Frequency of hypercholesterolemia after cardiac transplantation

MISCELLANEOUS

Frequency of Hypercholesterolemia Transplantation

After Cardiac

Jonathan S. Stamler, MD, Douglas E. Vaughan, MD, M. Audrey Rudd, PhD, Gilbert H. Mudge, MD, James Kirshenbaum, MD, Pia Young, RN, R. Wayne Alexander, MD, PhD, and Joseph Loscalzo, MD, PhD

Cardiac transplant patients are prone to accelerated coronary atherosclerosis. The mechanism by which this process occurs is not yet known, although immunologically mediated arterial injury is thought to play a primary role in its pathogenesis. Despite immunosuppressive potency, patients treated with cyclosporin A remain at significant risk for the development of accelerated atherosclerosis. It is hypothesized that cyclosporin A’s hepatotoxic effects might contribute to the atherosclerotic process by impairing low density lipoprotein hepatic clearance in transplant patients, which would be reflected in a more atherogenic lipoprotein profile. To test this hypothesis, serum cholesterol levels were analyzed after transplantation. Significant and progressive increases in total cholesterol and in the total-to-high density lipoprotein cholesterol ratio were found. This atherogenic lipoprotein profile may contribute to accelerated atherosclerosis in cardiac transplant patients treated with cyclosporin A. (Am J Cardiol 1988;62:1268-1272)

ardiac transplant patientsare proneto accelerated coronary atherosclerosis,which remains a significant cause of patient morbidity and graft failure.1-3The mechanism by which this processoccurs is not well understood.Evidencefrom animal transplantation models of mismatched heterotopic cardiac allografts and other experimental models of immunologic injury suggeststhat immunologically mediated arterial injury may interact with a high cholesterol diet to produce cholesterol-filled atheromatous plaques, histologically similar to lesions seenin man.4-7Data from Hess et al* support the view that hypercholesterolemiapredisposesto coronary artery diseasein cardiac transplant patients, particularly in the setting of immunologically mediated endothelial cytotoxicity. Cyclosporin A, the most active immunosuppressant available, is far more successfulthan its predecessorsat preventing rejection; however,patients treated with this drug remain at significant risk for the development of atherosclerosis.3The acceleratedatheromatousresponse in patients treated with cyclosporin A might be affected by its cytotoxic effects on both the endothelial cell9 and the hepatocyte.rO-l2The latter is manifested primarily by impaired low density lipoprotein (LDL) hepatic clearance. Accordingly, in this study we hypothesize that administration of cyclosporin A may be associated with impaired LDL metabolism and the consequentdevelopment of a more atherogenic lipoprotein profile. To this end, we report significant and progressiveincreases in cholesterol levels after transplantation predominantly owing to dramatic increasesin LDL cholesterol in patients treated with cyclosporin A.

C

METHODS Analysis of stored sera: Sera obtained before and at

From the Vascular and Cardiology Divisions, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts. Manuscript received June 6, 1988; revised manuscript received and accepted August 3, 1988. Address for reprints: Joseph Loscalzo, MD, PhD, Department of Medicine, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115.

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frequent intervals after transplantation were stored at -2OOC. Sera were thawed and triglyceride, total cholesterol and high density lipoprotein (HDL) cholesterol analyseswere performed on samplesfrom 20 patients. Total cholesterol and triglyceride determinations were performed using cholesterol oxidase13 and lipase14 methods, respectively; HDL cholesterol determinations were performed using standard methods with heparinMnC12 precipitation techniques.The availability of patients’ sera representedthe sole criterion for inclusion in this phase of the study. Retrospective chart review: Serum samples stored for prolonged periods may be subject to lipoprotein degradation.15 Thus, in order to confirm any temporal

-

formed on 25 (20 male, 5 female) aged-matched(39 f 14 years) transplant patients. Sixteen patients had nonischemic cardiomyopathy and 9 patients had ischemic cardiomyopathy. Immunosuppressantswere used as described before. Twelve patients were treated with azathioprine, on average6 months postoperatively, 5 with /3 blockers, 6 with diuretics, 12 with calcium antagonists, 2 with converting enzymeinhibitors, 2 with niacin and 1 with insulin. Significant increases in total cholesterol were noted by 3 weeks; they peaked at 3 months and persistedfor 2 years of follow-up (Table I). This temporal sequenceis essentially identical to that observedin our serum analysis of total cholesterol, thus confirming our findings and extending our observationsto 2 years after transplantation. LIVERFUNCTION TESTABNORMALITIES: Eighteen of 25 RESULTS Sera analysis: PATIENT POPULATION: Twenty patients patients developedliver enzyme elevations after trans(17 male, 3 female) with a mean age (* standard devi- plantation. Elevations were frequently of the cholestatic ation) of 41 f 14 years were included in the analysis. type, tended to cluster (occurring within 3 months of Transplantation was performed for nonischemiccardio- transplantation or after 7 months) and often recurred in myopathy in 13 patients and ischemic cardiomyopathy the same patient (Table I). in 7. Cyclosporin A was administered to all patients throughout their posttransplantation coursein dosescal- DISCUSSION The data presentedhere reveal a time-dependentinculated to produce trough serum levels between 100 and 150 rig/ml. Corticosteroid therapy was begun upon creasein total cholesterol levels in heart transplant patransplantation in all patients. At discharge, prednisone tients treated with cyclosporin A. These changes are was initiated at 0.5 mg/kg/day and tapered gradually predominantly a result of significant increases(approxito 0.15 to 0.20 mg/kg/day by 1 year. Azathioprine was mately 150%)in LDL cholesterol.Total cholesterol and added in the event of resistant rejection, and 13 patients LDL cholesterol becomeelevatedby the secondweek of received this drug beginning on average 6 months cyclosporin A therapy, peak at approximately 3 months (range 2 to 13) postoperatively. Antihypertensive thera- and remain elevated thereafter. Because LDL levels py was used frequently: 5 patients were treated with p have been shown to correlate directly with risk of coroblockers, 7 with diuretics, 9 with calcium antagonists nary disease,16this change in lipoprotein profile oband 2 with angiotensin converting enzyme inhibitors, servedwith cyclosporine A therapy is likely to contribTwo patients were treated for diabeteswith insulin and ute to atherosclerosisin transplant patients. There is little information available on lipid profiles 4 received niacin for hypercholesterolemia. CHOLESTEROL DETERMINATIONS: Significant elevations in cardiac transplant patients. In a preliminary commuin LDL and total cholesterol occurred by 2 weekspost- nication, Becker et alI7 noted increasesin total cholesoperatively, peaked at approximately 3 months and re- terol that also are predominantly a reflection of changes mained elevated throughout the first year of transplan- in LDL cholesterol, This supports our findings. In contation (Table I). A trend toward elevation of triglycer- trast to Gao et al,ls Becker et al noted no elevation of ide levels was seen by 3 months but it did not reach significance. A similar increasein HDL cholesterol was observedby 3 months postoperatively; it normalized by 1 year. Total cholesterol/HDL cholesterol increased progressively throughout the first year of transplantation. Posttransplantation trends in total and LDL cholesterol, measured more frequently, are shown in Figure 1. CORRELATION OFCYCLOSPORIN A LEVELS ANDPREDNISONE DOSAGES WITHCHOLESTEROL: Cyclosporin A dosages circles: total cholesterol triangles: LDL cholesterol were monitored and changed frequently, reflecting the / great variation in individual cyclosporin A levels over I t short periods. Cyclosporin A trough levels correlated 01”“’ ““““. 1 0 5 10 15 poorly with absolute total cholesterol values (r = 0.17) TIME (months) or with changes in cholesterol (r = 0.30) at 1 year. Prednisonedosages(mg/kg) also correlated poorly with FIGURE 1. Changes in total and low density lipoprotein total cholesterol levels at 1 year (r = 0.34). cholesterol with time after cardiac transplantation as deterChart reviews: To confirm the observedtemporal el- mined by sera analysis. Values at 0 indicate values before aaevation in cholesterol postoperatively as determined by ministration of immunosuppressants. Each point represents sera analysis of frozen samples,a chart review was per- the mean 5 standard error of the mean of 20 patient samples. change in cholesterol postoperatively as determined by sera analysis of frozen samples,and to evaluate any possible relation between these changesand the use of immunosuppressiveagents, a retrospective chart review was performed on 25 transplant patients, Total cholesterol levels were well documented;however, a complete lipoprotein profile was not available on these patients. Statistical analysis: Cholesterol and triglyceride levels were determined at frequent intervals after transplantation. The Student’s t test was performed at each interval comparing mean values with perioperative values. A linear regression analysis was used to correlate levels and dosagesof immunosuppressantswith cholesterol values. P values 10.05 were consideredsignificant.

-“------(J 1 +-----A

i 1

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AND CARDIAC

TRANSPLANTATION

triglyceride levels.Neither study gives detailed information on immunosuppressivetherapy or a proposedexplanation for these observations. Much of the available information addressing changesin lipoprotein profiles after transplantation has TABLE

I Lipid Profiles

After Cardiac

been ascertainedfrom renal transplant patients; in such patients posttransplant changes in cholesterol may refleet the effect of prolonged metabolic derangementsas well as immunosuppressivetherapy.19-23There is, however, a consensusthat steroid usage,by promoting insu-

Transplantation

Retrospective Chart Analysis of Total Cholesterol (mg/dl) Pt

Periopt

3 months

1 year

2 years

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Mean f SD p value

123 208 163 126

243 247 227 268 -

232 294 212 227 274

234 312

263 149 232 214 161 -

321 239 271 334 252 273 296

263 274 341 291 293 295 25% 215 263 372 285 230 267 286 266 -

252 310 297 241 270 307 217

282 380 228 275f44
256 388 227 281 f 46
Pt 26 27 2% 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Mean k SD D value

1270

276 181 213 213 164 136 168 255 184 200 306 164 195 f 50 -

Sera Analysis (mg/dl)f Total Cholesterol 1 2 3

157 161 166 126 117 112 123 146 130 94 166 10% 109 8% 124 12% f 25 -

30% 299 302 331 240 267 252 331 204 231 301 233 273 f 38
135 204 249 253 213

177 254 220 162

LDL Cholesterol 1 2 103 154 91 78 93

116 147 124

HDL Cholesterol i 2

3 115 184 126

38 62 51 61 34 45 25 49 47 21 40 33 23 29 34 39 f 12

103

32 18% 260 193

204 264

200 241 232 287 155 255 177 291 221 *45
THE AMERICAN

273 285 285 207 229 250 234 f41
JOURNAL

64 43 68 75 35 57 61 53 63 41 70 62 f 19 -

92 144 109 113 153 154 183 94 183 119 175 135 f 30
OF CARDIOLOGY

124

151 146 165 214 131 178 182 151 f 33
VOLUME

62

43 27 58 76 76 43 76 67 46 44 64 59 40 45 29 72 54 f 17 <0.02

Lrver Function Abnormalities” +tt tt+ t ++ + 0 0 + 0 + + + 0 +++ 0 0 +++ ++ + 0 +++ + +t+ + 0

301

300 237 348 -

3 2% 34 80

36 56

41 56 61 51 50 26 32 46 f 16 NS

Triglycerides 1 2 140 103 109 165 93 120 151 110 240 81 326 108 113 92 99 137 f 65 -

195 113 298 151 66 266 19% 154 205 219 71 227 107 136 145 220 173 f 67 NS

3 171 180 72

Total/HDL Cholesterol 1 2 3 4.3 7.6 4.1 2.6 3.3

113 120 361 355 295 99 131 127 179 183 f 99 NS

2.1 3.4 2.5 4.9 3.0 2.8 4.5 4.2 3.3 4.7 3.0 3.6 3.5 f 0.8

4.3 3.3 2.8 4.4 3.4 3.6

6.3 7.5 2.8

2.5 3.6

6.4 4.3 5.5 3.6 4.9 3.9 5.7 6.1 4.0 4.5 f 1.2 <0.02

4.9 4.7 5.6 4.1 8.8 7.8

lin resistance, is primarily responsible for lipid abnormalities induced in transplant patients.20,22,24 Although at times conflicting, the bulk of the evidence gleaned from experiencewith renal transplant patients suggests that corticosteroids induce increasesin triglyceride and HDL cholesterol with either no effect or a lowering effect24on LDL cholesterol. The effect of prednisone on cholesterol is seenwithin 48 hours, is dependenton dose and dosing interval and is reversed by lowering the dose.19%22,24 The pattern we have observed-a marked increase in LDL cholesterol that is maintained during the usual gradual decreasein prednisone dosageafter transplantation-is therefore not compatible with a steroid effect. That these changes in cholesterol may reflect an azathioprine effect is also unlikely. This immunosuppressantwas used in approximately half of the study patients well after increasesin cholesterol had occurred. In addition, in both animal and human transplantation, azathioprine has not been associatedwith significant increasesin serum cholestero1.25,26 Increases in LDL and very low density lipoprotein cholesterol have been reported in animal models of cyclosporin A-induced toxicity.12 In 10 renal transplant recipients, theseanimal findings were substantiatedby a decreasein triglyceride and total cholesterol noted after conversion from cyclosporin A to azathioprine.26The mechanism responsiblefor these changesin lipoprotein metabolism is not known; however,cyclosporin A hepatotoxicity is a well-established side effect of this drug, and the liver is primarily responsiblefor the metabolism of LDL.27 The cholestatic pattern with associatedmild hepatocellular enzyme elevations seen in many of our patients, the tendency for elevations to cluster early, (within the first 3 months of transplantation) and late (after 7 months) and the recurrence of enzyme elevations in individual patients are all signsof hepatotoxicity previously attributed to cyclosporin APi It is therefore possiblethat cyclosporin A hepatotoxicity results in defective LDL clearance that manifests as an atherogenie lipoprotein profile. Early recovery of LDL cholesterol after transplantation may be in part a reflection of improved nutritional status or liver perfusion, or both, accompanying the general hemodynamic benefits of transplantation in these generally very ill patients. These nutritional and hemodynamic benefits, however, cannot explain the evolution of an atherogenic lipoprotein profile accompanying continuing cyclosporin A use; neither can it explain the unanimous elevation in serum cholesterol seen after transplantation in this young patient population, the majority of whom did not have ischemic heart diseasebefore transplantation. Similar transient increasesin serum HDL cholesterol are noted after transplantation as well. A significant increase in HDL cholesterol is seen by 3 to 6 weeks postoperatively, which normalizes by approximately 6 months. Again, a prednisone-mediatedeffect is the most likely explanation for this temporal sequence,24although a cyclosporin A-mediated effect cannot be excluded. It is important to realize that any benefits derived from these elevations in HDL cholesterol are far

outweighed by the much greater simultaneouselevation in LDL cholesterol. This is reflected by the progressive increase in the total-to-HDL cholesterol ratio after transplantation (Table I), imparting a steadily increasing risk of coronary artery disease.i6 The cellular and biochemical mechanismsby which cyclosporin A induces changes in lipoprotein metabolism are not yet known. It has been suggestedthat its immunosuppressive,as well as its toxic, properties are related to its inherent hydrophobicity that facilitates solubilization within the lipid bilayer and an alteration of membrane fluidity. 27 The physical properties of cell membranesare important in the modification of membrane function mediated by cell surface receptors28and changes in membrane fluidity specifically affect LDL clearance.29We have preliminary data to suggestthat cyclosporin A decreasesmembranefluidity and that this is accompaniedby depressedcellular clearance of LDL in a hepatocyte cell line. We therefore suggestthat decreasedmembrane-mediatedcellular clearance of LDL by a high affinity specific receptor pathway primarily in the hepatocyte,leadsto increasedplasma levelsof LDL, predisposingto atherogenesis. Although we believe that cyclosporin A contributes to the observedchangein lipoprotein profile after transplantation, we acknowledgethat the evidenceis indirect and that we are unable to exclude contributing factors such as diet, medication and other cardiac risk factors. In support of our hypothesis, however, we emphasize that these changes in cholesterol levels in patients receiving cyclosporin A, confirmed by both serum analysis and chart review, are different from those seen with other immunosuppressiveregimens.‘9-26In addition, lipid-altering antihypertensive medication was usually added well after changesin cholesterol were first noted. We also note that we have provided no direct proof that cyclosporin A promotes atherosclerosis.Although the usual risk factors have not correlated with atherosclerosis in cardiac transplant patients,1-3,‘8the observed changesin serum cholesterol after transplantation were not previously appreciated,and the long-term follow-up necessaryto determine the effects of these changeson atherosclerosisis not yet available. In the rabbit allograft, the addition of a high cholesterol diet transforms intimal lesions into cholesterol-filled atheromatous plaques,’ and the interaction between hypercholesterolemia and immune-mediated endothelial injury has also been suggestedto predisposeto acceleratedatherosclerosis in man.8 We suspectthat the significant increases in LDL cholesterol in patients treated with cyclosporin A will prove to contribute to the complex entity of accelerated atherosclerosisin these patients, and we suggest that a rigorous lipid-lowering program be instituted as part of conventional care in these patients.

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Trento A, BahnsonHT. Developmentof coronary artery diseasein cardiac transplant patientsreceiving immunosuppressivetherapy with cyclosporinand prednisow. Circulation

1987;76:827-834.

4. Jar&son SW, Burton NA, Bieber CP, Reitz BA, Oyer PE, Stinson EB, Shumway NE. Survival of cardiac allografts in rats treated with cyclosporin A. Surg Forum 1979;30:289-291.

5. Minick R, Murphy GE. Experimental induction of atheroarteriosclerosisby the synergy of allergic injury to arteries and lipid-rich diet II. Am J Pathol 1973;73;265-300. 6. Hardin WJ, Minick R, Murphy GE. Experimental induction of atheroarteriosclerosisby the synergy of allergic injury to arteries and lipid-rich diet. Am J Pathol 1973;73:301-348.

7. Alonso DR, Stark PK, Minick CR. Studieson the pathogenesisof atheroarteriosclerosisinducedin rabbit cardiac allografts by the synergyof graft rejectionand hypercholesterolemia.Am .I Pathol 1977;87:415-435. 8. HessML, Hastillo A, Mohanakumar T, Cowley MJ, Vetrovac G, Szentpetery S, Wolfgang TC, Lower RA. Accelerated atherosclerosisin cardiac transplantation: role of cytotoxic B-cell antibodies and hyperlipidemia. Circulation 1983; 68(suppl II).%94-U-101.

9. Zoja C, Furci L, Ghilardi F, Zilio P, Benigini A, Remuzz G. Cyclosporininduced endothelial cell injury. Lab Inuest 1986;55:455-462. 10. Klintmalm GBG, Iwatsuki S, Starzl TE. Cyclosporin A hepatoxicity in 66 renal allograft recipients. Transplantation 1981;32:488-489. 11. Starzl TE, Weil R, Iwatsuki S, Klintmalm G, Schroter GPJ, Keep LJ, Iwaki Y, Terasaki PI, Porter KM. The useof cyclosporinA and prednisonein cadaver kidney transplantation. Surg Gynecoi Obstet 1980;151:17-26. 12. Ryffel B, DonatschP, Madorin M, Matter BE, Ruttimann G, SchonH, Stall R, Wilson J. Toxicological evaluation of cyclosporin A. Arch Toxicol l983;53: 107-141.

13. Meiattini F, PrencipeL, Bardelli F, Giannini G, Tarli P. The 4-hydroxybenzoate/4-aminophenazonechromogenicsystemusedin the enzymatic determination of serum cholesterol. Clin Chem 1978;24:2161-2165. 14. BucoloG, David H. Quantitative determinationof serumtriglycerides by use of enzymes.Clin Chem 1973;19:476-482. 15. Naito HK, David JA. Laboratory considerations:determinationof cholesterol, tryglyceride, phospholipidand other lipids in blood tissues.In: Story JA, ed. Lipid Research Methodology. New York: Alan R Liss, 1984:9-l 1. 16. Dawber TR. The FraminghamStudy: The Epidemiologyof Atherosclerotic

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Disease.Cambridge, Massachusetts: Harvard University Press, 1980. 17. BeckerDM, Mark&is BS, SensionM, Vitalis S, BaughmanK, PearsonTA. Hyperlipidemia and hypertension following heart transplantation: potential causesof coronary atherosclerosis(abstr). JACC 1986;7;9A. 18. Gao SZ, SchroederJ, Alderman E, Hunt S, Silverman J, Weiderhold V, Stinson E. Clinical and laboratory correlates of accelerated coronary vascular diseasein the cardiac transplant patient (abstr). Circulation 1986;74(suppl I&II219.

19. Curtis JJ, Galla JH, Woodford SY, Lucas BA, Luke RG. Effects of alternate-day prednisoneon plasma lipids in renal transplant recipients. Kidney Int 1982;22:42-47.

20. Chan MK, Varghese Z, Moorehead JF. Lipid abnormalities in uremia, dialysis and transplantation. Kidney Int 1981;19:625-637. 21. Lacour B, Roullet JB, BeyneP, Kris H, Thevenin M, Trueke T. Comparison of severalatherogenicity indicesby the analysisof serumlipoprotein composition in patients with chronic renal failure with or without hemodialysis,and in renal transplant patients. J Clin Chem Clin Biochem 1985;23:805-810. 22. Ibels LS, Alfrey AC, Weil R. Hyperlipidemia in adult, pediatric and diabetic renal transplant recipients.Am .I Med 1978;64:634-642. 23. C&ran DC, Steiner G, Wilson MD, FentonMD. Hyperlipidemia after renal transplantation: natural history and pathophysiology.Ann Intern Med 1979;91; 554-559. 24. Zimmerman J, Fainaru M, EisenbergS. The effects of prednisonetherapy on plasmalipoproteins and apolipoproteins:a prospectivestudy. Metabolism 1984; 33:521-526.

25. Worth WS, Miller NL, Taylor PD. Liver transplantation effects on canine plasma lipids. Nature 1966;5044;78-79. 26. Harris KPG, RussellGI, Parvin SP, Veitch PS,Walls J. Metabolic effects of conversionfrom cyclosporin to azathioprine in renal transplant recipients. Eur Dialysis and Transplant Association-Eur

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27. Haynes M, Fuller L, Haynes DM, Miller J. Cyclosporin partitions into phospholipidvesiclesand disrupts membranearchitecture. Immunol Lett 1985, 11:343-349.

29. Jost P, Griffith OH, Capaldi RA, Vanderkooi G. Evidence for boundary lipids in membranes.Proc Nat1 Acad Sci USA 1973;70:480-483. 29. Loscalzo J, Freedman J, Rudd MA, Barsky-VassermanI, Vaughan DE. Unsaturated fatty acids enhancelow-density lipoprotein uptake and degradation by peripheral blood mononuclearcells. Atherosclerosis 1987;7:450-455.