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BACKGROUND: Sphingomyelin (SM) and cholesterol are 2 key lipid partners on cell membranes and on lipoproteins. Many studies have indicated the influence of cholesterol on SM metabolism. This study examined the influence of SM biosynthesis on cholesterol metabolism. METHODS: Inducible global Sms1 KO (knockout)/global Sms2 KO mice were prepared to evaluate the effect of whole-body SM biosynthesis deficiency on lipoprotein metabolism. Tissue cholesterol, SM, ceramide, and glucosylceramide levels were measured. Triglyceride production rate and LDL (low-density lipoprotein) catabolism were measured. Lipid rafts were isolated and LDL receptor mass and function were evaluated. Also, the effects of exogenous sphingolipids on hepatocytes were investigated. RESULTS: We found that total SMS (SM synthase) depletion significantly reduced plasma SM levels. Also, the total deficiency significantly induced plasma cholesterol, apoB (apolipoprotein B), and apoE (apolipoprotein E) levels. Importantly, total SMS deficiency, but not SMS2 deficiency, dramatically decreased LDL receptors in the liver and attenuated LDL uptake through the receptor. Further, we found that total SMS deficiency greatly reduced LDL receptors in the lipid rafts, which contained significantly lower SM and significantly higher glucosylceramide, as well as cholesterol. Furthermore, we treated primary hepatocytes and Huh7 cells (a human hepatoma cell line) with SM, ceramide, or glucosylceramide, and we found that only SM could upregulate LDL receptor levels in a dose-dependent fashion. CONCLUSIONS: Whole-body SM biosynthesis plays an important role in LDL cholesterol catabolism. The total SMS deficiency, but not SMS2 deficiency, reduces LDL uptake and causes LDL cholesterol accumulation in the circulation. Given the fact that serum SM level is a risk factor for cardiovascular diseases, inhibiting SMS2 but not SMS1 should be the desirable approach.

Sphingomyelin synthase-related protein (SMSr), a member in SMS gene family, has no SM synthase activity. Although SMSr is conserved throughout the animal kingdom and ubiquitously expressed in all tested mammalian tissues, its biochemical activity in vivo is unknown till very recently. We found that SMSr is a phosphatidylethanolamine-specific phospholipase C (PE-PLC) which is involved in tissue PE steady-state regulation. We also found that Sms1/Sms2 double knockout (KO)-mediated glucosylceramide accumulation caused liver impairment, inflammation, and fibrosis. Importantly, these pathologic phenotypes could be reversed, at least partially, in Sms1/Sms2/Smsr triple KO mouse livers. Based on RNA-seq, we noticed that about 60 pro-inflammatory and fibrosis genes, including TGFb1, TNFa, IL-1a, IL-6, and collagen 1a1, were significantly upregulated in Sms1/2 double KO mouse liver compared with wild type mouse liver, while the triple KO mice could reduce them. The observations were confirmed by real-time PCRs. Further, we found that Sms1/Sms2/Smsr triple KO mice had much less liver PE-PLC activity and higher PE levels compared with the double KO mice. We also measured plasma TGFb1 whose signaling in hepatocytes participated in NASH and liver fibrosis and found the triple KO mice had significantly lower active TGFβ1 in the circulation compared with the double KO mice. Moreover, we treated the double KO mice with PE (i.p.) (10 mg/kg) every day for 7 days to mimic the situation of SMSr deficiency and we found that the active form of TGFβ1 in the plasma was also dramatically reduced in PE-treated mice compared with vehicle. These findings implicated SMSr/PE-PLC deficiency-mediated PE accumulation can prevent the development of NASH and liver fibrosis. Thus, SMSr could be drug target for the treatment of both.

Lysophosphatidylcholine acyltransferase (LPCAT3) is the major isoform of phosphatidylcholine remodeling enzyme in adipose tissue. To study the impact of LPCAT3 on adipose tissue lipid metabolism and its metabolic consequences, we prepared adipocyte-specific Lpcat3 gene knockout (KO) mice using the Cre-Loxp approach. We found LPCAT3 deficiency caused a significant reduction of LPCAT3 mRNA and activity by about 60%, and a significant reduction of polyunsaturated phosphatidylcholines in the plasma membrane. Further, the deficiency increased insulin sensitivity, reflected by glucose and insulin tolerance studies. Importantly, LPCAT3 deficiency promoted insulin-dependent insulin receptor kinase tyrosine phosphorylation and Akt phosphorylation through influencing membrane lipid rafts. Furthermore, the deficiency protected mice from high-fat, high-caloric diet-induced insulin resistance. Taken together, our findings identify LPCAT3 as one of the major players in maintaining cell membrane structure and illustrate the manipulation of cell membrane phospholipid saturation can be a new strategy to treat insulin resistance.

Background Inflammation is recognized as an important contributor of ischemia/reperfusion (I/R) damage after ischemic stroke. Sphingomyelin synthase 2 ( SMS 2), the key enzyme for the biosynthesis of sphingomyelin, can function as a critical mediator of inflammation. In the present study, we investigated the role of SMS 2 in a mouse model of cerebral I/R. Methods and Results Cerebral I/R was induced by 60‐minute transient middle cerebral artery occlusion in SMS 2 knockout ( SMS 2 ‐/‐ ) mice and wild‐type mice. Brain injury was determined by neurological deficits and infarct volume at 24 and 72 hours after transient middle cerebral artery occlusion. Microglia activation and inflammatory factors were detected by immunofluorescence staining, flow cytometry, western blot, and RT ‐ PCR . SMS 2 deficiency significantly improved neurological function and minimized infarct volume at 72 hours after transient middle cerebral artery occlusion. The neuroprotective effects of SMS 2 deficiency were associated with (1) suppression of microglia activation through Toll‐like receptor 4/nuclear factor kappa‐light‐chain‐enhancer of activated B cells pathway and (2) downregulation of the level of galactin‐3 and other proinflammatory cytokines. The mechanisms underlying the beneficial effects of SMS 2 deficiency may include altering sphingomyelin components in lipid raft fractions, thus impairing the recruitment of Toll‐like receptor 4 to lipid rafts and subsequently reducing Toll‐like receptor 4/myeloid differentiation factor 2 complex formation on the surface of microglia. Conclusions SMS 2 deficiency ameliorated inflammatory injury after cerebral I/R in mice, and SMS 2 may be a key modulator of Toll‐like receptor 4/nuclear factor kappa‐light‐chain‐enhancer of activated B cells activation by disturbing the membrane component homeostasis during cerebral I/R.

Background: The combination of Traditional Chinese medicine and Western medicine (TCM+WM) has been widely used in the treatment of glomerulosclerosis, but the results are still controversial. This study will assess the clinical efficacy of TCM+WM for glomerulosclerosis and provide evidence-based medical data via meta-analysis. Method: The MEDLINE, EMBASE, PubMed, Cochrane Central Registry of Controlled Trials, and multiple Chinese databases (Wan Fang, CNKI, and VIP) were searched for randomized controlled trials (RCT) that compared the effects of WM and TCM+WM. Review Manager 5.3 software was used for the meta-analysis of selected studies, and appropriate tests were performed to determine the quality, heterogeneity and sensitivity of these studies. Results: Sixteen RCTs met the inclusion criteria and were selected for the analysis. Compared with the placebo or WM-treated glomerulosclerosis patients, TCM+WM intervention significantly improved renal function indices including 24-hour urine protein quantity (24 h U-Pro), serum creatinine (Scr), blood urea nitrogen (BUN), creatinine clearance (Ccr). In addition, the serum albumin (ALB), triglyceride (TG), and cholesterol (CHOL) levels were also significantly improved (P

TGF-beta 1, via Smad-dependent or Smad-independent signaling, has a central role in the pathogenesis of renal fibrosis. This pathway has been recognized as a potential target for antifibrotic therapy. Here, we identified GQ5, a small molecular phenolic compound isolated from the dried resin of Toxicodendron vernicifluum, as a potent and selective inhibitor of TGF-beta 1-induced Smad3 phosphorylation. In TGF-beta 1-stimulated renal tubular epithelial cells and interstitial fibroblast cells, GQ5 inhibited the interaction of Smad3 with TGF-beta type I receptor (T beta RI) by blocking binding of Smad3 to SARA, suppressed subsequent phosphorylation of Smad3, reduced nuclear translocation of Smad2, Smad3, and Smad4, and downregulated the transcription of major fibrotic genes such as alpha-smooth muscle actin (alpha-SMA), collagen I, and fibronectin. Notably, intraperitoneal administration of GQ5 in rats immediately after unilateral ureteral obstruction (UUO) selectively inhibited Smad3 phosphorylation in UUO kidneys, suppressed renal expression of alpha-SMA, collagen I, and fibronectin, and resulted in impressive renal protection after obstructive injury. Late administration of GQ5 also effectively attenuated fibrotic lesions in obstructive nephropathy. In conclusion, our results suggest that GQ5 hinders' renal fibrosis in rats by selective inhibition of TGF-beta 1-induced Smad3 phosphorylation.

Background— Emerging clinical evidence demonstrates high prevalence of QTc prolongation and complex ventricular arrhythmias in patients with anti-Ro antibody (anti-Ro Ab)–positive autoimmune diseases. We tested the hypothesis that anti-Ro Abs target the HERG (human ether-a-go-go–related gene) K + channel, which conducts the rapidly activating delayed K + current, I Kr , thereby causing delayed repolarization seen as QT interval prolongation on the ECG. Methods and Results— Anti-Ro Ab–positive sera, purified IgG, and affinity-purified anti-52kDa Ro Abs from patients with autoimmune diseases and QTc prolongation were tested on I Kr using HEK293 cells expressing HERG channel and native cardiac myocytes. Electrophysiological and biochemical data demonstrate that anti-Ro Abs inhibit I Kr to prolong action potential duration by directly binding to the HERG channel protein. The 52-kDa Ro antigen–immunized guinea pigs showed QTc prolongation on ECG after developing high titers of anti-Ro Abs, which inhibited native I Kr and cross-reacted with guinea pig ERG channel. Conclusions— The data establish that anti-Ro Abs from patients with autoimmune diseases inhibit I Kr by cross-reacting with the HERG channel likely at the pore region where homology between anti–52-kDa Ro antigen and HERG channel is present. The animal model of autoimmune-associated QTc prolongation is the first to provide strong evidence for a pathogenic role of anti-Ro Abs in the development of QTc prolongation. It is proposed that adult patients with anti-Ro Abs may benefit from routine ECG screening and that those with QTc prolongation should receive counseling about drugs that may increase the risk for life-threatening arrhythmias.

Enhanced interaction between vascular endothelial cells and circulating leukocytes, as a result of transcriptional activation of cell adhesion molecules (CAM), helps establish a proinflammatory milieu contributing to the pathogenesis of chronic hypoxia-induced pulmonary hypertension. The molecular switch that dictates CAM transactivation is not clearly defined. Our goal was to determine the involvement of the transcriptional modulator megakaryocytic leukemia 1 (MKL1), also known as myocardin-related transcription factor A (MRTF-A), in CAM transactivation and the underlying mechanism. We report here that compared with wild-type littermates, MKL1/MRTF-A knockout mice were more resistant to the development of hypoxia-induced pulmonary hypertension when exposed to low oxygen pressure. Notably, CAM induction in knockout mice was significantly attenuated with a concomitant reduction of leukocyte adhesion. In cultured vascular endothelial cells, overexpression of MKL1/MRTF-A enhanced, whereas depletion of MKL1/MRTF-A dampened, hypoxia-induced CAM transactivation. In response to hypoxia, MKL1/MRTF-A formed a complex with NF-κB on the CAM promoters. Of interest, MKL1/MRTF-A was responsible for recruiting a histone H3 lysine 4 methyltransferase complex to the CAM promoters. Finally, endothelial-specific silencing of ASH2 and WDR5, 2 key components of the histone H3 lysine 4 methyltransferase complex, ameliorated hypoxia-induced pulmonary hypertension in mice. In conclusion, our data suggest that MKL1/MRTF-A, by coordinating key epigenetic alterations on CAM promoters, provides a critical link to hypoxia-induced endothelial malfunction and contributes to the pathogenesis of hypoxia-induced pulmonary hypertension.

Objective— Phospholipid transfer protein (PLTP) is highly expressed in adipose tissues. Thus, the effect of adipose tissue PLTP on plasma lipoprotein metabolism was examined. Approach and Results— We crossed PLTP-Flox-ΔNeo and adipocyte protein 2 (aP2)-Cre recombinase (Cre) transgenic mice to create PLTP-Flox-ΔNeo/aP2-Cre mice that have a 90 and a 60% reduction in PLTP mRNA in adipose tissue and macrophages, respectively. PLTP ablation resulted in a significant reduction in plasma PLTP activity (22%), high-density lipoprotein-cholesterol (21%), high-density lipoprotein-phospholipid (20%), and apolipoprotein A-I (33%) levels, but had no effect on nonhigh-density lipoprotein levels in comparison with those of PLTP-Flox-ΔNeo controls. To eliminate possible effects of PLTP ablation by macrophages, we lethally irradiated PLTP-Flox-ΔNeo/aP2-Cre mice and PLTP-Flox-ΔNeo mice, and then transplanted wild-type mouse bone marrow into them to create wild-type→PLTP-Flox-ΔNeo/aP2-Cre and wild-type→PLTP-Flox-ΔNeo mice. Thus, we constructed a mouse model (wild-type→PLTP-Flox-ΔNeo/aP2-Cre) with PLTP deficiency in adipocytes but not in macrophages. These knockout mice also showed significant decreases in plasma PLTP activity (19%) and cholesterol (18%), phospholipid (17%), and apolipoprotein A-I (26%) levels. To further investigate the mechanisms behind the reduction in plasma apolipoprotein A-I and high-density lipoprotein lipids, we measured apolipoprotein A-I–mediated cholesterol efflux in adipose tissue explants and found that endogenous and exogenous PLTP significantly increased cholesterol efflux from the explants. Conclusions— Adipocyte PLTP plays a small but significant role in plasma PLTP activity and promotes cholesterol efflux from adipose tissues.

Rationale: Endothelial dysfunction inflicted by inflammation is found in a host of cardiovascular pathologies. One hallmark event in this process is the aggregation and adhesion of leukocyte to the vessel wall mediated by the upregulation of adhesion molecules (CAM) in endothelial cells at the transcriptional level. The epigenetic modulator(s) of CAM transactivation and its underlying pathophysiological relevance remain poorly defined. Objective: Our goal was to determine the involvement of Brahma related gene 1 (Brg1) and Brahma (Brm) in CAM transactivation and its relevance in the pathogenesis of atherosclerosis. Methods and Results: In the present study, we report that proinflammatory stimuli augmented the expression of Brg1 and Brm in vitro in cultured endothelial cells and in vivo in arteries isolated from rodents. Overexpression of Brg1 and Brm promoted while knockdown of Brg1 and Brm abrogated transactivation of adhesion molecules and leukocyte adhesion induced by inflammatory signals. Brg1 and Brm interacted with and were recruited to the CAM promoters by nuclear factor κB/p65. Conversely, depletion of Brg1 and Brm disrupted the kinetics of p65 binding on CAM promoters and crippled CAM activation. Silencing of Brg1 and Brm also altered key epigenetic changes associated with CAM transactivation. Of intrigue, 17β-estradiol antagonized both the expression and activity of Brg1/Brm. Most importantly, endothelial-targeted elimination of Brg1/Brm conferred atheroprotective effects to Apoe -/- mice on a Western diet. Conclusions: Our data suggest that Brg1 and Brm integrate various proinflammatory cues into CAM transactivation and endothelial malfunction and, as such, may serve as potential therapeutic targets in treating inflammation-related cardiovascular diseases.

Objective— The liver is one of the critical organs for lipoprotein metabolism and a major source for phospholipid transfer protein (PLTP) expression. The effect of liver-specific PLTP deficiency on plasma lipoprotein production and metabolism in mice was investigated. Approach and Results— We created a liver-specific PLTP-deficient mouse model. We measured plasma high-density lipoprotein (HDL) and apolipoprotein B (apoB)–containing lipoprotein (or non-HDL) levels and their production rates. We found that hepatic ablation of PLTP leads to a significant decrease in plasma PLTP activity, HDL lipids, non-HDL lipids, apoAI, and apoB levels. In addition, nuclear magnetic resonance examination of lipoproteins showed that the deficiency decreases HDL and apoB-containing lipoprotein particle numbers, as well as very low-density lipoprotein particle size, which was confirmed by electron microscopy. Moreover, HDL particles from the deficient mice are lipid-poor ones. To unravel the mechanism, we evaluated the apoB and triglyceride production rates. We found that hepatic PLTP deficiency significantly decreases apoB and triglyceride secretion rates. To investigate the role of liver PLTP on HDL production, we set up primary hepatocyte culture studies and found that the PLTP-deficient hepatocytes produce less nascent HDL. Furthermore, we found that exogenous PLTP promotes nascent HDL production through an ATP-binding cassette A 1-mediated pathway. Conclusions— Liver-specific PLTP deficiency significantly reduces plasma HDL and apoB-containing lipoprotein levels. Reduction of production rates of both particles is one of the mechanisms.

Objective— Sphingolipid de novo biosynthesis is related to nonalcoholic fatty liver disease or hepatic steatosis. However, the mechanism is still unclear. Sphingomyelin synthase (SMS), using ceramide as one of the substrates to produce sphingomyelin, sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. SMS2 is the major isoform in liver. Approach and Results— To investigate the relationship between liver SMS2 activity–mediated sphingolipid changes and hepatic steatosis, we used 2 mouse models: Sms2 liver-specific transgenic and Sms2 knockout mice. We found that Sms2 liver-specific transgenic livers have lower ceramide and higher sphingomyelin, whereas Sms2 knockout livers have higher ceramide and lower sphingomyelin. We also found that liver Sms2 overexpression promoted fatty acid uptake and liver steatosis, whereas Sms2 deficiency had an opposite effect in comparison with their respective controls. Importantly, the exogenous ceramide supplementation to Huh7 cells, a human hepatoma cell line, reduced the expression of peroxisome proliferator-activated receptor γ2 and its target genes, Cd36 and Fsp27 . Peroxisome proliferator-activated receptor γ reporter analysis confirmed this phenomenon. Furthermore, peroxisome proliferator-activated receptor γ antagonist treatment significantly decreased triglyceride accumulation in Sms2 liver-specific transgenic liver. Conclusions— We attributed these effects to ceramide that can suppress peroxisome proliferator-activated receptor γ2, thus reducing the expression of Cd36 and Fsp27 and reducing liver steatosis. After all, SMS2 inhibition in the liver could diminish liver steatosis.

Background and objectives: Sphingolipids, particularly sphingosine 1-phosphate (S1P), play an important role in the cardiovascular homeostasis. Recently, we revealed that endothelial de novo biosynthesis of sphingolipids is very important to control vascular functions and blood pressure. We discovered that in blood vessels, particularly in endothelial cells, Nogo-B, a membrane protein of the endoplasmic reticulum, inhibits serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of de novo production of sphingolipids, to impact vascular tone and blood pressure. Indeed, mice lacking Nogo-B are protected from angiotensin II-induced hypertension, and pharmacological inhibition of SPT by myriocin reinstates high blood pressure in absence of Nogo-B, suggesting that the upregulation of SPT activity exerts anti-hypertensive functions. Thus, the goal of this study is to investigate the role of SPT in vascular functions and blood pressure regulation by using novel genetic mouse models. Methods: The SBP was evaluated in 14 weeks old mice heterozygous for Sptlc2 ( Sptlc2 +/- ) or lacking Sptlc2 specifically in endothelial cells (ECKO Sptlc2 ) and smooth muscle cells (SMCKO Sptlc2 ) by using tail-cuff system. Vascular reactivity of isolated mesenteric arteries was assessed ex-vivo by using the pressure myograph system. Results: Sptlc2 +/- , ECKO Sptlc2 and SMCKO Sptlc2 mice were hypertensive compared to their respective controls ( Sptlc2 +/- 128.9±2.6 vs. WT 112.1±2.6 mmHg; ECKO Sptlc2 125.5±1.8, SMCKO Sptlc2 127.2±0.6 vs. Sptlc2 f/f 106±0.84 mmHg) and developed endothelial dysfunction as shown by the impaired vasodilation in response to acetylcholine (EC 50 Sptlc2 +/- 1.48x10 -6 M vs. WT 4.46x10 -7 M; Emax ECKO Sptlc2 73.2±3.3% vs. Sptlc2 f/f 95.3±1.1%), as well as to flow (Emax: Sptlc2 +/- 23.3±1.4 μm vs. WT 42.9±4.4 μm; ECKO Sptlc2 19.9±0.9 μm vs. Sptlc2 f/f 41.3±3.1 μm). Conclusion: This study demonstrates the important role of SPT, thus the de novo production of sphingolipids, in controlling blood flow and pressure homeostasis, and provides the ground for the development of alternative therapeutic strategies to manage high blood pressure.

Objective— Sphingomyelin synthase (SMS) catalyzes the conversion of ceramide to sphingomyelin and sits at the crossroads of sphingolipid biosynthesis. SMS has 2 isoforms: SMS1 and SMS2. Although they have the same SMS activity, they are different enzymes with distinguishable subcellular localizations and cell expression patterns. It is conceivable that these differences could yield different consequences, in terms of sphingolipid metabolism and its related atherogenesis. Methods and Results— We created Sms1 gene knockout mice and found that Sms1 deficiency significantly decreased plasma, liver, and macrophage sphingomyelin (59%, 45%, and 54%, respectively), but only had a marginal effect on ceramide levels. Surprisingly, we found that Sms1 deficiency dramatically increased glucosylceramide and GM3 levels in plasma, liver, and macrophages (4- to 12-fold), whereas Sms2 deficiency had no such effect. We evaluated the total SMS activity in tissues and found that Sms1 deficiency causes 77% reduction in SMS activity in macrophages, indicating SMS1 is the major SMS in macrophages. Moreover, Sms1 -deficient macrophages have a significantly higher glucosylceramide synthase activity. We also found that Sms1 deficiency significantly attenuated toll-like 4 receptor–mediated nuclear factor-κB and mitogen-activated protein kinase activation after lipopolysaccharide treatment. To evaluate atherogenicity, we transplanted Sms1 knockout mouse bone marrow into low-density lipoprotein receptor knockout mice ( Sms1 –/– →Ldlr –/– ). After 3 months on a western diet, these animals showed a significant decrease of atherosclerotic lesions in the root and the entire aorta (35% and 44%, P P →Ldlr –/– mice. Conclusion— Sms1 deficiency decreases sphingomyelin, but dramatically increases the levels of glycosphingolipids. Atherosclerosis in Sms1 –/– →Ldlr –/– mice is significantly decreased.

High-density lipoprotein (HDL) has been proposed to have several antiatherosclerotic properties, including the ability to mediate macrophage cholesterol efflux, antioxidant capacity, antiinflammatory properties, nitric oxide–promoting activity, and ability to transport proteins with their own intrinsic biological activities.1 HDL particles are critical acceptors of cholesterol from lipid-laden macrophages and thereby participate in the maintenance of net cholesterol balance in the arterial wall and in the reduction of proinflammatory responses by arterial cholesterol-loaded macrophages. The pathways that regulate HDL-mediated macrophage cholesterol efflux and disposition of cholesterol involve cell membrane–bound transporters, plasma lipid acceptors, plasma proteins and enzymes, and hepatic cellular receptors (Figure 1). From the earliest proposed concept for HDL-mediated cholesterol efflux,2,3 the concentration of the cholesterol content in HDL particles has been considered a surrogate measurement for the efficiency of the “reverse cholesterol transport” (RCT) process; however, macrophage-derived cholesterol represents a minor component of the cholesterol transported by HDL particles.4–7 One important pathway for cholesterol-mediated efflux from macrophage foam cells involves interaction between the ATP-binding cassette transporter A1 (ABCA1) and cholesterol-deficient and phospholipid-depleted apolipoprotein (apo) A-I complexes (pre-β migrating HDL or very small HDL [HDL-VS]; Figure 2).1,8 Subsequently, the ATP-binding cassette transporter G1 (ABCG1) mediates macrophage cholesterol efflux through interactions (Figure 3) with spherical, cholesterol-containing α-HDL particles (small HDL [HDL-S], medium HDL [HDL-M], large HDL [HDL-L], and very large (HDL-VL).1 In contrast, the scavenger receptor class B type I (SR-BI) is a multifunctional receptor that mediates bidirectional lipid transport in the macrophage, which is dependent on the content of cholesterol in lipid-laden macrophages. A more established role for SR-BI in cholesterol trafficking involves selective uptake of cholesteryl esters from mature HDL by the liver. Recent studies suggest that polymorphisms in SR-BI contribute to the functional capacity of this cholesterol …

Objective— We used the sphingomyelin (SM) synthase 2 ( Sms2 ) gene knockout (KO) approach to test our hypothesis that selectively decreasing plasma lipoprotein SM can play an important role in preventing atherosclerosis. Methods and Results— The sphingolipid de novo synthesis pathway is considered a promising target for pharmacological intervention in atherosclerosis. However, its potential is hampered because the substance’s atherogenic mechanism is not completely understood. We prepared Sms2 and apolipoprotein E ( Apoe ) double-KO mice. They showed a significant decrease in plasma lipoprotein SM levels (35%, P P Sms2 KO/ Apoe KO mice showed a significant reduction in atherosclerotic lesions of the aortic arch and root (52%, P Sms2 KO/ Apoe KO brachiocephalic artery contained significantly less SM, ceramide, free cholesterol, and cholesteryl ester (35%, 32%, 58%, and 60%, respectively; P Apoe KO brachiocephalic artery. Conclusion— Decreasing plasma SM levels through decreasing SMS2 activity could become a promising treatment for atherosclerosis.

Background: Thyroid cancer is the most common endocrine tumor. Our previous studies have demonstrated that curcumin can induce apoptosis in human papillary thyroid carcinoma BCPAP cells. However, the underlined mechanism has not been clearly elucidated. Endoplasmic reticulum (ER) is a major organelle for synthesis, maturation, and folding proteins as well as a large store for Ca2+. Overcoming chronically activated ER stress by triggering pro-apoptotic pathways of the unfolded protein response (UPR) is a novel strategy for cancer therapeutics. Our study aimed to uncover the ER stress pathway involved in the apoptosis caused by curcumin. Methods: BCPAP cells were treated with different doses of curcumin (12.5–50 μM). Annexin V/PI double staining was used to determine cell apoptosis. Rhod-2/AM calcium fluorescence probe assay was performed to measure the calcium level of endoplasmic reticulum. Western blot was used to examine the expression of ER stress marker C/EBP homologous protein 10 (CHOP) and glucose-regulated protein 78 (GRP78). X-box binding protein1 (XBP-1) spliced form was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Results: Curcumin significantly inhibited anchorage-independent cell growth and induced apoptosis in BCPAP cells. Curcumin induced ER stress and UPR responses in a dose- and time-dependent manner, and the chemical chaperone 4-phenylbutyrate (4-PBA) partially reversed the antigrowth activity of curcumin. Moreover, curcumin significantly increased inositol-requiring enzyme 1α (IRE1α) phosphorylation and XBP-1 mRNA splicing to induce a subsets of ER chaperones. Increased cleavage of activating transcription factor 6 (ATF6), which enhances expression of its downstream target CHOP was also observed. Furthermore, curcumin induced intracellular Ca2+ influx through inhibition of the sarco-endoplasmic reticulum ATPase 2A (SERCA2) pump. The increased cytosolic Ca2+ then bound to calmodulin to activate calcium/calmodulin-dependent protein kinase II (CaMKII) signaling, leading to mitochondrial apoptosis pathway activation. Ca2+ chelator BAPTA partially reversed curcumin-induced ER stress and growth suppression, confirming the possible involvement of calcium homeostasis disruption in this response. Conclusions: Curcumin inhibits thyroid cancer cell growth, at least partially, through ER stress-associated apoptosis. Our observations provoked that ER stress activation may be a promising therapeutic target for thyroid cancer treatment.

Objective— A high plasma sphingomyelin level has been associated with subclinical atherosclerosis, coronary artery disease, and worse prognosis in subjects with acute coronary syndromes. We wanted to assess the predictive value of plasma sphingomyelin levels for incident coronary heart disease (CHD) events in the Multi-Ethnic Study of Atherosclerosis. Methods and Results— The plasma sphingomyelin level was measured in 6809 of 6814 subjects (age, mean±SD, 62.2±10.2 years) participating in the Multi-Ethnic Study of Atherosclerosis, a population-based cohort study of adults free of clinical cardiovascular disease at baseline recruited at 6 clinic sites in the United States. The subjects consisted of 52.8% females, 38.5% whites, 11.8% Chinese, 27.8% blacks, and 21.9% Hispanics. Cox proportional hazard analysis was used to examine the association between plasma sphingomyelin level and 5 years of adjudicated incident CHD events, including myocardial infarction, resuscitated cardiac arrest, angina, CHD-related death, and revascularization (coronary artery bypass grafting or percutaneous transluminal coronary angioplasty). The mean±SD plasma sphingomyelin level was 48.0±16.0 mg/dL. A total of 189 subjects had an adjudicated CHD event during the 5 years of follow-up. In the Kaplan-Meier analysis, subjects with a plasma sphingomyelin level higher than the sex-specific median had a similar event-free survival rate compared with subjects with a plasma sphingomyelin level at or less than the sex-specific median (97.16% versus 97.00%; log rank P =0.71). In the univariate Cox proportional hazard analysis, the plasma sphingomyelin level was not a predictor of an incident CHD event (hazard ratio, 0.992 [0.982–1.004]; P =0.09). In our multistage multivariate Cox proportional hazard models, a higher plasma sphingomyelin level had a modest negative association with incident CHD events when total cholesterol, high-density lipoprotein, and triglycerides were included in the model (hazard ratio, 0.985 [0.973–0.996]; P =0.008) and also in our full model after adjusting for age, sex, total cholesterol, high-density lipoprotein, triglycerides, diabetes mellitus, cigarette smoking, systolic and diastolic blood pressure, medication use for blood pressure, and 3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitor use (hazard ratio, 0.984 [0.973–0.996]; P =0.002). In other models, the plasma sphingomyelin level was not associated with incident CHD events. Conclusion— A high plasma sphingomyelin level is not associated with an increased risk of incident CHD in population-based adults free of clinical cardiovascular disease at baseline.

Background— It has been proposed that plasma sphingomyelin (SM) plays a very important role in plasma lipoprotein metabolism and atherosclerosis. Sphingomyelin synthase (SMS) is the last enzyme for SM de novo biosynthesis. Two SMS genes, SMS1 and SMS2, have been cloned and characterized. Methods and Results— To evaluate the in vivo role of SMS2 in SM metabolism, we prepared SMS2 knockout (KO) and SMS2 liver-specific transgenic (LTg) mice and studied their plasma SM and lipoprotein metabolism. On a chow diet, SMS2 KO mice showed a significant decrease in plasma SM levels (25%, P P P P P Conclusions— We therefore believe that regulation of liver SMS2 activity could become a promising treatment for atherosclerosis.

Background Cyclin B1 (CLB1) is necessary for mitotic initiation in mammalian cells and plays important roles in cancer development. Therefore, a potential strategy in cancer therapy is to suppress the activity of CLB1 by delivering antisense constructs of CLB1 into tumor cells. In previous CLB1 studies, antisense constructs with a short half life were often used and these constructs might not persistently inhibit CLB1. Methods We successfully created a recombinant plasmid encoding the full-length antisense cDNA of mouse cyclin B1 (AS-mCLB1) and transfected this construct to the murine Lewis lung carcinoma (LL/2) and CT-26 colon carcinoma (CT-26) cells. We isolated clones of LL/2 and CT-26 transfectants with stable expression of AS-mCLB1. Reverse transcriptional polymerase chain reaction (RT-PCR) and Western blot were applied to detect the expression of the mRNA and protein levels of CLB1. To further test the efficacy of this strategy in vivo, AS-mCLB1-expressing LL/2 and CT-26 transfectants were implanted into mice. Results We found the expression of the mRNA and protein levels of CLB1 decrease in these transfectants. The inhibition of CLB1 caused prominent G1 arrest, abnormal morphology, retarded cell growth and an increase in apoptosis. In AS-mCLB1-expressing LL/2 and CT-26 transfectants implanted mice, tumorigenicity was effectively suppressed compared with the controls. In addition, the expression of AS-mCLB1 also significantly increases the survival duration of implanted animals. Conclusion AS-mCLB1 is likely to be useful in future cancer therapy, which may be associated with its ability to down-regulate the expression of CLB1 and then induce G1 arrest and apoptosis in tumor cells.

Objective— Phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism and atherosclerosis. PLTP gene knockout (KO) mice show significant reduction of plasma cholesterol levels. Because small intestine is one of the major tissue expressing PLTP, we hypothesize that PLTP deficient small intestine absorbs less cholesterol, thus contributing to the diminishing of cholesterol levels in the plasma. Methods and Results— We used dual-labeled cholesterol/sitostanol feeding approach to study cholesterol absorption in PLTP KO and WT mice. We found that PLTP KO mice absorb significant less cholesterol than WT mice. Primary enterocytes isolated from PLTP KO enterocytes took up significant less cholesterol. Moreover, we observed that Niemann-Pick C1-like 1 (NPC1L1) mRNA levels were significantly decreased in the small intestine of PLTP KO mice. Next, we studied the secretion of cholesterol by enterocytes. The amounts of cholesterol transported to plasma and liver were significantly reduced in PLTP KO mice, compared with WT animals. Studies with isolated PLTP KO enterocytes revealed that the secretion of cholesterol via chylomicron and intestinal-HDL was significantly reduced. Furthermore, ATP-binding cassette transporters (ABC) A1 mRNA and microsomal triglyceride transfer protein (MTP) activity levels were significantly decreased in PLTP KO small intestine. Conclusion— These results indicate that PLTP deficiency results in reduced cholesterol uptake as well as secretion by the intestine. We suggest that PLTP could be a useful target to lower plasma cholesterol levels, thus reducing atherosclerosis.

Recent studies indicated that protective function of HDL may be more representative than its concentration. Apolipoprotein AI (apoAI), the major protein of HDL, is nitrosylated in vivo to nitrated apoAI (NT-apoAI) that might cause dysfunction. We measured plasma NT-apoAI and apoAI levels in 777 patients with coronary artery disease (CAD) by ELISA, and found that median NT-apoAI/apoAI ratio was significantly higher in diabetes mellitus (DM) (n=327) versus non-diabetic patients (n=450). Further analysis indicated that DM, thiobarbituric acid-reactive substances and C-reactive protein levels were independent predictors of higher NT-apoAI/apoAI ratio. There was negative correlation between NT-apoAI/apoAI and use of anti-platelet and lipid lowering drugs. The cholesterol efflux capacity of plasma from 67 individuals with differing NT-apoAI but similar apoAI levels from macrophages in vitro was negatively correlated with NT-apoAI/apoAI ratio. In conclusion, higher NT-apoAI/apoAI ratio is significantly associated with DM in this relatively large German cohort with CAD and may contribute to associated complications by reducing cholesterol efflux capacity.

Objective— The aim of the present study was to assess the effect of α-tocopherol, the main vitamin E isomer on phosphatidylserine (PS) exposure at the surface of circulating erythrocytes, and to determine consequences on erythrocyte properties. Methods and Results— In vitro α-tocopherol enrichment of isolated erythrocytes significantly decreased PS externalization as assessed by lower Annexin V-fluorescein isothiocyanate labeling. Plasma phospholipid transfer protein (PLTP) transfers vitamin E, and both α-and γ-tocopherol accumulated in circulating erythrocytes from PLTP-deficient homozygous (PLTP −/− ) mice as compared with wild-type mice. In agreement with in vitro studies, vitamin E–enriched erythrocytes from PLTP −/− mice displayed fewer externalized PS molecules than wild-type controls (−64%, P −/− erythrocytes was accompanied by impairment of their procoagulant properties, with a 20% increase in clotting time as compared with wild-type controls ( P −/− mice compared with wild-type mice. Conclusions— Vitamin E modifies PS externalization in circulating erythrocytes, thus modulating in vivo their PS-dependent procoagulant properties.

Objective— Plasma phospholipid transfer protein (PLTP) mediates both net transfer and exchange of phospholipids between different lipoproteins. Animal studies have shown that it is closely related to the development of atherosclerosis. PLTP-deficient mice have demonstrated increased antioxidation potential as well as a decrease in apolipoprotein B secretion and atherosclerotic lesions. In humans, high PLTP is associated with type II diabetes and obesity. Methods and Results— To assess the relationship between PLTP activity and coronary artery disease (CAD), a novel, high-throughput method to measure plasma PLTP activity was used, relating it to CAD in 1102 cases and 444 controls. This demonstrated that PLTP activity in patients with CAD was significantly higher than in controls (25.5 versus 22.4 pmol/μL per h; P Conclusions— These findings indicate that PLTP activity is positively and independently related to CAD and suggest that (1) prospective studies to evaluate this relationship are warranted and (2) PLTP should be considered a therapeutic target.

Objective— Plasma phospholipid transfer protein (PLTP) is involved in the metabolism of HDL and apolipoprotein B (apoB)-containing lipoproteins. Atherosclerosis susceptibility is decreased in mice with PLTP deficiency that is associated with decreased liver production of apoB-containing lipoproteins and increase in their antioxidant. To investigate additionally the effect of PLTP on the development of atherosclerosis, we overexpressed PLTP in mice. Methods and Results— PLTP was overexpressed in apoE knockout mice using an adenovirus-associated virus (AAV)-mediated system. Plasma PLTP activity was 1.3- to 2-fold higher in mice injected with AAV-PLTP than in mice injected with control AAV-GFP, and PLTP levels were sustained during the experiment period (4 months). We show that 2-fold increased PLTP activity results in (1) a decrease in HDL cholesterol, HDL phospholipid, and apoAI levels; (2) a decrease in vitamin E contents in total plasma and in individual lipoprotein fractions; (3) an increase in lipoprotein oxidizability as assessed by copper-induced formation of conjugated dienes; (4) an increase in autoantibodies against oxidized apoB-containing particles; and (5) an increase in atherosclerosis lesions in proximal aorta. Conclusions— These observations indicate that elevated plasma PLTP levels constitute a novel, long-term risk factor for atherosclerosis.

Increased phospholipid transfer protein (PLTP) activity has been found to be associated with diabetes, obesity, and metabolic syndrome in humans. However, whether or not PLTP has a direct effect on insulin sensitivity and obesity is largely unknown. Here we analyzed the effect by using PLTP knockout (PLTP-/-) mouse model. Although, PLTP-/- mice have normal body-weight-gain under chow diet, these mice were protected from high-fat-diet-induced obesity and insulin resistance, compared with wild type mice. In order to understand the mechanism, we evaluated insulin receptor and Akt activation and found that PLTP deficiency significantly enhanced phosphorylated insulin receptor and Akt levels in high-fat-diet fed mouse livers, adipose tissues, and muscles after insulin stimulation, while total Akt and insulin receptor levels were unchanged. Moreover we found that the deficiency induced significantly more GLUT4 immunostaining in the plasma membranes of adipocytes and muscle cells after insulin stimulation. Finally, we found that PLTP deficient hepatocytes had less sphingomyelin and free cholesterol in the plasma membrane and lipid raft than that of controls and this may provide a molecular basis for PLTP deficiency-mediated increasing in insulin sensitivity. We have concluded that PLTP deficiency leads to an improvement in tissue and whole-body insulin sensitivity in high-fat-diet induced insulin resistance mice model. Foundation:National Natural Science Foundation of China (# 81070247, 81170785) and Taishan Scholar Foundation of Shandong Province.

Abstract —Recently, the class B, type I scavenger receptor (SR-BI) has been shown to mediate the selective uptake of high density lipoprotein (HDL) cholesteryl esters (CEs), ie, lipid uptake independent of HDL holoparticle uptake. In vivo, this selective uptake delivers CEs to the liver for excretion and to steroidogenic tissues for hormone synthesis. Probucol, a hydrophobic antioxidant drug, lowers plasma cholesterol in humans and rodents and may inhibit progression of atherosclerosis and postangioplasty restenosis. In this study, the effect of probucol on HDL selective CE uptake was investigated in mice and in cells expressing SR-BI. Probucol feeding lowered plasma HDL cholesterol and markedly increased selective CE uptake from HDL in the liver and adrenal glands. However, probucol did not alter SR-BI protein levels in membranes from these organs. When incubated with control Chinese hamster ovary (CHO) cells, HDL isolated from probucol-treated mice (P-HDL) and HDL from control mice (C-HDL) showed similar low selective uptake of CEs. However, when incubated with SR-BI–transfected CHO cells, P-HDL showed a 2-fold increase in selective uptake compared with C-HDL. In an adrenal cell line (Y1-BS1), which expresses SR-BI in an adrenocorticotropic hormone–inducible manner, P-HDL showed significantly greater selective CE uptake than did C-HDL, and the differential response was amplified by adrenocorticotropic hormone treatment. In contrast to P-HDL, incorporation of this compound into HDL in vitro did not result in stimulation of selective CE uptake by SR-BI–transfected CHO cells, even though a significant mass of probucol could be detected in the HDL preparation. The specific interaction of P-HDL with SR-BI in cell culture could be observed after only 24 hours of probucol feeding, when there were minimal changes in HDL size and composition. Thus, probucol or one of its metabolites increases selective CE uptake in vivo by modifying HDL in a way that causes enhanced interaction with SR-BI. The increased interaction of P-HDL with SR-BI in the liver and arterial wall may be partly responsible for the effects of probucol on atherosclerosis and restenosis.

Clock is a key transcription factor that controls circadian regulation. Here, we show that dominant-negative Clock mutant protein (Clock Δ19/Δ19 ) enhances hyperlipidemia and atherosclerosis in Apoe -/- mice. Clk [[Unable to Display Character: ∆]]19/[[Unable to Display Character: ∆]]19 Apoe -/- mice display hypercholesterolemia, show enhanced cholesterol absorption and express more NPC1L1, ACAT2 and MTP in the intestine. Macrophages from Clk [[Unable to Display Character: ∆]]19/[[Unable to Display Character: ∆]]19 Apoe -/- mice express high levels of scavenger receptors and take up more modified lipoproteins compared to Apoe -/- mice; but they express low levels of ABCA1 and are defective in cholesterol efflux. Molecular studies reveal that Clock regulates ABCA1 expression in macrophages by modulating USF2 expression. Thus, Clock Δ19/Δ19 protein enhances atherosclerosis by increasing intestinal cholesterol absorption by enterocytes, augmenting uptake of modified lipoproteins by macrophages and reducing cholesterol efflux from macrophages. These studies establish that circadian Clock plays a pivotal role in the regulation of cholesterol metabolism and atherosclerosis.

Rationale and Objective: The F11 Receptor (F11R; aka Junctional Adhesion Molecule-A, JAM-A) is a cell adhesion protein present on the surface of circulating platelets and within tight junctions of endothelial cells (EC). We reported evidence for the critical role of F11R in the adhesion of platelets to cytokine-inflamed endothelium. Because platelet adhesion to an inflamed endothelium is one of the early steps of plaque formation in non-denuded blood vessels, we concluded that exposure of de novo synthesized F11R molecules on the luminal surface of EC is a crucial step in early stages of atherogenesis, leading to atherosclerosis, heart attacks and strokes. Utilizing recombinant F11R protein and F11R peptides, we have demonstrated that the expression of F11R on the luminal surface of inflamed EC initiates platelet adhesion to the inflamed EC thru binding of F11R molecules and this binding is inhibited by F11R antagonists. The present report provides new in-vivo evidence for the critical role of F11R antagonists in the formation of atherosclerotic plaques in apoE -/- mice. Methods and Results: The effect of F11R peptide on the formation of atherosclerotic plaques was determined in apoE -/- mice from 12 to 20 weeks of age. Fourteen apoE -/- mice were obtained from the Jackson Laboratory and fed a Western-type diet. Seven mice were injected with an F11R peptide (4.3 mg/100μl) every day for either a 3-, 4- or a 5- month period. Control animals were injected with diluent for the same period of time. The aortae and hearts were dissected and the aortic arch was photographed. An aortic lesion en face assay and morphometric lesion analysis was performed. Mice treated with the F11R peptide demonstrated healthy appearance with significantly decreased atherosclerotic lesion size as compared to unhealthy, control animals with large lesion sizes. Conclusions: F11R peptide inhibits the development of plaque formation in Apo E -/- mice, an animal model of atherosclerosis. The results of combined in vitro and in vivo studies provide information that development of small peptide analogs or peptidiomimetics which antagonize the pathological activity of F11R can be used as therapeutic agents for the prevention of atherosclerosis and thrombosis leading to heart attacks and strokes.

Phospholipids undergo extensive remodeling by the Lands’ Cycle after de novo biosynthesis. Enzymes involved in phospholipid biosynthesis have been studied extensively but not those involved in remodeling. One key enzyme in the Lands’ Cycle is fatty acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) which utilizes lysophosphatidylcholine (LysoPC) and fatty acyl-CoA to produce various phosphatidylcholine (PC) species. Four isoforms of LPCAT have been identified. We found, in this study, that LPCAT3 is the major hepatic isoform and its knockdown significantly reduces hepatic LPCAT activity. We also found that adenovirus-shRNA-mediated LPCAT3 knockdown in mice significantly increases different LysoPC with 16:0, 18:0, 18:1, and 18:2 fatty acids and reduces certain PC species in the liver. More importantly, LPCAT3 knockdown significantly reduces hepatic triglyceride levels but induces plasma triglyceride and apoB levels. Lipoprotein production studies indicated that LPCAT3 deficiency enhanced triglyceride-rich apoB-containing lipoprotein assembly and secretion. Further, the knockdown mice had significantly higher microsomal triglyceride transfer protein (MTP) mRNA and protein levels. Mechanistic studies in hepatoma cells revealed that LysoPC enhances secretion of apoB but not apoA-I in a concentration dependent manner. Moreover, LysoPC increased MTP mRNA, protein, and activity levels. In short, these results indicate that hepatic LPCAT3 modulates VLDL production by regulating LysoPC levels and MTP expression.

Rationale: High fasting serum lipid levels are significant risk factors for atherosclerosis. However, the contributions of postprandial excursions in serum lipoproteins to atherogenesis are less well characterized. Objective: This study aims to delineate whether changes in intestinal lipid absorption associated with loss of inositol requiring enzyme 1β (Ire1β) would affect the development of hyperlipidemia and atherosclerosis in Apoe -/- mice. Methods and Results: We used Ire1β deficient mice to assess the contribution of intestinal lipid absorption to atherosclerosis. Here we show that Ire1b -/- /Apoe -/- mice contain higher levels of intestinal microsomal triglyceride transfer protein, absorb more lipids, develop hyperlipidemia, and have higher levels of atherosclerotic plaques compared to Apoe -/- mice when fed chow and western diets. In contrast, plasma cytokines were similar in Ire1b -/- /Apoe -/- and Apoe -/- mice. Conclusions: These studies indicate that Ire1β regulates intestinal lipid absorption and that increased intestinal lipoprotein production contributes to atherosclerosis.

Atherosclerosis is the leading cause of death in western countries. Lipoprotein metabolism is closely related with the disease. ApoB-containing lipoproteins (BLp) are atherogenic particles, while, high density lipoproteins (HDL) are anti-atherogenic. Phospholipids Transfer Protein (PLTP) activity has important impact on BLp and HDL homeostasis. Animal studies, including general knock out (KO) and transgenic approaches, have shown pro-atherogenic properties for PLTP. However, the tissue specific function of PLTP remains mostly unknown. To address the impact of liver-specific expression of PLTP on lipoprotein metabolism, we have created two mouse models which either express PLTP specifically in the liver on a PLTP-null background or do not express PLTP in the liver (liver-specific PLTP KO) in a wild type background. In both models, our findings show that liver-specific PLTP is responsible for 25-30% of total plasma PLTP activity. Moreover, liver-specific PLTP expression model shows a remarkable increase in plasma levels of BLp, whereas liver-specific PLTP KO mice show significant reduction of BLp in plasma in compare to control group. These results further show the significant contribution of PLTP in BLp production. We also found that HDL levels are slightly increased in the first model, while in liver-specific PLTP KO animals HDL levels are significantly decreased. In order to unravel the mechanism, we evaluated the apoB and triglyceride production rates after blocking the clearance of the particles. We found that liver-specific PLTP increases apoB (both apo48 and apoB100) and triglyceride (TG) secretion rates. Additionally, the study of the hepatocyte microsomal luminal content lipidation in the first model showed that in the presence of PLTP BLp lipidation, which reflects as higher level of TG in microsomal luminal content, is significantly higher than the control group. Our results indicate that liver-generated PLTP promotes atherogenic lipoprotein production and provides a rationale for liver-specific PLTP inhibition as a therapeutical approach for the treatment of atherosclerosis.

The gene prostate tumor overexpressed 1 (PTOV1) was first found to be upregulated in prostate cancer. This upregulation increased tumor cell proliferation, retinoic acid resistance, and migration. This study investigated the expression and prognostic significance of PTOV1 in hepatocellular carcinoma (HCC). Real-time Polymerase Chain Reaction and western blot analysis were performed to examine PTOV1 expression in 11 HCC cell lines and 2 normal hepatic cell lines. PTOV1 expression levels were also determined in 8 pairs of tissue samples taken from primary HCC tumors and the matched adjacent noncancerous liver tissue from the same patient. Immunohistochemistry assays assessed PTOV1 protein expression in paraffin-embedded clinical samples taken from 215 HCC patients. The correlation of PTOV1 expression with the clinicopathological parameters was evaluated along with the prognostic impact of PTOV1 expression in these HCC patients. PTOV1 mRNA and protein were overexpressed in HCC cell lines compared with normal liver cell lines and were overexpressed in primary HCC samples compared with the matched noncancerous liver tissue samples. In the paraffin-embedded tissue samples from 215 HCC patients, PTOV1 protein expression was significantly correlated with T classification, N classification, clinical stage, and serum α-fetoprotein. HCC patients with higher PTOV1 expression had shorter survival times than patients with lower PTOV1 expression. Our study demonstrated that PTOV1 overexpression is correlated with increased aggressiveness of HCC and could be a prognostic biomarker for patients with HCC.