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    Estudo da fibrose no músculo esquelético mediada por receptores de angiotensina e tratamento do músculo lesionado por prp e terapia gênica
    (Universidade Federal de São Paulo (UNIFESP), 2015-12-17) Stilhano, Roberta Sessa [UNIFESP]; Han, Sang Won Han [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    process, which frequently occurs in cases of deeper muscle injury, leads to imperfect tissue regeneration. The current therapies have a low efficacy for prevention or treatment of the complications caused by fibrosis, such as atrophy, contraction and pain followed by functional loss and post-traumatic fibrosis. Hypertension is a contributing factor in fibrosis, and angiotensin II (Ang II), which is the main responsible for vasoconstriction, seems to trigger signaling fibrosis. There is communication between TGFB1 and Ang II signaling pathway in cardiac muscle. In parallel, there is a discordant discussion of GM-CSF role in skeletal muscle fibrosis. To better understand the fibrotic process occurring after injury in skeletal muscle, first of all, we established a muscle injury model followed by suture, which produces a deep lesion developing fibrosis largely muscle. To evaluate the possible role of the AT1 and GM-CSF in promoting fibrosis, multiple vectors were constructed, including, Lv-mirAT1a expressing a microRNA to silence the expression of AT1a protein and Lv-GM-CSF expressing GM-CSF. Considering that the injection of lentiviral solution in skeletal muscle may not be efficient for transduction of these vectors and cause leakage to other tissues, an alginate hydrogel was formulated for carrying lentivetores. The animals treated with alginate hydrogel loaded with Lv-GM-CSF and Lv-mirAT1a drastically reduced muscle fibrosis. Thus, it was inferred that the AT1a should participate in the fibrotic process of skeletal muscle and its inhibition is a good strategy for reducing post-injury fibrosis. On the other hand, the effect of the expression of GM-CSF in the control of fibrosis varied depending on the inflammatory progression, or its expression and the onset of inflammation-accentuated fibrosis, but expression in late stage drastically reduced fibrosis, confirming the results of our previous work. The mediators of fibrosis, such as angiotensin and growth factors are present in plasma and in sports medicine, the platelet rich plasma (PRP) is widely used to treat various types of lesions, including muscle injury. In an attempt to correlate the above studies with these factors in PRP, the molecular and cellular content of that preparation was evaluated. Two methods for preparation of PRP were established in the literature database and were named LPRP and PPRP. The main difference between the two preparations is the presence of leukocytes (LPRP) or not (PPRP), but the molecular and cellular contents thereof vary markedly PRPs. NIH3T3 cell lines (fibroblasts) and C2C12 (myoblast) grown with 1% PRP, and 10% FBS promoted cell proliferation in different patterns. The expression gene expression profile of 6 growth factors were analyzed and showed that each PRP for each cell type expression level of these genes is significantly variable. In conclusion, it was demonstrated that AT1 participates in skeletal muscle fibrosis and the inhibition of AT1 via microRNA is a good alternative to reduce fibrosis. Forced expression of GM-CSF early in the muscle injury promotes the formation of fibrosis, but their expression days later drastically reduces fibrosis. For late expression of GM-CSF via lentivetor contained in loco, the alginate hydrogel formulation with lentivetor developed here was ideal. As for PRPs, leukocytes contained in LPRP takes the PRP formation with different quality and amount of cells and molecules, hence its effect on the target cells were different.