Artificial Organs 17(12):985-995, Blackwell Scientific Publications, Inc., Boston 0 1993 International Society for Artificial Organs

Endothelium: The Next Frontier in Biocompatibility

Kai von Appen, *Peter Ivanovich, “Salim Mujais, and tHorst Klinkmann

University of Rostock and German Academic Exchange Service, Rostock and Bonn, Germany; *Northwestern University and the Department of Veteran Affairs Lakeside Medical Center, Chicago, Illinois, U.S.A.; and

flnternational Faculty for Artijicial Organs, Bologna, Italy

Abstract: Vascular endothelium plays a central role in two specific functional systems. It controls vascular tone, hemostasis, and substance transport. The endothelium is the “docking station” for trapping, deactivation, and re- generation of activated blood compounds and provides the principal clearance mechanism for biologically active mediators released by different cell types. The second function is a regenerational one. During the period be- tween insults (or between dialysis sessions), the endothe- lium has to restore the “first line of defense,” that is, to regenerate the injured athrombogenic surface of the ves- sel wall and its antioxidative potential, defoliate damaged endothelial cells, and interpolated new ones. These two important endothelial activities are required over and above its basic functions. Future research in artificial or- gans must take into account that continuous or intermit- tent blood-membrane contact creates an altered endothe- lial response. These altered responses may result in adaptional reactions that may differ substantially in the

Endothelial cells, nature’s border guards, line the vascular tree to serve as an interface between circu- lating blood elements and all tissues and organs. They subserve a variety of complex function that stand in contrast to their apparently simple histo- logic profile. Our knowledge of the function of en- dothelial cells and their role in disease has grown substantially in the last decade. This background will be utilized in the present review to examine the possible alternations in endothelial cell structure and function evoked by uremia and its dialytic ther- apy. To view things in context during hemodialysis, we should envisage two interfaces: one, the blood- dialyzer interface of the extracorporeal circuit; the

Received July 1993. Address correspondence and reprint request to Dr. P.

Ivanovich at Department of Veteran’s Affairs, 333 East Huron Street, Suite 853, Chicago, IL 60611, U.S.A.

acutely ill patient on continuous venovenous hemofiltra- tion (CVVH) or in a stable patient on maintenance hemo- dialysis. By a reduction in such factors as immediate or delayed cell-cell interactions (direct or indirect), it may be possible to influence the long-term outcome of chronic hemodialysis patients. Other research should strive to enhance those factors of endothelial function that are es- sential in the defensive and restorative properties of en- dothelial tissue. This is especially important in such con- tinuous therapies as CVVH, long-term membrane oxygenation, and artificial heart and blood vessels. Cur- rently, there are more unanswered questions than possi- ble answers concerning endothelial functions in long-term hernodialysis patients, but it is clear that excluding endo- thelial cell behavior from investigation of extracorporeal therapy in the future would be a substantial omission. Key Words: Vascular endothelium-Hemodialysis- Biocompatibility .

other, the intracorporeal blood-endothelial inter- face. Only when the latter interface is altered during dialysis do changes in the extracorporeal circuit have relevance to organ function beyond the circu- lating blood elements.

Endothelial cells are highly “plastic” cells capa- ble of transforming their shape, cell membrane structure and fenestration, cell functions, and se- cretion patterns in response to changes in location, tension forces created by bloodstream, and contact surface (1-4). They are also complex cells with a variety of functions (Table 1). The different pro- cesses occurring in endothelial cells are usually un- der physiologic conditions in a state of equilibrium, that is, factors with opposing effects are produced in the same cell and usually in a synchronized and closely interdependent fashion (e.g., endothelium- derived relaxing factor [EDRF] and endothelium- derived contracting factor [EDCF]). Disturbances

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986 K . VON APPEN ET AL.

TABLE 1. Functions of the endothelium and substances synthesized by endothelial cells

HemosCdsis I Antithrombotic and fibrinolytic properties

Protein S Heparan sulfate proteoglycan Prostacyclin Thrombomodulin, adenosine diphosphatase

Plasminogen-activator inhibitor (PAI- 1) Factor V von Willebrand factor Platelet-activating factor (PAF) High molecular weight kininogen

Regulation of vessel tone, contractility Prostacyclin EDRF EDC F

2 Activation and perpetuation of coagulation in vessel injury

Immune system, inflammatory mechanisms Syntheses of cytokines, prostaglandins, and growth factors Expression of antigen structures (e.g., major histocompati-

bility complex I and 11)

Lipids, proteins, carbohydrates Metabolic functions

Energy and substrate transport Cellular proliferation and vessel wall reconstruction after

injuries, matrix proteins Growth-promoting factors, fibronectin

Synthesis and breakdown of hormones and paracrine Endocrine function

active substances

Sources: Nabel, 1991 (1); Ware and Heistad, 1993 (5); Gry- glewski, 1990 ( 6 ) .

of this dynamic equilibrium may lead to a new equi- librium state without morphological or functional changes or in pathologic states to the sequel of cell damage or deteriorated cell function.,

Hemodialysis (HD) induces a variety of changes in the circulating components either because of its intended function (clearing of solutes and correc- tion of volume) or because of changes related to biocompatibility (blood-dialyzer interface). Endo- thelial cells so exposed to those disequilibrium situ- ations must achieve a new balance level. These changes take place against the background of possi- bly impaired cell metabolism caused by the uremic syndrome itself. The situation is made more com- plex by the coexistence of other factors in uremic subjects capable of altering endothelial cell function such as arterial hypertension, hyperlipidemia, dia- betes mellitus, and cigarette smoking. Table 2 lists the most important factors influencing endothelial cells during HD.

Most of the above-mentioned factors characteriz- ing artificial-organ-related interactions have been included in the term “biocompatibility” ; that is, the less the activation of various systems is, the more biocompatible a material, compound, system, or treatment mode is claimed to be (14). Previous in-

vestigations have been focused on dialyzer mem- brane-blood interactions. These data have been used as determinants of biocompatibility. It appears to us that the cellular and plasmatic interactions especially with endothelial cells after the biomate- rial-induced activation of the former systems will prove to be an important aspect of treatment-re- lated biocompatibility assessment.

Alterations in circulating elements (cells and plas- matic systems) occurring at the blood-dialyzer in- terface will have their initial intracorporeal effect at the level of the endothelial cell layer, which is in the first line of defense to those changes and serves as a target for activated blood cells and plasmatic sub- stances. The information we have about this “bat- tlefield” inside blood vessels is rather limited. Con- sidering the increasing potential longevity on hemodialysis and the recurring nature of these in- teractions, it is important to elucidate that their na- ture and significance for true biocompatibility must lie in the blood-endothelium interface as much as in the blood-dialyzer interface, and design of new biocompatible membranes should be influenced by the reactions of endothelial cells.

The main clinical signs of endothelial cell dys- function consist of acute changes in vascular tone

TABLE 2. Factors injluencing endothelial cell function during hemodialysis

Changes of extracellular fluid volume and hemodynamic condi-

Changes in blood composition tions

Blood osmolarity shifts Acid/base composition (pH, bicarbonate) Electrolyte concentrations (Na, K, Ca, Mg) Temperature changes (through the dialysate)

Release of cytokines Release of proteolytic enzymes Enhanced oxidative metabolism (oxidative burst) Changes in cell membrane receptor expression Augmented adhesion characteristics, enhanced phagocytosis

Release of vasoactive substances form a-granules

Activation of blood leukocytes

Activation of platelets

Thromboxane Az P-Thromboglobulin Adenosine diphosphate Serotonin Histamine Thrombin

Aggregation, adhesion Activation of the plasmatic coagulation system Activation of the plasma complement system Changes in lipid metabolism

Activation of lipoprotein lipase by heparin Enhanced lipolysis and lipid peroxidation

Sources: Vane et al., 1990 (7); Saran and Bors, 1989 (8); Paul et al., 1990 (9); Hegbrant et al., 1992 (10); Seyfert et al., 1991 (11); Schaefer et al., 1991 (12); Tetta et al., 1991 (13).

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ENDOTHELIUM AND BIOCOMPATIBILITY 987

* uremic celY metabolism * hemostasis * hypertension * cen adhesion

hyperlipidemia * diabetes mellitus * metabolic function

detoxification treatme

* control of vessel tone

* cell proliferation

FIG. 1. Aggressive and defensive factors in relation to endothelial function in a patient on maintenance hernodialysis.

(vasospasm), enhanced blood cell adhesion, throm- bus formation, and as a long-term sequel, arterio- sclerosis with vessel stenosis.

In the following, we will examine the specific re- actions of the endothelium in response to intermit- tent hemodialysis treatment (Fig. 1) and illustrate the complexity of the endothelial cells’ reactivity and the interplay of their various functions.

VASCULAR TONE

Endothelin-1 is the most important vasoconstric- tor generated locally by endothelial cells. Three mo- lecular forms of the peptide are known (15). The effect of the peptide depends on the ratio of its syn- thesis and breakdown. This ratio is disturbed under various pathologic conditions such as renal failure, hypertension, cardiac failure, chronic respiratory diseases, and sepsis (16,17). Although circulating levels of the peptide are elevated in renal failure (18-20), blood levels represent the “spill” pool and may not be a very representative index of the tis- sue-specific changes in concentration. Further, the functions of the peptide are wider than its vasoregu- latory role (21). One recently elucidated nonvasore- gulatory function of endothelin is its stimulation of leukocyte adhesion to endothelial cells (22). This observation underlines the complexity of those me- diator substances and the manifold interactions be- tween the mediator systems, which makes it nearly impossible to evaluate isolated branches of the sys- tem. The interdependence of opposing factors is also maintained in disease states. Experimental models of vascular disease have shown that states associated with elevated levels of EDCF also have increased levels of nitric oxide (NO) as an EDRF (23).

The action of the vasodilator acetylcholine is par- tially endothelium dependent as the hormone trig- gers endothelial cells via muscarinic receptors to release EDRF. This vasodilator effect is dependent on intact endothelial cell function and is impaired when a disease process affects the endothelium such as in atherosclerotic disease where the vasodi- lator response to acetylcholine is absent in disease segments of the circulation (24,25). Normal endo- thelial function with intact ability to release EDRF is a prerequisite for maintenance of a normal vascu- lar tone, and an impairment of such a status may contribute to the generation of hypertension (7). In uremia, the EDRF synthesis may be impaired not only by the uremic syndrome itself, but also by hy- pertension, arteriosclerosis, hyperlipidemia, and di- abetes mellitus (increasing number of older patients receiving HD).

Recently, an endogenous compound that specifi- cally inhibits EDRF synthesis and is normally ex- creted in the urine has been isolated from the serum of maintenance HD patients (26).

The EDRF balance in uremia is further influenced by such factors as the increase in the EDRF break- down by highly reactive oxygen radical species (27,28). During HD treatment in chronic uremic pa- tients, the oxygen radical synthesis is enhanced while the enzymatic and nonenzymatic antioxida- tive potential in uremia is simultaneously impaired (29).

A modification of the interleukin hypothesis would posit that an increase in the production of the cytokine interleukin- 1 (IL-1) would induce EDRF release by endothelial or vascular smooth muscle cells with a resulting rapid decrease in blood pres- sure. The increase in IL-1 either can be due to biomaterial-related blood cell activation (30) or can

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988 K . VON APPEN ET AL.

follow the transfer of bacterial endotoxins (lipopo- lysaccharides) from the dialysate compartment into the bloodstream (i.e., backfiltration). In physiologi- cal situations, EDRF (NO) acts only locally be- cause it is immediately inactivated by hemoglobin or superoxide anions when released into the blood- stream.

Another vasodilatory substance, which acts mainly as a paracrine hormone and is secreted by endothelial cells, is prostacyclin. It acts in concert with NO. Common to both substances is their pre- dominantly paracrine mechanism of action and their short half-life (7). Prostacyclin synthesis is mark- edly enhanced as a sequel of cell membrane dam- age. Prostacyclin as a vasodilatory mediator is ca- pable of substituting for NO in case of endothelial cell damage and reduced production of NO by the injured cells.

HEMOSTASIS AND COAGULATION

Both the plasmatic coagulation system and plate- lets are heavily influenced during extracorporeal de- toxification treatment. The clotting phenomenon in- side dialyzer capillaries and blood lines has been a widely known feature since the first days of HD treatment and became one of the first parameters of biocompatibility.

A description of all the alterations in the coagula- tion system occurring during dialysis is beyond the scope of this article. We focus instead on those ele- ments that have a direct bearing on the endothe- hum. Activation of the coagulation cascade and thrombocytes by extracorporeal therapy is a pro- cess with repercussion both on the extracorporeal circuit and within the blood vessels of the treated patient. For patients undergoing intermittent acute or chronic HD, the activation of the hemostatic sys- tem is necessary during repeated but not long-last- ing events, and the opportunity for restoration of the preintervention balance state between treatment sessions is readily available. During intermittent HD treatments, the balance between anticoagula- tory and procoagulatory systems will deviate in the latter direction. The main activating factors are the blood-biomaterial contact and the influence of cyto- kines (IL-1, tumor necrosis factor) and bacterial en- dotoxins (31). In addition, other factors capable of disturbing endothelial cell function factors such as hypoxia and mechanical injury (e.g., blood flow dis- turbances) may enhance the thrombogenic ten- dency. A peculiar case arises during continuous ex- tracorporeal treatment modes, such as continuous arteriovenous hemofiltration (CAVH) and continu-

ous venovenous hemofiltration (CVVH), that re- quire prolonged anticoagulation (with heparin or citrate) in close connection with enhanced coagula- tion and thrombotic processes. The latter treat- ments are often applied in patients with multiorgan failure including disseminated intravascular coagu- lation. Clotting of the extracorporeal system, ex- change of the components of the system at regular intervals, and the underlying disease state itself have been found to be associated with an exhaus- tion or depletion of the hemostatic system that can play a crucial role in determining patient survival or demise. This complex question has not been prop- erly investigated especially concerning the role of the endothelium and its reserves.

The endothelial cells express their antithrombotic properties by synthesis of three main mediator groups; first, substances that are embedded in the luminal cell surface coating (heparan sulfate pro- teoglycans, thrombomodulin); second, substances that are secreted into the bloodstream (prostacy- clin, EDRF) and influence plasmatic coagulation factors, platelets, and vascular smooth muscle cells; and third, substances that stay inside the en- dothelial cells and act from there (13-hydroxy-9,Il- octadecodienoic acid) (32). In addition, other sub- stances, synthesized mainly by liver cells (e.g., antithrombin III), are extracted from plasma by spe- cific binding sites on endothelial cell membranes and are included into the surface coating layer composed from a large variety of mainly endo- thelium-derived substance, including proteins, mucopolysaccharides, and others. Among the mucopolysaccharides are heparin-like substances that substantially enhance the antithrombogenic action of antithrombin I11 (33). They act in the same manner as heparin, and they are the components subject to possible exhaustion (34) in critically ill patients on continuous extracorporeal treatment modes (CAVH, CVVH).

As described for other mediator systems, a dis- equilibrium situation created by enhanced thrombin production causes a stimulation of plasminogen ac- tivator release by endothelial cells (35) as well as an inhibition of plasmin activator inhibitor. Activated thrombin in circulation is quickly transferred into the thrombin-antithrombin complex and effectively bound to high-affinity sites on endothelial cell mem- branes in a reversible manner (36). This membrane- bound complex can be internalized into endothelial cells by pinocytosis. Activated by biomaterial con- tact and further stimulated by released mediators, platelets tend to aggregate and are attracted by en- dothelial surface. Platelet degranulation is followed

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by the release of a-granular proteins (adenosine di- phosphate, serotonin, thromboxane A2, histamine) that cause vasoconstriction and induce the process of thrombus formation, change endothelial cell me- tabolism, and influence leukocyte behavior (5) . The endothelial cell surface has specific binding sites for coagulation factors (VII, IX, X) and for activated forms of the latter (IXa, Xa). By specific binding (reversible or covalent) as well as synthesis or acti- vation of other mediators, the endothelium is capa- ble of controlling inhibition (under physiological conditions) or activation (during endothelial pertur- bation) of coagulation processes.

Perturbed endothelial cells lose their antithrom- botic characteristics and become activators of both the plasmatic coagulation system (intrinsic and ex- trinsic pathways) and platelet aggregation. The same process is initiated during endothelial cell re- traction and exposure of the subendothelial base- ment membrane of blood compounds, so that differ- ent mechanisms of activated coagulation and platelet aggregation during HD are responsible for activated clotting mechanisms. The release of tissue factor from endothelial cells can also be enhanced without cell damage under the influence of bacterial endotoxins (37).

From recent information about cell-cell interac- tions and the role of different cell surface receptors, it is apparent that platelet activation and aggrega- tion can be caused by leukocytes. The mechanism of signal transduction between different cell types has a characteristic pattern during inflammatory processes, vascular disorders, and hemorrhagic events (38-40). Activated thrombin is also capable of enhancing further the platelet-leukocyte aggrega- tion. Various cell adhesion molecules and their spe- cific ligands expressed on platelets, leukocytes, and endothelial cells show a high structural similarity.

Another recent important finding describes the close interactions between leukocytes and platelets in exchanging intermediate products of arachidonic acid metabolism in both directions. Each of the two cell types is capable of synthesizing leukotrienes and lipoxines from precursors synthesized and re- leased by the other population (41).

If one assumes that the platelet and leukocyte surface receptor expression changes during HD are stepwise and interdependent processes, then the “ignition” and the enhancement of thrombotic and coagulatory phenomenona are potentiated by leu- kocyte and endothelial cell activation.

Summarizing the available information, one can assume that the endothelium functions as a “buffer” inhibiting or dampening the activation of

coagulation pathways occurring in the extracorpo- real system. In case of endothelial cell damage or dysfunction (e.g., under influence of mediators), en- dothelial cells can change their role and manifest prothrombotic properties. It can be hypothesized that future extracorporeal therapy should not only be as nonthrombogenic as possible, but also less injurious to the endothelium and endothelial cell function.

LEUKOCYTE ADHESION AND CELL INTERACTIONS

Leukocyte adhesion to endothelial cells is a well- known physiological phenomenon, which is con- trolled by surface receptors on the cell membranes of both cell types (42) and represents the first step in a chain of interactions during leukocyte migration to the extravascular compartment. The adhesion process can be dramatically increased during vari- ous pathological processes, changing cell mem- brane receptor expression.

The development of leukocyte sequestration in the pulmonary circulation is a well-known phenom- enon in HD and depends directly on the biocom- patibility of the dialyzer membrane; that is, the less biocompatible the membrane is, the more pro- nounced will be the leukopenia as a sequel to white blood cell trapping in pulmonary vessels (43-45).

Hemodialysis is not the only situation that in- duces pulmonary leukocyte sequestration. A simi- lar behavior can be seen during acute respiratory distress syndrome, sepsis, and under the influence of some snake toxins (cobra venom). Common to all these clinical pathologies is an activation of the complement system in close connection with the generation of oxygen-derived free radicals from ac- tivated phagocytic cells (46). In acute respiratory distress syndrome, the adherent leukocytes contrib- ute to a focal destruction of endothelial cells and lung interstitium (47) and an enhanced vascular per- meability to blood macromolecules such as albumin and immunoglobulins (48).

Complement activation and the effect of pro- teolytic enzymes in concert with oxygen radicals generated from activated leukocytes are necessary to cause cell injury. These occur regularly during HD. Their extent depends on the biocompatibility of the applied synthetic materials in the circuit and special treatment conditions. Sequestration of leu- kocytes especially in pulmonary capillaries high- lights the heterogeneity in structure and function of the endothelium as an organ. The trapping of leuko- cytes and their adhesion to the capillary wall is not a

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990 K . VON APPEN ET AL.

mechanical process, but a complicated cell-cell in- teraction mediated by specific adhesion molecule domains on the cell membrane surface (42,49-51). Most of those interactions must still be character- ized by future investigations. Increasingly, recent publications report that each blood cell population has different groups of adhesion molecules with li- gands on cells from the same population as well as on other cell types (homotypic, heterotypic adhe- sion). An extended role with different control func- tions is played by the endothelium (42,4932-66). The activation of adhesion molecules and the resul- tant binding of leukocytes to endothelial cells is a mandatory step for leukocyte degranulation in- duced by hemodialysis membranes (67) (Fig. 2). In the future development of synthetic membranes for HD, one should consider the degree of leukocyte surface receptor activation including adhesion mol- ecules during the blood-biomaterial contact.

Karlsson et al. (68) reported that free plasma en- zymatic antioxidative capacity in the form of super- oxide dismutase (SOD) is rapidly bound to the membrane of endothelial cells by heparan sulfate. This selective binding behavior possibly highlights the endothelial cell’s ability to react immediately to oxidative stress and to down-regulate activated leu- kocytes that produce free oxygen species during ox- idative burst after contact with foreign surfaces dur- ing HD. In this fashion, reactive oxygen species are inactivated on the surface of endothelial cells before initiating harmful injury to the endothelial cell mem- brane, including membrane phospholipid peroxida- tion with subsequent membrane damage.

Endothelial cell injury caused by oxygen-derived radicals from activated leukocytes has been investi- gated and is a well-known process (69). The pro- longed specific messenger action induced by acti- vated oxygen species, however, is known only in hypothetical terms (8,70). According to recent in- vestigations, the superoxide anion radical has an estimated lifetime and diffusion path length that are 100 or even 1,000 times greater than those of other activated oxygen species. In contrast to other radi- cal species, this radical has no relevant destructive potential during leukocyte interaction with target cells. The superoxide anion radical inactivating en- zyme SOD, located mainly intracellularly , has been extensively investigated and in uremic syndrome has been shown to be deficient. In animal experi- ments and cell-culture settings, it has been demon- strated that numerous cell transformations take place many cell cycles after an injuring irradia- tion procedure in the absence of the enzyme SOD (8).

C

D

\

Activated leukocycte released substances ! cytokines, leukotrienes

proteolytlc enzymes reactive oxygen species

FIG. 2. Interactions between activated polymorphonuclear leukocytes (PMN)/monocytes and endothelial cells (ap- proach, adhesion, dissociation). A: Activated PMN cell after biomaterial contact; expression of adhesion molecules on the cell membrane, activated intracellular granules. 8: Ad- herent to the endothelium PMN cell; tight contact zone be- tween the cell membranes with release of leukocyte-specific substances, initiation of injuring effects to the endothelial cell membrane and induction of endothelial defense mecha- nisms. C: Resting endothelial cell with a basic level expres- sion of adhesion molecules. D: Activated endothelial cell with additional expressed adhesion molecules on the luminal cell membrane (following first steps of cell-cell interactions) and up-regulated cellular defense mechanisms (antioxida- tive, antiproteolytic, anticoagulatory).

Superoxide anion radicals are synthesized not only by leukocytes but also by endothelial and smooth muscle cells. By releasing superoxide anion radicals, those cells modify the surface markers of low-density lipoprotein (LDL) particles in such a manner that recognition by the macrophage recep- tor is altered (see below, “Atherosclerosis”). Su-

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peroxide anion radical is involved in the production of arachidonic-acid-derived chemoattractants by phagocytes (71). It participates in the regulation of platelet aggregation and platelet adhesion to endo- thelium.

These specific messenger actions taken together with the ability of superoxide anion radical to de- grade EDRF (see above, “Vascular Tone”) impli- cate HD as one form of powerful oxidative stress that induces and accelerates changes in cell-to-cell interactions that last hours or even days after treat- ment and create signals in following messenger sys- tems that change balanced metabolic states (e.g., LDL metabolism) or include the sequel of cell transformations (carcinogenesis).

During experiments with endothelial cell cul- tures, Kutsumi et al. (72) found that by increasing concentrations of LDL cholesterol in the culture supernatant there followed an enhanced expression of intracellular adhesion molecules (ICAM-1, ELAM-1, PECAM-1) on the monolayer endothelial cells which more than doubled leukocyte adhesion to the cultured cells. This observation can shed light on uremia-specific cell-cell interactions, should it be corroborated in vivo. Then, it is likely that concur- rent disease states and uremia-related hyperlipide- mia could influence and modify substantially the in- teractions between the patient and long-term artificial organ treatment. The improved lipoprotein metabolism in HD patients treated for several months with biocompatible HD membranes (73) may not be explained exclusively by the better elim- ination of toxic uremic metabolites by the high-flux device, but by biomaterial-related improved endo- thelial clearance of lipoproteins.

The accumulation of P2-microglobulin (&m) in uremia is thought to be caused by impaired renal elimination (glomerular filtration and tubular reab- sorption with subsequent breakdown to amino acid residues), which is responsible for more than 95% of P2-m clearance from blood. A positive balance of Pz-m occurs because of permanently or repeatedly activated human lymphocyte antigen-rich cells (leu- kocytes), that synthesize and release Pz-m and the near zero renal excretory function. The protein and its fragmented or otherwise altered forms (with dif- ferent isoelectric properties) will distribute to the extracellular fluid and aggregate in locations of chronic inflammation or that are predisposed by chemical environment (pH, local substance compo- sition, and concentration) and surrounding cells.

Canaud et al. (74) conducted a trial to enhance P2-m elimination from the uremic patient by daily hemofiltration treatments with highly permeable

membranes and observed an induction of further substantial leukocyte activation with repeated cyto- kine release and induction of P2-m synthesis.

Considering the fact that activated leukocytes are usually adherent to the endothelium or have mi- grated through this cell layer to the vessel wall or further, the main part of the secreted &microglo- bulin or its incomplete parts will be distributed out- side the plasma volume and will not be able to in- duce measurable concentration changes in plasma. The history of uremic amyloidosis lasts many years in most patients. The enhanced P2-m synthesis as a sign of monocyte activation can doubtless explain the development of amyloidosis in peritoneal dialy- sis patients and that during hemofiltration too.

Following this hypothesis, further, there would be no reason to enhance greatly P2-m elimination during HD with highly permeable membranes if those membranes include the sequel of leukocyte activation. Further, it would be valuable to investi- gate the post-HD monocyte-endothelium interac- tion for the synthesis of P2-m and its transfer to other tissues. It is even possible that the first step of P2-m structure alteration is performed by proteases inside endothelial cells.

The interactions between platelets and endothe- lial cells have been discussed. Here we will point out only one more important observation support- ing the very close interactions between various cel- lular and plasmatic compounds of the blood. Acti- vated in the extracorporeal circuit, platelets release active substances (e.g., serotonin) that induce and amplify the adherence of leukocytes to the endothe- lial surface and cause cell damage (75).

ATHEROSCLEROSIS

The harmful effects of many injuring processes involve the presence of two or three factors simulta- neously at the site of prospective injury (“binary weapon”). For example, proteolytic enzymes will act effectively only if their inhibitors have been in- activated by hydrogen peroxide or halide products of oxidation reactions (76). All these conditions can easily be created by activated granulocytes entering the blood vessel system after contact with synthetic materials and adhering to the endothelial cell layer coating the vessel wall. This implicates an injury mechanism of endothelial cells that cannot be esti- mated by investigations of postdialyzer concentra- tions of plasma compounds and blood cell activa- tion levels because a main part of impairing reactions will happen only during the interaction of blood compounds with target cells (Fig. 2).

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992 K . VON APPEN ET AL.

New equilibrium states will be achieved for some of the compounds of the system hours or even days after the termination of HD (Fig. 3), but for other compounds, the uremic syndrome itself is such a powerful injuring factor that diminution of the ure- mic state by detoxification treatment is not able to correct the deviation or shift away from physiologi- cal conditions.

There are two extreme forms of HD induced de- viations from a physiological "equilibrium state." One of them is the well-known urea curve; that is, the more time elapsed since the last treatment, the more of the substance accumulates, and physiologi- cal level is approximated at the end of the next treatment procedure. The second type, illustrated in Fig. 3, represents an activation of substance syn- thesis by the treatment itself and a slow reversal of the activation process. During the next treatment session, activation is repeated.

The disequilibrium level after a regular, repeated detoxification procedure is possibly different for each involved metabolic and cellular system and is influenced by independent factors. An example is the level of oxidative processes in peripheral leuko- cytes, one that is markedly enhanced before HD (9). There are other examples of unphysiological activated leukocyte and thrombocyte functions in patients with chronic renal insufficiency which are present even before the initiation of maintenance

Changes in lipid metabolism and enhanced arteri- osclerosis processes are commonly observed in HD patients. It is uncertain if the endothelial cells are primarily altered by oxidized LDL particles on their

HD (9,77-80).

1. serum urea concentration 2. leukocyte activation level t

w HD 1 HD 2

time

HD .. - HD-sessions - "urea kinetic like behnviour"

"leucoq te xtirarion - like beha\ iour"

FIG. 3. Example of equilibrium changes during chronic he- rnodialysis.

cell surface or if arteriosclerotic plaques and foam cells develop because the new equilibrium is not able to metabolize the lipoprotein particles. There is increasing evidence that impaired lipid metabolism in uremia and potential injurious effects during HD are capable of impairing endothelial and smooth muscle cell functions without detectable athero- sclerotic lesions (81).

One of the most important prerequisites for ather- osclerosis processes is the damage of endothelial cells accompanied by injury to the athrombogenic surface layer at the lurninal side. Retraction of en- dothelial cell uncovering parts of the subendothe- lium, adhesion of platelets to this region, and re- lease of mediators are followed by fibrin deposition, underlying smooth muscle cell injury, and prolifera- tion as well as intimal thickening. Factors injuring the endothelium during HD can be divided into me- chanical factors (shear stress) and others. The latter include complement activation, bacterial endotox- ins, hyperlipidemia, and cell adhesion (platelets, leukocytes) (82). The localization of adherent blood monocytes to the endothelium and their number de- pend on such factors as activation degree in the extracorporeal circuit, hemodynamic stress, endo- thelial injury, and hypercholesterolemia (83,84). Those monocytes migrate through endothelial intra- cellular junctions and further differentiate inside the vessel wall to become macrophages. The LDL par- ticles in the bloodstream are permanently filtered from the vessel lumen through the vessel wall to enter the lymphatic system. The amount of filtered LDL depends on their concentration and endothe- lial permeability. Those two latter factors are changed in uremia and HD and may be one explana- tion for the LDL accumulation in the subendothelial layer, where the particles are internalized by mac- rophages and to a smaller extent by smooth muscle cells. LDL particles are powerful mitogens (82). In- ternalized into macrophages after recognition by the scavenger receptor, LDL particles will accumu- late, transforming these cells into foam cells. The incorporated LDL is modified and has been found mainly in the oxidized molecular form. On the back- ground of the uremic state including the presence of hypercholesterolemia, enhanced endothelial perme- ability, impaired macrophage function, and ele- vated oxidation processes (85), the blood vessel- wall during HD is exposed to all necessary conditions for an enhanced atherosclerosis. Once more, the closed connection between arterioscle- rotic processes on one side and disturbances of ves- sel tone or accelerated blood coagulation on the other side shall be emphasized (86,87).

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Acknowledgments: The work on this project was sup- ported by a German Acedemic Exchange Service Grant.

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