Dual pattern of cholesterol-induced decoupling of residue-residue interactions of Kir2.2.

Cholesterol is a negative regulator of a variety of ion channels. We have previously shown that cholesterol suppresses Kir2.2 channels via residue-residue uncoupling on the inter-subunit interfaces within the close state of the channels (3JYC). In this study, we extend this analysis to the other known structure of Kir2.2 that is closer to the open state of Kir2.2 channels (3SPI) and provide additional analysis of the residue distances between the uncoupled residues and cholesterol binding domains in the two conformation states of the channels. We found that the general phenomenon of cholesterol binding leading to uncoupling between specific residues is conserved in both channel states but the specific pattern of the uncoupling residues is distinct between the two states and implies different mechanisms. Specifically, we found that cholesterol binding in the 3SPI state results in an uncoupling of residues in three distinct regions; the transmembrane domain, membrane-cytosolic interface, and the cytosolic domain, with the first two regions forming an envelope around PI(4,5)P2 and cholesterol binding sites and the distal region overlapping with the subunit-subunit interface characterized in our previous study of the disengaged state. We also found that this uncoupling is dependent upon the number of cholesterol molecules bound to the channel. We further generated a mutant channel Kir2.2P187V with a single point mutation in a residue proximal to the PI(4,5)P2 binding site, which is predicted to be uncoupled from other residues in its vicinity upon cholesterol binding and found that this mutation abrogates the sensitivity of Kir2.2 to cholesterol changes in the membrane. These findings suggest that cholesterol binding to this conformation state of Kir2.2 channels may destabilize the PI(4,5)P2 interactions with the channels while in the disengaged state the destabilization occurs where the subunits interact. These findings give insight into the structural mechanistic basis for the functional effects of cholesterol binding to the Kir2.2 channel.

Cholesterol regulation of mechanosensitive ion channels.

The purpose of this review is to evaluate the role of cholesterol in regulating mechanosensitive ion channels. Ion channels discussed in this review are sensitive to two types of mechanical signals, fluid shear stress and/or membrane stretch. Cholesterol regulates the channels primarily in two ways: 1) indirectly through localizing the channels into cholesterol-rich membrane domains where they interact with accessory proteins and/or 2) direct binding of cholesterol to the channel at specified putative binding sites. Cholesterol may also regulate channel function via changes of the biophysical properties of the membrane bilayer. Changes in cholesterol affect both mechanosensitivity and basal channel function. We focus on four mechanosensitive ion channels in this review Piezo, Kir2, TRPV4, and VRAC channels. Piezo channels were shown to be regulated by auxiliary proteins that enhance channel function in high cholesterol domains. The direct binding mechanism was shown in Kir2.1 and TRPV4 where cholesterol inhibits channel function. Finally, cholesterol regulation of VRAC was attributed to changes in the physical properties of lipid bilayer. Additional studies should be performed to determine the physiological implications of these sterol effects in complex cellular environments.

Erratum: Piezo1 induces endothelial responses to shear stress via soluble adenylyl Cyclase-IP3R2 circuit.

[This corrects the article DOI: 10.1016/j.isci.2023.106661.].

Anoctamin-1 is induced by TGF-ß and contributes to lung myofibroblast differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-ß (TGF-ß) is one of the most established drivers of fibrotic processes. TGF-ß promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-ß robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle α-actin (SMA)-positive myofibroblasts. TGF-ß-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-ß signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-ß treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-ß-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-ß-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-ß; and 2) ANO1 mediates TGF-ß-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity.NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-ß (TGF-ß) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

Membrane Cholesterol Interactions with Proteins in Hypercholesterolemia-Induced Endothelial Dysfunction.

PURPOSE OF REVIEW: The goal of this review is to highlight work identifying mechanisms driving hypercholesterolemia-mediated endothelial dysfunction. We specifically focus on cholesterol-protein interactions and address specific questions related to the impact of hypercholesterolemia on cellular cholesterol and vascular endothelial function. We describe key approaches used to determine the effects of cholesterol-protein interactions in mediating endothelial dysfunction under dyslipidemic conditions. RECENT FINDINGS: The benefits of removing the cholesterol surplus on endothelial function in models of hypercholesterolemia is clear. However, specific mechanisms driving cholesterol-induced endothelial dysfunction need to be determined. In this review, we detail the latest findings describing cholesterol-mediated endothelial dysfunction, highlighting our studies indicating that cholesterol suppresses endothelial Kir2.1 channels as a major underlying mechanism. The findings detailed in this review support the targeting of cholesterol-induced suppression of proteins in restoring endothelial function in dyslipidemic conditions. The identification of similar mechanisms regarding other cholesterol-endothelial protein interactions is warranted.

Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-beta (TGF-ß) is one of the most established drivers of fibrotic processes. TGF-ß promotes transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-ß robustly upregulates the expression of the calcium-activated chloride channel Anoctamin-1 (ANO1) in human lung fibroblasts (HLF) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle alpha-actin (SMA)-positive myofibroblasts. TGF-ß-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1 and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate pro-fibrotic TGF-ß signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-ß treatment of HLF results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-ß-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that (i) ANO1 is a TGF-ß-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-ß; and (ii) ANO1 mediates TGF-ß-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein, but independent of WNK1 kinase activity.

Piezo1 induces endothelial responses to shear stress via soluble adenylyl Cyclase-IP3R2 circuit.

Endothelial cells (ECs) continuously sense and adapt to changes in shear stress generated by blood flow. Here, we show that the activation of the mechanosensitive channel Piezo1 by defined shear forces induces Ca2+ entry into the endoplasmic reticulum (ER) via the ER Ca2+ ATPase pump. This entry is followed by inositol trisphosphate receptor 2 (IP3R2)-elicited ER Ca2+ release into the cytosol. The mechanism of ER Ca2+ release involves the generation of cAMP by soluble adenylyl cyclase (sAC), leading to IP3R2-evoked Ca2+ gating. Depleting sAC or IP3R2 prevents ER Ca2+ release and blocks EC alignment in the direction of flow. Overexpression of constitutively active Akt1 restores the shear-induced alignment of ECs lacking Piezo1 or IP3R2, as well as the flow-induced vasodilation in endothelial restricted Piezo1 knockout mice. These studies describe an unknown Piezo1-cAMP-IP3R2 circuit as an essential mechanism activating Akt signaling and inducing adaptive changes in ECs to laminar flow.

Impairment of endothelial glycocalyx in atherosclerosis and obesity.

Endothelial glycocalyx is a negatively charged gel-like layer located on the apical surface of endothelial cells. It serves as a selective two-way physical barrier between the flowing blood and the endothelium, which regulates the access of macromolecules and of blood cells to the endothelial surface. In addition, endothelial glycocalyx plays a major role in sensing mechanical signals generated by the blood flow and transducing these signals to maintain endothelial functions; Thus, dysfunction or disruption of endothelial glycocalyx in pathological condition leads to endothelial dysfunction and contributes to the development of vascular diseases. In this review, we discuss the impact of atherosclerosis with the following viewpoints: (i) hypercholesterolemic effects on endothelial glycocalyx degradation in animal models and human patients, (ii) disruption of endothelial glycocalyx by atherogenic lipoproteins, (iii) proatherogenic disturbed flow effects on endothelial glycocalyx degradation, (iv) pathological consequences of the loss of glycocalyx integrity in atherogenesis, and (v) therapeutic effect of glycocalyx supplementation on atherosclerosis development. Additionally, we also discuss recent studies in pathological effects of obesity on the disruption of endothelial glycocalyx.

Caveolin-1 is a primary determinant of endothelial stiffening associated with dyslipidemia, disturbed flow, and ageing.

Endothelial stiffness is emerging as a major determinant in endothelial function. Here, we analyzed the role of caveolin-1 (Cav-1) in determining the stiffness of endothelial cells (EC) exposed to oxidized low density lipoprotein (oxLDL) under static and hemodynamic conditions in vitro and of aortic endothelium in vivo in mouse models of dyslipidemia and ageing. Elastic moduli of cultured ECs and of the endothelial monolayer of freshly isolated mouse aortas were measured using atomic force microscopy (AFM). We found that a loss of Cav-1 abrogates the uptake of oxLDL and oxLDL-induced endothelial stiffening, as well as endothelial stiffening induced by disturbed flow (DF), which was also oxLDL dependent. Mechanistically, Cav-1 is required for the expression of CD36 (cluster of differentiation 36) scavenger receptor. Genetic deletion of Cav-1 abrogated endothelial stiffening observed in the DF region of the aortic arch, and induced by a high fat diet (4-6 weeks) and significantly blunted endothelial stiffening that develops with advanced age. This effect was independent of stiffening of the sub-endothelium layer. Additionally, Cav-1 expression significantly increased with age. No differences in elastic modulus were observed between the sexes in advanced aged wild type and Cav-1 knockout mice. Taken together, this study demonstrates that Cav-1 plays a critical role in endothelial stiffening induced by oxLDL in vitro and by dyslipidemia, disturbed flow and ageing in vivo.

Cholesterol-induced suppression of Kir2 channels is mediated by decoupling at the inter-subunit interfaces.

Cholesterol is a major regulator of multiple types of ion channels. Although there is increasing information about cholesterol binding sites, the molecular mechanisms through which cholesterol binding alters channel function are virtually unknown. In this study, we used a combination of Martini coarse-grained simulations, a network theory-based analysis, and electrophysiology to determine the effect of cholesterol on the dynamic structure of the Kir2.2 channel. We found that increasing membrane cholesterol reduced the likelihood of contact between specific regions of the cytoplasmic and transmembrane domains of the channel, most prominently at the subunit-subunit interfaces of the cytosolic domains. This decrease in contact was mediated by pairwise interactions of specific residues and correlated to the stoichiometry of cholesterol binding events. The predictions of the model were tested by site-directed mutagenesis of two identified residues-V265 and H222-and high throughput electrophysiology.

OxLDL induces membrane structure rearrangement leading to biomechanics alteration and migration deficiency in macrophage.

Cholesterol sequestration from plasma membrane has been shown to induce lipid packing disruption, causing actin cytoskeleton reorganization and polymerization, increasing cell stiffness and inducing lysosomal exocytosis in non-professional phagocytes. Similarly, oxidized form of low-density lipoprotein (oxLDL) has also been shown to disrupt lipid organization and packing in endothelial cells, leading to biomechanics alterations that interfere with membrane injury and repair. For macrophages, much is known about oxLDL effects in cell activation, cytokine production and foam cell formation. However, little is known about its impact in the organization of macrophage membrane structured domains and cellular mechanics, the focus of the present study. Treatment of bone marrow-derived macrophages (BMDM) with oxLDL not only altered membrane structure, and potentially the distribution of raft domains, but also induced actin rearrangement, diffuse integrin distribution and cell shrinkage, similarly to observed upon treatment of these cells with MßCD. Those alterations led to decreased migration efficiency. For both treatments, higher co-localization of actin cytoskeleton and GM1 was observed, indicating a similar mechanism of action involving raft-like domain dynamics. Lastly, like MßCD treatment, oxLDL also induced lysosomal spreading in BMDM. We propose that OxLDL induced re-organization of membrane/cytoskeleton complex in macrophages can be attributed to the insertion of oxysterols into the membrane, which lead to changes in lipid organization and disruption of membrane structure, similar to the effect of cholesterol depletion by MßCD treatment. These results indicate that oxLDL can induce physical alterations in the complex membrane/cytoskeleton of macrophages, leading to significant biomechanical changes that compromise cell behavior.

Effective Parameters Controlling Sterol Transfer: A Time-Resolved Small-Angle Neutron Scattering Study.

Though cholesterol is the most prevalent and essential sterol in mammalian cellular membranes, its precursors, post-synthesis cholesterol products, as well as its oxidized derivatives play many other important physiological roles. Using a non-invasive in situ technique, time-resolved small angle neutron scattering, we report on the rate of membrane desorption and corresponding activation energy for this process for a series of sterol precursors and post-synthesis cholesterol products that vary from cholesterol by the number and position of double bonds in B ring of cholesterol's steroid core. In addition, we report on sterols that have oxidation modifications in ring A and ring B of the steroid core. We find that sterols that differ in position or the number of double bonds in ring B have similar time and energy characteristics, while oxysterols have faster transfer rates and lower activation energies than cholesterol in a manner generally consistent with known sterol characteristics, like Log P, the n-octanol/water partitioning coefficient. We find, however, that membrane/water partitioning which is dependent on lipid-sterol interactions is a better predictor, shown by the correlation of the sterols' tilt modulus with both the desorption rates and activation energy.

Differential effects of obesity on visceral versus subcutaneous adipose arteries: role of shear-activated Kir2.1 and alterations to the glycocalyx.

Obesity imposes well-established deficits to endothelial function. We recently showed that obesity-induced endothelial dysfunction was mediated by disruption of the glycocalyx and a loss of Kir channel flow sensitivity. However, obesity-induced endothelial dysfunction is not observed in all vascular beds: visceral adipose arteries (VAAs), but not subcutaneous adipose arteries (SAAs), exhibit endothelial dysfunction. To determine whether differences in SAA versus VAA endothelial function observed in obesity are attributed to differential impairment of Kir channels and alterations to the glycocalyx, mice were fed a normal rodent diet, or a high-fat Western diet to induce obesity. Flow-induced vasodilation (FIV) was measured ex vivo. Functional downregulation of endothelial Kir2.1 was accomplished by transducing adipose arteries from mice and obese humans with adenovirus containing a dominant-negative Kir2.1 construct. Kir function was tested in freshly isolated endothelial cells seeded in a flow chamber for electrophysiological recordings under fluid shear. Atomic force microscopy was used to assess biophysical properties of the glycocalyx. Endothelial dysfunction was observed in VAAs of obese mice and humans. Downregulating Kir2.1 blunted FIV in SAAs, but had no effect on VAAs, from obese mice and humans. Obesity abolished Kir shear sensitivity in VAA endothelial cells and significantly altered the VAA glycocalyx. In contrast, Kir shear sensitivity was observed in SAA endothelial cells from obese mice and effects on SAA glycocalyx were less pronounced. We reveal distinct differences in Kir function and alterations to the glycocalyx that we propose contribute to the dichotomy in SAA versus VAA endothelial function with obesity.NEW & NOTEWORTHY We identified a role for endothelial Kir2.1 in the differences observed in VAA versus SAA endothelial function with obesity. The endothelial glycocalyx, a regulator of Kir activation by shear, is unequally perturbed in VAAs as compared with SAAs, which we propose results in a near complete loss of VAA endothelial Kir shear sensitivity and endothelial dysfunction. We propose that these differences underly the preserved endothelial function of SAA in obese mice and humans.

Cholesterol-Induced Suppression of Endothelial Kir Channels Is a Driver of Impairment of Arteriolar Flow-Induced Vasodilation in Humans.

Dyslipidemia-induced endothelial dysfunction is an important factor in the progression of cardiovascular disease; however, the underlying mechanisms are unclear. Our recent studies demonstrated that flow-induced vasodilation (FIV) is regulated by inwardly rectifying K+ channels (Kir2.1) in resistance arteries. Furthermore, we showed that hypercholesterolemia inhibits Kir2.1-dependent vasodilation. In this study, we introduced 2 new mouse models: (1) endothelial-specific deletion of Kir2.1 to demonstrate the role of endothelial Kir2.1 in FIV and (2) cholesterol-insensitive Kir2.1 mutant to determine the Kir2.1 regulation in FIV under hypercholesterolemia. FIV was significantly reduced in endothelial-specific Kir2.1 knock-out mouse mesenteric arteries compared with control groups. In cholesterol-insensitive Kir2.1 mutant mice, Kir2.1 currents were not affected by cyclodextrin and FIV was restored in cells and arteries, respectively, with a hypercholesterolemic background. To extend our observations to humans, 16 healthy subjects were recruited with LDL (low-density lipoprotein)-cholesterol ranging from 51 to 153 mg/dL and FIV was assessed in resistance arteries isolated from gluteal adipose. Resistance arteries from participants with >100 mg/dL LDL (high-LDL) exhibited reduced FIV as compared with those participants with <100 mg/dL LDL (low-LDL). A significant negative correlation was observed between LDL cholesterol and FIV in high-LDL. Expressing dominant-negative Kir2.1 in endothelium blunted FIV in arteries from low-LDL but had no further effect on FIV in arteries from high-LDL. The Kir2.1-dependent vasodilation more negatively correlated to LDL cholesterol in high-LDL. Overexpressing wild-type Kir2.1 in endothelium fully recovered FIV in arteries from participants with high-LDL. Our data suggest that cholesterol-induced suppression of Kir2.1 is a major mechanism underlying endothelial dysfunction in hypercholesterolemia.

Paradigms of endothelial stiffening in cardiovascular disease and vascular aging.

Endothelial cells, the inner lining of the blood vessels, are well-known to play a critical role in vascular function, while endothelial dysfunction due to different cardiovascular risk factors or accumulation of disruptive mechanisms that arise with aging lead to cardiovascular disease. In this review, we focus on endothelial stiffness, a fundamental biomechanical property that reflects cell resistance to deformation. In the first part of the review, we describe the mechanisms that determine endothelial stiffness, including RhoA-dependent contractile response, actin architecture and crosslinking, as well as the contributions of the intermediate filaments, vimentin and lamin. Then, we review the factors that induce endothelial stiffening, with the emphasis on mechanical signals, such as fluid shear stress, stretch and stiffness of the extracellular matrix, which are well-known to control endothelial biomechanics. We also describe in detail the contribution of lipid factors, particularly oxidized lipids, that were also shown to be crucial in regulation of endothelial stiffness. Furthermore, we discuss the relative contributions of these two mechanisms of endothelial stiffening in vasculature in cardiovascular disease and aging. Finally, we present the current state of knowledge about the role of endothelial stiffening in the disruption of endothelial cell-cell junctions that are responsible for the maintenance of the endothelial barrier.

Evaluating membrane structure by Laurdan imaging: Disruption of lipid packing by oxidized lipids.

Impact of different lipids on membrane structure/lipid order is critical for multiple biological processes. Laurdan microscopy provides a unique tool to assess this property in heterogeneous biological membranes. This review describes the general principles of the approach and its application in model membranes and cells. It also provides an in-depth discussion of the insights obtained using Laurdan microscopy to evaluate the differential effects of cholesterol, oxysterols and oxidized phospholipids on lipid packing of ordered and disordered domains in vascular endothelial cells.

Lipoprotein-Induced Increases in Cholesterol and 7-Ketocholesterol Result in Opposite Molecular-Scale Biophysical Effects on Membrane Structure.

Under hypercholesterolemic conditions, exposure of cells to lipoproteins results in a subtle membrane increase in the levels of cholesterol and 7-ketocholesterol, as compared to normal conditions. The effect of these physiologically relevant concentration increases on multicomponent bilayer membranes was investigated using coarse-grained molecular dynamics simulations. Significant changes in the structural and dynamic properties of the bilayer membranes resulted from these subtle increases in sterol levels, with both sterol species inducing decreases in the lateral area and inhibiting lateral diffusion to varying extents. Cholesterol and 7-ketocholesterol, however, exhibited opposite effects on lipid packing and orientation. The results from this study indicate that the subtle increases in membrane sterol levels induced by exposure to lipoproteins result in molecular-scale biophysical perturbation of membrane structure.

Noncanonical function of long myosin light chain kinase in increasing ER-PM junctions and augmentation of SOCE.

Increased endothelial permeability leads to excessive exudation of plasma proteins and leukocytes in the interstitium, which characterizes several vascular diseases including acute lung injury. The myosin light chain kinase long (MYLK-L) isoform is canonically known to regulate the endothelial permeability by phosphorylating myosin light chain (MLC-P). Compared to the short MYLK isoform, MYLK-L contains an additional stretch of ~919 amino acid at the N-terminus of unknown function. We show that thapsigargin and thrombin-induced SOCE was markedly reduced in Mylk-L-/- endothelial cells (EC) or MYLK-L-depleted human EC. These agonists also failed to increase endothelial permeability in MYLK-L-depleted EC and Mylk-L-/- lungs, thus demonstrating the novel role of MYLK-L-induced SOCE in increasing vascular permeability. MYLK-L augmented SOCE by increasing endoplasmic reticulum (ER)-plasma membrane (PM) junctions and STIM1 translocation to these junctions. Transduction of N-MYLK domain (amino acids 1-919 devoid of catalytic activity) into Mylk-L-/- EC rescued SOCE to the level seen in control EC in a STIM1-dependent manner. N-MYLK-induced SOCE augmented endothelial permeability without MLC-P via an actin-binding motif, DVRGLL. Liposomal-mediated delivery of N-MYLK mutant but not ∆DVRGLL-N-MYLK mutant in Mylk-L-/- mice rescued vascular permeability increase in response to endotoxin, indicating that targeting of DVRGLL motif within MYLK-L may limit SOCE-induced vascular hyperpermeability.