Nikki Feirt

Blockade of receptor for advanced glycation end product (RAGE) attenuates ischemia and reperfusion injury to the liver in mice

Hepatic ischemia/reperfusion (I/R) injury associated with liver transplantation and hepatic resection is characterized by hepatocellular damage and a deleterious inflammatory response. In this study, we examined whether receptor for advanced glycation end product (RAGE) activation is linked to mechanisms accentuating inflammation on I/R in a murine model of total hepatic ischemia. Animals treated with soluble RAGE (sRAGE), the extracellular ligand‐binding domain of RAGE, displayed increased survival after total hepatic I/R compared with vehicle treatment. TUNEL assay and histologic analysis revealed that blockade of RAGE was highly protective against hepatocellular death and necrosis on I/R; in parallel, proliferating cell nuclear antigen was enhanced in livers of mice treated with sRAGE. Rapid activation of p38, p44/42, stress‐activated protein kinase and c‐Jun N‐terminal kinase mitogen‐activated protein kinases, signal transducer and activator of transcription‐3, and nuclear translocation of activator protein‐1 was evident at early times on I/R. In the remnants of sRAGE‐treated livers, however, activation of each of these signaling and transcription factor pathways was strikingly decreased. sRAGE‐treated remnants displayed enhanced activation of nuclear factor κB, in parallel with increased transcripts for the proregenerative cytokine, tumor necrosis factor‐α. In conclusion, these data suggest that RAGE modulates hepatic I/R injury, at least in part by activation of key signaling pathways linked to proinflammatory and cell death‐promoting responses. We propose that blockade of this pathway may represent a novel strategy to attenuate injury in hepatic I/R and to facilitate regeneration. (HEPATOLOGY 2004;39:422–432.)

Antifibrotic effects of a tissue inhibitor of metalloproteinase-1 antibody on established liver fibrosis in rats.

Liver fibrosis is characterized by increased synthesis, and decreased degradation, of extracellular matrix (ECM) within the injured tissue. Decreased ECM degradation results, in part, from increased expression of tissue inhibitor of metalloproteinase‐1 (TIMP‐1), which blocks matrix metalloproteinase (MMP) activity. TIMP‐1 is also involved in promoting survival of activated hepatic stellate cells (HSCs), a major source of ECM. This study examined the effects of blocking TIMP‐1 activity in a clinically relevant model of established liver fibrosis. Rats were treated with carbon tetrachloride (CCl4), or olive oil control, for 6 weeks; 24 days into the treatment, the rats were administered a neutralizing anti–TIMP‐1 antibody derived from a fully human combinatorial antibody library (HuCAL), PBS, or an isotype control antibody. Livers from CCl4‐treated rats exhibited substantial damage, including bridging fibrosis, inflammation, and extensive expression of smooth muscle α‐actin (α‐SMA). Compared to controls, rats administered anti–TIMP‐1 showed a reduction in collagen accumulation by histological examination and hydroxyproline content. Administration of anti–TIMP‐1 resulted in a marked decrease in α‐SMA staining. Zymography analysis showed antibody treatment decreased the activity of MMP‐2. In conclusion, administration of a TIMP‐1 antibody attenuated CCl4‐induced liver fibrosis and decreased HSC activation and MMP‐2 activity. (HEPATOLOGY 2004.)

Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

The Notch family of cell surface receptors and its ligands are highly conserved proteins that regulate cell fate determination, including those involved in mammalian vascular development. We report that Notch induces VEGFR-3 expression in vitro in human endothelial cells and in vivo in mice. In vitro, Notch in complex with the DNA-binding protein CBF-1/suppressor of hairless/Lag1 (CSL) bound the VEGFR-3 promoter and transactivated VEGFR-3 specifically in endothelial cells. Through induction of VEGFR-3, Notch increased endothelial cell responsiveness to VEGF-C, promoting endothelial cell survival and morphological changes. In vivo, VEGFR-3 was upregulated in endothelial cells with active Notch signaling. Mice heterozygous for null alleles of both Notch1 and VEGFR-3 had significantly reduced viability and displayed midgestational vascular patterning defects analogous to Notch1 nullizygous embryos. We found that Notch1 and Notch4 were expressed in normal and tumor lymphatic endothelial cells and that Notch1 was activated in lymphatic endothelium of invasive mammary micropapillary carcinomas. These results demonstrate that Notch1 and VEGFR-3 interact genetically, that Notch directly induces VEGFR-3 in blood endothelial cells to regulate vascular development, and that Notch may function in tumor lymphangiogenesis.

RAGE limits regeneration after massive liver injury by coordinated suppression of TNF-α and NF-κB

The exquisite ability of the liver to regenerate is finite. Identification of mechanisms that limit regeneration after massive injury holds the key to expanding the limits of liver transplantation and salvaging livers and hosts overwhelmed by carcinoma and toxic insults. Receptor for advanced glycation endproducts (RAGE) is up-regulated in liver remnants selectively after massive (85%) versus partial (70%) hepatectomy, principally in mononuclear phagocyte-derived dendritic cells (MPDDCs). Blockade of RAGE, using pharmacological antagonists or transgenic mice in which a signaling-deficient RAGE mutant is expressed in cells of mononuclear phagocyte lineage, significantly increases survival after massive liver resection. In the first hours after massive resection, remnants retrieved from RAGE-blocked mice displayed increased activated NF-κB, principally in hepatocytes, and enhanced expression of regeneration-promoting cytokines, TNF-α and IL-6, and the antiinflammatory cytokine, IL-10. Hepatocyte proliferation was increased by RAGE blockade, in parallel with significantly reduced apoptosis. These data highlight central roles for RAGE and MPDDCs in modulation of cell death–promoting mechanisms in massive hepatectomy and suggest that RAGE blockade is a novel strategy to promote regeneration in the massively injured liver.

NF-κB regulation of endothelial cell function during LPS-induced toxemia and cancer

The transcription factor NF-kappaB is an important regulator of homeostatic growth and inflammation. Although gene-targeting studies have revealed important roles for NF-kappaB, they have been complicated by component redundancy and lethal phenotypes. To examine the role of NF-kappaB in endothelial tissues, Tie2 promoter/enhancer-IkappaBalpha(S32A/S36A) transgenic mice were generated. These mice grew normally but exhibited enhanced sensitivity to LPS-induced toxemia, notable for an increase in vascular permeability and apoptosis. Moreover, B16-BL6 tumors grew significantly more aggressively in transgenic mice, underscoring a new role for NF-kappaB in the homeostatic response to cancer. Tumor vasculature in transgenic mice was extensive and disorganized. This correlated with a marked loss in tight junction formation and suggests that NF-kappaB plays an important role in the maintenance of vascular integrity and response to stress.

Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses

Blocking CCR8 inhibits entry of metastases from the collecting lymphatic vessel into the lymph node.

Blockade of the receptor for advanced glycation end products attenuates acetaminophen-induced hepatotoxicity in mice.

Background and Aim:  Severe injury to the liver, such as that induced by toxic doses of acetaminophen, triggers a cascade of events leading to hepatocyte death. It is hypothesized that activation of the receptor for advanced glycation end products (RAGE) might contribute to acetaminophen‐induced liver toxicity by virtue of its ability to generate reactive oxygen species, at least in part via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and thereby activate downstream signaling pathways leading to cellular injury.

Sustained VEGF blockade results in microenvironmental sequestration of VEGF by tumors and persistent VEGF receptor-2 activation.

Vascular endothelial growth factor (VEGF) blockade has been validated clinically as a treatment for human cancers, yet virtually all patients eventually develop progressive disease during therapy. In order to dissect this phenomenon, we examined the effect of sustained VEGF blockade in a model of advanced pediatric cancer. Treatment of late-stage hepatoblastoma xenografts resulted in the initial collapse of the vasculature and significant tumor regression. However, during sustained treatment, vessels recovered, concurrent with a striking increase in tumor expression of perlecan, a heparan sulfate proteoglycan. Whereas VEGF mRNA was expressed at the periphery of surviving clusters of tumor cells, both secreted VEGF and perlecan accumulated circumferential to central vessels. Vascular expression of heparanase, VEGF receptor-2 ligand binding, and receptor activation were concurrently maintained despite circulating unbound VEGF Trap. Endothelial survival signaling via Akt persisted. These findings provide a novel mechanism for vascular survival during sustained VEGF blockade and indicate a role for extracellular matrix molecules that sequester and release biologically active VEGF. (Mol Cancer Res 2008;6(1):1–9)

Utility of liver allograft biopsy obtained at procurement

Extended‐donor criteria (EDC) liver allografts potentiate the role of procurement biopsy in organ utilization. To expedite allocation, histologic evaluation is routinely performed upon frozen‐section (FS) specimens by local pathologists. This descriptive study compares FS reports by local pathologists with permanent‐section (PS) evaluation by dedicated hepatopathologists, identifies histologic characteristics underrepresented by FS evaluation, and evaluates the efficacy of a biopsy decision analysis based on organ visualization. Fifty‐two liver transplants using EDC allografts evaluated by FS with PS were studied. Pathologic worksheets created by an organ procurement organization were applied in 34 FS. PS analysis included 7 staining procedures for 8 histologic criteria. PS from 56 additional allografts determined not to require donor biopsy were also analyzed. A high correlation was observed between FS and PS. Underestimation of steatosis by FS was associated with allograft dysfunction. Surgical assessment of cholestasis, congestion, and steatosis was accurate whereas inflammation, necrosis, and fibrosis were underestimated in allografts suffering parenchymal injury. In conclusion, the correlation between FS and PS is high, and significant discrepancies are rare. Biopsy is not a prerequisite for EDC utilization but is suggested in hepatitis C, hypernatremia, donation after cardiac death, or multiple EDC indications. Implementation of a universal FS worksheet could standardize histologic reporting and facilitate data collection, allocation, and research. Liver Transpl 2008.

Discovery of diffuse biliary microhamartomas during liver procurement

James V. Guarrera, Barbara J. Alkofer, Nikki Feirt, Rodrigo Sandoval, Benjamin Samstein, Elton T. Smith Jr, David Marshman, Chris Cogswell, Jason Vannatta, Robert S. Brown Jr, Jean C. Emond, and John F. Renz Center for Liver Disease and Transplantation and Department of Pathology, Columbia University Medical Center, New York, NY; Department of Pathology, Carilion Roanoke Memorial Hospital, Roanoke, VA; and LifeNet, Roanoke, VA