Pflügers Archiv - European Journal of Physiology

, Volume 465, Issue 9, pp 1293–1302 | Cite as

Specificity in the participation of connexin proteins in flow-induced endothelial gap junction communication

Molecular and Cellular Mechanisms of Disease

Abstract

Endothelial cell (EC) dysfunction and atherosclerotic plaque formation coincide with human circulatory regions where blood flow is altered (disturbed). In areas of undisturbed uniform blood flow, including the majority of the vasculature, the vessel wall is relatively atherosclerotic lesion-resistant with normal endothelium. The molecular mechanisms of blood flow regulation of EC function and atherogenesis are unclear. We hypothesize that EC dysfunction potentiating atherosclerosis is related to disturbed flow (DF)-induced EC gap junctional intercellular communication (GJIC) changes via the gap junction connexin (Cx) 37, 40, and 43 proteins, which are involved in EC proliferation and vasoactivity that are known to be altered in atherosclerosis. We investigated human EC GJIC using an in vitro model of the hemodynamic features found in atherosclerotic-prone DF regions in vivo. Using dye transfer assays, Cx-specific mimetic peptide inhibitors, proliferation assays, and immunocytochemistry, we correlated functional GJIC via gap junction channels formed by hemichannels composed of the two most abundant endothelial Cx—Cx40 and Cx43—to EC proliferation and expression of vasoactive endothelial-type nitric oxide synthase (eNOS). We found that, in uniform flow conditions, substantial GJIC was conducted through gap junctions containing Cx40 hemichannels and correlated to a nonproliferative EC phenotype and membrane localization of eNOS, similar to physiological conditions. In DF, GJIC was largely attained through Cx43 hemichannel-containing gap junctions, EC phenotype was proliferative (attributed to loss of contact inhibition), and intracellular eNOS was more abundant than membrane eNOS, typical of atherosclerotic sites in vivo. This is the first in vitro study to demonstrate local hemodynamically defined Cx protein specificity in human EC GJIC with a potential role in endothelial dysfunction characteristic of early atherosclerosis.

Keywords

Disturbed flow Endothelial cells Gap junctional communication Cell proliferation Endothelial-type nitric oxide synthase 

References

  1. 1.
    Alonso F, Boittin FX, Beny JL, Haefliger JA (2010) Loss of connexin40 is associated with decreased endothelium-dependent relaxations and eNOS levels in the mouse aorta. Am J Physiol Heart Circ Physiol 299:H1365–H1373PubMedCrossRefGoogle Scholar
  2. 2.
    Bao X, Clark CB, Frangos JA (2000) Temporal gradient in shear-induced signaling pathway: involvement of MAP kinase, c-fos, and connexin43. Am J Physiol Heart Circ Physiol 278:H1598–H1605PubMedGoogle Scholar
  3. 3.
    Barber KM, Pinero A, Truskey GA (1998) Effects of recirculating flow on U-937 cell adhesion to human umbilical vein endothelial cells. Am J Physiol 275:H591–H599PubMedGoogle Scholar
  4. 4.
    Bastide B, Neyses L, Ganten D, Paul M, Willecke K, Traub O (1993) Gap junction protein connexin40 is preferentially expressed in vascular endothelium and conductive bundles of rat myocardium and is increased under hypertensive conditions. Circ Res 73:1138–1149PubMedCrossRefGoogle Scholar
  5. 5.
    Braet K, Aspeslagh S, Vandamme W, Willecke K, Martin PE, Evans WH, Leybaert L (2003) Pharmacological sensitivity of ATP release triggered by photoliberation of inositol-1,4,5-trisphosphate and zero extracellular calcium in brain endothelial cells. J Cell Physiol 197:205–213PubMedCrossRefGoogle Scholar
  6. 6.
    Burns MP, DePaola N (2005) Flow-conditioned HUVECs support clustered leukocyte adhesion by coexpressing ICAM-1 and E-selectin. Am J Physiol Heart Circ Physiol 288:H194–H204PubMedCrossRefGoogle Scholar
  7. 7.
    Caro CG, Dumoulin CL, Graham JM, Parker KH, Souza SP (1992) Secondary flow in the human common carotid artery imaged by MR angiography. J Biomech Eng 114:147–149PubMedCrossRefGoogle Scholar
  8. 8.
    Chaytor AT, Evans WH, Griffith TM (1998) Central role of heterocellular gap junctional communication in endothelium-dependent relaxations of rabbit arteries. J Physiol 508(Pt 2):561–573PubMedCrossRefGoogle Scholar
  9. 9.
    Chaytor AT, Martin PE, Edwards DH, Griffith TM (2001) Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery. Am J Physiol Heart Circ Physiol 280(6):H2441–H2450PubMedGoogle Scholar
  10. 10.
    Chen CN, Chang SF, Lee PL, Chang K, Chen LJ, Usami S, Chien S, Chiu JJ (2006) Neutrophils, lymphocytes, and monocytes exhibit diverse behaviors in transendothelial and subendothelial migrations under coculture with smooth muscle cells in disturbed flow. Blood 107:1933–1942PubMedCrossRefGoogle Scholar
  11. 11.
    Chiu JJ, Chien S (2011) Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives. Physiol Rev 91:327–387PubMedCrossRefGoogle Scholar
  12. 12.
    Chiu JJ, Chen CN, Lee PL, Yang CT, Chuang HS, Chien S, Usami S (2003) Analysis of the effect of disturbed flow on monocytic adhesion to endothelial cells. J Biomech 36:1883–1895PubMedCrossRefGoogle Scholar
  13. 13.
    Christ GJ, Spray DC, El-Sabban M, Moore LK, Brink PR (1996) Gap junctions in vascular tissues. Evaluating the role of intercellular communication in the modulation of vasomotor tone. Circ Res 79:631–646PubMedCrossRefGoogle Scholar
  14. 14.
    Cowan DB, Lye SJ, Langille BL (1998) Regulation of vascular connexin43 gene expression by mechanical loads. Circ Res 82:786–793PubMedCrossRefGoogle Scholar
  15. 15.
    de Wit C, Roos F, Bolz SS, Kirchhoff S, Kruger O, Willecke K, Pohl U (2000) Impaired conduction of vasodilation along arterioles in connexin40-deficient mice. Circ Res 86:649–655PubMedCrossRefGoogle Scholar
  16. 16.
    de Wit C, Roos F, Bolz SS, Pohl U (2003) Lack of vascular connexin 40 is associated with hypertension and irregular arteriolar vasomotion. Physiol Genomics 13:169–177PubMedGoogle Scholar
  17. 17.
    DePaola N, Gimbrone MA Jr, Davies PF, Dewey CF Jr (1992) Vascular endothelium responds to fluid shear stress gradients. Arterioscler Thromb 12:1254–1257PubMedCrossRefGoogle Scholar
  18. 18.
    DePaola N, Davies PF, Pritchard WF Jr, Florez L, Harbeck N, Polacek DC (1999) Spatial and temporal regulation of gap junction connexin43 in vascular endothelial cells exposed to controlled disturbed flows in vitro. Proc Natl Acad Sci U S A 96:3154–3159PubMedCrossRefGoogle Scholar
  19. 19.
    Desplantez T, Verma V, Leybaert L, Evans WH, Weingart R (2012) Gap26, a connexin mimetic peptide, inhibits currents carried by connexin43 hemichannels and gap junction channels. Pharmacol Res 65:546–552PubMedCrossRefGoogle Scholar
  20. 20.
    Ebong EE, Kim S, DePaola N (2006) Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels. Am J Physiol Heart Circ Physiol 290:H2015–H2023PubMedCrossRefGoogle Scholar
  21. 21.
    Evans WH, Leybaert L (2007) Mimetic peptides as blockers of connexin channel-facilitated intercellular communication. Cell Commun Adhes 14:265–273PubMedCrossRefGoogle Scholar
  22. 22.
    Figueroa XF, Duling BR (2009) Gap junctions in the control of vascular function. Antioxid Redox Signal 11:251–266PubMedCrossRefGoogle Scholar
  23. 23.
    Gabriels JE, Paul DL (1998) Connexin43 is highly localized to sites of disturbed flow in rat aortic endothelium but connexin37 and connexin40 are more uniformly distributed. Circ Res 83:636–643PubMedCrossRefGoogle Scholar
  24. 24.
    Griffith TM, Chaytor AT, Edwards DH (2004) The obligatory link: role of gap junctional communication in endothelium-dependent smooth muscle hyperpolarization. Pharmacol Res 49:551–564PubMedCrossRefGoogle Scholar
  25. 25.
    Grunwald J, Robenek H, Mey J, Hauss WH (1982) In vivo and in vitro cellular changes in experimental hypertension: electronmicroscopic and morphometric studies of aortic smooth muscle cells. Exp Mol Pathol 36:164–176PubMedCrossRefGoogle Scholar
  26. 26.
    Haefliger JA, Castillo E, Waeber G, Bergonzelli GE, Aubert JF, Sutter E, Nicod P, Waeber B, Meda P (1997) Hypertension increases connexin43 in a tissue-specific manner. Circulation 95:1007–1014PubMedCrossRefGoogle Scholar
  27. 27.
    Hoffmann A, Gloe T, Pohl U, Zahler S (2003) Nitric oxide enhances de novo formation of endothelial gap junctions. Cardiovasc Res 60:421–430PubMedCrossRefGoogle Scholar
  28. 28.
    Hutcheson IR, Chaytor AT, Evans WH, Griffith TM (1999) Nitric oxide-independent relaxations to acetylcholine and A23187 involve different routes of heterocellular communication. Role of Gap junctions and phospholipase A2. Circ Res 84:53–63PubMedCrossRefGoogle Scholar
  29. 29.
    Johnson TL, Nerem RM (2007) Endothelial connexin 37, connexin 40, and connexin 43 respond uniquely to substrate and shear stress. Endothelium 14:215–226PubMedGoogle Scholar
  30. 30.
    Johnstone S, Isakson B, Locke D (2009) Biological and biophysical properties of vascular connexin channels. Int Rev Cell Mol Biol 278:69–118PubMedCrossRefGoogle Scholar
  31. 31.
    Kwak BR, Pepper MS, Gros DB, Meda P (2001) Inhibition of endothelial wound repair by dominant negative connexin inhibitors. Mol Biol Cell 12:831–845PubMedCrossRefGoogle Scholar
  32. 32.
    Kwak BR, Mulhaupt F, Veillard N, Gros DB, Mach F (2002) Altered pattern of vascular connexin expression in atherosclerotic plaques. Arterioscler Thromb Vasc Biol 22:225–230PubMedCrossRefGoogle Scholar
  33. 33.
    Kwak BR, Silacci P, Stergiopulos N, Hayoz D, Meda P (2005) Shear stress and cyclic circumferential stretch, but not pressure, alter connexin43 expression in endothelial cells. Cell Commun Adhes 12:261–270PubMedCrossRefGoogle Scholar
  34. 34.
    Larson DM, Wrobleski MJ, Sagar GD, Westphale EM, Beyer EC (1997) Differential regulation of connexin43 and connexin37 in endothelial cells by cell density, growth, and TGF-beta1. Am J Physiol 272:C405–C415PubMedGoogle Scholar
  35. 35.
    Little TL, Xia J, Duling BR (1995) Dye tracers define differential endothelial and smooth muscle coupling patterns within the arteriolar wall. Circ Res 76:498–504PubMedCrossRefGoogle Scholar
  36. 36.
    Maday Y, Patera AT (1989) Spectral element methods for Navier–Stokes equation. In: Noor AK, Oden JT (eds) State-of-the-art surveys on computational mechanics. American Society of Mechanical Engineers, New York, pp 71–143Google Scholar
  37. 37.
    Pepper MS, Spray DC, Chanson M, Montesano R, Orci L, Meda P (1989) Junctional communication is induced in migrating capillary endothelial cells. J Cell Biol 109:3027–3038PubMedCrossRefGoogle Scholar
  38. 38.
    Pepper MS, Montesano R, El Aoumari A, Gros D, Orci L, Meda P (1992) Coupling and connexin 43 expression in microvascular and large vessel endothelial cells. Am J Physiol 262:1246–1257Google Scholar
  39. 39.
    Pfenniger A, Chanson M, Kwak BR (2012) Connexins in atherosclerosis. Biochim Biophys Acta 1828(1):157–166. doi:10.1016/j.bbamem.2012.05.011 Google Scholar
  40. 40.
    Pfenniger A, Wong C, Sutter E, Cuhlmann S, Dunoyer-Geindre S, Mach F, Horrevoets AJ, Evans PC, Krams R, Kwak BR (2012) Shear stress modulates the expression of the atheroprotective protein Cx37 in endothelial cells. J Mol Cell Cardiol 53:299–309PubMedCrossRefGoogle Scholar
  41. 41.
    Phelps JE, DePaola N (2000) Spatial variations in endothelial barrier function in disturbed flows in vitro. Am J Physiol Heart Circ Physiol 278:H469–H476PubMedGoogle Scholar
  42. 42.
    Polacek D, Bech F, McKinsey JF, Davies PF (1997) Connexin43 gene expression in the rabbit arterial wall: effects of hypercholesterolemia, balloon injury and their combination. J Vasc Res 34:19–30PubMedCrossRefGoogle Scholar
  43. 43.
    Rozental R, Srinivas M, Spray DC (2001) How to close a gap junction channel. Efficacies and potencies of uncoupling agents. Methods Mol Biol 154:447–476PubMedGoogle Scholar
  44. 44.
    Segal SS, Duling BR (1987) Propagation of vasodilation in resistance vessels of the hamster: development and review of a working hypothesis. Circ Res 61:II20–II25PubMedGoogle Scholar
  45. 45.
    Segal SS, Beny JL (1992) Intracellular recording and dye transfer in arterioles during blood flow control. Am J Physiol 263:H1–H7PubMedGoogle Scholar
  46. 46.
    Skilbeck C, Westwood SM, Walker PG, David T, Nash GB (2001) Dependence of adhesive behavior of neutrophils on local fluid dynamics in a region with recirculating flow. Biorheology 38:213–227PubMedGoogle Scholar
  47. 47.
    Tardy Y, Resnick N, Nagel T, Gimbrone MA Jr, Dewey CF Jr (1997) Shear stress gradients remodel endothelial monolayers in vitro via a cell proliferation–migration–loss cycle. Arterioscler Thromb Vasc Biol 17:3102–3106PubMedCrossRefGoogle Scholar
  48. 48.
    Tsai ML, Watts SW, Loch-Caruso R, Webb RC (1995) The role of gap junctional communication in contractile oscillations in arteries from normotensive and hypertensive rats. J Hypertens 13:1123–1133PubMedCrossRefGoogle Scholar
  49. 49.
    van Rijen HV, van Veen TA, Hermans MM, Jongsma HJ (2000) Human connexin40 gap junction channels are modulated by cAMP. Cardiovasc Res 45:941–951PubMedCrossRefGoogle Scholar
  50. 50.
    Vorderwulbecke BJ, Maroski J, Fiedorowicz K, Da Silva-Azevedo L, Marki A, Pries AR, Zakrzewicz A (2011) Regulation of endothelial connexin40 expression by shear stress. Am J Physiol Heart Circ Physiol 302:H143–H152PubMedCrossRefGoogle Scholar
  51. 51.
    Wang HH, Kung CI, Tseng YY, Lin YC, Chen CH, Tsai CH, Yeh HI (2008) Activation of endothelial cells to pathological status by down-regulation of connexin43. Cardiovasc Res 79:509–518PubMedCrossRefGoogle Scholar
  52. 52.
    Wedgwood S, Mitchell CJ, Fineman JR, Black SM (2003) Developmental differences in the shear stress-induced expression of endothelial NO synthase: changing role of AP-1. Am J Physiol Lung Cell Mol Physiol 284:L650–L662PubMedGoogle Scholar
  53. 53.
    Wright CS, van Steensel MA, Hodgins MB, Martin PE (2009) Connexin mimetic peptides improve cell migration rates of human epidermal keratinocytes and dermal fibroblasts in vitro. Wound Repair and Regeneration 17:240–249PubMedCrossRefGoogle Scholar
  54. 54.
    Xie HQ, Hu VW (1994) Modulation of gap junctions in senescent endothelial cells. Exp Cell Res 214:172–176PubMedCrossRefGoogle Scholar
  55. 55.
    Yeh HI, Rothery S, Dupont E, Coppen SR, Severs NJ (1998) Individual gap junction plaques contain multiple connexins in arterial endothelium. Circ Res 83:1248–1263PubMedCrossRefGoogle Scholar
  56. 56.
    Zarins CK, Giddens DP, Bharadvaj BK, Sottiurai VS, Mabon RF, Glagov S (1983) Carotid bifurcation atherosclerosis. Quantitative correlation of plaque localization with flow velocity profiles and wall shear stress. Circ Res 53:502–514PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Department of Biomedical Engineering and Center of Biotechnology and Interdisciplinary StudiesRensselaer Polytechnic InstituteTroyUSA
  2. 2.Department of Biomedical EngineeringThe City College of New YorkNew YorkUSA
  3. 3.Department of NeuroscienceAlbert Einstein College of MedicineBronxUSA
  4. 4.Department of Biomedical Engineering and the Pritzker Institute of Biomedical Science and EngineeringIllinois Institute of TechnologyChicagoUSA
  5. 5.Armour College of EngineeringIllinois Institute of TechnologyChicagoUSA

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