The antiangiogenic 16K prolactin impairs functional tumor neovascularization by inhibiting vessel maturation

Background

Angiogenesis, the formation of new blood vessels from existing vasculature, plays an essential role in tumor growth, invasion, and metastasis. 16K hPRL, the antiangiogenic 16-kDa N-terminal fragment of human prolactin was shown to prevent tumor growth and metastasis by modifying tumor vessel morphology.

Methodology/Principal Findings

Here we investigated the effect of 16K hPRL on tumor vessel maturation and on the related signaling pathways. We show that 16K hPRL treatment leads, in a murine B16-F10 tumor model, to a dysfunctional tumor vasculature with reduced pericyte coverage, and disruption of the PDGF-B/PDGFR-B, Ang/Tie2, and Delta/Notch pathways. In an aortic ring assay, 16K hPRL impairs endothelial cell and pericyte outgrowth from the vascular ring. In addition, 16K hPRL prevents pericyte migration to endothelial cells. This event was independent of a direct inhibitory effect of 16K hPRL on pericyte viability, proliferation, or migration. In endothelial cell-pericyte cocultures, we found 16K hPRL to disturb Notch signaling.

Conclusions/Significance

Taken together, our data show that 16K hPRL impairs functional tumor neovascularization by inhibiting vessel maturation and for the first time that an endogenous antiangiogenic agent disturbs Notch signaling. These findings provide new insights into the mechanisms of 16K hPRL action and highlight its potential for use in anticancer therapy.     

The antiangiogenic factor 16K PRL induces programmed cell death in endothelial cells by caspase activation

We asked whether the antiangiogenic action of 16K human PRL (hPRL), in addition to blocking mitogen-induced vascular endothelial cell proliferation, involved activation of programmed cell death. Treatment with recombinant 16K hPRL increased DNA fragmentation in cultured bovine brain capillary endothelial (BBE) and human umbilical vein endothelial (HUVE) cells in a time- and dose-dependent fashion, independent of the serum concentration. The activation of apoptosis by 16K hPRL was specific for endothelial cells, and the activity of the peptide could be inhibited by heat denaturation, trypsin digestion, and immunoneutralization, but not by treatment with the endotoxin blocker, polymyxin-B. 16K hPRL-induced apoptosis was correlated with the rapid activation of caspases 1 and 3 and was blocked by pharmacological inhibition of caspase activity. Caspase activation was followed by inactivation of two caspase substrates, poly(ADP-ribose) polymerase (PARP) and the inhibitor of caspase-activated deoxyribonuclease (DNase) (ICAD). Furthermore, 16K hPRL increased the conversion of Bcl-X to its proapoptotic form, suggesting that the Bcl-2 protein family may also be involved in 16K hPRL-induced apoptosis. These findings support the hypothesis that the antiangiogenic action of 16K hPRL includes the activation of programmed cell death of vascular endothelial cells.     

Down-regulation of cell adhesion molecules LFA-1 and ICAM-1 after in vitro treatment with the anti-TNF-alpha agent thalidomide.

The tumor necrosis factor-alpha (TNF-alpha) inhibitor thalidomide is known to be a potent modulator of host immunity, a potential treatment for autoimmune disorders such as rheumatoid arthritis (RA) and a treatment for complications of HIV-1 infection. RA is an autoimmune disease of the joints that has been associated with hyperactivity of lymphocytes and other leukocytes, over-expression of pro-inflammatory cytokines (TNF-alpha and IL-1) and chronic debilitating inflammation. Thalidomide may play a role in RA treatment by altering leukocyte function through down-modulation of cell adhesion molecules necessary for leukocyte migration to inflammatory sites. The present study investigates down-regulation of cell adhesion molecules (ICAM-1 and LFA-1) and decreases in cell-cell contacts between human T leukemic (CEM) cells and human umbilical vein endothelial cells (HUVEC) after thalidomide exposure. CEM cells were cultured in RPMI 1640 medium with 0, 10 or 50 microg/ml thalidomide, stained with fluorescent monoclonal antibodies specific to ICAM-1 and LFA-1 and expression was measured with flow cytometry. For cell-cell adhesion measurements, monolayers of HUVEC cultured in Kaign's F-12 medium were incubated with thalidomide treated CEM cells stained with calcein AM. Specific cell adhesion between the two cell types was visualized with fluorescence microscopy. Thalidomide treatment significantly reduced cell adhesion molecule expression in a dose-dependent fashion and inhibited HUVEC/CEM cell adhesion. These data support the hypothesis that thalidomide has modulatory actions on leukocyte functions through expression of cell adhesion molecules.     

The antiangiogenic factor 16K human prolactin induces caspase-dependent apoptosis by a mechanism that requires activation of nuclear factor-kappaB

We have previously shown that the 16-kDa N-terminal fragment of human prolactin (16K hPRL) has antiangiogenic properties, including the ability to induce apoptosis in vascular endothelial cells. Here, we examined whether the nuclear factor-kappaB (NF-kappaB) signaling pathway was involved in mediating the apoptotic action of 16K hPRL in bovine adrenal cortex capillary endothelial cells. In a dose-dependent manner, treatment with 16K hPRL induced inhibitor kappaB-alpha degradation permitting translocation of NF-kappaB to the nucleus and reporter gene activation. Inhibition of NF-kappaB activation by overexpression of a nondegradable inhibitor kappaB-alpha mutant or treatment with NF-kappaB inhibitors blocked 16K hPRL-induced apoptosis. Treatment with 16K hPRL activated the initiator caspases-8 and -9 and the effector caspase-3, all of which were essential for stimulation of DNA fragmentation. This activation of the caspase cascade by 16K hPRL was also NF-kappaB dependent. These findings support the conclusion that NF-kappaB signaling plays a central role in 16K hPRL-induced apoptosis in vascular endothelial cells.     

Cathepsin D processes human prolactin into multiple 16K-like N-terminal fragments: study of their antiangiogenic properties and physiological relevance

16K prolactin (PRL) is the name given to the 16-kDa N-terminal fragment obtained by proteolysis of rat PRL by tissue extracts or cell lysates, in which cathepsin D was identified as the candidate protease. Based on its antiangiogenic activity, 16K PRL is potentially a physiological inhibitor of tumor growth. Full-length human PRL (hPRL) was reported to be resistant to cathepsin D, suggesting that antiangiogenic 16K PRL may be physiologically irrelevant in humans. In this study, we show that hPRL can be cleaved by cathepsin D or mammary cell extracts under the same conditions as described earlier for rat PRL, although with lower efficiency. In contrast to the rat hormone, hPRL proteolysis generates three 16K-like fragments, which were identified by N-terminal sequencing and mass spectrometry as corresponding to amino acids 1-132 (15 kDa), 1-147 (16.5 kDa), and 1-150 (17 kDa). Biochemical and mutagenetic studies showed that the species-specific digestion pattern is due to subtle differences in primary and tertiary structures of rat and human hormones. The antiangiogenic activity of N-terminal hPRL fragments was assessed by the inhibition of growth factor-induced thymidine uptake and MAPK activation in bovine umbilical endothelial cells. Finally, an N-terminal hPRL fragment comigrating with the proteolytic 17-kDa fragment was identified in human pituitary adenomas, suggesting that the physiological relevance of antiangiogenic N-terminal hPRL fragments needs to be reevaluated in humans.     

Cyclosporine A enhances leukocyte binding by human intestinal microvascular endothelial cells through inhibition of p38 MAPK and iNOS. Paradoxical proinflammatory effect on the microvascular endothelium

The calcineurin inhibitor cyclosporine A (CsA) modulates leukocyte cytokine production but may also effect nonimmune cells, including microvascular endothelial cells, which regulate the inflammatory process through leukocyte recruitment. We hypothesized that CsA would promote a proinflammatory phenotype in human intestinal microvascular endothelial cells (HIMEC), by inhibiting inducible nitric-oxide synthase (iNOS, NOS2)-derived NO, normally an important mechanism in limiting endothelial activation and leukocyte adhesion. Primary cultures of HIMEC were used to assess CsA effects on endothelial activation, leukocyte interaction, and the expression of iNOS as well as cell adhesion molecules. CsA significantly increased leukocyte binding to activated HIMEC, but paradoxically decreased endothelial expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1). In contrast, CsA completely inhibited the expression of iNOS in tumor necrosis factor-alpha/lipopolysaccharide-activated HIMEC. CsA blocked p38 MAPK phosphorylation in activated HIMEC, a key pathway in iNOS expression, but failed to inhibit NFkappaB activation. These studies demonstrate that CsA exerts a proinflammatory effect on HIMEC by blocking iNOS expression. CsA exerts a proinflammatory effect on the microvascular endothelium, and this drug-induced endothelial dysfunction may help explain its lack of efficacy in the long-term treatment of chronically active inflammatory bowel disease.     

Tumor cell surface alpha 4 beta 1 integrin mediates adhesion to vascular endothelium: demonstration of an interaction with the N-terminal domains of INCAM-110/VCAM-1.

Hematogenous metastasis involves adhesive interactions between blood-borne tumor cells and the vessel wall. By the use of in vitro assays, the adhesion of human melanoma, osteosarcoma, and kidney carcinoma (but not colon carcinoma) cell lines was shown to involve the cytokine-inducible endothelial cell surface protein inducible cell adhesion molecule 110 (INCAM-110) and the alpha 4 beta 1 integrin, molecules normally involved in endothelial-leukocyte interactions. Tumor adhesion to human endothelial cell monolayers was increased 1.9- to 8.2-fold by endothelial activation with the cytokine tumor necrosis factor (TNF) and inhibited by the anti-INCAM-110 monoclonal antibody (mAb) E1/6. Each of these tumor cells expressed members of the beta 1 integrin family of adhesion molecules, and antibodies to the alpha 4 and beta 1 integrin subunits inhibited tumor-endothelial adhesion (48-87% inhibition). A cDNA encompassing the three N-terminal Ig-like domains of vascular cell adhesion molecule 1 (VCAM-1) encoded a protein recognized by the anti-INCAM-110 mAb E1/6 and, when captured onto plastic, supported melanoma cell adhesion by an alpha 4 integrin-dependent mechanism. In contrast to mAb E1/6, a second anti-INCAM-110 mAb Hu8/4 neither inhibited adhesion to activated endothelium nor bound the first three Ig-like domains of INCAM-110/VCAM-1. These data indicate that the adherence of several human tumors to activated endothelium is mediated by an interaction of alpha 4 beta 1 integrin and the N-terminal Ig-like domains of endothelial INCAM-110/VCAM-1. Tumor acquisition of the alpha 4 integrin subunit and endothelial expression of INCAM-110 may affect the frequency and distribution of metastasis.     

The inhibitory effect of alacepril, an angiotensin-converting enzyme inhibitor, on endothelial inflammatory response induced by oxysterol and TNF-alpha

The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-alpha. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-alpha-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-alpha. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-alpha.     

Studies on the mechanisms responsible for inhibition of experimental metastasis of B16-F10 murine melanoma by pentoxifylline

Pentoxifylline (PTX), a methylxanthine derivative widely used as a hemorheological agent in the treatment of peripheral vascular disease, was studied to unveil the mechanisms responsible for its inhibitory action on B16-F10 experimental metastasis. In vitro pretreatment of B16-F10 cells with noncytotoxic concentrations of PTX significantly inhibited their adhesion to reconstituted basement membrane Matrigel® and type IV collagen as well as the relative activity of secreted 92 kD metalloproteinase. However, PTX pretreatment of B16-F10 cells did not affect their in vitro invasiveness. Heterotypic organ adhesion assays carried out with B16-F10 cells and suspended organ tissues demonstrated that pretreatment with noncytotoxic concentrations of PTX of both, tumor cells or lung tissue, brought about a dose-dependent inhibition of melanoma cell adhesion to lung. Immunohistochemical studies using antibodies against CD31 adhesion molecule (PECAM-1) revealed that B16-F10 cells adhere to lung endothelial cells. Our results suggest that PTX may exert its inhibitory effect on tumor lodgment, and as a consequence of that on experimental metastases, through an inhibitory action on cell adhesion molecules.     

Expression level of neural cell adhesion molecule (NCAM) inversely correlates with the ability of neuroblastoma cells to adhere to endothelium in vitro

The precise function of cell adhesion molecules in the hematogenous phase of neuroblastoma metastasis is poorly understood. The aim of this study was to investigate whether neural cell adhesion molecule (NCAM) modulates neuroblastoma cell (NB) adhesion and transendothelial penetration in a coculture model. Our data, assessed on 11 NB cell lines, demonstrate an inverse correlation between NCAM expression and NB cell adhesion. Transfection of the NB cell line UKF-NB-4 with a cDNA encoding the human NCAM-140 kD isoform enhanced NCAM expression and the amount of tumor cell aggregates, reduced the amount of single tumor cells, and diminished initial NB cell adhesion to an endothelial cell monolayer. Treatment of UKF-NB-4 with NCAM antisense oligonucleotides reduced NCAM surface level, increased the number of single tumor cells, and induced up-regulation of NB cell adhesion to endothelium. Modulation of NCAM expression had no effect on transendothelial penetration. Fluorescence analysis revealed a down-regulation of NCAM in single tumor cells, prior to NB adhesion. The data support the view that low levels of NCAM are necessary for NB cells to leave a tumor cell aggregate and adhere to endothelial cells.     

1,4-dihydroxyxanthone modulates the adhesive property of endothelial cells by inhibiting intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin

Cell adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1) and E-selectin, play important roles in the recruitment of leukocytes to the site of inflammation. Blocking the expression of these molecules or preventing their interaction with the receptors has been shown to be important in controlling various inflammatory diseases. These cell adhesion molecules are induced on endothelial cells by various proinflammatory cytokines like IL-1beta and TNF-alpha and also by bacterial LPS. We demonstrate here that 1,4-Dihydroxyxanthone (1,4 DHX) inhibits the expression of cell adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin, on endothelial cells in a concentration and time dependent manner. The inhibition by 1,4 DHX is reversible. On further analysis, our results also show that 1,4 DHX inhibits the adhesion of peripheral neutrophils to the endothelial cell monolayers. 1,4 DHX, therefore, could be used as a novel target for controlling various pathological conditions associated with upregulation of endothelial leukocyte adhesion molecules.     

The inhibitory effect of alacepril,an angiotensin-converting enzyme inhibitor,on endothelial inflammatory response induced by oxysterol and TNF-α

Abstract

The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-α. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-α-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-α. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-α.     

Adhesion properties of adhesion-regulating molecule 1 protein on endothelial cells

Numerous adhesion molecules have been described, and the molecular mechanisms of lymphocyte trafficking across the endothelium is starting to be elucidated. Identification of the molecules involved in the organoselectivity of this process would help in the targeting of drug therapy to specific tissues. Adhesion-regulating molecule-1 (ARM-1) is an adhesion-regulating molecule previously identified on T cells. It does not belong to any known families of adhesion molecules. In this study, we show the presence of ARM-1 in endothelial cells, the adhesion partners of lymphocytes. ARM-1 mRNA was found to be differentially expressed in endothelial cell lines of various tissue origin and lymphocyte cell lines. Interestingly, ARM-1 is absent from skin endothelial cells. In our assay, skin endothelial cells display a distinct capacity to mediate adhesion of activated T lymphocytes. Overexpression of ARM-1 in skin endothelial cells increased adhesion of CEMT4 and NK lymphocytes, confirming that ARM-1 also regulates adhesion in endothelial cells. We also show that ARM-1 is a cytosolic protein associated with the plasma membrane. However, no cell surface expression of the protein was observed. These results suggest an indirect role of ARM-1 in adhesion rather than a direct role as an adhesion molecule itself.     

A novel role for interleukin-18 in adhesion molecule induction through NF kappa B and phosphatidylinositol (PI) 3-kinase-dependent signal transduction pathways

Interleukin-18 (IL-18) is a novel proinflammatory cytokine found in serum and joints of patients with rheumatoid arthritis (RA). We studied a novel role for IL-18 in mediating cell adhesion, a vital component of the inflammation found in RA and other inflammatory diseases. We examined the expression of cellular cell adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) on endothelial cells and RA synovial fibroblasts using flow cytometry. Adhesion of the monocyte-like cell line HL-60 to endothelial cells was determined by immunofluorescence. IL-18 significantly enhanced ICAM-1 and VCAM-1 expression on endothelial cells and RA synovial fibroblasts. In addition, IL-18 induced E-selectin expression on endothelial cells and promoted the adhesion of HL-60 cells to IL-18-stimulated endothelial cells. Neutralizing anti-VCAM-1 and anti-E-selectin could completely inhibit HL-60 adherence to endothelial cells. IL-18-induced adhesion molecule expression appears to be mediated through nuclear factor kappa B (NF kappa B) and phosphatidyl-inositol 3 kinase (PI 3-kinase) since addition of inhibitors to either NF kappa B (pyrrolidine dithiocarbamate and N-acetyl-l-cysteine) or PI 3-kinase (LY294002) inhibited RA synovial fibroblast VCAM-1 expression by 50 to 60%. Addition of both inhibitors resulted in inhibition of VCAM-1 expression by 85%. In conclusion, the ability of IL-18 to induce adhesion molecule expression on endothelial cells and RA synovial fibroblasts indicates that IL-18 may contribute to RA joint inflammation by enhancing the recruitment of leukocytes into the joint. IL-18 requires NF kappa B as well as PI 3-kinase to induce VCAM-1 on RA synovial fibroblasts, suggesting that there may be two distinct pathways in IL-18-induced adhesion molecule expression.     

16K human prolactin inhibits vascular endothelial growth factor-induced activation of Ras in capillary endothelial cells.

Signaling pathways mediating the antiangiogenic action of 16K human (h)PRL include inhibition of vascular endothelial growth factor (VEGF)-induced activation of the mitogen-activated protein kinases (MAPK). To determine at which step 16K hPRL acts to inhibit VEGF-induced MAPK activation, we assessed more proximal events in the signaling cascade. 16K hPRL treatment blocked VEGF-induced Raf-1 activation as well as its translocation to the plasma membrane. 16K hPRL indirectly increased cAMP levels; however, the blockade of Raf-1 activation was not dependent on the stimulation of cAMP-dependent protein kinase (PKA), but rather on the inhibition of the GTP-bound Ras. The VEGF-induced tyrosine phosphorylation of the VEGF receptor, Flk-1, and its association with the Shc/Grb2/Ras-GAP (guanosine triphosphatase-activating protein) complex were unaffected by 16K hPRL treatment. In contrast, 16K hPRL prevented the VEGF-induced phosphorylation and dissociation of Sos from Grb2 at 5 min, consistent with inhibition by 16K hPRL of the MEK/MAPK feedback on Sos. The inhibition of Ras activation was paralleled by the increased phosphorylation of 120 kDa proteins comigrating with Ras-GAP. Taken together, these findings show that 16K hPRL inhibits the VEGF-induced Ras activation; this antagonism represents a novel and potentially important mechanism for the control of angiogenesis.     

Inhibitory effects of red wine extracts on endothelial-dependent adhesive interactions with monocytes induced by oxysterols

Red wine polyphenolic compounds have been demonstrated to possess antioxidant properties, and several studies have suggested that they might constitute a relevant dietary factor in the protection from coronary heart disease. The aim of the present study is to examine whether red wine extracts (RWE) can ameliorate oxysterol-induced endothelial response, and whether inhibition of adhesion molecule expression is involved in monocyte adhesion to endothelial cells. Surface expression and mRNA levels of adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1) were determined by ELISA and RT-PCR performed on human aortic endothelial cells (HAEC) monolayers stimulated with 7beta-hydroxycholesterol or 25-hydroxycholesterol. Incubation of HAEC with oxysterols (10 microM) increased expression of adhesion molecules in a time-dependent manner. Pretreatment of HAEC with RWE at final concentrations of 1, 10, and 100 ng/ml significantly inhibited the increase of surface protein expression and mRNA levels. Adherence of monocytes to oxysterol-stimulated HAEC was increased compared to that of unstimulated cells. Treatment of HAEC with RWE significantly inhibited adherence of monocytes. These results suggest that RWE works as an anti-atherogenic agent through the inhibition of endothelial-dependent adhesive interactions with monocytes induced by oxysterols.     

Anti-angiogenesis: making the tumor vulnerable to the immune system

Ongoing angiogenesis has been shown to possess immune suppressive activity through several mechanisms. One of these mechanisms is the suppression of adhesion receptors, such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin-adhesion molecules involved in leukocyte interactions-on the vascular endothelium. This phenomenon, when happening to the tumor endothelium, supports tumor growth due to escape from immunity. Since angiogenesis has this immune suppressive effect, it has been hypothesized that inhibition of angiogenesis may circumvent this problem. In vitro and in vivo data now show that several angiogenesis inhibitors are able to normalize endothelial adhesion molecule expression in tumor blood vessels, restore leukocyte vessel wall interactions, and enhance the inflammatory infiltrate in tumors. It is suggested that such angiogenesis inhibitors can make tumors more vulnerable for the immune system and may therefore be applied to facilitate immunotherapy approaches for the treatment of cancer.