Pimonidazole Immunostaining Research Articles

Imaging Hypoxia to Predict Primary Neuronal Cell Damage in Branch Retinal Artery Occlusion.

To develop a reliable method to generate a mouse model of branch retinal artery occlusion (BRAO) using laser-induced thrombosis of a major artery in the mouse retina. Also, to develop a reliable method to detect retinal hypoxia as predictive biomarker for the risk of neuronal cell damage in BRAO. A reliable and reproducible model of laser-induced BRAO was developed in mouse retina using Rose Bengal. To characterize retinal hypoxia in BRAO, pimonidazole immunostaining and HYPOX-4 molecular imaging methods were used. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) was used to characterize neuronal cell damage in the BRAO retina. Expression of mRNA in retinal tissues from BRAO and age-matched control retinas were analyzed using qRT-PCR. Occlusion of a branch retinal artery near the optic nerve head (ONH) caused a pattern of retinal tissue hypoxia covering about 12.5% of the entire retina. TUNEL-positive cells were localized in all layers in BRAO retinal tissue cross sections. In addition, qRT-PCR data analysis suggests that BRAO is associated with both inflammation and hypoxia. This study provides a reliable method for BRAO in mouse retina and demonstrates the utility of molecular imaging method to detect retinal hypoxia as predictive biomarker for the risk of neuronal cell damage in BRAO. In addition, our data suggest that BRAO retinas are associated with inflammation and also associated with hypoxia-related neuronal cell damage. Imaging areas of retinal hypoxia may provide accurate diagnosis, evaluating retinal tissue injury from BRAO.

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.

Optimizing Bifurcated Channels within an Anisotropic Scaffold for Engineering Vascularized Oriented Tissues.

Despite progress in engineering both vascularized tissues and oriented tissues, the fabrication of 3D vascularized oriented tissues remains a challenge due to an inability to successfully integrate vascular and anisotropic structures that can support mass transfer and guide cell alignment, respectively. More importantly, there is a lack of an effective approach to guiding the scaffold design bearing both structural features. Here, an approach is presented to optimize the bifurcated channels within an anisotropic scaffold based on oxygen transport simulation and biological experiments. The oxygen transport simulation is performed using the experimentally measured effective oxygen diffusion coefficient and hydraulic permeability of the anisotropic scaffolds, which are also seeded with muscle precursor cells and cultured in a custom-made perfusion bioreactor. Symmetric bifurcation model is used as fractal unit to design the channel network based on biomimetic principles. The bifurcation level of channel network is further optimized based on the oxygen transport simulation, which is then validated by DNA quantification assay and pimonidazole immunostaining. This study provides a practical guide to optimizing bifurcated channels in anisotropic scaffolds for oriented tissue engineering.

Endothelin receptor B affects the perfusion of newborn intestine: possible mechanism of necrotizing enterocolitis development.

Necrotizing enterocolitis (NEC) is one of the most severe gastrointestinal diseases in infancy. Hypoxia is known as one of the major risk factors for the development of NEC. Endothelin, known to regulate vasoconstriction, has two receptors (A and B). However, the role of endothelin receptor B (EDNRB) in neonatal intestinal injury remains unclear. We aimed to investigate whether EDNRB is involved in NEC pathophysiology. Following ethical approval (#44032), EDNRB hetero knockout mice pups (EDNRB±) and their wild-type (WT) littermates were studied. NEC was induced from postnatal day 5-9 (P5-P9) by hypoxia, gavage feeding of formula and administration of lipopolysaccharide. On P9, the ileum was harvested. NEC induction in WT mice was associated with mucosal injury. However, EDNRB± NEC mice had reduced mucosal injury. Similarly, EDNRB± mice had significantly lower expression of IL-6 mRNA compared to WT NEC mice. Pimonidazole immunostaining was also significantly lower in EDNRB± compared to WT NEC, suggesting reduced tissue hypoxia. Partial knockout of EDNRB results in reduced NEC severity and reduced tissue hypoxia. Intestinal perfusion and hypoxia are important elements of NEC pathogenesis. These findings are relevant to the understanding of NEC pathophysiology and to the development of novel preventive strategies for NEC.

Hypoxia protects the liver from Small For Size Syndrome: A lesson learned from the associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) procedure in rats.

Portal hyperperfusion and "dearterialization" of the liver remnant are the main pathogenic mechanisms for Small For Size syndrome (SFSS). Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) induces rapid remnant hypertrophy. We hypothesized a similar increase in portal pressure/flow into the future liver remnant in ALPPS and SFSS-setting hepatectomies. In a rodent model, ALPPS was compared to SFSS-setting hepatectomy. We assessed mortality, remnant hypertrophy, hepatocyte proliferation, portal and hepatic artery flow, hypoxia-induced response, and liver sinusoidal morphology. SFSS-hepatectomy rats were subjected to local (hepatic artery ligation) or systemic (Dimethyloxalylglycine) hypoxia. ALLPS prevented mortality in SFSS-setting hepatectomies. Portal hyperperfusion per liver mass was similar in ALLPS and SFSS. Compared to SFSS, efficient arterial perfusion of the remnant was significantly lower in ALPPS causing pronounced hypoxia confirmed by pimonidazole immunostaining, activation of hypoxia sensors and upregulation of neo-angiogenic genes. Liver sinusoids, larger in ALPPS, collapsed in SFSS. Induction of hypoxia in SFSS reduced mortality. Hypoxia had no impact on hepatocyte proliferation but contributed to the integrity of sinusoidal morphology. ALPPS hemodynamically differ from SFSS by a much lower arterial flow in ALPPS's FLR. We show that the ensuing hypoxic response is essential for the function of the regenerating liver by preserving sinusoidal morphology.

Targeting Enox1 in tumor stroma increases the efficacy of fractionated radiotherapy.

The goal of this investigation was to clarify the question of whether targeting Enox1 in tumor stroma would synergistically enhance the survival of tumor-bearing mice treated with fractionated radiotherapy. Enox1, a NADH oxidase, is expressed in tumor vasculature and stroma. However, it is not expressed in many tumor types, including HT-29 colorectal carcinoma cells. Pharmacological inhibition of Enox1 in endothelial cells inhibited repair of DNA double strand breaks, as measured by γH2AX and 53BP1 foci formation, as well as neutral comet assays. For 4 consecutive days athymic mice bearing HT-29 hindlimb xenografts were injected with a small molecule inhibitor of Enox1 or solvent control. Tumors were then administered 2 Gy of x-rays. On day 5 tumors were administered a single ‘top-up’ fraction of 30 Gy, the purpose of which was to amplify intrinsic differences in the radiation fractionation regimen produced by Enox1 targeting. Pharmacological targeting of Enox1 resulted in 80% of the tumor-bearing mice surviving at 90 days compared to only 40% of tumor-bearing mice treated with solvent control. The increase in survival was not a consequence of reoxygenation, as measured by pimonidazole immunostaining. These results are interpreted to indicate that targeting of Enox1 in tumor stroma significantly enhances the effectiveness of 2 Gy fractionated radiotherapy and identifies Enox1 as a potential therapeutic target.

Abstract B88: Pharmacodynamic changes from the TH-302, gemcitabine, and nab-paclitaxel triplet combination in a xenograft model of pancreatic cancer

Abstract Background: Tumors often consist of hypoxic regions which are resistant to chemo- and radiotherapy. TH-302 (T) is an investigational prodrug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. We previously reported that the triplet combination of gemcitabine (G), nab-paclitaxel (nP) and T exhibited superior efficacy, without evidence of additive toxicity, compared to the G and nP doublet combination in preclinical pancreatic ductal adenocarcinoma (PDAC) models. These studies provided a translational rationale for combining G, nP, and T in the clinical setting to assess efficacy and safety, and a Phase 1 trial of G + nP + T in pancreatic cancer is currently open (NCT02047500). We used the PANC-1 PDAC xenograft model to investigate the pharmacodynamic (PD) changes resulting from combining T with G and nP and to explore the mechanism of action and biomarkers. Methods: The PANC-1 ectopic xenograft model was established by s.c. implantation of PANC-1 cells into the flank of nude mice. When the tumor size was 500 mm3, 5 animals in each group were treated with vehicle (V), T alone at 50 mg/kg, ip, G at 60 mg/kg ip + nP at 30 mg/kg, iv, or the triplet combination of G + nP + T, all with a Q3Dx5 regimen. Tumor samples were collected 24 h after the last treatment and were evaluated for hypoxia by immunostaining for pimonidazole (Pimo), and endogenous carbonic anhydrase IX (CA-IX). Tumor cell proliferation was assessed by Ki67, DNA damage by gamma histone 2AX (γH2AX) immunostaining, apoptosis by an in situ TUNEL assay, and tumor stromal density by alpha-smooth muscle actin (α-SMA) immunostaining. In a separate study, PANC-1-bearing animals (10 per group), treated with the same doses and regimens, were used to investigate the anti-tumor activity by quantifying tumor growth kinetics. Results: Two days after the last treatment, tumor size change% in V, T, G + nP and G + nP + T groups was 238%, 130%, -34%, and -51% respectively. The tumor size reduction achieved with the triplet treatment was significant compared with V, T or G + nP (p<0.05). The necrotic region in G + nP + T as measured by H&E staining was increased compared with all other groups. In the non-necrotic region the number of γH2AX-positive cells, as measured by immunostaining, was 31.3/field in G + nP + T, and was significantly increased compared with all other groups (3.6, 19.7 and 19.2/field in the V, T, and G + nP groups, p<0.05). Apoptosis was found in both stromal and tumor cells, and the number of apoptotic cells was significantly increased by the triple combination treatment compared with V, T or G + nP doublet (p<0.05). Ki67-positive cells and thus cell proliferation were significantly reduced after all drug treatments, and to a greater magnitude in the G + nP + T triplet group (p<0.05, compared with all other groups). T alone did not reduce the hypoxic fraction (HF) as measured by morphometrics of the Pimo staining to a significant degree (10.6 ± 1.1% vs. 12.4 ± 2% in V); however, by adding T to the G + nP doublet, the triplet significantly reduced the hypoxic level, with an HF of 2.5 ± 0.5%, compared with 7.1 ± 1.7% in the G + nP group (p<0.05). There was no co-localization between Pimo-positive and CA-IX-positive cells, and the α-SMA staining, and G + nP doublet significantly reduced α-SMA expression, compared with V (p<0.05), but no further reduction was observed in the G + nP + T group. Conclusions: In xenograft model of pancreatic cancer, the triplet combination of G + nP + T exhibits superior efficacy to T alone or G + nP doublet, and is associated with an increased inhibition of tumor cell proliferation, a reduction of the hypoxic fraction, and an enhancement of DNA damage, apoptosis, and necrosis. Citation Format: Jessica D. Sun, Qian Liu, Dharmendra Ahluwalia, Wenwu Li, Yan Wang, Charles P. Hart. Pharmacodynamic changes from the TH-302, gemcitabine, and nab-paclitaxel triplet combination in a xenograft model of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B88.

Uterine artery dysfunction in pregnant ACE2 knockout mice is associated with placental hypoxia and reduced umbilical blood flow velocity.

Angiotensin-converting enzyme 2 (ACE2) knockout is associated with reduced fetal weight at late gestation; however, whether uteroplacental vascular and/or hemodynamic disturbances underlie this growth-restricted phenotype is unknown. Uterine artery reactivity and flow velocities, umbilical flow velocities, trophoblast invasion, and placental hypoxia were determined in ACE2 knockout (KO) and C57Bl/6 wild-type (WT) mice at day 14 of gestation. Although systolic blood pressure was higher in pregnant ACE2 KO vs. WT mice (102.3 ± 5.1 vs. 85.1 ± 1.9 mmHg, n = 5-6), the magnitude of difference was similar to that observed in nonpregnant ACE2 KO vs. WT mice. Maternal urinary protein excretion, serum creatinine, and kidney or heart weights were not different in ACE2 KO vs. WT. Fetal weight and pup-to-placental weight ratio were lower in ACE2 KO vs. WT mice. A higher sensitivity to Ang II [pD2 8.64 ± 0.04 vs. 8.5 ± 0.03 (-log EC50)] and greater maximal contraction to phenylephrine (169.0 ± 9.0 vs. 139.0 ± 7.0% KMAX), were associated with lower immunostaining for Ang II receptor 2 and fibrinoid content of the uterine artery in ACE2 KO mice. Uterine artery flow velocities and trophoblast invasion were similar between study groups. In contrast, umbilical artery peak systolic velocities (60.2 ± 4.5 vs. 75.1 ± 4.5 mm/s) and the resistance index measured using VEVO 2100 ultrasound were lower in the ACE2 KO vs. WT mice. Immunostaining for pimonidazole, a marker of hypoxia, and hypoxia-inducible factor-2α were higher in the trophospongium and placental labyrinth of the ACE2 KO vs. WT. In summary, placental hypoxia and uterine artery dysfunction develop before major growth of the fetus occurs and may explain the fetal growth restricted phenotype.

Severe blunt muscle trauma in rats: only marginal hypoxia in the injured area.

BackgroundAfter severe muscle trauma, hypoxia due to microvascular perfusion failure is generally believed to further increase local injury and to impair healing. However, detailed analysis of hypoxia at the cellular level is missing. Therefore, in the present work, spectroscopic measurements of microvascular blood flow and O2 supply were combined with immunological detection of hypoxic cells to estimate O2 conditions within the injured muscle area.Materials and MethodsSevere blunt muscle trauma was induced in the right Musculus gastrocnemius of male Wistar rats by a standardized “weight-drop” device. Microvascular blood flow, relative hemoglobin amount, and hemoglobin O2 saturation were determined by laser Doppler and white-light spectroscopy. Hypoxic cells were detected by histologic evaluation of covalent binding of pimonidazole and expression of HIF-1α.ResultsDirectly after trauma and until the end of experiment (480 minutes), microvascular blood flow and relative hemoglobin amount were clearly increased. In contrast to blood flow and relative hemoglobin amount, there was no immediate but a delayed increase of microvascular hemoglobin O2 saturation. Pimonidazole immunostaining revealed a hypoxic fraction (percentage area of pimonidazole-labelled muscle cells within the injured area) between 8 to 3%. There was almost no HIF-1α expression detectable in the muscle cells under each condition studied.ConclusionsIn the early phase (up to 8 hours) after severe blunt muscle trauma, the overall microvascular perfusion of the injured area and thus its O2 supply is clearly increased. This increased O2 supply is obviously sufficient to ensure normoxic (or even hyperoxic) conditions in the vast majority of the cells.

OC-0320: Relationships between pimonidazole immunostaining and gene expression in prostate cancer patients

OC-0320: Relationships between pimonidazole immunostaining and gene expression in prostate cancer patients

Wound hypoxia in deep tissue after incision in rats.

Our previous studies using rat models of incisional pain have shown that tissue lactate levels increase and pH decreases for several days after incision, suggesting the presence of an ischemic-like condition. The purpose of this study was to evaluate the time course and the extent of tissue hypoxia that develops in incised muscle and skin. We directly measured oxygen tension at several time points after incisions of the gastrocnemius muscle, the paraspinal skin, and the plantar hindpaw in anesthetized rats using an oxygen-sensitive microelectrode. In vivo hypoxia of the incised tissues was also evaluated immunohistochemically using a hypoxia marker, pimonidazole hydrochloride. To minimize intersubject variability, unincised contralateral tissues were used as a control. Tissue oxygen tension was decreased in both skeletal muscle and skin compared with control, for several days after incision. When measured directly, oxygen tension decreased immediately and remained low for several days after incisions. Pimonidazole immunostaining revealed hypoxic areas in incised muscle and skin for several days. By postoperative day 10, tissue oxygen tension recovered to that of control tissue. These results support the evidence that a hypoxic condition is present in deep tissue after incisions and that an ischemic-like mechanism may contribute to postoperative pain.

Abstract 5583: Response biomarkers for the hypoxia-targeted drug TH-302: imaging, plasma, and tissue.

Abstract Biomarkers that assess the therapeutic response to anticancer treatments may aid in tailoring therapeutic regimens to individuals. Ideally, the relative contributory effect of each agent in a muti-agent regimen can be discerned. Hypoxia is a prevalent feature of solid tumors, associated with treatment failure, poor prognosis, and increased metastasis. TH-302, a hypoxia-activated prodrug of the DNA cross-linker bromo isophosphoramide mustard, has demonstrated broad efficacy in preclinical models and is exhibiting promising activity in multiple ongoing clinical trials, both as a monotherapy and in combination therapy regimens. Three fundamental biomarker platforms were assessed in this preclinical study: positron emission tomography (PET) imaging employing the tracer [18F]-HX4; immunohistochemistry of isolated tumor tissue employing antibodies directed against the exogenous hypoxia biomarker pimonidazole (PIMO) and the endogenous hypoxia biomarker carbonic anhydrase IX (CA-IX); and ELISA-based quantification of circulating plasma CA-IX. Both HX4 and PIMO utilize the same 2-nitroimidazole hypoxia-sensitive trigger for their activity and specificity as TH-302 utilizes. CA-IX is up-regulated by two distinct hypoxia sensing pathways: hypoxia-inducible factor 1; and the unfolded protein response. The human H460 non-small cell lung cancer human tumor ectopic xenograft model was employed for these studies. When the tumor size was 300-600 mm3, a single dose of TH-302 (150 mg/kg, ip) or vehicle was administered. This TH-302 treatment regimen yields a tumor growth inhibition of 58%, TGD1000 of 10 d, and maximum body weight loss of 2%. [18F]-HX4 uptake was assessed by PET/CT imaging and the tumor to muscle ratio (T/M) was determined. Animals were scanned pre- and post-treatment and the T/M ratio was significantly reduced 72 hours after TH-302 treatment (from 3.3 ± 0.3 to 2.1 ± 0.2, p<0.005). A slight increase in T/M was observed in the vehicle-treated animals (3.3 ± 0.4 to 3.6 ± 0.2, p>0.05). Blood was collected from the animals pre- and post- treatment via retro-orbital vein. Plasma CA-IX changed from baseline 165 ± 13 to 104 ± 9.3 pg/ml 72 hours after TH-302 dosing, a 37% reduction (p<0.05; n=10 per group). No change was observed in the vehicle-treated group. For tissue biomarker analysis, tumors were harvested 72 hours after TH-302 treatment, and tumor hypoxia was assessed by semi-quantitative morphometric analysis of the PIMO immunostaining at that time. The hypoxic fraction (HF) was 13 ± 1.7% in the vehicle group (n=5) and 5.8 ± 0.5% in the TH-302 treatment group (n=6, p<0.05). Strong co-localization of CA-IX and PIMO expression was observed in consecutive sections. These preclinical results highlight the promise of using TH-302 response biomarkers tested in the clinical setting for their ability to help guide TH-302 treatment decisions after the initiation of therapy. Citation Format: Jessica D. Sun, Qian Liu, Dharmendra Ahluwalia, Yan Wang, Anna-Katrin Szardenings, Luis F. Gomez, Hartmuth Kolb, Karen A. Pierce, Sheryl Brown-Shimer, Walter A. Carney, Charles P. Hart. Response biomarkers for the hypoxia-targeted drug TH-302: imaging, plasma, and tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5583. doi:10.1158/1538-7445.AM2013-5583

Chronic activation of vasopressin V2 receptor signalling lowers renal medullary oxygen levels in rats

In the present study, we aimed to elucidate the effects of chronic vasopressin administration on renal medullary oxygen levels. Adult Sprague Dawley or vasopressin-deficient Brattleboro rats were treated with the vasopressin V2 receptor agonist, desmopressin (5ng/h; 3d), or its vehicle via osmotic minipumps. Immunostaining for pimonidazole and the transcription factor HIF-1α (hypoxia-inducible factor-1α) were used to identify hypoxic areas. Activation of HIF-target gene expression following desmopressin treatment was studied by microarray analysis. Pimonidazole staining was detected in the outer and inner medulla of desmopressin-treated rats, whereas staining in control animals was weak or absent. HIF-1α immunostaining demonstrated nuclear accumulation in the papilla of desmopressin-treated animals, whereas no staining was observed in the controls. Gene expression analysis revealed significant enrichment of HIF-target genes in the group of desmopressin-regulated gene products (P=2.6*10(-21) ). Regulated products included insulin-like growth factor binding proteins 1 and 3, angiopoietin 2, fibronectin, cathepsin D, hexokinase 2 and cyclooxygenase 2. Our results demonstrate that an activation of the renal urine concentrating mechanism by desmopressin causes renal medullary hypoxia and an upregulation of hypoxia-inducible gene expression.

Type 2 Diabetes Mellitus in Mice Aggravates the Renal Impact of Hemorrhagic Shock

The objectives of this study were to determine whether type 2 diabetic mice would exhibit a more severe renal impact of hemorrhagic shock (HS) based on a recently described model of acute kidney injury and to determine the impact of HS on renal responses to hypoxia. We induced HS or sham procedure in type 2 diabetic and obese db/db mice. Creatininemia, glomerular filtration rate, urine output, histologic injury score, and kidney inductible molecule 1 mRNA were used to investigate the renal impact of HS. Tissular hypoxia and its impact were quantified using pimonidazole immunostaining and mRNA of hypoxic inducible factor, vascular endothelial growth factor receptors 1 and 2, Tie-2, endothelial nitric oxide synthase, and inducible nitric oxide synthase. Diabetic mice exhibiting mild diabetic nephropathy express hypoxic signals at baseline. The renal impact of HS was more severe in diabetic mice, with a worsening of tissular hypoxia and an altered response to hypoxia. Furthermore, endothelial nitric oxide synthase was highly overexpressed in diabetic shocked mice when compared with nondiabetic shocked mice. Renal impact of HS in type 2 diabetic mice is more intense than in nondiabetic ones. Preexisting hypoxia during diabetes could result in a renal preconditioning that modifies endothelial and tissular responses to acute kidney injury.

64Cu-ATSM and 18FDG PET uptake and 64Cu-ATSM autoradiography in spontaneous canine tumors: comparison with pimonidazole hypoxia immunohistochemistry

BackgroundThe aim of this study was to compare 64Cu-diacetyl-bis(N4-methylsemicarbazone) (64Cu-ATSM) and 18FDG PET uptake characteristics and 64Cu-ATSM autoradiography to pimonidazole immunohistochemistry in spontaneous canine sarcomas and carcinomas.MethodsBiopsies were collected from individual tumors between approximately 3 and 25 hours after the intravenous injection of 64Cu-ATSM and pimonidazole. 64Cu-ATSM autoradiography and pimonidazole immunostaining was performed on sectioned biopsies. Acquired 64Cu-ATSM autoradiography and pimonidazole images were rescaled, aligned and their distribution patterns compared. 64Cu-ATSM and 18FDG PET/CT scans were performed in a concurrent study and uptake characteristics were obtained for tumors where available.ResultsMaximum pimonidazole pixel value and mean pimonidazole labeled fraction was found to be strongly correlated to 18FDG PET uptake levels, whereas more varying results were obtained for the comparison to 64Cu-ATSM. In the case of the latter, uptake at scans performed 3 h post injection (pi) generally showed strong positive correlated to pimonidazole uptake.Comparison of distribution patterns of pimonidazole immunohistochemistry and 64Cu-ATSM autoradiography yielded varying results. Significant positive correlations were mainly found in sections displaying a heterogeneous distribution of tracers.ConclusionsTumors with high levels of pimonidazole staining generally displayed high uptake of 18FDG and 64Cu-ATSM (3 h pi.). Similar regional distribution of 64Cu-ATSM and pimonidazole was observed in most heterogeneous tumor regions. However, tumor and hypoxia level dependent differences may exist with regard to the hypoxia specificity of 64Cu-ATSM in canine tumors.

Abstract 5054: Hypoxia-activated prodrug TH-302 enhances antitumor activity of antiangiogenics in preclinical models

Abstract Antiangiogenics have revolutionized cancer therapy. Angiogenesis is a critical step in the establishment, growth, and metastasis of solid tumors. However, relative to other cancer drugs, the dramatic delays in disease progression after antiangiogenic therapy are tempered by the smaller differences in overall survival time. Such differences have led to the hypothesis that antiangiogenic therapy increases tumor hypoxia and leads to the emergence of an aggressive cancer phenotype. TH-302, a 2-nitroimidazole triggered hypoxia-activated prodrug, releases bromo-isophosphoramide mustard (Br-IPM), which induces DNA crosslinking in hypoxic cells. TH-302 has been shown in human tumor xenograft models to target the hypoxic compartment selectively and reduce its volume. A series of studies have been conducted to investigate whether TH-302 and antiangiogenic combination have improved efficacy. Xenograft models were established by s.c. implantation of 4 x106 786-O human renal cell carcinoma (RCC) or 1×106 H460 human non-small cell lung cancer (NSCLC) cells into the flanks of nude mice. Tumor hypoxia was detected by pimonidazole immunostaining, and morphometric analysis was performed to determine the hypoxic fraction. When tumor size was approximately 100-150mm3, sunitinib or sorafenib was administered daily. TH-302 administration began one week after antiangiogenics administration. Sunitinib and sorafenib increased hypoxia in both the RCC and NSCLC xenografts in a dose- and time-dependent manner. In the RCC model, sunitinib activity was potentiated with TH-302: TGI increased from 38% with 40mg/kg sunitinib monotherapy to 75% with 50 mg/kg TH-302. In the NSCLC model, sunitinib (20, 40 or 80mg/kg QDx21) activity was potentiated with TH-302 (50mg/kg, QDx5 x2wk) from 78% TGI (sunitinib monotherapy) to 97% TGI with TH-302 combination therapy. In another study, sunitinib was administered for 33 days, and TH-302 was administered for 4 weeks. With this longer dosing regimen, antitumor activity increased. Medium dose sunitinib (40mg/kg) in combination with TH-302 reached 99% TGI and delayed tumor growth to 500mm3 for 29 days. A complementary effect of TH-302 in combination with sorafenib was also observed (94% TGI for combination therapy versus less than 80% TGI for either monotherapy in the NSCLC model). Importantly, body weight loss, a toxicity indicator, was not significantly increased with TH-302 in combination with any of the antiangiogenics in these studies. In summary, TH-302 potentiates the anti-tumor efficacy of sunitinib and sorafenib by selectively targeting the antiangiogenic-induced increase in tumor hypoxia. These studies provide a translational rationale for combining TH-302 with antiangiogenics to increase the treatment benefit in not only the approved indication (RCC) but also other indications, such as NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5054. doi:10.1158/1538-7445.AM2011-5054

Vasopressin treatment causes widespread hypoxia in the renal medulla of Brattleboro rats

In the present study we aimed to elucidate the effects of chronic vasopressin (AVP) administration on renal medullary oxygen levels in AVP-deficient Brattleboro rats (DI rats). To this end, adult DI rats were treated for 3 days with the V2 AVP receptor agonist desmopressin (dDAVP, 5ng/h; 3d) or its vehicle via osmotic minipump. Pimonidazole immunostaining was employed to study oxygen distribution throughout the kidney. In addition, outer medullary gene expression was determined by microarray analysis. dDAVP treatment resulted in a significant drop in urine output (−94 +/− 24% compared to controls, p < .05). Pimonidazole staining was detected in medullary rays of the cortex and in the outer as well as in the inner medulla of dDAVP-treated rats whereas signal in control animals was weak or absent. Gene expression analysis revealed an upregulation of several known target genes for hypoxia-inducible factors which included hexokinase 2, phosphofructokinase, heme oxygenase 1, insulin like growth factor (IGF) 1 and IGF binding proteins 1 and 3. Our results suggest that an activation of the renal urine concentration mechanism by dDAVP causes renal medullary hypoxia and an upregulation of hypoxia-inducible target gene expression. This study was supported by the German Research Foundation (FOR 667)

Stretch-Induced Uterine Myocyte Differentiation During Rat Pregnancy: Involvement of Caspase Activation1

Proliferation, differentiation, and apoptosis are three major processes by which the pregnant uterus maintains homeostasis to accommodate the growing fetus. We demonstrated previously that caspase activation in the pregnant rat myometrium at midgestation coincides with the transition from uterine hyperplasia to hypertrophy. We hypothesized that this transition was induced by stasis of myometrial blood flow (and subsequent hypoxia/ischaemia insult) resulting from acute myometrial stretch induced by a growing embryo. Therefore, we measured the expression of active caspase 3 and two hypoxia markers (transcription factor HIF1A and pimonidazole hydrochloride) in pregnant rat myometrium. To investigate the effect of gravidity we used unilaterally pregnant rats. Caspase 3 was activated only in the gravid horn of the unilaterally pregnant animals on Gestational Days 12-15. This activation was associated with high levels of HIF1A and pimonidazole immunostaining, which were limited to the circular myometrial layer of the gravid horn, indicative of hypoxia within this tissue. To isolate the effect of myometrial stretch applied by the growing fetus, we inserted an expandable polymer tube (intra-uterine expandable tube [IUET]) into the empty horn of Day 13 and Day 20 unilaterally pregnant rats. Tissue was collected 2, 14, and 24 h later. In the IUET-stretched empty horn, cleaved caspase 3 was activated at midgestation (Day 14), but not at late gestation (Day 21). We speculate that hypoxia resulting from mechanical stretch may activate caspase 3 within the pregnant myometrium only in the context of a specific endocrine environment.

Imaging of HIF-1-Active Tumor Hypoxia Using a Protein Effectively Delivered to and Specifically Stabilized in HIF-1-Active Tumor Cells

Hypoxia-inducible factor-1 (HIF-1) plays an important role in malignant tumor progression and in the development of resistance to radiotherapy. We designed a novel fusion protein (PTD-ODD-SAV [POS]) consisting of a protein transduction domain (PTD), streptavidin (SAV), and a portion of the oxygen-dependent degradation domain (ODD) of HIF-1alpha that confers the same oxygen-dependent regulation as HIF-1alpha on POS. (3-(123/125)I-iodobenzoyl)norbiotinamide ((123/125)I-IBB) was conjugated to the SAV moiety of POS to synthesize (123/125)I-IBB-labeled POS ((123/125)I-IPOS). The purpose of this study was to evaluate the feasibility of (123)I-IPOS as an imaging probe for HIF-1-active tumor hypoxia. After a 24-h incubation of (125)I-IPOS with various tumor cell lines under either normoxic (20% O(2)) or hypoxic (0.1% O(2)) conditions, the intracellular radioactivity was investigated. Then, the biodistribution of (123/125)I-IPOS was examined with tumor-implanted mice, and an in vivo imaging study was performed. The tumoral accumulation of (125)I-IPOS was compared with HIF-1 activity using the mice carrying tumors with the HIF-1-dependent luciferase reporter gene. Furthermore, the intratumoral localization of (125)I-IPOS was examined by the autoradiographic study, and then the same slide was subjected to immunostaining for pimonidazole, which is the hypoxic marker. The ratios of radioactivity in hypoxic cells to that in normoxic cells were more than 2. These results indicate incorporation of (125)I-IPOS into these cells and degradation of (125)I-IPOS by normoxic tumor cells. In the biodistribution study, (125)I-IPOS accumulated in the tumor (1.4 +/- 0.3 percentage injected dose per gram) 24 h after administration. At that time, (125)I-IPOS showed high tumor-to-blood and tumor-to-muscle ratios (5.1 +/- 0.3 and 14.0 +/- 3.9, respectively). The tumors were clearly visualized by in vivo imaging 24 h after (123)I-IPOS injection (tumor-to-muscle ratio was 9.6). The tumoral accumulation of (125)I-IPOS correlated with HIF-1 activity (R = 0.71, P < 0.05), and its intratumoral distribution coincided with the hypoxic regions. (123)I-IPOS is a potential probe for the imaging of HIF-1 activity in tumors. Given the role of HIF-1 in tumor biology, its detection may be considered an indicator of aggressive cancer phenotypes.

Perindopril attenuates tubular hypoxia and inflammation in an experimental model of diabetic nephropathy in transgenic Ren‐2 rats

Renal hypoxia plays a role in the development of diabetic nephropathy, and may be mediated by overactivity of the renin-angiotensin-aldosterone system (RAAS). In this study the localization of cellular hypoxia in an experimental model of diabetic nephropathy was assessed, and the effect of the angiotensin-converting enzyme inhibitor perindopril on hypoxia evaluated. Female Sprague-Dawley rats heterozygous for the Ren-2 gene were randomized to three groups (n = 8 per group)--controls, diabetes or diabetes + perindopril. Diabetes was induced by injection of streptozotocin at 6 weeks of age. Perindopril was administered at a dose of 2 mg/kg daily from 6 weeks. Subjects were culled after 16 weeks. Areas of tissue hypoxia were localized using immunohistochemistry to detect pimonidazole uptake. Diabetic rat kidneys were characterized by increases in tubulointerstitial collagen deposition compared with controls. Tubular hypoxia was significantly greater in diabetic rats, indicated by a 2.5-fold increase in the proportional area of pimonidazole immunostaining (P < 0.001). Immunohistochemical staining for pimonidazole co-localized with osteopontin, and was associated with higher numbers of ED-1-positive cells (macrophages) within the tubulointerstitium. Treatment with perindopril ameliorated structural changes of diabetic nephropathy and reduced the amount of pimonidazole and ED-1 immunostaining to levels similar to that of controls. In diabetic Ren-2 rats the development of diabetic nephropathy was associated with tubular hypoxia. Co-localization of osteopontin with hypoxic cells suggests that tubular hypoxia may be involved in the pathogenesis of diabetic nephropathy. The degree of hypoxia and fibrosis was attenuated by treatment with perindopril.