Optimization of transdermal deferoxamine leads to enhanced efficacy in healing skin wounds.

Chronic wounds remain a significant burden to both the healthcare system and individual patients, indicating an urgent need for new interventions. Deferoxamine (DFO), an iron-chelating agent clinically used to treat iron toxicity, has been shown to reduce oxidative stress and increase hypoxia-inducible factor-1 alpha (HIF-1α) activation, thereby promoting neovascularization and enhancing regeneration in chronic wounds. However due to its short half-life and adverse side effects associated with systemic absorption, there is a pressing need for targeted DFO delivery. We recently published a preclinical proof of concept drug delivery system (TDDS) which showed that transdermally applied DFO is effective in improving chronic wound healing. Here we present an enhanced TDDS (eTDDS) comprised exclusively of FDA-compliant constituents to optimize drug release and expedite clinical translation. We evaluate the eTDDS to the original TDDS and compare this with other commonly used delivery methods including DFO drip-on and polymer spray applications. The eTDDS displayed excellent physicochemical characteristics and markedly improved DFO delivery into human skin when compared to other topical application techniques. We demonstrate an accelerated wound healing response with the eTDDS treatment resulting in significantly increased wound vascularity, dermal thickness, collagen deposition and tensile strength. Together, these findings highlight the immediate clinical potential of DFO eTDDS to treating diabetic wounds. Further, the topical drug delivery platform has important implications for targeted pharmacologic therapy of a wide range of cutaneous diseases.

Age-associated intracellular superoxide dismutase deficiency potentiates dermal fibroblast dysfunction during wound healing.

Reactive oxygen species (ROS) impair wound healing through destructive oxidation of intracellular proteins, lipids and nucleic acids. Intracellular superoxide dismutase (SOD1) regulates ROS levels and plays a critical role in tissue homoeostasis. Recent evidence suggests that age-associated wound healing impairments may partially result from decreased SOD1 expression. We investigated the mechanistic basis by which increased oxidative stress links to age-associated impaired wound healing. Fibroblasts were isolated from unwounded skin of young and aged mice, and myofibroblast differentiation was assessed by measuring α-smooth muscle actin and collagen gel contraction. Excisional wounds were created on young and aged mice to study the healing rate, ROS levels and SOD1 expression. A mechanistic link between oxidative stress and fibroblast function was explored by assessing the TGF-ß1 signalling pathway components in young and aged mice. Age-related wounds displayed reduced myofibroblast differentiation and delayed wound healing, consistent with a decrease in the in vitro capacity for fibroblast-myofibroblast transition following oxidative stress. Young fibroblasts with normal SOD1 expression exhibited increased phosphorylation of ERK in response to elevated ROS. In contrast, aged fibroblasts with reduced SOD1 expression displayed a reduced capacity to modulate intracellular ROS. Collectively, age-associated wound healing impairments are associated with fibroblast dysfunction that is likely the result of decreased SOD1 expression and subsequent dysregulation of intracellular ROS. Strategies targeting these mechanisms may suggest a new therapeutic approach in the treatment of chronic non-healing wounds in the aged population.

Small molecule inhibition of dipeptidyl peptidase-4 enhances bone marrow progenitor cell function and angiogenesis in diabetic wounds.

In diabetes, stromal cell-derived factor-1 (SDF-1) expression and progenitor cell recruitment are reduced. Dipeptidyl peptidase-4 (DPP-4) inhibits SDF-1 expression and progenitor cell recruitment. Here we examined the impact of the DPP-4 inhibitor, MK0626, on progenitor cell kinetics in the context of wound healing. Wildtype (WT) murine fibroblasts cultured under high-glucose to reproduce a diabetic microenvironment were exposed to MK0626, glipizide, or no treatment, and SDF-1 expression was measured with ELISA. Diabetic mice received MK0626, glipizide, or no treatment for 6 weeks and then were wounded. Immunohistochemistry was used to quantify neovascularization and SDF-1 expression. Gene expression was measured at the RNA and protein level using quantitative polymerase chain reaction and ELISA, respectively. Flow cytometry was used to characterize bone marrow-derived mesenchymal progenitor cell (BM-MPC) population recruitment to wounds. BM-MPC gene expression was assayed using microfluidic single cell analysis. WT murine fibroblasts exposed to MK0626 demonstrated increased SDF-1 expression. MK0626 treatment significantly accelerated wound healing and increased wound vascularity, SDF-1 expression, and dermal thickness in diabetic wounds. MK0626 treatment increased the number of BM-MPCs present in bone marrow and in diabetic wounds. MK0626 had no effect on BM-MPC population dynamics. BM-MPCs harvested from MK0626-treated mice exhibited increased chemotaxis in response to SDF-1 when compared to diabetic controls. Treatment with a DPP-4 inhibitor significantly improved wound healing, angiogenesis, and endogenous progenitor cell recruitment in the setting of diabetes.

The Role of Focal Adhesion Kinase in Keratinocyte Fibrogenic Gene Expression.

Abnormal skin scarring causes functional impairment, psychological stress, and high socioeconomic cost. Evidence shows that altered mechanotransduction pathways have been linked to both inflammation and fibrosis, and that focal adhesion kinase (FAK) is a key mediator of these processes. We investigated the importance of keratinocyte FAK at the single cell level in key fibrogenic pathways critical for scar formation. Keratinocytes were isolated from wildtype and keratinocyte-specific FAK-deleted mice, cultured, and sorted into single cells. Keratinocytes were evaluated using a microfluidic-based platform for high-resolution transcriptional analysis. Partitive clustering, gene enrichment analysis, and network modeling were applied to characterize the significance of FAK on regulating keratinocyte subpopulations and fibrogenic pathways important for scar formation. Considerable transcriptional heterogeneity was observed within the keratinocyte populations. FAK-deleted keratinocytes demonstrated increased expression of genes integral to mechanotransduction and extracellular matrix production, including Igtbl, Mmpla, and Col4a1. Transcriptional activities upon FAK deletion were not identical across all single keratinocytes, resulting in higher frequency of a minor subpopulation characterized by a matrix-remodeling profile compared to wildtype keratinocyte population. The importance of keratinocyte FAK signaling gene expression was revealed. A minor subpopulation of keratinocytes characterized by a matrix-modulating profile may be a keratinocyte subset important for mechanotransduction and scar formation.

Ultrasound-assisted liposuction provides a source for functional adipose-derived stromal cells.

BACKGROUND AIMS: Regenerative medicine employs human mesenchymal stromal cells (MSCs) for their multi-lineage plasticity and their pro-regenerative cytokine secretome. Adipose-derived mesenchymal stromal cells (ASCs) are concentrated in fat tissue, and the ease of harvest via liposuction makes them a particularly interesting cell source. However, there are various liposuction methods, and few have been assessed regarding their impact on ASC functionality. Here we study the impact of the two most popular ultrasound-assisted liposuction (UAL) devices currently in clinical use, VASER (Solta Medical) and Lysonix 3000 (Mentor) on ASCs. METHODS: After lipoaspirate harvest and processing, we sorted for ASCs using fluorescent-assisted cell sorting based on an established surface marker profile (CD34+CD31-CD45-). ASC yield, viability, osteogenic and adipogenic differentiation capacity and in vivo regenerative performance were assessed. RESULTS: Both UAL samples demonstrated equivalent ASC yield and viability. VASER UAL ASCs showed higher osteogenic and adipogenic marker expression, but a comparable differentiation capacity was observed. Soft tissue healing and neovascularization were significantly enhanced via both UAL-derived ASCs in vivo, and there was no significant difference between the cell therapy groups. CONCLUSIONS: Taken together, our data suggest that UAL allows safe and efficient harvesting of the mesenchymal stromal cellular fraction of adipose tissue and that cells harvested via this approach are suitable for cell therapy and tissue engineering applications.

Comparison of the Hydroxylase Inhibitor Dimethyloxalylglycine and the Iron Chelator Deferoxamine in Diabetic and Aged Wound Healing.

BACKGROUND: A hallmark of diabetes mellitus is the breakdown of almost every reparative process in the human body, leading to critical impairments of wound healing. Stabilization and activity of the transcription factor hypoxia-inducible factor (HIF)-1α is impaired in diabetes, leading to deficits in new blood vessel formation in response to injury. In this article, the authors compare the effectiveness of two promising small-molecule therapeutics, the hydroxylase inhibitor dimethyloxalylglycine and the iron chelator deferoxamine, for attenuating diabetes-associated deficits in cutaneous wound healing by enhancing HIF-1α activation. METHODS: HIF-1α stabilization, phosphorylation, and transactivation were measured in murine fibroblasts cultured under normoxic or hypoxic and low-glucose or high-glucose conditions following treatment with deferoxamine or dimethyloxalylglycine. In addition, diabetic wound healing and neovascularization were evaluated in db/db mice treated with topical solutions of either deferoxamine or dimethyloxalylglycine, and the efficacy of these molecules was also compared in aged mice. RESULTS: The authors show that deferoxamine stabilizes HIF-1α expression and improves HIF-1α transactivity in hypoxic and hyperglycemic states in vitro, whereas the effects of dimethyloxalylglycine are significantly blunted under hyperglycemic hypoxic conditions. In vivo, both dimethyloxalylglycine and deferoxamine enhance wound healing and vascularity in aged mice, but only deferoxamine universally augmented wound healing and neovascularization in the setting of both advanced age and diabetes. CONCLUSION: This first direct comparison of deferoxamine and dimethyloxalylglycine in the treatment of impaired wound healing suggests significant therapeutic potential for topical deferoxamine treatment in ischemic and diabetic disease.

Microfluidic single-cell transcriptional analysis rationally identifies novel surface marker profiles to enhance cell-based therapies.

Current progenitor cell therapies have only modest efficacy, which has limited their clinical adoption. This may be the result of a cellular heterogeneity that decreases the number of functional progenitors delivered to diseased tissue, and prevents correction of underlying pathologic cell population disruptions. Here, we develop a high-resolution method of identifying phenotypically distinct progenitor cell subpopulations via single-cell transcriptional analysis and advanced bioinformatics. When combined with high-throughput cell surface marker screening, this approach facilitates the rational selection of surface markers for prospective isolation of cell subpopulations with desired transcriptional profiles. We establish the usefulness of this platform in costly and highly morbid diabetic wounds by identifying a subpopulation of progenitor cells that is dysfunctional in the diabetic state, and normalizes diabetic wound healing rates following allogeneic application. We believe this work presents a logical framework for the development of targeted cell therapies that can be customized to any clinical application.

Suction assisted liposuction does not impair the regenerative potential of adipose derived stem cells.

BACKGROUND: Adipose-derived stem cells (ASCs) have been identified as a population of multipotent cells with promising applications in tissue engineering and regenerative medicine. ASCs are abundant in fat tissue, which can be safely harvested through the minimally invasive procedure of liposuction. However, there exist a variety of different harvesting methods, with unclear impact on ASC regenerative potential. The aim of this study was thus to compare the functionality of ASCs derived from the common technique of suction-assisted lipoaspiration (SAL) versus resection. METHODS: Human adipose tissue was obtained from paired abdominoplasty and SAL samples from three female donors, and was processed to isolate the stromal vascular fraction. Fluorescence-activated cell sorting was used to determine ASC yield, and cell viability was assayed. Adipogenic and osteogenic differentiation capacity were assessed in vitro using phenotypic staining and quantification of gene expression. Finally, ASCs were applied in an in vivo model of tissue repair to evaluate their regenerative potential. RESULTS: SAL specimens provided significantly fewer ASCs when compared to excised fat tissue, however, with equivalent viability. SAL-derived ASCs demonstrated greater expression of the adipogenic markers FABP-4 and LPL, although this did not result in a difference in adipogenic differentiation. There were no differences detected in osteogenic differentiation capacity as measured by alkaline phosphatase, mineralization or osteogenic gene expression. Both SAL- and resection-derived ASCs enhanced significantly cutaneous healing and vascularization in vivo, with no significant difference between the two groups. CONCLUSION: SAL provides viable ASCs with full capacity for multi-lineage differentiation and tissue regeneration, and is an effective method of obtaining ASCs for cell-based therapies.

Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.

The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

Challenges and Opportunities in Drug Delivery for Wound Healing.

Significance: Chronic wounds remain a significant public health problem. Alterations in normal physiological processes caused by aging or diabetes lead to impaired tissue repair and the development of chronic and nonhealing wounds. Understanding the unique features of the wound environment will be required to develop new therapeutics that impact these disabling conditions. New drug-delivery systems (DDSs) may enhance current and future therapies for this challenging clinical problem. Recent Advances: Historically, physical barriers and biological degradation limited the efficacy of DDSs in wound healing. In aiming at improving and optimizing drug delivery, recent data suggest that combinations of delivery mechanisms, such as hydrogels, small molecules, RNA interference (RNAi), as well as growth factor and stem cell-based therapies (biologics), could offer exciting new opportunities for improving tissue repair. Critical Issues: The lack of effective therapeutic approaches to combat the significant disability associated with chronic wounds has become an area of increasing clinical concern. However, the unique challenges of the wound environment have limited the development of effective therapeutic options for clinical use. Future Directions: New platforms presented in this review may provide clinicians and scientists with an improved understanding of the alternatives for drug delivery in wound care, which may facilitate the development of new therapeutic approaches for patients.

Adipose-Derived Stem Cell-Seeded Hydrogels Increase Endogenous Progenitor Cell Recruitment and Neovascularization in Wounds.

Adipose-derived mesenchymal stem cells (ASCs) are appealing for cell-based wound therapies because of their accessibility and ease of harvest, but their utility is limited by poor cell survival within the harsh wound microenvironment. In prior work, our laboratory has demonstrated that seeding ASCs within a soft pullulan-collagen hydrogel enhances ASC survival and improves wound healing. To more fully understand the mechanism of this therapy, we examined whether ASC-seeded hydrogels were able to modulate the recruitment and/or functionality of endogenous progenitor cells. Employing a parabiosis model and fluorescence-activated cell sorting analysis, we demonstrate that application of ASC-seeded hydrogels to wounds, when compared with injected ASCs or a noncell control, increased the recruitment of provascular circulating bone marrow-derived mesenchymal progenitor cells (BM-MPCs). BM-MPCs comprised 23.0% of recruited circulating progenitor cells in wounds treated with ASC-seeded hydrogels versus 8.4% and 2.1% in those treated with controls, p < 0.05. Exploring the potential for functional modulation of BM-MPCs, we demonstrate a statistically significant increase in BM-MPC migration, proliferation, and tubulization when exposed to hydrogel-seeded ASC-conditioned medium versus control ASC-conditioned medium (73.8% vs. 51.4% scratch assay closure; 9.1% vs. 1.4% proliferation rate; 10.2 vs. 5.5 tubules/HPF; p < 0.05 for all assays). BM-MPC expression of genes related to cell stemness and angiogenesis was also significantly increased following exposure to hydrogel-seeded ASC-conditioned medium (p < 0.05). These data suggest that ASC-seeded hydrogels improve both progenitor cell recruitment and functionality to effect greater neovascularization.

Murine Dermal Fibroblast Isolation by FACS.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin(-)) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Stem Cells in Wound Healing: The Future of Regenerative Medicine? A Mini-Review.

The increased risk of disease and decreased capacity to respond to tissue insult in the setting of aging results from complex changes in homeostatic mechanisms, including the regulation of oxidative stress and cellular heterogeneity. In aged skin, the healing capacity is markedly diminished resulting in a high risk for chronic wounds. Stem cell-based therapies have the potential to enhance cutaneous regeneration, largely through trophic and paracrine activity. Candidate cell populations for therapeutic application include adult mesenchymal stem cells, embryonic stem cells and induced pluripotent stem cells. Autologous cell-based approaches are ideal to minimize immune rejection but may be limited by the declining cellular function associated with aging. One strategy to overcome age-related impairments in various stem cell populations is to identify and enrich with functionally superior stem cell subsets via single cell transcriptomics. Another approach is to optimize cell delivery to the harsh environment of aged wounds via scaffold-based cell applications to enhance engraftment and paracrine activity of therapeutic stem cells. In this review, we shed light on challenges and recent advances surrounding stem cell therapies for wound healing and discuss limitations for their clinical adoption.

Extracellular superoxide dismutase deficiency impairs wound healing in advanced age by reducing neovascularization and fibroblast function.

Advanced age is characterized by impairments in wound healing, and evidence is accumulating that this may be due in part to a concomitant increase in oxidative stress. Extended exposure to reactive oxygen species (ROS) is thought to lead to cellular dysfunction and organismal death via the destructive oxidation of intra-cellular proteins, lipids and nucleic acids. Extracellular superoxide dismutase (ecSOD/SOD3) is a prime antioxidant enzyme in the extracellular space that eliminates ROS. Here, we demonstrate that reduced SOD3 levels contribute to healing impairments in aged mice. These impairments include delayed wound closure, reduced neovascularization, impaired fibroblast proliferation and increased neutrophil recruitment. We further establish that SOD3 KO and aged fibroblasts both display reduced production of TGF-ß1, leading to decreased differentiation of fibroblasts into myofibroblasts. Taken together, these results suggest that wound healing impairments in ageing are associated with increased levels of ROS, decreased SOD3 expression and impaired extracellular oxidative stress regulation. Our results identify SOD3 as a possible target to correct age-related cellular dysfunction in wound healing.

Cell-Assisted Lipotransfer Improves Volume Retention in Irradiated Recipient Sites and Rescues Radiation-Induced Skin Changes.

Radiation therapy is not only a mainstay in the treatment of many malignancies but also results in collateral obliteration of microvasculature and dermal/subcutaneous fibrosis. Soft tissue reconstruction of hypovascular, irradiated recipient sites through fat grafting remains challenging; however, a coincident improvement in surrounding skin quality has been noted. Cell-assisted lipotransfer (CAL), the enrichment of fat with additional adipose-derived stem cells (ASCs) from the stromal vascular fraction, has been shown to improve fat volume retention, and enhanced outcomes may also be achieved with CAL at irradiated sites. Supplementing fat grafts with additional ASCs may also augment the regenerative effect on radiation-damaged skin. In this study, we demonstrate the ability for CAL to enhance fat graft volume retention when placed beneath the irradiated scalps of immunocompromised mice. Histologic metrics of fat graft survival were also appreciated, with improved structural qualities and vascularity. Finally, rehabilitation of radiation-induced soft tissue changes were also noted, as enhanced amelioration of dermal thickness, collagen content, skin vascularity, and biomechanical measures were all observed with CAL compared to unsupplemented fat grafts. Supplementation of fat grafts with ASCs therefore shows promise for reconstruction of complex soft tissue defects following adjuvant radiotherapy.

Ultrasound-Assisted Liposuction Does Not Compromise the Regenerative Potential of Adipose-Derived Stem Cells.

Human mesenchymal stem cells (MSCs) have recently become a focus of regenerative medicine, both for their multilineage differentiation capacity and their excretion of proregenerative cytokines. Adipose-derived mesenchymal stem cells (ASCs) are of particular interest because of their abundance in fat tissue and the ease of harvest via liposuction. However, little is known about the impact of different liposuction methods on the functionality of ASCs. Here we evaluate the regenerative abilities of ASCs harvested via a third-generation ultrasound-assisted liposuction (UAL) device versus ASCs obtained via standard suction-assisted lipoaspiration (SAL). Lipoaspirates were sorted using fluorescent assisted cell sorting based on an established surface-marker profile (CD34+/CD31-/CD45-), to obtain viable ASCs. Yield and viability were compared and the differentiation capacities of the ASCs were assessed. Finally, the regenerative potential of ASCs was examined using an in vivo model of tissue regeneration. UAL- and SAL-derived samples demonstrated equivalent ASC yield and viability, and UAL ASCs were not impaired in their osteogenic, adipogenic, or chondrogenic differentiation capacity. Equally, quantitative real-time polymerase chain reaction showed comparable expression of most osteogenic, adipogenic, and key regenerative genes between both ASC groups. Cutaneous regeneration and neovascularization were significantly enhanced in mice treated with ASCs obtained by either UAL or SAL compared with controls, but there were no significant differences in healing between cell-therapy groups. We conclude that UAL is a successful method of obtaining fully functional ASCs for regenerative medicine purposes. Cells harvested with this alternative approach to liposuction are suitable for cell therapy and tissue engineering applications. Significance: Adipose-derived mesenchymal stem cells (ASCs) are an appealing source of therapeutic progenitor cells because of their multipotency, diverse cytokine profile, and ease of harvest via liposuction. Alternative approaches to classical suction-assisted liposuction are gaining popularity; however, little evidence exists regarding the impact of different liposuction methods on the regenerative functionality of ASCs. Human ASC characteristics and regenerative capacity were assessed when harvested via ultrasound-assisted (UAL) versus standard suction-assisted liposuction. ASCs obtained via UAL were of equal quality when directly compared with the current gold standard harvest method. UAL is an adjunctive source of fully functional mesenchymal stem cells for applications in basic research and clinical therapy.

Fibroblast-Specific Deletion of Hypoxia Inducible Factor-1 Critically Impairs Murine Cutaneous Neovascularization and Wound Healing.

BACKGROUND: Diabetes and aging are known risk factors for impaired neovascularization in response to ischemic insult, resulting in chronic wounds, and poor outcomes following myocardial infarction and cerebrovascular injury. Hypoxia-inducible factor (HIF)-1α, has been identified as a critical regulator of the response to ischemic injury and is dysfunctional in diabetic and elderly patients. To better understand the role of this master hypoxia regulator within cutaneous tissue, the authors generated and evaluated a fibroblast-specific HIF-1α knockout mouse model. METHODS: The authors generated floxed HIF-1 mice (HIF-1) by introducing loxP sites around exon 1 of the HIF-1 allele in C57BL/6J mice. Fibroblast-restricted HIF-1α knockout (FbKO) mice were generated by breeding our HIF-1 with tamoxifen-inducible Col1a2-Cre mice (Col1a2-CreER). HIF-1α knockout was evaluated on a DNA, RNA, and protein level. Knockout and wild-type mice were subjected to ischemic flap and wound healing models, and CD31 immunohistochemistry was performed to assess vascularity of healed wounds. RESULTS: Quantitative real-time polymerase chain reaction of FbKO skin demonstrated significantly reduced Hif1 and Vegfa expression compared with wild-type. This finding was confirmed at the protein level (p < 0.05). HIF-1α knockout mice showed significantly impaired revascularization of ischemic tissue and wound closure and vascularity (p < 0.05). CONCLUSIONS: Loss of HIF-1α from fibroblasts results in delayed wound healing, reduced wound vascularity, and significant impairment in the ischemic neovascular response. These findings provide new insight into the importance of cell-specific responses to hypoxia during cutaneous neovascularization.

Wnt signaling induces epithelial differentiation during cutaneous wound healing.

Cutaneous wound repair in adult mammals typically does not regenerate original dermal architecture. Skin that has undergone repair following injury is not identical to intact uninjured skin. This disparity may be caused by differences in the mechanisms that regulate postnatal cutaneous wound repair compared to embryonic skin development and thus we seek a deeper understanding of the role that Wnt signaling plays in the mechanisms of skin repair in both fetal and adult wounds. The influence of secreted Wnt signaling proteins in tissue homeostasis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. Wnt signaling is activated by wounding and participates in every subsequent stage of the healing process from the control of inflammation and programmed cell death, to the mobilization of stem cell reservoirs within the wound site. Endogenous Wnt signaling augmentation represents an attractive option to aid in the restoration of cutaneous wounds, as the complex mechanisms of the Wnt pathway have been increasingly investigated over the years. In this review, we summarize recent data elucidating the roles that Wnt signaling plays in cutaneous wound healing process.

dsRNA Released by Tissue Damage Activates TLR3 to Drive Skin Regeneration.

Regeneration of skin and hair follicles after wounding--a process known as wound-induced hair neogenesis (WIHN)--is a rare example of adult organogenesis in mammals. As such, WIHN provides a unique model system for deciphering mechanisms underlying mammalian regeneration. Here, we show that dsRNA, which is released from damaged skin, activates Toll-Like Receptor 3 (TLR3) and its downstream effectors IL-6 and STAT3 to promote hair follicle regeneration. Conversely, TLR3-deficient animals fail to initiate WIHN. TLR3 activation promotes expression of hair follicle stem cell markers and induces elements of the core hair morphogenetic program, including ectodysplasin A receptor (EDAR) and the Wnt and Shh pathways. Our results therefore show that dsRNA and TLR3 link the earliest events of mammalian skin wounding to regeneration and suggest potential therapeutic approaches for promoting hair neogenesis.

Exercise induces stromal cell-derived factor-1α-mediated release of endothelial progenitor cells with increased vasculogenic function.

BACKGROUND: Endothelial progenitor cells have been shown to traffic to and incorporate into ischemic tissues, where they participate in new blood vessel formation, a process termed vasculogenesis. Previous investigation has demonstrated that endothelial progenitor cells appear to mobilize from bone marrow to the peripheral circulation after exercise. In this study, the authors investigate potential etiologic factors driving this mobilization and investigate whether the mobilized endothelial progenitor cells are the same as those present at baseline. METHODS: Healthy volunteers (n = 5) performed a monitored 30-minute run to maintain a heart rate greater than 140 beats/min. Venous blood samples were collected before, 10 minutes after, and 24 hours after exercise. Endothelial progenitor cells were isolated and evaluated. RESULTS: Plasma levels of stromal cell-derived factor-1α significantly increased nearly two-fold immediately after exercise, with a nearly four-fold increase in circulating endothelial progenitor cells 24 hours later. The endothelial progenitor cells isolated following exercise demonstrated increased colony formation, proliferation, differentiation, and secretion of angiogenic cytokines. Postexercise endothelial progenitor cells also exhibited a more robust response to hypoxic stimulation. CONCLUSIONS: Exercise appears to mobilize endothelial progenitor cells and augment their function by means of stromal cell-derived factor 1α-dependent signaling. The population of endothelial progenitor cells mobilized following exercise is primed for vasculogenesis with increased capacity for proliferation, differentiation, secretion of cytokines, and responsiveness to hypoxia. Given the evidence demonstrating positive regenerative effects of exercise, this may be one possible mechanism for its benefits.