Oncogenic activation of JAK3-STAT signaling confers clinical sensitivity to PRN371, a novel selective and potent JAK3 inhibitor, in natural killer/T-cell lymphoma.

Aberrant activation of the JAK3-STAT signaling pathway is a characteristic feature of many hematological malignancies. In particular, hyperactivity of this cascade has been observed in natural killer/T-cell lymphoma (NKTL) cases. Although the first-in-class JAK3 inhibitor tofacitinib blocks JAK3 activity in NKTL both in vitro and in vivo, its clinical utilization in cancer therapy has been limited by the pan-JAK inhibition activity. To improve the therapeutic efficacy of JAK3 inhibition in NKTL, we have developed a highly selective and durable JAK3 inhibitor PRN371 that potently inhibits JAK3 activity over the other JAK family members JAK1, JAK2, and TYK2. PRN371 effectively suppresses NKTL cell proliferation and induces apoptosis through abrogation of the JAK3-STAT signaling. Moreover, the activity of PRN371 has a more durable inhibition on JAK3 compared to tofacitinib in vitro, leading to significant tumor growth inhibition in a NKTL xenograft model harboring JAK3 activating mutation. These findings provide a novel therapeutic approach for the treatment of NKTL.

Cyclic pressure modulates endothelial barrier function.

Although numerous studies have documented the importance of mechanical forces in regulating many endothelial cell functions, the effects of these physical stimuli on endothelial barrier function are not well characterized. The present study used a custom-designed, cyclic pressure system to expose human umbilical vein endothelial cells (HUVECs) to physiologically relevant sinusoidal pressures and demonstrated that exposure to 140/100, but not to 60/20, mm Hg cyclic pressure at 1 Hz for 18 h resulted in a significant (p <.05) reduction in transendothelial permeability to albumin. Moreover, these cyclic pressure-selective changes in HUVEC barrier function occurred concomitantly with redistribution of intracellular tight junction protein zona occludens (ZO)-1 and reorientation of the F-actin cytoskeleton. In contrast, exposure of HUVECs to cyclic pressure had no affect on localization of adherens junctions proteins, vascular endothelial (VE)-cadherin, and beta-catenin. These results, therefore, provide the first evidence that select levels of cyclic pressure, a mechanical force pertinent to the hemodynamic vascular milieu, modulates the endothelial barrier function concomitant with an altered distribution of tight junction component, ZO-1.

Vascular endothelial cell growth factor-driven endothelial tube formation is mediated by vascular endothelial cell growth factor receptor-2, a kinase insert domain-containing receptor.

Vascular endothelial cell growth factor (VEGF) binds to 2 related receptor tyrosine kinases, known as kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt-1). The KDR has been shown to mediate VEGF-stimulated endothelial cell mitogenesis, migration, and permeability. The Flt-1 receptor has been suggested to mediate VEGF-stimulated endothelial branching morphogenesis, a process whereby endothelial cells, in the presence of a 3D milieu composed of extracellular matrix components and a mixture of growth factors, undergo a morphological transition into a tubular network with many lumina. In the present study, we have used 2 independent endothelial cell tube formation models and highly selective VEGF mutants for the KDR and Flt-1 receptors. We demonstrate that KDR, not Flt-1, stimulation is responsible for the induction of endothelial tubulogenesis. In addition, we demonstrate a modulatory role for Flt-1 in VEGF-mediated tube formation. We also report that VEGF-driven endothelial tube formation is inhibited by selective inhibitors of mitogen-activated protein kinase activation and p38 protein kinase.

Endothelial expression of vascular cell adhesion molecule-1 correlates with metastatic pattern in spontaneous melanoma.

OBJECTIVE: Adhesive interactions between tumor cell surface receptors and endothelial cell adhesion molecules are thought to contribute to tumor cell arrest and extravasation during hematogenous metastasis. Recent reports suggest that melanoma cell integrin alpha4beta1 (very late antigen-4, VLA-4) interaction with the inducible cell adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1), is critical for tumor cell arrest. However, no information is available regarding microvascular VCAM-1 expression during spontaneous melanoma metastasis. The objectives of this study were to evaluate VCAM-1 expression in pulmonary and extrapulmonary vascular beds during melanoma progression, and to determine whether there is an organ-specific profile for VCAM-1 expression which corresponds with the clinical pattern of melanoma metastasis. METHODS: The dual-radiolabeled monoclonal antibody (mAb) technique for quantification of VCAM-1 in different vascular beds was applied to a physiological model of melanoma (B16-BL6) metastasis. Measurements of VCAM-1 were obtained when primary tumors reached 5 mm in size, and every 7 days following removal of the primary lesion. Histological examinations were performed, and mice were placed into 2 groups, based on the presence (+colonies) or absence (-colonies) of pulmonary metastases. VCAM-1 measurements obtained from several organ systems were then compared between these 2 groups of mice. Localization of VCAM-1 was achieved through immunohistochemical staining of tissues. Plasma collected from each experimental animal, as well as melanoma-conditioned media, was assayed to determine levels of the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1alpha (IL-1alpha). RESULTS: Data collected from the dual-radiolabeled mAb technique indicate that 3 weeks following removal of the primary lesion, there is a tendency for VCAM-1 expression to increase in cardiac, hepatic, and cerebral vascular beds. Four weeks following primary resection, when pulmonary metastatic burden was maximal, VCAM-1 was significantly upregulated in each of these vascular beds. Results obtained from the lung indicate that VCAM-1 remains unchanged in pulmonary vessels at all time points examined. Immunohistochemical staining of heart and brain support the radiolabeled mAb measurements, and reveals that these organs exhibit an inflammatory phenotype in mice with heavy pulmonary tumor burden. Furthermore, 25% of these mice had histological evidence of melanoma metastases in heart and brain. Transplantation of liver fragments from mice with advanced pulmonary metastases into subcutaneous tissue of donor mice resulted in the formation of melanotic outgrowths. Plasma levels of the cytokines TNF-alpha and IL-1alpha were negligible in both groups of mice. CONCLUSIONS: Our results indicate that upregulation of VCAM-1 is not a prerequisite for the formation of pulmonary metastases during spontaneous melanoma metastases. However, once lung metastases become well established, organ-specific increases in VCAM-1 expression become apparent. Furthermore, these organ-specific increments in VCAM-1 expression correspond with documented clinical patterns of melanoma metastasis. The enhanced expression of VCAM-1 is independent of systemic levels of TNF-alpha and IL-1alpha, but may be the result of melanoma-induced alterations at the local level, as we found evidence of melanoma cell occupation in heart, brain, and liver in pulmonary metastases-bearing mice.

Gene profiling techniques and their application in angiogenesis and vascular development.

The analysis of gene expression in specific tissues and physiological processes has evolved over the last 20 years from the painstaking identification of selected genes to the relatively efficient and open-ended surveying of potentially all genes expressed in a tissue. Current art for gene discovery includes the use of large-scale arrays of cDNA sequences or oligonucleotides, and molecular 'tagging' techniques such as GeneCalling and SAGE. Common to each of these techniques is a reliance on the increasingly comprehensive databases of human and mouse EST and full-length gene sequences. Early efforts to characterize candidate genes were limited by their narrow scope, while current efforts are confounded by the enormous volume of data returned. Sophisticated software tools are an integral part of the analysis, helping to organize information into coherent groups with temporal or functional similarity. These techniques, in conjunction with the continued analysis of human genetic syndromes, transgenic, and knockout mice, have driven genetic analysis of angiogenesis and vascular development from describing which individual genes are involved to defining the outlines of regulatory networks.

Hepatocyte growth factor enhances vascular endothelial growth factor-induced angiogenesis in vitro and in vivo.

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis in both physiological and pathological processes. Hepatocyte growth factor (HGF) is a mesenchyme-derived mitogen that also stimulates cell migration, and branching and/or tubular morphogenesis of epithelial and endothelial cells. In the present study, we tested the hypothesis that simultaneous administration of HGF and VEGF would synergistically promote new blood vessel formation. HGF acted in concert with VEGF to promote human endothelial cell survival and tubulogenesis in 3-D type I collagen gels, a response that did not occur with either growth factor alone. The synergistic effects of VEGF and HGF on endothelial survival correlated with greatly augmented mRNA levels for the anti-apoptotic genes Bcl-2 and A1. Co-culture experiments with human neonatal dermal fibroblasts and human umbilical vein endothelial cells demonstrated that neonatal dermal fibroblasts, in combination with VEGF, stimulated human umbilical vein endothelial cells tubulogenesis through the paracrine secretion of HGF. Finally, in vivo experiments demonstrated that the combination of HGF and VEGF increased neovascularization in the rat corneal assay greater than either growth factor alone. We suggest that combination therapy using HGF and VEGF co-administration may provide a more effective strategy to achieve therapeutic angiogenesis.

Receptor-selective variants of human vascular endothelial growth factor. Generation and characterization.

Vascular endothelial growth factor (VEGF) is a pleiotropic factor that exerts a multitude of biological effects through its interaction with two receptor tyrosine kinases, fms-like tyrosine kinase (Flt-1) or VEGF receptor 1 and kinase insert domain-containing receptor (KDR) or VEGF receptor 2. Whereas it is commonly accepted that KDR is responsible for the proliferative activities of VEGF, considerable controversy and uncertainty exist about the role of the individual receptors in eliciting many of the other effects. Based on a comprehensive mutational analysis of the receptor-binding site of VEGF, an Flt-1-selective variant was created containing four substitutions from the wild-type protein. This variant bound with wild-type affinity to Flt-1, was at least 470-fold reduced in binding to KDR, and had no activity in cell-based assays measuring autophosphorylation of KDR or proliferation of primary human vascular endothelial cells. Using a competitive phage display strategy, two KDR-selective variants were discovered with three and four changes from wild-type, respectively. Both variants had approximately wild-type affinity for KDR, were about 2000-fold reduced in binding to Flt-1, and showed activity comparable with the wild-type protein in KDR autophosphorylation and endothelial cell proliferation assays. These variants will serve as useful reagents in elucidating the roles of Flt-1 and KDR.

Gene expression profiling in an in vitro model of angiogenesis.

In the present study we have used a novel, comprehensive mRNA profiling technique (GeneCalling) for determining differential gene expression profiles of human endothelial cells undergoing differentiation into tubelike structures. One hundred fifteen cDNA fragments were identified and shown to represent 90 distinct genes. Although some of the genes identified have previously been implicated in angiogenesis, potential roles for many new genes, including OX-40, white protein homolog, KIAA0188, a homolog of angiopoietin-2, ADAMTS-4 (aggrecanase-1), and stanniocalcin were revealed. Support for the biological significance was confirmed by the abrogation of the changes in the expression of angiogenesis inhibitors and in situ hybridization studies. This study has significantly extends the molecular fingerprint of the changes in gene expression that occur during endothelial differentiation and provides new insights into the potential role of a number of new molecules in angiogenesis.

Functional roles for PECAM-1 (CD31) and VE-cadherin (CD144) in tube assembly and lumen formation in three-dimensional collagen gels.

Various in vitro models have been described that emulate one or more of the processes involved in angiogenesis in vivo. In the present study endothelial cells were cultured in three-dimensional type I collagen lattices in the presence of a mixture of basic fibroblast growth factor, vascular endothelial cell growth factor, and phorbol myristate acetate. Under these conditions, the endothelial cells rapidly assemble into an interconnected network of tube-like structures with a high frequency of intercellular canals or lumens. The formation of the networks and lumens was completely blocked by cycloheximide and by actinomycin D. Monoclonal antibodies directed against CD31 or vascular endothelial cadherin (VE-cadherin) inhibited the formation of endothelial tubes. A subtle difference in the morphology of cells treated with anti-CD31 versus anti-VE-cadherin was noted; namely, cells incubated in the presence of CD31 antibodies were rounded or formed attenuated tube-like structures, both of which were characterized by a single, large intra- or intercellular vacuole. In contrast, tube formation by cells incubated in the presence of VE-cadherin antibodies was also impaired and, most notably, demonstrated a reduction in either vacuole formation or vacuole fusion, depending upon the monoclonal antibody used. We suggest that the two endothelial-junction-associated proteins, CD31 and VE-cadherin, play different roles in the process of tube formation. CD31 appears to be required for cell elongation, migration, and/or invasion in the gels as well as for cell-cell association to form the network structures. VE-cadherin also appears to be required for cell-cell association, but additionally appears to play some role in the process of vacuolization or vacuole fusion leading to intercellular lumen formation.

Ischemia-reperfusion induced microvascular responses in LDL-receptor -/- mice.

The objective of this study was to determine whether the microvascular responses to ischemia and reperfusion (I/R) are altered in an animal model of atherosclerosis, the low-density lipoprotein-receptor knockout (LDLr -/-) mouse. Intravital video microscopy was used to monitor venular wall shear rate, leukocytes rolling velocity, the number of rolling, adherent and emigrated leukocytes, and albumin leakage in cremasteric postcapillary venules of wild-type (B6129) and LDLr -/- mice exposed to 60 min of ischemia and 60 min of reperfusion. The postcapillary venules of LDLr -/- mice exhibited two- to threefold larger increments in the number of adherent leukocytes and a more profound albumin leakage response to I/R than venules in wild-type mice. The exaggerated inflammatory responses noted in LDLr -/- mice placed on a normal diet were not exacerbated by a high-cholesterol diet. Treatment of LDLr -/- mice with either a platelet-activating factor (PAF) receptor antagonist (WEB-2086) or a monoclonal antibody (YN-1) against the endothelial cell adhesion molecule, intercellular adhesion molecule 1 (ICAM-1), markedly attenuated the I/R-induced leukocyte adherence and albumin leakage. These findings indicate that atherogenic mice are more vulnerable to the deleterious microvascular effects of I/R and that PAF-mediated, ICAM-1-dependent leukocyte adhesion contributes to this exaggerated response to I/R.

Distinct ICAM-1 forms and expression pathways in synovial microvascular endothelial cells.

Human synovial endothelial cell (HSE) intracellular adhesion molecule-1 (ICAM-1) is upregulated maximally by synergy of tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma). Such synergy is not as pronounced in human umbilical vein endothelium (HUVE). ICAM surface staining and ELISA detection reflected similar levels on HUVE and HSE cells, yet mRNA levels were much higher in HSE cells in response to TNF alpha/IFN gamma. To correlate protein and mRNA levels of ICAM-1, both cell types were permeabilized and stained with a monoclonal antibody against ICAM-1. HSE cells displayed a distinct vesicular cytoplasmic staining for ICAM while HUVE cells were devoid of such stained vesicles upon staining with the antibody. ICAM-1 immunostaining of HSE cytoplasmic vesicles appeared enhanced in cells treated with TNF alpha/IFN gamma and monensin, an endosomal processing inhibitor. Monensin inhibited HSE cell surface expression of ICAM-1 routinely up to 70%, while HUVE cell expression was unaffected. In addition, monensin also inhibited soluble ICAM-1 release from HSE cells while not effecting HUVE cells. Immunoprecipitation of ICAM-1 followed by gel electrophoresis indicated that HUVE and HSE cell ICAMs are expressed in cell-specific forms. These results define distinct forms and distinct secretory pathways for ICAM-1 in HSE cells and HUVE cells that indicate functional differences between these human endothelia.

Peroxisome proliferator-activated receptor gamma ligands are potent inhibitors of angiogenesis in vitro and in vivo.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that functions as a transcription factor to mediate ligand-dependent transcriptional regulation. Activation of PPARgamma by the naturally occurring ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), or members of a new class of oral antidiabetic agents, e.g. BRL49653 and ciglitizone, has been linked to adipocyte differentiation, regulation of glucose homeostasis, inhibition of macrophage and monocyte activation, and inhibition of tumor cell proliferation. Here we report that human umbilical vein endothelial cells (HUVEC) express PPARgamma mRNA and protein. Activation of PPARgamma by the specific ligands 15d-PGJ2, BRL49653, or ciglitizone, dose dependently suppresses HUVEC differentiation into tube-like structures in three-dimensional collagen gels. In contrast, specific PPARalpha and -beta ligands do not affect tube formation although mRNA for these receptors are expressed in HUVEC. PPARgamma ligands also inhibit the proliferative response of HUVEC to exogenous growth factors. Treatment of HUVEC with 15d-PGJ2 also reduced mRNA levels of vascular endothelial cell growth factor receptors 1 (Flt-1) and 2 (Flk/KDR) and urokinase plasminogen activator and increased plasminogen activator inhibitor-1 (PAI-1) mRNA. Finally, administration of 15d-PGJ2 inhibited vascular endothelial cell growth factor-induced angiogenesis in the rat cornea. These observations demonstrate that PPARgamma ligands are potent inhibitors of angiogenesis in vitro and in vivo, and suggest that PPARgamma may be an important molecular target for the development of small-molecule inhibitors of angiogenesis.

Exposure of human vascular endothelial cells to sustained hydrostatic pressure stimulates proliferation. Involvement of the alphaV integrins.

The present study investigated the effects of sustained hydrostatic pressure (SHP; up to 4 cm H2O) on human umbilical vein endothelial cell (HUVEC) proliferation, focal adhesion plaque (FAP) organization, and integrin expression. Exposure of HUVECs to SHP stimulated cell proliferation and a selective increase in the expression of integrin subunit alphaV. The increase in alphaV was observed as early as 4 hours after exposure to pressure and preceded detectable increases in the bromodeoxyuridine labeling index. Laser confocal microscopy studies demonstrated colocalization of the alphaV integrin to FAPs. The individual FAPs in pressure-treated cells demonstrated a reduced area and increased aspect ratio and were localized to both peripheral and more central regions of the cells, in contrast to the predilection for the cell periphery in cells maintained under control pressure conditions. The pressure-induced changes in alphaV distribution had functional consequences on the cells: adhesivity of the cells to vitronectin was increased, and alphaV antagonists blocked the pressure-induced proliferative response. Thus, the present study suggests a role for alphaV integrins in the mechanotransduction of pressure by endothelial cells.

Anti-inflammatory drugs and endothelial cell adhesion molecule expression in murine vascular beds.

BACKGROUND: Inflammatory bowel diseases (IBD) are characterised by an intense infiltration of leucocytes that is mediated by adhesion molecules expressed on the surface of activated endothelial cells. AIMS: To determine whether drugs used in the treatment of IBD, specifically dexamethasone (DEX), 5-aminosalicylic acid (5-ASA), methotrexate (MTX), and 6-mercaptopurine (6-MP), alter the expression of endothelial cell adhesion molecules (ECAMs). METHODS: The expression of P-selectin, E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular CAM 1 (VCAM-1) in different vascular beds of C57Bl/6J mice was measured using the dual radiolabelled monoclonal antibody technique. RESULTS: Lipopolysaccharide (LPS) elicited a profound increase in the expression of all ECAMs in the mesentery, small intestine, caecum, and distal colon. The LPS induced increase in CAM expression was not significantly affected by prior treatment with either MTX or 6-MP. However, pretreatment with either DEX or 5-ASA significantly attenuated LPS induced increases in expression of P- and E-selectin, and VCAM-1 in the majority of tissues evaluated. DEX also blunted the LPS induced increase in ICAM-1 expression in the caecum and distal colon. DEX, but not 5-ASA, largely abolished the rise in plasma tumour necrosis factor alpha elicited by LPS. CONCLUSIONS: These findings suggest that DEX and 5-ASA may exert their beneficial therapeutic action in IBD, at least in part, by inhibiting the expression of ECAMs which mediate leucocyte adhesion and transmigration in the microvasculature.

Nuclear integration of glucocorticoid receptor and nuclear factor-kappaB signaling by CREB-binding protein and steroid receptor coactivator-1.

The p65 (RelA) component of nuclear factor-kappaB (NF-kappaB) and the glucocorticoid receptor (GR) mutually repress each other's ability to activate transcription. Both of these transcriptional activators depend upon the coactivators CREB-binding protein (CBP) and steroid receptor coactivator-1 (SRC-1) for maximal activity. Here we show that increased levels of CBP relieves the inhibition of glucocorticoid-mediated repression of NF-kappaB activity and the NF-kappaB-mediated repression of GR activity. SRC-1 can relieve the NF-kappaB-mediated repression of GR activity. We propose that cross-talk between the p65 component of NF-kappaB and glucocorticoid receptors is due, at least in part, to nuclear competition for limiting amounts of the coactivators CBP and SRC-1, thus providing a novel mechanism for decreasing expression of genes involved in the inflammatory response.

Synovial fibroblasts and the sphingomyelinase pathway: sphingomyelin turnover and ceramide generation are not signaling mechanisms for the actions of tumor necrosis factor-alpha.

The activation of sphingomyelinase and the generation of ceramide has been proposed to mediate tumor necrosis factor-alpha (TNF-alpha)-induced nuclear factor (NF)-kappaB activation through its second messenger ceramide. Ceramide may also be an important regulator of cell growth, senescence, and apoptosis. Aberrant cell proliferation and apoptosis have been implicated in the rampant fibroblast proliferation and pannus formation characteristic of rheumatoid arthritis. However, the role of TNF-alpha and the sphingomyelinase pathway in the process have not been determined. The objective of this study was to determine whether TNF-alpha activates the sphingomyelin pathway in human synovial fibroblasts (HSF) and the potential role of ceramide in HSF proliferation and apoptosis. Cultured human synovial fibroblasts were stimulated with exogenous TNF-alpha, sphingomyelinase, and ceramide. Apoptosis was assessed by cell morphology and annexin V labeling. NF-kappaB and stress kinase pathway activation were determined by immunoblotting techniques. Sphingomyelinase activation was determined by quantitation of sphingomyelin and ceramide radioactivity in [14C]serine-prelabeled HSF cells. The addition of TNF-alpha (50 ng/ml) to HSF did not elicit detectable sphingomyelinase activation. TNF-alpha was shown to activate NF-kappaB (p65 translocation and degradation of IkappaBalpha) and the stress kinase pathway (phosphorylation of ATF-2, p38, and c-jun). In contrast, exogenous ceramide had no effect on these signaling pathways nor did ceramide stimulate the generation of interleukin-6 or interleukin-8. High concentrations of ceramide (> or =25 micromol/L) were cytotoxic, whereas lower concentrations of ceramide inhibited cell cycle progression. Thus, although TNF-alpha stimulates the NF-kappaB and stress kinase pathways in HSF, these effects of TNF-alpha are not associated with sphingomyelinase turnover or induction of apoptosis.

Lymphocytes mediate TNF-alpha-induced endothelial cell adhesion molecule expression: studies on SCID and RAG-1 mutant mice.

TNF-alpha is known to elicit a rapid increase in the expression of specific endothelial cell adhesion molecules (ECAMs) within different vascular beds. The aim of this study was to determine whether lymphocytes contribute to the increased ECAM expression elicited by TNF-alpha. A dual radiolabeled mAb technique was used to quantify constitutive and TNF-alpha-induced expression of ICAM-1, VCAM-1, E-selectin, and P-selectin in different vascular beds (lung, heart, stomach, mesentery, small intestine, large intestine, and muscle) in wild-type and SCID mice. In reconstitution experiments, either whole splenocytes, T cell-enriched splenocytes, or B cell-enriched splenocytes were injected into SCID mice 48 h before TNF-alpha administration. Although the constitutive expression of ECAMs differed only slightly between wild-type and SCID mice, TNF-alpha-induced ECAM expression was markedly blunted in SCID mice compared with wild-type mice. This blunted response to TNF-alpha was also demonstrated for VCAM-1 in recombination activating gene (RAG)-1 mutant mice. Reconstitution studies revealed that administration of 50 x 10(6) splenocytes in SCID mice at 48 h before cytokine treatment restored the TNF-alpha-induced expression of VCAM-1 to levels normally observed in wild-type mice. Reconstitution with T cell- but not B cell-enriched splenocytes, also restored the TNF-alpha-induced expression of VCAM-1 in SCID mice to wild-type levels. These results implicate circulating T lymphocytes as modulators of the increased ECAM expression elicited by TNF-alpha.