Endothelial Cell Phenotype Research Articles

Hyaluronic acid-functionalized poly-lactic acid (PLA) microfibers regulate vascular endothelial cell proliferation and phenotypic shape expression

This work was designed to evaluate the efficacy of hyaluronic acid (HA) functionalized tubular poly-lactic acid (PLA) microfibers in directing the luminal pre-endothelialization of vascular endothelial cells (ECs). Tubular HA/PLA microfibers with hierarchical architecture were prepared by electrospinning and chemical cross-linking process. A layer of HA microfibrous film coating was fixed on the inner wall surface of the tubular HA/PLA microfibers, resulting in higher anisotropy wettability and relatively lower surface energy and roughness. We confirmed that HA coating on PLA microfibers surface have reduced hemolytic activity and coagulation degree. Mouse vascular ECs exhibited surface-dependent differences in cell elongation and proliferation (HA/PLA > PLA). Compared with PLA microfibers, the gene expression levels of platelet EC adhesion molecule-1 (PECAM-1/CD31) and vascular endothelial growth factor (VEGF) in ECs of HA/PLA microfibers surface were up-regulated. Immunostaining analysis revealed that the surface of HA/PLA nanofibers supported the expression of mature vascular EC phenotype CD31 protein. In vitro co-culture analysis showed that the luminal pre-endothelialization induced vascular smooth muscle cells (SMCs) to maintain their phenotypic shape and establish natural behavior patterns in the hierarchical tubular scaffold. These studies indicate that the biophysical cues of scaffolds are potent regulators of vascular EC endothelialization.

Key molecular alterations in endothelial cells in human glioblastoma uncovered through single-cell RNA sequencing.

Passage of systemically delivered pharmacological agents into the brain is largely blocked by the blood-brain-barrier (BBB), an organotypic specialization of brain endothelial cells (ECs). Tumor vessels in glioblastoma (GBM), the most common malignant brain tumor in humans, are abnormally permeable, but this phenotype is heterogeneous and may differ between the tumor’s center and invasive front. Here, through single-cell RNA sequencing (scRNA-seq) of freshly isolated ECs from human glioblastoma and paired tumor peripheral tissues, we have constructed a molecular atlas of human brain ECs providing unprecedented molecular insight into the heterogeneity of the human BBB and its molecular alteration in glioblastoma. We identified 5 distinct EC phenotypes representing different states of EC activation and BBB impairment, and associated with different anatomical locations within and around the tumor. This unique data resource provides key information for designing rational therapeutic regimens and optimizing drug delivery.

Differential procoagulatory response of microvascular, arterial and venous endothelial cells upon inflammation in vitro

IntroductionInflammation induces a procoagulant phenotype of endothelial cells (EC) with the exposure of tissue factor (TF), a potent initiator of the extrinsic coagulation cascade. Although systemic inflammation affects the whole vascular system, thrombotic lesions occur particularly in microcirculation. This raises the question of whether TF-procoagulant activity (TF-PCA) differs between EC from arterial, venous, and microvascular beds. Materials and methodsFunctional coagulation tests, including TF-PCA, and inflammatory responses were investigated on arterial, venous and microvascular endothelial cells. Interleukin-6 (IL-6) and TF-levels were determined in cohort of 59 septic patients. ResultsWe found that tumor necrosis factor alpha (TNFα), lipopolysaccharide, and interleukin-1β induce a solid, dose-dependent increase in TF-PCA, which is highest in microvascular EC. A positive correlation of interleukin-6 (IL-6) with TF levels was observed in a cohort of 59 septic patients. In contrast, TF-PCA was independent of IL-6 concentrations in vitro. Re-analysis of publicly available gene expression data revealed that among the top 50 genes annotated to coagulation, TF is one of three regulated genes common to the three investigated EC subtypes. The response to inflammatory stimuli in terms of exposure of leukocyte-endothelial- and platelet-endothelial adhesion molecules (E-selectin and PECAM-1), remodeling of adherens junctions, co-exposure of negatively charged surfaces nor breakdown of the glycocalyx was comparable between the EC subtypes and did not explain the higher TF-PCA on microvascular cells. We found that the ratio of TF and TFPI exposure on the endothelial membrane significantly differs between the EC subtypes. ConclusionsThese findings indicate that the ratio of TF to its inhibitor TFPI is a determinant of endothelial TF-PCA, which is most pronounced on microvascular endothelial cells and might explain why the microvascular system is particularly susceptible to inflammation-induced thrombosis.

Cancer Cell-Derived Extracellular Vesicles Are Prothrombotic in an in-Vitro Vessel Model

Introduction:The risk of thrombosis is markedly increased in patients with cancer, and cancer-associated thrombosis (CAT) is associated with significant morbidity and mortality. It is likely that several mechanisms contribute to the development of CAT, although these may vary among different types of cancers and under different conditions. While genetically-engineered mice may be valuable models for studying thrombosis, an in-vitro model of human vasculature would enable study of CAT mechanisms more rapidly and with less expense; however, at this point such models are not well-developed. Extracellular vesicles (EV) are cell-derived particles, most of which range from <100 nM to 500 nM in diameter. These may arise from cancer cells, stromal cells, or vascular cells, including platelets, endothelial cells and leukocytes, and circulate at elevated concentrations in patients with cancer. EV may induce thrombosis through a variety of mechanisms, including expression of tissue factor, contact activation, and potentially other mechanisms including transfer of RNA or proteins to vascular cells, enhancing their prothrombotic activity.Methods:To develop a system for evaluating the prothrombotic potential of EV on a microscale, we used the Cellix microfludic system, with which we can assess cellular adhesion to the wall of a glass capillary tube through which fluid is driven by a peristaltic pump. In these experiments human umbilical vein endothelial cells (HUVEC) were cultured in fibronectin-coated glass capillary tubes, forming an intact monolayer (Figure, top left panel). After washing non-adherent endothelial cells from the vessel, the endothelial cell-lined vessels were exposed to EV derived from cancer cell lines (U87 glioblastoma or HT29 colorectal carcinoma), or primary human fibroblasts, for four hours prior to flowing of normal human whole blood, to which calcein-AM had been added to label platelets, through the vessel at a shear rate of 67 dynes/cm2. TNFα was used as a positive control to induce an adhesive endothelial cell phenotype. In some experiments, EV were added directly to whole blood, or both preincubated with endothelial cells and added to whole blood before perfusion through the system.Results:As expected (see Figure), we observed that preincubation of TNFα with HUVEC-lined vessels significantly increased platelet adhesion to the endothelial cell monolayer (~100 arbitrary fluorescence units, AFU). In addition, either preincubation of endothelial cells with U87 or HT29-derived exosomes (Figure), or addition of cancer cell-derived exosomes to whole blood also led to markedly-increased rates of platelet adhesion (50-80 AFU) to the endothelial monolayer, and in some cases caused complete occlusion of the vessel and cessation of flow (e.g. in vitro thrombus formation). Normal fibroblast-derived EV also stimulated platelet adhesion but to a much lower extent than cancer cell-derived EV (~20 AFU). Addition of cancer cell-derived EV to both the microfluidics vessel and whole blood almost universally caused vessel occlusion. In further studies, we also found that whole blood from Lewis lung carcinoma-bearing mice was more prothrombotic in this system than that from non-tumor-bearing littermates (~60% higher), and that the absence of high molecular weight kininogen (studied using blood from tumor bearing and non-tumor-bearing kininogen deficient mice) partially protected again vascular occlusion in this model.Conclusion:These studies suggest that cancer cell-derived EV are more prothrombotic than EV derived from non-transformed cells in this model vessel lined by human endothelial cells. Additional work is needed to further define the underlying mechanism by which this occurs, but involvement of the contact activation system is likely, particularly based on preliminary results using blood from tumor-bearing kininogen deficient mice. This microfluidics system will be of significant value in future translational and mechanistic studies focused on mechanisms of CAT. [Display omitted] DisclosuresKhorana:Janssen Scientific Affairs, LLC: Consultancy, Honoraria, Research Funding; Leo: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria; Halozyme: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Roche: Consultancy, Honoraria.

KLF2 Mediates the Suppressive Effect of Laminar Flow on Vascular Calcification by Inhibiting Endothelial BMP/SMAD1/5 Signaling.

[Figure: see text].

The Impact of the Renin-Angiotensin-Aldosterone System on Inflammation, Coagulation, and Atherothrombotic Complications, and to Aggravated COVID-19.

Atherosclerosis is considered a disease caused by a chronic inflammation, associated with endothelial dysfunction, and several mediators of inflammation are up-regulated in subjects with atherosclerotic disease. Healthy, intact endothelium exhibits an antithrombotic, protective surface between the vascular lumen and vascular smooth muscle cells in the vessel wall. Oxidative stress is an imbalance between anti- and prooxidants, with a subsequent increase of reactive oxygen species, leading to tissue damage. The renin-angiotensin-aldosterone system is of vital importance in the pathobiology of vascular disease. Convincing data indicate that angiotensin II accelerates hypertension and augments the production of reactive oxygen species. This leads to the generation of a proinflammatory phenotype in human endothelial and vascular smooth muscle cells by the up-regulation of adhesion molecules, chemokines and cytokines. In addition, angiotensin II also seems to increase thrombin generation, possibly via a direct impact on tissue factor. However, the mechanism of cross-talk between inflammation and haemostasis can also contribute to prothrombotic states in inflammatory environments. Thus, blocking of the renin-angiotensin-aldosterone system might be an approach to reduce both inflammatory and thrombotic complications in high-risk patients. During COVID-19, the renin-angiotensin-aldosterone system may be activated. The levels of angiotensin II could contribute to the ongoing inflammation, which might result in a cytokine storm, a complication that significantly impairs prognosis. At the outbreak of COVID-19 concerns were raised about the use of angiotensin converting enzyme inhibitors and angiotensin receptor blocker drugs in patients with COVID-19 and hypertension or other cardiovascular comorbidities. However, the present evidence is in favor of continuing to use of these drugs. Based on experimental evidence, blocking the renin-angiotensin-aldosterone system might even exert a potentially protective influence in the setting of COVID-19.

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies.

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

FC 105LITHIUM PRESERVES PERITONEAL MEMBRANE INTEGRITY BY REDUCING MESOTHELIAL CELL ΑB-CRYSTALLIN

Abstract Background and Aims Renal replacement therapy by peritoneal dialysis (PD) is limited in use and duration by progressive impairment of peritoneal membrane integrity and homeostasis. Preservation of peritoneal membrane integrity during chronic PD remains an urgent but long-unmet medical need. PD therapy failure results from peritoneal fibrosis and angiogenesis caused by hypertonic PD fluid (PDF)-induced mesothelial cytotoxicity. The incompletely defined pathophysiological mechanisms involved confound informed selection of therapeutic targets. Addition of cytoprotective agents to PDF have been shown to counteract pathophysiological mechanisms induced by current PDF. Lithium is a well described inhibitor of glycogen synthase kinase 3β and has recently been shown to also have nephroprotective effects in low doses. Here, we aim to characterize icodextrin-based, PDF-induced cellular injury with a combined omics approach and to investigate the effects of LiCl on the PD-induced observed molecular perturbations. Method To investigate mechanisms of acute cellular damage by PDF we chose an in vitro model of primary omental-derived peritoneal mesothelial cells with direct exposure to icodextrin-based PDF, followed by short-term or extended recovery for detection of short-term and long-term changes in transcriptome, proteome, and cell injury. 0, 2.5 or 10 mM LiCl were added to the PDF. In-vitro findings were validated in peritoneal biopsies (n=41) from pediatric PD and CDK5 patients or healthy controls and peritoneal effluents from adult and pediatric PD patients (n=27) or ascites samples (n=4) as control. For in-vivo experiments, healthy and uremic mice (C57/Bl6, female) were chronically exposed to PD-fluid without or with the addition of 5 mM LiCl via an implanted catheter. In-vivo overexpression of CRYAB was induced by i.p. injection of an adenoviral vector. All animal experiments and use of patient samples were approved by the local ethics committees and performed according to animal protection laws or the Declaration of Helsinki, respectively. Results LiCl significantly improved mesothelial cell survival in a dose-dependent manner. Combined transcriptomic and proteomic characterization of icodextrin-based PDF-induced mesothelial cell injury identified αB-crystallin as the mesothelial cell protein most significantly and consistently counter-regulated by LiCl. In-vitro and in-vivo overexpression of αB-crystallin triggered a fibrotic phenotype and PDF-like upregulation of vascular endothelial growth factor (VEGF), CD31-positive cells, and TGFβ-independent activation of TGFβ-regulated targets. In contrast, αB-crystallin knock-down decreased VEGF expression and early mesothelial-to-mesenchymal transition (MMT). LiCl reduced VEGF release and counteracted fibrosis- and angiogenesis-associated processes. αB-crystallin in patient-derived mesothelial cells was specifically upregulated in response to PDF and increased in peritoneal mesothelial cells from pediatric PD patient biopsies, correlating with markers of angiogenesis and fibrosis. Conclusion The cytoprotective effects of LiCl-supplemented PDF may be explained by counter-regulation of PD-induced angiogenesis via the novel target αB-crystallin. Reduction of mesothelial cell damage, peritoneal fibrosis and VEGF suggests therapeutic potential of this intervention. Repurposing LiCl as a cytoprotective PDF additive may offer a translatable therapeutic strategy to combat peritoneal membrane deterioration during PD therapy. Further study of LiCl-supplemented PDF is merited as a realistic approach to improving treatment longevity and patient outcomes during PD treatment.

Hyaluronan (HA) Immobilized on Surfaces via Self-Assembled Monolayers of HA-Binding Peptide Modulates Endothelial Cell Spreading and Migration through Focal Adhesion.

The extracellular matrix (ECM) modulates a multitude of cell functions, and this regulation is provided by key ECM components forming a complex network. Hyaluronic acid (HA) is an abundant component of the ECM that binds to proteins and influences various activities of endothelial cells (ECs). Although the effect of soluble HA on cell spreading has been studied, the impact of peptide-bound HA has not yet been investigated in great detail. We aim to comprehensively study the roles of immobilized HA on the regulation of EC behavior compared to the more conventional use of soluble HA. A 2D model surface formed by self-assembled monolayers (SAMs) of a HA-binding peptide (Pep-1) is used as an anchor for HA immobilization. Mixed SAMs, consisting of thiolated Pep-1 and 1-octanethiol, are prepared and characterized by using ellipsometry and contact angle measurement. Full density Pep-1 SAMs are more hydrophilic and bind more HA than mixed SAMs. Cell spreading and migration are enhanced by immobilized low molecular weight (LMW) HA, which also facilitates cell alignment and elongation under laminar flow conditions and potentially drives directional migration. This effect is not mediated by the expression of CD44, and immobilized LMW HA is found to accelerate the assembly of focal adhesions. Such biomimetic surfaces provide new insights into the role of HA in regulating the spreading and phenotype of endothelial cells.

Raman imaging-based phenotyping of murine primary endothelial cells to identify disease-associated biochemical alterations

Raman spectroscopy is successfully becoming an analytical tool used to characterize alterations in the biochemical composition of cells. In this work, we identify the features of Raman spectra of murine primary endothelial cells (EC) isolated from lungs, heart, liver, brain, kidney and aorta of normal mice, as well as from heart, lung and liver in a murine model of heart failure (HF) in Tgαq*44 mice. Primary cells were measured in suspension immediately after their isolation. Raman images showed that isolated primary EC were elliptical or circular, and did not show organ-specific spectral features for any of the studied organ, i.e. lungs, heart, liver, brain, kidney and aorta. Principal Component Analysis pairwise analysis of primary endothelial cells from FVB mice and Tgαq*44 mice revealed an increased protein content in EC isolated from the heart and increased lipid content in EC isolated from the lung in Tgαq*44 mice. No significant differences were found in the EC isolated from the liver using the same chemometric procedure. To our knowledge, this is the first report in which Raman spectroscopy has been used to characterize the biochemical phenotype of primary murine EC with developing HF. This pilot study shows that Raman-based analysis of freshly isolated primary EC did not revealed organ-specific features, however disease-associated changes were found in the coronary and pulmonary EC in the early stage of heart failure in Tgαq*44 mice.

Role of interleukin-6 in human umbilical vein endothelial cell to mesenchymal cell transformation

Role of interleukin-6 in human umbilical vein endothelial cell to mesenchymal cell transformation

Laminar flow substantially affects the morphology and functional phenotype of glomerular endothelial cells.

Shear stress induced by laminar blood flow has a profound effect on the morphology and functional phenotype of macrovascular endothelial cells. The influence of laminar flow on the glomerular microvascular endothelium, however, remains largely elusive. The glomerular endothelium, including its glycocalyx, is a crucial part of the glomerular filtration barrier, which is involved in blood filtration. We therefore investigated the influence of laminar flow-induced shear stress on the glomerular endothelium. Conditionally immortalized mouse glomerular endothelial cells were cultured for 7 days under a laminar flow of 5 dyn/cm2 to mimic the glomerular blood flow. The cells were subsequently analysed for changes in morphology, expression of shear stress-responsive genes, nitric oxide production, glycocalyx composition, expression of anti-oxidant genes and the inflammatory response. Culture under laminar flow resulted in cytoskeletal rearrangement and cell alignment compared to static conditions. Moreover, production of nitric oxide was increased and the expression of the main functional component of the glycocalyx, Heparan Sulfate, was enhanced in response to shear stress. Furthermore, glomerular endothelial cells demonstrated a quiescent phenotype under flow, characterized by a decreased expression of the pro-inflammatory gene ICAM-1 and increased expression of the anti-oxidant enzymes HO-1 and NQO1. Upon exposure to the inflammatory stimulus TNFα, however, glomerular endothelial cells cultured under laminar flow showed an enhanced inflammatory response. In conclusion, laminar flow extensively affects the morphology and functional phenotype of glomerular endothelial cells in culture. Furthermore, glomerular endothelial cells respond differently to shear stress compared to macrovascular endothelium. To improve the translation of future in vitro studies with glomerular endothelial cells to the in vivo situation, it appears therefore crucial to culture glomerular endothelial cells under physiological flow conditions.

Endothelial cell plasticity at the single-cell level

The vascular endothelium is characterized by a remarkable level of plasticity, which is the driving force not only of physiological repair/remodeling of adult tissues but also of pathological angiogenesis. The resulting heterogeneity of endothelial cells (ECs) makes targeting the endothelium challenging, no less because many EC phenotypes are yet to be identified and functionally inventorized. Efforts to map the vasculature at the single-cell level have been instrumental to capture the diversity of EC types and states at a remarkable depth in both normal and pathological states. Here, we discuss new EC subtypes and functions emerging from recent single-cell studies in health and disease. Interestingly, such studies revealed distinct metabolic gene signatures in different EC phenotypes, which deserve further consideration for therapy. We highlight how this metabolic targeting strategy could potentially be used to promote (for tissue repair) or block (in tumor) angiogenesis in a tissue or even vascular bed-specific manner.

Metformin Modulates Doxorubicin‐induced Senescence Phenotype in Endothelial Cells

Background Doxorubicin (DOX) is a cardiotoxic chemotherapeutic agent. In addition to cardiotoxicity, DOX treatment is associated with endothelial dysfunction and increased markers of vascular aging in cancer survivors, contributing to DOX-induced cardiovascular complications. Metformin, an anti-diabetic drug, has been shown to have anti-aging effects in different models of aging. Therefore, in our current work, we first established an in vitro model of DOX-induced senescence in EA.hy926 human endothelial-derived cells and Human Umbilical Vein Endothelial Cells (HUVECs). Then, we identified the effects of metformin on DOX-induced senescence phenotype. Methods Both HUVECs and EA.hy926 human endothelial-derived cells were treated with increasing DOX concentrations for 24 hours. Thereafter, DOX was removed and cells were incubated in DOX free media for another 72-120 hours. Protein expression of senescence markers were measured using Western blot analysis. Cell cycle analysis was assessed using flow cytometry. Senescence-associated beta-galactosidase (SA-beta GAL) activity was measured by SA-beta GAL assay. Next, cells were incubated with DOX +/- increasing metformin concentrations. Effects of metformin on DOX-induced senescence markers and cell cycle were assessed. To delineate the molecular mechanisms, the effects of metformin on major signaling pathways were determined. Results DOX significantly induced expression of senescence markers in endothelial cells in a concentration-dependent manner. Moreover, DOX caused cell cycle arrest in the G2/M phase and increased SA-beta GAL staining. Metformin corrected DOX-induced upregulation of senescence markers, which was associated with a significant inhibition of the JNK pathway. Intriguingly, metformin had no effect on DOX-induced cell cycle arrest and slightly corrected the increased expression of SA-beta GAL. Conclusions Our current work shows that metformin modulates DOX-induced senescence phenotype in endothelial cells, suggesting it may protect against DOX-induced vascular aging and endothelial dysfunction.

Computational Evaluation of Wall Shear Stress Experienced by Endothelial Tip Cells along Capillary Sprouts

Shear stress is commonly implicated as a regulator of angiogenesis. However, critical questions remain unanswered. Do endothelial cells within a capillary sprout lumen experience shear stress? Can tip cells that extend past the sprout lumen experience shear stress? A full understanding of how shear stresses influence endothelial cell phenotype and function during angiogenesis requires the identification of local stress distribution along capillary sprouts. However, the actual shear stress magnitudes experienced by endothelial cells remain unknown. Motivated by the hypothesis that endothelial tip cells can experience physiological shear stress, the objective of this study was to estimate shear stress magnitudes due to local interstitial flow along endothelial tip cells that extend past capillary sprout lumens. A computational fluid dynamics model was used to estimate flows within a blind-ended vessel, transendothelial flow across the vessel wall, and flow within the surrounding perivascular space. The two-dimensional axisymmetric model consisted of four regions: vessel lumen, vessel wall, perivascular space, and interstitium. The vessel wall was assumed to be porous media with 0.3 μm thickness. Blood plasma was represented as an incompressible Newtonian fluid with density and viscosity equal to 1050 kg/m3 and 1.2 cP, respectfully. Shear stresses along the wall of the tip cell extending past the lumen were calculated for varying sprout lengths (50, 200, and 400 μm), perivascular space channel width (1, 4, and 9 μm), and hydraulic conductivities (1.35 and 6.75 x10-10 ms-1 Pa-1). Model parameters and geometric dimensions were selected based on previously reported literature values. Velocity profiles confirmed transmural flow across the vessel wall, nearly parabolic flow within the perivascular space, and velocity gradients along the tip cell. Increasing sprout length, increasing permeability, and decreasing perivascular space increased shear stress values with the perivascular space parameter being the major influencer. Maximum shear stresses along tip cells were consistently higher than the stresses within the sprout lumen. Wall shear stresses within the lumen ranged from 0.02 to 0.52 dyne/cm2 at the sprout entrance and linearly decreased to near zero along the sprout length. Tip cell wall shear stress due to interstitial flow ranged from 0.19 to 4.65 dyne/cm2. The results suggest that wall shear stress values along tip cells can reach physiological relevant values depending on parameter combinations. Our computational estimates motivate new questions related to whether increased shear stresses along tip cells versus luminal cells contribute phenotypic and function signatures of tip cells.

Circulating endothelial cells transiently increase in peripheral blood after kidney transplantation

The diagnosis of kidney allograft rejection is based on late histological and clinical markers. Early, specific and minimally-invasive biomarkers may improve rejection diagnosis. Endothelial cells (EC) are one of the earliest targets in kidney transplant rejection. We investigated whether circulating EC (cEC) could serve as an earlier and less invasive biomarker for allograft rejection. Blood was collected from a cohort of 51 kidney transplant recipients before and at multiple timepoints after transplantation, including during a for cause biopsy. The number and phenotype of EC was assessed by flow-cytometric analysis. Unbiased selection of EC was done using principal component (PCA) analysis. Paired analysis revealed a transient cEC increase of 2.1-fold on the third day post-transplant, recovering to preoperative levels at seventh day post-transplant and onwards. Analysis of HLA subtype demonstrated that cEC mainly originate from the recipient. cEC levels were not associated with allograft rejection, allograft function or other allograft pathologies. However, cEC in patients with allograft rejection and increased levels of cEC showed elevated levels of KIM-1 (kidney injury marker-1). These findings indicate that cEC numbers and phenotype are affected after kidney transplantation but may not improve rejection diagnosis.

In Vitro Investigation of Vascular Permeability in Endothelial Cells from Human Artery, Vein and Lung Microvessels at Steady-State and Anaphylactic Conditions.

Human anaphylactic reactions largely involve an increase in vascular permeability, which is mainly controlled by endothelial cells (ECs). Due to the acute and serious nature of human anaphylaxis, in vivo studies of blood vessels must be replaced or supplemented with in vitro models. Therefore, we used a macromolecular tracer assay (MMTA) to investigate the EC permeability of three phenotypes of human ECs: artery (HAECs), vein (HSVECs) and microvessels from lung (HMLECs). ECs were stimulated with two fast-acting anaphylactic mediators (histamine and platelet-activating factor (PAF)) and one longer-lasting mediator (thrombin). At steady-state conditions, HSVEC monolayers were the most permeable and HMLEC the least (15.8% and 8.3% after 60 min, respectively). No response was found in ECs from artery or vein to any stimuli. ECs from microvessels reacted to stimulation with thrombin and also demonstrated a tendency of increased permeability for PAF. There was no reaction for histamine. This was not caused by missing receptor expression, as all three EC phenotypes expressed receptors for both PAF and histamine. The scarce response to fast-acting mediators illustrates that the MMTA is not suitable for investigating EC permeability to anaphylactic mediators.

Single cell morphological metrics and cytoskeletal alignment regulate VCAM-1 protein expression

In the initial stages of atherosclerosis, vascular adhesion molecule-1 (VCAM-1) is a surface protein that mediates leukocyte adhesion to the endothelium’s luminal surface. VCAM-1 expression is upregulated on endothelial cells (ECs) under pro-inflammatory conditions and is known to be modulated by fluid shear stress (FSS). High, pulsatile FSS induces endothelial elongation and cytoskeletal alignment and downregulates pro-inflammatory induced VCAM-1 expression, which is associated with an athero-protective EC phenotype. In contrast, athero-prone ECs under low, oscillatory FSS fail to elongate and maintain a cobblestone morphology with random cytoskeletal alignment, while VCAM-1 expression is upregulated. Whether EC shape and cytoskeletal alignment play a role in the regulation of VCAM-1 protein expression independent of FSS has not been previously determined. The goal of this study was to determine the effect of EC morphology, specifically cell elongation and alignment, and cytoskeletal alignment on VCAM-1 protein expression using topographical micropatterning of an endothelial monolayer and single cell image analysis techniques. Elongated ECs with an aligned cytoskeleton significantly downregulated VCAM-1 protein expression in the absence of FSS compared to planar controls. In addition, linear correlations between morphological metrics and protein expression showed that actin alignment had a significantly stronger effect on VCAM-1 expression than cell elongation. Functionally, monocytic U937 cells statically adhered less on micropatterns compared to planar substrates, in a VCAM-1 dependent manner. Therefore, endothelial cellular elongation and alignment as well as cytoskeletal alignment regulate VCAM-1 protein expression and immunogenic functions to produce a less inflammatory phenotype in the absence of hemodynamic effects.

Matrix stiffness primes lymphatic tube formation directed by vascular endothelial growth factor-C.

Dysfunction of the lymphatic system is associated with a wide range of disease phenotypes. The restoration of dysfunctional lymphatic vessels has been hypothesized as an innovative method to rescue healthy phenotypes in diseased states including neurological conditions, metabolic syndromes, and cardiovascular disease. Compared to the vascular system, little is known about the molecular regulation that controls lymphatic tube morphogenesis. Using synthetic hyaluronic acid (HA) hydrogels as a chemically and mechanically tunable system to preserve lymphatic endothelial cell (LECs) phenotypes, we demonstrate that low matrix elasticity primes lymphatic cord-like structure (CLS) formation directed by a high concentration of vascular endothelial growth factor-C (VEGF-C). Decreasing the substrate stiffness results in the upregulation of key lymphatic markers, including PROX-1, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and VEGFR-3. Consequently, higher levels of VEGFR-3 enable stimulation of LECs with VEGF-C which is required to both activate matrix metalloproteinases (MMPs) and facilitate LEC migration. Both of these steps are critical in establishing CLS formation in vitro. With decreases in substrate elasticity, we observe increased MMP expression and increased cellular elongation, as well as formation of intracellular vacuoles, which can further merge into coalescent vacuoles. RNAi studies demonstrate that MMP-14 is required to enable CLS formation and that LECs sense matrix stiffness through YAP/TAZ mechanosensors leading to the activation of their downstream target genes. Collectively, we show that by tuning both the matrix stiffness and VEGF-C concentration, the signaling pathways of CLS formation can be regulated in a synthetic matrix, resulting in lymphatic networks which will be useful for the study of lymphatic biology and future approaches in tissue regeneration.

Electrospun PCL-Based Vascular Grafts: In Vitro Tests.

Background: Electrospun fibers have attracted a lot of attention from researchers due to their several characteristics, such as a very thin diameter, three-dimensional topography, large surface area, flexible surface, good mechanical characteristics, suitable for widespread applications. Indeed, electro-spinning offers many benefits, such as great surface-to-volume ratio, adjustable porosity, and the ability of imitating the tissue extra-cellular matrix. Methods: we processed Poly ε-caprolactone (PCL) via electrospinning for the production of bilayered tubular scaffolds for vascular tissue engineering application. Endothelial cells and fibroblasts were seeded into the two side of the scaffolds: endothelial cells onto the inner side composed of PCL/Gelatin fibers able to mimic the inner surface of the vessels, and fibroblasts onto the outer side only exposing PCL fibers. Extracellular matrix production and organization has been performed by means of classical immunofluorescence against collagen type I fibers, Scanning Electron-Microscopy (SEM) has been performed in order to evaluated ultrastructural morphology, gene expression by means gene expression has been performed to evaluate the phenotype of endothelial cells and fibroblasts. Results and conclusion: results confirmed that both cells population are able to conserve their phenotype colonizing the surface supporting the hypothesis that PCL scaffolds based on electrospun fibers should be a good candidate for vascular surgery.