Coronary intraplaque hemorrhage evokes a novel atheroprotective macrophage phenotype.

Intraplaque hemorrhage accelerates atherosclerosis via oxidant stress and contributes to lesion development and destabilization. Normally, macrophages scavenge hemoglobin-haptoglobin (HbHp) complexes via CD163, and this process provokes the secretion of the anti-inflammatory atheroprotective cytokine interleukin (IL)-10. We therefore tested the hypothesis that HbHp complexes may drive monocyte differentiation to an atheroprotective phenotype. Examination of the macrophage phenotype in hemorrhaged atherosclerotic plaques revealed a novel hemorrhage-associated macrophage population (HA-mac), defined by high levels of CD163, but low levels of human leukocyte antigen-DR. HA-mac contained more iron, a pro-oxidant catalyst, but paradoxically had less oxidative injury, measured by 8-oxo-guanosine content. Differentiating monocytes with HbHp complexes reproduced the CD163(high) human leukocyte antigen-DR(low) HA-mac phenotype in vitro. These in vitro HA-mac cells cleared Hb more quickly, and consistently showed less hydrogen peroxide release, highly reactive oxygen species and oxidant stress, and increased survival. Differentiation to HA-mac was prevented by neutralizing IL-10 antibodies, indicating that IL-10 mediates an autocrine feedback mechanism in this system. Nonlinear dynamic modeling showed that an IL-10/CD163-positive feedback loop drove a discrete HA-mac lineage. Simulations further indicated an all-or-none switch to HA-mac at threshold levels of HbHp, and this conversion was experimentally verified. These data demonstrate the creation of a novel atheroprotective (HA-mac) macrophage subpopulation in response to intraplaque hemorrhage and raise the possibility that therapeutically reproducing this macrophage phenotype may be cardio-protective in cases of atherosclerosis.

Effect of aprotinin and recombinant variants on platelet protease-activated receptor 1 activation.

BACKGROUND: Thrombin generated during cardiopulmonary bypass activates the high-affinity thrombin receptor, protease-activated receptor 1 (PAR1), causing platelet dysfunction and excessive bleeding. The serine protease inhibitor aprotinin protects platelets against thrombin-mediated PAR1 activation in vitro and in vivo. Here we have investigated three novel recombinant aprotinin variants with specific modifications to the active site lysine at amino acid position 15 (arginine-15, arginine-15-alanine-17, and valine-15-leucine-17) for their effect on PAR1-mediated platelet aggregation in vitro. METHODS: Aggregation studies were carried out using washed human platelets (n = 9) or platelet rich plasma (n = 7) from healthy volunteers activated with 1 or 5 nM thrombin. Recombinant aprotinin variants were used at the molar equivalent to 50 KIU/mL of the parent compound. The PAR1-specific antagonist peptide, FLLRN, was used at 500 microM. RESULTS: Platelet aggregation at low concentrations of thrombin (1 nM) was mediated exclusively through PAR1, as shown by inhibition of aggregation in the presence of FLLRN. At 1 nM thrombin, the mean percentage +/- SD aggregation of washed platelets was 68.6% +/- 12.3%. This was suppressed by each aprotinin variant at the 50 KIU/mL equivalent dose: arginine-15 (23.0% +/- 17.5%, p < 0.001); arginine-15-alanine-17 (33.3% +/- 22.9%, p < 0.01); aprotinin (37.5% +/- 19.4%, p < 0.05); valine-15-leucine-17 (50.0% +/- 16.1%, not significant)). At 5 nM thrombin, which activates both high (PAR1) and low-affinity (PAR4) thrombin receptors on platelets, FLLRN and aprotinin failed to block aggregation: this finding indicates that aprotinin selectively targeted PAR1. In platelet-rich plasma, aggregation at 1 nM thrombin was 77.1% +/- 10.0%, and this was inhibited in the following order: arginine-15 (30.1% +/- 9.6%, p < 0.001); arginine-15-alanine-17 (52.3% +/- 9.7%, p > 0.001); aprotinin (55.9% +/- 6.2%, p > 0.001); valine-15-leucine-17 (73.7% +/- 7.1%, not significant). CONCLUSIONS: Aprotinin variants differentially inhibit PAR1-mediated platelet aggregation. With more understanding of the mechanisms of action of aprotinin and its derivatives, safer and more efficacious aprotinin variants may become available for clinical use.

Proinflammatory activation of macrophages by basic calcium phosphate crystals via protein kinase C and MAP kinase pathways: a vicious cycle of inflammation and arterial calcification?

Basic calcium phosphate (BCP) crystal deposition underlies the development of arterial calcification. Inflammatory macrophages colocalize with BCP deposits in developing atherosclerotic lesions and in vitro can promote calcification through the release of TNF alpha. Here we have investigated whether BCP crystals can elicit a proinflammatory response from monocyte-macrophages. BCP microcrystals were internalized into vacuoles of human monocyte-derived macrophages in vitro. This was associated with secretion of proinflammatory cytokines (TNFalpha, IL-1beta and IL-8) capable of activating cultured endothelial cells and promoting capture of flowing leukocytes under shear flow. Critical roles for PKC, ERK1/2, JNK, but not p38 intracellular signaling pathways were identified in the secretion of TNF alpha, with activation of ERK1/2 but not JNK being dependent on upstream activation of PKC. Using confocal microscopy and adenoviral transfection approaches, we determined a specific role for the PKC-alpha isozyme. The response of macrophages to BCP crystals suggests that pathological calcification is not merely a passive consequence of chronic inflammatory disease but may lead to a positive feed-back loop of calcification and inflammation driving disease progression.