Pulmonary 5-HT2B receptor expression in fibrotic interstitial lung diseases.

Pulmonary fibrosis is a severe condition in interstitial lung diseases (ILD) such as idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-ILD, where the underlying mechanism is not well defined and with no curative treatments available. Serotonin (5-HT) signaling via the 5-HT2B receptor has been recognized as a promising preclinical target for fibrosis. Despite this, the involvement of the 5-HT2B receptor in fibrotic ILD is widely unexplored. This work highlights the spatial pulmonary distribution of the 5-HT2B receptor in patients with IPF and systemic sclerosis-ILD. We show that the 5-HT2B receptor is located in typical pathological structures e.g. honeycomb cysts and weakly in fibroblast foci. Together with immunohistochemistry and immunofluorescence stainings of patient derived distal lung tissues, we identified cell targets for 5-HT2B receptor interference in type II alveolar epithelial cells, endothelial cells and M2 macrophages. Our results emphasize the role of 5-HT2B receptor as a target in lung fibrosis, warranting further consideration in targeting fibrotic ILDs.

Targeting serotonin receptor 2B inhibits TGFß induced differentiation of human vascular smooth muscle cells.

Vascular Smooth Muscle Cells (VSMCs) are known to be the key drivers of intimal thickening which contribute to early progression of atherosclerosis. VSMCs are the major producers of extracellular matrix within the vessel wall and in response to atherogenic stimuli they could modify the type of matrix proteins produced. Serotonin receptor 2B (5-HT2B receptor/HTR2B) has been implicated in several chronic fibrotic and vascular diseases. Although studies have successfully demonstrated the efficacy of HTR2B blockade in attenuating fibrotic disease, the role of 5-HT2B receptor in TGFß mediated VSMC differentiation remain largely unknown. In the present study, we investigated the potential of targeting the 5-HT2B receptor to prevent TGFß induced VSMCs differentiation. Our results showed that 5-HT2B receptors are expressed in human atherosclerotic lesion and HTR2B expression positively correlated to the VSMCs markers. We show that AM1125, a selective 5-HT2B receptor inhibitor, significantly inhibits TGFß1 induced production of collagen and CTGF. The investigation of underlying mechanisms indicated that 5-HT2B receptor antagonism blocks phospho-Smad2 mediated downstream signaling of TGFß1 in vascular smooth muscle cells. Collectively, the HTR2B/TGF-ß1/Phospho-Smad2 pathway plays a critical role in the regulation of VSMCs differentiation. Our findings might serve 5-HT2B receptor as a therapeutic target to limit TGF-ß1 induced VSMC differentiation.

Pathological Insight into 5-HT2B Receptor Activation in Fibrosing Interstitial Lung Diseases.

Interstitial lung disease (ILD) encompasses a heterogeneous group of more than 200 conditions, of which primarily idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonia, hypersensitivity pneumonitis, ILD associated with autoimmune diseases and sarcoidosis may present a progressive fibrosing (PF) phenotype. Despite different aetiology and histopathological patterns, the PF-ILDs have similarities regarding disease mechanisms with self-sustaining fibrosis, which suggests that the diseases may share common pathogenetic pathways. Previous studies show an enhanced activation of serotonergic signaling in pulmonary fibrosis, and the serotonin (5-HT)2 receptors have been implicated to have important roles in observed profibrotic actions. Our research findings in support by others, demonstrate antifibrotic effects with 5-HT2B receptor antagonists, alleviating several key events common for the fibrotic diseases such as myofibroblast differentiation and connective tissue deposition. In this review, we will address the potential role of 5-HT and in particular the 5-HT2B receptors in three PF-ILDs: ILD associated with systemic sclerosis (SSc-ILD), ILD associated with rheumatoid arthritis (RA-ILD) and IPF. Highlighting the converging pathways in these diseases discloses the 5-HT2B receptor as a potential disease target for PF-ILDs, which today have an urgent unmet need for therapeutic strategies.

Effects of 5-Hydroxytryptamine Class 2 Receptor Antagonists on Bronchoconstriction and Pulmonary Remodeling Processes.

Serotonin [5-hydroxytryptamine (5-HT)] is associated with several chronic pulmonary diseases, recognizing 5-HT2 receptor antagonists as potential inhibitors of tissue remodeling. However, the effects of 5-HT2 receptors, especially 5-HT2B receptors on airway function and remodeling, are unclear. We investigated the role of 5-HT2B receptors on airway smooth muscle contractility and remodeling processes. Murine precision-cut lung slices were pretreated with 5-HT2B receptor antagonists (EXT5, EXT9, RS 127445, and PRX 08066), as well as ketanserin (5-HT2A/2C receptor antagonist) (1, 10 µmol/L), before addition of cumulative concentrations of 5-HT to induce bronchoconstriction. Remodeling effects after treatment with 10 µmol/L 5-HT and 5-HT2 receptor antagonists were further studied in distal lung tissue by examining release of profibrotic transforming growth factor (TGF)-ß1 and proliferation of human bronchial smooth muscle cells (HBSMCs). 5-HT-induced bronchoconstriction was significantly reduced by EXT5, EXT9, and ketanserin, but not by RS 127445 or PRX 08066. The 5-HT2B receptor antagonists significantly reduced TGF-ß1 release. 5-HT, in combination with TGF-ß1, increased proliferation of HBSMCs, a process reduced by EXT5 and EXT9. Our results indicate that EXT5 and EXT9 may relieve bronchoconstriction in murine airways and serve as an add-on effect in attenuating pulmonary remodeling by improving airway function. The antiproliferative effect on HBSMCs and the inhibition of TGF-ß1 release further support a role of 5-HT2B receptors in pathologic remodeling processes.

Pulmonary fibrosis in vivo displays increased p21 expression reduced by 5-HT2B receptor antagonists in vitro - a potential pathway affecting proliferation.

Serotonin (5-hydroxytryptamine) has repeatedly been associated with the development of fibrotic disorders such as pulmonary fibrosis. By blocking the binding of 5-HT to 5-HT2B receptors with receptor antagonists, several pro-fibrotic mechanisms can be inhibited. Bleomycin-induced pulmonary fibrosis is a model used to evaluate pathological mechanisms and pharmacological interventions. Previously we have shown attenuated fibrosis in systemic bleomycin-treated mice following treatment with two 5-HT2B receptor antagonists (EXT5 and EXT9). Our aim is to further identify cellular effects and signaling pathways associated with the anti-fibrotic effects of EXT5/9. Gene expressions in lung tissues from systemic bleomycin-treated mice were examined, revealing significant increased expression of Cdkn1α (a gene coding for p21), particularly in distal regions of the lung. In vitro studies in human lung fibroblasts revealed increased levels of p21 (p = 0.0032) and pAkt (p = 0.12) following treatment with 5-HT (10 µM). The induction of p21 and pAkt appears to be regulated by 5-HT2B receptors, with diminished protein levels following EXT9-treatment (p21 p = 0.0024, pAkt p = 0.15). Additionally, 5-HT induced fibroblast proliferation, an event significantly reduced by EXT5 (10 µM) and EXT9 (10 µM). In conclusion, our results suggest that 5-HT2B receptor antagonism attenuates pulmonary fibrosis in part by anti-proliferative effects, associated with inhibited pAkt/p21 signaling pathway.

5-HT2B receptor antagonists attenuate myofibroblast differentiation and subsequent fibrotic responses in vitro and in vivo.

Pulmonary fibrosis is characterized by excessive accumulation of connective tissue, along with activated extracellular matrix (ECM)-producing cells, myofibroblasts. The pathological mechanisms are not well known, however serotonin (5-HT) and 5-HT class 2 (5-HT2) receptors have been associated with fibrosis. The aim of the present study was to investigate the role of 5-HT2B receptors in fibrosis, using small molecular 5-HT2B receptor antagonists EXT5 and EXT9, with slightly different receptor affinity. Myofibroblast differentiation [production of alpha-smooth muscle actin (α-SMA)] and ECM synthesis were quantified in vitro, and the effects of the receptor antagonists were evaluated. Pulmonary fibrosis was also modeled in mice by subcutaneous bleomycin administrations (under light isoflurane anesthesia), and the effects of receptor antagonists on tissue density, collagen-producing cells, myofibroblasts and decorin expression were investigated. In addition, cytokine expression was analyzed in serum. Lung fibroblasts displayed an increased α-SMA (P < 0.05) and total proteoglycan production (P < 0.01) when cultured with TGF-ß1 together with 5-HT, which were significantly reduced with both receptor antagonists. Following treatment with EXT5 or EXT9, tissue density, expression of decorin, number of collagen-producing cells, and myofibroblasts were significantly decreased in vivo compared to bleomycin-treated mice. Receptor antagonization also significantly reduced systemic levels of TNF-α and IL-1ß, indicating a role in systemic inflammation. In conclusion, 5-HT2B receptor antagonists have potential to prevent myofibroblast differentiation, in vitro and in vivo, with subsequent effect on matrix deposition. The attenuating effects of 5-HT2B receptor antagonists on fibrotic tissue remodeling suggest these receptors as novel targets for the treatment of pulmonary fibrosis.

Type I interferon-mediated skewing of the serotonin synthesis is associated with severe disease in systemic lupus erythematosus.

Serotonin, a highly pro-inflammatory molecule released by activated platelets, is formed by tryptophan. Tryptophan is also needed in the production of kynurenine, a process mediated by the type I interferon (IFN)-regulated rate-limiting enzyme indoleamine 2,3-dioxygenase (IDO). The aim of this study was to investigate levels of serotonin in patients with the autoimmune disease systemic lupus erythematosus (SLE), association to clinical phenotype and possible involvement of IDO in regulation of serotonin synthesis. Serotonin levels were measured in serum and plasma from patients with SLE (n=148) and healthy volunteers (n=79) by liquid chromatography and ELISA, as well as intracellularly in platelets by flow cytometry. We found that SLE patients had decreased serotonin levels in serum (p=0.01) and platelets (p<0.0001) as compared to healthy individuals. SLE patients with ongoing type I IFN activity, as determined by an in-house reporter assay, had decreased serum levels of serotonin (p=0.0008) as well as increased IDO activity (p<0.0001), as determined by the kynurenine/tryptophan ratio measured by liquid chromatography. Furthermore, SLE sera induced IDO expression in WISH cells in a type I IFN-dependent manner (p=0.008). Also platelet activation contributed to reduce overall availability of serotonin levels in platelets and serum (p<0.05). Decreased serum serotonin levels were associated with severe SLE with presence of anti-dsDNA antibodies and nephritis. In all, reduced serum serotonin levels in SLE patients were related to severe disease phenotype, including nephritis, suggesting involvement of important immunopathological processes. Further, our data suggest that type I IFNs, present in SLE sera, are able to up-regulate IDO expression, which may lead to decreased serum serotonin levels.

The skeletal phenotype of chondroadherin deficient mice.

Chondroadherin, a leucine rich repeat extracellular matrix protein with functions in cell to matrix interactions, binds cells via their α2ß1 integrin as well as via cell surface proteoglycans, providing for different sets of signals to the cell. Additionally, the protein acts as an anchor to the matrix by binding tightly to collagens type I and II as well as type VI. We generated mice with inactivated chondroadherin gene to provide integrated studies of the role of the protein. The null mice presented distinct phenotypes with affected cartilage as well as bone. At 3-6 weeks of age the epiphyseal growth plate was widened most pronounced in the proliferative zone. The proteome of the femoral head articular cartilage at 4 months of age showed some distinct differences, with increased deposition of cartilage intermediate layer protein 1 and fibronectin in the chondroadherin deficient mice, more pronounced in the female. Other proteins show decreased levels in the deficient mice, particularly pronounced for matrilin-1, thrombospondin-1 and notably the members of the α1-antitrypsin family of proteinase inhibitors as well as for a member of the bone morphogenetic protein growth factor family. Thus, cartilage homeostasis is distinctly altered. The bone phenotype was expressed in several ways. The number of bone sialoprotein mRNA expressing cells in the proximal tibial metaphysic was decreased and the osteoid surface was increased possibly indicating a change in mineral metabolism. Micro-CT revealed lower cortical thickness and increased structure model index, i.e. the amount of plates and rods composing the bone trabeculas. The structural changes were paralleled by loss of function, where the null mice showed lower femoral neck failure load and tibial strength during mechanical testing at 4 months of age. The skeletal phenotype points at a role for chondroadherin in both bone and cartilage homeostasis, however, without leading to altered longitudinal growth.

Identification and characterization of the integrin alpha2beta1 binding motif in chondroadherin mediating cell attachment.

Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin α(2)ß(1) and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same α(2)ß(1) receptor. We identified a cell binding motif, CQLRGLRRWLEAK(318) by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK(318) remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the α(2)ß(1) integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC(326), mimicking the structural constraints of this sequence in the intact protein, showed similar efficiency in inhibiting binding to chondroadherin. The unique peptide motif responsible for cellular binding is primarily located in the octamer sequence LRRWLEAK(318). Binding of cells to the active peptide or to chondroadherin immobilized on cell culture plates rapidly induces intracellular signaling (i.e. ERK phosphorylation). Thus, chondroadherin interaction with cells may be central for maintaining the adult chondrocyte phenotype and cartilage homeostasis. The peptides, particularly the more stable cyclic peptide, open new opportunities to modulate cell behavior in situations of tissue pathology.

Purification, crystallization and preliminary X-ray diffraction analysis of human chondroadherin.

Chondroadherin is a cartilage matrix protein that is known to mediate the adhesion of isolated chondrocytes. Its protein core is composed of 11 leucine-rich repeats flanked by cysteine-rich domains at the N- and C-terminal ends. Recombinant human chondroadherin was crystallized using the sitting-drop vapour-diffusion method. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 56.4, b = 111.3, c = 128.5 A, beta = 92.2, and are most likely to contain four molecules in the asymmetric unit. The crystals diffracted to at least 2.3 A using synchrotron radiation, but structure determination using molecular replacement has so far been unsuccessful.

Biglycan organizes collagen VI into hexagonal-like networks resembling tissue structures.

The ability of the leucine-rich repeat (LRR) proteins biglycan, decorin, and chondroadherin to interact with collagen VI and influence its assembly to supramolecular structures was studied by electron microscopy and surface plasmon resonance measurements in the BIAcore 2000 system. Biglycan showed a unique ability to organize collagen VI into extensive hexagonal-like networks over a time period of only a few minutes. Only the intact molecule, substituted with two dermatan sulfate chains, had this capacity. Intact decorin, with one dermatan sulfate chain only, was considerably less efficient, and aggregates of organized collagen VI were found only after several hours. Chondroadherin without glycosaminoglycan substitutions did not induce any ordered collagen VI organization. However, all three related LRR proteins were shown to interact with collagen VI using electron microscopy and surface plasmon resonance. Biglycan and decorin were exclusively found close to the N-terminal parts of the collagen VI tetramers, whereas chondroadherin was shown to bind close to both the N- and C-terminal parts of collagen VI. In the formed hexagonal networks, biglycan was localized to the intra-network junctions of the collagen VI filaments. This was demonstrated by electron microscopy after negative staining of gold-labeled biglycan in aggregation experiments with collagen VI.