Immunohistochemical analysis of nestin, CD34 and TGFβ3 in facial tissue of children with complete unilateral and bilateral cleft lip and palate

OBJECTIVE: This study aimed to evaluate levels of expression of nestin, CD34 and transforming growth factor β3 (TGFβ3) in facial tissue of children with complete unilateral (CU) and complete bilateral (CB) cleft lip and palate (CLP). MATERIALS AND METHODS: Twenty-nine CLP patients were enrolled in this study (18 boys and 11 girls). Tissue samples were collected during primary and repeated plastic surgery correction for CU (n=10) or CB (n=19) cleft palate (age range 3 months - 9 years, 6 months). Immunohistochemistry was used to assess levels of nestin, CD34 and TGFβ3. Positively stained cells were graded semi-quantitatively. Data were analyzed to compare cell counts between CUCLP and CBCLP, and CLP at an age before and during primary dentition and CLP in mixed dentition age patients using the Mann Whitney U-test (P<0.05). RESULTS: Moderate to abundant numbers of nestin immunoreactive structures were observed in the oral mucosa. CD34 antibodies labeled all microvessels in lamina propria of the CLP affected tissue, while low numbers of TGFβ3 positive cells were scattered in the connective tissue. There were no statistically significant differences between the study groups. CONCLUSION: Expression of nestin in complete unilateral and bilateral cleft lip and palate affected soft tissue indicates a potential increase of tissue regeneration. CD34 positive oral mucosa cells suggest increased angiogenesis, while the sporadic expression of TGFβ3 indicates an insignificant role in the maintenance and growth of cleft affected oral mucosa stem or progenitor cells. Nevertheless, scarce expression of TGFβ3 suggests a role in cleft morphopathogenesis.publishersversionPeer reviewe

MMPs and TIMPs expression in facial tissue of children with cleft lip and palate

Publisher Copyright: © 2016, PALACKY UNIV. All rights reserved.Background and Aims. Morphogenesis of the upper lip and palate is a complex process involving highly regulated interactions between epithelial and mesenchymal cells. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) in cleft lip palate (CLP) aetiology. This study investigated whether expression of MMP-2, MMP-8, MMP-9, TIMP-2, and TIMP-4, which are essential for the upper lip and palate fusion, is dysregulated in children with CLP. Methods. Oral mucosa tissue samples were obtained from patients with complete unilateral (CU) CLP (n = 25) and complete bilateral (CB) CLP (n = 19) during corrective plastic surgery and in unaffected control subjects (n = 10). MMPs and TIMPs expression was assessed by immunohistochemistry, and the data were analyzed using the Kruskal – Wallis test with the Bonferroni correction. Results. In CLP patients, MMP-2, TIMP-2 immunoreactivity in the oral mucosa was seen to have a few to abundant structures, but the overall number of MMP-2, TIMP-2-positive structures was greater than that in controls (P < 0.01). The total number of TIMP-4, MMP-9-positive cells showed a significant decrease in the CBCLP compared with that of CUCLP (P < 0.001). MMP-8 expression trends in the CLP group were similar to those of the control group. Conclusions. The results suggest that TIMP-4 and MMP-9 are the main ECM remodeling regulatory proteins expressed in CUCLP affected tissues of the oral mucosa. The increased expression of MMP-2 and TIMP-2 in CLP tissues implicates these factors in the regulation of cell migration during ECM turnover independently of different types of clefts. Investigation of MMP and TIMP expression in tissue samples from patients with CLP appears to be a promising approach to the etiopathogenesis of CLP.publishersversionPeer reviewe

Functional Morphology of Tissues in Ontogenetic Aspect in Children with Complete Bilateral Cleft Lip and Palate. Doctoral Thesis

Promocijas darbs izstrādāts: Rīgas Stradiņa universitātes Anatomijas un antropoloģijas institūtā, Morfoloģijas katedrā. Aizstāvēšana: 2016. gada 9. decembrī plkst. 15.00 Rīgas Stradiņa universitātes Medicīnas promocijas padomes atklātā sēdē Rīgā, Dzirciema ielā 16, Hipokrāta auditorijā.Lūpu un aukslēju šķeltnes ir viena no izplatītākajām iedzimtajām anomālijām, kas bērnam izraisa estētiskus un funkcionālus traucējumus. Abpusēja caurejoša šķeltne ir smaga patoloģija, tās ārstēšanai tiek lietotas daudzetapu plastiskas ķirurģiskas korekcijas, brūces slēdzas un veidojas rētaudi, kas var negatīvi ietekmēt sejas un mutes dobuma audu augšanu. Mūsdienās morfopatoģenētiskai sejas šķeltņu skarto audu izpētei ir būtiska nozīme, lai izprastu šīs embrionāli determinētās patoloģijas etiomorfopatoģenēzi un pēcoperācijas audu remodelācijas potenciālu. Sejas un mutes dobuma attīstības pamatā ir vairāki precīzi koordinēti procesi – šūnu proliferācija, diferenciācija, migrācija, programmēta šūnu nāve, ekstracelulārās matrices sintēze un degradācija, vietējās audu aizsardzības faktoru bāzes veidošanās. Minētos procesus embrija audos koordinē dažādas signālmolekulas un augšanas faktori. Noteikti gēni, ārvides un teratogēnie faktori var izraisīt kādas noteiktas signālmolekulas un/vai augšanas faktoru trūkumu vai pārlieku lielu to esamību, kā rezultātā var attīstīties sejas šķeltnes. Pētījuma mērķis bija specifisku signālmolekulu un šūnu nāves noteikšana šķeltņu skartajos audos, kā arī to faktoru noteikšana, kuri visbūtiskāk raksturo abpusējas caurejošas šķeltnes morfopatoģenēzi ontoģenētiskajā aspektā. Pētījumā tika iekļauti 46 pacienti: 22 bērniem bija abpusēja caurejoša lūpas, alveolārā izauguma un aukslēju šķeltne, 24 bērniem – vienpusēja caurejoša lūpas, alveolārā izauguma un aukslēju šķeltne. Ar imūnhistoķīmisko metodi tika izpētīts un analizēts matrices metaloproteināzes-2 (MMP-2), matrices metaloproteināzes-8 (MMP-8), matrices metaloproteināzes-9 (MMP-9), matrices metaloproteināzes-2 audu inhibitora (TIMP-2), matrices metaloproteināzes-4 audu inhibitora (TIMP-4), gēnu proteīnu MSX1, IRF6, PAX9, RYK, vaskulārā endotēlija augšanas faktora (VEGF), transformējošā augšanas faktora beta-3 (TGFβ3), transmembranozā glikoproteīna CD34 (CD34), proteīngēnvielas 9.5 (PGP 9.5), nestīna, proliferācijas marķiera Ki-67 (Ki-67), osteokalcīna (OC), osteopontīna (OP), osteoprotegerīna (OPG) un kaula morfoģenētiskā proteīna 2/4 (BMP2/4) imūnreaktīvo šūnu un nervšķiedru relatīvais daudzums. Izmantojot TUNEL metodi, tika izpētīta apoptotisko šūnu klātbūtne un sadalījums. Kopumā smagākajam šķeltnes veidam – abpusējai caurejošai lūpas, alveolārā izauguma un aukslēju šķeltnei – tika konstatēta samazināta transkripcijas faktora IRF6, MSX1 un PAX9, augšanas faktora TGFβ3 un VEGF klātbūtne, kā arī samazināta šūnu proliferācija un apoptoze mīkstajos audos, bet balstaudos samazināta OPN, OPG, MMP-2, TIMP-2, BMP2/4 un TGFβ3 klātbūtne. Pārliecinoši identificējām, ka morfoloģiskās audu izmaiņas ir smagākas pacientiem ar abpusēju šķeltni. Atkārtotu operāciju laikā vieniem un tiem pašiem pacientiem darbā novērojām palielinātu MSX1, MMP-9, TIMP-4, TGFβ3, Ki-67 un samazinātu VEGF ekspresiju.Promocijas darbs veikts ar ESF projekta “Atbalsts doktorantiem studiju programmas apguvei un zinātniskā grāda ieguvei Rīgas Stradiņa universitātē” finansiālu atbalstu, vienošanās Nr. 2009/0147/1DP/1.1.2.1.2/09/IPIA/VIAA/009

Audu funkcionālā morfoloģija bērniem ar abpusēju caurejošu lūpas, alveolārā izauguma un aukslēju šķeltni ontoģenētiskā aspektā. Promocijas darba kopsavilkums

The Doctoral Thesis was carried out at the Department of Morphology, Institute of Anatomy and Anthropology, Rīga Stradiņš University, Latvia. Defence: at the public session of the Doctoral Council of Medicine on 9 December 2016 at 15.00 in Hippocrates Lecture Theatre, 16 Dzirciema Street, Rīga Stradiņš University.Cleft lip and palate is one of the most common congenital pathologies, causing esthetic and functional disorders in a child. Complete bilateral cleft lip and palate is a grave pathology, in its treatment multigrade plastic surgical corrections are needed, resulting in the closure of the wound and formation of scar tissue, which can negatively affect the growth of the facial and oral tissues. Nowadays the morphogenetic study of facial cleft-affected tissues is of great significance, in order to understand the etiomorphogenesis of this embrionally determined pathology and post-operation tissue remodeling potential. The development of the face and oral cavity contains the following specifically coordinated processes – tissue proliferation, differentiation, migration, programmed tissue death, synthesis and degradation of extracellular matrix, development of the basis of local protection factors. The processes mentioned in embryonic tissues are coordinated by different signal molecules and growth factors. Specific genes, external and teratogenic factors can cause the lack or excessive presence of a certain signal molecule and/or growth factor, resulting in the development of the facial cleft. The aim of the research was to study specific signaling molecules and tissue death determination in the cleft-affected tissues, as well as determination of those factors, which most essentially characterize morphopathogenesis of bilateral cleft lip and palate in the ontogenetic aspect. 46 patients were included in the research: 22 children had bilateral complete cleft lip, alveolar process and palate, 24 children – unilateral complete cleft lip, alveolar process and palate. By means of immunohistochemical method there were studied and analyzed matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-8 (MMP-8), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), tissue inhibitor of matrix metalloproteinase-4 (TIMP-4), gene protein MSX1, IRF6, PAX9, RYK, vascular endothelial growth factor (VEGF), transformating growth factor beta-3 (TGFβ3), transmembranous glycoprotein CD34 (CD34), protein gene product 9.5 (PGP 9.5), nestin, proliferation marker Ki-67 (Ki-67), osteocalcin (OC), osteopontin (OP), osteoprotegerin (OPG) and relative amount of bone morphogenetic protein 2/4 (BMP2/4) immunoreactive structures. By TUNEL method was studied the presence and distribution of apoptotic cells. All in all, in the most severe type of the cleft – bilateral cleft lip and palate there was found the presence of decreased transcription factor IRF6, MSX1 and PAX9, the growth factor TGFβ3 and VEGF, as well as the decreased cell proliferation and apoptosis in the soft tissues, but in the supporting tissues – the decreased OPN, OPG, MMP-2, TIMP-2, BMP2/4 and TGFβ3 presence. It was conclusively proved, that morphological tissue changes were more severe in patients with bilateral clefts. During repeated operations, one and the same patients we were identified an increased MSX1, MMP-9, TIMP-4, TGFβ3, Ki-67 and decreased VEGF expression.The Doctoral Thesis was carried out with “Support for Doctoral Students in Mastering the Study Programme and Acquisition of a Scientific Degree in Rīga Stradiņš University”, agreement No 2009/0147/1DP/1.1.2.1.2/09/IPIA/VIAA/009”