Ablation of endothelial VEGFR1 improves metabolic dysfunction by inducing adipose tissue browning.

Angiogenesis plays an instrumental role in the modulation of adipose tissue mass and metabolism. Targeting adipose vasculature provides an outstanding opportunity for treatment of obesity and metabolic disorders. Here, we report the physiological functions of VEGFR1 in the modulation of adipose angiogenesis, obesity, and global metabolism. Pharmacological inhibition and genetic deletion of endothelial VEGFR1 augmented adipose angiogenesis and browning of subcutaneous white adipose tissue, leading to elevated thermogenesis. In a diet-induced obesity model, endothelial-VEGFR1 deficiency demonstrated a potent anti-obesity effect by improving global metabolism. Along with metabolic changes, fatty liver and insulin sensitivity were also markedly improved in VEGFR1-deficient high fat diet (HFD)-fed mice. Together, our data indicate that targeting of VEGFR1 provides an exciting new opportunity for treatment of obesity and metabolic diseases, such as liver steatosis and type 2 diabetes.

Intracrine VEGF signalling mediates colorectal cancer cell migration and invasion.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) are key regulators of angiogenesis, affecting endothelial cell survival and function. However, the effect of VEGF-VEGFR signalling on tumour cell function is not well understood. Our previous studies in colorectal cancer (CRC) cells have demonstrated an intracrine VEGF/VEGFR1 signalling mechanism that mediates CRC cell survival and chemo-sensitivity. Since extracellular VEGF signalling regulates migration of endothelial cells and various tumour cells, we attempted to determine whether intracrine VEGF signalling affects CRC cell motility. METHODS: Migration and invasion of CRC cells, with and without VEGF or VEGFR1 depletion, were assayed using transwell migration chambers. Changes in cell morphology, epithelial-mesenchymal transition (EMT) markers, and markers of cell motility were assessed by immunostaining and western blot. RESULTS: Depletion of intracellular VEGF and VEGFR1 in multiple CRC cell lines led to strong inhibition of migration and invasion of CRC cells. Except for Twist, there were no significant differences in markers of EMT between control and VEGF/VEGFR1-depleted CRC cells. However, VEGF/VEGFR1-depleted CRC cells demonstrated a significant reduction in levels of phosphorylated focal adhesion kinase and its upstream regulators pcMET and pEGFR. CONCLUSIONS: Inhibition of intracrine VEGF signalling strongly inhibits CRC cell migration and invasion by regulating proteins involved in cell motility.

The Role of Vascular Endothelial Growth Factor Receptor-1 Signaling in the Recovery from Ischemia.

Vascular endothelial growth factor (VEGF) is one of the most potent angiogenesis stimulators. VEGF binds to VEGF receptor 1 (VEGFR1), inducing angiogenesis through the receptor's tyrosine kinase domain (TK), but the mechanism is not well understood. We investigated the role of VEGFR1 tyrosine kinase signaling in angiogenesis using the ischemic hind limb model. Relative to control mice, blood flow recovery was significantly impaired in mice treated with VEGFA-neutralizing antibody. VEGFR1 tyrosine kinase knockout mice (TK-/-) had delayed blood flow recovery from ischemia and impaired angiogenesis, and this phenotype was unaffected by treatment with a VEGFR2 inhibitor. Compared to wild type mice (WT), TK-/- mice had no change in the plasma level of VEGF, but the plasma levels of stromal-derived cell factor 1 (SDF-1) and stem cell factor, as well as the bone marrow (BM) level of pro-matrix metalloproteinase-9 (pro-MMP-9), were significantly reduced. The recruitment of cells expressing VEGFR1 and C-X-C chemokine receptor type 4 (CXCR4) into peripheral blood and ischemic muscles was also suppressed. Furthermore, WT transplanted with TK-/- BM significantly impaired blood flow recovery more than WT transplanted with WT BM. These results suggest that VEGFR1-TK signaling facilitates angiogenesis by recruiting CXCR4+VEGFR1+ cells from BM.

Increased cardiac remodeling in cardiac-specific Flt-1 receptor knockout mice with pressure overload.

Vascular endothelial growth factor (VEGF) inhibition has previously been shown to have damaging effects on the heart. Because the role of Flt-1 (a phosphotyrosine kinase receptor for VEGF) in cardiac function and hypertrophy is unclear, we generated mice lacking Flt-1 only in their cardiomyocytes (Flt-1 KO). The hearts from 8- to 10-week-old mice were measured by using echocardiography and histology. No significant differences were seen in fraction shortening, cross-sectional area of cardiomyocytes, and interstitial collagen fraction between littermate controls and KO mice at baseline. To test the hypothesis that Flt-1 is involved in cardiac remodeling, we performed transverse aorta constriction (TAC) by ligating the transverse ascending aorta. Four weeks after TAC, echocardiography of the mice was performed, and the hearts were excised for pathological analysis and Western blotting. No difference in mortality was found between Flt-1 KO mice and controls; however, KO mice showed a greater cardiomyocyte cross-sectional area and interstitial collagen fraction than controls. Western blotting indicated that AKT was activated less in Flt-1 KO hearts after TAC compared with that in control hearts. Thus, Flt-1 deletion in cardiomyocytes increased hypertrophy, fibrosis, and regression of AKT phosphorylation. Our study suggests that Flt-1 plays a critical role in cardiac hypertrophy induced by pressure overload via the activation of AKT, which seems to be cardioprotective.

VEGFR1-positive macrophages facilitate liver repair and sinusoidal reconstruction after hepatic ischemia/reperfusion injury.

Liver repair after acute liver injury is characterized by hepatocyte proliferation, removal of necrotic tissue, and restoration of hepatocellular and hepatic microvascular architecture. Macrophage recruitment is essential for liver tissue repair and recovery from injury; however, the underlying mechanisms are unclear. Signaling through vascular endothelial growth factor receptor 1 (VEGFR1) is suggested to play a role in macrophage migration and angiogenesis. The aim of the present study was to examine the role of VEGFR1 in liver repair and sinusoidal reconstruction after hepatic ischemia/reperfusion (I/R). VEGFR1 tyrosine kinase knockout mice (VEGFR1 TK-/- mice) and wild-type (WT) mice were subjected to hepatic warm I/R, and the processes of liver repair and sinusoidal reconstruction were examined. Compared with WT mice, VEGFR1 TK-/- mice exhibited delayed liver repair after hepatic I/R. VEGFR1-expressing macrophages recruited to the injured liver showed reduced expression of epidermal growth factor (EGF). VEGFR1 TK-/- mice also showed evidence of sustained sinusoidal functional and structural damage, and reduced expression of pro-angiogenic factors. Treatment of VEGFR1 TK-/- mice with EGF attenuated hepatoceullar and sinusoidal injury during hepatic I/R. VEGFR1 TK-/- bone marrow (BM) chimeric mice showed impaired liver repair and sinusoidal reconstruction, and reduced recruitment of VEGFR1-expressing macrophages to the injured liver. VEGFR1-macrophages recruited to the liver during hepatic I/R contribute to liver repair and sinusoidal reconstruction. VEGFR1 activation is a potential therapeutic strategy for promoting liver repair and sinusoidal restoration after acute liver injury.

Suppressed soluble Fms-like tyrosine kinase-1 production aggravates atherosclerosis in chronic kidney disease.

Patients with chronic kidney disease (CKD) die of cardiovascular diseases for unknown reasons. Blood vessel formation in plaques and its relationship with plaque stability could be involved with signaling through the Flt-1 receptor and its ligands, vascular endothelial growth factor, and the closely related placental growth factor (PlGF). Flt-1 also exists as a circulating regulatory splice variant short-inhibitory form (sFlt-1) that serves as a decoy receptor, thereby inactivating PlGF. Heparin releases sFlt-1 by displacing the sFlt-1 heparin-binding site from heparin sulfate proteoglycans. Heparin could provide diagnostic inference or could also induce an antiangiogenic state. In the present study, postheparin sFlt-1 levels were lower in CKD patients than in control subjects. More importantly, sFlt-1 levels were inversely related to atherosclerosis in CKD patients, and this correlation was more robust after heparin injection, as verified by subsequent cardiovascular events. Knockout of apolipoprotein E (ApoE) and/or sFlt-1 showed that the absence of sFlt-1 worsened atherogenesis in ApoE-deficient mice. Thus, the relationship between atherosclerosis and PlGF signaling, as regulated by sFlt-1, underscores the underappreciated role of heparin in sFlt-1 release. These clinical and experimental data suggest that novel avenues into CKD-dependent atherosclerosis and its detection are warranted.

Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1.

Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life. DOI:http://dx.doi.org/10.7554/eLife.00324.001.

Elevated vascular endothelial growth factor receptor-2 abundance contributes to increased angiogenesis in vascular endothelial growth factor receptor-1-deficient mice.

BACKGROUND: Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is a potential therapeutic target for cardiovascular diseases, but its role in angiogenesis remains controversial. Whereas germline Vegfr-1(-/-) embryos die of abnormal vascular development in association with excessive endothelial differentiation, mice lacking only the kinase domain appear healthy. METHODS AND RESULTS: We performed Cre-loxP-mediated knockout to abrogate the expression of all known VEGFR-1 functional domains in neonatal and adult mice and analyzed developmental, pathophysiological, and molecular consequences. VEGFR-1 deficiency promoted tip cell formation and endothelial cell proliferation and facilitated angiogenesis of blood vessels that matured and perfused properly. Vascular permeability was normal at the basal level but elevated in response to high doses of exogenous VEGF-A. In the postinfarct ischemic cardiomyopathy model, VEGFR-1 deficiency supported robust angiogenesis and protected against myocardial infarction. VEGFR-1 knockout led to abundant accumulation of VEGFR-2 at the protein level, increased VEGFR-2 tyrosine phosphorylation transiently, and enhanced serine phosphorylation of Akt and ERK. Interestingly, increased angiogenesis, tip cell formation, vascular permeability, VEGFR-2 accumulation, and Akt phosphorylation could be partially rescued or suppressed by one or more of the following manipulations, including injection of the VEGFR-2 selective inhibitor SU1498, anti-VEGF-A, or introduction of Vegfr-2(+/-) heterozygosity into Vegfr-1 somatic knockout mice. CONCLUSIONS: Upregulation of VEGFR-2 abundance at the protein level contributes in part to increased angiogenesis in VEGFR-1-deficient mice.

Semaphorin-PlexinD1 signaling limits angiogenic potential via the VEGF decoy receptor sFlt1.

Sprouting angiogenesis expands the embryonic vasculature enabling survival and homeostasis. Yet how the angiogenic capacity to form sprouts is allocated among endothelial cells (ECs) to guarantee the reproducible anatomy of stereotypical vascular beds remains unclear. Here we show that Sema-PlxnD1 signaling, previously implicated in sprout guidance, represses angiogenic potential to ensure the proper abundance and stereotypical distribution of the trunk's segmental arteries (SeAs). We find that Sema-PlxnD1 signaling exerts this effect by antagonizing the proangiogenic activity of vascular endothelial growth factor (VEGF). Specifically, Sema-PlxnD1 signaling ensures the proper endothelial abundance of soluble flt1 (sflt1), an alternatively spliced form of the VEGF receptor Flt1 encoding a potent secreted decoy. Hence, Sema-PlxnD1 signaling regulates distinct but related aspects of angiogenesis: the spatial allocation of angiogenic capacity within a primary vessel and sprout guidance.

Regulation of angiogenesis by a non-canonical Wnt-Flt1 pathway in myeloid cells.

Myeloid cells are a feature of most tissues. Here we show that during development, retinal myeloid cells (RMCs) produce Wnt ligands to regulate blood vessel branching. In the mouse retina, where angiogenesis occurs postnatally, somatic deletion in RMCs of the Wnt ligand transporter Wntless results in increased angiogenesis in the deeper layers. We also show that mutation of Wnt5a and Wnt11 results in increased angiogenesis and that these ligands elicit RMC responses via a non-canonical Wnt pathway. Using cultured myeloid-like cells and RMC somatic deletion of Flt1, we show that an effector of Wnt-dependent suppression of angiogenesis by RMCs is Flt1, a naturally occurring inhibitor of vascular endothelial growth factor (VEGF). These findings indicate that resident myeloid cells can use a non-canonical, Wnt-Flt1 pathway to suppress angiogenic branching.

VEGFR-1 signaling regulates the homing of bone marrow-derived cells in a mouse stroke model.

Vascular endothelial growth factor receptor 1 (VEGFR-1) is highly expressed in endothelial cells and regulates developmental angiogenesis by acting as a decoy receptor and trapping VEGF-A. Vascular endothelial growth factor receptor 1 is also expressed in monocytes and macrophages; mice lacking the VEGFR-1 tyrosine kinase (TK) domain (VEGFR-1 TK mice) display impaired macrophage function. Because macrophages are recruited to sites of cerebral ischemic infarcts, we hypothesized that lack of VEGFR-1 TK in bone marrow(BM) cells would affect the outcome in an experimental stroke model. We performed BM transplantation experiments in C57BL/6J mice using VEGFR-1 TK and VEGFR-1 TK mice as BM donors and analyzed cell infiltration after cerebral ischemia. There was reduced initial recruitment of VEGFR-1 TK myeloid cells into the infarcted tissue and reduced postischemic angiogenesis at 3days postischemia. By 10 days, the numbers of infiltrating cells and the densities of vessels in the infarct peri-infarct zone were similar for both groups. Neither infarct size at 3 and 10 days postischemia nor neurological performance at 24 hours was different between the experimental groups. These results support a role of VEGFR-1 signaling in the early regulation of BM infiltration and angiogenesis after brain ischemia.

VEGFR1-activity-independent metastasis formation.

Molecules such as vascular endothelial growth factor (VEGF) or placental growth factor-critical regulators of tumour angiogenesis-are also thought to mobilize into blood circulation bone marrow-derived cells (BMDCs), which may subsequently be recruited to tumours and facilitate tumour growth and metastasis. A study has suggested that BMDCs form 'metastatic niches' in lungs before arrival of cancer cells, and showed that pharmacological inhibition of VEGF receptor 1 (VEGFR1, also known as Flt1)-cognate receptor for VEGF and placental growth factor-prevented BMDC infiltration in lungs and 'metastatic niche' formation. Here we report that blockade of VEGFR1 activity does not affect the rate of spontaneous metastasis formation in a clinically relevant and widely used preclinical model. Therefore, alternative pathways probably mediate the priming of tissues for metastasis.

VEGFR-1 regulates adult olfactory bulb neurogenesis and migration of neural progenitors in the rostral migratory stream in vivo.

The generation of new neurons in the olfactory bulb (OB) persists into adulthood and is a multistep process that includes proliferation, fate choice, migration, survival, and differentiation. Neural precursor cells destined to form olfactory interneurons arise in the subventricular zone (SVZ) and migrate along the rostral migratory stream (RMS) to the OB. Recently, some factors classically known from their effects on the vascular system have been found to influence different steps of adult neurogenesis. In the present study, we report a modulatory function for the vascular endothelial growth factor receptor-1 (VEGFR-1) in adult olfactory neurogenesis. We identified expression of VEGFR-1 in GFAP-positive cells within regions involved in neurogenesis of the adult mouse brain. To determine functions for VEGFR-1 in adult neurogenesis, we compared neural progenitor cell proliferation, migration, and differentiation from wild-type and VEGFR-1 signaling-deficient mice (Flt-1TK(-/-) mice). Our data show that VEGFR-1 signaling is involved in the regulation of proliferation of neuronal progenitor cells within the SVZ, migration along the RMS, and in neuronal differentiation and anatomical composition of interneuron subtypes within the OB. RMS migration in Flt-1TK(-/-) mice was altered mainly as a result of increased levels of its ligand VEGF-A, which results in an increased phosphorylation of VEGFR-2 in neuronal progenitor cells within the SVZ and the RMS. This study reveals that proper RMS migration is dependent on endogenous VEGF-A protein.

Soluble Flt-1 gene transfer ameliorates neointima formation after wire injury in flt-1 tyrosine kinase-deficient mice.

OBJECTIVE: We have demonstrated that vascular endothelial growth factor (VEGF) expression is upregulated in injured vascular wall, and blockade of VEGF inhibited monocyte infiltration and neointima formation in several animal models. In the present study, we aimed to clarify relative role of two VEGF receptors, flt-1 versus flk-1/KDR, in neointima formation after injury using flt-1 tyrosine kinase-deficient (Flt-1 TK(-/-)) mice and soluble Flt-1(sFlt-1) gene transfer. METHODS AND RESULTS: Neointima formation was comparable between wild-type and Flt-1 TK(-/-) mice 28 days after intraluminal wire injury in femoral arteries. By contrast, neointima formation was significantly suppressed by sFlt-1 gene transfer into Flt-1 TK(-/-) mice that blocks VEGF action on flk-1 (intima/media ratio: 2.8+/-0.4 versus 1.4+/-0.4, P<0.05). The inhibition of neointima formation was preceded by significant reduction of monocyte chemoattractant protein (MCP-1) expression in vascular smooth muscle cells (VSMCs) and monocyte infiltration 7 days after injury. Gene transfer of sFlt-1 or treatment of flk-1-specific antibody significantly inhibited VEGF-induced MCP-1 expression determined by RT-PCR in cultured aortic tissue and VSMCs. MCP-1-induced chemotaxis was equivalent between wild-type and Flt-1 TK(-/-) mice. CONCLUSIONS: These results suggest that endogenous VEGF accelerates neointima formation through flk-1 by regulating MCP-1 expression in VSMCs and macrophage-mediated inflammation in injured vascular wall in murine model of wire injury.

Vascular endothelial growth factor-A specifies formation of native collaterals and regulates collateral growth in ischemia.

The density of native (preexisting) collaterals and their capacity to enlarge into large conduit arteries in ischemia (arteriogenesis) are major determinants of the severity of tissue injury in occlusive disease. Mechanisms directing arteriogenesis remain unclear. Moreover, nothing is known about how native collaterals form in healthy tissue. Evidence suggests vascular endothelial growth factor (VEGF), which is important in embryonic vascular patterning and ischemic angiogenesis, may contribute to native collateral formation and arteriogenesis. Therefore, we examined mice heterozygous for VEGF receptor-1 (VEGFR-1(+/-)), VEGF receptor-2 (VEGFR-2(+/-)), and overexpressing (VEGF(hi/+)) and underexpressing VEGF-A (VEGF(lo/+)). Recovery from hindlimb ischemia was followed for 21 days after femoral artery ligation. All statements below are P<0.05. Compared to wild-type mice, VEGFR-2(+/-) showed similar: ischemic scores, recovery of hindlimb perfusion, pericollateral leukocytes, collateral enlargement, and angiogenesis. In contrast, VEGFR-1(+/-) showed impaired: perfusion recovery, pericollateral leukocytes, collateral enlargement, worse ischemic scores, and comparable angiogenesis. Compared to wild-type mice, VEGF(lo/+) had 2-fold lower perfusion immediately after ligation (suggesting fewer native collaterals which was confirmed by angiography) and blunted recovery of perfusion. VEGF(hi/+) mice had 3-fold greater perfusion immediately after ligation, more native collaterals, and improved recovery of perfusion. These differences were confirmed in the cerebral pial cortical circulation where, compared to VEGF(hi/+) mice, VEGF(lo/+) formed fewer collaterals during the perinatal period when adult density was established, and had 2-fold larger infarctions after middle cerebral artery ligation. Our findings indicate VEGF and VEGFR-1 are determinants of arteriogenesis. Moreover, we describe the first signaling molecule, VEGF-A, that specifies formation of native collaterals in healthy tissues.

Vascular endothelial growth factor receptor-1 regulates postnatal angiogenesis through inhibition of the excessive activation of Akt.

Vascular endothelial growth factor (VEGF) binds both VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2). Activation of VEGFR-2 is thought to play a major role in the regulation of endothelial function by VEGF. Recently, specific ligands for VEGFR-1 have been reported to have beneficial effects when used to treat ischemic diseases. However, the role of VEGFR-1 in angiogenesis is not fully understood. In this study, we showed that VEGFR-1 performs "fine tuning" of VEGF signaling to induce neovascularization. We examined the effects of retroviral vectors expressing a small interference RNA that targeted either the VEGFR-1 gene or the VEGFR-2 gene. Deletion of either VEGFR-1 or VEGFR-2 reduced the ability of endothelial cells to form capillaries. Deletion of VEGFR-1 markedly reduced endothelial cell proliferation and induced premature senescence of endothelial cells. In contrast, deletion of VEGFR-2 significantly impaired endothelial cell survival. When VEGFR-1 expression was blocked, VEGF constitutively activated Akt signals and thus induced endothelial cell senescence via a p53-dependent pathway. VEGFR-1(+/-) mice exhibited an increase of endothelial Akt activity and showed an impaired neovascularization in response to ischemia, and this impairment was ameliorated in VEGFR-1(+/-) Akt1(+/-) mice. These results suggest that VEGFR-1 plays a critical role in the maintenance of endothelial integrity by modulating the VEGF/Akt signaling pathway.

VEGFR1 tyrosine kinase signaling promotes lymphangiogenesis as well as angiogenesis indirectly via macrophage recruitment.

OBJECTIVE: Angiogenesis and lymphangiogenesis are complex phenomena that involve the interplay of several growth factors and receptors. Recently, we have demonstrated that in Keratin-14 (K14) promoter-driven Vegf-A transgenic (Tg) mice, not only angiogenesis but also lymphangiogenesis is stimulated. However, the mechanism by which VEGFR1 is involved in lymphangiogenesis remains unclear. METHODS AND RESULTS: To examine how important the tyrosine kinase (TK) of VEGFR1 is in lymphangiogenesis in K14 Vegf-A Tg mice, we crossed the K14 Vegf-A Tg mice with VEGFR1-TK-deficient mice to generate double mutant K14 Vegf-A Tg Vegfr1 tk(-/-) mice. K14 Vegf-A Tg Vegfr1 tk(-/-) mice exhibit a remarkable decrease in lymphangiogensis as well as angiogenesis in subcutaneous tissues. To address the mechanism underlying the decrease in lymphangiogensis, we investigated the recruitment of monocyte-macrophage-lineage cells into the skin. The recruitment of VEGFR1-expressing macrophages driven by VEGF-A was reduced in K14 Vegf-A Tg Vegfr1 tk(-/-) mice. Vegf-A Tg mice that received VEGFR1-TK-deficient bone marrow showed a reduction of macrophage recruitment, lymphangiogenesis and angiogenesis compared with those in K14 Vegf-A Tg mice. CONCLUSIONS: VEGFR1 signaling promotes lymphangiogenesis as well as angiogenesis mainly by increasing bone marrow-derived macrophage recruitment.

Flt-1 tyrosine kinase-deficient homozygous mice result in decreased trabecular bone volume with reduced osteogenic potential.

To clarify the role of Fms-like tyrosine kinase-1 (Flt-1) signaling in bone dynamics, we examined C57BL/6J mice, aged 6, 9 and 16 weeks, with disruption of the flt1 tyrosine kinase domain gene (flt1(TK-/-)) and compared with age-matched wild-type (flt1(TK+/+)) mice. Dynamic histomorphometric analysis confirmed a significant decrease in the values of mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS) in the trabecular bone of the proximal tibiae of flt1(TK-/-) mice compared with those in flt1(TK+/+) mice. The value of trabecular bone volume (BV/TV) was also significantly reduced in flt1(TK-/-) mice compared with that in flt1(TK+/+) mice. The values of osteoclast surface (Oc.S/BS) and osteoclast number (Oc.N/BS) in flt1(TK-/-) mice were somewhat lower than those in flt1(TK+/+) mice. The values of bending load of the femur significantly decreased in flt1(TK-/-) mice. In addition, serum osteocalcin significantly decreased in flt1(TK-/-) mice compared with those in flt1(TK+/+) mice. Furthermore, there was a significant decreased mineralization of bone marrow stromal cultures from flt1(TK-/-) mice. These findings demonstrate that flt1(TK-/-) mice show lower trabecular bone volume than flt1(TK+/+) mice, providing powerful evidence that vascular endothelial growth factor signal pathway through the Flt-1 tyrosine kinase domain could be implicated in osteoblast development.