Vascular Calcification: In Vitro Evidence for the Role of Inorganic Phosphate
Cecilia M. Giachelli
Bioengineering Department, University of Washington, Seattle, Washington
Correspondence to Dr. Cecilia M. Giachelli, Bioengineering Department, Box 351720, University of Washington, Seattle WA 98195-1720. Phone: 206-543-0205; Fax: 206-616-9763;
ABSTRACT. Uremic patients are prone to widespread ectopic extraskeletalcalcification resulting from an imbalance of systemic inorganicphosphate (Pi). There can be serious consequences of this process,particularly when it results in the calcification of the vasculature.A recent study examined the response of cultured human aorticsmooth muscle cells to varying levels of extracellular Pi. Cellsthat were exposed to Pi levels similar to those seen in uremicpatients (>1.4 mmol/L) showed dose-dependent increases incell culture calcium deposition. The results of this study alsodefined the role of elevated phosphate in transforming the vascularphenotype of these cells to an osteogenic phenotype, such thata predisposition for calcification was created. Pi-induced changesincluded increased expression of the osteogenic markers osteocalcinand core-binding factor-1 genes, the latter of which is considereda "master gene" critical for osteoblast differentiation. Thesechanges occur early after exposure to high phosphate levelsand seem to be mediated by a sodium-dependent phosphate co-transporter,Pit-1 (Glvr-1). Calcification of vascular cells also seems tooccur in the absence of a mineral imbalance but in the presenceof platelet-derived growth factor, a potent atherogenic factor.Taken together, these data suggest that calcification of vascularcells can occur early in a phosphate-rich environment similarto that seen in patients with renal failure and in a platelet-derivedgrowth factor–rich atherosclerotic region under normalphosphorus conditions. From a clinical viewpoint, it seems thatearly control or prevention of hyperphosphatemia may reducecoronary calcification and its associated morbidity and mortalityfor patients on dialysis. E-mail: ceci@u.washington.edu
Cardiovascular disease is prominent in ESRD (chronic kidneydisease stage 5). Nearly half of the deaths in dialysis patientsin 1999 were attributed to cardiovascular causes (1). It hasalso been documented that hyperphosphatemia is prevalent inpatients with chronic renal failure (2) and that hyperphosphatemiais linked to increased risk of cardiovascular mortality in thesepatients (3–5). The cellular and molecular correlatesof this linkage are now being elucidated and indicate that hyperphosphatemiacan lead to vascular calcification or deposition of calciumphosphate mineral, generally hydroxyapatite, in cardiovasculartissues such as arteries, cardiac muscle, and heart valves,including prosthetic valves.
Elevated serum phosphate levels in uremic patients have beenhighly correlated with vascular calcification (5). High levelsof calcium and phosphate can induce vascular calcification (6).Uremic patients are prone to ectopic calcification (3), definedas inappropriate mineralization in soft tissues (7). Ectopiccalcification can be metastatic or dystrophic. Uremic patientsare predisposed to metastatic calcification (3), defined asa systemic mineral imbalance associated with widespread ectopiccalcification (7). This predisposition occurs when the calcium-phosphorusproduct is elevated (3). Ectopic calcification presents a particularclinical problem when it occurs in the vasculature of uremicpatients, and it contributes to both the morbidity and mortalityassociated with ESRD (3,4).
Vascular calcification has been related to an increased riskof cardiovascular morbidities and complications such as atheroscleroticplaque burden (4,8,9), myocardial infarction (10,11), coronaryartery disease (12,13), postangioplasty dissection (14), andincreased ischemic episodes in peripheral vascular disease (15).It has also been found to be a powerful independent marker ofcoronary heart disease events in patients with diabetes (12).Recent studies also indicate that coronary calcification maybe predictive of or associated with sudden cardiac death (16,17).Indeed, both the Framingham risk index and coronary calcificationscore as measured by electron beam computed tomography havebeen shown to have prognostic value for cardiovascular events(17). Finally, strong associations among arterial calcification,stiffness, pulse pressure, and mortality in dialysis patientshave been noted and likely further contribute to the high ratesof cardiac and peripheral ischemic disease and left ventricularhypertrophy in this population (18–20).
In light of these risks, it is important to limit vascular calcificationin the dialysis population. Understanding the role of phosphateand improving our ability to manage hyperphosphatemia is anessential part of this effort. This article reviews data froma cellular and mechanistic viewpoint to explain the events thatregulate the entry of inorganic phosphate (Pi) into vascularcells, the subsequent genetic and biochemical response withinthese cells, and the physiologic or pathologic outcome of suchresponses. These data clearly define a role for phosphate inthe mineralization of aortic smooth muscle cell (SMC) and providebiochemical evidence for the role of hyperphosphatemia in transformingvascular cells into osteoblast-like cells, thus increasing therisk of calcification and cardiovascular disease.
Effect of Excess Pi in Vascular Cell Culture Media
Recent evidence dispels the classically held view that vascularcalcification is a passive, degenerative, end-stage processof vascular disease. Evidence now points to vascular calcificationas an actively regulated process akin to bone mineralization(7). Both pro- and anticalcifying mechanisms have been foundto play an active role in mineral deposition in vascular cells.Exploring the hypothesis that human aortic SMC (HSMC) in culturewould respond to elevated Pi levels by increasing pro-mineralizationfactors, we have examined the response of cultured HSMC to increasingconcentrations of Pi in the medium and found that cells thatare exposed to physiologic levels of Pi (1.4 mmol/L) grow normallyand do not undergo mineralization (21). In contrast, cells thatare grown in the presence of higher Pi concentrations (up to2 mmol/L) similar to those seen in individuals with hyperphosphatemia,show an increased deposition of calcium into vascular cells.This deposition occurs in a time- and dose-dependent manner(Figure 1). No spontaneous deposition of calcium occurred inthe calcification media or in media supplemented up to 10 mmol/LPi, indicating that vascular cells (or cell-derived matrix)were essential for mineralization. After 10 d of culture atPi >1.4 mmol/L, calcified cells developed granular depositsthat were identified as phosphate-containing material by positivevon Kossa staining. The granules were primarily associated withextracellular matrix, with the greatest accumulation occurringin areas of cell multilayering. Transmission electron microscopyand electron diffraction of specific sites verified an apatiticmineral phase, matrix vesicles, and calcified collagen fibers.The calcification seemed to be a general effect of vascularsmooth cells, because cells from various sources—primaryand immortalized human fetal and adult tissues and aortic andcoronary atherosclerotic plaque—all exhibit similar behavior(6,21). These data provided evidence to support the tenet thatcalcification of vascular SMC could occur with increasing frequencyin an environment of increasing phosphate concentration. Thisaccumulation of calcium can increase with time and can resultin calcium deposition primarily in the extracellular matrixof vascular cells regardless of age of tissue and vascular cellorigin.
Figure 1. Effect of increasing extracellular (medium) inorganic phosphate concentrations on calcium deposition in human aortic smooth muscle cells in culture. Reprinted with permission from Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H, Giachelli CM: Phosphate regulation of vascular smooth muscle cell calcification. Circ Res 87: E10–E17, 2000.
Mechanistic Evidence for Hyperphosphatemia-Induced Calcification
Results from several in vitro studies have indicated that Pistimulates SMC to undergo phenotypic changes that predisposethem to calcification (21–23). The transcription factorcore-binding factor-1 (Cbfa-1) has been found to regulate osteocalcin,osteopontin, and type I collagen gene expression. This factoris an absolute requirement for osteoblast differentiation (24).The expression of the osteogenic markers osteocalcin and Cbfa-1is strongly induced in the presence of elevated phosphate (21).In cell culture studies, induction of these osteogenic markersoccurred as early as 24 h after treatment with Pi at a concentrationof 2 mmol/L. Similarly, in a study of bovine aortic SMC, mineralizationof SMC in culture was associated with the dramatic loss of smoothmuscle–specific gene expression (smooth muscle lineagemarkers SM22 and smooth muscle actin) in the presence of anorganic phosphate donor, glycerophosphate (6). A third study(23) has shown that medial cells from calcified arteries ofmatrix gla-protein (a calcification inhibitor) null mice expresshigh levels of osteopontin and Cbfa-1, as well as decreasedlevels of smooth muscle actin, when compared with SMC fromnoncalcified wild-type blood vessels. It is interesting thatevidence for similar expression patterns in calcified humanarteries have recently been reported. Osteopontin levels wereincreased (25,26) and smooth muscle actin levels were decreased(25) in calcified medial layers of cutaneous blood vessels inpatients with calcific uremic arteriolopathy. Furthermore, stainingfor osteopontin and other bone matrix molecules was stronglycorrelated with medial calcification in epigastric arteriesof dialysis patients (27). These data support the concept thatSMC undergo phenotypic conversion to osteogenic cell type inthe presence of hyperphosphatemia in both animals and humans.Recently, in situ hybridization and immunostaining have shownthat Cbfa-1 and osteopontin are selectively expressed in themedia and intima of calcified but not uncalcified inferior epigastricarteries from uremic patients (28). In addition, pooled uremicsera and nonpooled control human sera were found to induce theexpression of Cbfa-1 in bovine vascular SMC in a time-dependent,non–phosphorus-mediated mechanism. As in the bone, Cbfa-1seems to be a key regulatory factor in vascular calcification,being upregulated by uremic toxins in dialysis patients.
It seems that the effects of hyperphosphatemia are mediatedby a sodium-dependent phosphate co-transporter (NPC) that facilitatesentry of Pi into vascular cells (21). Three types of NPC havebeen defined in humans. Types I and II are most common in thekidney and intestine. Type III is expressed throughout the body(29). PCR and Northern blot analysis have identified the NPCin HSMC as Pit-1 (Glvr-1), which is a type III NPC. No transcriptswere found for any other NPC in HSMC in culture (21).
For determining whether the phenotypic change seen in SMC culturecalcification was regulated by the NPC system, the NPC-specificinhibitor phosphonoformic acid (PFA) was added to the culturemedium. Gene expression was noted in the presence and in theabsence of PFA. PFA almost completely inhibited Pi uptake inHSMC. The phosphate-induced expression of osteogenes (as indicatedby osteocalcin and Cbfa-1 markers) was also inhibited by PFAin a dose-dependent manner. A second NPC inhibitor, arsenate,was also found to inhibit the expression of osteogenes, validatingthe hypothesis that Pi entry into cells and the subsequent activationof osteogenic genes is dependent on NPC-regulated cell entry(6,21).
Once the role of NPC in hyperphosphatemia-induced calcificationwas established, it was important to determine whether theseproteins would also affect calcification in the absence of amineral imbalance. Such calcification commonly occurs in atheroscleroticlesions. The atherogenic stimulus platelet-derived growth factor(PDGF) was studied in HSMC culture to address this question.It was found that PDGF increases the velocity of but not affinityfor phosphate uptake in HSMC culture. It also induces the expressionof Pit-1 mRNA and calcification of HSMC in a time- and dose-dependentmanner. This PDGF-mediated calcification seems to occur evenunder normal phosphate conditions (6). These data provide amechanistic basis for the increased calcification associatedwith atherosclerotic lesions, which typically contain high PDGFlevels even when phosphate levels are within normal limits (30).
Phosphate Regulation of SMC Mineralization: An Overview
On the basis of the data reviewed here, it is proposed thatextracellular Pi is moved into intracellular compartments viaNPC-mediated pathways (Figure 2). This intracellular movementis increased during hyperphosphatemia as seen in uremic patients(or during PDGF stimulation as seen in atherosclerotic lesions)and leads to the accumulation of intracellular phosphate. Bypathways that have not yet been fully elucidated, the increasedintracellular phosphate serves as a signal for osteogenic geneexpression (Cbfa-1 and downstream targets osteopontin and osteocalcin)and as a suppressor of HSMC-specific gene expression, resultingin increased secretion of mineral-nucleating molecules (matrixvesicles, calcium-binding proteins, alkaline phosphatase, andcollagen-rich extracellular matrix). These factors combine totransform the cell to be susceptible to vascular calcification(6).
Figure 2. Regulation of human smooth muscle cell mineralization by phosphate ion. Reprinted from Giachelli CM, Jono S, Shioi A, Nishizawa Y, Mori K, Morii H: Vascular calcification and inorganic phosphate. Am J Kidney Dis 38: S34–S37, 2001, with permission from the National Kidney Foundation.
The emerging picture of soft tissue calcification seems to beone that is actively regulated. The focus of this article hasbeen on the active inducers of calcification. It should be addedthat definitive data on the presence of local and systemic inhibitorsof calcification have been accumulating over the past severalyears and come largely from studies involving gene knockoutmice. These data implicate the involvement of several gene productsin ectopic calcification (Table 1) (31–37) and suggestthat the matrix gla-protein gene, osteoprotegrin, and osteopontinmay serve as natural inhibitors of cardiovascular calcificationthat may be either constitutively expressed or induced to preventectopic calcification. Indeed, vascular calcification may involvean active and dynamic balance of procalcifying and anticalcifyingmechanisms.
Uremic patients are prone to widespread ectopic and metastaticcalcification as a result of mineral imbalances, particularlythe imbalance of Pi. Serious consequences occur as a resultof this. The in vitro data reviewed here provide biochemicalevidence for the role of hyperphosphatemia in transforming cellsfrom a vascular phenotype to an osteogenic phenotype, creatinga predisposition for calcification. These changes occur earlyafter exposure to high Pi levels and continue to accumulateover time and with increasing Pi concentrations.
It seems that the phenotypic transformation of HSMC in responseto hyperphosphatemia is mediated by the NPC Pit-1, which predisposesSMC to undergo mineralization. Smooth muscle–specificgene expression may be downregulated, whereas osteoblast orchondrocyte-like gene expression may be upregulated throughupregulation of Cbfa-1 and its downstream genes, thus promotingmineralization. Pit-1 also seems to be able to affect calcificationin the absence of a systemic mineral imbalance. In light ofthis in vitro evidence, early control or prevention of hyperphosphatemiamay be key in reducing coronary calcification and the resultingmorbidity and mortality as a result of cardiovascular diseasefor patients on dialysis.
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Top
Abstract
Introduction
smooth muscle actin) in the presence of an organic phosphate donor, 
glycerophosphate (6). A third study (23) has shown that medial cells from calcified arteries of matrix gla-protein (a calcification inhibitor) null mice express high levels of osteopontin and Cbfa-1, as well as decreased levels of 
