long-term-intravital-immunofluorescence-imaging-tissue-matrix

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

École Polytechnique Fédérale de Lausanne

Inhibition of angiogenesis could be a treatment strategy for diseases such as cancer, rheumatoid arthritis, and diabetic retinopathy. PP2 is a pharmacological inhibitor of Src family kinases and was found to inhibit FGF-2 induced angiogenesis in vivo. Experiments in vitro showed that PP2 inhibited invasive growth and sprouting of both endothelial and vascular smooth muscle cells into a fibrin matrix. PP2 inhibited the formation of lamellopodia and expression of kinase inactive c-Src reduced phosphorylation of cortactin and paxillin, suggesting a model in which Src kinases are involved in organization of the actin cytoskeleton. Consequently, endothelial cells expressing kinase inactive c-Src failed to spread and form cord-like structures on a collagen matrix. These data suggest that pharmacological inactivation of Src family kinases inhibits FGF-2 stimulated angiogenesis by interference with organization of the actin cytoskeleton in both endothelial and vascular smooth muscle cells, which affects cell migration.

Inactivation of Src family kinases inhibits angiogenesis in vivo: implications for a mechanism involving organization of the actin cytoskeleton.

Migration, proliferation and invasive growth of myofibroblasts are key cellular events during formation of granulation tissue in situations of wound healing, arteriosclerosis and tumor growth. To study the invasive phenotype of myofibroblasts, we established an assay where arterial tissue from chicken embryos was embedded in fibrin gels and stimulated with growth factors. Addition of serum, PDGF-BB and FGF-2, but not VEGF-A, resulted in an outgrowth of cellular sprouts with a pattern that was similar to the organization of cells invading a provisional matrix in an in vivo model of wound healing using the chicken chorioallantoic membrane. Sprouting cells were defined as myofibroblasts based on being alpha-smooth muscle actin-positive but desmin-negative. There was no contribution of endothelial cells in outgrowing sprouts. The acquired myofibroblastic phenotype was stable since sprout-derived cells resumed sprouting in a growth factor-independent manner when re-embedded as spheroids in a fibrin matrix. Invasive growth and sprouting of vascular smooth muscle cells was not limited to chicken cells since a similar response was seen when spheroids composed of purified primary human aortic smooth muscle cells were embedded in fibrin. Finally, a technique for flat visualization of the three-dimensional sprouting and a quantification method is described. This ex vivo model allows quantitative analysis of invasive growth and differentiation of vascular smooth muscle cells and fibroblasts into myofibroblasts.

An ex vivo model for functional studies of myofibroblasts.

Interferon (IFN)-alpha has been used in the treatment of neuroendocrine (NE) tumors; however, the feasibility of IFN-alpha gene therapy has not been evaluated in NE tumor cells. In this study, human IFN-alpha2 (hIFN-alpha2) gene has been transferred into a NE tumor cell line BON. hIFN-alpha2-expressing BON cells were subcutaneously inoculated in nude mice. The results demonstrated that hIFN-alpha2 exerted significant antiproliferative effects on NE tumor cell lines (BON and LCC18) and other tumor cell lines (CA46 and SW480) as well as porcine aorta cell line. Furthermore, hIFN-alpha2 demonstrated its antineovascular activity in mice tumor and a direct antiangiogenic effect in chicken chorioallantoic membrane assay. hIFN-alpha2-expressing BON cells had a stable and long-term expression. Mice implanted with hIFN-alpha2-expressing BON cells showed a lower incidence, a delayed development and a significantly longer doubling time of the tumor compared to both wild-type (WT) and vector group. In addition, IFN-alpha significantly inhibited cell adhesion of WT BON cells. hIFN-alpha2-expressing BON tumors had a high level of hIFN-alpha2 protein. Finally, mice implanted with a mixture of WT and hIFN-alpha2-expressing BON cells (1:1) presented a delayed tumor development and had an even lower incidence of tumors than those implanted with hIFN-alpha2-expressing BON cells only. The doubling time of tumor was also longest in the mixture group. Our data suggest that hIFN-alpha2 gene therapy might be possible to be used as a new treatment for NE tumor patients. Further studies on the regulation of hIFN-alpha expression are needed, especially in combination with other cytokines, which could lead to a better understanding and improvements of hIFN-alpha gene therapy.

Efficient human interferon-alpha gene transfer to neuroendocrine tumor cells with long-term and stable expression.

Formation of new vessels in granulation tissue during wound healing has been assumed to occur solely through sprouting angiogenesis. In contrast, we show here that neovascularization can be accomplished by nonangiogenic expansion of preexisting vessels. Using neovascularization models based on the chick chorioallantoic membrane and the healing mouse cornea, we found that tissue tension generated by activated fibroblasts or myofibroblasts during wound contraction mediated and directed translocation of the vasculature. These mechanical forces pulled vessels from the preexisting vascular bed as vascular loops with functional circulation that expanded as an integral part of the growing granulation tissue through vessel enlargement and elongation. Blockade of vascular endothelial growth factor receptor-2 confirmed that biomechanical forces were sufficient to mediate the initial vascular growth independently of endothelial sprouting or proliferation. The neovascular network was further remodeled by splitting, sprouting and regression of individual vessels. This model explains the rapid appearance of large functional vessels in granulation tissue during wound healing.

Biomechanical regulation of blood vessel growth during tissue vascularization.

Growth of new blood vessels (angiogenesis) is essential for embryo development as well as for wound healing and progression of a number of diseases such as cancer, inflammatory conditions, eye diseases, psoriasis, and rheumatoid arthritis in the adult. Current paradigms explain blood vessel growth entirely by sprouting angiogenesis or by vessel splitting through so called intussusceptive angiogenesis. However, these mechanisms are mainly derived from experiments on the developing embryo while less is known about angiogenesis in the adult during, e.g., wound healing, tumor growth, and inflammation. Recently we showed that blood vessel growth in the adult can be induced and directed by mechanical forces that naturally develop during healing or remodeling of tissues. In contrast to sprouting and intussusception, the new biomechanical hypothesis assumes that functional blood vessels are passively translocated which, if found generic, may drastically change the approach for developing anti- and pro-angiogenic therapies in the treatment of a variety of diseases.

A new mechanism of blood vessel growth - hope for new treatment strategies.

[Blood vessel growth by matrix contraction].

Modern functional genomic approaches may help to better understand the molecular events involved in tissue morphogenesis and to identify molecular signatures and pathways. We have recently applied transcriptomic profiling to evidence molecular signatures in the development of the normal chicken chorioallantoic membrane (CAM) and in tumor engrafted on the CAM. We have now extended our studies by performing a transcriptome analysis in the "wound model" of the chicken CAM, which is another relevant model of tissue morphogenesis.

Gene signatures in wound tissue as evidenced by molecular profiling in the chick embryo model.

Therapeutic regulation of tissue vascularization has appeared as an attractive approach to treat a number of human diseases. In vivo neovascularization assays that reflect physiological and pathological formation of neovessels are important in this effort. In this report we present an assay where the effects of activators and inhibitors of angiogenesis can be quantitatively and qualitatively measured. A provisional matrix composed of collagen I and fibrin was formed in a plastic cylinder and implanted onto the chick chorioallantoic membrane. A nylon mesh separated the implanted matrix from the underlying tissue to distinguish new from pre-existing vessels. Vascularization of the matrix in response to fibroblast growth factor-2 or platelet-derived growth factor-BB was scored in a double-blinded manner, or vessel density was measured using a semi-automated image analysis procedure. Thalidomide, fumagillin, U0126 and TGFβ inhibited neovessel growth while hydrocortisone exerted a negative and wortmannin a toxic effect on the pre-existing vasculature. This quantitative, inexpensive and rapid in vivo angiogenesis assay might be a valuable tool in screening and characterizing factors that influence wound or tumor induced vascularization and in assessing their effects on the normal vasculature.

An in vivo neovascularization assay for screening regulators of angiogenesis and assessing their effects on pre-existing vessels.

The clear cell subtype of renal carcinoma (CCRCC) is highly vascularized and despite a slow progression rate, it is potentially a highly aggressive tumor. Although a doubling of median progression-free survival in CCRCC patients treated by targeted therapies has been observed, the fact that tumors escape after anti-VEGF treatment suggests alternative pathways. The chick chorioallantoic membrane (CAM) is a well-established model, which allows in vivo studies of tumor angiogenesis and the testing of anti-angiogenic molecules. However, only a few data exist on CCRCC grafted onto CAM. We aimed to validate herein the CAM as a suitable model for studying the development of CCRCC and the interactions with the surrounding stroma. Our study uses both CCRCC cell lines and fresh tumor samples after surgical resection. We demonstrate that in both cases CCRCC can be grafted onto the CAM, to survive and to induce an angiogenic process. We further provide insights into the transcriptional regulation of the model by performing a differential analysis of tumor-derived and stroma-derived transcripts.

The experimental renal cell carcinoma model in the chick embryo.

Visualizing the dynamic behaviors of immune cells in living tissue has dramatically increased our understanding of how cells interact with their surroundings, contributing important insights into mechanisms of leukocyte trafficking, tumor cell invasion, and T cell education by dendritic cells, among others. Despite substantial advances with various intravital imaging techniques including two-photon microscopy and the generation of multitudes of reporter mice, there is a growing need to assess cell interactions in the context of specific extracellular matrix composition and microvascular functions, and as well, simpler and more widely accessible methods are needed to image cell behaviors in the context of living tissue physiology. Here we present an antibody-based method for intravital imaging of cell interactions with the blood, lymphatic, and the extracellular matrix compartments of the living dermis while simultaneously assessing capillary permeability and lymphatic drainage function. Using the exposed dorsal ear of the anesthetized mouse and a fluorescence stereomicroscope, such events can be imaged in the context of specific extracellular matrix proteins, or matrix-bound chemokine stores. We developed and optimized the method to minimize tissue damage to the ear, rapidly immunostain for multiple extracellular or cell surface receptors of interest, minimize immunotoxicity with pre-blocking Fcγ receptors and phototoxicity with extracellular antioxidants, and highlight the major dermal tissue structures with basement membrane markers. We demonstrate differential migration behaviors of bone marrow-derived dendritic cells, blood-circulating leukocytes, and dermal dendritic cells, with the latter entering sparse CCL21-positive areas of pre-collecting lymphatic vessels. This new method allows simultaneous imaging of cells and tissue structures, microvascular function, and extracellular microenvironment in multiple skin locations for 12 hours or more, with the flexibility of immunolabeling in addition to genetic-based fluorescent reporters.

Intravital immunofluorescence for visualizing the microcirculatory and immune microenvironments in the mouse ear dermis.

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments.

Mapping the extracellular and membrane proteome associated with the vasculature and the stroma in the embryo.

Lymphatic vessels transport fluid, antigens, and immune cells to the lymph nodes to orchestrate adaptive immunity and maintain peripheral tolerance. Lymphangiogenesis has been associated with inflammation, cancer metastasis, autoimmunity, tolerance and transplant rejection, and thus, targeted lymphatic ablation is a potential therapeutic strategy for treating or preventing such events. Here we define conditions that lead to specific and local closure of the lymphatic vasculature using photodynamic therapy (PDT). Lymphatic-specific PDT was performed by irradiation of the photosensitizer verteporfin that effectively accumulates within collecting lymphatic vessels after local intradermal injection. We found that anti-lymphatic PDT induced necrosis of endothelial cells and pericytes, which preceded the functional occlusion of lymphatic collectors. This was specific to lymphatic vessels at low verteporfin dose, while higher doses also affected local blood vessels. In contrast, light dose (fluence) did not affect blood vessel perfusion, but did affect regeneration time of occluded lymphatic vessels. Lymphatic vessels eventually regenerated by recanalization of blocked collectors, with a characteristic hyperplasia of peri-lymphatic smooth muscle cells. The restoration of lymphatic function occurred with minimal remodeling of non-lymphatic tissue. Thus, anti-lymphatic PDT allows control of lymphatic ablation and regeneration by alteration of light fluence and photosensitizer dose.

Optimization and regeneration kinetics of lymphatic-specific photodynamic therapy in the mouse dermis.

Lymphatic vessels surround follicles within the ovary, but their roles in folliculogenesis and pregnancy, as well as the necessity of lymphangiogenesis in follicle maturation and health, are undefined. We used systemic delivery of mF4-31C1, a specific antagonist vascular endothelial growth factor receptor 3 (VEGFR-3) antibody to block lymphangiogenesis in mice. VEGFR-3 neutralization for 2 weeks before mating blocked ovarian lymphangiogenesis at all stages of follicle maturation, most notably around corpora lutea, without significantly affecting follicular blood angiogenesis. The numbers of oocytes ovulated, fertilized, and implanted in the uterus were normal in these mice; however, pregnancies were unsuccessful because of retarded fetal growth and miscarriage. Fewer patent secondary follicles were isolated from treated ovaries, and isolated blastocysts exhibited reduced cell densities. Embryos from VEGFR-3-neutralized dams developed normally when transferred to untreated surrogates. Conversely, normal embryos transferred into mF4-31C1-treated dams led to the same fetal deficiencies observed with in situ gestation. Although no significant changes were measured in uterine blood or lymphatic vascular densities, VEGFR-3 neutralization reduced serum and ovarian estradiol concentrations during gestation. VEGFR-3-mediated lymphangiogenesis thus appears to modulate the folliculogenic microenvironment and may be necessary for maintenance of hormone levels during pregnancy; both of these are novel roles for the lymphatic vasculature.

VEGFR-3 neutralization inhibits ovarian lymphangiogenesis, follicle maturation, and murine pregnancy.

Growth factors (GFs) are critical in tissue repair, but their translation to clinical use has been modest. Physiologically, GF interactions with extracellular matrix (ECM) components facilitate localized and spatially regulated signaling; therefore, we reasoned that the lack of ECM binding in their clinically used forms could underlie the limited translation. We discovered that a domain in placenta growth factor-2 (PlGF-2(123-144)) binds exceptionally strongly and promiscuously to ECM proteins. By fusing this domain to the GFs vascular endothelial growth factor-A, platelet-derived growth factor-BB, and bone morphogenetic protein-2, we generated engineered GF variants with super-affinity to the ECM. These ECM super-affinity GFs induced repair in rodent models of chronic wounds and bone defects that was greatly enhanced as compared to treatment with the wild-type GFs, demonstrating that this approach may be useful in several regenerative medicine applications.

Article	Year
Inactivation of Src family kinases inhibits angiogenesis in vivo: implications for a mechanism involving organization of the actin cytoskeleton. Experimental cell research\| PubMed ID: 14597409	2003
An ex vivo model for functional studies of myofibroblasts. Laboratory investigation; a journal of technical methods and pathology\| PubMed ID: 15723087	2005
Efficient human interferon-alpha gene transfer to neuroendocrine tumor cells with long-term and stable expression. Neuroendocrinology\| PubMed ID: 16721032	2005
Biomechanical regulation of blood vessel growth during tissue vascularization. Nature medicine\| PubMed ID: 19483693	2009
A new mechanism of blood vessel growth - hope for new treatment strategies. Discovery medicine\| PubMed ID: 19772838	2009
[Blood vessel growth by matrix contraction]. Médecine sciences : M/S\| PubMed ID: 20819701	2010
Gene signatures in wound tissue as evidenced by molecular profiling in the chick embryo model. BMC genomics\| PubMed ID: 20840761	2010
An in vivo neovascularization assay for screening regulators of angiogenesis and assessing their effects on pre-existing vessels. Angiogenesis\| PubMed ID: 22918697	2012
The experimental renal cell carcinoma model in the chick embryo. Angiogenesis\| PubMed ID: 23076651	2013
Intravital immunofluorescence for visualizing the microcirculatory and immune microenvironments in the mouse ear dermis. PloS one\| PubMed ID: 23451163	2013
Mapping the extracellular and membrane proteome associated with the vasculature and the stroma in the embryo. Molecular & cellular proteomics : MCP\| PubMed ID: 23674615	2013
Optimization and regeneration kinetics of lymphatic-specific photodynamic therapy in the mouse dermis. Angiogenesis\| PubMed ID: 23892627	2014
VEGFR-3 neutralization inhibits ovarian lymphangiogenesis, follicle maturation, and murine pregnancy. The American journal of pathology\| PubMed ID: 24036251	2013
Growth factors engineered for super-affinity to the extracellular matrix enhance tissue healing. Science (New York, N.Y.)\| PubMed ID: 24558160	2014

Witold W. Kilarski

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