Rat Fat Pad Endothelial Cells Research Articles

Matrix Stiffness Affects Glycocalyx Expression in Cultured Endothelial Cells.

Rationale: The endothelial cell glycocalyx (GCX) is a mechanosensor that plays a key role in protecting against vascular diseases. We have previously shown that age/disease mediated matrix stiffness inhibits the glycocalyx glycosaminoglycan heparan sulfate and its core protein Glypican 1 in human umbilical vein endothelial cells, rat fat pad endothelial cells and in a mouse model of age-mediated stiffness. Glypican 1 inhibition resulted in enhanced endothelial cell dysfunction. Endothelial cell culture typically occurs on stiff matrices such as plastic or glass. For the study of the endothelial GCX specifically it is important to culture cells on soft matrices to preserve GCX expression. To test the generality of this statement, we hypothesized that stiff matrices inhibit GCX expression and consequently endothelial cell function in additional cell types: bovine aortic endothelial cells, mouse aortic endothelial cell and mouse brain endothelial cells.Methods and Results: All cell types cultured on glass showed reduced GCX heparan sulfate expression compared to cells cultured on either soft polyacrylamide (PA) gels of a substrate stiffness of 2.5 kPa (mimicking the stiffness of young, healthy arteries) or on either stiff gels 10 kPa (mimicking the stiffness of old, diseased arteries). Specific cell types showed reduced expression of GCX protein Glypican 1 (4 of 5 cell types) and hyaluronic acid (2 of 5 cell types) on glass vs soft gels.Conclusion: Matrix stiffness affects GCX expression in endothelial cells. Therefore, the study of the endothelial glycocalyx on stiff matrices (glass/plastic) is not recommended for specific cell types.

Disturbed flow disrupts eNOS and caveolin‐1 expression and colocalization via glycocalyx degradation

Atherosclerosis is the main cause of cardiovascular diseases such as coronary artery and carotid artery disease and is known to occur in specific, atheroprone areas of the vasculature characterized by disturbed flow patterns. The endothelial glycocalyx, a mechanotransductive carbohydrate layer that lines the endothelium, helps maintain vascular homeostasis through flow‐induced regulation of vasculoprotective molecules such as nitric oxide. Areas of atherosclerosis development have been shown to contain diminished glycocalyx expression; however, it is unclear whether glycocalyx degradation contributes to atherosclerosis or is a result of its progression. Here, we investigated the effects of disturbed flow on glycocalyx‐mediated nitric oxide regulation in rat fat pad endothelial cells, which robustly express glycocalyx components. Using a custom parallel‐plate flow chamber, we found that disturbed flow (DF) patterns characteristic of atheroprone regions of the vasculature decrease glycocalyx coverage and thickness in comparison to uniform flow (UF). This decrease was accompanied by a reduction in endothelial nitric oxide synthase (eNOS) expression. For the first time, we discovered that DF also decreased the expression of caveolin‐1 (cav‐1), a core protein of caveolae microvesicles that assist in eNOS activation. Additionally, fluorescence microscopy also demonstrated decreased cav‐1 membrane and upstream distribution as well as decreased eNOS‐cav‐1 colocalization in areas of disturbed flow. Finally, UF exposure upon enzymatic degradation of heparan sulfate, a major glycocalyx component, resulted in DF‐like expression colocalization of eNOS and cav‐1, implicating the glycocalyx in eNOS and cav‐1 regulation. These findings provide new evidence to the body of information on glycocalyx‐mediated endothelial regulation, which collectively suggests that glycocalyx degradation may contribute to atherosclerosis development.Support or Funding InformationThis study was funded by National Institute of Health Grant K01 HL125499, National Science Foundation Grant DGE 1451070, Northeastern University Provost Start‐up funds, and an Engineering Dean's Fellowship provided by Northeastern University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial Glycocalyx-Mediated Nitric Oxide Production in Response to Selective AFM Pulling

Nitric oxide (NO) is a regulatory molecule in the vascular system and its inhibition due to endothelial injury contributes to cardiovascular disease. The glycocalyx is a thin layer of glycolipids, glycoproteins, and proteoglycans on the surface of mammalian epithelial cells. Extracellular forces are transmitted through the glycocalyx to initiate intracellular signaling pathways. In endothelial cells (ECs), previous studies have shown the glycocalyx to be a significant mediator of NO production; degradation of the endothelial glycocalyx layer (EGL) drastically reduces EC production of NO in response to fluid shear stress. However, the specific EGL components involved in this process are not well established. Recent work using short-hairpin RNA approaches in vitro suggest that the proteoglycan glypican-1, not syndecan-1, is the dominant core protein mediating shear-induced NO production. We utilized atomic force microscopy (AFM) to apply force selectively to components of the EGL of confluent rat fat pad ECs (RFPECs), including proteoglycans and glycosaminoglycans, to observe how each component individually contributes to force-induced production of NO. 4,5-diaminofluorescein diacetate, a cell-permeable fluorescent molecule, was used to detect changes in intracellular NO production. Antibody-coated AFM probes exhibited strong surface binding to RFPEC monolayers, with 100–300 pN mean adhesion forces. AFM pulling on glypican-1 and heparan sulfate for 10 min caused significantly increased NO production, whereas pulling on syndecan-1, CD44, hyaluronic acid, and with control probes did not. We conclude that AFM pulling can be used to activate EGL-mediated NO production and that the heparan sulfate proteoglycan glypican-1 is a primary mechanosensor for shear-induced NO production.

Endothelial glycocalyx conditions influence nanoparticle uptake for passive targeting.

Cardiovascular diseases are facilitated by endothelial cell (EC) dysfunction and coincide with EC glycocalyx coat shedding. These diseases may be prevented by delivering medications to affected vascular regions using circulating nanoparticle (NP) drug carriers. The objective of the present study was to observe how the delivery of 10 nm polyethylene glycol-coated gold NPs (PEG-AuNP) to ECs is impacted by glycocalyx structure on the EC surface. Rat fat pad endothelial cells were chosen for their robust glycocalyx, verified by fluorescent immunolabeling of adsorbed albumin and integrated heparan sulfate (HS) chains. Confocal fluorescent imaging revealed a ~3 µm thick glycocalyx layer, covering 75% of the ECs and containing abundant HS. This healthy glycocalyx hindered the uptake of PEG-AuNP as expected because glycocalyx pores are typically 7 nm wide. Additional glycocalyx models tested included: a collapsed glycocalyx obtained by culturing cells in reduced protein media, a degraded glycocalyx obtained by applying heparinase III enzyme to specifically cleave HS, and a recovered glycocalyx obtained by supplementing exogenous HS into the media after enzyme degradation. The collapsed glycocalyx waŝ2 µm thick with unchanged EC coverage and sustained HS content. The degraded glycocalyx showed similar changes in EC thickness and coverage but its HS thickness was reduced to 0.7 µm and spanned only 10% of the original EC surface. Both dysfunctional models retained six- to sevenfold more PEG-AuNP compared to the healthy glycocalyx. The collapsed glycocalyx permitted NPs to cross the glycocalyx into intracellular spaces, whereas the degraded glycocalyx trapped the PEG-AuNP within the glycocalyx. The repaired glycocalyx model partially restored HS thickness to 1.2 µm and 44% coverage of the ECs, but it was able to reverse the NP uptake back to baseline levels. In summary, this study showed that the glycocalyx structure is critical for NP uptake by ECs and may serve as a passive pathway for delivering NPs to dysfunctional ECs.

Shape and surface effects on the cytotoxicity of nanoparticles: Gold nanospheres versus gold nanostars.

Gold nanoparticles are materials with unique optical properties that have made them very attractive for numerous biomedical applications. With the increasing discovery of techniques to synthesize novel nanoparticles such as star-shaped gold nanoparticles for biomedical applications, the safety and performance of these new nanomaterials must be systematically assessed before use. In this study, gold nanostars (AuNSTs) with multibranched surface structures were synthesized, and their influence on the cytotoxicity of human skin fibroblasts and rat fat pad endothelial cells (RFPECs) were assessed and compared with that of gold nanospheres (AuNSPs) with unbranched surfaces. Results showed that the AuNSPs with diameters of approximately 61.46 nm showed greater toxicity with fibroblast cells and RFPECs compared with the synthesized AuNSTs with diameters of approximately 33.69 nm. The AuNSPs were lethal at concentrations of 40 μg/mL for both cell lines, whereas the AuNSTs were less toxic at higher concentrations (400 μg/mL). The calculated IC50 (50% inhibitory concentration) values of the AuNSPs exposed to fibroblast cells were greater at 1 and 4 days of culture (26.4 and 27.7 μg/mL, respectively) compared with the RFPECs (13.6 and 13.8 μg/mL, respectively), indicating that the AuNSPs have a greater toxicity to endothelial cells. It was proposed that possible factors that could be promoting the reduced toxicity effects of the AuNSTs to fibroblast cells and RFPECs, compared with the AuNSPs may be size, surface chemistry, and shape of the gold nanoparticles. The reduced cell toxicity observed with the AuNSTs suggests that AuNSTs may be a promising material for use in biomedical applications.

Toxicity studies using mammalian cells and impedance spectroscopy method

This research presents a study of cell-based Electric Cell-substrate Impedance Sensing (ECIS) sensors employed for testing toxicants in real-time. Mammalian cells are exposed to toxicants and information about cell viability are obtained from impedance measurements. The toxicants used are nicotine, phenol, ammonia and aldicarb. Two different adherent mammalian cells lines and cell culturewares were used to optimize the toxicity study. The cell lines used in this study were: bovine aortic endothelial cells (BAEC) and rat fat pad endothelial cells (RFPEC). The BAECs generated higher impedance values and formed cell monolayers at a faster rate compared to RFPECs. The two cell culturewares used in this study were simple open cell-culture wells and enclosed cell culturewares with microfluidic perfusion barriers. Experimental results indicated that the open wells are not suited for toxicity experiments. The turbulent flow created by pipetting media and toxicants in open cell culture wells disrupts the formation of the cell monolayer. When enclosed culture chambers were used, the flow of the cell culture medium and toxicant solutions were laminar on the cell monolayer. Both finite element simulations and experimental results showed that enclosed perfusion chamber have better performance due to the laminar flow of the solutions. The enclosed perfusion chamber also significantly shortens the response time of the cells when exposed to toxicants compared to the open cell culture wells. The experimental results demonstrated that ECIS biosensors seeded with BAECs inside enclosed cell culture chambers successfully assesses the effect of toxicants on mammalian cells in real-time.

Sphingosine-1-phosphate protects endothelial glycocalyx by inhibiting syndecan-1 shedding

Endothelial cells (ECs) are covered by a surface glycocalyx layer that forms part of the barrier and mechanosensing functions of the blood-tissue interface. Removal of albumin in bathing media induces collapse or shedding of the glycocalyx. The electrostatic interaction between arginine residues on albumin, and negatively charged glycosaminoglycans (GAGs) in the glycocalyx have been hypothesized to stabilize the glycocalyx structure. Because albumin is one of the primary carriers of the phospholipid sphingosine-1-phosphate (S1P), we evaluated the alternate hypothesis that S1P, acting via S1P1 receptors, plays the primary role in stabilizing the endothelial glycocalyx. Using confocal microscopy on rat fat-pad ECs, we demonstrated that heparan sulfate (HS), chondroitin sulfate (CS), and ectodomain of syndecan-1 were shed from the endothelial cell surface after removal of plasma protein but were retained in the presence of S1P at concentrations of >100 nM. S1P1 receptor antagonism abolished the protection of the glycocalyx by S1P and plasma proteins. S1P reduced GAGs released after removal of plasma protein. The mechanism of protection from loss of glycocalyx components by S1P-dependent pathways was shown to be suppression of metalloproteinase (MMP) activity. General inhibition of MMPs protected against loss of CS and syndecan-1. Specific inhibition of MMP-9 and MMP-13 protected against CS loss. We conclude that S1P plays a critical role in protecting the glycocalyx via S1P1 and inhibits the protease activity-dependent shedding of CS, HS, and the syndecan-1 ectodomain. Our results provide new insight into the role for S1P in protecting the glycocalyx and maintaining vascular homeostasis.

Microarray analysis of the mammalian thromboxane receptor-Trypanosoma cruzi interaction

Trypanosoma cruzi, the etiological agent of Chagas disease, causes vasculopathy and cardiomyopathy in humans and is associated with elevated levels of several vasoactive molecules such as nitric oxide, endothelin-1 and thromboxane A2 (TXA2). Parasite derived TXA2 modulates vasculopathy and other pathophysiological features of Chagasic cardiomyopathy. Previously, we demonstrated that in response to infection with T. cruzi, TXA2 receptor (TP) null mice displayed increased parasitemia; mortality and cardiac pathology compared with wild type (WT) and TXA2 synthase null mice. In order to further study the role of TXA2-TP signaling in the development of Chagas disease, GeneChip microarrays were used to detect transcriptome changes in rat fat pad endothelial cells (RFP-ECs) which is incapable of TXA2 signaling (TP null) to that of control (wild type) and RFP-EC with reconstituted TP expression. Genes that were significantly regulated due to infection were identified using a time course of 2, 18 and 48 hrs post infection. We identified several key genes such as suppressor of cytokine signaling (SOCS-5), several cytokines (CSF-1, CXCF ligands), and MAP kinases (MAPK-1, Janus kinase) that were upregulated in the absence of TP signaling. These data underscore the importance of the interaction of the parasite with mammalian TP and may explain the increased mortality and cardiovascular pathology observed in infected TP null mice.

Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation

To establish the role of vascular endothelial (VE)-cadherin in the regulation of endothelial cell functions, we investigated the effect of phosphorylation of a VE-cadherin site sought to be involved in p120-catenin binding on vascular permeability and endothelial cell migration. To this end, we introduced either wild-type VE-cadherin or Y658 phosphomimetic (Y658E) or dephosphomimetic (Y658F) VE-cadherin mutant constructs into an endothelial cell line (rat fat pad endothelial cells) lacking endogenous VE-cadherin. Remarkably, neither wild-type- nor Y658E VE-cadherin was retained at cell-cell contacts because of p120-catenin preferential binding to N-cadherin, resulting in the targeting of N-cadherin to cell-cell junctions and the exclusion of VE-cadherin. However, Y658F VE-cadherin was able to bind p120-catenin and to localize at adherence junctions displacing N-cadherin. This resulted in an enhanced barrier function and a complete abrogation of Rac1 activation and lamellipodia formation, thereby inhibiting cell migration. These findings demonstrate that VE-cadherin, through the regulation of Y658 phosphorylation, competes for junctional localization with N-cadherin and controls vascular permeability and endothelial cell migration.

The Endothelial Glycocalyx In Vitro: Its Structure and The Role of Heparan Sulfate and Glypican‐1 in eNOS Activation by Flow

The endothelial glycocalyx (GCX) mediates flow‐induced nitric oxide release via heparan sulfate (HS), but the GCX structure is unclear and the specific HS core protein(s) involved in this mechanotransduction is unknown. Our study tests the hypotheses that flow regulates GCX thickness and organization and that the HS glypican‐1 core protein mediates flow‐induced activation of endothelial nitric oxide synthase (eNOS). Monolayers of bovine aortic and rat fat pad endothelial cells with intact GCX, enzymatically degraded HS, or RNA‐silenced glypican‐1 were exposed to 15 dyne/cm2 uniform shear stress for 3 hr. Confocal immunocytochemistry and cryo‐transmission electron microscopy revealed a GCX that was 2.5 to 4.0 μm thick. Sheared GCX was well organized and aligned perpendicular to the cell surface, while unsheared GCX was disorganized. Western blot and confocal microscopy demonstrate that when the GCX is intact shear stress increases eNOS activation (ser1177 phosphorylation) and membrane localization, which are blocked by HS degradation. Baseline and flow‐induced levels of activated eNOS are attenuated when glypican‐1 is silenced. This work further elucidates GCX structure and role in endothelial cell mechanotransduction. Supported by NIH: 5T32HL007675, HL73732, HL57093.

The Egr-1 gene is induced by epidermal growth factor in ECV304 cells and primary endothelial cells.

The early growth response (Egr)-1 transcription factor serves to couple changes in the extracellular environment to alterations in gene expression. An understanding of the mechanisms that underlie Egr-1 gene regulation should provide important insights into how environmental signals are transduced by endothelial cells. The aim of the present study was to determine whether epidermal growth factor (EGF) induces Egr-1 expression in endothelial cells. In ECV304 cells, Egr-1 mRNA and protein levels were increased in response to EGF. In stable transfection assays, the 1,200-bp promoter of the mouse Egr-1 gene contained information for EGF response via a protein kinase C-independent, mitogen-activated protein kinase-dependent pathway. The endogenous Egr-1 gene was similarly responsive to EGF in primary human umbilical vein endothelial cells, human coronary artery endothelial cells, and rat fat pad endothelial cells, but not in bovine aortic endothelial cells, calf pulmonary artery endothelial cells, or PY-4-1 endothelial cells. Together, these results suggest that the Egr-1 gene is responsive to EGF in a subset of endothelial cells.