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CCR2 deficiency in monocytes impairs angiogenesis and functional recovery after ischemic stroke in mice.

ABSTRACT: Inflammatory Ly6C^hiCCR2⁺ monocytes infiltrate the brain after stroke but their functions are not entirely clear. We report that CCR2⁺ monocytes and CCR2⁺ lymphocytes infiltrate the brain after permanent ischemia. To underscore the role of CCR2⁺ monocytes, we generated mice with selective CCR2 deletion in monocytes. One day post-ischemia, these mice showed less infiltrating monocytes and reduced expression of pro-inflammatory cytokines, markers of alternatively macrophage activation, and angiogenesis. Accordingly, Ly6C^hi monocytes sorted from the brain of wild type mice 24 h post-ischemia expressed pro-inflammatory genes, M2 genes, and pro-angiogenic genes. Flow cytometry showed heterogeneous phenotypes within the infiltrating Ly6C^hiCCR2⁺ monocytes, including a subgroup of Arginase-1⁺ cells. Mice with CCR2-deficient monocytes displayed a delayed inflammatory rebound 15 days post-ischemia that was not found in wild type mice. Furthermore, they showed reduced angiogenesis and worse behavioral performance. Administration of CCR2^+/+ bone-marrow monocytes to mice with CCR2-deficient monocytes did not improve the behavioral performance suggesting that immature bone-marrow monocytes lack pro-reparative functions. The results show that CCR2⁺ monocytes contribute to acute post-ischemic inflammation and participate in functional recovery. The study unravels heterogeneity in the population of CCR2⁺ monocytes infiltrating the ischemic brain and suggests that pro-reparative monocyte subsets promote functional recovery after ischemic stroke.

SUBMITTER: Pedragosa J

PROVIDER: S-EPMC7687030 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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CCR2 deficiency in monocytes impairs angiogenesis and functional recovery after ischemic stroke in mice.

Pedragosa Jordi J Miró-Mur Francesc F Otxoa-de-Amezaga Amaia A Justicia Carles C Ruíz-Jaén Francisca F Ponsaerts Peter P Pasparakis Manolis M Planas Anna M AM

Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 20200309 1_suppl

Inflammatory Ly6ChiCCR2+ monocytes infiltrate the brain after stroke but their functions are not entirely clear. We report that CCR2+ monocytes and CCR2+ lymphocytes infiltrate the brain after permanent ischemia. To underscore the role of CCR2+ monocytes, we generated mice with selective CCR2 deletion in monocytes. One day post-ischemia, these mice showed less infiltrating monocytes and reduced expression of pro-inflammatory cytokines, marke ...[more]

PMID: 32151226

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Project description:Brain injury from stroke is typically considered an event exclusive to the CNS, but injury progression and repair processes are profoundly influenced by peripheral immunity. Stroke stimulates an acute inflammatory response that results in a massive infiltration of peripheral immune cells into the ischemic area. While these cells contribute to the development of brain injury, their recruitment has been considered as a key step for tissue repair. The paradoxical role of inflammatory monocytes in stroke raises the possibility that the manipulation of peripheral immune cells before infiltration into the brain could influence stroke outcome. One such manipulation is remote ischemic limb conditioning (RLC), which triggers an endogenous tolerance mechanism. We observed that mice subjected to poststroke RLC shifted circulating monocytes to a CCR2+ proinflammatory monocyte subset and had reduced acute brain injury, swelling, and improved motor/gait function in chronic stroke. The RLC benefits were observed regardless of injury severity, with a greater shift to a CCR2+ subset in severe stroke. Adoptive transfer of CCR2-deficient monocytes abolished RLC-mediated protection. The study demonstrates the importance of RLC-induced shift of monocytes to a CCR2+ proinflammatory subset in attenuating acute injury and promoting functional recovery in chronic stroke. The defined immune-mediated mechanism underlying RLC benefits allows for an evidence-based framework for the development of immune-based therapeutic strategies for stroke patients.SIGNIFICANCE STATEMENT Stroke is the leading cause of physical disability worldwide but has few treatment options for patients. Because remote ischemic limb conditioning (RLC) elicits endogenous tolerance in neither an organ- nor a tissue-specific manner, the immune system has been considered a mediator for an RLC-related benefit. Application of RLC after stroke increased a proinflammatory CCR2+ monocyte subset in the blood and the brain. RLC reduced acute stroke injury and promoted motor/gait function during the recovery phase. The RLC benefits were absent in mice that received CCR2-deficient monocytes. This preclinical study shows the importance of CCR2+ proinflammatory monocytes in RLC benefits in stroke and provides a therapeutic RLC platform as a novel immune strategy to improve outcomes in stroke patients.

Project description:Background and purposeEnhanced angiogenesis facilitates neurovascular remodeling processes and promotes brain functional recovery after stroke. Previous studies from our laboratory demonstrated that valproate (VPA), a histone deacetylase inhibitor, protects against experimental brain ischemia. The present study investigated whether VPA could enhance angiogenesis and promote long-term functional recovery after ischemic stroke.MethodsMale rats underwent middle cerebral artery occlusion for 60 minutes followed by reperfusion for up to 14 days. Assessed parameters were: locomotor function through the Rotarod test; infarct volume through T2-weighted MRI; microvessel density through immunohistochemistry; relative cerebral blood flow through perfusion-weighted imaging; protein levels of proangiogenic factors through Western blotting; and matrix metalloproteinase-2/9 activities through gelatin zymography.ResultsPostischemic VPA treatment robustly improved the Rotarod performance of middle cerebral artery occlusion rats on Days 7 and 14 after ischemia and significantly reduced brain infarction on Day 14. Concurrently, VPA markedly enhanced microvessel density, facilitated endothelial cell proliferation, and increased relative cerebral blood flow in the ipsilateral cortex. The transcription factor hypoxia-inducible factor-1α and its downstream proangiogenic factors, vascular endothelial growth factor and matrix metalloproteinase-2/9, were upregulated after middle cerebral artery occlusion and significantly potentiated by VPA in the ipsilateral cortex. Acetylation of histone-H3 and H4 was robustly increased by chronic VPA treatment. The beneficial effects of VPA on Rotarod performance and microvessel density were abolished by hypoxia-inducible factor-1α inhibition.ConclusionsChronic VPA treatment enhances angiogenesis and promotes functional recovery after brain ischemia. These effects may involve histone deacetylase inhibition and upregulation of hypoxia-inducible factor-1α and its downstream proangiogenic factors vascular endothelial growth factor and matrix metalloproteinase-2/9.

Project description:Ischemic stroke remains a serious threat to human life. Generation of neuronal and vascular cells is an endogenous regenerative mechanism in the adult brain, which may contribute to tissue repair after stroke. However, the regenerative activity is typically insufficient for significant therapeutic effects after brain injuries. Pyruvate kinase isoform M2 (PKM2) is a key regulator for energy metabolism. PKM2 also has nonmetabolic roles involving regulations of gene expression, cell proliferation, and migration in cancer cells as well as noncancerous cells. In a focal ischemic stroke mouse model, recombinant PKM2 (rPKM2) administration (160 ng/kg, intranasal delivery) at 1 h after stroke showed the significant effect of a reduced infarct volume of more the 60%. Delayed treatment of rPKM2, however, lost the acute neuroprotective effect. We then tested a novel hypothesis that delayed treatment of PKM2 might show proregenerative effects for long-term functional recovery and this chronic action could be mediated by its downstream STAT3 signaling. rPKM2 (160 ng/kg) was delivered to the brain using noninvasive intranasal administration 24 h after the stroke and repeated every other day. Western blot analysis revealed that, 7 days after the stroke, the levels of PKM2 and phosphorylated STAT3 and the expression of angiogenic factors VEGF, Ang-1, and Tie-2 in the peri-infarct region were significantly increased in the rPKM2 treatment group compared with those of the stroke vehicle group. To label proliferating cells, 5-bromo-2'-deoxyuridine (BrdU, 50 mg/kg, i.p.) was injected every day starting 3 days after stroke. At 14 days after stroke, immunohistochemistry showed that rPKM2 increased cell homing of doublecortin (DCX)-positive neuroblasts to the ischemic cortex. In neural progenitor cell (NPC) cultures, rPKM2 (0.4-4 nM) increased the expression of integrin β1 and the activation/phosphorylation of focal adhesion kinase (FAK). A mediator role of FAK in PKM2-promoted cell migration was verified in FAK-knockout fibroblast cultures. In the peri-infarct region of the brain, increased numbers of Glut-1/BrdU and NeuN/BrdU double-positive cells indicated enhanced angiogenesis and neurogenesis, respectively, compared to stroke vehicle mice. Using Laser Doppler imaging, we observed better recovery of the local blood flow in the peri-infarct region of rPKM2-treated mice 14 days after stroke. Meanwhile, rPKM2 improved the sensorimotor functional recovery measured by the adhesive removal test. Inhibiting the STAT3 phosphorylation/activation by the STAT3 inhibitor, BP-1-102 (3 mg/kg/day, o.g.), abolished all beneficial effects of rPKM2 in the stroke mice. Taken together, this investigation provides the first evidence demonstrating that early treatment of rPKM2 shows an acute neuroprotective effect against ischemic brain damage, whereas delayed rPKM2 treatment promotes regenerative activities in the poststroke brain leading to better functional recovery. The underlying mechanism involves activation of the STAT3 and FAK signals in the poststroke brain.

Project description:BACKGROUND AND PURPOSE:Although onset-to-treatment time is associated with early clinical recovery in acute ischemic stroke (AIS) patients treated with intravenous tissue plasminogen activator (tPA), the effect of the timing of tPA-induced recanalization on functional outcomes remains debatable. METHODS:We conducted a multicenter, prospective observational cohort study to determine whether early (within 1-hour from tPA-bolus) complete or partial recanalization assessed during 2-hour real-time transcranial Doppler monitoring is associated with improved outcomes in patients with proximal occlusions. Outcome events included dramatic clinical recovery (DCR) within 2 and 24-hours from tPA-bolus, 3-month mortality, favorable functional outcome (FFO) and functional independence (FI) defined as modified Rankin Scale (mRS) scores of 0-1 and 0-2 respectively. RESULTS:We enrolled 480 AIS patients (mean age 66±15 years, 60% men, baseline National Institutes of Health Stroke Scale score 15). Patients with early recanalization (53%) had significantly (P<0.001) higher rates of DCR at 2-hour (54% vs. 10%) and 24-hour (63% vs. 22%), 3-month FFO (67% vs. 28%) and FI (81% vs. 39%). Three-month mortality rates (6% vs. 17%) and distribution of 3-month mRS scores were significantly lower in the early recanalization group. After adjusting for potential confounders, early recanalization was independently associated with higher odds of 3-month FFO (odds ratio [OR], 6.19; 95% confidence interval [CI], 3.88 to 9.88) and lower likelihood of 3-month mortality (OR, 0.34; 95% CI, 0.17 to 0.67). Onset to treatment time correlated to the elapsed time between tPA-bolus and recanalization (unstandardized linear regression coefficient, 0.13; 95% CI, 0.06 to 0.19). CONCLUSIONS:Earlier tPA treatment after stroke onset is associated with faster tPA-induced recanalization. Earlier onset-to-recanalization time. RESULTS:in improved functional recovery and survival in AIS patients with proximal intracranial occlusions.

Dataset Information

CCR2 deficiency in monocytes impairs angiogenesis and functional recovery after ischemic stroke in mice.

Publications

CCR2 deficiency in monocytes impairs angiogenesis and functional recovery after ischemic stroke in mice.

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