Cholesteryl Ester Influx Research Articles

High Density Lipoprotein-like Nanoparticles Synergize with Akt and Btk Inhibitors to Induce Cell Death in Diffuse Large B Cell Lymphomas

Introduction: We previously reported that our bio-inspired, synthetic high-density lipoprotein-like nanoparticles (HDL NP) induced apoptosis in B cell lymphoma cells expressing scavenger receptor type B1 (SCARB1), the high-affinity receptor for cholesterol-rich HDLs. HDL NPs consist of a 5nm gold nanoparticle core surface functionalized with the HDL-defining apolipoprotein A1 and a phospholipid bilayer, and bind specifically to SCARB1, inducing the efflux of free cholesterol and inhibiting cholesteryl ester influx. SCARB1 is overexpressed in a subset of follicular and diffuse large B cell lymphomas (DLBCL), and resides in cholesterol-rich plasma membrane microdomains called lipid rafts, similar to the B cell receptor (BCR) and its associated signaling kinases. Upon binding to natural HDL, SCARB1 activates a number of pro-survival signaling kinases, including Akt and PI3K. Both Akt and PI3K are also involved in B cell receptor-mediated signaling in germinal center-derived (GC) DLBCL, through tonic BCR signaling, and activated B cell (ABC) DLBCL, through chronic active BCR signaling. Additionally, PI3K was recently shown to play a role in recruitment and activation of Btk, a crucial survival kinase downstream of the BCR. We hypothesized that small molecule inhibitors against pro-survival kinases, specifically Akt and Btk, will synergize with HDL NPs against B cell lymphomas.Methods: Burkitt's lymphoma (Ramos), GC DLBCL (SUDHL4) and ABC DLBCL (TMD8 and HBL-1) cell lines were treated with the Akt inhibitor GDC-0068 or the Btk inhibitor Ibrutinib, in the absence or presence of HDL NPs, and synergy was calculated using the Calcusyn software. Phos-flow was used to assay for changes in the phosphorylation status of Akt and Btk.Results: The Burkitt’s lymphoma and GC DLBCL cell lines were more sensitive to HDL NP induced cell death compared to the ABC DLBCL cell lines (Ramos HDL NP IC50 = 1.5nM; SUDHL4 HDL NP IC50 = 2.1nM; TMD8 HDL NP IC50 = 31.4nM; HBL-1 HDL NP IC50 = 89nM). HDL NPs synergized with GDC-0068 in the Ramos, SUDHL4 and TMD8 cell lines (all combination indexes < 1). Correspondingly, HDL NPs dose-dependently decreased phosphorylation of Akt in Ramos and TMD8 cells. Ibrutinib synergized with the HDL NPs in all cell lines tested (all combination indexes < 1). In TMD8 cells, HDL NPs decreased p-Btk levels comparable to treatment with 10nM Ibrutinib. Addition of the PI3K inhibitor Pilaralisib (XL147) demonstrated mild synergy in the Ramos cell line, but not the SUDHL4, TMD8 or HBL-1 cell lines (all combination index values >1). Treatment of Ramos and SUDHL4 cells with an inhibitor of PTEN, a phosphatase responsible for acting in opposition to PI3K leading to inactivation of Akt, rescued the cells from HDL NP-induced cell death. TMD8 cells treated with the PTEN inhibitor demonstrated a smaller increase in survival when HDL NPs were applied, suggesting that PI3K may not play a major role in HDL NP-induced cell death in activated B cell DLBCLs. PTEN activity is influenced by the level of cholesterol and cholesteryl esters present in the cell, with increasing levels correlating with decreased PTEN activity. Cholesterol levels were higher in the ABC DLBCL cell lines compared to the other B cell lymphoma cell lines. HDL NPs significantly reduced the cholesterol content of Ramos cells, but not the TMD8 or HBL-1 cells, suggesting that the ability of the HDL NPs to alter cellular cholesterol homeostasis correlates with their ability to induce lymphoma cell death.Conclusion: HDL NPs demonstrated synergy with inhibitors to the pro-survival kinases Akt and Btk, suggesting that HDL NPs act to disrupt second messenger signaling pathways in lymphoma cells by directly altering signaling through SCARB1, modulating cellular cholesterol homeostasis, and/or through disruption of membrane raft organization. HDL NPs represent an innovative, targeted therapeutic, with great potential, to add to existing combination chemotherapy regimens. DisclosuresThaxton:Aurasense: Equity Ownership, Patents & Royalties: The patent for the HDL NPs has been licensed to Aurasense, a biotech company co-founded by C. Shad Thaxton..

Evaluation of cholesteryl ester transfer in the seminiferous tubule cells of immature rats in vivo and in vitro.

Sertoli cells and germ cells are separated from the interstitial blood capillaries by an extracellular matrix and the peritubular cells, which constitute a barrier to the movement of plasma lipoproteins. The present study was undertaken to evaluate in vivo and in vitro the high density lipoprotein (HDL) cholesteryl ester transfer from plasma to seminiferous tubule cells in the testis of 30-day-old rats. Firstly, the transfer of HDL cholesteryl oleate from plasma to testicular compartments was evaluated and, secondly, the role of apolipoproteins A-I and E in the uptake of cholesteryl ester by Sertoli cells was investigated. At 2 h after the administration of HDL reconstituted with [3H]cholesteryl ester, dimyristoyl phosphatidylcholine and apolipoproteins, the tissue space in the interstitial cells (740 +/- 60 microliters g-1 cell protein) was fourfold higher than that in the seminiferous tubule cells (170 +/- 10 microliters g-1). Sertoli cells were isolated and incubated with [3H]cholesteryl ester HDL reconstituted with apolipoprotein A-I or E to evaluate the mechanisms of cholesteryl ester influx. At the same apolipoprotein concentration (50 micrograms apolipoprotein ml-1 medium), the uptake of [3H]cholesteryl oleate from phospholipid-apolipoprotein E vesicles was twofold higher than that with phospholipid-apolipoprotein A-I vesicles. The presence of heparin reduced the uptake of cholesteryl ester from apolipoprotein E vesicles but not with apolipoprotein A-I vesicles, indicating that uptake of apolipoprotein A-I vesicles via a secretion of apolipoprotein E by the cells themselves was not involved. These results demonstrate that plasma lipoprotein cholesterol is able to cross the testis lamina propria and that Sertoli cells take up cholesteryl ester for seminiferous tubule cell metabolism mainly via an apolipoprotein E pathway.

Evaluation of cholesteryl ester transfer in the seminiferous tubule cells of immature rats in vivo and in vitro

Sertoli cells and germ cells are separated from the interstitial blood capillaries by an extracellular matrix and the peritubular cells, which constitute a barrier to the movement of plasma lipoproteins. The present study was undertaken to evaluate in vivo and in vitro the high density lipoprotein (HDL) cholesteryl ester transfer from plasma to seminiferous tubule cells in the testis of 30-day-old rats. Firstly, the transfer of HDL cholesteryl oleate from plasma to testicular compartments was evaluated and, secondly, the role of apolipoproteins A-I and E in the uptake of cholesteryl ester by Sertoli cells was investigated. At 2 h after the administration of HDL reconstituted with [3H]cholesteryl ester, dimyristoyl phosphatidylcholine and apolipoproteins, the tissue space in the interstitial cells (740 +/- 60 microliters g-1 cell protein) was fourfold higher than that in the seminiferous tubule cells (170 +/- 10 microliters g-1). Sertoli cells were isolated and incubated with [3H]cholesteryl ester HDL reconstituted with apolipoprotein A-I or E to evaluate the mechanisms of cholesteryl ester influx. At the same apolipoprotein concentration (50 micrograms apolipoprotein ml-1 medium), the uptake of [3H]cholesteryl oleate from phospholipid-apolipoprotein E vesicles was twofold higher than that with phospholipid-apolipoprotein A-I vesicles. The presence of heparin reduced the uptake of cholesteryl ester from apolipoprotein E vesicles but not with apolipoprotein A-I vesicles, indicating that uptake of apolipoprotein A-I vesicles via a secretion of apolipoprotein E by the cells themselves was not involved. These results demonstrate that plasma lipoprotein cholesterol is able to cross the testis lamina propria and that Sertoli cells take up cholesteryl ester for seminiferous tubule cell metabolism mainly via an apolipoprotein E pathway.

Accelerated cholesterol accumulation in homologous arterial transplants in cholesterol-fed rabbits. A surgical model to study transplantation atherosclerosis.

Accelerated coronary artery disease has become a major complication to heart transplantation in humans. Therefore, we have developed a surgical model in the rabbit, with transplantation of the thoracic aorta as a bypass graft onto the abdominal aorta of another rabbit. The model permits the study of cholesterol metabolism in transplanted arteries. The graft did not accumulate cholesterol for as long as 298 days, provided that the rabbits were normocholesterolemic, i.e., with plasma cholesterol levels of 0.3-0.7 mmol/l. However, after a few weeks of cholesterol feeding resulting in plasma cholesterol levels of 2-5 mmol/l, the homologous graft accumulated cholesterol compared with intact aortic tissue in the rabbits and also compared with autologous aortic grafts. The intimal clearance of plasma cholesteryl ester, mainly high density lipoprotein cholesteryl ester, in the luminal layer of the aortic graft was 60-150 nl x cm-2 x hr-1 1-2 hours after transplantation. The intimal clearance in the corresponding intact thoracic aorta of the recipient animal was 5-20 nl x cm-2 x hr-1. The values were 1,500-3,000 nl x cm-2 x hr-1 51-298 days after transplantation, while the intimal clearance of the rabbit's own aorta remained unchanged. A pronounced increase in plasma lipoprotein permeability is thus an early event in transplanted arteries. It results in a higher cholesteryl ester influx that leads to cholesterol accumulation in the artery, but only if the rabbits are fed a cholesterol-enriched diet. This rabbit model may be useful in the search for interventional measures to prevent or diminish the accelerated coronary artery disease in transplanted hearts in humans.

The arterial barrier to lipoprotein influx in the hypercholesterolemic rabbit: 2. Long-term studies in deendothelialized and reendothelialized aortas

These studies consider whether a mild aortic injury that does not increase cholesteryl ester influx during the first few days promotes atheromatosis in the hypercholesterolemic rabbit. The cholesteryl ester influx in uninjured, deendothelialized, and reendothelialized aorta was also measured in order to account for the different cholesteryl ester contents in these areas. By 32–33 days after localized aortic injury which was made after 5–7 days of cholesterol feeding, uninjured (control) areas of the thoracic aortas had accumulated 48 μg/cm 2 each of esterified and nonesterified cholesterol due to continued cholesterol feeding. However, the previously injured deendothelialized and'reendothelialized areas of the aortas had accumulated 6 and 10 times as much esterified cholesterol, and 2 and 5 times as much nonesterified cholesterol, respectively, as the adjacent uninjured areas. Esterified cholesterol influx was low during the second day after injury but increased with time so that 30–31 days later the esterified cholesterol influx in deendothelialized and reendothelialized aorta was respectively 44 and 7 times as great as the 0.1 μg/h/cm 2 in the adjacent uninjured aorta. However, expressed per mg aortic cholesteryl ester, cholesteryl ester influx in reendothelialized aorta was no greater than in noninjured aorta. These studies suggest that an injury that does not initially increase cholesteryl ester influx can promote atheromatosis and that the higher rate of cholesteryl ester influx that develops with time in reendothelialized areas is closely related to its increased cholesteryl ester content.

The arterial barrier to lipoprotein influx in the hypercholesterolemic rabbit: 1. Studies during the first two days after mild aortic injury

These studies examine the hypothesis that removal of aortic endothelium eliminates a barrier to lipoprotein cholesterol influx. The aortas of rabbits fed a cholesterol-rich diet from 7 to 16 days before balloon injury were studied 1 or 2 days after deendothelialization of specific areas of the aorta. By this design the aortic sterol content was near normal on the day of injury, but areas of injured and noninjured aorta were exposed to identical levels of elevated plasma cholesterol. Measuring the arterial [ 3H]- and [ 14C] cholesterol fractions accumulated during 2 different intervals after dosage in the same animals permitted calculation of total influx and fractional loss of aortic cholesterol. During the first 2 days after deendothelialization, total (unidirectional) cholesteryl ester influx in deendothelialized aorta was similar to that in adjacent uninjured aorta, but total influx of nonesterified cholesterol was increased. The calculated increase in influx of nonesterified cholesterol was not a result of an increase in hydrolysis of entered cholesteryl ester but probably represents increased exchange of labeled cholesterol between artery and plasma. These results suggest that subendothelial layers of the aorta of short-term cholesterol-fed rabbits function as significant barriers to lipoprotein influx and that processes other than increased permeation by lipoproteins initiate injury-induced lesions.

In vivo transfer of cholesteryl ester from high and low density plasma lipoproteins into human aortic tissue.

For the study of cholesteryl ester transfer from different plasma lipoproteins into human aortic tissue, patients scheduled for reconstructive aortic surgery were intravenously injected with autologous in vitro labeled lipoproteins 20 to 24 hours before aortic intima-media samples were obtained during the operation. The injectate contained high density lipoproteins (d greater than 1.063) labeled with 3H-cholesteryl ester and lipoproteins of lower density (d less than 1.063) labeled with 14C-cholesteryl ester or lipoproteins with the opposite labeling. In 16 aortic tissue samples (some with visible atherosclerosis) from 11 normocholesterolemic patients, the aortic influx of total cholesteryl ester was 1 to 50 nmol x cm-2 x day-1. Some 39% +/- 3% (mean +/- SEM) of the influx was derived from high density lipoproteins, which in plasma accounted for only 22% +/- 2% (mean +/- SEM) of the esterified cholesterol. The findings suggest that: 1) esterified cholesterol from the two lipoprotein fractions in plasma enter the aortic intima by the same mechanism, and 2) influx of cholesteryl ester from the smaller, high density lipoproteins is greater than influx from the larger, lower density lipoproteins considering their concentrations in plasma. In some patients, the cholesterol content in the intima-media tissue with no visible atherosclerosis corresponded to only a few months of continuous cholesteryl ester influx. This time is short considering the age of the patients and, therefore, indicates that removal of esterified cholesterol from the intima-media is of major importance in preventing cholesterol deposition in the arterial wall.

Enhanced accumulation and turnover of esterified cholesterol in injured rabbit aorta.

Arterial injury exacerbates experimental atherogenesis. This report evaluates cholesteryl ester influx and loss during the first 5 days after arterial injury. Selected areas of aortas from rabbits made hypercholesterolemic by 12 to 16 days of cholesterol feeding were injured with a balloon catheter. This allowed measurements and a relatively precise comparison of cholesterol and cholesteryl ester influx into the uninjured and injured arterial wall within the same animal. The animals received oral doses of 3H- or 14C-cholesterol either just before injury or 1 day later. Most animals were given the other isotope of cholesterol 1 day before sacrifice, which took place 2 to 5 days after injury. Measurement of accumulated labelled esterified cholesterol within the same animal during two different time periods allowed the estimation of total influx and fractional loss of entered esterified cholesterol within the artery. Between 2 and 5 days after injury, total influx into areas of injured artery averaged 30 to 60 times the total influx into the adjacent uninjured areas. By the fifth day after injury, the esterified cholesterol content was about 10 times greater in injured areas than in adjacent, uninjured areas. The nonesterified cholesterol content of injured areas was about 70% greater than that of adjacent noninjured areas. Total influx and fractional loss of arterial cholesteryl ester calculated from radioactivity data could account for 117 +/- 6 (mean +/- SEM) percent of the cholesteryl ester mass that accumulated during the interval from injury to sacrifice. The total influx rates and daily increments in cholesteryl ester content of injured areas were positively related.

In vivo transfer of cholesterol from plasma into human aortic tissue

It has recently become possible to measure the in vivo flux of cholesteryl ester from plasma into human aortic tissue by use of labeled cholesterol in patients undergoing reconstructive aortic surgery. For the ascending thoracic aorta without visible atherosclerotic lesions the influx was 4.5 +/- 1.4 nmol X cm-2 X day-1 (means +/- SEM, n = 9). For the abdominal aorta with severe atherosclerosis the influx of cholesteryl ester was 45 +/- 5 nmol X cm-2 X day-1 (n = 12). In both types of tissues the influx of cholesteryl ester from HDL was 2-3 times higher than the influx of cholesteryl ester from LDL and VLDL compared with the concentration of these fractions in plasma. This is in accordance with an aortic influx-mechanism which depends on the sizes and the concentration of the lipoproteins in plasma. The transfer of plasma lipoproteins into human aortic tissue shows a number of similarities with the transfer of plasma lipoproteins into the aortic wall of cholesterol-fed rabbits and also with the transfer of other plasma macromolecules across various capillaries. The cholesterol content in intima-media tissue without lesions corresponded in some of the patients to less than one year of continuous influx of cholesteryl ester from plasma. This time is short compared with the age of the patients. It suggests that removal of cholesterol from the aortic wall represents a major importance in prevention of cholesterol accumulation in that tissue.

In vivo flux of plasma cholesterol into human abdominal aorta with advanced atherosclerosis.

To measure the flux of free and esterified cholesterol from plasma into abdominal aortic tissue that had severe atherosclerotic lesions, we intravenously injected two autologous plasma samples containing radioactive cholesterol into patients scheduled for reconstructive arterial surgery. After the injections, blood samples were collected for calculation of the exposure of arterial tissue to labeled free and esterified plasma cholesterol. When tissue specimens were removed a few days after the injection, the aortic influx was determined by the simultaneous use of two differently labeled species of cholesterol. The flux of free and esterified cholesterol into 51 tissue specimens from the abdominal aorta of 12 normocholesterolemic patients was 41 +/- 3 and 45 +/- 3 nmol X cm-2 X day-1 (mean +/- SE), respectively, with 7% to 30% hydrolysis and 8% to 24% esterification of the labeled sterols in the atherosclerotic tissue. The influx was up to 100 times greater than the influx into nonatherosclerotic ascending aorta previously measured in other patients. The cholesterol content of the atherosclerotic tissue corresponded to 16 +/- 3 months (mean +/- SE) of continuous cholesteryl ester influx. Unless counteracted by cholesteryl ester efflux from the plaque, this influx provides enough cholesteryl ester from plasma to cause rapid lesion progression.

Effect of 17 beta estradiol on aortic cholesterol content and metabolism in cholesterol-fed rabbits.

Administration of estrogen to cholesterol-fed rabbits dramatically retarded arterial lesion development despite its lack of effect on plasma cholesterol concentration and on lipoprotein patterns. Cholesteryl ester influx into the aortic wall was also much lower in the estrogen-treated animals and paralleled the aortic cholesterol content in treated and untreated animals; the fraction of aortic cholesteryl ester lost by efflux was the same in treated and untreated animals. The fraction of newly entered cholesteryl ester hydrolyzed by aorta was significantly reduced in the estrogen-treated animals. Low cholesteryl ester influx and relatively less hydrolysis of cholesteryl ester by the aorta may be indicative of reduced internalization of plasma cholesteryl ester by aortic cells, which may in turn account for the reduced atherogenesis in the estrogen-treated rabbits.

Influx, efflux, and hydrolysis of cholesteryl ester in atheromatous lesions of cholesterol-fed rabbits.

Total plasma lipoproteins were labeled with radioactive cholesteryl ester or cholesteryl ether by transfer of these lipids from phosphatidylcholine vesicles in the presence of plasma lipid transfer activity. Intravenous injection of these preparations into hypercholesterolemic rabbits showed disappearance curves identical to those of in vivo labeled lipoproteins. Disappearance of cholesteryl ester and ether were similar during the first 24 hours, but they diverged at later time intervals, indicating recirculation of labeled cholesteryl ester. Lipoproteins labeled with cholesteryl ether were injected at 25 days, 7 days and 1 day before sacrifice of the rabbits. The maximal loss of labeled ether from the aortas during a 24-hour period ranged from 1.6% to 8.9% of the labeled ether taken up from plasma. Hydrolysis of cholesteryl ester by the artery during 24 hours averaged 35% of the calculated cholesteryl ester influx. After hydrolysis, cholesteryl ester fatty acid appeared to be esterified more rapidly than the cholesterol moiety of the cholesteryl ester.

No effect of nifedipine on atherogenesis in cholesterol-fed rabbits.

It was recently reported that the calcium antagonist nifedipine suppresses aortic cholesterol accumulation in cholesterol-fed rabbits without reducing hypercholesterolemia. We extended this study on plasma lipoprotein levels and aortic influx of cholesteryl ester. We gave 40 mg per day of nifedipine orally to 17 rabbits fed a 2% cholesterol diet for 8 weeks. For the same period of time 15 control rabbits received placebo capsules and the same diet. During the study, total cholesterol, HDL, LDL, and VLDL cholesterol concentrations in plasma were not significantly different in the experimental and control animals. At the end of the study we found no difference in the two groups in accumulation of cholesterol in the intima media of the proximal thoracic aorta, the distal thoracic aorta, and the corresponding media layers. Furthermore, aortic influx of free cholesterol, cholesteryl ester, and albumin from plasma measured by radioactive tracers was not significantly affected by nifedipine.

Use of 3H-cholesteryl linoleyl ether for the quantitation of plasma cholesteryl ester influx into the aortic wall in hypercholesterolemic rabbits.

In this study use was made of 3H-cholesteryl linoleyl ether CLE), a nondegradable analogue of cholesteryl ester (CE) to measure plasma lipoprotein CE influx into rabbit aorta. Autologous serum labeled with 3H-CLE was injected into seven hypercholesterolemic rabbits, and more than 90% of the label was recovered in the plasma compartment 10 minutes after injection. Between 4 hours and 3 days the label was cleared from the circulation with a t1/2 of about 24 hours. Between 4 and 24 hours the lipoproteins isolated at d less than 1.006, d less than 1.019, and d less than 1.063 approached similar specific activity, assuming that 3H-CLE had mixed with the lipoprotein CE pool. The rabbits were killed 7 to 14 days after injection when plasma radioactivity decreased to less than 0.03% of injected dose/ml. Total recovery of the CLE ranged from 70% to 95% and 48% to 72% were found in the liver. The minimum influx of plasma CE into the aortic intima was determined by dividing the label found in the artery by the mean specific activity of the labeled compound in the plasma. The minimum influx into regions with atheromatous involvement ranged from 0.8 to 3.4 micrograms CE/cm2/hr. The rate of influx was highly correlated with the amount of CE mass in the intima and media indicating that the bulk of aortic CE is derived from plasma lipoprotein CE. The method described might be useful in distinguishing between possible effects of "antiatherogenic" drugs on plasma CE influx into the aortic wall from an effect on intracellular CE hydrolysis and subsequent efflux of free cholesterol from the artery.

Arterial influx of esterified cholesterol from two plasma lipoprotein fractions and its hydrolysis in vtvo in hype rcholesterolemic rabbits

Arterial influx of esterified cholesterol from 2 plasma lipoprotein fractions, d < 1.019 and d > 1.019, and influx of plasma free cholesterol were determined in each of 15 hypercholesterolemic rabbits with approximately the same plasma cholesterol concentrations but with different extents of arterial lesions. The procedure consisted of injecting intravenously into recipient rabbits [ 14C]- or [ 3H]cholesterol-labeled lipoproteins prepared from donor rabbits. The esterified cholesterol of one lipoprotein fraction was labeled primarily with one isotope and that of the other lipoprotein fraction was labeled with the other isotope. Thoracic aortas were removed 4–6 h after lipoprotein injections. The arterial influx of esterified cholesterol was up to 50 times higher in rabbits with maximal lesions than in those with minimal cholesterol deposits. The arterial influx of cholesteryl ester derived from d < 1.019 lipoproteins was about equal to that derived from the d > 1.019 fraction. The amount of cholesteryl ester in plasma d < 1.019 was approximately 3 times that in d > 1.019. Thus, per unit of cholesteryl ester concentration the d < 1.019 lipoproteins delivered about 1 3 as much cholesteryl ester to the artery as the lipoproteins in the higher density fractions. Some 5–40% of plasma esterified cholesterol which had entered the artery was hydrolyzed in the artery during the experimental period. The influx of free cholesterol that could not be accounted for by the influx of intact plasma lipoproteins was 5–80% of the free cholesterol influx. This excess probably represents free cholesterol influx by an exchange between the plasma lipoproteins and the intimal surface of the artery.