Cynthia St. Hilaire

The A2B adenosine receptor protects against inflammation and excessive vascular adhesion

Adenosine has been described as playing a role in the control of inflammation, but it has not been certain which of its receptors mediate this effect. Here, we generated an A2B adenosine receptor-knockout/reporter gene-knock-in (A2BAR-knockout/reporter gene-knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-alpha, and a consequent downregulation of IkappaB-alpha are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A2BAR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A2BAR-knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A2BAR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A2BARs regulate these processes. Hence, we identify the A2BAR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target.

TGF-β Signaling Mediates Endothelial-to-Mesenchymal Transition (EndMT) During Vein Graft Remodeling

In vivo endothelial cell fate mapping demonstrates that TGF-β signaling is a central pathway regulating the endothelial-to-mesenchymal transition (EndMT) during vein graft remodeling. Negative Remodeling In coronary bypass surgery, veins are grafted to arteries, in hopes of generating a functional vessel. Although a routine procedure, grafting can result in a negative remodeling process—with a poorly understood underlying mechanism. Here, Cooley and colleagues linked vein graft stenosis (blood vessel narrowing) and negative remodeling to a process called the endothelial-to-mesenchymal transition (EndMT). Although well known during development, the presence of EndMT in the vasculature is less documented and therefore represents a possible new target in preventing graft failure. The authors tracked endothelial cells in mice using yellow fluorescent protein (YFP), and saw that these cells lining the vessel walls contributed to arterial thickening (neointima formation) after vein grafting by first converting to mesenchymal cells. EndMT occurred via transforming growth factor–β (TGF-β) signaling, specifically through intermediates Smad2/3 and Slug. Knowing the pathway at play is important for translation to the clinic because therapeutics can be designed to target these signaling molecules. Indeed, the authors found that blocking TGF-β with an antibody or knocking down Smad2 in vivo in mice prevented EndMT. The mesenchymal transition was also noted in failed vein grafts taken from patients, suggesting that EndMT is also present in humans and contributes to graft failure and restenosis. More testing is required in human samples to confirm the mouse data, but EndMT appears to be a viable target for improving graft outcomes after surgery in patients. Veins grafted into an arterial environment undergo a complex vascular remodeling process. Pathologic vascular remodeling often results in stenosed or occluded conduit grafts. Understanding this complex process is important for improving the outcome of patients with coronary and peripheral artery disease undergoing surgical revascularization. Using in vivo murine cell lineage–tracing models, we show that endothelial-derived cells contribute to neointimal formation through endothelial-to-mesenchymal transition (EndMT), which is dependent on early activation of the Smad2/3-Slug signaling pathway. Antagonism of transforming growth factor–β (TGF-β) signaling by TGF-β neutralizing antibody, short hairpin RNA–mediated Smad3 or Smad2 knockdown, Smad3 haploinsufficiency, or endothelial cell–specific Smad2 deletion resulted in decreased EndMT and less neointimal formation compared to controls. Histological examination of postmortem human vein graft tissue corroborated the changes observed in our mouse vein graft model, suggesting that EndMT is operative during human vein graft remodeling. These data establish that EndMT is an important mechanism underlying neointimal formation in interpositional vein grafts, and identifies the TGF-β–Smad2/3–Slug signaling pathway as a potential therapeutic target to prevent clinical vein graft stenosis.

Stat3-dependent acute Rantes production in vascular smooth muscle cells modulates inflammation following arterial injury in mice

Inflammation is a key component of arterial injury, with VSMC proliferation and neointimal formation serving as the final outcomes of this process. However, the acute events transpiring immediately after arterial injury that establish the blueprint for this inflammatory program are largely unknown. We therefore studied these events in mice and found that immediately following arterial injury, medial VSMCs upregulated Rantes in an acute manner dependent on Stat3 and NF-kappaB (p65 subunit). This led to early T cell and macrophage recruitment, processes also under the regulation of the cyclin-dependent kinase inhibitor p21Cip1. Unique to VSMCs, Rantes production was initiated by Tnf-alpha, but not by Il-6/gp130. This Rantes production was dependent on the binding of a p65/Stat3 complex to NF-kappaB-binding sites within the Rantes promoter, with shRNA knockdown of either Stat3 or p65 markedly attenuating Rantes production. In vivo, acute NF-kappaB and Stat3 activation in medial VSMCs was identified, with acute Rantes production after injury substantially reduced in Tnfa-/- mice compared with controls. Finally, we generated mice with SMC-specific conditional Stat3 deficiency and confirmed the Stat3 dependence of acute Rantes production by VSMCs. Together, these observations unify inflammatory events after vascular injury, demonstrating that VSMCs orchestrate the arterial inflammatory response program via acute Rantes production and subsequent inflammatory cell recruitment.

Deregulated Aurora-B induced tetraploidy promotes tumorigenesis

High expression of Aurora‐B has been observed in various cancers, and inhibition of this kinase has been shown to halt cellular proliferation. However, the mechanism of effect of Aurora‐B on cellular transformation has not been fully explored. Here, we show that overexpression of Aurora‐B in murine epithelial cells promotes generation of tetraploids. In search of a related mechanism, spectral karyotyping was carried out, showing premature chromatid separation (PCS). Of interest, PCS is a hallmark of Roberts syndrome, which also involves cellular polyploidy and aneu‐ ploidy. Sorted tetraploid Aurora‐B‐overexpressing cells promoted significant mammary epithelial cancers when injected into nude mice, as compared to injection of nonfractionated cells, suggesting that tetraploidy is an important mediator of Aurora‐B‐induced tumorigenesis. Comparative chromosome hybridization performed on DNA derived from tetraploid cell‐induced tumors indicates amplifications and deletions of regions throughout the genome, which include tumor‐promoting or tumorsuppressing genes, respectively. Thus, sustained expression of Aurora‐B induces tetraploidy, which, in turn, facilitates genomic instability and tumor development in a xenograft model.— Nguyen, H. G., Makitalo, M., Yang, D., Chinnappan, D., St. Hilaire, C., Ravid, K. Deregulated Aurora‐B induced tetraploidy promotes tumorigenesis. FASEBJ. 23, 2741–2748 (2009)

Mechanisms of induction of adenosine receptor genes and its functional significance.

Adenosine is a metabolite generated and released from cells, particularly under injury or stress. It elicits protective or damaging responses via signaling through the adenosine receptors, including the adenylyl cyclase inhibitory A1 and A3, and the adenylyl cyclase stimulatory A2A and A2B. Multiple adenosine receptor types, including stimulatory and inhibitory, can be found in the same cell, suggesting that a careful balance of adenosine receptor expression in a particular cell is necessary for a specific adenosine‐induced response. This balance could be controlled by differential expression of the adenosine receptor genes under different stimuli. Here, we have reviewed an array of studies that have characterized basal or induced expression of the adenosine receptors and common as well as distinct mechanisms of effect, in hopes that ongoing studies on this topic will further elucidate detailed mechanisms of adenosine receptor regulation, leading to potential therapeutic applications. J. Cell. Physiol. 218: 35–44, 2009.

TNF-alpha upregulates the A2B adenosine receptor gene: the role of NAD(P)H oxidase 4

Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-alpha) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A(2B) adenosine receptor (A(2B)AR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-alpha. Here, we show a regulatory loop by which TNF-alpha upregulates the A(2B)AR in VSMC in vitro and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A(2B)AR, and Nox inhibitors dampen the effect of TNF-alpha. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A(2B)AR.

Increased polyploidy in aortic vascular smooth muscle cells during aging is marked by cellular senescence.

We previously reported that the frequency of polyploid aortic vascular smooth muscle cells (VSMC) serves as a biomarker of aging. Cellular senescence of somatic cells is another marker of aging that is characterized by the inability to undergo cell division. Here, we examined whether polyploidy is associated with the development of cellular senescence in vivo. Analysis of aortic tissue preparations from young and old Brown Norway rats showed that expression of senescence markers such as p16INK4a and senescence‐associated β‐galactosidase activity are detected primarily in the old tissues. VSMC from p16INK4a knockout and control mice display similar levels of polyploid cells. Intriguingly, senescence markers are expressed in most, but not all, polyploid VSMC. Moreover, the polyploid cells exhibit limited proliferative capacity in comparison to their diploid counterparts. This study is the first to demonstrate in vivo that polyploid VSMC adopt a senescent phenotype.

B‐Myb regulates the A2B adenosine receptor in vascular smooth muscle cells

The A2B adenosine receptor (A2BAR) has been described to control various vascular functions, including inhibition of smooth muscle cell proliferation. Here, we sought to understand the regulation of A2BAR gene expression in aortic vascular smooth muscle cells (VSMCs), with a focus on the proliferation phase. Assays with A2BAR‐β‐gal aortic VSMCs, in which the endogenous A2BAR gene promoter drives the expression of prokaryotic β‐galactosidase (β‐gal) instead of the endogenous A2BAR gene, show that β‐gal expression is upregulated when the cells are induced to exit from cell cycle arrest. Similarly, the level of A2BAR mRNA is upregulated in proliferating primary aortic VSMCs. In search of related mechanisms, it was noted that the A2BAR gene promoter contains several putative binding sites for the proliferation‐inducing transcription factor, B‐Myb. Using a clone of the 5′ region upstream of the mouse A2BAR gene linked to a reporter gene, B‐Myb site deletion mutants were generated. It was determined that B‐Myb upregulates the A2BAR gene promoter, and specific promoter binding sites were identified as functional. In accordance, B‐Myb also elevates endogenous A2BAR mRNA and receptor activity, and this activity decreases cell proliferation. Our data are novel in that they show that this proliferation‐inhibiting A2BAR is itself an inducible receptor regulated by B‐Myb. J. Cell. Biochem. 103: 1962–1974, 2007.

Increased activity of TNAP compensates for reduced adenosine production and promotes ectopic calcification in the genetic disease ACDC

Patient-derived induced pluripotent stem cells reveal treatment strategies for a rare genetic form of arterial calcification. Understanding vascular calcification ACDC is a rare genetic vascular calcification disease caused by loss of CD73, a secreted enzyme that converts adenosine monophosphate (AMP) to adenosine. Cells from ACDC patients have a compensatory increase in the phosphatase TNAP, which primarily catalyzes the conversion of pyrophosphate to inorganic phosphate but can also convert AMP to adenosine. Jin et al. generated induced pluripotent stem cells (iPSCs) from ACDC patients. Although in culture, these cells generated adenosine from AMP, the cells had decreased amounts of pyrophosphate, which inhibits calcification. ACDC patient–derived cells showed increased activation of the mTOR pathway, which promotes calcification. When injected into mice, the ACDC patient–derived iPSCs formed calcified teratomas. Treating mice bearing these teratomas with an adenosine receptor agonist, the mTOR inhibitor rapamycin, or etidronate (a drug that is structurally similar to pyrophosphate) reduced calcification in the teratomas, suggesting multiple potential strategies for treating ectopic calcification in ACDC patients and thereby alleviating the pain and peripheral ischemia associated with the disease. ACDC (arterial calcification due to deficiency of CD73) is an autosomal recessive disease resulting from loss-of-function mutations in NT5E, which encodes CD73, a 5′-ectonucleotidase that converts extracellular adenosine monophosphate to adenosine. ACDC patients display progressive calcification of lower extremity arteries, causing limb ischemia. Tissue-nonspecific alkaline phosphatase (TNAP), which converts pyrophosphate (PPi) to inorganic phosphate (Pi), and extracellular purine metabolism play important roles in other inherited forms of vascular calcification. Compared to cells from healthy subjects, induced pluripotent stem cell–derived mesenchymal stromal cells (iMSCs) from ACDC patients displayed accelerated calcification and increased TNAP activity when cultured under conditions that promote osteogenesis. TNAP activity generated adenosine in iMSCs derived from ACDC patients but not in iMSCs from control subjects, which have CD73. In response to osteogenic stimulation, ACDC patient–derived iMSCs had decreased amounts of the TNAP substrate PPi, an inhibitor of extracellular matrix calcification, and exhibited increased activation of AKT, mechanistic target of rapamycin (mTOR), and the 70-kDa ribosomal protein S6 kinase (p70S6K), a pathway that promotes calcification. In vivo, teratomas derived from ACDC patient cells showed extensive calcification and increased TNAP activity. Treating mice bearing these teratomas with an A2b adenosine receptor agonist, the mTOR inhibitor rapamycin, or the bisphosphonate etidronate reduced calcification. These results show that an increase of TNAP activity in ACDC contributes to ectopic calcification by disrupting the extracellular balance of PPi and Pi and identify potential therapeutic targets for ACDC.

Bidirectional Translation in Cardiovascular Calcification

Over the past decade, we have witnessed an explosion of fundamental research aimed at understanding mechanisms contributing to cardiovascular calcification. As highlighted in recent reviews, numerous animal models and patient group studies have lent key insights into mechanisms and processes underlying pathological remodeling of soft tissues,1 including activation of signaling cascades related to bone morphogenetic proteins,2 Wnt/β-catenin,3,4 matrix γ-carboxyglutamate (Gla) protein (MGP),5,6 transforming growth factor (TGF)-β, phosphate signaling,7,8 and various downstream targets. Although there are compelling data supporting the biological importance of these pathways, harnessing these mechanisms for the development of therapeutics has not yet been realized. Many pathways play an integral role in bone homeostasis, making systemic targeting a nonviable therapeutic approach. In this Recent Highlights focused on cardiovascular calcification, we have drawn from the pool of recent publications in ATVB and other leading journals that focus on genetic and nongenetic upstream modulators of ectopic calcification pathways, and we posit that interventions aimed at reducing their impact may be more readily translated to clinical therapies for patients. A greater understanding of the key local and systemic cofactors, initiators, and outcomes will create a complementary approach to advancing both science and medicine. We further argue that identification of biomarkers that are prognostic not only for the presence of vascular calcification (VC) but also for the rate of progression of VC will be instrumental in the early identification and appropriate management of patients in the future. Unlike metastatic calcification—caused by elevated levels of calcium in the blood—cardiovascular calcification is most often attributed to injury or maladaptive cellular responses to stress. Although in vitro studies and genetically modified model organisms can serve as useful platforms to understand the biology of disease phenotypes, the reality is that >85% of drugs stemming from …