Christina Lisk

Nrf2 Activation: A potential strategy for the prevention of Acute Mountain Sickness

Reactive oxygen species (ROS) formed during acute high altitude exposure contribute to cerebral vascular leak and development of acute mountain sickness (AMS). Nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) is a transcription factor that regulates expression of greater than 90% of antioxidant genes, but prophylactic treatment with Nrf2 activators has not yet been tested as an AMS therapy. We hypothesized that prophylactic activation of the antioxidant genome with Nrf2 activators would attenuate high-altitude-induced ROS formation and cerebral vascular leak and that some drugs currently used to treat AMS symptoms have an additional trait of Nrf2 activation. Drugs commonly used to treat AMS were screened with a luciferase reporter cell system for their effectiveness to activate Nrf2, as well as being tested for their ability to decrease high altitude cerebral vascular leak in vivo. Compounds that showed favorable results for Nrf2 activation from our screen and attenuated high altitude cerebral vascular leak in vivo were further tested in brain microvascular endothelial cells (BMECs) to determine if they attenuated hypoxia-induced ROS production and monolayer permeability. Of nine drugs tested, with the exception of dexamethasone, only drugs that showed the ability to activate Nrf2 (Protandim, methazolamide, nifedipine, amlodipine, ambrisentan, and sitaxentan) decreased high-altitude-induced cerebral vascular leak in vivo. In vitro, Nrf2 activation in BMECs before 24h hypoxia exposure attenuated hypoxic-induced hydrogen peroxide production and permeability. Prophylactic Nrf2 activation is effective at reducing brain vascular leak from acute high altitude exposures. Compared to acetazolamide, methazolamide may offer better protection against AMS. Nifedipine, in addition to its known vasodilatory activities in the lung and protection against high altitude pulmonary edema, may provide protection against brain vascular leak as well.

Free hemoglobin induction of pulmonary vascular disease: evidence for an inflammatory mechanism.

Cell-free hemoglobin (Hb) exposure may be a pathogenic mediator in the development of pulmonary arterial hypertension (PAH), and when combined with chronic hypoxia the potential for exacerbation of PAH and vascular remodeling is likely more pronounced. We hypothesized that Hb may contribute to hypoxia-driven PAH collectively as a prooxidant, inflammatory, and nitric oxide (NO) scavenger. Using programmable micropump technology, we exposed male Sprague-Dawley rats housed under room air or hypoxia to 12 or 30 mg per day Hb for 3, 5, and 7 wk. Blood pressure, cardiac output, right ventricular hypertrophy, and indexes of pulmonary vascular remodeling were evaluated. Additionally, markers of oxidative stress, NO bioavailability and inflammation were determined. Hb increased pulmonary arterial (PA) pressure, pulmonary vessel wall stiffening, and right heart hypertrophy with temporal and dose dependence in both room air and hypoxic cohorts. Hb induced a modest increase in plasma oxidative stress markers (malondialdehyde and 4-hydroxynonenal), no change in NO bioavailability, and increased lung ICAM protein expression. Treatment with the antioxidant Tempol attenuated Hb-induced pulmonary arterial wall thickening, but not PA pressures or ICAM expression. Chronic exposure to low plasma Hb concentrations (range = 3-10 μM) lasting up to 7 wk in rodents induces pulmonary vascular disease via inflammation and to a lesser extent by Hb-mediated oxidation. Tempol demonstrated a modest effect on the attenuation of Hb-induced pulmonary vascular disease. NO bioavailability was found to be of minimal importance in this model.

Hemoglobin-induced lung vascular oxidation, inflammation, and remodeling contribute to the progression of hypoxic pulmonary hypertension and is attenuated in rats with repeated-dose haptoglobin administration.

Haptoglobin (Hp) is an approved treatment in Japan for trauma, burns, and massive transfusion-related hemolysis. Additional case reports suggest uses in other acute hemolytic events that lead to acute kidney injury. However, Hps protective effects on the pulmonary vasculature have not been evaluated within the context of mitigating the consequences of chronic hemoglobin (Hb) exposure in the progression of pulmonary hypertension (PH) secondary to hemolytic diseases. This study was performed to assess the utility of chronic Hp therapy in a preclinical model of Hb and hypoxia-mediated PH. Rats were simultaneously exposed to chronic Hb infusion (35 mg per day) and hypobaric hypoxia for 5 weeks in the presence or absence of Hp treatment (90 mg/kg twice a week). Hp inhibited the Hb plus hypoxia-mediated nonheme iron accumulation in lung and heart tissue, pulmonary vascular inflammation and resistance, and right-ventricular hypertrophy, which suggests a positive impact on impeding the progression of PH. In addition, Hp therapy was associated with a reduction in critical mediators of PH, including lung adventitial macrophage population and endothelial ICAM-1 expression. By preventing Hb-mediated pathology, Hp infusions: (1) demonstrate a critical role for Hb in vascular remodeling associated with hypoxia and (2) suggest a novel therapy for chronic hemolysis-associated PH.

Aberrant Chloride Intracellular Channel 4 Expression Contributes to Endothelial Dysfunction in Pulmonary Arterial Hypertension

Background— Chloride intracellular channel 4 (CLIC4) is highly expressed in the endothelium of remodeled pulmonary vessels and plexiform lesions of patients with pulmonary arterial hypertension. CLIC4 regulates vasculogenesis through endothelial tube formation. Aberrant CLIC4 expression may contribute to the vascular pathology of pulmonary arterial hypertension. Methods and Results— CLIC4 protein expression was increased in plasma and blood-derived endothelial cells from patients with idiopathic pulmonary arterial hypertension and in the pulmonary vascular endothelium of 3 rat models of pulmonary hypertension. CLIC4 gene deletion markedly attenuated the development of chronic hypoxia-induced pulmonary hypertension in mice. Adenoviral overexpression of CLIC4 in cultured human pulmonary artery endothelial cells compromised pulmonary endothelial barrier function and enhanced their survival and angiogenic capacity, whereas CLIC4 shRNA had an inhibitory effect. Similarly, inhibition of CLIC4 expression in blood-derived endothelial cells from patients with idiopathic pulmonary arterial hypertension attenuated the abnormal angiogenic behavior that characterizes these cells. The mechanism of CLIC4 effects involves p65-mediated activation of nuclear factor-&kgr;B, followed by stabilization of hypoxia-inducible factor-1&agr; and increased downstream production of vascular endothelial growth factor and endothelin-1. Conclusion— Increased CLIC4 expression is an early manifestation and mediator of endothelial dysfunction in pulmonary hypertension.

Hemoglobin-Induced Endothelial Cell Permeability Is Controlled, in Part, via a Myeloid Differentiation Primary Response Gene–88–Dependent Signaling Mechanism

The release of hemoglobin (Hb) with hemolysis causes vascular dysfunction. New evidence implicates Hb-induced NF-κB and hypoxia inducible factor (HIF) activation, which may be under the control of a Toll-like receptor (TLR)-signaling pathway. Nearly all TLR-signaling pathways activate the myeloid differentiation primary response gene-88 (MyD88) that regulates NF-κB. We hypothesized that the differing transition states of Hb influence endothelial cell permeability via NF-κB activation and HIF regulation through a MyD88-dependent pathway. In cultured human dermal microvascular endothelial cells (HMECs-1), we examined the effects of Hb in the ferrous (HbFe(2+)), ferric (HbFe(3+)), and ferryl (HbFe(4+)) transition states on NF-κB and HIF activity, HIF-1α and HIF-2α mRNA up-regulation, and monolayer permeability, in the presence or absence of TLR4, MyD88, NF-κB, or HIF inhibition, as well as superoxide dismutase (SOD) and catalase. Our data showed that cell-free Hb, in each transition state, induced NF-κB and HIF activity, up-regulated HIF-1α and HIF-2α mRNA, and increased HMEC-1 permeability. The blockade of either MyD88 or NF-κB, but not TLR4, attenuated Hb-induced HIF activity, the up-regulation HIF-1 and HIF-2α mRNA, and HMEC-1 permeability. The inhibition of HIF activity exerted less of an effect on Hb-induced monolayer permeability. Moreover, SOD and catalase attenuated NF-κB, HIF activity, and monolayer permeability. Our results demonstrate that Hb-induced NF-κB and HIF are regulated by two mechanisms, either MyD88 activation or Hb transition state-induced ROS formation, that influence HMEC-1 permeability.

A retrospective study of acute mountain sickness on Mt. Kilimanjaro using trekking company data.

BACKGROUND High altitude illnesses (HAI) are a risk factor for any individual who is exposed to a significant increase in altitude. To learn more about the epidemiology of HAI, we sought to determine if health records from a commercial trekking company could provide novel data on the prevalence of HAI, as well as efficacy data regarding common HAI therapeutics. METHODS Health parameters from 917 tourists ascending Mt. Kilimanjaro over a 10-yr period were analyzed for meaningful data. RESULTS Of all subjects, 70% experienced at least one instance of a symptom related to HAI (headache, nausea, vomiting, diarrhea, or loss of appetite) during the trek. Acetazolamide was used at least once by 90% of subjects and, of those who used acetazolamide, 92% began taking it on day 1 of the ascent. Acetazolamide was found to improve oxygen saturation 1.2% above 9842.5 ft (3000 m). Dexamethasone use 12 h prior to ascending above 18,996 ft (5790 m) decreased the probability of a subject exhibiting at least one AMS symptom at that altitude. DISCUSSION The prevalence of AMS symptoms was not reduced by taking 2 extra days to reach the summit of Mt. Kilimanjaro. Prophylactic acetazolamide modestly improved oxygen saturation; however, it did not reduce symptoms. Therapeutic dexamethasone, especially at higher altitudes, was effective at reducing symptoms. We conclude that meaningful high altitude physiological data can be obtained from private trekking companies.

Hemoglobin induced cell trauma indirectly influences endothelial TLR9 activity resulting in pulmonary vascular smooth muscle cell activation.

It is now well established that both inherited and acquired forms of hemolytic disease can promote pulmonary vascular disease consequent of free hemoglobin (Hb) induced NO scavenging, elevations in reactive oxygen species and lipid peroxidation. It has recently been reported that oxidative stress can activate NFkB through a toll-like receptor 9 (TLR9) mediated pathway; further, TLR9 can be activated by either nuclear or mitochondrial DNA liberated by stress induced cellular trauma. We hypothesis that Hb induced lipid peroxidation and subsequent endothelial cell trauma is linked to TLR9 activation, resulting in IL-6 mediated pulmonary smooth muscle cell proliferation. We examined the effects of Hb on rat pulmonary artery endothelial and smooth muscle cells (rPAEC and rPASMC, respectively), and then utilized TLR9 and IL6 inhibitors, as well as the Hb and heme binding proteins (haptoglobin (Hp) and hemopexin (Hpx), respectively) to further elucidate the aforementioned mediators. Further, we explored the effects of Hb in vivo utilizing endothelial cell (EC) specific myeloid differentiation primary response gene-88 (MyD88) and TLR9 null mice. Our data show that oxidized Hb induces lipid peroxidation, cellular toxicity (5.5 ± 1.7 fold; p≤0.04), increased TLR9 activation (60%; p = 0.01), and up regulated IL6 expression (1.75±0.3 fold; p = 0.04) in rPAEC. Rat PASMC exhibited a more proliferative state (13 ± 1%; p = 0.01) when co-cultured with Hb activated rPAEC. These effects were attenuated with the sequestration of Hb or heme by Hp and Hpx as well as with TLR9 an IL-6 inhibition. Moreover, in both EC-MyD88 and TLR9 null mice Hb-infusion resulted in less lung IL-6 expression compared to WT cohorts. These results demonstrate that Hb-induced lipid peroxidation can initiate a modest TLR9 mediated inflammatory response, subsequently generating an activated SMC phenotype.