Current Neurovascular Research

ISSN: 1567-2026

Current Neurovascular Research
Volume 3, Number 4, November 2006

Contents

Cellular Balance, Genes, and the Huang Ti Nei Ching Su Wen Pp. 247-248
K. Maiese
[Abstract]


ORIGINAL ARTICLES


NRF2-Dependent Glutamate-L-Cysteine Ligase Catalytic Subunit Expression Mediates Insulin Protection Against Hyperglycemia- Induced Brain Endothelial Cell Apoptosis Pp. 249-261
M. Okouchi, N. Okayama, J.S. Alexander and T. Yee Aw
[Abstract]


Exercise Preconditioning Reduces Brain Damage and Inhibits TNF-α Receptor Expression after Hypoxia/Reoxygenation: An In Vivo and In Vitro Study Pp. 263-271
Y-H. Ding, M.l Mrizek, Q. Lai, Y. Wu, R. Reyes, Jr., J. Li, W.W. Davis and Y. Ding
[Abstract]


Dehydroepiandrosterone Treatment Alters Lipid/Phospholipid Profiles of Rat Brain and Liver Mitochondria Pp. 273-279
S.S. Katyare, H.R. Modi and M.A. Patel
[Abstract]


Derivation of Motor Neurons from three Clonal Human Embryonic Stem Cell Lines Pp. 281-288
U-M. Lim, K.S. Sidhu and B.E. Tuch
[Abstract]


Classic β-Amyloid Deposits Cluster Around Large Diameter Blood Vessels Rather than Capillaries in Sporadic Alzheimer's Disease Pp. 289-294
R.A. Armstrong
[Abstract]


REVIEW ARTICLES


Neuronal Semaphorins Regulate a Primary Immune Response Pp. 295-305
S. Moretti, A. Procopio, M. Boemi and A. Catalano
[Abstract]


Central Nervous System Circuitry and Peripheral Neural Sympathetic Activity Responsible for Essential Hypertension Pp. 307-325
F. Lechin and B. van der Dijs
[Abstract]




Abstracts

[Back to top]
Cellular Balance, Genes, and the Huang Ti Nei Ching Su Wen

The development of alternative therapies as well as strategies directed to prevent the onset of new illnesses is rapidly taking hold in Western Medicine and is evident not only at a more "grass roots" level with individual care takers, but also on a national scale with the introduction of centers such as the National Center for Complementary and Alternative Medicine of the National Institutes of Health. Interestingly, the origins of such medical disciplines may have begun with the great text of ancient Chinese Medicine known as Huang Ti Nei Ching Su Wen "The Yellow Emperor's Classic of Internal Medicine". The Nei Ching is considered by many to be one of the oldest if not the first medical text, but precise dating and documentation of its initial introduction remain in question. From the beginning of the existence of the text, the author of the book was believed to be one of China's initial rulers during the final centuries B.C.E., the Yellow Emperor Huang Ti, who is worshipped as the father of Chinese Medicine. Since multiple authors are believed to have contributed to the work over a course of the next hundreds of years, the present text with its translation into a Western interpretation may possibly stray by a large extent from the original version.

The Nei Ching begins with a discussion between the Yellow Emperor Huang Ti and his Minister Ch'i Pai to introduce the philosophy of Chinese Medicine, such as the concepts of the five agents doctrine and the opposing effects of the Yin and Yang that states opposite ends of a spectrum are vital for change as well as internal balance. These concepts are believed to illuminate the visionary aspects of the work that focused upon the climate, the environment, and behavior as precipitants of disease and the primary natural laws that govern illness. Furthermore, the objective to preserve and protect an individual's well being began to surface with descriptions that outline methods to normalize emotions, employ balanced diets, maintain personal hygiene, consume clean water and fresh food, and to incorporate regular exercise into one's daily regimen.

Many of the teachings of the Nei Ching appear to have seamlessly transcended time, especially with today's health care and the intense focus upon disease prevention. For example, bolstered by sophisticated clinical and basic research studies, regular exercise is recommended as preventative therapy for all individuals, irrespective of one's age. The incorporation of daily physical activity into an individual's daily schedule has been shown, or at least strongly suggestive for some conditions, to prevent a multitude of disorders that range from cardiovascular disease to dementia and may significantly reduce the risk of premature death. Physical fitness can involve aerobic exercise that requires the body to transport and use oxygen during exercise or anaerobic exercise that produces energy in the absence of oxygen. Although it has been noted that an individual's well being can be improved with minimal energy expenditures as low as 700 kcal per week, an average energy expenditure of approximately 200 kcal per day is recommended for improved cardiac, musculoskeletal, and mental function that is adjusted for any pre-existing physical disabilities.

The advantages of daily exercise may be evident over time through an individual's enhanced cardiovascular performance, cognitive function, or weight management, but the initial benefits must begin at the cellular level to eventually be translated into improved clinical well being and the possible prevention of disease. In this issue of Current Neurovascular Research, both our original articles and review papers elucidate novel mechanisms that potentially are required for cellular homeostasis and overall clinical health maintenance. For example, when one considers preventive therapeutic strategies to block cell injury during disorders such as diabetes that can lead to chronic elevations in glucose, Okouchi et al. demonstrate that the cytoprotective effects of insulin to maintain human brain endothelial cell survival may rest heavily upon cellular parameters that regulate phosphatidylinositol 3-kinase signaling and nuclear NF-E2-related factor 2 translocation to maintain cellular redox balance. Interestingly, another preventive treatment modality necessary for diabetics in addition to the sometimes required insulin administration is a daily exercise program. Ding et al. show that one potential benefit from exercise, at least in a model of focal cerebral ischemia, occurs initially at the cellular level. In animals subjected to daily treadmill activity for thirty minutes over a three week course, brain infarct size is remarkably decreased. These effects correspond to the reduced expression of tumor necrosis factor-α receptors that are also present in human endothelial cells, suggesting that enhanced physical and metabolic activity directly alters cellular genetic immune mediated pathways that can be beneficial not only during normal physiology, but also lead to improved tolerance against neuronal and vascular insults of the brain.

Yet, one cannot assume that administration of a particular therapeutic regiment will alter intracellular pathways in a similar manner or to the same degree in every cell. Katyare et al. bring such knowledge to light with their investigation of dehydroepiandrosterone (DHEA). Given that DHEA levels are reduced with advancing age and may be associated with the onset of cognitive loss during disorders such as Alzheimer's disease, supplemental therapy with DHEA has been on occasion advocated for the prevention of cognitive loss in the elderly. In their paper, Katyare et al. illustrate that DHEA increases total phospholipids and cholesterol in brain mitochondria, but does not significantly alter total phospholipids or cholesterol in liver mitochondria. With such observations, it is important to note that what may be considered preventive or protective therapy for one organ system may sometimes not convey benefit or be adverse to other tissues. However, Lim et al. may shed further light on "cell specific" therapeutics and drug discovery with their description of the ability to target and differentiate human embryonic stem cell lines to an enriched population of spinal motor neurons. Furthermore, changes in intracellular signaling pathways most likely represent only one of a number of factors that can determine the course that leads either to disease protection or disease progression. For example, in the original work by Armstrong, the author provides evidence in patients with Alzheimer's disease of a close spatial relationship between amyloid plaque deposition and large diameter cerebral vessels. These observations may suggest impaired perivascular clearance of the brain or the diffusion of protein through a dysfunctional vascular system leading to the potentially toxic accumulation of amyloid, echoing the significance of environmental factors described in the Nei Ching, although in this case at the cellular level, that can play a significant role during disease states.

In the review article by Moretti et al., we gain further insight into the broad role of the immune system not only during exercise with acute brain injury as described above, but also during chronic inflammatory processes associated with multiple sclerosis and myelopathies. The authors offer an exciting perspective of the semaphorin system that controls a broad array of functions to include axonal guidance, endothelial immune function, and T cell modulation and suggest that targeting the semaphorin system may be applicable for a variety of disorders. Our subsequent review paper by Lechin and van der Dijs further complements this issue of Current Neurovascular Research. This extensive analysis of essential hypertension reinforces for us the intimate relationship among cellular signaling pathways, the anatomical structures, and the combined central nervous system circuitry that must be considered for both the prevention and treatment of any disorder.

Despite the complexity and sophistication of current investigative work that attempts to define disease processes and develop effective treatment strategies for these disorders, our essential roots appear to always return to basic principles that may very well have been outlined in the initial versions of the Nei Ching. Clearly the present concentration on preventive and complimentary therapies had its early beginnings with the Yellow Emperor Huang Ti. As we attempt to acquire further insight into a variety of disease mechanisms, one could argue that studies should not digress significantly from the early work of the Nei Ching with its timeless analysis of the Yin and Yang concept, the description of the human body and its close association to the environment, and the principal foundations offered for the prevention, diagnosis, and treatment of disease.


Kenneth Maiese
Editor-in-Chief


[Back to top]
NRF2-Dependent Glutamate-L-Cysteine Ligase Catalytic Subunit Expression Mediates Insulin Protection Against Hyperglycemia- Induced Brain Endothelial Cell Apoptosis
M. Okouchi, N. Okayama, J.S. Alexander and T. Yee Aw

Increased oxidative stress and susceptibility of brain endothelium are contributing factors in the development of central nervous system complications in neurodegenerative disorders in diabetes, Alzheimer’s and Parkinson’s disease. The molecular mechanisms underpinning the vulnerability of brain endothelial cells to chronic oxidative challenge have not been elucidated. Here, we investigated the oxidative susceptibility of human brain endothelial cells (IHEC) to chronic hyperglycemic stress and insulin signaling and cytoprotection. Chronic hyperglycemia exacerbated IHEC apoptosis in accordance with exaggerated cytosolic and mitochondrial glutathione and protein-thiol redox imbalance, and actin/Keap-1 S-glutathionylation. Insulin attenuated hyperglycemia-induced apoptosis via restored cytosolic and mitochondrial redox. Insulin stimulated glutamate-L-cysteine ligase (GCL) activity by activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling, increased serine phosphorylation and nuclear translocation of nuclear NF-E2-related factor 2 (Nrf2), and upregulation of Nrf2-dependent GCL-catalytic (GCLc) subunit expression. Expression of the GCL-modulatory subunit (GCLm) was unchanged. Inhibitors of insulin receptor tyrosine kinase, PI3K, Akt and mTOR abrogated insulin-induced Nrf2-mediated GCLc expression, redox balance, and IHEC survival. Collectively, these results demonstrate that human brain endothelial cells exhibit vulnerability to hyperglycemic stress which is associated with marked cytosolic and mitochondrial redox shifts. Activation of insulin signaling through PI3K/Akt/mTOR/Nrf2/ GCLc pathway affords significant cell protection by maintaining cellular redox balance.


[Back to top]
Exercise Preconditioning Reduces Brain Damage and Inhibits TNF-α Receptor Expression after Hypoxia/Reoxygenation: An In Vivo and In Vitro Study
Y-H. Ding, M.l Mrizek, Q. Lai, Y. Wu, R. Reyes, Jr., J. Li, W.W. Davis and Y. Ding

Exercise reduces ischemia and reperfusion injury in rat stroke models. We investigated whether gradual increases in tumor necrosis factor-α (TNF-α) reported during exercise down-regulates expression of TNF-α receptors I and II (TNFRI and II) in stroke, leading to reduced brain damage. Adult male Sprague Dawley rats were subjected to 30 minutes of exercise on a treadmill each day for 3 weeks. Then, stroke was induced by a 2-hour middle cerebral artery (MCA) occlusion using an intra-luminal filament. Expressions of TNFRI and II mRNA in the brain were detected using a real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Protein expressions of TNFRI and II were determined by enzyme-linked immunoabsorbant assay (ELISA) in serum and brain homogenates. Spatial distribution of TNF-α receptors in brain regions was determined with immunocytochemistry. In human umbilical vein endothelial cells (HUVEC), we addressed the causal effect of TNF-α pretreatment on TNF I and II expression using ELISA and real-time PCR. In exercised rats after stroke, brain infarct was significantly (p<0.01) reduced in the entire MCA supplied regions, associated with a mild expression of TNFRI and II mRNA and protein. The TNF-α receptors were restricted to the ischemic core. In contrast, a robust expression of TNFRI and II molecules was found in non-exercised rats subjected to similar ischemia/reperfusion insults. An in vitro study revealed a causal link between TNF-α pretreatment and reduced cellular expression of TNF-α receptors under hypoxic/reoxygenated conditions. Our results suggest that reduced-brain damage in ischemic rats after exercise preconditioning may be attributable to the reduced expression of TNF-α receptors. Chronically increased TNF-α expression was also found to reduce TNFI and II responding to acute ischemia/reperfusion insult.


[Back to top]
Dehydroepiandrosterone Treatment Alters Lipid/Phospholipid Profiles of Rat Brain and Liver Mitochondria
S.S. Katyare, H.R. Modi and M.A. Patel

Stimulation of mitochondrial function following treatment with dehydroepiandrosterone (DHEA) has been demonstrated. Since the activity of several electron transport chain components is dependent on specific lipid/phospholipid components, we examined the effects of DHEA treatment (0.1-2.0 mg/kg body weight for 7 consecutive days) on lipid/phospholipids profiles of rat brain and liver mitochondria. In the brain mitochondria, contents of both total phospholipids (TPL) and cholesterol (CHL) increased. The major effect on phospholipids profile was increase in the contents of lysophospholipids (Lyso) and sphingomyelin (SPM) component followed by phosphatidylinositol (PI) and phosphatidylserine (PS). The contents of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and diphosphatidylglycerol (DPG) were not affected. At the higher dose (2.0 mg) the observed effects declined. The TPL and CHL contents of liver mitochondria were generally unchanged by DHEA treatment. Under this condition the content of PI and PS increased. The contents of other phospholipid components were not changed. Our results suggest that the observed changes may complement the function of electron transport chain components.


[Back to top]

Derivation of Motor Neurons from three Clonal Human Embryonic Stem Cell Lines
U-M. Lim, K.S. Sidhu and B.E. Tuch

Human embryonic stem cells (hESC) demonstrate a remarkable proliferative and developmental potential and thus have huge therapeutic potential. To direct the differentiation of hESC to a specific lineage of high purity for cell transplantation is highly desirable. Here we describe a modified in vitro procedure to direct differentiation of three clonal hESC lines, hES 3.1, hES 3.2 and hES 3.3 efficiently to spinal motor neurons by using various differentiation factors namely retinoic acid (RA), sonic hedgehog (Shh), bone morphogenetic protein-2 (BMP-2) and Wnt3A. The highest number of motor neurons (58.0 ± 7.6%) were obtained by an early treatment of embryoid bodies with a combination of RA + Shh from all the clonal hESC lines combined. The hES 3.1 line, however, produced relatively more motor neurons (69.5 ± 11.8%) compared to other two hES clones, 3.2 (52.4 ± 13.1%) and 3.3 (52.3 ± 15.5%). Immunolocalisation studies revealed the expression of neuronal specific marker, β -tubulin and motor neuron specific marker, HB9/HLXB9 in all the three hESC clones after 45 days of differentiation. The RT-PCR analyses showed the presence of the neuron-specific genes. This modified differentiation protocol provides a mean of obtaining an enriched population of motor neurons from hESC for possible use in studies of lineage development, drug discovery and also as a potential cell therapy for motor neuron disease.


[Back to top]
Classic β-Amyloid Deposits Cluster Around Large Diameter Blood Vessels Rather than Capillaries in Sporadic Alzheimer's Disease
R.A. Armstrong

Various hypotheses could explain the relationship between β-amyloid (Aβ) deposition and the vasculature in Alzheimer’s disease (AD). Amyloid deposition may reduce capillary density, affect endothelial cells of blood vessels, result in diffusion from blood vessels, or interfere with the perivascular clearance mechanism. Hence, the spatial pattern of the classic ('cored') type of Aβ deposit was studied in the upper laminae (I,II/III) of the superior frontal gyrus in nine cases of sporadic AD (SAD). Sections were immunostained with antibodies against Aβ and with collagen IV to study the relationships between the spatial distribution of the classic deposits and the blood vessel profiles. Both the classic deposits and blood vessel profiles were distributed in clusters. In all cases, there was a positive spatial correlation between the clusters of the classic deposits and the larger diameter (>10 µm) blood vessel profiles and especially the vertically penetrating arterioles. In only 1 case, was there a significant spatial correlation between the clusters of the classic deposits and the smaller diameter (<10 μm) capillaries. There were no negative correlations between the density of Aβ deposits and the smaller diameter capillaries. In 9/11 cases, the clusters of the classic deposits were significantly larger than those of the clusters of the larger blood vessel profiles. In addition, the density of the classic deposits declined as a negative exponential function with distance from a vertically penetrating arteriole. These results suggest that the classic Aβ deposits cluster around the larger blood vessels in the upper laminae of the frontal cortex. This aggregation could result from diffusion of proteins from blood vessels or from overloading the system of perivascular clearance from the brain.


[Back to top]
Neuronal Semaphorins Regulate a Primary Immune Response
S. Moretti, A. Procopio, M. Boemi and A. Catalano

Semaphorins are involved in a wide range of biological processes, including axon guidance, neuronal migration, angiogenesis, cardio- and osteo-genesis. Recently they have also been found to be important for immune response. Sema3A reduces the activation of T cells through its cell-surface receptors, including members of the neuropilin and plexin families. By contrast, Sema4D (CD100), which is expressed on the surface of T, B and dendritic cells, increases B cell and dendritic cell function using either plexin B1 or CD72 as receptors. The transmembrane protein Sema4A is involved in the activation of immune cells through interactions with Tim-2. Emerging evidence also indicates that additional semaphorins and related molecules seem to function in the reciprocal stimulation of T cells and antigen-presenting cells (APCs). This paper discusses the functions of these semaphorins in the immune system, focusing on their roles in T cell–APC interactions.


[Back to top]
Central Nervous System Circuitry and Peripheral Neural Sympathetic Activity Responsible for Essential Hypertension
F. Lechin and B. van der Dijs

Both clinical and experimental studies dealing with patients affected by idiopathic or essential hypertension (EH) are devoted to the great deal of physiological, pharmacological and pathological as well as therapeutical issues of EH. However, most articles devoted to EH do not refer to the central nervous system mechanisms underlying this disease and the channels which allow that these mechanisms are funneled to the peripheral autonomic nervous system and trigger this cardiovascular disorder. In the present review article we attempted to reach this target devoted to the central nervous system circuitry involved in the cardiovascular pathophysiology. We postulated that EH depends on the predominance of the binomial A5 noradrenergic (NA) nucleus + median raphe serotonergic (5-HT) nucleus over the (A6)-NA + dorsal raphe-5HT nuclei. This hypothesis receives additional support from our results obtained throughout the neuropharmacological therapy of this type of neurophysiological disorder. Our therapeutical strategy is addressed to enhance the activity of the (A6)-NA + dorsal raphe-5HT binomial circuitry.