Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy.

Capillary occlusions are characteristic features of the early diabetic retinopathy and are presumed to initiate neovascularization. Activated leukocytes can cause microvascular occlusions and cell damage by release of cytotoxic products. To explore the role of leukocytes in capillary occlusion, nonperfusion, and neovascularization of diabetic retinopathy, a rat model was used, in which a diabetic state was induced by alloxan. Retina flat preparations were differentially stained for monocytes and granulocytes. Capillary occlusion, nonperfusion, and neovascularization were assessed microscopically in the center, midperiphery, and periphery of the retina. In contrast to control retinas, 2- to 9-month diabetic rats showed many capillary occlusions by leukocytes, especially monocytes, endothelial cell damage, extravascular macrophage accumulation, and tissue damage. The percentage of activated monocytes and granulocytes in the circulating blood of diabetic rats was greatly increased, and areas of capillary 'loss' and neovascularization in the retina coincided with sites of extravascular leukocytes. The authors' results suggest a potential role of monocytes and macrophages in the pathogenesis of diabetic retinopathy

An in Vivo Method for Visualizing Flow Dynamics of Cells within Corneal Lymphatics

Background: Monitoring the trafficking of specific cell populations within lymphatics could improve our understanding of processes such as transplant rejection and cancer metastasis. Current methods, however, lack appropriate image resolution for single-cell analysis or are incompatible with in vivo and longitudinal monitoring of lymphatics in their native state. We therefore sought to achieve high-resolution live imaging of the dynamic behavior of cells within lymph vessels in the rat cornea. Methods/Results: Inflammatory angiogenesis was induced by suture placement in corneas of Wistar rats. Pre- and up to 3 weeks post-induction, corneas were noninvasively examined by laser-scanning in vivo corneal confocal microscopy (IVCM) using only endogenous contrast. Lymph vessels and the cells harbored therein were documented by still images, real-time video, and 3D confocal stack reconstruction of live tissue. In vivo, conjunctival and corneal lymphatics were morphologically distinct, those with corneal location being one-quarter the diameter of those in the conjunctiva (p<0.001). Cells were recruited to initially empty pre-existing lymph vessels during the first day of inflammation and maintained a dense occupation of vessels for up to 7 days. A diverse population of cells (diameter range: 1.5–27.5 μm) with varying morphology was observed, and exhibited variable flow patterns and were transported singly and in clusters of at least 2–9 adherent cells. Conclusions: The in vivo microscopic technique presented enables lymph vessels and cell trafficking to be studied in high resolution in a minimally-perturbed physiologic milieu

Fine control of endothelial VEGFR-2 activation: caveolae as fluid shear stress shelters for membrane receptors.

Recent experimental evidence points to the possibility that cell surface-associated caveolae may participate in mechanotransduction. The particular shape of caveolae suggests that these structures serve to prevent exposure of putative mechanosensors residing within these membrane invaginations to shear stresses at magnitudes associated with initiation of cell signaling. Accordingly, we numerically analyzed the fluid flow in and around caveolae using the equation of motion for flow of plasma at low Reynolds numbers and assuming no slip-condition on the membrane. The plasma velocity inside a typical caveola and the shear stress acting on its membrane are markedly reduced compared to the outside membrane. Computation of the diffusion field in the vicinity of a caveola under flow, however, revealed a rapid equilibration of agonist concentration in the fluid inside a caveola with the outside plasma. Western blots and immunocytochemistry support the role of caveolae as shear stress shelters for putative membrane-bound mechanoreceptors such as flk-1. Our results, therefore, suggest that caveolae serve to reduce the fluid shear stress acting on receptors in their interior, while allowing rapid diffusion of ligands into the interior. This mechanism may permit differential control of flow and ligand activation of flk-1 receptor in the presence of ligands

Fine control of endothelial VEGFR-2 activation: caveolae as fluid shear stress shelters for membrane receptors.

Recent experimental evidence points to the possibility that cell surface-associated caveolae may participate in mechanotransduction. The particular shape of caveolae suggests that these structures serve to prevent exposure of putative mechanosensors residing within these membrane invaginations to shear stresses at magnitudes associated with initiation of cell signaling. Accordingly, we numerically analyzed the fluid flow in and around caveolae using the equation of motion for flow of plasma at low Reynolds numbers and assuming no slip-condition on the membrane. The plasma velocity inside a typical caveola and the shear stress acting on its membrane are markedly reduced compared to the outside membrane. Computation of the diffusion field in the vicinity of a caveola under flow, however, revealed a rapid equilibration of agonist concentration in the fluid inside a caveola with the outside plasma. Western blots and immunocytochemistry support the role of caveolae as shear stress shelters for putative membrane-bound mechanoreceptors such as flk-1. Our results, therefore, suggest that caveolae serve to reduce the fluid shear stress acting on receptors in their interior, while allowing rapid diffusion of ligands into the interior. This mechanism may permit differential control of flow and ligand activation of flk-1 receptor in the presence of ligands