Long‐Term Imaging of Wound Angiogenesis with Large Scale Optoacoustic Microscopy

Wound healing is a well-coordinated process, necessitating efficient formation of new blood vessels. Vascularization defects are therefore a major risk factor for chronic, non-healing wounds. The dynamics of mammalian tissue revascularization, vessel maturation, and remodeling remain poorly understood due to lack of suitable in vivo imaging tools. A label-free large-scale optoacoustic microscopy (LSOM) approach is developed for rapid, non-invasive, volumetric imaging of tissue regeneration over large areas spanning up to 50 mm with a depth penetration of 1.5 mm. Vascular networks in dorsal mouse skin and full-thickness excisional wounds are imaged with capillary resolution during the course of healing, revealing previously undocumented views of the angiogenesis process in an unperturbed wound environment. Development of an automatic analysis framework enables the identification of key features of wound angiogenesis, including vessel length, diameter, tortuosity, and angular alignment. The approach offers a versatile tool for preclinical research in tissue engineering and regenerative medicine, empowering label-free, longitudinal, high-throughput, and quantitative studies of the microcirculation in processes associated with normal and impaired vascular remodeling, and analysis of vascular responses to pharmacological interventions in vivo

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 promotes skin carcinogenesis by attraction and reprogramming of macrophages.

Activin has emerged as an important player in different types of cancer, but the underlying mechanisms are largely unknown. We show here that activin overexpression is an early event in murine and human skin tumorigenesis. This is functionally important, since activin promoted skin tumorigenesis in mice induced by the human papillomavirus 8 oncogenes. This was accompanied by depletion of epidermal γδ T cells and accumulation of regulatory T cells. Most importantly, activin increased the number of skin macrophages via attraction of blood monocytes, which was prevented by depletion of CCR2-positive monocytes. Gene expression profiling of macrophages from pre-tumorigenic skin and bioinformatics analysis demonstrated that activin induces a gene expression pattern in skin macrophages that resembles the phenotype of tumor-associated macrophages in different malignancies, thereby promoting angiogenesis, cell migration and proteolysis. The functional relevance of this finding was demonstrated by antibody-mediated depletion of macrophages, which strongly suppressed activin-induced skin tumor formation. These results demonstrate that activin induces skin carcinogenesis via attraction and reprogramming of macrophages and identify novel activin targets involved in tumor formation

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

Negative Regulators of Angiogenesis during Wound Healing

During dermal wound repair, hypoxia-driven proliferation results in dense but poorly-perfused, disorganized microvascular networks, similar to those in tumors. Concurrently, activated dermal fibroblasts generate an angio-permissive, provisional extra-cellular matrix (ECM). Unlike cancers, wounds naturally resolve via blood vessel regression and ECM maturation, which are essential for re-establishing tissue homeostasis. However, the mechanisms guiding wound resolution are poorly understood. In this Thesis, we explored the roles of two candidate endogenous anti-angiogenic factors, Sprouty-2 and Pigment Epithelium-Derived Factor (PEDF), in the regulation of wound resolution. Spatial and temporal expression patterns of the two factors were characterized in a reproducible model of excisional wound healing in the mouse. Sprouty-2 was found to be an intra-cellular protein associated with cells in the wound bed and its expression peaked during the proliferative phase of healing. PEDF was found to be a fibroblast-produced matricellular protein that localized with microvasculature and its expression peaked during the remodeling phase of healing. In a series of controlled in vivo experiments, wherein levels of Sprouty-2 or PEDF in wounds were altered via exogenous addition of recombinant proteins or antibody inhibition of endogenous proteins, the anti-angiogenic roles of candidate factors were established. Exogenous Sprouty-2 and PEDF were both found to decrease blood vessel density in the wound bed during the proliferative phase, while inhibition of endogenous PEDF delayed vessel regression during the remodeling phase. Exogenous PEDF further promoted vascular integrity and increased collagen maturity. These results demonstrated that Sprouty-2 and PEDF contribute to the resolution of healing wounds by causing regression of immature blood vessels, thus promoting a return to tissue homeostasis after injury. In this Thesis, we also introduced a novel systems biology approach to the study of the complex mechanisms of healing, particularly angiogenesis and ECM-remodeling. High-throughput, time-course gene expression data of skin and oral mucosal wounds was analyzed via biological network analysis in the Angiome, Matrisome and combined networks. Results demonstrated significant co-regulation between angiogenesis and ECM-remodeling, with regulatory differences observed in oral versus skin wounds. Systems methods efficiently combined database knowledge with high-throughput experimental data to approach comprehensive data-driven models of healing wounds

Comparison of ZrO₂ Particles and Polyaniline as Additives in Polystyrene-Based Sorbents for the Micro-Solid Phase Extraction of Psychoactive Drugs from Biofluids

The intensive development of extraction methods based on μ-SPE extraction contributes to the increased interest in the synthesis of new sorption materials. This work presents the characterization of polystyrene fibers and polystyrene fibers blended with ZrO2 particles or polyaniline obtained by electrospinning and their use in the extraction of selected psychoactive drugs from biological samples. The characteristic of produced fibers is made by performing SEM images, measuring average fiber diameter, and examining their sorption abilities. Among the fibers based on pure polystyrene, tested in the first stage, the best sorption properties are demonstrated for the fibers obtained from a polystyrene solution in DMF with a concentration of 17.5 wt%. In the next stage, this material was modified with synthesized ZrO2 particles and polyaniline. Among the tested materials, the sorbent based on polystyrene with polyaniline shows the best sorption properties of the tested substances. The use of this material in the μ-SPE in a needle enables the extraction of selected compounds from aqueous and biological samples such as urine and human plasma

Long‐Term Imaging of Wound Angiogenesis with Large Scale Optoacoustic Microscopy

Wound healing is a well-coordinated process, necessitating efficient formation of new blood vessels. Vascularization defects are therefore a major risk factor for chronic, non-healing wounds. The dynamics of mammalian tissue revascularization, vessel maturation, and remodeling remain poorly understood due to lack of suitable in vivo imaging tools. A label-free large-scale optoacoustic microscopy (LSOM) approach is developed for rapid, non-invasive, volumetric imaging of tissue regeneration over large areas spanning up to 50 mm with a depth penetration of 1.5 mm. Vascular networks in dorsal mouse skin and full-thickness excisional wounds are imaged with capillary resolution during the course of healing, revealing previously undocumented views of the angiogenesis process in an unperturbed wound environment. Development of an automatic analysis framework enables the identification of key features of wound angiogenesis, including vessel length, diameter, tortuosity, and angular alignment. The approach offers a versatile tool for preclinical research in tissue engineering and regenerative medicine, empowering label-free, longitudinal, high-throughput, and quantitative studies of the microcirculation in processes associated with normal and impaired vascular remodeling, and analysis of vascular responses to pharmacological interventions in vivo.ISSN:2198-384

A paracrine activin A-mDia2 axis promotes squamous carcinogenesis via fibroblast reprogramming

Cancer-associated fibroblasts (CAFs) are key regulators of tumorigenesis and promising targets for next-generation therapies. We discovered that cancer cell-derived activin A reprograms fibroblasts into pro-tumorigenic CAFs. Mechanistically, this occurs via Smad2-mediated transcriptional regulation of the formin mDia2, which directly promotes filopodia formation and cell migration. mDia2 also induces expression of CAF marker genes through prevention of p53 nuclear accumulation, resulting in the production of a pro-tumorigenic matrisome and secretome. The translational relevance of this finding is reflected by activin A overexpression in tumor cells and of mDia2 in the stroma of skin cancer and other malignancies and the correlation of high activin A/mDia2 levels with poor patient survival. Blockade of this signaling axis using inhibitors of activin, activin receptors, or mDia2 suppressed cancer cell malignancy and squamous carcinogenesis in 3D organotypic cultures, ex vivo, and in vivo, providing a rationale for pharmacological inhibition of activin A-mDia2 signaling in stratified cancer patients