Recent Advances in the Modification and Improvement of Bioprosthetic Heart Valves.

Valvular heart disease (VHD) has become a burden and a growing public health problem in humans, causing significant morbidity and mortality worldwide. An increasing number of patients with severe VHD need to undergo heart valve replacement surgery, and artificial heart valves are in high demand. However, allogeneic valves from donors are lacking and cannot meet clinical practice needs. A mechanical heart valve can activate the coagulation pathway after contact with blood after implantation in the cardiovascular system, leading to thrombosis. Therefore, bioprosthetic heart valves (BHVs) are still a promising way to solve this problem. However, there are still challenges in the use of BHVs. For example, their longevity is still unsatisfactory due to the defects, such as thrombosis, structural valve degeneration, calcification, insufficient re-endothelialization, and the inflammatory response. Therefore, strategies and methods are needed to effectively improve the biocompatibility and longevity of BHVs. This review describes the recent research advances in BHVs and strategies to improve their biocompatibility and longevity.

Clinical incidence and relevance of incomplete endothelialization in atrial fibrillation patients with Left Atrial Appendage Closure.

BACKGROUND: The objective of this study is to investigate the incidence, potential risk factors, and clinical outcomes of incomplete device endothelialization (IDE) in atrial fibrillation (AF) patients undergoing Watchman left atrial appendage closure (LAAC). METHODS: In this study, 68 AF patients who underwent successful implantation of the Watchman device without peri-device leak (PDL) during follow-up were included. The endothelialization status was assessed using Transesophageal echocardiography (TEE) and LAA computed tomography angiography (CTA) at 6 weeks and 6 months post-implantation. Adverse cerebro-cardiac events were documented at one-year follow-up. Baseline characteristics, including age, device sizes, and clinical indicators, were analyzed as potential predictors for IDE. RESULTS: IDE was observed in 70.6% and 67.6% of patients at 6 weeks and 6 months after implantation, respectively. Higher levels of high-density lipoprotein cholesterol (HDL-C) [odds ratio (OR): 15.109, 95% confidence interval (CI): 1.637-139.478, p = 0.017 and OR: 11.015, 95% CI: 1.365-88.896, p = 0.024] and lower aspartate aminotransferase (AST) (OR 0.924, 95% CI: 0.865-0.986, p = 0.017 and OR: 0.930, 95% CI: 0.874-0.990, p = 0.023) at baseline were found to be significantly associated with IDE at 6 weeks and 6 months, respectively, although no significant difference in adverse cerebro-cardiac events was noted between incomplete and complete DE groups during 1-year follow-up CONCLUSIONS: IDE is found to be a prevalent occurrence in humans following LAAC. Elevated HDL-C and reduced AST levels are shown to be linked to an increased risk of IDE after LAAC.

Multifunctional nanocoatings with synergistic controlled release of zinc ions and cytokines for precise modulation of vascular intimal reconstruction.

Vascular stent implantation remains the major therapeutic method for cardiovascular diseases currently. We here introduced crucial biological functional biological function factors (SDF-1α, VEGF) and vital metal ions (Zn2+) into the stent surface to explore their synergistic effect in the microenvironment. The combination of the different factors is known to effectively regulate cellular inflammatory response and selectively regulate cell biological behavior. Meanwhile, in the implemented method, VEGF and Zn2+ were loaded into heparin and poly-l-lysine (Hep-PLL) nanoparticles, ensuring a controlled release of functional molecules with a multi-factor synergistic effect and excellent biological functions in vitro and in vivo. Notably, after 150 days of implantation of the modified stent in rabbits, a thin and smooth new intima was obtained. This study offers a new idea for constructing a modified surface microenvironment and promoting tissue repair.

Biofabrication of engineered blood vessels for biomedical applications.

To successfully engineer large-sized tissues, establishing vascular structures is essential for providing oxygen, nutrients, growth factors and cells to prevent necrosis at the core of the tissue. The diameter scale of the biofabricated vasculatures should range from 100 to 1,000 µm to support the mm-size tissue while being controllably aligned and spaced within the diffusion limit of oxygen. In this review, insights regarding biofabrication considerations and techniques for engineered blood vessels will be presented. Initially, polymers of natural and synthetic origins can be selected, modified, and combined with each other to support maturation of vascular tissue while also being biocompatible. After they are shaped into scaffold structures by different fabrication techniques, surface properties such as physical topography, stiffness, and surface chemistry play a major role in the endothelialization process after transplantation. Furthermore, biological cues such as growth factors (GFs) and endothelial cells (ECs) can be incorporated into the fabricated structures. As variously reported, fabrication techniques, especially 3D printing by extrusion and 3D printing by photopolymerization, allow the construction of vessels at a high resolution with diameters in the desired range. Strategies to fabricate of stable tubular structures with defined channels will also be discussed. This paper provides an overview of the many advances in blood vessel engineering and combinations of different fabrication techniques up to the present time.

This review covers several aspects and advancements of engineered blood vessel biofabrication, which are essential for establishment of large-sized tissues in different areas of biomedical applications.

Effects of exenatide on coronary stent's endothelialization in subjects with type 2 diabetes: a randomized controlled trial. The Rebuild study.

BACKGROUND: Subjects with type 2 diabetes (T2D) have a higher risk of in-stent restenosis and stent thrombosis. The activation of the glucagon-like peptide-1 receptor (GLP-1R) has been suggested to induce several effects on the vasculature that may reduce the risk of stent failure following an angioplasty. The aim of this study is to evaluate the effect of the GLP-1R agonist exenatide on endothelialization of a modern drug-eluting stent (DES) in subjects with T2D. METHODS: 38 subjects with T2D who were eligible for revascularization with implantation of DES were randomized to treatment with exenatide (once weekly) plus standard treatment, or to standard treatment alone. After 12 weeks, a new coronary angiography was performed to evaluate the percentage of strut coverage (primary endpoint) and the presence of neo-atherosclerosis by optical coherence tomography. This study was approved by the Stockholm's Ethical Review Board. RESULTS: The two groups were well balanced regarding baseline clinical characteristics. Strut coverage was 95% (88.7-98.5%) in the exenatide group and 91.4% (88.8-98.5%) in the control group (p = 0.692). There were no significant differences between groups neither in the thickness of neo-intima (0.2 mm in both groups, p = 0.471), nor the maximal in-stent obstruction by neo-intima (15.5% in exenatide group vs 14.7% in control group, p = 0.801). No significant differences were detected in the rate of target lesion revascularization between groups (p = 0.224). CONCLUSION: Twelve weeks treatment with exenatide did not lead to a significantly better stent coverage in people with T2D. No significant differences in the occurrence of neo-atherosclerosis were detected between groups. TRIAL REGISTRATION: The study was registered at www. CLINICALTRIALS: gov (Rebuild Study, NCT02621489).

MicroRNA-17-5p, a novel endothelial cell modulator, controls vascular re-endothelialization and neointimal lesion formation.

BACKGROUND: The functions of miR-17-5p in tumorigenesis have been explored. However, their functionalities in arterial endothelial cells (ECs) have not been investigated. Besides, the issue of vascular remodelling is barely addressed. OBJECTIVES: The study aimed to determine the effect of overexpression or inhibition of miR-17-5p on arterial endothelial cells' (ECs) function and vascular remodelling in vitro and the rat carotid arteries model. METHODS: Quantitative RT-PCR analysis was performed to examine the expression of miR-17-5p. Then, gain-of-function and loss-of-function approaches were employed to investigate the functional roles of miR-17-5p in cultured human coronary artery endothelial cells (HCAECs); further, TargetScan software analysis and luciferase reporter activity assay were performed to investigate the potential mechanism. Lastly, the results of the cell segment were verified in a rat carotid artery balloon injury model by Western blot analysis, measurement of the vascular cGMP level and plasma 8-iso-prostaglandin F2 (8-iso-PGF2) testing. Moreover, morphometric analysis was implemented to detect the re-endothelialization and neointimal formation in rat carotid artery after balloon injury. RESULTS: This study firstly found that miR-17-5p expression was upregulated in the injured vascular walls and highly expressive in ECs; overexpression of miR-17-5p inhibited HCAECs' proliferation and migration, whereas miR-17-5p knockdown strengthened its proliferative and migratory roles, influenced inflammatory response, through regulating VEGRA and VEGFR2. It was found that miR-17-5p bind to VEGFA and VEGFR2 at the 3'UTR. Next, downregulation of miR-17-5p promotes re-endothelialization, and attenuates neointimal formation as measured by the I/M ratio (0.63±0.05 vs 1.45±0.06, antagomiR-17-5p vs. Lenti-NC, p < 0.05). In addition, the functional recovery of the endothelium was also accelerated by miR-17-5p knockdown. CONCLUSION: Our study suggests that miR-17-5p is a feasible strategy for the selective modulation of endothelialization and vascular remodelling through regulating VEGFA and VEGFR2.

IMP3 promotes re-endothelialization after arterial injury via increasing stability of VEGF mRNAhv.

IMP3, an RNA-binding protein (RBP) that participates in the process of post-transcriptional modifications of mRNA transcripts, is capable of altering cellular functions, and in some cases, be involved in specific disease progression. We aimed to investigate whether IMP3 has the ability to regulate the functional properties of endothelial cells and re-endothelialization in response to arterial injury. Wire injury was introduced to the right carotid arteries of wildtype C57/BL6 mice. As a result, IMPs' expressions were up-regulated in the induced arterial lesions, and IMP3 was the most up-regulated RNA among other IMPs. We overexpressed IMP3 before the wire-injured surgery using adeno-associated virus AAV2-IMP3. In vivo studies confirmed that IMP3 overexpression accelerated the progress of re-endothelialization after arterial injury. In vitro, endothelial cells were transfected with either ad-IMP3 or Si-IMP3, cell functional studies showed that IMP3 could promote endothelial cell proliferation and migration, while reducing apoptosis. Mechanistic studies also revealed that IMP3 could enhance VEGF mRNA stability and therefore up-regulate activities of VEGF/PI3K/Akt signalling pathway. Our data indicated that IMP3 promotes re-endothelialization after arterial injury and regulates endothelial cell proliferation, migration and apoptosis via increasing stability of VEGF mRNA and activation of VEGF/PI3K/Akt signalling pathway.

GDF11 alleviates neointimal hyperplasia in a rat model of artery injury by regulating endothelial NLRP3 inflammasome activation and rapid re-endothelialization.

BACKGROUND: Neointimal hyperplasia induced by interventional surgery can lead to progressive obliteration of the vascular lumen, which has become a major factor affecting prognosis. The rate of re-endothelialization is known to be inversely related to neointima formation. Growth differentiation factor 11 (GDF11) is a secreted protein with anti-inflammatory, antioxidant, and antiaging properties. Recent reports have indicated that GDF11 can improve vascular remodeling by maintaining the differentiated phenotypes of vascular smooth muscle cells. However, it is not known whether and how GDF11 promotes re-endothelialization in vascular injury. The present study was performed to clarify the influence of GDF11 on re-endothelialization after vascular injury. METHODS: An adult Sprague-Dawley rat model of common carotid artery balloon dilatation injury was surgically established. A recombinant adenovirus carrying GDF11 was delivered into the common carotid artery to overexpress GDF11. Vascular re-endothelialization and neointima formation were assessed in harvested carotid arteries through histomolecular analysis. CCK-8 analysis, LDH release and Western blotting were performed to investigate the effects of GDF11 on endothelial NLRP3 inflammasome activation and relevant signaling pathways in vitro. RESULTS: GDF11 significantly enhanced re-endothelialization and reduced neointima formation in rats with balloon-dilatation injury by suppressing the activation of the NLRP3 inflammasome. Administration of an endoplasmic reticulum stress (ER stress) inhibitor, 4PBA, attenuated endothelial NLRP3 inflammasome activation induced by lysophosphatidylcholine. In addition, upregulation of LOX-1 expression involved elevated ER stress and could result in endothelial NLRP3 inflammasome activation. Moreover, GDF11 significantly inhibited NLRP3 inflammasome-mediated endothelial cell pyroptosis by negatively regulating LOX-1-dependent ER stress. CONCLUSIONS: We conclude that GDF11 improves re-endothelialization and can attenuate vascular remodeling by reducing endothelial NLRP3 inflammasome activation. These findings shed light on new treatment strategies to promote re-endothelialization based on GDF11 as a future target.

Peripheral blood derived endothelial colony forming cells as suitable cell source for pre-endothelialization of arterial vascular grafts under dynamic flow conditions.

In regenerative medicine, autologous peripheral blood derived endothelial colony forming cells (PB-derived ECFC) represent a promising source of endothelial cells (EC) for pre-endothelialization of arterial tissue engineered vascular grafts (TEVG) since they are readily attainable, can easily be isolated and possess a high proliferation potential. The aim of this study was to compare the phenotype of PB-derived ECFC with arterial and venous model cells such as human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) under dynamic cell culture conditions to find a suitable cell source of EC for pre-endothelialization. In this study PB-derived ECFC were cultivated over 24 h under a high pulsatile shear stress (20 dyn/cm2, 1 Hz) and subsequently analyzed. ECFC oriented and elongated in the direction of flow and expressed similar anti-thrombotic and endothelial differentiation markers compared to HAEC. There were significant differences observable in gene expression levels of CD31, CD34 and NOTCH4 between ECFC and HUVEC. These results therefore suggest an arterial phenotype for PB-derived ECFC both under static and flow conditions, and this was supported by NOTCH4 protein expression profiles. ECFC also significantly up-regulated gene expression levels of anti-thrombotic genes such as krueppel-like factor 2, endothelial nitric oxide synthase 3 and thrombomodulin under shear stress cultivation as compared to static conditions. Dynamically cultured PB-derived ECFC therefore may be a promising cell source for pre-endothelialization of arterial TEVGs.

Is cell regeneration and infiltration a double edged sword for porcine aortic valve deterioration? A large cohort of histopathological analysis.

BACKGROUND AND OBJECTIVE: Bioprostheses are the most common prostheses used for valve replacement in the Western medicine. The major flaw of bioprostheses is the occurrence of structural valve deterioration (SVD). This study aimed to assess the pathological features of porcine aortic valve (PAV)-SVD based on histomorphological and immunopathological characteristics of a large cohort of patients. METHODS: Histopathological data of 109 cases with resected PAV were collected. The type and amount of infiltrated cells were evaluated in the different types of bioprosthetic SVD by immunohistochemical staining. RESULTS: The most common cause of SVD was calcification, leaflet tear, and dehiscence (23.9%, 19.3%, and 18.3%, respectively). Immunohistochemical staining demonstrated that macrophages were infiltrated in the calcified, lacerated and dehiscence PAV, in which both M1 and M2 macrophages were existed in the calcified PAV. Importantly, the higher content of M1 macrophages and less content of M2 macrophages were found in the lacerated and dehiscence PAV, and MMP-1 expression was mainly found in the lacerated PAV. The endothelialization rate of leaflet dehiscence was higher than that of calcified and lacerated leaflets. A large number of CD31+/CD11b+ cells was aggregated in the spongy layer in the lacerated and dehiscence PAV. CONCLUSION: Cell regeneration and infiltration is a double edged sword for the PAV deterioration. Macrophage infiltration is involved in the different types of SVD, while only MMP-1 expression is involved in lacerated leaflets. The macrophage subtype of circulating angiogenic cells in dehiscence and tear PAV could be identified, which could reserve macrophages in the PAV-SVD.

Endothelialization and Inflammatory Reactions After Intracardiac Device Implantation.

BACKGROUND: Due to the advances in catheter-based interventional techniques, a wide range of heart diseases can now be treated with a purely interventional approach. Little is yet known regarding biological effects at the intracardiac implantation site or the effects on endothelialization and vascular inflammation in an in vivo environment. Detailed knowledge of ongoing vascular response, the process of endothelialization, and possible systemic inflammatory reactions after implantation is crucial for the clinical routine, since implants usually remain in the body for a lifetime. METHODS: For this narrative review, we conducted an extensive profound PubMed analysis of the current literature on the endothelialization processes of intracardially implanted devices, such as persistent foramen ovale (PFO) occluders, atrial septal defect (ASD) occluders, left atrial appendage (LAA) occluders, transcatheter aortic valve implantations (TAVIs), and leadless pacemakers. Additionally, the known biological activities of common metallic and synthetic components of intracardiac devices in an "in vivo" setting have been evaluated. RESULTS: Nitinol, an alloy of nickel and titanium, is by far the most commonly used material found in intracardiac devices. Although allergies to both components are known, implantation can be performed safely in the vast majority of patients. Depending on the device used, endothelialization can be expected within a time frame of 3-6 months. For those patients with a known allergy, gold coating may be considered as a viable alternative. CONCLUSION: Based on our analysis, we conclude that the vast majority of devices are made of a material that is both safe to implant and nontoxic in long-term treatment according to the current knowledge. The literature on the respective duration of endothelialization of individual devices however is highly divergent.

A Novel Knitted Polytetrafluoroethylene Patch for Cardiovascular Surgery.

Polytetrafluoroethylene (PTFE) is widely used in cardiovascular surgeries; however, postoperative complications such as thrombosis, calcification, and neointimal hyperplasia are yet to be resolved in patients. We developed two types of novel knitted PTFE patches and evaluated them using a swine model. Both patches were composed of knitted PTFE impregnated with micro-PTFE particles, and one of them was pressed after PTFE impregnation. Twenty micromini pigs were used in this study. After left lateral thoracotomy, the new patches (n = 8 for each type of patch) were implanted into the descending aorta and left atrium for the high- and low-pressure models, respectively. Clinically used expanded PTFE (ePTFE) patches were used as the control material (n = 4). The patches were explanted and histopathologically examined at 4, 12, and 24 weeks after implantation. A tensile test was also applied to the high-pressure model at 12 and 24 weeks. As a result, there was no significant difference noted in the tensile test, intimal hyperplasia thickness, or endothelialization among the three patches. In contrast, the degree of macrophage infiltration into the patches and the degree of macrophage, lymphocyte, and granulocyte infiltration outside the patches were lower in the new patches than in the control ePTFE. The degree of cellular infiltration outside new patches decreased over time. There were no significant differences between the two new patch types in these results. In conclusion, our novel knitted PTFE patch showed noninferiority in durability and intensity and less inflammatory responses than a clinically used ePTFE patch.

Importance of ß2AR elevation for re-endothelialization capacity mediated by late endothelial progenitor cells in hypertensive patients.

Dysfunction of late endothelial progenitor cells (EPCs) has been suggested to be associated with hypertension. ß2-Adrenergic receptor (ß2AR) is a novel and key target for EPC homing. Here, we proposed that attenuated ß2AR signaling contributes to EPCs dysfunction, whereas enhanced ß2AR signaling restores EPCs' functions in hypertension. EPCs derived from hypertensive patients exhibited reduced cell number, impaired in vitro migratory and adhesion abilities, and impaired re-endothelialization after transplantation in nude mice with carotid artery injury. ß2AR expression of EPCs from hypertensive patients was markedly downregulated, whereas the phosphorylation of the p38 mitogen-activated protein kinase (p38-MAPK) was elevated. The cleaved caspase-3 levels were elevated in EPCs. The overexpression of ß2AR in EPCs from hypertensive patients inhibited p38-MAPK signaling, whereas it enhanced in vitro EPC proliferation, migration, and adhesion and in vivo re-endothelialization. The ß2AR-mediated effects were attenuated by treating the EPCs with a neutralizing monoclonal antibody against ß2AR, which could be partially antagonized by the p38-MAPK inhibitor SB203580. Moreover, shear stress stimulation, a classic nonpharmacological intervention, increased the phosphorylation levels of ß2AR and enhanced the in vitro and in vivo functions of EPCs from hypertensive patients. Collectively, the current investigation demonstrated that impaired ß2AR/p38-MAPK/caspase-3 signaling at least partially reduced the re-endothelialization capacity of EPCs from hypertensive patients. Restoration of ß2AR expression and shear stress treatment could improve their endothelial repair capacity by regulating the p38-MAPK/caspase-3 signaling pathway. The clinical significance of ß2AR in endothelium repair still requires further investigation.NEW & NOTEWORTHY Impaired ß2-adrenergic receptor (ß2AR) expression with an elevation of p38-MAPK/caspase-3 signaling at least partially contributes to the decline of re-endothelialization capacity of late endothelial progenitor cells (EPCs) from hypertensive patients. ß2AR gene transfer and shear stress treatment improve the late EPC-mediated enhancement of the re-endothelialization capacity in hypertensive patients through activating ß2AR/p38-MAPK/caspase-3 signaling. The present study is the first to reveal the potential molecular mechanism of the impaired endothelium-reparative capacity of late EPCs in hypertension after vascular injury and strongly suggests that ß2AR is a novel and crucial therapeutic target for increasing EPC-mediated re-endothelialization capacity in hypertension.

Value of detecting peri-device leak and incomplete endothelialization by cardiac CT angiography in atrial fibrillation patients post Watchman LAAC combined with radiofrequency ablation.

OBJECTIVES: To explore the value of detecting the peri-device leak (PDL) and device endothelialization after left atrial appendage closure (LAAC) by cardiac computed tomography (CT) in patients with atrial fibrillation (AF), who underwent Watchman LAAC combined with radiofrequency ablation of atrial fibrillation (AFCA). METHODS: Patients with symptomatic drug-refractory atrial fibrillation at high risk of stroke (CHA2 DS2 -VASc Score ≥ 2), who underwent Watchman LAAC combined with AFCA in our center from March 2017 to December 2018 were enrolled. Maximum diameter of LAA orifice was determined by preoperative CCTA. A standardized view of Watchman device was obtained by postoperative CCTA multiplannar reconstruction to evaluate the PDL and device endothelialization. RESULTS: Approximately 84 patients post successful LAAC and AFCA were enrolled in this study. The satisfactory LAA occlusion rate was 100%. There was no death, bleeding, stroke, and device-related thrombus (DRT) events. At 6-month postprocedure, CCTA images evidenced complete endothelialization in 44 patients (no contrast enhancement in LAA); contrast enhancement in LAA and visible PDL in 33 patients; contrast enhancement in LAA but without PDL in seven patients (incomplete device endothelialization). Maximum diameter of LAA orifice could independently predict the occurrence of PDL (odds ratio, 1.31; 95% confidence interval, 1.11-1.55; p = .002), sensitivity was 69.7% and specificity was 80.4% with the cutoff value of maximum diameter of LAA orifice more than 28.2 mm on predicting PDL. CONCLUSIONS: CCTA is feasible to evaluate PDL and device endothelialization after LAAC. The maximum diameter of LAA orifice derived from CT can independently predict the occurrence of post-LAAC PDL.

A pre-conditioning protocol of peripheral blood derived endothelial colony forming cells for endothelialization of tissue engineered constructs.

In regenerative medicine, autologous endothelial colony forming cells (ECFCs) bear the greatest potential to be used for surface endothelialization of tissue engineered constructs, as they are easily attainable and possess a high proliferation rate. The aim of this study was to develop a standardized pre-conditioning protocol under dynamic conditions simulating the physiology of human circulation to improve the formation of a flow resistant monolayer of ECFCs and to enhance the antithrombogenicity of the endothelial cells. The main focus of the study was to consequently compare the cellular behavior under a steady laminar flow against a pulsatile flow. Mononuclear cells were isolated out of peripheral blood (PB) buffy coats and plated on uncoated tissue culture flasks in anticipation of guidelines for Advanced Therapy Medicinal Products. ECFCs were identified by typical surface markers such as CD31, CD146 and VE-Cadherin. To explore the effects of dynamic cultivation, ECFCs and human umbilical vein endothelial cells were comparatively cultured under either laminar or pulsatile (1 Hz) flow conditions with different grades of shear stress (5 dyn/cm2versus 20 dyn/cm2). High shear stress of 20 dyn/cm2 led to a significant upregulation of the antithrombotic gene marker thrombomodulin in both cell types, but only ECFCs orientated and elongated significantly after shear stress application forming a confluent endothelial cell layer. The work therefore documents a suitable protocol to pre-condition PB-derived ECFCs for sustainable endothelialization of blood contacting surfaces and provides essential knowledge for future cultivations in bioreactor systems.

PERK Inhibition Promotes Post-angioplasty Re-endothelialization via Modulating SMC Phenotype Changes.

BACKGROUND: Drug-eluting stents impair post-angioplasty re-endothelialization thus compromising restenosis prevention while heightening thrombotic risks. We recently found that inhibition of protein kinase RNA-like endoplasmic reticulum kinase (PERK) effectively mitigated both restenosis and thrombosis in rodent models. This motivated us to determine how PERK inhibition impacts re-endothelialization. METHODS: Re-endothelialization was evaluated in endothelial-denuded rat carotid arteries after balloon angioplasty and periadventitial administration of PERK inhibitor in a hydrogel. To study whether PERK in smooth muscle cells (SMCs) regulates re-endothelialization by paracrinally influencing endothelial cells (ECs), denuded arteries exposing SMCs were lentiviral-infected to silence PERK; in vitro, the extracellular vesicles isolated from the medium of PDGF-activated, PERK-upregulating human primary SMCs were transferred to human primary ECs. RESULTS: Treatment with PERK inhibitor versus vehicle control accelerated re-endothelialization in denuded arteries. PERK-specific silencing in the denuded arterial wall (mainly SMCs) also enhanced re-endothelialization compared to scrambled shRNA control. In vitro, while medium transfer from PDGF-activated SMCs impaired EC viability and increased the mRNA levels of dysfunctional EC markers, either PERK inhibition or silencing in donor SMCs mitigated these EC changes. Furthermore, CXCL10, a paracrine cytokine detrimental to ECs, was increased by PDGF activation and decreased after PERK inhibition or silencing in SMCs. CONCLUSIONS: Attenuating PERK activity pharmacologically or genetically provides an approach to accelerating post-angioplasty re-endothelialization in rats. The mechanism may involve paracrine factors regulated by PERK in SMCs that impact neighboring ECs. This study rationalizes future development of PERK-targeted endothelium-friendly vascular interventions.

Digital 3D Topographic Microscopy: Bridging the Gaps Between Macroscopy, Microscopy and Scanning Electron Microscopy.

Re-endothelialization of vascular lumen after endovascular procedures is a critical healing milestone and is subjected to routine pathological evaluation during preclinical safety assessment of new cardiovascular devices. Gross evaluation, microscopic evaluation, and scanning electron microscopy (SEM) are the methods of choice for evaluation of vascular surfaces. In this article, we present a new digital imaging approach of surface topography herein referred to as topographical digital microscopy (TDM) that is able to meet the objectives of endovascular healing assessment in a single instrumental platform combined with the same sample preparation techniques as for histology or SEM. This platform is taking advantage of digitally managed illumination, X-Y stitching, and Z-stacking to enable direct optical imaging of tissue surfaces at levels of details ranging from the macroscopic to the cellular level. This technique is enabled by advances in digital optical microscopy and provides images in color and 3 dimensions that can help in the analysis, especially in distinguishing biologically meaningful observations from technical preparation artifacts and in visualizing surface topography.

Reduced plasma level of basic fibroblast growth factor is associated with incomplete device endothelialization at six months following left atrial appendage closure.

OBJECTIVES: To investigate whether inflammatory and growth factors (IGFs) were associated with incomplete device endothelialization (IDE) at 6 months after successful left atrial appendage closure (LAAC). BACKGROUND: IDE after LAAC is correlated with device-related thrombus (DRT) formation and subsequent thromboembolic events. However, biomarkers for early detection of IDE remain lacking. METHODS: Plasma levels of IGFs including basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), stromal cell derived factor (SDF)-1a, transforming growth factor (TGF)-ß1, vascular growth factor receptor-1 (VEGF-R1) and von Willebrand factor (vWF) were determined using ELISA kits in 55 consecutive patients with atrial fibrillation (AF) at 6 months after LAAC with Watchman devices. The status of device endothelialization was assessed by transesophageal echocardiography and cardiac CT. RESULTS: IDE and complete device endothelialization(CDE)were detected in 38 and 17 patients, respectively. Among the six IGFs, only plasma level of bFGF was significantly lower in patients with IDE compared to those with CDE (303.49 ± 246.84 vs. 556.31 ± 197.84 pg/ml, p < 0.001). C-statistics of plasma bFGF for discriminating patients with IDE from those with CDE was 0.785 (95 % CI: 0.663-0.907, p < 0.001), with a cut-off value of 440.52pg/ml (sensitivity 0.765; specificity 0.789). Multivariate logistic regression model showed that lower bFGF was an independent factor for IDE (OR: 11.752, 95 % CI: 2.869-48.144, P = 0.001). bFGF improved the classification of patients (NRI: 0.677,95 % CI: 0.320-1.033, p = 0.004). CONCLUSIONS: Reduced plasma bFGF level confers an increased risk for IDE after LAAC. Further prospective studies are warranted to examine if bFGF could serve as a biomarker for IDE post LAAC.

Re-endothelialization of non-detergent decellularized porcine vessels.

Tissue engineering utilizes an interdisciplinary approach to generate constructs for the treatment and repair of diseased organs. Generation of small vessels as vascular grafts or as envisioned central vessel for vascularized constructs is still a challenge. Here, the decellularization of porcine vessels by a non-detergent based protocol was developed and investigated. Perfusion-decellularization with sodium hydroxide solution resulted in removal of cellular material throughout the whole length of the vessel while preserving structural and mechanical integrity. A re-endothelialization of the retrieved matrix with human umbilical vein endothelial cells and cardiac endothelial cells was achieved through rotation-based seeding employing a custom-made bioreactor. A confluent monolayer was detected on the entire luminal surface. Thus, a non-detergent-based decellularization method allowing the re-endothelialization of the luminal surface was developed in this study, thereby paving the way for future implementation of the resulting construct as vascular graft or as central vessel for tissue engineered constructs in need of a perfusion system with readily available anastomosis sites.

Void-free 3D Bioprinting for In-situ Endothelialization and Microfluidic Perfusion.

Two major challenges of 3D bioprinting are the retention of structural fidelity and efficient endothelialization for tissue vascularization. We address both of these issues by introducing a versatile 3D bioprinting strategy, in which a templating bioink is deposited layer-by-layer alongside a matrix bioink to establish void-free multimaterial structures. After crosslinking the matrix phase, the templating phase is sacrificed to create a well-defined 3D network of interconnected tubular channels. This void-free 3D printing (VF-3DP) approach circumvents the traditional concerns of structural collapse, deformation and oxygen inhibition, moreover, it can be readily used to print materials that are widely considered "unprintable". By pre-loading endothelial cells into the templating bioink, the inner surface of the channels can be efficiently cellularized with a confluent endothelial layer. This in-situ endothelialization method can be used to produce endothelium with a far greater uniformity than can be achieved using the conventional post-seeding approach. This VF-3DP approach can also be extended beyond tissue fabrication and towards customized hydrogel-based microfluidics and self-supported perfusable hydrogel constructs.