Dimethylarginine Dimethylaminohydrolase-1 Transgenic Mice Are Not Protected from Ischemic Stroke

Background

Methylated arginines are endogenous analogues of L-arginine, the substrate for nitric oxide (NO) synthase. Asymmetric dimethylarginine (ADMA) interferes with NO formation, causing endothelial dysfunction. ADMA is a predictor of cardiovascular events and mortality in humans. It is eliminated primarily by enzymatic activity of dimethylarginine dimethylaminohydrolase (DDAH).

Methodology/Principal Findings

We investigated whether human DDAH-1 (hDDAH-1) transgenicity protects from ischemic tissue damage in temporary middle cerebral artery occlusion (tMCAO) in mice. Infarct sizes did not significantly differ between hDDAH-1 transgenic (TG) mice and wild-type littermates (WT). As expected, ADMA plasma concentrations were significantly decreased, cerebral hDDAH expression and protein significantly increased in transgenic animals. Interestingly, neither brain tissue DDAH activity nor ADMA concentrations were different between TG and WT mice. In contrast, muscular DDAH activity was generally lower than in brain but significantly increased in TG mice.

Conclusion/Significance

Our study demonstrates that hDDAH-1 transgenic mice are not protected from ischemic cerebral tissue damage in tMCAO. This lack of protection is due to high basal cerebral DDAH activity, which is not further increasable by transgenic overexpression of DDAH.     

Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS^-/-) are protected against ALI. In both wild-type and eNOS^-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS^-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS^-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS^-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS^-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr³⁴. Finally, we found that the reduction in NOS uncoupling in eNOS^-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.     

Striatal Malonate Lesions Are Attenuated in Neuronal Nitric Oxide Synthase Knockout Mice

Abstract: Intrastriatal administration of the reversible succinate dehydrogenase inhibitor malonate produces both energy depletion and striatal lesions by a secondary excitotoxic mechanism. To investigate the role of nitric oxide (NO^•) in the pathogenesis of the lesions we examined malonate toxicity in mice in which the genes for neuronal nitric oxide synthase (nNOS) or endothelial nitric oxide synthase (eNOS) were disrupted. Malonate striatal lesions were significantly attenuated in the nNOS mutant mice, and they were significantly increased in the eNOS mutant mice. Malonate-induced increases in levels of 2,3- and 2,5-dihydroxybenzoic acid/salicylate, markers of hydroxyl radical generation, were significantly attenuated in the nNOS knockout mice. Malonate-induced increases in 3-nitrotyrosine, a marker for peroxynitrite-mediated damage, were blocked in the nNOS mice, whereas a significant increase occurred in the eNOS mice. These findings show that NO^• produced by nNOS results in generation of peroxynitrite, which plays a role in malonate neurotoxicity.     

Impaired de Novo Choline Synthesis Explains Why Phosphatidylethanolamine N-Methyltransferase-deficient Mice Are Protected from Diet-induced Obesity

Phosphatidylcholine (PC) is synthesized from choline via the CDP-choline pathway. Liver cells can also synthesize PC via the sequential methylation of phosphatidylethanolamine, catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). The current study investigates whether or not hepatic PC biosynthesis is linked to diet-induced obesity. Pemt^+/+ mice fed a high fat diet for 10 weeks increased in body mass by 60% and displayed insulin resistance, whereas Pemt^−/− mice did not. Compared with Pemt^+/+ mice, Pemt^−/− mice had increased energy expenditure and maintained normal peripheral insulin sensitivity; however, they developed hepatomegaly and steatosis. In contrast, mice with impaired biosynthesis of PC via the CDP-choline pathway in liver became obese when fed a high fat diet. We, therefore, hypothesized that insufficient choline, rather than decreased hepatic phosphatidylcholine, was responsible for the lack of weight gain in Pemt^−/− mice despite the presence of 1.3 g of choline/kg high fat diet. Supplementation with an additional 2.7 g of choline (but not betaine)/kg of diet normalized energy metabolism, weight gain, and insulin resistance in high fat diet-fed Pemt^−/− mice. Furthermore, Pemt^+/+ mice that were fed a choline-deficient diet had increased oxygen consumption, had improved glucose tolerance, and gained less weight. Thus, de novo synthesis of choline via PEMT has a previously unappreciated role in regulating whole body energy metabolism.     

Mice Rescued from Severe Malaria Are Protected against Renal Injury during a Second Kidney Insult

Malaria is a worldwide disease that leads to 1 million deaths per year. Plasmodium falciparum is the species responsible for the most severe form of malaria leading to different complications. Beyond the development of cerebral malaria, impairment of renal function is a mortality indicator in infected patients. Treatment with antimalarial drugs can increase survival, however the long-term effects of malaria on renal disease, even after treatment with antimalarials, are unknown. The aim of this study was to evaluate the effect of antimalarial drug treatment on renal function in a murine model of severe malaria and then evaluate kidney susceptibility to a second renal insult. Initially, mice infected with Plasmodium berghei ANKA achieved 20% parasitemia on day 5 post infection, which was completely abolished after treatment with 25 mg/kg artesunate and 40 mg/kg mefloquine. The treatment also decreased plasma creatinine levels by 43% and partially reversed the reduction in the glomerular filtration rate induced by infection. The urinary protein/creatinine ratio, collagen deposition, and size of the interstitial space decreased by 75%, 40%, and 20%, respectively, with drugs compared with untreated infected animals. In infected-treated mice that underwent a second renal insult, the plasma creatinine level decreased by 60% and the glomerular filtration rate increased compared with infected animals treated only with antimalarials. The number of glomerular cells, collagen deposition and the size of the interstitial space decreased by 20%, 39.4%, and 41.3%, respectively, in the infected group that underwent a second renal insult compared with the infected-treated groups. These functional and structural data show that renal injury observed in a murine model of severe malaria is partially reversed after antimalarial drug treatment, making the kidney less susceptible to a second renal insult.     

B Cell Deficient Mice Are Protected from Biliary Obstruction in the Rotavirus-Induced Mouse Model of Biliary Atresia

A leading theory regarding the pathogenesis of biliary atresia (BA) is that bile duct injury is initiated by a virus infection, followed by an autoimmune response targeting bile ducts. In experimental models of autoimmune diseases, B cells have been shown to play an important role. The aim of this study was to determine the role of B cells in the development of biliary obstruction in the Rhesus rotavirus (RRV)-induced mouse model of BA. Wild-type (WT) and B cell-deficient (Ig-α^-/-) mice received RRV shortly after birth. Ig-α^-/- RRV-infected mice had significantly increased disease-free survival rate compared to WT RRV-infected BA mice (76.8% vs. 17.5%). In stark contrast to the RRV-infected BA mice, the RRV-infected Ig-α^-/- mice did not have hyperbilirubinemia or bile duct obstruction. The RRV-infected Ig-α^-/- mice had significantly less liver inflammation and Th1 cytokine production compared to RRV-infected WT mice. In addition, Ig-α^-/- mice had significantly increased numbers of regulatory T cells (Tregs) at baseline and after RRV infection compared to WT mice. However, depletion of Tregs in Ig-α^-/- mice did not induce biliary obstruction, indicating that the expanded Tregs in the Ig-α^-/- mice were not the sole reason for protection from disease. Conclusion: B cell deficient Ig-α^-/- mice are protected from biliary obstruction in the RRV-induced mouse model of BA, indicating a primary role of B cells in mediating disease pathology. The mechanism of protection may involve lack of B cell antigen presentation, which impairs T-cell activation and Th1 inflammation. Immune modulators that inhibit B cell function may be a new strategy for treatment of BA.     

Mice Lacking C1q Are Protected from High Fat Diet-induced Hepatic Insulin Resistance and Impaired Glucose Homeostasis

Complement activation is implicated in the development of obesity and insulin resistance, and loss of signaling by the anaphylatoxin C3a prevents obesity-induced insulin resistance in mice. Here we have identified C1q in the classical pathway as required for activation of complement in response to high fat diets. After 8 weeks of high fat diet, wild-type mice became obese and developed glucose intolerance. This was associated with increased apoptotic cell death and accumulation of complement activation products (C3b/iC3b/C3c) in liver and adipose tissue. Previous studies have shown that high fat diet-induced apoptosis is dependent on Bid; here we report that Bid-mediated apoptosis was required for complement activation in adipose and liver. Although C1qa deficiency had no effect on high fat diet-induced apoptosis, accumulation of complement activation products and the metabolic complications of high fat diet-induced obesity were dependent on C1q. When wild-type mice were fed a high fat diet for only 3 days, hepatic insulin resistance was associated with the accumulation of C3b/iC3b/C3c in the liver. Mice deficient in C3a receptor were protected against this early high fat diet-induced hepatic insulin resistance, whereas mice deficient in the negative complement regulator CD55/DAF were more sensitive to the high fat diet. C1qa^−/− mice were also protected from high fat diet-induced hepatic insulin resistance and complement activation. Evidence of complement activation was also detected in adipose tissue of obese women compared with lean women. Together, these studies reveal an important role for C1q in the classical pathway of complement activation in the development of high fat diet-induced insulin resistance.     

Neurotransmitter and Immunomodulatory Actions of VIP and PACAP: Lessons from Knockout Mice

Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylyl Cyclase Activating Peptide (PACAP) are two closely related neuropeptides in the secretin family. They are widely expressed in the central and peripheral nervous systems, where they are classically thought to act as neurotransmitters or neuromodulators. They interact with high affinity receptors to regulate numerous behaviors as well as gastrointestinal, endocrine, cardiopulmonary, reproductive and immune functions. The recent generation of mice that specifically lack or overexpress VIP, PACAP or their receptors has yielded much new knowledge and enabled investigators to better understand the biological roles of these peptides and their impact on health. In this review, we attempt to summarize the major findings, but focus in greatest detail on the circadian and immune functions.Australian Peptite Conference Issue.     

Hyperlipidemia-Associated Renal Damage Decreases Klotho Expression in Kidneys from ApoE Knockout Mice

Background

Klotho is a renal protein with anti-aging properties that is downregulated in conditions related to kidney injury. Hyperlipidemia accelerates the progression of renal damage, but the mechanisms of the deleterious effects of hyperlipidemia remain unclear.

Methods

We evaluated whether hyperlipidemia modulates Klotho expression in kidneys from C57BL/6 and hyperlipidemic apolipoprotein E knockout (ApoE KO) mice fed with a normal chow diet (ND) or a Western-type high cholesterol-fat diet (HC) for 5 to 10 weeks, respectively.

Results

In ApoE KO mice, the HC diet increased serum and renal cholesterol levels, kidney injury severity, kidney macrophage infiltration and inflammatory chemokine expression. A significant reduction in Klotho mRNA and protein expression was observed in kidneys from hypercholesteromic ApoE KO mice fed a HC diet as compared with controls, both at 5 and 10 weeks. In order to study the mechanism involved in Klotho down-regulation, murine tubular epithelial cells were treated with ox-LDL. Oxidized-LDL were effectively uptaken by tubular cells and decreased both Klotho mRNA and protein expression in a time- and dose-dependent manner in these cells. Finally, NF-κB and ERK inhibitors prevented ox-LDL-induced Klotho downregulation.

Conclusion

Our results suggest that hyperlipidemia-associated kidney injury decreases renal expression of Klotho. Therefore, Klotho could be a key element explaining the relationship between hyperlipidemia and aging with renal disease.     

Uncoupling between Inflammatory and Fibrotic Responses to Silica: Evidence from MyD88 Knockout Mice

The exact implication of innate immunity in granuloma formation and irreversible lung fibrosis remains to be determined. In this study, we examined the lung inflammatory and fibrotic responses to silica in MyD88-knockout (KO) mice. In comparison to wild-type (WT) mice, we found that MyD88-KO animals developed attenuated lung inflammation, neutrophil accumulation and IL-1β release in response to silica. Granuloma formation was also less pronounced in MyD88-KO mice after silica. This limited inflammatory response was not accompanied by a concomitant attenuation of lung collagen accumulation after silica. Histological analyses revealed that while pulmonary fibrosis was localized in granulomas in WT animals, it was diffusely distributed throughout the parenchyma in MyD88-KO mice. Robust collagen accumulation was also observed in mice KO for several other components of innate immunity (IL-1R, IL-1, ASC, NALP3, IL-18R, IL-33R, TRIF, and TLR2-3-4,). We additionally show that pulmonary fibrosis in MyD88-KO mice was associated with the accumulation of pro-fibrotic regulatory T lymphocytes (T regs) and pro-fibrotic cytokine expression (TGF-β, IL-10 and PDGF-B), not with T helper (Th) 17 cell influx. Our findings indicate that the activation of MyD88-related innate immunity is central in the establishment of particle-induced lung inflammatory and granuloma responses. The development of lung fibrosis appears uncoupled from inflammation and may be orchestrated by a T reg-associated pathway.     

Pain Related Channels Are Differentially Expressed in Neuronal and Non-Neuronal Cells of Glabrous Skin of Fabry Knockout Male Mice

Fabry disease (FD) is one of the X-linked lysosomal storage disorders caused by deficient functioning of the alpha-galactosidase A (α-GalA) enzyme. The α-GalA deficiency leads to multi-systemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide in the endothelium and vascular smooth muscles. A hallmark symptom of FD patients is peripheral pain that appears in the early stage of the disease. Pain in FD patients is a peripheral small-fiber idiopathic neuropathy, with intra-epidermal fiber density and integrity being used for diagnosing FD in humans. However, the molecular correlates underlying pain sensation in FD remain elusive. Here, we have employed the α-GalA gene KO mouse as a model of FD in rodents to investigate molecular changes in their peripheral nervous system that may account for their algesic symptoms. The α-GalA null mice display neuropathic pain as evidenced by thermal hyperalgesia and mechanical allodynia, with histological analyses showing alterations in cutaneous innervation. Additionally, KO mice showed a decreased and scattered pattern of neuronal terminations consistent with the reduction in neuronal terminations in skin biopsies of patients with small fiber neuropathies. At the molecular level KO animals showed an increase in the expression of TRPV1 and Nav1.8, and a decrease in the expression of TRPM8. Notably, these alterations are observed in young animals. Taken together, our findings imply that the α-GalA KO mouse is a good model in which to study the peripheral small fiber neuropathy exhibited by FD patients, and provides molecular evidence for a hyperexcitability of small nociceptors in FD.