Vitamina D: a influência na matriz extracelular do tecido conjuntivo de sustentação dos órgãos pélvicos

• Ano de defesa: 2019
• Autor(a) principal: Kemp, Marta Maria [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Extracellular-Matrix; Pelvic Organ Prolapse; Animal Study; Molecular Biology; Hormone Therapy; Matriz Extracelular; Prolapso Genital; Estudo Em Animais; Biologia Molecular; Terapia Hormonal
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=8105359; https://repositorio.unifesp.br/handle/11600/60057

Hypothesis/aims of study: Vitamin D deficiency is largely spoken of as being associated with several different diseases. Most of the associations are inferred by the levels of 25(OH)D in the plasma, which has no direct correlation to the activity of its active metabolites, i.e. 1,25(OH)D, since it follows the patterns of a steroid hormone, with negative and positive feedbacks and other molecular interactions, instead of behaving as a vitamin. Pelvic organ prolapse has also been linked to lower serum levels of vitamin D, although the exact mechanism for this increase in risk is not totally understood. One of the potential mechanisms for this increased risk is the influence vitamin D has on the architecture of the extracellular matrix (ECM) main component of the structures of the pelvic support apparatus and responsible for its mechanical properties. The aim of this study was to examine the arrangement of collagen fibers, the most prevalent fibrous protein present in the ECM at the endopelvic fascia, and other structural components of the ECM and cytoskeleton of the endopelvic fascia, to capture any different morphological pattern between subjects with a normal diet and those under a vitamin D supplementation. Collagen quantification was also performed. Study design, materials and methods: This hypothesis was investigated using female healthy adult Wistar rats (8 weeks old, approximately 200-220g). The treatment group received cholecalciferol diluted in arachid oil by gavage (in the dose 37.5mcg/Kg/day, equivalent to 1,500Ui/Kg/day), and the control group received only the oil, for 18 consecutive days. Twenty-four hours after the last gavage procedure, euthanasia was performed and a surgical procedure was undertaken extracting, as a bloc, the lower third of the uterus with the uterosacral ligament, the upper half of the vagina with the connective tissue surrounding and the bladder with proximal urethra. Samples were immersed in Tissue-Tek and cut in 30 and 50 micrometer thick slices and placed on the sheets for microscopic evaluation with SHG (second harmonic generation) technique, which requires no tissue preparation. For the purpose of interpretation, the rats were also separated into groups according to the estrous phase: high estrogenic influence (proestrus and estrus) and low estrogenic influence (metaestrus and diestrus) identified by cytology and histology under conventional light microscopy. Collagen by Picrosirius and antibodies for other extracellular structural components were also performed. Tissues were analyzed using Confocal Microscope (TCS SP8 CARS from Leica) and F and Epi-SHG detectors were used to detect SHG signals from collagen fibers. Images are represented as maximum intensity projections, corresponding to the Z-series of confocal stacks and were collected by tile scan and processed with Leica LAS AF software and, posteriorly, manipulated with ImageJ software. Results: We found that collagen fibers have a consistently undulated pattern and more random disposition in the xvi treated group whereas in the control group it assumes a more taut and oriented appearance, when under high estrogenic influence. The collagen signal from SHG was statistically more intense in the treated group compared to the control group also under a high estrogenic environment. The extracellular components alpha-actin, heparan sulfate and glicoconjugates containing sialic acid and N-acetylgkucosamine residues also showed clear different images when comparing control and treated groups. Conclusion: These findings suggest that there is a difference in the morphological presentation of the extracellular matrix of subjects under high dose intake of vitamin D compared to subjects under normal intake of the vitamin, pointing to a probable change in the mechanical properties of the tissue and that there might be an interaction of the metabolic pathways of the two steroid hormones cholecalciferol and estrogen at least regarding the amount of collagen deposited in the tissue. Not only the collagen, other components of the ECM presented differently in the treated and control groups. Oral high dose intake of vitamin D changes the framework and composition of the extracellular matrix at the level of the endopelvic fascia, which can impact the properties and resilience of the tissue. In summary, new research are needed in the biomechanical field to approach a clinical significance but we have shown a tangible relation between vitamin D exposure and collagen fiber architecture.