TRANSACTIONS OF THE AMERICAN CLINICAL AND CLIMATOLOGICAL ASSOCIATION, VOL. 128, 2017
The Molecular Basis of Portal Hypertension DON C. ROCKEY, MD Charleston, South Carolina
Abstract Cirrhosis leads to portal hypertension and vascular abnormalities in multiple vascular beds. There is intense vasoconstriction in the liver and the kidneys, but also vasodilation in the other vascular beds, including the periphery, lungs, brain, and mesentery. The derangement in each of these beds leads to specific clinical disease. The vasoconstrictive phenotype in the liver ultimately leads to clinical portal hypertension, and is caused by an imbalance of vasoconstrictive and vasorelaxing molecules, which will be the focus of this review.
Sinusoidal blood flow The functional vascular unit of the liver is the hepatic sinusoid, made up of sinusoidal endothelial cells (SECs) and hepatic stellate cells (HSCs); both of these cells are important in the regulation of sinusoidal blood flow (1–3). Stellate cells have an anatomic orientation in the hepatic sinusoid that is reminiscent of tissue pericytes, possessing intricate cytoplasmic processes that are tightly associated with SECs (4,5). This close anatomic relationship of these two sinusoidal cell types suggests that they are linked functionally, perhaps via paracrine signaling. In virtually all forms of liver disease, intrahepatic resistance is elevated. Portal pressure is proportional to resistance and flow according to the hydraulic equivalent of Ohm’s law: ∆P = Q × R where ∆P is the change in pressure along a vessel, Q the flow in the vessel, and R the resistance to flow. Elevated intrahepatic resistance occurs early in the disease process, leading to a classic hyperdynamic circulatory mesenteric blood flow pattern. This leads to increased blood flow, and thus patients with cirrhosis typically have both increased intrahepatic resistance and increased flow through the splanchnic system, each contributing to portal hypertension. Although the level of increased Correspondence and reprint requests: Don C. Rockey, MD, Department of Internal Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 803, MSC 623, Charleston, South Carolina 29425, Tel: 843-792-2914, E-mail: [email protected]. Potential Conflicts of Interest: None disclosed.
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resistance to flow in cirrhosis varies to some degree with different types of liver disease (i.e., pre-, intra-, or post-sinusoidal resistance), most forms of liver disease lead to so-called “sinusoidal” portal hypertension in which elevated resistance is derived from the sinusoidal areas. This pathophysiology is also important clinically in that portal pressure is one of the best predictors of clinical outcome (6–8). In liver disease, portal hypertension typically occurs in parallel with fibrosis, the latter a common result of injury to the liver [and typical of injury to essentially all of the parenchymal organs and skin (9)]. It is notable that portal hypertension is usually proportional to fibrosis. This is particularly true in advanced fibrosis, where intrahepatic resistance is likely also mediated by architectural distortion of the liver and its lobules. Intrahepatic resistance is likely p articularly affected by changes in cellular and molecular interplay early in the liver injury process. Thus, intrahepatic resistance may be considered in terms of two major compartments — fixed and modulable compartments, with the fixed component related to advanced fibrosis and scarring, and the modulable component related to cells and molecules within the liver; this latter component is likely to be more active in early portal hypertension.
Molecular Mediators of Portal Hypertension Multiple vascular mediators have been implicated in the vasoconstrictive phenotype typical of intrahepatic portal hypertension. Most prominent of these appears to be endothelin-1 (ET-1), typically produced locally by the endothelium, with autocrine effects of endothelial cells and paracrine effects on local smooth muscle cells. Other vasoconstrictors such as epinephrine, angiotensin II, and others have been identified as important in the liver. The most prominent vasorelaxing agent in the liver is nitric oxide (NO), which stimulates cyclic guanosine monophosphate (cGMP) and leads to myosin dephosphorylation and inactivation of the actin-myosin contractile apparatus in smooth muscle cells. In essentially all forms of intrahepatic liver disease, especially early in the disease process, there is a predictable and measurable increase in intrahepatic resistance — most often mediated by contraction of sinusoidal cells such as hepatic stellate cells and smooth muscle cells. We have also identified a unique molecular pathobiology in SECs. The liver cellular microcirculation in the sinusoid (including sinusoidal endothelial cells and stellate cells) appears to parallel the more
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general vascular system of capillaries. Our laboratory has used in vivo microscopy to study the hepatic sinusoid, sinusoidal dynamics, and sinusoidal blood flow. We and others find that sinusoidal blood flow is dynamic and that it can be modulated by classic vasoactive compounds (10–15). Most notably, exposure of the sinusoid to the vasoconstrictor ET-1 leads to sinusoidal constriction, whereas NO leads to dilation of the sinusoids (16). Stellate Cells The hepatic stellate cell, pericyte like in its appearance, possesses a repertoire of contractile proteins that mediate contractility; we have shown that they express an abundance of contractile proteins, typical of those found in other contractile cells (i.e., such as smooth muscle cells). These are most prominent after liver injury and become highly expressed after liver injury and after they have transformed from a quiescent (normal) to an activated (injured) state. Stellate cells possess multiple actin isoforms, including both cytoplasmic forms (alpha and beta) as well as smooth muscle specific actin — known as smooth muscle alpha actin (17). Not only do stellate cells express abundant actin isoforms, but they also express a multitude of other smooth muscle specific proteins. Remarkably, stellate cells also appear to have characteristic transcription programs that lead to a smooth muscle profile (18). An important feature of stellate cell activation and the transition to a smooth muscle phenotype is that it is associated with important functional consequences. Specifically, the acquisition of smooth muscle specific alpha actin leads to a contractile phenotype (19). The contractile phenotype has been reproduced in a variety of different systems, including in isolated cells, on silicone gel matrices, in collagen lattice assays, and others (13,20,21). It is important to note that the greater the degree of injury and activation, the great the increase in expression of smooth muscle proteins, and the greater the degree of contraction (13). Additionally, contractile signaling pathways in stellate cell appear to parallel many of those found in smooth muscle cells, and include calcium dependent and calcium independent pathways (22–25). A calcium-independent contraction mechanism through the rho-kinase pathway appears to be prominent. Other important players include myosin light chain kinase, 17-kDa protein kinase C–potentiated protein phosphatase 1 inhibitor protein (CPI-17), and MLC phosphatase targeting subunit 1 (MYPT1). Stellate cell contraction signaling appears to be extremely complicated, and is now an active area of investigation.
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Mechanisms that induce relaxation appear to be less complicated, and involve primarily stimulation of cGMP and myosin dephosphorylation. Molecules That Stimulate Stellate Cell Contractility Although multiple vasoactive molecules including angiotensin II, adenosine, arginine vasopressin, adrenomedullin, eicosanoids, and others have been reported to stimulate stellate cell contractility, the most prominent agents include the family of endothelins (2). NO appears to be the major inhibitor of stellate cell contractility (26) (see below), but other compounds have also been identified (2). The endothelins, 21 amino acid peptides known as ET-1, ET-2, and ET-3 (27), are among the most potent vasoconstrictors known. They bind to two known G-protein coupled receptors (GPCRs), termed endothelin A (ETA) and endothelin B (ETB) receptors (28). ETA receptors are classically found on vascular smooth muscle cells, whereas ETB receptors are typically found on endothelial cells. ETA receptors typically mediate contraction and vasoconstriction on smooth muscle cells, whereas ETB receptors on endothelial cells stimulate endothelial cell NO synthase (eNOS) and NO release, leading to smooth muscle cell relaxation and vasodilation. In most vascular biology situations, endothelins are produced by endothelial cells and exert paracrine effects on adjacent smooth muscle cells and autocrine effects on endothelial cells themselves. We have shown a unique biology for the endothelins in the liver. In the normal liver, ET-1 is produced largely by SECs (similar to endothelial cells in the peripheral vasculature), whereas after liver injury and stellate cell activation, the cellular source shifts to HSCs. This remarkable biology is regulated by a series of remarkable events. Our laboratory has shown that ET-1 production can be controlled at the transcriptional and/or post-transcriptional level in HSCs. ET-1 is produced as a precursor peptide, known as preproendothelin-1 (approximately 200 amino acid residues). This initial product is cleaved by furin-like enzymes to intermediates known as big endothelin (38-41 amino acid residues). Big endothelins, which have little or no biologic activity, are cleaved by the highly specific endothelin converting enzyme (ECE) to yield the biologically active, mature 21 amino acid peptide. Two major isoforms of ECE, termed ECE-1 and ECE-2 have been identified (29,30). ECE-1 mRNA also undergoes extensive alternative splicing to lead to a number of variants (30,31). We have shown that in injured HSCs, preproendothelin-1 mRNA expression can be stimulated by many different
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factors such as cytokines including transforming growth factor beta (TGFβ), tumor necrosis factor alpha (TNFα), and extracellular matrix proteins such as fibronectin (32–35). We have also shown that ET-1 levels are controlled at the transcriptional level as well as at a posttranscriptional level by ECE-1 (32,33,36). The importance of the endothelins in liver disease became evident after initial reports showed that circulating ET-1/ET-3 levels were elevated in patients with cirrhosis (37–39). Our laboratory (and others) showed that the source of ET-1 in patients with cirrhosis is the injured liver itself (40–42). The mechanism underlying the increased production of ET-1 in the injured liver is remarkable, and is due to a shift in the production of ET-1 from SECs to stellate cells (36). Underlying mechanisms leading to abnormally increased ET-1 synthesis in stellate cells includes regulation at both by transcriptional control of precursor, preproendothelin-1 mRNA synthesis, as well as by regulation of ECE. Extracellular matrix components such as fibronectin (34), cytokines such as TNFα (35), ET-1 itself (32), and other factors (43) may stimulate preproendothelin-1 production. Perhaps even more remarkable is that ECE-1 expression is upregulated after stellate cell activation by a unique mechanism involving p ost-transcriptional stabilization of ECE-1 via de novo production of proteins that stabilize the ECE-1 mRNA (33,36). TGFβ in particular is important because it causes production of at least two proteins that bind to the ECE-1 3’ untranslated region to stabilize ECE-1 mRNA and thus cause protein levels to increase (33). Increased ET-1 production after stellate cell activation and liver injury leads to enhanced contractility of HSCs (by virtue of ET-1’s effect on ET or ET receptors on stellate cells), via an autocrine effect of A B ET-1 on stellate cells themselves, leading in turn to enhanced c ellular contraction and to increased sinusoidal resistance (10,11,13–15). Together, the increased production of endothelin as well as enhanced endothelin receptor expression on stellate cells in the injured liver contributes to enhanced contractility. Mediators That Stimulate Stellate Cell Relaxation NO is the most potent vasorelaxing compound for stellate cells. NO is produced by one of three NO synthase isoforms. In the liver, the inducible isoform (iNOS) is upregulated in response to inflammatory stimuli in essentially all cells (26). The major functional nitric oxide synthase (NOS) isoform in liver under most conditions is the endothelial isoform
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(eNOS). Our laboratory was the first to identify eNOS specifically in SECs (44), and notably, eNOS expression itself appears to be remarkably stable after injury to SECs. eNOS function is controlled largely by a multitude of post-translational (enzymatic) modifications and protein-protein interactions (including post-translational acylation, phosphorylation, and S- nitrosylation) (45–65, see also below under endothelial cells). The Ca++/calmodulin system was one of the first post-translational modifications of eNOS identified. Further, eNOS is palmitoylated; a biochemical feature that appears to be important in cellular trafficking and concomitant functionality of eNOS (66). In SECs, eNOS phosphorylation appears to be one of the most critical determinants of eNOS function, consistent with other systems (66–72). eNOS is phosphorylated at multiple sites (Ser1177, Ser633, Ser615, Thr495, Ser114; these sites refer to the human eNOS sequence, which differs from other species). eNOS interacts with a number of proteins (caveolin-1, HSP-90, Akt, dynamin, and GRK2), GIT1 (see below), and interaction with many of these leads to changes in eNOS phosphorylation and enzymatic activity (51,53 55,73–78). Endothelial Cells SECs exhibit several unique properties in the liver sinusoid. A classic feature is their fenestration, which has been implicated in transport of proteins from the blood to the SEC. The other particularly notable property of SECs is their close physical proximity in the hepatic sinusoid to the hepatic stellate cell. This has led to the presumption that there are paracrine effects of SECs on hepatic stellate cells. In particular, NO released by SECs presumably has effects on HSCs, including inhibition of their contraction, and theoretically maintenance of a lowpressure system in the sinusoidal bed. The seminal finding that NO production in the injured liver is dramatically reduced (despite constant expression of eNOS protein) after liver injury (44) led to the conclusion that this dysfunctional eNOS contributes to increased intrahepatic resistance in liver injury. Our laboratory has thus embarked on a series of investigations to uncover the mechanism(s) leading to eNOS dysfunction. We have shown that eNOS phosphorylation is reduced in SECs after injury, and that the mechanism for this was related to enhanced GPCR desensitization. Since it is has been well established that ET-1 signals through ETB receptors to stimulate eNOS and NO production, we initially began studies to evaluate putative compounds in the GPCR signaling
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pathway. We found that after liver injury, there is no change in total Akt expression, but that there is a dramatic reduction in phosphoAKT (76,77). Manipulation of phospho-AKT led to parallel changes in phospho-eNOS (no change in total eNOS) and phosphorylation-induced changes in eNOS function. In addition, in liver injury models, overexpressing dominant active (myristoylated) Akt led to increases in NO production, and reduced portal pressure to near normal levels. We have also explored the GPCR desensitization pathway, postulating that G-protein coupled receptor kinases, which dampen signaling (79,80), may be involved in desensitization of AKT and eNOS activation after liver injury. We specifically focused on GPCR kinase-2 (GRK2), a classic GPCR interacting protein in our system. First, we found there to be dramatic upregulation of GRK2 in SECs liver injury (Figure 1). We also showed that overexpression of GRK2 in cultured SECs or in
Fig. 1. Nitric oxide (NO) signaling in normal and injured livers. NO synthesis is triggered via a pathway involving phosphatidylinositol-3-OH kinase (PI-3-K) and the serine-threonine kinase, Akt. Akt phosphorylation, stimulated by factors such as shear stress and growth factors, leads to endothelial NO synthase (eNOS) phosphorylation and NO production. G protein receptor kinase 2 (GRK2) interacts with, and influences Akt phosphorylation, and thus regulates endothelial NO synthase (eNOS) phosphorylation and activity. In the injured liver, GRK2 expression is upregulated and dampens Akt signaling, in turn reducing eNOS phosphorylation and NO production. eNOS is subject to other post-translational effects in the injured liver. Reproduced from Nature Medicine 11(9):952–8; Figure 6.
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vivo in mouse models of liver injury caused a reduction in SEC-mediated production of NO as well as profound increase in portal pressure, respectively. Blocking GRK2 had the opposite effect both in vitro and in vivo in portal hypertension after liver injury. The data point to a dysregulated GPCR desensitization system in SECs, leading to reduced NO production and contributing to portal hypertension (Figure 1). We have extended this working model to include a new eNOS interacting protein, known as G protein-coupled receptor kinase i nteractor-1 (GIT1), which appears to interact directly with eNOS (78,81). The expression pattern of these two proteins overlaps substantially when examined in situ. In addition, biochemical evidence points to a strong interaction (78,81). We have also shown that GIT1 expression is dramatically reduced in SECs after liver injury (78,81). Although we are currently trying to better understand the mechanism by with GIT1 is reduced after injury, as well as how it interacts with eNOS, we find that we are able to rescue the reduced eNOS phosphorylation (and reduced NO production) phenotype found in injured SECs by overexpression of full length GIT-1. Our working model is that GIT1 functions as a scaffolding protein, and as well stimulates phosphorylation of eNOS. Thus, ongoing work in the laboratory is focused on understanding the specifics of the eNOS and GIT1 interaction.
Summary Our working model is that after liver injury, intrahepatic vasoconstrictors such as ET-1 are increased, whereas vasodilators such as NO are reduced. Based on the work highlighted here, we propose that after liver injury, SECs are injured, leading to impaired eNOS function, and what we have coined a “sinusoidal endothelialopathy” or an “endothelialopathy in liver disease” (Figure 2). This work has focused on 2 major areas — the first being stellate cell biology, and the second surrounding highly abnormal endothelial function after liver injury. The later has become a major focus, having revolved primarily around the finding that synthesis of the potent vasodilator, NO, by SECs is dramatically reduced following liver injury. It is also notable that correction of impaired NO synthesis in SECs in the sinusoid improves portal hypertension in the injured liver (77,82–85), and perfusion of the injured liver with NO donors also corrects this defect (16,86). One mechanism leading to the observed phenotype is upregulation of GRK2, which impairs AKT and eNOS phosphorylation and activity and down regulation of GIT1, which reduces eNOS phosphorylation
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Fig. 2. Changes in the hepatic sinusoid in response to injury. (A) A simplified version of a portal tract (left), central vein (right), and hepatic sinusoidal morphology with associated cell types within the sinusoid. (B) The sinusoid in response to liver injury; the upper panel depicts a cross-sectional image, whereas the lower panel shows a 3-dimensional view. In the normal state (left), sinusoidal endothelial cells produce NO supporting a low resistance state. In response to liver injury, extensive changes occur in the sinusoid. Liver injury (right) leads to both morphological as well as molecular abnormalities in sinusoidal endothelial cells and stellate cells. For example, endothelial cells become defenestrated, develop a defect in NO production, and concurrently increase production of other proteins such as endothelin and fibronectin that can contribute to stellate cell activation. Upon their activation, stellate cells develop an enhanced contractile phenotype and produce increased extracellular matrix. A number of paracrine and autocrine interactions occur between sinusoidal endothelial cells and stellate cells with some of the putative molecules and their associated functions as shown (gray box). Abbreviations: HSC, hepatic stellate cells; LSEC, liver sinusoidal endothelial cell; PDGF, platelet-derived growth factor; TGFβ, transforming growth factor β ; NO, nitric oxide. Reproduced from Journal of Hepatology 61:912–24; Figure 2.
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and activity. The result is impaired eNOS function and reduced NO production in the sinusoid. From a clinical standpoint, in the normal liver, there is low intrahepatic resistance, whereas after injury, activated and contractile stellate cells signaling through ET-1, constrict the sinusoid. This model provides obvious opportunities for translation to the clinic. In patients with portal hypertension, it would be ideal to inhibit vasoconstrictors, or to replace vasodilators (perhaps by optimizing eNOS function). Future efforts will ideally lead to targeted molecular approaches that correct the underlying defects associated with the abnormal regulation of vasoconstrictor and/or vasodilator molecules in patients with liver disease.
acknowledgments The author wishes to thank the lab members who have made so many contributions to the science presented.
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52. Feron O, Michel JB, Sase K, et al. Dynamic regulation of endothelial nitric oxide synthase: complementary roles of dual acylation and caveolin interactions. Biochemistry 1998;37:193–200. 53. Garcia-Cardena G, Fan R, Shah V, et al. Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature 1998;392:821–4. 54. Feron O, Dessy C, Moniotte S, et al. Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase. J Clin Invest 1999;103:897–905. 55. Fulton D, Gratton JP, McCabe TJ, et al. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 1999;399:597–601. 56. Fulton D, Gratton JP, Sessa WC. Post-translational control of endothelial nitric oxide synthase: why isn’t calcium/calmodulin enough? J Pharmacol Exp Ther 2001;299:818–24. 57. Fulton D, Babbitt R, Zoellner S, et al. Targeting of endothelial nitric-oxide synthase to the cytoplasmic face of the Golgi complex or plasma membrane regulates Akt- versus calcium-dependent mechanisms for nitric oxide release. J Biol Chem 2004;279:30349–57. 58. Fulton D, Church JE, Ruan L, et al. Src kinase activates endothelial nitric-oxide synthase by phosphorylating Tyr-83. J Biol Chem 2005;280:35943–52. 59. Church JE, Fulton D. Differences in eNOS activity because of subcellular localization are dictated by phosphorylation state rather than the local calcium environment. J Biol Chem 2006;281:1477–88. 60. Mookerjee RP, Wiesenthal A, Icking A, et al. Increased gene and protein expression of the novel eNOS regulatory protein NOSTRIN and a variant in alcoholic hepatitis. Gastroenterology 2007;132:2533–41. 61. Lei H, Venkatakrishnan A, Yu S, et al. Protein kinase A-dependent translocation of Hsp90 alpha impairs endothelial nitric-oxide synthase activity in high glucose and diabetes. J Biol Chem 2007;282:9364–71. 62. Li L, Hisamoto K, Kim KH, et al. Variant estrogen receptor-c-Src molecular interdependence and c-Src structural requirements for endothelial NO synthase activation. Proc Natl Acad Sci U S A 2007;104:16468–73. 63. Duval M, Le Boeuf F, Huot J, et al. Src-mediated phosphorylation of Hsp90 in response to vascular endothelial growth factor (VEGF) is required for VEGF receptor-2 signaling to endothelial NO synthase. Mol Biol Cell 2007;18:4659–68. 64. Sugimoto M, Nakayama M, Goto TM, et al. Rho-kinase phosphorylates eNOS at threonine 495 in endothelial cells. Biochem Biophys Res Commun 2007;361:462–7. 65. Dudzinski DM, Michel T. Life history of eNOS: partners and pathways. Cardiovasc Res 2007;75:247–60. 66. Gonzalez E, Kou R, Lin AJ, et al. Subcellular targeting and agonist-induced site-specific phosphorylation of endothelial nitric-oxide synthase. J Biol Chem 2002;277:39554–60. 67. Kureishi Y, Luo Z, Shiojima I, et al. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals [see comments]. Nat Med 2000;6:1004–10. 68. Igarashi J, Michel T. Sphingosine 1-phosphate and isoform-specific activation of phosphoinositide 3-kinase beta. Evidence for divergence and convergence of receptor-regulated endothelial nitric-oxide synthase signaling pathways. J Biol Chem 2001;276:36281–8. 69. Igarashi J, Bernier SG, Michel T. Sphingosine 1-phosphate and activation of endothelial nitric-oxide synthase. differential regulation of Akt and MAP kinase pathways
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by EDG and bradykinin receptors in vascular endothelial cells. J Biol Chem 2001;276:12420–6. 70. Kou R, Greif D, Michel T. Dephosphorylation of endothelial nitric-oxide synthase by vascular endothelial growth factor. Implications for the vascular responses to cyclosporin A. J Biol Chem 2002;277:29669–73. 71. Greif DM, Kou R, Michel T. Site-specific dephosphorylation of endothelial nitric oxide synthase by protein phosphatase 2A: evidence for crosstalk between phosphorylation sites. Biochemistry 2002;41:15845–53. 72. Mount PF, Kemp BE, Power DA. Regulation of endothelial and myocardial NO synthesis by multi-site eNOS phosphorylation. J Mol Cell Cardiol 2007;42:271–9. 73. Michel JB, Feron O, Sase K, et al. Caveolin versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase. J Biol Chem 1997;272:25907–12. 74. Marrero MB, Venema VJ, Ju H, et al. Endothelial nitric oxide synthase interactions with G-protein-coupled receptors. Biochem J 1999;343 (Pt 2):335–40. 75. Cao S, Yao J, McCabe TJ, et al. Direct interaction between endothelial nitric-oxide synthase and dynamin-2. Implications for nitric-oxide synthase function. J Biol Chem 2001;276:14249–56. 76. Liu S, Premont RT, Kontos CD, et al. Endothelin-1 activates endothelial cell nitricoxide synthase via heterotrimeric G-protein betagamma subunit signaling to protein kinase B/Akt. J Biol Chem 2003;278:49929–35. 77. Liu S, Premont RT, Kontos CD, et al. A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension. Nat Med 2005;11:952–8. 78. Liu S, Premont RT, Rockey DC. G-protein-coupled receptor kinase interactor-1 (GIT1) is a new endothelial nitric-oxide synthase (eNOS) interactor with functional effects on vascular homeostasis. J Biol Chem 2012;287:12309–20. 79. Premont RT, Claing A, Vitale N, et al. beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase- activating protein. Proc Natl Acad Sci U S A 1998;95:14082–7. 80. Pierce KL, Premont RT, Lefkowitz RJ. Seven-transmembrane receptors. Nat Rev Mol Cell Biol 2002;3:639–50. 81. Liu S, Premont RT, Rockey DC. eNOS is activated through GIT1 tyrosine phosphorylation and Src. J Biol Chem 2014;289(26):18163–74. 82. Yu Q, Shao R, Qian HS, et al. Gene transfer of the neuronal NO synthase isoform to cirrhotic rat liver ameliorates portal hypertension. J Clin Invest 2000; 105:741–8. 83. Morales-Ruiz M, Cejudo-Martn P, Fernandez-Varo G, et al. Transduction of the liver with activated Akt normalizes portal pressure in cirrhotic rats. Gastroenterology 2003;125:522–31. 84. Theodorakis NG, Wang YN, Skill NJ, et al. The role of nitric oxide synthase isoforms in extrahepatic portal hypertension: studies in gene-knockout mice. G astroenterology 2003;124:1500–8. 85. Abraldes JG, Rodriguez-Vilarrupla A, Graupera M, et al. Simvastatin treatment improves liver sinusoidal endothelial dysfunction in CCl4 cirrhotic rats. J Hepatol 2007;46:1040–6. 86. Loureiro-Silva MR, Cadelina GW, Iwakiri Y, et al. A liver-specific nitric oxide donor improves the intra-hepatic vascular response to both portal blood flow increase and methoxamine in cirrhotic rats. J Hepatol 2003;39:940–6.
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DISCUSSION Schiffman, Providence: I know a little bit about venous sinus endothelial cells of the spleen and they have actin and myosin filaments at their base and can open and shut to allow passage of all sorts of cells from the red pulp into the venous circulation. The basement membrane is actually fenestrated and it looks like chicken wire. But there’s no cell like the stellate cell. The endothelial cell in the spleen is also a phagocytic cell. It’s a lining cell and also microphage-like cell and I wonder if there is any morphologic correlate of this phagocytic function in the liver endothelial cell? Rockey, Charleston: Not that I am aware of in the endothelial cell. The liver as you know is very resourceful and has the whole population of phagocytic cells known as Kupffer cells. Stellate cells actually do have some phagocytic capability and have been shown to phagocytose small particles and exosomes. But I’m not aware of a function in the endothelial cells. Bodenheimer, New York City: Don, thank you very much for this condensed version of your life! The question I have is about this malleable concept of portal hypertension. Does this play a greater role in early liver disease or developing fibrosis, as opposed to the late established diseases and will that foretell more pharmacologic intervention early on? Rockey, Charleston: I think that’s a great question and I think it’s exactly where we are going in this disease. As clinicians, we’re focused on the patient with severe portal hypertension who has bled and who has end-stage disease, with a hard, scarred liver. But we need to be here early in the disease where the patient initially presented with early fibrosis and early liver injury. Where I think a lot of these molecules and cells are in play. So, we need to come up with better measures, non-invasive measures, of portal pressures so we can assess exactly where we are clinically. Zeidel, Boston: Wonderful talk Don, terrific. I just want to ask you to speculate a little bit about “cart and horse.” So, you’re looking at portal pressure and you’re looking at injuries and changes that reduce, may alter the blood flow, but at the same time we have fibrosis going on in the parenchymal cells. What is the relationship between the two? Is intervening in one part of this necessarily going to help much if we don’t fix the other part? Rockey, Charleston: A great question and I think about this a lot. I actually believe that portal hypertension in and of itself is somewhat like essential hypertension. It is pathogenic in the disease process, and this is the speculative part, likely fuels this fibrogenic potential. I don’t have any proof of that. We’re thinking about how we can stretch cells and modulate them to, recapitulate the physiology in the sinusoid to answer that exact question. I think it’s probably very important. Vierling, Houston: Terrific talk Don, and congratulations on all the progress that you have made against a lot of different forces which have taken the field of portal hypertension in different directions. I think you’ve really stood alone in this arena. I congratulate you for it. I have two brief questions: the first is about NASH, a sub type of NASH, along with alcoholic steatohepatitis. You have an interesting pathway toward the formation of cirrhosis, the micronodular cirrhosis, especially with alcohol. This tends to have a colonization of the space of Disse from the terminal hepatic venule. I am thinking of that difference obviously from post-necrotic cirrhosis portal based septal fibrosis, and bridging fibrosis leading to cirrhosis. I am wondering on your speculation of the consequences of those different paradigms of fibrosis in terms of the relative contribution of the vasoconstrictive property that would occur with the activated stellate cell. The second question is that all of these things always boil down early on in the disease to issues
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of inflammation. I am very interested to know what would be the chemokine or cytokine receptor contributions to the events you’ve shown molecularly. Rockey, Charleston: You have a lot there. Thanks very much for your comments. Second question first: what about inflammation? And so, I think in those forms of liver disease, inflammation is a major player and as you well know there are many companies and investigators that are targeting primarily inflammation as a way to not only inhibit fibrosis, but affect portal hypertension. I think that’s a key pathway. In terms of your question about the differences in capillarization or the differences in the different types of liver disease and the biology, I think there may be some differences. Hepatitis C may be different than alcohol and alcohol may be different from NASH which may be different from cystic fibrosis. Cystic fibrosis causes a pretty bland injury, yet those patients develop fibrosis and portal hypertension. I don’t know the answer to that, but I think that is something we need to be thinking about. Is the biology the same or different with the different diseases and I just don’t know. It’ll give me something in the next 20 years to work on. Palmer, New York City: Just a simple question: what’s going on with iNOS starting all this business? Is there any cross talk between the iNOS and eNOS systems? Or are they totally separate? Rockey, Charleston: I touched on this very briefly and I’m sorry I was moving fast. iNOS is found in the liver: It’s found primarily in the setting of acute inflammation, usually very prominent with inflammation, and it’s found primarily in Kupffer cells not in the other non-parenchymal cells. I am not aware of much cross talk between iNOS and eNOS.
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The Molecular Basis of Portal Hypertension DON C. ROCKEY, MD Charleston, South Carolina
Abstract Cirrhosis leads to portal hypertension and vascular abnormalities in multiple vascular beds. There is intense vasoconstriction in the liver and the kidneys, but also vasodilation in the other vascular beds, including the periphery, lungs, brain, and mesentery. The derangement in each of these beds leads to specific clinical disease. The vasoconstrictive phenotype in the liver ultimately leads to clinical portal hypertension, and is caused by an imbalance of vasoconstrictive and vasorelaxing molecules, which will be the focus of this review.
Sinusoidal blood flow The functional vascular unit of the liver is the hepatic sinusoid, made up of sinusoidal endothelial cells (SECs) and hepatic stellate cells (HSCs); both of these cells are important in the regulation of sinusoidal blood flow (1–3). Stellate cells have an anatomic orientation in the hepatic sinusoid that is reminiscent of tissue pericytes, possessing intricate cytoplasmic processes that are tightly associated with SECs (4,5). This close anatomic relationship of these two sinusoidal cell types suggests that they are linked functionally, perhaps via paracrine signaling. In virtually all forms of liver disease, intrahepatic resistance is elevated. Portal pressure is proportional to resistance and flow according to the hydraulic equivalent of Ohm’s law: ∆P = Q × R where ∆P is the change in pressure along a vessel, Q the flow in the vessel, and R the resistance to flow. Elevated intrahepatic resistance occurs early in the disease process, leading to a classic hyperdynamic circulatory mesenteric blood flow pattern. This leads to increased blood flow, and thus patients with cirrhosis typically have both increased intrahepatic resistance and increased flow through the splanchnic system, each contributing to portal hypertension. Although the level of increased Correspondence and reprint requests: Don C. Rockey, MD, Department of Internal Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Suite 803, MSC 623, Charleston, South Carolina 29425, Tel: 843-792-2914, E-mail: [email protected]. Potential Conflicts of Interest: None disclosed.
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resistance to flow in cirrhosis varies to some degree with different types of liver disease (i.e., pre-, intra-, or post-sinusoidal resistance), most forms of liver disease lead to so-called “sinusoidal” portal hypertension in which elevated resistance is derived from the sinusoidal areas. This pathophysiology is also important clinically in that portal pressure is one of the best predictors of clinical outcome (6–8). In liver disease, portal hypertension typically occurs in parallel with fibrosis, the latter a common result of injury to the liver [and typical of injury to essentially all of the parenchymal organs and skin (9)]. It is notable that portal hypertension is usually proportional to fibrosis. This is particularly true in advanced fibrosis, where intrahepatic resistance is likely also mediated by architectural distortion of the liver and its lobules. Intrahepatic resistance is likely p articularly affected by changes in cellular and molecular interplay early in the liver injury process. Thus, intrahepatic resistance may be considered in terms of two major compartments — fixed and modulable compartments, with the fixed component related to advanced fibrosis and scarring, and the modulable component related to cells and molecules within the liver; this latter component is likely to be more active in early portal hypertension.
Molecular Mediators of Portal Hypertension Multiple vascular mediators have been implicated in the vasoconstrictive phenotype typical of intrahepatic portal hypertension. Most prominent of these appears to be endothelin-1 (ET-1), typically produced locally by the endothelium, with autocrine effects of endothelial cells and paracrine effects on local smooth muscle cells. Other vasoconstrictors such as epinephrine, angiotensin II, and others have been identified as important in the liver. The most prominent vasorelaxing agent in the liver is nitric oxide (NO), which stimulates cyclic guanosine monophosphate (cGMP) and leads to myosin dephosphorylation and inactivation of the actin-myosin contractile apparatus in smooth muscle cells. In essentially all forms of intrahepatic liver disease, especially early in the disease process, there is a predictable and measurable increase in intrahepatic resistance — most often mediated by contraction of sinusoidal cells such as hepatic stellate cells and smooth muscle cells. We have also identified a unique molecular pathobiology in SECs. The liver cellular microcirculation in the sinusoid (including sinusoidal endothelial cells and stellate cells) appears to parallel the more
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general vascular system of capillaries. Our laboratory has used in vivo microscopy to study the hepatic sinusoid, sinusoidal dynamics, and sinusoidal blood flow. We and others find that sinusoidal blood flow is dynamic and that it can be modulated by classic vasoactive compounds (10–15). Most notably, exposure of the sinusoid to the vasoconstrictor ET-1 leads to sinusoidal constriction, whereas NO leads to dilation of the sinusoids (16). Stellate Cells The hepatic stellate cell, pericyte like in its appearance, possesses a repertoire of contractile proteins that mediate contractility; we have shown that they express an abundance of contractile proteins, typical of those found in other contractile cells (i.e., such as smooth muscle cells). These are most prominent after liver injury and become highly expressed after liver injury and after they have transformed from a quiescent (normal) to an activated (injured) state. Stellate cells possess multiple actin isoforms, including both cytoplasmic forms (alpha and beta) as well as smooth muscle specific actin — known as smooth muscle alpha actin (17). Not only do stellate cells express abundant actin isoforms, but they also express a multitude of other smooth muscle specific proteins. Remarkably, stellate cells also appear to have characteristic transcription programs that lead to a smooth muscle profile (18). An important feature of stellate cell activation and the transition to a smooth muscle phenotype is that it is associated with important functional consequences. Specifically, the acquisition of smooth muscle specific alpha actin leads to a contractile phenotype (19). The contractile phenotype has been reproduced in a variety of different systems, including in isolated cells, on silicone gel matrices, in collagen lattice assays, and others (13,20,21). It is important to note that the greater the degree of injury and activation, the great the increase in expression of smooth muscle proteins, and the greater the degree of contraction (13). Additionally, contractile signaling pathways in stellate cell appear to parallel many of those found in smooth muscle cells, and include calcium dependent and calcium independent pathways (22–25). A calcium-independent contraction mechanism through the rho-kinase pathway appears to be prominent. Other important players include myosin light chain kinase, 17-kDa protein kinase C–potentiated protein phosphatase 1 inhibitor protein (CPI-17), and MLC phosphatase targeting subunit 1 (MYPT1). Stellate cell contraction signaling appears to be extremely complicated, and is now an active area of investigation.
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Mechanisms that induce relaxation appear to be less complicated, and involve primarily stimulation of cGMP and myosin dephosphorylation. Molecules That Stimulate Stellate Cell Contractility Although multiple vasoactive molecules including angiotensin II, adenosine, arginine vasopressin, adrenomedullin, eicosanoids, and others have been reported to stimulate stellate cell contractility, the most prominent agents include the family of endothelins (2). NO appears to be the major inhibitor of stellate cell contractility (26) (see below), but other compounds have also been identified (2). The endothelins, 21 amino acid peptides known as ET-1, ET-2, and ET-3 (27), are among the most potent vasoconstrictors known. They bind to two known G-protein coupled receptors (GPCRs), termed endothelin A (ETA) and endothelin B (ETB) receptors (28). ETA receptors are classically found on vascular smooth muscle cells, whereas ETB receptors are typically found on endothelial cells. ETA receptors typically mediate contraction and vasoconstriction on smooth muscle cells, whereas ETB receptors on endothelial cells stimulate endothelial cell NO synthase (eNOS) and NO release, leading to smooth muscle cell relaxation and vasodilation. In most vascular biology situations, endothelins are produced by endothelial cells and exert paracrine effects on adjacent smooth muscle cells and autocrine effects on endothelial cells themselves. We have shown a unique biology for the endothelins in the liver. In the normal liver, ET-1 is produced largely by SECs (similar to endothelial cells in the peripheral vasculature), whereas after liver injury and stellate cell activation, the cellular source shifts to HSCs. This remarkable biology is regulated by a series of remarkable events. Our laboratory has shown that ET-1 production can be controlled at the transcriptional and/or post-transcriptional level in HSCs. ET-1 is produced as a precursor peptide, known as preproendothelin-1 (approximately 200 amino acid residues). This initial product is cleaved by furin-like enzymes to intermediates known as big endothelin (38-41 amino acid residues). Big endothelins, which have little or no biologic activity, are cleaved by the highly specific endothelin converting enzyme (ECE) to yield the biologically active, mature 21 amino acid peptide. Two major isoforms of ECE, termed ECE-1 and ECE-2 have been identified (29,30). ECE-1 mRNA also undergoes extensive alternative splicing to lead to a number of variants (30,31). We have shown that in injured HSCs, preproendothelin-1 mRNA expression can be stimulated by many different
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factors such as cytokines including transforming growth factor beta (TGFβ), tumor necrosis factor alpha (TNFα), and extracellular matrix proteins such as fibronectin (32–35). We have also shown that ET-1 levels are controlled at the transcriptional level as well as at a posttranscriptional level by ECE-1 (32,33,36). The importance of the endothelins in liver disease became evident after initial reports showed that circulating ET-1/ET-3 levels were elevated in patients with cirrhosis (37–39). Our laboratory (and others) showed that the source of ET-1 in patients with cirrhosis is the injured liver itself (40–42). The mechanism underlying the increased production of ET-1 in the injured liver is remarkable, and is due to a shift in the production of ET-1 from SECs to stellate cells (36). Underlying mechanisms leading to abnormally increased ET-1 synthesis in stellate cells includes regulation at both by transcriptional control of precursor, preproendothelin-1 mRNA synthesis, as well as by regulation of ECE. Extracellular matrix components such as fibronectin (34), cytokines such as TNFα (35), ET-1 itself (32), and other factors (43) may stimulate preproendothelin-1 production. Perhaps even more remarkable is that ECE-1 expression is upregulated after stellate cell activation by a unique mechanism involving p ost-transcriptional stabilization of ECE-1 via de novo production of proteins that stabilize the ECE-1 mRNA (33,36). TGFβ in particular is important because it causes production of at least two proteins that bind to the ECE-1 3’ untranslated region to stabilize ECE-1 mRNA and thus cause protein levels to increase (33). Increased ET-1 production after stellate cell activation and liver injury leads to enhanced contractility of HSCs (by virtue of ET-1’s effect on ET or ET receptors on stellate cells), via an autocrine effect of A B ET-1 on stellate cells themselves, leading in turn to enhanced c ellular contraction and to increased sinusoidal resistance (10,11,13–15). Together, the increased production of endothelin as well as enhanced endothelin receptor expression on stellate cells in the injured liver contributes to enhanced contractility. Mediators That Stimulate Stellate Cell Relaxation NO is the most potent vasorelaxing compound for stellate cells. NO is produced by one of three NO synthase isoforms. In the liver, the inducible isoform (iNOS) is upregulated in response to inflammatory stimuli in essentially all cells (26). The major functional nitric oxide synthase (NOS) isoform in liver under most conditions is the endothelial isoform
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(eNOS). Our laboratory was the first to identify eNOS specifically in SECs (44), and notably, eNOS expression itself appears to be remarkably stable after injury to SECs. eNOS function is controlled largely by a multitude of post-translational (enzymatic) modifications and protein-protein interactions (including post-translational acylation, phosphorylation, and S- nitrosylation) (45–65, see also below under endothelial cells). The Ca++/calmodulin system was one of the first post-translational modifications of eNOS identified. Further, eNOS is palmitoylated; a biochemical feature that appears to be important in cellular trafficking and concomitant functionality of eNOS (66). In SECs, eNOS phosphorylation appears to be one of the most critical determinants of eNOS function, consistent with other systems (66–72). eNOS is phosphorylated at multiple sites (Ser1177, Ser633, Ser615, Thr495, Ser114; these sites refer to the human eNOS sequence, which differs from other species). eNOS interacts with a number of proteins (caveolin-1, HSP-90, Akt, dynamin, and GRK2), GIT1 (see below), and interaction with many of these leads to changes in eNOS phosphorylation and enzymatic activity (51,53 55,73–78). Endothelial Cells SECs exhibit several unique properties in the liver sinusoid. A classic feature is their fenestration, which has been implicated in transport of proteins from the blood to the SEC. The other particularly notable property of SECs is their close physical proximity in the hepatic sinusoid to the hepatic stellate cell. This has led to the presumption that there are paracrine effects of SECs on hepatic stellate cells. In particular, NO released by SECs presumably has effects on HSCs, including inhibition of their contraction, and theoretically maintenance of a lowpressure system in the sinusoidal bed. The seminal finding that NO production in the injured liver is dramatically reduced (despite constant expression of eNOS protein) after liver injury (44) led to the conclusion that this dysfunctional eNOS contributes to increased intrahepatic resistance in liver injury. Our laboratory has thus embarked on a series of investigations to uncover the mechanism(s) leading to eNOS dysfunction. We have shown that eNOS phosphorylation is reduced in SECs after injury, and that the mechanism for this was related to enhanced GPCR desensitization. Since it is has been well established that ET-1 signals through ETB receptors to stimulate eNOS and NO production, we initially began studies to evaluate putative compounds in the GPCR signaling
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pathway. We found that after liver injury, there is no change in total Akt expression, but that there is a dramatic reduction in phosphoAKT (76,77). Manipulation of phospho-AKT led to parallel changes in phospho-eNOS (no change in total eNOS) and phosphorylation-induced changes in eNOS function. In addition, in liver injury models, overexpressing dominant active (myristoylated) Akt led to increases in NO production, and reduced portal pressure to near normal levels. We have also explored the GPCR desensitization pathway, postulating that G-protein coupled receptor kinases, which dampen signaling (79,80), may be involved in desensitization of AKT and eNOS activation after liver injury. We specifically focused on GPCR kinase-2 (GRK2), a classic GPCR interacting protein in our system. First, we found there to be dramatic upregulation of GRK2 in SECs liver injury (Figure 1). We also showed that overexpression of GRK2 in cultured SECs or in
Fig. 1. Nitric oxide (NO) signaling in normal and injured livers. NO synthesis is triggered via a pathway involving phosphatidylinositol-3-OH kinase (PI-3-K) and the serine-threonine kinase, Akt. Akt phosphorylation, stimulated by factors such as shear stress and growth factors, leads to endothelial NO synthase (eNOS) phosphorylation and NO production. G protein receptor kinase 2 (GRK2) interacts with, and influences Akt phosphorylation, and thus regulates endothelial NO synthase (eNOS) phosphorylation and activity. In the injured liver, GRK2 expression is upregulated and dampens Akt signaling, in turn reducing eNOS phosphorylation and NO production. eNOS is subject to other post-translational effects in the injured liver. Reproduced from Nature Medicine 11(9):952–8; Figure 6.
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vivo in mouse models of liver injury caused a reduction in SEC-mediated production of NO as well as profound increase in portal pressure, respectively. Blocking GRK2 had the opposite effect both in vitro and in vivo in portal hypertension after liver injury. The data point to a dysregulated GPCR desensitization system in SECs, leading to reduced NO production and contributing to portal hypertension (Figure 1). We have extended this working model to include a new eNOS interacting protein, known as G protein-coupled receptor kinase i nteractor-1 (GIT1), which appears to interact directly with eNOS (78,81). The expression pattern of these two proteins overlaps substantially when examined in situ. In addition, biochemical evidence points to a strong interaction (78,81). We have also shown that GIT1 expression is dramatically reduced in SECs after liver injury (78,81). Although we are currently trying to better understand the mechanism by with GIT1 is reduced after injury, as well as how it interacts with eNOS, we find that we are able to rescue the reduced eNOS phosphorylation (and reduced NO production) phenotype found in injured SECs by overexpression of full length GIT-1. Our working model is that GIT1 functions as a scaffolding protein, and as well stimulates phosphorylation of eNOS. Thus, ongoing work in the laboratory is focused on understanding the specifics of the eNOS and GIT1 interaction.
Summary Our working model is that after liver injury, intrahepatic vasoconstrictors such as ET-1 are increased, whereas vasodilators such as NO are reduced. Based on the work highlighted here, we propose that after liver injury, SECs are injured, leading to impaired eNOS function, and what we have coined a “sinusoidal endothelialopathy” or an “endothelialopathy in liver disease” (Figure 2). This work has focused on 2 major areas — the first being stellate cell biology, and the second surrounding highly abnormal endothelial function after liver injury. The later has become a major focus, having revolved primarily around the finding that synthesis of the potent vasodilator, NO, by SECs is dramatically reduced following liver injury. It is also notable that correction of impaired NO synthesis in SECs in the sinusoid improves portal hypertension in the injured liver (77,82–85), and perfusion of the injured liver with NO donors also corrects this defect (16,86). One mechanism leading to the observed phenotype is upregulation of GRK2, which impairs AKT and eNOS phosphorylation and activity and down regulation of GIT1, which reduces eNOS phosphorylation
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Fig. 2. Changes in the hepatic sinusoid in response to injury. (A) A simplified version of a portal tract (left), central vein (right), and hepatic sinusoidal morphology with associated cell types within the sinusoid. (B) The sinusoid in response to liver injury; the upper panel depicts a cross-sectional image, whereas the lower panel shows a 3-dimensional view. In the normal state (left), sinusoidal endothelial cells produce NO supporting a low resistance state. In response to liver injury, extensive changes occur in the sinusoid. Liver injury (right) leads to both morphological as well as molecular abnormalities in sinusoidal endothelial cells and stellate cells. For example, endothelial cells become defenestrated, develop a defect in NO production, and concurrently increase production of other proteins such as endothelin and fibronectin that can contribute to stellate cell activation. Upon their activation, stellate cells develop an enhanced contractile phenotype and produce increased extracellular matrix. A number of paracrine and autocrine interactions occur between sinusoidal endothelial cells and stellate cells with some of the putative molecules and their associated functions as shown (gray box). Abbreviations: HSC, hepatic stellate cells; LSEC, liver sinusoidal endothelial cell; PDGF, platelet-derived growth factor; TGFβ, transforming growth factor β ; NO, nitric oxide. Reproduced from Journal of Hepatology 61:912–24; Figure 2.
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and activity. The result is impaired eNOS function and reduced NO production in the sinusoid. From a clinical standpoint, in the normal liver, there is low intrahepatic resistance, whereas after injury, activated and contractile stellate cells signaling through ET-1, constrict the sinusoid. This model provides obvious opportunities for translation to the clinic. In patients with portal hypertension, it would be ideal to inhibit vasoconstrictors, or to replace vasodilators (perhaps by optimizing eNOS function). Future efforts will ideally lead to targeted molecular approaches that correct the underlying defects associated with the abnormal regulation of vasoconstrictor and/or vasodilator molecules in patients with liver disease.
acknowledgments The author wishes to thank the lab members who have made so many contributions to the science presented.
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DISCUSSION Schiffman, Providence: I know a little bit about venous sinus endothelial cells of the spleen and they have actin and myosin filaments at their base and can open and shut to allow passage of all sorts of cells from the red pulp into the venous circulation. The basement membrane is actually fenestrated and it looks like chicken wire. But there’s no cell like the stellate cell. The endothelial cell in the spleen is also a phagocytic cell. It’s a lining cell and also microphage-like cell and I wonder if there is any morphologic correlate of this phagocytic function in the liver endothelial cell? Rockey, Charleston: Not that I am aware of in the endothelial cell. The liver as you know is very resourceful and has the whole population of phagocytic cells known as Kupffer cells. Stellate cells actually do have some phagocytic capability and have been shown to phagocytose small particles and exosomes. But I’m not aware of a function in the endothelial cells. Bodenheimer, New York City: Don, thank you very much for this condensed version of your life! The question I have is about this malleable concept of portal hypertension. Does this play a greater role in early liver disease or developing fibrosis, as opposed to the late established diseases and will that foretell more pharmacologic intervention early on? Rockey, Charleston: I think that’s a great question and I think it’s exactly where we are going in this disease. As clinicians, we’re focused on the patient with severe portal hypertension who has bled and who has end-stage disease, with a hard, scarred liver. But we need to be here early in the disease where the patient initially presented with early fibrosis and early liver injury. Where I think a lot of these molecules and cells are in play. So, we need to come up with better measures, non-invasive measures, of portal pressures so we can assess exactly where we are clinically. Zeidel, Boston: Wonderful talk Don, terrific. I just want to ask you to speculate a little bit about “cart and horse.” So, you’re looking at portal pressure and you’re looking at injuries and changes that reduce, may alter the blood flow, but at the same time we have fibrosis going on in the parenchymal cells. What is the relationship between the two? Is intervening in one part of this necessarily going to help much if we don’t fix the other part? Rockey, Charleston: A great question and I think about this a lot. I actually believe that portal hypertension in and of itself is somewhat like essential hypertension. It is pathogenic in the disease process, and this is the speculative part, likely fuels this fibrogenic potential. I don’t have any proof of that. We’re thinking about how we can stretch cells and modulate them to, recapitulate the physiology in the sinusoid to answer that exact question. I think it’s probably very important. Vierling, Houston: Terrific talk Don, and congratulations on all the progress that you have made against a lot of different forces which have taken the field of portal hypertension in different directions. I think you’ve really stood alone in this arena. I congratulate you for it. I have two brief questions: the first is about NASH, a sub type of NASH, along with alcoholic steatohepatitis. You have an interesting pathway toward the formation of cirrhosis, the micronodular cirrhosis, especially with alcohol. This tends to have a colonization of the space of Disse from the terminal hepatic venule. I am thinking of that difference obviously from post-necrotic cirrhosis portal based septal fibrosis, and bridging fibrosis leading to cirrhosis. I am wondering on your speculation of the consequences of those different paradigms of fibrosis in terms of the relative contribution of the vasoconstrictive property that would occur with the activated stellate cell. The second question is that all of these things always boil down early on in the disease to issues
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of inflammation. I am very interested to know what would be the chemokine or cytokine receptor contributions to the events you’ve shown molecularly. Rockey, Charleston: You have a lot there. Thanks very much for your comments. Second question first: what about inflammation? And so, I think in those forms of liver disease, inflammation is a major player and as you well know there are many companies and investigators that are targeting primarily inflammation as a way to not only inhibit fibrosis, but affect portal hypertension. I think that’s a key pathway. In terms of your question about the differences in capillarization or the differences in the different types of liver disease and the biology, I think there may be some differences. Hepatitis C may be different than alcohol and alcohol may be different from NASH which may be different from cystic fibrosis. Cystic fibrosis causes a pretty bland injury, yet those patients develop fibrosis and portal hypertension. I don’t know the answer to that, but I think that is something we need to be thinking about. Is the biology the same or different with the different diseases and I just don’t know. It’ll give me something in the next 20 years to work on. Palmer, New York City: Just a simple question: what’s going on with iNOS starting all this business? Is there any cross talk between the iNOS and eNOS systems? Or are they totally separate? Rockey, Charleston: I touched on this very briefly and I’m sorry I was moving fast. iNOS is found in the liver: It’s found primarily in the setting of acute inflammation, usually very prominent with inflammation, and it’s found primarily in Kupffer cells not in the other non-parenchymal cells. I am not aware of much cross talk between iNOS and eNOS.
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