotic plaques in the aorta was increased in ApoE-/-knockout mice injected with SRY-EVs relative to the control mice. Moreover, the area of lipid vacuoles(oil red staining) was also increased in the vascular wall of ApoE-/-mice treated with SRY-EVs. To elucidate the functional role of SRY on ICAM-1 and CD11-a in vivo, we measured, by ELISA, the levels of CD11-a in monocytes and ICAM-1 in the vessels of the mice, and found increased levels of CD11-a in monocytes and ICAM-1 in the vessels from ApoE-/- mice injected with SRY-EVs.1.3 Conclusion1.3.1 SRY gene fragment in plasma EVs from male. EVs from male patients with CAD had higher SRY GCN. And leukocytes had higher SRY GCN and mRNA and protein expression in male CAD patients.1.3.2 SRY-EVs regulated the adhesion between the monocytes and endothelial cells by CD11-a and ICAM-1.1.3.3 The SRY gene in plasma EVs transferred to vascular endothelial cells may play an important role in the pathogenesis of atherosclerosis.Backgrounds: Ischemia-reperfusion injury(IRI) of kidney is clinically important complex pathophysiological process functioning in many ways involving many molecules and multiple factors. The various factors does not function alone, and they work together, which is key for structure and function of the kidneys from normal to injury and irreversible progress on organ failure and function impairment. Blood in kidney accounts for 20% of heart output volume, so kidney is high perfusion organ. When no sufficient blood is provided for kidney, the tissues and organs of kidney without sufficient blood could be recovered by oxygen offer after the following blood supply through infusion and rehydration, however, it not only could cause inflammation reaction of parenchymal cell and influence the survive and function of ischemia organs, but also result in systemic inflammatory response syndrome involving remote organs, such as heart, liver, lung, kidney, small intestine and brain, thus brings about more serious organs impairment and function obstacle. Clinically, for the hypovolemic shock, and some surgeries with temporarily blocking in blood flow to kidney, the recovery reperfusion of kidney is likely to cause kidney IRI. Serious IRI may lead to acute renal failure(ARF) and its mortality rate is greater than 50%. Kidney IRI is also the main non-antigen-dependent factor that clinically affects the short and long term survival of kidney, the severity degree and subsequent reactions of which will influence short-, long-term survive of transplanted kidney and functional status. How to prevent and relieve kidney IRI is always an important topic in kidney care and it is increasingly becoming the focus for clinic and research.Tirofiban, a new reversible non-peptide platelet surface glycoproteins GP IIb/Ⅲa receptor antagonist, is non-peptide tyrosine derivatives from Integrin family and can competitively inhibit the binding of Fibrinogen with platelet glycoproteins IIb/Ma receptor, platelet aggregation and thrombus formation. Tirofiban has a very short half-life antiplateletand relatively long plasma half-life. GPIIb/Ⅲa platelet aggregation receptor antagonist blocks the final pathway of platelet aggregation and can reduce the functional time of platelet-dependent blood flow without any complication, so it is considered to be one of the most effective antiplatelet drugs. The function of Tirofiban include anti-inflammation, anti-apoptosis, anti-cell proliferation and antioxidant. But there is no report whether Tirofiban has a protective effect on renal renal ischemia injury. So our research applied the technologies of Western blot, TUNEL cell apoptosis and ELISA to observe the serum concentration of muscle anhydride urea nitrogen, the expression of Bax and BCL-2 and Bax/Bcl-2 ratio in kidney organs and cell apoptosis in kidney and explored the protection effects and function mechanism of Tirofiban for the kidney IRI of rat, which will provide theoretical basis for clinical prevention and treatment of kidney injury.Objective: To investigate the effects of Tirofiba on ischemia-reperfusion injury(IRI) of rat and explore the pharmacological mechanism in kidney protection of Tirofiba from the aspects of SOD activity, MDA content and histopathological changes in kidney tissues and organs.Methods: We randomly selected 20 male SD rats,weighing 250-260 g and randomly divided them into four groups: Sham-operation Group(control), Sham+Tirofiban Group(Group T), renal ischemia injury(IR team), ischemia-reperfusion of kidney(Group IR) and ischemia-reperfusion of kidney + Tirofiban(IR+T). Each group included 5 rats. The 24 h model of ischemia-reperfusion of kidney was based on the method of resecting right kidney tissues and removing artery clamps after 45 min of clipping the left kidney artery by bulldog clamp to restore the blood supply. Control group: opening the abdominal cavity, separating tissues surrounding the kidney pedicle without occlusion of bilateral pedicle; T group: Tirofiban supply(dose of 200μg/kg); IR: reperfusion immediately after 45 min of clipping right kidney arteriovenous and samples were collected after 24h; IR+T: 15 min the same dose of Tirofiban was given before clipping left kidney pedicle. After the model construction, the serum and kidney tissues samples were collected. The muscle anhydride SCR, LDH, MDA, SOD, Bax and Bcl-2 expression and Bax/Bcl-2 ratio in kidney organs, the indexes for cell apoptosis in kidney organs were detected. Also, pathology results od kidney under HE staining observation and changes of kidney structure after IRI by electron microscope were tested.Results:1.Tirofiban could improve the renal failure caused by kidney IRIFirstly, we established IRI model on SD rat and observed the protection effects of Tirofiban on function injury of kidney caused by IRI. IRI brought about increased serum level of AST, urea nitrogen and blood muscle anhydride and serious damage in kidney function. Additionally, HE staining illustrated the visible damage in renal tubular epithelial cell, vacuolar degeneration and necrosis, tube cavity expansion, serious capillary congestion and hemorrhagic spot of kidney tubules and kidney parenchyma, erythrocyte aggregation, necrotizing precipitation, glomerulus degeneration, serious edema in kidney interstitial and swelling necrosis degeneration. With Tirofiban pretreatment, serum level of AST, urea nitrogen and blood muscle anhydride were dramatically decreased and the pathology score was reduced. The pathological biopsy indicated that there were a small amount of cell necrosis. Besides, the symptoms that the damage degree of kidney tubules was relieved, inflammatory cell infiltration was not obvious and simple renal interstitial edema and hyperemia were observed. The serum markers and pathology score confirmed Tirofiban had protective effects on kidney IRI.2.Tirofiban could reduce cell apoptosis induced by IRITo further analyze the level changes of cell apoptosis in kidney tissues, we adopted the TUNEL staining and apoptosis protein Western blot methods. In TUNEL staining test, that there were a very few separate apoptosis cells in control group. For IR group, there were many TUNEL positive cells, distributing mainly outer pith distal convoluted tubules, a few in proximal tubule of right kidney cortex and kidney tubules. For IR+T group, a few TUNEL positive cells were observed. Meanwhile, the increased level of caspase-3and Bax proteins, reduced content of Bcl-2 was observed in IR group, compared to control group. While, the significantly decreased expression of caspase-3 and Bax proteins and increased level of Bcl-2 were observed after the Tirofiban pretreatment.3.Tirofiban could relieve oxidative stress reaction caused by IRIIn order to reveal the protective mechanism of Tirofiban for renal IRI, we detected the oxidative stress level in SD rats, serum level of SOD, GSH, MDA, MPO and inflammatory factors(IL-1βand TNF-β). Compared to the IR group, the treatment in IR+T group could significantly reduce the serum level of MDA, MPO and inflammatory factors(IL-1βandTNF-β) of rats with ischemia-reperfusion and remarkablely increase the level of SOD and GSH, which indicates that Tirofiban may show protection function through the process of oxidative stress response.4.Tirofiban shows protective effects on IRI by NO pathwayPrevious researches suggest that the degree of kidney oxidative stress is closely related to nitric oxide synthase, so we analyzed the relationship between nitric oxide synthase and oxidative stress. In order to clarify if NO plays a key role in protection process of Tirofiban on IRI, endothelial nitric oxide synthase(e NOS) and inducible nitric oxide synthase(i NOS) levels were detected. Contrary results were obtained in IR group and IR+T group.In order to clarify the roles of e NOS and i NOS, we established anoxia-reoxygenation cell models with kidney endothelial cells(NRK52E cell). Then NG-Nitro-l-arginine methyl este r(L-NAME) and methyl isothiourea(SMT), the inhibitors of e NOS 和 i NOS, respectively, were added into cells. L-NAME could block the protection function of Tirofiban on kidney endothelial cells. This result demonstrated that Tirofiban exhibited protection effects on kidney with e NOS during IRI process.Conclusions: 1.Tirofiban could improve IRI and contribute to the recovery of kidney function. 2.Tirofiban has remarkablely anti-injury, anti-apoptosis and antioxidant effects in ischemia-reperfusion kidney tissues. 3.The protective effects of Tirofiban on IRI is done through NO signaling pathway. 4.The data discrepancies in each group may result from the reperfusion injury and the specific expression with agents. Further research on the issue will help for understanding the molecular mechanism of Tirofiban in protecting IRI.