Expression of human apolipoprotein A-II in apolipoprotein E-deficient mice induces features of familial combined hyperlipidemia

Familial combined hyperlipidemia (FCHL) is a common inherited hyperlipidemia and a major risk factor for atherothrombotic cardiovascular disease. The cause(s) leading to FCHL are largely unknown, but the existence of unidentified "major" genes that would increase VLDL production and of "modifier" genes that would influence the phenotype of the disease has been proposed. Expression of apolipoprotein A-II (apoA-II), a high density lipoprotein (HDL) of unknown function, in transgenic mice produced increased concentration of apoB-containing lipoproteins and decreased HDL. Here we show that expression of human apoA-II in apoE-deficient mice induces a dose-dependent increase in VLDL, resulting in plasma triglyceride elevations of up to 24-fold in a mouse line that has 2-fold the concentration of human apoA-II of normolipidemic humans, as well as other well-known characteristics of FCHL: increased concentrations of cholesterol, triglyceride, and apoB in very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL), reduced HDL cholesterol, normal lipoprotein lipase and hepatic lipase activities, increased production of VLDL triglycerides, and increased susceptibility to atherosclerosis. However, FCHL patients do not have plasma concentrations of human apoA-II as high as those of apoE-deficient mice overexpressing human apoA-II, and the apoA-II gene has not been linked to FCHL in genome-wide scans. Therefore, the apoA-II gene could be a "modifier" FCHL gene influencing the phenotype of the disease in some individuals through unkown mechanisms including an action on a "major" FCHL gene. We conclude that apoE-deficient mice overexpressing human apoA-II constitute useful animal models with which to study the mechanisms leading to overproduction of VLDL, and that apoA-II may function to regulate VLDL production.     

Altered immune responses in apolipoprotein E-deficient mice

Apolipoprotein E (apoE) is a 34 kDa glycosylated protein with multiple biological properties. In addition to its role in cholesterol transport, apoE has in vitro immunomodulatory properties. Recent data suggest that these immunomodulatory effects of apoE may be biologically relevant, and apoE-deficient mice have altered immune responses after bacterial inoculation and increased susceptibility to endotoxemia induced by lipopolysaccharide (LPS). To better understand the mechanism by which apoE-modulates immune responses, we tested the role of human apoE isoforms in assays of human T cell proliferation, and analyzed the immune responses of apoE-deficient mice. Both the E3 and E4 isoforms of apoE induced similar suppression of human lymphocyte function in assays of T cell proliferation, including mitogenic responses to phytohaemagglutin (PHA), stimulation of the T cell receptor with alphaCD3, and antigen-specific response to tetanus toxoid. ApoE-deficient mice showed no quantitative differences in thymic, splenic, or bone marrow lymphocyte populations, nor were there in vitro abnormalities in splenocyte proliferation after stimulation with alphaCD3 to suggest an inherent T cell defect in apoE-deficient mice. ApoE deficient animals, however, had significantly higher levels of antigen-specific IgM after immunization with tetanus toxoid, and impaired delayed type hypersensitivity responses as compared to control C57-BL/6 mice.These results support a growing body of evidence demonstrating an interplay between lipid metabolism and immune responses, and suggest that apoE plays a biologically relevant role in regulating humoral and cell-mediated immunity.     

Diet and atherosclerosis in apolipoprotein E-deficient mice

Atherosclerosis is a multifactorial, long-lasting process in humans. Accordingly, animal models in which more rapid changes occur can be useful for the study of this process. Among such models are apolipoprotein E-deficient (apoE-/-) mice, which give insight into the human process. ApoE-/- mice show impaired clearing of plasma lipoproteins and develop atherosclerosis in a short time, and hence they are an excellent model in which to assess the impact of dietary factors. This review considers lipid metabolism and inflammation as well as nutritional constituents affecting atherosclerosis, with reference to apoE-/- mice, and discusses the mechanisms through which they act.     

Iron-ion radiation accelerates atherosclerosis in apolipoprotein E-deficient mice

Radiation exposure from a number of terrestrial sources is associated with an increased risk for atherosclerosis. Recently, concern over whether exposure to cosmic radiation might pose a similar risk for astronauts has increased. To address this question, we examined the effect of 2 to 5 Gy iron ions ((56)Fe), a particularly damaging component of cosmic radiation, targeted to specific arterial sites in male apolipoprotein E-deficient (apoE(-/-)) mice. Radiation accelerated the development of atherosclerosis in irradiated portions of the aorta independent of any systemic effects on plasma lipid profiles or circulating leukocytes. Further, radiation exposure resulted in a more rapid progression of advanced aortic root lesions, characterized by larger necrotic cores associated with greater numbers of apoptotic macrophages and reduced lesional collagen compared to sham-treated mice. Intima media thickening of the carotid arteries was also exacerbated. Exposure to (56)Fe ions can therefore accelerate the development of atherosclerotic lesions and promote their progression to an advanced stage characterized by compositional changes indicative of increased thrombogenicity and instability. We conclude that the potential consequences of radiation exposure for astronauts on prolonged deep-space missions are a major concern. Knowledge gained from further studies with animal models should lead to a better understanding of the pathophysiological effects of accelerated ion radiation to better estimate atherogenic risk and develop appropriate countermeasures to mitigate its damaging effects.     

Macrophage-derived apolipoprotein E ameliorates dyslipidemia and atherosclerosis in obese apolipoprotein E-deficient mice

Previous studies have demonstrated that macrophage-derived apolipoprotein E (apoE) reduces atherosclerotic lesion formation in lean apoE-deficient ((-/-)) mice. apoE has also been demonstrated to play a role in adipocyte differentiation and lipid accumulation. Because the prevalence of obesity has grown to epidemic proportions, we sought to determine whether macrophage-derived apoE could impact atherosclerotic lesion formation or adipose tissue expansion and inflammation in obese apoE(-/-) mice. To this end, we transplanted obese leptin-deficient (ob/ob) apoE(-/-) mice with bone marrow from either ob/ob;apoE(-/-) or ob/ob;apoE(+/+) donors. There were no differences in body weight, total body adipose tissue, or visceral fat pad mass between recipient groups. The presence of macrophage-apoE had no impact on adipose tissue macrophage content or inflammatory cytokine expression. Recipients of apoE(+/+) marrow demonstrated 3.7-fold lower plasma cholesterol (P < 0.001) and 1.7-fold lower plasma triglyceride levels (P < 0.01) by 12 wk after transplantation even though apoE was present in plasma at concentrations <10% of wild-type levels. The reduced plasma lipids reflected a dramatic decrease in very low density lipoprotein and a mild increase in high-density lipoprotein levels. Atherosclerotic lesion area was >10-fold lower in recipients of ob/ob;apoE(+/+) marrow (P < 0.005). Similar results were seen in leptin receptor-deficient (db/db) apoE(-/-) mice. Finally, when bone marrow transplantation was performed in 4-mo-old ob/ob;apoE(-/-) and db/db;apoE(-/-) mice with preexisting lesions, recipients of apoE(+/+) marrow had a 2.8-fold lower lesion area than controls (P = 0.0002). These results demonstrate that macrophage-derived apoE does not impact adipose tissue expansion or inflammatory status; however, even very low levels of macrophage-derived apoE are capable of reducing plasma lipids and atherosclerotic lesion area in obese mice.     

Mulberry leaf powder prevents atherosclerosis in apolipoprotein E-deficient mice

Mulberry is commonly used to feed silkworms. Here we examined whether a dietary intake of mulberry leaf (ML) could affect atherogenesis in vivo and in vitro. Apolipoprotein E-deficient mice were fed either normal chow (control group) or a diet containing 1% ML powder (ML group) from 6 weeks of age. The mice were sacrificed after 12 weeks. The susceptibility of plasma lipoprotein to oxidation was assessed using diene formation. A significant increase in the lag time of lipoprotein oxidation was detected in the ML group compared with the control group. Furthermore, the ML group showed a 40% reduction in atherosclerotic lesion size in the aortae compared with the control. We also examined the direct anti-oxidative activity of ML in vitro. Aqueous extract of ML had a strong scavenging effect on 1,1-diphenyl-2-picrylhydrazyl and inhibited lipoprotein oxidation. These results confirm that ML contains anti-oxidative substances that might help prevent atherosclerosis.     

Increased vascular biosynthesis of tetrahydrobiopterin in apolipoprotein E-deficient mice

Previous studies suggested that loss of tetrahydrobiopterin (BH(4)) may play an important role in the pathogenesis of vascular endothelial dysfunction induced by diabetes and hypertension. In contrast, controversial results have been reported regarding BH(4) metabolism in experimental models of atherosclerosis. Therefore, the present study was designed to characterize the expression and activity of GTP-cyclohydrolase I, a rate-limiting enzyme in biosynthesis of BH(4), during atherogenesis. BH(4) levels were significantly increased in atherosclerotic aortas of apolipoprotein E (apoE)-deficient mice as compared with wild-type mice after 5 mo of Western diet treatment. This increase was further significantly enhanced in apoE-deficient mice fed for 9 and 14 mo. Removal of the endothelium almost eliminated BH(4) in wild-type mice but not in apoE-deficient mice, suggesting that a major component of increased BH(4) synthesis is localized in the vascular media of apoE-deficient mice. Oxidative products of BH(4) were low and did not differ between wild-type and apoE-deficient mice over the course of this study. Increased protein expression and enzymatic activity of GTP-cyclohydrolase I were detected in aortas of apoE-deficient mice (P < 0.05), providing molecular mechanisms responsible for elevation of vascular BH(4). In contrast to aortas, we did not detect any change in levels of BH(4) and in GTP-cyclohydrolase I expression in the brain. Our results demonstrate selective increase of intracellular BH(4) levels via elevation of GTP-cyclohydrolase I activity in vascular tissue of apoE-deficient mice.     

Increased aortic stiffness assessed by pulse wave velocity in apolipoprotein E-deficient mice

Atherosclerosis develops and progresses spontaneously in apolipoprotein E-knockout (apoE-KO) mice. A direct consequence of atherosclerosis is an increase in vascular stiffness. Pulse wave velocity (PWV) has been used to assess the stiffness of large vessels and was found to be increased in patients with atherosclerosis. In the present study, aortic stiffness was assessed by PWV in 4- and 13-mo-old apoE-KO mice and age-matched controls (C57BL/6J). In 13-mo-old apoE-KO mice with extensive atherosclerotic lesions in the aorta (61 +/- 4%), PWV increased significantly (3.8 +/- 0.2 m/s) compared with controls (2.9 +/- 0.2 m/s). Endothelial nitric oxide (EDNO)-mediated vasorelaxation in response to ACh was markedly diminished in the aortic rings isolated from 13-mo-old apoE-KO mice compared with age-matched controls. In contrast, in 4-mo-old apoE-KO mice with only moderate atherosclerotic lesions in the aorta (23 +/- 5%), there were no significant changes in PWV and EDNO-mediated relaxation compared with controls. Blood pressure was not different among the four groups of mice. There were no significant differences in endothelium-independent vascular responses to sodium nitroprusside among different groups investigated. Histological evaluation revealed focal fragmentation of the elastic laminae in the aortic walls of 13-mo-old apoE-KO mice. These results demonstrate for the first time that aortic stiffness determined by PWV increases in 13-mo-old apoE-KO mice. Endothelial dysfunction and elastic destruction in vascular wall caused by atherosclerosis may have contributed.     

Loss of lysophospholipase 3 increases atherosclerosis in apolipoprotein E-deficient mice

Human LCAT-like lysophospholipase (LLPL), or lysophospholipase 3, was first identified in vitro, in foam cells derived from THP-1 cells. We demonstrated that LLPL was present in foam cells in the severe atherosclerotic lesions that develop in apolipoprotein E-null (apoE(-/-)) mice. This indicated that LLPL might affect lipid metabolisms in foam cells and, therefore, atherogenesis. Accordingly, we created LLPL-knockout mice by gene targeting and crossed them with apoE(-/-) mice. We showed that the absence of LLPL increased lesion formation markedly in apoE(-/-) mice but had little effect on the plasma-lipid profile. In addition, LLPL-deficient peritoneal macrophages were more sensitive to apoptosis induced by exposure to oxidized low-density lipoprotein. LLPL might provide a link between apoptosis in macrophages and atherogenesis. Our data demonstrate that LLPL activity is anti-atherogenic and indicate that the regulation of this enzyme might be a novel drug target for the treatment of atherosclerosis.     

Homozygous disruption of Pctp modulates atherosclerosis in apolipoprotein E-deficient mice

Phosphatidylcholine transfer protein (PC-TP) is a cytosolic phospholipid binding protein and a member of the steroidogenic acute regulatory-related transfer domain superfamily. Its tissue distribution includes liver and macrophages. PC-TP regulates hepatic lipid metabolism, and its absence in cholesterol-loaded macrophages is associated with reduced ATP binding cassette transporter A1-mediated lipid efflux and increased susceptibility to apoptosis induced by unesterified cholesterol. To explore a role for PC-TP in atherosclerosis, we prepared PC-TP-deficient/apolipoprotein E-deficient (Pctp(-/-)/Apoe(-/-)) mice and littermate Apoe(-/-) controls. At 16 weeks, atherosclerosis was increased in chow-fed male, but not female, Pctp(-/-)/Apoe(-/-) mice. This effect was associated with increases in plasma lipid concentrations. By contrast, no differences in atherosclerosis were observed between male or female Pctp(-/-)/Apoe(-/-) mice and Apoe(-/-) controls fed a Western-type diet for 16 weeks. At 24 weeks, atherosclerosis in chow-fed male Pctp(-/-)/Apoe(-/-) mice tended to be reduced in proportion to plasma cholesterol. The attenuation of atherosclerosis in female Pctp(-/-)/Apoe(-/-) mice fed chow or the Western-type diet for 24 weeks was not attributable to changes in plasma cholesterol or triglyceride concentrations. These findings suggest that PC-TP modulates the development of atherosclerosis, in part by regulating plasma lipid concentrations.     

Suppression of atherogenesis by overexpression of glutathione peroxidase-4 in apolipoprotein E-deficient mice

Accumulation of oxidized lipids in the arterial wall contributes to atherosclerosis. Glutathione peroxidase-4 (GPx4) is a hydroperoxide scavenger that removes oxidative modifications from lipids such as free fatty acids, cholesterols, and phospholipids. Here, we set out to assess the effects of GPx4 overexpression on atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. The results revealed that atherosclerotic lesions in the aortic tree and aortic sinus of ApoE(-/-) mice overexpressing GPx4 (hGPx4Tg/ApoE(-/-)) were significantly smaller than those of ApoE(-/-) control mice. GPx4 overexpression also diminished signs of advanced lesions in the aortic sinus, as seen by a decreased occurrence of fibrous caps and acellular areas among hGPx4Tg/ApoE(-/-) animals. This delay of atherosclerosis in hGPx4Tg/ApoE(-/-) mice correlated with reduced aortic F(2)-isoprostane levels (R(2)=0.75, p<0.01). In addition, overexpression of GPx4 lessened atherogenic events induced by the oxidized lipids lysophosphatidylcholine and 7-ketocholesterol, including upregulated expression of adhesion molecules in endothelial cells and adhesion of monocytes to endothelial cells, as well as endothelial necrosis and apoptosis. These results suggest that overexpression of GPx4 inhibits the development of atherosclerosis by decreasing lipid peroxidation and inhibiting the sensitivity of vascular cells to oxidized lipids.     

Polydatin alleviates high-fat diet induced atherosclerosis in apolipoprotein E-deficient mice by autophagic restoration

BackgroundPolydatin has been reported to possess remarkable anti-atherosclerotic activities. However, there are different opinions on its regulatory mechanisms. It remains unclear whether the anti-atherosclerotic mechanism of polydatin is related to its autophagic restoration or not. The aim of this study was to explore the question.MethodsUsing atherosclerotic model induced by high-fat diet in apolipoprotein E-deficient mice, the investigation was performed with polydatin alone or in combination with autophagic inhibitor or inducer intervention. Inhibitory sites of polydatin to PI3K were identified by molecular docking.ResultsPolydatin can significantly inhibit PI3K/Akt/mTOR pathway proteins expression, improve autophagic dysfunction and reduce atherosclerotic lesions. These effects could be antagonized and reinforced by adding autophagic inhibitor and inducer, respectively. Inhibitory sites of polydatin to PI3K were found to be ASP-810, SER-854, VAL-851, LEU-807, SER-774, LYS-802, ASP-933, SER-919, ASN-920, PHE-930, MEF-922, GLN-859 of PI3Kα.ConclusionsThe mechanism of polydatin to alleviate atherosclerotic lesions was achieved by autophagic restoration.     

Arresting progressive atherosclerosis by immunization with an anti-glycosaminoglycan monoclonal antibody in apolipoprotein E-deficient mice

Atherogenesis is associated with the early retention of low-density lipoproteins (LDL) in the arterial intima by interaction with glycosaminoglycan (GAG)-side chains of proteoglycans. Retained LDL undergo reactive oxygen species-mediated oxidation. Oxidized LDL trigger oxidative stress (OS) and inflammation, contributing to atherosclerosis development. Recently, we reported the preventive anti-atherogenic properties of the chimeric mouse/human monoclonal antibody (mAb) chP3R99-LALA, which were related to the induction of anti-chondroitin sulfate antibody response able to inhibit chondroitin sulfate dependent LDL-enhanced oxidation. In the present work, we aimed at further investigating the impact of chP3R99-LALA mAb vaccination on progressive atherosclerosis in apolipoprotein E-deficient mice (apoE^−/−) fed with a high-fat high-cholesterol diet receiving 5 doses (50 µg) of the antibody subcutaneously, when ~5% of the aortic area was covered by lesions. Therapeutic immunization with chP3R99-LALA mAb halted atherosclerotic lesions progression. In addition, aortic OS was modulated, as shown by a significant (p<0.05) reduction of lipid and protein oxidation, preservation of antioxidant enzymes activity and reduced glutathione, together with a decrease of nitric oxide levels. chP3R99-LALA mAb immunization also regulated aortic NF-κB activation, diminishing the proinflammatory IL1-β and TNF-α gene expression as well as the infiltration of macrophages into the arterial wall. The therapeutic immunization of apoE^−/− with progressive atheromas and persistent hypercholesterolemia using chP3R99-LALA mAb arrested further development of lesions, accompanied by a decrease of aortic OS and NF-κB-regulated pro-inflammatory cytokine gene expression. These results contribute to broaden the potential use of this anti-GAG antibody-based immunotherapy as a novel approach to target atherosclerosis at different phases of progression.     

Evidence that dendritic cells infiltrate atherosclerotic lesions in apolipoprotein E-deficient mice

Earlier we reported that atherosclerotic lesions of apoE-deficient mice contained cells which stained positively with anti-S-100 antibody and that cells exhibiting the ultrastructural features of dendritic cells were present in the aortic lesions. These observations suggested that dendritic cells might be involved in mouse atherosclerosis. By employing DEC-205 and MIDC-8 antibodies specific for dendritic cells, the present study has established that dendritic cells indeed accumulate in atherosclerotic lesions of apoE-deficient mice. Finding dendritic cells infiltrating atherosclerotic lesions in apoE-deficient mice offers the possibility of investigating the migratory routes of dendritic cells and their involvement in T-cell activation.     

The continuous administration of aspirin attenuates atherosclerosis in apolipoprotein E-deficient mice

Aspirin reduces the incidence of thrombotic occlusive events. Classically this has been thought to be due to the platelet inhibitory action of aspirin but it has recently been shown that inflammation plays a predominant role in the initiation and progression of lesions in atherosclerosis. In humans, treatment with aspirin reduces cardiovascular risk and slows carotid plaque growth in a dose-dependent fashion. We have explored this issue in Apo E-deficient mice on a high-fat, high cholesterol diet which provided these animals with a continuous administration of 500 microg/day of acetylsalicylic acid in the drinking water. After 10 weeks of treatment, the size of the atherosclerotic lesion at the aortic sinus had reduced by 35%. At the end of the trial there were no significant changes in either plasma lipids or in the quantitative distribution among lipoproteins. Likewise, the total antioxidant status and the resistance of plasma to oxidation in vitro was similar and there was no change in the distribution of iron deposits and in the relative composition of plasma pro-oxidants and antioxidants, or in the concentration of plasma in ferritin. Therefore, it is our hypothesis that the antiinflammatory effect is responsible for the reduction in lesion size. We propose that antiinflammatory molecules which do not cause gastrointestinal complications should be tested in humans to determine long-term efficacy in the attenuation of atherosclerosis.     

Combined application of rapamycin and atorvastatin improves lipid metabolism in apolipoprotein E-deficient mice with chronic kidney disease

Atherosclerosis arising from the pro-inflammatory conditions associated with chronic kidney disease (CKD) increases major cardiovascular morbidity and mortality. Rapamycin (RAPA) is known to inhibit atherosclerosis under CKD and non-CKD conditions, but it can cause dyslipidemia; thus, the co-application of lipid-lowering agents is recommended. Atorvastatin (ATV) has been widely used to reduce serum lipids levels, but its synergistic effect with RAPA in CKD remains unclear. Here, we analyzed the effect of their combined treatment on atherosclerosis stimulated by CKD in apolipoprotein E-deficient (ApoE^−/−) mice. Oil Red O staining revealed that treatment with RAPA and RAPA＋ ATV, but not ATV alone, significantly decreased the atherosclerotic lesions in the aorta and aortic sinus, compared to those seen in the control (CKD) group. The co-administration of RAPA and ATV improved the serum lipid profile and raised the expression levels of proteins involved in reverse cholesterol transport (LXRα, CYP7A1, ABCG1, PPARγ, ApoA1) in the liver. The CKD group showed increased levels of various genes encoding atherosclerosis-promoting cytokines in the spleen (Tnf-α, Il-6 and Il-1β) and aorta (Tnf-α and Il-4), and these increases were attenuated by RAPA treatment. ATV and RAPA＋ATV decreased the levels of Tnf-α and Il-1β in the spleen, but not in the aorta. Together, these results indicate that, in CKD-induced ApoE^−/− mice, RAPA significantly reduces the development of atherosclerosis by regulating the expression of inflammatory cytokines and the co-application of ATV improves lipid metabolism.     

Effect of diets on lipoprotein concentrations in heterozygous apolipoprotein E-deficient mice

Loss of apolipoprotein E synthesis causes increased serum cholesterol concentrations and the sensitivity to high-fat diet in mice. We analyzed the changes in lipoprotein and hepatic structures in apolipoprotein E-deficient mice kept on control diet and cholesterol diets. Basal cholesterolemia of heterozygous (+/-) mice (2.2+/-0.28 mmol/l) was the same compared to wild-type (+/+) mice (2.3+/-0.15 mmol/l), but was lower compared to homozygous (-/-) mice (10.3+/-1.40 mmol/l). In +/- mice, cholesterolemia rose to 3.2 mmol/l on cholesterol diet and to 9 mmol/l on cholate diet, to 3 mmol/l and 3.6 mmol/l in +/+ mice, and to 23.4 mmol/l and 70.5 mmol/l in -/- mice, respectively. While the ratio of cholesterol/triglyceride concentrations in VLDL, IDL and LDL fractions was not increased in +/- mice and +/+ mice, it was increased in -/- mice on control diet. On the cholesterol diet, this ratio rose and was dramatically increased by cholate diet in all groups of mice. Even though cholate supplementation increased cholesterol concentration, it led to substantial toxic changes in hepatic morphology of all animals. In conclusion, one functional apo E allele in +/- mice is effective in keeping serum cholesterol concentrations in normal range on a control diet, but not on the cholesterol and cholate diets.     

Decreased expression of klotho gene in uremic atherosclerosis in apolipoprotein E-deficient mice

Chronic renal failure (CRF) markedly accelerates the development of atherosclerosis, but the pathogenesis of uremic atherosclerosis remains to be elucidated. The klotho gene, predominantly expressed in the kidney, plays a key role in regulating aging and the development of age-related diseases in mammals. A loss of klotho results in multiple aging-like phenotypes including atherosclerosis. This study examines the relationship between the klotho expression and the development of accelerated atherosclerosis in uremic state.Eight-week-old apolipoprotein E-deficient (apo-E^−/−) male mice underwent 5/6 partial kidney ablation to induce CRF or sham-operation. At 6 wk after nephrectomy, CRF mice showed significantly increased aortic plaque area fraction, aortic root plaque area and aortic cholesterol content as compared with non-CRF mice. Serum urea, total cholesterol and triglyceride concentrations were significantly higher in CRF apo-E^−/− mice compared with non-CRF controls. Moreover, the expression of renal klotho gene and the serum levels of klotho protein were markedly decreased in CRF mice compared with controls.These results suggested that CRF favored atherosclerosis in apo-E^−/− mice and uremic atherosclerosis was accompanied by down-regulation of klotho expression.