Simultaneous fluorescence imaging of distinct nerve and blood vessel patterns in dual Thy1-YFP and Flt1-DsRed transgenic mice.

Blood vessels and nerve tissues are critical to the development and functionality of many vital organs. However, little is currently known about their interdependency during development and after injury. In this study, dual fluorescence transgenic reporter mice were utilized to observe blood vessels and nervous tissues in organs postnatally. Thy1-YFP and Flt1-DsRed (TYFD) mice were interbred to achieve dual fluorescence in the offspring, with Thy1-YFP yellow fluorescence expressed primarily in nerves, and Flt1-DsRed fluorescence expressed selectively in blood vessels. Using this dual fluorescent mouse strain, we were able to visualize the networks of nervous and vascular tissue simultaneously in various organ systems both in the physiological state and after injury. Using ex vivo high-resolution imaging in this dual fluorescent strain, we characterized the organizational patterns of both nervous and vascular systems in a diverse set of organs and tissues. In the cornea, we also observed the dynamic patterns of nerve and blood vessel networks following epithelial debridement injury. These findings highlight the versatility of this dual fluorescent strain for characterizing the relationship between nerve and blood vessel growth and organization.

Transgenic models for investigating the nervous system: Currently available neurofluorescent reporters and potential neuronal markers.

Recombinant DNA technologies have enabled the development of transgenic animal models for use in studying a myriad of diseases and biological states. By placing fluorescent reporters under the direct regulation of the promoter region of specific marker proteins, these models can localize and characterize very specific cell types. One important application of transgenic species is the study of the cytoarchitecture of the nervous system. Neurofluorescent reporters can be used to study the structural patterns of nerves in the central or peripheral nervous system in vivo, as well as phenomena involving embryologic or adult neurogenesis, injury, degeneration, and recovery. Furthermore, crucial molecular factors can also be screened via the transgenic approach, which may eventually play a major role in the development of therapeutic strategies against diseases like Alzheimer's or Parkinson's. This review describes currently available reporters and their uses in the literature as well as potential neural markers that can be leveraged to create additional, robust transgenic models for future studies.

Quantification of Angiogenesis and Lymphangiogenesis in the Dual ex vivo Aortic and Thoracic Duct Assay.

BACKGROUND: Lymphatic vessel formation (lymphangiogenesis) plays important roles in cancer metastasis, organ rejection, and lymphedema, but the underlying molecular events remain unclear. Furthermore, despite significant overlap in the molecular families involved in angiogenesis and lymphangiogenesis, little is known about the crosstalk between these processes. The ex vivo aortic ring assay and lymphatic ring assay have enabled detailed studies of vessel sprouting, but harvesting and imaging clear thoracic duct samples remain challenging. Here we present a modified ex vivo dual aortic ring and thoracic duct assay using tissues from dual fluorescence reporter Prox1- GFP/Flt1-DsRed (PGFD) mice, which permit simultaneous visualization of blood and lymphatic endothelial cells. OBJECTIVE: To characterize the concurrent sprouting of intrinsically fluorescent blood and lymphatic vessels from harvested aorta and thoracic duct samples. METHODS: Dual aorta and thoracic duct specimens were harvested from PGFD mice, grown in six types of endothelial cell growth media (one control, five that each lack a specific growth factor), and visualized by confocal fluorescence microscopy. Linear mixed models were used to compare the extent of vessel growth and sprouting over a 28-day period. RESULTS: Angiogenesis occurred prior to lymphangiogenesis in our assay. The control medium generally induced superior growth of both vessel types compared with the different modified media formulations. The greatest decrease in lymphangiogenesis was observed in vascular endothelial growth factor-C (VEGF-C)-devoid medium, suggesting the importance of VEGF-C in lymphangiogenesis. CONCLUSION: The modified ex vivo dual aortic ring and thoracic duct assay represents a powerful tool for studying angiogenesis and lymphangiogenesis in concert.

Application of corneal injury models in dual fluorescent reporter transgenic mice to understand the roles of the cornea and limbus in angiogenic and lymphangiogenic privilege.

The role of the corneal epithelium and limbus in corneal avascularity and pathological neovascularization (NV) is not well understood. To investigate the contributions of the corneal and limbal epithelia in angiogenic and lymphangiogenic privilege, we designed five injury models involving debridement of different portions of the cornea and limbus and applied them to the dual-fluorescence reporter Prox1-GFP/Flt1-DsRed mouse, which permits in vivo imaging of blood and lymphatic vessels via fluorescence microscopy. Debridement of the whole cornea resulted in significant hemangiogenesis (HA) and lymphangiogenesis (LA), while that of the whole limbus yielded minimal corneal HA or LA. Following hemilimbal plus whole corneal debridement, corneal NV occurred only through the non-injured aspect of the limbus. Overall, these results suggest that the integrity of the corneal epithelium is important for (lymph)angiogenic privilege, whereas the limbus does not act as a physical or physiologic barrier to invading vessels. In CDh5-CreERT2VEGFR2lox/PGFD mice, conditional deletion of vascular endothelial growth factor receptor 2 in vascular endothelial cells abolished injury-induced HA and LA, demonstrating the utility of this transgenic mouse line for identifying important factors in the process of neovascularization.

Fluorescent reporter transgenic mice for in vivo live imaging of angiogenesis and lymphangiogenesis.

The study of lymphangiogenesis is an emerging science that has revealed the lymphatic system as a central player in many pathological conditions including cancer metastasis, lymphedema, and organ graft rejection. A thorough understanding of the mechanisms of lymphatic growth will play a key role in the development of therapeutic strategies against these conditions. Despite the known potential of this field, the study of lymphatics has historically lagged behind that of hemangiogenesis. Until recently, significant strides in lymphatic studies were impeded by a lack of lymphatic-specific markers and suitable experimental models compared to those of the more immediately visible blood vasculature. Lymphangiogenesis has also been shown to be a key phenomenon in developmental biological processes, such as cell proliferation, guided migration, differentiation, and cell-to-cell communication, making lymphatic-specific visualization techniques highly desirable and desperately needed. Imaging modalities including immunohistochemistry and in situ hybridization are limited by the need to sacrifice animal models for tissue harvesting at every experimental time point. Moreover, the processes of mounting and staining harvested tissues may introduce artifacts that can confound results. These traditional methods for investigating lymphatic and blood vasculature are associated with several problems including animal variability (e.g., between mice) when replicating lymphatic growth environments and the cost concerns of prolonged, labor-intensive studies, all of which complicate the study of dynamic lymphatic processes. With the discovery of lymphatic-specific markers, researchers have been able to develop several lymphatic and blood vessel-specific, promoter-driven, fluorescent-reporter transgenic mice for visualization of lymphatics in vivo and in vitro. For instance, GFP, mOrange, tdTomato, and other fluorescent proteins can be expressed under control of a lymphatic-specific marker like Prospero-related homeobox 1 (Prox1), which is a highly conserved transcription factor for determining embryonic organogenesis in vertebrates that is implicated in lymphangiogenesis as well as several human cancers. Importantly, Prox1-null mouse embryos develop without lymphatic vessels. In human adults, Prox1 maintains lymphatic endothelial cells and upregulates proteins associated with lymphangiogenesis (e.g., VEGFR-3) and downregulates angiogenesis-associated gene expression (e.g., STAT6). To visualize lymphatic development in the context of angiogenesis, dual fluorescent-transgenic reporters, like Prox1-GFP/Flt1-DsRed mice, have been bred to characterize lymphatic and blood vessels simultaneously in vivo. In this review, we discuss the trends in lymphatic visualization and the potential usage of transgenic breeds in hemangiogenesis and lymphangiogenesis research to understand spatial and temporal correlations between vascular development and pathological progression.

Potential lymphangiogenesis therapies: Learning from current antiangiogenesis therapies-A review.

In recent years, lymphangiogenesis, the process of lymphatic vessel formation from existing lymph vessels, has been demonstrated to have a significant role in diverse pathologies, including cancer metastasis, organ graft rejection, and lymphedema. Our understanding of the mechanisms of lymphangiogenesis has advanced on the heels of studies demonstrating vascular endothelial growth factor C as a central pro-lymphangiogenic regulator and others identifying multiple lymphatic endothelial biomarkers. Despite these breakthroughs and a growing appreciation of the signaling events that govern the lymphangiogenic process, there are no FDA-approved drugs that target lymphangiogenesis. In this review, we reflect on the lessons available from the development of antiangiogenic therapies (26 FDA-approved drugs to date), review current lymphangiogenesis research including nanotechnology in therapeutic drug delivery and imaging, and discuss molecules in the lymphangiogenic pathway that are promising therapeutic targets.

Angiogenesis and lymphangiogenesis in corneal transplantation-A review.

Corneal transplantation has been proven effective for returning the gift of sight to those affected by corneal disorders such as opacity, injury, and infections that are a leading cause of blindness. Immune privilege plays an important role in the success of corneal transplantation procedures; however, immune rejection reactions do occur, and they, in conjunction with a shortage of corneal donor tissue, continue to pose major challenges. Corneal immune privilege is important to the success of corneal transplantation and closely related to the avascular nature of the cornea. Corneal avascularity may be disrupted by the processes of angiogenesis and lymphangiogenesis, and for this reason, these phenomena have been a focus of research in recent years. Through this research, therapies addressing certain rejection reactions related to angiogenesis have been developed and implemented. Corneal donor tissue shortages also have been addressed by the development of new materials to replace the human donor cornea. These advancements, along with other improvements in the corneal transplantation procedure, have contributed to an improved success rate for corneal transplantation. We summarize recent developments and improvements in corneal transplantation, including the current understanding of angiogenesis mechanisms, the anti-angiogenic and anti-lymphangiogenic factors identified to date, and the new materials being used. Additionally, we discuss future directions for research in corneal transplantation.