Atherosclerosis begins when apo-B 100-containing lipoproteins (LDL, Lp(a), and remnants) infiltrate the artery wall at sites of endothelial activation. This focal perturbation in endothelial barrier function occurs beyond branch points, where low laminar flow combined with high oscillatory shear leads to reduced eNOS (generates nitric oxide) and Nrf-2 (antioxidant enzyme transcription) expression, combined with upregulated NADPH oxidase, xanthine oxidase, and Ang II activity. An intimal local environment is thus created where reactive oxygen and nitrogen derived species (ROS and RNS) are not counterbalanced by nitric oxide and intrinsic enzymatic antioxidant defenses. As flotsam and jetsam accumulate beyond a bend in the river, so will atherosclerosis initiate beyond arterial branch points.

Lipid particles traverse the endothelial cell monolayer via diffusion. The greater the number of lipid particles, and the smaller their particle size, the greater will be the level of passive lipid translocation. Systemic hypertension favors this process, as will other factors, including toxins, that promote systemic endothelial activation (adaptive in our response to true infection but when maladaptively present in atherosclerosis we term this phenomenon endothelial dysfunction).

This initial lipid intimal translocation is not by itself immunopathologic, as apo-B 100 is composed of amino acid sequences to which the innate and acquired immune system is not intolerant. LDL itself is food. It is a transport vehicle for the biosynthetic raw material cholesterol. Every cell of the intima expresses the molecular LDL receptor, at a level commensurate with its perceived need for free cholesterol. If the cell perceives a need for free cholesterol, the receptor will be expressed. If not, the LDL receptor is not expressed. LDL will not be taken up (your stomach is full so you put down your knife and fork).

Once oxidized, LDL cannot be taken up via the molecular LDL receptor; it is no longer biochemically useful. Thus, LDL carries with it antioxidant protection (lipid-soluble antioxidants such as tocopherol, beta-carotene, and coenzyme Q₁₀, which can be “recharged” by water-soluble vitamin C). This defense is typically sufficient to protect LDL within the relatively reductive plasma environment. The extracellular intimal compartment, however, is more pro-oxidative (a thousand times more so than plasma). Standard LDL antioxidant defense levels may not provide sufficient protection. This should not be an issue, as “unneeded” LDL particles, those not ligated by an open LDL receptor, will freely diffuse back into the circulation, and find an open LDL receptor elsewhere. In the absence of infection/inflammation, LDL will be internalized by cells only when and if it is needed for biosynthetic activity (steroid hormones, bile salts, vitamin D synthesis, etc.).

Physiologic intimal LDL give and take, a demand and supply phenomenon, has worked well for Man over our 4-million-year history. Mother Nature promotes hyperlipidemia and focal infiltration of lipids beyond homeostatic synthetic need only when these processes are needed to defend against Man’s natural predator, which is infection. Primitive Man experienced oxidative and inflammatory stress only in relation to infection. Modern Man experiences a pseudoinfectious pathophysiology in a progressive, age-related fashion, in relation to our ROS-generating diet, lifestyle, expanding waist line, and cumulative toxin burden—thus lipid metabolism is deranged!

To fight infection, we need more cholesterol (leukocyte cell membrane synthesis), we need ROS and RNS “bullets” (fired by WBCs to kill the invaders), and we need an activated endothelium (expressing adhesion molecules and elaborating chemotactic signals to pull immune cells into the breach). HMG Co-A reductase, the rate-limiting enzyme in cholesterol biosynthesis, thus upregulates, generating copious quantities of needed cholesterol, along with isoprenoid signaling molecules, which upregulate NADPH Oxidase, our most powerful superoxide generator, and downregulate eNOS, activating the endothelium. Oxidative stress means that “we are at war”!

Oxidative and inflammatory byproducts of Western living, false flags for chronic infection, thus lead to an age-related increase in circulating cholesterol, a progressively activated and thus leaky endothelium, and a biochemical milieu characterized by oxidative stress and dysregulated (Th1/Th17 rich and Treg poor) immune activation.

Thus, we have hyperlipidemia and endothelial activation, particularly beyond branch points. Lipids will infiltrate the endothelium at these stress points, but again, if they are not needed, if there are no open LDL receptors, then they will diffuse out. With respect to pathological atherosclerosis, the first thing to “really go wrong” is lipid retention within the subendothelial space. Retained lipid particles cannot diffuse out and are thus subject to pre-atherosclerotic oxidative modification.

LDL cholesterol is composed of cholesterol and cholesterol ester (food), containing a small number of single-use nonenzymatic antioxidants (protectors), surrounded by the apo-B 100 protein, which is studded with phosphatidylcholine molecules. The rheological characteristics of a given LDL particle relate to the status of its surface phospholipids (the initial composition of which, in turn, relates to relative dietary intake of saturated vs. unsaturated fatty acids).

Phospholipase enzymes, the expression of which is upregulated in relation to systemic and focal inflammation (which itself relates to systemic and focal oxidative stress; as we will discuss), can alter LDL surface phospholipids, causing them to clump together, and bind irreversibly to intimal proteoglycan (rich in chondroitin sulfate) matrix material. Sphingomyelinase (S-Smase), degrades LDL surface phospholipids, and this alteration leads to LDL particle adhesion and clumping. Lipoprotein lipase (LPL), present on the luminal endothelial surface, breaks large lipid particles down into smaller versions, favoring their diffusion across the endothelium (working in our favor within the liver, helping to clear circulating lipid particles, but working against us at vulnerable arterial branch points). All cells of the intima (endothelial cells, vascular smooth muscle cells, and especially activated mononuclear cells) can synthesize LPL. The role of this subendothelial LPL is to bind LDL to proteoglycan. Bound LDL cannot diffuse back out into the circulation. By generating LPL, intimal cells can “keep their food close at hand.” Phospholipase A2 (PLA2) splits a fatty acid (typically arachidonic acid in Industrialized Man) from the second carbon position of LDL surface phosphatidylcholine, to generate lysophosphatidylcholine (LysoPc) and one arachidonic acid molecules. The latter is subject to enzymatic (cyclooxygenase) and nonenzymatic (oxidative) conversion into downstream pro-inflammatory mediators (such as thromboxane A2). Lysophosphatidylcholine itself leads to endothelial activation. Its presence within the subendothelial space means that “something is wrong.” LysoPc (and other downstream products of phospholipid degradation such as diacylglycerol and platelet-activating factor) stimulates endothelial cells to express adhesion molecules (VCAM and ICAM), such that circulating monocytes can be drawn in to investigate this initial perturbation of intimal homeostasis (from Mother Nature’s perspective, likely another infectious breach). As with the case of S-Smase alteration, LDL particles containing PLA2-modified membrane phospholipids will adhere and clump together on proteoglycan (Figure 3.1). Clumped LDL particles will no longer “fit” into an open LDL receptor. They may appear as “debris” and thus are subject to ligation
by the scavenger receptor of resident tissue macrophages (roving monocytes that have wandered into the subintimal space looking for microbes or cellular debris), with subsequent endocytosis, followed by macrophage activation.

Altered (termed minimally oxidized, or MM-LDL) and now immobilized LDL particles will be subject to further oxidative alteration by free radical species generated within the subintimal space (initially with superoxide generated during normal oxidative metabolism and later with superoxide and secondary ROS/RNS species generated by pathologically upregulated NADPH oxidase [NOX], xanthine oxidase [XO], angiotensin II—angiotensin receptor type 1 (AT1R) trafficking, and HMG Co-A reductase expression).

Carbon to carbon double bonds on LDL particle surface area are polyunsaturated fatty acids (PUFAs), susceptible to oxidative alteration, convert into reactive aldehydes, which form adducts with exposed lysine and arginine of the apolipoprotein B100, altering LDL particle configuration and charge. The now heightened negative charge of the oxidized LDL particle (oxLDL) allows tighter binding to the positive charge of proteoglycan sulfate and carboxyl groups as well as to the scavenger receptor of tissue mononuclear cells, favoring its immobilization and uptake, respectively. Of greater consequence, this “acquired mutation” of apo-B 100 creates a protein sequence, or more precisely a protein shape, to which developing T lymphocytes were not exposed to during their maturation within the thymus. Oxidized apo-B 100 thus appears as a foreign molecule, an invader, a microbial “look alike” that must be killed or neutralized by the full force of Mother Nature’s anti-infectious defense mechanisms.

At this point we have trapped LDL molecules that are being oxidized, and an activated, nitric acid-poor, endothelial surface that is nonspecifically pulling in monocytes (termed macrophages upon entrance into the intima). Macrophages, as do all cells of the intima, bear LDL receptors. Oxidized LDL is not ligated by the native LDL receptor; it no longer fits. Rather the scavenger receptor expressed by mononuclear cells recognizes oxidized LDL as a microbe or cellular debris. Oxidized LDL is thus taken in. Having captured this nonnative and thus threatening particle, the macrophage activates. We kill microbes with superoxide and downstream ROS and RNS, and thus their production within the macrophage is increased. More scavenger receptors are elaborated. The activated macrophage will also express nonphagocytic threat receptors (such as TLR4, the Toll-like receptor, and cytokine receptors), such that it can better sense its local environment and carry out its search and destroy mission. ROS that cross into the local environment serve to hasten oxidation of adjacent immobilized but not-yet-oxidized LDL particles. The activated macrophage releases chemotactic molecules (such as MCP-1, monocyte chemotactic protein), creating a chemoattractant trail to lead mononuclear cells that ligated endothelial adhesion molecules to actively translocate to the point of perceived infection, and activation signals, such as MCSF (monocyte colony stimulating factor), stimulating the translocating mononuclear cells to “lock and load,” upregulating ROS/RNS generation and scavenger and threat receptor expression.

Intimal infection has been diagnosed. The endothelium further activates. Monocytes, along with second wave monocular defenders (dendritic cells and T memory cells), swarm in, more cholesterol is oxidized, and more oxidized cholesterol is phagocytosed (Figure 3.2). At this point, the activated macrophages contain more cholesterol than it needs for biosynthetic purposes, and thus its native LDL receptor should be withdrawn. That is not the case. As will be discussed in more detail later, inflammatory cytokines, the products of nuclear factor kappa beta (NF-κB) translocation, stimulate the conversion of free cholesterol into cholesterol ester (storing fuel for the infectious winter to come). As expression of the native LDL receptor relates to the level of free, not esterified cholesterol within the cell, the LDL receptor remains expressed even though the activated macrophage is chock full of phagocytosed oxidized LDL. The activated macrophage, eager to nonspecifically rid the local environment of all potential threats, not just the threat it perceives from oxidized LDL, begins to pinocytose adjacent molecules, including nonoxidized LDL particles.

We now have an activated macrophage, loaded to the gills with oxidized cholesterol, sending out distress signals to attract and activate fellow first-line defenders. Although later arriving, innate immune defenders can leave this inflamed intimal environment to warn the adaptive immune system as to the specific nature of the oxLDL threat, the early arriving and now cholesterol choked macrophage inactivates, transforming into a lipid-laden foam cell. Apoptosis and coalescences of foam cells create the fatty streak, the initial histologic manifestation of atherosclerosis.

This process, of course, is reversible. When a microbial threat has been neutralized, a wave of nonactivated macrophages (M2 macrophages) will infiltrate the previously inflamed region and phagocytose the dead first-line defenders, allowing function and histology to return to normal (termed catabasis). This process is programmed to occur. When Mother Nature initiates an inflammatory response, the biological clock begins to tick, and a few days later the immune response shifts from search and destroy to inflammation resolution, as the cytokine milieu shifts from Th1/Th17 (IL-1β, TNFα, IL-6, IL-17, IL-23) to Treg (IL-10 and TGF-β). This programmed shift from infiltration to catabasis will occur, of course, only if the infectious threat is indeed neutralized. If microbes continue to breach the endothelial barrier, the inflammatory response is not called off, and inflammation resolution will not occur. In the setting of human atherosclerosis, if the level of lipid particles diffusing into vulnerable endothelial sites decreases, then the foam cells will be resorbed. In contrast, if cholesterol infiltration continues (the situation in Industrialized Man), then catabasis will not occur, more foam cells will be created, apoptosis
will give way to tissue necrosis, and we now have a mass of coalesced lipid droplets and crystals within the vascular wall, the lipid core of the developing plaque.

Even before we get to this point, of atheroma development, and well afterward, we do have a means of removing cholesterol, and reversing LDL oxidation, within the intimal environment. Mother Nature (evolution or the creator, depending on your perspective) knew that our corrective response to endothelial microbial breach in the setting of infection-induced hyperlipidemia could lead to macrophage lipid engorgement and subsequent apoptosis. Thus she (or he, again depending on your perspective) created the HDL system. The HDL-associated enzyme lecithin cholesterol acyl-transferase (LCAT) esterifies free or deesterified cholesterol from mononuclear and intimal cells with linoleic acid, and then loads up the newly formed cholesterol-linoleate into the HDL particle, for transport back to the liver. Paraoxonase, another HDL-associated enzyme, removes lipid peroxides from mononuclear cells, intimal cells, and lipoproteins, essentially reversing LDL oxidation (and why stimulating HDL reverse cholesterol transport and antioxidant function protects against disease progression and adverse events).

Anatomic atherosclerosis begins with the fatty streak. If cholesterol levels fall, then catabasis and reverse cholesterol transport can occur, and the fatty streak will resorb. If cholesterol continues to breach the endothelium, then more mononuclear cells will be brought in, and the fatty streak gives way to the slowly growing atheroma. The focal absence of endothelial-derived nitric oxide allows vascular smooth muscle cells to proliferate and migrate into the endothelial zone, adding bulk to the atheroma. At some point the lesion will become visible on angiography.

So far, we have confined ourselves to the innate immune response, how pattern receptors on mononuclear cells react to perceived environmental threat. But atherosclerosis is a maladaptive response of the acquired immune system to perceived infection of the arterial wall with oxidized LDL and other perceived nonnative entities. How do we go from LDL oxidation to the Th1/Th17-rich, Treg-poor intimal environment where interferon-gamma stimulates macrophages to release matrix metalloproteinases to degrade the fibrous cap and precipitate an acute coronary event? A review of immune system dynamics is thus warranted.

Immune system effector cells, both innate and adaptive, originate within the bone marrow from a common hematopoietic progenitor stem cell line. Innate immune cells sense threat in a nonspecific fashion; they respond to abnormal shapes.
They sound an alarm and stimulate an adaptive or acquired immune response against a specific invader (in true infection, a snippet of bacterial protein; in atherosclerosis, initially an 8- to 20-amino-acid-long snippet of oxidized apo-B 100).

Innate immune cells sample the environment. A large surface area facilitates this function, and thus they send out cytoplasmic pseudopods (hence the generic term dendritic cells), coated with receptors that recognize nonnative shapes. PAMPs (pathogen-activated molecular receptors) recognize lipopolysaccharide of gram-negative microbes and like molecules expressed by gram-positive bacteria. DAMPs (damage-activated molecular receptors) recognize debris of apoptotic cells or particulate matter and oxLDL. In fact, oxLDL is the most common DAMP. Monocytes serve as circulating dendritic cells. Upon entry into the intima or other tissue compartments they transform into macrophages. Kupffer cells (liver), microglia (brain), Langerhans cells (skin) are all dendritic cells that have specialized to sense threats found within these specific tissue environments.

So, a microbe breaches our skin, or an oxidized LDL protein forms within the intima. PAMPs on the first in dendritic cell ligate the nonnative shape in question. The structure is then phagocytosed into a lysosome, where it is degraded into its component parts, including snippets of its protein structure. Having captured an invader, the roving dendritic cell now migrates to the nearest lymph node or lymph organ (liver or spleen), to inform the adaptive immune system as to the specific threat faced, aiming to initiate a rapidly amplifying immune response (antibodies and cytokine elaborating T helper cells) directed against the specific invader. In this process the dendritic cell, functionally an antigen capture cell, converts itself into an antigen-presenting cell (APC), prepared to present what it caught on a MHC II molecule, to display co-stimulatory molecules, and to secrete co-stimulatory cytokines, all in an effort to awaken a naïve T helper cell, to serve its one and only function, to activate, proliferate, and then direct an overwhelming immune response to a specific threat.

The lymphocyte population of the acquired immune response consists of B cells and T cells. B cells generate antibodies, only in response to T cell instruction, aiming to immobilize or damage invaders that are not destroyed via the mechanism of macrophage phagocytosis. B cells play little role in atherosclerosis and will not be discussed further. Nascent T cells leave the bone marrow, and migrate to the thymus, where they are “educated.” Within the thymus, T cells will be exposed to antigenic determinants (8- to 20-amino-acid-long snippets of native protein) that they will see within their lives within the specific human. T cells that react with normal proteins will be culled via apoptosis. The trillion different T cells that survive thymic maturation can “read” antigenic determinants presented on MHC molecules. No surviving T cells should react to a self-molecule (clinical auto-immunity occurs only when a self-molecule has been altered, such as in myocardial infarction, or when chronic, unrelenting oxidative and inflammatory stress overstimulates the immune system, such that it begins to indiscriminately misrecognize self as foreign). Following thymic maturation, naïve, nonactivated T cells migrate from the thymus to peripheral lymph organs, where they do nothing until they are awakened from biochemical slumber by an activated innate immune cell that bears an antigenic determinant, on an MHC II molecule, that is a specific match for the T cell receptor of the resting T cell.

Innate immune cells thus awaken dormant T helper cells by presenting antigenic determinants on an MHC II molecule. What are MHC molecules? What are T cell receptors? How does the acquired immune system activate to legitimate infection, or pathologically to oxidized LDL or troponin?

MHC (major histocompatibility) molecules are flag poles that can fly different flags. Cell membrane-bound MHC molecules “present” 12- to 20-amino-acid-long protein snippets, designed to inform adjacent or roving T cells as to the proteins contained within them.

Nonimmune, somatic cells bear MHC I molecules. Their job is to inform and activate natural killer T cells and CD8 self-surveillance T cells if their cell has undergone malignant transformation or viral invasion. Our cells generate specific proteins, in relation to which genes are being transcribed within the nucleus, when specific mRNA molecules are translated into specific amino acid sequences at the level of the ribosome. Transcribed proteins contribute to cell structure and function. After a period of time each cell protein will be degraded, thus allowing for an adaptive turn over in cellular protein, in relation to which genes are being translated, related to differing intracellular conditions, related to differing signals the cell is receiving from its outside local and systemic environment. Intracellular proteins are degraded within proteosomes, which will cut the protein into 12- to 20-amino-acid-long antigenic determinants, which are then attached to MHC I molecules to be expressed on the cell membrane. CD8 and T killer cells are monitoring this situation. If all intracellular proteins are native, then the antigenic determinants presented on MHC I molecules are something that they have seen before, something to which they are tolerant, and no action is taken.

However, if the cell has been hijacked, say by a virus, and is now cranking out nonnative, viral proteins, or if the cell has undergone malignant conversion and is generating inappropriate proteins, then nonnative antigenic determinants will be expressed on the MHC I molecules. Roving natural killer and CD8 T cells will recognize this anomaly, bind to the now rogue cell within an immune synapse, and then destroy it (clinical cancer occurs only when this monitoring process breaks down).

Cells of the innate immune, first-line defenders, also bear MHC II molecules, designed to “show off” the microbes that they have captured. Say you are a Kupffer cell in the liver or a macrophage or dendritic cell that has just phagocytosed an oxidized LDL molecule. Pattern receptor ligation with these entities tells you that something is wrong, that some form
of invasion has occurred, so you gobble up the target entity, begin to degrade it within your phagolysosomes, and then leave the site of antigenic capture and make your way to the nearest lymph organ, to “sound the alarm.” As your travel you transform yourself from an antigen capture cell into an APC. You mount antigenic determinants derived from the bacteria or oxLDL protein that you took in on to an MHC II molecule, and “show it off” to resting T cells within the lymph organ. If your antigenic determinant is an exact match to the configuration of the T cell receptor on one of the trillion T helper cells previously released by the thymus, then an acquired immune synapse will occur.

The initial immune synapse, at least 100 “lock in key” MHC II-antigenic determinant-T cell receptor molecular couplings between the APC and naïve T cell, is necessary to initiate an acquired immune response directed against the specific antigenic determinant (and the microbe from which it was derived), but by itself is not sufficient. The APC must elaborate co-stimulator molecules and co-stimulatory cytokines to “fully wake up” the resting naïve T cell. Stated otherwise, the APC must be “alarmed and angry” if it to fully “lock and load” the resting T cell (in this fashion, we will not mount an acquired immune response to a normal protein that was accidentally internalized by a first-line defender). Interleukins released by the APC awakens the slumbering naïve T cell. As it is activating, the previously naïve T cell will likewise release cytokines to further activate the APC to stimulate its own clonal proliferation. The greater the level of mutual co-stimulation, the greater will be the level of T cell activation and clonal proliferation.

CD80 and CD86 (often referred to as B7) on the APC will interact with CD28 on the resting T cell. The now activating T cell will express CD40L, which will interact with CD40 on the APC. CD4 on the T cell (T helper cells are all CD4 while T cells that eliminate rogue native cells are termed CD8) coordinates the interaction between an antigenic determinant bearing MHC II molecule on the APC and the TCR (T cell receptor) on the resting T cell.

Mother Nature thus created a number of checks and balances (which we can understand and therapeutically manipulate) with respect to whether or not the immune system will activate against a specific antigenic determinant, as well as to the direction and magnitude of any specific immune response to follow.

Naïve T cells activate into one of four lineages. If an antigen capture cell is bearing news of a large invader, such as a parasite, that cannot be phagocytosed by immune cells, then it will elaborate cytokine IL-4, which directs the activating T cell to differentiate within the Th2 lineage. Th2 cells instruct specific clones of B cells to generate antibodies to neutralize and kill the critter. If the first-line defender instead has captured a bacterium which can be phagocytosed by mononuclear cells, then it will activate within the Th1 or Th17 framework. These T helper cells secrete stimulatory cytokines and bear membrane signaling molecules designed to activate mononuclear cells to phagocytose and kill bacterial invaders. Not all antigens captured by innate immune cells are an appropriate target of an immune response. Antigens derived from inhaled or ingested molecules ideally should not lead to an immune response; in this situation we would become immunologically reactive to molecules that we breathe in or take in within our diet. Thus, the biochemical milieu within immune organs in the upper respiratory and GI tract promotes activation of T cells into the Treg lineage. Treg cell membrane-expressed CTLA-4 binds tightly to co-stimulatory molecules expressed by the APC, turning down the APC’s activation state. Soluble Treg-generated cytokines such as IL-10 and TGF-β turn down APC and T helper cell activity (stable plaques contain Treg cells; this inhibition is lost in unstable lesions).

Above and beyond the characteristic of the protein snippet presented to the resting, inactive T cell, and the location at which the immune synapse is occurring, the internal biochemical milieu of the lymph organ plays a role in determining the route into which the T cell will develop. Vitamin D, for example, alters the characteristics of the APC, such that the co-stimulating molecules that it elaborates will encourage the newly stimulated T helper cell to take an immune downregulating Treg course (consider the link between vitamin D sufficiency/insufficiency and the incidence of multiple sclerosis, which involves autoimmune attack against self-protein).

The cytokine and redox status of the lymph organ will greatly influence the lineage of a newly activated T cell (Figure 3.3). One’s immune history, in a sense, determines one’s immune future. If you are a chronic allergy sufferer, you bear Th2 cells that secrete IL-4 and stimulate B cells to make antibodies (unfortunately for you) to the pollen that you just inhaled (not enough Treg cells and too many Th2 cells and thus allergy occurs). Allergy begets more allergy, as the more IL-4 within the lymph organs, the more likely will you mount an undesirable Th2 response to newly presented protein snippets. Conversely, lots of IL-12 within the lymph organ skews new immune responses to newly presented antigenic determinants down the Th1 pathway. If you suffer from recurrent bacterial invasion, as did primitive man, then
the lymph node is rich in IL-12, and you tend toward a Th1 response. Recurrent infection adds IL-17 to the mix, stimulating T cells to mature within the Th17 lineage (essentially a more powerful version of the TH1 cell).

Oxidative stress trumps all other T lineage determinants and promotes maturation down the Th1 and Th17 pathways. The immune response to oxidized LDL, and other abnormal intimal proteins, as well as to troponin and other myocardial molecules to which a deleterious T cell response occurs in heart failure, is Th1 and Th17 driven. CV disease is characterized by skewing of the immune response, toward Th1/Th17 and away from Th2/Treg. Oxidative stress initially skews the immune response, these T cells release inflammatory mediators that lead to more oxidative stress, and the viscous, self-stimulating cycle of oxidative stress and immune dysregulation that drives CV disease follows.

Once activated by an upregulated innate immune cell that bears an antigenic determinant that matches its T cell receptor, the T cell assumes its role as a T effector cell. The cell line proliferates (generating IL-2 and a high affinity IL-2 receptor, leading to oligoclonal proliferation of this specific T helper cell line) and the daughter cells migrate to the periphery, aiming to kill their target (microbe in infection and oxidized LDL in atherosclerosis) at the site of initial stimulation of the innate immune system and at all other points in the body.

The previously naïve T cell bearing a T cell receptor specific for a protein snippet of a microbe (in this example bacteria or a snippet of oxLDL) has activated and proliferated. Millions if not billions of these activated T helper cells are soon swarming the circulation, seeking to help macrophages and other innate defenders phagocytose and kill the invaders (before they can kill us).

How do the T cells know where to go? Although the initially activated (within the lymph organ closest to the site of initial infection) Th1 helper cell can access any site within the vasculature, it makes more sense if they can be drawn into the region where the innate immune system initially identified the focal breach. This will be an area where activated immune cells (macrophages and dendritic cells) have generated ROS/RNS and inflammatory cytokines, such that the local endothelium has activated, generating chemokines (MCP-1) to pull circulating mononuclear cells toward the site of infection as well as adhesion molecules (ICAM and VCAM) to tether the mononuclear cells to the site of endothelial activation, making it easier for the cells to enter the subintimal space (where their target, invading bacteria or oxLDL snippets, reside). The first T helper cell bearing a T cell receptor specific to a protein snippet derived from the invader (microbe or oxLDL) has now been drawn into the area of infection.

Along with the newly activated T helper cell line, our circulation also contains T memory cells, allowing the acquired immune system to rapidly respond to recurrent infection. After a microbe has been eradicated (a joint effort between the innate and acquired immune systems), local ROS/RNS generation tails off, T cells specific to the target cease to proliferate, and most then die of senescence. A few persist, as T memory cells. They “remember” the threat, continue to bear TCRs specific to the threat (the specific antigenic determinant), and exist in a dormant state. However, upon reinfection, reexposure to “their” remembered antigenic determinant, they can fire up rapidly, undergo oligoclonal proliferation, and “return to the breach.” With your initial exposure to a specific microbe you may be sick for a week, the time needed to mount an effective acquired immune response to the specific organism. Upon reexposure, you may be sick for 1 to 2 days or not at all, as you now possess an at least partial “immunity” to the invader.

Vaccination creates T memory cells to specific potential invaders. Antigenic determinants derived from microbes or inactivated microbes are administered along with adjuvants (pro-inflammatory substances such a thimerosal) that stimulate innate immune cells to activate, internalize the exogenous protein, and then transform into an APC and initiate an acquired immune response to the potential invader. If the CDC accurately predicts which flu strains will dominate in a given year, then they will create a flu vaccine that generates T memory cells that will recognize and eradicate the incoming viruses. If they predict incorrectly, the flu vaccine will not protect you, as the flu virus is constantly mutating, trying to outwit mankind’s defenses (our T memory cell repertoire).

The circulation contains T memory cells, related to prior invasion, and newly activated T helper cells specific for the new infection (or pseudoinfection with oxLDL in atherosclerosis or altered myocardial molecules in heart failure). All of these T cells, along with circulating dendritic cells, will be attracted to and pulled into a region of endothelial activation, where infection resides, or (pathologically) at coronary branch points where ROS/RNS generation has activated the endothelium, allowing entry of LDL molecules that subsequently became trapped, oxidized, and internalized by mononuclear cells. All of these infiltrating T cells will sample the local intimal environment. If there are no activated intimal cells (macrophages or dendritic cells initially but within the oxidative milieu of an activated plaque smooth muscle and endothelial cells can transform into APCs) expressing “their” specific antigenic determinant, then they will return to the circulation, to see if they can be useful elsewhere.

If instead they undergo a second, lock in key interaction, with an intimal dendritic cell expressing “their” specific antigenic determinant, then a secondary, immune reactivation will occur. The job of the locally reactivated T helper cell is to help other cells in its vicinity to destroy the invader. Cell to cell contact (CD40L-CD40 or membrane bound TNF-TNF receptor) or secretory (interferon-gamma, IL-1β, and TNF) stimulate the APC into a phagocytic frenzy, generating more MHC II molecules and more lethal ROS/RNS. Although the interaction between the T helper cell and the APC is specific to a specific antigenic determinant, the cytokines released as a result of this antigenic determinant-specific interaction
will affect the general inflammatory status of all cells in the vicinity. The greater the number of individual skirmishes (T helper cells activating macrophages to kill a specific target), the greater will be the potential to damage, and potentially scar, the tissue battlefield.

Thus, the number and type of immunoreactive antigenic determinants within a local intimal environment (and the corresponding number and type of ligating T cells) will determine its overall activation state. If the molecules within the intima are normal, self in nature, then its T cell repertoire will consist of Treg cells, which release IL-10 and TGF-β, which downregulate, or temper the level of immune activation. An activated plaque contains few Treg cells, and 20 to 40 clones of T helper cells that have undergone local oligomeric proliferation in response to reactivation against 20 to 40 specific antigenic determinants. The local milieu will be inflammatory (Th1 and Th17 cytokines) and oxidative (these cytokines stimulate endothelial, smooth muscle cells, and infiltrating mononuclear cells to generate ROS/RNS).

20 to 40 clones of T helper cells reacting to 20 to 40 specific antigenic determinants! What are the determinants, from what proteins were they derived, and how did they get into the intima? We can qualitatively identify the T cell repertoire in plasma, within a plaque, within any region of the body. The T cells are harvested, and in vitro reacted with antigenic determinants derived from specific microbes or specific proteins. If T cells proliferation occurs (you measure radiolabeled thymidine incorporation), you can infer that T cells bearing TCRs specific for that microbe/protein snippet were present in the tissue sampled (and if you are dealing with an atherosclerotic plaque, that the same microbe/protein snippet was present within the plaque, at some time point). In the absence of infection, plasma will contain small numbers of T memory cells specific to any infection that you have experienced, a bell-shaped curve or polyclonal distribution of lymphocytes. If you contract an infection, say pneumonia, you will see skewing of the T cell repertoire, with oligoclonal proliferation of T memory cells specific to the invader or previously naïve, newly activated T helper cells reacting to a newly identified microbial antigenic determinant. As the infection clears, T cells specific to the microbe will die of senescence, and the polyclonal distribution will return. The T cell repertoire within a plaque relates to the age of the lesion, infectious activity elsewhere in the body, and local and systemic levels of ROS/RNS and cytokine milieu. 10% of the T cells within any plaque will react to oxLDL. A similar number will react to intimal structural proteins such as beta-2 glycoprotein and heat shock protein (HSP). Stressed cells, human and microbial, translocate mitochondrial HSPs to the cell membrane, aiming to stabilize their cytoskeleton (you are under attack, so circle your wagons). Intimal HSP expression is negligible within the healthy intima, low level within an early lesion, and extensive within a complicated lesion. But these are self-proteins. Why should they be subject to an acquired immune response? These proteins can be oxidized, rendered immunogenic, just as in the case of oxidized LDL. Also, in the presence of overwhelming ROS/RNS/cytokine stimulation, the frenzied acquired immune system can “break tolerance,” misrecognizing self-proteins (that are at the wrong place at the wrong time) as nonself (consider the link between intestinal hyperpermeability and autoimmune disease; with chronic cytokine stimulation the immune system starts making mistakes).

If you carry out incidental atherectomy of a noninflamed, nonculprit, 60% RCA narrowing in your patient who presents with chest pain and ST segment depression on the basis of an inflamed 95% LAD lesion, you will find that the T cell repertoire and immune histology of these two lesions are quite different. To paraphrase Tip O’Neil, atherosclerotic immune activation is local. The smooth nonculprit narrowing will contain macrophages, dendritic cells, and T helper cells, certainly some specific for oxLDL protein and other altered intimal proteins. Within a stable plaque, there will also be Treg cells, elaborating IL-10 and TGBF, aiming to neutralize the interferon-gamma and other Th1 cytokines being released from plaque Th1 helper cells, keeping plaque inflammation in check. Nitric oxide inhibits vascular smooth muscle cell (VSMC) growth and proliferation. Nitric oxide was lost long age, and proliferated VSMCs, along with fibroblasts, have generated a stabilizing fibrous cap, sequestering lesional atheromatous gruel and inflammatory activity from the vessel lumen. Such lesions, if they obstruct the lumen sufficiently, may produce effort-induced ischemia. If adjacent vessels are not severely diseased, and if local nitric oxide is available, then the pressure differential between the patent and diseased vessel will lead to the elaboration of a protective collateral network. Stable plaques, characterized by a low ROS/RNS/inflammatory cytokine burden, will not rupture/erode to precipitate ischemic injury (as a therapeutic corollary, if we can convert an inflamed plaque into a stable plaque, ischemic event risk will attenuate).

The culprit plaque demonstrates a quite different immune histology. Along with T helper cells specific for oxLDL and other altered intimal proteins, you will find 20 to 40 clones of T cells specific to a wide variety of microbes. You are aware of the link between infectious history and atherosclerosis. The greater the number of microbes to which you display immune experience (reactive T cells or IgG antibody levels), the greater is your atherosclerotic risk (and risk of disease recurrence following revascularization).

How does this link work? Are the microbes invading the intima, or is the link indirect? Recall that an active plaque is constantly elaborating chemotactic signals and adhesion molecules, aiming to pull in mononuclear help (Mother Nature is fighting what she perceives as infection, initially with oxLDL). Let us say you bear a chronic bacterial infection (gum disease being the most common). Mononuclear cells infiltrate the gum tissue, gobble up the corresponding microbes, and digest them within
phagolysosomes. Some hightail it to the nearest lymph node, generating a proliferation of Th1/Th17 cells specific to the invader, while some return to the circulation. If that mononuclear cell happens to traverse a coronary vessel containing an active plaque, it may be nonspecifically pulled into the lesion. There it may display gum microbe antigenic determinants to gum microbe sensitized T cells (T memory cells) that have also been nonspecifically pulled into the lesion, reactivating them to battle mode. As long as gum disease is present and the plaque remains active, more and more gum bacterial antigenic material will enter the plaque, and thus the immune system will be mounting an inflammatory response against gum disease within the active plaque. Nearly all plaques contain T cells sensitized to gingival invaders such as Porphyromonas gingivalis. Common pulmonary pathogens such as Chlamydia pneumoniae and Mycoplasma pneumoniae, frequently encountered viruses such as EBV and CMV, and GI and GU microbes will also be represented. In fact, virtually any infection can do this, such as bacterial, viral, fungal, TB, parasite, and other infections. As long as the plaque is active, focal infection elsewhere will contribute to plaque immune dysregulation. Keep this principle in mind the next time you attend a meeting in Las Vegas, where you might be tempted to interact with the wrong sort of people without proper protection. Do you want those sorts of microbial proteins within your coronary intima? The link between infection and atherosclerosis led to antibiotic intervention trials, which for the most part were unsuccessful. This is because the bacteria within the intima are long dead. Within the coronary vasculature T cells are reactivating to their antigenic determinants, not to the microbes themselves. Preventing chronic infection (and thus chronic immune stimulation) by killing the microbes where they reside, however, will be helpful (thus resolution of gum disease reduces your coronary risk). Also, recurrent bacterial infection will lead to skewing of the immune response toward Th1/Th17 and away from Th2/Treg, creating a cytokine milieu that drives atherosclerosis and heart failure. Heat shock protein (HSP) molecular mimicry is another link between extravascular infection and atherosclerosis. The HSP concept has worked well in evolution, for man and microbes. The HSP amino acid sequence of man and his common pathogens are little different. In response to infection, let us say with C. pneumonia, we will mount an immune response against antigenic determinants derived for this organism. The invaders feel the heat, and thus elaborate HSPs on their outer surface. We then respond by generating an immune response against C. pneumonia HSP, hastening clearance of the invader. Healthy endothelial cells do not express HSP; unhealthy cells (smokers, diabetics, individuals bearing a toxin burden) do, as do atherosclerotic cells, at a level commensurate with the degree of local immune dysregulation. Smokers experience more MIs in the winter than in the summer, in part related to immune cross-reactivity between endothelial and microbial HSPs.

The atheromatous core of a stable plaque is contained by a fibrous cap. In response to dietary/lifestyle change and/or appropriate pharmaceutical/nutraceutical intervention, ROS/RNS generation will have curtailed, Treg cells will be neutralizing Th1/Th17 activity, and endothelial nitric oxide production may return. This plaque will not progress and it will not activate.

The active or vulnerable plaque demonstrates quite dissimilar cytology and histology. The plaque will be infiltrated, particularly at its shoulder region, with activated mononuclear cells, all elaborating pro-inflammatory cytokines, ROS, and RNS. Plaque-derived chemokines are pulling in mononuclear cells bearing antigenic determinants from distant sites; foreign wars are now being conducted locally, and we see oligoclonal proliferation of 20 to 40 clones or Th1/Th17 cell lines within the plaque. Ongoing, intense inflammation leads to Treg cell regression (we do not want Treg cells turning down our Th1/Th17 defenses when we are fighting chronic infection). The character of the T cell response also changes. Recall that initial cell activation and T memory cell reactivation requires “lock in key” co-stimulatory as well specific antigenic determinant-T cell receptor interaction. In response to chronic re-activation to its specific antigenic determinant, T helper cells mutate such that their co-stimulatory receptor, CD 28, drops off. These cells (CD4+CD28^null) spontaneously express IL-12 receptors and may activate in response to their antigenic determinate, or simply to IL-12. Thus, immune activation elsewhere, which might lead to IL-12 generation, can activate these cells within the unstable plaque (another link between winter infection and MI). CD4+CD28^null cells are long-lived, spontaneously generate interferon-gamma, and are cytotoxic to endothelial cells. These cells may egress the activated plaque, may infiltrate distant intimal sites, and “splash” the vasculature with interferon-gamma. If you bear a 50% carotid plaque, it is far more likely to itself activate over the 2 years following an ACS (acute coronary event) than over a corresponding time period pre-ACS (you can think of this as metastatic plaque activation). The greater the percentage of plaque and circulating T cells that are CD4+CD28^null in character, the greater is the likelihood of subsequent ACS (and conversely, the greater will be your gain with anti-atherosclerotic therapy).

Large plaques may neovascularize. Hemorrhage within the plaque releases free iron, which catalyzes the Fenton reaction (discussed in more detail later), converting relatively benign H₂O₂ into 1000-fold more damaging hydroxyl anion. ROS-stimulated NLRP3 pathway activation (discussed in the Colchicine and NLRP3 section) also plays a role in plaque activation. “Frustrated phagocytosis” of crystalline cholesterol leads to a polymorphonuclear cell-led inflammatory response that activates NF-κB-generated pro-cytokines, precipitating plaque activation.

Atherosclerosis begins at points of intimal oxidative stress. Oxidative stress drives plaque progression and activation. Our goal, therapeutically, is not just to lower cholesterol
(which rises with age in relation to oxidative status), but to lower oxidative stress, and with it inflammation and the dysregulated, Th1/Th17-skewed immune response that drives vascular disease. A discussion of oxidative stress, how it relates to atherosclerotic risk factors, and how to attenuate it is thus in order.

Generation of superoxide (SO) and its second messenger hydrogen peroxide (H₂O₂) is constitutive and necessary for normal cellular function. Their synthesis can be physiologically upregulated to deal with infection and trauma.

Oxidative stress (OS) occurs when the production of these reactive oxygen species (ROS) is chronically and inappropriately increased, overwhelming our innate enzyme-based and dietary small molecule antioxidant defenses, allowing their conversion into more long-lived and damaging ROS and reactive nitrogen species (RNS) such as peroxynitrite (ONOO), hydroxyl (OH), and hypochlorous acid (HOCL) (Figure 3.4). When underdefended intimal ROS/RNS generation leads to endothelial activation, lipid trapping, and oxidation, and secondary Th1/Th17 skewed immune dysregulation, we experience intimal oxidative distress, followed by atherosclerosis and its sequelae.

Free radical species, ROS and RNS, are reactive electrophiles; they are electronically imbalanced. ROS such as SO contain an unpaired electron within their outer orbital shell. Absent a neutralizing electron donor (an antioxidant), a ROS will snatch an electron from an adjacent structure, quenching its electron thirst, but creating a new radical species. A biochemical chain reaction occurs, with resultant damage to cellular lipids, proteins, and nucleic acids.

Antioxidant enzymes (mineral dependent) convert a ROS to a less toxic metabolite. “Spent” antioxidant enzymes must then be recharged by a secondary antioxidant enzyme, or be resynthesized. Diet-derived antioxidant molecules “donate” an electron to the ROS, quenching its electron thirst, rendering it nonreactive. “Spent” antioxidants can be recharged by other antioxidants. Mother Nature designed us to maintain a dynamic and adaptive balance between ROS generation and antioxidant neutralization.

Primitive Man did not experience inappropriate or chronic oxidative stress. When faced with infection, microbicidal ROS/RNS generation was transiently increased, allowing for threat eradication. Modern Man is experiencing chronic oxidative stress, rarely because of chronic infection, but endemically because of errors of modern living, such as diabesity, intestinal hyperpermeability, chronic emotional stress, and age-related accumulation of radical-producing metal and organic pollutant toxins, phenomena not anticipated by evolution. Risk factors for atherosclerosis are either a cause or a consequence of inappropriate ROS/RNS generation.

The primary source of constitutive SO production is the mitochondria, where 1% to 2% of inhaled oxygen is incompletely reduced to SO, designated as a free radical as SO contains an unpaired electron in it outer orbital shell (molecular oxygen contains two, and is chemically far less reactive). Housekeeping enzyme systems (protein generation, digestion, and nutrient assimilation) generate lesser quantities of SO. SO is short-lived, but if not converted into H₂O₂ by superoxide dismutase (SOD) it can react with nitric oxide (NO) to generate peroxynitrite (ONOO).

Intimal ONOO generation, outside of our response to infection, is 100% maladaptive, as are the other “atherosclerotic bastard” descendants of SO and H₂O₂ (OH, HOCL, and lipid radicals). They fill no homeostatic need, they are not neutralized by endogenous antioxidant enzymes, they oxidize indiscriminately, and in this manner they initiate and drive atherosclerosis, malignancy, and neurodegenerative disease states.

As stated above, ROS generation is constitutive, a necessary byproduct of oxidative metabolism. Low-level H₂O₂ trafficking is also necessary for multiple cellular housekeeping functions, including protein folding, and appropriate cell growth and differentiation. Mother Nature generates and needs ROS, at a low level tonically, and at a high level transiently, when dealing with infection. Keeping SO and H₂O₂ under control, and preventing their conversion into ONOO, OH, and HOCL, is the responsibility of our mineral-based antioxidant defense enzymes, backed up, as needed, by diet-derived small molecule antioxidants.

Superoxide dismutase (SOD), present as three-cell compartment-specific isomers, rapidly converts SO into H₂O₂. If SO generation is not pathologically (or appropriately in infection) upregulated, SOD will convert SO into H₂O₂, and ONOO will not be generated. Copper and zinc-dependent SOD1 neutralizes SO within the cytoplasm. Manganese-dependent SOD2 degrades SO that is generated within the mitochondria. SOD3 (often referred to as extracellular SOD) is present on the endothelial surface or released into the intimal space; SOD3 protects these compartments against NO to ONOO conversion. Adequate mineral nutriture (not the rule in Western Man) is obviously important here. Some, but not all, diet-derived antioxidants, if at the right place at the right time, may assist in SO neutralization (vitamin C, coenzyme Q₁₀, and melatonin but not vitamin E).

Conversion of SO into H₂O₂ is thus a good thing, protecting against ONOO formation, and we do need low-concentration H₂O₂ for homeostatic signaling. Excessive SO to H₂O₂ trafficking must also be dealt with, and here Mother Nature has provided a redundant defense.

Single-purpose and single-use catalase (CAT) rapidly neutralizes H₂O₂ into H₂O. New CAT enzymes then must be transcribed via the Nrf-2 pathway. Dual-purpose glutathione peroxidase (GPX) transfers an electron from reduced glutathione (GSH) to H₂O₂, also generating H₂O (GPX can also neutralize lipid radicals). Now “spent” or oxidized glutathione (GSSG) is “recharged” into GSH by glutathione reductase (GSR), with an electron derived from NADPH, itself generated within the pentose phosphate biochemical pathway. A similar enzyme system, peroxiredoxin (PRX), also neutralizes H₂O and is “recharged” with an electron transferred from NADPH by the thioredoxin (TRX) system.

If our enzymatic and small molecule antioxidant defense systems cannot contain SO and H₂O₂, then bad things happen. Arachidonic acid (split off from LDL phosphatidylcholine by PLA₂) can be nonenzymatically converted into pro-inflammatory isoprostane molecules. The Fenton reaction, catalyzed by reduced iron (Fe²⁺) or copper (Cu²⁺), converts SO and H₂O₂ into hydroxyl anion (OH), a ROS with 1000-fold more destructive power (why we wish to avoid iron overload, why premenopausal status confers protection against atherosclerosis, and why low-dose vitamin C, which reduces Fe³⁺ to Fe²⁺, might work against us in the situation of iron overload). OH readily abstracts an electron from the double bond of cell membrane or organelle membrane polyunsaturated fatty acids, to create a lipid peroxide radical, which reacts with an adjacent double bond, setting up a membrane damaging chain reaction (generating oxLDL, malondialdehyde, hydroxyenol, and other lab markers, and mediators, of oxidative distress).

Lipophilic vitamin E (tocopherols and tocotrienols), found within the cell or organelle membrane, will react with lipid peroxides, fortunately 1000-fold faster than the lipid radical can react with an adjacent double bond, breaking the chain reaction. Spent vitamin E will then be recharged by hydrophilic vitamin C within the circulation or cytoplasm, and spent vitamin C can be recharged by diet-derived bioflavonoids.

Myeloperoxidase (MPO) converts H₂O₂ into microbicidal hypochlorous acid (HOCL). HOCL, essentially bleach, is great to have around during infection, but in the setting of inappropriate oxidative stress, is particularly damaging. MPO expression is upregulated, in turn, by upregulated H₂O₂ expression, because, as you appreciate, Mother Nature regards high-level H₂O₂ expression as a sign of infection. If MPO remains chronically elevated because of chronic infections, it will cause HDL to become dysfunctional, increase CHD plaque rupture, open calcium channels in the arteries, and increase blood pressure.

Paraoxonase (generated in the liver and associated with HDL within the circulation) excises oxidized regions within cell membranes, and within LDL and HDL particles. Paraoxonase thus mediates the antioxidant, or oxLDL neutralizing effect, of HDL. MPO is a physiologic antagonist of PON, degrading PON and oxidizing HDL, whereas PON degrades MPO and reverses lipoprotein oxidation. PON activity is uniquely upregulated by pomegranate juice, in part mediating the vasoprotective properties of this diet-derived polyphenol antioxidant.

The cartoons below describe the appropriate systemic response to infection, and how oxidative and inflammatory cues, artifacts of Western living, have hijacked these systems to generate what we refer to as risk factors for atherosclerosis (Figure 3.5). Let us explore these links, starting with endothelial dysfunction.

Endothelial dysfunction is a key determinant of outcome in all stages of atherosclerosis and in heart failure (Figure 3.6). This is because endothelial dysfunction is both a sign and a mediator of intimal and myocardial oxidative stress, the key driving force in CV disease. Before we discuss endothelial activation, an adaptive response to infection, let us discuss how endothelial chemistry works in a physiologic, reductive environment (stated otherwise, when Mother Nature does not perceive infection). Endothelial nitric oxide synthase (eNOS, or NOS3) converts arginine into nitric oxide (NO), which then diffuses within the cell and across the endothelial cell membrane, to activate soluble guanylate cyclase (sGC) in adjacent platelets and vascular smooth muscle cells. sGC in turn activates cAMP-dependent processes, which lead to
vascular smooth muscle relaxation, with subsequent vasodilatation, and reduced platelet adherence and activation. NO inhibits vascular smooth muscle cell proliferation and hypertrophy (preventing hypertension and muscle cell encroachment within the intima). NO restrains the endothelial cell from elaborating adhesion molecules and chemoattractant signals that would pull leukocytes into the intima. NO also inhibits nuclear translocation of NF-kB, our primary inflammation amplification pathway, and generator of nascent IL-1β and IL-18, and activation of the NLRP3 inflammasome,
which activates the nascent cytokines and generates an inhibitor of endothelial tone. NADPH oxidase (NOX), the key pathologic source of intimal superoxide (SO), is directly inhibited by NO. When eNOS is functioning properly, we maintain appropriate vasodilation, platelets are not sticky, and we inhibit enzyme systems that would, if unrestrained, lead to intimal oxidative stress, inflammation, atherosclerosis, and plaque destabilization.

Within an oxidative intimal environment, not only are these vascular protections lost, but eNOS itself becomes an SO generator (Figure 3.7). Adaptively, in infection, and maladaptively, in the setting of chronic, acquired oxidative stress, SO generation is upregulated, outpacing the rate at which it can be converted to H₂O₂ by SOD. SO reacts with NO three times more rapidly than it does with SOD, creating the long-lived pro-oxidant peroxynitrite (ONOO). ONOO is not constitutively generated; it has no physiologic role other than to kill microbes. Thus, ONOO is not subject to degradation by our endogenous antioxidant enzymes systems. ONOO is not neutralized by vit E, but it can be neutralized by taurine, N-acetylcysteine, methyl-folate, and pharmaceutically, by hydralazine. ONOO, a reactive nitrogen species (RNS), will activate (via tyrosine nitration) proinflammatory enzyme systems. With respect to endothelial function, ONOO degrades BH4, the key co-factor of NOS. NOS then splits into its monomeric form, stops generating NO, and instead starts converting oxygen into SO. Restraint on NOX is lost, leading to more SO, which along with the SO generated by now uncoupled eNOS combines with what little NO we have left to form more ONOO. Restraint of NF-κB (and a related inflammation amplification pathway, activator protein-1) is lost; thus, pro-inflammatory cytokines and
endothelial adhesion molecules and chemokines will be elaborated. Restraint on the NLRP3 inflammasome is lost. This enzyme complex (discussed in the Colchicine and NLRP3 section) promotes maturation of nascent NF-κB-generated IL-1β and IL-8, unleashing their inflammatory potential, and directly compromises endothelial tone. This intimal metabolic shift works in our favor when we are dealing with infection (the leading cause of death in Primitive Man), particularly if trauma is involved (saber tooth tiger bite). Here you want vasoconstriction, platelet activation, endothelial activation within the infected region, and bullets (cytokines, SO, ONOO, HOCL, and OH). iNOS, or inducible NOS, is present in phagocytic cells and upregulates its response to infection, here generating NO to combine with SO to form ONOO, to help kill the invaders. Our clinical problem, in the treatment of CV disease, is that Mother Nature interprets oxidative stress as a sign of infection, and thus chronic, inappropriate oxidative stress converts eNOS into a mediator of inflammatory atherosclerosis.

eNOS activity is modulated by two other redox sensitive phenomena: differential shear stress and the ADMA to arginine ratio. Physiologic laminar shear stress (5-20 dynes/cm²) upregulates transcription of eNOS and Nrf-2 (which controls transcription of our innate antioxidant enzymes). Low laminar flow, just beyond branch points and on bends, creates focal points of reduced NO and antioxidant intimal protection. Oscillatory shear, present beyond branch points, upregulates expression of intimal SO-generating enzymes systems (NADPH oxidase and xanthine oxidase). Focal insufficiency of NO in the presence of excessive SO thus explains the focal initiation of atherosclerosis just beyond branch points, and secondarily along curves. Saphenous vein grafts last, on average, 7 years, whereas 90% of left internal mammary (LIMA) grafts remain patent at 10 years, also in relation to differential intimal redox status. The LIMA is a long straightaway, and thus nonhypertensive flow creates a reductive, atherosclerosis-resistive LIMA intimal environment. The thin wall of the saphenous vein is designed for nonpulsatile venous flow; SO is not generated here and little NO is needed. Placing the SV within the high-pressure arterial circuit leads to upregulated SO generation, and thus rapidly developing SV graft atherosclerosis. Physiologic laminar flow promotes a reductive intimal environment, whereas excessive laminar shear (>20 dynes/cm²), on the basis of hypertension, turns up NADPH oxidase, creating an oxidative intimal environment, essentially the link between hypertension and atherosclerosis.

eNOS enzymatic activity is also governed by the ratio between its raw material, the amino acid arginine, and its physiologic, competitive inhibitor, asymmetric dimethylarginine (ADMA). ADMA is generated at a constant, constitutive rate, in relation to protein degradation. ADMA is metabolized by dimethylarginine dimethylaminohydrolase (DDAH). When DDAH is functioning normally, ADMA is broken down rapidly, the ADMA to arginine ratio is low, and if eNOS has not been oxidatively inhibited, arginine will be converted into NO. DDAH is inhibited by essentially all atherosclerotic risk factors, via the common mechanism of (you guessed it) oxidative stress. The more risk factors you bear, the more compromised will be DDAH function, the greater will be your ADMA to arginine ratio, the less NO you will generate. It is not the absolute level of ADMA that governs eNOS activity, but rather the ratio of ADMA to arginine; an imbalance here can be rectified with arginine supplementation.

Cumbersome methodologies have been utilized to measure endothelial function (intracoronary acetyl choline, forearm plethysmography, brachial artery flow-mediated vasodilation). Peripheral artery tonometry (EndoPAT) provides a low-cost (typically insurance-covered) office assessment of endothelial tone. Endothelial dysfunction is covered in greater detail elsewhere, but from the redox perspective we can address endothelial dysfunction with targeted antioxidant support (taurine and methyl-folate to neutralize ONOO and vit C and N-acetylcysteine to neutralize SO), neutralization of intimal SO generators (ARBs, HMG Co-A reductase inhibitors, berberine, or hydralazine to downregulate NADPH oxidase, and allopurinol to inhibit xanthine oxidase), and arginine (2-4 g tid). As we normalize endothelial function, we will also be turning down NF-κB, AP-1, NLRP3, and NADPH oxidase activity.

Microbes divide rapidly, and thus we need to keep up, with the rapid generation of microbicidal, pro-inflammatory cytokines and endothelial adhesion molecules. Our primary pathway of inflammation amplification is the nuclear factor kappa beta (NF-κB) transcription pathway. Under resting, reductive conditions, NF-κB is sequestered within the cytoplasm, complexed with IKBα (inhibitor of kappa beta). Ligation of threat receptors on immune response cells activates a chain reaction of serine kinases (ERK ½, p38MAPK, c-Jun N-terminal kinase), which converge to activate inhibitor of kappa beta kinase (IKK), which degrades IKBα, allowing NF-κB to translocate to the nucleus and transcribe the appropriate defense molecules. LPS (bacterial cell wall lipopolysaccharide) appropriately activates this pathway, as will molecules found in the presence of legitimate infection (IL-1β, Ang II, CD40L).

These “threat molecules” (and other NF-κB agonists such as free fatty acids) upregulate pathologically, in relation to the pro-oxidant and pro-inflammatory cues present in Western Man (leaky gut, visceral adiposity, toxins, etc.) such that NF-κB is constantly on the move (NF-κB upregulates 10-fold within unstable plaques), cranking out pro-atherogenic molecules.

NO upregulates IKBα expression, and thus blunts NF-κB trafficking within the intima. This restraint is lost in the presence of endothelial dysfunction (Figure 3.8). H₂O₂, in excess, will activate IKK to degrade IKBα, shunting NF-κB to the nucleus. Stated otherwise, you do not need real infection, or
the presence of infection signaling molecules, to activate the pro-inflammatory genes under NF-κB control; all you need is oxidative stress. Why? Primitive Man experienced oxidative stress only in relation to infection. Thus, our physiology interprets oxidative stress as “another infection” and translocates NF-κB, leading to the generation of hundreds of proinflammatory mediators.

Although episodic oxidative stress typically triggers a balancing, rebound generation of antioxidant molecules (via Nrf-2, to be discussed later), a downstream action of NF-κB is to inhibit Nrf-2 translocation (in legitimate infection we do not want antioxidant molecules; we want ROS/RNS bullets).

When circulating markers of inflammation, such as CRP or fibrinogen, are elevated, we often intervene with anti-inflammatory therapies, such as fish oil or turmeric. While these agents are helpful, a more expedient and complete approach would be to neutralize oxidative stress, as oxidative stress precedes and drives inflammatory stress.

In response to legitimate infection, HMG-Co-A reductase (the rate-limiting step in cholesterol generation) upregulates. This makes sense, as you need cholesterol to manufacture cell membranes for the rapidly proliferating leukocytic defenders. It also helps if you can activate the endothelium, making it easier for the defenders to access infected tissues, and to generate ROS/RNS bullets to kill the microbes.

HMG Co-A reductase generates cholesterol through a system of intermediate molecules, including the isoprenoids farnesyl and geranylgeranyl pyrophosphate. They activate (via prenylation) the signaling molecules Rac, Rho, and Ras, which in turn upregulate SO production, activate the endothelium, and upregulate Ang II receptor expression. These actions generate more ROS, which in turn promotes NF-κB translocation, to generate more proinflammatory cytokines (a virtuous cycle to deal with infection).

This system worked great for Primitive Man. Genomic hyperlipidemia thus arose as a defense against perinatal sepsis, the leading cause of death in the history of mankind. A minority of us carry these traits, accounting for the infrequently encountered situation of familial hyperlipidemia (good for your ancestors but bad for you). As a corollary, if your bear sickle cell genes, you will not die of malaria (Mother Nature never does anything without a good reason).

In the rest of us HMG Co-A reductase is not genomically upregulated, and when we are young our cholesterol levels are low. However, in Western Man, cholesterol values start to rise in our 20s, and continue to rise with aging. Why is this occurring? What factors determine the level of expression of HMG Co-A reductase?

HMG-Co-A reductase transcription is governed by the cell’s perceived need for free cholesterol. When intracellular free cholesterol is adequate, the free cholesterol sensor Insig sequesters SREBP (sterol regulatory element binding protein) within the endoplasmic reticulum (Figure 3.9). When free cholesterol is low, Insig releases SCAP (Sterol Regulating Element Binding Protein Cleavage Activating Protein) to transport SREBP to the Golgi apparatus, where it is enzymatically modified, promoting its translocation into the nucleus, where it binds the sterol regulatory element (SRE), the promoter site for the three key genomic sequences involved in cholesterol homeostasis.

Activation of the SRE leads to transcription of HMG Co-A reductase, such that the cell can generate more cholesterol, and of LDL receptor protein, enabling the cell to pull more cholesterol out of the circulation. Cholesterol biosynthesis and uptake occurs mainly in the liver, and as Mother Nature does not want the liver to hog all the cholesterol, SRE activation also leads to the generation of PCSK9 (Proprotein Convertase Subtilisin/Kexin type 9), a counterbalancing protein that degrades the hepatic LDL receptor, maintaining serum cholesterol to meet the needs of other cells.

In the presence of legitimate infection (lipopolysaccharide) or inflammation, acetyl Co-A acyl transferase (ACAT) expression upregulates, promoting cholesterol esterification (to store cholesterol raw material for the upcoming infectious winter). As Insig senses only free cholesterol, HMG Co-A reductase and PCSK9 transcription increase, even though total intracellular cholesterol is rising. Intracellular and circulating cholesterol thus rise with age. This does not relate directly to dietary cholesterol (in our 20s we consumed pizza and beer and our cholesterol values were low) but rather to the progressive accumulation of pro-oxidant and proinflammatory phenomena (visceral fat, leaky gut, metals, organic pollutants, etc.) that lead to maladaptive HMG Co-A reductase and PCSK9 transcription.

HMG Co-A reductase does not generate cholesterol; it generates mevalonate, which converts to cholesterol, through the intermediate isoprenoid molecules farnesyl and geranyl-geranyl pyrophosphate (Figure 3.10). These molecules activate (via prenylation) a series of GTPase signaling molecules that activate pathways critical to the response to infection, but which in our patients add to their oxidative/inflammatory burden.