Interleukin-15 modulates the response of cortical neurons to ischemia.

OBJECTIVE: Stroke is a major cause of death and disability in the United States. Current acute stroke therapy consists of clot-dissolving drugs, catheter-based interventions and physical rehabilitation. To date, there are no therapies that directly enhance neuronal survival after a stroke. Previous work from our lab demonstrated that Interleukin-15 (IL-15) peptide could rescue cardiomyocytes subjected to hypoxia. We sought to extend these findings to cortical neurons since IL-15 has been implicated to have an important role in neuronal homeostasis. METHODS: We have evaluated the effect of IL-15 peptide on primary cortical neurons derived from embryonic rats in vitro under conditions of anoxia and glucose deprivation, and in vivo following middle cerebral artery occlusion. RESULTS: IL-15 administration rescued neuronal cells subjected to anoxia coupled with glucose deprivation (AGD), as well as with reoxygenation. A hallmark of stroke is the ischemic microenvironment and associated oxidative stress, which results in DNA damage and ER stress, both of which contribute to neuronal cell damage and death. The expression of anoxia, ER stress, and DNA damage factors/markers was evaluated via western blot and correlated with the cellular survival effects of IL-15 in vitro. In addition, IL-15 effect of alleviating ER stress and increasing cell survival was also observed in vivo. INTERPRETATION: Our data indicate, for the first time, that administration of the pleiotropic factor IL-15 reduces neuronal cell death during AGD, which correlates with modulation of multiple cellular stress pathways.

Administration of Interleukin-15 Peptide Improves Cardiac Function in a Mouse Model of Myocardial Infarction.

Interleukin-15 is a pleotropic factor, capable of modulating metabolism, survival, proliferation, and differentiation in many different cell types. The rationale behind this study relates to previous work demonstrating that IL-15 is a major factor present in stem cell extracts, which protects cardiomyocytes subjected to hypoxic stress in vitro. The objective of this current study was to assess whether administration of IL-15 peptide will also show protective effects in vivo. The data indicate that administration of IL-15 reduces cell death, increases vascularity, decreases scar size, and significantly improves left ventricular ejection fraction in a mouse model of myocardial infarction.

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.

Activating transcription factor 4.

Activating transcription factor 4 (ATF4) belongs to the ATF/CREB (activating transcription factor/cyclic AMP response element binding protein) family of basic region-leucine zipper (bZip) transcription factors, which have the consensus binding site cAMP responsive element (CRE). ATF4 has numerous dimerization partners. ATF4 is induced by stress signals including anoxia/hypoxia, endoplasmic reticulum stress, amino acid deprivation, and oxidative stress. ATF4 expression is regulated transcriptionally, translationally via the PERK pathway of eIF2alpha phosphorylation, and posttranslationally by phosphorylation, which targets ATF4 to proteasomal degradation. ATF4 regulates the expression of genes involved in oxidative stress, amino acid synthesis, differentiation, metastasis and angiogenesis. Transgenic studies have demonstrated ATF4 to be involved in hematopoiesis, lens and skeletal development, fertility, proliferation, differentiation, and long-term memory. ATF4 expression is upregulated in cancer. Since ATF4 is induced by tumour microenvironmental factors, and regulates processes relevant to cancer progression, it might serve as a potential therapeutic target in cancer.

TGF-ß1/CD105 signaling controls vascular network formation within growth factor sequestering hyaluronic acid hydrogels.

Cell-based strategies for the treatment of ischemic diseases are at the forefront of tissue engineering and regenerative medicine. Cell therapies purportedly can play a key role in the neovascularization of ischemic tissue; however, low survival and poor cell engraftment with the host vasculature following implantation limits their potential to treat ischemic diseases. To overcome these limitations, we previously developed a growth factor sequestering hyaluronic acid (HyA)-based hydrogel that enhanced transplanted mouse cardiosphere-derived cell survival and formation of vasculature that anastomosed with host vessels. In this work, we examined the mechanism by which HyA hydrogels presenting transforming growth factor beta-1 (TGF-ß1) promoted proliferation of more clinically relevant human cardiosphere-derived cells (hCDC), and their formation of vascular-like networks in vitro. We observed hCDC proliferation and enhanced formation of vascular-like networks occurred in the presence of TGF-ß1. Furthermore, production of nitric oxide (NO), VEGF, and a host of angiogenic factors were increased in the presence of TGF-ß1. This response was dependent on the co-activity of CD105 (Endoglin) with the TGF-ßR2 receptor, demonstrating its role in the process of angiogenic differentiation and vascular organization of hCDC. These results demonstrated that hCDC form vascular-like networks in vitro, and that the induction of vascular networks by hCDC within growth factor sequestering HyA hydrogels was mediated by TGF-ß1/CD105 signaling.

Expression of bone morphogenetic protein 2 in breast cancer cells inhibits hypoxic cell death.

BMP-2 is involved in the fetal and postnatal development of the mammary gland but has also been detected in breast cancer cells. To clarify the biological role of BMP-2 in breast cancer, we used the human breast cancer cell line MCF-7. Incubation with BMP-2 under serum-free conditions induced activation of the mitogen activated protein kinases (MAPKs) ERK1/2 and the basic helix-loop-helix transcription factors Id-1, proteins that can protect from apoptosis. Stably transfected MCF-7 cells overexpressing BMP-2 revealed significantly increased resistance to hypoxia-induced apoptosis compared to empty vector controls. Cytoplasmic BMP-2/4 protein expression was detected in carcinoma cells of 81 samples of invasive breast cancer in contrast to adjacent normal mammary epithelial cells. BMP-2/4 expression did not correlate with common prognostic parameters and was not associated with relapse-free or overall survival. We conclude that BMP-2/4 expression is reactivated in invasive breast cancer and part of an autocrine/paracrine mechanism rescuing malignant cells from hypoxic cell death via activation of the MAPK and Id-1 pathway.

Bone morphogenetic protein 2 (BMP-2) and induction of tumor angiogenesis.

PURPOSE: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family and play an important role in the regulation of embryonic vasculogenesis but their role in postnatal angiogenesis remains to be clarified. In this study we investigated a possible role of BMP-2 in the promotion of tumor angiogenesis. METHODS: We studied the effect of BMP-2 on human dermal microvascular endothelial cells (HDMECs) and examined a possible angiogenic activity of BMP-2 with the mouse sponge assay. The effect of BMP-2 overexpression on tumor vascularization was also analyzed in xenografts of human BMP-2 transfected MCF-7 breast cancer cells (MCF-7/BMP2) in mice. RESULTS: BMP receptor activation selectively induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) in contrast to the ERK1/2 MAP kinases. In keeping with this finding, BMP-2 had no significant effect on endothelial cell proliferation but promoted HDMEC tube formation in the matrigel assay. The transcription factor inhibitor of differentiation 1 (Id1), which is known to play an important role in neovascularization of tumors, was confirmed as a BMP target in HDMECs. Immunohistochemical analysis of sponge sections revealed that BMP-2 induced vascularization and showed an additive enhancement of angiogenesis with VEGF. In the murine breast cancer xenograft model, human MCF-7 cells with stable overexpression of BMP-2 developed vascularized tumors while empty vector control MCF-7 cells failed to form tumors. CONCLUSIONS: We conclude that activation of the BMP pathway by BMP-2 can promote vascularization and might be involved in tumor angiogenesis possibly by stimulating the Id1 and p38 MAPK pathway.

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.

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.

Use of a mouse in vitro fertilization model to understand the developmental origins of health and disease hypothesis.

The Developmental Origins of Health and Disease hypothesis holds that alterations to homeostasis during critical periods of development can predispose individuals to adult-onset chronic diseases such as diabetes and metabolic syndrome. It remains controversial whether preimplantation embryo manipulation, clinically used to treat patients with infertility, disturbs homeostasis and affects long-term growth and metabolism. To address this controversy, we have assessed the effects of in vitro fertilization (IVF) on postnatal physiology in mice. We demonstrate that IVF and embryo culture, even under conditions considered optimal for mouse embryo culture, alter postnatal growth trajectory, fat accumulation, and glucose metabolism in adult mice. Unbiased metabolic profiling in serum and microarray analysis of pancreatic islets and insulin sensitive tissues (liver, skeletal muscle, and adipose tissue) revealed broad changes in metabolic homeostasis, characterized by systemic oxidative stress and mitochondrial dysfunction. Adopting a candidate approach, we identify thioredoxin-interacting protein (TXNIP), a key molecule involved in integrating cellular nutritional and oxidative states with metabolic response, as a marker for preimplantation stress and demonstrate tissue-specific epigenetic and transcriptional TXNIP misregulation in selected adult tissues. Importantly, dysregulation of TXNIP expression is associated with enrichment for H4 acetylation at the Txnip promoter that persists from the blastocyst stage through adulthood in adipose tissue. Our data support the vulnerability of preimplantation embryos to environmental disturbance and demonstrate that conception by IVF can reprogram metabolic homeostasis through metabolic, transcriptional, and epigenetic mechanisms with lasting effects for adult growth and fitness. This study has wide clinical relevance and underscores the importance of continued follow-up of IVF-conceived offspring.

ECM-dependent HIF induction directs trophoblast stem cell fate via LIMK1-mediated cytoskeletal rearrangement.

The Hypoxia-inducible Factor (HIF) family of transcriptional regulators coordinates the expression of dozens of genes in response to oxygen deprivation. Mammalian development occurs in a hypoxic environment and HIF-null mice therefore die in utero due to multiple embryonic and placental defects. Mouse embryonic stem cells do not differentiate into placental cells; therefore, trophoblast stem cells (TSCs) are used to study mouse placental development. Consistent with a requirement for HIF activity during placental development in utero, TSCs derived from HIF-null mice exhibit severe differentiation defects and fail to form trophoblast giant cells (TGCs) in vitro. Interestingly, differentiating TSCs induce HIF activity independent of oxygen tension via unclear mechanisms. Here, we show that altering the extracellular matrix (ECM) composition upon which TSCs are cultured changes their differentiation potential from TGCs to multinucleated syncytiotropholasts (SynTs) and blocks oxygen-independent HIF induction. We further find that modulation of Mitogen Activated Protein Kinase Kinase-1/2 (MAP2K1/2, MEK-1/2) signaling by ECM composition is responsible for this effect. In the absence of ECM-dependent cues, hypoxia-signaling pathways activate this MAPK cascade to drive HIF induction and redirect TSC fate along the TGC lineage. In addition, we show that integrity of the microtubule and actin cytoskeleton is critical for TGC fate determination. HIF-2α ensures TSC cytoskeletal integrity and promotes invasive TGC formation by interacting with c-MYC to induce non-canonical expression of Lim domain kinase 1-an enzyme that regulates microtubule and actin stability, as well as cell invasion. Thus, we find that HIF can integrate positional and metabolic cues from within the TSC niche to regulate placental development by modulating the cellular cytoskeleton via non-canonical gene expression.

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.

Anoxic induction of ATF-4 through HIF-1-independent pathways of protein stabilization in human cancer cells.

Hypoxia is a key factor in tumor development, contributing to angiogenesis and radiotherapy resistance. Hypoxia-inducible factor-1 (HIF-1) is a major transcription factor regulating the response of cancer cells to hypoxia. However, tumors also contain areas of more severe oxygen depletion, or anoxia. Mechanisms for survival under anoxia are HIF-1alpha independent in Caenorhabditis elegans and, thus, differ from the hypoxic response. Here we report a differential response of cancer cells to hypoxia and anoxia by demonstrating the induction of activating transcription factor-4 (ATF-4) and growth arrest DNA damage 153 (GADD153) protein specifically in anoxia and the lack of induction in hypoxia. By applying RNAi, ATF-4 induction in anoxia was shown to be independent of HIF-1alpha, and desferrioxamine mesylate (DFO) and cobalt chloride induced HIF-1alpha but not ATF-4 or GADD153. Furthermore, the inductive response of ATF-4 and GADD153 was not related to alterations in or arrest of mitochondrial respiration and was independent of von Hippel-Lindau (VHL) disease mutations. In reoxygenated anoxic cells, ATF-4 had a half-life of less than 5 minutes; adding the proteasome inhibitor to normoxic cells up-regulated ATF-4 protein. Extracts from primary human tumors demonstrated more ATF-4 expression in tumors near necrotic areas. Thus, this study demonstrates a novel HIF-1alpha-independent anoxic mechanism that regulates ATF-4 induction at the protein stability level in tumor cells.

Expression of HIF-1alpha by human macrophages: implications for the use of macrophages in hypoxia-regulated cancer gene therapy.

Large numbers of monocytes extravasate from the blood into human tumours, where they differentiate into macrophages. In both breast and prostate carcinomas, these cells accumulate in areas of low oxygen tension (hypoxia), where they respond to hypoxia with the up-regulation of one or more hypoxia-inducible factors (HIFs). These then accumulate in the nucleus and bind to short DNA sequences called hypoxia-response elements (HREs) near or in such oxygen-sensitive genes as that encoding the pro-angiogenic factor vascular endothelial growth factor (VEGF). This stimulates gene expression and could explain why, in part, macrophages express abundant VEGF only in avascular, hypoxic areas of breast carcinomas. It also suggests that macrophages could be used to deliver HRE-regulated therapeutic genes specifically to hypoxic tumour areas. A recent study suggested that hypoxic macrophages accumulate HIF-2 rather than HIF-1, prompting the search for HRE constructs that optimally bind HIF-2 for use in macrophage-based gene therapy protocols. However, the present study shows that human macrophages accumulate higher levels of HIF-1 than HIF-2 when exposed to tumour-specific levels of hypoxia in vitro; that macrophages in human tumours express abundant HIF-1; and that expression from HRE-driven reporter constructs in the human macrophage-like cell line MonoMac 6 correlates more closely with HIF-1 than with HIF-2 up-regulation under hypoxia. Taken together, these findings suggest that HIF-1 may be the major hypoxia-inducible transcription factor in macrophages and that HIF-1-regulated constructs are likely to be effective in macrophage delivery of hypoxia-regulated gene therapy to human tumours.