Nina Zidar

MicroRNAs miR-1, miR-133a, miR-133b and miR-208 Are Dysregulated in Human Myocardial Infarction

Objectives: MicroRNAs (miRNAs) are noncoding single-stranded RNA molecules that regulate gene expression in physiological functions, development and disease. In recent studies, three miRNAs have been described as muscle or cardiac specific: miR-1, miR-133, and miR-208, being involved in heart development and disease; but there are limited data on their role in human myocardial infarction (MI). We therefore analyzed their expression in human MI. Methods: Autopsy samples of infarcted heart tissue from 50 patients with MI, 8 healthy trauma victims and 9 fetuses that died in utero were included. miRNAs miR-1, miR-133a/b and miR-208 were analyzed using quantitative real-time polymerase chain reaction. Results:miR-208 was upregulated, whereas miR-1 and miR-133a were downregulated in MI compared to healthy adult and fetal hearts. All four tested miRNAs were downregulated in fetal hearts compared to healthy adult hearts. Conclusions: Our study showed the involvement of muscle- and/or cardiac-specific miRNAs miR-1, miR-133a/b and miR-208 in human MI. The most significant finding was upregulation of miR-208 and downregulation of miR-1 and miR-133a in MI compared to healthy adult hearts. Some patterns of miRNA expression were similar in MI and fetal hearts, supporting the concept of cardiac gene reprogramming in the remodeling of the heart.

MicroRNA microarray expression profiling in human myocardial infarction.

MicroRNAs (miRNAs), small non-coding RNA molecules, are negative regulators of gene expression. Recent studies have indicated their role in various forms of cardiovascular disease. In spite of the number of miRNA microarray analyses performed, little is known about the genome-wide miRNA expression pattern in human myocardial infarction (MI). Using miRNA microarrays and bioinformatic analysis, miRNA expression was analyzed on human MI and foetal hearts compared to healthy adult hearts, to determine whether there is any similar expression pattern between MI and foetal hearts, and to identified miRNAs that have not previously been described as dysregulated in cardiovascular diseases. Of 719 miRNAs analyzed, ∼ 50% were expressed in human hearts, 77 miRNAs were absent from all tested tissues and 57 were confidently dysregulated in at least one tested group. Some expression patterns appeared to be similar in MI and foetal hearts. Bioinformatic analysis revealed 10 miRNAs as dysregulated in MI not yet related to cardiovascular disease, and 5 miRNAs previously described only in animal models of cardiovascular diseases. Finally, qRT-PCR analysis confirmed dysregulation of 7 miRNAs, miR-150, miR-186, miR-210, miR-451, and muscle-specific, miR-1 and miR-133a/b; all of these are believed to be involved in various physiological and pathological processes.

Effect of intrauterine growth retardation on the clinical course and prognosis of IgA glomerulonephritis in children.

Intrauterine growth retardation (IUGR) resulting in a reduced number of nephrons is one of the nonimmune mechanisms that have been recently proposed as contributing to the progression of renal diseases. The purpose of our study was to determine whether IUGR has any effect on the clinical course and prognosis of IgA glomerulonephritis (IgA GN) in children. Fifty children with biopsy-proven IgA GN, who were followed for at least 3 years, were included. Six of the 50 children (12%) had signs of IUGR at birth, defined as birth weight below the 10th percentile for gestational age. There were no significant differences in initial clinical presentation between children with IUGR and those without IUGR. However, in kidney biopsy specimens, we found a significantly higher mean percentage of sclerotic glomeruli in children with IUGR than in those without IUGR (33 vs. 13%, p < 0.015). At the end of the follow-up period, we observed a significantly higher incidence of arterial hypertension in children with IUGR than in those without IUGR (50 vs. 11%, p < 0.05). Other differences between the two groups of children were not statistically significant. In conclusion, our study demonstrated an increased risk of the development of arterial hypertension and glomerulosclerosis in children with IgA GN who had suffered from IUGR with a birth weight below the 10th percentile for gestational age. IUGR may therefore help to identify early in the course of IgA GN those children who are at higher risk of an unfavorable course.

Cyclooxygenase in normal human tissues – is COX-1 really a constitutive isoform, and COX-2 an inducible isoform?

Cyclooxygenase (COX) is a key enzyme in prostanoid synthesis. It exists in two isoforms, COX‐1 and COX‐2. COX‐1 is referred to as a ‘constitutive isoform’, and is considered to be expressed in most tissues under basal conditions. In contrast, COX‐2 is referred to as an ‘inducible isoform’, which is believed to be undetectable in most normal tissues, but can be up‐regulated during various conditions, many of them pathological. Even though the role of COX in homeostasis and disease in now well appreciated, controversial information is available concerning the distribution of COX isoforms in normal human tissues. There is mounting evidence that it is much more complex than generally believed. Our aim was therefore to analyse the expression and distribution of COX isoforms in normal human tissues, using immunohistochemistry, Western blotting and real‐time RT‐PCR. Autopsy samples from 20 healthy trauma victims and samples from 48 biopsy surgical specimens were included. COX‐1 was found in blood vessels, interstitial cells, smooth muscle cells, platelets and mesothelial cells. In contrast, COX‐2 was found predominantly in the parenchymal cells of many tissues, with few exceptions, for example the heart. Our results confirm the hypothesis that the distribution of COX isoforms in healthy tissues is much more complex than generally believed. This and previous studies indicate that both isoforms, not only COX‐1, are present in many normal human tissues, and that both isoforms, not only COX‐2, are up‐regulated in various pathological conditions. We may have to revise the concept of ‘constitutive’ and ‘inducible’ COX isoforms.

The Ljubljana classification: a practical strategy for the diagnosis of laryngeal precancerous lesions.

Summary: There is no internationally accepted classification of epithelial hyperplastic laryngeal lesions (EHLL). The majority of current classifications follow criteria similar to those commonly used for cervical epithelial lesions. However, the different etiology of laryngeal cancer and its particular clinical and histologic features necessitate a grading system more appropriate to this region. The Ljubljana classification of EHLL was devised in 1971 to cater to this requirement. Detailed criteria for histologic grading in this classification were formulated by a working group on EHLL of the European Society of Pathology in 1999. The system recognizes four grades: simple and abnormal hyperplasia are benign categories; atypical hyperplasia (“risky” epithelium) is potentially malignant, and carcinoma in situ actually malignant. The main features by which the proposed grading system differs from other classifications are: 1. the distinction between benign and potentially malignant lesions: 2. the positive separation of carcinoma in situ from atypical hyperplasia; 3. the lack of prognostic significance for any surface keratin layer. The eventual outcome of EHLL patients so graded justifies the proposal for separating the lesions into a benign group, showing malignant transformation in only 0.9% of cases, from a potentially malignant group showing malignant transformation in 11% of cases. For diagnostically difficult cases, supplementary techniques such as those using morphometry, immunohistochemical and molecular biology are advised to improve the accuracy of diagnosis and predictions of their biological behavior.

Current review on squamous intraepithelial lesions of the larynx

Squamous intraepithelial lesions (SILs) of the larynx, clinically usually defined as leukoplakia and chronic laryngitis, have remained the main controversial topic in laryngeal pathology for decades as regards classification, histological diagnosis and treatment. SILs are caused by smoking and alcohol abuse. There is also mounting evidence that gastroesophageal reflux is a potential aetiological factor. Human papillomavirus infection seems to play little if any role in laryngeal carcinogenesis.

Contribution to the pathogenesis of radiation-induced injury to large arteries

We report a case of a 35-year-old man who died of a brain infarct 20 months after radiotherapy for carcinoma of the tonsil with metastases to the cervical lymph nodes. Histology revealed mild atherosclerosis, necrotizing vasculitis, and occlusive thrombosis of the internal carotid artery. Significant changes were observed in the vasa vasorum: swelling and detachment of the endothelium, subendothelial oedema, hyaline change, fibrinoid necrosis of the vessel walls with mononuclear cellular infiltration, accompanied by focal haemorrhages and chronic inflammation in the periadventitial soft tissue. We believe that these changes of the vasa vasorum and necrotizing vasculitis are causally related and that vasculitis represents focal ischaemic necroses with inflammatory reaction. Our findings support the hypothesis, based on experimental studies, that injury to the vasa vasorum is an important mechanism in the development of radiation-induced vasculopathy of large arteries. They also suggest an evolution of the injury to the vasa vasorum and periadventitial tissue from the early lesions described in our patient, to late stages resulting in dense periadventitial fibrosis as reported previously. We suggest that injury to the vasa vasorum and the consequent ischaemic lesions of the arterial wall are morphological features distinguishing radiation-induced arterial injury from spontaneous atherosclerosis.

Expression of Ki-67 antigen and proliferative cell nuclear antigen in benign and malignant epithelial lesions of the larynx

In an attempt to analyse the proliferative activity in benign and malignant laryngeal epithelial lesions, and to determine the relationship to their histologic grade, we studied the expression of proliferative cell nuclear antigen (PCNA) and Ki-67 antigen on 20 squamous carcinomas, and on 30 biopsies of epithelial hyperplasia categorized according to the Kambic-Lenart classification into simple, abnormal, and atypical hyperplasias. In simple hyperplasia, both antibodies stained the nuclei of the occasional cells in the basal layer. In abnormal hyperplasia (mild dyplasia), positive cells occupied up to a third, and in atypical hyperplasia (moderate and severe dysplasia) they occupied from two-thirds to the entire epithelial thickness. In squamous carcinoma, we have found a statistically significant correlation between its grade and the percentage of Ki-67-(p < 0.01) and PCNA-(p < 0.00001) positive cells. Our results suggest that the proliferative fraction progressively increases with the degree of epithelial hyperplasia and the grade of carcinoma. We conclude that the patterns of immunoreactivity to PCNA and Ki-67 antigen correspond to the histologic grade of both benign and malignant epithelial lesions of the larynx. This method should be regarded as a useful adjunct to traditional histological techniques allowing more objective grading of benign and malignant epithelial lesions.

Cadherin–catenin complex and transcription factor Snail-1 in spindle cell carcinoma of the head and neck

Spindle cell carcinoma (SpCC) is a biphasic tumor composed of squamous cell carcinoma (SCC) and a malignant spindle cell component. There is mounting evidence that SpCC is a monoclonal neoplasm originating from a stem cell giving rise to both components. We tested the hypothesis that spindle cell phenotype might be related to the cadherin–catenin complex, which forms adherens junctions between cells. We analyzed the immunohistochemical expression of E- and N-cadherin, α-, β- and γ-catenin, and Snail-1, a transcription repressor of E-cadherin, in 30 cases of SpCC, and 30 cases of SCC of the head and neck. In SpCC, cadherin and catenin expression was similar in the SCC component, whereas in the spindle cell component, loss of E-cadherin and neo-expression of N-cadherin was found in 19 cases, loss of cadherins in seven, and their co-expression in four cases. Catenin expression were altered in 18 SpCCs. Snail-1 was found in 19 SpCC cases. In SCC, E-cadherin and catenins were expressed in all cases, and N-cadherin focally in five cases. Snail-1 was observed in the stroma. To summarize, in SpCC, there is an altered expression of the cadherin–catenin complex, associated with morphological transition from epithelial to spindle cell phenotype. These features are reminiscent of epithelial–mesenchymal transition (EMT). Our study thus indicates that EMT might play an important role in the pathogenesis of SpCC. This conclusion is further supported by our finding of Snail-1 expression, a potent inducer of EMT, in more than half SpCC cases.

Down-regulation of microRNAs of the miR-200 family and miR-205, and an altered expression of classic and desmosomal cadherins in spindle cell carcinoma of the head and neck--hallmark of epithelial-mesenchymal transition.

MicroRNAs are small, noncoding RNAs that regulate gene expression by posttranscriptional regulation of target genes. miR-200 family and miR-205 have been shown experimentally to regulate epithelial-mesenchymal transition. As epithelial-mesenchymal transition is the postulated pathogenetic mechanism in spindle cell carcinoma, we analyzed the expression of these microRNAs in spindle cell carcinoma of the head and neck in comparison to conventional squamous cell carcinoma of similar location and stage. We also analyzed the expression of classic and desmosomal cadherins, which are believed to be important targets during epithelial-mesenchymal transition. Forty-five cases of spindle cell carcinoma and 45 cases of squamous cell carcinoma of the head and neck were analyzed using real-time polymerase chain reaction for microRNAs, and immunohistochemistry for classic cadherins (E- and N-cadherins) and desmosomal cadherins. We found a significant down-regulation of the miR-200 family and miR-205, loss of desmosomal cadherins, and an altered expression of classic cadherins in spindle cell carcinoma in comparison to squamous cell carcinoma. Down-regulation of the miR-200 family and miR-205 strongly supports the postulated role of epithelial-mesenchymal transition in spindle cell carcinoma. These microRNAs act on transcription repressors that were also up-regulated in our cases of spindle cell carcinoma, both on mRNA and on protein levels. The result is not only an altered expression of classic cadherins in adherens junctions but also a complete loss of desmosomal cadherins.