Thomas N. Wight

Human tissue-engineered blood vessels for adult arterial revascularization.

There is a crucial need for alternatives to native vein or artery for vascular surgery. The clinical efficacy of synthetic, allogeneic or xenogeneic vessels has been limited by thrombosis, rejection, chronic inflammation and poor mechanical properties. Using adult human fibroblasts extracted from skin biopsies harvested from individuals with advanced cardiovascular disease, we constructed tissue-engineered blood vessels (TEBVs) that serve as arterial bypass grafts in long-term animal models. These TEBVs have mechanical properties similar to human blood vessels, without relying upon synthetic or exogenous scaffolding. The TEBVs are antithrombogenic and mechanically stable for 8 months in vivo. Histological analysis showed complete tissue integration and formation of vasa vasorum. The endothelium was confluent and positive for von Willebrand factor. A smooth muscle–specific α-actin–positive cell population developed within the TEBV, suggesting regeneration of a vascular media. Electron microscopy showed an endothelial basement membrane, elastogenesis and a complex collagen network. These results indicate that a completely biological and clinically relevant TEBV can be assembled exclusively from an individuals own cells.

Versican: a versatile extracellular matrix proteoglycan in cell biology

Versican is a large extracellular matrix proteoglycan that is present in a variety of tissues. Successful cloning of the gene in man, mouse, cow and chicken has revealed the existence of at least four splice variants of versican, which differ in the size of the core protein and the number of glycosaminoglycan chains. The highly interactive nature of versican provides a basis for its importance as a structural molecule, creating loose and hydrated matrices during key events in development and disease; and by interacting either directly with cells or indirectly with molecules that associate with cells to, in part, regulate cell adhesion and survival, cell proliferation, cell migration and extracellular matrix assembly. Several studies within the past two years have confirmed a significant role for versican in regulating cell phenotype.

Formation of Hyaluronan- and Versican-Rich Pericellular Matrix Is Required for Proliferation and Migration of Vascular Smooth Muscle Cells

The accumulation of hyaluronan (HA) and the HA-binding proteoglycan versican around smooth muscle cells in lesions of atherosclerosis suggests that together these molecules play an important role in the events of atherogenesis. In this study we have examined the formation of HA- and versican-rich pericellular matrices by human aortic smooth muscle cells in vitro, using a particle-exclusion assay, and the role of the pericellular matrix in cell proliferation and migration. The structural dependence of the pericellular matrix on HA can be demonstrated by the complete removal of the matrix with Streptomyces hyaluronidase. The presence of versican in the pericellular matrix was confirmed immunocytochemically. By electron microscopy, the cell coat was seen as a tangled network of hyaluronidase-sensitive filaments decorated with ruthenium red-positive proteoglycan granules. Ninety percent of migrating cells in wounded cultures, and virtually all mitotic cells, displayed abundant HA- and versican-rich coats. Time-lapse video imaging revealed that HA- and versican-rich pericellular matrix formation is dynamic and rapid, and coordinated specifically with cell detachment and mitotic cell rounding. HA oligosaccharides, which inhibit the binding of HA to the cell surface and prevent pericellular matrix formation, significantly reduced proliferation and migration in response to platelet-derived growth factor, whereas larger HA fragments and high molecular weight HA had no effect. Treatment with HA oligosaccharides also led to changes in cell shape from a typical fusiform morphology to a more spread and flattened appearance. These data suggest that organization of HA- and versican-rich pericellular matrices may facilitate migration and mitosis by diminishing cell surface adhesivity and affecting cell shape through steric exclusion and the viscous properties of HA proteoglycan gels.

Cell biology of arterial proteoglycans.

Although proteoglycans constitute a minor component of vascular tissue, these molecules have been shown to influence a number of arterial properties such as viscoelasticity, permeability, lipid metabolism, hemostasis, and thrombosis. A hallmark of early and late atherosclerosis is the accumulation of proteoglycans in the intimal lesions. Yet, it is not clear why this accumulation occurs. This article reviews the classes of proteoglycans synthesized by the two major cell types of the arterial wall--the endothelial and smooth muscle cell. Detailed consideration is then given to the modulation of proteoglycan metabolism and the role that proteoglycans play in a number of cellular events such as adhesion, migration, and proliferation--important processes in both the development and the pathogenesis of blood vessels. Last, the involvement of proteoglycans in two critical vascular wall processes--hemostasis and lipid metabolism--is reviewed, because these events pertain to atherogenesis. This review emphasizes the importance of proteoglycans in regulating several key events in normal and pathophysiological processes in the vascular tissue.

Early Human Atherosclerosis Accumulation of Lipid and Proteoglycans in Intimal Thickenings Followed by Macrophage Infiltration

Objective—The present study was designed to clarify the morphological features of early human atherosclerosis and to determine whether specific extracellular matrix proteoglycans play a role in early atherogenesis. Methods and Results—Step and serial sections were obtained from right coronary arteries with no or early atherosclerosis. Atherosclerosis was classified into 4 grades according to the amount of lipid deposition. Coronary arteries with Grade 0 showed diffuse intimal thickening (DIT) with no lipid deposits. The extracellular matrix proteoglycans, biglycan and decorin, were localized in the outer layer of DIT. Most cases of Grade 1 and Grade 2 exhibited fatty streaks with extracellular lipids colocalizing with biglycan and decorin in the outer layer of the intima. As lipid grades increased, macrophages increased in number and were present in the deeper layers. Most cases of Grade 3 exhibited pathologic intimal thickening (PIT) with extracellular lipids underneath a layer of foam cell macrophages. Conclusions—In early human coronary atherosclerosis, fatty streaks develop via extracellular deposition of lipids associated with specific types of proteoglycans in the outer layer of preexisting DIT. As the amount of the lipid increases in fatty streaks, macrophages infiltrate toward the deposited lipid to form PIT with foam cells.

Proteoglycans in Atherosclerosis and Restenosis: Key Roles for Versican

The proteoglycan versican is one of several extracellular matrix (ECM) molecules that accumulate in lesions of atherosclerosis and restenosis. Its unique structural features create a highly interactive molecule that binds growth factors, enzymes, lipoproteins, and a variety of other ECM components to influence fundamental events involved in vascular disease. Versican is one of the principal genes that is upregulated after vascular injury and is a prominent component in stented and nonstented restenotic lesions. The synthesis of versican is highly regulated by specific growth factors and cytokines and the principal source of versican is the smooth muscle cell. Versican interacts with hyaluronan, a long chain glycosaminoglycan, to create expanded viscoelastic pericellular matrices that are required for arterial smooth muscle cell (ASMC) proliferation and migration. Versican is also prominent in advanced lesions of atherosclerosis, at the borders of lipid-filled necrotic cores as well as at the plaque-thrombus interface, suggesting roles in lipid accumulation, inflammation, and thrombosis. Versican influences the assembly of ECM and controls elastic fiber fibrillogenesis, which is of fundamental importance in ECM remodeling during vascular disease. Collectively, these studies highlight the critical importance of this specific ECM component in atherosclerosis and restenosis.

The role of proteoglycans in cell adhesion, migration and proliferation.

Proteoglycans comprise a part of the extracellular matrix that participates in the molecular events that regulate cell adhesion, migration and proliferation. Their structural diversity and tissue distribution suggest a functional versatility not generally encountered for other extracellular matrix components. This versatility is mainly dictated by their molecular interactions and their ability to regulate the activity of key molecules involved in several biological events. This molecular cooperativity either promotes or inhibits cell adhesion, migration and proliferation. A growing number of studies indicate that proteoglycans can play a direct role in these cellular events by functioning either as receptors or as ligands for molecules that are required for these events to occur. Such studies support a role for proteoglycans as important effectors of cellular processes that constitute the basis of development and disease.

Intimal fibromuscular hyperplasia at the venous anastomosis of PTFE grafts in hemodialysis patients. Clinical, immunocytochemical, light and electron microscopic assessment.

Failure of arteriovenous communications used for chronic hemodialysis was studied during sequential 5-year periods after placement of either endogenous Brescia-Cimino (B-C) fistulas (50 patients) or polytetrafluoroethylene (PTFE, Gore-Tex) grafts (66 patients). Venous stenosis near the anastomosis was the reason for failure in 45% of PTFE grafts compared with 16% of B-C fistulas (p less than 0.001). Failure occurred, on average, 16 months after PTFE graft placement compared with 22 for B-C fistulas (p = NS). Proximal vein segments removed from five failed and two functioning PTFE graft communications were studied using light and electron microscopy and immunocytochemical techniques. All venous segments removed during surgical shunt repair exhibited a marked intimal hyperplasia. The intimal cellular component was almost exclusively smooth muscle. Accumulation of intracellular lipid droplets was not seen. Foam cells as well as extracellular lipid deposits were absent; macrophages and lymphocytes were absent from the zone of proliferation. Ultrastructural examination revealed a large proportion of extracellular matrix surrounding smooth muscle cells in the neointima. Collagen and elastin were present in the extracellular matrix, in greatest concentration deeper in the intima. Closer to the lumen, most of the extracellular volume consisted of proteoglycan. Hemosiderin was absent from the lesions as were consistent signs of luminal and intimal fibrin. Uniform intimal gradients of actin, collagen, and proteoglycan suggest that this is a steadily progressive, rather than episodic, proliferative response. These clinical and histologic observations and an analysis of hemodynamic stresses support the postulate that upstream release of platelet-derived growth factor, and possibly, shear-induced intimal injury stimulate this response. This myointimal proliferative process provides a readily accessible model of fibromuscular hyperplasia in humans; its understanding may lead to effective methods for its prevention and may provide clues to the pathogenesis of arteriosclerosis.

Identification of the principal proteoglycan-binding site in LDL. A single-point mutation in apo-B100 severely affects proteoglycan interaction without affecting LDL receptor binding.

The subendothelial retention of LDLs through their interaction with proteoglycans has been proposed to be a key process in the pathogenesis of atherosclerosis. In vitro studies have identified eight clusters of basic amino acids in delipidated apo-B100, the protein moiety of LDL, that bind the negatively charged proteoglycans. To determine which of these sites is functional on the surface of LDL particles, we analyzed the proteoglycan-binding activity of recombinant human LDL isolated from transgenic mice. Substitution of neutral amino acids for the basic amino acids residues in site B (residues 3359-3369) abolished both the receptor-binding and the proteoglycan-binding activities of the recombinant LDL. Chemical modification of the remaining basic residues caused only a marginal further reduction in proteoglycan binding, indicating that site B is the primary proteoglycan-binding site of LDL. Although site B was essential for normal receptor-binding and proteoglycan-binding activities, these activities could be separated in recombinant LDL containing single-point mutation. Recombinant LDL with a K3363E mutation, in which a glutamic acid had been inserted into the basic cluster RKR in site B, had normal receptor binding but interacted defectively with proteoglycans; in contrast, another mutant LDL, R3500Q, displayed defective receptor binding but interacted normally with proteoglycans. LDL with normal receptor-binding activity but with severely impaired proteoglycan binding will be a unique resource for analyzing the importance of LDL- proteoglycan interaction in atherogenesis. If the subendothelial retention of LDL by proteoglycans is the initial event in early atherosclerosis, then LDL with defective proteoglycan binding may have little or no atherogenic potential.

Perlecan Binds to the β-Amyloid Proteins (Aβ) of Alzheimer's Disease, Accelerates Aβ Fibril Formation, and Maintains Aβ Fibril Stability

Abstract: Perlecan is a specific heparan sulfate proteoglycan that accumulates in the fibrillar β‐amyloid (Aβ) deposits of Alzheimers disease. Perlecan purified from the Engelbreth‐Holm‐Swarm tumor was used to define perlecans interactions with Aβ and its effects on Aβ fibril formation. Using a solid‐phase binding immunoassay, freshly solubilized full‐length Aβ peptides bound immobilized perlecan at two sites, representing both high‐affinity [KD = ∼5.8 × 10−11M for Aβ (1–40); KD = ∼6.5 × 10−12M for Aβ (1–42)] and lower‐affinity [KD = 3.5 × 10−8M for Aβ (1–40); KD = 4.3 × 10−8M for Aβ (1–42)] interactions. An increase in the binding capacity of Aβ (1–40) to perlecan correlated with an increase in Aβ amyloid fibril formation during a 1‐week incubation period. The high‐capacity binding of Aβ (1–40) to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans and was completely abolished by heparin, but not by chondroitin‐4‐sulfate. Using a thioflavin T fluorometry assay, perlecan accelerated the rate of Aβ (1–40) amyloid fibril formation, causing a significant increase in Aβ fibril assembly over a 2‐week incubation period at 1 h (2.8‐fold increase), 1 day (3.6‐fold increase), and 3 days (2.8‐fold increase) in comparison with Aβ (1–40) alone. Perlecan also initially accelerated the formation of Aβ (1–42) fibrils within 1 h and maintained significantly higher levels of Aβ (1–42) thioflavin T fluorescence throughout a 2‐week experimental period in comparison with Aβ (1–42) alone, suggesting perlecans ability to maintain amyloid fibril stability. Perlecans effects on Aβ (1–40) fibril formation and maintenance of Aβ (1–42) fibril stability occurred in a dose‐dependent manner and was also mediated primarily by perlecans glycosaminoglycan chains. Perlecan was the most effective enhancer and accelerator of Aβ fibril formation when compared directly with other amyloid plaque components, including apolipoprotein E, α1‐antichymotrypsin, P component, C1q, and C3. This study, therefore, demonstrates that perlecan not only binds to the predominant isoforms of Aβ, but also accelerates Aβ fibril formation and stabilizes amyloid fibrils once formed, confirming pivotal roles for perlecan in the pathogenesis of Aβ amyloidosis in Alzheimers disease.