Tomofumi Fushima

Reduced Uterine Perfusion Pressure (RUPP) Model of Preeclampsia in Mice

Preeclampsia (PE) is a pregnancy-induced hypertension with proteinuria that typically develops after 20 weeks of gestation. A reduction in uterine blood flow causes placental ischemia and placental release of anti-angiogenic factors such as sFlt-1 followed by PE. Although the reduced uterine perfusion pressure (RUPP) model is widely used in rats, investigating the role of genes on PE using genetically engineered animals has been problematic because it has been difficult to make a useful RUPP model in mice. To establish a RUPP model of PE in mice, we bilaterally ligated ovarian vessels distal to ovarian branches, uterine vessels, or both in ICR-strain mice at 14.5 days post coitum (dpc). Consequently, these mice had elevated BP, increased urinary albumin excretion, severe endotheliosis, and mesangial expansion. They also had an increased incidence of miscarriage and premature delivery. Embryonic weight at 18.5 dpc was significantly lower than that in sham mice. The closer to the ligation site the embryos were, the higher the resorption rate and the lower the embryonic weight. The phenotype was more severe in the order of ligation at the ovarian vessels < uterine vessels < both. Unlike the RUPP models described in the literature, this model did not constrict the abdominal aorta, which allowed BP to be measured with a tail cuff. This novel RUPP model in mice should be useful for investigating the pathogenesis of PE in genetically engineered mice and for evaluating new therapies for PE.

Coagulation Factor Xa and Protease-Activated Receptor 2 as Novel Therapeutic Targets for Diabetic Nephropathy

Objective—The role of hypercoagulability in the pathogenesis of diabetic nephropathy (DN) remains elusive. We recently reported the increased infiltration of macrophages expressing tissue factor in diabetic kidney glomeruli; tissue factor activates coagulation factor X (FX) to FXa, which in turn stimulates protease-activated receptor 2 (PAR2) and causes inflammation. Approach and Results—Here, we demonstrated that diabetes mellitus increased renal FX mRNA, urinary FXa activity, and FX expression in glomerular macrophages. Administration of an oral FXa inhibitor, edoxaban, ameliorated DN with concomitant reductions in the expression of PARs (Par1 and Par2) and of proinflammatory and profibrotic genes. Diabetes mellitus induced PAR2, and lack of Par2 ameliorated DN. FXa or PAR2 agonist increased inflammatory cytokines in endothelial cells and podocytes in vitro. Conclusions—We conclude that enhanced FXa and PAR2 exacerbate DN and that both are promising targets for preventing DN. Alleviating inflammation is probably more important than inhibiting coagulation per se when treating kidney diseases using anticoagulants.

Nicotinamide benefits both mothers and pups in two contrasting mouse models of preeclampsia

Significance Preeclampsia (PE), high blood pressure and protein in the urine in the last third of pregnancy, complicates about 1 in 20 human pregnancies, and it is one of the leading causes of pregnancy-related maternal deaths. The only definitive treatment, induced delivery, invariably results in premature babies. Blood pressure-lowering drugs help, but results in preventing preterm delivery and correcting the fetal growth restriction (FGR) that also occurs in PE have been disappointing. Here we show that feeding high doses of nicotinamide, a vitamin, improves the maternal condition, prolongs pregnancies, and prevents FGR in mice having PE-like conditions due to two contrasting causes. Because nicotinamide benefits both mothers and pups, it merits evaluation for preventing or treating PE in humans. Preeclampsia (PE) complicates ∼5% of human pregnancies and is one of the leading causes of pregnancy-related maternal deaths. The only definitive treatment, induced delivery, invariably results in prematurity, and in severe early-onset cases may lead to fetal death. Many currently available antihypertensive drugs are teratogenic and therefore precluded from use. Nonteratogenic antihypertensives help control maternal blood pressure in PE, but results in preventing preterm delivery and correcting fetal growth restriction (FGR) that also occurs in PE have been disappointing. Here we show that dietary nicotinamide, a nonteratogenic amide of vitamin B3, improves the maternal condition, prolongs pregnancies, and prevents FGR in two contrasting mouse models of PE. The first is caused by endotheliosis due to excess levels in the mothers of a soluble form of the receptor for vascular endothelial growth factor (VEGF), which binds to and inactivates VEGF. The second is caused by genetic absence of Ankiryn-repeat-and-SOCS-box–containing-protein 4, a factor that contributes to the differentiation of trophoblast stem cells into the giant trophoblast cells necessary for embryo implantation in mice; its absence leads to impaired placental development. In both models, fetal production of ATP is impaired and FGR is observed. We show here that nicotinamide decreases blood pressure and endotheliosis in the mothers, probably by inhibiting ADP ribosyl cyclase (ADPRC), and prevents FGR, probably by normalizing fetal ATP synthesis via the nucleotide salvage pathway. Because nicotinamide benefits both dams and pups, it merits evaluation for preventing or treating PE in humans.

Protease–activated receptor 2 exacerbates adenine–induced renal tubulointerstitial injury in mice

Hypercoagulability is associated with chronic kidney disease (CKD). Tissue factor/factor VIIa complex and factor Xa in the coagulation cascade are known to activate protease-activated receptor 2 (PAR2), and to cause inflammation and tissue injury. Although PAR2 is highly expressed in the kidney, it is unclear whether PAR2 plays a pathogenic role in CKD. To test this, we fed the mice lacking Par2 (F2rl1-/-) and wild type (F2rl1+/+) mice with adenine diet to induce tubulointerstitial injury, a hallmark of CKD. Adenine-treated mice showed severe renal dysfunction, tubular atrophy, and fibrosis. Fibrin deposition and the expression of tissue factor and PARs markedly increased in their kidneys. Lack of Par2 attenuated renal histological damage and reduced the expression levels of genes related to inflammation, fibrosis, and oxidative stress. Our data indicate that PAR2 is critically important in the pathogenesis of adenine-induced tubular injury. PAR2 antagonists under development could be useful to treat and prevent CKD.

OS 21-08 NOVEL REDUCED UTERINE PERFUSION PRESSURE (RUPP) MODEL OF PREECLAMPSIA IN MICE.

Objective: Preeclampsia (PE) is a pregnancy-induced hypertension with proteinuria that typically develops after 20 weeks of gestation. A reduction in uterine blood flow causes placental ischemia and placental release of anti-angiogenic factors such as soluble fms-like tyrosine kinase (sFlt-1) followed by PE. Although the reduced uterine perfusion pressure (RUPP) model is widely used in rats, investigating the genetics of PE has been problematic because it has been difficult to make a useful RUPP model in mice. The aim of the present study is to establish a novel PE model using an improved RUPP method in mice. Design and Method: As shown in the Figure 1B, we bilaterally ligated ovarian vessels distal to ovarian branches, uterine vessels, or both in ICR-strain mice at 14.5 days post coitum (dpc). BP, renal phenotype and pregnancy outcome were analyzed. Results: Unlike the RUPP models described in the literature, this model did not constrict the abdominal aorta, which allowed BP to be measured with a tail cuff. RUPP in mice had elevated BP, increased urinary albumin excretion, severe endotheliosis, and mesangial expansion. They also had an increased incidence of miscarriage and premature delivery. Embryonic weight at 18.5 dpc was significantly lower than that in sham mice. The closer to the ligation site the embryos were, the higher the resorption rate and the lower the embryonic weight. The phenotype was more severe in the order of ligation at the ovarian vessels < uterine vessels < both. Conclusions: We developed a novel RUPP mouse model that recapitulates the phenotype of PE. This model is expected to be useful for investigating pathogenesis of PE in genetically engineered mice and for evaluating new therapies for PE.

Nicotinamide ameliorates a preeclampsia-like condition in mice with reduced uterine perfusion pressure

Preeclampsia (PE) is pregnancy-induced hypertension with proteinuria that typically develops after 20 wk of gestation. Antihypertensives currently used for PE reduce blood pressure of PE mothers but do not prevent preterm delivery and do not alleviate fetal growth restriction (FGR) associated with PE. We have recently shown that the activation of the endothelin (ET) system exacerbates PE. However, ET receptor antagonists are teratogenic and not suitable for pregnant women. The vitamin B3 nicotinamide (Nam) inhibits vasoconstriction by ET and is generally considered safe and harmless to babies. Nam also alleviates oxidative stress, which exacerbates PE and FGR. The aim of the present study was to evaluate therapeutic effects of Nam on the PE-like phenotype using a reduced uterine perfusion pressure (RUPP) model in mice that we have recently developed. We bilaterally ligated uterine vessels of pregnant mice and administered Nam or water daily by gavage. Nam improved maternal hypertension, proteinuria, and glomerular endotheliosis in RUPP mice. Moreover, Nam prolonged pregnancies and improved survival and growth of the embryos in RUPP PE mice. In conclusion, Nam alleviates the PE-like phenotype and FGR in the murine RUPP model. Nam could help treat maternal hypertension and FGR in human PE.

Vitamin B 3 Nicotinamide: A Promising Candidate for Treating Preeclampsia and Improving Fetal Growth

Up to 8% of pregnant women suffer from preeclampsia (PE), a deadly disease characterized by high blood pressure (BP), blood vessel damage, called endotheliosis (vascular endothelial swelling with narrowing of capillary lumen), and high levels of protein in the urine. PE is often associated with premature delivery, which is a risk factor of cardiovascular and metabolic diseases among the offspring. Accordingly, establishing drug treatments of PE is in immediate needs. Currently, many of anti-hypertensive drugs cause malformation of the fetuses and are contraindicated for pregnant women. Anti-hypertensive drugs that are allowed to be used for treating pregnant women could lower BP of the mothers and reduce the risk of maternal death due to cardiovascular diseases such as cerebral hemorrhage. However, these anti-hypertensives do not improve endotheliosis and proteinuria. In fact, they reduce blood supply to the placentae and fetuses, which could lead to fetal growth restriction (FGR) and fetal and neonatal death. Until now, the only treatment for preeclamptic women has been delivery of the baby and placenta. Using three mechanistically different mouse models of PE, we have found that vitamin B3 nicotinamide (Nam) is the first safe drug that alleviates PE, and that Nam also alleviates or prevents miscarriage, prolongs pregnancy period, and improves the growth of the fetuses in mice with PE. Importantly, Nam has been used for pregnant and nursing women who have difficulty in taking sufficient meal. Nam could help treat or prevent PE and FGR associated with PE, if the treatment works in humans.

Hepatic dysfunction and thrombocytopenia induced by excess sFlt1 in mice lacking endothelial nitric oxide synthase

Liver dysfunction is a major problem in patients with severe preeclampsia (PE), hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome, or in patients receiving anti-vascular endothelial growth factor (VEGF) therapy. Excessive soluble fms-like tyrosine kinase 1 (sFlt1) that antagonizes VEGF has been implicated in the pathogenesis of PE. VEGF increases the expression of endothelial nitric oxide synthase (eNOS) and activates it. eNOS polymorphisms that cause reduced NO production are associated with PE. The aim of this study was to clarify the role on hepatic function by excess sFlt1 in the absence of eNOS gene product. We first overexpressed sFlt1 using adenovirus in eNOS−/− and eNOS+/+ mice. Excessive sFlt1 and lack of eNOS synergistically increased plasma levels of liver transaminases, exacerbated infiltration of inflammatory cells, elevated expression levels of cytokines in the liver, and aggravated oxidative stress and coagulation abnormalities. Lack of eNOS in the presence of excess sFlt1 also induced thrombocytopenia, whereas eNOS+/+ mice with excess sFlt1 alone showed no or modest liver phenotype. Taken together, excessive sFlt1 and lack of eNOS synergistically induce hepatic dysfunction and thrombocytopenia, suggesting a novel role for VEGF and nitric oxide signaling in hepatocyte-endothelial cross-talk in health and in liver injury states.

Relationship Between Short Term Variability (STV) and Onset of Cerebral Hemorrhage at Ischemia–Reperfusion Load in Fetal Growth Restricted (FGR) Mice

Fetal growth restriction (FGR) is a risk factor exacerbating a poor neurological prognosis at birth. A disease exacerbating a poor neurological prognosis is cerebral palsy. One of the cause of this disease is cerebral hemorrhage including intraventricular hemorrhage. It is believed to be caused by an inability to autoregulate cerebral blood flow as well as immaturity of cerebral vessels. Therefore, if we can evaluate the function of autonomic nerve, cerebral hemorrhage risk can be predicted beforehand and appropriate delivery management may be possible. Here dysfunction of autonomic nerve in mouse FGR fetuses was evaluated and the relationship with cerebral hemorrhage incidence when applying hypoxic load to resemble the brain condition at the time of delivery was examined. Furthermore, FGR incidence on cerebral nerve development and differentiation was examined at the gene expression level. FGR model fetuses were prepared by ligating uterine arteries to reduce placental blood flow. To compare autonomic nerve function in FGR mice with that in control mice, fetal short term variability (STV) was measured from electrocardiograms. In the FGR group, a significant decrease in the STV was observed and dysfunction of cardiac autonomic control was confirmed. Among genes related to nerve development and differentiation, Ntrk and Neuregulin 1, which are necessary for neural differentiation and plasticity, were expressed at reduced levels in FGR fetuses. Under normal conditions, Neurogenin 1 and Neurogenin 2 are expressed mid-embryogenesis and are related to neural differentiation, but they are not expressed during late embryonic development. The expression of these two genes increased in FGR fetuses, suggesting that neural differentiation is delayed with FGR. Uterine and ovarian arteries were clipped and periodically opened to give a hypoxic load mimicking the time of labor, and the bleeding rate significantly increased in the FGR group. This suggests that FGR deteriorates cardiac autonomic control, which becomes a risk factor for cerebral hemorrhage onset at birth. This study demonstrated that cerebral hemorrhage risk may be evaluated before parturition for FGR management by evaluating the STV. Further, this study suggests that choosing an appropriate delivery timing and delivery method contributes to neurological prognosis improvement.

OS 21-05 PIVOTAL ROLE OF HEME OXYGENASE IN AMELIORATION BY NICOTINAMIDE OF FETAL GROWTH RESTRICTION ASSOCIATED WITH PREECLAMPSIA.

Objective: Preeclampsia (PE) is pregnancy-induced hypertension with proteinuria. It causes maternal death or fetal growth restriction (FGR). Although high BP can be managed with antihypertensive drugs, there is no effective treatment of FGR associated with PE. We have clarified that nicotinamide (Nam) alleviates PE-like condition and FGR induced by soluble fms-like tyrosine kinase-1 (sFlt-1) in mice. But the mechanism of how Nam works is unclear. Because Nam induces cytoprotective heme oxygenase 1 (HO-1), our aim is to clarify whether HO-1 contributes to therapeutic effect of Nam against FGR associated with PE. Design and Method: We used chromium (III) mesoporphyrin (CrMP) to inhibit HO. We divided pregnant ICR wild type mice into 6 groups; control, Nam, sFlt-1, sFlt-1 + Nam, sFlt-1 + CrMP, sFlt-1 + Nam + CrMP. sFlt-1 was overproduced by adenovirus administered at 14.5 dpc. Nam and CrMP was given daily from 14.5 dpc and 12.5 dpc respectively. Fetuses and placentae were harvested on 18.5 dpc for analysis. Results: Nam improved PE-like conditions and FGR induced by sFlt-1. CrMP 2 &mgr;mol/kg/d exacerbated FGR and abolished its alleviation by Nam. CrMP 1.5 &mgr;mol/kg/d did not affect FGR but Nam still alleviated FGR. Pregnancies in mice overproducing sFlt-1 continued longer with Nam, and CrMP abolished this effect of Nam. Conclusions: HO plays a pivotal role in alleviation by Nam of FGR associated with PE.