Mark Warnock

Enzymes and Receptors of Prostaglandin Pathways with Arachidonic Acid-derived Versus Eicosapentaenoic Acid-derived Substrates and Products

Dietary fish oil containing ω3 highly unsaturated fatty acids has cardioprotective and anti-inflammatory effects. Prostaglandins (PGs) and thromboxanes are produced in vivo both from the ω6 fatty acid arachidonic acid (AA) and the ω3 fatty acid eicosapentaenoic acid (EPA). Certain beneficial effects of fish oil may result from altered PG metabolism resulting from increases in the EPA/AA ratios of precursor phospholipids. Here we report in vitro specificities of prostanoid enzymes and receptors toward EPA-derived, 3-series versus AA-derived, 2-series prostanoid substrates and products. The largest difference was seen with PG endoperoxide H synthase (PGHS)-1. Under optimal conditions purified PGHS-1 oxygenates EPA with only 10% of the efficiency of AA, and EPA significantly inhibits AA oxygenation by PGHS-1. Two- to 3-fold higher activities or potencies with 2-series versus 3-series compounds were observed with PGHS-2, PGD synthases, microsomal PGE synthase-1 and EP1, EP2, EP3, and FP receptors. Our most surprising observation was that AA oxygenation by PGHS-2 is only modestly inhibited by EPA (i.e. PGHS-2 exhibits a marked preference for AA when EPA and AA are tested together). Also unexpectedly, TxA3 is about equipotent to TxA2 at the TPα receptor. Our biochemical data predict that increasing phospholipid EPA/AA ratios in cells would dampen prostanoid signaling with the largest effects being on PGHS-1 pathways involving PGD, PGE, and PGF. Production of 2-series prostanoids from AA by PGHS-2 would be expected to decrease in proportion to the compensatory decrease in the AA content of phospholipids that would result from increased incorporation of ω3 fatty acids such as EPA.

Visceral Adipose Tissue Inflammation Accelerates Atherosclerosis in Apolipoprotein E–Deficient Mice

Background— Fat inflammation may play an important role in comorbidities associated with obesity such as atherosclerosis. Methods and Results— To first establish feasibility of fat transplantation, epididymal fat pads were harvested from wild-type C57BL/6J mice and transplanted into leptin-deficient (Lepob/ob) mice. Fat transplantation produced physiological leptin levels and prevented obesity and infertility in Lepob/ob mice. However, the transplanted fat depots were associated with chronically increased macrophage infiltration with characteristics identical to those observed in fat harvested from obese animals. The inflammation in transplanted adipose depots was regulated by the same factors that have been implicated in endogenous fat inflammation such as monocyte chemoattractant protein-1. To determine whether this inflamed adipose depot could affect vascular disease in mice, epididymal fat depots were transplanted into atherosclerosis-prone apolipoprotein E-deficient ApoE−/− mice. Plasma from ApoE−/− mice receiving fat transplants contained increased leptin, resistin, and monocyte chemoattractant protein-1 compared with plasma from sham-operated ApoE−/− mice. Furthermore, mice transplanted with visceral fat developed significantly more atherosclerosis compared with sham-operated animals, whereas transplants with subcutaneous fat did not affect atherosclerosis despite a similar degree of fat inflammation. Treatment of transplanted ApoE−/− mice with pioglitazone decreased macrophage content of the transplanted visceral fat pad and reduced plasma monocyte chemoattractant protein-1. Importantly, pioglitazone also reduced atherosclerosis triggered by inflammatory visceral fat but had no protective effect on atherosclerosis in the absence of the visceral fat transplantation. Conclusions— Our results indicate that visceral adipose-related inflammation accelerates atherosclerosis in mice. Drugs such as thiazolidinediones might be a useful strategy to specifically attenuate the vascular disease induced by visceral inflammatory fat.

Mechanism of inactivation of plasminogen activator inhibitor-1 by a small molecule inhibitor

The inactivation of plasminogen activator inhibitor-1 (PAI-1) by the small molecule PAI-1 inhibitor PAI-039 (tiplaxtinin) has been investigated using enzymatic analysis, direct binding studies, site-directed mutagenesis, and molecular modeling studies. Previously PAI-039 has been shown to exhibit in vivo activity in various animal models, but the mechanism of inhibition is unknown. PAI-039 bound specifically to the active conformation of PAI-1 and exhibited reversible inactivation of PAI-1 in vitro. SDS-PAGE indicated that PAI-039 inactivated PAI-1 predominantly through induction of PAI-1 substrate behavior. Preincubation of PAI-1 with vitronectin, but not bovine serum albumin, blocked PAI-039 activity while analysis of the reciprocal experiment demonstrated that preincubation of PAI-1 with PAI-039 blocked the binding of PAI-1 to vitronectin. Together, these data suggest that the site of interaction of the drug on PAI-1 is inaccessible when PAI-1 is bound to vitronectin and may overlap with the PAI-1 vitronectin binding domain. This was confirmed by site-directed mutagenesis and molecular modeling studies, which suggest that the binding epitope for PAI-039 is localized adjacent to the previously identified interaction site for vitronectin. Thus, these studies provide a detailed characterization of the mechanism of inhibition of PAI-1 by PAI-039 against free, but not vitronectin-bound PAI-1, suggesting for the first time a novel pool of PAI-1 exists that is vulnerable to inhibition by inactivators that bind at the vitronectin binding site.

Characterization of a Novel Class of Polyphenolic Inhibitors of Plasminogen Activator Inhibitor-1

Plasminogen activator inhibitor type 1, (PAI-1) the primary inhibitor of the tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, has been implicated in a wide range of pathological processes, making it an attractive target for pharmacologic inhibition. Currently available small-molecule inhibitors of PAI-1 bind with relatively low affinity and do not inactivate PAI-1 in the presence of its cofactor, vitronectin. To search for novel PAI-1 inhibitors with improved potencies and new mechanisms of action, we screened a library selected to provide a range of biological activities and structural diversity. Five potential PAI-1 inhibitors were identified, and all were polyphenolic compounds including two related, naturally occurring plant polyphenols that were structurally similar to compounds previously shown to provide cardiovascular benefit in vivo. Unique second generation compounds were synthesized and characterized, and several showed IC50 values for PAI-1 between 10 and 200 nm. This represents an enhanced potency of 10–1000-fold over previously reported PAI-1 inactivators. Inhibition of PAI-1 by these compounds was reversible, and their primary mechanism of action was to block the initial association of PAI-1 with a protease. Consistent with this mechanism and in contrast to previously described PAI-1 inactivators, these compounds inactivate PAI-1 in the presence of vitronectin. Two of the compounds showed efficacy in ex vivo plasma and one blocked PAI-1 activity in vivo in mice. These data describe a novel family of high affinity PAI-1-inactivating compounds with improved characteristics and in vivo efficacy, and suggest that the known cardiovascular benefits of dietary polyphenols may derive in part from their inactivation of PAI-1.

The vitronectin-binding function of PAI-1 exacerbates lung fibrosis in mice

Plasminogen activator inhibitor-1 (PAI-1) is increased in the lungs of patients with pulmonary fibrosis, and animal studies have shown that experimental manipulations of PAI-1 levels directly influence the extent of scarring that follows lung injury. PAI-1 has 2 known properties that could potentiate fibrosis, namely an antiprotease activity that inhibits the generation of plasmin, and a vitronectin-binding function that interferes with cell adhesion to this extracellular matrix protein. To determine the relative importance of each PAI-1 function in lung fibrogenesis, we administered mutant PAI-1 proteins that possessed either intact antiprotease or vitronectin-binding activity to bleomycin-injured mice genetically deficient in PAI-1. We found that the vitronectin-binding capacity of PAI-1 was the primary determinant required for its ability to exacerbate lung scarring induced by intratracheal bleomycin administration. The critical role of the vitronectin-binding function of PAI-1 in fibrosis was confirmed in the bleomycin model using mice genetically modified to express the mutant PAI-1 proteins. We conclude that the vitronectin-binding function of PAI-1 is necessary and sufficient in its ability to exacerbate fibrotic processes in the lung.

Imatinib treatment reduces brain injury in a murine model of traumatic brain injury

Current therapies for Traumatic brain injury (TBI) focus on stabilizing individuals and on preventing further damage from the secondary consequences of TBI. A major complication of TBI is cerebral edema, which can be caused by the loss of blood brain barrier (BBB) integrity. Recent studies in several CNS pathologies have shown that activation of latent platelet derived growth factor-CC (PDGF-CC) within the brain can promote BBB permeability through PDGF receptor α (PDGFRα) signaling, and that blocking this pathway improves outcomes. In this study we examine the efficacy for the treatment of TBI of an FDA approved antagonist of the PDGFRα, Imatinib. Using a murine model we show that Imatinib treatment, begun 45 min after TBI and given twice daily for 5 days, significantly reduces BBB dysfunction. This is associated with significantly reduced lesion size 24 h, 7 days, and 21 days after TBI, reduced cerebral edema, determined from apparent diffusion co-efficient (ADC) measurements, and with the preservation of cognitive function. Finally, analysis of cerebrospinal fluid (CSF) from human TBI patients suggests a possible correlation between high PDGF-CC levels and increased injury severity. Thus, our data suggests a novel strategy for the treatment of TBI with an existing FDA approved antagonist of the PDGFRα.

Thrombostatin FM compounds: direct thrombin inhibitors - mechanism of action in vitro and in vivo.

Summary.  Background: Novel pentapeptides called Thrombostatin FM compounds consisting mostly of D‐isomers and unusual amino acids were prepared based upon the stable angiotensin converting enzyme breakdown product of bradykinin – RPPGF. Methods and Results: These peptides are direct thrombin inhibitors prolonging the thrombin clotting time, activated partial thromboplastin time, and prothrombin time at ≥0.78, 1.6, and 1.6 μm, respectively. They competitively inhibit α‐thrombin‐induced cleavage of a chromogenic substrate at 4.4–8.2 μm. They do not significantly inhibit plasma kallikrein, factor (F) XIIa, FXIa, FIXa, FVIIa‐TF, FXa, plasmin or cathepsin G. One form, FM19 [rOicPaF(p‐Me)], blocks α‐thrombin‐induced calcium flux in fibroblasts with an IC50 of 6.9 ± 1.2 μm. FM19 achieved 100% inhibition of threshold α‐ or γ‐thrombin‐induced platelet aggregation at 8.4 ± 4.7 μm and 16 ± 4 μm, respectively. The crystal structure of thrombin in complex with FM19 shows that the N‐terminal D‐Arg retrobinds into the S1 pocket, its second residue Oic interacts with His‐57, Tyr‐60a and Trp‐60d, and its C‐terminal p‐methyl Phe engages thrombin’s aryl binding site composed of Ile‐174, Trp‐215, and Leu‐99. When administered intraperitoneal, intraduodenal, or orally to mice, FM19 prolongs thrombin clotting times and delays carotid artery thrombosis. Conclusion: FM19, a low affinity reversible direct thrombin inhibitor, might be useful as an add‐on agent to address an unmet need in platelet inhibition in acute coronary syndromes in diabetics and others who with all current antiplatelet therapy still have reactive platelets.

Mapping the interaction of bradykinin 1-5 with the exodomain of human protease activated receptor 4

The angiotensin converting enzyme breakdown product of bradykinin, bradykinin 1–5 (RPPGF), inhibits thrombin‐induced human or mouse platelet aggregation. RPPGF binds to the exodomain of human protease‐activated receptor 1 (PAR1). Studies determined if RPPGF also binds to the exodomain of human PAR4. RPPGF binds to a peptide of the thrombin cleavage site on PAR4. Recombinant wild‐type and mutated exodomain of human PAR4 was prepared. The N‐terminal arginine on RPPGF binds to the P2 position or proline46 on PAR4 to block thrombin cleavage. These data indicate that RPPGF influences thrombin activity by binding to the thrombin cleavage site on both PAR4 and PAR1.

Synthesis of novel peptide inhibitors of thrombin-induced platelet activation

Inhibitors of the activation of platelet aggregation have promise as important therapeutic agents for the management of acute coronary syndrome (ACS). Platelet activation by thrombin, a serine protease, occurs by binding to and cleavage of the extracellular N‐terminal domains of protease‐activated receptors 1 and 4 (PAR1 and PAR4). The proteolysis of the PARs exposes new tethered ligands that then signal through transmembrane domains to initiate platelet activation as a downstream effect. A pentapeptide cleavage product of bradykinin with the sequence Arg‐Pro‐Pro‐Gly‐Phe serves as a thrombin inhibitor by blocking α‐ and γ‐thrombin‐induced platelet aggregation. Analogs of RPPGF have been prepared that result in improved inhibition of thrombin activation of platelets. Specific amino acid residues required for activity against platelet aggregation have been identified, and a lead compound, rOicPaPhe(p‐Me)‐NH2 (FM19), has been developed. FM19, which completely inhibits threshold γ‐thrombin‐induced platelet aggregation at a concentration of 16 ± 4 μm, represents an important lead compound in the development of inhibitors of thrombin‐mediated platelet aggregation for treatment of ACS.

Taming Neonatal Hypoxic–Ischemic Brain Injury by Intranasal Delivery of Plasminogen Activator Inhibitor-1

Background and Purpose— Plasminogen activator inhibitor-I (PAI-1), a ≈50-kDa serine protease inhibitor, markedly reduces the extravascular toxicity of tissue-type plasminogen activator in experimental hypoxic–ischemic (HI) brain injury of newborns. However, the current treatment with PAI-1 requires intracerebroventricle injection to cross the blood–brain barrier, which is an invasive procedure of limited clinical potential. Thus, we tested whether intranasal administration of PAI-1 can bypass blood–brain barrier and mitigate neonatal HI brain injury. Methods— Rat pups were subjected to HI, with or without lipopolysaccharide pre-exposure, followed by intranasal delivery of a stable-mutant form of PAI-1 (CPAI). Results— Immunoblotting showed that CPAI sequentially entered the olfactory bulbs and cerebral cortex after intranasal delivery and reduced ≈75% of brain atrophy in HI or lipopolysaccharide-sensitized HI injury. Mechanistically, CPAI attenuated HI-induced plasminogen activators and lipopolysaccharide/HI-induced nuclear factor-&kgr;B signaling, neuroinflammation, and blood–brain barrier permeability. Conclusions— Intranasal delivery of CPAI is an effective treatment of experimental HI brain injury of newborns. Clinical application of this experimental therapy merits further investigation.