Common haplotypes at the CFH locus and low-frequency variants in CFHR2 and CFHR5 associate with systemic FHR concentrations and age-related macular degeneration.

Age-related macular degeneration (AMD) is the principal cause of blindness in the elderly population. A strong effect on AMD risk has been reported for genetic variants at the CFH locus, encompassing complement factor H (CFH) and the complement-factor-H-related (CFHR) genes, but the underlying mechanisms are not fully understood. We aimed to dissect the role of factor H (FH) and FH-related (FHR) proteins in AMD in a cohort of 202 controls and 216 individuals with AMD. We detected elevated systemic levels of FHR-1 (p = 1.84 × 10-6), FHR-2 (p = 1.47 × 10-4), FHR-3 (p = 1.05 × 10-5) and FHR-4A (p = 1.22 × 10-2) in AMD, whereas FH concentrations remained unchanged. Common AMD genetic variants and haplotypes at the CFH locus strongly associated with FHR protein concentrations (e.g., FH p.Tyr402His and FHR-2 concentrations, p = 3.68 × 10-17), whereas the association with FH concentrations was limited. Furthermore, in an International AMD Genomics Consortium cohort of 17,596 controls and 15,894 individuals with AMD, we found that low-frequency and rare protein-altering CFHR2 and CFHR5 variants associated with AMD independently of all previously reported genome-wide association study (GWAS) signals (p = 5.03 × 10-3 and p = 2.81 × 10-6, respectively). Low-frequency variants in CFHR2 and CFHR5 led to reduced or absent FHR-2 and FHR-5 concentrations (e.g., p.Cys72Tyr in CFHR2 and FHR-2, p = 2.46 × 10-16). Finally, we showed localization of FHR-2 and FHR-5 in the choriocapillaris and in drusen. Our study identifies FHR proteins as key proteins in the AMD disease mechanism. Consequently, therapies that modulate FHR proteins might be effective for treating or preventing progression of AMD. Such therapies could target specific individuals with AMD on the basis of their genotypes at the CFH locus.

Trimerized apolipoprotein A-I (TripA) forms lipoproteins, activates lecithin: cholesterol acyltransferase, elicits lipid efflux, and is transported through aortic endothelial cells.

Apolipoprotein A-I (apoA-I) exerts many potentially anti-atherogenic properties and is therefore attractive for prevention and therapy of coronary heart disease. Since induction of apoA-I production by small molecules has turned out as difficult, application of exogenous apoA-I is pursued as an alternative therapeutic option. To counteract fast renal filtration of apoA-I, a trimeric high-molecular weight variant of apoA-I (TripA) was produced by recombinant technology. We compared TripA and apoA-I for important properties in reverse cholesterol transport. Reconstituted high-density lipoproteins (rHDL) containing TripA or apoA-I together with palmitoyl-2-oleyl-phosphatidylcholine (POPC) differed slightly by size. Compared to apoA-I, TripA activated lecithin:cholesterol acyltransferase (LCAT) with similar maximal velocity but concentration leading to half maximal velocity was slightly reduced (K(m)=2.1±0.3µg/mL vs. 0.59±0.06µg/mL). Both in the lipid-free form and as part of rHDL, TripA elicited cholesterol efflux from THP1-derived macrophages with similar kinetic parameters and response to liver-X-receptor activation as apoA-I. Lipid-free TripA is bound and transported by aortic endothelial cells through mechanisms which are competed by apoA-I and TripA and inhibited by knock-down of ATP-binding cassette transporter (ABC) A1. Pre-formed TripA/POPC particles were bound and transported by endothelial cells through mechanisms which are competed by excess native HDL as well as reconstituted HDL containing either apoA-I or TripA and which involve ABCG1 and scavenger receptor B1 (SR-BI). In conclusion, apoA-I and TripA show similar in vitro properties which are important for reverse cholesterol transport. These findings are important for further development of TripA as an anti-atherosclerotic drug.

Molecular recognition at the active site of factor Xa: cation-π interactions, stacking on planar peptide surfaces, and replacement of structural water.

Factor Xa, a serine protease from the blood coagulation cascade, is an ideal enzyme for molecular recognition studies, as its active site is highly shape-persistent and features distinct, concave sub-pockets. We developed a family of non-peptidic, small-molecule inhibitors with a central tricyclic core orienting a neutral heterocyclic substituent into the S1 pocket and a quaternary ammonium ion into the aromatic box in the S4 pocket. The substituents were systematically varied to investigate cation-π interactions in the S4 pocket, optimal heterocyclic stacking on the flat peptide walls lining the S1 pocket, and potential water replacements in both the S1 and the S4 pockets. Structure-activity relationships were established to reveal and quantify contributions to the binding free enthalpy, resulting from single-atom replacements or positional changes in the ligands. A series of high-affinity ligands with inhibitory constants down to K(i)=2 nM were obtained and their proposed binding geometries confirmed by X-ray co-crystal structures of protein-ligand complexes.

Discovery of a factor Xa inhibitor (3R,4R)-1-(2,2-difluoro-ethyl)-pyrrolidine-3,4-dicarboxylic acid 3-[(5-chloro-pyridin-2-yl)-amide] 4-[[2-fluoro-4-(2-oxo-2H-pyridin-1-yl)-phenyl]-amide] as a clinical candidate.

A series of (3R,4R)-pyrrolidine-3,4-dicarboxylic acid amides was investigated with respect to their factor Xa inhibitory activity, selectivity, pharmacokinetic properties, and ex vivo antithrombotic activity. The clinical candidate from this series, R1663, exhibits excellent selectivity against a panel of serine proteases and good pharmacokinetic properties in rats and monkeys. A Phase I clinical study with R1663 has been finalized.

Differential Effects of the Mitochondria-Active Tetrapeptide SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH2) and Its Peptidase-Targeted Prodrugs in Experimental Acute Kidney Injury.

The mitochondria-active tetrapeptide SS-31 can control oxidative tissue damage in kidney diseases. To investigate other potential beneficial nephroprotective effects of SS-31, in vivo murine models of acute tubular injury and glomerular damage were developed. Reduction of acute kidney injury was demonstrated in mice treated with SS-31. The expression of mRNAs involved in acute inflammatory and oxidative stress responses in the diseased kidneys confirmed that SS-31 could regulate these pathways in our in vivo models. Furthermore, ex vivo histoenzymography of mouse kidneys showed that aminopeptidase A (APA), the enzyme involved in the processing of angiotensin (Ang) II to Ang III, was induced in the diseased kidneys, and its activity was inhibited by SS-31. As the renin-angiotensin system (RAS) is a main regulator of kidney functions, the modulation of Ang receptors (ATR) and APA by SS-31 was further investigated using mRNAs extracted from diseased kidneys. Following acute tubular and/or glomerular damage, the expression of the AT1R mRNA was upregulated, which could be selectively downregulated upon SS-31 administration to the animals. At the same time, SS-31 was able to increase the expression of the AT2R, which may contribute to limit renal damage. Consequently, SS-31-based prodrugs were developed as substrates and/or inhibitors for APA and were screened using cells expressing high levels of APA, showing its selective regulation by α-Glu-SS-31. Thus, a link between SS-31 and the RAS opens new therapeutic implications for SS-31 in kidney diseases.

Immunogenicity, Inflammation, and Lipid Accumulation in Cynomolgus Monkeys Infused with a Lipidated Tetranectin-ApoA-I Fusion Protein.

High density lipoprotein (HDL)-targeted therapies, which promote cholesterol efflux from cells, are currently in development for reducing cardiovascular events in acute coronary syndrome. Human apolipoprotein A-I (apoA-I), the major HDL protein, was fused to the trimerization domain of tetranectin (TN) and complexed with phospholipids to generate a HDL mimetic (lipidated TN-ApoA-I) with reduced renal clearance and enhanced efficacy. Cynomolgus monkeys received 24-h intravenous infusions of control, 100 mg/kg or 400 mg/kg lipidated TN-ApoA-I every 4 days for 3 weeks, followed by a 6-week recovery period. After multiple infusions of lipidated TN-ApoA-I, clinical condition deteriorated and was accompanied by changes indicative of a progressive inflammatory response; increased levels of cytokines, C-reactive protein and vascular/perivascular infiltrates in multiple tissues. Rapid formation of antidrug antibodies occurred in all animals receiving lipidated TN-ApoA-I. Enhanced drug clearance corresponding to a relative lack of high molecular weight immune complexes in blood, suggestive of preferred removal/clearance, was observed in some animals. Expected dose-dependent increases in serum lipids were accompanied by vacuolated monocytes/macrophages in multiple organs, which in the glomeruli were shown to be CD68-positive, contain lipid and co-localized with granular IgG deposits. Lipid accumulation may have been a direct result of a high drug load, possibly enhanced by immune complex formation, inflammation, and altered lipid metabolism. Noteworthy was the inter- individual inconsistency in the severity of clinical and histopathologic findings, drug clearance and inflammatory markers. In conclusion, multiple infusions of lipidated TN-ApoA-I resulted in high immunogenicity, lipid accumulation and were not well tolerated in nonhuman primates.

Effect of compounds affecting ABCA1 expression and CETP activity on the HDL pathway involved in intestinal absorption of lutein and zeaxanthin.

The antioxidant xanthophylls lutein and zeaxanthin are absorbed from the diet in a process involving lipoprotein formation. Selective mechanisms exist for their intestinal uptake and tissue-selective distribution, but these are poorly understood. We investigated the role of high-density lipoprotein (HDL), apolipoprotein (apo) A1 and ATP-binding cassette transporter (ABC) A1 in intestinal uptake of lutein in a human polarized intestinal cell culture and a hamster model. Animals received dietary lutein and zeaxanthin and either a liver X receptor (LXR) agonist or statin, which up- or down-regulate intestinal ABCA1 expression, respectively. The role of HDL was studied following treatment with the cholesteryl ester transfer protein (CETP) modulator dalcetrapib or the CETP inhibitor anacetrapib. In vitro, intestinal ABCA1 at the basolateral surface of enterocytes transferred lutein and zeaxanthin to apoA1, not to mature HDL. In hamsters, plasma lutein and zeaxanthin levels were markedly increased with the LXR agonist and decreased with simvastatin. Dalcetrapib, but not anacetrapib, increased plasma and liver lutein and zeaxanthin levels. ABCA1 expression and apoA1 acceptor activity are important initial steps in intestinal uptake and maintenance of lutein and zeaxanthin levels by an HDL-dependent pathway. Their absorption may be improved by physiological and pharmacological interventions affecting HDL metabolism.

rHDL administration increases reverse cholesterol transport in mice, but is not additive on top of ezetimibe or cholestyramine treatment.

OBJECTIVE: Promoting reverse cholesterol transport (RCT) is a major atheroprotective property of HDL. The present study explored the effect of stimulating the first step of RCT (cholesterol efflux from macrophages) alone or in combination with stimulating the last step of RCT (fecal sterol excretion). METHODS AND RESULTS: Reconstituted HDL (rHDL) was injected into wild-type mice either with or without administration of the cholesterol absorption inhibitor ezetimibe or the bile acid sequestrant cholestyramine. Single dose administration of rHDL (100 mg apoA-I/kg) resulted in an early (4 h) increase in plasma free cholesterol levels (p < 0.001), without affecting hepatic cholesterol levels or fecal mass sterol excretion. rHDL injection also increased [(3)H]cholesterol appearance in plasma at an early time-point (4 h) after intraperitoneal administration of [(3)H]cholesterol-labeled mouse macrophage foam cells and fecal radioactivity excretion indicating completed RCT was increased by 26% (p < 0.05). Ezetimibe treatment inhibited intestinal cholesterol absorption by 74% (p < 0.01), but also the bile acid sequestrant cholestyramine decreased cholesterol absorption significantly (24%, p < 0.01). Consequently, ezetimibe increased RCT 2.1-fold (p < 0.001) primarily within fecal neutral sterols, while cholestyramine increased RCT by 3.6-fold (p < 0.001), primarily within bile acids (p < 0.001), but also within neutral sterols (p < 0.001). However, no additive effects of both intestinal sterol uptake inhibitors were observed on top of rHDL administration. CONCLUSION: These data demonstrate that increasing the first step of RCT by rHDL administration results in transient cholesterol mobilization from macrophages to plasma. This effect is not further enhanced by stimulating the last step of RCT, fecal sterol excretion.

Hydrophilic interaction chromatography of intact, soluble proteins.

The separation of intact proteins by means of Hydrophilic Interaction Chromatography (HILIC) was demonstrated with human apoA-I, recombinant human apoM, and equine cytochrome C. Five different commercially available HILIC columns were compared. Using one of these columns, different glycosylated isoforms of apoM were separated from each other and from the aglyco-form.