Intravital Two-Photon Microscopy of Immune Cell Dynamics in Corneal Lymphatic Vessels

BACKGROUND: The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years. METHODOLOGY/PRINCIPAL FINDINGS: We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1-5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces. CONCLUSIONS: To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions

Bone marrow-derived cells in ocular neovascularization: contribution and mechanisms

Ocular neovascularization often leads to severe vision loss. The role of bone marrow-derived cells (BMCs) in the development of ocular neovascularization, and its significance, is increasingly being recognized. In this review, we discuss their contribution and the potential mechanisms that mediate the effect of BMCs on the progression of ocular neovascularization. The sequence of events by which BMCs participate in ocular neovascularization can be roughly divided into four phases, i.e., mobilization, migration, adhesion and differentiation. This process is delicately regulated and liable to be affected by multiple factors. Cytokines such as vascular endothelial growth factor, granulocyte colony-stimulating factor and erythropoietin are involved in the mobilization of BMCs. Studies have also demonstrated a key role of cytokines such as stromal cell-derived factor-1, tumor necrosis factor-α, as well as vascular endothelial growth factor, in regulating the migration of BMCs. The adhesion of BMCs is mainly regulated by vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and vascular endothelial cadherin. However, the mechanisms regulating the differentiation of BMCs are largely unknown at present. In addition, BMCs secrete cytokines that interact with the microenvironment of ocular neovascularization; their contribution to ocular neovascularization, especially choroidal neovascularization, can be aggravated by several risk factors. An extensive regulatory network is thought to modulate the role of BMCs in the development of ocular neovascularization. A comprehensive understanding of the involved mechanisms will help in the development of novel therapeutic strategies related to BMCs. In this review, we have limited the discussion to the recent progress in this field, especially the research conducted at our laboratory

Factors regulating capillary remodeling in a reversible model of inflammatory corneal angiogenesis

Newly formed microcapillary networks arising in adult organisms by angiogenic and inflammatory stimuli contribute to pathologies such as corneal and retinal blindness, tumor growth, and metastasis. Therapeutic inhibition of pathologic angiogenesis has focused on targeting the VEGF pathway, while comparatively little attention has been given to remodeling of the new microcapillaries into a stabilized, functional, and persistent vascular network. Here, we used a novel reversible model of inflammatory angiogenesis in the rat cornea to investigate endogenous factors rapidly invoked to remodel, normalize and regress microcapillaries as part of the natural response to regain corneal avascularity. Rapid reversal of an inflammatory angiogenic stimulus suppressed granulocytic activity, enhanced recruitment of remodelling macrophages, induced capillary intussusception, and enriched pathways and processes involving immune cells, chemokines, morphogenesis, axonal guidance, and cell motility, adhesion, and cytoskeletal functions. Whole transcriptome gene expression analysis revealed suppression of numerous inflammatory and angiogenic factors and enhancement of endogenous inhibitors. Many of the identified genes function independently of VEGF and represent potentially new targets for molecular control of the critical process of microvascular remodeling and regression in the cornea.Funding Agencies|Bayer HealthCare AB, Solna, Sweden; Swedish Research Council [2012-2472]</p

Optimality and sub-optimality in a bacterial growth law

Organisms adjust their gene expression to improve fitness in diverse environments. But finding the optimal expression in each environment presents a challenge. We ask how good cells are at finding such optima by studying the control of carbon catabolism genes in Escherichia coli. Bacteria show a growth law: growth rate on different carbon sources declines linearly with the steady-state expression of carbon catabolic genes. We experimentally modulate gene expression to ask if this growth law always maximizes growth rate, as has been suggested by theory. We find that the growth law is optimal in many conditions, including a range of perturbations to lactose uptake, but provides sub-optimal growth on several other carbon sources. Combining theory and experiment, we genetically re-engineer E. coli to make sub-optimal conditions into optimal ones and vice versa. We conclude that the carbon growth law is not always optimal, but represents a practical heuristic that often works but sometimes fails

Quantifying the benefit of a proteome reserve in fluctuating environments

Fast-growing bacteria produce many proteins in excess of what seems optimal for exponential growth. Here, the authors present a mathematical model and experimental evidence supporting that this overexpression serves as a strategic reserve to quickly meet demand upon sudden improvement in growth conditions