The plasmid encoding HSP47 enhances collagen expression and promotes skin wound healing in an alloxan-induced diabetic model

The objective of this work was initially to investigate the effects on skin wound healing process by local injection of HSP47 recombinant plasmid in an alloxan-induced diabetic rat model and assess the possibility and utility of gene therapy based on HSP47 plasmid to improve the diabetic skin wound healing. Rats were injected intraperitoneally with alloxan (120 mg/kg) to induce diabetes. The fragment containing the rat 47 kDa heat shock protein (HSP47) gene lacking its own promoter was cloned into plasmids containing a promoter and green fluorescent protein (GFP). The resulting gene constructs were first tested in vitro using 3T3 fibroblast cell line and subsequently in vivo after inducing wounds with alloxan in diabetic rats. Immunohistochemistry, quantitative fluorescent RT-PCR, and Western blotting 3-5 days after plasmid injection were performed to measure the expression changes of HSP47 and collagen I. The results demonstrate an increase of HSP47 levels in vitro in 3T3 fibroblast cells and in vivo in diabetic rat after treatment with plasmids expressing HSP47. The level of collagen I around the wound during the repair process was higher in the treated group than that in the control group, indicating that the constructs may have use in human gene therapy in cases of impaired skin wound healing in diabetes.     

Cytoplasmic Domain Interactions of Syndecan-1 and Syndecan-4 with α6β4 Integrin Mediate Human Epidermal Growth Factor Receptor (HER1 and HER2)-dependent Motility and Survival

Epithelial cells are highly dependent during wound healing and tumorigenesis on the α6β4 integrin and its association with receptor tyrosine kinases. Previous work showed that phosphorylation of the β4 subunit upon matrix engagement depends on the matrix receptor syndecan (Sdc)-1 engaging the cytoplasmic domain of the β4 integrin and coupling of the integrin to human epidermal growth factor receptor-2 (HER2). In this study, HER2-dependent migration activated by matrix engagement is compared with migration stimulated by EGF. We find that whereas HER2-dependent migration depends on Sdc1, EGF-dependent migration depends on a complex consisting of human epidermal growth factor receptor-1 (HER1, commonly known as EGFR), α6β4, and Sdc4. The two syndecans recognize distinct sites at the extreme C terminus of the β4 integrin cytoplasmic domain. The binding motif in Sdc1 is QEEXYX, composed in part by its syndecan-specific variable (V) region and in part by the second conserved (C2) region that it shares with other syndecans. A cell-penetrating peptide containing this sequence competes for HER2-dependent epithelial migration and carcinoma survival, although it is without effect on the EGFR-stimulated mechanism. β4 mutants bearing mutations specific for Sdc1 and Sdc4 recognition act as dominant negative mutants to block cell spreading or cell migration that depends on HER2 or EGFR, respectively. The interaction of the α6β4 integrin with the syndecans appears critical for it to be utilized as a signaling platform; migration depends on α3β1 integrin binding to laminin 332 (LN332; also known as laminin 5), whereas antibodies that block α6β4 binding are without effect. These findings indicate that specific syndecan family members are likely to have key roles in α6β4 integrin activation by receptor tyrosine kinases.     

In vitro study of the impact of mechanical tension on the dermal fibroblast phenotype in the context of skin wound healing

Skin wound healing is finely regulated by both matrix synthesis and degradation which are governed by dermal fibroblast activity. Actually, fibroblasts synthesize numerous extracellular matrix proteins (i.e., collagens), remodeling enzymes and their inhibitors. Moreover, they differentiate into myofibroblasts and are able to develop endogenous forces at the wound site. Such forces are crucial during skin wound healing and have been widely investigated. However, few studies have focused on the effect of exogenous mechanical tension on the dermal fibroblast phenotype, which is the objective of the present paper. To this end, an exogenous, defined, cyclic and uniaxial mechanical strain was applied to fibroblasts cultured as scratch-wounded monolayers. Results showed that fibroblasts? response was characterized by both an increase in procollagen type-I and TIMP-1 synthesis, and a decrease in MMP-1 synthesis. The monitoring of scratch-wounded monolayers did not show any decrease in kinetics of the filling up when mechanical tension was applied. Additional results obtained with proliferating fibroblasts and confluent monolayer indicated that mechanical tension-induced response of fibroblasts depends on their culture conditions. In conclusion, mechanical tension leads to the differentiation of dermal fibroblasts and may increase their wound-healing capacities. So, the exogenous uniaxial and cyclic mechanical tension reported in the present study may be considered in order to improve skin wound healing.