Transgenic system for conditional induction and rescue of chronic myocardial hibernation provides insights into genomic programs of hibernation

A key energy-saving adaptation to chronic hypoxia that enables cardiomyocytes to withstand severe ischemic insults is hibernation, i.e., a reversible arrest of contractile function. Whereas hibernating cardiomyocytes represent the critical reserve of dysfunctional cells that can be potentially rescued, a lack of a suitable animal model has hampered insights on this medically important condition. We developed a transgenic mouse system for conditional induction of long-term hibernation and a system to rescue hibernating cardiomyocytes at will. Via myocardium-specific induction (and, in turn, deinduction) of a VEGF-sequestering soluble receptor, we show that VEGF is indispensable for adjusting the coronary vasculature to match increased oxygen consumption and exploit this finding to generate a hypoperfused heart. Importantly, ensuing ischemia is tunable to a level at which large cohorts of cardiomyocytes are driven to enter a hibernation mode, without cardiac cell death. Relieving the VEGF blockade even months later resulted in rapid revascularization and full recovery of contractile function. Furthermore, we show that left ventricular remodeling associated with hibernation is also fully reversible. The unique opportunity to uncouple hibernation from other ischemic heart phenotypes (e.g., infarction) was used to determine the genetic program of hibernation; uncovering hypoxia-inducible factor target genes associated with metabolic adjustments and induced expression of several cardioprotective genes. Autophagy, specifically self-digestion of mitochondria, was identified as a key prosurvival mechanism in hibernating cardiomyocytes. This system may lend itself for examining the potential utility of treatments to rescue dysfunctional cardiomyocytes and reverse maladaptive remodeling.

Perioperative fluid management: comparison of high, medium and low fluid volume on tissue oxygen pressure in the small bowel and colon

BACKGROUND AND OBJECTIVE: Insufficient blood flow and oxygenation in the intestinal tract is associated with increased incidence of postoperative complications after bowel surgery. High fluid volume administration may prevent occult regional hypoperfusion and intestinal tissue hypoxia. We tested the hypothesis that high intraoperative fluid volume administration increases intestinal wall tissue oxygen pressure during laparotomy. METHODS: In all, 27 pigs were anaesthetized, ventilated and randomly assigned to one of the three treatment groups (n = 9 in each) receiving low (3 mL kg-1 h-1), medium (7 mL kg-1 h-1) or high (20 mL kg-1 h-1) fluid volume treatment with lactated Ringer's solution. All animals received 30% and 100% inspired oxygen in random order. Cardiac index was measured with thermodilution and tissue oxygen pressure with a micro-oximetry system in the jejunum and colon wall and subcutaneous tissue. RESULTS: Groups receiving low and medium fluid volume treatment had similar systemic haemodynamics. The high fluid volume group had significantly higher mean arterial pressure, cardiac index and subcutaneous tissue oxygenation. Tissue oxygen pressures in the jejunum and colon were comparable in all three groups. CONCLUSIONS: The three different fluid volume regimens tested did not affect tissue oxygen pressure in the jejunum and colon, suggesting efficient autoregulation of intestinal blood flow in healthy subjects undergoing uncomplicated abdominal surgery.

Effects of continuous remifentanil administration on intra-operative subcutaneous tissue oxygen tension

Surgical stress response markedly increases sympathetic nerve activity and catecholamine concentrations. This may contribute to peripheral vasoconstriction, reduced wound perfusion and subsequent tissue hypoxia. Opioids are known to depress the hypothalamic-adrenal response to surgery in a dose-dependent manner. We tested the hypothesis that continuous remifentanil administration produces improved subcutaneous tissue oxygen tension compared to fentanyl bolus administration. Forty-six patients undergoing major abdominal surgery were randomly assigned to receive either fentanyl bolus administration or continuous remifentanil infusion. Mean subcutaneous tissue oxygen values over the entire intra-operative period were significantly higher in the remifentanil group, when compared to the fentanyl group: 8 (2) kPa vs 6.7 (1.5) kPa, % CI difference: - 2.3 kPa to - 0.3 kPa, p = 0.013. Continuous intra-operative opioid administration may blunt vasoconstriction caused by surgical stress and adrenergic responses more than an equi-effective anaesthetic regimen based on smaller-dose bolus opioid administration.

Multiple pathways of neuroprotection against oxidative stress and excitotoxic injury in immature primary hippocampal neurons

In the immature brain hydrogen peroxide accumulates after excitotoxic hypoxia-ischemia and is neurotoxic. Immature hippocampal neurons were exposed to N-methyl-D-aspartate (NMDA), a glutamate agonist, and hydrogen peroxide (H(2)O(2)) and the effects of free radical scavenging and transition metal chelation on neurotoxicity were studied. alpha-Phenyl-N-tert.-butylnitrone (PBN), a known superoxide scavenger, attenuated both H(2)O(2) and NMDA mediated toxicity. Treatment with desferrioxamine (DFX), an iron chelator, at the time of exposure to H(2)O(2) was ineffective, but pretreatment was protective. DFX also protected against NMDA toxicity. TPEN, a metal chelator with higher affinities for a broad spectrum of transition metal ions, also protected against H(2)O(2) toxicity but was ineffective against NMDA induced toxicity. These data suggest that during exposure to free radical and glutamate agonists, the presence of iron and other free metal ions contribute to neuronal cell death. In the immature nervous system this neuronal injury can be attenuated by free radical scavengers and metal chelators.

Hypoxic expansion promotes the chondrogenic potential of articular chondrocytes

For cell-based cartilage repair strategies, an ex vivo expansion phase is required to obtain sufficient numbers of cells needed for therapy. Although recent reports demonstrated the central role of oxygen for the function and differentiation of chondrocytes, a beneficial effect of low oxygen concentrations during the expansion of the cells to further improve their chondrogenic capacity has not been investigated.Therefore, freshly harvested bovine articular chondrocytes were grown in two-dimensional monolayer cultures at 1.5% and 21% O2 and redifferentiation was subsequently induced in three-dimensional micromass cultures at 1.5%, 5%, and 21% O2. Cells expanded at 1.5% O2 were characterized by low citrate synthase (aerobic energy metabolism)--and high LDH (anaerobic energy metabolism-activities,suggesting an anaerobic energy metabolism. Collagen type II mRNA was twofold higher in cells expanded at 1.5% as compared to expansion at 21% O2. Micromass cultures grown at 21% O2 showed up to a twofold increase in the tissue content of glycosaminoglycans when formed with cells expanded at 1.5% instead of 21% O2. However, no differences in the levels of transcripts and in the staining for collagen type II protein were observed in these micromass cultures. Hypoxia (1.5% and 5% O2) applied during micromass cultures gave rise to tissues with low contents of glycosaminoglycans only. In vivo, the chondrocytes are adapted to a hypoxic environment. Taking this into account, by applying 1.5% O2 in the expansion phase in the course of cell-based cartilage repair strategies, may result in a repair tissue with higher quality by increasing the content of glycosaminoglycans.

Association between elevated brain tissue glycerol levels and poor outcome following severe traumatic brain injury

OBJECT: Glycerol is considered to be a marker of cell membrane degradation and thus cellular lysis. Recently, it has become feasible to measure via microdialysis cerebral extracellular fluid (ECF) glycerol concentrations at the patient's bedside. Therefore the aim of this study was to investigate the ECF concentration and time course of glycerol after severe traumatic brain injury (TBI) and its relationship to patient outcome and other monitoring parameters. METHODS: As soon as possible after injury for up to 4 days, 76 severely head-injured patients were monitored using a microdialysis probe (cerebral glycerol) and a Neurotrend sensor (brain tissue PO2) in uninjured brain tissue confirmed by computerized tomography scanning. The mean brain tissue glycerol concentration in all monitored patients decreased significantly from 206 +/- 31 micromol/L on Day 1 to 9 +/- 3 micromol/L on Day 4 after injury (p < 0.0001). Note, however, that there was no significant difference in the time course between patients with a favorable outcome (Glasgow Outcome Scale [GOS] Scores 4 and 5) and those with an unfavorable outcome (GOS Scores 1-3). Significantly increased glycerol concentrations were observed when brain tissue PO2 was less than 10 mm Hg or when cerebral perfusion pressure was less than 70 mm Hg. CONCLUSIONS: Based on results in the present study one can infer that microdialysate glycerol is a marker of severe tissue damage, as seen immediately after brain injury or during profound tissue hypoxia. Given that brain tissue glycerol levels do not yet add new clinically significant information, however, routine monitoring of this parameter following traumatic brain injury needs further validation.

Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after treatment cessation. To assess the responses to irradiation and vascular endothelial growth factor-receptor tyrosine kinase inhibition (by the vascular endothelial growth factor tyrosine kinase inhibitor PTK787/ZK222854), mammary carcinoma allografts were investigated by vascular casting; electron, light, and confocal microscopy; and immunoblotting. Irradiation and anti-angiogenic therapy had similar effects on the tumor vasculature. Both treatments reduced tumor vascularization, particularly in the tumor medulla. After cessation of therapy, the tumor vasculature expanded predominantly by intussusception with a plexus composed of enlarged sinusoidal-like vessels containing multiple transluminal tissue pillars. Tumor revascularization originated from preserved alpha-smooth muscle actin-positive vessels in the tumor cortex. Quantification revealed that recovery was characterized by an angiogenic switch from sprouting to intussusception. Up-regulated alpha-smooth muscle actin-expression during recovery reflected the recruitment of alpha-smooth muscle actin-positive cells for intussusception as part of the angio-adaptive mechanism. Tumor recovery was associated with a dramatic decrease (by 30% to 40%) in the intratumoral microvascular density, probably as a result of intussusceptive pruning and, surprisingly, with only a minimal reduction of the total microvascular (exchange) area. Therefore, the vascular supply to the tumor was not severely compromised, as demonstrated by hypoxia-inducible factor-1alpha expression. Both irradiation and anti-angiogenic therapy cause a switch from sprouting to intussusceptive angiogenesis, representing an escape mechanism and accounting for the development of resistance, as well as rapid recovery, after cessation of therapy.

Temporary hypoxic stress protects the liver from Fas-mediated apoptosis and ischemia reperfusion injury

Molecular responses to hypoxia restore oxygen homeostasis and promote cell survival, and are mainly regulated through the activation of the hypoxia-inducible transcription factor (HIF)-1 and its target genes. In this study we questioned whether surgically depleting the liver s arterial blood supply, by clamping the hepatic artery (HA), would be sufficient to mount a hypoxia-driven molecular response, the up-regulation of hepatoprotective genes and thereby protect the liver from subsequent damaging insults.;;The HA of normal male Balb/c mice was clamped with a micro vascular clip for 2 hours. The liver s saturated oxygen concentration (SO2) was measured using an O2C surface probe (LEA-Medizintechnik) and interstitial fluid was collected with microdialysis membranes to monitor tissue damage. Mice without clamping served as sham operated controls. Interstitial fluid was assessed for lactate pyruvate (L/P) and glycerol content and the mRNA of hepatoprotective genes was analyzed by real time PCR. Subsequently, mice received either a tail vein injection of anti-Fas antibody (Jo2, 0.2 mg/kg) or the liver was made ischemic (60min) followed by 6 hours reperfusion. Caspase 3-activity and cleaved lamin A were used to assess apoptosis. In separate groups, animal were monitored for survival.;;After 30min of clamping the HA the SO2 of the liver decreased and remained at a reduced level for up to 2 hours, without an increase in L/P ratio or glycerol release. We demonstrate the activation of a hypoxia-inducible signaling pathway by the stabilization of HIF-1 protein (Western blot) and by an increase of its target gene, Epo, mRNA. There was an up-regulation of the hepatoprotective genes IL-6, IGFBP-1, HO-1 and A20 mRNA. When subsequently injected with Jo2, animals preconditioned with HA clamping, had a significantly decreased caspase-3 activity (avg21044 vs. avg3637; p=0.001, T-test) and there were fewer positive cells for cleaved Lamin A. The survival probability (10.5 hours, n=12) of mice with HA clamping was significantly higher (3.2 hours, n=13; p=0.014, Logrank test). Likewise, survival after 60 minutes of partial hepatic ischemia and 6 hours of reperfusion was reduced from 86% in mice with pretreatment by HA clamping to 56% in sham treated controls.;;This study demonstrates that a localized hypoxic stress can be achieved by surgically removing the livers arterial blood supply. Furthermore it can stimulate a hepatoprotective response that protects the liver against Fas-mediated apoptosis and ischemia-reperfusion injury. Our findings offer an innovative approach to induce hepatoprotective genes to defend the liver against subsequent insults.

High incidence of optic disc swelling at very high altitudes

OBJECTIVES: To determine the incidence of optic disc swelling as a possible indicator of cerebral edema in a large group of healthy mountaineers exposed to very high altitudes and to correlate these findings with various clinical and environmental factors and occurrence of acute mountain sickness and high-altitude cerebral edema. METHODS: This multidisciplinary, prospective, observational cohort study was performed in 2005 within the scope of a medical research expedition to Muztagh Ata (7546 m [24,751 ft]) in Western Xinjiang Province, China. Twenty-seven healthy mountaineers aged 26 to 62 years participated. Medical examinations were performed in Switzerland 1 month before and 4 1/2 months after the expedition. Ophthalmologic examinations were performed at 4 high camps (maximum elevation, 6865 m [22,517 ft]). Optic disc status was documented using digital photography. Further assessments included arterial oxygen saturation and cerebral acute mountain sickness scores. RESULTS: Sixteen of 27 study subjects (59%) exhibited optic disc swelling during their stay at high altitudes, with complete regression on return to lowlands. Significant correlation was noted between optic disc swelling and lower arterial oxygen saturation (odds ratio, 0.86 per percentage of arterial oxygen saturation; 95% confidence interval, 0.81-0.92; P < .001), younger age (odds ratio, 0.95 per year; 95% confidence interval, 0.90-0.99; P = .03), and higher cerebral acute mountain sickness scores (odds ratio, 2.32 per 0.1 point; 95% confidence interval, 1.48-3.63; P < .001). CONCLUSION: Optic disc swelling occurs frequently in high-altitude climbers and is correlated with peripheral oxygen saturation and symptoms of acute mountain sickness. It is most likely the result of hypoxia-induced brain volume increase.

Expression and hypoxic regulation of the endothelin system in endocrine cells of human and rat pancreatic islets

CONTEXT: The success of pancreatic islet transplantation depends largely on the capacity of the islet graft to survive the initial phase immediately after transplantation until revascularization is completed. Endothelin-1 (ET-1) is a strong vasoconstrictor which has been involved in solid organ graft failure but is also known to be a potent mitogenic/anti-apoptotic factor which could also potentially enhance the survival of the transplanted islets. OBJECTIVE: Characterization of the endothelin system with regard to a potential endothelin agonist/antagonist treatment. DESIGN: Regulated expression of the endothelin system in human and rat pancreatic islets and beta-cell lines was assessed by means of immunohistochemistry, competition binding studies, western blot, RT-PCR, real-time PCR and transplant studies. RESULTS: ET-1, ETA- and ETB-receptor immunoreactivity was identified in the endocrine cells of human and rat pancreatic islets. The corresponding mRNA was detectable in rat beta-cell lines and isolated rat and human pancreatic islets. Competition binding studies on rat islets revealed binding sites for both receptor types. ET-1 stimulated the phosphorylation of mitogen-activated protein kinase, which was prevented by ETA- and ETB-receptor antagonists. After exposure to hypoxia equal to post-transplant environment oxygen tension, mRNA levels of ET-1 and ETB-receptor of human islets were robustly induced whereas ETA-receptor mRNA did not show significant changes. Immunostaining signals for ET-1 and ETA-receptor of transplanted rat islets were markedly decreased when compared to native pancreatic sections. CONCLUSIONS: In pancreatic islets, ET-1 and its receptors are differentially expressed by hypoxia and after transplantation. Our results provide the biological basis for the study of the potential use of endothelin agonists/antagonists to improve islet transplantation outcome.

Pharmacological inhibition of integrin alphavbeta3 aggravates experimental liver fibrosis and suppresses hepatic angiogenesis

The vitronectin receptor integrin alphavbeta3 promotes angiogenesis by mediating migration and proliferation of endothelial cells, but also drives fibrogenic activation of hepatic stellate cells (HSCs) in vitro. Expecting antifibrotic synergism, we studied the effect of alphavbeta3 inhibition in two in vivo models of liver fibrogenesis. Liver fibrosis was induced in rats by way of bile duct ligation (BDL) for 6 weeks or thioacetamide (TAA) injections for 12 weeks. A specific alphavbeta3 (alphavbeta5) inhibitor (Cilengitide) was given intraperitoneally twice daily at 15 mg/kg during BDL or after TAA administration. Liver collagen was determined as hydroxyproline, and gene expression was quantified by way of quantitative polymerase chain reaction. Liver angiogenesis, macrophage infiltration, and hypoxia were assessed by way of CD31, CD68 and hypoxia-inducible factor-1alpha immunostaining. Cilengitide decreased overall vessel formation. This was significant in portal areas of BDL and septal areas of TAA fibrotic rats and was associated with a significant increase of liver collagen by 31% (BDL) and 27% (TAA), and up-regulation of profibrogenic genes and matrix metalloproteinase-13. Treatment increased gamma glutamyl transpeptidase in both models, while other serum markers remained unchanged. alphavbeta3 inhibition resulted in mild liver hypoxia, as evidenced by up-regulation of hypoxia-inducible genes. Liver infiltration by macrophages/Kupffer cells was not affected, although increases in tumor necrosis factor alpha, interleukin-18, and cyclooxygenase-2 messenger RNA indicated modest macrophage activation. CONCLUSION: Specific inhibition of integrin alphavbeta3 (alphavbeta5) in vivo decreased angiogenesis but worsened biliary (BDL) and septal (TAA) fibrosis, despite its antifibrogenic effect on HSCs in vitro. Angiogenesis inhibitors should be used with caution in patients with hepatic fibrosis.

VHL inactivation is an important pathway for the development of malignant sporadic pancreatic endocrine tumors

A small subset of familial pancreatic endocrine tumors (PET) arises in patients with von Hippel-Lindau syndrome and these tumors may have an adverse outcome compared to other familial PET. Sporadic PET rarely harbors somatic VHL mutations, but the chromosomal location of the VHL gene is frequently deleted in sporadic PET. A subset of sporadic PET shows active hypoxia signals on mRNA and protein level. To identify the frequency of functionally relevant VHL inactivation in sporadic PET and to examine a possible prognostic significance we correlated epigenetic and genetic VHL alterations with hypoxia signals. VHL mutations were absent in all 37 PETs examined. In 2 out of 35 informative PET (6%) methylation of the VHL promoter region was detected and VHL deletion by fluorescence in situ hybridization was found in 14 out of 79 PET (18%). Hypoxia inducible factor 1alpha (HIF1-alpha), carbonic anhydrase 9 (CA-9), and glucose transporter 1 (GLUT-1) protein was expressed in 19, 27, and 30% of the 152 PETs examined. Protein expression of the HIF1-alpha downstream target CA-9 correlated significantly with the expression of CA-9 RNA (P<0.001), VHL RNA (P<0.05), and VHL deletion (P<0.001) as well as with HIF1-alpha (P<0.005) and GLUT-1 immunohistochemistry (P<0.001). These PET with VHL alterations and signs of hypoxia signalling were characterized by a significantly shortened disease-free survival. We conclude that VHL gene impairment by promoter methylation and VHL deletion in nearly 25% of PET leads to the activation of the HIF-pathway. Our data suggest that VHL inactivation and consecutive hypoxia signals may be a mechanism for the development of sporadic PET with an adverse outcome.

Hemoglobin vesicles improve wound healing and tissue survival in critically ischemic skin in mice

Local hypoxia, as due to trauma, surgery, or arterial occlusive disease, may severely jeopardize the survival of the affected tissue and its wound-healing capacity. Initially developed to replace blood transfusions, artificial oxygen carriers have emerged as oxygen therapeutics in such conditions. The aim of this study was to target primary wound healing and survival in critically ischemic skin by the systemic application of left-shifted liposomal hemoglobin vesicles (HbVs). This was tested in bilateral, cranially based dorsal skin flaps in mice treated with a HbV solution with an oxygen affinity that was increased to a P(50) (partial oxygen tension at which the hemoglobin becomes 50% saturated with oxygen) of 9 mmHg. Twenty percent of the total blood volume of the HbV solution was injected immediately and 24 h after surgery. On the first postoperative day, oxygen saturation in the critically ischemic middle flap portions was increased from 23% (untreated control) to 39% in the HbV-treated animals (P < 0.05). Six days postoperatively, flap tissue survival was increased from 33% (control) to 57% (P < 0.01) and primary healing of the ischemic wound margins from 6.6 to 12.7 mm (P < 0.05) after HbV injection. In addition, higher capillary counts and endothelial nitric oxide synthase expression (both P < 0.01) were found in the immunostained flap tissue. We conclude that left-shifted HbVs may ameliorate the survival and primary wound healing in critically ischemic skin, possibly mediated by endothelial nitric oxide synthase-induced neovascularization.

Erythropoietin enhances oxygenation in critically perfused tissue through modulation of nitric oxide synthase

The aim of this study was to investigate the effect of human recombinant erythropoietin (EPO) on the microcirculation and oxygenation of critically ischemic tissue and to elucidate the role of endothelial NO synthase in EPO-mediated tissue protection. Island flaps were dissected from the back skin of anesthetized male Syrian golden hamsters including a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. Before ischemia, animals received an injection of epoetin beta at a dose of 5,000 U/kg body weight with (n = 7) or without (n = 7) blocking NO synthase by 30 mg/kg body weight L-NAME (Nomega-nitro-L-arginine methyl ester hydrochloride). Saline-treated animals served as control (n = 7). Ischemic tissue damage was characterized by severe hypoperfusion and inflammation, hypoxia, and accumulation of apoptotic cell nuclei after 5 h of collateralization. Erythropoietin pretreatment increased arteriolar and venular blood flow by 33% and 37%, respectively (P < 0.05), and attenuated leukocytic inflammation by approximately 75% (P < 0.05). Furthermore, partial tissue oxygen tension in the ischemic tissue increased from 8.2 to 15.8 mmHg (P < 0.05), which was paralleled by a 21% increased density of patent capillaries (P < 0.05) and a 50% reduced apoptotic cell count (P < 0.05). The improved microcirculation and oxygenation were associated with a 2.2-fold (P < 0.05) increase of endothelial NO synthase protein expression. Of interest, L-NAME completely abolished all the beneficial effects of EPO pretreatment. Our study demonstrates that, in critically ischemic and hypoxic collateralized tissue, EPO pretreatment improves tissue perfusion and oxygenation in vivo. This effect may be attributed to NO-dependent vasodilative effects and anti-inflammatory actions on the altered vascular endothelium.

TLR2 and TLR4 agonists induce production of the vasoactive peptide endothelin-1 by human dendritic cells

Endothelin-1 (ET-1) is mainly secreted by endothelial cells and acts as a potent vasoconstrictor. In addition ET-1 has also been shown to have pleiotropic effects on a variety of other systems including adaptive immunity. There are two main ET-1 receptors, ET(A) and ET(B), which have different tissue and functional distributions. Dendritic cells (DC) are pivotal antigen-presenting cells linking the innate with the adaptive immune system. DC are sentinels expressing pattern-recognition receptors, e.g. the toll-like receptors (TLR) for detecting danger signals released from pathogens or tissue injury. Here we show for the first time that stimulation of human monocyte-derived DC with exogenous as well as endogenous selective TLR4 and TLR2 agonists induces the production of ET-1 in a dose- and time-dependent manner. 'Alternative' activation of DC in the presence of 1alpha,25-dihydroxyvitamin D(3) results in a marked potentiation of the endothelin response, whereas prostaglandin E(2) or dexamethasone do not increase ET-1 production. Furthermore, chetomin, an inhibitor of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha), prevents TLR-mediated secretion of ET-1. Surprisingly, stimulation of human monocytes with LPS does not lead to secretion of detectable amounts of ET-1. These results suggest a role of ET-1 as an important player in human DC biology and innate immunity in general.