A High Salt Diet Alters Pressure-Induced Mechanical Activity of the Rat Lymphatics with Enhancement of Myogenic Characteristics

Abstract

Background:

The lymphatic system has become a new player for pathogenesis in salt-sensitive hypertension animals. A high salt diet (HSD) evokes accumulation of Na⁺ in the skin of rodents. In response to increase in Na⁺-proteoglycan complex, infiltrated macrophages stimulate secretion of vascular endothelial growth factor (VEGF)-C. Macrophage-derived VEGF-C increases density of the dermal lymph capillaries, indicating that lymphangiogenesis is advantageous to hypertensive animals by buffering elevated blood pressure. However, the effects of a high salt diet (HSD) on changes in mechanical activity of collecting lymph vessels, which directly connect with lymph capillaries, have not yet been determined.

Methods and Results:

Changes in mechanical activity of isolated collecting lymphatics in normal salt diet (NSD) and HSD rats in response to increase in intraluminal pressures were measured by video-microscopy. HSD vessels had smaller % active diameters (maximum and minimum) and higher amplitude compared with NSD vessels. The frequency of lymphatic oscillation was better maintained in HSD rats than in NSD. Lymphatic pump efficiency including stroke volume index (SVI), frequency times SVI, and amplitude times frequency in HSD rats were significantly higher than those of NSD. Thus, a HSD enhances the resistance to pressure-induced decreases in lymphatic pump efficiency.

Conclusions:

The present ex vivo study suggest that collecting lymphatics of rats enhance myogenic activity and lymphatic pump efficiency to compensate for increase in lymph flow and/or pressure after 2 weeks salt loading.

Introduction

The lymphatic system is important for the transport of extracellular fluids and macromolecular substances from tissues and organs under physiological conditions. Additionally, the lymphatic system plays a significant role in inflammation, immunity, and tumor metastasis in pathophysiological states.^1,2 Body fluids that escape from the capillary to the interstitial space enter into lymph capillaries, initial sites for lymph formation. Collecting lymphatics connected to lymph capillaries contribute to the collection of lymph into the systemic circulation via subclavian veins. Collecting lymph vessels work as a series of lymphatic pumps to propel the lymph fluid centripetally by rhythmic constriction and dilation. Smooth muscle cells in the vessels of humans and animals have unique characteristics that exhibit spontaneous activity, and serve as the main machinery of the pump in and ex vivo.^1,3

Hypertension is a major risk factor for cardiovascular diseases, and high salt intake is known to be closely associated with its pathogenesis and development in salt-sensitive patients.⁴ Salt loading, via inefficient renal excretion of sodium, often results in the expansion of circulating body fluid volumes and sodium retention. Machnik et al. demonstrated that a high salt diet (HSD) caused hyperplasia of lymph capillaries in rodent ear skins and proposed that the subcutaneous lymphatic system, working as a buffering system of body fluids, may be associated with blood pressure regulation in salt sensitive hypertension.⁵ However, the functional role of lymph transporters in collecting lymphatics connecting with lymph capillaries during high salt conditions remains uncharacterized. Several important issues regarding salt-induced hypertension and the lymphatic system need to be addressed. Does lymph conveyance by collecting lymphatics increase with a HSD? Can lymphatic smooth muscle enhance contractility during HSD?

Addressing these questions will provide new insights on the physiological and pathophysiological role of the lymphatic system in salt-sensitive hypertension, possibly leading to novel prevention and treatment for hypertension and cardiovascular diseases. Therefore, in the present study we focused on examination of the effect of intraluminal pressure on the mechanical activity of isolated rat lymphatic vessels following a normal salt diet (NSD) and a HSD.

Materials and Methods

Animals

Male Sprague-Dawley rats (n=24, Tokyo Jikken Dobutsu, Tokyo, Japan) were divided into NSD (n=12) and HSD (n=12) groups. NSD rats were fed a standard pellet diet and water for 7 weeks. HSD rats were fed a NSD for 5 weeks and then received a HSD (8% (w/v) NaCl) and 1% (w/v) saline for 2 weeks.^5,6 The Animal Ethics Committee of The University of Tokyo School of Medicine approved all experimental protocols, in accordance with the principles and guidelines on animal care of the Physiological Society of Japan.

In vivo studies: Blood pressure and heart rate measurements

Arterial blood pressures and heart rate of NSD (n=5) and HSD (n=5) rats were measured by a tail-cuff method (BP-98A-L, Natsume).

Ex vivo studies: Tissue preparations

Rats were sacrificed by a forceful impact to the head, followed by decapitation. Afferent lymphatics (5 mm in length) with the iliac lymph nodes^7,8 were excised and placed in a Petri dish containing cold (4°C) Krebs-bicarbonate solution (in mM: 120.0 NaCl, 5.9 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 NaH₂PO₄, 5.0 glucose, and 25.0 NaHCO₃). With microsurgical instruments and an operating microscope, afferent lymphatics were isolated and then transferred to a 10 mL organ chamber with two glass micropipettes containing Krebs-bicarbonate solution. After each lymph vessel was mounted on a pipette (proximal) and secured with sutures, the perfusion pressure was raised to 4 cmH₂O to flush out and clear the vessel. Subsequently, the distal end of the vessel was mounted on the outflow micropipette (distal). The proximal and distal micropipettes were connected through Tygon tubing with a 10 mL syringe and a stopcock, respectively. Krebs-bicarbonate solution by bubbling with 5% (v/v) CO₂-10% (v/v) O₂–85% (v/v) N₂ was perfused extraluminally over the lymphatics within the organ chamber. The flow rate of the superfused solution was kept at 2.5 mL/min throughout the experiment. After cannulation of the lymphatics, the chamber was transferred to the stage of a microscope (Olympus BH-2, Tokyo, Japan). The lymphatics were then warmed slowly to 37°C and allowed to equilibrate for≈60 min.

Measurement of mechanical characteristics of lymphatic vessels

To measure the mechanical activity of isolated lymphatic vessels we used a video-microscopy system, as previously described.^7,8 An objective lens (X10) and a monochrome charge-coupled device camera (KOCOM, KCB-270A, Korea) were used to obtain images of the lymphatics, which were displayed on a monochrome television monitor (Hamamatsu Photonics, C1846, Hamamatsu, Japan). The diameter of the lymphatics was measured with a diameter-detection device with an edge-detection method. The vessels used in the present study contained one valve and we measured its diameter at a nonvalvular region.^7,8 They were recorded on a DVD recorder (Pioneer, DVR55, Tokyo, Japan) and a direct-writing oscillograph (Sanei-Sokki, Recti 8K, Tokyo, Japan).

In the present study, we measured the passive diameter (PD; μm), active diameter (maximum diameter; Dmax μm, and minimum diameter; Dmin μm) and frequency min⁻¹ of lymphatic activity in NSD and HSD rats. We calculated amplitude (Dmax – Dmin), amplitude times frequency, ejection fraction [EF; (πDmax²-πDmin²)/πDmax²], stroke volume index (SVI; πDmax²-πDmin²), and frequency times SVI.⁹

Effect of intraluminal pressure on lymphatic function in NSD and HSD vessels

After the equilibration period at an intraluminal pressure of 5 cmH₂O, intraluminal pressure was increased from 3 to 19 cmH₂O in 2 cmH₂O steps by elevation of a 10 mL syringe connected to the inflow tubing, while the outflow tubing was closed with a stopcock throughout the experiment. Each level of pressure was maintained for≈15 min to allow the vessels to exhibit stable and spontaneous diameter oscillations. At the end of each experiment, Krebs-bicarbonate solution was changed to a Ca²⁺-free Krebs-bicarbonate solution that also contained ethylene diamine tetraacetic acid (EDTA; 1 mM) and nifedipine (a blocker of Ca²⁺ channels, 10 μM). Lymphatic vessels were incubated with Ca²⁺-free solution for≈20 min and then the pressure steps were repeated and the PDs were measured at each pressure value.^7,8

Drugs

All salts and EDTA were obtained from Wako (Osaka, Japan); nifedipine was from Sigma Aldrich (St. Louis, MO, USA).

Statistics

Changes in the diameter and amplitude of vessels following increases in intraluminal pressure were expressed as a percentage of the corresponding PD (100× diameter in the presence of extracellular Ca²⁺/diameter in the absence of extracellular Ca²⁺).^7,8 Data are presented as the mean±standard error of mean, and n indicates the number of vessels. Significant differences (p<0.05) were determined by unpaired and paired Student's t-test, as appropriate.

Results

Effects of HSD on blood pressure and heart rate of rats

Arterial blood pressures of HSD rats (systolic: 158±6 mmHg and mean: 122±8 mmHg, n=5) at 7 weeks were significantly higher than those of NSD (systolic: 125±5 mmHg and mean: 99±2 mmHg, n=5). Also, heart rate of HSD rats (444±10 beats/min) was significantly higher than that of NSD (396±17 beats/min, n=5).

Characteristics of passive and active diameters of lymphatic vessels in NSD and HSD rats: Response to elevated intraluminal pressure

To examine the effect of salt loading on physiological parameters of lymphatic vessels, we first analyzed PDs of lymphatic vessels in NSD and HSD rats in response to increases in intraluminal pressure.

In response to elevated intraluminal pressure from 3 to 19 cmH₂O, increments in PD of NSD and HSD lymphatic vessels ranged between 290±22 μm to 308±24 μm (n=7) and 385±33 μm to 406±35 μm (n=7), respectively. The PD of HSD vessels were significantly larger than those of NSD vessels over the range of intraluminal pressures we tested (Fig. 1A).

FIG. 1.

Changes in passive diameter (PD; μm, A), maximum diameter (Dmax; μm, B), and minimum diameter (Dmin; μm, C) of lymphatics in response to increases in intraluminal pressure. Intraluminal pressure-induced changes in % maximum diameter (% Dmax, D) and % minimum diameter (% Dmin, E) expressed as a percentage of the corresponding passive diameter of lymphatics. Open and closed circles indicate vessels isolated from NSD (n=7) and HSD (n=7) rats, respectively. *p<0.05 from the vessels of NSD.

We also measured the active maximum (Dmax) and minimum (Dmin) diameters of lymphatic vessels from NSD and HSD rats in response to the increases in intraluminal pressures to calculate the % Dmax and % Dmin, parameters for myogenic reactivity and contractility in the presence of extracellular Ca²⁺. In response to elevated intraluminal pressure from 3 to 19 cmH₂O, Dmax of lymphatic vessels in NSD (from 258±20 μm to 284±26 μm, n=7) and HSD (from 328±33 μm to 343±34 μm, n=7) rats did not show clear constriction, with no significant difference at the corresponding intraluminal pressure between NSD and HSD (Fig. 1B). In response to elevated intraluminal pressure from 3 to 19 cmH₂O, Dmin of lymphatic vessels in NSD and HSD rats ranged between 114±15 μm to 279±30 μm (n=7) and 149±24 μm to 322±36 μm (n=7), respectively, with no significant difference at the corresponding intraluminal pressure between NSD and HSD (Fig.1C). Thus, a HSD for 2 weeks induced PD augmentation of lymphatic vessels with no significant pressure-induced changes in their active diameters, suggesting enhanced lymphatic myogenic activity.

Characteristics of % diameters of lymphatic vessels in NSD and HSD rats: responses to elevation of intraluminal pressure

Next, we calculated % Dmax and % Dmin, standard parameters for normalized myogenic activity in ex vivo vessels in response to elevation of intraluminal pressure (100× diameter in the Ca²⁺ Krebs-bicarbonate solution/diameter in the Ca²⁺-free Krebs-bicarbonate solution).^7,8

Both the % Dmax and % Dmin of lymphatic vessels in HSD rats were significantly lower than that of NSD rats at intraluminal pressures of 7, 9, and 19 cmH₂O for % Dmax (Fig. 1D) and 9, 11, 13, and 19 cmH₂O for % Dmin (Fig. 1E). Also we analyzed slopes of relationships between pressures and % diameters, which enabled us to evaluate myogenic activity of isolated microvessels (arterioles, venules, and lymphatics) by linear regression analyses at a selected pressure range.^7,10,11 There were no significant differences of the slopes of % Dmax between NSD (-0.25±0.14, n=7) and HSD (-0.63±0.26, n=7) rats at a pressure range between 5 and 9 mH₂O. The slope values of % Dmin in HSD (3.30±0.72, n=7) rats were significantly smaller than those of NSD (5.74±0.81, n=7) at a pressure range between 5 and 9 mH₂O. These results suggest that a HSD for 2 weeks caused an increase in myogenic activity (decreased % Dmax and decreased % Dmin) of lymphatic vessels due to significant enlargement of PD and insignificant increase in Dmax and Dmin, and a significant reduction of the slopes of % Dmin more clearly indicates that 2 weeks salt loading enhanced myogenic activity of lymphatic vessels against increase in intraluminal pressures.

Inotropic and chronotropic characteristics of lymphatic vessels in NSD and HSD rats: Responses to elevated intraluminal pressure

To further clarify the effect of HSD on inotropism and chronotropism of lymphatic pump activity, we calculated the amplitude, EF, frequency, SVI, frequency times SVI, and amplitude times frequency of lymphatic vessels from NSD and HSD rats in response to increases in intraluminal pressure.

Elevation of intraluminal pressure from 3 to 19 cmH₂O reduced amplitude of lymphatics both in NSD (from 144±9 μm to 5±3 μm, n=7) and HSD (179±14 μm to 21±5 μm, n=7) vessels. The amplitude value of HSD vessels was significantly larger than that of NSD vessels at intraluminal pressure 3 to 19 cmH₂O, except for 5 and 15 cmH₂O (Fig. 2A).There were no significant differences of the % amplitude normalized by PD between NSD and HSD rats (Fig. 2B). Elevation of intraluminal pressure from 3 to 19 cmH₂O reduced EF of lymphatics both in NSD (from 0.80±0.03 to 0.04±0.02, n=7) and HSD (0.80±0.03 to 0.13±0.03, n=7) vessels. The EF value of HSD vessels was slightly larger than that of NSD vessels and there was significant difference of EF between NSD and HSD only at 19 cmH₂O (Fig. 2C). These results suggest that large amplitude of lymph vessels in HSD rats depends on PD enlargement and enhanced myogenic activity, indicating that a HSD prevented pressure-dependent decreases in amplitude and EF of lymphatic vessels.

FIG. 2.

Changes in amplitude (μm, A) of lymphatics in response to increases in intraluminal pressure. Intraluminal pressure-induced changes in % amplitude (B) expressed as a percentage of the corresponding passive diameter of lymphatics. Intraluminal pressure-induced changes in ejection fraction (C) of lymphatic activity. Open and closed circles indicate vessels isolated from NSD (n=7) and HSD (n=7) rats, respectively. *p<0.05 from the vessels of NSD.

Spontaneous oscillations of lymphatic diameter are known to normally occur and often disappear in response to high intraluminal pressure.^7,8 In this study, spontaneous oscillations were seen in three out of seven NSD vessels at intraluminal pressures ranging from 3 to 19 cmH₂O, while oscillations terminated in the remaining four NSD vessels at 7 cmH₂O and the higher intraluminal pressure. However, HSD lymphatic vessels (six out of seven preparations) kept oscillating even at higher ranges of intraluminal pressure. Therefore, the error bar values were quite high in the frequency of vessels at the higher intraluminal pressure. In response to elevations in intraluminal pressures from 3 to 9 cmH₂O, increments in the frequency of lymphatic contractions in NSD and HSD vessels ranged between 15±2 min⁻¹ to 21±4 min⁻¹ (n=7) and 17±3 min⁻¹ to 23±1 min⁻¹ (n=7), respectively. In spite of oscillatory termination in some vessels in NSD lymphatic vessels and stabilized HSD lymphatic vessels, significant differences in the frequency of lymphatic contractions were only seen at an intraluminal pressure of 11 cmH₂O between NSD (13±5 min⁻¹) and HSD (24±1 min⁻¹) rats (Fig. 3A).

FIG. 3.

Intraluminal pressure-induced changes in frequency (min⁻¹, A), stroke volume index (SVI x 10³, B), frequency times SVI times [x10³ (min⁻¹), C], and amplitude times frequency (μm x min⁻¹, D) of lymphatic activity. Open and closed circles indicate vessels isolated from NSD (n=7) and HSD (n=7) rats, respectively. *p<0.05 from the vessels of NSD.

The increment of intraluminal pressure reduced SVI, frequency times SVI, and amplitude times frequency of lymphatic vessels in NSD and HSD rats. Values of SVI, frequency times SVI, and amplitude times frequency of HSD vessels were significantly larger than those of NSD vessels, indicating that pump efficiency was better maintained in HSD than NSD rats (Fig. 3B,C,D).

Discussion

HSD enhances myogenic activity of lymphatic vessels

The pumping activity of afferent lymphatics isolated from the iliac lymph nodes of NSD and HSD rats both decreased in response to the elevation of intraluminal pressure mainly due to decreased amplitude and EF. Parameters for myogenic activity included % Dmax and % Dmin, which were calculated from maximum and minimum lymphatic diameters, respectively, normalized to PD at corresponding intraluminal pressures.^7,8 We found that both % Dmax and % Dmin values in HSD rats were smaller than those in NSD rats, and indicating that HSD enhanced myogenic activity of lymphatic vessels. The slopes of relationships between pressure and % Dmin of HSD lymphatics were significantly lower than those of NSD. Amplitude and SVI of lymphatic vessel in HSD rats were also significantly larger when compared with NSD vessels. These results indicated that a HSD for 2 weeks enhanced lymphatic myogenic activity. Because an acute intravenous infusion of physiological salt solution into rats was reported to increase lymphatic flow and pressure in the mesenteries,¹² we presume that treatment with a HSD expands plasma volume in rats and subsequently increases plasma exchange rates at capillary sites. These excess changes in microcirculatory fluid movements may promote lymph production and lymphatic transport. Thus, enhanced myogenic activity in HSD lymphatic vessels appears to compensate for increasing lymph flow and/or lymph pressure. In the present study, the PD of HSD vessels were significantly larger than those of NSD over the range of intraluminal pressures, suggesting that a HSD for 2 weeks also may cause a change in passive characteristics of lymphatic vessels that are most likely due to augmentation of lymphatic drainage.

Lymph nodes are perfused by the lymphatic system with blood circulation. Afferent and efferent lymphatics are inlets and outlets, respectively, of the lymphatic pathway. Afferent lymphatic pressure and flow are greatly influenced by hemodynamic changes of the lymph nodes in vivo.¹³ Under hyper-dynamic circumstances, afferent lymphatics strenuously inject intraluminal lymph into the lymph nodes competing against the resistance. Enhancement of myogenic activity in afferent lymphatics is also one of the compensatory responses to maintain the lymph nodal microcirculation. Lymphatic vessels are adaptable to various physiological stimuli, including short-term exercise¹⁴ or microgravity circumstances¹⁵ by enhancing myogenic constrictor tone or inactivating the lymphatic pump. In contrast to a HSD, oral feeding of a high fructose diet causes impairment to intrinsic contractility of mesenteric collecting lymphatic vessels in a rat model of metabolic syndrome.¹⁶ Also in model of guinea pig ileitis 2,4,6-trinitrobenzenesulfonic acid (TNBS), a well-accepted model of intestinal inflammation, altered lymphatic contractile function with endogenous prostanoids dependent mechanisms.¹⁷

Elevation of intraluminal pressure is known to increase the frequency of lymphatic vasomotion accompanied by reduction of amplitude, EF, and SVI.^7,8 Although the frequency of lymphatics of HSD rats at pressures ranging from 3 to 19 cmH₂O was slightly higher than that of NSD, the lymphatics of HSD rats exhibited significant high frequency only at pressure of 11 cmH₂O. At pressure range used in the present study, HSD lymphatic vessels were more persistently oscillatory and resistant to termination than NSD lymphatic vessels. Although amplitude of lymphatics in HSD rats was significantly larger than that of NSD rats, normalized amplitude (% amplitude) of vessels was not significantly different between NSD and HSD rats. When we calculated amplitude times frequency as a pump efficiency similar to frequency times SVI, HSD lymphatics demonstrated significant higher values than NSD, suggesting that HSD augmented minute pump activity of lymphatics. Collectively, these results suggest that HSD not only affects inotropism of lymphatics but also chronotropism, and that the lymphatic smooth muscle of HSD rats can adapt to produce phasic contraction and relaxation against higher pressure loading, and that HSD volumetrically elicits lymphatic minute pump efficiency associated with enhancement of SVI, frequency times SVI and amplitude times frequency of the vessels.

Lymphatic activity in hypertensive animal models

Although several studies have shown that oxidative stress¹⁸ or aging¹⁹ affects the function of mesenteric collecting lymphatics in rats, there are relatively few reports regarding the role of the lymphatic system in hypertensive animals.¹⁸ In addition, there is little information about the contractility of collecting lymphatic vessels and regulation of lymph transport in hypertensive rat models. Machnik et al. used Sprague Dawley rats to investigate the effects of a HSD on lymphangiogenesis in the skin and observed that mean arterial pressure of HSD rats was significantly higher than that of low salt animals.⁵ Consistently, we confirmed elevation of mean blood pressure in HSD rats following their protocol when compared with NSD rats. Additionally, 2 weeks HSD caused tachycardia in rats compared to the NSD group, suggesting that HSD rats were under hyper hemodynamic condition. Machnik et al. revealed that a HSD induces secretion of vascular endothelial growth factor (VEGF-C) from infiltrated macrophages in rodent ear skins and that secreted VEGF-C elicits lymphangiogenesis.⁵ Another report by Wiig et al. suggests that HSD also elevated osmolality of ear skin that associated with lymphangiogenesis.²⁰ Such architectural adaptation of lymph capillaries to high salt loading could attenuate and buffer blood pressure elevation. However, their studies did not clarify functional changes or adaptation of lymphatic inotropism and chronotropism. Collecting lymphatic vessels connecting to lymph capillaries should transport lymph to thoracic ducts and finally return body fluids to the venous system through active and passive transport mechanisms. If orchestra of lymph capillary and collecting lymphatics cannot work to lymph transport, edema would be present in peripheral tissues including ear skins.

By using near-infrared fluorescence imaging, Kwon et al. indirectly measured lymphatic function and architecture in rats and mice under anesthetized conditions, demonstrating that HSD for 2 weeks significantly dilated the diameters of lymphatics in the hind limbs and increased the oscillating frequency of lymphatic contraction.⁶ However, intraluminal pressure and flow rate in lymphatics have never been directly measured in conscious hypertensive or HSD rats. Owing to technical difficulties of direct and precise measurement of lymphatic dynamics in vivo, we selected an ex vivo approach to investigate details of lymphatic function under controlled conditions. In the present study, we used afferent lymphatic vessels isolated from the iliac lymph nodes of rats, which are lymphatic vessels that mainly drain lymph from hind limbs. In in vivo study by using anesthetized Sprague-Dawley rats intraluminal pressure of collecting lymphatics in the mesentery were 5.5±2.5 cmH₂O (end-diastolic pressure) and 9.9±3.4 H₂O (peak pressure).¹² In spite of no reports regarding intraluminal pressure of rat iliac lymphatics, we presume that the present experimental conditions may be a hyper-pressurizing system. Because the isolated lymphatic vessel was pressurized by pressure column connected with inflow tubing and a stopcock blocked its outflow end in the present study,^7,8 we speculate that outflow resistance of afferent lymphatics may change during HSD since lymph nodes are closely associated with lymphatic function.²¹ If HSD expands plasma circulation though lymph nodes, the nodal swelling may increase outflow resistance against afferent lymphatics. One limitation of this study is that the HSD-induced functional changes in lymphatics we observed may not be general to the whole lymphatic system because there are regional variations of contractile activity and possible heterogeneous functional modulations in collecting lymphatics.²²

Results from the current study were incorporated with previously known mechanisms for HSD-induced morphological and functional lymphatic changes (Fig. 4),supporting a concept that collecting lymphatic vessels play an important role in preventing edema in lower extremities during HSD conditions^1,6 or possibly to buffer blood pressure elevation. In²³ and ex vivo²⁴ human studies indicate that intrinsic spontaneous contraction of lymphatic vessels is a notably important factor to drain lymph from peripheral tissues to venous system via thoracic ducts, because peripheral lymph vessels located under heart could transport lymph against the hydrostatic pressure gradient created by gravity.²⁵ Pathological abnormalities including degeneration of smooth muscle cells in the wall of lymphatics have been reported in human extremity lymphedema.²⁶ Kopp et al. reported that hypertensive patients had increased tissue Na⁺ content in calf muscles compared with normotensive controls revealed by ²³Na magnetic resonance imaging,²⁷ indicating that spatial heterogeneities of Na⁺ accumulation clearly exist among species, genders, or aging. Although in the present we underlined alternation of mechanical activity in rat lymphatic tissue preparations after 2 weeks salt loading, we did not show mechanisms of functional changes in the cells, including smooth muscle and endothelium. Further investigation will be needed to identify mechanisms of the lymphatic system under a HSD.

FIG. 4.

Schema of the suggested alteration in micro- and lymph-circulation during NSD (left) and HSD (right) conditions. A HSD increases perfusion plasma volume and the exchange rate of plasma and interstitial fluids that facilitate lymph formation at lymph capillaries (LC) with lymphangiogenesis.^3,18 Collecting lymphatics (CL) connected to lymph capillaries drain lymph overproduced by HSD. This surplus lymph flow and/or pressure stretch the wall of collecting lymphatics. Finally, the mechanical and physical factors transduce biological functions of lymphatic smooth muscle (SM).

Footnotes

Acknowledgment

The authors would like to thank Novartis Pharma K.K. for their financial support.

Author Disclosure Statement

No competing financial interests exist. This study was supported in part by a grant-in aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 22500403 to RM, No. 21590948 to MI).

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