Morphogenetic messages are in the extracellular matrix: biotechnology from bench to bedside

2000 ◽  
Vol 28 (4) ◽  
pp. 345-349 ◽  
Author(s):  
A. H. Reddi
Keyword(s):  
Extracellular Matrix ◽  
Solid State ◽  
Affinity Chromatography ◽  
Bone Morphogenetic Proteins ◽  
Binding Proteins ◽  
Type Iv Collagen ◽  
Origin And Evolution ◽  
High Affinity ◽  
Type Iv

The origin and evolution of multicellular metazoa was accompanied by the appearance of extracellular matrix. The demineralized extracellular matrix of bone is enriched in morphogenetic proteins that induce bone. Bone morphogenetic proteins (BMPs) are intimately bound to collagens. BMP-4 has high affinity for type-IV collagen, and other binding proteins such as noggin and chordin. Soluble morphogens are kept in the solid state by extracellular matrix. In this sense Nature used the principles of affinity matrices long before humans patented the principle of affinity chromatography.

scholarly journals Isolation and characterization of the neutrophil-binding proteins for platelet-derived adherence-inhibiting factor

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1884-1890 ◽  
Author(s):  
K Iwabuchi ◽  
I Nagaoka ◽  
A Someya ◽  
T Yamashita
Keyword(s):  
Affinity Chromatography ◽  
Binding Sites ◽  
Binding Proteins ◽  
Type Iv Collagen ◽  
Affinity Binding ◽  
Collagen Binding ◽  
Type Iv ◽  
Isolation And Characterization ◽  
Inhibiting Factor ◽  
Sepharose Column

Abstract Guinea pig neutrophils adhered to adherence-inhibiting factor (AIF)- coated plastic; the adherence was completely inhibited by the addition of AIF, but partly inhibited by type IV collagen. Binding of 125I- labeled AIF to neutrophils was inhibited by unlabeled AIF, but partly inhibited by type IV collagen. Scatchard analysis showed that neutrophils have two classes of binding sites for AIF, high-affinity binding sites (kd = 5.0 pmol/L) numbering 500 per cell and low-affinity binding sites (kd = 860 pmol/L) numbering 6,400 per cell. Type IV collagen increased the kd of low-affinity binding sites. We have isolated and characterized the AIF-binding sites. We have isolated and characterized the AIF-binding proteins. Using AIF affinity chromatography, the radioactive fraction containing six proteins of molecular mass 45, 63, 87, 90 to 105, 145, and 195 Kd was isolated from 125I surface-labeled neutrophil extracts. This radioactive fraction was further separated into two fractions using type IV collagen affinity chromatography, ie, one fraction was adsorbed on the type IV collagen column and contained the 45-, 63-, and 87-Kd proteins, whereas another fraction was not adsorbed on the column and contained the 45-, 63-, 90- to 105-, 145-, and 195-Kd proteins. To isolate the type IV collagen- binding proteins, 125I surface-labeled neutrophil extracts were applied to a type IV collagen-Sepharose column; the isolated radioactive fraction contained the 45-, 63-, and 87-Kd proteins and bound to an AIF- Sepharose column. Taken together, these results suggest that the AIF- binding proteins, which bind to type IV collagen, are the type IV collagen-binding proteins of neutrophils.

scholarly journals Isolation and characterization of the neutrophil-binding proteins for platelet-derived adherence-inhibiting factor

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1884-1890
Author(s):  
K Iwabuchi ◽  
I Nagaoka ◽  
A Someya ◽  
T Yamashita
Keyword(s):  
Affinity Chromatography ◽  
Binding Sites ◽  
Binding Proteins ◽  
Type Iv Collagen ◽  
Affinity Binding ◽  
Collagen Binding ◽  
Type Iv ◽  
Isolation And Characterization ◽  
Inhibiting Factor ◽  
Sepharose Column

Guinea pig neutrophils adhered to adherence-inhibiting factor (AIF)- coated plastic; the adherence was completely inhibited by the addition of AIF, but partly inhibited by type IV collagen. Binding of 125I- labeled AIF to neutrophils was inhibited by unlabeled AIF, but partly inhibited by type IV collagen. Scatchard analysis showed that neutrophils have two classes of binding sites for AIF, high-affinity binding sites (kd = 5.0 pmol/L) numbering 500 per cell and low-affinity binding sites (kd = 860 pmol/L) numbering 6,400 per cell. Type IV collagen increased the kd of low-affinity binding sites. We have isolated and characterized the AIF-binding sites. We have isolated and characterized the AIF-binding proteins. Using AIF affinity chromatography, the radioactive fraction containing six proteins of molecular mass 45, 63, 87, 90 to 105, 145, and 195 Kd was isolated from 125I surface-labeled neutrophil extracts. This radioactive fraction was further separated into two fractions using type IV collagen affinity chromatography, ie, one fraction was adsorbed on the type IV collagen column and contained the 45-, 63-, and 87-Kd proteins, whereas another fraction was not adsorbed on the column and contained the 45-, 63-, 90- to 105-, 145-, and 195-Kd proteins. To isolate the type IV collagen- binding proteins, 125I surface-labeled neutrophil extracts were applied to a type IV collagen-Sepharose column; the isolated radioactive fraction contained the 45-, 63-, and 87-Kd proteins and bound to an AIF- Sepharose column. Taken together, these results suggest that the AIF- binding proteins, which bind to type IV collagen, are the type IV collagen-binding proteins of neutrophils.

scholarly journals Type IV collagen-binding proteins of neutrophils: possible involvement of L-selectin in the neutrophil binding to type IV collagen

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 365-372 ◽  
Author(s):  
K Iwabuchi ◽  
I Nagaoka ◽  
A Someya ◽  
T Yamashita
Keyword(s):  
Sialic Acid ◽  
Affinity Chromatography ◽  
Binding Proteins ◽  
Type Iv Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Collagen Binding ◽  
Type Iv ◽  
Sepharose Column ◽  
Neutrophil Adherence

Abstract To isolate type IV collagen-binding proteins, 125I-labeled human- neutrophil extracts were chromatographed on a type IV collagen- Sepharose column. The affinity chromatography-separated fraction contained the four radioactive proteins with apparent molecular masses of 28, 49, 67, and 95 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis indicated that the 95-kD proteins contained both L-selectin and nonspecific cross-reacting antigen 90 (NCA90), and that the 67-kD protein was the 67-kD elastin/laminin-binding protein (67BP). The data obtained with the type IV collagen-affinity chromatography and the immunoaffinity chromatographies using anti-L-selectin and anti-NCA90 monoclonal antibodies (MoAbs) have shown that L-selectin is closely associated with 67BP and the 49-kD protein, and that NCA90 is associated with 67BP, the 28-kD and 49-kD proteins. Among these binding proteins, sialic acid residues were contained in 67BP, L-selectin, and NCA90, but not in the 28-kD and 49-kD proteins. Sialidase treatment completely abolished both the binding affinity of the type IV collagen-binding proteins to type IV collagen and the neutrophil adherence to type IV collagen-coated plastic. Thus, the sialic acid residues of 67BP, L- selectin, and NCA90 seem to be important for the binding of neutrophils to type IV collagen. Furthermore, L-selectin IgG chimeric protein directly bound to type IV collagen-Sepharose column, and anti-L- selectin MoAb DREG56 inhibited the neutrophil adherence to type IV collagen-coated plastic by 51%. These observations suggest that L- selectin likely plays a role in the neutrophil binding to type IV collagen, although neutrophils have several kinds of adhesion molecules for type IV collagen such as L-selectin, NCA90, 67BP, and the 28-kD and 49-kD proteins.

scholarly journals Type IV collagen-binding proteins of neutrophils: possible involvement of L-selectin in the neutrophil binding to type IV collagen

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 365-372
Author(s):  
K Iwabuchi ◽  
I Nagaoka ◽  
A Someya ◽  
T Yamashita
Keyword(s):  
Sialic Acid ◽  
Affinity Chromatography ◽  
Binding Proteins ◽  
Type Iv Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Collagen Binding ◽  
Type Iv ◽  
Sepharose Column ◽  
Neutrophil Adherence

To isolate type IV collagen-binding proteins, 125I-labeled human- neutrophil extracts were chromatographed on a type IV collagen- Sepharose column. The affinity chromatography-separated fraction contained the four radioactive proteins with apparent molecular masses of 28, 49, 67, and 95 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis indicated that the 95-kD proteins contained both L-selectin and nonspecific cross-reacting antigen 90 (NCA90), and that the 67-kD protein was the 67-kD elastin/laminin-binding protein (67BP). The data obtained with the type IV collagen-affinity chromatography and the immunoaffinity chromatographies using anti-L-selectin and anti-NCA90 monoclonal antibodies (MoAbs) have shown that L-selectin is closely associated with 67BP and the 49-kD protein, and that NCA90 is associated with 67BP, the 28-kD and 49-kD proteins. Among these binding proteins, sialic acid residues were contained in 67BP, L-selectin, and NCA90, but not in the 28-kD and 49-kD proteins. Sialidase treatment completely abolished both the binding affinity of the type IV collagen-binding proteins to type IV collagen and the neutrophil adherence to type IV collagen-coated plastic. Thus, the sialic acid residues of 67BP, L- selectin, and NCA90 seem to be important for the binding of neutrophils to type IV collagen. Furthermore, L-selectin IgG chimeric protein directly bound to type IV collagen-Sepharose column, and anti-L- selectin MoAb DREG56 inhibited the neutrophil adherence to type IV collagen-coated plastic by 51%. These observations suggest that L- selectin likely plays a role in the neutrophil binding to type IV collagen, although neutrophils have several kinds of adhesion molecules for type IV collagen such as L-selectin, NCA90, 67BP, and the 28-kD and 49-kD proteins.

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 High affinity immunoreactive FGF receptors in the extracellular matrix of vascular endothelial cells--implications for the modulation of FGF-2.

1995 ◽  
Vol 128 (6) ◽  
pp. 1221-1228 ◽  
Author(s):  
A Hanneken ◽  
P A Maher ◽  
A Baird
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Protein Interactions ◽  
Binding Proteins ◽  
Vascular Endothelial Cells ◽  
Extracellular Domain ◽  
Cytoplasmic Domain ◽  
Basement Membranes ◽  
Vascular Endothelial ◽  
High Affinity

We recently characterized three FGF-binding proteins (FGF-BPs) which are soluble forms of the extracellular domains of the high affinity FGF receptors (Hanneken, A. M., W. Ying, N. Ling, and A. Baird. Proc. Natl. Acad. Sci. USA. 1994. 91:9170-9174). These proteins circulate in blood and have been proposed to modulate the biological activity of the FGF family of proteins. Immunohistochemical studies now demonstrate that these soluble, truncated FGF receptors are also present in the basement membranes of retinal vascular endothelial cells. These immunoreactive proteins can be detected with antibodies raised to the extracellular domain of FGFR-1 but not with antibodies raised to either the juxtamembrane domain or the cytoplasmic domain of FGFR-1. Western blotting of human retinal extracts with the antibody raised to the extracellular domain of FGFR-1 detects specific, low molecular mass proteins at 85 kD and 55 kD, corresponding in size to the FGF-BPs, which are not detected with antibodies against the cytoplasmic domain of the receptor. The interaction of this receptor with the extracellular matrix is not dependent on the presence of FGF-2. Immunoreactive receptors are still detected in vascular basement membranes after the removal of FGF-2 with heparitinase. In addition, the recombinant extracellular domain of FGFR-1 continues to bind to corneal endothelial cell matrix after endogenous FGF-2 has been removed with 2 M NaCl. Acid treatment, which has been shown to disrupt protein interactions with the extracellular matrix, leads to a significant reduction in the presence of the matrix form of the FGF receptor. This loss can be restored with exogenous incubations of the recombinant extracellular domain of FGFR-1. This report is the first demonstration that a truncated form of a high affinity growth factor receptor can be localized to the extracellular matrix. These findings add to the list of binding proteins associated with the extracellular matrix (IGFBP-5) and suggest a potentially new regulatory mechanism for controlling the biological availability of FGF, and other peptide growth factors, in the extracellular matrix.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document