Cunning Pathogen Tactics: Neutrophil Movement Influenced by Bacterial Attractants in the Experimental Model

Albumin-bound fatty acids induce mitochondrial oxidant stress and impair antioxidant responses in proximal tubular cells

Quantitative characterization of endothelial cell morphologies depending on shear stress in different blood vessels of domestic pigs using a focused ion beam and high resolution scanning electron microscopy (FIB-SEM).

Endothelial cell (EC) morphology and functions can be highly impacted by the mechanical stresses that the cells experience in vivo. In most areas in the vasculature, ECs are continuously exposed to unsteady blood flow-induced shear stress and vasodilation-contraction-induced tensile stress/strain simultaneously. Investigations on how ECs respond to combined shear stress and tensile strain will help us to better understand how an altered mechanical environment affects EC mechanotransduction, dysfunction, and associated cardiovascular disease development. In the present study, a programmable shearing and stretching device that can apply dynamic fluid shear stress and cyclic tensile strain simultaneously to cultured ECs was developed. Flow and stress/strain conditions in the device were simulated using a fluid structure interaction (FSI) model. To characterize the performance of this device and the effect of combined shear stress-tensile strain on EC morphology, human coronary artery ECs (HCAECs) were exposed to concurrent shear stress and cyclic tensile strain in the device. Changes in EC morphology were evaluated through cell elongation, cell alignment, and cell junctional actin accumulation. Results obtained from the numerical simulation indicated that in the "in-plane" area of the device, both fluid shear stress and biaxial tensile strain were uniform. Results obtained from the in vitro experiments demonstrated that shear stress, alone or combined with cyclic tensile strain, induced significant cell elongation. While biaxial tensile strain alone did not induce any appreciable change in EC elongation. Fluid shear stress and cyclic tensile strain had different effects on EC actin filament alignment and accumulation. By combining various fluid shear stress and cyclic tensile strain conditions, this device can provide a physiologically relevant mechanical environment to study EC responses to physiological and pathological mechanical stimulation.

Blood vessels are permanently subjected to mechanical forces in the form of stretch, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow and shear stress. Significant variations in mechanical forces, of physiological or physiopathological nature, occur in vivo. These are accompanied by phenotypical modulation of smooth muscle cells and endothelial cells, producing structural modifications of the arterial wall. In all the cases, vascular remodelling can be allotted to a modification of the tensional strain or shear, and underlie a trend to reestablish baseline mechanical conditions. Vascular cells are equipped with numerous receptors that allow them to detect and respond to the mechanical forces generated by pressure and shear stress. The cytoskeleton and other structural components have an established role in mechanotransduction, being able to transmit and modulate tension within the cell via focal adhesion sites, integrins, cellular junctions and the extracellular matrix. Mechanical forces also initiate complex signal transduction cascades, including nuclear factor-kappaB and mitogen-activated protein kinase pathways, leading to functional changes within the cell.

Neovascularization is important repair mechanism in response to ischemic injury and its process is in part dependent on reactive oxygen species (ROS). IQGAP1, an actin-binding scaffold protein, is key regulator for actin cytoskeleton and motility. We demonstrated that IQGAP1 is expressed in endothelial cells (ECs) and mediates VEGF-induced ROS production and migration of cultured ECs. However, its role in postnatal neovascularization is unknown. Here we show that neovascularization induced by hindlimb ischemia is significantly inhibited in IQGAP1-deficient mice as evaluated by laser Doppler blood flow (38%), capillary density (48%) and alpha-actin positive arterioles (68%). In wild-type (WT) mice, western and immunofluorescence analysis reveal that IQGAP1 protein expression in ischemic tissues is significantly increased at Mac3+ macrophages and lectin+ capillary-like ECs at 3 days and 7 days after ischemia, respectively, which is associated with increased superoxide production. Of note, macrophage infiltration is important for post-ischemic revascularization. Mice lacking IQGAP1 show significant decrease in the number of infiltrated Mac3-positive macrophage and superoxide production in ischemic muscles (30% and 52%, respectively) as compared to WT mice. Numbers of inflammatory cells and cKit+/Flk1+ endothelial progenitor cells in peripheral blood are not affected in these knockout mice. Moreover, thioglycollate-induced peritoneal leukocyte recruitment and accumulation are significantly inhibited in IQGAP1-deficient mice (53%), whereas white blood cell number is not changed. In vitro, peritonitis-elicited (activated) macrophages obtained from IQGAP1-deficient mice show impaired polarization of actin, as visualized by phalloidin staining as well as complete inhibition of migration towards stromal derived factor-1 and VEGF, as measured by modified Boyden-chambered method. In summary, IQGAP1 plays a key role in reparative neovascularization in response to tissue ischemia by regulating not only ECs but also macrophage migration/infiltration as well as ROS production in the vessel growth area. Thus, IQGAP1 is a potential therapeutic target to promote neovascularization in ischemic cardiovascular diseases. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Changes in endothelial cell (EC) morphology occur at sites of physiological lymphocyte traffic and in areas of chronic inflammation. Previous studies have shown that EC shape changes also occur in vitro following exposure of EC monolayers to peripheral blood mononuclear cell (PBMC)-derived conditioned media (CM). In the present study, quantitative image analysis is used to define the cell of origin of the elongating factor(s), to examine changes in EC proliferation and function accompanying PBMC-induced human EC elongation and to identify the active PBMC-derived products responsible for this elongation. By separating mononuclear cells into subpopulations (macrophages, B cells and T cells) and adding conditioned media derived from these subpopulations to cultured ECs, the macrophage (M phi) is shown to be the primary cell of origin of the elongating factor(s). Furthermore, EC elongation is accompanied by both a dose-dependent decrease in cellular proliferation and an increase in prostacyclin production. These findings suggest that PBMC-induced changes in EC morphology may be associated with a shift from a proliferative state to a more secretory phase of the EC cycle. Finally, using recombinant factors it is shown that TNF alpha acting in combination with IL-1 may be the active PBMC-derived products which contribute to EC elongation.

Vascular dysfunction in response to reactive oxygen species (ROS) plays an important role in the development and progression of atherosclerotic lesions. In most cells, mitochondria are the major source of cellular ROS during aerobic respiration. Under most conditions the rates of ROS formation and elimination are balanced through mechanisms that sense relative ROS levels. However, a chronic imbalance in redox homeostasis is believed to contribute to various chronic diseases, including atherosclerosis. Uncoupling protein-2 (UCP2) is a mitochondrial inner membrane protein shown to be a negative regulator of macrophage ROS production. In response to a cholesterol-containing atherogenic diet, C57BL/6J mice significantly increased expression of UCP2 in the aorta, while mice lacking UCP2, in the absence of any other genetic modification, displayed significant endothelial dysfunction following the atherogenic diet. Compared with wild-type mice, Ucp2(-/-) mice had decreased endothelial nitric oxide synthase, an increase in vascular cell adhesion molecule-1 expression, increased ROS production, and an impaired ability to increase total antioxidant capacity. These changes in Ucp2(-/-) mice were associated with increased aortic macrophage infiltration and more numerous and larger atherosclerotic lesions. These data establish that in the vasculature UCP2 functions as an adaptive antioxidant defense to protect against the development of atherosclerosis in response to a fat and cholesterol diet.

<p dir="ltr"><b>Abstract</b></p><p dir="ltr">Diabetes is a significant global public health issue<a href="" target="_blank"> with implications for vascular</a> endothelial cells (ECs) dysfunction and the subsequent development and advancement of diabetic complications. This study aims to compare the cellular and molecular properties of the aorta in normal and streptozotocin (STZ)-induced diabetic mice, with a focus on elucidating potential mechanism underlying EC dysfunction. Here, we performed a <a href="" target="_blank">single-cell RNA sequencing</a> survey of <a href="" target="_blank">32,573</a> cells from the aorta of normal and STZ-induced diabetic mice. We found a compendium of 10 distinct cell types, mainly ECs, smooth muscle cells (SMCs), fibroblast, pericyte, immune cells and stromal cells. <a href="" target="_blank">As the diabetes condition progressed, we observed a subpopulation of aortic ECs that exhibited significantly elevated expression of </a><a href="" target="_blank">complement</a> (C) molecule C1qa compared to their healthy counterparts. This increased expression of C1qa was found to induce reactive oxygen species (ROS) production, facilitate EC migration and increased permeability, and impair the vasodilation within the aortic segment of mice. Furthermore, AAV-Tie2-shRNA-C1qa was administered into diabetic mice by <a href="" target="_blank">tail vein injection</a>, showing that inhibition of C1qa in the endothelium led to a reduction in ROS production, decreased vascular permeability, and improved vasodilation. Collectively, these findings highlight the crucial involvement of C1qa in <a href="" target="_blank">endothelial dysfunction associated with diabetes</a>.</p><p><br></p><p><br></p><p dir="ltr"><b>Article Highlights</b></p><p dir="ltr">The molecular mechanisms and cellular heterogeneity of C1q in endothelial cells, which regulate vascular function, remain unclear.</p><p dir="ltr">C1q is involved in the regulation of vascular function.</p><p dir="ltr">C1q in vascular <a href="" target="_blank">endothelial</a> cells can regulate vascular function, including permeability, immunity and diastolic function.</p><p dir="ltr">C1q represents a promising therapeutic target for the treatment of endothelial dysfunction associated with diabetes.</p>

Intersectins (ITSNs) are multidomain adaptor proteins implicated in endocytosis, regulation of actin polymerization, and Ras/MAPK signaling. We have previously shown that ITSN-1s is required for caveolae fission and internalization in endothelial cells (ECs). In the present study, using small interfering RNA to knock down ITSN-1s protein expression, we demonstrate a novel role of ITSN-1s as a key antiapoptotic protein. Knockdown of ITSN-1s in ECs activated the mitochondrial pathway of apoptosis as determined by genomic DNA fragmentation, extensive mitochondrial fission, activation of the proapoptotic proteins BAK and BAX, and cytochrome c efflux from mitochondria. ITSN-1 knockdown acts as a proapoptotic signal that causes mitochondrial outer membrane permeabilization, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. These effects were secondary to decreased activation of Erk1/2 and its direct activator MEK. Bcl-X(L) overexpression prevented BAX activation and the apoptotic ECs death induced by suppression of ITSN-1s. Our findings demonstrate a novel role of ITSN-1s as a negative regulator of the mitochondrial pathway-dependent apoptosis secondary to activation of the Erk1/2 survival signaling pathway.

Pleiotrophin-induced endothelial cell migration is regulated by xanthine oxidase-mediated generation of reactive oxygen species

UVB Radiation Induces Apoptosis in Keratinocytes by Activating a Pathway Linked to “BLT2-Reactive Oxygen Species”

Vascular dysfunction is closely related to and caused by oxidative stress and chronic inflammation. Oxidative stress may be augmented by inflammation due to overproduction of reactive oxygen species (ROS) and concomitant impairments in ROS clearance. Premenopausal women experience a lower incidence of vascular dysfunction compared with age‐matched men. This sex difference is, in part, attributed to differences in estrogen and androgen concentrations and actions on endothelial cells. However, limited knowledge exists regarding sex differences in ROS production and ROS clearance in endothelial cells following induced inflammation. Because male human umbilical vein endothelial cells (HUVECs) are exposed to higher androgen concentrations in umbilical cord blood than female HUVECs, we sought to evaluate sex differences in ROS production and superoxide dismutase (SOD) activity (a major contributor to ROS clearance) in male and female HUVECs. We hypothesized that female HUVECs would exhibit lower ROS production and higher SOD activity than male HUVECs. Inflammation was induced in HUVEC cell lines (n=4/group) using tumor necrosis factor‐alpha (TNF‐α, 50ng/ml). Cell lysate samples from time‐matched control and TNF‐α treatments were collected at 4 (4H) and 24 hours (24H) post‐stimulus. Fluorescence detection was used to quantify ROS production and viable cells alive at 4H and 24H post‐stimulus in cells stained for ROS (CellROX) and living cells (Hoechst). Data are presented as a ratio of ROS to live cells (CellROX/Hoechst ratio). Cell lysate samples were subsequently assayed for SOD activity and normalized to protein concentration. TNF‐α treatment significantly increased ROS production in all HUVECs at 24H compared with 4H (CellROX/Hoechst ratio‐ 4H TNF‐α: 1.37 ± 0.21, 24H TNF‐α: 1.44 ± 0.20, p=0.0015). Notably, female HUVECs exhibited significantly lower ROS production than male HUVECs in time‐matched Control samples and following TNF‐α‐ induced inflammation (CellROX/Hoechst ratio‐ Female HUVECs: 4H TNF‐α: 1.20 ± 0.17, 24H TNF‐α: 1.33 ± 0.14, Male HUVECs: 4H TNF‐α: 1.32 ± 0.14, 24H TNF‐α: 1.56 ± 0.20; p=0.0040). Further, female HUVECs exhibited significant increases in SOD activity with increased exposure time to TNF‐α‐ induced inflammation while male HUVECs did not (female HUVECs: 4H TNF‐α: 2.93 ± 1.10, 24H TNF‐α: 4.82 ± 1.60, p=0.0098; male HUVECs: 4H TNF‐α: 3.30 ± 1.87, 24H TNF‐α: 3.72 ± 1.06 U/mg; p<0.05). Compared with female HUVECs, male HUVECs exhibit a pro‐inflammatory state following induced inflammation, potentially due to differences in exposure to androgen concentrations in fetal umbilical cord blood.

Induction of apoptosis and modulation of production of reactive oxygen species in human endothelial cells by diphenyleneiodonium

Positive Role of CCAAT/Enhancer-Binding Protein Homologous Protein, a Transcription Factor Involved in the Endoplasmic Reticulum Stress Response in the Development of Colitis

The expression of the NADPH oxidase subunit p22phox is regulated by a redox-sensitive pathway in endothelial cells