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A novel heart derived inhibitor of vascular cell proliferation. Purification and biological activity
1Max-Planck-Institute for Clinical and Physiological Research, Department of Experimental Cardiology, Bad Nauheim, Federal Republic of Germany.
Insights
Researchers discovered a novel 11 kD protein inhibitor in bovine heart tissue that suppresses vascular cell proliferation. This finding suggests a new mechanism for regulating heart angiogenesis and vascular cell growth.
Area of Science:
- Cardiovascular Biology
- Molecular Biology
- Biochemistry
Background:
- Adult heart tissue contains mitogenic growth factors for vascular cells.
- Angiogenesis is typically absent in the normal heart, suggesting the presence of inhibitory factors.
Purpose of the Study:
- To isolate and characterize potential growth inhibitors from bovine heart extracts.
- To investigate the role of these inhibitors in regulating vascular cell proliferation.
Main Methods:
- Protein purification using ammonium sulfate precipitation, cation exchange, hydroxylapatite chromatography, ultrafiltration, and gelfiltration.
- Biochemical characterization including SDS-PAGE, isoelectric focusing, and amino acid sequencing.
- Assay of [3H]-thymidine incorporation to measure cell proliferation inhibition.
Main Results:
- An 11 kD protein inhibitor of vascular cell proliferation was isolated.
- The inhibitor reversibly suppressed [3H]-thymidine incorporation in bovine aortic endothelial and smooth muscle cells.
- The protein is heat and acetic acid stable, hydrophilic, and shows no homology to known growth inhibitors.
Conclusions:
- A novel endogenous inhibitor of vascular cell proliferation exists in the bovine heart.
- This inhibitor may counterbalance mitogenic factors, regulating vascular cell proliferation and angiogenesis in the heart.
- Vascular cell proliferation is likely regulated by a balance between growth factors and inhibitors.
Abstract:
Recently, growth factors with mitogenic properties for vascular wall cells have been isolated from adult heart tissue. Since angiogenesis in the heart typically does not occur under normal physiological conditions, despite the presence of many growth factors, we hypothesized the existence of growth inhibitors. To test this hypothesis, we subjected whole bovine heart extracts to a series of protein purification steps in search of such an inhibitor. The purification procedure consisted of ammonium sulfate precipitation followed by cation exchange chromatography, hydroxylapatite chromatography, ultrafiltration and gelfiltration. We isolated a small protein, which is an inhibitor of cell proliferation from the bovine heart. The inhibitor reversibly suppressed [3H]-thymidine incorporation into nuclei of bovine aortic endothelial and smooth muscle cells. The moiety responsible for the inhibitory activity was identified biochemically (SDS Page, isoelectric focusing, HPEC) as an 11 kD protein with an isoelectric point of 7. The substance is a heat and acetic acid stable protein which does not bind to reversed phase columns because of its hydrophilic character. The inhibitor has no affinity to heparin sepharose. The inhibitory activity was destroyed by hydrolysis. No homology to any hitherto structurally investigated growth inhibitor was observed using the chemical determination of the amino acid sequence by microsequencing after previous trypsin digestion. We conclude that the described growth inhibitor may counteract the activity of mitogens that are abundantly present in normal heart. Vascular cell proliferation may be regulated by inhibition or production of the inhibitor.