Selective NLRP3 Inflammasome Inhibitor MCC950 Suppresses Inflammation and Facilitates Healing in Vascular Materials.

Minimally invasive interventions using drug-eluting stents or balloons are a first-line treatment for certain occlusive cardiovascular diseases, but the major long-term cause of failure is neointimal hyperplasia (NIH). The drugs eluted from these devices are non-specific anti-proliferative drugs, such as paclitaxel (PTX) or sirolimus (SMS), which do not address the underlying inflammation. MCC950 is a selective inhibitor of the NLRP3-inflammasome, which drives sterile inflammation commonly observed in NIH. Additionally, in contrast to broad-spectrum anti-inflammatory drugs, MCC950 does not compromise global immune function due this selective activity. In this study, MCC950 is found to not impact the viability, integrity, or function of human coronary endothelial cells, in contrast to the non-specific anti-proliferative effects of PTX and SMS. Using an in vitro model of NLRP3-mediated inflammation in murine macrophages, MCC950 reduced IL-1ß expression, which is a key driver of NIH. In an in vivo mouse model of NIH in vascular grafts, MCC950 significantly enhanced re-endothelialization and reduced NIH compared to PTX or SMS. These findings show the effectiveness of a targeted anti-inflammatory drug-elution strategy with significant implications for cardiovascular device intervention.

Evaluation of the Immune Response to Chitosan-graft-poly(caprolactone) Biopolymer Scaffolds.

Biomimetic scaffolds recreating key elements of the architecture and biological activity of the extracellular matrix have enormous potential for soft tissue engineering applications. Combining appropriate mechanical properties with select biological cues presents a challenge for bioengineering, as natural materials are most bioactive but can lack mechanical integrity, while synthetic polymers have strength but are often biologically inert. Blends of synthetic and natural materials, aiming to combine the benefits of each, have shown promise but inherently require a compromise, diluting down favorable properties in each polymer to accommodate the other. Here, we electrospun a material comprising chitosan, a natural polysaccharide, and polycaprolactone (PCL), one of the most widely studied synthetic polymers used in materials engineering. In contrast to a classical blend, here PCL was chemically grafted onto the chitosan backbone to create chitosan-graft-polycaprolactone (CS-g-PCL) and then combined further with unmodified PCL to generate scaffolds with discreet chitosan functionalization. These small amounts of chitosan led to significant changes in scaffold architecture and surface chemistry, reducing the fiber diameter, pore size, and hydrophobicity. Interestingly, all CS-g-PCL-containing blends were stronger than control PCL, though with reduced elongation. In in vitro assessments, increasing the CS-g-PCL content led to significant improvements in in vitro blood compatibility compared to PCL alone while increasing fibroblast attachment and proliferation. In a mouse subcutaneous implantation model, a higher CS-g-PCL content improved the immune response to the implants. Macrophages in tissues surrounding CS-g-PCL scaffolds decreased proportionately to the chitosan content by up to 65%, with a corresponding decrease in pro-inflammatory cytokines. These results suggest that CS-g-PCL is a promising hybrid material comprising natural and synthetic polymers with tailorable mechanical and biological properties, justifying further development and in vivo evaluation.

Endothelial thioredoxin interacting protein (TXNIP) modulates endothelium-dependent vasorelaxation in hyperglycemia.

Endothelial dysfunction, hallmarked by an imbalance between vasoconstriction and vasorelaxation, is associated with diabetes. Thioredoxin Interacting protein (TXNIP), controlled by an exquisitely glucose sensitive gene, is increasingly recognized for its role in diabetes. However, the role of TXNIP in modulating diabetes-related endothelial dysfunction remains unclear. To elucidate the role of TXNIP, we generated two novel mouse strains; endothelial-specific TXNIP knockout (EKO) and a Tet-O inducible, endothelial-specific TXNIP overexpression (EKI). Hyperglycemia was induced by streptozotocin (STZ) treatment in floxed control (fl/fl) and EKO mice. Doxycycline (DOX) was given to EKI mice to induce endothelial TXNIP overexpression. The ablation of endothelial TXNIP improved glucose tolerance in EKO mice. Acetylcholine-induced, endothelium-dependent vasorelaxation was impaired in STZ-treated fl/fl mice while this STZ impaired vasorelaxation was attenuated in EKO mice. Hyperglycemia induction of NLRP3 and reductions in Akt and eNOS phosphorylation were also mitigated in EKO mice. Overexpression of endothelial TXNIP did not impair glucose tolerance in DOX-treated EKI mice, however induction of endothelial TXNIP led to impaired vasorelaxation in EKI mice. This was associated with increased NLRP3 and reduced Akt and eNOS activation. In conclusion, deletion of endothelial TXNIP is protective against and overexpression of endothelial TXNIP induces endothelial dysfunction; thus, endothelial TXNIP plays a critical role in modulating endothelial dysfunction.

Synthetic vascular graft with spatially distinct architecture for rapid biomimetic cell organisation in a perfusion bioreactor.

Access to lab-grown fully functional blood vessels would provide an invaluable resource to vascular medicine. The complex architecture and cellular makeup of native vessels, however, makes this extremely challenging to reproducein vitro. Bioreactor systems have helped advanced research in this area by replicating many of the physiological conditions necessary for full-scale tissue growth outside of the body. A key element underpinning these technologies are 3D vascular graft templates which serve as temporary scaffolds to direct cell growth into similar cellular architectures observed in native vessels. Grafts further engineered with appropriate physical cues to accommodate the multiple cell types that reside within native vessels may help improve the production efficiency and physiological accuracy of bioreactor-grown vessel substitutes. Here, we engineered two distinct scaffold architectures into an electrospun vascular graft aiming to encourage the spatial organisation of human vascular endothelial cells (hCAECs) in a continuous luminal monolayer, co-cultured with human fibroblasts (hFBs) populating the graft wall. Using an electrospun composite of polycaprolactone and gelatin, we evaluated physical parameters including fibre diameter, fibre alignment, and porosity, that best mimicked the spatial composition and growth of hCAECs and hFBs in native vessels. Upon identifying the optimal scaffold architectures for each cell type, we constructed a custom-designed mandrel that combined these distinct architectures into a single vascular graft during a single electrospinning processing run. When connected to a perfusion bioreactor system, the dual architecture graft spatially oriented hCAECs and hFBs into the graft wall and lumen, respectively, directly from circulation. This biomimetic cell organisation was consistent with positive graft remodelling with significant collagen deposition in the graft wall. These findings demonstrate the influence of architectural cues to direct cell growth within vascular graft templates and the future potential of these approaches to more accurately and efficiency produce blood vessel substitutes in bioreactor systems.

Bioengineering artificial blood vessels from natural materials.

Bioengineering an effective, small diameter (<6 mm) artificial vascular graft for use in bypass surgery when autologous grafts are unavailable remains a persistent challenge. Commercially available grafts are typically made from plastics, which have high strength but lack elasticity and present a foreign surface that triggers undesirable biological responses. Tissue engineered grafts, leveraging decellularized animal vessels or derived de novo from long-term cell culture, have dominated recent research, but failed to meet clinical expectations. More effective constructs that are readily translatable are urgently needed. Recent advances in natural materials have made the production of robust acellular conduits feasible and their use increasingly attractive. Here, we identify a subset of natural materials with potential to generate durable, small diameter vascular grafts.

Bioengineering silk into blood vessels.

The rising incidence of cardiovascular disease has increased the demand for small diameter (<6 mm) synthetic vascular grafts for use in bypass surgery. Clinically available synthetic grafts (polyethylene terephthalate and expanded polytetrafluorethylene) are incredibly strong, but also highly hydrophobic and inelastic, leading to high rates of failure when used for small diameter bypass. The poor clinical outcomes of commercial synthetic grafts in this setting have driven significant research in search of new materials that retain favourable mechanical properties but offer improved biocompatibility. Over the last several decades, silk fibroin derived from Bombyx mori silkworms has emerged as a promising biomaterial for use in vascular applications. Progress has been driven by advances in silk manufacturing practices which have allowed unprecedented control over silk strength, architecture, and the ensuing biological response. Silk can now be manufactured to mimic the mechanical properties of native arteries, rapidly recover the native endothelial cell layer lining vessels, and direct positive vascular remodelling through the regulation of local inflammatory responses. This review summarises the advances in silk purification, processing and functionalisation which have allowed the production of robust vascular grafts with promise for future clinical application.

Macrophage Polarization as a Novel Therapeutic Target for Endovascular Intervention in Peripheral Artery Disease.

Peripheral artery disease (PAD) has a significant impact on human health, affecting 200 million people globally. Advanced PAD severely diminishes quality of life, affecting mobility, and in its most severe form leads to limb amputation and death. Treatment of PAD is among the least effective of all endovascular procedures in terms of long-term efficacy. Chronic inflammation is a key driver of PAD; however, stents and coated balloons eluting antiproliferative drugs are most commonly used. As a result, neither stents nor coated balloons produce durable clinical outcomes in the superficial femoral artery, and both have recently been associated with significantly increased mortality. This review summarizes the most common clinical approaches and limitations to treating PAD and highlights the necessity to address the underlying causes of inflammation, identifying macrophages as a novel therapeutic target in the next generation of endovascular PAD intervention.

Silk Fibroin Scaffold Architecture Regulates Inflammatory Responses and Engraftment of Bone Marrow-Mononuclear Cells.

Despite being one of the most clinically trialed cell therapies, bone marrow-mononuclear cell (BM-MNC) infusion has largely failed to fulfill its clinical promise. Implanting biomimetic scaffolds at sites of injury prior to BM-MNC infusion is a promising approach to enhance BM-MNC engraftment and therapeutic function. Here, it is demonstrated that scaffold architecture can be leveraged to regulate the immune responses that drive BM-MNC engraftment. Silk scaffolds with thin fibers and low porosity (LP) impairs immune activation in vitro compared with thicker fiber, high porosity (HP) scaffolds. Using the authors' established in vivo bioluminescent BM-MNC tracking model, they showed that BM-MNCs home to and engraft in greater numbers in HP scaffolds over 14 days. Histological analysis reveals thicker fibrous capsule formation, with enhanced collagen deposition in HP compared to LP scaffolds consistent with substantially more native CD68+ macrophages and CD4+ T cells, driven by their elevated pro-inflammatory M1 and Th1 phenotypes, respectively. These results suggest that implant architecture impacts local inflammation that drives differential engraftment and remodeling behavior of infused BM-MNC. These findings inform the future design of biomimetic scaffolds that may better enhance the clinical effectiveness of BM-MNC infusion therapy.

Immobilized Macrophage Colony-Stimulating Factor (M-CSF) Regulates the Foreign Body Response to Implanted Materials.

The functionality and durability of implanted biomaterials are often compromised by an exaggerated foreign body reaction (FBR). M1/M2 polarization of macrophages is a critical regulator of scaffold-induced FBR. Macrophage colony-stimulating factor (M-CSF), a hematopoietic growth factor, induces macrophages into an M2-like polarized state, leading to immunoregulation and promoting tissue repair. In the present study, we explored the immunomodulatory effects of surface bound M-CSF on poly-l-lactic acid (PLLA)-induced FBR. M-CSF was immobilized on the surface of PLLA via plasma immersion ion implantation (PIII). M-CSF functionalized PLLA, PLLA-only, and PLLA+PIII were assessed in an IL-1ß luciferase reporter mouse to detect real-time levels of IL-1ß expression, reflecting acute inflammation in vivo. Additionally, these different treated scaffolds were implanted subcutaneously into wild-type mice to explore the effect of M-CSF in polarization of M2-like macrophages (CD68+/CD206+), related cytokines (pro-inflammatory: IL-1ß, TNF and MCP-1; anti-inflammatory: IL-10 and TGF-ß), and angiogenesis (CD31) by immunofluorescent staining. Our data demonstrated that IL-1ß activity in M-CSF functionalized scaffolds was ∼50% reduced compared to PLLA-only at day 1 (p < 0.01) and day 2 (p < 0.05) post-implantation. There were >2.6-fold more CD206+ macrophages in M-CSF functionalized PLLA compared to PLLA-only at day 7 (p < 0.001), along with higher levels of IL-10 at both day 7 (p < 0.05) and day 14 (p < 0.01), and TGF-ß at day 3 (p < 0.05), day 7 (p < 0.05), and day 14 (p < 0.001). Lower levels of pro-inflammatory cytokines were also detected in M-CSF functionalized PLLA in the early phase of the immune response compared to PLLA-only: a ∼58% decrease at day 3 in IL-1ß; a ∼91% decrease at day 3 and a ∼66% decrease at day 7 in TNF; and a ∼60% decrease at day 7 in MCP-1. Moreover, enhanced angiogenesis inside and on/near the scaffold was observed in M-CSF functionalized PLLA compared to PLLA-only at day 3 (p < 0.05) and day 7 (p < 0.05), respectively. Overall, M-CSF functionalized PLLA enhanced CD206+ macrophage polarization and angiogenesis, consistent with lower levels of pro-inflammatory cytokines and higher levels of anti-inflammatory cytokines in early stages of the host response, indicating potential immunoregulatory functions on the local environment.

Altered processing enhances the efficacy of small-diameter silk fibroin vascular grafts.

Current synthetic vascular grafts are not suitable for use in low-diameter applications. Silk fibroin is a promising natural graft material which may be an effective alternative. In this study, we compared two electrospun silk grafts with different manufacturing processes, using either water or hexafluoroisopropanol (HFIP) as solvent. This resulted in markedly different Young's modulus, ultimate tensile strength and burst pressure, with HFIP spun grafts observed to have thicker fibres, and greater stiffness and strength relative to water spun. Assessment in a rat abdominal aorta grafting model showed significantly faster endothelialisation of the HFIP spun graft relative to water spun. Neointimal hyperplasia in the HFIP graft also stabilised significantly earlier, correlated with an earlier SMC phenotype switch from synthetic to contractile, increasing extracellular matrix protein density. An initial examination of the macrophage response showed that HFIP spun conduits promoted an anti-inflammatory M2 phenotype at early timepoints while reducing the pro-inflammatory M1 phenotype relative to water spun grafts. These observations demonstrate the important role of the manufacturing process and physical graft properties in determining the physiological response. Our study is the first to comprehensively study these differences for silk in a long-term rodent model.

Rapid Endothelialization of Off-the-Shelf Small Diameter Silk Vascular Grafts.

Synthetic vascular grafts for small diameter revascularization are lacking. Clinically available conduits expanded polytetrafluorethylene and Dacron fail acutely due to thrombosis and in the longer term from neointimal hyperplasia. We report the bioengineering of a cell-free, silk-based vascular graft. In vitro we demonstrate strong, elastic silk conduits that support rapid endothelial cell attachment and spreading while simultaneously resisting blood clot and fibrin network formation. In vivo rat studies show complete graft patency at all time points, rapid endothelialization, and stabilization and contraction of neointimal hyperplasia. These studies show the potential of silk as an off-the-shelf small diameter vascular graft.

Glomeruli or interstitium targeted by inter-renal injections supplemented by electroporation: Still a useful tool in renal research.

BACKGROUND: Studies concerning proteins are always a crucial part of renal research. As a result of current technologies, scientists have mastered several techniques for generating genetically modified animals. However, in most cases, accessing these animals is still time-consuming and often expensive. This makes the alteration of protein expression by in vivo plasmid transfection an easily-accessible alternative. However, there is still no comprehensive study describing where plasmids would be expressed when they are injected into the kidneys. METHODS: We injected pEGFP-N1 into rats via intra-/inter-renal channels and detected green fluorescent protein (GFP) by immunohistochemistry and immunofluorescence to localize plasmid expression. RESULTS: Seven days post-injection, we found that GFP was expressed in the glomeruli when pEGFP-N1 was injected via the renal artery or vein enhanced by electroporation and in the interstitium following injection via the ureter. Other channels, including intraperitoneal, subcapsule and parenchymal injection, only led to scattered expression within the kidneys. CONCLUSIONS: The present study provides evidence that plasmid transfection via the renal vessels is suitable for glomeruli research and that transfection via the ureter is appropriate for studies regarding interstitium lesions. Additionally, we provide evidence that plasmid transfection on live animals is still an applicable and useful tool, as well as being cost-effective and facile.

Usp2-69 overexpression slows down the progression of rat anti-Thy1.1 nephritis.

Mesangial proliferative glomerulonephritis is characterized by proliferation of mesangial cells (MCs) and transforming growth factor-ß (TGF-ß)-dependent stimulation of abnormal extracellular matrix (ECM) accumulation. We previously showed that Decorin--a leucine-rich proteoglycan inhibiting the progression of glomerulonephritis and glomerular sclerosis--can be degraded by the ubiquitin-proteasome pathway and deubiquitinated and stabilized by ubiquitin-specific processing protease 2-69(Usp2-69). Usp2-69 is highly expressed in the kidney and has been implicated in the regulation of cell proliferation and apoptosis. However, its role in mesangial proliferative glomerulonephritis remains unclear. Here, we explored the effect of Usp2-69 on MC proliferation and ECM deposition by transfecting Usp2-69 plasmid into rat anti-Thy1.1 nephritis model and into cultured MCs, as well as detected Usp2-69 and Decorin in rat anti-Thy1.1 nephritis model by western blot. Overexpressing Usp2-69 at the early stage, but not advanced stage, of anti-Thy1.1 nephritis alleviated cell proliferation and ECM deposition, which was shown by decreased Ki-67, Collagen IV and Fibronectin detected by immunohistochemistry. Overexpression also increased Decorin and decreased TGF-ß1 and Collagen IV both in vitro and in vivo. In conclusion, our findings suggest that Usp2-69 overexpression alleviates the progression of rat anti-Thy1.1 nephritis and, therefore, that exogenous plasmid injection via the renal artery enhanced by electrotransfer technology could be a promising avenue for glomerular disease research.