Transition from androgenic to neurosteroidal action of 5α-androstane-3α, 17ß-diol through the type A γ-aminobutyric acid receptor in prostate cancer progression.

Androgen ablation is the standard of care prescribed to patients with advanced or metastatic prostate cancer (PCa) to slow down disease progression. Unfortunately, a majority of PCa patients under androgen ablation progress to castration-resistant prostate cancer (CRPC). Several mechanisms including alternative intra-prostatic androgen production and androgen-independent androgen receptor (AR) activation have been proposed for CRPC progression. Aldo-keto reductase family 1 member C3 (AKR1C3), a multi-functional steroid metabolizing enzyme, is specifically expressed in the cytoplasm of PCa cells; and positive immunoreactivity of the type A γ-aminobutyric acid receptor (GABAAR), an ionotropic receptor and ligand-gated ion channel, is detected on the membrane of PCa cells. We studied a total of 72 radical prostatectomy cases by immunohistochemistry, and identified that 21 cases exhibited positive immunoreactivities for both AKR1C3 and GABAAR. In the dual positive cancer cases, AKR1C3 and GABAAR subunit α1 were either expressed in the same cells or in neighboring cells. Among several possible substrates, AKR1C3 reduces 5α-dihydrotesterone (DHT) to form 5α-androstane-3α, 17ß-diol (3α-diol). 3α-diol is a neurosteroid that acts as a positive allosteric modulator of the GABAAR in the central nervous system (CNS). We examined the hypothesis that 3α-diol-regulated pathological effects in the prostate are GABAAR-dependent, but are independent of the AR. In GABAAR-positive, AR-negative human PCa PC-3 cells, 3α-diol significantly stimulated cell growth in culture and the in ovo chorioallantoic membrane (CAM) xenograft model. 3α-diol also up-regulated expression of the epidermal growth factor (EGF) family of growth factors and activation of EGF receptor (EGFR) and Src as measured by quantitative polymerase chain reaction and immunoblotting, respectively. Inclusion of GABAAR antagonists reversed 3α-diol-stimulated tumor cell growth, expression of EGF family members, and activation of EGFR and Src to the level observed in untreated cells. Results from the present study suggest that 3α-diol may act as an alternative intra-prostatic neurosteroid that activates AR-independent PCa progression. The involvement of AKR1C3-mediated steroid metabolisms in modulating GABAAR activation and promoting PCa progression requires continued studies.

A small molecule with anticancer and antimetastatic activities induces rapid mitochondrial-associated necrosis in breast cancer.

Therapy for treatment-resistant breast cancer provides limited options and the response rates are low. Therefore, the development of therapies with alternative chemotherapeutic strategies is necessary. AG311 (5-[(4-methylphenyl)thio]-9H-pyrimido[4,5-b]indole-2,4-diamine), a small molecule, is being investigated in preclinical and mechanistic studies for treatment of resistant breast cancer through necrosis, an alternative cell death mechanism. In vitro, AG311 induces rapid necrosis in numerous cancer cell lines as evidenced by loss of membrane integrity, ATP depletion, HMGB1 (high-mobility group protein B1) translocation, nuclear swelling, and stable membrane blebbing in breast cancer cells. Within minutes, exposure to AG311 also results in mitochondrial depolarization, superoxide production, and increased intracellular calcium levels. Additionally, upregulation of mitochondrial oxidative phosphorylation results in sensitization to AG311. This AG311-induced cell death can be partially prevented by treatment with the mitochondrial calcium uniporter inhibitor, Ru360 [(µ)[(HCO2)(NH3)4Ru]2OCl3], or an antioxidant, lipoic acid. Additionally, AG311 does not increase apoptotic markers such as cleavage of poly (ADP-ribose) polymerase (PARP) or caspase-3 and -7 activity. Importantly, in vivo studies in two orthotopic breast cancer mouse models (xenograft and allograft) demonstrate that AG311 retards tumor growth and reduces lung metastases better than clinically used agents and has no gross or histopathological toxicity. Together, these data suggest that AG311 is a first-in-class antitumor and antimetastatic agent inducing necrosis in breast cancer tumors, likely through the mitochondria.

Discovery of antitubulin agents with antiangiogenic activity as single entities with multitarget chemotherapy potential.

Antiangiogenic agents (AA) are cytostatic, and their utility in cancer chemotherapy lies in their combination with cytotoxic chemotherapeutic agents. Clinical combinations of vascular endothelial growth factor receptor-2 (VEGFR2) inhibitors with antitubulin agents have been particularly successful. We have discovered a novel, potentially important analogue, that combines potent VEGFR2 inhibitory activity (comparable to that of sunitinib) with potent antitubulin activity (comparable to that of combretastatin A-4 (CA)) in a single molecule, with GI50 values of 10(-7) M across the entire NCI 60 tumor cell panel. It potently inhibited tubulin assembly and circumvented the most clinically relevant tumor resistance mechanisms (P-glycoprotein and ß-III tubulin expression) to antimicrotubule agents. The compound is freely water-soluble as its HCl salt and afforded excellent antitumor activity in vivo, superior to docetaxel, sunitinib, or Temozolomide, without any toxicity.

Development and characterization of a preclinical model of breast cancer lung micrometastatic to macrometastatic progression.

Most cancer patients die with metastatic disease, thus, good models that recapitulate the natural process of metastasis including a dormancy period with micrometastatic cells would be beneficial in developing treatment strategies. Herein we report a model of natural metastasis that balances time to complete experiments with a reasonable dormancy period, which can be used to better study metastatic progression. The basis for the model is a 4T1 triple negative syngeneic breast cancer model without resection of the primary tumor. A cell titration from 500 to 15,000 GFP tagged 4T1 cells implanted into fat pad number four of immune proficient eight week female BALB/cJ mice optimized speed of the model while possessing metastatic processes including dormancy and beginning of reactivation. The frequency of primary tumors was less than 50% in animals implanted with 500-1500 cells. Although implantation with over 10,000 cells resulted in 100% primary tumor development, the tumors and macrometastases formed were highly aggressive, lacked dormancy, and offered no opportunity for treatment. Implantation of 7,500 cells resulted in >90% tumor take by 10 days; in 30-60 micrometastases in the lung (with many animals also having 2-30 brain micrometastases) two weeks post-implantation, with the first small macrometastases present at five weeks; many animals displaying macrometastases at five weeks and animals becoming moribund by six weeks post-implantation. Using the optimum of 7,500 cells the efficacy of a chemotherapeutic agent for breast cancer, doxorubicin, given at its maximal tolerated dose (MTD; 1 mg/kg weekly) was tested for an effect on metastasis. Doxorubicin treatment significantly reduced primary tumor growth and lung micrometastases but the number of macrometastases at experiment end was not significantly affected. This model should prove useful for development of drugs to target metastasis and to study the biology of metastasis.

The design and discovery of water soluble 4-substituted-2,6-dimethylfuro[2,3-d]pyrimidines as multitargeted receptor tyrosine kinase inhibitors and microtubule targeting antitumor agents.

The design, synthesis and biological evaluations of fourteen 4-substituted 2,6-dimethylfuro[2,3-d]pyrimidines are reported. Four compounds (11-13, 15) inhibit vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived growth factor receptor ß (PDGFR-ß), and target tubulin leading to cytotoxicity. Compound 11 has nanomolar potency, comparable to sunitinib and semaxinib, against tumor cell lines overexpressing VEGFR-2 and PDGFR-ß. Further, 11 binds at the colchicine site on tubulin, depolymerizes cellular microtubules and inhibits purified tubulin assembly and overcomes both ßIII-tubulin and P-glycoprotein-mediated drug resistance, and initiates mitotic arrest leading to apoptosis. In vivo, its HCl salt, 21, reduced tumor size and vascularity in xenograft and allograft murine models and was superior to docetaxel and sunitinib, without overt toxicity. Thus 21 affords potential combination chemotherapy in a single agent.

Solid phase synthesis and biological evaluation of probestin as an angiogenesis inhibitor.

Probestin is a potent aminopeptidase N (APN) inhibitor originally isolated from the bacterial culture broth. Here, we report probestin synthesis by solid phase peptide synthesis (SPPS) method and evaluated its activity to inhibit angiogenesis using a chicken embryo chorioallantoic membrane (CAM) assay and a CAM tumor xenograft model. Results from these studies demonstrate that probestin inhibits the angiogenic activity and tumor growth.

N2-Trimethylacetyl substituted and unsubstituted-N4-phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines: design, cellular receptor tyrosine kinase inhibitory activities and in vivo evaluation as antiangiogenic, antimetastatic and antitumor agents.

Six novel N(4)-phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines and their N(2)-trimethylacetyl substituted analogs were synthesized as receptor tyrosine kinase (RTK) inhibitors. A microwave-mediated Sonogashira reaction was used as a key step for the synthesis of these compounds. Biological evaluation, in whole cell assays, showed that some analogs had remarkable inhibitory activity against a variety of RTKs and in particular cytotoxic activity against A431 tumor cells in culture. The inhibitory data against RTKs in this study demonstrated that variation of the 4-anilino substituents of these analogs dictates both potency and specificity of inhibitory activity against various RTKs. The study also supported the hypothesis that interaction of substituents on the 2-amino group with hydrophobic site-II provides an increase in potency. Compound 8 of this series was selected for evaluation in vivo in a B16-F10 syngeneic mouse tumor model and exhibited significant reduction in tumor growth rate, in tumor vascular density and in metastases to the lung compared to the control.

Synthesis and biological activity of 5-chloro-N4-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamines as vascular endothelial growth factor receptor-2 inhibitors and antiangiogenic agents.

Inhibition of receptor tyrosine kinase (RTK) signaling pathways is an important area for the development of novel anticancer agents. Numerous multikinase inhibitors (MKIs) have been recently approved for the treatment of cancer. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the principal mediator of tumor angiogenesis. In an effort to develop ATP-competitive VEGFR-2 selective inhibitors the 5-chloro-N(4)-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold was designed. The synthesis of the target compounds involved N-(4,5-dichloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethylpropanamide) as a common intermediate. A nucleophilic displacement of the 4-chloro group of the common intermediate by appropriately substituted anilines afforded the target compounds. Biological evaluation indicated that compound 5 is a potent and selective VEGFR-2 inhibitor comparable to sunitinib and semaxinib.

Novel tricyclic indeno[2,1-d]pyrimidines with dual antiangiogenic and cytotoxic activities as potent antitumor agents.

We designed, synthesized and evaluated 13 novel tricyclic indeno[2,1-d]pyrimidines as RTK inhibitors. These analogues were synthesized via a Dieckmann condensation of 1,2-phenylenediacetonitrile followed by cyclocondensation with guanidine carbonate to afford the 2-amino-3,9-dihydro-indeno[2,1-d]pyrimidin-4-one. Sulfonation of the 4-position followed by displacement with appropriately substituted anilines afforded the target compounds. These compounds were potent inhibitors of platelet-derived growth factor receptor ß (PDGFRß) and inhibited angiogenesis in the chicken embryo chorioallantonic membrane (CAM) assay compared to standards. In addition, compound 7 had a two digit nanomolar GI(50) against nine tumor cell lines, a submicromolar GI(50) against 29 of other tumor cell lines in the preclinical NCI 60 tumor cell line panel. Compound 7 also demonstrated significant in vivo inhibition of tumor growth and angiogenesis in a B16-F10 syngeneic mouse melanoma model.

N4-(3-Bromophenyl)-7-(substituted benzyl) pyrrolo[2,3-d]pyrimidines as potent multiple receptor tyrosine kinase inhibitors: design, synthesis, and in vivo evaluation.

With the goal of developing multitargeted receptor tyrosine kinase inhibitors that display potent inhibition against PDGFRß and VEGFR-2 we designed and synthesized eleven N(4)-(3-bromophenyl)-7-(substitutedbenzyl) pyrrolo[2,3-d]pyrimidines 9a-19a. These compounds were obtained from the key intermediate N(4)-(3-bromophenyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine 29. Various arylmethyl groups were regiospecifically attached at the N7 of 29 via sodium hydride induced alkylation with substituted arylmethyl halides. Compounds 11a and 19a were potent dual inhibitors of PDGFRß and VEGFR-2. In a COLO-205, in vivo tumor mouse model 11a demonstrated inhibition of tumor growth, metastasis, and tumor angiogenesis that was better than or comparable to the standard compound TSU-68 (SU6668, 8).

N4-Aryl-6-substitutedphenylmethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as receptor tyrosine kinase inhibitors.

Six novel N(4)-substitutedphenyl-6-substitutedphenylmethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines were synthesized as multiple receptor tyrosine kinase (RTK) inhibitors and antitumor agents. An improvement in the inhibitory potency against epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor 1 (VEGFR-1) and vascular endothelial growth factor receptor 2 (VEGFR-2) assays and in the A431 cellular proliferation assay was observed for compounds 8-13 over the previously reported 5-7. Three compounds (8, 9 and 13) demonstrated potent, multiple RTK inhibition and were more potent or equipotent compared to the lead compounds 5 and 7 and the standard compounds. Compounds 10 and 12 showed potent inhibition of VEGFR-2 over EGFR, platelet-derived growth factor receptor-ß (PDGFR-ß) and VEGFR-1. The results indicate that the RTK inhibitory profile could be modulated with slight variations to the N(4)-aryl-6-substitutedphenylmethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamino scaffold.

Synthesis and biological activity of N(4)-phenylsubstituted-6-(2,4-dichloro phenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as vascular endothelial growth factor receptor-2 inhibitors and antiangiogenic and antitumor agents.

A series of eight N(4)-phenylsubstituted-6-(2,4-dichlorophenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines 8-15 were synthesized as vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors with varied substitutions in the phenyl ring of the 4-anilino moiety. In addition, five N(4)-phenylsubstituted-6-phenylmethylsubstituted-7H-pyrrolo[2,3-d]pyrimidin-4-amines 16-20 were synthesized to evaluate the importance of the 2-NH(2) moiety for multiple receptor tyrosine kinase (RTK) inhibition. Cyclocondensation of alpha-halomethylbenzylketones with 2,6-diamino-4-hydroxypyrimidine afforded 2-amino-6-(2,4-dichlorophenylmethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, 23 and reaction of alpha-bromomethylbenzylketones with ethylamidinoacetate followed by cyclocondensation with formamide afforded the 6-phenylmethylsubstituted-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-ones, 40-42, respectively. Chlorination of the 4-position and displacement with appropriate anilines afforded the target compounds 8-20. Compounds 8, 10 and 14 were potent VEGFR-2 inhibitors and were 100-fold, 40-fold and 8-fold more potent than the standard semaxanib, respectively. Previously synthesized multiple RTK inhibitor, 5 and the VEGFR-2 inhibitor 8 from this study, were chosen for further evaluation in a mouse orthotopic model of melanoma and showed significant inhibition of tumor growth, angiogenesis and metastasis.

Chronic exposure to arsenic in the drinking water alters the expression of immune response genes in mouse lung.

BACKGROUND: Chronic exposure to drinking water arsenic is a significant worldwide environmental health concern. Exposure to As is associated with an increased risk of lung disease, which may make it a unique toxicant, because lung toxicity is usually associated with inhalation rather than ingestion. OBJECTIVES: The goal of this study was to examine mRNA and protein expression changes in the lungs of mice exposed chronically to environmentally relevant concentrations of As in the food or drinking water, specifically examining the hypothesis that As may preferentially affect gene and protein expression related to immune function as part of its mechanism of toxicant action. METHODS: C57BL/6J mice fed a casein-based AIN-76A defined diet were exposed to 10 or 100 ppb As in drinking water or food for 5-6 weeks. RESULTS: Whole genome transcriptome profiling of animal lungs revealed significant alterations in the expression of many genes with functions in cell adhesion and migration, channels, receptors, differentiation and proliferation, and, most strikingly, aspects of the innate immune response. Confirmation of mRNA and protein expression changes in key genes of this response revealed that genes for interleukin 1beta, interleukin 1 receptor, a number of toll-like receptors, and several cytokines and cytokine receptors were significantly altered in the lungs of As-exposed mice. CONCLUSIONS: These findings indicate that chronic low-dose As exposure at the current U.S. drinking-water standard can elicit effects on the regulation of innate immunity, which may contribute to altered disease risk, particularly in lung.

Clinical review 2: The "metabolic memory": is more than just tight glucose control necessary to prevent diabetic complications?

CONTEXT: The concept of a "metabolic memory," that is of diabetic vascular stresses persisting after glucose normalization, has been supported both in the laboratory and in the clinic and in both type 1 and type 2 diabetes. EVIDENCE ACQUISITION: Using PubMed, we searched for publications on diabetic micro- and macrovascular complications using terms such as persistence, prolongation, sustained, and "memory" and focusing on the mechanistic basis behind this metabolic memory. EVIDENCE SYNTHESIS: We found that as early as the mid-1980s this memory phenomenon was described in diabetic animals and isolated cells exposed to high glucose followed by normalized glucose and then, beginning around 2002, in results from large clinical trials such as the Diabetes Complications and Control Trial-Epidemiology of Diabetes Interventions and Complications and the United Kingdom Prospective Diabetes Study. Furthermore, mechanisms for propagating this memory appear focused on the nonenzymatic glycation of cellular proteins and lipids and on an excess of cellular reactive oxygen and nitrogen species, in particular originating at the level of glycated mitochondrial proteins and perhaps acting in concert with one another to maintain stress signaling independent of glucose levels. CONCLUSIONS: The emergence of this metabolic memory suggests the need for early aggressive treatment aiming to "normalize" metabolic control together perhaps with the addition of agents which reduce cellular reactive species and glycation in order to minimize long-term diabetic complications.

Design, synthesis and biological evaluation of substituted pyrrolo[2,3-d]pyrimidines as multiple receptor tyrosine kinase inhibitors and antiangiogenic agents.

Direct and indirect involvement of receptor tyrosine kinases (RTKs) in tumor growth and metastasis makes them ideal targets for anticancer therapy. A paradigm shift from inhibition of single RTK to inhibition of multiple RTKs has been recently demonstrated. We designed and synthesized eight N(4)-phenylsubstituted-6-(2-phenylethylsubstituted)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as homologated series of our previously published RTK inhibitors. We reasoned that increased flexibility of the side chain, which determines potency and selectivity, would improve the spectrum of RTK inhibition. These compounds were synthesized using a bis-electrophilic cyclization to afford substituted pyrrolo[2,3-d]pyrimidines followed by chlorination and substitution at the 4-position with various anilines. Five additional compounds of this series were previously reported by Gangjee et al.(1) with activities against IGFR only. Their synthesis, characterization and biological activities against a variety of other RTKs are reported in this study for the first time. The biological evaluation, in whole cell assays, showed several analogs had remarkable inhibitory activity against epithelial growth factor receptor (EGFR), vascular endothelial growth factor receptor-1 (VEGFR-1), platelet-derived growth factor receptor-beta (PDGFR-beta), the growth of A431 cells in culture, and in the chicken embryo chorioallantoic membrane (CAM) angiogenesis assay. The inhibitory data against the RTKs in this study demonstrate that variation of the 6-ethylaryl substituents as well as the N(4)-phenyl substituents of these analogs does indeed control both the potency and specificity of inhibitory activity against RTKs. In addition, homologation of the chain length of the 6-substituent from a methylene to an ethyl increases the spectrum of RTK inhibition. New multi-RTK inhibitors (8, 12) and potent inhibitors of angiogenesis (15, 19) were identified with the best compound, N(4)-(3-trifluromethylphenyl)-6-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (15), with an IC(50) value of 30nM in the CAM angiogenesis inhibition assay.

Laboratory diet profoundly alters gene expression and confounds genomic analysis in mouse liver and lung.

Nutritional studies in laboratory animals have long shown that various dietary components can contribute to altered gene expression and metabolism, but diet alone has not been considered in whole animal genomic studies. In this study, global gene expression changes in mice fed either a non-purified chow or a purified diet were investigated and background metal levels in the two diets were measured by ICP-MS. C57BL/6J mice were raised for 5 weeks on either the cereal-based, non-purified LRD-5001 diet or the purified, casein-based AIN-76A diet, as part of a larger study examining the effects of low dose arsenic (As) in the diet or drinking water. Affymetrix Mouse Whole Genome 430 2.0 microarrays were used to assess gene expression changes in the liver and lung. Microarray analysis revealed that animals fed the LRD-5001 diet displayed a significantly higher hepatic expression of Phase I and II metabolism genes as well as other metabolic genes. The LRD-5001 diet masked the As-induced gene expression changes that were clearly seen in the animals fed the AIN-76A diet when each dietary group was exposed to 100 ppb As in drinking water. Trace metal analysis revealed that the LRD-5001 diet contained a mixture of inorganic and organic As at a total concentration of 390 ppb, while the AIN-76A diet contained approximately 20 ppb. These findings indicate that the use of non-purified diets may profoundly alter observable patterns of change induced by arsenic and, likely, by other experimental treatments, particularly, altering gene and protein expression.

Oscillating glucose is more deleterious to endothelial function and oxidative stress than mean glucose in normal and type 2 diabetic patients.

OBJECTIVE: To explore the possibility that oscillating glucose may outweigh A1C levels in determining the risk for cardiovascular diabetes complications. RESEARCH DESIGN AND METHODS: A euinsulinemic hyperglycemic clamp at 5, 10, and 15 mmol/l glucose was given in increasing steps as a single "spike" or oscillating between basal and high levels over 24 h in normal subjects and type 2 diabetic patients. Flow-mediated dilatation, a marker of endothelial function, and plasma 3-nitrotyrosine and 24-h urinary excretion rates of free 8-iso PGF2 alpha, two markers of oxidative stress, were measured over 48 h postclamp. RESULTS: Glucose at two different levels (10 and 15 mmol/l) resulted in a concentration-dependent fasting blood glucose-independent induction of both endothelial dysfunction and oxidative stress in both normal and type 2 diabetic patients. Oscillating glucose between 5 and 15 mmol/l every 6 h for 24 h resulted in further significant increases in endothelial dysfunction and oxidative stress compared with either continuous 10 or 15 mmol/l glucose. CONCLUSIONS: These data suggest that oscillating glucose can have more deleterious effects than constant high glucose on endothelial function and oxidative stress, two key players in favoring cardiovascular complications in diabetes. Concomitant vitamin C infusion can reverse this impairment.

Exposure to arsenic at levels found inU.S. drinking water modifies expression in the mouse lung.

The mechanisms of action of drinking water arsenic in the lung and the threshold for biologic effects remain controversial. Our study utilizes Affymetrix 22,690 transcript oligonucleotide microarrays to assess the long-term effects of increasing doses of drinking water arsenic on expression levels in the mouse lung. Mice were exposed at levels commonly found in contaminated drinking water wells in the United States (0, 0.1, 1 ppb), as well as the 50 ppb former maximum contaminant level, for 5 weeks. The expression profiles revealed modification of a number of important signaling pathways, many with corroborating evidence of arsenic responsiveness. We observed statistically significant expression changes for transcripts involved in angiogenesis, lipid metabolism, oxygen transport, apoptosis, cell cycle, and immune response. Validation by reverse transcription-PCR and immunoblot assays confirmed expression changes for a subset of transcripts. These data identify arsenic-modified signaling pathways that will help guide investigations into mechanisms of arsenic's health effects and clarify the threshold for biologic effects and potential disease risk.

A long-term "memory" of HIF induction in response to chronic mild decreased oxygen after oxygen normalization.

BACKGROUND: Endothelial dysfunction (ED) is functionally characterized by decreased vasorelaxation, increased thrombosis, increased inflammation, and altered angiogenic potential, has been intimately associated with the progression and severity of cardiovascular disease. Patients with compromised cardiac function oftentimes have a state of chronic mild decreased oxygen at the level of the vasculature and organs, which has been shown to exacerbate ED. Hypoxia inducible factor (HIF) is a transcription factor complex shown to be the master regulator of the cellular response to decreased oxygen levels and many HIF target genes have been shown to be associated with ED. METHODS: Human endothelial and aortic smooth muscle cells were exposed either to A) normoxia (21% O2) for three weeks, or to B) mild decreased oxygen (15% O2) for three weeks to mimic blood oxygen levels in patients with heart failure, or to C) mild decreased oxygen for two weeks followed by one week of normoxia ("memory" treatment). Levels of HIF signaling genes (HIF-1alpha, HIF-2alpha, VEGF, BNIP3, GLUT-1, PAI-1 and iNOS) were measured both at the protein and mRNA levels. RESULTS: It was found that chronic exposure to mild decreased oxygen resulted in significantly increased HIF signaling. There was also a "memory" of HIF-1alpha and HIF target gene induction when oxygen levels were normalized for one week, and this "memory" could be interrupted by adding a small molecule HIF inhibitor to the last week of normalized oxygen. Finally, levels of ubiquitylated HIF-1alpha were reduced in response to chronic mild decreased oxygen and were not full restored after oxygen normalization. CONCLUSION: These data suggest that HIF signaling may be contributing to the pathogenesis of endothelial dysfunction and that normalization of oxygen levels may not be enough to reduce vascular stress.

Mutant p53 facilitates pro-angiogenic, hyperproliferative phenotype in response to chronic relative hypoxia.

There is much controversy in the literature regarding the role of p53 status response on hypoxia inducible factor (HIF) signaling in response to chronic relative hypoxia (CRH). The goal of this paper was to methodically examine this response in isogenically matched tumor cells. We report that p53-mutant (MUT) cells, versus p53-wild-type (WT) cells, showed decreased apoptosis, increased cell proliferation with higher basal HIF-1alpha levels in response to CRH. In addition, we found increased HIF-mediated transactivation and increased VEGF release with decreased HIF-1alpha/p53 and HIF-1alpha/MDM-2 partnering in p53-MUT versus p53-WT cells in response to CRH.