Exclusive expression of KANK4 promotes myofibroblast mobility in keloid tissues.

Keloids are characterized by abnormal wound healing with excessive accumulation of extracellular matrix. Myofibroblasts are the primary contributor to extracellular matrix secretion, playing an essential role in the wound healing process. However, the differences between myofibroblasts involved in keloid formation and normal wound healing remain unclear. To identify the specific characteristics of keloid myofibroblasts, we initially assessed the expression levels of well-established myofibroblast markers, α-smooth muscle actin (α-SMA) and transgelin (TAGLN), in scar and keloid tissues (n = 63 and 51, respectively). Although myofibroblasts were present in significant quantities in keloids and immature scars, they were absent in mature scars. Next, we conducted RNA sequencing using myofibroblast-rich areas from keloids and immature scars to investigate the difference in RNA expression profiles among myofibroblasts. Among significantly upregulated 112 genes, KN motif and ankyrin repeat domains 4 (KANK4) was identified as a specifically upregulated gene in keloids. Immunohistochemical analysis showed that KANK4 protein was expressed in myofibroblasts in keloid tissues; however, it was not expressed in any myofibroblasts in immature scar tissues. Overexpression of KANK4 enhanced cell mobility in keloid myofibroblasts. Our results suggest that the KANK4-mediated increase in myofibroblast mobility contributes to keloid pathogenesis.

Photo-crosslinkable polyester microneedles as sustained drug release systems toward hypertrophic scar treatment.

Burn injuries can result in a significant inflammatory response, often leading to hypertrophic scarring (HTS). Local drug therapies e.g. corticoid injections are advised to treat HTS, although they are invasive, operator-dependent, extremely painful and do not permit extended drug release. Polymer-based microneedle (MN) arrays can offer a viable alternative to standard care, while allowing for direct, painless dermal drug delivery with tailorable drug release profile. In the current study, we synthesized photo-crosslinkable, acrylate-endcapped urethane-based poly(ε-caprolactone) (AUP-PCL) toward the fabrication of MNs. Physico-chemical characterization (1H-NMR, evaluation of swelling, gel fraction) of the developed polymer was performed and confirmed successful acrylation of PCL-diol. Subsequently, AUP-PCL, and commercially available PCL-based microneedle arrays were fabricated for comparative evaluation of the constructs. Hydrocortisone was chosen as model drug. To enhance the drug release efficiency of the MNs, Brij®35, a nonionic surfactant was exploited. The thermal properties of the MNs were evaluated via differential scanning calorimetry. Compression testing of the arrays confirmed that the MNs stay intact upon applying a load of 7 N, which correlates to the standard dermal insertion force of MNs. The drug release profile of the arrays was evaluated, suggesting that the developed PCL arrays can offer efficient drug delivery for up to two days, while the AUP-PCL arrays can provide a release up to three weeks. Finally, the insertion of MN arrays into skin samples was performed, followed by histological analysis demonstrating the AUP-PCL MNs outperforming the PCL arrays upon providing pyramidical-shaped perforations through the epidermal layer of the skin.

AUP-PCL MN arrays provide long-term transdermal drug delivery of hydrocortisoneAUP-PCL-based MN arrays provide superior drug release profiles compared to PCL MNsEffective skin penetration AUP-PCL-based MNs on skin was achieved.

[Influences and mechanism of extracellular vesicles from dermal papilla cells of mice on human hypertrophic scar fibroblasts].

Objective: To investigate the influences and mechanism of extracellular vesicles from dermal papilla cells (DPC-EVs) of mice on human hypertrophic scar fibroblasts (HSFs). Methods: The study was an experimental research. The primary dermal papilla cells (DPCs) of whiskers were extracted from 10 6-week-old male C57BL/6J mice and identified successfully. The DPC-EVs were extracted from the 3rd to 5th passage DPCs by ultracentrifugation, and the morphology was observed through transmission electron microscope and the particle diameter was detected by nanoparticle tracking analyzer (n=3) at 24 h after culture. The 3rd passage of HSFs were divided into DPC-EV group and phosphate buffer solution (PBS) group, which were cultured with DPC-EVs and PBS, respectively. The cell scratch test was performed and cell migration rate at 24 h after scratching was calculated (n=5). The cell proliferation levels at 0 (after 12 h of starvation treatment and before adding DPC-EVs or PBS), 24, 48, 72, and 96 h after culture were detected by using cell counting kit 8 (n=4). The protein expressions of α-smooth muscle actin (α-SMA) and collagen typeⅠ (ColⅠ) in cells at 24 h after culture were detected by immunofluorescence method and Western blotting, and the protein expression of Krüppel-like factor 4 (KLF4) in cells at 24 h after culture was detected by Western blotting. After the 3rd passage of HSFs were cultured with DPC-EVs for 24 h, the cells were divided into blank control group, KLF4 knockdown group, and KLF4 overexpression group according to the random number table. The cells in blank control group were only routinely cultured for 48 h. The cells in KLF4 knockdown group and KLF4 overexpression group were incubated with KLF4 knockdown virus for 24 h, then the cells in KLF4 knockdown group were routinely cultured for 24 h while the cells in KLF4 overexpression group were incubated with KLF4 overexpression virus for 24 h. The protein expressions of KLF4, α-SMA, and ColⅠ in cells were detected by Western blotting at 48 h after culture. Results: At 24 h after culture, the extracted DPC-EVs showed vesicular structure with an average particle diameter of 108.8 nm. At 24 h after scratching, the migration rate of HSFs in PBS group was (54±10)%, which was significantly higher than (29±8)% in DPC-EV group (t=4.37, P<0.05). At 48, 72, and 96 h after culture, the proliferation levels of HSFs in DPC-EV group were significantly lower than those in PBS group (with t values of 4.06, 5.76, and 6.41, respectively, P<0.05). At 24 h after culture, the protein expressions of α-SMA and ColⅠ of HSFs in DPC-EV group were significantly lower than those in PBS group, while the protein expression of KLF4 was significantly higher than that in PBS group. At 48 h after culture, compared with those in blank control group, the protein expression of KLF4 of HSFs in KLF4 knockdown group was down-regulated, while the protein expressions of α-SMA and ColⅠ were both up-regulated; compared with those in KLF4 knockdown group, the protein expression of KLF4 of HSFs in KLF4 overexpression group was up-regulated, while the protein expressions of ColⅠ and α-SMA were down-regulated. Conclusions: The DPC-EVs of mice can inhibit the proliferation and migration of human HSFs and significantly inhibit the expressions of fibrosis markers α-SMA and ColⅠ in human HSFs by activating KLF4.

Global proteomic analysis reveals lysine succinylation is involved in the pathogenesis of hypertrophic scar.

Lysine succinylation (Ksucc) is a recently identified posttranslational modification that is involved in many diseases. This study examined the role of Ksucc in the pathogenesis of hypertrophic scar (HS). The presence of Ksucc in human skin was measured by immunoblotting. Ksucc occurs in many skin proteins ranging from 25 to 250 kDa, and higher levels of Ksucc are found in HS skin than in normal skin. An immunoaffinity approach coupled with LC-MS/MS was used to characterize the first succinylome of human skin, and 159 Ksucc sites in 79 proteins were identified. Among these, there were 38 increased succinylated sites in 29 proteins but no decreased succinylated sites in HS compared with normal skin. A parallel reaction monitoring assay was performed to validate the results of the succinylome and showed that the levels of Ksucc in decorin and collagens, which are involved in the pathogenesis of HS, were increased in HS than in normal skin. In addition, increasing the level of Ksucc enhanced cell proliferation and upregulated the expression of fibrosis markers (α-SMA, COL1, and COL3) in human skin fibroblasts. Our results provide global insights into the functional role of Ksucc in hypertrophic scarring.

Parecoxib decreases cellular growth and Bcl-2 protein levels in primary cultures of keloid fibroblasts.

Keloids seem to overexpress cyclo-oxygenase-2 (COX-2), suggesting a role in its deregulated pathway in inducing an altered epithelial-mesenchymal interaction, which may be responsible for the overgrowth of dermal components resulting in scars or keloid lesions. This study aimed to evaluate the effect of Parecoxib, a COX-2 inhibitor, on cell growth in fibroblast primary cultures obtained from human keloid tissues. Tissue explants were obtained from patients who underwent intralesional excision of untreated keloids; central fractions were isolated from keloid tissues and used for establishing distinct primary cultures. Appropriate aliquots of Parecoxib, a COX-2 inhibitor were diluted to obtain the concentration used in the experimental protocols in vitro (1, 10 or 100 µM). Treatment with Parecoxib (at all concentrations) caused a significant decrease in cellular growth from 24 hours onwards, and with a maximum at 72 hours (P < .02). Moreover, at 72 hours Parecoxib significantly reduced cellular vitality. Parecoxib treatment also induced an increase in fragmented nuclei with a maximum effect at 100 µM and a significant decrease in Bcl-2 and an increase in activated caspase-3 protein levels at 72 hours compared with control untreated cultures. Our findings suggest a potential use of the COX-2 inhibitor, Parecoxib, as the therapy for keloids.

Differential Photosensitivity of Fibroblasts Obtained from Normal Skin and Hypertrophic Scar Tissues.

It is unclear whether normal human skin tissue or abnormal scarring are photoreceptive. Therefore, this study investigated photosensitivity in normal skin tissue and hypertrophic scars. The expression of opsins, which are photoreceptor proteins, in normal dermal fibroblasts (NDFs) and hypertrophic scar fibroblasts (HSFs) was examined. After exposure to blue light (BL), changes in the expression levels of αSMA and clock-related genes, specifically PER2 and BMAL1, were examined in both fibroblast types. Opsins were expressed in both fibroblast types, with OPN3 exhibiting the highest expression levels. After peripheral circadian rhythm disruption, BL induced rhythm formation in NDFs. In contrast, although HSFs showed changes in clock-related gene expression levels, no distinct rhythm formation was observed. The expression level of αSMA was significantly higher in HSFs and decreased to the same level as that in NDFs upon BL exposure. When OPN3 knocked-down HSFs were exposed to BL, the reduction in αSMA expression was inhibited. This study showed that BL exposure directly triggers peripheral circadian synchronization in NDFs but not in HSFs. OPN3-mediated BL exposure inhibited HSFs. Although the current results did not elucidate the relationship between peripheral circadian rhythms and hypertrophic scars, they show that BL can be applied for the prevention and treatment of hypertrophic scars and keloids.

PTEN hinders the formation of scars by regulating the levels of proteins in the extracellular matrix and promoting the apoptosis of dermal fibroblasts through Bcl-xL.

Hypertrophic scar (HS) is a dermatological condition characterized by an excessive accumulation of proteins in the extracellular matrix (ECM) and an elevated cell count. The development of HS is thought to be linked to the disruption of dermal fibroblast proliferation and apoptosis. The processes of cell proliferation and apoptosis are notably influenced by PTEN. However, the precise mechanisms by which PTEN regulates hypertrophic scar fibroblasts (HSFs) and its overall role in scar formation are still not fully understood. The objective of this study was to investigate the influence of PTEN on hypertrophic scars(HS) and its function in the regulation of scar formation, with the aim of identifying a pivotal molecular target for scar treatment. Our results demonstrate that the overexpression of PTEN (AdPTEN) significantly suppressed the expression of type I collagen (Col I), type III collagen (Col III), and alpha smooth muscle actin (α-SMA) in HSFs. Furthermore, it was observed that the introduction of AdPTEN resulted in the suppression of Bcl-xL expression, which consequently led to an increase in the apoptosis of HSFs. Similarly, in the inhibition of collagens expression and subsequent increase in HSF apoptosis were also observed upon silencing Bcl-xL (sibcl-xL). Additionally, the in vitro model demonstrated that both AdPTEN and sibcl-xL were effective in reducing the contraction of FPCL. The findings of our study provide validation for the role of PTEN in inhibiting the development of hypertrophic scars (HS) by modulating the expression of extracellular matrix (ECM) proteins and promoting apoptosis in hypertrophic scar fibroblasts (HSFs) via Bcl-xL. These results indicate that PTEN and Bcl-xL may hold promise as potential molecular targets for therapeutic interventions aimed at managing hypertrophic scars.

Effects of miR-214 on adenosine A2A receptor and carboxymethyl chitosan nanoparticles on the function of keloid fibroblasts and their mechanisms.

This work prepared and investigated the impact of carboxymethyl chitosan nanoparticles (MC-NPs) on the proliferative capability of keloid fibroblasts (KFBs) while analyzing the mechanistic roles of miR-214 and adenosine A2A receptor (A2AR) in fibroblasts within hypertrophic scars. MC-NPs were synthesized through ion cross-linking, were characterized using transmission electron microscopy (TEM) and laser particle size scattering. The influence of MC-NPs on the proliferation capacity of KFBs was assessed using the MTT method. Changes in the expression levels of miR-214 and A2AR in KFBs, normal skin fibroblasts (NFBs), hypertrophic scar tissue, and normal skin tissue were analyzed. KFBs were categorized into anti-miR-214, anti-miR-NC, miR-214 mimics, miR-NC, si-A2AR, si-con, anti-miR-214+ si-con, and anti-miR-214+ si-A2AR groups. Bioinformatics target prediction was conducted to explore the interaction between miR-214 and A2AR. Real-time quantitative PCR and immunoblotting (WB) were employed to detect the expression levels of miR-214, A2AR, apoptotic protein Bax, and TGF-ß in different cells. Cell counting kit-8 (CCK8) and flow cytometry were employed to assess cell proliferation activity and apoptosis. The results indicated that MC-NPs exhibited spherical particles with an average diameter of 236.47 ± 4.98 nm. The cell OD value in the MC-NPs group was lower than that in KFBs (P < 0.05). The mRNA levels of miR-214 in KFBs and hypertrophic scar tissue were lower than those in NFBs and normal tissue (P < 0.001), while the mRNA and protein levels of A2AR were significantly elevated (P < 0.05). Compared to the control group and anti-miR-NC, the anti-miR-214 group showed significantly increased cell OD values and Bcl-2 protein expression (P < 0.001), decreased levels of apoptotic gene Bax protein, TGF-ß gene mRNA, and protein expression (P < 0.001). Continuous complementary binding sites were identified between miR-214 and A2AR. Compared to the control group, the si-A2AR group exhibited a significant decrease in A2AR gene mRNA and protein expression levels (P < 0.001), reduced cell viability (P < 0.001), increased apoptosis rate (P < 0.001), and a significant elevation in TGF-ß protein expression (P < 0.001). miR-214 targetedly regulated the expression of A2AR, inducing changes in TGF-ß content, promoting the proliferation of keloid fibroblasts, and inhibiting cell apoptosis.

Hydroxypyridone anti-fungals selectively induce myofibroblast apoptosis in an in vitro model of hypertrophic scars.

Hypertrophic scars are a common complication of burn injuries, yet there are no medications to prevent their formation. During scar formation, resident fibroblasts are transformed to myofibroblasts which become resistant to apoptosis. Previously, we have shown that hydroxypyridone anti-fungals can inhibit transformation of fibroblasts, isolated from hypertrophic scars, to myofibroblasts. This study aimed to investigate if these drugs can also target myofibroblast persistence. Primary human dermal fibroblasts, derived from burn scar tissue, were exposed to transforming growth factor beta-1 (TGF-ß1) for 72 h to induce myofibroblast transformation. The cells were then incubated with three hydroxypyridone anti-fungals (ciclopirox, ciclopirox ethanolamine and piroctone olamine; 0.03-300 µM) for a further 72 h. The In-Cell ELISA method was utilised to quantify myofibroblast transformation by measuring alpha-smooth muscle actin (α-SMA) expression and DRAQ5 staining, to measure cell viability. TUNEL staining was utilised to assess if the drugs could induce apoptosis. When given to established myofibroblasts, the three hydroxypyridones did not reverse myofibroblast transformation, but instead elicited a concentration-dependent decrease in cell viability. TUNEL staining confirmed that the hydroxypyridone anti-fungals induced apoptosis in established myofibroblasts. This is the first study to show that hydroxypyridone anti-fungals are capable of inducing apoptosis in established myofibroblasts. Together with our previous results, we suggest that hydroxypyridone anti-fungals can prevent scar formation by preventing the formation of new myofibroblasts and by reducing the number of existing myofibroblasts.

TEM1/endosialin/CD248 promotes pathologic scarring and TGF-ß activity through its receptor stability in dermal fibroblasts.

BACKGROUND: Pathologic scars, including keloids and hypertrophic scars, represent a common form of exaggerated cutaneous scarring that is difficult to prevent or treat effectively. Additionally, the pathobiology of pathologic scars remains poorly understood. We aim at investigating the impact of TEM1 (also known as endosialin or CD248), which is a glycosylated type I transmembrane protein, on development of pathologic scars. METHODS: To investigate the expression of TEM1, we utilized immunofluorescence staining, Western blotting, and single-cell RNA-sequencing (scRNA-seq) techniques. We conducted in vitro cell culture experiments and an in vivo stretch-induced scar mouse model to study the involvement of TEM1 in TGF-ß-mediated responses in pathologic scars. RESULTS: The levels of the protein TEM1 are elevated in both hypertrophic scars and keloids in comparison to normal skin. A re-analysis of scRNA-seq datasets reveals that a major profibrotic subpopulation of keloid and hypertrophic scar fibroblasts greatly expresses TEM1, with expression increasing during fibroblast activation. TEM1 promotes activation, proliferation, and ECM production in human dermal fibroblasts by enhancing TGF-ß1 signaling through binding with and stabilizing TGF-ß receptors. Global deletion of Tem1 markedly reduces the amount of ECM synthesis and inflammation in a scar in a mouse model of stretch-induced pathologic scarring. The intralesional administration of ontuxizumab, a humanized IgG monoclonal antibody targeting TEM1, significantly decreased both the size and collagen density of keloids. CONCLUSIONS: Our data indicate that TEM1 plays a role in pathologic scarring, with its synergistic effect on the TGF-ß signaling contributing to dermal fibroblast activation. Targeting TEM1 may represent a novel therapeutic approach in reducing the morbidity of pathologic scars.

MiR-141-3p-Functionalized Exosomes Loaded in Dissolvable Microneedle Arrays for Hypertrophic Scar Treatment.

Hypertrophic scar (HS) is a common fibroproliferative disease caused by abnormal wound healing after deep skin injury. However, the existing approaches have unsatisfactory therapeutic effects, which promote the exploration of newer and more effective strategies. MiRNA-modified functional exosomes delivered by dissolvable microneedle arrays (DMNAs) are expected to provide new hope for HS treatment. In this study, a miRNA, miR-141-3p, which is downregulated in skin scar tissues and in hypertrophic scar fibroblasts (HSFs), is identified. MiR-141-3p mimics inhibit the proliferation, migration, and myofibroblast transdifferentiation of HSFs in vitro by targeting TGF-ß2 to suppress the TGF-ß2/Smad pathway. Subsequently, the engineered exosomes encapsulating miR-141-3p (miR-141-3pOE -Exos) are isolated from adipose-derived mesenchymal stem cells transfected with Lv-miR-141-3p. MiR-141-3pOE -Exos show the same inhibitive effects as miR-141-3p mimics on the pathological behaviors of HSFs in vitro. The DMNAs for sustained release of miR-141-3pOE -Exos are further fabricated in vivo. MiR-141OE -Exos@DMNAs effectively decrease the thickness of HS and improve fibroblast distribution and collagen fiber arrangement, and downregulate the expression of α-SMA, COL-1, FN, TGF-ß2, and p-Smad2/3 in the HS tissue. Overall, a promising, effective, and convenient exosome@DMNA-based miRNA delivery strategy for HS treatment is provided.

Comprehensive modular analyses of scar subtypes illuminate underlying molecular mechanisms and potential therapeutic targets.

Pathological scarring resulting from traumas and wounds, such as hypertrophic scars and keloids, pose significant aesthetic, functional and psychological challenges. This study provides a comprehensive transcriptomic analysis of these conditions, aiming to illuminate underlying molecular mechanisms and potential therapeutic targets. We employed a co-expression and module analysis tool to identify significant gene clusters associated with distinct pathophysiological processes and mechanisms, notably lipid metabolism, sebum production, cellular energy metabolism and skin barrier function. This examination yielded critical insights into several skin conditions including folliculitis, skin fibrosis, fibrosarcoma and congenital ichthyosis. Particular attention was paid to Module Cluster (MCluster) 3, encompassing genes like BLK, TRPV1 and GABRD, all displaying high expression and potential implications in immune modulation. Preliminary immunohistochemistry validation supported these findings, showing elevated expression of these genes in non-fibrotic samples rich in immune activity. The complex interplay of different cell types in scar formation, such as fibroblasts, myofibroblasts, keratinocytes and mast cells, was also explored, revealing promising therapeutic strategies. This study underscores the promise of targeted gene therapy for pathological scars, paving the way for more personalised therapeutic approaches. The results necessitate further research to fully ascertain the roles of these identified genes and pathways in skin disease pathogenesis and potential therapeutics. Nonetheless, our work forms a strong foundation for a new era of personalised medicine for patients suffering from pathological scarring.

Transdermal Transfersome Nanogels Control Hypertrophic Scar Formation via Synergy of Macrophage Phenotype-Switching and Anti-Fibrosis Effect.

Hypertrophic scar (HS), which results from prolonged inflammation and excessive fibrosis in re-epithelialized wounds, is one of the most common clinical challenges. Consequently, sophisticated transdermal transfersome nanogels (TA/Fu-TS) are prepared to control HS formation by synergistically inhibiting inflammation and suppressing fibrosis. TA/Fu-TSs have unique structures comprising hydrophobic triamcinolone acetonide (TA) in lipid multilayers and hydrophilic 5-fluorouracil in aqueous cores, and perform satisfactorily with regard to transdermal co-delivery to macrophages and HS fibroblasts in emerging HS tissues. According to the in vitro/vivo results, TA/Fu-TSs not only promote macrophage phenotype-switching to inhibit inflammation by interleukin-related pathways, but also suppress fibrosis to remodel extracellular matrix by collagen-related pathways. Therefore, TA/Fu-TSs overcome prolonged inflammation and excessive fibrosis in emerging HS tissues, and provide an effective therapeutic strategy for controlling HS formation via their synergy of macrophage phenotype-switching and anti-fibrosis effect.

Effect of Silicone Patch Containing Metal-organic Framework on Hypertrophic Scar Suppression.

BACKGROUND/AIM: Hypertrophic scars (HS) are an abnormal cutaneous condition of wound healing characterized by excessive fibrosis and disrupted collagen deposition. This study assessed the potential of a silicone patch embedded with chemically stable zirconium-based metal-organic frameworks (MOF)-808 structures to mitigate HS formation using a rabbit ear model. MATERIALS AND METHODS: A silicone patch was strategically engineered by incorporating Zr-MOF-808, a composite structure comprising metal ions and organic ligands. Structural integrity of the Zr-MOF-808 silicone patch was validated using scanning electron microscopy and X-ray diffraction analysis. The animals were divided into three groups: a control, no treatment group (Group 1), a silicone patch treatment group (Group 2), and a group treated with a 0.2% loaded Zr-MOF-808 silicone patch (Group 3). HS suppression effects were quantified using scar elevation index (SEI), dorsal skin thickness measurements, and myofibroblast protein expression. RESULTS: Histopathological examination of post-treatment HS samples revealed substantial reductions in SEI (34.6%) and epidermal thickness (49.5%) in Group 3. Scar hyperplasia was significantly diminished by 53.5% (p<0.05), while collagen density declined by 15.7% in Group 3 compared to Group 1. Western blot analysis of protein markers, including TGF-ß1, collagen-1, and α-SMA, exhibited diminished levels by 8.8%, 12%, and 21.3%, respectively, in Group 3, and substantially higher levels by 21.9%, 27%, and 39.9%, respectively, in Group 2. On the 35th day post-wound generation, Zr-MOF-808-treated models exhibited smoother, less conspicuous, and flatter scars. CONCLUSION: Zr-MOF-808-loaded silicone patch reduced HS formation in rabbit ear models by inducing the proliferation and remodeling of the wound healing process.

The effects of Shikonin on the hypertrophic scar of rabbit ears via the TLR4/NF-κB signaling pathway.

As a traditional Chinese medicine, Zihuang Shengji Ointment has obvious effects on promoting postoperative wound healing and reducing scar formation in clinical application. Shikonin is the major phytochemical in Zihuang Shengji Ointment. As a kind of naphquinone compound with anti-tumor, anti-viral, anti-inflammatory, anti-bacterial and other biological activities extracted from Lithospermum erythrorhizon, shikonin exerts an important role in many diseases. Shikonin has impacts on the development of hypertrophic scars (HS), however, these effects are yet mostly unknown. As a result, we created the Newland white rabbit ear HS model, administered shikonin to it, and then assessed scar hypertrophy using HE and VG staining. The degree of scarring is assessed by HI, NA, as well as AA. The expression levels of collagen I, collagen III, as well as α-SMA as well as fibroblast proliferation, are also measured using real-time PCR, immunohistochemistry, and western blot. TUNEL tests are used to assess fibroblast apoptosis. In our work, HE staining and VG staining showed that the shikonin-treated group had normal bundles of collagen fibers and regular fibroblasts. Shikonin suppresses the production of HS, according to histopathological features, HI, NA, and AA measures. Shikonin also causes fibroblast apoptosis and lowers the production of α-SMA, collagen I, as well as collagen III in the HS rat. Notably, we discover that NF-κB activation and TLR4 activity are inhibited by shikonin. Overall, the results show that the signaling pathway of TLR4/NF-κB is modulated by shikonin's inhibitory effect on scar formation, which represses the levels of collagen I, collagen III, α-SMA, as well as fibroblasts.

Shikonin promotes hypertrophic scar repair by autophagy of hypertrophic scar-derived fibroblasts.

PURPOSE: To investigate the Shikonin (SHI) induce autophagy of hypertrophic scar-derived fibroblasts (HSFs) and the mechanism of which in repairing hypertrophic scar. METHODS: This study showed that SHI induced autophagy from HSFs and repaired skin scars through the AMPK/mTOR pathway. Alamar Blue and Sirius red were used to identify cell activity and collagen. Electron microscopy, label-free quantitative proteomic analysis, fluorescence and other methods were used to identify autophagy. The differences in the expression of autophagy and AMPK/mTOR pathway-related proteins after SHI treatment were quantitatively analyzed by Western blots. A quantitative real-time polymerase chain reaction assay was used to detect the expression of LC3, AMPK and ULK after adding chloroquine (CQ) autophagy inhibitor. RESULTS: After treatment with SHI for 24 hours, it was found that the viability of HSFs was significantly reduced, the protein expression of LC3-II/LC3-I and Beclin1 increased, while the protein expression of P62 decreased. The expression of phosphorylated AMPK increased and expression of phosphorylated mTOR decreased. After the use of CQ, the cell autophagy caused by SHI was blocked. The key genes LC3 and P62 were then reexamined by immunohistochemistry using a porcine full-thickness burn hypertrophic scar model, and the results verified that SHI could induce autophagy in vivo. CONCLUSIONS: These findings suggested that SHI promoted autophagy of HSFs cells, and the potential mechanism may be related to the AMPK/mTOR signal pathway, which provided new insights for the treatment of hypertrophic scars.

Heterogeneous response to TGF-ß1/3 isoforms in fibroblasts of different origins: implications for wound healing and tumorigenesis.

Identification of therapeutic targets for treating fibrotic diseases and cancer remains challenging. Our study aimed to investigate the effects of TGF-ß1 and TGF-ß3 on myofibroblast differentiation and extracellular matrix deposition in different types of fibroblasts, including normal/dermal, cancer-associated, and scar-derived fibroblasts. When comparing the phenotype and signaling pathways activation we observed extreme heterogeneity of studied markers across different fibroblast populations, even within those isolated from the same tissue. Specifically, the presence of myofibroblast and deposition of extracellular matrix were dependent on the origin of the fibroblasts and the type of treatment they received (TGF-ß1 vs. TGF-ß3). In parallel, we detected activation of canonical signaling (pSMAD2/3) across all studied fibroblasts, albeit to various extents. Treatment with TGF-ß1 and TGF-ß3 resulted in the activation of canonical and several non-canonical pathways, including AKT, ERK, and ROCK. Among studied cells, cancer-associated fibroblasts displayed the most heterogenic response to TGF-ß1/3 treatments. In general, TGF-ß1 demonstrated a more potent activation of signaling pathways compared to TGF-ß3, whereas TGF-ß3 exhibited rather an inhibitory effect in keloid- and hypertrophic scar-derived fibroblasts suggesting its clinical potential for scar treatment. In summary, our study has implications for comprehending the role of TGF-ß signaling in fibroblast biology, fibrotic diseases, and cancer. Future research should focus on unraveling the mechanisms beyond differential fibroblast responses to TGF-ß isomers considering inherent fibroblast heterogeneity.

Altered actin dynamics is possibly implicated in the inhibition of mechanical stimulation-induced dermal fibroblast differentiation into myofibroblasts.

The formation of hypertrophic scars and keloids is strongly associated with mechanical stimulation, and myofibroblasts are known to play a major role in abnormal scar formation. Wounds in patients with neurofibromatosis type 1 (NF1) become inconspicuous and lack the tendency to form abnormal scars. We hypothesized that there would be a unique response to mechanical stimulation and subsequent scar formation in NF1. To test this hypothesis, we investigated the molecular mechanisms of differentiation into myofibroblasts in NF1-derived fibroblasts and neurofibromin-depleted fibroblasts and examined actin dynamics, which is involved in fibroblast differentiation, with a focus on the pathway linking LIMK2/cofilin to actin dynamics. In normal fibroblasts, expression of α-smooth muscle actin (α-SMA), a marker of myofibroblasts, significantly increased after mechanical stimulation, whereas in NF1-derived and neurofibromin-depleted fibroblasts, α-SMA expression did not change. Phosphorylation of cofilin and subsequent actin polymerization did not increase in NF1-derived and neurofibromin-depleted fibroblasts after mechanical stimulation. Finally, in normal fibroblasts treated with Jasplakinolide, an actin stabilizer, α-SMA expression did not change after mechanical stimulation. Therefore, when neurofibromin was dysfunctional or depleted, subsequent actin polymerization did not occur in response to mechanical stimulation, which may have led to the unchanged expression of α-SMA. We believe this molecular pathway can be a potential therapeutic target for the treatment of abnormal scars.

Effects of ultrashort wave diathermy on skin wounds in rabbit ears.

PURPOSE: Ultrashort wave diathermy (USWD) is commonly used in diseases associated with osteoarticular and soft tissue injuries. However, while accelerating wound healing and preventing joint stiffness, there have been few reports on whether it leads to excessive hypertrophic scarring. The aim was to investigate the effects of different doses of USWD on hypertrophic scars. MATERIALS AND METHODS: A rabbit model of hypertrophic scars was used to determine which dose of USWD reduced scar hyperplasia. The scar thickness was calculated using Sirius red staining. All protein expression levels were determined by western blotting, including fibrosis, collagen deposition, and neoangiogenesis related proteins. Subsequently, flow cytometry and ELISAs were used to determine the proportions of macrophage and inflammatory levels. RESULTS: The wounds with USWD in histopathology showed the dermis was more markedly thickened in the 120 mA group, whereas the wounds with the 60 mA were less raised, comparing with the 0 mA; all detected protein levels were increased significantly, the 120 mA group comparing with the others, including heat shock, fibrosis, and neoangiogenesis, whereas the collagen deposition relative protein levels were decreased, the 60 mA group comparing with Sham group; Finally, in the proportion of macrophages and inflammatory levels the 120 mA group were the highest, and the group Sham was lower than group 60 mA. CONCLUSIONS: In hypertrophic scars, the 60 mA USWD could relieve scar formation and inflammatory reactions; however, higher doses could result in opposite consequences.

Anti-Fibrotic Effects of RF Electric Currents.

Hypertrophic scars and keloids are two different manifestations of excessive dermal fibrosis and are caused by an alteration in the normal wound-healing process. Treatment with radiofrequency (RF)-based therapies has proven to be useful in reducing hypertrophic scars. In this study, the effect of one of these radiofrequency therapies, Capacitive Resistive Electrical Transfer Therapy (CRET) on biomarkers of skin fibrosis was investigated. For this, in cultures of human myofibroblasts treated with CRET therapy or sham-treated, proliferation (XTT Assay), apoptosis (TUNEL Assay), and cell migration (Wound Closure Assay) were analyzed. Furthermore, in these cultures the expression and/or localization of extracellular matrix proteins such as α-SMA, Col I, Col III (immunofluorescence), metalloproteinases MMP1 and MMP9, MAP kinase ERK1/2, and the transcription factor NFκB were also investigated (immunoblot). The results have revealed that CRET decreases the expression of extracellular matrix proteins, modifies the expression of the metalloproteinase MMP9, and reduces the activation of NFκB with respect to controls, suggesting that this therapy could be useful for the treatment of fibrotic pathologies.