CC chemokine ligand 3 and receptors 1 and 5 gene expression in recurrent aphthous stomatitis

Objective. The aim of this study was to investigate the local and systemic expression of CC-chemokine ligand 3 (CCL3) and its receptors (CCR1 and CCR5) in tissue samples and peripheral blood mononuclear cells of recurrent aphthous stomatitis (RAS) patients. Study Design. This case-control study enrolled 29 patients presenting severe RAS manifestations and 20 non-RAS patients proportionally matched by sex and age. Total RNA was extracted from biopsy specimens and peripheral blood mononuclear cells for quatitative reverse-transcription polymerase chain reaction. The data obtained by relative quantification were evaluated by the 2(-Delta Delta Ct) method, normalized by the expression of an endogenous control, and analyzed by Student t test. Results. The results demonstrated overexpression in RAS tissue samples of all of the chemokines evaluated compared with healthy oral mucosa, whereas the blood samples showed only CCR1 overexpression in RAS patients. Conclusions. These findings suggest that the increased expression of CCL3, CCR1, and CCR5 may influence the immune response in RAS by T(H)1 cytokine polarization. (Oral Surg Oral Med Oral Pathol Oral Radiol 2012;114:93-98)Fundacao de Amparo a Pesquisa do Estado de Sao PauloFundacao de Amparo a Pesquisa do Estado de Sao Paulo [06/57505-3

Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue

<div><p>The initiation or progression of periodontitis might involve a local renin-angiotensin system (RAS) in periodontal tissue. The aim of this study was to further characterize the local RAS in human and rat periodontal tissues between healthy and periodontally-affected tissue. Components of the RAS were investigated using <i>in vitro</i>, <i>ex vivo</i> and <i>in vivo</i> experiments involving both human and Wistar rat periodontium. Although not upregulated when challenged with <i>P</i>. <i>gingivalis</i>-lipopolysaccharide, human gingival and periodontal ligament fibroblasts expressed RAS components. Likewise, healthy and inflamed human gingiva expressed RAS components, some of which were shown to be functional, yet no differences in expression were found between healthy and diseased gingiva. However, in inflamed tissue the immunoreactivity was greater for the AT₁R compared to AT₂R in fibroblasts. When compared to healthy tissue, ACE activity was increased in human gingiva from volunteers with gingivitis. Human-gingiva homogenates generated Ang II, Ang 1-9 and Ang 1-7 when incubated with precursors. In gingiva homogenates, Ang II formation from Ang I was nearly abolished only when captopril and chymostatin were combined. Ang 1-7 formation was significantly greater when human gingiva homogenates were incubated with chymostatin alone compared to incubation without any inhibitor, only captopril, or captopril and chymostatin. In rat gingiva, RAS components were also found; their expression was not different between healthy and experimentally induced periodontitis (EP) groups. However, renin inhibition (aliskiren) and an AT₁R antagonist (losartan) significantly blocked EP-alveolar-bone loss in rats. Collectively, these data are consistent with the hypothesis that a local RAS system is not only present but is also functional in both human and rat periodontal tissue. Furthermore, blocking AT₁R and renin can significantly prevent periodontal bone loss induced by EP in rats.</p></div

Renin-Angiotensin System Components in Human Gingiva.

<p><b>A)</b> qPCR analysis of the following RAS components extracted from human gingiva from healthy (open bars), gingivitis (gray bars) and periodontitis (black bars) groups: angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin converting enzyme 2 (ACE-2), renin, angiotensin II receptor type 1 (AT₁R), and Mas receptor (MasR), (n = 7). Graphs displays relative expression levels of the target mRNA relative to RPL-13 mRNA. The means were compared using a 1-way ANOVA and Tukey’s test. <b>B)</b> Immunoreactivity of AT₁R and AT₂R in the gingiva from healthy volunteers (open bars) and volunteers with gingivitis and periodontitis combined (black bars). The means were compared using a 1-way ANOVA and Ang with gingivitis and periodontitis is indicated by *. <b>C)</b> Representative photographs of immunoreactivity for either AT₁R (Ca and Ce) or AT₂R (Ce and Cf) in the gingiva from either healthy volunteers (Ca and Cd) or volunteers with gingivitis and periodontitis (Ce and Cf). Photographs Cb and Cd are from tissue incubated with nonimmune serum in Healthy Tissue. Scale bars indicate a distance of 20μm.</p

Catalog numbers for primers used for human qPCR—RAS genes assays (Applied Biosystems).

<p>Catalog numbers for primers used for human qPCR—RAS genes assays (Applied Biosystems).</p

Bone Loss in Rats with Experimentally Induced Periodontitis.

<p><b>A)</b> Graph with representative images indicating the amount of area measured between the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) of the first molar after 14 d of experimentally induced periodontal disease (EP) or fictitious induction (sham). Rats were treated with vehicle (water), losartan (50 mg/kg), aliskiren (30 mg/kg) or enalapril (10 mg/kg). All groups n = 5. Statistical significance was determined by ANOVA with interaction analysis and Tukey’s test. Statistical significance (<i>p</i>-value < 0.05) is indicated by * <i>vs</i>. periodontal disease rats treated with water. <b>B to F)</b> Representative images of each of the following: sham group treated with water <b>(B)</b>, EP group treated with water <b>(C)</b>, aliskiren <b>(D)</b>, losartan <b>(E</b>) or enalapril <b>(F</b>). The samples pictured have the following respective CEJ to ABC area: (B) 2.1 mm², (C) 3.3 mm², (D) 2.1 mm², (E) 2.1 mm², (F) 3.1 mm².</p

Renin-Angiotensin System Components in the Gingiva Tissue of Rats with Experimentally Induced Periodontitis.

<p><b>A)</b> Results of qPCR analysis for mRNA of various RAS components extracted from the rat gingiva of either sham surgery (sham) or experimentally induced periodontitis (EP) for 14 days; tested RAS components included the following: angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin converting enzyme 2 (ACE-2), angiotensin II receptor type 1A (AT_1AR), angiotensin II receptor type 1B (AT_1BR), angiotensin II receptor type 2 (AT₂R), and Mas receptor (MasR). Graph displays expression levels of the target mRNA relative to β-actin mRNA from 5 rats in duplicate (n = 5). Solid bars represent the means with SD of sham group; whereas, open bars represent animals with EP. A one-way ANOVA and Tukey’s test with statistical significance set at <i>p</i>-value < 0.05 was used. <b>B)</b> All images are at 10x magnification and scale bars indicate a distance of 100 μm. Immunoreactivity (IR) for renin (panels a to d), ACE (panels e to h), AT₁R (panels i to l) or AT₂R (panels m to p) in rat gingiva and bone tissue. Brown staining indicates positive IR. Black arrows indicate some of the positive IR in the positive controls. Negative <b>(panel a)</b> and positive <b>(panel b)</b> control for renin in rat kidney. <b>Panel c)</b> negative control for renin in mandible of rat with 14 d of EP treated with water. <b>Panel d)</b> immunoreactivity for renin in mandible of rat with 14 d of EP treated with water. Negative <b>(panel e)</b> and positive <b>(panel f)</b> control for ACE in a rat kidney. <b>Panel g)</b> negative control for ACE in mandible of rat with 14 d of EP treated with water. <b>Panel h)</b> immunoreactivity for ACE in mandible of rat with 14 d of EP treated with water. Negative <b>(panel i)</b> and positive <b>(panel j)</b> control for AT₁Rs in a rat adrenal gland. <b>Panel k)</b> negative control for AT₁Rs in mandible of rat with 14 d of EP treated with water. <b>Panel l)</b> Immunoreactivity for AT₁Rs in mandible of rat with 14 d of EP treated with water. Negative <b>(panel m)</b> and positive <b>(panel n)</b> control for AT₂Rs in a rat adrenal gland. <b>Panel o)</b> negative control for AT₂Rs in mandible of rat with 14 d of EP treated with water. <b>Panel p)</b> immunoreactivity for AT₂Rs in mandible of rat with 14 d of EP treated with water. <b>C)</b> Table indicating location of immunoreactivity observed in 2 sections of 5 rats with 14 d of EP treated with water for renin, ACE, AT₁Rs and AT₂Rs in different mandibular regions: (-) indicates negative immunoreactivity, (+) indicates positive immunoreactivity and (++) indicates abundant immunoreactivity.</p

Schematic Representation of the Renin-Angiotensin System.

<p>Angiotensin-I converting enzyme, ACE; angiotensin I converting enzyme 2, ACE-2; angiotensin II 1 receptor, AT₁R; angiotensin II 2 receptor, AT₂R; aminopeptidase, AP; aminopeptidase A, APA; aminopeptidase N, APN; carboxypeptidase, CPP; endopeptidase, EP; Mas receptor, MasR; Mas-related gene type D receptor, MrgDR; neprilysin, NEP; (pre)prorenin receptor, PRR.</p

ACE Activity and Generation of Renin-Angiotensin System Components from Human Gingiva.

<p><b>A)</b> Fluorimetric assay of ACE activity in gingiva homogenates from donors with healthy gingiva (open bar), gingivitis (gray bar) or periodontitis (black bar). The means were compared using a 1-way ANOVA and Tukey’s test. * indicates significant difference from healthy group. <b>B to E)</b> HPLC assay of homogenates from all three groups after incubation with either captopril (10μM), chymostatin (100μM), both captopril (10μM) and chymostatin (100μM), or nothing. * indicates significant differences from all other healthy groups (open bars); † indicates significant differences from all other gingivitis groups (gray bars); ‡ indicates significant differences from all other periodontitis groups (black bars); unless otherwise noted with brackets (<i>e</i>.<i>g</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134601#pone.0134601.g003" target="_blank">Fig 3C</a>). <b>B)</b> Indicates the amount of Ang II formed when incubated with Ang I. <b>C)</b> Indicates the amount of Ang 1–9 formed when incubated with Ang I. <b>D)</b> Indicates the amount of Ang 1–7 formed when incubated with Ang I. <b>E)</b> Indicates the amount of Ang 1–7 formed when incubated with Ang II.</p

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC