Pimonidazole Adducts Research Articles

Persistence and expansion of hypoxia detected by pimonidazole adduct immunostaining during progression of diabetic nephropathy in diabetic mice.

Hypoxia and oxidative stress are considered to be underlying factors in the deterioration of renal function and pathogenesis in acute kidney injury (AKI) and chronic kidney disease, including diabetic nephropathy (DN). However, the long-term role of hypoxia in DN is unknown. Here, we investigated the distribution, severity, and time course of hypoxia during DN development in our well-established severely diabetic transgenic (Tg) DN mouse model that mimics human DN up to 80 wk of age, using pimonidazole adduct immunohistochemistry. The relationship between pimonidazole adduct distribution and hypoxia-inducible factor (HIF) expression was also examined. We found 1) persistent pimonidazole immunostaining mainly in the outer zone of the outer medulla, extending into the inner zone, 2) significant expansion of area and intensity up to 40 wk of age, and 3) characteristic subcellular localization mainly at apical sites in vesicular form by laser scanning microscopy of thin slices. The distribution of pimonidazole adducts was different from that of HIF reported previously, indicating that hypoxia does not directly contribute to persistent abnormal HIF expression. These results suggest that pimonidazole adducts produced under low [Formula: see text] conditions are sustained by a mechanism distinct from direct ischemia. We propose that in the long course of DN development, persistent hyperfiltration and hyperexcretion of glucose, albumin, and water increase metabolism and energy expenditure in the tubules, and such chronic stimulation leads to relative ischemia and local hypoxia, which may contribute in part to the loss of nephrons.NEW & NOTEWORTHY This study provides new insights into hypoxia during the long course of diabetic nephropathy development. Hypoxia was persistently localized only in limited areas and its distribution differed significantly from that of hypoxia-inducible factors. These findings suggests that in the long course of diabetic nephropathy development, increased energy requirements and limited blood supply may lead to relative ischemia and induction of local and persistent hypoxia, which may contribute in part to the loss of nephrons.

Pancreatic stellate cells promote pancreatic β-cell death through exosomal microRNA transfer in hypoxia

Hypoxia in pancreatic islets (islet hypoxia) can occur in type 2 diabetes mellitus. Previously, our in vitro experiments demonstrated that pancreatic stellate cells (PSCs) within the islet are activated in hypoxia, promoting pancreatic β-cell death. Here, we aimed to demonstrate the in vivo activation of intra-islet PSCs and investigate the mechanism of PSC-induced β-cell death in hypoxia. A novel in vivo model of islet hypoxia was established by injecting fluorescent microspheres into a carotid artery of Balb/c mice (Microsphere mice). The intraperitoneal glucose tolerance (IPGTT) was performed, and pancreatic tissues were stained for insulin expression after tissue clearing. Pimonidazole staining was also performed in the pancreas to detect the presence of hypoxia in islets. Next, primary PSCs were isolated and cultured from Balb/c mice. Exosomes were isolated from culture media from PSCs cultured in hypoxia (1% oxygen). MicroRNAs (miRNAs) were prepared from exosomes from PSCs, and miRNA expression profiles were analyzed by miRNA sequencing. Several miRNAs were overexpressed in islets using miRNA mimics. Two weeks after injection of microspheres, the Microsphere mice showed worsening of glucose tolerance in IPGTT. Later, cataracts were developed in the eyes of the mice. The pancreas showed that the areas, perimeters, and diameters of insulin-positive cells decreased in Microsphere mice. Pimonidazole adducts were detected in the islets of these mice, indicating the presence of islet hypoxia. In addition, α-smooth muscle actin-positive cell numbers per islet were higher in Microsphere mice, confirming the in vivo activation of intra-islet PSCs in hypoxia. Mouse islets incubated with exosomes isolated from PSCs cultured in hypoxia showed a decrease in cell viability. The exosomes contained a variety of miRNAs, of which miR-23a-3p was found to notably increase β-cell death through apoptosis. Together, our in vivo and in vitro data provide evidence to support that PSCs within the islets are activated in hypoxia and promote β-cell death through exosomal miRNA transfer, which may contribute to the progression of type 2 diabetes mellitus.

Phthalimide Analogs Enhance Genotoxicity of Cyclophosphamide and Inhibit Its Associated Hypoxia.

Cyclophosphamide (CP) is a mutagen that is used in cancer chemotherapy, due to its genotoxicity and as an immunosuppressive agent. Thalidomide (TH) is another cancer chemotherapeutic drug. In this study, the cytogenotoxicity and hypoxia modulatory activities of two phthalimide analogs of TH have been evaluated with/without CP. Both analogs have increased CP-stimulated chromosomal aberrations than those induced by TH, including gaps, breaks/fragments, deletions, multiple aberrations, and tetraploidy. The analogs have elevated the cytotoxic effect of CP by inhibiting the mitotic activity, in which analog 2 showed higher mitosis inhibition. CP has induced binucleated and polynucleated bone marrow cells (BMCs), while micronuclei (MN) are absent. TH and analogs have elevated the CP-stimulated binucleated BMCs, while only analogs have increased the CP-induced polynucleated BMCs and inhibited the mononucleated BMCs. MN-BMCs were shown together with mononucleated, binucleated, and polynucleated cells in the CP group. Both analogs have elevated mononucleated and polynucleated MN-BMCs, whereas in presence of CP, TH and analogs have enhanced mononucleated and binucleated MN-BMCs. The analogs significantly induce DNA fragmentation in a comet assay, where analog 1 is the strongest inducer. The treatment of mice with CP has resulted in a high hypoxia status as indicated by high pimonidazole adducts and high HIF-1α and HIF-2α concentrations in lymphocytes. Analogs/CP-treated mice showed low pimonidazole adducts. Both analogs have inhibited HIF-1α concentration but not HIF-2α. Taken together, the study findings suggest that both analogs have a higher potential to induce CP-genotoxicity than TH and that both analogs inhibit CP-hypoxia via the HIF-1α-dependent mechanism, in which analog 1 is a more potent anti-hypoxic agent than analog 2. Analog 1 is suggested as an adjacent CP-complementary agent to induce CP-genotoxicity and to inhibit CP-associated hypoxia.

Decreased Choroidal Blood Perfusion Induces Myopia in Guinea Pigs.

PurposeThe development of myopia in guinea pigs can be inhibited by attenuating scleral hypoxia by increasing choroidal blood perfusion (ChBP). In this study, we reduced ChBP through surgical and pharmacological methods to determine the effect on myopia development. We also determined whether ChBP was reduced by quinpirole, a drug that enhances form-deprivation myopia (FDM).MethodsChBP was reduced in the right eyes of guinea pigs via transection of the temporal ciliary arteries or daily injections of phenylephrine into the inferior peribulbar space for one week during normal ocular growth. Other guinea pigs were subjected to two weeks of monocular FDM—with facemasks, along with daily injections of quinpirole, a dopamine D2 receptor agonist, to enhance the FDM. Changes in refraction, axial length, ChBP, and choroidal thickness (ChT) were measured in both treated and fellow eyes of the treatment and control groups. Scleral hypoxia labeling with pimonidazole adducts and α-smooth muscle actin (α-SMA) protein were also measured.ResultsSurgical and pharmacological reduction of ChBP induced myopia development in the treated eyes. These treatments rendered the scleral hypoxia and increased scleral α-SMA expression. Furthermore, quinpirole injections, which increased the magnitude of myopia, augmented the FDM-associated reductions in ChBP and ChT and increased the levels of scleral hypoxia and α-SMA protein.ConclusionsDecreased ChBP in guinea pigs leads to scleral hypoxia and scleral myofibroblast transdifferentiation with increased α-SMA expression, ultimately resulting in myopia development. In future clinical trials, ChBP reduction can serve as a potential biomarker for early detection of myopia development.

Retinal hypoxia and angiogenesis with methamphetamine

Central retinal artery occlusion, retinopathy, and retinal neovascularization have been reported in methamphetamine (METH) abusers. In the current study, we investigated whether METH induces retinal neovascularization in a mouse model, and if so, whether the neovascularization is associated with increased hypoxia, hypoxia-inducible factor 1α (HIF-1α), and vascular endothelial growth factor (VEGF). Mice were administrated METH by intraperitoneal injection over a 26-day period, or injected with saline as a vehicle control. The number of retinal arterioles and venules were counted using in vivo live imaging following infusion with fluorescein isothiocyanate-dextran. Excised retinas were stained with griffonia simplicifolia lectin I and flat mounted for a measurement of vascularity (length of vessels per tissue area) with AngioTool. Retinal hypoxia was examined by formation of pimonidazole adducts with an anti-pimonidazole antibody, and HIF-1α and VEGFa protein levels in the retina were detected by immunoblot. METH administration increased vascularity (including the number of arterioles) measured on Day 26. Retinal VEGFa protein level was not changed in METH-treated mice on Day 5, but was increased on Day 12 and Day 26. Hypoxia (pimonidazole adduct formation) was increased in retinas of METH-treated mice on Day 12 and Day 26, as were HIF-1α protein expression levels. These results indicate that METH administration induces hypoxia, HIF-1α, VEGFa, and angiogenesis in the retina.

Increased Choroidal Blood Perfusion Can Inhibit Form Deprivation Myopia in Guinea Pigs.

PurposeIn guinea pigs, choroidal thickness (ChT) and choroidal blood perfusion (ChBP) simultaneously decrease in experimental myopia, and both increase during recovery. However, the causal relationship between ChBP and myopia requires further investigation. In this study, we examined the changes of ChBP with three different antimyopia treatments. We also actively increased ChBP to examine the direct effect on myopia development in guinea pigs.MethodsExperiment 1: Guinea pigs wore occluders on the right eye for two weeks to induce form-deprivation myopia (FDM). Simultaneously they received daily antimyopia treatments: peribulbar injections of atropine or apomorphine or exposure to intense light. Experiment 2: The vasodilator prazosin was injected daily into the form-deprivation eyes to increase ChBP during the two-week induction of FDM. Other FDM animals received appropriate control treatments. Changes in refraction, axial length, ChBP, ChT, and hypoxia-labeled pimonidazole adducts in the sclera were measured.ResultsThe antimyopia treatments atropine, apomorphine, and intense light all significantly inhibited myopia development and the decrease in ChBP. The treatments also reduced scleral hypoxia, as indicated by the decrease in hypoxic signals. Furthermore, actively increasing ChBP with prazosin inhibited the progression of myopia, as well as the increase in axial length and scleral hypoxia.ConclusionsOur data strongly indicate that increased ChBP attenuates scleral hypoxia, and thereby inhibits the development of myopia. Thus ChBP may be a promising target for myopia retardation. As such, it can serve as an immediate predictor of myopia development as well as a long-term marker of it.

Inhibition of Oxidative Phosphorylation Reverses Bone Marrow Hypoxia Visualized in Imageable Syngeneic B-ALL Mouse Model.

Abnormally low level of interstitial oxygen, or hypoxia, is a hallmark of tumor microenvironment and a known promoter of cancer chemoresistance. Inside a solid tumor mass, the hypoxia stems largely from inadequate supply of oxygenated blood through sparse or misshapen tumor vasculature whilst oxygen utilization rates are low in typical tumor's glycolytic metabolism. In acute leukemias, however, markers of intracellular hypoxia such as increased pimonidazole adduct staining and HIF-1α stabilization are observed in advanced leukemic bone marrows (BM) despite an increase in BM vasculogenesis. We utilized intravital fast scanning two-photon phosphorescence lifetime imaging microscopy (FaST-PLIM) in a BCR-ABL B-ALL mouse model to image the extracellular oxygen concentrations (pO2) in leukemic BM, and we related the extracellular oxygen levels to intracellular hypoxia, vascular markers and local leukemia burden. We observed a transient increase in BM pO2 in initial disease stages with intermediate leukemia BM burden, which correlated with an expansion of blood-carrying vascular network in the BM. Yet, we also observed increased formation of intracellular pimonidazole adducts in leukemic BM at the same time. This intermediate stage was followed by a significant decrease of extracellular pO2 and further increase of intracellular hypoxia as leukemia cellularity overwhelmed BM in disease end-stage. Remarkably, treatment of leukemic mice with IACS-010759, a pharmacological inhibitor of mitochondrial Complex I, substantially increased pO2 in the BM with advanced B-ALL, and it alleviated intracellular hypoxia reported by pimonidazole staining. High rates of oxygen consumption by B-ALL cells were confirmed by Seahorse assay including in ex vivo cells. Our results suggest that B-ALL expansion in BM is associated with intense oxidative phosphorylation (OxPhos) leading to the onset of metabolic BM hypoxia despite increased BM vascularization. Targeting mitochondrial respiration may be a novel approach to counteract BM hypoxia in B-ALL and, possibly, tumor hypoxia in other OxPhos-reliant malignancies.

Methamphetamine‐induced retinal hyperpermeability, hypoxia, and vascularization

PurposeMethamphetamine (METH), a highly addictive stimulant of neurotransmitters induces central retinal artery occlusion with retinal atrophy and neovascularization. We previously have identified METH‐induced loss of endothelial surface molecules including platelet and endothelial cell adhesion molecule‐1 (PECAM‐1) and glycocalyx in mice. However, the pathophysiologic pathway of METH‐induced retinal vascular dysfunction has been insufficiently investigated. The purpose of this study is to investigate the METH‐induced alteration of the retinal vascular circulation.Materials and methodsC57BL/6J male and female mice were administrated progressively increasing doses of METH (0 to 6 mg/kg) by repetitive intraperitoneal injections (4 times per day) for 4 weeks. Norepinephrine levels in plasma were measured by ELISA. Cross‐sections of the retina were stained with hematoxylin and eosin. Levels of hypoxia in the retina were determined by immunostaining of pimonidazole adducts with Hypoxyprobe. Retinal blood vessels were visualized with fluorescent plasma tracers injected via femoral vein injection for live imaging, and immunostained with griffonia simplicifolia lectin 1 (GSL‐1) for retinal flatmounts. Expression of various proteins was determined by immunoblot.ResultsThe level of pimonidazole adduct was increased in METH‐treated retinas, compared with saline‐treated retinas, indicating METH‐induced retinal hypoxia. Plasma norepinephrine levels, which can promote hypoxia‐inducible factor 1a (HIF‐1a) at the transcriptional level, were elevated by METH, compared with saline treatment at 1 week (2.2±0.9 vs 7.8±1.6 ng/mL, P<0.01), 2 weeks (0.8±0.3 vs 5.5±1.8 ng/mL, P<0.05), and 4 weeks (0.9±0.2 vs 2.0±0.4 ng/mL, P<0.05). Retinal protein expression levels of HIF‐1a and angiogenic proteins, such as vascular endothelial growth factor a (VEGFa), erythroblast transformation‐specific (ETS)‐related gene (ERG), and Protein C‐ets‐1 (ETS‐1) were increased in the METH‐treated retinas. Counts of retinal arterioles and capillaries were higher in METH‐treated mice than in saline‐treated mice. Moreover, protein concentration in the vitreous humor and immunoreactivity of immunoglobulin in retina, which are measures of retinal permeability, were increased by METH treatment.ConclusionOur present data suggest that METH is associated with retinal neovascularization and hyperpermeability with increases in hypoxia, plasma norepinephrine, and HIF‐1a activation.Support or Funding InformationThis work was supported by funding from the National Institute of Health (NIH) EY025632.

Decreased β-Cell Proliferation and Vascular Density in a Subpopulation of Low-Oxygenated Male Rat Islets.

Low-oxygenated and dormant islets with a capacity to become activated when needed may play a crucial role in the complex machinery behind glucose homeostasis. We hypothesized that low-oxygenated islets, when not functionally challenged, do not rapidly cycle between activation and inactivation but are a stable population that remain low-oxygenated. As this was confirmed, we aimed to characterize these islets with regard to cell composition, vascular density, and endocrine cell proliferation. The 2-nitroimidazole low-oxygenation marker pimonidazole was administered as a single or repeated dose to Wistar Furth rats. The stability of oxygen status of islets was evaluated by immunohistochemistry as the number of islets with incorporated pimonidazole adducts after one or repeated pimonidazole injections. Adjacent sections were evaluated for islet cell composition, vascular density, and endocrine cell proliferation. Single and repeated pimonidazole injections over an 8-hour period yielded accumulation of pimonidazole adducts in the same islets. An average of 30% of all islets was in all cases positively stained for pimonidazole adducts. These islets showed a similar endocrine cell composition as other islets but had lower vascular density and β-cell proliferation. In conclusion, low-oxygenated islets were found to be a stable subpopulation of islets for at least 8 hours. Although they have previously been observed to be less functionally active, their islet cell composition was similar to that of other islets. Consistent with their lower oxygenation, they had fewer blood vessels than other islets. Notably, β-cell regeneration preferentially occurred in better-oxygenated islets.

Detection of cellular hypoxia by pimonidazole adduct immunohistochemistry in kidney disease: methodological pitfalls and their solution.

Pimonidazole adduct immunohistochemistry is one of the few available methods for assessing renal tissue hypoxia at the cellular level. It appears to be prone to artifactual false positive staining under some circumstances. Here, we assessed the nature of this false positive staining and, having determined how to avoid it, reexamined the nature of cellular hypoxia in rat models of kidney disease. When a mouse-derived anti-pimonidazole primary antibody was used, two types of staining were observed. First, there was diffuse staining of the cytoplasm of tubular epithelial cells, which was largely absent when the primary antibody was omitted from the incubation protocol or in tissues known not to contain pimonidazole adducts. Second, there was staining of the apical membranes of tubular epithelial cells, debris within the lumen of renal tubules, including tubular casts, and the interstitium; this latter staining was present even when the primary antibody was omitted from the incubation protocol. Such false positive staining was particularly prominent in acutely injured kidneys. It could not be avoided by preincubation of sections with a mouse IgG blocking reagent. Furthermore, preadsorption of the secondary antibody against rat Ig abolished all staining; however, when a rabbit-derived polyclonal anti-pimonidazole primary antibody was used, the false positive staining was largely avoided. Using this method, we confirmed the presence of hypoxia, localized mainly to the tubular epithelium, in the acute phase of severe renal ischemia-reperfusion injury, adenine-induced chronic kidney disease, and polycystic kidney disease. We conclude that this new method provides improved detection of renal cellular hypoxia.

Reoxygenation and Repopulation of Tumor Cells after Ablative Hypofractionated Radiotherapy (SBRT and SRS) in Murine Tumors.

In this work, we investigated the change in tumor microenvironment caused by semi-ablative high-dose irradiation and its implication on tumor cell survival, reoxygenation of hypoxic cells and repopulation in FSaII tumors grown subcutaneously in the hind legs of C3H mice. Tumors were exposed to 10-30 Gy of X-ray radiation in a single exposure, and the vascularity and blood perfusion were assessed based on the levels of CD31 expression and Hoechst 33342 perfusion, respectively. The tumor hypoxia was assessed by staining for pimonidazole adduct formation and the expression of hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase 9 (CA9). Tumor cell survival was determined using in vivo-in vitro excision assay method. The proportion of hypoxic cells in the tumor was determined from the surviving cell fraction in tumors exposed to a test dose under aerobic and hypoxic conditions. Radiation expsoure markedly reduced the functional vascularity and blood perfusion, and profoundly increased the expression of HIF-1α and CA9 pointing to an increase in tumor hypoxia. The overall clonogenic cell survival progressively decreased during 2-5 days postirradiation, most likely due to the radiation-induced vascular dysfunction. In turn, the proportion of surviving hypoxic cells decreased over several days postirradiation, presumably due to reoxygenation of hypoxic cells. The oxygen supplied through small fractions of blood vessels that survived the high-dose exposure, together with a reduction of oxygen consumption due to massive cell death, appeared to be the cause of the reoxygenation of hypoxic cells. The surviving tumor cells then subsequently repopulated. The findings from this study using a murine tumor model suggest that the efficacy of stereotactic body radiotherapy (SBRT) and stereotactic radiosurgery (SRS) may be significantly improved by allowing an inter-fraction time for reoxygenation while avoiding repopulation.

Absence of renal hypoxia in the subacute phase of severe renal ischemia-reperfusion injury.

Tissue hypoxia has been proposed as an important event in renal ischemia-reperfusion injury (IRI), particularly during the period of ischemia and in the immediate hours following reperfusion. However, little is known about renal oxygenation during the subacute phase of IRI. We employed four different methods to assess the temporal and spatial changes in tissue oxygenation during the subacute phase (24 h and 5 days after reperfusion) of a severe form of renal IRI in rats. We hypothesized that the kidney is hypoxic 24 h and 5 days after an hour of bilateral renal ischemia, driven by a disturbed balance between renal oxygen delivery (Do2) and oxygen consumption (V̇o2). Renal Do2 was not significantly reduced in the subacute phase of IRI. In contrast, renal V̇o2 was 55% less 24 h after reperfusion and 49% less 5 days after reperfusion than after sham ischemia. Inner medullary tissue Po2, measured by radiotelemetry, was 25 ± 12% (mean ± SE) greater 24 h after ischemia than after sham ischemia. By 5 days after reperfusion, tissue Po2 was similar to that in rats subjected to sham ischemia. Tissue Po2 measured by Clark electrode was consistently greater 24 h, but not 5 days, after ischemia than after sham ischemia. Cellular hypoxia, assessed by pimonidazole adduct immunohistochemistry, was largely absent at both time points, and tissue levels of hypoxia-inducible factors were downregulated following renal ischemia. Thus, in this model of severe IRI, tissue hypoxia does not appear to be an obligatory event during the subacute phase, likely because of the markedly reduced oxygen consumption.

Improved Beta-Cell Differentiation and Function in Isolated Islets Established in Dynamic Culture to Reduce Hypoxia

Background: Pancreatic islet transplantation effectively prevents severe hypoglycaemia in type 1 diabetes. Restoration of normal β-cell functional mass remains elusive and we have recently demonstrated loss of β-cell end-differentiation in clinical islet recipients. We hypothesised that reducing islet hypoxia in culture would restore β-cell differentiation and function. Methods: MIN6 pseudo-islets (PI) and primary human islets (HI) were cultured in either static (SC) or horizontal rotation (RC) culture for 7 days. Hypoxia was detected by pimonidazole (Pim) adduct formation and hypoxia-induced gene expression (qPCR). β-cell end-differentiation was assessed by UCN3 expression and IF staining (urocortin-3+ insulin+ cells/total insulin+ cells) with function assayed by glucose-stimulated insulin secretion (GSIS). Results: Hypoxia was confirmed in established PI (70±1.0% Pim+). This was reduced in rotation culture (Pim+: 15±1.5% RC vs. 80±2.5% SC; p<0.05) with reduced hypoxia-induced gene expression compared with adherent MIN6 (LDHA: 6.0±2.1 RC vs. 86.0±7.8 fold SC; MCT4: RC 2.9±0.4 vs. 54.6±0.6 fold SC; p<0.05). RC enhanced UCN3 gene expression (4.2±0.5 RC vs. 2.2±0.2 fold SC; p<0.05) and increased UCN3/insulin co-staining (80±1.2% RC vs. 30±2.4% SC; p<0.0001) with enhanced GSIS stimulation index (4.1±0.1 RC vs. 2.8±0.1 SC; p<0.0001). Hypoxia-induced expression was reduced in HI RC compared with freshly isolated islets (LDHA: 0.3±0.1 RC vs. 1.8±0.04 fold SC; MCT4: 0.08±0.01 RC vs. 1.1±0.2 fold SC; p<0.05) with increased UCN3 expression (5.9±1.6 RC vs. 3.3±0.9 fold SC; p<0.05) and enhanced GSIS stimulation index (4.1±0.9 RC vs. 3.0±1.2 SC; p<0.05). Conclusion: Hypoxia in established PI and isolated HI was reduced over 7 days in dynamic culture. This was associated with enhanced β-cell differentiation and function providing a potential approach to maximise optimal functional mass in islet transplantation. Disclosure N.A. Al-Jahdhami: None. S.J. Anderson: None. A. Aldibbiat: None. J.A. Shaw: Advisory Panel; Self; Novo Nordisk A/S.

Evaluation of renal oxygen homeostasis in a preclinical animal model to elucidate difference in blood quality after transfusion.

Red blood cell (RBC) oxygen (O2 ) delivery may be impacted at the tissue, cellular, and molecular levels after storage duration, preservation strategies, and pathogen reduction. Collectively, the preclinical measurement of arterial and venous PO2 , systemic blood flow, tissue hypoxia-inducible factors (HIFs), pimonidazole adduction, and erythropoietin (EPO) regulation can serve to elucidate differential RBC quality after storage and processing. Donor guinea pig blood was collected, leukoreduced, and stored at 4°C in AS-3 for 1 (fresh) or 14 (stored) days. RBC variables-2,3-diphosphoglycerate, adenosine triphosphate, hemoglobin, morphology, deformability, and in vivo recovery at 24 hours-were measured at each storage duration. Recipient guinea pigs were exchange transfused until 80% volume replacement was achieved. Arterial and venous blood gases, systemic blood flow, renal HIF-1α and HIF-2α, renal EPO mRNA, plasma EPO, and renal tissue pimonidazole adduction were measured after transfusion. RBC variables declined significantly with storage; however, hemolysis and in vivo recovery remained within the allowable limits for human blood storage. Posttransfusion arterial and venous PO2 and systemic blood flow decreased, and renal HIFs, EPO mRNA, and pimonidazole adducts increased. Subsequently, EPO accumulated in plasma indicating decreased O2 availability in the kidneys. Conversely, all variables remained at basal levels in the fresh blood group. The evaluation of renal O2 homeostasis after transfusion represents an effective approach to defining RBC quality between predicate and novel processing. Methods are adapted from standardized techniques and ideal for preclinical evaluation.

Cyclosporine A induces endothelin-converting enzyme-1: Studies invivo and invitro.

Cyclosporine A (CsA) induces renal vasoconstriction and hypoxia and enhances the expression of endothelin-1 (ET-1) pro-hormone (pre-pro-ET-1), plausibly leading to a feed-forward loop of renal vasoconstriction, hypoxia and enhanced synthesis of the potent vasoconstrictor ET-1. Endothelin-converting enzyme (ECE)-1 cleaves big endothelin to generate endothelin (ET)-1 and is upregulated by hypoxia via hypoxia-inducible factor (HIF). We hypothesized that in addition to the direct induction of ET-1 synthesis, CsA might also intensify renal ECE-1 expression, thus contributing to enhanced ET-1 synthesis following CsA. CsA was administered to Sprague Dawley rats (120mg/kg/SC) for 4days, and renal HIF and ECE-1 expression were assessed with Western blots and immunostaining. Human umbilical vein endothelial cells (HUVEC) and proximal tubular cell line (HK-2) were subjected to CsA, and ECE-1 induction was evaluated using real-time mRNA PCR and Western blots. Cyclosporine A intensified renal parenchymal ECE-1 expression in the rat kidney, particularly in distal nephron segments, along with renal hypoxia (detected by pimonidazole adducts) and HIF expression, in line with our recent observations showing episodic hypoxia in mice subjected to CsA. Furthermore, in cultured normoxic HUVEC and HK-2 cells, CsA dose-dependently induced both pre-pro-ET-1 and ECE-1 mRNA and protein expression, with enhanced ET-1 generation. CsA induces ECE-1 via both hypoxic and non-hypoxic pathways. ECE-1 may contribute to increased renal ET-1 generation following CsA, participating in a feed-forward loop of renal parenchymal hypoxia and ET synthesis.

Characterisation of fibrosis in chemically-induced rat mammary carcinomas using multi-modal endogenous contrast MRI on a 1.5T clinical platform

ObjectivesTo determine the ability of multi-parametric, endogenous contrast MRI to detect and quantify fibrosis in a chemically-induced rat model of mammary carcinoma.MethodsFemale Sprague-Dawley rats (n=18) were administered with N-methyl-N-nitrosourea; resulting mammary carcinomas underwent nine-b-value diffusion-weighted (DWI), ultrashort-echo (UTE) and magnetisation transfer (MT) magnetic resonance imaging (MRI) on a clinical 1.5T platform, and associated quantitative MR parameters were calculated. Excised tumours were histologically assessed for degree of necrosis, collagen, hypoxia and microvessel density. Significance level adjusted for multiple comparisons was p=0.0125.ResultsSignificant correlations were found between MT parameters and degree of picrosirius red staining (r > 0.85, p < 0.0002 for ka and δ, r < -0.75, p < 0.001 for T1 and T1s, Pearson), indicating that MT is sensitive to collagen content in mammary carcinoma. Picrosirius red also correlated with the DWI parameter fD* (r=0.801, p=0.0004) and conventional gradient-echo T2* (r=-0.660, p=0.0055). Percentage necrosis correlated moderately with ultrashort/conventional-echo signal ratio (r=0.620, p=0.0105). Pimonidazole adduct (hypoxia) and CD31 (microvessel density) staining did not correlate with any MR parameter assessed.ConclusionsMagnetisation transfer MRI successfully detects collagen content in mammary carcinoma, supporting inclusion of MT imaging to identify fibrosis, a prognostic marker, in clinical breast MRI examinations.Key Points• Magnetisation transfer imaging is sensitive to collagen content in mammary carcinoma.• Magnetisation transfer imaging to detect fibrosis in mammary carcinoma fibrosis is feasible.• IVIM diffusion does not correlate with microvessel density in preclinical mammary carcinoma.

Cyclosporin a induces renal episodic hypoxia

Cyclosporin A (CsA) causes renal toxicity. The underlying mechanisms are incompletely understood, but may involve renal hypoxia and hypoxia-inducible factors (Hifs). We sought for hypoxia and Hif in mouse kidneys with CsA-induced toxicity, assessed their time course, Hif-mediated responses and the impact of interventional Hif upregulation. Mice received CsA or its solvent cremophore for up to 6weeks. Low salt diet (Na+ ↓) was given in combination with CsA to enhance toxicity. We assessed fine morphology, renal function, blood oxygen level-dependent magnetic resonance imaging under room air and following changes in breathing gas composition which correlate with vascular reactivity, pimonidazole adducts (which indicate O2 tensions below 10mmHg), Hif-α proteins, as well as expression of Hif target genes. Stable Hif upregulation was achieved by inducible, Pax8-rtTA-based knockout of von Hippel-Lindau protein (Vhl-KO), which is crucial for Hif-α degradation. Cyclosporin A transiently increased renal deoxyhaemoglobin (R2*). Augmented vascular reactivity was observed at 2h, but decreased at 24h after CsA treatment. Na+ ↓/CsA provoked chronic renal failure with tubular degeneration and interstitial fibrosis. Nephron segments at risk for injury accumulated pimonidazole adducts, as well as Hif-α proteins. Remarkably, Hif target gene expression remained unchanged, while factor-inhibiting Hif (Fih) was enhanced. Na+ ↓/CsA/Vhl-KO aggravated morpho-functional outcome of chronic renal CsA toxicity. Cyclosporin A provokes episodic hypoxia in nephron segments most susceptible to chronic CsA toxicity. Fih is upregulated and likely blocks further Hif activity. Continuous tubular Hif upregulation via Vhl-KO worsens the outcome of chronic CsA-induced renal toxicity.

Immunohistochemical evidence of tissue hypoxia and astrogliosis in the rostral ventrolateral medulla of spontaneously hypertensive rats

Increased activity of the sympathetic nervous system has been highlighted as a key factor that contributes to the development and maintenance of arterial hypertension. However, the factors that precipitate sustained increases in sympathetic activity remain poorly understood. Resting tissue oxygen partial pressure (PtO2) in the brainstem of anesthetized spontaneously hypertensive rats (SHRs) has been shown to be lower than in normotensive rats despite normal levels of arterial PO2. A hypoxic environment in the brainstem has been postulated to activate astroglial signalling mechanisms in the rostral ventrolateral medulla (RVLM) which in turn increase the excitability of presympathetic neuronal networks. In this study, we assessed the expression of indirect markers of tissue hypoxia and astroglial cell activation in the RVLM of SHRs and age-matched normotensive Wistar rats. Immunohistochemical labelling for hypoxia-induced factor-1α (HIF-1α) and bound pimonidazole adducts revealed the presence of tissue hypoxia in the RVLM of SHRs. Double immunostaining showed co-localization of bound pimonidazole labelling in putative presympathetic C1 neurons and in astroglial cells. Quantification of glial fibrillary acidic protein (GFAP) immunofluorescence showed relatively higher number of astrocytes and increased GFAP mean grey value density, whilst semi-quantitative analysis of skeletonized GFAP-immunoreactive processes revealed greater % area covered by astrocytic processes in the RVLM of adult SHRs. In conclusion, the morphological findings of tissue hypoxia and astrogliosis within brainstem presympathetic neuronal networks in the SHR support previous observations, showing that low brainstem PtO2 and increased astroglial signalling in the RVLM play an important role in pathological sympathoexcitation associated with the development of arterial hypertension.

Renal cellular hypoxia in adenine-induced chronic kidney disease.

We determined whether adenine-induced chronic kidney disease (CKD) in rats is associated with renal tissue hypoxia. Adenine (100mg) or its vehicle was administered to male Sprague-Dawley rats, daily by oral gavage, over a 15-day period. Renal function was assessed before, and 7 and 14days after, adenine treatment commenced, by collection of a 24-hour urine sample and a blood sample from the tail vein. On day 15, arterial pressure was measured in conscious rats via the tail artery. Renal tissue hypoxia was then assessed by pimonidazole adduct immunohistochemistry and fibrosis was assessed by staining tissue with picrosirius red and Masson's trichrome. CKD was evident within 7days of commencing adenine treatment, as demonstrated by increased urinary albumin to creatinine ratio (30±12-fold). By day 14 of adenine treatment plasma creatinine concentration was more than 7-fold greater, and plasma urea more than 5-fold greater, than their baseline levels. On day 15, adenine-treated rats had slightly elevated mean arterial pressure (8mmHg), anaemia and renomegaly. Kidneys of adenine-treated rats were characterised by the presence of tubular casts, dilated tubules, expansion of the interstitial space, accumulation of collagen, and tubulointerstitial hypoxia. Pimonidazole staining (hypoxia) co-localised with fibrosis and was present in both patent and occluded tubules. We conclude that renal tissue hypoxia develops rapidly in adenine-induced CKD. This model, therefore, should prove useful for examination of the temporal and spatial relationships between tubulointerstitial hypoxia and the development of CKD, and thus the testing of the 'chronic hypoxia hypothesis'.

Growth hormone receptor antagonism suppresses tumour regrowth after radiotherapy in an endometrial cancer xenograft model

Human GH expression is associated with poor survival outcomes for endometrial cancer patients, enhanced oncogenicity of endometrial cancer cells and reduced sensitivity to ionising radiation in vitro, suggesting that GH is a potential target for anticancer therapy. However, whether GH receptor inhibition sensitises to radiotherapy in vivo has not been tested. In the current study, we evaluated whether the GH receptor antagonist, pegvisomant (Pfizer), sensitises to radiotherapy in vivo in an endometrial tumour xenograft model. Subcutaneous administration of pegvisomant (20 or 100 mg/kg/day, s.c.) reduced serum IGF1 levels by 23% and 68%, respectively, compared to vehicle treated controls. RL95-2 xenografts grown in immunodeficient NIH-III mice were treated with vehicle or pegvisomant (100 mg/kg/day), with or without fractionated gamma radiation (10 × 2.5 Gy over 5 days). When combined with radiation, pegvisomant significantly increased the median time tumours took to reach 3× the pre-radiation treatment volume (49 days versus 72 days; p = 0.001). Immunohistochemistry studies demonstrated that 100 mg/kg pegvisomant every second day was sufficient to abrogate MAP Kinase signalling throughout the tumour. In addition, treatment with pegvisomant increased hypoxic regions in irradiated tumours, as determined by immunohistochemical detection of pimonidazole adducts, and decreased the area of CD31 labelling in unirradiated tumours, suggesting an anti-vascular effect. Pegvisomant did not affect intratumoral staining for HIF1α, VEGF-A, CD11b, or phospho-EGFR. Our results suggest that blockade of the human GH receptor may improve the response of GH and/or IGF1-responsive endometrial tumours to radiation.