Delivery of EVs from HSCs to...
The role of LSECs-released EVs...
Liver fibrosis is a common pathogenic process of primary biliary cholangitis, hepatitis B virus, alcohol or nonalcoholic steatohepatitis and cirrhosis caused by multiple risk factors like excessive bile acids, alcohol consumption, overloaded lipids and virus infection,the formation of fibrous scar [1, 2]. Regardless of the causes of hepatic fibrosis, patients will meet the stage of elevated serum liver transaminase values like AST and ALT, excessive buildup of bilirubin and abnormal imaging of liver parenchyma. In the clinic, liver biopsy is usually performed as a common invasive procedure to accurately assess the severity of different liver diseases but with a lot of complications including but not limited to pain, hemorrhage and bile peritonitis [3]. On the other hand, although several non-invasive strategies, such as MRI fibroscans, have been applied for the diagnosis of liver fibrosis, they may be more suitable and accurate for diagnosing advanced fibrotic disorders [4]. What is worse, problems with poor targeting and severe side effects limit the clinical application of existing drugs in treating liver fibrosisexcavate the pathogenesis of liver fibrosis and discover novel drug targets for fibrotic liver diseases.
side the liver, which can interact with each other and synergistically trigger a distinct cascade of fibrotic responses. Hepatocytes are usually damaged first and spark inflammation by releasing a variety of signaling substancesactivate resident liver immune cells and recruit bone marrow-derived immune cells into the injured sites [5]. Meanwhile, injured cholangiocytes adjacent to hepatocytes can also release  to a pathological reparative reaction [6]. Under normal conditiontitute fenestrated capillaries that offer a channel for transporting resident macrophages and natural killer (NK) cells; once damaged or influenced by injured adjacent cells, LSECs start dedifferentiation towards a capillarized phenotype, accompanied with the release of soluble factors that delivery to neighboring cells, such as hepatic stellate cells (HSCs). The involve, Wncontribute to HSCs transdifferentiate from a quiescent state to an activated state (i.e., myofibroblasts), becoming the core factor driving fibrosis [7-9]. Under the stimulation of the above factors, HSCs not only produce ECM but also aggravate liver inflammation by secreting cytokines, emphasizing emerging molecular and cellular signals that drive fibrotic liver injury. Finally, large amounts of ECM components continuously occupy the empty space left by dead cells to induce excessive repair in the liver, which may be initially beneficial, but in turn, prevent damaged liver cells from returning to normal function and then construct a vicious circle [10]. Collectively, intercellular interactions among liver cells are multidirectional and work in concert to affect the progression of liver fibrosis. Emerging studies have highlighted the potential of extracellular vesicles (EVs) in the diagnosis and therapy of liver fibrosis via modulating intercellular communication networks in the liver.
Schematic overview of EV biogenesis and cargo. When the donor cells are stimulated, the plasma membranes invaginate to form early endosomes and gradually form late endosomes. The late endosome membranes then invaginate while encapsulating informative material (containing proteins, nucleic acids, lipids, etc.) to mature into multivesicular bodies (MVBs). Eventually, the MVBs fuse with the cell membrane releasing the inner vesicles into the extracellular environment to form exosomes. Oand subsequent release of vesicles into the extracellular space form microvesicles. During apoptosis the cytosolic membrane crumples and invaginates, dividing and enveloping the cytoplasm and containing DNA material and organelles to form apoptotic vesicles.
Typically, intercellular communication is roughly classified into following three categories: (i) direct cell contact that mediated by sets of adhesion molecules, (ii) gap junction by establishing tunneling nanotubules between neighboring cells and (iii) transmission of extracellular chemical signal depending on mechanism of ligand-receptor binding in the endocrine manner [11, 12]. As membrane-bound vesicles with the structure of typical lipid bilayer, EVs encapsulate signaling molecules and thus afford a more efficient modality of transmitting intercellular signaling during cell-to-cell crosstalk (Figure 1). After originated from either healthy or stressed cells, EVs deliver signaling substances including DNAs, RNlipids and proteins into recipient cells [13]. Interestingly, nucleic acids or proteins specific derived from damaged organelles such as mitochondrial DNA (mtDNA) can also be encapsulated by EVs [14]. EVs containing virus-coding RNA and proteins are also reported to secret from the infected cells and transfer to the target cells [15]. Based on their different biogenesis, EVss, ranging from 30 nm to 1,000 nm in diameter [16]. Inward budding of endosomal membrane forms intraluminal vesicles that are the precursor of exosomes and are encased by multivesicular endosomes. After maturation, exosomes are released through the fusion with the plasma membrane of multivesicular bodies [17]. Unlike exosomes, microvesicles are only formed by the outward budding at the plasma membrane [18]. Characteristically, large-scale blebbing of plasma membrane makes cells dissociate into apoptotic bodies when cells undergo apoptosis, autophagy and programmed necrosis [19]. With the gradual deepening research of the cellular communication mediated by EVs, increasing evidence show that EVchronic diseases.
To date, although a few studies have generally summarized the functions of EVs played in different liver diseases, a systematic understanding of potential effects and underlying mechanism of delivering substances among multiple liver cells is still lacking and the complexity and bidirectionality of EVs existing in livers, which may be associated with the direction of material transfer between cells and the stage of fibrosis, has been neglected. Thus, a review from perspectives of donor and recipient cells of EVs will help future researchers to conduct more in-depth studies of EVs-mediated communications. Herein, we comprehensively summarized and discussed recent findings focusingVs and substance transmitting information via EVs among different liver cells. We also probed into the role of EVs in the initiation and progression of fibrotic liver diseases and emphasized the therapeutic potential of targeting EVs in the clinic.
Given that hepatocyte injury is a ubiquitous event of liver fibrosis, question about how hepatocytes exert the regulation of neighboring liver cells via EVs will be gradually unveiled by many lines of evidence (Figure 2).
Schematic diagram of hepatocytes-centered EVs network during liver fibrotic diseases. The EVs secreted by hepatocytes contain a variety of contents that are delivered to the quiescent HSCs to activate them. Activated HSCs then secrete cytokines that stimulate immune cells to exacerbate liver fibrosis. Both liver resident macrophages (kupffer cells) and BMDM receive EVs from hepatocytes and transdifferentiate into M1-type pro-inflammatory macrophages. Hepatocytes deliver EVs to LSECs to disrupt the integrity of the hepatic blood sinusoidal endothelium and accelerate capillarization to worsen liver fibrosis. Hepatocytes also autocrine EVs to deliver injury signals. Further studies are needed to investigate how cholangiocytes and immune cells other than NK cells communicate with hepatocytes via EVs. The solid line indicates that this has now been reported and the dotted line indicates that it has not been reported.
The repertoire of HSC functions is recently found to be much more diverse, since they can act as antigen presenting cells (APC), express pattern recognition receptors (PRRs) and respond to hepatocyte-derived DAMPs [20]. Furthermore, as is known, a wide variety of stimulus including drugs, virus and lipids can trigger the transmission of EVs between hepatocytes and HSCs, suggesting thatelivery of information, thus affecting the progression of liver fibrosis [21]AKT signaling pathway and hedgehog signaling pathway are dominant pro-fibrotic signaling pathways by regulating the transcription of fibrosis-related genes in livers [22]. According to our latest study, mtDNA-enriched EVs released from oxidative stressed hepatocytes induced by either carbon tetrachloride (CCl4) or acetaminophen directly activated HSCs and drived liver scarring by upregulating the transcription of fibrotic genes including Acta2, Col1a1 and Fibronectin [5]. Although we did not explicitly investigate how these mtDNA-enriched EVs regulated gene transcription because of the mtDNA loop structure, there are many other studies linking this change to miRNAs, another kind of non-coding RNAs being recognized to regulate a variety of target genes. Recent study showed that after infected with hepatitis C virus (HCV), miR-192 was released from hepatocytes via exosomes and further transmitted into HSCs, resulting in the trans-differentiation of HSCs into myofibroblasts, which was mediated by the production of TGF-β1 and subsequently upregulation of fibrogenic markers such as Acta2 and Col1a1 [23]. Similarly, Devhare PB et al. reported that exosomes-containing miR-19a released from hepatocytes in the same injured situation were also delivered into HSCs and depleted suppressor of cytokine signaling 3 (SOCS3 TGF-β signaling pathway, which in turn promoted the HSC activation [24]. Moreover, HCV-derived EVs were reported to contain lower levels of antifibrogenic miRNAs including miR204-5p, miR181a-5p, miR143-5p, miR93-5p and miR122-5p, and higher levels of miRNAs associated with HCV-related immunopathogenesis including miR-222-3p, miR-146a, miR-150-5p, miR-30c, miR-378a-3p and miR-20a-5p, which induced fibrogenic marker expression (Fn-1, Acta2, Col1a1 and Tgfb1) and HSC activation [25, 26]. Lipids generally deposit in hepatocytes as an adaptive response and further increase free fatty acids (FFAs), while the dysregulation of lipid homeostasis transmits damage signals to other liver cells via the release of EVs. Lee YS et al. further confirmed the significant functions of miR-192 during lipids-induced fibrosis. Notably, palmitic acid increased the number of isolated exosomes from hepatocytes and then directly transfected exosomal miR-192 into HSCs to enhance the expression of fibrosis-associated genes including TGFb1, Acta2 and Col1a1, which accelerated the progress[27]. As another pro-fibrotic pathway, AKT signaling can be negatively regulated by the that contributes to the proliferation of HSCs [28]. Exosomal miR-1297 secreted from lipotoxic hepatocytes was reported to decrease the mRNA and protein levels of PTEN and increased the phosphorylation of PI3K and AKT in HSCs, which then triggered HSC activation and proliferation [29]. In addition, exosomal miR-21 released by hepatoma cells converted normal HSCs to cancer-associated fibroblasts (CAF) by directly downregulating PTEN and then activating 3-phosphoinositide-dependent kinase 1 (PDK1)/AKT signaling pathway. Once activated, CAF further promoted angiogenesis and fibrotic injury by secreting2 (MMP2), MMP9 and TGF-β [30]. It is worth noting that miRNAs not only can be transported via EVs, but also be regulated in the recipient cells by other substances delivered from EVs. Exosomes containinge derived from hepatocytes treated with arsenite and were further transported into HSCs to promote their activation through the downregulation of miR-26b and subsequent upregulation of Col1a2 [31]. In addition, activation of HSCs has a strong correlation with hepatocellular carcinoma (HCC) occurrence and development. As an oncogene in HCC, smoothened (SMO) was packaged in HCC-derived exosomes, transmitted to HSCs and triggered HSC activation by increasing GLI family zinc finger 1 expression and activating hedgehog pathway. Furthermore, SMO led to the transcriptional activation of miRNA let-7b host gene, which in turns, upregulated the expression of SMO and formed a profibrotic and vicious feedback loop [32].
An additional point deserved to be mentioned is that hepatic pathological events caused by exosomes delivering from hepatocytes to HSCs may be not only limited to these two types of cells. A previous study confirmed that exosomes derived from CCl4-treated hepatocytes specifically activated toll-like receptor 3 (TLR3) and upregulated interleukin-17A (IL-17A) expression in HSCs, leading to the production of IL-17, IL-1β and IL-23 at the early stage of fibrotic liver injury. In addition, these increased cytokines in exosome-treated HSCs could subsequently promote the recruitment of γδT cells and boosted IL-17A production in γδT cells by increasing the[33]. Considering that the apoptosis and exhaustion of HSCs are closely associated with the activation other innate T cells, we speculate more extensive changes happened in the 'bystander' immune cells during the communication between hepatocytes and HSCs.
Under pathological conditions, hepatocytes produce different chemokines at the site of injury, recruiting and triggering more immune cells to induce local damage reactions [34]. As a vital category of immune cells, macrophages ubiquitously exist and are susceptible to be affected by hepatocyte-derived EVs due to their chemotactic activities [35]. Typically, macrophages can be roughly divided into liver-resident macrophages (kupffer cells) or recruited macrophages based on their origin. Besides, two dynamic active states of macrophages are usually portrayed as classical activated M1 and alternative activated M2 phenotypes, the former of which promote inflammation and the latter ones accelerate fibrosis progression [36]. Numerous studies have proved that, regardless of the source or phenotypes, macrophages have to take risks of EVs released by injured hepatocytes. It has been reported that kupffer cells were evoked by EVs released from FFAs- and cobalt chloride-stimulated hepatocytes and further secreted pro-inflammatory cytokines to promote liver fibrosis [37]. Saha et al. intensively studied those data and found that EVs secreted from primary hepatocytes of mice with fibrotic liver disease could increase the percentages of kupffer cells (inflammatory M1 type) and infiltrating monocytes by up (HSP90) [38]. IEVs released from lipotoxic hepatocytes also play a vital role in the activation and transformation of kupffer cells. Under the stimulation of large amounts of alcohol, exosomal CD40 ligands were released from hepatocytes as results of caspase 3/7 activation and were further delivered into kupffer cells, in which promoted the polarization of M1-type inflammatory phenotype and triggered continuous inflammation [39]. A subsequent study further explored the possible mechanisms and demonstrated that exosomal miR-192-5p released from palmitate-induced lipotoxic hepatocytes promoted the pro-inflammatory M1 phenotype activation, the differentiation of THP-1 macrophages and liver fibrosis at least partially by downregulating the Rictor/AKT/Forkhead box transcription factor O1 signaling pathway [40].
Comparatively, exosomes from impaired hepatocytes appeared to affect peripheral monocytes in a more complex manner than those from in situ liver macrophages. It was reported that the activatedpromoted the transcription of serine palmitoyl transferase genes in mouse hepatocytes via upregulating X-box binding protein 1 (XBP1), resulting in ceramide biosynthesis and release of ceramide-enriched inflammatory EVs, which recruited bone marrow-derived macrophages (BMDMs) to the liver, resulting in inflammation and fibrotic injury in mice with diet-induced steatohepatitis [41]. Palmitate treatment also promoted tfrom hepatocytes by activating mixed lineage kinase 3 (MLK3) signaling cascade. Subsequently, the lipotoxic hepatocyte-derived EVs were delivered into BMDMs, and promoted macrophage chemotaxis into the liver in a CXCL10-dependant manner [42]. In addition to affecting the chemotaxis of BMDMs, EVs secreted from hepatocytes also have impact on the BMDM activation. Either palmitate or its active metabolite lysophophatidylcholine could promote the release of death receptor 5 (DR5) ligand-bearing EV from injured hepatocytes by activating DR5-caspase 8-caspase 3-Rho-associated kinase 1 (ROCK1) proapoptotic signaling. These lipotoxic EVs further induced BMDM (M1-like phenotype) activation by increasing the gene expression and release of pro-fibrotic cytokines in a receptor-interacting protein kinase 1-dependent manner, accelerating the progression of fibrotic liver injury and inflammation [43]. These findings clearly link injured hepatocytes-released EVs to a profibrotic macrophage response.
In addition to the direct dialogue with macrophages, hepatocytes also affect many other immune cells like multifarious T-cells that communicate with macrophages and influence the fibrotic response in the liver. A recent study revealed that under alcoholic stress, hepatocytes were damaged first and secreted mitochondrial double-stranded RNA-enriched exosomes, which were uptaken by kupffer cells and stimulated IL-1β production through the activation of TLR3. The IL-1β produced by those injured kupffer cells further promoted the IL-17A production in γδ T cells in a short time and increased IL-17A production in CD4+ T cells and liver fibrosis after chronic alcohol consumption [44]. In addition to CD4+ T cells, CD8+ T cells are another crucial type of T lymphocyte involved in intercellular communication-mediated immune responses. Moreover, hepatoma cells-derived exosomal miR-23a-3p were uptaken by THP-1 monocytes-derived macrophages, which subsequently decreased PTEN expression and elevated AKT phosphorylation and PD-L1 expression in macrophages. The macrophages further decreased the ratio of CD8+ T cells, promoted T cell apoptosis and fibrotic liver damage [45]. Recently, Liu et al. also identified the interaction between hepatocytes and NK cells and demonstrated that hepatic depletion of FBP1 derepressed an zeste homolog 2 (EZH2)-dependent transcriptional program to inhibit PKLR expression. This led to the reduced transmission of PKLR-enriched EVs from hepatocytes to NK cells, thus dampened the function of EV-targeted NK cells and promoted liver fibrosis and tumorigenesis [46]. Collectively, at present, the research on the intercellular transmission of hepatocyte-derived EVs mainly focuses on their interaction with immune cells represented by macrophages and associated T cells, prompting the specific EVs to be activated and produce diverse cytokines to disturb immune microenvironment and thus accelerate hepatic fibrogenesis.
Following LSECs injury, the pathological generation of intrahepatic vessels and progressive microvascular dysfunction will increase hepatic vascular resistancethat regarded as a major complication of liver fibrosis [47]. Previous study reported that hedgehog ligands-enriched EVs released from myofibroblastic HSCs altered the phenotype of neighboring LSECs and promoted vascular remodeling by upregulating the expressions of SEC activation markers including B-Actin, annexin V and keratin 19 (CK19) and inducing the release of vasoactive factors like nitric oxide (NO) [48]. Numerous reports of fibrotic liver diseases pointed out that significant changes in miRNA profiles played a destructive effect on hepatic sinusoid since individual miRNA simultaneously regulated multiple target genes. As a molecular cargo of EVs secreted by hepatoma cells, miR-103 was reported to significantly disrupt endothelial barrier integrity by suppressing adherent molecules including VE-Cadherin, p120-catenin and zonula occludens 1 (ZO-1) in LSECs, which increased vascular permeability and promoted fibrotic liver injury [49]. Althot obtained from the liver, they are commonly used in in vitro experiments as a replacement of LSECs due to their relative ease of isolation and similarity in characteristics to LSEC. MiR-1 was enriched in exosomes secreted by steatotic hepatocytes and caused inflammatory and fibrotic injury in HUVECs through the following two pathways: on the one hand, exosomal miR-1 activatedh endothelial inflammation and fibrosis, especially for vascular cell adhesion molecule-1; on the other hand, it suppressed the expression of the zinc finger protein Krüppel-like factor 4 (KLF4) in HUVECs [50]. It is well-known thatfunction as important intracellular signaling molecules in TGF-β signaling and are only substrates for TGF-β intracellular kinase, which is the reason why SMADs are critically linked to the development of liver fibrosis [51]. Notably, KLF4 silencing effectively 'hijacks' SMAD promoters to inhibited TGF-β transcription [52]. Considering the close relationship between KLF4 and SMAD, the researchers also examined the role of SMAD between hepatocytes and HUVEC. Exosomal miR-210 was released from hepatoma cells and transferred to HUVECs, there. Furthermore, exosomal miR-210 increased the microvascular density and promoted liver fibrosis in vivo, which was primarily due to the inhibition of SMAD4 and STAT6 expression [53]. Our prior studies identified that lncRNA H19 rapidly and highly expressed in the liver of patients with liver fibrosis and was closely[54]. Interestingly, exosomal lncRNA H19 was reported to promote HUVECs proliferation and adhere to CD90+ cancer stem cells (CSCs) by regulating intercellular cell adhesion molecule-1 phenotype and further regulate hepatic material exchange microenvironment. In addition, after entered HUVECs, exogenous H19 exosomes activated the production and secretion of VEGF and thus increased tubular-like structures in the liver sinusoids, leading to the pathological neovascular response and liver fibrosis [55].
In addition to miRNAs and lncRNAs, hepatoma cells can also deliver protein signals via EVs to LSECs and accelerate fibrosis progression. Lysyl oxidase like 4 (LOXL4), regarded as extracellular copper-dependent enzymes that involved in ECM cross-linking, was found to be secreted by HCC cells and partly assembled by exosomes to increase the adhesion of cellular matrix via phosphorylation of Src and focal adhesion kinase. In addition, HCC-derived exosomes transferred LOXL4 proteins to both HCC cells and HUVECs and promoted angiogenesis and liver fibrosis in a paracrine manner [56]. Another research suggested that HCC cell-derived exosomes promoted the angiogenesis of HUVECs by transmitting angiopoietin-2 (ANGPT2) to HUVECs, which in turn, promoted the progression of liver fibrosis. Of note, exosomal ANGPT2 increased the phosphorylation of AKT, endothelial nitric oxide synthases and β-catenin rather than activated the classic ANGPT2/tunica interna endothelial cell kinase (Tie2) pathway in HUVECs [57].
Our recent study found that mtDNA-EVs secreted by damaged hepatocytes aggravated hepatocyte death in the CCl4-induced hepatic fibrosis mouse model, thus releasing more immunogenic DAMPs and accelerating innate immune response and fibrogenesis in an autocrine manner [5]. However, few studies exist evaluating how damaged liver cells affect other hepatocytes to influence the progression of liver fibrosis, which is worthy of further study.
An important proportion of HCC cases are developed from hepatic fibrosis or cirrhosis [58]. It is worthy to note that HCC cells also regulate the fibrotic process through the transfer of EVs and feed back to favor their own growth at the same time. Seo W. noticed this phenomenon and first demonstrated that aldthe development of alcohol-related liver fibrosis in both patients and mouse models developed with liver fibrosis and HCC. Mechanically speaking, a large number of harmful mtDNA were released by ALDH2-deficient hepatocytes through EVs after chronic alcohol exposure, which could be transferred to adjacent HCC cells and subsequently activate multiple carcinogenic pathways such as c-Jun N-terminal kinase (JNK), STAT3, B-cell lymphoma-2 and PDZ-binding motif (TAZ), thus promoting fibrotic liver injury and tumor growth [59]. More interestingly, exosomal miRNA-25-5p from HCC cells (HuH-7 or HCCLM3) were able to transfer to anoikis-resistant HCC cells, a kind of liver circulating tumor cells (CTCs) being particularly prone to distant metastases. The mRNA profiles of anoikis-resistant HCC cells revealed the significant reduced leucine-rich repeat containing 7 (LRRC7) expressions since extracellular mature exosomal miR-25-5p directly targeted its 5' UTR, which facilitated the trans-endothelial migration of CTCs in a dose dependent manner [60]. HCC cells received self-derived miRNAs (such as miR-423-5p and miR-21-5p)-enriched exosomes to promote cell growth, migration and fibrosis. Moreover, Vps4A utilized exosomes as mediators to downregulate the secretion and uptake of miRNAs in hepatoma cells through the suppression of PI3K/AKT signaling, leading to significant repression of HCC metastasis and fibrotic liver damage [61].
Interestingly, despite being the principal fibrogenic cell type in the liver, there are few studies on intercellular communication between HSCs and themselves (Figure 3). Notably, compared with paracrine mode, whether HSCs could secrete substances to mediate self-change is an interesting and emerging topic. In addition to the TGF-β we are familiar with, platelet-derived growth factor (PDGF)-BB has also been reported to be an essential factor that activates HSCs. Being activated by PDGF-BB, HSCs secreted phosphorylated tyrosine 720-dependent PDGF receptor α (PDGFRα)-enriched EVs to neighboring HSCs. Moreover, the phosphorylated tyrosine 720-recruited Src homology 2 domain tyrosine phosphatase 2 (SHP2) was also enriched in PDGFRα-loaded EVs and then these EVs were translocated into recipient HSC, triggering the transcription of downstream pro-fibrotic genes (Col1a1 and Acta2) and resulting in the deterioration of liver fibrosis [62]. Similarity, Gao et al. also demonstrated that PDGF- and SHP2-stimulated HSCs released nano-sized vesicles enriched with fibrogenic proteins includie2. Then the secreted vesicles further induced HSC migration by upregulating mTOR and Rho-associated protein kinase 1 (ROCK1) signaling and inhibiting autophagy [63]. Given that HSC is the key cell type in the hepatic fibrogenesis processautocrine-dependent activation of HSC in different types of liver fibrosis injury, which is of great significance for the targeted inhibition of HSCs.
Exosomes derived from HSCs not only act as mediators of HSCs self-communication but also participate in the dialogue between HSCs and liver nonparenchymal cells. It has been reported that miR19b- and miR200-enriched EVs released from activated-HSCs were transmitted and colocalized to macrophage cell membranes, stimulated the release of pro-fibrotic cytokines (like IL-6 and tumor necrosis factor-α) from these macrophages, prompting hepatic inflammation and fibrosis progression [64]. In addition, under stimulation of Hif-1, an oxygen-sensitive transcription factor, activated HSCs released glycolysis-related proteins (GLUT1 and PKM2) via exosomes to accelerate glycolysis in liver nonparenchymal cells under hypoxic conditions. Quiescent HSCs highly proliferated due to metabolic phenotype alteration after accepting exosomes [65], while other recipient cells like kupffer cells and LSECs warranted further elucidation.
It has been reported that HSCs phagocytosed apoptotic bodies from hepatocyte, suggesting that the fate of hepatocytes is closely related to HSCs [66]. As demonstrated by Coulouarn and his colleagues, after coculturing with activated HSC, HepaRG turned into the migratory and prominent inflammatory phenotype by improv-10 and IL-8) and growth factors (AREG, EREG) and upregulation of acute phase proteins (CP and SAA1) [67]. Surprisingly, recent studies have found that EVs secreted by activated HSC can be an effective intervention strategy for suppressing HCC cells growth. HSC-derived EVs loaded with miR-335-5p were uptaken by cancerous hepatocytes and consistently downregulated 13 downstream mRNA targets of miR-335-5p to inhibit hepatocyte proliferation in vitro. Besides, exosomal miR-335-5p was indeed utilized by cancer cells to slow the growth of HCC cells after being artificially injected to tumor tissue in vivo, suggesting miR-335-5p as a potential HCC therapeutics [68]. Moreover, LX-2 cells could rapidly shuttle exosomal MiR-214 to hepatocytes that suppressed connective tissue growth factor (CCN2) 3'-UTR activity and expression of CCN2 and its downstream targets such as α-SMA and collagen, inhibiting fibrogenic signaling [69]. More interestingly, a recent study reported that exosomal miRNAs from donor cells are uptaken by recipient cells and further reduces the release of homologous miRNA from recipient cells. Exosomal miR-30a-3p derived from HSCs directly inhibited the migration and invasion of HCCs and indirectly reduced EV-containing miR-30a-3p released from HCC cells by downregulating synaptosome-associated protein 23. Reduction of EV released from HCC cells formed a positive feedback loop to increase miR-30a-3p accumulation in HSCs, which in turn, enhanced the role of miR-30a-3p played in reducing the migration of HCC cells [70].
Schematic diagram of HSCs-centered EVs network. Activated HSCs deliver EVs to quiescent HSCs to activate the liver fibrosis process. Kupffer cells, LSECs and hepatocytes all receive EVs secreted by HSCs, whereas the effect of EVs released by HSCs on cholangiocytes and other immune cells has not been identified and deserves further investigation, since HSC activation is an important link in the initiation of liver fibrosis. Solid lines indicate that this has now been reported and dashed lines indicate that it has not yet been reported.
Just as hepatocytes generally secrete exosomes to LSECs, hepatocytes can also internalize exosomes derived from LSECs, supporting the relevance between hepatocytes and LSEC during the progression of hepatic fibrosis. A variety of studies have already pointed that LSECs also release exosomes targeting cancerous hepatocytes and thus directly accelerate liver fibrosis. Yes-associated protein (YAP) is a critical downstream target in the Hippo signaling pathway, which may be involved in liver fibrosis by regulating HSC proliferation and apoptosis [71]. Several studies have shown that the clinical application of YAP inhibitor verteporfin induced the spread of HCC, of which possible mechanism was further revealed by the study of Yang L and colleagues. Under the deficiency of YAP caused by intervention with small interfering RNAs and verteporfin, vascular endothelial cells exhibited a higher level of further promoted the release of exosomes transferring to HCC cells. Notably, these YAP1-deficiency exosomes containing lncRNA MALAT1 increased the invasion and migration of HCC  [72]. As mentioned earlier, exosomal MALAT1 released by hepatocytes can directly deliver to the HSCs, so whether MALAT1 released from LSECs via EVs also targets and regulates HSCs may be a direction worth exploring in the future.
Meanwhile, unlike 'bad' exosomes secreted by injured or cancerous hepatocytes targeting LSECs during liver fibrosis; 'good' exosomes might be also produced from virus infected LSECs and further deliver to hepatocytes and restore liver function. Giugliano S. first demonstrated that HCV induced the broad transcription of Type I and Type III interferons (IFN) in human LSECs that internalized HCV in a clathrinid-dependent manner. Both Type I and Type III IFNs stimulated human LSECs to release exosomes, which abrogated HCV infection in uninfected human LSECs via robustly upregulating the transcription of Type III IFNs and viperine. Subsequently, LSECs leaked-exosomes dose-dependently upregulated the transcription of IFN-stimulated genes to inhibit of HCV replication in hepatocytes and promoted anti-viral responses, consequently leading to the prevention of liver fibrosis [73].
HSCs situate in the subendothelial space of Disse and are anatomically adjacent to LSECs [74]. Therefore, the neighboring intercellular communication between liver endothelial cells and HSCs through different transmission modes especially for EVs cannot be ignored. Initially, the possibility of crosstalk between LSECs and HSCs was observed to be associated with sphingosine kinase 1 (SphK1). Endothelial cells-derived exosomes expressed fibronectin on membrane surface and then adhered with α5β1-integrin on HSCs to enter the HSCs through dynamin 2-dependent endocytosis. SphK1 entrapped within endothelial cell-derived exosomes activated AKT phosphorylation while promoted pathological HSC migration and aggravated fibrogenesis [75]. Another study further found that natural products influenced the above pathways to alleviate the process of liver fibrosis, thus clarifying the importance of SphK1 in LSEC-to-HSC communication. Salidroside subdued the release of exosomal SphK1 from LSECs and lessened HSC migration and activation by inhibiting AKT phosphorylation. Additionally, it inhibited JNK phosphorylation to reduce mitochondria-mediated hepatocyte apoptosis and apoptotic irritants release, which subsequently decreased the secondary damage to HSCs [76]. Most recently, Wu et al. first reported that adipocyte fatty acid binding protein (A-FABP) from LSECs exacerbated t activating HSCs. Mechanistically, the enhancement of intracellular A-FABP in LSECs activated Hedgehog signaling pathway to potentiate LSEC capillarization. In addition, A-FABP released from the conditional medium (containing an amount of EVs) of LSECs was phagocytized by HSCs in a paracrine manner and then activated the JNK/c-Jun signaling to increase the transactivation of TGFβ1, leading to the perpetuation of HSC activation [77]. Although Wu et al. didn't distinguish the pro-fibrotic effects of LSEC-released A-FABP on HSCs are dependent on EVs or other ingredients in conditional medium, they still provided a critical future research direction for this field (Figure 4).
Our results and a growing number of other studies all point out that cholangiocytes are the primary targets of exogenous stimulus especially for bile acids and play a very important role in the whole spectrum of liver fibrosis [78]es of EVs isolated from cholangiocytes on liver fibrosis likely due to few quantities and isolation difficulties (Figure 5). Under the co-stimulation of estrogen and taurocholate (TCA), lncRNA H19 was markedly induced and enriched in exosomes released from cholangiocytes, which were further uptaken by hepatocytes. Furthermore, we found that overexpressed lncRNA H19 suppressed the level of small heterodimer partner (SHP) in hepatocytes by inhibiting its promoter activity and mRNA stability, thereby disrupting bile acid metabolism and resulting in liver fibrosis [54].
On the account of strong correlation between hepatocytes and HSCs, we further probed whether lncRNA H19-riched exosomes secreted from cholangiocytes directly regulated the activation or proliferation of HSC and thus affected collagen deposition. As expected, our results showed that cholangiocytes-derived exosomal lncRNA H19 was rapidly uptaken by HSCs and HSC-derived fibroblasts under TCA challenge, which subsequently upregulated the expressions of fibrotic genes to activate HSCs and promoted the trans-differentiation from HSCs to fibroblasts. In addition, exosomal lncRNA H19 also directly promoted HSC proliferation by enhancing G1/S cell cycle transition, leading to the exacerbation of liver fibrosis [79]. Meanwhile, it has been shown that lncRNA H19 was increased in serum EVs from patients with biliary atresia and fibrosis and restrained the bioavailability of miRNA let-7 family to promote bile ducts proliferation and BDL-induced liver fibrosis [80lncRNA H19-riched exosomes affect the transcription of fibrosis genes in HSCs by influencing other transcription factors or miRNAs such as let-7.
During processes of various cholangiopathies, injured cholangiocytes increase the release of various inflammatory and fibrogenic mediators and engage in adjusting barrier function of endothelial cells. An elegant study has shown the potential of intercellular communication between cholangiocytes and LSECs via EVs. Cholangiocarcinoma-associated circular RNA 1 were wrapped in EVs secreted by cholangiocytes and transferred to vascular endothelial cells to increase the expression of SH3 domain-containing GRB2-like protein 2 and trimethylation of promoter histone 3 lysine 27, which inhibited the expressions of ZO-1 and occludin andr cells, leading to the destruction of vascular endothelial barrier integrity, angiogenesis and inflammatory reaction during liver fibrosis [81].
Schematic diagram of LSECs-centered EVs network. LSEC-secreted EVs are poorly studied and focus on hepatocytes and HSCs. EVs delivering to hepatocytes both are 'good' or 'bad', with the former inhibiting HCV replication in hepatocytes and the latter activating oncogenic hepatocyte migration. LSECs release pro-fibrotic EVs to HSCs, which can be inhibited by the natural product salidroside. However, the effect of EVs secreted by LSEC on themselves,explored yetashed line indicates that it has not been reported.
Schematic diagram of cholangiocytes-centered EVs network. The cholangiocytes can release lncRNA H19 into hepatocytes, HSCs and macrophages (liver-resident and bone marrow-derived), causing hepatocyte injury, HSC proliferation activation, and macrophage differentiation to the M1 phenotype and exacerbating hepatic fibrosis. Cholangiocyte-derived exosomes are also transmitted to LSECs, which results in the disruption of hepatic blood sinusoidal endothelial barrier by increasing cell permeabilityIn addition to LSEC, immune cells including kupffer cells are also recruited to the injured biliary ductal milieu, promoting cholestasis and fibrotic liver injury. We continued to delve into this question about how lncRNA H19 influenced the pathological microenvironment and liver fibrosis via EVs. Notably, we first demonstrated that lncRNA H19-enriched exosomes enhanced the active M1 polarization of kupffer cells and promoted the recruitment and differentiation of BMDMs through the activation of CCL-2/CCR-2 signaling pathways [82]. These results indicated that cholangiocyte-secreted H19-exosomes played a critical role in promoting macrophage activation and hepatic inflammation in cholestatic liver diseases. The current status of little research on the communication of EVs between cholangiocytes and immune cells led us to propose that CCL-2/CCR-2 signaling may induce the release of proinflammatory EV from cholangiocytes, which further activate immune cells by a chemokine-dependent process. In addition to kupffer cells, whether EVs participate in theother immune cells such as myeloid and lymphoid cells regulated by CCL2 by driving janus kinase 2 (JAK2)/STAT3, mitogen-activated protein kinases or PI3K signaling associated with cell survival remains to be further investigated.
In addition to above hepatic parenchymal and non-parenchymal cells, the liver also recruits and activates many innate and acquired immune cells from themselves or bone marrow when injury occurs. These recruited immune cells regulate the immune/inflammatory and the tissue repair responses in the acute phase, while trigger the inflammatory and the tissue excessive repair responses alternately in the chronic inflammatory phase, evoking secondary tissue damage. Indeed, recent accumulating evidence suggests that immune cells secreted therapeutic EVs to rescue dying hepatocytes. TLR3-activated macrophages were reported to transmit the anti-fibrotic and anti-inflammatory factor miR-29 to infected hepatocytes through exosomes, thus suppressing virus replication and fibrotic liver damage [83]. Recombination signal binding protein-Jκ (RBPJ) is a critical switch for Notch signaling that is. He et al. demonstrated that exosomes loaded with RBPJ decoy oligodeoxynucleotides (ODNs) by tail vein administration could be primarily taken up by macrophages and blocked Notch signaling, thereby ameliorating hepatic inflammation and fibrosis in CCl4- or BDL-induced mouse model [84]. Contradictorily, Zhang et al. discovered that RBPJ-overexpressed macrophages secreted abundant curative EVs to respond liver malignant diseases resulting from progressive fibrosis. When RBPJ-overexpressed macrophages were transplanted into the nude mice, the severity and volume of malignant liver lesions were prone to alleviate and shrink because exosomal Hsa_circ_0091570 from macrophages was delivered into HCC cells and acted as a ceRNA of miR-499b-5p to result in the repression of JAM3 [85]. Therefore, the multifaceted effect of RBPJ expressed in macrophages during liver diseases are associated with the different of clinical disease stages. We hypothesized that inhibition of RBPJ in the early stage of disease is a possible therapeutic approach to fibrotic liver diseases, while the inhibition of RBPJ in the late stage of the disease may lead to unsatisfactory treatment effect.
It is generally believedxcessive hepatic inflammation and subsequent liver fibrosis. Contrary to what is well accepted, interestingly, Hou et al. identified that macrophages and neutrophil infiltration alleviated tissue fibrotic remodeling during initial phase of fibrotic response in NASH. IL-6 activated EVs biogenesis- and trafficking-related genes (Hrs, Stam1 and Vta1) to release a large number of EVs containing miR-223 from myeloid cells via STAT3 activation. After IL-6 knockout combined with high-fat diet, neutrophils and infiltrating macrophages were drastically decreased in liver, which reduced miR-223 secretion and thus upregulated TAZ expression to promote NASH and liver fibrosis [86]. Soon after, He et al. found additional evidence about the essential role of neutrophils played in liver fibrosis. FFAs were able to activate miR-223 transcription through the up-regulation of purine-rich PU-box-binding protein 1 (PU.1) expression in neutrophils and increase the level of apolipoprotein E on neutrophil-derived EVs surface. These neutrophil-specific EVs were then selectively uptaken by steatotic hepatocytes via to mitigate hepatic inflammation and fibrosis [87]. Thus, EVs secreted by immune cells reduced the release of pro-inflammatory or fibrotic factors from damaged hepatocytes as well as induced apoptosis of irreparably dysfunctional hepatocytes. Moreover, whether multiple T cells, as the main acting cells of acquired immunity, have above regulatory effects on hepatocytes, is quite interesting and worth studying in the future.
Emerging evidences have indicated that immune cells also release extracellular signals to directly regulate HSC activation. A recent clinical study found that NK cells were positively correlated with better liver function in HCV-associated liver fibrosis, revealing that increased NK cells might slow down the fibrogenesis [88]. EVs isolated from NK cells not only distinctly mitigated TGF-β1-reduced proliferation and activation of HSCs in vitro, which was reversed by a specific exosome inhibitor GW4869, but also alleviated in the CCl4-induced fibrotic mouse model [89]. Wang et al. further provided more direct evidence for this specific phenomenon and found that NK cells could rapidly transfer exosomal miR-233 to HSCs that suppressed autophagy by inhibiting the mRNA expression of autophagy marker ATG7, and consequently decreased the number of TGF-β-activated HSCs [90]. Unlike protective effects induced by NK cells, recent research pointed out that macrophages secreted pathogenic EVs to activate HSCs and thus brought about a damaging effect on the liver. Stimulated with lipopolysaccharide, the miRNA profile of THP-1 macrophages-secreted exosomes were significantly altered with increased levels of miR-155-3p and miR-103-3p and reduced levels of miR-20a-3p and miR-106b-5p. Notably, miR-103-3p-enriched exosomes promoted the proliferation and activation of HSCs via the inhibition of KLF4 expression [91]. Since hepatocyte-derived exosomes have been previously mentioned to inhibit KLF4 expression in HSCs, we speculate that KLF4 may be an important potential target for intercellular communication by EVs in the liver. Therefore, we conjecture whether EVs secreted by immune cells enter LSECs and activate endothelial inflammation in the liver sinusoids by regulating KLF, which can be a point for future research (Figure 6).
In this review, we comprehensively summarized both pathogenic and protective effects of EVs on different fibrotic liver diseases by describing in detail the substances encapsulated in EVs secreted or excreted by different cells in the liver, and how these substances affect the target cells in the liver (Figure 2-6). Based on the summary of EV transmission by specific liver cells, the 'good' or 'bad' EVs that transmit intercellular signals between two deterministic liver cells could be more obviously identified, thus helping researchers to delve into the fundamental rules of EV-mediated signaling in the liver.
Schematic diagram of immune cells-centered EVs network. Immune cells primarily secrete therapeutic EVs to alleviate liver disease. Macrophages and neutrophils deliver EVs to hepatocytes to reduce the development of fibrosis and cancerous lesions. Activated HSCs receive NK cell-derived EVs to activate autophagy. Macrophages, in contrast, deliver pathogenic EVs to HSCs and promote HSC activation by inhibiting KLF4 expression. Confirmatory studies about EVs delivery from immune cells to themselves, cholangiocytes or LSECs have not been reported, which should be considered for further study.
Taken as a whole, there are more studies related to hepatocytes affecting HSCs and immune cells via EVs but only few studies about cholangiocytes and LSECs affecting other cells in the liver. On the one hand, growing experimental evidence showed that cholangiocytes injury is essential for myofibroblast differentiation of portal fibroblasts and HSCs, suggesting a non-negligible role of cholangiocytes in the pathogenesis of hepatic fibrosis [92]. In addition, cholangiocytes recruited immune cellsMeanwhile, damaged cholangiocytes are also reported to express pattern-recognizing molecules to sense inflammatory mediators secreted by themselves and further regulate their survival and function [6]. On the other hand, LSECs represent the first barrier orchestrating liver responses to pathogens, tissue injury and various trauma. Once injured, abnormal phenotypic changes of LSEC characterized by the loss of fenestrae and formation of large gaps and basement membrane contribute to liver fibrosis progression. Of note, the above two cell types are physically close to each other and have been reported to release pathogenic substances into the hepatic environment, however, few studies have made direct evidence about which targeted liver cells are these substances released into. Moreover, it also remains to be determined whether these damaged cells interfered with the process of cellular communication by altering their own physiological properties or promoting the release of pathological signals. Therefore, we suggest that more researchers should pay greater attention on the intercellular communication among cholangiocytes, immune cells and LSECs in the liver. At the same time, we speculate that the reason why these cells have been troublesomely explored may be related to the less content and distribution in the liver and the technical difficulty of the extraction method. Therefore, optimizing current available extraction techniques or making full use of single-cell sequencing may provide effective means of assisting EV-related research in the future.
Among numerous studies reviewed here, hepatocytes represent the 'spotlight cells' of cell-to-cell communication in the liver. Once liver injured, hepatocytes first respond to harmful stimulus and appear structural and functional disorders. Hepatocyte injury is typically driven by cell apoptosis or necrosis thatthe activation of death receptors. Ruptured hepatocyte may initiate the release of multiple damage signals, and a portion of which can be delivered to normal hepatocytes and non-parenchymal cells via EVs. Since there are far more hepatocytes than other liver cells, an imbalanced ratio of injury signals is flooded to the receptor cells, which allows various liver cells to rapidly receive stimulus and then activates fibrotic signaling cascades. The elimination of EVs from damaged hepatocytes or the enhancement of EVs production artificially in normal hepatocytes might provide effective methods for treating fibrotic liver diseases. Although the effects of immune cells have been defined at two separate states, including anti-inflammatory and pro-inflammatory states, the interaction between immune cells and hepatocytes mainly results in the alleviation of fibrotic liver injury. In summary, selective amplification of intercellular signals released by immune cells via EVs is a new alternative way to reduce fibrotic liver disease and participate in the occurrence of many diseases by binding to the 3'untranslated region or open reading frame transcriptional direction of their target mRNAs. Under the normal condition, the susceptible degradation and inefficient cellular uptaken characteristics of miRNA make it difficult to reach the receipt cells. However, because of neutral sphingomyelinase 2 or (and) heterogeneous nuclear ribonucleoproteins activation by its specific sequences [93, 94], miRNAs can be entrapped within EVs with higher stability, and can be effectively delivered to distant receptor cells and regulate multiple biological processes. In addition, the ability of intracellular miRNA to be loaded into exosomes was also enhanced owing to the direct binding of sP) to specific miRNAs enriched in exosomes sharing hEXO motif [95].or retained in cells remain largely unknown. Recently, researchers have made a breakthrough in this field by demonstrating that miRNAs contain sequencing sequences that determine their secretion or cell retention in small EVs (sEVs), and that different cell types use specific sequencing sequences to determine the corresponding sEV miRNA profiles. Insertion or deletion of these sequencing sequences in miRNAs can increase or decrease the intracellular production or secretion of exosomes and other sEVs, therefore, the code possessed by miRNAs is an important factor in linking circulating exosomal miRNAs to tissues of origin [96]. Similar to other diseases, the contents of exosome transmitted between cells are mainly miRNAs and few easily decomposed proteases in the pathological environment of fibrotic liver diseases. Contrasting the available studies, we surprisingly find that miRNAs are primarily focusing on miR-103, miR-1297, miR-192, miR-25, miR210, miR-233, miR-19, miR-30 and miR-335, especially for miR-233 and miR-192, since the former is a pivotal miRNA of lipid metabolism and hepatic inflammation and the latter is expressed at higher level in the liver. Therefore, researchers can shed light more on miRNA to explore contents of EVs transmitting biological signals between liver cells in future studies.
Prior studies have shown that plasma EVs from healthy donors protect myocardium from ischemia-reperfusion injury [97]. In addition, serum EVs have been reported to maintain homeostasis under normal conditions and also alleviate acute hindlimb ischemic injury in mouse model [98], suggesting the rich therapeutic value of serum EVs for treating multiple pathological injuries. In recent years, since EVs function as conduits for intercellular molecular transfer, the therapeutic effects of EVs have increasingly aroused the interests of researchers in liver diseases. It has been reported that EVs from healthy donors possess anti-fibrotic properties. EVs derived from normal mouse serum have been reported to inhibit CCl4 or thioacetic acid-induced fibrotic liver injury by inhibiting fibrogenic- and HSC activation-related gene expression in mice [97]. Similarly, human serum EVs have been reported to have similar therapeutic effects as mouse serum EVs [97]. Notablyrelatively high in EVs based on summary of reviewed above. We thus speculate that the suppression of miRNAs in EVs results in a therapeutic action targeting HSC fibrogenesis. So, it will be of great value to understand the function of EVs in hepatic intercellular communication and their potential as carriers of biomarkers to aid disease diagnosis and prognosis. At the same time, some researchers have found that EVs derived from red blood cells are natural liver drug carriers because of their characteristics on liver accumulation [99]. Exosomal membrane protein CD9 was fused with RNA binding protein to encapsulate target miRNAs in exosomes and relief liver injury caused by CCl4 in vivo [100], suggesting that the construction of artificial liver targeted EVs may be applied in clinical practice. With the rapid development of isolation, detection and artificially-modification technology of EVs, the researches and clinical applications of EVs will be much more in-depth, revealing the irreplaceability of EV in the treatment of fibrotic liver disorders and their complications.
In the current review, we provide an update information on the contents of the intercellularly delivered EVs as well as their roles between different liver cells. Furthermore, our work points out the shortcomings of current EV research and proposes approaches for improving the rigorousness of future studies. We expect that these intercellular EVs will have the potential to become diagnostic and therapeutic targets in the near future, which will greatly influence the scenery of hepatology and other medical specialties.
BMDM: bone marrow-derived macrophages; CXCL10: C-X-C motif ligand 10 EVs: extracellular vesicles; HSC: hepatic stellate cellFN: interferon; IL-17A: interleukin-17A; LSEC: liver sinusoidal endothelial cell; mtDNA: mitochondrial DNA; MMP2: matrix metalloproteinase 2; MVBs: multivesicular bodies; PDGF: platelet-derived growth factor; PTEN:; SMAD: small mothers against decapentaplegicLR3: toll-like receptor 32274186 and 82004045 to XL,);O. ZYYCXTD-C-202006 to XG and XL NO. 7212174 to XL);(Grant NO. 2020-QNRC2-01 to XL).
Dr. X. Li conceived the original idea. Dr. Y. Li, Dr. J. Wu and Dr. R. Liu wrote and revised the paper. Dr. Y. Li and Ms. Y. Zhang8. Alshehade S, Alshawsh MA, Murugaiyah V, Asif M, Alshehade O, Almoustafa H. et al. The role of protein kinases as key driversprogression: New insights and future directions. Life Sci. 2022: 120732.
Corresponding author: Xiaojiaoyang Li, Ph.D., E-mail: xiaojiaoyang.liedu.cn.


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Unlike the BBC’s Panorama (1), The British Medical Journal (BMJ) gives space to a wide range of views on mental health and behavioural disorders. An article on adult ADD / ADHD last week is an example of how polarized debates can become. One side repeated, as fact, Panorama’s “unfair and not open-minded” opinion on the poor long-term outcome for medication in ADD / ADHD (2).
The BMJ published my own comment online (3), pointing out the need to test and monitor new nurse-led adult ADD / ADHD diagnosis and treatment (4). Of course, the Department of Health is quite right to look for the most cost-effective ways of delivering services, and nurses bring their own distinct and valuable skills to a wide range of clinical settings. But I have already had two conversations with specialist NHS nurses working with children and teenagers, who were concerned at being pushed into taking on too much responsibility too soon.
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Here are the stories of nearly two dozen species recently declared extinct.
Alex Bonk
Oct 15 · 10 min read
There’s never been a time in human history when so many of the world’s species were vanishing before our eyes extinct, from a tiny catfish and several colorful mussels to some of the world’s most spectacular birds. Without swift and powerful action, we risk losing a million more species in the coming decades to the wildlife extinction crisis.
Here are the stories of the Lost 23:
Illustration of Bachman’s warbler by Louis Agassiz Fuertes
1. The last time anyone saw a Bachman’s warbler was in Cuba in 1981. For decades, though, it was considered one of the rarest songbirds in North America, living mostly in swampy forests of the Southeast. A Lutheran minister named John Bachman, a friend of John James Audubon, provided the first written description of the warbler in 1832 from a specimen found near Charleston, South Carolina. They were small, yellow and black birds with a charming, if monotone call, sometimes sounding like tsip or zee-e-eep. Extinction was due to logging and collection.
2. The Guam bridled white-eye was impossible to miss if you knew what to look for. It was a little green, yellow and white bird with a short beak and a distinctive white ring around its eyes. Known as the Nosa’ by the Chamoru people, it was considered one of the most common birds on the island in the early 1900s, inhabiting forests, grasslands and even swamps.  Guam in the 1950s marked the beginning of the end for the bridled white-eye. The last individual of the subspecies was seen in 1983, one year befor.
3. The little Mariana fruit bat wasn’t all that little, actually — it had a two-foot wingspan. And loved to dine on tropical fruits. Also known as a flying fox, this bat lived on the island of Guam and was noticeable for the gold on the side of its neck and yellowish-brown fur on its head. It was last seen in 1968, and went extinct because of habitat loss, invasive brown treesnakes and hunting.
4. In Ohio, there used to be a small, shy catfish called the Scioto madtom. By day, it hid under rocks and vegetation. At night, it came out to quietly feed along the stream bottom. Only 18 individuals were ever collected — so it was extremely rare. It was last seen in 1957 and went extinct because of agricultural runoff and silt that spilled into Big Darby Creek, where many endangered species are currently under dire threat from sprawl from the city Columbus, which is polluting this very special habitat.
Photo of ivory-billed woodpecker by Arthur A. Allen
5. So stunning was the ivory-billed woodpecker that it was sometimes referred to as the “Lord God Bird.” The third-largest woodpecker in the world, it once flew in the old-growth forests of 13 Southeast states. Often it could be seen picking beetles out of dead and dying trees or soaring through the forests with its 30-inch wingspan. It had a nasal-sounding call some compared to a tin horn or clarinet. The last verified sighting was in 1944 in an area known as the Singer Tract; pleas made to the Chicago Mill and Lumber Company, which owned the timber rights to the land, were ignored and the area was logged — driving the Lord God bird to extinction.
Photo of San Marcos River by WisdomFromIntrospect (CC BY-SA 3.0)
6. Small but mighty, the San Marcos gambusia made its home in a half-mile, slow-flowing section of the upper San Marcos River in Texas. It was only an inch long and earned its living eating insects. Unlike many other fish, the San Marcos gambusia It was already extremely rare by the tim1980, and was last seen in the wild in 1983, driven extinct by people’s overuse of its water.
Photo of Kaua’i ‘akialoa specimen by Hiart, Bishop Museum, Honolulu
7. The Kauaʻi ʻakialoa was a Hawaiian honeycreeper that lived only on the island of Kauaʻi. It had an extraordinarily long, downcurved bill, which spanned about of a third of its body length and was used to obtain nectar and insects. Never very common, the ʻakialoa was last seen in 1967. It went extinct because of habitat loss and introduced mosquitos that spread avian diseases.
Illustration of Kaua’i nukupu’u by John Gerrard Keulemans
8. The Kauaʻi nukupuʻu was a stout, short-tailed honeycreeper — extremely elusive but delightful to find. Males of the species were a stunning combination of bright yellow, olive and white, with a black mask and bill. The females were mostly olive-gray-white, with yellowish highlights in the face, wings and tail. Although there have been sporadic unconfirmed reports of it over the past century, this bird was likely extinct sometime shortly after 1899, the date of the last confirmed sighting. It was lost to clearing of its lowland forest habitats for agriculture.
Illustration of Kaua’i ʻōʻō by John Gerrard Keulemans
9. The Kauaʻi ʻōʻō had a distinctive, bell-like call that cut through the forests of Kauaʻi. This small black-and-yellow songbird was common in the late 1800s and considered rare by the 1920s. It ultimately went extinctrats, pigs and mosquitos. The Kauaʻi ʻōʻō was the last surviving member of the Mohoidae family, a group of honey-eating birds, and represents. Its call was last heard in 1987.
Illustration of kāmaʻo (top) by John Gerrard Keulemans
10. Kāmaʻo: In the 1880s, the kāmaʻo was the most common bird on Kauaʻi. Sometimes called the large Kauaʻi thrush, it lived mostly in the forest and feasted primarily on fruit and insects. It grew to be about 8 inches long with a brownish body and a gray belly. By 1928 it had mostly disappeared from the island’s outer forests and by 1980 there were just 24 left. The last one was seen in 1987. Extinction was mostly due to habitat loss and introduced mosquitos.
11. In former times, a keen listener on the island of Maui would recognize the lovely call of the Maui ākepa: a quivering whistle ending in a long trill. Already extremely rare by the early 1900s, this 4-inch, dusty-green songbird with a small cross bill was seen only once in 1970, the year it was protected — and just five times after that. The last sighting was 1988, the last hearing in 1995.
12. The Maui nukupuʻu was a small bird found in the wet, high-elevation forests of Maui. Tenacious, this bird used its inch-long bill to peck for insects in the bark of ʻōhiʻa lehua and koa trees. It was feared extinct by 1896, but there were unconfirmed sightings for decades, and a research expedition recorded seeing the bird in 1967 in the Kīpahulu Valley. It was protected in 1970 in the hopes that it might make a resurgence, but the last confirmed sighting is said to have been in 1996.
Illustration of kākāwahie by John Gerrard Keulemans
13. Also known as the Molokaʻi creeper, the kākāwahie was a striking bird with bright orange or red feathers that some said resembled flames. In fact, Hawaiians traditionally used the kākāwahie’s bright plumage in the capes and leis of royalty. If that wasn’t enough, its call sounded like someone cutting wood. The bird largely vanished before it could be thoroughly studied. There were a few sightings in the early 1960s, with the last confirmed report in 1963. Extinction was due to mosquito spread disease, habitat loss and invasive species.
Photo of poʻouli by Hawai’i DLNR Division of Forestry and Wildlife
14. The poʻouli, or black-faced honeycreeper, was once thought to exist in the hundreds on Maui. It inhabited only the very wet, easternmost side of the island and was considered an extremely quiet bird. Extremely rare by the early 1900s, it suffered from habitat loss, disease and a decline in the native tree snails that served as a key food. In 2004 the last three poʻouli were brought into captivity in an effort to save them, but they didn’t survive.
15. On the Hawaiian island of Lānaʻi, there used to be a pretty flowering plant in the mint family that lived in steep, wet canyons. It had white flowers that were occasionally tinged with purple. It was so rare and barely noticed that it was only ever assigned a Latin name: Phyllostegia glabra var. lanaiensis. It was last seen in 1914.
Photo of flat pigtoe mussel by Todd Amacker Conservation Visuals
16. The flat pigtoe mussel inhabited the Tombigbee River in Mississippi and Alabama. It had a preference for clean, fast-flowing water and tended toward shallow stretches of the river, where the bottom was silt-free. The flat pigtoe was just over a couple of inches across, with dark lines on the outer shell and blueish-white inner shells. It went extinct because of dams: T lock and dam system, whose construction began in 1972, sealed its fate. It didn’t get protection under the Endangered Species Act until 1987, seven years after it was last seen.
Photo of green-blossom pearly mussel by Todd Amacker Conservation Visuals
17. The green-blossom pearly mussel was an elusive species, spotted just a handful of times, mostly before 1918. It had an elliptical outer shell — smooth and shiny, with fine green rays — and its inner shell varied from white to salmon-red. Muskrat middens reveal that the green-blossom pearly mussel lived in the Clinch, Powell, Holston and Nolichucky rivers in Tennessee and Virginia. Dam building ruined some of its most important habitat in the headwaters of the Tennessee River; then pollution from coal mining drove it over the edge of extinction. It was last seen alive in the Clinch River in 1982.
Photo of southern acornshell mussel by Todd Amacker Conservation Visuals
18. The southern acornshell mussel was small, smooth, shiny and yellow. It lived in freshwater stream riffles in Alabama, Georgia and Tennessee, mostly in the Coosa River system and the Cahaba River. It was identified as endangered in 1971, but its Endangered Species Act protection wasn’t finalized until 1993. Meanwhile extensive damming had already driven it to extinction.
Photo of stirrup9. The stirrupshell mussel had a way of standing out in a crowd, with its bumpy and ridged shell with zig-zagging lines. It lived in Alabama in the fast-flowing waters of the Black Warrior and Alabama rivers, as well as in the Tombigbee River in Mississippi. Scientists put it on a list for Endangered Species Act protection in 1971, but it wasn’t protected until 1987, 7 years after the last live individual was encountered. Extinction was prompted by dams, river channeling and sedimentation that smothered its habitat.
Photo of female turgid20. The turgid-blossom pearly mussel was once relatively common in the clean, fresh waters flowing out of the southern Appalachian Mountains in Tennessee and Alabama and the Ozarks in Arkansas. It was yellow and fairly small, at 1.5 inches. The elliptical males were shaped so differently from the rounder females that scientists once thought they were separate species, four years after it was last seen alive in the Duck River. These mussels could live to be 50 years of age but were driven extinct by dams that cut off the fast-flowing waters they needed to survive.
Photo of tubercle1. The tubercled-blossom pearly mussel was once widespread and abundant, making its loss even more tragic because it was known from more than 18 major rivers — it ranged from Ontario, Canada, through seven states south to Alabama. This 4-inch long bumpy mussel was wiped out due to dams and pollution from coal mining, logging, industry and agriculture. But it was last seen in 1969: The Endangered Species Act and Clean Water Act came too late to throw it a lifeline.
Photo of upland combshell by Todd Amacker Conservation Visuals
22. The upland combshell rarely exceeded 2.5 inches and sometimes came in a shape described as “rhomboidal.” It had a tawny shell that was occasionally sprinkled with small green dots or broken green rays. The upland combshell was historically found in the Black Warrior and Cahaba river drainages in Alabama and the Coosa River drainage in Alabama, Georgia and Tennessee. Scientists identified it as endangered in 1971, but it wasn’t1993, five years after the last live individual was seen. Declining without protection, dams and pollution drove it over the edge.
Photo of yellow3. The yellow-blossom pearly mussel made its home in shallow, fast-moving waterways in Alabama and Tennessee, including in the Cumberland and Tennessee River systems. It had an elliptical shell that was shiny and sometimes honey-colored. A lucky viewer might spot one with green rays shooting across its shell. Lost to dams and pollution, it was last seen in 1967 in the Little Tennessee Rive
We’re at a critical moment in stemming the global wildlife extinction crisis. It can be done but it takes leadership, courage, vision and action.
The best way to honor the Lost 23 is to get involved. Check out our plan for addressing the extinction crisis and how you can help Save Life on Earth.
Because life is good.
Digital Director, Center for Biological Diversity


articipating in the celebration of World Water Day on March 22 under the title "Water and Climate Change and how they are closely related".


Good tenants can be hard to come by. As a property owner, you will find during the rental process that once you find a good tenant, you willResidential property owners have found that a good tenant pays their rent on time, keeps the home clean and in good shape, which creates a quality relationship between the tenant and landlord. When it comes to property leasing, you want to work with trustworthy and friendly individuals. Once you find such renters, you have to do your part to ensure they…!Experience When searching for commercial…
Have you dreamed of owning property, perhaps several investment properties, in order to have a steady stream of income?How wonderful would it be to make sound i and make a living from the monthly rental income?Many people do just that and in the process of becoming a landlord have discovered many pitfalls and mistakes along the way. It is important to learn what is really involved in owning rental property before contacting a real estate agent to begin the property search. Check out the 8 issues we cover below to ensure you do…
Using real estate as rental property is a process that many individuals use in order to make money. Residential real estate, vacation homes and even commercial property can be a solid investment when used as rental property. Property renting can provide a steady stream of income but what do you do when your property is vacated?While monthly rent payments will pay the bills, it can be detrimental to your finances when you lose a regular tenant. Below are a few tips on how to deal with vacant real estate properties to maintain your investments. Assess Your Property To begin,…
As a property owner, you understand the day to day struggle to keep track of your many investments. If you own property that is currently being used in a residential setting, such as vacation rentals or residential rental, then you may have found yourself overwhelmed at having to deal with tenants or renters as well as the property upkeep. Thankfully, a residential management service is a quality solution to help you with the managing of your many residential properties. Qualified individuals will work for you, providing essential services to your many residential properties, such as regular upkeep, dealing with tenants…


Removals Wanstead, E11 ~ Get Up to 40% Off!
The question then, for the people of Victoria Dock, Leytonstone and Lower Clapton?


"For three days, CEDIA will offer by offeringting for regulations that improve our industry, and hearing from insightful speakers," she says. It's simply the premier gathering of the brightest minds in home technology, bar none."
"A lot of that work can be a 'secret' to many in the industry, but we don't intend it to be a secret," admits Beckner. "So this is our opportunity to not only share how CEDIA is protecting our business, but how our attendees can use their voices to influence policymakers and be part of the solution."
"Our goal is to lead the industry with training on markets and emerging technologies," says Beckner. "This is a volunteer-led program designed to encourage discussion and networking that will create new connections between peers."


The machine, weighing over 70 tons, was dismantled into several elements - the heaviest two weighing 42 and 22 tons - and delivered on five trucks.
Assembly and commissioning of the plant will take place in the next three weeks.
Utz currently operates 28 injection moulding machines in Bremgarten.
For more information about the transport please watch the video.
Additional information can be found at production.


The property is built on two levels, with large terraces and spacious bedrooms that offer natural lightlarge terrace, the garden amorning sun reflected on the sea, with Gibraltar in the distance. Open plan, also with sea views.


Dentures are a common solution used for replacing missing teeth, whether a section of teeth or an entire arch. These removable prosthetic teeth provide some advantages, like restoring your abilities to eat and speak while also improvingFor them to function properly, however, they need to be cared for. Florida Prosthodontics is here to help.
When removing and cleaning your dentures, it is important that they be handled with care. While strong, they can be easily broken if they are dropped. Before removing your dentures, lay a folded towel on the bathroom, just in case your dentures are accidentally dropped.
Your dentures need to be carefully and completely cleaned each night. This should be done using a soft-bristled toothbrush and either a prosthetic teeth. cause serious issues in your mouth. Make sure to clean your entire denture, including the base and around any metalsoak in water or a denture cleaning solution overnight. Leaving your dentures out while you sleep your gums an opportunity to rest. Dentures need to be soaked to prevent them from drying out and warping, which can significantly affect their fit. Make sure you do not use hot water, as hot water can cause warping as well. Store your soaking dentures in a safe location away from curious pets and out of reach of young children.
After cleaning your f you do have healthy teeth remaining, it is important to brush and floss these teeth as normal. You should also gently brush your gums and your other intraoral tissues. Taking care of your mouth is essential for protecting your gums and your healthy teeth, preventingWhat If My Dentures Need Maintenance?
If something feels off about your dentures, they chip or break, the teeth come loose, wires bend, or they do not fit ile you can find denture repair kits for sale, it is important that you do not attempt to fix your dentures on your own. Attempting your repairs can affect the fit of your dentures, which can harm your mouth, or you can cause further damage that might then require denture replacement. If you notice any damage or a change in the way your dentures fit, it is important that you give us a call right away.
Taking care of your dentures is crucial for ensuring that they do their jobdentures, call Florida Pros.


"Tyler needs to start…He's a starting pitcher prospect for us and who knows where he might fit out of the pen?
"I can pull somebody from the pen. That dynamic is going to kind of change each day, I think. I think it's more you've kind of got an idea of who it won't be. Right now, it's the only thing I know for sure."


Is the diplomat involved in a cigarette case?
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are obliged to revenge them as far as possible rather than conclude a treaty with the offenders. Furthermore, there is the Hellenicity, consisting in the same blood and the same language, the common shrines of gods and cult and the same way of life, which the Athenians should not betray (Herodotus, Histories, 8.144.3; Hinge’s underling).
Thus, Hinge argues, “language is quintessential to Herodotus’ concept of ethnicity” (68). In this Hinge is arguing against Jonathan Hall, who in his Ethnic Identity in Greek Antiquity holds the view “that language played only a minor role in the formation of ethnic groups” (Hinge and Krasilnikoff, “Introduction,” 11). Hinge argues that while it may have mattered “what sort of Greek you are” ? whether one was a Spartan, an Argive, or an Athenian ? in the Greek homeland, once the colonization of the eighth and seventh centuries BCE got underway, “a Greek identity” began to emerge “in opposition to the non-Greek natives in Cyprus, Egypt, Libya, Sicily, Italy or Scythia. The otherness of those ‘Barbarians’ and the complete unintelligibility of their languages, which were frequently compared to the chirping of birds, made the existence of a specific Hellenic identity obvious” (Hinge, 69). This identity, as Hinge emphasizes, “is not natural per se, but a cultural construction” that has its origins in the Mycenaean Age and that leads to “the creation of a Koine.” That Koine displaced local dialects, Hinge argues, was not just a way to bridge various local languages and dialects, “but the symptom of a new identity, and not only a symptom, but also a most powerful contribution to that identity” (77).
In his paperersen takes a quite different approach from Bilde’s, for he is not interested in well-known writers like Philo nor is he interested in “the empirical subject matter of Alexandrian Jewry” (Petersen, 116); rather, Petersen’s paper is much more ambitious and is focused on the theoretical problem of how to reconstruct past cultures. Petersen begins by briefly sketching out the history of Jewish people in Alexandria. Then, he examines “Alexandrian Judaism with close attention to a number of theoretical problems that are infrequently mentioned in the predominant strands of scholarship.” Finally, Petersen concludes by offering “a theoretically viable way of reconstructing ancient cultures in a manner that is simultaneously theoretically adequate to the acknowledgement of the confined nature of the sources, and to current insights withinof how to speak and to conceive of culture.” Petersen is critical of approaches to culture that assume one individual, such as Philo, Aristeas, or Artapanus, can speak for or represent a particular culture or subculture. While contemporary scholarship seems to understand this, Petersen maintains that even though many contemporary scholars acknowledge this problem, they proceed to deal with their sources without considering the consequences of taking “one trajectory of thought” as the embodiment of an entire cultural entity. Indeed, “the banalities of culture and


O’ROURKE; ELECTRICITY CREDIT WILL BE WIPED OUT
Sinn Féin spokesperson on says announcements of electricity and gas price increases mean that the government’s €200 electricity credit will be wiped out before it arrives.
Today, Bord Gáis Energy announced price increases for customers to be introduced from 15th April which will amount to a 27% increase on the average electricity bill and a 39% increase on the average gas bi
O’Rourke told Meath Live it is clear that existing government measures to mitigate energy price increases don’t go far enough.
He claimed, “Runaway fuel and electricity costs are reaching crisis point, putting ordinary workers and families under financial pressurng.
“It is clear that existing measures announced by government don’t go anywhere near far enough. This morning, the Minister for Finance said there were no further plans to support struggling families in advance of the Budget in October. This afternoon, Bord Gáis announced price hikes that will wipe of the government’s €200 electricity credit before it arrives. The government obviously do not appreciate the scale of the problem.
“One of these core proposals is to extend eligibility for a fuel allowance. The current criteria are far too limited and mean that many people who need support are being let down by this government.
“Sinn Féin’s proposals would ensure a further 50,000 households would have access to these much-needed supports. We also outlined the need to introduce a discretionary fund of €15m to assist households with energy costs. It is clear that there is an urgent need for this and that it must include people struggling to afford to heat and electricity. This will be vital in ensuring people aren’t plunged into debt and left at the mercy of moneylenders as they struggle to meet these sky-high costs.
“Far too often we see this government act too little too late. Rather than appreciating the urgency of the cost of living crisis, this government is constantly out of touch and on the back foot. I am urging the government to grasp the urgency of the cost of living crisis and act now without delay.
“There can be no half measures like we saw last week with the government’s late and lackluster plans on fuel costs. The government must act urgently, decisively, and deliver for people now.
“Families are facing huge pressures. No one should be faced with the choice of heating their home or feeding their family. Too often, the government turns its back on this crisis and fails to support those who need it. This cannot be allowed to continuefamilies get a much-needed break. It is vital that we put ordinary workers and families first and ensure they get the supports they need now.”
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DUNBOYNE MAN SAYS, ‘I’LL PUT MY BODY ON THE LINE’ IN LIDL ROW


There's some move to have CABI in other parts of Fairfax County, but I haven't heard of anything in Falls Church yet. My guess is that it will progress outward from Arlington in a westerly direction similarly to how it spread to Alexandria. I think it is only a matter of time.
Fairfax is planning to add stations in Reston but not this year. Maybe in a year or two. They have also mentioned Tysons Corner and Merrifield-Mosaic as possible locations, but even farther out on the timeline.
I haven't heard anything about the City of Falls Church joining Capital Bikeshare. They would have to join in order to have any stations within the city limits.
Anyway, if you are a resident of Falls Church, I'd suggest that you contact your local officials and voice your support for bikeshare. I'm sure the officials are aware of CaBi but they may not be aware of the level of local support for bike stations in Falls Church. You can also add bike station suggestions on the crowdsourcing map. There's a link to the map on the CaBi site. (Go to the Station Map page. Then click on the "Suggest a Station Location" link above the main map.) Even though FC does not participate in CaBi, if there are a lot of votes and suggestions for FC stations on the crowdsourcing map, that can help to indicate the level of interest in FC bike stations.
Last edited by PotomacCyclist; 01-26-2014 at 09:27 AM.


Why Is He Not A Goblin?
"Boy!" Harry grimaced. Uncle Vernon didn't have to yell; you could hear him from a mile away but for some reason, he liked to bellow. "We're taking Dudley to the cinema!Stay in your cupboard!" Harry rolled his eyes; his uncle seemed to have forgotten he'd locked him in and said loudly back, "Yes, Uncle Vernon."
He heard the door slam, and sat down on the bed whilst letting out a sigh. He hated his life here at his 'home.' Wouldn't it be nice if the Dursleys actually treated him as part of the family?
He crept out with Petunia immediately barking orders at him. "Clean the kitchen. And make yourself a sandwich or something; we've already eaten."
"Oh, darn it, Vernon, I've left my purse in the restaurant!" Harry heard Aunt Petunia say unhappily from her seat in the living room. "Can you drive me there?"
"Of course, Pet. But what about the children?"
Harry's heart was speeding up; would he be able to go out and visit somewhere, if only for a minute or two?
"We could leave them here, I suppose. Maybe get Mrs Figg to watch them for five minutes or so. I'll ring her and see if it's alright."
Putting the phone down, Aunt Petunia exhaled noisily. "She's twisted her ankle so she can't come across to look after them but she offered to watch them from the window. It should be alright, surely; we'll only be gone for about five minutes."
"Right you are, Pet." Uncle Vernon said. "Dudley, we'll be out for about five minutes and Mrs Figg will be watching from across the road." There was no answer except for a grunt of acknowledgement from Dudley. "Boy, I'll let you stay out of your cupboard just this once, but if anything bad happens, you're going straight back in there, you hear me!"
"I went to see Labyrinth today." Dudley said conversationally. Harry knew nothing good would come of this; it was never a good sign when Dudley started speaking to him instead of punching him. "It was about these goblins who take wished away people." Dudley now had a predatory look in his eyes and was moving closer and closer to Harry. "Y'know, I want to wish you away and see if it works."!
Feeling a rage suddenly sweep over him, he spat, "When Mum and Dad get home, you're gonna be in so much trouble!"
"Dudley Dursley, I presume."
"You're him. The Goblin King." Dudley realised. "But you're not real. You can't be real. You're magic and magic doesn't exist. Mum said so."
"Why is it every mortal must ask me who I am when they appear to know the answer?" Jareth remarked to no-one in particular. "But why tell me I am not real when you can so clearly see that is false. Why do you not believe in magic, Dudley Dursley," Jareth said softly. "Why is it that your mother does not believe in magic?When it has proven itself by taking away your cousin to the Goblin Kingdom."
Jareth continued. "Do you want your cousin back?" At some indiscernible point, he had taken out a crystal ball and was juggling it without ever taking his eyes off Dudley. Hse mismatched eyes watching him, seeming to pierce through to his very soul. "You could have this instead of Harry. It gives you your dreams."
"If I want my cousin back," Dudley said slowly, "then I have to come with you and run your Labyrinth."
"Yes," Jareth confirmed, "but you don't have to." His tone was silky and almost hypnotic as he continued, juggling the crystal with Dudley's eyes following every movement. "You can take the crystal, take your dreams with it and all I ask is that you forget your cousin, you forget the freak, so that he becomes one of us. You'll never have to worry about him again; your family would never have to worry ever again. You could be completely normal."
"Vernon, the boy mustn't know. Nor should Dudley. But we have to keep him here. For our safety. Otherwise, they'll turn up here and use their things to make us forget. They can make us forget everything. Or they'll kill us. Lily told me about one of them; he was evil and he took pleasure in killing normal people like us!"
"No, the boy must stay!I don't care if we have to worry about the secret coming out or having attention directed towards us but we need him for our safety."
It was this more than anything that made him agree to Jareth's offer. "You can have the freak, but I want the ball."
Author's Note: So what did you think?A) Good?B) Bad?C) Fantastic D) Awful?or E) You have a similar/another opinion that you'd love to put in a review?