Nrf2 is a key regulator of the antioxidant defense system, and pharmacological Nrf2 activation is a promising strategy for cancer prevention and promotion of tissue repair. Here we show, however, that activation of Nrf2 in fibroblasts induces cellular senescence. Using a combination of transcriptomics, matrix proteomics, chromatin immunoprecipitation and bioinformatics we demonstrate that fibroblasts with activated Nrf2 deposit a senescence-promoting matrix, with plasminogen activator inhibitor-1 being a key inducer of the senescence program. In vivo, activation of Nrf2 in fibroblasts promoted re-epithelialization of skin wounds, but also skin tumorigenesis. The pro-tumorigenic activity is of general relevance, since Nrf2 activation in skin fibroblasts induced the expression of genes characteristic for cancer-associated fibroblasts from different mouse and human tumors. Therefore, activated Nrf2 qualifies as a marker of the cancer-associated fibroblast phenotype. These data highlight the bright and the dark sides of Nrf2 and the need for time-controlled activation of this transcription factor.

Physiological angiogenesis refers to a naturally occurring process of blood vessel growth and regression, and it occurs as an integral component of tissue repair and regeneration. During wound healing, sprouting and branching results in an extensive yet immature and leaky neovascular network that ultimately resolves by systematic pruning of extraneous vessels to yield a stable, well-perfused vascular network ideally suited to maintain tissue homeostasis. While the molecular mechanisms of blood vessel growth have been explored in numerous cell and animal models in remarkable detail, the endogenous factors that prevent further angiogenesis and control vessel regression have not received much attention and are largely unknown. In this review, we introduce the relevant literature from various disciplines to fill the gaps in the current limited understanding of the major molecular and biomechanical inducers of vascular regression. The processes are described in the context of endothelial cell biology during wound healing: hypoxia-driven activation and sprouting followed by apoptosis or maturation of cells comprising the vasculature. We discuss and integrate the likely roles of a variety of endogenous factors, including oxygen availability, vessel perfusion and shear stress, intracellular negative feedback mechanisms (Spry2, vasohibin), soluble cytokines (CXCL10), matrix-binding proteins (TSP, PEDF), protein cleavage products (angiostatin, vasostatin), matrix-derived anti-angiogenic peptides (endostatin, arresten, canstatin, tumstatin), and the biomechanical properties of remodeling the extra-cellular matrix itself. These factors aid in the spatio-temporal control of blood vessel pruning by inducing specific anti-angiogenic signaling pathways in activated endothelial cells, pathways which compete with pro-angiogenic and maturation signals in the resolving wound. Gaining more insight into these mechanisms is bound to shed light on unresolved questions regarding scar formation, tissue regeneration, and increase our understanding of the many diseases with angiogenic phenotypes, especially cancer.

Wietecha MS, Król MJ, Michalczyk ER, Chen L, Gettins PG, DiPietro LA. Pigment epithelium-derived factor as a multifunctional regulator of wound healing. Am J Physiol Heart Circ Physiol 309: H812-H826, 2015. First published July 10, 2015; doi:10.1152/ajpheart.00153.2015.-During dermal wound repair, hypoxia-driven proliferation results in dense but highly permeable, disorganized microvascular networks, similar to those in solid tumors. Concurrently, activated dermal fibroblasts generate an angiopermissive, provisional extracellular matrix (ECM). Unlike cancers, wounds naturally resolve via blood vessel regression and ECM maturation, which are essential for reestablishing tissue homeostasis. Mechanisms guiding wound resolution are poorly understood; one candidate regulator is pigment epithelium-derived factor (PEDF), a secreted glycoprotein. PEDF is a potent antiangiogenic in models of pathological angiogenesis and a promising cancer and cardiovascular disease therapeutic, but little is known about its physiological function. To examine the roles of PEDF in physiological wound repair, we used a reproducible model of excisional skin wound healing in BALB/c mice. We show that PEDF is abundant in unwounded and healing skin, is produced primarily by dermal fibroblasts, binds to resident microvascular endothelial cells, and accumulates in dermal ECM and epidermis. PEDF transcript and protein levels were low during the inflammatory and proliferative phases of healing but increased in quantity and colocalization with microvasculature during wound resolution. Local antibody inhibition of endogenous PEDF delayed vessel regression and collagen maturation during the remodeling phase. Treatment of wounds with intradermal injections of exogenous, recombinant PEDF inhibited nascent angiogenesis by repressing endothelial proliferation, promoted vascular integrity and function, and increased collagen maturity. These results demonstrate that PEDF contributes to the resolution of healing wounds by causing regression of immature blood vessels and stimulating maturation of the vascular microenvironment, thus promoting a return to tissue homeostasis after injury.

Matrix deposition is essential for wound repair, but when excessive, leads to hypertrophic scars and fibrosis. The factors that control matrix deposition in skin wounds have only partially been identified and the consequences of matrix alterations for the mechanical properties of wounds are largely unknown. Here, we report how a single diffusible factor, activin A, affects the healing process across scales. Bioinformatics analysis of wound fibroblast transcriptome data combined with biochemical and histopathological analyses of wounds and functional in vitro studies identify that activin promotes pro-fibrotic gene expression signatures and processes, including glycoprotein and proteoglycan biosynthesis, collagen deposition, and altered collagen cross-linking. As a consequence, activin strongly reduces the wound and scar deformability, as identified by a non-invasive in vivo method for biomechanical analysis. These results provide mechanistic insight into the roles of activin in wound repair and fibrosis and identify the functional consequences of alterations in the wound matrisome at the biomechanical level.

Angiogenesis is regulated by signals received by receptor tyrosine kinases such as vascular endothelial growth factor receptors. Mammalian Sprouty (Spry) proteins are known to function by specifically antagonizing the activation of the mitogen-activated protein kinase signaling pathway by receptor tyrosine kinases, a pathway known to promote angiogenesis. To examine the role of Spry2 in the regulation of angiogenesis during wound repair, we used a model of murine dermal wound healing. Full-thickness excisional wounds (3 mm) were made on the dorsum of anesthetized adult female FVB mice. Samples were harvested at multiple time points postwounding and analyzed using real-time RT-PCR, Western blot analysis, and immunofluorescent histochemistry. Spry2 mRNA and protein levels in the wound bed increased significantly during the resolving phases of healing, coincident with the onset of vascular regression in this wound model. In another experiment, intracellular levels of Spry2 or its dominant-negative mutant (Y55F) were elevated by a topical application to the wounds of controlled-release gel containing cell permeable, transactivator of transcription-tagged Spry2, Spry2 Y55F , or green fluorescent protein (as control). Wound samples were analyzed for vascularity using CD31 immunofluorescent histochemistry as well as for total and phospho-Erk1/2 protein content. The treatment of wounds with Spry2 resulted in a significant decrease in vascularity and a reduced abundance of phospho-Erk1/2 compared with wounds treated with the green fluorescent protein control. In contrast, the wounds treated with the dominant-negative Spry2 Y55F exhibited a moderate increase in vascularity and elevated phospho-Erk1/2 content. These results indicate that endogenous Spry2 functions to downregulate angiogenesis in the healing murine skin wound, potentially by inhibiting the mitogenactivated protein kinase signaling pathway.