The inhibitory effects of rosiglitazone on cardiac hypertrophy through modulating the renin‐angiotensin system in diet‐induced hypercholesterolemic rats

Cardiac hypertrophy is not only an adaptational state before heart failure but also is an independent risk factor for ischemia, arrhythmia, and sudden death. However, the direct effects of hypercholesterolemia on the myocardium and mechanisms are not completely understood. It has been demonstrated that peroxisome proliferator‐activated receptor‐γ (PPARγ) ligand agonists attenuate cardiac hypertrophy through anti‐inflammatory effects. The present study investigated the effects of PPARγ agonists on hypercholesterolemia‐dependent, renin‐angiotensin‐system‐related cardiac hypertrophy. The findings showed that left ventricular hypertrophy, eminent cardiomyocyte hypertrophy, and lipid deposits in myocardium were observed in the rats fed a cholesterol‐rich diet for 6 months, while these characteristic pathological alterations and the increase in angiotensin II (ANG II) level and over‐expression of angiotensin II type 1 receptor (AT₁R) in the left ventricular tissues induced by the cholesterol‐rich diet were significantly suppressed to equal extents by rosiglitazone and irbesartan. In contrast, expression of angiotensin II type 2 receptor (AT₂R) was upregulated by these two drugs. In addition, lipid metabolism was markedly improved. The above findings suggest that the cardioprotection of the PPARγ agonist against cardiac hypertrophy evoked by hypercholesterolemia in rats is mediated partially by the improvement of lipid profile, the reduction of ANG II level in the local tissue along with the downregulation of AT₁R expression, and upregulation of AT₂R expression. Copyright © 2009 John Wiley & Sons, Ltd.     

Sub-terminal sequences modulating IS30 transposition in vivo and in vitro

Inverted repeats of insertion sequences (ISs) are indispensable for transposition. We demonstrate that sub-terminal sequences adjacent to the inverted repeats of IS30 are also required for optimal transposition activity. We have developed a cell-free recombination system and showed that the transposase catalyses formation of a figure-of-eight transposition intermediate, where a 2 bp long single strand bridge holds the inverted repeat sequences (IRs) together. This is the first demonstration of the figure-of-eight structure in a non-IS3 family element, suggesting that this mechanism is likely more widely adopted among IS families. We show that the absence of sub-terminal IS30 sequences negatively influences figure-of-eight production both in vivo and in vitro. These regions enhance IR-IR junction formation and IR-targeting events in vivo. Enhancer elements have been identified within 51 bp internal to IRL and 17 bp internal to IRR. In the right end, a decanucleotide, 5′-GAGATAATTG-3′, is responsible for wild-type activity, while in the left end, a complex assembly of repetitive elements is required. Functioning of the 10 bp element in the right end is position-dependent and the repetitive elements in the left end act cooperatively and may influence bendability of the end. In vitro kinetic experiments suggest that the sub-terminal enhancers may, at least partly, be transposase-dependent. Such enhancers may reflect a subtle regulatory mechanism for IS30 transposition.     

Nonspecific immune modulating effects of ascites fluid and hyperimmune sera in vivo

To determine whether the appearance of interferon (IFN) and the modulation of humoral responses observed following injection of irradiated tumor cells were mediated by suppressor cells, the effects of in vivo injection of I-Js specific antibodies were studied. We found that anti-I-J-containing, as well as normal ascites fluids, obtained after repeated ip injection of complete Freund's adjuvant, contain a factor which (a) induces the appearance of serum IFN, (b) enhances the response to SRBC, and (c) suppresses the response to TNP-Ficoll, when injected 1 day before antigen. This effect is not immunologically specific, is probably not caused by intact Ig, and does not appear to be mediated by T cells. Although the nature of the factor(s) responsible for the observed results has not been fully clarified, we report these findings now as a cautionary note for the interpretation of studies where in vivo injection of unfractionated ascites fluids containing monoclonal antibodies are used.     

Ginsenoside Rb1 exerts anti-inflammatory effects in vitro and in vivo by modulating toll-like receptor 4 dimerization and NF-kB/MAPKs signaling pathways

BackgoundGinsenoside Rb1, the main active constituent of Panax ginseng, displays significant anti-inflammatory activity, although the mechanism has not been clearly unraveled. In this study, Rb1’s mechanism of anti-inflammatory effects were investigated.MethodsThe flow cytometry and enzyme-linked immunosorbent assay (ELISA) were empolyed to detect pro-inflammatory cytokines release. The related protein and gene expression was investigated by western blotting and qRT-PCR. The dimerization of TLR4 was measured by co-immunoprecipitation and molecular docking assays. Cellular thermal shift assay was used for the determination of the binding of Rb1 and TLR4. For animal moldels, LPS- or cantharidin-induced acute kidney injury, LPS-induced septic death, and dimethyl benzene-induced ear edema were employed to investigate Rb1’s anti-inflammatory activity in vivo.ResultsRb1 significantly decreased inflammatory cytokines release in LPS-stimulated RAW264.7 cells and BMDMs, as well as COX-2 and iNOS amounts. Rb1 reduced LPS-associated calcium influx, ROS production, and NO generation. The NF-κB and MAPK axes participated in Rb1’s anti-inflammatory effects. Molecular docking simulation indicated Rb1 bound to TLR4 to prevent TLR4 dimerization, as confirmed by co-immunoprecipitation and cellular thermal shift assay. Furthermore, MyD88 recruitment and TAK1 expression were altered by reduced TLR4 dimerization, indicating the TLR4-MyD88-NF-κB/MAPK pathways contributed to Rb1’s anti-inflammatory process. In animal models, Rb1 markedly alleviated LPS- or cantharidin-induced acute kidney injury, rescued LPS-induced septic mice from death, and inhibited dimethyl benzene-induced mouse ear edema.ConclusionOverall, these findings demonstrate Rb1 exhibits marked anti-inflammatory effects, suggesting Rb1 represents an optimal molecule for treating inflammatory diseases.     

Upregulation of the vascular NAD(P)H-oxidase isoforms Nox1 and Nox4 by the renin-angiotensin system in vitro and in vivo.

In different cardiovascular disease states, oxidative stress decreases the bioavailability of endothelial NO, resulting in endothelial dysfunction. An important molecular source of reactive oxygen species is the enzyme family of NAD(P)H oxidases (Nox). Here we provide evidence that the vascular Nox isoforms Nox1 and Nox4 appear to be involved in vascular oxidative stress in response to risk factors like angiotensin II (Ang II) in vitro as well as in vivo. Nox mRNA and protein levels were quantified by real-time RT-PCR and Western blotting, respectively. Nox1 and Nox4 were expressed in the vascular smooth muscle cell (VSMC) line A7r5 and aortas and kidneys of rats. Upon exposure of A7r5 cells to Ang II (1 microM, 4 h), Nox1 and Nox4 mRNA levels were increased 6-fold and 4-fold, respectively. Neither the vasoconstrictor endothelin 1 (up to 500 nM, 1-24 h) nor lipopolysaccharide (up to 100 ng/ml, 1-24 h) had any effect on Nox1 and Nox4 expression in these cells. Consistent with these observations made in vitro, aortas and kidneys of transgenic hypertensive rats overexpressing the Ren2 gene [TGR(mRen2)27] had significantly higher amounts of Nox1 and Nox4 mRNA and of Nox4 protein compared to tissues from normotensive wild-type animals. In conclusion, Nox4 and Nox1 are upregulated by the renin-angiotensin system. Increased superoxide production by upregulated vascular Nox isoforms may diminish the effectiveness of NO and thus contribute to the development of vascular diseases. Nox1 and Nox4 could be targeted therapeutically to reduce vascular reactive oxygen species production and thereby increase the bioavailability of NO.     

Cytotoxic effects of nanoparticles assessed in vitro and in vivo

An increasing number of applications is being developed for the use of nanoparticles in various fields. We investigated possible toxicities of nanoparticles in cell culture and in mice. Nanoparticles tested were Zn (300 nm), Fe (100 nm), and Si (10-20, 40-50, and 90-110 nm). The cell lines used were brain, liver, stomach, and lung from humans. In the presence of nanopaticles, mitochodrial activity decreased zero to 15%. DNA contents decreased zero to 20%, and glutathione production increased zero to 15%. None of them showed a dose dependency. Plasma membrane permeability was not altered by nanoparticles. In the case of Si, different sizes of the nanoparticles did not affect cytotoxicity. The cytotoxicity was also shown to be similar in the presence of micro-sized (45 microm) Si particles. Organs from mice fed with nanoparticles showed nonspecific hemorrhage, lymphocytic infiltration, and medullary congestion. A treatment with the micro-sized particle showed similar results, suggesting that the acute in vivo toxicity was not altered by nano-sized particles.     

Anti-allergic effects of His-Ala-Gln tripeptide in vitro and in vivo

We examined the inhibitory effects of HAQ (His-Ala-Gln) peptide on type-1 allergy in vitro and in vivo. HAQ peptide inhibited β-hexosaminidase release and intracellular Ca²⁺ levels of rat basophilic leukemia RBL-2H3 cells. Oral administration of a HAQ peptide-added diet (1 mg/mouse/administration) to C3H/HeJ mice for 14 days led to significant suppression of allergic symptoms, but did not reduce allergen-specific IgE or IgG₁.     

Responses to dehydration in the one-humped camel and effects of blocking the renin-angiotensin system

Our objectives were to compare the levels of circulating electrolytes, hormones, and renal function during 20 days of dehydration in camels versus the level in non-dehydrated camels and to record the effect of blocking angiotensin II AT1 receptors with losartan during dehydration. Dehydration induced significant increments in serum sodium, creatinine, urea, a substantial fall in body weight, and a doubling in plasma arginine vasopressin (AVP) levels. Plasma aldosterone, however, was unaltered compared with time-matched controls. Losartan significantly enhanced the effect of dehydration to reduce body weight and increase serum levels of creatinine and urea, whilst also impairing the rise in plasma AVP and reducing aldosterone levels. We conclude that dehydration in the camel induces substantial increments in serum sodium, creatinine, urea and AVP levels; that aldosterone levels are altered little by dehydration; that blockade of angiotensin II type 1 receptors enhances the dehydration-induced fall in body weight and increase in serum creatinine and urea levels whilst reducing aldosterone and attenuating the rise in plasma AVP.     

Comutagenic and coclastogenic effects of selenium in vitro and in vivo

The comutagenic activity of selenium was investigated using in vitro and in vivo techniques, including the liquid suspension modification of the standard Salmonella/microsome mutagenicity assay, the metaphase analysis of chromosome aberrations in CHO cells and in mouse bone marrow as well as the micronucleus assay in mouse bone marrow. 4 h growth of S. typhimurium TA1535 in a nutrient broth containing 2.9 x 10(-5) M but not 1.16 x 10(-5) M Na2SeO3 caused an up to 10-fold increase of the number of N-methylnitrosourea (MNU, 2.0-2.5 mM)-induced his+ revertants and an up to 2-fold elevation of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 1.48 x 10(-5))-induced mutation rate. Pretreatment of bacteria with Na2SeO3 alone had no effect on the spontaneous mutation level. The combined treatment of CHO cells with MNNG (1.25 x 10(-5) M) or tobacco smoke (TS, 2-3 puffs generated by a cigarette inhalation machine) plus Na2SeO3 (0.58-1.16 x 10(-5) M) starting 2 h and 4 h before the MNNG or TS treatment respectively resulted in a 2-3-fold increase in the percent of metaphases with chromosome aberrations. Furthermore, treatment for 7-14 days of male BDF1 (C57Bl x DBA2) or CC57W mice with Na2SeO3, added to the drinking water at a concentration of 10 ppm, potentiated by 2-3 times the chromosome-damaging activity of urethane (0.5-1.0 g/kg, i.p.) in mouse bone marrow, as measured by the formation of micronuclei or chromosome aberrations. In addition, Na2SeO3 increased up to 43.8% the number of micronucleated polychromatic erythrocytes (MNPCE) induced by mitomycin C (MMC, 1.5 mg/kg, i.p.) in BDF1 mouse bone marrow. Treatment of mice with Na2SeO3 alone had no effect on the spontaneous level of MNPCE. All these findings are consistent with a comutagenic and coclastogenic activity of selenium both in prokaryotes and in eukaryotes, in vitro as well as in vivo after pretreatment of target cells with the trace element.     

Growth and mitogenic effects of arylphorin in vivo and in vitro

In insects, developmental responses are organ- and tissue-specific. In previous studies of insect midgut cells in primary tissue cultures, growth-promoting and differentiation factors were identified from the growth media, hemolymph, and fat body. Recently, it was determined that the mitogenic effect of a Manduca sexta fat body extract on midgut stem cells of Heliothis virescens was due to the presence of monomeric alpha-arylphorin. Here we report that in primary midgut cell cultures, this same arylphorin stimulates stem cell proliferation in the lepidopterans M. sexta and Spodoptera littoralis, and in the beetle Leptinotarsa decemlineata. Studies using S. littoralis cells confirm that the mitogenic effect is due to free alpha-arylphorin subunits. In addition, feeding artificial diets containing arylphorin increased the growth rates of several insect species. When tested against continuous cell lines, including some with midgut and fat body origins, arylphorin had no effect; however, a cell line derived from Lymantria dispar fat body grew more rapidly in medium containing a chymotryptic digest of arylphorin.     

Unequal effects of renin-angiotensin system inhibitors in acute cardiac dysfunction induced by isoproterenol

Several clinical trials have demonstrated that angiotensin-converting enzyme inhibitor (ACEI) and angiotensin II type 1 receptor blocker (ARB) are equally effective in the treatment of chronic heart failure. However, this has not been confirmed for acute cardiac dysfunction. We examined whether ACEI or ARB prevents isoproterenol-induced acute left ventricular (LV) dysfunction in dogs. LV dysfunction induced by a large dose of isoproterenol (1 microg.kg(-1).min(-1), 3-h infusion) was compared in dogs treated with ACEI (temocaprilat) or ARB (olmesartan). Atrial pacing induced a constant heart rate and use of adjustable aortic banding provided a nearly constant afterload. LV systolic function (LV dP/dt, fractional shortening, and ejection fraction) and diastolic function (tau and LV end-diastolic pressure) were significantly deteriorated after isoproterenol infusion. The LV dysfunction was almost totally prevented by ARB but was only partially prevented by ACEI. The partial effect of ACEI was complemented by cotreatment with HOE-140, a bradykinin B2 receptor antagonist. At baseline, the response to low doses of isoproterenol was significantly attenuated by ACEI but not by ARB, and the ACEI-induced attenuation was totally abolished by cotreatment with HOE-140. The response to isoproterenol was significantly attenuated after 3 h of excess isoproterenol loading, and it was almost completely preserved by ARB but not by ACEI. In conclusion, acute LV dysfunction and beta-adrenergic desensitization induced by excess isoproterenol administration were almost totally prevented by ARB but only partially prevented by ACEI. These differences were attributable at least in part to bradykinin pathways activated by ACEI administration in acute LV dysfunction.     

Cytogenetic effects of cigarette smoke condensates in vitro and in vivo

Summary Various cigarette smoke condensates (CSC) were analyzed with respect to the induction of sister-chromatid exchanges (SCE) in human lymphocytes in vitro. CSC from a reference cigarette, from three different tobaccos of the reference cigarette, and from a British cigarette induced similar SCE frequencies. CSC from the reference cigarette did not induce SCE in Chinese hamster bone marrow cells in vivo.     

Anti-angiogenic effects of conjugated docosahexaenoic acid in vitro and in vivo

The anti-angiogenic effects of conjugated docosahexaenoic acid (CDHA), which was prepared by an alkaline treatment of docosahexaenoic acid and contained conjugated double bonds, were investigated in vitro and in vivo. CDHA inhibited tube formation by the bovine aortic endothelial cell (BAEC), and also inhibited the proliferation of BAEC at a concentration of CDHA that suppressed tube formation, but did not influence cell migration. The inhibition of BAEC growth caused by CDHA was accompanied by a marked change in cellular morphology. Nuclear condensation and brightness were observed in Hoechst 33342-stained cells treated with CDHA, indicating that CDHA induced apoptosis in BAEC. We also evaluated the angiogenesis inhibition of CDHA in vivo. The vessel formation which was triggered by tumor cells was clearly suppressed in mice orally given CDHA. Our findings suggest that CDHA has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.