- 254-742-3033
D. Zawieja
Texas A&M Health Science Center, Medical Physiology, Faculty Member
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... more
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 lymphatic's endothelial cell and matrix proteins. Functi...
Research Interests: Gap Junctions, Mass Spectrometry, Aging, Biology, Medicine, and 15 moreBiological Sciences, Endothelial Cells, Animals, Male, Glycosylation, Lymphatics, Homeostasis, Amino Acid Sequence, Lymphatic System, Extracellular Matrix Proteins, Lymph nodes, Connexins, glycocalyx, Mesentery, and Medical and Health Sciences
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Studies of microvascular, tissue and lymphatic transport using microphotometric and microfluorometric techniques are potentially subject to artifact due to variations in tissue specimen thickness. Absorbance techniques utilize the... more
Studies of microvascular, tissue and lymphatic transport using microphotometric and microfluorometric techniques are potentially subject to artifact due to variations in tissue specimen thickness. Absorbance techniques utilize the Lambert-Beer law in which A = log I0/I = act, where A is absorbance, I0 is incident light intensity, I is transmitted intensity, a is an absorbance coefficient, c is concentration of substance, and t is path length. If differences in t are known to be present, then inferences of changes in c from changes in A become suspect. In microfluorometry the amount of light gathered is proportional to the number of fluorochromes in the effective cuvette, which is determined by the microscope's numerical aperture and the sample thickness. If variations in thickness are known to occur, the effective cuvette volume may be changing; therefore, inferences of changes in fluorochrome concentration from changes in intensity become suspect. Existing data suggest that rat...
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Lymph samples were obtained from villus initial and mesenteric prenodal collecting lymphatics of the anesthetized rat using standard micropuncture techniques. The protein concentrations of the lymph samples were determined using a... more
Lymph samples were obtained from villus initial and mesenteric prenodal collecting lymphatics of the anesthetized rat using standard micropuncture techniques. The protein concentrations of the lymph samples were determined using a fluorometric microassay. These procedures were performed on preparations either superfused with a modified Krebs solution or covered with paraffin oil. The protein-concentrating ability of the intestinal lymphatics was evaluated to test the osmotic theory of lymph formation. The mean initial lymph protein concentration in superfused preparations was 2.08 g/dl (SE = 0.20). The lymph protein concentration in collecting vessels in superfused preparations was 2.20 g/dl (SE = 0.19). The protein concentration from initial lymphatics in oil-covered preparation was 2.48 g/dl (SE = 0.17). The lymph protein concentration in collecting vessels in oil-covered preparations was 2.32 g/dl (SE = 0.15). The difference between initial and collecting lymph protein concentrat...
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Previous studies in the literature indicate that intraenteric placement of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) evokes an intestinal inflammatory response characterized by an accumulation of... more
Previous studies in the literature indicate that intraenteric placement of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) evokes an intestinal inflammatory response characterized by an accumulation of interstitial fluid and increased lymph flow. Furthermore, it is known that movement of lymph away from the intestine is dependent on the rhythmic pumping of lymph by collecting lymphatics in the mesentery. The purpose of the present study was to determine whether the f-Met-Leu-Phe-induced increase in lymph formation is countered by an increase in lymphatic pump efficiency. Male Sprague-Dawley rats were anesthetized, and a segment of ileum with adjacent mesentery was exteriorized. The mesentery was positioned over an optical window, and a 100-microns collecting lymphatic was selected for study. The preparation was transferred to a video microscope, and the activity of the lymphatic pump was monitored under control conditions and during intraluminal infus...
Research Interests: Physiology, Medicine, Animals, Male, Medical Physiology, and 4 moreIntestines, Rats, Rat, and Lymphatic System
This study reports measurements of porcine neutrophil dynamics in isolated microvessels. Porcine coronary venules and arterioles were isolated, cannulated, and perfused with fluorescently labeled neutrophils at a series of flow... more
This study reports measurements of porcine neutrophil dynamics in isolated microvessels. Porcine coronary venules and arterioles were isolated, cannulated, and perfused with fluorescently labeled neutrophils at a series of flow velocities. In venules (62.50 +/- 5.41 microns diam) under control conditions, rolling neutrophils were often observed at intraluminal flow velocities ranging from 600 to 6,000 microns/s, and the rolling fraction varied inversely as a function of flow velocity. There was no significant adherence under the control conditions at any of the various flow velocities. Pretreatment of the neutrophils with human recombinant complement 5a (C5a, 10(-8) M) increased adherence at low flow velocities but did not alter the rolling fraction. In contrast to venules, rolling neutrophils were not observed in arterioles (58.80 +/- 5.6 microns diam). Furthermore, neutrophils that were pretreated with C5a did not adhere to the arteriolar endothelium even at low flow velocities. W...
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The propagation and coordination of lymphatic contractions were studied in the mesentery of the rat small intestine using in situ microscopic observation. Indexes of lymphatic diameter were simultaneously measured at two adjacent... more
The propagation and coordination of lymphatic contractions were studied in the mesentery of the rat small intestine using in situ microscopic observation. Indexes of lymphatic diameter were simultaneously measured at two adjacent lymphangions in spontaneously contracting lymphatics (n = 51). Diameter index, contraction frequency, and the percentage of the intersegmental contractions that were propagated and coordinated (PP) were determined at both sites. The conduction velocity of the contractile activity and the percentage of the coordinated contractions that were propagated both antegrade to the direction of lymph flow and retrograde to the flow stream were determined. The results indicate that 1) 80-90% of the lymphatic contractions in the vessels we evaluated were propagated, 2) the wave of contractile activity propagated both centrally and peripherally, and 3) the conduction velocity of the contractile activity was approximately 4-8 mm/s. We tested the hypothesis that gap junct...
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Vascular smooth muscle responds to stretch with an increase in active force development. To investigate the role of Ca2+ in this response, we used the fluorescent dye fura-2 to quantitate changes in cytosolic Ca2+ in single, vascular... more
Vascular smooth muscle responds to stretch with an increase in active force development. To investigate the role of Ca2+ in this response, we used the fluorescent dye fura-2 to quantitate changes in cytosolic Ca2+ in single, vascular smooth muscle cells during rapid stretch. Cells were enzymatically dispersed from pig coronary arteries, loaded with fura-2/AM, and studied using a digital-imaging microscope. Stretch of individual cells was accomplished by attachment with suction to two patch-type micropipettes to apply force to the ends of the cell. Stretch induced the release of Ca2+ from intracellular stores as well Ca2+ influx across the plasma membrane. In physiological saline solution containing 1.5 mM Ca2+, intracellular calcium increased with cell stretch in a sigmoidal fashion. This relationship was shifted upward in 10 mM Ca2+ bath solution and abolished after several minutes in Ca(2+)-free solution. The dihydropyridine Ca2+ channel blocker nifedipine, in doses sufficient to ...
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In this study, we hypothesized that histaminergic increases in venular permeability result from a cascade triggered by activation of phospholipase C (PLC), inducing the synthesis of nitric oxide (NO) and activating guanylate cyclase. The... more
In this study, we hypothesized that histaminergic increases in venular permeability result from a cascade triggered by activation of phospholipase C (PLC), inducing the synthesis of nitric oxide (NO) and activating guanylate cyclase. The apparent permeability coefficient to albumin (Pa) was measured in isolated porcine coronary venules subjected to constant flow and hydrostatic and oncotic pressures. Histamine (2.5, 5, and 10 microM) transiently and progressively increased Pa. The PLC inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC; 100 microM) decreased baseline permeability and abolished the effect of histamine. The NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 10 microM) and the guanylate cyclase inhibitor 6-anilinoquinoline-5,8-quinone (LY 83583; 10 microM) also blocked the histamine-induced hyperpermeability. L-Arginine (3 mM) reversed the inhibition by L-NMMA. NG-monomethyl-D-arginine did not influence the effect of histamine. Furthermore, sodium nitrop...
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Lymph nodes (LNs) are positioned strategically throughout the body as critical mediators of lymph filtration and immune response. Lymph carries cytokines, antigens, and cells to the downstream LNs, and their effective delivery to the... more
Lymph nodes (LNs) are positioned strategically throughout the body as critical mediators of lymph filtration and immune response. Lymph carries cytokines, antigens, and cells to the downstream LNs, and their effective delivery to the correct location within the LN directly impacts the quality and quantity of immune response. Despite the importance of this system, the flow patterns in LN have never been quantified, in part because experimental characterization is so difficult. To achieve a more quantitative knowledge of LN flow, a computational flow model has been developed based on the mouse popliteal LN, allowing for a parameter sensitivity analysis to identify the important system characteristics. This model suggests that about 90% of the lymph takes a peripheral path via the subcapsular and medullary sinuses, while fluid perfusing deeper into the paracortex is sequestered by parenchymal blood vessels. Fluid absorption by these blood vessels under baseline conditions was driven ma...
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The lymphatics have emerged as critical players both in the progression and resolution of inflammation. The goal of this study was to identify specific microRNAs (miRNAs) that regulate lymphatic inflammatory processes. Rat mesenteric... more
The lymphatics have emerged as critical players both in the progression and resolution of inflammation. The goal of this study was to identify specific microRNAs (miRNAs) that regulate lymphatic inflammatory processes. Rat mesenteric lymphatic endothelial cells (LECs) were exposed to the proinflammatory cytokine tumor necrosis factor-α (TNF-α) for different time points (2hr, 24hr and 96hr) and miRNA profiling was carried out by Real time PCR arrays. Our data demonstrate a specific set of miRNAs that are differentially expressed (>1.8 fold and/or p<0.05) in LECs in response to TNF-α and are involved in inflammation, angiogenesis, endothelial to mesenchymal transition (EndMT), cellular proliferation and senescence. We further characterized the expression of miR-9 that was induced in LECs and in inflamed rat mesenteric lymphatics. Our results showed that miR-9 overexpression, significantly repressed NF-κB expression, thereby suppressing inflammation but promoted LEC tube formatio...
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The lymphatic system is vital to a proper maintenance of fluid and solute homeostasis. Collecting lymphatics are composed of actively contracting tubular vessels segmented by bulbous sinus regions that encapsulate bi-leaflet check valves.... more
The lymphatic system is vital to a proper maintenance of fluid and solute homeostasis. Collecting lymphatics are composed of actively contracting tubular vessels segmented by bulbous sinus regions that encapsulate bi-leaflet check valves. Valve resistance to forward flow strongly influences pumping performance. However, because of the sub-millimeter size of the vessels with flow rates typically <1ml/h and pressures of a few cmH2O, resistance is difficult to measure experimentally. Using a newly defined idealized geometry, we employed an uncoupled approach where the solid leaflet deflections of the open valve were computed and lymph flow calculations were subsequently performed. We sought to understand: 1) the effect of sinus and leaflet size on the resulting deflections experienced by the valve leaflets and 2) the effects on valve resistance to forward flow of the fully open valve. For geometries with sinus-to-root diameter ratios…
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Diabetes in pregnancy is associated with microvascular complications and a higher incidence of preeclampsia. The regulatory signaling pathways involving nitric oxide, cGMP, and cGMP-dependent protein kinase (PKG) have been shown to be... more
Diabetes in pregnancy is associated with microvascular complications and a higher incidence of preeclampsia. The regulatory signaling pathways involving nitric oxide, cGMP, and cGMP-dependent protein kinase (PKG) have been shown to be down-regulated under diabetic conditions and contribute to the pathogenesis of vascular complications in diabetes. The present study was undertaken to investigate how high glucose concentrations regulate PKG expression in cytotrophoblast cells (CTBs). Human CTBs (Sw. 71) were treated with 45, 135, 225, 495, or 945 mg/dL glucose for 48 h. Some cells were pretreated with a p38 inhibitor (10 μM SB203580) or 10 μM rosiglitazone. After treatment, the cell lysates were subjected to measure the expression of protein kinase G1α (PKG1α), protein kinase G1β (PKG1β), soluble guanylate cyclase 1α (sGC1α), and soluble guanylate cyclase 1 β (sGC1β) by Western blot. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test. The...
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The lymphatic system is a transport system that has important roles in fluid/macromolecule homeostasis, lipid absorption, metastasis and immune function. It accomplishes these roles via the generation of a regulated lymph circulation... more
The lymphatic system is a transport system that has important roles in fluid/macromolecule homeostasis, lipid absorption, metastasis and immune function. It accomplishes these roles via the generation of a regulated lymph circulation which is dependent upon valves and pumps to overcome the normal fluid pressure gradients. Lymphatic contractility plays crucial roles in the regulation and generation of lymph transport. Whereas our understanding of lymphatic contractility in humans is somewhat limited, a number of studies both in situ and in vitro have provided important insights into the presence and modulation of lymphatic contractility. These studies have clearly demonstrated that lymphatic vessels from a number of different human tissues possess both tonic and phasic changes in contractility. These changes in contractility are presumably involved in the generation and regulation of lymph flow. It has been shown that human lymphatic contractility can be influenced by a number of neu...
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Embedded into the wall of collecting lymphatic vessels and trunks, the lymphatic smooth muscles are cardinal to the functions of the lymphatic system. Their intrinsic contractile property--the intrinsic lymph pump--through rhythmical and... more
Embedded into the wall of collecting lymphatic vessels and trunks, the lymphatic smooth muscles are cardinal to the functions of the lymphatic system. Their intrinsic contractile property--the intrinsic lymph pump--through rhythmical and phasic contractions of the vessels, represents the principal mechanism by which lymph flow is generated. Through changes in tonic constrictions, lymphatic smooth muscles also modulate lymph flow resistance. Lymphatic smooth muscles are sensitive to physical and chemical stimuli, mediating changes in their activity and modulating lymphatic drainage. Because lymphatic smooth muscles play such an important role in fluid transport, their dysfunction may be a component of many inflammatory disease states. This review presents recent findings on the physiology and cellular biology of lymphatic smooth muscles and discusses the importance of these cells for the function of the lymphatic system in physiological and pathophysiological situations.
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Research Interests: Physiology, Chemistry, Rheology, Enzyme Inhibitors, Medicine, and 15 moreBiological Sciences, Smooth muscle, Nitric oxide, Animals, Male, Pressure, Rats, Homeostasis, Nitric Oxide Synthase, Muscle contraction, Muscle Relaxation, Lymphatic System, Thoracic Duct, Medical and Health Sciences, and In Vitro Techniques
The shear stress applied to lymphatic endothelial cells (LEC) by lymph flow changes dramatically under normal conditions as well as in response to disease conditions and immune reactions. In general, LEC are known to regulate the... more
The shear stress applied to lymphatic endothelial cells (LEC) by lymph flow changes dramatically under normal conditions as well as in response to disease conditions and immune reactions. In general, LEC are known to regulate the contraction frequency and strength of lymphatic pumping in response to shear stress. Intracellular calcium concentration, [Ca(2+)]i, is an important factor that regulates lymphatic contraction characteristics. In this study, we measured changes in the [Ca(2+)]i under different shear stress levels, and determined the source of this calcium signal. Briefly, human dermal LEC were cultured in custom-made micro-channels for 3 days before loading with 2μM Fura-2 AM, a ratiometric calcium dye to measure [Ca(2+)]i. Step-changes in shear stress resulted in a rapid increase in [Ca(2+)]i followed by a gradual return to the basal level and sometimes below the initial baseline (45.2±2.2 nM). The [Ca(2+)]i reached a peak at 126.2±5.6 nM for 10 dyn/cm(2) stimulus, whereas...
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Our objective was to devise a system to automatically correct for stress relaxation of isometric rat mesenteric lymphatics (90-120 microm, ID). Stress relaxation is a particular problem in isometric studies of highly distensible vessels... more
Our objective was to devise a system to automatically correct for stress relaxation of isometric rat mesenteric lymphatics (90-120 microm, ID). Stress relaxation is a particular problem in isometric studies of highly distensible vessels and is evident as a time-dependent, secondary decline in force after an abrupt length increase. Because the phasic contraction pattern of lymphatics is exquisitely sensitive to changes in preload, stress relaxation makes stable contraction patterns difficult to achieve and analyze. A DMT wire myograph was modified to accommodate an Inchworm piezo stack in series with a standard micrometer drive to permit automated control of vessel caliber/force. The force output of the myograph was digitized and computer algorithms were devised to servo control force by changing vessel diameter. The system was tested on passive lymphatics, passive small veins, and lymphatics exhibiting spontaneous force transients. The software was designed to temporarily disable servo control during a spontaneous force transient. For both active and passive lymphatics, stable preloads were very well maintained, indicating that the system was adequately compensating for stress relaxation. The method works well with isometric rat mesenteric lymphatics without disturbing spontaneous activity. It should be applicable to arterial, venous, and lymphatic vessels (80-500 microm in diameter) isolated from other tissues and species.
Research Interests: Materials Science, Research Design, Microcirculation, Research, Medicine, and 14 moreMethods, Automation, Animals, Vasoconstriction, Lymphatics, Rats, Muscle contraction, Physiological Stress Markers, Lymphatic System, Isometric Contraction, Vein, Mesentery, Stress Physiological, and Medical and Health Sciences
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Experiments were designed to determine if the effect of preload on the spontaneous contractile activity of rat mesenteric lymphatics would be different under isometric and isobaric conditions. Isobaric protocols on cannulated, pressurized... more
Experiments were designed to determine if the effect of preload on the spontaneous contractile activity of rat mesenteric lymphatics would be different under isometric and isobaric conditions. Isobaric protocols on cannulated, pressurized rat mesenteric lymphatics ( approximately 120 microm, ID) measured the effects of lumenal pressure on contraction amplitude, frequency, and rate of diameter change (dD/dt). Analogous protocols were conducted using a wire myograph to determine the effects of passive force (preload) on the characteristics of spontaneous, isometric force transients. Servo-control systems allowed responses to ramp or step increases in preload/pressure to be tested. Under isobaric conditions, contraction amplitude was maximal at the lowest pressure tested (1 cm H(2)O), and progressively declined with pressure elevation to 10 cm H(2)O. Changes in dD/dt followed a similar pattern. In contrast, contraction frequency progressively increased with pressure. Under isometric conditions, the amplitude of spontaneous force transients was maximal at preloads of 0.3-0.4 mN, with dF/dt following a similar pattern; the average peak amplitude was approximately 15% of the maximal active contractile force developed during agonist stimulation. The frequency of the transients increased with preload from 0.05 to 0.3 mN, and remained constant at higher preloads. The amplitude-frequency product, an estimate of lymphatic pumping ability, reached a maximum value at 0.4 mN in isometric vessels and at 6 cm H(2)O in isobaric vessels. Isometric and isobaric methods yielded qualitatively similar indices of spontaneous contractile activity. However, the ranges of amplitude and frequency changes were much greater under isobaric conditions (3- to 5-fold) than under isometric conditions (50-80%).
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Research Interests: Immunology, Fluorescence Microscopy, Immunohistochemistry, Smooth muscle, Endothelial Cells, and 13 moreAnimals, Male, Polymerase Chain Reaction, Beta-Catenin, Glycoproteins, Integrins, Rats, Nitric Oxide Synthase, Endothelial cell, Time Factors, Lymphatic System, Vascular endothelial growth factor receptor, and reverse transcriptase polymerase chain reaction
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Research Interests: Immunology, Medicine, Femur, Animals, Male, and 5 morePressure, Mechanical Stress, Rats, Thoracic Duct, and Isometric Contraction
Research Interests: Microcirculation, Enzyme Inhibitors, Serotonin, Permeability, Nitric oxide, and 15 moreAnimals, PKC, Green Fluorescent Protein, Vascular, Enzyme, Nitric Oxide Synthase, Endothelial cell, Swine, Transfection, Vasodilation, Bradykinin, Arterioles, Protein Kinase C, Medical and Health Sciences, and Coronary Vessels
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Research Interests: Physiology, Kinetics, Aging, Calcium, Mitochondria, and 15 moreCaffeine, Animals, Calcium channels, Male, Basal forebrain, Potassium, Medical Physiology, Neurons, Laser Scanning Confocal Microscopy, Calcium Homeostasis, Buffers, Rat, Mitochondrial Membrane Potential, Biochemistry and cell biology, and In Vitro Techniques
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The ends of striated muscle tropomyosin (TM) are integral for thin filament cooperativity, determining the cooperative unit size and regulating the affinity of TM for actin. We hypothesized that altering the α-TM carboxy terminal overlap... more
The ends of striated muscle tropomyosin (TM) are integral for thin filament cooperativity, determining the cooperative unit size and regulating the affinity of TM for actin. We hypothesized that altering the α-TM carboxy terminal overlap end to the β-TM counterpart would affect the amino-terminal association, which would alter the end-to-end interactions of TM molecules in the thin filament regulatory strand and affect the mechanisms of cardiac muscle contraction. To test this hypothesis, we generated transgenic (TG) mouse lines that express a mutant form of α-TM in which the first 275 residues are from α-TM and the last nine amino acids are from β-TM (α-TM9aaΔβ). Molecular analyses show that endogenous α-TM mRNA and protein are nearly completely replaced with α-TM9aaΔβ. Working heart preparations data show that the rates of contraction and relaxation are reduced in α-TM9aaΔβ hearts. Left ventricular pressure and time to peak pressure are also reduced (−12% and −13%, respectively). ...
Research Interests: Physiology, Chemistry, Calcium, Medicine, Western blotting, and 15 moreTransgenic Mice, Mutation, Mice, Animals, Heart rate, Heart, Calcium Signaling, American, Medical Physiology, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Actin Cytoskeleton, Amino Acids, Protein Conformation, Tropomyosin, and Papillary Muscles
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... more
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 (−d D/d t)] during spontaneous ...
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During spaceflight the normal head-to-foot hydrostatic pressure gradients are eliminated and body fluids shift toward the head, resulting in a diminished fluid volume in the legs and an increased fluid volume in the head, neck, and upper... more
During spaceflight the normal head-to-foot hydrostatic pressure gradients are eliminated and body fluids shift toward the head, resulting in a diminished fluid volume in the legs and an increased fluid volume in the head, neck, and upper extremities. Lymphatic function is important in the maintenance of normal tissue fluid volume, but it is not clear how microgravity influences lymphatic pumping. We performed a detailed evaluation of the influence of simulated microgravity on lymphatic diameter, wall thickness, elastance, tone, and other measures of phasic contractility in isolated lymphatics. Head-down tail suspension (HDT) rats were used to simulate the effects of microgravity. Animals were exposed to HDT for 2 wk, after which data were collected and compared with the control non-HDT group. Lymphatics from four regional lymphatic beds (thoracic duct, cervical, mesenteric, and femoral lymphatics) were isolated, cannulated, and pressurized. Input and output pressures were adjusted t...
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Research Interests: Computer Science, Biomedical Engineering, Medical Image Processing, Medicine, Biomedical Optics, and 15 moreVideo microscopy, Animals, Optical microscopy, Optical physics, High Speed, Optometry and Ophthalmology, Rats, Reproducibility of Results, Biological clocks, Sensitivity and Specificity, Equipment Design, Equipment Failure Analysis, Pulsatile Flow, Lymphatic System, and Signal transduction networks
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Transport of tissue-derived lymphatic fluid and clearance by draining lymph nodes are pivotal for maintenance of fluid homeostasis in the body and for immune-surveillance of the self-and non-self-proteomes. Yet a quantitative analysis of... more
Transport of tissue-derived lymphatic fluid and clearance by draining lymph nodes are pivotal for maintenance of fluid homeostasis in the body and for immune-surveillance of the self-and non-self-proteomes. Yet a quantitative analysis of nodal filtration of the tissue-derived proteome present in lymphatic fluid has not been reported. Here we quantified the efficiency of nodal clearance of the composite proteomic load using label-free and isotope-labeling proteomic analysis of pre-nodal and post-nodal samples collected by direct cannulation. These results were extended by quantitation of the filtration efficiency of fluorophore-labeled proteins, bacteria, and beads infused at physiological flow rates into pre-nodal lymphatic collectors and collected by post-nodal cannulation. We developed a linear model of nodal filtration efficiency dependent on pre-nodal protein concentrations and molecular weight, and uncovered criteria for disposing the proteome incoming from defined anatomical districts under physiological conditions. These findings are pivotal to understanding the maximal antigenic load sustainable by a draining node, and promote understanding of pathogen spreading and nodal filtration of tumor metastasis, potentially helping to improve design of vaccination protocols, immunization strategies and drug delivery. Blood circulating throughout the capillary bed is not in direct contact with the cellular layers of each parenchymal tissue. Thus lipids, proteins, and small molecules need to extravasate in order to provide cellular nutrients and to hydrate tissue cells 1,2. Extravasation is mediated by hydrostatic pressure inside the blood capillaries and by the Starling forces that drive the ultrafiltration process, moving proteins, macromolecules, and associated water into the interstitial space. A fraction of the extravasated fluid will be absorbed back into the capillary bed, but most will remain in the interstitial tissue 1,2. These products of capillary extravasation, combined with secreted products deriving from cellular metabolism and catabolism, make up the interstitial fluid that baths every parenchymal organ 3. Under physiological conditions, in humans, around 8–10 liters of interstitial fluid are formed daily, which need to be returned to the blood circulation to prevent tissue edema 1. However, the vast majority of the interstitial fluid will not directly be reabsorbed into the blood system, but rather will be collected into the lymphatic capillar-ies as lymph, and will pass through one or more of the 600–800 draining lymph nodes disseminated throughout the human body, before circulating into the thoracic duct and then the vena cava 2. There are several possible explanations why interstitial fluid does not drain directly into the general blood circulation but instead is filtered through the lymph nodes. First, lymphatic passage through the nodes ensures that tissue-invading pathogens do not directly enter into the bloodstream but can be captured by dendritic cells and macrophages residing in the lymph node. Second, the collection of products of tissue remodeling, cellular secretion/processing, and extracellular debris by lymphatic fluid ensures that nodal immune cells are constantly exposed to the self-proteome from each parenchymal organ, helping to maintain peripheral tolerance 2,4–7. Third, immune cells patrolling peripheral tissues can use lymph flow as a fast and direct conduit to lymph nodes. Fourth, lymph composition at different times and locations can vary widely in protein concentration, electrolytes composition , pH and cellular composition, as opposed to blood, where these parameters are tightly controlled. Thus, the lymph, as observed in both physiological and pathological conditions, can withstand changes occurring in Published: xx xx xxxx OPEN
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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... more
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 lymphatic's endothelial cell and matrix proteins. Functi...
Research Interests: Gap Junctions, Mass Spectrometry, Aging, Proteomics, Biological Sciences, and 15 moreEndothelial Cells, Mice, Animals, Male, Staphylococcus aureus, Glycosylation, Lymphatics, Rats, Homeostasis, Amino Acid Sequence, Proteome, Extracellular Matrix Proteins, Lymph nodes, Molecular Sequence Data, and Connexins
Preeclampsia (preE) is a hypertensive disorder of pregnancy. Cardiotonic steroids (CTS) are endogenous inhibitors of Na ? /K ? ATPase, and at least one CTS, marinobufagenin (MBG), is elevated in a rat model of preE prior to the... more
Preeclampsia (preE) is a hypertensive disorder of pregnancy. Cardiotonic steroids (CTS) are endogenous inhibitors of Na ? /K ? ATPase, and at least one CTS, marinobufagenin (MBG), is elevated in a rat model of preE prior to the development of the syndrome. MBG and oua-bain impair cytotrophoblast (CTB) cell function, which is critical for placental development. We evaluated the effect of a CTS, cinobufotalin (CINO), on CTB cell function in vitro. CINO at C1 nM inhibited CTB cell proliferation, migration, and invasion (p \ 0.05), but had no effect on cell viability. There was a higher (p \ 0.05) percentage of G0/ G1 phase cells in groups treated with CINO at C1 nM. CINO caused an increase in stress signaling p38 MAPK and a positive annexin-V staining in CTB cells, indicating the activation of apoptotic signaling. However, the CINO-induced apoptotic signaling was prevented by p38 inhibition. These data demonstrate that CINO impairs CTB cell function via cell cycle arrest and apoptotic signaling.
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We describe the development of an optical flow visualization method for resolving the flow velocity vector field in lymphatic vessels in vitro. The aim is to develop an experimental protocol for accurately estimating flow parameters, such... more
We describe the development of an optical flow visualization method for resolving the flow velocity vector field in lymphatic vessels in vitro. The aim is to develop an experimental protocol for accurately estimating flow parameters, such as flow rate and shear stresses, with high spatial and temporal resolution. Previous studies in situ have relied on lymphocytes as tracers, but their low density resulted in a reduced spatial resolution whereas the assumption that the flow was fully developed in order to determine the flow parameters of interest may not be valid, especially in the vicinity of the valves, where the flow is undoubtedly more complex. To overcome these issues, we have applied the time-resolved microparticle image velocimetry (μ-PIV) technique, a well-established method that can provide increased spatial and temporal resolution that this transient flow demands. To that end, we have developed a custom light source, utilizing high-power light-emitting diodes, and associated control and image processing software. This paper reports the performance of the system and the results of a series of preliminary experiments performed on vessels isolated from rat mesenteries, demonstrating, for the first time, the successful application of the μ-PIV technique in these vessels.
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Diabetes in pregnancy is associated with mi-crovascular complications and a higher incidence of preeclampsia. The regulatory signaling pathways involving nitric oxide, cGMP, and cGMP-dependent protein kinase (PKG) have been shown to be... more
Diabetes in pregnancy is associated with mi-crovascular complications and a higher incidence of preeclampsia. The regulatory signaling pathways involving nitric oxide, cGMP, and cGMP-dependent protein kinase (PKG) have been shown to be down-regulated under diabetic conditions and contribute to the pathogenesis of vascular complications in diabetes. The present study was undertaken to investigate how high glucose concentrations regulate PKG expression in cytotrophoblast cells (CTBs). Human CTBs (Sw. 71) were treated with 45, 135, 225, 495, or 945 mg/dL glucose for 48 h. Some cells were pretreated with a p38 inhibitor (10 lM SB203580) or 10 lM rosiglitazone. After treatment, the cell lysates were subjected to measure the expression of protein kinase G1a (PKG1a), protein kinase G1b (PKG1b), soluble guanylate cyclase 1a (sGC1a), and soluble guanylate cyclase 1 b (sGC1b) by Western blot. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test. The expressions of PKG1a, PKG1b, sGC1a, and sGC1b were significantly down-regulated (p \ 0.05) in CTBs treated with [135 mg/ dL glucose compared to basal (45 mg/dL). The hyper-glycemia-induced down-regulation of cGMP and cGMP-dependent PKG were attenuated by the SB203580 or rosiglitazone pretreatment. Exposure of CTBs to excess glucose down-regulates cGMP and cGMP-dependent PKG, contributing to the development of vascular complications in diabetic mothers during pregnancy. The attenuation of hy-perglycemia-induced down-regulation of PKG proteins by SB203580 or rosiglitazone pretreatment further suggests the involvement of stress signaling mechanisms in this process.