Perlecan Domain-V Enhances Neurogenic Brain Repair After Stroke in Mice.

The extracellular matrix fragment perlecan domain V is neuroprotective and functionally restorative following experimental stroke. As neurogenesis is an important component of chronic post-stroke repair, and previous studies have implicated perlecan in developmental neurogenesis, we hypothesized that domain V could have a broad therapeutic window by enhancing neurogenesis after stroke. We demonstrated that domain V is chronically increased in the brains of human stroke patients, suggesting that it is present during post-stroke neurogenic periods. Furthermore, perlecan deficient mice had significantly less neuroblast precursor cells after experimental stroke. Seven-day delayed domain V administration enhanced neurogenesis and restored peri-infarct excitatory synaptic drive to neocortical layer 2/3 pyramidal neurons after experimental stroke. Domain V's effects were inhibited by blockade of α2ß1 integrin, suggesting the importance of α2ß1 integrin to neurogenesis and domain V neurogenic effects. Our results demonstrate that perlecan plays a previously unrecognized role in post-stroke neurogenesis and that delayed DV administration after experimental stroke enhances neurogenesis and improves recovery in an α2ß1 integrin-mediated fashion. We conclude that domain V is a clinically relevant neuroprotective and neuroreparative novel stroke therapy with a broad therapeutic window.

The potential role of perlecan domain V as novel therapy in vascular dementia.

Vascular dementia (VaD) is the second most common cause of dementia and leads to a decline in cognitive thinking via conditions that lead to blockage or reduced blood flow to the brain. It is a poorly understood disease, and the changes that occur are often linked to other types of dementia such as Alzheimer's disease. To date, there are no approved therapies or drugs to treat the symptoms of VaD, even though there is some evidence of drugs approved for Alzheimer's that might have some benefit in patients diagnosed with VaD. The altered blood flow that precedes VaD may result in compensatory mechanisms, such as angiogenesis, to increase blood flow in the brain. Angiogenesis, the process of new blood vessel formations from pre-existing ones, involves several pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and is regulated by a variety of growth factors from neurons, astrocytes, and pericytes in the brain as well the extracellular matrix (ECM). The ECM highly regulates angiogenesis and other processes in the brain. One such ECM component is the heparan sulfate proteoglycan perlecan and its bioactive region, Domain V (DV). Here we discuss the potential role of DV as a novel therapy to treat VaD.

Investigating the role of perlecan domain V in post-ischemic cerebral angiogenesis.

Cerebral angiogenesis is an important process for physiological events such as brain development, but it also occurs in pathological conditions such as stroke. Defined as the generation of new blood vessels from preexisting vasculature, angiogenesis after ischemic stroke is important to limit the subsequent neuronal injury and death, as well as contribute to neurorepair. However, current therapies for ischemic stroke are largely focused on reestablishing uninterrupted blood flow, an important but inherently risky proposition. Furthermore, these therapies can have limited efficacy due to narrow therapeutic windows, and in the case of mechanical clot removal, are invasive procedures. Therefore, better stroke therapies are needed. Since the brain possesses mechanisms, including angiogenesis, to attempt self-repair after injury, it may prove beneficial to look at how such mechanisms are regulated to identify potential targets for new and improved stroke therapies. Perlecan domain V (DV), an endogenous extracellular matrix protein fragment, may represent one such therapeutic target. Key to its appeal is that perlecan DV is endogenously and persistently generated in the brain after stroke and has significant angio-modulatory properties. These, and other properties, have been therapeutically manipulated to improve experimental stroke outcomes, suggesting that DV could represent a promising new stroke therapy. Here we discuss a novel approach to studying DV-mediated angiogenesis in vitro using a coculture model.

MG624, an α7-nAChR antagonist, inhibits angiogenesis via the Egr-1/FGF2 pathway.

Small cell lung cancer (SCLC) demonstrates a strong etiological association with smoking. Although cigarette smoke is a mixture of about 4,000 compounds, nicotine is the addictive component of cigarette smoke. Several convergent studies have shown that nicotine promotes angiogenesis in lung cancers via the α7-nicotinic acetylcholine receptor (α7-nAChR) on endothelial cells. Therefore, we conjectured that α7-nAChR antagonists may attenuate nicotine-induced angiogenesis and be useful for the treatment of human SCLC. For the first time, our study explores the anti-angiogenic activity of MG624, a small-molecule α7-nAChR antagonist, in several experimental models of angiogenesis. We observed that MG624 potently suppressed the proliferation of primary human microvascular endothelial cells of the lung (HMEC-Ls). Furthermore, MG624 displayed robust anti-angiogenic activity in the Matrigel, rat aortic ring and rat retinal explant assays. The anti-angiogenic activity of MG624 was assessed by two in vivo models, namely the chicken chorioallantoic membrane model and the nude mice model. In both of these experimental models, MG624 inhibited angiogenesis of human SCLC tumors. Most importantly, the administration of MG624 was not associated with any toxic side effects, lethargy or discomfort in the mice. The anti-angiogenic activity of MG624 was mediated via the suppression of nicotine-induced FGF2 levels in HMEC-Ls. MG624 decreased nicotine-induced early growth response gene 1 (Egr-1) levels in HMEC-Ls, and reduced the levels of Egr-1 on the FGF2 promoter. Consequently, this process decreased FGF2 levels and angiogenesis. Our findings suggest that the anti-angiogenic effects of MG624 could be useful in anti-angiogenic therapy of human SCLCs.

The α7-nicotinic acetylcholine receptor and MMP-2/-9 pathway mediate the proangiogenic effect of nicotine in human retinal endothelial cells.

PURPOSE: Nicotine, the active component of cigarette smoke, has been found to stimulate angiogenesis in several experimental systems. In this study, the Matrigel duplex assay (Matrigel; BD Biosciences, Franklin Lakes, NJ) and the rat retinal explant assay were used to explore the molecular mechanisms underlying the proangiogenic effects of nicotine in endothelial cells. METHODS: Western blot analysis was performed to determine the nicotinic acetylcholine receptor (nAChR) subtypes expressed on primary human retinal microvascular endothelial cells (HRMECs). The angiogenic effect of nicotine in the retina was evaluated with the duplex assay. The results obtained from the assay were confirmed by the rat retinal explant angiogenesis assay. ELISAs were used to measure MMP-2, -9, and -13 levels in HRMEC culture supernatants. The role of α7-nAChRs in nicotine-induced angiogenesis was examined by siRNA techniques. RESULTS: Nicotine-induced angiogenesis required nAChR function and was associated with the upregulation of MMP-2 and -9 in HRMECs. Specifically, α7-nAChRs mediated the stimulatory effects of nicotine on retinal angiogenesis and MMP levels. Treatment of HRMECs with α7-nAChR antagonists ablated nicotine-induced angiogenesis. The inhibitory actions of α7-nAChR antagonists correlated with the suppression of MMP-2 and -9 levels in HRMECs. CONCLUSIONS: The α7-nAChR is vital for the proangiogenic activity of nicotine. The α7-nAChRs expressed on HRMECs upregulate levels of MMP-2 and -9, which stimulate retinal angiogenesis. The data also suggest that α7-nAChR antagonists could be useful agents for the therapy of angiogenesis-related retinal diseases.

Thimerosal induces apoptosis in a neuroblastoma model via the cJun N-terminal kinase pathway.

The cJun N-terminal kinase (JNK)-signaling pathway is activated in response to a variety of stimuli, including environmental insults, and has been implicated in neuronal apoptosis. In this study, we investigated the role that the JNK pathway plays in neurotoxicity caused by thimerosal, an ethylmercury-containing preservative. SK-N-SH cells treated with thimerosal (0-10 microM) showed an increase in the phosphorylated (active) form of JNK and cJun with 5 and 10 microM thimerosal treatment at 2 and 4 h. To examine activator protein-1 (AP-1) transcription, cells were transfected with a pGL2 vector containing four AP-1 consensus sequences and then treated with thimerosal (0-2.5 microM) for 24 h. Luciferase studies showed an increase in AP-1 transcriptional activity upon thimerosal administration. To determine the components of the AP-1 complex, cells were transfected with a dominant negative to either cFos (A-Fos) or cJun (TAM67). Reporter analysis showed that TAM67, but not A-Fos, decreased AP-1 transcriptional activity, indicating a role for cJun in this pathway. To assess which components are essential to apoptosis, cells were treated with a cell-permeable JNK inhibitor II (SP600125) or transfected with TAM67, and the downstream effectors of apoptosis were analyzed. Cells pretreated with SP600125 showed decreases in activation of caspases 9 and 3, decreases in degradation of poly(ADP-ribose) polymerase (PARP), and decreased levels of proapoptotic Bim, in comparison to cells treated with thimerosal alone. However, cells transfected with TAM67 showed no changes in those same components. Taken together, these results indicate that thimerosal-induced neurotoxicity occurs through the JNK-signaling pathway, independent of cJun activation, leading ultimately to apoptotic cell death.