INTRODUCTION

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THE MAIN FUNCTION of lymphatic vessels is to promote the central movement of lymph and thereby return it to the systemic circulation and to maintain interstitial homeostasis (1, 10, 25, 27, 29). The factors participating in the governing of lymph flow are still not clearly established. The centripetal transport of lymph depends on the rate of lymph formation, the spontaneous constriction and dilation of lymphatic vessels (1, 2, 3, 21, 22), and the pressure drop between the interstitial space and the central veins (10, 21, 25, 27, 29, 30). In addition, passive forces, such as the skeletal muscle pump (11, 24), respiratory movements (26), and arterial pulsation (32) contribute to the propelling of lymph centrally.

The combined effect of the rate of lymph formation and active spontaneous contraction and relaxation of bovine (18), sheep (9, 17), rat (3, 34), guinea pig (31), and human (26) lymphatic vessels on the transport of lymph has already been characterized. Earlier in vivo studies showed that, during edema, first there is a linear increase in total lymph flow followed by a plateau phase (29, 30), despite a further increase in driving pressure. On the basis of subsequent studies, Guyton et al. (10) suggested that, in edema, the plateau phase in the interstitial pressure-lymph flow relationship is due to the compression of lymphatic vessels by the surrounding tissues and not to changes in lymphatic pumping capacity itself. Other studies, however, suggested that the plateau in lymph flow after an increase in capillary filtration and edema formation may result from the high outflow cannula resistance in the experiments (8). Thus the roles of the lymphatic wall and/or specific experimental circumstances in the development of the plateau phase in lymph flow during edema are still not clarified.

In vivo studies suggest that during high lymph flow conditions, elicited by hemodilution, the contribution of the lymphatic pump to the edema safety factor declines (3, 30). This conclusion was based on the assumption that total lymph flow exceeded lymphatic pumping capacity. Such a dissociation between lymph flow and pumping capacity of lymph vessels has been observed by others as well (13, 17). A decline in pumping activity could also be due to an increase in lymphatic resistance (hindrance); however, this hypothesis has not yet been tested.

The combined effect of changes of intraluminal pressure and flow on lymphatic activity during increased formation of lymph has been examined previously (13, 17). Recent in vitro studies demonstrated that increases in intraluminal pressure (in the absence of intraluminal flow) have a substantive effect on spontaneous diameter oscillations of lymph microvessels (19) and induce a myogenic tone, the magnitude of which is similar to that present in coronary (16) and skeletal muscle (7) venules.

Previous studies showed that increases in perfusate flow (in the presence of constant intraluminal pressure) increase the diameter of arterioles (15) and venules (6, 14, 16) in an endothelium-dependent manner. The sole effect of changes in perfusate flow on the spontaneous vasomotor activity of lymphatics is not known. It is known, however, that the endothelium of lymphatics releases, in response to various agonists, vasoactive substances that are able to modulate the tone of the vessels (19, 21, 22, 32). Endothelium-derived nitric oxide (20, 23) and various prostaglandins (12, 13, 20) were shown to be released in response to naturally occurring vasoactive agents, modulating the frequency and amplitude of spontaneous dilation and contraction of isolated large and small lymph vessels. Thus it is likely that increases in lymph flow are sensed by the lymphatic endothelium, eliciting changes in the release of endothelial factors and affecting, in turn, the vasomotion of microlymphatics.

The present studies were conducted to test the hypothesis that, in the presence of constant intraluminal (and transmural) pressure, the vasomotor activity of small lymphatic vessels will change in response to increases in intraluminal flow and that the mechanism responsible for this change is intrinsic to the lymphatic wall.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Eleven-week-old male Wistar rats weighing ~360 g were anesthetized with pentobarbital sodium (50 mg/kg ip). The iliac lymph nodes and their afferent lymph vessels were exposed by an incision of the abdomen, excised, and placed on a petri dish containing cold (4°C) Krebs solution (pH 7.4). The Krebs solution contained (in mM) 120.0 NaCl, 5.9 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 5.5 glucose, and 25.0 NaHCO₃. With the use of microsurgical instruments and an operating microscope, lymph microvessels (n = 55; ~140-160 µm active maximum diameter, ~3 mm in length) were isolated and transferred to a vessel chamber (10 ml) containing Krebs solution and two glass micropipettes. The proximal (inflow) and distal (outflow) micropipettes were connected with Tygon tubing to a pressure transducer (Statham P23DC, Gould, MA) and a 50-ml syringe. A physiological salt solution (PSS), bubbled with a gas mixture of 10% O₂, 5% CO₂, and 85% N₂ (pH 7.4), was superfused over the vessel. The total volume of the chamber and superfusion circuit was 80 ml, and the rate of flow of the superfusion solution was 20 ml/min. After the lymph microvessels were mounted on the primary pipette and secured with sutures, perfusion pressure was raised to 4 cmH₂O to flush out and clear the vessels. Then the distal end of the vessels was mounted to the outflow micropipette.

As described previously, perfusing arterioles (15), venules (7), and lymphatics (19) with air results in morphological change, functional impairment, or loss of the endothelial cell layer. The lymphatic endothelium was removed carefully by infusing 0.1 ml of air into the lumen of the lymph microvessels within a period of 2 min. They were then flushed with 0.2 ml of PSS solution. After cannulation of a lymph microvessel, the vessel chamber was transferred to the stage of an intravital microscope (Olympus BH-2). The vessel was then warmed slowly to 37°C and allowed to equilibrate for 60 min at a perfusion pressure of 6 cmH₂O. The changes in minimum diameter (D_min, µm), maximum diameter (D_max, µm), and oscillation frequency (FRE, oscillations/min) of lymph microvessels in response to flow or agonists were measured with an image-shearing monitor (Instrumentation for Physiology and Medicine, IPM model 908), calibrated with a stage micrometer, and recorded on a chart recorder (Grass model 7WC8G). To ensure that rhythmic changes in diameter were followed accurately, the experiments were also recorded on videotape (Sony, SL-HF 1000, SuperBeta) for later replay.

After the equilibration period, the vasoactive function of lymphatic endothelium and smooth muscle was assessed by changes in the diameter of the lymph microvessels with or without endothelium (19, 20) in response to test doses of acetylcholine (10⁶ M) and sodium nitroprusside (10⁷ M). Then the vessel was washed for 20 min with Krebs solution until the amplitude and the frequency of vasomotion returned to control levels. Intraluminal flow was established by increases of the pressure gradient across the vessel. To initiate flow, pressure differences (P_diff) were increased from 0 to 6 cmH₂O, corresponding to a perfusate flow of 0 to 3.5 µl/min. The inflow syringe was elevated in steps of 0.5 cmH₂O, while the outflow syringe was moved in an opposite direction in steps of 0.5 cmH₂O, providing increments of 1 cmH₂O P_diff. Because oscillation in the diameter of lymph microvessels may cause small changes in the volume of the vessels, we employed 50-ml syringes as reservoirs to minimize the changes in pressure. The mean intraluminal pressure was kept constant throughout the experiment (6 cmH₂O). Each level of flow was maintained for 5-10 min to allow the vessels to exhibit stable and spontaneous diameter oscillations. Two groups of vessels were studied, one with and another without the endothelium. Effects of indomethacin (Indo; an inhibitor of cyclooxygenase; 10⁵ M), N-nitro-L-arginine (L-NNA; 10⁵ M), or 10⁶ M SQ-29,548, a selective antagonist of PGH₂-thromboxane A₂ (PGH₂/TxA₂) receptors, on flow-induced responses of lymphatics were examined. At the end of each experiment, the PSS was changed to a Ca²⁺-free PSS that also contained EDTA (1.0 mM). Lymph microvessels were incubated with Ca²⁺-free PSS for 20 min, then the pressure steps were repeated and the passive diameters (PD) were measured at corresponding flow values. All salts and drugs were obtained from Sigma and J. T. Baker and were prepared in PSS on the day of the experiment.

The contractile activity of lymph microvessels was characterized by changes in D_max and D_min (µm) and oscillation frequency (oscillations/min). Data are presented as means ±SE. Vessels that did not exhibit vasomotion were not included in the study. Differences were considered significant at P < 0.05 and were determined by analysis of variance, followed by a multiple comparison test (Duncan's post hoc test) and unpaired Student's t-test, as appropriate.

	RESULTS

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Effect of increases in intraluminal perfusion on lymphatic vasomotion. Figure 1 depicts an actual recording of changes in diameter oscillation of an isolated lymph microvessel with intact endothelium in the presence of 0 and 4 cmH₂O pressure gradient (P_diff) corresponding to 0 and 2.7 µl/min perfusate flow. At 0 µl/min perfusate flow, the D_max of the lymph microvessel was 157 µm and D_min was 52 µm, providing a close to 100-µm amplitude in spontaneous change in diameter. In response to an increase in perfusate flow, D_max, D_min, and the amplitude of vasomotion decreased.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Original record of spontaneous vasomotion of isolated lymphatics in response to perfusion flow elicited by an increase in pressure gradient from 0 cmH2O (A) to 4 cmH2O (B), respectively.

Data from these experiments are summarized in Fig. 2. At 0 cmH₂O pressure difference, the D_max and D_min of lymphatic vessels were 157.5 ± 5.4 and 91.9 ± 5.3 µm, respectively. Figure 2A shows that in the presence of endothelium, increases in perfusion pressure gradient from 0 to 4 cmH₂O elicited a significant reduction in D_max and D_min (to 90.9 ± 5.4 and 66.3 ± 3.6 µm, respectively), hence a consequent reduction in the amplitude of diameter oscillation reaching a minimum at 4 cmH₂O. On the other hand, increases in perfusate flow caused a significant increase in the oscillation frequency of lymph microvessels from 18.0 ± 0.7 to 23.4 ± 1.1 oscillations/min (Fig. 2B). The passive diameters of lymph microvessels obtained in Ca²⁺-free solution (216.0 ± 7.1 µm) did not change in response to increases in perfusate flow (Fig. 2A).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effect of increases in perfusion (pressure gradient) on passive diameter (), maximum diameter (Dmax; ), and minimum diameter (Dmin; ) of lymphatics (A) and on frequency of lymphatic vasomotion (B). * Significant difference from pressure gradient of 0 cmH2O (P < 0.05, n = 20).

Removal of endothelium (Fig. 3) abolished the reduction in D_max (from 65.7 ± 4.9 to 0.4 ± 4.6 µm; Fig. 3A) and D_min (from 23.9 ± 3.6 to 4.9 ± 5.1 µm; Fig. 3B) and also the increase in the frequency of vasomotion (from 5.5 ± 0.8 to 1.9 ± 0.9 min¹; Fig. 3C) in response to increases in pressure gradient (above data at P_diff 4 cmH₂O). It is of note that at higher pressure differences the reduction of D_max and D_min and the increases in the frequency of vasomotion were tempered in the presence of endothelium.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effects of removal of endothelium on perfusion (pressure gradient)-induced changes in Dmax (A), Dmin (B), and frequency (FRE) (C) of lymphatics. * Significant difference between endothelium-intact (, n = 17) and endothelium-denuded (, n = 8) lymphatics (P < 0.05). , Change.

Endothelial factors mediating perfusion-induced lymphatic responses. In Fig. 4 changes in diameter of lymph microvessels in the absence and presence of the nitric oxide synthase inhibitor L-NNA are summarized. The data show that the presence of L-NNA in the bath solution did not affect significantly the changes in vasomotion of lymph microvessels in response to elevation of the perfusion pressure gradient, yet it elicited a significant reduction in the perfusion-induced increase in the frequency of vasomotion.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effects of N-nitro-L-arginine (, n = 11) on perfusion (pressure gradient)-induced changes in Dmax (A), Dmin (B), and FRE (C) of lymphatics. * Significant difference from control (, P < 0.05, n = 17).

In Fig. 5, original records depict the changes in vasomotion of a lymphatic vessel at 0 and 4 cmH₂O P_diff, corresponding to 0 and 2.9 µl/min perfusion flow, in control conditions and during inhibition of prostaglandin synthesis with indomethacin or inhibition of PGH₂/TxA₂ receptors by SQ-29,548. Unlike in Fig. 1, in the presence of Indo or SQ-29,564 in the bath solution, the decrease in D_max and D_min and the increase in frequency in response to increases in pressure gradient were abolished. Summary data of these experiments, depicted in Fig. 6, show that the presence of indomethacin or SQ-29,564 inhibited the decrease in D_max (Fig. 6A) and D_min (Fig. 6B) and the increase in frequency of vasomotion (Fig. 6C) in response to increases in perfusate flow.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Original record of effect of indomethacin (A) and SQ-29,548 (B) perfusion (pressure gradient)-induced responses of isolated lymphatics.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effects of Indo (, n = 8) and SQ-29,548 (, n = 8) on perfusion (pressure gradient)-induced changes in Dmax (A), Dmin (B), and FRE (C) of lymphatics. * Significant difference from control (; P < 0.05, n = 17).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The new findings of the present study are that in the presence of constant intraluminal pressure, increases in intraluminal flow reduce D_max, D_min, and the amplitude and increase the frequency of spontaneous diameter oscillations of isolated lymph vessels of rats. The changes in lymphatic vasomotion to increased perfusion are mediated by the endothelium-derived constrictor factors PGH₂ / TxA₂.

Modulation of spontaneous changes in lymphatic diameter by increased perfusion. In addition to passive forces and spontaneous contraction and relaxation of smooth muscle of lymphatic vessels, the flow of lymph is generated primarily by the pressure drop between the interstitial space and central veins (1, 2, 10, 26, 27, 29). Previous in vivo studies investigated the effect of changes in input and/or output pressure on lymphatic flow. In these conditions, however, not only lymph flow but also intraluminal pressure changed, which may have affected substantively the spontaneous vasomotion of lymphatics (2, 3, 9, 13, 17-19) and, consequently, the results of these studies. In our previous study, we found that increases in intraluminal pressure (in the absence of perfusate flow) activate a myogenic response, which reduces the passive distension of the lymphatic wall, thereby limiting the increase in diameter. Thus, to a degree, the pressure-sensitive myogenic mechanism is likely to contribute to the regulation of lymph flow.

Previous studies revealed that increases in intraluminal flow affect the diameter of arterioles (15) and venules (5, 14, 16), eliciting dilation in most instances. In larger vessels, an increase in vascular tone in response to flow was also observed (4). Thus, in theory, the tone of lymph vessels can also be affected by forces related to changes in intraluminal flow. The structure and function of lymphatic vessels are clearly different from those of blood vessels. Lymphatics demonstrate a spontaneous oscillation in diameter, whereas the diameter of blood vessels appears to be more stable. The effect of several-fold increases in lymph flow, elicited by various experimental interventions, on the vasomotion of lymphatics has been studied in vivo but only in conditions when both intraluminal pressure and flow changed simultaneously (2, 3, 10, 13, 27, 29). Also, it is quite difficult to distinguish intrinsic from extrinsic factors that may influence lymph flow, especially in edematous conditions when tissue pressure also changes (8, 9, 29). Thus, in the present study, we aimed to investigate and characterize solely, the effect of an increase in intraluminal flow on the vasomotion of lymphatics while the intraluminal pressure was held constant. The in vitro system used by our study provided the conditions in which the response of lymphatics to an elevation of intraluminal perfusion could be studied without the confounding effects of intraluminal and/or interstitial pressure and metabolic factors originating from parenchymal tissue.

We found that in the presence of constant intraluminal pressure, increases in perfusate flow elicited significant decreases in D_max, D_min, and the amplitude of vasomotion of lymphatics. It is important to note that the passive diameter of lymphatics obtained in the absence of extracellular Ca²⁺ did not change in response to increases in flow, a further indication that the changes in diameter were active responses and not due to changes in perfusion pressure or other factors.

Previous in vivo studies showed a significant increase in lymph flow elicited by edema; however, these studies also revealed that at higher flow rates pumping activity declines (2, 3, 10, 13, 29). Subsequent studies revealed segmental differences in vasomotion of lymphatic vessels in response to increases in lymph flow (2). It was found that the smaller lymphatic vessels increase their end-diastolic and end-systolic diameter (corresponding to D_max and D_min) in response to hemodilution-induced increases in lymph flow. In contrast, the end-diastolic and end-systolic diameters of larger lymphatic vessels did not increase, even at higher flow rates (2), which, together with the reduction in frequency, limited substantially the calculated ejection fraction and stroke volume of the lymphatics (2). The present in vitro findings show that if the distending effect of increases in intraluminal pressure is absent, then the diameter of lymphatics decreases in response to increases in perfusion. Interestingly, higher perfusion rates have smaller effects on D_max and D_min and frequency of oscillation, a phenomenon for which, as yet, we have no ready explanation. Nevertheless, in the present experiments, intraluminal flow was established by increasing the inflow and decreasing the outflow pressures; therefore, outflow resistance did not limit the increase in perfusion, yet the activity of lymphatics declined. Thus the plateau phase in lymph flow observed previously during edema (1, 9, 10) could be due to an active response of the lymphatic wall, a response that may contribute to the regulation of lymph circulation, especially during high lymph production. It seems, therefore, that lymphatic vessels possess two intrinsic vasomotor mechanisms, one sensitive to pressure (19) and another sensitive to flow. In in vivo conditions (1, 2, 10, 29, 30), the presence of other mechanisms may well mask these functions. The present findings show that the flow (perfusion)-dependent behavior of lymph microvessels is clearly different from that of arterioles and venules and suggest that the underlying mechanisms evoking this response are also different.

Role of endothelium in mediation of perfusion-induced response of lymph microvessels. Earlier findings demonstrated that arachidonate metabolites affect lymphatic contractility (12). Studies in the canine thoracic duct (23) and bovine (33) and guinea pig (31) lymph vessels showed that in response to various stimuli, the endothelium of lymphatics also produces nonprostanoid factor(s) that affect the contractile state of smooth muscle. We have also found significant increases in the diameter of lymphatics in response to acetylcholine, owing to the release of nitric oxide, and significant decreases in their diameter in response to exogenous arachidonic acid, which is due to the release of PGH₂/TxA₂ from the endothelium (19, 20).

In the present study, after removal of the endothelium, increases in perfusion did not elicit significant changes in the vasomotion of isolated lymph microvessels, indicating that endothelial factors mediate the perfusion-induced constrictor response. Our data also suggest that increases in perfusion did not elicit the release of nitric oxide, because L-NNA did not affect the response; instead, increases in perfusion stimulated prostanoid synthesis. It remains an intriguing question why constrictor prostanoids are produced in lymphatic vessels and what stimuli and signal transduction mechanisms are responsible for such an effect. It is possible that PGH₂/TxA₂ released to changes in flow may stimulate the pacemaker activity of lymphatic smooth muscle, affecting the frequency and consequently the amplitude of vasomotion (20, 31). On the other hand, the constrictor prostaglandins derived from the endothelium of lymphatics may also serve to counterbalance the effect of dilator factors, which are known to be released in inflammatory conditions (1, 12). Interestingly, it has been observed previously that vessels from low-pressure segments of the circulatory system, such as pulmonary and venular circulations, tend to produce more constrictor factors (such as PGH₂ and endothelin) than arterioles and that these factors can contribute significantly to the tone of these vessels (6, 7, 28).

In summary, our findings indicate that increases in perfusate flow significantly reduce the spontaneous vasomotor activity of microlymphatics by eliciting release of PGH₂/TxA₂ from the endothelium, a mechanism that in vivo could control their pumping capacity. Thus the intrinsic mechanism in the lymphatic endothelium that is sensitive to changes in intraluminal flow may contribute importantly to the local control of lymphatic resistance.