Impact of Suramin on Key Pathological Features of Sporadic Alzheimer's Disease-Derived Forebrain Neurons.

Background: Alzheimer's disease (AD) is characterized by disrupted proteostasis and macroautophagy (hereafter "autophagy"). The pharmacological agent suramin has known autophagy modulation properties with potential efficacy in mitigating AD neuronal pathology. Objective: In the present work, we investigate the impact of forebrain neuron exposure to suramin on the Akt/mTOR signaling pathway, a major regulator of autophagy, in comparison with rapamycin and chloroquine. We further investigate the effect of suramin on several AD-related biomarkers in sporadic AD (sAD)-derived forebrain neurons. Methods: Neurons differentiated from ReNcell neural progenitors were used to assess the impact of suramin on the Akt/mTOR signaling pathway relative to the autophagy inducer rapamycin and autophagy inhibitor chloroquine. Mature forebrain neurons were differentiated from induced pluripotent stem cells (iPSCs) sourced from a late-onset sAD patient and treated with 100µM suramin for 72 h, followed by assessments for amyloid-ß, phosphorylated tau, oxidative/nitrosative stress, and synaptic puncta density. Results: Suramin treatment of sAD-derived neurons partially ameliorated the increased p-Tau(S199)/Tau ratio, and fully remediated the increased glutathione to oxidized nitric oxide ratio, observed in untreated sAD-derived neurons relative to healthy controls. These positive results may be due in part to the distinct increases in Akt/mTOR pathway mediator p-p70S6K noted with suramin treatment of both ReNcell-derived and iPSC-derived neurons. Longer term neuronal markers, such as synaptic puncta density, were unaffected by suramin treatment. Conclusions: These findings provide initial evidence supporting the potential of suramin to reduce the degree of dysregulation in sAD-derived forebrain neurons in part via the modulation of autophagy.

Effects of Stromal Cell Conditioned Medium and Antipurinergic Treatment on Macrophage Phenotype.

The immunomodulatory capacity of the human mesenchymal stromal cell (MSC) secretome has been a critical driver for the development of cell-free MSC products, such as conditioned medium (CM), for regenerative medicine applications. This is particularly true as cell-free MSC products present several advantages over direct autologous or allogeneic MSC delivery with respect to safety, manufacturability, and defined potency. Recently, significant effort has been placed into creating novel MSC CM formulations with an immunomodulatory capacity tailored for specific regenerative contexts. For instance, the immunoregulatory nature of MSC CM has previously been tuned through a number of cytokine-priming strategies. Herein, we propose an alternate method to tailor the immunomodulatory "phenotype" of cytokine-primed MSC CM through coupling with the pharmacological agent, suramin. Suramin interferes with the signaling of purines including extracellular adenosine triphosphate (ATP), which plays a critical role in the activation of the innate immune system after injury. Toward this end, human THP-1-derived macrophages were activated to a proinflammatory phenotype and treated with (1) unprimed/native MSC CM, (2) interferon-γ/tumor necrosis factor α-primed MSC CM (primed CM), (3) suramin alone, or (4) primed MSC CM and suramin (primed CM/suramin). Markers of key macrophage functions-cytokine secretion, autophagy, oxidative stress modulation, and activation/migration-were assessed. Consistent with previous literature, primed CM elevated macrophage secretion of several proinflammatory and pleiotropic cytokines relative to native CM; whereas addition of suramin imparted consistent shifts in terms of TNFα (↓), interleukin-10 (↓), and hepatocyte growth factor (↑) irrespective of CM. In addition, both primed CM and suramin, individually and combined, increased reactive oxygen species production relative to native CM, and addition of suramin to primed CM shifted levels of CX3CL1, a factor involved in ATP-associated macrophage regulation. Varimax rotation assessment of the secreted cytokine profiles confirmed that primed CM/suramin resulted in a THP-1 phenotypic shift away from the lipopolysaccharide-activated proinflammatory state that was distinct from that of primed CM or native CM alone. This altered primed CM/suramin-associated phenotype may prove beneficial for healing in certain regenerative contexts. These results may inform future work coupling antipurinergic treatments with MSC-derived therapies in regenerative medicine applications.

Ultra-High Modulus Hydrogels Mimicking Cartilage of the Human Body.

The human body is comprised of numerous types of cartilage with a range of high moduli, despite their high hydration. Owing to the limitations of cartilage tissue healing and biological grafting procedures, synthetic replacements have emerged but are limited by poorly matched moduli. While conventional hydrogels can achieve similar hydration to cartilage tissues, their moduli are substantially inferior. Herein, triple network (TN) hydrogels are prepared to synergistically leverage intra-network electrostatic repulsive and hydrophobic interactions, as well as inter-network electrostatic attractive interactions. They are comprised of an anionic 1st network, a neutral 2nd network (capable of hydrophobic associations), and a cationic 3rd network. Collectively, these interactions act synergistically as effective, yet dynamic crosslinks. By tuning the concentration of the cationic 3rd network, these TN hydrogels achieve high moduli of ≈1.5 to ≈3.5 MPa without diminishing cartilage-like water contents (≈80%), strengths, or toughness values. This unprecedented combination of properties poises these TN hydrogels as cartilage substitutes in applications spanning articulating joints, intervertebral discs (IVDs), trachea, and temporomandibular joint disc (TMJ).

Cannabidiol-Driven Alterations to Inflammatory Protein Landscape of Lipopolysaccharide-Activated Macrophages In Vitro May Be Mediated by Autophagy and Oxidative Stress.

Background: The nonpsychotropic phytocannabinoid cannabidiol (CBD) presents itself as a potentially safe and effective anti-inflammatory treatment relative to clinical standards. In this present study, we compare the capacity of CBD to the corticosteroid dexamethasone (Dex) in altering the secreted protein landscape of activated macrophages and speculate upon the mechanism underpinning these alterations. Materials and Methods: Human THP-1 monocytes were differentiated into macrophages (THP-1 derived macrophages [tMACs]), activated with lipopolysaccharide (LPS), and then treated with 5, 10, 25, 50, or 100 µM CBD or 10 µM Dex for 24 h. Following treatment, cytotoxicity of CBD and protein expression levels from culture supernatants and from whole cell lysates were assessed for secreted and intracellular proteins, respectively. Results: High concentration (50 and 100 µM) CBD treatments exhibit a cytotoxic effect on LPS-activated tMACs following the 24-h treatment. Relative to the LPS-activated and untreated control (M[LPS]), both 25 µM CBD and 10 µM Dex reduced expression of pro-inflammatory markers-tumor necrosis factor alpha, interleukin 1 beta, and regulated on activation, normal T cell expressed and secreted (RANTES)-as well as the pleiotropic marker interleukin-6 (IL-6). A similar trend was observed for anti-inflammatory markers interleukin-10 and vascular endothelial growth factor (VEGF). Dex further reduced secreted levels of monocyte chemoattractant protein-1 in addition to suppressing IL-6 and VEGF beyond treatments with CBD. The anti-inflammatory capacity of 25 µM CBD was concurrent with reduction in levels of phosphorylated mammalian target of rapamycin Ser 2448, endothelial nitric oxide synthase, and induction of cyclooxygenase 2 relative to M(LPS). This could suggest that the observed effects on macrophage immune profile may be conferred through inhibition of mammalian target of rapamycin complex 1 and ensuing induction of autophagy. Conclusion: Cumulatively, these data demonstrate cytotoxicity of high concentration CBD treatment. The data reported herein largely agree with other literature demonstrating the anti-inflammatory effects of CBD. However, there is discrepancy within literature surrounding efficacious concentrations and effects of CBD on specific secreted proteins. These data expand upon previous work investigating the effects of CBD on inflammatory protein expression in macrophages, as well as provide insight into the mechanism by which these effects are conferred.

Interferon-Gamma Stimulated Murine Macrophages In Vitro: Impact of Ionic Composition and Osmolarity and Therapeutic Implications.

Background: Injections of osmolytes are promising immunomodulatory treatments for medical benefit, although the rationale and underlying mechanisms are often lacking. The goals of the present study were twofold: (1) to clarify the anti-inflammatory role of the potassium ion and (2) to begin to decouple the effects that ionic strength, ionic species, and osmolarity have on macrophage biology. Materials and Methods: RAW 264.7 murine macrophages were encapsulated in three-dimensional, poly(ethylene glycol) diacrylate hydrogels and activated with interferon-gamma to yield M(IFN). Gene and protein profiles were made of M(IFN) exposed to different hyperosmolar treatments (80 mM potassium gluconate, 80 mM sodium gluconate, and 160 mM sucrose). Results: Relative to M(IFN), all hyperosmolar treatments suppressed expression of pro-inflammatory markers (nitric oxide synthase-2 [NOS-2], tumor necrosis factor-alpha, monocyte chemoattractant protein-1 [MCP-1]) and increased messenger RNA (mRNA) expression of the pleiotropic and angiogenic markers interleukin-6 (IL-6) and vascular endothelial growth factor-A (VEGF), respectively. Ionic osmolytes also demonstrated a greater level of change compared to the nonionic treatments, with mRNA levels of IL-6 the most significantly affected. M(IFN) exposed to K+ exhibited the lowest levels of NOS-2 and MCP-1, and this ion limited IL-6 release induced by osmolarity. Conclusion: Cumulatively, these data suggest that osmolyte composition, ionic strength, and osmolarity are all parameters that can influence therapeutic outcomes. Future work is necessary to further decouple and mechanistically understand the influence that these biophysical parameters have on cell biology, including their impact on other macrophage functions, intracellular osmolyte composition, and cellular and organellular membrane potentials.