Coordination between cytoskeletal organization, cell contraction and extracellular matrix development, is depended on LOX for aneurysm prevention.

Distinct, seemingly independent, cellular pathways affecting intracellular machineries or extracellular matrix (ECM) deposition and organization, have been implicated in aneurysm formation. One of the key genes associated with the pathology in both humans and mice is Lysyl oxidase (LOX), a secreted ECM-modifying enzyme, highly expressed in medial vascular smooth muscle cells. To dissect the mechanisms leading to aneurysm development, we conditionally deleted Lox in smooth muscle cells. We find that cytoskeletal organization is lost following Lox deletion. Cell culture assays and in vivo analyses demonstrate a cell-autonomous role for LOX affecting myosin light chain phosphorylation and cytoskeletal assembly resulting in irregular smooth muscle contraction. These results not only highlight new intracellular roles for LOX, but notably they link between multiple processes leading to aneurysm formation suggesting LOX coordinates ECM development, cytoskeletal organization and cell contraction required for media development and function.

Brain-Targeted Liposomes Loaded with Monoclonal Antibodies Reduce Alpha-Synuclein Aggregation and Improve Behavioral Symptoms in Parkinson's Disease.

Monoclonal antibodies (mAbs) hold promise in treating Parkinson's disease (PD), although poor delivery to the brain hinders their therapeutic application. In the current study, it is demonstrated that brain-targeted liposomes (BTL) enhance the delivery of mAbs across the blood-brain-barrier (BBB) and into neurons, thereby allowing the intracellular and extracellular treatment of the PD brain. BTL are decorated with transferrin to improve brain targeting through overexpressed transferrin-receptors on the BBB during PD. BTL are loaded with SynO4, a mAb that inhibits alpha-synuclein (AS) aggregation, a pathological hallmark of PD. It is shown that 100-nm BTL cross human BBB models intact and are taken up by primary neurons. Within neurons, SynO4 is released from the nanoparticles and bound to its target, thereby reducing AS aggregation, and enhancing neuronal viability. In vivo, intravenous BTL administration results in a sevenfold increase in mAbs in brain cells, decreasing AS aggregation and neuroinflammation. Treatment with BTL also improve behavioral motor function and learning ability in mice, with a favorable safety profile. Accordingly, targeted nanotechnologies offer a valuable platform for drug delivery to treat brain neurodegeneration.

Initiation of fibronectin fibrillogenesis is an enzyme-dependent process.

Fibronectin fibrillogenesis and mechanosensing both depend on integrin-mediated force transmission to the extracellular matrix. However, force transmission is in itself dependent on fibrillogenesis, and fibronectin fibrils are found in soft embryos where high forces cannot be applied, suggesting that force cannot be the sole initiator of fibrillogenesis. Here, we identify a nucleation step prior to force transmission, driven by fibronectin oxidation mediated by lysyl oxidase enzyme family members. This oxidation induces fibronectin clustering, which promotes early adhesion, alters cellular response to soft matrices, and enhances force transmission to the matrix. In contrast, absence of fibronectin oxidation abrogates fibrillogenesis, perturbs cell-matrix adhesion, and compromises mechanosensation. Moreover, fibronectin oxidation promotes cancer cell colony formation in soft agar as well as collective and single-cell migration. These results reveal a force-independent enzyme-dependent mechanism that initiates fibronectin fibrillogenesis, establishing a critical step in cell adhesion and mechanosensing.

The biophysical property of the limbal niche maintains stemness through YAP.

The cell fate decisions of stem cells (SCs) largely depend on signals from their microenvironment (niche). However, very little is known about how biochemical niche cues control cell behavior in vivo. To address this question, we focused on the corneal epithelial SC model in which the SC niche, known as the limbus, is spatially segregated from the differentiation compartment. We report that the unique biomechanical property of the limbus supports the nuclear localization and function of Yes-associated protein (YAP), a putative mediator of the mechanotransduction pathway. Perturbation of tissue stiffness or YAP activity affects SC function as well as tissue integrity under homeostasis and significantly inhibited the regeneration of the SC population following SC depletion. In vitro experiments revealed that substrates with the rigidity of the corneal differentiation compartment inhibit nuclear YAP localization and induce differentiation, a mechanism that is mediated by the TGFß-SMAD2/3 pathway. Taken together, these results indicate that SC sense biomechanical niche signals and that manipulation of mechano-sensory machinery or its downstream biochemical output may bear fruits in SC expansion for regenerative therapy.

Lysyl Oxidases: Orchestrators of Cellular Behavior and ECM Remodeling and Homeostasis.

Lysyl oxidases have long been considered key secreted extracellular matrix modifying enzymes. As such, their activity has been associated with the crosslinking of collagens and elastin, and as a result, they have been linked to multiple developmental and pathological processes. However, numerous lines of evidence also demonstrated that members of this enzyme family are localized and are active within the cytoplasm or cell nuclei, where they regulate and participate in distinct cellular events. In this review, we focus on a few of these events and highlight the intracellular role these enzymes play. Close examination of these events, suggest that the intracellular activities of lysyl oxidases is mostly observed in processes where concomitant changes in the extracellular matrix takes place. Here, we suggest that the LOX family members act in the relay between changes in the cells' environment and the intracellular processes that promote them or that follow.

T Cells Promote Metastasis by Regulating Extracellular Matrix Remodeling following Chemotherapy.

Metastasis is the main cause of cancer-related mortality. Despite intense efforts to understand the mechanisms underlying the metastatic process, treatment of metastatic cancer is still challenging. Here we describe a chemotherapy-induced, host-mediated mechanism that promotes remodeling of the extracellular matrix (ECM), ultimately facilitating cancer cell seeding and metastasis. Paclitaxel (PTX) chemotherapy enhanced rapid ECM remodeling and mechanostructural changes in the lungs of tumor-free mice, and the protein expression and activity of the ECM remodeling enzyme lysyl oxidase (LOX) increased in response to PTX. A chimeric mouse model harboring genetic LOX depletion revealed chemotherapy-induced ECM remodeling was mediated by CD8+ T cells expressing LOX. Consistently, adoptive transfer of CD8+ T cells, but not CD4+ T cells or B cells, from PTX-treated mice to naïve immunodeprived mice induced pulmonary ECM remodeling. Lastly, in a clinically relevant metastatic breast carcinoma model, LOX inhibition counteracted the metastasis-promoting, ECM-related effects of PTX. This study highlights the role of immune cells in regulating ECM and metastasis following chemotherapy, suggesting that inhibiting chemotherapy-induced ECM remodeling represents a potential therapeutic strategy for metastatic cancer. SIGNIFICANCE: Chemotherapy induces prometastatic pulmonary ECM remodeling by upregulating LOX in T cells, which can be targeted with LOX inhibitors to suppress metastasis.See related commentary by Kolonin and Woodward, p. 197.

Lysyl oxidase interactions with transforming growth factor-ß during angiogenesis are mediated by endothelin 1.

Crosstalk between multiple components underlies the formation of mature vessels. Although the players involved in angiogenesis have been identified, mechanisms underlying the crosstalk between them are still unclear. Using the ex vivo aortic ring assay, we set out to dissect the interactions between two key angiogenic signaling pathways, vascular endothelial growth factor (VEGF) and transforming growth factor ß (TGFß), with members of the lysyl oxidase (LOX) family of matrix modifying enzymes. We find an interplay between VEGF, TGFß, and the LOXs is essential for the formation of mature vascular smooth muscle cells (vSMC)-coated vessels. RNA sequencing analysis further identified an interaction with the endothelin-1 pathway. Our work implicates endothelin-1 downstream of TGFß in vascular maturation and demonstrate the complexity of processes involved in generating vSMC-coated vessels.

Fibroblast fusion to the muscle fiber regulates myotendinous junction formation.

Vertebrate muscles and tendons are derived from distinct embryonic origins yet they must interact in order to facilitate muscle contraction and body movements. How robust muscle tendon junctions (MTJs) form to be able to withstand contraction forces is still not understood. Using techniques at a single cell resolution we reexamine the classical view of distinct identities for the tissues composing the musculoskeletal system. We identify fibroblasts that have switched on a myogenic program and demonstrate these dual identity cells fuse into the developing muscle fibers along the MTJs facilitating the introduction of fibroblast-specific transcripts into the elongating myofibers. We suggest this mechanism resulting in a hybrid muscle fiber, primarily along the fiber tips, enables a smooth transition from muscle fiber characteristics towards tendon features essential for forming robust MTJs. We propose that dual characteristics of junctional cells could be a common mechanism for generating stable interactions between tissues throughout the musculoskeletal system.

Intracellular Role for the Matrix-Modifying Enzyme Lox in Regulating Transcription Factor Subcellular Localization and Activity in Muscle Regeneration.

Integration of extracellular matrix (ECM)-derived cues into transcriptional programs is essential primarily in rapidly morphing environments, such as regenerating tissues. Here, we demonstrate that lysyl oxidase (Lox), known for its ECM-modifying activities, primarily collagen crosslinking, also directly regulates transcription factor (TF) localization. Using genetic and pharmacological strategies, we highlight an intracellular role for Lox in myogenic progenitors essential for muscle regeneration. We show that Lox interacts with, and directly oxidizes, vestigial-like 3 (Vgll3), a transcriptional co-activator acting with Mef2 and transcriptional enhancer factor (TEF) TFs. This enzymatic activity is required for Vgll3 cytoplasmic-to-nuclear translocation in regulation of myogenic differentiation. Our work highlights an additional mechanism for TF subcellular localization facilitating integration of ECM organization with transcriptional output during myogenic differentiation. Modulating this integration mechanism could affect the balance between ECM organization and cell differentiation and serve as a basis for novel therapeutic strategies targeting fibrotic pathologies.

Interactions between lysyl oxidases and ADAMTS proteins suggest a novel crosstalk between two extracellular matrix families.

The extracellular matrix (ECM) regulates numerous cellular events in addition to providing structural integrity. Among several protein and enzyme families implicated in functions of the ECM, the lysyl oxidases and ADAMTS proteins are known to participate in microfibril and elastic fiber formation as well as ECM-associated signaling. A yeast two-hybrid screen to identify lysyl oxidase (LOX) binding proteins identified ADAMTSL4 as a potential interactor. We demonstrate here that several members of the LOX and ADAMTS families interact with one another. Upon investigating the interaction between LOX and ADAMTSL2 we found that the absence or inhibition of Lox affected ADAMTSL2 molecular forms and reduced its tissue levels. Thus, ADAMTSL2 stability and inter-molecular complexes may depend on the activity of lysyl oxidases.

SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium.

Mutations in key transcription factors SOX2 and P63 were linked with developmental defects and postnatal abnormalities such as corneal opacification, neovascularization, and blindness. The latter phenotypes suggest that SOX2 and P63 may be involved in corneal epithelial regeneration. Although P63 has been shown to be a key regulator of limbal stem cells, the expression pattern and function of SOX2 in the adult cornea remained unclear. Here, we show that SOX2 regulates P63 to control corneal epithelial stem/progenitor cell function. SOX2 and P63 were co-expressed in the stem/progenitor cell compartments of the murine cornea in vivo and in undifferentiated human limbal epithelial stem/progenitor cells in vitro. In line, a new consensus site that allows SOX2-mediated regulation of P63 enhancer was identified while repression of SOX2 reduced P63 expression, suggesting that SOX2 is upstream to P63. Importantly, knockdown of SOX2 significantly attenuated cell proliferation, long-term colony-forming potential of stem/progenitor cells, and induced robust cell differentiation. However, this effect was reverted by forced expression of P63, suggesting that SOX2 acts, at least in part, through P63. Finally, miR-450b was identified as a direct repressor of SOX2 that was required for SOX2/P63 downregulation and cell differentiation. Altogether, we propose that SOX2/P63 pathway is an essential regulator of corneal stem/progenitor cells while mutations in SOX2 or P63 may disrupt epithelial regeneration, leading to loss of corneal transparency and blindness. Stem Cells 2019;37:417-429.

Building functional units of movement-generation and movement-sensation in the embryo.

The musculoskeletal and proprioceptive sensory systems exhibit intricate crosstalk between force generation, force sensation and force transmission, all of which are critical for coordinated animal locomotion. Recent developmental studies of the musculoskeletal and proprioceptive units of the invertebrate Drosophila embryo, have revealed several common molecular and structural principles mediating the formation of each of these systems. These common principles, as well as the differences between the developmental programs of the two systems, are discussed. Interestingly, a molecular pathway triggered by the Neuregulin/Vein ligand-dependent activation of the epidermal growth factor receptor (EGFR) pathway, which upregulates the early growth response (EGR)-like transcription factor Stripe, is utilized not only by the Drosophila muscle-tendon and proprioceptive organ-ectoderm attachment, but also by their vertebrate counterparts. An additional theme that has been observed during the development of the musculoskeletal system in both invertebrates and vertebrates is the functional importance of the extracellular matrix and its adhesion receptors. The contribution of mechanical forces to proper junction formation between muscles and tendons and between the sensory cap/ligament cells and their epidermal attachment cells is discussed. The structural and genetic similarities between the musculoskeletal and the proprioceptive systems offer new perspectives as to their common developmental nature.

Blocking Surgically Induced Lysyl Oxidase Activity Reduces the Risk of Lung Metastases.

Surgery remains the most successful curative treatment for cancer. However, some patients with early-stage disease who undergo surgery eventually succumb to distant metastasis. Here, we show that in response to surgery, the lungs become more vulnerable to metastasis due to extracellular matrix remodeling. Mice that undergo surgery or that are preconditioned with plasma from donor mice that underwent surgery succumb to lung metastases earlier than controls. Increased lysyl oxidase (LOX) activity and expression, fibrillary collagen crosslinking, and focal adhesion signaling contribute to this effect, with the hypoxic surgical site serving as the source of LOX. Furthermore, the lungs of recipient mice injected with plasma from post-surgical colorectal cancer patients are more prone to metastatic seeding than mice injected with baseline plasma. Downregulation of LOX activity or levels reduces lung metastasis after surgery and increases survival, highlighting the potential of LOX inhibition in reducing the risk of metastasis following surgery.

Heparanase 2 Attenuates Head and Neck Tumor Vascularity and Growth.

The endoglycosidase heparanase specifically cleaves the heparan sulfate (HS) side chains on proteoglycans, an activity that has been implicated strongly in tumor metastasis and angiogenesis. Heparanase-2 (Hpa2) is a close homolog of heparanase that lacks intrinsic HS-degrading activity but retains the capacity to bind HS with high affinity. In head and neck cancer patients, Hpa2 expression was markedly elevated, correlating with prolonged time to disease recurrence and inversely correlating with tumor cell dissemination to regional lymph nodes, suggesting that Hpa2 functions as a tumor suppressor. The molecular mechanism associated with favorable prognosis following Hpa2 induction is unclear. Here we provide evidence that Hpa2 overexpression in head and neck cancer cells markedly reduces tumor growth. Restrained tumor growth was associated with a prominent decrease in tumor vascularity (blood and lymph vessels), likely due to reduced Id1 expression, a transcription factor highly implicated in VEGF-A and VEGF-C gene regulation. We also noted that tumors produced by Hpa2-overexpressing cells are abundantly decorated with stromal cells and collagen deposition, correlating with a marked increase in lysyl oxidase expression. Notably, heparanase enzymatic activity was unimpaired in cells overexpressing Hpa2, suggesting that reduced tumor growth is not caused by heparanase regulation. Moreover, growth of tumor xenografts by Hpa2-overexpressing cells was unaffected by administration of a mAb that targets the heparin-binding domain of Hpa2, implying that Hpa2 function does not rely on heparanase or heparan sulfate. Cancer Res; 76(9); 2791-801. ©2016 AACR.

Localized LoxL3-Dependent Fibronectin Oxidation Regulates Myofiber Stretch and Integrin-Mediated Adhesion.

For muscles to function, myofibers have to stretch and anchor at the myotendinous junction (MTJ), a region rich in extracellular matrix (ECM). Integrin signaling is required for MTJ formation, and mutations affecting the cascade lead to muscular dystrophies in mice and humans. Underlying mechanisms for integrin activation at the MTJ and ECM modifications regulating its signaling are unclear. We show that lysyl oxidase-like 3 (LoxL3) is a key regulator of integrin signaling that ensures localized control of the cascade. In LoxL3 mutants, myofibers anchor prematurely or overshoot to adjacent somites, and are loose and lack tension. We find that LoxL3 complexes with and directly oxidizes Fibronectin (FN), an ECM scaffold protein and integrin ligand enriched at the MTJ. We identify a mechanism whereby localized LoxL3 secretion from myofiber termini oxidizes FN, enabling enhanced integrin activation at the tips of myofibers and ensuring correct positioning and anchoring of myofibers along the MTJ.

Muscle composition is regulated by a Lox-TGFß feedback loop.

Muscle is an integrated tissue composed of distinct cell types and extracellular matrix. While much emphasis has been placed on the factors required for the specification of the cells that comprise muscle, little is known about the crosstalk between them that enables the development of a patterned and functional tissue. We find in mice that deletion of lysyl oxidase (Lox), an extracellular enzyme regulating collagen maturation and organization, uncouples the balance between the amount of myofibers and that of muscle connective tissue (MCT). We show that Lox secreted from the myofibers attenuates TGFß signaling, an inhibitor of myofiber differentiation and promoter of MCT development. We further demonstrate that a TGFß-Lox feedback loop between the MCT and myofibers maintains the dynamic developmental homeostasis between muscle components while also regulating MCT organization. Our results allow a better understanding of diseases such as Duchenne muscular dystrophy, in which LOX and TGFß signaling have been implicated and the balance between muscle constituents is disturbed.

"Soft" tissue patterning: muscles and tendons of the limb take their form.

The musculoskeletal system grants our bodies a vast range of movements. Because it is mainly composed of easily identifiable components, it serves as an ideal model to study patterning of the specific tissues that make up the organ. Surprisingly, although critical for the function of the musculoskeletal system, understanding of the embryonic processes that regulate muscle and tendon patterning is very limited. The recent identification of specific markers and the reagents stemming from them has revealed some of the molecular events regulating patterning of these soft tissues. This review will focus on some of the current work, with an emphasis on the roles of the muscle connective tissue, and discuss several key points that addressing them will advance our understanding of these patterning events.

Tbx4 and tbx5 acting in connective tissue are required for limb muscle and tendon patterning.

Proper functioning of the musculoskeletal system requires the precise integration of bones, muscles, and tendons. Complex morphogenetic events ensure that these elements are linked together in the appropriate three-dimensional configuration. It has been difficult, however, to tease apart the mechanisms that regulate tissue morphogenesis. We find that deletion of Tbx5 in forelimbs (or Tbx4 in hindlimbs) specifically affects muscle and tendon patterning without disrupting skeletal development, thus suggesting that distinct cues regulate these processes. We identify muscle connective tissue as the site of action of these transcription factors and show that N-Cadherin and beta-Catenin are key downstream effectors acting in muscle connective tissue and regulating soft-tissue morphogenesis. In humans, TBX5 mutations lead to Holt-Oram syndrome, which is characterized by forelimb musculoskeletal defects. Our results suggest that a focus on connective tissue is required to understand the etiology of diseases affecting soft tissue formation.

Tbx5 is dispensable for forelimb outgrowth.

Tbx5 is essential for initiation of the forelimb, and its deletion in mice results in the failure of forelimb formation. Misexpression of dominant-negative forms of Tbx5 results in limb truncations, suggesting Tbx5 is also required for forelimb outgrowth. Here we show that Tbx5 is expressed throughout the limb mesenchyme in progenitors of cartilage, tendon and muscle. Using a tamoxifeninducible Cre transgenic line, we map the time frame during which Tbx5 is required for limb development. We show that deletion of Tbx5 subsequent to limb initiation does not impair limb outgrowth. Furthermore, we distinguish two distinct phases of limb development: a Tbx5-dependent limb initiation phase, followed by a Tbx5-independent limb outgrowth phase. In humans, mutations in the T-box transcription factor TBX5 are associated with the dominant disorder Holt-Oram syndrome (HOS), which is characterised by malformations in the forelimb and heart. Our results demonstrate a short temporal requirement for Tbx5 during early limb development, and suggest that the defects found in HOS arise as a result of disrupted TBX5 function during this narrow time window.

EGFR signaling attenuates Groucho-dependent repression to antagonize Notch transcriptional output.

Crosstalk between signaling pathways is crucial for the generation of complex and varied transcriptional networks. Antagonism between the EGF-receptor (EGFR) and Notch pathways in particular is well documented, although the underlying mechanism is poorly understood. The global corepressor Groucho (Gro) and its transducin-like Enhancer-of-split (TLE) mammalian homologs mediate repression by a myriad of repressors, including effectors of the Notch, Wnt (Wg) and TGF-beta (Dpp) signaling cascades. Given that there are genetic interactions between gro and components of the EGFR pathway (ref. 9 and P.H. et al., unpublished results), we tested whether Gro is at a crossroad between this and other pathways. Here we show that phosphorylation of Gro in response to MAPK activation weakens its repressor capacity, attenuating Gro-dependent transcriptional silencing by the Enhancer-of-split proteins, effectors of the Notch cascade. Thus, Gro is a new junction between signaling pathways, enabling EGFR signaling to antagonize transcriptional output by Notch and potentially other Gro-dependent pathways.