Simultaneous silencing of TGF-β1 and COX-2 reduces human skin hypertrophic scar through activation of fibroblast apoptosis

Abstract

Excessive skin scars due to elective operations or trauma represent a challenging clinical problem. Pathophysiology of hypertrophic scars entails a prolonged inflammatory and proliferative phase of wound healing. Over expression of TGF-β1 and COX-2 play key regulatory roles of the aberrant fibrogenic responses and proinflammatory mediators. When we silenced TGF-β1 and COX-2 expression simultaneously in primary human fibroblasts, a marked increase in the apoptotic cell population occurred in contrast to those only treated with either TGF-β1 or COX-2 siRNA alone. Furthermore, using human hypertrophic scar and skin graft implant models in mice, we observed significant size reductions of the implanted tissues following intra-scar administration of TGF-β1/COX-2 specific siRNA combination packaged with Histidine Lysine Polymer (HKP). Gene expression analyses of those treated tissues revealed silencing of the target gene along with down regulations of pro-fibrotic factors such as α-SMA, hydroxyproline acid, Collagen 1 and Collagen 3. Using TUNEL assay detection, we found that the human fibroblasts in the implanted tissues treated with the TGF-β1/COX-2siRNAs combination exhibited significant apoptotic activity. Therefore we conclude that a synergistic effect of the TGF-β1/COX-2siRNAs combination contributed to the size reductions of the hypertrophic scar implants, through activation of fibroblast apoptosis and re-balancing between scar tissue deposition and degradation.

Inhibition of Wound TGF Beta-1 by Celecoxib: A Possible Therapeutic Route for Scar Free Wound

“…While the reduction of inflammatory response with early wound closure achieved by Celecoxib application at early time point could be attributed to its direct anti-inflammatory effect [62][63][64]. The improved scar quality could be attributed to an indirect antifibrotic effect of Celecoxib through reducing wound TGF Beta-1 [61,62] with subsequent reduction in collagen deposition and scar formation [89].…”

Biological approaches for hypertrophic scars

“…Activation of TGF‐β1 assembles and TGF‐β‐receptor complex activated the Smad2/3 and JNK signalling in fibroblasts and exhibited the biological behaviour of myofibroblasts . On the contrary, silencing TGF‐β1 resulted in lower collagen synthesis and alleviative scarring which suggested it was a potential therapeutic target for limiting scar formation . High levels of TGF‐β1 may stimulate the activation of detrimental myofibroblasts, but blocking TGF‐β1 completely shows spontaneous skin inflammation and defective vasculogenesis in TGF‐β1 knockout mouse, which indicated that maintaining a certain level of TGF‐β1 in wounds was essential.…”

“…149 On the contrary, silencing TGF-β1 resulted in lower collagen synthesis and alleviative scarring which suggested it was a potential therapeutic target for limiting scar formation. 150 High levels of TGF-β1 may stimulate the activation of detrimental myofibroblasts, but blocking TGF-β1 completely shows spontaneous skin inflammation and defective vasculogenesis in TGF-β1 knockout mouse, 151 which indicated that maintaining a certain level of TGF-β1 in wounds was essential. What we need to do now is to pay attention to the expression level of TGF-β1 in scarfree repair wounds, and to the temporal and spatial dynamics of TGF-β1 to control the homeostasis of reepithelialisation, vascularization, and inflammation.…”

Effect of n-3 long-chain polyunsaturated fatty acids on wound healing using animal models – a review

“…Periodically collected samples of the granulation tissue from wound sites using predominantly a rodent model are usually evaluated (Nascimento and Costa 2006;Gercek et al 2007;Otranto et al 2010;dos Santos Rosa et al 2014;Ramanathan et al 2017;Sathyanarayanan et al 2017;Zhou et al 2017). As far as histological preparations are concerned, samples of the healing tissue including ca 2 mm of skin surrounding the wound are usually taken, fixed in 10% buffered formalin, dehydrated by a gradual alcohol series, cleared in xylene, embedded in paraffin blocks, sectioned into a size of (usually) 5 µm thickness, stained with haematoxylin-eosin (H&E) and observed under light microscopy.…”

“…Rabbit polyclonal IgG (Ramanathan et al 2017) or mouse monoclonal antibody (Otranto et al 2010;dos Santos Rosa et al 2014) are usually used as a primary antibody for immunostaining. Goat anti-rabbit antibody (Zhou et al 2017) and biotinylated anti-rat antibody (dos Santos Rosa et al 2014) are frequently applied secondary antibodies in this context. The last mentioned authors (dos Santos Rosa et al 2014) also used an anti-mouse secondary antibody conjugated with Alexa Fluor 647 for the detection of epithelial cadherin (E-cadherin) when evaluating cutaneous wound healing in mice.…”