Rescue of Holoprosencephaly in Fetal Alcohol-Exposed Cdon Mutant Mice by Reduced Gene Dosage of Ptch1

Holoprosencephaly (HPE) is a commonly occurring developmental defect in which midline patterning of the forebrain and midface is disrupted. Sonic hedgehog (SHH) signaling is required during multiple stages of rostroventral midline development, and heterozygous mutations in SHH pathway components are associated with HPE. However, clinical presentation of HPE is highly variable, and carriers of heterozygous mutations often lack apparent defects. It is therefore thought that such mutations must interact with more common modifiers, genetic and/or environmental. We have modeled this scenario in mice. Cdon mutant mice have a largely subthreshold defect in SHH signaling, rendering them sensitive to a wide spectrum of HPE phenotypes by additional hits that are themselves insufficient to produce HPE, including transient in utero exposure to ethanol. These variable HPE phenotypes may arise in embryos that fail to reach a threshold level of SHH signaling at a specific developmental stage. To provide evidence for this possibility, here we tested the effect of removing one copy of the negative regulator Ptch1 from Cdon^−/− embryos and compared their response to ethanol with that of Cdon^−/−;Ptch1^+/+ embryos. Ptch1 heterozygosity decreased the penetrance of HPE in this system by >75%. The major effect of reduced Ptch1 gene dosage was on penetrance, as those Cdon^−/−;Ptch1^+/− embryos that displayed HPE did not show major differences in phenotype from Cdon^−/−;Ptch1^+/+ embryos with ethanol-induced HPE. Our findings are consistent with the notion that even in an etiologically complex model of HPE, the level of SHH pathway activity is rate-limiting. Furthermore, the clinical outcome of an individual carrying a SHH pathway mutation will likely reflect the sum effect of both deleterious and protective modifier alleles and their interaction with non-genetic risk factors like fetal alcohol exposure.     

An Enhanced Immune Response of Mclk1 +/? Mutant Mice Is Associated with Partial Protection from Fibrosis,Cancer and the Development of Biomarkers of Aging

The immune response is essential for survival by destroying microorganisms and pre-cancerous cells. However, inflammation, one aspect of this response, can result in short- and long-term deleterious side-effects. Mclk1 ^+/− mutant mice can be long-lived despite displaying a hair-trigger inflammatory response and chronically activated macrophages as a result of high mitochondrial ROS generation. Here we ask whether this phenotype is beneficial or simply tolerated. We used models of infection by Salmonella serovars and found that Mclk1 ^+/− mutants mount a stronger immune response, control bacterial proliferation better, and are resistant to cell and tissue damage resulting from the response, including fibrosis and types of oxidative damage that are considered to be biomarkers of aging. Moreover, these same types of tissue damage were found to be low in untreated 23 months-old mutants. We also examined the initiation of tumour growth after transplantation of mouse LLC1 carcinoma cells into Mclk1 ^+/− mutants, as well as during spontaneous tumorigenesis in Mclk1 ^+/− Trp53 ^+/− double mutants. Tumour latency was increased by the Mclk1 ^+/− genotype in both models. Furthermore, we used the transplantation model to show that splenic CD8+ T lymphocytes from Mclk1 ^+/− graft recipients show enhanced cytotoxicity against LLC1 cells in vitro. Mclk1 ^+/− mutants thus display an association of an enhanced immune response with partial protection from age-dependent processes and from pathologies similar to those that are found with increased frequency during the aging process. This suggests that the immune phenotype of these mutants might contribute to their longevity. We discuss how these findings suggest a broader view of how the immune response might impact the aging process.     

Twisted gastrulation limits apoptosis in the distal region of the mandibular arch in mice

The mandibular arch (BA1) is critical for craniofacial development. The distal region of BA1, which gives rise to most of the mandible, is dependent upon an optimal level of bone morphogenetic protein (BMP) signaling. BMP activity is modulated in the extracellular space by BMP-binding proteins such as Twisted gastrulation (TWSG1). Twsg1^−/− mice have a spectrum of craniofacial phenotypes, including mandibular defects that range from micrognathia to agnathia. At E9.5, the distal region of the mutant BA1 was prematurely and variably fused with loss of distal markers eHand and Msx1. Expression of proximal markers Fgf8 and Barx1 was expanded across the fused BA1. The expression of Bmp4 and Msx2 was preserved in the distal region, but shifted ventrally. While wild type embryos showed a gradient of BMP signaling with higher activity in the distal region of BA1, this gradient was disrupted and shifted ventrally in the mutants. Thus, loss of TWSG1 results in disruption of the BMP4 gradient at the level of signaling activity as well as mRNA expression. Altered distribution of BMP signaling leads to a shift in gene expression and increase in apoptosis. The extent of apoptosis may account for the variable degree of mandibular defects in Twsg1 mutants.     

Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage,despite rapid and sustained mitochondrial DNA depletion,altered energetics and growth

DNA polymerase gamma (POLG) is essential for replication and repair of mitochondrial DNA (mtDNA). Mutations in POLG cause mtDNA instability and a diverse range of poorly understood human diseases. Here, we created a unique Polg animal model, by modifying polg within the critical and highly conserved polymerase domain in zebrafish. polg^+/− offspring were indistinguishable from WT siblings in multiple phenotypic and biochemical measures. However, polg^−/− mutants developed severe mtDNA depletion by one week post-fertilization (wpf), developed slowly and had regenerative defects, yet surprisingly survived up to 4 wpf. An in vivo mtDNA polymerase activity assay utilizing ethidium bromide (EtBr) to deplete mtDNA, showed that polg^+/− and WT zebrafish fully recover mtDNA content two weeks post-EtBr removal. EtBr further reduced already low levels of mtDNA in polg^−/− animals, but mtDNA content did not recover following release from EtBr. Despite significantly decreased respiration that corresponded with tissue-specific levels of mtDNA, polg^−/− animals had WT levels of ATP and no increase in lactate. This zebrafish model of mitochondrial disease now provides unique opportunities for studying mtDNA instability from multiple angles, as polg^−/− mutants can survive to juvenile stage, rather than lose viability in embryogenesis as seen in Polg mutant mice.     

Retinoic acid exerts sexually dimorphic effects on muscle energy metabolism and function

The retinol dehydrogenase Rdh10 catalyzes the rate-limiting reaction that converts retinol into retinoic acid (RA), an autacoid that regulates energy balance and reduces adiposity. Skeletal muscle contributes to preventing adiposity, by consuming nearly half the energy of a typical human. We report sexually dimorphic differences in energy metabolism and muscle function in Rdh10+/− mice. Relative to wild-type (WT) controls, Rdh10+/− males fed a high-fat diet decrease reliance on fatty-acid oxidation and experience glucose intolerance and insulin resistance. Running endurance decreases 40%. Rdh10+/− females fed this diet increase fatty acid oxidation and experience neither glucose intolerance nor insulin resistance. Running endurance increases 220%. We therefore assessed RA function in the mixed-fiber type gastrocnemius muscles (GM), which contribute to running, rather than standing, and are similar to human GM. RA levels in Rdh10+/− male GM decrease 38% relative to WT. Rdh10+/− male GM increase expression of Myog and reduce Eif6 mRNAs, which reduce and enhance running endurance, respectively. Cox5A, complex IV activity, and ATP decrease. Increased centralized nuclei reveal existence of muscle malady and/or repair in GM fibers. Comparatively, RA in Rdh10+/− female GM decreases by less than half the male decrease, from a more modest decrease in Rdh10 and an increase in the estrogen-induced retinol dehydrogenase Dhrs9. Myog mRNA decreases. Cox5A, complex IV activity, and ATP increase. Centralized GM nuclei do not increase. We conclude that Rdh10/RA affects whole body energy use and insulin resistance partially through sexual dimorphic effects on skeletal muscle gene expression, structure, and mitochondria activity.     

The Canonical Wnt Signal Restricts the Glycogen Synthase Kinase 3/Fbw7-Dependent Ubiquitination and Degradation of Eya1 Phosphatase

Haploinsufficiency of Eya1 causes the branchio-oto-renal (BOR) syndrome, and abnormally high levels of Eya1 are linked to breast cancer progression and poor prognosis. Therefore, regulation of Eya1 activity is key to its tissue-specific functions and oncogenic activities. Here, we show that Eya1 is posttranslationally modified by ubiquitin and that its ubiquitination level is self-limited to prevent premature degradation. Eya1 has an evolutionarily conserved CDC4 phosphodegron (CPD) signal, a target site of glycogen synthase kinase 3 (GSK3) kinase and Fbw7 ubiquitin ligase, which is required for Eya1 ubiquitination. Genetic deletion of Fbw7 and pharmacological inhibition of GSK3 significantly decrease Eya1 ubiquitination. Conversely, activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the canonical Wnt signal suppresses Eya1 ubiquitination. Compound Eya1^+/−; Wnt9b^+/− mutants exhibit an increased penetrance of renal defect, indicating that they function in the same genetic pathway in vivo. Together, these findings reveal that the canonical Wnt and PI3K/Akt signal pathways restrain the GSK3/Fbw7-dependent Eya1 ubiquitination, and they further suggest that dysregulation of this novel axis contributes to tumorigenesis.     

Heterozygosity for Fibrinogen Results in Efficient Resolution of Kidney Ischemia Reperfusion Injury

Fibrinogen (Fg) has been recognized to play a central role in coagulation, inflammation and tissue regeneration. Several studies have used Fg deficient mice (Fg^−/−) in comparison with heterozygous mice (Fg^+/−) to point the proinflammatory role of Fg in diverse pathological conditions and disease states. Although Fg^+/− mice are considered ‘normal’, plasma Fg is reduced to ∼75% of the normal circulating levels present in wild type mice (Fg^+/+). We report that this reduction in Fg protein production in the Fg^+/− mice is enough to protect them from kidney ischemia reperfusion injury (IRI) as assessed by tubular injury, kidney dysfunction, necrosis, apoptosis and inflammatory immune cell infiltration. Mechanistically, we observed binding of Fg to ICAM-1 in kidney tissues of Fg^+/+ mice at 24 h following IRI as compared to a complete absence of binding observed in the Fg^+/− and Fg^−/− mice. Raf-1 and ERK were highly activated as evident by significantly higher phosphorylation in the Fg^+/+ kidneys at 24 h following IRI as compared to Fg^+/− and Fg^−/− mice kidneys. On the other hand Cyclin D1 and pRb, indicating higher cell proliferation, were significantly increased in the Fg^+/− and Fg^−/− as compared to Fg^+/+ kidneys. These data suggest that Fg heterozygosity allows maintenance of a critical balance of Fg that enables regression of initial injury and promotes faster resolution of kidney damage.     

Quantification of seminolipid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt mice

Seminolipid, also known as sulfogalactosylglycerolipid (SGG), plays important roles in male reproduction. Therefore, an accurate and sensitive method for SGG quantification in testes and sperm is needed. Here we compare SGG quantitation by the traditional colorimetric Azure A assay with LC-ESI-MS/MS using multiple reaction monitoring (MRM). Inclusion of deuterated SGG as the internal standard endowed accuracy to the MRM method. The results showed reasonable agreement between the two procedures for purified samples, but for crude lipid extracts, the colorimetric assay significantly overestimated the SGG content. Using ESI-MS/MS MRM, C16:0-alkyl/C16:0-acyl SGG of Cgt^+/− mice was quantified to be 406.06 ± 23.63 μg/g testis and 0.13 ± 0.02 μg/million sperm, corresponding to 78% and 87% of the wild-type values, respectively. CGT (ceramide galactosyltransferase) is a critical enzyme in the SGG biosynthesis pathway. Cgt^−/− males depleted of SGG are infertile due to spermatogenesis arrest. However, Cgt^+/− males sire offspring. The higher than 50% expression level of SGG in Cgt^+/− animals, compared with the wild-type expression, might be partly due to compensatory translation of the active CGT enzyme. The results also indicated that 78% of SGG levels in Cgt^+/− mice were sufficient for normal spermatogenesis.     

The Amount of Keratins Matters for Stress Protection of the Colonic Epithelium

Keratins (K) are important for epithelial stress protection as evidenced by keratin mutations predisposing to human liver diseases and possibly inflammatory bowel diseases. A role for K8 in the colon is supported by the ulcerative colitis-phenotype with epithelial hyperproliferation and abnormal ion transport in K8-knockout (K8^−/−) mice. The heterozygote knockout (K8^+/−) colon appears normal but displays a partial ion transport-defect. Characterizing the colonic phenotype we show that K8^+/− colon expresses ~50% less keratins compared to K8 wild type (K8^+/+) but de novo K7 expression is observed in the top-most cells of the K8^+/− and K8^−/− crypts. The K8^+/− colonic crypts are significantly longer due to increased epithelial hyperproliferation, but display no defects in apoptosis or inflammation in contrast to K8^−/−. When exposed to colitis using the dextran sulphate sodium-model, K8^+/− mice showed higher disease sensitivity and delayed recovery compared to K8^+/+ littermates. Therefore, the K8^+/− mild colonic phenotype correlates with decreased keratin levels and increased sensitivity to experimental colitis, suggesting that a sufficient amount of keratin is needed for efficient stress protection in the colonic epithelia.     

Acid Sphingomyelinase Gene Knockout Ameliorates Hyperhomocysteinemic Glomerular Injury in Mice Lacking Cystathionine-β-Synthase

Acid sphingomyelinase (ASM) has been implicated in the development of hyperhomocysteinemia (hHcys)-induced glomerular oxidative stress and injury. However, it remains unknown whether genetically engineering of ASM gene produces beneficial or detrimental action on hHcys-induced glomerular injury. The present study generated and characterized the mice lacking cystathionine β-synthase (Cbs) and Asm mouse gene by cross breeding Cbs^+/− and Asm^+/− mice. Given that the homozygotes of Cbs^−/−/Asm^−/− mice could not survive for 3 weeks. Cbs^+/−/Asm^+/+, Cbs^+/−/Asm^+/− and Cbs^+/−/Asm^−/− as well as their Cbs wild type littermates were used to study the role of Asm^−/− under a background of Cbs^+/− with hHcys. HPLC analysis revealed that plasma Hcys level was significantly elevated in Cbs heterozygous (Cbs^+/−) mice with different copies of Asm gene compared to Cbs^+/+ mice with different Asm gene copies. Cbs^+/−/Asm^+/+ mice had significantly increased renal Asm activity, ceramide production and O₂.⁻ level compared to Cbs^+/+/Asm^+/+, while Cbs^+/−/Asm^−/− mice showed significantly reduced renal Asm activity, ceramide production and O₂.⁻ level due to increased plasma Hcys levels. Confocal microscopy demonstrated that colocalization of podocin with ceramide was much lower in Cbs^+/−/Asm^−/− mice compared to Cbs^+/−/Asm^+/+ mice, which was accompanied by a reduced glomerular damage index, albuminuria and proteinuria in Cbs^+/−/Asm^−/− mice. Immunofluorescent analyses of the podocin, nephrin and desmin expression also illustrated less podocyte damages in the glomeruli from Cbs^+/−/Asm^−/− mice compared to Cbs^+/−/Asm^+/+ mice. In in vitro studies of podocytes, hHcys-enhanced O₂.⁻ production, desmin expression, and ceramide production as well as decreases in VEGF level and podocin expression in podocytes were substantially attenuated by prior treatment with amitriptyline, an Asm inhibitor. In conclusion, Asm gene knockout or corresponding enzyme inhibition protects the podocytes and glomeruli from hHcys-induced oxidative stress and injury.     

c-Fms Signaling Mediates Neurofibromatosis Type-1 Osteoclast Gain-In-Functions

Skeletal abnormalities including osteoporosis and osteopenia occur frequently in both pediatric and adult neurofibromatosis type 1 (NF1) patients. NF1 (Nf1) haploinsufficient osteoclasts and osteoclast progenitors derived from both NF1 patients and Nf1^+/− mice exhibit increased differentiation, migration, and bone resorptive capacity in vitro, mediated by hyperactivation of p21^Ras in response to limiting concentrations of macrophage-colony stimulating factor (M-CSF). Here, we show that M-CSF binding to its receptor, c-Fms, results in increased c-Fms activation in Nf1^{+/ −} osteoclast progenitors, mediating multiple gain-in-functions through the downstream effectors Erk1/2 and p90RSK. PLX3397, a potent and selective c-Fms inhibitor, attenuated M-CSF mediated Nf1^+/− osteoclast migration by 50%, adhesion by 70%, and pit formation by 60%. In vivo, we administered PLX3397 to Nf1 ^+/− osteoporotic mice induced by ovariectomy (OVX) and evaluated changes in bone mass and skeletal architecture. We found that PLX3397 prevented bone loss in Nf1^+/−-OVX mice by reducing osteoclast differentiation and bone resorptive activity in vivo. Collectively, these results implicate the M-CSF/c-Fms signaling axis as a critical pathway underlying the aberrant functioning of Nf1 haploinsufficient osteoclasts and may provide a potential therapeutic target for treating NF1 associated osteoporosis and osteopenia.     

Hidden Disease Susceptibility and Sexual Dimorphism in the Heterozygous Knockout of Cyp51 from Cholesterol Synthesis

We examined the genotype-phenotype interactions of Cyp51^+/− mice carrying one functional allele of lanosterol 14α-demethylase from cholesterol biosynthesis. No distinct developmental or morphological abnormalities were observed by routine visual inspection of Cyp51^+/− and Cyp51^+/+ mice and fertility was similar. We further collected a large data-set from female and male Cyp51^+/− mice and controls fed for 16 weeks with three diets and applied linear regression modeling. We used 3 predictor variables (genotype, sex, diet), and 39 response variables corresponding to the organ characteristics (7), plasma parameters (7), and hepatic gene expression (25). We observed significant differences between Cyp51^+/− and wild-type mice in organ characteristics and blood lipid profile. Hepatomegaly was observed in Cyp51^+/− males, together with elevated total and low-density lipoprotein cholesterol. Cyp51^+/− females fed high-fat, high-cholesterol diet were leaner and had elevated plasma corticosterone compared to controls. We observed elevated hepatocyte apoptosis, mitosis and lipid infiltration in heterozygous knockouts of both sexes. The Cyp51^+/− females had a modified lipid storage homeostasis protecting them from weight-gain when fed high-fat high-cholesterol diet. Malfunction of one Cyp51 allele therefore initiates disease pathways towards cholesterol-linked liver pathologies and sex-dependent response to dietary challenge.     

Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I

Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α₂β₂ tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1^−/−) leads to embryonic lethality in mouse, whereas P4ha1^+/− mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2^−/− mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1^+/−;P4ha2^−/− mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the T_m of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1^+/−;P4ha2^−/− mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2^−/− mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype.     

Increased Corneal Epithelial Turnover Contributes to Abnormal Homeostasis in the Pax6+/? Mouse Model of Aniridia

We aimed to test previous predictions that limbal epithelial stem cells (LESCs) are quantitatively deficient or qualitatively defective in Pax6^+/− mice and decline with age in wild-type (WT) mice. Consistent with previous studies, corneal epithelial stripe patterns coarsened with age in WT mosaics. Mosaic patterns were also coarser in Pax6^+/− mosaics than WT at 15 weeks but not at 3 weeks, which excludes a developmental explanation and strengthens the prediction that Pax6^+/− mice have a LESC-deficiency. To investigate how Pax6 genotype and age affected corneal homeostasis, we compared corneal epithelial cell turnover and label-retaining cells (LRCs; putative LESCs) in Pax6^+/− and WT mice at 15 and 30 weeks. Limbal BrdU-LRC numbers were not reduced in the older WT mice, so this analysis failed to support the predicted age-related decline in slow-cycling LESC numbers in WT corneas. Similarly, limbal BrdU-LRC numbers were not reduced in Pax6^+/− heterozygotes but BrdU-LRCs were also present in Pax6^+/− corneas. It seems likely that Pax6^+/− LRCs are not exclusively stem cells and some may be terminally differentiated CD31-positive blood vessel cells, which invade the Pax6^+/− cornea. It was not, therefore, possible to use this approach to test the prediction that Pax6^+/− corneas had fewer LESCs than WT. However, short-term BrdU labelling showed that basal to suprabasal movement (leading to cell loss) occurred more rapidly in Pax6^+/− than WT mice. This implies that epithelial cell loss is higher in Pax6^+/− mice. If increased corneal epithelial cell loss exceeds the cell production capacity it could cause corneal homeostasis to become unstable, resulting in progressive corneal deterioration. Although it remains unclear whether Pax6^+/− mice have LESC-deficiency, we suggest that features of corneal deterioration, that are often taken as evidence of LESC-deficiency, might occur in the absence of stem cell deficiency if corneal homeostasis is destabilised by excessive cell loss.     

A mouse model of inherited choline kinase β-deficiency presents with specific cardiac abnormalities and a predisposition to arrhythmia

The CHKB gene encodes choline kinase β, which catalyzes the first step in the biosynthetic pathway for the major phospholipid phosphatidylcholine. Homozygous loss-of-function variants in human CHKB are associated with a congenital muscular dystrophy. Dilated cardiomyopathy is present in some CHKB patients and can cause heart failure and death. Mechanisms underlying a cardiac phenotype due to decreased CHKB levels are not well characterized. We determined that there is cardiac hypertrophy in Chkb^−/− mice along with a decrease in left ventricle size, internal diameter, and stroke volume compared with wildtype and Chkb^+/− mice. Unlike wildtype mice, 60% of the Chkb^+/− and all Chkb^−/− mice tested displayed arrhythmic events when challenged with isoproterenol. Lipidomic analysis revealed that the major change in lipid level in Chkb^+/− and Chkb^−/− hearts was an increase in the arrhythmogenic lipid acylcarnitine. An increase in acylcarnitine level is also associated with a defect in the ability of mitochondria to use fatty acids for energy and we observed that mitochondria from Chkb^−/− hearts had abnormal cristae and inefficient electron transport chain activity. Atrial natriuretic peptide (ANP) is a hormone produced by the heart that protects against the development of heart failure including ventricular conduction defects. We determined that there was a decrease in expression of ANP, its receptor NPRA, as well as ventricular conduction system markers in Chkb^+/− and Chkb^−/− mice.     

Hypoxia-Induced Endothelial Progenitor Cell Function Is Blunted in Angiotensinogen Knockout Mice

Angiotensinogen (AGT), the precursor of angiotensin I, is known to be involved in tumor angiogenesis and associated with the pathogenesis of coronary atherosclerosis. This study was undertaken to determine the role played by AGT in endothelial progenitor cells (EPCs) in tumor progression and metastasis. It was found that the number of EPC colonies formed by AGT heterozygous knockout (AGT^+/−) cells was less than that formed by wild-type (WT) cells, and that the migration and tube formation abilities of AGT^+/− EPCs were significantly lower than those of WT EPCs. In addition, the gene expressions of vascular endothelial growth factor (VEGF), Flk1, angiopoietin (Ang)-1, Ang-2, Tie-2, stromal derived factor (SDF)-1, C-X-C chemokine receptor type 4 (CXCR4), and of endothelial nitric oxide synthase (eNOS) were suppressed in AGT^+/− EPCs. Furthermore, the expressions of hypoxia-inducible factor (HIF)-1α and -2α were downregulated in AGT^+/− early EPCs under hypoxic conditions, suggesting a blunting of response to hypoxia. Moreover, the activation of Akt/eNOS signaling pathways induced by VEGF, epithelial growth factor (EGF), or SDF-1α were suppressed in AGT^+/− EPCs. In AGT^+/− mice, the incorporation of EPCs into the tumor vasculature was significantly reduced, and lung tumor growth and melanoma metastasis were attenuated. In conclusion, AGT is required for hypoxia-induced vasculogenesis.