Targeting OLFML3 in Colorectal Cancer Suppresses Tumor Growth and Angiogenesis, and Increases the Efficacy of Anti-PD1 Based Immunotherapy.

The role of the proangiogenic factor olfactomedin-like 3 (OLFML3) in cancer is unclear. To characterize OLFML3 expression in human cancer and its role during tumor development, we undertook tissue expression studies, gene expression analyses of patient tumor samples, in vivo studies in mouse cancer models, and in vitro coculture experiments. OLFML3 was expressed at high levels, mainly in blood vessels, in multiple human cancers. We focused on colorectal cancer (CRC), as elevated expression of OLFML3 mRNA correlated with shorter relapse-free survival, higher tumor grade, and angiogenic microsatellite stable consensus molecular subtype 4 (CMS4). Treatment of multiple in vivo tumor models with OLFML3-blocking antibodies and deletion of the Olfml3 gene from mice decreased lymphangiogenesis, pericyte coverage, and tumor growth. Antibody-mediated blockade of OLFML3 and deletion of host Olfml3 decreased the recruitment of tumor-promoting tumor-associated macrophages and increased infiltration of the tumor microenvironment by NKT cells. Importantly, targeting OLFML3 increased the antitumor efficacy of anti-PD-1 checkpoint inhibitor therapy. Taken together, the results demonstrate that OLFML3 is a promising candidate therapeutic target for CRC.

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

RATIONALE: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. OBJECTIVE: To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. METHODS AND RESULTS: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. CONCLUSIONS: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

Glatiramer acetate reduces infarct volume in diabetic mice with cerebral ischemia and prevents long-term memory loss.

Stroke is currently the second leading cause of death in industrialized countries and the second cause of dementia after Alzheimer's disease. Diabetes is an independent risk factor for stroke that exacerbates the severity of lesions, disability and cognitive decline. There is increasing evidence that sustained brain inflammation may account for this long-term prejudicial outcome in diabetic patients in particular. We sought to demonstrate that experimental permanent middle cerebral artery occlusion (pMCAo) in the diabetic mouse aggravates stroke, induces cognitive decline, and is associated with exacerbated brain inflammation, and that these effects can be alleviated and/or prevented by the immunomodulator, glatiramer acetate (GA). Male diabetic C57Bl6 mice (streptozotocin IP) subjected to permanent middle cerebral artery occlusion (pMCAo), were treated by the immunomodulator, GA (Copaxone®) (1 mg/kg daily, sc) until 3 or 7 days post stroke. Infarct volume, brain pro- and anti-inflammatory mediators, microglial/macrophage density, and neurogenesis were monitored during the first week post stroke. Neurological sensorimotor deficit, spatial memory and brain deposits of Aß40 and Aß42 were assessed until six weeks post stroke. In diabetic mice with pMCAo, proinflammatory mediators (IL-1ß, MCP1, TNFα and CD68) were significantly higher than in non-diabetic mice. In GA-treated mice, the infarct volume was reduced by 30% at D3 and by 40% at D7 post stroke (P < 0.05), sensorimotor recovery was accelerated as early as D3, and long-term memory loss was prevented. Moreover, proinflammatory mediators significantly decreased between D3 (COX2) and D7 (CD32, TNFα, IL-1ß), and neurogenesis was significantly increased at D7. Moreover, GA abrogates the accumulation of insoluble Aß40. This work is the first one to evidence that the immunomodulatory drug GA reduces infarct volume and proinflammatory mediators, enhances early neurogenesis, accelerates sensorimotor recovery, and prevents long-term memory loss in diabetic mice with pMCAo.

Diabetic Ephrin-B2-Stimulated Peripheral Blood Mononuclear Cells Enhance Poststroke Recovery in Mice.

Clinical trials of cell therapy in stroke favor autologous cell transplantation. To date, feasibility studies have used bone marrow-derived mononuclear cells, but harvesting bone marrow cells is invasive thus complicating bedside treatment. We investigated the therapeutic potential of peripheral blood-derived mononuclear cells (PB-MNC) harvested from diabetic patients and stimulated by ephrin-B2 (PB-MNC+) (500,000 cells), injected intravenously 18-24 hours after induced cerebral ischemia in mice. Infarct volume, neurological deficit, neurogenesis, angiogenesis, and inflammation were investigated as were the potential mechanisms of PB-MNC+ cells in poststroke neurorepair. At D3, infarct volume was reduced by 60% and 49% compared to unstimulated PB-MNC and PBS-treated mice, respectively. Compared to PBS, injection of PB-MNC+ increased cell proliferation in the peri-infarct area and the subventricular zone, decreased microglia/macrophage cell density, and upregulated TGF-ß expression. At D14, microvessel density was decreased and functional recovery was enhanced compared to PBS-treated mice, whereas plasma levels of BDNF, a major regulator of neuroplasticity, were increased in mice treated with PB-MNC+ compared to the other two groups. Cell transcriptional analysis showed that ephrin-B2 induced phenotype switching of PB-MNC by upregulating genes controlling cell proliferation, inflammation, and angiogenesis, as confirmed by adhesion and Matrigel assays. Conclusions. This feasibility study suggests that PB-MNC+ transplantation poststroke could be a promising approach but warrants further investigation. If confirmed, this rapid, noninvasive bedside cell therapy strategy could be applied to stroke patients at the acute phase.

Angiogenic factor-driven inflammation promotes extravasation of human proangiogenic monocytes to tumours.

Recruitment of circulating monocytes is critical for tumour angiogenesis. However, how human monocyte subpopulations extravasate to tumours is unclear. Here we show mechanisms of extravasation of human CD14dimCD16+ patrolling and CD14+CD16+ intermediate proangiogenic monocytes (HPMo), using human tumour xenograft models and live imaging of transmigration. IFNγ promotes an increase of the chemokine CX3CL1 on vessel lumen, imposing continuous crawling to HPMo and making these monocytes insensitive to chemokines required for their extravasation. Expression of the angiogenic factor VEGF and the inflammatory cytokine TNF by tumour cells enables HPMo extravasation by inducing GATA3-mediated repression of CX3CL1 expression. Recruited HPMo boosts angiogenesis by secreting MMP9 leading to release of matrix-bound VEGF-A, which amplifies the entry of more HPMo into tumours. Uncovering the extravasation cascade of HPMo sets the stage for future tumour therapies.

Inactivation of Nitric Oxide Synthesis Exacerbates the Development of Alzheimer Disease Pathology in APPPS1 Mice (Amyloid Precursor Protein/Presenilin-1).

The epidemiological link between hypertension and Alzheimer disease is established. We previously reported that hypertension aggravates the Alzheimer-like pathology in APPPS1 mice (amyloid precursor protein/presenilin-1, mouse model of Alzheimer disease) with angiotensin II-induced hypertension, in relation with hypertension and nitric oxide deficiency. To provide further insights into the role of nitric oxide in the hypertension-Alzheimer disease cross-talk, we studied the effects of nitric oxide blockade in APPPS1 mice using N(ω)-nitro-l-arginine methyl ester (l-NAME) alone or in combination with hydralazine, to normalize blood pressure. Compared with normotensive APPPS1 mice, those with l-NAME-induced hypertension had greater amyloid burden ( P<0.05), increased cortical amyloid angiopathy ( P<0.01), decreased regional microvascular density ( P<0.05), and deficient long-term spatial reference memory ( P<0.001). Blood pressure normalization with hydralazine did not protect APPPS1 mice from l-NAME-induced deterioration except for cortical amyloid angiopathy, linked to hypertension-induced arterial wall remodeling. By testing the cerebrovascular response to hypercapnic breathing, we evidenced early functional impairment of cerebral vasomotor activity in APPPS1 mice. Whereas in control wild-type normotensive mice, carbon dioxide breathing resulted in 15±1.3% increase in the mean blood flow velocity ( P<0.001), paradoxical mild decrease (1.5±0.4%) was recorded in normotensive APPPS1 mice ( P<0.001). Carbon dioxide-induced decrease in mean blood flow velocity was not significantly modified in l-NAME-treated hypertensive APPPS1 mice (2.5±1.2%) and partly reversed to mild vasodilation by hydralazine (3.2±1.5%, P<0.01). These results suggest that impaired nitric oxide bioavailability exacerbates the pathophysiology of Alzheimer disease, essentially impacting amyloid load and cognitive impairment, independently of l-NAME-induced hypertension. Only cerebral amyloid angiopathy seems to be dependent on hypertension.

Diabetic microangiopathy: impact of impaired cerebral vasoreactivity and delayed angiogenesis after permanent middle cerebral artery occlusion on stroke damage and cerebral repair in mice.

Diabetes increases the risk of stroke by three, increases related mortality, and delays recovery. We aimed to characterize functional and structural alterations in cerebral microvasculature before and after experimental cerebral ischemia in a mouse model of type 1 diabetes. We hypothesized that preexisting brain microvascular disease in patients with diabetes might partly explain increased stroke severity and impact on outcome. Diabetes was induced in 4-week-old C57Bl/6J mice by intraperitoneal injections of streptozotocin (60 mg/kg). After 8 weeks of diabetes, the vasoreactivity of the neurovascular network to CO2 was abolished and was not reversed by nitric oxide (NO) donor administration; endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS) mRNA, phospho-eNOS protein, nNOS, and phospho-nNOS protein were significantly decreased; angiogenic and vessel maturation factors (vascular endothelial growth factor a [VEGFa], angiopoietin 1 (Ang1), Ang2, transforming growth factor-ß [TGF-ß], and platelet-derived growth factor-ß [PDGF-ß]) and blood-brain barrier (BBB) occludin and zona occludens 1 (ZO-1) expression were significantly decreased; and microvessel density was increased without changes in ultrastructural imaging. After permanent focal cerebral ischemia induction, infarct volume and neurological deficit were significantly increased at D1 and D7, and neuronal death (TUNEL+ / NeuN+ cells) and BBB permeability (extravasation of Evans blue) at D1. At D7, CD31+ / Ki67+ double-immunolabeled cells and VEGFa and Ang2 expression were significantly increased, indicating delayed angiogenesis. We show that cerebral microangiopathy thus partly explains stroke severity in diabetes.

Hypertension accelerates the progression of Alzheimer-like pathology in a mouse model of the disease.

Cerebrovascular impairment is frequent in patients with Alzheimer disease and is believed to influence clinical manifestation and severity of the disease. Cardiovascular risk factors, especially hypertension, have been associated with higher risk of developing Alzheimer disease. To investigate the mechanisms underlying the hypertension, Alzheimer disease cross talk, we established a mouse model of dual pathology by infusing hypertensive doses of angiotensin II into transgenic APPPS1 mice overexpressing mutated human amyloid precursor and presenilin 1 proteins. At 4.5 months, at the early stage of disease progression, only hypertensive APPPS1 mice presented impairment of temporal order memory performance in the episodic-like memory task. This cognitive deficit was associated with an increased number of cortical amyloid deposits (223±5 versus 207±5 plaques/mm(2); P<0.05) and a 2-fold increase in soluble amyloid levels in the brain and in plasma. Hypertensive APPPS1 mice presented several cerebrovascular alterations, including a 25% reduction in cerebral microvessel density and a 30% to 40% increase in cerebral vascular amyloid deposits, as well as a decrease in vascular endothelial growth factor A expression in the brain, compared with normotensive APPPS1 mice. Moreover, the brain levels of nitric oxide synthase 1 and 3 and the nitrite/nitrate levels were reduced in hypertensive APPPS1 mice (by 49%, 34%, and 33%, respectively, compared with wild-type mice; P<0.05). Our results indicate that hypertension accelerates the development of Alzheimer disease-related structural and functional alterations, partially through cerebral vasculature impairment and reduced nitric oxide production.

Smooth muscle cell phenotypic switching in stroke.

Disruption of cerebral blood flow after stroke induces cerebral tissue injury through multiple mechanisms that are not yet fully understood. Smooth muscle cells (SMCs) in blood vessel walls play a key role in cerebral blood flow control. Cerebral ischemia triggers these cells to switch to a phenotype that will be either detrimental or beneficial to brain repair. Moreover, SMC can be primarily affected genetically or by toxic metabolic molecules. After stroke, this pathological phenotype has an impact on the incidence, pattern, severity, and outcome of the cerebral ischemic disease. Although little research has been conducted on the pathological role and molecular mechanisms of SMC in cerebrovascular ischemic diseases, some therapeutic targets have already been identified and could be considered for further pharmacological development. We examine these different aspects in this review.

MFGE8 inhibits inflammasome-induced IL-1ß production and limits postischemic cerebral injury.

Milk fat globule-EGF 8 (MFGE8) plays important, nonredundant roles in several biological processes, including apoptotic cell clearance, angiogenesis, and adaptive immunity. Several recent studies have reported a potential role for MFGE8 in regulation of the innate immune response; however, the precise mechanisms underlying this role are poorly understood. Here, we show that MFGE8 is an endogenous inhibitor of inflammasome-induced IL-1ß production. MFGE8 inhibited necrotic cell-induced and ATP-dependent IL-1ß production by macrophages through mediation of integrin ß(3) and P2X7 receptor interactions in primed cells. Itgb3 deficiency in macrophages abrogated the inhibitory effect of MFGE8 on ATP-induced IL-1ß production. In a setting of postischemic cerebral injury in mice, MFGE8 deficiency was associated with enhanced IL-1ß production and larger infarct size; the latter was abolished after treatment with IL-1 receptor antagonist. MFGE8 supplementation significantly dampened caspase-1 activation and IL-1ß production and reduced infarct size in wild-type mice, but did not limit cerebral necrosis in Il1b-, Itgb3-, or P2rx7-deficient animals. In conclusion, we demonstrated that MFGE8 regulates innate immunity through inhibition of inflammasome-induced IL-1ß production.

Glatiramer Acetate administration does not reduce damage after cerebral ischemia in mice.

Inflammation plays a key role in ischemic stroke pathophysiology: microglial/macrophage cells and type-1 helper cells (Th1) seem deleterious, while type-2 helper cells (Th2) and regulatory T cells (Treg) seem protective. CD4 Th0 differentiation is modulated by microglial cytokine secretion. Glatiramer Acetate (GA) is an immunomodulatory drug that has been approved for the treatment of human multiple sclerosis by means of a number of mechanisms: reduced microglial activation and pro-inflammatory cytokine production, Th0 differentiation shifting from Th2 to Th2 and Treg with anti-inflammatory cytokine production and increased neurogenesis. We induced permanent (pMCAo) or transient middle cerebral artery occlusion (tMCAo) and GA (2 mg) or vehicle was injected subcutaneously immediately after cerebral ischemia. Mice were sacrificed at D3 to measure neurological deficit, infarct volume, microglial cell density and qPCR of TNFα and IL-1ß (pro-inflammatory microglial cytokines), IFNγ (Th2 cytokine), IL-4 (Th2 cytokine), TGFß and IL-10 (Treg cytokines), and at D7 to evaluate neurological deficit, infarct volume and neurogenesis assessment. We showed that in GA-treated pMCAo mice, infarct volume, microglial cell density and cytokine secretion were not significantly modified at D3, while neurogenesis was enhanced at D7 without significant infarct volume reduction. In GA-treated tMCAo mice, microglial pro-inflammatory cytokines IL-1ß and TNFα were significantly decreased without modification of microglial/macrophage cell density, cytokine secretion, neurological deficit or infarct volume at D3, or modification of neurological deficit, neurogenesis or infarct volume at D7. In conclusion, Glatiramer Acetate administered after cerebral ischemia does not reduce infarct volume or improve neurological deficit in mice despite a significant increase in neurogenesis in pMCAo and a microglial pro-inflammatory cytokine reduction in tMCAo.