Targeted polyelectrolyte complex micelles treat vascular complications in vivo.

Vascular disease is a leading cause of morbidity and mortality in the United States and globally. Pathological vascular remodeling, such as atherosclerosis and stenosis, largely develop at arterial sites of curvature, branching, and bifurcation, where disturbed blood flow activates vascular endothelium. Current pharmacological treatments of vascular complications principally target systemic risk factors. Improvements are needed. We previously devised a targeted polyelectrolyte complex micelle to deliver therapeutic nucleotides to inflamed endothelium in vitro by displaying the peptide VHPKQHR targeting vascular cell adhesion molecule 1 (VCAM-1) on the periphery of the micelle. This paper explores whether this targeted nanomedicine strategy effectively treats vascular complications in vivo. Disturbed flow-induced microRNA-92a (miR-92a) has been linked to endothelial dysfunction. We have engineered a transgenic line (miR-92aEC-TG /Apoe-/- ) establishing that selective miR-92a overexpression in adult vascular endothelium causally promotes atherosclerosis in Apoe-/- mice. We tested the therapeutic effectiveness of the VCAM-1-targeting polyelectrolyte complex micelles to deliver miR-92a inhibitors and treat pathological vascular remodeling in vivo. VCAM-1-targeting micelles preferentially delivered miRNA inhibitors to inflamed endothelial cells in vitro and in vivo. The therapeutic effectiveness of anti-miR-92a therapy in treating atherosclerosis and stenosis in Apoe-/- mice is markedly enhanced by the VCAM-1-targeting polyelectrolyte complex micelles. These results demonstrate a proof of concept to devise polyelectrolyte complex micelle-based targeted nanomedicine approaches treating vascular complications in vivo.

Genetic variant at coronary artery disease and ischemic stroke locus 1p32.2 regulates endothelial responses to hemodynamics.

Biomechanical cues dynamically control major cellular processes, but whether genetic variants actively participate in mechanosensing mechanisms remains unexplored. Vascular homeostasis is tightly regulated by hemodynamics. Exposure to disturbed blood flow at arterial sites of branching and bifurcation causes constitutive activation of vascular endothelium contributing to atherosclerosis, the major cause of coronary artery disease (CAD) and ischemic stroke (IS). Conversely, unidirectional flow promotes quiescent endothelium. Genome-wide association studies (GWAS) have identified chromosome 1p32.2 as strongly associated with CAD/IS; however, the causal mechanism related to this locus remains unknown. Using statistical analyses, assay of transposase accessible chromatin with whole-genome sequencing (ATAC-seq), H3K27ac/H3K4me2 ChIP with whole-genome sequencing (ChIP-seq), and CRISPR interference in human aortic endothelial cells (HAECs), our results demonstrate that rs17114036, a common noncoding polymorphism at 1p32.2, is located in an endothelial enhancer dynamically regulated by hemodynamics. CRISPR-Cas9-based genome editing shows that rs17114036-containing region promotes endothelial quiescence under unidirectional shear stress by regulating phospholipid phosphatase 3 (PLPP3). Chromatin accessibility quantitative trait locus (caQTL) mapping using HAECs from 56 donors, allelic imbalance assay from 7 donors, and luciferase assays demonstrate that CAD/IS-protective allele at rs17114036 in PLPP3 intron 5 confers increased endothelial enhancer activity. ChIP-PCR and luciferase assays show that CAD/IS-protective allele at rs17114036 creates a binding site for transcription factor Krüppel-like factor 2 (KLF2), which increases the enhancer activity under unidirectional flow. These results demonstrate that a human SNP contributes to critical endothelial mechanotransduction mechanisms and suggest that human haplotypes and related cis-regulatory elements provide a previously unappreciated layer of regulatory control in cellular mechanosensing mechanisms.

Proatherogenic Flow Increases Endothelial Stiffness via Enhanced CD36-Mediated Uptake of Oxidized Low-Density Lipoproteins.

OBJECTIVE: Disturbed flow (DF) is well-known to induce endothelial dysfunction and synergistically with plasma dyslipidemia facilitate plaque formation. Little is known, however, about the synergistic impact of DF and dyslipidemia on endothelial biomechanics. Our goal was to determine the impact of DF on endothelial stiffness and evaluate the role of dyslipidemia/oxLDL (oxidized low-density lipoprotein) in this process. APPROACH AND RESULTS: Endothelial elastic modulus of intact mouse aortas ex vivo and of human aortic endothelial cells exposed to laminar flow or DF was measured using atomic force microscopy. Endothelial monolayer of the aortic arch is found to be significantly stiffer than the descending aorta (4.2+1.1 versus 2.5+0.2 kPa for aortic arch versus descending aorta) in mice maintained on low-fat diet. This effect is significantly exacerbated by short-term high-fat diet (8.7+2.5 versus 4.5+1.2 kPa for aortic arch versus descending aorta). Exposure of human aortic endothelial cells to DF in vitro resulted in 50% increase in oxLDL uptake and significant endothelial stiffening in the presence but not in the absence of oxLDL. DF also increased the expression of oxLDL receptor CD36 (cluster of differentiation 36), whereas downregulation of CD36 abrogated DF-induced endothelial oxLDL uptake and stiffening. Furthermore, genetic deficiency of CD36 abrogated endothelial stiffening in the aortic arch in vivo in mice fed either low-fat diet or high-fat diet. We also show that the loss of endothelial stiffening in CD36 knockout aortas is not mediated by the loss of CD36 in circulating cells. CONCLUSIONS: DF facilitates endothelial CD36-dependent uptake of oxidized lipids resulting in local increase of endothelial stiffness in proatherogenic areas of the aorta.

TIFA as a crucial mediator for NLRP3 inflammasome.

Toll-like receptor-mediated NF-κB activation is a major innate immune reaction of vascular endothelial cells (ECs) in response to prooxidative and proinflammatory stimuli. We identified that TNF-α receptor-associated factor-interacting protein with a forkhead-associated domain (TIFA) is a regulator of priming (signal 1) and activating (signal 2) signals of nucleotide oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome in ECs. Oxidative and inflammatory stresses such as atheroprone flow and hyperlipidemia induce and activate TIFA in vitro and in vivo. For the priming of signal 1, sterol regulatory element-binding protein 2 transactivates TIFA, which in turn induces NF-κB activation and augments the transcription of NLRP3 inflammasome components. For the activation of signal 2, Akt is involved in TIFA Thr9 phosphorylation, which is essential for TIFA-TIFA homophilic oligomerization. Thr9 phosphorylation-dependent TIFA oligomerization facilitates the higher-order assembly of NLRP3 inflammasome, as indicated by the interaction between TIFA and caspase-1 in the activated ECs. Our results suggest that TIFA is a crucial mediator in the endothelial innate immune response by potentiating and amplifying NLRP3 inflammasome via augmenting signals 1 and 2.

Molecular-Scale Biophysical Modulation of an Endothelial Membrane by Oxidized Phospholipids.

The influence of two bioactive oxidized phospholipids on model bilayer properties, membrane packing, and endothelial cell biomechanics was investigated computationally and experimentally. The truncated tail phospholipids, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), are two major oxidation products of the unsaturated phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine. A combination of coarse-grained molecular dynamics simulations, Laurdan multiphoton imaging, and atomic force microscopy microindentation experiments was used to determine the impact of POVPC and PGPC on the structure of a multicomponent phospholipid bilayer and to assess the consequences of their incorporation on membrane packing and endothelial cell stiffness. Molecular simulations predicted differential bilayer perturbation effects of the two oxidized phospholipids based on the chemical identities of their truncated tails, including decreased bilayer packing, decreased bilayer bending modulus, and increased water penetration. Disruption of lipid order was consistent with Laurdan imaging results indicating that POVPC and PGPC decrease the lipid packing of both ordered and disordered membrane domains. Computational predictions of a larger membrane perturbation effect by PGPC correspond to greater stiffness of PGPC-treated endothelial cells observed by measuring cellular elastic moduli using atomic force microscopy. Our results suggest that disruptions in membrane structure by oxidized phospholipids play a role in the regulation of overall endothelial cell stiffness.

Thy-1 interaction with Fas in lipid rafts regulates fibroblast apoptosis and lung injury resolution.

Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1-/-) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1-/- mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis.

AMP-Activated Protein Kinase and Sirtuin 1 Coregulation of Cortactin Contributes to Endothelial Function.

OBJECTIVE: Cortactin translocates to the cell periphery in vascular endothelial cells (ECs) on cortical-actin assembly in response to pulsatile shear stress. Because cortactin has putative sites for AMP-activated protein kinase (AMPK) phosphorylation and sirtuin 1 (SIRT1) deacetylation, we examined the hypothesis that AMPK and SIRT1 coregulate cortactin dynamics in response to shear stress. APPROACH AND RESULTS: Analysis of the ability of AMPK to phosphorylate recombinant cortactin and oligopeptides whose sequences matched AMPK consensus sequences in cortactin pointed to Thr-401 as the site of AMPK phosphorylation. Mass spectrometry confirmed Thr-401 as the site of AMPK phosphorylation. Immunoblot analysis with AMPK siRNA and SIRT1 siRNA in human umbilical vein ECs and EC-specific AMPKα2 knockout mice showed that AMPK phosphorylation of cortactin primes SIRT1 deacetylation in response to shear stress. Immunoblot analyses with cortactin siRNA in human umbilical vein ECs, phospho-deficient T401A and phospho-mimetic T401D mutant, or aceto-deficient (9K/R) and aceto-mimetic (9K/Q) showed that cortactin regulates endothelial nitric oxide synthase activity. Confocal imaging and sucrose-density gradient analyses revealed that the phosphorylated/deacetylated cortactin translocates to the EC periphery facilitating endothelial nitric oxide synthase translocation from lipid to nonlipid raft domains. Knockdown of cortactin in vitro or genetic reduction of cortactin expression in vivo in mice substantially decreased the endothelial nitric oxide synthase-derived NO bioavailability. In vivo, atherosclerotic lesions increase in ApoE-/-/cortactin+/- mice, when compared with ApoE-/-/cortactin+/+ littermates. CONCLUSIONS: AMPK phosphorylation of cortactin followed by SIRT1 deacetylation modulates the interaction of cortactin and cortical-actin in response to shear stress. Functionally, this AMPK/SIRT1 coregulated cortactin-F-actin dynamics is required for endothelial nitric oxide synthase subcellular translocation/activation and is atheroprotective.

Sterol regulatory element binding protein 2 activation of NLRP3 inflammasome in endothelium mediates hemodynamic-induced atherosclerosis susceptibility.

BACKGROUND: The molecular basis for the focal nature of atherosclerotic lesions is poorly understood. Here, we explored whether disturbed flow patterns activate an innate immune response to form the NLRP3 inflammasome scaffold in vascular endothelial cells via sterol regulatory element binding protein 2 (SREBP2). METHODS AND RESULTS: Oscillatory flow activates SREBP2 and induces NLRP3 inflammasome in endothelial cells. The underlying mechanisms involve SREBP2 transactivating NADPH oxidase 2 and NLRP3. Consistently, SREBP2, NADPH oxidase 2, and NLRP3 levels were elevated in atheroprone areas of mouse aortas, suggesting that the SREBP2-activated NLRP3 inflammasome causes functionally disturbed endothelium with increased inflammation. Mimicking the effect of atheroprone flow, endothelial cell-specific overexpression of the activated form of SREBP2 synergized with hyperlipidemia to increase atherosclerosis in the atheroresistant areas of mouse aortas. CONCLUSIONS: Atheroprone flow induces NLRP3 inflammasome in endothelium through SREBP2 activation. This increased innate immunity in endothelium synergizes with hyperlipidemia to cause topographical distribution of atherosclerotic lesions.

The role of oxysterols in control of endothelial stiffness.

Endothelial dysfunction is a key step in atherosclerosis development. Our recent studies suggested that oxLDL-induced increase in endothelial stiffness plays a major role in dyslipidemia-induced endothelial dysfunction. In this study, we identify oxysterols, as the major component of oxLDL, responsible for the increase in endothelial stiffness. Using Atomic Force Microscopy to measure endothelial elastic modulus, we show that endothelial stiffness increases with progressive oxidation of LDL and that the two lipid fractions that contribute to endothelial stiffening are oxysterols and oxidized phosphatidylcholines, with oxysterols having the dominant effect. Furthermore, endothelial elastic modulus increases as a linear function of oxysterol content of oxLDL. Specific oxysterols, however, have differential effects on endothelial stiffness with 7-ketocholesterol and 7α-hydroxycholesterol, the two major oxysterols in oxLDL, having the strongest effects. 27-hydroxycholesterol, found in atherosclerotic lesions, also induces endothelial stiffening. For all oxysterols, endothelial stiffening is reversible by enriching the cells with cholesterol. oxLDL-induced stiffening is accompanied by incorporation of oxysterols into endothelial cells. We find significant accumulation of three oxysterols, 7α-hydroxycholesterol, 7ß-hydroxycholesterol, and 7-ketocholesterol, in mouse aortas of dyslipidemic ApoE⁻/⁻ mice at the early stage of atherosclerosis. Remarkably, these are the same oxysterols we have identified to induce endothelial stiffening.

Cholesterol regulates prokaryotic Kir channel by direct binding to channel protein.

Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by direct binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a K(D) of 390µM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3ß-17 ß-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol-KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol-KirBac1.1 binding results in full recovery of the channel activity. Collectively, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.

Targeting mechanosensitive endothelial TXNDC5 to stabilize eNOS and reduce atherosclerosis in vivo.

Although atherosclerosis preferentially develops at arterial curvatures and bifurcations where disturbed flow (DF) activates endothelium, therapies targeting flow-dependent mechanosensing pathways in the vasculature are unavailable. Here, we provided experimental evidence demonstrating a previously unidentified causal role of DF-induced endothelial TXNDC5 (thioredoxin domain containing 5) in atherosclerosis. TXNDC5 was increased in human and mouse atherosclerotic lesions and induced in endothelium subjected to DF. Endothelium-specific Txndc5 deletion markedly reduced atherosclerosis in ApoE-/- mice. Mechanistically, DF-induced TXNDC5 increases proteasome-mediated degradation of heat shock factor 1, leading to reduced heat shock protein 90 and accelerated eNOS (endothelial nitric oxide synthase) protein degradation. Moreover, nanoparticles formulated to deliver Txndc5-targeting CRISPR-Cas9 plasmids driven by an endothelium-specific promoter (CDH5) significantly increase eNOS protein and reduce atherosclerosis in ApoE-/- mice. These results delineate a new molecular paradigm that DF-induced endothelial TXNDC5 promotes atherosclerosis and establish a proof of concept of targeting endothelial mechanosensitive pathways in vivo against atherosclerosis.

Single-cell metabolic imaging reveals a SLC2A3-dependent glycolytic burst in motile endothelial cells.

Single-cell motility is spatially heterogeneous and driven by metabolic energy. Directly linking cell motility to cell metabolism is technically challenging but biologically important. Here, we use single-cell metabolic imaging to measure glycolysis in individual endothelial cells with genetically encoded biosensors capable of deciphering metabolic heterogeneity at subcellular resolution. We show that cellular glycolysis fuels endothelial activation, migration and contraction and that sites of high lactate production colocalize with active cytoskeletal remodelling within an endothelial cell. Mechanistically, RhoA induces endothelial glycolysis for the phosphorylation of cofilin and myosin light chain in order to reorganize the cytoskeleton and thus control cell motility; RhoA activation triggers a glycolytic burst through the translocation of the glucose transporter SLC2A3/GLUT3 to fuel the cellular contractile machinery, as demonstrated across multiple endothelial cell types. Our data indicate that Rho-GTPase signalling coordinates energy metabolism with cytoskeleton remodelling to regulate endothelial cell motility.

oxLDL-induced decrease in lipid order of membrane domains is inversely correlated with endothelial stiffness and network formation.

Oxidized low-density lipoprotein (oxLDL) is a major factor in development of atherosclerosis. Our earlier studies have shown that exposure of endothelial cells (EC) to oxLDL increases EC stiffness, facilitates the ability of the cells to generate force, and facilitates EC network formation in three-dimensional collagen gels. In this study, we show that oxLDL induces a decrease in lipid order of membrane domains and that this effect is inversely correlated with endothelial stiffness, contractility, and network formation. Local lipid packing of cell membrane domains was assessed by Laurdan two-photon imaging, endothelial stiffness was assessed by measuring cellular elastic modulus using atomic force microscopy, cell contractility was estimated by measuring the ability of the cells to contract collagen gels, and EC angiogenic potential was estimated by visualizing endothelial networks within the same gels. The impact of oxLDL on endothelial biomechanics and network formation is fully reversed by supplying the cells with a surplus of cholesterol. Furthermore, exposing the cells to 7-keto-cholesterol, a major oxysterol component of oxLDL, or to another cholesterol analog, androstenol, also results in disruption of lipid order of membrane domains and an increase in cell stiffness. On the basis of these observations, we suggest that disruption of lipid packing of cholesterol-rich membrane domains plays a key role in oxLDL-induced changes in endothelial biomechanics.

Fibroblast-enriched endoplasmic reticulum protein TXNDC5 promotes pulmonary fibrosis by augmenting TGFß signaling through TGFBR1 stabilization.

Pulmonary fibrosis (PF) is a major public health problem with limited therapeutic options. There is a clear need to identify novel mediators of PF to develop effective therapeutics. Here we show that an ER protein disulfide isomerase, thioredoxin domain containing 5 (TXNDC5), is highly upregulated in the lung tissues from both patients with idiopathic pulmonary fibrosis and a mouse model of bleomycin (BLM)-induced PF. Global deletion of Txndc5 markedly reduces the extent of PF and preserves lung function in mice following BLM treatment. Mechanistic investigations demonstrate that TXNDC5 promotes fibrogenesis by enhancing TGFß1 signaling through direct binding with and stabilization of TGFBR1 in lung fibroblasts. Moreover, TGFß1 stimulation is shown to upregulate TXNDC5 via ER stress/ATF6-dependent transcriptional control in lung fibroblasts. Inducing fibroblast-specific deletion of Txndc5 mitigates the progression of BLM-induced PF and lung function deterioration. Targeting TXNDC5, therefore, could be a novel therapeutic approach against PF.

Thy-1 dependent uptake of mesenchymal stem cell-derived extracellular vesicles blocks myofibroblastic differentiation.

Bone marrow-derived mesenchymal stem cells (MSC) have been promoted for multiple therapeutic applications. Many beneficial effects of MSCs are paracrine, dependent on extracellular vesicles (EVs). Although MSC-derived EVs (mEVs) are beneficial for acute lung injury and pulmonary fibrosis, mechanisms of mEV uptake by lung fibroblasts and their effects on myofibroblastic differentiation have not been established. We demonstrate that mEVs, but not fibroblast EVs (fEVs), suppress TGFß1-induced myofibroblastic differentiation of normal and idiopathic pulmonary fibrosis (IPF) lung fibroblasts. MEVs display increased time- and dose-dependent cellular uptake compared to fEVs. Removal or blocking of Thy-1, or blocking Thy-1-beta integrin interactions, decreased mEV uptake and prevented suppression of myofibroblastic differentiation. MicroRNAs (miRs) 199a/b-3p, 21-5p, 630, 22-3p, 196a-5p, 199b-5p, 34a-5p and 148a-3p are selectively packaged in mEVs. In silico analyses indicated that IPF lung fibroblasts have increased expression of genes that are targets of mEV-enriched miRs. MiR-630 mimics blocked TGFß1 induction of CDH2 in normal and IPF fibroblasts, and antagomiR-630 abrogated the effect of mEV on CDH2 expression. These data suggest that the interaction of Thy-1 with beta integrins mediates mEV uptake by lung fibroblasts, which blocks myofibroblastic differentiation, and that mEVs are enriched for miRs that target profibrotic genes up-regulated in IPF fibroblasts.

Regulation of SIRT1 by oxidative stress-responsive miRNAs and a systematic approach to identify its role in the endothelium.

SIGNIFICANCE: Oxidative stress is a common denominator of various risk factors contributing to endothelial dysfunction and vascular diseases. Accumulated evidence suggests that sirtuin 1 (SIRT1) expression and/or activity is impaired by supraphysiological levels of oxidative stress, which in turn disrupts endothelial homeostasis. RECENT ADVANCES: Several microRNAs (miRNAs) are induced by oxidative stress and termed as oxidative stress-responsive miRNAs. They may play a role linking the imbalanced redox state with dysregulated SIRT1. CRITICAL ISSUES: This review summarizes recent findings on oxidative stress-responsive miRNAs and their involvement in SIRT1 regulation. Because of the unique characteristics of miRNAs, research in this new area requires an integrative approach that combines bioinformatics and experimental validation. Thus, a research strategy is discussed to identify the SIRT1-regulating miRNAs under oxidative stress and their functional outcomes in relation to endothelial dysfunction. Additionally, the miRNAs implicated in vascular diseases such as atherosclerosis and abdominal aortic aneurysms are discussed along with the translational potential and challenges of using miRNAs and its analogs as therapeutic agents. FUTURE DIRECTIONS: Although at its infancy, research on oxidative stress-responsive miRNAs and their regulation of SIRT1 may provide new insights in understanding vascular disorders. Moreover, systematic approaches integrating in silico, in vitro, and in vivo observations can be useful tools in revealing the pathways modulating endothelial biology.

Oxidized low-density lipoprotein alters the effect of matrix stiffness on the formation of endothelial networks and capillary lumens.

Abstract Formation of new blood vessels is essential for vascular repair and remodeling, and it is known that biomechanical properties of extracellular matrix play a major role in this process. Our earlier studies have also shown that exposing endothelial cells to oxidized modification of low-density lipoproteins (oxLDL) increases endothelial stiffness and facilitates their ability to form cellular networks, suggesting that it facilitates endothelial angiogenic potential. The goal of this study, therefore, was to test the interrelationship between matrix stiffness and oxLDL in the regulation of angiogenesis. Our results show that, as expected, an increase in matrix stiffness inhibited endothelial network formation and that exposure to oxLDL significantly facilitated this process. We also show, however, that oxLDL-induced facilitation of endothelial networks was observed only in stiff (3 mg/mL) but not in soft (1 mg/mL) collagen gels, resulting in blunting the effect of matrix stiffness. Also unexpectedly, we show that an increase in matrix stiffness results in a significant increase in the number of capillary lumens that are formed by single cells or pairs of cells, suggesting that while endothelial connectivity is impaired, formation of single-cell lumens is facilitated. oxLDL facilitates lumen formation, but this effect is also matrix dependent and is observed only in soft gels and not in stiff gels. Finally, an increase in both matrix stiffness and oxLDL exposure results in changes in capillary morphology, with the formation of larger capillary lumens. Overall, our study suggests that oxLDL plays an important role in formation of new capillaries and their morphology and that this effect is critically dependent on the extracellular environment's compliance, thereby underlining the importance of the interdependence of these parameters.

Impact of oxLDL on Cholesterol-Rich Membrane Rafts.

Numerous studies have demonstrated that cholesterol-rich membrane rafts play critical roles in multiple cellular functions. However, the impact of the lipoproteins on the structure, integrity and cholesterol composition of these domains is not well understood. This paper focuses on oxidized low-density lipoproteins (oxLDLs) that are strongly implicated in the development of the cardiovascular disease and whose impact on membrane cholesterol and on membrane rafts has been highly controversial. More specifically, we discuss three major criteria for the impact of oxLDL on membrane rafts: distribution of different membrane raft markers, changes in membrane cholesterol composition, and changes in lipid packing of different membrane domains. We also propose a model to reconcile the controversy regarding the relationship between oxLDL, membrane cholesterol, and the integrity of cholesterol-rich membrane domains.

Endothelial actin and cell stiffness is modulated by substrate stiffness in 2D and 3D.

There is a growing appreciation of the profound effects that passive mechanical properties, especially the stiffness of the local environment, can have on cellular functions. Many experiments are conducted in a 2D geometry (i.e., cells grown on top of substrates of varying stiffness), which is a simplification of the 3D environment often experienced by cells in vivo. To determine how matrix dimensionality might modulate the effect of matrix stiffness on actin and cell stiffness, endothelial cells were cultured on top of and within substrates of various stiffnesses. Endothelial cells were cultured within compliant (1.0-1.5mg/ml, 124+/-8 to 202+/-27Pa) and stiff (3.0mg/ml, 502+/-48Pa) type-I collagen gels. Cells elongated and formed microvascular-like networks in both sets of gels as seen in previous studies. Cells in stiffer gels exhibited more pronounced stress fibers and approximately 1.5-fold greater staining for actin. As actin is a major determinant of a cell's mechanical properties, we hypothesized that cells in stiff gels will themselves be stiffer. To test this hypothesis, cells were isolated from the gels and their stiffness was assessed using micropipette aspiration. Cells isolated from relatively compliant gels were 1.9-fold more compliant than cells isolated from relatively stiff gels (p<0.05). Similarly, cells cultured on top of 1700Pa polyacrylamide gels were 2.0-fold more compliant that those cultured on 9000Pa (p<0.05). These data demonstrate that extracellular substrate stiffness regulates endothelial stiffness in both three- and two-dimensional environments, though the range of stiffnesses that cells respond to vary significantly in different environments.