World-Wide Efficacy of Bone Marrow Derived Mesenchymal Stromal Cells in Preclinical Ischemic Stroke Models: Systematic Review and Meta-Analysis

Background: Following extensive, positive results in pre-clinical experiments, Bone Marrow Derived-Mesenchymal Stromal Cells (BM-MSCs) are now being tested as a novel therapy for ischemic stroke in ongoing clinical trials. However, multiple critical questions relating to their translational application remain to be clarified. We performed a comprehensive, systematic review and meta-analysis of pre-clinical studies to evaluate the efficacy of BM-MSCs on functional outcomes after ischemic stroke, as well as the independent role of translational factors on their effect size.Methods: We systematically reviewed the literature and identified articles using BM-MSCs in animal models of focal ischemic stroke. After abstraction of all relevant data, we performed a meta-analysis to estimate the combined effect size of behavioral endpoints after BM-MSC administration. To describe the effect size across many behavioral outcomes, we divided these outcomes into four categories: (1) Composite scores, (2) Motor Tests, (3) Sensorimotor Tests, and (4) Cognitive Tests. We also performed a meta-regression analysis for measuring the effect of individual characteristics of BM-MSC administration on the effect size.Results: Our results from 141 articles indicate a significant beneficial effect on composite, motor, and sensorimotor outcomes after treatment with BM-MSCs compared to control groups. We found no major differences in treatment effect based on delivery route, dose, fresh vs. frozen preparation, or passage number. There were no consistent findings supporting a difference in treatment effect based on time windows from acute periods (0–6 h) vs. later windows (2–7 days). Furthermore, these positive treatment effects on functional outcome were consistent across different labs in different parts of the world as well as over the last 18 years. There was a negative correlation between publication year and impact factor.Conclusions: Our results show worldwide efficacy of BM-MSCs in improving functional outcomes in pre-clinical animal models of stroke and support testing these cells in clinical trials in various ranges of time windows using different delivery routes. The continued growing number of publications showing functional benefit of BM-MSCs are now adding limited value to an oversaturated literature spanning 18 years. Researchers should focus on identifying definitive mechanisms on how BM-MSCs lead to benefit in stroke models

Prodrugs of a 1-Hydroxy-2-Oxopiperidin-3-Yl Phosphonate Enolase Inhibitor for the Treatment of ENO1-Deleted Cancers

Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). A previous work described the sustained tumor regression activities of a substrate-competitive phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX) (5), and its bis-pivaloyoxymethyl prodrug, POMHEX (6), in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma [Nature Metabolism 2020, 2, 1423-1426]. Due to poor pharmacokinetics of bis-ester prodrugs, this study was undertaken to identify potential non-esterase prodrugs for further development. Whereas phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, McGuigan prodrugs were not. Other strategies, including cycloSal and lipid prodrugs of 5, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over 6. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation

Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine.

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP\u27s substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion

Ongoing Secondary Degeneration of the Limbic System in Patients With Ischemic Stroke: A Longitudinal MRI Study

Purpose: Ongoing post-stroke structural degeneration and neuronal loss preceding neuropsychological symptoms such as cognitive decline and depression are poorly understood. Various substructures of the limbic system have been linked to cognitive impairment. In this longitudinal study, we investigated the post-stroke macro- and micro-structural integrity of the limbic system using structural and diffusion tensor magnetic resonance imaging.Materials and Methods: Nineteen ischemic stroke patients (11 men, 8 women, average age 53.4 ± 12.3, range 18–75 years), with lesions remote from the limbic system, were serially imaged three times over 1 year. Structural and diffusion-tensor images (DTI) were obtained on a 3.0 T MRI system. The cortical thickness, subcortical volume, mean diffusivity (MD), and fractional anisotropy (FA) were measured in eight different regions of the limbic system. The National Institutes of Health Stroke Scale (NIHSS) was used for clinical assessment. A mixed model for multiple factors was used for statistical analysis, and p-values <0.05 was considered significant.Results: All patients demonstrated improved NIHSS values over time. The ipsilesional subcortical volumes of the thalamus, hippocampus, and amygdala significantly decreased (p < 0.05) and MD significantly increased (p < 0.05). The ipsilesional cortical thickness of the entorhinal and perirhinal cortices was significantly smaller than the contralesional hemisphere at 12 months (p < 0.05). The cortical thickness of the cingulate gyrus at 12 months was significantly decreased at the caudal and isthmus regions as compared to the 1 month assessment (p < 0.05). The cingulum fibers had elevated MD at the ipsilesional caudal-anterior and posterior regions compared to the corresponding contralesional regions.Conclusion: Despite the decreasing NIHSS scores, we found ongoing unilateral neuronal loss/secondary degeneration in the limbic system, irrespective of the lesion location. These results suggest a possible anatomical basis for post stroke psychiatric complications

Mesenchymal Stem Cell Derived Extracellular Vesicles for Repairing the Neurovascular Unit after Ischemic Stroke

Ischemic stroke is a debilitating disease and one of the leading causes of long-term disability. During the early phase after ischemic stroke, the blood-brain barrier (BBB) exhibits increased permeability and disruption, leading to an influx of immune cells and inflammatory molecules that exacerbate the damage to the brain tissue. Mesenchymal stem cells have been investigated as a promising therapy to improve the recovery after ischemic stroke. The therapeutic effects imparted by MSCs are mostly paracrine. Recently, the role of extracellular vesicles released by these MSCs have been studied as possible carriers of information to the brain. This review focuses on the potential of MSC derived EVs to repair the components of the neurovascular unit (NVU) controlling the BBB, in order to promote overall recovery from stroke. Here, we review the techniques for increasing the effectiveness of MSC-based therapeutics, such as improved homing capabilities, bioengineering protein expression, modified culture conditions, and customizing the contents of EVs. Combining multiple techniques targeting NVU repair may provide the basis for improved future stroke treatment paradigms

Cryopreservation of Bone Marrow Mononuclear Cells Alters Their Viability and Subpopulation Composition but Not Their Treatment Effects in a Rodent Stroke Model

The systemic administration of autologous bone marrow (BM) derived mononuclear cells (MNCs) is under investigation as a novel therapeutic modality for the treatment of ischemic stroke. Autologous applications raise the possibility that MNCs could potentially be stored as a banked source. There have been no studies that investigate the effects of cryopreservation of BM-MNCs on their functional abilities in stroke models. In the present study, C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAo) for 60 minutes and then divided into two treatment groups: fresh MNCs versus cryopreserved MNCs. BM-MNCs were collected at 22 hours after MCAo and were stored in liquid nitrogen for 12 months in cryopreserved MNCs group. BM-MNCs cellular viability, composition, and phenotype of the various subpopulations of mice BM-MNCs were evaluated by flow cytometry, and the behavioral recovery of stroke animals was tested with freshly harvested MNCs versus cryopreserved MNCs by corner test and ladder rung test. We found that long-term cryopreservation negatively impacts the cellular viability of bone marrow MNCs. Cryopreservation also alters the cellular composition of various subpopulations within the MNCs. However, despite the changes observed in cryopreserved cells, both fresh and frozen MNCs have similar beneficial effect on behavioral and histological outcomes

Medications for Hypertension Change the Secretome Profile from Marrow Stromal Cells and Peripheral Blood Monocytes

Marrow stromal cells (MSCs) are in different stages of clinical trials for stroke patients. MSCs are proposed to promote recovery through the release of secretomes that modulate the function of beneficial immune cells. The majority of stroke patients have comorbidities including hypertension, for which they are prescribed antihypertensive medications that might affect the function of MSCs, when they are administered in stroke patients. Here, we studied the effects of common antihypertensive medications on the secretomes of human MSCs and their modulation of human monocytes (Mo) derived from stroke patients. MTT assay was used to assess the proliferation of MSCs after they were exposed to increased levels of antihypertensive medications. MSCs were exposed to the following medications: atenolol, captopril, and losartan. Monocytes were isolated from stroke patients with NIHSS ranging from 11 to 20 and from healthy controls. MSC-Mo cocultures were established, and a secretome profile was analyzed using the Magpix Multiplex cytokine array from Luminex technology. The linear mixed-effect model was used for statistical analysis. All analyses were performed using SAS 9.4, and p values less than 0.05 were considered significant. At clinically relevant levels, there was no change in MSC proliferation after exposure to atenolol, captopril, or losartan. Atenolol increased IL-1RA in stroke-Mo and decreased IL-8 secretion from MSCs indicating an anti-inflammatory effect of atenolol on secretomes of these cells. Captopril increased IL-8 from stroke-Mo and increased IL-6, IL-8, and MCP-1 secretions from MSCs. Captopril also increased IL-6 secretion from cocultures of stroke-Mo and MSCs indicating a strong proinflammatory effect on MSCs and their interaction with Mo. Atenolol increased the secretion of IL-8 and MCP-1 while captopril increased the secretion of IL-6 and MCP-1 from MSCs. Losartan decreased the release of IL-6 from MSCs. Losartan reduced MCP-1 and TNF-α from stroke-Mo and reduced IL-8 from cocultures of stroke-Mo and MSCs. Our results show that antihypertensive medications such as atenolol, captopril, and losartan, at concentrations comparable to doses prescribed for patients hospitalized for acute stroke, modulate the secretome profile of MSCs and their modulatory effects on target immune cells. Our results suggest that stroke trials involving the use of intravenous MSCs should consider the effect of these antihypertensive drugs administered to stroke patients

ENOblock Does Not Inhibit the Activity of the Glycolytic Enzyme Enolase

<div><p>Inhibition of glycolysis is of great potential for the treatment of cancer. However, inhibitors of glycolytic enzymes with favorable pharmacological profiles have not been forthcoming. Due to the nature of their active sites, most high-affinity transition-state analogue inhibitors of glycolysis enzymes are highly polar with poor cell permeability. A recent publication reported a novel, non-active site inhibitor of the glycolytic enzyme Enolase, termed ENOblock (N-[2-[2-2-aminoethoxy)ethoxy]ethyl]4-4-cyclohexylmethyl)amino]6-4-fluorophenyl)methyl]amino]1,3,5-triazin-2-yl]amino]benzeneacetamide). This would present a major advance, as this is heterocyclic and fully cell permeable molecule. Here, we present evidence that ENOblock does not inhibit Enolase enzymatic activity <i>in vitro</i> as measured by three different assays, including a novel ³¹P NMR based method which avoids complications associated with optical interferences in the UV range. Indeed, we note that due to strong UV absorbance, ENOblock interferes with the direct spectrophotometric detection of the product of Enolase, phosphoenolpyruvate. Unlike established Enolase inhibitors, ENOblock does not show selective toxicity to <i>ENO1</i>-deleted glioma cells in culture. While our data do not dispute the biological effects previously attributed to ENOblock, they indicate that such effects must be caused by mechanisms other than direct inhibition of Enolase enzymatic activity.</p></div

Non-selective toxicity of ENOBlock to ENO1-deleted glioma cells.

<p>A representative plate of cancer cells treated with ENOblock is shown in panel <b>a</b>, with quantification shown in panel <b>b</b> A plate treated with SF2312 is shown in panel <b>c</b>, with quantification shown in panel <b>d</b>. Cell were treated for 7 days. <b>(b, d)</b> D423 <i>ENO1</i>-deleted (red diamonds), D423 <i>ENO1</i>-rescued (blue squares) and LN319 <i>ENO1</i> WT (grey circles) were treated with the indicated doses of ENOblock in panel <b>b</b> (N = 4 ± S.D) or SF2312 in panel <b>d</b> (N = 4 ± S.D). Cell density was quantified by crystal violet and expressed relative to vehicle control as a function of inhibitor concentration. At high concentrations, SF2312 selectively killed D423 <i>ENO1</i>-deleted cells as compared to D423 <i>ENO1</i>-rescued cells (p<0.05, Repeated Measures one-way ANOVA with Bonferroni correction). ENOblock failed to show such selectivity regardless of dose.</p