Zeshaan N. Maan

Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential

Fibroblasts in fibrosis Excess fibrous connective tissue, similar to scarring, forms during the repair of injuries. Fibroblasts are known to be involved, but their role is poorly characterized. Rinkevich et al. identify two lineages of dermal fibroblasts in the dorsal skin of mice (see the Perspective by Sennett and Rendl). A fibrogenic lineage, defined by embryonic expression of Engrailed-1, plays a central role in dermal development, wound healing, radiation-induced fibrosis, and cancer stroma formation. Targeted inhibition of this lineage results in reduced melanoma growth and scar formation, with no effect on the structural integrity of the healed skin, thus indicating therapeutic approaches for treating fibrotic disease. Science, this issue 10.1126/science.aaa2151; see also p. 284 An embryonic fibroblast lineage deposits connective tissue in wounds. [Also see Perspective by Sennett and Rendl] INTRODUCTION Fibroblasts are the predominant cell type that synthesizes and remodels the extracellular matrix in organs during both embryonic and adult life and are central to the fibrotic response across a range of pathologic states. Morphologically, they are most commonly defined as elongated, spindle-shaped cells that readily adhere to and migrate over tissue culture substrates. However, fibroblasts exhibit a variety of shapes and sizes, depending on the physiologic or pathologic state of the host tissue, and represent a heterogeneous population of cells with diverse features that remain largely undefined. In cutaneous tissues, fibroblasts display considerable functional variation during wound repair, depending on developmental time, and between anatomic sites. For example, wounds in the oral cavity remodel with minimal scar formation, whereas scar tissue deposition within cutaneous wounds is substantial. The mechanisms underlying this diversity of regenerative responses in cutaneous tissues have remained largely underexplored. RATIONALE The effective development of treatments for fibrosis depends on a mechanistic understanding of its pathogenesis. The identification and characterization of distinct lineages of fibroblasts, based on functional role, hold potential value for developing therapeutic approaches to fibrosis. We employed a nonselective depletion-based fluorescence-activated cell sorting strategy to isolate fibroblasts from a murine model that labels a particular lineage of cells based on the gene expression of Engrailed-1 (En1) in its embryonic progenitors. Using this reporter mouse, we reveal the presence of at least two functionally distinct embryonic fibroblast lineages in murine dorsal skin and characterize a single lineage that plays a primary role in connective tissue formation. RESULTS Genetic lineage tracing and transplantation assays demonstrate that a single somitic-derived fibroblast lineage that is defined by embryonic expression of En1 is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Reciprocal transplantation of distinct fibroblast lineages between the dorsal back and oral cavity induces ectopic dermal architectures that mimic their place of origin rather than their site of transplantation. Lineage-specific cell ablation using transgenic-mediated expression of the simian diphtheria toxin receptor in conjunction with localized administration of diphtheria toxin leads to diminished connective tissue deposition in wounds and significantly reduces melanoma growth in the dorsal skin of mice. Tensile strength testing reveals that, although scar formation is significantly reduced in wounds treated with diphtheria toxin to ablate the En1 lineage, as compared with control wounds, tensile strength in lineage-ablated wounds is not significantly affected. Using flow cytometry and in silico approaches, we identify CD26/dipeptidyl peptidase-4 (DPP4) as a surface marker that allows for the isolation of this fibrogenic, scar-forming lineage. Small molecule–based inhibition of CD26/DPP4 enzymatic activity in the wound bed of wild-type mice during wound healing results in diminished cutaneous scarring after excisional wounding. CONCLUSION We have identified multiple lineages of fibroblasts in the dorsal skin. Among these, we have characterized a single lineage responsible for the fibrotic response to injury in the dorsal skin of mice and demonstrated that targeted inhibition of this lineage results in reduced scar formation with no effect on the structural integrity of the healed skin. Furthermore, these studies demonstrate that intra- and intersite diversity of dermal architectures are set embryonically and are maintained postnatally by distinct lineages of fibroblasts in different anatomic locations. These results hold promise for the development of therapeutic approaches to fibrotic disease, wound healing, and cancer progression in humans. Schematic showing reduced scarring with targeted ablation/inhibition of En1 fibroblasts. Fibroblasts derived from embryonic precursors expressing En1 are responsible for most connective tissue deposition in skin fibrosis. Targeted ablation/inhibition of this lineage leads to a reduction in fibrosis during wound repair and tumor stroma formation. These findings may lead to the elimination of scarring and other types of fibrotic tissue disease. Green cells, En1-positive fibroblasts; red cells, En1-negative fibroblasts. CREDIT: SILHOUETTES FROM PHYLOPIC.ORG Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined. We reveal the presence of at least two fibroblast lineages in murine dorsal skin. Lineage tracing and transplantation assays demonstrate that a single fibroblast lineage is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Lineage-specific cell ablation leads to diminished connective tissue deposition in wounds and reduces melanoma growth. Using flow cytometry, we identify CD26/DPP4 as a surface marker that allows isolation of this lineage. Small molecule–based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. Identification and isolation of these lineages hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.

Aging disrupts cell subpopulation dynamics and diminishes the function of mesenchymal stem cells

Advanced age is associated with an increased risk of vascular morbidity, attributable in part to impairments in new blood vessel formation. Mesenchymal stem cells (MSCs) have previously been shown to play an important role in neovascularization and deficiencies in these cells have been described in aged patients. Here we utilize single cell transcriptional analysis to determine the effect of aging on MSC population dynamics. We identify an age-related depletion of a subpopulation of MSCs characterized by a pro-vascular transcriptional profile. Supporting this finding, we demonstrate that aged MSCs are also significantly compromised in their ability to support vascular network formation in vitro and in vivo. Finally, aged MSCs are unable to rescue age-associated impairments in cutaneous wound healing. Taken together, these data suggest that age-related changes in MSC population dynamics result in impaired therapeutic potential of aged progenitor cells. These findings have critical implications for therapeutic cell source decisions (autologous versus allogeneic) and indicate the necessity of strategies to improve functionality of aged MSCs.

Diabetes impairs the angiogenic potential of adipose-derived stem cells by selectively depleting cellular subpopulations

IntroductionPathophysiologic changes associated with diabetes impair new blood vessel formation and wound healing. Mesenchymal stem cells derived from adipose tissue (ASCs) have been used clinically to promote healing, although it remains unclear whether diabetes impairs their functional and therapeutic capacity.MethodsIn this study, we examined the impact of diabetes on the murine ASC niche as well as on the potential of isolated cells to promote neovascularization in vitro and in vivo. A novel single-cell analytical approach was used to interrogate ASC heterogeneity and subpopulation dynamics in this pathologic setting.ResultsOur results demonstrate that diabetes alters the ASC niche in situ and that diabetic ASCs are compromised in their ability to establish a vascular network both in vitro and in vivo. Moreover, these diabetic cells were ineffective in promoting soft tissue neovascularization and wound healing. Single-cell transcriptional analysis identified a subpopulation of cells which was diminished in both type 1 and type 2 models of diabetes. These cells were characterized by the high expression of genes known to be important for new blood vessel growth.ConclusionsPerturbations in specific cellular subpopulations, visible only on a single-cell level, represent a previously unreported mechanism for the dysfunction of diabetic ASCs. These data suggest that the utility of autologous ASCs for cell-based therapies in patients with diabetes may be limited and that interventions to improve cell function before application are warranted.

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.

Mechanotransduction and fibrosis

Scarring and tissue fibrosis represent a significant source of morbidity in the United States. Despite considerable research focused on elucidating the mechanisms underlying cutaneous scar formation, effective clinical therapies are still in the early stages of development. A thorough understanding of the various signaling pathways involved is essential to formulate strategies to combat fibrosis and scarring. While initial efforts focused primarily on the biochemical mechanisms involved in scar formation, more recent research has revealed a central role for mechanical forces in modulating these pathways. Mechanotransduction, which refers to the mechanisms by which mechanical forces are converted to biochemical stimuli, has been closely linked to inflammation and fibrosis and is believed to play a critical role in scarring. This review provides an overview of our current understanding of the mechanisms underlying scar formation, with an emphasis on the relationship between mechanotransduction pathways and their therapeutic implications.

Scarless wound healing: chasing the holy grail.

Summary: Over 100 million patients acquire scars in the industrialized world each year, primarily as a result of elective operations. Although undefined, the global incidence of scarring is even larger, extending to significant numbers of burn and other trauma-related wounds. Scars have the potential to exert a profound psychological and physical impact on the individual. Beyond aesthetic considerations and potential disfigurement, scarring can result in restriction of movement and reduced quality of life. The formation of a scar following skin injury is a consequence of wound healing occurring through reparative rather than regenerative mechanisms. In this article, the authors review the basic stages of wound healing; differences between adult and fetal wound healing; various mechanical, genetic, and pharmacologic strategies to reduce scarring; and the biology of skin stem/progenitor cells that may hold the key to scarless regeneration.

Tissue Engineering and Regenerative Repair in Wound Healing

Wound healing is a highly evolved defense mechanism against infection and further injury. It is a complex process involving multiple cell types and biological pathways. Mammalian adult cutaneous wound healing is mediated by a fibroproliferative response leading to scar formation. In contrast, early to mid-gestational fetal cutaneous wound healing is more akin to regeneration and occurs without scar formation. This early observation has led to extensive research seeking to unlock the mechanism underlying fetal scarless regenerative repair. Building upon recent advances in biomaterials and stem cell applications, tissue engineering approaches are working towards a recapitulation of this phenomenon. In this review, we describe the elements that distinguish fetal scarless and adult scarring wound healing, and discuss current trends in tissue engineering aimed at achieving scarless tissue regeneration.

Transdermal deferoxamine prevents pressure-induced diabetic ulcers

Significance Diabetes is the leading cause of nontraumatic amputations. There are no effective therapies to prevent diabetic ulcer formation and only modestly effective technologies to help with their healing. To enhance diabetic wound healing we designed a transdermal delivery system containing the FDA-approved small molecule deferoxamine, an iron chelator that increases defective hypoxia inducible factor-1 alpha transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. This system overcomes the challenge of delivering hydrophilic molecules through the normally impermeable stratum corneum and both prevents diabetic ulcer formation and improves the healing of existing diabetic wounds. This represents a prophylactic pharmacological agent to prevent ulcer formation that is rapidly translatable into the clinic and has the potential to ultimately transform the care and prevention of diabetic complications. There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.

Wound healing: an update

Wounds, both chronic and acute, continue to be a tremendous socioeconomic burden. As such, technologies drawn from many disciplines within science and engineering are constantly being incorporated into innovative wound healing therapies. While many of these therapies are experimental, they have resulted in new insights into the pathophysiology of wound healing, and in turn the development of more specialized treatments for both normal and abnormal wound healing states. Herein, we review some of the emerging technologies that are currently being developed to aid and improve wound healing after cutaneous injury.

Diabetes Irreversibly Depletes Bone Marrow-Derived Mesenchymal Progenitor Cell Subpopulations

Diabetic vascular pathology is largely attributable to impairments in tissue recovery from hypoxia. Circulating progenitor cells have been postulated to play a role in ischemic recovery, and deficiencies in these cells have been well described in diabetic patients. Here, we examine bone marrow–derived mesenchymal progenitor cells (BM-MPCs) that have previously been shown to be important for new blood vessel formation and demonstrate significant deficits in the context of diabetes. Further, we determine that this dysfunction is attributable to intrinsic defects in diabetic BM-MPCs that are not correctable by restoring glucose homeostasis. We identify two transcriptionally distinct subpopulations that are selectively depleted by both type 1 and type 2 diabetes, and these subpopulations have provasculogenic expression profiles, suggesting that they are vascular progenitor cells. These results suggest that the clinically observed deficits in progenitor cells may be attributable to selective and irreversible depletion of progenitor cell subsets in patients with diabetes.