Marta Scatena

Osteopontin: A Multifunctional Molecule Regulating Chronic Inflammation and Vascular Disease

Osteopontin (OPN) is a multifunctional molecule highly expressed in chronic inflammatory and autoimmune diseases, and it is specifically localized in and around inflammatory cells. OPN is a secreted adhesive molecule, and it is thought to aid in the recruitment of monocytes-macrophages and to regulate cytokine production in macrophages, dendritic cells, and T-cells. OPN has been classified as T-helper 1 cytokine and thus believed to exacerbate inflammation in several chronic inflammatory diseases, including atherosclerosis. Besides proinflammatory functions, physiologically OPN is a potent inhibitor of mineralization, it prevents ectopic calcium deposits and is a potent inducible inhibitor of vascular calcification. Clinically, OPN plasma levels have been found associated with various inflammatory diseases, including cardiovascular burden. It is thus imperative to dissect the OPN proinflammatory and anticalcific functions. OPN recruitment functions of inflammatory cells are thought to be mediated through its adhesive domains, especially the arginine-glycine-aspartate (RGD) sequence that interacts with several integrin heterodimers. However, the integrin receptors and intracellular pathways mediating OPN effects on immune cells are not well established. Furthermore, several studies show that OPN is cleaved by at least 2 classes of proteases: thrombin and matrix-metalloproteases (MMPs). Most importantly, at least in vitro, fragments generated by cleavage not only maintain OPN adhesive functions but also expose new active domains that may impart new activities. The role for OPN proteolytic fragments in vivo is almost completely unexplored. We believe that further knowledge of the effects of OPN fragments on cell responses might help in designing therapeutics targeting inflammatory and cardiovascular diseases.

The role of osteopontin in inflammatory processes.

Osteopontin (OPN) is a matricellular protein that mediates diverse biological functions. OPN is involved in normal physiological processes and is implicated in the pathogenesis of a variety of disease states, including atherosclerosis, glomerulonephritis, cancer, and several chronic inflammatory diseases. Through interactions with several integrins, OPN mediates cell migration, adhesion, and survival in many cell types. OPN also functions as a Th1 cytokine, promotes cell-mediated immune responses, and plays a role in chronic inflammatory and autoimmune diseases. Besides its function in inflammation, OPN is also a regulator of biomineralization and a potent inhibitor of vascular calcification.

Apoptosis overrides survival signals through a caspase-mediated dominant-negative NF-|[kgr]|B loop

The transcription factor NF-κB 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-κB, whereas apoptotic cells show caspase-mediated cleavage of the NF-κB p65/RelA 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-κB, 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-κB inactivation.

VEGF induces differentiation of functional endothelium from human embryonic stem cells: implications for tissue engineering

Objective—Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs. Methods and Results—To enhance endothelial cell differentiation above a baseline of ≈2% in embryoid body (EB) spontaneous differentiation, 3 alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10 to 14. Continuous VEGF treatment resulted in a 4- to 5-fold enrichment of CD31+ cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31+ cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNF&agr;, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells. Conclusions—VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. This enrichment method increases endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants.

PEG-cross-linked heparin is an affinity hydrogel for sustained release of vascular endothelial growth factor.

An affinity-based controlled release system for growth factors having heparin-binding domains was prepared using a cross-linked heparin gel. The heparin gel was made by reacting hydrazide-functionalized heparin (Hep-ADH) with the N-hydroxysuccinimidyl ester of poly(ethylene glycol)-bis-butanoic acid (SBA-PEG-SBA). The degree of cross-linking could be controlled by defining the stoichiometry of hydrazide modification and the PEG cross-linker addition. The release of vascular endothelial growth factor (VEGF) was characterized as a heparin-binding growth factor. VEGF was directly injected into the heparin gel and the loaded VEGF displayed a slow, controlled release over 3 weeks with little initial burst phase. The biological activity of the released VEGF was measured with a proliferation assay utilizing human umbilical vein endothelial cells. The released VEGF maintained its biological activity at all time points investigated. The heparin gel with loaded VEGF was implanted sub-cutaneously in the dorsal region of mice. A significantly increased density of the endothelial cell marker platelet endothelial adhesion molecule (PECAM-1) was observed in histological specimens of the tissues surrounding the implanted gel.

Myosin Heavy-Chain Isoform Composition and Distribution in Developing and Adult Human Aortic Smooth Muscle

The myosin heavy-chain (MHC) composition of developing and adult human aortic smooth muscle (SM) was studied by SDS-polyacrylamide gel electrophoresis, Western blotting and indirect immunofluorescence using a panel of anti-MHC antibodies. On 5% SDS gels, three bands of 204, 200 and 196 kDa apparent molecular mass were identified in fetal, infant and adult stages of development. In the extracts from thoracic aorta (upper level), the 204, and 200-kDa bands (designated as SM-1 and SM-2, respectively) were recognized by SM-G4 and SMMS-1 antibodies, raised against a SM antigen, whereas the 196-kDa band was reactive with nonmuscle (NM)-F6 and NM-G2 antiplatelet MHC antibodies. Western blotting and immunofluorescence tests performed on bovine brain and other human NM tissues using NM-F6 and NM-G2 indicated that antigenic targets of the two antibodies resembled that of so-called IIB and IIA NM myosin found in the bovine system, respectively. In the aortic media, SM-1 was expressed throughout development, while SM-2 was upregulated during late fetal and postnatal development. Similarly, the 196-kDa band showed two distinct patterns of immunoreactivity with the anti-NM-MHC antibodies: with NM-G2, antigenicity was equal at all the developmental stages examined, whereas with NM-F6, it diminished during postnatal development. In the upper level, the cellular distribution of NM-G2 and NM-F6 immunoreactivities was similar in the early fetus but quite distinct at later stages of development. In infant and adult subjects, SM cells (SMC) reactive with NM-F6 accumulated predominantly within the intimal layer as well as in some areas of the underlying media as cell foci, whereas NM-G2 homogeneously stained the two layers. In the aorta near the diaphragm (lower level), both antibodies stained the thickened intima but not the underlying media. These data are consistent with the existence of developmental, stage-specific molecular and cellular transitions during vascular SMC maturation in human aortic media. In addition, these data suggest that IIB-like myosin may be expressed in SMC involved specifically in intimal thickening.

Rat aorta-derived mural precursor cells express the Tie2 receptor and respond directly to stimulation by angiopoietins

Recent studies have implicated the Tie2 tyrosine-kinase receptor and its main ligands - angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) - as crucial regulators of mural cell recruitment during angiogenesis. Angiopoietin-mediated activation of Tie2 promotes perivascular mural cell assembly, but the mechanisms regulating this process are poorly understood because differentiated mural cells do not have the Tie2 receptor, which is reportedly expressed only in endothelial cells. There is also no direct evidence that Tie2 activation results in production of mural cell chemoattractants by the endothelium. In the rat aorta model of angiogenesis, developing microvessels recruit mural cells from the intimal/subintimal layers of the aortic wall. Ang-1 and Ang-2 promote angiogenesis in this system, stimulating branching morphogenesis and mural cell assembly. Mural precursor cells (MPCs) isolated with a nonenzymatic method from the intimal aspect of the rat aorta were positive for smooth muscle cell markers (α-smooth muscle actin and calponin) and negative for endothelial markers (factor-VIII-related antigen and CD31). These cells responded chemotactically to Ang-1 and Ang-2, and secreted MMP-2 when treated with these factors. Western-blot analysis, immunocytochemistry and RT-PCR demonstrated that MPCs express the Tie2 receptor. Immunoprecipitation showed phosphorylation of MPC Tie2 on tyrosine residues upon stimulation with Ang-1 or Ang-2. Surface expression of Tie2 was further demonstrated by isolating Tie2+/α-smooth muscle actin+ MPCs from primary aortic outgrowths with anti-Tie2-IgG-coated magnetic beads. Immunostaining of the rat aorta confirmed expression of Tie2 not only in endothelial cells but also in nonendothelial mesenchymal cells located in the aortic intimal/subintimal layers, which are the source of MPCs. These data indicate that the aortic wall contains Tie2+ nonendothelial mesenchymal cells and suggest that Tie2-related recruitment of mural cells during angiogenesis may occur through angiopoietin-mediated direct stimulation of these cells.

The αvβ3 Integrin, NF-κB, Osteoprotegerin Endothelial Cell Survival Pathway

Abstract The growth and survival of many cell types requires integrin-mediated adhesion to the extracellular matrix (ECM). Physiologically, the prerequisite of cell–ECM adhesion interaction for cell cycle progression and cell survival is likely to be important in tissue morphology and regression as a mechanism to regulate tissue architecture and cell number. Pathologically, anchorage-dependent survival may limit tumor invasion and metastasis. Endothelial cells are anchorage-dependent cells, and many ECM molecules interacting with different classes of integrins promote their survival. It has became clear, however, that during the angiogenesis process the α v β 3 integrin plays a fundamental role in maintaining endothelial cell viability. The downstream signals regulating this process are becoming clarified, and new functions are described for molecules involved in apparently distant systems.The growth and survival of many cell types requires integrin-mediated adhesion to the extracellular matrix (ECM). Physiologically, the prerequisite of cell-ECM adhesion interaction for cell cycle progression and cell survival is likely to be important in tissue morphology and regression as a mechanism to regulate tissue architecture and cell number. Pathologically, anchorage-dependent survival may limit tumor invasion and metastasis. Endothelial cells are anchorage-dependent cells, and many ECM molecules interacting with different classes of integrins promote their survival. It has became clear, however, that during the angiogenesis process the alpha(v)beta(3) integrin plays a fundamental role in maintaining endothelial cell viability. The downstream signals regulating this process are becoming clarified, and new functions are described for molecules involved in apparently distant systems.

Osteoprotegerin and RANKL differentially regulate angiogenesis and endothelial cell function

Osteoprotegerin (OPG) a soluble tumor necrosis factor receptor family molecule protects endothelial cells from apoptosis in vitro and promotes neovascularization in vivo. In this study, we assessed the role of OPG and its ligands, receptor activator of nuclear factor-κB ligand (RANKL) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL), in microvessel formation using the rat aortic ring model of angiogenesis. OPG was found to promote a twofold increase in angiogenic sprouting in the aortic ring model, and this effect was inhibited by pre-incubation with a fivefold molar excess of either RANKL or TRAIL. While TRAIL had no effect upon angiogenesis on its own, RANKL was found to potently inhibit basal and vascular endothelial growth factor-induced angiogenesis. OPG increased the rate of endothelial cell proliferation in sprouting microvessels; in contrast, RANKL inhibited proliferation. RANKL was found to induce endothelial apoptosis at days 6, 7, and 10 in the aortic ring model and after incubation with human umbilical vein endothelial cells (HUVECs). Signaling studies showed that OPG induced ERK1/2 and Akt phosphorylation in HUVECs while RANKL had no effect. Our results indicate that OPG is a positive regulator of microvessel formation, while RANKL is an angiogenic inhibitor due to effects on regulation of endothelial cell proliferation, apoptosis, and signaling.

RANKL enhances macrophage paracrine pro-calcific activity in high phosphate-treated smooth muscle cells: dependence on IL-6 and TNF-α.

Background: Vascular calcification is highly correlated with cardiovascular disease (CVD) morbidity and mortality, and it is associated with inflammation. Receptor activator of NF-ĸB ligand (RANKL) inhibition in vivo has been shown to reduce vascular calcification in a mouse model of atherosclerosis. Therefore, we tested the hypothesis that RANKL regulates smooth muscle cell (SMC) calcification by modulating macrophage production of pro-calcific cytokines. Methods: We used a bone marrow-derived macrophage (BMDM)/SMC co-culture system and examined the effects of RANKL on BMDM activation and SMC matrix calcification. Results: Treatment with RANKL alone did not stimulate SMC calcification induced by elevated phosphate. BMDMs differentiated with macrophage colony-stimulating factor and placed in co-culture with SMCs increased phosphate-induced SMC calcification. RANKL added to the BMDM/SMC co-cultures further enhanced SMC calcification. Treatment of BMDMs with RANKL resulted in increased expression of IL-6 and TNF-α. Thus, increased expression of these pro-calcific cytokines in macrophages may mediate RANKL-induced SMC calcification in a paracrine fashion. Addition of neutralizing IL-6 and TNF-α antibodies together with RANKL treatment significantly reduced the RANKL induction of SMC calcification. Conclusion: RANKL activation of pro-inflammatory and pro-calcific pathways in macrophages may contribute to vascular calcification and inflammation.