mTOR-Dependent Autophagy Machinery Is Inhibited in Fibroblasts of Keloid

Objectives:Mechanistic target of rapamycin(mTOR)activation has been identified in keloid.This study aimed to identify the role of mTOR-dependent autophagy activity in keloid.Methods:We detected the expression of specific proteins representing mTOR activity and baseline autophagy levels in keloid tissues(KTs)and primary keloid fibroblasts(KFs)using immunohistochemical staining and western blotting.Simultaneously,the formation of acid vesicles was assessed by acridine orange staining in KFs.To investigate whether mTOR-dependent pathway mediated the regulation of autophagy machinery in keloid,we first validated whether mTOR inhibitors,rapamycin(100 nmol/L)and KU-0063794(5mmol/L),could inhibit mTOR activity in KFs by western blotting.Then we explored the reverse effects on autophagy activity induced by mTOR inhibitors in the presence of lysosomal protease inhibitors by western blotting.Results:It demonstrated elevated expression of mTOR,S6,and their activated forms in KTs,and an elevated expression of p-S6 Ser235/236 in KFs,suggesting mTOR was activated in keloid.Less LC3 and Beclin1 were expressed in the cytoplasm of KFs,whereas Ubiquitin was abundantly expressed in KTs compared with extra-lesional tissues.In addition,at the cellular level,an impeded conversion of LC3-I to LC3-II was shown in KFs and the formation of acid vesicles were also decreased in KFs compared with normal fibroblasts(NFs),indicating that autophagy activity is defective in keloid.mTOR inhibitors,Rapamycin(E-64d+pepstatin vs.rapamycin+E-64d+pepstatin:[0.88±0.35]vs.[1.56±0.46],F=5.56,P=0.049)and KU-0063794(E-64d+pepstatin vs.KU-0063794+E-64d+pepstatin:[0.92±0.22]vs.[1.51±0.25],F=25.88,P=0.011)can reverse the inhibition effect on autophagy of KFs while inhibiting mTOR activity.Conclusion:Autophagy machinery is inhibited in keloid which is regulated by mTOR-dependent pathway.

Mediation of Anti-Keloid Effects of mTOR Inhibitors by Autophagy-Independent Machinery

Objective:Blocking mechanistic target of rapamycin(mTOR)activation with mTOR inhibitors has promising therapeutic potential for keloids.However,the precise mechanism of mTOR inhibitors remains unclear.This study was aimed to investigate the role of autophagy machinery in the anti-keloid effects of mTOR inhibitors.Methods:We first validated the biological effects induced by the mTOR inhibitors rapamycin(100 nmol/L)and KU-0063794(5μmol/L)on the proliferation,apoptosis,migration,and collagen synthesis of keloid fibroblasts(KFs)derived from Han Chinese persons through a Cell Counting Kit-8 assay,5-Bromo-2’-deoxyuridine incorporation,Annexin V/propidium iodide staining,migration,and western blotting.To explore whether autophagy machinery is involved in the anti-keloid effects of mTOR inhibitors,we first blocked the autophagy activation induced by rapamycin and KU-0063794 with a pharmacological autophagy inhibitor(wortmannin)or by silencing the key autophagy gene(ATG5),and we then re-evaluated these biological effects on KFs.Results:Blocking mTOR activation with either rapamycin or KU-0063794 completely inhibited proliferation,migration,and collagen synthesis of primary KFs but did not affect apoptosis.Incubating KFs with the autophagy inhibitor wortmannin or performingATG5 silencing abrogated the subsequent activation of autophagic activity induced by rapamycin(rapamycin+E-64d+pepstatinvs.rapamycin+wortmannin+E-64d+pepstatin:1.88±0.38vs.1.02±0.35,F=6.86,P=0.013),(non-sense control+rapamycinvs.ATG5 small interfering RNA+rapamycin:1.46±0.18vs.0.75±0.20,respectively;F=7.68,P=0.01)or KU-0063794(KU-0063794+E-64d+pepstatinvs.KU-0063794+wortmannin+E-64d+pepstatin:1.65±0.35vs.0.76±0.17,F=10.01,P=0.004),(NC+KU-0063794vs.ATG5 small interfering RNA+KU-0063794:1.59±0.50vs.0.77±0.09,F=5.93,P=0.02)as evidenced by decreased accumulation of LC3-II.However,blockage of autophagy induction in mTOR inhibitor-treated KFs with both methods did not disturb their anti-keloid effects,such as inhibition of cell viability,cell migration,and collagen synthesis(P>0.05 each).Conclusion:Blocking mTOR activation with the mTOR inhibitors rapamycin and KU-0063794 showed anti-keloid effects in KFs.Restoration of autophagy inhibition by mTOR inhibitors does not contribute to their anti-keloid effects.

Clinical Application of Platelet-Rich Fibrin in Dermatology

Platelet-rich fibrin(PRF)is a second-generation platelet concentrate obtained from autologous blood.PRF is composed of abundant platelets,leucocytes,and a high concentration of various growth factors and fibrinogen.The composition and three-dimensional structure of PRF enable it to effectively make cells migrate and proliferate,playing an important role in tissue repair.Furthermore,the easy preparation and low cost of PRF make it a good treatment option.Numerous articles have been published about the application of PRF in clinical practice,however,the application of PRF in dermatology has not been comprehensively reviewed.The objective of this review article was to discuss various applications of PRF in dermatology,including healing chronic wounds,treating androgenic alopecia,skin rejuvenation,autologous fat transplantation,and treating vitiligo.PRF is a promising dermatologic treatment,but lacks a standardized protocol regarding its methods of attainment and use,which needs more investigations.

Skin Organoid Research Progress and Potential Applications

Skin diseases were characterized by various types and high incidence,which seriously affect people’s health.At present,skin pathogenesis research and the therapeutic drug development for skin diseases are limited by the lack of reasonable research models that recapitulate the development of skin diseases.Organoids are three-dimensionally cultured cell populations derived from skin stem cells,which exhibits the ability of multicell assembly and the similar histological characteristics with the living tissues and organs.This article reviews the establishment of normal skin organoids and skin tumor organoids,and summarizes the application of skin organoids in the evaluation of drug sensitivity,pathological mechanism research,and individualized treatment.In addition,the advantages and limitations of organoids in skin disease research are also discussed,which provides a basis for revealing the pathogenesis of skin diseases and developing preventive and therapeutic drugs for skin diseases.

Treatment of Nevoid Basal Cell Carcinoma Syndrome by Surgery Combined With ALA-PDT: A Case Report

Introduction:Nevoid basal cell carcinoma syndrome is an autosomal dominant disorder characterized by developmental malformations and carcinogenic activity.Multi-systemic anomalies may occur in this syndrome,such as odentogenic keratocysts of the mandible and postnatal tumors,especially multiple basal cell carcinoma.Case presentation:A 60 year old man presented with systemic plaques and nodules for more than 30 years.Cutaneous examination revealed that invasive erythema and black papules scattered on the face,trunk and limbs.He underwent extended surgical excision of lesions at multiple sites.No new lesions were found in the treated areas during a 3-year follow-up.Topical 5-aminolevulinic acid-mediated photodynamic therapy(ALA-PDT)was given as a palliative treatment for ulcerated tumors on the right lower eyelid and bleeding of the right temporal lesion.After application of ALA-PDT,the ulcers almost healed and the recurring hemorrhage ceased.Discussion:The management of multiple tumors in patients with nevoid basal cell carcinoma syndrome is extremely difficult,especially in the face.Surgical excision is the standard method,but the procedure can be applied in limited sites and may result in significant disfigurement and the difficulty of wound recovery.PDT can play a significant role in combination therapy for tumors that are extremely difficult to remove completely by surgical excision.Conclusion:We present a rare case manifested as multiple basal cell carcinoma and palmar pits who treated by surgery combined with topical 5-aminolevulinic acid-mediated photodynamic therapy.The combined therapy plays a complementary role in the treatment of nevoid basal cell carcinoma syndrome.