Hypoxic Gradient Research Articles

Abstract 593: Hypoxia is Present in the Macrophage-rich Center of Human Carotid Atherosclerotic Plaques

Intraplaque neovascularization is linked to plaque instability and thought to be stimulated by hypoxia. However, hypoxia has not been demonstrated yet in human atherosclerosis. The hypoxia marker pimonidazole was administrated intravenously 2 hours prior to carotid endarterectomy in 6 symptomatic patients to evaluate the presence of hypoxia. Subsequent immunohistochemistry of the operatively removed atherosclerotic plaques demonstrated the presence of hypoxia, especially in the macrophage-rich center of the lesions. Notably, two hypoxic gradients were observed: hypoxia was very strong in the center of the plaque, but almost absent close to the main artery lumen and in the media. hypoxia was most intense in segments with advanced atheroma and almost absent in segments containing only diffuse intimal thickening. Hypoxia strongly correlated with CD68 immunoreactivity (ρ= 0.7, p=0.000), neovascularization (ρ= 0.6, p=0.000) and the presence of a thrombus (ρ= 0.4, p=0.009). In addition, hypoxia co-localized with expression of HIF1α and VEGF . To exclude that pimonidazole immunoreactivity in the atherosclerotic plaque was the result of surgery-induced ischemia, arterial wall segments were collected at two time-points: directly after incision of the carotid artery and directly following excision of the plaque. Pimonidazole immunoreactivity in these two pieces was not different, suggesting that hypoxia and pimonidazole adducts were already present in the plaques before surgery . To show that pimonidazole reactivity was hypoxia-specific and independent of reactive oxygen species, human THP-1 macrophages were exposed to normoxia (20% O 2 ), hypoxia (0.2% O 2 ) and/or H 2 O 2 (100 μM) in the presence of pimonidazole. Indeed, flow cytometry only showed pimonidazole-positive cells after hypoxic exposure. This is the first study proving direct evidence of the existence of hypoxia in advanced human atherosclerotic lesions, most prominently in the macrophage-rich center. Also, hypoxia was associated with the expression of HIF1α, VEGF and intraplaque microvessels, suggesting its involvement in the regulation of human intraplaque neovascularization.

Angiogenesis and vasculogenesis: Inducing the growth of new blood vessels and wound healing by stimulation of bone marrow–derived progenitor cell mobilization and homing

During embryonic development, the vasculature is among the first organs to form and is in charge of maintaining metabolic homeostasis by supplying oxygen and nutrients and removing waste products. As one would expect, blood vessels are critical not only for organ growth in the embryo but also for repair of wounded tissue in the adult. An imbalance in angiogenesis (a time-honored term that globally refers to the growth of new blood vessels) contributes to the pathogenesis of numerous malignant, inflammatory, ischemic, infectious, immune, and wound-healing disorders. This review focuses on the central role of the growth of new blood vessels in ischemic and diabetic wound healing and defines the most current nomenclature that describes the neovascularization process in wounds. There are now two well-defined, distinct, yet interrelated processes for the formation of postnatal new blood vessels, angiogenesis, and vasculogenesis. Reviewed are recent new data on vasculogenesis that promise to advance the field of wound healing.

Endothelial Progenitor Cell Release into Circulation Is Triggered by Hyperoxia‐Induced Increases in Bone Marrow Nitric Oxide

Endothelial progenitor cells (EPC) are known to contribute to wound healing, but the physiologic triggers for their mobilization are often insufficient to induce complete wound healing in the presence of severe ischemia. EPC trafficking is known to be regulated by hypoxic gradients and induced by vascular endothelial growth factor-mediated increases in bone marrow nitric oxide (NO). Hyperbaric oxygen (HBO) enhances wound healing, although the mechanisms for its therapeutic effects are incompletely understood. It is known that HBO increases nitric oxide levels in perivascular tissues via stimulation of nitric oxide synthase (NOS). Here we show that HBO increases bone marrow NO in vivo thereby increasing release of EPC into circulation. These effects are inhibited by pretreatment with the NOS inhibitor l-nitroarginine methyl ester (l-NAME). HBO-mediated mobilization of EPC is associated with increased lower limb spontaneous circulatory recovery after femoral ligation and enhanced closure of ischemic wounds, and these effects on limb perfusion and wound healing are also inhibited by l-NAME pretreatment. These data show that EPC mobilization into circulation is triggered by hyperoxia through induction of bone marrow NO with resulting enhancement in ischemic limb perfusion and wound healing.

Predictive computer simulations of tumor drug response demonstrate that 3-D hypoxic gradients significantly increase drug resistance

2071 Background: We created a three-dimensional physiologically based computer (in-silico) model of cancer based on a description of biological events at the cellular scale with input variables determined from patient specific information, such as in-vitro drug response experiments and in-vivo tumor imaging, with the long term goal of individualized treatment selection. The central hypothesis is that such a model that incorporates basic tumor growth kinetics information is capable of representing and predicting tumor response to chemotherapy. Methods: We measured in-vitro tumor growth and drug response for Doxorubicin sensitive and resistant MCF-7 breast cancer cells through trypan blue exclusion counts, tridiated thymidine incorporation, and the XTT assay. We used these results of parameter-based statistics to define input variables to our in-silico model of cancer, and ran computer simulations to measure the drug response predicted by the model. Results: The computer model could accurately predict the in-vitro response of drug sensitive and resistant MCF-7 breast cancer cells. The model also predicted that gradients of oxygen and nutrient in a tumor microenvironment, whether naturally occurring or induced by treatment, and which in previous work we found could increase the invasive capability of tumor cells and destabilize tumor morphology, could also contribute to acquired drug resistance by increasing the population of quiescent cells. Conclusions: We demonstrated that a rigorously, experimentally calibrated computer model of cancer is accurately predictive of in-vitro tumor response to chemotherapeutic drugs, and established that this model offers a means to quantitatively study tumor drug response. We did this through a grounds-up physical representation of tumor biology, not by fitting to experimental data. This validation begins the path to computational modeling and more efficient prediction of in-vivo tumor response to chemotherapy. No significant financial relationships to disclose.

The Oxygen Sensor Factor-Inhibiting Hypoxia-Inducible Factor-1 Controls Expression of Distinct Genes through the Bifunctional Transcriptional Character of Hypoxia-Inducible Factor-1α

The function of the hypoxia-inducible factor-1 (HIF-1), the key transcription factor involved in cellular adaptation to hypoxia, is restricted to low oxygen tension (pO(2)). As such, this transcription factor is central in modulating the tumor microenvironment, sensing nutrient availability, and controlling anaerobic glycolysis, intracellular pH, and cell survival. Degradation and inhibition of the limiting HIF-1alpha subunit are intimately connected in normoxia. Hydroxylation of two proline residues by prolyl hydroxylase domain (PHD) 2 protein earmarks the protein for degradation, whereas hydroxylation of an asparagine residue by factor-inhibiting HIF-1 (FIH-1 or FIH) reduces its transcriptional activity. Indeed, silencing of either PHD2 or FIH in normoxia partially induced hypoxic genes, whereas combined PHD2/FIH silencing generated a full hypoxic gene response. Given the fact that HIF-1alpha possesses two transcriptional activation domains [TAD; NH(2)-terminal (N-TAD) and COOH-terminal (C-TAD)], we hypothesized on a possible bifunctional activity of HIF-1alpha that could be discriminated by FIH, an inhibitor of the C-TAD. In human cell lines engineered to overexpress or silence FIH in response to tetracycline, we show by quantitative reverse transcription-PCR that a set of hypoxic genes (ca9, phd3, pgk1, and bnip3) respond differently toward FIH expression. This finding, extended to 26 hypoxia-induced genes, indicates differential gene expression by the N-TAD and C-TAD in response to the hypoxic gradient. We propose that the oxygen-sensitive attenuator FIH, together with two distinct TADs, is central in setting the gene expression repertoire dictated by the cell pO(2).

Spatial Heterogeneity and Oxygen Dependence of Glucose Consumption in R3230Ac and Fibrosarcomas of the Fischer 344 Rat

To examine the oxygen-dependence of glucose consumption in solid tumors, we monitored gradients of glucose, lactate, and hypoxia in R3230Ac and FSA tumors growing in Fischer 344 rats. Bioluminescence imaging, detection of Hoechst 33342, and immunostaining of the hypoxia marker EF5 [2-8-N-(2,2,3,3,3-pentafluoropropyl)acetamide] were done in serial tumor slices. Glucose and lactate levels were also determined in liver and blood. Cells were further tested for glucose consumption and lactate production in vitro. In both tumor types, EF5 staining indicated similar maximum levels of hypoxia; the most intense staining occurred in perinecrotic regions. Glucose concentrations were highest in liver, declined from blood to tumor edge, further into vital tumor regions, and were lowest close to necrosis. Glucose was significantly lower in FSA than in R3230Ac tumors. Glucose concentrations in R3230Ac tumors were consistently higher in nonhypoxic than in hypoxic areas, with maximum values equal to systemic blood levels. Glucose in FSA tumors was close to zero, regardless of the presence or absence of hypoxia. Lactate did not differ significantly between the tumor types. FSA cells in culture showed a trend towards higher aerobic glucose consumption versus R3230Ac. Both cell lines increased their lactate production to similar levels under hypoxia. We conclude that both R3230Ac and FSA tumors retain the Pasteur effect, i.e., hypoxia triggers increased glycolysis. However, our results imply that increased aerobic glucose utilization leads to low glucose levels in FSA and a situation where supply limits uptake. This explains the repeatedly observed correlation between tumor blood flow and 18F-deoxyglucose uptake.

Transient Changes in Oxygen Tension Inhibit Osteogenic Differentiation and Runx2 Expression in Osteoblasts

Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O(2) (ambient oxygen), 2% O(2) (hypoxia), and <0.02% O(2) (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts.

Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1

The trafficking of circulating stem and progenitor cells to areas of tissue damage is poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1 or CXCL12) mediates homing of stem cells to bone marrow by binding to CXCR4 on circulating cells. SDF-1 and CXCR4 are expressed in complementary patterns during embryonic organogenesis and guide primordial stem cells to sites of rapid vascular expansion. However, the regulation of SDF-1 and its physiological role in peripheral tissue repair remain incompletely understood. Here we show that SDF-1 gene expression is regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1) in endothelial cells, resulting in selective in vivo expression of SDF-1 in ischemic tissue in direct proportion to reduced oxygen tension. HIF-1-induced SDF-1 expression increases the adhesion, migration and homing of circulating CXCR4-positive progenitor cells to ischemic tissue. Blockade of SDF-1 in ischemic tissue or CXCR4 on circulating cells prevents progenitor cell recruitment to sites of injury. Discrete regions of hypoxia in the bone marrow compartment also show increased SDF-1 expression and progenitor cell tropism. These data show that the recruitment of CXCR4-positive progenitor cells to regenerating tissues is mediated by hypoxic gradients via HIF-1-induced expression of SDF-1.

Improved Immunohistochemical Method for Detecting Hypoxia Gradients in Mouse Tissues and Tumors

We describe an improved immunohistochemical procedure for detecting regions of hypoxia in normal organs and tumors in mice. The method employs a primary fluorescein-conjugated mouse monoclonal antibody directed against pimonidazole protein adducts that are created in hypoxic tissues and a secondary mouse anti-fluorescein antibody that is conjugated to horseradish peroxidase. Using these reagents, we clearly visualized the regions of relative hypoxia in implanted tumors in mice as well as in normal organs such as liver and kidney. Significantly, the resulting tissue sections were remarkably free of the background staining that is characteristically observed when rodent antibodies are used to detect antigens in rodent tissues.

Changes in oxygen tension modulate osteoblast gene expression and long-term differentiation

Introduction: Disruption of blood flow with bony and soft-tissue injury results in a hypoxic gradient within the wound microenvironment. As a first step to understanding osteoblast behavior following hypoxic insult, we perform large-scale transcriptional profiling of hypoxic and anoxic osteoblasts. In addition, we investigate long-term osteoblast differentiation after brief hypoxic exposure. Methods: RNA from murine calvarial osteoblasts cultured in 21%, 2%, or 0% O2 for up to 24 hours was used for microarray analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Osteoblasts exposed to 24 hours of hypoxia were differentiated for 28 days and compared to non-hypoxic cultures via qRT-PCR and bone-nodule staining by the Von Kossa method. Results: Osteoblast exposure to 0% or 2% O2 for 24 hours resulted in statistically significant expression changes for approximately 700 genes in each group. Of these, only 89 genes were shared by both groups. We observed differential regulation of osteogenic and angiogenic markers, transcription and growth factors, and inflammatory signaling modulators. As expected, both conditions induced anaerobic glycolysis, but only anoxia elevated heat-shock response elements. Surprisingly, osteoblasts exposed to 24 hours of anoxia demonstrated diminished bone nodule formation at 28 days. Conclusions: Gene expression changes in osteoblasts within the hypoxic post-injury microenvironment remain largely unknown. Transcriptional profiling reveals distinct patterns of expression at 0% versus 2% O2 relative to ambient oxygen. Furthermore, in vitro osteoblast differentiation is diminished even after brief anoxic insult. Together with induction of angiogenic and inflammatory genes, this underscores the involvement of additional cell types for successful bone repair.

Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients.

Current theories of CF pathogenesis predict different predisposing "local environmental" conditions and sites of bacterial infection within CF airways. Here we show that, in CF patients with established lung disease, Pseudomonas aeruginosa was located within hypoxic mucopurulent masses in airway lumens. In vitro studies revealed that CF-specific increases in epithelial O(2) consumption, linked to increased airway surface liquid (ASL) volume absorption and mucus stasis, generated steep hypoxic gradients within thickened mucus on CF epithelial surfaces prior to infection. Motile P. aeruginosa deposited on CF airway surfaces penetrated into hypoxic mucus zones and responded to this environment with increased alginate production. With P. aeruginosa growth in oxygen restricted environments, local hypoxia was exacerbated and frank anaerobiosis, as detected in vivo, resulted. These studies indicate that novel therapies for CF include removal of hypoxic mucus plaques and antibiotics effective against P. aeruginosa adapted to anaerobic environments.

Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients

Current theories of CF pathogenesis predict different predisposing “local environmental” conditions and sites of bacterial infection within CF airways. Here we show that, in CF patients with established lung disease, Psuedomonas aeruginosa was located within hypoxic mucopurulent masses in airway lumens. In vitro studies revealed that CF-specific increases in epithelial O2 consumption, linked to increased airway surface liquid (ASL) volume absorption and mucus stasis, generated steep hypoxic gradients within thickened mucus on CF epithelial surfaces prior to infection. Motile P. aeruginosa deposited on CF airway surfaces penetrated into hypoxic mucus zones and responded to this environment with increased alginate production. With P. aeruginosa growth in oxygen restricted environments, local hypoxia was exacerbated and frank anaerobiosis, as detected in vivo, resulted. These studies indicate that novel therapies for CF include removal of hypoxic mucus plaques and antibiotics effective against P. aeruginosa adapted to anaerobic environments.

Response of Benthic Fauna and Changing Sediment Redox Profiles over a Hypoxic Gradient

The Koljöfjord is an enclosed, stratified fjord on the Swedish west coast with hypoxic/anoxic bottom water during most of the year. In the winter 1999/2000, the water in the entire fjord was re-oxygenated after a period of stagnation, but the following summer oxygen concentrations declined to below 1ml l−1between 20 and 40m depths. The objectives of this study were to investigate the structure of benthic communities along a depth gradient of declining oxygen concentrations and the impact of fauna on sediment redox conditions. The vertical distribution of the fauna in the sediment was restricted to the upper few centimetres. Dominant species at most stations were the burrower Capitella capitata and the tube-builder Pseudopolydora antennata. The species found in the fjord are probably not particularly tolerant towards hypoxia, but they have life-history traits that facilitate a rapid colonization following improved oxygen conditions. The depth of the Redox potential discontinuity (RPD) layer, a recognizable division zone between oxidized (sub-oxic) and reduced chemical conditions, is dependent on infaunal activity, e.g. burrows, tubes and feeding voids. Measurement of apparent RPD (aRPD) from sediment profile images (SPIs) compared well to electrode measurement of RPD. We conclude that a digital analysis of aRPD from images has many advantages compared to RPD measurements by electrodes.

Hypoxia Regulates Osteoblast Gene Expression

Vascular disruption secondary to fracture creates a hypoxic gradient of injury wherein the oxygen tension at the center of the wound is very low. In vivo this hypoxic microenvironment stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and osteoblasts. In order to begin to dissect this complex system, we have examined the effects of hypoxia on isolated osteoblast gene expression in vitro. Understanding gene expression in this system may facilitate the development of targeted therapeutic modalities designed to accelerate fracture repair and reduce complications. Using an established model of in vitro hypoxia, we have analyzed the expression of genes involved in bone matrix production and turnover. Subconfluent neonatal rat calvarial osteoblasts were exposed to hypoxia (pO2 = 35–40 mm Hg) and total cellular RNA was collected at 0, 3, 6, 24, and 48 h. Northern analysis was used to analyze the expression patterns of (1) transforming growth factors (TGFs)-β1, -β2, and -β3 and their type I receptor; (2) collagens I and III; and (3) tissue inhibitor of metalloproteinase-1. We have demonstrated a marked elevation of TGF-β1 gene expression within 3 h of hypoxia. Although neither TGF-β2 nor TGF-β3 expression was affected by hypoxia, the TGF-β type I receptor was substantially upregulated within 6 h. In addition, extracellular matrix scaffolding molecules (collagens I and III) were markedly, but differentially, upregulated. Finally, we have demonstrated that the expression of an inhibitor of extracellular matrix turnover, the tissue inhibitor of metalloproteinase-1, was strikingly decreased in response to hypoxia. These results imply that hypoxia can affect osseous healing by altering the expression of cytokines, bone-specific extracellular matrix molecules, and their regulators.

Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro.

Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.

Vascular endothelial growth factor expression coincides with coronary vasculogenesis and angiogenesis.

Vascular endothelial growth factor (VEGF) plays an important role in early embryonic vasculogenesis. To establish its temporal expression and localization in the heart during development, we studied rat hearts from the first embryonic day (E) of myocardial vascular tube formation through the early postnatal period. Ventricular VEGF immunoreactivity was noted in the epicardium and the thin underlying myocardium in E10 ventricles. During the earliest stages of vascularization (E13-E16) immunoreactivity was highest in the compact myocardium nearest the epicardium, and subsequently (E18 and thereafter) became more evenly distributed transmurally. By birth (E22) immunoreactivity was most intense around microvessels. Similarly, VEGF mRNA localization, demonstrated by in situ hybridization, was initially highest near the epicardium and then became more evenly distributed transmurally by late gestation. Within the interventricular septum, the highest expression occurred in the middle of the wall where it correlated with the greatest vascularization. Northern blot analysis showed that from E12 through the first 10 days of postnatal life, VEGF was two to three times higher than in the adult. Western blot analysis showed that VEGF tended to be higher in the atria than the ventricles, and negligible in the outflow tract. Our data indicate that VEGF localization and expression 1) correspond to the pattern of vascularization in the embryonic/fetal heart, and 2) remain high during the early postnatal period when capillary proliferation is high. Because VEGF is stimulated by hypoxia, its preferential mRNA expression near the epicardium, that is, farthest from the ventricular lumen and the O2 source, fits with the hypothesis that a hypoxic gradient is a driving force in the transmural vascularization process.

Sectional analysis of tumor colony growth in the soft-agar assay: effects of oxygen.

Soft-agar clonogenicity of L1210 mouse leukemia cells and of xenografts of a human melanoma and a carcinoma of the cervix was studied sectionally by the sizes of the colonies grown under hypoxic gradients and aerobic condition. Soft-agar plating efficiency was increased in cultured L1210 cells with decreasing oxygen concentrations. The growth of both cultured L1210 cells and their BDF1 ascites was better in 5% oxygen than in 20% oxygen. Although soft-agar colony development of both melanoma and cervical carcinoma was significantly better in 5% oxygen, the former has a secondary preference for a hypoxic atmosphere and the latter, for an aerobic condition.