Faton Agani

Hypoxia, HIF-1, and the pathophysiology of common human diseases

Hypoxia plays a fundamental role in the pathophysiology of common causes of mortality, including ischemic heart disease, stroke, cancer, chronic lung disease, and congestive heart failure. In these disease states, hypoxia induces changes in gene expression in target organs that either fail to result in adequate adaptation or directly contribute to disease pathogenesis. Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that is expressed in response to cellular hypoxia and mediates multiple cellular and systemic homeostatic responses to hypoxia. Recent studies have provided evidence that important pathophysiological responses to hypoxia in pulmonary hypertension, myocardial ischemia, and cancer are mediated by HIF-1. Pharmacologic and gene therapy strategies designed to modulate HIF-1 activity may represent a novel and effective therapeutic approach to these common disorders.

The Role of Mitochondria in the Regulation of Hypoxia-inducible Factor 1 Expression during Hypoxia

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that regulates transcriptional activation of several genes responsive to the lack of oxygen, including erythropoietin, vascular endothelial growth factor, glycolytic enzymes, and glucose transporters. Because the involvement of mitochondria in the regulation of HIF-1 has been postulated, we tested the effects of mitochondrial electron transport chain deficiency on HIF-1 protein expression and DNA binding in hypoxic cells. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) inhibits electron transport chain at the level of complex I. MPTP is first converted to a pharmacologically active metabolite 1-methyl-4-phenylpyridinum (MPP+). MPP+ effectively inhibited both complex I activity and hypoxic accumulation of HIF-1α protein in dopaminergic cell lines PC12 and CATH.a. In C57BL/6 mice, a single dose of MPTP (15 mg/kg, intraperitoneal) inhibited complex I activity and HIF-1α protein accumulation in the striatum in response to a subsequent hypoxic challenge (8% O2, 4 h). In a genetic model system, 40% complex I-inhibited human-ape xenomitochondrial cybrids, hypoxic induction of HIF-1α was severely reduced, and HIF-1 DNA binding was diminished. However, succinate, the mitochondrial complex II substrate, restored the hypoxic response in cybrid cells, suggesting that electron transport chain activity is required for activation of HIF-1. A partial complex I deficiency and a mild reduction in intact cell oxygen consumption effectively prevented hypoxic induction of HIF-1α protein.

Nitric oxide in the sensory function of the carotid body

Recent studies suggest that nitric oxide (NO) may act as a chemical messenger in the nervous system. Since neurotransmitters are considered necessary for the sensory function of the carotid body, and molecular O2 is a co-factor for NO synthesis, we examined whether (a) chemoreceptor tissue also synthesizes NO and if so, (b) does endogenous NO affect chemosensory activity. Experiments were performed on carotid bodies obtained from anesthetized cats (n = 20). Distribution of nitric oxide synthase (NOS), an enzyme that catalyzes the formation of NO was examined using NADPH-diaphorase histochemistry. Many nerve plexuses innervating the chemoreceptor tissue were positive for NADPH-diaphorase, indicating that the nerve fibers are the primary source of NO production in the carotid body. Radiometric analysis of NOS activity of the chemoreceptor tissue averaged 1.94 pmol [3H]citrulline/min/mg protein. NOS activity was significantly less in low pO2 reaction medium than in room air controls. Chemosensory activity in vitro increased in a dose-dependent manner in response to L-omega-nitro arginine (L-NNA), an inhibitor of NOS activity. The effects of NOS inhibitor were enantiomer selective as evidenced by reversal of the responses by L- but not D-arginine. These observations imply that endogenous NO is inhibitory to carotid body sensory activity. cGMP levels of L-NNA-treated carotid bodies were significantly less than untreated controls, suggesting that the actions of NO are coupled to the cGMP second messenger system, as elsewhere in the nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Cardiovascular Development and Physiology by Hypoxia‐Inducible Factor 1a

Abstract: Hypoxia is an essential pathophysiologic component of ischemic cardiovascular disease. A better understanding of the molecular mechanisms underlying adaptive responses to hypoxia may lead to novel therapeutic strategies. Hypoxia‐inducible factor 1 (HIF‐1) is a heterodimeric basic‐helix‐loop‐helix‐PAS domain transcription factor that mediates changes in gene expression in response to changes in O2 concentration. Genes that are transcriptionally activated by HIF‐1 in hypoxic cells encode proteins that increase O2 delivery or allow metabolic adaptation to limited O2 availability. HIF‐1 target genes include those encoding vascular endothelial growth factor (VEGF), erythropoietin, glucose transporters, and glycolytic enzymes. In anemic fetal sheep, increased myocardial vascularization was associated with concomitant increases in the expression of HIF‐1 and VEGF. Expression of HIF‐1 target genes was not induced by hypoxia in embryonic stem cells lacking expression of the O2‐regulated HIF‐1αa subunit. Mouse embryos lacking HIF‐1α expression arrested in their development by E9.0 and died by E10.5 with cardiovascular malformations and massive cell death throughout the embryo. These studies indicate that HIF‐1 functions as a master regulator of O2 homeostasis that controls the establishment of essential physiologic systems during embryogenesis as well as their subsequent utilization during fetal and postnatal life.

Cadmium Increases HIF-1 and VEGF Expression through ROS, ERK, and AKT Signaling Pathways and Induces Malignant Transformation of Human Bronchial Epithelial Cells

Cadmium is categorized as a human carcinogen especially involved in lung cancers. Angiogenesis is considered a fundamental requirement for tumorigenesis, but the mechanisms underlying the tumor angiogenesis induced by cadmium are poorly understood. Using in vitro and in vivo models, we investigated the angiogenic mechanisms of cadmium in human bronchial epithelial cells and tumor formation. Our results demonstrated that cadmium (CdCl(2)) activated extracellular signal-regulated kinases (ERK) and AKT signaling and elevated the expression of a key downstream proangiogenic molecule hypoxia-inducible factor-1 (HIF-1) in immortalized human lung epithelial BEAS-2B cells. Cadmium also induced reactive oxygen species (ROS) production, which could be inhibited by ROS scavengers, catalase and diphenyleneiodonium chloride. Inhibition of ROS generation also attenuated ERK, AKT, p70S6K1 activation, and HIF-1α expression. Similar results were obtained in normal human bronchial epithelial (NHBE) cells, showing that cadmium induced HIF-1 expression via ROS/ERK/AKT signaling pathway. Furthermore, cadmium induced vascular endothelial growth factor expression and transcriptional activation through ROS, ERK, and AKT pathways. Finally, cadmium transformed human bronchial epithelial cells in culture; the transformed cells induced tube formation in vitro, angiogenesis on chicken chorioallantoic membrane, and formed tumors in nude mice. Taken together, the results of this study provide explanation for the role and molecular mechanisms of cadmium in promoting angiogenesis in lung epithelial cells and malignant transformation and will be helpful for improved occupational protection, prevention, as well as chemotherapy of human lung cancers caused by heavy metal cadmium.

Oxygen-independent regulation of HIF-1: novel involvement of PI3K/AKT/mTOR pathway in cancer.

Studies on erythropoietin regulation led to discovery of hypoxia-inducible factor 1 (HIF-1), a transcription factor which is central component of oxygen sensing mechanism in mammalian cells. The number of HIF-1 and hypoxia-regulated target genes has grown exponentially and includes genes that encode proteins with roles in erythropoiesis, angiogenesis, glycolytic pathway, glucose transport, metastasis, and cell survival. Thus, HIF-1 claimed the role of the master that orchestrates cellular responses to oxygen deprivation. In addition, HIF-1 is also activated or influenced through oxygen-independent mechanisms via growth factors, deregulated oncogenes, and/or tumor suppressors. Whereas HIF prolyl hydroxylases (PHDs) regulate HIF-1 (and subsequently identified HIF-2) during hypoxia, the PI3K, AKT and MAPK pathways mediate primarily non-hypoxic HIF regulation. Here we will focus primarily on pathways that lead to HIF activation via PI3K/AKT, and mTOR/p70S6K1. In addition, recent studies have revealed novel factors and mechanisms that regulate oxygen-independent HIF-1α and HIF-2α degradation. HIFs play important roles in many processes in health and disease. Consequently, HIFs and pathways (PI3K/AKT and mTOR/p70S6K1) that lead to normoxic HIF activation are considered potential therapeutic targets in these pathologies.

Reactive oxygen species regulate ERBB2 and ERBB3 expression via miR-199a/125b and DNA methylation.

Overexpression of ERBB2 or ERBB3 is associated with cancer development and poor prognosis. In this study, we show that reactive oxygen species (ROS) induce both ERBB2 and ERBB3 expression in vitro and in vivo. We also identify that miR‐199a and miR‐125b target ERBB2 and/or ERBB3 in ovarian cancer cells, and demonstrate that ROS inhibit miR‐199a and miR‐125b expression through increasing the promoter methylation of the miR‐199a and miR‐125b genes by DNA methyltransferase 1. These findings reveal that ERBB2 and ERBB3 expression is regulated by ROS via miR‐199a and miR‐125b downregulation and DNA hypermethylation.

Mdm2 and HIF-1α interaction in tumor cells during hypoxia

The interaction between HIF‐1α, Mdm2, and p53 proteins during hypoxia has received recent attention. Here, we investigated the consequences of interaction between HIF‐1α and Mdm2 under hypoxic conditions. Endogenous HIF‐1α and Mdm2 proteins were co‐immunoprecipitated from lysates of hypoxic HCT116 p53WT and p53−/− cells, suggesting that association of these two proteins is a p53‐independent event. The cellular Mdm2 protein content was not significantly altered in hypoxic tumor cells. Overexpression of Mdm2 resulted in an increase in HIF‐1α protein content in hypoxic cells and increased hypoxia‐induced vascular endothelial growth factor (VEGF) transcriptional activation. These results point toward a novel and p53‐independent function of Mdm2 to promote tumor cell adaptations to hypoxia by interacting with and promoting HIF‐1 activation.

Effects of Hyperoxia on Nitric Oxide Synthase Expression, Nitric Oxide Activity, and Lung Injury in Rat Pups

Although hyperoxic exposure is an important contributor to the development of bronchopulmonary dysplasia and nitric oxide (NO) has been implicated in the pulmonary response to oxygen, the role of NO in mediating chronic neonatal lung injury is unclear. Therefore, rat pups were exposed to normoxia or hyperoxia (>95% O2) from d 21 to 29. After the rats were killed, their lungs were removed for analysis of nitric oxide synthase (NOS) expression, NO activity as measured by 3′,5′-cyclic guanosine monophosphate (cGMP) assay, and lung pathology. Hyperoxia caused 5-fold and 2-fold increases in inducible (i) NOS and endothelial (e) NOS levels, respectively. NO activity was assessed by measuring cGMP levels after normoxic or hyperoxic exposure in the presence and absence of NOS blockade with either aminoguanidine (AG) or Nω-nitro-L-arginine (L-NNA). cGMP levels were elevated in hyperoxic versus normoxic rats (287 ± 15 versus 106 ± 9 pmol/mg protein, respectively, p < 0.001), and this increase in cGMP was attenuated after NOS blockade with either AG or L-NNA. Hyperoxic exposure significantly increased lung/body weight ratios and induced histologic changes of interstitial and alveolar edema; however, these hyperoxia-induced histologic changes were not altered by NOS blockade with AG or L-NNA. We conclude that hyperoxic exposure of rat pups up-regulated both iNOS and eNOS and increased NO activity as measured by cGMP levels derived from both iNOS and eNOS. Blockade of NOS reduced cGMP levels in the hyperoxic rat pups; however, it did not seem to reverse the pathologic consequences of hyperoxic exposure.

Heterogeneity in cytosolic calcium responses to hypoxia in carotid body cells.

Previous investigators have reported that intracellular pH responds to hypoxia with a heterogenous pattern in individual glomus cells of the carotid body. The aim of the present study was to examine whether hypoxia had similar effects on cytosolic calcium ([Ca2+]i) in glomus cells, and if so, whether a heterogenous response pattern is also seen in other cell types. Experiments were performed on glomus cells from adult rat carotid bodies, rat pheochromocytoma (PC12) and vascular smooth muscle (A7r5) cells. Changes in [Ca2+]i in individual cells were determined by fluorescence imaging using Fura-2. Glomus cells were identified by catecholamine fluorescence. [Ca2+]i in glomus cells increased in response to hypoxia (pO2 = 35 +/- 8 mmHg; 5 min), whereas hypoxia induced decreases in [Ca2+]i were not seen. Increases in [Ca2+]i were observed in 20% of the isolated cells and strings of cells, but clustered glomus cells never responded. The magnitude of the calcium change in responding cells was proportional to the hypoxic stimulus. Under a given hypoxic challenge, there were marked variations in the response pattern between glomus cells. The response pattern characteristic of any given cell was reproducible. At comparable levels of hypoxia, PC12 cells also responded with an increase in [Ca2+]i with a heterogenous response pattern similar to that seen in glomus cells. In contrast, increases in [Ca2+]i in A7r5 cells could be seen only with sustained hypoxia (approximately 20 min), and little heterogeneity in the response patterns was evident. These results demonstrate that: (a) hypoxia increases cytosolic calcium in glomus cells; (b) response patterns were heterogeneous in individual cells; and (c) the pattern of the hypoxia-induced changes in [Ca2+]i is cell specific. These results suggest that hypoxia-induced increases in [Ca2+]i are faster in secretory than in non-secretory cells.