Engineering macrophages to control the inflammatory response and angiogenesis.

Macrophage (MΦ) dysregulation is increasingly becoming recognized as a risk factor for a number of inflammatory complications including atherosclerosis, cancer, and the host response elicited by biomedical devices. It is still unclear what roles the pro-inflammatory (M1) MΦ and pro-healing (M2) MΦ phenotypes play during the healing process. However, it has been shown that a local overabundance of M1 MΦs can potentially lead to a chronically inflamed state of the tissue; while a local over-exuberant M2 MΦ response can lead to tissue fibrosis and even promote tumorigenesis. These notions strengthen the argument that the tight temporal regulation of this phenotype balance is necessary to promote inflammatory resolution that leads to tissue homeostasis. In this study, we have engineered pro-inflammatory MΦs, MΦ-cTLR4 cells, which can be activated to a M1-like MΦ phenotype with a small molecule, the chemical inducer of dimerization (CID) drug. The MΦ-cTLR4 cells when activated with the CID drug, express increased levels of TNFα, IL-6, and iNOS. Activated MΦ-cTLR4 cells stay stimulated for at least 48h; once the CID drug is withdrawn, the MΦ-cTLR4 cells return to baseline state within 18h. Further, in vitro CID-activated MΦ-cTLR4 cells induce upregulation of VCAM-1 and ICAM-1 on endothelial cells (EC) in a TNFα-dependent manner. With the ability to specifically modulate the MФ-cTLR4 cells with the presence or absence of a small molecule, we now have the tool necessary to observe a primarily M1 MФ response during inflammation. By isolating this phase of the wound healing response, it may be possible to determine conditions for ideal healing.

Apoptosis overrides survival signals through a caspase-mediated dominant-negative NF-kappa B loop.

The transcription factor NF-kappa B is an important regulator of gene expression during immune and inflammatory responses, and can also protect against apoptosis. Here we show that endothelial cells undergo apoptosis when deprived of growth factors. Surviving viable cells exhibit increased activity of NF-kappa B, whereas apoptotic cells show caspase-mediated cleavage of the NF-kappa B p65/ReIA subunit. This cleavage leads to loss of carboxy-terminal transactivation domains and a transcriptionally inactive p65 molecule. The truncated p65 acts as a dominant-negative inhibitor of NF-kappa B, promoting apoptosis, whereas an uncleavable, caspase-resistant p65 protects the cells from apoptosis. The generation of a dominant-negative fragment of p65 during apoptosis may be an efficient pro-apoptotic feedback mechanism between caspase activation and NF-kappa B inactivation.

NF-kappaB mediates alphavbeta3 integrin-induced endothelial cell survival.

The alphavbeta3 integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-kappaB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and beta3 integrin ligation rapidly increased NF-kappaB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a beta1-integrin ligand) did not induce NF-kappaB activity. The alphavbeta3 integrin was most important for osteopontin-mediated NF-kappaB induction and survival, since adding a neutralizing anti-beta3 integrin antibody blocked NF-kappaB activity and induced endothelial cell death when cells were plated on osteopontin. NF-kappaB was required for osteopontin- and vitronectin-induced survival since inhibition of NF-kappaB activity with nonphosphorylatable IkappaB completely blocked the protective effect of osteopontin and vitronectin. In contrast, NF-kappaB was not required for fibronectin, laminin, and collagen type I-induced survival. Activation of NF-kappaB by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NF-kappaB reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NF-kappaB activation. These studies identify NF-kappaB as an important signaling molecule in alphavbeta3 integrin-mediated endothelial cell survival.

Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques.

In an earlier report, we used differential cloning to identify genes that might be critical in controlling arterial neointima formation (Giachelli, C., N. Bae, D. Lombardi, M. Majesky, and S. Schwartz. 1991. Biochem. Biophys. Res. Commun. 177:867-873). In this study, we sequenced the complete cDNA and conclusively identified one of these genes, 2B7, as rat osteopontin. Using immunochemistry and in situ hybridization, we found that medial smooth muscle cells (SMC) in uninjured arteries contained very low levels of osteopontin protein and mRNA. Injury to either the adult rat aorta or carotid artery using a balloon catheter initiated a qualitatively similar time-dependent increase in both osteopontin protein and mRNA in arterial SMC. Expression was transient and highly localized to neointimal SMC during the proliferative and migratory phases of arterial injury, suggesting a possible role for osteopontin in these processes. In vitro, basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and angiotensin II (AII), all proteins implicated in the rat arterial injury response, elevated osteopontin expression in confluent vascular SMC. Finally, we found that osteopontin was a novel component of the human atherosclerotic plaque found most strikingly associated with calcified deposits. These data implicate osteopontin as a potentially important mediator of arterial neointima formation as well as dystrophic calcification that often accompanies this process.

The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro.

Osteopontin is an arginine-glycine-aspartate containing acidic glycoprotein postulated to mediate adhesion, migration, and biomineralization in diverse tissues. The mechanisms explaining this multifunctionality are not well understood, although it is known that one osteopontin receptor is the alpha v beta 3 integrin. In this work, we studied human smooth muscle cells varying in alpha v beta 3 levels to identify additional osteopontin receptors. We report that, in addition to alpha v beta 3, both alpha v beta 5 and alpha v beta 1 are osteopontin receptors. Moreover, the presence or absence of alpha v beta 3 on the cell surface altered the adhesive and migratory responses of smooth muscle cells to osteopontin. Adhesion of alpha v beta 3-deficient cell populations to osteopontin was only half that of cells containing alpha v beta 3, and migration toward an osteopontin gradient in the Boyden chamber was dependent on cell surface alpha v beta 3. Although alpha v beta 3-deficient smooth muscle cells were unable to migrate to osteopontin, they did migrate significantly in response to vitronectin and fibronectin. These findings represent the first description of alpha v beta 5 and alpha v beta 1 as osteopontin receptors and suggest that, while adhesion to osteopontin is supported by integrins containing beta 1, beta 3, and beta 5, migration in response to osteopontin appears to depend on alpha v beta 3. Thus, interaction with distinct receptors is one mechanism by which osteopontin may initiate multiple functions.

Osteopontin is produced by rat cardiac fibroblasts and mediates A(II)-induced DNA synthesis and collagen gel contraction.

Angiotensin II (AII) is a critical factor in cardiac remodeling which involves hypertrophy, fibroblast proliferation, and extracellular matrix production. However, little is known about the mechanism by which AII accelerates these responses. Osteopontin is an acidic phosphoprotein with RGD (arginine-glycine-aspartate) sequences that are involved in the vascular smooth muscle cell remodeling process. We identified the presence of osteopontin mRNA and protein in cultured rat cardiac fibroblasts and its prominent regulation by AII (10(-11) M). Osteopontin message levels were increased fourfold (P < 0.01) and protein fivefold (P < 0.05) at 24 h after addition of AII (10(-7) M). This response was inhibited by the AT1 receptor blocker, losartan. Osteopontin mRNA levels were increased in hypertrophied ventricles from animals with renovascular hypertension (1.6-fold, P < 0.05) and aortic banding (2.9-fold, P < 0.05). To examine the function of osteopontin, we determined its effects on (a) the ability of cardiac fibroblasts to contract three-dimensional collagen gels and (b) cardiac fibroblast growth. A monoclonal antibody against osteopontin partially blocked AII-induced three-dimensional collagen gel contraction by cardiac fibroblasts (64+/-4 vs. 86+/-5% in the presence of antibody, P < 0.05), while osteopontin itself promoted contraction of the gels by fibroblasts (71+/-5%, P < 0.05 compared with control). Either a monoclonal antibody against beta3 integrin which is a ligand for osteopontin or the RGD peptide blocked both AII and osteopontin-induced collagen gel contraction. Thus, the osteopontin RGD sequence binds to beta3 integrins on the fibroblast to promote fibroblast binding to collagen. All induced a threefold increase in DNA synthesis of cardiac fibroblasts, which was completely blocked by antibodies against osteopontin and beta3 integrin, or by RGD peptide, but not by controls. Thus, All-induced growth of cardiac fibroblasts also requires osteopontin engagement of the beta3 integrin. Taken together, these results provide the first evidence that osteopontin is a potentially important mediator of AII regulation of cardiac fibroblast behavior in the cardiac remodeling process.

The role of osteoprotegerin and tumor necrosis factor-related apoptosis-inducing ligand in human microvascular endothelial cell survival.

Endothelial cell survival and antiapoptotic pathways, including those stimulated by extracellular matrix, are critical regulators of vasculogenesis, angiogenesis, endothelial repair, and shear-stress-induced endothelial activation. One of these pathways is mediated by alpha(v)beta(3) integrin ligation, downstream activation of nuclear factor-kappaB, and subsequent up-regulation of osteoprotegerin (OPG). In this study, the mechanism by which OPG protects endothelial cells from death was examined. Serum-starved human microvascular endothelial cells (HMECs) plated on the alpha(v)beta(3) ligand osteopontin were protected from cell death. Immunoprecipitation experiments indicated that OPG formed a complex with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HMECs under these conditions. Furthermore, inhibitors of TRAIL, including recombinant soluble TRAIL receptors and a neutralizing antibody against TRAIL, blocked apoptosis of serum-starved HMECs plated on the nonintegrin attachment factor poly-d-lysine. Whereas TRAIL was unable to induce apoptosis in HMECs plated on osteopontin, the addition of recombinant TRAIL did increase the percentage of apoptotic HMECs plated on poly-d-lysine. This evidence indicates that OPG blocks endothelial cell apoptosis through binding TRAIL and preventing its interaction with death-inducing TRAIL-receptors

Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers.

Bovine aortic smooth muscle cell (BASMC) cultures undergo mineralization on addition of the organic phosphate donor, beta-glycerophosphate (betaGP). Mineralization is characterized by apatite deposition on collagen fibrils and the presence of matrix vesicles, as has been described in calcified vascular lesions in vivo as well as in bone and teeth. In the present study, we used this model to investigate the molecular mechanisms driving vascular calcification. We found that BASMCs lost their lineage markers, SM22alpha and smooth muscle alpha-actin, within 10 days of being placed under calcifying conditions. Conversely, the cells gained an osteogenic phenotype as indicated by an increase in expression and DNA-binding activity of the transcription factor, core binding factor alpha1 (Cbfa1). Moreover, genes containing the Cbfa1 binding site, OSE2, including osteopontin, osteocalcin, and alkaline phosphatase were elevated. The relevance of these in vitro findings to vascular calcification in vivo was further studied in matrix GLA protein null (MGP(-/-)) mice whose arteries spontaneously calcify. We found that arterial calcification was associated with a similar loss in smooth muscle markers and a gain of osteopontin and Cbfa1 expression. These data demonstrate a novel association of vascular calcification with smooth muscle cell phenotypic transition, in which several osteogenic proteins including osteopontin, osteocalcin, and the bone determining factor Cbfa1 are gained. The findings suggest a positive role for SMCs in promoting vascular calcification.

Phosphate regulation of vascular smooth muscle cell calcification.

Vascular calcification is a common finding in atherosclerosis and a serious problem in diabetic and uremic patients. Because of the correlation of hyperphosphatemia and vascular calcification, the ability of extracellular inorganic phosphate levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro was examined. HSMCs cultured in media containing normal physiological levels of inorganic phosphate (1.4 mmol/L) did not mineralize. In contrast, HSMCs cultured in media containing phosphate levels comparable to those seen in hyperphosphatemic individuals (>1.4 mmol/L) showed dose-dependent increases in mineral deposition. Mechanistic studies revealed that elevated phosphate treatment of HSMCs also enhanced the expression of the osteoblastic differentiation markers osteocalcin and Cbfa-1. The effects of elevated phosphate on HSMCs were mediated by a sodium-dependent phosphate cotransporter (NPC), as indicated by the ability of the specific NPC inhibitor phosphonoformic acid, to dose dependently inhibit phosphate-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. With the use of polymerase chain reaction and Northern blot analyses, the NPC in HSMCs was identified as Pit-1 (Glvr-1), a member of the novel type III NPCs. These data suggest that elevated phosphate may directly stimulate HSMCs to undergo phenotypic changes that predispose to calcification and offer a novel explanation of the phenomenon of vascular calcification under hyperphosphatemic conditions. The full text of this article is available at http://www.circresaha.org.

Upregulation of osteopontin expression in renal cortex of streptozotocin-induced diabetic rats is mediated by bradykinin.

The model of streptozotocin (STZ)-induced diabetes in Wistar rats was used to study the expression of osteopontin during development of diabetic nephropathy. Diabetes was confirmed by serum glucose levels exceeding 16 mmol/l during the experimental period of 12 weeks. During this period of time, diabetic nephropathy developed, as characterized by a reduced glomerular filtration rate (2.7 +/- 0.3 ml/min in controls vs. 1.7 +/- 0.1 ml/min in diabetic rats) and proteinuria (8.3 +/- 1.7 mg/24 h in controls vs. 22.0 +/- 4 mg/24 h in diabetic rats). Northern blot analysis revealed a time-dependent upregulation of renal cortical osteopontin expression reaching 138 +/- 6% of control levels after 2 weeks and 290 +/- 30% (mean +/- SE, n = 6-9) after 12 weeks. By immunostaining, the increased osteopontin expression could be located to the tubular epithelium of the renal cortex. Chronic treatment of animals with ramipril (3 mg/kg) during the 12-week experimental period led to a further increase in osteopontin mRNA expression in diabetic animals, amounting to 570 +/- 73% (mean +/- SE, n = 6) of controls. Increased levels of osteopontin were not associated with accumulation of monocyte/macrophages that were identified by the cell type specific monoclonal antibody ED-1. The increased osteopontin expression in ramipril-pretreated rats was abolished by application of the bradykinin B2-receptor antagonist, icatibant (0.5 mg/kg). In addition, increased osteopontin expression in diabetic rats, which did not receive any treatment after STZ injection, could as well be reduced by icatibant given for the final 2 weeks of the experimental period. These data suggest that a strong bradykinin B2-receptor-mediated upregulation of osteopontin occurs during the pathogenesis of experimental diabetic nephropathy in rats.

Molecular and cellular biology of osteopontin Potential role in cardiovascular disease.

Molecular biology has greatly enhanced our ability to identify molecules expressed as a part of the vascular response to injury. Among these, osteopontin is of particular interest. Osteopontin is a multifunctional adhesive glycoprotein implicated in the pathogenesis of a growing number of cardiovascular lesions, including restenosis, atherosclerosis, aortic valve calcification, and repair of myocardial wounds. In this review, we focus on structural, functional, and regulatory properties of osteopontin, which might be particularly relevant to cardiovascular lesion development.

Angiotensin II induction of osteopontin expression and DNA replication in rat arteries.

We recently identified the adhesive protein osteopontin as a novel smooth muscle cell product overexpressed in rat developing neointima and human atheroma. Although osteopontin is a candidate stimulant for intimal lesion progression because of its chemotactic and calcium binding functions, factors controlling osteopontin expression in arteries remain poorly defined. In vitro, smooth muscle cell expression of osteopontin is associated with cell cycle transit or alterations in cell phenotype, and it is increased by angiotensin II (Ang II) stimulation. In the present studies, we investigated both osteopontin expression and DNA replication in the arterial wall in response to chronic Ang II infusion in vivo. Rat carotid arteries with or without intimal thickening (induced by balloon catheterization) were examined. Ang II (250 ng/kg per minute) or vehicle was coinfused with bromodeoxyuridine (to label replicating DNA in vivo) for 2 weeks beginning 4 weeks after injury. With Ang II, smooth muscle cells overexpressed osteopontin as shown by protein immunohistochemistry, in situ hybridization, and Northern blot analyses. Osteopontin mRNA levels were increased markedly (approximately fivefold) in the normal artery media and injured artery neointima, but levels remained low in the injured artery media, in positive correlation (R2 = 0.88, P < .001) with DNA replication in the smooth muscle layers, further suggesting that osteopontin may be a growth-associated, phenotype-dependent gene for smooth muscle cells. However, osteopontin expression in neointima was not restricted to areas showing DNA replication, suggesting a nonobligatory association. Ang II induced severe hypertension. Arterial osteopontin expression was increased also by chronic catecholamine infusion, a model of vascular growth stimulation showing labile pressure elevations. Osteopontin induction in smooth muscle cells may contribute to Ang II-dependent intimal lesion progression and vascular remodeling events associated with renovascular diseases or hyperadrenergic disorders.

Osteopontin: a versatile regulator of inflammation and biomineralization.

Osteopontin is a secreted glycoprotein with a multidomain structure and functions characteristic of a matricellular protein. Osteopontin interacts with cell surface receptors via arginine-glycine-aspartate (RGD)- and non-RGD containing adhesive domains, in addition to binding to components of the structural extracellular matrix. While normally expressed in bone and kidney, osteopontin levels are elevated during wound healing and inflammation in most tissues studied to date. Since 1986, over one thousand studies have been published on osteopontin, including recent experiments in osteopontin-deficient mice. These studies reveal osteopontin as a cell adhesive, signaling, migratory, and survival stimulus for various mesenchymal, epithelial, and inflammatory cells, in addition to being a potent regulator of osseous and ectopic calcification. Based on these reports, a general picture of osteopontin as an important regulator of inflammation and biomineralization is emerging. A common denominator in osteopontin function in these situations is its ability to regulate the function of macrophage and macrophage-derived cells (i.e. osteoclasts). While we have learned much about osteopontin and the processes it appears to regulate over the past decade, many questions regarding this important multifunctional protein remain unanswered and provide important directions for future studies.

In vivo stimulation of vascular plasminogen activator inhibitor-1 production by very low-density lipoprotein involves transcription factor binding to a VLDL-responsive element.

High plasma levels of plasminogen activator inhibitor-1 (PAI-1) are associated with an increased risk of cardiovascular disease. There is also a close relation between high plasma levels of PAI-1 and hypertriglyceridemia. Cell culture studies have shown that very low density lipoprotein (VLDL) increases the production and secretion of PAI-1 in endothelial cells and hepatocytes, suggesting a possible mechanism for this association. To determine whether VLDL stimulates PAI-1 production in vascular cells also in vivo, Sprague-Dawley rats were injected intravenously with 6 mg/kg of VLDL (derived from human subjects with type IV hyperlipidemia). Previous studies have demonstrated that this results in an accumulation of human VLDL in the aorta and other arteries followed by increased nuclear factor-kappa B (NF-kappaB) activation. Endothelial, but not smooth muscle cells, showed a basal PAI-1 mRNA and protein expression as assessed by in situ hybridization and immunohistochemistry, respectively. Six to twenty-four hours after the VLDL injection, lipoprotein particle accumulation was seen in the aortic wall, which was accompanied by increasing PAI-1 mRNA and protein expression in endothelial and smooth muscle cells. Within the rat PAI-1 promoter we identified a sequence located at -589 to -571 with 74% homology with the recently described VLDL responsive element in the human PAI-1 promoter and located adjacent to a 4-guanosine motif presumably corresponding to the human 4G/5G polymorphism. Transient transfection studies showed that VLDL exerts its stimulatory effects on rat PAI-1 gene expression in vascular cells by interaction with promoter sequences located within bp -656 and -505. Electrophoretic mobility shift assays showed that VLDL increases the binding of as yet incompletely characterized factors to this response element. Taken together these observations support a direct influence of VLDL on vascular PAI-1 gene expression ill vivo. This stimulation is exerted on the level of PAI-1 gene transcription, and involves transcription factor binding to a VLDL responsive element adjacent to a 4G motif within the PAI-1 promoter.

Vascular calcification and inorganic phosphate.

Vascular calcification is highly correlated with elevated serum phosphate levels in uremic patients. To shed light on this process, we examined the ability of extracellular inorganic phosphate (Pi) levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro. When cultured in media containing normal physiological levels of Pi (1.4 mmol/L Pi), HSMC grew in monolayers and did not mineralize. In contrast, HSMC cultured in media containing Pi levels comparable to those seen in hyperphosphatemic individuals (>1.4 mmol/L), showed dose-dependent increases in cell culture calcium deposition. Mechanistic studies showed that elevated Pi treatment of HSMC also enhanced the expression of the osteogenic markers, osteocalcin and Cbfa-1. The effects of elevated Pi on HSMC were mediated by a sodium-dependent phosphate cotransporter (NPC), as indicated by the ability of the specific NPC inhibitor, phosphonoformic acid (PFA), to dose-dependently inhibit Pi-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. Using polymerase chain reaction and Northern blot analyses, the NPC in HSMC was identified as Pit-1 (Glvr-1), a member of the type III NPCs. Interestingly, platelet-derived growth factor-BB (PDGF-BB), a potent atherogenic stimulus, increased the maximum velocity (Vmax) but not the affinity (Km) of phosphate uptake, enhanced the expression of Pit-1 mRNA, and induced HSMC culture calcification in a time- and dose-dependent manner. Importantly, in the presence of PDGF, HSMC culture calcification occurred under normophosphatemic conditions. These data suggest that elevated Pi may directly stimulate HSMC to undergo phenotypic changes that predispose to calcification and may help explain both the phenomena of human metastatic calcification under hyperphosphatemic conditions as well as increased calcification in PDGF-rich atherosclerotic lesions.

Osteopontin expression in cardiovascular diseases.

Adhesive interactions are recognized requirements for cellular proliferation, migration and differentiation during normal morphogenesis as well as disease. By differential cloning, osteopontin was identified as an adhesive protein upregulated during vascular remodeling and neointima formation in both rat models and human vascular diseases including atherosclerosis and restenosis. In functional studies, purified osteopontin promoted adhesion, focal contact formation, and migration of vascular smooth muscle and endothelial cells. Utilizing neutralizing antibodies, three integrin-type receptors, alpha v beta 3, alpha v beta 1, and alpha v beta 5 were found to support cellular adhesion to osteopontin. In contrast, only cells containing the alpha v beta 3 integrin could migrate towards an osteopontin gradient, demonstrating for the first time that different functions of osteopontin are mediated via distinct receptors. These results suggest a model whereby osteopontin, via its integrin-type receptors, contributes to vascular remodeling during development and disease by facilitating smooth muscle migration and simultaneously promoting endothelial coverage of the affected area.

Quantitative interrogation of micropatterned biomolecules by surface force microscopy.

Synthetic biomaterials are widely used in medical implants with success in improving and extending quality of life. However, these materials were not originally designed to interact with cells through specific signaling pathways. As a result, the interaction with the body is mediated through passive adsorption of a disorganized protein monolayer. Next generation biomaterials have been proposed to be active in modifying the biological response of the host through the incorporation of specific biorecognition moieties. An important tool in the development of these novel active biomaterials is the scanning force microscope (SFM). The SFM allows for interrogation of bioactive biomaterials in mapping or spectroscopic modes. In this work, micropatterned protein surfaces were prepared using biomolecules implicated in wound healing. The surfaces were imaged via SFM and the specific binding forces between surface associated biomolecules and antibody functionalized tips were quantified.

Inducers and inhibitors of biomineralization: lessons from pathological calcification.

OBJECTIVES: Ectopic calcification is a common response to soft tissue injury and systemic mineral imbalance and can lead to devastating clinical consequences when present in joints, heart valves and blood vessels. We have hypothesized that mineralization of matrices in any tissue is normally controlled by a balance between procalcific and anticalcific regulatory proteins such that abnormal deposition of apatite is avoided. Alterations in this balance induced by injury, disease or genetic deficiency are postulated to induce ectopic mineral deposition. Over the past several years, we have developed in vitro and in vivo models of ectopic calcification to investigate potential inducers and inhibitors of this process. RESULTS: Osteopontin, a secreted phosphoprotein, has emerged as a major inhibitor of ectopic mineralization. Osteopontin is a potent inhibitor of vascular cell calcification in vitro and mice lacking osteopontin are highly susceptible to ectopic calcification. Furthermore, osteopontin treatment of biomaterials protected against ectopic mineralization. Our studies indicate that in addition to inhibiting apatite crystal initiation and growth, osteopontin stimulates resorption of ectopic calcification via peripheral macrophages and giant cells. In contrast, inorganic phosphate has emerged as a major inducer of mineralization in these systems. Elevated inorganic phosphate (Pi) was shown to induce smooth muscle cell matrix calcification with morphological properties similar to those observed in calcified human valves and atherosclerotic plaques. Furthermore, mineralization induced by inorganic phosphate was dependent on the activity of the sodium-dependent phosphate cotransporter, Pit-1. CONCLUSIONS: These studies implicate controlled, transcellular transport of Pi as a major requirement for matrix calcification.

Ectopic calcification: new concepts in cellular regulation.

Ectopic calcification is a common response to soft tissue injury and systemic mineral imbalance, and can lead to devastating clinical consequences when present in heart valves or blood vessels. It is hypothesized that mesenchymal and inflammatory cells normally maintain the balance between procalcific and anti-calcific regulatory proteins in soft tissues such that ectopic deposition of apatite is avoided. Alterations in this balance induced by injury or disease is postulated to induce ectopic apatite deposition. We have developed in vitro and in vivo models of ectopic calcification and found that: 1) elevated extracellular phosphate levels induce smooth muscle culture mineralization morphologically similar to that observed in calcified human valves and atherosclerotic plaques, 2) sodium-dependent phosphate cotransporter function is required for smooth muscle cell culture mineralization, 3) smooth muscle cell culture mineralization is associated with a dramatic loss of smooth muscle-specific gene expression and gain of osteoblast-like properties, including expression of osteoblast differentiation factor, Cbfa-1, and 4) osteopontin, a secreted phosphoprotein abundant in macrophages found in human calcified atherosclerotic and valvular lesions, is a potent inhibitor of ectopic calcification in vitro and in vivo. These studies suggest that both constitutively expressed (surveillance) and inducible (damage control) mineralization inhibitory molecules are produced by mesenchymal and inflammatory cells to control ectopic calcification.

Renal expression of genes that promote interstitial inflammation and fibrosis in rats with protein-overload proteinuria.

Rats with significant proteinuria induced by daily injections of bovine serum albumin develop interstitial inflammation and fibrosis. The present study was designed to investigate the molecular basis of interstitial monocyte (Mø) recruitment and early interstitial fibrosis. Groups of rats were sacrificed after one, two and three weeks. Despite an increase in interstitial Mø at week 1, whole kidney mRNA levels were not elevated for monocyte chemoattractant protein-1 (MCP-1), osteopontin or vascular cell adhesion molecule-1 (VCAM-1). Only osteopontin mRNA levels were significantly elevated in the renal cortex at four days. At two and three weeks, MCP-1 and osteopontin mRNA levels were increased and the proteins showed distinct tubular patterns of distribution. By immunostaining increased expression of VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) was restricted to their presence or the surface of the interstitial inflammatory cells. TGF-beta 1 mRNA levels were increased at weeks 1, 2 and 3 (2.1, 2.9, 3.6x); interstitial and occasional cortical tubular cells expressed TGF-beta 1 mRNA and protein. There was a progressive rise in the number of cortical interstitial fields with increased staining for collagen (col) 1 (18, 29, 44%), col III (39, 61, 63%), col IV (7, 13, 29%), laminin (4, 10, 30%), fibronectin (14, 28, 37%), tenascin (19, 22, 14%) and in total renal col measured biochemically (1.1, 1.4, 2.0x) at weeks 1, 2 and 3, respectively. Renal matrix protein mRNA levels were variable and not always predictive of fibrosis. Only col I and tenascin levels were increased at week 1; all matrix protein mRNA levels except col IV were increased at week 2; but only tenascin, laminin and col IV mRNA levels remained elevated at three weeks. Plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metallo-proteinases (TIMP)-1 mRNA levels were significantly increased at two weeks. During the three weeks there was no change in urokinase, stromelysin or TIMP-3 mRNA levels. These results suggest that both increased matrix protein synthesis and altered matrix remodeling/degradation contribute to the final interstitial fibrogenic process in rats with protein-overload proteinuria. Mø, one of the sources of TGF-beta 1, infiltrate the interstitium by complex recruitment mechanisms which may depend in part on osteopontin, ICAM-1 and VCAM-1 expression.