Cardiovascular diseases are a major concern these days all around the world. Atherosclerosis (AS) is among the most common pathological changes in ischemic cardiovascular and cerebrovascular disease. While elevated cholesterol is the root cause, the innate immunity driven inflammation is also a key factor in the atherosclerosis development and progression. Early diagnosis can be very effective in saving lives, and this is where biomarkers can play a very vital role. In this article, we will discuss the role of granulocytes in cardiovascular diseases and as a novel source of biomarkers for diagnosing cardiovascular diseases.

Introduction

Cardiovascular diseases (CVDs) have been established as the leading cause of deaths in the developed countries. Atherosclerosis is undoubtedly regarded as the major underlying cause of the cardiovascular diseases. Atherosclerosis is a systemic and multifactorial process. It is a chronic state of inflammation, hyperlipidemia and oxidative stress that commences quite early in life, but the clinical manifestations (symptoms) occur only during the late stages in adulthood. Therefore the need of identifying patients at early stages of atherosclerotic development is very important.

Biomarkers

A biomarker (biological marker) is a naturally occurring molecule, gene, or characteristic by which a particular disease or pathological (or physiological) process can be detected. During the last two decades a lot of biomarkers, derived from blood (including plasma and serum) and plaque tissue, have been discovered to identify patients at risk of cardiovascular diseases. But unfortunately, they are still proving to be inadequate to improve the treatment on an individual basis. The blood-based biomarkers are a little bit limited in reflecting the exact state of disease progression while the plaque-based biomarkers are not the easiest ones to access despite being the source of novel biomarkers. Keeping this in mind, the granulocytes could serve as a great source of novel biomarkers for predicting both primary and secondary cardiovascular events.

Granulocytes in Cardiovascular Diseases

Granulocytes are a type of white blood cells that primarily function by protecting against foreign organisms. There are three types of granulocyte cells, viz. neutrophils, eosinophils, and basophils. Granulocytes have a very short span of life. Granulocytes contain a wide variety of known and unknown proteins. They get produced continuously from stem cells, gets rapidly recruited during tissue injury, and the potential of granulocyte proteins with functional and phenotypic heterogeneity in CVD is really a goldmine that is yet to be explored completely. This review will focus on the role of granulocytes in cardiovascular diseases and also their utility as an unexplored pool of predictive biomarkers.

Atherosclerosis and Cardiovascular Diseases (CVD)

Arteries carry blood from the heart to the rest of the body. They are lined with a thin layer of cells (endothelium) that keeps them smooth and allows blood to flow easily.

Atherosclerosis is the condition where the arteries become narrowed and hardened due to a buildup of plaque around the artery wall. Atherosclerosis is the usual cause of cardiovascular diseases like heart attacks, strokes, and peripheral vascular disease.

Atherosclerosis starts when the endothelium becomes damaged, allowing the harmful (bad) type of cholesterol (LDL) to build up in the artery wall.

As an immunogenic response, the human body sends a type of white blood cell (neutrophils) to clean up this cholesterol. But, sometimes, the cells get stuck at the affected site. Over the time, plaque starts forming. Plaque is made of cholesterol, macrophages, calcium, and other substances from the blood.

Sometimes, the plaque grows to a certain size and clogs up the artery, disrupting the flow of blood around the body. This makes deep vein clots more likely, which can result in life-threatening conditions. It can be usually picked up on if the patient is already experiencing symptoms of peripheral artery disease because the arteries are already narrowing at the extremities, it can signal fatty deposits in arteries throughout the system. For patients, understanding peripheral artery disease management can help to mitigate the development of Atherosclerosis.

In some cases, the plaque eventually, breaks open. If this happens, platelets gather in the affected area and can stick together, forming blood clots. This can block the artery, leading to life-threatening complications, such as stroke and heart attack.

Hypercholesterolemia (hyperlipidemia) is the major factor for inducing inflammation, oxidative stress, and atherosclerosis.

Neutrophils in Cardiovascular Diseases

Neutrophils, also known as polymorphonuclear leukocytes (PMNs) are the most abundant population of white blood cells. The neutrophils form the first line of innate immune defense and the main initiator of inflammation. They are classically considered as beneficial for host protection, but an imbalance in auto-immunity can turn the neutrophils detrimental against the host.

A lot of research in the 2000s was done to understand the role of neutrophils in atherosclerosis. Neutrophils are a key source of inflammation and oxidative stress during atherosclerosis. They develop distinctive phenotypes and subsets under physiological and pathological conditions. Currently, studies are being done to evaluate the potential and clinically relevant functional endpoints of the different neutrophil phenotypes during different stages of the atherosclerotic disease.

Neutrophil Recruitment

Neutrophils are the most common cell type that gets recruited rapidly to the damaged tissue and the subsequent activation triggers the release of other pro-inflammatory and anti-inflammatory mediators in the adjacent areas and also get activated. Ghasemzadeh and Hosseini (2013) did an extensive review of the neutrophil recruitment at the damaged site. The ‘extravasation’ or recruitment of the neutrophils to the ‘injured vessel’ is primarily platelet-dependent, while that at ‘inflamed vessel’ is platelet-dependent. However, emerging studies do challenge that platelets also play a role at the inflamed site.

Neutrophil recruitment at the endothelium is mainly driven by either pro-inflammatory agents or ischemia-reperfusion (I-R) injury. The neutrophil recruitment process is a multi-step cascade and is tightly regulated by various signaling pathways. The recruitment involves capturing and rolling (or rolling adhesion), priming, arrest, firm adhesion and reverse transmigration.

The capturing and rolling of neutrophils are the initial step during recruitment where selectins (L, E, and P) play vital roles in adhesion. While P-selectin is expressed on activated endothelium and platelets, E-selectin is localized to inflamed endothelium only. L-selectin is the only selectin expressed constitutively on circulating neutrophils. P-selectin glycoprotein-1 (PSGL-1), which is the main receptor for selectins, is expressed on neutrophils only.

Monoclonal antibody targeted to PSGL-1 has been reported to abolish the rolling effect completely. After binding to L-selectin to the PSGL-1 receptor on the neutrophil cell surface, the PSGL-1-L-selectin complex interacts with P-selectin or E-selectin on inflamed endothelium. This leads to the initiation of the priming process of Beta-2-integrin on neutrophils. The first step is the Src kinase pathway induction that leads to macrophage antigen-1 (aka Mac-1, a major leukocyte integrin) activation.

Following the capture (tethering) and rolling, proinflammatory stimuli like platelet-activating factor (PAF) and CCX chemokines come in to play by triggering cell arrest and ultimately integrin activation. During the arrest phase, chemokines interact with the G-protein coupled receptors (GPCRs) on the neutrophil surface.

Neutrophils express a numerous GPCRs that include receptors for sensing tissue injury (formyl peptide receptors), and receptors for lipid mediators and a wide range of chemo-attractants like leukotriene B4, PAF, complement factor C5a, chemokines (CXCR1, CXCR2, CCR1, CCR2). Besides chemotaxis, the GPCRs are also responsible for causing exocytosis and subsequent priming, and ROS production, an important neutrophilic response that plays a vital role in atherosclerosis.

The GPCR signaling on neutrophils occurs via activation of two parallel pathways, viz. PLC-beta2/3 and PI3-K-gamma. The downstream pathways including the Src-family kinases that mediate neutrophil adhesion o platelets are still a matter of intense studies.

After activation of cells by chemokines, the beta-2-integrins and Mac-1 undergo conformational changes. Mac-1, a key mediator in the adhesion process, is an E-selectin ligand and is expressed on neutrophils. Mac-1 mediates firm adhesion by interacting with platelet surface fibrinogen, GPIb, ICAM-1, and ICAM-2. Following a firm arrest on the cell, ICAM-1 and ICAM-2 facilitate neutrophils for transmigration through the vessel wall.

After being recruited to the damaged sites, neutrophils were classically considered to be phagocytosed by macrophages. But, the current theory is that they are capable of undergoing reverse transendothelial cell migration (rTEM).

Neutrophil-Platelet Interaction

Following neutrophil recruitment and adhesion at the site of developing thrombi or ischemia, platelet activation starts. The platelet activation hallmarks the release of numerous proinflammatory molecules including cytokines (e.g. IL-8, RANTES), inflammatory lipids PAF, arachidonic acid metabolites) and shed proteins (P-selectin). These proinflammatory molecules are also supposed to regulate neutrophil adhesion and activation.

MPO: Role in Neutrophil activation and CVDs

After being activated the neutrophils exerts a massive release of myeloperoxidase (MPO) into the circulation. MPO is a hemoprotein, which is stored inside the azurophilic granules. The neutrophil recruitment and activation processes have been discussed quite in detail earlier in this review. On the top of that, emerging studies add that MPO is also very likely to play a role in recruiting the neutrophils.

In the normal state, the electrostatic repulsion between the neutrophils and the endothelium prevents any interaction. But, in response to the pro-inflammatory stimuli, the highly cationic MPO gets released and facilitates neutrophil-endothelium interaction and thereby prompts rolling, adhesion and ultimately transmigration through the vessel wall. Additionally, MPO up-regulates Mac-1 expression on neutrophils. In the endothelium, MPO triggers enhanced expression of cytokines and chemokines, and MPO-derived oxidants augment increased production of adhesion molecules both on neutrophils and endothelium.

Apart from promoting neutrophil recruitment, MPO participates in oxidative stress that ultimately accelerates atherosclerosis. Studies reported huge accumulation of MPO at the unstable coronary plaques and ruptured plaques of both human patients and atherosclerotic animal models. MPO-derived hypochlorous acid (HOCl) activates metalloproteinases (MMPs), which weakens the fibrous cap of the atherosclerotic plaque and thereby increases the chance of plaque rupture. MPO also actively participates in oxidation of lipoproteins, which is a key factor for development and progression of atherosclerosis and reduction of NO bioavailability leading to endothelial dysfunction. Besides, MPO also plays pivotal roles in the pathogenesis of atrial fibrillation and left ventricular remodeling.

MPO: Biomarker for CVDs

There are quite a few clinical studies that reported elevated levels of plasma MPO in patients with cardiovascular diseases in comparison to healthy controls. Circulating MPO levels have been found to be a useful marker both for prediction of adverse events and prognosis as well in coronary artery disease (CAD), acute coronary syndromes (CAS), acute myocardial infarction (AMI), stroke, reperfusion injury. MPO content has been found to be elevated in STEMI patients, although not in case of non-STEMI or unstable angina. In fact, MPO was also found to be more effective than other markers including gold-standard troponin to identify patients at high risk for adverse cardiac events including cardiac death.

However, there are also studies that have put question marks over the utility of circulating MPO as a biomarker for diagnosis of MI and CAD. The elevated levels of plasma MPO are not specific for cardiac events only. There are also doubts if subjects with MPO deficiency bear any added advantage regarding protection from cardiac events. Another limitation is lack of a standardized assay for measuring MPO in plasma. In this respect, the MPO content in neutrophils could prove to a useful marker. The MPO content in the neutrophils do not change with age or gender in healthy subjects and has been found to reduce drastically in subjects with ACS.





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