Journal: Pharmaceutics

Article Title: Continuous NPWT Regulates Fibrosis in Murine Diabetic Wound Healing

doi: 10.3390/pharmaceutics14102125

Figure Lengend Snippet: YAP, TAZ, En1 and CD26 levels. ( a ) Immunofluorescent staining of the wound bed. anti-YAP, anti-TAZ, anti-En1 and anti-CD26 with DAPI stained sections of the wound bed of the two groups on day 10. Scale bar = 100 µm. ( b ) Relative fluorescence intensity of YAP. YAP presence significantly differed between the two groups (NPWT: 2.3 ± 1.3 RFI vs. control:1 ± 0.6; p = 0.04). ( c ) Percentage of TAZ + ve cells. There were no differences in the presence of TAZ between the two groups. ( d ) Percentage of En1 + ve cells. En1 was significantly lower in the NPWT group (NPWT:2.8 ± 2.6% En1 + cells vs. control:37 ± 32% En1 + cells; p = 0.01). ( e ) Percentage of CD26 + ve cells. CD26 was significantly lower in the NPWT group (NPWT: 2.8 ± 2.6% CD26 + cells vs. control:37 ± 32% CD26 + cells; p < 0.0001). Number of circles/squares per bar equals sample size ( n = 7–10) with each circle/square representing the average of three HPF measurements. Where ns equals p > 0.05, * equals p ≤ 0.05, *** equals p ≤ 0.001 and **** equals p ≤ 0.0001.

Article Snippet: For immunofluorescent staining, de-paraffinized and rehydrated cross-sections were probed with the antibodies against YAP (1:200; PA1-46189, Invitrogen, Waltham, MA, USA), PDZ-binding motif (TAZ 1:300, NB110-58359SS, Novus Biologicals, Centennial, CO, USA), En1 (1:200, BS-11744R, Bioss Antibodies, Woburn, MA, USA), α-SMA (1:100; NBP1-30894, Novus Biologicals, Centennial, CO, USA), CD26 (1:150, 559652, BD Biosciences, Haryana, India), Fibronectin (1:400, NBP1-91258SS, Novus Biologicals, Centennial, CO, USA), Ki67 (1 ug/mL, ab15580, Abcam, Cambridge, UK), Heat shock protein 47 (Hsp47, 1:200, ab109117, Abcam, Cambridge, UK), Vimentin (1:200, ab92547, Abcam, Cambridge, UK) or S100A4 (1:200, ab197896, Abcam, Cambridge, UK) for 16 h at 4 °C.

Techniques: Staining, Fluorescence

Journal: Pharmaceutics

Article Title: Continuous NPWT Regulates Fibrosis in Murine Diabetic Wound Healing

doi: 10.3390/pharmaceutics14102125

Figure Lengend Snippet: Proposed mechanism of action of NPWT in fibrosis. The normal fibrotic response that occurs during wound healing, and which becomes upregulated in hypertrophic scarring, is summarized in this schematic. Mechanotransduction, in the form of increased tension between the cell and the extracellular matrix, is hypothesized to lead to an upregulation of YAP. Within the cytoplasm, YAP is bound to a-catenin via 14-3-3 which prevents its nuclear sequestration. Fibrosis-promoting signals upregulated caspase-3 which cleaves a-catenin, and allows YAP to translocate into the nucleus. YAP is then able to promote transcription of En1 which induces a switch of the fibroblast phenotype, from pro-regenerative to pro-fibrotic. The fibrotic response results in an increase in the conversion of pro-collagen to collagen. The increased collagen deposition and increase in scarring then further amplifies the loop. In this study, we demonstrate that although NPWT increased the expression of YAP, En1 was decreased. The decrease in Caspase-3 would help explain this de-coupling, as decreased caspase-3 would result in decreased cleavage of a-catenin, decreased nuclear sequestration of YAP and decreased En1 transcription, as identified in our study. A decreased fibrotic response would result in decreased collagen formation. Hsp47 which is required for pro-collagen to be converted to collagen was also identified as decreased in this study, further supporting the decoupling mechanism. YSP, yes-associated protein; EN1, Engrailed-1; RhoA–ROCK, RhoA and Rho-associated protein kinase.

Article Snippet: For immunofluorescent staining, de-paraffinized and rehydrated cross-sections were probed with the antibodies against YAP (1:200; PA1-46189, Invitrogen, Waltham, MA, USA), PDZ-binding motif (TAZ 1:300, NB110-58359SS, Novus Biologicals, Centennial, CO, USA), En1 (1:200, BS-11744R, Bioss Antibodies, Woburn, MA, USA), α-SMA (1:100; NBP1-30894, Novus Biologicals, Centennial, CO, USA), CD26 (1:150, 559652, BD Biosciences, Haryana, India), Fibronectin (1:400, NBP1-91258SS, Novus Biologicals, Centennial, CO, USA), Ki67 (1 ug/mL, ab15580, Abcam, Cambridge, UK), Heat shock protein 47 (Hsp47, 1:200, ab109117, Abcam, Cambridge, UK), Vimentin (1:200, ab92547, Abcam, Cambridge, UK) or S100A4 (1:200, ab197896, Abcam, Cambridge, UK) for 16 h at 4 °C.

Techniques: Expressing