Actinomyces capricornis sp. nov., isolated from the oral cavity of a Japanese serow.

A novel Gram-positive, facultatively anaerobic, rod-shaped, nonspore forming, nonmotile organism was isolated from a Japanese serow oral cavity. Designated strain MAS-1T , it is most closely related to Actinomyces bowdenii DSM 15435T , with which it shares 98.07% sequence homology in the 16S ribosomal RNA gene. The primarily detected cellular fatty acids in strain MAS-1T were C16:0 and C18:1 w9c. The predominant respiratory quinone was MK-9 (H4 ). The major polar lipids were phosphatidylcholines, phosphatidylinositols, and glycophospholipids. The genomic DNA GC content of the isolate was 71.3 mol%. The digital DNA-DNA hybridization and average nucleotide identity values between MAS-1T and its related species were 23.5%-39.5% and 82.11%-91.01%, respectively, which were below the threshold (70% and 95%, respectively) for species delineation, indicating that strain MAS-1T represents a novel species. Strain MAS-1T can be differentiated from A. bowdenii by their reactions to naphthol-AS-BI-phosphohydrolase, α-galactosidase, ß-galactosidase, and N-acetyl-ß-glucosaminidase, as well as differing acid production from glycogen. Based on the results of genotypic, phenotypic, and biochemical analyses, herein it is proposed that the identified bacteria can be classified as a novel species, Actinomyces capricornis sp. nov., strain MAS-1T (=JCM 34236T = DSM 111732T ).

Oral challenge with Streptococcus sanguinis induces aortic inflammation and accelerates atherosclerosis in spontaneously hyperlipidemic mice.

Atherosclerosis is exacerbated by periodontal pathogens, which induce vascular inflammation after entering the bloodstream. Among oral indigenous bacteria, Streptococcus sanguinis and S. anginosus are related to systemic disorders, such as infective endocarditis and abscess, and are sometimes detected in human atherosclerotic plaques or blood. Thus, these oral streptococci may contribute to the progression of atherosclerosis. To test this hypothesis, apolipoprotein E-deficient spontaneously hyperlipidemic mice were intraorally challenged with S. sanguinis or S. anginosus. Atherosclerotic plaque formation increased significantly in the S. sanguinis-challenged group compared with the carboxymethylcellulose-treated control group. Expression levels of mRNAs of proinflammatory cytokines in the aorta and levels of atherosclerosis-related mediators in blood increased upon S. sanguinis challenge. Adaptor molecule TNF receptor-associated factor 6 was also enhanced in the aorta when mice were challenged with S. sanguinis. Furthermore, challenge with S. anginosus induced systemic inflammation, but inflammation-related mRNA expression levels in the aorta only increased slightly and were accompanied by minimal expansion of the lesion area. By contrast, with the exception of IL-1α, the expression levels of inflammation-related genes did not change in gingival tissues of both bacteria- and sham-challenged groups. These results reveal that S. sanguinis causes aortic inflammation that leads to accelerated progression of atherosclerosis.

Pseudopropionibacterium rubrum sp. nov., a novel red-pigmented species isolated from human gingival sulcus.

In this study, Strain [corrected] SK-1(T), a novel gram-positive, pleomorphic, rod-shaped, non-spore forming, non-motile organism, designated SK-1T , was isolated from human gingival sulcus and found to produce acetic acid, propionic acid, lactic acid, and succinic acid as end products of glucose fermentation. Strain SK-1T is most closely related to Pseudopropionibacterium (Propionibacterium) propionicum with sequence homologies of the 16S rRNA and RNA polymerase ß subunit (rpoB) genes of 96.6% and 93.1%, respectively. The genomic DNA G + C content of the isolate was 61.8 mol%. On the basis of the sequence data of the 16S rRNA and housekeeping (rpoB) genes, a novel taxon is here proposed, Pseudopropionibacterium rubrum sp. nov. (type strain SK-1T = JCM 31317T = DSM 100122T ). The 16S rRNA and rpoB gene sequences of strain SK-1T have been deposited in the DDBJ under the accession numbers LC002971 and LC102236, respectively.

Streptococcusdentiloxodontae sp. nov., isolated from the oral cavity of elephants.

A Gram-stain-positive, catalase-negative, coccus-shaped organism was isolated from oral cavity samples collected from healthy elephants. The isolated strain, NUM 2404T, was tentatively identified as a streptococcal species based on the results of biochemical tests. Although a comparative 16S rRNA gene sequence analysis suggested the classification of this organism into the genus Streptococcus, it did not correspond to any recognized species of the genus. Strain NUM 2404T was related most closely to Streptococcus saliviloxodontae NUM 6306T with 95.8 % 16S rRNA gene sequence similarity, but the phylogenetic tree reconstructed based on the 16S rRNA gene sequence showed that NUM 2404T clustered with Streptococcus mutans NCTC 10449T and Streptococcus troglodytae TKU 31T. Comparative sequence analysis based on two housekeeping genes, groEL, which encodes the 60 kDa heat-shock protein, and rpoB, encoding the ß subunit of RNA polymerase, of NUM 2404T indicated that it was most closely related to those of Streptococcus orisratti A63T and Streptococcus sobrinus ATCC 33478T with 82.7 and 85.1 % sequence similarities, respectively. On the basis of genotypic and phenotypic differences, it is proposed that the novel isolate be classified in the genus Streptococcus as representative of a novel species, Streptococcus dentiloxodontae sp. nov. The type strain is NUM 2404T (=JCM 19284T=DSM 27381T).

Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Serotype g Strain NUM4039 (JCM 30399).

Aggregatibacter actinomycetemcomitans is considered to be a major etiological agent of aggressive periodontitis and includes serotype a to g strains. We herein report the first complete genome sequence of A. actinomycetemcomitans serotype g strain NUM4039. The genome is 2,382,853 bp in length with a G+C content of 44.34%.

A selective medium for the isolation of Microbacterium species in oral cavities.

The genus Microbacterium has been isolated from the environment, dairy goods, and human clinical specimens. Although, in our previous studies, some Microbacterium species were infrequently detected in oral samples collected from humans, there is currently no report that these organisms, which are capable of causing serious systemic infections, were isolated from the human oral cavity. The aim of the present study was to develop a selective medium to isolate the representative Microbacterium species most frequently detected in human clinical specimens, and reveal the distribution of individual Microbacterium species in the oral cavity. The growth recoveries of representative Microbacterium species on the selective medium, designated as MSM, were sufficient. Moreover, the growth of other representative oral bacteria was markedly inhibited on the selective medium. The proportion of Microbacterium species in the saliva samples of 60 subjects, 20 of whom were removable denture wearers, was then examined. The proportion of these organisms was also examined in environmental samples obtained by swabbing 20 washstands. PCR primers were designed for representative Microbacterium species. The genus Microbacterium was detected in 45% of the saliva and denture plaque samples collected from the twenty removable denture wearers, but was absent in the saliva of the forty non-denture wearers. On the other hand, these organisms were detected in all environmental samples. The genus Microbacterium accounted for 0.00003%, 0.0001%, and 12.6% of the total cultivable bacteria number on the BHI medium in the saliva and denture plaque samples of removable denture wearers and in the environmental samples, respectively. The most predominant Microbacterium species in all positive samples was Microbacterium oxydans. These results indicated that the genus Microbacterium was not a part of the normal flora in the human oral cavity, except for subjects wearing dentures that were contaminated by the environment, and the selective medium, designated as MSM, was useful for isolating Microbacterium species, which are frequently encountered in human clinical specimens, from the various samples.

Streptococcus oriloxodontae sp. nov., isolated from the oral cavities of elephants.

Two strains were isolated from oral cavity samples of healthy elephants. The isolates were Gram-positive, catalase-negative, coccus-shaped organisms that were tentatively identified as a streptococcal species based on the results of biochemical tests. Comparative 16S rRNA gene sequence analysis suggested classification of these organisms in the genus Streptococcus with Streptococcus criceti ATCC 19642(T) and Streptococcus orisuis NUM 1001(T) as their closest phylogenetic neighbours with 98.2 and 96.9% gene sequence similarity, respectively. When multi-locus sequence analysis using four housekeeping genes, groEL, rpoB, gyrB and sodA, was carried out, similarity of concatenated sequences of the four housekeeping genes from the new isolates and Streptococcus mutans was 89.7%. DNA-DNA hybridization experiments suggested that the new isolates were distinct from S. criceti and other species of the genus Streptococcus. On the basis of genotypic and phenotypic differences, it is proposed that the novel isolates are classified in the genus Streptococcus as representatives of Streptococcus oriloxodontae sp. nov. The type strain of S. oriloxodontae is NUM 2101(T) ( =JCM 19285(T) =DSM 27377(T)).

Streptococcus loxodontisalivarius sp. nov. and Streptococcus saliviloxodontae sp. nov., isolated from oral cavities of elephants.

Four Gram-stain-positive, catalase-negative, coccoid-shaped organisms were isolated from elephant oral cavities. The isolates were tentatively identified as streptococcal species based on the results of biochemical tests. Comparative 16S rRNA gene sequencing studies confirmed the organisms to be members of the genus Streptococcus. Two isolates (NUM 6304(T) and NUM 6312) were related most closely to Streptococcus salivarius with 96.8 % and 93.1 % similarity based on the 16S rRNA gene and the RNA polymerase ß subunit encoding gene (rpoB), respectively, and to Streptococcus vestibularis with 83.7 % similarity based on the 60 kDa heat-shock protein gene (groEL). The other two isolates (NUM 6306(T) and NUM 6318) were related most closely to S. vestibularis with 97.0 % and 82.9 % similarity based on the 16S rRNA and groEL genes, respectively, and to S. salivarius with 93.5 % similarity based on the rpoB gene. Based on phylogenetic and phenotypic evidence, these isolates are suggested to represent novel species of the genus Streptococcus, for which the names Streptococcus loxodontisalivarius sp. nov. (type strain NUM 6304(T) = JCM 19287(T) = DSM 27382(T)) and Streptococcus saliviloxodontae sp. nov. (type strain NUM 6306(T) = JCM 19288(T) = DSM 27513(T)) are proposed.

A selective medium for the isolation of Corynebacterium species in oral cavities.

Corynebacterium matruchotii is a microbial inhabitant in the oral cavity of humans and is associated with the formation of dental calculi. C. matruchotii forms highly specific morphological units, which are referred to as corn-cobs. Although other Corynebacterium species have frequently been isolated from the oral cavity of humans, their distribution has not been reported as extensively. The aim of the present study was to develop a selective medium to isolate the genus Corynebacterium and examine the distribution Corynebacterium species in the oral cavity of humans. The growth recoveries of representative Corynebacterium species on the selective medium were sufficient. Moreover, the growth of other representative oral bacteria was markedly inhibited on the selective medium. The proportion of Corynebacterium species in saliva samples collected from 20 subjects was examined. PCR primers were designed for the oral Corynebacterium species. C. matruchotii and Corynebacterium durum accounted for 0.3% and 1.5% of the total cultivable bacteria number on the BHI medium from saliva samples, respectively. The selective medium could distinguish C. matruchotii from C. durum by each colony color using differences in acid production from galactose. The selective medium, designated OCM, was useful for isolating oral Corynebacterium species.

Establishment of a convenient sandwich-ELISA for direct quantification of glucosyltransferase-I: application for dual diagnosis of dental caries.

Previously, we established a convenient enzyme-linked immunosorbent assay (ELISA) system targeting glucosyltransferase (GTF)-B derived from Streptococcus mutans for diagnosing caries risk. However, it has been reported that S. sobrinus possesses high cariogenicity and is more frequently detected in highly caries-susceptible patients than S. mutans is. S. sobrinus can secrete GTF-I, an important cariogenic factor for dental plaque formation, as well as S. mutans GTF-B. Therefore, in this study, we developed another feasible ELISA system targeting S. sobrinus GTF-I that would ensure caries risk determination by combined GTF-I and GTF-B levels. A readily measurable sandwich-ELISA system was devised, which consisted of monoclonal and polyclonal antibodies against GTF-I. The developed sandwich-ELISA system quantified the purified GTF-I with sensitivity and specificity, and a positive correlation was observed between the amount of GTF-I extracted from clinical plaque samples and S. sobrinus levels. Furthermore, high levels of GTF-I and GTF-B were detected using the sandwich-ELISA system in caries-susceptible subjects. These results indicate that the sandwich-ELISA system against GTF-I developed in this study is useful, and that the dual detection of the caries risk factors GTF-I and GTF-B is helpful for predicting caries risk.

Sequence and phylogenetic analyses of the water-soluble glucan synthesizing-glucosyltransferase genes of Streptococcus dentirousetti.

Two tandemly aligned glucosyltransferase (GTF) genes whose gene products are responsible for water-soluble glucan synthesis were isolated from Streptococcus dentirousetti NUM1303 and sequenced. One of the GTF genes of S. dentirousetti consisted of a 4110 bp open reading frame (ORF) that encoded for a 1369 amino acid protein and was revealed to be a S. sobrinus gtfS homolog. The percent similarity of amino acid sequences of the GTF-S from S. dentirousetti compared to those from S. sobrinus was 99%. In addition, a putative gtfT was found in tandem in the downstream region of the S. dentirousetti gtfS. The gtfT of S. dentirousetti consisted of a 4527 bp ORF encoding for 1508 amino acids. The similarity of amino acid sequences of the GTF-T from S. dentirousetti and S. sobrinus was 94%. Phylogenetic analysis based on amino acid sequences from other related streptococcal GTFs suggested that both GTF-S and GTF-T of S. dentirousetti are closely related to S. sobrinus.

A feasible enzyme-linked immunosorbent assay system using monoclonal and polyclonal antibodies against glucosyltransferase-B from Streptococcus mutans.

Streptococcus mutans has been considered the principal etiological agent of dental caries in humans. S. mutans can secrete three kinds of glucosyltransferases (GTFs). One of these, GTF-B, which synthesizes water-insoluble glucans from sucrose, has been considered to be one of the most important factors of cariogenic dental plaque formation. Therefore, determination of whether GTF-B is present in plaque and saliva samples may contribute to the evaluation of individual virulence potential (caries risk). The aim of this study was to develop a feasible enzyme-linked immunosorbent assay (ELISA) for the routine quantification of GTF-B in plaque-derived cultures and clinical samples, and to apply this assay to an epidemiological study. To determine the presence of GTF-B in plaque samples, a sandwich-ELISA was devised, consisting of mouse monoclonal and rabbit polyclonal antibodies against GTF-B and a horseradish peroxidase-conjugated anti-rabbit antibody. The developed ELISA allowed for quantification of the amounts of purified GTF-B with satisfactory sensitivity and specificity; this method was not affected by other components such as plaque and saliva. Plaque samples from healthy volunteers were examined using this ELISA method and microbial analysis to apply the assay to an epidemiological study. A correlation was observed between the amount of extracted GTF-B and S. mutans levels as determined by ELISA and cultivated with Mitis Salivarius Bacitracin agar plates derived from plaque samples, although there were some exceptions. In this regard, this ELISA system has the advantage of estimating both the individual numbers of S. mutans and the productivity of GTF-B, namely, the cariogenic potential of S. mutans simultaneously. These results indicate that this ELISA method is a useful tool for the diagnosis of caries risk.

Gibbsiella dentisursi sp. nov., isolated from the bear oral cavity.

A Gram-negative, rod-shaped, non-spore forming and non-motile bacterium, designated strain NUM 1720(T) , was isolated from the oral cavity of bears. Based on 16S rRNA gene sequence similarity, strain NUM 1720(T) was shown to be related to Gibbsiella quercinecans (99.4%). The gyrB and rpoB gene sequences of strain NUM 1720T showed 98.0% and 98.2% similarity with those of G. quercinecans. The DNA-DNA hybridization value of strain NUM 1720(T) with G. quercinecans was 63.8%. The G + C content of the genomic DNA of the isolates was 55.0 mol%. Fatty acid analysis data supported the affiliation of strain NUM 1720(T) to the genus Gibbsiella. The major menaquinone and ubiquinone were MK-8 and Q-8, respectively. Strain NUM 1720(T) can be differed from G. quercinecans by the reactions to acetoin, inositol and D-arabinose. Strain NUM 1720(T) therefore represents a novel species, for which the name Gibbsiella dentisursi sp. nov. is proposed, with type strain NUM 1720(T) (= JCM 17201(T) = DSM 23818(T)).

Streptococcus ursoris sp. nov., isolated from the oral cavities of bears.

Three Gram-positive, catalase-negative, coccus-shaped organisms were isolated from the oral cavities of bears. The isolates were tentatively identified as a streptococcal species based on the results of biochemical tests. Comparative 16S rRNA gene sequencing studies confirmed that the organisms were members of the genus Streptococcus, but they did not correspond to any recognized species of the genus. The nearest phylogenetic relative of the new isolates was Streptococcus ratti ATCC 19645(T) (98.6 %), however, DNA-DNA hybridization analysis showed that the isolates displayed less than 15 % DNA-DNA relatedness with the type strain of S. ratti. Colonies of the novel strains grown on mitis salivarius agar showed an extracellular polysaccharide-producing colony morphology. Based on phenotypic and phylogenetic evidence, it is proposed that the novel isolates are classified in the genus Streptococcus as Streptococcus ursoris sp. nov. The type strain of S. ursoris is NUM 1615(T) (=JCM 16316(T)=DSM 22768(T)).

Sequence and phylogenetic analyses of novel glucosyltransferase genes of mutans streptococci isolated from pig oral cavity.

Nucleotide sequences of water-insoluble glucan-producing glucosyltransferase (gtf) genes of new mutans streptococci isolated from pig oral cavity, Streptococcus orisuis JCM14035, and of Streptococcus criceti HS-6 were determined. The gtf gene of S. orisuis JCM14035 consisted of a 4,401 bp ORF encoding for a 1,466 amino acids, and was revealed to belong to the gtfI group. The percent homology of amino acid sequence of the GTF-I from S. orisuis and S. criceti are 95.0%, however, this score ranges from 77.0% to 78.0% when compared to Streptococcus sobrinus 6715. The deduced N-terminal amino acid sequence was considered responsible for the secretion of GTF-I in S. orisuis JCM14035 and S. criceti HS-6 with high similarity to known GTF proteins from other streptococci. In addition, two other conserved regions, i.e., N-terminal putative catalytic-site and C-terminal glucan binding domain, were also found in GTF-Is of S. orisuis JCM14035 and S. criceti HS-6. Phylogenetic analysis suggested that S. orisuis JCM14035 and S. criceti HS-6, closely related to each other, resemble S. sobrinus and S. downei based on the amino acid sequences of the GTFs.

HMG-CoA reductase inhibition improves anti-aging klotho protein expression and arteriosclerosis in rats with chronic inhibition of nitric oxide synthesis.

BACKGROUND: The klotho gene and its protein product are mainly expressed in the kidney. The klotho protein induces suppression of multiple aging-related phenotypes, and homozygous klotho gene mutant mice display various senescent morbidity. Chronic inhibition of nitric oxide synthase (NOS) induces arteriosclerosis, while HMG-CoA reductase inhibitors (statins) have pleiotropic vascular protective effects besides cholesterol lowering. Therefore, the present studies were performed to determine whether chronic NOS blockade would affect anti-ageing klotho protein expression. In addition, the effects of statins on klotho protein expression and arteriosclerosis in these rats were investigated. METHODS: Forty-two rats were divided into 6 groups as follows: (1) control, (2) NOS blockade, (3) atorvastatin (10 mg/kg/day), (4) pitavastatin (3 mg/kg/day), (5) NOS blockade+atorvastatin, (6) NOS blockade+pitavastatin. To induce arteriosclerosis further, a cuff was placed around the left femoral artery in each rat. After 4 weeks observation, rats were killed and renal klotho expression and the level of arteriosclerosis were examined. RESULTS: The rats of chronic NOS inhibition developed hypertension, while statin treatment did not affect blood pressure in the rats with or without NOS blockade. Despite statin treatment, plasma levels of lipids did not differ among 6 groups. Immunohistochemical staining revealed that klotho protein was localized in the renal tubules. Chronic NOS inhibition markedly reduced renal klotho protein expression, while treatment with atorvastatin or pitavastatin completely prevented the reduction of klotho expression induced by NOS inhibition. In addition, statin treatment significantly improved arteriosclerotic lesions induced by NOS inhibition and cuff placement. CONCLUSION: Since statin treatment did not alter blood pressure or serum lipid profiles, a novel vascular protective effect of statins via enhancing anti-aging klotho protein expression is suggested.

Isolated lymphoid follicles are not IgA inductive sites for recombinant Salmonella.

In this study, we investigated whether isolated lymphoid follicles (ILF) play a role in the regulation of intestinal IgA antibody (Ab) responses. The transfer of wild type (WT) bone marrow (BM) to lymphotoxin-alpha-deficient (LTalpha(-/-)) mice resulted in the formation of mature ILF containing T cells, B cells, and FDC clusters in the absence of mesenteric lymph nodes and Peyer's patches. Although the ILF restored total IgA Abs in the intestine, antigen (Ag)-specific IgA responses were not induced after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin. Moreover, Ag-specific cell proliferation was not detected in the ILF. Interestingly, no IgA anti-LPS Abs were detected in the fecal extracts of LTalpha(-/-) mice reconstituted with WT BM. On the basis of these findings, ILF can be presumed to play a role in the production of IgA Abs, but lymphoid nodules are not inductive sites for the regulation of Ag-specific intestinal IgA responses to recombinant Salmonella.

Augmentation of pulse wave velocity precedes vascular structural changes of the aorta in rats treated with N(omega)-nitro-L-arginine methyl ester.

We examined the relationship between structural changes of the aorta and pulse wave velocity (PWV), and the effects of antihypertensive treatments on PWV in N(omega)-nitro-L-arginine methyl ester (L-NAME)-treated rats. Twelve-week-old Wistar-Kyoto (WKY) rats were divided into the following groups, all of which received drug treatment in their drinking water: an untreated control group (n = 36), an L-NAME-treated group (0.7 mg/ml) (n = 32), an L-NAME and angiotensin converting enzyme (ACE) inhibitor (ACEI)-treated group (imidapril: 0.4 mg/ml) (n = 8), and an L-NAME and hydralazine-treated group (0.2 mg/ml) (n = 10). PWV was measured at the same blood pressure (BP) level as in the control group and the wall-to-lumen ratio of the thoracic aorta was evaluated in all groups. In the L-NAME group, PWV increased compared with the value in the control group, at the same time that BP was increasing. After the third day of treatment, PWV was higher in the L-NAME group than in the control group after adjusting BP to the control level, while the wall-to-lumen ratios were equal between the two groups. After the first week of treatment, not only the adjusted PWV, but also the wall-to-lumen ratios were greater in the L-NAME group than in the control group. With administration of antihypertensive agents, both PWV and the thickening of the aortic wall were reduced, but there was no significant difference between the ACEI and hydralazine-treated groups. In conclusion, in a rat model of nitric oxide (NO) synthesis inhibition, the increase in PWV preceded the vascular structural changes, while antihypertensive treatment reduced both changes. There was no significant difference between treatments with ACEI and hydralazine in this model.

Expression of the gtfI gene from Streptococcus sobrinus in Streptococcus anginosus using integration-mediated transformation system.

We have constructed a Streptococcus anginosus transformant expressing the gtfI gene from Streptococcus sobrinus, using a previously developed integration-mediated transformation system to introduce foreign genes onto the oral streptococcal chromosome, and attempted to evaluate the gene expression. In this system, one cloning plasmid and three pACYC184 derivatives, anchor, heterodimer, and integration plasmids were used for the construction of a series of integrants via homologous recombination. A portion of S. sobrinus gtfI gene devoid of approximately 1 kb of the 5'-region derived from pMD39 was cloned into the integration plasmid and introduced onto the S. anginosus chromosome. Next, the polymerase chain reaction product corresponding to 2.0 kb of the 5'-region of the gtfI gene from S. sobrinus chromosome was further cloned into the cloning plasmid, and the intact gtfI gene was reconstructed following integration. The final S. anginosus integrant successfully secreted the enzymatically active gtfI gene products and extracellular enzyme was characterized. This enzyme produced water-insoluble glucans and glucan-forming activity was stimulated by the addition of dextranT10. When this integrant was grown in Todd-Hewitt broth supplemented with sucrose, the integrant adhered to the glass surface in vitro and this integrant exhibited the different colony morphology on Mitis-Salivarius agar plates compared to S. sobrinus and S. anginosus. These observations strongly suggest that the construction of S. anginosus integrant expressing S. sobrinus gtfI gene using this transformation system may be an effective means of analysis of cariogenic biofilm formation.

HMG-CoA reductase inhibitors up-regulate anti-aging klotho mRNA via RhoA inactivation in IMCD3 cells.

OBJECTIVE: Klotho is thought to play a critical role in the development of age-related disorders including arteriosclerosis. Statins may exert vascular protective effects, independent of the lowering of plasma cholesterol levels. We investigated the impact of statins on mRNA expression of the age-suppressor gene, klotho in mIMCD3 cells. METHODS AND RESULTS: Klotho mRNA levels were evaluated with real-time RT-PCR. Atorvastatin and pitavastatin increased the expression of klotho mRNA in a dose-dependent manner. This stimulatory effect was abolished by the addition of mevalonate, GGPP and FPP, essential molecules for isoprenylation of the small GTPase Rho. As was the case with the statin treatment, inhibition of Rho-kinase by Y27632 up-regulated klotho mRNA. In contrast to the statin treatment, stimulation with angiotensin II down-regulated klotho mRNA expression without obvious morphological changes. Furthermore, pretreatment with atorvastatin blunted the angiotensin II-induced response and ameliorated the decrease in klotho mRNA expression towards basal levels. RhoA activity was further evaluated by detection of its translocation. Angiotensin II activated RhoA, whereas statins potently inactivated RhoA and blocked RhoA activation by angiotensin II. CONCLUSION: Statins inactivate the RhoA pathway, resulting in over-expression of klotho mRNA, which may contribute to the novel pleiotropic effects of statins towards vascular protection.