Ribera J, Pauta M, Melgar-Lesmes P, Tugues S, Fernandez-Varo G, Held KF, et al. Increased nitric oxide production in lymphatic endothelial cells causes impairment of lymphatic drainage in cirrhotic rats. Gut 2013;62:138-145. (Reprinted with permission.)
The lymphatic network plays a major role in maintaining tissue fluid homoeostasis. However, the role of the lymphatic system in the pathogenesis of ascites and edema 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 edema observed in cirrhosis. 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). 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. Thus, up regulation of eNOS in the LyECs of CH rats causes long-term lymphatic remodeling, which is characterized 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 lymphatic system is a major accessory route, carrying large particulates from interstitial spaces into the blood circulation. The lymph is formed by the result of the net filtration pressure across the capillary basement membrane in the tissues as determined by the Starling forces. The rate of lymph formation, respiration, and skeletal muscle movement primarily determine the lymphatic flow rate. The endothelial cells of the lymphatic collecting duct are covered by smooth muscle cells which contract and act as intrinsic lymphatic pump, hence facilitating lymph flow. When there is an increased interstitial fluid pressure, the overlapping junctions are thrown open and the lymphatic capillaries become hyperpermeable to carry the excess fluid away from interstitium back to circulation. The interplay and homeostasis and structural similarities between the lymphatic and the circulatory systems are an area of great interest and little is known in the context of portal circulation.
Iwakiri and Groszmann described that the hyperdynamic circulation in portal hypertension is a progressive vasodilatory syndrome with nitric oxide (NO) playing a central pathognomonic role. Flow shear stress causes raised endothelial nitric oxide synthase (eNOS) activity followed by NO-induced splanchnic vasodilatation. This leads to effective arterial hypovolemia and increased filtration pressure as exemplified by Starling forces with consequent interstitial edema manifesting as ascites. In a seminal article, Dumont and Mulholland showed by thoracic duct cannulation that the rate of thoracic lymph flow was 3 to 6 times higher in cirrhosis patients compared to a normal rate of 1 mL per minute in a noncirrhosis patient. In a patient with gross ascites, after thoracic duct cannulation these workers were able to drain hemorrhagic lymphatic fluid at 6-7 mL per minute and could drain the entire ascites in 6 hours. Subsequently, they showed that the thoracic duct pressures ranged from 15-70 cm of saline (normal, 4-6 cm saline), highest in an acute variceal bleed patient in whom, interestingly, the bleed stopped once thoracic lymph drainage was established and the bleed resumed once the lymph drainage was stopped. The splenic pulp pressure (a measure of portal pressure) fell by 10 cm after lymphatic drainage. These data indicate that in portal hypertension the lymphatic system is a high-pressure system with impeded flow, which could be restored on thoracic duct cannulation.
Hence, one can hypothesize that in portal hypertension there is likely to be some sort of a pump failure or a functional outflow obstruction in the lymphatic system which contributes to ascites. Understanding and managing these situations in the lymphatic system could ameliorate development of ascites. Similar to vascular system in portal hypertension, the lymphatic system is also in a progressive vasodilatory state with resultant hyperdynamic circulation. Since a negative interstitial pressure cannot be maintained, interstitial edema and ascites develops. Lymphatic vasculature is believed to be formed by budding from the preexisting veins, with a contribution from mesenchymal progenitors, after expression of Prox-1 and action of vascular endothelial growth factor (VEGF)-C and VEGF-D on VEGFR-3 receptors.[1, 4] This suggests that there are close similarities in vascular responsiveness in blood vessels and lymphatic system. Fernandez-Varo et al. showed that increased eNOS activity led to vascular remodeling and consequent circulatory dysfunction in cirrhosis and reversal with the use of the NOS inhibitor L-NAME. A similar effect of NO on lymphatic flow in circulation by way of action on the lymphatic pump in collecting ducts has been shown.
Ribera et al., in an elegant animal study, have shown that lymphatic dysfunction in cirrhosis patients was due to increased NO production and there was improvement with use of L-NMMA, an NO synthase inhibitor. Fluorescent lymphangiography showed that there was impaired lymphatic drainage in cirrhotic rats compared to control rats, both in peripheral and splanchnic circulation. Expression of eNOS was significantly higher in lymphatic endothelial cells (LyECs) from cirrhotic rats. The effect of NOS inhibition on lymphatic drainage was studied by dividing cirrhotic rats into two groups; one group received L-NMMA (0.5 mg/kg/day) and the other received midodrine (5 mg/kg/week) for 1 week. On lymphangiography, there was improved peripheral and splanchnic lymphatic drainage with L-NMMA compared to midodrine (an oral alphamimetic agent that acts directly on the peripheral alphareceptors and works as vasoconstrictor). Although both treatments increased mean arterial pressure equally, there was an independent functional improvement in lymphatic drainage only with L-NMMA and not with midodrine. A magnetic resonance imaging (MRI) scan showed reduced ascites pre- and post-L-NMMA use (6.2 ± 1.5 mL at day 0 to 2.0 ± 0.1 mL at day 7, P < 0.05) without any change with use of midodrine. An important observation suggestive of lymphatic pump failure was that podoplanin-positive lymphatic vessels coated with smooth muscle cells were significantly less in cirrhotic rats than in the control rats (55.2 ± 6.8% versus 2.7 ± 1.1% of smooth muscle cell [SMC] coverage, P < 0.05). By using L-NMMA, the SMC coverage improved from 2.7 ± 1.1% to 16 ± 1.2% (P < 0.05). The inhibitory effect of NO on SMC proliferation was non-cGMP-mediated, as shown by lack of effect on addition of soluble guanylate cyclase inhibitor. This study clearly explains the concept of lymphatic pump failure due to reduced SMC proliferation and function in portal hypertension secondary to raised eNOS activity. The authors provided clear data that this led to impaired lymphatic drainage in cirrhotic animals with portal hypertension. These changes in the lymphatic circulatory bed could be reversed with the use of the NOS inhibitor L-NMMA, resulting in improved lymphatic drainage and regression of ascites.
In portal hypertension, there is a relative NO deficiency in the liver resulting in raised intrahepatic resistance and a contrasting situation of NO excess in systemic circulation. Various nitrovasodilators have been tried to cause a therapeutic intrahepatic vasodilation, namely, NCX-1000; nitroflurbiprofen, an NO-releasing cyclooxygenase inhibitor; atorvastatin, an high mobility group (HMG) CoA inhibitor causing inhibition of hepatic RhoA/Rho-kinase signaling and activating eNOS in cirrhotic rats. Isosorbide mononitrate in combination with nonselective beta blockers has been shown to have a greater portal pressure-reducing effect. However, isosorbide mononitrate should be used cautiously in patients with renal impairment and cirrhosis, specially those with ascites. Martin et al. showed that NOS inhibition in cirrhotic rats with ascites by L-NAME for 1 week improved renal sodium and free water excretion by improvement in hemodynamics. On the contrary, Graebe et al. showed that chronic NOS inhibition by L-NAME led to marked increase in NHE3 exchanger and Na+ K+ ATPase protein levels in proximal convoluted tubules. This leads to sodium and water retention in the kidney and can worsen ascites. In brief, the use of nitrovasodilators can worsen systemic hemodynamics and probably lymphatic drainage. It is apparently a “catch-22” situation and much more needs to be learned about the two circulatory beds. In the current study, the use of L-NMMA led to suppression of the NO production in the LySECs and improved the lymphatic drainage with reduction in ascites. It is not known whether reduction in NO in the lymphatic system by L-NMMA is concurrently occurring in the portal circulation to the same degree and does it lead to increased vascular resistance and raised portal pressure. If the data provided by the Spanish group stands the test of time in human studies, the drugs working in one area may not be suitable for the other regional bed and we would need different strategies for the two circulatory beds (Fig. 1).
Ribera et al. have resurrected the forgotten importance of lymphatic circulation in cirrhotic portal hypertension and also have rekindled the interest in NO and NO donors and inhibitors in the management of portal hypertension. They need to be complimented for having provided cellular and molecular insights into the pathophysiology of lymphatic dysfunction in portal hypertension by ascertaining a central role of NO to intrinsic lymphatic pump failure. It would be interesting to study the role of other vasodilatory molecules such as carbon monoxide and H2S in these two regional beds. The current study brings enthusiasm to the field of portal hypertension and has opened new vistas for further investigations and better therapeutic horizons.
Shiv Kumar Sarin, M.D., D.M., F.N.A., D.Sc.
Chandan Kumar, M.D.
Department of Hepatology
Institute of Liver and Biliary Sciences
New Delhi, India