Fig 1.
Topical BRAF inhibition accelerates wound healing in an excisional diabetic wound model.
(a) Representative images of vehicle-treated and vemurafenib (vemurafenib)-treated wounds on day 0 and day 7. (b) Wound closure scores of vehicle-treated and vemurafenib-treated mice on day 0 and day 7 (*P = 0.04, n = 6). (c) Representative H&E and pERK and Ki67 staining IHC images in the presence and absence of vemurafenib by day 7. (d) Box plots representing the quantification of pERK+ (*P = 0.01) and Ki67+ (NS) cells, for control wounds versus treated wounds on day 7. NS, not significant; H&E, hematoxylin and eosin; IHC, immunohistochemistry. Error bars in b and d, mean ± s.d. Bar graphs represent one experiment with 6 replicates per group.
Fig 2.
Topical vemurafenib promotes wound closure in diabetic wounds by Day 16.
(a) Representative images of vehicle- and vemurafenib-treated wounds on day 0 and day 16. (b) Epidermal+dermal thickness quantification of vehicle- and vemurafenib-treated wounds by Day 16 (*P = 0.02). (c) Representative H&E, pERK and Ki67 staining IHC images in the presence and absence of vemurafenib by day 16. (d) Scatter plots representing the quantification of pErk+ (P = 0.04), Ki67 (*P = 0.015); for control wounds versus treated wounds on day 16. (e) Heatmap of representative genes that are differentially expressed either the vemurafenib-treated or control wounds on Day 16. (f) Pathway/gene ontology enrichment analyses of the full list of differentially expressed genes (at least 1.5-fold difference) using ENRICHR. Enriched gene sets (adjusted P ≤ 0.1, Jensen: Jensen tissues database, MGA: mouse gene atlas, ARCHS4: tissue RNAseq dataset, GO: gene ontology database) are listed. NS, not significant; H&E, hematoxylin and eosin; IHC, immunohistochemistry. Error bars in b and d, mean ± s.d. Bar graphs represent one experiment with 5 replicates per group.
Fig 3.
Topical vemurafenib promotes hair follicle regeneration in db/db wounds.
(a) Representative H&E images of vehicle-treated and vemurafenib (vemurafenib)-treated wounds on day 0 and day 16 in diabetic mice. (b) Scatter plots representing the quantification of hair follicles within the wound area (****P = 0.0001) for control wounds versus treated wounds on day 16 in diabetic mice. (c) Representative Sox-9 staining IHC images in the presence and absence of vemurafenib in db/db mice wounds by day 16. (d) Scatter plot representing the quantification of Sox-9+ cells (*P = 0.02, n = 4) for control wounds versus treated wounds on day 16. (e) Representative CD34, K15 and DAPI staining IF images in the presence and absence of vemurafenib in db/db mice wounds by Day 16. (f) Scatter plots representing the quantification of CD34+ K15+ cells for control wounds versus treated wounds (*P = 0.02). Error bars in b, d and f mean ± s.d. Bar graphs represent one experiment with 4–9 replicates per group.
Fig 4.
Topical vemurafenib promotes hair follicle regeneration in Balb/c wounds.
(a) Representative H&E images of vehicle-treated and vemurafenib (vemurafenib)-treated wounds on day 0 and day 14 in Balb/c mice. (b) Scatter plots representing the quantification of hair follicles within the wound area (*P = 0.02) for control wounds versus treated wounds on day 14 in Balb/c mice. (c) Representative Sox-9 staining IHC images in the presence and absence of vemurafenib in Balb/c mice wounds by day 14. (d) Scatter plots representing the quantification of Sox-9+ cells (*P = 0.02, n = 4) for control wounds versus treated wounds on day 14. (e) Representative CD34, K15 and DAPI staining immunofluorescent images in the presence and absence of vemurafenib in Balb/c mice wounds by day 14. (f) Scatter plots representing the quantification of K15+ with surrounding CD34+ stroma for control wounds versus treated wounds (*P = 0.02). Error bars in b, d and f mean ± s.d. Bar graphs represent one experiment with 4–5 replicates per group.
Fig 5.
Topical vemurafenib enhances FZD7 and promotes β-catenin signaling which is reversed by MEK inhibition.
(a and c) Representative FZD7 staining IHC images in the presence and absence of vemurafenib in (a) db/db mice and (c) Balb/c wounds by day 16. (b and d) Scatter plots representing the quantification of FZD7+ cells (*P = 0.02, n = 4) for control wounds versus treated db/db (b) and Balb/c wounds (d) on day 16. (e) Representative images of Sox-9 IHC staining of vehicle-, vemurafenib-, vemurafenib plus trametinib (T), trametinib alone (T) treated C3H wounds on day 6 (f) Scatter plots representing the quantification of SOX-9+ cells (P = 0.02; Vehicle vs vemurafenib and vemurafenib vs V+T, n = 4) for control wounds versus treated C3H wounds on day 6. (g) Representative images of β-catenin IHC staining of vehicle-, vemurafenib-, vemurafenib plus trametinib (V+T), trametinib alone (T)- treated C3H wounds on day 6. (h) Scatter plots representing the quantification of β-catenin+ cells (*P = 0.02, vemurafenib vs V+T, n = 4; ns, vehicle vs vemurafenib and vemurafenib vs T) for control wounds versus treated C3H wounds on day 6. (i) Representative images of active β-catenin IHC staining of vehicle-, vemurafenib-, vemurafenib plus trametinib (V+T), trametinib alone (T)- treated C3H wounds on day 6. (j) Scatter plots representing the quantification of active β-catenin + cells (*P = 0.02; Vehicle vs vemurafenib and vemurafenib vs V+T; n = 4) for control wounds versus treated C3H wounds on day 6. (k) Representative images of TCF1/TCF7 IHC staining of vehicle-, vemurafenib-, vemurafenib plus trametinib (T), trametinib (T)- treated wounds on day 6. (l) Scatter plots representing the quantification of TCF1/TCF7+ cells (*P = 0.02; vehicle vs vemurafenib and vemurafenib vs V+T; n = 4) for control wounds versus treated C3H wounds on day 6. Error bars in b, d, f, h, j and l mean ± s.d. Bar graphs represent one experiment with 3–4 replicates per group.
Fig 6.
Evidence of hair follicle neogenesis.
(a and b) Skin adjacent to a wound demonstrates growth of normal hair follicles and sebaceous glands, embedding in the subcutaneous adipose tissue whereas skin in the center of the wound demonstrates regeneration of many hair follicles, some with sebaceous glands with random orientation in the dermis, directly underlying a hyperplastic, recently reepithelialized epidermis. (c and d) Sox9 and active β-catenin immunohistochemistry showed adjacent skin with typical appearance of hair follicles (left of dotted lines) and wounded tissue (right of dotted lines) with atypical hair follicles and germs expressing Sox9 and active β-catenin in different orientation from normal hair follicles. (e and f) High power view showing the distinct appearance of hair follicles in normal adjacent skin (e) and within the center of a wound (f).