Stellate cell-specific adhesion molecule protocadherin 7 regulates sinusoidal contraction.

BACKGROUND AND AIMS: Sustained inflammation and hepatocyte injury in chronic liver disease activate HSCs to transdifferentiate into fibrogenic, contractile myofibroblasts. We investigated the role of protocadherin 7 (PCDH7), a cadherin family member not previously characterized in the liver, whose expression is restricted to HSCs. APPROACH AND RESULTS: We created a PCDH7 fl/fl mouse line, which was crossed to lecithin retinol acyltransferase-Cre mice to generate HSC-specific PCDH7 knockout animals. HSC contraction in vivo was tested in response to the HSC-selective vasoconstrictor endothelin-1 using intravital multiphoton microscopy. To establish a PCDH7 null HSC line, cells were isolated from PCDH7 fl/fl mice and infected with adenovirus-expressing Cre. Hepatic expression of PCDH7 was strictly restricted to HSCs. Knockout of PCDH7 in vivo abrogated HSC-mediated sinusoidal contraction in response to endothelin-1. In cultured HSCs, loss of PCDH7 markedly attenuated contractility within collagen gels and led to altered gene expression in pathways governing adhesion and vasoregulation. Loss of contractility in PCDH7 knockout cells was impaired Rho-GTPase signaling, as demonstrated by altered gene expression, reduced assembly of F-actin fibers, and loss of focal adhesions. CONCLUSIONS: The stellate cell-specific cadherin, PCDH7, is a novel regulator of HSC contractility whose loss leads to cytoskeletal remodeling and sinusoidal relaxation.

The mitochondrial calcium uniporter mediates mitochondrial Fe2+ uptake and hepatotoxicity after acetaminophen.

Acetaminophen (APAP) overdose disrupts hepatocellular lysosomes, which release ferrous iron (Fe2+) that translocates into mitochondria putatively via the mitochondrial calcium uniporter (MCU) to induce oxidative/nitrative stress, the mitochondrial permeability transition (MPT), and hepatotoxicity. To investigate how MCU deficiency affects mitochondrial Fe2+ uptake and hepatotoxicity after APAP overdose, global MCU knockout (KO), hepatocyte specific (hs) MCU KO, and wildtype (WT) mice were treated with an overdose of APAP both in vivo and in vitro. Compared to strain-specific WT mice, serum ALT decreased by 88 and 56%, respectively, in global and hsMCU KO mice at 24 h after APAP (300 mg/kg). Hepatic necrosis also decreased by 84 and 56%. By contrast, when MCU was knocked out in Kupffer cells, ALT release and necrosis were unchanged after overdose APAP. Intravital multiphoton microscopy confirmed loss of viability and mitochondrial depolarization in pericentral hepatocytes of WT mice, which was decreased in MCU KO mice. CYP2E1 expression, hepatic APAP-protein adduct formation, and JNK activation revealed that APAP metabolism was equivalent between WT and MCU KO mice. In cultured hepatocytes after APAP, loss of cell viability decreased in hsMCU KO compared to WT hepatocytes. Using fructose plus glycine to prevent cell killing, mitochondrial Fe2+ increased progressively after APAP, as revealed with mitoferrofluor (MFF), a mitochondrial Fe2+ indicator. By contrast in hsMCU KO hepatocytes, mitochondrial Fe2+ uptake after APAP was suppressed. Rhod-2 measurements showed that Ca2+ did not increase in mitochondria after APAP in either WT or KO hepatocytes. In conclusion, MCU mediates uptake of Fe2+ into mitochondria after APAP and plays a central role in mitochondrial depolarization and cell death during APAP-induced hepatotoxicity.

A new fluorescent sensor mitoferrofluor indicates the presence of chelatable iron in polarized and depolarized mitochondria.

Mitochondrial chelatable iron contributes to the severity of several injury processes, including ischemia/reperfusion, oxidative stress, and drug toxicity. However, methods to measure this species in living cells are lacking. To measure mitochondrial chelatable iron in living cells, here we synthesized a new fluorescent indicator, mitoferrofluor (MFF). We designed cationic MFF to accumulate electrophoretically in polarized mitochondria, where a reactive group then forms covalent adducts with mitochondrial proteins to retain MFF even after subsequent depolarization. We also show in cell-free medium that Fe2+ (and Cu2+), but not Fe3+, Ca2+, or other biologically relevant divalent cations, strongly quenched MFF fluorescence. Using confocal microscopy, we demonstrate in hepatocytes that red MFF fluorescence colocalized with the green fluorescence of the mitochondrial membrane potential (ΔΨm) indicator, rhodamine 123 (Rh123), indicating selective accumulation into the mitochondria. Unlike Rh123, mitochondria retained MFF after ΔΨm collapse. Furthermore, intracellular delivery of iron with membrane-permeant Fe3+/8-hydroxyquinoline (FeHQ) quenched MFF fluorescence by ∼80% in hepatocytes and other cell lines, which was substantially restored by the membrane-permeant transition metal chelator pyridoxal isonicotinoyl hydrazone. We also show FeHQ quenched the fluorescence of cytosolically coloaded calcein, another Fe2+ indicator, confirming that Fe3+ in FeHQ undergoes intracellular reduction to Fe2+. Finally, MFF fluorescence did not change after addition of the calcium mobilizer thapsigargin, which shows MFF is insensitive to physiologically relevant increases of mitochondrial Ca2+. In conclusion, the new sensor reagent MFF fluorescence is an indicator of mitochondrial chelatable Fe2+ in normal hepatocytes with polarized mitochondria as well as in cells undergoing loss of ΔΨm.

Strategy for optical data encryption and decryption using a D-A type stimuli-responsive AIE material.

In this study, we report a new donor-acceptor (D-A) type stimuli-responsive material, (E)-4-(((9-ethyl-9H-carbazol-2-yl)methylene)amino)benzoic acid (C1), which possesses both aggregation-induced emission (AIE) and intramolecular charge transfer (ICT) natures. It glows green photoluminescence which changes into yellow color in response to mechanical stimuli, and fumigation in volatile organic compounds (VOCs) can switch the emission back to the initial state with high reversibility. In addition, the C1 film glows yellow-orange light, but turns into blue emission under continuous fumigation in ethyl acetate vapor. However the vapochromism behaves different when the C1 film is smeared: The emission of the smeared film is similar to the unsmeared but changes into cyan color after fumigation. The differences in vapochromism between smeared and unsmeared film can be easily distinguished by naked eyes. As revealed by SEM, the as-prepared film undergoes a morphology change from ill-shaped particles to microspheres in response to organic vapor, while the smeared film with scratched surface changes into dendritic patterns. According to the morphology study, the vapochromic luminescence can be ascribed to the physical adsorption of ethyl acetate vapor and the resulting change in the ICT process. In light of the unique vapochromism of C1, a new encryption-decryption technique for data recording was developed. Information can be recorded on the C1 film by mechanical writing and simultaneously concealed. It can be only accessed via fumigation in organic vapor, demonstrating a reliable steganography technology.

Aldehyde dehydrogenase-2 activation decreases acetaminophen hepatotoxicity by prevention of mitochondrial depolarization.

Oxidative stress contributes to acetaminophen (APAP) hepatotoxicity. Since lipid peroxidation produces reactive aldehydes, we investigated whether activation of mitochondrial aldehyde dehydrogenase-2 (ALDH2) with Alda-1 decreases liver injury after APAP. Male C57BL/6 mice fasted overnight received Alda-1 (20 mg/kg, i.p.) or vehicle 30 min before APAP (300 mg/kg, i.p.). Blood and livers were collected 2 or 24 h after APAP. Intravital multiphoton microscopy of rhodamine 123 (Rh123) and propidium iodide (PI) fluorescence was conducted 6 h after APAP administration to detect mitochondrial polarization status and cell death. 4-Hydroxynonenal protein adducts were present in 0.1% of tissue area without APAP treatment but increased to 7% 2 h after APAP treatment, which Alda-1 blunted to 1%. Serum alanine and aspartate aminotransferases increased to 7594 and 9768 U/L at 24 h respectively, which decreased ≥72% by Alda-1. Alda-1 also decreased centrilobular necrosis at 24 h after APAP from 47% of lobular areas to 21%. N-acetyl-p-benzoquinone imine protein adduct formation and c-Jun-N-terminal kinase phosphorylation increased after APAP as expected, but Alda-1 did not alter these changes. Without APAP, no mitochondrial depolarization was detected by intravital microscopy. At 6 h after APAP, 62% of tissue area showed depolarization, which decreased to 33.5% with Alda-1. Cell death as detected by PI labeling increased from 0 to 6.8 cells per 30× field 6 h after APAP, which decreased to 0.6 cells by Alda-1. In conclusion, aldehydes are important mediators of APAP hepatotoxicity. Accelerated aldehyde degradation by ALDH2 activation with Alda-1 decreases APAP hepatotoxicity by protection against mitochondrial dysfunction.

Suppression of iron mobilization from lysosomes to mitochondria attenuates liver injury after acetaminophen overdose in vivo in mice: Protection by minocycline.

Acetaminophen (APAP) overdose causes hepatotoxicity involving mitochondrial dysfunction. Previous studies showed that translocation of Fe2+ from lysosomes into mitochondria by the mitochondrial Ca2+ uniporter (MCU) promotes the mitochondrial permeability transition (MPT) after APAP. Here, our Aim was to assess protection by iron chelation and MCU inhibition against APAP hepatotoxicity in mice. C57BL/6 mice and hepatocytes were administered toxic doses of APAP with and without starch-desferal (an iron chelator), minocycline (MCU inhibitor), or N-acetylcysteine (NAC). In mice, starch-desferal and minocycline pretreatment decreased ALT and liver necrosis after APAP by >60%. At 24 h after APAP, loss of fluorescence of mitochondrial rhodamine 123 occurred in pericentral hepatocytes often accompanied by propidium iodide labeling, indicating mitochondrial depolarization and cell death. Starch-desferal and minocycline pretreatment decreased mitochondrial depolarization and cell death by more than half. In cultured hepatocytes, cell killing at 10 h after APAP decreased from 83% to 49%, 35% and 27%, respectively, by 1 h posttreatment with minocycline, NAC, and minocycline plus NAC. With 4 h posttreatment in vivo, minocycline and minocycline plus NAC decreased ALT and necrosis by ~20% and ~50%, respectively, but NAC alone was not effective. In conclusion, minocycline and starch-desferal decrease mitochondrial dysfunction and severe liver injury after APAP overdose, suggesting that the MPT is likely triggered by iron uptake into mitochondria through MCU. In vivo, minocycline and minocycline plus NAC posttreatment after APAP protect at later time points than NAC alone, indicating that minocycline has a longer window of efficacy than NAC.

Irbesartan in Marfan syndrome (AIMS): a double-blind, placebo-controlled randomised trial.

BACKGROUND: Irbesartan, a long acting selective angiotensin-1 receptor inhibitor, in Marfan syndrome might reduce aortic dilatation, which is associated with dissection and rupture. We aimed to determine the effects of irbesartan on the rate of aortic dilatation in children and adults with Marfan syndrome. METHODS: We did a placebo-controlled, double-blind randomised trial at 22 centres in the UK. Individuals aged 6-40 years with clinically confirmed Marfan syndrome were eligible for inclusion. Study participants were all given 75 mg open label irbesartan once daily, then randomly assigned to 150 mg of irbesartan (increased to 300 mg as tolerated) or matching placebo. Aortic diameter was measured by echocardiography at baseline and then annually. All images were analysed by a core laboratory blinded to treatment allocation. The primary endpoint was the rate of aortic root dilatation. This trial is registered with ISRCTN, number ISRCTN90011794. FINDINGS: Between March 14, 2012, and May 1, 2015, 192 participants were recruited and randomly assigned to irbesartan (n=104) or placebo (n=88), and all were followed for up to 5 years. Median age at recruitment was 18 years (IQR 12-28), 99 (52%) were female, mean blood pressure was 110/65 mm Hg (SDs 16 and 12), and 108 (56%) were taking ß blockers. Mean baseline aortic root diameter was 34·4 mm in the irbesartan group (SD 5·8) and placebo group (5·5). The mean rate of aortic root dilatation was 0·53 mm per year (95% CI 0·39 to 0·67) in the irbesartan group compared with 0·74 mm per year (0·60 to 0·89) in the placebo group, with a difference in means of -0·22 mm per year (-0·41 to -0·02, p=0·030). The rate of change in aortic Z score was also reduced by irbesartan (difference in means -0·10 per year, 95% CI -0·19 to -0·01, p=0·035). Irbesartan was well tolerated with no observed differences in rates of serious adverse events. INTERPRETATION: Irbesartan is associated with a reduction in the rate of aortic dilatation in children and young adults with Marfan syndrome and could reduce the incidence of aortic complications. FUNDING: British Heart Foundation, the UK Marfan Trust, the UK Marfan Association.

Translocation of iron from lysosomes to mitochondria during acetaminophen-induced hepatocellular injury: Protection by starch-desferal and minocycline.

Acetaminophen (APAP) overdose causes hepatotoxicity involving mitochondrial dysfunction and the mitochondrial permeability transition (MPT). Iron is a critical catalyst for ROS formation, and reactive oxygen species (ROS) play an important role in APAP-induced hepatotoxicity. Previous studies show that APAP disrupts lysosomes, which release ferrous iron (Fe(2+)) into the cytosol to trigger the MPT and cell killing. Here, our aim was to investigate whether iron released from lysosomes after APAP is then taken up into mitochondria via the mitochondrial electrogenic Ca(2+), Fe(2+) uniporter (MCFU) to cause mitochondrial dysfunction and cell death. Hepatocytes were isolated from fasted male C57BL/6 mice. Necrotic cell killing was assessed by propidium iodide fluorimetry. Mitochondrial membrane potential (ΔΨ) was visualized by confocal microscopy of rhodamine 123 (Rh123) and tetramethylrhodamine methylester (TMRM). Chelatable Fe(2+) was monitored by quenching of calcein (cytosol) and mitoferrofluor (MFF, mitochondria). ROS generation was monitored by confocal microscopy of MitoSox Red and plate reader fluorimetry of chloromethyldihydrodichlorofluorescein diacetate (cmH2DCF-DA). Administered 1h before APAP (10mM), the lysosomally targeted iron chelator, starch-desferal (1mM), and the MCFU inhibitors, Ru360 (100nM) and minocycline (4µM), decreased cell killing from 83% to 41%, 57% and 53%, respectively, after 10h. Progressive quenching of calcein and MFF began after ~4h, signifying increased cytosolic and mitochondrial chelatable Fe(2+). Mitochondria then depolarized after ~10h. Dipyridyl, a membrane-permeable iron chelator, dequenched calcein and MFF fluorescence after APAP. Starch-desferal, but not Ru360 and minocycline, suppressed cytosolic calcein quenching, whereas starch-desferal, Ru360 and minocycline all suppressed mitochondrial MFF quenching and mitochondrial depolarization. Starch-desferal, Ru360 and minocycline also each decreased ROS formation. Moreover, minocycline 1h after APAP decreased cell killing by half. In conclusion, release of Fe(2+) from lysosomes followed by uptake into mitochondria via MCFU occurs during APAP hepatotoxicity. Mitochondrial iron then catalyzes toxic hydroxyl radical formation, which triggers the MPT and cell killing. The efficacy of minocycline post-treatment shows minocycline as a possible therapeutic agent against APAP hepatotoxicity.

Why some tumours trigger neovascularisation and others don't: the story thus far.

BACKGROUND: Angiogenesis is not essential for tumours to develop and expand, as cancer can also grow in a non-angiogenic fashion, but why this type of growth occurs is unknown. Surprisingly, our data from mRNA transcription profiling did not show any differences in the classical angiogenic pathways, but differences were observed in mitochondrial metabolic pathways, suggesting a key role for metabolic reprogramming. We then validated these results with mRNA profiling by investigating differential protein expression via immunohistochemistry in angiogenic and non-angiogenic non-small cell lung cancers (NSCLCs). METHODS: Immunohistochemical staining for 35 angiogenesis- and hypoxia-related biomarkers were performed on a collection of 194 angiogenic and 73 non-angiogenic NSCLCs arranged on tissue microarrays. Sequencing of P53 was performed with frozen tissue samples of NSCLC. RESULTS: The non-angiogenic tumours were distinguished from the angiogenic ones by having higher levels of proteins associated with ephrin pathways, mitochondria, cell biogenesis, and hypoxia-inducible factor 1 (HIF1) regulation by oxygen and transcription of HIF-controlled genes but lower levels of proteins involved in the stroma, cell-cell signaling and adhesion, integrins, and Delta-Notch and epidermal growth factor (EGF)-related signaling. However, proteins classically associated with angiogenesis were present in both types of tumours at very comparable levels. Cytoplasmic expression of P53 was strongly associated with non-angiogenic tumours. A pilot investigation showed that P53 mutations were observed in 32.0% of angiogenic cases but in 71.4% of non-angiogenic tumours. CONCLUSIONS: Our observations thus far indicate that both angiogenic and non-angiogenic tumours experience hypoxia/HIF and vascular endothelial growth factor (VEGF) pathway protein expression in a comparable fashion. However, angiogenesis does not ensue in the non-angiogenic tumours. Surprisingly, metabolic reprogramming seems to distinguish these two types of neoplastic growth. On the basis of these results, we raise the hypothesis that in some, but not in all cases, initial tissue remodeling and/or inflammation could be one of the secondary steps necessary to trigger angiogenesis. In the non-angiogenic tumours, in which neovascularisation fails to occur, HIF pathway activation could be the driving force toward metabolic reprogramming.

Low Dose Acetaminophen Induces Reversible Mitochondrial Dysfunction Associated with Transient c-Jun N-Terminal Kinase Activation in Mouse Liver.

Acetaminophen (APAP) overdose causes hepatotoxicity involving mitochondrial dysfunction and c-jun N-terminal kinase (JNK) activation. Because the safe limit of APAP dosing is controversial, our aim was to evaluate the role of the mitochondrial permeability transition (MPT) and JNK in mitochondrial dysfunction after APAP dosing considered nontoxic by criteria of serum alanine aminotransferase (ALT) release and histological necrosis in vivo. C57BL/6 mice were given APAP with and without the MPT inhibitor, N-methyl-4-isoleucine cyclosporin (NIM811), or the JNK inhibitor, SP600125. Fat droplet formation, cell viability, and mitochondrial function in vivo were monitored by intravital multiphoton microscopy. Serum ALT, liver histology, total JNK, and activated phospho(p)JNK were also assessed. High APAP (300 mg/kg) caused ALT release, necrosis, irreversible mitochondrial dysfunction, and hepatocellular death. By contrast, lower APAP (150 mg/kg) caused reversible mitochondrial dysfunction and fat droplet formation in hepatocytes without ALT release or necrosis. Mitochondrial protein N-acetyl-p-benzoquinone imine adducts correlated with early JNK activation, but irreversible mitochondrial depolarization and necrosis at high dose were associated with sustained JNK activation and translocation to mitochondria. NIM811 prevented cell death and/or mitochondrial depolarization after both high and low dose APAP. After low dose, SP600125 decreased mitochondrial depolarization. In conclusion, low dose APAP produces reversible MPT-dependent mitochondrial dysfunction and steatosis in hepatocytes without causing ALT release or necrosis, whereas high dose leads to irreversible mitochondrial dysfunction and cell death associated with sustained JNK activation. Thus, nontoxic APAP has the potential to cause transient mitochondrial dysfunction that may synergize with other stresses to promote liver damage and steatosis.

Gene Signatures Stratify Computed Tomography Screening Detected Lung Cancer in High-Risk Populations.

BACKGROUND: Although screening programmes of smokers have detected resectable early lung cancers more frequently than expected, their efficacy in reducing mortality remains debatable. To elucidate the biological features of computed tomography (CT) screening detected lung cancer, we examined the mRNA signatures on tumours according to the year of detection, stage and survival. METHODS: Gene expression profiles were analysed on 28 patients (INT-IEO training cohort) and 24 patients of Multicentre Italian Lung Detection (MILD validation cohort). The gene signatures generated from the training set were validated on the MILD set and a public deposited DNA microarray data set (GSE11969). Expression of selected genes and proteins was validated by real-time RT-PCR and immunohistochemistry. Enriched core pathway and pathway networks were explored by GeneSpring GX10. FINDINGS: A 239-gene signature was identified according to the year of tumour detection in the training INT-IEO set and correlated with the patients' outcomes. These signatures divided the MILD patients into two distinct survival groups independently of tumour stage, size, histopathological type and screening year. The signatures can also predict survival in the clinically detected cancers (GSE11969). Pathway analyses revealed tumours detected in later years enrichment of the PI3K/PTEN/AKT pathway, with up-regulation of PDPK1, ITGB1 and down-regulation of FOXO1A. Analysis of normal lung tissue from INT-IEO cohort produced signatures distinguishing patients with early from late detected tumours. INTERPRETATION: The distinct pattern of "indolent" and "aggressive" tumour exists in CT-screening detected lung cancer according to the gene expression profiles. The early development of an aggressive phenotype may account for the lack of mortality reduction by screening observed in some cohorts.

Minocycline and doxycycline, but not tetracycline, mitigate liver and kidney injury after hemorrhagic shock/resuscitation.

BACKGROUND: Despite recovery of hemodynamics by fluid resuscitation after hemorrhage, development of the systemic inflammatory response and multiple organ dysfunction syndromes can nonetheless lead to death. Minocycline and doxycycline are tetracycline derivatives that are protective in models of hypoxic, ischemic, and oxidative stress. Our aim was to determine whether minocycline and doxycycline protect liver and kidney and improve survival in a mouse model of hemorrhagic shock and resuscitation. METHODS: Mice were hemorrhaged to 30 mmHg for 3 h and then resuscitated with shed blood followed by half the shed volume of lactated Ringer's solution containing tetracycline (10 mg/kg), minocycline (10 mg/kg), doxycycline (5 mg/kg), or vehicle. For pretreatment plus posttreatment, drugs were administered intraperitoneally prior to hemorrhage followed by second equal dose in Ringer's solution after blood resuscitation. Blood and tissue were harvested after 6 h. RESULTS: Serum alanine aminotransferase (ALT) increased to 1,988 and 1,878 U/L after posttreatment with vehicle and tetracycline, respectively, whereas minocycline and doxycycline posttreatment decreased ALT to 857 and 863 U/L. Pretreatment plus posttreatment with minocycline and doxycycline also decreased ALT to 849 and 834 U/L. After vehicle, blood creatinine increased to 134 µM, which minocycline and doxycycline posttreatment decreased to 59 and 56 µM. Minocycline and doxycycline pretreatment plus posttreatment decreased creatinine similarly. Minocycline and doxycycline also decreased necrosis and apoptosis in liver and apoptosis in both liver and kidney, the latter assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) and caspase 3 activation. Lastly after 4.5 h of hemorrhage followed by resuscitation, minocycline and doxycycline (but not tetracycline) posttreatment improved 1-week survival from 38% (vehicle) to 69% and 67%, respectively. CONCLUSION: Minocycline and doxycycline were similarly protective when given before as after blood resuscitation and might therefore have clinical efficacy to mitigate liver and kidney injury after resuscitated hemorrhage.

Cyclosporin A in left ventricular remodeling after myocardial infarction.

Recent studies suggest that an increase in apoptosis within the myocardium may be a contributing factor for the progression of late adverse left ventricular (LV) remodeling following myocardial infarction (MI). Given that apoptosis is often triggered by induction of the mitochondrial permeability transition (MPT) pore, the goal of this study was to evaluate the therapeutic efficacy of cyclosporin A (CsA), an MPT blocker, to prevent cells from undergoing apoptosis and consequently attenuate late LV remodeling post-MI. MI was induced in C57BL/6 mice and then randomized to either vehicle or CsA groups. Beginning 48 h after surgery after infarction had already occurred, mice were gavaged with CsA (2 mg/kg) or vehicle once daily. LV end-diastolic volume and LV ejection fraction were assessed by echocardiography before MI induction and terminally at either 7 days (n = 7) or 28 days (n = 8) post-MI. LV end-diastolic volume increased and LV ejection fraction decreased in all MI groups with no difference between the CsA-treated and untreated groups. After vehicle and CsA, areas of necrosis were present at 7 and 28 days post-MI with no difference between treatment groups. Caspase-3 activity and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling in distal nonnecrotic LV both increased after MI but were lower in CsA-treated mice compared with vehicle (P < 0.05). In conclusion, CsA decreased apoptosis occurring late after MI, confirming involvement of a CsA-sensitive MPT in the cell death. However, CsA-mediated reduction in apoptosis in non-MI myocardium was not beneficial against late pump dysfunction occurring during post-MI remodeling.

Vessel co-option in primary human tumors and metastases: an obstacle to effective anti-angiogenic treatment?

Angiogenesis has been regarded as essential for tumor growth and progression. Studies of many human tumors, however, suggest that their microcirculation may be provided by nonsprouting vessels and that a variety of tumors can grow and metastasize without angiogenesis. Vessel co-option, where tumor cells migrate along the preexisting vessels of the host organ, is regarded as an alternative tumor blood supply. Vessel co-option may occur in many malignancies, but so far mostly reported in highly vascularized tissues such as brain, lung, and liver. In primary and metastatic lung cancer and liver metastasis from different primary origins, as much as 10-30% of the tumors are reported to use this alternative blood supply. In addition, vessel co-option is introduced as a potential explanation of antiangiogenic drug resistance, although the impact of vessel co-option in this clinical setting is still to be further explored. In this review we discuss tumor vessel co-option with specific examples of vessel co-option in primary and secondary tumors and a consideration of the clinical implications of this alternative tumor blood supply.

Tumor necrosis factor receptor-associated protein 1(TRAP1) regulates genes involved in cell cycle and metastases.

Tumor necrosis factor (TNF) receptor-associated protein 1(TRAP1/HSP75) is a heat shock protein, highly homologous to HSP90, which acts as a molecular chaperone to retinoblastoma protein (Rb) during cellular stress although the current literature suggests that this protein could have additional functions. The aim of this study was to identify the pathways regulated by TRAP1. TRAP1 was silenced by siRNA in A549 cells and re-expressed by stable transfection in MDA231 cells. After growing the cells for 16h under normoxic or hypoxic conditions, oligonucleotide microarrays were employed to detect differentially expressed genes. In TRAP1 positive cells there are high levels of cell proliferation promoting genes coding for G protein coupled receptors, cell adhesion genes and genes associated with Rho-kinase pathways. In TRAP1 negative cells there are higher levels of genes involved in cell motility and metastatic spread. Pathway map analysis shows that TRAP1 controls cell cycle activity through the tumor necrosis factor pathways. Our data suggest that in many tumors TRAP1 could activate proliferation whilst inhibiting metastatic spread.

Identification of potential therapeutic targets by gene-expression profiling in pancreatic endocrine tumors.

BACKGROUND & AIMS: Prediction of biological behavior in pancreatic endocrine tumors (PET) is difficult on histologic criteria alone. The aim of this study was to characterize PET gene expression by complementary DNA (cDNA) microarray and identify specific differences in gene-expression profiles between nonmetastatic and metastatic tumors. METHODS: We studied 24 well-differentiated PETs corresponding to 12 benign tumors and to 12 carcinomas with metastasis. Total RNAs were extracted followed by microarray hybridization, imaging scan procedure, and statistical anaylsis. PET tissue arrays were constructed from 129 archival tumors for immunohistochemical validation of microarray data. RESULTS: A total of 123 transcripts were found to separate nonmetastatic from metastatic PETs (ie, 72 up-regulated and 51 down-regulated genes in malignant tumors). Some of these genes were involved in pathways related to (1) angiogenesis and remodeling (CD34, cadherin-5, E-selectin, semaphorin E, and fibrillin), (2) signal transduction through tyrosine kinases (tyrosine kinase-2, platelet-derived growth factor-Rbeta, MKK4, and discoidin domain receptor-1), (3) calcium-dependent cell signaling (transient receptor potential cation channel-1, calcium channel voltage-dependent beta2, neurocalcin delta, and GABA-A receptor gamma2), and (4) response to drug (MDR1 and carcinoembryonic antigen-related cell adhesion molecule 6). By using tissue arrays, we confirmed the differential expression of CD34 (P = .0008), E-selectin (P = .003), MKK4 (P = .0001), and MDR1 (P = .0003) in metastatic vs nonmetastatic PETs. CONCLUSIONS: This study provides insight into tumorigenic pathways in PET. Some of the genes identified are potentially new molecular markers for the detection and treatment of these tumors.

Gene expression signature for angiogenic and nonangiogenic non-small-cell lung cancer.

Angiogenesis is regarded as essential for tumour growth. However, we have demonstrated that some other aggressive non-small-cell lung carcinomas (n-SCLC) do not have angiogenesis. In this study, using cDNA microarray analysis, we demonstrate that angiogenic and nonangiogenic tumour types can be distinguished by their gene expression profiles. Tissue samples from 42 n-SCLC patients were obtained with consent. In all, 12 tumours were nonangiogenic and 30 angiogenic. The two groups were matched by age, sex, smoking and tumour stage. Total RNAs were extracted followed by microarray hybridization and image scan procedure. Data were analysed using GeneSpring 5.1 software. A total of 62 genes were found to be able to separate angiogenic from nonangiogenic tumours. Nonangiogenic tumours have higher levels of genes concerned with mitochondrial metabolism, mRNA transcription, protein synthesis and the cell cycle. Angiogenic tumours have higher levels of genes coding for membrane vesicles, integrins, remodelling, angiogenesis and apoptosis. These results further support our first finding that nonangiogenic lung tumours are fast-growing tumours filling the alveoli in the absence of vascular remodelling. We raise the hypothesis that in nonangiogenic tumours, hypoxia leads to a higher activation of the mitochondrial respiratory chain, which allows tumour growth without triggering angiogenesis.

Lung cancers detected by screening with spiral computed tomography have a malignant phenotype when analyzed by cDNA microarray.

PURPOSE: Spiral computed tomography (CT) can detect lung cancer at an early stage, but the malignant potential is unknown. The question is, as follows: do these small lesions have the same lethal potential as do symptomatic tumors? EXPERIMENTAL DESIGN: We used a cDNA microarray platform and compared the gene expression profile of spiral CT-detected lung carcinomas with a matched case-control population of patients presenting with symptomatic lung cancer. RESULTS: CT-detected and symptomatic tumors have shown a comparable gene expression profile. Correspondence analysis has demonstrated that nine genes were differentially expressed, although with a high variability across the samples that prevented distinguishing the two groups of tumors. Analysis of these nine genes has suggested that early-detected tumors have higher levels of retinoic acid production and higher expression levels of caveolin 2, matrix Gla, and cystatin A, which are already known to be lost during tumor progression. CONCLUSIONS: All of the tumors observed are histologically malignant according to the WHO Classification. Early lung cancers that are detected by screening have a gene expression pattern similar to, but not identical to, that of symptomatic lung carcinomas.