Arginase reciprocally regulates nitric oxide synthase activity and contributes to endothelial dysfunction in aging blood vessels.

BACKGROUND: Although abnormal L-arginine NO signaling contributes to endothelial dysfunction in the aging cardiovascular system, the biochemical mechanisms remain controversial. L-arginine, the NO synthase (NOS) precursor, is also a substrate for arginase. We tested the hypotheses that arginase reciprocally regulates NOS by modulating L-arginine bioavailability and that arginase is upregulated in aging vasculature, contributing to depressed endothelial function. METHODS AND RESULTS: Inhibition of arginase with (S)-(2-boronoethyl)-L-cysteine, HCl (BEC) produced vasodilation in aortic rings from young (Y) adult rats (maximum effect, 46.4+/-9.4% at 10(-5) mol/L, P<0.01). Similar vasorelaxation was elicited with the additional arginase inhibitors N-hydroxy-nor-L-arginine (nor-NOHA) and difluoromethylornithine (DFMO). This effect required intact endothelium and was prevented by 1H-oxadiazole quinoxalin-1-one (P<0.05 and P<0.001, respectively), a soluble guanylyl cyclase inhibitor. DFMO-elicited vasodilation was greater in old (O) compared with Y rat aortic rings (60+/-6% versus 39+/-6%, P<0.05). In addition, BEC restored depressed L-arginine (10(-4) mol/L)-dependent vasorelaxant responses in O rings to those of Y. Arginase activity and expression were increased in O rings, whereas NOS activity and cyclic GMP levels were decreased. BEC and DFMO suppressed arginase activity and restored NOS activity and cyclic GMP levels in O vessels to those of Y. CONCLUSIONS: These findings demonstrate that arginase modulates NOS activity, likely by regulating intracellular L-arginine availability. Arginase upregulation contributes to endothelial dysfunction of aging and may therefore be a therapeutic target.

Perinatal exposure to genistein alters reproductive development and aggressive behavior in male mice.

Exposure to endocrine disrupting chemicals adversely affects reproductive development and behavior in males. The goal of this study was to determine if exposure to genistein, an isoflavone found in soy, during early periods of sex differentiation alters reproductive development and behavior in male mice. Female C57BL/6 mice were fed a phytoestrogen-free diet supplemented with 0, 5 or 300 mg/kg of genistein throughout gestation and lactation. Anogenital distance (AGD) and body mass of male offspring was measured weekly from postnatal days 2-21, timing of preputial separation was assessed at puberty, and in adulthood, reproductive organ masses, sperm and testosterone production, and reproductive and aggressive behaviors were assessed. Exposure to genistein resulted in smaller AGD are reduced body mass, with the low-dose diet exerting a greater effect. Timing of preputial separation, adult reproductive behavior, sperm concentrations and testosterone production were not influenced by genistein treatment at either dose. Aggressive behaviors were decreased, whereas defensive behaviors were increased, in males that received the low-dose genistein diet. Exposure to genistein during critical periods of sex differentiation results in concurrent and persistent demasculinization in male mice. Phenotypic and behavioral abnormalities induced by genistein showed a non-monotonic response, where treatment with a low dose exerted a greater effect than treatment with a high dose of genistein. Given the popularity of soy infant formulas, the influence isoflavone exposure on reproductive and behavioral health in boys and men should be considered.

Deficiency of different nitric oxide synthase isoforms activates divergent transcriptional programs in cardiac hypertrophy.

Decreased nitric oxide synthase (NOS) activity induces left ventricular hypertrophy (LVH), but the transcriptional pathways mediating this effect are unknown. We hypothesized that specific NOS isoform deletion (NOS3 or NOS1) would activate different transcriptional programs in LVH. We analyzed cardiac expression profiles (Affymetrix MG-U74A) from NOS-/- mice using robust multi-array average (RMA). Of 12,422 genes analyzed, 47 genes were differentially expressed in NOS3-/- and 67 in NOS1(-/-) hearts compared with wild type (WT). Only 16 showed similar changes in both NOS-/- strains, most notably decreased heat-shock proteins (HSP10, 40, 70, 86, 105). Hypertrophied NOS1-/- hearts had unique features, including decreased myocyte-enriched calcineurin interacting protein and paradoxical downregulation of fetal isoforms (alpha-skeletal actin and brain natriuretic peptide). Cluster analyses demonstrated that NOS1 deletion caused more pronounced changes in the myocardial transcriptome than did NOS3 deletion, despite similar cardiac phenotypes. These findings suggest that the transcriptional basis for LVH varies depending on the inciting biochemical stimulus. In addition, NOS isoforms appear to play distinct roles in modulating cardiac structure.

Involvement of gonadal steroids and gamma interferon in sex differences in response to blood-stage malaria infection.

To examine the hormonal and immunological mechanisms that mediate sex differences in susceptibility to malaria infection, intact and gonadectomized (gdx) C57BL/6 mice were inoculated with Plasmodium chabaudi AS-infected erythrocytes, and the responses to infection were monitored. In addition to reduced mortality, intact females recovered from infection-induced weigh loss and anemia faster than intact males. Expression microarrays and real-time reverse transcription-PCR revealed that gonadally intact females exhibited higher expression of interleukin-10 (IL-10), IL-15Ralpha, IL-12Rbeta, Gadd45gamma, gamma interferon (IFN-gamma), CCL3, CXCL10, CCR5, and several IFN-inducible genes in white blood cells and produced more IFN-gamma than did intact males and gdx females, with these differences being most pronounced during peak parasitemia. Intact females also had higher anti-P. chabaudi immunoglobulin G (IgG) and IgG1 responses than either intact males or gdx females. To further examine the effector mechanisms mediating sex differences in response to P. chabaudi infection, responses to infection were compared among male and female wild-type (WT), T-cell-deficient (TCRbetadelta-/-), B-cell-deficient (microMT), combined T- and B-cell-deficient (RAG1), and IFN-gamma knockout (IFN-gamma-/-) mice. Males were 3.5 times more likely to die from malaria infection than females, with these differences being most pronounced among TCRbetadelta-/-, microMT, and RAG1 mice. Male mice also exhibited more severe weight loss, anemia, and hypothermia, and higher peak parasitemia than females during infection, with WT, RAG1, TCRbetadelta-/-, and microMT mice exhibiting the most pronounced sexual dimorphism. The absence of IFN-gamma reduced the sex difference in mortality and was more detrimental to females than males. These data suggest that differential transcription and translation of IFN-gamma, that is influenced by estrogens, may mediate sex differences in response to malaria.

Alteration in host cell tropism limits the efficacy of immunization with a surface protein of malaria merozoites.

Immunization with Plasmodium yoelii merozoite surface protein-8 (PyMSP-8) has been shown to protect mice against lethal P. yoelii 17XL malaria. Here we demonstrate that PyMSP-8-specific antibodies preferentially suppress P. yoelii 17XL growth in mature erythrocytes compared to growth in reticulocytes and do not suppress the growth of nonlethal P. yoelii 17X, a parasite that primarily replicates in reticulocytes. The protection against normocyte-associated P. yoelii malaria parasites is mediated by antibodies that recognize conformational epitopes of PyMSP-8 that are nonpolymorphic. We examined changes in gene expression in reticulocyte-restricted P. yoelii 17XL parasites that escaped neutralization by PyMSP-8-specific antibodies using P. yoelii DNA microarrays. Of interest, Pymsp-8 gene expression decreased, while the expression of msp-1, msp-7, and several rhoptry protein genes increased. Breakthrough parasites also exhibited increases in the expression of a subset of yir and Pyst-a genes that are predicted to encode polymorphic antigens expressed on the surface of infected erythrocytes. These data suggest that changes in the expression of parasite proteins expressed on the merozoite surface, as well as the surface of infected erythrocytes, may alter host cell tropism and contribute to the ability of malaria parasites to evade merozoite-specific, neutralizing antibodies.

Differential expression of immunoregulatory genes in male and female Norway rats following infection with Seoul virus.

Males of many species are more susceptible than females to infections caused by parasites, bacteria, fungi, and viruses. Following inoculation with Seoul virus, male rats have more virus present in target organs and shed virus longer than females. The goal of this study was to test the hypothesis that variation in the expression of genes associated with immune function mediates sex differences in hantavirus infection. Using DNA microarrays, we examined changes in gene expression in lung tissue during the early (when animals are viremic and shedding virus; Day 15 post-inoculation (p.i.)) and late (animals have low levels of infectious virus, but high antibody titers; Day 40 p.i.) phases of infection in adult male and female rats. After normalizing the gene expression levels from infected animals to the gene expression levels from same-sex uninfected controls, our data revealed that 1,813 genes were differentially expressed between the sexes during infection. The expression of key transcriptional factors (e.g., eIF-2 alpha, NF-kappa B, IRF-1, NF-IL-6, and STAT6) and genes that encode for proinflammatory (e.g., TNF alpha R, IL-1R, and IL-1RAcP), antiviral (e.g., IFN gamma R and Mx proteins), T cell (e.g., CD3 and TCR), and Ig superfamily (e.g., IgM, IgG, and MHC class I and II) proteins was higher in females than males. Conversely, males had higher expression of heat shock protein genes (e.g., hsp70) suggesting that cellular stress is elevated in males. These data provide candidate genes and cellular pathways that may underlie sex differences in responses to Seoul virus and possibly other hemorrhagic fever viruses.