HEPATOLOGY, Vol. 62, No. 4, 2015
3. Ito K, Groudine M. A new member of the cationic amino acid transporter family is preferentially expressed in adult mouse brain. J Biol Chem 1997;272:26780-26786. 4. Vekony N, Wolf S, Boissel JP, Gnauert K, Closs EI. Human cationic amino acid transporter hCAT-3 is preferentially expressed in peripheral tissues. Biochemistry 2001;40:12387-12394. 5. Closs EI, Rinkes IH, Bader A, Yarmush ML, Cunningham JM. Retroviral infection and the expression of cationic amino acid transporters in rodent hepatocytes. J Virol 1993;67:2097-2102.
CORRESPONDENCE
RNA knockdown in mice. After knockdown of Slc7a3 with two small interfering RNAs in mice, representative western blotting assays for the brain were conducted (results shown in fig. 7D of our article).6 We do not think the western blotting results of CAT3 knockdown in mice are off-target effects. However, further experiments are needed to address whether CAT-2A plays a role in the mouse liver under starvation. QILIN GU, PH.D.1,2 ZONGBIN CUI, PH.D.1 1 Institute of Hydrobiology Chinese Academy of Sciences Wuhan, China 2 University of Chinese Academy of Sciences Beijing, China
C 2015 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27763 Potential conflict of interest: Nothing to report.
Reply: Dr. Closs has raised some interesting questions that are associated with the production and distribution of nitric oxide in the body. Several plasma membrane proteins including cationic amino acid transporter-1 (CAT-1), CAT-2A, CAT-2B, and CAT-3 have been identified to function as selective transporters of arginine.1 Expression of CAT1 is ubiquitous,2,3 and CAT-2 has two transcripts, CAT2A and CAT2B, which are expressed in hepatocytes and macrophages, respectively4; CAT-3 is mainly expressed in the brain of adult mice.5 Their corresponding homologs in zebrafish are Slc7a1, Slc7a2, and Slc7a3a.6 It appears that Slc7a3a plays a key role in intracellular transport of arginine because the defective function of Slc7a3a cannot be compensated by Slc7a1 and Slc7a2 in fasted slc7a3a-null mutants. In addition, Slc7a3a is not expressed in zebrafish liver, so the liver is not the major organ for the production of nitric oxide; however, nitric oxide produced in other organs is soluble in body fluid and blood and thus can be delivered from the blood vessel to all tissues including the liver in zebrafish.6 Thus, the decreased production of nitric oxide in other tissues of the whole body due to the failure of arginine transport can also impair the adenosine monophosphate–activated protein kinase signaling that controls the lipid oxidation and synthesis in hepatocytes. Indeed, cyclic guanosine monophosphate levels in Slc7a3-knockdown mice under starvation are significantly lower than those in the siCtrl-group (see fig. 7F).6 Western blotting assays were performed to detect the expression of Slc7a3 in several tissues including brain, lung, heart, liver, stomach, intestine, and muscle after adenovirus-based small interfering
1327
References 1. Ignarro LJ. Nitric Oxide: Biology and Pathobiology. San Diego, CA: Academic Press, 2000. 2. Kim JW, Closs EI, Albritton LM, Cunningham JM. Transport of cationic amino acids by the mouse ecotropic retrovirus receptor. Nature 1991;352:725-728. 3. Wang H, Kavanaugh MP, North RA, Kabat D. Cell-surface receptor for ecotropic murine retroviruses is a basic amino-acid transporter. Nature 1991;352(6337):729-731. 4. Closs EI, Lyons CR, Kelly C, Cunningham JM. Characterization of the third member of the MCAT family of cationic amino acid transporters. Identification of a domain that determines the transport properties of the MCAT proteins. J Biol Chem 1993;268(28):20796-20800. 5. Ito K, Groudine M. A new member of the cationic amino acid transporter family is preferentially expressed in adult mouse brain. J Biol Chem 1997;272(42):26780-26786. 6. Gu Q, Yang X, Lin L, Li S, Li Q, Zhong S, et al. Genetic ablation of solute carrier family 7a3a leads to hepatic steatosis in zebrafish during fasting. HEPATOLOGY 2014;60:1929-1941.
C 2015 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27759 Potential conflict of interest: Nothing to report.
Transplantation for Hepatocellular Carcinoma: More Haste, Less Speed? To the Editor: We read with great interest the recent article by Halazun et al.1 As with what the Chinese proverb says “more haste less speed,” the authors provided evidence that expediting patients with hepatocellular carcinoma (HCC) to transplant at too fast a rate may adversely affect patient outcomes. The results of this study are in marked contrast to the traditional assumption that early transplantation for HCC patients would produce better survival outcomes. We appreciate the authors’ comprehensive analyses using three patient cohorts. However, we would like to raise the following comments. In this study, by comparing with the short waiting time regions (SWTRs), the long waiting time regions (LWTRs) had significantly better survivals, and the authors assumed that the better results were owing to differences in tumor biology between the two groups. The biggest limitation of this study is the lack of the data on tumor biology based on pathological examination, such as macro- and microscopic vascular invasions,
satellites, and tumor differentiation, which have been demonstrated to be independently associated with recurrence-free or overall survivals after transplantation for HCC.2 It is uncertain whether by enrolling these important confounding variables into the survival analyses the survival differences between the LWTRs and the SWTRs would disappear. The authors gave another explanation for the better survivals in the LWTRs: There was a higher proportion of patients who received transarterial chemoembolization (TACE) during the waiting period in this cohort. However, we should recognize that the decision to perform TACE or not varies among not only various regions and transplant centers, but also various surgeons and even individual patients with different waiting time in one same transplant center. Additionally, as a matter of fact, this study did not identify the significance of pretransplant TACE on long-term survivals after transplantation for HCC by multivariate analyses. The propensity-score–matched analysis has generally been used in retrospective observational studies. This analysis offers
1328
CORRESPONDENCE
investigators the ability to balance two groups of patients across all putative risk factors, and it facilitates easy inspection of achieved balance across the measured variables.3 Therefore, it is suggested that this method be applied in this study to reduce any potential bias coming from confounding variables between the LWTRs and the SWTRs, including the variable of pretransplant TACE use during the waiting period, as well as the variables of mean donor age, expanded criteria donor, and T3 tumor at listing (which all have a P value
3. Ito K, Groudine M. A new member of the cationic amino acid transporter family is preferentially expressed in adult mouse brain. J Biol Chem 1997;272:26780-26786. 4. Vekony N, Wolf S, Boissel JP, Gnauert K, Closs EI. Human cationic amino acid transporter hCAT-3 is preferentially expressed in peripheral tissues. Biochemistry 2001;40:12387-12394. 5. Closs EI, Rinkes IH, Bader A, Yarmush ML, Cunningham JM. Retroviral infection and the expression of cationic amino acid transporters in rodent hepatocytes. J Virol 1993;67:2097-2102.
CORRESPONDENCE
RNA knockdown in mice. After knockdown of Slc7a3 with two small interfering RNAs in mice, representative western blotting assays for the brain were conducted (results shown in fig. 7D of our article).6 We do not think the western blotting results of CAT3 knockdown in mice are off-target effects. However, further experiments are needed to address whether CAT-2A plays a role in the mouse liver under starvation. QILIN GU, PH.D.1,2 ZONGBIN CUI, PH.D.1 1 Institute of Hydrobiology Chinese Academy of Sciences Wuhan, China 2 University of Chinese Academy of Sciences Beijing, China
C 2015 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27763 Potential conflict of interest: Nothing to report.
Reply: Dr. Closs has raised some interesting questions that are associated with the production and distribution of nitric oxide in the body. Several plasma membrane proteins including cationic amino acid transporter-1 (CAT-1), CAT-2A, CAT-2B, and CAT-3 have been identified to function as selective transporters of arginine.1 Expression of CAT1 is ubiquitous,2,3 and CAT-2 has two transcripts, CAT2A and CAT2B, which are expressed in hepatocytes and macrophages, respectively4; CAT-3 is mainly expressed in the brain of adult mice.5 Their corresponding homologs in zebrafish are Slc7a1, Slc7a2, and Slc7a3a.6 It appears that Slc7a3a plays a key role in intracellular transport of arginine because the defective function of Slc7a3a cannot be compensated by Slc7a1 and Slc7a2 in fasted slc7a3a-null mutants. In addition, Slc7a3a is not expressed in zebrafish liver, so the liver is not the major organ for the production of nitric oxide; however, nitric oxide produced in other organs is soluble in body fluid and blood and thus can be delivered from the blood vessel to all tissues including the liver in zebrafish.6 Thus, the decreased production of nitric oxide in other tissues of the whole body due to the failure of arginine transport can also impair the adenosine monophosphate–activated protein kinase signaling that controls the lipid oxidation and synthesis in hepatocytes. Indeed, cyclic guanosine monophosphate levels in Slc7a3-knockdown mice under starvation are significantly lower than those in the siCtrl-group (see fig. 7F).6 Western blotting assays were performed to detect the expression of Slc7a3 in several tissues including brain, lung, heart, liver, stomach, intestine, and muscle after adenovirus-based small interfering
1327
References 1. Ignarro LJ. Nitric Oxide: Biology and Pathobiology. San Diego, CA: Academic Press, 2000. 2. Kim JW, Closs EI, Albritton LM, Cunningham JM. Transport of cationic amino acids by the mouse ecotropic retrovirus receptor. Nature 1991;352:725-728. 3. Wang H, Kavanaugh MP, North RA, Kabat D. Cell-surface receptor for ecotropic murine retroviruses is a basic amino-acid transporter. Nature 1991;352(6337):729-731. 4. Closs EI, Lyons CR, Kelly C, Cunningham JM. Characterization of the third member of the MCAT family of cationic amino acid transporters. Identification of a domain that determines the transport properties of the MCAT proteins. J Biol Chem 1993;268(28):20796-20800. 5. Ito K, Groudine M. A new member of the cationic amino acid transporter family is preferentially expressed in adult mouse brain. J Biol Chem 1997;272(42):26780-26786. 6. Gu Q, Yang X, Lin L, Li S, Li Q, Zhong S, et al. Genetic ablation of solute carrier family 7a3a leads to hepatic steatosis in zebrafish during fasting. HEPATOLOGY 2014;60:1929-1941.
C 2015 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27759 Potential conflict of interest: Nothing to report.
Transplantation for Hepatocellular Carcinoma: More Haste, Less Speed? To the Editor: We read with great interest the recent article by Halazun et al.1 As with what the Chinese proverb says “more haste less speed,” the authors provided evidence that expediting patients with hepatocellular carcinoma (HCC) to transplant at too fast a rate may adversely affect patient outcomes. The results of this study are in marked contrast to the traditional assumption that early transplantation for HCC patients would produce better survival outcomes. We appreciate the authors’ comprehensive analyses using three patient cohorts. However, we would like to raise the following comments. In this study, by comparing with the short waiting time regions (SWTRs), the long waiting time regions (LWTRs) had significantly better survivals, and the authors assumed that the better results were owing to differences in tumor biology between the two groups. The biggest limitation of this study is the lack of the data on tumor biology based on pathological examination, such as macro- and microscopic vascular invasions,
satellites, and tumor differentiation, which have been demonstrated to be independently associated with recurrence-free or overall survivals after transplantation for HCC.2 It is uncertain whether by enrolling these important confounding variables into the survival analyses the survival differences between the LWTRs and the SWTRs would disappear. The authors gave another explanation for the better survivals in the LWTRs: There was a higher proportion of patients who received transarterial chemoembolization (TACE) during the waiting period in this cohort. However, we should recognize that the decision to perform TACE or not varies among not only various regions and transplant centers, but also various surgeons and even individual patients with different waiting time in one same transplant center. Additionally, as a matter of fact, this study did not identify the significance of pretransplant TACE on long-term survivals after transplantation for HCC by multivariate analyses. The propensity-score–matched analysis has generally been used in retrospective observational studies. This analysis offers
1328
CORRESPONDENCE
investigators the ability to balance two groups of patients across all putative risk factors, and it facilitates easy inspection of achieved balance across the measured variables.3 Therefore, it is suggested that this method be applied in this study to reduce any potential bias coming from confounding variables between the LWTRs and the SWTRs, including the variable of pretransplant TACE use during the waiting period, as well as the variables of mean donor age, expanded criteria donor, and T3 tumor at listing (which all have a P value
