Mohammad Ghazizadeh

Prognostic significance of vascular endothelial growth factor expression in human ovarian carcinoma

The influence of vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) on prognosis and the relationship between VEGF expression and MVD in ovarian carcinoma are not well defined. We studied VEGF expression in parallel with MVD by immunohistochemistry in 94 ovarian tumours (64 malignant, 13 borderline, and 17 benign) and correlated the results with the clinicopathologic prognostic factors of the disease to clarify their significance in this disease. Assessment of VEGF mRNA isoforms by RT-PCR was also performed. Of the malignant, borderline, and benign ovarian tumours respectively, two (3%), four (31%) and 16 (94%) were negative, 31 (48%), seven (54%) and one (6%) had low expressions, and 31 (48%), two (15%) and none (0%) had high expressions of VEGF. There were significant associations between the VEGF expression and disease stage (P = 0.002), histologic grade (P = 0.0004), and patient outcome (P = 0.0002). MVD did not correlate significantly with the clinicopathologic parameters. Likewise, no correlation was found between MVD and VEGF expression. The survival of patients with high VEGF expression was significantly worse than that of patients with low and negative VEGF expression (P = 0.0004). Multivariate analysis revealed that disease stage and VEGF expression were significant and independent prognostic indicators of overall survival time (P = 0.008 and P = 0.006 respectively). These findings suggest that in conjunction with the established clinicopathologic prognostic parameters of ovarian carcinoma, VEGF expression may enhance the predictability of patients at high risk for tumour progression who are potential candidates for further aggressive therapy.

Reversing the effects of Formalin fixation with citraconic anhydride and heat: A universal antigen retrieval method

Formalin is a commonly used fixative for tissue preservation in pathology laboratories. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking. To achieve a universal antigen retrieval method for immunohistochemistry under a constant condition, we developed a new method in which the effects of formalin fixation were reversed with citraconic anhydride (a reversible protein cross-linking agent) plus heating. Formalin-fixed, paraffin-embedded tissues from various organs were examined for immunohistochemical localization of a wide variety of antigens. Deparaffinized tissue sections were placed in an electric kitchen pot containing 0.05% citraconic anhydride solution, pH 7.4, and the pot was set at “keep warm” temperature mode of 98C for 45 min. This mode allowed heating the sections at a constant temperature. The sections were then washed in buffer solution and immunostained using a labeled streptavidinbiotin method using an automated stainer. In general, formalin-fixed tissues demonstrated specific immunostainings comparable to that in fresh frozen tissues and significantly more enhanced than after conventional antigen retrieval methods. In particular, even difficult-to-detect antigens such as CD4, cyclin D1, granzyme β, bcl-6, CD25, and lambda chain revealed distinct immunostainings. Different classes of antigens such as cellular markers and receptors, as well as cytoplasmic and nuclear proteins, consistently produced enhanced reactions. This method provides efficient antigen retrieval for successful immunostaining of a wide variety of antigens under an optimized condition. It also allows standardization of immunohistochemistry for formalin-fixed tissues in pathology laboratories, eliminating inter-laboratory discrepancies in results for accurate clinical and research studies.

Protease-activated receptor (PAR)-1 and PAR-2 participate in the cell growth of alveolar capillary endothelium in primary lung adenocarcinomas.

Cell growth can be induced via elicitation of protease‐activated receptors (PAR) with serine proteases such as thrombin and trypsin.

Immunohistochemical and ultrastructural studies of basal cells, Clara cells and bronchiolar cuboidal cells in normal human airways

Immunohlstochemical studies were made of the distribution of various cytokeratins (CK), Clara cell secretory protein (CC10), surfactant proteln A (SP‐A) and type VII collagen in normal human alrways. Electron microscopic studies were made to Identify hemldesmosomes and anchoring fibrils on the basal surfaces of the epithelial cells. CK19 was detected In all epithelial cells, and CK17 in all basal cells. CK14 was coaxpressed in a few basal cells, and this coexpression was decreased in the distal airways. Two types of basal cells were recognized. One type, found mainly In large airways, was characterized by abundant intermediate filaments and well‐developed hemidesmosomes and anchoring fibrils. The second type contained few intermediate filaments and poorly developed hemidesmosomes and anchoring fibrils. Reactivity for type VII collagen was found along the basement membrane throughout the airways, but not in the alveoli. Clara cells were reactive for CC10 and CK17, but not for CK14 and SP‐A. The bronchiolar cuboidal cells in the respiratory bronchloles were positive only for CK19. Surfactant proteln A was present only in type II alveolar eplthellal cells. Thus, two types of basal cells are present in always, and the bronchiolar cuboidal cells appear distinct from these basal cells, Clara cells and type II alveolar epithelial cells.

Potential role of the Slit/Robo signal pathway in angiogenesis

Intensive investigations on angiogenesis and vasculogenesis have increased our understanding of molecular mechanisms of blood vessel formation during pathologic and developmental conditions. However, endothelial cells (ECs), the main component of vasculature, are heterogeneous, as revealed by our phenotypic and molecular biological studies in the laboratory, and it is still hard to adequately understand the molecular mechanisms of angiogenesis and vasculogenesis. Indeed, there are several major ligand/receptor signal pathways: VEGF/VEGFR, Jagged-1/Notch, Wnt ligand/frizzled receptor, and ephrin/Eph; each of which having distinct and independent roles during vascular formation. In this review, we focus on the angiogenic effect of the Slit and Robo signal pathway that was formally known as neuronal axon guidance. Among the existing vascular signals, this pathway is the most recently found ligand/receptor vascular signal, and may play important physiological roles as other major receptor/ligand signals do. Here, we briefly address: (1) the background of Slit and Robo families; (2) expression patterns of Slit and Robo; (3) functional roles of the Slit/Robo pathway in vascular formation; and (4) confronting tasks of this novel vascular pathway in the near future. Together, a summary of these data suggest the essential role of the Slit/Robo pathway in angiogenesis, and may explain why multiple vascular signals exist in heterogenic endothelial cells.

Mosaic-like distribution of endothelial cell antigens in capillaries and juxta-alveolar microvessels in the normal human lung

The distribution patterns of endothelial cell antigens, including thrombomodulin and von Willebrand factor (vWf), were studied in normal lung tissues obtained from distant areas of solitary nodules (seven adenocarcinomas and four hamartomas). By single immunoalkaline phosphatase and dual immunofluorescence stainings, the plasma membranes of alveolar capillary endothelium showed linear distribution of thrombomodulin, but their cytoplasm was rarely reactive for vWf (thrombomodulin‐dominant pattern). Microvessels with a diameter larger than 10 μm located in the connective tissue zones demonstrated band‐like reaction for vWf in their cytoplasm, and their plasma membranes often lacked reactivity for thrombomodulin (vWf‐dominant pattern). The juxta‐alveolar microvessels located along the borders between the alveolar‐ and connective‐tissue zones showed mosaic‐like pattern of distribution for these antigens. The pulmonary venules and peribronchial microvessels measuring up to 40 μm in diameter, demonstrated the expression of thrombomodulin along the plasma membrane, and that of vWf in the cytoplasm. Capillaries of the bronchial circulation were also characterized by mosaic‐like pattern of distribution. Both antigens were often expressed in a single cytoplasmic segment. The heterogeneous distribution pattern of these antigens suggests topographic difference in endothelial cell function to maintain coagulatory and anticoagulatory balance in the normal human lung.

Oolong tea extract as a substitute for uranyl acetate in staining of ultrathin sections

In conventional transmission electron microscopy, uranyl acetate staining is used to enhance the cellular components. However, uranyl acetate is considered a radioactive material that is very toxic  if  ingested or inhaled and subject to restrictions in many countries. In an attempt to introduce a substitute for uranyl acetate, we evaluated oolong tea extract (OTE) for staining of ultrathin sections. Tissue sections from normal rat liver representing an ideal model organ were processed according to a routine electron microscopic fixation and embedding procedure. Serial ultrathin sections were cut and processed with either routine double electron staining or 0.2% OTE staining for 30–40 min at room temperature followed by lead citrate staining (OTE staining method). Transmission electron microscopy observations revealed that all sub‐cellular structures in hepatocytes were clearly visible with OTE staining and the quality of staining was highly compatible with those of routine double staining methods. It is suggested that OTE could be used as a non‐radioactive and hazard‐free substitute for uranyl acetate in transmission electron microscopy staining.

Role of cdk4, p16INK4, and Rb Expression in the Prognosis of Bronchioloalveolar Carcinomas

Background: The p16INK4 protein has been identified as a potent inhibitor of cyclin-dependent kinase (cdk)4 by blocking cdk4-mediated phosphorylation of the tumor suppressor retinoblastoma (Rb) protein, thus allowing Rb-mediated growth suppression. Objectives: Loss of p16INK4 has been associated with a poor cancer prognosis, but its potential significance in bronchioloalveolar carcinomas (BACs) has not been explored. Methods: We examined immunohistochemical expression of p16INK4, cdk4, and Rb proteins in 38 BACs and correlated their expression levels with known clinicopathological features of the disease. Results: All BACs expressed cdk4, while 89 and 82% expressed p16INK4 and Rb proteins, respectively. None of the clinicopathological factors correlated with p16INK4, cdk4, or Rb expression separately. A low p16INK4/cdk4 ratio was significantly associated with a high disease stage (p = 0.04), and the ratio tended to be lower in mucinous than nonmucinous tumors. BACs with a low p16INK4/cdk4 ratio showed significantly higher Rb expression levels (p = 0.02). Univariable survival analyses showed a significantly lower 5-year survival probability in patients with a high stage (p = 0.002) or low p16INK4/cdk4 ratio (p = 0.01). Conclusions: The results suggest a role of the cdk4/p16INK4 pathway in the prognosis of BACs. Further studies are warranted to clarify whether a low p16INK4/cdk4 ratio may identify tumors that are destined to behave unfavorably.

Correlation of Autophagy Type in Podocytes with Histopathological Diagnosis of IgA Nephropathy

Objective: IgA nephropathy (IgA-N) frequently leads to progressive renal failure, thus estimation of the degree of progression is important for patient management. Autophagy is a mechanism that facilitates clearance of waste products to preserve renal function. The aim of this study was to assess autophagy in podocytes in children with progressive IgA-N at initial diagnosis by electron microscopy and investigate the relationship between the types of autophagy and severity of the disease. Methods: Renal biopsies from 16 children with established progressive IgA-N were examined by light and transmission electron microscopy with reference to autophagy types in the podocytes and histopathological diagnosis of IgA-N. Results: Two autophagy types were found. Type I rarely transformed to autophagic vacuoles and did not dissolve, thus possibly impairing cell function. However, type II frequently transformed to autophagosomes and autophagic vacuoles thus facilitating protein and lipid clearance. Of the 16 children studied, 8 (50%) with type I autophagy at initial diagnosis showed focal proliferative glomerulosclerosis (GN) of mild type (3 cases, 37.5%), mild/moderate type (2 cases, 25%) and moderate type (3 cases, 37.5%). In contrast, the remaining 8 children with type II autophagy at initial diagnosis showed focal proliferative GN of mild type in 7 (87.5%) and mild/moderate type in 1 (12.5%) case. Conclusion: In IgA-N children, the occurrence of type I autophagy is correlated with histopathologically more progressive disease, possibly reflecting a tendency to a poorer prognosis.

Silver staining of nucleolar organizer regions in prostatic lesions

Variations in the number of silver‐stained nucleolar organizer region‐associated proteins (AgNORs) were studied in paraffin sections of 42 benign prostatic lesions, comprising four cases of granulomatous prostatitis, five of squamous or transitional metaplasia, eight of atypical and 25 of regular hyperplasia, and 37 of prostatic adenocarcinoma, with their metastases. There was a significant difference between the mean AgNOR counts of the benign and malignant prostatic lesions (1.58±0.26 v. 4.34±1.53; P<0.01). The mean AgNOR counts significantly increased with increasing Gleasons grade (P<0.01) and clinical stage (P<0.05) of the tumours. AgNOR counting may contribute to the conventional diagnostic and prognostic indices of cancer of the prostate.