PhotoImmunoNanoTherapy reveals an anticancer role for sphingosine kinase 2 and dihydrosphingosine-1-phosphate.

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.

Altered sphingolipid metabolism in patients with metastatic pancreatic cancer.

Although numerous genetic mutations and amplifications have been identified in pancreatic cancer, much of the molecular pathogenesis of the disease remains undefined. While proteomic and transcriptomic analyses have been utilized to probe and characterize pancreatic tumors, lipidomic analyses have not been applied to identify perturbations in pancreatic cancer patient samples. Thus, we utilized a mass spectrometry-based lipidomic approach, focused towards the sphingolipid class of lipids, to quantify changes in human pancreatic cancer tumor and plasma specimens. Subgroup analysis revealed that patients with positive lymph node metastasis have a markedly higher level of ceramide species (C16:0 and C24:1) in their tumor specimens compared to pancreatic cancer patients without nodal disease or to patients with pancreatitis. Also of interest, ceramide metabolites, including phosphorylated (sphingosine- and sphinganine-1-phosphate) and glycosylated (cerebroside) species were elevated in the plasma, but not the pancreas, of pancreatic cancer patients with nodal disease. Analysis of plasma level of cytokine and growth factors revealed that IL-6, IL-8, CCL11 (eotaxin), EGF and IP10 (interferon inducible protein 10, CXCL10) were elevated in patients with positive lymph nodes metastasis, but that only IP10 and EGF directly correlated with several sphingolipid changes. Taken together, these data indicate that sphingolipid metabolism is altered in human pancreatic cancer and associated with advanced disease. Assessing plasma and/or tissue sphingolipids could potentially risk stratify patients in the clinical setting.

Selective integrin subunit reduction disrupts fibronectin extracellular matrix deposition and fibrillin 1 gene expression.

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 ß3-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.

Osteosarcoma in a Marfan patient with a novel premature termination codon in the FBN1 gene.

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.

Human embryonic and mesenchymal stem cells express different nuclear proteomes.

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.

Bone and soft connective tissue alterations result from loss of fibrillin-2 expression.

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.

Arg-Gly-Asp-containing domains of fibrillins-1 and -2 distinctly regulate lung fibroblast migration.

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.

Tendon injury response: assessment of biomechanical properties, tissue morphology and viability following flexor digitorum profundus tendon transection.

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<0.05). Morphologically, at 7 and 21 days the cut surface had a smooth appearance and the end of the injured tendon was increased in thickness by 30% and 50%, respectively (p<0.05). Histologically, we observed increased cellularity and dramatic fibroblast proliferation within the injured tendon stump; there was no evidence of decreased cell viability. We conclude that during the interval from 0 to 21 days after FDP insertion-site injury, tendons cells are viable, proliferative and synthesizing new matrix. This leads to increased tendon size and enhanced resistance to suture pullout. These findings offer a scientific rationale supporting the clinical practice of surgical re-attachment within the first 3 weeks after injury.

Identification of a major microfibril-associated glycoprotein-1-binding domain in fibrillin-2.

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.

Tendon cell array isolation reveals a previously unknown fibrillin-2-containing macromolecular assembly.

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 microm in length.

Fibrillin-1 and -2 contain heparin-binding sites important for matrix deposition and that support cell attachment.

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.

Tendon cells produce gelatinases in response to type I collagen attachment.

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.

Distribution of the elastic fiber and associated proteins in flexor tendon reflects function.

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.