Martha E. O'Donnell - US grants

The main goal of the present proposal is to investigate Na-K-Cl cotransport of vascular endothelial cells with respect to its role in endothelial cell function. The endothelium is known to play a central role in maintenance of vascular homeostasis. In this regard, endothelial cells have been shown to participate in regulating vascular processes such as thrombosis, vasoconstriction, and smooth muscle cell proliferation. The endothelium also provides a vitally important selective permeability barrier between the blood and interstitium. Modulation of the endothelial barrier permeability to macromolecules such as albumin has been shown to be a major determinant of fluid distribution between vascular and extravascular spaces. Thus, the functional importance of the endothelium has been well documented. However, little is known about the involvement of ion transporters in endothelial cell function. Previous studies from the applicant's laboratory have established that endothelial cells exhibit a prominent Na-K-Cl cotransport system that is regulated by a variety of vasoactive agents. These findings suggest that Na-K-Cl contransport may play an essential role in normal endothelial cell function. Two physiological roles have been demonstrated for Na-K-Cl cotransport in other cell types: volume regulation in both nonepithelial and epithelial cells; and vectorial transport of ions across epithelia. As an initial approach to determining the functional significance of the prominent Na-K-Cl cotransport system in endothelial cells, the proposed project will investigate the involvement of the cotransporter in endothelial cell volume regulation. Cell volume regulation has been hypothesized to be critical for maintenance of the endothelial permeability barrier. Thus, the influence of endothelial cell volume on macromolecular permeability of the barrier will also be evaluated. Specifically, the present proposal has two primary aims: 1) to test the hypothesis that Na-K-Cl cotransport participates in endothelial cell volume regulation; and 2) to test the hypothesis that alteration of endothelial cell volume influences endothelial permeability to macromolecules. In these studies, Na-K-Cl cotransport activity and intracellular volume of a variety of cultured endothelial cells will be evaluated. Macromolecular permeability will be assessed for albumin using two systems: 1) an in vitro system of endothelial monolayers grown on microporous membrane cell culture chamber inserts; and 2) an in vivo system of perfused hamster mesentery microvessels. The proposed studies should provide information regarding the role of plasma membrane ion transport systems in endothelial cell function. This information could lead to new therapeutic approaches to the treatment and prevention of vascular pathophysiological conditions such as hypertension and atherosclerosis.

[unreadable] DESCRIPTION (provided by applicant): The long term goal of this project is to identify blood-brain barrier (BBB) ion transporters that mediate ischemia induced brain edema, a major cause of brain damage in stroke. During the early hours of cerebral ischemia, brain edema formation occurs in the presence of an intact BBB. In this process, BBB endothelial cells transport Na and Cl from blood into brain interstitium, with osmotically obliged water following. The specific ion transporters responsible are unknown, however BBB luminal Na and Cl transporters appear to play a key role. Much evidence indicates that hypoxia, which rapidly develops during ischemia, aglycemia occurring as glucose is depleted, and also centrally-released vasopressin are mediators of ischemia-induced brain edema formation. A novel aspect of this proposed project is the preliminary finding that a Na-K-Cl cotransporter appears to be localized in the luminal membrane of brain microvessel endothelial cells and that vasopressin, hypoxia and aglycemia stimulate activity of the cotransporter. This has led to the central hypothesis that a Na-K-Cl cotransporter, located at the luminal membrane of the BBB, is stimulated during ischemia to increase transport of Na and Cl with osmotically obliged water from blood to brain, causing edema formation. The present project has three specific aims. The first aim is to test the hypothesis that Na-K-Cl cotransport is present in luminal membranes of cerebral microvascular endothelial cells (CMEC). These studies will evaluate bovine brain microvessel luminal and abluminal membrane preparations for cotransport activity by radioisotopic flux analyses. Also, the in situ distribution of the cotransporter will be examined by immunoelectron microscopy of brain sections. The second aim is to test the hypothesis that Na-K-CI cotransport of BBB endothelial cells is stimulated by agents that mediate ischemia-induced cerebral edema. Here, the effects of hypoxia, aglycemia and vasopressin on cotransport activity will be examined in cultured human and bovine CMEC and freshly isolated bovine cerebral microvessels. The third aim is to test the hypothesis that inhibition of Na-K-Cl cotransport activity attenuates ischemia-induced brain edema. To do this, the effect of inhibiting the cotransporter on ischemia-induced changes in rat brain Na and water will be examined by nuclear magnetic resonance methods, which allow in vivo changes in brain Na and water to be followed in real time. The proposed studies should reveal whether therapeutic approaches aimed at blocking BBB Na-K-Cl cotransporter activity may be of value for attenuating ischemia-induced brain edema.

This proposal is a request for funds toward the support of a Gordon Research Conference to be held in July 2002. The intended meeting is the second Gordon Research Conference on Barriers of the Central Nervous System. This Gordon Research Conference was initiated as a catalyst for interaction among researchers from a variety f disciplines working on issues of blood-brain- and blood-CSF barriers in health disease. Research in this area is already leading to development of new therapeutic approaches for th treatment of stroke, head trauma, neurodegeneration, brain inflammation and brain tumors, to name a fe3w. Although portions of these areas of investigation are represented at several national and international research meetings, there have been few opportunities for scientists working in the field to come together with a specific concentrated focus on issues related to barriers of the CNS. The subtitle of the upcoming second Barriers of the CNS Gordon Research Conference is "Blood-Brain CSF barriers, CNS Homeostasis and Drug Delivery to the Brain". The specific focus of this conference will include discussion of a number of the problems and tissues faced by the Pharmaceutical industry in targeting therapeutic drugs to the CNS as well as discussion of broader current major developments of the blood- brain-barrier and blood-CSF barrier field. Most of these problems concern overcoming the blood-brain barrier to drugs. To this end a number of eminent scientists from academia and from the pharmaceutical industry, are being brought together for an exciting "leading edge" meeting. The intention is to foster the development of new ideas and their application to fundamental knowledge and the treatment of human and animal disease. Speakers have been selected on the basis of their eminence in the field and their communication skills. TO this end they are drawn from a wide geographical background. Chairs of the sessions have been selected on the basis of their considerable eminence and experience in the field and a known ability to communicate and debate well. In addition the presentation of posters is being vigorously encouraged especially by young postdoctoral and research students. It is expected that a considerable proportion of any NIH grant will be used to fund the attendance of young scientists and chairs. It is also intended to have three open workshops/debates during the meeting. One is already in place on "Microdialysis". The maximum permitted attendance at this meeting is 150 scientists.

[unreadable] DESCRIPTION (provided by applicant): This proposal is a request for funds toward support of the 3rd "Barriers of the CNS" Gordon Research Conference to be held June 27th to July 2nd 2004 in Tilton, NH. Funds are also requested for support of the 4th and 5th conferences, to be held in 2006 and 2008. This Gordon Research Conference was initiated as a catalyst for interaction among researchers from a variety of disciplines working on issues of blood-brain-barrier (BBB) and blood-CSF barrier in health and disease. Research in this area is already leading to development of new therapeutic approaches for the treatment of stroke, head trauma, neurodegeneration, brain inflammation and brain tumors, to name a few. Although portions of these areas of investigation are represented at several national and international research meetings, there have been few opportunities for scientists working in the field to come together with a specific concentrated focus on issues related to barriers of the CNS. The upcoming 2004 conference will include issues related to the physiology and pathophysiology of the BBB and blood-CSF barriers as well as the delivery of drug and gene therapies to the CNS. A new aspect of the 2004 meeting will be consideration of the neurovascular unit and how BBB cells interact with other cells in the neural environment. It is especially noteworthy that blood-brain barrier research has been identified by NIH as a critical area for development. Consistent with this, the mission of the Barriers of the CNS Gordon Research Conferences is to provide a forum for scientists with a variety of backgrounds and experience to share unpublished findings and work together to identify new strategies for attacking the many remaining unanswered questions with respect to the BBB and blood/CSF barriers. To this end, eminent scientists from both academia and the pharmaceutical industry are being brought together for an exciting "leading edge" meeting. Speakers have been selected on the basis of their eminence in the field and their communication skills. They are drawn from a wide geographical background. Chairs of the sessions have been selected on the basis of their considerable eminence and experience in the field and a known ability to communicate and debate well. In addition the presentation of posters is being vigorously encouraged especially by young postdoctoral and research students. It is expected that a considerable proportion of any NIH grant will be used to fund the attendance of young scientists and chairs. Two open workshops/debates to occur during the meeting are also planned. The maximum permitted attendance at this meeting is 150 scientists. [unreadable] [unreadable]

The cessation of reproductive function is one of the most dramatic and fairly rapid endocrine changes in the life of a female and carries profound and broad repercussions. At one time, it was thought that decreasing estradiol concentration, resulting from exhaustion of ovarian follicles, caused the menopause. Now, it is clear that many factors interact to bring about declining reproductive function and the cessation of reproductive cycles. Furthermore, we are beginning to appreciate the most- reproductive state affects multiple functions that extend beyond the traditional reproductive axis. An increasing number and an increasing proportion of women will liver a larger fraction of their lives in the postmenopausal state. Therefore, it is critical that we deepened our understanding of the factors that regulate the transition to acyclicity and, conversely, the impact of transition to acyclicity on the function of multiple systems. The program is organized into five research projects that are supported by one Core. The projects are integrated and coordinated to test the following hypotheses (1) changing estrogen responsiveness governs the health and function of ovarian follicles during aging, (2) age-related and estradiol-dependent alterations in the pattern of expression in the ovarian metalloproteinases and their inhibitors result in abnormalities in follicular development and ovulation, (3) aging and/or the cumulative exposure to estradiol regulate changes in somatotroph and lactotroph function, (4) alterations in glutamatergic and adrenergic regulation of GnRH neurons cause changes in their ability to induce LH surges; and (5) estradiol plays a critical protective role in the brain; therefore, declining estradiol concentrations lead to an increased vulnerability to brain injury and neurodegeneration. An Administrative/Animal ore supports all of the projects, providing organizational planning, integrating results from each project into a more comprehensive overview, and maintaining all animal records and coordinating their optical endocrine treatments and use. We submit this program project proposal because we share interests in interdependent aspects of reproductive aging. A common threat that weaves through all of the projects is our interests in the role of estradiol in aging. Finally, this mechanism of support will permit us to use aging animals in the most optimal manner, allowing us to compare data and economize on the costs of aging animals.

DESCRIPTION (provided by applicant): The long term goal of this project is to identify blood-brain barrier (BBB) ion transporters that mediate ischemia-induced brain edema. During the early hours of ischemic stroke, edema forms in the presence of an intact BBB by a process involving BBB transport of Na and Cl from blood into brain. Our studies have shown that Na-K-Cl cotransport, present in the luminal BBB membrane, is stimulated by ischemic factors, including hypoxia, aglycemia, vasopressin (AVP) and that inhibiting the cotransporter reduces edema in a rat model of stroke. Thus, the BBB Na-K-Cl cotransporter appears to be a major contributor to ischemia-induced edema. We have now found in preliminary studies that a BBB Na/H exchanger also appears to participate in ischemia- induced edema formation which suggests the exciting possibility of an additional BBB target for reduction of edema during the early hours of stroke. Our hypothesis is that, in addition to the cotransporter, a luminal BBB Na/H exchanger is stimulated during ischemia to increase transport of Na from blood into brain. The first aim is to determine whether Na/H exchange is present at the luminal BBB membrane and is stimulated by ischemic factors. We will use immunoelectron microscopy to evaluate BBB Na/H exchange protein in situ and microspectrofluorometry to assess ischemic factor effects on CMEC Na/H exchange activity. The second aim is to determine whether inhibition of the BBB Na/H exchanger attenuates ischemia-induced edema. Here, we will examine the effect of BBB Na/H exchange inhibition on ischemia-induced changes in rat brain Na and water, using nuclear magnetic resonance methods. We will also assess the efficacy of Na/H exchange and Na-K-Cl cotransport inhibitors for reduction of cerebral edema when administered after the onset of ischemia. The third aim of this project is to evaluate the signaling pathways by which ischemia stimulates BBB Na-K-Cl cotransporter and Na/H exchanger activities. We will start by evaluating the roles of AMP kinase, p38 MAP kinase and intracellular [Ca] in hypoxia, aglycemia and AVP-induced stimulation of the cotransporter and exchanger for reasons detailed in the application . However, our studies will also include an initial assessment of protein kinase C, ERK1/2 MAP kinase and JNK MAP kinase. For these studies we will use cerebral microvascular endothelial cells and perfusion-fixed rat brain to evaluate ischemia-induced activation of the kinases by Western blot and confocal immunofluorescence, respectively. We will also evaluate the effects of kinase inhibitors on ischemia (hypoxia, aglycemia and AVP) stimulated cotransporter activity (radioisotope flux) and Na/H exchanger activity (spectrofluorometry and radioisotope flux). PUBLIC HEALTH RELEVANCE: Ischemia-induced edema is a major cause of brain damage in stroke, a leading cause of death in the U.S. The proposed studies will reveal whether therapeutic approaches aimed at preventing ischemia stimulation of BBB Na-K-Cl cotransporter and/or Na/H exchange activity may be of value for attenuating stroke-induced brain edema. The fact that luminal-facing BBB Na transporters appear to contribute to edema formation and are readily accessible to intravenously administered drugs makes them a therapeutic target that we must investigate.