Evaluation of the effect of topical and systemic ozone application in periodontitis: an experimental study in rats

Abstract Objective: The goal of the present study was to determine the effect of systemic and topical ozone application on alveolar bone loss (ABL) by evaluating the effect of Hypoxia-inducible factor −1 alpha (HIF-1-α) and receptor activator of NF-kB ligand (RANKL)-positive cells on histopathological and immunohistochemical changes in a rat periodontitis model. Methodology: Thirty male Wistar rats were divided into three groups: 1) Group C (control group); 2) Group SO (systemic ozone group) and 3) Group TO (topical ozone group). Experimental periodontitis was induced with a 3/0 silk suture placed at the mandibular left first molars of rats, and the suture was removed 14 days later. Ozone gas was injected intraperitoneally (0.7 mg/kg) in SO group. Topical ozone application protocol was performed using an ozone generator at 80% concentration (4th grade) 90- degree probe for the duration of 30 s. Both ozone applications were carried out for two weeks at intervals of two days. Histomorphometric and immunohistochemical analysis were performed. Results: ABL was significantly lower in Group SO compared to Group C (p: 0.0052). HIF-1α- positive cells were significantly lower in Group TO than in Group C (p: 0.0043). RANKL-positive cells were significantly lower in Group SO and in Group TO compared to the control group (p: 0.0033, p: 0.0075, respectively). Conclusion: Both ozone applications decreased RANKL-positive cell counts, TO application decreased HIF-1-α positive cells counts, and SO application was found to be more effective in reducing ABL compared to control group.

Evaluation of prophylactic and therapeutic effects of sildenafil on acute radiation proctitis in rats

Abstract Purpose: To investigate the prophylactic and therapeutical effects of sildenafil in a model of acute radiation proctitis (ARP). Methods: All experimental procedures of this study was examined by histopathological, immunohistochemical and transmission electron microscopic analysis. Results: Our histopathological evaluations indicated significant increases in lesion severity, cryptic apsis, cryptitis, cryptic distortion, reactive atypia and infiltration depth of the control (proctitis) group. While the prophylaxis group and the treatment group had significantly lower scores. High-dose group showed similar results as prophylaxis group. Histopathological findings of the prophylaxis group was more significant than the treatment group. Immunoreactivities of IL-1β, FGF-2, TNF- α and HIF-1α increased in the control group especially in the epithelial and cryptic regions. On the contrary, sildenafil application caused significant decreases of inflammatory markers in all treatment groups, specifically better results in the prophylaxis group. Conclusion: The sildenafil has anti-inflammatory effects on ARP, as well as protective effects against ARP and the protective effect of sildenafil surpasses its therapeutic effect histopathologically.

Mechanism of hif-1α mediated hypoxia-induced permeability changes in bladder endothelial cells

This study aimed to investigate the mechanism of hypoxia-inducible factor-1 alpha (HIF-1α) mediated hypoxia-induced permeability changes in bladder endothelial cells. Models of in vitro hypoxic cell culture of bladder cancer, bladder cancer cells with low HIF-1α expression and HIF-1α RNA interference (RNAi) expression vector were established. Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of HIF-1α and vascular endothelial growth factor (VEGF) in each group. Bladder cell permeability was determined. Results showed that protein and mRNA expression of HIF-1α and VEGF at 3 and 12 h of hypoxia were significantly higher than normal control (P<0.05), and peaked at 12 h. HIF-1α and VEGF expression in the hypoxic group and hypoxic+3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) group were significantly higher than normal control (P<0.05), while expression in the hypoxic+YC-1 group was significantly lower than the hypoxic group (P<0.05). Bladder cell permeability in the hypoxic and hypoxic+YC-1 group were significantly increased compared to normal control (P<0.05), while in the hypoxic+YC-1 group was significantly decreased compared to the hypoxic group (P<0.05). Most of the cells in the stably transfected HIF-1α RNAi expression vector pcDNA6.2-GW/EmGFP-miR-siHIF-1α expressed green fluorescence protein (GFP) under fluorescence microscope. pcDNA6.2-GW/EmGFP-miR-siHIF-1α could significantly inhibit HIF-1α gene expression (P<0.05). HIF-1α and VEGF expression in the hypoxic group and siHIF-1α hypoxic group were significantly higher than normal group (P<0.05), while expression in the siHIF-1α hypoxic group was significantly lower than the hypoxic group (P<0.05). Findings suggest that HIF-1α is an important factor in the increase of bladder cancer cell permeability.

Analysis of various potential prognostic markers and survival data in clear cell renal cell carcinoma

ABSTRACT Purpose Clear cell renal cell cancers frequently harbor Von Hippel-Lindau gene mutations, leading to stabilization of the hypoxia-inducible factors (HIFs) and their target genes. In this study, we investigated the relationship between vascular endothelial growth factor (VEGF), HIF-1α, HIF-2α, p53 positivity, microvessel density, and Ki-67 rates with prognostic histopathologic factors (Fuhrman nuclear grade, stage, and sarcomatoid differentiation) and survival in clear cell renal cell carcinomas. Material and Methods Seventy-two nephrectomy specimens diagnosed as clear cell renal cell carcinoma between 2000 and 2012 were reevaluated. Immunohistochemically VEGF, HIF-1α, HIF-2α, p53, CD34 (for microvessel density evaluation), and Ki-67 antibodies were applied to the tumor areas. The relationships of these antibodies with prognostic factors and survival rates were evaluated with statistical analyses. Results Mean survival time was 105.6 months in patients with ccRCC. Patients with high expression of VEGF, HIF-1α and HIF-2α positivity, a high Ki-67 proliferation index, and a high microvessel density evaluation score had a shorter survival time (p<0.05). Conclusions Our findings supported that with the use of these immunohistochemical markers, prognosis of renal cell carcinoma may be predicted at the first step of patient management. New treatment modalities targeted to HIF-1α and HIF-2α might be planned as well as VEGF-targeted therapies in the management of clear cell renal cell carcinomas.

Inflammation induced by increased frequency of intermittent hypoxia is attenuated by tempol administration

The levels of serum inflammatory cytokines and the activation of nuclear factor kappa B (NF-κB) and hypoxia inducible factor-1α (HIF-1α) in heart tissues in response to different frequencies of intermittent hypoxia (IH) and the antioxidant tempol were evaluated. Wistar rats (64 males, 200-220 g) were randomly divided into 6 experimental groups and 2 control groups. Four groups were exposed to IH 10, 20, 30, or 40 times/h. The other 2 experimental groups were challenged with IH (30 times/h) plus tempol, either beginning on day 0 (IH30T0) or on day 29 (IH30T29). After 6 weeks of challenge, serum levels of tumor necrosis factor (TNF)-α, intracellular adhesion molecule (ICAM)-1, and interleukin-10 were measured, and western blot analysis was used to detect NF-κB p65 and HIF-1α in myocardial tissues. Serum levels of TNF-α and ICAM-1 and myocardial expression of NF-κB p65 and HIF-1α were all significantly higher in IH rats than in controls (P<0.001). Increased IH frequency resulted in more significant changes. Administration of tempol in IH rats significantly reduced levels of TNF-α, ICAM-1, NF-κB and HIF-1α compared with the non-tempol-treated group (F=16.936, P<0.001). IH induced an inflammatory response in a frequency-dependent manner. Additionally, HIF-1α and NF-κB were increased following IH administration. Importantly, tempol treatment attenuated this effect.

Nitric oxide ineerferes with hypoxia signaling during colonic inflamaation / A interferência do óxido nítrico na sinalização da hipóxia durante a inflamação colônica

Context Intestinal inflammation can induce a local reduction in oxygen levels that triggers an adaptive response centered on the expression of hypoxia-inducible factors (HIFs). Nitric oxide, a well-described inflammatory mediator, may interfere with hypoxia signaling. Objectives We aimed to evaluate the role of nitric oxide in hypoxia signaling during colonic inflammation. Methods Colitis was induced by single (acute) or repeated (reactivated colitis) trinitrobenzenosulfonic acid administration in rats. In addition, one group of rats with reactivated colitis was also treated with Nw-Nitro-L-arginine methyl ester hydrochloride to block nitric oxide synthase. Colitis was assessed by macroscopic score and myeloperoxidase activity in the colon samples. Hypoxia was determined using the oxygen-dependent probe, pimonidazole. The expression of HIF-1α and HIF-induced factors (vascular endothelial growth factor - VEGF and apelin) was assessed using Western blotting. Results The single or repeated administration of trinitrobenzenosulfonic acid to rats induced colitis which was characterized by a high macroscopic score and myeloperoxidase activity. Hypoxia was observed with both protocols. During acute colitis, HIF-1α expression was not increased, but VEGF and apelin were increased. HIF-1α expression was inhibited during reactivated colitis, and VEGF and apelin were not increased. Nw-Nitro-L-arginine methyl ester hydrochloride blockade during reactivated colitis restored HIF-1α, VEGF and apelin expression. Conclusions Nitric oxide could interfere with hypoxia signaling during reactivated colitis inflammation modifying the expression of proteins regulated by HIF-1α. .

Contexto A inflamação intestinal pode induzir uma redução local nos níveis de oxigênio e ativar uma resposta adaptativa relacionada à expressão de fatores induzíveis por hipóxia (HIFs). O óxido nítrico, um mediador inflamatório bem descrito, pode interferir com a sinalização de hipóxia. Objetivos O objetivo foi avaliar o papel do óxido nítrico na sinalização de hipóxia durante a inflamação colônica. Métodos A colite foi induzida em ratos pela administração única (aguda) ou repetida (com reativações) de ácido trinitrobenzenosulfônico. Adicionalmente, um grupo de ratos de colite com reativações foi também tratado com Nw-Nitro-L-arginina metil éster para inibir a óxido nítrico sintase. A colite foi avaliada através do escore macroscópico e da atividade de mieloperoxidase em amostras de cólon. A hipóxia foi determinada usando uma sonda dependente de oxigênio, o pimonidazol. A expressão de HIF-1α e de fatores induzidos pelo HIF (factor de crescimento endotelial vascular - VEGF e apelina) foi avaliada pela técnica de Western blotting. Resultados A administração única ou repetida de ácido trinitrobenzenosulfônico a ratos induziu colite que foi caracterizada por um alto escore macroscópico e alta atividade de mieloperoxidase. Hipóxia foi observada em ambos os protocolos. Durante a colite aguda, a expressão de HIF-1α não aumentou, enquanto a de VEGF e apelina aumentou. A expressão de HIF-1α esteve inibida durante a colite com reativações e, a expressão de VEGF e apelina não se modificou. O bloqueio com Nw-Nitro-L-arginina metil éster durante a colite com reativações restabeleceu a expressão de HIF-1α, VEGF e ...

Differential expression of hypoxia-inducible factor 1α in non-small cell lung cancer and small cell lung cancer

OBJECTIVES: The aim of this study was to compare the expression of hypoxia-inducible factor 1α and vascular endothelial growth factor in small cell lung cancer and subtypes of non-small cell lung cancer and examine their relationships with clinicopathologic factors, response to treatment and survival. METHODS: We examined samples obtained by bronchial endoscopic biopsy from 55 patients with inoperable lung cancer (16 with adenocarcinoma, 17 with squamous cell carcinoma, and 22 with small cell lung cancer). Hypoxiainducible factor 1α and vascular endothelial growth factor were detected using immunohistochemistry. The diagnosis, treatment, and follow-up of patients were conducted according to the standard practice. RESULTS: A significant difference (p=0.022) in hypoxia-inducible factor 1α expression was observed between nonsmall cell lung cancer (75.8% positive) and small cell lung cancer (45.5% positive). The frequency of hypoxiainducible factor 1α nuclear expression was 88.2% in squamous cell carcinoma, 62.5% in adenocarcinoma, and 45.5% in small cell lung cancer. A significant correlation was observed between hypoxia-inducible factor 1α and vascular endothelial growth factor expression (Fisher's exact test, p=0.001) when all types of lung cancer were examined, either collectively or separately. CONCLUSIONS: The expression of hypoxia-inducible factor-1α differs significantly between subtypes of lung cancer. These findings could help elucidate the biology of the different types of non-operable lung carcinomas and have implications for the design of new therapeutic approaches for lung cancer.

Immunohistochemical profile of HIF-1α, VEGF-A, VEGFR2 and MMP9 proteins in tegumentary leishmaniasis / Estudo da expressão imunohistoquímica das proteínas HIF-1α, VEGF-A, VEGFR2 e MMP9 em leishmaniose tegumentar americana

BACKGROUND: Leishmaniasis is one of the most important infectious diseases worldwide. Our study can provide more knowledge about angiogenic and hypoxic events in leishmaniasis. We attempted to verify whether the HIF-1 α protein expression may be associated to VEGF-A, VEGFR2 and MMP9 in leishmanial lesions. OBJECTIVES: Besides understanding the pathway, we performed the correlation of VEGF-A, VEGFR2 and MMP9 proteins. METHODS: In this study, we gathered 54 paraffin blocks taken from skin lesions in patients from northern Minas Gerais, Brazil, with confirmed diagnosis of tegumentary leishmaniasis. Immunohistochemistry was used to evaluate the expression of the proteins. The expression of HIF-1α was categorized into two groups according to the median: HIF-1 α lower and HIF-1 α higher. RESULTS: We observed increase of VEGFR2 and MMP9 protein expressions in HIF-1 α higher group of epithelial cells. Spearman analyses in epithelial cells showed correlation between VEGF-A and MMP9, VEGFR2 and MMP9 protein expression. CONCLUSIONS: HIF-1 α higher group showed increase of VEGFR2 and MMP9 proteins. In epithelial cells, VEGF-A was correlated to MMP9 protein. Furthermore, considering leukocyte cells, VEGFR2 was negatively correlated to MMP9 protein levels. This pathway possibly prepares the cells for a higher activity in a hypoxic or an angiogenic microenvironment. Other in vitro and in vivo studies may clarify the activation mechanism and the response from the proteins HIF-1 α, VEGFR2 and MMP-9 in tegumentary leishmaniasis.

FUNDAMENTOS: A leishmaniose é uma das mais importantes doenças infecciosas em todo o mundo. Em leishmaniose, tem sido sugerido que muitas características da lesão está associado a eventos de hipóxia, podendo este ter um papel fundamental na evolução da doença. OBJETIVO: O presente estudo pode fornecer dados acerca do fenômeno hipóxia e da angiogênese em leishmaniose tegumentar americana. Buscou-se verificar se a expressão da proteína HIF-1 α associa-se à expressão das proteínas VEGF-A, VEGFR2 e MMP9 em lesões de Leishmania sp. MÉTODOS: Neste estudo retrospectivo, foram utilizados 54 blocos de parafina de lesões de leishmaniose tegumentar americana de pacientes do norte de Minas Gerais, Brasil, com diagnóstico confirmado de leishmaniose tegumentar americana. A técnica de imunohistoquimica foi utilizada para avaliação da expressão das proteínas. A expressão da HIF-1α foi categorizada em dois grupos de acordo com a mediana: HIF-1 α abaixo e HIF-1 α acima da mediana. RESULTADOS: Observamos aumento das expressões das proteínas VEGFR2 e MMP9 no grupo HIF-1 α acima da mediana. A análise de Spearman demonstrou correlação entre as proteínas VEGF-A e MMP9, VEGFR2 e MMP9. CONCLUSÃO: Os dados aqui apresentados indicam uma alta expressão da proteína HIF-1 α em LTA. O grupo HIF-1α acima da mediana apresentou maior expressão das proteínas VEGFR2 e MMP9. Foi demonstrada correlação entre as proteínas VEGF-A e MMP9, VEGFR2 e MMP9. Outros estudos in vitro e in vivo devem ser realizados a fim de esclarecer o mecanismo de ativação e resposta das proteínas HIF-1 α, VEGFR2 e MMP-9 em leishmaniose tegumentar americana.

The effect of alpha-blocker treatment on bladder hypoxia inducible factor-1 alpha regulation during lower urinary tract obstruction

AIMS: To determine whether á1-blocker treatment, in chronic bladder outlet obstruction (BOO), influences bladder tissue ischemia. Materials and methods: This prospective study included 60 patients with BOO, of which 40 were under á1-blocker medication and 20 without treatment. Patients underwent transurethral resection of the prostate (TURP) or suprapubic prostatectomy (SPP). Ten patients with non-muscle invasive bladder cancer underwent transurethral resection of the bladder tumor and served as the control group. Tissue specimens were immunohistochemically stained for hypoxia inducible factor-1á (HIF-1á). Results: Bladder tissue from obstructed subjects showed high immunoreactivity to HIF-1á. The specimens from the control group, showed no or weak, mainly cytoplasmic immunoreactivity to HIF-1á. Patients under á -blocker treatment did not differ in the number of HIF-1á positive cells compared to subjects with no treatment (median number 86.8 [20-150] and 88.6 [0-175], respectively) (p > 0.05). The lowest bladder pressure at which HIF-1á was up regulated, was detected at detrusor pressure Qmax (PdetQmax) = 60 cm H2O. Conclusions: Treatment with á-blockers in obstructed patients considered as non-responders, does not result in HIF-1á down regulation, thus bladder continues to be under chronic stress.