Biological Functions And Molecular Mechanisms Of LIGHT In The Pathogenesis Of Preeclampsia

Part1LIGHT plays an important role in the pathophysiology of preeclampsiaBackground:Preeclarnpsia (PE), a prevalent complication of pregnancy, is believed to be the leading cause of mortality of mom and fetus. The major clinical symptoms of PE are hypertension and proteinuria. The current treatment for PE is limited because underlying pathophysiologic mechanisms remain undefined. However, PE is associated with maternal abnormal immune response and excessive inflammatory. By using unbiased sensitive ELISA array, we demonstrated an overall inflammatory response in the circulation of PE patients. Unexpectedly, we found LIGHT, a novel Tumor Necrosis Factor Super-family member14, is significantly increased in PE patients. Recently, LIGHT has emerged as a key contributor to multiple immune-associated diseases. LIGHT transmembrane receptors, Herpes Virus Entry Mediator (HVEM/TNFRSF14) and Lymphotoxin β Receptor (LTβR/TNFRSF3), mediate biological functions of LIGHT in different diseases. Based on our preliminary studies, we will further determine the biological function and mechanism of LIGHT in PE.Objective:To investigate LIGHT expression in preeclamptic patients; to determine whether LIGHT can induce hypertension and proteinuria in pregnant mice and non-pregnant mice; to determine whether LIGHT can induce placental and renal damage in pregnant mice in order to confirm the effect of LIGHT on PE.Methods:1. Collect serum and placental tissue from preeclamptic patients and normotensive pregnant women; measure LIGHT level in serum by ELISA; detect LIGHT and receptors expression in human placental tissue by immunohistochemistry and western blot;2. inject recombinant mouse LIGHT into pregnant mice and non-pregnant mice, all the animals were co-treated with neutralizing antibodies which against LTβR or HVEM. Monitor blood pressure every day, collect24h urine by using metabolic cages and measure proteinuria by ELISA; collect placentas and kidneys from pregnant mice, assess placental and renal damage by H&E and PAS staining.Result:1. LIGHT protein levels are increased in both the circulation and placental tissue of PE patients. The protein levels of LTβR and HVEM are significantly increased in placental tissue from PE patients compared with normotensive pregnant women;2. Blood pressure is increased significantly in both pregnant mice and non-pregnant mice injected with LIGHT relative to control mice injected with saline; however, LIGHT induces proteinuria in pregnant mice but not non-pregnant mice; LIGHT induces placental and renal damage in pregnant mice; elevated blood pressure and urinary protein levels seen in LIGHT injected pregnant mice were significantly attenuated by infusion of anti-LTβR mAb or anti-HVEM mAb; blocking LTβR or HVEM both dramatically attenuate LIGHT induced placental and renal damage in pregnant mice; blocking LTβR or HVEM significantly reduce high blood pressure induced by LIGHT.Conclusion:1. LIGHT protein levels are increased in both the circulation and placental tissue in PE;2. LIGHT, signaling through LTβR and HVEM, can induce PE features including hypertension, proteinuria, placental and kidney damage in pregnant mice;3. Pregnancy is required for LIGHT-induced proteinuria. Part2Downstream mechanisms in LIGHT induced PEBackground:PE, a life-threatening complication of pregnancy, appears7-8%of the first pregnancy. Although the underlying mechanisms of PE are unclear, PE has been considered as an autoimmune disease, a series of inflammatory factors are involved in the pathophysiology of PE. LIGHT, a novel TNFSF member, is considered to play an important role in multiple immune-associated diseases. We have demonstrated:1. LIGHT protein levels are increased in both the circulation and placental tissue in PE.2. LIGHT, signaling through LTβR and HVEM, can induce PE features including hypertension, proteinuria, placental and kidney damage in pregnant mice.3. Pregnancy is required for LIGHT-induced proteinuria. Here we will further determine the downstream mechanisms in LIGHT induced PE.Objective:1. To investigate circulating sFlt-1and ET-1level in pregnant mice which infused with LIGHT;2. to determine whether LIGHT can induce placental tissue trophoblast cells apoptosis in pregnant mice, in order to determine the underlying mechanisms in LIGHT-induced PE.Methods:Inject recombinant mouse LIGHT into pregnant mice and non-pregnant mice, all the animals were co-treated with neutralizing antibodies which against LTβR or HVEM. Collect serum from pregnant mice and non-pregnant mice, measure sFlt-1and ET-1by ELISA; collect placentas from pregnant mice, detect placental tissue trophoblast cell apoptosis by TUNEL assay.Result:1. Circulating sFlt-1levels were remarkably increased in LIGHT-injected pregnant mice compared with control group;2. elevated sFlt-1levels in the circulation of LIGHT injected pregnant mice were significantly down-regulated by co-treatment with anti-LTβR mAb or anti-HVEM mAb. In contrast, there were no significant differences in the low levels of circulating sFlt-1between the LIGHT injected non-pregnant mice with or without co-injection of anti-LTβR and anti-HVEM;3. LIGHT injection resulted in elevated circulating ET-1in pregnant mice and non-pregnant mice, compared to saline injected pregnant and non-pregnant mice. Co-injection with anti-LTβR or anti-HVEM significantly inhibited LIGHT mediated induction of ET-1;4. LIGHT injection significantly increase apoptosis in the labyrinth zone of placentas from pregnant mice compared to placentas from control mice; co-injection with anti-LTβR or anti-HVEM significantly inhibited LIGHT mediated placental trophoblast cell apoptosis.Conclusion:1. Circulating sFlt-1is a downstream mediator induced by LIGHT via HVEM and LTβR activation in pregnant mice but not non-pregnant mice;2. Circulating ET-1is a common downstream mediator induced by LIGHT via HVEM and LTβR activation in both pregnant mice and non-pregnant mice;3. LIGHT, signaling via HVEM and LTβR, induces placental trophoblast cell apoptosis in pregnant mice. Part3The biological functions and mechanisms of DcR3in PEBackground:PE is a complication of pregnancy which is associated with abnormal immune response. A series of inflammatory factors are involved in the pathogenesis of the disease. LIGHT, a novel TNFSF member, is considered to play an important role in multiple immune-associated diseases. Decoy Receptor3(DcR3, TNFRSF6B) is a soluble form of LIGHT receptor which competitively combines with LIGHT and interrupts interactions between LIGHT and transmembrane receptors LTβR/HVEM. Numerous studies indicated that DcR3functions as an immune-suppressor. Our preliminary studies have proved that LIGHT perfusion can induce PE features in pregnant mice; however, the biological function of DcR3in PE is still undefined.Objective:1. To investigate trophoblast cells apoptosis of human villous explants (HVEx) in vitro which is incubated with LIGHT;2. To measure the sFlt-1level in the supernatant of LIGHT-incubated HVEx;3. To determine whether DcR3can attenuate LIGHT induced trophoblast cells apoptosis and sFlt-1secretion in HVEx.Methods:HVEx, collected from normotensive pregnant women after full-term delivery, were quickly incubated with LIGHT and DcR3for24hours. Collect HVEx tissue and investigate trophoblast cells apoptosis by TUNEL assay; collect supernatant and measure sFlt-1by ELISA.Result:1. sFlt-1levels in the supernatant were remarkably increased in the group of LIGHT-incubated HVEx compared with control group;2. elevated sFlt-1levels in the supernatant of LIGHT-incubated HVEx were significantly down-regulated by co-treatment with DcR3;3. LIGHT treatment significantly increases trophoblast cells apoptosis in cultured HVEx;4. co-treatment with DcR3significantly inhibited LIGHT mediated trophoblast cell apoptosis in HVEx in vitro.Conclusion:1. LIGHT can induce sFlt-1secretion from cultured HVEx;2. LIGHT can induce trophblast cells apoptosis in cultured HVEx;3. DcR3can significantly attenuate LIGHT induced sFlt-1secretion and trophblast cells apoptosis in HVEx in vitro.