Replication of association between polymorphisms of the pancreatic ATP-sensitive potassium channel and susceptibility to type 2 diabetes in two Russian urban populations

The KCNJ11 and ABCC8 genes encode components of the pancreatic ATP-sensitive potassium (KATP) channel. Previously, we reported association of the KCNJ11 E23K and ABCC8 R1273R G/A variants with type 2 diabetes (T2D) in a small Russian population sample (n=244). Here we replicated association between these genetic variants and T2D in a larger cohort (588 diabetic and 597 non-diabetic subjects). Using the ANCOVA analysis, Odds Ratios (ORs) and relationships between the carriage of a genotype and biochemical parameters of the patients were assessed and then adjusted for confounders (age, gender, HbA1c, hypertension, and obesity). The KCNJ11 K23 variant and the ABCC8 R1273R allele A showed association with higher risk of T2D (adjusted OR of 1.41 and 2.03, P<0.0001, respectively). Diabetic patients homozygous for K/K had lower 2h insulin (P_adjusted=0.044). The ABCC8 A/A variant was associated with increased 2h serum insulin in diabetic and non-diabetic subjects (P_adjusted=0.027 and 0.033, respectively). The carriage of the risk variant K/K of KCNJ11 E23K or A/A of ABCC8 G/A R1273R was associated with reduced response to nonsulfonylurea and sulfonylurea blockers of the pancreatic KATP channel. Adjusted attributable population risk was 3.0% (KCNJ11 E23K) and 4.8% (ABCC8 G/A) suggesting for the modest effects of these genetic variants on diabetes susceptibility.     

A novel KCNJ11 mutation associated with congenital hyperinsulinism reduces the intrinsic open probability of beta-cell ATP-sensitive potassium channels

The beta-cell ATP-sensitive potassium (KATP) channel controls insulin secretion by linking glucose metabolism to membrane excitability. Loss of KATP channel function due to mutations in ABCC8 or KCNJ11, genes that encode the sulfonylurea receptor 1 or the inward rectifier Kir6.2 subunit of the channel, is a major cause of congenital hyperinsulinism. Here, we report identification of a novel KCNJ11 mutation associated with the disease that renders a missense mutation, F55L, in the Kir6.2 protein. Mutant channels reconstituted in COS cells exhibited a wild-type-like surface expression level and normal sensitivity to ATP, MgADP, and diazoxide. However, the intrinsic open probability of the mutant channel was greatly reduced, by approximately 10-fold. This low open probability defect could be reversed by application of phosphatidylinositol 4,5-bisphosphates or oleoyl-CoA to the cytoplasmic face of the channel, indicating that reduced channel response to membrane phospholipids and/or long chain acyl-CoAs underlies the low intrinsic open probability in the mutant. Our findings reveal a novel molecular mechanism for loss of KATP channel function and congenital hyperinsulinism and support the importance of phospholipids and/or long chain acyl-CoAs in setting the physiological activity of beta-cell KATP channels. The F55L mutation is located in the slide helix of Kir6.2. Several permanent neonatal diabetes-associated mutations found in the same structure have the opposite effect of increasing intrinsic channel open probability. Our results also highlight the critical role of the Kir6.2 slide helix in determining the intrinsic open probability of KATP channels.     

KATP channel knockout worsens myocardial calcium stress load in vivo and impairs recovery in stunned heart

Gene knockout of the KCNJ11-encoded Kir6.2 ATP-sensitive K(+) (K(ATP)) channel implicates this stress-response element in the safeguard of cardiac homeostasis under imposed demand. K(ATP) channels are abundant in ventricular sarcolemma, where subunit expression appears to vary between the sexes. A limitation, however, in establishing the full significance of K(ATP) channels in the intact organism has been the inability to monitor in vivo the contribution of the channel to intracellular calcium handling and the superimposed effect of sex that ultimately defines heart function. Here, in vivo manganese-enhanced cardiac magnetic resonance imaging revealed, under dobutamine stress, a significantly greater accumulation of calcium in both male and female K(ATP) channel knockout (Kir6.2-KO) mice compared with sex- and age-matched wild-type (WT) counterparts, with greatest calcium load in Kir6.2-KO females. This translated, poststress, into a sustained contracture manifested by reduced end-diastolic volumes in K(ATP) channel-deficient mice. In response to ischemia-induced stunning, male and female Kir6.2-KO hearts demonstrated accelerated time to contracture and increased peak contracture compared with WT. The outcome on reperfusion, in both male and female Kir6.2-KO hearts, was a transient reduction in systolic performance, measured as rate-pressure product compared with WT, with protracted increase in left ventricular end-diastolic pressure, exaggerated in female knockout hearts, despite comparable leakage of creatine kinase across groups. Kir6.2-KO hearts were rescued from diastolic dysfunction by agents that target alternative pathways of calcium handling. Thus K(ATP) channel deficit confers a greater susceptibility to calcium overload in vivo, accentuated in female hearts, impairing contractile recovery under various conditions of high metabolic demand.     

Crucial role of peroxiredoxin III in placental antioxidant defense of mice

We observed frequent stillbirth in peroxiredoxin III (PrxIII) knockout maternal mice. Quantitative real time PCR (qRT-PCR) and Western-blot analysis revealed increased oxidative stress in placentas that were deficient in PrxIII. We did not find significant difference between PrxIII knockout maternal mice and wild-type littermates in hematological parameters, fetal number, and embryonic development. Nevertheless, we noticed enhanced expression of PrxI in erythrocytes of pregnant knockout mice. Our results provided in vivo evidence that PrxIII played a crucial role in placental antioxidant defense. Up-regulation of PrxI might provide a compensation that protected erythrocytes against oxidative damage.     

Permanent neonatal diabetes caused by dominant, recessive, or compound heterozygous SUR1 mutations with opposite functional effects

Heterozygous activating mutations in the KCNJ11 gene encoding the pore-forming Kir6.2 subunit of the pancreatic beta cell K(ATP) channel are the most common cause of permanent neonatal diabetes (PNDM). Patients with PNDM due to a heterozygous activating mutation in the ABCC8 gene encoding the SUR1 regulatory subunit of the K(ATP) channel have recently been reported. We studied a cohort of 59 patients with permanent diabetes who received a diagnosis before 6 mo of age and who did not have a KCNJ11 mutation. ABCC8 gene mutations were identified in 16 of 59 patients and included 8 patients with heterozygous de novo mutations. A recessive mode of inheritance was observed in eight patients with homozygous, mosaic, or compound heterozygous mutations. Functional studies of selected mutations showed a reduced response to ATP consistent with an activating mutation that results in reduced insulin secretion. A novel mutational mechanism was observed in which a heterozygous activating mutation resulted in PNDM only when a second, loss-of-function mutation was also present.     

KATP channel subunits are expressed in the epididymal epithelium in several mammalian species

Adenosine triphosphate-sensitive K(++) (K(ATP)) channels are poorly characterized in the reproductive tract. The present study was designed to evaluate the putative expression of K(ATP) channel subunits (Kir6.x and SURx) in the epididymis from different mammalian species. Immunohistochemical, Western blot, and RT-PCR techniques were used. A positive immunostaining for Kir6.2 (KCNJ11) and SUR2 (ABCC9) was observed by immunoenzymatic and immunofluorescent approaches in the principal epithelial cells throughout all regions of the rat and mouse epididymis. Double labeling with anti-aquaporin 9 (AQP9) and anti-Kir6.2 (KCNJ11) confirmed their colocalization in the principal cells. No immunostaining could be demonstrated for Kir6.1 (KCNJ8) and SUR1 (ABCC8) subunits. Under higher magnification, the immunostaining for Kir6.2 (KCNJ11) exhibited a cytoplasmic labeling that was more intense at the level of the Golgi apparatus along the whole epididymis. A similar pattern was observed for SUR2 (ABCC9), although in the latter case, the Golgi labeling appeared to be region specific. Spermatozoa in epididymal tubules from rodents also immunostained for Kir6.2 (KCNJ11) and SUR2 (ABCC9). Western blot analysis of epididymal total protein and crude membrane extracts from adult and prepubertal rats confirmed the presence of Kir6.2 (KCNJ11). SUR2 (ABCC9) protein expression was detected in adult epididymal extracts. Furthermore, RT-PCR established the presence of Kir6.2 (KCNJ11) and SUR2 (ABCC9) mRNA in prepubertal and adult mouse epididymis. Indirect immunofluorescence also documented the presence of Kir6.2 (KCNJ11) and SUR2 (ABCC9) in the epididymal epithelium, as well as in spermatozoa, of canine, feline, bovine, and human origin. These data demonstrate the presence of the K(ATP) channel subunits, Kir6.2 (KCNJ11) and SUR2 (ABCC9), in epididymal epithelial cells and spermatozoa from several mammalian species. Although their physiological roles need to be fully characterized, it is tempting to propose that such types of K(++) channels might be involved in protein secretion and fluid-electrolyte transport occurring along the epididymal epithelium, leading to spermatozoa maturation.     

Role of TASK2 potassium channels regarding volume regulation in primary cultures of mouse proximal tubules

Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using beta-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 microM 293B, but blocked by 500 microM quinidine and 10 microM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.     

Identification and functional characterization of Kir2.6 mutations associated with non-familial hypokalemic periodic paralysis

Hypokalemic periodic paralysis (hypoKPP) is characterized by episodic flaccid paralysis of muscle and acute hypokalemia during attacks. Familial forms of hypoKPP are predominantly caused by mutations of either voltage-gated Ca(2+) or Na(+) channels. The pathogenic gene mutation in non-familial hypoKPP, consisting mainly of thyrotoxic periodic paralysis (TPP) and sporadic periodic paralysis (SPP), is largely unknown. Recently, mutations in KCNJ18, which encodes a skeletal muscle-specific inwardly rectifying K(+) channel Kir2.6, were reported in some TPP patients. Whether mutations of Kir2.6 occur in other patients with non-familial hypoKPP and how mutations of the channel predispose patients to paralysis are unknown. Here, we report one conserved heterozygous mutation in KCNJ18 in two TPP patients and two separate heterozygous mutations in two SPP patients. These mutations result in V168M, R43C, and A200P amino acid substitution of Kir2.6, respectively. Compared with the wild type channel, whole-cell currents of R43C and V168M mutants were reduced by ～78 and 43%, respectively. No current was detected for the A200P mutant. Single channel conductance and open probability were reduced for R43C and V168M, respectively. Biotinylation assays showed reduced cell surface abundance for R43C and A200P. All three mutants exerted dominant negative inhibition on wild type Kir2.6 as well as wild type Kir2.1, another Kir channel expressed in the skeletal muscle. Thus, mutations of Kir2.6 are associated with SPP as well as TPP. We suggest that decreased outward K(+) current from hypofunction of Kir2.6 predisposes the sarcolemma to hypokalemia-induced paradoxical depolarization during attacks, which in turn leads to Na(+) channel inactivation and inexcitability of muscles.     

Ischemic shortening of action potential duration as a result of KATP channel opening attenuates myocardial stunning by reducing calcium influx

Action potential duration (APD) shortening due to opening of sarcolemmal ATP-dependent potassium (KATP) channels has been postulated to protect the myocardium against postischemic damage by reducing Ca²⁺ influx. This hypothesis was assessed, assuming that increased postischemic stunning due to KATP channel inhibition with glibenclamide could be reverted by the addition of the Ca²⁺ channel blocker diltiazem. Percent wall thickening fraction (% WTh, conscious sheep) and APD (open-chest sheep) were obtained from the following groups: control: 12 min ischemia by anterior descending coronary artery occlusion followed by 2 h reperfusion; glibenclamide: same as control, with glibenclamide (0.4 mg/kg) infused 30 min before ischemia; diltiazem: same as control, with diltiazem (100 g/kg) administered prior to ischemia; glibenclamide+diltiazem: both drugs infused as in glibenclamide and diltiazem groups. APD was reduced in control ischemia. Conversely, KATP-channel blockade by glibenclamide lengthened APD and increased postischemic stunning (p < 0.01 vs. control); glibenclamide+diltiazem did not shorten APD but enhanced functional recovery (p < 0.01 vs. glibenclamide). Ca²⁺ channel blockade improvement of increased stunning provoked by KATP channel inhibition supports the hypothesis that APD shortening due to opening of KATP channels protects against postischemic stunning by limiting Ca²⁺ influx.     

Potent inhibition of L-type Ca currents by a Rad variant associated with congestive heart failure

Ca²⁺ influx via L-type voltage-gated Ca²⁺ channels supports the plateau phase of ventricular action potentials and is the trigger for excitation–contraction (EC) coupling in the myocardium. Rad, a member of the RGK (Rem, Rem2, Rad, Gem/Kir) family of monomeric G proteins, regulates ventricular action potential duration and EC coupling gain through its ability to inhibit cardiac L-type channel activity. In this study, we have investigated the potential dysfunction of a naturally occurring Rad variant (Q66P) that has been associated with congestive heart failure in humans. Specifically, we have tested whether Rad Q66P limits, or even eliminates, the inhibitory actions of Rad on Ca_V1.2 and Ca_V1.3, the two L-type channel isoforms known to be expressed in the heart. We have found that mouse Rad Q65P (the murine equivalent of human Rad Q66P) inhibits L-type currents conducted by Ca_V1.2 or Ca_V1.3 channels as potently as wild-type Rad (>95% inhibition of both channels). In addition, Rad Q65P attenuates the gating movement of both channels as effectively as wild-type Rad, indicating that the Q65P substitution does not differentially impair any of the three described modes of L-type channel inhibition by RGK proteins. Thus, we conclude that if Rad Q66P contributes to cardiomyopathy, it does so via a mechanism that is not related to its ability to inhibit L-type channel-dependent processes per se. However, our results do not rule out the possibility that decreased expression, mistargeting or altered regulation of Rad Q66P may reduce the RGK protein’s efficacy in vivo.     

Carcinoembryonic antigen-related cell adhesion molecule 10 expressed specifically early in pregnancy in the decidua is dispensable for normal murine development

The carcinoembryonic antigen (CEA) family consists of a large group of evolutionarily and structurally divergent glycoproteins. The murine CEACAM9 and CEACAM11-related proteins as well as the pregnancy-specific glycoproteins (PSG) are secreted members of the CEA family which are differentially expressed in fetal trophoblast cell populations during placental development. PSG are essential for a successful pregnancy, possibly by protecting the semiallotypic fetus from the maternal immune system. In contrast, Ceacam10 mRNA, coding for a protein identical in structure with CEACAM11-related proteins, is expressed in the maternal decidua surrounding the implantation site of the conceptus only during early stages of gestation between day 6.5 and day 10.5 postcoitum. To determine its role during murine development, we inactivated Ceacam10. Ceacam10(-/-) mice developed, like the previously established Ceacam9(-/-) mice, indistinguishably from wild-type littermates with respect to sex ratio, weight gain, and fertility. However, a small but significant reduction of the litter size by 23% was observed in Ceacam10(-/-) matings. Furthermore, combining the Ceacam9 and Ceacam10 null alleles, both located on chromosome 7, by meiotic recombination and subsequent mating of heterozygotes carrying both knockout alleles on one chromosome yielded wild-type and double knockout offspring at the expected Mendelian ratio. Taken together, both Ceacam10 and Ceacam9, alone or in combination, are not essential for either murine placental and embryonic development or for adult life.     

Sur domains that associate with and gate KATP pores define a novel gatekeeper

Structure-function analyses of K+ channels identify a common pore architecture whose gating depends on diverse signal sensing elements. The "gatekeepers" of the long, ATP-inhibited KIR6.0 pores of KATP channels are ABC proteins, SURs, receptors for channel opening and closing drugs. Several competing models for SUR/KIR coupling exist. We show that SUR TMD0, the N-terminal bundle of five transmembrane helices, specifically associates with KIR6.2, forcing nearly silent pores to burst like native KATP channels and enhancing surface expression. Inclusion of adjacent submembrane residues of L0, the linker between TMD0 and the stimulatory nucleotide- and drug-binding ABC core, generates constitutively active channels, whereas additional cytoplasmic residues counterbalance this activation establishing a relationship between the mean open and burst times of intact pores. SUR fragments, lacking TMD0, fail to modulate KIR. TMD0 is thus the domain that anchors SUR to the KIR pore. Consistent with data on chimeric ABCC/KIRs and a modeled channel structure, we propose that interactions of TMD0-L0 with the outer helix and N terminus of KIR bidirectionally modulate gating. The results explain and predict pathologies associated with alteration of the 5' ends of clustered ABCC8 (9)/KCNJ11 (8) genes.     

Nucleotide-gated KATP channels integrated with creatine and adenylate kinases: Amplification,tuning and sensing of energetic signals in the compartmentalized cellular environment

Transmission of energetic signals to membrane sensors, such as the ATP-sensitive K+ (KATP) channel, is vital for cellular adaptation to stress. Yet, cell compartmentation implies diffusional hindrances that hamper direct reception of cytosolic energetic signals. With high intracellular ATP levels, KATP channels may sense not bulk cytosolic, but rather local submembrane nucleotide concentrations set by membrane ATPases and phosphotransfer enzymes. Here, we analyzed the role of adenylate kinase and creatine kinase phosphotransfer reactions in energetic signal transmission over the strong diffusional barrier in the submembrane compartment, and translation of such signals into a nucleotide response detectable by KATP channels. Facilitated diffusion provided by creatine kinase and adenylate kinase phosphotransfer dissipated nucleotide gradients imposed by membrane ATPases, and shunted diffusional restrictions. Energetic signals, simulated as deviation of bulk ATP from its basal level, were amplified into an augmented nucleotide response in the submembrane space due to failure under stress of creatine kinase to facilitate nucleotide diffusion. Tuning of creatine kinase-dependent amplification of the nucleotide response was provided by adenylate kinase capable of adjusting the ATP/ADP ratio in the submembrane compartment securing adequate KATP channel response in accord with cellular metabolic demand. Thus, complementation between creatine kinase and adenylate kinase systems, here predicted by modeling and further supported experimentally, provides a mechanistic basis for metabolic sensor function governed by alterations in intracellular phosphotransfer fluxes.     

Differential gene expression in the EphA4 knockout spinal cord and analysis of the inflammatory response following spinal cord injury

Mice lacking the axon guidance molecule EphA4 have been shown to exhibit extensive axonal regeneration and functional recovery following spinal cord injury. To assess mechanisms by which EphA4 may modify the response to neural injury a microarray was performed on spinal cord tissue from mice with spinal cord injury and sham injured controls. RNA was purified from spinal cords of adult EphA4 knockout and wild-type mice four days following lumbar spinal cord hemisection or laminectomy only and was hybridised to Affymetrix All-Exon Array 1.0 GeneChips?. While subsequent analyses indicated that several pathways were altered in EphA4 knockout mice, of particular interest was the attenuated expression of a number of inflammatory genes, including Arginase 1, expression of which was lower in injured EphA4 knockout compared to wild-type mice. Immunohistological analyses of different cellular components of the immune response were then performed in injured EphA4 knockout and wildtype spinal cords. While numbers of infiltrating CD3+ T cells were low in the hemisection model, a robust CD11b+ macrophage/microglial response was observed post-injury. There was no difference in the overall number or spread of macrophages/activated microglia in injured EphA4 knockout compared to wild-type spinal cords at 2, 4 or 14 days post-injury, however a lower proportion of Arginase-1 immunoreactive macrophages/activated microglia was observed in EphA4 knockout spinal cords at 4 days post-injury. Subtle alterations in the neuroinflammatory response in injured EphA4 knockout spinal cords may contribute to the regeneration and recovery observed in these mice following injury.     

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

Zona Occludens-2 Is Critical for Blood–Testis Barrier Integrity and Male Fertility

Tight junction integral membrane proteins such as claudins and occludin are tethered to the actin cytoskeleton by adaptor proteins, notably the closely related zonula occludens (ZO) proteins ZO-1, ZO-2, and ZO-3. All three ZO proteins have recently been inactivated in mice. Although ZO-3 knockout mice lack an obvious phenotype, animals deficient in ZO-1 or ZO-2 show early embryonic lethality. Here, we rescue the embryonic lethality of ZO-2 knockout mice by injecting ZO-2(−/−) embryonic stem (ES) cells into wild-type blastocysts to generate viable ZO-2 chimera. ZO-2(−/−) ES cells contribute extensively to different tissues of the chimera, consistent with an extraembryonic requirement for ZO-2 rather than a critical role in epiblast development. Adult chimera present a set of phenotypes in different organs. In particular, male ZO-2 chimera show reduced fertility and pathological changes in the testis. Lanthanum tracer experiments show a compromised blood–testis barrier. Expression levels of ZO-1, ZO-3, claudin-11, and occludin are not apparently affected. ZO-1 and occludin still localize to the blood–testis barrier region, but claudin-11 is less well restricted and the localization of connexin-43 is perturbed. The critical role of ZO-2 for male fertility and blood–testis barrier integrity thus provides a first example for a nonredundant role of an individual ZO protein in adult mice.     

Abnormal angiogenesis in Foxo1 (Fkhr)-deficient mice

Members of the Foxo family, Foxo1 (Fkhr), Foxo3 (Fkhrl1), and Foxo4 (Afx), are mammalian homologs of daf-16, which influences life span and energy metabolism in Caenorhabditis elegans. Mammalian FOXO proteins also play important roles in cell cycle arrest, apoptosis, stress resistance, and energy metabolism. In this study, we generated Foxo1-deficient mice to investigate the physiological role of FOXO1. The Foxo1-deficient mice died around embryonic day 11 because of defects in the branchial arches and remarkably impaired vascular development of embryos and yolk sacs. In vitro differentiation of embryonic stem cells demonstrated that endothelial cells derived from wild-type and Foxo1-deficient embryonic stem cells were able to produce comparable numbers of colonies supported by a layer of OP9 stromal cells. Although the morphology of the endothelial cell colonies was identical in both genotypes in the absence of exogenous vascular endothelial growth factor (VEGF), Foxo1-deficient endothelial cells showed a markedly different morphological response compared with wild-type endothelial cells in the presence of exogenous VEGF. These results suggest that Foxo1 is essential to the ability of endothelial cells to respond properly to a high dose of VEGF, thereby playing a critical role in normal vascular development.