Immunohistochemical study of the plasminogen activator system in benign epithelial odontogenic lesions

Abstract: The aim of this study was to analyze and compare the immunohistochemical expression of plasminogen activator system (PAS) proteins (uPA, uPAR, and PAI-1) in ameloblastomas (AMBs), odontogenic keratocysts (OKCs), and dental follicles (DFs) representing normal odontogenic tissue, as well as to investigate possible correlations between these proteins. Twenty AMBs, 20 OKCs, and 10 DFs were selected for immunohistochemical analysis. In each case, the immunoexpression of uPA, uPAR, and PAI-1 was evaluated semiquantitatively based on the percentage of positivity in odontogenic epithelial and connective tissue cells. The epithelial immunoexpression of uPA was significantly lower in AMBs when compared to OKCs (p = 0.001) and DFs (p = 0.029). Significantly higher epithelial immunostaining for uPAR was observed in AMBs when compared to OKCs (p < 0.001). There were no significant differences in the epithelial immunoexpression of PAI-1 between AMBs and OKCs (p = 1.000). The correlations found for the expression of the studied proteins were not statistically significant (p > 0.05). However, the epithelial and connective tissue expressions of uPAR have a strong positive and statistically significant correlation in AMBs. The present results suggest that uPA is involved in the pathogenesis of OKCs and that uPAR may participate in tumorigenesis in AMBs. The high percentage of PAI-1-positive cells suggests a possible role for this protein in the development of AMBs and OKCs. Furthermore, the studied proteins do not seem to act synergistically in AMBs, OKCs, and DFs.

Epidermal growth factor receptor regulates fibrinolytic pathway elements in cervical cancer: functional and prognostic implications

Epidermal growth factor receptor (EGFR) signaling and components of the fibrinolytic system, including urokinase-type plasminogen activator (uPA) and thrombomodulin (TM), have been implicated in tumor progression. In the present study, we employed cBioPortal platform (http://www.cbioportal.org/), cancer cell lines, and an in vivo model of immunocompromised mice to evaluate a possible cooperation between EGFR signaling, uPA, and TM expression/function in the context of cervical cancer. cBioPortal analysis revealed that EGFR, uPA, and TM are positively correlated in tumor samples of cervical cancer patients, showing a negative prognostic impact. Aggressive human cervical cancer cells (CASKI) presented higher gene expression levels of EGFR, uPA, and TM compared to its less aggressive counterpart (C-33A cells). EGFR induces uPA expression in CASKI cells through both PI3K-Akt and MEK1/2-ERK1/2 downstream effectors, whereas TM expression induced by EGFR was dependent on PI3K/Akt signaling alone. uPA induced cell-morphology modifications and cell migration in an EGFR-dependent and -independent manner, respectively. Finally, treatment with cetuximab reduced in vivo CASKI xenografted-tumor growth in nude mice, and decreased intratumoral uPA expression, while TM expression was unaltered. In conclusion, we showed that EGFR signaling regulated expression of the fibrinolytic system component uPA in both in vitro and in vivo settings, while uPA also participated in cell-morphology modifications and migration in a human cervical cancer model.

Estudo da imunoexpressão de proteínas do sistema ativador de plasminogênio em lesões benignas do epitélio odontogênico / Study of immunoexpression of plasminogen activator system proteins in benign lesions of the odontogenic epithelium

Introdução: Cistos e tumores odontogênicos são lesões originadas a partir de tecidos que formam os elementos dentários e apresentam diferentes comportamentos biológicos. Dentre os diversos elementos que podem estar associados ao crescimento cístico e tumoral, encontram-se as enzimas necessárias para degradação da matriz extracelular (MEC). O sistema ativador de plasminogênio (SAP) é responsável pela regulação da remodelação da MEC através da conversão do plasminogênio em plasmina. Além disto, diversos estudos têm sugerido associações entre o SAP e vários fatores na evolução de neoplasias malignas, como a transição epitélio-mesênquima, proliferação, migração, adesão celular e disseminação metastática. Entretanto, poucos trabalhos foram realizados avaliando a influência do SAP em lesões odontogênicas. Objetivo: Avaliar e comparar a expressão imuno-histoquímica das proteínas do SAP, o ativador do plasminogênio tipo uroquinase (uPA), seu receptor (uPAR) e o inibidor (PAI-1) em ameloblastomas (AMBs), ceratocistos odontogênicos (COs) e tecidos odontogênicos normais, os folículos dentários (FDs), além de investigar possíveis correlações entre as proteínas estudadas. Materiais e métodos: As células epiteliais odontogênicas foram analisadas, de forma semi-quantitativa, a partir de fotomicrografias de 5 campos representativos de cada caso, com ampliação de 400x, sendo atribuídos escores de 0 a 4 de acordo com o percentual de células positivas. Após a análise dos 5 campos, foi obtida a mediana dos escores, sendo gerado o escore de imunomarcação do caso. Os dados foram submetidos à análise estatística por meio dos testes de Kruskal-Wallis (KW), Mann-Whitney (U) e correlação de Spearman (r), com o nível de significância estabelecido em 5% (p<0,05). Resultados: A imunoexpressão de uPA foi significativamente menor em AMBs, quando comparados com COs (p=0,001) e FDs (p=0,029), enquanto que a imunomarcação de uPAR em AMBs foi significativamente maior em comparação aos COs (p<0,001). Não houve diferenças significativas na imunoexpressão do PAI-1 entre os grupos estudados (p=0,775). Também não foram encontradas correlações estatisticamente significativas entre as proteínas avaliadas (p>0,05). Conclusões: Os resultados do presente estudo sugerem que o uPA esteja envolvido no crescimento dos COs, enquanto que o uPAR participe do processo de tumorigênese dos AMBs, contudo, o PAI-1 parece não contribuir, de forma direta, na patogênese de AMBs e COs. No contexto das lesões estudadas, as proteínas do SAP parecem não atuar sinergicamente (AU).

Background: Odontogenic cysts and tumors are lesions originated from odontogenic tissues and exhibit different biological behaviors. Among the various elements that may be associated with cystic and tumor growth are the enzymes required for extracellular matrix (ECM) degradation. The plasminogen activator system (PAS) is responsible for regulating ECM remodeling by converting plasminogen to plasmin. In addition, several studies have suggested associations between PAS and other factors in the evolution of malignant neoplasms, such as epithelial-mesenchymal transition, proliferation, migration, cell adhesion, and metastatic dissemination. However, just a few studies have evaluated the influence of PAS on odontogenic lesions. Aim: To evaluate and to compare the immunohistochemical expression of PAS proteins, the urokinase-type plasminogen activator (uPA), its receptor (uPAR) and inhibitor (PAI-1) in ameloblastomas (AMBs), odontogenic keratocysts (OKCs) and normal odontogenic tissues, dental follicles (FDs), in addition to investigating possible correlations between these proteins. Materials and methods: Odontogenic epithelial cells were analyzed, in a semi-quantitative way, using photomicrographs of 5 representative fields of each case, with 400x magnification, with scores ranging from 0 to 4 according to the percentage of positive cells. After the analysis of 5 fields, the median of the scores was obtained, generating the immunostaining score of the case. The data were submitted to statistical analysis using Kruskal-Wallis (KW) and Mann-Whitney (U) tests and Spearman's correlation (r), with significance level set at 5% (p<0.05). Results: For uPA, the immunoexpression was significantly lower in AMBs, when compared to OKs (p=0.001) and DFs (p=0.029). While, uPAR immunostaining in AMBs was significantly higher compared to OKs (p<0.001). There were no significant differences in PAI-1 immunoexpression between the groups studied (p=0.775). There were also no statistically significant correlations between the evaluated proteins (p>0.05). Conclusions: The results of this study suggest that uPA is involved in growth of OKCs, while uPAR participates in tumorigenesis process of AMBs, however, PAI1 does not seem to contribute, directly, to the pathogenesis of AMBs and OKCs. In the context of the studied lesions, SAP proteins do not seem to act synergistically (AU).

In IgA Nephropathy, Glomerulosclerosis Is Associated with Increased Urinary CD80 Excretion and Urokinase-Type Plasminogen Activator Receptor-Positive Podocyturia.

BACKGROUND: Podocyturia may determine the evolution to podocytopenia, glomerulosclerosis, and renal failure. According to the Oxford classification of IgA nephropathy (IgAN), the S1 lesion describes glomerulosclerosis. Urokinase-type plasminogen activator receptor (uPAR) participates in podocyte attachment, while CD80 increases in glomerulosclerosis. We measured uPAR-positive urinary podocytes and urinary CD80 (uCD80) in controls and in IgAN subjects with M1E0S0T0 and M1E0S1T0 Oxford scores to assess a potential association between podocyturia, inflammation, and glomerulosclerosis. METHODS: The groups were as follows: controls (G1), n = 20 and IgAN group (G2), n = 39, subdivided into M1E0S0T0 (G2A), n = 21 and M1E0S1T0 (G2B), n = 18. Among the included variables, we determined uPAR-positive podocytes/gram of urinary creatinine (gUrCr) and uCD80 ng/gUrCr. Biopsies with interstitial fibrosis and tubular atrophy <10% were included. RESULTS: Groups were not different in age and gender; urinary protein-creatinine (uP/C) ratio, Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) equation, uPAR-positive podocytes/gUrCr, and uCD80 were significantly increased in G2 versus G1. G2A and G2B were not different in age, gender, hypertension, and follow-up. G2B displayed significantly higher uP/C, uPAR-positive podocytes, uCD80, and lower CKD-EPI versus G2A. Strong significant correlations were encountered between uCD80 and podocyturia in G2A and G2B. However, when G1 was compared to G2A and G2B separately, the differences with respect to uP/C, uPAR-positive podocytes, and podocyturia were significantly stronger versus G2B than versus G2A. CONCLUSIONS: IgAN presents elevated uCD80 excretion and uPAR-positive podocyturia, while CD80 correlates with podocyturia. Glomerulosclerosis (S1) at the time of biopsy is associated with higher uP/C, lower renal function, increased uPAR-positive podocyturia, and CD80 excretion, and is independent of M1. In IgAN, uPAR may participate in podocyte detachment.

Expressão imuno-histoquímica do ativador de plasminogênio do tipo uroquinase e seu receptor em carcinoma epidermoide de língua oral e sua relação com parâmetros clínico-patológicos / Immunohistochemical expression of plasminogen activator of the urokinase type and its receptor in squamous cell carcinoma of tongue and their relationship with clinicopathological parameters

O carcinoma epidermoide oral (CEO) é a neoplasia maligna mais comum da cavidade oral, apresentando uma alta taxa de mortalidade. Devido a isto, a descoberta de biomarcadores que facilitem a compreensão do comportamento biológico desse tumor e aprimorem o tratamento é necessário. O ativador de plasminogênio do tipo uroquinase (uPA) e o seu receptor, uPAR, têm se destacado por atuarem na proteólise de estruturas da membrana basal e matriz extracelular, facilitando a invasão tumoral. O presente estudo se propôs a avaliar a imunoexpressão dessas proteínas em 46 casos de carcinoma epidermoide de língua oral (CELO). Esses resultados foram relacionados com a presença de metástase, estadiamento clínico TNM, recidiva locoregional, desfecho da lesão e gradação histológica de malignidade. A imunomarcação de cada caso foi avaliada semiquantitativamente, tanto no front de invasão como no centro do tumor, na qual foram atribuídos os escores: 0 (0% de células positivas), 1(1-10% de células positivas), 2 (11-50% de células positivas), 3 (mais de 50% de células positivas). A expressão do uPA foi observada em 93,5% dos casos no front de invasão, com predomínio do escore 2 (34,8%), e em 67,9% dos casos no centro do tumor, com predomínio do escore 1 (32,6%). De modo geral, os parâmetros clínicos não exerceram influência na imunoexpressão do uPA. Em relação à gradação histológica, foi observada uma maior expressão de uPA nos casos de alto grau de malignidade em relação aos de baixo grau de malignidade (p=0,05). Quando analisado em relação aos parâmetros morfológicos, foi identificado uma maior expressão do uPA nos casos de pior padrão de invasão (p=0,03).

A expressão do uPAR foi observada em 73,9% dos casos no front de invasão, com predomínio do escore 1 (45,65%), e em 47,5% dos casos no centro do tumor, com predomínio do escore 0 (54,35%). Embora não tenham sido observadas significâncias estatísticas em relação à metástase linfonodal, estadiamento clínico TNM, desfecho e gradação histológica, houve uma maior expressão do uPAR nos casos com recidiva locoregional em relação aos sem recidiva (p=0,04). Em relação à análise da localização tumoral, foi observada uma maior expressão de uPA e uPAR no front de invasão em relação ao centro do tumor (p<0,001). Na correlação entre uPA e uPAR, não foi observada significância estatística. Com base nestes resultados, sugere-se que o uPA e uPAR estejam envolvidos na progressão do CELO, atuando principalmente na região mais profunda do tumor. (AU)

Squamous cell carcinoma (SCC ) is the most common malignancy of the oral cavity (OSCC), with a high mortality rate. Due to this, the discovery of biomarkers that facilitate the understanding of the biological behavior of the tumor and improve treatment is necessary. Urokinase type plasminogen activator (uPA) and its receptor, uPAR, are responsible for the proteolysis of structures of the basement membrana and extracellular matrix, facilitating tumor invasion. This study aims to assess the immuno expression of these proteins in 46 cases of squamous cell carcinoma of the oral tongue (OTSCC). These results were related to the presence of metastasis, clinical TNM staging, locoregional recurrence, outcome of the lesion and histological grading. Immunostaining of each case was evaluated semiquantitatively, in the front of invasion and center of the tumor, in which scores were assigned: 0 (0% of positive cells), 1 (1-10% of positive cells), 2 (11 -50% positive cells) and 3 (more than 50% positive cells). The expression of uPA was observed in 93.5% (n=43) of the cases in the front of invasion, with predominance of score 2 (n=16; 34.8%) and in 67.9% (n=31) of the cases in the center of the tumor, with predominance of score 1 (n=15; 32.6%). Overall, the immunoexpression of uPA was not associated with clinical parameters. Regarding the malignant histological grading, a higher expression of uPA was observed in cases of high-grade malignancy comp ared to low-grade malignancy (p=0.05).

Regarding the morphological parameters, increased expression of uPA was observed in the worst mode of invasion (p=0.03 ). The expression of uPAR was observed in 73.9% of cases in the front of invasion, with a predominance of score 1 (n=21; 45.6 %), and in 47.5% (n=21) of the cases in the center of the tumor, with a predominance of score 0 (n=25; 54.4%). Although no statistical differences were observed in relation to lymph node metastasis, clinical TNM staging, outcome, and histological grading, there was a higher expression of uPAR in cases with locoregional recurrence (p=0.04). Regarding the tumor intra -localization, it was observed an increased expression of uPA and uPAR at the front of invasion in relation to the center of the tumor (p<0.001). Regarding the correlation between uPA and uPAR, there was no statistical sign ificance. Based on these results, it is suggested that uPA and uPAR are involved in the progression of CELO, mainly in the deeper region of the tumor. (AU)

Fibrinólisis como tratamiento en la trombosis de la vena subclavia: nuestra experiencia en causas primarias y secundarias / Fibrinolysis as Treatment in Subclavian Vein Thrombosis: Our Experience in Primary and Secondary Causes

La trombosis de la vena subclavia reviste especial gravedad por las secuelas funcionales y las posibles complicaciones sistémicas que puede desencadenar cuando no se diagnostica a tiempo. Aunque en muchos casos la presentación clínica puede ser diagnóstica, siempre se requieren pruebas de imagen, ya sean no invasivas, como la ecografía con Doppler color, o invasivas, como la flebografía, que es considerada el patrón de oro, ya que demuestra el trombo y confirma la permeabilidad de la circulación colateral. El objetivo de este estudio es demostrar los resultados de la fibrinólisis por catéter y comprobar que su tratamiento agresivo está justificado para evitar secuelas incapacitantes, especialmente en gente joven. Entre el 1 enero de 2006 y el 31 de diciembre de 2012 se atendieron cinco pacientes diagnosticados con trombosis en la vena subclavia, y fueron tratados con fibrinólisis endovenosa con uroquinasa a 100.000 UI/hora. El criterio para escoger los pacientes a trombolizar, fue el tiempo de evolución menor de 6 días y las características agudas del trombo (trombo hipoecogénico-homogéneo, que ocasiona aumento del calibre venoso), así como las características del paciente, edad y repercusión clínica. En nuestra limitada experiencia hemos tenido un éxito del 80 %, con resolución de la sintomatología en el 100 % en trombólisis con uroquinasa, por lo cual la recomendamos como el manejo inicial de las trombosis subclavias, siempre y cuando cumplan las indicaciones y haya ausencia de contraindicaciones absolutas o relativas para trombólisis.

Subclavian vein thrombosis can be particularly serious due to the functional consequences and possible systemic complications that can be triggered when not diagnosed early. Although in many cases the clinical presentation may be diagnostic, imaging is always required, either non-invasive (Doppler ultrasound) and / or invasive, as is the case with venography which is considered the gold standard because it shows the thrombus and confirms the permeability of the collateral circulation. The purpose of the study is to demonstrate the results of catheter directed fibrinolysis and suggests that aggressive treatment of this condition is justified to prevent the possible disabling sequelae, particularly in young people. During the period between January 1, 2006 to December 31, 2012, 5 patients were treated for thrombosis in the subclavian vein with catheter directed thrombolysis with urokinase at 100,000 UI/hour. The selection criteria for thrombolysis, was the time of evolution (less than 6 days) and acute characteristics of thrombus in ultrasound (hypo echogenic-homogenous thrombus, which causes an increase in venous caliber), as well as the characteristics of the patient, the age, and clinical repercussions. In our limited experience we had a success rate of 80% due to the dissolution of the thrombus, with a 100% resolution of symptoms in thrombolysis with urokinase; so we recommend it as the initial management of the subclavian thrombosis as long as the patients are symptomatic and have not contraindications to thrombolysis.

Inhibition of nitric oxide is a good therapeutic target for bladder tumors that express iNOS.

Bladder cancer is the second cause of death for urological tumors in man. When the tumor is nonmuscle invasive, transurethral resection is curative. On the other hand, radical cystectomy is the treatment chosen for patients with invasive tumors, but still under treatment, these patients have high risk of dying, by the development of metastatic disease within 5 years. It is therefore important to identify a new therapeutic target to avoid tumor recurrences and tumor progression. Nitric oxide (NO) is an important biological messenger known to influence several types of cancers. In bladder cancer, production of NO and expression and activity of inducible NO synthase was associated to recurrence and progression. The objective of this work was to analyze if inhibition of nitric oxide production could be considered a therapeutic target for bladder tumors expressing iNOS. Using a bladder cancer murine model with different invasiveness grade we have demonstrated that NO inhibition was able to inhibit growth of bladder tumors expressing iNOS. Furthermore, invasive properties of MB49-I orthotopic growth was inhibited using NO inhibitors. This paper also shows that levels of NO in urine can be correlated with tumor size. In conclusion, inhibition of NO could be considered as a therapeutic target that prevents tumor growth and progression. Also, urine NO levels may be useful for measuring tumor growth.

Primary focal and segmental glomerulosclerosis and soluble factor urokinase-type plasminogen activator receptor.

Primary focal and segmental glomerulosclerosis (FSGS) may be due to genetic or acquired etiologies and is a common cause of nephrotic syndrome with high morbidity that often leads to end-stage renal failure. The different available therapeutic approaches are unsuccessful, in part due to partially deciphered heterogeneous and complex pathophysiological mechanisms. Moreover, the term FSGS, even in its primary form, comprises a histological description shared by a number of different causes with completely different molecular pathways of disease. This review focuses on the latest developments regarding the pathophysiology of primary acquired FSGS caused by soluble factor urokinase type plasminogen activator receptor, a circulating permeability factor involved in proteinuria and edema formation, and describes recent advances with potential success in therapy.