Joel Pachter - US grants

[unreadable] DESCRIPTION (provided by applicant): The issue of microvascular endothelial heterogeneity greatly impacts vascular function in both health and disease, and potentially severely complicates the use of tissue culture-based models to study endothelial behavior. Yet, despite this topic having been explored in considerable depth in peripheral vascular beds, surprisingly little attention has yet been focused on the microvascular axis of central nervous system (CNS). Unrelated reports describing particular genes have, nonetheless, provocatively suggested that a diversity in endothelial gene expression may exist at the segmental level (i.e., between arterioles, capillaries and venules) as well as the regional level of the CNS, and approximate or surpass that found in the periphery. Given the preeminence of the microvascular network in forming both the blood-brain barrier and blood- spinal cord barrier, and the significant microvascular involvement in inflammatory, infectious, degenerative and traumatic conditions of the CNS, it is critical that there be a systematic and detailed evaluation of this clinically important issue. It is of further importance that such analysis be performed on microvascular tissue in situ," as endothelial cell gene expression is exquisitely prone to environmental modulation. Accordingly, we propose the following Specific Aims: 1) To validate and optimize the approach of coupling laser capture microdissection (LCM) of brain microvascular endothelial cells, with global gene expression profiling by DMA microarray analysis. This will include demonstrating feasibility, by maximizing the percentage of transcripts detected (P call rate) in capillaries only, and establishing reproducibility, by identifying both technical and biological sources of variance in microarray data; and 2) To use the LCM/microarray approach to compare the global gene expression profiles of endothelial cells from capillaries, venules and arterioles. The R21 format chosen is specifically designed for projects that are exploratory and may involve considerable risk, but lead to the development of novel techniques that could have major impact on a field of research. In this regard, results obtained here will set the stage for evaluating regional gene expression by the neurovascular unit (i.e., endothelial cells and intimately associated neural cells) along the CNS microvascular tree in both health and disease. In turn, these studies will enable formulation of more precise endothelial models to study the molecular basis of physiological and pathophysiological processes of the cerebral microvasculature. [unreadable] [unreadable] [unreadable]

While the entry of leukocytes into the central nervous system (CNS) is fundamental to the pathogenesis of many neuroinflammatory conditions, mechanisms regulating the last and most critical step of this process, transendothelial migration (TEM), remain obscure. But two recent series of findings from separate fields could reveal novel and vital clues to how and where TEM takes place. One, ectopic expression of tight junction (TJ) proteins has been described on circulating leukocytes in patients with relapses of the neuroinflammatory disease multiple sclerosis (MS). Elevated expression of TJ proteins by leukocytes in inflammatory conditions, as well as by stem and malignant cells, might endow these cells with heightened ability to cross tissue barriers ? possibly by enabling transient interactions with endothelial TJ proteins via a ?zipper? mechanism. Two, nano- sized extracellular vesicles (EVs), e.g., exosomes and microvesicles, bearing junctional proteins and shed by endothelial cells have been reported. EVs are elevated in blood during inflammation (including MS), shuttle protein and RNA between cells, and interact with various immune cells to alter their adhesion and migration properties. These collective findings could suggest EVs transfer TJ proteins to leukocytes and serve as the missing links functionally connecting TJ proteins on leukocytes, TEM and neuroinflammation. Specifically, by transferring TJ cargo from endothelial cells to leukocytes in a juxtacrine manner, EVs might enable adherent leukocytes to transiently engage corresponding vascular TJ proteins and, thereby, foster TEM across the highly restrictive endothelium of the blood-brain barrier (BBB). Using state-of-the-art technologies, we will focus on leukocytes displaying claudin 5 (CLN-5) ? a major TJ protein of the BBB ? and test the following hypothesis: Leukocytes exploit CLN-5 and endothelial-derived EVs via novel interactions to extravasate across the BBB during neuroinflammation. Aim 1 will use high-resolution 3D fluorescence imaging and FACS to detect CLN-5+-leukocytes and identify their immunophenotypes and activation states at various times in the blood and different CNS regions of mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. Aim 2, will use novel endothelial conditional, eGFP-CLN-5 mice or CLN-5 knockdown mice to determine if leukocytes express CLN-5 endogenously or acquire it exogenously from endothelial cells. Aim 3, will use 3D fluorescence imaging/FACS to analyze transfer of CLN-5 cargo (protein and/or mRNA) from endothelial-derived EVs to leukocytes, and a novel CLN-5 peptidomimetic, Pep5, to establish if this action is CLN-5-dependent. Aim 4, will use a recognized in vitro BBB model and Pep5 to examine the functional role(s) of leukocyte CLN-5 and EVs in promoting TEM. These studies will be the first systematical and methodical approach to address the relationship between leukocyte TJ proteins and EVs, and their concerted role in neuroinflammation. As similar processes might generically operate in other instances of cell extravasation, these studies should reveal common pathogenic mechanisms and highlight new, therapeutic approaches that target EVs. !

Abstract Neurodegenerative diseases and brain cancers are challenging to treat due to the presence of blood brain barrier (BBB), which is formed by tight junctions between endothelial cells in the microvasculature of the brain and prevents most of the therapeutics from access to the brain tissues. Among several reported approaches, ultrasound (US) has been demonstrated to be the most effective and safe method to facilitate the BBB opening. External US is however limited in efficacy to small animals whose skull bone is thin. In the case of humans, the thick skull bone absorbs more than 90% of US energy, requiring large and bulky arrays of external US transducers, which often consumes several hours of stimulation and requires tedious MRI monitoring during the sonication. Moreover, this extensive process is only useful for a single-time stimulation while research has shown the opening of BBB requires repetitive application of US. Implanted ultrasound transducers have thus emerged as an excellent alternative that can be easily used to repeatedly induce low-intensity sonication deep inside brain tissue at a precise brain location. Indeed, a commercial ultrasound transducer, termed as SonocloudTM, has been clinically tested for brain implantation and shown a great potential to facilitate BBB drug-delivery without any US- induced damage to underlying brain tissue. Unfortunately, commercial transducers, including the Sonocloud, rely on conventional piezoelectric materials (mostly ceramics of PZT or Lead Zirconate Titanate), which contain toxic elements such as Lead and are non-degradable. The conventional US transducers therefore require invasive brain-surgery for removal, raising a significant safety concern. In this regard, the PI?s group has developed a new biodegradable and biocompatible piezoelectric material, based on a common medical polymer of Poly-L-Lactide (PLLA). We were also successful to employ the material for creating the first biodegradable ultrasonic transducer. Toward the end goal of using this novel transducer for BBB drug-delivery, here we propose to study the safety of this device for long-term implantation in the brain and assess the ability of the transducer to open the BBB, which then facilitates the delivery of drug models into the brain tissue. Our main hypothesis is that the transducer, made of common medical materials, which have been used extensively for many FDA-approved implants, will be highly biocompatible and eventually self-vanish to avoid invasive, surgical removal, while providing an excellent performance for BBB opening to deliver multi-sized drugs into the brain during its defined functional-lifetime. To demonstrate the premise, this proposal will have three different aims. Aim 1 is to characterize the output acoustic pressure and functional lifetime of our biodegradable US transducer. Aim 2 is to study long-term biocompatibility of the US transducer inside the rodent brain. And Aim 3 is to assess BBB disruption (BBBD) and demonstrate the delivery of drug models into the brain tissue in vivo.