isolation-culture-characterization-primary-dermal-fibroblasts-from

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...

Southern Medical University

Keloids are benign fibroproliferative diseases with abnormally proliferated bulges beyond the edge of the skin lesions, and they are characterized by uncontrolled fibroblast proliferation and excessive extracellular matrix deposition in the dermis. However, the definite mechanisms that increase fibroblast proliferation and collagen deposition in keloids remain unclear. Thrombospondin 1 (TSP1) has been suggested to play an important role in wound healing and fibrotic disorders, but its role in keloids is unknown. In this study, we aimed to clarify the specific role of TSP1 in keloids and explore the potential mechanism. Our results demonstrated that TSP1 was highly expressed in keloid lesions compared to normal skin. Knockdown of TSP1 in keloid fibroblasts decreased cell proliferation and collagen I deposition. Exogenous TSP1 treatment increased cell proliferation and collagen I deposition in normal fibroblasts. We further investigated the underlying mechanism and found that TSP1 promoted fibroblast proliferation and extracellular matrix deposition by upregulating the IL6/JAK2/STAT3 pathway. Moreover, we verified that TSP1 expression was positively correlated with IL6/STAT3 signalling activity in keloids. Taken together, our findings indicate that TSP1 promotes keloid development via the IL6/JAK2/STAT3 signalling pathway and blocking TSP1 may represent a potential strategy for keloid therapy.

TSP1 promotes fibroblast proliferation and extracellular matrix deposition via the IL6/JAK2/STAT3 signalling pathway in keloids.

Cystatin M/E (encoded by the CST6 gene) is a cysteine protease inhibitor, that exerts regulatory and protective effects against uncontrolled proteolysis mainly by directly regulating cathepsin V, cathepsin L, and legumain activities. Previous studies have suggested that CST6 may exert a regulatory role in epidermal differentiation and hair follicle formation by inhibiting the activity of respective cognate target proteases. However, until recently, studies have revealed that loss- or gain-of-function of the CST6 gene causes dry skin with hypotrichosis in humans. Here, we reported two siblings of Chinese origin with dry skin, desquamation and abnormal keratosis without hypotrichosis. By applying whole-exome sequencing, we identified homozygous loss-of-function mutation c.251G > A (p.Gly84Asp) in the CST6 gene as the underlying genetic cause. Further fluorimetric enzyme assays demonstrated the mutant cystatin M/E protein lost its inhibitory function on the protease activity of cathepsins. Moreover, the corresponding mutation in mice resulted in excessive cornification, desquamation, impaired skin barrier function, and abnormal proliferation and differentiation of keratinocytes. In conclusion, the homozygous missense mutation c.251G > A in CST6 gene resulted in dry skin, desquamation, as well as abnormal keratosis of the skin, promoting our understanding of the role of protease-antiprotease balance in human skin disorders.

Loss-of-function mutations in CST6 cause dry skin, desquamation and abnormal keratosis without hypotrichosis.

Keloids represent one extreme of aberrant dermal wound healing and are characterized by fibroblast hyperproliferation and excessive deposition of extracellular matrix. Genetics is a major factor for predisposition to keloids and genome-wide association study has identified a single-nucleotide polymorphism (SNP) rs873549 at 1q41 as a susceptibility locus. The SNP rs873549, and the SNPs in strong linkage disequilibrium (LD) with rs873549, may be involved in keloid development. However, the functional significance of these SNPs in keloid pathogenesis remains elusive.

Risk single-nucleotide polymorphism-mediated enhancer-promoter interaction drives keloids through long noncoding RNA down expressed in keloids.

CD80 is an important co-stimulatory molecule that participates in the immune response. Soluble CD80 can induce T cell activation and overcome PDL1-mediated immune suppression. In this study, we aimed to construct recombinant  for oral delivery of the soluble CD80 (hsCD80) protein or the fusion protein containing the cholera toxin B subunit (CTB) and hsCD80 (CTB-hsCD80) under the control of the nisin-inducible expression system. The recombinant  expressed and secreted hsCD80 or CTB-hsCD80 fusion proteins after induction by nisin in vitro and in the enteric cavity. Additionally, the CTB-hsCD80 fusion protein showed uptake by intestinal epithelial cells, was cleaved by the furin protease, and was released as free hsCD80 protein into the blood circulation. Orally administered hsCD80 and CTB-hsCD80 containing  increased the proportion of activated T cells in the spleen and intestinal epithelium, inhibited tumor growth, and prolonged the survival of tumor-bearing mice. The hsCD80-containing L.  showed greater therapeutic effects on primary colonic adenoma in APC mice and completely suppressed tumor growth. Further, recombinant CTB-hsCD80 in  was more efficient than hsCD80-containing bacteria in inhibiting the growth of xenografted colon cancer and melanoma cells. hsCD80 engineered probiotics may serve as a promising new approach for antitumor immunotherapy, especially for colorectal cancer.

Article	Year
TSP1 promotes fibroblast proliferation and extracellular matrix deposition via the IL6/JAK2/STAT3 signalling pathway in keloids. Experimental dermatology\| PubMed ID: 35661430	2022
Loss-of-function mutations in CST6 cause dry skin, desquamation and abnormal keratosis without hypotrichosis. Clinical genetics\| PubMed ID: 36371786	2023
Risk single-nucleotide polymorphism-mediated enhancer-promoter interaction drives keloids through long noncoding RNA down expressed in keloids. The British journal of dermatology\| PubMed ID: 36689521	2023
Soluble CD80 oral delivery by recombinant Lactococcus suppresses tumor growth by enhancing antitumor immunity. Bioengineering & translational medicine\| PubMed ID: 37476068	2023

Xueyan Xu

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