► Triply n/i/eNOS−/− mice exhibit accelerated arteriosclerosis after carotid ligation. ► WT mice transplanted with n/i/eNOS−/− bone marrow also show similar acceleration. ► The whole endogenous NOS ...system exerts a crucial vasculoprotective role. ► The NOS system in the bone marrow is involved in this vasculoprotective mechanism.
Although all three nitric oxide (NO) synthases (nNOS, iNOS, and eNOS) are expressed in injured arteries, it remains to be elucidated the role of the NOSs in their entirety in the vascular lesion formation. We addressed this issue in mice deficient in all NOS genes. Vascular injury was induced by permanent ligation of a unilateral carotid artery in wild-type (WT), singly, and triply NOS−/− mice. Two weeks after the procedure, constrictive vascular remodeling and neointimal formation were recognized in the ligated arteries. While constrictive remodeling was noted in the nNOS−/− and iNOS−/− genotypes, it was most accelerated in the n/i/eNOS−/− genotype. While neointimal formation was evident in the eNOS−/− and nNOS−/− genotypes, it was also most aggravated in the n/i/eNOS−/− genotype. Those lesions were reversed by long-term treatment with isosorbide dinitrate, a NO donor. Finally, we examined the involvement of bone marrow-derived cells in the vascular lesion formation. Bone marrow from the WT, singly, or triply NOS−/− mice was transplanted into the WT mice, and then the carotid ligation was performed. Intriguingly, constrictive remodeling and neointimal formation were both similarly most exacerbated in the case of the n/i/eNOS−/− bone marrow transplantation. These results indicate that the complete disruption of all the NOS genes causes markedly accelerated vascular lesion formation caused by blood flow disruption in mice in vivo, demonstrating the crucial vasculoprotective role of the whole endogenous NOS system. Our findings also suggest that the NOS system in bone marrow-derived cells may be involved in this vasculoprotective mechanism.
Abstract Background Raf is one of the downstream effectors of Ras GTPases. The induction of Raf in the epidermis causes the proliferation of keratinocytes and epidermal hyperplasia. However, skin ...inflammation accompanying Ras-induced epidermal reactions has not been fully delineated. Objective The aim of this study was to characterize inflammatory reactions induced by epidermal-specific Raf expression and to elucidate its role in skin inflammation. Methods K14-Raf:ER transgenic mice, in which the 4-hydroxytamoxifen (4OHT)-responsive mutant estrogen receptor (ER) ligand binding domain-Raf fusion gene was expressed under control of the keratin 14 promoter, were used to characterize inflammatory reactions induced by Raf expression in the epidermis. Results A single topical application of 4OHT induced the expression of phosphorylated extracellular signal-related kinase 1/2 and elicited neutrophil-dominant inflammatory infiltrates in the skin. The Raf expression also rapidly induced the production of several cytokines and chemokines, including VEGF and CXCL1, by keratinocytes and in mouse skin in vivo . Furthermore, CD4-positive cells from regional lymph nodes had the potential to differentiate into IFNγ- and IL17-producing cells. Treatment with an anti-Gr-1 antibody diminished the Raf-induced cutaneous inflammation and partially reversed the epidermal hyperplasia and hyperkeratosis. Conclusion Activation of the Raf signaling pathway is involved in the epidermal hyperplasia and the neutrophil-dominant cutaneous inflammatory reactions which are characteristics of psoriasis.
The Food Safety Commission (FSC) of Japan, established in July 2003, has its own initiative to conduct risk assessments on food stuffs known as “self-tasking assessment”. Within this framework, the ...FSC decided to conduct a risk assessment of beef and beef offal imported into Japan from countries with no previous BSE reports; thus, a methodology was formed to suit to this purpose. This methodology was partly based on the previous assessments of Japanese domestic beef and beef imported from U.S.A./Canada, but some modifications were made. Other organizations’ assessment methods, such as those used for BSE status assessment in live cattle by the OIE and EFSA’s GBR, were also consulted. In this review, the authors introduce this alternative methodology, which reflects (1) the risk of live cattle in the assessed country including temporal risks of BSE invasion and domestic propagation, with the assessment results verified by surveillance data, and (2) the risk of beef and beef offal consisting of cumulative BSE risk by types of slaughtering and meat production processes implemented and the status of mechanically recovered meat production. Other possible influencing factors such as atypical BSE cases were also reviewed. The key characteristic of the current assessment is a combination of the time-sequential risk level of live cattle and qualitative risk level of meat production at present in an assessed country.
Abstract We investigated the effect of subtotal nephrectomy on the incidence of acute myocardial infarction (AMI) in mice deficient in all three nitric oxide synthases (NOSs). Two-thirds nephrectomy ...(NX) was performed on male triple NOSs−/− mice. The 2/3NX caused sudden cardiac death due to AMI in the triple NOSs−/− mice as early as 4 months after the surgery. The 2/3NX triple NOSs−/− mice exhibited electrocardiographic ST-segment elevation, reduced heart rate variability, echocardiographic regional wall motion abnormality, and accelerated coronary arteriosclerotic lesion formation. Cardiovascular risk factors (hypertension, hypercholesterolemia, and hyperglycemia), an increased number of circulating bone marrow-derived vascular smooth muscle cell (VSMC) progenitor cells (a pro-arteriosclerotic factor), and cardiac up-regulation of stromal cell-derived factor (SDF)-1α (a chemotactic factor of the progenitor cells) were noted in the 2/3NX triple NOSs−/− mice and were associated with significant increases in plasma angiotensin II levels (a marker of renin–angiotensin system activation) and urinary 8-isoprostane levels (a marker of oxidative stress). Importantly, combined treatment with a clinical dosage of an angiotensin II type 1 receptor blocker, irbesartan, and a calcium channel antagonist, amlodipine, markedly prevented coronary arteriosclerotic lesion formation and the incidence of AMI and improved the prognosis of those mice, along with ameliorating all those pro-arteriosclerotic parameters. The 2/3NX triple NOSs−/− mouse is a new experimentally useful model of AMI. Renin–angiotensin system activation, oxidative stress, cardiovascular risk factors, and SDF-1α-induced recruitment of bone marrow-derived VSMC progenitor cells appear to be involved in the pathogenesis of AMI in this model.
Monomeric carbonyl reductase 3 (CBR3) is a member of the short-chain dehydrogenase/reductase family. CBR3 exhibits much lower activity than monomeric carbonyl reductase 1 (CBR1) in humans and Chinese ...hamsters although they are highly homologous to each other in amino acid sequence levels. In the present study, we first cloned the CBR3 gene of rat origin (rCBR3), and characterized its enzymatic activity. rCBR3 also exhibited a limited catalytic efficiency similarly to the other CBR3 orthologues of humans and Chinese hamsters. Among the CBR3 orthologues, the human enzyme showed considerably lower activity. Compared with the amino acid sequences of CBR1 and CBR3 among humans, rats, Chinese hamsters, and mice, the tryptophan residue at position 230 is highly conserved while human CBR3 possesses rigid amino acid, proline, at that position instead. Thus, the Trp-230 was expected to be one of the important residues for catalysis since it locates in the hinge region at the substrate-binding loop. The substitution of tryptophan for proline in hCBR3 failed to affect the enzymatic characteristics. Similarly, the substitution of proline for tryptophan in either Chinese hamster CBR3 (CHCR3) or rCBR3 showed no significant change in the catalytic properties. These results suggest that limited catalytic efficiency of carbonyl reductase activity of CBR3 is a common property among animal species, and the substitution of the amino acid residue at position 230 alone has no apparent impact on their enzymatic activities.
OBJECTIVE—We demonstrated recently that neuronal NO synthase (NOS) is expressed in arteriosclerotic lesions and exerts important vasculoprotective effects in vivo. In this study, we examined the ...molecular mechanism(s) for vascular neuronal NOS (nNOS) expression.
METHODS AND RESULTS—In cultured rat aortic smooth muscle cells, treatment with platelet-derived growth factor (PDGF) selectively upregulated nNOS expression but not inducible NOS (iNOS) or endothelial NOS (eNOS) expression. Treatment with PDGF also significantly caused activation of mitogen-activated protein kinase (MAPK) family, including extracellular signal-regulated kinase (ERK), p38MAPK, and c-Jun N-terminal kinase (JNK). ERK kinase (MAPK kinase MEK) inhibitors inhibited PDGF-induced nNOS expression, whereas a p38MAPK inhibitor or JNK inhibitor was without effects. Importantly, gene transfer of MEK per se elicited nNOS induction, and gene transfer of dominant-negative MEK abolished PDGF-induced nNOS expression. In isolated aortas of wild-type, eNOS, and iNOS mice, but not in those of nNOS mice, treatment with PDGF significantly enhanced nNOS expression and nitrite plus nitrate production, both of which were again attenuated by a MEK inhibitor.
CONCLUSIONS—These results provide the first evidence that vascular nNOS expression is upregulated selectively in response to PDGF through the MEK/ERK pathway. Upregulated nNOS may play an important compensatory role under arteriosclerotic/inflammatory conditions associated with eNOS dysfunction to maintain vascular homeostasis.
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