Expression And Function Of Transient Receptor Potential Channel Canonical Type 3 In Hypertrophic Scar

Background and Aim:Hypertrophic scars?HTS?often lead to dysfunction,damaged appearance and psychological discomfort,and the current clinical treatments are not optimal.Myofibroblasts are overabundant in HTS tissue after burn injury.Myofibroblast proliferation is initiated by the formation of granulation tissue and the recruitment of activating myofibroblasts,which play central roles in extracellular matrix?ECM?deposition,re-epithelialization and eventual wound closure.The major components of HTS tissue are collagen-1?Col1a1?and fibronectin,which mediate ECM remodelling,and?-smooth muscle actin??SMA?-expressing myofibroblasts,which provide contractile strength.Myofibroblast transdifferentiation associated with HTS formation plays a central role during wound healing.However,the molecular mechanisms remain unclear.Transforming growth factor??TGF??is a cytokine that may promote myofibroblast transdifferentiation during acute tissue injury.TGF?plays important roles in regulating proliferation and differentiation as well as in tissue fibrosis.TGF?induces de novo synthesis of?SMA fibres that enhance contractility and increases the protein expression of the ECM proteins collagen and fibronectin after injury.Mammals express the TGF?isoforms TGF?1-TGF?3.TGF?1 regulates the proliferation of keratinocytes and dermal fibroblasts,including in chronic ulcers.Notably,Smad proteins are among the most important intracellular signal transduction proteins downstream of the TGF?superfamily.TGF?1 acts through a heterodimeric receptor at the plasma membrane that phosphorylates the transcription factors Smad2 and Smad3.Disruption of the TGF?1/Smad3 signalling pathway due to loss of Smad3 confers resistance to tissue fibrosis in the skin,kidneys,lungs and liver.Mitochondrial Ca²⁺uptake is critical for the regulation of numerous cellular processes and of energy metabolism,but Ca²⁺overload in the mitochondrial matrix impairs mitochondrial function and leads to reactive oxygen species?ROS?generation.Mitochondrial pyruvate dehydrogenase?PDH?and several electron transport complexes are associated with changes in mitochondrial Ca²⁺homeostasis.NADPH oxidase?Nox?4utilizes electrons from NADPH to generate superoxides,and suppression of Nox4 has been found to decrease myofibroblast formation and fibrosis in several tissue injury models.Recently,TGF?-induced myofibroblast transformation has been shown to be associated with the ROS-producing enzyme Nox4.However,the mechanisms of mitochondrial Ca²⁺overload through ROS and the role of the Nox4/Smad3 signalling pathway in regulating myofibroblast transformation remain unknown.Transient receptor potential?TRP?channels play important regulatory roles in cellular Ca²⁺homeostasis,growth,migration and inflammatory mechanisms.TRP channels are involved in dermatological disorders,but the function of TRP channels in myofibroblast transdifferentiation is poorly understood.TRPC6 is known to activate the Ca²⁺-responsive protein phosphatase calcineurin to induce myofibroblast transdifferentiation and dermal wound healing,and TRPA1 promotes cardiac myofibroblast transdifferentiation after myocardial infarction injury.Our previous studies have also revealed that enhancement of TRPC3 is associated with increased migration of monocytes and with elevated mitochondrial Ca²⁺uptake and ROS generation in the vasculature in hypertension.However,little is known about whether TGF?1 can regulate mitochondrial Ca²⁺and ROS production in the mitochondrial respiratory chain and elevate myofibroblast transdifferentiation by targeting TRPC3.Therefore,we hypothesized that TGF?1 enhances TRPC3-mediated mitochondrial Ca²⁺uptake and ROS production,ultimately promoting myofibroblast transdifferentiation during wound healing and increasing HTS formation.This study includes two parts:1.Expression of TRPC3 in human hypertrophic scars.2.The effect of TRPC3 in regulating fibroblast transdifferentiation into myofibroblasts promotes hypertrophic scars formation.We performed The Vancouver Scar Scale on the patients with skin hyperplastic scar included in the clinical study,and signed the informed consent for the patients who needed skin scar resection and were included in this study.Fresh scar tissue specimens and surrounding normal skin tissues were collected during scar resection,and immunohistochemistry,qRT-PCR and Western Blot were used to determine the expression levels of TRPC3 and TGF?1 in hypertrophic scar tissues and normal skin tissues.At the same time,using the primary human skin fibroblasts,detection of fibroblasts cytoplasm calcium ion concentration([Ca²⁺]_i)and mitochondrial calcium intake([Ca²⁺]_m),TRPC3 in the role of hyperplastic scar,and TRPC3 via NOX4/pSmad signaling pathways regulate fibroblast differentiation to fibroblasts,reveal TRPC3 participation mechanism of hyperplastic scar formation.This study provides a new intervention target for the prevention and treatment of skin scar.Material and Methods:The experiments include in vivo and in vitro tests.In the first part,human hypertrophic scar tissue and normal skin tissue as main objects to verify the TRPC3 and TGF?1 protein or mRNA expression levels.In the second part,primary cultured fibroblasts from human normal skin tissue,Trpc3^-/-mice and Trpc3^+/+mice to demonstrate the expression and function of TRPC3 in regulating mitochondrial functions and ROS generation.In vivo experiments,open wounds were created on the backs of Trpc3^+/+and Trpc3^-/-mice,in order to verify the role of TRPC3 in the regulation of myofibroblast transdifferentiation,and those proteins expression of?SMA,TGF?1,fibronectin and Col1a1 in wound granulation tissues during wound healing.Meanwhile,verify TRPC3 deficiency antagonizes myofibroblast transdifferentiation and hypertrophic scar formation.The main experimental methods include:1.Real-time quantitative PCR or immunohistochemistry were used to measure TRPC3 and TGF?1 mRNA or protein expression levels in hypertrophic scar tissue and normal skin tissue.2.Cellular and mitochondrial calcium uptake were measured using Fluorescence Master System and Multiskan Spectrum treated with above factors.3.High resolution respirometry and other parameters of redox were used to measure the mitochondrial respiratory functions,ATP and ROS generations.4.Open wounds on the back of mouse were created to measure wound healing rate and immunoblotting were used to detect the expression of TRPC3,?SMA,TGF?1,fibronectin,Col1a1 and Nox4/pSmad2/3pathway in wound granulation tissues.Results:1.Increased TRPC3 and TGF?1 protein and mRNA expression levels in fibroblasts from Hypertrophic scars tissues compared with normal skin tissues.2.Activation of TRPC3 by TGF?1 treatment elevated myofibroblast transdifferentiation by increasing myofibroblast marker,?SMA expression.Inhibition of TRPC3 by its specific inhibitor,Pyr3,significantly decreased myofibroblast transdifferentiation by inhibition of?SMA in cultured human fibroblasts.3.Increased TRPC3-mediated mitochondrial calcium uptake and ROS production,but decreased ATP content in TGF?1-treated human fibroblasts,whereas inhibition of TRPC3 significantly reversed these effects.This beneficial effect was associated with improvement of mitochondrial respiratory function through recovering the activity of pyruvate dehydrogenase?PDH?.4.In vivo,Trpc3^-/-mice exhibited significantly attenuated myofibroblast transdifferentiation by decreased?SMA protein expression in wound granulation tissues after 6 days wounded.Furthermore,TGF?1-induced Store-Operated Calcium Entry?SOCE?significantly decreased in fibroblasts from Trpc3^-/-compared with Trpc3^+/+mice.In addition,Trpc3^-/-mice significantly decreased Nox4 and phosphorylated Smad2/3 proteins expression in wound granulation tissues.Conclusions:1.Increased TRPC3 and TGF?1 expression are observed in hypertrophic scars tissues.2.Enhanced TRPC3 by TGF?1-treated in cultured human fibroblasts,which promotes mitochondrial calcium uptake,ROS production and myofibroblast transdifferentiation.whereas inhibition of TRPC3significantly reversed these effects.3.Trpc3^-/-could decreased TGF?1-induced SOCE and attenuated hypertrophic scars.Inhibition of the TRPC3-mediated Nox4/pSmad2/3pathway may be a useful strategy to limit hypertrophic scars formation after injury.