Negative Pressure Wound Therapy Regulates Yes-Associated Protein and Engrailed-1 Activity to Modulate Scarring in Diabetic Wounds

Background: Hypertrophic and keloid scars are debilitating sequelae of surgery that can have significant functional and aesthetic morbidity, substantially impacting patients’ quality of life. Such scarring may be managed through modification of the wound microenvironment via mechanical forces such as Negative Pressure Wound Therapy (NPWT).

In this study, we investigated the pathways underlying scarring and fibrosis in murine diabetic wounds treated with continuous NPWT.Methods:20 diabetic (db/db) mice received a 1x1cm2 full-thickness excisional dorsal skin wound. 10 mice received continuous NPWT (-125 mmHg, 24hours, 7 days; NPWT group), and 10 mice group received occlusive covering (Control group). The wounds were monitored for 10 days, and then the wound tissue was harvested for histological and molecular analysis.

Results: Immunofluorescent staining showed that the NPWT group displayed significantly lower Engrailed-1 expression (En1; 10±4 vs 2±1 En1+/HPF; p=0.0001), and significantly higher collagen deposition (34±4 vs 20±4 RFI; p< 0.0001), Yes-Associated Protein expression (YAP; 2±1 vs 1±1 RFI; p=0.03), cellular proliferation (15±7 vs 5±2 RFI; p=0.002), and fibronectin deposition (2±1 vs 1±1 RFI; p=0.03) of the wound bed. Furthermore, western blotting demonstrated that the NPWT group downregulated Caspase-3 (p=0.03) while upregulating YAP (p=0.02) and RhoA (p=0.03) compared to the Control group.

Conclusions:It is classically believed that the mechanical signaling transcriptional regulator YAP induces En1 fibroblasts and is involved in fibrosis. Past research has also illustrated that increased caspase-3 is linked to keloid formation. In this study, we demonstrated that NPWT affects cell proliferation and apoptosis, which may modulate scarring. More specifically, we found that NPWT promotes upregulation of YAP and downregulation of En1, implying that YAP and En1 may be uncoupled by the administration of mechanical forces such as NPWT. We also observed that NPWT decreased caspase-3. Thus, NPWT may limit scarring through its effects on fibroblast turnover.