Publications

Scholarly Journals--Published

• Long-term hypoxia uncouples Ca2+ and eNOS in bradykinin-mediated pulmonary arterial relaxation. Bradykinin-induced activation of the pulmonary endothelium triggers a rise in intracellular Ca2+ that activates nitric oxide (NO)-dependent vasorelaxation. Chronic hypoxia is commonly associated with increased pulmonary vascular tone, which can cause pulmonary hypertension in responsive individuals. In the present study, we tested the hypothesis that long-term high-altitude hypoxia (LTH) diminishes bradykinin-induced Ca2+ signals and inhibits endothelial nitric oxide synthase (eNOS), prostacyclin (PGI2), and large-conductance K+ (BKCa) channels in sheep, which are moderately responsive to LTH, resulting in decreased pulmonary arterial vasorelaxation. Pulmonary arteries were isolated from ewes kept near sea level (720 m) or at high altitude (3,801 m) for >100 days. Vessel force was measured with wire myography and endothelial intracellular Ca2+with confocal microscopy. eNOS was inhibited with 100 μM NG-nitro-l-arginine methyl ester (l-NAME), PGI2production was inhibited with 10 µM indomethacin that inhibits cyclooxygenase, and BKCa channels were blocked with 1 mM tetraethylammonium. Bradykinin-induced endothelial Ca2+ signals increased following LTH, but bradykinin relaxation decreased. Furthermore, some vessels contracted in response to bradykinin after LTH. l-NAME sensitivity decreased, suggesting that eNOS dysfunction played a role in uncoupling Ca2+ signals and bradykinin relaxation. The Ca2+ ionophore A-23187 (10 µM) elicited an enhanced Ca2+ response following LTH while relaxation was unchanged although l-NAME sensitivity increased. Additionally, BKCa function decreased during bradykinin relaxation following LTH. Western analysis showed that BKCa α-subunit expression was increased by LTH while that for the β1 subunit was unchanged. Overall, these results suggest that those even moderately responsive to LTH can have impaired endothelial function. (06/2018) (link)
• Silpanisong J, Kim D, Williams JM, Adeoye OO, Thorpe RB, Pearce WJ. Chronic hypoxia alters fetal cerebrovascular responses to endothelin-1. Am J Physiol Cell Physiol. 2017 Aug 1;313(2):C207-C218. doi: 10.1152/ajpcell.00241.2016. Epub 2017 May 3 PMID: 28566491 In utero hypoxia influences the structure and function of most fetal arteries, including those of the developing cerebral circulation. Whereas the signals that initiate this hypoxic remodeling remain uncertain, these appear to be distinct from the mechanisms that maintain the remodeled vascular state. The present study explores the hypothesis that chronic hypoxia elicits sustained changes in fetal cerebrovascular reactivity to endothelin-1 (ET-1), a potent vascular contractant and mitogen. In fetal lambs, chronic hypoxia (3,820-m altitude for the last 110 days of gestation) had no significant effect on plasma ET-1 levels or ETA receptor density in cerebral arteries but enhanced contractile responses to ET-1 in an ETA-dependent manner. In organ culture (24 h), 10 nM ET-1 increased medial thicknesses less in hypoxic than in normoxic arteries, and these increases were ablated by inhibition of PKC (chelerythrine) in both normoxic and hypoxic arteries but were attenuated by inhibition of CaMKII (KN93) and p38 (SB203580) in normoxic but not hypoxic arteries. As indicated by Ki-67 immunostaining, ET-1 increased medial thicknesses via hypertrophy. Measurements of colocalization between MLCK and SMαA revealed that organ culture with ET-1 also promoted contractile dedifferentiation in normoxic, but not hypoxic, arteries through mechanisms attenuated by inhibitors of PKC, CaMKII, and p38. These results support the hypothesis that chronic hypoxia elicits sustained changes in fetal cerebrovascular reactivity to ET-1 through pathways dependent upon PKC, CaMKII, and p38 that cause increased ET-1-mediated contractility, decreased ET-1-mediated smooth muscle hypertrophy, and a depressed ability of ET-1 to promote contractile dedifferentiation. (08/2017) (link)
• Thorpe RB, Hubbell MC, Silpanisong J, Williams JM, Pearce WJ. Chronic hypoxia attenuates the vasodilator efficacy of protein kinase G in fetal and adult ovine cerebral arteries. Am J Physiol Heart Circ Physiol. 2017 Jul 1;313(1):H207-H219. doi: 10.1152/ajpheart.00480.2016. Epub 2017 May 26. PMID: 28550175 Long-term hypoxia (LTH) attenuates nitric oxide-induced vasorelaxation in ovine middle cerebral arteries. Because cGMP-dependent protein kinase (PKG) is an important mediator of NO signaling in vascular smooth muscle, we tested the hypothesis that LTH diminishes the ability of PKG to interact with target proteins and cause vasorelaxation. Prominent among proteins that regulate vascular tone is the large-conductance Ca2+-sensitive K+ (BK) channel, which is a substrate for PKG and is responsive to phosphorylation on multiple serine/threonine residues. Given the influence of these proteins, we also examined whether LTH attenuates PKG and BK channel protein abundances and PKG activity. Middle cerebral arteries were harvested from normoxic and hypoxic (altitude of 3,820 m for 110 days) fetal and adult sheep. These arteries were denuded and equilibrated with 95% O2-5% CO2 in the presence of N-nitro-l-arginine methyl ester (l-NAME) to inhibit potential confounding influences of events upstream from PKG. Expression and activity of PKG-I were not significantly affected by chronic hypoxia in either fetal or adult arteries. Pretreatment with the BK inhibitor iberiotoxin attenuated vasorelaxation induced by 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate in normoxic but not LTH arteries. The spatial proximities of PKG with BK channel α- and β1-proteins were examined using confocal microscopy, which revealed a strong dissociation of PKG with these proteins after LTH. These results support our hypothesis that hypoxia reduces the ability of PKG to attenuate vasoconstriction in part through suppression of the ability of PKG to associate with and thereby activate BK channels in arterial smooth muscle.NEW & NOTEWORTHY Using measurements of contractility, protein abundance, kinase activity, and confocal colocalization in fetal and adult ovine cerebral arteries, the present study demonstrates that long-term hypoxia diminishes the ability of cGMP-dependent protein kinase (PKG) to cause vasorelaxation through suppression of its colocalization and interaction with large-conductance Ca2+-sensitive K+ (BK) channel proteins in cerebrovascular smooth muscle. These experiments are among the first to demonstrate hypoxic changes in BK subunit abundances in fetal cerebral arteries and also introduce the use of advanced methods of confocal colocalization to study interaction between PKG and its targets. (05/2017) (link)
• Blum-Johnston C, Thorpe R B, Wee C, Romero M, Brunelle A R, . . . Wilson S M. (2015). Developmental acceleration of bradykinin-dependent relaxation by prenatal chronic hypoxia impedes normal development after birth. Am J Physiol Lung Cell Mol Physiol, , ajplung.00340.2015. Bradykinin-induced activation of the pulmonary endothelium triggers nitric oxide (NO) production and other signals that cause vasorelaxation, including stimulation of large-conductance K+ (BKCa) channels in myocytes that hyperpolarize the plasma membrane and decrease intracellular Ca2+. Intrauterine chronic hypoxia (CH) may reduce vasorelaxation in the fetal transition, and contribute to pulmonary hypertension of the newborn. Thus, we examined the effects of maturation and CH on the role of BKCa channels during bradykinin-induced vasorelaxation by examining endothelial Ca2+ signals, wire-myography and Western Immunoblot on pulmonary arteries (PA) isolated from near term fetal (~ 140 days gestation) and newborn, 9 to 19 days old, sheep that lived in normoxia at 700 m or in CH at high altitude (3,801 m) for >100 days. CH enhanced bradykinin-induced relaxation of fetal vessels but decreased relaxation in newborns. Endothelial Ca2+ responses decreased with maturation but increased with CH. Bradykinin-dependent relaxation was sensitive to 100 muM LNAME or 10 muM ODQ, supporting roles for endothelial nitric oxide synthase and soluble guanylate cyclase activation. Indomethacin blocked relaxation in CH vessels suggesting upregulation of PLA2 pathways. BKCa channel inhibition with 1 mM TEA reduced bradykinin-induced vasorelaxation in the normoxic newborn and fetal CH vessels. Maturation reduced whole-cell BKCa alpha1 subunit expression but increased beta1 expression. These results suggest that CH amplifies the contribution of BKCa channels to bradykinin-induced vasorelaxation in fetal sheep, but stunts further development of this vasodilatory pathway in newborns. This involves complex changes in multiple components of the bradykinin-signaling axes. (12/2015) (link)
• Thorpe R B, Stockman S L, Williams J M, Lincoln T M, & Pearce W J. (2013). Hypoxic depression of PKG-mediated inhibition of serotonergic contraction in ovine carotid arteries. American Journal of Physiology-Regulatory Integrative and Comparative Physiology, 304(9), R734-R743. Chronic hypoxia attenuates soluble guanylate cyclase-induced vasorelaxation in serotonin (5-HT)-contracted ovine carotid arteries. Because protein kinase G (PKG) mediates many effects of soluble guanylate cyclase activation through phosphorylation of multiple kinase targets in vascular smooth muscle, we tested the hypothesis that chronic hypoxia reduces the ability of PKG to phosphorylate its target proteins, which attenuates the ability of PKG to induce vasorelaxation. We also tested the hypothesis that hypoxia attenuates PKG expression and/or activity. Arteries from normoxic and chronically hypoxic (altitude of 3,820 m for 110 days) fetal and adult sheep were denuded of endothelium and equilibrated with 95% O-2-5% CO2 in the presence of nitro-L-arginine methyl ester (L-NAME) and N-G-nitro-L-arginine (L-NNA) to inhibit residual endothelial nitric oxide synthase. Concentration-response relations for 5-HT were determined in the presence of prazosin to minimize activation of alpha-adrenergic receptors. The PKG activator 8-(p-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCTP-cGMP) reduced agonist binding affinity of the 5-HT receptor in a concentration-dependent manner that was attenuated by hypoxia. Expression and activity of PKG-I was not significantly affected by chronic hypoxia in either fetal or adult arteries, although PKG-I abundance was greater in fetal arteries. Pretreatment with the large conductance calcium-sensitive potassium channel (BK) inhibitor iberiotoxin attenuated the vasorelaxation induced by 8-pCPT-cGMP in normoxic but not chronically hypoxic arteries. These results support the hypothesis that hypoxia attenuates the vasorelaxant effects of PKG through suppression of the ability of PKG to activate large conductance calcium-sensitive potassium channels in arterial smooth muscle. The results also reveal that this hypoxic effect is greater in fetal than adult arteries and that chronic maternal hypoxia can profoundly affect fetal vascular function. (05/2013) (link)

Abstract

• "High Altitude Hypoxia Impacts Omega-3 Fatty Acid Metabolites in Plasma of Fetal and Newborn Sheep", Vanessa Lopez, Michael La Frano, Remy Bosviel, John Newman, Richard Thorpe, Oliver Feihn, Lubo Zhang, and Sean M. Wilson.  The FASEB Journal, Vol. 32, No. 1 supplement, April 2018 Perinatal hypoxia has profound effect on an infant’s development, with physiological impairment of multiple organ systems. The present study explored the effect of hypoxia due to long term high altitude exposure during gestation and after birth on lipid mediators by measuring the metabolites that foreshadow oxidative stress and inflammation, which are primary drivers of dysfunction. We tested the hypothesis that long term hypoxia reduces the amount of oxylipin and endocannabinoid production, which are important mediators of oxidative stress and inflammation. To test this hypothesis pregnant sheep and newborn lambs were housed at a low altitude (700 m) or exposed to an altitude of 3,800 meters starting gestation day 30. UPLC-MS/MS analysis was used to investigate the lipid mediator composition in plasma collected from veins of near-term fetal and newborn animals. The results show that hypoxia causes an overwhelming effect on omega-3 fatty acids and their derivatives, which are crucial in late-stage fetal development. Several of the omega-3 fatty acids reduced with hypoxia have well known effects on inflammation, vascular tone, and oxidative stress. Furthermore, we tracked the origin of the identified oxylipins and endocannabinoids by tracing their pathways. The cytochrome P450 (CYP) pathway enzymes and the subsequent activity of soluble epoxide hydrolase (sEH) are prominent synthesizers of the oxylipins such as epoxyeicosatrienoic acids (EETs) and epoxyoctadecenoic acids (EpOMEs). Previous studies have suggested the role of EETs and EpOMEs in vasodilation, a feature in inflammation. Based on these findings we demonstrated that a majority of the affected oxylipins were produced from CYP and sEH enzymes, which are important to vascular function. These findings provide novel insight into our understanding of lipid metabolites to hypoxia-induced dysfunction in the developing fetus and newborn. In the long term this may help us develop novel therapies that target inflammatory pathways induced by pre and post-natal hypoxia. (04/2018) (link)
• Thorpe R B, Williams J M, & Pearce W J. (2012). Hypoxia attenuates the ability of cyclic guanine-dependent protein kinase (PKG) to increase BK channel activity. Faseb Journal, 26, . Chronic hypoxia attenuates soluble guanylate cyclase-induced vasorelaxation in serotonin (5-HT)-contracted ovine carotid arteries. Because protein kinase G (PKG) mediates many effects of soluble guanylate cyclase activation through phosphorylation of multiple kinase targets in vascular smooth muscle, we tested the hypothesis that chronic hypoxia reduces the ability of PKG to phosphorylate its target proteins, which attenuates the ability of PKG to induce vasorelaxation. We also tested the hypothesis that hypoxia attenuates PKG expression and/or activity. Arteries from normoxic and chronically hypoxic (altitude of 3,820 m for 110 days) fetal and adult sheep were denuded of endothelium and equilibrated with 95% O2-5% CO2 in the presence of nitro-l-arginine methyl ester (l-NAME) and N(G)-nitro-l-arginine (l-NNA) to inhibit residual endothelial nitric oxide synthase. Concentration-response relations for 5-HT were determined in the presence of prazosin to minimize activation of α-adrenergic receptors. The PKG activator 8-(p-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCTP-cGMP) reduced agonist binding affinity of the 5-HT receptor in a concentration-dependent manner that was attenuated by hypoxia. Expression and activity of PKG-I was not significantly affected by chronic hypoxia in either fetal or adult arteries, although PKG-I abundance was greater in fetal arteries. Pretreatment with the large conductance calcium-sensitive potassium channel (BK) inhibitor iberiotoxin attenuated the vasorelaxation induced by 8-pCPT-cGMP in normoxic but not chronically hypoxic arteries. These results support the hypothesis that hypoxia attenuates the vasorelaxant effects of PKG through suppression of the ability of PKG to activate large conductance calcium-sensitive potassium channels in arterial smooth muscle. The results also reveal that this hypoxic effect is greater in fetal than adult arteries and that chronic maternal hypoxia can profoundly affect fetal vascular function. (04/2012)