Megan S. Lord

Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells.

Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture.

Heparan Sulfate-Dependent Signaling of Fibroblast Growth Factor 18 by Chondrocyte-Derived Perlecan

Perlecan is a large multidomain proteoglycan that is essential for normal cartilage development. In this study, perlecan was localized in the pericellular matrix of hypertrophic chondrocytes in developing human cartilage rudiments. Perlecan immunopurified from medium conditioned by cultured human fetal chondrocytes was found to be substituted with heparan sulfate (HS), chondroitin sulfate (CS), and keratan sulfate (KS). Ligand and carbohydrate engagement (LACE) assays demonstrated that immunopurified chondrocyte-derived perlecan formed HS-dependent ternary complexes with fibroblast growth factor (FGF) 2 and either FGF receptors (FGFRs) 1 or 3; however, these complexes were not biologically active in the BaF32 cell system. Chondrocyte-derived perlecan also formed HS-dependent ternary complexes with FGF18 and FGFR3. The proliferation of BaF32 cells expressing FGFR3 was promoted by chondrocyte-derived perlecan in the presence of FGF18, and this activity was reduced by digestion of the HS with either heparinase III or mammalian heparanase. These data suggest that FGF2 and -18 bind to discrete structures on the HS chains attached to chondrocyte-derived perlecan which modulate the growth factor activities. The presence and activity of mammalian heparanase may be important in the turnover of HS and subsequent signaling required for the establishment and maintenance of functional osteo-chondral junctions in long bone growth.

The Structure, Location, and Function of Perlecan, a Prominent Pericellular Proteoglycan of Fetal, Postnatal, and Mature Hyaline Cartilages

The aim of this study was to immunolocalize perlecan in human fetal, postnatal, and mature hyaline cartilages and to determine information on the structure and function of chondrocyte perlecan. Perlecan is a prominent component of human fetal (12-14 week) finger, toe, knee, and elbow cartilages; it was localized diffusely in the interterritorial extracellular matrix, densely in the pericellular matrix around chondrocytes, and to small blood vessels in the joint capsules and perichondrium. Aggrecan had a more intense distribution in the marginal regions of the joint rudiments and in para-articular structures. Perlecan also had a strong pericellular localization pattern in postnatal (2-7 month) and mature (55-64 year) femoral cartilages, whereas aggrecan had a prominent extracellular matrix distribution in these tissues. Western blotting identified multiple perlecan core protein species in extracts of the postnatal and mature cartilages, some of which were substituted with heparan sulfate and/or chondroitin sulfate and some were devoid of glycosaminoglycan substitution. Some perlecan core proteins were smaller than intact perlecan, suggesting that proteolytic processing or alternative splicing had occurred. Surface plasmon resonance and quartz crystal microbalance with dissipation experiments demonstrated that chondrocyte perlecan bound fibroblast growth factor (FGF)-1 and -9 less efficiently than endothelial cell perlecan. The latter perlecan supported the proliferation of Baf-32 cells transfected with FGFR3c equally well with FGF-1 and -9, whereas chondrocyte perlecan only supported Baf-32 cell proliferation with FGF-9. The function of perlecan therefore may not be universal but may vary with its cellular origin and presumably its structure.

Cellular uptake and reactive oxygen species modulation of cerium oxide nanoparticles in human monocyte cell line U937

Cerium oxide nanoparticles (nanoceria) are promising materials for intracellular oxygen free radical scavenging providing a potential therapy for reactive oxygen species (ROS)-mediated inflammatory processes. In this study rhombohedral-shaped nanoceria were synthesized by flame spray pyrolysis with tuneable particle diameters between 3 and 94 nm by changing the liquid precursor flow rate. Monocytes and macrophages are major players in inflammatory processes as their production of ROS species has important downstream effects on cell signalling. Therefore, this study examined the ability of the nanoceria to be internalised by the human monocytic cell line, U937, and scavenge intracellular ROS. U937 cells activated in the presence of phorbol 12-myristate 13-acetate (PMA) were found to be more responsive to the nanoceria than U937 cells, which may not be surprising given the role of monocyte/macrophages in phagocytosing foreign material. The smaller particles were found to contain more crystal lattice defects with which to scavenge ROS, however a greater proportion of both the U937 and activated U937 cell populations responded to the larger particles. Hence all nanoceria particle sizes examined in this study were equally effective in scavenging intracellular ROS.

The modulation of platelet adhesion and activation by chitosan through plasma and extracellular matrix proteins.

Chitosan has been shown to promote initial wound closure events to prevent blood loss. Platelet adhesion and activation are crucial early events in these processes after traumatic bleeding leading to thrombus formation. Platelet adhesion to chitosan was found to be enhanced in the presence of adsorbed plasma and extracellular matrix proteins and was found to be primarily mediated by α(IIb)β(3) integrins, while α(2)β(1) integrins were found to be involved in platelet adhesion to collagen and perlecan. Platelets were found to be activated by chitosan, as shown by an increase in the expression of α(IIb)β(3) integrins and P-selectin, while the extent of activation was modulated by the presence of proteins including perlecan and fibrinogen. Collagen-coated chitosan was found to activate platelets to the same extent as either chitosan or collagen alone. These data support the role of plasma and extracellular matrix proteins in promoting chitosan mediated platelet adhesion and activation supporting the hypothesis that chitosan promotes wound healing via these interactions.

Scaling deterministic lateral displacement arrays for high throughput and dilution-free enrichment of leukocytes

A disposable device for fractionation of blood into its components that is simple to operate and provides throughput of greater than 1 mL min−1 is highly sought after in medical diagnostics and therapies. This paper describes a device with parallel deterministic lateral displacement devices for enrichment of leukocytes from blood. We show capture of 98% and approximately ten-fold enrichment of leukocytes in whole blood. We demonstrate scaling up through the integration of six parallel devices to achieve a flow rate of 115 µL of undiluted blood per minute per atmosphere of applied pressure.

The role of vascular-derived perlecan in modulating cell adhesion, proliferation and growth factor signaling.

Smooth muscle cell proliferation can be inhibited by heparan sulfate proteoglycans whereas the removal or digestion of heparan sulfate from perlecan promotes their proliferation. In this study we characterized the glycosaminoglycan side chains of perlecan isolated from either primary human coronary artery smooth muscle or endothelial cells and determined their roles in mediating cell adhesion and proliferation, and in fibroblast growth factor (FGF) binding and signaling. Smooth muscle cell perlecan was decorated with both heparan sulfate and chondroitin sulfate, whereas endothelial perlecan contained exclusively heparan sulfate chains. Smooth muscle cells bound to the protein core of perlecan only when the glycosaminoglycans were removed, and this binding involved a novel site in domain III as well as domain V/endorepellin and the α2β1 integrin. In contrast, endothelial cells adhered to the protein core of perlecan in the presence of glycosaminoglycans. Smooth muscle cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains and promoted the signaling of FGF2 but not FGF1. Also endothelial cell perlecan bound both FGF1 and FGF2 via its heparan sulfate chains, but in contrast, promoted the signaling of both growth factors. Based on this differential bioactivity, we propose that perlecan synthesized by smooth muscle cells differs from that synthesized by endothelial cells by possessing different signaling capabilities, primarily, but not exclusively, due to a differential glycanation. The end result is a differential modulation of cell adhesion, proliferation and growth factor signaling in these two key cellular constituents of blood vessels.

Extracellular matrix remodelling during cell adhesion monitored by the quartz crystal microbalance.

A cells ability to remodel adsorbed protein layers on surfaces is influenced by the nature of the protein layer itself. Remodelling is often required to accomplish cellular adhesion and extracellular matrix formation which forms the basis for cell spreading, increased adhesion and expression of different phenotypes. The adhesion of NIH3T3 (EGFP) fibroblasts to serum protein (albumin or fibronectin) precoated tantalum (Ta) and oxidised polystyrene (PS(ox)) surfaces was examined using the quartz crystal microbalance with dissipation (QCM-D) monitoring and fluorescence microscopy. The cells were either untreated or treated with cycloheximide to examine the contribution of endogenous protein production during cell adhesion to the QCM-D response over a period of 2h. Following adsorption of albumin onto Ta and PS(ox) there was no difference detected between the response to seeding untreated and cycloheximide treated cells. The QCM-D was able to detect differences in the untreated cellular responses to fibronectin versus serum precoated Ta and PS(ox) substrates, while cycloheximide treatment of the cells produced the same QCM-D response for fibronectin and serum precoatings on each of the materials. This confirmed that the process of matrix remodelling by the cells is dependent on the underlying substrate and the preadsorbed proteins and that the QCM-D response is dominated by changes in the underlying protein layer. Changes in dissipation correspond to the development of the actin cytoskeleton as visualised by actin staining.

Mast Cells Produce Novel Shorter Forms of Perlecan That Contain Functional Endorepellin: A ROLE IN ANGIOGENESIS AND WOUND HEALING*

Background: Mast cells modulate events in wound healing. Results: Shorter forms of perlecan are produced by mast cells via proteolytic processing and alternative splicing, which contain domain V and functional endorepellin. Conclusion: The production of these shorter forms modulates endothelial cell adhesion, proliferation, and migration. Significance: Mast cells produce specific forms of perlecan that affect endothelial cell behavior. Mast cells are derived from hematopoietic progenitors that are known to migrate to and reside within connective and mucosal tissues, where they differentiate and respond to various stimuli by releasing pro-inflammatory mediators, including histamine, growth factors, and proteases. This study demonstrated that primary human mast cells as well as the rat and human mast cell lines, RBL-2H3 and HMC-1, produce the heparan sulfate proteoglycan, perlecan, with a molecular mass of 640 kDa as well as smaller molecular mass species of 300 and 130 kDa. Utilizing domain-specific antibodies coupled with N-terminal sequencing, it was confirmed that both forms contained the C-terminal module of the protein core known as endorepellin, which were generated by mast cell-derived proteases. Domain-specific RT-PCR experiments demonstrated that transcripts corresponding to domains I and V, including endorepellin, were present; however, mRNA transcripts corresponding to regions of domain III were not present, suggesting that these cells were capable of producing spliced forms of the protein core. Fractions from mast cell cultures that were enriched for these fragments were shown to bind endothelial cells via the α2β1 integrin and stimulate the migration of cells in “scratch assays,” both activities of which were inhibited by incubation with either anti-endorepellin or anti-perlecan antibodies. This study shows for the first time that mast cells secrete and process the extracellular proteoglycan perlecan into fragments containing the endorepellin C-terminal region that regulate angiogenesis and matrix turnover, which are both key events in wound healing.

The modulation of platelet and endothelial cell adhesion to vascular graft materials by perlecan.

Controlled neo-endothelialisation is critical to the patency of small diameter vascular grafts. Endothelialisation and platelet adhesion to purified endothelial cell-derived perlecan, the major heparan sulfate (HS) proteoglycan in basement membranes, were investigated using in vivo and in vitro assays. Expanded polytetrafluoroethylene (ePTFE) vascular grafts were coated with perlecan and tested in an ovine carotid interposition model for a period of 6 weeks and assessed using light and scanning microscopy. Enhanced endothelial cell growth and reduced platelet adhesion were observed on the perlecan coated grafts when compared to uncoated controls implanted in the same sheep (n=5). Perlecan was also found to stimulate endothelial cell proliferation in vitro over a period of 6 days in the presence of plasma proteins and fibroblastic growth factor 2 (FGF-2), however in the absence of FGF-2 endothelial cell growth could not be maintained during this period. Perlecan was found to be anti-adhesive for platelets, however after removal of the HS chains attached to perlecan, platelet adhesion and aggregation were supported. These results suggest a role for HS chains of perlecan in improving graft patency by selectively promoting endothelial cell proliferation while modulating platelet adhesion.