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Biomedical subjects

David C Zawieja

Publications and source records attributed to David C Zawieja.

At least 19 recordsLinked to original sources

Length-tension relationships of small arteries, veins, and lymphatics from the rat mesenteric microcirculation.

The passive and active length-tension relationships of isolated rat mesenteric lymphatics ( approximately 150 microm ID), and adjacent small arteries ( approximately 240 microm) and veins ( approximately 275 microm) were compared under isometric conditions using a wire myograph. About 60% of the lymphatic vessels developed spontaneous contractions in physiological saline solution at nominal preload. To maximally activate smooth muscle, 145 mM K(+) + 5 x 10(-5) M norepinephrine was used for arteries, and 145 mM K(+) + 1 x 10(-6) M substance P was used for lymphatics and veins. In response, arteries exhibited monotonic force development to a plateau level, whereas lymphatics and veins showed biphasic force development, consisting of a transient force peak followed by partial relaxation to a plateau over approximately 5 min. The passive and the active length-tension curves were similar in shape among all three vessels. However, the maximal active tension of arteries (3.4 +/- 0.42 mN/mm) was significantly greater than peak active tension (0.59 +/- 0.04 mN/mm) or plateau tension (0.20 +/- 0.04 mN/mm) in small veins and greater than peak active tension (0.34 +/- 0.02 mN/mm) or plateau tension (0.21 +/- 0.02 mN/mm) in lymphatics. Maximal active medial wall stress was similar between lymphatics and veins but was approximately fivefold higher in small arteries. For lymphatics, the pressure calculated from the optimal preload was significantly higher than that found previously in isobaric studies of isolated lymphatics, suggesting the capacity to operate at higher than normal pressures for increased responsiveness. Our results represent the first mechanical comparisons of arterial, venous, and lymphatic vessels in the same vasculature.

Animals↗

Image correlation algorithm for measuring lymphocyte velocity and diameter changes in contracting microlymphatics.

Efforts have recently been made to estimate wall shear stress throughout the contractile cycle of mesenteric rat lymphatics with a high speed video microscopy system. This was prompted by reports in the literature that lymphatic pumping is related to wall shear stress. While one can estimate wall shear stress by tracking lymphocyte velocity, it is prohibitively tedious to manually track particles over a reasonable time frame for a good number of experiments. To overcome this, an image correlation method similar to digital particle imaging velocimetry was developed and tested on contracting lymphatics to measure both vessel diameter and fluid velocity. The program tracked temporal fluctuations in spatially averaged velocity with a standard error of prediction of 0.4 mm/s. From these studies we have measured velocities ranging from -2 to 4 mm/s. Diameter changes were also measured with a standard error of 7 microm. These algorithms and techniques could be beneficial for investigating various changes in contractile behavior as a function of changes in velocity and wall shear stress.

Algorithms↗

Intrinsic pump-conduit behavior of lymphangions.

Lymphangions, segments of lymphatic vessels bounded by valves, have characteristics of both ventricles and arteries. They can act primarily like pumps when actively transporting lymph against a pressure gradient. They also can act as conduit vessels when passively transporting lymph down a pressure gradient. This duality has implications for clinical treatment of several types of edema, since the strategy to optimize lymph flow may depend on whether it is most beneficial for lymphangions to act as pumps or conduits. To address this duality, we employed a simple computational model of a contracting lymphangion, predicted the flows at both positive and negative axial pressure gradients, and validated the results with in vitro experiments on bovine mesenteric vessels. This model illustrates that contraction increases flow for normal axial pressure gradients. With edema, limb elevation, or external compression, however, the pressure gradient might reverse, and lymph may flow passively down a pressure gradient. In such cases, the valves may be forced open during the entire contraction cycle. The vessel thus acts as a conduit, and contraction has the effect of increasing resistance to passive flow, thus inhibiting flow rather than promoting it. This analysis may explain a possible physiological benefit of the observed flow-mediated inhibition of the lymphatic pump at high flow rates.

Animals↗

Contraction-initiated NO-dependent lymphatic relaxation: a self-regulatory mechanism in rat thoracic duct.

The objectives of this study were to evaluate the physiological importance of the flow and shear generated by phasic contractions of lymphatic vessels and the mechanisms responsible for the influences of such shear on lymphatic pumping. Lymphatic segments of the rat thoracic duct were isolated, cannulated and pressurized. The diastolic diameters were measured in phasically non-active segments. The diastolic and systolic diameters, half-relaxation time (HRT), contraction frequency, ejection fraction and fractional pump flow were determined in phasically active segments. Since imposed flow was excluded, flow and shear occurred only as a result of the intrinsic contractions in phasically active segments whereas in phasically non-active segments contraction-generated flow and shear were absent. The influences of incrementally increased transmural pressure (from 1 to 5 cmH(2)O) were examined in control conditions and after NO synthase blockade (l-NAME 10(-4) m) or cyclooxygenase blockade (indomethacin 10(-5) m). The spontaneous phasic contractions produced a flow-dependent diastolic relaxation. This reduction of the lymphatic tone is a regulatory mechanism that maintains pumping in thoracic duct in an energy-saving/efficient mode: it improves diastolic filling (enhanced lusitropy - lowering HRT), makes lymphatic contractions stronger (enhanced inotropy - higher contraction amplitude) and propels more fluid forward during each contraction (elevated ejection fraction) while decreasing contraction frequency (reduced chronotropy). The findings also demonstrated that the NO pathway, not the cyclooxygenase pathway is responsible for this reduction of lymphatic tone and is the prevailing pathway responsible for the self-regulatory adjustment of thoracic duct pumping to changes in lymph flow pattern.

Animals↗

Signaling pathways mediating VEGF165-induced calcium transients and membrane depolarization in human endothelial cells.

Cytosolic calcium and membrane potential were monitored simultaneously in quiescent human umbilical vein endothelial cells (HUVEC) exposed to vascular endothelial growth factor (VEGF)165 using the fluorescent indicators indo-1 AM and DiSBAC2(3), respectively. Application of VEGF165 to cells elicits a rapid rise in cytosolic calcium followed by a slower decline toward control values. Peak calcium is associated with a slight membrane hyperpolarization; however, as calcium falls toward control, a strong depolarization develops and is sustained throughout a 10-min period of VEGF165 stimulation. Both the VEGF165-mediated rise in cytosolic calcium and membrane depolarization are eliminated by inhibitors of VEGFR-2, tyrosine kinase, src kinase and inositol-1,4,5 triphosphate-operated calcium channels. Calcium entry, which is initially facilitated by transient hyperpolarization, is restricted by a substantial, sustained depolarization that developed during the downstroke of the calcium spike. Inhibition of plasmalemmal calcium channels diminished the magnitude and duration of the calcium spike, suggesting that extracellular calcium influx, secondary to stores release, is a significant component of the calcium transient. Inhibition of chloride channels substantially reduced membrane depolarization. In addition, the depolarization is modulated by PI3 kinase in a ras-independent manner. In summary, intracellular calcium and membrane potential are influenced by several key signaling cascades of VEGFR-2 activation in HUVEC.

Calcium Signaling↗

Changes in end-to-end interactions of tropomyosin affect mouse cardiac muscle dynamics.

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 alpha-TM carboxy terminal overlap end to the beta-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 alpha-TM in which the first 275 residues are from alpha-TM and the last nine amino acids are from beta-TM (alpha-TM9aaDeltabeta). Molecular analyses show that endogenous alpha-TM mRNA and protein are nearly completely replaced with alpha-TM9aaDeltabeta. Working heart preparations data show that the rates of contraction and relaxation are reduced in alpha-TM9aaDeltabeta hearts. Left ventricular pressure and time to peak pressure are also reduced (-12% and -13%, respectively). The ratio of maximum to minimum first derivatives of change in left ventricular systolic pressure with respect to time (ratio of +dP/dt to -dP/dt, respectively) is increased, but tau is not changed significantly. Force-intracellular calcium concentration ([Ca2+]i) measurements from intact papillary fibers demonstrate that alpha-TM9aaDeltabeta TG fibers produce less force per given [Ca2+]i compared with nontransgenic fibers. Taken together, the data demonstrate that the rate of contraction is primarily affected in TM TG hearts. Protein docking studies show that in the mutant molecule, the overall carbon backbone is perturbed about 1.5 A, indicating that end-to-end interactions are altered. These results demonstrate that the localized flexibility present in the coiled-coil structures of TM isoforms is different, and that plays an important role in interacting with neighboring thin filament regulatory proteins and with differentially modulating the myofilament activation processes.

Actin Cytoskeleton↗

Inhibition of active lymph pump by simulated microgravity in rats.

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 to apply a range of transmural pressures and flows to the lymphatics. Simulated microgravity caused a potent inhibition of pressure/stretch-stimulated pumping in all four groups of lymphatics. The greatest inhibition was found in cervical lymphatics. These findings presumably are correlated to the cephalic fluid shifts that occur in HDT rats as well as those observed during spaceflight. Flow-dependent pump inhibition was increased after HDT, especially in the thoracic duct. Mesenteric lymphatics were less strongly influenced by HDT, which may support the idea that lymph hydrodynamic conditions in the mesenteric lymphatic during HDT are not dramatically altered.

Animals↗

Molecular profile and proliferative responses of rat lymphatic endothelial cells in culture.

BACKGROUND: Lymphangiogenesis plays an important role in metastasis of many solid tumors. To study lymphangiogenesis under controlled conditions, an in vitro model is needed. The goal of this work was to establish such an in vitro model by determining a molecular profile of rat mesenteric lymphatic endothelial cells (RMLEC) and characterizing their proliferative responses to angiogenic and lymphangiogenic factors, such as vascular endothelial growth factor A and C (VEGF-A and VEGF-C). METHODS AND RESULTS: RMLEC strongly expressed most lymphatic-specific markers, including Prox-1, LYVE-1, and VEGFR-3. Proliferation of RMLEC was serum and heparin dependent. In the presence of low (2%) serum concentration, exogenously added VEGF-A and VEGFC stimulated RMLEC in a linear and dose-dependent manner. This effect was abrogated by anti-VEGF-A and VEGF-C antibodies, as well as by soluble Tie-2 and Flt-4 fusion proteins. Abrogation was reversed by VEGF-A, suggesting that this factor as an important regulator of lymphangiogenesis. CONCLUSIONS: Cultured RMLEC preserved a molecular profile consistent with the phenotype of lymphatic endothelium in vivo and respond to either VEGF-A or VEGF-C factors. VEGFA was able to rescue RMLEC proliferation inhibited by a neutralizing VEGF-C antibody or soluble Tie-2 fusion protein. These results support the existence of cross-talk among angiogenic and lymphangiogenic factors. This work established experimental conditions that allow in vitro modeling of lymphatic endothelial responses to lymphangiogenic regulators. Preliminary results using this model suggest that VEGF-A, VEGF-C, and angiopoietins work in concert to promote lymphangiogenesis in vivo.

Angiotensins↗

Pulmonary air embolization inhibits lung lymph flow by increasing lymphatic outflow pressure.

BACKGROUND: Air embolization of the pulmonary vascular tree increases pulmonary microvascular filtration and induces pulmonary edema formation. Flow from cannulated pulmonary lymphatic vessels increases significantly following air embolization. However, in the intact animal, lymph flows into the venous system and the magnitude of lymph flow is directly affected by systemic venous pressure. We hypothesized that pulmonary air embolization would lead to systemic venous hypertension and that this increase in lymphatic outflow pressure would prevent an increase in pulmonary lymph flow. METHODS AND RESULTS: Pulmonary air embolization was induced in dogs under general anesthesia. Flow from cannulated pulmonary lymphatic vessels was recorded for lymphatic outflow pressure set equal to atmospheric pressure (Q(LA)) and for outflow pressure set equal to systemic venous pressure (Q(LV)) both before and after embolization. Air embolization resulted in significant increases in systemic venous pressure from 6.4 +/- 0.3 to 12.4 +/- 1.2 mm Hg and in QLA from 48 +/- 9 to 175 +/- 29 microL . min(1). However, embolization did not increase Q(LV) (10 +/- 2 vs. 3 +/- 3 microl . min(1)). CONCLUSIONS: Pulmonary air embolization impedes pulmonary lymph flow by increasing systemic venous pressure and, thereby, contributes to pulmonary edema formation.

Animals↗

Automated measurement of diameter and contraction waves of cannulated lymphatic microvessels.

BACKGROUND: Studies of lymphatic function often employ collecting lymphatic vessels that exhibit large-amplitude, spontaneous contractions. Data from such preparations have been analyzed using cardiac pump analogies that require accurate determination of vascular dimensions, including external (OD) and internal (ID) diameters. These measurements would be facilitated by an accurate automated measurement system. METHODS AND RESULTS: A computer-based diameter tracking system was developed specifically for lymphatic vessels, with advantages over previous automated systems. The system also permits continuous diameter tracking at two axial locations, enabling the measurement and analysis of contraction wave conduction. The method was validated using spontaneously contracting segments of rat thoracic duct which sometimes exhibited conducted contraction waves. In such preparations, conduction wave velocity was modulated by the axial flow rate and could be easily measured by the tracking system. CONCLUSIONS: The method offers improvement and increased convenience over manual diameter measurements in lymphatic vessels, with little or no sacrifice in accuracy. It should be a useful tool for general studies of collecting lymphatic function as well as for the analysis of contraction wave conduction and coordination.

Algorithms↗

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.

Cells, Cultured↗

Charged residue alterations in the inner-core domain and carboxy-terminus of alpha-tropomyosin differentially affect mouse cardiac muscle contractility.

Two important charge differences between the alpha- and beta-tropomyosin (TM) isoforms are the exchange of a serine residue in the inner-core region at position 229, and a histidine residue at the carboxy-terminal end at position 276, with glutamic acid and asparagine, respectively. We have recently shown that altering these two residues in alpha-TM to their beta-TM counterparts in transgenic (TG) mouse hearts causes a depression in both +dP/dt and -dP/dt and a decrease in calcium sensitivity. In this study, we address whether independent charge changes at these two residues in alpha-TM modulate cardiac function differentially. To test this hypothesis we generated two TG lines: alpha-TMSer229Glu and alpha-TMHis276Asn. Molecular analyses show that 98% of native alpha-TM is replaced by mutated protein in alpha-TM229 hearts whereas alpha-TM276 hearts show 82% replacement with the mutated protein. Isolated working heart data show that alpha-TM229 TG hearts exhibit a significant decrease in both +dP/dt (7%) and -dP/dt (8%) compared with nontransgenics (NTGs) and time to peak pressure (TPP) is also reduced in alpha-TM229 hearts. alpha-TM276 hearts show a decrease only in -dP/dt (14%) and TPP is increased. pCa(2+)-tension relationships in skinned fibre preparations indicate decreased calcium sensitivity in alpha-TM229 but no change in alpha-TM276 preparations. Force-[Ca(2+)](IC) measurements from intact papillary fibres indicate that alpha-TM276 fibres produce more force per given [Ca(2+)](IC) when compared to NTG fibres, while alpha-TM229 fibres produce less force per given [Ca(2+)](IC). These data demonstrate that changing charged residues at either the inner-core domain or the carboxyl end of TM alters sarcomeric performance differently, suggesting that the function of TM is compartmentalized along its length.

Animals↗

Roles of phosphorylation of myosin binding protein-C and troponin I in mouse cardiac muscle twitch dynamics.

A normal heart increases its contractile force with increasing heart rate. Although calcium handling and myofibrillar proteins have been implicated in maintaining this positive force-frequency relationship (FFR), the exact mechanisms by which it occurs have not been addressed. In this study, we have developed an analytical method to define the calcium-force loop data, which characterizes the function of the contractile proteins in response to calcium that is independent of the calcium handling proteins. Results demonstrate that increasing the stimulation frequency causes increased force production per unit calcium concentration and decreased frequency-dependent calcium sensitivity during the relaxation phase. We hypothesize that phosphorylation of myosin binding protein-C (MyBP-C) and troponin I (TnI) acts coordinately to change the rates of force generation and relaxation, respectively. To test this hypothesis, we performed simultaneous calcium and force measurements on stimulated intact mouse papillary bundles before and after inhibition of MyBP-C and TnI phosphorylation using the calcium/calmodulin kinase II (CaMK2) inhibitor autocamtide-2 related inhibitory peptide, or the protein kinase A (PKA) inhibitor 14-22 amide. CaMK2 inhibition reduced both MyBP-C and TnI phosphorylation and decreased active force without changing the magnitude of the [Ca(2+)](i) transient. This reduced the normalized change in force per change in calcium by 19-39%. Data analyses demonstrated that CaMK2 inhibition changed the myofilament characteristics via a crossbridge feedback mechanism. These results strongly suggest that the phosphorylation of MyBP-C and TnI contributes significantly to the rates of force development and relaxation.

Animals↗

Lymphatic smooth muscle: the motor unit of lymph drainage.

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.

Humans↗

Reduced mitochondrial buffering of voltage-gated calcium influx in aged rat basal forebrain neurons.

Alterations of neuronal Ca(2+) homeostatic mechanisms could be responsible for many of the cognitive deficits associated with aging in mammals. Mitochondrial participation in Ca(2+) signaling is now recognized as a prominent feature in neuronal physiology. We combined voltage-clamp electrophysiology with Ca(2+)-sensitive ratiometric microfluorimetry and laser scanning confocal microscopy to investigate the participation in Ca(2+) buffering of in situ mitochondria in acutely dissociated basal forebrain neurons from young and aged F344 rats. By pharmacologically blocking mitochondrial Ca(2+) uptake, we determined that mitochondria were not involved in rapid buffering of small Ca(2+) influx through voltage-gated Ca(2+) channels (VGCCs) in the somatic compartment. For larger Ca(2+) influx, aged mitochondria showed a significant buffering deficit. Evidence obtained with the potentiometric indicator, JC-1, suggests a significantly reduced mitochondrial membrane potential in aged neurons. These results support the interpretation that there is a fundamental difference in the way young and aged neurons buffer Ca(2+), and a corresponding difference in the quality of the Ca(2+) signal experienced by young and aged neurons for different intensities of cytoplasmic Ca(2+) influx.

Aging↗

Regional variations of contractile activity in isolated rat lymphatics.

OBJECTIVE: To evaluate lymphatic contractile activity in different regions of the lymphatic system in a single animal model (the rat thoracic duct, mesenteric, cervical, and femoral lymphatics) in response to changes in lymph pressure and flow. METHODS: The systolic and diastolic diameters of isolated, cannulated, and pressurized lymphatic vessels were measured. Contraction frequency, ejection fraction, and fractional pump flow were determined. The influences of incrementally increased transmural pressure (from 1 to 9 cm H2O) and imposed flow (from 1 to 5 cm H2O transaxial pressure gradient) were investigated. RESULTS: The authors determined regional differences in lymphatic contractility in response to pressure and imposed flow. They found the highest pumping (at the optimal pressure levels) in mesenteric lymphatics and lowest pumping in thoracic duct. All lymphatics had their optimal pumping conditions at low levels of transmural pressure. Different degrees of the flow-induced inhibition of the pump were observed in the different types of lymphatics. During high flow, the active lymph pumps in thoracic duct and cervical lymphatics were almost completely abolished, whereas mesenteric and femoral lymphatics still exhibited significant active pumping. CONCLUSIONS: The active lymph pumps in different regions of the rat body express variable relative strengths and sensitivities that are predetermined by different hydrodynamic factors and regional outflow resistances in their respective locations.

Animals↗

Effects of substance P on mesenteric lymphatic contractility in the rat.

BACKGROUND: Substance P (SP) is associated with lymphatic tissue and is a putative mediator of inflammation. The lymph pump is one of the major "safety factors" preventing edema and its activity is altered by inflammatory mediators. The impact of SP on lymphatics was studied in the rat mesentery. METHODS AND RESULTS: Rats were prepared for in situ lymphatic observation using intravital videomicroscopic techniques. Sections of the small intestine and mesentery were exteriorized and superfused. Lymphatic diameters were measured and pumping activity was determined from the lymphatic diameter tracing. Lymph pump parameters evaluated included diastolic diameter, systolic diameter, contraction frequency, stroke volume, and lymph pump flow. After a control period, the tissues were exposed to SP (10(-9), 10(-8), 10(-7), 10(-6) M). SP reduced both diameters and increased contraction frequency in a concentration-dependent manner. SP (1.0 microM) produced the following changes (% of control): reductions in diastolic diameter (50%), systolic diameter (55%), and stroke volume (63%); a large increase in contraction frequency (661%); and a modest increase in lymph pump flow (44%). SP also stimulated vessels that were not contracting phasically to develop typical contraction patterns. CONCLUSIONS: Although SP produced a tonic constriction of the lymphatics, lymph pump flow was maintained or slightly elevated via an increase in contraction frequency. SP also induced pumping activity in lymphatics that were previously quiescent, thereby further stimulating flow. These effects may serve to minimize the formation of edema in face of the inflammatory edemagenic conditions produced by SP.

Animals↗

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.

Animals↗