Abstract 235: Sonic Hedgehog Induces Angiogenesis via Rho Kinase-dependent MMP-9 and Osteopontin Expression

The morphogen Sonic Hedgehog (Shh) is known to promote neovascularization in adults via indirect induction of pro-angiogenic cytokine expression by fibroblasts. Direct effects of Shh on endothelial cell (EC) function and angiogenesis, however, have not been characterized. Accordingly, we performed a series of in vitro and in vivo studies to evaluate the direct effects of Shh on EC and to investigate certain mechanisms by which Shh modulates angiogenesis. Our data disclose that Shh promotes capillary morphogenesis (tube length on Matrigel ™ increased to 271±50% of the length in untreated cells), induces EC migration (191±35%) and increases EC expression of matrix metalloproteinase 9 (MMP-9) and osteopontin (OPN), which are shown to be essential for Shh-induced angiogenesis both in vitro and in vivo . Shh effects in ECs, however, occur while Gli-dependent transcription is not modulated. Furthemore, our studies show that changes in gene expression and EC migration are mediated by Shh induction of Rho. The Rho dependence of Shh-induced EC angiogenic activity is documented in vitro using dominant negative constructs for RhoA and Rho kinase (ROCK), showing that RhoA and ROCK blockade attenuates Shh induced migration and tube formation, as well as the Shh induced expression of MMP-9 and OPN. In vivo in the mouse corneal angiogenesis model pharmacologic inhibition of ROCK blocks Shh induced angiogenesis, confirming the ROCK dependence of this process. Furthermore, in MMP-9 and OPN null mice Shh induced angiogenesis also blocked, indicating that Shh induced angiogenesis is also dependent on MMP-9 and OPN expression. These data elucidate an entirely novel, “non-classical” pathway by which Shh directly modulates EC phenotype and angiogenic activity

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Hyperoxia, reactive oxygen species, and hyperventilation: oxygen sensitivity of brain stem neurons

Journal of Applied Physiology ◽

10.1152/japplphysiol.00892.2003 ◽

2004 ◽

Vol 96 (2) ◽

pp. 784-791 ◽

Cited By ~ 103

Author(s):

Jay B. Dean ◽

Daniel K. Mulkey ◽

Richard A. Henderson ◽

Stephanie J. Potter ◽

Robert W. Putnam

Keyword(s):

Oxidative Stress ◽

Brain Stem ◽

Brain Slices ◽

Respiratory Control ◽

In Vivo Studies ◽

Future Research ◽

Direct Effects ◽

Underlying Assumption

Hyperoxia is a popular model of oxidative stress. However, hyperoxic gas mixtures are routinely used for chemical denervation of peripheral O2 receptors in in vivo studies of respiratory control. The underlying assumption whenever using hyperoxia is that there are no direct effects of molecular O2 and reactive O2 species (ROS) on brain stem function. In addition, control superfusates used routinely for in vitro studies of neurons in brain slices are, in fact, hyperoxic. Again, the assumption is that there are no direct effects of O2 and ROS on neuronal activity. Research contradicts this assumption by demonstrating that O2 has central effects on the brain stem respiratory centers and several effects on neurons in respiratory control areas; these need to be considered whenever hyperoxia is used. This mini-review summarizes the long-recognized, but seldom acknowledged, paradox of respiratory control known as hyperoxic hyperventilation. Several proposed mechanisms are discussed, including the recent hypothesis that hyperoxic hyperventilation is initiated by increased production of ROS during hyperoxia, which directly stimulates central CO2 chemoreceptors in the solitary complex. Hyperoxic hyperventilation may provide clues into the fundamental role of redox signaling and ROS in central control of breathing; moreover, oxidative stress may play a role in respiratory control dysfunction. The practical implications of brain stem O2 and ROS sensitivity are also considered relative to the present uses of hyperoxia in respiratory control research in humans, animals, and brain stem tissues. Recommendations for future research are also proposed.

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Dopamine Modulates Excitability of Basolateral Amygdala Neurons In Vitro

Journal of Neurophysiology ◽

10.1152/jn.00843.2004 ◽

2005 ◽

Vol 93 (3) ◽

pp. 1598-1610 ◽

Cited By ~ 104

Author(s):

Sven Kröner ◽

J. Amiel Rosenkranz ◽

Anthony A. Grace ◽

German Barrionuevo

Keyword(s):

Basolateral Amygdala ◽

Neuronal Response ◽

Brain Slices ◽

Receptor Activation ◽

D1 Receptor ◽

In Vivo Studies ◽

Projection Neurons ◽

Direct Effects

The amygdala plays a role in affective behaviors, which are modulated by the dopamine (DA) innervation of the basolateral amygdala complex (BLA). Although in vivo studies indicate that activation of DA receptors alters BLA neuronal activity, it is unclear whether DA exerts direct effects on BLA neurons or whether it acts via indirect effects on BLA afferents. Using whole cell patch-clamp recordings in rat brain slices, we investigated the site and mechanisms through which DA regulates the excitability of BLA neurons. Dopamine enhanced the excitability of BLA projection neurons in response to somatic current injections via a postsynaptic effect. Dopamine D1 receptor activation increased excitability and evoked firing, whereas D2 receptor activation increased input resistance. Current- and voltage-clamp experiments in projection neurons showed that D1 receptor activation enhanced excitability by modulating a 4-aminopyridine- and α-dendrotoxin-sensitive, slowly inactivating K+ current. Furthermore, DA and D1 receptor activation increased evoked firing in fast-spiking BLA interneurons. Consistent with a postsynaptic modulation of interneuron excitability, DA also increased the frequency of spontaneous inhibitory postsynaptic currents recorded in projection neurons without changing release of GABA. These data demonstrate that DA exerts direct effects on BLA projection neurons and indirect actions via modulation of interneurons that may work in concert to enhance the neuronal response to large, suprathreshold inputs, while suppressing weaker inputs.

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High glucose promotes nascent nephron apoptosis via NF-κB and p53 pathways

AJP Renal Physiology ◽

10.1152/ajprenal.00361.2010 ◽

2011 ◽

Vol 300 (1) ◽

pp. F147-F156 ◽

Cited By ~ 16

Author(s):

Yun-Wen Chen ◽

Isabelle Chenier ◽

Shiao-Ying Chang ◽

Stella Tran ◽

Julie R. Ingelfinger ◽

...

Keyword(s):

High Glucose ◽

Nuclear Translocation ◽

Dominant Negative ◽

In Vivo Studies ◽

Ros Generation ◽

Small Interfering Rnas ◽

Kidney Size ◽

P53 Signaling

A hyperglycemic environment in utero reduces kidney size and nephron number due to nascent nephron apoptosis. However, the underlying mechanisms are incompletely understood. The present study investigated whether the nascent nephron apoptosis promoted by high glucose is mediated via the transcription factor NF-κB and p53 signaling pathways. Neonatal mouse kidneys from the offspring of nondiabetic, diabetic, and insulin-treated diabetic dams were used for in vivo studies, and MK4 cells, an embryonic metanephric mesenchymal (MM) cell line, were used for in vitro studies. Neonatal kidneys of the offspring of diabetic mothers exhibited an increased number of apoptotic cells and reactive oxygen species (ROS) generation, enhanced NF-κB activation, and nuclear translocation of its subunits (p50 and p65 subunits) as well as phosphorylation (Ser 15) of p53 compared with kidneys of offspring of nondiabetic mothers. Insulin treatment of diabetic dams normalized these parameters in the offspring. In vitro, high-glucose (25 mM) induced ROS generation and significantly increased MK4 cell apoptosis and caspase-3 activity via activation of NF-κB pathway, with p53 phosphorylation and nuclear translocation compared with normal glucose (5 mM). These changes in a high-glucose milieu were prevented by transient transfection of small interfering RNAs for dominant negative IκBα or IKK or p53. Our data demonstrate that high glucose-induced nascent nephron apoptosis is mediated, at least in part, via ROS generation and the activation of NF-κB and p53 pathways.

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Mediator Modulates Gli3-Dependent Sonic Hedgehog Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.00443-06 ◽

2006 ◽

Vol 26 (23) ◽

pp. 8667-8682 ◽

Cited By ~ 78

Author(s):

Haiying Zhou ◽

Seokjoong Kim ◽

Shunsuke Ishii ◽

Thomas G. Boyer

Keyword(s):

Gene Transcription ◽

Sonic Hedgehog ◽

Target Gene ◽

Dominant Negative ◽

Transactivation Domain ◽

Functional Interaction ◽

Shh Signaling ◽

Target Gene Transcription

ABSTRACT The physiological and pathological manifestations of Sonic hedgehog (Shh) signaling arise from the specification of unique transcriptional programs dependent upon key nuclear effectors of the Ci/Gli family of transcription factors. However, the underlying mechanism by which Gli proteins regulate target gene transcription in the nucleus remains poorly understood. Here, we identify and characterize a physical and functional interaction between Gli3 and the MED12 subunit within the RNA polymerase II transcriptional Mediator. We show that Gli3 binds to MED12 and intact Mediator both in vitro and in vivo through a Gli3 transactivation domain (MBD; MED12/Mediator-binding domain) whose activity derives from concerted functional interactions with both Mediator and the histone acetyltransferase CBP. Analysis of MBD truncation mutants revealed an excellent correlation between the in vivo activation strength of an MBD derivative and its ability to bind MED12 and intact Mediator in vitro, indicative of a critical functional interaction between the Gli3 MBD and the MED12 interface in Mediator. Disruption of the Gli3-MED12 interaction through dominant-negative interference inhibited, while RNA interference-mediated MED12 depletion enhanced, both MBD transactivation function and Gli3 target gene induction in response to Shh signaling. We propose that activated Gli3 physically targets the MED12 interface within Mediator in order to functionally reverse Mediator-dependent suppression of Shh target gene transcription. These findings thus link MED12 to the modulation of Gli3-dependent Shh signaling and further implicate Mediator in a broad range of developmental and pathological processes driven by Shh signal transduction.

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Emerging and diverse roles of protein kinase C in immune cell signalling

Biochemical Journal ◽

10.1042/bj20031406 ◽

2003 ◽

Vol 376 (3) ◽

pp. 545-552 ◽

Cited By ~ 193

Author(s):

Seng-Lai TAN ◽

Peter J. PARKER

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Immune Responses ◽

Immune Cell ◽

Cell Signalling ◽

Dominant Negative ◽

In Vivo Studies ◽

Kinase C

Members of the protein kinase C (PKC) family are expressed in many different cell types, where they are known to regulate a wide variety of cellular processes that impact on cell growth and differentiation, cytoskeletal remodelling and gene expression in the response to diverse stimuli. The broad tissue distribution and redundancy of in vitro function have often hampered the identification of definitive roles for each PKC family member. However, recent in vivo studies of PKC isoenzyme-selective knockout and transgenic mice have highlighted distinct functions of individual PKCs in the immune system. These genetic analyses, along with biochemical studies utilizing PKC isoenzyme-specific cDNA (wild-type, constitutively active and dominant-negative), antisense oligonucleotides (ASO), RNA interference (RNAi), and pharmacological inhibitors, indicate that PKC-regulated signalling pathways play a significant role in many aspects of immune responses, from development, differentiation, activation and survival of lymphocytes to macrophage activation. The importance of PKCs in cellular immune responses suggests that improved understanding of the molecular events that govern their actions could point to new avenues for development of treatments for immune disorders.

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Gene transfer of dominant negative Rho kinase suppresses neointimal formation after balloon injury in pigs

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.278.6.h1744 ◽

2000 ◽

Vol 278 (6) ◽

pp. H1744-H1750 ◽

Cited By ~ 58

Author(s):

Yasuhiro Eto ◽

Hiroaki Shimokawa ◽

Junko Hiroki ◽

Kunio Morishige ◽

Tadashi Kandabashi ◽

...

Keyword(s):

Gene Transfer ◽

Rho Kinase ◽

Dominant Negative ◽

Balloon Injury ◽

Cellular Functions ◽

Neointimal Formation ◽

Stenotic Lesion ◽

Dominant Negative Form

Restenosis after angioplasty still remains a major problem for which neointimal formation appears to play an important role. Recent studies in vitro suggested that Rho kinase, a target protein of Rho, is important in various cellular functions. We thus examined whether Rho kinase is involved in the restenotic changes after balloon injury. In vivo gene transfer was performed immediately after balloon injury in both sides of the porcine femoral arteries with adenoviral vector encoding either a dominant negative form of Rho kinase (AdDNRhoK) or β-galactosidase (AdLacZ) as a control. One week after the transfer, immunohistochemistry confirmed the successful gene expression in the vessel wall, whereas 2 wk after the transfer, Western blotting showed the functional upregulation of Rho kinase at the AdLacZ site and its suppression at the AdDNRhoK site. Angiography showed the development of a stenotic lesion at the AdLacZ site where histological neointimal formation was noted, whereas those changes were significantly suppressed at the AdDNRhoK site. These results indicate that Rho kinase is involved in the pathogenesis of neointimal formation after balloon injury in vivo.

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Role of the glucocorticoid receptor in glomerular disease

AJP Renal Physiology ◽

10.1152/ajprenal.00217.2019 ◽

2019 ◽

Vol 317 (1) ◽

pp. F133-F136

Author(s):

Julie E. Goodwin

Keyword(s):

Risk Assessment ◽

Therapeutic Benefit ◽

In Vivo Studies ◽

Direct Effects ◽

Glomerular Diseases ◽

Benefit Ratio ◽

Do No Harm

Glucocorticoids are potent anti-inflammatory agents that are commonly used in the treatment of various glomerular diseases. Data from in vitro and in vivo studies, in both animals and humans, convincingly demonstrate that glucocorticoids have many beneficial direct effects on glomeruli, including podocytes, suggesting that, in theory, systemic administration is not necessary to achieve therapeutic benefit. Indeed, it is increasingly recognized that systemic steroids often have an unfavorable risk-to-benefit ratio. As we move into an age of personalized medicine, strategies to develop targeted steroid delivery systems and individualized risk assessment algorithms are desirable in clinicians’ efforts to “first, do no harm.”

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