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Identification of non heparin-binding endothelial cell growth factor from rat myofibroblasts
N Sato1, N Tsuruoka, M Yamamoto
1Department of Biochemistry, National Defense Medical College, Tokorozawa, Japan.
Abstract:
Myofibroblasts (Mfs) from rat fat tissues produced a potent endothelial cell growth factor (Mf-ECGF). The growth factor activity found in the conditioned media from primary cultures of Mfs, was labile to heat (80 degrees C for 10 min) and proteinase (trypsin), and did not bind to heparin in the presence of 0.2 M NaCl. Mf-ECGF was partially purified 4760-fold with a recovery of 25% from serum-free conditioned media by sequential carboxymethyl (CM) ion-exchange column chromatography and gel filtration. This Mf-ECGF activity was recovered from the 40 kD region of a non-reducing SDS-PAGE, and from the pH region between 6.5 and 7 of isoelectric focusing, with recoveries of 20% and 65%, respectively. These results indicated that a major portion of ECGF activity in the conditioned media was clearly distinct from other well-known endothelial cell growth factors including fibroblast growth factors (FGFs).
Insights
Rat fat myofibroblasts secrete a potent endothelial cell growth factor (Mf-ECGF). This novel Mf-ECGF is distinct from known growth factors and shows unique biochemical properties.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Myofibroblasts (Mfs) are key cells in tissue repair and remodeling.
- Endothelial cell growth factors (ECGFs) are crucial for angiogenesis.
- Characterizing novel ECGFs can reveal new insights into vascular biology.
Purpose of the Study:
- To isolate and characterize a novel endothelial cell growth factor produced by rat fat myofibroblasts.
- To determine the biochemical properties and molecular weight of the myofibroblast-derived ECGF (Mf-ECGF).
- To compare Mf-ECGF with other known endothelial cell growth factors.
Main Methods:
- Primary culture of rat fat myofibroblasts and collection of conditioned media.
- Partial purification of Mf-ECGF using sequential carboxymethyl ion-exchange chromatography and gel filtration.
- Characterization of Mf-ECGF stability (heat, proteinase) and binding properties (heparin).
- Determination of molecular weight by SDS-PAGE and isoelectric point by isoelectric focusing.
Main Results:
- A potent endothelial cell growth factor (Mf-ECGF) was identified in conditioned media from rat fat myofibroblasts.
- Mf-ECGF activity was heat-labile and sensitive to trypsin, and did not bind to heparin.
- Partial purification yielded a 4760-fold increase in Mf-ECGF activity with 25% recovery.
- SDS-PAGE indicated an approximate molecular weight of 40 kD, and isoelectric focusing showed a pI between 6.5 and 7.
- Mf-ECGF exhibited distinct properties compared to known endothelial cell growth factors, including fibroblast growth factors (FGFs).
Conclusions:
- Rat fat myofibroblasts produce a unique and potent endothelial cell growth factor.
- Mf-ECGF possesses distinct biochemical characteristics differentiating it from other known ECGFs.
- This finding suggests a novel pathway for regulating angiogenesis involving myofibroblast-derived factors.