Lymphatic Cannulation for Lymph Sampling and Molecular Delivery.

Unlike the blood, the interstitial fluid and the deriving lymph are directly bathing the cellular layer of each organ. As such, composition analysis of the lymphatic fluid can provide more precise biochemical and cellular information on an organ's health and be a valuable resource for biomarker discovery. In this study, we describe a protocol for cannulation of mouse and rat lymphatic collectors that is suitable for the following: the "omic" sampling of pre- and postnodal lymph, collected from different anatomical districts; the phenotyping of immune cells circulating between parenchymal organs and draining lymph nodes; injection of known amounts of molecules for quantitative immunological studies of nodal trafficking and/or clearance; and monitoring an organ's biochemical omic changes in pathological conditions. Our data indicate that probing the lymphatic fluid can provide an accurate snapshot of an organ's physiology/pathology, making it an ideal target for liquid biopsy.

Collecting lymphatic vessel permeability facilitates adipose tissue inflammation and distribution of antigen to lymph node-homing adipose tissue dendritic cells.

Collecting lymphatic vessels (CLVs), surrounded by fat and endowed with contractile muscle and valves, transport lymph from tissues after it is absorbed into lymphatic capillaries. CLVs are not known to participate in immune responses. In this study, we observed that the inherent permeability of CLVs allowed broad distribution of lymph components within surrounding fat for uptake by adjacent macrophages and dendritic cells (DCs) that actively interacted with CLVs. Endocytosis of lymph-derived Ags by these cells supported recall T cell responses in the fat and also generated Ag-bearing DCs for emigration into adjacent lymph nodes (LNs). Enhanced recruitment of DCs to inflammation-reactive LNs significantly relied on adipose tissue DCs to maintain sufficient numbers of Ag-bearing DCs as the LN expanded. Thus, CLVs coordinate inflammation and immunity within adipose depots and foster the generation of an unexpected pool of APCs for Ag transport into the adjacent LN.

The position- and lymphatic lumen-controlled tissue chambers to study live lymphatic vessels and surrounding tissues ex vivo.

BACKGROUND: Until now, there has been no tool available to provide lymphatic researchers the ability to perform experiments in tissue explants containing lymphatic vessels under tissue position- and lymphatic lumen-controlled conditions. METHODS AND RESULTS: In this article we provide technical details and description of the method of using the newly developed and implemented the position- and lymphatic lumen-controlled tissue chambers to study live lymphatic vessels and surrounding tissues ex vivo. In this study, we, for the first time, performed detailed comparative analysis of the contractile and pumping activity of rat mesenteric lymphatic vessels (MLVs) situated within tissue explants mounted in new tissue chambers and isolated, cannulated, and pressurized rat MLVs maintained in isolated vessel setups. We found no significant differences of the effects of both transmural pressure- and wall shear stress sensitivities of MLVs in tissue chambers and isolated MLVs. CONCLUSIONS: We conclude that this new experimental tool, a position- and lymphatic lumen-controlled tissue chamber, allows precise investigation of lymphatic function of MLVs interacting with elements of the tissue microenvironment. This method provides an important new set of experimental tools to investigate lymphatic function.

An immunological fingerprint differentiates muscular lymphatics from arteries and veins.

The principal function of the lymphatic system is to transport lymph from the interstitium to the nodes and then from the nodes to the blood. In doing so lymphatics play important roles in fluid homeostasis, macromolecular/antigen transport and immune cell trafficking. To better understand the genes that contribute to their unique physiology, we compared the transcriptional profile of muscular lymphatics (prenodal mesenteric microlymphatics and large, postnodal thoracic duct) to axillary and mesenteric arteries and veins isolated from rats. Clustering of the differentially expressed genes demonstrated that the lymph versus blood vessel differences were more profound than between blood vessels, particularly the microvessels. Gene ontology functional category analysis indicated that microlymphatics were enriched in antigen processing/presentation, IgE receptor signaling, catabolic processes, translation and ribosome; while they were diminished in oxygen transport, regulation of cell proliferation, glycolysis and inhibition of adenylate cyclase activity by G-proteins. We evaluated the differentially expressed microarray genes/products by qPCR and/or immunofluorescence. Immunofluorescence documented that multiple MHC class II antigen presentation proteins were highly expressed by an antigen-presenting cell (APC) type found resident within the lymphatic wall. These APCs also expressed CD86, a co-stimulatory protein necessary for T-cell activation. We evaluated the distribution and phenotype of APCs within the pre and postnodal lymphatic network. This study documents a novel population of APCs resident within the walls of muscular, prenodal lymphatics that indicates novel roles in antigen sampling and immune responses. In conclusion, these prenodal lymphatics exhibit a unique profile that distinguishes them from blood vessels and highlights the role of the lymphatic system as an immunovascular system linking the parenchymal interstitium, lymph nodes and the blood.

Lymphatic muscle cells in rat mesenteric lymphatic vessels of various ages.

BACKGROUND: Recent studies on aging-associated changes in mesenteric lymph flow in situ demonstrated predominance of the severe negative chronotropic effect of aging on the contractility of aged mesenteric lymphatic vessels (MLV). At the same time, contraction amplitude of the aged vessels was only slightly diminished by aging and can be rapidly stimulated within 5-15 minutes. However, the detailed quantitative evaluation of potential aging-associated changes in muscle cells investiture in MLV has never been performed. METHODS AND RESULTS: In this study we, for the first time, performed detailed evaluation of muscle cells investiture in MLV in reference to the position of lymphatic valve in different zones of lymphangion within various age groups (3-mo, 9-mo and 24-mo Fischer-344 rats). Using visual and quantitative analyses of the images of MLV immunohistochemically labeled for actin, we confirmed that the zones located close upstream (pre-valve zones) and above lymphatic valves (valve zones) possess the lowest investiture of lymphatic muscle cells. Most of the high muscle cells investiture zones exist downstream to the lymphatic valve (post-valve zones). The muscle cells investiture of these zones is not affected by aging, while pre-valve and valve zones demonstrate significant aging-associated decrease in muscle cells investiture. CONCLUSIONS: The low muscle cells investiture zones in lymphatic vessels consist of predominantly longitudinally oriented muscle cells which are positioned in pre-valve and valve zones and connect adjacent lymphangions. These cells may provide important functional impact on the biomechanics of the lymphatic valve gating and electrical coupling between lymphangions, while their aging-associated changes may delimit adaptive reserves of aged lymphatic vessels.

Evidence of increased oxidative stress in aged mesenteric lymphatic vessels.

BACKGROUND: We have previously shown that aging is associated with weakened rat mesenteric lymphatic vessel (MLV) contractility. However, the specific mechanisms contributing to this aging-associated contractile degeneration remain unknown. Aging is often associated with elevations in oxidative stress, and reactive oxygen species (ROS) have been shown to reduce the contractility of MLV. Thus in the present study, we sought to assess whether aging is associated with increased levels of oxidative stress and oxidative damage in MLV. METHODS AND RESULTS: MLV were isolated from 9-mo- and 24-mo-old Fischer-344 rats and subjected to the following experimental techniques: measurement of total superoxide dismutase (SOD) activity; estimation of lipid peroxidation levels via measurement of thiobarbituric acid reactive substances (TBARS); detection of superoxide and mitochondrial ROS in live MLV; Western blot analysis, and immunohistochemical labeling of the SOD isoforms and nitro-tyrosine proteins. We found that aging is associated with increased levels of cellular superoxide and mitochondrial ROS concomitant with a reduction in Cu/Zn-SOD protein expression and total SOD enzymatic activity in MLV. This increase in oxidative stress and decrease in antioxidant activity was associated with evidence of increased lipid (as indicated by TBARS) and protein (as indicated by nitro-tyrosine labeling) oxidative damage. CONCLUSIONS: Thus for the first time, we demonstrate that aging-associated increases in oxidative stress and oxidative damage is indeed present in the walls of MLV and may contribute to the aging-associated lymphatic pump dysfunction we previously reported.

Isolation and preparation of RNA from rat blood and lymphatic microvessels for use in microarray analysis.

DNA microarray methodologies have proven to be an indispensable tool for genome-wide transcriptional profiling of organs, tissues, and cells. Here, we present a protocol for the optimized isolation and preparation of RNA from rat microvessels (including arteries, veins, and lymphatics) for subsequent use in two-color microarray analysis. The investigation of wide-ranging vessel sizes from all three vessel lineages necessitates an RNA isolation strategy that can effectively isolate high-quality RNA from varying and often very small quantities (<1 mg) of fibrous vessel tissue. Additionally, the lack of sample biomass necessitates the use of amplification strategies to generate enough RNA for use in microarray analysis. While the methods presented here were developed for use with two-color microarray analysis, the procedures and general concepts are applicable to most fluorescence-based microarray platforms.

Aging-associated alterations in contractility of rat mesenteric lymphatic vessels.

OBJECTIVE: To evaluate the age-related changes in pumping of mesenteric lymphatic vessels in 9- and 24-month-old male Fisher-344 rats. METHODS: Lymphatic diameters, contraction amplitude, contraction frequency, and fractional pump flow were determined in isolated MLV before and after l-NAME application. RESULTS: The data demonstrate a severe weakening of the lymphatic pump in aged MLV including diminished lymphatic contraction amplitude, contraction frequency, and as a result, lymphatic pump activity. The data also suggest that the imposed flow gradient-generated shear-dependent relaxation does not exist in aged rat MLV, and the sensitivity of both adult and aged MLV to such shear cannot be eliminated by nitric oxide (NO) synthases blockade. CONCLUSIONS: These data provide new evidence of lymphatic regional heterogeneity for both adult and aged MLV. In MLV, a constant interplay between the tonic and phasic components of the myogenic response and the shear-dependent release of NO predominantly determine the level of contractile activity; the existence of another shear-dependent, but NO-independent regulatory mechanism is probably present. Aging remarkably weakens MLV contractility, which would predispose this lymphatic network to lower total lymph flow in resting conditions and limit the ability to respond to an edemagenic challenge in the elderly.

Indocyanine green and lymphatic imaging: current problems.

BACKGROUND: Near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) has recently been presented as a comparatively easy and informative technique to image lymphatic channels in vivo. However, no data or references have been provided concerning the impact of ICG application on normal lymphatic contractility and lymph transport. Thus, the imaging agent and/or the method of administration may introduce a significant artifact. METHODS AND RESULTS: Standard pharmacological tests were performed to investigate the influence of ICG on the spontaneous contractility of isolated, cannulated, and pressurized rat mesenteric lymphatic vessels. The data demonstrate that non-irradiated ICG dramatically and dynamically influences the contractility of rat lymphatic vessels in both a dose- and diluent-dependent manner with low ICG concentrations principally altering contractile frequency and higher ICG concentrations completely blocking lymphatic contractility. CONCLUSIONS: Currently, both researchers and doctors should exercise caution in extrapolating the data obtained with ICG imaging to normal lymphatic function regardless of whether it was obtained in mice, pigs, or humans. Careful and extended pharmacological tests must be performed to evaluate the mechanism of action of ICG on the contractility and physiology of lymphatic vessels with consideration of dose, diluent, and duration of irradiation.

Transforming growth factor-beta signaling in hypertensive remodeling of porcine aorta.

A porcine aortic coarctation model was used to examine regulation of gene expression in early hypertensive vascular remodeling. Aortic segments were collected proximal (high pressure) and distal (low pressure) to the coarctation after 2 wk of sustained hypertension (mean arterial pressure>150 mmHg). Porcine 10K oligoarrays used for gene expression profiling of the two regions of aorta revealed downregulation of cytoskeletal and upregulation of extracellular region genes relative to the whole genome. A genomic database search for transforming growth factor-beta (TGF-beta) control elements showed that 19% of the genes that changed expression due to hypertension contained putative TGF-beta control elements. Real-time RT-PCR and microarray analysis showed no change in expression of TGF-beta1, TGF-beta2, TGF-beta3, or bone morphogenetic proteins-2 and -4, yet immunohistochemical staining for phosphorylated SMAD2, an indicator of TGF-beta signaling, and for phosphorylated SMAD1/5/8, an indicator of signaling through the bone morphogenetic proteins, showed the highest percentage of positively stained cells in the proximal aortic segments of occluded animals. For TGF-beta signaling, this increase was significantly different than for sham-operated controls. Western blot analysis showed no difference in total TGF-beta1 protein levels with respect to treatment or aortic segment. Immunohistochemistry showed that the protein levels of latency-associated peptide was decreased in proximal segments of occluded animals. Collectively, these results suggest that activation of TGF-beta, but not altered expression, may be a major mechanism regulating early hypertensive vascular remodeling.

Microarray analysis of VEGF-C responsive genes in human lymphatic endothelial cells.

Vascular endothelial growth factor-C (VEGF-C) is considered one of the most important factors influencing lymphatic endothelial cell biology. The goal of this work was to characterize the gene expression response by lymphatic endothelial cells (LECs) to VEGF-C. Primary cultures of human microvascular LECs were exposed to 100 ng/mL VEGF-C for 30 minutes and 6 hours, and their lysates were evaluated by microarray analysis to determine changes in mRNA expression induced by VEGF-C. Characteristic of a response to a growth factor stimulus, the largest number of differentially expressed genes were transcription factors and cell cycle related. A number of genes known to be important in angiogenesis, tumorigenesis and tumor invasion, and the transport of proteins, solutes, and lipids were also affected. Interestingly, a number of genes related to lipid metabolism as well as neurogenesis and neurodegeneration were also responsive to VEGF-C stimulation. Further analysis of these genes may not only provide insight into the molecular mechanisms underlying lymphangiogenesis and associated pathogenesis, but may also identify other important roles of VEGF-C.

Lymphatic muscle: a review of contractile function.

A recent surge in lymphangiogenesis research has led to a greater understanding of lymphatic endothelial cell biology. However, a general understanding of lymphatic muscle cell biology lags far behind its endothelial counterpart. Lymphatics at the level of the collecting vessels and higher contain muscular walls capable of both tonic and phasic contractions, which both generate and regulate lymph flow. Because lymphatic contraction is crucial to lymphatic function, a solid understanding of lymphatic muscle development and function is necessary to understand lymphatic biology. This review summarizes the current body of lymphatic muscle research and addresses important questions that are currently unanswered.