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Tumor-associated inflammation mediates the development of a systemic immunosuppressive milieu that is a major obstacle to effective treatment of cancer. Inflammation has been shown to promote the systemic expansion of immature myeloid cells which have been shown to exert immunosuppressive activity in laboratory models of cancer as well as cancer patients. Consequentially, significant effort is underway toward the development of therapies that decrease tumor-associated inflammation and immunosuppressive cells. The current study demonstrated that a previously described deep tissue imaging modality, which utilized indocyanine green-loaded calcium phosphosilicate nanoparticles (ICG-CPSNPs), could be utilized as an immunoregulatory agent. The theranostic application of ICG-CPSNPs as photosensitizers for photodynamic therapy was shown to block tumor growth in murine models of breast cancer, pancreatic cancer, and metastatic osteosarcoma by decreasing inflammation-expanded immature myeloid cells. Therefore, this therapeutic modality was termed PhotoImmunoNanoTherapy. As phosphorylated sphingolipid metabolites have been shown to have immunomodulatory roles, it was hypothesized that the reduction of immature myeloid cells by PhotoImmunoNanoTherapy was dependent upon bioactive sphingolipids. Mechanistically, PhotoImmunoNanoTherapy induced a sphingosine kinase 2-dependent increase in sphingosine-1-phosphate and dihydrosphingosine-1-phosphate. Furthermore, dihydrosphingosine-1-phosphate was shown to selectively abrogate myeloid lineage cells while concomitantly allowing the expansion of lymphocytes that exerted an antitumor effect. Collectively, these findings revealed that PhotoImmunoNanoTherapy, utilizing the novel nontoxic theranostic agent ICG-CPSNP, can decrease tumor-associated inflammation and immature myeloid cells in a sphingosine kinase 2-dependent manner. These findings further defined a novel myeloid regulatory role for dihydrosphingosine-1-phosphate. PhotoImmunoNanoTherapy holds the potential to be a revolutionary treatment for cancers with inflammatory and immunosuppressive phenotypes.

Integrins are transmembrane receptors that can specifically bind extracellular matrix (ECM) proteins. Assembly of the ECM protein fibronectin into fibrils has been shown to be a cell-mediated process that requires integrins. Like fibronectin, fibrillin 1 is an ECM glycoprotein that can assemble into fibrils, but the role of integrins in fibril formation is not understood. To investigate the role of integrins in fibrillin 1 ECM deposition, cells that normally produce and assemble fibrillin 1 fibers in vitro were stably transfected with plasmid constructs encoding short interfering RNAs that target specific integrin subunits. Cells that were deficient in α2- and β3-integrin subunits produced and deposited fibronectin normally, but cells that were deficient for α5 and αV were unable to elaborate a fibronectin matrix, although they continued to produce and secrete the protein. Surprisingly, the cells that were unable to elaborate a fibronectin matrix also lost fibrillin 1 gene expression.

Nano-/microtextured, free standing, flexible, thin film substrates of parylene C were fabricated by physicochemical vapour deposition of parylene C on silicon wafers with a spin coated layer of soap solution. The thin film substrates were engineered with different volumetric fibrous morphologies, ranging from 100 nm to 100 μm in thickness, and had top surface texture on nano- to micrometre length scales. The bottom surfaces of the thin film substrates are morphological negatives of the top surfaces of the soap coatings and can assist in the integration with implant surfaces. The attachment and growth of human osteosarcoma cells on the top surfaces of thin film substrates were successfully demonstrated.

We designed a two-dimensional (2D) morphology–composition gradient panel, which has a gradient in surface morphology at constant chemical composition along its length and a gradient in surface chemical composition without change of surface morphology in the orthogonal direction. The panels, which exhibit a 2D gradient in surface wettability, were fabricated by thermolysis of parylene-C followed by oxygen-plasma treatment for various times. As a demonstration study, binding of three different biotinylated serum proteins (B-IgG, B-A, and B-TF) to the 2D panel was investigated. The 2D gradient panels will facilitate development of optimized binding surfaces for various biotechnological applications.

Although the polymeric form of parylene-C is used in many medical devices, the mechanistic nature of cellular attachment to polymeric parylene-C is not clear. We examined the effects of (i) substrate morphology, (ii) surface wettability and (iii) presence of serum proteins on fibroblast attachment. A physicochemical vapor deposition technique was implemented to deposit flat parylene-C substrates as well as fibrous substrates of three different morphologies: slanted columnar, chevronic and chiral. Flat parylene-C surfaces were moderately hydrophobic while fibrous substrates were superhydrophobic. Pretreatment with oxygen plasma changed the substrate surfaces from hydrophobic to superhydrophilic. The attachment efficiency of human fibroblast cells to the flat and three fibrous thin-film parylene-C substrates was investigated. Fibroblast attachment was better on fibrous substrates than on flat substrates, and oxygen plasma pretreatment facilitated fibroblast attachment on all four morphologies. Serum proteins also facilitated cell attachment on all substrates. The combination of oxygen plasma pre-treatment and serum proteins increased fibroblast adhesion in an additive manner on flat, but not on fibrous parylene-C substrates. The morphology of cell–substrate interactions differed between fibrous and flat parylene-C substrates.

Human embryonic stem cells (hESCs) are characterized by their immortality and pluripotency. Human mesenchymal stem cells (hMSC), on the other hand, have limited self-renewal and differentiation capabilities. The underlying molecular differences that account for this characteristic self-renewal and plasticity are, however, poorly understood. This study reports a nuclear proteomic analysis of human embryonic and bone marrow-derived mesenchymal stem cells. Our proteomic screen highlighted a 5-fold difference in the expression of Reptin52. We show, using two-dimensional difference gel electrophoresis (2-DIGE), western analysis, and quantitative reverse transcriptase polymerase chain reaction, that Reptin52 is more abundantly expressed in hESC than hMSC. Moreover, we observed differential expression of Pontin52 and beta-catenin-proteins known to interact with Reptin52. This difference in the expression of Reptin52 and Pontin52 (known regulators of beta-catenin) further supports a role for Wnt signaling in stem cell self-renewal and proliferation.

Fibrillins 1, 2 and 3 make up a family of genes that encode large, cysteine-rich extracellular matrix glycoproteins found in connective tissues, lung, blood vessels and other extensible tissues. Fibrillins 1 and 2 have both overlapping as well as separate distributions in human embryonic and adult tissues. Fibrillin-containing microfibrils are known to modulate morphogenetic events by proper targeting of growth factors to the extracellular matrix. Mutation of the fibrillin-2 gene causes a genetic disorder, congenital contractural arachnodactyly (CCA), that results in flexion contractures. Previously, we have shown a distinct fibrillin-2 distribution in the pericellular matrix of interior tenocytes and later demonstrated a unique fibrillin-2 containing structure that runs along the tendon cell arrays in the canine flexor tendon. We hypothesized that loss of these fibrillin-2 containing structures might affect normal tendon development. To test our hypothesis, connective tissues from mice null for fibrillin-2 gene expression were studied. Murine flexor digitorum longus tendons were evaluated for total collagen content, and the intermolecular collagen cross-links hydroxylysyl and lysyl pyridinoline. The results show decreased collagen cross-links in fibrillin-2 null mice, however total collagen content remained the same when compared to wild type. Bone morphology was studied using micro computed tomography (CT). Fibrillin-2 null mice display a focal area of decreased bone length in the extremities as compared to wild type mice. Together, these results demonstrate a role for fibrillin-2 in bone and soft connective tissue morphological and biochemical processes.

Insertion site injuries of the flexor digitorum profundus (FDP) tendon often present for delayed treatment. Apart from gross observations made at the time of surgery, the changes that occur in the flexor tendon stump during the interval from injury to repair are unknown. These changes may include tendon softening and loss of viability, which may contribute to the poor outcomes observed clinically and experimentally. Thirty-eight FDP tendons from 23 adult dogs were transected sharply from their insertions on the distal phalanges and were not repaired. Dogs were allowed full weight bearing and were euthanized 7 or 21 days after injury. Biomechanical testing indicated that the resistance of injured tendons to pullout of a Kessler-type suture was not different from control tendons at 7 days and was increased at 21 days by 25% (p

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight protein associated with extracellular matrix microfibrils. Biochemical studies have shown that MAGP-1 undergoes several posttranslational modifications that may influence its associations with other microfibrillar components. To identify the sites in the molecule where posttranslational modifications occur, we expressed MAGP-1 constructs containing various point mutations as well as front and back half truncations in CHO cells. Characterization of transiently expressed protein showed that MAGP-1 undergoes O-linked glycosylation and tyrosine sulfation at sites in its amino-terminal half. This region of the protein also served as a major amine acceptor site for transglutaminase and mediated self-assembly into high molecular weight multimers through a glutamine-rich sequence. Fine mapping of the modification sites through mutational analysis demonstrated that Gln20 is a major amine acceptor site for the transglutaminase reaction and confirmed that a canonical tyrosine sulfation consensus sequence is the site of MAGP-1 sulfation. Our results also show that O-glycosylation occurs at more than one site in the molecule.

Fibrous Parylene-C films with tilted columnar morphology were deposited with a physicochemical version of the oblique-angle deposition (OAD) technique. Advancing contact angles increased from 103.9° (smooth hydrophobic surface) to 168.5° (rough superhydrophobic surface) as the thickness of the film increased from ∼ 100 nm to ∼ 60 μm, reflecting changes in the morphology of the top surface. Treatment with oxygen plasma can render these fibrous films completely water wettable. These techniques allow tailoring of surface wettability to meet the demands of specific biomedical applications.

Alignment of tropoelastin molecules during the process of elastogenesis is thought to require fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, fibrillin-1 and fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of fibrillin-2 and fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking.

Aggregation of fibrillin molecules via disulphide bonds is postulated to be an early step in microfibril assembly. By expressing fragments of fibrillin 1 and fibrillin 2 in a mammalian expression system, we found that the N-terminal region of each protein directs the formation of homodimers and that disulphide bonds stabilize this interaction. A large fragment of fibrillin 1 containing much of the region downstream from the N-terminus remained as a monomer when expressed in the same cell system, indicating that this region of the protein lacks dimerization domains. This finding also confirms that the overexpression of fibrillin fragments does not in itself lead to spurious dimer formation. Pulse-chase analysis demonstrated that dimer formation occurred intracellularly, suggesting that the process of fibrillin aggregation is initiated early after biosynthesis of the molecules. These findings also implicate the N-terminal region of fibrillin 1 and fibrillin 2 in directing the formation of a dimer intermediate that aggregates to form the functional microfibril.

Osteosarcoma is a malignant neoplasm of mesenchymal origin that is presumed to arise from osteoblasts. Considered a rare tumor, approximately 1000 cases of osteosarcoma are diagnosed in the United States each year, and osteosarcoma of the foot is rarer still. Marfan syndrome (MFS) is a rare genetic disorder that affects 1 in 5000 individuals and is caused by mutations in the fibrillin 1 (FBN1) gene. MFS phenotype affects several body systems, including soft connective tissue and bone. Here we report, for the first time, an individual with MFS that was treated for osteosarcoma. Surgically resected tissue was used to initiate an osteosarcoma cell line (PSU-OS-M) that exhibits attachment-independent growth and loss of contact inhibition in vitro. Genomic DNA was isolated from the tumor cells, and primers that anneal to intronic regions were used to amplify and sequence all 65 coding exons of the FBN1 gene. A two base pair insertion that results in a novel premature termination codon (PTC) was found in exon 52. Protein from the normal allele is detectable in PSU-OS-M cell-conditioned medium, but protein from the mutant allele was not detectable. Immunofluorescent microscopy demonstrates that PSU-OS-M cells can assemble fibrillin 1 microfibrils in culture, and fibronectin assembly is normal. As such, the PSU-OS-M cell line is to our knowledge the first oncogenically transformed cell line with a mutant fibrillin gene and may serve as a useful tool for studying molecular mechanisms of MFS and nonsense-mediated decay.

Development of the extracellular matrix is a critical feature of alveolar formation and actively involves pulmonary interstitial fibroblasts. The elastic fiber network is an interconnected system of load-bearing fibers that also influences the behavior of adjacent cells, particularly the interstitial lung fibroblasts (LF). We hypothesized that discrete domains of fibrillins-1 and -2 interact with LF integrins and direct their migration in the presence of platelet-derived growth factor (PDGF)-A. Surfaces coated with recombinant peptides lacking or including an arginine-glycine-aspartic acid (RGD) motif were used to study LF migration across porous filters and on protein-coated glass. Exon 24 of fibrillin-2 (Fib2 24), which encodes for an RGD-containing transforming growth factor-beta-binding (TB) domain, stimulated migration with greater directional persistence and more effectively stimulated trans-filter migration at low concentrations. Exons 36-44 of fibrillin-1 (Fib1 36-44), which include epidermal growth factor-like domains and an RGD-containing TB domain, induce more lamlellipodia and more widespread remodeling of the leading edge, resulting in greater migration velocity than did Fib2 24. Distinct structural features in regions that surround the RGD motifs may differentially regulate how the PDGF receptor-alpha promotes integrin distribution and actin filament remodeling at the cell's leading edge. Understanding how fibrillins regulate LF migration may help elucidate how the elastic fiber system could be restored as an interconnected unit, which fails to occur in emphysematous lungs.

Nanoengineered parylene-C sculptured thin films (STFs) are deposited on glass and silicon substrates using a direct one-step growth technique. The deposited STFs support fibroblast cell attachment and proliferation in vitro, which is an early indication of biocompatibility and bioactivity of this emerging class of biomaterials. Surface modification of endoprostheses of the small joints of the hand, which heal with fibrous stabilization, may be greatly enhanced by such nanoengineered biomaterials.

Using yeast two-hybrid, ligand blotting, and solid phase binding assays, we have shown that microfibril-associated glycoprotein-1 (MAGP-1) interacts with the 8-cysteine motif of fibrillin-2 encoded by exon 24. Binding to this sequence was demonstrated for full-length MAGP-1 as well as for the MAGP-1 matrix-binding domain encoded by exons 7 and 8. The matrix-binding domain, but not the full-length protein, also bound to regions of fibrillin-2 defined by exons 16 and 17, exon 20, and exons 23 and 24. Interestingly, no binding was detected to sequences near the N or C terminus where MAGP-1 and MAGP-2, respectively, were shown to interact with fibrillin-1. The localization of MAGP-1 binding to the 8-Cys domain encoded by exon 24 suggests that the bead structure of microfibrils consists of exon 24 and portions of the central region of fibrillin-2. Exon 24 in fibrillin lies in the region of the molecule where mutations produce the most severe phenotypes associated with Marfan syndrome (fibrillin-1) and congenital contractural arachnodactyly (fibrillin-2). It is possible that these mutations alter the ability of fibrillin to bind MAGP-1, which may contribute to the severity of the disease.

Purpose Recent in vivo canine studies have shown incomplete restoration of the flexor digitorum profundus (FDP) insertion site after transection and repair to the cortical surface of the distal phalanx. Previous biomechanical analyses of tendon to bone surface repair have suggested that repair site gap formation of greater than 3 mm occurs frequently under physiologic loads. A recent ex vivo investigation into a novel repair of the FDP tendon into a bone tunnel in the distal phalanx showed improved tensile properties with a decrease in repair site gap formation. Time-zero data, however, do not always accurately reflect in vivo responses. The repair response of the FDP tendon when placed in an osseous compartment is not known. The purpose of this study was to analyze the histologic and vascular anatomic properties of the FDP insertion site after transection and repair in a bone tunnel within the distal phalanx. Methods Twenty-six FDP tendon to bone repairs were performed in 13 adult mongrel dogs after insertion site transection. The tendons were repaired in a bone tunnel in the distal phalanx. Vascular analysis of the tendon and repair site was performed by using a modified Spalteholtz technique and routine hematoxylin-eosin staining was used to assess histologic properties of the repair. Results In normal specimens the vascular analysis showed that there was a distal network of vessels extending 1- to 2-cm proximal to the FDP insertion site. At 10 days after repair the distal tendon segment tendon remained avascular. By 21 days after repair there was proximal migration of an unorganized reticular network of tendon surface vessels with sparse intratendinous communications. At 6 weeks after repair the structure of the distal tendon vascular network resembled that of normals. The vascular response of the tendon within the bone tunnel followed a similar time frame. Histologic analysis showed an inflammatory reaction in the bone tunnel leading to a progressive degradation of that portion of the FDP tendon that resided in the tunnel. Tendon necrosis was not seen. Conclusions The FDP tendon, after insertion site transection and repair in a bone tunnel, undergoes a process of neovascularization and revascularization over a period of 6 weeks. There is a progressive loss of tendon parenchyma within the bone tunnel and the suture tracks appeared to serve as conduits for the ingrowth of inflammatory tissue. Restoration of the normal 4-zone tendon-bone interface was not seen. Although ex vivo biomechanical assessment of tendon repair in a bone tunnel appears promising, the repair response in vivo may not be favorable for tendon to bone healing. The progressive tendon degeneration that was observed here may have detrimental effects on repair site tensile properties, increasing the potential for early failure.

Fibrillin-1 and −2 are large modular extracellular matrix glycoproteins found in many vertebrate organ systems and are known to be key components of the elastic fibre. In the present study, we identify a new heparin-binding region in fibrillin-2 between exons 18 and 24. Additionally, we have narrowed the location of heparin-binding activity previously identified in fibrillin-1 to the last 17 residues of the mature proteolytically processed protein. This domain demonstrated higher activity as a multimer than as a monomer. The fibrillin-1 C-terminal site supported cell attachment in each of nine cell types tested. Attachment was shown to be mediated by cell-surface heparan sulphate proteoglycans. Fibrillin-1 has been shown previously to have heparin-binding activity that is important for matrix deposition of the molecule by fibroblasts. This function in deposition was confirmed in two additional fibrillin-producing cell types (osteosarcoma and epithelial cells) for the deposition of both fibrillin-1 and −2 into the extracellular matrix.

Cells that carry out wound healing must be able to perform catabolic as well as anabolic functions. As the tendon is a tissue rich in extracellular matrix (ECM) proteins, we hypothesized that cells which participate in tendon healing should be able to produce proteases that would allow the remodeling of such a tissue. To this end, we assessed the ability of endotenon cells isolated from canine flexor digitorum profundus tendon and from surrounding parietal sheath to produce the gelatinases MMP-2 and MMP-9. Endotenon and sheath cells cultured in vitro on polystyrene produced small amounts of MMP-2 and MMP-9 was not detectable. When cultured on polystyrene coated with type I collagen, the cells upregulated MMP-2 production and MMP-9 production was induced. No other ECM protein elicited this response nor did other cell lines respond in this way after attachment to type I collagen. The two gelatinases were identified by immunological methods, ability to bind gelatin, size, metal ion requirement, serine protease inhibitor insensitivity, and APMA activation. For cells grown on collagen-coated plastic, gelatinase upregulation was proportional to the amount of ligand present until saturation was reached. For any group of fresh tendon cells, MMP-2 and MMP-9 upregulation was greater in a three dimensional collagen gel than the highest response from the same group under two dimensional culture conditions. Attachment of the cells to type I collagen increased the ratio of active to inactive MMP-2. Dexamethasone inhibited the upregulation of both MMP-2 and MMP-9. These results show that ECM proteins can influence both the production and the state of activation of these matrix metalloproteinases.

The elastic fiber is known to be an important component of skin, lung, and vasculature. Much less is known about the distribution of elastin and elastic fiber-related proteins in connective tissues, yet genetic defects of elastic fiber constituents can lead to deficiencies in these tissues. For the first time, we determine the distribution of elastin, fibrillins 1 and 2, and microfibril-associated glycoproteins (MAGPs) 1 and 2 in the flexor digitorum profundus (FDP) tendon. Three functionally distinct regions of the FDP tendon, the fibrocartilagenous (FC) region, avascular/tensional (AV/T) region, and insertion region, were evaluated by immunohistochemical methods for these five proteins. Biochemical analysis of desmosine content, an elastin-specific cross-link, demonstrated the presence of elastin in each region, and this was verified histochemically. The fibrillins were found with elastin and also pericellularly with internal fibroblasts where elastin was not detected. Although there was overlapping distribution, fibrillin 2 was more prominent in the interior of the tendon while fibrillin 1 was prominent in outer cell layers that contained elastic fibers. Both MAGP-1 and -2 were found throughout the tendon, although the greatest abundance was near the tendon insertion to bone. Surprisingly, MAGP-1 demonstrated a filamentous appearance within the fibrocartilage that did not correspond to the fibrillin 1 or 2 or MAGP-2 staining pattern. Lastly, we have shown that a vincular membrane located along the dorsal surface of the tendon near the insertion has a very high elastin content and a unique interface with the tendon that consists of an elastic anchor within the tendon body.

To investigate the processing and general properties of the fibrillin-1 carboxyl-terminal domain, three protein expression constructs have been developed as follows: one without the domain, one with the domain, and one with a mutation near the putative proteolytic processing site. The constructs have been expressed in two eukaryotic model systems, baculoviral and CHO-K1. Post-translational modifications that normally occur in fibrillin-1, including glycosylation, signal peptide cleavage, and carboxyl-terminal processing, occur in the three constructs in both cell systems. Amino-terminal sequencing of secreted protein revealed leader sequence processing at two sites, a primary site between Gly-24/Ala-25 and a secondary site of Ala-27/Asn-28. Processing of the carboxyl-terminal domain could be observed by migration differences in SDS-polyacrylamide gel electrophoresis and was evident in both mammalian and insect cells. Immunological identification by Western blotting confirmed the loss of the expected region. The failure of both cell systems to process the mutant construct shows that the multi-basic sequence is the site of proteolytic processing. Cleavage of the fibrillin-1 carboxyl-terminal domain occurred intracellularly in CHO-K1 cells in an early secretory pathway compartment as demonstrated by studies with secretion blocking agents. This finding, taken with the multi-basic nature of the cleavage site and observed calcium sensitivity of cleavage, suggests that the processing enzyme is a secretory pathway resident furin-like protease.

Within tendon, between collagen fascicles, cells are organized in linear arrays surrounded by a specialized environment of extracellular matrix (ECM) proteins that are largely unidentified. Our goal was to identify interfascicular, pericellular ECM components and provide additional resolution to the organization of the pericellular matrix. To this end, we employed a combination of enzymatic digestion, mechanical disruption, and differential sedimentation to demonstrate for the first time that it possible to liberate living linear tendon cell arrays from whole tendon. Here, we identify type VI collagen, versican, and fibrillin-2 as components of the immediate pericellular ECM of linearly arrayed tendon cells. Additionally, a unique fibrillin-2-containing macromolecular assembly is described in detail for the first time. This new structure is unlike any previously described fibrillin-containing macromolecular assembly. Having a largely constant diameter, it runs axially along tendon cell arrays and can exceed 1000 μm in length.