Sudichhya Shrestha

Inhibition of Arthritis in the Lewis Rat by Apolipoprotein A-I and Reconstituted High-Density Lipoproteins

Objective— This study questions whether high-density lipoproteins (HDLs) and apolipoprotein A-I inhibit joint inflammation in streptococcal cell wall peptidoglycan-polysaccharide (PG-PS)–induced arthritis in female Lewis rats. Approach and Results— Administration of PG-PS to female Lewis rats caused acute joint inflammation after 4 days, followed by remission by day 8. The animals subsequently developed chronic joint inflammation that persisted until euthanasia at day 21. Treatment with apolipoprotein A-I 24 hours before and 24 hours after PG-PS administration reduced the acute and chronic joint inflammation. Treatment with apolipoprotein A-I at days 7, 9, and 11 after PG-PS administration reduced the chronic joint inflammation. Treatment with apolipoprotein A-I or reconstituted HDLs consisting of apolipoprotein A-I complexed with phosphatidylcholine 24 hours before and at days 1, 7, 9, and 11 after PG-PS administration reduced acute and chronic joint inflammation. Treatment with apolipoprotein A-I also reduced the inflammatory white blood cell count, synovial fluid proinflammatory cytokine levels, synovial tissue macrophage accumulation, as well as toll-like receptor 2, and inflammatory cytokine expression. At the molecular level, preincubation of human monocyte–derived macrophages with apolipoprotein A-I or reconstituted HDLs before PG-PS stimulation inhibited the PG-PS–induced increase in toll-like receptor 2 and myeloid differentiation primary response gene (88) mRNA levels, nuclear factor-&kgr;B activation, and proinflammatory cytokine production. The effects of apolipoprotein A-I and reconstituted HDLs were abolished by transfecting the human monocyte–derived macrophages with ATP-binding cassette transporter A1 or G1 siRNA. Conclusions— Apolipoprotein A-I and reconstituted HDLs attenuate PG-PS–induced arthritis in the rat. Studies in human monocyte–derived macrophages indicate that this benefit may be because of the inhibition of toll-like receptor 2 expression and decreased nuclear factor-&kgr;B activation in macrophages.

Cholesteryl Ester Transfer Protein Inhibition Enhances Endothelial Repair and Improves Endothelial Function in the Rabbit

Objective— High-density lipoproteins (HDLs) can potentially protect against atherosclerosis by multiple mechanisms, including enhancement of endothelial repair and improvement of endothelial function. This study asks if increasing HDL levels by inhibiting cholesteryl ester transfer protein activity with the anacetrapib analog, des-fluoro-anacetrapib, enhances endothelial repair and improves endothelial function in New Zealand White rabbits with balloon injury of the abdominal aorta. Approach and Results— New Zealand White rabbits received chow or chow supplemented with 0.07% or 0.14% (wt/wt) des-fluoro-anacetrapib for 8 weeks. Endothelial denudation of the abdominal aorta was carried out after 2 weeks. The animals were euthanized 6 weeks postinjury. Treatment with 0.07% and 0.14% des-fluoro-anacetrapib reduced cholesteryl ester transfer protein activity by 81±4.9% and 92±12%, increased plasma apolipoprotein A–I levels by 1.4±0.1-fold and 1.5±0.1-fold, increased plasma HDL-cholesterol levels by 1.8±0.2-fold and 1.9±0.1-fold, reduced intimal hyperplasia by 37±11% and 51±10%, and inhibited vascular cell proliferation by 25±6.1% and 35±6.7%, respectively. Re-endothelialization of the injured aorta increased from 43±6.7% (control) to 69±6.6% and 76±7.7% in the 0.07% and 0.14% des-fluoro-anacetrapib-treated animals, respectively. Aortic ring relaxation and guanosine 3′,5′-cyclic monophosphate production in response to acetylcholine were also improved. Incubation of HDLs from the des-fluoro-anacetrapib-treated animals with human coronary artery endothelial cells increased cell proliferation and migration relative to control. These effects were abolished by knockdown of scavenger receptor-B1 and PDZ domain-containing protein 1 and by pharmacological inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt. Conclusions— Increasing HDL levels by inhibiting cholesteryl ester transfer protein reduces intimal thickening and regenerates functional endothelium in damaged New Zealand White rabbit aortas in an scavenger receptor-B1-dependent and phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt-dependent manner.

High-Density Lipoprotein-Associated miR-223 Is Altered after Diet-Induced Weight Loss in Overweight and Obese Males.

Background and Aims microRNAs (miRNAs) are small, endogenous non-coding RNAs that regulate metabolic processes, including obesity. The levels of circulating miRNAs are affected by metabolic changes in obesity, as well as in diet-induced weight loss. Circulating miRNAs are transported by high-density lipoproteins (HDL) but the regulation of HDL-associated miRNAs after diet-induced weight loss has not been studied. We aim to determine if HDL-associated miR-16, miR-17, miR-126, miR-222 and miR-223 levels are altered by diet-induced weight loss in overweight and obese males. Methods HDL were isolated from 47 subjects following 12 weeks weight loss comparing a high protein diet (HP, 30% of energy) with a normal protein diet (NP, 20% of energy). HDL-associated miRNAs (miR-16, miR-17, miR-126, miR-222 and miR-223) at baseline and after 12 weeks of weight loss were quantified by TaqMan miRNA assays. HDL particle sizes were determined by non-denaturing polyacrylamide gradient gel electrophoresis. Serum concentrations of human HDL constituents were measured immunoturbidometrically or enzymatically. Results miR-16, miR-17, miR-126, miR-222 and miR-223 were present on HDL from overweight and obese subjects at baseline and after 12 weeks of the HP and NP weight loss diets. The HP diet induced a significant decrease in HDL-associated miR-223 levels (p = 0.015), which positively correlated with changes in body weight (r = 0.488, p = 0.032). Changes in miR-223 levels were not associated to changes in HDL composition or size. Conclusion HDL-associated miR-223 levels are significantly decreased after HP diet-induced weight loss in overweight and obese males. This is the first study reporting changes in HDL-associated miRNA levels with diet-induced weight loss.

Impact of Perturbed Pancreatic β-Cell Cholesterol Homeostasis on Adipose Tissue and Skeletal Muscle Metabolism

Elevated pancreatic β-cell cholesterol levels impair insulin secretion and reduce plasma insulin levels. This study establishes that low plasma insulin levels have a detrimental effect on two major insulin target tissues: adipose tissue and skeletal muscle. Mice with increased β-cell cholesterol levels were generated by conditional deletion of the ATP-binding cassette transporters, ABCA1 and ABCG1, in β-cells (β-DKO mice). Insulin secretion was impaired in these mice under basal and high-glucose conditions, and glucose disposal was shifted from skeletal muscle to adipose tissue. The β-DKO mice also had increased body fat and adipose tissue macrophage content, elevated plasma interleukin-6 and MCP-1 levels, and decreased skeletal muscle mass. They were not, however, insulin resistant. The adipose tissue expansion and reduced skeletal muscle mass, but not the systemic inflammation or increased adipose tissue macrophage content, were reversed when plasma insulin levels were normalized by insulin supplementation. These studies identify a mechanism by which perturbation of β-cell cholesterol homeostasis and impaired insulin secretion increase adiposity, reduce skeletal muscle mass, and cause systemic inflammation. They further identify β-cell dysfunction as a potential therapeutic target in people at increased risk of developing type 2 diabetes.

Increasing HDL levels by inhibiting cholesteryl ester transfer protein activity in rabbits with hindlimb ischemia is associated with increased angiogenesis

BACKGROUND High density lipoprotein (HDL) infusions increase new blood vessel formation (angiogenesis) in rodents with ischemic injury. This study asks if increasing HDL levels by inhibiting cholesteryl ester transfer protein (CETP) activity increases angiogenesis in New Zealand White (NZW) rabbits with hindlimb ischemia. METHODS AND RESULTS NZW rabbits were maintained for 6weeks on chow or chow supplemented with 0.07% or 0.14% (wt/wt) of the CETP inhibitor, des-fluoro-anacetrapib. The left femoral artery was ligated after 2weeks of des-fluoro-anacetrapib treatment. The animals were sacrificed 4weeks after femoral artery ligation. Treatment with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib reduced CETP activity by 63±12% and 81±8.6%, increased plasma apoA-I levels by 1.3±0.1- and 1.4±0.1-fold, and increased plasma HDL-cholesterol levels by 1.4±0.1- and 1.7±0.2-fold, respectively. Treatment with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib increased the number of collateral arteries by 60±16% and 84±27%, and arteriole wall area in the ischemic hindlimbs by 84±16% and 94±13%, respectively. Capillary density in the ischemic hindlimb adductor muscle increased from 1.1±0.2 (control) to 2.1±0.3 and 2.2±0.4 in the 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib-treated animals, respectively. Incubation of HDLs from des-fluoro-anacetrapib-treated animals with human coronary artery endothelial cells at apoA-I concentrations comparable with their plasma levels increased tubule network formation. These effects were abolished by knockdown of scavenger receptor-B1 (SR-B1) and PDZK1, and pharmacological inhibition of PI3K/Akt. CONCLUSION Increasing HDL levels by inhibiting CETP activity is associated with increased collateral blood vessel formation in NZW rabbits with hindlimb ischemia in an SR-B1- and PI3K/Akt-dependent manner.

Cholesteryl Ester Transfer Protein and its Inhibitors

Most of the cholesterol in plasma is in an esterified form that is generated in potentially cardioprotective HDLs. Cholesteryl ester transfer protein (CETP) mediates bidirectional transfers of cholesteryl esters (CEs) and triglycerides (TGs) between plasma lipoproteins. Because CE originates in HDLs and TG enters the plasma as a component of VLDLs, activity of CETP results in a net mass transfer of CE from HDLs to VLDLs and LDLs, and of TG from VLDLs to LDLs and HDLs. As inhibition of CETP activity increases the concentration of HDL-cholesterol and decreases the concentration of VLDL- and LDL-cholesterol, it has the potential to reduce atherosclerotic CVD. This has led to the development of anti-CETP neutralizing monoclonal antibodies, vaccines, and antisense oligonucleotides. Small molecule inhibitors of CETP have also been developed and four of them have been studied in large scale cardiovascular clinical outcome trials. This review describes the structure of CETP and its mechanism of action. Details of its regulation and nonlipid transporting functions are discussed, and the results of the large scale clinical outcome trials of small molecule CETP inhibitors are summarized.

Reduction of In-Stent Restenosis by Cholesteryl Ester Transfer Protein Inhibition

Objective— Angioplasty and stent implantation, the most common treatment for atherosclerotic lesions, have a significant failure rate because of restenosis. This study asks whether increasing plasma high-density lipoprotein (HDL) levels by inhibiting cholesteryl ester transfer protein activity with the anacetrapib analog, des-fluoro-anacetrapib, prevents stent-induced neointimal hyperplasia. Approach and Results— New Zealand White rabbits received normal chow or chow supplemented with 0.14% (wt/wt) des-fluoro-anacetrapib for 6 weeks. Iliac artery endothelial denudation and bare metal steel stent deployment were performed after 2 weeks of des-fluoro-anacetrapib treatment. The animals were euthanized 4 weeks poststent deployment. Relative to control, dietary supplementation with des-fluoro-anacetrapib reduced plasma cholesteryl ester transfer protein activity and increased plasma apolipoprotein A-I and HDL cholesterol levels by 53±6.3% and 120±19%, respectively. Non-HDL cholesterol levels were unaffected. Des-fluoro-anacetrapib treatment reduced the intimal area of the stented arteries by 43±5.6% (P<0.001), the media area was unchanged, and the arterial lumen area increased by 12±2.4% (P<0.05). Des-fluoro-anacetrapib treatment inhibited vascular smooth muscle cell proliferation by 41±4.5% (P<0.001). Incubation of isolated HDLs from des-fluoro-anacetrapib–treated animals with human aortic smooth muscle cells at apolipoprotein A-I concentrations comparable to their plasma levels inhibited cell proliferation and migration. These effects were dependent on scavenger receptor-B1, the adaptor protein PDZ domain-containing protein 1, and phosphatidylinositol-3-kinase/Akt activation. HDLs from des-fluoro-anacetrapib–treated animals also inhibited proinflammatory cytokine-induced human aortic smooth muscle cell proliferation and stent-induced vascular inflammation. Conclusions— Inhibiting cholesteryl ester transfer protein activity in New Zealand White rabbits with iliac artery balloon injury and stent deployment increases HDL levels, inhibits vascular smooth muscle cell proliferation, and reduces neointimal hyperplasia in an scavenger receptor-B1, PDZ domain-containing protein 1– and phosphatidylinositol-3-kinase/Akt-dependent manner.

Transcoronary gradients of HDL-associated MicroRNAs in unstable coronary artery disease

AIMS MicroRNAs (miRNAs) are transported on high-density lipoproteins (HDLs) and HDL-associated miRNAs are involved in intercellular communication. We explored HDL-associated miRNAs concentration gradients across the coronary circulation in stable and unstable coronary artery disease patients and whether changes in the transcoronary gradient were associated with changes in HDL composition and size. METHODS Acute coronary syndrome (ACS, n=17) patients, those with stable coronary artery disease (stable CAD, n=19) and control subjects without CAD (n=6) were studied. HDLs were isolated from plasma obtained from the coronary sinus (CS), aortic root (arterial blood) and right atrium (venous blood). HDL-associated miRNAs (miR-16, miR-20a, miR-92a, miR-126, miR-222 and miR-223) were quantified by TaqMan miRNA assays. HDL particle sizes were determined by non-denaturing polyacrylamide gradient gel electrophoresis. HDL composition was measured immunoturbidometrically or enzymatically. RESULTS A concentration gradient across the coronary circulation was observed for all the HDL-associated miRNAs. In ACS patients, there was a significant inverse transcoronary gradient for HDL-associated miR-16, miR-92a and miR-223 (p<0.05) compared to patients with stable CAD. Changes in HDL-miRNA transcoronary gradients were not associated with changes in HDL composition or size. CONCLUSION HDLs are depleted of miR-16, miR-92a and miR-223 during the transcoronary passage in patients with ACS compared to patients with stable CAD.

Erratum. Impact of Perturbed Pancreatic β-Cell Cholesterol Homeostasis on Adipose Tissue and Skeletal Muscle Metabolism. Diabetes 2016;65:3610-3620.

In the article listed above, a funding source was erroneously omitted. This work was partially …