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(Circulation Research. 2005;96:9.)
© 2005 American Heart Association, Inc.

Editorials

Cross-Linking Vasomotor Tone and Vascular Remodeling

A Novel Function for Tissue Transglutaminase?

B. Lowell Langille, Dorota Dajnowiec

From the Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada.

Correspondence to B. Lowell Langille, PhD, Toronto General Hospital, 200 Elizabeth St, Toronto, ON M5G 2C4, Canada. E-mail langille{at}uhnres.utoronto.ca

Key Words: extracellular matrix • nitric oxide • G-protein • fibronectin

Tightly regulated remodeling of arterial tissues is essential for survival in all vertebrate species. Arterial remodeling tunes development of the vasculature to the changing metabolic demands of embryonic tissues, it accommodates the extraordinary and diverse adjustments of cardiovascular function that occur at birth in both mother and neonate, and it adapts vascular structure to a host of physiological changes in adults. Arterial tissue remodeling also greatly influences the progression of the most important vascular pathologies including atherosclerosis, hypertension, and restenosis.¹

Mechanical forces, particularly blood flow-derived shear stress, are potent stimuli for arterial remodeling. Large increases in shear stress elicit immediate vasodilation that is largely NO-dependent,² and then a subsequent growth and remodeling selectively deliver new tissue in the circumferential direction to further increase vessel diameter. Conversely, decreased blood flow rate stimulates reduction in arterial diameter during some phases of development, in the postpartum maternal circulation, and in occlusive vascular pathologies.¹ Early vasoregulation can be particularly important when shear stress is reduced because increases in vasomotor tone can cause substantial vasoconstriction, even in conduit arteries, and true remodeling often serves only to entrench the reduction in vessel diameter.³

The entrenchment of reduced vessel diameter during inward remodeling has important implications for the reversal of vascular pathologies after therapeutic interventions including bypass graft implantation, angioplasty, stenting, and antihypertensive therapy. But what does "remodeling" mean in this context? What causes the resetting of resting arterial diameter, and how do smooth muscle cells ultimately achieve a capacity to narrow diameter below that which previously characterized maximal constriction?³ Two recent studies have shed light on novel mechanisms by which constrictor responses can be converted to structural remodeling.

Martinez-Lemus et al⁴ found that maintenance of norepinephrine-induced constriction for only 4 hours was sufficient to at least partially entrench the diameter reduction caused by the drug. Surprisingly, they also found that the in situ lengths of many mural cells (>50% of them) returned toward normal levels over this time, even though vessel constriction was fully sustained. These findings suggest that active restoration of normal cell length contributed to entrenchment of reduced diameter (see Figure) and they raise intriguing questions concerning the cell biology that underlies cellular elongation in the context of fixed vessel diameter. For example, what is the nature of the cytoskeletal reorganization that drives cell elongation and how does it proceed while vasomotor tone is sustained? What is the role of matrix metalloproteinases or other degradative enzymes in allowing cells to elongate through surrounding extracellular matrix? Finally, how do these motile cells reorganize adhesion complexes that link them to surrounding cells and matrix?

View larger version (26K): [in this window] [in a new window]   Some functions of tTG and their relationship to inward arterial remodeling. Dashed arrows show pathways that are suppressed when shear stress is reduced. Lower endothelial NO production increases vasomotor tone and disinhibits tTG activity. The capacity of tTG to cross-link extracellular matrix proteins then may entrench reductions in vessel diameter that are initiated by increased smooth muscle tone. Contraction-related increases in [Ca2+]i provides additional activation of tTG, which will activate the RhoA/ROCK2 pathway, to amplify vasoconstriction, while suppressing G-protein function of the enzyme and regulation of the phospholipase Cd1 (PLC1) pathway. Additional signaling is outlined in the text. Also shown is the cell elongation (gray arrows) without vessel enlargement that follows persistent vasoconstriction.4

Tissue Transglutaminase As a Mediator of Inward Arterial Remodeling

Extracellular matrix bears most of the arterial wall tension generated by blood pressure; therefore, matrix remodeling is required for adjustment of resting vessel diameter. Turnover of elastin, collagens, and other wall matrix may participate; however, Bakker et al present, in this issue of Circulation Research, data supporting an intriguing alternative mechanism.⁵ These authors, like Martinez-Lemus et al,⁴ previously showed that persistent vasoconstriction leads to entrenchment of reduced diameter.⁶ Their new evidence indicates that tissue transglutaminase (tTG) participates in this inward remodeling, possibly through the capacity of tTG to cross-link extracellular matrix proteins. Accordingly, inhibitors of tTG prevent the entrenchment of vessel narrowing after endothelin treatment; furthermore, exogenous, or upregulation of endogenous, tTG enhanced both arterial remodeling and the capacity of vascular smooth muscle to contract collagen gels. Finally, tTG inhibition suppressed inward remodeling because of reduced blood flow in mesenteric arterioles in vivo.

Transglutaminases are calcium-dependent enzymes that display diverse intracellular and extracellular activities, including protein cross-linking, matrix protein binding, functioning as a G-protein, and protein modification through amine incorporation or deamidation.⁷ A role of tTG in arterial remodeling was foreshadowed by evidence that it is constitutively expressed at high levels in endothelial and vascular smooth muscle cells⁸ and that it participates in angiogenesis during wound healing.⁹ The capacity of tTG to form highly stable cross-links of extracellular matrix and related proteins, including fibronectin, vitronectin, osteonectin, osteopontin, and collagens,^10,11 may be most relevant to entrenchment of inward remodeling. Bakker et al suggest that collagen cross-linking is critical, given that remodeling became evident at pressures that mechanically load collagen.

It is noteworthy, however, that cross-links formed by tTG are extremely stable and their effects are normally reversed only by turnover of the cross-linked proteins. These properties are not readily reconciled with observations that long-term narrowing of arteries attributable to reduced shear stress is rapidly reversed after flow restoration¹² and that vascular extracellular matrix proteins are normally very stable.¹³ It is possible that flow restoration induces unusually rapid degradation/resynthesis of these proteins; alternatively, cross-links produced by tTG may represent a rapid but transient means of entrenching inward remodeling that becomes supplanted over time by gradual matrix turnover. A third possibility is that tTG functions, other than matrix cross-linking, are critical in inward remodeling of vessels.

Multiple Functions of Tissue tTG May Participate in Arterial Remodeling

In the absence of calcium, tTG acts as a G-Protein,¹⁴ with GTPGDP exchange coupling tTG to multiple receptors, including -adrenoreceptors, to disinhibit PLC1 and thereby upregulate IP3 production.⁷ The high intracellular Ca²⁺ levels that accompany flow-related constriction will downregulate this function and possibly provide some negative feedback control over vasomotor tone, but the net effect of tTG activity on vasoregulation is probably enhanced constriction. This is because Ca²⁺-activated intracellular tTG enhances RhoA/ROCK-2 kinase binding and ROCK-2 autophosphorylation, which facilitates smooth muscle cell contraction.^7,15 Beyond influences on contractile function, intracellular tTG can translocate to the nucleus where its multiple activities may directly participate in regulation of gene transcription or they may induce chromatin modifications, so that expression of genes that are important in tissue remodeling may be affected significantly by tTG activity.^16,17

In addition to cross-linking matrix proteins, extracellular tTG associates with ß-integrins, where it acts as a coreceptor for fibronectin and participates in enhancing both cell adhesion and cell motility.^18,19 One intriguing possibility is that tTG-regulated motility contributes to the elongation of smooth muscle cells during persistent vasoconstriction that was reported by Martinez-Lemus et al.⁴

tTG activity is also fundamental to appropriate handling of cellular products of apoptosis.^7,11 Expression of tTG, and its activation by a massive increase in [Ca²⁺]_i during apoptosis, leads to extensive cross-linking of cellular proteins that stabilize cell structure and limit the release of toxic or proinflammatory substances until apoptotic bodies are phagocytosed by neighboring cells or macrophages. This function may be particularly important to inward remodeling because of flow reduction during some phases of development when apoptosis rates can be very high.²¹

Regulation of Tissue tTG Expression and Function

tTG is constitutively expressed in vascular cells⁸; however, the promoter region of the gene has functional response elements that respond to pathways that are shear-sensitive in the endothelium (transforming growth factor-ß, nuclear factor-B); therefore, expression may be modulated during remodeling. In addition, there are several modes of posttranslational regulation of tTG.

Withdrawal of NO participates in acute vasoconstriction when flow rates are reduced (D.D. and B.L.L., 2004, unpublished observation), and it is a key initiator of flow-related inward arterial remodeling.²² It is therefore of particular interest that NO is a potent inhibitor of tTG activity, through Ca²⁺-sensitive nitrosylation of multiple cysteine residues.^23,24 An attractive hypothesis is that suppression of NO production by reduced shear stress leads to tTG activation that first amplifies constriction, through Rho/Rock signaling, and then entrenches vessel narrowing (see Figure).

Other modes of tTG regulation may also prevail. Interestingly, the cell surface–associated tTG that interacts with integrins and fibronectin is subject to hydrolysis by the activity of matrix metalloproteinase-2, which is activated locally by membrane type 1–matrix metalloproteinase, an activity that is upregulated during flow-related inward remodeling.^25,26

Conclusion

Arterial remodeling has been the focus of intense research by vascular biologist over the last decade, but the concept has remained clouded by a paucity of mechanistic insights into how the process is achieved. Recent work has suggested novel directions for future study concerning the roles of cell contractile function, cell motility, and expression of matrix modifying enzymes. If a role for tTG in remodeling is confirmed, then there should be much interest in elucidating the relative importance of the disparate functions of this enzyme in vascular tissue reorganization. Furthermore, laboratories investigating other mechanically loaded tissues, eg, lung, heart, and the peripartum uterus, will undoubtedly wish to pursue the role of tTG in comparable remodeling responses. Finally, vascular biologists will be particularly interested in how this enzyme influences pathological arterial remodeling and how its roles in these processes might be modulated therapeutically.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

See related article, pages 119–126

References

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