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The nonhuman primate (NHP) has always been a limited resource for pharmaceutical research with ongoing efforts to conserve. This is due to their inherent biological properties, the growth in biotherapeutics and other modalities, and their use in small molecule drug development. The SARS-CoV-2 pandemic has significantly impacted the availability of NHPs due to the immediate need for NHPs to develop COVID-19 vaccines and treatments and the China NHP export ban; thus, accelerating the need to further replace, reduce and refine (3Rs) NHP use. The impact of the NHP shortage on drug development led DruSafe, BioSafe, and the United States (U.S.) Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) to discuss this issue at their 2021 annual meeting. This meeting identified areas to further the 3Rs in NHP use within the current nonclinical safety evaluation regulatory framework and highlighted the need to continue advancing alternative methods towards the aspirational goal to replace use of NHPs in the long term. Alignment across global health authorities is necessary for implementation of approaches that fall outside existing guidelines. This article captures the proceedings from this meeting highlighting current best practices and areas for 3Rs in NHP use.

The development of cancer in humans is caused by irreversible modifications of the genome through genetic or epigenetic alterations resulting in the acquisition of multiple heritable abnormal cellular phenotypes. We have investigated DNA methylation and gene expression alterations in mammary glands of Sprague Dawley rats induced by treatment with the weight-loss drug lorcaserin, a drug that increased the cancer incidence in a human clinical trial (CAMELLIA-TIMI 61) and in rats. Female Sprague Dawley rats received lorcaserin by gavage 7 days/week for 12 weeks, at doses previously used in a 2-year rodent cancer bioassay (0, 30, and 100 mg/kg bw/day). DNA methylation changes were investigated in mammary glands, a target organ for lorcaserin carcinogenicity in rats, by reduced representation bisulfite sequencing. Gene expression changes were analyzed by RNA sequencing. Lorcaserin exposure resulted in dose-dependent DNA methylation alterations in the mammary glands, as evidenced by the presence of 1832 and 2352 significantly differentially methylated CpG (dmCpG) sites in the 30 and 100 mg/kg bw/day treatment groups, respectively, as compared to the control group, among which 593 sites were hypomethylated or hypermethylated in common. Similar to DNA methylation changes, lorcaserin treatment resulted in dose-dependent changes in gene expression. Pathway enrichment analysis of the dmCpG-containing genes demonstrated a strong representation of genes associated with cell morphology, cellular function and maintenance, and cellular growth and proliferation. Analysis of dmCpG sites indicated an increase in the number of hypermethylated sites compared to the number of hypomethylated sites in both 30 (1.9 fold) and 100 (2.6 fold) mg/kg bw/day treatment groups. Furthermore, while the number of hypomethylated CpG sites did not differ between treatment groups, the number of hypermethylated CpG sites in rats treated with 100 mg/kg bw/day of lorcaserin was 1.4 times greater than in rats treated with 30 mg/kg bw/day. By combining the analysis of differentially expressed genes and dmCpG sites, we identified 13 genes (11 hypermethylated and 2 hypomethylated) the expression of which was inversely correlated with DNA methylation in the mammary glands of rats treated with 100 mg/kg bw/day of lorcaserin. We also identified 41 hypermethylated and 17 hypomethylated genes containing more than two dmCpG sites. In summary, we have demonstrated that lorcaserin induces extensive DNA methylation and transcriptomic changes in mammary glands at early preneoplastic stages of lorcaserin-induced rat carcinogenesis. These findings suggest the importance of these non-genotoxic alterations in the carcinogenicity of lorcaserin. Citation Format: Volodymyr P. Tryndyak, Rose A. Willett, Todd Bourcier, Frederick A. Beland, Igor P. Pogribny. Exposure-related DNA methylation and gene expression changes in mammary glands of Sprague Dawley rats treated with lorcaserin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6017.

Nonclinical testing of human pharmaceuticals is conducted to assess the safety of compounds to be studied in human clinical trials and for marketing of new drugs. Although there is no exact number and type of nonclinical studies required for safety assessments, as there is inherent flexibility for each new compound, the traditional approach is outlined in various FDA and ICH guidance documents and involves a combination of in vitro assays and whole animal testing methods. Recent advances in science have led to the emergence of numerous new approach methodologies (NAMs) for nonclinical testing that are currently being used in various aspects of drug development. Traditional nonclinical testing methods can predict clinical outcomes, although improvements in these methods that can increase predictivity of clinical outcomes are encouraged and needed. This paper discusses FDA/CDER's view on the opportunities and challenges of using NAMs in drug development especially for regulatory purposes, and also includes examples where NAMs are currently being used in nonclinical safety assessments and where they may supplement and/or enhance current testing methods. FDA/CDER also encourages communication with stakeholders regarding NAMs and is committed to exploring the use of NAMs to improve regulatory efficiency and potentially expedite drug development.

After evaluating a safety signal regarding pancreatitis and pancreatic cancer in patients using incretin-based drugs, the Food and Drug Administration and the European Medicines Agency conclude that assertions of a causal association are inconsistent with the data. With approximately 25.8 million diabetic patients in the United States and 33 million in the European Union alone, the growing prevalence of diabetes worldwide poses a major public health challenge. Both the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) are committed to ensuring the safety of drug products marketed for the treatment of diabetes, and postmarketing reports of pancreatitis and pancreatic cancer in patients taking certain antidiabetic medications have been of concern to both agencies. Working in parallel, the agencies have reviewed nonclinical toxicology studies, clinical trial data, and epidemiologic data pertaining to blood glucose-lowering ...

The ICH initiated talks in June 2012 to revise regulatory guidance for carcinogenicity assessment of pharmaceutical products, stimulated in part by a proposal called Negative for Endocrine, Genotoxicity, and Chronic Study Associated Histopathologic Risk Factors for Carcinogenicity in the Rat (NEGCARC) from the Pharmaceutical Research and Manufacturing Association (PhRMA). The 2012 STP Town Hall Meeting focused on the need for change in carcinogenicity assessment strategies for pharmaceuticals. Dr. Todd Bourcier from the Division of Endocrine and Metabolic Products, U.S. FDA and a member of the FDA’s Alternative Carcinogenicity Assessment Committee, was the guest speaker and a panelist. Dr. Bourcier is also one of FDA’s representatives to the ICH S1 Expert Working Group that is discussing changes to regulatory guidelines for carcinogenicity assessment. Drs. Carl Alden and Dan Morton also participated in the panel discussion.

Cancer risk assessment is a major part of establishing the safety profile of investigational pharmaceuticals intended for chronic use. Because the nonclinical carcinogenicity assessment program is typically conducted in the later stage of clinical development and requires extensive resources, careful consideration must be given to its design, timing, conduct, interpretation, and reporting in order to protect patients. Positive results in carcinogenicity studies are not uncommon and can have a significant impact on the overall development program. Toxicologists interpreting carcinogenicity results and their relevance to human safety should use a multifaceted approach including statistical and exposure considerations. While for some investigational products the human relevance of a positive result can readily be discounted, others require follow-up assessments in order to characterize their relevance to human risk. Ultimately, even if a positive signal is concluded to be relevant to humans and occur at low or no margins of clinical exposures, its impact on the overall development program is shaped by the clinical indication, targeted population and its demonstrated therapeutic benefits. This chapter discusses some practical factors to consider when establishing whether a carcinogenicity study is ‘positive’, and establishing the path forward by determining the impact of positive findings on patient safety and the overall clinical development program, both from a Sponsor and FDA perspective.

The mannose-binding lectin (MBL), a circulating pattern recognition molecule, recognizes a wide range of infectious agents with resultant initiation of the complement cascade in an Ab-independent manner. MBL recognizes infectious non-self and altered self in the guise of apoptotic and necrotic cells. In this study, we demonstrate that mice lacking MBL, and hence are devoid of MBL-dependent lectin pathway activation but have fully active alternative and classical complement pathways, are protected from cardiac reperfusion injury with resultant preservation of cardiac function. Significantly, mice that lack a major component of the classical complement pathway initiation complex (C1q) but have an intact MBL complement pathway, are not protected from injury. These results suggest that the MBL-dependent pathway of complement activation is a key regulator of myocardial reperfusion ischemic injury. MBL is an example of a pattern recognition molecule that plays a dual role in modifying inflammatory responses to sterile and infectious injury.

Background—Myocardial ischemia and reperfusion-induced tissue injury involve a robust inflammatory response, but the proximal events in reperfusion injury remain incompletely defined. Toll-like receptor 4 (TLR4) is a proximal signaling receptor in innate immune responses to lipopolysaccharide of Gram-negative pathogens. TLR4 is also expressed in the heart and vasculature, but a role for TLR4 in the myocardial response to injury separate from microbial pathogens has not been examined. This study assessed the role of TLR4 in myocardial infarction and inflammation in a murine model of ischemia-reperfusion injury.Methods and Results—Myocardial ischemia-reperfusion (MIR) was performed on 2 strains of TLR4-deficient mice (C57/BL10 ScCr and C3H/HeJ) and controls (C57/BL10 ScSn and C3H/OuJ). Mice were subjected to 1 hour of coronary ligation, followed by 24 hours of reperfusion. TLR4-deficient mice sustained significantly smaller infarctions compared with control mice given similar areas at risk. Fewer neutrophils infiltrated the myocardium of TLR4-deficient Cr mice after MIR, indicated by less myeloperoxidase activity and fewer CD45/GR1-positive cells. The myocardium of TLR4-deficient Cr mice contained fewer lipid peroxides and less complement deposition compared with control mice after MIR. Serum levels of interleukin-12, interferon-γ, and endotoxin were not increased after ischemia-reperfusion. Neutrophil trafficking in the peritoneum was similar in all strains after injection of thioglycollate.Conclusions—TLR4-deficient mice sustain smaller infarctions and exhibit less inflammation after myocardial ischemia-reperfusion injury. The data suggest that in addition to its role in innate immune responses, TLR4 serves a proinflammatory role in murine myocardial ischemia-reperfusion injury.

Strong evidence links cardiomyocyte loss to the pathology of some forms of heart failure. Both necrotic and apoptotic modes of cell death have been invoked as the mechanism underlying progressive cardiomyocyte dropout. Nitric oxide (NO) has received particular attention as a candidate reactive oxygen intermediate that influences not only cardiac function, but also cell death elicited by both apoptotic and necrotic mechanisms. NO is produced by resident cardiac cells under stress, and is produced in large quantities by activated immune cells that infiltrate the injured heart. A review of the literature, however, reveals that the actions of NO on apoptotic cell death are complex, especially in the context of heart disease, and that the practical contribution of NO to cell death in heart disease is yet to be defined.

Growing evidence from patients with heart failure and from experimental animal models implicates effectors of innate immunity in the pathogenesis of this syndrome. The expression of the innate immunity signaling protein, Toll-like receptor 4 (TLR4), is increased in cardiac myocytes in situ and in failing myocardium, but the mechanism by which TLRs may be activated in the failing heart remains unclear. We report that TLR2, which is expressed in cardiac myocytes, participates in the response of these cells to oxidative stress, a major contributor to the pathogenesis of cardiac dysfunction. Hydrogen peroxide increased nuclear factor kappaB (NF-kappaB) activation in Chinese hamster ovary fibroblasts that overexpress TLR2 but not in normal or TLR4-overexpressing Chinese hamster ovary cells, an effect that was abrogated by an alpha-TLR2 antibody. In neonatal rat ventricular myocytes, the alpha-TLR2 antibody inhibited hydrogen peroxide-induced nuclear translocation of NF-kappaB and activator protein-1 (AP-1). Inhibition of TLR2 had no effect on tumor necrosis factor alpha-induced NF-kappaB or AP-1 activation, on the DNA binding of the basal transcription factor Oct-1, or on hydrogen peroxide-induced phosphorylation of p38 MAP kinase. Importantly, oxidative stress-induced cytotoxicity was enhanced by blocking TLR2. Given the importance of cytotoxicity and apoptosis to the pathology of the ischemic heart, an anti-apoptotic effect of TLR2 in cardiac myocytes exposed to elevated levels of ROS may limit further cardiac dysfunction.

In the lesions of atherosclerosis, vascular smooth muscle cells (SMC) display many functions characteristic of cytokine activation that likely contribute importantly to ongoing inflammation during human atherogenesis. The transcription factor nuclear factor kappa-B (NFkappaB) often mediates the effects of cytokines on target cells, but the identity of Rel family members important in human SMC activation remains uncertain. In vitro, human SMC express multiple Rel family members. Of these, dimers of p65 and p50, but not a putative SMC-Rel, comprise basal and inducible NFkappaB binding activities. SMC express two inhibitor proteins IkappaBbeta and IkappaBalpha. Interleukin-1beta stimulation caused transient loss of IkappaBalpha and a sustained decrease of IkappaBbeta that correlated with increased and persistent levels of p65/p50 protein and binding activity in the nucleus. SMC cultured under serum-free conditions displayed little NFkappaB activity, but addition of serum or platelet-derived growth factor did activate NFkappaB. In situ analyses showed no evidence for basal NFkappaB activity in SMC in vivo as nonatherosclerotic arteries did not contain nuclear p65 or p50 protein. However, the nuclei of intimal SMC within human atheroma did contain both Rel proteins. We conclude that (i) dimers of p65 and p50, but not SMC-Rel, comprise NFkappaB complexes in human SMC; (ii) stimulatory components in serum activate NFkappaB and likely account for previously reported "constitutive" NFkappaB activity in cultured SMC; and (iii) exposure to inflammatory cytokines may produce prolonged NFkappaB activation in SMC because of sustained decreases in the inhibitory subunit IkappaB-beta.

A high intake of the omega-3 fatty acid docosahexaenoate [docosahexaenoic acid (DHA)] has been associated with systemic antiinflammatory effects and cardiovascular protection. Cyclooxygenase (COX)-2 is responsible for the overproduction of prostaglandins (PG) at inflammatory sites, and its expression is increased in atheroma. We studied the effects of DHA on COX-2 expression and activity in human saphenous vein endothelial cells challenged with proinflammatory stimuli. Aor=24-h exposure to DHA reduced COX-2 expression and activity induced by IL-1, without affecting COX-1 expression. DHA effect depended on the NF-kappaB-binding site in the COX-2 promoter. EMSAs confirmed that DHA attenuated NF-kappaB activation. Because MAPK, PKC, and NAD(P)H oxidase all participate in IL-1-mediated COX-2 expression, we also tested whether these enzymes were involved in DHA effects. Western blots showed that DHA blocked nuclear p65 NF-kappaB subunit translocation by decreasing cytokine-stimulated reactive oxygen species and ERK1/2 activation by effects on both NAD(P)H oxidase and PKCepsilon activities. Finally, to address the question whether DHA itself or DHA-derived products were responsible for these effects, we inhibited the most important enzymes involved in polyunsaturated fatty acid metabolism, showing that 15-lipoxygenase-1 products mediate part of DHA effects. These studies provide a mechanistic basis for antiinflammatory and possibly plaque-stabilizing effects of DHA.

The complement system is involved in several aspects of the pathophysiology of myocardial ischemia and infarction. Initially a role for complement in ischemic heart disease was inferred from the deposition of complement components within the myocardium of experimental models of myocardial infarction. Further animal models demonstrated that depletion or inhibition of complement prior to myocardial ischemia/reperfusion (MI/R) can reduce complement-mediated tissue injury. Recently, in vivo examination of naturally occurring complement inhibitors and monoclonal antibodies directed at specific complement components has confirmed complement dependent injury following MI/R. Current research provides intriguing evidence on the initiating pathways and the possible methods of complement regulation in the management of MI/R injury. This chapter focuses on many of the studies demonstrating complement activation and deposition in MI/R, the functional consequences of complement activation following MI/R, the initial and recent anti-complement therapies used in vivo and the current insight of the mechanisms of complement activation following MI/R.

The nuclear factor-kappa B (NFκB) family of transcription factors has emerged as a signaling pathway that figures prominently in a cell's initial response to a plethora of inflammatory stimuli. Modified lipids, oxidative stress, bacterial endotoxins, growth factors and cytokines, such as platelet-derived growth factor (PDGF) and interleukin-1 (IL-1), are among the stimuli that free NFκB dimers from their cytosolic inhibitor proteins leading to nuclear translocation of NFκB and transactivation of target genes (1-3). The smooth muscle cells (SMC) of the vasculature express components of this pathway and are activated by these stimuli, promoting dysregulation of gene expression by cells within the atherosclerotic vessel. Indeed, NFκB proteins have been localized within the nuclei of vascular SMC at sites of human atherosclerotic lesions, suggesting a role for NFκB in activation of this cell type in vivo (4-6). NFκB participates in dysregulated gene expression not only by SMC but also by endothelial cells and macrophages, prominent cell types within atherosclerotic lesions, thus much attention has focused on the workings of this signaling pathway within vascular cells and its role in atherogenesis.

Most micro-organisms that are encountered daily by a healthy individual are detected initially by defense mechanisms that are not antigen-specific, a response that is mediated by the innate immune system. In contrast to the adaptive immunity, where specific antigen receptors are generated by somatic hypermutation and selection, the innate immune systems uses germline encoded proteins that recognize specific patterns shared by groups of pathogens, but not the host. These receptors are called “pattern recognition receptors” (“PRRs”) and recognize largely invariant “pathogen-associated molecular patterns” (“PAMPs”), as for example lipopolysaccharides of bacteria or double-stranded RNA of viruses [1]. This review will focus on the toll-like receptor (TLR) family, a class of pattern recognition receptors of major importance [2].

Strong evidence links cardiomyocyte loss to the pathology of some forms of heart failure. Both necrotic and apoptotic modes of cell death have been invoked as the mechanism underlying progressive cardiomyocyte dropout. Nitric oxide (NO) has received particular attention as a candidate reactive oxygen intermediate that influences not only cardiac function, but also cell death elicited by both apoptotic and necrotic mechanisms. NO is produced by resident cardiac cells under stress, and is produced in large quantities by activated immune cells that infiltrate the injured heart. A review of the literature, however, reveals that the actions of NO on apoptotic cell death are complex, especially in the context of heart disease, and that the practical contribution of NO to cell death in heart disease is yet to be defined.

Abstract —The clinical benefit of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may derive from a qualitative, functional change in atherosclerotic lesions in addition to their lipid-lowering properties. We examined whether statins altered expression of the major determinants of fibrinolytic balance, plasminogen activator inhibitor-1 (PAI-1), and tissue-type plasminogen activator (tPA) in human vascular smooth muscle (SMC) and endothelial (EC) cells. Simvastatin reduced levels of PAI-1 antigen released from SMCs and ECs stimulated with platelet-derived growth factor or transforming growth factor-β (IC 50 ≈1 μmol/L). Levels of EC-derived tPA increased 2-fold over the same concentrations of simvastatin that inhibited release of PAI-1. Simvastatin’s inhibitory effect was mimicked by C3 exoenzyme and prevented by geranylgeranyl pyrophosphate, but not by farnesyl pyrophosphate, suggesting the involvement of geranylgeranyl-modified intermediates. Decreased PAI-1 antigen was correlated with reduced mRNA transcription and activity of the PAI-1 promoter. By inhibiting expression of PAI-1 from SMCs and ECs while increasing expression of tPA from ECs, simvastatin may alter the local fibrinolytic balance within the vessel wall toward increased fibrinolytic capacity that, in turn, would reduce thrombotic risk after plaque rupture.

IntroductionThrombosis underlies most acute manifestations of atherosclerosis and likely is an important contributor to the evolution of atherosclerotic lesions. In the coronary arteries, thrombosis precipitates most episodes of unstable angina and acute myocardial infarction (both Q-wave and non Q- wave). But, of course, atherosclerosis is a systemic disease that affects many arterial beds. Thus, the thrombotic complications of atherosclerosis also cause many strokes and acute exacerbations of peripheral vascular disease that place limbs in jeopardy.Many studies have clarified the microanatomy of fatal coronary thrombosis. Flow limiting stenoses were formerly considered the cause of the acute coronary syndromes. Recent clinical observations, however, have taught us that most myocardial infarctions result from thrombi that occur on the substrate of a lesion that does not cause a critical narrowing of a coronary artery. We now appreciate that a physical disruption of the atheromatous plaque most often provokes thrombus formation. Plaque disruption takes two major forms: a frank fissuring of the plaque’s fibrous cap and a superficial erosion of the intimal surface (Table 1). In the case of a ruptured cap, blood coagulation factors come into contact with the plaque’s lipid core, which is rich in tissue factor and considered the major procoagulant in this situation. In the case of superficial erosion, platelets can contact subendothelial basement membrane and collagen within the plaque, which may trigger platelet aggregation. Additionally, smooth muscle cells underlying the endothelium can also express tissue factor, further contributing to thrombus formation.Excellent evidence suggests that plaque rupture occurs frequently, even in asymptomatic individuals. Consequently, most arterial thrombi do not produce clinical manifestations. Presumably, most mural thrombi either fail to occlude the vessel, as they do not propagate, or are evanescent. A healing mural thrombus, although clinically silent, may still contribute to plaque growth. Platelets contain potent fibrogenic mediators, such as platelet-derived growth factor (a smooth muscle cell chemoattractant) and transforming growth factor (TGF)-β (a strong stimulus to smooth muscle collagen gene expression). Thrombin and activated factor X can stimulate smooth muscle cell proliferation by direct and/or indirect routes. These mediators may link healing mural thrombi in arteries to intimal growth and the formation of stenotic lesions.Why do some plaque disruptions lead to an occlusive thrombus that may cause sudden death, while other disruptions pass unnoticed? The locally prevailing hemostatic and fibrinolytic balance likely proves decisive in determining the fate of a given disruption of an atheroma. Much evidence has recently accumulated regarding the factors that regulate these regional balances. This chapter will selectively summarize certain of those recent findings. It will also provide some mechanistic insight into how contemporary therapies may act to reduce the thrombotic complications that cause the most dreaded and dramatic complications of atherosclerosis.

Abstract —Plasminogen activator inhibitor type-1 (PAI-1) is a major physiological inhibitor of fibrinolysis, with its plasma levels correlating with the risk for myocardial infarction and venous thrombosis. The regulation of PAI-1 transcription by endothelial cells (ECs), a major source of PAI-1, remains incompletely understood. Adipocytes also produce PAI-1, suggesting possible common regulatory pathways between adipocytes and ECs. Peroxisomal proliferator-activated receptor-γ (PPAR)γ is a ligand-activated transcription factor that regulates gene expression in response to various mediators such as 15-deoxy- Δ12,14 -prostaglandin J 2 (15d-PGJ 2 ) and oxidized linoleic acid (9- and 13-HODE). The present study tested the hypotheses that human ECs express PPARγ and that this transcriptional activator regulates PAI-1 expression in this cell type. We found that human ECs contain both PPARγ mRNA and protein. Immunohistochemistry of human carotid arteries also revealed the presence of PPARγ in ECs. Bovine ECs transfected with a PPAR response element (PPRE)–luciferase construct responded to stimulation by the PPARγ agonist 15d-PGJ 2 in a concentration-dependent manner, suggesting a functional PPARγ in ECs. Treatment of human ECs with 15d-PGJ 2 , 9( S )-HODE, or 13( S )-HODE augmented PAI-1 mRNA and protein expression, whereas multiple PPARα activators did not change PAI-1 levels. Introduction of increasing amounts of a PPARγ expression construct in human fibroblasts enhanced PAI-1 secretion from these cells in proportion to the amount of transfected DNA. Thus, ECs express functionally active PPARγ that regulates PAI-1 expression in ECs. Our results establish a role for PPARγ in the regulation of EC gene expression, with important implications for the clinical links between obesity and atherosclerosis.

Both chlamydial and human heat shock protein 60s (HSP 60), which colocalize in human atheroma, may contribute to inflammation during atherogenesis. We tested the hypothesis that chlamydial or human HSP 60 activates human endothelial cells (ECs), smooth muscle cells (SMCs), and monocyte-derived macrophages. We examined the expression of adhesion molecules such as endothelial-leukocyte adhesion molecule-1 (E-selectin), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), and the production of the proinflammatory cytokine interleukin-6 (IL-6). We also tested whether either HSP 60 induces nuclear factor-kappaB (NF-kappaB), which contributes to the gene expression of these molecules. Either chlamydial or human HSP 60 induced E-selectin, ICAM-1, and VCAM-1 expression on ECs similar to levels induced by Escherichia coli lipopolysaccharide (LPS). Each HSP 60 also significantly induced IL-6 production by ECs, SMCs, and macrophages to an extent similar to that induced by E. coli LPS, as assessed by enzyme-linked immunosorbent assay (ELISA). In ECs, either HSP 60 triggered activation of NF-kappaB complexes containing p65 and p50 Rel proteins. Heat treatment abolished all these effects, but did not alter the ability of E. coli LPS to induce these functions. Chlamydial and human HSP 60s therefore activate human vascular cell functions relevant to atherogenesis and lesional complications. These findings help to elucidate the mechanisms by which a chronic asymptomatic chlamydial infection might contribute to the pathophysiology of atheroma.

Increasing evidence supports involvement of inflammation and immunity in atherogenesis. We report here that CD40 ligand (CD40L), an immunoregulatory signaling molecule heretofore considered largely restricted to recently activated CD4+T lymphocytes, is expressed by human vascular endothelial cells (EC), smooth muscle cells (SMC), and human macrophagesin vitro, and is coexpressed with its receptor CD40 on all three cells types in human atherosclerotic lesionsin situ. Cultured human vascular EC, SMC, and human macrophages all constitutively expressed CD40L mRNA as well as protein. Stimulation with interleukin 1β, tumor necrosis factor α, or interferon γ increased surface levels andde novosynthesis of CD40L on all three cell types. CD40L expressed on EC, SMC, and macrophages exhibited biological activity, as it induced B7.2 expression on B cells. Human vascular SMC also constitutively expressed CD40, the receptor for CD40L, and through CD40 signaling, human recombinant CD40L induced expression of proinflammatory cytokines in these cells, identifying SMC as a target for CD40L. Human atherosclerotic lesions (n= 8) showed expression of immunoreactive CD40L on EC, SMC, and macrophages, while normal arterial tissues (n= 5) contained no CD40L. In atheroma CD40L+cells often also expressed CD40. These observations establish human vascular EC, SMC, and human macrophages as a novel source of CD40L, and point to T cell-independent CD40 signaling, and a broader function of this pathway in regulation of nonimmune cells, as illustrated here by potential autocrine and paracrine activation during atherogenesis.

Activated macrophages release cytokines and growth factors that may contribute to the growth of vascular smooth muscle cells in injured blood vessels. In the present study, we investigated the interactions between interleukin-1 beta (IL-1 beta) and basic fibroblast growth factor (FGF-2) in primary rat aortic smooth muscle cells, relative to their effects on DNA synthesis and cell proliferation. We report that femtomolar levels of IL-1 beta, which alone were non-mitogenic or weakly mitogenic, synergistically increased FGF-2-induced [3H]thymidine incorporation and cell proliferation. The potentiating effect of IL-1 beta extended to PDGF-AB and EGF, but not to IGF-1-induced thymidine incorporation. An antagonist of the IL-1 receptor, IL-1ra, blocked the co-mitogenic effect of IL-1 beta. Stimulation of cells with FGF-2 and IL-1 beta increased both DNA content and proliferation, an observation that was consistent with the thymidine incorporation experiments. An inhibitor of NO synthase, N5-iminoethyl L-ornithine (L-NIO), did not block the co-mitogenic effect of IL-1 beta, despite effective inhibition of NO synthase activity, suggesting that the synergistic interaction between IL-1 beta and FGF-2 was independent of the NO/cGMP pathway. The mechanism of co-mitogenesis appeared to be independent of the intermediacy of PDGF-AA, IL-6, and prostanoids, and was not associated with increased levels of c-fos mRNA, FGF receptor-1 protein, or FGF-2-induced early and delayed tyrosine phosphorylation events. We conclude that IL-1 beta interacts with FGF-2 to amplify the proliferation of primary rat aortic smooth muscle cells, an effect that may be important in vascular smooth muscle cell proliferation following vascular injury.