automated-measurement-microcirculatory-blood-flow-velocity-pulmonary

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

University of Colorado Denver

Hypoxia increases pulmonary vascular leak, which is regulated in part by neutral endopeptidase (NEP). NEP is a cell-surface metalloprotease that degrades several vasoactive peptides, including endothelin-1 (ET-1) and atrial natriuretic peptide (ANP). We therefore hypothesized that NEP attenuates high altitude-induced pulmonary vascular leak. Wild-type and NEP null mice were exposed to a simulated high altitude (HA) of 6,728 m (22,000 ft; P(B) = 328 mmHg) or remained at the relatively low altitude (LA) of 1,500 m (4,920 ft; P(B) = 640 mmHg) for 24 h. Plasma ANP and ET-1 concentrations, right ventricular pressure (P(RV)), and indexes of lung injury were recorded. At HA, lung wet weight-to-body weight increased in all animals, but was greatest in the NEP wild-type mice. Vascular leak, as measured by Evans blue dye, increased only in the NEP wild-type mice at HA. P(RV) increased in both genotypes at HA. Plasma ANP concentrations increased at HA in both genotypes, but plasma ET-1 concentrations were elevated only in the NEP null mice at HA. Correlations between lung wet weight-to-body weight versus P(RV) (r = 0.56; p = 0.0136) and ANP versus P(RV) (r = -0.54; p = 0.02) were noted. We conclude that NEP null mice exposed to HA have a greater rise in ANP versus ET-1 plasma concentration, decreased pulmonary vascular pressure, and reduced high altitude-induced pulmonary vascular leak.

Neutral endopeptidase null mice are less susceptible to high altitude-induced pulmonary vascular leak.

The mechanisms of noncardiogenic pulmonary edema syndromes, such as neurogenic and high-altitude pulmonary edema, remain unclear even after years of study. Previous attempts to develop an animal model for these illnesses have used increased intracranial pressure or whole-body hypoxia. We hypothesized that a cerebral insult induced with a venous hypoxic blood infusion to the brain would trigger neurogenic pulmonary edema in a canine model.

Pulmonary edema induced by cerebral hypoxic insult in a canine model.

Hemoglobin-based oxygen carriers (HBOC) have been primarily studied for blood loss treatment. More recently infusions of HBOC in euvolemic subjects have been proposed for a wide variety of potential therapies in which increased tissue oxygenation would be beneficial. However, compared with the exchange transfusion models to study blood loss, less is known about HBOC oxygen delivery and vasoacitvity when it is infused in euvolemic subjects. We hypothesized that HBOC [polymerized bovine hemoglobin (PBvHb)] infusion creating hypervolemia would increase oxygen delivery to tissues during acute global hypoxia. Vascular oxygen content and hemodynamics were determined after euvolemic rats were infused with 3 ml of either lactated Ringer or PBvHb solution (13 g/dl, 1.3 g/kg) during acute hypoxia (FIO2 = 10%, 4 h) or normoxia (FIO2 = 21%) exposure. Our data demonstrated that compared with Ringer-infused animals, in hypoxia and normoxia, PBvHb treatment improved oxygen content but raised mean arterial pressure, lowered stroke volume, heart rate, and cardiac index, which resulted in a net reduction in blood flow and oxygen delivery to the tissues. The PBvHb vasoactive effect was similar in magnitude and direction as to the Ringer-infused animals treated with a nitric oxide synthase inhibitor nitro-l-arginine, suggesting the PBvHb effect is mediated via nitric oxide scavenging. We conclude that infusion of PBvHb is not likely to be useful in treating global hypoxia under these conditions.

Polymerized bovine hemoglobin decreases oxygen delivery during normoxia and acute hypoxia in the rat.

Acute hypoxia causes pulmonary vascular leak and is involved in the pathogenesis of pulmonary edema associated with inflammation, acute altitude exposure, and other critical illnesses. Reactive oxygen species, HIF-1, and VEGF have all been implicated in various hypoxic pathologies, yet the ROS-HIF-1-VEGF pathway in pulmonary vascular leak has not been defined. We hypothesized that the ROS-HIF-1-VEGF pathway has an important role in producing hypoxia-induced pulmonary vascular leak. Human pulmonary artery endothelial cell (HPAEC) monolayers were exposed to either normoxia (21% O(2)) or acute hypoxia (3% O(2)) for 24 h and monolayer permeability and H(2)O(2), nuclear HIF-1alpha, and cytosolic VEGF levels were determined. HPAEC were treated with antioxidant cocktail (AO; ascorbate, glutathione, and alpha-tocopherol), HIF-1 siRNA, or the VEGF soluble binding protein fms-like tyrosine kinase-1 (sFlt-1) to delineate the role of the ROS-HIF-1-VEGF pathway in hypoxia-induced HPAEC leak. Additionally, mice exposed to hypobaric hypoxia (18,000 ft, 10% O(2)) were treated with the same antioxidant to determine if in vitro responses corresponded to in vivo hypoxia stress. Hypoxia increased albumin permeativity, H(2)O(2) production, and nuclear HIF-1alpha and cytosolic VEGF concentration. Treatment with an AO lowered the hypoxia-induced HPAEC monolayer permeability as well as the elevation of HIF-1alpha and VEGF. Treatment of hypoxia-induced HPAEC with either an siRNA designed against HIF-1alpha or the VEGF antagonist sFlt-1 decreased monolayer permeability. Mice treated with AO and exposed to hypobaric hypoxia (18,000 ft, 10% O(2)) had less pulmonary vascular leak than those that were untreated. Our data suggest that hypoxia-induced permeability is due, in part, to the ROS-HIF-1alpha-VEGF pathway.

A potential role for reactive oxygen species and the HIF-1alpha-VEGF pathway in hypoxia-induced pulmonary vascular leak.

Hemoglobin-based oxygen carriers (HBOC) of several types scavenge nitric oxide from the vasculature resulting in vasoconstriction and hypertension, both systemic and pulmonary. Phosphodiesterase-5 (PDE5) inhibitors promote nitric oxide activity and enhance vasodilation. The purpose of this study was to determine whether combined therapy of glutaraldehyde-polymerized bovine hemoglobin (HBOC) with a PDE5 inhibitor would counter the negative hemodynamic consequences of HBOC therapy alone, resulting in improved hemodynamics and oxygen delivery.

Glutaraldehyde-polymerized bovine hemoglobin and phosphodiesterase-5 inhibition.

Small molecule histone deacetylase (HDAC) inhibitors block adverse cardiac remodeling in animal models of heart failure. The efficacious compounds target class I, class IIb and, to a lesser extent, class IIa HDACs. It is hypothesized that a selective inhibitor of a specific HDAC class (or an isoform within that class) will provide a favorable therapeutic window for the treatment of heart failure, although the optimal selectivity profile for such a compound remains unknown. Genetic studies have suggested that class I HDACs promote pathological cardiac remodeling, while class IIa HDACs are protective. In contrast, nothing is known about the function or regulation of class IIb HDACs in the heart. We developed assays to quantify catalytic activity of distinct HDAC classes in left and right ventricular cardiac tissue from animal models of hypertensive heart disease. Class I and IIa HDAC activity was elevated in some but not all diseased tissues. In contrast, catalytic activity of the class IIb HDAC, HDAC6, was consistently increased in stressed myocardium, but not in a model of physiologic hypertrophy. HDAC6 catalytic activity was also induced by diverse extracellular stimuli in cultured cardiac myocytes and fibroblasts. These findings suggest an unforeseen role for HDAC6 in the heart, and highlight the need for pre-clinical evaluation of HDAC6-selective inhibitors to determine whether this HDAC isoform is pathological or protective in the setting of cardiovascular disease.

Cardiac HDAC6 catalytic activity is induced in response to chronic hypertension.

Histone deacetylase (HDAC) inhibitors are efficacious in models of hypertension-induced left ventricular heart failure. The consequences of HDAC inhibition in the context of pulmonary hypertension with associated right ventricular cardiac remodeling are poorly understood.

Selective class I histone deacetylase inhibition suppresses hypoxia-induced cardiopulmonary remodeling through an antiproliferative 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.

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

Increased production of reactive oxygen species has been implicated in the pathogenesis of cardiovascular disease (CVD), and enhanced endogenous antioxidants have been proposed as a mechanism for regulating redox balance. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcriptional regulator of phase II antioxidant enzymes, and activation of Nrf2 has been suggested to be an important step in attenuating oxidative stress associated with CVD. A well-defined combination of five widely studied medicinal plants derived from botanical sources (Bacopa monniera, Silybum marianum (milk thistle), Withania somnifera (Ashwagandha), Camellia sinensis (green tea), and Curcuma longa (turmeric)) has been shown to activate Nrf2 and induce phase II enzymes through the antioxidant response element. The purpose of these experiments was to determine if treatment of cardiomyocytes with this phytochemical composition, marketed as Protandim, activates Nrf2, induces phase II detoxification enzymes, and protects cardiomyocytes from oxidant-induced apoptosis in a Nrf2-dependent manner. In cultured HL-1 cardiomyocytes, phytochemical treatment was associated with nuclear accumulation of Nrf2, significant induction of phase II enzymes, and concomitant protection against hydrogen peroxide-induced apoptosis. The protection against oxidant stress was abolished when Nrf2 was silenced by shRNA, suggesting that our phytochemical treatment worked through the Nrf2 pathway. Interestingly, phytochemical treatment was found to be a more robust activator of Nrf2 than oxidant treatment, supporting the use of the phytochemicals as a potential treatment to increase antioxidant defenses and protect heart cells against an oxidative challenge.

Upregulation of phase II enzymes through phytochemical activation of Nrf2 protects cardiomyocytes against oxidant stress.

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.

Hemoglobin-induced endothelial cell permeability is controlled, in part, via a myeloid differentiation primary response gene-88-dependent signaling mechanism.

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.

Nrf2 activation: a potential strategy for the prevention of acute mountain sickness.

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.

Aberrant chloride intracellular channel 4 expression contributes to endothelial dysfunction in pulmonary arterial hypertension.

Excess superoxide has been implicated in pulmonary hypertension (PH). We previously found that lung overexpression of the antioxidant extracellular superoxide dismutase (EC-SOD) attenuates PH and vascular remodeling. Though comprising only a small fraction of total SOD activity in most tissues, EC-SOD is abundant in arteries. We hypothesize that the selective loss of vascular EC-SOD promotes hypoxia-induced PH through redox-sensitive signaling pathways. EC-SOD(loxp/loxp)× Tg(cre/SMMHC) mice (SMC EC-SOD KO) received tamoxifen to conditionally deplete smooth muscle cell (SMC)-derived EC-SOD. Mice were exposed to hypobaric hypoxia for 35 days and PH was assessed by RV systolic pressure measurements and RV hypertrophy. Vascular remodeling was evaluated by morphometric analysis and two-photon microscopy for collagen. We examined lung cyclic GMP content, soluble guanylate cyclase expression and activity, phosphodiesterase5 expression and activity, expression of total and active endothelial nitric oxide synthase and GTP cyclohydrolase-1 (GTPCH-1), the rate-limiting enzyme in tetrahydrobiopterin synthesis. Knockout of SMC EC-SOD selectively decreased pulmonary artery EC-SOD without altering total lung EC-SOD. PH and vascular remodeling induced by chronic hypoxia was augmented in SMC EC-SOD KO. Depletion of SMC EC-SOD did not impact content or activity of lung sGC or PDE5, though it blunted the hypoxia-induced increase in cGMP. Though total eNOS was not altered, active eNOS and GTPCH-1 decreased with hypoxia only in SMC EC-SOD KO. We conclude that the localized loss of pulmonary artery EC-SOD augments chronic hypoxic PH. In addition to oxidative inactivation of NO, deletion of EC-SOD seems to reduce eNOS activity, further compromising pulmonary vascular function.

Article	Year
Neutral endopeptidase null mice are less susceptible to high altitude-induced pulmonary vascular leak. High altitude medicine & biology\| PubMed ID: 16351565	2005
Pulmonary edema induced by cerebral hypoxic insult in a canine model. Aviation, space, and environmental medicine\| PubMed ID: 18500043	2008
Polymerized bovine hemoglobin decreases oxygen delivery during normoxia and acute hypoxia in the rat. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 18567708	2008
A potential role for reactive oxygen species and the HIF-1alpha-VEGF pathway in hypoxia-induced pulmonary vascular leak. Free radical biology & medicine\| PubMed ID: 19358884	2009
Glutaraldehyde-polymerized bovine hemoglobin and phosphodiesterase-5 inhibition. Critical care medicine\| PubMed ID: 19384199	2009
Cardiac HDAC6 catalytic activity is induced in response to chronic hypertension. Journal of molecular and cellular cardiology\| PubMed ID: 21539845	2011
Selective class I histone deacetylase inhibition suppresses hypoxia-induced cardiopulmonary remodeling through an antiproliferative mechanism. Circulation research\| PubMed ID: 22282194	2012
Free hemoglobin induction of pulmonary vascular disease: evidence for an inflammatory mechanism. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 22728465	2012
Upregulation of phase II enzymes through phytochemical activation of Nrf2 protects cardiomyocytes against oxidant stress. Free radical biology & medicine\| PubMed ID: 23201694	2013
Hemoglobin-induced endothelial cell permeability is controlled, in part, via a myeloid differentiation primary response gene-88-dependent signaling mechanism. American journal of respiratory cell and molecular biology\| PubMed ID: 23713977	2013
Nrf2 activation: a potential strategy for the prevention of acute mountain sickness. Free radical biology & medicine\| PubMed ID: 23722164	2013
Aberrant chloride intracellular channel 4 expression contributes to endothelial dysfunction in pulmonary arterial hypertension. Circulation\| PubMed ID: 24503951	2014
Selective depletion of vascular EC-SOD augments chronic hypoxic pulmonary hypertension. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 25326578	2014

David C. Irwin

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