Anatoliy A. Gashev

Phasic contractions of rat mesenteric lymphatics increase basal and phasic nitric oxide generation in vivo

Multiple investigators have shown interdependence of lymphatic contractions on nitric oxide (NO) activity by pharmacological and traumatic suppression of endothelial NO synthase (eNOS). We demonstrated that lymphatic diastolic relaxation is particularly sensitive to NO from the lymphatic endothelium. The predicted mechanism is shear forces produced by the lymph flow during phasic pumping, activating eNOS in the lymphatic endothelium to produce NO. We measured [NO] during phasic contractions using microelectrodes on in situ mesenteric lymphatics in anesthetized rats under basal conditions and with an intravenous saline bolus (0.5 ml/100 g) or infusion (0.5 ml x 100 g(-1) x h(-1)). Under basal conditions, [NO] measured on the tubular portions of the lymphatics was approximately 200-250 nM, slightly higher than in the adjacent adipocyte microvasculature, whereas [NO] measured on the lymphatic bulb surface was approximately 400 nM. Immunohistochemistry of eNOS in isolated lympathics indicated a much greater expression in the lymph valves and surrounding bulb area than in the tubular regions. During phasic lymphatic contractions, the valve and tubular [NO] increased with each contraction, and during intravenous saline infusion, [NO] increased in proportion to the contraction frequency and, presumably, lymph flow. The partial blockade of eNOS over approximately 1 cm length with N(omega)-nitro-L-arginine methyl ester lowered the [NO]. These in vivo data document for the first time that both valvular and tubular lymphatic segments increase NO generation during each phasic contraction and that [NO] summated with increased contraction frequency. The combined data predict regional variations in eNOS and [NO] in the tubular and valve areas, plus the summated NO responses dependent on contraction frequency provide for a complex relaxation mechanism involving NO.

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).

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.

Involvement of histamine in endothelium-dependent relaxation of mesenteric lymphatic vessels

The knowledge of the basic principles of lymphatic function, still remains, to a large degree, rudimentary and will require significant research efforts. Recent studies of the physiology of the MLVs suggested the presence of an EDRF other than NO. In this study, we tested the hypothesis that lymphatic endothelium‐derived histamine relaxes MLVs.

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

BACKGROUNDnRecent 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.nnnMETHODS AND RESULTSnIn 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.nnnCONCLUSIONSnThe 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.

Regional heterogeneity of length-tension relationships in rat lymph vessels.

BACKGROUNDnHeterogeneity of the length-tension relationships in lymph vessels has never been evaluated systematically.nnnMETHODS AND RESULTSnIn this study we measured the length-tension relationships in lymph vessels from three different regions of the rat: thoracic duct, cervical, and femoral lymph vessels, and compared the results to our previous measurements of rat mesenteric lymph vessels. We performed isometric force measurements on activated and passive lymph vessel segments using a small-vessel wire myograph. We found that all groups of vessels had relatively broad plateaus in their active tension versus length relationships, suggesting that they are adapted to generate near-maximal tensions over a relatively wide range of preloads (at least 0.85-1.05 L(0)). Thoracic duct exhibited the flattest active tension curve, particularly for peak active tension, in which there was less than a 5% change in peak active tension from 0.75 to 1.30 of optimal length. Femoral lymph vessels were able to withstand the highest estimated pressures, followed by mesenteric and cervical vessels and then thoracic duct.nnnCONCLUSIONSnWe conclude that lymph vessels effectively adapt their contractile force to the particular hydrodynamic conditions (transmural pressures and intraluminal flows) that exist in different regions of the lymphatic system.

Aging-related anatomical and biochemical changes in lymphatic collectors impair lymph transport, fluid homeostasis, and pathogen clearance

The role of lymphatic vessels is to transport fluid, soluble molecules, and immune cells to the draining lymph nodes. Here, we analyze how the aging process affects the functionality of the lymphatic collectors and the dynamics of lymph flow. Ultrastructural, biochemical, and proteomic analysis indicates a loss of matrix proteins, and smooth muscle cells in aged collectors resulting in a decrease in contraction frequency, systolic lymph flow velocity, and pumping activity, as measured in vivo in lymphatic collectors. Functionally, this impairment also translated into a reduced ability for in vivo bacterial transport as determined by time‐lapse microscopy. Ultrastructural and proteomic analysis also indicates a decrease in the thickness of the endothelial cell glycocalyx and loss of gap junction proteins in aged lymph collectors. Redox proteomic analysis mapped an aging‐related increase in the glycation and carboxylation of lymphatics endothelial cell and matrix proteins. Functionally, these modifications translate into apparent hyperpermeability of the lymphatics with pathogen escaping from the collectors into the surrounding tissue and a decreased ability to control tissue fluid homeostasis. Altogether, our data provide a mechanistic analysis of how the anatomical and biochemical changes, occurring in aged lymphatic vessels, compromise lymph flow, tissue fluid homeostasis, and pathogen transport.

An Automated Method to Control Preload by Compensation for Stress Relaxation in Spontaneously Contracting, Isometric Rat Mesenteric Lymphatics

ABSTRACT

Maximum shortening velocity of lymphatic muscle approaches that of striated muscle

Lymphatic muscle (LM) is widely considered to be a type of vascular smooth muscle, even though LM cells uniquely express contractile proteins from both smooth muscle and cardiac muscle. We tested the hypothesis that LM exhibits an unloaded maximum shortening velocity (Vmax) intermediate between that of smooth muscle and cardiac muscle. Single lymphatic vessels were dissected from the rat mesentery, mounted in a servo-controlled wire myograph, and subjected to isotonic quick release protocols during spontaneous or agonist-evoked contractions. After maximal activation, isotonic quick releases were performed at both the peak and plateau phases of contraction. Vmax was 0.48 ± 0.04 lengths (L)/s at the peak: 2.3 times higher than that of mesenteric arteries and 11.4 times higher than mesenteric veins. In cannulated, pressurized lymphatic vessels, shortening velocity was determined from the maximal rate of constriction [rate of change in internal diameter (-dD/dt)] during spontaneous contractions at optimal preload and minimal afterload; peak -dD/dt exceeded that obtained during any of the isotonic quick release protocols (2.14 ± 0.30 L/s). Peak -dD/dt declined with pressure elevation or activation using substance P. Thus, isotonic methods yielded Vmax values for LM in the mid to high end (0.48 L/s) of those the recorded for phasic smooth muscle (0.05-0.5 L/s), whereas isobaric measurements yielded values (>2.0 L/s) that overlapped the midrange of values for cardiac muscle (0.6-3.3 L/s). Our results challenge the dogma that LM is classical vascular smooth muscle, and its unusually high Vmax is consistent with the expression of cardiac muscle contractile proteins in the lymphatic vessel wall.

Demonstration and Analysis of the Suction Effect for Pumping Lymph from Tissue Beds at Subatmospheric Pressure

Many tissues exhibit subatmospheric interstitial pressures under normal physiologic conditions. The mechanisms by which the lymphatic system extracts fluid from these tissues against the overall pressure gradient are unknown. We address this important physiologic issue by combining experimental measurements of contractile function and pressure generation with a previously validated mathematical model. We provide definitive evidence for the existence of ‘suction pressure’ in collecting lymphatic vessels, which manifests as a transient drop in pressure downstream of the inlet valve following contraction. This suction opens the inlet valve and is required for filling in the presence of low upstream pressure. Positive transmural pressure is required for this suction, providing the energy required to reopen the vessel. Alternatively, external vessel tethering can serve the same purpose when the transmural pressure is negative. Suction is transmitted upstream, allowing fluid to be drawn in through initial lymphatics. Because suction plays a major role in fluid entry to the lymphatics and is affected by interstitial pressure, our results introduce the phenomenon as another important factor to consider in the study of lymphoedema and its treatment.