Pathogenesis of Atherosclerosis

Various immunodeficiencies

Hyperinflammatory but inadequate immune response

Clinical picture of (Hemophagocytic lymphohistiocytosis) HLH

HLH
Variable course of disease Rapidly progressive leading to death within weeks Transient improvements with unspecific therapies Disappearance of symptoms without therapy Disappearance of symptoms with immunosuppressive/immunomodulatory drugs

HLH
Classification

Genetic, primary HLH

FHLH
Perforin mutations (chr.10) Chromosom 9 linkage

Acquired, secondary HLH

Exogenous agents - infectious organisms, toxins (VAHS, IAHS)

Unknown mutations

HLH

Endogenous products - tissue damage - radical stress - metabolic products Rheumatic disorders

- Immune deficiencies
CHS Griscelli syndrome XLP SCID Malignancies

HLH Diagnostic criteria Histiocyte Society 1991 Clinical Fever > 38.5 Splenomegaly Laboratory Cytopenia of => 2/3 cell lines Hypertriglyceridemia and/or hypofibrinogenemia Histopathology Hemophagocytosis in bone marrow or spleen or liver or lymphnode Strong supportive evidence are spinal fluid pleocytosis, liver histology resembling chronic persistent hepatitis, low natural killer cell activity

HLH Therapy h Cytostatic and immunsuppressive/ immunomodulatory drugs: Corticosteroids, Cyclosporin A, Etoposide Immunoglobulins, Antithymocyte globulin h Bone marrow transplantation Prognosis In 20% no response to therapy After BMT 60-70% relapse-free survival

Introduction
Arteriosclerosis
Thickening and loss of elasticity of arterial walls Hardening of the arteries Greatest morbidity and mortality of all human diseases via Narrowing Weakening

Atherosclerotic Disease
Prevalence
In US there are 6 million with CAD 3 million Americans have had strokes

Mortality
1.5 million deaths/yr in US due to myocardial infarction 0.5 million deaths/yr in US due to strokes

Three patterns of arteriosclerosis

Atherosclerosis
The dominant pattern of arteriosclerosis Primarily affects the elastic (aorta, carotid, iliac) and large to medium sized muscular arteries (coronary, popliteal)

Monckeberg medial calcific sclerosis Arteriolosclerosis small arteries and arterioles (hypertension and DM)

Non-Modifiable Risk Factors

Age
A dominant influence Atherosclerosis begins in the young, but does not precipitate organ injury until later in life

Gender
Men more prone than women, but by age 60-70 about equal frequency

Family History
Familial cluster of risk factors Genetic differences

Modifiable Risk Factors (potentially controllable)

Hyperlipidemia Hypertension Cigarette smoking Diabetes Mellitus Elevated Homocysteine Factors that affect hemostasis and thrombosis Infections: Herpes virus; Chlamydia pneumoniae Obesity, sedentary lifestyle, stress

AHA Classification of atherosclerosis

Fig. 11.7

Normal Artery

Normal Artery

Atherosclerosis
A disease of the intima A disease of the intima A disease of the intima Atheromas, atheromatous/fibrofatty plaques, fibrous plaques Narrowing/occlusion; weakness of wall

Major components of plaque

Cells (SMC, macrophages and other WBC) ECM (collagen, elastin, and PGs) Lipid = Cholesterol (Intra/extracellular) (Often calcification)

Two major processes in plaque formation

Intimal thickening (SMC proliferation and ECM synthesis) Lipid accumulation

Consequences of plaque formation

Generalized Narrowing/Occlusion Rupture Emboli Leading to specific problems:
Myocardial and cerebral infarcts Aortic aneurysms Peripheral vascular disease

Fatty Streak-Aorta

Fatty Streak-Coronary Artery

Altered Vessel Function

Vessel change
Plaque narrows lumen Wall weakened Thrombosis Breaking loose of plaque Loss of elasticity

Consequence
Ischemia, turbulence Aneurysms, vessel rupture Narrowing, ischemia, embolization Athero-embolization Increase systolic blood pressure

Late Changes
Calcification
An example of dystrophic calcification

Cracking, ulceration, rupture

Usually occurs at edge of plaque

Thrombus formation
Caused by endothelial injury,ulceration, turbulence Organization of thrombus More thrombus

Encroachment
Weakens vessel wall

Bleeding
Ulceration, cracking and angiogenesis

ATHEROSCLEROSIS: Pathology, Pathogenesis, Complications, Natural History

Fibrous Plaques

Complicated Lesions

Fibrous cap Cholesterol clefts

Elastin membrane destroyed

Neovas. Calcification Inflam. cells

Hemorrhage into Plaque

Ulceration/Hemorrhage/Cholesterol Crystals

Complicated Lesion/Calcification

Foam Cells/Cholesterol Crystals

Cholesterol Crystals/Foam Cells

Thrombosis/Complicated Lesion

Complicated Lesion/Ulceration/Thrombosis

Aortic Aneurysm

Aortic Aneurysm

Pathogenesis of Atherosclerosis
Cause?
Current hypothesis: Response to Injury
Initiated by endothelial dysfunction

Disease of the intima

Intimal thickening Intra- and extra-cellular lipid accumulation Chronic Inflammation

Basic Lesion: is termed atheroma, fibro-fatty plaque, or atheromatous plaque

Response to injury hypothesis

* Injury to the endothelium (dysfunctional endothelium) * Chronic inflammatory response * Migration of SMC from media to intima * Proliferation of SMC in intima Excess production of ECM Enhanced lipid accumulation

Response to injury hypothesis (I)

1. Chronic EC injury (subtle?)
EC dysfunction Increased permeability Leukocyte adhesion (via VCAM-1) Thrombotic potential

Response to injury hypothesis (II)

2. Accumulation of LDL (cholesterol) 3. Oxidation of lesional LDL 4. Adhesion & migration of blood monocytes; transformation into macrophages and foam cells 5. Adhesion of platelets 6. Release of factors from platelets, macrophages and ECs

Response to injury hypothesis (III)

7. Migration of SMC from media to intima 8. Proliferation of SMC 9. ECM production by SMC 10. Enhanced lipid accumulation
Intracellular (SMC and macrophages) Extracellular

Endothelial dysfunction
Induced by oxidized LDL, can be worsened by cigarette smoking, and can be reversed with correction of hyperlipidemia by diet or by therapy with a statin, which increases the bioavailability of nitric oxide, with ACE inhibitors, or with antioxidants such as vitamin C or flavonoids contained in red wine
Anderson et al., 1996; Harison et al 1987; John et al., 1998; Mancini et al 1996; Levine et al 1996

Endothelial dysfunction
Expression of VCAM-1 on endothelial surfaces is an early, and necessary, step in the pathogenesis of atherosclerosis. Increased cellular adhesion and associated endothelial dysfunction then sets the stage for the recruitment of inflammatory cells, release of cytokines and recruitment of lipid into the atherosclerotic plaque.
Li et al., 1993

Dyslipidaemia
Lipid abnormalities play a critical role in the development of atherosclerosis Early experiments demonstrated accelerated atherosclerosis with a high cholesterol diet. Epidemiologic studies showed increasing incidence of atherosclerosis when serum cholesterol above 3.9 mM

Dyslipidaemia
High levels of LDL and low levels of HDL important risk factors for atherosclerosis Macrophage uptake of LDL may initially be adaptive response, which prevents LDL-induced endothelial injury However, cholesterol accumulation in foam cells leads to mitochondrial dysfunction, apoptosis, and necrosis, with resultant release of cellular proteases, inflammatory cytokines, and prothrombotic molecules

Dyslipidaemia
Oxidized LDL can cause disruption of the endothelial cell surface, promote inflammatory and immune changes via cytokine release from macrophages and antibody production and increase platelet aggregation

Dyslipidaemia
HDL, in contrast to LDL, has antiatherogenic properties that include reverse cholesterol transport, maintenance of endothelial function, protection against thrombosis, and maintenance of low blood viscosity through a permissive action on red cell deformability

Inflammation
Best evidence supporting the importance of inflammation in the pathogenesis of atherosclerosis comes from the observation that markers of increased or decreased systemic inflammation are directly associated with the risk of atherosclerosis

Inflammation and chronic endothelial injury

VCAM-1 expression increases recruitment of monocytes and T-cells to sites of endothelial injury Subsequent release of MCP-1 by leukocytes magnifies the inflammatory cascade by recruiting additional leukocytes, activating leukocytes in the media, and causing recruitment and proliferation of smooth muscle cells

Atherosclerosis is initiated when leucocytes adhere to the endothelium as a result of expression of adhesive proteins.

Crowther et al., 2005

Inflammation and chronic endothelial injury

Monocytes adhere to the endothelium and then migrate through the endothelium and basement membrane by elaborating enzymes, including locally activated matrix metaloproteinases (MMP) that degrade the connective tissue matrix
Crowther et al., 2005

Leucocytes than cross the endothelial barrier and begin to accumulate

Crowther et al., 2005

Inflammation and chronic endothelial injury

Macrophages both release additional cytokines and begin to migrate through the endothelial surface into media of the vessel. This process is further enhanced by the local release of M-CSF, which causes monocytic proliferation Local activation of monocytes leads to both cytokine-mediated progression of atherosclerosis, and oxidation of LDL
Crowther et al., 2005

Monocytes within the subendothelial space subsequently orchestrate the development of atherosclerosis through cytokine release. Crowther et al., 2005

Inflammation and chronic endothelial injury

CD40L elaborated within the plaque has been shown to increase the expression of tissue factor in atherosclerotic plaques anti-CD40L abrogates evolution of established atherosclerotic lesions in animal models
Schonbeck et al., 2000

Clinically apparent disease if first noted as a result of the accumulation of foam cells. Crowther et al., 2005

Inflammatory mediators
Include IL-1, TNF and , IL-6, M-CSF, MCP-1, IL-18 and CD-40L. The impact of these mediators is diverse and includes mitogenesis, intracellular matrix proliferation, angiogenesis and foam cell development
Crowther et al., 2005

The clinically important lesion is characterized by intimal narrowing, many foam cells, neovascularization and flow limiting narrowing. However, this stage of the disease is sufficiently advanced that treatments aimed at it do not impact the pathogenesis of the underlying disorder.

CRP
CRP may be only a marker of inflammation and thrombotic risk CRP binds to LDL, allowing LDL to be taken up by macrophages without the need for modification CRP induces adhesion molecule expression and production of IL-6 and MCP-1 in human endothelial cells; these effects might enhance a local inflammatory response within the atherosclerotic plaque by the recruitment of monocytes and lymphocytes

CRP
The proinflammatory and prothrombotic effects of CRP on monocytes and endothelial cells in vivo by subjecting wild-type mice, which do not express CRP, and human CRP-transgenic (CRPtg) mice to two models of arterial injury. In an arterial injury model complete thrombotic occlusion of the femoral artery at 28 days was seen in 17% of wild-type mice compared with 75% of CRPtg arteries.
Danenberg et al., 2003

Multivariable-adjusted relative risks of cardiovascular disease according to levels of CRP and the estimated 10-year risk based on the Framingham Risk Score. CHD indicates coronary heart disease. Data from Ridker and colleagues

IL-1 and TNF-alpha

Enhance expression of cell surface molecules such as ICAM-1, VCAM-1, CD40, CD40L, and selectins on endothelial cells, smooth muscle cells, & macrophages Induce cell proliferation, contribute to the production of ROS, stimulate matrix metalloproteinases, & induce TF expression

Leukocyte activation
mRNA profiles showing increased levels of most inflammatory mRNAs in individuals with prior AMI

Toll-like receptor 4
Polymorphisms in the toll-like receptor 4 gene that confer differences in the inflammatory response to Gram negative pathogens Carriers of the Asp299Gly polymorphism, compared to wild-type alleles, have reduced circulating levels of inflammatory markers, including CRP, adhesion molecules, and IL-6, and a reduced incidence of carotid atherosclerosis

Molecular Targets to address chronic inflammation

Peroxisome proliferator-activated receptors (PPARs) have emerged as important antiatherogeneic targets Endothelial specific roles of PPAR- include
inhibition of adhesion molecules, including VCAM-1 increased endothelial NO release reduced foam cell formation reduced uptake of glycated LDL and triglyceride-rich remnant lipoproteins

PPARs
Ligands of PPAR- include fatty acids and the oral hypoglycemic drugs belonging to the glitazone family PPAR- is expressed in numerous cell types found within the atherosclerotic lesion, including endothelial cells, smooth musclecells, macrophages, and T cells

Potential anti-atherogenic activities of peroxisome proliferator-activated receptors (PPARs) 1. Increased nitric oxide synthesis and release 2. Decreased recruitment of T cells 3. Reduced angiogenesis 4. Inhibition of smooth muscle cell (SMC) migration 5. Decreased SMC expression of matrix-degrading enzymes 6. Decreased macrophage-dependent expression of matrix metalloproteinase (MMP)-9 and osteopontin 7. Enhanced release of the interleukin-1 receptor antagonist 8. Enhanced reverse cholesterol transport

Lucas et al., 2006

Angiogenesis
Angiogenic signaling and proliferation of microvessels within the plaque is only now beginning to be understood Plaque hemorrhage is likely attributable to bleeding from fragile microvessels that proliferate within the plaque itself, presumably in response to local angiogenic stimuli.

Angiogenesis
Kockx et al identified intraplaque hemorrhage from microvessels triggering macrophage activation and foam cell formation in carotid lesions These authors propose that intraplaque microhemorrhage may initiate platelet and erythrocyte deposition, lead to iron deposition, activate macrophages and contribute to foam cell formation.
Kockx et al.,2003

Angiogenesis
The importance of angiogenesis in the pathogenesis of plaque growth was recently bolstered by the finding that intra-plaque microvessels were an independent predictor of plaque rupture The potential importance of angiogenesis in the development of atherosclerosis is found in experiments that demonstrate that antiangiogenic therapy reduced atherosclerotic lesion development in a placebo controlled trial in atherosclerosis prone mice
Moreno et al., 2004; Chew

Moreno et al., 2006

General features of insulin signal transduction pathways. PI 3-kinase branch of insulin signaling regulates GLUT4 translocation and glucose uptake in skeletal muscle and NO production and vasodilation in vascular endothelium. MAPkinase branch of insulin signaling generally regulates growth and mitogenesis and controls secretion of ET-1 in vascular Endothelium Kim et al., 2006

Kim et al., 2006

Shared and interacting mechanisms of glucotoxicity, lipotoxicity, and inflammation underlie reciprocal relationships between insulin resistance and endothelial dysfunction that contribute to linkage between metabolic and cardiovascular diseases. CAD indicates coronary artery disease

Mechanisms for the contribution of insulin resistance to atherosclerosis. VSMC indicates vascular smooth muscle cell; CHF, congestive heart failure. Kim et al., 2006

Tissue factor: A key regulator of coagulation. Tissue factor (TF) is a key initiator of the coagulation cascade. Formation of a complex with factor VIIa (FVIIa) leads to activation of factor IX (FIX) and factor X (FX), resulting in thrombin generation and, ultimately, clot formation. Tissue factor pathway inhibitor (TFPI), the endogenous inhibitor of TF activity, is synthesized and secreted mainly by endothelial cells. TFPI binds to FXa and thereby inhibits TF/FVIIa activity.

Steffel et al; 2006

Induction of tissue factor expression and activity. Induction of tissue factor (TF) is exemplified in an endothelial cell. Various mediators induce TF expression through activation of their receptors. Induction of TF primarily occurs at the transcriptional level, resulting in an increase in TF mRNA and, eventually, TF protein expression. TF is distributed in three cellular pools as cytoplasmatic TF, surface TF, and encrypted TF. Moreover, TF-containing microparticles are released from the cell. Alternative splicing results in a soluble secreted form of TF (asTF). IL-1 indicates interleukin-1; LPS, lipopolysaccharide; TNF-, tumor necrosis factor ; VEGF, vascular endothelial growth factor; HB1B, histamine HB1B-receptor; 5-HTB2aB, 5-hydroxytryptamineB2aB receptor; IL1-R, interleukin-1 receptor; TLR-4, toll-like receptor 4; PAR, proteaseSteffel et al; 2006 activated receptor; KDR, VEGF receptor-2.

Tissue factor in the atherosclerotic plaque. In the inflammatory environment of atherosclerotic plaques, tissue factor (TF) is present at high levels in endothelial cells, vascular smooth muscle cells, macrophages/foam cells, and in the necrotic core. TF induction is exemplified by selected mediators in endothelial cells (EC, left panel), macrophages (M, middle panel), and vascular smooth muscle cells (VSMC, right panel). On plaque rupture, highly procoagulant material including TF-containing microparticles is released into the blood, leading to rapid initiation of coagulation, platelet aggregation, and, ultimately, thrombus formation with vessel occlusion. Steffel et al; 2006

Therapeutic approaches. Several therapeutic strategies have been developed to specifically interfere with the action of tissue factor (TF). Molecular approaches such as ribozymes or antisense oligonucleotides specifically inhibit TF production. Monoclonal or polyclonal anti-TF antibodies directly target and inactivate the TF protein. Site-inactivated factor VIIa (FVIIai) binds to TF but lacks catalytic activity for conversion of factor X (FX) or factor IX (FIX). Recombinant tissue factor pathway inhibitor (rTFPI) interferes with the activity of the TF/FVIIa complex by binding to the active site of factor Xa (FXa), leading to formation of a quaternary inhibitory complex with TF/FVIIa. Similarly, recombinant nematode anticoagulant protein c2 (rNAPc2) interferes with the TF/FVIIa complex by binding to FXa or FX before formation of a quarternary inhibitory complex with TF/FVIIa.

Response to Injury

Endothelial Dysfunction

Initiation of Fatty Streak

Fatty Streak

Fibro-fatty Atheroma

Summary of Atherosclerotic Process

Multifactorial process (risk factors) Initiated by endothelial dysfunction Up regulation of endothelial and leukocyte adhesion molecules Macrophage diapedesis LDL transcytosis LDL oxidation Foam cells Recruitment and proliferation of smooth muscle cells (synthesis of connective tissue proteins) Formation and organization of arterial thrombi

Is Atherosclerosis Reversible
Primate experiments
High fat diet discontinued; atherosclerotic lesions regress

Humans
Decrease fat and caloric intake (wars, famine, wasting disease), atheromas decrease. Angiography after cholesterol lowering, plaque size decreases

What has to happen for plaques to regress?

LDL lowered Mac ingest lipids Reverse cholesterol transport, depends on HDL