Atherosclerosis: The Atherogenic Process

Inflammation plays a role in each stage of the atherogenic process, from its initiation to its manifestation as acute coronary syndromes. Dr. Peter Libby of Brigham and Women’s Hospital reviewed evidence from his laboratory and others that have led to this current understanding.

Inflammation in the initiation of atherosclerotic disease

Inflammation plays an essential role in the pathobiology of a normal arterial wall becoming an atherosclerotic plaque. Adhesion is the first step, in which overexpression of leukocyte adhesion molecules recruits blood inflammatory cells to the early lesion. The activated endothelium expresses specific adhesion molecules.

Vascular cell adhesion molecule-1 (VCAM-1), among others, is induced by proinflammatory mediators or by exposure to cardiovascular (CV) risk factors. Libby’s group showed that VCAM-1 is present as early as 1 week after the start of an atherogenic diet in experimental rabbits. At 3 weeks, leukocytes adhering to endothelial cells were seen.

Chemoattraction is the next step in this process. Monocyte chemoattractant protein-1 (MCP-1), produced by cells present in the early atherosclerotic lesion, directs migration of the leukocyte adhesion molecules into the arterial intima. Other chemoattractants are also involved. Libby and colleagues showed markedly less atherosclerosis in LDL receptor-deficient mice with the alleles of the gene encoding MCP inactivated, compared to wild-type mice of the same age, background, and consuming the same diet for the same length of time. This provided causal evidence for the role of MCP-1 in atherosclerosis.

The maturation of the monocyte into a macrophage is the next step in atherogenesis. After entering the arterial intima, the blood monocyte changes into a tissue macrophage, which express scavenger receptors that allow the macrophage to engulf modified lipoproteins and then become a foam cell, the hallmark of the early atherosclerotic plaque. As the monocytes mature into macrophages, they can divide and increase the number of inflammatory cells in the atherosclerotic plaque. After the macrophage is present and multiplied in the arterial wall, there is an amplification and sustaining of the process, Libby stated, because the “professional” phagocyte can elaborate many pro-oxidant and pro-inflammatory mediators.

What are the signals that cause the transition from monocyte to macrophage, elicit scavenger receptor expression, and promote cell division? Macrophage colony-stimulating factor (MCSF) was shown by Libby and colleagues and others to induce scavenger receptor expression on mononuclear phagocytes. Also, they and others showed augmented expression of MCSF in human and experimental atherosclerosis.

Libby’s group showed a gene dosage-dependent decrease in atherogenesis by inactivating MCSF genes. In LDL-deficient mice, with both MCSF alleles there was considerable fatty lesion circumferentially in the aortic root; with 1 MCSF allele, markedly less lesion; and with no MCSF allele a marked and striking reduction in lesion formation.

T-lymphocytes also exchange mediators with the cells in the immune response, such as the macrophage, and help to modulate and control the atherogenic process. A trio of gamma interferon-inducible chemokines interact with the receptor CXCR3, which is expressed exclusively on T-cells in the atherosclerotic plaque. After the lymphocyte is recruited into the intima, it can exchange messages with the macrophage and modulate the atherogenic process in several ways.

Other chemokines involved in atherogenesis include interleukin-8 which binds to CXCR2. And the chemoattractant eotaxin, which interacts with the chemokine receptor CCR3, is important in recruiting mast cells to the plaque, as shown by Libby, Lee, and colleagues. Libby and colleagues, along with Luster and colleagues, showed that the chemokine stromal derived factor-1 interacts with the chemokine receptor CXCR4, and can cause platelets to aggregate; this property was not seen with the other two dozen chemokines tested.

Inflammation and the silent, clinically stable phase of atherogenesis

The pro-inflammatory signaling pathway CD40-CD40 ligand dyad is involved with lesion progression during the long but silent clinically stable phase of atherogenesis. Libby and colleagues identified this cell surface-associated cytokine in the atherosclerotic plaque. CD40 signaling contributes to promoting atherosclerosis and its complications in a number of ways. Ligation of CD40 on human mononuclear phagocytes can induce the potent procoagulant tissue factor, important in later phases of atherogenesis. This tissue factor induction is not seen with other soluble cytokines, such as interleukin-1 or tumor necrosis factor alpha.

Libby’s group showed in early experiments that inhibition of the CD40 ligand blocks the initiation of atherosclerosis. Work by others in gene knockout animals also showed that blocking CD40 signaling interrupts the initiation of atherosclerosis.

The progression of atherosclerosis also can be stopped by blocking CD40 signaling, as shown by Libby and colleagues in another series of experiments. Libby stated these experiments are one illustration that inflammation is important in the progression of atherosclerosis.

Inflammation and acute coronary syndromes

Unstable angina or acute myocardial infarction (AMI) are the most dramatic and clinical presentation of atherosclerosis. Atherosclerotic plaque rupture causes most fatal AMIs, because of a fissure in the fibrous cap of the plaque. Microanatomical characteristics of these plaques include a large number of inflammatory cells, rich in macrophages, thin fibrous cap, and relative lack of smooth muscle cells.

To test whether or not inflammation contributed to thrombotic complications, Libby and colleagues studied the metabolism of the collagen comprising the fibrous cap. The structural integrity of the plaque’s fibrous cap depends on interstitial collagen fibrils synthesized by smooth muscle cells.

Nearly 15 years ago, they showed an increase in collagen synthesis by smooth muscle cells in culture when they are exposed to platelet-derived growth factor or transforming growth factor beta, which are released by platelets during thrombosis. This step is very important in healing, and provided a positive control for the experiments.

Most noteworthy, Libby stated, new collagen synthesis by smooth muscle cells was nearly halted when the cells were exposed to interferon gamma, a pro-inflammatory cytokine known to operate during atherogenesis. They also showed that even after maximal stimulation of the smooth muscle cells with transforming growth factor beta to increase collagen synthesis, it is possible to return collagen synthesis to baseline levels with interferon gamma. Further, messenger RNA levels of the procollagens decreased when exposed to interferon gamma.

Do inflammatory mediators augment the breakdown of interstitial collagen? Work by this group showed that although the normal artery does not express collagenolytic enzymes, there is overexpression of these enzymes in human atherosclerotic plaques. They showed overexpression of matrix metalloproteinase-1 (MMP-1) and MMP-13, and others showed overexpression of MMP-8. Thus, all three known interstitial collagenases are overexpressed in the atherosclerotic plaque. Further, Libby’s group co-localized the overexpression of these collagenases with the appearance of the neoepitope with the antibody directed to that cleaved collagen, carboxyl terminus, surrounding the area of intact collagen.

Some two-thirds to three-quarters of AMIs are because of disruption of the fibrous cap related to low levels of collagen. The collagen is doubly regulated when inflammation is present in the intima and the decreased synthesis sets the stage for plaque rupture. The CD40 ligand induces tissue factor procoagulant. The stage for coronary thrombosis is set with the intimal inflammation, weakened fibrous cap, and heightened procoagulant activity.

Direct evidence linking collagenases with the regulation of collagen content of the atheroma was provided by experiments in genetically-modified mice performed by Libby and colleagues. In compound mutant mice, fed an atherogenic diet they showed that animals with the mutant collagen resisted the collagenases and there was more circumferential collagen accumulation in the intima. All of their analyses showed that in the animals with the collagenase-resistent form of collagen had greater collagen accumulation compared to the wild-type animals. In a follow-up series of experiments they showed that MMP-13 is one of the important collagenases involved in the accumulation of collagen in the atherosclerotic plaque.

MMP-13-deficient mice were also shown to have more and thicker collagen fibers and more homogenous concentration of angles of orientation, compared to MMP-13 wild-type mice. Thus, MMP-13 contributes to the accumulation and the architecture of collagen in atherosclerotic plaques.

Ongoing work by this group is attempting to understand the molecular pathways involved in the economy of collage, a crucial feature in the stability of atherosclerotic plaques. They are also looking at various aspects of the metalloproteinase system and their inhibitors to understand the regulation of extracellular matrix accumulation in atherogenesis. They hope to gain further insight into how this crucial structural component of the atherosclerotic plaque undergoes regulation, particularly in relation to inflammation.