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1.
J Clin Invest ; 134(10)2024 May 15.
Article in English | MEDLINE | ID: mdl-38747292

ABSTRACT

Cerebral small vessel disease (cSVD) encompasses a heterogeneous group of age-related small vessel pathologies that affect multiple regions. Disease manifestations range from lesions incidentally detected on neuroimaging (white matter hyperintensities, small deep infarcts, microbleeds, or enlarged perivascular spaces) to severe disability and cognitive impairment. cSVD accounts for approximately 25% of ischemic strokes and the vast majority of spontaneous intracerebral hemorrhage and is also the most important vascular contributor to dementia. Despite its high prevalence and potentially long therapeutic window, there are still no mechanism-based treatments. Here, we provide an overview of the recent advances in this field. We summarize recent data highlighting the remarkable continuum between monogenic and multifactorial cSVDs involving NOTCH3, HTRA1, and COL4A1/A2 genes. Taking a vessel-centric view, we discuss possible cause-and-effect relationships between risk factors, structural and functional vessel changes, and disease manifestations, underscoring some major knowledge gaps. Although endothelial dysfunction is rightly considered a central feature of cSVD, the contributions of smooth muscle cells, pericytes, and other perivascular cells warrant continued investigation.


Subject(s)
Cerebral Small Vessel Diseases , Collagen Type IV , Receptor, Notch3 , Humans , Cerebral Small Vessel Diseases/genetics , Cerebral Small Vessel Diseases/physiopathology , Cerebral Small Vessel Diseases/pathology , Receptor, Notch3/genetics , Receptor, Notch3/metabolism , Collagen Type IV/genetics , Collagen Type IV/metabolism , High-Temperature Requirement A Serine Peptidase 1/genetics , High-Temperature Requirement A Serine Peptidase 1/metabolism , Animals
2.
Invest Ophthalmol Vis Sci ; 65(5): 15, 2024 May 01.
Article in English | MEDLINE | ID: mdl-38717426

ABSTRACT

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.


Subject(s)
Collagen Type IV , Glaucoma , Receptor, Transforming Growth Factor-beta Type II , Signal Transduction , Transforming Growth Factor beta , Animals , Mice , Anterior Eye Segment/metabolism , Anterior Eye Segment/pathology , Collagen Type IV/metabolism , Collagen Type IV/genetics , Disease Models, Animal , Glaucoma/metabolism , Glaucoma/genetics , Glaucoma/pathology , Intraocular Pressure/physiology , Mice, Inbred C57BL , Mutation , Optic Nerve/pathology , Optic Nerve/metabolism , Optic Nerve Diseases/metabolism , Optic Nerve Diseases/genetics , Phenotype , Receptor, Transforming Growth Factor-beta Type II/genetics , Receptor, Transforming Growth Factor-beta Type II/metabolism , Retinal Ganglion Cells/pathology , Retinal Ganglion Cells/metabolism , Signal Transduction/physiology , Slit Lamp Microscopy , Tomography, Optical Coherence , Tonometry, Ocular , Transforming Growth Factor beta/metabolism
3.
J Mol Histol ; 55(3): 371-378, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38703340

ABSTRACT

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.


Subject(s)
Adenocarcinoma , Collagen Type IV , Collagen Type I , Extracellular Matrix , Laminin , Neoplasm Grading , Prostatic Neoplasms , Humans , Male , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Laminin/metabolism , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Collagen Type IV/metabolism , Collagen Type I/metabolism , Extracellular Matrix/metabolism , Aged , Middle Aged
4.
JCI Insight ; 9(11)2024 May 07.
Article in English | MEDLINE | ID: mdl-38713515

ABSTRACT

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.


Subject(s)
Collagen Type IV , Endothelial Cells , Hypertension, Portal , Liver Cirrhosis , Liver , Hypertension, Portal/metabolism , Hypertension, Portal/pathology , Hypertension, Portal/genetics , Animals , Collagen Type IV/metabolism , Collagen Type IV/genetics , Mice , Endothelial Cells/metabolism , Endothelial Cells/pathology , Humans , Liver Cirrhosis/pathology , Liver Cirrhosis/metabolism , Liver Cirrhosis/genetics , Liver/pathology , Liver/metabolism , Liver/blood supply , Male , NF-kappa B/metabolism , Mice, Inbred C57BL , Epigenesis, Genetic
5.
Life Sci Alliance ; 7(6)2024 Jun.
Article in English | MEDLINE | ID: mdl-38561223

ABSTRACT

Glomerular filtration relies on the type IV collagen (ColIV) network of the glomerular basement membrane, namely, in the triple helical molecules containing the α3, α4, and α5 chains of ColIV. Loss of function mutations in the genes encoding these chains (Col4a3, Col4a4, and Col4a5) is associated with the loss of renal function observed in Alport syndrome (AS). Precise understanding of the cellular basis for the patho-mechanism remains unknown and a specific therapy for this disease does not currently exist. Here, we generated a novel allele for the conditional deletion of Col4a3 in different glomerular cell types in mice. We found that podocytes specifically generate α3 chains in the developing glomerular basement membrane, and that its absence is sufficient to impair glomerular filtration as seen in AS. Next, we show that horizontal gene transfer, enhanced by TGFß1 and using allogenic bone marrow-derived mesenchymal stem cells and induced pluripotent stem cells, rescues Col4a3 expression and revive kidney function in Col4a3-deficient AS mice. Our proof-of-concept study supports that horizontal gene transfer such as cell fusion enables cell-based therapy in Alport syndrome.


Subject(s)
Nephritis, Hereditary , Podocytes , Mice , Animals , Nephritis, Hereditary/genetics , Nephritis, Hereditary/metabolism , Podocytes/metabolism , Collagen Type IV/genetics , Collagen Type IV/metabolism , Glomerular Basement Membrane/metabolism , Stem Cells/metabolism
6.
JAMA Netw Open ; 7(4): e247034, 2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38630472

ABSTRACT

Importance: Cerebral small vessel diseases (CSVDs) account for one-fifth of stroke cases. Numerous familial cases remain unresolved after routine screening of known CSVD genes. Objective: To identify novel genes and mechanisms associated with familial CSVD. Design, Setting, and Participants: This 2-stage study involved linkage analysis and a case-control study; linkage analysis and whole exome and genome sequencing were used to identify candidate gene variants in 2 large families with CSVD (9 patients with CSVD). Then, a case-control analysis was conducted on 246 unrelated probands, including probands from these 2 families and 244 additional probands. All probands (clinical onset

Subject(s)
3' Untranslated Regions , Cerebral Small Vessel Diseases , Collagen Type IV , Adult , Female , Humans , Middle Aged , 3' Untranslated Regions/genetics , Alleles , Case-Control Studies , Cerebral Small Vessel Diseases/genetics , Collagen Type IV/metabolism , Protein Isoforms , Mutagenesis, Insertional
7.
Nature ; 628(8009): 863-871, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38570687

ABSTRACT

Vertebrate organs require locally adapted blood vessels1,2. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals3-5. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-ß-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.


Subject(s)
Brain , Neovascularization, Physiologic , Animals , Basement Membrane/metabolism , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/cytology , Brain/cytology , Brain/blood supply , Brain/metabolism , Cell Movement , Collagen Type IV/metabolism , CRISPR-Cas Systems/genetics , Endothelial Cells/metabolism , Endothelial Cells/cytology , Meninges/cytology , Meninges/blood supply , Meninges/metabolism , Organ Specificity , Wnt Proteins/metabolism , Wnt Signaling Pathway , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/metabolism , Zebrafish Proteins/genetics
8.
Int Angiol ; 43(2): 229-239, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38619205

ABSTRACT

BACKGROUND: Varicose veins affect approximately 25% of people in industrialized countries. METHODS: The study aimed at detecting apoptotic cells and histopathological changes in varicose vein walls. Patients (N.=41) with varicose veins and 30 control group patients were divided into two groups according to their age (younger and older than 50 years). Apoptosis was determined by the TUNEL assay, elastin and collagen IV expression by immunohistochemistry and ultrastructural changes by transmission electron microscopy. RESULTS: The results show that the number of apoptotic cells in the layers of varicose veins increased, in particular in a group of patients aged over 50 years. In the varicose veins as compared to control veins the elastic fibers were found to be thinner, more fragmented and disorderly arranged. Elastin and collagen IV expression was found to decline in the intima and the media of varicose veins in both age groups. Electron microscopy demonstrated hypertrophy and degeneration of smooth muscle cells. Furthermore, cells with ultrastructural feature of apoptosis were noted. In the disorganized and expanded extracellular matrix membrane-bound vesicles, ghost bodies with different size and electron density were observed. Ghost bodies seem to bud off from smooth muscle cells and are likely to be involved in extracellular matrix remodeling as they are seen in close contact with collagen fibers. CONCLUSIONS: The study demonstrates increase of apoptotic cells in the wall of varicose veins along with vein wall structural abnormalities including alterations of smooth muscle cells and decline of elastin and collagen IV expression.


Subject(s)
Apoptosis , Elastin , Microscopy, Electron, Transmission , Myocytes, Smooth Muscle , Saphenous Vein , Varicose Veins , Humans , Saphenous Vein/ultrastructure , Saphenous Vein/pathology , Saphenous Vein/metabolism , Middle Aged , Elastin/metabolism , Varicose Veins/pathology , Varicose Veins/metabolism , Female , Adult , Male , Myocytes, Smooth Muscle/ultrastructure , Myocytes, Smooth Muscle/pathology , Myocytes, Smooth Muscle/metabolism , Aged , Case-Control Studies , Collagen Type IV/metabolism , Muscle, Smooth, Vascular/ultrastructure , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/metabolism , Immunohistochemistry , Venous Insufficiency/pathology , Venous Insufficiency/metabolism , Young Adult , Age Factors , Elastic Tissue/ultrastructure , Elastic Tissue/metabolism , Elastic Tissue/pathology
9.
Redox Biol ; 71: 103102, 2024 May.
Article in English | MEDLINE | ID: mdl-38430684

ABSTRACT

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin ß1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/ß1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor ß1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.


Subject(s)
Bromates , Extracellular Matrix Proteins , Pulmonary Fibrosis , Humans , Animals , Mice , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Bromides/adverse effects , Bromides/metabolism , Laminin/genetics , Laminin/metabolism , Extracellular Matrix/metabolism , Lung/metabolism , Peroxidasin , Collagen Type IV/metabolism , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/metabolism , Tyrosine/metabolism
10.
Clin Transl Med ; 14(3): e1611, 2024 03.
Article in English | MEDLINE | ID: mdl-38481388

ABSTRACT

BACKGROUND: Breast phyllodes tumours (PTs) are a unique type of fibroepithelial neoplasms with metastatic potential and recurrence tendency. However, the precise nature of heterogeneity in breast PTs remains poorly understood. This study aimed to elucidate the cell subpopulations composition and spatial structure and investigate diagnostic markers in the pathogenesis of PTs. METHODS: We applied single-cell RNA sequencing and spatial transcriptomes on tumours and adjacent normal tissues for integration analysis. Immunofluorescence experiments were conducted to verify the tissue distribution of cells. Tumour cells from patients with PTs were cultured to validate the function of genes. To validate the heterogeneity, the epithelial and stromal components of tumour tissues were separated using laser capture microdissection, and microproteomics data were obtained using data-independent acquisition mass spectrometry. The diagnostic value of genes was assessed using immunohistochemistry staining. RESULTS: Tumour stromal cells harboured seven subpopulations. Among them, a population of widely distributed cancer-associated fibroblast-like stroma cells exhibited strong communications with epithelial progenitors which underwent a mesenchymal transition. We identified two stromal subpopulations sharing epithelial progenitors and mesenchymal markers. They were inferred to further differentiate into transcriptionally active stromal subpopulations continuously expressing COL4A1/2. The binding of COL4A1/2 with ITGA1/B1 facilitated a growth pattern from the stroma towards the surrounding glands. Furthermore, we found consistent transcriptional changes between intratumoural heterogeneity and inter-patient heterogeneity by performing microproteomics studies on 30 samples from 11 PTs. The immunohistochemical assessment of 97 independent cohorts identified that COL4A1/2 and CSRP1 could aid in accurate diagnosis and grading. CONCLUSIONS: Our study demonstrates that COL4A1/2 shapes the spatial structure of stromal cell differentiation and has important clinical implications for accurate diagnosis of breast PTs.


Subject(s)
Breast Neoplasms , Phyllodes Tumor , Humans , Female , Phyllodes Tumor/diagnosis , Phyllodes Tumor/genetics , Phyllodes Tumor/metabolism , Transcriptome/genetics , Stromal Cells/metabolism , Cell Differentiation/genetics , Breast Neoplasms/diagnosis , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Collagen Type IV/genetics , Collagen Type IV/metabolism
11.
J Dermatol Sci ; 113(3): 130-137, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38431439

ABSTRACT

BACKGROUND: "Curved hair" caused by acquired factors is considered to have adverse cosmetic effects, but the detailed mechanism behind curved hair remains obscure. OBJECTIVE: We attempted to clarify the causes of curved hair that appeared to have occurred via acquired factors. METHODS: Outer root sheath cells (ORSC) isolated from plucked human hair follicles were used to evaluate the expression of type IV collagen. Straight and curved hairs with hair follicle tissue attached were also collected from the same individuals and subjected to morphological, immunohistochemical, and gene expression analyses. RESULTS: The amount of type IV collagen increased upon inducing endoplasmic reticulum stress in ORSC. Meanwhile, in curved hair follicle tissue, the gene expression of type IV collagen decreased. In addition, the curved hair follicle tissue obtained from participants in their 30 s to 50 s had distorted shapes compared with that of straight hair from the same individuals. It was also observed that hair matrix cells based on multiple hair germs fused to eventually form a single hair follicle and hair shaft. In curved hair follicle tissue, KRT71 protein, a marker of inner root sheath differentiation, was unevenly distributed and there was elevated expression of Dickkopf-1 (DKK1) protein, an inhibitor of the Wnt signaling pathway. CONCLUSION: Our study revealed the fusion of hair matrix cells during hair follicle regeneration as a cause of acquired curved hair. We consider that such fusion causes hair follicle tissue to abnormally differentiate, resulting in asymmetric hair follicle shapes and curved hair.


Subject(s)
Collagen Type IV , Hair Follicle , Humans , Hair Follicle/metabolism , Collagen Type IV/metabolism , Hair , Cell Differentiation
12.
Am J Pathol ; 194(5): 641-655, 2024 May.
Article in English | MEDLINE | ID: mdl-38309427

ABSTRACT

Alport syndrome is an inherited kidney disease, which can lead to glomerulosclerosis and fibrosis, as well as end-stage kidney disease in children and adults. Platelet-derived growth factor-D (PDGF-D) mediates glomerulosclerosis and interstitial fibrosis in various models of kidney disease, prompting investigation of its role in a murine model of Alport syndrome. In vitro, PDGF-D induced proliferation and profibrotic activation of conditionally immortalized human parietal epithelial cells. In Col4a3-/- mice, a model of Alport syndrome, PDGF-D mRNA and protein were significantly up-regulated compared with non-diseased wild-type mice. To analyze the therapeutic potential of PDGF-D inhibition, Col4a3-/- mice were treated with a PDGF-D neutralizing antibody. Surprisingly, PDGF-D antibody treatment had no effect on renal function, glomerulosclerosis, fibrosis, or other indices of kidney injury compared with control treatment with unspecific IgG. To characterize the role of PDGF-D in disease development, Col4a3-/- mice with a constitutive genetic deletion of Pdgfd were generated and analyzed. No difference in pathologic features or kidney function was observed in Col4a3-/-Pdgfd-/- mice compared with Col4a3-/-Pdgfd+/+ littermates, confirming the antibody treatment data. Mechanistically, lack of proteolytic PDGF-D activation in Col4a3-/- mice might explain the lack of effects in vivo. In conclusion, despite its established role in kidney fibrosis, PDGF-D, without further activation, does not mediate the development and progression of Alport syndrome in mice.


Subject(s)
Nephritis, Hereditary , Animals , Mice , Collagen Type IV/genetics , Collagen Type IV/metabolism , Fibrosis , Kidney/pathology , Mice, Knockout , Nephritis, Hereditary/genetics , Nephritis, Hereditary/metabolism , Nephritis, Hereditary/pathology , Platelet-Derived Growth Factor/metabolism , Platelet-Derived Growth Factor/pharmacology , Platelet-Derived Growth Factor/therapeutic use
13.
BMC Pulm Med ; 24(1): 75, 2024 Feb 08.
Article in English | MEDLINE | ID: mdl-38331745

ABSTRACT

BACKGROUND: Collagen type IV alpha 1 chain (COL4A1) in the basement membrane is an important component during lung development, as suggested from animal models where COL4A1 has been shown to regulate alveolarization and angiogenesis. Less is known about its role in human lung development. Our aim was to study COL4A1 expression in preterm infants with different lung maturational and clinical features. METHODS: COL4A1 expression in 115 lung samples from newborn infants (21-41 weeks' gestational age; 0-228 days' postnatal age [PNA]) was studied by immunohistochemistry combined with digital image analysis. Cluster analysis was performed to find subgroups according to immunohistologic and clinical data. RESULTS: Patients were automatically categorized into 4 Groups depending on their COL4A1 expression. Expression of COL4A1 was mainly extracellular in Group 1, low in Group 2, intracellular in Group 3, and both extra- and intracellular in Group 4. Intracellular/extracellular ratio of COL4A1 expression related to PNA showed a distinctive postnatal maturational pattern on days 1-7, where intracellular expression of COL4A1 was overrepresented in extremely preterm infants. CONCLUSIONS: COL4A1 expression seems to be highly dynamic during the postnatal life due to a possible rapid remodeling of the basement membrane. Intracellular accumulation of COL4A1 in the lungs of extremely premature infants occurs more frequently between 1 and 7 postnatal days than during the first 24 hours. In view of the lung arrest described in extremely preterm infants, the pathological and/or developmental role of postnatally increased intracellular COL4A1 as marker for basement membrane turnover, needs to be further investigated.


Subject(s)
Collagen Type IV , Infant, Premature , Infant, Newborn , Animals , Humans , Collagen Type IV/genetics , Collagen Type IV/metabolism , Mutation , Basement Membrane/metabolism , Lung/metabolism
14.
Exp Eye Res ; 240: 109797, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38246333

ABSTRACT

Nephronectin (Npnt) is an extracellular matrix (ECM) protein with pleiotropic functions during organogenesis, disease, and homeostasis. Although the ECM plays a crucial role during development and homeostasis of the adult cornea, little is known about the expression of Npnt in the mammalian cornea. Here, we investigated the expression of Npnt during early embryonic and postnatal development, and in adult mouse corneas. We combined ultrastructural and immunohistochemical analyses to study the early formation of the Descemet's membrane and how the expression of Npnt relates to key basement membrane proteins. Our section in situ hybridization and immunohistochemical analyses revealed that Npnt mRNA is expressed by the nascent corneal endothelial cells at embryonic day (E) 14.5, whereas the protein is localized in the adjacent extracellular matrix. These expression patterns were maintained in the corneal endothelium and Descemet's membrane throughout development and in adult corneas. Ultrastructural analysis revealed discontinuous electron dense regions of protein aggregates at E18.5 that was separated from the endothelial layer by an electron lucent space. At birth (postnatal day, P0), the Descemet's membrane was a single layer, which continuously thickened throughout P4, P8, P10, and P14. Npnt was localized to the Descemet's membrane by E18.5 and overlapped with Collagens IV and VIII, Laminin, and Perlecan. However, the proteins subsequently shifted and formed distinct layers in the adult cornea, whereby Npnt localized between two Collagen VIII bands and anterior to Collagen IV but overlapped with Laminin and Perlecan. Combined, our results reveal the expression of Npnt in the mouse cornea and define its spatiotemporal localization relative to key basement membrane proteins during the formation of the Descemet's membrane and in the adult cornea. Understanding the spatiotemporal expression of Npnt is important for future studies to elucidate its function in the mammalian cornea.


Subject(s)
Descemet Membrane , Endothelial Cells , Extracellular Matrix Proteins , Animals , Mice , Collagen Type IV/metabolism , Cornea/metabolism , Descemet Membrane/metabolism , Endothelial Cells/metabolism , Homeostasis , Laminin/metabolism , Mammals , Membrane Proteins/metabolism
15.
J Biochem ; 175(5): 539-549, 2024 Apr 29.
Article in English | MEDLINE | ID: mdl-38167713

ABSTRACT

Mural cell adhesion is important for the localization of basement membrane components during angiogenesis, and cell-cell interactions are thought to be critical for basement membrane formation. Type IV collagen, a component of the basement membrane, and non-triple helical type IV collagen α1 chain (NTH α1(IV)) co-localize in the basement membrane of neovascular vessels. However, it remains unclear how type IV collagen and NTH α1(IV) are produced around the basement membrane. In the present study, we developed a de novo angiogenesis model using human umbilical vein endothelial cell spheroids and TIG-1 fibroblast cells and demonstrated that NTH α1(IV), probably with α1(IV) chain before forming triple helix molecule, was localized in the fibroblasts in contact with vascular endothelial cells. This localization was disrupted by DAPT, a Notch signaling inhibitor. DAPT treatment also reduced type IV collagen and NTH α1(IV) secretion in TIG-1 fibroblasts, along with diminished COL4A1 and COL4A2 gene expression. Downregulation of Notch3 in TIG-1 fibroblasts decreased the secretion of type IV collagen and NTH α1(IV). Taken together, these findings suggest that heterogeneous and homogeneous intercellular Notch signaling via Notch3 induces type IV collagen and NTH α1(IV) expression in fibroblasts and contributes to basement membrane formation in neovascular vessels.


Subject(s)
Collagen Type IV , Human Umbilical Vein Endothelial Cells , Neovascularization, Physiologic , Receptors, Notch , Signal Transduction , Collagen Type IV/metabolism , Humans , Receptors, Notch/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Fibroblasts/metabolism , Receptor, Notch3/metabolism , Receptor, Notch3/genetics , Basement Membrane/metabolism , Angiogenesis
16.
Sci Rep ; 14(1): 1641, 2024 01 18.
Article in English | MEDLINE | ID: mdl-38238446

ABSTRACT

Neutrophils play a significant role in sustaining chronic inflammation in Inflammatory Bowel Disease. The intestinal basement membrane acts as a barrier for immunological homeostasis, where the α3 and α4 chains of type IV collagen are expressed on the mucosal surface. We wanted to develop a biomarker reflecting early tissue injury, providing an opportunity for intervention. Two competitive enzyme-linked immunosorbent assays (ELISAs) quantifying human neutrophil elastase (HNE) degraded neo-epitopes of COL4A3 and COL4A4 were developed and investigated in two observational cohorts (n = 161, n = 100). A biomarker of MMP-mediated degradation of COL4A1 (C4M) was used for comparison. In Cohort 1, patients with mild endoscopic ulcerative colitis showed elevated levels of C4A3-HNE compared to those with severe disease. C4M had a strong positive correlation with disease activity. C4A3-HNE/C4M provided superior discrimination between mild and severe endoscopic disease and negatively correlated to disease activity. In Cohort 2, C4A4-HNE and C4A4-HNE/C4M showed similar trends. C4A3-HNE and C4A4-HNE possibly reflect early intestinal tissue injury. Combining the markers with a biomarker of another α-chain of the same collagen provides information on two distinct stages of mucosal damage. These biomarkers may be used to monitor disease flare-up in patients in remission, reducing the need for frequent endoscopic procedures.


Subject(s)
Colitis, Ulcerative , Humans , Colitis, Ulcerative/metabolism , Collagen Type IV/metabolism , Neutrophils/metabolism , Basement Membrane/metabolism , Biomarkers/metabolism
17.
Genesis ; 62(1): e23539, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37501352

ABSTRACT

Classical collagen type IV comprising of a heterotrimer of two collagen IV alpha 1 chains and one collagen IV alpha 2 chain is the principal type of collagen synthesized by endothelial cells (EC) and is a major constituent of vascular basement membranes. In mouse and man, mutations in genes that encode collagen IV alpha 1 and alpha 2 result in vascular dysfunction. In addition, mutations in genes that encode the Ephrin receptor B4 (EPHB4) and the p120 Ras GTPase-activating protein (RASA1) that cause increased activation of the Ras mitogen-activated protein kinase (MAPK) signaling pathway in EC result in vascular dysfunction as a consequence of impaired export of collagen IV. To understand the pathogenesis of collagen IV-related vascular diseases and phenotypes it is necessary to identify at which times collagen IV is actively synthesized by EC. For this purpose, we used CRISPR/Cas9 targeting in mice to include immediately after the terminal Col4a1 codon a sequence that specifies a P2A peptide followed by enhanced green fluorescent protein (eGFP). Analysis of eGFP expression in Col4a1-P2A-eGFP mice revealed active embryonic EC synthesis of collagen IV alpha 1 through mid to late gestation followed by a sharp decline before birth. These results provide a contextual framework for understanding the basis for the varied vascular abnormalities resulting from perturbation of EC expression and export of functional collagen IV.


Subject(s)
Collagen Type IV , Endothelial Cells , Humans , Female , Pregnancy , Endothelial Cells/metabolism , Collagen Type IV/genetics , Collagen Type IV/metabolism , Green Fluorescent Proteins , Embryonic Development , p120 GTPase Activating Protein/genetics , p120 GTPase Activating Protein/metabolism
18.
J Thromb Haemost ; 22(3): 700-708, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38072379

ABSTRACT

BACKGROUND: Factor (F)IX can bind to type IV collagen in the endothelial basement membrane and diffuse into extravascular spaces. Previous studies in rodents have reported a large biodistribution of FIX. OBJECTIVES: The aim of the study was to evaluate the potential hemostatic activity of extravascular FIX and its role in protecting against joint bleeds. METHODS: The capacity of 4 different FIX molecules (plasma-derived and recombinant) to bind type I and type IV collagen was studied here. FIX molecules were also administered intravenously at doses of 50 to 3000 IU/kg in FIX knockout mice. RESULTS: A specific FIX signal was detected in immunohistochemistry in the liver as well as in muscles and knee joints with recombinant FIX molecules injected at 1000 and 3000 IU/kg but not at the usual clinical doses of 50 to 100 IU/kg, while plasma-derived FIX generated a FIX signal at all doses, including 50 IU/kg. Such a signal was also detected after five 100 IU/kg daily infusions of recombinant FIX, suggesting that FIX can accumulate in the extravascular space during prophylaxis. The extravascular procoagulant activity of FIX, assessed in saphenous vein bleeding assays, was significantly higher in hemophilia B mice after these 5 days of prophylaxis compared to a single infusion of 100 IU/kg of FIX and assessment of FIX activity 7 days later. CONCLUSION: Taken together, these results show that in individuals with severe hemophilia B receiving regular prophylaxis with FIX, extravascular accumulation of FIX over time may have a significant impact on the coagulation capacity and protection toward bleeding.


Subject(s)
Hemophilia B , Hemostatics , Mice , Animals , Factor IX/metabolism , Hemophilia B/drug therapy , Hemostatics/therapeutic use , Collagen Type IV/metabolism , Tissue Distribution , Hemorrhage/prevention & control , Hemorrhage/drug therapy , Mice, Knockout
19.
Poult Sci ; 103(1): 103179, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37931400

ABSTRACT

Collagen type IV (COL4) is one of the major components of animals' and humans' basement membranes of several tissues, such as skeletal muscles and vascular endothelia. Alterations in COL4 assembly and secretion are associated to muscular disorders in humans and animals among which growth-related abnormalities such as white striping and wooden breast affecting Pectoralis major muscles (PMs) in modern fast-growing (FG) chickens. Considering the high prevalence of these myopathies in FG broilers and that a worsening is observed as the bird slaughter age is increased, the present study was intended to evaluate the distribution and the expression level of COL4 protein and its coding genes in PMs of FG broilers at different stages of muscle development (i.e., 7, 14, 21, 28, 35, and 42 d of age). Medium-growing (MG) chickens have been considered as the control group in consideration of the lower selection pressure on breast muscle growth rate and hypertrophy. Briefly, 5 PM/sampling time/genotype were selected for western blot, immunohistochemistry (IHC), and gene expression analyses. The normalized expression levels of COL4 coding genes showed an overexpression of COL4A2 in FG than MG at d 28, as well as a significant decrease in its expression over their rearing period. Overall, results obtained through the gene expression analysis suggested that selection for the hypertrophic growth of FG broilers may have led to an altered regulation of fibroblast proliferation and COL4 synthesis. Moreover, western blot and IHC analyses suggested an altered secretion and/or degradation of COL4 protein in FG broilers, as evidenced by the fluctuating trend of 2 bands observed in FG over time. In view of the above, the present research supports the evidence about a potential aberrant synthesis and/or degradation of COL4 and corroborates the hypothesis regarding a likely involvement of COL4 in the series of events underlying the growth-related abnormalities in modern FG broilers.


Subject(s)
Muscular Diseases , Poultry Diseases , Humans , Animals , Pectoralis Muscles/metabolism , Chickens/physiology , Collagen Type IV/metabolism , Poultry Diseases/genetics , Poultry Diseases/metabolism , Muscle, Skeletal/metabolism , Muscular Diseases/genetics , Muscular Diseases/veterinary , Muscular Diseases/metabolism , Meat/analysis
20.
Matrix Biol ; 125: 1-11, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38000777

ABSTRACT

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membranes that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved throughout the animal kingdom, indicating they are important, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a new CRISPR-generated catalytic null, and found that homozygotes were mostly lethal with 13 % viable escapers. Mouse mutants also show semi-lethality, with Mendelian analysis demonstrating ∼50 % lethality and ∼50 % escapers. Despite the strong mutations, the homozygous fly and mouse escapers had low but detectable levels of Col4 crosslinking, indicating the existence of inefficient alternative crosslinking mechanisms, probably responsible for the viable escapers. Fly mutant phenotypes are consistent with decreased basement membrane stiffness. Interestingly, we found that even after basement membranes are assembled and crosslinked in wild-type animals, continuing Peroxidasin activity is required in adults to maintain tissue stiffness over time. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.


Subject(s)
Peroxidase , Peroxidasin , Animals , Mice , Basement Membrane/metabolism , Collagen Type IV/metabolism , Drosophila/metabolism , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/metabolism , Peroxidase/genetics
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