**3. Mechanisms of CsA-Induced hyperlipidemia – What we learn from cell and animal studies**

As the effects of CsA in humans are confounded by many factors such as other medication, obesity, insulin resistance and nutritional status, cell and animal studies are useful to elucidate the mechanism(s) of CsA-induced hyperlipidemia. Figure 1 depicts the reported CsA-effects on VLDL, LDL and HDL metabolism.

### **3.1. VLDL**

#### *3.1.1. Effects of CsA on VLDL synthesis and secretion*

CsA decreased apoB translocation over the endoplasmic reticulum (ER) membrane in the human liver cell line HepG2 [47]. It was suggested that this was due to a reduction in the efficiency of lipid transfer by inhibition of MTP, however whether MTP activity is inhibited by CsA was not investigated. These findings are in line with the report from Kaptein et al. [48], which showed that CsA inhibits VLDL and apoB secretion from HepG2 cells, by posttranslational mechanisms. In contrast, in mice, CsA increased the rate of hepatic VLDL secretion *in vivo*, while total apoB secretion was unaffected [49]. No effect of CsA on levels of VLDL receptors in either adipose tissue or skeletal muscle were found [50] suggesting that VLDL uptake may not be affected by CsA. There are no studies that we are aware of studying the effect of CsA on *in vivo* VLDL synthesis in humans.

#### *3.1.2. VLDL metabolism*

Inhibition of lipolysis by CsA could contribute to increased plasma VLDL and reduced HDL concentrations. Various studies have investigated lipase activity in patients, but results may be confounded by co-treatment with steroids. HL activity was increased in cardiac transplant patients and correlated with CsA dose while lipoprotein lipase (LPL) activity was decreased in these patients [51]. Others have shown decreased HL as well as LPL activity in kidney transplant recipients [52]. More directly, Tory et al [53] showed suppression of LPL activity in plasma from normolipidemic subjects treated with CsA, while in rats, CsA doseand time-dependent decreased plasma LPL activity [24]. In addition, LPL abundance in skeletal muscle and adipose tissue was decreased in rats [50]. These latter studies suggested CsA can inhibit LPL activity independently of steroids. Although the precise mechanism of CsA-inhibited LPL activity is unknown, it helps to explain increased triglyceride levels observed after CsA treatment.

Some studies show reduced cholesteryl ester transfer protein (CETP) activity in transplant recipients [54]. In contrast, CsA directly added to human plasma *ex vivo* increased CETP activity [53]. These apparently anomalous results may relate to differences between the direct effects of CsA on CETP itself and indirect effects secondary to changes in the concentrations of other lipoproteins, but remain unexplained. Since CETP transfers cholesteryl ester from HDL to apoB-containing lipoproteins with reciprocal transfer of triglycerides, any effect of CsA on CETP activity could be expected to have major effects on plasma lipoprotein profiles.

#### **3.2. LDL**

342 Lipoproteins – Role in Health and Diseases

**cell and animal studies** 

*3.1.2. VLDL metabolism* 

observed after CsA treatment.

**3.1. VLDL** 

CsA-effects on VLDL, LDL and HDL metabolism.

*3.1.1. Effects of CsA on VLDL synthesis and secretion* 

studying the effect of CsA on *in vivo* VLDL synthesis in humans.

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].

**3. Mechanisms of CsA-Induced hyperlipidemia – What we learn from** 

As the effects of CsA in humans are confounded by many factors such as other medication, obesity, insulin resistance and nutritional status, cell and animal studies are useful to elucidate the mechanism(s) of CsA-induced hyperlipidemia. Figure 1 depicts the reported

CsA decreased apoB translocation over the endoplasmic reticulum (ER) membrane in the human liver cell line HepG2 [47]. It was suggested that this was due to a reduction in the efficiency of lipid transfer by inhibition of MTP, however whether MTP activity is inhibited by CsA was not investigated. These findings are in line with the report from Kaptein et al. [48], which showed that CsA inhibits VLDL and apoB secretion from HepG2 cells, by posttranslational mechanisms. In contrast, in mice, CsA increased the rate of hepatic VLDL secretion *in vivo*, while total apoB secretion was unaffected [49]. No effect of CsA on levels of VLDL receptors in either adipose tissue or skeletal muscle were found [50] suggesting that VLDL uptake may not be affected by CsA. There are no studies that we are aware of

Inhibition of lipolysis by CsA could contribute to increased plasma VLDL and reduced HDL concentrations. Various studies have investigated lipase activity in patients, but results may be confounded by co-treatment with steroids. HL activity was increased in cardiac transplant patients and correlated with CsA dose while lipoprotein lipase (LPL) activity was decreased in these patients [51]. Others have shown decreased HL as well as LPL activity in kidney transplant recipients [52]. More directly, Tory et al [53] showed suppression of LPL activity in plasma from normolipidemic subjects treated with CsA, while in rats, CsA doseand time-dependent decreased plasma LPL activity [24]. In addition, LPL abundance in skeletal muscle and adipose tissue was decreased in rats [50]. These latter studies suggested CsA can inhibit LPL activity independently of steroids. Although the precise mechanism of CsA-inhibited LPL activity is unknown, it helps to explain increased triglyceride levels

#### *3.2.1. LDL synthesis and catabolism*

We have recently reviewed this literature in detail [55]. There appear to be conflicting conclusions arising from *in vitro* and *in vivo* studies. One of the key discrepancies is the role of LDLr expression and LDL clearance by the liver in mediating CsA-hyperlipidemia. In general, *in vitro* studies are consistent with a role for decreased LDL receptor expression or activity in liver cells after exposure to CsA [48,56]. *In vivo* studies however, show mixed effects, with no effect or an increase in hepatic LDLr protein or mRNA levels [49,50]. Similarly 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoAr), the rate limiting enzyme in cholesterol synthesis, mRNA levels were upregulated in HepG2 cells and mouse liver after CsA, but hepatic HMG-CoA reductase protein levels in rat liver were unaffected by CsA treatment [49,50,57]. In rats, CsA decreased the fractional catabolic rate of LDL [58]. One very important consideration is the difference in concentrations of CsA used in *in vitro* studies relative to those achieved *in vivo* under normal transplant immunosuppression. *In vitro* studies commonly use concentrations of 10 μg/ml whereas plasma levels of CsA in humans and in animal studies are typically in the order of 100 ng/ml. This apparent 10-fold difference in concentration may underestimate the difference in effective concentrations tested *in vivo* and *in vitro* studies because of the complicating effects of *in vivo* hyperlipidemia, which under some circumstances can lessen the effective concentration of CsA delivered to some tissues [46].

#### **3.3. HDL**

CsA effects on plasma HDL and HDL subclasses may be mediated by effects on the synthesis and/or formation of HDL as well as by effecting remodeling of HDL through changes in lipase and/or CETP activity (see 3.1.2)

#### *3.3.1. Effect of CsA on HDL synthesis and formation*

*In vitro* studies have indicated that CsA potently inhibits ABCA1 activity thereby inhibiting apoA-I lipidation, the first step in HDL formation [59,60,61]. This was associated with decreased ABCA1 turnover and an increase in total and cell-surface levels of ABCA1 [59]. Uptake, Internalization and re-secreton of apoA-I were however decreased by CsA,

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 body system NB.

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