Anuran Chatterjee

D-series resolvin attenuates vascular smooth muscle cell activation and neointimal hyperplasia following vascular injury

Recent evidence suggests that specialized lipid mediators derived from polyunsaturated fatty acids control resolution of inflammation, but little is known about resolution pathways in vascular injury. We sought to determine the actions of D‐series resolvin (RvD) on vascular smooth muscle cell (VSMC) phenotype and vascular injury. Human VSMCs were treated with RvD1 and RvD2, and phenotype was assessed by proliferation, migration, monocyte adhesion, superoxide production, and gene expression assays. A rabbit model of arterial angioplasty with local delivery of RvD2 (10 nM vs. vehicle control) was employed to examine effects on vascular injury in vivo. Local generation of proresolving lipid mediators (LC‐MS/MS) and expression of RvD receptors in the vessel wall were assessed. RvD1 and RvD2 produced dose‐dependent inhibition of VSMC proliferation, migration, monocyte adhesion, superoxide production, and proinflammatory gene expression (IC50≈0.1–1 nM). In balloon‐injured rabbit arteries, cell proliferation (51%) and leukocyte recruitment (41%) were reduced at 3 d, and neointimal hyperplasia was attenuated (29%) at 28 d by RvD2. We demonstrate endogenous biosynthesis of proresolving lipid mediators and expression of receptors for RvD1 in the artery wall. RvDs broadly reduce VSMC responses and modulate vascular injury, suggesting that local activation of resolution mechanisms expedites vascular homeostasis.—Miyahara, T., Runge, S., Chatterjee, A., Chen, M., Mottola, G., Fitzgerald, J. M., Serhan, C. N., Conte, M. S. D‐series resolvin attenuates vascular smooth muscle cell activation and neointimal hyperplasia following vascular injury. FASEB J. 27, 2220–2232 (2013). www.fasebj.org

The pro-resolving lipid mediator maresin 1 (MaR1) attenuates inflammatory signaling pathways in vascular smooth muscle and endothelial cells.

Objective Inflammation and its resolution are central to vascular injury and repair. Maresins comprise a new family of bioactive lipid mediators synthesized from docosahexaenoic acid, an ω-3 polyunsaturated fatty acid. They have been found to exert anti-inflammatory and pro-resolving responses in macrophages, neutrophils and bronchial epithelial cells and impart beneficial actions in murine models of peritonitis and colitis. We investigated the impact of maresin-1 (MaR1) on tumor necrosis factor alpha (TNF-α) induced inflammatory responses in human vascular endothelial (EC) and smooth muscle cells (VSMC). Methods Primary cultures of human saphenous vein EC and VSMC were employed. We tested the naturally occurring MaR1 as modulator of TNF-α effects, with examination of monocyte adhesion, oxidant stress, and intracellular inflammatory signaling pathways. Results MaR1 attenuated TNF-α induced monocyte adhesion and reactive oxygen species (ROS) generation in both EC and VSMC, associated with down-regulated expression (cell surface) of the adhesion molecule E-selectin (in EC) and NADPH-oxidases (NOX4, NOX1, NOX2). MaR1 attenuated TNF-α induced release of pro-inflammatory mediators by EC and VSMC. MaR1 caused an attenuation of TNF-α induced NF-κB activation in both cell types associated with inhibition of I-κ Kinase (IKK) phosphorylation, IκB-α degradation and nuclear translocation of the NF- κB p65 subunit. MaR1 also caused a time-dependent increase in intracellular cyclic AMP (cAMP) in both naive and TNF-α stimulated VSMC and EC. Conclusions MaR1 has broad anti-inflammatory actions on EC and VSMC, which may be partly mediated through up-regulation of cAMP and down-regulation of the transcription factor NF-κB. The results suggest that the pro-resolving lipid mediator MaR1 exerts homeostatic actions on vascular cells that counteract pro-inflammatory signals. These findings may have direct relevance for acute and chronic states of vascular inflammation.

Systemic delivery of proresolving lipid mediators resolvin D2 and maresin 1 attenuates intimal hyperplasia in mice

Vascular injury induces a potent inflammatory response that influences vessel remodeling and patency, limiting long‐term benefits of cardiovascular interventions such as angioplasty. Specialized proresolving lipid mediators (SPMs) derived from ω‐3 polyunsaturated fatty acids [eicosapentaenoic acid and docosahexaenoic acid (DHA)] orchestrate resolution in diverse settings of acute inflammation. We hypothesized that systemic administration of DHA‐derived SPMs [resolvin D2 (RvD2) and maresin 1 (MaR1)] would influence vessel remodeling in a mouse model of arterial neointima formation (carotid ligation). In vitro, SPM treatment inhibited mouse aortic smooth muscle cell migration (IC50 ≅ 1 nM) to a PDGF gradient and reduced TNF‐α‐stimulated p65 translocation, superoxide production, and proinflammatory gene expression (MCP‐1). In vivo, adult FVB mice underwent unilateral carotid artery ligation with administration of RvD2, MaR1, or vehicle (100 ng by intraperitoneal injection at 0, 1, 3, 5, and 7 d after ligation). In ligated carotid arteries at 4 d, SPM treatment was associated with reduced cell proliferation and neutrophil and macrophage recruitment and increased polarization of M2 macrophages in the arterial wall. Neointimal hyperplasia (at 14 d) was notably attenuated in RvD2 (62%)‐ and MaR1 (67%)‐treated mice, respectively. Modulation of resolution pathways may offer new opportunities to regulate the vascular injury response and promote vascular homeostasis.—Akagi, D., Chen, M., Toy, R., Chatterjee, A., Conte, M. S. Systemic delivery of proresolving lipid mediators resolvin D2 and maresin 1 attenuates intimal hyperplasia in mice. FASEB J. 29, 2504‐2513 (2015). www.fasebj.org

D-series resolvins inhibit murine abdominal aortic aneurysm formation and increase M2 macrophage polarization

The role of resolvins in abdominal aortic aneurysm (AAA) has not been established. We hypothesized that treatmentwithD‐series resolvins (RvD2 or RvD1)would attenuate murine AAA formation through alterations in macrophage polarization and cytokine expression. Male C57/B6 mice (n = 9 per group) 8 to 12 wk old received RvD2 (100 ng/kg/treatment), RvD1 (100 ng/kg/treatment), or vehicle only every third day beginning 3 d before abdominal aorticperfusionwith elastase asprevention. Aortaswere collected 14 d after elas taseperfusion. Cytokine analysis (n = 5 per group) or confocal microscopy (n = 4 per group) was performed. In a separate experiment, RvD2 was provided tomicewith small AAAs 3 d after elastase treatment (n = 8 per group). Additionally, apolipoprotein E knockoutmice treatedwith angiotensin II (1000 ng/kg) were treated with RvD2 or vehicle alone (n = 10 per group) in a nonsurgical model of AAA. To determine the effect of RvD2 on macrophage polarization, confocal staining for macrophages, M1 and M2 macrophage subtypes, α‐actin, and DAPI was performed. Mean aortic dilationwas 966 13% for vehicle‐treated mice ≤57 × 9.7% for RvD2‐treated mice, and 61 ± 11% for RvD1‐treated mice (P < 0.0001). Proinflammatory cytokines macrophage chemotactic protein 1, C‐X‐C motif ligand 1, and IL‐1β were significantly elevated in control animals compared to RvD2‐ and RvD1‐treated animals (P < 0.05), resulting in a reduction of matrix metalloproteinase 2 and 9 activity in resolvin‐treated mice in both elastase and angiotensin II models. Treatment of existing small AAAs with RvD2 demonstrated a 25%reduction in aneurysm size at d 14 compared to vehicle alone (P = 0.018). Confocal histology demonstrated a prevalence of M2 macrophages within the aortic medium in mice treated with RvD2. Resolvin D2 exhibits a potent protective effect against experimental AAA formation. Treatment with RvD2 significantly influences macrophage polarization and decreases several important proinflammatory cytokines. Resolvins and the alteration of macrophage polarization represent potential future targets for prevention of AAA.—Pope, N.H., Salmon, M.,Davis, J. P.,Chatterjee, A., Su, G., Conte, M. S.,Ailawadi, G., Upchurch, G. R., Jr. D‐series resolvins inhibit murine abdominal aortic aneurysm formation and increase M2 macrophage polarization. FASEB J. 30, 4192–4201 (2016). www.fasebj.org

Perivascular delivery of resolvin D1 inhibits neointimal hyperplasia in a rat model of arterial injury

Objective: Lipid mediators derived from omega‐3 polyunsaturated fatty acids such as resolvin D1 (RvD1) accelerate the resolution of inflammation and have potential as vascular therapeutics. The objective of this study was to evaluate local perivascular delivery of RvD1 as a means to attenuate neointimal hyperplasia in a rat model of arterial injury. Methods: Smooth muscle cells were harvested from rat aortas to study the effects of RvD1 on rat arterial vascular smooth muscle cell responses in vitro, with focus on inflammation, proliferation, migration, cytoskeletal changes, and cytotoxicity. The safety and efficacy of perivascular delivery of RvD1 through thin biodegradable three‐layered poly(lactic‐co‐glycolic acid) wraps or 25% Pluronic F127 gels were studied in a rat model of carotid angioplasty. A total of 200 ng of RvD1 was loaded into each construct for perivascular delivery after injury. Morphometric and histologic analyses were performed 3 and 14 days after injury. Results: RvD1 attenuated rat arterial vascular smooth muscle cell inflammatory pathways, proliferation, migration, and mitogen‐induced cytoskeletal changes in vitro, without evidence of cytotoxicity. RvD1‐loaded wraps reduced neointimal formation after carotid angioplasty by 59% vs no‐wrap controls (P = .001) and by 45% vs vehicle‐wrap controls (P = .002). RvD1‐loaded Pluronic gels similarly reduced neointimal formation by 49% vs no‐gel controls (P = .02) and by 52% vs vehicle‐gel controls (P = .02). No group was associated with infection, thrombosis, or negative vessel remodeling. Wraps were found to be easier to apply than gel constructs. Ki67 proliferation index was significantly lower in RvD1‐loaded wrap‐treated arteries compared with both no‐wrap and vehicle‐wrap controls at both 3 and 14 days after injury (65% vs no‐wrap group and 70% vs vehicle‐wrap group at day 3, 49% vs both control groups at day 14; P < .05). Similarly, oxidative stress (30% and 29%; P < .05) and nuclear factor &kgr;B activation (42% and 45%; P < .05) were significantly lower in the RvD1‐loaded wrap group compared with both no‐wrap and vehicle‐wrap controls at 3 days after injury. Conclusions: Local perivascular delivery of RvD1 attenuates formation of neointimal hyperplasia without associated toxicity in a rat model of carotid angioplasty. This effect is likely due to attenuation of inflammatory pathways as well as decreased arterial smooth muscle cell proliferation and migration. Clinical Relevance: Failure of peripheral vascular interventions remains the single greatest challenge in management of vascular disease, with failure rates approaching 50% or greater within 2 years for many interventions. In contrast to the currently available cytotoxic drugs used on vascular devices that delay vessel healing, novel endogenous “proresolving” lipid mediators (such as resolvin D1) have potential to reduce neointimal hyperplasia by accelerating homeostasis. This study provides proof‐of‐concept for local perivascular delivery of proresolving agents to improve the healing response after acute vascular injury.

Unidirectional and sustained delivery of the proresolving lipid mediator resolvin D1 from a biodegradable thin film device.

Resolvin D1 (RvD1) belongs to a family of endogenously derived proresolving lipid mediators that have been shown to attenuate inflammation, activate proresolution signaling, and promote homeostasis and recovery from tissue injury. In this study we present a poly(lactic-co-glycolic acid) (PLGA) based thin-film device composed of layers of varying ratios of lactic and glycolic acid that elutes RvD1 unidirectionally to target tissues. The device demonstrated sustained release in vitro for 56 days with an initial burst of release over 14 days. The asymmetric design of the device released 98% of RvD1 through the layer with the lowest molar ratio of lactic acid to glycolic acid, and the remainder through the opposite side. We validated structural integrity of RvD1 released from the device by mass spectrometry and investigated its bioactivity on human vascular endothelial (EC) and smooth muscle cells (VSMC). RvD1 released from the device attenuated VSMC migration, proliferation, and TNF-α induced NF-κB activation, without evidence of cytotoxicity. Delivery of RvD1 to blood vessels was demonstrated ex vivo in a flow chamber system using perfused rabbit aortas and in vivo in a rat carotid artery model, with the devices applied as an adventitial wrap. Our results demonstrate a novel approach for sustained, local delivery of Resolvin D1 to vascular tissue at therapeutically relevant levels.

Aspirin-triggered resolvin D1 attenuates PDGF-induced vascular smooth muscle cell migration via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway

Background and objectives Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration. Methods VSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity. Results Treatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor. Conclusions Our results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

Perivascular delivery of resolvin D1 inhibits neointimal hyperplasia in a rabbit vein graft model

Objective: Inflammation is a key driver of excessive neointimal hyperplasia within vein grafts. Recent work demonstrates that specialized proresolving lipid mediators biosynthesized from omega‐3 polyunsaturated fatty acids, such as resolvin D1 (RvD1), actively orchestrate the process of inflammation resolution. We investigated the effects of local perivascular delivery of RvD1 in a rabbit vein graft model. Methods: Ipsilateral jugular veins were implanted as carotid interposition grafts through an anastomotic cuff technique in New Zealand white rabbits (3–4 kg; N = 80). RvD1 (1 &mgr;g) was delivered to the vein bypass grafts in a perivascular fashion, using either 25% Pluronic F127 gel (Sigma‐Aldrich, St. Louis, Mo) or a thin bilayered poly(lactic‐co‐glycolic acid) (PLGA) film. No treatment (bypass only) and vehicle‐loaded Pluronic gels or PLGA films served as controls. Delivery of RvD1 to venous tissue was evaluated 3 days later by liquid chromatography‐tandem mass spectrometry. Total leukocyte infiltration, macrophage infiltration, and cell proliferation were evaluated by immunohistochemistry. Elastin and trichrome staining was performed on grafts harvested at 28 days after bypass to evaluate neointimal hyperplasia and vein graft remodeling. Results: Perivascular treatments did not influence rates of graft thrombosis (23%), major wound complications (4%), or death (3%). Leukocyte (CD45) and macrophage (RAM11) infiltration was significantly reduced in the RvD1 treatment groups vs controls at 3 days (60%‐72% reduction; P < .01). Cellular proliferation (Ki67 index) was also significantly lower in RvD1‐treated vs control grafts at 3 days (40%‐50% reduction; P < .01). Treatment of vein grafts with RvD1‐loaded gels reduced neointimal thickness at 28 days by 61% vs bypass only (P < .001) and by 63% vs vehicle gel (P < .001). RvD1‐loaded PLGA films reduced neointimal formation at 28 days by 50% vs bypass only (P < .001). RvD1 treatment was also associated with reduced collagen deposition in vein grafts at 28 days. Conclusions: Local perivascular delivery of RvD1 attenuates vein graft hyperplasia without associated toxicity in a rabbit carotid bypass model. This effect appears to be mediated by both reduced leukocyte recruitment and decreased cell proliferation within the graft. Perivascular PLGA films may also impart protection through biomechanical scaffolding in this venous arterialization model. Our studies provide further support for the potential therapeutic role of specialized proresolving lipid mediators such as D‐series resolvins in modulating vascular injury and repair. Clinical Relevance: Autologous vein bypass grafts are the most durable means for revascularization in peripheral vascular disease; however, midterm and long‐term outcomes are limited by vein graft hyperplasia with associated vein graft failure. Endogenous proresolving lipid mediators such as resolvin D1 have the potential to attenuate vein graft hyperplasia by accelerating repair. This study provides proof of concept for local delivery of resolvin D1 to reduce inflammation and to improve the healing response after vein bypass grafting.