Sanjeev K. Sahni

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.

Nuclear Factor κB Protects against Host Cell Apoptosis during Rickettsia rickettsii Infection by Inhibiting Activation of Apical and Effector Caspases and Maintaining Mitochondrial Integrity

ABSTRACT Apoptotic host cell death is a critical determinant in the progression of microbial infections and outcome of resultant diseases. The potentially fatal human infection caused by Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, involves the vascular endothelium of various organ systems of the host. Earlier studies have shown that survival of endothelial cells (EC) during this infection depends on their ability to activate the transcription factor nuclear factor κB (NF-κB). Here, we investigated the involvement of caspase cascades and associated signaling pathways in regulation of host cell apoptosis by NF-κB. Infection of cultured human EC with R. rickettsii with simultaneous inhibition of NF-κB induced the activation of apical caspases 8 and 9 and also the executioner enzyme, caspase 3, whereas infection alone had no significant effect. Inhibition of either caspase-8 or caspase-9 with specific cell-permeating peptide inhibitors caused a significant decline in the extent of apoptosis, confirming their importance. The peak caspase-3 activity occurred at 12 h postinfection and led to cleavage of poly(ADP-ribose) polymerase, followed by DNA fragmentation and apoptosis. However, the activities of caspases 6 and 7, other important downstream executioners, remained unchanged. Caspase-9 activation was mediated through the mitochondrial pathway of apoptosis, as evidenced by loss of transmembrane potential and cytoplasmic release of cytochrome c. These findings suggest that activation of NF-κB is required for maintenance of mitochondrial integrity of host cells and protection against infection-induced apoptotic death by preventing activation of caspase-9- and caspase-8-mediated pathways. Targeted inhibition of NF-κB may therefore be exploited to enhance the clearance of infections with R. rickettsii and other intracellular pathogens with similar survival strategies.

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.

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.

NF-κB Activation during Rickettsia rickettsii Infection of Endothelial Cells Involves the Activation of Catalytic IκB Kinases IKKα and IKKβ and Phosphorylation-Proteolysis of the Inhibitor Protein IκBα

ABSTRACT Rocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacterium Rickettsia rickettsii, is associated with widespread infection of the vascular endothelium. R. rickettsii infection induces a biphasic pattern of the nuclear factor-κB (NF-κB) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h. To elucidate the underlying mechanisms, we investigated the expression of NF-κB subunits, p65 and p50, and IκB proteins, IκBα and IκBβ. The transcript and protein levels of p50, p65, and IκBβ remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IκBα at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IκBα mRNA. The level of IκBα mRNA gradually returned toward baseline, whereas that of total IκBα protein remained lower than the corresponding controls. The activities of IKKα and IKKβ, the catalytic subunits of IκB kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected and R. rickettsii-infected ECs, revealed significant increases at 2 h after infection. The activation of IKK and early phase of NF-κB response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae. The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKKα and IKKβ, leading to attenuation of nuclear translocation of NF-κB. Also, increased activity of IKKα was evident later during the infection, coinciding with the late phase of NF-κB activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R. rickettsii-induced NF-κB activation. Since NF-κB is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.

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.

Activation of p38 stress‐activated protein kinase during Rickettsia rickettsii infection of human endothelial cells: role in the induction of chemokine response

Rickettsia rickettsii, a Gram‐negative and obligate intracellular bacterium, preferentially infects the vascular endothelium during human infections leading to inflammation and dysfunction. The aim of this study was to determine whether R. rickettsii infection of endothelial cells (EC) activates p38 and/or c‐jun N‐terminal kinases (JNK) mitogen‐activated protein (MAP) kinase, key regulatory proteins that control the response to inflammatory stimuli. We show that infection of cultured human EC results in the dose‐dependent activation of p38, as assessed by increased phosphorylation and activity, without affecting the status of JNK. Rickettsia inactivation by heat or formaldehyde abolished the activation of p38 kinase and inhibition of cellular invasion by infection at low temperature, pre‐treatment of host EC with cytochalasin D, or pre‐incubation of rickettsiae with an irreversible phospholipase inhibitor led to a diminished p38 phosphorylation, suggesting requirement of invasion by viable rickettsiae for this host cell response. SB 203580, a p38‐specific inhibitor, had no effect on infection‐induced activation of the ubiquitous transcriptional regulator nuclear factor‐kappa B, but effectively reduced the expression and secretion of important chemoattractant cytokines interleukin (IL)‐8 and monocyte chemoattractant protein (MCP)‐1 by R. rickettsii‐infected EC. Selective inhibition of p38 activity may be exploited as an anti‐inflammatory target to prevent rickettsial vasculitis and to develop new and improved chemotherapeutic agents.

Rickettsia rickettsii Infection of Human Macrovascular and Microvascular Endothelial Cells Reveals Activation of Both Common and Cell Type-Specific Host Response Mechanisms

ABSTRACT Although inflammation and altered barrier functions of the vasculature, due predominantly to the infection of endothelial cell lining of small and medium-sized blood vessels, represent salient pathological features of human rickettsioses, the interactions between pathogenic rickettsiae and microvascular endothelial cells remain poorly understood. We have investigated the activation of nuclear transcription factor-kappa B (NF-κB) and p38 mitogen-activated protein (MAP) kinase, expression of heme oxygenase 1 (HO-1) and cyclooxygenase 2 (COX-2), and secretion of chemokines and prostaglandins after Rickettsia rickettsii infection of human cerebral, dermal, and pulmonary microvascular endothelial cells in comparison with pulmonary artery cells of macrovascular origin. NF-κB and p38 kinase activation and increased HO-1 mRNA expression were clearly evident in all cell types, along with relatively similar susceptibility to R. rickettsii infection in vitro but considerable variations in the intensities/kinetics of the aforementioned host responses. As expected, the overall activation profiles of macrovascular endothelial cells derived from human pulmonary artery and umbilical vein were nearly identical. Interestingly, cerebral endothelial cells displayed a marked refractoriness in chemokine production and secretion, while all other cell types secreted various levels of interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to infection. A unique feature of all microvascular endothelial cells was the lack of induced COX-2 expression and resultant inability to secrete prostaglandin E2 after R. rickettsii infection. Comparative evaluation thus yields the first experimental evidence for the activation of both common and unique cell type-specific host response mechanisms in macrovascular and microvascular endothelial cells infected with R. rickettsii, a prototypical species known to cause Rocky Mountain spotted fever in humans.