Recent Submissions

  • Dissecting the Roles of Reactive Oxygen Species in Cardiovascular Disease

    Wang, Yusi; Vascular Biology Center (2015-09)
    Cardiovascular disease remains the leading cause of death in the USA. While much has been learned about the root causes, the underlying mechanisms remain incompletely understood. In particular, elevated levels of reactive oxygen species (ROS) have been observed in the vasculature of blood vessels from animal models and humans with hypertension, atherosclerosis and diabetes. The importance of ROS to cardiovascular disease and the mechanisms by which it alters the function of cells of the cardiovascular system are the goals of this dissertation.
  • Molecular Mechanisms of High Glucose-Induced Vascular Endothelial Growth Factor Expression in Retinal Endothelial Cells

    Platt, Daniel H.; Vascular Biology Center (2004-10)
    Studies in diabetic patients, experimental animal models and tissue culture cells treated with high glucose have shown a close association between pathologic vascular growth, over-expression o f the angiogenic factor vascular endothelial growth factor .5 (VEGF) and oxidative stress. Studies o f diabetic patients and high glucose treated cells have also shown increased levels o f tyrosine nitration, a marker for the formation o f the reactive nitrogen species peroxynitrite. Excess formation o f reactive oxygen/nitrogen species has been shown to activate two transcription factors that regulate the expression o f VEGF, hypoxia-inducible factor-1 (HIF-1) and signal transducer and activator o f transcription 3 (STAT3). These observations suggest that diabetes causes increases in VEGF expression due to the effects o f high glucose in stimulating the formation o f peroxynitrite, which leads to the activation o f the transcription factors HIF-1 and/or STAT3 and increases in VEGF expression. This hypothesis was tested by experiments using primary cultures o f retinal endothelial cells treated with peroxynitrite or high glucose. Both treatments increased VEGF mRNA and protein levels. Further, pretreatment with the specific peroxynitrite decomposition catalyst FeTPPs blocked the increase in VEGF expression. To determine if HIF-1 and/or STAT3 play a role in the peroxynitrite-induced VEGF expression, studies were done to analyze the activation patterns o f both transcription factors. These studies showed that peroxynitrite had no effect on the activation or nuclear translocation o f HIF-1 a , but did induce a rapid activation and nuclear translocation o f STAT3. To further explore the role o f STAT3 in the VEGF expression, cells were treated with peroxynitrite or high glucose in the presence or absence o f an adenoviral vector expressing dominant-negative STAT3. Overexpression o f the dominant-negative STAT3 blocked the effects o f either peroxynitrite or high glucose in increasing VEGF mRNA. Further, treatment with FeTPPS blocked the effects o f high glucose in stimulating activation o f STAT3. A non-receptor tyrosine kinase, cSrc, has been shown to play a role in the activation o f STAT3 as well as the induction o f VEGF expression during tumor angiogenesis. To determine if cSrc plays a role in STAT3 regulated VEGF transcriptional activation, retinal endothelial cells were transduced with an adenovirus over-expressing a constitutively active Src (vSrc). The vSrc transduction induced activation o f STAT3 and increased VEGF expression. Further, FeTPPs blocked the effects o f peroxynitrite and high glucose in stimulating activation o f cSrc. Additionally, the Src inhibitor PP1 blocked the effects o f peroxynitrite and high glucose in increasing VEGF mRNA and protein expression. This work is the first to show that 1) high glucose-induced peroxynitrite formation increases VEGF expression, 2) STAT3 activation by high glucose-induced peroxynitrite formation regulates VEGF expression and 3) cSrc activation by high glucose-induced peroxynitrite formation activates STAT3 and increases VEGF expression.
  • Post-translational regulation of NADPH Oxidase 5 (Nox5) mediated via Phosphorylation and SUMOylation

    Pandey, Deepesh; Vascular Biology Center (2011-03)
    Increased levels of reactive oxygen species (ROS) are a hallmark of cardiovascular disease and are most prominently observed in blood vessels from humans and animals with diabetes, atherosclerosis and hypertension [1]. The NADPH oxidase (Nox) family of enzymes is comprised of seven members, Nox 1-5 and Duox 1 and 2 [2] has been shown to be a major source of ROS including superoxide (O2") and hydrogen peroxide (H2O2) in vascular cells [3]. Nox5 was the most recent of the conventional Nox enzymes to be identified and because it has been lost from rodent genomes (mice and rats) which have become our primary models for experimentation, very little is known about the molecular regulation and functional significance of Nox5. Our first goal was to determine whether Nox5 and its splice variants a, P, 8, y andNox5 Short (e) are expressed in human blood vessels. We detected Nox5 mRNA and protein expression in human blood vessels, smooth muscle cells and endothelial cells, but not in fibroblasts. The primary splice variants of Nox5 detected were a and P whereas 5 and y were undetected. We also found that Nox5 a and p were active and produced extracellular superoxide and H2O2, while Nox5, 5, y and 8 did not produce measurable ROS. As much as we lack knowledge about functional significance of Nox5, we are not so far ahead in understanding its molecular regulation. The mechanisms controlling the activity of NADPH oxidase 5 (Nox5) are unique in that they appear to be independent of the protein: protein interactions that coordinate the activation of other Nox isoforms [4]. Instead, the primary driving force for Nox5 activity is calcium [5]. While calcium is absolutely required for Nox5 activity, discrepancies between the amount of calcium needed to initiate ROS production versus that measured inside cells has led to the discovery by our laboratory and others that the calcium sensitivity of Nox5 can be modified by the specific phosphorylation of serine/threonine residues in response to the protein kinase C (PKC)-agonist, PMA resulting in a sustained activation of Nox5 at resting levels of calcium [6, 7]. However, the specific kinase(s) mediating the phosphorylation and activation of Nox5 are not known and their identification was the goal of our study. Using pharmacological inhibitors, dominant negative mutants and knockdown of endogenous genes (MEK1, MEK2 and CAMKIIa) using siRNA approach, we demonstrated that MEK1/2-ERK1/2 and CAMKIIa signaling pathways can positively regulate Nox5 activity by inducing the specific phosphorylation of S498 and S475, respectively. While much attention has been given to the mechanisms that positively regulate Nox activity, little is known about mechanisms that suppress Nox function. Cellular stress arising from changes in osmotic pressure, heat, cold etc are potent stimuli for protein SUMOylation. Importantly, oxidative stress arising from increased ROS is one of the best recognized stimuli for regulating protein SUMOylation [8, 9]. Hence, we investigated whether SUMO could influence the activity of Nox and thus limit the damaging effects of these molecules. We found that SUMO-1 and the SUMO-specific conjugating enzyme, UBC-9 potently suppressed the activity of Nox5 as well as other Nox isoforms (Noxl, 2, 3 and 4). We also found that co-expression of SUMO-1 does not result in the SUMOylation of Nox5 and that mutation of predicted sites of SUMOylation and conserved lysines on Nox5 failed to prevent the SUMO-1 driven inhibition of ROS production. In summary, we have identified the expression of Nox5 and more specifically the and p splice variants in human blood vessels and tissues. Our data suggest that Nox5 a and p are the only variants capable of producing ROS in human blood vessels, but also that the inactive variants can function as dominant negatives. Additionally, we have shown that MAPK and CAMKIIa signaling pathways positively regulate Nox5 activity via changes in phosphorylation whereas SUMO-1 negatively regulates activity through a yet to be defined mechanism.
  • The Role of PTP-1B in Vascular Insulin Resistance

    Ketsawatsomkron, Pimonrat; Vascular Biology Center (2008-02)
    Recent studies have suggested that insulin resistance in the vasculature can be linked to cardiovascular complications. However, the mechanism of insulin resistance which occurs in blood vessels is not well understood. Previous studies have shown that Protein Tyrosine Phosphatase -IB (PTP-1B) is a negative regulator of insulin signaling, however, the role of PTP-1B in regulating insulin signaling in the vasculature has never been explored. We hypothesized that PTP-1B plays an important role in vascular insulin resistance both in vitro and in vivo. For in vitro experiments, we utilized the model of angiotensin II (Ang II)-induced insulin resistance in vascular smooth muscle cells (VSMC) and hypothesized that Ang II-induced activation of PTP-1B is the underlying mechanism. Using standard Western techniques, we found that Ang II significantly inhibited insulin-induced phosphorylation of IRS-1 and Akt, downstream members the insulin-induced anti-mitogenic pathway. Furthermore, Ang II enhanced the insulin-induced activation of p42/p44 MAPK, a mitogenic pathway. In addition, we found that PTP-1B is involved in the insulin-induced blockade of Ang II-induced VSMC growth. Finally, we also showed that Ang II induced activation of PTP-1B in VSMC was through a PKA/JAK2 dependent mechanism. Therefore, from these in vitro studies, we conclude that Ang II modulates both anti-mitogenic and mitogenic pathways stimulated by insulin via activation of PTP-1B. For the in vivo studies, we hypothesized that PTP- 1B is an underlying mechanism of vascular insulin resistance in animal models. Experiments were conducted on PTP-1B knockout (PTP-1B KO) mice compared to wild type (WT) mice in different insulin resistant conditions. In high fat feeding induced obesity, we showed that the activation of Akt following insulin stimulation ex vivo was significantly decreased in high fat fed WT mice which was restored by deletion of PTP- 1B. However, the expression of PTP-1B was not different between WT mice on either regular or high fat diet. We concluded that PTP-1B partly plays a role in vascular insulin resistance in high fat fed model. We next examined the roles of PTP-1B and vascular insulin resistance in a new double transgenic obese model. We showed here that the expression of PTP-1B was increased significantly in obese control mice (K^HPTP-IB) compared to lean control mice. Activation of Akt following insulin injection was impaired in aorta of obese KdbHpTP-iB mice and was not restored by deletion of PTP-1B. Therefore, our data suggest that other insulin induced signaling molecules in the aortamay be involved in the regulation of Akt and not PTP-1B. Overall, our studies in this thesis suggest both an in vivo and in vitro contributionof PTP-1B to vascular insulin resistance. The overall goal of the study was to determine the significance of PTP-1B in the development of vascular insulin resistance particularly in vascular smooth muscle cell (VSMC). We hypothesized that PTP-1B plays an important role in vascular insulin resistance both in vitro and in vivo.
  • The role of the transcription factor, Sox18, in pulmonary endothelial barrier function

    Gross, Christine M; Vascular Biology Center (2014-12)
    Pulmonary endothelial cells form a continuous monolayer on the luminal surface of the lung vasculature. These cells provide a surface for gas exchange and importantly regulate vascular tone. Despite being constantly exposed to hemodynamic forces and/or vasoactive agents, the endothelium also maintains a selectively permeable monolayer under physiologic conditions. However, little is known about the transcriptional events in the pulmonary endothelium that regulate the paracellular barrier under normal conditions or when the cells are exposed to pathological factors such as increased shear stress from congenital heart abnormalities (shunt), lipopolysaccharide (LPS) from the outer membrane of gram negative bacteria, or increased cyclic stretch from mechanical ventilation. Shear stress has been shown to increase, while LPS and cyclic strain have been shown to decrease, alveolar-capillary barrier function. The transcription factor, Sox18, is known to play a key role in regulating vascular development. Here, in ovine pulmonary arterial endothelial cells (PAEC) subjected to physiologic levels of laminar flow (20 dyn/cm2), we identified an increase in trans-endothelial resistance (TER) that correlated with an increase in Sox18 expression. Further, we found that shear stress up-regulated the cellular tight junction protein, Claudin-5, in a Sox18 dependent manner, and Claudin-5 depletion abolished the Sox18 mediated increase in TER in response to shear stress. Utilizing peripheral lung tissue of 4 week old shunt lambs with increased pulmonary blood flow, we found that both Sox18 and Claudin-5 mRNA and protein levels were elevated. In contrast, in human lung microvascular endothelial cells (HLMVEC) exposed to LPS (1EU/ml) for 4 h, the mRNA and protein levels of Sox18 and Claudin-5 were decreased in an NF-κB (p65) and HDAC dependent manner. Sox18 over-expression prevented the LPS dependent loss of TER. Interestingly, this barrier protective effect of Sox18 was abolished by Claudin-5 silencing. In mice given an intratracheal instillation of LPS (2mg/kg, 24 h), we found that the over-expression of Sox18 in the pulmonary vasculature significantly increased Claudin-5 expression and attenuated the LPS mediated increase in lung vascular leak, inflammatory cell infiltration, and inflammatory cytokines in the bronchoalveolar lavage fluid. Sox18 gene delivery also increased oxygen saturation and improved lung function in LPS exposed mice. Similarly, in mice ventilated with high tidal volumes (HTV; 30 ml/kg, 75 bpm, 0.5 FiO2) for 8 h, Sox18 and Claudin-5 protein levels were reduced. However, Sox18 over-expression significantly increased Claudin-5 expression and improved lung function in HTV exposed mice. Together, our study demonstrates that Sox18 is an important regulator of pulmonary endothelial barrier function.
  • Mechanisms of Vessel Obliteration in Oxygen-Induced Retinopathy

    Gu, Xiaolin; Vascular Biology Center (2001-11)
    The overall goal of this study was to explore the possible molecular mediators of vaso-obliteration in retinopathy o f prematurity. Vaso-obliteration is the early hyperoxiainduced pathology. It leads to the later relative hypoxia in the retina tissue, because the insufficient blood supply cannot meet the increasing demands o f oxygen from the developing retina. Such retinal hypoxia then causes the blinding outcome through the formation o f neovessels and subsequent vitreous bleeding and fibrotic change in both retina and vitreous. Therefore, identification o f the possible mediators o f hyperoxiainduced vaso-obliteration will help us to understand more about the pathogenesis o f ROP and provide new and better strategies of treating and preventing this disease. Previous studies have shown that administration o f exogenous antioxidants can attenuate retinopathy in certain animal models and that hyperoxia is able to upregulate the expression and activity o f eNOS in vascular endothelial cells (Liao et al., 1995; North et al., 1996; Phelan and Faller, 1996). Hyperoxia also increases formation o f O2 ' which rapidly combines with NO to form the highly reactive oxidant ONOO*. Therefore, it is hypothesized that the NO and O2 'derived oxidant, ONOO', play an important role in the initial vascular injury leading to obliteration of the developing retinal capillaries in oxygen-induced retinopathy (OIR). It is further proposed that ONOO' causes vascular injury by modifying the critical intracellular signaling pathway that controls endothelial cell survival (Fig 5). This hypothesis has been tested by accomplishing the following specific aims: 1. Establish the OIR mouse model for ROP. Analyze NOS expression and assay the formation of NO and ONOO' in the vaso-obliteration phase o f OIR. 2. Determine whether deletion o f the eNOS or iNOS gene alters the vaso-obliteration phase o f OIR. If so, determine whether the gene deletion also reduces ONOO' formation in the vaso-obliteration phase o f OIR. 3. Test whether or not pharmacological inhibition o f NOS reduces vascular obliteration in wild-type mice with OIR. 4. Establish a tissue culture model for oxygen-induced endothelial cell injury. Determine the effect o f hyperoxia on endothelial cell survival and test whether the effects are mediated by NO, O2 ', and /or ONOO'. 5. Test whether ONOO' alters the signal transduction pathway for endothelial cell survival by altering the activity o f PI3K/AKT.
  • Insights into the Arginine Paradox and the Role of Arginase in Diabetic Retinopathy

    Elms, Shawn; Vascular Biology Center (2012-12)
    Reduced production of nitric oxide (NO) is one of the first indications of endothelial dysfunction and precedes the development of many cardiovascular diseases. Arginase has been shown to be upregulated in cardiovascular disease and has been proposed as a mechanism to account for diminished NO production. Arginases consume L-arginine, the substrate for nitric oxide synthase (NOS), and L-arginine depletion is thought to reduce NOS-derived NO. However, this simple relationship is complicated by the L-arginine paradox. The paradox addresses the phenomenon that L-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis yet increasing Larginine externally drives increased production of NO. One mechanism proposed to explain this is compartmentalization of intracellular L-arginine into distinct pools. In the current study we investigated this concept by targeting eNOS and arginase to different locations within the cell. We first showed that supplemental L-arginine and L-citrulline dose-dependently increased NO production in a manner independent of the location of eNOS within the cell. Cytosolic arginase-1 (ArgI) and mitochondrial arginase-2 (Argil) inhibited eNOS activity equally regardless of where in the cell eNOS was expressed. Similarly, targeting ArgI to different regions of the cell did not modify its ability to inhibit NO formation. These results argue against compartmentalization as the mechanism by which arginase inhibit eNOS. Further studies showed that arginasedependent inhibition of NO formation was prevented pharmacologically with arginase inhibitors. Also, arginase inhibition of NO production was absent in a catalytically inactive arginase mutant. Arginase did not co-immunoprecipitate with eNOS and the metabolic products of arginase or downstream enzymes did not contribute to reduced NO formation. Because of previous studies in animals and cell culture supporting the role of ArgI specifically in vascular dysfunction, we aimed to investigate the role of ArgI in the retinal vascular dysfunction of diabetic retinopathy (DR). Our hypothesis was that ArgI could be a mediator in the vascular dysfunction associated with DR. While using a mouse funduscope to image the retinal vasculature, we infused acetylcholine or sodium nitroprasside intravenously into diabetic or normoglycemic control mice and measured vessel relaxation. Endothelium-dependent retinal vasorelaxation was impaired in diabetic mice (40% of control). Diabetic mice hemizygous for arginase-1 (Argl+/") had improved function of the retinal vessels (71% of control). Endothelium-independent vasorelaxation was similar in diabetic and control, Argl+/' and wild type mice, indicating that the diabetes effect was specifically an endothelial issue and not one of smooth muscle dysfunction. Arginase inhibitors were shown to be effective in improving vascular function and reducing arginase activity. Further experiments were conducted in isolated central retinal arteries of diabetic and control rats, which recapitulated the results found in the mouse. We found that pharmacologic inhibition in both mice and rats or partial knock out of ArgI in mice resulted in improvement in the retinal vascular dysfunction associated with DR. We conclude that ArgI is a potential player in the retinal vascular dysfunction of DR.
  • From Adipokines to Atherosclerosis: The Role of Adipose Tissue in Inflammation and Etiology of Vascular Disease

    Bundy, Vanessa; Vascular Biology Center (2007-04)
    The prevalence of overweight and obese has steadily increased over the years among males and females of all ages, all racial and ethnic groups, and all educational levels. Recent studies have established adipose tissue as a dynamic, endocrine organ with the capacity to secrete a number of adipokines which may act directly upon the vasculature to stimulate adhesion molecule expression and exacerbate vascular disease. Our aim was to elucidate the associations of vasoactive pro- and anti- inflammatory factors, including adhesion molecules, with adiposity, blood pressure and endothelial function, and to distinguish race and sex variations in these relationships. To accomplish this, we expanded upon existing measurements within a Georgia Prevention Institute cross-sectional study entitled Lifestyle, Adiposity & Cardiovascular Health in Youths (LACHY) by adding two cardiovascular disease risk factor domains: inflammation and vascular adhesion. Our model included measurements of adiposity, adiponectin, C-reactive protein, leptin, insulin, resistin, tumor necrosis factor-a, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, blood pressure and endothelial-dependent arterial dilation. Our findings include numerous race and sex differences in the concentration of circulating risk factors along with significant interactions between them and measurements of adiposity. However, we did not find circulating cardiovascular disease risk factors or their concentration differences to be significantly associated with blood pressure or endothelial function. We believe this to be largely due to the fact that our subjects were young and apparently healthy at time of measurement. Overall, our findings provide insight into the relationships between adiposity, inflammation and cardiovascular outcomes in black and white, male and female adolescents. Future studies are needed to further elucidate these relationships and how they may change over time.
  • The Role of SFLT-1 in Corneal Avascularity

    Ambati, Balamurali K.; Vascular Biology Center (2008-03)
    Ocular vascular compartmentalization is manifested by certain structures richly vascularized adjacent to normally avascular ones (e.g., the limbus next to the cornea or the retina next to the vitreous) and is necessary for optical transparency in the visual axis. Vision-threatening corneal angiogenesis can be caused by disease, aging, infection, or trauma. The basis of the cornea's avascularity has long been obscure. Although the absence of blood vessels in the cornea was known to the ancients such as Susruta (Sharma 2001) and Galen (Magnus 1999) millennia ago, only in the last century were angiostatic substances postulated to underpin corneal avascularity (Meyer & Chaffee 1940). Because of its avascularity and ease of accessibility the cornea has been a proving ground for testing antiangiogenic strategies for over 30 years (Gimbrone et al. 1974). Despite the cornea's widespread use as a readout template for anti-angiogenesis testing, the molecular foundations of corneal avascularity remain unclear. In the last decade, numerous anti-angiogenic molecules such as angiostatin, endostatin, interleukin-1 receptor antagonist, pigment epithelium derived factor, and thrombospondins were identified in the cornea (Chang et al. 2001). None of these molecules, however, is singly requisite for corneal avascularity because mice deficient in any of them retain normal corneal avascularity (Wiegand et al. 2004; Cursiefen et al. 2004; Bugge et al. 1995; Fukai et al. 2002; Hirsch et al. 1996), supporting the view of multiply redundant mechanisms of corneal avascularity. This study aims to elucidate the role of sFlt-1 in the preservation of corneal avascularity. This project applies RNA interference, a promising new efficient and specific molecular technology that targets specific mRNAs, to elucidate mechanisms of normal corneal avascularity. Other strategies employed include genomic and protein-based deletion of target molecules and cross-species protein expression profiles. We hypothesize that corneal avascularity is maintained at least in part by extracellular soluble VEGF receptor-1 (sVEGFR-1, also known as and henceforth referred to as sFlt-1).
  • Vascular dysfunction in pulmonary hypertension: Role of protein kinase G-1a nitration

    Aggarwal, Saurbh; Vascular Biology Center (2011-06)
    Pulmonary hypertension is a common and debilitating complication of pulmonary, cardiac, and extrathoracic pathologies. The development of pulmonary hypertension is associated with elevated pulmonary arterial pressure (PAP), and increased vascular remodeling. Pulmonary vascular tone is regulated through the activation of protein kinase G-la (PKG-la), which is the isoform predominantly found in the lungs, via a complex signaling pathway that involves nitric oxide (NO), natriuretic peptides (NP), and cyclic guanosine monophosphate (cGMP). Vascular injury secondary to increased reactive oxygen (ROS) and nitrogen species (RNS) in pulmonary hypertension disrupts these regulatory mechanisms, potentiating the development of vascular dysfunction. However, the molecular mechanisms underlying this dysfunction are not completely understood and were the focus of this study.
  • Notch3 Signaling Mediates Heterotypic Cell Interactions During Blood Vessel Formation

    Liu, Hua; Vascular Biology Center (2010-11)
    Blood vessel formation is essential for embryogenesis, wound healing, menstruation, and pregnancy [1, 2]. While much emphasis has been placed on understanding the initial event of endothelial-tube formation, relatively little attention has been paid to the interactions of endothelial cells and the surrounding mural cells (pericytes, smooth muscle cells and fibroblasts). Increasing evidence suggests that the communication of endothelial cells and mural cells is crucial for the assembly, subsequent maturation, and stabilization of blood vessels [3-5]. Abnormal interactions between these two cell types have been implicated in many pathological conditions, including tumor angiogenesis, diabetic microangiopathy, tissue calcification and stroke. However, the molecules mediating the heterotypic interaction are still largely unknown. Our previous studies have shown that in a three-dimensional (3-D) angiogenesis assay, mural cells enhance blood vessel formation and directly interact with endothelial cells [6]. During this process, Notch3 is one gene that is strongly induced in mural cells upon coculture with endothelial cells [6]. Notch3, the causative gene of the neurovascular disorder CADASIL [7], belongs to an evolutionarily conserved family of transmembrane receptors that are known to govern cell fate determination in diverse cell types [8]. Given that Notch receptors and ligands are expressed on both endothelial and mural cells and - 2 - Notch3 is upregulated in mural cells by coculturing with endothelial cells, it is reasonable to assume that the Notch3 receptor might regulate the association of endothelial and mural cells through receptor-ligand interaction during blood vessel formation. The goal of my thesis is to investigate how Notch3 gene expression is regulated in mural cells by endothelial cells and whether the Notch3 receptor is involved in the communication between endothelial and mural cells during blood vessel formation. To achieve these goals, three aims were proposed: Specific Aim 1: To define how Notch3 expression in mural cells is upregulated by endothelial cells. Specific Aim 2: To determine if endothelial cell-induced Notch3 expression is critical for mural cell differentiation. Specific Aim 3: To determine whether Notch3 expression in mural cells modulates blood vessel formation under both physiological and pathological conditions.
  • eNOS Regulation by Phosphorylation and Protein-Protein Interactions

    Li, Chunying; Vascular Biology Center (2006-08)
    Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO). Protein phosphorylation and protein-protein interactions are two major mechanisms for eNOS regulation at the post-translational level, three aspects of which have been investigated in this study. The first aspect of eNOS regulation that we have examined is whether endostatin (ES) is a novel eNOS-activating agonist responsible for stimulating multi-site eNOS phosphorylation in endothelial cells. We show that ES induces acute endothelial NO release accompanied by eNOS phosphorylation events in cultured bovine aortic endothelial cells (BAECs). ES also induces relaxation of rat aortic rings. The second aspect of eNOS regulation that we have examined is the role of individual eNOS serine and threonine phosphorylation sites in the regulation of eNOS activity in BAECs. We mutated all five Thr- and Ser- sites of eNOS phosphorylation to aspartate or alanine and overexpressed the proteins in BAECs using adenoviral-mediated gene transfer. We show that mimicking phosphorylation of Ser-116 and Thr-497 is inhibitory, and mimicking phosphorylation of Ser-617, Ser-635 and Ser-1179 is stimulatory. Mimicking phosphorylation of Ser-635 and Ser-1179 together does not show synergistic effects on endothelial NO release. In addition, removal of any of the five Ser/Thr phosphorylation sites does not affect thapsigargin- or VEGF-stimulated NO release. A final aspect of eNOS regulation that we have investigated is the role of protein-protein interactions of eNOS with the CAT (cationic amino acid transporter)-1 arginine transporter. We show that eNOS interacts directly with CAT-1 and that overexpression of CAT-1 proteins in BAECs results in significant increases in NO release which is not altered by the CAT-1 inhibitor, L-lysine, suggesting that NO production in this in vitro model is independent of CAT-1 mediated arginine transport. Furthermore, eNOS enzymatic activity is increased in lysates of CAT-1-overexpressing cells accompanied by increased eNOS association with CAT-1, alterations of eNOS phosphorylation and eNOS association with caveolin-1. The present study adds to the knowledge of the regulation of eNOS by multi-site phosphorylation and protein-protein interactions.
  • HSP90 Inhibitors in Sepsis and Sepsis-induced Acute Lung Injury

    Chatterjee, Anuran; Vascular Biology Center (2007-06)
    Severe sepsis is the leading cause of death for patients in intensive care units. Patients with severe sepsis develop multiple organ failure, including acute lung injury, resulting from a dysregulated inflammatory response. Inhibitors of the ubiquitous chaperone, heat shock protein 90 (hsp90), block the activity of certain pro-inflammatory mediators, in vitro. We hypothesized that hsp90 inhibitors may ameliorate the inflammation and acute lung injury associated with severe sepsis. Male C57Bl/6 mice received either one of two hsp90 inhibitors, radicicol or 17-allylaminodemethoxygeldanamycin (17-AAG), at 24, 12, 6 and 0 hr before receiving a lethal dose of endotoxin (6.75 x 104 EU / g body weight). Outcomes included survival and parameters of systemic inflammation (plasma cytokine, chemokine and nitrite/nitrate levels), pulmonary inflammation (lung NF-κB and myeloperoxidase activities, inducible nitric oxide or iNOS expression, iNOS-hsp90 complex formation, leukocyte infiltration), and lung injury (pulmonary capillary leak, expression of endothelial specific cell-adhesion or adherens junction proteins, lung function). Mice pre-treated with vehicle and receiving endotoxin exhibited 100% 24-hr lethality, dramatic increase in all parameters of systemic and pulmonary inflammation, reduced lung function and increased capillary leak associated with reduced expression of functional adherens junction proteins. In comparison, mice receiving either radicicol or 17-AAG prior to endotoxin, exhibited prolonged survival, reduced or abolished increases in systemic and pulmonary inflammatory parameters, normal lung function and attenuated capillary leak with restored expression of adherens junction proteins. Additionally, in an in vitro model of endotoxin and activated neutrophil-induced endothelial barrier dysfunction, we show that pre-incubation of neutrophils or endothelial cells or both with hsp90 inhibitors impart a profound barrier protective effect, which is mediated, at-least in part, through reduced activation of pp60Src kinase (an hsp90 client protein) and phosphorylation of the focal adhesion protein paxillin, a pp60Src kinase substrate. Hsp90 inhibitors are drugs already in use clinically as adjunct cancer treatment. Therefore, these findings point to a potential clinical use of these drugs in sepsis and sepsis induced ALI.