miR-125b-5p delivered by adipose-derived stem cell exosomes alleviates hypertrophic scarring by suppressing Smad2.

Background: Hypertrophic scarring is the most serious and unmet challenge following burn and trauma injury and often leads to pain, itching and even loss of function. However, the demand for ideal scar prevention and treatment is difficult to satisfy. We aimed to discover the effects and mechanisms of adipose-derived stem cell (ADSC) exosomes in hypertrophic scarring. Methods: ADSC exosomes were isolated from the culture supernatant of ADSCs and identified by nanoparticle tracking analysis, transmission electron microscopy and western blotting. The effect of ADSC exosomes on wound healing and scar formation was detected by the wound model of BALB/c mice. We isolated myofibroblasts from hypertrophic scar tissue and detected the cell viability, proliferation and migration of myofibroblasts. In addition, collagen formation and fibrosis-related molecules were also detected. To further disclose the mechanism of ADSC exosomes on fibrosis in myofibroblasts, we detected the expression of Smad2 in hypertrophic scar tissue and normal skin and the regulatory mechanism of ADSC exosomes on Smad2. Injection of bleomycin was performed in male BALB/c mice to establish an in vivo fibrosis model while ADSC exosomes were administered to observe their protective effect. The tissue injury of mice was observed via hematoxylin and eosin and Masson staining and related testing. Results: In this study, we found that ADSC exosomes could not only speed up wound healing and improve healing quality but also prevent scar formation. ADSC exosomes inhibited expression of fibrosis-related molecules such as α-smooth muscle actin, collagen I (COL1) and COL3 and inhibited the transdifferentiation of myofibroblasts. In addition, we verified that Smad2 is highly expressed in both hypertrophic scar tissue and hypertrophic fibroblasts, while ADSC exosomes downregulated the expression of Smad2 in hypertrophic fibroblasts. Further regulatory mechanism analysis revealed that microRNA-125b-5p (miR-125b-5p) is highly expressed in ADSC exosomes and binds to the 3' untranslated region of Smad2, thus inhibiting its expression. In vivo experiments also revealed that ADSC exosomes could alleviate bleomycin-induced skin fibrosis and downregulate the expression of Smad2. Conclusions: We found that ADSC exosomes could alleviate hypertrophic scars via the suppression of Smad2 by the specific delivery of miR-125b-5p.

Exosomes derived from adipose tissue-derived stem cells alleviated H2O2-induced oxidative stress and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells by inhibition of the mir-486-3p/Sirt6/Smad signaling pathway.

Hypertrophic scar (HS) is characterized by excessive collagen deposition and myofibroblasts activation. Endothelial-to-mesenchymal transition (EndoMT) and oxidative stress were pivotal in skin fibrosis process. Exosomes derived from adipose tissue-derived stem cells (ADSC-Exo) have the potential to attenuate EndoMT and inhibit fibrosis. The study revealed reactive oxygen species (ROS) levels were increased during EndoMT occurrence of dermal vasculature of HS. The morphology of endothelial cells exposure to H2O2, serving as an in vitro model of oxidative stress damage, transitioned from a cobblestone-like appearance to a spindle-like shape. Additionally, the levels of endothelial markers decreased in H2O2-treated endothelial cell, while the expression of fibrotic markers increased. Furthermore, H2O2 facilitated the accumulation of ROS, inhibited cell proliferation, retarded its migration and suppressed tube formation in endothelial cell. However, ADSC-Exo counteracted the biological effects induced by H2O2. Subsequently, miRNAs sequencing analysis revealed the significance of mir-486-3p in endothelial cell exposed to H2O2 and ADSC-Exo. Mir-486-3p overexpression enhanced the acceleration of EndoMT, its inhibitors represented the attenuation of EndoMT. Meanwhile, the target regulatory relationship was observed between mir-486-3p and Sirt6, whereby Sirt6 exerted its anti-EndoMT effect through Smad2/3 signaling pathway. Besides, our research had successfully demonstrated the impact of ADSC-Exo and mir-486-3p on animal models. These findings of our study collectively elucidated that ADSC-Exo effectively alleviated H2O2-induced ROS and EndoMT by inhibiting the mir-486-3p/Sirt6/Smad axis.

Extracellular vesicles from 3D cultured dermal papilla cells improve wound healing via Krüppel-like factor 4/vascular endothelial growth factor A -driven angiogenesis.

Background: Non-healing wounds are an intractable problem of major clinical relevance. Evidence has shown that dermal papilla cells (DPCs) may regulate the wound-healing process by secreting extracellular vesicles (EVs). However, low isolation efficiency and restricted cell viability hinder the applications of DPC-EVs in wound healing. In this study, we aimed to develop novel 3D-DPC spheroids (tdDPCs) based on self-feeder 3D culture and to evaluate the roles of tdDPC-EVs in stimulating angiogenesis and skin wound healing. Methods: To address the current limitations of DPC-EVs, we previously developed a self-feeder 3D culture method to construct tdDPCs. DPCs and tdDPCs were identified using immunofluorescence staining and flow cytometry. Subsequently, we extracted EVs from the cells and compared the effects of DPC-EVs and tdDPC-EVs on human umbilical vein endothelial cells (HUVECs) in vitro using immunofluorescence staining, a scratch-wound assay and a Transwell assay. We simultaneously established a murine model of full-thickness skin injury and evaluated the effects of DPC-EVs and tdDPC-EVs on wound-healing efficiency in vivo using laser Doppler, as well as hematoxylin and eosin, Masson, CD31 and α-SMA staining. To elucidate the underlying mechanism, we conducted RNA sequencing (RNA-seq) of tdDPC-EV- and phosphate-buffered saline-treated HUVECs. To validate the RNA-seq data, we constructed knockdown and overexpression vectors of Krüppel-like factor 4 (KLF4). Western blotting, a scratch-wound assay, a Transwell assay and a tubule-formation test were performed to detect the protein expression, cell migration and lumen-formation ability of KLF4 and vascular endothelial growth factor A (VEGFA) in HUVECs incubated with tdDPC-EVs after KLF4 knockdown or overexpression. Dual-luciferase reporter gene assays were conducted to verify the activation effect of KLF4 on VEGFA. Results: We successfully cultured tdDPCs and extracted EVs from DPCs and tdDPCs. The tdDPC-EVs significantly promoted the proliferation, lumen formation and migration of HUVECs. Unlike DPC-EVs, tdDPC-EVs exhibited significant advantages in terms of promoting angiogenesis, accelerating wound healing and enhancing wound-healing efficiency both in vitro and in vivo. Bioinformatics analysis and further functional experiments verified that the tdDPC-EV-regulated KLF4/VEGFA axis is pivotal in accelerating wound healing. Conclusions: 3D cultivation can be utilized as an innovative optimization strategy to effectively develop DPC-derived EVs for the treatment of skin wounds. tdDPC-EVs significantly enhance wound healing via KLF4/VEGFA-driven angiogenesis.

miR-125b-5p in adipose derived stem cells exosome alleviates pulmonary microvascular endothelial cells ferroptosis via Keap1/Nrf2/GPX4 in sepsis lung injury.

BACKGROUND: Sepsis is a fatal disease with a high rate of morbidity and mortality, during which acute lung injury is the earliest and most serious complication. Injury of pulmonary microvascular endothelial cells (PMVECs) induced by excessive inflammation plays an important role in sepsis acute lung injury. This study is meant to explore the protective effect and mechanism of ADSCs exosomes on excessive inflammation PMVECs injury. RESULTS: We successfully isolated ADSCs exosomes, the characteristic of which were confirmed. ADSCs exosomes reduced excessive inflammatory response induced ROS accumulation and cell injury in PMVECs. Besides, ADSCs exosomes inhibited excessive inflammatory response induced ferroptosis while upregulated expression of GPX4 in PMVECs. And further GPX4 inhibition experiments revealed that ADSCs exosomes alleviated inflammatory response induced ferroptosis via upregulating GPX4. Meanwhile, ADSCs exosomes could increase the expression and nucleus translocation of Nrf2, while decrease the expression of Keap1. miRNA analysis and further inhibition experiments verified that specific delivery of miR-125b-5p by ADSCs exosomes inhibited Keap1 and alleviated ferroptosis. In CLP induced sepsis model, ADSCs exosomes could relieve the lung tissue injury and reduced the death rate. Besides, ADSCs exosomes alleviated oxidative stress injury and ferroptosis of lung tissue, while remarkably increase expression of Nrf2 and GPX4. CONCLUSION: Collectively, we illustrated a novel potentially therapeutic mechanism that miR-125b-5p in ADSCs exosomes could alleviate the inflammation induced PMVECs ferroptosis in sepsis induced acute lung injury via regulating Keap1/Nrf2/GPX4 expression, hence improve the acute lung injury in sepsis.

Exosomes Derived from Adipose Mesenchymal Stem Cells Promote Diabetic Chronic Wound Healing through SIRT3/SOD2.

Chronic wounds resulting from diabetes are a major health concern in both industrialized and developing countries, representing one of the leading causes of disability and death. This study aimed to investigate the effect of adipose mesenchymal stem cell-derived exosomes (ADSC-exos) on diabetic wounds and the mechanism underlying this effect. The results showed that ADSC-exos could improve oxidative stress and secretion of inflammatory cytokines in diabetic wounds, thereby increasing periwound vascularization and accelerating wound healing. At the cellular level, ADSC-exos reduced reactive oxygen species (ROS) generation in human umbilical vein endothelial cells (HUVECs) and improved mitochondrial function in a high-glucose environment. Moreover, the Western blot analysis showed that the high-glucose environment decreased Sirtuin 3 (SIRT3) expression, while exosome treatment increased SIRT3 expression. The activity of superoxide dismutase 2 (SOD2) was enhanced, and the level of inflammatory cytokines was decreased. Further, SIRT3 interference experiments indicated that the effects of ADSC-exos on oxidative stress and angiogenesis were partly dependent on SIRT3. After SIRT3 was inhibited, ROS production increased, while mitochondrial membrane potential and SOD2 activity decreased. These findings confirmed that ADSC-exos could improve the level of high-glucose-induced oxidative stress, promote angiogenesis, and reduce mitochondrial functional impairment and the inflammatory response by regulating SIRT3/SOD2, thus promoting diabetic wound healing.