Focal Hypoxia Research Articles

Tubulovascular protection from protease-activated receptor-1 depletion during AKI-to-CKD transition.

Thromboembolic events are prevalent in chronic kidney disease (CKD) patients due to increased thrombin generation leading to a hypercoagulable state. We previously demonstrated that inhibition of protease-activated receptor-1 (PAR-1) by vorapaxar reduces kidney fibrosis. We used an animal model of unilateral ischemia-reperfusion injury-induced CKD to explore the tubulovascular crosstalk mechanisms of PAR-1 in acute kidney injury (AKI)-to-CKD transition. During the early phase of AKI, PAR-1-deficient mice exhibited reduced kidney inflammation, vascular injury, and preserved endothelial integrity and capillary permeability. During the transition phase to CKD, PAR-1 deficiency preserved kidney function and diminished tubulointerstitial fibrosis via downregulated transforming growth factor-β/Smad signaling. Maladaptive repair in the microvasculature after AKI further exacerbated focal hypoxia with capillary rarefaction, which was rescued by stabilization of hypoxia-inducible factor and increased tubular vascular endothelial growth factor A in PAR-1-deficient mice. Chronic inflammation was also prevented with reduced kidney infiltration by both M1- and M2-polarized macrophages. In thrombin-induced human dermal microvascular endothelial cells (HDMECs), PAR-1 mediated vascular injury through activation of NF-κB and ERK MAPK pathways. Gene silencing of PAR-1 exerted microvascular protection via a tubulovascular crosstalk mechanism during hypoxia in HDMECs. Finally, pharmacologic blockade of PAR-1 with vorapaxar improved kidney morphology, promoted vascular regenerative capacity, and reduced inflammation and fibrosis depending on the time of initiation. Our findings elucidate a detrimental role of PAR-1 in vascular dysfunction and profibrotic responses upon tissue injury during AKI-to-CKD transition and provide an attractive therapeutic strategy for post-injury repair in AKI.

Effect of VEGF on neurological impairment and prognosis of acute cerebral infarction patients: A retrospective case-control study.

Due to the complex pathological mechanism of acute cerebral infarction, the role of vascular endothelial growth factor (VEGF) on the disease is not clear. Therefore, a retrospective case-control study was performed to explore the effect of VEGF on neurological impairment and prognosis of acute cerebral infarction patients. A total of 100 patients with acute cerebral infarction admitted to our hospital from April 2021 to April 2022 were selected. Blood samples from all patients would be routinely collected to detect the expression of serum VEGF. Pearson chi-square, Spearman correlation and univariate Logistic regression were used to analyze the clinical data to explore the relationship between VEGF expression and basic information, stroke degree, quality of life, and prognosis of patients. To determine whether VEGF can provide relevant basis for the early prevention and prognostic treatment of acute cerebral infarction. And multivariate logistic regression was used to calculate the odds ratio between each variable and VEGF expression. Pearson chi-square test and Spearman correlation coefficient showed that sex, degree of stroke, limb convulsions, loss of consciousness, hemiplegia, aphasia, mental functioning score, overall quality of life score, and short-term prognosis were significantly correlated with VEGF expression in 100 patients. Univariate logistic regression was used to describe the ORs and 95% confidence interval of subjects at the univariate level, and the degree of stroke (OR = 83.333, P < 0.001), tic of limbs (OR = 26.316, P < 0.001), loss of consciousness (OR = 23.256, P < 0.001), hemiplegia (OR = 62.500, P < 0.001), aphasia (OR = 76.923, P < 0.001), mental functioning score (OR = 7.937, P < 0.001), overall quality of life score (OR = 5.464, P < 0.001), short-term prognosis (OR = 37.037, P < 0.001) was significantly correlated with the high expression of VEGF. The level of serum VEGF was positively correlated with neurological impairment degree and prognosis in patients with acute cerebral infarction, the more severe the degree of stroke and the worse the prognosis.

Ginsenoside Rb1 improves energy metabolism after spinal cord injury.

Mitochondrial damage caused by oxidative stress and energy deficiency induced by focal ischemia and hypoxia are important factors that aggravate diseases. Studies have shown that ginsenoside Rb1 has neurotrophic and neuroprotective effects. However, whether it influences energy metabolism after spinal cord injury remains unclear. In this study, we treated mouse and cell models of spinal cord injury with ginsenoside Rb1. We found that ginsenoside Rb1 remarkably inhibited neuronal oxidative stress, protected mitochondria, promoted neuronal metabolic reprogramming, increased glycolytic activity and ATP production, and promoted the survival of motor neurons in the anterior horn and the recovery of motor function in the hind limb. Because sirtuin 3 regulates glycolysis and oxidative stress, mouse and cell models of spinal cord injury were treated with the sirtuin 3 inhibitor 3-TYP. When Sirt3 expression was suppressed, we found that the therapeutic effects of ginsenoside Rb1 on spinal cord injury were remarkably inhibited. Therefore, ginsenoside Rb1 is considered a potential drug for the treatment of spinal cord injury, and its therapeutic effects are closely related to sirtuin 3.

Electrochemically induced in vitro focal hypoxia in human neurons.

Focalised hypoxia is widely prevalent in diseases such as stroke, cardiac arrest, and dementia. While in some cases hypoxia improves cellular functions, it mostly induces or exacerbates pathological changes. The lack of methodologies that can simulate focal acute hypoxia, in either animal or cell culture, impedes our understanding of the cellular consequences of hypoxia. To address this gap, an electrochemical localised oxygen scavenging system (eLOS), is reported, providing an innovative platform for spatiotemporal in vitro hypoxia modulation. The electrochemical system is modelled showing O2 flux patterns and localised O2 scavenging and hypoxia regions, as a function of distance from the electrode and surrounding flux barriers, allowing an effective focal hypoxia tool to be designed for in vitro cell culture study. O2 concentration is reduced in an electrochemically defined targeted area from normoxia to hypoxia in about 6 min depending on the O2-flux boundaries. As a result, a cell culture-well was designed, where localised O2 scavenging could be induced. The impact of localised hypoxia was demonstrated on human neural progenitor cells (hNPCs) and it was shown that miniature focal hypoxic insults can be induced, that evoke time-dependent HIF-1α transcription factor accumulation. This transcription is “patterned” across the culture according to the electrochemically induced spatiotemporal hypoxia gradient. A basic lacunar infarct model was also developed through the application of eLOS in a purpose designed microfluidic device. Miniature focal hypoxic insults were induced in cellular processes of fully oxygenated cell bodies, such as the axons of human cortical neurons. The results demonstrate experimentally that localised axonal hypoxic stress can lead to significant increase of neuronal death, despite the neurons remaining at normoxia. This suggests that focal hypoxic insult to axons alone is sufficient to impact surrounding neurons and may provide an in vitro model to study the impact of microinfarcts occurring in the deep cerebral white matter, as well as providing a promising tool for wider understanding of acute hypoxic insults with potential to uncover its pathophysiology in multiple diseases.

QS34. Prostaglandin F2a Analog Focally Depletes Adipose Volume Without Concurrent Cytolysis

Purpose: Adipose is one of the most mutable and commonly treated tissues in plastic surgery. Adipose tissue is often the thickest and most variable of the cutaneous layers and is a key contributor to smooth contour, feature, and expression. Unwanted focal adiposity is a common complaint and can arise physiologically, pathologically as lipomas, or iatrogenically after fat grafting. While some indications are amenable to liposuction or resection, not all patients want invasive procedures. Non-invasive ablative procedures have arisen to fill this need including cryolipolysis and the injectable cytolytic deoxycholic acid (Kybella). This agent, however, caries significant comorbid risk. In this study, we investigated repurposing a potent FDA approved prostaglandin F2α (PGF2a) analogue, Latanoprost, currently used as first-line treatment for glaucoma, as an alternative adipolytic agent for small volume focal adipose reduction. Methods: Human adipose obtained under IRB exemption was incubated for 14-days and treated with Kybella and either low- or high-dose soluble Latanoprost every 48-hours. Mature adipocytes and the stromal vascular fractions were collected via enzymatic dissolution and size, count, morphology, and viability were assessed using Calcein-AM/PI imaging. Additionally, single administration of Kybella (500µl) or Latanoprost (1.5 or 150mcg) was performed in vitro to murine fat pads and maintained 2-weeks. These samples were harvested and evaluated via H&E, Pentachrome to assess inguinal fat volume, and fibrotic tissue architecture. Samples were further interrogated via immunofluorescence against Perilipin, Hypoxia Inducible Factor 1-alpha (Hif1a), Uncoupling Protein 1 (UCP1), and hexokinase 2 to assess the variable contributions of Latanoprost therapy to white/brown adipose ratio, focal hypoxia, and metabolic flux. Results: Incubation of lipoaspirate with Kybella resulted in total tissue dissolution and loss of viability. In contrast, Latanoprost did not negatively affect viability of stromal cells or adipocytes. In vivo, both 150 mcg Latanoprost and Kybella significantly decreased inguinal fat pad volume at 2-weeks. Kybella treatment resulted in consistent dermal lesions and histologic evidence of inflammation that were not present in Latanoprost treatment. Furthermore, Latanoprost and vehicle-control treated samples did not significantly vary in presence of fibrotic collagen, Hif1a activation, or presence of UCP1-positive brown fat. Conclusion: Single administration of soluble Latanoprost safely reduced fat pad volume at two weeks post injection in vivo with no ex vivo loss of viability in human tissues. This supports a potential application for reduction in focal adiposity without undesirable cytolysis and future studies will be conducted to optimize dose and determine duration of the effect.

(IMCS First Place Best Paper Award) Local Hypoxia System as a Sensing Interface of Cellular Responses

Oxygen concentration is one of the critical factors tightly controlled in each part of an organism to regulate the cells’ role and function. Yet, changes in tissue oxygenation often occur in small focal areas with aging or as a result of an injury. Despite the serious health implications of focal hypoxia, no methodologies exist that can model oxygen supply system accordingly in animal models or in vitro models. Therefore, we designed a novel local hypoxia system as a sensing interface for in vitro cell culture. The oxygen removal efficiencies measured by Clark electrodes showed that O2 concentration can be reduced from 20.9% / 140mmHg or 5% / 35mmHg and maintained at hypoxia (< 1.5% / 10mmHg) in 15 or 8 minutes respectively, compared with 3 hours needed by conventional incubator and hypoxic chambers. The induced hypoxia stimulated localized time-dependent HIF-1α transcription factor nuclear accumulation at the region of interest on human neural progenitor cells. This new culture system can robustly generate a hypoxia gradient in the same culture dish for the first time and provides the opportunity to decipher the impact of focal hypoxia, the function of localised oxygen homeostasis and the pathophysiology of multiple diseases.

Local Hypoxia System As a Sensing Interface of Cellular Responses

Oxygen concentration is one of the critical factors tightly controlled in each part of an organism to regulate the cells’ role and function. Yet, changes in tissue oxygenation often occur in small focal areas with aging or as a result of an injury. Despite the serious health implications of focal hypoxia, no methodologies exist that can model oxygen supply system accordingly in animal models or in vitro models. Therefore, we designed a novel local hypoxia system as a sensing interface for in vitro cell culture. The oxygen removal efficiencies measured by Clark electrodes showed that O2 concentration can be reduced from 20.9% / 140mmHg or 5% / 35mmHg and maintained at hypoxia (< 1.5% / 10mmHg) in 15 or 8 minutes respectively. The induced hypoxia stimulated localized time-dependent HIF-1α transcription factor nuclear accumulation at the region of interest on human neural progenitor cells. This is the first system capable of robustly generating normoxia and hypoxia side-by-side on the same culture dish for comparative study. The new culture system provides the opportunity to decipher the pathophysiology of hypoxia-related diseases including stroke, dementia and cardiovascular complications.

Convulsive Stress Mimics Brain Hypoxia and Promotes the P-Glycoprotein (P-gp) and Erythropoietin Receptor Overexpression. Recombinant Human Erythropoietin Effect on P-gp Activity.

Erythropoietin (EPO) is not only a hormone that promotes erythropoiesis but also has a neuroprotective effect on neurons attributed to its known anti-apoptotic action. Previously, our group has demonstrated that recombinant-human EPO (rHu-EPO) can protect neurons and recovery motor activity in a chemical focal brain hypoxia model (Merelli et al., 2011). We and others also have reported that repetitive seizures can mimic a hypoxic- like condition by HIF-1α nuclear translocation and high neuronal expression P-gp. Here, we report that a single 20-min status epilepticus (SE) induces P-gp and EPO-R expression in cortical pyramidal neurons and only P-gp expression in astrocytes. In vitro, excitotoxic stress (300 μM glutamate, 5 min), can also induce the expression of EPO-R and P-gp simultaneously with both HIF-1α and NFkB nuclear translocation in primary cortical neurons. Primary astrocytes exposed to chemical hypoxia with CoCl2 (0.3 mM, 6 h) increased P-gp expression as well as an increased efflux of Rhodamine 123 (Rho123) that is a P-gp substrate. Tariquidar, a specific 3er generation P-gp-blocker was used as an efflux inhibitor control. Astrocytes treated with rHu-EPO showed a significant recovery of the Rho123 retention in a similar way as seen by Tariquidar, demonstrating for first time that rHu-EPO can inhibit the P-gp-dependent efflux activity. Taking together, these data suggest that stimulation of EPO depending signaling system could not only play a central role in brain cell protection, but this system could be a new tool for reverse the pharmacoresistant phenotype in refractory epilepsy as well as in other pharmacoresistant hypoxic brain diseases expressing P-gp.

Pharmacokinetics and Scintigraphic Imaging of the Hypoxia-Imaging Agent [123I]IAZA in Healthy Adults Following Exercise-Based Cardiac Stress †.

The objective of this work is to evaluate the potential effect of cardiac stress exercise on the accumulation of [123I]IAZA, a radiopharmaceutical used to image focal tissue hypoxia, in otherwise normal myocardium in healthy volunteers, and to determine the impact of exercise on [123I]IAZA pharmacokinetics. The underlying goal is to establish a rational basis and a baseline for studies of focal myocardial hypoxia in cardiac patients using [123I]IAZA. Three healthy male volunteers ran the ‘Bruce’ treadmill protocol, a clinically-accepted protocol designed to expose myocardial ischemia in patients. The ‘Bruce’ criterion heart rate is 85% of [220–age]. Approximately one minute before reaching this level, [123I]IAZA (5.0 mCi/0.85 mg) was administered as a slow (1–3 min) single intravenous (i.v.) injection via an indwelling venous catheter. The volunteer continued running for an additional 1 min before being transferred to a gamma camera. Serum samples were collected from the arm contralateral to the administration site at pre-determined intervals from 1 min to 45 h post injection and were analyzed by radio HPLC. Pharmacokinetic (PK) parameters were derived for [123I]IAZA and total radioactivity (total[123I]) using compartmental and noncompartmental analyses. Whole-body planar scintigraphic images were acquired from 0.75 to 24 h after dosing. PK data and scintigraphic images were compared to previously published [123I]IAZA data from healthy volunteers rest. Following exercise stress, both [123I]IAZA and total[123I] exhibited bi-exponential decline profiles, with rapid distribution phases [half-lives (t1/2α) of 1.2 and 1.4 min, respectively], followed by slower elimination phases [t1/2β of 195 and 290 min, respectively]. Total body clearance (CLTB) and the steady state volume of distribution (Vss) were 0.647 L/kg and 185 mL/min, respectively, for [123I]IAZA and 0.785 L/kg and 135 mL/min, respectively, for total[123I]. The t1/2β, CLTB and Vss values were comparable to those reported previously for rested volunteers. The t1/2α was approximately 4-fold shorter for [123I]IAZA and approximately 3-fold shorter for total[123I] under exercise relative to rested subjects. The heart region was visualized in early whole body scintigraphic images, but later images showed no accumulated radioactivity in this region, and no differences from images reported for rested volunteers were apparent. Minimal uptake of radiotracer in myocardium and skeletal muscle was consistent with uptake in non-stressed myocardium. Whole-body scintigrams for [123I]IAZA in exercise-stressed healthy volunteers were indistinguishable from images of non-exercised volunteers. There was no evidence of hypoxia-dependent binding in exercised but otherwise healthy myocardium, supporting the conclusion that exercise stress at Bruce protocol intensity does not induce measurable myocardial hypoxia. Effects of exercise on PK parameters were minimal; specifically, the t1/2α was shortened, reflecting increased cardiac output associated with exercise. It is concluded that because [123I]IAZA was not metabolically bound in exercise-stressed myocardium, a stress test will not create elevated myocardial background that would mask regions of myocardial perfusion deficiency. [123I]IAZA would therefore be suitable for the detection of viable, hypoxic myocardium in patients undergoing stress-test-based diagnosis.

Lipopolysaccharide Associates with Amyloid Plaques, Neurons and Oligodendrocytes in Alzheimer's Disease Brain: A Review.

This review proposes that lipopolysaccharide (LPS, found in the wall of all Gram-negative bacteria) could play a role in causing sporadic Alzheimer’s disease (AD). This is based in part upon recent studies showing that: Gram-negative E. coli bacteria can form extracellular amyloid; bacterial-encoded 16S rRNA is present in all human brains with over 70% being Gram-negative bacteria; ultrastructural analyses have shown microbes in erythrocytes of AD patients; blood LPS levels in AD patients are 3-fold the levels in control; LPS combined with focal cerebral ischemia and hypoxia produced amyloid-like plaques and myelin injury in adult rat cortex. Moreover, Gram-negative bacterial LPS was found in aging control and AD brains, though LPS levels were much higher in AD brains. In addition, LPS co-localized with amyloid plaques, peri-vascular amyloid, neurons, and oligodendrocytes in AD brains. Based upon the postulate LPS caused oligodendrocyte injury, degraded Myelin Basic Protein (dMBP) levels were found to be much higher in AD compared to control brains. Immunofluorescence showed that the dMBP co-localized with β amyloid (Aβ) and LPS in amyloid plaques in AD brain, and dMBP and other myelin molecules were found in the walls of vesicles in periventricular White Matter (WM). These data led to the hypothesis that LPS acts on leukocyte and microglial TLR4-CD14/TLR2 receptors to produce NFkB mediated increases of cytokines which increase Aβ levels, damage oligodendrocytes and produce myelin injury found in AD brain. Since Aβ1–42 is also an agonist for TLR4 receptors, this could produce a vicious cycle that accounts for the relentless progression of AD. Thus, LPS, the TLR4 receptor complex, and Gram-negative bacteria might be treatment or prevention targets for sporadic AD.

Galectin-1 inhibition attenuates profibrotic signaling in hypoxia-induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by lung remodeling arising from epithelial injury, aberrant fibroblast growth, and excessive deposition of extracellular matrix. Repeated epithelial injury elicits abnormal wound repair and lung remodeling, often associated with alveolar collapse and edema, leading to focal hypoxia. Here, we demonstrate that hypoxia is a physiological insult that contributes to pulmonary fibrosis (PF) and define its molecular roles in profibrotic activation of lung epithelial cells. Hypoxia increased transcription of profibrotic genes and altered the proteomic signatures of lung epithelial cells. Network analysis of the hypoxic epithelial proteome revealed a crosstalk between transforming growth factor-β1 and FAK1 (focal adhesion kinase-1) signaling, which regulated transcription of galectin-1, a profibrotic molecule. Galectin-1 physically interacted with and activated FAK1 in lung epithelial cells. We developed a novel model of exacerbated PF wherein hypoxia, as a secondary insult, caused PF in mice injured with subclinical levels of bleomycin. Hypoxia elevated expression of phosphorylated FAK1, galectin-1, and α-smooth muscle actin and reduced caspase-3 activation, suggesting aberrant injury repair. Galectin-1 inhibition caused apoptosis in the lung parenchyma and reduced FAK1 activation, preventing the development of hypoxia-induced PF. Galectin-1 inhibition also attenuated fibrosis-associated lung function decline. Further, galectin-1 transcript levels were increased in the lungs of IPF patients. In summary, we have identified a profibrotic role of galectin-1 in hypoxia signaling driving PF.

The principal pathways involved in the invivo modulation of hypoxic pulmonary vasoconstriction, pulmonary arterial remodelling and pulmonary hypertension.

Hypoxic pulmonary vasoconstriction (HPV) serves to optimize ventilation-perfusion matching in focal hypoxia and thereby enhances pulmonary gas exchange. During global hypoxia, however, HPV induces general pulmonary vasoconstriction, which may lead to pulmonary hypertension (PH), impaired exercise capacity, right-heart failure and pulmonary oedema at high altitude. In chronic hypoxia, generalized HPV together with hypoxic pulmonary arterial remodelling, contribute to the development of PH. The present article reviews the principal pathways in the invivo modulation of HPV, hypoxic pulmonary arterial remodelling and PH with primary focus on the endothelin-1, nitric oxide, cyclooxygenase and adenine nucleotide pathways. In summary, endothelin-1 and thromboxane A2 may enhance, whereas nitric oxide and prostacyclin may moderate, HPV as well as hypoxic pulmonary arterial remodelling and PH. The production of prostacyclin seems to be coupled primarily to cyclooxygenase-1 in acute hypoxia, but to cyclooxygenase-2 in chronic hypoxia. The potential role of adenine nucleotides in modulating HPV is unclear, but warrants further study. Additional modulators of the pulmonary vascular responses to hypoxia may include angiotensin II, histamine, serotonin/5-hydroxytryptamine, leukotrienes and epoxyeicosatrienoic acids. Drugs targeting these pathways may reduce acute and/or chronic hypoxic PH. Endothelin receptor antagonists and phosphodiesterase-5 inhibitors may additionally improve exercise capacity in hypoxia. Importantly, the modulation of the pulmonary vascular responses to hypoxia varies between species and individuals, with hypoxic duration and age. The review also define how drugs targeting the endothelin-1, nitric oxide, cyclooxygenase and adenine nucleotide pathways may improve pulmonary haemodynamics, but also impair pulmonary gas exchange by interference with HPV in chronic lung diseases.

Oleanolic acid: a promising neuroprotective agent for cerebral ischemia.

Oleanolic acid: a promising neuroprotective agent for cerebral ischemia.

Further evidence for the neuroprotective role of oleanolic acid in a model of focal brain hypoxia in rats

Ischemic brain injury is a dynamic process involving oxidative stress, inflammation, cell death and the activation of endogenous adaptive and regenerative mechanisms depending on the activation of transcription factors such as hypoxia-inducible factor 1-alpha. Accordingly, we have previously described a new focal hypoxia model by direct intracerebral cobalt chloride injection. In turn, oleanolic acid, a plant-derived triterpenoid, has been extensively used in Asian countries for its anti-inflammatory and anti-tumor properties. A variety of novel pharmacological effects have been attributed to this triterpenoid, including beneficial effects on neurodegenerative disorders – including experimental autoimmune encephalomyelitis – due to its immunomodulatory activities at systemic level, as well as within the central nervous system. In this context, we hypothesize that this triterpenoid may be capable of exerting neuroprotective effects in ischemic brain, suppressing glial activities that contribute to neurotoxicity while promoting those that support neuronal survival. In order to test this hypothesis, we used the intraperitoneal administration of oleanoic acid in adult rats for seven days previous to focal cortical hypoxia induced by cobalt chloride brain injection. We analyzed the neuroprotective effect of oleanoic acid from a morphological point of view, focusing on neuronal survival and glial reaction.

Stanniocalcin-1 is induced by hypoxia inducible factor in rat alveolar epithelial cells

Alveolar type II (ATII) cells remain differentiated and express surfactant proteins when cultured at an air–liquid (A/L) interface. When cultured under submerged conditions, ATII cells dedifferentiate and change their gene expression profile. We have previously shown that gene expression under submerged conditions is regulated by hypoxia inducible factor (HIF) signaling due to focal hypoxia resulting from ATII cell metabolism. Herein, we sought to further define gene expression changes in ATII cells cultured under submerged conditions. We performed a genome wide microarray on RNA extracted from rat ATII cells cultured under submerged conditions for 24–48h after switching from an A/L interface. We found significant alterations in gene expression, including upregulation of the HIF target genes stanniocalcin-1 (STC1), tyrosine hydroxylase (Th), enolase (Eno) 2, and matrix metalloproteinase (MMP) 13, and we verified upregulation of these genes by RT-PCR. Because STC1, a highly evolutionarily conserved glycoprotein with anti-inflammatory, anti-apoptotic, anti-oxidant, and wound healing properties, is widely expressed in the lung, we further explored the potential functions of STC1 in the alveolar epithelium. We found that STC1 was induced by hypoxia and HIF in rat ATII cells, and this induction occurred rapidly and reversibly. We also showed that recombinant human STC1 (rhSTC1) enhanced cell motility with extended lamellipodia formation in alveolar epithelial cell (AEC) monolayers but did not inhibit the oxidative damage induced by LPS. We also confirmed that STC1 was upregulated by hypoxia and HIF in human lung epithelial cells. In this study, we have found that several HIF target genes including STC1 are upregulated in AECs by a submerged condition, that STC1 is regulated by hypoxia and HIF, that this regulation is rapidly and reversibly, and that STC1 enhances wound healing moderately in AEC monolayers. However, STC1 did not inhibit oxidative damage in rat AECs stimulated by LPS in vitro. Therefore, alterations in gene expression by ATII cells under submerged conditions including STC1 were largely induced by hypoxia and HIF, which may be relevant to our understanding of the pathogenesis of various lung diseases in which the alveolar epithelium is exposed to relative hypoxia.

Microwave-assisted radiosynthesis of the hypoxia marker 1-α-D-(5- deoxy-5-[18F]fluoroarabinofuranosyl)-2-nitroimidazole ([18F]FAZA).

The routine manufacture of most short-lived positron-emitting radiopharmaceuticals (PERs) involves conventional heating to accelerate the radiolabeling process. Nucleophilic radiofluorination reactions are generally slow at lower temperatures, and are accompanied by thermal decomposition of both precursor and product at higher temperatures. This necessitates HPLC purification and results in lower recovered radiochemical yields (rRCYs). [(18)F]FAZA, a PER for clinical imaging of focal tissue hypoxia, is routinely manufactured in-house in 3-12% rRCY using a Health Canada approved conventional heating procedure. The microwave-assisted (MW) radiosynthesis of [(18)F]FAZA is now reported. Manual (MRDS) and automated (ASU) reagent delivery systems coupled to a commercial MW unit were built in-house. The MW unit controlled power, irradiation time and monitored reaction temperature (Tmax control), while the acetylAZA tosylate precursor and QMA Accel(TM) cartridge eluent reagents (K2CO3, K2.2.2) were dispensed by the MRDS or ASU. The radiofluorination yields (RFYs) and the chemical and radiochemical TLC profiles of the post-labeling reaction mixtures were compared to those obtained using the conventional heating production method and to those reported for optimized literature methods. MW RFYs for [(18)F]FAZA reached >76% (n=3) in 3 min. Post-labeling analysis of the MW-assisted reaction mixtures demonstrated cleaner UV and radiochemical TLC profiles than those obtained from conventional heating in routine production runs; the relatively clean MW reactions allowed rapid HPLC isolation of [(18)F]FAZA in overall rRCYs of 55±4%. In practical terms, the MW process provided only small gains in reaction time and RFY, but produced only a few secondary impurities, thereby improving the rRCY in comparison to conventional heating methods. These findings provide a rationale for adaptation of the MW-assisted method for the routine production of clinical [(18)F]FAZA.

Reactive Changes in the Cellular Elements of the Rat Brain in Different Conditions of Circulatory Hypoxia

The aim of the present work was to identify structural, spatial, and quantitative changes in elements of the paranigral nucleus (PNN) of the midbrain and anterior cingulate field (ACF) of the telencephalon in different conditions of circulatory hypoxia. The anterior medial part of the PNN and layers V–VI of the ACF were studied in healthy rats seven days (n = 4) after occlusion of both common carotid arteries and in intact (control 1, n = 4) and sham-operated (control 2, n = 4) rats. Histological sections stained with cresyl violet by the Nissl method and processed for detection of glial fibrillary acidic protein and Iba1 protein were used to identify the ratio of essentially unchanged, hypochromic, pyknomorphic, and ghost neurons, along with the numbers of astrocytes, oligodendrocytes, microgliocytes, and endotheliocytes. The areas of neurons and gliocyte bodies were measured, along with the distances from these cells to capillary walls, and gliocyte:neuron indexes were calculated. The results showed that after exposure to different conditions of circulatory hypoxia, the cellular elements of the brain showed a spectrum of characteristic pathological changes. Neurons showed signs of nuclear pyknosis, lysis, and conversion to “ghost” cells. Cells in the nuclear zone of hypoxia tended to undergo death or pyknosis. Neurons located outside the hypoxic area and exposed only to the humoral actions of the responses of the glutamate-calcium cascade often showed acute swelling. The reactions of microgliocytes, with minor increases in numbers and structural signs of activation, represented early diffuse signs of focal hypoxia in the forebrain. Endotheliocyte proliferation seen at seven days of ischemia was not associated with the chain of cascade reactions, but occurred only in the focus of hypoxia. The concentrations of viable neurons and astrocytes close to blood capillaries and the increase in the number of satellite types of glial cells represent an adaptive mechanism and a constitute a condition for cell survival in the conditions of various types of cerebral ischemia.

Proposed Toxic and Hypoxic Impairment of a Brainstem Locus in Autism

Electrophysiological findings implicate site-specific impairment of the nucleus tractus solitarius (NTS) in autism. This invites hypothetical consideration of a large role for this small brainstem structure as the basis for seemingly disjointed behavioral and somatic features of autism. The NTS is the brain’s point of entry for visceral afference, its relay for vagal reflexes, and its integration center for autonomic control of circulatory, immunological, gastrointestinal, and laryngeal function. The NTS facilitates normal cerebrovascular perfusion, and is the seminal point for an ascending noradrenergic system that modulates many complex behaviors. Microvascular configuration predisposes the NTS to focal hypoxia. A subregion—the “pNTS”—permits exposure to all blood-borne neurotoxins, including those that do not readily transit the blood-brain barrier. Impairment of acetylcholinesterase (mercury and cadmium cations, nitrates/nitrites, organophosphates, monosodium glutamate), competition for hemoglobin (carbon monoxide, nitrates/nitrites), and higher blood viscosity (net systemic oxidative stress) are suggested to potentiate microcirculatory insufficiency of the NTS, and thus autism.

P57 Hydrogen sulfide differentially regulates hepatic blood flow at sinusoidal and extrasinusoidal sites during endotoxemia

Background The predominant effect of Hydrogen sulfide (HSS) on the vasculature is considered to be vasodilation. The vasodilatory action of H2S occurs through hyperpolarization of vascular smooth muscle cells via KATP channel opening. H2S has been shown to improve tissue perfusion in disease states, such as ischemia/reperfusion injury, where blood flow is inadequate. During sepsis, liver injury results in increased sensitivity to Endothelin 1 (ET-1) resulting in sinusoidal hyperconstriction and heterogeneous hepatic blood flow. Unlike the other gaseous vasodilators NO and CO which ameliorate liver injury by improving sinusoidal blood flow, H2S has been shown to be deleterious during sepsis. Although a vasodilatory effect of H2S has been reported on overall hepatic portal resistance, functional liver blood flow is determined by sinusoidal constriction mediate by contraction of hepatic stellate cells; the effects of H2S at this site has not been studied. Therefore, we investigated the vasoactive effect of H2S on hepatic blood flow during endotoxemia using modulators of portal venular (phenylephrine) and sinusoidal (ET-1) vessels. Methods Portal vascular response was determined using an isolated perfused rat liver. Total portal flow was maintained constant and portal resistance estimated by monitoring inflow pressure. For determination of the response to endotoxemia, rats were administered 1 mg/kg E. coli endotoxin 6 h prior to liver isolation. Sinusoidal response was determined using intravital microscopy. The liver microcirculation was viewed using NADH fluorescence for contrast and sinusoid diameter was determined off-line using morphmetric software (PTI). Results H2S inhibited the increased portal pressure response to PE infusion (20μM) from (4.7 ± 0.3 vs 3.2 ± 0.3, P Conclusion Our findings suggest H2S has both dilator and constrictor effects in the hepatic circulation with the constrictor effects leading to the heterogeneous perfusion and focal hypoxia previously observed in sepsis and endotoxemia. These sinusoid constrictor effects are likely to be a major contributor to the deleterious effects of H2S observed in sepsis.

Hypoxia‐inducible factor‐2α regulates intestinal inflammation and colon cancer

A combination of a disturbed function of the intestinal epithelial barrier and a dysregulation of the mucosal immune system are critical in the pathogenesis of Inflammatory bowel disease (IBD). Moreover, IBD is a major risk factor for colorectal cancer. Significant increases in focal hypoxia and its associated transcription factors have been observed during the pathogenesis of colitis and colon cancer. Regulation of hypoxia‐mediated genes is dependent on the nuclear transcription factor, hypoxia‐inducible factor (HIF)1α and HIF2α. HIF1α has been shown to be critical in maintaining the integrity of intestinal epithelial barrier during the pathogenesis of colitis. However, the role of HIF2α in inflammatory lesions in the intestine and colorectal cancer remains less clear. Here we developed novel mouse models with intestine‐specific gain‐of‐function and loss‐of‐function of HIF2α to investigate the role of HIF2α in experimental colitis and colon tumorigenesis. Activation of HIF2α potentiated inflammation, whereas deletion of HIF2α attenuated inflammation in an acute model of colitis. Moreover, HIF2α was critical in the apoptotic response following tissue injury. Since, chronic inflammation is major risk factor for colon cancer and HIF2α was shown to be critical in intestinal inflammation and apoptosis, studies assessing the role of HIF2α in colon carcinogenesis were also performed. The data demonstrated that activation of HIF2α increases colon tumor multiplicity, incidence, and progression. Together, our data suggest an important role of HIF2α as a molecular link between intestinal inflammation and cancer.