Intact Endothelial Cells Research Articles

Oxidative stress alters mitochondrial homeostasis in isolated brain capillaries

BackgroundNeurovascular deficits and blood-brain barrier (BBB) dysfunction are major hallmarks of brain trauma and neurodegenerative diseases. Oxidative stress is a prominent contributor to neurovascular unit (NVU) dysfunction and can propagate BBB disruption. Oxidative damage results in an imbalance of mitochondrial homeostasis, which can further drive functional impairment of brain capillaries. To this end, we developed a method to track mitochondrial-related changes after oxidative stress in the context of neurovascular pathophysiology as a critical endophenotype of neurodegenerative diseases.MethodsTo study brain capillary-specific mitochondrial function and dynamics in response to oxidative stress, we developed an ex vivo model in which we used isolated brain capillaries from transgenic mice that express dendra2 green specifically in mitochondria (mtD2g). Isolated brain capillaries were incubated with 2,2’-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) or hydrogen peroxide (H2O2) to induce oxidative stress through lipid peroxidation. Following the oxidative insult, mitochondrial bioenergetics were measured using the Seahorse XFe96 flux analyzer, and mitochondrial dynamics were measured using confocal microscopy with Imaris software.ResultsWe optimized brain capillary isolation with intact endothelial cell tight-junction and pericyte integrity. Further, we demonstrate consistency of the capillary isolation process and cellular enrichment of the isolated capillaries. Mitochondrial bioenergetics and morphology assessments were optimized in isolated brain capillaries. Finally, we found that oxidative stress significantly decreased mitochondrial respiration and altered mitochondrial morphology in brain capillaries, including mitochondrial volume and count.ConclusionsFollowing ex vivo isolation of brain capillaries, we confirmed the stability of mitochondrial parameters, demonstrating the feasibility of this newly developed platform. We also demonstrated that oxidative stress has profound effects on mitochondrial homeostasis in isolated brain capillaries. This novel method can be used to evaluate pharmacological interventions to target oxidative stress or mitochondrial dysfunction in cerebral small vessel disease and neurovascular pathophysiology as major players in neurodegenerative disease.

Isolation of Mouse Pancreatic Endothelial Cells.

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.

Phenotyping adult mice with inducible depletion of sequestosome-1 (p62) in endothelial cells

Macroautophagy (autophagy) maintains intracellular homeostasis by targeting damaged / dysregulated organelles for their transport to and degradation by the lysosome. Autophagy regulated gene 3 ( Atg3) enables autophagosome maturation which is required for the process of autophagy to occur. We reported that inducible depletion of Atg3 specifically in endothelial cells (ECs) impairs arterial function in the absence of metabolic disruptions (Cho et al., Cardiovasc Res, 2023). Sequestosome-1 (p62/SQSTM1) tethers proteins into the autophagic pathway for subsequent lysosomal degradation. The contribution from EC p62 to arterial and metabolic function is unknown and we addressed this. Adult male and female C57BL/6 mice with tamoxifen inducible depletion of p62 specifically in ECs ( p62EC−/−) were compared to littermate controls ( p62EC+/+). p62/18S mRNA expression was lower (p<0.05) in ECs but not vascular smooth muscle cells (VSM) from p62EC−/− [n=6] vs. p62EC+/+ [n=9] mice, whereas Beclin-1/18S was similar in ECs and VSM from both groups. Neither body composition, glucose disposal nor insulin tolerance were impacted by EC specific p62 depletion. Vascular contractile responses to: (i) phenylephrine mediated α1-activation and (ii) potassium chloride evoked L-type Ca2+ channel opening; and relaxation responses to (iii) acetylcholine (endothelium-dependent) and (iv) sodium nitroprusside (endothelium-independent); were similar between groups in mesenteric resistance arteries (isometric myography) and middle cerebral arteries (isobaric myography). Further, intrinsic arterial stiffness was not different between p62EC−/− [n=6] and p62EC+/+ [n=9] mice i.e., neither the elastic modulus nor stress vs. strain curves generated using segments of thoracic aorta were altered. These findings indicate EC specific p62 depletion does not impact arterial function or the indexes of systemic metabolism we measured. Because p62 is upregulated during cellular stress, it is possible that a challenge (e.g., hypoxia, ischemia) might be required to unmask a protective role for intact EC p62 and studies are ongoing to test this. Supported by AHA23PRE1025910 (SM); Rural and Underserved Utah Training Experience T32 (CG), Pacific Islander Research Internship Program (LG), NIHR01HL149870-01A1 (SB) and NIHRO1HL141540 (JDS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The bisphosphonates alendronate and zoledronate induce adaptations of aerobic metabolism in permanent human endothelial cells

Nitrogen-containing bisphosphonates (NBPs), compounds that are widely used in the treatment of bone disorders, may cause side effects related to endothelial dysfunction. The aim of our study was to investigate the effects of chronic 6-day exposure to two common bone-preserving drugs, alendronate and zoledronate, on endothelial function and oxidative metabolism of cultured human endothelial cells (EA.hy926). NBPs reduced cell viability, induced oxidative stress and a pro-inflammatory state and downregulated the prenylation-dependent ERK1/2 signaling pathway in endothelial cells. In addition, NBPs induced increased anaerobic respiration and slightly increased oxidative mitochondrial capacity, affecting mitochondrial turnover through reduced mitochondrial fission. Moreover, by blocking the mevalonate pathway, NBPs caused a significant decrease in the level of coenzyme Q10, thereby depriving endothelial cells of an important antioxidant and mitochondrial electron carrier. This resulted in increased formation of reactive oxygen species (ROS), upregulation of antioxidant enzymes, and impairment of mitochondrial respiratory function. A general decrease in mitochondrial respiration occurred with stronger reducing fuels (pyruvate and glutamate) in NBP-treated intact endothelial cells, and significantly reduced phosphorylating respiration was observed during the oxidation of succinate and especially malate in NBP-treated permeabilized endothelial cells. The observed changes in oxidative metabolism caused a decrease in ATP levels and an increase in oxygen levels in NBP-treated cells. Thus, NBPs modulate the energy metabolism of endothelial cells, leading to alterations in the cellular energy state, coenzyme Q10 redox balance, mitochondrial respiratory function, and mitochondrial turnover.

Corneal optical density: Structural basis, measurements, influencing factors, and roles in refractive surgery.

The cornea is the main refractive medium of the human eye, and its clarity is critical to visual acuity. Corneal optical density (COD) is an important index to describe corneal transparency. Intact corneal epithelial and endothelial cells, regular arrangement of collagen fibers in the stroma, and normal substance metabolism are all integral for the cornea to maintain its transparency. In the last two decades, the Pentacam Scheimpflug imaging system has emerged as a breakthrough for the measurement of COD (also called corneal densitometry). It has been found that a wide variety of factors such as age, refractive status, and corneal diseases can affect COD. Different corneal refractive surgery methods also change COD in different corneal regions and layers and affect visual acuity following the surgery. Thus, COD has gradually become a significant indicator to evaluate corneal health, one on which the attention of clinicians has been increasingly focused.

KIR channel regulation of electrical conduction along cerebrovascular endothelium: Enhanced modulation during Alzheimer's disease.

Endothelial cell (EC) coupling occurs through gap junctions and underlies cerebral blood flow regulation governed by inward-rectifying (KIR ) channels. This study addressed effects of KIR channel activity on EC coupling before and during Alzheimer's disease (AD). Intact EC tubes (width: ~90-100 μm; length: ~0.5 mm) were freshly isolated from posterior cerebral arteries of young Pre-AD (1-3 months) and aged AD (13-18 months) male and female 3xTg-AD mice. Dual intracellular microelectrodes applied simultaneous current injections (±0.5-3 nA) and membrane potential (Vm ) recordings in ECs at distance ~400 μm. Elevated extracellular potassium ([K+ ]E ; 8-15 mmol L-1 ; reference, 5 mmol L-1 ) activated KIR channels. Conducted Vm (∆Vm ) responses ranged from ~-30 to 30 mV in response to -3 to +3 nA (linear regression, R2 ≥0.99) while lacking rectification for charge polarity or axial direction of spread. Conduction slope decreased ~10-20% during 15 mmol L-1 [K+ ]E in Pre-AD males and AD females. 15 mmol L-1 [K+ ]E decreased conduction at lower thresholds in AD animals (~±20 mV) versus Pre-AD (~±25 mV). AD increased (~10-15%) conducted hyperpolarization during 8-12 mmol L-1 [K+ ]E . Brain endothelial KIR channel activity modulates bidirectional spread of vasoreactive signals with enhanced regulation of EC coupling during AD pathology.

Mitochondrial ATP Production is Required for Endothelial Cell Control of Vascular Tone.

Arteries and veins are lined by nonproliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis, rather than mitochondrial oxidative phosphorylation, to fuel each of these energy-demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells, and little is known about energy production in endothelial cells within the fully formed vascular wall. Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial respiration disrupts endothelial control of vascular tone. Basal, mechanically activated, and agonist-evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. Agonist-evoked calcium activity was also inhibited by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. The role for mitochondria in endothelial cell energy production is independent of species, sex, or vascular bed. These data show that a mitochondrial ATP supply is necessary for calcium-dependent, nitric oxide-mediated endothelial control of vascular tone, and identifies the critical role of endothelial mitochondrial energy production in fueling perfused blood vessel function.

Calciprotein Particles Cause Physiologically Significant Pro-Inflammatory Response in Endothelial Cells and Systemic Circulation

Calciprotein particles (CPPs) represent an inherent mineral buffering system responsible for the scavenging of excessive Ca2+ and PO43- ions in order to prevent extraskeletal calcification, although contributing to the development of endothelial dysfunction during the circulation in the bloodstream. Here, we performed label-free proteomic profiling to identify the functional consequences of CPP internalisation by endothelial cells (ECs) and found molecular signatures of significant disturbances in mitochondrial and lysosomal physiology, including oxidative stress, vacuolar acidification, accelerated proteolysis, Ca2+ cytosolic elevation, and mitochondrial outer membrane permeabilisation. Incubation of intact ECs with conditioned medium from CPP-treated ECs caused their pro-inflammatory activation manifested by vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) upregulation and elevated release of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1/ C-C motif ligand 2 (MCP-1/CCL2). Among the blood cells, monocytes were exclusively responsible for CPP internalisation. As compared to the co-incubation of donor blood with CPPs in the flow culture system, intravenous administration of CPPs to Wistar rats caused a considerably higher production of chemokines, indicating the major role of monocytes in CPP-triggered inflammation. Upregulation of sICAM-1 and IL-8 also suggested a notable contribution of endothelial dysfunction to systemic inflammatory response after CPP injections. Collectively, our results demonstrate the pathophysiological significance of CPPs and highlight the need for the development of anti-CPP therapies.

Mitochondrial oxidative phosphorylation fuels endothelial control of blood vessel tone

Arteries and veins are lined by non‐proliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis to fuel each of these energy‐demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells in angiogenesis, and little is known about energy production in endothelial cells within the fully‐formed vascular wall.Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial oxidative phosphorylation at mitochondrial complex V disrupts calcium‐dependent, nitric oxide‐mediated endothelial cell control of vascular tone. Basal, mechanically‐activated, and agonist‐evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. This effect is mimicked by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. The role for endothelial cell energy production is independent of species, sex, or vascular bed.These data show that mitochondrial ATP is necessary for the obligatory role of endothelial cells in the control of blood vessel diameter, and validate the idea of targeting endothelial cell metabolism to treat endothelial cell dysfunction in cardiovascular disease.

Activating P2Y1receptors improves function in arteries with repressed autophagy

Objective and hypothesisThe importance of endothelial cell (EC) autophagy to vascular homeostasis in the context of health and disease is evolving. Earlier we reported that intact EC autophagy is requisite to maintain shear‐stress‐induced nitric oxide (NO) generation via glycolysis‐dependent purinergic signaling to eNOS. Here we illustrate the translational and functional significance of these findings.Methods and ResultsFirst, we assessed translational relevance using older male humans and mice that exhibit blunted EC autophagy and impaired arterial function vs. adult controls. Active hyperemia evoked by rhythmic handgrip exercise elevated radial artery shear rate similarly from baseline in adult (23±1 y) and older (68±2 y) subjects for 60‐min. Compared to baseline, indexes of autophagy initiation, p‐eNOSS1177 activation, and NO generation, occurred in radial artery ECs obtained from adult but not older volunteers. Regarding mice, indexes of autophagy and p‐eNOSS1177 activation were robust in ECs from adult (7 ± 1 months) but not older (23 ± 1 months) animals that completed 60‐min treadmill‐running. Further, results concerning the extracellular acidification rate (ECAR; Seahorse Bioanalyzer) indicate glycolysis and glycolytic capacity were elevated in response to 20 dyn/cm2 laminar shear stress x 45‐min in primary arterial ECs obtained from adult but not older mice. We next questioned whether the inability to initiate EC autophagy, glycolysis, and p‐eNOSS1177 precipitates dysfunction in arteries from older vs. adult mice. Compromised intraluminal flow‐mediated vasodilation displayed by arteries from older vs. adult mice was recapitulated in vessels from adult mice by : (i) NO synthase inhibition; (ii) acute autophagy impairment using 3‐methyladenine (3‐MA); (iii) EC Atg3 depletion (Atg3EC‐/‐ mice); (iv) purinergic 2Y1‐receptor (P2Y1‐R) blockade; and (v) germline depletion of P2Y1‐Rs. Importantly, P2Y1‐R activation using 2‐methylthio‐ADP (2‐Me‐ADP) improved vasodilatory capacity in arteries from : (i) adult mice treated with 3‐MA; (ii) adult Atg3EC‐/‐ mice; and (iii) older animals with repressed EC autophagy.ConclusionsArterial dysfunction concurrent with pharmacological, genetic, and age‐associated EC autophagy disruption is improved by activating P2Y1‐Rs.

Activating P2Y1 receptors improves function in arteries with repressed autophagy.

The importance of endothelial cell (EC) autophagy to vascular homeostasis in the context of health and disease is evolving. Earlier, we reported that intact EC autophagy is requisite to maintain shear-stress-induced nitric oxide (NO) generation via glycolysis-dependent purinergic signalling to endothelial NO synthase (eNOS). Here, we illustrate the translational and functional significance of these findings. First, we assessed translational relevance using older male humans and mice that exhibit blunted EC autophagy and impaired arterial function vs. adult controls. Active hyperaemia evoked by rhythmic handgrip exercise-elevated radial artery shear-rate similarly from baseline in adult and older subjects for 60 min. Compared with baseline, indexes of autophagy initiation, p-eNOSS1177 activation, and NO generation, occurred in radial artery ECs obtained from adult but not older volunteers. Regarding mice, indexes of autophagy and p-eNOSS1177 activation were robust in ECs from adult but not older animals that completed 60-min treadmill-running. Furthermore, 20 dyne • cm2 laminar shear stress × 45-min increased autophagic flux, glycolysis, ATP production, and p-eNOSS1177 in primary arterial ECs obtained from adult but not older mice. Concerning functional relevance, we next questioned whether the inability to initiate EC autophagy, glycolysis, and p-eNOSS1177in vitro precipitates arterial dysfunction ex vivo. Compromised intraluminal flow-mediated vasodilation displayed by arteries from older vs. adult mice was recapitulated in vessels from adult mice by (i) NO synthase inhibition; (ii) acute autophagy impairment using 3-methyladenine (3-MA); (iii) EC Atg3 depletion (iecAtg3KO mice); (iv) purinergic 2Y1-receptor (P2Y1-R) blockade; and (v) germline depletion of P2Y1-Rs. Importantly, P2Y1-R activation using 2-methylthio-ADP (2-Me-ADP) improved vasodilatory capacity in arteries from (i) adult mice treated with 3-MA; (ii) adult iecAtg3KO mice; and (iii) older animals with repressed EC autophagy. Arterial dysfunction concurrent with pharmacological, genetic, and age-associated EC autophagy compromise is improved by activating P2Y1-Rs.

Endothelial Injury in Diving: Atomic Force-, Electronic-, and Light-Microscopy Study of the Ovine Decompressed Blood Vessels.

We suggested that the nanobubbles, which appear at the active hydrophobic spots (AHSs) at the luminal aspect of the blood vessels, are the gas micronuclei from which the decompression bubbles evolve and the endothelial injury during the decompression is due to the tearing off the cell membranes with the detaching bubbles. Ovine blood vessels were stretched over the polycarbonate plates or glass microscopic slides and were exposed under saline to the hyperbaric pressure (1,013 kPa, 19 h). Following decompression, the blood vessels were photographed for the identification (by bubble formation) of the AHS. Nanobubbles could not be demonstrated at the AHS by using the atomic force microscopy (AFM) because of the roughness of the surface, which disabled the close contact of the probe. In the electron microscopy, no endothelial cells were observed in the samples from the area near to the AHS, but the underlying elastin layer of the intima was observed adjacent to the media. Some intact endothelial cells were observed only in the locations far from an AHS. In the optical microscopy, no endothelial cells were observed in the blood vessels in close proximity to the AHS and in some sections, debris or a detached cluster of the endothelial cells were observed. Intact endothelial cells could be found at the sites distant from an AHS. This study supports the assumption, where the detached bubbles tear off the endothelial cells and cause the initial endothelial injury following the decompression.

High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells.

The influence of glucose and palmitic acid (PA) on mitochondrial respiration and emission of hydrogen peroxide (H2O2) was determined in skeletal muscle‐derived microvascular endothelial cells. Measurements were assessed in intact and permeabilized (cells treated with 0.025% saponin) low passage endothelial cells with acute‐or prolonged (3 days) incubation with regular (1.7 mM) or elevated (2.2 mM) PA concentrations and regular (5 mM) or elevated (11 mM) glucose concentrations. In intact cells, acute incubation with 1.7 mM PA alone or with 1.7 mM PA + 5 mM glucose (p < .001) led to a lower mitochondrial respiration (p < 0.01) and markedly higher H2O2/O2 emission (p < 0.05) than with 5 mM glucose alone. Prolonged incubation of intact cells with 1.7 mM PA +5 mM glucose led to 34% (p < 0.05) lower respiration and 2.5‐fold higher H2O2/O2 emission (p < 0.01) than incubation with 5 mM glucose alone. Prolonged incubation of intact cells with elevated glucose led to 60% lower (p < 0.05) mitochondrial respiration and 4.6‐fold higher H2O2/O2 production than incubation with 5 mM glucose in intact cells (p < 0.001). All effects observed in intact cells were present also in permeabilized cells (State 2). In conclusion, our results show that acute and prolonged lipid availability, as well as prolonged hyperglycemia, induces mitochondrial dysfunction as evidenced by lower mitochondrial respiration and enhanced H2O2/O2 emission. Elevated plasma substrate availability may lead to microvascular dysfunction in skeletal muscle by impairing endothelial mitochondrial function.

Eruptive xanthoma model reveals endothelial cells internalize and metabolize chylomicrons, leading to extravascular triglyceride accumulation.

Although tissue uptake of fatty acids from chylomicrons is primarily via lipoprotein lipase (LpL) hydrolysis of triglycerides (TGs), studies of patients with genetic LpL deficiency suggest additional pathways deliver dietary lipids to tissues. Despite an intact endothelial cell (EC) barrier, hyperchylomicronemic patients accumulate chylomicron-derived lipids within skin macrophages, leading to the clinical finding eruptive xanthomas. We explored whether an LpL-independent pathway exists for transfer of circulating lipids across the EC barrier. We found that LpL-deficient mice had a marked increase in aortic EC lipid droplets before and after a fat gavage. Cultured ECs internalized chylomicrons, which were hydrolyzed within lysosomes. The products of this hydrolysis fueled lipid droplet biogenesis in ECs and triggered lipid accumulation in cocultured macrophages. EC chylomicron uptake was inhibited by competition with HDL and knockdown of the scavenger receptor-BI (SR-BI). In vivo, SR-BI knockdown reduced TG accumulation in aortic ECs and skin macrophages of LpL-deficient mice. Thus, ECs internalize chylomicrons, metabolize them in lysosomes, and either store or release their lipids. This latter process may allow accumulation of TGs within skin macrophages and illustrates a pathway that might be responsible for creation of eruptive xanthomas.

Gold Nanoparticles Affect Pericyte Biology and Capillary Tube Formation.

Gold nanoparticles (AuNPs) are used for diagnostic and therapeutic purposes, especially antiangiogenesis, which are accomplished via inhibition of endothelial cell proliferation, migration, and tube formation. However, no research has been performed on the effects of AuNPs in pericytes, which play vital roles in endothelial cell functions and capillary tube formation during physiological and pathological processes. Therefore, the effects of AuNPs on the morphology and functions of pericytes need to be elucidated. This study treated human placental pericytes in monoculture with 20 nm AuNPs at a concentration of 30 ppm. Ki-67 and platelet-derived growth factor receptor-β (PDGFR-β) mRNA expression was measured using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell migration was assessed by Transwell migration assay. The fine structures of pericytes were observed by transmission electron microscopy. In addition, 30 ppm AuNP-treated pericytes and intact human umbilical vein endothelial cells were cocultured on Matrigel to form three-dimensional (3D) capillary tubes. The results demonstrated that AuNPs significantly inhibited proliferation, reduced PDGFR-β mRNA expression, and decreased migration in pericytes. Ultrastructural analysis of pericytes revealed AuNPs in late endosomes, autolysosomes, and mitochondria. Remarkably, many mitochondria were swollen or damaged. Additionally, capillary tube formation was reduced. We found that numerous pericytes on 3D capillary tubes were round and did not extend their processes along the tubes, which resulted in more incomplete tube formation in the treatment group compared with the control group. In summary, AuNPs can affect pericyte proliferation, PDGFR-β mRNA expression, migration, morphology, and capillary tube formation. The findings highlight the possible application of AuNPs in pericyte-targeted therapy for antiangiogenesis.

Lysophosphatidylcholine in phospholipase A2-modified LDL triggers secretion of angiopoietin 2

Background and aimsSecretory phospholipase A2 (PLA2) hydrolyzes LDL phospholipids generating modified LDL particles (PLA2-LDL) with increased atherogenic properties. Exocytosis of Weibel–Palade bodies (WPB) releases angiopoietin 2 (Ang2) and externalizes P-selectin, which both play important roles in vascular inflammation.Here, we investigated the effects of PLA2-LDL on exocytosis of WPBs. MethodsHuman coronary artery endothelial cells (HCAECs) were stimulated with PLA2- LDL, and its uptake and effect on Ang2 release, leukocyte adhesion, and intracellular calcium levels were measured. The effects of PLA2-LDL on Ang2 release and WPB exocytosis were measured in and ex vivo in mice. ResultsExposure of HCAECs to PLA2-LDL triggered Ang2 secretion and promoted leukocyte-HCAEC interaction. Lysophosphatidylcholine was identified as a critical component of PLA2-LDL regulating the WPB exocytosis, which was mediated by cell-surface proteoglycans, phospholipase C, intracellular calcium, and cytoskeletal remodeling. PLA2-LDL also induced murine endothelial WPB exocytosis in blood vessels in and ex vivo, as evidenced by secretion of Ang2 in vivo, P-selectin translocation to plasma membrane in intact endothelial cells in thoracic artery and tracheal vessels, and reduced Ang2 staining in tracheal endothelial cells. Finally, in contrast to normal human coronary arteries, in which Ang2 was present only in the endothelial layer, at sites of advanced atherosclerotic lesions, Ang2 was detected also in the intima, media, and adventitia. ConclusionsOur studies reveal PLA2-LDL as a potent agonist of endothelial WPB exocytosis, resulting in increased secretion of Ang2 and translocation of P-selectin. The results provide mechanistic insight into PLA2-LDL-dependent promotion of vascular inflammation and atherosclerosis.

Effects of the micro/nanostructure of electrospun zein fibres on cells in simulated blood flow environment

In order to prevent thrombosis, reduce intima hyperplasia, and to maintain long-term patency after implantation of an artificial blood vessel, the formation of intact endothelial cells layer on an inner surface of graft is desirable. The present study aimed to improve endothelial cell adhesion by regulating the morphology of the inner surface of artificial blood vessels. Zein fibre membranes with three fibre diameters (small, ~100 nm; medium, ~500 nm; and large, ~1000 nm) were constructed by electrospinning. A flow chamber device was designed to simulate the blood flow environment. The morphology and adhesion of human umbilical vein fusion cells (EA.hy926) on the surface of the fibre membranes were studied under a shear stress of approximately 15 dynes/cm2. The results showed that oriented electrospun zein fibre surfaces with both medium- and large-diameter fibres can regulate the morphology of endothelial cells (EA.hy926), which are aligned by the fibre direction. The three fibre membranes improved the adhesion of endothelial cells significantly compared to that on the flat membrane. When the fibre direction was fixed parallel to the fluid direction, the medium-diameter oriented-fibre membrane could significantly improve the ability endothelial cells to resist shear stress, and there was a significant difference at 1, 2 and 4 h time points compared with the shear stress resistance on the small-diameter and large-diameter oriented-fibre membranes. When the fibre direction was perpendicular to the fluid direction, again the medium-diameter oriented-fibre membrane improved the ability of endothelial cells to resist shear stress significantly at 1 and 2 h time points. It was concluded that by changing the diameter and arrangement of electrospun fibres, cell morphology control and shear stress resistance can be achieved.

Ligation of HLA Class I Molecules Induces YAP Activation through Src in Human Endothelial Cells.

Ab cross-linking of HLA class I (HLA I) molecules on the surface of endothelial cells (EC) triggers proliferative and prosurvival intracellular signaling, which is implicated in the process of chronic allograft rejection, also known as transplant vasculopathy. Despite the importance of Ab-mediated rejection in transplantation, the mechanisms involved remain incompletely understood. In this study, we examined the regulation of yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in human ECs challenged with Abs that bind HLA I. In unstimulated ECs, YAP localized mainly in the cytoplasm. Stimulation of these cells with Ab W6/32 induced marked translocation of YAP to the nucleus. The nuclear import of YAP was associated with a rapid decrease in YAP phosphorylation at Ser127 and Ser397, sites targeted by LATS1/2 and with the expression of YAP-regulated genes, including connective tissue growth factor (CTGF), and cysteine-rich angiogenic inducer 61 (CYR61). Transfection of small interfering RNAs targeting YAP/TAZ blocked the migration of ECs stimulated by ligation of HLA I, indicating that YAP mediates the increase in EC migration induced by HLA I ligation. Treatment of intact ECs with Src family inhibitors induced cytoplasmic localization of YAP in unstimulated ECs and, strikingly, blocked the nuclear import of YAP induced by Ab-induced HLA I activation in these cells and the increase in the expression of the YAP-regulated genes CTGF and CYR61 induced by HLA I stimulation. Our results identify the Src/YAP axis as a key player in promoting the proliferation and migration of ECs that are critical in the pathogenesis of transplant vasculopathy.

Is Intact Endothelial Cell Autophagy Required for Training‐Induced Vascular Adaptations?

Macroautophagy is operational during basal conditions to maintain intracellular organelle and protein quality control, but is upregulated during cellular stress (e.g, dynamic exercise) to adapt to changing nutritional and energy demands. We tested the hypothesis that intact endothelial cell (EC) autophagy is required to observe training‐induced vascular adaptations. Rationale for this hypothesis was provided by an earlier report that obese mice with germline, whole body mutation of a protein requisite for autophagy i.e., Bcl2‐AAA mice were refractory to training‐induced improvements concerning glucose homeostasis (He et al., Nature, 2012). First, we demonstrated that : (i) workload achieved during a maximal treadmill test; (ii) soleus muscle citrate synthase activity; (iii) mRNA and protein expression of vascular autophagy; and (iv) intraluminal flow‐mediated vasodilatory responses (FMD) of femoral arteries examined ex vivo, were greater (all p&lt;0.05) in ~ 4‐month old male C57Bl/6 mice that completed 10‐weeks of treadmill‐training vs. age‐matched sedentary animals (n=10 mice per group). These findings indicate that our efficacious training protocol improves vascular autophagy and arterial function in adult mice. Next, ~ 4‐month old male mice on a C75Bl/6 background with inducible Cre/LoxP‐based impairment of autophagy‐related gene 3 (Atg3) specifically in ECs (iecAtg3KO mice) and their Cre negative littermates (WT) were treated with tamoxifen. Two weeks later, one cohort of iecAtg3KO mice initiated a treadmill training program (ETR; 10–60 min per day × 0–20% grade × 6 days per week × 10 weeks) or maintained familiarity with the treadmill by running 10 min per day × 5% grade × 1 day per week (SED). Two cohorts of WT littermates were treated identically. After 10‐weeks efficacy of the training protocol was established as described earlier and verification of our mutant was assessed. With regard to the latter, primary ECs obtained from the carotid artery and aorta indicated Atg3 mRNA and Atg3 protein, respectively, was minimal (p&lt;0.05) in iecAtg3KO vs. WT mice, whereas vascular smooth muscle cell Atg3 was similar between groups. As expected, intraluminal FMD responses were greater (p&lt;0.05) in WT‐ETR vs. WT‐SED mice, while vascular smooth muscle responses to sodium nitroprusside were not different between groups. Further, as anticipated, intraluminal FMD was blunted (p&lt;0.05) in iecAtg3KO‐SED vs. WT‐SED mice, indicating the importance of intact EC autophagy to FMD. Contrary to our hypothesis, however, training‐induced vascular adaptations indeed were observed (p&lt;0.05) in iecAtg3KO‐ETR vs. iecAtg3KO‐SED mice, while vascular smooth muscle responses were not different between groups. These findings indicate that intact EC Atg3 is not necessary for training‐induced vascular adaptations to occur.Support or Funding InformationCR [APS UGRF, University of Utah (UU) Undergraduate Research Opportunities Program (UROP)]; JMC (UU Graduate Research Fellowship); SKP (AHA 17POST33670663); KL (APS STRIDE, UU UROP), LT (UU UROP); SB (NIDDK R01‐DK‐098646‐01A1, R01‐DK‐099110, AHA 16GRNT30990018), JDS (AHA16GRNT31050004, NIH RO3AGO52848, NIHRO1HL141540).

Microfluidic Prototyping by Xurography to Engineer Fully‐lumenized Microvessels In Vitro

Vascular permeability (VP) is a measure of the barrier function (BF) of an intact endothelial cell (EC) monolayer. This parameter is especially important in physiology, as deregulation of permeability leads to leaky blood vessels with a transvascular imbalance of chemical gradients, fluidic pressure, and in some instances excessive trafficking of blood‐borne cells into the surrounding tissue. Regulation of VP is a highly coordinated process. However, monitoring VP in vivo can be challenging as it typically requires invasive and labor intensive intravital microscopy. Here we present an in vitro alternative by developing a microfluidic model of a blood vessel analog that permits characterization of endothelial BF under the influence of different extracellular matrix (ECM) and perivascular cellular constituents. Our model relies on the manufacturing process known as xurography, an inexpensive and rapid print‐and‐peel prototyping method for assembling multiple layers of polymers. This method has been recently applied to bypass the need for high‐end fabrication facilities that are not readily available at all research institutions. However, to date most experimental platforms manufactured with xurography have focused on biochemical studies with few cell culture‐based applications. Briefly, after using a cutting plotter to form a channel (.8 mm x 8 mm) on a PDMS layer (~ 400 um thick), the device is constructed through a series of bindings to other PDMS layers (3 x ~ 250 um) and biopsy punching that form two regions. The inlet and outlet ports for cell seeding (2mm and 4mm) are located at the extremes of the model. An area for ECM hydrogels is allocated in the middle, with a PDMS rod placed in the center (Fig 1). After the construction, collagen type I at 6mg/ml is inserted into the central region. Once polymerized the PDMS rod is pulled to leave a lumen, permitting the addition of ECs into the formed ductal structure. In order to assess BF, the vessels were stained and imaged with confocal microscopy for the intercellular adherent junction molecule, VE‐cadherin as well as cytoskeletal proteins. To quantify VP, a fluorescent tracer molecule was infused in the vessel, and this parameter was determined by measuring changes of intensity as the dye permeated from the vessel to the surrounding hydrogel. Importantly, our results indicated the vessel analog demonstrated VP on the same order of magnitude as similar studies with ECs growing in static conditions (Fig 2). Notably, the microdevice allows the addition of cells that can support EC function. These findings will be fundamental as we explore the crosstalk between cells that serve in regulating blood vessel BF. Moreover, the inexpensive fabrication process can be adapted seamlessly by research institutions across the nation to probe further into vascular biology.Support or Funding InformationFunding was provided by the National Heart, Lung, and Blood Institute (R01HL141941) and The Mark Foundation for Cancer Research ASPIRE Award. MC‐M acknowledges funding from a NIH Diversity Supplement (R01HL141941).Microfluidic model design and Confocal Imaging ‐ A) Assembly of microfluidic vessel analogue through binding of layers after fabrication. B) Staining for cytoskeletal protein, cell nuclei, and VE‐cadherin.Figure 1Apparent Permeability: Quantification of Vascular Permeability. Images of fluorescent dye permeating through the endothelial cell wall (time point ‐ 0 to 5 min.)Figure 2