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Research over the past few years has provided fascinating results indicating that biglycan, besides being a ubiquitous structural component of the extracellular matrix (ECM), may act as a signaling molecule. Proteolytically released from the ECM, biglycan acts as a danger signal signifying tissue stress or injury. As a ligand of innate immunity receptors and activator of the inflammasome, biglycan stimulates multifunctional proinflammatory signaling linking the innate to the adaptive immune response. By clustering several types of receptors on the cell surface and orchestrating their downstream signaling events, biglycan is capable to autonomously trigger sterile inflammation and to potentiate the inflammatory response to microbial invasion. Besides operating in a broad biological context, biglycan also displays tissue-specific affinities to certain receptors and structural components, thereby playing a crucial role in bone formation, muscle integrity, and synapse stability at the neuromuscular junction. This review attempts to provide a concise summary of recent data regarding the involvement of biglycan in the regulation of inflammation and the musculoskeletal system, pointing out both a signaling and a structural role for this proteoglycan. The potential of biglycan as a novel therapeutic target or agent for the treatment of inflammatory diseases and skeletal muscular dystrophies is also addressed.
In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif) ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs) and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1) in a TLR4- and Toll/interleukin (IL)-1R domain-containing adaptor inducing interferon (IFN)-β (TRIF)-dependent manner in murine primary macrophages. We provide genetic and pharmacological proof that SphK1 is a crucial downstream mediator of biglycan-triggered CCL2 and CCL5 mRNA and protein expression. This is selectively driven by biglycan/SphK1-dependent phosphorylation of the nuclear factor NF-κB p65 subunit, extracellular signal-regulated kinase (Erk)1/2 and p38 mitogen-activated protein kinases. Importantly, in vivo overexpression of soluble biglycan causes Sphk1-dependent enhancement of renal CCL2 and CCL5 and macrophage recruitment into the kidney. Our findings describe the crosstalk between biglycan- and SphK1-driven extracellular matrix- and lipid-signaling. Thus, SphK1 may represent a new target for therapeutic intervention in biglycan-evoked inflammatory conditions.
Activation of the tumor-associated stroma to support tumor growth is a common feature observed in different cancer entities. This principle is exemplified by cancer-associated fibroblasts (CAFs), which are educated by the tumor to shape its development across all stages. CAFs can alter the extracellular matrix (ECM) and secrete a variety of different molecules. In that manner they have the capability to affect activation, survival, proliferation, and migration of other stromal cells and cancer cell themselves. Alteration of the ECM, desmoplasia, is a common feature of breast cancer, indicating a prominent role for CAFs in shaping tumor development in the mammary gland. In this review, we summarize the multiple roles CAFs play in mammary carcinoma. We discuss experimental and clinical strategies to interfere with CAFs function in breast cancer. Moreover, we highlight the issues arising from CAFs heterogeneity and the need for further research to identify CAFs subpopulation(s) that can be targeted to improve breast cancer therapy.
Inflammation is a highly regulated biological response of the immune system that is triggered by assaulting pathogens or endogenous alarmins. It is now well established that some soluble extracellular matrix constituents, such as small leucine-rich proteoglycans (SLRPs), can act as danger signals and trigger aseptic inflammation by interacting with innate immune receptors. SLRP inflammatory signaling cascade goes far beyond its canonical function. By choosing specific innate immune receptors, coreceptors, and adaptor molecules, SLRPs promote a switch between pro- and anti-inflammatory signaling, thereby determining disease resolution or chronification. Moreover, by orchestrating signaling through various receptors, SLRPs fine-tune inflammation and, despite their structural homology, regulate inflammatory processes in a molecule-specific manner. Hence, the overarching theme of this review is to highlight the molecular and functional specificity of biglycan-, decorin-, lumican-, and fibromodulin-mediated signaling in inflammatory and autoimmune diseases.
Osteoarthritis (OA) is a slow-progressing joint disease, leading to the degradation and remodeling of the cartilage extracellular matrix (ECM). The usually quiescent chondrocytes become reactivated and accumulate in cell clusters, become hypertrophic, and intensively produce not only degrading enzymes, but also ECM proteins, like the cartilage oligomeric matrix protein (COMP) and thrombospondin-4 (TSP-4). To date, the functional roles of these newly synthesized proteins in articular cartilage are still elusive. Therefore, we analyzed the involvement of both proteins in OA specific processes in in vitro studies, using porcine chondrocytes, isolated from femoral condyles. The effect of COMP and TSP-4 on chondrocyte migration was investigated in transwell assays and their potential to modulate the chondrocyte phenotype, protein synthesis and matrix formation by immunofluorescence staining and immunoblot. Our results demonstrate that COMP could attract chondrocytes and may contribute to a repopulation of damaged cartilage areas, while TSP-4 did not affect this process. In contrast, both proteins similarly promoted the synthesis and matrix formation of collagen II, IX, XII and proteoglycans, but inhibited that of collagen I and X, resulting in a stabilized chondrocyte phenotype. These data suggest that COMP and TSP-4 activate mechanisms to protect and repair the ECM in articular cartilage.
The manifestation of chronic back pain depends on structural, psychosocial, occupational and genetic influences. Heritability estimates for back pain range from 30% to 45%. Genetic influences are caused by genes affecting intervertebral disc degeneration or the immune response and genes involved in pain perception, signalling and psychological processing. This inter-individual variability which is partly due to genetic differences would require an individualized pain management to prevent the transition from acute to chronic back pain or improve the outcome. The genetic profile may help to define patients at high risk for chronic pain. We summarize genetic factors that (i) impact on intervertebral disc stability, namely Collagen IX, COL9A3, COL11A1, COL11A2, COL1A1, aggrecan (AGAN), cartilage intermediate layer protein, vitamin D receptor, metalloproteinsase-3 (MMP3), MMP9, and thrombospondin-2, (ii) modify inflammation, namely interleukin-1 (IL-1) locus genes and IL-6 and (iii) and pain signalling namely guanine triphosphate (GTP) cyclohydrolase 1, catechol-O-methyltransferase, μ opioid receptor (OPMR1), melanocortin 1 receptor (MC1R), transient receptor potential channel A1 and fatty acid amide hydrolase and analgesic drug metabolism (cytochrome P450 [CYP]2D6, CYP2C9).
The human blood–brain barrier (BBB) represents the interface of microvasculature and the central nervous system, regulating the transport of nutrients and protecting the brain from external threats. To gain a deeper understanding of (patho)physiological processes affecting the BBB, sophisticated models mimicking the in vivo situation are required. Currently, most in vitro models are cultivated on stiff, semipermeable, and non-biodegradable Transwell® membrane inserts, not adequately mimicking the complexity of the extracellular environment of the native human BBB. To overcome these disadvantages, we developed three-dimensional electrospun scaffolds resembling the natural structure of the human extracellular matrix. The polymer fibers of the scaffold imitate collagen fibrils of the human basement membrane, exhibiting excellent wettability and biomechanical properties, thus facilitating cell adhesion, proliferation, and migration. Cultivation of human induced pluripotent stem cells (hiPSCs) on these scaffolds enabled the development of a physiological BBB phenotype monitored via the formation of tight junctions and validated by the paracellular permeability of sodium fluorescein, further accentuating the non-linearity of TEER and barrier permeability. The novel in vitro model of the BBB forms a tight endothelial barrier, offering a platform to study barrier functions in a (patho)physiologically relevant context.
Pretubulysin (PT), a biosynthetic precursor of the myxobacterial compound tubulysin D, was recently identified as a novel microtubule-targeting agent (MTA) causing microtubule destabilization. MTAs are the most frequently used chemotherapeutic drugs. They are well studied regarding their direct cytotoxic effects against various tumors as well as for their anti-angiogenic and vascular-disrupting action addressing endothelial cells of the tumor vasculature. However, the impact of MTAs on endothelial cells of the non-tumor vasculature has been largely neglected, although tumor cell interactions with the healthy endothelium play a crucial role in the process of cancer metastasis. Besides their use as potent anti-cancer drugs, some MTAs such as colchicine are traditionally used or recommended for the therapy of inflammatory diseases. Here, too, the role of endothelial cells has been largely neglected, although the endothelium is crucially involved in regulating the process of inflammation.
In the present study, the impact of PT on tumor-endothelial cell interactions was therefore analyzed in vitro to gain insights into the mechanism underlying its anti-metastatic effect that was recently confirmed in vivo. In the second part of this work, the influence of PT and other MTAs, namely the microtubule-destabilizing compounds vincristine (VIN) and colchicine (COL) and the microtubule-stabilizing drug paclitaxel (PAC), on leukocyte-endothelial cell interactions was investigated in vitro and in vivo (only PT). It is important to mention that in all in vitro experiments solely endothelial cells and not tumor cells or leukocytes were treated with the MTAs to strictly focus on the role of the endothelium in the action of these compounds.
The impact of PT on tumor-endothelial cell interactions was analyzed in vitro by cell adhesion and transendothelial migration assays as well as immunocytochemistry using the breast cancer cell line MDA-MB-231 and primary human umbilical vein endothelial cells (HUVECs). The treatment of HUVECs with PT increased the adhesion of MDA cells onto the endothelial monolayer, whereas their transendothelial migration was reduced by the compound. Thereafter, the influence of PT on the endothelial cell adhesion molecules (CAMs) E-selectin, N-cadherin, ICAM-1, VCAM-1 and galectin-3 and on the CXCL12/CXCR4 chemokine system was examined, since they might be involved in the PT-triggered tumor cell adhesion. Interestingly, although PT induced the upregulation of ICAM-1, VCAM-1, N-cadherin and CXCL12, cell adhesion assays using neutralizing antibodies or the CXCL12 inhibitor AMD3100 revealed that all these molecules were dispensable for the PT-evoked tumor cell adhesion. As PT induces the formation of interendothelial gaps and MDA cells might adhere onto components of the underlying extracellular matrix (ECM), the precise location of MDA cells attached to the PT-treated endothelial monolayer was investigated. Instead of a direct interaction between tumor and endothelial cells, this work showed that MDA cells preferred to adhere to the ECM component collagen that was exposed within PT-triggered endothelial gaps. Both the PT-evoked increase in tumor cell adhesion onto and the decrease in trans-endothelial migration were completely abolished when β1-integrins were blocked on MDA cells. Similar results were obtained when endothelial cells were treated with VIN and COL but not PAC, indicating that the observed effects of PT depend on its microtubule-destabilizing activity.
The impact of PT, VIN, COL and PAC on leukocyte-endothelial cell interactions was analyzed in vivo (only PT) by intravital microscopy of the mouse cremaster muscle and in vitro by cell adhesion assays using the monocyte-like cell line THP-1 and TNFα-activated human dermal microvascular endothelial cells (HMEC-1). While PT did not affect the rolling of leukocytes on the endothelium, their firm adhesion onto and transmigration through the activated endothelium was reduced by PT in vivo. In accordance, the treatment of HMEC-1 with PT, VIN and COL decreased the TNFα-induced adhesion of THP-1 cells onto the endothelial monolayer, whereas PAC had no influence on this process. Thereafter, the influence of PT, VIN, COL and PAC on endothelial ICAM-1 and VCAM-1 was examined, since these molecules are substantially involved in the firm adhesion of leukocytes onto the endothelium. The cell surface protein expression of ICAM-1 and VCAM-1 was reduced by PT, VIN and COL in activated endothelial cells, whereas PAC did only slightly affect the TNFα-induced upregulation of VCAM-1. As the pro-inflammatory transcription factor NFκB plays a crucial role in the TNFα-induced expression of these CAMs, the impact of the MTAs on the NFκB promotor activity was investigated. While PT, VIN and COL decreased the activation of NFκB in activated endothelial cells, PAC did not affect this process. However, in contrast to the strong effects regarding the cell surface protein expression of ICAM-1 and VCAM-1, the effects of PT, VIN and COL on the NFκB activity was rather low. Thus, the used MTAs might also affect other relevant signaling pathways and/or the intracellular transport of CAMs might be influenced by the impact of the MTAs on the microtubule network.
Taken together, the current study provides – at least in part – an explanation for the anti-metastatic potential of PT and gives first insights into the use of PT and VIN as anti-inflammatory drugs. Moreover, this work highlights the endothelium as an attractive target for the development of new anti-cancer and anti-inflammatory drugs.
Patients with type 2 diabetes (T2D) are threatened by excessive cardiovascular morbidity and mortality. While accelerated arterial stiffening may represent a critical mechanistic factor driving cardiovascular risk in T2D, specific therapies to contain the underlying diabetic arterial remodeling have been elusive. The present translational study investigates the role of microRNA-29b (miR-29b) as a driver and therapeutic target of diabetic aortic remodeling and stiffening. Using a murine model (db/db mice), as well as human aortic tissue samples, we find that diabetic aortic remodeling and stiffening is associated with medial fibrosis, as well as fragmentation of aortic elastic layers. miR-29b is significantly downregulated in T2D and miR-29b repression is sufficient to induce both aortic medial fibrosis and elastin breakdown through upregulation of its direct target genes COL1A1 and MMP2 thereby increasing aortic stiffness. Moreover, antioxidant treatment restores aortic miR-29b levels and counteracts diabetic aortic remodeling. Concluding, we identify miR-29b as a comprehensive—and therefore powerful—regulator of aortic remodeling and stiffening in T2D that moreover qualifies as a (redox-sensitive) target for therapeutic intervention.