J. Brandon Dixon

Transmural Flow Modulates Cell and Fluid Transport Functions of Lymphatic Endothelium

Rationale: Lymphatic transport of peripheral interstitial fluid and dendritic cells (DCs) is important for both adaptive immunity and maintenance of tolerance to self-antigens. Lymphatic drainage can change rapidly and dramatically on tissue injury or inflammation, and therefore increased fluid flow may serve as an important early cue for inflammation; however, the effects of transmural flow on lymphatic function are unknown. Objective: Here we tested the hypothesis that lymph drainage regulates the fluid and cell transport functions of lymphatic endothelium. Methods and Results: Using in vitro and in vivo models, we demonstrated that lymphatic endothelium is sensitive to low levels of transmural flow. Basal-to-luminal flow (0.1 and 1 &mgr;m/sec) increased lymphatic permeability, dextran transport, and aquaporin-2 expression, as well as DC transmigration into lymphatics. The latter was associated with increased lymphatic expression of the DC homing chemokine CCL21 and the adhesion molecules intercellular adhesion molecule-1 and E-selectin. In addition, transmural flow induced delocalization and downregulation of vascular endothelial cadherin and PECAM-1 (platelet/endothelial cell adhesion molecule-1). Flow-enhanced DC transmigration could be reversed by blocking CCR7, intercellular adhesion molecule-1, or E-selectin. In an experimental model of lymphedema, where lymphatic drainage is greatly reduced or absent, lymphatic endothelial expression of CCL21 was nearly absent. Conclusions: These findings introduce transmural flow as an important regulator of lymphatic endothelial function and suggest that flow might serve as an early inflammatory signal for lymphatics, causing them to regulate transport functions to facilitate the delivery of soluble antigens and DCs to lymph nodes.

Lymphatic lipid transport: sewer or subway?

The lymphatics began receiving attention in the scientific community as early as 1622, when Gasparo Aselli noted the appearance of milky-white vessels in the mesentery of a well-fed dog. Since this time, the lymphatic system has been historically regarded as the sewer of the vasculature, passively draining fluid and proteins from the interstitial spaces (along with lipid from the gut) into the blood. Recent reports, however, suggest that the lymphatic role in lipid transport is an active and intricate process, and that when lymphatic function is compromised, there are systemic consequences to lipid metabolism and transport. This review highlights these recent findings, and suggests future directions for understanding the interplay between lymphatic and lipid biology in health and disease.

Measuring microlymphatic flow using fast video microscopy

Despite advances in the measurement of lymphatic function, little is known about the actual velocities of flow in microlymphatic ( approximately 100 mum diam) vessels. In this work, video microscopy and particle tracking methods are adapted and integrated with an ultra-high-speed imaging camera to obtain measurements of lymph velocities throughout the entire lymphatic contraction cycle in the ratmesentery, something that previous systems were incapable of measuring. To determine the systems accuracy, calibration experiments are conducted across the hypothesized physiologically significant range of velocities for microlymphatic flow (up to 15 mmsec). The system shows high accuracy, less than 2% error, when comparing actual with measured velocities. Microspheres flowing through 140-mum-diam tubing are imaged to demonstrate the systems ability to determine flow rates in these small vessels by measuring particle velocities. To demonstrate biological applicability, mesenteric microlymphatics in loops of the small intestine of three male Sprague-Dawley rats are exteriorized and imaged with the high-speed system at a rate of 500 framessec for several contraction sequences. Lymph velocity fluctuates cyclically with the vessel wall contractions, ranging from -1 to 7 mmsec. These rates are higher than would be possible with standard video microscopy (3.75 mmsec maximum).

A Tissue-Engineered Model of the Intestinal Lacteal for Evaluating Lipid Transport by Lymphatics

Lacteals are the entry point of all dietary lipids into the circulation, yet little is known about the active regulation of lipid uptake by these lymphatic vessels, and there lacks in vitro models to study the lacteal—enterocyte interface. We describe an in vitro model of the human intestinal microenvironment containing differentiated Caco‐2 cells and lymphatic endothelial cells (LECs). We characterize the model for fatty acid, lipoprotein, albumin, and dextran transport, and compare to qualitative uptake of fatty acids into lacteals in vivo. We demonstrate relevant morphological features of both cell types and strongly polarized transport of fatty acid in the intestinal‐to‐lymphatic direction. We found much higher transport rates of lipid than of dextran or albumin across the lymphatic endothelial monolayer, suggesting most lipid transport is active and intracellular. This was confirmed with confocal imaging of Bodipy, a fluorescent fatty acid, along with transmission electron microscopy. Since our model recapitulates crucial aspects of the in vivo lymphatic–enterocyte interface, it is useful for studying the biology of lipid transport by lymphatics and as a tool for screening drugs and nanoparticles that target intestinal lymphatics. Biotechnol. Bioeng. 2009;103: 1224–1235.

Sensitivity analysis of near-infrared functional lymphatic imaging

Near-infrared imaging of lymphatic drainage of injected indocyanine green (ICG) has emerged as a new technology for clinical imaging of lymphatic architecture and quantification of vessel function, yet the imaging capabilities of this approach have yet to be quantitatively characterized. We seek to quantify its capabilities as a diagnostic tool for lymphatic disease. Imaging is performed in a tissue phantom for sensitivity analysis and in hairless rats for in vivo testing. To demonstrate the efficacy of this imaging approach to quantifying immediate functional changes in lymphatics, we investigate the effects of a topically applied nitric oxide (NO) donor glyceryl trinitrate ointment. Premixing ICG with albumin induces greater fluorescence intensity, with the ideal concentration being 150 μg/mL ICG and 60 g/L albumin. ICG fluorescence can be detected at a concentration of 150 μg/mL as deep as 6 mm with our system, but spatial resolution deteriorates below 3 mm, skewing measurements of vessel geometry. NO treatment slows lymphatic transport, which is reflected in increased transport time, reduced packet frequency, reduced packet velocity, and reduced effective contraction length. NIR imaging may be an alternative to invasive procedures measuring lymphatic function in vivo in real time.

Constitutive Modeling of Mouse Carotid Arteries Using Experimentally Measured Microstructural Parameters

Changes in the local mechanical environment and tissue mechanical properties affect the biological activity of cells and play a key role in a variety of diseases, such as cancer, arthritis, nephropathy, and cardiovascular disease. Constitutive relations have long been used to predict the local mechanical environment within biological tissues and to investigate the relationship between biological responses and mechanical stimuli. Recent constitutive relations for soft tissues consider both material and structural properties by incorporating parameters that describe microstructural organization, such as fiber distributions, fiber angles, fiber crimping, and constituent volume fractions. The recently developed technique of imaging the microstructure of a single artery as it undergoes multiple deformations provides quantitative structural data that can reduce the number of estimated parameters by using parameters that are truly experimentally intractable. Here, we employed nonlinear multiphoton microscopy to quantify collagen fiber organization in mouse carotid arteries and incorporated measured fiber distribution data into structurally motivated constitutive relations. Microscopy results demonstrate that collagen fibers deform in an affine manner over physiologically relevant deformations. The incorporation of measured fiber angle distributions into constitutive relations improves the models predictive accuracy and does not significantly reduce the goodness of fit. The use of measured structural parameters rather than estimated structural parameters promises to improve the predictive capabilities of the local mechanical environment, and to extend the utility of intravital imaging methods for estimating the mechanical behavior of tissues using in situ structural information.

A multichamber fluidic device for 3D cultures under interstitial flow with live imaging: Development, characterization, and applications

Interstitial flow is an important biophysical cue that can affect capillary morphogenesis, tumor cell migration, and fibroblast remodeling of the extracellular matrix, among others. Current models that incorporate interstitial flow and that are suitable for live imaging lack the ability to perform multiple simultaneous experiments, for example, to compare effects of growth factors, extracellular matrix composition, etc. We present a nine‐chamber radial flow device that allows simultaneous 3D fluidic experiments for relatively long‐term culture with live imaging capabilities. Flow velocity profiles were characterized by fluorescence recovery after photobleaching (FRAP) for flow uniformity and estimating the hydraulic conductivity. We demonstrate lymphatic and blood capillary morphogenesis in fibrin gels over 10 days, comparing flow with static conditions as well as the effects of an engineered variant of VEGF that binds fibrin via Factor XIII. We also demonstrate the culture of contractile fibroblasts and co‐cultures with tumor cells for modeling the tumor microenvironment. Therefore, this device is useful for studies of capillary morphogenesis, cell migration, contractile cells like fibroblasts, and multicellular cultures, all under interstitial flow. Biotechnol. Bioeng. 2010;105: 982–991.

Low-cost microcontroller platform for studying lymphatic biomechanics in vitro.

The pumping innate to collecting lymphatic vessels routinely exposes the endothelium to oscillatory wall shear stress and other dynamic forces. However, studying the mechanical sensitivity of the lymphatic endothelium remains a difficult task due to limitations of commercial or custom systems to apply a variety of time-varying stresses in vitro. Current biomechanical in vitro testing devices are very expensive, limited in capability, or highly complex; rendering them largely inaccessible to the endothelial cell biology community. To address these shortcomings, the authors propose a reliable, low-cost platform for augmenting the capabilities of commercially available pumps to produce a wide variety of flow rate waveforms. In particular, the Arduino Uno, a microcontroller development board, is used to provide open-loop control of a digital peristaltic pump using precisely timed serial commands. In addition, the flexibility of this platform is further demonstrated through its support of a custom-built cell-straining device capable of producing oscillatory strains with varying amplitudes and frequencies. Hence, this microcontroller development board is shown to be an inexpensive, precise, and easy-to-use tool for supplementing in vitro assays to quantify the effects of biomechanical forces on lymphatic endothelial cells.

Mechanisms of chylomicron uptake into lacteals.

Right from birth, the lymphatics play a crucial role in dietary functions. A majority of the lipid absorbed from the newborns lipid‐rich diet enters the blood circulation through the lymphatic system, which transports triglyceride‐loaded particles known as chylomicrons from the villi of the small intestine to the venous circulation near the heart. In light of the significance of this role, as well as the fact that lipid transport from the gut was one of the earliest discovered functions of the lymphatic vasculature, it is surprising that so little is known about how chylomicrons initially gain access to the lymphatic vessel. This review will focus on the current mechanisms thought to be important in this process and highlight important questions that need to be answered in the future.

Dual-wavelength polarimetry for monitoring glucose in the presence of varying birefringence

In a continuing effort to develop a noninvasive means of monitoring glucose levels using the aqueous humor of the eye, a dual-wavelength system is developed to show that varying birefringence, similar to what is seen with a moving cornea, can be compensated. In this work, a dual-wavelength, closed-loop system is designed and a model is developed to extract the glucose concentration information. The system and model are tested using various concentrations of glucose in a birefringent test cell subject to motion artifact. The results show that for a static, nonmoving sample, glucose can be predicted to within 10 mg/dl for the entire physiologic range (0 to 600 mg/dl) for either laser wavelength (523 or 635 nm). In the presence of moving birefringence, each individual wavelength produces standard errors on the order of a few thousand mg/dL. However, when the two wavelengths are combined into the developed model, this error is less than 20 mg/dL. The approach shows that multiple wavelengths can be used to drastically reduce the error in the presence of a moving birefringent sample and thus may have the potential to be used to noninvasively monitor glucose levels in vivo in the presence of moving corneal birefringence.