Pamela Y. Johnson

Decorin Promotes Aortic Smooth Muscle Cell Calcification and Colocalizes to Calcified Regions in Human Atherosclerotic Lesions

Objective—Ectopic calcification localized to the intima of atherosclerotic plaque is a risk marker for cardiovascular events and increases the risk of aortic dissection during angioplasty. A variety of extracellular matrix molecules such as collagen type 1, bone sialoprotein, and osteopontin are known to regulate the biomineralization of bone and ectopic vascular calcification. In the present study, it was investigated whether decorin, a small leucine-rich proteoglycan expressed in bone and atherosclerotic plaque, is involved in arterial calcification. Methods and ResultsCalcification was induced in cultured bovine aortic smooth muscle cell (BASMC) by the addition of β-glycerophosphate or inorganic phosphate. Northern and Western analysis revealed that decorin expression was strongly upregulated in mineralizing BASMC. Furthermore, overexpression of decorin using a retroviral expression vector resulted in a 3- to 4-fold elevation of calcium deposited on the BASMC monolayer. Increased calcification in response to decorin could also be mimicked by adding exogenous decorin to the cultures. In addition, human coronary atherosclerotic lesions taken from sudden-death patients showed marked colocalization of calcium deposits with decorin. Conclusions—Decorin induces calcification of arterial smooth muscle cell cultures and colocalizes to mineral deposition in human atherosclerotic plaque, suggesting that decorin functions as promoter of intimal calcification.

Organization of hyaluronan and versican in the extracellular matrix of human fibroblasts treated with the viral mimetic poly I:C.

We have examined structural details of hyaluronan- and versican-rich peri-cellular matrices in human lung fibroblasts, as well as fixation effects after treatment with the viral mimetic, poly I:C. Lateral aggregation of hyaluronan chains was promoted by acid-ethanol-formalin fixation compared with a network appearance with formalin alone. However, hyaluronidase-sensitive cable structures were seen in live cells, suggesting that they are not a fixation artifact. With all fixatives, versican and hyaluronan probes bound alternately along strands extending from the plasma membrane. However, a yellow colocalization signal required aggregation/overlap of several hyaluronan/versican strands and was more pronounced after acid-ethanol-formalin fixation. In addition to the main cell surface, hyaluronan and versican were also associated with fine actin-positive membrane protrusions, retraction fibers, and surface blebs. After wounding plus treatment with poly I:C, cells displayed larger hyaluronan coats and cable-like structures, as well as more membrane protrusions. However, treated cells did not migrate and had increased stress fibers compared with control wounded cells. Deposition of hyaluronan into cable-like structures in response to poly I:C was diminished but still apparent following actin filament disruption with cytochalasin D, suggesting that the protrusions only partially facilitate cable formation. As seen by scanning electron microscopy, the membrane protrusions may participate in poly I:C-induced binding of monocytes to hyaluronan- and versican-rich matrices. These results suggest that poly I:C-induced hyaluronan- and versican-rich cable structures are not deposited during migration, and that cellular protrusions partially contribute to hyaluronan cable formation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Polyinosine-polycytidylic acid stimulates versican accumulation in the extracellular matrix promoting monocyte adhesion.

Viral infections are known to exacerbate asthma and other lung diseases in which chronic inflammatory processes are implicated, but the mechanism is not well understood. The viral mimetic, polyinosine-polycytidylic acid, causes accumulation of a versican- and hyaluronan-enriched extracellular matrix (ECM) by human lung fibroblasts with increased capacity for monocyte adhesion. The fivefold increase in versican retention in this ECM is due to altered compartmentalization, with decreased degradation of cell layer-associated versican, rather than an increase in total accumulation in the culture. This is consistent with decreased mRNA levels for all of the versican splice variants. Reduced versican degradation is further supported by low levels of the epitope, DPEAAE, a product of versican digestion by a disintegrin-like and metallopeptidase with thrombospondin type 1 motif enzymes, in the ECM. The distribution of hyaluronan is similarly altered with a 3.5-fold increase in the cell layer. Pulse-chase studies of radiolabeled hyaluronan show a 50% reduction in the rate of loss from the cell layer over 24 hours. Formation of monocyte-retaining, hyaluronidase-sensitive ECMs can be blocked by the presence of anti-versican antibodies. In comparison, human lung fibroblasts treated with the cytokines, IL-1beta plus TNF-alpha, synthesize increased amounts of hyaluronan, but do not retain it or versican in the ECM, which, in turn, does not retain monocytes. These results highlight an important role for versican in the hyaluronan-dependent binding of monocytes to the ECM of lung fibroblasts stimulated with polyinosine-polycytidylic acid.

Hyaluronan and Hyaluronan Binding Proteins Accumulate in both Human Type 1 Diabetic Islets and Lymphoid Tissues and Associate with Inflammatory Cells in Insulitis

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor–stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes.

A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease

The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4(-/-) mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.

Synthesis and Organization of Hyaluronan and Versican by Embryonic Stem Cells Undergoing Embryoid Body Differentiation

Embryonic stem cells (ESCs) provide a convenient model to probe the molecular and cellular dynamics of developmental cell morphogenesis. ESC differentiation in vitro via embryoid bodies (EBs) recapitulates many aspects of early stages of development, including the epithelial–mesenchymal transition (EMT) of pluripotent cells into more differentiated progeny. Hyaluronan and versican are important extracellular mediators of EMT processes, yet the temporal expression and spatial distribution of these extracellular matrix (ECM) molecules during EB differentiation remains undefined. Thus, the objective of this study was to evaluate the synthesis and organization of hyaluronan and versican by using murine ESCs during EB differentiation. Hyaluronan and versican (V0 and V1 isoforms), visualized by immunohistochemistry and evaluated biochemically, accumulated within EBs during the course of differentiation. Interestingly, increasing amounts of a 70-kDa proteolytic fragment of versican were also detected over time, along with ADAMTS-1 and −5 protein expression. ESCs expressed each of the hyaluronan synthases (HAS) −1, −2, and −3 and versican splice variants (V0, V1, V2, and V3) throughout EB differentiation, but HAS-2, V0, and V1 were expressed at significantly increased levels at each time point examined. Hyaluronan and versican exhibited overlapping expression patterns within EBs in regions of low cell density, and versican expression was excluded from clusters of epithelial (cytokeratin-positive) cells but was enriched within the vicinity of mesenchymal (N-cadherin-positive) cells. These results indicate that hyaluronan and versican synthesized by ESCs within EB microenviron-ments are associated with EMT processes and furthermore suggest that endogenously produced ECM molecules play a role in ESC differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Proteolytic Cleavage of Versican and Involvement of ADAMTS-1 in VEGF-A/VPF-Induced Pathological Angiogenesis

Malignant tumors and chronic inflammatory diseases induce angiogenesis by overexpressing vascular endothelial growth factor A (VEGF-A/VPF). VEGF-A-induced pathological angiogenesis can be mimicked in immunoincompetent mice with an adenoviral vector expressing VEGF-A164 (Ad-VEGF-A164). The initial step is generation of greatly enlarged “mother” vessels (MV) from preexisting normal venules by a process involving degradation of their rigid basement membranes. Immunohistochemical and Western blot analyses revealed that versican, an extracellular matrix component in the basement membranes of venules, is degraded early in the course of MV formation, resulting in the appearance of a versican N-terminal DPEAAE fragment associated with MV endothelial cells. The protease ADAMTS-1, known to cleave versican near its N terminus to generate DPEAAE, is also upregulated by VEGF-A in parallel with MV formation and localizes to the endothelium of the developing MV. The authors also show that MMP-15 (MT-2 MMP), a protease that activates ADAMTS-1, is upregulated by VEGF-A in endothelial cells in vitro and in vivo. These data suggest VEGF-A initiates MV formation, in part, by inducing the expression of endothelial cell proteases such as ADAMTS-1 and MMP-15 that act in concert to degrade venular basement membrane versican. Thus, versican is actively processed during the early course of VEGF-A-induced pathological angiogenesis.

Athsq1 Is an Atherosclerosis Modifier Locus With Dramatic Effects on Lesion Area and Prominent Accumulation of Versican

Objective—Susceptibility to atherosclerosis is genetically complex, and modifier genes that do not operate via traditional risk factors are largely unknown. A mouse genetics approach can simplify the genetic analysis and provide tools for mechanistic studies. Methods and Results—We previously identified atherosclerosis susceptibility QTL (Athsq1) on chromosome 4 acting independently of systemic risk factors. We now report confirmation of this locus in congenic strains carrying the MOLF-derived susceptibility allele in the C57BL/6J-Ldlr−/− genetic background. Homozygous congenic mice exhibited up to 4.5-fold greater lesion area compared to noncongenic littermates (P<0.0001). Analysis of extracellular matrix composition revealed prominent accumulation of versican, a presumed proatherogenic matrix component abundant in human lesions but almost absent in the widely-used C57BL/6 murine atherosclerosis model. The results of a bone marrow transplantation experiment suggested that both accelerated lesion development and versican accumulation are mediated, at least in part, by macrophages. Interestingly, comparative mapping revealed that the Athsq1 congenic interval contains the mouse region homologous to a widely-replicated CHD locus on human chromosome 9p21. Conclusion—These studies confirm the proatherogenic activity of a novel gene(s) in the MOLF-derived Athsq1 locus and provide in vivo evidence for a causative role of versican in lesion development.

Retrovirally Mediated Overexpression of Glycosaminoglycan-Deficient Biglycan in Arterial Smooth Muscle Cells Induces Tropoelastin Synthesis and Elastic Fiber Formation in Vitro and in Neointimae after Vascular Injury

Galactosamine-containing glycosaminoglycans (GAGs), such as the chondroitin sulfate chains of the proteoglycan versican, have been shown to inhibit elastogenesis. Another proteoglycan that may influence elastogenesis is biglycan, which possesses two GAG chains. To assess the importance of these chains on elastogenesis in blood vessels, rat aortic smooth muscle cells were transduced with a GAG-deficient biglycan cDNA-containing retroviral vector (LmBSN). Control cells were transduced with either biglycan or empty vector. Transduced cells were characterized in vitro and then seeded into balloon-injured rat carotid arteries to determine the effects on neointimal structure. Cultured cells overexpressing LmBSN showed marked up-regulation of tropoelastin and fibulin-5 mRNAs, increased amounts of desmosine and insoluble elastin, and increased deposition of elastic fibers as compared with empty vector- and biglycan-transduced cells. Conversely, collagen alpha(1) synthesis and the deposition of collagen fibers were both markedly decreased in LmBSN cultures. In vivo, neointimae formed from cells that overexpressed LmBSN and showed increased deposits of elastin that aggregated into parallel nascent fibers, generally arranged circumferentially. Neointimae that formed from cells with biglycan or empty vector contained fewer and less aggregated deposits of elastin. These findings suggest that the GAG chains of biglycan serve as inhibitors of elastin synthesis and assembly, and that biglycan can act as an important modulator of the composition of the extracellular matrix of blood vessels.

Overexpression of Decorin by Rat Arterial Smooth Muscle Cells Enhances Contraction of Type I Collagen In Vitro

Objective—Overexpression of decorin reduces neointimal thickening in balloon-injured carotid arteries of rats by decreasing the volume of neointimal extracellular matrix (ECM). We examined the hypothesis that decorin regulates ECM volume by stimulating cell-mediated contraction of collagen-rich ECMs. Methods and Results—Rat arterial smooth muscle cells (ASMCs) transduced with bovine decorin cDNA by retroviral transfection (LDSN) exhibited enhanced contraction of collagen gels in vitro when compared with vector-only transduced (LXSN) cells. Addition of recombinant decorin to LXSN or LDSN cells did not stimulate contraction of collagen gels. Enhanced contraction of collagen by LDSN cells was unaffected by the metalloproteinase inhibitor GM6001. LDSN cells exhibited increased expression of type I collagen mRNA when compared with that of LXSN cells. Correspondingly, collagen gel contraction by LDSN cells was reduced by inhibition of collagen synthesis by 3,4-l-dehydroproline (L-DHP). Antibodies to &agr;1&bgr;1-integrin, but not to &agr;2&bgr;1-integrin, blocked collagen contraction by both LXSN and LDSN cells. However, LXSN and LDSN cells expressed similar levels of &agr;1- and &bgr;1-integrin mRNAs. Conclusions—Decorin synthesized de novo by ASMCs increases type I collagen synthesis and enhances contraction of collagen gels. Regulated synthesis of decorin may be a useful therapeutic approach to reduce ECM volume in vascular disease.