Cleft Palate Formation in Fetal Br Mice with Midfacial Retrusion: Tenascin, Fibronectin, Laminin, and Type IV Collagen Immunolocalization

1998 ◽  
Vol 35 (1) ◽  
pp. 65-76 ◽  
Author(s):  
G.D. Singh ◽  
J. Johnston ◽  
W. Ma ◽  
S. Lozanoff
Keyword(s):  
Extracellular Matrix ◽  
Cleft Palate ◽  
Type Iv Collagen ◽  
Craniofacial Morphology ◽  
Homogeneous Distribution ◽  
Secondary Antibody ◽  
Type Iv ◽  
Rabbit Igg ◽  
Secondary Palate ◽  
Immunohistological Analysis

Objective This study tested the hypothesis that altered craniofacial morphology does not affect the expression of extracellular matrix (ECM) molecules such as fibronectin (FN), laminin (LN), type IV collagen, and tenascin-C (TN) but is associated with failure of palatal shelf elevation and fusion concomitant with cleft palate formation. Design To test this hypothesis, a comparative immunohistological analysis of FN, LN, type IV collagen, and TN was undertaken on brachyrrhine (Br/Br) mice and normal (+/+) fetuses during secondary palate formation. Normal and Br/Br fetuses were collected at gestational days E13 and E14 (representing prefusion stages) and E15 and E18 (representing postfusion stages). Cryostat palatal sections (8 μm) were postfixed in methanol, washed, and stained with primary antibody. All sections were washed and coated with secondary antibody (swine-anti-rabbit IgG) and mounted with citifluor. Results Immunohistological analysis showed that LN and type IV collagen were located near the presumptive medial epithelial seam (MES) or edge (MEE) in +/+ or Br/Br fetuses, respectively. Fibronectin showed a homogeneous distribution at all stages in both groups of mice. In contrast, TN became localized below the presumptive MES or MEE in both groups of mice at E14. In +/+ animals at E15, TN dissipated and became confined to the oral basement membrane by E18. At E15 and E18 in cleft Br/Br mutants, TN stained beneath the MEE. Conclusion Although the distributions of ECM molecules are similar during normal and cleft palatogenesis, differences in TN expression are associated with cleft palate formation.

scholarly journals Regulation of angiotensin II receptors and extracellular matrix turnover in human retinal pigment epithelium: role of angiotensin II

2008 ◽  
Vol 295 (6) ◽  
pp. C1633-C1646 ◽  
Author(s):  
Gary E. Striker ◽  
Francoiçe Praddaude ◽  
Oscar Alcazar ◽  
Scott W. Cousins ◽  
Maria E. Marin-Castaño
Keyword(s):  
Extracellular Matrix ◽  
Angiotensin Ii ◽  
Type Iv Collagen ◽  
Pigment Epithelium ◽  
Receptor Expression ◽  
Age Related Macular Degeneration ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Ang Ii ◽  
Type Iv

The early stage of age-related macular degeneration (AMD) is characterized by the formation of subretinal pigment epithelium (RPE) deposits as a result of the dysregulation in the turnover of extracellular matrix (ECM) molecules. However, the mechanism involved remains unclear. Hypertension (HTN) is an important risk factor for AMD, and angiotensin II (ANG II) is the most important hormone associated with HTN. However, the relevance of ANG II receptors and ANG II effects on RPE have not been investigated yet. Therefore, the expression and regulation of ANG II receptors as well as the ECM turnover were studied in human RPE. ANG II receptors were expressed and upregulated by ANG II in human RPE. This regulation resulted in functional receptor expression, since an increase in intracellular concentration of calcium was observed upon ANG II stimulation. ANG II also increased matrix metalloproteinase (MMP)-2 activity and MMP-14 at the mRNA and protein levels as well as type IV collagen degradation. These ANG II effects were abolished in the presence of the ANG II receptor subtype 1 (AT1) receptor antagonist candesartan. In contrast, ANG II decreased type IV collagen via both AT1 and AT2 receptors, suggesting a synergistic effect of the two receptor subtypes. In conclusion, we have confirmed the presence of ANG II receptors in human RPE and their regulation by ANG II as well as the regulation of ECM molecules via ANG II receptors. Our data support the hypothesis that ANG II may exert biological function in RPE through ANG II receptors and that ANG II may cause dysregulation of molecules that play a major role in the turnover of ECM in RPE basement membrane and Bruch's membrane, suggesting a pathogenic mechanism to explain the link between HTN and AMD.

scholarly journals Agrin-induced reorganization of extracellular matrix components on cultured myotubes: relationship to AChR aggregation.

1990 ◽  
Vol 111 (3) ◽  
pp. 1161-1170 ◽  
Author(s):  
R M Nitkin ◽  
T C Rothschild
Keyword(s):  
Extracellular Matrix ◽  
Type Iv Collagen ◽  
Neuromuscular Junctions ◽  
Synaptic Organization ◽  
Type Iv ◽  
Extracellular Matrix Components ◽  
Cultured Myotubes ◽  
Matrix Components ◽  
Induced Aggregation

Agrin, an extracellular matrix-associated protein extracted from synapse-rich tissues, induces the accumulation of acetylcholine receptors (AChRs) and other synaptic components into discrete patches on cultured myotubes. The appearance of agrin-like molecules at neuromuscular junctions suggests that it may direct synaptic organization in vivo. In the present study we examined the role of extracellular matrix components in agrin-induced differentiation. We used immunohistochemical techniques to visualize the spatial and temporal distribution of laminin, a heparan sulfate proteoglycan (HSPG), fibronectin, and type IV collagen on cultured chick myotubes during agrin-induced aggregation of AChRs. Myotubes displayed significant amounts of laminin and HSPG, lesser amounts of type IV collagen, and little, if any, fibronectin. Agrin treatment caused cell surface laminin and HSPG to patch, while collagen and fibronectin distributions were generally unaffected. Many of the agrin-induced laminin and HSPG patches colocalized with AChR patches, raising the possibility of a causal relationship between matrix patching and AChR accumulations. However, patching of AChRs (complete within a few hours) preceded that of laminin or HSPG (not complete until 15-20 h), making it unlikely that matrix accumulations initiate AChR patching at agrin-induced sites. Conversely, when AChR patching was blocked by treatment with anti-AChR antibody mAb 35, agrin was still able to effect patching of laminin and HSPG. Taken together, these findings suggest that agrin-induced accumulations of AChR and laminin/HSPG are not mechanistically linked.

Production of type IV collagen and 72-kDa gelatinase by human endothelial cells cultured in high glucose. Effects of a protein kinase C inhibitor, GF 109203X

1996 ◽  
Vol 74 (5) ◽  
pp. 659-667 ◽  
Author(s):  
Anne-Marie Grigorova-Borsos ◽  
Ahmed Bakillah ◽  
Paul Urios ◽  
Valérie Leblond ◽  
Raymonde Guillot ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
High Glucose ◽  
Glucose Concentration ◽  
Type Iv Collagen ◽  
Culture Supernatant ◽  
Total Proteins ◽  
Gelatinase Activity ◽  
Type Iv ◽  
Kinase C ◽  
Gf 109203X

Since diabetic microangiopathy and macroangiopathy are characterized by type IV collagen accumulation in vascular basement membranes, it was of interest to study type IV collagen production and type IV collagenase secretion by endothelial cells (EC) cultured in high glucose and to evaluate the role of protein kinase C (PKC) activation in the alterations induced by high glucose. Primary cultures of human umbilical vein EC were exposed to high glucose concentration for 3 days at the beginning of confluence. The number of EC decreased with glucose concentration from 5 to 50 mM. At 16.7 mM glucose concentration, the amount of type IV collagen, determined by a two-step ELISA, increased in the culture supernatant and in the insoluble fraction associated with the extracellular matrix and cells; proline incorporation was more markedly elevated in the collagenous than in the total proteins of the culture supernatant and of the extracellular matrix and cell extracts. Gelatin zymography of the culture supernatant showed that EC mainly produce a 72-kDa gelatinase known to degrade type IV collagen. At 16.7 mM glucose concentration, total gelatinase activity per millilitre of culture supernatant was reduced and the 72-kDa gelatinase activity measured on the zymogram scan was lowered. When EC were exposed to 16.7 mM glucose, the specific PKC inhibitor GF 109203X corrected the increases in type IV collagen concentration and in proline incorporation into the collagenous or total proteins present in the culture supernatant or in the extract of the insoluble fraction, including the extracellular matrix and cells. Our results show that soluble and insoluble type IV collagen accumulation by EC cultured at high glucose concentration is not only associated with increased synthesis of the collagenous and total proteins but also with decreased total 72-kDa gelatinase activity in the extracellular fluid. The observed effects of GF 109203X are in favour of the involvement of PKC activation in the type IV collagen accumulation.Key words: human umbilical vein endothelial cells, high glucose, type IV collagen, type IV collagenase, protein kinase C inhibitor

scholarly journals Modulation of extracellular matrix biosynthesis by bovine retinal pericytes in vitro: effects of the substratum and cell density

1990 ◽  
Vol 96 (1) ◽  
pp. 159-169
Author(s):  
A.E. Canfield ◽  
T.D. Allen ◽  
M.E. Grant ◽  
S.L. Schor ◽  
A.M. Schor
Keyword(s):  
Extracellular Matrix ◽  
Type Iv Collagen ◽  
Type I Collagen ◽  
Single Cells ◽  
Collagen Gel ◽  
Type Iii Collagen ◽  
Type I ◽  
Experimental Conditions ◽  
Type Iv ◽  
Retinal Pericytes

Bovine retinal pericytes plated on a two-dimensional substratum display a characteristic stellate morphology. In post-confluent cultures these cells aggregate spontaneously to form multicellular nodules. The same cells plated within a three-dimensional collagen matrix display an elongated sprouting morphology. Sprouting pericytes may be embedded within a gel either as individual cells or as multicellular aggregates. We have compared the nature of the matrix proteins synthesised by pericytes displaying these different phenotypes. Stellate pericytes cultured on plastic dishes synthesised predominantly type I collagen, some type III collagen and only traces of type IV collagen. The same collagen types were secreted when nodules had formed in postconfluent cultures on plastic, and by sprouting cells plated as single cells within the collagen gel. By contrast, sprouting pericytes plated as aggregates within the collagen gel secreted increased levels of type IV collagen and reduced amounts of type I collagen. Fibronectin was synthesized by pericytes under all experimental conditions examined; thrombospondin was produced in relatively large amounts by cells grown on plastic dishes, whereas only trace amounts could be detected in the medium when the cells were cultured within a collagen gel matrix. Transmission electron microscopy revealed that pericyte aggregates within a collagen gel contained cells in close apposition surrounded by a dense extracellular matrix. In contrast, cells in the centre of a nodule on plastic appeared to be separated from each other by loose extracellular material. These results suggest that the morphological and biosynthetic phenotypes of retinal pericytes are modulated by cell-matrix and/or cell-cell interactions.

scholarly journals Formation of basement membranes in the embryonic kidney: an immunohistological study

1981 ◽  
Vol 91 (1) ◽  
pp. 1-10 ◽  
Author(s):  
P Ekblom
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Type Iv Collagen ◽  
Basement Membranes ◽  
Glomerular Endothelial Cell ◽  
Type Iv ◽  
Endothelial Cell Differentiation ◽  
Inductive Interaction ◽  
Immunohistological Study

Specific antibodies to laminin, type IV collagen, basement-membrane proteoglycan, and fibronectin have been used in immunofluorescence microscopy to study the development of basement membranes of the embryonic kidney. Kidney tubules are known to form from the nephrogenic mesenchyme as a result of an inductive tissue interaction. This involves a change in the composition of the extracellular matrix. The undifferentiated mesenchyme expresses in the composition of the extracellular matrix. The undifferentiated mesenchyme expresses fibronectin but no detectable laminin, type IV collagen, or basement-membrane proteoglycan. During the inductive interaction, basement-membrane specific components (laminin, type IV collagen, basement membrane proteoglycan) become detectable in the induced area, whereas fibronectin is lost. While the differentiation to epithelial cells of the kidney requires an inductive interaction, the development of the vasculature seems to involve an ingrowth of cells which throughout development deposits basement-membrane specific components, as well as fibronectin. These cells form the endothelium and possibly also the mesangium of the glomerulus, and contribute to the formation of the glomerular basement membrane. An analysis of differentiation of the kidney mesenchyme in vitro in the absence of circulation supports these conclusions. Because a continuity with vasculature is required for glomerular endothelial cell differentiation, it is possible that these cells are derived from outside vasculature.

scholarly journals Cells that emerge from embryonic explants produce fibers of type IV collagen.

1985 ◽  
Vol 101 (4) ◽  
pp. 1175-1181 ◽  
Author(s):  
J M Chen ◽  
C D Little
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Type Iv Collagen ◽  
Polyclonal Antibodies ◽  
Mouse Lung ◽  
Embryonic Mouse ◽  
Type Iv ◽  
Collagen Substratum

Double immunofluorescence staining experiments designed to examine the synthesis and deposition of collagen types I and IV in cultured explants of embryonic mouse lung revealed the presence of connective tissue-like fibers that were immunoreactive with anti-type IV collagen antibodies. This observation is contrary to the widely accepted belief that type IV collagen is found only in sheet-like arrangements beneath epithelia or as a sheath-like layer enveloping bundles of nerve or muscle cells. The extracellular matrix produced by cells that migrate from embryonic mouse lung rudiments in vitro was examined by double indirect immunofluorescence microscopy. Affinity-purified monospecific polyclonal antibodies were used to examine cells after growth on glass or native collagen substrata. The data show that embryonic mesenchymal cells can produce organized fibers of type IV collagen that are not contained within a basement membrane, and that embryonic epithelial cells deposit fibers and strands of type IV collagen beneath their basal surface when grown on glass; however, when grown on a rat tail collagen substratum the epithelial cells produce a fine meshwork. To our knowledge this work represents the first report that type IV collagen can be organized by cells into a fibrous extracellular matrix that is not a basement membrane.

scholarly journals Blood-based extracellular matrix biomarkers are correlated with clinical outcome after PD-1 inhibition in patients with metastatic melanoma

2020 ◽  
Vol 8 (2) ◽  
pp. e001193
Author(s):  
Daan P Hurkmans ◽  
Christina Jensen ◽  
Stijn L W Koolen ◽  
Joachim Aerts ◽  
Morten Asser Karsdal ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Clinical Outcome ◽  
Metastatic Melanoma ◽  
Type Iv Collagen ◽  
Type Iii Collagen ◽  
Cox Regression Analysis ◽  
High Baseline ◽  
Type Iii ◽  
Type Iv ◽  
M1 Macrophage

BackgroundImmune checkpoint inhibitors that target the programmed cell death protein 1 (PD-1) receptor induce a response in only a subgroup of patients with metastatic melanoma. Previous research suggests that transforming growth factor beta signaling and a collagen-rich peritumoral stroma (tumor fibrosis), may negatively interfere with the interaction between T cells and tumor cells and thereby contribute to resistance mechanisms by immune-exclusion, while increased tumor infiltration of M1-like macrophages enhances T cell activity. Hence, the current study aimed to assess the relationship between blood-based markers of collagen or vimentin turnover (reflecting M1 macrophage activity) and clinical outcome in patients with metastatic melanoma after PD-1 inhibition.MethodsPatients with metastatic melanoma who were treated with anti-PD-1 monotherapy between May 2016 and March 2019 were included in a prospective observational study. N-terminal pro-peptide of type III collagen (PRO-C3) cross-linked N-terminal pro-peptides of type III collagen (PC3X), matrix metalloprotease (MMP)-degraded type III (C3M) and type IV collagen (C4M), granzyme B-degraded type IV collagen and citrullinated and MMP-degraded vimentin (VICM) were measured with immunoassays in serum before (n=107), and 6 weeks after the first administration of immunotherapy (n=94). The association between biomarker levels and overall survival (OS) or progression-free survival (PFS) was assessed.ResultsMultivariate Cox regression analysis identified high baseline PRO-C3 (Q4) and PC3X (Q4) as independent variables of worse PFS (PRO-C3: HR=1.81, 95% CI=1.06 to 3.10, p=0.030 and PC3X: HR=1.86, 95% CI=1.09 to 3.18, p=0.023). High baseline PRO-C3 was also independently related to worse OS (HR=2.08, 95% CI=1.06 to 4.09, p=0.035), whereas a high C3M/PRO-C3 ratio was related to improved OS (HR=0.42, 95% CI=0.20 to 0.90, p=0.025). An increase in VICM (p<0.0001; in 56% of the patients) was observed after 6 weeks of treatment, and an increase in VICM was independently associated with improved OS (HR=0.28, 95% CI=0.10 to 0.77, p=0.014).ConclusionsBlood-based biomarkers reflecting excessive type III collagen turnover were associated with worse OS and PFS after PD-1 inhibition in metastatic melanoma. Moreover, an increase in VICM levels after 6 weeks of treatment was associated with improved OS. These findings suggest that type III collagen and vimentin turnover contribute to resistance/response mechanisms of PD-1 inhibitors and hold promise of assessing extracellular matrix-derived and stroma-derived components to predict immunotherapy response.

scholarly journals Binding of human plasminogen to basement-membrane (type IV) collagen

1992 ◽  
Vol 284 (1) ◽  
pp. 103-108 ◽  
Author(s):  
M S Stack ◽  
T L Moser ◽  
S V Pizzo
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Type Iv Collagen ◽  
Specific Interaction ◽  
Serine Proteinase ◽  
Type Iv ◽  
Basement Membrane Collagen ◽  
Extracellular Matrix Remodelling ◽  
Membrane Type ◽  
Protein Components

Plasminogen, the zymogen form of the serine proteinase plasmin, has been implicated in numerous physiological and pathological processes involving extracellular-matrix remodelling. We have previously demonstrated that the activation of plasminogen catalysed by tissue plasminogen activator is dramatically stimulated in the presence of basement-membrane-specific type IV collagen [Stack, Gonzalez-Gronow & Pizzo (1990) Biochemistry 29, 4966-4970]. The present paper describes the binding of plasminogen to type IV collagen. Plasminogen binds to both the alpha 1(IV) and alpha 2(IV) chains of basement-membrane collagen, with binding to the alpha 2(IV) chain preferentially inhibited by 6-aminohexanoic acid. This binding is specific and saturable, with Kd,app. values of 11.5 and 12.7 nM for collagen and gelatin respectively. Although collagen also binds to immobilized plasminogen, this interaction is unaffected by 6-aminohexanoic acid. Limited elastase proteolysis of plasminogen generated distinct collagen-binding fragments, which were identified as the kringle 1-3 and kringle 4 domains. No binding of collagen to mini-plasminogen was observed. These studies demonstrate a specific interaction between plasminogen and type IV collagen and provide further evidence for regulation of plasminogen activation by protein components of the extracellular matrix.

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