Increased nitric oxide production in lymphatic ...

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Gut Online First, published on January 20, 2012 as 10.1136/gutjnl-2011-300703 Cirrhosis

ORIGINAL ARTICLE

Increased nitric oxide production in lymphatic endothelial cells causes impairment of lymphatic drainage in cirrhotic rats Jordi Ribera,1 Montse Pauta,1 Pedro Melgar-Lesmes,1 So`nia Tugues,1,2 Guillermo Fernańdez-Varo,1 Kara F Held,3 Guadalupe Soria,4 Rau´l Tudela,4,5 Anna M Planas,4,6 Carlos Fernańdez-Hernando,7 Vicente Arroyo,8 Wladimiro Jimeńez,1,9 Manuel Morales-Ruiz1 < Additional materials are

published online only. To view these files please visit the journal online (http://gut.bmj. com/content/early/recent). For numbered affiliations see end of article. Correspondence to Dr Manuel Morales-Ruiz, Department of Biochemistry and Molecular Genetics, Hospital Clinic Universitari, Villarroel 170, Barcelona 08036, Spain; [email protected] Revised 3 December 2011 Accepted 21 December 2011

ABSTRACT Background and aim The lymphatic network plays a major role in maintaining tissue fluid homoeostasis. Therefore several pathological conditions associated with oedema formation result in deficient lymphatic function. However, the role of the lymphatic system in the pathogenesis of ascites and oedema formation in cirrhosis has not been fully clarified. The aim of this study was to investigate whether the inability of the lymphatic system to drain tissue exudate contributes to the oedema observed in cirrhosis. Methods Cirrhosis was induced in rats by CCl4 inhalation. Lymphatic drainage was evaluated using fluorescent lymphangiography. Expression of endothelial nitric oxide synthase (eNOS) was measured in primary lymphatic endothelial cells (LyECs). Inhibition of eNOS activity in cirrhotic rats with ascites (CH) was carried out by L-NG-methyl-L-arginine (L-NMMA) treatment (0.5 mg/kg/day). Results The (CH) rats had impaired lymphatic drainage in the splanchnic and peripheral regions compared with the control (CT) rats. LyECs isolated from the CH rats showed a significant increase in eNOS and nitric oxide (NO) production. In addition, the lymphatic vessels of the CH rats showed a significant reduction in smooth muscle cell (SMC) coverage compared with the CT rats. CH rats treated with L-NMMA for 7 days showed a significant improvement in lymphatic drainage and a significant reduction in ascites volume, which were associated with increased plasma volume. This beneficial effect of L-NMMA inhibition was also associated with a significant increase in lymphatic SMC coverage. Conclusions The upregulation of eNOS in the LyECs of CH rats causes long-term lymphatic remodelling, which is characterised by a loss of SMC lymphatic coverage. The amelioration of this lymphatic abnormality by chronic eNOS inhibition results in improved lymphatic drainage and reduced ascites.

The prognostic expectations for cirrhotic patients are poor because of the severe complications these patients experience. One of the most common complications is ascites, which is associated with increased morbidity and mortality.1 Some of the mechanisms that contribute to the pathogenesis of ascites include increased intravascular hydrostatic pressure, enlarged splanchnic arterial vasculature

Significance of this study What is already known about this subject? < Under pathological conditions, four derange-

ments contribute to the formation of oedema: (1) an increase in intravascular hydrostatic pressure; (2) a decrease in plasma oncotic pressure; (3) renal retention of salt and water; and (4) impairment of lymphatic drainage. < There is abundant literature establishing the presence of most of these mechanisms in cirrhosis and their role in the consequent oedema and ascites. However, few studies have investigated whether there is impaired lymphatic functionality in cirrhosis. < Lymphatic transport is promoted by extrinsic and intrinsic forces. The intrinsic forces are derived from the contractility of the smooth muscle cells (SMCs) of the collecting lymphatic vessels. Regarding the molecular mechanisms governing the intrinsic contractility of the collecting lymphatic vessels, there is evidence that the lymphatic endothelial cells control lymph flow by regulating the contractility of SMCs via the production of nitric oxide (NO). < Increased endothelial nitric oxide synthase (eNOS) activity in the blood vessels of cirrhotic rats, along with its effect on vascular remodelling, has already been demonstrated. However, the changes in lymphatic NO production and its effect on lymphatic remodelling have not previously been investigated in cirrhosis.

and decreased plasma oncotic pressure.2e4 These changes result in excessive fluid filtration, which is exacerbated by the concomitant transformation of the hepatic microvasculature into a capillarised and defenestrated endothelium.5 6 In this scenario, the increase in intrahepatic pressure is transmitted upstream by the blood to the splanchnic organs, resulting in further oedema.7 As the liver disease progresses, the antidiuretic and antinatriuretic properties of certain neuroendocrine systems, which are activated secondary to the arterial vasodilation, worsen the oedematous condition and

Ribera J, Pauta M, Melgar-Lesmes P, et their al. Gut (2012). doi:10.1136/gutjnl-2011-300703 1 of 8 Copyright Article author (or employer) 2012. Produced by BMJ Publishing Group Ltd (& BSG) under licence.


Cirrhosis

Significance of this study What are the new findings? < Cirrhosis is accompanied by impairment of lymphatic drainage

of the splanchnic and peripheral regions. < The cirrhotic lymphatic endothelial cells overexpress eNOS

and overproduce NO. overproduction causes lymphatic remodelling. The remodelling process is characterised by a decrease in coverage of the lymphatic vessels by pericytes or SMCs. < Long-term inhibition of eNOS activity with L-NMMA corrected lymphatic abnormalities, resulting in an increase in coverage of the lymphatic vessels by pericytes, an improvement in lymphatic drainage, and a significant decrease in ascites volume. < NO

How might it impact on clinical practice in the foreseeable future? < Our observations have implications for the pathophysiology of ascites and oedema formation in cirrhosis. We demonstrate that cirrhosis is accompanied by impairment of the lymphatic drainage. In addition, we show that NO overproduction causes lymphatic remodelling. Both lymphatic abnormalities are reversed by L-NMMA treatment, resulting in improved lymphatic drainage from the peritoneal cavity and reduced ascites. Therefore eNOS inhibition is a new target for correcting lymphatic dysfunction in cirrhosis.

also investigated whether NO overproduction by the LyECs may be responsible for lymphatic impairment in cirrhosis.

MATERIALS AND METHODS The experimental cirrhosis model The study was performed in cirrhotic (CH) and control (CT) male adult Wistar rats (CharleseRiver, Saint Aubin les Elseuf, France), following the guidelines of the investigation and ethics committees of the Hospital Clinic. Cirrhosis was induced by inhalation of CCl4, as described previously.13

Isolation of LyECs Freshly isolated primary LyECs were obtained from the mesentery of the CT and CH rats. Briefly, the atrium was cannulated and the vascular system perfused with normal saline solution. Mesenteric lymphatic tissue mucosa was harvested, placed on 35 mm plates containing ice-cold phosphate-buffered saline, and cut into small (1 mm) fragments. The fragments were incubated in 0.25% collagenase A (Roche Diagnostics, Basel, Switzerland) at 378C. The suspension was passed through 100 mm nylon mesh and centrifuged at 1800 rpm for 4 min at 48C. The cell pellet was resuspended in Hank’s balanced salt solution. The LyECs were isolated using rabbit antibody to rat podoplanin (Sigma Chemical, St Louis, Missouri, USA) in a 1:100 dilution as the primary antibody and microbeads coupled with a secondary goat antirabbit antibody (MACS system, Miltenyi Biotec, BergischGladbach, Germany). The cells were grown in Dulbecco’s modified Eagle medium that was supplemented with 20% fetal calf serum, 50 U/ml penicillin and 50 mg/ml streptomycin.

Lymphangiography 8

cause ascites. All of these mechanisms contribute to the onset and maintenance of oedema and ascites. However, the role of the lymphatic system in the pathogenesis of ascites formation has not been fully clarified. The lymphatic capillaries are formed by a thin layer of nonfenestrated lymphatic endothelial cells (LyECs) without basement membranes that form closed-ended structures. Perivascular smooth muscle cells (SMCs) and pericytes are absent in lymphatic capillaries but are present in the collecting lymphatic vessels that conduct lymph within the lymphatic system.9 In the absence of a muscular pumping organ, such as the heart in the cardiovascular system, lymphatic transport is promoted by extrinsic and intrinsic forces. The intrinsic forces are derived from the contractility of the SMCs of the collecting lymphatic vessels, which has been proposed as one of the major driving forces of lymph circulation.10 Regarding the molecular mechanisms governing the intrinsic contractility of the collecting lymphatic vessels, there is evidence that the LyECs control lymph flow by regulating the contractility of SMCs via the production of nitric oxide (NO).11 In cirrhotic patients, the lymphatic system helps to prevent ascites accumulation by reabsorbing excess fluid in the liver and splanchnic regions. Lymph flow is increased as a consequence,12 which stimulates hepatic lymphangiogenesis.13 However, this compensatory mechanism is not sufficient to prevent ascites formation in decompensated cirrhotic patients. This lymphatic deficiency supports the hypothesis that advanced liver disease is associated with impaired lymphatic function. Therefore our study aimed to investigate whether the inability of the lymphatic system to drain tissue exudate contributes to the oedema observed in cirrhosis. In addition, increased eNOS activity in the blood vessels of cirrhotic rats, along with its effect on vascular remodelling, has already been demonstrated.14 15 Therefore we 2 of 8

To evaluate lymphatic drainage in peripheral areas, 0.3 ml fluorescein isothiocyanate (FITC)-Dextran 2000 kDa or 20 kDa (Sigma Chemical) was subcutaneously injected into the ears, tails and footpads of the CT and CH rats. Immediately after the injection, fluorescent images of the subcutaneous lymphatic drainage were visualised using a fluorescence stereomicroscope system (Leica Microsystems, Heerbrugg, Switzerland). For the splanchnic areas, a long-chain fatty acid (BODIPY FL C16; Invitrogen, San Diego, California, USA) was administered intragastrically. After 2 h, fluorescent images of the mesenteric lymphatic drainage were obtained.

L-NG-methyl-L-arginine (L-NMMA) and midodrine treatments The CH rats were treated intragastrically with L-NMMA (Calbiochem, Gibbstown, New Jersey, USA) at a dose of 0.5 mg/ kg/day for 1 week, or midodrine (Sigma Chemical) at a dose of 5 mg/kg/day for 1 week. The untreated CH rats were given the dosing vehicle intragastrically as a treatment control.

Immunohistochemistry, immunocytofluorescence and whole-mount immunofluorescence staining For immunohistochemistry, mesentery samples were fixed in 10% buffered formaldehyde solution and embedded in paraffin. Immunolabelling was performed using a rabbit antibody to rat podoplanin (Sigma Chemical). Immunolabelled cells were detected with the Dako LSAB2 System, HRP (Dako, Glostrup, Denmark). Immunoreactivity was visualised using a light microscope (Nikon Eclipse E600, Kawasaki, Kanagawa, Japan). For the immunocytofluorescence analyses, LyECs were fixed with 4% paraformaldehyde. The cells were incubated with one of the following primary antibodies for 1 h: rabbit anti-rat podoplanin, mouse anti-rat CD31 (BD Pharmigen, Franklin Lakes, New Jersey, USA) or goat anti-rat CD34 (LifeSpan Biosciences, Seattle, Washington, USA). The respective secondary Ribera J, Pauta M, Melgar-Lesmes P, et al. Gut (2012). doi:10.1136/gutjnl-2011-300703


Cirrhosis antibodies were Cy3-conjugated donkey anti-rabbit (Jackson ImmunoResearch, Newmarket, Suffolk, UK), Alexa Fluor 488conjugated goat anti-mouse and Alexa Fluor 488-conjugated donkey anti-goat (Molecular Probes, Invitrogen, San Diego, California, USA). Immunofluorescence was visualised with an immunofluorescence microscope system (Nikon Eclipse E600). For whole-mount immunofluorescence staining, ear tissue was fixed overnight in a solution containing 80% methanol and 20% dimethyl sulphoxide at 208C and permeabilized in Trisbuffered saline (TBS) containing 2% bovine serum albumin and 0.1% Triton for 5 h. The tissue was incubated with rabbit antibody to rat podoplanin at a 1:200 dilution overnight, washed four times with TBS, and blocked again with TBS containing 2% BSA and 0.1% Triton for 3 h. Finally, the tissue was incubated with anti-rabbit Cy3 (Jackson Immunoresearch) at a 1:300 dilution overnight and washed eight times with TBS. Immunofluorescence was visualised using an immunofluorescence microscope system (Nikon Eclipse E600).

Other measurements Mean arterial and portal pressures were measured as previously described.16 Sodium concentrations were measured using flame

photometry (IL 943; Instrumentation Laboratory, Lexington, Massachusetts, USA). Aldosterone was measured in rat plasma samples with a radioimmunoassay using a commercial kit according to the manufacturer ’s instructions (Coat-A-Count; DPC, Los Angeles, California, USA).

Statistical analysis Statistical differences were analysed using unpaired or paired Student t tests and analysis of variance models (with Tukey’s post hoc test) when appropriate. Differences were considered to be significant at a p value