Vijay H. Shah

Diverse Origin and Function of Cells With Endothelial Phenotype Obtained From Adult Human Blood

Cells with endothelial phenotype generated from adult peripheral blood have emerging diagnostic and therapeutic potential. This study examined the lineage relationship between, and angiogenic function of, early endothelial progenitor cells (EPCs) and late outgrowth endothelial cells (OECs) in culture. Culture conditions were established to support the generation of both EPCs and OECs from the same starting population of peripheral blood mononuclear cells (PBMCs). Utilizing differences in expression of the surface endotoxin receptor CD14, it was determined that the vast majority of EPCs arose from a CD14+ subpopulation of PBMCs but OECs developed exclusively from the CD14− fraction. Human OECs, but not EPCs, expressed key regulatory proteins endothelial nitric oxide synthase (eNOS) and caveolin-1. Moreover, OECs exhibited a markedly greater capacity for capillary morphogenesis in in vitro and in vivo matrigel models, tube formation by OECs being in part dependent on eNOS function. Collectively, these data indicate lineage and functional heterogeneity in the population of circulating cells capable of assuming an endothelial phenotype and provide rationale for the investigation of new cell-therapeutic approaches to ischemic cardiovascular disease.

The third gas: H2S regulates perfusion pressure in both the isolated and perfused normal rat liver and in cirrhosis

The regulation of sinusoidal resistance is dependent on the contraction of hepatic stellate cells (HSC) around sinusoidal endothelial cell (SEC) through paracrine cross‐talk of vasoconstrictor and vasodilator agents. Hydrogen sulfide (H2S), a recently discovered gas neurotransmitter, is a putative vasodilator whose role in hepatic vascular regulation and portal hypertension is unexplored. Four‐week bile duct–ligated (BDL) rats with cirrhosis and control rats were treated daily with NaHS (56 μmol/kg) for 5 days. Isolated livers were perfused first with NaHS for 20 minutes and then with norepinephrine (NE) and the intrahepatic resistance studied. In normal rats and animals with cirrhosis, administration of NE resulted in a dose‐dependent increase of portal pressure. This effect was attenuated by H2S treatment (P < .05). The H2S‐induced relaxation of hepatic microcirculation was attenuated by glibenclamide, an adenosine triphosphate (ATP)‐sensitive K+ channel inhibitor. L‐Cysteine, a substrate of cystathionine‐gamma‐lyase (CSE), decreased vasoconstriction in normal rat livers (P < .05) but failed to do so in livers with cirrhosis. BDL resulted in a downregulation of CSE mRNA/protein levels and activity (P < .05). Our in vitro data demonstrate that CSE is expressed in hepatocytes, HSCs, but not in sinusoidal endothelial cells (SEC). HSC activation downregulates CSE mRNA expression, resulting in a defective production of H2S and abrogation of relaxation induced by L‐cysteine. In conclusion, CSE‐derived H2S is involved in the maintenance of portal venous pressure. The reduction of CSE expression in the liver with cirrhosis contributes to the development of increased intrahepatic resistance and portal hypertension. (HEPATOLOGY 2005.)

MELD accurately predicts mortality in patients with alcoholic hepatitis

Assessing severity of disease in patients with alcoholic hepatitis (AH) is useful for predicting mortality, guiding treatment decisions, and stratifying patients for therapeutic trials. The traditional disease‐specific prognostic model used for this purpose is the Maddrey discriminant function (DF). The model for end‐stage liver disease (MELD) is a more recently developed scoring system that has been validated as an independent predictor of patient survival in candidates for liver transplantation. The aim of the present study was to examine the ability of MELD to predict mortality in patients with AH. A retrospective cohort study of 73 patients diagnosed with AH between 1995 and 2001 was performed at the Mayo Clinic in Rochester, Minnesota. MELD was the only independent predictor of mortality in patients with AH. MELD was comparable to DF in predicting 30‐day mortality (c‐statistic and 95% CI: 0.83 [0.71‐0.96] and 0.74 [0.62‐0.87] for MELD and DF, respectively, not significant) and 90‐day mortality (c‐statistic and 95% CI: 0.86 [0.77‐0.96] and 0.83 [0.74‐0.92] for MELD and DF, respectively, not significant). A MELD score of 21 had a sensitivity of 75% and a specificity of 75% in predicting 90‐day mortality in AH. In conclusion, MELD is useful for predicting 30‐day and 90‐day mortality in patients with AH and maintains some practical and statistical advantages over DF in predicting mortality rate in these patients. MELD is a useful clinical tool for gauging mortality and guiding treatment decisions in patients with AH, particularly those complicated by ascites and/or encephalopathy. (HEPATOLOGY 2005;41:353–358.)

Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat

BACKGROUND & AIMS A reduction in nitric oxide (NO) has been implicated as a cause of intrahepatic vasoconstriction in cirrhosis, but the regulatory mechanisms remain undefined. The aim of this study was to examine a contributory role for caveolin-1, a putative negative regulator of endothelial NO synthase, in mediating deficient intrahepatic NO production in the intact cirrhotic liver. METHODS Cirrhosis was induced by carbon tetrachloride inhalation. Flow regulation of NO production and perfusion pressure was examined in the perfused rat liver. Protein expression of endothelial NO synthase (eNOS), caveolin, and calmodulin was examined by Western blotting and immunohistochemistry. NOS activity and NO production were assessed by citrulline generation and chemiluminescence, respectively. Protein-protein interactions were examined using whole tissue protein immunoprecipitation. RESULTS In response to incremental increases in flow, cirrhotic animals produced significantly less NO(x) than control animals. NOS activity was significantly reduced in liver tissue from cirrhotic animals compared with control animals in the presence of similar eNOS protein levels. Deficient eNOS activity was associated with a severalfold increase in binding of eNOS with caveolin. Protein levels of caveolin-1 were markedly increased in the cirrhotic liver. CONCLUSIONS These studies provide evidence that enhanced expression and interaction of caveolin with eNOS contribute to impaired NO production, reduced NOS activity, and vasoconstriction in the intact cirrhotic liver.

A Randomized, Double-Blinded, Placebo-Controlled Multicenter Trial of Etanercept in the Treatment of Alcoholic Hepatitis

BACKGROUND & AIMS Alcoholic hepatitis is a cause of major morbidity and mortality that lacks effective therapies. Both experimental and clinical evidence indicate that the multifunctional cytokine tumor necrosis factor-alpha (TNF-alpha) contributes to pathogenesis and clinical sequelae of alcoholic hepatitis. A pilot study demonstrated that the TNF-alpha-neutralizing molecule etanercept could be an effective treatment for patients with alcoholic hepatitis. METHODS Forty-eight patients with moderate to severe alcoholic hepatitis (Model for End-Stage Liver Disease score > or = 15) were enrolled and randomized to groups that were given up to 6 subcutaneous injections of either etanercept or placebo for 3 weeks. Primary study end points included mortality at 1- and 6-month time points. RESULTS There were no significant baseline differences between the placebo and etanercept groups in demographics or disease severity parameters including age, gender, and Model for End-Stage Liver Disease score. The 1-month mortality rates of patients receiving placebo and etanercept were similar on an intention-to-treat basis (22.7% vs 36.4%, respectively; OR, 1.8; 95% CI, 0.5-6.5). The 6-month mortality rate was significantly higher in the etanercept group compared with the placebo group (57.7% vs 22.7%, respectively; OR, 4.6; 95% CI, 1.3-16.4; P = .017). Rates of infectious serious adverse events were significantly higher in the etanercept group compared with the placebo group (34.6% vs 9.1%, respectively, P = .04). CONCLUSIONS In patients with moderate to severe alcoholic hepatitis, etanercept was associated with a significantly higher mortality rate after 6 months, indicating that etanercept is not effective for the treatment of patients with alcoholic hepatitis.

Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking

Nitric oxide (NO) is a highly diffusible and short-lived physiological messenger. Despite its diffusible nature, NO modifies thiol groups of specific cysteine residues in target proteins and alters protein function via S-nitrosylation. Although intracellular S-nitrosylation is a specific posttranslational modification, the defined localization of an NO source (nitric oxide synthase, NOS) with protein S-nitrosylation has never been directly demonstrated. Endothelial NOS (eNOS) is localized mainly on the Golgi apparatus and in plasma membrane caveolae. Here, we show by using eNOS targeted to either the Golgi or the nucleus that S-nitrosylation is concentrated at the primary site of eNOS localization. Furthermore, localization of eNOS on the Golgi enhances overall Golgi protein S-nitrosylation, the specific S-nitrosylation of N-ethylmaleimide-sensitive factor and reduces the speed of protein transport from the endoplasmic reticulum to the plasma membrane in a reversible manner. These data indicate that local NOS action generates organelle-specific protein S-nitrosylation reactions that can regulate intracellular transport processes.

Sinusoidal remodeling and angiogenesis: A new function for the liver-specific pericyte?

Pericyte is a term for vascular mural cells that make specific focal contacts with the endothelium within the microvasculature.1 For a long time, the existence and role of pericytes were neglected, but during recent years these cells have gained increasing attention, not only as contractile cells but also as obligatory regulators of vascular development, stabilization, maturation, and remodeling. Intimate interactions between pericytes and endothelial cells are reflected by observations that impairment of either of these vessel wall cell types inevitably affects the other. Pericytes also reside in liver, where they maintain specialized functions. Hepatic stellate cells (HSCs) are thought to be the pericyte equivalent in the liver.2 Much of our understanding of HSC function is derived from the broader function of pericytes in vascular beds outside the liver. In liver, HSC are well established as collagen-producing cells but are being increasingly recognized for their role in angiogenesis and vascular remodeling, processes that are particularly important in cancer and portal hypertension. This review highlights new advances in pericyte biology that are relevant to angiogenesis and vascular remodeling and places them in the context of HSC and liver pathobiology.

Intrahepatic angiogenesis and sinusoidal remodeling in chronic liver disease: New targets for the treatment of portal hypertension?

Portal hypertension accounts for the majority of morbidity and mortality that is encountered in patients with cirrhosis. Portal hypertension is initiated in large part through increases in intrahepatic vascular resistance. Fibrosis, regenerative nodule formation, and intrahepatic vasoconstriction are classical mechanisms that account for increased intrahepatic vascular resistance in cirrhosis. Recent data suggest that intrahepatic angiogenesis and sinusoidal remodeling could also be involved in sinusoidal resistance, fibrosis, and portal hypertension. While angiogenesis is defined as the formation of new vessels deriving from existing ones, sinusoidal remodeling in its pathological form associated with cirrhosis is characterized by increased mural coverage of vessels by contractile HSC. Most attention on the mechanisms of these processes has focused on the liver sinusoidal endothelial cell (SEC), the hepatic stellate cell (HSC), and the paracrine signaling pathways between these two cell types. Interventions that target these vascular structural changes have beneficial effects on portal hypertension and fibrosis in some animal studies which has stimulated interest for pursuing parallel studies in humans with portal hypertension.

Impact of Hepatopulmonary Syndrome on Quality of Life and Survival in Liver Transplant Candidates

BACKGROUND & AIMS Hepatopulmonary syndrome (HPS) affects 10%-30% of patients with cirrhosis and portal hypertension, but the impact on functional status, quality of life, and survival is poorly defined. We assessed the impact of HPS in patients evaluated for liver transplantation. METHODS We performed a prospective multicenter cohort study of patients being evaluated for liver transplantation in 7 academic centers in the United States. Patients with HPS (defined as an increased alveolar-arterial oxygen gradient with intrapulmonary vasodilation) were compared with those without HPS in terms of demographics and clinical variables. New York Heart Association functional class, quality of life, and survival were assessed. RESULTS Seventy-two patients with HPS and 146 patients without HPS were compared. There were no differences in age, sex, or etiology or severity of liver disease between the groups; however, patients with HPS were less likely to have a history of smoking (P = .03). Patients with HPS had worse New York Heart Association functional class (P = .005) and had significantly worse quality of life in certain domains compared with patients without HPS. In addition, patients with HPS also had a significantly increased risk of death compared with patients without HPS despite adjustment for age, sex, race/ethnicity, Model for End-Stage Liver Disease score, and liver transplantation (adjusted hazard ratio = 2.41; 95% confidence interval, 1.31-4.41; P = .005). CONCLUSIONS HPS was associated with a significant increase in risk of death as well as worse functional status and quality of life in patients evaluated for liver transplantation.

A Histologic Scoring System for Prognosis of Patients With Alcoholic Hepatitis

BACKGROUND & AIMS There is no histologic classification system to determine prognoses of patients with alcoholic hepatitis (AH). We identified histologic features associated with disease severity and created a histologic scoring system to predict short-term (90-day) mortality. METHODS We analyzed data from 121 patients admitted to the Liver Unit (Hospital Clinic, Barcelona, Spain) from January 2000 to January 2008 with features of AH and developed a histologic scoring system to determine the risk of death using logistic regression. The system was tested and updated in a test set of 96 patients from 5 academic centers in the United States and Europe, and a semiquantitative scoring system called the Alcoholic Hepatitis Histologic Score (AHHS) was developed. The system was validated in an independent set of 109 patients. Interobserver agreement was evaluated by weighted κ statistical analysis. RESULTS The degree of fibrosis, degree of neutrophil infiltration, type of bilirubinostasis, and presence of megamitochondria were independently associated with 90-day mortality. We used these 4 parameters to develop the AHHS to identify patients with a low (0-3 points), moderate (4-5 points), or high (6-9 points) risk of death within 90 days (3%, 19%, and 51%, respectively; P < .0001). The AHHS estimated 90-day mortality in the training and test sets with an area under the receiver operating characteristic value of 0.77 (95% confidence interval, 0.71-0.83). Interrater agreement values were 0.65 for fibrosis, 0.86 for bilirubinostasis, 0.60 for neutrophil infiltration, and 0.46 for megamitochondria. Interestingly, the type of bilirubinostasis predicted the development of bacterial infections. CONCLUSIONS We identified histologic features associated with the severity of AH and developed a patient classification system that might be used in clinical decision making.