Skip to main content
SpringerLink
Log in

Exogenous HSP70 becomes cell associated, but not internalized, by stressed arterial smooth muscle cells

  • Growth, Differentiation, And Senescence
  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Summary

Cell death within atherosclerotic plaques leads to necrosis and rupture, resulting in vascular occlusion. We have previously demonstrated that addition of exogenous 70 kDa heat shock protein (HSP70) to arterial smooth muscle cells (aSMCs) in vitro can protect against toxins that may initiate necrosis. To determine whether exogenous HSP70 enters aSMCs or acts from outside cells to preserve viability, cultured rabbit aSMCs were stressed by serum deprivation and treated with fluorescently labeled (7-aminomethyl-4-coumarin-3-acetate) or125I-radiolabeled HSP70. Cell-associated HSP70 was analyzed using Western blotting, fluoresence spectroscopy, and gamma counting/autoradiogarphy. Surface binding of HSP70 to aSMCs was differentiated from uptake by using trypsin treatment to degrade non-internalized HSP70. Specificity of HSP70 binding was tested by inhibiting uptake of125I-HSP70 with excess unlabeled HSP70 or bovine serum albumin (BSA). The effect of unlabeled exogenous HSP70 on endogenous HSP synthesis was also tested. Exogenous HSP70 increased total cell-associated HSP70 2.9- to 3.6-fold over levels present in unstressed aSMCs. However, <5% of the exogenous HSP70 was trypsin-insensitive, indicating that bound HSP70 was not internalized. Binding of125I-HSP70 was inhibited by both unlabeled HSP70 and BSA, implying a non-specific interaction with the plasmalemma. Exogenous HSP70 significantly lowered overall protein synthesis by serum-deprived aSMCs, but it did not specifically inhibit synthesis of endogenous HSPs after heat shock. The results indicate that exogenous HSP70 protects viability of stressed aSMCs through interactions with the cell surface rather than via internalization.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Alder, G. M.; Austen, B. M.; Bashford, C. L., et al. Heat shock proteins induce pores in membranes. Biosci. Rep. 10:509–518; 1990.

    Article  PubMed  CAS  Google Scholar 

  2. Ambrose, J. A.; Winsters, S. L.; Stern, A., et al. Angiographic morphology and the pathogenesis of unstable angina pectoris. J. Am. Coll. Cardiol. 5:609–616; 1985.

    Article  PubMed  CAS  Google Scholar 

  3. Baler, R.; Welch, W. J.; Voellmy, R. Heat shock gene regulation by nascent polypeptides and denatured proteins: hsp70 as a potential autoregulatory factor. J. Cell Biol. 117:1151–1159; 1992.

    Article  PubMed  CAS  Google Scholar 

  4. Beckman, R. P.; Lovett, M.; Welch, W. J. Examining the function and regulation of hsp 70 in cells subjected to metabolic stress. J. Cell Biol. 117:1137–1150; 1992.

    Article  Google Scholar 

  5. Berberian, P. A.; Jenison, M. Renewed receptor function of enzymatically isolated arterial cells after short-term incubation. Proc. Soc. Exp. Biol. Med. 184:525; 1987.

    Google Scholar 

  6. Björnheden, T.; Bondjers, G. Oxygen consumption in aortic tissue from rabbits with diet-induced atherosclerosis. Arteriosclerosis 7:238–247; 1987.

    PubMed  Google Scholar 

  7. Bonner, W. M.; Laskey, R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83–88; 1974.

    Article  PubMed  CAS  Google Scholar 

  8. Carpenter, G. Receptors for epidermal growth factor and other polypeptide mitogens. Annu. Rev. Biochem. 56:881–914; 1987.

    Article  PubMed  CAS  Google Scholar 

  9. Casscells, W.; Lappi, D. A.; Olwin, B. B., et al. Elimination of smooth muscle cells in experimental restenosis: targeting of fibroblast growth factor receptors. Proc. Natl. Acad. Sci. USA 89:7159–7163; 1992.

    Article  PubMed  CAS  Google Scholar 

  10. Clerc, G. Atherosclerosis as an immune disease? Med. Hypotheses 36:24–26; 1991.

    Article  PubMed  CAS  Google Scholar 

  11. Crawford, D. W.; Blankenhorn, D. W. Arterial wall oxygenation, oxyradicals, and atherosclerosis. Atherosclerosis 89:97–108; 1991.

    Article  PubMed  CAS  Google Scholar 

  12. Deshaies, R. J.; Koch, B. D.; Schekman, R. The role of stress proteins in membrane biogenesis. Trends Biochem. Sci. 13:384–388; 1988.

    Article  PubMed  CAS  Google Scholar 

  13. Freshney, R. I. Disaggregation of the tissue and primary culture: enzymatic disaggregation. In: Freshney, R. I., ed. Culture of animal cells: a manual of basic technique. New York: Alan R. Liss Inc.; 1983:106–116.

    Google Scholar 

  14. Fuster, V.; Stein, B.; Ambrose, J. A., et al. Atherosclerotic plaque rupture and thrombosis: evolving concepts. Circulation 82(suppl.II):II47-II59; 1990.

    PubMed  CAS  Google Scholar 

  15. Guyton, J. R.; Black, B. L.; Seidel, C. L. Focal toxicity of oxysterols in vascular smooth muscle cell culture: a model of the atherosclerotic core region. Am. J. Pathol. 137:425–434; 1990.

    PubMed  CAS  Google Scholar 

  16. Halliwell, B. Lipid peroxidationin vivo andin vitro in relation to atherosclerosis: some fundamental questions. Agents Actions Suppl. 26:223–231; 1988.

    PubMed  CAS  Google Scholar 

  17. Haudenschild, C. C. Pathogenesis of atherosclerosis: state of the art. Cardiovasc. Drugs Ther. 4:993–1004; 1990.

    Article  PubMed  Google Scholar 

  18. Henrion, D.; Laher, I.; Laporte, R., et al. Angiotensin II amplifies arterial contractile response to norepinephrine without increasing Ca++ influx: role of protein kinase C. J. Pharmacol. Exp. Ther. 261:835–840; 1992.

    PubMed  CAS  Google Scholar 

  19. Hightower, L. E.; Guidon, P. T., Jr. Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble gliaaxon transfer proteins. J. Cell. Physiol. 138:257–266; 1982.

    Article  Google Scholar 

  20. Jiang, J. X.; London, E. Involvement of denaturation-like changes in Pseudomonas exotoxin A hydrophobicity and membrane penetration determined by characterization of pH and thermal transitions. Roles of two distinct conformationally altered states. J. Biol. Chem. 265:8636–8641; 1990.

    PubMed  CAS  Google Scholar 

  21. Johnson, A. D.; Berberian, P. A.; Bond, M. G. Effect of heat shock proteins on survival of isolated aortic cells from normal and atherosclerotic cynomolgus macaques. Atherosclerosis 84:111–119; 1990.

    Article  PubMed  CAS  Google Scholar 

  22. Khalfan, H.; Abuknesha, R.; Rand-Weaver, M., et al. Aminomethyl coumarin acetic acid: a new fluorescent labelling agent for proteins. Histochem. J. 18:497–499; 1986.

    Article  PubMed  CAS  Google Scholar 

  23. Libby, P.; Hansson, G. K. Involvement of the immune system in human atherogenesis: current knowledge and unanswered questions. Lab. Invest. 64:5–15; 1991.

    PubMed  CAS  Google Scholar 

  24. Mans, R. J.; Novelli, G. D. Measurement of the incorporation of radioactive amino acids into protein by a filter paper disk method. Arch. Biochem. Biophys. 94:48–53; 1968.

    Article  Google Scholar 

  25. Markwell, M. A. K. A new solid-state reagent to iodinate proteins. I. Conditions for the efficient labeling of antiserum. Anal. Biochem. 125:427–432; 1982.

    Article  PubMed  CAS  Google Scholar 

  26. Pettersen, K. S.; Boberg, K. M.; Stabursvik, A., et al. Toxicity of oxygenated cholesterol derivatives toward cultured human umbilical vein endothelial cells. Arterioscler. Thromb. 11:423–428; 1991.

    PubMed  CAS  Google Scholar 

  27. Sauro, M. D.; Zorn, N. E. Prolactin induces proliferation of vascular smooth muscle cells through a protein kinase C-dependent mechanism. J. Cell Physiol. 148:133–138; 1991.

    Article  PubMed  CAS  Google Scholar 

  28. Schiffrin, E. L.; Turgeon, A.; Tremblay, J., et al. Effects of ANP, angiotensin, vasopressin, and endothelin on ANP receptors in cultured rat vascular smooth muscle cells. Am. J. Physiol. 260(29):H58-H65; 1991.

    PubMed  CAS  Google Scholar 

  29. Thyberg, J.; Hedin, U.; Sjölund, M., et al. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis 10:966–990; 1990.

    PubMed  CAS  Google Scholar 

  30. Tytell, M.; Greenberg, S. G.; Lasek, R. J. Heat shock-like protein is transferred from glia to axon. Brain Res. 363:161–164; 1986.

    Article  PubMed  CAS  Google Scholar 

  31. Tytell, M.; Barbe, M. Synthesis and axonal transport of heat shock proteins. Neurol. Neurobiol. 25:473–492; 1987.

    CAS  Google Scholar 

  32. Walsh, K. A. Trypsinogens and trypsins of various species. Methods Enzymol. 19:41–63; 1970.

    Article  CAS  Google Scholar 

  33. Welch, W. J. The mammalian stress response: cell physiology and biochemistry of stress proteins. In: Morimoto, R. I.; Tissieres, A.; Georgopoulos, C., eds. Stress proteins in biology and medicine. Cold Spring Harbor, NY: Cold Spring Harbor Press; 1990:223–278.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Neurobiology and Anatomy, Bowman Gray School of Medicine of Wake Forest University, 27157, Winston-Salem, North Carolina

    A. Daniel Johnson & Michael Tytell

Authors
  1. A. Daniel Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Tytell
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Johnson, A.D., Tytell, M. Exogenous HSP70 becomes cell associated, but not internalized, by stressed arterial smooth muscle cells. In Vitro Cell Dev Biol - Animal 29, 807–812 (1993). https://doi.org/10.1007/BF02634348

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02634348

Key words

Search

Navigation