Darcy Lidington

Deafness and Stria Vascularis Defects in S1P2 Receptor-null Mice

The S1P2 receptor is a member of a family of G protein-coupled receptors that bind the extracellular sphingolipid metabolite sphingosine 1-phosphate with high affinity. The receptor is widely expressed and linked to multiple G protein signaling pathways, but its physiological function has remained elusive. Here we have demonstrated that S1P2 receptor expression is essential for proper functioning of the auditory and vestibular systems. Auditory brainstem response analysis revealed that S1P2 receptor-null mice were deaf by one month of age. These null mice exhibited multiple inner ear pathologies. However, some of the earliest cellular lesions in the cochlea were found within the stria vascularis, a barrier epithelium containing the primary vasculature of the inner ear. Between 2 and 4 weeks after birth, the basal and marginal epithelial cell barriers and the capillary bed within the stria vascularis of the S1P2 receptor-null mice showed markedly disturbed structures. JTE013, an S1P2 receptor-specific antagonist, blocked the S1P-induced vasoconstriction of the spiral modiolar artery, which supplies blood directly to the stria vascularis and protects its capillary bed from high perfusion pressure. Vascular disturbance within the stria vascularis is a potential mechanism that leads to deafness in the S1P2 receptor-null mice.

Role of Sphingosine-1-Phosphate Phosphohydrolase 1 in the Regulation of Resistance Artery Tone

Sphingosine-1-phosphate (S1P), which mediates pleiotropic actions within the vascular system, is a prominent regulator of microvascular tone. By virtue of its S1P-degrading function, we hypothesized that S1P-phosphohydrolase 1 (SPP1) is an important regulator of tone in resistance arteries. Hamster gracilis muscle resistance arteries express mRNA encoding SPP1. Overexpression of SPP1 (via transfection of a SPP1wt) reduced resting tone, Ca2+ sensitivity, and myogenic vasoconstriction, whereas reduced SPP1 expression (antisense oligonucleotides) yielded the opposite effects. Expression of a phosphatase-dead mutant of SPP1 (SPP1H208A) had no effect on any parameter tested, suggesting that catalytic activity of SPP1 is critical. The enhanced myogenic tone that follows overexpression of S1P-generating enzyme sphingosine kinase 1 (Sk1wt) was functionally antagonized by coexpression with SPP1wt but not SPP1H208A. SPP1 modulated vasoconstriction in response to 1 to 100 nmol/L exogenous S1P, a concentration range that was characterized as S1P2-dependent, based on the effect of S1P2 inhibition by antisense oligonucleotides and 1 &mgr;mol/L JTE013. Inhibition of the cystic fibrosis transmembrane regulator (CFTR) (1) restored S1P responses that were attenuated by SPP1wt overexpression; (2) enhanced myogenic vasoconstriction; but (3) had no effect on noradrenaline responses. We conclude that SPP1 is an endogenous regulator of resistance artery tone that functionally antagonizes the vascular effects of both Sk1wt and S1P2 receptor activation. SPP1 accesses extracellular S1P pools in a manner dependent on a functional CFTR transport protein. Our study assigns important roles to both SPP1 and CFTR in the physiological regulation of vascular tone, which influences both tissue perfusion and systemic blood pressure.

Sphingosine kinase functionally links elevated transmural pressure and increased reactive oxygen species formation in resistance arteries

Myogenic vasoconstriction, an intrinsic response to elevated transmural pressure (TMP), requires the activation of sphingosine kinase (Sk1) and the generation of reactive oxygen species (ROS). We hypothesized that pressure‐induced Sk1 signaling and ROS generation are functionally linked. Using a model of cannulated resistance arteries isolated from the hamster gracilis muscle, we monitored vessel diameter and smooth muscle cell (SMC) Ca2+i (Fura‐2) or ROS production (dichlorodihydrofluorescein). Elevation of TMP stimulated the translocation of a GFP‐tagged Sk1 fusion protein from the cytosol to the plasma membrane, indicative of enzymatic activation. Concurrently, elevation of TMP initiated a rapid and transient production of ROS, which was enhanced by expression of wild‐type Sk1 (hSkwt) and inhibited by its dominant‐negative mutant (hSkG82D). Exogenous sphingosine‐1‐phosphate (S1P) also stimulated ROS generation is isolated vessels. Chemical (1μmol/L DPI), peptide (gp91ds‐tat/gp91ds), and genetic (N17Rac) inhibition strategies indicated that NADPH oxidase was the source of the pressure‐induced ROS. NADPH oxidase inhibition attenuated myogenic vasoconstriction and reduced the apparent Ca2+ sensitivity of the SMC contractile apparatus, without affecting Ca2+‐independent, RhoA‐mediated vasoconstriction in response to exogenous S1P. Our results indicate a mandatory role for Sk1/S1P in mediating pressure‐induced, NADPH oxidase‐derived ROS formation. In turn, ROS generation appears to increase Ca2+ sensitivity, necessary for full myogenic vasoconstriction.

Exocrine specific expression of Connexin32 is dependent on the basic helix-loop-helix transcription factor Mist1

Gap junctions are intercellular channels that provide direct passage of small molecules between adjacent cells. In pancreatic acini, the connexin26 (Cx26) and connexin32 (Cx32) proteins form functional channels that coordinate the secretion of digestive enzymes. Although the function of Cx26/Cx32 gap junctions are well characterized, the regulatory circuits that control the spatial and temporal expression patterns of these connexin genes are not known. In an effort to identify the molecular pathways that regulate connexin gene expression, we examined Cx26 and Cx32 gene activities in mice lacking the basic helix-loop-helix transcription factor Mist1 (Mist1KO). Mist1, Cx26 and Cx32 are co-expressed in most exocrine cell types, and acinar cells from Mist1KO mice exhibit a highly disorganized cellular architecture and an altered pattern of expression for several genes involved in regulated exocytosis. Analysis of Mist1KO mice revealed a dramatic decrease in both connexin proteins, albeit through different molecular mechanisms. Cx32 gene transcription was greatly reduced in all Mist1KO exocrine cells, while Cx26 gene expression remained unaffected. However, in the absence of Cx32 protein, Cx26 did not participate in gap junction formation, leading to a complete lack of intercellular communication among Mist1KO acinar cells. Additional studies testing Mist1 gene constructs in pancreatic exocrine cells confirmed that Mist1 transcriptionally regulates expression of the Cx32 gene. We conclude that Mist1 functions as a positive regulator of Cx32 gene expression and, in its absence, acinar cell gap junctions and intercellular communication pathways become disrupted.

Neuronal Differentiation and Growth Control of Neuro-2a Cells After Retroviral Gene Delivery of Connexin43

Given the roles proposed for gap junctional intercellular communication in neuronal differentiation and growth control, we examined the effects of connexin43 (Cx43) expression in a neuroblastoma cell line. A vesicular stomatitis virus G protein (VSVG)-pseudotyped retrovector was engineered to co-express the green fluorescent protein (GFP) and Cx43 in the communication-deficient neuro-2a (N2a) cell line. The 293 GPG packaging cell line was used to produce VSVG-pseudotyped retrovectors coding for GFP, Cx43, or chimeric Cx43·GFP fusion protein. The titer of viral supernatant, as measured by flow cytometry for GFP fluorescence, was approximately 2.0 × 107 colony form units (CFU)/ml and was free of replication-competent retroviruses. After a 7-day treatment with retinoic acid (20 μm), N2a transformants (N2a-Cx43 and N2a-Cx43·GFP) maintained the expression of Cx43 and Cx43·GFP. Expression of both constructs resulted in functional coupling, as evidenced by electrophysiological and dye-injection analysis. Suppression of cell growth correlated with expression of both Cx43 or Cx43·GFP and retinoic acid treatment. Based on morphology and immunocytochemistry for neurofilament, no difference was observed in the differentiation of N2a cells compared with cells expressing Cx43 constructs. In conclusion, constitutive expression of Cx43 in N2a cells does not alter retinoic acid-induced neuronal differentiation but does enhance growth inhibition.

Tumor Necrosis Factor-α Enhances Microvascular Tone and Reduces Blood Flow in the Cochlea via Enhanced Sphingosine-1-Phosphate Signaling

Background and Purpose— We sought to demonstrate that tumor necrosis factor (TNF)-&agr;, via sphingosine-1-phosphate signaling, has the potential to alter cochlear blood flow and thus, cause ischemic hearing loss. Methods— We performed intravital fluorescence microscopy to measure blood flow and capillary diameter in anesthetized guinea pigs. To measure capillary diameter ex vivo, capillary beds from the gerbil spiral ligament were isolated from the cochlear lateral wall and maintained in an organ bath. Isolated gerbil spiral modiolar arteries, maintained and transfected in organ culture, were used to measure calcium sensitivity (calcium-tone relationship). In a clinical study, a total of 12 adult patients presenting with typical symptoms of sudden hearing loss who were not responsive or only partially responsive to prednisolone treatment were identified and selected for etanercept treatment. Etanercept (25 mg s.c.) was self-administered twice a week for 12 weeks. Results— TNF-&agr; induced a proconstrictive state throughout the cochlear microvasculature, which reduced capillary diameter and cochlear blood flow in vivo. In vitro isolated preparations of the spiral modiolar artery and spiral ligament capillaries confirmed these observations. Antagonizing sphingosine-1-phosphate receptor 2 subtype signaling (by 1 &mgr;mol/L JTE013) attenuated the effects of TNF-&agr; in all models. TNF-&agr; activated sphingosine kinase 1 (Sk1) and induced its translocation to the smooth muscle cell membrane. Expression of a dominant-negative Sk1 mutant (Sk1G82D) eliminated both baseline spiral modiolar artery calcium sensitivity and TNF-&agr; effects, whereas a nonphosphorylatable Sk1 mutant (Sk1S225A) blocked the effects of TNF-&agr; only. A small group of etanercept-treated, hearing loss patients recovered according to a 1-phase exponential decay (half-life=1.56±0.20 weeks), which matched the kinetics predicted for a vascular origin. Conclusions— TNF-&agr; indeed reduces cochlear blood flow via activation of vascular sphingosine-1-phosphate signaling. This integrates hearing loss into the family of ischemic microvascular pathologies, with implications for risk stratification, diagnosis, and treatment.

Endotoxin increases intercellular resistance in microvascular endothelial cells by a tyrosine kinase pathway.

Gap junction communication between microvascular endothelial cells has been proposed to contribute to the coordination of microvascular function. Septic shock may attenuate microvascular cell‐to‐cell communication. We hypothesized that lipopolysaccharide (LPS) attenuates communication between microvascular endothelial cells derived from rat hindlimb skeletal muscle. Endothelial cells grown in monolayers expressed mRNA for connexin 37, 40, and 43. The expression of connexin 43 protein was confirmed, but connexin 40 protein was not detected by immunocytochemistry or immunoblot analysis. Intercellular resistance between cells of the monolayer, calculated using a Bessel function model, was increased from 3.3 to 5.3 MΩ by LPS. The effect was seen after 1 h of exposure and required a minimum concentration of 10 ng/ml. Intercellular resistance returned to normal 1 h following removal of LPS. Neither the response to LPS, nor its reversal, was blocked by the protein synthesis inhibitor cycloheximide (10 μg/ml). Pretreatment of monolayers with the tyrosine kinase inhibitors PP‐2 (10 nM), lavendustin‐C (1 μM), and geldanamycin (200 nM) prevented this LPS response; geldanamycin was also able to reverse the response. Inhibitors of MAP kinases, PD 98059 (5 μM) and SB 202190 (5 μM), and PKC (500 nM bisindolylmaleimide I) were unable to block the LPS response. We propose that LPS attenuates cell‐to‐cell communication through a signaling pathway that is tyrosine kinase dependent. J. Cell. Physiol. 185:117–125, 2000.

Lipopolysaccharide-induced reductions in cellular coupling correlate with tyrosine phosphorylation of connexin 43

We have previously shown in cultured rat microvascular endothelial cells (RMEC) that lipopolysaccharide (LPS) stimulates a protein tyrosine kinase (PTK)‐dependent reduction in cellular coupling. We hypothesized that connexin 43 (Cx43) becomes phosphorylated following exposure to LPS. Cx43 was immunoprecipitated from control and LPS‐treated RMEC monolayers. Tyrosine phosphorylation of Cx43, detected by immunoblot, was found only in the LPS treatment. To verify these results, Cx43 was radiolabeled with [32P]‐orthophosphate. Radiolabeled Cx43 exhibited a slight increase in phosphorylation in response to LPS; phosphoamino acid analysis displayed equivalent amounts of phosphoserine in control and LPS treatments, but detected phosphotyrosine only in the LPS treatment. The PTK inhibitors PP‐2 (10 nM) and geldanamycin (200 nM) were found to block the response to LPS in terms of Cx43 tyrosine phosphorylation and cellular coupling. The phosphatase inhibitor BpV (1 μM) accentuated the effect of LPS, while the putative phosphatase activator C6‐ceramide prevented it. When measuring cell communication, phosphatase inhibition also blocked the reversal of the LPS response following LPS washout. We conclude that Cx43 is tyrosine phosphorylated following exposure to LPS and suggest that the LPS‐induced increase in intercellular resistance may be mediated by tyrosine phosphorylation of this connexin. Altering tyrosine kinase and phosphatase activities can modulate the LPS‐induced tyrosine phosphorylation of Cx43 and reductions in cellular coupling. J. Cell. Physiol. 193: 373–379, 2002.

Priming of hypoxia-inducible factor by neuronal nitric oxide synthase is essential for adaptive responses to severe anemia

Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS)−/− mice demonstrated increased mortality during anemia. Unlike wild-type (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS−/− mice. At the cellular level, anemia increased expression of HIF-1α protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS−/− mice, despite comparable reductions in tissue PO2. Paradoxically, nNOS−/− mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-α(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-α protein stability in vivo. S-nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S-nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia.

Tumor Necrosis Factor-α–Mediated Downregulation of the Cystic Fibrosis Transmembrane Conductance Regulator Drives Pathological Sphingosine-1-Phosphate Signaling in a Mouse Model of Heart Failure

Background— Sphingosine-1-phosphate (S1P) signaling is a central regulator of resistance artery tone. Therefore, S1P levels need to be tightly controlled through the delicate interplay of its generating enzyme sphingosine kinase 1 and its functional antagonist S1P phosphohydrolase-1. The intracellular localization of S1P phosphohydrolase-1 necessitates the import of extracellular S1P into the intracellular compartment before its degradation. The present investigation proposes that the cystic fibrosis transmembrane conductance regulator transports extracellular S1P and hence modulates microvascular S1P signaling in health and disease. Methods and Results— In cultured murine vascular smooth muscle cells in vitro and isolated murine mesenteric and posterior cerebral resistance arteries ex vivo, the cystic fibrosis transmembrane conductance regulator (1) is critical for S1P uptake; (2) modulates S1P-dependent responses; and (3) is downregulated in vitro and in vivo by tumor necrosis factor-&agr;, with significant functional consequences for S1P signaling and vascular tone. In heart failure, tumor necrosis factor-&agr; downregulates the cystic fibrosis transmembrane conductance regulator across several organs, including the heart, lung, and brain, suggesting that it is a fundamental mechanism with implications for systemic S1P effects. Conclusions— We identify the cystic fibrosis transmembrane conductance regulator as a critical regulatory site for S1P signaling; its tumor necrosis factor-&agr;–dependent downregulation in heart failure underlies an enhancement in microvascular tone. This molecular mechanism potentially represents a novel and highly strategic therapeutic target for cardiovascular conditions involving inflammation.