Latent transforming growth factor beta binding protein 4: A regulator of mitochondrial function in acute kidney injury.

Recently, latent transforming growth factor beta binding protein 4 (LTBP4) was implicated in the pathogenesis of renal damage through its modulation of mitochondrial dynamics. The seminal article written by Su et al. entitled "LTBP4 (Latent Transforming Growth Factor Beta Binding Protein 4) Protects Against Renal Fibrosis via Mitochondrial and Vascular Impacts" uncovers LTBP4's renoprotective role against acute kidney injury via modulating mitochondrial dynamics. Recently, LTBP4 has emerged as a driver in the mitochondrial-dependent modulation of age-related organ pathologies. This article aims to expand our understanding of LTBP4's diverse roles in these diseases in the context of these recent findings.

circWHSC1: A circular RNA piece in the human cancer puzzle.

Circular RNAs (circRNAs) are a group of non-coding RNAs with a closed loop shape, which are transcribed via non-canonical splicing. They are mainly formed by reverse splicing of a precursor mRNA. circWHSC1 (Hsa_circ_0001387), is a cancer-related circRNA that originated from the Wolf-Hirschhorn syndrome candidate 1 (WHSC1) gene on chromosome 4. circWHSC1 has been found to be overexpressed in different neoplastic conditions. circWHSC1 acts as a sponge for many different miRNAs, including miR-195-5p, miR-532-3p, miR-646, miR-142-3p, miR-7, miR-296-3p, miR-145, miR-1182, miR-212-5p, etc. It can also moderate several signaling pathways, including FASN/AMPK/mTOR, LTBP2, NPM1, HOXA1, TAB2, AKT3, hTERT, and MUC1. Studies have shown that circWHSC1 may leads to an increase in cell growth, tumor size, cell migration, invasion, and metastasis, but a reduction in apoptosis rates. Moreover, upregulation of CircWHSC1 has been associated with reduced patient's survival in different cancers, representing the function of this circRNA as a novel prognostic marker. Nevertheless, there are no reviews focusing on the relationship between circWHSC1 and cancers. Therefore, in the current review, we will first describe the oncogenic effect of circWHSC1 in various tissues according to the evidence from in vitro, in vivo, and human studies.

Aqueous Humor TGF-ß2 and Its Association With Intraocular Pressure in a Naturally Occurring Large Animal Model of Glaucoma.

Purpose: Transforming growth factor (TGF)-ß2 has been widely implicated in human glaucoma pathology. The purpose of this study was to determine the source of TGF-ß2 in aqueous humor (AH) and its relationship with intraocular pressure (IOP) in an inherited large animal model of glaucoma. Methods: Sixty-six glaucomatous cats homozygous for LTBP2 mutation, and 42 normal cats were studied. IOP was measured weekly by rebound tonometry. AH was collected by anterior chamber paracentesis from each eye under general anesthesia, and serum samples collected from venous blood concurrently. Concentrations of total, active and latent TGF-ß2 in AH and serum samples were measured by quantitative sandwich immunoassay. For comparisons between groups, unpaired t-test or Mann Whitney test were used, with P < 0.05 considered significant. The relationships between TGF-ß2 concentrations and IOP values were examined by Pearson's correlation coefficient and generalized estimating equation. Results: IOP and AH TGF-ß2 concentrations were significantly higher in glaucomatous than in normal cats. AH TGF-ß2 showed a significant, robust positive correlation with IOP in glaucomatous cats (r = 0.83, R2 = 0.70, P < 0.0001). Serum TGF-ß2 did not correlate with AH TGF-ß2 and was not significantly different between groups. TGF-ß2 mRNA and protein expression were significantly increased in local ocular tissues in glaucomatous cats. Conclusions: Enhanced, local ocular production of TGF-ß2 with a robust positive association with IOP was identified in this spontaneous feline glaucoma model, providing a foundation for preclinical testing of novel therapeutics to limit disease-associated AH TGF-ß2 elevation and signaling in glaucoma.

Immune suppressive signaling regulated by latent transforming growth factor beta binding protein 1 promotes metastasis in cervical cancer.

Although metastasis is the major cause of death in cervical cancer, the mechanism of metastasis is still unclear. The mRNA expression and protein level of latent transforming growth factor beta binding protein 1 (LTBP1) were detected in tumor tissues and paracancerous tissues from in-house samples. Cell proliferation, cell cycle, migration, and in vivo metastasis were determined after LTBP1 was knocked down. Then, 13 drugs were screened, and the changes in cell apoptosis and proliferation and tumor metastasis were detected after drug treatment in shRNA cells. In our in-house samples, LTBP1 was lowly expressed in cervical cancer tissues. After LTBP1 knockdown, cell proliferation was increased, and the ability of in vitro migration and in vivo metastasis was enhanced. At the same time, the proportion of myeloid derived suppressor cells (MDSC) in situ increased, the proportion of T cells decreased, and transforming growth factor beta-1 (TGFß1) signaling was activated. After carboplatin treatment, LTBP1 shRNA cell line apoptosis increased, metastasis in vivo was limited, and the proportion of MDSC in situ decreased. LTBP1 was lowly expressed in cervical cancer, and the inhibition of LTBP1 can improve the malignant degree of the tumor, and this process can be blocked by carboplatin.

Specific Overexpression of YAP in Vascular Smooth Muscle Attenuated Abdominal Aortic Aneurysm Formation by Activating Elastic Fiber Assembly via LTBP4.

Abdominal aortic aneurysm (AAA) is a fatal vascular disease. Vascular smooth muscle cells (VSMCs) play a crucial role in the pathogenesis of AAA. Increasing evidence has shown that Yes-associated protein (YAP) is involved in diverse vascular diseases. However, the role of YAP in AAA remains unclear. The current study aimed to determine the role of YAP in AAA formation and the underlying mechanism. We found that YAP expression in VSMCs was markedly decreased in human and experimental AAA samples. Furthermore, VSMC-specific YAP overexpression prevented several pathogenic factor-induced AAA. Mechanistically, YAP overexpression in VSMCs promoted latent transforming growth factor-ß binding protein 4 (LTBP4) expression, an important factor in elastic fiber assembly. Finally, silencing of LTBP4 in VSMCs abolished the protective role of YAP in AAA formation in vivo. Our results suggest that YAP promotes LTBP4-mediated elastic fibril assembly in VSMCs, which mitigates elastin degradation and AAA formation.

Association of polymorphisms rs2303729, rs10880, and rs1131620 of LTBP4 with sarcopenia in elderly patients with type 2 diabetes mellitus.

BACKGROUND: Latent TGFß binding protein 4 (LTBP4) modifies skeletal muscle function, and polymorphisms in this gene have been associated with a longer ambulation time in patients with Duchenne muscular dystrophy. However, no studies associate these polymorphisms with an acquired muscle condition. AIM: The study aims to determine whether three functional variants within the LTBP4 were associated with sarcopenia in patients with type 2 diabetes mellitus (T2DM). SUBJECTS AND METHODS: We performed an analysis with 144 elderly individuals with T2DM, including 101 without sarcopenia and 43 with sarcopenia. Polymorphism frequency was determined by real-time PCR allelic discrimination TaqMan assay. RESULTS: Under different genetic models, the univariant analysis did not show a significant association of any polymorphism with sarcopenia. But the multivariate model analysis showed that variant rs1131620 (OR 7.852, 95% CI 1.854-33.257, p = 0.005) was significantly associated with sarcopenia under a dominant model. Under the same analysis, the variants rs2303729 and rs10880 had a more discrete association (OR 3.537 95% CI 1.078-11.607, p = 0.037; OR 5.008, 95% CI 1.120-22.399, p = 0.035, respectively). CONCLUSIONS: Our study highlights the importance of studying LTBP4 polymorphisms associated with sarcopenia. These findings suggest that the rs1131620 polymorphism of the LTBP4 may be part of the observed sarcopenia process in patients with T2DM.

The landscape of cancer-associated fibroblasts in colorectal cancer liver metastases.

Rationale: Patients with colorectal cancer die mainly due to liver metastases (CRC-LM). Although the tumor microenvironment (TME) plays an important role in tumor development and therapeutic response, our understanding of the individual TME components, especially cancer-associated fibroblasts (CAFs), remains limited. Methods: We analyzed CRC-LM CAFs and cancer cells by single-cell transcriptomics and used bioinformatics for data analysis and integration with related available single-cell and bulk transcriptomic datasets. We validated key findings by RT-qPCR, western blotting, and immunofluorescence. Results: By single-cell transcriptomic analysis of 4,397 CAFs from six CRC-LM samples, we identified two main CAF populations, contractile CAFs and extracellular matrix (ECM)-remodeling/pro-angiogenic CAFs, and four subpopulations with distinct phenotypes. We found that ECM-remodeling/pro-angiogenic CAFs derive from portal resident fibroblasts. They associate with areas of strong desmoplastic reaction and Wnt signaling in low-proliferating tumor cells engulfed in a stiff extracellular matrix. By integrating public single-cell primary liver tumor data, we propose a model to explain how different liver malignancies recruit CAFs of different origins to this organ. Lastly, we found that LTBP2 plays an important role in modulating collagen biosynthesis, ECM organization, and adhesion pathways. We developed fully human antibodies against LTBP2 that depleted LTBP2+ CAFs in vitro. Conclusion: This study complements recent reports on CRC-LM CAF heterogeneity at the single-cell resolution. The number of sequenced CAFs was more than one order of magnitude larger compared to existing data. LTBP2 targeting by antibodies might create opportunities to deplete ECM-remodeling CAFs in CRC-LMs. This might be combined with other therapies, e.g., anti-angiogenic compounds as already done in CRC. Moreover, we showed that in intrahepatic cholangiocarcinoma, in which ECM-remodeling CAF proportion is similar to that of CRC-LM, several genes expressed by ECM-remodeling CAFs, such as LTBP2, were associated with survival.

ADAMTS6 cleaves the large latent TGFß complex and increases the mechanotension of cells to activate TGFß.

The ADAMTS superfamily is composed of secreted metalloproteases and structurally related non-catalytic ADAMTS-like proteins. A subset of this superfamily, including ADAMTS6, ADAMTS10 and ADAMTSL2, are involved in elastic fiber assembly and bind to fibrillin and other matrix molecules that regulate the extracellular bioavailability of the potent growth factor TGFß. Fibrillinopathies, that can also result from mutation of these ADAMTS/L proteins, have been linked to disrupted TGFß homeostasis. ADAMTS6 and ADAMTS10 are homologous metalloproteases with poorly characterized substrates where ADAMTS10 is thought to process fibrillin-2 and ADAMTS6 latent TGFß-binding protein (LTBP)-1. In order to understand the contribution of ADAMTS6, and these other members of the ADAMTS/L family, to TGFß homeostasis, we have analyzed the effects of ADAMTS6, ADAMTS10 and ADAMTSL2 expression on TGFß activation. We found that their expression increases TGFß activation in a dose dependent manner, following stimulation with mature TGFß1. For ADAMTS6, the catalytically active protease is required for effective TGFß activation, where ADAMTS6 cleaves LTBP3 as well as LTBP1, and binds to the large latent TGFß complexes of LTBP1 and LTBP3. Furthermore, ADAMTS6 expression increases the mechanotension of cells which results in inactivation of the Hippo Pathway, resulting in an increased translocation of YAP/TAZ complex to the nucleus. Together these findings suggest that when the balance of TGFß is perturbed ADAMTS6 can influence TGFß activation via two mechanisms. It directly cleaves the latent TGFß complexes and also acts indirectly, along with ADAMTS10 and ADAMTSL2, by altering the mechanotension of cells. Together this increases activation of TGFß from large latent complexes which may contribute to disease pathogenesis.

LTBP2 Knockdown Promotes Ferroptosis in Gastric Cancer Cells through p62-Keap1-Nrf2 Pathway.

Gastric cancer (GC) is one of the most common gastrointestinal malignancies. Ferroptosis is a new type of peroxidation-driven and iron-dependent cell death. However, the biological functions and exact regulatory mechanisms of ferroptosis in GC remain elusive. Here, we performed RNAi and gene transfection, cell viability assay, lipid peroxidation assay, reactive oxygen species (ROS) assay, glutathione assay, qRT-PCR, Western blotting, and transmission electron microscopy (TEM) to study ferroptosis in gastric cancer. The results revealed that silencing latent transforming growth factor ß binding proteins (LTBP2) can significantly inhibit GC cell proliferation and decrease cellular GSH levels, reduce GPX4 activity, and increase ROS generation and malondialdehyde (MDA) levels, leading to ferroptosis in GC cells. In addition, we demonstrate that suppression of LTBP2 could regulate the p62-Keap1-Nrf2 pathway, thereby downregulating the GPX4 and xCT expression and upregulating the PTGS2 and 4HNE expression. Our findings described a new role of LTBP2 in regulating ferroptosis, which heralds the prospect of ferroptosis-mediated cancer therapy.

Specificity of TGF-ß1 signal designated by LRRC33 and integrin αVß8.

Myeloid lineage cells present the latent form of transforming growth factor-ß1 (L-TGF-ß1) to the membrane using an anchor protein LRRC33. Integrin αVß8 activates extracellular L-TGF-ß1 to trigger the downstream signaling functions. However, the mechanism designating the specificity of TGF-ß1 presentation and activation remains incompletely understood. Here, we report cryo-EM structures of human L-TGF-ß1/LRRC33 and integrin αVß8/L-TGF-ß1 complexes. Combined with biochemical and cell-based analyses, we demonstrate that LRRC33 only presents L-TGF-ß1 but not the -ß2 or -ß3 isoforms due to difference of key residues on the growth factor domains. Moreover, we reveal a 2:2 binding mode of integrin αVß8 and L-TGF-ß1, which shows higher avidity and more efficient L-TGF-ß1 activation than previously reported 1:2 binding mode. We also uncover that the disulfide-linked loop of the integrin subunit ß8 determines its exquisite affinity to L-TGF-ß1. Together, our findings provide important insights into the specificity of TGF-ß1 signaling achieved by LRRC33 and integrin αVß8.

Fibrillin-1 deficiency in the outer perichondrium causes longitudinal bone overgrowth in mice with Marfan syndrome.

A disproportionate tall stature is the most evident manifestation in Marfan syndrome (MFS), a multisystem condition caused by mutations in the extracellular protein and TGFß modulator, fibrillin-1. Unlike cardiovascular manifestations, there has been little effort devoted to unravel the molecular mechanism responsible for long bone overgrowth in MFS. By combining the Cre-LoxP recombination system with metatarsal bone cultures, here we identify the outer layer of the perichondrium as the tissue responsible for long bone overgrowth in MFS mice. Analyses of differentially expressed genes in the fibrillin-1-deficient perichondrium predicted that loss of TGFß signaling may influence chondrogenesis in the neighboring epiphyseal growth plate (GP). Immunohistochemistry revealed that fibrillin-1 deficiency in the outer perichondrium is associated with decreased accumulation of latent TGFß-binding proteins (LTBPs)-3 and -4, and reduced levels of phosphorylated (activated) Smad2. Consistent with these findings, mutant metatarsal bones grown in vitro were longer and released less TGFß than the wild-type counterparts. Moreover, addition of recombinant TGFß1 normalized linear growth of mutant metatarsal bones. We conclude that longitudinal bone overgrowth in MFS is accounted for by diminished sequestration of LTBP-3 and LTBP-4 into the fibrillin-1-deficient matrix of the outer perichondrium, which results in less TGFß signaling locally and improper GP differentiation distally.

Elevated latent transforming growth factor beta binding protein 2 in endometriosis promotes endometrial stromal cell invasion and proliferation via the NF-kB signaling pathway.

Endometriosis, defined as the abnormal growth of functional endometrium outside the uterus, is characterized by the abnormal phenotype of endometrial cells. This study aimed to investigate the role of latent transforming growth factor beta binding protein 2 (LTBP2), an extracellular matrix protein, in the occurrence and development of endometriosis. Elevated LTBP2 expression levels were observed in endometrial tissues and serum of endometriosis patients and their area under the ROC curve (AUC) values for distinguishing endometriosis were 0.9044 and 0.9534, respectively. Overexpressing-LTBP2 could promote proliferation, migration, and invasion, whereas suppressing apoptosis of endometrial stromal cells (ESCs). Moreover, LTBP2 downregulation induced the opposite effect. The supernatant from ESCs overexpressing LTBP2 promoted the tube formation of human umbilical vein endothelial cells (HUVECs), thus indicating an angiogenic effect. Furthermore, overexpression of LTBP2 facilitated the inflammation and might promote endometriosis progression through the NF-kB signaling pathway. Conclusively, LTBP2 might be a potential target in the diagnosis and treatment of endometriosis.

LTBP1 promotes fibrillin incorporation into the extracellular matrix.

LTBP1 is a large extracellular matrix protein and an associated ligand of fibrillin-microfibrils. Knowledge of LTBP1 functions is largely limited to its role in targeting and sequestering TGFß growth factors within the extracellular matrix, thereby regulating their bioavailability. However, the recent description of a wide spectrum of phenotypes in multiple tissues in patients harboring LTBP1 pathogenic variants suggests a multifaceted role of the protein in the homeostasis of connective tissues. To better understand the human pathology caused by LTBP1 deficiency it is important to investigate its functional role in extracellular matrix formation. In this study, we show that LTBP1 coordinates the incorporation of fibrillin-1 and -2 into the extracellular matrix in vitro. We also demonstrate that this function is differentially exerted by the two isoforms, the short and long forms of LTBP1. Thereby our findings uncover a novel TGFß-independent LTBP1 function potentially contributing to the development of connective tissue disorders.

Elastic Fibre Proteins in Elastogenesis and Wound Healing.

As essential components of our connective tissues, elastic fibres give tissues such as major blood vessels, skin and the lungs their elasticity. Their formation is complex and co-ordinately regulated by multiple factors. In this review, we describe key players in elastogenesis: fibrillin-1, tropoelastin, latent TGFß binding protein-4, and fibulin-4 and -5. We summarise their roles in elastogenesis, discuss the effect of their mutations on relevant diseases, and describe their interactions involved in forming the elastic fibre network. Moreover, we look into their roles in wound repair for a better understanding of their potential application in tissue regeneration.

CircWHSC1 expedites cervical cancer progression via miR-532-3p/LTBP2 axis.

Dysregulated circRNAs have potential roles in the progression of various cancer types, including cervical cancer (CaCx). The carcinogenic roles of circRNA Wolf-Hirschhorn syndrome candidate gene-1 (circWHSC1) are described in the development of diverse cancers. The objective of this study was to investigate the expression and the underlying role of circWHSC1 in CaCx. The expression of circWHSC1 was detected by real-time PCR. After the suppression of circWHSC1 expression, the changes in the proliferation, migration, invasion, and apoptosis capacities were detected by CCK-8 assay, colony formation assay, Transwell assays, flow cytometry, and the determination of apoptosis-related proteins. The interplay among circWHSC1, miR-532-3p, and latent transforming growth factor-ß binding protein 2 (LTBP2) was confirmed by luciferase reporter and biotinylated RNA pull-down assays. A nude mice xenograft tumor model was established to evaluate the anti-tumorigenic role of circWHSC1 silencing in vivo. CircWHSC1 was overexpressed in CaCx tissues and cell lines and its high expression was inversely associated with the survival rate of patients with CaCx. CircWHSC1 silencing was capable of suppressing the proliferation, metastasis, and invasion of tumor cells and inducing apoptosis. Investigation to its molecular mechanism revealed that circWHSC1 functioned as a competitive endogenous RNA (ceRNA), mediating LTBP2 expression by targeting miR-532-3p. The in vivo experiments further confirmed the inhibition of tumor growth and metastasis by circWHSC1 knockdown. The circWHSC1-mediated miR-532-3p/LTBP2 signaling axis might be a novel therapeutic target for CaCx.

Latent TGFß complexes are transglutaminase cross-linked to fibrillin to facilitate TGFß activation.

TGFß superfamily members are potent growth factors in the extracellular matrix with essential roles in all aspects of cellular behaviour. Latent TGFß binding proteins (LTBPs) are co-expressed with TGFß, essential for correct folding and secretion of the growth factor, to form large latent complexes. These large latent complexes bind extracellular proteins such as fibrillin for sequestration of TGFß in the matrix, essential for normal tissue function, and dysregulated TGFß signalling is a hallmark of many fibrillinopathies. Transglutaminase-2 (TG2) cross-linking of LTBPs is known to play a role in TGFß activation but the underlying molecular mechanisms are not resolved. Here we show that fibrillin is a matrix substrate for TG2 and that TG2 cross-linked complexes can be formed between fibrillin and LTBP-1 and -3, and their latent TGFß complexes. The structure of the fibrillin-LTBP1 complex shows that the two elongated proteins interact in a perpendicular arrangement which would allow them to form distal interactions between the matrix and the cell surface. Formation of the cross-link with fibrillin does not change the interaction between latent TGFß and integrin αVß6 but does increase TGFß activation in cell-based assays. The activating effect may be due to direction of the latent complexes to the cell surface by fibrillin, as competition with heparan sulphate can ameliorate the activating effect. Together, these data support that TGFß activation can be enhanced by covalent tethering of LTBPs to the matrix via fibrillin.

Latent TGFß-binding proteins 1 and 3 protect the larval zebrafish outflow tract from aneurysmal dilatation.

Aortic root aneurysm is a common cause of morbidity and mortality in Loeys-Dietz and Marfan syndromes, where perturbations in transforming growth factor beta (TGFß) signaling play a causal or contributory role, respectively. Despite the advantages of cross-species disease modeling, animal models of aortic root aneurysm are largely restricted to genetically engineered mice. Here, we report that zebrafish devoid of the genes encoding latent-transforming growth factor beta-binding protein 1 and 3 (ltbp1 and ltbp3, respectively) develop rapid and severe aneurysm of the outflow tract (OFT), the aortic root equivalent. Similar to syndromic aneurysm tissue, the distended OFTs display evidence for paradoxical hyperactivated TGFß signaling. RNA-sequencing revealed significant overlap between the molecular signatures of disease tissue from mutant zebrafish and a mouse model of Marfan syndrome. Moreover, chemical inhibition of TGFß signaling in wild-type animals phenocopied mutants but chemical activation did not, demonstrating that TGFß signaling is protective against aneurysm. Human relevance is supported by recent studies implicating genetic lesions in LTBP3 and, potentially, LTBP1 as heritable causes of aortic root aneurysm. Ultimately, our data demonstrate that zebrafish can now be leveraged to interrogate thoracic aneurysmal disease and identify novel lead compounds through small-molecule suppressor screens. This article has an associated First Person interview with the first author of the paper.

Single-Nucleus Transcriptional Profiling of Chronic Kidney Disease after Cisplatin Nephrotoxicity.

Cisplatin induces both acute and chronic nephrotoxicity during chemotherapy in patients with cancer. Presented here is the first study of single-nucleus RNA sequencing (snRNA-seq) of cisplatin-induced nephrotoxicity. Repeated low-dose cisplatin treatment (RLDC) led to decreases in renal function and kidney weight in mice at 9 weeks. The kidneys of these mice showed tubular degeneration and dilation. snRNA-seq identified 16 cell types and 17 cell clusters in these kidneys. Cluster-by-cluster comparison demonstrated cell type-specific changes in gene expression and identified a unique proximal tubule (PT) injury/repair cluster that co-expressed the injury marker kidney injury molecule-1 (Kim1) and the proliferation marker Ki-67. Compared with control, post-RLDC kidneys had 424 differentially expressed genes in PT cells, including tubular transporters and cytochrome P450 enzymes involved in lipid metabolism. snRNA-seq also revealed transcriptional changes in potential PT injury markers (Krt222, Eda2r, Ltbp2, and Masp1) and repair marker (Bex4). RLDC induced inflammation and proinflammatory cytokines (RelB, TNF-α, Il7, Ccl2, and Cxcl2) and the expression of fibrosis markers (fibronectin, collagen I, connective tissue growth factor, vimentin, and α-smooth muscle actin). Together, these results provide new insights into RLDC-induced transcriptional changes at the single-cell level that may contribute to the development of chronic kidney problems in patients with cancer after cisplatin chemotherapy.

The role of LTBPs in TGF beta signaling.

The purpose of this review is to discuss the transforming growth factor beta (TGFB) binding proteins (LTBP) with respect to their participation in the activity of TGFB. We first describe pertinent aspects of the biology and cell function of the LTBPs. We then summarize the physiological consequences of LTBP loss in humans and mice. Finally, we consider a number of outstanding questions relating to LTBP function.

Compositional Analysis of Extracellular Aggregates in the Eyes of Patients With Exfoliation Syndrome and Exfoliation Glaucoma.

Purpose: Exfoliation syndrome (XFS) is a condition characterized by the production of insoluble fibrillar aggregates (exfoliation material; XFM) in the eye and elsewhere. Many patients with XFS progress to exfoliation glaucoma (XFG), a significant cause of global blindness. We used quantitative mass spectrometry to analyze the composition of XFM in lens capsule specimens and in aqueous humor (AH) samples from patients with XFS, patients with XFG and unaffected individuals. Methods: Pieces of lens capsule and samples of AH were obtained with consent from patients undergoing cataract surgery. Tryptic digests of capsule or AH were analyzed by high-performance liquid chromatography-mass spectrometry and relative differences between samples were quantified using the tandem mass tag technique. The distribution of XFM on the capsular surface was visualized by SEM and super-resolution light microscopy. Results: A small set of proteins was consistently upregulated in capsule samples from patients with XFS and patients with XFG, including microfibril components fibrillin-1, latent transforming growth factor-ß-binding protein-2 and latent transforming growth factor-ß-binding protein-3. Lysyl oxidase-like 1, a cross-linking enzyme associated with XFS in genetic studies, was an abundant XFM constituent. Ligands of the transforming growth factor-ß superfamily were prominent, including LEFTY2, a protein best known for its role in establishing the embryonic body axis. Elevated levels of LEFTY2 were also detected in AH from patients with XFG, a finding confirmed subsequently by ELISA. Conclusions: This analysis verified the presence of suspected XFM proteins and identified novel components. Quantitative comparisons between patient samples revealed a consistent XFM proteome characterized by strong expression of fibrillin-1, lysyl oxidase-like-1, and LEFTY2. Elevated levels of LEFTY2 in the AH of patients with XFG may serve as a biomarker for the disease.