{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,3]],"date-time":"2026-11-03T18:54:30Z","timestamp":1793732070875,"version":"4.6.1"},"reference-count":52,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2025,7,12]],"date-time":"2025-07-12T00:00:00Z","timestamp":1752278400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JCM"],"abstract":"<jats:p>Introduction: Unilateral cleft lip and palate (CLP) is a severe orofacial birth defect characterized by improper fusion of facial parts and disturbed orofacial functions. The defect manifests as a gap in the orofacial tissues that is accompanied by defective healing patterns and chronic inflammation. The immune system\u2019s defense factors modulate immunity, inflammation, and healing. Angiogenesis factors control blood-vessel formation. Therefore, these factors are vital in the immunological assessment and understanding of CLP morphopathogenesis. The aim of the study is to assess the distribution of vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF- \u03b21), the total macrophage population and the M2 subtype, heat-shock proteins (HSP) 60 and 70, and nuclear factor kappa B (NF-\u03baB) p50 and p65 subtypes in the affected tissue of children with CLP before and during primary dentition. Materials and Methods: Tissue samples were obtained from 15 patients aged from 3 to 8 months during veloplastic surgery. Five controls were used for comparison of data. Immunohistochemistry, light microscopy, semi-quantitative evaluation (from 0 to ++++), and statistics (Mann\u2013Whitney U test and Spearman\u2019s rank correlation) were used to evaluate the data for statistically significant differences and correlations between the groups. Results: Epithelial tissues affected by CLP presented with statistically significant increases in levels of VEGF (p = 0.007), total macrophages (p = 0.007), HSP60 (p = 0.001), NF-\u03baB p65 (p = 0.000), and p50 (p = 0.045), but with a decrease in M2 macrophages (p = 0.025). Blood vessels in CLP-affected tissues showed a statistically significant increase in levels of NF-\u03baB p65 (p = 0.003) and a statistically significant decrease in M2 numbers (p = 0.014). Connective tissue presented with no statistically significant differences. Spearman\u2019s rank correlation revealed multiple statistically significant correlations\u201426 positive and 5 negative. Conclusions: Statistically significant changes in levels of VEGF and both NF-\u03baB subtypes and numbers of total macrophages and M2 macrophages suggest a possible alteration of variable immune and inflammatory reactions and macrophage functions associated with the initiation and maintenance of the chronic process and the resulting damage.<\/jats:p>","DOI":"10.3390\/jcm14144952","type":"journal-article","created":{"date-parts":[[2025,7,14]],"date-time":"2025-07-14T10:55:41Z","timestamp":1752490541000},"page":"4952","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Characterization of Factors Associated with Tissue Immunity, Cellular Activity and Angiogenesis in Children with Unilateral Cleft Lip and Palate Before and During Primary Dentition: A Comparative Cross-Sectional Study"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-3927-3208","authenticated-orcid":false,"given":"Laura","family":"Ozola","sequence":"first","affiliation":[{"name":"Institute of Anatomy and Anthropology, Riga Stradins University, Kronvalda Boulevard 9, LV-1010 Riga, Latvia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9804-4666","authenticated-orcid":false,"given":"M\u0101ra","family":"Pilmane","sequence":"additional","affiliation":[{"name":"Institute of Anatomy and Anthropology, Riga Stradins University, Kronvalda Boulevard 9, LV-1010 Riga, Latvia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1097\/01.JAA.0000721644.06681.06","article-title":"An overview of cleft lip and palate","volume":"33","author":"Alois","year":"2020","journal-title":"JAAPA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1597\/01-083","article-title":"Clinical and microbiological evaluation of the periodontal status of children with unilateral complete cleft lip and palate","volume":"40","author":"Costa","year":"2003","journal-title":"Cleft Palate Craniofac. J."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"937","DOI":"10.1177\/154405910708601006","article-title":"VEGF: An essential mediator of both angiogenesis and endochondral ossification","volume":"86","author":"Dai","year":"2007","journal-title":"J. Dent. Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"S4","DOI":"10.1159\/000088478","article-title":"VEGF as a key mediator of angiogenesis in cancer","volume":"69","author":"Carmeliet","year":"2005","journal-title":"Oncology"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"699","DOI":"10.1111\/all.12403","article-title":"Transforming growth factor-beta 1 pathways in inflammatory airway diseases","volume":"69","author":"Yang","year":"2014","journal-title":"Allergy"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"274","DOI":"10.1002\/path.4119","article-title":"MicroRNAs, transforming growth factor beta-1, and tissue fibrosis","volume":"229","author":"Bowen","year":"2013","journal-title":"J. Pathol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1089\/gtmb.2015.0301","article-title":"Association Between the Transforming Growth Factor Beta 1 Gene Polymorphisms and Turkish Patients with Nonsyndromic Cleft Lip With\/Without Cleft Palate","volume":"20","author":"Oner","year":"2016","journal-title":"Genet. Test. Mol. Biomark."},{"key":"ref_8","first-page":"e42412","article-title":"Interplay of Transforming Growth Factor-Beta 1 and 3 in the Pathogenesis of Oral Submucous Fibrosis and Its Malignant Transformation: An Immunohistochemical Study","volume":"15","author":"Bansal","year":"2023","journal-title":"Cureus"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Alharbi, K.S., Fuloria, N.K., Fuloria, S., Rahman, S.B., Al-Malki, W.H., Javed Shaikh, M.A., Thangavelu, L., Singh, S.K., Rama Raju Allam, V.S., and Jha, N.K. (2021). Nuclear factor-kappa B and its role in inflammatory lung disease. Chem. Biol. Interact., 345.","DOI":"10.1016\/j.cbi.2021.109568"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1812","DOI":"10.1111\/febs.13627","article-title":"NFKB1: A suppressor of inflammation, ageing and cancer","volume":"283","author":"Cartwright","year":"2016","journal-title":"FEBS J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1016\/j.gendis.2020.06.005","article-title":"The Nuclear Factor Kappa B (NF-kB) signaling in cancer development and immune diseases","volume":"8","author":"Zinatizadeh","year":"2021","journal-title":"Genes Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1111\/imr.12224","article-title":"Tissue macrophage identity and self-renewal","volume":"262","author":"Gentek","year":"2014","journal-title":"Immunol. Rev."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1177\/0022034516677539","article-title":"Cellular and Molecular Pathways Leading to External Root Resorption","volume":"96","author":"Hartsfield","year":"2017","journal-title":"J. Dent. Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1106","DOI":"10.1172\/JCI93025","article-title":"CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis","volume":"128","author":"Guo","year":"2018","journal-title":"J. Clin. Investig."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"117243","DOI":"10.1016\/j.cca.2023.117243","article-title":"The clinical utility of CD163 in viral diseases","volume":"541","author":"Yap","year":"2023","journal-title":"Clin. Chim. Acta"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Lee, H.J., Hong, Y.J., and Kim, M. (2021). Angiogenesis in Chronic Inflammatory Skin Disorders. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms222112035"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Paiva, K.B.S., Maas, C.S., Dos Santos, P.M., Granjeiro, J.M., and Letra, A. (2019). Extracellular Matrix Composition and Remodeling: Current Perspectives on Secondary Palate Formation, Cleft Lip\/Palate, and Palatal Reconstruction. Front. Cell Dev. Biol., 7.","DOI":"10.3389\/fcell.2019.00340"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"d\u2019Amaro, R., Scheidegger, R., Blumer, S., Pazera, P., Katsaros, C., Graf, D., and Chiquet, M. (2012). Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis. Front. Physiol., 3.","DOI":"10.3389\/fphys.2012.00377"},{"key":"ref_19","first-page":"98","article-title":"Immunohistochemical analysis of nestin, CD34 and TGF\u03b23 in facial tissue of children with complete unilateral and bilateral cleft lip and palate","volume":"18","author":"Pilmane","year":"2016","journal-title":"Stomatologija"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Ozola, L., and Pilmane, M. (2024). Characterization of Tissue Immunity Defense Factors of the Lip in Primary Dentition Children with Bilateral Cleft Lip Palate. J. Pers. Med., 14.","DOI":"10.3390\/jpm14090965"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"101330","DOI":"10.1016\/j.smim.2019.101330","article-title":"IL-10 producing regulatory and helper T-cells in systemic lupus erythematosus","volume":"44","author":"Geginat","year":"2019","journal-title":"Semin. Immunol."},{"key":"ref_22","unstructured":"Suvarna, S.K., Layton, C., and Bancroft, J.D. (2019). Bancroft\u2019s Theory and Practice of Histological Techniques, Elsevier."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"De\u0146isova, A., Pilmane, M., and Ka\u017eoka, D. (2023). Antimicrobial Peptides and Interleukins in Cleft Soft Palate. Children, 10.","DOI":"10.3390\/children10071162"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Vaivads, M., Akota, I., and Pilmane, M. (2021). Cleft Candidate Genes and Their Products in Human Unilateral Cleft Lip Tissue. Diseases, 9.","DOI":"10.3390\/diseases9020026"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.prp.2018.10.029","article-title":"The evaluation of inflammatory, anti-inflammatory and regulatory factors contributing to the pathogenesis of COPD in airways","volume":"215","author":"Vitenberga","year":"2019","journal-title":"Pathol. Res. Pract."},{"key":"ref_26","unstructured":"Barton, B., and Peat, J. (2014). Medical Statistics: A Guide to SPSS, Data Analysis and Critical Appraisal, Wiley."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Vaivads, M., Akota, I., and Pilmane, M. (2023). Characterization of SHH, SOX3, WNT3A and WNT9B Proteins in Human Non-Syndromic Cleft Lip and Palate Tissue. Dent. J., 11.","DOI":"10.3390\/dj11060151"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1089\/wound.2018.0796","article-title":"Vascular Endothelial Growth Factor and Cutaneous Scarring","volume":"8","author":"Wilgus","year":"2019","journal-title":"Adv. Wound Care"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Boreak, N. (2021). Small Molecule \u201cSilmitasertib\u201d Repurposed as Inhibitor of Transforming Growth Factor Beta 1 for the Development of Therapeutics for Oral Submucous Fibrosis. Biomed. Res. Int., 2021.","DOI":"10.1155\/2021\/6631848"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Skytthe, M.K., Graversen, J.H., and Moestrup, S.K. (2020). Targeting of CD163(+) Macrophages in Inflammatory and Malignant Diseases. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21155497"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2352","DOI":"10.1089\/ars.2012.4834","article-title":"CD163 and inflammation: Biological, diagnostic, and therapeutic aspects","volume":"18","author":"Etzerodt","year":"2013","journal-title":"Antioxid. Redox Signal"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1650","DOI":"10.1016\/j.molimm.2010.02.008","article-title":"Scavenger receptor CD163, a Jack-of-all-trades and potential target for cell-directed therapy","volume":"47","author":"Delputte","year":"2010","journal-title":"Mol. Immunol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Wang, L., Fu, Y., Yu, B., Jiang, X., Liu, H., Liu, J., Zha, B., and Chu, Y. (2021). HSP70, a Novel Regulatory Molecule in B Cell-Mediated Suppression of Autoimmune Diseases. J. Mol. Biol., 433.","DOI":"10.1016\/j.jmb.2020.08.019"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1016\/j.imbio.2005.05.010","article-title":"The macrophage scavenger receptor CD163","volume":"210","author":"Fabriek","year":"2005","journal-title":"Immunobiology"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"90","DOI":"10.21037\/atm-22-6390","article-title":"Dental pulp stem cells induce anti-inflammatory phenotypic transformation of macrophages to enhance osteogenic potential via IL-6\/GP130\/STAT3 signaling","volume":"11","author":"Liu","year":"2023","journal-title":"Ann. Transl. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1105","DOI":"10.31557\/APJCP.2021.22.4.1105","article-title":"IL-4, IL-17 and CD163 Immunoexpression and IL-6 Gene Polymorphism in Chronic Hepatitis C Patients and Associated Hepatocellular Carcinoma","volume":"22","author":"Aboushousha","year":"2021","journal-title":"Asian Pac. J. Cancer Prev."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Ozola, L., and Pilmane, M. (2023). Local Defense Factors in Cleft-Affected Palate in Children before and during Milk Dentition Age: A Pilot Study. J. Pers. Med., 14.","DOI":"10.3390\/jpm14010027"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Tang, Y., Zhou, Y., Fan, S., and Wen, Q. (2022). The multiple roles and therapeutic potential of HSP60 in cancer. Biochem. Pharmacol., 201.","DOI":"10.1016\/j.bcp.2022.115096"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Singh, M.K., Shin, Y., Han, S., Ha, J., Tiwari, P.K., Kim, S.S., and Kang, I. (2024). Molecular Chaperonin HSP60: Current Understanding and Future Prospects. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25105483"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"16013","DOI":"10.1038\/npjregenmed.2016.13","article-title":"Extracellular HSP60 triggers tissue regeneration and wound healing by regulating inflammation and cell proliferation","volume":"1","author":"Pei","year":"2016","journal-title":"NPJ Regen. Med."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s12192-011-0290-6","article-title":"Regulation of survival gene hsp70","volume":"17","author":"Silver","year":"2012","journal-title":"Cell Stress. Chaperones"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"585","DOI":"10.5935\/0103-507X.20200084","article-title":"Oxidative stress and decreased tissue HSP70 are involved in the genesis of sepsis: HSP70 as a therapeutic target","volume":"32","author":"Sulzbacher","year":"2020","journal-title":"Rev. Bras. Ter. Intensiv."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Albakova, Z., Armeev, G.A., Kanevskiy, L.M., Kovalenko, E.I., and Sapozhnikov, A.M. (2020). HSP70 Multi-Functionality in Cancer. Cells, 9.","DOI":"10.3390\/cells9030587"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1007\/s12192-020-01068-2","article-title":"Autophagy and Hsp70 activation alleviate oral epithelial cell death induced by food-derived hypertonicity","volume":"25","author":"Yang","year":"2020","journal-title":"Cell Stress. Chaperones"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"534","DOI":"10.1111\/j.1346-8138.2009.00696.x","article-title":"Differential expression of phosphorylated extracellular signal-regulated kinase 1\/2, phosphorylated p38 mitogen-activated protein kinase and nuclear factor-kappaB p105\/p50 in chronic inflammatory skin diseases","volume":"36","author":"Wang","year":"2009","journal-title":"J. Dermatol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1080\/003655202317315971","article-title":"VEGF and tumour angiogenesis. Impact of surgery, wound healing, inflammation and blood transfusion","volume":"37","author":"Svendsen","year":"2002","journal-title":"Scand. J. Gastroenterol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"216709","DOI":"10.1016\/j.canlet.2024.216709","article-title":"S100A6 drives lymphatic metastasis of liver cancer via activation of the RAGE\/NF-kB\/VEGF-D pathway","volume":"587","author":"Chen","year":"2024","journal-title":"Cancer Lett."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"e592","DOI":"10.1002\/cpz1.592","article-title":"Heat-Shock Proteins","volume":"2","author":"Hagymasi","year":"2022","journal-title":"Curr. Protoc."},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Vaivads, M., Akota, I., and Pilmane, M. (2021). PAX7, PAX9 and RYK Expression in Cleft Affected Tissue. Medicina, 57.","DOI":"10.3390\/medicina57101075"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/j.mod.2018.02.003","article-title":"Genes and microRNAs associated with mouse cleft palate: A systematic review and bioinformatics analysis","volume":"150","author":"Suzuki","year":"2018","journal-title":"Mech. Dev."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Zheng, X., Zhao, X., Wang, Y., Chen, J., Wang, X., Peng, X., Ma, L., and Du, J. (2023). Inhibition of Cxcr4 Disrupts Mouse Embryonic Palatal Mesenchymal Cell Migration and Induces Cleft Palate Occurrence. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241612740"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/bs.ctdb.2021.12.003","article-title":"Mouse models in palate development and orofacial cleft research: Understanding the crucial role and regulation of epithelial integrity in facial and palate morphogenesis","volume":"148","author":"Lan","year":"2022","journal-title":"Curr. Top. Dev. Biol."}],"container-title":["Journal of Clinical Medicine"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2077-0383\/14\/14\/4952\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:09:11Z","timestamp":1760033351000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2077-0383\/14\/14\/4952"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,12]]},"references-count":52,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2025,7]]}},"alternative-id":["jcm14144952"],"URL":"https:\/\/doi.org\/10.3390\/jcm14144952","relation":{},"ISSN":["2077-0383"],"issn-type":[{"value":"2077-0383","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,7,12]]}}}