Mesenchymal stem cells (MSCs) secrete bioactive factors that exert diverse responses in vivo. In the present study, we explored mechanism how MSCs may lead to higher functional recovery in the animal stroke model.

Ceramide is a bioactive lipid that plays an important role in stress responses leading to apoptosis, cell growth arrest and differentiation. Ceramide production is due in part to sphingomyelin hydrolysis by sphingomyelinases. In brain, neutral sphingomyelinase 2 (nSMase2) is expressed in neurons and increases in its activity and expression have been associated with pro-inflammatory conditions observed in Alzheimer’s disease, multiple sclerosis and human immunodeficiency virus (HIV-1) patients. Increased nSMase2 activity translates into higher ceramide levels and neuronal cell death, which can be prevented by chemical or genetic inhibition of nSMase2 activity or expression. However, to date, there are no soluble, specific and potent small molecule inhibitor tool compounds for in vivo studies or as a starting point for medicinal chemistry optimization. Moreover, the majority of the known inhibitors were identified using bacterial, bovine or rat nSMase2. In an attempt to identify new inhibitor scaffolds, two activity assays were optimized as screening platform using the recombinant human enzyme. First, active hits were identified using a fluorescence-based high throughput compatible assay. Then, hits were confirmed using a 14C sphingomyelin-based direct activity assay. Pharmacologically active compounds and approved drugs were screened using this strategy which led to the identification of cambinol as a novel uncompetitive nSMase2 inhibitor (K i = 7 μM). The inhibitory activity of cambinol for nSMase2 was approximately 10-fold more potent than for its previously known target, silence information regulator 1 and 2 (SIRT1/2). Cambinol decreased tumor necrosis factor-α or interleukin-1 β-induced increases of ceramide and cell death in primary neurons. A preliminary study of cambinol structure and activity allowed the identification of the main structural features required for nSMase2 inhibition. Cambinol and its analogs may be useful as nSMase2 inhibitor tool compounds to prevent ceramide-dependent neurodegeneration.

ABSTRACT

Transplantation of mesenchymal stem cells (MSCs) has been shown to enhance the recovery of brain functions following ischemic injury. Although immune modulation has been suggested to be one of the mechanisms, the molecular mechanisms underlying improved recovery has not been clearly identified. Here, we report that MSCs secrete transforming growth factor-beta (TGF-β) to suppress immune propagation in the ischemic rat brain. Ischemic stroke caused global death of resident cells in the infarcted area, elevated the monocyte chemoattractant protein-1 (MCP-1) level, and evoked massive infiltration of circulating CD68+ immune cells through the impaired blood-brain barrier. Transplantation of MSCs at day 3 post-ischemia blocked the subsequent upregulation of MCP-1 in the ischemic area and the infiltration of additional CD68+ immune cells. MSC-conditioned media decreased the migration and MCP-1 production of freshly isolated immune cells in vitro, and this effect was blocked by an inhibitor of TGF-β signaling or an anti-TGF-β neutralizing antibody. Finally, transplantation of TGF-β1-silenced MSCs failed to attenuate the infiltration of CD68+ cells into the ischemic brain, and was associated with only minor improvements in motor function. These results indicate that TGF-β is key to the ability of MSCs to beneficially attenuate immune reactions in the ischemic brain. Our findings offer insight into the interactions between allogeneic MSCs and the host immune system, reinforcing the prospective clinical value of using MSCs in the treatment of neurological disorders involving inflammation-mediated secondary damage.