Comparing the efficacy in reducing brain injury of different neuroprotective agents following neonatal hypoxia-ischemia in newborn rats: a multi-drug randomized controlled screening trial.

Intrapartum hypoxia-ischemia leading to neonatal encephalopathy (NE) results in significant neonatal mortality and morbidity worldwide, with > 85% of cases occurring in low- and middle-income countries (LMIC). Therapeutic hypothermia (HT) is currently the only available safe and effective treatment of HIE in high-income countries (HIC); however, it has shown limited safety or efficacy in LMIC. Therefore, other therapies are urgently required. We aimed to compare the treatment effects of putative neuroprotective drug candidates following neonatal hypoxic-ischemic (HI) brain injury in an established P7 rat Vannucci model. We conducted the first multi-drug randomized controlled preclinical screening trial, investigating 25 potential therapeutic agents using a standardized experimental setting in which P7 rat pups were exposed to unilateral HI brain injury. The brains were analysed for unilateral hemispheric brain area loss after 7 days survival. Twenty animal experiments were performed. Eight of the 25 therapeutic agents significantly reduced brain area loss with the strongest treatment effect for Caffeine, Sonic Hedgehog Agonist (SAG) and Allopurinol, followed by Melatonin, Clemastine, ß-Hydroxybutyrate, Omegaven, and Iodide. The probability of efficacy was superior to that of HT for Caffeine, SAG, Allopurinol, Melatonin, Clemastine, ß-hydroxybutyrate, and Omegaven. We provide the results of the first systematic preclinical screening of potential neuroprotective treatments and present alternative single therapies that may be promising treatment options for HT in LMIC.

Human placental trophoblast progenitor cells (hTPCs) promote angiogenesis and neurogenesis after focal cerebral ischemia in rats.

INTRODUCTION: Reduction of blood flow below a threshold value in brain regions locally or globally is called cerebral ischemia and proper treatment requires either the restoration of normal blood flow and/or the administration of neuroprotective therapies. Human trophoblast progenitor cells (hTPCs) give rise to the placenta and are responsible for the invasion and vascular remodeling of the maternal vessels within the uterus. Here, we tested whether hTPCs promoted to differentiate along neural lineages may exhibit therapeutic properties in the setting of cerebral ischemia in vivo. MATERIALS AND METHODS: Cerebral ischemia was generated in rats via middle cerebral artery occlusion and, after 24 h, hTPCs were injected systemically via tail vein. Animals were sacrified at Day 3 or 11. RESULTS: TTC staining indicated that infarct volumes were smaller in hTPC treated animals. Visible myelin recovery was observed in the hTPC injected group with Luxol Fast Blue staining. On Day 11 after hTPC transplantation, DLX5 and VEGF expression, as well as 2 and 10 d after hTPC transplantation, NKX2.2 were significantly increased; while LHX6, Olig1, PDGFRα, VEGFR1 and VEGFR2 showed trends toward improved expression in brain tissue via immunoblot analysis. Neuron-like differentiated cells were positive for both NeuN and Cresyl Violet staining. CONCLUSION: Here, we demonstrate for the first time that hTPCs enhance the expression of angiogenic and neurogenic factors in rat brain after stroke. Transplantation of hTPCs could form the basis of novel therapeutic approaches for the treatment of stroke in the clinical setting.

HIF-1α promotes cellular growth in lymphatic endothelial cells exposed to chronically elevated pulmonary lymph flow.

Normal growth and development of lymphatic structures depends on mechanical forces created by accumulating interstitial fluid. However, prolonged exposure to pathologic mechanical stimuli generated by chronically elevated lymph flow results in lymphatic dysfunction. The mechanisms that transduce these mechanical forces are not fully understood. Our objective was to investigate molecular mechanisms that alter the growth and metabolism of isolated lymphatic endothelial cells (LECs) exposed to prolonged pathologically elevated lymph flow in vivo within the anatomic and physiologic context of a large animal model of congenital heart disease with increased pulmonary blood flow using in vitro approaches. To this end, late gestation fetal lambs underwent in utero placement of an aortopulmonary graft (shunt). Four weeks after birth, LECs were isolated and cultured from control and shunt lambs. Redox status and proliferation were quantified, and transcriptional profiling and metabolomic analyses were performed. Shunt LECs exhibited hyperproliferative growth driven by increased levels of Hypoxia Inducible Factor 1α (HIF-1α), along with upregulated expression of known HIF-1α target genes in response to mechanical stimuli and shear stress. Compared to control LECs, shunt LECs exhibited abnormal metabolism including abnormalities of glycolysis, the TCA cycle and aerobic respiration. In conclusion, LECs from lambs exposed in vivo to chronically increased pulmonary lymph flow are hyperproliferative, have enhanced expression of HIF-1α and its target genes, and demonstrate altered central carbon metabolism in vitro. Importantly, these findings suggest provocative therapeutic targets for patients with lymphatic abnormalities.

Complex interplay between autophagy and oxidative stress in the development of pulmonary disease.

The autophagic pathway involves the encapsulation of substrates in double-membraned vesicles, which are subsequently delivered to the lysosome for enzymatic degradation and recycling of metabolic precursors. Autophagy is a major cellular defense against oxidative stress, or related conditions that cause accumulation of damaged proteins or organelles. Selective forms of autophagy can maintain organelle populations or remove aggregated proteins. Dysregulation of redox homeostasis under pathological conditions results in excessive generation of reactive oxygen species (ROS), leading to oxidative stress and the associated oxidative damage of cellular components. Accumulating evidence indicates that autophagy is necessary to maintain redox homeostasis. ROS activates autophagy, which facilitates cellular adaptation and diminishes oxidative damage by degrading and recycling intracellular damaged macromolecules and dysfunctional organelles. The cellular responses triggered by oxidative stress include the altered regulation of signaling pathways that culminate in the regulation of autophagy. Current research suggests a central role for autophagy as a mammalian oxidative stress response and its interrelationship to other stress defense systems. Altered autophagy phenotypes have been observed in lung diseases such as chronic obstructive lung disease, acute lung injury, cystic fibrosis, idiopathic pulmonary fibrosis, and pulmonary arterial hypertension, and asthma. Understanding the mechanisms by which ROS regulate autophagy will provide novel therapeutic targets for lung diseases. This review highlights our current understanding on the interplay between ROS and autophagy in the development of pulmonary disease.

Defining the ATPome reveals cross-optimization of metabolic pathways.

Disrupted energy metabolism drives cell dysfunction and disease, but approaches to increase or preserve ATP are lacking. To generate a comprehensive metabolic map of genes and pathways that regulate cellular ATP-the ATPome-we conducted a genome-wide CRISPR interference/activation screen integrated with an ATP biosensor. We show that ATP level is modulated by distinct mechanisms that promote energy production or inhibit consumption. In our system HK2 is the greatest ATP consumer, indicating energy failure may not be a general deficiency in producing ATP, but rather failure to recoup the ATP cost of glycolysis and diversion of glucose metabolites to the pentose phosphate pathway. We identify systems-level reciprocal inhibition between the HIF1 pathway and mitochondria; glycolysis-promoting enzymes inhibit respiration even when there is no glycolytic ATP production, and vice versa. Consequently, suppressing alternative metabolism modes paradoxically increases energy levels under substrate restriction. This work reveals mechanisms of metabolic control, and identifies therapeutic targets to correct energy failure.

The Gag protein PEG10 binds to RNA and regulates trophoblast stem cell lineage specification.

Peg10 (paternally expressed gene 10) is an imprinted gene that is essential for placental development. It is thought to derive from a Ty3-gyspy LTR (long terminal repeat) retrotransposon and retains Gag and Pol-like domains. Here we show that the Gag domain of PEG10 can promote vesicle budding similar to the HIV p24 Gag protein. Expressed in a subset of mouse endocrine organs in addition to the placenta, PEG10 was identified as a substrate of the deubiquitinating enzyme USP9X. Consistent with PEG10 having a critical role in placental development, PEG10-deficient trophoblast stem cells (TSCs) exhibited impaired differentiation into placental lineages. PEG10 expressed in wild-type, differentiating TSCs was bound to many cellular RNAs including Hbegf (Heparin-binding EGF-like growth factor), which is known to play an important role in placentation. Expression of Hbegf was reduced in PEG10-deficient TSCs suggesting that PEG10 might bind to and stabilize RNAs that are critical for normal placental development.

Sonic Hedgehog Agonist Protects Against Complex Neonatal Cerebellar Injury.

The cerebellum undergoes rapid growth during the third trimester and is vulnerable to injury and deficient growth in infants born prematurely. Factors associated with preterm cerebellar hypoplasia include chronic lung disease and postnatal glucocorticoid administration. We modeled chronic hypoxemia and glucocorticoid administration in neonatal mice to study whole cerebellar and cell type-specific effects of dual exposure. Chronic neonatal hypoxia resulted in permanent cerebellar hypoplasia. This was compounded by administration of prednisolone as shown by greater volume loss and Purkinje cell death. In the setting of hypoxia and prednisolone, administration of a small molecule Smoothened-Hedgehog agonist (SAG) preserved cerebellar volume and protected against Purkinje cell death. Such protective effects were observed even when SAG was given as a one-time dose after dual insult. To model complex injury and determine cell type-specific roles for the hypoxia inducible factor (HIF) pathway, we performed conditional knockout of von Hippel Lindau (VHL) to hyperactivate HIF1α in cerebellar granule neuron precursors (CGNP) or Purkinje cells. Surprisingly, HIF activation in either cell type resulted in no cerebellar deficit. However, in mice administered prednisolone, HIF overactivation in CGNPs resulted in significant cerebellar hypoplasia, whereas HIF overactivation in Purkinje cells caused cell death. Together, these findings indicate that HIF primes both cell types for injury via glucocorticoids, and that hypoxia/HIF + postnatal glucocorticoid administration act on distinct cellular pathways to cause cerebellar injury. They further suggest that SAG is neuroprotective in the setting of complex neonatal cerebellar injury.

Metabolic Changes Precede the Development of Pulmonary Hypertension in the Monocrotaline Exposed Rat Lung.

There is increasing interest in the potential for metabolic profiling to evaluate the progression of pulmonary hypertension (PH). However, a detailed analysis of the metabolic changes in lungs at the early stage of PH, characterized by increased pulmonary artery pressure but prior to the development of right ventricle hypertrophy and failure, is lacking in a preclinical animal model of PH. Thus, we undertook a study using rats 14 days after exposure to monocrotaline (MCT), to determine whether we could identify early stage metabolic changes prior to the manifestation of developed PH. We observed changes in multiple pathways associated with the development of PH, including activated glycolysis, increased markers of proliferation, disruptions in carnitine homeostasis, increased inflammatory and fibrosis biomarkers, and a reduction in glutathione biosynthesis. Further, our global metabolic profile data compare favorably with prior work carried out in humans with PH. We conclude that despite the MCT-model not recapitulating all the structural changes associated with humans with advanced PH, including endothelial cell proliferation and the formation of plexiform lesions, it is very similar at a metabolic level. Thus, we suggest that despite its limitations it can still serve as a useful preclinical model for the study of PH.

HIGD1A Regulates Oxygen Consumption, ROS Production, and AMPK Activity during Glucose Deprivation to Modulate Cell Survival and Tumor Growth.

Hypoxia-inducible gene domain family member 1A (HIGD1A) is a survival factor induced by hypoxia-inducible factor 1 (HIF-1). HIF-1 regulates many responses to oxygen deprivation, but viable cells within hypoxic perinecrotic solid tumor regions frequently lack HIF-1α. HIGD1A is induced in these HIF-deficient extreme environments and interacts with the mitochondrial electron transport chain to repress oxygen consumption, enhance AMPK activity, and lower cellular ROS levels. Importantly, HIGD1A decreases tumor growth but promotes tumor cell survival in vivo. The human Higd1a gene is located on chromosome 3p22.1, where many tumor suppressor genes reside. Consistent with this, the Higd1a gene promoter is differentially methylated in human cancers, preventing its hypoxic induction. However, when hypoxic tumor cells are confronted with glucose deprivation, DNA methyltransferase activity is inhibited, enabling HIGD1A expression, metabolic adaptation, and possible dormancy induction. Our findings therefore reveal important new roles for this family of mitochondrial proteins in cancer biology.

HIGD1A-mediated dormancy and tumor survival.

Solid tumors contain regions of anoxia that are also glucose deprived. How cancer cells survive such extreme conditions remains unclear. Here, we discuss our recent findings that regulation of hypoxia inducible gene domain family member 1 A (HIGD1A) via epigenetic mechanisms during glucose starvation modulates oxygen consumption and reactive oxygen species production to enable tumor cell survival through the activation of dormancy mechanisms.

LIMK1 regulates human trophoblast invasion/differentiation and is down-regulated in preeclampsia.

Successful human pregnancy requires extensive invasion of maternal uterine tissues by the placenta. Invasive extravillous trophoblasts derived from cytotrophoblast progenitors remodel maternal arterioles to promote blood flow to the placenta. In the pregnancy complication preeclampsia, extravillous trophoblasts invasion and vessel remodeling are frequently impaired, likely contributing to fetal underperfusion and maternal hypertension. We recently demonstrated in mouse trophoblast stem cells that hypoxia-inducible factor-2 (HIF-2)-dependent Lim domain kinase 1 (LIMK1) expression regulates invasive trophoblast differentiation by modulating the trophoblast cytoskeleton. Interestingly, in humans, LIMK1 activity promotes tumor cell invasion by modulating actin and microtubule integrity, as well as by modulating matrix metalloprotease processing. Here, we tested whether HIF-2α and LIMK1 expression patterns suggested similar roles in the human placenta. We found that LIMK1 immunoreactivity mirrored HIF-2α in the human placenta in utero and that LIMK1 activity regulated human cytotrophoblast cytoskeletal integrity, matrix metallopeptidase-9 secretion, invasion, and differentiation in vitro. Importantly, we also found that LIMK1 levels are frequently diminished in the preeclampsia setting in vivo. Our results therefore validate the use of mouse trophoblast stem cells as a discovery platform for human placentation disorders and suggest that LIMK1 activity helps promote human placental development in utero.

Sexually dimorphic effect of in vitro fertilization (IVF) on adult mouse fat and liver metabolomes.

The preimplantation embryo is particularly vulnerable to environmental perturbation, such that nutritional and in vitro stresses restricted exclusively to this stage may alter growth and affect long-term metabolic health. This is particularly relevant to the over 5 million children conceived by in vitro fertilization (IVF). We previously reported that even optimized IVF conditions reprogram mouse postnatal growth, fat deposition, and glucose homeostasis in a sexually dimorphic fashion. To more clearly interrogate the metabolic changes associated with IVF in adulthood, we used nontargeted mass spectrometry to globally profile adult IVF- and in vivo-conceived liver and gonadal adipose tissues. There was a sex- and tissue-specific effect of IVF on adult metabolite signatures indicative of metabolic reprogramming and oxidative stress and reflective of the observed phenotypes. Additionally, we observed a striking effect of IVF on adult sexual dimorphism. Male-female differences in metabolite concentration were exaggerated in hepatic IVF tissue and significantly reduced in IVF adipose tissue, with the majority of changes affecting amino acid and lipid metabolites. We also observed female-specific changes in markers of oxidative stress and adipogenesis, including reduced glutathione, cysteine glutathione disulfide, ophthalmate, urate, and corticosterone. In summary, embryo manipulation and early developmental experiences can affect adult patterns of sexual dimorphism and metabolic physiology.

Oligodendrocyte-encoded HIF function couples postnatal myelination and white matter angiogenesis.

Myelin sheaths provide critical functional and trophic support for axons in white matter tracts of the brain. Oligodendrocyte precursor cells (OPCs) have extraordinary metabolic requirements during development as they differentiate to produce multiple myelin segments, implying that they must first secure adequate access to blood supply. However, mechanisms that coordinate myelination and angiogenesis are unclear. Here, we show that oxygen tension, mediated by OPC-encoded hypoxia-inducible factor (HIF) function, is an essential regulator of postnatal myelination. Constitutive HIF1/2α stabilization resulted in OPC maturation arrest through autocrine activation of canonical Wnt7a/7b. Surprisingly, such OPCs also show paracrine activity that induces excessive postnatal white matter angiogenesis in vivo and directly stimulates endothelial cell proliferation in vitro. Conversely, OPC-specific HIF1/2α loss of function leads to insufficient angiogenesis in corpus callosum and catastrophic axon loss. These findings indicate that OPC-intrinsic HIF signaling couples postnatal white matter angiogenesis, axon integrity, and the onset of myelination in mammalian forebrain.

Nuclear localization of the mitochondrial factor HIGD1A during metabolic stress.

Cellular stress responses are frequently governed by the subcellular localization of critical effector proteins. Apoptosis-inducing Factor (AIF) or Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH), for example, can translocate from mitochondria to the nucleus, where they modulate apoptotic death pathways. Hypoxia-inducible gene domain 1A (HIGD1A) is a mitochondrial protein regulated by Hypoxia-inducible Factor-1α (HIF1α). Here we show that while HIGD1A resides in mitochondria during physiological hypoxia, severe metabolic stress, such as glucose starvation coupled with hypoxia, in addition to DNA damage induced by etoposide, triggers its nuclear accumulation. We show that nuclear localization of HIGD1A overlaps with that of AIF, and is dependent on the presence of BAX and BAK. Furthermore, we show that AIF and HIGD1A physically interact. Additionally, we demonstrate that nuclear HIGD1A is a potential marker of metabolic stress in vivo, frequently observed in diverse pathological states such as myocardial infarction, hypoxic-ischemic encephalopathy (HIE), and different types of cancer. In summary, we demonstrate a novel nuclear localization of HIGD1A that is commonly observed in human disease processes in vivo.

Cytotrophoblast induction of arterial apoptosis and lymphangiogenesis in an in vivo model of human placentation.

We studied the vascular effects of invasive human cytotrophoblasts in vivo by transplanting placental villi to the fifth mammary fat pads or beneath the kidney capsules of Scid mice. Over 3 weeks, robust cytotrophoblast invasion was observed in both locations. The architecture of the mammary fat pad allowed for detailed analysis of the cells' interactions with resident murine blood vessels, which revealed specific induction of apoptosis in the endothelial cells and smooth muscle walls of the arterioles. This finding, and confirmation of the results in an in vitro coculture model, suggests that a parallel process is important for enabling cytotrophoblast endovascular invasion during human pregnancy. Cytotrophoblast invasion of the kidney parenchyma was accompanied by a robust lymphangiogenic response, while in vitro, the cells stimulated lymphatic endothelial cell migration via the actions of VEGF family members, FGF, and TNF-alpha. Immunolocalization analyses revealed that human pregnancy is associated with lymphangiogenesis in the decidua since lymphatic vessels were not a prominent feature of the nonpregnant endometrium. Thus, the placenta triggers the development of a decidual lymphatic circulation, which we theorize plays an important role in maintaining fluid balance during pregnancy, with possible implications for maternal-fetal immune cell trafficking.

Hypoxia-inducible factors 1alpha and 2alpha regulate trophoblast differentiation.

Placental development initially occurs in a low-oxygen (O2) or hypoxic environment. In this report we show that two hypoxia-inducible factors (HIFs), HIF1alpha and HIF2alpha, are essential for determining murine placental cell fates. HIF is a heterodimer composed of HIFalpha and HIFbeta (ARNT) subunits. Placentas from Arnt-/- and Hif1alpha-/- Hif2alpha-/- embryos exhibit defective placental vascularization and aberrant cell fate adoption. HIF regulation of Mash2 promotes spongiotrophoblast differentiation, a prerequisite for trophoblast giant cell differentiation. In the absence of Arnt or Hifalpha, trophoblast stem cells fail to generate these cell types and become labyrinthine trophoblasts instead. Therefore, HIF mediates placental morphogenesis, angiogenesis, and cell fate decisions, demonstrating that O2 tension is a critical regulator of trophoblast lineage determination. This novel genetic approach provides new insights into the role of O2 tension in the development of life-threatening pregnancy-related diseases such as preeclampsia.