The Molecular Mechanisms of the Acute Coronary Syndromes

Accumulated clinical evidence of the past 20 years along with clinical research has cast in doubt the traditional concept of the pathogenesis of atherosclerosis transitioning from chronic stable or asymptomatic disease to acute thrombotic.

Thrombotic complications result from physical disruption of the atherosclerotic plaque. Dr. Peter Libby, the Mikamo Lecturer for the 71st Annual Scientific Meeting of the Japanese Circulation Society reviewed study results related to two of the mechanisms causing this disruption, rupture of the plaque’s fibrous cap that frees pro-coagulant materials in the lipid core and superficial erosion of the endothelial monolayer.

Basic inflammation biology and mechanisms of ACS

Dr. Libby and his group believe that understanding the metabolism of collagen, i.e., the material structural component of the fibrous cap of the plaque, would provide insights into thrombotic complications.

They hypothesized that inflammatory mediators would decrease the synthesis of interstitial collagen, a product of the smooth muscle cells (SMCs) in the artery wall that lends strength to the fibrous cap. Libby’s group performed a metabolic labeling experiment that demonstrated de novo collagen synthesis from cultured human SMCs. Further, they showed that the inflammatory mediator gamma interferon can markedly inhibit the ability of SMCs to manufacture new collagen, even when stimulated by transforming growth factor-beta (TGF-b), the most potent stimulus of collagen synthesis. Co-incubation of TGF-b and interferon gamma reduced the rate of new collagen synthesis to baseline or below.

Figure 1. Interstitial collagenases are required to initiate proteolytic cleavage on the intact triple helical collagen molecule Click to enlarge

Interstitial collagen, a stable molecule, resists the proteolytic attack of most enzymes. Interstitial collagenases, most of which are members of the matrix metalloproteinase family (MMPs), are required to initiate proteolytic cleavage on the intact triple helical collagen molecule (Figure 1). This cleavage with MMPs begins the catabolic cascade of collagen. More than a dozen years ago, Libby and colleagues showed that macrophages in human atherosclerotic plaque trigger over-expression of MMP-1, the prototypical collagenase, in SMCs and in foam cells.

Thereafter they sought to determine whether the immunostainable excess of collagenase reflects a stoichiometric excess of active enzyme over its endogenous inhibitor. The major known endogenous inhibitors of MMPs are tissue inhibitors of matrix metalloproteinases (TIMPs). Libby’s group showed collagen degradation in the atherosclerotic plaque and co-localized the area of collagenolysis with MMP-1 and MMP-13, another interstitial collagenase. In other work, this group showed that MMP-8 is misnamed and that not only neutrophils but also endothelial cells, SMCs, and macrophages in human atherosclerotic plaques express this proteinase.

Model of molecular pathogenesis in ACS

Inflammation in the intima places collagen in double jeopardy, i.e., decreased synthesis and increased breakdown, and thereby sets the stage for ACS. In this model of molecular pathogenesis proposed by Libby and colleagues, when inflammation prevails in the intima, the T-cell can send a signal to the SMCs. Gamma interferon stops the new collagen synthesis necessary to repair and maintain the interstitial collagen in the fibrous cap of the plaque. The T-cell can also send signals to the macrophage, elevating the production of interstitial collagenases that can cleave this collagen.

Figure 2. The experimental protocol in the animal study providing direct evidence to link interstitial collagenase and regulation of collagen content in atheroma. Click to enlarge

Figure 3. MMP-13–deficient mice had more and better-organized collagen, as shown by advanced optical techniques. Click to enlarge

Libby and colleagues obtained direct evidence linking interstitial collagenases with the regulation of collagen content of atheroma through their work with genetically altered animals (Figure 2). In collagenase-resistant knockout mice, they showed accumulation of collagen. A converse experiment in a compound mutant mouse model that inactivated MMP-13, an important interstitial collagenase, showed greater accumulation of collagen in the intima, indicating that collagenase factors importantly in the economy of collagen in the experimental atherosclerotic plaque.

Additional studies with advanced optical techniques showed that MMP-13–deficient mice had more organization of fibers in the collagen, thicker collagen, and tighter distribution of the angular dispersion of fibers. Hence, the MMP-13–deficient mice had more, better-organized collagen (Figure 3). Thus, MMP-13 collagenase contributes to the accumulation and architecture of collagen in the atherosclerotic plaque.

Other experimental work in knockout mice by Libby and colleagues showed that MMP-14, an enzyme discovered in Japan, also influences collagen metabolism, probably through a combination of direct and indirect mechanisms. Collagenases critically influence collagen accumulation in mouse atheromata in vivo.

Mechanism of superficial erosion of endothelial monolayer

Superficial erosion of the endothelial monolayer provides another mechanism of plaque disruption, particularly in women and in persons with hypertriglyceridemia. Libby and colleagues hypothesized that apoptosis of endothelial cells in response to inflammatory mediators could heighten the risk of this superficial erosion. Further, that reactive oxygen species generated in response to inflammatory stimuli or inflammatory cells may promote endothelial cell apoptosis or the tethering of the endothelial cell to its subadjacent basement membrane.

This group showed the involvement of myeloperoxidase (MPO) in plaque disruption and thrombosis. MPO, an enzyme released by activated granulocytes, is a monocyte subpopulation. It binds the extracellular matrix at sites of inflammation and converts chloride ion plus peroxidase to hypochlorous acid, a potent oxidant and chlorinating species. Libby’s group provided histochemical evidence of the expression of MPO and hypochlorous acid modified proteins in plaque that had ruptured or was undergoing superficial erosion.

Further work by Sugiyama in collaboration with Libby’s group indicates that hypochlorous acid at concentrations relevant to sites of inflammation provokes programmed death of endothelial cells.  Increasing concentrations of hypochlorous acid caused laddering of the DNA in the endothelial cell structure and increased caspase-3, the machinery enzyme involved in the apoptotic pathway.

In this manner, oxidative stress, a process that is intimately related to inflammation, may sensitize endothelial monolayers to desquamation and thereby cause superficial erosion. Many matrix-degrading proteinases participate in the atherogenic process, including the remodeling that causes the compensatory enlargement responsible for hiding the disease.

Applying molecular imaging to vascular biology

Proteolysis, Libby’s group believes, can predispose toward plaque disruption and thrombosis. They are collaborating with another group specializing in molecular imaging to study the possibility of monitoring proteinase activity in vivo and apply that modality to vascular biology. They have taken key steps toward visualizing collagenase activity in vivo, including publishing early work with this technique in vivo and in vitro with MMP-2 and MMP-9, and performing unpublished work with MMP-13, a collagenase considered intimately involved in plaque biology.

Other work utilizing the Watanabe rabbit with diffuse atherosclerosis is in progress, using this imaging technology via a catheter-based system to visualize enzymes in situ in the intact living animal. With further development and validation, this technology could transfer effectively to humans. Molecular imaging hopefully can validate pathophysiologic hypotheses and provide a more efficient way to develop therapeutic agents.

Closing

Dr. Libby thanked the Japanese Circulation Society for the honor of giving the Mikamo Lecture and for closing the circle on the initial basic science studies of inflammation in vascular biology he presented 17 years ago at a Japan Atherosclerosis Society meeting in the same venue in Kobe. Dr. Libby also thanked Dr. Yokoyama, president of the meeting, the Japanese Circulation Society, and Japan’s vascular biology, cardiovascular medicine, and science communities for the rich collaboration and friendship over several decades.