Pengtao Li

Vascular endothelial growth factor signaling implicated in neuroprotective effects of placental growth factor in an in vitro ischemic model

Placental growth factor (PlGF) is involved in the angiopoiesis of the placental chorion and the maintenance of the placenta. Some additional roles of PlGF in other tissues have recently been described. Relatively little is known about PlGF expression in the CNS and the involvement of PlGF in cerebral ischemia injury. We examined the expression of PlGF in cerebral ischemia, utilizing a permanent middle cerebral artery occlusion (MCAO) model in the rat. PlGF expression and release from brain microvascular endothelial cells (BMECs) in response to oxygen and glucose deprivation (OGD) were examined in primary culture. To elucidate the effects of PlGF in cerebral ischemic injury, we investigated the effects of varying concentrations of PlGF upon neurons in an in vitro model of OGD. The effects of PlGF upon neuronal vascular endothelial growth factor receptor-1 (VEGFR-1) and vascular endothelial growth factor receptor-2 (VEGFR-2) expression were examined. We detected PlGF immunoreactivity mainly in the microvessels and interstitum of rat brain cortex after cerebral ischemic injury. In primary BMECs, PlGF expression and release were significantly higher under OGD conditions in culture. In primary cultures of rat cortical neurons, PlGF administration reduced cell death in an in vitro model of OGD. VEGFR-1 and VEGFR-2 were expressed in primary cortical neurons as measured by Western blotting. VEGFR-2 expression in primary neurons was significantly higher following PlGF administration. These data demonstrate that VEGFR-2 signaling may play a role in PlGF-mediated neuroprotection, and that PlGF may be a promising target for therapeutic intervention in ischemic injury.

The impact of paracrine signaling in brain microvascular endothelial cells on the survival of neurons

Neurons depend for survival on local neurotrophic factors which act in an autocrine/paracrine manner. However, the effect of paracrine signaling of brain microvascular endothelial cells (BMECs) under pathological conditions on neuron survival is not fully understood. In this study we cultured rat BMECs and cortical neurons. BMECs were cultured in oxygen-glucose-deprived (OGD) conditions to mimic cerebral ischemia in vitro. The conditioned media of normal BMECs or OGD-injured BMECs were used to culture normal or injured neurons. Neuron activity, free Ca(2+) concentration, NMDA receptor status, mitochondrial membrane potential and cytochrome C release level were determined. The results showed: mitochondrial activity of injured neurons was significantly increased and lactate dehydrogenase (LDH) leakage was decreased (P<0.05) by grown in conditioned medium of normal BMECs. Inversely, mitochondrial activity of normal or injured neurons was decreased and LDH leakage was significantly increased (P<0.05) by grown in conditioned medium of injured BMECs. The changes in free Ca(2+) concentration, NMDA receptor status, mitochondrial membrane potential and cytochrome C release level were consistent with the changes in neuronal activity. These findings suggest that the conditioned medium of normal BMECs has a neuroprotective effect. However, this protective effect was lost after BMECs injury; in fact, the conditioned medium became neurotoxic. Therefore, it appears that early recovery of BMECs might be helpful for neuron survival.

The interaction of amyloid β and the receptor for advanced glycation endproducts induces matrix metalloproteinase-2 expression in brain endothelial cells.

The pathological hallmarks of Alzheimer’s disease (AD) include formation of extracellular amyloid-β peptide (Aβ) and inflammatory responses. Numerous studies have reported that cerebral microvascular Aβ deposition promotes neuroinflammation in AD. Matrix metalloproteinases (MMPs) are involved in the cleavage of extracellular matrix proteins and regulation of growth factors, receptors, and adhesion molecules. Relatively little is known about the involvement of MMPs as inflammatory mediators in the pathological processes of AD. In this study, we explored the signaling pathway of MMP-2 up-regulation by Aβ in brain endothelial cells (BECs) of mice. Using Western blots, we found that inhibitors of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) significantly decreased Aβ-induced MMP-2 expression in BECs. Furthermore, antibody neutralization of the receptor for advanced glycation endproducts effectively blocked Aβ-induced activation of ERK and JNK and their contribution to elevated MMP-2 expression in BECs. Our results suggest that increased MMP-2 expression induced by the interaction of Aβ with RAGE in BECs may contribute to enhanced vascular inflammatory stress in Aβ-related vascular disorders, such as cerebral amyloid angiopathy and AD. This study offers new insights into neuroinflammation in the progression of AD.

Effect of Tong Luo Jiu Nao on Aβ-degrading enzymes in AD rat brains.

ETHNOPHARMACOLOGICAL RELEVANCE Tong Luo Jiu Nao (TLJN) is a modern Chinese formula based on Traditional Chinese Medicine theory that has been used to treat ischemic cerebral stroke and vascular dementia. TLJN belongs to the ethnopharmacological family of medicines. In this study, we investigated the mechanism of the TLJN effect on Alzheimers disease (AD). AIM OF THE STUDY To investigate the effect of TLJN on β-amyloid-degrading enzymes and learning and memory in the AD rat brain. MATERIALS AND METHODS AD rats whose disease was induced by Aβ(25-35) injection into the bilateral hippocampus CA1 region were subjected to intragastric administration of various preparations. The experimental animals were healthy male Sprague-Dawley rats which were randomly divided into normal, sham, model, TLJN min, TLJN max and donepezil hydrochloride groups. Spontaneous alternation and passive avoidance behavior, which are regarded as measures of spatial learning and memory, were investigated using Y-maze testing. Western blotting and immunohistochemistry were used to observe the therapeutic effect of TLJN on the deposits of amyloid plaque and on the expression of synaptophysin, insulin-degrading enzyme and neprilysin. RESULTS Y-maze results showed that the AD model group presented with spatial learning and memory impairments. Hematoxylin-eosin and Congo red staining indicated neuronal impairment and deposits of amyloid plaque in the model group and these results were consistent with their learning and memory deficits in the Y-maze. The TLJN-treated groups exhibited prolonged a cavity delitescence, decreased arm entries and improvement in learning and memory. Moreover, the structure of the neurons of the treated groups was restored and the expression of synaptophysin increased in both the hippocampus and cortex. In addition, their levels of insulin-degrading enzyme and neprilysin in the cortex and hippocampus were upregulated and the amyloid plaque was decreased. CONCLUSION TLJN can improve learning and memory, up-regulate insulin-degrading enzyme and neprilysin levels, promote the degrading of Aβ and clear amyloid plaque from the AD rat brain. In future, TLJN may have significant therapeutic potential in the treatment of AD patients.

Insulin-like growth factor-1 secreted by brain microvascular endothelial cells attenuates neuron injury upon ischemia

Insulin‐like growth factor (IGF)‐1 is essential for the development of the nervous system, and is present in many cell types. Relatively little is known about IGF‐1 expression in brain microvascular endothelial cells (BMECs). For in vivo studies, we examined the expression of IGF‐1 and insulin‐like growth factor‐binding protein (IGFBP)‐2 after focal cerebral ischemia for 12 h, 24 h, 3 days and 7 days, utilizing a permanent middle cerebral artery occlusion (MCAO) model in rats. For in vitro studies, we examined the levels of IGF‐1 and IGFBP‐2 in the culture medium or primary culture of BMECs injured by oxygen–glucose deprivation (OGD). Then, we elucidated the protective effects of IGF‐1 on cortical neurons injured by OGD and the possible mechanism. In addition, we investigated the effect of BMEC‐conditioned medium on IGF‐1 receptor expression in neurons. The results showed that IGF‐1 expression increased in serum and brain tissue, whereas IGFBP‐2 expression decreased in brain tissue of MCAO‐injured rats. In primary culture of BMECs, the expression levels of IGF‐1 and IGFBP‐2 were significantly higher under OGD conditions in culture. IGF‐1 administration improved neuron viability upon normoxia or OGD, and upregulated p‐Akt expression. This effect was reversed by LY294002, a specific inhibitor of the phosphoinositide 3‐kinase–Akt signaling pathway. Furthermore, conditioned medium from OGD‐treated BMECs substantially suppressed neuron viability and the expression of IGF‐1 receptor simultaneously. These data demonstrate that therapeutic strategies that prioritize the early recovery of the IGF‐1 system in BMECs might be promising in ischemic injury.

Brain microvascular endothelial cells mediate neuroprotective effects on ischemia/reperfusion neurons

AIM OF THE STUDY The permeability of the blood-brain barrier (BBB) is a bottleneck for the development of new cerebropathy medications because the medication must be transmitted across the BBB to achieve its curative function. To explore a new approach to the treatment of brain disease, this study investigated the mediating effects of brain microvascular endothelial cells (MVECs) on injured neurons. MATERIALS AND METHODS MVECs and cortical neurons were cultured and damage by cerebral ischemia/reperfusion (I/R) was simulated. The conditioned media from four groups of MVECs - normal cells (N-CM), normal cells treated with Tong Luo Jiu Nao (TLJN) (NT-CM), simulated cerebral I/R cells (I/R-CM), and simulated cells treated with TLJN (I/RTCM) - were then collected. These conditioned media were added to neuronal cultures and the viability of the neurons was examined. RESULTS The results demonstrated that N-CM could alleviate I/R damage to neurons, and this capacity could be improved by TLJN treatment. However, I/R-CM could cause damage to normal and I/R neurons, while I/RT-CM could significantly alleviate the damage to I/R neurons. CONCLUSIONS We propose that MVECs secrete active substances that influence the survival of neurons, and so MVECs may mediate a neuroprotective effect on ischemia/reperfusion neurons.

Tong Luo Jiu Nao injection, a traditional Chinese medicinal preparation, inhibits MIP-1β expression in brain microvascular endothelial cells injured by oxygen-glucose deprivation.

ETHNOPHARMACOLOGICAL RELEVANCE Tong Luo Jiu Nao injection (TLJN), a modern Chinese formula based on Traditional Chinese Medicine theory, has been used to treat ischemic stroke and vascular dementia. TLJN belongs to the ethnopharmacological family of medicines. AIM OF THE STUDY To investigate the protective effect of TLJN on oxygen-glucose deprivation (OGD) induced-injury of brain microvascular endothelial cells (BMECs). MATERIALS AND METHODS The model of OGD was established in the primarily cultured BMECs. TLJN was added to the OGD-injured BMECs for 6h. A series of assays were used to detect the effects of TLJN on: (i) MIP-1β content in BMECs conditioned media (CM) by ELISA; (ii) MIP-1β protein expression in BMECs by western blotting and immunocytochemistry; (iii) the expression of CCR5, receptor of MIP-1β, in BMECs by western blotting; (iv) the proliferative activity of microglial cells via the Cell Counting Kit-8 (CCK-8). RESULTS Our results showed that the OGD-injured BMECs presented with large amounts of secretion and expression of MIP-1β and up-regulation of CCR5. Also, the CM of OGD-injured BMECs remarkably increased the proliferative activity of microglial cells. The TLJN-treated BMECs exhibited a reduction of MIP-1β content in BMECs-CM and a down-regulation of MIP-1β and CCR5 expression. In addition, an inhibitory effect of CM of OGD-injured plus TLJN injection-treated BMECs on microglial proliferation was also found. CONCLUSION TLJN reduced the expression of MIP-1β and CCR5 in OGD-injured BMECs, and the CM of OGD-injured plus TLJN injection-treated BMECs inhibited the proliferative activity of microglial cells, suggesting the therapeutic potential of TLJN on ischemic cerebral vascular disease.

Impact of paracrine signals from brain microvascular endothelial cells on microglial proliferation and migration.

Neuronal survival can be influenced by activated microglia, but limited evidence exists on the effects of paracrine signaling from brain microvascular endothelial cells (BMECs) on microglial action. Therefore, we examined the effects of normal BMECs conditioned medium (BMECs-CM) on activated microglia induced by pro-inflammatory cytokine macrophage inflammatory protein-1beta (MIP-1β), a chemokine that released from ischemic BMECs and has been proved to stimulate microglial proliferation. Our results showed that BMECs-CM inhibited the proliferation and transmigration of microglia induced by MIP-1β. Moreover, BMECs-CM significantly suppressed the expression of the MIP-1β receptor, CCR5, and the phosphorylation of p38 and JNK (P<0.05). These findings suggest that BMECs-CM could inhibit MIP-1β-induced microglial activation. Future therapeutic strategies that prioritize the early recovery of BMECs could be beneficial for microglial inhibition and further increase neuronal survival.

High-dose chlorogenic acid induces inflammation reactions and oxidative stress injury in rats without implication of mast cell degranulation

ETHNOPHARMACOLOGICAL RELEVANCE Chlorogenic acid (CA) exits widely in those Chinese herbal injections that have antibacterial and antiphlogistic effects and belongs to the ethnopharmacological family of medicines. Chinese herbal injections containing high levels of CA have been reported to increase the adverse drug reactions, but the mechanism for which is still unclear. In this study, we investigated the mechanism of the CA derived adverse drug reactions. AIM OF THE STUDY The present study was to explore the potential role of CA in initiating inflammatory reaction and the underlying mechanism. MATERIALS AND METHODS Male Wistar rats were treated with different dosages of CA for different time period. The variables examined included microcirculation by intravital microscopy, histology of ileum tissue, expression of adhesion molecules CD11b and CD18 on leukocytes by flow cytometry, myeloperoxidase activity and maleic dialdehyde content in ileum tissue by spectrophotometry, activity of superoxide dismutase and catalase in serum by ELISA, and expression of NADPH oxidase subunits by PCR and Western blot. RESULTS High-dose CA increased the number of adherent leukocytes, generation of peroxides in the venular walls and induced albumin leakage from mesentery venules. High-dose CA induced changes also included an increase in maleic dialdehyde, myeloperoxidase, inflammatory cytokines and NADPH oxidase activities, and a decline in activity of superoxide dismutase and catalase. CONCLUSION High-dose, but not Low-dose CA induced inflammation reaction, and in this process an imbalance between oxidant and antioxidant mechanism may be involved, providing more information for better understanding the rationale behind the adverse effects of CA.

Neuroprotective effects of tongluojiunao in neurons exposed to oxygen and glucose deprivation.

ETHNOPHARMACOLOGICAL RELEVANCE Tongluojiunao (TLJN) is an herb extract that mainly contains ginsenoside Rg1 and geniposide, which are clinically used for treating ischemic damages in the brain. AIM OF THE STUDY In the stroke, cerebral ischemia followed by oxygen reperfusion induced apoptosis in hippocampal neurons, while extension of axons and dendrites in neurons may compensate for and repair damages of neuronal network in the hypoxia brain. In this study, we investigated whether TLJN can protect neurons against damages by ischemia in brain vasculature. MATERIALS AND METHODS We measured cell viability and lactate dehydrogenase (LDH) release from primary culture of rat hippocampal neurons before and after the neurons were deprived of oxygen and glucose (OGD). In addition, the effects were evaluated with cell viability and neurite outgrowth before or after OGD. RESULTS We found that TLJN could play a neuroprotective role to cultured primary rat hippocampal neurons under both normal and oxygen/glucose-deprivation (OGD) conditions. TLJN could protect both cultured primary rat hippocampal neurons and brain microvascular endothelial cells (BMECs) from cell death under both normal and oxygen/glucose-deprivation (OGD) conditions. Moreover, under the same conditions, BMECs-conditioned media pretreated by TNJN could also promote neuron viability and neurite outgrowth, indicating that TLJN stimulated BMECs to secret some neuroprotective/neurotrophic factors. CONCLUSION These findings suggest that TLJN has a marked neuroprotective and neurotrophic roles by either direct or indirect operation, and provide insight into the mechanism of clinical efficacy of this drug against stroke.