Claire A. Sand

A 28-kDa Splice Variant of NADPH Oxidase-4 Is Nuclear-Localized and Involved in Redox Signaling in Vascular Cells

Objective—Reactive oxygen species–generating nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase proteins (Noxs) are involved in cell differentiation, migration, and apoptosis. Nox4 is unique among Noxs in being constitutively active, and its subcellular localization may therefore be particularly important. In this study, we identified and characterized a novel nuclear-localized 28-kDa splice variant of Nox4 in vascular cells. Approach and Results—Nox4 immunoreactivity was noted in the nucleus and nucleolus of vascular smooth muscle cells and multiple other cell types by confocal microscopy. Cell fractionation, sequence analyses, and siRNA studies indicated that the nuclear-localized Nox4 is a 28-kDa splice variant, Nox4D, which lacks putative transmembrane domains. Nox4D overexpression resulted in significant NADPH-dependent reactive oxygen species production as detected by several different methods and caused increased phosphorylation of extracellular-signal-regulated kinase1/2 and the nuclear transcription factor Elk-1. Overexpression of Nox4D could also induce DNA damage as assessed by &ggr;-H2AX phosphorylation. These effects were inhibited by a single amino acid substitution in the Nox4D NADPH-binding region. Conclusions—Nox4D is a nuclear-localized and functionally active splice variant of Nox4 that may have important pathophysiologic effects through modulation of nuclear signaling and DNA damage.

Quantification of microcirculatory blood flow: a sensitive and clinically relevant prognostic marker in murine models of sepsis

Sepsis and sepsis-associated multiorgan failure represent the major cause of mortality in intensive care units worldwide. Cardiovascular dysfunction, a key component of sepsis pathogenesis, has received much research interest, although research translatability remains severely limited. There is a critical need for more comprehensive preclinical sepsis models, with more clinically relevant end points, such as microvascular perfusion. The purpose of this study was to compare microcirculatory blood flow measurements, using a novel application of laser speckle contrast imaging technology, with more traditional hemodynamic end points, as part of a multiparameter monitoring system in preclinical models of sepsis. Our aim, in measuring mesenteric blood flow, was to increase the prognostic sensitivity of preclinical studies. In two commonly used sepsis models (cecal ligation and puncture, and lipopolysaccharide), we demonstrate that blood pressure and cardiac output are compromised postsepsis, but subsequently stabilize over the 24-h recording period. In contrast, mesenteric blood flow continuously declines in a time-dependent manner and in parallel with the development of metabolic acidosis and organ dysfunction. Importantly, these microcirculatory perturbations are reversed by fluid resuscitation, a mainstay intervention associated with improved outcome in patients. These data suggest that global hemodynamics are maintained at the expense of the microcirculation and are, therefore, not sufficiently predictive of outcome. We demonstrate that microcirculatory blood flow is a more sensitive biomarker of sepsis syndrome progression and believe that incorporation of this biomarker into preclinical models will facilitate sophisticated proof-of-concept studies for novel sepsis interventions, providing more robust data on which to base future clinical trials.

Overexpression of GTP cyclohydrolase 1 feedback regulatory protein is protective in a murine model of septic shock

ABSTRACT Overproduction of nitric oxide (NO) by inducible NO synthase contributes toward refractory hypotension, impaired microvascular perfusion, and end-organ damage in septic shock patients. Tetrahydrobiopterin (BH4) is an essential NOS cofactor. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme for BH4 biosynthesis. Under inflammatory conditions, GCH1 activity and hence BH4 levels are increased, supporting pathological NOS activity. GCH1 activity can be controlled through allosteric interactions with GCH1 feedback regulatory protein (GFRP). We investigated whether overexpression of GFRP can regulate BH4 and NO production and attenuate cardiovascular dysfunction in sepsis. Sepsis was induced in mice conditionally overexpressing GFRP and wild-type littermates by cecal ligation and puncture. Blood pressure was monitored by radiotelemetry, and mesenteric blood flow was quantified by laser speckle contrast imaging. Blood biochemistry data were obtained using an iSTAT analyzer, and BH4 levels were measured in plasma and tissues by high-performance liquid chromatography. Increased BH4 and NO production and hypotension were observed in all mice, but the extents of these pathophysiological changes were attenuated in GFRP OE mice. Perturbations in blood biochemistry were similarly attenuated in GFRP OE compared with wild-type controls. These results suggest that GFRP overexpression regulates GCH1 activity during septic shock, which in turn limits BH4 bioavailability for iNOS. We conclude that the GCH1-GFRP axis is a critical regulator of BH4 and NO production and the cardiovascular derangements that occur in septic shock.

Blockade or deletion of transient receptor potential vanilloid 4 (TRPV4) is not protective in a murine model of sepsis

Sepsis is a systemic inflammatory response triggered by microbial infection that can cause cardiovascular collapse, insufficient tissue perfusion and multi-organ failure. The cation channel transient receptor potential vanilloid 4 (TRPV4) is expressed in vascular endothelium and causes vasodilatation, but excessive TRPV4 activation leads to profound hypotension and circulatory collapse - key features of sepsis pathogenesis. We hypothesised that loss of TRPV4 signaling would protect against cardiovascular dysfunction in a mouse model of sepsis (endotoxaemia). Multi-parameter monitoring of conscious systemic haemodynamics (by radiotelemetry probe), mesenteric microvascular blood flow (laser speckle contrast imaging) and blood biochemistry (iSTAT blood gas analysis) was carried out in wild type (WT) and TRPV4 knockout (KO) mice. Endotoxaemia was induced by a single intravenous injection of lipopolysaccharide (LPS; 12.5 mg/kg) and systemic haemodynamics monitored for 24 h. Blood flow recording was then conducted under terminal anaesthesia after which blood was obtained for haematological/biochemical analysis. No significant differences were observed in baseline haemodynamics or mesenteric blood flow. Naïve TRPV4 KO mice were significantly acidotic relative to WT counterparts. Following induction of sepsis, all mice became significantly hypotensive, though there was no significant difference in the degree of hypotension between TRPV4 WT and KO mice. TRPV4 KO mice exhibited a higher sepsis severity score. While septic WT mice became significantly hypernatraemic relative to the naïve state, this was not observed in septic KO mice. Mesenteric blood flow was inhibited by topical application of the TRPV4 agonist GSK1016790A in naïve WT mice, but enhanced 24 h following LPS injection. Contrary to the initial hypothesis, loss of TRPV4 signaling (either through gene deletion or pharmacological antagonism) did not attenuate sepsis-induced cardiovascular dysfunction: in fact, pathology appeared to be modestly exaggerated in mice lacking TRPV4. Local targeting of TRPV4 signalling may be more beneficial than global inhibition in sepsis treatment.

Dimethylarginine dimethylaminohydrolase 1 is involved in spinal nociceptive plasticity.

Abstract Activation of neuronal nitric oxide synthase, and consequent production of nitric oxide (NO), contributes to spinal hyperexcitability and enhanced pain sensation. All NOS isoforms are inhibited endogenously by asymmetric dimethylarginine, which itself is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). Inhibition of DDAH can indirectly attenuate NO production by elevating asymmetric dimethylarginine concentrations. Here, we show that the DDAH-1 isoform is constitutively active in the nervous system, specifically in the spinal dorsal horn. DDAH-1 was found to be expressed in sensory neurons within both the dorsal root ganglia and spinal dorsal horn; L-291 (NG–[2-Methoxyethyl]-L-arginine methyl ester), a DDAH-1 inhibitor, reduced NO synthesis in cultured dorsal root ganglia neurons. Spinal application of L-291 decreased N-methyl-D-aspartate–dependent postdischarge and windup of dorsal horn sensory neurons—2 measures of spinal hyperexcitability. Finally, spinal application of L-291 reduced both neuronal and behavioral measures of formalin-induced central sensitization. Thus, DDAH-1 may be a potential therapeutic target in neuronal disorders, such as chronic pain, where elevated NO is a contributing factor.

Vascular Expression of Transient Receptor Potential Vanilloid 1 (TRPV1)

Dear Editor, In the February 2014 issue of the Journal of Histochemistry & Cytochemistry, Tόth and colleagues published a report on Transient Receptor Potential Vanilloid 1 (TRPV1) expression and function in the rat vascular system (Tόth et al. 2014). The authors found that several commercially available anti-TRPV1 antibodies were not selective for TRPV1, and identified two that were considered to be individually selective for either neuronal or vascular—specifically smooth muscle—TRPV1. We have carried out similar studies on the role of TRPV1 in the mouse vascular system (which shares 95% nucleotide sequence homology with the rat), focusing specifically on endothelial cells. Like Tόth and colleagues, we found several commercially available anti-TRPV1 antibodies lacked specificity for TRPV1, highlighting the importance of conducting functional analysis of TRPV1 expression and activity. We found no evidence of functional TRPV1 expression in isolated murine endothelial cells and smooth muscle cells, despite numerous previous reports to the contrary. These data call into question much of what has been published on TRPV1 expression in the vasculature. The role—and indeed the very presence—of TRPV1 in vascular tissue remain highly contentious. While several groups have reported evidence of endothelial TRPV1 expression (Bratz et al. 2008; Fantozzi et al. 2003; Yang et al. 2010), others have failed to reproduce these findings (Cavanaugh et al. 2011; Marrelli et al. 2007). Many studies have relied on mRNA expression alone, and protein quantification has often been conducted in the absence of appropriate controls, or using antibodies that have not been validated in TRPV1 knockout (KO) tissue. We aimed to clarify these discrepancies using a combination of biochemical and functional analysis in a number of different endothelial cell lines. Using reverse transcription and PCR amplification of vascular cDNA, we found evidence— consistent with the observations of Tόth and colleagues—of TRPV1 mRNA expression in aortic lysates of wild type (WT), but not TRPV1 KO, mice and in freshly isolated and immortalized endothelial cells from three different species (Fig. 1A). TRPV4 expression, here used as a positive control, was clearly evident in all tissues (Fig. 1A), consistent with published reports (Baylie and Brayden 2011). In order to confirm protein expression, we used ACC-030 (Alomone Labs; Jerusalem, Israel), a widely used anti-TRPV1 antibody that, in previous reports, showed no immunoreactivity in samples from TRPV1 KO mice (Yang et al. 2010); albeit, this was in the absence of a protein loading control. We observed clear and distinct bands in aortic and dorsal root ganglia lysates of KO mice of identical origins to those used by Yang et al. (Fig. 1B), suggesting— in line with the results of Tόth and colleagues—that this antibody is not a suitable indicator of TRPV1 protein expression. Furthermore, these bands were observed at approximately 75 kDa, which is 20 kDa smaller than the predicted molecular weight of TRPV1 (Caterina et al. 1997); this is further evidence of non-selective immunoreactivity. We additionally tested three other commercially available antibodies (Table 1) that were similarly non-selective for TRPV1 (Fig. 1C–1E). Although TRPV1 KO mice are functional knockouts and retain the C-terminus of the TRPV1 gene against which most TRPV1 antibodies are raised, sequencing of TRPV1 KO cDNA 581014 JHCXXX10.1369/0022155415581014Sand et al.Vascular expression of TRPV1 research-article2015