Abha Sahni

Heparin inhibition of endothelial cell proliferation and organization is dependent on molecular weight.

Objective—Studies have shown improved survival in cancer patients treated with low molecular weight heparins (LMWHs). Tumors depend on an expanding vasculature, and heparins may affect vessel growth and function. We investigated the effect of heparins differing in Mr on selected endothelial cell properties. Methods and Results—Human umbilical vein endothelial cells were cultured with fibroblast growth factor-2 and heparins differing in Mr. Cell proliferation was assessed by [3H]thymidine incorporation, and vascular organization was assessed by in vitro assays. Maximum inhibition of 94±2% was observed with 6-kDa LMWH, greater than the inhibition seen with unfractionated heparin (58±8%) or 3-kDa LMWH (60±9%, P =0.02 for both). No inhibition of proliferation was observed with heparin tetrasaccharide, octasaccharide, or pentasaccharide (fondaparinux). Three- and 6-kDa fractions decreased endothelial tube formation in Matrigel to 58±15% and 67±9% (P <0.05), respectively, of that with fibroblast growth factor-2, whereas no inhibition was observed with unfractionated heparin, tetrasaccharide, pentasaccharide, or octasaccharide. LMWH (6 kDa) also inhibited vessel formation in a placental explant. Conclusions—Heparin inhibition of endothelial cell proliferation and organization requires a chain length of >8 saccharide units, with maximal inhibition at Mr of 6 kDa. This Mr dependence differs from that required for anticoagulant activity.

Fibrinogen synthesized by cancer cells augments the proliferative effect of fibroblast growth factor-2 (FGF-2).

Summary.  Background: Fibroblast growth factor (FGF)‐2 is a critical growth factor in normal and malignant cell proliferation and tumor‐associated angiogenesis. Fibrinogen and fibrin bind to FGF‐2 and modulate FGF‐2 functions. Furthermore, we have shown that extrahepatic epithelial cells are capable of endogenous production of fibrinogen.

Potentiation of Endothelial Cell Proliferation by Fibrin(ogen)-bound Fibroblast Growth Factor-2

Endothelial cell growth is stimulated by fibroblast growth factor-2 (FGF-2), and both adhesion and proliferation are modulated by interactions with fibrinogen and fibrin. Previous evidence indicates that FGF-2 binds specifically and with high affinity to fibrinogen and fibrin, suggesting that their effects on endothelial cells may be coordinated. In this study, we have, therefore, investigated the ability of FGF-2 bound to fibrinogen and fibrin to stimulate proliferation of endothelial cells. Human umbilical vein endothelial cells were cultured in the presence of FGF-2 with or without fibrinogen, and proliferation was assessed by microscopic examination of cultures, incorporation of [3H]thymidine and by cell counting. Cells cultured in the presence of both FGF-2 and fibrinogen proliferated more rapidly than those with FGF-2 alone and exhibited a decreased population doubling time. At concentrations of FGF-2 up to 150 ng/ml, there was greater endothelial cell proliferation in the presence of fibrinogen than in its absence with the most pronounced effect below 1 ng/ml. The maximum effect of fibrinogen was observed at a molar ratio of fibrinogen to FGF-2 of 2:1, corresponding to the maximum molar binding ratio. Endothelial cells proliferated when plated on fibrin or surface-immobilized fibrinogen with FGF-2, indicating that FGF-2 bound to surface-associated fibrin(ogen) retained activity. We conclude that fibrinogen- or fibrin-bound FGF-2 is able to support endothelial cell proliferation and that fibrinogen potentiates the proliferative capacity of FGF-2.

FGF-2 but not FGF-1 binds fibrin and supports prolonged endothelial cell growth

Summary.  Endothelial cell viability and growth are dependent on both polypeptide growth factors, and integrin‐mediated matrix interactions. We have now examined the ability of fibrin‐binding and non‐binding growth factors to support long‐term endothelial cell growth in the presence or absence of the soluble form. Endothelial cells were cultured on a fibrin surface, with or without FGF‐1 or FGF‐2, and proliferation was determined by 3H‐thymidine incorporation. Cells cultured on fibrin with no growth factor showed minimal proliferation up to 96 h. In contrast, when FGF‐2 was incorporated into fibrin, proliferation was increased 6.5 ± 0.6‐fold, equal to growth on a fibrin surface with FGF‐2 continually present in the medium. Thymidine incorporation was similar when cells were cultured on a fibrin surface that had been incubated with FGF‐2 and then the growth factor removed (8.6 ± 0.5‐fold). In contrast to results with FGF‐2, a surface of fibrin exposed to FGF‐1 supported minimal growth, whereas growth was comparable to either FGF‐1 or FGF‐2 present in the medium. Comparable results were observed when proliferation was quantitated by cell counting at times up to 48 h. Binding studies demonstrated no high‐affinity interaction of FGF‐1 with fibrinogen or fibrin. We conclude that FGF‐2 bound to fibrin supports prolonged endothelial cell growth as well as soluble FGF‐2, whereas FGF‐1 does not bind to fibrin and can support endothelial cell growth only if continually present in soluble form. Fibrin may serve as a matrix reservoir for FGF‐2 to support cell growth at sites of injury or thrombosis.

The VE-cadherin binding domain of fibrinogen induces endothelial barrier permeability and enhances transendothelial migration of malignant breast epithelial cells.

Fibrin deposition and exudation of plasma fibrinogen (Fg) have long been recognized as hallmarks of inflammation, cardiovascular disease and neoplasia. The Fg‐β15–42 domain binds to the endothelial cell adhesion molecule, VE‐cadherin, promoting endothelial cell proliferation, angiogenesis and leukocyte diapedesis. Furthermore, spontaneous blood‐borne and lymphatic metastasis of some types of tumor emboli requires plasma fibrin(ogen); however, the molecular mechanisms by which this occurs are poorly understood. We sought to determine whether Fg‐β15–42 and VE‐cadherin binding interactions promote endothelial barrier permeability and breast cancer cell transendothelial migration (TEM) using transwell insert culture systems. Synthetic peptides containing/missing residues β15–17 critical for Fg‐β15–42 binding to VE‐cadherin, and antibodies that bind to Fg‐β15–21 (T2G1) and VE‐cadherin (BV9) were used to induce or inhibit Fg‐mediated permeability and TEM. Fg induced dose‐dependent permeability of human umbilical vein and microvascular endothelial but not epithelial cell barriers. Maximal Fg‐induced endothelial permeability required Fg‐β15–42 and VE‐cadherin‐binding interactions involving Fg‐β15–17. Fg‐induced TEM of malignant MDA‐MB‐231 and MCF‐7 breast cancer cells also required Fg‐β15–42 and VE‐cadherin binding; however, such TEM was independent of E‐cadherin or estrogen receptor expression. In contrast, Fg did not induce TEM of nonmalignant MCF‐10A breast epithelial cells. Fg‐induced endothelial permeability was retained in the presence of MDA‐MB‐231 but inhibited in the presence of MCF‐10A cells. It is intriguing to speculate that loss of Fg‐β15–42 binding by premalignant breast epithelial cells serves as a molecular switch to induce a highly aggressive, metastatic breast cancer phenotype. Hence, Fg‐β15–42 represents a potential molecular target for therapeutic intervention of breast cancer metastasis.

Rickettsia rickettsii Infection of Cultured Human Endothelial Cells Induces Heme Oxygenase 1 Expression

ABSTRACT Existing evidence suggests that oxidative insults and antioxidant defense mechanisms play a critical role in the host cell response during infection of endothelial cells by Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. Heme oxygenase (HO), a rate-limiting enzyme in the pathway for heme catabolism, protects against oxidant damage in a variety of stress situations. Here, we report on the expression of the inducible and constitutive HO isozymes, HO-1 and HO-2, during R. rickettsii infection of endothelial cells. Steady-state levels for HO-1 mRNA were increased two- to threefold, as early as 4 h postinfection, whereas HO-2 mRNA was not affected. Induction of HO-1 mRNA was dependent on the dose of infection and occurred in a time-dependent manner, reaching maximal levels at 4 to 7 h. The increase in HO-1 mRNA occurred at the level of trancription as it was blocked by the transcriptional inhibitors, actinomycin D and α-amanitin. The eukaryotic protein synthesis inhibitor, cycloheximide, caused a >50% reduction in the infection-induced increase in HO-1 mRNA level, suggesting its dependence on de novo protein synthesis of host cell. The uptake of viable organisms appeared to be necessary, since inactivation of R. rickettsii by heat or formalin fixation, or incubation of cells with cytochalasin B to prevent entry resulted in marked inhibition of HO-1 response. N-Acetyl-l-cysteine, a known oxidant scavenger, inhibited the HO-1 induction by R. rickettsii. Finally, Western analysis with a specific monoclonal antibody revealed higher levels of HO-1 protein (∼32 kDa), confirming that changes in HO-1 mRNA levels were followed by increases in the levels of protein. The findings indicate that R. rickettsii infection induces HO-1 expression in host endothelial cells and suggest an important role for this enzyme in cellular response to infection, possibly by serving a protective function against oxidative injury.

Bcr Kinase Activation by Angiotensin II Inhibits Peroxisome Proliferator-Activated Receptor γ Transcriptional Activity in Vascular Smooth Muscle Cells

Bcr is a serine/threonine kinase activated by platelet-derived growth factor that is highly expressed in the neointima after vascular injury. Here, we demonstrate that Bcr is an important mediator of angiotensin (Ang) II and platelet-derived growth factor–mediated inflammatory responses in vascular smooth muscle cells (VSMCs). Among transcription factors that might regulate Ang II–mediated inflammatory responses we found that ligand-mediated peroxisome proliferator-activated receptor (PPAR)&ggr; transcriptional activity was significantly decreased by Ang II. Ang II increased Bcr expression and kinase activity. Overexpression of Bcr significantly inhibited PPAR&ggr; activity. In contrast, knockdown of Bcr using Bcr small interfering RNA and a dominant-negative form of Bcr (DN-Bcr) reversed Ang II–mediated inhibition of PPAR&ggr; activity significantly, suggesting the critical role of Bcr in Ang II–mediated inhibition of PPAR&ggr; activity. Point-mutation and in vitro kinase analyses showed that PPAR&ggr; was phosphorylated by Bcr at serine 82. Overexpression of wild-type Bcr kinase did not inhibit ligand-mediated PPAR&ggr;1 S82A mutant transcriptional activity, indicating that Bcr regulates PPAR&ggr; activity via S82 phosphorylation. DN-Bcr and Bcr small interfering RNA inhibited Ang II–mediated nuclear factor &kgr;B activation in VSMCs. DN-PPAR&ggr; reversed DN-Bcr–mediated inhibition of nuclear factor &kgr;B activation, suggesting that PPAR&ggr; is downstream from Bcr. Intimal proliferation in low-flow carotid arteries was decreased in Bcr knockout mice compared with wild-type mice, suggesting the critical role of Bcr kinase in VSMC proliferation in vivo, at least in part, via regulating PPAR&ggr;/nuclear factor &kgr;B transcriptional activity.

Infection of Human Endothelial Cells with Spotted Fever Group Rickettsiae Stimulates Cyclooxygenase 2 Expression and Release of Vasoactive Prostaglandins

ABSTRACT Rickettsiae, a diverse group of obligately intracellular gram-negative bacteria, include etiologic agents of the spotted fever and typhus groups of diseases. Rocky Mountain spotted fever and boutonneuse fever, due to Rickettsia rickettsii and R. conorii, respectively, are characterized by widespread infection of the vascular endothelium, microvascular injury, and vasculitis. Cultured human endothelial cells (EC) are highly susceptible to infection and respond by altering the expression of adhesion molecules, regulatory cytokines, and the antioxidant enzyme heme oxygenase (HO). In the vasculature, HO regulates the cyclooxygenase (COX) enzymes, among which the inducible isozyme COX-2 facilitates the synthesis of prostaglandins (PGs). Using in vitro and ex vivo models of infection, we demonstrate here that R. rickettsii infection of human EC causes robust induction of COX-2 mRNA and protein expression but has no apparent effect on the constitutive COX-1 isoform. Cells infected with viable rickettsiae consistently displayed significantly increased secretion of 6-keto-PGF1α and PGE2. R. rickettsii-induced COX-2 was sensitive to inhibitors of de novo transcription and the pyridinylimidazole-based compound SB 203580, suggesting that this transcriptional host cell response involves signaling through p38 mitogen-activated protein kinase. PG production by infected cells was abrogated by NS 398 (a selective COX-2 inhibitor) and indomethacin (a pan-COX inhibitor). Immunohistochemical staining of sections of infected umbilical cords and corresponding uninfected controls revealed comparatively more intense and abundant staining for COX-2 in infected endothelia. Induction of the endothelial COX-2 system and the resultant enhanced release of vasoactive PGs may contribute to the regulation of inflammatory responses and vascular permeability changes during spotted fever rickettsioses.

Fibrinogen regulates the expression of inflammatory chemokines through NF-κB activation of endothelial cells

The objective of this study was to characterize the role of fibrinogen in stimulating expression of inflammatory chemokines in endothelial cells through NF-kappaB activation. Human umbilical vein endothelial cells (HUVEC) were exposed to fibrinogen up to 3,000 microg/ml, and NF-kappaB activation was assessed using electrophoretic mobility shift assay (EMSA). Fibrinogen exposure resulted in a concentration dependent increase in NF-kappaB activation that reached a maximum at 1,000 microg/ml after 4 hours and was sustained up to 24 hours. The effect was inhibited by antibodies to alpha(v)beta(3) and alpha(5)beta(1) and by the GRGDS peptide, indicating integrin involvement. Preincubation with Mn(2+) lowered the fibrinogen concentration-dependence, consistent with integrin activation. Supershift assays demonstrated involvement of the p50, p65 and c-Rel components of NF-kappaB. Fibrinogen exposure also resulted in up-regulation of expression of monocyte chemoattractant protein-1 (MCP-1) and of interleukin-8 as shown by RNase protection assays and by real-time RT-PCR. Increased secretion of MCP-1 was confirmed by ELISA. Parthenolide, an IkappaB kinase inhibitor, prevented up-regulation of MCP-1 by fibrinogen, linking this response to NF-kappaB activation. From our findings, we conclude that fibrinogen regulates NF-kappaB activation and expression of inflammatory chemokines in endothelial cells and may be involved in mediating inflammatory processes.

Fibrinogen binding potentiates FGF‐2 but not VEGF induced expression of u‐PA, u‐PAR, and PAI‐1 in endothelial cells

Summary.   Endothelial cell responses at sites of injury occur in a fibrin matrix and are regulated by growth factors including those of the FGF and VEGF families. The pericellular proteolytic balance is important in these responses, and FGF‐2 and VEGF up‐regulate endothelial cell u‐PA, u‐PAR and PAI‐1. Because both VEGF and FGF‐2 bind to fibrinogen, we have examined the capacity of fibrinogen to modulate the up‐regulation of these proteins by FGF‐2 and VEGF. Confluent cultures of endothelial cells were exposed to FGF‐2, VEGF, and fibrinogen or to combinations of growth factors with fibrinogen. Changes in mRNA levels of u‐PA, u‐PAR and PAI‐1 were measured by Northern blot. FGF‐2 increased u‐PA, u‐PAR, and PAI‐1 mRNA, but there was a significantly greater induction when fibrinogen was added to FGF‐2 at all concentrations. The potentiation by fibrinogen was particularly evident at an FGF‐2 concentration of 0.1 ng mL−1, which resulted in non‐significant change in transcript levels by itself, but significantly increased up to 2.6‐fold with fibrinogen. VEGF also increased endothelial cell expression of u‐PA, u‐PAR and PAI‐1, but this effect was not potentiated by fibrinogen. Addition of LM609, a monoclonal antibody to αVβ3, significantly inhibited induction of u‐PA mRNA and activity by fibrinogen–bound FGF‐2 compared to FGF‐2. A monoclonal antibody to FGFR1 also inhibited u‐PA mRNA expression induced by fibrinogen‐bound FGF‐2. We conclude that fibrinogen increases the capacity of FGF‐2, but not of VEGF, to up‐regulate u‐PA, u‐PAR, and PAI‐1 in endothelial cells and that fibrinogen‐bound FGF‐2 requires αVβ3 binding to up‐regulate endothelial cell u‐PA.