Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in Mice.

Chronic kidney disease (CKD) is associated with various pathologic changes, including elevations in serum phosphate levels (hyperphosphatemia), vascular calcification, and skeletal muscle atrophy. Elevated phosphate can damage vascular smooth muscle cells and cause vascular calcification. Here, we determined whether high phosphate can also affect skeletal muscle cells and whether hyperphosphatemia, in the context of CKD or by itself, is associated with skeletal muscle atrophy. As models of hyperphosphatemia with CKD, we studied mice receiving an adenine-rich diet for 14 weeks and mice with deletion of Collagen 4a3 (Col4a3-/-). As models of hyperphosphatemia without CKD, we analyzed mice receiving a high-phosphate diet for three and six months as well as a genetic model for klotho deficiency (kl/kl). We found that adenine, Col4a3-/-, and kl/kl mice have reduced skeletal muscle mass and function and develop atrophy. Mice on a high-phosphate diet for six months also had lower skeletal muscle mass and function but no significant signs of atrophy, indicating less severe damage compared with the other three models. To determine the potential direct actions of phosphate on skeletal muscle, we cultured primary mouse myotubes in high phosphate concentrations, and we detected the induction of atrophy. We conclude that in experimental mouse models, hyperphosphatemia is sufficient to induce skeletal muscle atrophy and that, among various other factors, elevated phosphate levels might contribute to skeletal muscle injury in CKD.

COL4A2 enhances thyroid cancer cell proliferation through the AKT pathway.

Objectives: Thyroid cancer (THCA) is the most common malignant tumor in endocrine system and the incidence has been increasing worldwide. And the number of patients dying from THCA has also gradually risen because the incidence continues to increase, so the mechanisms related to effective targets is necessary to improve the survival. This study was to preliminarily investigate the effects of the COL4A2 gene on the regulation of thyroid cancer (THCA) cell proliferation and the associated pathways. Methods: Bioinformatics analysis revealed that COL4A2 was closely associated with cancer development. COL4A2 expression in THCA tissues was analyzed using immunohistochemistry, and survival information was determined via Kaplan‒Meier curves. The expression of COL4A2 and AKT pathway-related genes were analyzed using qPCR and western blot analyses. Colony formation as well as CCK-8 assays exhibited the cell proliferation level and cell activity, respectively. Downstream of COL4A2 was identified by Gene set enrichment analysis (GSEA). The effects of the COL4A2 and AKT pathways on THCA tumor growth in vivo were determined using a mouse model. Results: Bioinformatics analysis exhibited that COL4A2 plays a significant role in cancer and that the AKT pathway is downstream of COL4A2. THCA patients with high COL4A2 expression had shorter recurrence-free survival. Upregulation of COL4A2 gene expression in 2 THCA cell lines promoted tumor cell growth and activity. The use of AKT pathway blockers also restrained the growth and activity of the 2 THCA cell lines. The use of AKT pathway blockers reduced tumor volume and mass and prolonged mouse survival. Conclusions: COL4A2 can promote the growth as well as development of THCA through the AKT pathway and COL4A2 could be used as a target for THCA.

Chemical chaperones to the rescue of Alport syndrome?

Alport syndrome is a hereditary kidney disease caused by collagen IV mutations that interfere with the formation and deposition of the α3α4α5 protomer into the glomerular basement membrane. In this issue, Yu et al. show that the chemical chaperone tauroursodeoxycholic acid prevented kidney structural changes and function decline in mice with a pathogenic missense Col4a3 mutation by increasing mutant α3α4α5 protomer glomerular basement membrane deposition and preventing podocyte apoptosis induced by endoplasmic reticulum stress.

Collagen IV deficiency causes hypertrophic remodeling and endothelium-dependent hyperpolarization in small vessel disease with intracerebral hemorrhage.

BACKGROUND: Genetic variants in COL4A1 and COL4A2 (encoding collagen IV alpha chain 1/2) occur in genetic and sporadic forms of cerebral small vessel disease (CSVD), a leading cause of stroke, dementia and intracerebral haemorrhage (ICH). However, the molecular mechanisms of CSVD with ICH and COL4A1/COL4A2 variants remain obscure. METHODS: Vascular function and molecular investigations in mice with a Col4a1 missense mutation and heterozygous Col4a2 knock-out mice were combined with analysis of human brain endothelial cells harboring COL4A1/COL4A2 mutations, and brain tissue of patients with sporadic CSVD with ICH. FINDINGS: Col4a1 missense mutations cause early-onset CSVD independent of hypertension, with enhanced vasodilation of small arteries due to endothelial dysfunction, vascular wall thickening and reduced stiffness. Mechanistically, the early-onset dysregulated endothelium-dependent hyperpolarization (EDH) is due to reduced collagen IV levels with elevated activity and levels of endothelial Ca2+-sensitive K+ channels. This results in vasodilation via the Na/K pump in vascular smooth muscle cells. Our data support this endothelial dysfunction preceding development of CSVD-associated ICH is due to increased cytoplasmic Ca2+ levels in endothelial cells. Moreover, cerebral blood vessels of patients with sporadic CSVD show genotype-dependent mechanisms with wall thickening and lower collagen IV levels in those harboring common non-coding COL4A1/COL4A2 risk alleles. INTERPRETATION: COL4A1/COL4A2 variants act in genetic and sporadic CSVD with ICH via dysregulated EDH, and altered vascular wall thickness and biomechanics due to lower collagen IV levels and/or mutant collagen IV secretion. These data highlight EDH and collagen IV levels as potential treatment targets. FUNDING: MRC, Wellcome Trust, BHF.

Skeletal pathology in mouse models of Gould syndrome is partially alleviated by genetically reducing TGFß signaling.

Skeletal defects are hallmark features of many extracellular matrix (ECM) and collagen-related disorders. However, a biological function in bone has never been defined for the highly evolutionarily conserved type IV collagen. Collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) form α1α1α2 (IV) heterotrimers that represent a fundamental basement membrane constituent present in every organ of the body, including the skeleton. COL4A1 and COL4A2 mutations cause Gould syndrome, a variable and clinically heterogenous multisystem disorder generally characterized by the presence of cerebrovascular disease with ocular, renal, and muscular manifestations. We have previously identified elevated TGFß signaling as a pathological insult resulting from Col4a1 mutations and demonstrated that reducing TGFß signaling ameliorate ocular and cerebrovascular phenotypes in Col4a1 mutant mouse models of Gould syndrome. In this study, we describe the first characterization of skeletal defects in Col4a1 mutant mice that include a developmental delay in osteogenesis and structural, biomechanical and vascular alterations of mature bones. Using distinct mouse models, we show that allelic heterogeneity influences the presentation of skeletal pathology resulting from Col4a1 mutations. Importantly, we found that TGFß target gene expression is elevated in developing bones from Col4a1 mutant mice and show that genetically reducing TGFß signaling partially ameliorates skeletal manifestations. Collectively, these findings identify a novel and unsuspected role for type IV collagen in bone biology, expand the spectrum of manifestations associated with Gould syndrome to include skeletal abnormalities, and implicate elevated TGFß signaling in skeletal pathogenesis in Col4a1 mutant mice.

A distinct subset of urothelial cells with enhanced EMT features promotes chemotherapy resistance and cancer recurrence by increasing COL4A1-ITGB1 mediated angiogenesis.

Drug resistance and tumor recurrence remain clinical challenges in the treatment of urothelial carcinoma (UC). However, the underlying mechanism is not fully understood. Here, we performed single-cell RNA sequencing and identified a subset of urothelial cells with epithelial-mesenchymal transition (EMT) features (EMT-UC), which is significantly correlated with chemotherapy resistance and cancer recurrence. To validate the clinical significance of EMT-UC, we constructed EMT-UC like cells by introducing overexpression of two markers, Zinc Finger E-Box Binding Homeobox 1 (ZEB1) and Desmin (DES), and examined their histological distribution characteristics and malignant phenotypes. EMT-UC like cells were mainly enriched in UC tissues from patients with adverse prognosis and exhibited significantly elevated EMT, migration and gemcitabine tolerance in vitro. However, EMT-UC was not specifically identified from tumorous tissues, certain proportion of them were also identified in adjacent normal tissues. Tumorous EMT-UC highly expressed genes involved in malignant behaviors and exhibited adverse prognosis. Additionally, tumorous EMT-UC was associated with remodeled tumor microenvironment (TME), which exhibited high angiogenic and immunosuppressive potentials compared with the normal counterparts. Furthermore, a specific interaction of COL4A1 and ITGB1 was identified to be highly enriched in tumorous EMT-UC, and in the endothelial component. Targeting the interaction of COL4A1 and ITGB1 with specific antibodies significantly suppressed tumorous angiogenesis and alleviated gemcitabine resistance of UC. Overall, our findings demonstrated that the driven force of chemotherapy resistance and recurrence of UC was EMT-UC mediated COL4A1-ITGB1 interaction, providing a potential target for future UC treatment.

Mutations in fibulin-1 and collagen IV suppress the short healthspan of mig-17/ADAMTS mutants in Caenorhabditis elegans.

The ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family metalloprotease MIG-17 plays a crucial role in the migration of gonadal distal tip cells (DTCs) in Caenorhabditis elegans. MIG-17 is secreted from the body wall muscle cells and localizes to the basement membranes (BMs) of various tissues including the gonadal BM where it regulates DTC migration through its catalytic activity. Missense mutations in the BM protein genes, let-2/collagen IV a2 and fbl-1/fibulin-1, have been identified as suppressors of the gonadal defects observed in mig-17 mutants. Genetic analyses indicate that LET-2 and FBL-1 act downstream of MIG-17 to regulate DTC migration. In addition to the control of DTC migration, MIG-17 also plays a role in healthspan, but not in lifespan. Here, we examined whether let-2 and fbl-1 alleles can suppress the age-related phenotypes of mig-17 mutants. let-2(k196) fully and fbl-1(k201) partly, but not let-2(k193) and fbl-1(k206), suppressed the senescence defects of mig-17. Interestingly, fbl-1(k206), but not fbl-1(k201) or let-2 alleles, exhibited an extended lifespan compared to the wild type when combined with mig-17. These results reveal allele specific interactions between let-2 or fbl-1 and mig-17 in age-related phenotypes, indicating that basement membrane physiology plays an important role in organismal aging.

Heterogeneous fibroblasts contribute to fibrotic scar formation after spinal cord injury in mice and monkeys.

Spinal cord injury (SCI) leads to fibrotic scar formation at the lesion site, yet the heterogeneity of fibrotic scar remains elusive. Here we show the heterogeneity in distribution, origin, and function of fibroblasts within fibrotic scars after SCI in mice and female monkeys. Utilizing lineage tracing and single-cell RNA sequencing (scRNA-seq), we found that perivascular fibroblasts (PFs), and meningeal fibroblasts (MFs), rather than pericytes/vascular smooth cells (vSMCs), primarily contribute to fibrotic scar in both transection and crush SCI. Crabp2 + /Emb+ fibroblasts (CE-F) derived from meninges primarily localize in the central region of fibrotic scars, demonstrating enhanced cholesterol synthesis and secretion of type I collagen and fibronectin. In contrast, perivascular/pial Lama1 + /Lama2+ fibroblasts (LA-F) are predominantly found at the periphery of the lesion, expressing laminin and type IV collagen and functionally involved in angiogenesis and lipid transport. These findings may provide a comprehensive understanding for remodeling heterogeneous fibrotic scars after SCI.

Astaxanthin attenuates diabetic kidney injury through upregulation of autophagy in podocytes and pathological crosstalk with mesangial cells.

Objectives: Astaxanthin (ATX) is a strong antioxidant drug. This study aimed to investigate the effects of ATX on podocytes in diabetic nephropathy and the underlying renal protective mechanism of ATX, which leads to pathological crosstalk with mesangial cells.Methods: In this study, diabetic rats treated with ATX exhibited reduced 24-h urinary protein excretion and decreased blood glucose and lipid levels compared to vehicle-treated rats. Glomerular mesangial matrix expansion and renal tubular epithelial cell injury were also attenuated in ATX-treated diabetic rats compared to control rats.Results: ATX treatment markedly reduced the α-SMA and collagen IV levels in the kidneys of diabetic rats. Additionally, ATX downregulated autophagy levels. In vitro, compared with normal glucose, high glucose inhibited LC3-II expression and increased p62 expression, whereas ATX treatment reversed these changes. ATX treatment also inhibited α-SMA and collagen IV expression in cultured podocytes. Secreted factors (vascular endothelial growth factor B and transforming growth factor-ß) generated by high glucose-induced podocytes downregulated autophagy in human mesangial cells (HMCs); however, this downregulation was upregulated when podocytes were treated with ATX.Conclusions: The current study revealed that ATX attenuates diabetes-induced kidney injury likely through the upregulation of autophagic activity in podocytes and its antifibrotic effects. Crosstalk between podocytes and HMCs can cause renal injury in diabetes, but ATX treatment reversed this phenomenon.

Establishment of an induced pluripotent stem cell line from a patient with X-linked Alport syndrome carrying a hemizygous splicing variant (NM_033380; c.929[exon 16]delG) in the collagen type IV alpha 5 chain gene.

X-linked hereditary Alport syndrome (XLAS) type 1 (OMIM: 301050) results from a pathogenic variant in the collagen type IV alpha 5 chain (COL4A5) gene.A human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells of a 7-year-old male patient with XLAS using non-integrating episomal vector technique. The male donor had a heterozygous variant in the COL4A5 gene. The resulting iPSC line has a standard karyotype, can express pluripotent biomarkers, and is able to create germ layers in vivo. It can serve as a valuable cellular model for investigating the underlying mechanisms of XLAS.

Characterization of Ocular Morphology in Col4a3-/- Mice as a Murine Model for Alport Syndrome.

Purpose: The purpose of this study was to investigate the ocular morphological characteristics of Col4a3-/- mice as a model of Alport syndrome (AS) and the potential pathogenesis. Methods: The expression of collagen IV at 8, 12, and 21 weeks of age was evaluated by immunohistochemistry in wild-type (WT) and Col4a3-/- mice. Hematoxylin and eosin (H&E) staining and thickness measurements were performed to assess the thickness of anterior lens capsule and retina. Ultrastructure analysis of corneal epithelial basement membrane, anterior lens capsule, internal limiting membrane (ILM), and retinal pigment epithelium (RPE) basement membrane was performed using transmission electron microscopy. Finally, Müller cell activation was evaluated by glial fibrillary acidic protein (GFAP) expression. Results: Collagen IV was downregulated in the corneal epithelial basement membrane and ILM of Col4a3-/- mice. The hemidesmosomes of Col4a3-/- mice corneal epithelium became flat and less electron-dense than those of the WT group. Compared with those of the WT mice, the anterior lens capsules of Col4a3-/- mice were thinner. Abnormal structure was detected at the ILM Col4a3-/- mice, and the basal folds of the RPE basement membrane in Col4a3-/- mice were thicker and shorter. The retinas of Col4a3-/- mice were thinner than those of WT mice, especially within 1000 µm away from the optic nerve. GFAP expression enhanced in each age group of Col4a3-/- mice. Conclusions: Our results suggested that Col4a3-/- mice exhibit ocular anomalies similar to patients with AS. Additionally, Müller cells may be involved in AS retinal anomalies. Translational Relevance: This animal model could provide an opportunity to understand the underlying mechanisms of AS ocular disorders and to investigate potential new treatments.

AdamTS proteases control basement membrane heterogeneity and organ shape in Drosophila.

The basement membrane (BM) is an extracellular matrix that plays important roles in animal development. A spatial heterogeneity in composition and structural properties of the BM provide cells with vital cues for morphogenetic processes such as cell migration or cell polarization. Here, using the Drosophila egg chamber as a model system, we show that the BM becomes heterogeneous during development, with a reduction in Collagen IV density at the posterior pole and differences in the micropattern of aligned fiber-like structures. We identified two AdamTS matrix proteases required for the proper elongated shape of the egg chamber, yet the molecular mechanisms by which they act are different. Stall is required to establish BM heterogeneity by locally limiting Collagen IV protein density, whereas AdamTS-A alters the micropattern of fiber-like structures within the BM at the posterior pole. Our results suggest that AdamTS proteases control BM heterogeneity required for organ shape.

Diagnostic role of SPP1 and collagen IV in a rat model of type 2 diabetes mellitus with MASLD.

Type 2 diabetes mellitus combined with metabolic dysfunction-associated steatotic liver disease (MASLD) leads to an increasing incidence of liver injury year by year, and patients are at a significantly higher risk of developing cirrhosis or even liver failure. No drugs have emerged to specifically treat this disease. The aim of this study is to investigate the mechanisms and causative hub genes of type 2 diabetes combined with MASLD. The data were obtained through the GEO platform for bioinformatics analysis and validated by in vitro experiments to find the causative targets of type 2 diabetes mellitus combined with MASLD, which will provide some theoretical basis for the development of future therapeutic drugs. GSE23343 and GSE49541 were downloaded from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs) in type 2 diabetes mellitus combined with MASLD for functional enrichment analysis. And STRING database and Cytoscape software were used to construct Protein-Protein Interaction (PPI) and hub gene networks. And GO (gene ontology, GO) analysis and KEGG (Kyoto encyclopedia of genes and genomes, KEGG) enrichment analysis were performed on target genes. A total of 185 co-expressed DEGs were obtained by differential analysis, and 20 key genes involved in the development and progression of type 2 diabetes were finally screened. These 20 key genes were involved in 529 GO enrichment results and 20 KEGG enrichment results, and were mainly associated with ECM-receptor interaction, Focal adhesion, Human papillomavirus infection, PI3K-Akt signaling pathway, and the Toll-like receptor signaling pathway. A total of two target genes (SPP1, collagen IV) were found to be highly correlated with type 2 diabetes mellitus combined with MASLD. Real time PCR results showed that there was a significant difference in SPP1 and collagen IV mRNA expression among the three groups (P < 0.05). SPP1 and Collagen IV may be candidate biomarkers for type 2 diabetes mellitus combined with MASLD, as verified by bioinformatics screening and in vitro experiments. Our findings provide new targets for the treatment of type 2 diabetes combined with MASLD.

Comprehensive analysis of the expression, prognostic, and immune infiltration for COL4s in stomach adenocarcinoma.

BACKGROUND: Collagen (COL) genes, play a key role in tumor invasion and metastasis, are involved in tumor extracellular matrix (ECM)-receptor interactions and focal adhesion pathways. However, studies focusing on the diagnostic value of the COL4 family in stomach adenocarcinoma (STAD) are currently lacking. METHODS: The TCGA database was employed to retrieve the clinical features and RNA sequencing expression profiles of patients with STAD. We conducted an investigation to examine the expression disparities between STAD and adjacent normal tissues. Kaplan-Meier survival analysis was utilized to assess their prognostic significance, while Spearman correlation analysis was employed to determine their association with immune checkpoint genes and immunomodulatory molecules. Furthermore, GO and KEGG analyses were performed on the COL4s-related genes, revealing potential biological pathways through gene set enrichment analysis (GSEA). Subsequently, we explored the extent of immune infiltration of the COL4 family in STAD using the TIMER database. Lastly, the expression levels of the COL4 family in STAD were further validated through quantitative PCR (qPCR) and western blot techniques. RESULTS: The expression levels of COL4A1/2 were significantly upregulated, while COL4A5/6 were conspicuously downregulated in STAD. The survival analysis revealed that the upregulated COL4s indicated poorer overall survival, first progression and post-progression survival outcomes. Additionally, our findings demonstrated a positive correlation between the expressions of COL4A1/2/3/4 and the infiltration of immune cells, including CD8 + T cells, dendritic cells, macrophages, neutrophils and CD4 + T cells. Further correlation analysis uncovered a favorable association between the expression of COL4A1/2/3/4 and various crucial immunomodulatory molecules, immunological checkpoint molecules, and chemokines. Quantitative PCR analysis confirmed that the expression patterns of COL4A1/3/4/6 genes aligned with the finding from the TCGA database. However, gastric cancer cells exhibited downregulation of COL4A2. Consistently, the protein level of COL4A1 was elevated, whereas the protein level of COL4A2 was reduced in the gastric cancer cell lines. CONCLUSION: COL4s could potentially serve as biomarkers for diagnosing and predicting the prognosis of STAD.

Obesity Induces Temporally Regulated Alterations in the Extracellular Matrix That Drive Breast Tumor Invasion and Metastasis.

Obesity is associated with increased incidence and metastasis of triple-negative breast cancer, an aggressive breast cancer subtype. The extracellular matrix (ECM) is a major component of the tumor microenvironment that drives metastasis. To characterize the temporal effects of age and high-fat diet (HFD)-driven weight gain on the ECM, we injected allograft tumor cells at 4-week intervals into mammary fat pads of mice fed a control or HFD, assessing tumor growth and metastasis and evaluating the ECM composition of the mammary fat pads, lungs, and livers. Tumor growth was increased in obese mice after 12 weeks on HFD. Liver metastasis increased in obese mice only at 4 weeks, and elevated body weight correlated with increased metastasis to the lungs but not the liver. Whole decellularized ECM coupled with proteomics indicated that early stages of obesity were sufficient to induce changes in the ECM composition. Obesity led to an increased abundance of the proinvasive ECM proteins collagen IV and collagen VI in the mammary glands and enhanced the invasive capacity of cancer cells. Cells of stromal vascular fraction and adipose stem and progenitor cells were primarily responsible for secreting collagen IV and collagen VI, not adipocytes. Longer exposure to HFD increased the invasive potential of ECM isolated from the lungs and liver, with significant changes in ECM composition found in the liver with short-term HFD exposure. Together, these data suggest that changes in the breast, lungs, and liver ECM underlie some of the effects of obesity on triple-negative breast cancer incidence and metastasis. Significance: Organ-specific extracellular matrix changes in the primary tumor and metastatic microenvironment are mechanisms by which obesity contributes to breast cancer progression.

Evaluating neural crest cell migration in a Col4a1 mutant mouse model of ocular anterior segment dysgenesis.

The periocular mesenchyme (POM) is a transient migratory embryonic tissue derived from neural crest cells (NCCs) and paraxial mesoderm that gives rise to most of the structures in front of the eye. Morphogenetic defects of these structures can impair aqueous humor outflow, leading to elevated intraocular pressure and glaucoma. Mutations in collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause Gould syndrome - a multisystem disorder often characterized by variable cerebrovascular, ocular, renal, and neuromuscular manifestations. Approximately one-third of individuals with COL4A1 and COL4A2 mutations have ocular anterior segment dysgenesis (ASD), including congenital glaucoma resulting from abnormalities of POM-derived structures. POM differentiation has been a major focus of ASD research, but the underlying cellular mechanisms are still unclear. Moreover, earlier events including NCC migration and survival defects have been implicated in ASD; however, their roles are not as well understood. Vascular defects are among the most common consequences of COL4A1 and COL4A2 mutations and can influence NCC survival and migration. We therefore hypothesized that NCC migration might be impaired by COL4A1 and COL4A2 mutations. In this study, we used 3D confocal microscopy, gross morphology, and quantitative analyses to test NCC migration in Col4a1 mutant mice. We show that homozygous Col4a1 mutant embryos have severe embryonic growth retardation and lethality, and we identified a potential maternal effect on embryo development. Cerebrovascular defects in heterozygous Col4a1 mutant embryos were present as early as E9.0, showing abnormal cerebral vasculature plexus remodeling compared to controls. We detected abnormal NCC migration within the diencephalic stream and the POM in heterozygous Col4a1 mutants whereby mutant NCCs formed smaller diencephalic migratory streams and POMs. In these settings, migratory NCCs within the diencephalic stream and POM localize farther away from the developing vasculature. Our results show for the first time that Col4a1 mutations lead to cranial NCCs migratory defects in the context of early onset defective angiogenesis without affecting cell numbers, possibly impacting the relation between NCCs and the blood vessels during ASD development.

Immunohistochemical findings of lens capsules obtained from patients with dead bag syndrome.

PURPOSE: To investigate the extracellular matrix and cellular components in lens capsules extracted from patients with dead bag syndrome (DBS) through immunohistochemistry. SETTING: Department of Ophthalmology, Wakayama Medical University School of Medicine, Wakayama, Japan, and Department of Ophthalmology and Visual Sciences, John A. Moran Eye Center, University of Utah, Salt Lake City, Utah. DESIGN: Immunohistochemical experimental study. METHODS: 9 capsular bag specimens from DBS cases, as well as 2 control specimens from late-postoperative in-the-bag intraocular lens dislocation cases related to previous vitrectomy, pseudoexfoliation, and blunt trauma were included. They were processed for histopathology; unstained sections were obtained from each one and analyzed by immunohistochemistry targeting collagen type IV, laminin, vimentin, collagen type I, and fibronectin. RESULTS: Immunohistochemistry in DBS showed lens capsule stained for basement membrane components. The outer part of the anterior capsule that was split from the inner part was more markedly stained for type IV collagen as compared with the posterior part. Faint staining for fibrous posterior capsular opacification (PCO) components, for example, collagen type I and fibronectin, was detected in limited areas, but the major portion of the capsule was free from these components. Small spotty vimentin-positive materials, suggesting the presence of cell debris, were also detected in limited samples. CONCLUSIONS: Small amounts of fibrotic PCO components were detected in capsules extracted from patients with DBS, but their major parts were free from PCO components. Current findings suggest small amounts of lens epithelial cells were present after surgery and secreted fibrous components before undergoing cell death process.

Content and tissue expression of Collagen I, Collagen IV, and Laminin in the Extracellular Matrix in Prostate Adenocarcinoma.

Prostate cancer is one of the most common neoplasm in the male population. It is not known why some tumors become more aggressive than others. Although most studies show changes in the expression of cell adhesion molecules and the extracellular matrix correlated with the Gleason score, no study has objectively measured the tissue content of these molecules. This study aims to measure the content and tissue expression of collagen type I and IV and laminin in the extracellular matrix of patients with prostate adenocarcinoma and correlate these findings with the Gleason score and clinical characteristics. Forty-one patients who underwent radical prostate surgery at the Urology Department of a reference Hospital in Brazil between January 2015 and December 2020 were studied. The tissue protein content was estimated under light microscopy at a final magnification of 200 × . The mean collagen I score in prostate adenocarcinoma tissue samples was 7.16 ± 1.03 pixels/field. The mean type IV collagen score was 3.44 ± 0.61 pixels/field. The mean laminin score was 5.19 ± 0.79 pixels/field. The total Gleason score was correlated with both collagen and laminin. All the correlations were negative, which shows that the higher the collagen/laminin expression was, the lower the total Gleason score (p-value < 0,05). According to the Pearson correlation analysis, age has no statistical relationship with collagen and laminin content. PSA, in turn, showed a correlation only with laminin, but r = -0.378 (p = 0.015). Among the associated diseases and lifestyle habits, there is only statistical significance in the comparison of alcoholism for collagen I. For collagen IV and laminin, no statistical significance was obtained with the clinical variables analyzed.

TGFß Signaling Dysregulation May Contribute to COL4A1-Related Glaucomatous Optic Nerve Damage.

Purpose: Mutations in the genes encoding type IV collagen alpha 1 (COL4A1) and alpha 2 (COL4A2) cause a multisystem disorder that includes ocular anterior segment dysgenesis (ASD) and glaucoma. We previously showed that transforming growth factor beta (TGFß) signaling was elevated in developing anterior segments from Col4a1 mutant mice and that reducing TGFß signaling ameliorated ASD, supporting a role for the TGFß pathway in disease pathogenesis. Here, we tested whether altered TGFß signaling also contributes to glaucoma-related phenotypes in Col4a1 mutant mice. Methods: To test the role of TGFß signaling in glaucoma-relevant phenotypes, we genetically reduced TGFß signaling using mice with mutated Tgfbr2, which encodes the common receptor for all TGFß ligands in Col4a1+/G1344D mice. We performed slit-lamp biomicroscopy and optical coherence tomography for qualitative and quantitative analyses of anterior and posterior ocular segments, histological analyses of ocular tissues and optic nerves, and intraocular pressure assessments using rebound tonometry. Results: Col4a1+/G1344D mice showed defects of the ocular drainage structures, including iridocorneal adhesions, and phenotypes consistent with glaucomatous neurodegeneration, including thinning of the nerve fiber layer, retinal ganglion cell loss, optic nerve head excavation, and optic nerve degeneration. We found that reducing TGFß receptor 2 (TGFBR2) was protective for ASD, ameliorated ocular drainage structure defects, and protected against glaucomatous neurodegeneration in Col4a1+/G1344D mice. Conclusions: Our results suggest that elevated TGFß signaling contributes to glaucomatous neurodegeneration in Col4a1 mutant mice.

Liver sinusoidal endothelial cells contribute to portal hypertension through collagen type IV-driven sinusoidal remodeling.

Portal hypertension (PHTN) is a severe complication of liver cirrhosis and is associated with intrahepatic sinusoidal remodeling induced by sinusoidal resistance and angiogenesis. Collagen type IV (COL4), a major component of basement membrane, forms in liver sinusoids upon chronic liver injury. However, the role, cellular source, and expression regulation of COL4 in liver diseases are unknown. Here, we examined how COL4 is produced and how it regulates sinusoidal remodeling in fibrosis and PHTN. Human cirrhotic liver sample RNA sequencing showed increased COL4 expression, which was further verified via immunofluorescence staining. Single-cell RNA sequencing identified liver sinusoidal endothelial cells (LSECs) as the predominant source of COL4 upregulation in mouse fibrotic liver. In addition, COL4 was upregulated in a TNF-α/NF-κB-dependent manner through an epigenetic mechanism in LSECs in vitro. Indeed, by utilizing a CRISPRi-dCas9-KRAB epigenome-editing approach, epigenetic repression of the enhancer-promoter interaction showed silencing of COL4 gene expression. LSEC-specific COL4 gene mutation or repression in vivo abrogated sinusoidal resistance and angiogenesis, which thereby alleviated sinusoidal remodeling and PHTN. Our findings reveal that LSECs promote sinusoidal remodeling and PHTN during liver fibrosis through COL4 deposition.