A Combination of Atorvastatin and Aspirin Enhances the Pro-Regenerative Interactions of Marrow Stromal Cells and Stroke-Derived Monocytes In Vitro.

Background and Purpose: Marrow stromal cells (MSCs) are being tested in clinical trials for stroke patients. MSCs appear to promote recovery through secretomes that promote modulation of immune cells, including myeloid phagocytes. Many stroke patients have comorbidities such as metabolic syndrome, hypertension, hypercholesterolemia, and diabetes for which they are prescribed medications that might affect the function of MSCs and monocytes (Mo) when they are administered in stroke patients. We studied the effects of the two most commonly prescribed stroke medications, statin and statin plus aspirin, on the secretomes of MSCs and their modulation of Mo derived from stroke patients. Methods: Human MSCs, Mo and their co-cultures were exposed to atorvastatin or atorvastatin plus aspirin followed by secretome analysis at 24 h. Monocytes were isolated from healthy controls as well as stroke patients with NIHSS ranging from 11 to 20. Secretome composition was measured using multiplex immunoassay. We used MTT assay to measure proliferation of monocytes. The mixed model was used to analyze experimental data. p-values less than 0.05 were considered significant. Results: Atorvastatin and aspirin combination increased the release of IL-1RA from stroke Mo. In MSCs, atorvastatin and aspirin combination reduced the release of pro-inflammatory cytokines such as IL-6, IL-8, MCP-1 and IFN-γ. Atorvastatin alone reduced the release of IL-6, IL-8 and MCP-1 from co-cultures of stroke monocytes and MSCs. Combination of atorvastatin and aspirin had additive effect on reducing the secretion of IL-6 from co-cultures of stroke Mo and MSCs. Conclusion: Atorvastatin, alone and in combination with aspirin can promote anti-inflammatory effect by modulating the secretome profile of Mo and MSCs. Our results suggest that stroke trials involving the use of intravenous MSCs should consider the effect of aspirin and atorvastatin, both of which are administered to the majority of hospitalized ischemic stroke patients.

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.

Aspirin in stroke patients modifies the immunomodulatory interactions of marrow stromal cells and monocytes.

BACKGROUND AND OBJECTIVE: Most stroke patients are prescribed aspirin (ASA) to adjust blood coagulability. Marrow stromal cells (MSCs) are being tested in clinical trials for stroke patients who likely are prescribed aspirin. One of the principal mechanisms of action of MSCs and ASA is modulation of the inflammatory response, including those mediated by monocytes (Mo). Thus, here we tested if aspirin can modify anti-inflammatory properties of MSCs or Mo alone, and in combination. METHODS: Mo were isolated at 24 h of stroke onset from ischemic stroke patients with NIHSS ranging from 11 to 20 or from healthy controls. Human bone marrow-derived MSCs from healthy subjects were used at passage 3. Mo, MSCs, and MSCs-Mo co-cultures were exposed to ASA at clinically relevant doses. The secretome profile of inflammatory mediators was measured using Magpix multiplex cytokine array. Viability was measured using MTT assay. Linear mixed effect model was used for statistical analysis. RESULTS: Overall Mo from control subjects exposed to ASA showed increased secretion of IL-1RA, IL-8, MCP-1, and TNF-α and Mo from stroke patients showed greater release of IL-1RA and MCP-1. In MSCs-Mo co-cultures, ASA added to co-cultures of control Mo reduced fractalkine secretion while it increased the fractalkine secretion when added to Mo from stroke patients. In addition, in co-cultures independent of Mo origin, ASA reduced IL-6, IL-8, MCP-1, and TNF-α. CONCLUSIONS: Aspirin in acute stroke patients may modulate the secretome profile of Mo and MSCs, thus potentially modulating immune and inflammatory responses associated with stroke. Our results suggest that stroke trials involving the use of intravenous MSCs should consider the effect of aspirin as a confounding factor.

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.

Endothelial-Specific EphA4 Negatively Regulates Native Pial Collateral Formation and Re-Perfusion following Hindlimb Ischemia.

Leptomeningeal anastomoses play a critical role in regulating vascular re-perfusion following obstruction, however, the mechanisms regulating their development remains under investingation. Our current findings indicate that EphA4 receptor is a novel negative regulator of collaterogenesis. We demonstrate that EphA4 is highly expressed on pial arteriole collaterals at post-natal day (P) 1 and 7, then significantly reduced by P21. Endothelial cell (EC)-specific loss of EphA4, EphA4f/f/Tie2::Cre (KO), resulted in an increase in the density but not diameter of pial collaterals compared to WT mice. ECs isolated from KO mice displayed a 3-fold increase in proliferation, enhanced migration, tube formation and elevated levels of phospho(p)-Akt compared to WT ECs. Attenuating p-Akt, using LY294002, reduced the proliferative and migration effects in the KO ECs. RNAseq analysis also revealed altered expression patterns for genes that regulate cell proliferation, vascular development, extracellular matrix and immune-mediate responses, namely MCP-1, MMP2 and angiopoietin-1. Lastly, we show that induction of hindlimb ischemia resulted in accelerated re-perfusion, collateral remodeling and reduced tissue necrosis in the absence of EC-specific EphA4 compared to WT mice. These findings demonstrate a novel role for EphA4 in the early development of the pial collateral network and suggests a role in regulating vascular remodeling after obstruction.