Geoffrey D. Wool

An apoA-I mimetic peptide containing a proline residue has greater in vivo HDL binding and anti-inflammatory ability than the 4F peptide.

Modifying apolipoprotein (apo) A-I mimetic peptides to include a proline-punctuated &agr;-helical repeat increases their anti-inflammatory properties as well as allows better mimicry of full-length apoA-I function. This study compares the following mimetics, either acetylated or biotinylated (b): 4F (18mer) and 4F-proline-4F (37mer, Pro). b4F interacts with both mouse HDL (moHDL) and LDL in vitro. b4F in vivo plasma clearance kinetics are not affected by mouse HDL level. Administration of biotinylated peptides to mice demonstrates that b4F does not associate with lipoproteins smaller than LDL in vivo, though it does associate with fractions containing free hemoglobin (Hb). In contrast, bPro specifically interacts with HDL. b4F and bPro show opposite binding responses to HDL by surface plasmon resonance. Administration of acetylated Pro to apoE−/− mice significantly decreases plasma serum amyloid A levels, while acetylated 4F does not have this ability. In contrast to previous reports that inferred that 4F associates with HDL in vivo, we systematically examined this potential interaction and demonstrated that b4F does not interact with HDL in vivo but rather elutes with Hb-containing plasma fractions. bPro, however, specifically binds to moHDL in vivo. In addition, the number of amphipathic &agr;-helices and their linker influences the anti-inflammatory effects of apoA-I mimetic peptides in vivo.

Apoprotein A-I mimetic peptides and their potential anti-atherogenic mechanisms of action.

Purpose of review Peptides that resemble in physicochemical properties the helices of apoprotein A-I, the major protein of atheroprotective HDL, show promise for the treatment of atherosclerosis-related vascular disease. The properties and promise of these so-called mimetic peptides will be explored in this review. Recent findings Both HDL and mimetic peptides are able to scavenge and sequester oxidized lipids and hence protect endothelial cells and arteries from the pro-inflammatory action of oxidized LDL. Active mimetic peptides have an amphipathic α-helical secondary structure, whose hydrophobic face is particularly important for its bioactivity. The most frequently employed peptide is 4F. The comparative bioactivity of variants of 4F, particularly tandem helical peptides, has been explored. The recent observation of the very high affinity of bioactive peptides for oxidized fatty acids and phospholipids provides a likely mechanism for the action of these peptides in inhibiting early atherosclerosis formation. It is not clear that these peptides alone are effective in reversing established atherosclerosis, although they may achieve this outcome in synergy with statin therapy. Summary Recent observations of mimetic peptides have pointed to promising therapies for patients with cardiovascular disease. The peptides appear to be well tolerated and effective in promoting the anti-inflammatory properties of HDL.

4F Peptide reduces nascent atherosclerosis and induces natural antibody production in apolipoprotein E-null mice

Our objective was to contrast the effect of apolipoprotein (apo) A‐I mimetic peptides, such as 4F and 4F‐Pro‐4F (Pro), on nascent and mature atherosclerotic lesions and on levels of antibodies against oxidation‐specific epitopes. Chow‐fed apoE–/– mice were injected intraperitoneally with either the 4F pep‐tide or a tandem helix apoA‐I mimetic peptide (Pro) every other day. Mice treated with 4F, but not Pro, for 4 wk starting at 10 wk of age showed a dramatic decrease in atherosclerosis at 2 arterial sites. However, neither peptide was effective in mice treated for 8 wk starting at 20 wk of age; lesions were larger and more mature at this time point. Peptide treatment caused increased production of antibodies against oxidation‐specific epitopes, including a disproportionate induction of the IgM natural antibody (NAb) E06/ T15 to oxidized phospholipids. In summary, 4F, but not the tandem peptide Pro, effectively inhibited early atherogenesis but was ineffective against more mature lesions. Two different apoA‐I mimetic peptides increased titers of natural antibodies against oxidation‐specific epitopes.—Wool, G. D., Cabana, V. G., Lukens, J., Shaw, P. X., Binder, C. J., Witztum, J. L., Reardon, C. A., Getz, G. S. 4F Peptide reduces nascent atherosclerosis and induces natural antibody production in apolipoprotein E‐null mice. FASEB J. 25, 290–300 (2011). www.fasebj.org

HDL apolipoprotein-related peptides in the treatment of atherosclerosis and other inflammatory disorders.

Elevations of HDL levels or modifying the inflammatory properties of HDL are being evaluated as possible treatment of atherosclerosis, the underlying mechanism responsible for most cardiovascular diseases. A promising approach is the use of small HDL apoprotein-related mimetic peptides. A number of peptides mimicking the repeating amphipathic α-helical structure in apoA-I, the major apoprotein in HDL, have been examined in vitro and in animal models. Several peptides have been shown to reduce early atherosclerotic lesions, but not more mature lesions unless coadministered with statins. These peptides also influence the vascular biology of the vessel wall and protect against other acute and chronic inflammatory diseases. The biologically active peptides are capable of reducing the pro-inflammatory properties of LDL and HDL, likely due to their high affinity for oxidized lipids. They are also capable of influencing other processes, including ABCA1 mediated activation of JAK-2 in macrophages, which may contribute to their anti-atherogenic function. The initial studies involved monomeric 18 amino acid peptides, but tandem peptides are being investigated for their anti-atherogenic and anti-inflammatory properties as they more closely resemble the repeating structure of apoA-I. Peptides based on other HDL associated proteins such as apoE, apoJ and SAA have also been studied. Their mechanism of action appears to be distinct from the apoA-I based mimetics.

Biological Properties of Apolipoprotein A-I Mimetic Peptides

Apolipoprotein A-I (apoA-I) mimetic peptides resemble the physiochemical properties of the helices of apoA-I and show promise for the treatment of atherosclerotic vascular diseases and other chronic inflammatory disorders. These peptides have numerous properties, such as the ability to remodel high-density lipoprotein, sequester oxidized lipids, promote cholesterol efflux, and activate an anti-inflammatory process in macrophages, any or all of which may contribute to their antiatherogenic properties. In murine models, the 4F peptide attenuates early atherosclerosis but seems to require the addition of statins to influence more mature lesions. A recently developed method for the oral delivery of the peptides that protects them from proteolysis will facilitate further research on the mechanism of action of these peptides. This review focuses on the properties of the 4F peptide, although numerous apoA-I mimetics are under investigation and a single “best” peptide that mimics all of the properties of the antiatherogenic protein apoA-I has not been identified.

A superior method for cell block preparation for fine-needle aspiration biopsies.

Cell block (CB) techniques for fine‐needle aspiration biopsies (FNABs) vary. A direct comparison of CB techniques with statistical validation was performed to identify the best method.

Bone marrow necrosis: ten-year retrospective review of bone marrow biopsy specimens.

OBJECTIVES Bone marrow can undergo necrosis for many different causes; malignant causes are reported to be more frequent. METHODS We undertook a 10-year retrospective review of all bone marrow biopsy specimens with bone marrow necrosis at our institution. RESULTS Identified cases represented approximately 0.3% of our bone marrow cases. Most identified bone marrow cases with necrosis were involved by metastatic tumor or hematolymphoid malignancy (90% of total) in relatively equal proportions. In those cases of bone marrow necrosis with hematolymphoid malignancy, lymphoid disease predominated and the necrosis was often seen in the setting of chemotherapy. In metastatic tumor cases, necrosis seemed to enrich in prostate adenocarcinoma and Ewing sarcoma/primitive neuroectodermal tumor; neuroblastoma showed much less necrosis. Ten percent of patients with bone marrow necrosis had no underlying malignancy, and the associated causes varied. CONCLUSIONS The causes of bone marrow necrosis are diverse but should always prompt careful assessment for malignancy and infectious etiology.

Mimetic peptides of human apoA-I helix 10 get together to lower lipids and ameliorate atherosclerosis: is the action in the gut?

This issue of the Journal of Lipid Research contains an intriguing paper from the Scripps Research Institute by Zhao and colleagues (1) investigating the in vivo anti-atherosclerotic properties of HDL-like nanoparticles containing α-helical amphipathic peptides. The group uses 23-amino acid peptides representing a modified tenth helix of human apoA-I (amino acid residues 221-241, modified sequence = CGVLESFKASFLSALEEWTKKLQ). This last helix of apoA-I is associated with initial lipid binding and cholesterol efflux by the apoprotein (2, 3). The two conservative amino acid substitutions were designed to increase amphiphilicity and spectral properties. The use of the tenth helix sequence as a peptide model by Zhao et al. is novel and is in contrast to the development of the 18A peptide and subsequently the 4F peptide, which were designed as idealized class A amphipathic helices without sequence homology to a natural apoprotein (4). Zhao et al. formed HDL-like nanoparticles with their peptide mimetics of human apoA-I helix 10 by incubation with DMPC multilamellar vesicles without utilizing cholate. The HDL-like nanoparticles come in two flavors, those with monomeric 23-mer peptide and those with peptide trimers; that is, three 23-mer peptide monomers each linked to a small uncharged organic scaffold to generate a multivalent construct. To contrast this report with the majority of work in the apoA-I mimetic peptide field, peptide mimetics such 18A and 4F have generally been investigated as monomers and administered to animals as bare peptide without associated lipid (5) with some notable exceptions (6, 7). While tandem linked dimeric peptides have previously been investigated (5), there is no previous description of the anti-atherosclerotic properties of trimeric peptides. The Scripps group had previously shown that these nanoparticles were 9–12 nm discs that incorporated into and remodeled HDL in vitro and mediated cholesterol efflux in culture. It similarly remodeled HDL in vivo following intraperitoneal injection (8). The complexing of peptide with DMPC provided a degree of protease protection in vitro relative to lipid-free peptide. The nanoparticles with trimeric peptides were more effective at incorporating into HDL, mediating cholesterol efflux, and also were more resistant to proteolysis than the nanoparticles with monomeric peptide. The improved ability of nanoparticles with peptide multimers to interact with HDL and efflux cholesterol mirrors what our group and others have reported with tandem symmetric or asymmetric dimeric apoA-I mimetic peptides with proline linkers (9–11). Using the LDL-receptor null mouse model of atherosclerosis, the mice were switched to Western-type diet and simultaneously treated with the nanoparticles. The Scripps group shows striking efficacy in decreasing both nonHDL-C (plasma total cholesterol reduced ∼40% after 2 weeks of treatment) and aortic atherosclerosis (whole aorta and sinus lesion reductions ∼50% after 10 weeks of treatment) by either the intraperitoneal or oral route of administration of the nanoparticles. This is in striking contrast to the effect of the well-described 4F and other apoA-I mimetic peptides, which can decrease atherosclerotic lesion size without a consistent significant effect on plasma lipids (5). The use of this human apoA-I tenth helix mimetic seems to provide lipid lowering abilities not typically seen in apoA-I mimetic peptides. Of note, previous mimetic reports generally used lipid-free peptide rather than the peptide-lipid particles reported here. Prior to the current report, mimetic peptides based on apoE (12–14), which contain motifs for binding the LDL-receptor or scavenger receptors, were the exception in the mimetic peptide field in that they show both lipid lowering and anti-atherosclerotic effect. However, lipid lowering may not be the primary mechanism by which the nanoparticles are anti-atherogenic. Zhao et al. show that their HDL-like nanoparticles have oral efficacy in lipid lowering only at a younger age (12 weeks, i.e., two weeks of treatment) but not after a more extended treatment (at 20 weeks). At 20 weeks of age, the mice given nanoparticles by the oral route did not exhibit significantly lower plasma lipids. Nevertheless these mice had notably reduced atherosclerotic lesions. Of note, despite these nanoparticles being atheroprotective by the oral route, they could not be detected by pharmacokinetic studies of the plasma using sensitive LC-MS selected ion monitoring techniques. Thus plasma concentrations and lipid lowering do not seem to be the major bases for the anti-atherogenicity of PO-administered nanoparticles. Additionally, nanoparticles with monomeric peptide showed similar in vivo potency as those with trimeric peptide, despite previous work by the Scripps group showing improved plasma pharmacokinetics and in vivo HDL remodeling by the trimeric peptide nanoparticles (8). What then might be the basis for the anti-atherogenicity of the nanoparticles? The effectiveness of 4F peptides at preventing atherosclerosis is generally thought to be due to binding oxidized lipid (15) or reverse cholesterol transport (16) as opposed to plasma lipid lowering. In the current study, plasma 15(S)-HETE levels were not influenced by the nanoparticle treatment and in vivo macrophage to feces reverse cholesterol transport was not investigated, although these nanoparticles were previously shown to mediate efficient cholesterol efflux from macrophages in culture (8). However, oral administration shows no detectable nanoparticle in plasma so there likely is not significant lipoprotein association/remodeling or reverse cholesterol transport promoted by the nanoparticles in this set of mice. In addition, nanoparticles with the monomeric peptide are more susceptible to proteolysis but are still effective orally. Overall, the current study suggests that the in vitro and short-term in vivo nanoparticle properties investigated previously by the authors are unlikely to play a significant role in the in vivo atheroprotection. All these findings lend further credence to the hypothesis that the gut is the site of action for apoA-I mimetic peptides (17). This has been highlighted recently by the Fogelman group (18, 19) in experiments using transgenic tomato feeds as a source of peptide administration where anti-inflammatory, lipid lowering, and anti-atherosclerotic effects were seen but intact 6F peptide was not found in the plasma. Further investigations of the effect of nanoparticle treatment on gut lipid absorption, lysophosphatidic acid accumulation, enterocyte lipid esterification and transporter function, and chyle composition obviously are called for. Perhaps work by the Fogelman group with 6F-transgenic tomato feeds and the current work showing equivalency of peptide via IP and oral administration (without detectable peptide plasma levels after oral dosing) should hasten workers in the field of atherosclerosis to pay as much attention to the gut as they do to other tissues where atheroprotective mechanisms may be operating. The basis for the resistance of these peptides to gut proteases also needs exploration. The Scripps group point out several ways in which their peptides behave similarly to the 4F/6F peptide studies, despite their differences in primary amino acid sequence, including that they are effective in vivo despite the lack of detection of the peptides in the plasma. However, the use of an α-helix involved in initial apoA-I lipid binding would otherwise be expected to behave very differently from the 4F-type peptides. For instance, nanoparticle treatment did not effectively reduce plasma 15-S-HETE and showed mixed effectiveness at reducing plasma serum amyloid A, two features commonly reported for the 4F and 6F peptides (15, 18, 20). The binding of oxidized lipids by these nanoparticles has not been studied by the Scripps group, but lipid-peptide complexes may not be expected to bind oxidized lipid with as high affinity as lipid-free peptides. The current study illustrates that apoA-I mimetic peptides with different structural properties may exert their protective effects via different mechanisms. While it is well known that apoA-I has many putative functions in atheroprotection, it is becoming apparent that this is also the case for mimetic peptides. Additionally, this work points out a possible disconnect between apparently anti-atherogenic functionality in vitro (such as cholesterol efflux in culture) and the actual atheroprotective effect in vivo. This work also continues to show the dispensability of lipid lowering for atheroprotection by apoA-I mimetic peptides. Intravenous infusions of HDL, HDL-like particles, and delipidated apoA-I have been reported to be effective in reducing atherosclerotic lesions in animal models (21) although the results have been less robust in human studies (22). The current report and work by the Fogelman group have raised the exciting possibility of a future orally available agent targeted at improving HDL function, likely through a gut-mediated mechanism of action. While recent therapeutic trials of pharmacologic HDL-C raising agents have been disappointing (23, 24), the ongoing work with apoA-I peptides continues to show promise.

The management of anticoagulation in patients undergoing therapeutic plasma exchange: A concise review

We surveyed multiple apheresis centers represented by the authors for their clinical approach to the management of anticoagulation issues during therapeutic plasma exchange (TPE). We present the results of their practices and a review of the pertinent literature. As plasma is removed during TPE, replacement with all or partial non‐plasma‐containing fluids (eg, 5% albumin) may lead to significant changes in hemostasis. These changes are amplified in patients who are receiving anticoagulation. We discuss various anticoagulants as well as the monitoring and adjustment of anticoagulation before, during, and after TPE. No single guideline can be applied, but rather, patients must be monitored individually, taking into account their often complex clinical conditions and medication profiles.

Therapeutic plasma exchange as part of multimodal treatment of acquired hemophilia in a patient with concurrent acute intracerebral bleed and pulmonary embolism

Autoantibodies against Factor VIII (FVIII) define the rare but life‐threatening bleeding disorder acquired hemophilia A (AHA). Correction of FVIII deficiency and eradication of the factor inhibitor are the ultimate therapeutic goals in this disorder. Bypassing agents such as recombinant factor VIIa (rFVIIa) or FVIII inhibitor bypassing agent are often used to control coagulopathy before the inhibitor is eradicated. Bypassing agents carry a risk of thrombosis, however.