Prevention of neointimal hyperplasia after coronary artery bypass graft via local delivery of sirolimus and rosuvastatin: network pharmacology and in vivo validation.

BACKGROUND: Coronary artery bypass graft (CABG) is generally used to treat complex coronary artery disease. Treatment success is affected by neointimal hyperplasia (NIH) of graft and anastomotic sites. Although sirolimus and rosuvastatin individually inhibit NIH progression, the efficacy of combination treatment remains unknown. METHODS: We identified cross-targets associated with CABG, sirolimus, and rosuvastatin by using databases including DisGeNET and GeneCards. GO and KEGG pathway enrichment analyses were conducted using R studio, and target proteins were mapped in PPI networks using Metascape and Cytoscape. For in vivo validation, we established a balloon-injured rabbit model by inducing NIH and applied a localized perivascular drug delivery device containing sirolimus and rosuvastatin. The outcomes were evaluated at 1, 2, and 4 weeks post-surgery. RESULTS: We identified 115 shared targets between sirolimus and CABG among databases, 23 between rosuvastatin and CABG, and 96 among all three. TNF, AKT1, and MMP9 were identified as shared targets. Network pharmacology predicted the stages of NIH progression and the corresponding signaling pathways linked to sirolimus (acute stage, IL6/STAT3 signaling) and rosuvastatin (chronic stage, Akt/MMP9 signaling). In vivo experiments demonstrated that the combination of sirolimus and rosuvastatin significantly suppressed NIH progression. This combination treatment also markedly decreased the expression of inflammation and Akt signaling pathway-related proteins, which was consistent with the predictions from network pharmacology analysis. CONCLUSIONS: Sirolimus and rosuvastatin inhibited pro-inflammatory cytokine production during the acute stage and regulated Akt/mTOR/NF-κB/STAT3 signaling in the chronic stage of NIH progression. These potential synergistic mechanisms may optimize treatment strategies to improve long-term patency after CABG.

Effect of sequential release of sirolimus and rosuvastatin using silk fibroin microneedle to prevent intimal hyperplasia.

Intimal hyperplasia (IH) is a major cause of vascular restenosis after bypass surgery, which progresses as a series of processes from the acute to chronic stage in response to endothelial damage during bypass grafting. A strategic localized drug delivery system that reflects the pathophysiology of IH and minimizes systemic side effects is necessary. In this study, the sequential release of sirolimus, a mechanistic target of rapamycin (mTOR) inhibitor, and statin, an HMG-COA inhibitor, was realized as a silk fibroin-based microneedle device in vivo. The released sirolimus in the acute stage reduced neointima (NI) and vascular fibrosis through mTOR inhibition. Furthermore, rosuvastatin, which was continuously released from the acute to chronic stage, reduced vascular stiffness and apoptosis through the inactivation of Yes-associated protein (YAP). The sequential release of sirolimus and rosuvastatin confirmed the synergistic treatment effects on vascular inflammation, VSMC proliferation, and ECM degradation remodeling through the inhibition of transforming growth factor (TGF)-beta/NF-κB pathway. These results demonstrate the therapeutic effect on preventing restenosis with sufficient vascular elasticity and significantly reduced IH in response to endothelial damage. Therefore, this study suggests a promising strategy for treating coronary artery disease through localized drug delivery of customized drug combinations.

Sirolimus-Embedded Silk Microneedle Wrap to Prevent Neointimal Hyperplasia in Vein Graft Model.

We investigated the role of a sirolimus-embedded silk microneedle (MN) wrap as an external vascular device for drug delivery efficacy, inhibition of neointimal hyperplasia, and vascular remodeling. Using dogs, a vein graft model was developed to interpose the carotid or femoral artery with the jugular or femoral vein. The control group contained four dogs with only interposed grafts; the intervention group contained four dogs with vein grafts in which sirolimus-embedded silk-MN wraps were applied. After 12-weeks post-implantation, 15 vein grafts in each group were explanted and analyzed. Vein grafts applied with the rhodamine B-embedded silk-MN wrap showed far higher fluorescent signals than those without the wrap. The diameter of vein grafts in the intervention group decreased or remained stable without dilatation; however, it increased in the control group. The intervention group had femoral vein grafts with a significantly lower mean neointima-to-media ratio, and had vein grafts with an intima layer showing a significantly lower collagen density ratio than the control group. In conclusion, sirolimus-embedded silk-MN wrap in a vein graft model successfully delivered the drug to the intimal layer of the vein grafts. It prevented vein graft dilatation, avoiding shear stress and decreasing wall tension, and it inhibited neointimal hyperplasia.

In situ diagnosis and simultaneous treatment of cardiac diseases using a single-device platform.

The in situ diagnosis of cardiac activities with simultaneous therapeutic electrical stimulation of the heart is key to preventing cardiac arrhythmia. Here, we present an unconventional single-device platform that enables in situ monitoring even in a wet condition and control of beating heart motions without interferences to the recording signal. This platform consists of the active-matrix array of pressure-sensitive transistors for detecting cardiac beatings, biocompatible, low-impedance electrodes for cardiac stimulations, and an alginate-based hydrogel adhesive for attaching this platform conformally to the epicardium. In contrast to conventional electrophysiological sensing using electrodes, the pressure-sensitive transistors measured mechanophysiological characteristics by monitoring the spatiotemporal distributions of cardiac pressures during heart beating motions. In vivo tests show mechanophysiological readings having good correlation with electrocardiography and negligible interference with the electrical artifacts caused during cardiac stimulations. This platform can therapeutically synchronize the rhythm of abnormal heartbeats through efficient pacing of cardiac arrhythmia.

Time-dependent pathobiological and physiological changes of implanted vein grafts in a canine model.

Although autologous vein grafting is essential, the high vein failure rate and specific clinical interventions are not clear, so a potential treatment is critically needed; thus, complex analyses of the relationship between pathobiological and physiological processes in preclinical are essential. The interposition of the femoral vein was performed in a canine model. Maximized expansion and velocity were measured at 8 weeks post-implantation, and a relative decrease was observed at 12 weeks. However, NI formation and NI/Media ratio significantly increased time dependently, and differences between the mechanical properties were observed. Additionally, RhoA-mediated TNF-α induced by rapid structural changes and high shear stress was confirmed. After adaptation to the arterial environment, vascular remodeling occurred by SMC proliferation and differentiation, apoptosis and autophagy were induced through YAP activity without vasodilation and RhoA activity. Our results show that understanding pathobiological processes in which time-dependent physiological changes contribute to vein failure can lead to a potential strategy. The implanted vein graft within the arterial environment undergoes pathobiological processes through RhoA and YAP activity, leading to pathophysiological changes.

Highly flexible and porous silk fibroin microneedle wraps for perivascular drug delivery.

Various perivascular drug delivery techniques have been demonstrated for localized post-treatment of intimal hyperplasia: a vascular inflammatory response caused by endothelial damages. Although most perivascular devices have focused on controlling the delivery duration of anti-proliferation drug, the confined and unidirectional delivery of the drug to the target tissue has become increasingly important. In addition, careful attention should also be paid to the luminal stability and the adequate exchange of vascular protein or cell between the blood vessel and extravascular tissue to avoid any side effect from the long-term application of any perivascular device. Here, a highly flexible and porous silk fibroin microneedle wrap (Silk MN wrap) is proposed to directly inject antiproliferative drug to the anastomosis sites while ensuring sufficient vascular exchanges. Drug-embedded silk MNs were transfer-molded on a highly flexible and porous silk wrap. The enhanced cell compatibility, molecular permeability, and flexibility of silk MN wrap guaranteed the structural integrity of blood vessels. Silk wrap successfully supported the silk MNs and induced multiple MN penetration to the target tissue. Over 28 days, silk MN wrap significantly inhibited intimal hyperplasia with a 62.1% reduction in neointimal formation.

Enhanced Biocompatibility of Multi-Layered, 3D Bio-Printed Artificial Vessels Composed of Autologous Mesenchymal Stem Cells.

Artificial vessels capable of long-term patency are essential clinical tools in vascular surgery that involves small vessels. On-going attempts to develop artificial vessels that complements restenosis have not been entirely successful. Here, we report on the fabrication of small-sized artificial vessels using a three-dimensional bio-printer. The fabrication employed biodegradable polycaprolactone and autologous MSCs harvested from the bone-marrow of canines. The MSCs were cultured and differentiated into endothelial-like cells. After confirming differentiation, artificial vessels comprising three-layers were constructed and implanted into the arteries of canines. The autologous MSCs printed on artificial vessels (cell-derived group) maintained a 64.3% patency (9 of 14 grafts) compared with artificial vessels without cells (control group, 54.5% patency (6 of 11 grafts)). The cell-derived vessels (61.9 cells/mm2 ± 14.3) had more endothelial cells on their inner surfaces than the control vessels (21 cells/mm2 ± 11.3). Moreover, the control vessels showed acute inflammation on the porous structures of the implanted artificial vessels, whereas the cell-derived vessels exhibited fibrinous clots with little to no inflammation. We concluded that the minimal rejection of these artificial vessels by the immune system was due to the use of autologous MSCs. We anticipate that this study will be of value in the field of tissue-engineering in clinical practice.

Transfer-molded wrappable microneedle meshes for perivascular drug delivery.

After surgical procedures such as coronary/peripheral bypass grafting or endarterectomy for the treatment of organ ischemia derived from atherosclerosis, intimal hyperplasia (IH) which leads to restenosis or occlusion at the site of graft anastomosis frequently occurs. In order to inhibit IH caused by abnormal growth of smooth muscle cells (SMCs) in tunica media, various perivascular drug delivery devices are reported for delivery of anti-proliferation drugs into vascular tissue. However, there still remain conflicting requirements such as local and unidirectional delivery vs device porosity, and conformal tight device installation vs pulsatile expansion and constriction of blood vessels. In this study, a biodegradable microneedle (MN) array is developed on a flexible woven surgical mesh using a transfer molding method. Mechanical properties of 'wrappable' MN meshes are investigated and compared to the properties of blood vessels. Ex vivo and in vivo animal studies demonstrate enhanced drug delivery efficiency, efficacy for IH reduction, and safety of MN mesh. In particular, MN mesh showed significantly reduced neointiamal formation (11.1%) compared to other competitive groups (23.7 and 22.2%) after 4-week in vivo animal study. Additionally, wrappable MN meshes effectively suppressed side effects such as IH due to mechanical constriction, loss of toxic drug to the surroundings, and cell death that were frequently observed with other previous perivascular drug delivery devices.

A Biodegradable Microneedle Cuff for Comparison of Drug Effects through Perivascular Delivery to Balloon-Injured Arteries.

Restenosis at a vascular anastomosis site is a major cause of graft failure and is difficult to prevent by conventional treatment. Perivascular drug delivery has advantages as drugs can be diffused to tunica media and subintima while minimizing the direct effect on endothelium. This in vivo study investigated the comparative effectiveness of paclitaxel, sirolimus, and sunitinib using a perivascular biodegradable microneedle cuff. A total of 31 New Zealand white rabbits were used. Rhodamine was used to visualize drug distribution (n = 3). Sirolimus- (n = 7), sunitinib- (n = 7), and paclitaxel-loaded (n = 7) microneedle cuffs were placed at balloon-injured abdominal aortae and compared to drug-free cuffs (n = 7). Basic histological structures were not affected by microneedle devices, and vascular wall thickness of the device-only group was similar to that of normal artery. Quantitative analysis revealed significantly decreased neointima formation in all drug-treated groups (p < 0.001). However, the tunica media layer of the paclitaxel-treated group was significantly thinner than that of other groups and also showed the highest apoptotic ratio (p < 0.001). Proliferating cell nuclear antigen (PCNA)-positive cells were significantly reduced in all drug-treated groups. Sirolimus or sunitinib appeared to be more appropriate for microneedle devices capable of slow drug release because vascular wall thickness was minimally affected.

Exovascular application of epigallocatechin-3-O-gallate-releasing electrospun poly(L-lactide glycolic acid) fiber sheets to reduce intimal hyperplasia in injured abdominal aorta.

Intimal hyperplasia is an excessive ingrowth of tissue resulting in chronic structural lesions commonly found at sites of atherosclerotic lesions, arterial angioplasty, vascular graft anastomoses, and other vascular abnormalities. Epigallocatechin-3-O-gallate (EGCG) was shown to elicit antioxidant, anti-proliferative, and anti-thrombogenic effects. In this study, we used an electrospinning technique to synthesize EGCG-eluting biodegradable poly(L-lactide glycolic acid) (PLGA) fiber sheets for local delivery of EGCG and investigated the effect of their exovascular application on intimal hyperplasia following balloon-induced abdominal aorta injury in a rabbit experimental model. The morphology of the composite sheets was characterized using scanning electron microscopy and Fourier transform-infrared spectroscopy. EGCG was loaded and dispersed into the PLGA-based electrospun fibers. The EGCG-loaded PLGA sheets exhibited sustained EGCG release following the initial 24 h of burst release in phosphate-buffered saline. In vivo studies demonstrated significant inhibition of intimal hyperplasia following the application of the EGCG-eluting electrospun PLGA fiber sheets, compared with vehicle PLGA controls. In conclusion, our results show that exovascular application of EGCG-eluting PLGA electrospun fiber sheets may provide a useful system for the effective local delivery of drugs for the prevention of intimal hyperplasia.

Perivascular biodegradable microneedle cuff for reduction of neointima formation after vascular injury.

Restenosis often occurs at the site of vascular grafting and may become fatal for patients. Restenosis at anastomosis sites is due to neointimal hyperplasia (NH) and difficult to treat with conventional treatments. Such abnormal growth of smooth muscle cells in tunica media of vascular tissue can be reduced by delivering anti-proliferation drugs such as paclitaxel (PTX) to the inner vascular layer. Drug eluting stents (DES) or drug eluting balloon (DEB) have been developed to treat such vascular diseases. However, they are less efficient in drug delivery due to the drug loss to blood stream and inadequate to be applied to re-stenotic area in the presence of stent or anastomosis sites. Recently, we have introduced microneedle cuff (MNC) as perivascular delivery devices to achieve high delivery efficiency to tunica media. In this study, we investigated in vivo microneedle insertion and efficacy in treating NH using a rabbit balloon injury model. Microneedle shape was optimized for reliable insertion into tunica media layer. Uniform distribution of PTX in tunica media delivered by MNC devices was also confirmed. Animal study demonstrated significant NH reduction by MNC treatments and much higher delivery efficiency than flat type devices.

Spatially discrete thermal drawing of biodegradable microneedles for vascular drug delivery.

Spatially discrete thermal drawing is introduced as a novel method for the fabrication of biodegradable microneedles with ultra-sharp tip ends. This method provides the enhanced control of microneedle shapes by spatially controlling the temperature of drawn polymer as well as drawing steps and speeds. Particular focus is given on the formation of sharp tip ends of microneedles at the end of thermal drawing. Previous works relied on the fracture of polymer neck by fast drawing that often causes uncontrolled shapes of microneedle tips. Instead, this approach utilizes the surface energy of heated polymer to form ultra-sharp tip ends. We have investigated the effect of such temperature control, drawing speed, and drawing steps in thermal drawing process on the final shape of microneedles using biodegradable polymers. XRD analysis was performed to analyze the effect of thermal cycle on the biodegradable polymer. Load-displacement measurement also showed the dependency of mechanical strengths of microneedles on the microneedle shapes. Ex vivo vascular tissue insertion and drug delivery demonstrated microneedle insertion to tunica media layer of canine aorta and drug distribution in the tissue layer.

Curved biodegradable microneedles for vascular drug delivery.

Curved biodegradable microneedles for application to the outer surface of blood vessels are produced to enhance drug delivery to vascular tissues suffering from hyperplasia or atherosclerosis. Spatially discrete thermal drawing and post-annealing processes are employed to fabricate microneedles on a curved surface. Insertion of microneedles into arteries in vivo and ex vivo is demonstrated, and their mechanical properties and drug-delivery function are studied.