Targeting Axl favors an antitumorigenic microenvironment that enhances immunotherapy responses by decreasing Hif-1α levels.

Hypoxia is an important phenomenon in solid tumors that contributes to metastasis, tumor microenvironment (TME) deregulation, and resistance to therapies. The receptor tyrosine kinase AXL is an HIF target, but its roles during hypoxic stress leading to the TME deregulation are not well defined. We report here that the mammary gland-specific deletion of Axl in a HER2+ mouse model of breast cancer leads to a normalization of the blood vessels, a proinflammatory TME, and a reduction of lung metastases by dampening the hypoxic response in tumor cells. During hypoxia, interfering with AXL reduces HIF-1α levels altering the hypoxic response leading to a reduction of hypoxia-induced epithelial-to-mesenchymal transition (EMT), invasion, and production of key cytokines for macrophages behaviors. These observations suggest that inhibition of Axl generates a suitable setting to increase immunotherapy. Accordingly, combining pharmacological inhibition of Axl with anti-PD-1 in a preclinical model of HER2+ breast cancer reduces the primary tumor and metastatic burdens, suggesting a potential therapeutic approach to manage HER2+ patients whose tumors present high hypoxic features.

Selective HIF-1 Regulation under Nonhypoxic Conditions by the p42/p44 MAP Kinase Inhibitor PD184161.

Hypoxia-inducible factor-1 (HIF-1) is a key gene regulator for cellular adaptation to low oxygen. In addition to hypoxia, several nonhypoxic stimuli, including hormones and growth factors, are essential for cell-specific HIF-1 regulation. Our studies have highlighted angiotensin II (AngII), a vasoactive hormone, as a potent HIF-1 activator in vascular smooth muscle cells (VSMC). AngII increases HIF-1 transcriptional activity by modulating specific signaling pathways. In VSMC, p42/p44 mitogen-activated protein kinase (MAPK) pathway activation is essential for HIF-1-mediated transcription during AngII treatment. The present study shows that PD184161, a potent MEK1/2 inhibitor, is an HIF-1 blocker in Ang II-treated VSMC. Unlike PD98059, a widely-used MEK1/2 inhibitor, we found that PD184161 blocked AngII-driven HIF-1α protein induction in a dose-dependent manner. Interestingly, the effect of PD184161 was specific to nonhypoxic activators, since HIF-1α induction by hypoxia (1% O2) was unaffected under similar conditions. VSMC treatment with MG132, a proteasome inhibitor, indicated that PD184161 influenced HIF-1α protein stability. PD184161 also increased HIF-1α binding to von Hippel-Lindau tumor suppressor protein, an E3 ligase component and an indication of HIF-1α hydroxylation. Finally, we show that PD184161 blocked mitochondrial ROS (mtROS) production and cellular ATP levels, at the same time enhancing ascorbate availability in AngII-treated VSMC. Taken together, our study indicates that, independently of p42/p44 MAPK activation, PD184161 blocks mtROS generation by AngII, leading to re-establishment of cellular ascorbate levels, increased VHL binding, and decreased HIF-1α stability. Therefore, this study reveals a previously unsuspected role for PD184161 as an HIF-1 inhibitor in VSMC under nonhypoxic conditions.

Hypoxia-inducible factor-1 plays a role in phosphate-induced vascular smooth muscle cell calcification.

Medial vascular calcification is a common complication of chronic kidney disease (CKD). Although elevated inorganic phosphate stimulates vascular smooth muscle cell (VSMC) osteogenic transdifferentiation and calcification, the mechanisms involved in their calcification during CKD are not fully defined. Because hypoxic gene activation is linked to CKD and stimulates bone cell osteogenic differentiation, we used in vivo and in vitro rodent models to define the role of hypoxic signaling during elevated inorganic phosphate-induced VSMC calcification. Cell mineralization studies showed that elevated inorganic phosphate rapidly induced VSMC calcification. Hypoxia strongly enhanced elevated inorganic phosphate-induced VSMC calcification and osteogenic transdifferentiation, as seen by osteogenic marker expression. Hypoxia-inducible factor-1 (HIF-1), the key hypoxic transcription factor, was essential for enhanced VSMC calcification. Targeting HIF-1 expression in murine VSMC blocked calcification in hypoxia with elevated inorganic phosphate while HIF-1 activators, including clinically used FG-4592/Roxadustat, recreated a procalcifying environment. Elevated inorganic phosphate rapidly activated HIF-1, even in normal oxygenation; an effect mediated by HIF-1α subunit stabilization. Thus, hypoxia synergizes with elevated inorganic phosphate to enhance VSMC osteogenic transdifferentiation. Our work identifies HIF-1 as an early CKD-related pathological event, prospective marker, and potential target against vascular calcification in CKD-relevant conditions.

The Werner syndrome gene product (WRN): a repressor of hypoxia-inducible factor-1 activity.

Werner syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of genes essential for adaptation to low oxygen conditions. HIF-1 is also implicated in the molecular mechanisms of ageing. Here, we show that the cellular depletion of WRN protein (by siRNA targeting) leads to increased HIF-1 complex stabilization and activation. HIF-1 activation in the absence of WRN involves the generation of mitochondrial reactive oxygen species (mtROS) since SkQ1, a mitochondrial-targeted antioxidant, and stigmatellin, an inhibitor of mitochondrial complex III, blocked increased HIF-1 levels. Ascorbate, an essential co-factor involved in HIF-1 stability, was decreased in WRN-depleted cells. Interestingly, expression levels of GLUT1, a known dehydroascorbic acid transporter, were also decreased in WRN-depleted cells. Ascorbate supplementation of WRN-depleted cells led to a dose-dependent inhibition of HIF-1 activation. These results indicate that WRN protein regulates HIF-1 activation by affecting mitochondrial ROS production and intracellular ascorbate levels. This work provides a novel mechanistic link between HIF-1 activity and different age-associated pathologies.

Antihypertensive treatment with hydrochlorothiazide-hydralazine combination aggravates medial vascular calcification in CKD rats with mineral bone disorder.

Background: Arterial stiffness and medial vascular calcification, leading to isolated systolic blood pressure (BP), are major cardiovascular risk factors in patients with chronic kidney disease (CKD) and mineral bone disorders (MBD). The impact of BP on MBD-induced medial vascular calcification in CKD remains uncertain. We investigated whether BP reduction improves arterial stiffness and medial vascular calcification in a rat model of CKD-MBD. Methods: CKD was induced in Wistar rats by subtotal nephrectomy. Then, MBD was generated by a Ca/P-rich diet with calcitriol supplementation to induce medial vascular calcification. Two antihypertensive treatments were evaluated: (1) the angiotensin AT1 receptor antagonist losartan, and (2) the combination of the thiazide diuretic hydrochlorothiazide and the direct vasodilator hydralazine (HCTZ/HY). After 5 weeks, mean BP (MBP), pulse pressure (PP), and pulse wave velocity (PWV) were determined. Vascular calcification was assessed in the thoracic aorta. Results: While MBP was similar in CKD-MBD and control CKD rats, PP and PWV were increased in CKD-MBD rats. The heightened arterial stiffness in CKD-MBD rats was associated with diffused medial calcification along the thoracic aorta. Although both losartan and HCTZ/HY reduced MBP in CKD-MBD rats, losartan did not affect PP and PWV nor medial vascular calcification, whereas HCTZ/HY, unexpectedly, further increased arterial stiffness and medial vascular calcification. Conclusion: In the rat model of CKD-MBD, antihypertensive treatment with losartan did not affect arterial stiffness or medial vascular calcification. However, HCTZ/HY treatment aggravated arterial stiffness and vascular calcification despite a similar reduction of MBP, suggesting a blood pressure-independent mechanism for vascular calcification.

Tumor necrosis factor inhibitors as novel therapeutic tools for vascular remodeling diseases.

Vascular remodeling diseases (VRDs) are characterized by enhanced inflammation and proliferative and apoptosis-resistant vascular smooth muscle cells (VSMCs). The sustainability of this phenotype has been attributed in part to the activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). There is evidence that circulating cytokines can act as HIF-1 activators in a variety of tissues, including VSMCs. Increased circulating tumor necrosis factor (TNF) levels have been associated with vascular diseases, but the mechanisms involved remain unknown. We hypothesized that increased circulating levels of TNF promotes VRDs by the activation of HIF-1, resulting in VSMC proliferation and resistance to apoptosis. Circulating TNF levels were significantly increased in patients with vascular diseases (n = 19) compared with healthy donors (n = 15). Using human carotid artery smooth muscle cells (CASMCs), we demonstrated that TNF (100 ng/ml) activates HIF-1 (HIF-1α expression), leading to increased CASMC proliferation (Ki-67 and PCNA staining) and resistance to mitochondrial-dependent apoptosis [tetramethylrhodamine methyl ester perchlorate (TMRM), terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL), annexin-V staining]. In vivo, TNF inhibition using polyethylene glycol coupled with TNF membrane receptor 1 (PEGsTNFR1), a soluble TNF receptor inhibiting circulating TNF, prevented carotid artery postinjury media remodeling and neointima development in rats. This effect was associated with lowered HIF-1 activation and decreased CASMC proliferation. In conclusion, we demonstrate for the first time that the inhibition of the TNF/Akt/HIF-1 axis prevents vascular remodeling. TNF inhibitors may therefore represent new and interesting therapeutic tools against VRDs.

Sphingosine-1-phosphate: a novel nonhypoxic activator of hypoxia-inducible factor-1 in vascular cells.

OBJECTIVE: Sphingosine-1-phosphate (S1P) is a potent bioactive phospholipid responsible for a variety of vascular cell responses. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of genes essential for adaptation to low oxygen. S1P and HIF-1 are both important mediators of vascular cell responses such as migation, proliferation, and survival. Studies have shown that nonhypoxic stimuli can activate HIF-1 in oxygenated conditions. Here, we attempt to determine whether S1P can modulate the vascular activation of HIF-1. METHODS AND RESULTS: We show that in vascular endothelial and smooth muscle cells, activation of the S1P type-2 receptor by S1P strongly increases HIF-1 alpha protein levels, the active subunit of HIF-1. This is achieved through pVHL-independent stabilization of HIF-1 alpha. We demonstrate that the HIF-1 nuclear complex, formed on S1P stimulation, is transcriptionally active and specifically binds to a hypoxia-responsive elements. Moreover, S1P activates the expression of genes known to be closely regulated by HIF-1. CONCLUSIONS: Our results identify S1P as a novel and potent nonhypoxic activator of HIF-1. We believe that understanding the role played by HIF-1 in S1P gene regulation will have a strong impact on different aspects of vascular biology.

The adenoviral protein E4orf4: a probing tool to decipher mechanical stress-induced nuclear envelope remodeling in tumor cells.

The human adenovirus (Ad) type 2/5 early region 4 (E4) ORF4 protein (E4orf4) exerts a remarkable tumor cell-selective killing activity in mammalian cells. This indicates that E4orf4 can target tumor cell-defining features and is a unique tool to probe cancer cell vulnerabilities. Recently, we found that E4orf4, through an interaction with the polarity protein PAR3, subverts nuclear envelope (NE) remodeling processes in a tumor cell-selective manner. In this Perspective, we outline mechanical signals that modify nuclear dynamics and tumor cell behavior to highlight potential mechanisms for E4orf4's tumoricidal activity. Through an analysis of E4orf4's cellular targets, we define a protein subnetwork that comprises phosphatase systems interconnected to polarity protein hubs, which could contribute to enhanced NE plasticity. We infer that elucidating E4orf4's protein network at a functional level could uncover key mechanisms of NE remodeling that define the tumor cell phenotype.

Adenoviral protein E4orf4 interacts with the polarity protein Par3 to induce nuclear rupture and tumor cell death.

The tumor cell-selective killing activity of the adenovirus type 2 early region 4 ORF4 (E4orf4) protein is poorly defined at the molecular level. Here, we show that the tumoricidal effect of E4orf4 is typified by changes in nuclear dynamics that depend on its interaction with the polarity protein Par3 and actomyosin contractility. Mechanistically, E4orf4 induced a high incidence of nuclear bleb formation and repetitive nuclear ruptures, which promoted nuclear efflux of E4orf4 and loss of nuclear integrity. This process was regulated by nucleocytoskeletal connections, Par3 clustering proximal to nuclear lamina folds, and retrograde movement of actin bundles that correlated with nuclear ruptures. Significantly, Par3 also regulated the incidence of spontaneous nuclear ruptures facilitated by the downmodulation of lamins. This work uncovered a novel role for Par3 in controlling the actin-dependent forces acting on the nuclear envelope to remodel nuclear shape, which might be a defining feature of tumor cells that is harnessed by E4orf4.

(2R)-[(4-Biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide (BiPS), a matrix metalloprotease inhibitor, is a novel and potent activator of hypoxia-inducible factors.

Hypoxia-inducible factors (HIFs) are unstable heterodimeric transcription factors and decisive elements for the transcriptional regulation of genes important in the adaptation to low-oxygen conditions. Hypoxia is the ubiquitous inducer of HIFs, stabilizing the alpha-subunit and permitting the formation of a functional HIF complex. Here, we identify (2R)-[(4-biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide (BiPS), a commercially available metalloprotease-2 and -9 inhibitor, as a rapid and potent inducer of HIFs. We show that in different cell lines, BiPS induces the HIF-alpha subunit by inhibiting its degradation through stabilization of its labile oxygen-dependent degradation domain. This is achieved through the inhibition of HIF-1alpha hydroxylation. The HIF-1 complex, formed after BiPS treatment, is capable of DNA binding and activation of HIF target genes, including the expression of vascular endothelial growth factor. Because novel HIF activators have generated considerable interest in the possible treatment of different ischemic diseases, we believe that BiPS and derivative molecules could have strong therapeutic potential.

Differential regulation of hypoxia-inducible factor-1 through receptor tyrosine kinase transactivation in vascular smooth muscle cells.

Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of many genes expressed in hypoxic conditions. In vascular smooth muscle cells, the vasoactive hormone angiotensin II (Ang II) is a very potent inducer and activator of HIF-1. As opposed to hypoxia, which induces HIF-1alpha by protein stabilization, Ang II induced HIF-1alpha through transcriptional and translational mechanisms. Interestingly, a number of intracellular signaling events triggered by Ang II are mediated by the transactivation of receptor tyrosine kinases. The major receptor tyrosine kinases shown to be transactivated by Ang II in vascular smooth muscle cells are the epidermal growth factor receptor and the IGF-I receptor. In this study, we demonstrate that the transactivation of both these receptor tyrosine kinases is involved in HIF-1 complex activation by Ang II. More interestingly, this modulation of HIF-1 is at different degrees and through different pathways. Our results show that transactivation of IGF-I receptor is essential for HIF-1alpha protein translation through phosphatidylinositol 3-kinase/p70S6 kinase pathway activation, and epidermal growth factor receptor transactivation is implicated in HIF-1 complex activation through the stimulation of the p42/p44 MAPK pathway. Our results therefore show that Ang II-induced receptor tyrosine kinase transactivation is essential in both the induction and activation of HIF-1. These findings identify novel and intricate signaling mechanisms involved in HIF-1 complex activation.

Endothelin type A receptor blockade reduces vascular calcification and inflammation in rats with chronic kidney disease.

OBJECTIVE: Arterial stiffness and calcification are nontraditional cardiovascular risk factors in chronic kidney disease (CKD). Using a rat model of CKD with mineral imbalance, medial vascular calcification has been associated with inflammation and increased endothelin-1 (ET-1) production. We therefore hypothesized that ET-1, through the endothelin type A (ETA) receptor, induces vascular inflammation, calcification and stiffness in CKD. METHODS: CKD was induced in Wistar rats by renal mass ablation. To induce medial vascular calcification, mineral imbalance was established with a identified as calcium-rich/phosphate-rich diet and vitamin D supplementation (Ca/P/VitD). One group of CKD + Ca/P/VitD rats was given the ETA receptor antagonist atrasentan (10 mg/kg/day) for 6 weeks. Hemodynamic parameters including SBP, pulse pressure (PP) and pulse wave velocity (PWV) were determined. Vascular calcification, smooth muscle cells osteoblastic differentiation and expression of inflammatory markers such as inflammatory cytokines and calgranulins S100A8 and S100A9 were assessed in the thoracic aorta. RESULTS: As compared with CKD control rats, CKD + Ca/P/VitD rats developed medal vascular calcification that was associated with increased SBP, PP and PWV. These changes were also associated with increased macrophage infiltration and expression of IL-6, calgranulins and osteoblastic markers. Treatment of CKD + Ca/P/VitD rats with atrasentan reduced vascular calcification, SBP, PP and PWV, macrophage infiltration and expression of IL-1ß, IL-6, tumor necrosis factor, calgranulins and osteoblastic markers. CONCLUSION: This study shows that ETA receptor blockade reduced vascular inflammation, smooth muscle cells differentiation, calcification and stiffness indicating a pivotal role for ET-1 in medial vascular calcification in this rat remnant kidney model of CKD with mineral imbalance. Therefore, the endothelin system may be a potential therapeutic target for improving cardiovascular morbidity in patients with CKD.

Hypoxia-inducible factor 1: regulation by hypoxic and non-hypoxic activators.

Oxygen availability is crucial for cellular metabolism. Hypoxia-inducible factor 1 (HIF-1) is the major oxygen homeostasis regulator. Under normoxic conditions, HIF-1 is rapidly degraded by the proteasome. However, under hypoxic conditions, HIF-1 is stabilized and permits the activation of genes essential to cellular adaptation to low oxygen conditions. These genes include the vascular endothelial growth factor (VEGF), erythropoietin and glucose transporter-1. There is increasing evidence showing that HIF-1 is also implicated in biological functions requiring its activation under normoxic conditions. Amongst others, growth factors and vascular hormones are implicated in this normoxic activation. In this review, we will focus on differences between hypoxic and non-hypoxic induction and activation of HIF-1. We will also discuss the biological functions of HIF-1 associated with these two induction pathways. The clear understanding of both HIF-1 activation mechanisms could have a major impact in cancer and vascular disease.

Inflammatory cytokines and reactive oxygen species as mediators of chronic kidney disease-related vascular calcification.

BACKGROUND: Vascular calcification, a regulated process in chronic kidney disease (CKD), requires vascular smooth muscle cell (VSMC) differentiation into osteoblast-like cells. This phenomenon can be enhanced by inflammatory cytokines and production of reactive oxygen species (ROS). In CKD rats with vascular calcification, we investigated whether inflammatory cytokines, ROS generation, and downstream signaling events are associated with CKD-related vascular calcification. METHODS: CKD was induced in male Wistar rats by renal mass ablation and vascular calcification was induced with a high calcium-phosphate diet and vitamin D supplementation (Ca/P/VitD). At week 3-6, hemodynamic parameters were determined and thoracic aorta was harvested for assessment of vascular calcification, macrophage infiltration, cytokines expression, VSMC differentiation, ROS generation, and related signaling pathway activation. RESULTS: CKD rats treated with Ca/P/VitD developed medial calcification of thoracic aorta and increased pulse pressure and aortic pulse wave velocity. VSMC differentiation was confirmed by increased bone morphogenetic protein-2 and osteocalcin expression and reduced α-smooth muscle actin expression. The expression of interleukin-1ß, interleukin-6, and tumor necrosis factor were also increased. The expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22(phox) and p47(phox) were increased, whereas the expression of antioxidant enzymes (SOD1, SOD2, Gpx1, and Prdx1) was reduced in CKD + Ca/P/VitD rats. Oxidized peroxiredoxin, a sensor of ROS generation, was significantly increased and ROS-sensitive signaling pathways were activated in the aorta from CKD + Ca/P/VitD rats. CONCLUSION: This study demonstrates a relationship between inflammation/ROS and arterial calcification in CKD and contributes to understanding of the complex pathways that mediate arterial calcification in CKD patients.

Transcriptional repression of hypoxia-inducible factor-1 (HIF-1) by the protein arginine methyltransferase PRMT1.

Hypoxia-inducible factors (HIF-1 and HIF-2) are essential mediators for the adaptive transcriptional response of cells and tissues to low-oxygen conditions. Under hypoxia or when cells are treated with various nonhypoxic stimuli, the active HIF-α subunits are mainly regulated through increased protein stabilization. For HIF-1α, it is clear that further transcriptional, translational, and posttranslational regulations are important for complete HIF-1 activity. Novel evidence links hypoxia and HIF-1 to arginine methylation, an important protein modification. These studies suggest that arginine methyltransferases may be important for hypoxic responses. Protein arginine methyltransferase 1 (PRMT1), the predominant arginine methyltransferase, can act as a transcriptional activator or repressor by modifying a diverse set of substrates. In this work, we show that PRMT1 is a repressor of both HIF-1 and HIF-2. The cellular depletion of PRMT1 by small interference RNA targeting leads to increased HIF transcriptional activity. This activation is the result of enhanced HIF-α subunit transcription, which allows increased HIF-α subunit availability. We provide evidence that PRMT1-dependent HIF-1α regulation is mediated through the activities of both specificity protein 1 (Sp1) and Sp3, two transcription factors known to control HIF-1α expression. This study therefore identifies PRMT1 as a novel regulator of HIF-1- and HIF-2-mediated responses.

The prolyl isomerase Pin1 regulates hypoxia-inducible transcription factor (HIF) activity.

Hypoxia-inducible transcription factor-1 (HIF-1) plays a decisive role in cell survival and adaptation to hypoxic stress by controlling the expression of genes involved in oxygen homeostasis. HIF-1 activity is fine-tuned through specific post-translational modifications of its essential HIF-1α subunit. Among these modifications, phosphorylation is important for HIF-1 transcriptional activity. Studies have shown that the mitogen-activated protein kinases, p42/p44 MAPKs, directly phosphorylate HIF-1α and increase HIF-1-mediated transcription. Pin1, a peptidyl-prolyl cis/trans isomerase, targets a number of proteins containing a phosphorylated Ser/Thr-Pro motif. Pin1 isomerization causes a change in target protein conformation which can modify their activity. Here, we identify Pin1 as an important HIF-1α partner. Immunoprecipitation and pull-down studies show that Pin1 interacts with HIF-1α. We demonstrate that the interaction between Pin1 and HIF-1α is regulated through p42/p44 MAPK pathway activation. By performing proteolysis studies, our results indicate that Pin1 catalytic activity generates a conformational change in HIF-1α. Finally, our work shows that Pin1 is required for gene-specific HIF-1 transcriptional activity. Our results indicate that the prolyl isomerase Pin1 regulates HIF-1 transcriptional activity by interacting with HIF-1α and promoting conformational changes in a p42/p44 MAPK phosphorylation-dependent manner.

Conserved molecular interactions within the HBO1 acetyltransferase complexes regulate cell proliferation.

Acetyltransferase complexes of the MYST family with distinct substrate specificities and functions maintain a conserved association with different ING tumor suppressor proteins. ING complexes containing the HBO1 acetylase are a major source of histone H3 and H4 acetylation in vivo and play critical roles in gene regulation and DNA replication. Here, our molecular dissection of HBO1/ING complexes unravels the protein domains required for their assembly and function. Multiple PHD finger domains present in different subunits bind the histone H3 N-terminal tail with a distinct specificity toward lysine 4 methylation status. We show that natively regulated association of the ING4/5 PHD domain with HBO1-JADE determines the growth inhibitory function of the complex, linked to its tumor suppressor activity. Functional genomic analyses indicate that the p53 pathway is a main target of the complex, at least in part through direct transcription regulation at the initiation site of p21/CDKN1A. These results demonstrate the importance of ING association with MYST acetyltransferases in controlling cell proliferation, a regulated link that accounts for the reported tumor suppressor activities of these complexes.

Hypoxia-inducible factor-1 activation in nonhypoxic conditions: the essential role of mitochondrial-derived reactive oxygen species.

Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor for responses to low oxygen. Different nonhypoxic stimuli, including hormones and growth factors, are also important HIF-1 activators in the vasculature. Angiotensin II (Ang II), the main effecter hormone in the renin-angiotensin system, is a potent HIF-1 activator in vascular smooth muscle cells (VSMCs). HIF-1 activation by Ang II involves intricate mechanisms of HIF-1α transcription, translation, and protein stabilization. Additionally, the generation of reactive oxygen species (ROS) is essential for HIF-1 activation during Ang II treatment. However, the role of the different VSMC ROS generators in HIF-1 activation by Ang II remains unclear. This work aims at elucidating this question. Surprisingly, repression of NADPH oxidase-generated ROS, using Vas2870, a specific inhibitor or a p22(phox) siRNA had no significant effect on HIF-1 accumulation by Ang II. In contrast, repression of mitochondrial-generated ROS, by complex III inhibition, by Rieske Fe-S protein siRNA, or by the mitochondrial-targeted antioxidant SkQ1, strikingly blocked HIF-1 accumulation. Furthermore, inhibition of mitochondrial-generated ROS abolished HIF-1α protein stability, HIF-1-dependent transcription and VSMC migration by Ang II. A large number of studies implicate NADPH oxidase-generated ROS in Ang II-mediated signaling pathways in VSMCs. However, our work points to mitochondrial-generated ROS as essential intermediates for HIF-1 activation in nonhypoxic conditions.

Effects of TGF-beta1 on endothelial factors.

This study investigated the mechanistic effect of transforming growth factor-beta1 (TGFbeta1) on the endothelial mediators: endothelin-1 (ET-1), prostacyclin (PGI(2)) and nitric oxide (NO) in the endothelial cell line 1G11. Endothelial cells were incubated with increasing concentrations of TGFbeta1 in the presence and absence of growth medium (deprived) or various inhibitors. In deprived cells, TGFbeta1 increased the release of PGI(2) (6-keto-PGF1alpha) concomitantly to an increase in COX-2 expression, whereas the production of ET-1 and NO metabolites was not affected. Either the removal of prior serum and heparin deprivation or NO synthase inhibition by L-NAME unmasked an inhibitory effect of TGFbeta1 on ET-1 production. Indomethacin abolished the TGFbeta1 inhibitory action on L-NAME-increased ET-1 production. These results show that TGFbeta1 induces an increase in production of PGI(2) that is consecutive to an induction of COX-2 in endothelial cells. This increase in PGI(2) partly accounts for the inhibitory action of TGFbeta1 on ET-1 secretion.

HIF-1 inhibition decreases systemic vascular remodelling diseases by promoting apoptosis through a hexokinase 2-dependent mechanism.

AIMS: Vascular remodelling diseases are characterized by the presence of proliferative and apoptosis-resistant vascular smooth muscle cells (VSMC). There is evidence that pro-proliferative and anti-apoptotic states are characterized by metabolic remodelling (a glycolytic phenotype with hyperpolarized mitochondria) involving Akt pathway activation by circulating growth factors. Hypoxia-inducible factor-1 (HIF-1) is involved in different vascular diseases. Since this transcription factor is implicated in metabolic responses, we hypothesized that HIF-1 activity could be involved in vascular remodelling in response to arterial injury. METHODS AND RESULTS: Our findings indicate that growth factors, such as platelet-derived growth factor (PDGF), activate the Akt pathway (measured by immunoblot) in human carotid artery VSMC. Activation of this pathway increased HIF-1 activation (measured by immunoblot), leading to increased glycolysis in VSMC. Expression and mitochondrial activity of hexokinase 2 (HXK2), a primary initiator of glycolysis, are increased during HIF-1 activation. The mitochondrial activity of HXK2 in VSMC led to the hyperpolarization of mitochondrial membrane potential (measured by tetramethylrhodamine methyl-ester perchlorate) and the suppression of apoptosis (measured by TUNEL assay and 3 activity), effects that are blocked by HIF-1 inhibition. Additionally, HIF-1 inhibition also decreased VSMC proliferation (proliferating cell nuclear antigen and Ki-67 assays). In vivo, we demonstrate that localized HIF-1 inhibition, using a dominant-negative HIF-1α adenoviral construct, prevented carotid artery post-injury remodelling in rats. CONCLUSION: We propose that HIF-1 is centrally involved in carotid artery remodelling in response to arterial injury and that localized inhibition of HIF-1 may be a novel therapeutic strategy to prevent carotid stenosis.