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

Lymphatic vessels are thought to contribute to metastasis primarily by serving as a transportation system. It is widely believed that tumor cells enter lymph nodes passively by the flow of lymph. We demonstrate that lymph node lymphatic sinuses control tumor cell entry into the lymph node, which requires active tumor cell migration. In human and mouse tissues, CCL1 protein is detected in lymph node lymphatic sinuses but not in the peripheral lymphatics. CCR8, the receptor for CCL1, is strongly expressed by human malignant melanoma. Tumor cell migration to lymphatic endothelial cells (LECs) in vitro is inhibited by blocking CCR8 or CCL1, and recombinant CCL1 promotes migration of CCR8(+) tumor cells. The proinflammatory mediators TNF, IL-1ß, and LPS increase CCL1 production by LECs and tumor cell migration to LECs. In a mouse model, blocking CCR8 with the soluble antagonist or knockdown with shRNA significantly decreased lymph node metastasis. Notably, inhibition of CCR8 led to the arrest of tumor cells in the collecting lymphatic vessels at the junction with the lymph node subcapsular sinus. These data identify a novel function for CCL1-CCR8 in metastasis and lymph node LECs as a critical checkpoint for the entry of metastases into the lymph nodes.

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.

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.

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.

Soluble Ig-like transcript 3 inhibits tumor allograft rejection in humanized SCID mice and T cell responses in cancer patients.

Attempts to enhance patients' immune responses to malignancies have been largely unsuccessful. We now describe an immune-escape mechanism mediated by the inhibitory receptor Ig-like transcript 3 (ILT3) that may be responsible for such failures. Using a humanized SCID mouse model, we demonstrate that soluble and membrane ILT3 induce CD8(+) T suppressor cells and prevent rejection of allogeneic tumor transplants. Furthermore, we found that patients with melanoma, and carcinomas of the colon, rectum, and pancreas produce the soluble ILT3 protein, which induces the differentiation of CD8(+) T suppressor cells and impairs T cell responses in MLC. These responses are restored by anti-ILT3 mAb or by depletion of soluble ILT3 from the serum. Immunohistochemical staining of biopsies from the tumors and metastatic lymph nodes suggests that CD68(+) tumor-associated macrophages represent the major source of soluble ILT3. Alternative splicing, resulting in the loss of the ILT3 transmembrane domain, may contribute to the release of ILT3 in the circulation. These data suggest that ILT3 depletion or blockade is crucial to the success of immunotherapy in cancer. In contrast, the inhibitory activity of soluble ILT3 on T cell alloreactivity in vitro and in vivo suggests the potential usefulness of rILT3 for immunosuppressive treatment of allograft recipients or patients with autoimmune diseases.

Single-chain bifunctional vascular endothelial growth factor (VEGF)-follicle-stimulating hormone (FSH)-C-terminal peptide (CTP) is superior to the combination therapy of recombinant VEGF plus FSH-CTP in stimulating angiogenesis during ovarian folliculogenesis.

Infertility technologies often employ exogenous gonadotropin therapy to increase antral follicle production. In an effort to enhance ovarian response, several long-acting FSH therapies have been developed including an FSH-C-terminal peptide (CTP), where the FSH subunits are linked by the CTP moiety from human chorionic gonadotropin, which is responsible for the increased half-life of human chorionic gonadotropin. We found that administration of FSH-CTP for ovarian hyperstimulation in rats blunted ovarian follicle vascular development. In women, reduced ovarian vasculature has been associated with lower pregnancy rates. We were interested in determining whether vascular endothelial growth factor (VEGF) therapy could enhance ovarian angiogenesis in FSH-CTP-treated rats. Coadministration of systemic FSH-CTP plus recombinant VEGF was compared with treatment with a novel, single-chain bifunctional VEGF-FSH-CTP (VFC) analog. For VFC, the FSH portion targets the protein to the ovary and stimulates follicle growth, whereas VEGF enhances local vascular development. Both in vitro and in vivo studies confirm the dual FSH and VEGF action of the VFC protein. Evaluation of ovarian follicle development demonstrates that administration of combination therapy using VEGF and FSH-CTP failed to increase follicle vasculature above levels seen with FSH-CTP monotherapy. However, treatment with VFC significantly increased follicle vascular development while concurrently increasing the number of large antral follicles produced. In conclusion, we report the production and characterization of a long-acting, bifunctional VEGF-FSH-CTP protein that is superior to combination therapy for enhancing VEGF activity in the ovary and stimulating follicular angiogenesis in rats.

NF-kappaB 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.

Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis.

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. However, their origin is still unknown. We tested the hypothesis that bone marrow (BM) contributes to the population of HSCs. METHODS: Chimeric mice transplanted with donor BM from collagen alpha1(I)-GFP+ reporter mice were subjected to the bile duct ligation (BDL)-induced liver injury. RESULTS: In response to injury, BM-derived collagen-expressing GFP+ cells were detected in liver tissues of chimeric mice. However, these cells were not activated HSCs in that they did not express alpha-smooth muscle actin or desmin and could not be isolated with the HSC fraction. Meanwhile, the majority of these BM-derived cells co-expressed collagen-GFP+ and CD45+, suggesting that these cells represent a unique population of fibrocytes. Consistent with their lymphoid origin, the number of GFP+CD45+ fibrocytes found in BM and spleen of chimeric mice increased in response to injury. Fibrocytes cultured in the presence of TGF-beta1 differentiated into SMA+desmin+ collagen-producing myofibroblasts, potentially contributing to liver fibrosis. CONCLUSIONS: In response to the BDL-induced liver injury: (i) HSCs do not originate in the BM; (ii) collagen-producing fibrocytes are recruited from the BM to damaged liver.

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. METHODS: A model was employed in which toxic doses of acetaminophen (1125 mg/kg) were administered to C57BL/6 mice. To block RAGE, mice received murine soluble (s) RAGE, the extracellular ligand binding domain of the receptor that acts as a decoy to interrupt ligand-RAGE signaling. RESULTS: Animals treated with sRAGE displayed increased survival compared with vehicle treatment, and markedly decreased hepatic necrosis. Consistent with an important role for RAGE-triggered oxidant stress in acetaminophen-induced injury, a significant reduction of nitrotyrosine protein adducts was observed in hepatic tissue in sRAGE-treated versus vehicle-treated mice receiving acetaminophen, in parallel with significantly increased levels of glutathione. In addition, pro-regenerative cytokines tumor necrosis factor-alpha and interleukin-6 were increased in sRAGE-treated versus vehicle-treated mice. CONCLUSION: These findings implicate RAGE-dependent mechanisms in acetaminophen-induced liver damage and suggest that blockade of this pathway may impart beneficial effects in toxin-induced liver injury.

Gastric bypass surgery improves metabolic and hepatic abnormalities associated with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Most patients with extreme obesity have nonalcoholic fatty liver disease (NAFLD). Although gastric bypass (GBP) surgery is the most common bariatric operation performed in obese patients in the United States, the effect of GBP surgery-induced weight loss on the metabolic and hepatic abnormalities associated with NAFLD are not clear. METHODS: Whole-body glucose, fatty acid and lipoprotein kinetics, liver histology, and hepatic cellular factors involved in inflammation and fibrogenesis were evaluated in 7 extremely obese subjects (body mass index, 58 +/- 4 kg/m(2)) before and 1 year after GBP surgery. RESULTS: At 1 year after surgery, subjects lost 29% +/- 5% of initial body weight (P < .01); palmitate rate of appearance in plasma, an index of adipose tissue lipolysis, decreased by 47% +/- 4% (P < .01); endogenous glucose production rate decreased by 27% +/- 7% (P < .01); and very-low-density lipoprotein-triglyceride secretion rate decreased by 44% +/- 9% (P < .05). In addition, GBP surgery-induced weight loss decreased hepatic steatosis but did not change standard histologic assessments of inflammation and fibrosis. However, there was a marked decrease in hepatic factors involved in regulating fibrogenesis (collagen-alpha1(I), transforming growth factor-beta1, alpha-smooth muscle actin, and tissue inhibitor of metalloproteinase 1 expression and alpha-smooth muscle actin content) and inflammation (macrophage chemoattractant protein 1 and interleukin 8 expression) (P < .05, compared with values before weight loss). CONCLUSIONS: These data demonstrate that weight loss induced by GBP surgery normalizes the metabolic abnormalities involved in the pathogenesis and pathophysiology of NAFLD and decreases the hepatic expression of factors involved in the progression of liver inflammation and fibrosis.

Inhibition of cyclooxygenase-2 disrupts tumor vascular mural cell recruitment and survival signaling.

Much evidence supports an important role for the inducible enzyme cyclooxygenase-2 (COX-2) in tumor angiogenesis. Previous studies have focused on the role of COX-2 in stimulating endothelial proliferation, with blockade of this enzyme impairing endothelial homeostasis. However, recent data suggest that COX-2 also regulates molecules implicated in endothelial trafficking with pericytes/vascular mural cells (VMC), an interaction crucial to vessel stability. We investigated the role of COX-2 in vascular assembly by testing the effect of the specific COX-2 inhibitor SC-236 in an orthotopic xenograft model of human Wilms' tumor. Tumor growth was significantly suppressed by SC-236 (78% at day 28, 55% at day 35). Perfusion studies and immunostaining showed a marked decrease in vasculature, particularly in small vessels. Specifically, SC-236 inhibited participation of VMC in xenograft vessels. SC-236-treated tumors developed segmentally dilated, architecturally erratic tumor vessels with decreased nascent pericytes and scant mature VMC. Although vascular endothelial growth factor expression was unchanged, expression of the chemokine receptor CXCR4 was decreased in tumor vessels, consistent with defective homing of vascular progenitor cells. Vascular expression of phosphorylated platelet-derived growth factor receptor-beta was also diminished, indicating impaired VMC-endothelial trafficking. Consistent with the key role of this interaction in vessel homeostasis, vascular cells in SC-236-treated tumors displayed markedly diminished phosphorylated Akt, indicating disrupted survival signaling. These results show that SC-236 causes defective vascular assembly by attenuating incorporation of VMC into tumor vessels, impairing endothelial survival, and raise the possibility that blockade of COX-2 may provide therapeutic synergies with antiangiogenic molecules that more selectively target endothelial cells.

RAGE limits regeneration after massive liver injury by coordinated suppression of TNF-alpha and NF-kappaB.

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-kappaB, principally in hepatocytes, and enhanced expression of regeneration-promoting cytokines, TNF-alpha 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.

Long natural history of recurrent granulosa cell tumor of the ovary 23 years after initial diagnosis: a case report and review of the literature.

BACKGROUND: Granulosa cell tumors (GCTs) of the ovary are rare, hormonally active neoplasms characterized by endocrine manifestations, an indolent course, and late relapse. CASE: We report a case of a prolonged history of ovarian GCT managed primarily with repeat surgical resections. CONCLUSION: This case illustrates the benefits of cytoreductive surgery for the management of recurrent disease, the use of serum tumor markers to help guide therapy, and the importance of extended follow-up.

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 (CCl(4)), 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 CCl(4)-treated rats exhibited substantial damage, including bridging fibrosis, inflammation, and extensive expression of smooth muscle alpha-actin (alpha-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 alpha-SMA staining. Zymography analysis showed antibody treatment decreased the activity of MMP-2. In conclusion, administration of a TIMP-1 antibody attenuated CCl(4)-induced liver fibrosis and decreased HSC activation and MMP-2 activity.

Notch in mammary gland development and breast cancer.

Notch signaling has been implicated in many processes including cell fate determination and oncogenesis. In mice, the Notch1 and Notch4 genes are both targets for insertion and rearrangement by the mouse mammary tumor virus and these mutations promote epithelial mammary tumorigenesis. Moreover, expression of a constitutively active form of Notch4 in mammary epithelial cells inhibits epithelial differentiation and leads to tumor formation in this organ. These data implicate the Notch pathway in breast tumorigenesis and provide the foundation for future experiments that will aid in our understanding of the role of Notch in human breast cancer development. Here, we review studies of mammary tumorigenesis induced by Notch in mouse and in vitro culture models providing evidence that Notch activation is a causal factor in human breast cancer.

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 kappaB, in parallel with increased transcripts for the proregenerative cytokine, tumor necrosis factor-alpha. 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.

Platelet-derived growth factor-AB limits the extent of myocardial infarction in a rat model: feasibility of restoring impaired angiogenic capacity in the aging heart.

BACKGROUND: Compared with younger patients, myocardial infarction in the elderly has been associated with less favorable clinical outcomes, which may be attributable to a decline in angiogenic capacity in the aging heart. METHODS AND RESULTS: To test the hypothesis that the functional phenotype of cardiac microvascular endothelial cells is maintained partly by a cardiac myocyte platelet-derived growth factor (PDGF)-B-induced paracrine pathway, we conducted in vitro studies with murine cardiac cells. These studies demonstrated that unlike young endothelial cells, endothelial cells of the aging heart do not express PDGF-B when cultured in the presence of cardiac myocytes. The functional significance of this endothelial dysregulation was assessed with an ex vivo pinnal cardiac allograft model to demonstrate that senescent cardiac angiogenic activity was depressed (2 of 17 allografts were viable in 18-month-old mice versus 19 of 20 in 3-month-old mice; P<0.01). PDGF-AB pretreatment specifically restored the viability of the cardiac allografts in the aging hosts (13 of 13 allografts were viable; P<0.01 versus 18-month-old controls). Finally, in vivo studies in rat hearts demonstrated that pretreatment by intramyocardial delivery of PDGF-AB promotes angiogenesis and minimizes the extent of myocardial infarction in the aging hearts after coronary ligation (myocardial infarction size: 10.0 +/- 7.0% of left ventricular area in PDGF pretreatment [n=7] versus 17.6 +/- 5.6% in control [n=5] groups; P<0.03). CONCLUSION: Aging hearts have impaired angiogenic function as a result of depressed PDGF-B production. Restoration of the dysregulated endothelial PDGF-mediated angiogenic pathway in the aging heart reverses the senescent impairment in cardioprotective angiogenic function and offers a foundation for developing novel therapies for cardiovascular disease in older individuals.