Thérèse Rachell Theodoro

Methylene blue photodynamic therapy in malignant melanoma decreases expression of proliferating cell nuclear antigen and heparanases

Background. Malignant melanoma (MM) is a very aggressive tumour. Although surgical excision of MM in the early stages has a very good prognosis, it often fails to completely inhibit tumour progression. Methylene blue photodynamic therapy (MB‐PDT) is a technique that induces tissue damage by reactive oxygen species (ROS).

Heparanase isoform expression and extracellular matrix remodeling in intervertebral disc degenerative disease

OBJECTIVE: To determine the molecules involved in extracellular matrix remodeling and to identify and quantify heparanase isoforms present in herniated and degenerative discs. INTRODUCTION: Heparanase is an endo-beta-glucuronidase that specifically acts upon the heparan sulfate chains of proteoglycans. However, heparanase expression in degenerative intervertebral discs has not yet been evaluated. Notably, previous studies demonstrated a correlation between changes in the heparan sulfate proteoglycan pattern and the degenerative process associated with intervertebral discs. METHODS: Twenty-nine samples of intervertebral degenerative discs, 23 samples of herniated discs and 12 samples of non-degenerative discs were analyzed. The expression of both heparanase isoforms (heparanase-1 and heparanase-2) was evaluated using immunohistochemistry and real-time RT-PCR analysis. RESULTS: Heparanase-1 and heparanase-2 expression levels were significantly higher in the herniated and degenerative discs in comparison to the control tissues, suggesting a possible role of these proteins in the intervertebral degenerative process. CONCLUSION: The overexpression of heparanase isoforms in the degenerative intervertebral discs and the herniated discs suggests a potential role of both proteins in the mediation of inflammatory processes and in extracellular matrix remodeling. The heparanase-2 isoform may be involved in normal metabolic processes, as evidenced by its higher expression in the control intervertebral discs relative to the expression of heparanase-1.

Heparanase expression and glycosaminoglycans profile in renal cell carcinoma

A better understanding of the molecular mechanisms of renal cell carcinogenesis could contribute to a decrease in the mortality rate of this disease. The aim of this study was to evaluate the glycosaminoglycans profile and heparanase expression in renal cell carcinoma. The study included 24 patients submitted to nephrectomy with confirmed pathological diagnosis of renal cell carcinoma. The majority of the samples (87.5%) were classified in the initial stage of renal cell carcinoma (clinical stages I and II). Heparanase messenger ribonucleic acid expression was evaluated by quantitative real‐time reverse transcription polymerase chain reaction, and sulfated glycosaminoglycans were identified and quantified by agarose gel electrophoresis of renal cell carcinoma samples or non‐neoplastic tissues obtained from the same patients (control group). The sulfated glycosaminoglycans and hyaluronic acid were analyzed in urine samples of the patients before and after surgery. The data showed a significant statistical increase in chondroitin sulfate, and a decrease in heparan sulfate and dermatan sulfate present in neoplastic tissues compared with non‐neoplastic tissues. Higher heparanase messenger ribonucleic acid expression in the neoplastic tissues was also shown, compared with the non‐neoplastic tissues. The urine glycosaminoglycans profile showed no significant difference between renal cell carcinoma and control samples. Extracellular matrix changes observed in the present study clarify that heparanase is possibly involved with heparan sulfate turnover, and that heparanase and the glycosaminoglycans can modulate initial events of renal cell carcinoma development.

The Immunoexpression of Heparanase 2 in Normal Epithelium, Intraepithelial, and Invasive Squamous Neoplasia of the Cervix

Objective Heparanase 2 (HPSE2) is expressed in various tissues, including the brain, intestine, prostate, breast, and endometrium. The aim of this study was to investigate the role of HPSE2 in cervical carcinogenesis, which has not been clarified to date. Materials and Methods The immunoexpression of HPSE2 in normal and neoplastic cervical squamous epithelia was determined using a semiquantitative (SQ) method and an index of expression (IE) method, using Image Lab Software. A total of 230 cervical tissue samples were analyzed and segregated into the following diagnostic groups: normal (27.4%), cervical intraepithelial neoplasia 1 (CIN 1, 15.2%), CIN 2 (16.5%), CIN 3 (15.2%), and invasive neoplasia (25.7%). The mean HPSE2 expression in the normal group was significantly lower than that of the other groups individually or combined (p < .001, for all combinations). The immunoexpression via the SQ method was significantly greater in the CIN 3 group compared with that in the CIN 1 group (p = .02). The mean immunoexpression of the high-grade squamous intraepithelial lesion groups was significantly greater than those of the normal and low-grade squamous intraepithelial lesion groups (p < .001) and lower compared with that of the invasive neoplasia group (p < .001). There were no statistically significant differences in the immunoexpression of HPSE2 among the different clinical states within the invasive neoplasia group. Conclusions The SQ method produced a greater sensitivity and specificity than did the index of expression method. There was a progressive increase in the mean HPSE2 immunoexpression according to the severity of the cervical lesion from the low-grade squamous intraepithelial lesion group to the invasive neoplasm group, whereas the normal group displayed the lowest level of expression. This is a novel study concerning HPSE2 in the cervix and cervical cancer carcinogenesis.

The Profile of Heparanase Expression Distinguishes Differentiated Thyroid Carcinoma from Benign Neoplasms

Introduction The search for a specific marker that could help to distinguish between differentiated thyroid carcinoma and benign lesions remains elusive in clinical practice. Heparanase (HPSE) is an endo-beta-glucoronidase implicated in the process of tumor invasion, and the heparanase-2 (HPSE2) modulates HPSE activity. The aim of this study was to evaluate the role of heparanases in the development and differential diagnosis of follicular pattern thyroid lesions. Methods HPSE and HPSE2 expression by qRT-PCR, immunohistochemistry evaluation, western blot analysis and HPSE enzymatic activity were evaluated. Results The expression of heparanases by qRT-PCR showed an increase of HPSE2 in thyroid carcinoma (P = 0.001). HPSE activity was found to be higher in the malignant neoplasms than in the benign tumors (P<0.0001). On Western blot analysis, HPSE2 isoforms were detected only in malignant tumors. The immunohistochemical assay allowed us to establish a distinct pattern for malignant and benign tumors. Carcinomas showed a typical combination of positive labeling for neoplastic cells and negative immunostaining in colloid, when compared to benign tumors (P<0.0001). The proposed diagnostic test presents sensitivity and negative predictive value of around 100%, showing itself to be an accurate test for distinguishing between malignant and benign lesions. Conclusions This study shows, for the first time, a distinct profile of HPSE expression in thyroid carcinoma suggesting its role in carcinogenesis.

Immunohistochemical expression of heparanases 1 and 2 in benign tissue and in invasive neoplasia of the endometrium: a case-control study.

Objectives Our purpose was to compare the expression of heparanase isoforms, in normal and in neoplastic endometrium. In a pioneering way, we sought to evaluate the expression of heparanase 1 (HPSE1) and heparanase 2 (HPSE2) in glandular and in stromal tissues. Methods This is a case-control study, conducted retrospectively in a public hospital, using paraffin blocks of endometrial tissue from patients admitted from 2002 to 2011 with and without endometrial cancer, with regard to the immunohistochemical expression of HPSE1 and HPSE2. The paraffin blocks were used for tissue microarray analysis and immunohistochemistry study in glandular and stromal tissues. Results In the study period, 195 participants were enrolled, 75 with and 120 without cancer. There was no significant difference between them regarding HPSE1 expression, both in gland and in stromal tissues. Heparanase 1 expression in the glandular tissue was more frequent among those with high-grade carcinoma, compared with patients with carcinoma type I. The difference in the expression of HPSE2 was significant between groups: it was less frequent in the controls than in the patients with cancer in the glandular tissue. In the stromal tissue, HPSE2 expression was significantly higher in the controls than in the patients with cancer and different when patients of the secretory endometrium subgroup were compared with those with hypotrophic, proliferative endometriums or with architectural disorders. No significant difference was found in the heparanase expressions in patients with cancer according to prognosis factors. Conclusions Heparanase 1 is more intensely expressed in the glandular tissue of high-grade compared with type I carcinomas. Heparanase 2 is more intensely expressed in the glandular tissue of cancer than in nonneoplastic endometrium, whereas the HPSE2 expression in the stromal tissue is higher in the nonneoplastic controls compared with the group of patients with cancer mainly in the secretory endometrium. This suggests that HPSE2 might be stimulated by progesterone, with a possible antineoplastic role, antagonist to HPSE1, to be further investigated.

Cells involved in extracellular matrix remodeling after acute myocardial infarction.

Objective Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Methods Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. Results There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. Conclusion The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct.

E-Cadherin, beta-catenin and HER2 expression in prostate cancer tissues with perineural invasion and their correlation with Gleason score: A preliminary study

Prostate cancer (PC) is the most common malignant tumor in men. Early identification of prostate cancer may result in improved cure rates and increased life expectancies. Gleason score and clinical range at the time of diagnosis are important factors to predict prognosis and outcome after therapy but additional accurate and reliable biomarkers are warranted. Few biomarkers of prostate cancer have been successfully implemented and used in clinical practice. In this study, we sought to determine the expression of E-cadherin, beta-catenin and human epidermal receptor (HER2) in biopsy specimens of prostate cancer with perineural invasion, and correlate them with Gleason score in order to verify the relationship between those markers and prostate cancer process. Our study demonstrated abnormal expression of E-cadherin, beta-catenin and HER2. On the other hand, our results showed no correlation between Gleason score and the expression of those markers in invasive prostate cancer tissues. Other different biomarkers remain to be identified, that potentially could improve the evaluation of prognostic of the patient. Key words: Biomarkers, biopsy specimens, Gleason score, perineural invasion, prostate cancer.

Extracellular matrix alterations after blood instillation in tunica albuginea of rats

The cause of Peyronie’s disease (PD) is still not completely understood. The objective of this study, therefore, was to analyze the histological and biochemical alterations that occur after the instillation of blood in the tunica albuginea (TA) of rats with an emphasis on the remodeling process of ECM. Thirty male Wistar rats were divided into 4 groups: two control groups with instillation of distilled water in TA followed by penectomy after 15 days or 45 days, respectively and two experimental groups with instillation of blood in TA followed by penectomy after 15 days or 45 days, respectively. Histological, immunofluorescent and immunohistochemical analyses were performed. The higher presence of fibrotic tissue in rats injected with blood demonstrated alterations in TA similar to inflammation found in PD. The increased expression of TGF-β, MMP9, HPSE, and biglycan associated with the decreased expression of syndecan-1 and aggrecan in the experimental groups suggested an enhancement in the remodeling of ECM. The results contribute to show that blood instillation on TA appears to trigger alterations in the ECM similar to the ones found in inflammatory diseases such as PD.

PD45-12 NEW EXPERIMENTAL MODEL IN RATS FOR PEYRONIE'S DISEASE: CHANGES OF COMPONENTS OF THE EXTRACELLULAR MATRIX

INTRODUCTION AND OBJECTIVES: Why phenotypes of Peyronie’s disease can span from mild fibrotic plaques to severely calcified plaques is poorly understood. Mineralized Peyronie’s plaque represents the most severe manifestation, occurs in a subset of patients, and often requires complex surgical excision and reconstruction. The development of analytical high resolution X-ray microscopy techniques may shed new insights on the pathophysiologic mechanisms on plaque-forming processes. The purpose of this study was to characterize the structure of mineralized Peyronie’s plaque and evaluate its association with the adjacent softer extracellular matrix. METHODS: Surgically excised Peyronie’s plaque using a tunica-sparing approach were imaged using high resolution micro-CT with a spatial resolution of 2 mm. Reconstructed tomograms were used to segment regions of different mineral densities by using intensity differences with Avizo 3D software. RESULTS: A representative figure demonstrates micro-CT imaging demonstrating virtual sections and 3D reconstructions. Mineral density of the plaque ranged from 1000 to 1380 mg/cc and was similar to known mineral density of alveolar bone (1200 mg/cc). The plaque was heterogenous in nature and similar to alveolar bone. Both contain endosteal-like spaces (alveoli) encircled by extracellular matrix of lower mineral densities followed by higher mineral densities in regions farther away from alveoli. Endosteal-like spaces were suggestive of regions containing vasculature. CONCLUSIONS: Calcified Peyronie’s plaque demonstrated heterogeneity both in structure and mineral density. The structure of the pathologic plaque is highly analogous to non-pathologic alveolar bone suggesting shared biomineralization pathways. Understanding the structural and elemental properties of mineralized plaque may help elucidate interventions that can slow or mitigate the ossification process in the tunica albuginea.