Blockage of the adenosine A2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension.

Sustained pressure overload and fibrosis of the right ventricle (RV) are the leading causes of mortality in pulmonary arterial hypertension (PAH). Although the role of adenosine in PAH has been attributed to the control of pulmonary vascular tone, cardiac reserve, and inflammatory processes, the involvement of the nucleoside in RV remodelling remains poorly understood. Conflicting results exist on targeting the low-affinity adenosine A2B receptor (A2BAR) for the treatment of PAH mostly because it displays dual roles in acute vs. chronic lung diseases. Herein, we investigated the role of the A2BAR in the viability/proliferation and collagen production by cardiac fibroblasts (CFs) isolated from RVs of rats with monocrotaline (MCT)-induced PAH. CFs from MCT-treated rats display higher cell viability/proliferation capacity and overexpress A2BAR compared to the cells from healthy littermates. The enzymatically stable adenosine analogue, 5'-N-ethylcarboxamidoadenosine (NECA, 1-30 µM), concentration-dependently increased growth, and type I collagen production by CFs originated from control and PAH rats, but its effects were more prominent in cells from rats with PAH. Blockage of the A2BAR with PSB603 (100 nM), but not of the A2AAR with SCH442416 (100 nM), attenuated the proliferative effect of NECA in CFs from PAH rats. The A2AAR agonist, CGS21680 (3 and 10 nM), was virtually devoid of effect. Overall, data suggest that adenosine signalling via A2BAR may contribute to RV overgrowth secondary to PAH. Therefore, blockage of the A2AAR may be a valuable therapeutic alternative to mitigate cardiac remodelling and prevent right heart failure in PAH patients.

The Systemic Immune Response to Collagen-Induced Arthritis and the Impact of Bone Injury in Inflammatory Conditions.

Rheumatoid arthritis (RA) is a systemic disease that affects the osteoarticular system, associated with bone fragility and increased risk of fractures. Herein, we aimed to characterize the systemic impact of the rat collagen-induced arthritis (CIA) model and explore its combination with femoral bone defect (FD). The impact of CIA on endogenous mesenchymal stem/stromal cells (MSC) was also investigated. CIA induction led to enlarged, more proliferative, spleen and draining lymph nodes, with altered proportion of lymphoid populations. Upon FD, CIA animals increased the systemic myeloid cell proportions, and their expression of co-stimulatory molecules CD40 and CD86. Screening plasma cytokine/chemokine levels showed increased tumor necrosis factor-α (TNF-α), Interleukin (IL)-17, IL-4, IL-5, and IL-12 in CIA, and IL-2 and IL-6 increased in CIA and CIA+FD, while Fractalkine and Leptin were decreased in both groups. CIA-derived MSC showed lower metabolic activity and proliferation, and significantly increased osteogenic and chondrogenic differentiation markers. Exposure of control-MSC to TNF-α partially mimicked the CIA-MSC phenotype in vitro. In conclusion, inflammatory conditions of CIA led to alterations in systemic immune cell proportions, circulating mediators, and in endogenous MSC. CIA animals respond to FD, and the combined model can be used to study the mechanisms of bone repair in inflammatory conditions.

A bioinspired multifunctional hydrogel patch targeting inflammation and regeneration in chronic intestinal wounds.

Healing of intestinal chronic wounds remains a major challenge as current therapies are ineffective in promoting proper regeneration of the damaged intestinal wall. An innovative concept, based on a bioinspired multifunctional alginate-melanin hybrid 3D scaffold, to target both inflammatory and regenerative processes, is proposed herein. Hydrogel-entrapped melanin nanoparticles demonstrated free-radical scavenging activity, supported by the neutralization of free-radicals in solution (90%), and the in vitro capture of reactive oxygen species (ROS) produced by stimulated macrophages in an inflammatory-mimicking environment. Notably, scaffolds could be reused (at least 3 times), while maintaining these properties. The extracellular matrix (ECM)-inspired biomaterial, containing protease-sensitive and integrin-binding domains, exhibited remarkable ability for cell colonisation. Human intestinal fibroblasts and epithelial cells (Caco-2) co-seeded on lyophilized scaffolds were able to invade/colonize the construct and produce endogenous ECM, key for neo-tissue formation and re-epithelialization. Scaffolds presented tuneable mechanical properties and could be used both in hydrated and freeze-dried states, maintaining their performance upon rehydration, which are attractive features for clinical application. Collectively, our results highlight the potential of biofunctionalized alginate-melanin hybrid 3D scaffolds as multi-therapeutic patches for modulating inflammation and tissue regeneration in chronic intestinal wounds, which address a major but still unmet clinical need. The proposed multi-therapeutic strategy may potentially be extended to the treatment of other chronic wounds.

Osteoclasts degrade fibrinogen scaffolds and induce mesenchymal stem/stromal osteogenic differentiation.

Fibrinogen (Fg) is a pro-inflammatory protein with pro-healing properties. Previous work showed that fibrinogen 3D scaffolds (Fg-3D) promote bone regeneration, but the cellular players were not identified. Osteoclasts are bone resorbing cells that promote bone remodeling in close crosstalk with osteoblasts. Herein, the capacity of osteoclasts differentiated on Fg-3D to degrade the scaffolds and promote osteoblast differentiation was evaluated in vitro. Fg-3D scaffolds were prepared by freeze-drying and osteoclasts were differentiated from primary human peripheral blood monocytes. Results obtained showed osteoclasts expressing the enzymes cathepsin K and tartrate resistant acid phosphatase colonizing Fg-3D scaffolds. Osteoclasts were able to significantly degrade Fg-3D, reducing the scaffold's area, and increasing D-dimer concentration, a Fg degradation product, in their culture media. Osteoclast conditioned media from the first week of differentiation promoted significantly stronger human primary mesenchymal stem/stromal cell (MSC) osteogenic differentiation, evaluated by alkaline phosphatase activity. Moreover, week 1 osteoclast conditioned media promoted earlier MSC osteogenic differentiation, than chemical osteogenesis inductors. TGF-ß1 was found increased in osteoclast conditioned media from week 1, when compared to week 3 of differentiation. Taken together, our results suggest that osteoclasts are able to differentiate and degrade Fg-3D, producing factors like TGF-ß1 that promote MSC osteogenic differentiation.

Fibrinogen and magnesium combination biomaterials modulate macrophage phenotype, NF-kB signaling and crosstalk with mesenchymal stem/stromal cells.

Macrophage behavior upon biomaterial implantation conditions the inflammatory response and subsequent tissue repair. The hypothesis behind this work was that fibrinogen (Fg) and magnesium (Mg) biomaterials, used in combination (FgMg) could act synergistically to modulate macrophage activation, promoting a pro-regenerative phenotype. Materials were characterized by scanning electron microscopy, Fg and Mg degradation products were quantified by atomic absorption spectroscopy and ELISA. Whole blood immune cells and primary human monocyte-derived macrophages were exposed to the biomaterials extracts in unstimulated (M0) or pro-inflammatory LPS or LPS-IFNγ (M1) conditions. Macrophage phenotype was evaluated by flow cytometry, cytokines secreted by whole blood cells and macrophages were measured by ELISA, and signaling pathways were probed by Western blotting. The secretomes of macrophages preconditioned with biomaterials extracts were incubated with human mesenchymal stem/stromal cells (MSC) and their effect on osteogenic differentiation was evaluated via Alkaline Phosphatase (ALP) activity and alizarin red staining. Scaffolds of Fg, alone or in the FgMg combination, presented similar 3D porous architectures. Extracts from FgMg materials reduced LPS-induced TNF-α secretion by innate immune cells, and macrophage M1 polarization upon LPS-IFNγ stimulation, resulting in lower cell surface CD86 expression, lower NFκB p65 phosphorylation and reduced TNF-α secretion. Moreover, while biomaterial extracts per se did not enhance MSC osteogenic differentiation, macrophage secretome, particularly from cells exposed to FgMg extracts, increased MSC ALP activity and alizarin red staining, compared with extracts alone. These findings suggest that the combination of Fg and Mg synergistically influences macrophage pro-inflammatory activation and crosstalk with MSC. STATEMENT OF SIGNIFICANCE: Modulating macrophage phenotype by degradable and bioactive biomaterials is an increasingly explored strategy to promote tissue repair/regeneration. Fibrinogen (Fg) and magnesium (Mg)-based materials have been explored in this context. Previous work from our group showed that monocytes interact with fibrinogen adsorbed onto chitosan surfaces through TLR4 and that fibrinogen scaffolds promote in vivo bone regeneration. Also, magnesium ions have been reported to modulate macrophage pro-inflammatory M1 stimulation and to promote bone repair. Here we report, for the first time, the combination of Fg and Mg materials, hypothesizing that it could act synergistically on macrophages, directing them towards a pro-regenerative phenotype. As a first step towards proving/disproving our hypothesis we used extracts obtained from Fg, Mg and FgMg multilayer constructs. We observed that FgMg extracts led to a reduction in the polarization of macrophages towards a pro-inflammatory phenotype. Also, the secretome of macrophages exposed to extracts of the combination material promoted the expression of osteogenic markers by MSCs.

Macrophages Down-Regulate Gene Expression of Intervertebral Disc Degenerative Markers Under a Pro-inflammatory Microenvironment.

Low back pain is a highly prevalent clinical problem and intervertebral disc (IVD) degeneration is now accepted as the major pathophysiological mechanism responsible for this condition. Accumulating evidence suggests that inflammation plays a crucial role in the progression of human IVD degeneration, with macrophages being pointed as the key immune cell players in this process since their infiltration in degenerated IVD samples has been extensively demonstrated. Since they are highly plastic, macrophages can play different roles depending on the microenvironmental cues. The study of inflammation associated with IVD degeneration has been somehow neglected and one of the reasons is related with lack of adequate models. To overcome this, we established and characterized a new model of IVD organ culture under pro-inflammatory conditions to further dissect the role of macrophages in IVD associated immune response. For that, human monocyte-derived macrophages were co-cultured either with bovine caudal IVD punches in the presence of the pro-inflammatory cytokine IL-1ß, or IVD-conditioned medium (CM), to investigate how IVD-produced factors influence macrophage phenotype. After 72 h, metabolic activity, gene expression and cytokine profile of macrophages and IVD cells were measured. Our results show that macrophages and IVDs remain metabolically active in the presence of IL-1ß, significantly upregulate CCR7 gene expression and increase production of IL-6 on macrophages. When treating macrophages with IL-1ß-IVD-CM, CCR7 upregulation follows the same trend, while for IL-6 an opposite effect was observed. On the other hand, macrophages interfere with IVD ECM remodeling, decreasing MMP3 expression and downregulating aggrecan and collagen II gene expression in the presence of IL-1ß. Overall, the co-culture model established in this study can be considered a suitable approach to address the cellular and molecular pathways that regulate macrophage-IVD crosstalk, suggesting that degenerated IVD tissue tends to polarize human macrophages toward a more pro-inflammatory profile, which seems to aggravate IVD degeneration. This model could be used to improve the knowledge of the mechanisms that link IVD degeneration and the immune response.

Is the adenosine A2B 'biased' receptor a valuable target for the treatment of pulmonary arterial hypertension?

Pulmonary arterial hypertension (PAH) is a maladaptive disorder characterized by increased pulmonary vascular resistance leading to right ventricular failure and death. Adenosine released by injured tissues, such as the lung and heart, influences tissue remodeling through the activation of adenosine receptors. Evidence regarding activation of the low-affinity A2BAR by adenosine points towards pivotal roles of this receptor in processes associated with both acute and chronic lung diseases. Conflicting results exist concerning the beneficial or detrimental roles of the A2B 'biased' receptor in right ventricular failure secondary to PAH. In this review, we discuss the pros and cons of manipulating A2BARs as a putative therapeutic target in PAH.