Cultures Of Human Endothelial Cells Research Articles

Hydroxyapatite/tricalcium phosphate (HA/beta-TCP) scaffold combined with bone and endothelial cells as a potential candidate for oral and maxillofacial bone regeneration.

Cell-based scaffolds have been proposed as useful for tissue repair and bone regeneration in oral and maxillofacial tissue engineering. However, vascularization, a key player in bone regeneration, is often inadequate. To improve this, the use of biphasic calcium phosphate biomaterial (30% hydroxyapatite (HA)-70% tricalcium phosphate (TCP) mixture) HA/beta-TCP-scaffold with defined micron-channel sizes and pore sizes capable of driving angiogenesis, may prove helpful. Therefore, our study aimed to combine the HA/beta-TCP scaffold with in vitro differentiated cells, especially endothelial cells, well-known players in angiogenesis. The HA/beta-TCP-scaffold was combined with primary cultures of human osteoblasts (hOBs), osteoclasts (hOCs), and endothelial cells (Human Umbilical Vein Endothelial Cells, HUVECs). Cells were co-cultured with HA/beta-TCP-scaffold in normoxia (20% oxygen tension) for 14 days. Then, we evaluated: cell adhesion/morphology (Scanning Electron Microscopy and Confocal Microscopy); cell viability (Calcein AM/Propidium Iodide Assay); the presence of multinucleated hOCs (Tartrate Resistant- Acid Phosphatase assay); expression of specific markers of both osteo- and vasculogenic differentiation (Immunohistochemistry). All cell types were viable and well adhered to the entire scaffold surface. Interestingly, bone matrix formation and newly formed vessel-like structures were both detected. Although further investigation is needed, our data proved that differentiated cells combined in vitro with HA/beta-TCP-scaffold prior to implantation could represent a useful strategy for future applications in bone regeneration. Especially, the use of customized micro-vascularized cell-based scaffold would fall within the innovative "personalized medicine” approach.

RGMa Participates in the Blood-Brain Barrier Dysfunction Through BMP/BMPR/YAP Signaling in Multiple Sclerosis.

The infiltration of inflammatory cells into the central nervous system (CNS) through the dysfunctional blood–brain barrier (BBB) was critical in the early stages of MS. However, the mechanisms underlying BBB dysfunction remain unknown. Repulsive guidance molecule-a (RGMa) is involved in the pathogenesis of multiple sclerosis (MS), but its role needs to be further explored. This study aimed to evaluate whether RMGa regulates BBB permeability in endothelial cells and MS, and if so, what mechanism may be involved. We created an experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice and a human brain microvascular endothelial cell (HBMEC) culture. The permeability of the BBB is measured in response to various interventions. Our results showed that RGMa is expressed in the endothelial cells in HBMECs and EAE mice. RGMa and its signaling counterpart, bone morphogenetic protein 2 (BMP2)/bone morphogenetic protein receptor type II (BMPRII), were gradually increased as the disease progressed. Moreover, as EAE progressed and the BBB was disrupted, the downstream effector, yes-associated protein (YAP), as well as the tight junctional proteins zonula occludens 1 (ZO-1) and claudin-5, decreased significantly. The permeability assay revealed that lentivirus-induced RGMa overexpression in HBMECs caused a significant breakdown of the BBB, whereas RGMa knockdown significantly strengthens the integrity of the BBB. Furthermore, specifically activating BMPR II or inhibiting YAP based on RGMa knockdown results in a significant decrease of ZO-1 and claudin-5 in vitro. On the contrary, inhibition of BMPR II or activation of YAP after upregulating RGMa prevents the downregulation of ZO-1 and claudin-5 in HBMECs. In addition, serum-soluble RGMa (sRGMa) levels were significantly higher in MS patients, particularly in MS patients with Gd+ lesions, indicating that the BBB has been disrupted. In conclusion, this study shows that RGMa causes BBB dysfunction in endothelial cells via BMP2/BMPR II/YAP, resulting in BBB integrity disruption in MS and that it could be a novel therapeutic target for BBB permeability in MS.

Resolvin E1 attenuates doxorubicin-induced endothelial senescence by modulating NLRP3 inflammasome activation

Endothelial cell senescence contributes to chronic inflammation and endothelial dysfunction, while favoring cardiovascular disorders and frailty. Senescent cells acquire a pro-inflammatory secretory phenotype that further propagates inflammation and senescence to neighboring cells. Cell senescence can be provoked by plethora of stressors, including inflammatory molecules and chemotherapeutic drugs. Doxorubicin (Doxo) is a powerful anthracycline anticancer drug whose clinical application is constrained by a dose-limiting cardiovascular toxicity. We here investigated whether cell senescence can contribute to the vascular damage elicited by Doxo. In human umbilical vein endothelial cells (HUVEC) cultures, Doxo (10–100 nM) increased the number of SA-β-gal positive cells and the levels of γH2AX, p21 and p53, used as markers of senescence. Moreover, we identified Doxo-induced senescence to be mediated by the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, a key player of the immune innate system capable of releasing interleukin (IL)-1β. In fact, IL-1β itself mimicked the stimulatory action of Doxo on both NLRP3 activation and cellular senescence, while the pharmacological blockade of IL-1 receptors markedly attenuated the pro-senescence effects of Doxo. In search of additional pharmacological strategies to attenuate Doxo-induced endothelial senescence, we identified resolvin E1 (RvE1), an endogenous pro-resolving mediator, as capable of reducing cell senescence induced by both Doxo and IL-1β by interfering with the increased expression of pP65, NLRP3, and pro-IL-1β proteins and with the formation of active NLRP3 inflammasome complexes. Overall, RvE1 and the blockade of the NLRP3 inflammasome-IL-1β axis may offer a novel therapeutic approach against Doxo-induced cardiovascular toxicity and subsequent sequelae.

Genetic deletion of endothelial Caveolin‐1 is protective against metabolic disease

In metabolic disease, the endothelium is exposed to a substantial increase in fatty acid concentrations creating a pathological role for endothelial cells (ECs) as lipid metabolizing cells. One mechanism whereby lipids may enter ECs is by caveolin‐1 (Cav1)‐mediated endocytosis. Because both ECs and adipocytes both have abundant Cav1 and both can readily take up lipids, we wanted to differentiate the role of Cav1 between the two cell types. For these reasons, we used an inducible EC‐specific Cav1 knockout mouse (Cav1fl/fl/Cdh5‐CreERT2+). Using these mice, we induced Cav1 genetic deletion from endothelium at 6 weeks, and began a high fat diet (HFD; 60% fat) to induce metabolic disease at 8 weeks. Normal chow fed littermates were used as controls. All mice were examined at 20 weeks (i.e., 12 weeks of HFD). Thus, we had four cohorts: Cre+ HFD, Cre‐ HFD, Cre+ normal chow, and Cre‐ normal chow all injected with tamoxifen. All mice regardless of genotype had comparable weight gain within diet groups, and gonadal fat pad mass. Food consumption was similar between groups, and electron microscopy was used to confirm presence of caveola in adipocytes, but not endothelium. Initial experiments examining plasma lipids found HFD Cre+ mice had both increased serum cholesterol (175mg/dL) and LDL (75mg/dL) compared to HFD Cre‐ controls (cholesterol, 125mg/dL; LDL, 60mg/dL). When these mice were challenged with a bolus of insulin, the blood glucose of Cre+ HFD mice dropped significantly resembling an insulin tolerance curve from normal chow fed controls. Similarly, Cre+ HFD mice were protected from hyperglycemia after bolus glucose injections, resulting in a glucose tolerance phenotype similar to that of a normal chow fed mouse (HFD Cre+ AUC 3000 vs. HFD Cre‐ AUC 60000). There was no difference in Cre+ or Cre‐ mice on normal chow diets with serum lipids or after challenge with insulin or glucose. It isn’t clear why the insulin and glucose protection correspond with significantly increased lipids in the Cre+ HFD mice. For these reasons, we began investigating the possible mechanisms with primary cultures of Human Adipose Microvascular Endothelial Cells (HAMECs) and knocked down Cav1 with siRNA. In monolayers of HAMEC without any other cell type present, we did not observe altered lipid uptake after addition of 50µM of exogenous fatty acids with a 70% knockdown of Cav1 protein. Additionally, HAMECs seahorse assays were used to determine if there were changes in metabolic function when Cav1 was knocked down. This work provides initial evidence for the unique metabolic roles of endothelial Cav1 during periods of high lipid circulation.

Different Contribution of Monocyte- and Platelet-Derived Microvesicles to Endothelial Behavior.

Several contributions of circulating microvesicles (MVs) to the endothelial dysfunction have been reported in the past; a head-to-head comparison of platelet- and monocyte–derived MVs has however never been performed. To this aim, we assessed the involvement of these MVs in vessel damage related processes, i.e., oxidative stress, inflammation, and leukocyte-endothelial adhesion. Platelets and monocytes isolated from healthy subjects (HS, n = 15) were stimulated with TRAP-6 and LPS to release MVs that were added to human vascular endothelial cell (hECV) culture to evaluate superoxide anion production, inflammatory markers (IL-6, TNFα, NF-κB mRNA expression), and hECV adhesiveness. The effects of the MVs-induced from HS were compared to those induced by MVs spontaneously released from cells of patients with ST-segment elevation myocardial infarction (STEMI, n = 7). MVs released by HS-activated cells triggered a threefold increase in oxidative burst in a concentration-dependent manner. Only MVs released from monocytes doubled IL-6, TNFα, and NF-κB mRNA expression and monocyte-endothelial adhesion. Interestingly, the effects of the MVs isolated from STEMI-monocytes were not superimposable to previous ones except for adhesion to hECV. Conversely, MVs released from STEMI-platelets sustained both redox state and inflammatory phenotype. These data provide evidence that MVs released from activated and/or pathologic platelets and monocytes differently affect endothelial behavior, highlighting platelet-MVs as causative factors of impaired endothelial function in the acute phase of STEMI.

Topical Nanoemulsion of a Runt-related Transcription Factor 1 Inhibitor for the Treatment of Pathologic Ocular Angiogenesis.

Purpose:To test the efficacy of runt-related transcription factor 1 (RUNX1) inhibition with topical nanoemulsion containing Ro5-3335 (eNano-Ro5) in experimental ocular neovascularization.Design:Preclinical experimental study.Participants:In vitro primary culture human retinal endothelial cell (HREC) culture. C57BL/6J 6- to 10-week-old male and female mice.Methods:We evaluated the effect of eNano-Ro5 in cell proliferation, cell toxicity, and migration of HRECs. We used an alkali burn model of corneal neovascularization and a laser-induced model of choroidal neovascularization to test in vivo efficacy of eNano-Ro5 in pathologic angiogenesis in mice. We used mass spectrometry to measure penetration of Ro5-3335 released from the nanoemulsion in ocular tissues.Main Outcome Measures:Neovascular area.Results:RUNX1 inhibition reduced cell proliferation and migration in vitro. Mass spectrometry analysis revealed detectable levels of the active RUNX1 small-molecule inhibitor Ro5-3335 in the anterior and posterior segment of the mice eyes. Topical treatment with eNano-Ro5 significantly reduced corneal neovascularization and improved corneal wound healing after alkali burn. Choroidal neovascularization lesion size and leakage were significantly reduced after treatment with topical eNano-Ro5.Conclusions:Topical treatment with eNano-Ro5 is an effective and viable platform to deliver a small-molecule RUNX1 inhibitor. This route of administration offers advantages that could improve the management and outcomes of these sight-threatening conditions. Topical noninvasive delivery of RUNX1 inhibitor could be beneficial for many patients with pathologic ocular neovascularization.

Engineering pre-vascularized bone-like tissue from human mesenchymal stem cells through simulating endochondral ossification

Currently, most in vitro engineered bone tissues do not contain viable blood vessel systems, so the vascularization depends on post-implantation angiogenesis from the host, which is often insufficient for repairing large bone defects. In this study, we aimed to create pre-vascularized bone-like tissue from human bone marrow-derived mesenchymal stem cells (HBMSCs) within the self-generated extracellular matrix by simulating the developmental endochondral ossification. Afterward, a three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs)/HBMSCs was introduced to cover bone-like constructs surface for vascularization. Lastly, the pre-vascularized bone-like tissues were subcutaneously implanted into mice and the quality of newly formed blood vessels and bones were later assessed. We particularly examined whether the pre-existing HUVECs/HBMSCs vascular networks within the implants were able to integrate with the host’s blood vessels and facilitate bone formation. Our results showed that this developmentally informed procedure resulted in a robust osteogenic differentiation of HBMSCs. Moreover, the bone-like constructs markedly promoted HUVEC/HBMSCs network formation in vitro. After 28 days of implantation in mice, the experimental group, in which bone-like constructs were pre-vascularized with HUVEC/HBMSCs networks, exhibited significantly more functional blood vessels than the control group that contained HUVEC and HBMSC single cells. Interestingly, increased levels of bone formation and absorption markers were also observed in the pre-vascularized bone-like constructs. Taken together, these findings demonstrated the potential of pre-vascularized bone-like constructs in repairing bone defects.

Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

Activation of the coagulation cascade favours metastatic spread, but antithrombotic therapy might also have detrimental effects on cancer progression. In this study, we characterized the effects of dabigatran, a direct reversible thrombin inhibitor, on the pulmonary endothelial barrier and metastatic spread in a murine model of breast cancer metastasis. Dabigatran etexilate (100 mg kg−1) was administered to mice twice daily by oral gavage. Pulmonary metastasis, pulmonary endothelium permeability in vivo, and platelet reactivity were evaluated after intravenous injection of 4T1 breast cancer cells into BALB/c mice. The effect of dabigatran on platelet-dependent protection of pulmonary endothelial barrier in the presence of an inflammatory stimulus was also verified in vitro using human lung microvascular endothelial cell (HLMVEC) cultures. Dabigatran-treated mice harbored more metastases in their lungs and displayed increased pulmonary endothelium permeability after cancer cell injection. It was not associated with altered lung fibrin deposition, changes in INFγ, or complement activation. In the in vitro model of the pulmonary endothelial barrier, dabigatran inhibited platelet-mediated protection of pulmonary endothelium. In a murine model of breast cancer metastasis, dabigatran treatment promoted pulmonary metastasis by the inhibition of platelet-dependent protection of pulmonary endothelial barrier integrity.

Uremic Toxin Lanthionine Induces Endothelial Cell Mineralization In Vitro.

Vascular calcification (VC) is a pathological event caused by the unusual deposition of minerals in the vascular system, representing the leading cause of cardiovascular mortality in chronic kidney disease (CKD). In CKD, the deregulation of calcium and phosphate metabolism, along with the effect of several uremic toxins, act as key processes conveying altered mineralization. In this work, we tested the ability of lanthionine, a novel uremic toxin, to promote calcification in human endothelial cell cultures (Ea.hy926). We evaluated the effects of lanthionine, at a concentration similar to that actually detected in CKD patients, alone and under pro-calcifying culture conditions using calcium and phosphate. In pro-calcific culture conditions, lanthionine increased both the intracellular and extracellular calcium content and induced the expression of Bone Morphogenetic Protein 2 (BMP2) and RUNX Family Transcription Factor 2 (RUNX2). Lanthionine treatment, in pro-calcifying conditions, raised levels of tissue-nonspecific alkaline phosphatase (ALPL), whose expression also overlapped with Dickkopf WNT Signaling Pathway Inhibitor 1 (DKK1) gene expression, suggesting a possible role of the latter gene in the activation of ALPL. In addition, treatment with lanthionine alone or in combination with calcium and phosphate reduced Inorganic Pyrophosphate Transport Regulator (ANKH) gene expression, a protective factor toward the mineralizing process. Moreover, lanthionine in a pro-calcifying condition induced the activation of ERK1/2, which is not associated with an increase in DKK1 protein levels. Our data underscored a link between mineral disease and the alterations of sulfur amino acid metabolisms at a cell and molecular level. These results set the basis for the understanding of the link between uremic toxins and mineral-bone disorder during CKD progression.

Adult Human Vascular Smooth Muscle Cells on 3D Silk Fibroin Nonwovens Release Exosomes Enriched in Angiogenic and Growth-Promoting Factors.

Background. Our earlier works showed the quick vascularization of mouse skin grafted Bombyx mori 3D silk fibroin nonwoven scaffolds (3D-SFnws) and the release of exosomes enriched in angiogenic/growth factors (AGFs) from in vitro 3D-SFnws-stuck human dermal fibroblasts (HDFs). Here, we explored whether coronary artery adult human smooth muscle cells (AHSMCs) also release AGFs-enriched exosomes when cultured on 3D-SFnws in vitro. Methods. Media with exosome-depleted FBS served for AHSMCs and human endothelial cells (HECs) cultures on 3D-SFnws or polystyrene. Biochemical methods and double-antibody arrays assessed cell growth, metabolism, and intracellular TGF-β and NF-κB signalling pathways activation. AGFs conveyed by CD9+/CD81+ exosomes released from AHSMCs were double-antibody array analysed and their angiogenic power evaluated on HECs in vitro. Results. AHSMCs grew and consumed D-glucose more intensely and showed a stronger phosphorylation/activation of TAK-1, SMAD-1/-2/-4/-5, ATF-2, c-JUN, ATM, CREB, and an IκBα phosphorylation/inactivation on SFnws vs. polystyrene, consistent overall with a proliferative/secretory phenotype. SFnws-stuck AHSMCs also released exosomes richer in IL-1α/-2/-4/-6/-8; bFGF; GM-CSF; and GRO-α/-β/-γ, which strongly stimulated HECs’ growth, migration, and tubes/nodes assembly in vitro. Conclusions. Altogether, the intensified AGFs exosomal release from 3D-SFnws-attached AHSMCs and HDFs could advance grafts’ colonization, vascularization, and take in vivo—noteworthy assets for prospective clinical applications.

Spatially arranged encapsulation of stem cell spheroids within hydrogels for the regulation of spheroid fusion and cell migration

Mesenchymal stem cell spheroids have been encapsulated in hydrogels for various applications because spheroids demonstrate higher cell activity than individual cells in suspension. However, there is limited information on the effect of distance between spheroids (inter-spheroid distance) on fusion or migration in a hydrogel. In this study, we developed temperature-responsive hydrogels with surface microwell patterns to culture adipose-derived stem cell (ASC) spheroids and deliver them into a Matrigel for the investigation of the effect of inter-spheroid distance on spheroid behavior. The ASC spheroids were encapsulated successfully in a Matrigel, denoted as sandwich culture, with a specific inter-spheroid distance ranging from 100 to 400 µm. Interestingly, ASCs migrated from the host spheroid and formed a bridge-like structure between spheroids, denoted as a cellular bridge, only when the inter-spheroid distance was 200 µm. Thus, we performed a sandwich culture of human umbilical vein endothelial cells (HUVECs) and ASCs in co-cultured spheroids in the Matrigel to create a homogeneous endothelial cell network in the hydrogel. The HUVECs sprouted through the ASC cellular bridge and directly interacted with the adjacent spheroid when the inter-spheroid distance was 200 µm. Similar results were obtained from an in vivo study. Thus, our study suggests the appropriate inter-spheroid distance for effective spheroid encapsulation in a hydrogel. Statement of significanceRecently, spheroid-based 3D tissue culture techniques such as spheroid encapsulation or 3D printing are being intensively investigated for various purposes. However, there is limited research regarding the effect of the inter-spheroid distance on spheroid communication. Here, we demonstrate a spatially arranged spheroid encapsulation method within a Matrigel by using a temperature-responsive hydrogel. Human adipose-derived stem cell spheroids are encapsulated with a precisely controlled inter-spheroid distance from 100 to 400 µm and show different tendencies in cell migration and spheroid fusion. Our results suggest that the inter-spheroid distance affects spheroid communication, and thus, the inter-spheroid distance needs to be considered carefully according to the purpose.

Histone deacetylase inhibitors (HDACi) increase expression of KCa2.3 (SK3) in primary microvascular endothelial cells.

The small conductance calcium-activated potassium channel (KCa2.3) has long been recognized for its role in mediating vasorelaxation through the endothelium-derived hyperpolarization (EDH) response. Histone deacetylases (HDACs) have been implicated as potential modulators of blood pressure and histone deacetylase inhibitors (HDACi) are being explored as therapeutics for hypertension. Herein, we show that HDACi increase KCa2.3 expression when heterologously expressed in HEK cells and endogenously expressed in primary cultures of human umbilical vein endothelial cells (HUVECs) and human intestinal microvascular endothelial cells (HIMECs). When primary endothelial cells were exposed to HDACi, KCa2.3 transcripts, subunits, and functional current are increased. Quantitative RT-PCR (qPCR) demonstrated increased KCa2.3 mRNA following HDACi, confirming transcriptional regulation of KCa2.3 by HDACs. By using pharmacological agents selective for different classes of HDACs, we discriminated between cytoplasmic and epigenetic modulation of KCa2.3. Biochemical analysis revealed an association between the cytoplasmic HDAC6 and KCa2.3 in immunoprecipitation studies. Specifically inhibiting HDAC6 increases expression of KCa2.3. In addition to increasing the expression of KCa2.3, we show that nonspecific inhibition of HDACs causes an increase in the expression of the molecular chaperone Hsp70 in endothelial cells. When Hsp70 is inhibited in the presence of HDACi, the magnitude of the increase in KCa2.3 expression is diminished. Finally, we show a slower rate of endocytosis of KCa2.3 as a result of exposure of primary endothelial cells to HDACi. These data provide the first demonstrated approach to increase KCa2.3 channel number in endothelial cells and may partially account for the mechanism by which HDACi induce vasorelaxation.

Simultaneous Measurement of Endothelial Cell Proliferation and Cell Cycle Stage Using Flow Cytometry.

Endothelial cell proliferation rate is an important indicator of vascular health. Being able to detect the rate of endothelial cell proliferation, or cell cycle disturbances after intervention is a valuable tool for analysing any beneficial or detrimental effects of treatments in vitro. Here, we describe a straightforward flow cytometric-based method of proliferation and cell cycle tracking that can be performed on human endothelial cells in culture over several days.

Rapid assembly of PMMA microfluidic devices with PETE membranes for studying the endothelium

Biomicrofluidic devices and organ-on-a-chip (OOC) systems with integrated membranes are often fabricated from two different thermoplastic materials but bonding of such dissimilar thermoplastics remains challenging to manufacture at scale. Here, we present a method to bond poly(methylmethacrylate) layers to a polyethylene terephthalate porous membrane to create membrane-based microfluidic devices for biological barrier modeling. By combining milling, laser cutting and chlorocarbon-based solvent bonding supported by retention grooves, we achieved a fabrication rate of 36 devices in 5 h. Chlorocarbon-based solvent bonding resulted in bond strength of ~10 J/m2 and did not adversely affect the membrane pore structure or the channel cross-sectional shape. The bonded devices were found to support long term culture of human endothelial cells that developed expected morphology and cell-cell adhesion contacts as evidenced by immunofluorescent labeling of VE-cadherin. Barrier permeability was measured to be 3.38 × 106 cm/s for 10 kDa dextran using a sampling-based method compatible with mass spectrometry and scintillation techniques and was in agreement with literature. To validate the devices for cell migration experiments, THP-1 monocytes were introduced into devices with confluent endothelial monolayers. Monocytes adhered to and migrated through the endothelium. Activation of the endothelium with TNF-α prior to introducing monocytes significantly increased monocyte adhesion. Overall, the rapid device fabrication method achieved medium-volume production rates and was found to support both cell culture and experiments associated with measuring barrier and endothelial function. This fabrication method has potential to both accelerate biomicrofluidics and OOC research in the lab and accelerate development of commercialized microfluidic membrane devices.

Fabrication of a novel Three-Dimensional porous PCL/PLA tissue engineering scaffold with high connectivity for endothelial cell migration

As an artificial extracellular matrix, tissue engineering scaffolds promote tissue regeneration and repair. Therefore, the scaffold’s internal structure is crucial for cell adhesion, growth, and migration. In this paper, based on the supercritical CO2 foaming technology, a novel three-dimensional (3D) porous polycaprolactone/polylactic acid tissue engineering scaffold induced by heterogeneous nanofibres was prepared by introducing the second phase of polylactic acid electrospinning nanofibres. The scanning electron microscopy results showed that the prepared scaffolds exhibited obvious nanofibre structures and 3D reticular pore structures. The influence of foaming temperature on pore morphology was also systematically explored. Tensile tests and water contact angle tests showed that the novel scaffolds exhibited good mechanical properties and hydrophilicity. The results of cross-sectional human umbilical endothelial cell (HUVEC) culture showed that the scaffolds were biocompatible and significantly promote the adhesion, migration and permeability of HUVECs. Single-cell migration assays showed that the 3D porous scaffolds effectively promote HUVEC migration. Therefore, 3D porous scaffolds have enormous application potential in the field of vascular patches.

Association of 5-aminolevulinic acid with intraoperative hypotension in malignant glioma surgery

BackgroundUse of 5-aminolevulinic acid for photodynamic malignant tumor diagnosis reportedly causes intraoperative hypotension (systolic blood pressure < 70 mmHg) during urologic surgery. However, its association with intraoperative hypotension in malignant glioma surgery and underlying mechanisms has not yet been elucidated.. This study aimed to investigate whether 5-aminolevulinic acid administration is associated with intraoperative hypotension in malignant glioma surgery and explore the mechanisms of 5-aminolevulinic acid-induced hypotension in vitro. MethodsIn this retrospective multicenter cohort study, we investigated intracellular nitric oxide as a candidate mediator of hypotension in response to 5-aminolevulinic acid in vitro in human umbilical vein endothelial cell cultures. ResultsOf 142 patients, 94 underwent 5-aminolevulinic acid-guided surgery. Systolic blood pressure was significantly lower throughout surgery with 5-aminolevulinic acid administration. 5-Aminolevulinic acid administration was an independent risk factor for intraoperative hypotension according to multivariable logistic regression analysis (89% vs. 56%; odds ratio = 6.72, 95% confidence interval [2.05–22.1], P = 002). In subgroup analysis of the 5-aminolevulinic acid group, increasing age and use of renin-angiotensin system inhibitors had a synergistic effect with 5-aminolevulinic acid on decreased blood pressure. In the vascular endothelial cell culture study, 5-aminolevulinic acid induced a significant increase in intracellular nitric oxide generation. Conclusions5-Aminolevulinic acid administration was associated with intraoperative hypotension in malignant glioma surgery, with increasing age and use of renin-angiotensin system inhibitors boosting the blood pressure-lowering effect of 5-aminolevulinic acid. According to in vitro results, the low blood pressure induced by 5-aminolevulinic acid may be mediated by a nitric oxide increase in vascular endothelial cells.

Incremental Experience in In Vitro Primary Culture of Human Pulmonary Arterial Endothelial Cells Harvested from Swan-Ganz Pulmonary Arterial Catheters.

Pulmonary arterial hypertension (PAH) is a devastating condition affecting the pulmonary microvascular wall and endothelium, resulting in their partial or total obstruction. Despite a combination of expensive vasodilatory therapies, mortality remains high. Personalized therapeutic approaches, based on access to patient material to unravel patient specificities, could move the field forward. An innovative technique involving harvesting pulmonary arterial endothelial cells (PAECs) at the time of diagnosis was recently described. The aim of the present study was to fine-tune the initial technique and to phenotype the evolution of PAECs in vitro subcultures. PAECs were harvested from Swan-Ganz pulmonary arterial catheters during routine diagnostic or follow up right heart catheterization. Collected PAECs were phenotyped by flow cytometry and immunofluorescence focusing on endothelial-specific markers. We highlight the ability to harvest patients’ PAECs and to maintain them for up to 7–12 subcultures. By tracking the endothelial phenotype, we observed that PAECs could maintain an endothelial phenotype for several weeks in culture. The present study highlights the unique opportunity to obtain homogeneous subcultures of primary PAECs from patients at diagnosis and follow-up. In addition, it opens promising perspectives regarding tailored precision medicine for patients suffering from rare pulmonary vascular diseases.

Genetic inhibition of RIPK3 ameliorates functional outcome in controlled cortical impact independent of necroptosis

Traumatic brain injury (TBI) is a leading cause of death and disability with no specific effective therapy, in part because disease driving mechanisms remain to be elucidated. Receptor interacting protein kinases (RIPKs) are serine/threonine kinases that assemble multi-molecular complexes that induce apoptosis, necroptosis, inflammasome and nuclear factor kappa B activation. Prior studies using pharmacological inhibitors implicated necroptosis in the pathogenesis of TBI and stroke, but these studies cannot be used to conclusively demonstrate a role for necroptosis because of the possibility of off target effects. Using a model of cerebral contusion and RIPK3 and mixed lineage kinase like knockout (MLKL−/−) mice, we found evidence for activation of RIPK3 and MLKL and assembly of a RIPK1-RIPK3-MLKL necrosome complex in pericontusional brain tissue. Phosphorylated forms of RIPK3 and MLKL were detected in endothelium, CD11b + immune cells, and neurons, and RIPK3 was upregulated and activated in three-dimensional human endothelial cell cultures subjected to CCI. RIPK3−/− and MLKL−/− mice had reduced blood-brain barrier damage at 24 h (p < 0.05), but no differences in neuronal death (6 h, p = ns in CA1, CA3 and DG), brain edema (24 h, p = ns), or lesion size (4 weeks, p = ns) after CCI. RIPK3−/−, but not MLKL−/− mice, were protected against postinjury motor and cognitive deficits at 1–4 weeks (RIPK3−/− vs WT: p < 0.05 for group in wire grip, Morris water maze hidden platform trials, p < 0.05 for novel object recognition test, p < 0.01 for rotarod test). RIPK3−/− mice had reduced infiltrating leukocytes (p < 0.05 vs WT in CD11b + cells, microglia and macrophages), HMGB1 release and interleukin-1 beta activation at 24–48 h (p < 0.01) after CCI. Our data indicate that RIPK3 contributes to functional outcome after cerebral contusion by mechanisms involving inflammation but independent of necroptosis.

SARS-CoV-2 infection negatively affects ovarian function in ART patients

Several organs, such as the heart, breasts, intestine, testes, and ovaries, have been reported to be target tissues of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To date, no studies have demonstrated SARS-CoV-2 infection in the female reproductive system. In the present study, we investigated the effects of SARS-CoV-2 infection on ovarian function by comparing follicular fluid (FF) from control and recovered coronavirus disease 2019 (COVID-19) patients and by evaluating the influence of these FF on human endothelial and non-luteinized granulosa cell cultures. Our results showed that most FFs (91.3%) from screened post COVID-19 patients were positive for IgG antibodies against SARS-CoV-2. Additionally, patients with higher levels of IgG against SARS-CoV-2 had lower numbers of retrieved oocytes. While VEGF and IL-1β were significantly lower in post COVID-19 FF, IL-10 did not differ from that in control FF. Moreover, in COV434 cells stimulated with FF from post COVID-19 patients, steroidogenic acute regulatory protein (StAR), estrogen-receptor β (Erβ), and vascular endothelial growth factor (VEGF) expression were significantly decreased, whereas estrogen-receptor α (ERα) and 3β-hydroxysteroid dehydrogenase (3β-HSD) did not change. In endothelial cells stimulated with post COVID-19 FF, we observed a decrease in cell migration without changes in protein expression of certain angiogenic factors. Both cell types showed a significantly higher γH2AX expression when exposed to post COVID-19 FF. In conclusion, our results describe for the first time that the SARS-CoV-2 infection adversely affects the follicular microenvironment, thus dysregulating ovarian function.

Mimicking chronic hyperglycaemic conditions in primary human endothelial cell cultures using pathophysiological concentrations of glucose in prediabetes and type 2 diabetes

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)