Hypoxia-induced Hyperpermeability Research Articles

Novel HIF-1-target gene isthmin1 contributes to hypoxia-induced hyperpermeability of pulmonary microvascular endothelial cells monolayers.

Hypoxia-induced pulmonary microvascular endothelial cell (PMVEC) monolayers hyperpermeability is vital for vascular leakage, which participates in vascular diseases, such as acute lung injury (ALI) and high-altitude pulmonary edema (HAPE). We previously observed that PMVEC permeability was markedly elevated in hypoxia when cocultured with primary type II alveolar epithelial cells (AECII) in which isthmin1 (ISM1) was highly upregulated. However, whether the upregulation of ISM1 plays a role in hypoxia-induced PMVEC hyperpermeability is unclear. In this study, we assessed the role of AECII-derived ISM1 in hypoxia-induced PMVEC hyperpermeability with an AECII/PMVEC coculture system and uncovered the underlying mechanism whereby hypoxia stimulates ISM1 gene expression. We found that ISM1 gene expression was upregulated in cultured AECII cells exposed to hypoxia (3% O2) and that AECII-derived ISM1 participated in hypoxia-induced hyperpermeability of PMVEC monolayers, as small interference RNA (siRNA)-mediated knockdown of ISM1 in AECII markedly attenuated the increase in PMVEC permeability in coculture system under hypoxia. In addition, we confirmed that ISM1 was regulated by hypoxia-inducible factor-1α (HIF1α) according to the evidence that silencing of HIF1α inhibited the hypoxia-mediated upregulation of ISM1. Mechanismly, overexpression of HIF1α transcriptionally activated ISM1 gene expression by directly binding to the conserved regulatory elements upstream of the ism1 locus. We identified a novel HIF-1-target gene ISM1, which involves in hyperpermeability of pulmonary microvascular endothelial cell monolayers under hypoxia. Our in vitro cell experiments implied that the upregulated ISM1 derived from alveolar epithelium might be a vital modulator in hypoxia-induced endothelial hyperpermeability and thereby implicates with hypoxic pulmonary-related diseases.

Effects of Sphingosine‐1‐Phosphate on Endothelial Barrier Function Following Hypoxic Injury and Hemorrhagic Shock

Our lab has previously demonstrated that S1P reduces hemorrhagic shock and resuscitation (HSR)‐induced microvascular hyperpermeability. HSR can cause poor perfusion and hypoxia in the gut, leading to endothelial apoptosis and injury to the endothelial surface layer (glycocalyx). In the current study our objective was to investigate the extent to which S1P can prevent apoptotic signaling, shedding of the (glycocalyx), and elevated microvascular permeability in response to HSR in vivo or hypoxia in a cultured endothelial cell model. For our in vivo studies, we used a rat model of HSR, in which S1P was administered in the intravenous resuscitation fluid. The mesenteric microvasculature was dissected and protein was extracted for the assessment of pro‐apoptotic proteins by western blot. We also performed immunofluorescence of the mesenteric microvasculature to evaluate paracellular junction protein interaction. As an indicator of glycocalyx health, we used an intravenous injection of FITC‐labeled lectin and measured the amount of lectin bound to the microvascular endothelium. For the in vitro studies, we placed cultured primary endothelial cells in a low‐oxygen environment and applied S1P during the hypoxic challenge. We tested whether S1P has the potential to protect barrier function of endothelial cells grown on Transwell membranes (0.4 μm pores) from hypoxia‐induced hyperpermeability by determining permeability to FITC‐albumin. We have found that HSR causes an increase in pro‐apoptotic protein BAK in vivo, but S1P fails to blunt this increase. However, S1P seems to decrease levels of cytosolic cytochrome c compared to HSR‐treated rats. Furthermore, we found that the amount of lectin bound to the mesenteric microvasculature is higher in HSR animals treated with S1P compared to HSR only. In our in vitro studies, we found that S1P prevents hypoxia‐induced permeability in different types of endothelial cells, characterized by a decrease in endothelial permeability. Taken together, our results suggest that S1P has the potential to protect microvascular barrier dysfunction caused by HSR by preventing glycocalyx shedding. However, how S1P reduces the activation of apoptotic cascade following HSR remains elusive.Support or Funding InformationSupported by NIH grants R01HL098215 and R01GM120774.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypoxia-induced hyperpermeability of rat glomerular endothelial cells involves HIF-2α mediated changes in the expression of occludin and ZO-1.

This study aimed to investigate the role of hypoxia-inducible factor-2α (HIF-2α) in the expression of tight junction proteins and permeability alterations in rat glomerular endothelial cells (rGENCs) under hypoxia conditions. The expression level of HIF-2α and tight junction proteins (occludin and ZO-1) in rGENCs were examined following 5% oxygen density exposure at different treatment times. HIF-2α lentivirus transfection was used to knockdown HIF-2α expression. Cells were divided into four groups: 1) control group (rGENCs were cultured under normal oxygen conditions), 2) hypoxia group (rGENCs were cultured under hypoxic conditions), 3) negative control group (rGENCs were infected with HIF-2α lentivirus negative control vectors and cultured under hypoxic conditions), and 4) Len group (rGENCs were transfected with HIF-2α lentivirus and cultured under hypoxic conditions). The hypoxia, negative control, and Len groups were kept in a hypoxic chamber (5% O2, 5% CO2, and 90% N2) for 24 h and the total content of occludin and ZO-1, and the permeability of rGENCs were assessed. With increasing hypoxia time, the expression of HIF-2α gradually increased, while the expression of occludin decreased, with a significant difference between groups. ZO-1 expression gradually decreased under hypoxia conditions, but the difference between the 24 and 48 h groups was not significant. The permeability of cells increased following 24-h exposure to hypoxia compared to the control group (P<0.01). The knockdown of HIF-2α expression significantly increased occludin and ZO-1 content compared with hypoxia and negative control groups (P<0.01), while permeability was reduced (P<0.01). Hypoxia increased HIF-2α content, inducing permeability of rGENCs through the reduced expression of occludin and ZO-1.

Hypoxia Modulates Epithelial Permeability via Regulation of Vascular Endothelial Growth Factor in Airway Epithelia.

Hypoxia resulting from occlusion of the sinus ostium is known to be one of the major pathogenic mechanisms of sinusitis. Hypoxia-inducible factor (HIF)-1 is a widely known transcription factor that induces the cellular response to hypoxic conditions and activates the transcription of several genes, including vascular endothelial growth factor (VEGF). We hypothesized that induced permeability caused by hypoxia is a major pathophysiologic mechanism of upper airway diseases, such as sinusitis. The aim of this study was to investigate the mechanism of hypoxia-induced hyperpermeability, which mediates increased paracellular permeability and enhanced microbial invasiveness in the airway epithelium. We show that expression of VEGF mRNA and protein and HIF-1α protein increased as a function of time under hypoxia in normal human nasal epithelial cells. Our results also indicate that VEGF expression was induced by transfection with a mammalian expression vector encoding HIF-1 but down-regulated by transfection with small interfering RNA specific for HIF-1α under hypoxic conditions. Results of a transepithelial permeability assay measuring transepithelial electrical resistance indicated that permeability was increased as a function of time under hypoxia and was rescued by anti-VEGF monoclonal antibody (bevacizumab) and small interfering RNA specific for HIF-1α. We detected up-regulated HIF-1α and VEGF expression in mucosal epithelium samples from patients with sinusitis compared with normal mucosal epithelium using Western blotting and immunohistochemical staining. In conclusion, we suggest that the hypoxia-HIF-1α-VEGF axis plays an important role in hyperpermeability of airway epithelial cells, implying a role in the pathophysiology of upper respiratory tract diseases, such as sinusitis.

Role of PKCβII and PKCδ in blood–brain barrier permeability during aglycemic hypoxia

Blood–brain barrier (BBB) dysfunction contributes to the pathophysiology of cerebrovascular diseases such as stroke. In the present study, we investigated the role of PKC isoforms in aglycemic hypoxia-induced hyperpermeability using an in vitro model of the BBB consisting of mouse bEnd.3 cells. PKCβII and PKCδ isoforms were activated during aglycemic hypoxia. CGP53353, a specific PKCβII inhibitor, significantly attenuated aglycemic hypoxia-induced BBB hyperpermeability and disruption of occludin and zonula occludens-1 (ZO-1), indicating a deleterious role of PKCβII in the regulation of BBB permeability during aglycemic hypoxia. Conversely, rottlerin, a specific PKCδ inhibitor, exacerbated BBB hyperpermeability and tight junction (TJ) disruption during aglycemic hypoxia, indicating a protective role of PKCδ against aglycemic hypoxia-induced BBB hyperpermeability. Furthermore, disruption of TJ proteins during aglycemic hypoxia was attenuated by PKCβII DN and PKCδ WT overexpression, and aggravated by PKCβII WT and PKCδ DN overexpression. These results suggest that PKCβII and PKCδ counter-regulate BBB permeability during aglycemic hypoxia.

Flt‐1, but not Flk‐1 mediates hyperpermeability through activation of the PI3‐K/Akt pathway

Vascular endothelial growth factor (VEGF), a potent mediator of endothelial proliferation and migration, has an important role also in brain edema formation during hypoxia and ischemia. VEGF binds to the tyrosine kinase receptors Flt-1 and Flk-1. Yet, their relative importance for hypoxia-induced hyperpermeability is not well understood. We used an in vitro blood-brain barrier (BBB) model consisting of porcine brain microvascular endothelial cells (BMEC) to determine the role of Flt-1 in VEGF-induced endothelial cell (EC) barrier dysfunction. Soluble Flt-1 abolished hypoxia/VEGF-induced hyperpermeability. Furthermore, selective antisense oligonucleotides to Flt-1, but not to Flk-1, inhibited hypoxia-induced permeability changes. Consistent with these data, addition of the receptor-specific homolog placenta-derived growth factor, which binds Flt-1 but not Flk-1, increased endothelial permeability to the same extent as VEGF, whereas adding VEGF-E, a viral VEGF molecule from the orf virus family activating Flk-1 and neuropilin-1, but not Flt-1, did not show any effect. Using the carcinoma submandibular gland cell line (CSG), only expressing Flt-1, it was demonstrated that activation of Flt-1 is sufficient to induce hyperpermeability by hypoxia and VEGF. Hyperpermeability, induced by hypoxia/VEGF, depends on activation of phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), nitric oxide synthase (NOS) and protein kinase G (PKG). The activation of the PI3-K/Akt pathway by hypoxia was confirmed using an in vivo mice hypoxia model. These results demonstrate that hypoxia/VEGF-induced hyperpermeability can be mediated by activation of Flt-1 independently on the presence of Flk-1 and indicate a central role for activation of the PI3-K/Akt pathway, followed by induction of NOS and PKG activity.

In-vitro-Effekte von An�sthetika auf die Blut-Hirn-Schranke

The blood-brain barrier (BBB) forms a selective barrier between blood and brain and regulates the passage of most molecules. Pathological conditions such as ischemia lead to breakdown of the BBB. Vascular endothelial growth factor (VEGF) has been shown to be responsible for hypoxia-induced hyperpermeability of the BBB in vivo as well as in vitro. To eliminate factors which alter the permeability of the BBB in vivo, an in vitro model was used to test the effects of intravenous and volatile anesthetics on the permeability and on VEGF expression during normoxia and hypoxia. The in vitro model of the BBB consisted of primary cultures of porcine brain microvascular endothelial cells (BMEC). The permeability was measured by the paracellular passage of [3H]inulin across the BMEC monolayer and the expression of VEGF was determined by northern blot analysis. All intravenous and volatile anesthetics tested (etomidate, ketamine, fentanyl, propofol, midazolam, sodium-gamma-hydroxybutyrate as well as halothane, enflurane, isoflurane, sevoflurane, desflurane) did not alter the permeability of the BBB or the expression of VEGF in vitro. Hypoxia (2 vol%) increased the permeability and the VEGF expression significantly which was not altered in the presence of the anesthetics. The in vitro model represents a suitable model of the BBB to investigate direct effects of anesthetics on functions of the BBB independent of hemodynamic factors.

Hypoxia-Induced Hyperpermeability in Brain Microvessel Endothelial Cells Involves VEGF-Mediated Changes in the Expression of Zonula Occludens-1

In vivo, hypoxia is known to damage the blood–brain barrier (BBB) leading to the development of vasogenic brain edema. Primary cultures of porcine brain derived microvascular endothelial cells were used as an in vitro BBB model to evaluate the mechanisms by which hypoxia regulates paracellular permeability. Paracellular passage across endothelial cell monolayers is regulated by specialized intercellular structures like the tight junctions (TJ). Zonula occludens-1 (ZO-1), a protein of the TJ, lines the cytoplasmic face of intact TJ. The continuity of the ZO-1 expression was disrupted during 24 h of hypoxia which correlated with a decrease of the protein level to 32 ± 8% and with a twofold increase in the phosphorylation of ZO-1 in comparison to values determined at the start of the experiment. The localization and expression level of ZO-1 were maintained during hypoxia in the presence of a polyclonal antibody to vascular endothelial growth factor (VEGF) demonstrating that hypoxia-induced changes of the ZO-1 expression are mediated by VEGF. The effect of hypoxia on the ZO-1 distribution probably is not tissue- or cell-specific because similar changes of ZO-1 distribution were observed when the rat brain endothelial cell line RBE4 or the murine epithelial cell line CSG was used. Furthermore, ZO-1 changes correlated with small changes in actin distribution. These results suggest that hypoxia increases the paracellular flux across the cell monolayer via the release of VEGF, which in turn leads to the dislocalization, decreased expression, and enhanced phosphorylation of ZO-1.

In vitro effects of dexamethasone on hypoxia-induced hyperpermeability and expression of vascular endothelial growth factor

Clinically, dexamethasone is known to reduce cerebral edema. To further investigate the mechanism of this neuroprotection, an in vitro model of brain-derived microvessel endothelial cells (BME cells) was used to investigate the effect of dexamethasone on hypoxia-induced hyperpermeability. Furthermore, the expression of vascular endothelial growth factor (VEGF), which is known to be the mediator of hypoxia-induced hyperpermeability, was evaluated. Dexamethasone (40 μg/ml=100 μM) decreased hypoxia-induced permeability and VEGF expression significantly during time periods of more than 3 h. The time dependence of the dexamethasone effect correlated with a changed mechanism by which hypoxia induced VEGF expression. This was deduced because hypoxia-induced hyperpermeability and VEGF mRNA level were decreased in the presence of an antisense oligonucleotide coding for a region which binds a mRNA stabilizing protein, but only up to 3 h of hypoxia. Furthermore, during this time period the half-life of VEGF mRNA was increased. Results suggest that dexamethasone only decreases transcriptional-induced VEGF expression and that this may be related to the efficacy of dexamethasone to treat brain edema.

Effect of astroglial cells on hypoxia-induced permeability in PBMEC cells

An in vitro model of the blood-brain barrier (BBB), consisting of porcine brain-derived microvascular endothelial cells (PBMEC), was used to evaluate the effect of astrocytes in the BBB disruption during hypoxia. Hypoxia-induced hyperpermeability was decreased significantly in a coculture model of astroglia cells, either astrocytes or C6 glioma cells, with PBMEC and, to the same extent, when glia cell-conditioned medium was used. Corresponding to effects on hypoxia-induced hyperpermeability, astrocyte- and C6 cell-conditioned medium diminished hypoxia-induced vascular endothelial growth factor (VEGF) mRNA and protein expression, which recently was shown to be responsible for hypoxia-induced permeability changes in vitro. The effect on hypoxia-induced hyperpermeability and VEGF expression was specific for astroglia cells because conditioned medium from bovine smooth muscle cells (BSMC) did not show any effect. Immunocytochemistry revealed that 24 h of hypoxia disrupted the continuity of the tight junction protein, zonula occludens-1 (ZO-1), which lines the cytoplasmic face of intact tight junctions. These changes were prevented when hypoxia was performed in glia cell-conditioned medium. Results suggest that astrocytes protect the BBB from hypoxia-induced paracellular permeability changes by decreasing hypoxia-induced VEGF expression in microvascular endothelial cells.

Hypothermia abolishes hypoxia-induced hyperpermeability in brain microvessel endothelial cells

The effect of mild (32°C) and deep (22°C) hypothermia on hypoxia-induced hyperpermeability was examined using an in vitro model of brain derived microvascular endothelial cells (BMEC). It was shown that hypoxia-induced hyperpermeability to inulin across the BMEC monolayer was completely abolished at 32°C and 22°C for up to 24 h of hypoxia. During normoxia, no influence of hypothermia on BMEC monolayer permeability was observed. The hypoxia-induced decrease of the cyclic AMP level after 6 h was abolished at 32°C as well as at 22°C of hypoxia. But after 24 h of hypoxia, hypothermia did no longer prevent the hypoxia-induced decrease of the cAMP level, which suggests that the effect of hypothermia on hypoxia-induced hyperpermeability is not caused by maintenance of the cAMP level. Because vascular endothelial growth factor (VEGF) has been shown to be the mediator of hypoxia-induced permeability changes of BMEC via the release of nitric oxide (NO), the effect of hypothermia on the VEGF expression was evaluated. During normoxia, hypothermia did not change the VEGF expression significantly but the hypoxia-induced increase in VEGF mRNA and protein expression was completely abolished at 32°C and 22°C respectively. Accordingly, the hypoxia-induced increase of the cGMP level was depressed by hypothermia, which demonstrates that also the amount of NO released during hypoxia is decreased at lower temperatures. Results suggest that deep as well as mild hypothermia decreased hypoxia-induced hyperpermeability by lowering the expression of the permeability-increasing protein VEGF and with it the release of NO.

Hypoxia induces permeability in brain microvessel endothelial cells via VEGF and NO.

In this study, an in vitro model of the blood-brain barrier, consisting of porcine brain-derived microvascular endothelial cells (BMEC), was used to evaluate the mechanism of hypoxia-induced hyperpermeability. We show that hypoxia-induced permeability in BMEC was completely abolished by a neutralizing antibody to vascular endothelial growth factor (VEGF). In contrast, under normoxic conditions, addition of VEGF up to 100 ng/ml did not alter monolayer barrier function. Treatment with either hypoxia or VEGF under normoxic conditions induced a twofold increase in VEGF binding sites and VEGF receptor 1 (Flt-1) mRNA expression in BMEC. Hypoxia-induced permeability also was prevented by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine, suggesting that NO is involved in hypoxia-induced permeability changes, which was confirmed by measurements of the cGMP level. During normoxia, treatment with VEGF (5 ng/ml) increased permeability as well as cGMP content in the presence of several antioxidants. These results suggest that hypoxia-induced permeability in vitro is mediated by the VEGF/VEGF receptor system in an autocrine manner and is essentially dependent on reducing conditions stabilizing the second messenger NO as the mediator of changes in barrier function of BMEC.

Cytosolic ionized Ca2+ modulates chemical hypoxia-induced hyperpermeability in intestinal epithelial monolayers.

We reported previously that ATP depletion induced by glycolytic inhibition or cellular hypoxia increases the permeability of intestinal epithelial monolayers [N. Unno, M. J. Menconi, A. L. Salzman, M. Smith, S. Hagen, Y. Ge, R. M. Ezzell, and M. P. Fink. Am. J. Physiol. 270 (Gastrointest. Liver Physiol. 33): G1010-G1021, 1996]. In the present study, we examined the effects of the Ca2+ ionophore A-23187 or the intracellular ionized Ca2+ concentration ([Ca2+]i) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM on the permeability of enterocytic (T84) monolayers depleted of ATP by metabolic inhibition. Permeability was assessed by measuring transepithelial electrical resistance and the transepithelial flux of fluorescein sulfonic acid. Although neither A-23187 nor BAPTA-AM affected ATP depletion, A-23187 augmented, whereas BAPTA-AM ameliorated, chemical hypoxia-induced hyperpermeability. BAPTA-AM ameliorated chemical hypoxia-induced cytoskeletal derangements. Monolayers subjected to chemical hypoxia but incubated in a low (i.e., 100 microM) [Ca2+] environment showed preservation of junctional integrity, whereas voltage-dependent Ca2+ channel blockers (NiCl2 or verapamil) failed to ameliorate chemical hypoxia-induced hyperpermeability. ATP depletion induces hyperpermeability in intestinal epithelial monolayers via a [Ca2+]i-dependent mechanism. Increased [Ca2+]i under these conditions reflects leakage of Ca2+ from the extracellular milieu via a mechanism unrelated to voltage-dependent Ca2+ channels.