Larissa A Shimoda

Johns Hopkins University, Baltimore, Maryland, United States

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Publications (72)392.3 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Hypoxia is a common finding in advanced human tumors and is often associated with metastatic dissemination and poor prognosis. Cancer cells adapt to hypoxia by utilizing physiological adaptation pathways that promote a switch from oxidative to glycolytic metabolism. This promotes the conversion of glucose into lactate while limiting its transformation into acetyl coenzyme A (acetyl-CoA). The uptake of glucose and the glycolytic flux are increased under hypoxic conditions, mostly owing to the upregulation of genes encoding glucose transporters and glycolytic enzymes, a process that depends on hypoxia-inducible factor 1 (HIF-1). The reduced delivery of acetyl-CoA to the tricarboxylic acid cycle leads to a switch from glucose to glutamine as the major substrate for fatty acid synthesis in hypoxic cells. In addition, hypoxia induces (1) the HIF-1-dependent expression of BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) and BNIP3-like (BNIP3L), which trigger mitochondrial autophagy, thereby decreasing the oxidative metabolism of both fatty acids and glucose, and (2) the expression of the sodium-hydrogen exchanger NHE1, which maintains an alkaline intracellular pH. Here, we present a compendium of methods to study hypoxia-induced metabolic alterations.
    Methods in enzymology. 01/2014; 542:425-55.
  • Larissa A Shimoda, Steven S Laurie
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    ABSTRACT: In the lung, acute reductions in oxygen lead to hypoxic pulmonary vasoconstriction, whereas prolonged exposures to hypoxia result in sustained vasoconstriction, pulmonary vascular remodeling and the development of pulmonary hypertension. Data from both human subjects and animal models implicates a role for hypoxia-inducible factors (HIFs), oxygen-sensitive transcription factors, in pulmonary vascular responses to both acute and chronic hypoxia. In this review, we discuss work from our lab and others supporting a role for HIF in modulating hypoxic pulmonary vasoconstriction and mediating hypoxia-induced pulmonary hypertension, identify some of the downstream targets of HIF, and assess the potential to pharmacologically target the HIF system.
    Journal of Applied Physiology 12/2013; · 3.48 Impact Factor
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    ABSTRACT: Increased catalytic activity of cystathionine ß-synthase (CBS) was recently shown to mediate vasodilation of the cerebral microcirculation, which is initiated within minutes after the onset of acute hypoxia. To test whether chronic hypoxia was a stimulus for increased CBS expression, U87-MG human glioblastoma and PC12 rat pheochromocytoma cells were exposed to 1% or 20% O2 for 24 to 72 h. CBS mRNA and protein expression were increased in hypoxic cells. Hypoxic induction of CBS expression was abrogated in cells transfected with vector encoding short hairpin RNA targeting hypoxia-inducible factor (HIF) 1alpha or 2alpha. Exposure of rats to hypobaric hypoxia (0.35 atm) for 3 d induced increased Cbs mRNA, protein, and catalytic activity in the cerebral cortex and cerebellum, which was blocked by administration of the HIF inhibitor digoxin. HIF binding sites, located 0.8 and 1.2 kb 5' to the transcription start site of the human CBS and rat Cbs genes, respectively, were identified by chromatin immunoprecipitation assays. A 49-bp human sequence, which encompassed an inverted repeat of the core HIF binding site, functioned as a hypoxia response element in luciferase reporter transcription assays. Thus, HIFs mediate tissue-specific CBS expression, which may augment cerebral vasodilation as an adaptive response to chronic hypoxia.
    Biochemical Journal 12/2013; · 4.65 Impact Factor
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    ABSTRACT: Both pulmonary arterial smooth muscle cell (PASMC) proliferation and migration are important contributors to the vascular remodeling that occurs during development of pulmonary hypertension. We previously demonstrated that aquaporin 1 (AQP1), a member of the water channel family of proteins, was expressed in PASMCs and necessary for hypoxia-induced migration; however, the mechanism by which AQP1 controls this response is unclear. The C-terminal tail of AQP1 contains putative calcium (EF-hand) and protein binding sites. Thus, we wanted to explore whether the C-terminal tail or EF-hand motif of AQP1 was required for migration and proliferation. Rat PASMCs were isolated from distal pulmonary arteries, and proliferation and migration were measured using BrdU incorporation and transwell filters, respectively. To deplete AQP1, PASMCs were transfected with AQP1 siRNA (siAQP1) or non-targeting siRNA. Knockdown of AQP1 reduced basal proliferation and hypoxia-induced migration and proliferation in PASMCs. In subsequent experiments, wild-type AQP1, AQP1 lacking the entire cytoplasmic C-terminal tail or AQP1 with a mutation in the EF-hand motif were expressed in PASMCs using adenoviral constructs. For all AQP1 constructs, infection increased AQP1 protein levels, water permeability and the change in cell volume induced by hypotonic challenge. Infection with wild-type and EF-hand mutated AQP1, but not C-terminal deleted AQP1, increased PASMC migration and proliferation. Our results suggest that AQP1 controls both proliferation and migration in PASMCs and that the mechanism requires the C-terminal tail of the protein but is independent of water transport or the EF-hand motif.
    American Journal of Respiratory Cell and Molecular Biology 12/2013; · 4.15 Impact Factor
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    ABSTRACT: Apoptosis is a key pathologic feature in acute lung injury (ALI). Animal studies have demonstrated that pathways regulating apoptosis are necessary in the development of ALI and that activation of p38 MAP kinase is linked to the initiation of the apoptotic cascade. In this study we assessed the role of the mitogen activated protein kinase-activated protein kinase 2 (MK2), one of p38 MAP kinase's immediate downstream effectors, in the development of apoptosis in an animal model of LPS-induced pulmonary vascular permeability. Our results indicate that wild-type (WT) mice exposed to LPS demonstrate increased apoptosis as evidenced by cleavage of caspase 3 and PARP1 and increased TUNEL staining, which is accompanied by increases in markers of vascular permeability. In contrast, MK2(-/-) mice are protected from pulmonary vascular permeability and apoptosis in response to LPS. While there was no difference in activation of caspase 3 in MK2(-/-) compared to WT mice, interestingly, cleaved caspase 3 translocated to the nucleus in WT mice while it remained in the cytosol of MK2(-/-) mice in response to LPS. In separate experiments, LPS-induced apoptosis in human lung microvascular endothelial cells was also associated with nuclear translocation of cleaved caspase 3 and apoptosis, which were both prevented by MK2 silencing. In conclusion, our data suggest that MK2 plays a critical role in the development of apoptosis and pulmonary vascular permeability and its effects on apoptosis are in part related to its ability to regulate nuclear translocation of cleaved caspase 3.
    American Journal of Respiratory Cell and Molecular Biology 12/2013; · 4.15 Impact Factor
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    ABSTRACT: Numerous cellular responses to hypoxia are mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 plays a central role in the pathogenesis of hypoxic pulmonary hypertension. Under certain conditions, HIF-1 may utilize feed-forward mechanisms to amplify its activity. Since hypoxia increases endothelin-1 (ET-1) levels in the lung, we hypothesized that during moderate, prolonged hypoxia ET-1 might contribute to HIF-1 signaling in pulmonary arterial smooth muscle cells (PASMCs). Primary cultures of rat PASMCs were treated with ET-1 or exposed to moderate, prolonged hypoxia (4% O(2) for 60 h). Levels of the oxygen-sensitive HIF-1α subunit and expression of HIF target genes were increased in both hypoxic cells and cells treated with ET-1. Both hypoxia and ET-1 also increased HIF-1α mRNA expression and decreased mRNA and protein expression of prolyl hydroxylase 2 (PHD2), which is the protein responsible for targeting HIF-1α for O(2)-dependent degradation. The induction of HIF-1α by moderate, prolonged hypoxia was blocked by BQ-123, an antagonist of ET-1 receptor subtype A . The effects of ET-1 were mediated by increased intracellular calcium, generation of reactive oxygen species and ERK1/2 activation. Neither ET-1 nor moderate hypoxia induced the expression of HIF-1α or HIF target genes in aortic smooth muscle cells. These results suggest that ET-1 induces a PASMC-specific increase in HIF-1α levels by upregulation of HIF-1α synthesis and downregulation of PHD2-mediated degradation, thereby amplifying the induction of HIF-1α in PASMCs during moderate, prolonged hypoxia.
    AJP Lung Cellular and Molecular Physiology 02/2013; · 3.52 Impact Factor
  • Larissa A Shimoda, Steven S Laurie
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    ABSTRACT: Pulmonary hypertension is a complex, progressive condition arising from a variety of genetic and pathogenic causes. Patients present with a spectrum of histologic and pathophysiological features, likely reflecting the diversity in underlying pathogenesis. It is widely recognized that structural alterations in the vascular wall contribute to all forms of pulmonary hypertension. Features characteristic of the remodeled vasculature in patients with pulmonary hypertension include increased stiffening of the elastic proximal pulmonary arteries, thickening of the intimal and/or medial layer of muscular arteries, development of vaso-occlusive lesions, and the appearance of cells expressing smooth muscle-specific markers in normally non-muscular small diameter vessels, resulting from proliferation and migration of pulmonary arterial smooth muscle cells and cellular transdifferentiation. The development of several animal models of pulmonary hypertension has provided the means to explore the mechanistic underpinnings of pulmonary vascular remodeling, although none of the experimental models currently used entirely replicates the pulmonary arterial hypertension observed in patients. Herein, we provide an overview of the histological abnormalities observed in humans with pulmonary hypertension and in preclinical models and discuss insights gained regarding several key signaling pathways contributing to the remodeling process. In particular, we will focus on the roles of ion homeostasis, endothelin-1, serotonin, bone morphogenetic proteins, Rho kinase, and hypoxia-inducible factor 1 in pulmonary arterial smooth muscle and endothelial cells, highlighting areas of cross-talk between these pathways and potentials for therapeutic targeting.
    Journal of Molecular Medicine 01/2013; · 4.77 Impact Factor
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    ABSTRACT: Obstructive sleep apnea is associated with insulin resistance, glucose intolerance, and type 2 diabetes mellitus. Although several studies have suggested that intermittent hypoxia in obstructive sleep apnea may induce abnormalities in glucose homeostasis, it remains to be determined whether these abnormalities improve after discontinuation of the exposure. The objective of this study was to delineate the effects of intermittent hypoxia on glucose homeostasis, beta cell function, and liver glucose metabolism and to investigate whether the impairments improve after the hypoxic exposure is discontinued. C57BL6/J mice were exposed to 14 days of intermittent hypoxia, 14 days of intermittent air, or 7 days of intermittent hypoxia followed by 7 days of intermittent air (recovery paradigm). Glucose and insulin tolerance tests were performed to estimate whole-body insulin sensitivity and calculate measures of beta cell function. Oxidative stress in pancreatic tissue and glucose output from isolated hepatocytes were also assessed. Intermittent hypoxia increased fasting glucose levels and worsened glucose tolerance by 67% and 27%, respectively. Furthermore, intermittent hypoxia exposure was associated with impairments in insulin sensitivity and beta cell function, an increase in liver glycogen, higher hepatocyte glucose output, and an increase in oxidative stress in the pancreas. While fasting glucose levels and hepatic glucose output normalized after discontinuation of the hypoxic exposure, glucose intolerance, insulin resistance, and impairments in beta cell function persisted. Intermittent hypoxia induces insulin resistance, impairs beta cell function, enhances hepatocyte glucose output, and increases oxidative stress in the pancreas. Cessation of the hypoxic exposure does not fully reverse the observed changes in glucose metabolism. Polak J; Shimoda LA; Drager LF; Undem C; McHugh H; Polotsky VY; Punjabi NM. Intermittent hypoxia impairs glucose homeostasis in C57BL6/J mice: partial improvement with cessation of the exposure. SLEEP 2013;36(10):1483-1490.
    Sleep 01/2013; 36(10):1483-1490. · 5.10 Impact Factor
  • Larissa A Shimoda
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    ABSTRACT: When exposed to chronic hypoxia (CH), the pulmonary circulation responds with enhanced contraction and vascular remodeling, resulting in elevated pulmonary arterial pressures. Our work has identified CH-induced alterations in the expression and activity of several ion channels and transporters in pulmonary vascular smooth muscle that contribute to the development of hypoxic pulmonary hypertension and uncovered a critical role for the transcription factor hypoxia-inducible factor-1 (HIF-1) in mediating these responses. Current work is focused on the regulation of HIF in the chronically hypoxic lung and evaluating the potential for pharmacological inhibitors of HIF to prevent, reverse or slow the progression of pulmonary hypertension.
    Journal of Applied Physiology 08/2012; · 3.48 Impact Factor
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    ABSTRACT: Previous studies have suggested that exstrophic bladder smooth muscle cells grown in culture show contractility similar to that of normal bladder smooth muscle cells. Despite this similar contractility, other cellular characteristics may vary between exstrophic and normal bladder smooth muscle cells. Primary cultures of bladder smooth muscle cells were established from patients with bladder exstrophy (14) and vesicoureteral reflux as a control (10). Expression of smooth muscle specific α-actin and heavy chain myosin was determined with immunohistochemistry. Response of smooth muscle cells to high potassium Krebs solution or acetylcholine (0.1 mM) was assessed using a calcium sensitive fluorescent dye. Intracellular calcium concentration was measured after 48 hours in basal media. Cell migration in basal media during 24 hours was determined using transwell assays. Baseline proliferation and response to 10% fetal bovine serum were assessed with bromodeoxyuridine incorporation assays. More than 95% of exstrophy and control smooth muscle cells stained positive for actin and myosin. Functional integrity was verified in each exstrophy and control cell line by response to high potassium Krebs solution or acetylcholine. The intracellular calcium concentration was lower in exstrophy smooth muscle cells than in control smooth muscle cells (71 vs 136 nM, p <0.001). More exstrophy cells migrated than control cells (37% vs 18%, p = 0.004). There was no statistically significant difference in proliferation between exstrophy and control smooth muscle cells in basal or growth media. Cultured exstrophy smooth muscle cells demonstrate some differences in baseline characteristics compared to control cells. Differences in migration and intracellular calcium may have implications for in vivo detrusor function and tissue engineering.
    The Journal of urology 08/2012; 188(4 Suppl):1521-7. · 4.02 Impact Factor
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    ABSTRACT: Transforming growth factor-β1 regulates extracellular matrix composition, and impacts function and proliferation in multiple cell types, including bladder smooth muscle cells. In this study we evaluated the response to transforming growth factor-β1 in cultured exstrophy and control bladder smooth muscle cells. Primary bladder smooth muscle cell cultures were established from patients with bladder exstrophy or vesicoureteral reflux. Smooth muscle specific α-actin and heavy chain myosin expression was determined using immunohistochemistry. Cell migration, intracellular calcium concentration and proliferation were determined after incubation for 24 to 48 hours in basal media, with or without transforming growth factor-β1 (0.001 to 3 nM) or transforming growth factor-β1 receptor inhibitor SB 431542 (10 μM). Cultured exstrophy and control smooth muscle cells stained positive for α-actin and heavy chain myosin. Exstrophy smooth muscle cells demonstrated increased migration compared to control smooth muscle cells at baseline (38% vs 20%, p = 0.01). Transforming growth factor-β1 increased control smooth muscle cell migration while SB 431542 decreased exstrophy smooth muscle cell migration. Control cells had a higher intracellular calcium concentration, which decreased significantly when exposed to SB 431542. Transforming growth factor-β1 did not cause significant changes in intracellular calcium concentration. Inhibition of transforming growth factor-β1 receptors decreased proliferation in exstrophy and control smooth muscle cells, but exogenous transforming growth factor-β1 did not impact proliferation. Our results suggest that there are distinct differences in bladder smooth muscle cell function between control and exstrophy cases which persist in culture. Although resting intracellular calcium concentration was higher in control cells, proliferation rates were similar in both cell types, indicating that lower intracellular calcium concentration did not impact growth potential. In contrast, enhanced migration was observed in exstrophy cells, possibly due to excess transforming growth factor-β1 signaling, but seemingly independent of increases in intracellular calcium concentration.
    The Journal of urology 08/2012; 188(4 Suppl):1528-34. · 4.02 Impact Factor
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    ABSTRACT: Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration ([Ca(2+)](i)) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca(2+) dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, [Ca(2+)](i), and migration in PASMCs. Blockade of Ca(2+) entry through voltage-dependent Ca(2+) or nonselective cation channels prevented the hypoxia-induced increase in PASMC [Ca(2+)](i), AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in [Ca(2+)](i) that results in increased AQP1 protein levels and cell migration.
    AJP Lung Cellular and Molecular Physiology 06/2012; 303(4):L343-53. · 3.52 Impact Factor
  • Jian Wang, Larissa A Shimoda, J T Sylvester
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    ABSTRACT: In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.
    AJP Lung Cellular and Molecular Physiology 05/2012; 303(2):L161-8. · 3.52 Impact Factor
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    ABSTRACT: Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca(2+)](i)) occurring through activation of voltage-dependent Ca(2+) channels (VDCC) even though ET-1-induced depolarization via inhibition of K(+) channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca(2+)](i) in PASMCs from rats exposed to CH (10% O(2), 3 wk) using the Ca(2+)-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca(2+)](i) by >70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca(2+)](i) to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca(2+)](i) in PASMCs occurs via Ca(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.
    AJP Lung Cellular and Molecular Physiology 03/2012; 302(10):L1128-39. · 3.52 Impact Factor
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    ABSTRACT: Chronic hypoxia is an inciting factor for the development of pulmonary arterial hypertension. The mechanisms involved in the development of hypoxic pulmonary hypertension (HPH) include hypoxia-inducible factor 1 (HIF-1)-dependent transactivation of genes controlling pulmonary arterial smooth muscle cell (PASMC) intracellular calcium concentration ([Ca(2+)](i)) and pH. Recently, digoxin was shown to inhibit HIF-1 transcriptional activity. In this study, we tested the hypothesis that digoxin could prevent and reverse the development of HPH. Mice were injected daily with saline or digoxin and exposed to room air or ambient hypoxia for 3 wk. Treatment with digoxin attenuated the development of right ventricle (RV) hypertrophy and prevented the pulmonary vascular remodeling and increases in PASMC [Ca(2+)](i), pH, and RV pressure that occur in mice exposed to chronic hypoxia. When started after pulmonary hypertension was established, digoxin attenuated the hypoxia-induced increases in RV pressure and PASMC pH and [Ca(2+)](i). These preclinical data support a role for HIF-1 inhibitors in the treatment of HPH.
    Proceedings of the National Academy of Sciences 01/2012; 109(4):1239-44. · 9.74 Impact Factor
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    Clark Undem, Eon J Rios, Julie Maylor, Larissa A Shimoda
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    ABSTRACT: Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pH(i)), typically due to activation of Na(+)/H(+) exchange (NHE). In the systemic vasculature, ET-1 increases pH(i), Na(+)/H(+) exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pH(i) and smooth muscle growth in the lung; however, the effect of ET-1 on basal pH(i) and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3-5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pH(i) and NHE activity induced by increasing concentrations of ET-1 (10(-10) to 10(-8) M). We found that application of exogenous ET-1 increased pH(i) and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pH(i) or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pH(i).
    PLoS ONE 01/2012; 7(9):e46303. · 3.73 Impact Factor
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    ABSTRACT: It has been known for more than 60 years, and suspected for over 100, that alveolar hypoxia causes pulmonary vasoconstriction by means of mechanisms local to the lung. For the last 20 years, it has been clear that the essential sensor, transduction, and effector mechanisms responsible for hypoxic pulmonary vasoconstriction (HPV) reside in the pulmonary arterial smooth muscle cell. The main focus of this review is the cellular and molecular work performed to clarify these intrinsic mechanisms and to determine how they are facilitated and inhibited by the extrinsic influences of other cells. Because the interaction of intrinsic and extrinsic mechanisms is likely to shape expression of HPV in vivo, we relate results obtained in cells to HPV in more intact preparations, such as intact and isolated lungs and isolated pulmonary vessels. Finally, we evaluate evidence regarding the contribution of HPV to the physiological and pathophysiological processes involved in the transition from fetal to neonatal life, pulmonary gas exchange, high-altitude pulmonary edema, and pulmonary hypertension. Although understanding of HPV has advanced significantly, major areas of ignorance and uncertainty await resolution.
    Physiological Reviews 01/2012; 92(1):367-520. · 30.17 Impact Factor
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    ABSTRACT: Pulmonary hypertension (PH) is a lethal syndrome associated with the pathogenic remodeling of the pulmonary vasculature and the emergence of apoptosis-resistant cells. Apoptosis repressor with caspase recruitment domain (ARC) is an inhibitor of multiple forms of cell death known to be abundantly expressed in striated muscle. We show for the first time that ARC is expressed in arterial smooth muscle cells of the pulmonary vasculature and is markedly upregulated in several experimental models of PH. In this study, we test the hypothesis that ARC expression is essential for the development of chronic hypoxia-induced PH. Experiments in which cells or mice were rendered ARC-deficient revealed that ARC not only protected pulmonary arterial smooth muscle cells from hypoxia-induced death, but also facilitated growth factor-induced proliferation and hypertrophy and hypoxia-induced downregulation of selective voltage-gated potassium channels, the latter a hallmark of the syndrome in humans. Moreover, ARC-deficient mice exhibited diminished vascular remodeling, increased apoptosis, and decreased proliferation in response to chronic hypoxia, resulting in marked protection from PH in vivo. Patients with PH have significantly increased ARC expression not only in remodeled vessels but also in the lumen-occluding lesions associated with severe disease. These data show that ARC, previously unlinked to pulmonary hypertension, is a critical determinant of vascular remodeling in this syndrome.
    Circulation 11/2011; 124(23):2533-42. · 15.20 Impact Factor
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    ABSTRACT: T cell differentiation into distinct functional effector and inhibitory subsets is regulated, in part, by the cytokine environment present at the time of antigen recognition. Here, we show that hypoxia-inducible factor 1 (HIF-1), a key metabolic sensor, regulates the balance between regulatory T cell (T(reg)) and T(H)17 differentiation. HIF-1 enhances T(H)17 development through direct transcriptional activation of RORγt and via tertiary complex formation with RORγt and p300 recruitment to the IL-17 promoter, thereby regulating T(H)17 signature genes. Concurrently, HIF-1 attenuates T(reg) development by binding Foxp3 and targeting it for proteasomal degradation. Importantly, this regulation occurs under both normoxic and hypoxic conditions. Mice with HIF-1α-deficient T cells are resistant to induction of T(H)17-dependent experimental autoimmune encephalitis associated with diminished T(H)17 and increased T(reg) cells. These findings highlight the importance of metabolic cues in T cell fate determination and suggest that metabolic modulation could ameliorate certain T cell-based immune pathologies.
    Cell 09/2011; 146(5):772-84. · 31.96 Impact Factor
  • Letitia Weigand, Larissa A Shimoda, J T Sylvester
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    ABSTRACT: Hypoxic contraction of pulmonary arterial smooth muscle is thought to require increases in both intracellular Ca(2+) concentration ([Ca(2+)](i)) and myofilament Ca(2+) sensitivity, which may or may not be endothelium-dependent. To examine the effects of hypoxia and endothelium on Ca(2+) sensitivity in pulmonary arterial smooth muscle, we measured the relation between [Ca(2+)](i) and isometric force at 37°C during normoxia (21% O(2)-5% CO(2)) and after 30 min of hypoxia (1% O(2)-5% CO(2)) in endothelium-intact (E+) and -denuded (E-) rat distal intrapulmonary arteries (IPA) permeabilized with staphylococcal α-toxin. Endothelial denudation enhanced Ca(2+) sensitivity during normoxia but did not alter the effects of hypoxia, which shifted the [Ca(2+)](i)-force relation to higher force in E+ and E- IPA. Neither hypoxia nor endothelial denudation altered Ca(2+) sensitivity in mesenteric arteries. In E+ and E- IPA, hypoxic enhancement of Ca(2+) sensitivity was abolished by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (30 μM), which shifted normoxic [Ca(2+)](i)-force relations to higher force. In E- IPA, the Rho kinase antagonist Y-27632 (10 μM) shifted the normoxic [Ca(2+)](i)-force relation to lower force but did not alter the effects of hypoxia. These results suggest that acute hypoxia enhanced myofilament Ca(2+) sensitivity in rat IPA by decreasing nitric oxide production and/or activity in smooth muscle, thereby revealing a high basal level of Ca(2+) sensitivity, due in part to Rho kinase, which otherwise did not contribute to Ca(2+) sensitization by hypoxia.
    AJP Lung Cellular and Molecular Physiology 06/2011; 301(3):L380-7. · 3.52 Impact Factor

Publication Stats

3k Citations
392.30 Total Impact Points

Institutions

  • 1999–2014
    • Johns Hopkins University
      • • Division of Pulmonary and Critical Care Medicine
      • • Department of Medicine
      Baltimore, Maryland, United States
  • 2000–2011
    • Johns Hopkins Medicine
      • • Division of Pulmonary and Critical Care Medicine
      • • Department of Medicine
      Baltimore, MD, United States
  • 2004
    • University of Maryland, Baltimore
      • Department of Medicine
      Baltimore, MD, United States