Endothelial Cell Phenotype Research Articles

Eimeria bovis infections induce G1 cell cycle arrest and a senescence-like phenotype in endothelial host cells.

Apicomplexan parasites are well-known to modulate their host cells at diverse functional levels. As such, apicomplexan-induced alteration of host cellular cell cycle was described and appeared dependent on both, parasite species and host cell type. As a striking evidence of species-specific reactions, we here show that Eimeria bovis drives primary bovine umbilical vein endothelial cells (BUVECs) into a senescence-like phenotype during merogony I. In line with senescence characteristics, E. bovis induces a phenotypic change in host cell nuclei being characterized by nucleolar fusion and heterochromatin-enriched peripheries. By fibrillarin staining we confirm nucleoli sizes to be increased and their number per nucleus to be reduced in E. bovis-infected BUVECs. Additionally, nuclei of E. bovis-infected BUVECs showed enhanced signals for HH3K9me2 as heterochromatin marker thereby indicating an infection-induced change in heterochromatin transition. Furthermore, E. bovis-infected BUVECs show an enhanced β-galactosidase activity, which is a well-known marker of senescence. Referring to cell cycle progression, protein abundance profiles in E. bovis-infected endothelial cells revealed an up-regulation of cyclin E1 thereby indicating a cell cycle arrest at G1/S transition, signifying a senescence key feature. Similarly, abundance of G2 phase-specific cyclin B1 was found to be downregulated at the late phase of macromeront formation. Overall, these data indicate that the slow proliferative intracellular parasite E. bovis drives its host endothelial cells in a senescence-like status. So far, it remains to be elucidated whether this phenomenon indeed reflects an intentionally induced mechanism to profit from host cell-derived energy and metabolites present in a non-dividing cellular status.

The Protective Role of Bioactive Quinones in Stress-induced Senescence Phenotype of Endothelial Cells Exposed to Cigarette Smoke Extract.

Endothelial dysfunction represents the initial stage in atherosclerotic lesion development which occurs physiologically during aging, but external factors like diet, sedentary lifestyle, smoking accelerate it. Since cigarette smoking promotes oxidative stress and cell damage, we developed an in vitro model of endothelial dysfunction using vascular cells exposed to chemicals present in cigarette smoke, to help elucidate the protective effects of anti-inflammatory and antioxidant agents, such as ubiquinol and vitamin K, that play a fundamental role in vascular health. Treatment of both young and senescent Human Umbilical Vein Endothelial Cells (HUVECs) for 24 h with cigarette smoke extract (CSE) decreased cellular viability, induced apoptosis via reactive oxygen species (ROS) imbalance and mitochondrial dysfunction and promoted an inflammatory response. Moreover, the senescence marker SA-β-galactosidase was observed in both young CSE-exposed and in senescent HUVECs suggesting that CSE exposure accelerates aging in endothelial cells. Supplementation with 10 µM ubiquinol and menaquinone-7 (MK7) counteracted oxidative stress and inflammation, resulting in improved viability, decreased apoptosis and reduced SA-β-galactosidase, but were ineffective against CSE-induced mitochondrial permeability transition pore opening. Other K vitamins tested like menaquinone-4 (MK4) and menaquinone-1 (K1) were less protective. In conclusion, CSE exposure was able to promote a stress-induced senescent phenotype in young endothelial cells likely contributing to endothelial dysfunction in vivo. Furthermore, the molecular changes encountered could be offset by ubiquinol and menaquinone-7 supplementation, the latter resulting the most bioactive K vitamin in counteracting CSE-induced damage.

Flow-Induced Transcriptomic Remodeling of Endothelial Cells Derived From Human Induced Pluripotent Stem Cells.

The vascular system is essential for the development and function of all organs and tissues in our body. The molecular signature and phenotype of endothelial cells (EC) are greatly affected by blood flow-induced shear stress, which is a vital component of vascular development and homeostasis. Recent advances in differentiation of ECs from human induced pluripotent stem cells (hiPSC) have enabled development of in vitro experimental models of the vasculature containing cells from healthy individuals or from patients harboring genetic variants or diseases of interest. Here we have used hiPSC-derived ECs and bulk- and single-cell RNA sequencing to study the effect of flow on the transcriptomic landscape of hiPSC-ECs and their heterogeneity. We demonstrate that hiPS-ECs are plastic and they adapt to flow by expressing known flow-induced genes. Single-cell RNA sequencing showed that flow induced a more homogenous and homeostatically more stable EC population compared to static cultures, as genes related to cell polarization, barrier formation and glucose and fatty acid transport were induced. The hiPS-ECs increased both arterial and venous markers when exposed to flow. Interestingly, while in general there was a greater increase in the venous markers, one cluster with more arterial-like hiPS-ECs was detected. Single-cell RNA sequencing revealed that not all hiPS-ECs are similar even after sorting, but exposing them to flow increases their homogeneity. Since hiPS-ECs resemble immature ECs and demonstrate high plasticity in response to flow, they provide an excellent model to study vascular development.

Sunitinib facilitates metastatic breast cancer spreading by inducing endothelial cell senescence

BackgroundSunitinib, a receptor tyrosine kinase (RTK) inhibitor that targets multiple receptors such as vascular endothelial growth factor receptors (VEGFRs), was approved for cancer treatment in 2006. However, it was unsuccessful in treating certain cancers, particularly metastatic breast cancer (MBC), and the mechanism underlying this “sunitinib resistance” remains unclear. Herein, we investigated whether the sunitinib-associated inferior survival benefit in MBC was due to sunitinib-induced endothelial cell (EC) injury or EC senescence.Methods4T1 murine breast cancer cells were used as the main breast tumor model for it produces a highly metastatic solid tumor that can spontaneously metastasize to the lung, which closely mimics highly metastatic human breast cancer. Senescence-associated β-galactosidase (SA-β-Gal, immunohistochemistry [IHC]-staining), P16, P53, and P57 (immunoblotting) were used as markers of cell senescence. A protein array containing 25 senescence-associated chemokines and the transwell chemotaxis assay were used to examine whether sunitinib increases inflammatory chemokine secretion which attracts tumor cells via chemokinesis. Flow cytometry and IHC were used to detect whether the sunitinib-induced senescent ECs recruit cancer-associated inflammatory myeloid cells. Finally, the spontaneous metastatic model was used to monitor whether sunitinib causes the formation of “pre-metastatic niche” which promotes MBC to metastasize to the lungs.ResultsWe demonstrated that sunitinib induced a senescence-like endothelial cell (EC) phenotype. Inflammatory chemokine secretion and VCAM1 expression were significantly increased in senescent ECs, resulting in tumor cell (TC) chemotaxis and TC/EC interactions. Meanwhile, EC senescence caused loosening of EC junctions, facilitating TC transmigration through the endothelial barrier. Sunitinib-induced senescent ECs also recruited cancer-associated myeloid cells to form a “pre-metastatic niche”-like microenvironment. Alterations at the molecular level and in the tissue environment ultimately led to an increase in distant metastasis.ConclusionAlthough sunitinib was designed to target the EC directly, the increase in tumor metastasis may ironically be due to sunitinib “correctly” playing its role. Our findings suggest that we should carefully weigh the pros and cons before using sunitinib and other antiangiogenic drugs that directly target the ECs.

The Signature Amino Acid Residue Serine 31 of HIV-1C Tat Potentiates an Activated Phenotype in Endothelial Cells.

The natural cysteine to serine variation at position 31 of Tat in HIV-1C disrupts the dicysteine motif attenuating the chemokine function of Tat. We ask if there exists a trade-off in terms of a gain of function for HIV-1C Tat due to this natural variation. We constructed two Tat-expression vectors encoding Tat proteins discordant for the serine 31 residue (CS-Tat vs. CC-Tat), expressed the proteins in Jurkat cells under doxycycline control, and performed the whole transcriptome analysis to compare the early events of Tat-induced host gene expression. Our analysis delineated a significant enrichment of pathways and gene ontologies associated with the angiogenic signaling events in CS-Tat stable cells. Subsequently, we validated and compared angiogenic signaling events induced by CS- vs. CC-Tat using human umbilical vein endothelial cells (HUVEC) and the human cerebral microvascular endothelial cell line (hCMEC/D3). CS-Tat significantly enhanced the production of CCL2 from HUVEC and induced an activated phenotype in endothelial cells conferring on them enhanced migration, invasion, and in vitro morphogenesis potential. The ability of CS-Tat to induce the activated phenotype in endothelial cells could be of significance, especially in the context of HIV-associated cardiovascular and neuronal disorders. The findings from the present study are likely to help appreciate the functional significance of the SAR (signature amino acid residues) influencing the unique biological properties.

The protective role of estrogen on endothelial and glycocalyx barriers after shock conditions: A microfluidic study

BackgroundSexual dimorphism has been demonstrated after major trauma and hemorrhage shock with protective effects related to female sex or estrogen. Traumatic endotheliopathy is an important component of trauma-induced coagulopathy. Components of endothelial barrier dysfunction include degradation of the endothelial glycocalyx and endothelial cellular injury. Estrogen modulates endothelial function via its membrane and cellular receptors. The effects of estrogen on the vascular endothelial barrier after trauma and hemorrhage shock are, however, unknown. This topic was studied in an in vitro model under flow conditions. MethodsMonolayers of human umbilical vein endothelial cells were established in microfluidic flow devices. After overnight perfusion, cell monolayers were subjected to normoxic or hypoxic perfusion and then treated with either estrogen (as estradiol), testosterone (as dihydrotestosterone), or media alone. Endothelial activation/injury was indexed by soluble thrombomodulin and glycocalyx degradation by syndecan-1 and hyaluronic acid shedding as well as measurement of the thickness of the glycocalyx layer. The coagulation phenotype of the human umbilical vein endothelial cells was indexed by the relative values of the activities of tissue plasminogen activator and plasminogen activator inhibitor-1. Vascular endothelial growth factor was measured in cell culture supernatants using a solid-phase enzyme-linked immunosorbent assay. ResultsTreatment with estrogen but not testosterone mitigated the adverse effect of shock on endothelial and glycocalyx barrier properties. Our biomimetic model suggests a beneficial effect of estrogen administration after trauma and hemorrhage shock on the glycocalyx and endothelial barriers. ConclusionEarly estrogen treatment after trauma and hemorrhage shock may be a useful adjunct to mitigating the development of traumatic endotheliopathy.

Targeting HIF2α-ARNT hetero-dimerisation as a novel therapeutic strategy for pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a destructive disease of the pulmonary vasculature often leading to right heart failure and death. Current therapeutic intervention strategies only slow disease progression. The role of aberrant hypoxia-inducible factor (HIF)2α stability and function in the initiation and development of pulmonary hypertension (PH) has been an area of intense interest for nearly two decades.Here we determine the effect of a novel HIF2α inhibitor (PT2567) on PH disease initiation and progression, using two pre-clinical models of PH. Haemodynamic measurements were performed, followed by collection of heart, lung and blood for pathological, gene expression and biochemical analysis. Blood outgrowth endothelial cells from idiopathic PAH patients were used to determine the impact of HIF2α-inhibition on endothelial function.Global inhibition of HIF2a reduced pulmonary vascular haemodynamics and pulmonary vascular remodelling in both su5416/hypoxia prevention and intervention models. PT2567 intervention reduced the expression of PH-associated target genes in both lung and cardiac tissues and restored plasma nitrite concentration. Treatment of monocrotaline-exposed rodents with PT2567 reduced the impact on cardiovascular haemodynamics and promoted a survival advantage. In vitro, loss of HIF2α signalling in human pulmonary arterial endothelial cells suppresses target genes associated with inflammation, and PT2567 reduced the hyperproliferative phenotype and overactive arginase activity in blood outgrowth endothelial cells from idiopathic PAH patients. These data suggest that targeting HIF2α hetero-dimerisation with an orally bioavailable compound could offer a new therapeutic approach for PAH. Future studies are required to determine the role of HIF in the heterogeneous PAH population.

Effects of syndecan-4 gene silencing by micro RNA interference in anoikis resistant endothelial cells: Syndecan-4 silencing and anoikis resistance.

The cell's resistance to cell death by adhesion loss to extracellular matrix (anoikis), contributes to tumor progression and metastasis. Various adhesion molecules are involved in the anoikis resistance, including the syndecan-4 (SDC4), a heparan sulfate proteoglycan (HSPG) present on the cell surface. Changes in the expression of SDC4 have been observed in tumor and transformed cells, indicating its involvement in cancer. In previous works, we demonstrated that acquisition of anoikis resistance resistance by blocking adhesion to the substrate up-regulates syndecan-4 expression in endothelial cells. This study investigates the role of SDC4 in the transformed phenotype of anoikis resistant endothelial cells. Anoikis-resistant endothelial cells (Adh1-EC) were transfected with micro RNA interference (miR RNAi) targeted against syndecan-4. The effect of SDC4 silencing was analyzed by real-time PCR, western blotting and immunofluorescence. Transfection with miRNA-SDC4 resulted in a sequence-specific decrease in syndecan-4 mRNA and protein levels. Furthermore, we observed a reduction in the number of heparan and chondroitin sulfate chains in the cell extract and culture medium. The SDC4 silencing led to downregulation of proliferative and invasive capacity and angiogenic abilities of anoikis-resistant endothelial cells. Compared with the parental cells (Adh1-EC), SDC4 silenced cells (SDC4 miR-Syn-4-1-Adh1-EC e miR-Syn-4-2-Adh1-EC) exhibited an increase in adhesion to collagen and laminin and also in the apoptosis rate. Moreover, transfection with miRNA-SDC4 caused a decrease in the number of cells in the S phase of the cell cycle. This is accompanied by an increase in the heparan sulfate synthesis after 12 h of simulation with fetal calf serum (FCS). SDC4 silencing cells are more dependent of growth factors present in the FCS to synthesize heparan sulfate than parental cells. Similar data were obtained for the wild-type cell line (EC). Our results indicated that downregulation of SDC4 expression reverses the transformed phenotype of anoikis resistant endothelial cells. These and other findings suggest that syndecan-4 is suitable for pharmacological intervention, making it an attractive target for cancer therapy.

ANTI-IL-6 ANTIBODY REDUCES ENDOTHELIAL MEDIATED PRO-INFLAMMATORY RESPONSES AND AFTER BINDING OF HLA-SPECIFIC DSA TO ENDOTHELIAL CELLS

Endothelial cell activation and microvascular inflammation are hallmarks of antibody-mediated rejection and expression of activation associated transcripts by intragraft endothelial cells is observed whether or not complement activation is indicated. HLA expression is strongly upregulated by the allograft microvasculature during rejection and is a target for Donor Specific Antibody (DSA) binding. DSA specific for HLA antigens are prevalent, are deleterious to the allograft and both complement-dependent and -independent pathways of DSA-mediated damage have been reported. We have developed an in vitro model of human microvascular endothelial cells to reveal that inflammation-induced HLA-DR expression results in the activation and differentiation of allogeneic CD4+-T into both IL-6-dependent pro-inflammatory Th17 and anti-inflammatory Treg (Taflin C. 2011). Endothelial binding of HLA-II-specific antibodies further increased IL-6 secretion and amplification of pro-inflammatory Th17 and Th1 (Lion J. 2016, Cross AR. 2019). In the current study we have evaluated whether a humanized monoclonal anti-IL-6 antibody (clazakizumab) can alter the outcome of HLA-specific antibody binding to endothelial cells. Human microvascular endothelial cells cells (HMEC-1 and primary renal glomerular cells) were cultured in the presence or absence of non-HLA matched PBMC as described (Taflin C 2011). Soluble factors were assesed by ELISA assay and CD4+-T sub-populations identified by intracellular cytokine labelling. C4d and C5b-9 deposition on endothelial cells were measured in the presence or absence of complement-containing human AB serum. Antibodies specific for HLA antigens were either monoclonal or purified DSA from transplant patient serum or plasma-exchange products. When endothelial cells were co-cultured with PBMC from non-HLA matched donors, addition of clazakizumab strongly reduced IL-6 at 24hrs and the decrease was maintained even after 72hrs. MCP-1 levels were also reduced. Activation of endothelial cells by HLA class I and II antibodies prior to co-culture further increased IL-6 and this was also decreased by clazakizumab. The cell-surface phenotype of endothelial cells in co-cultures was unaltered by clazakizumab whereas differentiation of Th17 and Th1 were both decreased (particularly IFNY+Th1). HLA-specific antibody binding activated complement, and clazakizumab did not have a direct effect on C4d or C5b-9 deposition on endothelial cells Neither steady-state expression of the complement regulatory proteins CD46, CD55 and CD59 nor their expression under conditions of complement activation were altered by clazakizumab. These data indicate that anti-IL-6 antibody may limit endothelial damage mediated by DSA binding to HLA class I or II. The results reveal the ability of clazakizumab to alter endothelial immunogenicity leading to reduced differentiation of pro-inflammatory CD4+-T. Moreover the decreased level of MCP-1 may limit recuitment of myeloid cells within the allograft. Société Française de Transplantation. EU-COST 17138. Vitaeris Inc. Vaincre la Mucoviscidose.

Exacerbated inflammatory signaling underlies aberrant response to BMP9 in pulmonary arterial hypertension lung endothelial cells

Imbalanced transforming growth factor beta (TGFβ) and bone morphogenetic protein (BMP) signaling are postulated to favor a pathological pulmonary endothelial cell (EC) phenotype in pulmonary arterial hypertension (PAH). BMP9 is shown to reinstate BMP receptor type-II (BMPR2) levels and thereby mitigate hemodynamic and vascular abnormalities in several animal models of pulmonary hypertension (PH). Yet, responses of the pulmonary endothelium of PAH patients to BMP9 are unknown. Therefore, we treated primary PAH patient-derived and healthy pulmonary ECs with BMP9 and observed that stimulation induces transient transcriptional signaling associated with the process of endothelial-to-mesenchymal transition (EndMT). However, solely PAH pulmonary ECs showed signs of a mesenchymal trans-differentiation characterized by a loss of VE-cadherin, induction of transgelin (SM22α), and reorganization of the cytoskeleton. In the PAH cells, a prolonged EndMT signaling was found accompanied by sustained elevation of pro-inflammatory, pro-hypoxic, and pro-apoptotic signaling. Herein we identified interleukin-6 (IL6)-dependent signaling to be the central mediator required for the BMP9-induced phenotypic change in PAH pulmonary ECs. Furthermore, we were able to target the BMP9-induced EndMT process by an IL6 capturing antibody that normalized autocrine IL6 levels, prevented mesenchymal transformation, and maintained a functional EC phenotype in PAH pulmonary ECs. In conclusion, our results show that the BMP9-induced aberrant EndMT in PAH pulmonary ECs is dependent on exacerbated pro-inflammatory signaling mediated through IL6.

Evaluation of the Influence of Wall Shear Stress Features from Newly Fashioned Arteriovenous Fistulae on Endothelial Cell Phenotypes In Vitro

Objective: Patency rates for autogenous arteriovenous fistulae (AVFs) range between 60% and 80% at two years. The wall shear stress (WSS) is the frictional force per unit area exerted at the interface of the flowing blood and the endothelium and is a potent stimulus capable of evoking differential phenotypes in endothelial cells (ECs). The aim of this study was to investigate the influence of WSS on EC phenotypes. Methods: The intraluminal pressures and waveforms from the arterial and venous limbs of 14 newly fashioned human AVFs (four radiocephalic, 10 brachiocephalic) were measured using a microcatheter transducer and pull through technique. Human umbilical vein endothelial cells (HUVECs) were exposed for 24 hours to 10 idealised physiological waveforms (using calculations from in vivo experiments) with WSS peak magnitude 0.5 – 2.5 Pa, temporal wall shear stress gradient (t-WSSG) from 2 to 20 Pa/s, and from 0 to 0.5 of the oscillatory shear index value. Differential protein and gene expression was evaluated using reverse transcription polymerase chain reaction, western blot, and immunofluorescence staining. Results: In vitro results indicate that WSS waveforms with a peak magnitude of 1.5 or 2.5 Pa can induce a protective phenotype in ECs in culture (Krüppel-like factor-2 (KLF-2), KLF-4, Interleukin-8 (IL-8), and vascular cell adhesion molecule -1 (VCAM-1) expression). By presenting a reverse phase or decreasing time averaged WSS of the waveforms with a peak of 1.5 or 2.5 Pa, the flow induced protective effect is maintained. A WSS peak of 1.5 Pa is shown to be sufficient to induce protective pathways. Pro-inflammatory gene analysis showed no differences in the expression of two subunits of nuclear factor kappa B. Conclusion: The differential effects of WSS profiles can induce protective or pro-inflammatory EC profiles. These results suggest that geometrical variations may influence AVF patency rates and outcomes.

Long-Term Cryopreservation Preserves Blood-Brain Barrier Phenotype of iPSC-Derived Brain Microvascular Endothelial Cells and Three-Dimensional Microvessels.

Brain microvascular endothelial cells derived from induced pluripotent stem cells (dhBMECs) are a scalable and reproducible resource for studies of the human blood-brain barrier, including mechanisms and strategies for drug delivery. Confluent monolayers of dhBMECs recapitulate key in vivo functions including tight junctions to limit paracellular permeability and efflux and nutrient transport to regulate transcellular permeability. Techniques for cryopreservation of dhBMECs have been reported; however, functional validation studies after long-term cryopreservation have not been extensively performed. Here, we characterize dhBMECs after 1 year of cryopreservation using selective purification on extracellular matrix-treated surfaces and ROCK inhibition. One-year cryopreserved dhBMECs maintain functionality of tight junctions, efflux pumps, and nutrient transporters with stable protein localization and gene expression. Cryopreservation is associated with a decrease in the yield of adherent cells and unique responses to cell stress, resulting in altered paracellular permeability of Lucifer yellow. Additionally, cryopreserved dhBMECs reliably form functional three-dimensional microvessels independent of cryopreservation length, with permeabilities lower than non-cryopreserved two-dimensional models. Long-term cryopreservation of dhBMECs offers key advantages including increased scalability, reduced batch-to-batch effects, the ability to conduct well-controlled follow up studies, and support of multisite collaboration from the same cell stock, all while maintaining phenotype for screening pharmaceutical agents.

Plasma microparticles of sickle patients during crisis or taking hydroxyurea modify endothelium inflammatory properties

AbstractMicroparticles (MPs) are submicron extracellular vesicles exposing phosphatidylserine (PS), detected at high concentration in the circulation of sickle cell anemia (SS) patients. Several groups studied the biological effects of MPs generated ex vivo. Here, we analyzed for the first time the impact of circulating MPs on endothelial cells (ECs) from 60 sickle cell disease (SCD) patients. MPs were collected from SCD patients and compared with MPs isolated from healthy individuals (AA). Other plasma MPs were purified from SS patients before and 2 years after the onset of hydroxyurea (HU) treatment or during a vaso-occlusive crisis and at steady-state. Compared with AA MPs, SS MPs increased EC ICAM-1 messenger RNA and protein levels, as well as neutrophil adhesion. We showed that ICAM-1 overexpression was primarily caused by MPs derived from erythrocytes, rather than from platelets, and that it was abolished by MP PS capping using annexin V. MPs from SS patients treated with HU were less efficient to induce a proinflammatory phenotype in ECs compared with MPs collected before therapy. In contrast, MPs released during crisis increased ICAM-1 and neutrophil adhesion levels, in a PS-dependent manner, compared with MPs collected at steady-state. Furthermore, neutrophil adhesion was abolished by a blocking anti–ICAM-1 antibody. Our study provides evidence that MPs play a key role in SCD pathophysiology by triggering a proinflammatory phenotype of ECs. We also uncover a new mode of action for HU and identify potential therapeutics: annexin V and anti–ICAM-1 antibodies.

Designer Descemet Membranes Containing PDLLA and Functionalized Gelatins as Corneal Endothelial Scaffold.

Corneal blindness is the fourth leading cause of visual impairment. Of specific interest is blindness due to a dysfunctional corneal endothelium which can only be treated by transplanting healthy tissue from a deceased donor. Unfortunately, corneal supply does not meet the demand with only one donor for every 70 patients. Therefore, there is a huge interest in tissue engineering of grafts consisting of an ultra-thin scaffold seeded with cultured endothelial cells. The present research describes the fabrication of such artificial Descemet membranes based on the combination of a biodegradable amorphous polyester (poly (d,l-lactic acid)) and crosslinkable gelatins. Four different crosslinkable gelatin derivatives are compared in terms of processing, membrane quality, and function, as well as biological performance in the presence of corneal endothelial cells. The membranes are fabricated through multi-step spincoating, including a sacrificial layer to allow for straightforward membrane detachment after production. As a consequence, ultrathin (<1µm), highly transparent (>90%), semi-permeable membranes could be obtained with high biological potential. The membranes supported the characteristic morphology and correct phenotype of corneal endothelial cells while exhibiting similar proliferation rates as the positive control. As a consequence, the proposed membranes prove to be a promising synthetic alternative to donor tissue.

Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3.

Loss-of-function variants in CCM1/KRIT1, CCM2, and CCM3/PDCD10 are associated with autosomal dominant cerebral cavernous malformations (CCMs). CRISPR/Cas9-mediated CCM3 inactivation in human endothelial cells (ECs) has been shown to induce profound defects in cell-cell interaction as well as actin cytoskeleton organization. We here show that CCM3 inactivation impairs fibronectin expression and consequently leads to reduced fibers in the extracellular matrix. Despite the complexity and high molecular weight of fibronectin fibrils, our in vitro model allowed us to reveal that fibronectin supplementation restored aberrant spheroid formation as well as altered EC morphology, and suppressed actin stress fiber formation. Yet, fibronectin replacement neither enhanced the stability of tube-like structures nor inhibited the survival advantage of CCM3-/- ECs. Importantly, CRISPR/Cas9-mediated introduction of biallelic loss-of-function variants into either CCM1 or CCM2 demonstrated that the impaired production of a functional fibronectin matrix is a common feature of CCM1-, CCM2-, and CCM3-deficient ECs.

O-GlcNAcylation Mediates Glucose-Induced Alterations in Endothelial Cell Phenotype in Human Diabetes Mellitus.

BackgroundPosttranslational protein modification with O‐linked N‐acetylglucosamine (O‐GlcNAc) is linked to high glucose levels in type 2 diabetes mellitus (T2DM) and may alter cellular function. We sought to elucidate the involvement of O‐GlcNAc modification in endothelial dysfunction in patients with T2DM.Methods and ResultsFreshly isolated endothelial cells obtained by J‐wire biopsy from a forearm vein of patients with T2DM (n=18) was compared with controls (n=10). Endothelial O‐GlcNAc levels were 1.8‐ford higher in T2DM patients than in nondiabetic controls (P=0.003). Higher endothelial O‐GlcNAc levels correlated with serum fasting blood glucose level (r=0.433, P=0.024) and hemoglobin A1c (r=0.418, P=0.042). In endothelial cells from patients with T2DM, normal glucose conditions (24 hours at 5 mmol/L) lowered O‐GlcNAc levels and restored insulin‐mediated activation of endothelial nitric oxide synthase, whereas high glucose conditions (30 mmol/L) maintained both O‐GlcNAc levels and impaired insulin action. Treatment of endothelial cells with Thiamet G, an O‐GlcNAcase inhibitor, increased O‐GlcNAc levels and blunted the improvement of insulin‐mediated endothelial nitric oxide synthase phosphorylation by glucose normalization.ConclusionsTaken together, our findings indicate a role for O‐GlcNAc modification in the dynamic, glucose‐induced impairment of endothelial nitric oxide synthase activation in endothelial cells from patients with T2DM. O‐GlcNAc protein modification may be a treatment target for vascular dysfunction in T2DM.

Mesenchymal Behavior of the Endothelium Promoted by SMAD6 Downregulation Is Associated With Brain Arteriovenous Malformation Microhemorrhage.

In unruptured brain arteriovenous malformations (bAVMs), microhemorrhage portends a higher risk of future rupture and may represent a transitional state along the continuum of destabilization. Exploration of the molecular and cellular mechanisms of microhemorrhage will provide a possible target for medical treatment to prevent bAVM bleeding. We performed RNA sequencing analysis on 34 unruptured bAVM surgical samples. Functional pathway analysis was performed to identify potential signals associated with the microhemorrhagic phenotype. Candidate gene was then investigated in bAVM specimens by immunohistochemical staining. Several functional assays were used to investigate the effects of candidate genes on the phenotypic properties of cultured human umbilical vein endothelial cells. Then, Masson trichrome staining and immunofluorescence staining were used to evaluate the phenotypic and molecular changes in bAVM tissue. Via RNA sequencing, we identified differential gene expression between 18 microhemorrhagic bAVMs and 16 nonmicrohemorrhagic bAVMs. TGFβ (transforming growth factor-beta)/BMP (bone morphogenetic protein) signaling was associated with the bAVM microhemorrhage group when SMAD6 (SMAD family member 6) was downregulated. Immunohistochemical staining showed that the vascular endothelium of microhemorrhagic bAVMs exhibited decreased SMAD6 expression. Functional assays revealed that SMAD6 downregulation promoted the formation of endothelial cell tubes with deficient cell-cell junctions and facilitated the acquisition of mesenchymal behavior by endothelial cells. Masson trichrome and immunofluorescence staining demonstrated that mesenchymal phenotype of endothelial cells is promoted in microhemorrhagic bAVMs. TGFβ/BMP signaling mediated by SMAD6 in vascular endothelial cells is associated with microhemorrhagic bAVMs, and mesenchymal behavior of endothelial cells induced by SMAD6 downregulation is related with bAVM microhemorrhage.

Development of a human in vitro blood\u2013brain tumor barrier model of diffuse intrinsic pontine glioma to better understand the chemoresistance

BackgroundPediatric diffuse intrinsic pontine glioma (DIPG) represents one of the most devastating and lethal brain tumors in children with a median survival of 12 months. The high mortality rate can be explained by the ineligibility of patients to surgical resection due to the diffuse growth pattern and midline localization of the tumor. While the therapeutic strategies are unfortunately palliative, the blood–brain barrier (BBB) is suspected to be responsible for the treatment inefficiency. Located at the brain capillary endothelial cells (ECs), the BBB has specific properties to tightly control and restrict the access of molecules to the brain parenchyma including chemotherapeutic compounds. However, these BBB specific properties can be modified in a pathological environment, thus modulating brain exposure to therapeutic drugs. Hence, this study aimed at developing a syngeneic human blood–brain tumor barrier model to understand how the presence of DIPG impacts the structure and function of brain capillary ECs.MethodsA human syngeneic in vitro BBB model consisting of a triple culture of human (ECs) (differentiated from CD34+-stem cells), pericytes and astrocytes was developed. Once validated in terms of BBB phenotype, this model was adapted to develop a blood–brain tumor barrier (BBTB) model specific to pediatric DIPG by replacing the astrocytes by DIPG-007, -013 and -014 cells. The physical and metabolic properties of the BBTB ECs were analyzed and compared to the BBB ECs. The permeability of both models to chemotherapeutic compounds was evaluated.ResultsIn line with clinical observation, the integrity of the BBTB ECs remained intact until 7 days of incubation. Both transcriptional expression and activity of efflux transporters were not strongly modified by the presence of DIPG. The permeability of ECs to the chemotherapeutic drugs temozolomide and panobinostat was not affected by the DIPG environment.ConclusionsThis original human BBTB model allows a better understanding of the influence of DIPG on the BBTB ECs phenotype. Our data reveal that the chemoresistance described for DIPG does not come from the development of a “super BBB”. These results, validated by the absence of modification of drug transport through the BBTB ECs, point out the importance of understanding the implication of the different protagonists in the pathology to have a chance to significantly improve treatment efficiency.

Topography-driven alterations in endothelial cell phenotype and contact guidance

Understanding how endothelial cell phenotype is affected by topography could improve the design of new tools for tissue engineering as many tissue engineering approaches make use of topography-mediated cell stimulation. Therefore, we cultured human pulmonary microvascular endothelial cells (ECs) on a directional topographical gradient to screen the EC vascular-like network formation and alignment response to nano to microsized topographies. The cell response was evaluated by microscopy. We found that ECs formed unstable vascular-like networks that aggregated in the smaller topographies and flat parts whereas ECs themselves aligned on the larger topographies. Subsequently, we designed a mixed topography where we could explore the network formation and proliferative properties of these ECs by live imaging for three days. Vascular-like network formation continued to be unstable on the topography and were only produced on the flat areas and a fibronectin coating did not improve the network stability. However, an instructive adipose tissue-derived stromal cell (ASC) coating provided the correct environment to sustain the vascular-like networks, which were still affected by the topography underneath. It was concluded that large microsized topographies inhibit vascular endothelial network formation but not proliferation and flat and nano/microsized topographies allow formation of early networks that can be stabilized by using an ASC instructive layer.

Transport and Preservation Comparison of Preloaded and Prestripped-Only DMEK Grafts

This study compares the effect of the transport of conventionally prestripped Descemet membrane endothelial keratoplasty (DMEK) tissue with the DMEK revolutionary advanced Preloadable Injection Device (RAPID) preloaded transport system from Geuder AG (Heidelberg, Germany). Endothelial cell loss, tissue integrity, endothelial cell phenotype, and viability were assessed and compared. Twelve DMEK grafts were prestripped by the cornea bank and transported using the following 2 conditions: conventional flask (n = 6) or a preloaded transport cartridge (DMEK RAPID, n = 6). After transport, tissues were analyzed for cell density; denuded areas; immunolocalization of corneal endothelial markers, such as ZO-1, CD166, and Na/K ATPase; histology analysis; and cell viability staining with Hoechst, calcein AM, and ethidium homodimer. Endothelial cell loss (10.35% vs. 9.15%) did not differ between transport conditions. Histological analysis confirmed the integrity of the Descemet membrane and endothelial cell layer with both transport conditions. Similarly, the corneal endothelial cell mosaic was conserved in both conditions. The ZO-1 tight junctions confirmed the integrity of the confluent corneal endothelial cell monolayer. CD166 and Na/K ATPase detection with immunofluorescence was also comparable. A similar percentage of dead cells was reported in both conditions (18.1% vs. 16.73%). Moreover, the surface covered with calcein-positive cells (59.02% vs. 61.95%) did not differ between transport conditions. Our results suggest that DMEK grafts can be prestripped or preloaded into a novel transport cartridge and shipped to the clinic with comparable endothelial cell loss, phenotypical marker expression, and viability to the conventional prestripped donor tissue.