Wound Healing In Aged Mice Research Articles

Improving dermal fibroblast-to-epidermis communications and aging wound repair through extracellular vesicle-mediated delivery of Gstm2 mRNA

Skin aging is characterized by the disruption of skin homeostasis and impaired skin injury repair. Treatment of aging skin has long been limited by the unclear intervention targets and delivery techniques. Engineering extracellular vesicles (EVs) as an upgraded version of natural EVs holds great potential in regenerative medicine. In this study, we found that the expression of the critical antioxidant and detoxification gene Gstm2 was significantly reduced in aging skin. Thus, we constructed the skin primary fibroblasts-derived EVs encapsulating Gstm2 mRNA (EVsGstm2), and found that EVsGstm2 could significantly improve skin homeostasis and accelerate wound healing in aged mice. Mechanistically, we found that EVsGstm2 alleviated oxidative stress damage of aging dermal fibroblasts by modulating mitochondrial oxidative phosphorylation, and promoted dermal fibroblasts to regulate skin epidermal cell function by paracrine secretion of Nascent Polypeptide-Associated Complex Alpha subunit (NACA). Furthermore, we confirmed that NACA is a novel skin epidermal cell protective molecule that regulates skin epidermal cell turnover through the ROS-ERK-ETS-Cyclin D pathway. Our findings demonstrate the feasibility and efficacy of EVs-mediated delivery of Gstm2 for aged skin treatment and unveil novel roles of GSTM2 and NACA for improving aging skin.Graphical

Downregulated SPESP1-driven fibroblast senescence decreases wound healing in aged mice.

Human dermal fibroblasts (HDFs) are essential in the processes of skin ageing and wound healing. However, the underlying mechanism of HDFs in skin healing of the elderly has not been well defined. This study aims to elucidate the mechanisms of HDFs senescence and how senescent HDFs affect wound healing in aged skin. The expression and function of sperm equatorial segment protein 1 (SPESP1) in skin ageing were evaluated via in vivo and in vitro experiments. To delve into the potential molecular mechanisms by which SPESP1 influences skin ageing, a combination of techniques was employed, including proteomics, RNA sequencing, immunoprecipitation, chromatin immunoprecipitation and liquid chromatography-mass spectrometry analyses. Clearance of senescent cells by dasatinib plus quercetin (D+Q) was investigated to explore the role of SPESP1-induced senescent HDFs in wound healing. Here, we define the critical role of SPESP1 in ameliorating HDFs senescence and retarding the skin ageing process. Mechanistic studies demonstrate that SPESP1 directly binds to methyl-binding protein, leading to Decorin demethylation and subsequently upregulation of its expression. Moreover, SPESP1 knockdown delays wound healing in young mice and SPESP1 overexpression induces wound healing in old mice. Notably, pharmacogenetic clearance of senescent cells by D+Q improved wound healing in SPESP1 knockdown skin. Taken together, these findings reveal the critical role of SPESP1 in skin ageing and wound healing, expecting to facilitate the development of anti-ageing strategies and improve wound healing in the elderly.

Sustained activation of NLRP3 inflammasome contributes to delayed wound healing in aged mice

The cutaneous wounds in the elderly heal poorly, resulting in medical and economic burdens posed by defect repairing. Age-related delayed wound healing is associated with persistent inflammation and insufficient ECM deposition. The NLRP3 inflammasome has been proven to be a critical regulator of age-related inflammatory diseases, as well as impaired wound healing. Here, we create a 6 mm full-thickness cutaneous wound on the back of young and aged mice. Compared with young mice, aged counterparts display a retardation in wound healing, accompanied by increased activation of NLRP3 inflammasome. The application of the NLRP3 inhibitor (MCC950) ameliorates wound healing in aged mice. MCC950 inhibits sustained inflammation and reduces pyroptotic cell death in fibroblasts by blocking the abnormal activation of the NLRP3 inflammasome. Our findings illuminate that the NLRP3 inflammasome is a previously unrecognized regulator of aged wound healing and may be a potential target for the therapeutic strategy of delayed wound healing with aging.

Loading Nanoceria Improves Extracellular Vesicle Membrane Integrity and Therapy to Wounds in Aged Mice.

Wound healing is a programmed process through which tissue restores its integrity after an injury. Advancing age is a risk factor for delayed cutaneous wound healing; however, ideal therapeutic approaches for aged wound have not been developed yet. By dissecting the harsh microenvironment of aged wound, we propose an integrated chemical and biological strategy to mitigate two main hostile factors including oxidative stress and ischemia. Mesenchymal stem cell-derived extracellular vesicles (EVs) are a rising star in regenerative medicine due to their powerful facilitation in tissue repair and regeneration. However, the fragile lipid membrane limits their function under the oxidative stress microenvironment. Nanoceria is an antioxidative nanozyme; here, we reveal that nanoceria-loaded EVs derived from mesenchymal stem cells facilitate cutaneous wound healing in aged mice. DG-CeO2 was prepared via coating CeO2 covalently with d-glucose to promote their cellular endocytosis. DG-CeO2 was packaged into EVs under optimized hypoxic conditions (DG-CeO2 EVsHyp). We further demonstrated that DG-CeO2 EVsHyp had favorable biocompatibility and antioxidative and proangiogenic effects during the cutaneous wound healing in both young and aged mice. Further evidence revealed that DG-CeO2 EVsHyp-transferred miR-92a-3p/125b-5p and their targets associated with aging degeneration may be the potential mechanisms. Collectively, these findings highlight that nanoceria-loaded EVs released by engineered stem cells may represent a potential therapeutic approach for tissue regeneration in aged population.

Young fibroblast-derived exosomal microRNA-125b transfers beneficial effects on aged cutaneous wound healing

Aged skin wounds heal poorly, resulting in medical, economic, and social burdens posed by nonhealing wounds. Age-related defects in repair are associated with reduced myofibroblasts and dysfunctional extracellular matrix (ECM) deposition. Bidirectional cell-cell communication involving exosome-borne cargo such as micro RNAs (miRs) has emerged as a critical mechanism for wound healing and aged tissue regeneration. Here we report that at the wound edge, aged fibroblasts display reduced migration and differentiation into myofibroblasts, with impaired ECM deposition, when compared with young tissue. Proper activation of fibroblasts to myofibroblasts may alleviate age-related defects in wound healing. Herein, an exosome-guided cell technique was performed to induce effective wound healing. Supplementing wounds with exosomes isolated from young mouse wound-edge fibroblasts (exosomesYoung) significantly improved the abundance of myofibroblasts and wound healing in aged mice and caused fibroblasts to migrate and transition to myofibroblasts in vitro. To determine the underlying mechanism, we found that exosomal transfer of miR-125b to fibroblasts inhibited sirtuin 7 (Sirt7), thus accelerating myofibroblast differentiation and wound healing in aged mice. Notably, after epidermal inhibition of miR-125b or overexpression of Sirt7 in fibroblasts, migration and myofibroblast transition were perturbed. Our findings thus reveal that miR-125b is transferred through exosomes from young fibroblasts to old fibroblasts contributes to promoting fibroblast migration and transition to counteract aging, suggesting a potential avenue for anti-aging interventions in wound healing.Graphical

Human embryonic stem cell-derived exosomes promote pressure ulcer healing in aged mice by rejuvenating senescent endothelial cells

Background & Aim Background:Angiogenesis, as an important endogenous repair mechanism, plays crucial roles in wound healing and tissue regeneration. However, this process is impaired in the elderly due to aging-related vascular endothelial dysfunction. This study was aimed to explore the pro-angiogenic effects of exosomes from human embryonic stem cells (ESC-Exos) in aged mice of pressure-induced ulcer model and the underlying mechanism. Methods, Results & Conclusion Methods: Pressure ulcer wounds were created on the back of D-galactose-induced aging mice. ESC-Exos were locally applied onto the wound beds, with PBS as control. The effects of ESC-Exos on wound healing were analyzed by measuring wound closure rates, histological and immunofluorescence analyses.The anti-aging effect of ESC-Exos on vascular endothelial cells was tested in an in vitro D-galactose-induced HUVEC senescence model. Results ESC-Exos could accelerate wound closure, enhance angiogenesis,and ameliorate the senescence of vascular endothelial cells significantly. ESC-Exos could rejuvenate the senescence of endothelial cells and recover compromised proliferation, migratory capacity, and tube formation in vitro. This recovery was Nrf2-activation-dependent, since cotreatment with Nrf2 inhibitor Brusatol could abolish the rejuvenative effects of ESC-Exos. Further study revealed that miR-200a was highly enriched in ESC-Exos and played a crucial role in ESC-Exos-mediated rejuvenation through downregulating Keap1, which negatively regulated Nrf2 expression. Conclusions ESC-Exos ameliorate endothelial senescence by activating Nrf2 and recover aging-related angiogenic dysfunction, thereby accelerate wound healing in aged mice. ESC-Exos might be a useful and natural nano-biomaterial for aging-related diseases therapy.

Small extracellular vesicles secreted by urine-derived stem cells enhanced wound healing in aged mice by ameliorating cellular senescence

Regeneration of chronic skin wounds in tissue is still a key challenge in regenerative medicine because of the accumulation of senescent cells and increasing secretion of "senescence-associated secretory phenotype"-(SASP) in the wound site. Recently, some studies have reported that small extracellular vesicles (sEVs) derived from stem cells can alleviate cellular senescence with very low risk of tumorigenesis and immune responses. As our previous studies have shown that urine-derived stem cells (USCs) can be obtained easily and noninvasively and sEVs derived from USCs (USC-sEVs) have capabilities of regenerating tissue injuries, using USC-sEVs to enhance chronic skin wound healing in aged tissue might be a feasible and efficient strategy. Therefore, in this study, the USC-sEVs were collected and firstly loaded in a human acellular amniotic membrane (HAAM) for controlled releasing and locating the USC-sEVs in the wound site before they were implanted into a chronic skin wound in aged mice. In vivo results showed that the USC-sEVs in HAAM could effectively accelerate the wound healing by ameliorating cellular senescence and reducing the secretion of SASP in the aged skin wounds. To elucidate the mechanism, USC-sEVs were used to in vitro culture human dermal fibroblasts (HDFs) and results showed that USC-sEVs could rejuvenate senescent fibroblasts by reversing the aging phenotypes of senescent HDFs and efficiently reducing the secretion of SASP after they activated the Sirt1 pathway. Therefore, USC-sEVs are efficient for enhancing wound healing in aged mice by ameliorating cellular senescence.

The exercise cytokine interleukin-15 rescues slow wound healing in aged mice

Impaired wound healing in elderly individuals increases infection risk and prolongs surgical recovery, but current treatment options are limited. Low doses of interleukin-15 (IL-15) that mimic exercise responses in the circulation improve skin structure and increase mitochondria in uninjured aged skin, suggesting that IL-15 is an essential mitochondrial signal for healing that is lost during aging. Here we used gene microarray analysis of old and young murine epidermal stem cells and demonstrate that aging results in a gene signature characteristic of bioenergetic dysfunction. Intravenous IL-15 treatment rescued chronological aging-induced healing defects and restored youthful wound closure in old, sedentary mice. Additionally, exercise-mediated improvements in the healing of aged skin depend upon circulating IL-15. We show that IL-15 induces signal transducer and activator of transcription 3 (STAT3) signaling characteristic of young animals, reduces markers of growth arrest, and increases keratinocyte and fibroblast growth. Moreover, exercise or exercise-mimicking IL-15 treatment rescued the age-associated decrease in epidermal mitochondrial complex IV activity. Overall, these results indicate that IL-15 or its analogs represent promising therapies for treating impaired wound healing in elderly patients.

Local and transient inhibition of p21 expression ameliorates age‐related delayed wound healing

Nonhealing chronic wounds in the constantly growing elderly population represent a major public health problem with high socioeconomic burden. Yet, the underlying mechanism of age-related impairment of wound healing remains elusive. Here, we show that the number of dermal cells expressing cyclin-dependent kinase inhibitor p21 was elevated upon skin injury, particularly in aged population, in both man and mouse. The nuclear expression of p21 in activated wound fibroblasts delayed the onset of the proliferation phase of wound healing in a p53-independent manner. Further, the local and transient inhibition of p21 expression by in vivo delivered p21-targeting siRNA ameliorated the delayed wound healing in aged mice. Our results suggest that the increased number of p21+ wound fibroblasts enforces the age-related compromised healing, and targeting p21 creates potential clinical avenues to promote wound healing in aged population.

Human embryonic stem cell-derived exosomes promote pressure ulcer healing in aged mice by rejuvenating senescent endothelial cells

BackgroundAngiogenesis, as an endogenous repair mechanism, plays crucial roles in wound healing and tissue regeneration. However, this process is impaired in the elderly due to aging-related vascular endothelial dysfunction. This study was aimed to explore the pro-angiogenic effects of exosomes from human embryonic stem cells (ESC-Exos) in aged mice of pressure-induced ulcer model and the underlying mechanism.MethodsPressure ulcer wounds were created on the back of d-galactose-induced aging mice. ESC-Exos were locally applied onto the wound beds, with PBS as control. The effects of ESC-Exos on wound healing were analyzed by measuring wound closure rates, histological and immunofluorescence analyses. Then, the anti-aging effect of ESC-Exos on vascular endothelial cells was tested in an in vitro d-galactose-induced HUVEC senescence model.ResultsESC-Exos could accelerate wound closure and enhance angiogenesis, and the senescence of vascular endothelial cells was significantly ameliorated after ESC-Exos treatment. In vitro, ESC-Exos could rejuvenate the senescence of endothelial cells and recover compromised proliferation, migratory capacity, and tube formation. This recovery was Nrf2-activation-dependent, since cotreatment with Nrf2 inhibitor Brusatol could abolish the rejuvenative effects of ESC-Exos. Further study revealed that miR-200a was highly enriched in ESC-Exos and played a crucial role in ESC-Exos-mediated rejuvenation through downregulating Keap1, which negatively regulates Nrf2 expression.ConclusionsESC-Exos ameliorate endothelial senescence by activating Nrf2 and recover aging-related angiogenic dysfunction, thereby accelerating wound healing in aged mice. ESC-Exos might be a natural nano-biomaterial for aging-related diseases therapy.

Human Embryonic Stem Cells Derived Exosomes Promote Tissue Regeneration in Aged Mice by Rejuvenating Senescent Endothelial Cells

Background: Angiogenesis, as an endogenous repair mechanism, plays crucial roles in wound healing and tissue regeneration. However, this process is impaired in the elderly due to aging-related vascular endothelial dysfunction. This study was aimed to explore the pro-angiogenic effects of exosomes from human embryonic stem cells (ESC-Exos) in aged mice of pressure-induced ulcer model and the underlying mechanism. Methods: Pressure ulcer wounds were created on the back of D-galactose-induced aging mice. ESC-Exos were locally applied onto the wound beds once a day, with PBS as control. The effects of ESC-Exos on wound healing were analyzed by measuring wound closure rates, histological and immunofluorescence analyses. Then the anti-aging effect of ESC-Exos on vascular endothelial cells was tested in an in vitro D-galactose-induced HUVEC senescence model. Findings: ESC-Exos could accelerate wound closure and enhance angiogenesis, and the senescence of vascular endothelial cells was significantly ameliorated after ESC-Exos treatment. In vitro, ESC-Exos could rejuvenate the senescence of endothelial cells and recover compromised proliferation, migratory capacity, and tube formation. This recovery was Nrf2-activation-dependent, since cotreatment with Nrf2 inhibitor Brusatol could abolish the rejuvenative effects of ESC-Exos. Further study revealed that miR-200a was highly enriched in ESC-Exos and played a crucial role in ESC-Exos-mediated rejuvenation through downregulating Keap1, which negatively regulates Nrf2 expression. Interpretation: ESC-Exos ameliorate endothelial senescence by activating Nrf2, and recover aging-related angiogenic dysfunction, thereby accelerate wound healing in aged mice. ESC-Exos might be a natural nano-biomaterial for aging-related diseases therapy. Funding: National Natural Science Fund (81472152 and 81572223). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: Animal care and experimental procedures were approved by the Animal Research Committee of the Sixth People’s Hospital at the Shanghai Jiao Tong University.

MiR-21 ameliorates age-associated skin wound healing defects in mice.

The cellular and molecular mechanisms responsible for the age-associated delay of cutaneous wound healing are still not well understood. Previous studies have shown that miR-21 plays key roles during skin wound healing. We presumed that dysregulation of miR-21 may be involved in age-associated defects in wound healing and that miR-21 may be one potential therapeutic target by which to ameliorate wound defects in elderly subjects. Circular full thickness excisional wounds were made on the dorsal skin of young (2-month-old) and aged (12-month-old) female mice. The wound healing rates were quantified and compared between wild-type and miR-21 knock-in mice. Both histologic and morphometric analyses of the wounds were evaluated. Furthermore, the expression patterns of miR-21 during wound healing in both young and aged mice were assessed by in situ hybridization. The effects of topical miR-21 overexpression on wound healing in aged mice were estimated by both wound closure quantification and histological analyses. Aged miR-21 knock-in female mice showed significantly improved wound healing compared to their wild-type counterparts with respect to mature granulation tissue, smaller wound width and thinner epidermis. The expression patterns of miR-21 showed that miR-21 levels were insufficient for repairing granulation tissue in aged mice. Intradermal injection of miR-21 plasmid around wounds could upregulate miR-21 levels during wound healing and ameliorate age-associated skin wound defects. The results of the present study reveal that the upregulation of miR-21 levels could improve wound repair in aged mice, which suggests that a therapeutic strategy targeting miR-21 expression in age-associated wound healing may be feasible.

Combination of HIF-1α gene transfection and HIF-1-activated bone marrow-derived angiogenic cell infusion improves burn wound healing in aged mice

Impaired burn wound healing in the elderly represents a major clinical problem. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator that orchestrates the cellular response to hypoxia. Its actions in dermal wounds promote angiogenesis and improve healing. In a murine burn wound model, aged mice had impaired wound healing associated with reduced levels of HIF-1. When gene therapy with HIF-1 alone did not correct these deficits, we explored the potential benefit of HIF-1 gene therapy combined with the intravenous infusion of bone marrow-derived angiogenic cells (BMDACs) cultured with dimethyloxalylglycine (DMOG). DMOG is known to reduce oxidative degradation of HIF-1. The mice treated with a plasmid DNA construct expressing a stabilized mutant form of HIF-1α (CA5-HIF-1α)+BMDACs had more rapid wound closure. By day 17, there were more mice with completely closed wounds in the treated group (χ(2), P=0.05). The dermal blood flow measured by laser Doppler showed significantly increased wound perfusion on day 11. Homing of BMDACs to the burn wound was dramatically enhanced by CA5-HIF-1α gene therapy. HIF-1α mRNA expression in the burn wound was increased after transfection with CA5-HIF-1α plasmid. Our findings offer insight into the pathophysiology of burns in the elderly and point to potential targets for developing new therapeutic strategies.

Exercise Speeds Cutaneous Wound Healing in High-Fat Diet-Induced Obese Mice

Obesity has been shown to impair cutaneous wound healing, which is associated with increased wound inflammation. Exercise is known to decrease obesity-associated inflammation and has been shown to speed cutaneous wound healing in aged mice. Therefore, we investigated whether treadmill exercise could speed cutaneous wound healing in obese, high-fat diet-fed mice. We fed female C57Bl/6J mice a high-fat diet (45% calories from fat) for 16 wk to induce a state of obesity and insulin resistance. Mice then ran on a treadmill for 3 d before excisional wounding. On day 4, mice were wounded 1 h after exercise. Mice then exercised for 5 d after wounding, and healing was assessed by photoplanimetry for 10 d. As described previously, obesity impaired wound healing, with significantly larger wound sizes measured from days 3 to day 10 after wounding (P < 0.05). Exercise did not improve healing in lean mice fed a normal chow diet. However, wound size was significantly smaller in exercised obese mice compared with their lean counterparts (P < 0.05 at day 1, day 4, and day 5 after wound). Surprisingly, we were unable to detect any differences in gene or protein expression of proinflammatory cytokines interleukin-1β and tumor necrosis factor-α or the anti-inflammatory cytokine interleukin-10 in the wounds. Likewise, there were no differences in gene expression of chemokines monocyte chemoattractant protein-1 and keratinocyte chemoattractant or of growth factor platelet-derived growth factor in wounds of exercise and sedentary mice. This suggests an effect of exercise independent of alterations in inflammation. Future work should focus on early events after wounding, including exercise effects on hemostasis and myofibroblast function.

HIF-1α gene therapy combined with HIF-1 activated bone marrow-derived angiogenic cell (BMDAC) therapy improves burn wound healing in aged mice

HIF-1α gene therapy combined with HIF-1 activated bone marrow-derived angiogenic cell (BMDAC) therapy improves burn wound healing in aged mice

Delayed wound healing in aged mice is associated with diminished mobilization of circulating angiogenic cells

Zhang, Xianjie MD, PhD; Sarkar, Kakali PhD; Andrikopoulou, Efstathia MD; Xing, Dongmei MD, PhD; Liu, Lixin PhD; Reinblatt, Maura MD; Laines, Raul Sebastian MD; Marti, Guy P. MD; Semenza, Gregg L. MD, PhD; Harmon, John W. MD, FACS Author Information

Neutrophil depletion delays wound repair in aged mice

One of the most important clinical problems in caring for elderly patients is treatment of pressure ulcers. One component of normal wound healing is the generation of an inflammatory reaction, which is characterized by the sequential infiltration of neutrophils, macrophages and lymphocytes. Neutrophils migrate early in the wound healing process. In aged C57BL/6 mice, wound healing is relatively inefficient. We examined the effects of neutrophil numbers on wound healing in both young and aged mice. We found that the depletion of neutrophils by anti-Gr-1 antibody dramatically delayed wound healing in aged mice. The depletion of neutrophils in young mice had less effect on the kinetics of wound healing. Intravenous G-CSF injection increased the migration of neutrophils to the wound site. While the rate of wound repair did not change significantly in young mice following G-CSF injection, it increased significantly in old mice.

Expression of fibroblast growth factors and their receptors during full-thickness skin wound healing in young and aged mice

The highly ordered process of wound healing involves the coordinated regulation of cell proliferation and migration and tissue remodeling, predominantly by polypeptide growth factors. Consequently, the slowing of wound healing that occurs in the aged may be related to changes in the activity of these various regulatory factors. To gain additional insight into these issues, we quantified the absolute copy numbers of mRNAs encoding all the fibroblast growth factors (FGFs), their receptors (FGFRs) and two other growth factors in the dorsal skin of young and aged mice during the healing of full-thickness skin excisional wounds. In young adult mice (8 weeks old), FGF7, FGF10 and FGF22 mRNAs were all strongly expressed in healthy skin, and levels of FGF7 and 10 but not 22 increased 2- to 3.5-fold over differing time courses after wounding. The levels of FGF9, 16, 18 and especially 23 mRNAs were moderate or low in healthy skin but increased 2- to 33-fold after wounding. Among the four FGFRs, expression of only FGFR1 mRNA was augmented during wound healing. Expression of transforming growth factor-beta and hepatocyte growth factor was also high in healthy skin and was upregulated during healing. Notably, in aged mice (35 weeks old), where healing proceeded more slowly than in the young, both the basal and wound-induced mRNA expression of most of these genes was reduced. While these results confirm the established notion that FGFR2 IIIB ligands (FGF7 and FGF10) are important for wound healing, they also suggest that decreased expression of multiple FGF ligands contributes to the slowing of wound healing in aged mice and indicate the potential importance of further study of the involvement of FGF9, 16, 18 and 23 in the wound healing process.