Sakine Simsekyilmaz

Neutrophil-Derived Cathelicidin Protects from Neointimal Hyperplasia

Cathelicidin-coated stents limit neointima formation. A Narrow Escape for Stents A heart-healthy diet may reduce your risk of atherosclerosis and heart attack, but what if the damage is already done? For severe cases of atherosclerosis, a cardiologist can implant a device called a stent to widen or even open a blocked artery. Ironically, however, stent implantation can activate an immune response, which leads to restenosis—a narrowing of the blood vessels that restricts flow. Stents that elute drugs such as antiproliferative or anti-inflammatory agents have decreased this risk, but at the same time, they increase the risk of blood clot formation. Now, Soehnlein et al. find that stents coated with an antimicrobial peptide produced by innate immune cells promote vascular healing. In an animal model of atherosclerosis, the authors observed that neutrophils could decrease vessel narrowing by secreting the antimicrobial peptide cathelicidin (mouse CRAMP, human LL-37). When deposited by neutrophils at the site of vascular injury, CRAMP recruited new blood vessel–producing cells and promoted the regrowth of vascular endothelial cells in damaged regions. The authors then translated this observation to clinically applicable technology by coating vascular stents with cathelicidin. Indeed, cathelicidin-coated stents reduced in-stent restenosis in their mouse model of atherosclerosis. Although it remains to be seen whether this effect will be reproduced in humans, cathelicidin coating may prevent stents from causing the very problem they’re supposed to treat and thus improve therapy for severe atherosclerosis. Percutaneous transluminal angioplasty with stent implantation is used to dilate arteries narrowed by atherosclerotic plaques and to revascularize coronary arteries occluded by atherothrombosis in myocardial infarction. Commonly applied drug-eluting stents release antiproliferative or anti-inflammatory agents to reduce the incidence of in-stent stenosis. However, these stents may still lead to in-stent stenosis; they also show increased rates of late stent thrombosis, an obstacle to optimal revascularization possibly related to endothelial recovery. Here, we examined the contribution of neutrophils and neutrophilic granule proteins to arterial healing after injury. We found that neutrophil-borne cathelicidin (mouse CRAMP, human LL-37) promoted reendothelization and thereby limited neointima formation after stent implantation. We then translated these findings to an animal model using a neutrophil-instructing, biofunctionalized, miniaturized Nitinol stent coated with LL-37. This stent reduced in-stent stenosis in a mouse model of atherosclerosis, suggesting that LL-37 may promote vascular healing after interventional therapy.

Differential roles of angiogenic chemokines in endothelial progenitor cell-induced angiogenesis

This study aimed to analyze the role of endothelial progenitor cell (EPC)-derived angiogenic factors and chemokines in the multistep process driving angiogenesis with a focus on the recently discovered macrophage migration inhibitory factor (MIF)/chemokine receptor axis. Primary murine and murine embryonic EPCs (eEPCs) were analyzed for the expression of angiogenic/chemokines and components of the MIF/CXC chemokine receptor axis, focusing on the influence of hypoxic versus normoxic stimulation. Hypoxia induced an upregulation of CXCR2 and CXCR4 but not CD74 on EPCs and triggered the secretion of CXCL12, CXCL1, MIF, and vascular endothelial growth factor (VEGF). These factors stimulated the transmigration activity and adhesive capacity of EPCs, with MIF and VEGF exhibiting the strongest effects under hypoxia. MIF-, VEGF-, CXCL12-, and CXCL1-stimulated EPCs enhanced tube formation, with MIF and VEGF exhibiting again the strongest effect following hypoxia. Tube formation following in vivo implantation utilizing angiogenic factor-loaded Matrigel plugs was only promoted by VEGF. Coloading of plugs with eEPCs led to enhanced tube formation only by CXCL12, whereas MIF was the only factor which induced differentiation towards an endothelial and smooth muscle cell (SMC) phenotype, indicating an angiogenic and differentiation capacity in vivo. Surprisingly, CXCL12, a chemoattractant for smooth muscle progenitor cells, inhibited SMC differentiation. We have identified a role for EPC-derived proangiogenic MIF, VEGF and MIF receptors in EPC recruitment following hypoxia, EPC differentiation and subsequent tube and vessel formation, whereas CXCL12, a mediator of early EPC recruitment, does not contribute to the remodeling process. By discerning the contributions of key angiogenic chemokines and EPCs, these findings offer valuable mechanistic insight into mouse models of angiogenesis and help to define the intricate interplay between EPC-derived angiogenic cargo factors, EPCs, and the angiogenic target tissue.

Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice

Background— Atherosclerosis and vascular remodeling after injury are driven by inflammation and mononuclear cell infiltration. Extracellular RNA (eRNA) has recently been implicated to become enriched at sites of tissue damage and to act as a proinflammatory mediator. Here, we addressed the role of eRNA in high-fat diet–induced atherosclerosis and neointima formation after injury in atherosclerosis-prone mice. Methods and Results— The presence of eRNA was revealed in atherosclerotic lesions from high-fat diet–fed low-density lipoprotein receptor–deficient (Ldlr−/−) mice in a time-progressive fashion. RNase activity in plasma increased within the first 2 weeks (44±9 versus 70±7 mU/mg protein; P=0.0012), followed by a decrease to levels below baseline after 4 weeks of high-fat diet (44±9 versus 12±2 mU/mg protein; P<0.0001). Exposure of bone marrow–derived macrophages to eRNA resulted in a concentration-dependent upregulation of the proinflammatory mediators tumor necrosis factor-&agr;, arginase-2, interleukin-1&bgr;, interleukin-6, and interferon-&ggr;. In a model of accelerated atherosclerosis after arterial injury in apolipoprotein E–deficient (ApoE−/−) mice, treatment with RNase1 diminished the increased plasma level of eRNA evidenced after injury. Likewise, RNase1 administration reduced neointima formation in comparison with vehicle-treated ApoE−/− controls (25.0±6.2 versus 46.9±6.9×103 &mgr;m2, P=0.0339) and was associated with a significant decrease in plaque macrophage content. Functionally, RNase1 treatment impaired monocyte arrest on activated smooth muscle cells under flow conditions in vitro and inhibited leukocyte recruitment to injured carotid arteries in vivo. Conclusions— Because eRNA is associated with atherosclerotic lesions and contributes to inflammation-dependent plaque progression in atherosclerosis-prone mice, its targeting with RNase1 may serve as a new treatment option against atherosclerosis.

C5a Receptor Targeting in Neointima Formation After Arterial Injury in Atherosclerosis-Prone Mice

Background— Receptor binding of complement C5a leads to proinflammatory activation of many cell types, but the role of receptor-mediated action during arterial remodeling after injury has not been studied. In the present study, we examined the contribution of the C5a receptor (C5aR) to neointima formation in apolipoprotein E–deficient mice employing a C5aR antagonist (C5aRA) and a C5aR-blocking monoclonal antibody. Methods and Results— Mice fed an atherogenic diet were subjected to wire-induced endothelial denudation of the carotid artery and treated with C5aRA and anti-C5aR-blocking monoclonal antibody or vehicle control. Compared with controls, neointima formation was significantly reduced in mice receiving C5aRA or anti-C5aR-blocking monoclonal antibody for 1 week but not for 3 weeks, attributable to an increased content of vascular smooth muscle cells, whereas a marked decrease in monocyte and neutrophil content was associated with reduced vascular cell adhesion molecule-1. As assessed by immunohistochemistry, reverse transcription polymerase chain reaction, and flow cytometry, C5aR was expressed in lesional and cultured vascular smooth muscle cells, upregulated by injury or tumor necrosis factor-&agr;, and reduced by C5aRA. Plasma levels and neointimal plasminogen activator inhibitor-1 peaked 1 week after injury and were downregulated in C5aRA-treated mice. In vitro, C5a induced plasminogen activator inhibitor-1 expression in endothelial cells and vascular smooth muscle cells in a C5aRA-dependent manner, possibly accounting for higher vascular smooth muscle cell immigration. Conclusions— One-week treatment with C5aRA or anti-C5aR-blocking monoclonal antibody limited neointimal hyperplasia and inflammatory cell content and was associated with reduced vascular cell adhesion molecule-1 expression. However, treatment for 3 weeks failed to reduce but rather stabilized plaques, likely by reducing vascular plasminogen activator inhibitor-1 and increasing vascular smooth muscle cell migration.

Compartmentalized Protective and Detrimental Effects of Endogenous Macrophage Migration-Inhibitory Factor Mediated by CXCR2 in a Mouse Model of Myocardial Ischemia/Reperfusion

Objective—Here, we aimed to clarify the role of CXC chemokine receptor (CXCR) 2 in macrophage migration-inhibitory factor (MIF)–mediated effects after myocardial ischemia and reperfusion. As a pleiotropic chemokine-like cytokine, MIF has been identified to activate multiple receptors, including CD74 and CXCR2. In models of myocardial infarction, MIF exerts both proinflammatory effects and protective effects in cardiomyocytes. Similarly, CXCR2 displays opposing effects in resident versus circulating cells. Approach and Results—Using bone marrow transplantation, we generated chimeric mice with Cxcr2−/− bone marrow–derived inflammatory cells and wild-type (wt) resident cells (wt/Cxcr2−/−), Cxcr2−/− cardiomyocytes and wt bone marrow–derived cells (Cxcr2−/−/wt), and wt controls reconstituted with wt bone marrow (wt/wt). All groups were treated with anti-MIF or isotype control antibody before they underwent myocardial ischemia and reperfusion. Blocking MIF increased infarction size and impaired cardiac function in wt/wt and wt/CXCR2−/− mice but ameliorated functional parameters in Cxcr2−/−/wt mice, as analyzed by echocardiography and Langendorff perfusion. Neutrophil infiltration and angiogenesis were unaltered by MIF blockade or Cxcr2 deficiency. Monocyte infiltration was blunted in wt/Cxcr2−/− mice and reduced by MIF blockade in wt/wt and Cxcr2−/−/wt mice. Furthermore, MIF blockade attenuated collagen content in all groups in a CXCR2-independent manner. Conclusions—The compartmentalized and opposing effects of MIF after myocardial ischemia and reperfusion are largely mediated by CXCR2. Although MIF confers protective effects by improving myocardial healing and function through CXCR2 in resident cells, thereby complementing paracrine effects through CD74/AMP-activated protein kinase, it exerts detrimental effects on CXCR2-bearing inflammatory cells by increasing monocyte infiltration and impairing heart function. These dichotomous findings should be considered when developing novel therapeutic strategies to treat myocardial infarction.

Deficiency of Endothelial Cxcr4 Reduces Reendothelialization and Enhances Neointimal Hyperplasia After Vascular Injury in Atherosclerosis-Prone Mice

Objective—The Cxcl12/Cxcr4 chemokine ligand/receptor axis mediates the mobilization of smooth muscle cell progenitors, driving injury-induced neointimal hyperplasia. This study aimed to investigate the role of endothelial Cxcr4 in neointima formation. Approach and Results—&bgr;-Galactosidase staining using bone marrow x kinase (Bmx)-CreERT2 reporter mice and double immunofluorescence revealed an efficient and endothelial-specific deletion of Cxcr4 in Bmx-CreERT2+ compared with Bmx-CreERT2− Cxcr4-floxed apolipoprotein E–deficient (Apoe−/−) mice (referred to as Cxcr4EC-KOApoE−/− and Cxcr4EC-WT ApoE−/−, respectively). Endothelial Cxcr4 deficiency significantly increased wire injury–induced neointima formation in carotid arteries from Cxcr4EC-KOApoE−/− mice. The lesions displayed a higher number of macrophages, whereas the smooth muscle cell and collagen content were reduced. This was associated with a significant reduction in reendothelialization and endothelial cell proliferation in injured Cxcr4EC-KOApoE−/− carotids compared with Cxcr4EC-WTApoE−/− controls. Furthermore, stimulation of human aortic endothelial cells with chemokine (C-X-C motif) ligand 12 (CXCL12) significantly enhanced their wound-healing capacity in an in vitro scratch assay, an effect that could be reversed with the CXCR4 antagonist AMD3100. Also, flow cytometric analysis showed a reduced mobilization of Sca1+Flk1+Cd31+ and of Lin−Sca1+ progenitors in Cxcr4EC-KO ApoE−/− mice after vascular injury, although Cxcr4 surface expression was unaltered. No differences could be detected in plasma concentrations of Cxcl12, vascular endothelial growth factor, sphingosine 1-phosphate, or Flt3 (fms-related tyrosine kinase 3) ligand, all cytokines with an established role in progenitor cell mobilization. Nonetheless, double immunofluorescence revealed a significant reduction in local endothelial Cxcl12 staining in injured carotids from Cxcr4EC-KOApoE−/− mice. Conclusions—Endothelial Cxcr4 is crucial for efficient reendothelialization after vascular injury through endothelial wound healing and proliferation, and through the mobilization of Sca1+Flk1+Cd31+ cells, often referred to as circulating endothelial progenitor cells.

Compartmentalized Protective and Detrimental Effects of Endogenous Migration-Inhibitory Factor Mediated by CXCR2 in a Mouse Model of Myocardial Ischemia/Reperfusion

Objective—Here, we aimed to clarify the role of CXC chemokine receptor (CXCR) 2 in macrophage migration-inhibitory factor (MIF)–mediated effects after myocardial ischemia and reperfusion. As a pleiotropic chemokine-like cytokine, MIF has been identified to activate multiple receptors, including CD74 and CXCR2. In models of myocardial infarction, MIF exerts both proinflammatory effects and protective effects in cardiomyocytes. Similarly, CXCR2 displays opposing effects in resident versus circulating cells. Approach and Results—Using bone marrow transplantation, we generated chimeric mice with Cxcr2−/− bone marrow–derived inflammatory cells and wild-type (wt) resident cells (wt/Cxcr2−/−), Cxcr2−/− cardiomyocytes and wt bone marrow–derived cells (Cxcr2−/−/wt), and wt controls reconstituted with wt bone marrow (wt/wt). All groups were treated with anti-MIF or isotype control antibody before they underwent myocardial ischemia and reperfusion. Blocking MIF increased infarction size and impaired cardiac function in wt/wt and wt/CXCR2−/− mice but ameliorated functional parameters in Cxcr2−/−/wt mice, as analyzed by echocardiography and Langendorff perfusion. Neutrophil infiltration and angiogenesis were unaltered by MIF blockade or Cxcr2 deficiency. Monocyte infiltration was blunted in wt/Cxcr2−/− mice and reduced by MIF blockade in wt/wt and Cxcr2−/−/wt mice. Furthermore, MIF blockade attenuated collagen content in all groups in a CXCR2-independent manner. Conclusions—The compartmentalized and opposing effects of MIF after myocardial ischemia and reperfusion are largely mediated by CXCR2. Although MIF confers protective effects by improving myocardial healing and function through CXCR2 in resident cells, thereby complementing paracrine effects through CD74/AMP-activated protein kinase, it exerts detrimental effects on CXCR2-bearing inflammatory cells by increasing monocyte infiltration and impairing heart function. These dichotomous findings should be considered when developing novel therapeutic strategies to treat myocardial infarction.

Controlled intramyocardial release of engineered chemokines by biodegradable hydrogels as a treatment approach of myocardial infarction.

Myocardial infarction (MI) induces a complex inflammatory immune response, followed by the remodelling of the heart muscle and scar formation. The rapid regeneration of the blood vessel network system by the attraction of hematopoietic stem cells is beneficial for heart function. Despite the important role of chemokines in these processes, their use in clinical practice has so far been limited by their limited availability over a long time‐span in vivo. Here, a method is presented to increase physiological availability of chemokines at the site of injury over a defined time‐span and simultaneously control their release using biodegradable hydrogels. Two different biodegradable hydrogels were implemented, a fast degradable hydrogel (FDH) for delivering Met‐CCL5 over 24 hrs and a slow degradable hydrogel (SDH) for a gradual release of protease‐resistant CXCL12 (S4V) over 4 weeks. We demonstrate that the time‐controlled release using Met‐CCL5‐FDH and CXCL12 (S4V)‐SDH suppressed initial neutrophil infiltration, promoted neovascularization and reduced apoptosis in the infarcted myocardium. Thus, we were able to significantly preserve the cardiac function after MI. This study demonstrates that time‐controlled, biopolymer‐mediated delivery of chemokines represents a novel and feasible strategy to support the endogenous reparatory mechanisms after MI and may compliment cell‐based therapies.

Progress in interventional cardiology: challenges for the future

Cardiovascular disease is the leading cause of death in the western and developing countries. Percutaneous transluminal coronary interventions have become the most prevalent treatment option for coronary artery disease; however, due to serious complications, such as stent thrombosis and in-stent restenosis (ISR), the efficacy and safety of the procedure remain important issues to address. Strategies to overcome these aspects are under extensive investigation. In this review, we summarise relevant milestones during the time to overcome these limitations of coronary stents, such as the development of polymer-free drug-eluting stents (DES) to avoid pro-inflammatory response due to the polymer coating or the developement of stents with cell-directing drugs to, simultaneously, improve re-endothelialisation and inhibit ISR amongst other techniques most recently developed, which have not fully entered the clinical stage. Also the novel concept of fully biodegradable DES featured by the lack of a permanent foreign body promises to be a beneficial and applicable tool to restore a natural vessel with maintained vasomotion and to enable optional subsequent surgical revascularisation.

Response to Letter Regarding Article “Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice”

We acknowledge the interest and comments of Dr Chen et al regarding our findings that self-extracellular RNA (eRNA) significantly contributes to atherogenesis (as demonstrated in 2 established animal models) by inducing a prominent inflammatory response in situ and in bone marrow–derived macrophages (BMDM), as well.1 In particular, Chen et al question whether eRNA-dependent effects may have been mediated by Toll-like receptor (TLR)–related signaling, because they recently reported that BMDM responses toward the RNA analogue poly(IC) were significantly dampened in TLR3-deficient cells.2 During the past decade, our laboratory has characterized a number of new functions of eRNA in inflammation and cardiovascular diseases. In the indicated study, we aimed to characterize a causal role of natural eRNA, which may also serve as a cell-injury marker, in the onset and progression of atherosclerosis. It …