Activation of Smad2/3 signaling by low fluid shear stress mediates artery inward remodeling.

Endothelial cell (EC) sensing of wall fluid shear stress (FSS) from blood flow governs vessel remodeling to maintain FSS at a specific magnitude or set point in healthy vessels. Low FSS triggers inward remodeling to restore normal FSS but the regulatory mechanisms are unknown. In this paper, we describe the signaling network that governs inward artery remodeling. FSS induces Smad2/3 phosphorylation through the type I transforming growth factor (TGF)-ß family receptor Alk5 and the transmembrane protein Neuropilin-1, which together increase sensitivity to circulating bone morphogenetic protein (BMP)-9. Smad2/3 nuclear translocation and target gene expression but not phosphorylation are maximal at low FSS and suppressed at physiological high shear. Reducing flow by carotid ligation in rodents increases Smad2/3 nuclear localization, while the resultant inward remodeling is blocked by the EC-specific deletion of Alk5. The flow-activated MEKK3/Klf2 pathway mediates the suppression of Smad2/3 nuclear translocation at high FSS, mainly through the cyclin-dependent kinase (CDK)-2-dependent phosphosphorylation of the Smad linker region. Thus, low FSS activates Smad2/3, while higher FSS blocks nuclear translocation to induce inward artery remodeling, specifically at low FSS. These results are likely relevant to inward remodeling in atherosclerotic vessels, in which Smad2/3 is activated through TGF-ß signaling.

Comparison of small-diameter decellularized scaffolds from the aorta and carotid artery of pigs.

AIM: Tissue-specific extracellular matrix promotes tissue regeneration and repair. We aimed to identify the optimal decellularized matrices for tissue-engineered vascular graft (TEVG). METHODS: Decellularized aorta of fetal pigs (DAFP, n = 6, group A), decellularized aorta of adult pigs (DAAP, n = 6, group B), and decellularized carotid artery of adult pigs (DCAP, n = 6, group C) were prepared. Scaffolds were compared using histology and ultrastructure. Endothelial cell (EC) and myofibroblast (MFB) infiltration assessments were performed in vitro. Cell infiltration was measured in vivo. Biomechanical properties were also determined. RESULTS: Almost original cells were removed by the acellularization procedure, while the construction of the matrix basically remained. In vitro, monolayer ECs and multi-layer MFBs were formed onto the internal surface of the specimens after 3 weeks. In vivo, cell infiltration in group A significantly increased at the 6th and 8th week when compared with groups B and C (p < 0.01). The infiltrated cells were mainly MFBs and a few CD4+ T-lymphocytes/macrophages in the specimens. Groups A and B showed greater axial compliance than group C (p < 0.01). CONCLUSION: DAFP was the most suitable for use as a small-caliber vascular graft.

WD Repeat Domain 1 Deficiency Inhibits Neointima Formation in Mice Carotid Artery by Modulation of Smooth Muscle Cell Migration and Proliferation.

The migration, dedifferentiation, and proliferation of vascular smooth muscle cells (VSMCs) are responsible for intimal hyperplasia, but the mechanism of this process has not been elucidated. WD repeat domain 1 (WDR1) promotes actin-depolymerizing factor (ADF)/cofilin-mediated depolymerization of actin filaments (F-actin). The role of WDR1 in neointima formation and progression is still unknown. A model of intimal thickening was constructed by ligating the left common carotid artery in Wdr1 deletion mice, and H&E staining showed that Wdr1 deficiency significantly inhibits neointima formation. We also report that STAT3 promotes the proliferation and migration of VSMCs by directly promoting WDR1 transcription. Mechanistically, we clarified that WDR1 promotes the proliferation and migration of VSMCs and neointima formation is regulated by the activation of the JAK2/STAT3/WDR1 axis.

A near-infrared light-triggered shape-memory polymer for long-time fluorescence imaging in deep tissues.

Implanting a stent in the body through a minimally invasive operation and tracking its location in real-time is still a challenge. Herein, a near-infrared (NIR) light-triggered shape-memory polymer possessing a long-time fluorescence imaging function has been developed by cross-linking 6-arm poly(ethylene glycol)-poly(ε-caprolactone) using a croconate dye YHD798 as the chemical crosslinker and NIR-absorption perssad. Due to the extraordinary photothermal conversion property of YHD798, the temperature of the material raised from 20 °C to 120 °C under 808 nm near-infrared irradiation at 0.4 W cm-2, leading to shape recovery in 50 s in a programmed shape-transition process. YHD798 also exerted an aggregation-induced emission effect, endowing the polymer with a clear NIR fluorescence imaging function even when covered by a 2 mm pork slab and could be used for the real-time visualization of the implanted device fabricated from this polymer in deep tissues of the body. When a tubular stent that was fabricated from this polymer, was implanted into the carotid artery of a Sprague-Dawley rat, it could recover to its permanent shape under 808 nm laser irradiation in 60 s owing to the shape-memory function and facilitated NIR-I fluorescence imaging after implantation for a week owing to the croconate dye. This work provides a new strategy for designing and developing smart polymers with NIR-light-triggered shape-memory effect and long-term fluorescence imaging function for biomedical applications.

Salvia miltiorrhiza-derived miRNAs suppress vascular remodeling through regulating OTUD7B/KLF4/NMHC IIA axis.

Objective: Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are essential for vascular remodeling. Natural compounds with diterpene chinone or phenolic acid structure from Salvia miltiorrhiza, an eminent medicinal herb widely used to treat cardiovascular diseases in China, can effectively attenuate vascular remodeling induced by vascular injury. However, it remains unknown whether Salvia miltiorrhiza-derived miRNAs can protect VSMCs from injury by environmental stimuli. Here, we explored the role and underlying mechanisms of Salvia miltiorrhiza-derived Sal-miR-1 and 3 in the regulation of VSMC migration and monocyte adhesion to VSMCs induced by thrombin. Methods: A mouse model for intimal hyperplasia was established by the ligation of carotid artery and the injured carotid arteries were in situ-transfected with Sal-miR-1 and 3 using F-127 pluronic gel. The vascular protective effects of Sal-miR-1 and 3 were assessed via analysis of intimal hyperplasia with pathological morphology. VSMC migration and adhesion were analyzed by the wound healing, transwell membrane assays, and time-lapse imaging experiment. Using loss- and gain-of-function approaches, Sal-miR-1 and 3 regulation of OTUD7B/KLF4/NMHC IIA axis was investigated by using luciferase assay, co-immunoprecipitation, chromatin immunoprecipitation, western blotting, etc. Results:Salvia miltiorrhiza-derived Sal-miR-1 and 3 can enter the mouse body after intragastric administration, and significantly suppress intimal hyperplasia induced by carotid artery ligation. In cultured VSMCs, these two miRNAs inhibit thrombin-induced the migration of VSMCs and monocyte adhesion to VSMCs. Mechanistically, Sal-miR-1 and 3 abrogate OTUD7B upregulation by thrombin via binding to the different sites of the OTUD7B 3'UTR. Most importantly, OTUD7B downregulation by Sal-miR-1 and 3 attenuates KLF4 protein levels via decreasing its deubiquitylation, whereas decreased KLF4 relieves its repression of transcription of NMHC IIA gene and thus increases NMHC IIA expression levels. Further, increased NMHC IIA represses VSMC migration and monocyte adhesion to VSMCs via maintaining the contractile phenotype of VSMCs. Conclusions: Our studies not only found the novel bioactive components from Salvia miltiorrhiza but also clarified the molecular mechanism underlying Sal-miR-1 and 3 inhibition of VSMC migration and monocyte adhesion to VSMCs. These results add important knowledge to the pharmacological actions and bioactive components of Salvia miltiorrhiza. Sal-miR-1 and 3-regulated OTUD7B/KLF4/NMHC IIA axis may represent a therapeutic target for vascular remodeling.

Airway reconstruction using decellularized aortic xenografts in a dog model.

Tracheal reconstruction after extensive resection remains a challenge in thoracic surgery. Aortic allograft has been proposed to be a potential tracheal substitute. However, clinically, its application is limited for the shortage of autologous aortic segment. Whether xenogeneic aortic biosheets can be used as tracheal substitutes remains unknown. In the present study, we investigated the possibility in dog model. The results show that all dogs were survived without airway symptoms at 6 months after tracheal reconstruction with gently decellularized bovine carotid arteries. In the interior of engrafted areas, grafted patch integrated tightly with the residual native tracheal tissues and tracheal defects in the lumen were repaired smoothly without obvious inflammation, granulation, anastomotic leakage, or stenosis. In addition, histological and scanning electron microscopy examination showed that grafted patches were covered with ciliated columnar epithelium similar to epithelium in native trachea, which indicated successfully re-epithelialization of decellularized bovine carotid arteries in dogs. These findings provide preclinical investigation of xenogeneic aortic biosheets in serving as tracheal substitute in a dog model, which proposes that decellularized biosheets of bovine carotid may be a potential material for bioartificial tracheal graft.

Characterization of carotid endothelial cell proliferation on Au, Au/GO, and Au/rGO surfaces by electrical impedance spectroscopy.

To the best of the authors' knowledge, testing the biocompatibility of graphene coatings can be considered as the first to demonstrate human carotid endothelial cell (HCtAEC) proliferation on Au, graphene oxide-coated Au (Au/GO), and reduced graphene oxide-coated Au (Au/rGO) surfaces. We hypothesized that stent material modified with graphene (G)-based coatings could be used as electrodes for electrical impedance spectroscopy (EIS) in monitoring cell cultures, i.e., endothelialization. Alamar Blue cell viability assay and cell staining and cell counting with optical images were performed. For EIS analysis, an EIS sensor consisting of Au surface electrodes was produced by the photolithographic technique. Surface characterizations were performed by considering scanning electron microscope (SEM) and water contact angle analyses. Results showed that GO and rGO coatings did not prevent neither the electrical measurements nor the cell proliferation and that rGO had a positive effect on HCtAEC proliferation. The rate of increase of impedance change from day 1 to day 10 was nearly fivefold for all electrode surfaces. Alamar Blue assay performed to monitor cell proliferation rates between groups, and rGO has shown the highest Alamar Blue reduction value of 43.65 ± 8.79%. Graphical abstract.

Long noncoding RNA SNHG12 promotes vascular smooth muscle cell proliferation and migration via regulating miR-199a-5p/HIF-1α.

The dysregulation of proliferation and migration of vascular smooth muscle cells (VSMCs) contributes to atherosclerosis (AS) and accumulating reports indicate the crucial role of long noncoding RNA in AS. However, the role of small nucleolar RNA host gene 12 (SNHG12) in regulating the phenotypes of VSMCs and AS remains largely unknown. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to detect the expression levels of SNHG12 and miR-199a-5p in an in vivo AS model and VSMCs treated by oxidized low-density lipoprotein (ox-LDL). The proliferation ability, migration ability, and apoptosis of VSMCs were tested by cell counting kit-8, Transwell assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, respectively. StarBase database was used to predict the binding sites between miR-199a-5p and SNHG12. The interaction between miR-199a-5p and SNHG12 was validated by qRT-PCR, western blot, and luciferase reporter assay. Western blot was used to examine the effects of SNHG12 and miR-199a-5p on the expression of hypoxia-inducible factor 1α (HIF-1α). We found that the expression level of SNHG12 was significantly increased in the animal model and VSMCs treated by ox-LDL. Knockdown of SNHG12 suppressed the proliferation and migration abilities of VSMCs, while overexpression of SNHG12 had the opposite effects. Mechanically, we validated that miR-199a-5p was a target of SNHG12, and the target gene of miR-199a-5p, HIF-1α could be indirectly and positively regulated by SNHG12. In conclusion, SHNG12 targeting miR-199a-5p/HIF-1α contributed to the pathophysiological process of AS by regulating the phenotypes of VSMCs, and could be a potential therapy target for this disease.

Decellularized equine carotid artery layers as matrix for regenerated neurites of spiral ganglion neurons.

Today's best solution in compensating for sensorineural hearing loss is the cochlear implant, which electrically stimulates the spiral ganglion neurons in the inner ear. An optimum hearing impression is not ensured due to, among other reasons, a remaining anatomical gap between the spiral ganglion neurons and the implant electrodes. The gap could be bridged via pharmacologically triggered neurite growth toward the electrodes if biomaterials for neurite guidance could be provided. For this, we investigated the suitability of decellularized tissue. We compared three different layers (tunica adventitia, tunica media, and tunica intima) of decellularized equine carotid arteries in a preliminary approach. Rat spiral ganglia explants were cultured on decellularized equine carotid artery layers and neurite sprouting was assessed quantitatively. Generally, neurite outgrowth was possible and it was most prominent on the intima (in average 83 neurites per spiral ganglia explants, followed by the adventitia (62 neurites) and the lowest growth on the media (20 neurites). Thus, decellularized equine carotid arteries showed promising effects on neurite regeneration and can be developed further as efficient biomaterials for neural implants in hearing research.

Differentiation of Neural Crest Stem Cells in Response to Matrix Stiffness and TGF-ß1 in Vascular Regeneration.

The neural crest stem cells derived from human induced pluripotent stem cells (iPSC-NCSCs) are a valuable autologous cell source for tissue engineering and regenerative medicine. In this study, we investigated how iPSC-NCSCs could be regulated to regenerate arteries by microenvironmental factors, including the physical factor of matrix stiffness, and the chemical factor of transforming growth factor beta-1 (TGF-ß1). We found that, compared to soft substrate, stiff substrate drove iPSC-NCSCs differentiation into smooth muscle cells, which was further enhanced by TGF-ß1. To investigate the regulatory role of TGF-ß1 in vivo, we fabricated vascular grafts composed of electrospun nanofibrous scaffolds, collagen gel, iPSC-NCSCs, and TGF-ß1, and implanted them into athymic rats. The results showed that TGF-ß1 significantly promoted extracellular matrix synthesis and increased mechanical strength of vascular grafts. This study presents a proof of concept that iPSC-NCSCs can be used as a promising autologous cell source for vascular regeneration when combined with physical and chemical engineering.

Decellularization, cross-linking and heparin immobilization of porcine carotid arteries for tissue engineering vascular grafts.

Tissue engineering vascular grafts (TEVGs) have the potential to replace small-diameter grafts in bypass surgery which is good news for patients with cardiovascular disease. Decellularized arteries can be ideal TEVGs because their natural three-dimensional structures support the migration of host cells and vascular remodeling. There are many methods for decellularization without a standard protocol. In this study, a combination of Triton X-100 and sodium dodecyl sulfate (SDS) were used to prepare decellularized arteries. However, decellularization may damage the biochemical and mechanical properties to some degree. We used the cross-linking agents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to improve mechanical properties and immobilize heparin to inhibit thrombogenesis. Histological analysis, scanning electron microscopy, biomechanical properties test, determination of immobilized heparin, active partial thrombin time assay, and subcutaneous embedding experiment were used to evaluate the efficiency of decellularization and the efficacy of heparinized cross-linked vascular scaffold. Results showed 1% Triton X-100 combined with 0.3% SDS can decellularize successfully. EDC and NHS cross-linking can improve the mechanical properties, reduce the inflammatory reaction and slow the degradation time. Heparin immobilized on the scaffolds can inhibit thrombogenesis effectively. This study indicated the heparinized cross-linked vascular scaffolds may be ideal scaffolds for TEVGs.

Static spatial growth restriction micropatterning of endothelial colony forming cells influences their morphology and gene expression.

BACKGROUND: Endothelialization of small diameter synthetic vascular grafts is a potential solution to the thrombosis and intimal hyperplasia that plague current devices. Endothelial colony forming cells, which are blood-derived and similar to mature endothelial cells, are a potential cell source. Anisotropic spatial growth restriction micropatterning has been previously shown to affect the morphology and function of mature endothelial cells in a manner similar to unidirectional fluid shear stress. To date, endothelial colony forming cells have not been successfully micropatterned. This study addresses the hypothesis that micropatterning of endothelial colony forming cells will induce morphological elongation, cytoskeletal alignment, and changes in immunogenic and thrombogenic-related gene expression. METHODS: Spatially growth restrictive test surfaces with 25 µm-wide lanes alternating between collagen-I and a blocking polymer were created using microfluidics. Case-matched endothelial colony forming cells and control mature carotid endothelial cells were statically cultured on either micropatterned or non-patterned surfaces. Cell elongation was quantified using shape index. Using confocal microscopy, cytoskeletal alignment was visualized and density and apoptotic rate were determined. Gene expression was measured using quantitative PCR to measure KLF-2, eNOS, VCAM-1, and vWF. RESULTS: Endothelial colony forming cells were successfully micropatterned for up to 50 hours. Micropatterned cells displayed elongation and actin alignment. Micropatterning increased the packing densities of both cell types, but did not affect apoptotic rate, which was lower in endothelial colony forming cells. KLF-2 gene expression was increased in micropatterned relative to non-patterned endothelial colony forming cells after 50 hours. No significant differences were seen in the other genes tested. CONCLUSIONS: Endothelial colony forming cells can be durably micropatterned using spatial growth restriction. Micropatterning has a significant effect on the gross and subcellular morphologies of both cell types. Further study is required to fully understand the effect of micropatterning on endothelial colony forming cell gene expression.

[The transplantation of bone marrow mesenchymal stem cells pretreated with stromal cell-derived factor-1 alleviates the proliferation of endothelium and smooth muscle cells of carotid artery in rats].

Objective To investigating the effect of bone marrow mesenchymal stem cells (BMSCs) pretreated with stromal cell-derived factor-1 (SDF-1) on carotid stenosis in rats. Methods The plasmid carrying enhanced green fluorescent protein (EGFP) was transfected into BMSCs and then intravenously injected into rats. The rats were divided into carotid artery injury model control group, BMSCs transplantation group, and BMSCs pretreated with SDF-1 group. Two weeks after the cell transplantation, the injured vascular tissues were collected and EGFP expression was detected by immunofluorescence histochemistry to determine the homing of BMSCs. Four weeks after cell transplantation, the endothelialization of injured intima was observed by Evans blue staining, and CD31 expression in injured vessels was detected by the immunofluorescence technique. The neointimal hyperplasia of injured carotid arteries was observed by HE staining. The expression of proliferating cell nuclear antigen (PCNA) in injured vessels was detected by immunohistochemical staining. The protein level of vascular endothelial growth factor (VEGF) was tested by Western blot analysis. Results The transplantation of BMSCs pretreated with SDF-1 could effectively promote the homing of BMSCs to injured blood vessels, and promote the re-endothelialization of injured vessels. The neointimal area and neointimal area/medial area in the two BMSC-transplantation groups were both lower than those in the model control group, which were more significantly different from the BMSCs pretreated with SDF-1 group. The transplantation of BMSCs pretreated with SDF-1 could significantly increase the expression of CD31 and VEGF in injured intima and inhibit the expression of PCNA. Conclusion The transplantation of BMSCs pretreated with SDF-1 can inhibit the proliferation of smooth muscle cells in the media and reduce arterial stenosis by promoting the migration of BMSCs to the injured site and inducing the differentiation of BMSCs.

A vascular smooth muscle cell X-box binding protein 1 and transglutaminase 2 regulatory circuit limits neointimal hyperplasia.

Neointimal hyperplasia, stimulated by injury and certain vascular diseases, promotes artery obstruction and tissue ischemia. In vascular smooth muscle cell (VSMCs), multiple modulators of protein handling machinery regulate intimal hyperplasia. These include elements of the VSMC unfolded protein response to endoplasmic reticulum stress (UPRER), and transglutaminase 2 (TG2), which catalyzes post-translational protein modification. Previous results for deficiency of UPRER-specific mediator XBP1, and of TG2, have been significant, but in multiple instances contradictory, for effects on cultured VSMC function, and, using multiple models, for neointimal hyperplasia in vivo. Here, we engineered VSMC-specific deficiency of XBP1, and studied cultured VSMCs, and neointimal hyperplasia in response to carotid artery ligation in vivo. Intimal area almost doubled in Xbp1fl/fl SM22α-CRE+ mice 21 days post-ligation. Cultured murine Xbp1 deficient VSMCs migrated more in response to platelet derived growth factor (PDGF) than control VSMCs, and had an increased level of inositol-requiring enzyme 1α (Ire1α), a PDGF receptor-binding UPRER transmembrane endonuclease whose substrates include XBP1. Cultured XBP1-deficient VSMCs demonstrated decreased levels of TG2 protein, in association with increased TG2 polyubiquitination, but with increased TG transamidation catalytic activity. Moreover, IRE1α, and TG2-specific transamidation cross-links were increased in carotid artery neointima in Xbp1fl/fl SM22α-CRE+ mice. Cultured TG2-deficient VSMCs had decreased XBP1 associated with increased IRE1α, and increased migration in response to PDGF. Neointimal hyperplasia also was significantly increased in Tgm2fl/fl SM22α-CRE+ mice at 21 days after carotid ligation. In conclusion, a VSMC regulatory circuit between XBP1 and TG2 limits neointimal hyperplasia in response to carotid ligation.

Combination of freeze-thaw with detergents: A promising approach to the decellularization of porcine carotid arteries.

BACKGROUND: A tissue engineering technique based on use of the extracellular matrix (ECM) as a scaffold shows great potential for preparing small-caliber vascular grafts. Decellularization protocols are still not standardized for bioengineering. The effects of freeze-thaw cycles used for decellularization are unknown. OBJECTIVE: To evaluate the effects of freeze-thaw cycles on porcine carotid arteries during decellularization and to develop a promising protocol for preparing ECM scaffolds. METHODS: Porcine carotid arteries were decellularized with freeze-thaw cycles followed by three different chemical protocols. Histological analysis, scanning electron microscopy, mechanical tests and pore size measurement were performed to assess their effects on the ECM. RESULTS: The composition, structure, and mechanical properties were not significantly changed after freeze-thaw cycles, with the exception of endothelial cells loss. Freeze-thaw led to a porous structure within arteries. The use of Triton X-100 followed by sodium dodecyl sulfate (SDS) resulted in ECM scaffolds with well-preserved composition, structure, and mechanical properties, as well as with adequate porosity. CONCLUSIONS: As the initial step for decellularization, freeze-thaw had little impact on arteries. Decellularized porcine carotid arteries, prepared using freeze-thaw cycles followed by treatment with Triton X-100 and SDS, may serve as a promising biological scaffold as a tissue-engineered vascular graft.

circDiaph3 regulates rat vascular smooth muscle cell differentiation, proliferation, and migration.

Intimal hyperplasia is a reaction to vascular injury, which is the primary reason for vascular restenosis caused by the diagnostic or therapeutic procedure for cardiovascular diseases. Circular RNAs (circRNAs) are known to be associated with several cardiovascular conditions, but the expression of circRNAs in the neointima has not been reported in detail. In this study, we established the balloon-injured rat carotid artery model and detected the expression of circRNAs in the carotid arteries with a microarray. We found that the circRNA expression profile of the healthy carotid arteries and the injured arteries were significantly different. We investigated the role of rno-circ_005717 ( circDiaph3) in the differentiation of rat vascular smooth muscle cells (VSMCs). We found that knockdown of circDiaph3 up-regulated the level of diaphanous-related formin-3 and promoted the differentiation of VSMCs to contractile type. In addition, circDiaph3 up-regulated the transcription of Igf1r and supported the proliferation and migration of VSMCs. circDiaph3 could be a molecular target to combat intimal hyperplasia.-Xu, J.-Y., Chang, N.-B., Rong, Z.-H., Li, T., Xiao, L., Yao, Q.-P., Jiang, R., Jiang, J. circDiaph3 regulates rat vascular smooth muscle cell differentiation, proliferation, and migration.

Biofunctionalized Electrospun PCL-PIBMD/SF Vascular Grafts with PEG and Cell-Adhesive Peptides for Endothelialization.

Artificial small-caliber vascular grafts are still limited in clinical application because of thrombosis, restenosis, and occlusion. Herein, a small-caliber vascular graft (diameter 2 mm) is fabricated from poly(ε-caprolactone)-b-poly(isobutyl-morpholine-2,5-dione) (PCL-PIBMD) and silk fibroin (SF) by electrospinning technology and then biofunctionalized with low-fouling poly(ethylene glycol) (PEG) and two cell-adhesive peptide sequences (CREDVW and CAGW) with the purpose of enhancing antithrombogenic activity and endothelialization. The successful grafting of PEG and peptide sequences is confirmed by X-ray photoelectron spectroscopy. The suitable surface wettability of the modified vascular graft is testified by water contact angle analysis. The surface hemocompatibility is verified by platelet adhesion assays and protein adsorption assays, and the results demonstrate that both platelet adhesion and protein adsorption on the biofunctionalized surface are significantly reduced. In vitro studies demonstrate that the biofunctionalized surface with suitable hydrophilicity and cell-adhesive peptides can selectively promote the adhesion, spreading, and proliferation of human umbilical vein endothelial cells. More importantly, compared with control groups, this biofunctionalized small-caliber vascular graft shows high long-term patency and endothelialization after 10 weeks of implantation. The biofunctionalization with PEG and two cell-adhesive peptide sequences is an effective method to improve the endothelialization and long-term performance of synthetic vascular grafts.

Inhibitory effects of growth differentiation factor 11 on autophagy deficiency-induced dedifferentiation of arterial smooth muscle cells.

Growth differentiation factor (GDF)11 has been reported to reverse age-related cardiac hypertrophy in mice and cause youthful regeneration of cardiomyocytes. The present study attempted to test a hypothesis that GDF11 counteracts the pathologic dedifferentiation of mouse carotid arterial smooth muscle cells (CASMCs) due to deficient autophagy. By real-time RT-PCR and Western blot analysis, exogenously administrated GDF11 was found to promote CASMC differentiation with increased expression of various differentiation markers (α-smooth muscle actin, myogenin, myogenic differentiation, and myosin heavy chain) as well as decreased expression of dedifferentiation markers (vimentin and proliferating cell nuclear antigen). Upregulation of the GDF11 gene by trichostatin A (TSA) or CRISPR-cas9 activating plasmids also stimulated the differentiation of CASMCs. Either GDF11 or TSA treatment blocked 7-ketocholesterol-induced CASMC dedifferentiation and autophagosome accumulation as well as lysosome inhibitor bafilomycin-induced dedifferentiation and autophagosome accumulation. Moreover, in CASMCs from mice lacking the CD38 gene, an autophagy deficiency model in CASMCs, GDF11 also inhibited its phenotypic transition to dedifferentiation status. Correspondingly, TSA treatment was shown to decrease GDF11 expression and reverse CASMC dedifferentiation in the partial ligated carotid artery of mice. The inhibitory effects of TSA on dedifferentiation of CASMCs were accompanied by reduced autophagosome accumulation in the arterial wall, which was accompanied by attenuated neointima formation in partial ligated carotid arteries. We concluded that GDF11 promotes CASMC differentiation and prevents the phenotypic transition of these cells induced by autophagosome accumulation during different pathological stimulations, such as Western diet, lysosome function deficiency, and inflammation. NEW & NOTEWORTHY The present study demonstrates that growth differentiation factor (GDF)11 promotes autophagy and subsequent differentiation in carotid arterial smooth muscle cells. Upregulation of GDF11 counteracts dedifferentiation under different pathological conditions. These findings provide novel insights into the regulatory role of GDF11 in the counteracting of sclerotic arterial diseases and also suggest that activation or induction of GDF11 may be a new therapeutic strategy for the treatment or prevention of these diseases.

IL-1ß/MMP9 activation in primary human vascular smooth muscle-like cells: Exploring the role of TNFα and P2X7.

BACKGROUND: Vascular smooth muscle cells exhibit phenotypic plasticity in response to microenvironmental stimuli and contribute to vascular remodelling through mechanisms only partially understood. In atherosclerosis, P2X-purinoceptor7 (P2X7) has been related to interleukin-1ß (IL-1ß) and metalloproteinase 9 (MMP9). The hypoxia-inducible factor-1alpha (HIF1α) was associated to remodelling. Here the activation of IL-1ß and MMP9 was studied in relationship to P2X7 and HIF1α in cells exploited from human carotid plaque and internal mammary artery. METHODS AND RESULTS: Migrating cells expressed HIF1α-regulated canopy FGF-signalling regulator 2 and CD117, and led to primary cells with SMC-like phenotype (VSMC), P2X7+. We investigated in VSMC the effects of hypoxia, of treatment with tumour necrosis factor-α (TNFα) and/or with P2X7 antagonist, A740003. Quantitative RT-PCR showed that hypoxia unaffected IL-1ß and down-regulated MMP9 mRNAs, without activating HIF1α. TNFα increased IL-1ß mRNA via NLR Family Pyrin Domain-Containing 3, with production of proIL-1ß but no rise of mature IL-1ß. Zymography demonstrated that A740003 triggered MMP9 secretion from VSMC. Combination of A740003 with TNFα abrogated this effect. Combination was ineffective on IL-1ß activation elicited by TNFα, but down-regulated HIF1α mRNA. A740003 induced the intracellular P2X7 aggregation and differently perturbed lysosome and mitochondria network compared to TNFα. CONCLUSIONS: Cells migration from human arteries leads to partially differentiated VSMC analogous to neointimal cells within atherosclerotic lesions. Down-regulated HIF1α in stimulated VSMC translates in resilience in atherosclerotic lesions. P2X7-independent partial activation of IL-1ß elicited by TNFα underlines complexity of the cytokine secretion. Data also supported P2X7 as modulator of MMP9 secretion, important for atherosclerosis progression.

Facile preparation of a controlled-release tubular scaffold for blood vessel implantation.

Salvianic acid-loaded mesoporous silica nanoparticles into gelatin/polyurethane bilayered small-diameter tubular scaffold were prepared by thermally induced phase separation (TIPS) and electrospinning. Mesoporous silica nanoparticles (MSNs) were selected as carriers to load salvianic acid (SAL). The SAL-loaded MSNs (SAL@MSNs) with an optimized SAL loading efficiency of 10% was initially dispersed in gelatin solution and under a vacuum freeze-drying process as an inner layer of vascular scaffolds. Then, poly(ester-urethane)urea (C-PEEUU) nanofibers were electrospun outside the SAL@MSNs/Gelatin vascular scaffold to strengthen the spongy matrix. The loaded SAL within the MSNs/Gelatin/C-PEEUU bilayered small-diameter tubular scaffold showed a sustained release profile and good mechanical properties. In addition, the drug-loaded composite scaffold showed no unfavorable effects on the adhesion and proliferation of endothelial cells. Moreover, no intimal hyperplasia and acute thrombosis was observed in the short-term implantation in rabbit's carotid artery. We believe the SAL@MSNs/Gelatin/C-PEEUU bilayered vascular scaffolds have promise for vascular tissue engineering applications.