Antibodies to Gliomedin Cause Peripheral Demyelinating Neuropathy and the Dismantling of the Nodes of Ranvier

Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are conditions that affect peripheral nerves. The mechanisms that underlie demyelination in these neuropathies are unknown. Recently, we demonstrated that the node of Ranvier is the primary site of the immune attack in patients with GBS and CIDP. In particular, GBS patients have antibodies against gliomedin and neurofascin, two adhesion molecules that play a crucial role in the formation of nodes of Ranvier. We demonstrate that immunity toward gliomedin, but not neurofascin, induced a progressive neuropathy in Lewis rats characterized by conduction defects and demyelination in spinal nerves. The clinical symptoms closely followed the titers of anti-gliomedin IgG and were associated with an important deposition of IgG at nodes. Furthermore, passive transfer of antigliomedin IgG induced a severe demyelinating condition and conduction loss. In both active and passive models, the immune attack at nodes occasioned the loss of the nodal clusters for gliomedin, neurofascin-186, and voltage-gated sodium channels. These results indicate that primary immune reaction against gliomedin, a peripheral nervous system adhesion molecule, can be responsible for the initiation or progression of the demyelinating form of GBS. Furthermore, these autoantibodies affect saltatory propagation by dismantling nodal organization and sodium channel clusters. Antibodies reactive against nodal adhesion molecules thus likely participate in the pathologic process of GBS and CIDP.Guillain-Barré syndrome (GBS) is a group of inflammatory neuropathies that affect peripheral nerves. In Europe, acute inflammatory demyelinating polyneuropathy (AIDP) is the most common form of GBS. Autopsy and biopsy studies indicated that both humoral and cellular immune reaction against Schwann cell or axonal antigens are implicated in GBS etiology.¹ Early investigations have found that conduction defects closely correlate with myelin retraction and macrophage invasion in many patients.^{2, 3, 4, 5} Some GBS cases also involve acute demyelination without immune cell invasion and are primarily humorally mediated.^{6, 7} In particular, deposition of complement on the abaxonal surface of the Schwann cells has been shown during the early stage of GBS^{8, 9, 10} and in experimental allergic neuritis (EAN).¹¹ In a recent study, we demonstrated that nodes of Ranvier and paranodes are the targets of the immune attack in GBS and in chronic inflammatory demyelinating polyneuropathy (CIDP).¹² Notably, cell adhesion molecules (CAMs) at nodes or paranodes (gliomedin, neurofascin, and contactin) were recognized by IgG antibodies in patients with GBS or CIDP.^{12, 13} Autoantibodies against neurofascin and gliomedin were also detected in a rat model of AIDP and correlated with important conduction defects.¹⁴ This finding suggested that antibodies to nodal CAMs may participate to the pathogenesis of AIDP and CIDP. However, the exact mechanisms by which these humoral factors mediate demyelination and conduction defects are still elusive.Several CAMs are implicated in node formation and are responsible for the enrichment of voltage-gated sodium (Nav) channels at the nodes of Ranvier.¹⁵ At peripheral, nodes gliomedin and NrCAM are secreted into the nodal gap lumen and interact with neurofascin-186 (NF186) expressed at nodal axolemma.^{16, 17, 18, 19} This interaction is crucial for Nav channel aggregation at nodes.^{19, 20, 21} In addition, the paranodal axoglial junctions are made by the association of contactin and contactin-associated protein (Caspr) with neurofascin-155 (NF155), a variant expressed in glia.²² This adhesive junction forms a barrier to the lateral diffusion of nodal channels.^{19, 21, 23} In a rat model of AIDP, we found that the loss of NF186 and gliomedin at nodes preceded paranodal demyelination and the diffusion of Nav channels in demyelinated segments.¹⁴ This finding indicated that antibodies to nodal CAMs may participate to conduction defects by dismantling axoglial attachment at nodes and paranodes.We investigated whether immunity toward gliomedin and NF186 can trigger peripheral neuropathies and be responsible for demyelination in GBS patients. We found that immunization against gliomedin induced a biphasic condition associated with conduction loss and demyelination. Passive transfer of antibodies to gliomedin exacerbated the clinical signs of EAN and resulted in the disorganization of the nodes of Ranvier. Altogether, these results demonstrate that humoral immune response directed against nodal CAMs participates in conduction abnormalities in peripheral nerves and in the etiology of GBS and CIDP.     

The Glucagon-Like Peptide-1 Receptor Agonist Exendin 4 Has a Protective Role in Ischemic Injury of Lean and Steatotic Liver by Inhibiting Cell Death and Stimulating Lipolysis

Nonalcoholic fatty liver disease is an increasingly prevalent spectrum of conditions characterized by excess fat deposition within hepatocytes. Affected hepatocytes are known to be highly susceptible to ischemic insults, responding to injury with increased cell death, and commensurate liver dysfunction. Numerous clinical circumstances lead to hepatic ischemia. Mechanistically, specific means of reducing hepatic vulnerability to ischemia are of increasing clinical importance. In this study, we demonstrate that the glucagon-like peptide-1 receptor agonist Exendin 4 (Ex4) protects hepatocytes from ischemia reperfusion injury by mitigating necrosis and apoptosis. Importantly, this effect is more pronounced in steatotic livers, with significantly reducing cell death and facilitating the initiation of lipolysis. Ex4 treatment leads to increased lipid droplet fission, and phosphorylation of perilipin and hormone sensitive lipase – all hallmarks of lipolysis. Importantly, the protective effects of Ex4 are seen after a short course of perioperative treatment, potentially making this clinically relevant. Thus, we conclude that Ex4 has a role in protecting lean and fatty livers from ischemic injury. The rapidity of the effect and the clinical availability of Ex4 make this an attractive new therapeutic approach for treating fatty livers at the time of an ischemic insult.The incidence of obesity and fatty liver disease is increasing worldwide. Non alcoholic fatty liver disease (NAFLD) includes a spectrum of liver abnormalities ranging from simple steatosis with preserved synthetic function to end-stage liver disease requiring transplantation.^{1, 2} The cause of hepatic dysfunction related to steatosis remains incompletely defined.³ However, it is known that a steatotic liver has increased susceptibility to ischemic insults, such as those induced during liver resections and liver surgery,^{4, 5, 6} heart failure,⁷ and shock.⁸ In addition, steatotic livers are known to weather the ischemic insult of transplantation poorly,⁹ resulting in increased rates of primary nonfunction and initial graft dysfunction.^{10, 11} As such, fatty livers are routinely turned down for transplantation and this impacts transplant wait list morbidity and mortality.¹² Thus, liver steatosis contributes to the public health burden and methods to mollify the adverse effects of liver steatosis are relevant across a large spectrum of hepatic diseases.The inability of a steatotic liver to withstand ischemic insult is directly related to increased post ischemic cell death, which can occur through necrosis and apoptosis. The fundamental connection between intracellular fat and poor hepatic cell survival¹³ is incompletely understood. However, it has been suggested that methods that decrease intracellular fat reverse this susceptibility and the use of glucagon-like peptide-1 (GLP-1) analogues is one such approach. GLP-1 is secreted from the L cells of the small intestine and its cognate receptor (GLP-1R) is present in several organs, such as the pancreas, brain, heart, kidney, and liver. Although it is well known for its incretin action,¹⁴ it also has pleotropic effects.^{15, 16, 17, 18, 19} In the liver we have shown that GLP-1 or its homologue Exendin 4 (Ex4) acts directly on steatotic hepatocytes to decrease their lipid content.^{20, 21} In addition, a cytoprotective action of Ex4 with improvement in cell survival has also been reported.²² Thus, we hypothesize that anti-steatotic effects of Ex4 in hepatocytes and cytoprotective effects in other organs make it a rational target for investigation in steatotic livers undergoing ischemia reperfusion injury (IRI), a common clinical scenario in people with NAFLD. In this study, we explore the role of Ex4 in protecting against necrosis and apoptosis, the two forms of cell death encountered in hepatic IRI, and we provide evidence to show that Ex4 stimulates lipolysis with a short course of treatment. To our knowledge, this is the first study showing a direct and rapid action of Ex4 in acutely reversing the vulnerability of a steatotic liver to ischemic insults, supporting the investigation of Ex4 as a potential therapeutic agent for treatment of people with NAFLD undergoing ischemic injury and at the time of procurement of a fatty liver for transplantation.     

Interaction of MCM7 and RACK1 for Activation of MCM7 and Cell Growth

MCM7 is one of the pivotal DNA replication licensing factors in controlling DNA synthesis and cell entry into S phase. Its expression and DNA copy number are some of the most predictive factors for the growth and behavior of human malignancies. In this study, we identified that MCM7 interacts with the receptor for activated protein kinase C 1 (RACK1), a protein kinase C (PKC) adaptor, in vivo and in vitro. The RACK1 binding motif in MCM7 is located at the amino acid 221-248. Knocking down RACK1 significantly reduced MCM7 chromatin association, DNA synthesis, and cell cycle entry into S phase. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate dramatically decreased MCM7 DNA replication licensing and induced cell growth arrest. Activation of PKC induced redistribution of RACK1 from nucleus to cytoplasm and decreased RACK1-chromatin association. The MCM7 mutant that does not bind RACK1 has no DNA replication licensing or oncogenic transformation activity. As a result, this study demonstrates a novel signaling mechanism that critically controls DNA synthesis and cell cycle progression.Miniature chromosome maintenance (MCM) proteins were initially identified from autonomously replicating sequence in Saccharomyces cerevisiae. Mutations of some of these proteins, such as MCM7 or MCM3 result in loss of the large chunk of yeast chromosomes in yeast. MCM7 cDNA encodes a 543-amino acid protein and is ubiquitously expressed in all tissues. A large body of studies indicate that MCM7 is a critical component of DNA replication licensing complex in the yeast and xenopus.^1–4 Some studies suggest that MCM4, MCM6, and MCM7 complex contains DNA helicase activity.^5,6 DNA replication licensing complex is multimeric and phase specific. In yeast, DNA replication licensing proteins, such as MCM2-7 and several replication origin binding proteins, such as Cdc6, germinin, and Cdt1, form DNA replication licensing complex in G1 phase to enable DNA replication and to promote cell cycle entry into S phase. Initial implication of MCM7 involvement in human malignancies came from positive immunostaining of MCM7 in several human malignancies, including endometrial carcinoma,⁷ melanoma,⁸ esophageal adenocarcinoma,⁹ colorectal adenocarcinoma,¹⁰ oral squamous cell carcinoma,¹¹ glioblastoma,¹² and thyroid cancer.¹³ The first study addressing the oncogenic role of MCM7 in prostate cancer came from genome analysis of prostate cancer by performing a genome wide copy number analysis using biotin-labeled genome DNA on Affymetrix U95av2 chip.¹⁴ The DNA copy number of MCM7 was found to increase severalfold accompanied with a concomitant increase of MCM7 mRNA level. Subsequent validation analyses suggest that either copy number and/or protein level increase of MCM7 are associated with prostate cancer relapse and metastasis. Amplification of MCM7 was also found in esophageal carcinoma.⁹ The magnitude of MCM7 amplification correlates with the expression of MCM7, tumor grades, and the aggressiveness of esophageal cancer.⁹ It is presumed that amplification of MCM7 is the driving force of MCM7 overexpression in primary human malignancies. MCM7 is probably the primary target of Rb, the tumor suppressor that controls cell entry into S phase.¹⁵ There is growing evidence that other signaling pathways also regulate MCM7 activity.Receptor for activated protein kinase C 1 (RACK1), was initially identified as an adaptor of several protein kinase C (PKC) isoforms.¹⁶ The binding of RACK1 and PKC anchor PKC to its substrate to initiate second messenger signaling. It is suggested, according to recent studies that RACK1 interacts with a variety of other signaling molecules, including ras-GTPase activating protein,¹⁷ dynamin-1,¹⁸ src,¹⁹ integrins,²⁰ PTPμ,²¹ phosphodiesterase,²² hypoxia induced factor-1,²³ and so forth, that play an important role in several physiological processes, including, growth, hypoxia response, migration, adhesion, and cell differentiation. RACK1 only binds PKC activated by diacylglycerol or phorbol ester, but not quiescent PKC. In this study, we showed that RACK1 binds with MCM7 N-terminus. The MCM7/RACK1 interaction appears essential for DNA replication activity of MCM7.     

Cytoprotective Mitochondrial Chaperone TRAP-1 As a Novel Molecular Target in Localized and Metastatic Prostate Cancer

Molecular chaperones of the heat shock protein-90 (Hsp90) family promote cell survival, but the molecular requirements of this pathway in tumor progression are not understood. Here, we show that a mitochondria-localized Hsp90 chaperone, tumor necrosis factor receptor-associated protein-1 (TRAP-1), is abundantly and ubiquitously expressed in human high-grade prostatic intraepithelial neoplasia, Gleason grades 3 through 5 prostatic adenocarcinomas, and metastatic prostate cancer, but largely undetectable in normal prostate or benign prostatic hyperplasia in vivo. Prostate lesions formed in genetic models of the disease, including the transgenic adenocarcinoma of the mouse prostate and mice carrying prostate-specific deletion of the phosphatase tensin homolog tumor suppressor (Pten^pc−/−), also exhibit high levels of TRAP-1. Expression of TRAP-1 in nontransformed prostatic epithelial BPH-1 cells inhibited cell death, whereas silencing of TRAP-1 in androgen-independent PC3 or DU145 prostate cancer cells by small interfering RNA enhanced apoptosis. Targeting TRAP-1 with a novel class of mitochondria-directed Hsp90 inhibitors, ie, Gamitrinibs, caused rapid and complete killing of androgen-dependent or -independent prostate cancer, but not BPH-1 cells, whereas reintroduction of TRAP-1 in BPH-1 cells conferred sensitivity to Gamitrinib-induced cell death. These data identify TRAP-1 as a novel mitochondrial survival factor differentially expressed in localized and metastatic prostate cancer compared with normal prostate. Targeting this pathway with Gamitrinibs could be explored as novel molecular therapy in patients with advanced prostate cancer.Apart from skin tumors, prostate cancer is the most commonly diagnosed malignancy in men in the United States.¹ Despite progress in early diagnosis,² and prolongation of patient survival,³ the disease still carries significant morbidity and mortality, with its advanced and metastatic phase claiming over 30,000 deaths per year in the United States alone. Similar to the genetic heterogeneity of most epithelial malignancies, prostate cancer progresses through a stepwise acquisition of multiple molecular changes,⁴ of which insensitivity to androgen deprivation,⁵ emergence of an ‘osteomimetic’ phenotype responsible for metastatic tropism to the bone,⁶ and deregulated cell proliferation and cell survival,⁷ are pivotal traits.In this context, advanced prostate cancer is almost invariably associated with a heightened anti-apoptotic threshold,⁴ which may contribute to disease progression and resistance to therapy. This process often involves aberrant resistance to mitochondrial cell death,⁸ with reduced organelle permeability to solutes, and attenuated release of mitochondrial apoptogenic proteins in the cytosol.⁹ The regulators of such ‘mitochondrial permeability transition’ normally triggered by cell death stimuli are still largely elusive, but knockout data in mice have identified pro-apoptotic Bcl-2 family proteins and the mitochondrial matrix immunophilin, cyclophilin D, as pivotal effectors of this process, controlling the integrity of the mitochondrial outer membrane,⁸ and the opening a permeability transition pore,^10,11 respectively.Recent data have shown that molecular chaperones of the heat shock protein-90 (Hsp90) family,¹² may function as novel regulators of mitochondrial permeability transition,¹³ especially in tumor cells.¹⁴ Accordingly, Hsp90, and its ortholog, tumor necrosis factor receptor-associated protein-1 (TRAP-1) are abundantly localized to mitochondria of tumor, but not most normal cells, and antagonize cyclophilin D-dependent pore-forming function, potentially via a protein (re)folding mechanism.¹⁴ Consistent with a general role of Hsp90 as a drug target in prostate cancer,¹⁵ this mitochondria-compartmentalized cytoprotective pathway could provide a novel therapeutic target to enhance tumor cell apoptosis.¹⁴In the current study, we demonstrate that TRAP-1 is dramatically expressed in all lesions that comprise the entire natural history of human prostate cancer, as well as genetic disease models in rodents, but undetectable in the normal prostate. Importantly, we show that Gamitrinibs, a novel class of small molecule Hsp90 antagonists selectively engineered to target the pool of these chaperones in mitochondria,¹⁶ cause sudden prostate cancer cell death without affecting nontransformed prostatic epithelium.     

Genetic testing in asymptomatic minors Background considerations towards ESHG Recommendations

Although various guidelines and position papers have discussed, in the past, the ethical aspects of genetic testing in asymptomatic minors, the European Society of Human Genetics had not earlier endorsed any set of guidelines exclusively focused on this issue. This paper has served as a background document in preparation of the development of the policy recommendations of the Public and Professional Committee of the European Society of Human Genetics. This background paper first discusses some general considerations with regard to the provision of genetic tests to minors. It discusses the concept of best interests, participation of minors in health-care decisions, parents'' responsibilities to share genetic information, the role of clinical genetics and the health-care system in communication within the family. Second, it discusses, respectively, the presymptomatic and predictive genetic testing for adult-onset disorders, childhood-onset disorders and carrier testing.Although various guidelines and position papers have discussed, in the past, the ethical aspects of genetic testing in asymptomatic minors,^{1, 2} the European Society of Human Genetics had not earlier endorsed any set of guidelines exclusively focused on this issue. This background paper was preceded by an in-depth research on the topic by Eurogentest.³ Eurogentest (http://www.eurogentest.org aims to develop the necessary infrastructure, tools, resources, guidelines and procedures that will structure, harmonize and improve the overall quality of all the EU genetic services at the molecular, cytogenetic, biochemical and clinical level.⁴ Attention has also been paid to the provision of appropriate counselling related to genetic testing, the education of patients and professionals, as well as to the ethical, legal and social issues surrounding testing. The focus of the ethics unit of Eurogentest was oriented towards the study of the ethical issues related to genetic testing in minors. This work was the starting point for this background paper, which has been prepared and supported by different types of evidence. First, research has been performed on the existing recommendations regarding predictive genetic testing in minors¹ and carrier testing,² with the intention of identifying areas of agreement and disagreement. Second, the literature on medico–ethical and medico–legal aspects of predictive genetic testing in minors,⁵ carrier testing,^{6, 7} the position of minors⁸ and patient rights⁹ was studied. Third, a systematic literature review was performed to gather information regarding the attitudes of the different stakeholders (minors, health-care professionals, parents and relatives of the affected individuals) towards genetic testing in asymptomatic minors.^{10, 11} Fourth, the attitudes of European clinical geneticists regarding genetic testing in asymptomatic minors were gathered.^{12, 13, 14}In 2007, contacts were made with the Public and Professional Policy Committee of the European Society of Human Genetics with the aim of developing policy recommendations on the issue. On the basis of a decision of the PPPC meeting during the ESHG conference in Nice (June 2007), an ad hoc committee, consisting of Pascal Borry (Eurogentest), Kris Dierickx (Eurogentest), Angus Clarke, Gerry Evers-Kiebooms (PPPC) and Martina Cornel (PPPC), was created. This ad hoc committee met on 15 November 2007 to discuss a first draft of a background paper and recommendations that were prepared by Pascal Borry under the supervision of Kris Dierickx. A revised version was discussed during a PPPC meeting in Amsterdam (April 2008) and Barcelona (June 2008). In order not to repeat issues that have been discussed elsewhere, reference will often be made to the above-referenced publications.     

Biological sample collections from minors for genetic research: a systematic review of guidelines and position papers

Stored tissue samples are an important resource for epidemiological genetic research. Genetic research on biological material from minors can yield valuable information on the development and genesis of early-onset genetic disorders and the early interaction of environmental and genetic factors. The use of such tissue raises some specific ethical and governance questions, which are not completely covered by the discussion on biological materials from adults. We have retrieved 29 guidelines and position papers pertaining to the storage and use of biological tissue samples for genetic research, originating from 27 different organizations. Five documents have an international scope, three have an European scope and 21 have a national scope. We discovered that 11 of these documents did not contain a section on biological materials from minors. The content of the remaining 18 documents was categorized according to four themes: consent, principles of non-therapeutic research on vulnerable populations, ethics committee approval and difference between anonymous and identifiable samples. We found out that these themes are not consistently mentioned by each document, but that documents discussing the same themes were mostly in agreement with their recommendations. However, a systematic reflection on the ethical and policy issues arising from the participation of minors in biobank research is missing.Stored tissue sample collections for genetic research exist in different forms. Some of these collections provide a resource for potentially unlimited genetic research, and gather samples and data from specific populations. An example is the ‘UK biobank''.¹ Other collections are stored for research on a specific disease. Collections that were originally gathered for different purposes, for example blood spot cards for newborn screening, could be reused for genetic research.²Genetic research on biological material from minors and the associated medical records can yield valuable information on the development and genesis of early-onset genetic disorders and the early interaction of environmental and genetic factors. For example, Rasmussen³ describes the incorporation of DNA sample collections into the ‘National Birth Defects Prevention Study'' in the United States to identify the risk factors for birth defects. Studies such as the ‘Avon Longitudinal Study of Parents and Children'' in Bristol (children of the nineties) use genetic, phenotypic and environmental information of 14 000 babies from their conception onwards to study the interaction between these data.⁴An extensive ethical literature exists on the collection, storage and use of biological samples for genetic research. The overwhelming majority of these documents discuss issues of privacy, confidentiality, commercialization and consent.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, research on pediatric data raises specific ethical questions with regard to consent and privacy. For example, who should give consent to the inclusion of tissue and data from children? Is the general requirement that non-therapeutic research can only be done with children if it involves no more than minimal risk, applicable to biobank research? We shall review whether and how guidelines and policy documents discuss children in the context of storing biological samples and DNA for non-therapeutic research.     

Tissue Factor-Deficiency and Protease Activated Receptor-1-Deficiency Reduce Inflammation Elicited by Diet-Induced Steatohepatitis in Mice

Altered hepatic lipid homeostasis, hepatocellular injury, and inflammation are features of nonalcoholic steatohepatitis, which contributes significantly to liver-related morbidity and mortality in the Western population. A collection of inflammatory mediators have been implicated in the pathogenesis of steatohepatitis in mouse models. However, the pathways essential for coordination and amplification of hepatic inflammation and injury caused by steatosis are not completely understood. We tested the hypothesis that tissue factor (TF)-dependent thrombin generation and the thrombin receptor protease activated receptor-1 (PAR-1) contribute to liver inflammation induced by steatosis in mice. Wild-type C57Bl/6J mice fed a diet deficient in methionine and choline for 2 weeks manifested steatohepatitis characterized by increased serum alanine aminotransferase activity, macrovesicular hepatic steatosis, hepatic inflammatory gene expression, and lobular inflammation. Steatohepatitis progression was associated with thrombin generation and hepatic fibrin deposition. Coagulation cascade activation was significantly reduced in low TF mice, which express 1% of normal TF levels. Hepatic triglyceride accumulation was not affected in low TF mice or PAR-1-deficient mice. In contrast, biomarkers of hepatocellular injury, inflammatory gene induction, and hepatic accumulation of macrophages and neutrophils were greatly reduced by TF-deficiency and PAR-1-deficiency. The results suggest that TF-dependent thrombin generation and activation of PAR-1 amplify hepatic inflammation and injury during the pathogenesis of steatohepatitis.Non-alcoholic fatty liver disease (NAFLD) is increasingly appreciated as a hepatic feature of the metabolic syndrome. NAFLD may occur in 25% of the Western population and altered hepatic function increases the risk for developing diseases including diabetes and atherosclerosis.^1,2 The progression of simple hepatic steatosis to the more severe nonalcoholic steatohepatitis (NASH) contributes significantly to liver-related morbidity and mortality.³ Requisite histological features of NASH include macrovesicular hepatic steatosis, evidence of hepatocellular injury, and lobular inflammation.⁴ In a subset of patients with chronic steatohepatitis, stellate cell activation coordinates a fibrogenic response causing fibrosis and cirrhosis.⁵ Of importance, the mechanisms required for the progression of hepatic inflammation during steatohepatitis are not completely understood.Animal models used to define mechanisms of steatohepatitis have used genetic and dietary modification to induce various features of the disease.² In particular, feeding mice a diet deficient in methionine and choline (MCD diet) is an established model to study the progression of steatohepatitis and has been extensively used to study mechanisms of hepatic inflammation and fibrosis. Rodents fed an MCD diet for 2 weeks manifest a defect in hepatic β oxidation resulting in accumulation of triglyceride and the induction of steatohepatitis.^2,6,7 Prolonged feeding (>4 weeks) of the MCD diet activates hepatic stellate cells and increases collagen expression and deposition in the liver. Utilization of the MCD diet model has revealed the contribution of hepatic triglyceride,⁸ various inflammatory mediators,^9,10 nuclear receptors,^11,12 and signaling pathways¹³ in the manifestation of steatohepatitis.An important physiological process disrupted by chronic liver disease is blood coagulation. Several studies have indicated that the progression of liver disease is associated with altered blood coagulation.¹⁴ For example, steatosis in patients with the metabolic syndrome is associated with a shift in the balance of procoagulant and antifibrinolytic factors favoring coagulation.^15–17 This links the progression of NAFLD with increased risk of thrombotic complications associated with vascular disease and the metabolic syndrome. However, it is not clear whether the altered coagulation impacts progression of the liver pathology in patients with NAFLD or NASH.The coagulation cascade is initiated by tissue factor (TF), the transmembrane receptor for coagulation factor VIIa.¹⁸ TF is expressed by the normal liver,¹⁹ albeit at much lower levels compared with other tissues (eg, lung, heart).²⁰ Of importance, potent inducers of TF expression such as bacterial lipopolysaccharide and pro-inflammatory cytokines (eg, tumor necrosis factor [TNF]α, monocyte chemoattractant protein [MCP]-1) are linked to the pathogenesis of NAFLD and NASH in humans and animal models.^21–24 TF-dependent coagulation cascade activation leads to generation of the serine protease thrombin, which cleaves circulating fibrinogen to form fibrin. Thrombin also elicits intracellular signaling by activating the G-protein coupled receptor protease activated receptor-1 (PAR-1).²⁵ This TF–PAR-1 pathway has been shown to increase inflammation in other models of tissue injury.^26–29 However, the contribution of both TF and PAR-1 to coagulation and inflammation during steatohepatitis has not been determined.To this end, we characterized the procoagulant response associated with steatohepatitis induced in mice by a MCD diet. Furthermore, we used mice expressing 1% of normal TF levels (ie, low TF mice³⁰ and PAR-1-deficient mice³¹ to test the hypothesis that TF-dependent thrombin generation contributes to the pathogenesis of murine steatohepatitis by activating PAR-1.     

Non-invasive prenatal testing: ethical issues explored

This paper explores the ethical implications of introducing non-invasive prenatal diagnostic tests (NIPD tests) in prenatal screening for foetal abnormalities. NIPD tests are easy and safe and can be performed early in pregnancy. Precisely because of these features, it is feared that informed consent may become more difficult, that both testing and selective abortion will become ‘normalized'', and that there will be a trend towards accepting testing for minor abnormalities and non-medical traits as well. In our view, however, the real moral challenge of NIPD testing consists in the possibility of linking up a technique with these features (easy, safe and early) with new genomic technologies that allow prenatal diagnostic testing for a much broader range of abnormalities than is the case in current procedures. An increase in uptake and more selective abortions need not in itself be taken to signal a thoughtless acceptance of these procedures. However, combining this with considerably enlarging the scope of NIPD testing will indeed make informed consent more difficult and challenge the notion of prenatal screening as serving reproductive autonomy. If broad NIPD testing includes later-onset diseases, the ‘right not to know'' of the future child will become a new issue in the debate about prenatal screening. With regard to the controversial issue of selective abortion, it may make a morally relevant difference that after NIPD testing, abortion can be done early. A lower moral status may be attributed to the foetus at that moment, given the dominant opinion that the moral status of the foetus progressively increases with its development.Since the discovery of cell-free foetal DNA/RNA (cffDNA/RNA) in maternal plasma in 1997,¹ the possibility to use this cffDNA/RNA for non-invasive prenatal diagnosis (NIPD) has been investigated many times.^{2, 3, 4, 5, 6} cffDNA/RNA can be obtained from a maternal blood sample, as early as 4 weeks of gestation,⁷ but currently only reliably so from 7 weeks of gestation.⁴ This development holds the promise of NIPD testing early in pregnancy and without the small, but significant risk of foetal loss that the current invasive procedures of chorionic villus sampling (CVS) and amniocentesis (AP) carry. NIPD testing for the determination of a Y-signal for pregnancies at risk of X-linked disorders and for diagnosis of Rhesus factor status in RhD-negative women is now being translated into clinical practice.⁴ In many European countries, discussion about broader applications of NIPD testing can be expected in the coming years.^{8, 9} The feasibility of NIPD for trisomy 21, 13 and 18 has already been shown,² but large-scale independent studies are still needed. Sex-chromosomal abnormalities (eg, Turner syndrome (X0) and triple X syndrome (XXX)) could in principle be diagnosed by NIPD testing as well,⁴ if reliable quantitative tests become available in the future and the maternal ‘background'' can be excluded from testing. Even if accurate NIPD testing for the mentioned abnormalities becomes possible, the clinical utility of the test remains to be assessed. This includes balancing the benefits to the harms also with regard to its psychological, ethical, legal, social and economic implications.^{10, 11} The possible ethical implications of NIPD as a new approach to prenatal testing have so far been reviewed in a few publications.^{4, 8, 9, 12, 13, 14, 15, 16, 17} Apart from clear benefits related to avoiding the miscarriage risk of present invasive methods, important potential drawbacks have been mentioned as well. For one thing, proper counselling and informed consent is argued to become more challenging when offering NIPD testing. Moreover, there is a concern that the ease and safety of NIPD may lead to prenatal screening being increasingly conceived as a matter of course, both by those making the offer and by the women undergoing the test. Related to this is the concern that selective abortion of foetuses with minor abnormalities, the wrong sex or unwanted paternity, will become normalized.This paper aims to expand and refine these ethical evaluations and will add some new ethical perspectives with regard to possible implications of NIPD at present and in the future.In our view, it is not so much the fact that foetal material used for prenatal testing can be obtained early and non-invasively (allowing easy and safe testing) that would lead to moral challenges. Rather, it is the fact that a technology with these features would be open to being used for testing a potentially much broader range of abnormalities than those included in the presently used method of microscopic chromosome analysis (karyotyping).Although NIPD testing can also be applied in high genetic-risk families and for the management of pregnancy, the focus of this paper will primarily be on the application of NIPD testing in the screening context. The reason for this focus on prenatal screening is that in the near future, the question if, and if so, in what way NIPD testing is to be applied within prenatal screening strategies should be considered and discussed by policy makers, health care professionals and society at large.To avoid confusion, a preliminary remark is needed on terminology. In medicine, ‘screening'' is often used as referring to a kind of test for risk assessment or disease discovery. However, after the convention in normative and regulatory discourse, we will use ‘screening'' as referring to any systematic and unsolicited offer of predictive testing (using whatever types of test) involving individuals who themselves have no reason (yet) to seek medical help for the condition in question.¹⁸ In this broader sense, screening stands in contrast to ‘diagnosis'' as testing on indication.     

Endometrial Cancer Side-Population Cells Show Prominent Migration and Have a Potential to Differentiate into the Mesenchymal Cell Lineage

Cancer stem-like cell subpopulations, referred to as “side-population” (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and, uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, α-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into α-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage.Recently, adult stem cells have been identified in several mature tissues, such as the adult intestine,¹ skin,² muscle,³ blood,⁴ and the nervous system^5–7 A stem cell is an undifferentiated cell that is defined by its ability to both self-renew and to produce mature progeny cells.⁸ Stem cells are classified based on their developmental potential as totipotent, pluripotent, oligopotent, and unipotent. Adult somatic stem cells were originally thought to be tissue specific and only able to give rise to progeny cells corresponding to their tissue of origin. Recent studies, however, have shown that adult mammalian stem cells are able to differentiate across tissue lineage boundaries,^9,10 although this “plasticity” of adult somatic stem cells remains controversial.Stem cell subpopulations (“side-population” (SP) cells) have been identified in many mammals, including humans, based on the ability of these cells to efflux the fluorescent dye Hoechst 33342.¹¹ Recent evidence suggests that the SP phenotype is associated with a high expression level of the ATP-binding cassette transporter protein ABCG2/Bcrp1.¹² Most recently, established malignant cell lines, which have been maintained for many years in culture, have also been shown to contain SP cells as a minor subpopulation.¹³The human endometrium is a highly dynamic tissue undergoing cycles of growth, differentiation, shedding, and regeneration throughout the reproductive life of women. Endometrial adult stem/progenitor cells are likely responsible for endometrial regeneration.¹⁴ Rare populations of human endometrial epithelial and stromal colony-forming cells¹⁵ and SP cells^16,17 have been identified. Although coexpression of CD146 and PDGFRβ isolates a population of mesenchymal stem like cells from human endometrium,¹⁸ specific stem cell markers of endometrium remain unclear. Recently, Gotte et al¹⁹ demonstrated that the adult stem cell marker Musashi-1 was coexpressed with Notch-1 in a subpopulation of endometrial cells. Furthermore, they showed that telomerase and Musashi-1-expressing cells were significantly increased in proliferative endometrium, endometriosis, and endometrial carcinoma tissue, compared with secretary endometrium, suggesting the concept of a stem cell origin of endometriosis and endometrial carcinoma.Recent evidence suggests that cancer stem-like cells exist in several malignant tumors, such as leukemia^20,21 breast cancer,²² and brain tumors,²³ and that these stem cells express surface markers similar to those expressed by normal stem cells in each tissue.^20,24Development of endometrial carcinoma is associated with a variety of genetic alterations. For example, increased expression and activity of telomerase^25,26 and frequent dysregulation of signaling pathways have been observed in endometrial carcinoma. Some of these pathways are important determinants of stem cell activity (Wnt-β-catenin and PTEN).^27–29 These suggest a stem cell contribution to endometrial carcinoma development.Recently, we isolated SP cells from the human endometrium. These SP cells showed long-term proliferating capacity in cultures and produced both gland and stromal-like cells. Additionally, they were able to function as progenitor cells.¹⁶ In this study, we isolated and characterized SP cells from human endometrial cancer cells and from rat endometrial cells expressing oncogenic [¹²Val] human K-Ras protein and demonstrated their cancer stem-like cell phenotypes.     

Murine Cerebral Malaria Is Associated with a Vasospasm-Like Microcirculatory Dysfunction,and Survival upon Rescue Treatment Is Markedly Increased by Nimodipine

Brain hemodynamics in cerebral malaria (CM) is poorly understood, with apparently conflicting data showing microcirculatory hypoperfusion and normal or even increased blood flow in large arteries. Using intravital microscopy to assess the pial microvasculature through a closed cranial window in the murine model of CM by Plasmodium berghei ANKA, we show that murine CM is associated with marked decreases (mean: 60%) of pial arteriolar blood flow attributable to vasoconstriction and decreased blood velocity. Leukocyte sequestration further decreased perfusion by narrowing luminal diameters in the affected vessels and blocking capillaries. Remarkably, vascular collapse at various degrees was observed in 44% of mice with CM, which also presented more severe vasoconstriction. Coadministration of artemether and nimodipine, a calcium channel blocker used to treat postsubarachnoid hemorrhage vasospasm, to mice presenting CM markedly increased survival compared with artemether plus vehicle only. Administration of nimodipine induced vasodilation and increased pial blood flow. We conclude that vasoconstriction and vascular collapse play a role in murine CM pathogenesis and nimodipine holds potential as adjunctive therapy for CM.Cerebral malaria (CM) caused by Plasmodium falciparum claims the lives of nearly 1 million children every year.¹ Despite antimalarial treatment, 10% to 20% of patients die, and one in every four survivors develops neurological sequelae,^2,3 therefore adjunctive therapies are urgently needed. A number of clinical trials addressing potential adjunctive therapies for CM showed no proven benefits and some interventions were even deleterious,⁴ stressing the need for a better understanding of CM pathogenesis to develop effective therapies.An unresolved issue of CM pathogenesis regards the role of brain hemodynamic perturbations and ischemia. Sequestration of parasitized red blood cells (pRBCs) containing mature forms of the parasite in the brain microvasculature is a characteristic postmortem finding in human CM cases⁵ and together with rosetting⁶ and reduced RBC deformability⁷ may result in the obstruction of blood flow potentially leading to ischemia and hypoxia. In vivo studies of the microcirculation in human CM support this mechanism, with direct observation of retinal microvasculature showing impaired perfusion, retinal whitening, vascular occlusion, and ischemia.⁸ Accordingly, microvascular obstruction observed in the rectal mucosa of CM patients was proportional to the severity of the disease.⁹ In addition, hypovolemia, shock and intracranial hypertension, commonly associated with poor outcomes in CM,⁴ reduce tissue perfusion, and tissue hypoxia is one of the likely explanations for the acidosis frequently observed in severe malaria.^7,10 Ischemic damage has also been shown in children with CM and was associated with severe neurological sequelae.¹¹ On the other hand, transcranial Doppler sonography studies showed normal or even increased cerebral blood flow (CBF) velocities^12–15 in large arteries during CM, which associated with microcirculatory obstruction has been suggested to increase cerebral blood volume leading to intracranial hypertension.¹⁶ Alternatively, collateral flow has been proposed as a mechanism to reconcile the findings of normal or increased CBF velocities and impaired perfusion,¹⁷ an interpretation supported by findings of hyperdynamic flow in capillaries adjacent to obstructed vessels.⁹ Interventions that improve cerebral perfusion have been proposed to be beneficial in CM.^8,18The murine model of CM by Plasmodium berghei ANKA (PbA) shares many features with the human pathology,¹⁹ including the presence of multiple brain microhemorrhages and vascular obstruction, although the nature of the sequestered cell (leukocytes) differs. In murine CM, magnetic resonance imaging (MRI) and spectroscopy studies showed the presence of brain edema, decreased CBF, and ischemia.^20,21 Lack of resolution in MRI, however, precludes detailed studies of the microcirculation, which is a major target and player in CM pathogenesis. A few studies have addressed the in vivo microcirculatory changes in murine models of severe malaria,^22–24 however in sites other than the brain (cremaster muscle or skin). In the present work, we used for the first time brain intravital microscopy to follow the dynamic changes in the pial microcirculation during the course of PbA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow, and eventually vascular collapse, events similar to postsubarachnoid hemorrhage (SAH) vasospasm.²⁵ We also show that nimodipine, a calcium channel blocker used to treat post-SAH vasospasm,^25,26 markedly increased survival when given off-label to mice with CM as adjunctive therapy to artemether.     

Knockdown of Osteopontin Reduces the Inflammatory Response and Subsequent Size of Postsurgical Adhesions in a Murine Model

Adhesions between organs after abdominal surgery remain a significant unresolved clinical problem, causing considerable postoperative morbidity. Osteopontin (OPN) is a cytokine up-regulated after cell injury and tissue repair. Our previous studies have shown that blocking OPN expression at sites of cutaneous wounding resulted in reduced granulation tissue and scarring. We hypothesize that it may be possible to similarly reduce inflammation-associated fibrosis that causes small-bowel adhesions after abdominal surgery. By using a mouse model, we deliver OPN antisense oligodeoxynucleotides via Pluronic gel to the surface of injured, juxtaposed small bowel and show a significant reduction of inflammatory cell influx to the developing adhesion and a dramatic reduction in the resulting adhesion size. A significant reduction in α-smooth muscle actin expression and collagen deposition within the mature adhesion is also demonstrated. We see no impact on mortality, and the healing of serosal injury to intact bowel appeared normal given the reduced inflammatory response. Our studies suggest that dampening OPN levels might be a potentially important target for anti-adhesion therapeutics.The peritoneum is an extensive and complex organ consisting of a layer of mesothelial cells lining the peritoneal cavity and all organs within it.¹ One of the main functions of the peritoneum is to allow friction-free movement between abdominal viscera and the peritoneal wall.² Any surgery that breaches the peritoneal lining causes injury to the peritoneum, which responds by raising inflammatory signals that attract innate immune cells in parallel with a wound repair response and subsequent fibrosis.^3–5 This almost invariably results in permanent peritoneal adhesion formation.⁶ The result can be tethering of adjacent small-bowel loops that may lead to abdominal pain⁷ and/or bowel obstruction,⁸ which is a significant cause of postoperative morbidity in clinical practice. Readmission rates secondary to adhesional complications are as high as 5% to 10% after abdominal surgery.^9,10 Adhesion prevention options in clinical practice are limited to either barrier methods¹¹ or flotation fluids,¹² which use the concept of keeping damaged peritoneal surfaces separated during their healing process; however, these options are of limited effectiveness.^13,14 Pathophysiological manipulation of the cascade events leading to fibrosis has been investigated,^15–18 but none has led to a clinically usable product. Herein, we investigate whether therapeutic strategies used to block scar formation after skin healing might also be effective during peritoneal repair. Microarray studies of wound tissues from wild-type mice versus PU.1 mice (lacking neutrophils, macrophages, and mast cells) reveal an inflammation-dependent gene, osteopontin (OPN), that is expressed by wound granulation tissue fibroblasts, coincident with a skin wound inflammatory response.^19,20 PU.1 mice heal skin wounds without the standard inflammatory cascade, which results in less fibrosis and scarring at the healed wound site.¹⁹ OPN acts both as a secreted chemokine-like protein and as part of an intracellular signaling complex.²¹ It plays key roles in several processes associated with tissue repair, including cell adhesion, migration, and survival.^21,22 Short-term local knockdown of OPN in cutaneous wounds leads to decreased granulation tissue and reduced scar formation.²³ In this study, we investigate whether these effects are transferable to peritoneal repair and also might block i.p. fibrosis.     

Orally Administered Prion Protein Is Incorporated by M Cells and Spreads into Lymphoid Tissues with Macrophages in Prion Protein Knockout Mice

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases. Infection by the oral route is assumed to be important, although its pathogenesis is not understood. Using prion protein (PrP) knockout mice, we investigated the sequence of events during the invasion of orally administered PrPs through the intestinal mucosa and the spread into lymphoid tissues and the peripheral nervous system. Orally administered PrPs were incorporated by intestinal epitheliocytes in the follicle-associated epithelium and villi within 1 hour. PrP-positive cells accumulated in the subfollicle region of Peyer''s patches a few hours thereafter. PrP-positive cells spread toward the mesenteric lymph nodes and spleen after the accumulation of PrPs in the Peyer''s patches. The number of PrP molecules in the mesenteric lymph nodes and spleen peaked at 2 days and 6 days after inoculation, respectively. The epitheliocytes in the follicle-associated epithelium incorporating PrPs were annexin V-positive microfold cells and PrP-positive cells in Peyer''s patches and spleen were CD11b-positive and CD14-positive macrophages. Additionally, PrP-positive cells in Peyer''s patches and spleen were detected in the vicinity of peripheral nerve fibers in the early stages of infection. These results indicate that orally delivered PrPs were incorporated by microfold cells promptly after challenge and that macrophages might act as a transporter of incorporated PrPs from the Peyer''s patches to other lymphoid tissues and the peripheral nervous system.Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative diseases that infect humans and both wild and domestic animals. They include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle.¹ The common neuropathological features within the central nervous system (CNS) of TSEs are seen as a spongiform pathology, neuronal loss,² glial activation,³ and the accumulation of an abnormal and protease-resistant conformer of the scrapie-associated prion proteins (PrP-res or PrP^Sc),⁴ which are closely associated with the infection.⁵It has been reported that variant CJD in humans is most likely to have occurred because of the transmission of BSE after the consumption of beef contaminated with the BSE agent.⁶ Therefore, the oral route of TSE infection is widely assumed to be important under natural conditions. Many of the infectious agents accumulate in the gut-associated lymphoid tissues (GALT) after oral infection, such as the Peyer''s patches and mesenteric lymph nodes (MLN) before spreading to the CNS.⁷ It is necessary for the infectious agents to cross the intestinal epithelium before they can accumulate in the GALT. In addition, there are microfold cells (M cells) within the follicle-associated epithelium (FAE) that are specialized for the transepithelial transport of macromolecules and particles.⁸ One in vitro study has demonstrated that M cells actively transcytose the scrapie agents into the basolateral side of the epithelium.⁹ However, it is still a matter of controversy as to whether M cells may be involved in the in vivo transport of the infectious agents across the intestinal epithelium. After alimentary uptake of the infectious agents, they accumulate in the GALT and the lymphoreticular systems (eg, the spleen and other peripheral lymph nodes) long before they are detected in the CNS.¹⁰ As the GALT and the lymphoreticular systems are highly innervated, they are believed to be important sites for the infectious agents to gain contact with the nervous system (ie, neuroinvasion).¹¹ Once neuroinvasion occurs, the infectious agents reach their initial CNS target sites by spreading in a retrograde direction along efferent nerve fibers.¹²In the lymphoid tissues, it is believed that the macrophages, dendritic cells (DCs), and follicular dendritic cells (FDCs) are involved in the transportation and replication of the infectious agents. Macrophages are prevalent candidates for both spread¹³ and clearance¹⁴ of the infectious agents. DCs can capture and retain protein antigens in a nondegraded state.^15,16 These characteristics suggest that the macrophages and DCs may act as a transporter of the infectious agents from the gut to lymphoid tissues. FDCs express high levels of cellular PrPs (PrP^c), and therefore an early accumulation of PrP^Sc is seen in them.^17,18 Many studies of the alimentary pathogenesis of TSEs have been conducted to elucidate how infectious agents spread from the GALT to the CNS, although this has not been clearly determined yet. Therefore, the aim of the present study was to reveal the cells involved in the early stages of the pathogenesis of oral TSE infection, such as the sites of entry, spread, and neuroinvasion.     

Neutrophil Elastase Is Produced by Pulmonary Artery Smooth Muscle Cells and Is Linked to Neointimal Lesions

Previously, we reported that murine gammaherpesvirus-68 (M1-MHV-68) induces pulmonary artery (PA) neointimal lesions in S100A4-overexpressing, but not in wild-type (C57), mice. Lesions were associated with heightened lung elastase activity and PA elastin degradation. We now investigate a direct relationship between elastase and PA neointimal lesions, the nature and source of the enzyme, and its presence in clinical disease. We found an association exists between the percentage of PAs with neointimal lesions and elastin fragmentation in S100A4 mice 6 months after viral infection. Confocal microscopy documented the heightened susceptibility of S100A4 versus C57 PA elastin to degradation by elastase. A transient increase in lung elastase activity occurs in S100A4 mice, 7 days after M1-MHV-68, unrelated to inflammation or viral load and before neointimal lesions. Administration of recombinant elafin, an elastase-specific inhibitor, ameliorates early increases in serine elastase and attenuates later development of neointimal lesions. Neutrophils are the source of elevated elastase (NE) in the S100A4 lung, and NE mRNA and protein levels are greater in PA smooth muscle cells (SMC) from S100A4 mice than from C57 mice. Furthermore, elevated NE is observed in cultured PA SMC from idiopathic PA hypertension versus that in control lungs and localizes to neointimal lesions. Thus, PA SMC produce NE, and heightened production and activity of NE is linked to experimental and clinical pulmonary vascular disease.Pulmonary arterial (PA) neointimal lesions are observed in patients with PA hypertension that is idiopathic (IPAH) or associated with other medical conditions. These vascular abnormalities cause narrowing and even obliteration of the vessel lumen and contribute to the progressive increase in pulmonary vascular resistance that can lead to right ventricular failure (reviewed in Ref. ¹). Only a few murine or rodent models recapitulate this pathological feature, eg, mice exposed to ovalbumin or aspergillus² or to schistosomiasis,³ rats treated with the vascular endothelial receptor blocker Sugen 5416, exposed to chronic hypoxia and recovered in room air,⁴ mice that overexpress IL-6 and are subjected to chronic hypoxia,⁵ or mice that overexpress S100A4.⁶ The latter mice, when over 1 year of age, can on rare occasions “spontaneously” develop severe neointimal lesions.⁶ However, these lesions are observed consistently following infection with murine gammaherpesvirus-68 (MHV-68).⁷ S100A4 is also known as metastasin-1 (mts-1), and is a member of the calcium binding family of proteins that clusters on chromosome 1 and that has been related to cancer and inflammation.⁸The S100A4-overexpressing mouse infected with MHV-68 is relevant to clinical PAH. Increased immunoreactivity for S100A4 is observed in vascular lesions in patients with advanced PAH,⁶ and human immunodeficiency virus (HIV) and HHV-8/Kaposi''s sarcoma virus, the human homologue of MHV-68, have been implicated in clinical PAH. Specifically, the viral protein for HHV-8 has been detected in neointimal and plexiform lesions in lung tissues from some,⁹ albeit not all,¹⁰ series of PAH patients.Pulmonary vascular neointimal and plexiform lesions in S100A4 mice are associated with fragmentation of elastic laminae and with heightened activity of a serine elastase.⁷ Fragmentation of PA elastin has been observed in PAs of PAH patients,¹¹ and heightened activity of a serine elastase has been identified in the PA in a variety of experimental forms of PAH^12–15 and in cultured PA smooth muscle cells (SMC).^16–19 Moreover, inhibition of this elastase can attenuate or prevent^12–14 and even reverse¹⁵ experimental pulmonary vascular disease in rodents. In all rodent models where elastase inhibitors were used, the pulmonary vascular lesions were characterized by loss or increased muscularization of normally nonmuscular peripheral arteries at the alveolar wall and duct level, and medial hypertrophy of proximal muscular arteries. Neointimal lesion formation, however, was not present. Elastase inhibition should, however, attenuate these lesions since proliferation and migration of SMC in the neointima are likely the consequences of elastase-mediated release of growth factors from the extracellular matrix^16,17 and activation of growth factor receptors.²⁰We therefore hypothesized, and subsequently demonstrated in this study, that in the S100A4 overexpressing versus C57 mouse, heightened susceptibility of elastin to fragmentation, coupled to elevated serine elastase activity following M1-MHV-68 infection, can contribute to the development of neointimal lesions. We identified the elastase involved as neutrophil elastase (NE) produced by PA SMC, suggesting that it is the endogenous vascular elastase previously related to PAH in other experimental models.^12,16,18,19 Moreover, we showed that NE is produced in significantly greater amounts by cultured murine S100A4 versus C57 PA SMC, and by human PA SMC from IPAH versus control lungs, and we localized NE to neointimal and plexiform lesions in human lung specimens.