Adipocyte-Derived Versican and Macrophage-Derived Biglycan Control Adipose Tissue Inflammation in Obesity.

Obesity is characterized by adipose tissue inflammation. Because proteoglycans regulate inflammation, here we investigate their role in adipose tissue inflammation in obesity. We find that adipose tissue versican and biglycan increase in obesity. Versican is produced mainly by adipocytes and biglycan by adipose tissue macrophages. Both proteoglycans are also present in adipose tissue from obese human subjects undergoing gastric bypass surgery. Deletion of adipocyte-specific versican or macrophage-specific biglycan in mice reduces macrophage accumulation and chemokine and cytokine expression, although only adipocyte-specific versican deletion leads to sustained improvement in glucose tolerance. Macrophage-derived biglycan activates inflammatory genes in adipocytes. Versican expression increases in cultured adipocytes exposed to excess glucose, and adipocyte-conditioned medium stimulates inflammation in resident peritoneal macrophages, in part because of a versican breakdown product, versikine. These findings provide insights into the role of adipocyte- and macrophage-derived proteoglycans in adipose tissue inflammation in obesity.

Versican-A Critical Extracellular Matrix Regulator of Immunity and Inflammation.

The extracellular matrix (ECM) proteoglycan, versican increases along with other ECM versican binding molecules such as hyaluronan, tumor necrosis factor stimulated gene-6 (TSG-6), and inter alpha trypsin inhibitor (IαI) during inflammation in a number of different diseases such as cardiovascular and lung disease, autoimmune diseases, and several different cancers. These interactions form stable scaffolds which can act as "landing strips" for inflammatory cells as they invade tissue from the circulation. The increase in versican is often coincident with the invasion of leukocytes early in the inflammatory process. Versican interacts with inflammatory cells either indirectly via hyaluronan or directly via receptors such as CD44, P-selectin glycoprotein ligand-1 (PSGL-1), and toll-like receptors (TLRs) present on the surface of immune and non-immune cells. These interactions activate signaling pathways that promote the synthesis and secretion of inflammatory cytokines such as TNFα, IL-6, and NFκB. Versican also influences inflammation by interacting with a variety of growth factors and cytokines involved in regulating inflammation thereby influencing their bioavailability and bioactivity. Versican is produced by multiple cell types involved in the inflammatory process. Conditional total knockout of versican in a mouse model of lung inflammation demonstrated significant reduction in leukocyte invasion into the lung and reduced inflammatory cytokine expression. While versican produced by stromal cells tends to be pro-inflammatory, versican expressed by myeloid cells can create anti-inflammatory and immunosuppressive microenvironments. Inflammation in the tumor microenvironment often contains elevated levels of versican. Perturbing the accumulation of versican in tumors can inhibit inflammation and tumor progression in some cancers. Thus versican, as a component of the ECM impacts immunity and inflammation through regulating immune cell trafficking and activation. Versican is emerging as a potential target in the control of inflammation in a number of different diseases.

Respiratory Syncytial Virus Infection of Human Lung Fibroblasts Induces a Hyaluronan-Enriched Extracellular Matrix That Binds Mast Cells and Enhances Expression of Mast Cell Proteases.

Human lung fibroblasts (HLFs) treated with the viral mimetic polyinosine-polycytidylic acid (poly I:C) form an extracellular matrix (ECM) enriched in hyaluronan (HA) that avidly binds monocytes and lymphocytes. Mast cells are important innate immune cells in both asthma and acute respiratory infections including respiratory syncytial virus (RSV); however, the effect of RSV on HA dependent mast cell adhesion and/or function is unknown. To determine if RSV infection of HLFs leads to the formation of a HA-enriched ECM that binds and enhances mast cell activity primary HLFs were infected with RSV for 48 h prior to leukocyte binding studies using a fluorescently labeled human mast cell line (LUVA). Parallel HLFs were harvested for characterization of HA production by ELISA and size exclusion chromatography. In separate experiments, HLFs were infected as above for 48 h prior to adding LUVA cells to HLF wells. Co-cultures were incubated for 48 h at which point media and cell pellets were collected for analysis. The role of the hyaladherin tumor necrosis factor-stimulated gene 6 (TSG-6) was also assessed using siRNA knockdown. RSV infection of primary HLFs for 48 h enhanced HA-dependent LUVA binding assessed by quantitative fluorescent microscopy. This coincided with increased HLF HA synthase (HAS) 2 and HAS3 expression and decreased hyaluronidase (HYAL) 2 expression leading to increased HA accumulation in the HLF cell layer and the presence of larger HA fragments. Separately, LUVAs co-cultured with RSV-infected HLFs for 48 h displayed enhanced production of the mast cell proteases, chymase, and tryptase. Pre-treatment with the HA inhibitor 4-methylumbelliferone (4-MU) and neutralizing antibodies to CD44 (HA receptor) decreased mast cell protease expression in co-cultured LUVAs implicating a direct role for HA. TSG-6 expression was increased over the 48-h infection. Inhibition of HLF TSG-6 expression by siRNA knockdown led to decreased LUVA binding suggesting an important role for this hyaladherin for LUVA adhesion in the setting of RSV infection. In summary, RSV infection of HLFs contributes to inflammation via HA-dependent mechanisms that enhance mast cell binding as well as mast cell protease expression via direct interactions with the ECM.

Immune Checkpoint Ligand PD-L1 Is Upregulated in Pulmonary Lymphangioleiomyomatosis.

Pulmonary lymphangioleiomyomatosis (LAM) is a slow-progressing metastatic disease that is driven by mutations in the tumor suppressor tuberous sclerosis complex 1/2 (TSC1/2). Rapamycin inhibits LAM cell proliferation and is the only approved treatment, but it cannot cause the regression of existing lesions and can only stabilize the disease. However, in other cancers, immunotherapies such as checkpoint blockade against PD-1 and its ligand PD-L1 have shown promise in causing tumor regression and even curing some patients. Thus, we asked whether PD-L1 has a role in LAM progression. In vitro, PD-L1 expression in murine Tsc2-null cells is unaffected by mTOR inhibition with torin but can be upregulated by IFN-γ. Using immunohistochemistry and single-cell flow cytometry, we found increased PD-L1 expression both in human lung tissue from patients with LAM and in Tsc2-null lesions in a murine model of LAM. In this model, PD-L1 is highly expressed in the lung by antigen-presenting and stromal cells, and activated T cells expressing PD-1 infiltrate the affected lung. In vivo treatment with anti-PD-1 antibody significantly prolongs mouse survival in the model of LAM. Together, these data demonstrate that PD-1/PD-L1-mediated immunosuppression may occur in LAM, and suggest new opportunities for therapeutic targeting that may provide benefits beyond those of rapamycin.

Inhibition of versican expression by siRNA facilitates tropoelastin synthesis and elastic fiber formation by human SK-LMS-1 leiomyosarcoma smooth muscle cells in vitro and in vivo.

Versican is an extracellular matrix (ECM) molecule that interacts with other ECM components to influence ECM organization, stability, composition, and cell behavior. Versican is known to increase in a number of cancers, but little is known about how versican influences the amount and organization of the ECM components in the tumor microenvironment. In the present study, we modulated versican expression using siRNAs in the human leiomyosarcoma (LMS) smooth muscle cell line SK-LMS-1, and observed the formation of elastin and elastic fibers in vitro and also in vivo in a nude mouse tumor model. Constitutive siRNA-directed knockdown of versican in LMS cells resulted in increased levels of elastin, as shown by immunohistochemical staining of the cells in vitro, and by mRNA and protein analyses. Moreover, versican siRNA LMS cells, when injected into nude mice, generated smaller tumors that had significantly greater immunohistochemical and histochemical staining for elastin when compared to control tumors. Additionally, microarray analyses were used to determine the influence of versican isoform modulation on gene expression profiles, and to identify genes that influence and relate to the process of elastogenesis. cDNA microarray analysis and TaqMan low density array validation identified previously unreported genes associated with downregulation of versican and increased elastogenesis. These results highlight an important role for the proteoglycan versican in regulating the expression and assembly of elastin and the phenotype of LMS cells.

Postnatal Deletion of the Type II Transforming Growth Factor-ß Receptor in Smooth Muscle Cells Causes Severe Aortopathy in Mice.

OBJECTIVE: Prenatal deletion of the type II transforming growth factor-ß (TGF-ß) receptor (TBRII) prevents normal vascular morphogenesis and smooth muscle cell (SMC) differentiation, causing embryonic death. The role of TBRII in adult SMC is less well studied. Clarification of this role has important clinical implications because TBRII deletion should ablate TGF-ß signaling, and blockade of TGF-ß signaling is envisioned as a treatment for human aortopathies. We hypothesized that postnatal loss of SMC TBRII would cause aortopathy. APPROACH AND RESULTS: We generated mice with either of 2 tamoxifen-inducible SMC-specific Cre (SMC-CreER(T2)) alleles and homozygous floxed Tgfbr2 alleles. Mice were injected with tamoxifen, and their aortas examined 4 and 14 weeks later. Both SMC-CreER(T2) alleles efficiently and specifically rearranged a floxed reporter gene and efficiently rearranged a floxed Tgfbr2 allele, resulting in loss of aortic medial TBRII protein. Loss of SMC TBRII caused severe aortopathy, including hemorrhage, ulceration, dissection, dilation, accumulation of macrophage markers, elastolysis, abnormal proteoglycan accumulation, and aberrant SMC gene expression. All areas of the aorta were affected, with the most severe pathology in the ascending aorta. Cre-mediated loss of SMC TBRII in vitro ablated both canonical and noncanonical TGF-ß signaling and reproduced some of the gene expression abnormalities detected in vivo. CONCLUSIONS: SMC TBRII plays a critical role in maintaining postnatal aortic homeostasis. Loss of SMC TBRII disrupts TGF-ß signaling, acutely alters SMC gene expression, and rapidly results in severe and durable aortopathy. These results suggest that pharmacological blockade of TGF-ß signaling in humans could cause aortic disease rather than prevent it.

Expression of versican V3 by arterial smooth muscle cells alters tumor growth factor ß (TGFß)-, epidermal growth factor (EGF)-, and nuclear factor κB (NFκB)-dependent signaling pathways, creating a microenvironment that resists monocyte adhesion.

Monocyte/macrophage accumulation plays a critical role during progression of cardiovascular diseases, such as atherosclerosis. Our previous studies demonstrated that retrovirally mediated expression of the versican V3 splice variant (V3) by arterial smooth muscle cells (ASMCs) decreases monocyte adhesion in vitro and macrophage accumulation in a model of lipid-induced neointimal formation in vivo. We now demonstrate that V3-expressing ASMCs resist monocyte adhesion by altering the composition of the microenvironment surrounding the cells by affecting multiple signaling pathways. Reduction of monocyte adhesion to V3-expressing ASMCs is due to the generation of an extracellular matrix enriched in elastic fibers and depleted in hyaluronan, and reduction of the proinflammatory cell surface vascular cell adhesion molecule 1 (VCAM1). Blocking these changes reverses the protective effect of V3 on monocyte adhesion. The enhanced elastogenesis induced by V3 expression is mediated by TGFß signaling, whereas the reduction in hyaluronan cable formation induced by V3 expression is mediated by the blockade of epidermal growth factor receptor and NFκB activation pathways. In addition, expression of V3 by ASMCs induced a marked decrease in NFκB-responsive proinflammatory cell surface molecules that mediate monocyte adhesion, such as VCAM1. Overall, these results indicate that V3 expression by ASMCs creates a microenvironment resistant to monocyte adhesion via differentially regulating multiple signaling pathways.

Versican and the control of inflammation.

Versican is an extracellular matrix (ECM) proteoglycan that interacts with cells by binding to non-integrin and integrin receptors and to other ECM components that associate with the cell surface. Recent studies have shown also that versican interacts with myeloid and lymphoid cells promoting their adhesion and production of inflammatory cytokines. Versican is produced by stromal cells, as well as leukocytes, and is markedly increased in inflammation. Inflammatory agonists, such as double-stranded RNA mimetics (e.g., poly I:C), stimulate stromal cells, smooth muscle cells and fibroblasts, to produce fibrillar ECMs enriched in versican and hyaluronan (HA) that interact with leukocytes promoting their adhesion. Interference with the incorporation of versican into this ECM blocks monocyte adhesion and dampens the inflammatory response. Tumor cells also express elevated levels of versican which interact with myeloid cells to promote an inflammatory response, through stimulating cytokine release, and metastasis. In addition, myeloid cells, such as macrophages in tumors, synthesize versican which affects tumor cell phenotypes, inflammation, and subsequent metastasis. Versican, by binding to hyaluronan, influences T lymphocyte phenotypes and in part controls the ability of these cells to synthesize and secrete cytokines that influence the immune response. Collectively, these studies indicate that versican as an ECM molecule plays a central role in inflammation and as a result it is emerging as a potential target promising wide therapeutic benefits.

In situ force mapping of mammary gland transformation.

Tumor progression is characterized by an incremental stiffening of the tissue. The importance of tissue rigidity to cancer is appreciated, yet the contribution of specific tissue elements to tumor stiffening and their physiological significance remains unclear. We performed high-resolution atomic force microscopy indentation in live and snap-frozen fluorescently labeled mammary tissues to explore the origin of the tissue stiffening associated with mammary tumor development in PyMT mice. The tumor epithelium, the tumor-associated vasculature and the extracellular matrix all contributed to mammary gland stiffening as it transitioned from normal to invasive carcinoma. Consistent with the concept that extracellular matrix stiffness modifies cell tension, we found that isolated transformed mammary epithelial cells were intrinsically stiffer than their normal counterparts but that the malignant epithelium in situ was far stiffer than isolated breast tumor cells. Moreover, using an in situ vitrification approach, we determined that the extracellular matrix adjacent to the epithelium progressively stiffened as tissue evolved from normal through benign to an invasive state. Importantly, we also noted that there was significant mechanical heterogeneity within the transformed tissue both in the epithelium and the tumor-associated neovasculature. The vascular bed within the tumor core was substantially stiffer than the large patent vessels at the invasive front that are surrounded by the stiffest extracellular matrix. These findings clarify the contribution of individual mammary gland tissue elements to the altered biomechanical landscape of cancerous tissues and emphasize the importance of studying cancer cell evolution under conditions that preserve native interactions.

Neointima formed by arterial smooth muscle cells expressing versican variant V3 is resistant to lipid and macrophage accumulation.

OBJECTIVE: Extracellular matrix (ECM) of neointima formed following angioplasty contains elevated levels of versican, loosely arranged collagen, and fragmented deposits of elastin, features associated with lipid and macrophage accumulation. ECM with a low versican content, compact structure, and increased elastic fiber content can be achieved by expression of versican variant V3, which lacks chondroitin sulfate glycosaminoglycans. We hypothesized that V3-expressing arterial smooth muscle cells (ASMC) can be used to form a neointima resistant to lipid and macrophage accumulation associated with hypercholesterolemia. METHODS AND RESULTS: ASMC transduced with V3 cDNA were seeded into ballooned rabbit carotid arteries, and animals were fed a chow diet for 4 weeks, followed by a cholesterol-enriched diet for 4 weeks, achieving plasma cholesterol levels of 20 to 25 mmol/L. V3 neointimae at 8 weeks were compact, multilayered, and elastin enriched. They were significantly thinner (57%) than control neointimae; contained significantly more elastin (118%), less collagen (22%), and less lipid (76%); and showed significantly reduced macrophage infiltration (85%). Mechanistic studies demonstrated that oxidized low-density lipoprotein stimulated the formation of a monocyte-binding ECM, which was inhibited in the presence of V3 expressing ASMC. CONCLUSION: These results demonstrate that expression of V3 in vessel wall creates an elastin-rich neointimal matrix that in the presence of hyperlipidemia is resistant to lipid deposition and macrophage accumulation.

Effect of neutrophil adhesion on the mechanical properties of lung microvascular endothelial cells.

Neutrophil adhesion to pulmonary microvascular endothelial cells (ECs) initiates intracellular signaling, resulting in remodeling of F-actin cytoskeletal structure of ECs. The present study determined the mechanical properties of ECs and the changes induced by neutrophil adhesion by atomic force microscopy. The elastic moduli of ECs were compared before neutrophils were present, as soon as neutrophil adhesion was detected, and 1 minute later. ECs that were adjacent to those with adherent neutrophils were also evaluated. Neutrophil adhesion induced a decrease in the elastic moduli in the 6.25-µm rim of ECs surrounding adherent neutrophils as soon as firmly adherent neutrophils were detected, which was transient and lasted less than 1 minute. Adjacent ECs developed an increase in stiffness that was significant in the central regions of these cells. Intercellular adhesion molecule-1 crosslinking did not induce significant changes in the elastic modulus of ECs in either region, suggesting that crosslinking intercellular adhesion molecule-1 is not sufficient to induce the observed changes. Our results demonstrate that neutrophil adhesion induces regional changes in the stiffness of ECs.

Changes in the hyperelastic properties of endothelial cells induced by tumor necrosis factor-alpha.

Mechanical properties of living cells can be determined using atomic force microscopy (AFM). In this study, a novel analysis was developed to determine the mechanical properties of adherent monolayers of pulmonary microvascular endothelial cells (ECs) using AFM and finite element modeling, which considers both the finite thickness of ECs and their nonlinear elastic properties, as well as the large strain induced by AFM. Comparison of this model with the more traditional Hertzian model, which assumes linear elastic behavior, small strains, and infinite cell thickness, suggests that the new analysis can predict the mechanical response of ECs during AFM indentation better than Hertz's model, especially when using force-displacement data obtained from large indentations (>100 nm). The shear moduli and distensibility of ECs were greater when using small indentations (<100 nm) compared to large indentations (>100 nm). Tumor necrosis factor-alpha induced changes in the mechanical properties of ECs, which included a decrease in the average shear moduli that occurred in all regions of the ECs and an increase in distensibility in the central regions when measured using small indentations. These changes can be modeled as changes in a chain network structure within the ECs.

Methionine 35 oxidation reduces fibril assembly of the amyloid abeta-(1-42) peptide of Alzheimer's disease.

The major component of amyloid plaques in Alzheimer's disease (AD) is Abeta, a small peptide that has high propensity to assemble as aggregated beta-sheet structures. Using three well established techniques for studying amyloid structure, namely circular dichroism, thioflavin-T fluorescence, and atomic force microscopy, we demonstrate that oxidation of the Met-35 side chain to a methionine sulfoxide (Met-35(ox)) significantly hinders the rate of fibril formation for the 42-residue Abeta-(1-42) at physiological pH. Met-35(ox) also alters the characteristic Abeta fibril morphology and prevents formation of the protofibril, which is a key intermediate in beta-amyloidosis and the associated neurotoxicity. The implications of these results for the biological function and role of Abeta with oxidative stress in AD are discussed.