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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Molecular and cellular basis of the regulation of lymphatic contractility and lymphatic absorption
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Yu Wang a,c,1 , Wei Zhao b,1 , Lin Zhang a , Yong-Na Zhao a , Fei Li a , Zhen Zhang a , Yun-Dong Dai a , Wei-Feng Li a , Yan-Ning Qiao b , Cai-Ping Chen b , Ji-Min Gao a,∗∗ , Min-Sheng Zhu b,∗ a
Zhejiang Provincial Key Laboratory for Technology and Application of Model Organisms, School of Life Sciences, Wenzhou Medical College, Wenzhou, China Model Animal Research Center and MOE Key Laboratory of Animal Models of Disease, Nanjing University, Nanjing, China c Jinhua Municipal Central Hospital, Jinhua, Zhejiang, China b
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Article history: Received 13 November 2013 Received in revised form 22 April 2014 Accepted 5 May 2014 Available online xxx
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Keywords: Lymphatic vessel Myosin light chain kinase Smooth muscle Contractility, Absorption
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1. Introduction
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Lymphatic absorption is a highly regulated process driven by both an extrinsic mechanism (external force) and an intrinsic mechanism (lymphatic vessel contractility). The lymphatic muscle is a specialized smooth muscle with unique mechanical properties. To understand the molecular mechanism and relative contribution of smooth muscle contraction in lymphatic absorption, we analyzed mice with a smooth muscle-specific deletion of Mylk, a critical gene for smooth muscle contraction. Interestingly, the knockout mice were significantly resistant to anesthesia reagents. Upon injection in the feet with FITC-dextran, the mutant mice displayed a 2-fold delay of the absorption peak in the peripheral circulation. Examining the ear lymphatic vessels of the mutant mice revealed a reduction in the amount of fluid in the lumens of the lymphangions, suggesting an impairment of lymph formation. The Mylk-deficient lymphatic muscle exhibited a significant reduction of peristalsis and of myosin light chain phosphorylation in response to depolarization. We thus concluded that MLCK and myosin light chain phosphorylation are required for lymphatic vessel contraction. Lymphatic contractility is not an exclusive requirement for lymphatic absorption, and external force appears to be necessary for absorption. © 2014 Published by Elsevier Ltd.
Transportation of fluid, macromolecules, and immune cells via the lymphatic vascular system is fundamental for homeostasis in the body (Zawieja, 2005). Impairment of this process occurs during various pathological alterations, such as aging (Gasheva et al., 2007), inflammatory bowel disease (Wu et al., 2005) and other lymph-associated diseases (Lucci et al., 2007; Modi et al., 2007; Simon and Cody, 1992). This lymphatic vascular system is composed of a network of lymphatic vessels connecting the lymph nodes, the spleen and Peyer’s patches, which are found in the small
∗ Corresponding author at: Model Animal Research Center and MOE Key Laboratory of Animal Models of Disease, Nanjing University, 12 Xue-Fu Road, Pukou, Nanjing 210061, China. Tel.: +86 025 58641529. ∗ ∗ Corresponding author at: Zhejiang Provincial Key Laboratory for Technology and Application of Model Organisms, School of Life Sciences, Wenzhou Medical College, Wenzhou 325035, China. Tel.: +86 0577 86689779. E-mail addresses: [email protected] (J.-M. Gao), [email protected] (M.-S. Zhu). 1 These authors contributed equally to this work.
intestine and serve as the base of lymph fluid transportation (Davis et al., 2011; Mislin, 1964, 1976). Structurally, the lymphatic system contains initial and collecting lymphatic vessels. The former possess no muscle cells and transfers lymph liquid into collecting lymphatic vessels; the latter is composed of lymphangions with valves and muscle cells lining the vessel wall and driving unidirectional flow (Benoit et al., 1989). Current knowledge suggests that lymph vessels serve as passive thin-walled tubes that drain interstitial fluid back to the venous side of the blood circulation, and the unidirectional propulsion of lymph is mediated by one-way valves and intermittent external pressure provided by pulsating arteries, intestinal peristalsis, skeletal muscle contraction or respiratory movements (Aukland, 2005). However, accumulating evidence suggests that active pumping by lymphatic contraction is also important for lymph propulsion (Aukland, 2005), but the roles and relative importance of these passive and active mechanisms underlying lymphatic regulation remain unclear. Lymphatic muscle is a highly specialized type of smooth muscle that exhibits important differences from typical vascular or gastroenterological smooth muscle cells (Benoit et al., 1989; Muthuchamy et al., 2003; Zawieja, 1996). Interestingly, these
http://dx.doi.org/10.1016/j.biocel.2014.05.002 1357-2725/© 2014 Published by Elsevier Ltd.
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smooth muscle cells share many of contractile proteins with cardiac and skeletal muscles, thereby displaying phenotypic characteristics of cardiac and skeletal myocytes (Muthuchamy et al., 2003). One question is whether lymphatic muscle shares a similar regulatory mechanism with other smooth muscle cells. Although data from the application of non-specific pharmaceutical inhibitors suggests the involvement of smooth muscle signaling in lymphatic contraction (Nepiyushchikh et al., 2011; Wang et al., 2009; Zhanna et al., 2011), little is known about the regulation of this signaling from genetic evidence of lymphatic muscle contractility (Muthuchamy and Zawieja, 2008; Zawieja, 2005, 2009). Smooth muscle contraction is evoked by calcium signaling through depolarization and G protein-coupled receptor (GPCR) agonists that activate myosin light chain kinase (MLCK) through its binding to calmodulin (He et al., 2008, 2011a,b; Kamm and Stull, 2001; Somlyo and Somlyo, 2003). The activated kinase phosphorylates the regulatory light chain (RLC), resulting in the activation of actomyosin Mg-ATPase and cross-bridge cycling of force development in smooth muscle. RLC is dephosphorylated by myosin light chain phosphatase (MLCP), thereby initiating relaxation by returning myosin to an inhibited state. The relative activities of MLCK and MLCP are thought to be a primary determinant for the extent of RLC phosphorylation and the production of force (Kamm and Stull, 2001; Somlyo and Somlyo, 1994, 2003). However, other Ca2+ -independent kinases, such as integrin-linked kinase, RhoAassociated kinase, and zipper-interacting protein kinase, are also able to phosphorylate RLC (Ganitkevich et al., 2002; Ihara and MacDonald, 2007; Murthy, 2006; Sward et al., 2003), implying a calcium-independent mechanism involving smooth muscle contractility. Our previous reports show that MLCK is the primary kinase responsible for myosin light chain phosphorylation and is required for the force development of various smooth muscle tissues in the gut, airway, bladder and blood vascular system, etc. (He et al., 2008, 2011a,b; Zhang et al., 2010). The effects of pharmaceutical inhibitors of MLCK suggest a role for MLCK in lymphatic contractility, but there is a lack of loss-of-function evidence. We analyzed the lymphatic function of mice with a smooth musclespecific deletion of the Mylk gene.
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2. Materials and methods
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Mylkflox/flox mice (He et al., 2008) and SMA-Cre transgenic mice were maintained at the animal center of the Model Animal Research Center of Nanjing University. MLCKSMKO mice (Mylkflox/flox ; SMACre) were produced by crossing Mylkflox/flox mice with SMA-Cre transgenic mice. The genotyping strategy used is described in our previous report (He et al., 2011a,b). All animal procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee of the Model Animal Research Center of Nanjing University (Nanjing, China).
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2.2. Reagents
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FITC-dextran and anti-mouse MLCK antibody (K36) were purchased from Sigma. Human Prox-1 (Prospero-related homeobox 1) polyclonal Ab was purchased from R&D Systems (Cat. AF2727). Pecam-1 (rat anti-mouse CD31 ) was purchased from BD (Cat. 553370). Cy5-AffiniPure Donkey Anti-Goat IgG was purchased from Jacison (Cat. 705175147). Alexa Fluor® 594 Donkey Anti-Rat IgG (H+L) was purchased from Invitrogen (A21209). Anti-mouse phosphor-myosin light chain 2 (Ser19) was purchased from Cell Signaling Technology (#3675). Other chemicals and protein reagents were purchased from Sigma or MuCyte.
2.3. Western blot analysis Measurements of MLCK, RLC and other proteins were performed according to our previous protocol (He et al., 2008). Briefly, the mesentery including intestine were dissected from mice and maintained in H-T buffer; the lymphatic vessels around the mesenteric arteries were cleaned of adipose and connective tissue; the tissue was collected and frozen quickly with 10% trichloroacetic acid and 10 mM dithiothreitol in acetone precooled to slush at −80 ◦ C. After thorough homogenization, the sample pellet was centrifuged at 3000 × g twice for 2 min at 4 ◦ C, and the supernatant was removed. The sediment was washed three times with ether and dried to remove the residual ether. The dried protein powder was carefully and completely dissolved in 8 M urea. Equal amounts of protein were subjected to SDS-PAGE followed by protein transfer to a nitrocellulose membrane. The membrane was then probed with a primary antibody to MLCK (K36; Sigma) or phosphorylated RLC (Cell Signaling Technology) and then with corresponding secondary antibodies. The membrane was incubated in Super Signal West Pico Chemiluminescent Substrate (Thermo) before exposure to film. 2.4. Immunofluorescence For whole-mount staining, mice ears were dissected and removed the hair by using Hair removal cream. The ears were fixed in 4% paraformaldehyde at 4 ◦ C overnight, splitted and removed intermediated cartilage under microscope, and then attached to Sylgard plates with insect pins. The ears were blocked with 3% milk in 0.3% PBS (0.3% Triton X-100 in PBS) for 4 time at room temperature, and incubated with polyclonal antibodies against Prox-1 and Pecam-1 at 4 ◦ C overnight. Alexa 594 and CY5 conjugated secondary antibody were used for visualization. The samples were then mounted with Olympus FluoView 1000 and examined under an Olympus BX51 confocal microscope. 2.5. Measurement of the transport in the lymphatic vessel MLCKSMKO mice and their littermates were anesthetized with avertin (250 mg/kg i.p. injection), and then subcutaneously injected with 2.5 l of FITC-dextran (25 mg/ml) in two feet. Approximately 500 l of blood from the eyeballs was placed in tubes containing 10 l of 0.2 M EDTA at different time points after injection (1 h, 2 h, 3 h, 4 h), and this was centrifuged at 3000 × g for 10 min at room temperature. The resultant supernatants were collected and stored at −20 ◦ C in the dark. The FITC fluorescence was measured with a Synergy2 Multi-Mode Microplate Reader, with excitation at between 485 and 528 nm (BIO-TEK, Inc.). 2.6. Measurement of the lymphatics rhythmical movement frequency MLCKSMKO mice and their littermates were anesthetized with avertin (230 mg/kg i.p. injection), and then injected with 3% Evan’s Blue subcutaneously into two feet’s pads so as to visualize lymphatic vessel clearly. 15 min after injection, both of the left and right inguinal lymphatic vessels with blue color were collected and subjected to peristalsis measurement. The vessels with removal of adipose and connective tissues were incubated with HEPES-Tyrode (H-T) buffer (137.0 mmol/L NaCl, 2.7 mmol/L KCl, 1.0 mmol/L MgCl2 , 1.8 mmol/L CaCl2 , 10.0 mmol/L HEPES, and 5.6 mmol/L glucose, pH 7.4), and the peristalsis could be observed visually under a microscope within a few minutes. We started counting when peristalsis occurred and the frequency of peristalsis was calculated with a modified method reported previously (Aukland, 2005).
Please cite this article in press as: Wang Y, et al. Molecular and cellular basis of the regulation of lymphatic contractility and lymphatic absorption. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.05.002
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Q4 Fig. 1. Ablated expression of MLCK in lymphatic vessels. Mesenteric lymphatic vessels were isolated from P15-18 MLCKSMKO (KO) and control (CTR) mice and evaluated by Western blotting and immunofluorescence analysis. (A) Western blot of MLCK protein with a primary monoclonal antibody (K36) against MLCK. -Actin was used as the loading control. (B) Quantitation of MLCK protein (n = 3). **p < 0.01. (C) The pattern of collecting lymphatic vessels in the ear skin with Pecam-1 (red) and Prox-1 (white). Scale bars = 200 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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To abate MLCK expression in lymphatic smooth muscle, we crossed Mylkflox/flox mice with SMA-Cre mice (He et al., 2008). The resultant Mylkflox/flox ; SMA-Cre (MLCKSMKO ) and Mylkflox/+ ; SMA-Cre littermates were used as knockout (KO) and control (CTR) mice, respectively. In KO mice, specific deletion of MLCK in almost all
smooth muscle tissues was expected, while in CTR mice, hemizygosity for MLCK produced no apparent phenotypic effects [19]. Q2 Western blot analysis showed a significant reduction of MLCK expression in mutant mesenteric lymphatic vessels (Fig. 1A and B). However, approximately 34.26 ± 12.93% of MLCK protein could be detected in the KO tissue. This residual expression of MLCK protein detected by Western blot may be due to MLCK expression in the lymphatic endothelium. Interestingly, we could not detect the long isoform of MLCK in the endothelium-containing lymphatic
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Fig. 2. Macrophenotypes of MLCKSMKO mice. (A) MLCKSMKO (KO) and control (CTR) mice (P24) were euthanized and their abdomens were exposed. (B) Growth curves of body weight (n = 5). (C) Histological examination of the ileum and jejunum. Scale bars for C: upper row, 20 m; lower row, 4 m. *p < 0.05; **p < 0.01.
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vessels (Garcia, 1995). This result indicates that the endothelial cells in lymphatic vessels only express short MLCK, unlike the cultured endothelial cells expressing long MLCK in vitro. This observation is consistent with the expression pattern of MLCK in the endothelium of vascular vessels reported previously (Yu et al., 2012). We also examined the patterns of collecting lymphatic vessels in knockout mice ears by staining Prox-1 and Pecam-1 with a whole-mount immunofluorescence assay. The mutant ears had a comparable morphology of lymphatic network with that of control ears (Fig. 1C). Thus, deletion of MLCK in smooth muscle cells did not affect development and structure of lymphatic vessels.
control (141.25 ± 23.13 vs. 81.25 ± 11.61 s, p < 0.01) (Fig. 3). However, despite the delay in anesthetization, 100% of the knockout mice could be anesthetized in a similar manner to CTR mice. 3.3. Deletion of MLCK caused impaired absorption by the lymphatic system
3.2. MLCK-deficient mice displayed resistance to an anesthesia reagent
The MLCK deletion-induced resistance to anesthesia led us to investigate whether the anesthesia delay was caused by impaired absorption by the lymphatic system. We subcutaneously injected 5 l FITC-dextran into the feet of mice and then collected blood from their eyeballs at different time points. The fluorescent substance in the serum was quantified as shown in Fig. 4. One hour after injection, the fluorescence intensity of the serum samples
The knockout mice appeared to have significantly reduced gastrointestinal motility, low blood pressure and abolished responses to KCl depolarization and agonists, which is consistent with our previous reports (Fig. 2A) (He et al., 2008, 2011a,b; Zhang et al., 2010). The growth of the mutant mice was retarded from the 18th day after birth (Fig. 2B). The mutant small intestine exhibited edema with an enlarged lumen (Fig. 2A and C). Histological examination also showed edema around the smooth muscle tissue (Fig. 2C). To our surprise, it was difficult to anesthetize the knockout mice with a typical dose of an anesthesia reagent. To quantify this effect, we measured the time required to be anesthetize by avertin (230 mg/kg), a widely used anesthesia reagent for mice. Upon intraperitoneal injection with a typical dose of avertin, the control mice went to anesthetized status (laid down) for approximately 80 s. However, with the same dose, the knockout mice required approximately 140 s to achieve the same status, which was significantly (approximately 2-fold) longer than the
Fig. 3. MLCK-deficient mice displayed resistance to anesthesia. MLCKSMKO mice (KO) and control mice (CTR) (P18) received intraperitoneal injection of 2.5% avertin at a dose of 230 mg/kg body weight. The time required for complete anesthetization was recorded, and the average values were analyzed using a paired t-test. n = 3, *p < 0.05.
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Fig. 4. Delayed absorption of dextran in MLCK-deficient mice. MLCKSMKO (KO) and control (CTR) mice (P18) were injected individually with 5 l of FITC-dextran in the foot. Peripheral blood samples were collected for the measurement of fluorescence with a Synergy2 Multi-Mode Microplate Reader. The excitation wavelengths were between 485 and 528 nm. The y-axis indicates the relative fluorescence intensities. Each time point reflected data from three mice. *p < 0.05. KO: 1 h, n = 7; 2 h, n = 6; 3 h, n = 6; 4 h, n = 10; CTR: 1 h, n = 9; 2 h, n = 16; 3 h, n = 13; 4 h, n = 14.
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both from control and knockout mice were comparable. However, 2 h after injection, the fluorescence intensity in control mice was strikingly increased, while only a slight increase was observed in the knockout mice. Three hours after injection, the fluorescence intensity of the control mice was reduced, while that of mutant mice increased rapidly (Fig. 4). The absorption peak of the knockout mice appeared at the fourth hour after injection, which is approximately 2-fold longer than the control. This absorption delay was consistent with the anesthetization delay mentioned above. We conclude that the delayed distribution effect of MLCK deletion is caused by impaired lymphatic absorption. The movement of lymph, an essential process of lymphatic absorption, may be regulated by extrinsic compression from external forces and intrinsic compression from lymphatic contractility. To assess the contribution of smooth muscle to this process, we examined the movement of lymph in the ears and tail, where there is less tissue pressure (Aarli and Aukland, 1991). After injection of FITC-dextran in the ear, the collecting lymphatic was visualized under a fluorescence stereomicroscope. The lymphangions in the lymphatic vessels of control mice were even with clear valves. However, the mutant lymphangions appeared to be discontinuous (Fig. 5A and B). Magnification showed less liquid in the mutant lymphangions compared to the control, but the sizes of the valves were comparable. This morphological alteration indicated that mutant lymphangions had a weak pumping activity and a reduced ability to collect lymph fluid. Because the process of lymph absorption in the mouse ear is too fast for the velocity to be quantified, we injected FITC-dextran at the tail and measured the distance moved at different time points. 10 min after injection, the fluorescent substance in the tail of control mice had moved approximately 16.83 mm. However, the distance moved in the mutant tail was approximately 4.94 mm, and the average value was significantly shorter than control (p < 0.01) (Fig. 6A and B). These observations indicated a reduced capability of lymph absorption after MLCK deletion.
Fig. 5. Visualization of lymph flow in the mouse ear. MLCKSMKO (KO) and control (CTR) mice were injected with 1 l of FITC-dextran solution in the ear. (A) The visualized lymphatic vessels were photographed at different time points. The magnification shows the typical morphology of lymphangions, and the mutant vessels were found to exhibit many discontinuous lymphangions. (B) The percentage of discontinuous lymphangions was quantified. The average percentages are expressed as the mean value ± SEM (n = 5). **p < 0.01; ***p < 0.001.
3.4. Myosin light chain phosphorylation of lymphatic smooth muscle was inhibited by MLCK deletion MLCK mediates RLC phosphorylation, and RLC phosphorylation is required for smooth muscle contraction. The control inguinal lymphatic vessel displayed a peristalsis under resting conditions at rate of 7.7 ± 1.1 per minute, while of 1.5 ± 0.5 per minute for the mutant vessels (Fig. 7A). We measured RLC phosphorylation in lymphatic smooth muscle in response to KCl. Due to the difficulty of collecting enough lymphatic vessels, we only measured
Fig. 6. Lymph movement in the mouse tail. MLCKSMKO (KO) and control (CTR) mice were injected with 1 l of FITC-dextran solution. (A) The visualized moving lymph flow was monitored at different time points by scanning the tails. (B) The distances moved were measured by calculating the displacement between the origin and the broad edge of the fluorescence. The displacement is expressed as the mean ± SEM, n = 3. **p < 0.001.
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Fig. 7. Lymphatic smooth muscle contraction is relied on the phosphorylation of regulatory myosin light chain in response to depolarization. Lymph vessels were isolated from control (CTR) and MLCKSMKO mice and incubated in H-T buffer respectively. (A) Measurement of the rhythmical movement frequency of inguinales profundi lymphatics whose pulsatile contraction was easy to be visualized. (B) At 0, 10 and 20 s after stimulation with KCl, mesenteric lymph vessels were sampled and subjected to Western blotting analysis. The primary antibody was anti-phosphorylated RLC antibody. -Actin was used as the protein loading control. This result represents three independent experiments. The average percentages are expressed as the mean value ± SEM, n = 3 (MLCKSMKO mice) or n = 7 (CTR mice), *p < 0.05.
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phosphorylated RLC at three time points after stimulation (0, 10 and 20 s). In the control mesenteric lymphatic vessel, basal RLC phosphorylation could be clearly detected. The amount of phosphorylated RLC increased rapidly after stimulation with KCl and then declined. In knockout mesenteric lymphatic vessels, both basal and stimulation-evoked RLC phosphorylation were significantly inhibited (Fig. 7B), which was consistent with delayed lymphatic absorption in MLCK-deficient mice. This suggests a critical role of RLC phosphorylation of MLCK in lymphatic smooth muscle contraction. However, due to technological limitations, we could not directly record the force of the lymphatic smooth muscle of such a small animal.
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A contraction of a typical smooth muscle is evoked by KCl depolarization and agonists through calcium signaling, and the resultant elevation of cytosolic calcium causes MLCK activation. RLC phosphorylation by MLCK triggers ATPase activity of myosin, thereby initiating cross-bridge movement (Kamm and Stull, 2001). Lymphatic muscle is a specialized smooth muscle composed of different contractile proteins and contractile properties (Bridenbaugh et al., 2003; Sjoberg and Steen, 1991). One question is whether MLCK is required for lymphatic contraction in the same manner as for other smooth muscles. Although the observations from MLCK inhibitors with non-specific effect imply an involvement of MLCK in lymphatic contraction, the in vivo role of MLCK in lymphatic muscle is basically not elucidated particularly through genetic analysis. We found that deletion of MLCK abolished pulsatile contraction as
well as contractile responses to depolarization and agonists. Our loss-of-function evidence suggests that the contractility of lymphatic vessels is primarily attributed to the contraction of smooth muscle rather than to that of other muscle types. Signaling integration between MLCK and its RLC phosphorylation in the lymphatic smooth muscle cells is required for lymphatic contraction in the same manner as in other smooth muscle cells. Because MLCK is a strictly calcium/calmodulin-dependent myosin light chain kinase, we also emphasize the importance of a calcium-dependent rather than calcium-independent mechanism underlying lymphatic contraction. Lymphatic absorption includes lymph formation, draining, and lymph flow, which is a highly coordinated process. It is widely accepted that the lymph fluid is formed by entrance of interstitial fluid into collecting lymphatic vessel after a decrease in the luminal pressure of the lymphatic vessel and an increased external compression from movements or intermittent pressure (Gashev, 2008; Scallan et al., 2013; Smith, 1949). Lymph formation seems to be primarily dependent on external force through a passive mechanism (Aukland, 2005; Granger et al., 1984; Guyton et al., 1966). During this process, the relative importance of the suction mediated by muscular lymphatic contractility remains unclear (Aukland, 2005). We here found that inhibition of lymphatic contraction by MLCK deletion resulted in incomplete filling of lymph fluid in lymphangions, indicating a significant reduction of lymph formation. We thus suggest that the suction of muscular lymphatics is necessary for lymph formation in addition for lymph movement (Aukland, 2005). The intestinal edema phenotype observed in MLCK knockout mice also supports this proposal. A mass of evidence suggests that lymphatic absorption is driven by extrinsic (or passive) and intrinsic (or active) mechanisms. However, their relative contributions to lymphatic absorption remain unclear. Lymphatic absorption from the peritoneal cavity and feet to circulation is typical pathways reflecting such active and passive absorption mechanisms. Upon MLCK deletion, the absorption peaks in the peripheral circulation for anesthesia reagents absorbed from the peritoneal cavity and for FITC-dextran absorbed from the feet were approximately 2-fold longer compared to those in the control mice. However, the absorption process was not completely abolished. Therefore, we conclude that as a primary active mechanism, lymphatic contractility is not an exclusive requirement for lymphatic absorption. The passive mechanism seems to be necessary for the absorption process. Due to the delay of lymphatic absorption caused by MLCK deletion, effective inhibition of lymphatic smooth muscle contractility is expected to slow down absorption at least two folds. This is particularly useful for the development of a novel therapeutic strategy that would slow the distribution of a given toxin, allowing more time to elapse before receiving medical care. A successful example is the application of a nitric oxide donor, which causes a 3-fold reduction in the foot-to-groin lymph transit time of snake toxin (Saul et al., 2011). It is worth noting that inhibition of smooth muscle contraction only delays and does not abolish the absorption, so such a strategy can only be used as a first-aid medical approach. Conflict of interests The authors declare no conflict of interests regarding the publication of this article. Acknowledgements We thank Man Chu, Zhi Shang and Xu-Dong Cao of Labo- Q3 ratory of Vascular and Cancer Biology, Cyrus Tang Hematology Center, Soochow University, Suzhou, China. They provided the methods of immunofluorescence and tail injection. This study was
Please cite this article in press as: Wang Y, et al. Molecular and cellular basis of the regulation of lymphatic contractility and lymphatic absorption. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.05.002
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supported by National Key Scientific Research Program of China (Grant 2014CB964701) and the National Natural Science Foundation of China (31272311 to M.S.Z.).
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Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocel. 2014.05.002.
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Mislin H. On a spontaneous extrasystole in the pacemaker system of tunicate hearts (Ciona intestinalis L.). Experientia 1964;20:227–8. Mislin H. Active contractility of the lymphangion and coordination of lymphangion chains. Experientia 1976;32:820–2. Modi S, Stanton AW, Mortimer PS, Levick JR. Clinical assessment of human lymph flow using removal rate constants of interstitial macromolecules: a critical review of lymphoscintigraphy. Lymph Res Biol 2007;5:183–202. Murthy KS. Signaling for contraction and relaxation in smooth muscle of the gut. Annu Rev Physiol 2006;68:345–74. Muthuchamy M, Gashev A, Boswell N, Dawson N, Zawieja D. Molecular and functional analyses of the contractile apparatus in lymphatic muscle. FASEB J 2003;17:920–2. Muthuchamy M, Zawieja D. Molecular regulation of lymphatic contractility. Ann NY Acad Sci 2008;1131:89–99. Nepiyushchikh ZV, Chakraborty S, Wang W, Davis MJ, Zawieja DC, Muthuchamy M. Differential effects of myosin light chain kinase inhibition on contractility, force development and myosin light chain 20 phosphorylation of rat cervical and thoracic duct lymphatics. J Physiol (Lond) 2011;589:5415–29. Saul ME, Thomas PA, Dosen PJ, Isbister GK, O’Leary MA, Whyte IM, et al. A pharmacological approach to first aid treatment for snakebite. Nat Med 2011;17:809–11. Scallan JP, Wolpers JH, Davis MJ. Constriction of isolated collecting lymphatic vessels in response to acute increases in downstream pressure. J Physiol (Lond) 2013;591:443–59. Simon MS, Cody RL. Cellulitis after axillary lymph node dissection for carcinoma of the breast. Am J Med 1992;93:543–8. Sjoberg T, Steen S. Contractile properties of lymphatics from the human lower leg. Lymphology 1991;24:16–21. Smith RO. Lymphatic contractility: a possible intrinsic mechanism of lymphatic vessels for the transport of lymph. J Exp Med 1949;90:497–509. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 1994;372:231–6. Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: Modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 2003;83:1325–58. Sward K, Mita M, Wilson DP, Deng JT, Susnjar M, Walsh MP. The role of RhoA and Rho-associated kinase in vascular smooth muscle contraction. Curr Hypertens Rep 2003;5:66–72. Wang W, Nepiyushchikh Z, Zawieja DC, Chakraborty S, Zawieja SD, Gashev AA, et al. Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity. Am J Physiol Heart Circ Physiol 2009;297: H726–34. Wu TF, MacNaughton WK, von der Weid PY. Lymphatic vessel contractile activity and intestinal inflammation. Mem Inst Oswaldo Cruz 2005;100(Suppl. 1):107–10. Yu Y, Lv N, Lu Z, Zheng YY, Zhang WC, Chen C, et al. Deletion of myosin light chain kinase in endothelial cells has a minor effect on the lipopolysaccharideinduced increase in microvascular endothelium permeability in mice. FEBS J 2012;279:1485–94. Zawieja D. Lymphatic biology and the microcirculation: past, present and future. Microcirculation 2005;12:141–50. Zawieja DC. Lymphatic microcirculation. Microcirculation 1996;3:241–3. Zawieja DC. Contractile physiology of lymphatics. Lymph Res Biol 2009;7:87–96. Zhang WC, Peng YJ, Zhang GS, He WQ, Qiao YN, Dong YY, et al. Myosin light chain kinase is necessary for tonic airway smooth muscle contraction. J Biol Chem 2010;285:5522–31. Zhanna Y, Ester H, Lola W, Offer G, Shimon S, Shapira MY. Multidonor bone marrow transplantation improves donor engraftment and increases the graft versus tumor effect while decreasing graft-versus-host disease. Transpl Int 2011;24:194–200.
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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Molecular and cellular basis of the regulation of lymphatic contractility and lymphatic absorption
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Yu Wang a,c,1 , Wei Zhao b,1 , Lin Zhang a , Yong-Na Zhao a , Fei Li a , Zhen Zhang a , Yun-Dong Dai a , Wei-Feng Li a , Yan-Ning Qiao b , Cai-Ping Chen b , Ji-Min Gao a,∗∗ , Min-Sheng Zhu b,∗ a
Zhejiang Provincial Key Laboratory for Technology and Application of Model Organisms, School of Life Sciences, Wenzhou Medical College, Wenzhou, China Model Animal Research Center and MOE Key Laboratory of Animal Models of Disease, Nanjing University, Nanjing, China c Jinhua Municipal Central Hospital, Jinhua, Zhejiang, China b
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Article history: Received 13 November 2013 Received in revised form 22 April 2014 Accepted 5 May 2014 Available online xxx
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Keywords: Lymphatic vessel Myosin light chain kinase Smooth muscle Contractility, Absorption
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1. Introduction
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Lymphatic absorption is a highly regulated process driven by both an extrinsic mechanism (external force) and an intrinsic mechanism (lymphatic vessel contractility). The lymphatic muscle is a specialized smooth muscle with unique mechanical properties. To understand the molecular mechanism and relative contribution of smooth muscle contraction in lymphatic absorption, we analyzed mice with a smooth muscle-specific deletion of Mylk, a critical gene for smooth muscle contraction. Interestingly, the knockout mice were significantly resistant to anesthesia reagents. Upon injection in the feet with FITC-dextran, the mutant mice displayed a 2-fold delay of the absorption peak in the peripheral circulation. Examining the ear lymphatic vessels of the mutant mice revealed a reduction in the amount of fluid in the lumens of the lymphangions, suggesting an impairment of lymph formation. The Mylk-deficient lymphatic muscle exhibited a significant reduction of peristalsis and of myosin light chain phosphorylation in response to depolarization. We thus concluded that MLCK and myosin light chain phosphorylation are required for lymphatic vessel contraction. Lymphatic contractility is not an exclusive requirement for lymphatic absorption, and external force appears to be necessary for absorption. © 2014 Published by Elsevier Ltd.
Transportation of fluid, macromolecules, and immune cells via the lymphatic vascular system is fundamental for homeostasis in the body (Zawieja, 2005). Impairment of this process occurs during various pathological alterations, such as aging (Gasheva et al., 2007), inflammatory bowel disease (Wu et al., 2005) and other lymph-associated diseases (Lucci et al., 2007; Modi et al., 2007; Simon and Cody, 1992). This lymphatic vascular system is composed of a network of lymphatic vessels connecting the lymph nodes, the spleen and Peyer’s patches, which are found in the small
∗ Corresponding author at: Model Animal Research Center and MOE Key Laboratory of Animal Models of Disease, Nanjing University, 12 Xue-Fu Road, Pukou, Nanjing 210061, China. Tel.: +86 025 58641529. ∗ ∗ Corresponding author at: Zhejiang Provincial Key Laboratory for Technology and Application of Model Organisms, School of Life Sciences, Wenzhou Medical College, Wenzhou 325035, China. Tel.: +86 0577 86689779. E-mail addresses: [email protected] (J.-M. Gao), [email protected] (M.-S. Zhu). 1 These authors contributed equally to this work.
intestine and serve as the base of lymph fluid transportation (Davis et al., 2011; Mislin, 1964, 1976). Structurally, the lymphatic system contains initial and collecting lymphatic vessels. The former possess no muscle cells and transfers lymph liquid into collecting lymphatic vessels; the latter is composed of lymphangions with valves and muscle cells lining the vessel wall and driving unidirectional flow (Benoit et al., 1989). Current knowledge suggests that lymph vessels serve as passive thin-walled tubes that drain interstitial fluid back to the venous side of the blood circulation, and the unidirectional propulsion of lymph is mediated by one-way valves and intermittent external pressure provided by pulsating arteries, intestinal peristalsis, skeletal muscle contraction or respiratory movements (Aukland, 2005). However, accumulating evidence suggests that active pumping by lymphatic contraction is also important for lymph propulsion (Aukland, 2005), but the roles and relative importance of these passive and active mechanisms underlying lymphatic regulation remain unclear. Lymphatic muscle is a highly specialized type of smooth muscle that exhibits important differences from typical vascular or gastroenterological smooth muscle cells (Benoit et al., 1989; Muthuchamy et al., 2003; Zawieja, 1996). Interestingly, these
http://dx.doi.org/10.1016/j.biocel.2014.05.002 1357-2725/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Wang Y, et al. Molecular and cellular basis of the regulation of lymphatic contractility and lymphatic absorption. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.05.002
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smooth muscle cells share many of contractile proteins with cardiac and skeletal muscles, thereby displaying phenotypic characteristics of cardiac and skeletal myocytes (Muthuchamy et al., 2003). One question is whether lymphatic muscle shares a similar regulatory mechanism with other smooth muscle cells. Although data from the application of non-specific pharmaceutical inhibitors suggests the involvement of smooth muscle signaling in lymphatic contraction (Nepiyushchikh et al., 2011; Wang et al., 2009; Zhanna et al., 2011), little is known about the regulation of this signaling from genetic evidence of lymphatic muscle contractility (Muthuchamy and Zawieja, 2008; Zawieja, 2005, 2009). Smooth muscle contraction is evoked by calcium signaling through depolarization and G protein-coupled receptor (GPCR) agonists that activate myosin light chain kinase (MLCK) through its binding to calmodulin (He et al., 2008, 2011a,b; Kamm and Stull, 2001; Somlyo and Somlyo, 2003). The activated kinase phosphorylates the regulatory light chain (RLC), resulting in the activation of actomyosin Mg-ATPase and cross-bridge cycling of force development in smooth muscle. RLC is dephosphorylated by myosin light chain phosphatase (MLCP), thereby initiating relaxation by returning myosin to an inhibited state. The relative activities of MLCK and MLCP are thought to be a primary determinant for the extent of RLC phosphorylation and the production of force (Kamm and Stull, 2001; Somlyo and Somlyo, 1994, 2003). However, other Ca2+ -independent kinases, such as integrin-linked kinase, RhoAassociated kinase, and zipper-interacting protein kinase, are also able to phosphorylate RLC (Ganitkevich et al., 2002; Ihara and MacDonald, 2007; Murthy, 2006; Sward et al., 2003), implying a calcium-independent mechanism involving smooth muscle contractility. Our previous reports show that MLCK is the primary kinase responsible for myosin light chain phosphorylation and is required for the force development of various smooth muscle tissues in the gut, airway, bladder and blood vascular system, etc. (He et al., 2008, 2011a,b; Zhang et al., 2010). The effects of pharmaceutical inhibitors of MLCK suggest a role for MLCK in lymphatic contractility, but there is a lack of loss-of-function evidence. We analyzed the lymphatic function of mice with a smooth musclespecific deletion of the Mylk gene.
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Mylkflox/flox mice (He et al., 2008) and SMA-Cre transgenic mice were maintained at the animal center of the Model Animal Research Center of Nanjing University. MLCKSMKO mice (Mylkflox/flox ; SMACre) were produced by crossing Mylkflox/flox mice with SMA-Cre transgenic mice. The genotyping strategy used is described in our previous report (He et al., 2011a,b). All animal procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee of the Model Animal Research Center of Nanjing University (Nanjing, China).
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2.2. Reagents
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FITC-dextran and anti-mouse MLCK antibody (K36) were purchased from Sigma. Human Prox-1 (Prospero-related homeobox 1) polyclonal Ab was purchased from R&D Systems (Cat. AF2727). Pecam-1 (rat anti-mouse CD31 ) was purchased from BD (Cat. 553370). Cy5-AffiniPure Donkey Anti-Goat IgG was purchased from Jacison (Cat. 705175147). Alexa Fluor® 594 Donkey Anti-Rat IgG (H+L) was purchased from Invitrogen (A21209). Anti-mouse phosphor-myosin light chain 2 (Ser19) was purchased from Cell Signaling Technology (#3675). Other chemicals and protein reagents were purchased from Sigma or MuCyte.
2.3. Western blot analysis Measurements of MLCK, RLC and other proteins were performed according to our previous protocol (He et al., 2008). Briefly, the mesentery including intestine were dissected from mice and maintained in H-T buffer; the lymphatic vessels around the mesenteric arteries were cleaned of adipose and connective tissue; the tissue was collected and frozen quickly with 10% trichloroacetic acid and 10 mM dithiothreitol in acetone precooled to slush at −80 ◦ C. After thorough homogenization, the sample pellet was centrifuged at 3000 × g twice for 2 min at 4 ◦ C, and the supernatant was removed. The sediment was washed three times with ether and dried to remove the residual ether. The dried protein powder was carefully and completely dissolved in 8 M urea. Equal amounts of protein were subjected to SDS-PAGE followed by protein transfer to a nitrocellulose membrane. The membrane was then probed with a primary antibody to MLCK (K36; Sigma) or phosphorylated RLC (Cell Signaling Technology) and then with corresponding secondary antibodies. The membrane was incubated in Super Signal West Pico Chemiluminescent Substrate (Thermo) before exposure to film. 2.4. Immunofluorescence For whole-mount staining, mice ears were dissected and removed the hair by using Hair removal cream. The ears were fixed in 4% paraformaldehyde at 4 ◦ C overnight, splitted and removed intermediated cartilage under microscope, and then attached to Sylgard plates with insect pins. The ears were blocked with 3% milk in 0.3% PBS (0.3% Triton X-100 in PBS) for 4 time at room temperature, and incubated with polyclonal antibodies against Prox-1 and Pecam-1 at 4 ◦ C overnight. Alexa 594 and CY5 conjugated secondary antibody were used for visualization. The samples were then mounted with Olympus FluoView 1000 and examined under an Olympus BX51 confocal microscope. 2.5. Measurement of the transport in the lymphatic vessel MLCKSMKO mice and their littermates were anesthetized with avertin (250 mg/kg i.p. injection), and then subcutaneously injected with 2.5 l of FITC-dextran (25 mg/ml) in two feet. Approximately 500 l of blood from the eyeballs was placed in tubes containing 10 l of 0.2 M EDTA at different time points after injection (1 h, 2 h, 3 h, 4 h), and this was centrifuged at 3000 × g for 10 min at room temperature. The resultant supernatants were collected and stored at −20 ◦ C in the dark. The FITC fluorescence was measured with a Synergy2 Multi-Mode Microplate Reader, with excitation at between 485 and 528 nm (BIO-TEK, Inc.). 2.6. Measurement of the lymphatics rhythmical movement frequency MLCKSMKO mice and their littermates were anesthetized with avertin (230 mg/kg i.p. injection), and then injected with 3% Evan’s Blue subcutaneously into two feet’s pads so as to visualize lymphatic vessel clearly. 15 min after injection, both of the left and right inguinal lymphatic vessels with blue color were collected and subjected to peristalsis measurement. The vessels with removal of adipose and connective tissues were incubated with HEPES-Tyrode (H-T) buffer (137.0 mmol/L NaCl, 2.7 mmol/L KCl, 1.0 mmol/L MgCl2 , 1.8 mmol/L CaCl2 , 10.0 mmol/L HEPES, and 5.6 mmol/L glucose, pH 7.4), and the peristalsis could be observed visually under a microscope within a few minutes. We started counting when peristalsis occurred and the frequency of peristalsis was calculated with a modified method reported previously (Aukland, 2005).
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Q4 Fig. 1. Ablated expression of MLCK in lymphatic vessels. Mesenteric lymphatic vessels were isolated from P15-18 MLCKSMKO (KO) and control (CTR) mice and evaluated by Western blotting and immunofluorescence analysis. (A) Western blot of MLCK protein with a primary monoclonal antibody (K36) against MLCK. -Actin was used as the loading control. (B) Quantitation of MLCK protein (n = 3). **p < 0.01. (C) The pattern of collecting lymphatic vessels in the ear skin with Pecam-1 (red) and Prox-1 (white). Scale bars = 200 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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To abate MLCK expression in lymphatic smooth muscle, we crossed Mylkflox/flox mice with SMA-Cre mice (He et al., 2008). The resultant Mylkflox/flox ; SMA-Cre (MLCKSMKO ) and Mylkflox/+ ; SMA-Cre littermates were used as knockout (KO) and control (CTR) mice, respectively. In KO mice, specific deletion of MLCK in almost all
smooth muscle tissues was expected, while in CTR mice, hemizygosity for MLCK produced no apparent phenotypic effects [19]. Q2 Western blot analysis showed a significant reduction of MLCK expression in mutant mesenteric lymphatic vessels (Fig. 1A and B). However, approximately 34.26 ± 12.93% of MLCK protein could be detected in the KO tissue. This residual expression of MLCK protein detected by Western blot may be due to MLCK expression in the lymphatic endothelium. Interestingly, we could not detect the long isoform of MLCK in the endothelium-containing lymphatic
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Fig. 2. Macrophenotypes of MLCKSMKO mice. (A) MLCKSMKO (KO) and control (CTR) mice (P24) were euthanized and their abdomens were exposed. (B) Growth curves of body weight (n = 5). (C) Histological examination of the ileum and jejunum. Scale bars for C: upper row, 20 m; lower row, 4 m. *p < 0.05; **p < 0.01.
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vessels (Garcia, 1995). This result indicates that the endothelial cells in lymphatic vessels only express short MLCK, unlike the cultured endothelial cells expressing long MLCK in vitro. This observation is consistent with the expression pattern of MLCK in the endothelium of vascular vessels reported previously (Yu et al., 2012). We also examined the patterns of collecting lymphatic vessels in knockout mice ears by staining Prox-1 and Pecam-1 with a whole-mount immunofluorescence assay. The mutant ears had a comparable morphology of lymphatic network with that of control ears (Fig. 1C). Thus, deletion of MLCK in smooth muscle cells did not affect development and structure of lymphatic vessels.
control (141.25 ± 23.13 vs. 81.25 ± 11.61 s, p < 0.01) (Fig. 3). However, despite the delay in anesthetization, 100% of the knockout mice could be anesthetized in a similar manner to CTR mice. 3.3. Deletion of MLCK caused impaired absorption by the lymphatic system
3.2. MLCK-deficient mice displayed resistance to an anesthesia reagent
The MLCK deletion-induced resistance to anesthesia led us to investigate whether the anesthesia delay was caused by impaired absorption by the lymphatic system. We subcutaneously injected 5 l FITC-dextran into the feet of mice and then collected blood from their eyeballs at different time points. The fluorescent substance in the serum was quantified as shown in Fig. 4. One hour after injection, the fluorescence intensity of the serum samples
The knockout mice appeared to have significantly reduced gastrointestinal motility, low blood pressure and abolished responses to KCl depolarization and agonists, which is consistent with our previous reports (Fig. 2A) (He et al., 2008, 2011a,b; Zhang et al., 2010). The growth of the mutant mice was retarded from the 18th day after birth (Fig. 2B). The mutant small intestine exhibited edema with an enlarged lumen (Fig. 2A and C). Histological examination also showed edema around the smooth muscle tissue (Fig. 2C). To our surprise, it was difficult to anesthetize the knockout mice with a typical dose of an anesthesia reagent. To quantify this effect, we measured the time required to be anesthetize by avertin (230 mg/kg), a widely used anesthesia reagent for mice. Upon intraperitoneal injection with a typical dose of avertin, the control mice went to anesthetized status (laid down) for approximately 80 s. However, with the same dose, the knockout mice required approximately 140 s to achieve the same status, which was significantly (approximately 2-fold) longer than the
Fig. 3. MLCK-deficient mice displayed resistance to anesthesia. MLCKSMKO mice (KO) and control mice (CTR) (P18) received intraperitoneal injection of 2.5% avertin at a dose of 230 mg/kg body weight. The time required for complete anesthetization was recorded, and the average values were analyzed using a paired t-test. n = 3, *p < 0.05.
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Fig. 4. Delayed absorption of dextran in MLCK-deficient mice. MLCKSMKO (KO) and control (CTR) mice (P18) were injected individually with 5 l of FITC-dextran in the foot. Peripheral blood samples were collected for the measurement of fluorescence with a Synergy2 Multi-Mode Microplate Reader. The excitation wavelengths were between 485 and 528 nm. The y-axis indicates the relative fluorescence intensities. Each time point reflected data from three mice. *p < 0.05. KO: 1 h, n = 7; 2 h, n = 6; 3 h, n = 6; 4 h, n = 10; CTR: 1 h, n = 9; 2 h, n = 16; 3 h, n = 13; 4 h, n = 14.
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both from control and knockout mice were comparable. However, 2 h after injection, the fluorescence intensity in control mice was strikingly increased, while only a slight increase was observed in the knockout mice. Three hours after injection, the fluorescence intensity of the control mice was reduced, while that of mutant mice increased rapidly (Fig. 4). The absorption peak of the knockout mice appeared at the fourth hour after injection, which is approximately 2-fold longer than the control. This absorption delay was consistent with the anesthetization delay mentioned above. We conclude that the delayed distribution effect of MLCK deletion is caused by impaired lymphatic absorption. The movement of lymph, an essential process of lymphatic absorption, may be regulated by extrinsic compression from external forces and intrinsic compression from lymphatic contractility. To assess the contribution of smooth muscle to this process, we examined the movement of lymph in the ears and tail, where there is less tissue pressure (Aarli and Aukland, 1991). After injection of FITC-dextran in the ear, the collecting lymphatic was visualized under a fluorescence stereomicroscope. The lymphangions in the lymphatic vessels of control mice were even with clear valves. However, the mutant lymphangions appeared to be discontinuous (Fig. 5A and B). Magnification showed less liquid in the mutant lymphangions compared to the control, but the sizes of the valves were comparable. This morphological alteration indicated that mutant lymphangions had a weak pumping activity and a reduced ability to collect lymph fluid. Because the process of lymph absorption in the mouse ear is too fast for the velocity to be quantified, we injected FITC-dextran at the tail and measured the distance moved at different time points. 10 min after injection, the fluorescent substance in the tail of control mice had moved approximately 16.83 mm. However, the distance moved in the mutant tail was approximately 4.94 mm, and the average value was significantly shorter than control (p < 0.01) (Fig. 6A and B). These observations indicated a reduced capability of lymph absorption after MLCK deletion.
Fig. 5. Visualization of lymph flow in the mouse ear. MLCKSMKO (KO) and control (CTR) mice were injected with 1 l of FITC-dextran solution in the ear. (A) The visualized lymphatic vessels were photographed at different time points. The magnification shows the typical morphology of lymphangions, and the mutant vessels were found to exhibit many discontinuous lymphangions. (B) The percentage of discontinuous lymphangions was quantified. The average percentages are expressed as the mean value ± SEM (n = 5). **p < 0.01; ***p < 0.001.
3.4. Myosin light chain phosphorylation of lymphatic smooth muscle was inhibited by MLCK deletion MLCK mediates RLC phosphorylation, and RLC phosphorylation is required for smooth muscle contraction. The control inguinal lymphatic vessel displayed a peristalsis under resting conditions at rate of 7.7 ± 1.1 per minute, while of 1.5 ± 0.5 per minute for the mutant vessels (Fig. 7A). We measured RLC phosphorylation in lymphatic smooth muscle in response to KCl. Due to the difficulty of collecting enough lymphatic vessels, we only measured
Fig. 6. Lymph movement in the mouse tail. MLCKSMKO (KO) and control (CTR) mice were injected with 1 l of FITC-dextran solution. (A) The visualized moving lymph flow was monitored at different time points by scanning the tails. (B) The distances moved were measured by calculating the displacement between the origin and the broad edge of the fluorescence. The displacement is expressed as the mean ± SEM, n = 3. **p < 0.001.
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Fig. 7. Lymphatic smooth muscle contraction is relied on the phosphorylation of regulatory myosin light chain in response to depolarization. Lymph vessels were isolated from control (CTR) and MLCKSMKO mice and incubated in H-T buffer respectively. (A) Measurement of the rhythmical movement frequency of inguinales profundi lymphatics whose pulsatile contraction was easy to be visualized. (B) At 0, 10 and 20 s after stimulation with KCl, mesenteric lymph vessels were sampled and subjected to Western blotting analysis. The primary antibody was anti-phosphorylated RLC antibody. -Actin was used as the protein loading control. This result represents three independent experiments. The average percentages are expressed as the mean value ± SEM, n = 3 (MLCKSMKO mice) or n = 7 (CTR mice), *p < 0.05.
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phosphorylated RLC at three time points after stimulation (0, 10 and 20 s). In the control mesenteric lymphatic vessel, basal RLC phosphorylation could be clearly detected. The amount of phosphorylated RLC increased rapidly after stimulation with KCl and then declined. In knockout mesenteric lymphatic vessels, both basal and stimulation-evoked RLC phosphorylation were significantly inhibited (Fig. 7B), which was consistent with delayed lymphatic absorption in MLCK-deficient mice. This suggests a critical role of RLC phosphorylation of MLCK in lymphatic smooth muscle contraction. However, due to technological limitations, we could not directly record the force of the lymphatic smooth muscle of such a small animal.
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A contraction of a typical smooth muscle is evoked by KCl depolarization and agonists through calcium signaling, and the resultant elevation of cytosolic calcium causes MLCK activation. RLC phosphorylation by MLCK triggers ATPase activity of myosin, thereby initiating cross-bridge movement (Kamm and Stull, 2001). Lymphatic muscle is a specialized smooth muscle composed of different contractile proteins and contractile properties (Bridenbaugh et al., 2003; Sjoberg and Steen, 1991). One question is whether MLCK is required for lymphatic contraction in the same manner as for other smooth muscles. Although the observations from MLCK inhibitors with non-specific effect imply an involvement of MLCK in lymphatic contraction, the in vivo role of MLCK in lymphatic muscle is basically not elucidated particularly through genetic analysis. We found that deletion of MLCK abolished pulsatile contraction as
well as contractile responses to depolarization and agonists. Our loss-of-function evidence suggests that the contractility of lymphatic vessels is primarily attributed to the contraction of smooth muscle rather than to that of other muscle types. Signaling integration between MLCK and its RLC phosphorylation in the lymphatic smooth muscle cells is required for lymphatic contraction in the same manner as in other smooth muscle cells. Because MLCK is a strictly calcium/calmodulin-dependent myosin light chain kinase, we also emphasize the importance of a calcium-dependent rather than calcium-independent mechanism underlying lymphatic contraction. Lymphatic absorption includes lymph formation, draining, and lymph flow, which is a highly coordinated process. It is widely accepted that the lymph fluid is formed by entrance of interstitial fluid into collecting lymphatic vessel after a decrease in the luminal pressure of the lymphatic vessel and an increased external compression from movements or intermittent pressure (Gashev, 2008; Scallan et al., 2013; Smith, 1949). Lymph formation seems to be primarily dependent on external force through a passive mechanism (Aukland, 2005; Granger et al., 1984; Guyton et al., 1966). During this process, the relative importance of the suction mediated by muscular lymphatic contractility remains unclear (Aukland, 2005). We here found that inhibition of lymphatic contraction by MLCK deletion resulted in incomplete filling of lymph fluid in lymphangions, indicating a significant reduction of lymph formation. We thus suggest that the suction of muscular lymphatics is necessary for lymph formation in addition for lymph movement (Aukland, 2005). The intestinal edema phenotype observed in MLCK knockout mice also supports this proposal. A mass of evidence suggests that lymphatic absorption is driven by extrinsic (or passive) and intrinsic (or active) mechanisms. However, their relative contributions to lymphatic absorption remain unclear. Lymphatic absorption from the peritoneal cavity and feet to circulation is typical pathways reflecting such active and passive absorption mechanisms. Upon MLCK deletion, the absorption peaks in the peripheral circulation for anesthesia reagents absorbed from the peritoneal cavity and for FITC-dextran absorbed from the feet were approximately 2-fold longer compared to those in the control mice. However, the absorption process was not completely abolished. Therefore, we conclude that as a primary active mechanism, lymphatic contractility is not an exclusive requirement for lymphatic absorption. The passive mechanism seems to be necessary for the absorption process. Due to the delay of lymphatic absorption caused by MLCK deletion, effective inhibition of lymphatic smooth muscle contractility is expected to slow down absorption at least two folds. This is particularly useful for the development of a novel therapeutic strategy that would slow the distribution of a given toxin, allowing more time to elapse before receiving medical care. A successful example is the application of a nitric oxide donor, which causes a 3-fold reduction in the foot-to-groin lymph transit time of snake toxin (Saul et al., 2011). It is worth noting that inhibition of smooth muscle contraction only delays and does not abolish the absorption, so such a strategy can only be used as a first-aid medical approach. Conflict of interests The authors declare no conflict of interests regarding the publication of this article. Acknowledgements We thank Man Chu, Zhi Shang and Xu-Dong Cao of Labo- Q3 ratory of Vascular and Cancer Biology, Cyrus Tang Hematology Center, Soochow University, Suzhou, China. They provided the methods of immunofluorescence and tail injection. This study was
Please cite this article in press as: Wang Y, et al. Molecular and cellular basis of the regulation of lymphatic contractility and lymphatic absorption. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.05.002
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supported by National Key Scientific Research Program of China (Grant 2014CB964701) and the National Natural Science Foundation of China (31272311 to M.S.Z.).
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