*3.3.2. Effects on HDL metabolism*

As mentioned above (section 3.1.2), CsA directly suppresses LPL activity and increases CETP activity in human plasma and animals (section 3.1.2). LPL activity is strongly associated with plasma HDL2 concentrations [66], and decreased LPL levels in CsA treatment may therefore contribute to decreased HDL2 levels [4,25]. On the other hand, increased CETP activity will generate triglyceride-rich HDL, which is converted to smaller HDL3 particles by HL [66].

#### **3.4. Effects on bile acid synthesis and secretion**

#### *3.4.1. Effects on bile synthesis*

In liver, cholesterol is converted to bile acids by 7-hydroxylase (CYP7) or 27-hydroxylase (CYP27A1) [67]. In healthy humans, CYP7α is considered the predominantly pathway while CYP27A1 accounts for 10% of bile acid synthesis and subsequent formation of chenodeoxycholate. However inhibition of Cyp7α can increase the contribution of the CYP27A1 pathway [68]. *In vitro* studies show that CsA inhibits both CYP27A1 activity and subsequent formation of chenodeoxycholate in human and animal liver extracts and in primary hepatocyte cultures [57,69,70,71]. A CsA responsive element has been mapped on the CYP27A1 promoter [72], indicating that CsA affects transcription of the CYP27A1 gene directly. In most of the *in vitro* studies, CYP7α activity was not affected by CsA [69,70]. *In vivo,* in rat however, CsA decreased CYP7α protein levels [50], indicating that the predominant bile acid synthesis pathway may also be affected by CsA. The inhibitory effect of CsA on bile synthesis is suggested to contribute to increased plasma lipid concentrations in transplant recipients. Radioisotope studies performed in children after liver transplantation demonstrated that CsA treatment significantly inhibits bile salts synthesis rates, especially that of chenodeoxycholate and that bile acid synthesis rate inversely correlates with plasma cholesterol and triglyceride levels [73].

344 Lipoproteins – Role in Health and Diseases

*3.3.2. Effects on HDL metabolism* 

HDL3 particles by HL [66].

*3.4.1. Effects on bile synthesis* 

**3.4. Effects on bile acid synthesis and secretion** 

body system NB.

suggesting that ABCA1 trapped at the plasma membrane is dysfunctional [59,60]. *In vivo* studies using wild type C57Bl6 mice corroborated these *in vitro* findings. CsA lowered plasma HDL levels after 6 days of treatment [59]. A lowering in plasma HDL in mice was however not observed by others after long-term treatment of mice with CsA combined with a high fat diet [62]. As many aspects of lipid metabolism can be affected by CsA, it may be difficult to determine a causal effect on HDL levels via ABCA1 inhibition in an *in vivo* whole

Direct effects of CsA on the expression of ABCA1 and apoA-I have also been reported and may contribute to the changes in HDL formation. The target of immunosuppression by CsA, Nuclear Factor of activated T-cells, cytoplasmic 2 (NFATc2), was found to bind the mouse ABCA1 promoter and mediate CsA-inhibition of ABCA1 expression by inflammatory stimuli [63]. In addition CsA has been found to inhibit apoA-I gene expression in human HepG2 cells and rats [64]. A recent proteomic study in HepG2 cells showed that CsA decreased secretion levels of apoA-I suggesting that the transcriptional effects of CsA on

As mentioned above (section 3.1.2), CsA directly suppresses LPL activity and increases CETP activity in human plasma and animals (section 3.1.2). LPL activity is strongly associated with plasma HDL2 concentrations [66], and decreased LPL levels in CsA treatment may therefore contribute to decreased HDL2 levels [4,25]. On the other hand, increased CETP activity will generate triglyceride-rich HDL, which is converted to smaller

In liver, cholesterol is converted to bile acids by 7-hydroxylase (CYP7) or 27-hydroxylase (CYP27A1) [67]. In healthy humans, CYP7α is considered the predominantly pathway while CYP27A1 accounts for 10% of bile acid synthesis and subsequent formation of chenodeoxycholate. However inhibition of Cyp7α can increase the contribution of the CYP27A1 pathway [68]. *In vitro* studies show that CsA inhibits both CYP27A1 activity and subsequent formation of chenodeoxycholate in human and animal liver extracts and in primary hepatocyte cultures [57,69,70,71]. A CsA responsive element has been mapped on the CYP27A1 promoter [72], indicating that CsA affects transcription of the CYP27A1 gene directly. In most of the *in vitro* studies, CYP7α activity was not affected by CsA [69,70]. *In vivo,* in rat however, CsA decreased CYP7α protein levels [50], indicating that the predominant bile acid synthesis pathway may also be affected by CsA. The inhibitory effect of CsA on bile synthesis is suggested to contribute to increased plasma lipid concentrations in transplant recipients. Radioisotope studies performed in children after liver transplantation demonstrated that CsA treatment significantly inhibits bile salts synthesis

apoA-I expression may lead to decreased amounts of secreted apoA-I [65].

**Figure 1.** Mechanisms of CsA-mediated hyperlipidemia. Figure only displays pathways that are reported to be affected by CsA. 1) Inhibition of VLDL formation via inhibition of MTP, 2) Increased and decreased secretion of VLDL particles have been reported, 3) Decreased lipolysis of VLDL due to increased apoCIII and subsequent inhibition of LPL, 4) hypertriglyceridemia by increased CETP activity, 5) Increased LDL due to decreased LDLr expression as well as activity, 6) Increased liver FC content leading to decreased LDLr levels, 7) Increased and decreased levels of HMG-CoAr affecting cholesterol synthesis, 8/9) Inhibition of bile acid conversion via CYP27A1 or CYP7α leading to increased liver FC levels, however in most studies Cyp7 is not affected by CsA. NB: decreased CYP27A1 activity can increase HMG-CoAr levels via negative feedback, 10) Decreased flow of bile salts, cholesterol and phospholipids into bile, 11) Decreased expression and secretion of apoA-I, 12) Inhibition of ABCA1 expression, 13) inhibition of apoA-I lipidation via inhibition of ABCA1 activity 14) Stimulation of HL and CETP leads to increased formation of HDL2 to HDL3, however decreased HL activity has also been reported. VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; AI, apolipoprotein A-I, B, apolipoprotein B; CIII, apolipoprotein CIII; MTP, microsomal triglyceride transfer protein; LPL, lipoprotein lipase; HL, hepatic lipase; CETP, cholesteryl ester transfer protein; ABCA1, ATP-binding cassette transporter-1; SRB1, scavenger receptor class B1; LDLr, LDLreceptor; VLDLr, VLDLreceptor; PL, phospholipid; FC, free cholesterol; HMG-CoAr, 3-hydroxy-3-methyl-glutaryl-CoA reductase; CYP7, 7-hydroxylase; CYP27A1, 27-hydroxylase; MRD, multidrug resistance protein; BSEP, bile salt export protein.

The effects of CsA on CYP27A1 may relate to effects of CsA on cholesterol metabolism. 27 hydroxycholesterol is a potent negative feedback regulator of HMG-CoA reductase [74] and decreased CYP27A1 activity may therefore explain increased HMG-CoA reductase mRNA and cholesterol levels [57]. Although important in macrophages, it should be noted however that it is not clear whether such a feedback loop exists in liver cells [75]. Increased cholesterol synthesis could subsequently lead to downregulation of LDLr levels as observed in some CsA studies, also contributing to increased plasma cholesterol levels (see section 3.2.1).

Besides effects on bile acid synthesis CsA may affect bile flow. CsA treatment is associated with increased plasma bile acid concentrations and cholestasis in humans as well as in animal models [9,52,76]. Studies in rat indicate that bile flow and the secretion of bile salts, proteins and lipids into the bile are dose-dependently inhibited by CsA [52,76,77]. Interestingly, the changes in serum levels of bile acids are consistent with CsA-mediated inhibition of hepatocellular uptake of individual bile acids [78,79]. The inhibitory effect was greater for phosholipid secretion than that for cholesterol [80] and in some studies no inhibition of cholesterol excretion was observed [81], suggesting differential effects on transport mechanisms. Transport pumps involved in bile synthesis and secretion belong to the family of the ATP-binding cassette transporters which include, multidrug resistance proteins (MDR) and P-glycoprotein, and most of which are effectively inhibited by CsA [79,82]. Interestingly, comparison of the bile salt export pump (BSEP) activity from different species, showed that CsA inhibits bile salt transport with species and bile salt specific variation [83]. Rat BSEP was for example more effectively inhibited than mouse BSEP. Biliary cholesterol secretion is mediated via ABCG5 and ABCG8 [84]. Although both members of the ATPbinding cassette family, it has not been investigated whether CsA inhibits ABCG5/8 activity. As phospholipids are transported via MDR3, it is likely that differences in efficacy of CsA between inhibition of MRD3 and ABCG5/8 exist. It is clear that CsA can affect bile flow and secretion in cultured cells and animal models. It should be noted however, that in humans no inhibitory effect of CsA on secretion of bile acids and lipids or on bile composition after liver transplantation was observed [85]. Others have shown that although cholate synthesis was reduced by CsA, compensatory increased intestinal absorption counteracted this decrease [86]. It remains therefore unclear to what extent inhibition of bile flow and secretion by CsA are contributing to hypercholesteremia *in vivo*.
