Biology of monocyte interactions with the endothelium : the platelet factor - Chapter 1 Introduction and aim of study

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Biology of monocyte interactions with the endothelium : the platelet factor

da Costa Martins, P.A.

Publication date

2005

Link to publication

Citation for published version (APA):

da Costa Martins, P. A. (2005). Biology of monocyte interactions with the endothelium : the

platelet factor.

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Thrombosiss and atherosclerosis, in relation to cardiovascular diseases, are

majorr determinants of morbidity and mortality in the Western societies. Scientific

research,, so far, has focused mainly on non-cellular risk factors for both processes.

Forr example, deficient inhibition of coagulation and fibrinolysis have been delineated

ass causes for thrombosis, and aberrant lipid and/or cholesterol metabolism received

mostt attention as a cause for atherogenesis. However, these processes are

decisivelyy determined by cellular responses: firstly by the magnitude or chronicity of

endotheliall stimulation and, secondly, by the subsequent inflammatory and

hemostaticc response to this stimulation. The responses and interactions of

endotheliall cells with leukocytes and platelets are the main subject of this

introduction. .

Togetherr with platelets, inflammatory cells are known to play a role in thrombosis and

atherosclerosis.. In vivo adhesion and influx of phagocytes at and within the thrombus

hass been object of several studies

"

. The role of phagocytes can be advantageous

ass they are considered to play a role in the reorganization of the thrombus and

neededd to restore vascular integrity and new vessel formation. Moreover, the

presencee of inflammatory cells at these sites, where pathogen invasion is a risk,

mightt be beneficial. On the other hand, monocyte and lymphocyte infiltration can be

viewed,, pathologically, as the first inflammatory events in the development of

atherosclerosis.. As lipid-laden foam cells are derived from monocytes

'

, in a more

progressedd lesion, these foam cells correlate with a high risk of plaque rupture,

expressionn of tissue factor and subsequent activation of the coagulation cascade.

Thee current paradigm of atherosclerosis as an inflammatory process that promotes

lesionn development and progression has been established throughout the years. By

consideringg the importance of this inflammatory process, in this thesis we will show

evidencee of strong interaction between the hemostatic and the inflammatory

response,, as well as present new insights into the mechanism of recruitment of

leukocytess and platelets to the inflamed vessel wall. This general introduction briefly

describess the early hemostatic events after vessel wall injury and the cellular

adhesionn molecules that mediate adhesion of leukocytes and/or leukocyte-platelet

aggregatess under flow conditions.

Platelets/thrombii at the vessel wall induce colocalisation of leukocytes

PlateletPlatelet adhesion to injured vessel wall. Platelet function and, in particular,

plateletss capacity to adhere, to become activated, to form aggregates and to support

locall initiation of coagulation, play a central role in normal hemostasis. To avoid

harmfull thrombus formation, platelet activation usually only occurs at sites of

vascularr damage and, more drastically, when the subendothelial tissue is exposed. If

thee latter occurs in the arterial vascular bed, shear stress and red blood cell

-dependentt platelet margination will favor the formation of a thrombus consisting

mainlyy of platelets (white thrombus)

"

. Due to its shear-dependent functionality, Von

Willebrandd factor (vWF) plays a major role in thrombus formation as the most

importantt plasma and vascular glycoprotein mediating platelet adhesion

. In

contrast,, venous thrombi mostly consist of a loosely packed network of erythrocytes

andd islands of aggregated platelets reinforced/encapsulated by fibrin that can be

formedd at lower shear stresses ("red thrombus"). Vessel wall injury and subsequent

exposuree of the extracellular matrix (ECM) may be caused by disturbed blood flow,

hypertension,, platelet- or leukocyte-released products, bacteria, endotoxins, viruses,

smoking,, dietary lipids, homocystenemia, diabetes, other metabolic disorders, and

stresss

. Under these conditions, platelet-dependent hemostasis is initiated

(Figuree 1). Platelets readily adhere to vWF and collagen in the extracellular matrix

andd become activated. Subsequently, there is an increase in adhesivity, initiated by

collagenn and locally generated thrombin, and release of platelet-activating

substancess enabling platelet aggregation

.

bloodd flow

^ ^

tetheringg

thrombus

7 \\ . adhesion formation

CC -* endothelial cell

tt activated platelet

Figuree 1. Multistep adhesive interactions of platelets with vascular surfaces under flow.

Adhesionn process and molecules involved in platelet recruitment to subendothelial surfaces.

Activationn of endothelial cells, without ECM exposure, also support platelet

deposition.. However, these are mostly transient and short-termed interactions

leadingg to rolling of platelets over the vessel wall

. In contrast, activated

endotheliall cells with subsequently upregulated adhesion receptors are more

adhesivee for inflammatory cells than the sites of endothelial vascular wall damage.

Therefore,, the inflammatory and hemostatic responses are determined by the

conditionsconditions that disturb the integrity of the endothelial monolayer. The extreme end of

thesee processes can be mechanical denudation or plaque rupture, with endothelial

Platelet-derivedd microparticles at damage sites have been shown to increase the

adhesivee interactions of neutrophils, monocytes or monocytoid cells

with the

endothelium.. This indicates that platelets and platelet-derived products at sites of

vessell wall injury may not only influence thrombosis but also modulate subsequent

multicellularr interactions that can be of relevance for the early stages of

atherogenesis. .

LeukocyteLeukocyte adhesion to activated endothelium. Stimulation of endothelial

cellss by e.g. bacterial chemokines, leads to upregulation of adhesion molecules on

thee cell surface. However, leukocytes also need to be activated before stable firm

adhesionn and subsequent migration can take place. The presence of specific

adhesionn receptors and leukocyte-activating chemokines act as a kind of an area

codee and start a process of multistep adhesion and endothelial diapedesis of

leukocytess at these sites.

MultistepMultistep paradigm of leukocyte extravasation {Figure 2). The first short-term

interactionss between leukocytes and endothelial cells are called "primary tethering".

Subsequently,, the leukocytes slow down to a speed a thousand times lower than the

freee flowing cells, and show a rolling interaction with the endothelium. Rolling is

dependentt on a minimum of force exhibited by the blood stream, called "shear

stress"

.. Both primary tethering and rolling of leukocytes over the endothelium are

mediatedd by the selectin family of adhesion molecules (Figure 3A). E-selectin is, with

aa slight delay after stimulation (e.g. by TNF-a), upregulated on activated endothelial

cells.. L-selectin is constitutively present on leukocytes

"

and, in contrast to

E-selectin,, shed from the surface when leukocytes become activated

"

. P-selectin is

storedd in Weibel-Palade bodies of EC and a-granules present in platelets

"

.

P-selectinn is expressed transiently on the surface of these cells after stimulation with

variouss inflammatory stimuli e.g. histamine or thrombin on EC.

P-- and L-selectin are primarily responsible for tethering of flowing leukocytes to the

endotheliumm but they also support cell rolling. E-selectin is particularly important for

slowingg the rolling velocity of leukocytes after they have tethered through P- or

L-selectinn

. These slower rolling interactions increase the probability of firm adhesion,

necessaryy for final extravasation of leukocytes in different ways: A. by allowing firm

adhesionn via low-affinity receptor-ligand interactions and, B. by increasing the

leukocyte'ss probability of binding chemokines on the endothelial cell surface

.

Chemokiness will activate the leukocyte by transducing signals that intersect with

thosee produced by engagement of selectin ligands

, usually leading to increased

expressionn and/or increased affinity of receptors on the leukocyte membrane.

monocyte e

capturee rolling firm adhesion diapedesis

Figuree 2. Multistep model of leukocyte extravasation. At sites of inflammation, leukocytes

tether,, or form initial attachments, to vascular endothelial cells. They then roll, or move continuously withh rotational movements, on the vessel wall, until they finally arrest, spread and migrate between endotheliall cells into the surrounding tissues.

Firmm adhesion is mediated by integrins on the leukocytes that bind to their ligands on

thee endothelium, belonging to the immunoglobulin superfamily (Figure 3B). On

non-activated,, resting leukocytes, integrins maintain a conformationally inactive state.

Uponn stimulation by chemoattractants or other stimuli, integrins change their

conformation,, leading to increased affinity and/or avidity for their ligands. This

processs is called inside-out signaling

"

. Integrins, by interacting with their

respectivee endothelial receptor molecules (e.g. ICAM-1, -2 or VCAM-1) mediate firm

adhesionn of leukocytes to the vessel wall

~

. Subsequently, the adhering

leukocytess crawl to the site where endothelial cells are connected to each other and

migratee in between two adjacent cells to the inflammatory site. Additionally, the

bindingg of integrins to their ligands, by itself, leads to activation of intracellular

signalingg pathways in the leukocytes and in the endothelial cells (outside-in

signaling))

. In this way also endothelial cells are able to respond to inflammatory

conditions.. For example, it has been shown that outside-in signaling by adhesion of

neutrophilss to endothelial receptors increases the endothelial permeability by

disorganizingg endothelial cell-to-cell adherent junctions, thus facilitating

trans-endotheliall migration

. Another possible adhesion-regulated response may be the

migrationn through an endothelial cell instead of in between two adjacent endothelial

cellss as shown for neutrophils

. After passing the endothelium, the leukocytes will

interactt with the subendothelial matrix and migrate to the site of inflammation

.

AA B

PSGL-11 ESL-1

sLe

,, Le

sLe

, Le

L-selectin Mac-1 LFA-1 VLA-4

P-selectinn E-selectin MadCAM ICAM-1 ICAM-2 VCAM-1

GlyCAM M

sLe

Figuree 3. Selectins, integrins and respective ligands presented by leukocytes and endotheliall cells. A. Interactions of selectins with the respective cell-surface glycoconjugates mediate

tetheringg and rolling adhesion of leukocytes on the vessel wall. PSGL-1 binds with high affinity to P-selectinn but it can also bind E- and L-selectin. ESL-1 is a specific ligand for E-selectin. The ligands for L-selectinn on endothelial cells can be MadCAM, GlyCAM. B. p, and p2 integrins mediate firm adhesion

off leukocytes to the endothelium. Integrins are expressed by leukocytes and their ligands, from the immunoglobulinn superfamily, are expressed on endothelial cells.

LeukocyteLeukocyte adhesion to vessel wall-localized mixed thrombi: the platelet

factor.factor. Remarkably, the above-described multi-step process of leukocyte adhesion

too stimulated EC is very similar to their adhesion to a thrombus formed at a damaged

vascularr wall. In this respect, the thrombus seems to be a stronger adhesive

substratee than the activated endothelium. In vitro experiments have identified the

molecularr substrates of these interactions as specific cell adhesion molecules and

ligandss present on platelets and/or leukocytes

"

. Matrix glycoproteins in the

exposedd subendothelial tissue and other specific proteins within the thrombus (e.g.

fibrinn or vWF) can serve as adhesion molecules for both leukocytes and/or platelets.

Platelett and fibrin/fibrinogen deposition occur at sites of vascular damage. These

thrombii often become infiltrated with inflammatory cells. In vivo observations in

atherosclerosis,, vasculitis, and thrombosis have shown that platelets and large

numberss of leukocytes colocalise at these sites

'

'

. Colocalisation is probably

causedd by the fact that even minimal platelet deposition and activation at the injured

vessell wall result in high expression of P-selectin at the platelet surface, which can

servee as a strong adhesive surface for leukocytes

. Leukocytes are able to tether

overr platelet-rich thrombi and platelet aided-colocalisation of leukocytes has been

shownn not only in vitro, by means of perfusion systems

, but also in in-vivo grafts

inn mice

. The role of platelets and the (32-integrin Mac-1 in monocyte or PMN firm

adhesionn has been well established and involves a P-selectin-mediated stimulation

off Mac-1-dependent adhesion

. Leukocyte arrest by platelets can be mediated by

interactionss of leukocyte integrins with GPIIbllla-bound fibrinogen (Mac-1), ICAM-2

(LFA-1)) or vWF on platelets

"

. Similar to fibrinogen, also fibrin is a very potent

substratee for p2 integrin-mediated leukocyte adhesion

. Therefore, platelets and

fibrinn are synergistic/cooperative substrates supporting leukocyte rolling/tethering

interactionss and firm adhesion, respectively

. Additionally, oriented and

flow-formedd fibrin tracks have been described as very strong adhesive substrates for

leukocytes. .

AtAt high shear stress, and thus bond-disturbing and detachment-inducing conditions,

alll receptor-ligand interactions, in principle redundant, become more and more

criticall to maintain firm adhesion. At low shear stress or static conditions, redundancy

betweenn the several possible interactions allows several of these bonds to be absent

beforee overall adhesion is affected.

IncreaseIncrease in adhesion due to homotypic interactions between leukocytes.

Homotypicc aggregates or cluster-forming interactions can occur at thrombi or at the

endotheliall cell surface and are able to efficiently increase leukocyte adhesion

"

.. Under flow conditions, adhered monocytes can serve as an L-selectin-rich surface

thatt can slow down other leukocytes still in the circulation and lead to the formation

off cell clusters or strings elongated according to the flow direction

. These dusters

aree L-selectin dependent and are observed mainly when the local flow is increased.

Thee observed increase in adhesion is dependent on the cluster mechanism.

However,, even when L-selectin function on leukocytes is blocked, they still

aggregatee and interact with P-selectin glycoprotein ligand-1 (PSGL-l)-expressing

cells.. Platelet microparticles (PMPs) allow neutrophils to aggregate and interact with

PSGL-11 -expressing cells, indicating that leukocyte adhesion might be strengthened

byy the presence of PMPs

. PMPs are released from activated platelets and express

functionall adhesion receptors, including P-selectin, on their surface. In a similar way,

andd in agreement with the fact that leukocytes adhere to activated platelets on the

vascularr wall, activated platelets in the circulation might also bind leukocytes and

formm the so-called platelet-leukocyte complexes

.

Platelet-leukocytee aggregates in circulation

Underr hemodynamic conditions, the rolling and arrest of leukocytes on spread

plateletss or activated endothelium involves the transition from P-selectin-mediated

tetheringg to more stable (32-integrin - mediated interactions. However, stimulation of

plateletss in the circulation can also occur. These platelets might then adhere to other

adjacentt platelets to form platelet-platelet aggregates or to leukocytes and form

platelet-leukocytee aggregates (PLAs). PLA formation is mainly initiated via P-selectin,

expressedd on platelets that will bind PSGL-1 on leukocytes

(Figure 4A).

Circulatingg PLAs mostly consist of monocytes and granulocytes heterotypically

aggregatedd with platelets, rather than lymphocytes

"

. As said, platelets and

leukocytess conjugate primarily via bridgings of P-selectin - PSGL-1; however,

integrinmediatedd interactions via glycoprotein (GP) llb/llla fibrinogen

-CD11b/CD188

'

and CD36 (GPIV) - thrombospondin - CD36

also contribute.

PLAA formation seems to strongly increase upon platelet activation

and mildly

uponn leukocyte activation

"

. In fact, platelet activation is one of the major

characteristicss present throughout the atherosclerotic process. Circulating activated

plateletss have been found in patients with unstable atherosclerosis

"

, stable

coronaryy disease

and hypercholesterolemia

.

However,, platelet-leukocyte aggregates are not only considered as markers of

platelett activation and thus of cardiovascular disease, but also as having

(patho)physiologicall importance. By passive addition of platelet receptors to the

aggregatee and, possibly, also by active upregulation of adhesion molecules on the

leukocyte,, platelets bound to leukocytes facilitate leukocyte rolling, adhesion and

migrationn on/into the vessel wall in in-vitro assays

'

. in agreement with this

notion,, activated platelets affect endothelial inflammation and leukocyte-endothelial

interactionss and, therefore, the development of atherosclerotic lesions in

atherosclerosis-pronee animals

. In fact, P-selectin knock-out models show a

weakerr development of atherosclerosis

. Furthermore, activated platelets also bind

too circulating lymphocytes and may support lymphocyte homing to the lymph nodes

54 4

Thee in vivo circulation time and clearance of PLAs is still not well defined. Ley and

co-workerss

have shown that circulating PLAs, in mice, were no longer detectable

att 3 to 4 hours after a single injection of activated platelets. Moreover, the infusion of

activatedd platelets caused removal of leukocytes, preferentially monocytes, from the

circulation.. Since interactions of platelets with the vessel wall only occur in a

transientt way, leading to little platelet accumulation on the endothelial surface of

atheroscleroticc lesions

'

'

, P-selectin - mediated platelet-leukocyte interactions

seemm to be more stable

"

. Probably, PLA formation will cause monocytes and

neutrophilss to disappear from the circulation by adhesion of the complexes to

activated,, damaged vascular wall e.g. atherosclerotic lesions in carotid arteries

.

resting g

platelet t platelet t activation n

P-selectin n

Figuree 4. Translocation of P-selectin during platelet activation. P-selectin resides in the

alphaa granule membrane in resting platelets. Upon activation, specific proteins will form complexes andd will direct the granules to the plasma membrane. Recognition and fusion occurs and P-selectin is translocatedd to the plasma membrane where the extracellular domain can establish contact with adhesionn molecules on other cells (e.g. PSGL-1 on leukocytes). P-selectin binding to leukocyte receptorss leads to activation of proinflammatory and prothrombotic pathways by increasing the capacityy of the leukocyte to adhere secrete cytokines.

Changess in leukocyte phenotype by platelet binding and/or by paracrine

platelet-derivedd factors

Too bind P-selectin, PSGL-1 has to undergo several molecular modifications.

PSGL-11 is a type I transmembrane protein

with an extracellular domain

characterizedd by repeat units that include sites for O-linked glycosylation

<

.

Thesee are, presumably, the sites for the fucosylated, sialylated glucosamines that

aree critical for P-selectin recognition

~". Furthermore, PSGL-1 undergoes critical

sulfationn of specific tyrosine residues that is required for P-selectin recognition

'

,

Besidess their docking function in cell-cell interactions, both P-selectin and PSGL-1

aree also involved in signal transduction (Figure 4B). Leukocyte activation occurs

partiallyy through signaling via PSGL-1 and, therefore, platelets might be particularly

efficientt because of their high surface density of P-selectin

"

. PSGL-1 signaling

iss responsible for an array of intracellular events within the leukocyte that will modify

nott only the expression and affinity of adhesion molecules (e.g. integrins) but also

genee transcription within monocytes, e.g. tissue factor (TF)

Onn the observed time scale of several hours, tissue factor upregulation might not be

relevantt to local hemostasis, but after diapedesis and possibly macrophage

differentiation,, this monocyte-derived TF might become important in further

thrombogenesiss and wound healing. P-selectin - mediated signaling might

additionallyy prime monocytes towards pro-inflammatory responses

. While the

productionn of various cytokines is induced when leukocytes bind P-selectin through

PSGL-1,, mostly widely reported effects are Mac-1 integrin activation and, with it,

homotypicc neutrophil aggregation and neutrophil-platelet conjugation

'

. Hidari et

al..

observed that engagement of PSGL-1 induces tyrosine phosphorylation,

activatess mitogen-activated protein (MAP) kinases (ERK-1 and ERK-2) through MEK

(MAPP kinase kinase), and stimulates IL-8 secretion by neutrophils. Direct correlation

off these events with p2-integrin activation has been the subject of some controversy.

Somee reports concluded that PSGL-1 ligation is not sufficient to activate p2 integrins

onn neutrophils

while others

showed P-selectin induced p2 integrin-mediated

celll attachment to ICAM-1. Recently, Ma et al.

demonstrated that the binding of

P-selectinn to PSGL-1 results in a moderate clustering and a partial activation of Mac-1,

thuss enhancing adhesion and binding of human neutrophils to immobilized

fibrinogen.. P-selectin binding also promotes VLA-4-dependent adhesion of

monocytess to vascular cell adhesion molecule-1 (VCAM-1)

under flow conditions.

Anotherr interesting signaling pathway occurs via the CD40 ligand (CD40L),

expressedd on the surface of activated platelets

. Upon ligation, the cognate

receptorr CD40, which is present on B cells, monocytes, macrophages and

endotheliall cells, may trigger inflammatory reactions

by inducing expression of

tissuee factor and other adhesion molecules

'

.

Plateletss in complex with leukocytes are likely to have paracrine effects due to the

secretionn of a number of chemokines of both the CC and CXC subgroups

, most of

whichh are know to be stored in the a-granules

. The chemokine RANTES is

knownn to be a chemoattractant for lymphocytes, monocytes and eosinophils

-

-

andd to stimulate [Ca

]j transients in these cells

. As deposition of RANTES by

plateletss triggers shear-resistant monocyte arrest on inflamed or atherosclerotic

endotheliumm

, platelet-derived RANTES in the PLAs might act similarly and

supportt recruitment of monocytes from the circulation to the endothelium

.

RANTES,, in the context of PSGL-1 engagement by P-selectin, also induces MCP-1

andd IL-8 secretion from monocytes

. Similarly, exposure of monocytes to

P-selectinn and platelet activating factor (PAF) induces expression of the cytokines

TNFaa and MCP-1

.

Leukocytess may also synergistically influence platelet activation

. Leukocytes per

see and leukocyte released-02~ may enhance platelet adhesion. Furthermore,

leukocyte-releasedd substances, such as O2", platelet-activating factor (PFA),

elastase,, and cathepsin G, may induce platelet aggregation and secretion

.

Conversely,, unstimulated or weakly activated leukocytes may also attenuate platelet

aggregationn via leukocyte-released NO and/or ADPase

. Neutrophil-derived

elastasee may contribute to proteolysis of the GP Iba subunit

, which contains the

vonn Willebrand Factor binding site, and may thus influence platelet adhesion.

Inn conclusion, P-selectin - PSGL-1 interactions, together or not with other

cooperativee factors, seem to initiate a number of signaling cascades, in both

interactingg cells, that can modify or amplify a range of inflammatory and/or thrombotic

responses.. The total effect of these responses will depend on the inflammatory

stimulus,, the vascular bed, the inflammatory mediators presented, and the types of

leukocytee recruited to the site of injury. Indeed, after the formation of PLAs, the

subsequentt P-selectin - mediated leukocyte activation seems to compensate for the

sheddingg of L-selectin as an important player in leukocyte tethering and rolling

.

Leukocytee activation by P-selectin binding increases the affinity/avidity of the

leukocytee integrins and therefore leads to increased firm adhesion and migratory

capacityy of the PLA.

{Pathophysiologicall implications

Atherosclerosis,, widely recognized as an inflammatory disease, is

characterizedd by early accumulation of monocytes into the arterial wall

and the

mechanismss that modulate this early step are thus of great importance. P-selectin

wass shown to be particularly important in the pathogenesis of atherosclerosis in the

apolipoproteinn E-deficient mouse. PLAs, by presenting a stronger adhesive capacity

ass compared to "bare leukocytes", should be considered as proatherogenic particles

thatt a. might accelerate atherothrombotic disease and b. could thus be a target for

therapy.. In fact, the presence of PLAs often correlates with an adverse outcome of

patientss e.g. with unstable angina pectoris. PLA formation and platelet microparticles

aree also elevated in other platelet-activating conditions, and their pathophysiological

rolee seems to exceed the well-established role as local atherosclerosis amplifier and

triggerr of thrombosis. Increased populations of circulating, activated platelets have

beenn observed in conditions such as diabetes mellitus

, coronary restenosis

,

allergicc inflammation

and severe trauma or sepsis

. These platelets increase

theirr levels of activated glycoprotein alip3, thrombospondin and most importantly,

P-selectin.. In myeloproliferative syndromes, platelet activation

and PLA formation

aree associated with an increased risk of arterial/venous thrombotic events

. in

heparin-inducedd thrombocytopenia (HIT), a strong relation between the

complicationss of heparin therapy with elevated formation of PLA has been suggested

.. Furthermore, an increased formation of platelet-neutrophil coaggregates occurs

duringg dialysis

. This was reported to be due to increased expression of CD15s

(Lewis

)) on neutrophils and monocytes, which by interacting with CD62P (P-selectin)

willl play a major role in the transient leukocyte margination during hemodialysis

144,1455 |n addition a

|

platelet-P-selectinn and PLAs that might result from platelet stimulation by a damaged

cerebrall endothelium

.

Pharmacologicall modulation of PLA presence or function

Thee general assumption that inflammation is important in atherothrombosis

hass supported the therapeutic use of anti-inflammatory treatment in the prevention of

cardiovascularr disease

*

. indeed, there is evidence that blocking inflammation

couldd lower thrombosis and thus prevent acute coronary events.

Anticoagulationn and antiplatelet therapies are known to modulate vascular death risk.

However,, to what extent these therapies are effective in modulating coagulation- and

platelett activation - dependent inflammation, is unknown. This should be considered

whenn choosing for any classic or new therapeutic agent that blocks platelet activation

and/orr coagulation, e.g. aspirin, glycoprotein GPIIbllla inhibitors or COX inhibitors.

Aspirinn has become the standard antiplatelet agent for prevention of most ischemic

syndromes.. Mainly, long-term therapy with aspirin reduces the risk of critical

cardiovascularr and cerebrovascular events (death, stroke, myocardial infarction,

unstablee angina) by average 25% compared to placebo

. However, although

aspirinn is a widely used platelet aggregation inhibitor, there are some studies

showingg that aspirin treatment does not attenuate platelet or leukocyte activation

,

andd therefore its efficacy in preventing coronary events is being questioned. Other

widelyy used platelet inhibitors are the thienopyridines (ticlopidine and clopidogrel),

whichh affect platelet functionality through inhibition of ADP platelet activation.

Comparedd to aspirin these agents have been shown to offer convincing and clinically

significantt benefits with respect to reduction of ischemic events. More importantly,

clopidogrell was shown to reduce platelet - P-selectin expression and, subsequent,

platelet-leukocytee aggregate formation

, decrease serum level of soluble

ICAM-11 and diminish chemokinesis of monocytes

. Furthermore, the incorporation of GP

llb/lllaa receptor blockers into medical stabilization and mortality-reducing regimens

forr unstable angina has enhanced the safety and has reduced the number of

invasivee procedures. However, Hu and co-workers

showed that although

GPIIb/lllaa blockade attenuated PAF-induced platelet activation and PAF-induced

platelet-leukocytee aggregation, it also enhanced ADP- or TRAP-induced platelet

leukocytee aggregation. Therefore, definitive conclusions about the current role of GP

llb/lllaa inhibitors as inflammatory modulators are difficult to draw because many of

thee studies performed to support these agents show conflicting and inconsistent

results. .

Inn conclusion and although a tot of progress has been made throughout the years,

theree are still controversies and conflicting research results regarding the

developmentt of new therapies for management of patients at risk of vascular-related

disorders.. Regarding the fact that platelets as well as leukocytes, and most

importantly,, PLAs are important contributors to arterial thrombosis and

atherosclerosis,, inhibition of aggregate formation might become a potent tool in the

therapyy of cardiovascular diseases. Many of the therapies currently used have no

effectt on the formation of PLA. In fact, only few studies have focused on the effect of

therapyy on platelet interactions with leukocytes. Most of those studies only showed a

decreasee in platelet - P-selectin expression and subsequent PLA formation after

combiningg two or three different treatments

"

. However, the advantages of these

combinedd therapies have to be evaluated considering costs of therapy and possible

toxicityy associated with hematological risks. An efficient therapy might be the use of

P-selectin/PSGL-11 antagonists. It has been shown that mice deficient in P-selectin,

E-selectinn or ICAM-1 are protected from a variable degree from atherosclerosis in

murinee models

. P-selectin inhibition has been shown to have advantageous

resultss in enhancing thrombus resolution in rat models

and to decrease

inflammationn and thrombus formation in baboons

. This effect was shown to be

duee to a strong inhibition of leukocyte-endothelium interactions and subsequent

reductionn in the leukocyte infiltrate into the vessel wall. This new strategy might

becomee extremely useful in the treatment of cardiovascular disease in humans but

clinicall studies have to be awaited. However, the optimal therapy and its overall

effectivenesss are still a matter of debate.

Aimm of the study

Adhesionn of monocytes to the endothelium can be supported by

monocyte-monocytee interactions, resulting in the formation of cell aggregates at the vessel wall

(clusters).. Platelets that are bound to the injured vessel wall are also able to support

monocytee adhesion, and platelet activation leads to the formation of

platelet-monocytee complexes (PMC) in the circulation. We hypothesize that direct

interactionss between platelets and monocytes enhance the monocyte atherogenic

capacityy and that PMC formation might correlate with vascular disease by inducing

monocytee activation and adhesion to the vessel wall.

Thiss study focuses on the differentially modulated mechanisms by which monocyte

adhesion,, as primary event in atherosclerosis, can take place at the vessel wall. We

havee studied monocyte interactions with the endothelium and focused on the

influencee of PMC in this process. As a model, we studied monocyte/PMC rolling and

adhesionn to stimulated endothelial cells under physiological flow conditions, using an

in-vitroo perfusion system. In fact, flow is an important aspect of this study, because

primaryy tethering, rolling, secondary tethering and firm adhesion are steps of the

multistepp model that can only be properly studied under flow conditions

Wee used a flow chamber, depicted in Figure 5, to answer the following

questions; ;

What is the influence of platelet binding to monocytes on the monocyte

adhesiveadhesive capacity to the endothelium? The adhesion molecules that are involved in

platelet-monocytee complex formation and in the rolling and adhesion of

monocytes/PMCC to activated endothelium were characterized (Chapter II). The

presencee and new role of a specific adhesion molecule on endothelial cells were

discoveredd (Chapter III).

What is the influence of platelet binding to monocytes on the monocyte

activationactivation status? The changes that occur at the monocyte surface level upon

platelett binding, regarding expression of adhesion molecules and implicating

changess in the monocyte adhesive capacity, were characterized (Chapter IV).

Which other molecules or mechanisms might play an important role in

monocytemonocyte recruitment to the activated endothelium. Other important mechanisms

andd molecules, involved in leukocyte recruitment to endothelium have been

described.. Molecules such as LDL-receptor - related protein (Chapter V) and Lewis

(Chapterr VI) play an important role regulating/mediating leukocyte adhesion to the

endothelium. .

Figuree 5. In vitro flow chamber model. Coverslips coated with a confluent layer of

endotheliall cells are placed in the flow chamber (1). Leukocytes are added to the reservoir (2) and are pulledd into, and through, the flow chamber, over the endothelial cells, by a pump (3). On the microscopee (4), a video camera (5) is mounted and with a video recorder (6) films of the rolling or adherentt cells are recorded. A heater (8) on the incubation box (9) regulates and keeps the temperaturee at .

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