5-HT 1A Agonist Properties Contribute to a Robust Response to Vilazodone in the Novelty Suppressed Feeding Paradigm.

BACKGROUND: Differences in 5-HT 1A receptor function have been implicated in vulnerability to depression and in response to treatment. Adding 5-HT 1A partial agonists to selective serotonin reuptake inhibitors has been touted as a strategy to increase their efficacy. Here we use the novelty suppressed feeding paradigm to compare the effects of vilazodone, a high-potency selective serotonin reuptake inhibitor, with high affinity for 5-HT 1A receptors to the reference selective serotonin reuptake inhibitor fluoxetine across several mouse strains that differ in their response to selective serotonin reuptake inhibitors. METHODS: To confirm 5-HT 1A agonist activity, body temperature was measured after acute administration of vilazodone or fluoxetine, as administration of 5-HT 1A agonists induces hypothermia. We next used 3 strains of mice to examine the effects of the drugs on latency in the novelty suppressed feeding, a paradigm generally sensitive to chronic but not acute effects of antidepressants. RESULTS: Vilazodone induces robust hypothermia and blocks stress-induced hyperthermia in a 5-HT 1A -dependent manner, consistent with agonist effects at 5-HT 1A autoreceptors. In 129SvEv mice, vilazodone (10mg/kg/d) reduces the latency to eat in the novelty suppressed feeding test within 8 days, while no effect of fluoxetine (20mg/kg/d) was detected at that time. In contrast, both vilazodone and fluoxetine are effective at decreasing latency to eat in the novelty suppressed feeding paradigm in a strain with low autoreceptor levels. In mice with higher autoreceptor levels, no significant difference was detected between fluoxetine and vehicle ( P=. 8) or vilazodone and vehicle ( P =.06). CONCLUSION: In mice, vilazodone may offer advantages in time of onset and efficacy over a reference selective serotonin reuptake inhibitor in the novelty suppressed feeding test.

Characterization and molecular profiling of PSEN1 familial Alzheimer's disease iPSC-derived neural progenitors.

Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aß42 to Aß40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aß42/40, and have characterized novel expression changes.

Factors secreted by endothelial progenitor cells enhance neurorepair responses after cerebral ischemia in mice.

Cell therapy with endothelial progenitor cells (EPCs) has emerged as a promising strategy to regenerate the brain after stroke. Here, we aimed to investigate if treatment with EPCs or their secreted factors could potentiate angiogenesis and neurogenesis after permanent focal cerebral ischemia in a mouse model of ischemic stroke. BALB/C male mice were subjected to distal occlusion of the middle cerebral artery, and EPCs, cell-free conditioned media (CM) obtained from EPCs, or vehicle media were administered one day after ischemia. Magnetic resonance imaging (MRI) was performed at baseline to confirm that the lesions were similar between groups. Immunohistochemical and histological evaluation of the brain was performed to evaluate angio-neurogenesis and neurological outcome at two weeks. CM contained growth factors, such as VEGF, FGF-b and PDGF-bb. A significant increase in capillary density was noted in the peri-infarct areas of EPC- and CM-treated animals. Bielschowsky's staining revealed a significant increase in axonal rewiring in EPC-treated animals compared with shams, but not in CM-treated mice, in close proximity with DCX-positive migrating neuroblasts. At the functional level, post-ischemia forelimb strength was significantly improved in animals receiving EPCs or CM, but not in those receiving vehicle media. In conclusion, we demonstrate for the first time that the administration of EPC-secreted factors could become a safe and effective cell-free option to be considered in future therapeutic strategies for stroke.

The angiogenic gene profile of circulating endothelial progenitor cells from ischemic stroke patients.

BACKGROUND: The identification of circulating endothelial progenitor cells (EPCs) has introduced new possibilities for cell-based treatments for stroke. We tested the angiogenic gene expression of outgrowth endothelial cells (OECs), an EPC subtype capable to shape vessel structures. METHODS: OECs (at colony or mature stages) from ischemic stroke patients (n=8) were characterized using the RT2 ProfilerTM human angiogenesis PCR Array, and human microvascular endothelial cells (hCMEC/D3) were used as an expression reference of endothelial cells. RESULTS: Colony-OECs showed higher expression of CCL2, ID3, IGF-1, MMP9, TGFBR1, TNFAIP2, TNF and TGFB1. However, BAI-1, NRP2, THBS1, MMP2 and VEGFC expression was increased in mature-OECs (p<0.05). ID3 (p=0.008) and TGFBR1 (p=0.03) genes remained significantly overexpressed in colony-OECs compared to mature-OECs or hCMEC/D3. MMP9 levels were significantly increased in colony-OECs (p=0.025) compared to mature-OECs. Moreover, MMP-2, VEGF-C, THBS1 and NRP-2 gene expression was also significantly increased in mature-OECs compared to hCMEC/D3 (p<0.05). Some of these genes were positively validated by RT-PCR. CONCLUSION: Our study shows that OECs from stroke patients present higher levels of pro-angiogenic factors at early stages, decreasing in mature OECs when they become more similar to mature microvascular endothelial cells.

A large screening of angiogenesis biomarkers and their association with neurological outcome after ischemic stroke.

BACKGROUND: The induction of angiogenesis after stroke may enhance neurorestorative processes. Our aim was to examine the endogenous angiogenesis balance and their association with long-term clinical outcome in ischemic stroke patients. METHODS: A total of 109 stroke subjects were included in the study. Firstly, plasma samples were obtained from control subjects (n = 26) and tPA-treated stroke patients (n = 29) at baseline (within 3h of symptoms onset), 1, 2, 12, 24h after tPA treatment, at discharge and 3 months after the ischemic event. Angiogenic promoters (PDGF-AA, PDGF-BB, HGF, FGF, KGF, HB-EGF, TPO, VEGF, VEGFR-1, VEGFR-2 and SDF-1α) and inhibitors (endostatin, angiostatin, thrombospondin-1 and thrombospondin-2) were analyzed by Searchlight(®) technology or ELISA. Additionally, baseline and 24h endostatin plasma level was determined in a new set of stroke patients (n = 80). Clinical parameters (NIHSS, mRS, mortality and hemorrhagic transformation events) were assessed to evaluate outcome. RESULTS: Baseline PDGF-BB, endostatin and thrombospondin-2 levels were higher in stroke patients than in controls (p < 0.05). A pro-angiogenic balance was associated with lower NIHSS scores and less intracranial hemorrhagic complications. Interestingly, a high baseline endostatin level was associated to long-term functional dependency (mRS > 2; p = 0.004). Finally, a baseline endostatin cut-off point of 184 ng/mL was an independent predictor of functional dependency at three months in the multiple logistic regression with an odds ratio of 8.9 (95% CI: 2.7-28.8; p = 0.0002). CONCLUSIONS: Our results indicate that an early pro-angiogenic balance is associated with mild short-term neurological deficit, while an acute anti-angiogenesis status determined by high endostatin plasma level predicts a worse long-term functional outcome.

Mobilization, endothelial differentiation and functional capacity of endothelial progenitor cells after ischemic stroke.

Endothelial progenitor cells (EPCs) have introduced new possibilities for cell-based vasculogenesis treatment after stroke. In this study we quantified circulating levels of EPCs in stroke patients and in healthy controls, and evaluated the potential of EPCs to induce vasculogenesis in vitro. Blood was drawn from tPA-treated stroke patients and control subjects, and the circulating EPCs levels in each group were quantified by flow cytometry and cell culture assays. Immunophenotyping was performed using multiple markers (UEA-lectin, CD133, vWF and KDR) and tubulogenic function was assessed with the Matrigel® assay. The produced angiogenic factors were quantified by multiple ELISA and RT-PCR. Fluorescence-activated cell sorting (FACS) revealed higher levels of circulating CD133+/CD34+/KDR+/CD45+ cells in the acute strokes as compared to the control subjects (p=0.02). On the other hand, more EPCs grew in cell culture from subacute strokes (p=0.016) than from controls. The endothelial and progenitor lineages of the EPCs were confirmed by immunophenotyping. Interestingly, the appearance of outgrowth EPCs (OECs) correlated positively to stroke severity (p=0.013). Finally, greater capacity to induce vasculogenesis in vitro was found in EPCs from subacute strokes (p=0.03), which we attribute to a higher expression and secretion of angiogenic factors. Our results suggest an early EPC mobilization but an enhanced angiogenic function in the subacute phase of stroke. Nonetheless, development of cell-based therapy for stroke will require further studies to identify those EPCs with the greatest therapeutic potential.

Catecholamine-induced heart injury in mice: differential effects of isoproterenol and phenylephrine.

The involvement of catecholamines in stress-induced heart injury is well documented. However, the contribution of adrenergic receptor types is less understood. Both the profile of plasma marker enzyme activities (lactate dehydrogenase-1 and aspartate transaminase) and the distribution and morphology of the lesions observed in tissue sections of adrenaline-injected mice resembled those of stressed (restraint and cold exposed) mice. Next, we compared the effect of isoproterenol (beta-adrenergic agonist) and phenylephrine (alpha1-adrenergic agonist) on both heart function and tissue injury. In Langendorff-perfused rat hearts, alpha1-adrenergic receptors made a minor contribution to the tonic effect of adrenaline, as indicated by the lack of effect on the heart rate and the delayed negative inotropic effect of phenylephrine. However, in whole mice, phenylephrine but not isoproterenol, induced an increase of both lactate dehydrogenase-1 and aspartate transaminase activities. Hearts of phenylephrine-injected mice showed necrotic lesions in subendocardial areas of the left ventricle. In addition a scattered focal leukocyte infiltration around single apoptotic-like myocytes was observed in the ventricle wall. Hearts of isoproterenol-injected mice showed a similar number of apoptotic-like myocytes, but a much lower number of necrotic areas, than phenylephrine-injected animals. Our results suggest that the cardiotonic effect of catecholamines involves mainly the beta-adrenergic receptors. However, the acute catecholamine-induced heart injury involves mainly alpha1-adrenergic receptors.

Role of endogenous granulocyte-macrophage colony stimulating factor following stroke and relationship to neurological outcome.

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is a proinflammatory cytokine with neuroprotective and angiogenic properties demonstrated in animal models of cerebral ischemia but their role in human ischemic stroke is still unknown. Thus, our aim is to determine human GM-CSF plasma level in control subjects and stroke patients and its relationship to clinical outcome. Forty-three patients with middle cerebral artery occlusion who received thrombolytic therapy within the first three hours of stroke onset and nineteen healthy controls were included. Blood samples were drawn before tissue plasminogen activator (t-PA) treatment. In a group of thirteen strokes blood samples were also obtained one hour after t-PA treatment, at 24 hours of symptoms onset, at discharge and at three months. GM-CSF levels were determined by enzyme-linked immunosorbent assay (ELISA). Stroke severity and neurological outcome was assessed by National Institutes of Health Stroke Scale (NIHSS) and functional outcome were scored by modified Rankin Scale (mRS) at 3 months. Baseline GM-CSF level was significantly higher in stroke patients than in healthy controls (17.8 pg/ml vs 12.8 pg/ml); p<0.0001 and was positively correlated with NIHSS score at 12 hours (R=0.3, p=0.03). No association was detected with functional status at three months measured by mRS. Temporary profile of GM-CSF level in stroke patients gradually decreases from admission to three months. Higher plasma endogenous GM-CSF level is found in stroke patients compared to controls. However, no relation was found with a better outcome. Further research is necessary for elucidating the role of GM-CSF in ischemic stroke.