Vinit Karmali

Hemoglobin Directs Macrophage Differentiation and Prevents Foam Cell Formation in Human Atherosclerotic Plaques

OBJECTIVES The purpose of this study was to examine selective macrophage differentiation occurring in areas of intraplaque hemorrhage in human atherosclerosis. BACKGROUND Macrophage subsets are recognized in atherosclerosis, but the stimulus for and importance of differentiation programs remain unknown. METHODS We used freshly isolated human monocytes, a rabbit model, and human atherosclerotic plaques to analyze macrophage differentiation in response to hemorrhage. RESULTS Macrophages characterized by high expression of both mannose and CD163 receptors preferentially exist in atherosclerotic lesions at sites of intraplaque hemorrhage. These hemoglobin (Hb)-stimulated macrophages, M(Hb), are devoid of neutral lipids typical of foam cells. In vivo modeling of hemorrhage in the rabbit model demonstrated that sponges exposed to red cells showed an increase in mannose receptor-positive macrophages only when these cells contained Hb. Cultured human monocytes exposed to Hb:haptoglobin complexes, but not interleukin-4, expressed the M(Hb) phenotype and were characterized by their resistance to cholesterol loading and up-regulation of ATP-binding cassette (ABC) transporters. M(Hb) demonstrated increased ferroportin expression, reduced intracellular iron, and reactive oxygen species (ROS). Degradation of ferroportin using hepcidin increased ROS and inhibited ABCA1 expression and cholesterol efflux to apolipoprotein A-I, suggesting reduced ROS triggers these effects. Knockdown of liver X receptor alpha (LXRα) inhibited ABC transporter expression in M(Hb) and macrophages differentiated in the antioxidant superoxide dismutase. Last, LXRα luciferase reporter activity was increased in M(Hb) and significantly reduced by overnight treatment with hepcidin. Collectively, these data suggest that reduced ROS triggers LXRα activation and macrophage reverse cholesterol transport. CONCLUSIONS Hb is a stimulus for macrophage differentiation in human atherosclerotic plaques. A decrease in macrophage intracellular iron plays an important role in this nonfoam cell phenotype by reducing ROS, which drives transcription of ABC transporters through activation of LXRα. Reduction of macrophage intracellular iron may be a promising avenue to increase macrophage reverse cholesterol transport.

Pharmacological Suppression of Hepcidin Increases Macrophage Cholesterol Efflux and Reduces Foam Cell Formation and Atherosclerosis

Objective—We recently reported that lowering of macrophage free intracellular iron increases expression of cholesterol efflux transporters ABCA1 and ABCG1 by reducing generation of reactive oxygen species. In this study, we explored whether reducing macrophage intracellular iron levels via pharmacological suppression of hepcidin can increase macrophage-specific expression of cholesterol efflux transporters and reduce atherosclerosis. Methods and Results—To suppress hepcidin, increase expression of the iron exporter ferroportin, and reduce macrophage intracellular iron, we used a small molecule inhibitor of bone morphogenetic protein (BMP) signaling, LDN 193189 (LDN). LDN (10 mg/kg IP b.i.d.) was administered to mice, and its effects on atherosclerosis, intracellular iron, oxidative stress, lipid efflux, and foam cell formation were measured in plaques and peritoneal macrophages. Long-term LDN administration to apolipoprotein E−/− mice increased ABCA1 immunoreactivity within intraplaque macrophages by 3.7-fold (n=8; P=0.03), reduced Oil Red O–positive lipid area by 50% (n=8; P=0.02), and decreased total plaque area by 43% (n=8; P=0.001). LDN suppressed liver hepcidin transcription and increased macrophage ferroportin, lowering intracellular iron and hydrogen peroxide production. LDN treatment increased macrophage ABCA1 and ABCG1 expression, significantly raised cholesterol efflux to ApoA-1, and decreased foam cell formation. All preceding LDN-induced effects on cholesterol efflux were reversed by exogenous hepcidin administration, suggesting modulation of intracellular iron levels within macrophages as the mechanism by which LDN triggers these effects. Conclusion—These data suggest that pharmacological manipulation of iron homeostasis may be a promising target to increase macrophage reverse cholesterol transport and limit atherosclerosis.

CD163 interacts with TWEAK to regulate tissue regeneration after ischaemic injury.

Macrophages are an essential component of the immune response to ischaemic injury and play an important role in promoting inflammation and its resolution, which is necessary for tissue repair. The type I transmembrane glycoprotein CD163 is exclusively expressed on macrophages, where it acts as a receptor for haemoglobin:haptoglobin complexes. An extracellular portion of CD163 circulates in the blood as a soluble protein, for which no physiological function has so far been described. Here we show that during ischaemia, soluble CD163 functions as a decoy receptor for TWEAK, a secreted pro-inflammatory cytokine of the tumour necrosis factor family, to regulate TWEAK-induced activation of canonical nuclear factor-κB (NF-κB) and Notch signalling necessary for myogenic progenitor cell proliferation. Mice with deletion of CD163 have transiently elevated levels of TWEAK, which stimulate muscle satellite cell proliferation and tissue regeneration in their ischaemic and non-ischaemic limbs. These results reveal a role for soluble CD163 in regulating muscle regeneration after ischaemic injury.

Metformin impairs vascular endothelial recovery after stent placement in the setting of locally eluted mammalian target of rapamycin inhibitors via S6 kinase-dependent inhibition of cell proliferation.

OBJECTIVES This study sought to examine the effect of oral metformin (Mf) therapy on endothelialization in the setting of drug-eluting stents (DES). BACKGROUND Mf is a commonly used therapy in diabetic patients receiving DES. Mf and locally eluted mammalian target of rapamycin (mTOR) inhibitors used in DES have convergent molecular signaling; however, the impact of this drug interaction on stent endothelialization is unknown. METHODS We examined human endothelial aortic cells (HAECs) and a rabbit model of stenting to determine points on molecular convergence between these 2 agents and their impact on stent endothelialization. RESULTS Western blotting of HAECs treated with Mf and the mTOR inhibitor sirolimus and 14-day rabbit iliacs treated with the combination of zotarolimus-eluting stents (ZES) and oral Mf demonstrated greater inhibition of S6 kinase (S6K), a downstream effector of mTOR complex 1, than either treatment alone. HAEC proliferation was significantly inhibited by Mf or sirolimus treatments alone and further reduced when they were combined. Knockdown of S6K via short interfering RNA in HAECs impaired cell proliferation via a cyclin D1-dependent mechanism, whereas its overexpression rescued the antiproliferative effects of both agents. Last, endothelialization and endothelial cell proliferation at 14 days were assessed in rabbits receiving ZES or bare-metal stents and Mf or placebo by scanning electron microscopy and bromodeoxyuridine/CD31 labeling, respectively. Both endpoints were inhibited by ZES treatment alone and were further reduced by the combination of Mf and ZES. CONCLUSIONS Significant convergence of signaling occurs between Mf and locally delivered mTOR inhibitors at S6K. This further impairs endothelial recovery/proliferation via an S6K-dependent mechanism. Patients receiving Mf in combination with stents that elute mTOR inhibitors are potentially at increased risk of delayed endothelial healing and stent thrombosis.

Differential Healing After Sirolimus, Paclitaxel, and Bare Metal Stent Placement in Combination With Peroxisome Proliferator-Activator Receptor γ Agonists. Requirement for mTOR/Akt2 in PPARγ Activation

Rationale: Sirolimus-eluting coronary stents (SESs) and paclitaxel-eluting coronary stents (PESs) are used to reduce restenosis but have different sites of action. The molecular targets of sirolimus overlap with those of the peroxisome proliferator-activated receptor (PPAR)&ggr; agonist rosiglitazone (RSG) but the consequence of this interaction on endothelialization is unknown. Objective: Using the New Zealand white rabbit iliac model of stenting, we examined the effects of RSG on SESs, PESs, and bare metal stents endothelialization. Methods and Results: Animals receiving SESs, PESs, or bare metal stents and either RSG (3 mg/kg per day) or placebo were euthanized at 28 days, and arteries were evaluated by scanning electron microscopy. Fourteen-day organ culture and Western blotting of iliac arteries and tissue culture experiments were conducted. Endothelialization was significantly reduced by RSG in SESs but not in PESs or bare metal stents. Organ culture revealed reduced vascular endothelial growth factor in SESs receiving RSG compared to RSG animals receiving bare metal stent or PESs. Quantitative polymerase chain reaction in human aortic endothelial cells (HAECs) revealed that sirolimus (but not paclitaxel) inhibited RSG-induced vascular endothelial growth factor transcription. Western blotting demonstrated that inhibition of molecular signaling in SES+RSG–treated arteries was similar to findings in HAECs treated with RSG and small interfering RNA to PPAR&ggr;, suggesting that sirolimus inhibits PPAR&ggr;. Transfection of HAECs with mTOR (mammalian target of rapamycin) short hairpin RNA and with Akt2 small interfering RNA significantly inhibited RSG-mediated transcriptional upregulation of heme oxygenase-1, a PPAR&ggr; target gene. Chromatin immunoprecipitation assay demonstrated sirolimus interferes with binding of PPAR&ggr; to its response elements in heme oxygenase-1 promoter. Conclusions: mTOR/Akt2 is required for optimal PPAR&ggr; activation. Patients who receive SESs during concomitant RSG treatment may be at risk for delayed stent healing.

Sirolimus-FKBP12.6 Impairs Endothelial Barrier Function Through Protein Kinase C-α Activation and Disruption of the p120–Vascular Endothelial Cadherin Interaction

Objective—Sirolimus (SRL) is an immunosuppressant drug used to prevent rejection in organ transplantation and neointimal hyperplasia when delivered from drug-eluting stents. Major side effects of SRL include edema and local collection of intimal lipid deposits at drug-eluting stent sites, suggesting that SRL impairs endothelial barrier function (EBF). The aim of this study was to address the role of SRL on impaired EBF and the potential mechanisms involved. Approach and Results—Cultured human aortic endothelial cells (HAECs) and intact human and mouse endothelium was examined to determine the effect of SRL, which binds FKBP12.6 to inhibit the mammalian target of rapamycin, on EBF. EBF, measured by transendothelial electrical resistance, was impaired in HAECs when treated with SRL or small interfering RNA for FKBP12.6 and reversed when pretreated with ryanodine, a stabilizer of ryanodine receptor 2 intracellular calcium release channels. Intracellular calcium increased in HAECs treated with SRL and normalized with ryanodine pretreatment. SRL-treated HAECs demonstrated increases in protein kinase C-&agr; phosphorylation, a calcium sensitive serine/threonine kinase important in vascular endothelial (VE) cadherin barrier function through its interaction with p120-catenin (p120). Immunostaining of HAECs, human coronary and mouse aortic endothelium treated with SRL showed disruption of p120–VE cadherin interaction treated with SRL. SRL impairment of HAEC EBF was reduced with protein kinase C-&agr; small interfering RNA. Mice treated with SRL demonstrated increased vascular permeability by Evans blue albumin extravasation in the lungs, heart, and aorta. Conclusions—SRL-FKBP12.6 impairs EBF by activation of protein kinase C-&agr; and downstream disruption of the p120–VE cadherin interaction in vascular endothelium. These data suggest this mechanism may be an important contributor of SRL side effects related to impaired EBF.

Metformin impairs endothelialization after placement of Newer Generation Drug Eluting Stents

OBJECTIVES Metformin impairs endothelialization of drug eluting stents (DES) due to convergent signaling at the mammalian target of rapamycin (mTOR) pathway. We assessed whether metformin will continue to adversely affect stent endothelialization despite design improvements in newer generation DES. METHODS Rabbit iliac artery stenting with newer generation DES was performed followed by 14 days of either normal chow diet or one with metformin (100 mg/kg/day) added. Scanning electron microscopy was used to assess stent endothelialization after sacrifice. RESULTS In the metformin-treated group there was significantly less endothelialization compared to the placebo-treated group. Paclitaxel-eluting stents in placebo-treated group had the greatest degree of endothelialization with significantly less in its metformin-treated counterpart and all-limus eluting stent groups. CONCLUSIONS Metformin inhibited stent endothelialization in newer generation DES despite improvements in stent design. By impairing stent endothelialization, metformin may increase the risk for thrombotic complications after newer generation DES placement.

Hepcidin-ferroportin axis controls toll-like receptor 4 dependent macrophage inflammatory responses in human atherosclerotic plaques

OBJECTIVES Toll-like Receptor 4 (TLR4) is implicated in modulating inflammatory cytokines though its role in atherosclerosis remains uncertain. We have recently described a non-foam cell macrophage phenotype driven by ingestion of hemoglobin:haptoglobin complexes (HH), via the scavenger receptor CD163, characterized by reduced inflammatory cytokine production. In this study, we examined the role of iron metabolism in modulating TLR4 signaling in these cells. METHODS AND RESULTS Areas in human atherosclerotic plaque with non-foam cell, CD163 positive macrophages demonstrated reduced expression of tumor necrosis factor alpha (TNF-α) and interferon-beta (INF-β) compared to foam cells. Human macrophages differentiated in hemoglobin:haptoglobin (HH) complexes expressed the CD163 positive non-foam cell phenotype and demonstrated significantly less TNF-α and INF-β compared to control macrophages when exposed to oxidized LDL (oxLDL) or lipopolysaccharide (LPS). LPS stimulated expression of TNF-α and INF-β could be restored in HH macrophages by pretreatment with hepcidin, an endogenous suppressor of ferroportin1 (FPN), or by genetic suppression of FPN in macrophages derived from myeloid specific FPN knockout mice. LPS stimulated control macrophages demonstrated increase in TLR4 trafficking to lipid rafts; this response was suppressed in HH macrophages but was restored upon pretreatment with hepcidin. Using a pharmacologic hepcidin suppressor, we observed a decrease in cytokine expression and TLR4-lipid raft trafficking in LPS-stimulated in a murine macrophage model. CONCLUSION TLR4 dependent macrophage signaling is controlled via hepcidin-ferroportin1 axis by influencing TLR4-lipid raft interactions. Pharmacologic manipulation of iron metabolism may represent a promising approach to limiting TLR4-mediated inflammatory responses.

CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis

Intake of hemoglobin by the hemoglobin-haptoglobin receptor CD163 leads to a distinct alternative non–foam cell antiinflammatory macrophage phenotype that was previously considered atheroprotective. Here, we reveal an unexpected but important pathogenic role for these macrophages in atherosclerosis. Using human atherosclerotic samples, cultured cells, and a mouse model of advanced atherosclerosis, we investigated the role of intraplaque hemorrhage on macrophage function with respect to angiogenesis, vascular permeability, inflammation, and plaque progression. In human atherosclerotic lesions, CD163+ macrophages were associated with plaque progression, microvascularity, and a high level of HIF1&agr; and VEGF-A expression. We observed irregular vascular endothelial cadherin in intraplaque microvessels surrounded by CD163+ macrophages. Within these cells, activation of HIF1&agr; via inhibition of prolyl hydroxylases promoted VEGF-mediated increases in intraplaque angiogenesis, vascular permeability, and inflammatory cell recruitment. CD163+ macrophages increased intraplaque endothelial VCAM expression and plaque inflammation. Subjects with homozygous minor alleles of the SNP rs7136716 had elevated microvessel density, increased expression of CD163 in ruptured coronary plaques, and a higher risk of myocardial infarction and coronary heart disease in population cohorts. Thus, our findings highlight a nonlipid-driven mechanism by which alternative macrophages promote plaque angiogenesis, leakiness, inflammation, and progression via the CD163/HIF1&agr;/VEGF-A pathway.

Everolimus-Eluting Stents Improve Vascular Response in a Diabetic Animal Model

Background—Preclinical evaluation of the vascular response of drug-eluting stents is limited especially in the setting of diabetes mellitus preventing the evaluation of changes in drug-eluting stent design and eluted drugs after clinical use. Methods and Results—Cultured human aortic endothelial cells were used to assess the differences between sirolimus and its analog, everolimus, in the setting of hyperglycemia on various cellular functions necessary for endothelial recovery. A diabetic rabbit model of iliac artery stenting was used to compare histological and morphometric characteristics of the vascular response to everolimus-eluting, sirolimus-eluting, and bare metal stent placement. Under hyperglycemic conditions, sirolimus impaired human aortic endothelial cell barrier function, migration, and proliferation to a greater degree compared with everolimus. In our in vivo model of diabetes mellitus, endothelialization at 28 days was significantly lower and endothelial integrity was impaired in sirolimus-eluting stent compared with both everolimus-eluting and bare metal stents. Neointimal area, uncovered struts, and fibrin deposition were significantly higher in sirolimus-eluting compared with everolimus-eluting and bare metal stents. Conclusions—Use of everolimus-eluting stent results in improved vascular response in our preclinical models of diabetes mellitus.