Single-cell transcriptomic heterogeneity between conduit and resistance mesenteric arteries in rats.

The endothelium contains morphologically similar cells throughout the vasculature, but individual cells along the length of a single vascular tree or in different regional circulations function dissimilarly. When observations made in large arteries are extrapolated to explain the function of endothelial cells (ECs) in the resistance vasculature, only a fraction of these observations are consistent between artery sizes. To what extent endothelial (EC) and vascular smooth muscle cells (VSMCs) from different arteriolar segments of the same tissue differ phenotypically at the single-cell level remains unknown. Therefore, single-cell RNA-seq (10x Genomics) was performed using a 10X Genomics Chromium system. Cells were enzymatically digested from large (>300 µm) and small (<150 µm) mesenteric arteries from nine adult male Sprague-Dawley rats, pooled to create six samples (3 rats/sample, 3 samples/group). After normalized integration, the dataset was scaled before unsupervised cell clustering and cluster visualization using UMAP plots. Differential gene expression analysis allowed us to infer the biological identity of different clusters. Our analysis revealed 630 and 641 differentially expressed genes (DEGs) between conduit and resistance arteries for ECs and VSMCs, respectively. Gene ontology analysis (GO-Biological Processes, GOBP) of scRNA-seq data discovered 562 and 270 pathways for ECs and VSMCs, respectively, that differed between large and small arteries. We identified eight and seven unique ECs and VSMCs subpopulations, respectively, with DEGs and pathways identified for each cluster. These results and this dataset allow the discovery and support of novel hypotheses needed to identify mechanisms that determine the phenotypic heterogeneity between conduit and resistance arteries.

Systematic evaluation of the effect of different apolipoprotein A-I mimetic peptides on the performance of synthetic high-density lipoproteins in vitro and in vivo.

Synthetic high-density lipoproteins nanomedicine (sHDL) composed of apolipoprotein A-I (ApoA-I) mimetic peptides and lipids have shown very promising results for the treatment of various cardiovascular diseases. Numerous efforts have also been made to design different ApoA-I mimetic peptides to improve the potency of sHDL, especially the efficiency of reverse cholesterol transport. However, the way in which ApoA-I mimetic peptides affect the properties of sHDL, including stability, cholesterol efflux, cholesterol esterification, elimination in vivo, and the relationship of these properties, is still poorly understood. Revealing the effect of these factors on the potency of sHDL is important for the design of better ApoA-I mimetic peptides. In this study, three widely used ApoA-I mimetic peptides with different sequences, lengths, LCAT activation and lipid binding affinities were used for the preparation of sHDL and were evaluated in terms of physical/chemical properties, cholesterol efflux, cholesterol esterification, remodeling, and pharmacokinetics/pharmacodynamics. Our results showed that ApoA-I mimetic peptides with the highest cholesterol efflux and cholesterol esterification in vitro did not exhibit the highest cholesterol mobilization in vivo. Further analysis indicated that other factors, such as pharmacokinetics and remodeling of sHDL, need to be considered in order to predict the efficiency of cholesterol mobilization in vivo. Thus, our study highlights the importance of using the overall performance, rather than in vitro results alone, as the blueprint for the design and optimization of ApoA-I mimetic peptides.

Phospholipids impact the protective effects of HDL-mimetic nanodiscs against lipopolysaccharide-induced inflammation.

Aim: The impacts of synthetic high-density lipoprotein (sHDL) phospholipid components on anti-sepsis effects were investigated. Methods: sHDL composed with ApoA-I mimetic peptide (22A) and different phosphatidylcholines were prepared and characterized. Anti-inflammatory effects were investigated in vitro and in vivo on lipopolysaccharide (LPS)-induced inflammation models. Results: sHDLs composed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (22A-DMPC) most effectively neutralizes LPS, inhibits toll-like receptor 4 recruitment into lipid rafts, suppresses nuclear factor κB signaling and promotes activating transcription factor 3 activating. The lethal endotoxemia animal model showed the protective effects of 22A-DMPC. Conclusion: Phospholipid components affect the stability and fluidity of nanodiscs, impacting the anti-septic efficacy of sHDLs. 22A-DMPC presents the strongest LPS binding and anti-inflammatory effects in vitro and in vivo, suggesting a potential sepsis treatment.

Sepsis is triggered by endotoxins released by bacteria. These endotoxins trigger an exaggerated inflammatory response, leading to widespread inflammation and organ damage. Synthetic high-density lipoprotein (sHDL) is a potential treatment of sepsis by neutralizing endotoxins and regulating inflammatory responses. The phospholipid components of sHDL may affect the effectiveness of sHDL against sepsis. In this study, we prepared sHDLs with different phospholipids and compared their anti-septic effects on cells and in animal models. We found that sHDL made from DMPC presented the best anti-septic effects, possibly because DMPC-sHDL had the best fluidity at body temperature.

Role of Cholesterol in the Regulation of Hydrogen Sulfide Signaling within the Vascular Endothelium.

H2S is a gaseous signaling molecule enzymatically produced in mammals and H2S-producing enzymes are expressed throughout the vascular wall. We previously reported that H2S-induced vasodilation is mediated through transient receptor potential cation channel subfamily V member 4 (TRPV4) and large conductance (BKCa) potassium channels; however, regulators of this pathway have not been defined. Previous reports have shown that membrane cholesterol limits activity of TRPV4 and BKCa potassium channels. The current study examined the ability of endothelial cell (EC) plasma membrane (PM) cholesterol to regulate H2S-induced vasodilation. We hypothesized that EC PM cholesterol hinders H2S-mediated vasodilation in large mesenteric arteries. In pressurized, U46619 pre-constricted mesenteric arteries, decreasing EC PM cholesterol in large arteries using methyl-ß-cyclodextrin (MBCD, 100 µM) increased H2S-induced dilation (NaHS 10, 100 µM) but MBCD treatment had no effect in small arteries. Enface fluorescence showed EC PM cholesterol content is higher in large mesenteric arteries than in smaller arteries. The NaHS-induced vasodilation following MBCD treatment in large arteries was blocked by TRPV4 and BKCa channel inhibitors (GSK219384A, 300 nM and iberiotoxin, 100 nM, respectively). Immunohistochemistry of mesenteric artery cross-sections show that TRPV4 and BKCa are both present in EC of large and small arteries. Cholesterol supplementation into EC PM of small arteries abolished NaHS-induced vasodilation but the cholesterol enantiomer, epicholesterol, had no effect. Proximity ligation assay studies did not show a correlation between EC PM cholesterol content and the association of TRPV4 and BK. Collectively, these results demonstrate that EC PM cholesterol limits H2S-induced vasodilation through effects on EC TRPV4 and BKCa channels.

Endothelial cell membrane cholesterol content regulates the contribution of TRPV4 channels in ACh-induced vasodilation in rat gracilis arteries.

OBJECTIVE: Our previous work demonstrated that endothelial cell (EC) membrane cholesterol is reduced following 48 h of chronic hypoxia (CH). CH couples endothelial transient receptor potential subfamily V member 4 (TRPV4) channels to muscarinic receptor signaling through an endothelium-dependent hyperpolarization (EDH) pathway does not present in control animals. TRVPV4 channel activity has been shown to be regulated by membrane cholesterol. Hence, we hypothesize that acute manipulation of endothelial cell membrane cholesterol inversely determines the contribution of TRPV4 channels to endothelium-dependent vasodilation. METHODS: Male Sprague-Dawley rats were exposed to ambient atmospheric (atm.) pressure or 48-h of hypoxia (0.5 atm). Vasodilation to acetylcholine (ACh) was determined using pressure myography in gracilis arteries. EC membrane cholesterol was depleted using methyl-ß-cyclodextrin (MßCD) and supplemented with MßCD-cholesterol. RESULTS: Inhibiting TRPV4 did not affect ACh-induced vasodilation in normoxic controls. However, TRPV4 inhibition reduced resting diameter in control arteries suggesting basal activity. TRPV4 contributes to ACh-induced vasodilation in these arteries when EC membrane cholesterol is depleted. Inhibiting TRPV4 attenuated ACh-induced vasodilation in arteries from CH animals that exhibit lower EC membrane cholesterol than normoxic controls. EC cholesterol repletion in arteries from CH animals abolished the contribution of TRPV4 to ACh-induced vasodilation. CONCLUSION: Endothelial cell membrane cholesterol impedes the contribution of TRPV4 channels in EDH-mediated dilation. These results provide additional evidence for the importance of plasma membrane cholesterol content in regulating intracellular signaling and vascular function.

Replenishing HDL with synthetic HDL has multiple protective effects against sepsis in mice.

Sepsis is a major health issue with mortality exceeding 30% and few treatment options. We found that high-density lipoprotein cholesterol (HDL-C) abundance was reduced by 45% in septic patients compared to that in nonseptic patients. Furthermore, HDL-C abundance in nonsurviving septic patients was substantially lower than in those patients who survived. We therefore hypothesized that replenishing HDL might be a therapeutic approach for treating sepsis and found that supplementing HDL with synthetic HDL (sHDL) provided protection against sepsis in mice. In mice subjected to cecal ligation and puncture (CLP), infusing the sHDL ETC-642 increased plasma HDL-C amounts and improved the 7-day survival rate. Septic mice treated with sHDL showed improved kidney function and reduced inflammation, as indicated by marked decreases in the plasma concentrations of blood urea nitrogen (BUN) and the cytokines interleukin-6 (IL-6) and IL-10, respectively. We found that sHDL inhibited the ability of the endotoxins LPS and LPA to activate inflammatory pathways in RAW264.7 cells and HEK-Blue cells expressing the receptors TLR4 or TLR2 and NF-κB reporters. In addition, sHDL inhibited the activation of HUVECs by LPS, LTA, and TNF-α. Together, these data indicate that sHDL treatment protects mice from sepsis in multiple ways and that it might be an effective therapy for patients with sepsis.

Synthetic high-density lipoproteins delivering liver X receptor agonist prevent atherogenesis by enhancing reverse cholesterol transport.

Liver X nuclear receptor (LXR) agonists are promising anti-atherosclerotic agents that increase the expression of cholesterol transporters on atheroma macrophages leading to increased efflux of cholesterol to endogenous high-density lipoprotein (HDL) acceptors. HDL subsequently delivers effluxed cholesterol to the liver by the process of reverse cholesterol transport, resulting in reduction of atherosclerotic plaques. However, LXR agonists administration triggers undesirable liver steatosis and hypertriglyceridemia due to increased fatty acid and sterol synthesis. LXR-induced liver toxicity, poor drug aqueous solubility and low levels of endogenous HDL acceptors in target patient populations limit the clinical translation of LXR agonists. Here, we propose a dual-antiatherogenic strategy for administration of the LXR agonist, T0901317 (T1317), by encapsulating in synthetic HDL (sHDL) nanoparticles. sHDL had been clinically proven to serve as cholesterol acceptors, resulting in plaque reduction in atherosclerosis patients. In addition, the hydrophobic core and endogenous atheroma-targeting ability of sHDL allow for encapsulation of water-insoluble drugs and their subsequent delivery to atheroma. Several compositions of sHDL were tested to optimize both T1317 encapsulation efficiency and ability of T1317-sHDL to efflux cholesterol. Optimized T1317-sHDL exhibited more efficient cholesterol efflux from macrophages and enhanced atheroma-targeting relative to free drug. Most importantly, in an apolipoprotein E deficient (ApoE-/-) atherosclerosis progression murine model, T1317-sHDL showed superior inhibition of atherogenesis and reduced hypertriglyceridemia side effects in comparison to the free drug and blank sHDL. The T1317-sHDL pharmacological efficacy was observed at doses lower than those previously described for LXR agents, which may have additional safety benefits. In addition, the established clinical manufacturing, safety and efficacy of blank sHDL nanoparticles used in this study could facilitate future clinical translation of LXR-loaded sHDLs.

Synergetic Effect of rHDL and LXR Agonist on Reduction of Atherosclerosis in Mice.

High-density lipoproteins (HDLs) are unique in that they play an important role in the reverse cholesterol transport process. However, reconstituted HDL (rHDL) infusions have demonstrated limited beneficial effect in clinical practice. This is perhaps a consequence of the limited cholesterol efflux abilities of atheroma macrophages due to decreased expression of cholesterol transporters in advanced atheromas and following rHDL infusion treatment. Thus, we propose that a combination therapy of rHDL and a liver X receptor (LXR) agonist could maximize the therapeutic benefit of rHDL by upregulating ATP-binding cassette transporters A-1 (ABCA1) and ATP-binding cassette transporter G-1 (ABCG1), and enhancing cholesterol efflux to rHDL. In macrophages, rHDL downregulated the expression of ABCA1/G1 in a dose- and rHDL composition-dependent manner. Although LXR agonist, T0901317 (T1317), upregulated the expression of ABCA1 and ABCG1, the drug itself did not have any effect on cholesterol efflux (6.6 ± 0.5%) while the combination of rHDL and T1317 exhibited enhanced cholesterol efflux from [3H]-cholesterol loaded J774A.1 macrophages (23.3 ± 1.3%). Treatment with rHDL + T1317 significantly reduced the area of aortic plaque in ApoE-/- mice compared to PBS treated control animals (24.16 ± 1.42% vs. 31.59 ± 1.93%, p < 0.001), while neither rHDL nor T1317 treatment alone had a significant effect. Together, we show that rHDL paired with an LXR agonist can induce a synergetic effect in reducing atheroma burden. This synergy could lead to lower overall effective dose for both drugs, potentially overcoming the existing barriers in clinical development and renewing pharmaceutical interest in these two drug classes.

High-Density Lipoprotein in Lupus: Disease Biomarkers and Potential Therapeutic Strategy.

Systemic lupus erythematosus (SLE) patients exhibit accelerated development of atherosclerosis and increased incidents of cardiovascular disease (CVD) that cannot be explained by traditional risk factors alone. Accumulating evidence suggests that reduced levels of high-density lipoproteins (HDLs), along with altered HDL composition and function, may contribute to the accelerated atherosclerosis in SLE patients. Normally, HDLs play various atheroprotective roles through facilitating cholesterol efflux, inhibiting vascular inflammation, and scavenging oxidative species. However, systemic inflammation, oxidative stress, and autoimmunity in SLE patients induce changes in HDL size distribution and proteomic and lipidomic signatures. These compositional changes in HDLs result in the formation of proinflammatory, dysfunctional HDL. These lupus-altered HDLs have impaired antiatherogenic function with reduced cholesterol efflux capacities, impaired antioxidation abilities, and diminished antiinflammatory properties. In fact, dysfunctional HDL may promote atherogenesis by inducing inflammation. Thus, dysfunctional HDLs could be an important biomarker of accelerated atherosclerosis in lupus. Additionally, HDL-targeted therapies, especially infusion of reconstituted HDLs, may serve as a potential therapeutic intervention for SLE patients with CVD.

Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann-Pick diseases.

BACKGROUND: Niemann-Pick disease type C is a fatal and progressive neurodegenerative disorder characterized by the accumulation of unesterified cholesterol in late endosomes and lysosomes. We sought to develop new therapeutics for this disorder by harnessing the body's endogenous cholesterol scavenging particle, high-density lipoprotein (HDL). METHODS: Here we design, optimize, and define the mechanism of action of synthetic HDL (sHDL) nanoparticles. RESULTS: We demonstrate a dose-dependent rescue of cholesterol storage that is sensitive to sHDL lipid and peptide composition, enabling the identification of compounds with a range of therapeutic potency. Peripheral administration of sHDL to Npc1 I1061T homozygous mice mobilizes cholesterol, reduces serum bilirubin, reduces liver macrophage size, and corrects body weight deficits. Additionally, a single intraventricular injection into adult Npc1 I1061T brains significantly reduces cholesterol storage in Purkinje neurons. Since endogenous HDL is also a carrier of sphingomyelin, we tested the same sHDL formulation in the sphingomyelin storage disease Niemann-Pick type A. Utilizing stimulated Raman scattering microscopy to detect endogenous unlabeled lipids, we show significant rescue of Niemann-Pick type A lipid storage. CONCLUSIONS: Together, our data establish that sHDL nanoparticles are a potential new therapeutic avenue for Niemann-Pick diseases.

Survey of Clinical Translation of Cancer Nanomedicines-Lessons Learned from Successes and Failures.

In 1995, the year the first cancer nanomedicine, Doxil, was approved by the Food and Drug Administration (FDA), only 23 manuscripts appeared in a PubMed search for "nanoparticles for cancer" keywords. Now, over 25 000 manuscripts can be found using those same keywords, yet only 15 nanoparticle-based cancer nanomedicines are approved globally. Based on the clinicaltrials.gov database, a total of 75 cancer nanomedicines are under clinical investigation involving 190 clinical trials summarized here. In this Account, we focus on cancer nanomedicines that have been approved or reached clinical trials to understand this high attrition rate. We classify the various nanomedicines, summarize their clinical outcomes, and discuss possible reasons for product failures and discontinuation of product development efforts. Among ongoing and completed clinical trials, 91 (48 completed) are phase 1, 78 (59 completed) phase 2, and 21 (11 completed) phase 3. The success rate of phase 1 trials has been high-roughly 94%. Of those phase 1 trials with identified outcomes, 45 showed positive safety and efficacy results, with only one negative result (low efficacy) and two terminated due to adverse reactions. In some cases, findings from these trials have not only shown improved pharmacokinetics, but also avid drug accumulation within tumor tissues among active-targeting nanoparticles, including BIND-014, CALAA-01, and SGT-94. However, the success rate drops to ∼48% among completed phase 2 trials with identified outcomes (31 positive, 15 negative, and 4 terminated for toxicity or poor efficacy). A majority of failures in phase 2 trials were due to poor efficacy (15 of 19), rather than toxicity (4 of 19). Unfortunately, the success rate for phase 3 trials slumps to a mere ∼14%, with failures stemming from lack of efficacy. Although the chance of success for cancer nanomedicines starting from the proof-of-concept idea in the laboratory to valuable marketed product may seem daunting, we should not be discouraged. Despite low success rates, funding from the government, foundations, and research organizations are still strong-an estimated > $130 M spent by the National Institutes of Health (NIH) on R01s focused on nanomedicine in 2018 alone. In addition, the NIH created several special initiatives/programs, such as the National Cancer Institute (NCI) Alliance, to facilitate clinical translation of nanomedicines. Companies developing cancer nanomedicines raised diverse ranges of funds from venture capital, capital markets, and industry partnerships. In some cases, the development efforts resulted in regulatory approvals of cancer nanomedicines. In other cases, clinical failures and market pressure from improving standard of care products resulted in product terminations and business liquidation. Yet, recent approvals of nanomedicine products for orphan cancers and continuing development of nanoparticle based drugs for immune-oncology applications fuel continuing industrial and academic interest in cancer nanomedicines.

HDL in Endocrine Carcinomas: Biomarker, Drug Carrier, and Potential Therapeutic.

High-density lipoprotein (HDL) have long been studied for their protective role against cardiovascular diseases, however recently relationship between HDL and cancer came into focus. Several epidemiological studies have shown an inverse correlation between HDL-cholesterol (HDL-C) and cancer risk, and some have even implied that HDL-C can be used as a predictive measure for survival prognosis in for specific sub-population of certain types of cancer. HDL itself is an endogenous nanoparticle capable of removing excess cholesterol from the periphery and returning it to the liver for excretion. One of the main receptors for HDL, scavenger receptor type B-I (SR-BI), is highly upregulated in endocrine cancers, notably due to the high demand for cholesterol by cancer cells. Thus, the potential to exploit administration of cholesterol-free reconstituted or synthetic HDL (sHDL) to deplete cholesterol in endocrine cancer cell and stunt their growth of use chemotherapeutic drug loaded sHDL to target payload delivery to cancer cell has become increasingly attractive. This review focuses on the role of HDL and HDL-C in cancer and application of sHDLs as endocrine cancer therapeutics.

Battle of GLP-1 delivery technologies.

Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) belong to an important therapeutic class for treatment of type 2 diabetes. Six GLP-1 RAs, each utilizing a unique drug delivery strategy, are now approved by the Food and Drug Administration (FDA) and additional, novel GLP-1 RAs are still under development, making for a crowded marketplace and fierce competition among the manufacturers of these products. As rapid elimination is a major challenge for clinical application of GLP-1 RAs, various half-life extension strategies have been successfully employed including sequential modification, attachment of fatty-acid to peptide, fusion with human serum albumin, fusion with the fragment crystallizable (Fc) region of a monoclonal antibody, sustained drug delivery systems, and PEGylation. In this review, we discuss the scientific rationale of the various half-life extension strategies used for GLP-1 RA development. By analyzing and comparing different approved GLP-1 RAs and those in development, we focus on assessing how half-life extending strategies impact the pharmacokinetics, pharmacodynamics, safety, patient usability and ultimately, the commercial success of GLP-1 RA products. We also anticipate future GLP-1 RA development trends. Since similar drug delivery strategies are also applied for developing other therapeutic peptides, we expect this case study of GLP-1 RAs will provide generalizable concepts for the rational design of therapeutic peptides products with extended duration of action.

Synthetic High-Density Lipoprotein-Mediated Targeted Delivery of Liver X Receptors Agonist Promotes Atherosclerosis Regression.

Targeting at enhancing reverse cholesterol transport (RCT) is apromising strategy for treating atherosclerosis via infusion of reconstitute high density lipoprotein (HDL) as cholesterol acceptors or increase of cholesterol efflux by activation of macrophage liver X receptors (LXRs). However, systemic activation of LXRs triggers excessive lipogenesis in the liver and infusion of HDL downregulates cholesterol efflux from macrophages. Here we describe an enlightened strategy using phospholipid reconstituted apoA-I peptide (22A)-derived synthetic HDL (sHDL) to deliver LXR agonists to the atheroma and examine their effect on atherosclerosis regression in vivo. A synthetic LXR agonist, T0901317 (T1317) was encapsulated in sHDL nanoparticles (sHDL-T1317). Similar to the T1317 compound, the sHDL-T1317 nanoparticles upregulated the expression of ATP-binding cassette transporters and increased cholesterol efflux in macrophages in vitro and in vivo. The sHDL nanoparticles accumulated in the atherosclerotic plaques of ApoE-deficient mice. Moreover, a 6-week low-dose LXR agonist-sHDL treatment induced atherosclerosis regression while avoiding lipid accumulation in the liver. These findings identify LXR agonist loaded sHDL nanoparticles as a promising therapeutic approach to treat atherosclerosis by targeting RCT in a multifaceted manner: sHDL itself serving as both a drug carrier and cholesterol acceptor and the LXR agonist mediating upregulation of ABC transporters in the aorta.

Effect of Synthetic High Density Lipoproteins Modification with Polyethylene Glycol on Pharmacokinetics and Pharmacodynamics.

Synthetic high density lipoprotein nanoparticles (sHDLs) capable of mobilizing excess cholesterol from atherosclerotic arteries and delivering it to the liver for elimination have been shown to reduce plaque burden in patients. Unfortunately, sHDLs have a narrow therapeutic index and relative to the endogenous HDL shorter circulation half-life. Surface modification with polyethylene glycol (PEG) was investigated for its potential to extend sHDL circulation in vivo. Various amounts (2.5, 5, and 10%) and different chain lengths (2 and 5 kDa) of PEG-modified lipids were incorporated in sHDL's lipid membrane. Incorporating PEG did not reduce the ability of sHDL to facilitate cholesterol efflux, nor did it inhibit cholesterol uptake by the liver cells. By either adding more PEG or using PEG of longer chain lengths, the circulation half-life was extended. Addition of PEG also increased the area under the curve for the phospholipid component of sHDL (p < 0.05), but not for the apolipoprotein A-I peptide component of sHDL, suggesting sHDL is remodeled by endogenous lipoproteins in vivo. The extended phospholipid circulation led to a higher mobilization of plasma free cholesterol, a biomarker for facilitation of reverse cholesterol transport. The area under the cholesterol mobilization increased about 2-4-fold (p < 0.05), with greater increases observed for longer PEG chains and higher molar percentages of incorporated PEGylated lipids. Mobilized cholesterol was associated primarily with the HDL fraction, led to a transient increase in VLDL cholesterol, and returned to baseline 24 h postdose. Overall, PEGylation of sHDL led to beneficial changes in sHDL particle pharmacokinetic and pharmacodynamic behaviors.

Influence of route of administration and lipidation of apolipoprotein A-I peptide on pharmacokinetics and cholesterol mobilization.

apoA-I, apoA-I mimetic peptides, and their lipid complexes or reconstituted high-density lipoprotein (HDL) have been studied as treatments for various pathologies. However, consensus is lacking about the best method for administration, by intravenous (IV) or intraperitoneal (IP) routes, and formulation, as an HDL particle or in a lipid-free form. The objective of this study was to systematically examine peptide plasma levels, cholesterol mobilization, and lipoprotein remodeling in vivo following administration of lipid-free apoA-I peptide (22A) or phospholipid reconstituted 22A-sHDL by IV and IP routes. The mean circulation half-life was longer for 22A-sHDL (T1/2 = 6.27 h) than for free 22A (T1/2 = 3.81 h). The percentage of 22A absorbed by the vascular compartment after the IP dosing was ∼50% for both 22A and 22A-sHDL. The strongest pharmacologic response came from IV injection of 22A-sHDL, specifically a 5.3-fold transient increase in plasma-free cholesterol (FC) level compared with 1.3- and 1.8-fold FC increases for 22A-IV and 22A-sHDL-IP groups. Addition of either 22A or 22A-sHDL to rat plasma caused lipoprotein remodeling and appearance of a lipid-poor apoA-I. Hence, both the route of administration and the formulation of apoA-I peptide significantly affect its pharmacokinetics and pharmacodynamics.

Synthetic High-Density Lipoprotein (sHDL) Inhibits Steroid Production in HAC15 Adrenal Cells.

High density lipoprotein (HDL) transported cholesterol represents one of the sources of substrate for adrenal steroid production. Synthetic HDL (sHDL) particles represent a new therapeutic option to reduce atherosclerotic plaque burden by increasing cholesterol efflux from macrophage cells. The effects of the sHDL particles on steroidogenic cells have not been explored. sHDL, specifically ETC-642, was studied in HAC15 adrenocortical cells. Cells were treated with sHDL, forskolin, 22R-hydroxycholesterol, or pregnenolone. Experiments included time and concentration response curves, followed by steroid assay. Quantitative real-time RT-PCR was used to study mRNA of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, lanosterol 14-α-methylase, cholesterol side-chain cleavage enzyme, and steroid acute regulatory protein. Cholesterol assay was performed using cell culture media and cell lipid extracts from a dose response experiment. sHDL significantly inhibited production of cortisol. Inhibition occurred in a concentration- and time-dependent manner and in a concentration range of 3µM-50µM. Forskolin (10µM) stimulated cortisol production was also inhibited. Incubation with 22R-hydroxycholesterol (10µM) and pregnenolone (10µM) increased cortisol production, which was unaffected by sHDL treatment. sHDL increased transcript levels for the rate-limiting cholesterol biosynthetic enzyme, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase. Extracellular cholesterol assayed in culture media showed a positive correlation with increasing concentration of sHDL, whereas intracellular cholesterol decreased after treatment with sHDL. The current study suggests that sHDL inhibits HAC15 adrenal cell steroid production by efflux of cholesterol, leading to an overall decrease in steroid production and an adaptive rise in adrenal cholesterol biosynthesis.

HDL in sepsis - risk factor and therapeutic approach.

High-density lipoprotein (HDL) is a key component of circulating blood and plays essential roles in regulation of vascular endothelial function and immunity. Clinical data demonstrate that HDL levels drop by 40-70% in septic patients, which is associated with a poor prognosis. Experimental studies using Apolipoprotein A-I (ApoAI) null mice showed that HDL deficient mice are susceptible to septic death, and overexpressing ApoAI in mice to increase HDL levels protects against septic death. These clinical and animal studies support our hypothesis that a decrease in HDL level is a risk factor for sepsis, and raising circulating HDL levels may provide an efficient therapy for sepsis. In this review, we discuss the roles of HDL in sepsis and summarize the efforts of using synthetic HDL as a potential therapy for sepsis.

The effect of phospholipid composition of reconstituted HDL on its cholesterol efflux and anti-inflammatory properties.

The goal of this study was to understand how the reconstituted HDL (rHDL) phospholipid (PL) composition affects its cholesterol efflux and anti-inflammatory properties. An ApoA-I mimetic peptide, 5A, was combined with either SM or POPC. Both lipid formulations exhibited similar in vitro cholesterol efflux by ABCA1, but 5A-SM exhibited higher ABCG1- and SR-BI-mediated efflux relative to 5A-POPC (P < 0.05). Injection of both rHDLs in rats resulted in mobilization of plasma cholesterol, although the relative potency was 3-fold higher for the same doses of 5A-SM than for 5A-POPC. Formation of preß HDL was observed following incubation of rHDLs with both human and rat plasma in vitro, with 5A-SM inducing a higher extent of preß formation relative to 5A-POPC. Both rHDLs exhibited anti-inflammatory properties, but 5A-SM showed higher inhibition of TNF-α, IL-6, and IL-1ß release than did 5A-POPC (P < 0.05). Both 5A-SM and 5A-POPC showed reduction in total plaque area in ApoE(-/-) mice, but only 5A-SM showed a statistically significant reduction over placebo control and baseline (P < 0.01). The type of PL used to reconstitute peptide has significant influence on rHDL's anti-inflammatory and anti-atherosclerosis properties.