Human leukocyte antigen class I antibody-activated endothelium promotes CD206+ M2 macrophage polarization and MMP9 secretion through TLR4 signaling and P-selectin in a model of antibody-mediated rejection and allograft vasculopathy.

HLA donor-specific antibodies (DSA) elicit alloimmune responses against the graft vasculature, leading to endothelial cell (EC) activation and monocyte infiltration during antibody-mediated rejection (AMR). AMR promotes chronic inflammation and remodeling, leading to thickening of the arterial intima termed transplant vasculopathy or cardiac allograft vasculopathy (CAV) in heart transplants. Intragraft-recipient macrophages serve as a diagnostic marker in AMR; however, their polarization and function remain unclear. In this study, we utilized an in vitro Transwell coculture system to explore the mechanisms of monocyte-to-macrophage polarization induced by HLA I DSA-activated ECs. Anti-HLA I (IgG or F(ab')2) antibody-activated ECs induced the polarization of M2 macrophages with increased CD206 expression and MMP9 secretion. However, inhibition of TLR4 signaling or PSGL-1-P-selectin interactions significantly decreased both CD206 and MMP9. Monocyte adherence to Fc-P-selectin coated plates induced M2 macrophages with increased CD206 and MMP9. Moreover, Fc-receptor and IgG interactions synergistically enhanced active-MMP9 in conjunction with P-selectin. Transcriptomic analysis of arteries from DSA+CAV+ rejected cardiac allografts and multiplex-immunofluorescent staining illustrated the expression of CD68+CD206+CD163+MMP9+ M2 macrophages within the neointima of CAV-affected lesions. These findings reveal a novel mechanism linking HLA I antibody-activated endothelium to the generation of M2 macrophages which secrete vascular remodeling proteins contributing to AMR and CAV pathogenesis.

The size-dependent genotoxic potentials of titanium dioxide nanoparticles to endothelial cells.

Despite intensive research activities, there are still many major knowledge gaps over the potential adverse effects of titanium dioxide nanoparticles (TiO2 -NPs), one of the most widely produced and used nanoparticles, on human cardiovascular health and the underlying mechanisms. In the present study, alkaline comet assay and cytokinesis-block micronucleus test were employed to determine the genotoxic potentials of four sizes (100, 50, 30, and 10 nm) of anatase TiO2 -NPs to human umbilical vein endothelial cells (HUVECs) in culture. Also, the intracellular redox statuses were explored through the measurement of the levels of reactive oxygen species (ROS) and reduced glutathione (GSH) with kits, respectively. Meanwhile, the protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) were also detected by western blot. The results showed that at the exposed levels (1, 5, and 25 µg/mL), all the four sizes of TiO2 -NPs could elicit an increase of both DNA damage and MN frequency in HUVECs in culture, with a positive dose-dependent and negative size-dependent effect relationship (T100 < T50 < T30 < T10). Also, increased levels of intracellular ROS, but decreased levels of GSH, were found in all the TiO2 -NP-treated groups. Intriguingly, a very similar manner of dose-dependent and size-dependent effect relationship was observed between the ROS test and both comet assay and MN test, but contrary to that of GSH assay. Correspondingly, the levels of Nrf2 protein were also elevated in the TiO2 -NP-exposed HUVECs, with an inversely size-dependent effect relationship. These findings indicated that induction of oxidative stress and subsequent genotoxicity might be an important biological mechanism by which TiO2 -NP exposure would cause detrimental effects to human cardiovascular health.

The Toxicity of Nanoparticles to Human Endothelial Cells.

The use of nanoparticles (NPs) in commercially available products and as biomedicinal materials could lead to increasing contact of human blood vessels with NPs, and it is necessary to assess the potential adverse effects of NPs to cells lining blood vessels. Of them, endothelial cells (ECs) are of particular relevance as they play a crucial role in the regulation of function of blood vessels. In this book chapter, I discussed studies that used human ECs to study the toxicity and mechanisms of NPs. It has been shown that exposure of human ECs to NPs could lead to cytotoxicity, genotoxicity, endothelial activation and impaired NO signaling. Oxidative stress and inflammation induced by NPs have been suggested as the mechanisms associated with the toxicity of NPs to ECs, and a three-tier model has been proposed to explain the association between NP induced oxidative stress and toxicity. In recent years, dysfunction of autophagy (excessive autophagy induction) has also been suggested as one of the mechanisms associated with the toxicity of NPs to human ECs. In the future, it is necessary to use human ECs to assess the toxicity of NPs to better understand the potential adverse effects of NPs entering circulation.

The size-dependent apoptotic effect of titanium dioxide nanoparticles on endothelial cells by the intracellular pathway.

Concerns over the health risk of the widely distributed, commonly used titanium dioxide nanoparticles (nano-TiO2 ) are increasing worldwide. Yet, up-to-now, our understanding in their potential effects on the cardiovascular system is very limited and the toxicological mechanisms are still unclear. In the present study, the CCK-8 assay was performed to determine the cytotoxicity of four sizes (10, 30, 50, and 100 nm) of anatase nano-TiO2 on human umbilical vein endothelial cells (HUVECs) in culture, and the flow cytometry was employed to investigate the potential of these nano-TiO2 to induce the apoptosis of HUVECs. The apoptotic pathway was also probed through the determination of the protein expression and activation of p53, Bax, Bcl-2, caspases-9, -7, -3, and PARP by western blot. The results showed that at the administrative levels (1, 5, 25 µg/mL), all the four sizes of nano-TiO2 could significantly inhibit the viability of HUVECs and elicit significant apoptosis in them, compared with the negative control (P < .05, P < .01). Moreover, the apoptotic rates of HUVECs were increased respectively with the elevating levels and decreasing sizes of the administrative nano-TiO2 , showing a clear dose- and size-dependent effect relationships. Interestingly, the increasing phosphorylation of p53, decreasing ratio of Bcl-2/Bax, and enhancing activation of the downstream proteins caspase-9, -7, -3, and PARP, were also observed with the decreasing sizes of the administrative nano-TiO2 in the western blot, indicating that the intracellular approach of apoptosis, the p53-caspase pathway, is the major way of the nano-TiO2 -mediated apoptosis in HUVECs in culture and that the size is an important parameter that may determine the potential of nano-TiO2 to induce cellular response. In conclusion, these results suggested that high levels of nano-TiO2 exposure may pose potential risks to human cardiovascular health by inducing cardiovascular EC apoptosis.

Acidosis Activates Endoplasmic Reticulum Stress Pathways through GPR4 in Human Vascular Endothelial Cells.

Acidosis commonly exists in the tissue microenvironment of various pathophysiological conditions such as tumors, inflammation, ischemia, metabolic disease, and respiratory disease. For instance, the tumor microenvironment is characterized by acidosis and hypoxia due to tumor heterogeneity, aerobic glycolysis (the "Warburg effect"), and the defective vasculature that cannot efficiently deliver oxygen and nutrients or remove metabolic acid byproduct. How the acidic microenvironment affects the function of blood vessels, however, is not well defined. GPR4 (G protein-coupled receptor 4) is a member of the proton-sensing G protein-coupled receptors and it has high expression in endothelial cells (ECs). We have previously reported that acidosis induces a broad inflammatory response in ECs. Acidosis also increases the expression of several endoplasmic reticulum (ER) stress response genes such as CHOP (C/EBP homologous protein) and ATF3 (activating transcription factor 3). In the current study, we have examined acidosis/GPR4- induced ER stress pathways in human umbilical vein endothelial cells (HUVEC) and other types of ECs. All three arms of the ER stress/unfolded protein response (UPR) pathways were activated by acidosis in ECs as an increased expression of phosphorylated eIF2α (eukaryotic initiation factor 2α), phosphorylated IRE1α (inositol-requiring enzyme 1α), and cleaved ATF6 upon acidic pH treatment was observed. The expression of other downstream mediators of the UPR, such as ATF4, ATF3, and spliced XBP-1 (X box-binding protein 1), was also induced by acidosis. Through genetic and pharmacological approaches to modulate the expression level or activity of GPR4 in HUVEC, we found that GPR4 plays an important role in mediating the ER stress response induced by acidosis. As ER stress/UPR can cause inflammation and cell apoptosis, acidosis/GPR4-induced ER stress pathways in ECs may regulate vascular growth and inflammatory response in the acidic microenvironment.

SiNPs induce ferroptosis in HUVECs through p38 inhibiting NrF2 pathway.

Introduction: Despite of growing evidence linking silica nanoparticles (SiNPs), one of the global-top-three-produced and -used nanoparticle (NP), to human health risks, there remain many knowledge gaps over the adverse effects of SiNPs exposure on cardiovascular system and the underlying molecular mechanisms. Methods: In this study, the ferroptotic effects of SiNPs (20 nm; 0, 25, 50, and 100 µg/mL) on human umbilical vein endothelial cells (HUVECs) and the possible molecular mechanism were studied with the corresponding biochemical and molecular biology assays. Results and discussion: The results showed that at the tested concentrations, SiNPs could decrease HUVEC viability, but the deferoxamine mesylate (an iron ion chelator) might rescue this reduction of cell viability. Also, increased levels of intracellular reactive oxygen species and enhanced mRNA expression of lipid oxidation enzymes (ACSL4 and LPCAT3) with increase in lipid peroxidation (malondialdehyde), but decreased ratios of intracellular GSH/total-GSH and mitochondrial membrane potential as well as reduced enzymatic activities of anti-oxidative enzymes (CAT, SOD, and GSH-PX), were found in the SiNPs-treated HUVECs. Meanwhile, increase in p38 protein phosphorylation and decrease in NrF2 protein phosphorylation with reduced mRNA expressions of downstream anti-oxidative enzyme genes (CAT, SOD1, GSH-PX, and GPX4) was identified in the SiNPs-exposed HUVECs. These data indicated that SiNPs exposure might induce ferroptosis in HUVECs via p38 inhibiting NrF2 pathway. Ferroptosis of HUVECs will become a useful biomarker for assessing the cardiovascular health risks of environmental contaminants.

GEF-H1/RhoA signaling pathway mediates pro-inflammatory effects of NF-κB on CD40L-induced pulmonary endothelial cells.

One of the key targets of the inflammatory response in acute lung injury (ALI) is the human pulmonary micro-vascular endothelial cells (HPMVECs). Owing to its role in the activation of endothelial cells (ECs), CD40L figures prominently in the pathogenesis of ALI. Increasing evidences have showed that CD40L mediates inflammatory effects on ECs, at least in part, by triggering NF-κB-dependent gene expression. However, the mechanisms of such signal transmission remain unknown. In this study, we found that CD40L stimulated the transactivation of NF-κB and expression of its downstream cytokines in a p38 MAPK-dependent mechanism in HPMVECs. In addition, CD40L-mediated inflammatory effects might be correlated with the activation of the IKK/IκB/NF-κB pathway and nuclear translocation of NF-κB, being accompanied by dynamic cytoskeletal changes. GEF-H1/RhoA signaling is best known for its role in regulating cytoskeletal rearrangements. An interesting finding was that CD40L induced the activation of p38 and IKK/IκB, and the subsequent transactivation of NF-κB via GEF-H1/RhoA signaling. The critical role of GEF-H1/RhoA in CD40L-induced inflammatory responses in the lung was further confirmed in GEF-H1 and RhoA knockout mouse models, both of which were established by adeno-associated virus (AAV)-mediated delivery of sgRNAs into mice with EC-specific Cas9 expression. These results taken together suggested that p38 and IKK/IκB-mediated signaling pathways, both of which lied downstream of GEF-H1/RhoA, may coordinately regulate the transactivation of NF-κB in CD40L-activated HPMVECs. These findings may help to determine key pharmacological targets of intervention for CD40L-activated inflammatory effects associated with ALI.

Diabetic macrophage small extracellular vesicles-associated miR-503/IGF1R axis regulates endothelial cell function and affects wound healing.

Diabetic foot ulcer (DFU) is a break in the skin of the foot caused by diabetes. It is one of the most serious and debilitating complications of diabetes. The previous study suggested that dominant M1 polarization during DFU could be the leading reason behind impaired wound healing. This study concluded that macrophage M1 polarization predominates in DFU skin tissue. iNOS was increased in HG-induced M1-polarized macrophages; conversely, Arg-1 was decreased. Macrophage pellets after HG stimulation can impair endothelial cell (EC) function by inhibiting cell viability, tube formation and cell migration, indicating M1 macrophage-derived small extracellular vesicles (sEVs) -mediated HUVEC dysfunction. sEVs miR-503 was significantly upregulated in response to HG stimulation, but inhibition of miR-503 in HG-stimulated macrophages attenuated M1 macrophage-induced HUVEC dysfunction. ACO1 interacted with miR-503 and mediated the miR-503 package into sEVs. Under HG stimulation, sEVs miR-503 taken in by HUVECs targeted IGF1R in HUVECs and inhibited IGF1R expression. In HUVECs, miR-503 inhibition improved HG-caused HUVEC dysfunction, whereas IGF1R knockdown aggravated HUVEC dysfunction; IGF1R knockdown partially attenuated miR-503 inhibition effects on HUVECs. In the skin wound model in control or STZ-induced diabetic mice, miR-503-inhibited sEVs improved, whereas IGF1R knockdown further hindered wound healing. Therefore, it can be inferred from the results that the M1 macrophage-derived sEVs miR-503 targets IGF1R in HUVECs, inhibits IGF1R expression, leads to HUVEC dysfunction, and impedes wound healing in diabetic patients, while packaging miR-503 as an M1 macrophage-derived sEVs may be mediated by ACO1.

Endotheliopathy in the metabolic syndrome: Mechanisms and clinical implications.

The increasing prevalence of the metabolic syndrome (MetS) is a threat to global public health due to its lethal complications. Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the MetS characterized by hepatic steatosis, which is potentially progressive to the inflammatory and fibrotic nonalcoholic steatohepatitis (NASH). The adipose tissue (AT) is also a major metabolic organ responsible for the regulation of whole-body energy homeostasis, and thereby highly involved in the pathogenesis of the MetS. Recent studies suggest that endothelial cells (ECs) in the liver and AT are not just inert conduits but also crucial mediators in various biological processes via the interaction with other cell types in the microenvironment both under physiological and pathological conditions. Herein, we highlight the current knowledge of the role of the specialized liver sinusoidal endothelial cells (LSECs) in NAFLD pathophysiology. Next, we discuss the processes through which AT EC dysfunction leads to MetS progression, with a focus on inflammation and angiogenesis in the AT as well as on endothelial-to-mesenchymal transition of AT-ECs. In addition, we touch upon the function of ECs residing in other metabolic organs including the pancreatic islet and the gut, the dysregulation of which may also contribute to the MetS. Finally, we highlight potential EC-based therapeutic targets for human MetS, and NASH based on recent achievements in basic and clinical research and discuss how to approach unsolved problems in the field.

Role of endothelial cells in vascular calcification.

Vascular calcification (VC) is active and regulates extraosseous ossification progress, which is an independent predictor of cardiovascular disease (CVD) morbidity and mortality. Endothelial cells (ECs) line the innermost layer of blood vessels and directly respond to changes in flow shear stress and blood composition. Together with vascular smooth muscle cells, ECs maintain vascular homeostasis. Increased evidence shows that ECs have irreplaceable roles in VC due to their high plasticity. Endothelial progenitor cells, oxidative stress, inflammation, autocrine and paracrine functions, mechanotransduction, endothelial-to-mesenchymal transition (EndMT), and other factors prompt ECs to participate in VC. EndMT is a dedifferentiation process by which ECs lose their cell lineage and acquire other cell lineages; this progress coexists in both embryonic development and CVD. EndMT is regulated by several signaling molecules and transcription factors and ultimately mediates VC via osteogenic differentiation. The specific molecular mechanism of EndMT remains unclear. Can EndMT be reversed to treat VC? To address this and other questions, this study reviews the pathogenesis and research progress of VC, expounds the role of ECs in VC, and focuses on the regulatory factors underlying EndMT, with a view to providing new concepts for VC prevention and treatment.