Trained Memory of Human Uterine NK Cells Enhances Their Function in Subsequent Pregnancies.

Natural killer cells (NKs) are abundant in the human decidua, regulating trophoblast invasion and angiogenesis. Several diseases of poor placental development are associated with first pregnancies, so we thus looked to characterize differences in decidual NKs (dNKs) in first versus repeated pregnancies. We discovered a population found in repeated pregnancies, which has a unique transcriptome and epigenetic signature, and is characterized by high expression of the receptors NKG2C and LILRB1. We named these cells Pregnancy Trained decidual NK cells (PTdNKs). PTdNKs have open chromatin around the enhancers of IFNG and VEGFA. Activation of PTdNKs led to increased production and secretion of IFN-γ and VEGFα, with the latter supporting vascular sprouting and tumor growth. The precursors of PTdNKs seem to be found in the endometrium. Because repeated pregnancies are associated with improved placentation, we propose that PTdNKs, which are present primarily in repeated pregnancies, might be involved in proper placentation.

Identification of vascular cues contributing to cancer cell stemness and function.

Glioblastoma stem cells (GSCs) reside close to blood vessels (BVs) but vascular cues contributing to GSC stemness and the nature of GSC-BVs cross talk are not fully understood. Here, we dissected vascular cues influencing GSC gene expression and function to perfusion-based vascular cues, as well as to those requiring direct GSC-endothelial cell (EC) contacts. In light of our previous finding that perivascular tumor cells are metabolically different from tumor cells residing further downstream, cancer cells residing within a narrow, < 60 µm wide perivascular niche were isolated and confirmed to possess a superior tumor-initiation potential compared with those residing further downstream. To circumvent reliance on marker expression, perivascular GSCs were isolated from the respective locales based on their relative state of quiescence. Combined use of these procedures uncovered a large number of previously unrecognized differentially expressed GSC genes. We show that the unique metabolic milieu of the perivascular niche dominated by the highly restricted zone of mTOR activity is conducive for acquisition of GSC properties, primarily in the regulation of genes implicated in cell cycle control. A complementary role of vascular cues including those requiring direct glioma/EC contacts was revealed using glioma/EC co-cultures. Outstanding in the group of glioma cells impacted by nearby ECs were multiple genes responsible for maintaining GSCs in an undifferentiated state, a large fraction of which also relied on Notch-mediated signaling. Glioma-EC communication was found to be bidirectional, evidenced by extensive Notch-mediated EC reprogramming by contacting tumor cells, primarily metabolic EC reprogramming.

Age-Dependent Remarkable Regenerative Potential of the Dentate Gyrus Provided by Intrinsic Stem Cells.

Multiple insults to the brain lead to neuronal cell death, thus raising the question to what extent can lost neurons be replenished by adult neurogenesis. Here we focused on the hippocampus and especially the dentate gyrus (DG), a vulnerable brain region and one of the two sites where adult neuronal stem cells (NSCs) reside. While adult hippocampal neurogenesis was extensively studied with regard to its contribution to cognitive enhancement, we focused on their underestimated capability to repair a massively injured, nonfunctional DG. To address this issue, we inflicted substantial DG-specific damage in mice of either sex either by diphtheria toxin-based ablation of >50% of mature DG granule cells (GCs) or by prolonged brain-specific VEGF overexpression culminating in extensive, highly selective loss of DG GCs (thereby also reinforcing the notion of selective DG vulnerability). The neurogenic system promoted effective regeneration by increasing NSCs proliferation/survival rates, restoring a nearly original DG mass, promoting proper rewiring of regenerated neurons to their afferent and efferent partners, and regaining of lost spatial memory. Notably, concomitantly with the natural age-related decline in the levels of neurogenesis, the regenerative capacity of the hippocampus also subsided with age. The study thus revealed an unappreciated regenerative potential of the young DG and suggests hippocampal NSCs as a critical reservoir enabling recovery from catastrophic DG damage.SIGNIFICANCE STATEMENT Adult hippocampal neurogenesis has been extensively studied in the context of its role in cognitive enhancement, but whether, and to what extent can dentate gyrus (DG)-resident neural stem cells drive regeneration of an injured DG has remained unclear. Here we show that DG neurogenesis acts to replace lost neurons and restore lost functions even following massive (>50%) neuronal loss. Age-related decline of neurogenesis is paralleled by a progressive decline of regenerative capacity. Considering also the exceptional vulnerability of the DG to insults, these findings provide a further rationale for maintaining DG neurogenesis in adult life.

Modulation of MKNK2 alternative splicing by splice-switching oligonucleotides as a novel approach for glioblastoma treatment.

The gene encoding the kinase Mnk2 (MKNK2) is alternatively spliced to produce two isoforms-Mnk2a and Mnk2b. We previously showed that Mnk2a is downregulated in several types of cancer and acts as a tumor suppressor by activation of the p38-MAPK stress pathway, inducing apoptosis. Moreover, Mnk2a overexpression suppressed Ras-induced transformation in culture and in vivo. In contrast, the Mnk2b isoform acts as a pro-oncogenic factor. In this study, we designed modified-RNA antisense oligonucleotides and screened for those that specifically induce a strong switch in alternative splicing of the MKNK2 gene (splice switching oligonucleotides or SSOs), elevating the tumor suppressive isoform Mnk2a at the expense of the pro-oncogenic isoform Mnk2b. Induction of Mnk2a by SSOs in glioblastoma cells activated the p38-MAPK pathway, inhibited the oncogenic properties of the cells, re-sensitized the cells to chemotherapy and inhibited glioblastoma development in vivo. Moreover, inhibition of p38-MAPK partially rescued glioblastoma cells suggesting that most of the anti-oncogenic activity of the SSO is mediated by activation of this pathway. These results suggest that manipulation of MKNK2 alternative splicing by SSOs is a novel approach to inhibit glioblastoma tumorigenesis.

Unique role for dentate gyrus microglia in neuroblast survival and in VEGF-induced activation.

Neurogenic roles of microglia (MG) are thought to include an active role in adult hippocampal neurogenesis in addition to their established roles in pruning surplus dendrites and clearing dead neuroblasts. However, identification of such a role and its delineation in the neurogenic cascade is yet to be established. Using diphtheria toxin-aided MG ablation, we show that MG reduction in the DG-the site where neuronal stem cells (NSCs) reside-is sufficient to impede overall hippocampal neurogenesis due to reduced survival of newly formed neuroblasts. To examine whether MG residing in the hippocampal neurogenic zone are inherently different from MG residing elsewhere in the hippocampus, we compared growth factor responsiveness of DG MG with that of CA1 MG. Strikingly, transgenic induction of the potent neurogenic factor VEGF elicited robust on-site MG expansion and activation exclusively in the DG and despite eliciting a comparable angiogenic response in the CA1 and elsewhere. Temporally, DG-specific MG expansion preceded both angiogenic and neurogenic responses. Remarkably, even partial MG reduction during the process of VEGF-induced neurogenesis led to reducing the number of newly formed neuroblasts to the basal level. Transcriptomic analysis of MG retrieved from the naïve DG and CA1 uncovered a set of genes preferentially expressed in DG MG. Notably the tyrosine kinase Axl is exclusively expressed in naïve and VEGF-induced DG MG and its inhibition prevented neurogenesis augmentation by VEGF. Taken together, findings uncover inherent unique properties of DG MG of supporting both basal- and VEGF-induced adult hippocampal neurogenesis.

VEGF preconditioning leads to stem cell remodeling and attenuates age-related decay of adult hippocampal neurogenesis.

Several factors are known to enhance adult hippocampal neurogenesis but a factor capable of inducing a long-lasting neurogenic enhancement that attenuates age-related neurogenic decay has not been described. Here, we studied hippocampal neurogenesis following conditional VEGF induction in the adult brain and showed that a short episode of VEGF exposure withdrawn shortly after the generation of durable new vessels (but not under conditions where newly made vessels failed to persist) is sufficient for neurogenesis to proceed at a markedly elevated level for many months later. Continual neurogenic increase over several months was not accompanied by accelerated exhaustion of the neuronal stem cell (NSC) reserve, thereby allowing neurogenesis to proceed at a markedly elevated rate also in old mice. Neurogenic enhancement by VEGF preconditioning was, in part, attributed to rescue of age-related NSC quiescence. Remarkably, VEGF caused extensive NSC remodelling manifested in transition of the enigmatic NSC terminal arbor onto long cytoplasmic processes engaging with and spreading over even remote blood vessels, a configuration reminiscent of early postnatal "juvenile" NSCs. Together, these findings suggest that VEGF preconditioning might be harnessed for long-term neurogenic enhancement despite continued exposure to an "aged" systemic milieu.

Conditional islet hypovascularisation does not preclude beta cell expansion during pregnancy in mice.

AIMS/HYPOTHESIS: Endothelial-endocrine cell interactions and vascular endothelial growth factor (VEGF)-A signalling are deemed essential for maternal islet vascularisation, glucose control and beta cell expansion during mouse pregnancy. The aim of this study was to assess whether pregnancy-associated beta cell expansion was affected under conditions of islet hypovascularisation. METHODS: Soluble fms-like tyrosine kinase 1 (sFLT1), a VEGF-A decoy receptor, was conditionally overexpressed in maternal mouse beta cells from 1.5 to 14.5 days post coitum. Islet vascularisation, glycaemic control, beta cell proliferation, individual beta cell size and total beta cell volume were assessed in both pregnant mice and non-pregnant littermates. RESULTS: Conditional overexpression of sFLT1 in beta cells resulted in islet hypovascularisation and glucose intolerance in both pregnant and non-pregnant mice. In contrast to non-pregnant littermates, glucose intolerance in pregnant mice was transient. sFLT1 overexpression did not affect pregnancy-associated changes in beta cell proliferation, individual beta cell size or total beta cell volume. CONCLUSIONS/INTERPRETATION: Reduced intra-islet VEGF-A signalling results in maternal islet hypovascularisation and impaired glycaemic control but does not preclude beta cell expansion during mouse pregnancy.

Short-term overexpression of VEGF-A in mouse beta cells indirectly stimulates their proliferation and protects against diabetes.

AIMS/HYPOTHESIS: Vascular endothelial growth factor (VEGF) has been recognised by loss-of-function experiments as a pleiotropic factor with importance in embryonic pancreas development and postnatal beta cell function. Chronic, nonconditional overexpression of VEGF-A has a deleterious effect on beta cell development and function. We report, for the first time, a conditional gain-of-function study to evaluate the effect of transient VEGF-A overexpression by adult pancreatic beta cells on islet vasculature and beta cell proliferation and survival, under both normal physiological and injury conditions. METHODS: In a transgenicmouse strain, overexpressing VEGF-A in a doxycycline-inducible and beta cell-specific manner, we evaluated the ability of VEGF-A to affect islet vessel density, beta cell proliferation and protection of the adult beta cell mass from toxin-induced injury. RESULTS: Short-term VEGF-A overexpression resulted in islet hypervascularisation, increased beta cell proliferation and protection from toxin-mediated beta cell death, and thereby prevented the development of hyperglycaemia. Extended overexpression of VEGF-A led to impaired glucose tolerance, elevated fasting glycaemia and a decreased beta cell mass. CONCLUSIONS/INTERPRETATION: Overexpression of VEGF-A in beta cells time-dependently affects glycometabolic control and beta cell protection and proliferation. These data nourish further studies to examine the role of controlled VEGF delivery in (pre)clinical applications aimed at protecting and/or restoring the injured beta cell mass.

A perfusion-independent role of blood vessels in determining branching stereotypy of lung airways.

Blood vessels have been shown to play perfusion-independent roles in organogenesis. Here, we examined whether blood vessels determine branching stereotypy of the mouse lung airways in which coordinated branching of epithelial and vascular tubes culminates in their co-alignment. Using different ablative strategies to eliminate the lung vasculature, both in vivo and in lung explants, we show that proximity to the vasculature is indeed essential for patterning airway branching. Remarkably, although epithelial branching per se proceeded at a nearly normal rate, branching stereotypy was dramatically perturbed following vascular ablation. Specifically, branching events requiring a rotation to change the branching plane were selectively affected. This was evidenced by either the complete absence or the shallow angle of their projections, with both events contributing to an overall flat lung morphology. Vascular ablation also led to a high frequency of ectopic branching. Regain of vascularization fully rescued arrested airway branching and restored normal lung size and its three-dimensional architecture. This role of the vasculature is independent of perfusion, flow or blood-borne substances. Inhibition of normal branching resulting from vascular loss could be explained in part by perturbing the unique spatial expression pattern of the key branching mediator FGF10 and by misregulated expression of the branching regulators Shh and sprouty2. Together, these findings uncovered a novel role of the vasculature in organogenesis, namely, determining stereotypy of epithelial branching morphogenesis.

Blood vessels restrain pancreas branching, differentiation and growth.

How organ size and form are controlled during development is a major question in biology. Blood vessels have been shown to be essential for early development of the liver and pancreas, and are fundamental to normal and pathological tissue growth. Here, we report that, surprisingly, non-nutritional signals from blood vessels act to restrain pancreas growth. Elimination of endothelial cells increases the size of embryonic pancreatic buds. Conversely, VEGF-induced hypervascularization decreases pancreas size. The growth phenotype results from vascular restriction of pancreatic tip cell formation, lateral branching and differentiation of the pancreatic epithelium into endocrine and acinar cells. The effects are seen both in vivo and ex vivo, indicating a perfusion-independent mechanism. Thus, the vasculature controls pancreas morphogenesis and growth by reducing branching and differentiation of primitive epithelial cells.

Delineating multiple functions of VEGF-A in the adult brain.

Vascular endothelial growth factor-A (abbreviated throughout this review as VEGF) is mostly known for its angiogenic activity, for its activity as a vascular permeability factor, and for its vascular survival activity [1]. There is a growing body of evidence, however, that VEGF fulfills additional less 'traditional' functions in multiple organs, both during development, as well as homeostatic functions in fully developed organs. This review focuses on the multiple roles of VEGF in the adult brain and is less concerned with the roles played by VEGF during brain development, functions described elsewhere in this review series. Most functions of VEGF that are essential for proper brain development are, in fact, dispensable in the adult brain as was clearly demonstrated using a conditional brain-specific VEGF loss-of-function (LOF) approach. Thus, in contrast to VEGF LOF in the developing brain, a process which is detrimental for the growth and survival of blood vessels and leads to massive neuronal apoptosis [2-4], continued signaling by VEGF in the mature brain is no longer required for maintaining already established cerebral vasculature and its inhibition does not cause appreciable vessel regression, hypoxia or apoptosis [4-7]. Yet, VEGF continues to be expressed in the adult brain in a constitutive manner. Moreover, VEGF is expressed in the adult brain in a region-specific manner and in distinctive spatial patterns incompatible with an angiogenic role (see below), strongly suggesting angiogenesis-independent and possibly also perfusion-independent functions. Here we review current knowledge on some of these 'non-traditional', often unexpected homeostatic VEGF functions, including those unrelated to its effects on the brain vasculature. These effects could be mediated directly (on non-vascular cells expressing cognate VEGF receptors) or indirectly (via the endothelium). Experimental approaches aimed at distinguishing between these possibilities for each particular VEGF function will be described. This review is only concerned with homeostatic functions of VEGF in the normal, non-injured brain. The reader is referred elsewhere in this series for a review on VEGF actions in response to various forms of brain injury and/or brain pathology.

Reversible modulations of neuronal plasticity by VEGF.

Neurons, astrocytes, and blood vessels are organized in functional "neurovascular units" in which the vasculature can impact neuronal activity and, in turn, dynamically adjust to its change. Here we explored different mechanisms by which VEGF, a pleiotropic factor known to possess multiple activities vis-à-vis blood vessels and neurons, may affect adult neurogenesis and cognition. Conditional transgenic systems were used to reversibly overexpress VEGF or block endogenous VEGF in the hippocampus of adult mice. Importantly, this was done in settings that allowed the uncoupling of VEGF-promoted angiogenesis, neurogenesis, and memory. VEGF overexpression was found to augment all three processes, whereas VEGF blockade impaired memory without reducing hippocampal perfusion or neurogenesis. Pertinent to the general debate regarding the relative contribution of adult neurogenesis to memory, we found that memory gain by VEGF overexpression and memory impairment by VEGF blockade were already evident at early time points at which newly added neurons could not yet have become functional. Surprisingly, VEGF induction markedly increased in vivo long-term potentiation (LTP) responses in the dentate gyrus, and VEGF blockade completely abrogated LTP. Switching off ectopic VEGF production resulted in a return to a normal memory and LTP, indicating that ongoing VEGF is required to maintain increased plasticity. In summary, the study not only uncovered a surprising role for VEGF in neuronal plasticity, but also suggests that improved memory by VEGF is primarily a result of increasing plasticity of mature neurons rather than the contribution of newly added hippocampal neurons.

VEGF is required for dendritogenesis of newly born olfactory bulb interneurons.

The angiogenic factor vascular endothelial growth factor A (VEGF) has been shown to have a role in neurogenesis, but how it affects adult neurogenesis is not fully understood. To delineate a role for VEGF in successive stages of olfactory bulb (OB) neurogenesis, we used a conditional transgenic system to suppress VEGF signaling at the adult mouse sub-ventricular zone (SVZ), rostral migratory stream (RMS) and OB, which constitute the respective sites of birth, the migration route, and sites where newly born interneurons mature and integrate within the existing OB circuitry. Following the development of fluorescently tagged adult-born neurons, we show that sequestration of VEGF that is constitutively expressed by distinct types of resident OB neurons greatly impaired dendrite development in incoming SVZ-born neurons. This was evidenced by reduced dendritic spine density of granule cells and significantly shorter and less branched dendrites in periglomerular neurons. Notably, the vasculature and perfusion of the SVZ, RMS and OB were not adversely affected when VEGF suppression was delayed until after birth, thus uncoupling the effect of VEGF on dendritogenesis from its known role in vascular maintenance. Furthermore, a requirement for VEGF was specific to newly born neurons, as already established OB neurons were not damaged by VEGF inhibition. This study thus uncovered a surprising perfusion-independent role of VEGF in the adult brain, namely, an essential role in the maturation of adult-born neurons.

Local retention versus systemic release of soluble VEGF receptor-1 are mediated by heparin-binding and regulated by heparanase.

RATIONALE: The vascular endothelial growth factor (VEGF) decoy receptor soluble VEGF-R1 (sVEGF-R1) is thought to protect the cells that produce it from adverse VEGF signaling. To accomplish this role, a mechanism for pericellular retention of sVEGF-R1 is required. Local retention may also prevent the accumulation of high circulating levels of sVEGF-R1 and resulting interference with homeostatic VEGF functions in remote organs. OBJECTIVE: To reveal natural storage depots of sVEGF-R1 and determine mechanisms underlying its pericellular retention. To uncover natural mechanisms regulating its systemic release. METHODS AND RESULTS: We show that both the canonical and human-specific isoforms of sVEGF-R1 are strongly bound to heparin. sVEGF-R1 produced by vascular smooth muscle cells is stored in the vessel wall and can be displaced from isolated mouse aorta by heparin. Another major reservoir of sVEGF-R1 is the placenta. Heparin increases the level of sVEGF-R1 released by cultured human placental villi, and pregnant women treated with low molecular weight heparin showed markedly elevated levels of sVEGF-R1 in the circulation. Heparanase is expressed in human placenta at the same locales as sVEGF-R1, and its transgenic overexpression in mice resulted in a marked increase in the levels of circulating sVEGF-R1. Conversely, heparanase inhibition, by either a neutralizing antibody or by inhibition of its maturation, reduced the amounts of sVEGF-R1 released from human placental villi, indicating a natural role of heparanase in sVEGF-R1 release. CONCLUSIONS: Together, the findings uncover a new level of regulation governing sVEGF-R1 retention versus release and suggest that manipulations of the heparin/heparanase system could be harnessed for reducing unwarranted release of sVEGF-R1 in pathologies such as preeclampsia.

Vascular endothelial growth factor-induced neovascularization rescues cardiac function but not adverse remodeling at advanced ischemic heart disease.

OBJECTIVE: Proangiogenic therapy is a promising avenue for the treatment for chronic heart failure and a potentially powerful modality for reversing adverse cardiac remodeling. There is a concern, however, that adverse remodeling might enter an irreversible stage, and become refractory to treatments. The present study aims to determine whether neovascularization therapy is feasible at end stage heart failure and its capacity to reverse adverse cardiac remodeling during progressive disease stages. METHODS AND RESULTS: Using a conditional transgenic mouse system for generating escalating levels of myocardium-specific vascular deficit and resultant stepwise development of heart remodeling, we show that left ventricular dilatation and fibrosis precede ventricular hypertrophy, but that interstitial fibrosis is progressive and eventually results in heart failure. Vascular endothelial growth factor-mediated neovascularization was efficient even at the end stage of disease, and rescued compromised contractile function. Remarkably, remodeling was also fully reversed by neovascularization during early and late stages. Adverse remodeling could not be rescued, however, at the end stage of the disease, thus defining a point of no return and indentifying a critical level of fibrosis as the key determinant to be considered in intended reversal. CONCLUSIONS: The study supports the notion of a restricted golden time for remodeling reversal but not for vascular endothelial growth factor-induced neovascularization, which is feasible even during advanced disease stages.

Decidual NK cells regulate key developmental processes at the human fetal-maternal interface.

Human CD56(bright) NK cells accumulate in the maternal decidua during pregnancy and are found in direct contact with fetal trophoblasts. Several mechanisms have been proposed to explain the inability of NK cells to kill the semiallogeneic fetal cells. However, the actual functions of decidual NK (dNK) cells during pregnancy are mostly unknown. Here we show that dNK cells, but not peripheral blood-derived NK subsets, regulate trophoblast invasion both in vitro and in vivo by production of the interleukin-8 and interferon-inducible protein-10 chemokines. Furthermore, dNK cells are potent secretors of an array of angiogenic factors and induce vascular growth in the decidua. Notably, such functions are regulated by specific interactions between dNK-activating and dNK-inhibitory receptors and their ligands, uniquely expressed at the fetal-maternal interface. The overall results support a 'peaceful' model for reproductive immunology, in which elements of innate immunity have been incorporated in a constructive manner to support reproductive tissue development.

Decrease in VEGF expression induces intussusceptive vascular pruning.

OBJECTIVE: The concept of vascular pruning, the "cuting-off" of vessels, is gaining importance due to expansion of angio-modulating therapies. The proangiogenic effects of vascular endothelial growth factor (VEGF) are broadly described, but the mechanisms of structural alterations by its downregulation are not known. METHODS AND RESULTS: VEGF(165)-releasing hydrogels were applied onto the chick chorioallantoic membrane on embryonic day 10. The hydrogels, designed to completely degrade within 2 days, caused high-level VEGF presentation followed by abrupt VEGF withdrawal. Application of VEGF resulted in a pronounced angiogenic response within 24 hours. The drastic decrease in level of exogenous VEGF-A within 48 hours was corroborated by enzyme-linked immunosorbent assay. Following this VEGF withdrawal we observed vasculature adaptation by means of intussusception, including intussusceptive vascular pruning. As revealed on vascular casts and serial semithin sections, intussusceptive vascular pruning occurred by emergence of multiple eccentric pillars at bifurcations. Time-lapse in vivo microscopy has confirmed the de novo occurrence of transluminal pillars and their capability to induce pruning. Quantitative evaluation corroborated an extensive activation of intussusception associated with VEGF withdrawal. CONCLUSIONS: Diminution of VEGF level induces vascular tree regression by intussusceptive vascular pruning. This observation may allude to the mechanism underlying the "normalization" of tumor vasculature if treated with antiangiogenic drugs. The mechanism described here gives new insights into the understanding of the processes of vasculature regression and hence provides new and potentially viable targets for antiangiogenic and/or angio-modulating therapies during various pathological processes.

Vascular Endothelial Growth Factor Augments the Tolerance Towards Cerebral Stroke by Enhancing Neurovascular Repair Mechanism.

The breakdown of the blood-brain barrier (BBB) is a critical event in the development of secondary brain injury after stroke. Among the cellular hallmarks in the acute phase after stroke are a downregulation of tight-junction molecules and the loss of microvascular pericyte coverage and endothelial sealing. Thus, a rapid repair of blood vessel integrity and re-stabilization of the BBB is considered an important strategy to reduce secondary brain damage. However, the mechanisms underlying BBB disruption remain poorly understood. Especially, the role of VEGF in this context remains inconclusive. With the conditional and reversible VEGF expression systems, we studied the time windows of deleterious and beneficial VEGF actions on blood vessel integrity in mice. Using genetic systems for gain of function and loss of function experiments, we activated and inhibited VEGF signaling prior and simultaneously to ischemic stroke onset. In both scenarios, VEGF seems to play a vital role in containing the stroke-induced damage after cerebral ischemia. We report that the transgenic overexpression of VEGF (GOF) prior to the stroke stabilizes the vasculature and prevents blood-brain barrier disruption in young and aged animals after stroke. Whereas inhibition of signals for endogenous VEGF (LOF) prior to stroke results in bigger infarction with massive brain swelling and enhanced BBB permeability, furthermore, activating or blocking VEGF signaling after ischemic stroke onset had comparable effects on BBB repair and cerebral edema. VEGF can function as an anti-permeability factor, and a VEGF-based therapy in the context of stroke prevention and recovery has an enormous potential.

Metabolic homeostasis is maintained in myocardial hibernation by adaptive changes in the transcriptome and proteome.

A transgenic mouse model for conditional induction of long-term hibernation via myocardium-specific expression of a VEGF-sequestering soluble receptor allowed the dissection of the hibernation process into an initiation and a maintenance phase. The hypoxic initiation phase was characterized by peak levels of K(ATP) channel and glucose transporter 1 (GLUT1) expression. Glibenclamide, an inhibitor of K(ATP) channels, blocked GLUT1 induction. In the maintenance phase, tissue hypoxia and GLUT1 expression were reduced. Thus, we employed a combined "-omics" approach to resolve this cardioprotective adaptation process. Unguided bioinformatics analysis on the transcriptomic, proteomic and metabolomic datasets confirmed that anaerobic glycolysis was affected and that the observed enzymatic changes in cardiac metabolism were directly linked to hypoxia-inducible factor (HIF)-1 activation. Although metabolite concentrations were kept relatively constant, the combination of the proteomic and transcriptomic dataset improved the statistical confidence of the pathway analysis by 2 orders of magnitude. Importantly, proteomics revealed a reduced phosphorylation state of myosin light chain 2 and cardiac troponin I within the contractile apparatus of hibernating hearts in the absence of changes in protein abundance. Our study demonstrates how combining different "-omics" datasets aids in the identification of key biological pathways: chronic hypoxia resulted in a pronounced adaptive response at the transcript and the protein level to keep metabolite levels steady. This preservation of metabolic homeostasis is likely to contribute to the long-term survival of the hibernating myocardium.