Serine protease 35 regulates the fibroblast matrisome in response to hyperosmotic stress.

Hyperosmotic stress occurs in several diseases, but its long-term effects are largely unknown. We used sorbitol-treated human fibroblasts in 3D culture to study the consequences of hyperosmotic stress in the skin. Sorbitol regulated many genes, which help cells cope with the stress condition. The most robustly regulated gene encodes serine protease 35 (PRSS35). Its regulation by hyperosmotic stress was dependent on the kinases p38 and JNK and the transcription factors NFAT5 and ATF2. We identified different collagens and collagen-associated proteins as putative PRSS35 binding partners. This is functionally important because PRSS35 affected the extracellular matrix proteome, which limited cell proliferation. The in vivo relevance of these findings is reflected by the coexpression of PRSS35 and its binding partners in human skin wounds, where hyperosmotic stress occurs as a consequence of excessive water loss. These results identify PRSS35 as a key regulator of the matrisome under hyperosmotic stress conditions.

Phase-specific signatures of wound fibroblasts and matrix patterns define cancer-associated fibroblast subtypes.

Healing wounds and cancers present remarkable cellular and molecular parallels, but the specific roles of the healing phases are largely unknown. We developed a bioinformatics pipeline to identify genes and pathways that define distinct phases across the time-course of healing. Their comparison to cancer transcriptomes revealed that a resolution phase wound signature is associated with increased severity in skin cancer and enriches for extracellular matrix-related pathways. Comparisons of transcriptomes of early- and late-phase wound fibroblasts vs skin cancer-associated fibroblasts (CAFs) identified an "early wound" CAF subtype, which localizes to the inner tumor stroma and expresses collagen-related genes that are controlled by the RUNX2 transcription factor. A "late wound" CAF subtype localizes to the outer tumor stroma and expresses elastin-related genes. Matrix imaging of primary melanoma tissue microarrays validated these matrix signatures and identified collagen- vs elastin-rich niches within the tumor microenvironment, whose spatial organization predicts survival and recurrence. These results identify wound-regulated genes and matrix patterns with prognostic potential in skin cancer.

Activin-mediated alterations of the fibroblast transcriptome and matrisome control the biomechanical properties of skin wounds.

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.

Nrf2-Mediated Fibroblast Reprogramming Drives Cellular Senescence by Targeting the Matrisome.

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.

Pigment epithelium-derived factor as a multifunctional regulator of wound healing.

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.

U.S. dental students' attitudes toward research and science: impact of research experience.

This study aimed to provide a first nationwide assessment of dental students' attitudes toward the importance of research and its integration into the dental curriculum. For this purpose, the American Association for Dental Research National Student Research Group developed an online survey that was distributed to 89 percent of U.S. dental students in May 2012. The survey consisted of twenty-one Likert-type items divided into three groups: importance of research in dentistry, barriers to research involvement, and exposure to research in the dental curriculum. There were 733 responses (3.9 percent response rate), including students in all stages of education representing fifty-eight out of sixty-one dental schools. Age and race/ethnic distributions corresponded with U.S. dental school enrollees. Results showed that 63 percent of respondents had conducted research before matriculation, and of the 34 percent that participated in research during dental school, only 27 percent were newcomers. Respondents strongly agreed that scientific research enabled their progress in dentistry. Inadequate time in the curriculum was an obstacle they perceived to research involvement during dental school. Respondents agreed that dental curricula emphasize evidence-based practices but may be inadequately teaching biostatistics and research methodologies. Students with research experience tended to have stronger positive opinions about the importance of research in dental education. Efforts to foster research in schools have been well received by students, but several issues remain for enriching dental education through greater involvement of students in research.

Mechanisms of vessel regression: toward an understanding of the resolution of angiogenesis.

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.

Therapeutic Approaches to the Regulation of Wound Angiogenesis.

SIGNIFICANCE: Re-establishment of a functional vascular network is a critical component of successful wound repair. One of the most potent pro-angiogenic agents is vascular endothelial growth factor (VEGF), which, from a basic science and pre-clinical perspective, seems ideal for the therapeutic stimulation of blood vessel growth in non-healing wounds. CRITICAL ISSUES: Current strategies to improve the dysfunctional angiogenesis that occurs in non-healing wounds are inadequate with regard to the nature and magnitude of the clinical problem. However, VEGF therapy has so far been unsuccessful in promoting healing in the clinic. More effective means of delivery to the wound, which take into account the biochemical and spatio-temporal aspects of angiogenesis, may be necessary to realize VEGF's therapeutic potential. Reviewed approaches for the regulation of wound angiogenesis include: targeting regulators of intracellular VEGF signaling, making use of collagen-binding VEGF fusion proteins for increased retention in the wound, and implantation of heterogeneous scaffold systems for spatial control of angiogenesis with simultaneous use of VEGF and its inhibitor. FUTURE DIRECTIONS: To maximize efficacy of therapeutic VEGF, it may be necessary to also target its intracellular inhibitory mechanisms. Immobilizing VEGF to the wound matrix may increase its bioavailability and therapeutic efficacy. Gaining spatial control of angiogenesis opens up possibilities for advanced directed therapy. The reviewed studies present innovative approaches to in vivo directed modulation of angiogenesis utilizing VEGF biology which can, if taken further and validated in human subjects, have significant impact on clinical wound care in the future.

Sprouty2 downregulates angiogenesis during mouse skin wound healing.

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, Spry2Y55F, 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 Spry2Y55F 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 mitogen-activated protein kinase signaling pathway.