Isoprenylcysteine Carboxylmethyltransferase-Based Therapy for Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS, progeria) is a rare genetic disease characterized by premature aging and death in childhood for which there were no approved drugs for its treatment until last November, when lonafarnib obtained long-sought FDA approval. However, the benefits of lonafarnib in patients are limited, highlighting the need for new therapeutic strategies. Here, we validate the enzyme isoprenylcysteine carboxylmethyltransferase (ICMT) as a new therapeutic target for progeria with the development of a new series of potent inhibitors of this enzyme that exhibit an excellent antiprogeroid profile. Among them, compound UCM-13207 significantly improved the main hallmarks of progeria. Specifically, treatment of fibroblasts from progeroid mice with UCM-13207 delocalized progerin from the nuclear membrane, diminished its total protein levels, resulting in decreased DNA damage, and increased cellular viability. Importantly, these effects were also observed in patient-derived cells. Using the Lmna G609G/G609G progeroid mouse model, UCM-13207 showed an excellent in vivo efficacy by increasing body weight, enhancing grip strength, extending lifespan by 20%, and decreasing tissue senescence in multiple organs. Furthermore, UCM-13207 treatment led to an improvement of key cardiovascular hallmarks such as reduced progerin levels in aortic and endocardial tissue and increased number of vascular smooth muscle cells (VSMCs). The beneficial effects go well beyond the effects induced by other therapeutic strategies previously reported in the field, thus supporting the use of UCM-13207 as a new treatment for progeria.

Small-Molecule Therapeutic Perspectives for the Treatment of Progeria.

Hutchinson-Gilford progeria syndrome (HGPS), or progeria, is an extremely rare disorder that belongs to the class of laminopathies, diseases characterized by alterations in the genes that encode for the lamin proteins or for their associated interacting proteins. In particular, progeria is caused by a point mutation in the gene that codifies for the lamin A gene. This mutation ultimately leads to the biosynthesis of a mutated version of lamin A called progerin, which accumulates abnormally in the nuclear lamina. This accumulation elicits several alterations at the nuclear, cellular, and tissue levels that are phenotypically reflected in a systemic disorder with important alterations, mainly in the cardiovascular system, bones, skin, and overall growth, which results in premature death at an average age of 14.5 years. In 2020, lonafarnib became the first (and only) FDA approved drug for treating progeria. In this context, the present review focuses on the different therapeutic strategies currently under development, with special attention to the new small molecules described in recent years, which may represent the upcoming first-in-class drugs with new mechanisms of action endowed with effectiveness not only to treat but also to cure progeria.

A Potent Isoprenylcysteine Carboxylmethyltransferase (ICMT) Inhibitor Improves Survival in Ras-Driven Acute Myeloid Leukemia.

Blockade of Ras activity by inhibiting its post-translational methylation catalyzed by isoprenylcysteine carboxylmethyltransferase (ICMT) has been suggested as a promising antitumor strategy. However, the paucity of inhibitors has precluded the clinical validation of this approach. In this work we report a potent ICMT inhibitor, compound 3 [UCM-1336, IC50 = 2 µM], which is selective against the other enzymes involved in the post-translational modifications of Ras. Compound 3 significantly impairs the membrane association of the four Ras isoforms, leading to a decrease of Ras activity and to inhibition of Ras downstream signaling pathways. In addition, it induces cell death in a variety of Ras-mutated tumor cell lines and increases survival in an in vivo model of acute myeloid leukemia. Because ICMT inhibition impairs the activity of the four Ras isoforms regardless of its activating mutation, compound 3 surmounts many of the common limitations of available Ras inhibitors described so far. In addition, these results validate ICMT as a valuable target for the treatment of Ras-driven tumors.

Activation of Rac1 and RhoA Preserve Corneal Endothelial Barrier Function.

PURPOSE: The corneal endothelium is responsible for the correct hydration of the corneal stroma. Corneal endothelial cells have a low proliferative capacity, so preserving their barrier function under suboptimal conditions that cause osmotic imbalance, such as those arising from corneal pathologies, age, cryopreservation, and transplantation, is essential for maintaining corneal transparency. We have investigated the signaling induced by hyperosmotic shock that reversibly disrupts corneal endothelial barriers in human endothelial cells and in murine corneas. METHODS: Endothelial barrier properties were analyzed in vitro by electric cell substrate impedance sensing (ECIS) and confocal microscopy of the human endothelial cell line B4G12-HCEC, and, ex vivo, by confocal microscopy and stimulated emission-depletion (STED) super-resolution microscopy of murine corneas. Cell signaling in response to hyperosmotic stress, induced with an excess of sodium chloride, was investigated in B4G12-HCECs. Rho GTPase activity was detected by pulldown assays with recombinant GST proteins fused to the Rho binding domains of Rho effectors. RESULTS: Hyperosmotic stress increased actin polymerization and activated the Rho GTPases Rac1 and RhoA, but not Cdc42. Rac1- and RhoA-mediated pathway inhibition had a minor effect on barrier disruption but partially delayed barrier reformation after stress withdrawal. In contrast, Rac1 and RhoA activation enhanced constitutive endothelial barrier function and accelerated barrier repair. CONCLUSIONS: Our results indicate that Rac1 and RhoA activation do not mediate stress-induced cell contraction but are endothelial responses that act to restore and maintain barrier homeostasis. Therefore, pharmacological activation of these two GTPases could be a therapeutic strategy for preserving corneal endothelial barrier function.

RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border.

Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation.

Apicobasal polarity controls lymphocyte adhesion to hepatic epithelial cells.

Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1) adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α). We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery.

Microparticles in multiple sclerosis and clinically isolated syndrome: effect on endothelial barrier function.

BACKGROUND: Cell-derived microparticles are secreted in response to cell damage or dysfunction. Endothelial and platelet dysfunction are thought to contribute to the development of multiple sclerosis (MS). Our aim here is, first, to compare the presence of microparticles of endothelial and platelet origin in plasma from patients with different clinical forms of MS and with clinically isolated syndrome. Second, to investigate the effect of microparticles on endothelial barrier function. RESULTS: Platelet-poor plasma from 95 patients (12 with clinically isolated syndrome, 51 relapsing-remitting, 23 secondary progressive, 9 primary progressive) and 49 healthy controls were analyzed for the presence of platelet-derived and endothelium-derived microparticles by flow cytometry. The plasma concentration of platelet-derived and endothelium-derived microparticles increased in all clinical forms of MS and in clinically isolated syndrome versus controls. The response of endothelial barriers to purified microparticles was measured by electric cell-substrate impedance sensing. Microparticles from relapsing-remitting MS patients induced, at equivalent concentrations, a stronger disruption of endothelial barriers than those from healthy donors or from patients with clinically isolated syndrome. MS microparticles acted synergistically with the inflammatory mediator thrombin to disrupt the endothelial barrier function. CONCLUSIONS: Plasma microparticles should be considered not only as markers of early stages of MS, but also as pathological factors with the potential to increase endothelial permeability and leukocyte infiltration.

MYADM controls endothelial barrier function through ERM-dependent regulation of ICAM-1 expression.

The endothelium maintains a barrier between blood and tissue that becomes more permeable during inflammation. Membrane rafts are ordered assemblies of cholesterol, glycolipids, and proteins that modulate proinflammatory cell signaling and barrier function. In epithelial cells, the MAL family members MAL, MAL2, and myeloid-associated differentiation marker (MYADM) regulate the function and dynamics of ordered membrane domains. We analyzed the expression of these three proteins in human endothelial cells and found that only MYADM is expressed. MYADM was confined in ordered domains at the plasma membrane, where it partially colocalized with filamentous actin and cell-cell junctions. Small interfering RNA (siRNA)-mediated MYADM knockdown increased permeability, ICAM-1 expression, and leukocyte adhesion, all of which are features of an inflammatory response. Barrier function decrease in MYADM-silenced cells was dependent on ICAM-1 expression. Membrane domains and the underlying actin cytoskeleton can regulate each other and are connected by ezrin, radixin, and moesin (ERM) proteins. In endothelial cells, MYADM knockdown induced ERM activation. Triple-ERM knockdown partially inhibited ICAM-1 increase induced by MYADM siRNA. Importantly, ERM knockdown also reduced ICAM-1 expression in response to the proinflammatory cytokine tumor necrosis factor-α. MYADM therefore regulates the connection between the plasma membrane and the cortical cytoskeleton and so can control the endothelial inflammatory response.

Crosstalk between reticular adherens junctions and platelet endothelial cell adhesion molecule-1 regulates endothelial barrier function.

OBJECTIVE: Endothelial cells provide a barrier between the blood and tissues, which is reduced during inflammation to allow selective passage of molecules and cells. Adherens junctions (AJ) play a central role in regulating this barrier. We aim to investigate the role of a distinctive 3-dimensional reticular network of AJ found in the endothelium. METHODS AND RESULTS: In endothelial AJ, vascular endothelial-cadherin recruits the cytoplasmic proteins ß-catenin and p120-catenin. ß-catenin binds to α-catenin, which links AJ to actin filaments. AJ are usually described as linear structures along the actin-rich intercellular contacts. Here, we show that these AJ components can also be organized in reticular domains that contain low levels of actin. Reticular AJ are localized in areas where neighboring cells overlap and encompass the cell adhesion receptor platelet endothelial cell adhesion molecule-1 (PECAM-1). Superresolution microscopy revealed that PECAM-1 forms discrete structures distinct from and distributed along AJ, within the voids of reticular domains. Inflammatory tumor necrosis factor-α increases permeability by mechanisms that are independent of actomyosin-mediated tension and remain incompletely understood. Reticular AJ, but not actin-rich linear AJ, were disorganized by tumor necrosis factor-α. This correlated with PECAM-1 dispersal from cell borders. PECAM-1 inhibition with blocking antibodies or small interfering RNA specifically disrupted reticular AJ, leaving linear AJ intact. This disruption recapitulated typical tumor necrosis factor-α-induced alterations of barrier function, including increased ß-catenin phosphorylation, without altering the actomyosin cytoskeleton. CONCLUSIONS: We propose that reticular AJ act coordinately with PECAM-1 to maintain endothelial barrier function in regions of low actomyosin-mediated tension. Selective disruption of reticular AJ contributes to permeability increase in response to tumor necrosis factor-α.

Endothelial membrane reorganization during leukocyte extravasation.

Leukocyte trafficking from the bloodstream to inflamed tissues across the endothelial barrier is an essential response in innate immunity. Leukocyte adhesion, locomotion, and diapedesis induce signaling in endothelial cells and this is accompanied by a profound reorganization of the endothelial cell surfaces that is only starting to be unveiled. Here we review the current knowledge on the leukocyte-mediated alterations of endothelial membrane dynamics and their role in promoting leukocyte extravasation. The formation of protein- and lipid-mediated cell adhesion nanodomains at the endothelial apical surface, the extension of micrometric apical membrane docking structures, which are derived from microvilli and embrace adhered leukocytes, as well as the vesicle-trafficking pathways that are required for efficient leukocyte diapedesis, are discussed. The coordination between these different endothelial membrane-remodeling events probably provides the road map for transmigrating leukocytes to find exit points in the vessel wall, in a context of severe mechanical and inflammatory stress. A better understanding of how vascular endothelial cells respond to immune cell adhesion should enable new therapeutic strategies to be developed that can abrogate uncontrolled leukocyte extravasation in inflammatory diseases.

MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration.

Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes.

Adherens junctions connect stress fibres between adjacent endothelial cells.

BACKGROUND: Endothelial cell-cell junctions maintain endothelial integrity and regulate vascular morphogenesis and homeostasis. Cell-cell junctions are usually depicted with a linear morphology along the boundaries between adjacent cells and in contact with cortical F-actin. However, in the endothelium, cell-cell junctions are highly dynamic and morphologically heterogeneous. RESULTS: We report that endothelial cell-cell junctions can attach to the ends of stress fibres instead of to cortical F-actin, forming structures that we name discontinuous adherens junctions (AJ). Discontinuous AJ are highly dynamic and are increased in response to tumour necrosis factor (TNF)-alpha, correlating with the appearance of stress fibres. We show that vascular endothelial (VE)-cadherin/beta-catenin/alpha-catenin complexes in discontinuous AJ are linked to stress fibres. Moreover, discontinuous AJ connect stress fibres from adjacent cells independently of focal adhesions, of which there are very few in confluent endothelial cells, even in TNF-alpha-stimulated cells. RNAi-mediated knockdown of VE-cadherin, but not zonula occludens-1, reduces the linkage of stress fibres to cell-cell junctions, increases focal adhesions, and dramatically alters the distribution of these actin cables in confluent endothelial cells. CONCLUSIONS: Our results indicate that stress fibres from neighbouring cells are physically connected through discontinuous AJ, and that stress fibres can be stabilized by AJ-associated multi-protein complexes distinct from focal adhesions.