Hair follicle and interfollicular epidermal stem cells make varying contributions to wound regeneration

Upon wounding, multiple stem cell populations in the hair follicle (HF) and interfollicular epidermis (IFE) converge at the site of injury. Although these cells can contribute permanently to the regenerating epithelium, it remains unclear whether these contributions vary among cells originating from diverse compartments in the skin. By comparing the fates of several keratinocyte lineages, we observed here an initial decrease in both HF- and IFE-derived cells within the transient acanthotic layers of the regenerating epithelium. At the same time, the relative abundance of early-arriving IFE-derived cells specifically in the wound basal layer declined as later-arriving HF-derived cells entered the site of injury. Although laggard bulge-derived cells were typically constrained at the regenerative periphery, these cells persisted in the wound basal layer. Finally, suppressing Notch enabled IFE-derived cells to out-compete HF-derived cells. Taken together, these findings indicate that IFE-, HF- and bulge-derived cells make distinct contributions to regeneration over time. Furthermore, we speculate that extrinsic, non-genetic factors such as spatial constraint, distance from the wound, and basal versus suprabasal position may largely determine whether a cell ultimately persists.     

Epidermal stem cells: the cradle of epidermal determination, differentiation and wound healing

The field of epidermal stem cells has dramatically advanced in the last decade, leading to a better understanding of the molecular factors, signalling pathways and cellular events that identify and characterize stem cells, thus revealing their immense potential for therapeutic use. Furthermore, multipotent epidermal stem cells present the major advantage of easy accessibility with the discovery of their specific location within the bulge of the hair follicle. This review focuses on the most recent findings on epidermal stem cells, and their potential role in initial epidermal commitment, differentiation and wound healing processes in the skin.     

Exosomes from adipose‐derived stem cells and application to skin wound healing

Skin wound healing is an intractable problem that represents an urgent clinical need. To solve this problem, a large number of studies have focused on the use of exosomes (EXOs) derived from adipose‐derived stem cells (ADSCs). This review describes the mechanisms whereby ADSCs‐EXOs regulate wound healing and their clinical application. In the wound, ADSCs‐EXOs modulate immune responses and inflammation. They also promote angiogenesis, accelerate proliferation and re‐epithelization of skin cells, and regulate collagen remodelling which inhibits scar hyperplasia. Compared with ADSCs therapeutics, ADSCs‐EXOs have highly stability and are easily stored. Additionally, they are not rejected by the immune system and have a homing effect and their dosage can be easily controlled. ADSCs‐EXOs can improve fat grafting and promote wound healing in patients with diabetes mellitus. They can also act as a carrier and combined scaffold for treatment, leading to scarless cutaneous repair. Overall, ADSCs‐EXOs have the potential to be used in the clinic to promote wound healing.     

Attenuation corrected-optical coherence tomography for quantitative assessment of skin wound healing and scar morphology

Imaging the structural modifications of underlying tissues is vital to monitor wound healing. Optical coherence tomography (OCT) images high-resolution sub-surface information, but suffers a loss of intensity with depth, limiting quantification. Hence correcting the attenuation loss is important. We performed swept source-OCT of full-thickness excision wounds for 300 days in mice skin. We used single-scatter attenuation models to determine and correct the attenuation loss in the images. The phantom studies established the correspondence of corrected-OCT intensity (reflectivity) with matrix density and hydration. We histologically validated the corrected-OCT and measured the wound healing rate. We noted two distinct phases of healing—rapid and steady-state. We also detected two compartments in normal scars using corrected OCT that otherwise were not visible in the OCT scans. The OCT reflectivity in the scar compartments corresponded to distinct cell populations, mechanical properties and composition. OCT reflectivity has potential applications in evaluating the therapeutic efficacy of healing and characterizing scars.     

SIRT3 deficiency delays diabetic skin wound healing via oxidative stress and necroptosis enhancement

Sirtuin 3 (SIRT3) plays a vital role in several dermatological diseases. However, the role and detailed mechanism of SIRT3 in diabetic wound healing are unknown well yet. To explore possible involvement of SIRT3 and necroptosis in diabetic skin wound healing, SIRT3 knockout (KO) mice and 129S1/SvImJ wild‐type (WT) mice were injected with streptozotocin (STZ), and mice skin fibroblasts were exposed to high glucose (HG). It was found that SIRT3 expression decreased in the skin of diabetic patients. SIRT3 deficiency delayed healing rate, reduced blood supply and vascular endothelial growth factor expression, promoted superoxide production, increased malondialdehyde (MDA) levels, decreased total antioxidant capacity (T‐AOC), reduced superoxide dismutase (SOD) activity and aggravated ultrastructure disorder in skin wound of diabetic mice. SIRT3 deficiency inhibited mice skin fibroblasts migration with HG stimulation, which was restored by SIRT3 overexpression. SIRT3 deficiency also suppressed α‐smooth muscle actin (α‐SMA) expression, enhanced superoxide production but decreased mitochondrial membrane potential with HG stimulation after scratch. SIRT3 deficiency further elevated receptor‐interacting protein kinase 3 (RIPK3), RIPK1 and caspase 3 expression both in vitro and in vivo. Collectively, SIRT3 deficiency delayed skin wound healing in diabetes, the mechanism might be related to impaired mitochondria function, enhanced oxidative stress and increased necroptosis. This may provide a novel therapeutic target to accelerate diabetic skin wound healing.     

Granzyme B degrades extracellular matrix and contributes to delayed wound closure in apolipoprotein E knockout mice

Chronic inflammation and excessive protease activity have a major role in the persistence of non-healing wounds. Granzyme B (GzmB) is a serine protease expressed during chronic inflammation that, in conjunction with perforin, has a well-established role in initiating apoptotic cell death. GzmB is also capable of acting extracellularly, independent of perforin and can degrade several extracellular matrix (ECM) proteins that are critical during wound healing. We used apolipoprotein E (ApoE) knockout (AKO) mice as a novel model of chronic inflammation and impaired wound healing to investigate the role of GzmB in chronic wounds. Wild-type and AKO mice were grown to 7 weeks (young) or 37 weeks (old) of age on a regular chow or high-fat diet (HFD), given a 1-cm diameter full thickness wound on their mid dorsum and allowed to heal for 16 days. Old AKO mice fed a HFD exhibited reduced wound closure, delayed contraction, chronic inflammation and altered ECM remodeling. Conversely, GzmB/ApoE double knockout mice displayed improved wound closure and contraction rates. In addition, murine GzmB was found to degrade both fibronectin and vitronectin derived from healthy mouse granulation tissue. In addition, GzmB-mediated degradation of fibronectin generated a fragment similar in size to that observed in non-healing mouse wounds. These results provide the first direct evidence that GzmB contributes to chronic wound healing in part through degradation of ECM.     

Deletion of delta-opioid receptor in mice alters skin differentiation and delays wound healing

In addition to their well-known antinociceptive action, opioids can modulate non-neuronal functions, such as immune activity and physiology of different cell types. Several findings suggest that the delta-opioid receptor (DOR) and its endogenous ligands (enkephalins) are important players in cell differentiation and proliferation. Here we show the expression of DOR in mouse skin and human skin cultured fibroblasts and keratinocytes using RT-PCR. In DOR knock-out (KO) mice, a phenotype of thinner epidermis and higher expression of cell differentiation marker cytokeratin 10 (CK 10) were observed compared with wild type (WT). Using a burn wound model, significant wound healing delay (about 2 days) and severe epidermal hypertrophy were shown at the wound margin of DOR KO mice. This wound healing delay was further investigated by immunohistochemistry using markers for proliferation, differentiation, re-epithelialization, and dermal repair (CK 6, CK 10, and collagen IV). The levels of all these markers were increased in wounds of KO mice compared with WT. During the wound healing, the epidermal thickness in KO mice augments faster and exceeds that of the WT by day 3. These results suggest an essential role of DOR in skin differentiation, proliferation, and migration, factors that are important for wound healing.     

Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.     

Monotropein promotes angiogenesis and inhibits oxidative stress‐induced autophagy in endothelial progenitor cells to accelerate wound healing

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated viamTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.     

Anti‐aging pharmacology in cutaneous wound healing: effects of metformin,resveratrol, and rapamycin by local application

Cutaneous wounds are among the most common soft tissue injuries and are particularly hard to heal in aging. Caloric restriction (CR) is well documented to extend longevity; pharmacologically, profound rejuvenative effects of CR mimetics have been uncovered, especially metformin (MET), resveratrol (RSV), and rapamycin (RAPA). However, locally applied impacts and functional differences of these agents on wound healing remain to be established. Here, we discovered that chronic topical administration of MET and RSV, but not RAPA, accelerated wound healing with improved epidermis, hair follicles, and collagen deposition in young rodents, and MET exerted more profound effects. Furthermore, locally applied MET and RSV improved vascularization of the wound beds, which were attributed to stimulation of adenosine monophosphate‐activated protein kinase (AMPK) pathway, the key mediator of wound healing. Notably, in aged skin, AMPK pathway was inhibited, correlated with impaired vasculature and reduced healing ability. As therapeutic approaches, local treatments of MET and RSV prevented age‐related AMPK suppression and angiogenic inhibition in wound beds. Moreover, in aged rats, rejuvenative effects of topically applied MET and RSV on cell viability of wound beds were confirmed, of which MET showed more prominent anti‐aging effects. We further verified that only MET promoted wound healing and cutaneous integrity in aged skin. These findings clarified differential effects of CR‐based anti‐aging pharmacology in wound healing, identified critical angiogenic and rejuvenative mechanisms through AMPK pathway in both young and aged skin, and unraveled chronic local application of MET as the optimal and promising regenerative agent in treating cutaneous wound defects.     

Knock‐out of MicroRNA 145 impairs cardiac fibroblast function and wound healing post‐myocardial infarction

Prevention of infarct scar thinning and dilatation and stimulation of scar contracture can prevent progressive heart failure. Since microRNA 145 (miR‐145) plays an important role in cardiac fibroblast response to wound healing and cardiac repair after an myocardial infarction (MI), using a miR‐145 knock‐out (KO) mouse model, we evaluated contribution of down‐regulation of miR‐145 to cardiac fibroblast and myofibroblast function during adverse cardiac remodelling. Cardiac function decreased more and the infarct size was larger in miR‐145 KO than that in WT mice after MI and this phenomenon was accompanied by a decrease in cardiac fibroblast‐to‐myofibroblast differentiation. Quantification of collagen I and α‐SMA protein levels as well as wound contraction revealed that transdifferentiation of cardiac fibroblasts into myofibroblasts was lower in KO than WT mice. In vitro restoration of miR‐145 induced more differentiation of fibroblasts to myofibroblasts and this effect involved the target genes Klf4 and myocardin. MiR‐145 contributes to infarct scar contraction in the heart and the absence of miR‐145 contributes to dysfunction of cardiac fibroblast, resulting in greater infarct thinning and dilatation. Augmentation of miR‐145 could be an attractive target to prevent adverse cardiac remodelling after MI by enhancing the phenotypic switch of cardiac fibroblasts to myofibroblasts.     

Integrin–TGF‐β crosstalk in fibrosis,cancer and wound healing

Accumulating evidence indicates that there is extensive crosstalk between integrins and TGF‐β signalling. TGF‐β affects integrin‐mediated cell adhesion and migration by regulating the expression of integrins, their ligands and integrin‐associated proteins. Conversely, several integrins directly control TGF‐β activation. In addition, a number of integrins can interfere with both Smad‐dependent and Smad‐independent TGF‐β signalling in different ways, including the regulation of the expression of TGF‐β signalling pathway components, the physical association of integrins with TGF‐β receptors and the modulation of downstream effectors. Reciprocal TGF‐β–integrin signalling is implicated in normal physiology, as well as in a variety of pathological processes including systemic sclerosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and cancer; thus, integrins could provide attractive therapeutic targets to interfere with TGF‐β signalling in these processes.     

Laser‐induced capillary leakage for blood biomarker detection and vaccine delivery via the skin

Circulation system is the center for coordination and communication of all organs in our body. Examination of any change in its analytes or delivery of therapeutic drugs into the system consists of important medical practice in today's medicine. Two recent studies prove that brief illumination of skin with a low powered laser, at wavelengths preferentially absorbed by hemoglobin, increases the amount of circulating biomarkers in the epidermis and upper dermis by more than 1,000‐fold. When probe‐coated microneedle arrays are applied into laser‐treated skin, plasma blood biomarkers can be reliably, accurately, and sufficiently quantified in 15～30 min assays, with a maximal detection in one hr in a manner independent of penetration depth or a molecular mass of the biomarker. Moreover, the laser treatment permits a high efficient delivery of radiation‐attenuated malarial sporozoites (RAS) into the circulation, leading to robust immunity against malaria infections, whereas similar immunization at sham‐treated skin elicits poor immune responses. Thus this technology can potentially instruct designs of small, portable devices for onsite, in mobile clinics, or at home for point‐of‐care diagnosis and drug/vaccine delivery via the skin.

Laser‐induced capillary leakage ( a ) to induce extravasation of circualing molecules only ( b ) or facilitate entry of attenuated malaria sporozoites into the capillary ( c ). Skin illumination with a laser preferably absorbed by hemoglobin causes dilation of the capillary beneath the skin. The extravasated molecules can be sufficiently measured in the skin or guide sporozoites to enter the vessel.     

Positive effects of low-dose photodynamic therapy with aminolevulinic acid or its methyl ester in skin rejuvenation and wound healing: An update

In dermatology, photodynamic therapy (PDT) is widely used in skin tumors, infections, etc., because of the killing effect triggered by toxic reactive oxygen species (ROS). However, the ROS concentration is determined by various photosensitizer concentrations and formulations, as well as various irradiation parameters. Low-dose PDT leads to sufficiently low ROS level, which results in biological effects that are the exact opposite of the killing potency. Therefore, in recent years, low-dose PDT has been exploited in improving aging and wound. Low-dose ALA/MAL PDT might improve aging through promoting the proliferation of fibroblasts, blocking DNA damage, counteracting oxidative stress, inhibiting melanogenesis, and remodeling lymphatic vessels in aged skin. Promoting fibroblasts and epidermal stem cells proliferation and migration, promoting granulation tissue formation and angiogenesis and regulating the inflammatory process might be the mechanisms of low-dose ALA/MAL PDT in wound healing. Nevertheless, the positive effects of low-dose PDT have not been thoroughly investigated in dermatology, and high-quality studies are still needed to fill the relevant vacancy.     

The advanced glycation end-products (AGEs)/ROS/NLRP3 inflammasome axis contributes to delayed diabetic corneal wound healing and nerve regeneration

Diabetic keratopathy (DK) is an important diabetic complication at the ocular surface. Chronic low-grade inflammation mediated by the NLRP3 inflammasome promotes pathogenesis of diabetes and its complications. However, the effect of the NLRP3 inflammasome on DK pathogenesis remains elusive. Wild-type (WT) and Nlrp3 knockout (KO) C57BL/6 mice were used to establish a type I diabetes model by intraperitoneal injection of streptozotocin. The effect of the NLRP3 inflammasome on diabetic corneal wound healing and never regeneration was examined by a corneal epithelial abrasion model. Western blot, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and pharmacological treatment were performed to investigate the regulatory mechanism of advanced glycation end products (AGEs) on NLRP3 inflammasome activation and corneal wound healing in vivo. The cultured mouse corneal epithelial cells (TKE2) were used to evaluate the effect and mechanism of AGEs on NLRP3 inflammasome activation in vitro. We revealed that NLRP3 inflammasome-mediated inflammation and pyroptosis contributed to DK pathogenesis. Under physiological conditions, the NLRP3 inflammasome was required for corneal wound healing and nerve regeneration. However, under a diabetic scenario, sustained activation of the NLRP3 inflammasome resulted in postponed corneal wound healing and impaired nerve regeneration. Mechanistically, the accumulated AGEs promoted hyperactivation of the NLRP3 inflammasome through ROS production. Moreover, genetically and pharmacologically blocking the AGEs/ROS/NLRP3 inflammasome axis significantly expedited diabetic corneal epithelial wound closure and nerve regeneration. Our results revealed that AGEs-induced hyperactivation of the NLRP3 inflammasome resulted in delayed diabetic corneal wound healing and impaired nerve regeneration, which further highlighted the NLRP3 inflammasome as a promising target for DK treatment.