Matrix metalloproteinase-9 overexpression enhances vascular smooth muscle cell migration and alters remodeling in the injured rat carotid artery

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of atherosclerosis as well as intimal hyperplasia after vascular injury. We used Fischer rat smooth muscle cells (SMCs) overexpressing MMP-9 to determine the role of MMP-9 in migration and proliferation as well as in vessel remodeling after balloon denudation. Fischer rat SMCs were stably transfected with a cDNA for rat MMP-9 under the control of a tetracycline-regulatable promoter. In this system, MMP-9 was overexpressed in the absence, but not in the presence, of tetracycline. In vitro SMC migration was determined using a collagen invasion assay as well as a Boyden chamber assay. In vivo migration was determined by measuring the invasion into the medial and intimal layers of transduced SMCs seeded on the outside of the artery. Transduced SMCs were also seeded on the luminal surface, and the effect of local MMP-9 overexpression on vascular structure was measured morphometrically at intervals up to 28 days. MMP-9 overexpression enhanced SMC migration in both the collagen invasion assay and Boyden chamber in vitro, increased SMC migration into an arterial matrix in vivo, and altered vessel remodeling by increasing the vessel circumference, thinning the vessel wall and decreasing intimal matrix content. These results demonstrate that MMP-9 enhances vascular SMC migration in vitro and in vivo and alters postinjury vascular remodeling.     

Allogenic immune response promotes the accumulation of host-derived smooth muscle cells in transplant arteriosclerosis

OBJECTIVE: Smooth muscle cells (SMCs) involved in intimal hyperplasia during transplant vasculopathy are derived both from the graft and the host. Here, the role of an allogenic immune response in the accumulation of host-derived SMCs in the neointima was explored. METHODS: Infrarenal aorta was transplanted from female F344 to male Lewis rats with or without immunosuppression by cyclosporine A (CsA). Accumulation of host-derived SMCs and inflammatory cells in the grafts, SMC proliferation, and apoptosis were analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR) for the SRY gene. Finally, SMCs were seeded in an allogenic or isogenic manner after balloon injury to carotid arteries and SMC survival was estimated. RESULTS: Proliferating graft SMCs and infiltrating leukocytes were observed in the intima early after transplantation. In parallel, inflammatory cells and immunoglobulins infiltrated the media and apoptosis of medial SMCs occurred, leading to destruction of this layer. CsA decreased the number of SRY+ SMCs in the lesions, restricted medial destruction, and improved survival of allogenic SMCs after seeding in injured arteries. CONCLUSIONS: Development of intimal thickenings during transplant vasculopathy involves an allogenic immune response, which promotes accumulation of host-derived SMCs and apoptosis of resident graft SMCs.     

Contribution of monocyte-derived macrophages and smooth muscle cells to arterial foam cell formation

Smooth muscle cells (SMCs) are the main cell type in intimal thickenings and some stages of human atherosclerosis. Like monocyte-derived macrophages, SMCs accumulate excess lipids and contribute to the total intimal foam cell population. In contrast, apolipoprotein (Apo)E-deficient and LDL receptor-deficient mice develop atherosclerotic lesions that are macrophage- as opposed to SMC-rich. The lesser contribution of SMCs to lesion development in these mouse models has distracted attention away from the importance of SMC cholesterol homeostasis in the artery wall. Intimal SMCs accumulate excess amounts of cholesteryl esters when compared with medial layer SMCs, possibly explained by reduced ATP-binding cassette transporter A1 expression and ApoA-I binding to intimal-type SMCs. The aim of this review is to compare the relative contribution of monocyte-derived macrophages and SMCs to human vs. mouse atherosclerosis, and describe what is known about lipid uptake and removal mechanisms contributing to arterial macrophage and SMC foam cell formation. An increased understanding of the contribution of these cell types to lesion development will help to delineate their relative importance in atherogenesis and as potential therapeutic targets.     

Cholesterol homeostasis and high-density lipoprotein formation in arterial smooth muscle cells

The balance between lipid accumulation and removal from cells in the artery wall is a key factor in atherogenesis. Smooth muscle cells (SMCs) are the main cell type in human intimal thickenings and some stages of atherosclerosis; however, cholesterol homeostasis in these cells has received little attention when compared with intimal macrophages. In addition, it has been demonstrated that SMCs may change phenotype to express macrophage markers upon cholesterol loading, indicating that a large portion of what are considered monocyte-derived human macrophages in human lesions may actually have originated as SMCs. We have recently reported low expression of the key regulator of cholesterol removal to apolipoprotein A-I to form high-density lipoprotein particles, the adenosine-triphosphate-binding cassette transporter A1, in cultured intima-phenotype epithelioid rat SMCs, as well as reduced expression of adenosine-triphosphate-binding cassette transporter A1 in intimal as compared with medial SMCs in human coronary atheromas. These combined observations suggest that SMCs and SMC-derived macrophage-like cells may contribute a much larger amount of the excess cholesterol accumulated in the atherosclerotic intima than previously known. The aim of this review is to present the current state of knowledge of cholesterol homeostasis in arterial SMC and to frame questions requiring further study to understand the relative importance to SMCs in overall atheroma lipid metabolism.     

Blockade of the 4-1BB pathway attenuates graft arterial disease in cardiac allografts

4-1BB, a member of the tumor necrosis factor (TNF) receptor superfamily, binds the 4-1BB ligand (4-1BBL) and works as a costimulatory molecule and regulates T cell-mediated immune responses. Because T cell-mediated immunity is associated with graft arterial disease (GAD), we investigated the role of the 4-1BB pathway in the progression of GAD. Hearts from C57BL/6 mice were transplanted into Bm12 mice (class II mismatch). 4-1BB expression was induced on CD4(+) and CD8(+) splenocytes in allografts after cardiac transplantation. 4-1BBL was detected in the vessel wall of the rejecting cardiac allograft and in cultured smooth muscle cells (SMCs) stimulated with fetal calf serum. Recipients were injected intraperitoneally with 4-1BBIg every 7 days for 8 weeks. GAD was significantly attenuated by 4-1BBIg treatment (luminal occlusion, 15.4 +/- 3.1% versus control IgG treatment, 75.6 +/- 4.6%, P < 0.001). T-cell infiltration of cardiac allografts and expression of interferon-g , interleukin-6, and interleukin-15 in cardiac allografts were suppressed by 4-1BBIg treatment. Coculture of SMCs with sensitized splenocytes after transplantation induced SMC proliferation, and this was inhibited by addition of 4-1BBIg. The 4-1BB pathway regulates not only T-cell activation but also SMC proliferation. Blockade of the 4-1BB pathway is a promising strategy to prevent progression of GAD.     

Differences in retinol metabolism and proliferative response between neointimal and medial smooth muscle cells

Vascular disease is multifactorial and smooth muscle cells (SMCs) play a key role. Retinoids have been shown to influence many disease-promoting processes including proliferation and differentiation in the vessel wall. Phenotypic heterogeneity of vascular SMCs is a well-known phenomenon and phenotypic modulation of SMCs precedes intimal hyperplasia. The SMCs that constitute the intimal hyperplasia demonstrate a distinct phenotype and differ in gene expression compared to medial SMCs. Cellular retinol-binding protein-1 (CRBP-I), involved in retinoid metabolism, is highly expressed in intimal SMCs, indicating altered retinoid metabolism in this subset of cells. The aim of this study was to evaluate the metabolism of all-trans ROH (atROH), the circulating prohormone to active retinoids, in vascular SMCs of different phenotypes. The results show an increased uptake of atROH in intimal SMCs compared to medial SMCs as well as increased expression of the retinoid-metabolizing enzymes retinol dehydrogenase-5 and retinal dehydrogenase-1 and, in conjunction with this gene expression, increased production of all-trans retinoic acid (atRA). Furthermore, the retinoic acid-catabolizing enzyme CYP26A1 is expressed at higher levels in medial SMCs compared to intimal SMCs. Thus, both retinoid activation and deactivation processes are in operation. To analyze if the difference in ROH metabolism was also correlated to differences in the biological response to retinol, the effects of ROH on proliferation of SMCs with this phenotypic heterogeneity were studied. We found that intimal SMCs showed a dose- and time-dependent growth inhibition when treated with atROH in contrast to medial SMCs, in which atROH had a mitogenic effect. This study shows, for the first time, that (1) vascular SMCs are able to synthesize biologically active atRA from the prohormone atROH, (2) intimal SMCs have a higher capacity to internalize atROH and metabolize atROH into atRA compared to medial SMCs and (3) atROH inhibits growth of intimal SMCs, but induces medial SMC growth.     

Reduced neointima in vein grafts following a blockage of cell recruitment from the vein and the surrounding tissue

OBJECTIVE: Accumulation of intimal smooth muscle cells (SMC) is an important event in vein graft-stenosis. Different SMC sources have been reported, but their interrelations have been poorly studied. In a mouse vein graft model we investigated whether recipient-derived intimal SMCs are recruited from the surrounding tissue and whether blockage of SMC recruitment from the surrounding tissue and/or the donor vein will reduce neointimal formation. METHODS: To detect recipient-derived cells, wild-type veins were implanted into ROSA26 transgenic mice. To block cell recruitment from the surrounding tissue, implanted veins were isolated with a tube-shaped plastic film. To exclude vein-derived cells in the neointimal formation, acellular veins were implanted. RESULTS: In vein grafts isolated from the surrounding tissue the recipient contribution became minimal, but the total number of SMCs was not decreased. Acellular grafts contained an equal number of intimal SMCs as cellular controls after 4 weeks. Isolation of acellular grafts from the surrounding tissue decreased the number of intimal SMCs by 90%. CONCLUSIONS: Recipient-derived SMCs are mainly recruited from the surrounding tissue. Cell recruitment from either the vein or the surrounding tissue is enough to form a neointima. Therefore, a simultaneous inhibition of both these sources is needed to reduce accumulation of intimal SMCs.     

Role of basic fibroblast growth factor in vascular lesion formation

In the present study we investigated whether basic fibroblast growth factor (bFGF) plays a role in the proliferative response of smooth muscle cells (SMCs) to denuding injury. Rat carotid smooth muscle was found to express the mRNA for bFGF, and bFGF protein was found to be present in rat aorta by immunoblot analysis. Systemically administered bFGF was a potent mitogen for vascular SMCs in arteries denuded with a balloon catheter, increasing replication from 11.5% in controls to 54.8%. Denudation with a device (filament loop), which causes only minimal damage to medial SMCs, showed a similar increase in replication (1.3% versus 43.3%) after bFGF infusion. In unmanipulated vessels, however, SMCs were unresponsive to infused bFGF. Infusion of a "mitotoxin" (bFGF conjugated to saporin) caused a greater than 50% decrease in the number of viable SMCs in the arterial wall after balloon injury. Prolonged administration of bFGF (12 micrograms/day for 2 weeks) after balloon injury caused an approximately twofold increase in intimal thickening. These results show that bFGF, which is synthesized by the arterial wall, could be a potent mitogen for SMCs in vivo and suggest that any release of endogenous bFGF may be capable of stimulating SMC proliferation, which may subsequently lead to intimal lesion formation.     

Proliferation of smooth muscle cells after vascular injury is inhibited by an antibody against basic fibroblast growth factor.

Proliferation of smooth muscle cells (SMCs) represents an important event in vascular lesion formation. Despite the common belief that growth factors contribute to the development of the atherosclerotic plaque, until now there has been no direct evidence for a role of mitogens in the development of arterial lesions. Balloon catheter injury of the rat carotid artery is accompanied by death of medial SMCs and is typically followed by proliferation of SMCs with subsequent formation of an intimal lesion. Our hypothesis is that injury causes mitogens to be released from dead cells, which then stimulate cell proliferation. One such mitogen that may be important in this process is basic fibroblast growth factor (bFGF), which can be detected immunocytochemically in SMCs and endothelial cells of adult rat carotid arteries. Systemic injection of a neutralizing antibody against bFGF prior to balloon catheterization significantly decreased the induced SMC proliferation by approximately 80%. The intimal lesion that developed within 8 days after injury, however, was not significantly reduced. The results of this study support the concept that endogenous bFGF is the major mitogen controlling the growth of vascular smooth muscle cells following injury. These data may have implications for the observed failure of endarterectomy and angioplasty procedures.     

Arterial smooth muscle cell heterogeneity: implications for atherosclerosis and restenosis development

During atheromatous plaque formation or restenosis after angioplasty, smooth muscle cells (SMCs) migrate from the media toward the intima, where they proliferate and undergo phenotypic changes. The mechanisms that regulate these phenomena and, in particular, the phenotypic modulation of intimal SMCs have been the subject of numerous studies and much debate during recent years. One view is that any SMCs present in the media could undergo phenotypic modulation. Alternatively, the seminal observation of Benditt and Benditt that human atheromatous plaques have the features of a monoclonal or an oligoclonal lesion has led to the hypothesis that a predisposed, medial SMC subpopulation could play a crucial role in the production of intimal thickening. The presence of a distinct SMC population in the arterial wall implies that under normal conditions, SMCs are phenotypically heterogeneous. The concept of SMC heterogeneity is gaining wider acceptance, as shown by the increasing number of publications on this subject. In this review, we discuss the in vitro studies that demonstrate the presence of distinct SMC subpopulations in arteries of various species, including humans. Their specific features and their regulation will be highlighted. Finally, the relevance of an atheroma-prone phenotype to intimal thickening formation will be discussed.     

Increased intimal hyperplasia and smooth muscle cell proliferation in transgenic mice with heparan sulfate-deficient perlecan

Smooth muscle cell (SMC) proliferation is a critical process in vascular disease. Heparan sulfate (HS) proteoglycans inhibit SMC growth, but the role of endogenous counterparts in the vessel wall in control of SMC function is not known in detail. Perlecan is the major HS proteoglycans in SMC basement membranes and in vessel wall extracellular matrix (ECM). In this study, transgenic mice with HS-deficient perlecan were analyzed with respect to vascular phenotype and intimal lesion formation. Furthermore, SMC cultures were established and characterized with respect to morphology, immunocytochemical features, proteoglycan synthesis, proliferative capacity, and ECM binding of basic fibroblast growth factor (FGF-2). In vitro, mutant SMCs formed basement membranes with perlecan core protein, but with decreased levels of HS, they showed diminished secretion of HS-containing perlecan into the medium and a defective ECM-binding capacity of FGF-2. In vitro, mutant SMCs showed increased proliferation compared with wild-type cells, and in vivo, enhanced SMC proliferation and intimal hyperplasia were observed after flow cessation of the carotid artery in mutant mice. The results indicate that the endogenous HS side-chains of perlecan contribute to SMC growth control both in vitro and during intimal hyperplasia, possibly by sequestering heparin-binding mitogens such as FGF-2.     

Vascular remodeling in transplant vasculopathy

As therapeutic strategies to prevent acute rejection progressively improve, transplant vasculopathy (TV) constitutes the single most important limitation for long-term functioning of solid organ allografts. In TV, allograft arteries characteristically develop severe, diffuse intimal hyperplastic lesions that eventually compromise luminal flow and cause ischemic graft failure. Traditional immunosuppressive strategies that check acute allograft rejection do not prevent TV; indeed 50% of transplant recipients will have significant disease within five years of organ transplantation, and 90% will have significant TV a decade after their surgery. TV can involve the entire length of the transplanted arterial bed, including penetrating intraorgan arterioles. Indeed, the luminal narrowing of such penetrating vessels may be the most functionally significant because arterioles represent the major contributors to tissue vascular resistance. Because of the diffuseness of TV involvement in the allograft vascular bed, the only currently definitive therapy requires re-transplantation. Nevertheless, as we better understand the pathogenesis and critical mediators of these lesions, pharmacological advances can be anticipated. Other articles in this thematic review series focus on the specifics of the inciting injury, the cytokines and chemokines that drive TV development, and the nature of the recruited cells in TV lesions, as well as the pathogenic similarities between TV and other vascular lesions such as atherosclerosis. This review focuses on the mechanisms of vascular wall remodeling in TV, including the intimal accumulation of smooth muscle-like cells and associated extracellular matrix, medial smooth muscle cell degeneration, and adventitial fibrosis. A brief overview highlights the aneurysmal changes that can accrue when vessel wall inflammation has a cytokine profile distinct from the typical proinflammatory interferon-gamma-dominated milieu.     

Antisense Bcl-x oligonucleotide induces apoptosis and prevents arterial neointimal formation in murine cardiac allografts

OBJECTIVE: Cardiac allograft arteriosclerosis, which limits long-term survival of recipients, cannot be prevented by conservative therapies. The arteriopathy is characterized by diffuse intimal thickening comprised of proliferative smooth muscle cells (SMCs). Cell death is a prominent feature of atherosclerosis; Bcl-x is one of the anti-apoptotic mediators. METHODS: To test the hypothesis that antisense bcl-x oligodeoxynucleotide (ODN) is effective in preventing intimal hyperplasia through enhancing apoptosis after cardiac transplantation, we performed single intraluminal delivery of antisense bcl-x ODN into murine cardiac allografts (n = 9). DBA/2 (H-2d) hearts were transplanted into B10.D2 (H-2d) mice. Sense bcl-x ODN (n = 8) and no treatment (n = 8) studies were also performed. RESULTS: Allografts were harvested at 4 weeks after transplantation; all allografts kept beating throughout the period. Coronary intimal thickening had developed in nontreated and sense ODN transfected allografts at 4 weeks after transplantation with enhanced expression of Bcl-x and cell adhesion molecules, and suppressed apoptosis. However, antisense bcl-x ODN prevented neointimal formation through enhanced apoptosis. CONCLUSION: These results indicate that apoptosis of vascular SMCs induced by Bcl-x is associated with initial hyperplasia after heart transplantation. Antisense bcl-x ODN inhibits SMC proliferation by inducing apoptosis in graft coronary arteries.     

Common protective and diverse smooth muscle cell effects of AAV-mediated angiopoietin-1 and -2 expression in rat cardiac allograft vasculopathy

Angiopoietin-1 (Ang1) and Ang2 regulate the maintenance of normal vasculature by direct endothelial and indirect smooth muscle cell (SMC) effects. Dysfunction of vascular wall cells is considered central in cardiac allograft vasculopathy (CAV), where inflammation and arterial injury initiate subsequent intimal SMC proliferation. In this study, we investigated the effect of exogenous Ang1 and Ang2 in chronically rejecting rat cardiac allografts by intracoronary adeno-associated virus (AAV)-mediated gene transfer. Bioluminescent imaging of AAV-transfected syngeneic grafts revealed gradual and stable transgene expression in graft cardiomyocytes. In cardiac allografts, both AAV-Ang1 and AAV-Ang2 decreased inflammation and increased antiapoptotic Bcl-2 mRNA and Bcl-2/Bax ratio at 8 weeks. Only AAV-Ang2 decreased the development of CAV, whereas AAV-Ang1 activated arterial SMC and increased PDGF-A mRNA in the allograft. Collectively, our results show that exogenous Ang1 and Ang2 have similar antiinflammatory and antiapoptotic effects in cardiac allografts. Prolonged AAV-mediated Ang1 transgene expression also induced SMC activation, whereas AAV-Ang2 lacked the SMC activating effects and decreased CAV. Our results thus highlight the common protective and diverse SMC effects of Ang1 and Ang2 in cardiac allograft microenvironment and the importance of timing of angiopoietins to achieve therapeutic effects.     

Cellular pathology of atherosclerosis: smooth muscle cells prime cocultured endothelial cells for enhanced leukocyte adhesion

During the development of an atherosclerotic plaque, mononuclear leukocytes infiltrate the artery wall through vascular endothelial cells (ECs). At the same time, arterial smooth muscle cells (SMCs) change from the physiological contractile phenotype to the secretory phenotype and migrate into the plaque. We investigated whether secretory SMCs released cytokines that stimulated ECs in a manner leading to increased leukocyte recruitment and thus might accelerate atheroma formation. SMCs and ECs were established in coculture on the opposite sides of a porous membrane, and the cocultured cells were incorporated into a flow-based assay for studying leukocyte adhesion. We found that coculture primed ECs so that their response to the inflammatory cytokine tumor necrosis factor-alpha was amplified. ECs cocultured with SMCs supported greatly increased adhesion of flowing leukocytes and were sensitized to respond to tumor necrosis factor-alpha at concentrations 10 000 times lower than ECs cultured alone. In addition, coculture altered the endothelial selectin adhesion molecules used for leukocyte capture. EC priming was attributable to the cytokine transforming growth factor-beta(1), which was proteolytically activated to a biologically active form by the serine protease plasmin. These results suggest a new role for secretory SMCs in the development of atheromatous plaque. We propose that paracrine interaction between ECs and SMCs has the potential to amplify leukocyte recruitment to sites of atheroma and exacerbate the inflammatory processes believed to be at the heart of disease progression.     

Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury.

The migration and proliferation of smooth muscle cells (SMCs) within the intima of arteries following mechanical injury is thought to be initiated by vessel wall injury and release of growth factors, in particular the platelet-derived growth factor (PDGF). However, the mechanism by which SMC proliferation is regulated after platelet interaction with the vessel wall has ceased is unknown. Here we show that SMCs derived from the intima of injured rat arteries (intimal SMCs) are phenotypically distinct from SMCs from unmanipulated vessels (medial SMCs). Intimal SMCs secrete 5-fold greater amounts of PDGF-like activity into conditioned medium in culture, have fewer receptors for 125I-labeled PDGF, and are not mitogenically stimulated by exogenous purified PDGF. This study demonstrates that two SMC phenotypes can develop in the adult rat artery and suggests that SMC proliferation in vivo may be controlled, in part, by SMCs that produce PDGF-like molecules.     

Direct and indirect effects of pulsatile shear stress on the smooth muscle cell.

BACKGROUND: Anastomotic intimal hyperplasia is still an unsolved problem after small caliber prosthetic bypass grafting. Oscillatory turbulent flow occurs at the end to side anastomosis, and produces various effects on smooth muscle cells (SMCs) and endothelial cells (ECs), which compose intimal hyperplasia. We examined the influences of pulsatile oscillating shear stress on smooth muscle cells mitogenic activity induced by sheared endothelial cells. METHODS:1) Smooth muscle cells were cultured under three different pulsatile shear conditions (mean: 0, 6, and 60 dyne/cm2). 2) Endothelial cells were cultured under both static and sheared condition (mean: 60 dyne/cm2). Using the conditioned media from each well, SMCs were cultured under static and sheared conditions (60 dyne/cm2). Four groups of SMCs were devised by combining the two types of media and the two culture conditions. SMC colony spreading distances were measured as an index of combined migration and proliferation activity. An MTT assay and a cell counting assay were used to determine the proliferation activities of SMCs. RESULTS: 1) SMC spreading activity was suppressed by shear stress. SMC proliferative activity was stimulated by pulsatile turbulent shear stress. 2) SMC spreading activity was stimulated by mitogens derived from ECs under shear stress. However, this augmented SMC spreading activity was attenuated under sheared conditions. The mitogens derived from ECs under pulsatile shear stress had no effects on SMC proliferation activity. CONCLUSIONS: Pulsatile oscillating shear stress attenuates SMC migration activity induced by EC-denve mitogens and stimulates SMC proliferative activity.     

Changes of coronary artery morphology and distribution of smooth muscle cells during progression of coronary atherosclerosis after heart transplantation

To examine the changes in coronary artery morphology and the distribution of smooth muscle cells during the progression of coronary atherosclerosis after cardiac transplantation, intimal and medial tissues were evaluated and the density of smooth muscle cells in the media were measured 30 and 60 days after transplantation by microspectrophotometry from rats receiving both auto- and allo-transplantation. Transplanted animals were given cyclosporine A to prevent graft rejection. Signs of rejection were not seen in the animals receiving auto-transplants. Rejection gradually progressed after transplantation in animals receiving allografts. The intima of the coronary arteries in the allograft group was significantly thickened at both 30 and 60 days after transplantation. The total intimal area in the day 60 group was significantly increased relative to the day 30 group among animals receiving allo-transplantation. The medial area of the coronary arteries in the group receiving allotransplantation was significantly less than that of the auto-transplantation group at both 30 and 60 days after transplantation. Azocarmine G stain revealed that the principal component of the thickened intima was smooth muscle cells. Coronary arteries in the allotransplantation group had disruption of the internal elastic lamina. We therefore hypothesized that the smooth muscle cells (SMCs) in the intima are probably derived from the media. The density of SMCs in the media was measured by microspectrophotometry. The density of SMCs was significantly decreased in the allo-transplantation group relative to the autotransplantation group. We conclude that intimal thickening of the coronary arteries is due to SMC proliferation, the myocytes being from the media through disruption of the internal elastic lamina. This process is similar to the mechanism of the development of atherosclerosis.