Brant E. Isakson - US grants

DESCRIPTION (provided by applicant): Cell-cell communication between endothelial cells (EC) and smooth muscle cells (SMC), provided by gap junctions, coordinates vessel contractility and electrical conduction. Gene knockout (KO) of vessel gap junction proteins (connexins (Cx)40 and Cx43) yields dramatically different blood pressures and conduction. A possible reason for these results are 1) co-regulation of Cx40 and Cx43, which in effect would 2) alter the method of cell-cell communication by gap junctions in the vessel wall. Based on this idea, I hypothesize that contact between EC and SMC plays a key role in modulating connexin function and expression, and thus in modulating vascular reactivity. In order to elucidate this hypothesis, an in vitro system to model EC and SMC interactions similar to in vivo will be developed and utilized. This model, in which EC and SMC cells will be isolated from the same mouse and aortic sections, will be used to test methods of dye and second messenger transfer between EC and SMC. These results will be compared to similar experiments on in situ wild type and connexin KO animals. Combined, these methods will help delineate the physiological role of connexins between EC and SMC.

DESCRIPTION (provided by applicant): Vascular smooth muscle cells (VSMC) and endothelial cells (EC) in the resistance vessels are functionally linked, and the point of contact between the two cells, the myoendothelial junction (MEJ), plays a key role in many elements of vascular function. However, the location and size of the MEJ have made it extremely difficult to study in vivo. We have developed a model of the MEJ by co-culturing VSMC and EC that show connexin- dependent dye transfer and a coupled pool of Ca2+. Moreover, the mode of intercellular calcium signaling depends on which of the two cell types is stimulated, i.e. intercellular second messenger signaling is polarized, and this appears to mimic signaling patterns seen in vivo. We propose to determine the structural and molecular basis for the intercellular coupling that occurs at the MEJ, which we believe has important implications for the operation of the MEJ in vivo. We will determine which connexins and second messengers are involved in intercellular signaling, and will test whether polarization of calcium communication is determined by selective permeability of the gap junctions at the MEJ or differential expression of second messenger receptors at the MEJ in the two cell types. We propose 3 specific experimental aims: Specific Aim 1 - Use light and electron microscopy-based immunocytochemistry to assess and compare the in vivo and in vitro placement of: a.) connexins, b.) ryanodine receptors, and c.) inositol 1,4,5 triphosphate-receptors at the MEJ;Specific Aim 2 - Measure the effect of modification of the gap junctional connexin composition on: a.) Ca2+ and b.) inositol 1,4,5 triphosphate -mediated intercellular signaling;and lastly, Specific Aim 3 - Assess the effects of cell specific deletion of a) ryanodine receptors and b) inositol 1,4,5 triphosphate-receptors on polarized calcium signaling. Our experiments should enhance understanding of the coordination of VSMC and EC and will provide insights into basic questions of vasomotor control and a variety of pathophysiological responses. The methods by which vascular cells communicate are key for understanding vascular processes such as hypertension and control of blood flow. Our model and the experiments proposed offer the first opportunity to investigate the capabilities of the myoendothelial junction and to understand its role in the vessel wall. We propose to study the ways in which vascular cells utilize this structure to maintain vascular function.

Abstract During the course of metabolic syndrome, there is a significant increase in plasminogen activator inhibitor-1 (PAI-1); however the pathological role that PAI-1 plays during the course of the disease is not clear. We have recently identified PAI-1 as being key to regulation of myoendothelial junctions (MEJs), which are critical heterocellular structures linking the endothelium and smooth muscle in the resistance vasculature, coordinating communication between the two cell types. In mice with metabolic syndrome, we have shown that PAI-1 accumulates in MEJs and increases the total number of these structures, indicating a possible role in altering heterocellular communication. In order to understand how PAI-1 may alter MEJ formation, we used TNF-a to mimic the effect of metabolic syndrome and demonstrate significant increases in PAI-1 mRNA throughout endothelium, MEJ, and smooth muscle. However, after application of TNF-a, there was only a significant increase in PAI-1 protein at the MEJ. This indicates that PAI-1 may be locally translated at the MEJ. Recently, the serpine binding protein 1 (SERBP1) has been described as a novel RNA binding protein (RBP) specifically for PAI-1 mRNA that promotes stabilization of the mRNA for translation. However, an RBP requires anchoring to the cytoskeleton to remain in a specific area of the cell (e.g., the MEJ). To that end, we discovered a microtubule binding domain in the novel protein nicotinamide phosphoribosyltransferase (NAMPT), demonstrated to increase concurrently with PAI-1 during the course of metabolic syndrome, which we found to be localized to the MEJ. Therefore, we hypothesize that SERBP1 and NAMPT act together as a PAI-1 RBP complex to localize PAI-1 mRNA to the MEJ for the rapid and specific dissemination of PAI-1 protein. To test this hypothesis, we have put forth two specific aims: 1) PAI-1 mRNA is locally translated at the MEJ and 2) NAMPT regulates localization of SERBP1 to the MEJ. This proposal will focus on how PAI-1 is capable of being polarized to the MEJ using such techniques as FlAsH/ReAsH to examine nascent PAI-1 protein production and how RBP proteins could be anchored to the cytoskeleton using peptides against the microtubule binding domain of NAMPT. The aggregate of the experiments will provide for the first time a pathological mechanism for the effect of increased PAI-1 seen in metabolic syndrome.

Overall Program Project - Project Summary Inter-cellular communication between cells within a tissue environment is fundamentally important for many physiological processes. Channels and transmembrane transporters that conduct ions and other molecules across the plasma membrane in healthy living cells are also linked to pathologies of the cardiovascular and respiratory systems. Extracellular nucleltides (such as ATP) and their derivatires critically influence many aspects of vascular physiology such as vasoconstriction and blood pressure regulation, as well disease states such as metabolic syndromes. Recent exciting series of observations suggest that the pannexin proteins form channels on the plasma membrane, and by permeating ions and/or the release of nucleotides in a very regulated manner, these pannexin channels allow cells to communicate with other cells. Consistent with this, altered expression of pannexin channels have been linked to cardovascular and metabolic disorders. On an independent and inter-related set of observations, the pannexin channels also play a role in releasing nucleotides from early stage apoptotic cells that appear critical for communicating with phagocytes and in turn promoting prompt corpse removal. Since, failed clearance of dying cells is linked to atherosclerosis and airway inflammation, pannexin channels likely also play a role in regulating inflammation within tissues. The central hypothesis tested via this P01 application is that pannexin channels sit at a critical interphase between normal homeostasis within the cardiovascular system, and the disease states leading inflammation, atherosclerosis, and hypertension. The four projects that comprise this proposal address the role of pannexin channels as follows. Project 1 (Ravichandran) addresses the role of pannexin channels in cell death and recruitment of monocytes during atherosclerosis, cholesterol efflux, and in tissue inflammation; Project 2 (Isakson) addresses how pannexin channels in smooth muscle cells contribute to vasoconstriction in resistance vessels to regulate blood pressure and how this is altered in obesity; Project 3 (Leitinger) addresses how pannexin channels regulate adipocyte functions and the inflammation induced by dying adipocytes in obesity, insulin resistance and hypertension; Project 4 (Bayliss) addresses molecular mechanisms of pannexin channel activation in physiological and diseased states. With the combination of mouse models and ex vivo studies, and mechanistic approaches, and the preliminary identification of new compounds capable of altering Panx1 function, we expect to provide exciting new insights on pannexin channels and purinergic signaling in vascular physiology and hypertension, and provide the basis for novel treatment strategies targeting the regulated opening and closing of these channels in specific disease states. We expect this would have a broad impact to cardiovascular, metabolic, and respiratory diseases.

ABSTRACT It is well known that aged arterioles have increased constriction and the animals have an overall higher blood pressure. There have been several hypothesis that have arise as to the etiology of this process, with NO bioavailability being one of the most consistent issues in that the arterioles may make normal levels of NO via eNOS, but the ability to dilate has been severely diminished. The cause for this remains unknown. Recently we demonstrated that a potent NO scavenger, alpha globin, is uniquely expressed in endothelium of arterioles. Once more, preliminary data presented herein demonstrated an age-dependent increase in alpha globin that is consistent with movement of NO across the blood vessel wall. Because of this, we postulate that aged arterioles may develop increased alpha globin protein expression, providing a pathological ?sink? for NO, and decreasing the overall ability of these arterioles to dilate and increasing overall blood pressure. To accomplish this, we propose two aims: Aim 1 we will determine changes in alpha globin protein expression and function in aged mice. Aim 2 proposes that genetic deletion or modification of alpha globin could alter vascular reactivity and blood pressure in aged mice. This last aim uses the stable of mice generated by us to uniquely delete or over- express alpha globin specifically in the endothelium. Our exciting preliminary data demonstrates that alpha globin protein expression could be a key part of the vascular dysfunction observed in an aging vasculature. Our lab is unique in being able to tease the possible relationship between aging and alpha globin expression and function with the mice and reagents developed as part of the original R01.

|| ABSTRACT Breakdown of the endothelial cell barrier is considered a defining pathological hallmark of multiple diseases. Indeed, sepsis accounts for more hospital deaths per year than any other condition in the United States, and the disease is currently devoid of any targeted pharmacological intervention. Critical to understanding how inflammation affects vascular barrier function is that endothelial cells throughout the circulatory system are not homogenous. Inflammation specifically affects vascular permeability through effects on the venous endothelium, whereas the arterial endothelium is not susceptible to inflammation-induced permeability and instead primarily regulates blood pressure and angiogenesis. Thus, a mechanistic view of how venous endothelial barrier function is regulated is essential to human health and disease. Our current understanding of vascular barrier function does not account for endothelial heterogeneity and the unique cell adhesion and signaling pathways specific to each endothelial cell type. Purinergic signaling has been identified as a key regulator of endothelial permeability; however the means by which purine nucleotides are brought into and affect the local environment has never been identified. We hypothesize that venous endothelial barrier function is regulated by a localized purinergic signaling cascade that controls the stability and expression of tight junction proteins. We will use three aims to test this concept. In Aim 1, we will determine roles for Pannexin 1 in regulating venous endothelial permeability. This aim will use novel methods for ex vivo vein isolation and measure transendothelial resistance and dye movement from endothelial cell specific Panx1 knockout mice and endothelial cell specific Panx1 over-expressing mice, as well in vivo testing using the cecal ligation puncture (CLP) septic model. In Aim 2, we will measure the relative contributions of different Adenosine Receptors (ARs) on endothelial barrier function. We will determine the differential role of CD39 and CD73 on adenosine receptor activation, using endothelial cell specific A2A and A2B floxed mice, as well as the relative contribution of PKA or PKC upon activation. Lastly, in Aim 3, we will define roles for claudin-11 in venous endothelial barrier function and as a target for TRPV4-mediated disruption of tight junctions. This aim will utilize state of the art calcium imaging to determine a role for TRPV4 in regulation of claudin-11 production, trafficking, assembly and stability. Claudin-11 is a novel claudin isoform whose role in barrier function is only beginning to be elucidated. Molecular manipulation of claudins will be used to demonstrate that claudin-11 is required for veins to be sensitive to calcium fluxes because it has the unique capacity to bind calmodulin, whereas claudin-5 does not. The feasibility of accomplishing these aims is underscored by all proposed knockout mice being in hand, an IRB in place for human samples, and strong preliminary data.

PROJECT 2 PROJECT SUMMARY The fastest growing type of hypertension is sympathetically driven due to its close association with obesity, itself reaching epidemic proportions. In this form of hypertension, the effect of sympathetic nerve (SN) activity ? i.e., release of norepinephrine (NE) to bind ?-adrenergic receptors (?-AR) on smooth muscle cells (SMC) on SMC ? is enhanced. NE is a potent vasoconstrictor and can strongly increase blood pressure. Thus, the smooth muscle cells of resistance arteries is a major site for SN-driven hypertension. Our PPG has recently made important discoveries in understanding the ??AR-mediated vasoconstriction pathway, which have forced us to re-think the classical mechanism whereby sympathetic nerve induces SMC constriction. In resistance arteries, we found that ?-AR activation (and not other vasoconstriction receptor pathways) induced Pannexin 1 (Panx1) channel opening on SMC to release ATP. This work identifies a key functional role for Panx1-derived ATP, and raises new questions on the interaction between sympathetic nerve and SMCs. Furthermore, our PPG recently discovered that the potent anti-hypertensive drug spironolactone acts directly on Panx1 channels to lower blood pressure, independent of mineralocorticoid receptors. This work may have ?unmasked? Panx1 as an important additional component to the anti-hypertensive effects of spironolactone. Together, our published and preliminary data provide the premise for the hypothesis tested in this proposal: Pannexin 1 links the sympathetic nervous system to arterial function. We propose two aims to test this hypothesis. In Specific Aim 1, we hypothesize that Pannexin 1 channels regulate sympathetic nerve control of peripheral resistance in hypertension. This aim will incorporate models of sympathetic hypertension with Panx1 genetic knockout to determine if Panx1 intervention can reverse high blood pressure. Additional Panx1 over-expression models and new Panx1 pharmacological activators will help determine whether Panx1 can modulate blood pressure. In Specific Aim 2, we ask further on the communication between sympathetic nerves and smooth muscle cells. We will examine the purinergic signaling domain directly, by visualizing ATP release and stimulating sympathetic nerves directly. A translational component will compare our findings to humans with and without hypertension. The feasibility of accomplishing these aims is underscored by all proposed knockout mice being in hand, an IRB in place for human samples, and the strong preliminary data. The integration of Project 2 with other Projects on this P01 provides us with an opportunity to explore a novel pharmacological target for sympathetic nerve-driven hypertension that could not have been achieved alone.

|| ABSTRACT .. The International Symposium on Resistance Arteries (ISRA) meeting is the only one dedicated to resistance arteries. Resistance arteries are small caliber, muscularized blood vessels that are directly responsible for the peripheral resistance component of blood pressure, as well as autoregulation in the renal and cerebral circulations to ensure appropriate blood flow. Therefore, understanding how these types of arteries function in a homeostatic and/or pathological context is of direct relevance to the mission of NHLBI. More specifically, the meeting will include extensive discussions on the potential role of inflammatory cell cross-talk within resistance arteries, as well as the most recent advances in state-of-the-art imaging techniques, such as super-resolution and two-photon microscopy, and genetic models of human disease. Two internationally renowned keynote speakers will provide the bookends for this meeting--Dr. Berislav V. Zlokovic, Professor and Chair, Department of Physiology and Neuroscience, University of Southern California and Dr. Robert Balaban, Scientific Director NHLBI, NIH. Three energetic and young leaders in the field of resistance artery biology, with previous experience in conference organization, will be the organizers for the meeting, including Dr. Brant Isakson (Univ of Virginia), Dr. Scott Earley (Univ of Nevada-Reno), and Dr. Manuel Navedo (Univ of California-Davis) whom together have already secured the venue and some funding streams. There are other key features to this meeting that provide significant value-added components, including the fact the meeting is solely ?grassroots? in that it is a conference put on by scientist, and for scientists for over thirty years. In addition, the smaller size meeting (~120 attendees) ensures an intimate environment for easy and effective networking, exchange of ideas and establishment of new collaborations between post-docs, graduate students, and senior investigators. Indeed, our meeting has a plethora of confirmed speakers that are doing renowned resistance artery research from a diverse background of female, under-represented minorities, and new investigators. Overall, the 2020 ISRA meeting provides a unique conference venue for researchers focused on resistance arteries in health and disease, and provides opportunities for researchers of diverse backgrounds and experience to cultivate new collaborations.