**2. Hyperlipidemia in humans after solid organ transplantation**

Hyperlipidemia is observed in about 60% of kidney, liver, cardiac and bone marrow transplants after treatment with CsA (for review see [1,2]. There are multiple factors potentially contributing to hyperlipidemia in these patients, such as post-transplantation obesity, multiple drug therapy and diabetes. The concurrent use of steroids in particular, makes it hard to establish a direct contribution of CsA to dyslipidemia in humans, as corticosteroids are known to exacerbate hyperlipidemia in transplant recipients [3,4].

Studies investigating plasma lipids after CsA monotherapy are limited [4,5,6,7,8,9] and only a few studies have directly compared the combination of CsA therapy with low dose prednisolone with other immune suppressing strategies in combination with low dose steroids [10,11]. In general, these studies indicate that CsA treatment can independently lead to elevated plasma triglyceride and cholesterol levels in humans and that these effects are reversible upon cessation of immunosuppression therapy (Table 1). Animal studies (reviewed in [12]), where the effect of CsA can be studied in a more controlled background, indicate that CsA directly raises plasma lipid levels in rats, mice, guinea pigs and rabbits, and have proven that animals are valuable models to study mechanisms of CsA-induced hyperlipidemia.

© 2012 Kockx and Kritharides, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



ALG, Minnesota antilymphocyte globulin; aza, azathioprine; cort, corticosteroids; pred, prednisolone

TC, total cholesterol; TG, total triglyceride; LDL, low density lipoprotein; VLDL, very low density lipoprotein; HDL, high density lipoprotein; apo, apolipoprotein;

**Table 1.** Effect of CsA on plasma lipid parameters in humans

#### **2.1. Plasma VLDL**

338 Lipoproteins – Role in Health and Diseases

Monotherapy Amyotrophic

Monotherapy Autologous

Monotherapy Renal

Monotherapy Renal

Monotherapy and CsA/pred

*Treatment Patients Patient* 

lateral sclerosis

bone marrow transplants

transplants

transplants

Bone marrow transplants

Monotherapy Psoriasis 15 3 mnths TC (22%)

*number* 

36 2 mnths TC (21%)

13 32 days TC (26%)

59 3-6 and 12

mnths

58 >1 yr TC

180 100 days TC

*Duration Lipid effects Reference* 

[5]

[13]

[8]

[14]

[4]

[9]

LDL-C (31%) apoB (12%)

TG = HDL =

TC = LDL-C = apoB TG HDL-C apoA-I

LDL-C apoB TG VLDL-C = HDL-C HDL2-C = HDL3-C

LDL-C apoB TG

VLDL-trig VLDL-C = HDL HDL2 HDL3 = apoA-I

LDL-C (35%)

TG = VLDL-C = HDL-C =

LDL-C HDL-C TG = VLDL-C =

> Triglyceride-containing VLDL particles are produced in the liver via lipidation of apolipoprotein B (apoB) by microsomal triglyceride transfer protein (MTP), generating triglyceride-poor (VLDL2) as well as triglyceride-rich VLDL (VLDL1) particles, both of which can be secreted [15]. In plasma, VLDL is converted to intermediate-density lipoprotein (IDL) by lipoprotein lipase (LPL). IDL can be further hydrolyzed by lipases to low density lipoprotein (LDL). CsA increases plasma VLDL levels in transplant recipients and a concomitant increase in plasma apoB levels is observed [4,10,11]. It is unclear whether both plasma VLDL1 and VLDL2 levels are elevated. In contrast to LDL levels, plasma triglyceride and VLDL levels appear to increase only after long-term treatment with CsA (Table 1 and [8])

> Hypertriglyceridemia in transplant patients is associated with increased plasma apolipoprotein CIII (apoCIII) levels [16,17,18] and decreased lipase activity (see below). As apoCIII inhibits LPL and hepatic lipase (HL) as well as uptake of triglyceride lipoprotein in liver, the increase of apoCIII may be an important contributor to hypertriglyceridemia found in transplant patients.

#### **2.2. Plasma LDL**

Plasma LDL levels appear to be consistently elevated by CsA [4,5,6,7,9,10,13,14] even in patients where plasma VLDL levels are not altered [5,6,9,13]. A correlation between CsA levels and plasma LDL-C has been described in some studies [19], but was not observed in others [5,20]. Regulation of plasma LDL levels is complex, depending on hepatic VLDL production, subsequent lipolysis of VLDL, clearance of LDL via the LDL receptor (LDLr) in the liver and conversion into bile. CsA may affect LDL metabolism at several levels (section 3.2).

#### **2.3. Plasma HDL**

Total plasma HDL levels are inversely correlated with the risk of cardiovascular disease [21]. HDL particles are however heterogeneous in size and composition, and occur as HDL2a, HDL2b, HDL3a, HDL3b and HDL3c which are progressively smaller in diameter and contain higher protein to lipid ratios. The precise contribution of various HDL subclasses to cardiovascular disease is currently unclear [21,22]. Plasma HDL cholesterol levels are determined by production of nascent HDL particles in the liver and intestine, by plasma transfer reactions of lipids between HDL and lipolysed triglyceride lipoproteins such as VLDL or chylomicrons, hepatic uptake of HDL lipids via the scavenger receptor class B1 (SRB1) HDL receptor in the liver, and renal clearance of small, lipid-poor apoA-I particles. Nascent HDL particles are formed by lipidation of apolipoprotein A-I (apoA-I) via the ATP-binding cassette transporter-1 (ABCA1) located in cellular membranes, although ABCA1-independent pathways of apoA-I lipidation also exist [23]. The formed lipid-poor HDL particles acquire more lipid after interaction with ABCG1 and mature by the subsequent esterification of cholesterol by lecithin-acyl transferase (LCAT). Further remodeling occurs by phospholipid transfer protein (PLTP) generating HDL2. HDL2 can be converted into HDL3 by hydrolysis via lipases and by transfer of cholesteryl esters to triglyceride-containing lipoproteins with the reciprocal exchange for triglycerides, which is mediated by cholesteryl ester transfer protein (CETP).

Immunosuppressive therapy has been reported to increase, decrease or leave HDL levels unaffected [5,10,11,24]. Parallel changes in plasma apoA-I levels are usually observed. Increased HDL levels are observed in most transplant patients, but this is most likely related to the concomitant treatment with steroids, which are known to increase plasma HDL [3]. CsA may affect particular subclasses of HDL more than others. Independently of steroids, plasma HDL levels, especially the HDL3 subpopulation, were found to inversely relate to plasma CsA levels [19]. In a study of bone marrow transplant recipients CsA decreased total plasma HDL, and in particular HDL2 [4]. In rats, a similar decrease in plasma HDL and HDL2 levels was observed after CsA treatment [25]. A recent study performed in pediatric renal transplant recipients showed that although total plasma HDL levels were not changed with CsA treatment, the relative proportion of HDL2b decreased while the relative proportion of HDL3a, HDL3b and HDL3c increased [26]. This is important as decreased HLD2b with increased HDL3b is associated with an atherogenic lipoprotein phenotype characterized by increased triglycerides and small dense LDL [27]. This result also emphases that simple monitoring of total HDL cholesterol may be insufficient to understand the consequences of CsA on HDL biology.

#### **2.4. Plasma lipoprotein (a)**

340 Lipoproteins – Role in Health and Diseases

mediated by cholesteryl ester transfer protein (CETP).

consequences of CsA on HDL biology.

(section 3.2).

**2.3. Plasma HDL** 

levels and plasma LDL-C has been described in some studies [19], but was not observed in others [5,20]. Regulation of plasma LDL levels is complex, depending on hepatic VLDL production, subsequent lipolysis of VLDL, clearance of LDL via the LDL receptor (LDLr) in the liver and conversion into bile. CsA may affect LDL metabolism at several levels

Total plasma HDL levels are inversely correlated with the risk of cardiovascular disease [21]. HDL particles are however heterogeneous in size and composition, and occur as HDL2a, HDL2b, HDL3a, HDL3b and HDL3c which are progressively smaller in diameter and contain higher protein to lipid ratios. The precise contribution of various HDL subclasses to cardiovascular disease is currently unclear [21,22]. Plasma HDL cholesterol levels are determined by production of nascent HDL particles in the liver and intestine, by plasma transfer reactions of lipids between HDL and lipolysed triglyceride lipoproteins such as VLDL or chylomicrons, hepatic uptake of HDL lipids via the scavenger receptor class B1 (SRB1) HDL receptor in the liver, and renal clearance of small, lipid-poor apoA-I particles. Nascent HDL particles are formed by lipidation of apolipoprotein A-I (apoA-I) via the ATP-binding cassette transporter-1 (ABCA1) located in cellular membranes, although ABCA1-independent pathways of apoA-I lipidation also exist [23]. The formed lipid-poor HDL particles acquire more lipid after interaction with ABCG1 and mature by the subsequent esterification of cholesterol by lecithin-acyl transferase (LCAT). Further remodeling occurs by phospholipid transfer protein (PLTP) generating HDL2. HDL2 can be converted into HDL3 by hydrolysis via lipases and by transfer of cholesteryl esters to triglyceride-containing lipoproteins with the reciprocal exchange for triglycerides, which is

Immunosuppressive therapy has been reported to increase, decrease or leave HDL levels unaffected [5,10,11,24]. Parallel changes in plasma apoA-I levels are usually observed. Increased HDL levels are observed in most transplant patients, but this is most likely related to the concomitant treatment with steroids, which are known to increase plasma HDL [3]. CsA may affect particular subclasses of HDL more than others. Independently of steroids, plasma HDL levels, especially the HDL3 subpopulation, were found to inversely relate to plasma CsA levels [19]. In a study of bone marrow transplant recipients CsA decreased total plasma HDL, and in particular HDL2 [4]. In rats, a similar decrease in plasma HDL and HDL2 levels was observed after CsA treatment [25]. A recent study performed in pediatric renal transplant recipients showed that although total plasma HDL levels were not changed with CsA treatment, the relative proportion of HDL2b decreased while the relative proportion of HDL3a, HDL3b and HDL3c increased [26]. This is important as decreased HLD2b with increased HDL3b is associated with an atherogenic lipoprotein phenotype characterized by increased triglycerides and small dense LDL [27]. This result also emphases that simple monitoring of total HDL cholesterol may be insufficient to understand the Lipoprotein (a) [Lp(a)] is a LDL-like lipoprotein consisting of LDL with one molecule of apoB covalently linked to a molecule of apolipoprotein (a). Plasma Lp(a) levels, and especially certain genetic Lp(a) variants, are independently associated with an increased risk for CVD [28,29]. Elevated Lp(a) plasma levels have been observed in renal transplant studies [14,30] this was however, not observed by others [31]. Although some studies suggested normalization of elevated Lp(a) levels after successful transplantation due to improved kidney function [31,32], CsA treatment has been indicated to independently increase Lp(a) levels in renal transplant recipients [8,14,33]. The mechanisms by which CsA affect plasma Lp(a) levels are unexplored, but may involve similar mechanisms to that of elevation of plasma LDL levels. As the LDLr does not play a major role in the clearance of Lp(a), the mechanism however, is unlikely mediated via effects of CsA on the LDLr (see section 3.2.1).

#### **2.5. Qualitative differences in lipoproteins**

#### *2.5.1. Particle changes*

Elevated plasma triglyceride levels are associated with the formation of triglyceride rich LDL particles that are more atherogenic [34]. A high prevalence of smaller denser LDL particles is observed in transplant recipients [35] and appears to be associated with CsA therapy [26,36]. Inhibition of lipoprotein lipase (LPL) activity is associated with the formation of small dense LDL subclasses. As apoCIII inhibits lipase activity, increased plasma apoCIII levels observed with CsA-treatment may explain inhibited lipase activity and subsequent increase in small dense LDL particles [17]. In addition decreased lipase activity could contribute to decreased HDL2 subclasses observed, while effects on CETP by CsA may help explain increases in HDL3 subfractions (see section 2.3 and 3.1.2).

#### *2.5.2. Interaction of CsA with plasma lipoproteins*

In whole blood CsA is primarily transported bound to lipoproteins (33%) and erythrocytes (58%) and whole blood CsA levels correlate with lipoprotein levels [37,38]. *In vitro* and *in vivo* studies show that in serum from healthy patients 50-60% of CsA is bound to HDL, 20- 30% to LDL, 10-25% to VLDL with 10-15% bound to the non-lipoprotein proteins [39,40,41,42]. However, the proportion of CsA bound to the LDL and VLDL fractions increases in hyperlipidemic serum, without changing the amount bound to free protein [40,41], indicating that the distribution of CsA between the lipoprotein classes will change as plasma lipoprotein concentrations change. The binding of CsA to lipoprotein particles may also depend on lipoprotein composition. For example, Wasan et al. [41] showed that high triglyceride content of HDL was associated with a decreased percentage of CsA recovered in the HDL fraction and an increased percentage recovered in the VLDL fraction. Interestingly, treatment of patients with lipid lowering agents, such as statins have been reported to increase the unbound fraction of CsA and clearance of CsA in plasma [43].

Concerns have been raised about changes to the bioavailabilty and activity of CsA resulting from its binding to lipoproteins, especially as decreased CsA activity and increased toxicity have been observed in patients with hyperlipidemia [42,44]. CsA levels are higher in hyperlipidemic patients due to decreased clearance which was reversed after lipid-lowering with fibrates (reviewed in [37]). *In vitro* studies using skin fibroblasts indicate that CsA bound to LDL does not affect binding to cells via the LDLr, but uptake of CsA is inhibited [45]. These studies were confirmed in HepG2 and Jurkat Tcells which showed decreased uptake of CsA in the presence of LDL [40]. In line with these findings, uptake of CsA in tissues from rats was reduced when CsA was co-injected with lipoproteins [46].
