Intraprocedural endothelial cell seeding of arterial stents via biotin/avidin targeting mitigates in-stent restenosis.

Impaired endothelialization of endovascular stents has been established as a major cause of in-stent restenosis and late stent thrombosis. Attempts to enhance endothelialization of inner stent surfaces by pre-seeding the stents with endothelial cells in vitro prior to implantation are compromised by cell destruction during high-pressure stent deployment. Herein, we report on the novel stent endothelialization strategy of post-deployment seeding of biotin-modified endothelial cells to avidin-functionalized stents. Acquisition of an avidin monolayer on the stent surface was achieved by consecutive treatments of bare metal stents (BMS) with polyallylamine bisphosphonate, an amine-reactive biotinylation reagent and avidin. Biotin-modified endothelial cells retain growth characteristics of normal endothelium and can express reporter transgenes. Under physiological shear conditions, a 50-fold higher number of recirculating biotinylated cells attached to the avidin-modified metal surfaces compared to bare metal counterparts. Delivery of biotinylated endothelial cells to the carotid arterial segment containing the implanted avidin-modified stent in rats results in immediate cell binding to the stent struts and is associated with a 30% reduction of in-stent restenosis in comparison with BMS.

Stent-based delivery of AAV2 vectors encoding oxidation-resistant apoA1.

In-stent restenosis (ISR) complicates revascularization in the coronary and peripheral arteries. Apolipoprotein A1 (apoA1), the principal protein component of HDL possesses inherent anti-atherosclerotic and anti-restenotic properties. These beneficial traits are lost when wild type apoA1(WT) is subjected to oxidative modifications. We investigated whether local delivery of adeno-associated viral (AAV) vectors expressing oxidation-resistant apoA1(4WF) preserves apoA1 functionality. The efflux of 3H-cholesterol from macrophages to the media conditioned by endogenously produced apoA1(4WF) was 2.1-fold higher than for apoA1(WT) conditioned media in the presence of hypochlorous acid emulating conditions of oxidative stress. The proliferation of apoA1(WT)- and apoA1(4FW)-transduced rat aortic smooth muscle cells (SMC) was inhibited by 66% ± 10% and 65% ± 11%, respectively, in comparison with non-transduced SMC (p < 0.001). Conversely, the proliferation of apoA1(4FW)-transduced, but not apoA1(WT)-transduced rat blood outgrowth endothelial cells (BOEC) was increased 41% ± 5% (p < 0.001). Both apoA1 transduction conditions similarly inhibited basal and TNFα-induced reactive oxygen species in rat aortic endothelial cells (RAEC) and resulted in the reduced rat monocyte attachment to the TNFα-activated endothelium. AAV2-eGFP vectors immobilized reversibly on stainless steel mesh surfaces through the protein G/anti-AAV2 antibody coupling, efficiently transduced cells in culture modeling stent-based delivery. In vivo studies in normal pigs, deploying AAV2 gene delivery stents (GDS) preloaded with AAV2-eGFP in the coronary arteries demonstrated transduction of the stented arteries. However, implantation of GDS formulated with AAV2-apoA1(4WF) failed to prevent in-stent restenosis in the atherosclerotic vasculature of hypercholesterolemic diabetic pigs. It is concluded that stent delivery of AAV2-4WF while feasible, is not effective for mitigation of restenosis in the presence of severe atherosclerotic disease.

Adeno-Associated Viral Vector Immobilization and Local Delivery from Bare Metal Surfaces.

Spatially and temporally controlled delivery of biologicals, including gene vectors, represents an unmet need for regenerative medicine and gene therapy applications. Here we describe a method of reversible attachment of serotype 2 adeno-associated viral vectors (AAV2) to metal surfaces. This technique enables localized delivery of the vector to the target cell population in vitro and in vivo with the subsequent effective transduction of cells adjacent to the metal substrate. The underlying bioengineering approach employs coordination chemistry between the bisphosphonic groups of polyallylamine bisphosphonates and the metal atoms on the surface of metallic samples. Formation of a stable polybisphosphonate monolayer with plentiful allyl-derived amines allows for further chemical modification to consecutively append thiol-modified protein G, an anti-AAV2 antibody, and AAV2 particles. Herein we present a detailed protocols for the metal substrate modification, for the visualization of the metal surface-immobilized vector using direct and indirect fluorescent AAV2 labeling and scanning electron microscopy, for quantification of the surface-immobilized vector load with RT-PCR, and for the localized vector transduction in vitro and in vivo.

Loss of Protease-Activated Receptor 4 Prevents Inflammation Resolution and Predisposes the Heart to Cardiac Rupture After Myocardial Infarction.

BACKGROUND: Cardiac rupture is a major lethal complication of acute myocardial infarction (MI). Despite significant advances in reperfusion strategies, mortality from cardiac rupture remains high. Studies suggest that cardiac rupture can be accelerated by thrombolytic therapy, but the relevance of this risk factor remains controversial. METHODS: We analyzed protease-activated receptor 4 (Par4) expression in mouse hearts with MI and investigated the effects of Par4 deletion on cardiac remodeling and function after MI by echocardiography, quantitative immunohistochemistry, and flow cytometry. RESULTS: Par4 mRNA and protein levels were increased in mouse hearts after MI and in isolated cardiomyocytes in response to hypertrophic and inflammatory stimuli. Par4-deficient mice showed less myocyte apoptosis, reduced infarct size, and improved functional recovery after acute MI relative to wild-type (WT). Conversely, Par4-/- mice showed impaired cardiac function, greater rates of myocardial rupture, and increased mortality after chronic MI relative to WT. Pathological evaluation of hearts from Par4-/- mice demonstrated a greater infarct expansion, increased cardiac hemorrhage, and delayed neutrophil accumulation, which resulted in impaired post-MI healing compared with WT. Par4 deficiency also attenuated neutrophil apoptosis in vitro and after MI in vivo and impaired inflammation resolution in infarcted myocardium. Transfer of Par4-/- neutrophils, but not of Par4-/- platelets, in WT recipient mice delayed inflammation resolution, increased cardiac hemorrhage, and enhanced cardiac dysfunction. In parallel, adoptive transfer of WT neutrophils into Par4-/- mice restored inflammation resolution, reduced cardiac rupture incidence, and improved cardiac function after MI. CONCLUSIONS: These findings reveal essential roles of Par4 in neutrophil apoptosis and inflammation resolution during myocardial healing and point to Par4 inhibition as a potential therapy that should be limited to the acute phases of ischemic insult and avoided for long-term treatment after MI.

Cardiac Expression of Factor X Mediates Cardiac Hypertrophy and Fibrosis in Pressure Overload.

Activated factor X is a key component of the coagulation cascade, but whether it directly regulates pathological cardiac remodeling is unclear. In mice subjected to pressure overload stress, cardiac factor X mRNA expression and activity increased concurrently with cardiac hypertrophy, fibrosis, inflammation and diastolic dysfunction, and responses blocked with a low coagulation-independent dose of rivaroxaban. In vitro, neurohormone stressors increased activated factor X expression in both cardiac myocytes and fibroblasts, resulting in activated factor X-mediated activation of protease-activated receptors and pro-hypertrophic and -fibrotic responses, respectively. Thus, inhibition of cardiac-expressed activated factor X could provide an effective therapy for the prevention of adverse cardiac remodeling in hypertensive patients.

Dual inhibition of cathepsin G and chymase reduces myocyte death and improves cardiac remodeling after myocardial ischemia reperfusion injury.

Early reperfusion of ischemic cardiac tissue increases inflammatory cell infiltration which contributes to cardiomyocyte death and loss of cardiac function, referred to as ischemia/reperfusion (IR) injury. Neutrophil- and mast cell-derived proteases, cathepsin G (Cat.G) and chymase, are released early after IR, but their function is complicated by potentially redundant actions and targets. This study investigated whether a dual inhibition of Cat.G and chymase influences cardiomyocyte injury and wound healing after experimental IR in mice. Treatment with a dual Cat.G and chymase inhibitor (DCCI) immediately after reperfusion blocked cardiac Cat.G and chymase activity induced after IR, which resulted in decreased immune response in the infarcted heart. Mice treated with DCCI had less myocardial collagen deposition and showed preserved ventricular function at 1 and 7 days post-IR compared with vehicle-treated mice. DCCI treatment also significantly attenuated focal adhesion (FA) complex disruption and myocyte degeneration after IR. Treatment of isolated cardiomyocytes with Cat.G or chymase significantly promoted FA signaling downregulation, myofibril degeneration and myocyte apoptosis. Conversely, treatment of cardiac fibroblasts with Cat.G or chymase induced FA signaling activation and increased their migration and differentiation to myofibroblasts. These opposite responses in cardiomyocytes and fibroblasts were blocked by treatment with DCCI. These findings show that Cat.G and chymase are key mediators of myocyte apoptosis and fibroblast migration and differentiation that play a role in adverse cardiac remodeling and function post-IR. Thus, dual targeting of neutrophil- and mast cell-derived proteases could be used as a novel therapeutic strategy to reduce post-IR inflammation and improve cardiac remodeling.

Protease-activated receptor 4 deficiency offers cardioprotection after acute ischemia reperfusion injury.

Protease-activated receptor (PAR)4 is a low affinity thrombin receptor with less understood function relative to PAR1. PAR4 is involved in platelet activation and hemostasis, but its specific actions on myocyte growth and cardiac function remain unknown. This study examined the role of PAR4 deficiency on cardioprotection after myocardial ischemia-reperfusion (IR) injury in mice. When challenged by in vivo or ex vivo IR, PAR4 knockout (KO) mice exhibited increased tolerance to injury, which was manifest as reduced infarct size and a more robust functional recovery compared to wild-type mice. PAR4 KO mice also showed reduced cardiomyocyte apoptosis and putative signaling shifts in survival pathways in response to IR. Inhibition of PAR4 expression in isolated cardiomyocytes by shRNA offered protection against thrombin and PAR4-agonist peptide-induced apoptosis, while overexpression of wild-type PAR4 significantly enhanced the susceptibility of cardiomyocytes to apoptosis, even under low thrombin concentrations. Further studies implicate Src- and epidermal growth factor receptor-dependent activation of JNK on the proapoptotic effect of PAR4 in cardiomyocytes. These findings reveal a pivotal role for PAR4 as a regulator of cardiomyocyte survival and point to PAR4 inhibition as a therapeutic target offering cardioprotection after acute IR injury.