Immune mechanisms in fibrotic interstitial lung disease.

Fibrotic interstitial lung diseases (fILDs) have poor survival rates and lack effective therapies. Despite evidence for immune mechanisms in lung fibrosis, immunotherapies have been unsuccessful for major types of fILD. Here, we review immunological mechanisms in lung fibrosis that have the potential to impact clinical practice. We first examine innate immunity, which is broadly involved across fILD subtypes. We illustrate how innate immunity in fILD involves a complex interplay of multiple cell subpopulations and molecular pathways. We then review the growing evidence for adaptive immunity in lung fibrosis to provoke a re-examination of its role in clinical fILD. We close with future directions to address key knowledge gaps in fILD pathobiology: (1) longitudinal studies emphasizing early-stage clinical disease, (2) immune mechanisms of acute exacerbations, and (3) next-generation immunophenotyping integrating spatial, genetic, and single-cell approaches. Advances in these areas are essential for the future of precision medicine and immunotherapy in fILD.

Early cellular mechanisms of type I interferon-driven susceptibility to tuberculosis.

Mycobacterium tuberculosis (Mtb) causes 1.6 million deaths annually. Active tuberculosis correlates with a neutrophil-driven type I interferon (IFN) signature, but the cellular mechanisms underlying tuberculosis pathogenesis remain poorly understood. We found that interstitial macrophages (IMs) and plasmacytoid dendritic cells (pDCs) are dominant producers of type I IFN during Mtb infection in mice and non-human primates, and pDCs localize near human Mtb granulomas. Depletion of pDCs reduces Mtb burdens, implicating pDCs in tuberculosis pathogenesis. During IFN-driven disease, we observe abundant DNA-containing neutrophil extracellular traps (NETs) described to activate pDCs. Cell-type-specific disruption of the type I IFN receptor suggests that IFNs act on IMs to inhibit Mtb control. Single-cell RNA sequencing (scRNA-seq) indicates that type I IFN-responsive cells are defective in their response to IFNγ, a cytokine critical for Mtb control. We propose that pDC-derived type I IFNs act on IMs to permit bacterial replication, driving further neutrophil recruitment and active tuberculosis disease.

Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue.

The extracellular space (ECS) of the brain has an extremely complex spatial organization, which has defied conventional light microscopy. Consequently, despite a marked interest in the physiological roles of brain ECS, its structure and dynamics remain largely inaccessible for experimenters. We combined 3D-STED microscopy and fluorescent labeling of the extracellular fluid to develop super-resolution shadow imaging (SUSHI) of brain ECS in living organotypic brain slices. SUSHI enables quantitative analysis of ECS structure and reveals dynamics on multiple scales in response to a variety of physiological stimuli. Because SUSHI produces sharp negative images of all cellular structures, it also enables unbiased imaging of unlabeled brain cells with respect to their anatomical context. Moreover, the extracellular labeling strategy greatly alleviates problems of photobleaching and phototoxicity associated with traditional imaging approaches. As a straightforward variant of STED microscopy, SUSHI provides unprecedented access to the structure and dynamics of live brain ECS and neuropil.

Spontaneous Chitin Accumulation in Airways and Age-Related Fibrotic Lung Disease.

The environmentally widespread polysaccharide chitin is degraded and recycled by ubiquitous bacterial and fungal chitinases. Although vertebrates express active chitinases from evolutionarily conserved loci, their role in mammalian physiology is unclear. We show that distinct lung epithelial cells secrete acidic mammalian chitinase (AMCase), which is required for airway chitinase activity. AMCase-deficient mice exhibit premature morbidity and mortality, concomitant with accumulation of environmentally derived chitin polymers in the airways and expression of pro-fibrotic cytokines. Over time, these mice develop spontaneous pulmonary fibrosis, which is ameliorated by restoration of lung chitinase activity by genetic or therapeutic approaches. AMCase-deficient epithelial cells express fibrosis-associated gene sets linked with cell stress pathways. Mice with lung fibrosis due to telomere dysfunction and humans with interstitial lung disease also accumulate excess chitin polymers in their airways. These data suggest that altered chitin clearance could exacerbate fibrogenic pathways in the setting of lung diseases characterized by epithelial cell dysfunction.

Ca2+ dynamics in interstitial cells: foundational mechanisms for the motor patterns in the gastrointestinal tract.

The gastrointestinal (GI) tract displays multiple motor patterns that move nutrients and wastes through the body. Smooth muscle cells (SMCs) provide the forces necessary for GI motility, but interstitial cells, electrically coupled to SMCs, tune SMC excitability, transduce inputs from enteric motor neurons, and generate pacemaker activity that underlies major motor patterns, such as peristalsis and segmentation. The interstitial cells regulating SMCs are interstitial cells of Cajal (ICC) and PDGF receptor (PDGFR)α+ cells. Together these cells form the SIP syncytium. ICC and PDGFRα+ cells express signature Ca2+-dependent conductances: ICC express Ca2+-activated Cl- channels, encoded by Ano1, that generate inward current, and PDGFRα+ cells express Ca2+-activated K+ channels, encoded by Kcnn3, that generate outward current. The open probabilities of interstitial cell conductances are controlled by Ca2+ release from the endoplasmic reticulum. The resulting Ca2+ transients occur spontaneously in a stochastic manner. Ca2+ transients in ICC induce spontaneous transient inward currents and spontaneous transient depolarizations (STDs). Neurotransmission increases or decreases Ca2+ transients, and the resulting depolarizing or hyperpolarizing responses conduct to other cells in the SIP syncytium. In pacemaker ICC, STDs activate voltage-dependent Ca2+ influx, which initiates a cluster of Ca2+ transients and sustains activation of ANO1 channels and depolarization during slow waves. Regulation of GI motility has traditionally been described as neurogenic and myogenic. Recent advances in understanding Ca2+ handling mechanisms in interstitial cells and how these mechanisms influence motor patterns of the GI tract suggest that the term "myogenic" should be replaced by the term "SIPgenic," as this review discusses.

Biology of lung macrophages in health and disease.

Tissue-resident alveolar and interstitial macrophages and recruited macrophages are critical players in innate immunity and maintenance of lung homeostasis. Until recently, assessing the differential functional contributions of tissue-resident versus recruited macrophages has been challenging because they share overlapping cell surface markers, making it difficult to separate them using conventional methods. This review describes how scRNA-seq and spatial transcriptomics can separate these subpopulations and help unravel the complexity of macrophage biology in homeostasis and disease. First, we provide a guide to identifying and distinguishing lung macrophages from other mononuclear phagocytes in humans and mice. Second, we outline emerging concepts related to the development and function of the various lung macrophages in the alveolar, perivascular, and interstitial niches. Finally, we describe how different tissue states profoundly alter their functions, including acute and chronic lung disease, cancer, and aging.

The enteric nervous system.

Of all the organ systems in the body, the gastrointestinal tract is the most complicated in terms of the numbers of structures involved, each with different functions, and the numbers and types of signaling molecules utilized. The digestion of food and absorption of nutrients, electrolytes, and water occurs in a hostile luminal environment that contains a large and diverse microbiota. At the core of regulatory control of the digestive and defensive functions of the gastrointestinal tract is the enteric nervous system (ENS), a complex system of neurons and glia in the gut wall. In this review, we discuss 1) the intrinsic neural control of gut functions involved in digestion and 2) how the ENS interacts with the immune system, gut microbiota, and epithelium to maintain mucosal defense and barrier function. We highlight developments that have revolutionized our understanding of the physiology and pathophysiology of enteric neural control. These include a new understanding of the molecular architecture of the ENS, the organization and function of enteric motor circuits, and the roles of enteric glia. We explore the transduction of luminal stimuli by enteroendocrine cells, the regulation of intestinal barrier function by enteric neurons and glia, local immune control by the ENS, and the role of the gut microbiota in regulating the structure and function of the ENS. Multifunctional enteric neurons work together with enteric glial cells, macrophages, interstitial cells, and enteroendocrine cells integrating an array of signals to initiate outputs that are precisely regulated in space and time to control digestion and intestinal homeostasis.

Microtubule-binding protein MAP1B regulates interstitial axon branching of cortical neurons via the tubulin tyrosination cycle.

Regulation of directed axon guidance and branching during development is essential for the generation of neuronal networks. However, the molecular mechanisms that underlie interstitial (or collateral) axon branching in the mammalian brain remain unresolved. Here, we investigate interstitial axon branching in vivo using an approach for precise labeling of layer 2/3 callosal projection neurons (CPNs). This method allows for quantitative analysis of axonal morphology at high acuity and also manipulation of gene expression in well-defined temporal windows. We find that the GSK3ß serine/threonine kinase promotes interstitial axon branching in layer 2/3 CPNs by releasing MAP1B-mediated inhibition of axon branching. Further, we find that the tubulin tyrosination cycle is a key downstream component of GSK3ß/MAP1B signaling. These data suggest a cell-autonomous molecular regulation of cortical neuron axon morphology, in which GSK3ß can release a MAP1B-mediated brake on interstitial axon branching upstream of the posttranslational tubulin code.

Programming of Distinct Chemokine-Dependent and -Independent Search Strategies for Th1 and Th2 Cells Optimizes Function at Inflamed Sites.

T-helper (Th) cell differentiation drives specialized gene programs that dictate effector T cell function at sites of infection. Here, we have shown Th cell differentiation also imposes discrete motility gene programs that shape Th1 and Th2 cell navigation of the inflamed dermis. Th1 cells scanned a smaller tissue area in a G protein-coupled receptor (GPCR) and chemokine-dependent fashion, while Th2 cells scanned a larger tissue area independent of GPCR signals. Differential chemokine reliance for interstitial migration was linked to STAT6 transcription-factor-dependent programming of integrin αVß3 expression: Th2 cell differentiation led to high αVß3 expression relative to Th1 cells. Th1 and Th2 cell modes of motility could be switched simply by manipulating the amount of αVß3 on the cell surface. Deviating motility modes from those established during differentiation impaired effector function. Thus, programmed expression of αVß3 tunes effector T cell reliance on environmental cues for optimal exploration of inflamed tissues.

Curvature-mediated rapid extravasation and penetration of nanoparticles against interstitial fluid pressure for improved drug delivery.

Elevated interstitial fluid pressure (IFP) within pathological tissues (e.g., tumors, obstructed kidneys, and cirrhotic livers) creates a significant hindrance to the transport of nanomedicine, ultimately impairing the therapeutic efficiency. Among these tissues, solid tumors present the most challenging scenario. While several strategies through reducing tumor IFP have been devised to enhance nanoparticle delivery, few approaches focus on modulating the intrinsic properties of nanoparticles to effectively counteract IFP during extravasation and penetration, which are precisely the stages obstructed by elevated IFP. Herein, we propose an innovative solution by engineering nanoparticles with a fusiform shape of high curvature, enabling efficient surmounting of IFP barriers during extravasation and penetration within tumor tissues. Through experimental and theoretical analyses, we demonstrate that the elongated nanoparticles with the highest mean curvature outperform spherical and rod-shaped counterparts against elevated IFP, leading to superior intratumoral accumulation and antitumor efficacy. Super-resolution microscopy and molecular dynamics simulations uncover the underlying mechanisms in which the high curvature contributes to diminished drag force in surmounting high-pressure differentials during extravasation. Simultaneously, the facilitated rotational movement augments the hopping frequency during penetration. This study effectively addresses the limitations posed by high-pressure impediments, uncovers the mutual interactions between the physical properties of NPs and their environment, and presents a promising avenue for advancing cancer treatment through nanomedicine.

Autoimmunity against melanoma differentiation-associated gene 5 induces interstitial lung disease mimicking dermatomyositis in mice.

Anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive dermatomyositis (DM) is characterized by amyopathic DM with interstitial lung disease (ILD). Patients with anti-MDA5 antibody-associated ILD frequently develop rapidly progression and present high mortality rate in the acute phase. Here, we established a murine model of ILD mediated by autoimmunity against MDA5. Mice immunized with recombinant murine MDA5 whole protein, accompanied with complete Freund's adjuvant once a week for four times, developed MDA5-reactive T cells and anti-MDA5 antibodies. After acute lung injury induced by intranasal administration of polyinosinic-polycytidylic acid [poly (I:C)] mimicking viral infection, the MDA5-immunized mice developed fibrotic ILD representing prolonged respiratory inflammation accompanied by fibrotic changes 2 wk after poly (I:C)-administration, while the control mice had quickly and completely recovered from the respiratory inflammation. Treatment with anti-CD4 depleting antibody, but not anti-CD8 depleting antibody, suppressed the severity of MDA5-induced fibrotic ILD. Upregulation of interleukin (IL)-6 mRNA, which was temporarily observed in poly (I:C)-treated mice, was prolonged in MDA5-immunized mice. Treatment with anti-IL-6 receptor antibody ameliorated the MDA5-induced fibrotic ILD. These results suggested that autoimmunity against MDA5 exacerbates toll-like receptor 3-mediated acute lung injury, and prolongs inflammation resulting in the development of fibrotic ILD. IL-6 may play a key role initiating ILD in this model.

Immunological mechanisms underlying clinical phenotypes and noninvasive diagnosis of immune checkpoint inhibitor-induced kidney disease.

Immune checkpoint inhibitors (ICIs) have become a mainstay of cancer therapy, with over 80 FDA-approved indications. Used in a variety of settings and in combination with each other and with traditional chemotherapies, the hyperactive immune response induced by ICIs can often lead to immune-related adverse events in bystander normal tissues such as the kidneys, lungs, and the heart. In the kidneys, this immune-related adverse event manifests as acute interstitial nephritis (ICI-AIN). In the era of widespread ICI use, it becomes vital to understand the clinical manifestations of ICI-AIN and the importance of prompt diagnosis and management of these complications. In this review, we delve into the clinical phenotypes of ICI-AIN and how they differ from traditional drug-induced AIN. We also detail what is known about the mechanistic underpinnings of ICI-AIN and the important diagnostic and therapeutic implications behind harnessing those mechanisms to further our understanding of these events and to formulate effective treatment plans to manage ICI-AIN.

Hox11-expressing interstitial cells contribute to adult skeletal muscle at homeostasis.

Interstitial stromal cells play critical roles in muscle development, regeneration and repair and we have previously reported that Hoxa11 and Hoxd11 are expressed in the interstitial cells of muscles attached to the zeugopod, and are crucial for the proper embryonic patterning of these muscles. Hoxa11eGFP expression continues in a subset of muscle interstitial cells through adult stages. The induction of Hoxa11-CreERT2-mediated lineage reporting (Hoxa11iTom) at adult stages in mouse results in lineage induction only in the interstitial cells. However, Hoxa11iTom+ cells progressively contribute to muscle fibers at subsequent stages. The contribution to myofibers exceeds parallel Pax7-CreERT2-mediated lineage labeling. Nuclear-specific lineage labeling demonstrates that Hoxa11-expressing interstitial cells contribute nuclear contents to myofibers. Crucially, at no point after Hoxa11iTom induction are satellite cells lineage labeled. When examined in vitro, isolated Hoxa11iTom+ interstitial cells are not capable of forming myotubes, but Hoxa11iTom+ cells can contribute to differentiating myotubes, supporting Hox-expressing interstitial cells as a new population of muscle progenitors, but not stem cells. This work adds to a small but growing body of evidence that supports a satellite cell-independent source of muscle tissue in vivo.

Homeostatic chemokines as putative therapeutic targets in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal chronic interstitial lung disease (ILD) that affects lung mechanical functions and gas exchange. IPF is caused by increased fibroblast activity and collagen deposition that compromise the alveolar-capillary barrier. Identifying an effective therapy for IPF remains a clinical challenge. Chemokines are key proteins in cell communication that have functions in immunity as well as in tissue homeostasis, damage, and repair. Chemokine receptor signaling induces the activation and proliferation of lung-resident cells, including alveolar macrophages (AMs) and fibroblasts. AMs are an important source of chemokines and cytokines during IPF. We highlight the complexity of this system and, based on insights from genetic and transcriptomic studies, propose a new role for homeostatic chemokine imbalance in IPF, with implications for putative therapeutic targets.

Brainstem Neural Circuits Triggering Vertical Saccades and Fixation.

Neurons in the nucleus raphe interpositus have tonic activity that suppresses saccadic burst neurons (BNs) during eye fixations, and that is inhibited before and during saccades in all directions (omnipause neurons, OPNs). We have previously demonstrated via intracellular recording and anatomical staining in anesthetized cats of both sexes that OPNs are inhibited by BNs in the medullary reticular formation (horizontal inhibitory BNs, IBNs). These horizontal IBNs receive monosynaptic input from the caudal horizontal saccade area of the superior colliculus (SC), and then produce monosynaptic inhibition in OPNs, providing a mechanism to trigger saccades. However, it is well known that the neural circuits driving horizontal components of saccades are independent from the circuits driving vertical components. Thus, our previous results are unable to explain how purely vertical saccades are triggered. Here, we again apply intracellular recording to show that a disynaptic vertical IBN circuit exists, analogous to the horizontal circuit. Specifically, we show that stimulation of the SC rostral vertical saccade area produces disynaptic inhibition in OPNs, which is not abolished by midline section between the horizontal IBNs. This excludes the possibility that horizontal IBNs could be responsible for the OPN inhibition during vertical saccades. We then show that vertical IBNs in the interstitial nucleus of Cajal, which receive monosynaptic input from rostral SC, are responsible for the disynaptic inhibition of OPNs. These results indicate that a similarly functioning SC-IBN-OPN circuit exists for both the horizontal and vertical oculomotor pathways. These two IBN-mediated circuits are capable of triggering saccades in any direction.Significance Statement Saccades shift gaze to objects of interest, moving their image to the central retina, where it is maintained for detailed examination (fixation). During fixation, high gain saccade burst neurons (BNs) are tonically inhibited by omnipause neurons (OPNs). Our previous study showed that medullary horizontal inhibitory BNs (IBNs) activated from the caudal superior colliculus (SC) inhibit tonically active OPNs in order to initiate horizontal saccades. The present study addresses the source of OPN inhibition for vertical saccades. We find that OPNs monosynaptically inhibit vertical IBNs in the interstitial nucleus of Cajal during fixation. Those same vertical IBNs are activated by the rostral SC, and inhibit OPN activity to initiate vertical saccades.

Dynamic duo: Cell-extracellular matrix interactions in hair follicle development and regeneration.

Ectodermal organs, such as hair follicles, originate from simple epithelial and mesenchymal sheets through a complex developmental process driven by interactions between these cell types. This process involves dermal condensation, placode formation, bud morphogenesis, and organogenesis, and all of these processes require intricate interactions among various tissues. Recent research has emphasized the crucial role of reciprocal and dynamic interactions between cells and the extracellular matrix (ECM), referred to as the "dynamic duo", in the development of ectodermal organs. These interactions provide spatially and temporally changing biophysical and biochemical cues within tissues. Using the hair follicle as an example, this review highlights two types of cell-ECM adhesion units-focal adhesion-type and hemidesmosome-type adhesion units-that facilitate communication between epithelial and mesenchymal cells. This review further explores how these adhesion units, along with other cell-ECM interactions, evolve during hair follicle development and regeneration, underscoring their importance in guiding both developmental and regenerative processes.

Monoallelic pathogenic ALG5 variants cause atypical polycystic kidney disease and interstitial fibrosis.

Disorders of the autosomal dominant polycystic kidney disease (ADPKD) spectrum are characterized by the development of kidney cysts and progressive kidney function decline. PKD1 and PKD2, encoding polycystin (PC)1 and 2, are the two major genes associated with ADPKD; other genes include IFT140, GANAB, DNAJB11, and ALG9. Genetic testing remains inconclusive in ∼7% of the families. We performed whole-exome sequencing in a large multiplex genetically unresolved (GUR) family affected by ADPKD-like symptoms and identified a monoallelic frameshift variant (c.703_704delCA) in ALG5. ALG5 encodes an endoplasmic-reticulum-resident enzyme required for addition of glucose molecules to the assembling N-glycan precursors. To identify additional families, we screened a cohort of 1,213 families with ADPKD-like and/or autosomal-dominant tubulointerstitial kidney diseases (ADTKD), GUR (n = 137) or naive to genetic testing (n = 1,076), by targeted massively parallel sequencing, and we accessed Genomics England 100,000 Genomes Project data. Four additional families with pathogenic variants in ALG5 were identified. Clinical presentation was consistent in the 23 affected members, with non-enlarged cystic kidneys and few or no liver cysts; 8 subjects reached end-stage kidney disease from 62 to 91 years of age. We demonstrate that ALG5 haploinsufficiency is sufficient to alter the synthesis of the N-glycan chain in renal epithelial cells. We also show that ALG5 is required for PC1 maturation and membrane and ciliary localization and that heterozygous loss of ALG5 affects PC1 maturation. Overall, our results indicate that monoallelic variants of ALG5 lead to a disorder of the ADPKD-spectrum characterized by multiple small kidney cysts, progressive interstitial fibrosis, and kidney function decline.

Past and future of alveolar organoids for lung regenerative medicine.

The lung is regarded as having limited regenerative capacity, and there are few treatment options for refractory lung diseases, such as interstitial pneumonia. Lung transplantation is the final option available in some scenarios. Research in this area has been slow owing to the complex structure of the lung for efficient gas exchange between the alveolar spaces and capillaries as well as the difficulty in obtaining specimens from patients with progressive lung disease. However, basic research over the past decade in the field of mouse and human embryology using genetic lineage tracing techniques and stem cell biology using primary and pluripotent stem cell-derived alveolar organoids has begun to clarify the tissue response in various intractable lung diseases and the mechanisms underlying remodeling. Advancement in this area may expand potential therapeutic targets for alveolar regeneration, providing alternatives to lung transplantation, and contribute to the development of effective therapeutic methods that activate or repopulate stem cells in the lung. In this review, we cover research focused on alveolar epithelial cells and discuss methods expected to regenerate lungs that are damaged by diseases.

Interstitial cells of the sip syncytium regulate basal membrane potential in murine gastric corpus.

Smooth muscle cells (SMCs), Interstitial cells of Cajal (ICC) and Platelet-derived growth factor receptor α positive (PDGFRα+) cells form an integrated, electrical syncytium within the gastrointestinal (GI) muscular tissues known as the SIP syncytium. Immunohistochemical analysis of gastric corpus muscles showed that c-KIT+/ANO1+ ICC-IM and PDGFRα+ cells were closely apposed to one another in the same anatomical niches. We used intracellular microelectrode recording from corpus muscle bundles to characterize the roles of intramuscular ICC and PDGFRα+ cells in conditioning membrane potentials of gastric muscles. In muscle bundles, that have a relatively higher input impedance than larger muscle strips or sheets, we recorded an ongoing discharge of stochastic fluctuations in membrane potential, previously called unitary potentials or spontaneous transient depolarizations (STDs) and spontaneous transient hyperpolarizations (STHs). We reasoned that STDs should be blocked by antagonists of ANO1, the signature conductance of ICC. Activation of ANO1 has been shown to generate spontaneous transient inward currents (STICs), which are the basis for STDs. Ani9 reduced membrane noise and caused hyperpolarization, but this agent did not block the fluctuations in membrane potential quantitatively. Apamin, an antagonist of small conductance Ca2+-activated K+ channels (SK3), the signature conductance in PDGFRα+ cells, further reduced membrane noise and caused depolarization. Reversing the order of channel antagonists reversed the sequence of depolarization and hyperpolarization. These experiments show that the ongoing discharge of STDs and STHs by ICC and PDGFRα+ cells, respectively, exerts conditioning effects on membrane potentials in the SIP syncytium that would effectively regulate the excitability of SMCs.

Sex-dependent expression of galectin-1, a cardioprotective ß-galactoside-binding lectin, in human calcific aortic stenosis.

We aimed to analyze sex-related differences in galectin-1 (Gal-1), a ß-galactoside-binding lectin, in aortic stenosis (AS) and its association with the inflammatory and fibrocalcific progression of AS. Gal-1 was determined in serum and aortic valves (AVs) from control and AS donors by western blot and immunohistochemistry. Differences were validated by ELISA and qPCR in AS samples. In vitro experiments were conducted in primary cultured valve interstitial cells (VICs). Serum Gal-1 was not different neither between control and AS nor between men and women. There was no association between circulating and valvular Gal-1 levels. The expression of Gal-1 in stenotic AVs was higher in men than women, even after adjusting for confounding factors, and was associated with inflammation, oxidative stress, extracellular matrix remodeling, fibrosis, and osteogenesis. Gal-1 (LGALS1) mRNA was enhanced within fibrocalcific areas of stenotic AVs, especially in men. Secretion of Gal-1 was up-regulated over a time course of 2, 4, and 8 days in men's calcifying VICs, only peaking at day 4 in women's VICs. In vitro, Gal-1 was associated with similar mechanisms to those in our clinical cohort. ß-estradiol significantly up-regulated the activity of an LGALS1 promoter vector and the secretion of Gal-1, only in women's VICs. Supplementation with rGal-1 prevented the effects elicited by calcific challenge including the metabolic shift to glycolysis. In conclusion, Gal-1 is up-regulated in stenotic AVs and VICs from men in association with inflammation, oxidative stress, matrix remodeling, and osteogenesis. Estrogens can regulate Gal-1 expression with potential implications in post-menopause women. Exogenous rGal-1 can diminish calcific phenotypes in both women and men.