HIF1α-dependent uncoupling of glycolysis suppresses tumor cell proliferation.

Hypoxia-inducible factor-1α (HIF1α) attenuates mitochondrial activity while promoting glycolysis. However, lower glycolysis is compromised in human clear cell renal cell carcinomas, in which HIF1α acts as a tumor suppressor by inhibiting cell-autonomous proliferation. Here, we find that, unexpectedly, HIF1α suppresses lower glycolysis after the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step, leading to reduced lactate secretion in different tumor cell types when cells encounter a limited pyruvate supply such as that typically found in the tumor microenvironment in vivo. This is because HIF1α-dependent attenuation of mitochondrial oxygen consumption increases the NADH/NAD+ ratio that suppresses the activity of the NADH-sensitive GAPDH glycolytic enzyme. This is manifested when pyruvate supply is limited, since pyruvate acts as an electron acceptor that prevents the increment of the NADH/NAD+ ratio. Furthermore, this anti-glycolytic function provides a molecular basis to explain how HIF1α can suppress tumor cell proliferation by increasing the NADH/NAD+ ratio.

Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation.

Inhibition of the heterodimeric amino acid carrier SLC7A5/SLC3A2 (LAT1/CD98) has been widely studied in tumor biology but its role in physiological conditions remains largely unknown. Here we show that the SLC7A5/SLC3A2 heterodimer is constitutively present at different stages of erythroid differentiation but absent in mature erythrocytes. Administration of erythropoietin (EPO) further induces SLC7A5/SLC3A2 expression in circulating reticulocytes, as it also occurs in anemic conditions. Although Slc7a5 gene inactivation in the erythrocyte lineage does not compromise the total number of circulating red blood cells (RBCs), their size and hemoglobin content are significantly reduced accompanied by a diminished erythroblast mTORC1 activity. Furthermore circulating Slc7a5-deficient reticulocytes are characterized by lower transferrin receptor (CD71) expression as well as mitochondrial activity, suggesting a premature transition to mature RBCs. These data reveal that SLC7A5/SLC3A2 ensures adequate maturation of reticulocytes as well as the proper size and hemoglobin content of circulating RBCs.

Unique expression of the atypical mitochondrial subunit NDUFA4L2 in cerebral pericytes fine tunes HIF activity in response to hypoxia.

A central response to insufficient cerebral oxygen delivery is a profound reprograming of metabolism, which is mainly regulated by the Hypoxia Inducible Factor (HIF). Among other responses, HIF induces the expression of the atypical mitochondrial subunit NDUFA4L2. Surprisingly, NDUFA4L2 is constitutively expressed in the brain in non-hypoxic conditions. Analysis of publicly available single cell transcriptomic (scRNA-seq) data sets coupled with high-resolution multiplexed fluorescence RNA in situ hybridization (RNA F.I.S.H.) revealed that in the murine and human brain NDUFA4L2 is exclusively expressed in mural cells with the highest levels found in pericytes and declining along the arteriole-arterial smooth muscle cell axis. This pattern was mirrored by COX4I2, another atypical mitochondrial subunit. High NDUFA4L2 expression was also observed in human brain pericytes in vitro, decreasing when pericytes are muscularized and further induced by HIF stabilization in a PHD2/PHD3 dependent manner. In vivo, Vhl conditional inactivation in pericyte targeting Ng2-cre transgenic mice dramatically induced NDUFA4L2 expression. Finally NDUFA4L2 inactivation in pericytes increased oxygen consumption and therefore the degree of HIF pathway induction in hypoxia. In conclusion our work reveals that NDUFA4L2 together with COX4I2 is a key hypoxic-induced metabolic marker constitutively expressed in pericytes coupling mitochondrial oxygen consumption and cellular hypoxia response.

Inactivation of HIF-prolyl 4-hydroxylases 1, 2 and 3 in NG2-expressing cells induces HIF2-mediated neurovascular expansion independent of erythropoietin.

AIM: NG2 cells in the brain are comprised of pericytes and NG2 glia and play an important role in the execution of cerebral hypoxia responses, including the induction of erythropoietin (EPO) in pericytes. Oxygen-dependent angiogenic responses are regulated by hypoxia-inducible factor (HIF), the activity of which is controlled by prolyl 4-hydroxylase domain (PHD) dioxygenases and the von Hippel-Lindau (VHL) tumour suppressor. However, the role of NG2 cells in HIF-regulated cerebral vascular homeostasis is incompletely understood. METHODS: To examine the HIF/PHD/VHL axis in neurovascular homeostasis, we used a Cre-loxP-based genetic approach in mice and targeted Vhl, Epo, Phd1, Phd2, Phd3 and Hif2a in NG2 cells. Cerebral vasculature was assessed by immunofluorescence, RNA in situ hybridization, gene and protein expression analysis, gel zymography and in situ zymography. RESULTS: Vhl inactivation led to a significant increase in angiogenic gene and Epo expression. This was associated with EPO-independent expansion of capillary networks in cortex, striatum and hypothalamus, as well as pericyte proliferation. A comparable phenotype resulted from the combined inactivation of Phd2 and Phd3, but not from Phd2 inactivation alone. Concomitant PHD1 function loss led to further expansion of the neurovasculature. Genetic inactivation of Hif2a in Phd1/Phd2/Phd3 triple mutant mice resulted in normal cerebral vasculature. CONCLUSION: Our studies establish (a) that HIF2 activation in NG2 cells promotes neurovascular expansion and remodelling independently of EPO, (b) that HIF2 activity in NG2 cells is co-controlled by PHD2 and PHD3 and (c) that PHD1 modulates HIF2 transcriptional responses when PHD2 and PHD3 are inactive.

HIF1α Suppresses Tumor Cell Proliferation through Inhibition of Aspartate Biosynthesis.

Cellular aspartate drives cancer cell proliferation, but signaling pathways that rewire aspartate biosynthesis to control cell growth remain largely unknown. Hypoxia-inducible factor-1α (HIF1α) can suppress tumor cell proliferation. Here, we discovered that HIF1α acts as a direct repressor of aspartate biosynthesis involving the suppression of several key aspartate-producing proteins, including cytosolic glutamic-oxaloacetic transaminase-1 (GOT1) and mitochondrial GOT2. Accordingly, HIF1α suppresses aspartate production from both glutamine oxidation as well as the glutamine reductive pathway. Strikingly, the addition of aspartate to the culture medium is sufficient to relieve HIF1α-dependent repression of tumor cell proliferation. Furthermore, these key aspartate-producing players are specifically repressed in VHL-deficient human renal carcinomas, a paradigmatic tumor type in which HIF1α acts as a tumor suppressor, highlighting the in vivo relevance of these findings. In conclusion, we show that HIF1α inhibits cytosolic and mitochondrial aspartate biosynthesis and that this mechanism is the molecular basis for HIF1α tumor suppressor activity.

Pre-clinical assay of the tissue integration and mechanical adhesion of several types of cyanoacrylate adhesives in the fixation of lightweight polypropylene meshes for abdominal hernia repair.

INTRODUCTION: Lightweight (LW) polypropylene (PP) meshes better adapt to host tissue, causing less fibrosis and inflammatory responses than high-density meshes. Mesh fixation using tissue adhesives (TA) that replace conventional sutures may improve the process of hernia repair and tissue trauma. This preclinical study compares the behavior of different cyanoacrylate-based adhesives in the fixation of LW-PP meshes for hernia repair. METHODS: Partial abdominal wall defects were repaired using LW-PP Optilene meshes in New Zealand rabbits. The following groups were established according to the mesh fixation method: Suture (control), Glubran 2 (n-butyl), Ifabond (n-hexyl), SafetySeal (n-butyl) and Evobond (n-octyl). At 14, 90 and 180 days after surgery, the recovered implants were examined to assess the host tissue integration, the macrophage response and the biomechanical strength. RESULTS: All the groups showed optimal host tissue incorporation regardless of the fixation procedure. Significantly increased levels of collagen 1 and collagen 3 gene expression (p<0.001) were observed at 14 days compared to the medium- and long-term durations, where the Suture and Glubran groups showed the highest expression of collagen 1. All the adhesives increased the macrophage reaction (p<0.001) compared to sutures at all implant times. Maximal macrophage response was observed in the short-term Glubran group (p<0.01) compared to the rest of the groups. Although SafetySeal and Evobond did not reach the biomechanical resistance of sutures at 14 days, all the adhesives did reach this level in the medium- to long-term periods, providing significantly higher resistance (p<0.05). CONCLUSIONS: All the cyanoacrylates, despite inducing a significantly increased macrophage response versus sutures, showed optimal host tissue integration and long-term mechanical behavior; thus, they might be good choices for LW-PP mesh hernia repairs.

Sutures versus new cyanoacrylates in prosthetic abdominal wall repair: a preclinical long-term study.

BACKGROUND: As an alternative to sutures, meshes used for hernia repair can be fixed using cyanoacrylate-based adhesives. Attempts to improve these adhesives include alkyl-chain lengthening to reduce their toxicity. This preclinical study compares the long-term behavior of cyanoacrylates of different chain lengths already used in hernia repair and new ones for this application. MATERIALS AND METHODS: Partial abdominal wall defects were repaired using a Surgipro mesh in 18 New Zealand White rabbits, and groups were established according to the mesh fixation method: sutures (control), Glubran 2 (n-butyl), Ifabond (n-hexyl), and the new adhesives SafetySeal (n-butyl), and Evobond (n-octyl). Six months after surgery, recovered implants were examined to assess adhesive degradation, host tissue reaction, and biomechanical strength. RESULTS: All the cyanoacrylate groups showed good host tissue incorporation in the meshes. Macrophage responses to Glubran and Ifabond were quantitatively greater compared with sutures. Cell damage caused by the adhesives was similar, and only Glubran induced significantly more damage than sutures. Significantly lower collagen 1/3 messenger RNA expression was induced by Ifabond than the remaining fixation materials. No differences were observed in collagen expression except slightly reduced collagen I deposition in Glubran/Ifabond and collagen III deposition in the suture group. Mechanical strengths failed to vary between the suture and cyanoacrylate groups. CONCLUSIONS: All cyanoacrylates showed good long-term behavior and tolerance irrespective of their long or intermediate chain length. Cyanoacrylate residues persisted at 6 mo, indicating their incomplete degradation. Biomechanical strengths were similar both for the adhesives and sutures.

Host tissue response by the expression of collagen to cyanoacrylate adhesives used in implant fixation for abdominal hernia repair.

The less traumatic use of surgical adhesives rather than sutures for mesh fixation in hernia repair has started to gain popularity because they induce less host tissue damage and provoke less postoperative pain. This study examines the host tissue response to a new cyanoacrylate (CA) adhesive (n-octyl, OCA). Partial defects (3 × 5 cm) created in the rabbit anterior abdominal wall were repaired by mesh fixation using OCA, Glubran2®(n-butyl-CA), Ifabond®(n-hexyl-CA) or sutures. Samples were obtained at 14/90 days for morphology, collagens qRT-PCR/immunofluorescence and biomechanical studies. All meshes were successfully fixed. Seroma was detected mainly in the Glubran group at 14 days. Meshes fixed using all methods showed good host tissue incorporation. No signs of degradation of any of the adhesives were observed. At 14 days, collagen 1 and 3 mRNA expression levels were greater in the suture and OCA groups, and lower in Ifabond, with levels varying significantly in the latter group with respect to the others. By 90 days, expression levels had fallen in all groups, except for collagen 3 mRNA in Ifabond. Collagen I and III protein expression was marked in the suture and OCA groups at 90 days, but lower in Ifabond at both time points. Tensile strengths were similar across groups. Our findings indicate the similar behavior of the adhesives to sutures in terms of good tissue incorporation of the meshes and optimal repair zone strength. The lower seroma rate and similar collagenization to controls induced by OCA suggests its improved behavior over the other two glues. This article deals with a preclinical study to examine different aspects of the repair process in the host of three alkyl cyanoacrylates (n-butyl (GLUBRAN 2), n-hexyl (IFABOND), and n-octyl cyanoacrylate (EVOBOND)) compared to sutures (control), in the fixation of surgical meshes for hernia repair. It goes into detail about collagen deposition in the repair zone at short and medium term. The results obtained demonstrate lower seroma rate and similar collagenization to sutures induced by the n-octyl suggesting better behavior than the other two cyanoacrylates.

Effects of a novel NADPH oxidase inhibitor (S42909) on wound healing in an experimental ischemic excisional skin model.

Chronic wounds are a serious healthcare problem. As non-healing wounds involve continuous pathologic inflammatory stage, research is focused on anti-inflammatory treatments. Our objective was to analyze the effect of S42909, a potent NADPH oxidase inhibitor activity, with vascular anti-inflammatory properties. An ischemic rabbit ear ulcer model (24 New Zealand white rabbits) was used to evaluate the reepithelialization/contraction areas, anti-/pro-inflammatory cytokines mRNA (TGF-ß1/IL-10/IFN-γ/VEGF) by qRT-PCR, collagen I/III deposition, and neovascularization (TGF-ß1/VEGF) by morphological and immunohistochemical analyses. Three different doses were administered by gavage for 2 weeks: 10 and 30 mg/kg/d in self-microemulsion drug delivery system (SMEDDS) and 100 mg/kg/d in arabic gum. Each vehicle was used as control. No signs of infection or necrosis were found. Reepithelialization was almost complete whatever the groups reaching 95% at the dose of 100 mg/kg. Wound contraction was significantly reduced in all S42909-treated groups. A significant increase in anti-inflammatory cytokines TGF-ß1 mRNA and IL-10 mRNA was observed at the dose of 100 and 30 mg/kg/d, respectively. No changes were observed in pro-inflammatory factors INF-γ and VEGF mRNA. Ischemic skin wound areas had scarce expression of collagen I/III and showed rich glycosaminoglycans content. Treatment increased the collagen deposition and TGF-ß1 protein expression and decreased glycosaminoglycan content dose dependently; however, no effect in VEGF was appreciated. Therefore, our results indicate that S42909 improved healing process by dampening excessive inflammation and facilitating collagen deposition without wound contraction phenomena. S42909 might be a promising therapy to treat chronic wounds as venous leg ulcers.