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(Circulation Research. 2005;97:4.)
© 2005 American Heart Association, Inc.

Editorials

Pre–B-cell Colony-Enhancing Factor Regulates Vascular Smooth Muscle Maturation Through a NAD⁺-Dependent Mechanism

Recognition of a New Mechanism for Cell Diversity and Redox Regulation of Vascular Tone and Remodeling

Stephen L. Archer

From the Department of Medicine (Cardiology), Department of Physiology, and the Vascular Biology Group, University of Alberta, Edmonton, Canada.

Correspondence to Dr Stephen L. Archer, Heart and Stroke Chair in Cardiovascular Research, Chair, Cardiology Division, Department of Medicine, University of Alberta, WMC 2C2.36, 8440 112th St, Edmonton, Alberta, Canada, T6G 2B7. E-mail sarcher{at}cha.ab.ca

See related article, pages 25–34

Key Words: nicotinamide phosphoribosyltranferase (NAmPRTase) • survivin • redox signaling • NAD⁺-dependent histone deacetylase • pulmonary hypertension

	Introduction

Top Introduction What Is PBEF? How Does the Effect... PBEF in Pathophysiology What Are the Implications... Unanswered Questions Conclusions References

 
There is a growing recognition of diversity in the morphology and function of vascular smooth muscle cells (SMCs). Diversity is both visible, with SMCs of different shape coexisting in arteries, and functional, manifest through differences in ionic profile, oxygen-sensitivity, proliferative capacity, and apoptosis susceptibility. SMC diversity is evident temporally (differences within a segment during development) and geographically (differences in SMCs between functionally discrete vascular segments within a single circulation).¹ Moreover, many vibrant "vascular villages" display rich diversity within a single vascular segment, with heterogeneity in SMCs that exist side by side.^2,3

The article by Pickering’s group addresses the molecular mechanism underlying a form of SMC diversity that has morphological and functional aspects; namely the existence of synthetic versus contractile SMCs.⁴ It is an important contribution because, in addressing their own hypothesis, they reveal a mechanism that has relevance to several unresolved questions in vascular biology.⁴ How do blood vessels regulate the transition from fetus to adult? What is the molecular basis for SMC diversity? How does acute redox regulation of vascular tone translate to altered vascular structure in hypertension? By providing partial answers to these questions, their work has implications for hypoxic pulmonary vasoconstriction (HPV),^1,2 pulmonary hypertension,³ systemic hypertension, vascular repair, and atherosclerosis.

The Pickering group used 2 clonal SMC lines, derived from human mammary arteries, to study the molecular mechanism for the maturational conversion from a proliferative to a constrictive phenotype. One cell line mimicked the SMC in fetal or healing arteries, being morphologically "Rubenesque" and possessed a synthetic noncontractile phenotype; the other line of cells was svelte, contractile, and thus, mature⁴ (Figure). Triggered by removal of serum, the synthetic, proliferating cells become elongate, spindle-shaped, and developed a contractile phenotype accompanied by apoptosis resistance.

View larger version (43K): [in this window] [in a new window]   PBEF determines cell maturation by regulating NAD+ synthesis and NAD+-dependent histone deacetylase which controls SMC gene transcription and apoptosis.

The hunt for the mechanism of this shape-shifting began with microarray analysis which implicated a little-studied gene, pre–B-cell colony-enhancing factor (PBEF). The shift from proliferative to contractile phenotype was accompanied by upregulation of PBEF, and PBEF overexpression promoted the acquisition of a contractile, stable SMC phenotype. PBEF overexpression also increased markers of SMC maturation (eg, metavinculin) and suppressed apoptosis. This would presumably support the adult blood vessel becoming active in terms of tone and reduce its tendency to continuously grow or remodel. Knockdown of endogenous PBEF, achieved by retrovirally-delivered siRNA, increased SMC apoptosis and reduced the capacity of the synthetic SMC line to mature to a contractile state. In an ex vivo chimeric model of vasculogenesis, using a matrigel implant in SCID mice, PBEF-overexpressing SMCs had increased ability to invest primitive endothelial vessels, creating a more mature, vascular phenotype.⁴ A corollary they do not explore is that a reservoir of PBEF-negative cells in adults could serve as a healing reservoir for vascular remodeling in response to injury or disease.

What makes the article noteworthy is the revelation of the mechanism by which PBEF achieves maturational transformation. PBEF increased SMC nicotinamide phosphoribosyltransferase (NAmPRTase) activity and SMC nicotinamide adenine dinucleotide (NAD) content in the synthetic cell line. They showed that PBEF is itself a NAmPRTase, able to drive the production of NAD⁺. In elegant experiments, it was shown that PBEF-induced increases in NAD⁺ determined SMC phenotype and function. This occurred through activation of a NAD⁺-dependent histone deacetylase (HDAC) (Figure, A).

	What Is PBEF?

Top Introduction What Is PBEF? How Does the Effect... PBEF in Pathophysiology What Are the Implications... Unanswered Questions Conclusions References

 
PBEF was first isolated from an activated peripheral-blood lymphocyte cDNA library and was found to promote maturation of B-cell precursors.⁵ It is now known to be ubiquitously expressed in nonlymphoid tissues, including lung⁶ and human fetal membranes.⁷ At the genomic level, PBEF is composed of 11 exons and 10 introns, spanning 34.7 kb of genomic DNA.⁷ Although initially felt to be a cytokine,⁸ PBEF has no homology to known cytokines.⁷ On observing that PBEF expression was higher in the cytoplasm of proliferating cells versus the nucleus in confluent cells, Kitani concluded that PBEF was an intracellular, cell-cycle associated protein.⁹ The Pickering group used different evidence to come to a similar conclusion.⁴ First PBEF is not detectably secreted, even when the gene is overexpressed and its biological activity is augmented.⁴ Even more compelling is the phylogenetic similarity of PBEF to NAmPRTases in primitive organisms. These enzymes regulate prokaryotic NAD synthesis⁴ by catalyzing the condensation of nicotinamide with 5-phosphoribosyl-1-pyrophosphate to yield nicotinamide mononucleotide (NMN; Figure).¹⁰ van der Veer concluded that PBEF in human SMCs is also a NAmPRTase. In prokaryotes and eukaryotes, the resulting NMN is converted to NAD⁺ by nicotinamide mononucleotide adenylyltransferase-1 (Figure, B).

	How Does the Effect of PBEF on NAD+ Alter Gene Transcription and Apoptosis?

Top Introduction What Is PBEF? How Does the Effect... PBEF in Pathophysiology What Are the Implications... Unanswered Questions Conclusions References

 
The PBEF pathway supplies NAD⁺ for the downstream Sir2 family of transcription regulators and their mammalian orthologs (SIRT1-SIRT7).¹¹ Sir2 members classically suppress transcription in yeast but its orthologs are conserved in mammals.¹¹ They regulate transcription and apoptosis through their ability to deacetylate histones and nonhistone proteins (eg, p53), thereby controlling the longevity of cells and organisms (Figure, B). SIRT1 is a NAD⁺-dependent HDAC, which regulates SMC maturation and survival, presumably as a result of altered transcription, although this is not directly proved. Activation of SIRT-1 (HDAC) by PBEF promotes cell maturation and survival whereas inhibition (by sirtinol) impairs maturation. Activators of SERT-1 exist, including resveratrol, a polyphenol in red wine (Figure).

	PBEF in Pathophysiology

Top Introduction What Is PBEF? How Does the Effect... PBEF in Pathophysiology What Are the Implications... Unanswered Questions Conclusions References

 
These are early days for PBEF, and little is known about its role in human health and disease. Although not itself a cytokine, PBEF expression is upregulated by cytokines⁶ and phorbol esters,⁵ perhaps explaining its upregulation in sepsis¹² and acute lung injury (ALI).⁶ In septic patients, PBEF slows neutrophil apoptosis by inhibiting caspase-8 and -3.¹² PBEF expression is also elevated in human and experimental ALI. PBEF single nucleotide polymorphisms also confer higher risk of ALI.⁶ Thus, whatever the contribution of PBEF is to ALI mechanistically, PBEF may be a useful biomarker.

	What Are the Implications of This Work?

Top Introduction What Is PBEF? How Does the Effect... PBEF in Pathophysiology What Are the Implications... Unanswered Questions Conclusions References

 
Cellular redox state is defined as the balance of reduced/oxidized couples (eg, NADH/NAD, glutathione, GSH/GSSG) and production of reactive oxygen species (by the mitochondria and NADPH oxidases). In contrast to redox reactions, which cycle but do not consume NAD⁺, some reactions, such as those in the HDAC pathway, consume NAD⁺, releasing nicotinamide, from which NAD⁺ is regenerated via a salvage pathway (Figure, B). In the Redox Hypothesis of HPV, there is substantial evidence that the mitochondrial electron transport chain (ETC), and possibly NAD(P)H oxidases, control the SMC redox pool and thereby regulate vascular tone,^13,14 SMC proliferation, and apoptosis.¹⁴ PBEF is likely one of several regulators of NAD⁺ levels and the relative importance of PBEF versus these other regulators of NAD⁺/NADH ratios (mitochondria/oxdases,¹⁵ or cyclic-ADP ribose) is unexplored. The sensitivity of PBEF or HDAC themselves to redox stimuli (mitochondrial inhibition, hypoxia, NAD⁺) awaits description.

In the pulmonary circulation, the resistance arteries are composed of a homogenous SMC population which is resistant to cytokine- or hypoxia-induced proliferation.¹⁶ These spindle-shaped SMCs, which display O₂-sensitive Kv channels that initiate HPV,^1,2 resemble mature PBEF-enriched SMCs. Conversely, the conduit PA, which lacks HPV, is a SMC mosaic, with some BK_Ca channel-enriched cells resembling the synthetic cells.

Frid et al found 4 SMC populations in pulmonary arteries as delineated using a panel of markers. They noted proliferation in response to hypoxia or pulmonary hypertension occurred only in the meta-vinculin–negative SMC cells.¹⁷ In an interesting parallel, PBEF-overexpressing SMCs also displayed increased maturation markers. Perhaps the proliferative and ionic phenotypes of pulmonary artery SMCs correlate with diversity of PBEF expression/function?

	Unanswered Questions

Top Introduction What Is PBEF? How Does the Effect... PBEF in Pathophysiology What Are the Implications... Unanswered Questions Conclusions References

 
The role of PDEF in vivo remains unanswered. There is also a curious dissociation between apoptosis and maturation in this initial report. Surprisingly, PBEF-overexpressing cells have increased expression of maturation markers even when apoptosis was not suppressed. This may indicate separate PBEF-sensitive targets for maturation versus apoptosis pathways or reflect the influence of PBEF-independent apoptosis pathways.

Finally, PBEF is certainly not the only pathway controlling SMC proliferation and apoptosis. During pathological proliferation, as occurs in pulmonary hypertension, survivin, an apoptosis inhibiting protein, is induced and drives vascular remodeling. Survivin, a rehabilitated cancer protein, is now recognized to control SMC proliferation in human pulmonary arterial hypertension.^18,19 Survivin inhibition depolarizes SMC mitochondria, triggering beneficial apoptosis and remodeling.¹⁹ A logical starting point to examine the intersection of the PBEF and the mitochondrial–survivin pathways is at their intersection—the redox couples.

Finally, this article offers strategic lessons for young scientists. There are 20 articles cited in PubMed on PBEF, none relating to SMCs. To discover, one must venture into the unexplored. Although confirmation of the results of others is useful (up to a point), true novelty coupled with sufficient experimental depth to yield conclusive results won this article priority in the competition for scarce journal space.

	Conclusions

Top Introduction What Is PBEF? How Does the Effect... PBEF in Pathophysiology What Are the Implications... Unanswered Questions Conclusions References

 
There are lessons that can be borrowed for vascular biology from hematology-oncology. This reflects the convergence of the great pathways, which overarch artificial separations of science into disciplines. The vascular biologist should now be as interested in maturational and apoptotic proteins, such as PBEF⁴ and survivin,¹⁸ as they have been in the traditional pathways that regulate vascular tone.

	Acknowledgments

 
S.L.A. is a Canada Research Chair in Oxygen Sensing and Translational Cardiovascular Research and is supported by the Canada Foundation for Innovation, the Alberta Heart and Stroke Foundation, the Canadian Institutes for Health Research (CIHR), NIH-RO1-HL071115, and ABACUS (the Alberta Cardiovascular and Stroke Research Centre).

	Footnotes

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

	References

Top Introduction What Is PBEF? How Does the Effect... PBEF in Pathophysiology What Are the Implications... Unanswered Questions Conclusions References

 

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2. Archer SL, Huang JM, Reeve HL, Hampl V, Tolarova S, Michelakis E, Weir EK. Differential distribution of electrophysiologically distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia. Circ Res. 1996; 78: 431–442.[Abstract/Free Full Text]
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10. Rongvaux A, Shea RJ, Mulks MH, Gigot D, Urbain J, Leo O, Andris F. Pre-B-cell colony-enhancing factor, whose expression is up-regulated in activated lymphocytes, is a nicotinamide phosphoribosyltransferase, a cytosolic enzyme involved in NAD biosynthesis. Eur J Immunol. 2002; 32: 3225–3234.[CrossRef][Medline] [Order article via Infotrieve]
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Pre–B-Cell Colony–Enhancing Factor Regulates NAD⁺-Dependent Protein Deacetylase Activity and Promotes Vascular Smooth Muscle Cell Maturation

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