Boyeon Lee

Perlecan domain V is neuroprotective and proangiogenic following ischemic stroke in rodents

Stroke is the leading cause of long-term disability and the third leading cause of death in the United States. While most research thus far has focused on acute stroke treatment and neuroprotection, the exploitation of endogenous brain self-repair mechanisms may also yield therapeutic strategies. Here, we describe a distinct type of stroke treatment, the naturally occurring extracellular matrix fragment of perlecan, domain V, which we found had neuroprotective properties and enhanced post-stroke angiogenesis, a key component of brain repair, in rodent models of stroke. In both rat and mouse models, Western blot analysis revealed elevated levels of perlecan domain V. When systemically administered 24 hours after stroke, domain V was well tolerated, reached infarct and peri-infarct brain vasculature, and restored stroke-affected motor function to baseline pre-stroke levels in these multiple stroke models in both mice and rats. Post-stroke domain V administration increased VEGF levels via a mechanism involving brain endothelial cell α5β1 integrin, and the subsequent neuroprotective and angiogenic actions of domain V were in turn mediated via VEGFR. These results suggest that perlecan domain V represents a promising approach for stroke treatment.

The Pyrethroid Pesticide Esfenvalerate Suppresses the Afternoon Rise of Luteinizing Hormone and Delays Puberty in Female Rats

Background One of the most widely used classes of insecticides is the synthetic pyrethroids. Although pyrethroids are less acutely toxic to humans than to insects, in vitro studies have suggested that pyrethroids may be estrogenic. Objectives We assessed pubertal effects by orally administering 0.5, 1.0, and 5.0 mg/kg/day of the type II pyrethroid esfenvalerate (ESF) to female rats beginning on postnatal day (PND) 22 until vaginal opening. ESF administration suppresses serum estradiol and delays pubertal onset. Materials and methods To assess possible hypothalamic and/or pituitary effects, animals received 0.5 or 1.0 mg/kg ESF or corn oil on PNDs 22–29. On PND30, we drew three blood samples (200 μL) from each rat at 15-min intervals beginning at 1000 hours, and again at 1500 hours. To test hypothalamic responsiveness, after the third afternoon sample, all animals received an intravenous injection of N-methyl-d,l-aspartic acid (NMA; 40 mg/kg), and then we drew two more samples. We performed a second experiment as above except that animals received luteinizing hormone–releasing hormone (LHRH; 25 ng/rat) to test pituitary responsiveness. Results Basal levels of luteinizing hormone (LH) in the afternoon hours were higher in control animals than in animals treated with 1.0 mg/kg ESF (p < 0.05). Furthermore, NMA- and LHRH-stimulated LH release was similar in control and ESF-treated animals, indicating that both hypothalamic and pituitary responsiveness, respectively, were unaffected. Conclusions Although the hypothalamus is able to respond to exogenous stimuli, absence of a normal afternoon rise in LH would indicate a hypothalamic deficit in ESF-treated animals.

Manganese stimulates luteinizing hormone releasing hormone secretion in prepubertal female rats: hypothalamic site and mechanism of action

We have shown recently that Mn2+ stimulates gonadotropin secretion via an action at the hypothalamic level, and a diet supplemented with a low dose of the element is capable of advancing the time of female puberty. In this study, we used an in vitro approach to investigate the mechanism by which Mn2+ induces luteinizing hormone‐releasing hormone (LHRH) secretion from prepubertal female rats. The medial basal hypothalamus from 30‐day‐old rats was incubated in Locke solution for 30 min to assess basal LHRH secretion, then incubated with buffer alone or buffer plus either a nitric oxide synthase (NOS) inhibitor (N‐monomethyl‐l‐arginine (NMMA); 300 or 500 μm) or a soluble guanylyl cyclase (sGC) inhibitor (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ); 100 or 250 μm) for another 30 min. Finally, the incubation continued for a further 30 min, but in the presence of MnCl2 (50 or 250 μm) to assess the effect of the blockers on stimulated LHRH secretion. Both 50 and 250 μm MnCl2 stimulated LHRH release (P < 0.05 and P < 0.01, respectively). The addition of 300–500 μm NMMA to the medium did not block Mn2+‐stimulated release of LHRH, even with the higher dose of MnCl2. Furthermore, while 50, 100 and 250 μm MnCl2 all significantly induced LHRH release, the two lowest doses did not stimulate total nitrite released from the same tissue, an effect only observed with the highest dose. Taken together, these data suggest that Mn2+ is not an effective stimulator of NO. Conversely, inhibiting sGC with ODQ blocked the Mn2+‐stimulated secretion of LHRH in a dose‐dependent manner, indicating that GC is the site of action of Mn2+. Additionally, we showed that Mn2+ stimulated cGMP and LHRH from the same tissues, and that downstream blocking of protein kinase G formation with KT5823 (10 μm) inhibited Mn2+‐induced LHRH release. These data demonstrate that the principal action of Mn2+ within the hypothalamus is to activate sGC directly and/or as a cofactor with available NO, hence generating cGMP and resulting in prepubertal LHRH release.

Perlecan Domain V Induces VEGf Secretion in Brain Endothelial Cells through Integrin α5β1 and ERK-Dependent Signaling Pathways

Perlecan Domain V (DV) promotes brain angiogenesis by inducing VEGF release from brain endothelial cells (BECs) following stroke. In this study, we define the specific mechanism of DV interaction with the α5β1 integrin, identify the downstream signal transduction pathway, and further investigate the functional significance of resultant VEGF release. Interestingly, we found that the LG3 portion of DV, which has been suggested to possess most of DV’s angio-modulatory activity outside of the brain, binds poorly to α5β1 and induces less BEC proliferation compared to full length DV. Additionally, we implicate DV’s DGR sequence as an important element for the interaction of DV with α5β1. Furthermore, we investigated the importance of AKT and ERK signaling in DV-induced VEGF expression and secretion. We show that DV increases the phosphorylation of ERK, which leads to subsequent activation and stabilization of eIF4E and HIF-1α. Inhibition of ERK activity by U0126 suppressed DV-induced expression and secretion of VEGR in BECs. While DV was capable of phosphorylating AKT we show that AKT phosphorylation does not play a role in DV’s induction of VEGF expression or secretion using two separate inhibitors, LY294002 and Akt IV. Lastly, we demonstrate that VEGF activity is critical for DV increases in BEC proliferation, as well as angiogenesis in a BEC-neuronal co-culture system. Collectively, our findings expand our understanding of DV’s mechanism of action on BECs, and further support its potential as a novel stroke therapy.

Perlecan domain V modulates astrogliosis in vitro and after focal cerebral ischemia through multiple receptors and increased nerve growth factor release.

Astrogliosis constitutes part of the central nervous systems physiological response to injury. Considered for decades to be a major challenge for brain repair, recent studies have highlighted it as a promoter of such repair mechanisms. Recently, our group demonstrated the ability of perlecan domain V (DV) to be a novel potential stroke therapy by its neuroprotective effects. However, the potential for DV to modulate astrogliosis has not been investigated. The aim of this study is to better understand the relevance of DV to astrogliosis using both in vitro and in vivo rodent models. Notably, under basal conditions, astrocytes express all three DV receptors described in the literature: integrin α2β1, α5β1, and α‐dystroglycan (αDG). DV promoted astrocyte cell adhesion, cell migration as well as astrocyte stellation. Moreover, DV induced nerve growth factor (NGF) secretion through a αDG‐ and ERK‐dependent pathway. In contrast, α2β1 or α5β1 mediated DV antiproliferative effects in astrocytes. NGF production after DV treatment acted as a strong anti‐proliferative agent. Another remarkable effect of DV was that it decreased several markers of astrogliosis such as glial fibrillary acidic protein (GFAP), neurocan and phosphacan both in vitro and in vivo, suggesting the role of DV as a potential modulator of postinjury during late astrogliosis, and eventually the onset of glial scarring. Taken together, our study demonstrates the ability of DV to modulate key events of astrogliosis by promoting early astrogliosis and inhibiting glial scar formation, suggesting an additional therapeutic benefit of DV for recovery from stroke.

Oxygen-glucose deprivation and interleukin-1α trigger the release of perlecan LG3 by cells of neurovascular unit.

J. Neurochem. (2011) 119, 760–771.

Endostatin binds nerve growth factor and thereby inhibits neurite outgrowth and neuronal migration in-vitro.

Endostatin (ES), the C-terminal fragment of collagen XVIII known for its anti-angiogenic properties, is associated with neurological diseases in mammals. In this study, we investigated the effect of ES on nerve growth factor (NGF)-induced neuronal differentiation, migration, neuritogenesis, and neurite extension. ES partially inhibited PC12 cell differentiation and cerebellar granule cell migration. In addition, neurite outgrowth was inhibited in a concentration-dependent manner. This effect was also matrix-dependent, as we observed better inhibition on PC12 cells grown on collagen compared to laminin matrices. Furthermore, we observed partial NGF depletion by collagen and ES, but not by laminin suggesting that NGF-matrix interactions may be important for promoting neuritogenesis, competitive inhibition by ES or low affinity matrix impairs PC12 differentiation and neurite outgrowth. Finally, using a biosensor technique, we demonstrated a direct interaction between NGF and ES suggesting the mechanism of action of ES may involve NGF sequestration. In conclusion, our study demonstrates the inhibitory effect of ES on different steps of neurogenesis including cell differentiation and migration and neuritogenesis by NGF sequestration. Such sequestration may compromise brain repair following injury, but also may play important role in axon finding as well as a potent therapeutical target in diseases involving abnormal elevated neurotrophic growth factor levels. Taken together, this study raises the consideration of ES as a double-edge sword that carries both deleterious and putative therapeutical effects.

Perlecan domain V inhibits α2 integrin-mediated amyloid-β neurotoxicity

Amyloid-β (Aβ) peptide is a key component of amyloid plaques, one of the pathological features of Alzheimers disease. Another feature is pronounced cell loss in the brain leading to an enlargement of the ventricular area and a decrease in brain weight and volume. Aβ plaque deposition and neuronal toxicity can be modeled by treating human cortical neuronal cultures with Aβ and showing robust Aβ deposition and neurotoxicity that is mediated by α2β1 and αvβ1 integrins. The current study expands on these findings by showing that the domain V of perlecan, a known α2 integrin ligand, inhibits Aβ neurotoxicity in an α2 integrin-dependent manner. Additionally, Aβ binds more efficiently to cells expressing activated α2 integrin. Finally the inhibition of Aβ neurotoxicity with domain V is synergistic with inhibitors of αv integrin and β1 integrin. We propose that domain V and potentially other α2 integrin ligands could be a new therapeutic approach for inhibiting the Aβ plaque deposition and neurotoxicity observed in Alzheimers disease.

Perlecan domain V is upregulated in human brain arteriovenous malformation and could mediate the vascular endothelial growth factor effect in lesional tissue.

&NA; Brain arteriovenous malformation (BAVM), a rare but important cause of intracranial hemorrhage, has increased angiogenesis and inflammation as key components of the nidus of abnormal vessels and stroma that form the resected surgical specimen. Accordingly, both vascular endothelial growth factor (VEGF) and transforming growth factor‐[beta] have been implicated in the pathology of BAVM for their proangiogenic and vascular‐regulating roles. The C‐terminal fragment of the extracellular matrix component perlecan (domain V, DV) has been shown to be increased and through the [alpha]5[beta]1 integrin, to increase VEGF levels in and around areas of cerebral ischemic injury, another proangiogenic condition. We aimed to determine whether the concentrations of DV, DVs proangiogenic receptor [alpha]5[beta]1 integrin, or DVs antiangiogenic receptor [alpha]2[beta]1 integrin are elevated in human BAVM tissue. DV levels were increased in BAVM compared with control brain tissue from epileptic resection, as was [alpha]5[beta]1 integrin. In addition, [alpha]5[beta]1 integrin was preferentially increased and localized to endothelial cells compared with [alpha]2[beta]1 integrin. VEGF and transforming growth factor‐[beta] levels were also increased in BAVM compared with control tissue. Furthermore, increases in all components were strongly correlated. Excessive generation of proangiogenic DV in BAVM suggests that DV may participate in its pathology and may represent a future therapeutic target.

Oxygen-glucose deprivation and interleukin-1α trigger the release of perlecan LG3 by cells of neurovascular unit: Release of perlecan LG3 by neurovascular unit

J. Neurochem. (2011) 119, 760–771.