BDNF mediates the effects of testosterone on the survival of new neurons in an adult brain

New neurons are incorporated into the high vocal center (HVC), a nucleus of the adult canary (Serinus canaria) brain that plays a critical role in the acquisition and production of learned song. Recruitment of new neurons in the HVC is seasonally regulated and depends upon testosterone levels. We show here that brain-derived neurotrophic factor (BDNF) is present in the HVC of adult males but is not detectable in that of females, though the HVC of both sexes has BDNF receptors (TrkB). Testosterone treatment increases the levels of BDNF protein in the female HVC, and BDNF infused into the HVC of adult females triples the number of new neurons. Infusion of a neutralizing antibody to BDNF blocks the testosterone-induced increase in new neurons. Our results demonstrate that BDNF is involved in the regulation of neuronal replacement in the adult canary brain and suggest that the effects of testosterone are mediated through BDNF.     

Sexual Differences in Cell Proliferation in the Ventricular Zone,Cell Migration and Differentiation in the HVC of Juvenile Bengalese Finch

Song control nuclei have distinct sexual differences and thus are an ideal model to address how brain areas are sexually differentiated. Through a combination of histological analysis and electrical lesions, we first identified the ventricle site for HVC progenitor cells. We then found that there were significant sex differences in the cellular proliferation activity in the ventricular zone of the HVC, the number of migrating cells along the radial cells (positive immunoreactions to vimentin) and differentiation towards neurons. Through co-culturing of male and female slices containing the developing HVC in the same well, we found that the male slices could produce diffusible substances to masculinize the female HVC. By adding estrogen, an estrogen antagonist, brain-derived neurotrophic factor (BDNF) or its antibody into the culture medium, separately or in combination, we found that these diffusible substances may include estrogen and BDNF. Finally, we found that 1) estrogen-induced BDNF upregulation could be detected 48 hr after estrogen treatment and could not be blocked by a vascular endothelial growth factor (VEGF) receptor inhibitor and 2) the amount of VEGF mRNA expressed in the developing HVC and its adjacent area did not display any significant sex differences, as did the distribution of VEGF and laminin-expressing endothelial cells in the developing HVC. Because these findings are largely different from previous reports on the adult female HVC, it is suggested that our estrogen-induced BDNF up-regulation and the resultant sexual differentiation might not be mediated by VEGF and endothelial cells, but instead, may result from the direct effects of estrogen on BDNF.     

VEGF receptor antagonism blocks arteriogenesis, but only partially inhibits angiogenesis, in skeletal muscle of exercise-trained rats

Both collateral vessel enlargement (arteriogenesis) and capillary growth (angiogenesis) in skeletal muscle occur in response to exercise training. Vascular endothelial growth factor (VEGF) is implicated in both processes. Thus we examined the effect of a VEGF receptor (VEGF-R) inhibitor (ZD4190, AstraZeneca) on collateral-dependent blood flow in vivo and collateral artery size ex vivo (indicators of arteriogenesis) and capillary contacts per fiber (CCF; an index of angiogenesis) in skeletal muscle of both sedentary and exercise-trained rats 14 days after bilateral occlusion of the femoral arteries. Total daily treadmill run time increased appreciably from approximately 70 to approximately 100 min (at 15-20 m/min, twice per day) and produced a large (approximately 75%, P < 0.01) increase in calf muscle blood flow and a greater size of the collateral artery (wall cross-sectional area). ZD4190, which previously has been shown to inhibit the activity of VEGF-R2 and -R1 tyrosine kinase in vitro (IC50 = 30 and 700 nM, respectively), completely blocked the increase in collateral-dependent blood flow and inhibited collateral vessel enlargement. Thus exercise-stimulated collateral arteriogenesis appears to be completely dependent on VEGF-R signaling. Interestingly, enhanced mRNA expression of the VEGF family ligand placental growth factor (2- to 3.5-fold), VEGF-R1 (approximately 2-fold), and endothelial nitric oxide synthase (2- to 3.5-fold) in an isolated collateral artery implicates these factors as important in arteriogenesis. Training of ischemic muscle also induced angiogenesis, as shown by an increase (approximately 25%, P < 0.01) in CCF in white gastrocnemius muscle. VEGF-R inhibition only partially blocked (P < 0.01) but did not eliminate the increase (P < 0.01) in capillarity. Our findings indicate that VEGF-R tyrosine kinase activity is essential for collateral arteriogenesis and important for the angiogenesis induced in ischemic muscle by exercise training; however, other angiogenic stimuli are also important for angiogenesis in flow-limited active muscle.     

Aberrant,ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy.     

Fluid shear stress induces differentiation of circulating phenotype endothelial progenitor cells

Endothelial progenitor cells (EPCs) are mobilized from bone marrow to peripheral blood, and contribute to angiogenesis in tissue. In the process, EPCs are exposed to shear stress generated by blood flow and tissue fluid flow. Our previous study showed that shear stress induces differentiation of mature EPCs in adhesive phenotype into mature endothelial cells and, moreover, arterial endothelial cells. In this study we investigated whether immature EPCs in a circulating phenotype differentiate into mature EPCs in response to shear stress. When floating-circulating phenotype EPCs derived from ex vivo expanded human cord blood were exposed to controlled levels of shear stress in a flow-loading device, the bioactivities of adhesion, migration, proliferation, antiapoptosis, tube formation, and differentiated type of EPC colony formation increased. The surface protein expression rate of the endothelial markers VEGF receptor 1 (VEGF-R1) and -2 (VEGF-R2), VE-cadherin, Tie2, VCAM1, integrin α(v)/β(3), and E-selectin increased in shear-stressed EPCs. The VEGF-R1, VEGF-R2, VE-cadherin, and Tie2 protein increases were dependent on the magnitude of shear stress. The mRNA levels of VEGF-R1, VEGF-R2, VE-cadherin, Tie2, endothelial nitric oxide synthase, matrix metalloproteinase 9, and VEGF increased in shear-stressed EPCs. Inhibitor analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signal transduction pathway is a potent activator of adhesion, proliferation, tube formation, and differentiation in response to shear stress. Western blot analysis revealed that shear stress activated the VEGF-R2 phosphorylation in a ligand-independent manner. These results indicate that shear stress increases differentiation, adhesion, migration, proliferation, antiapoptosis, and vasculogenesis of circulating phenotype EPCs by activation of VEGF-R2 and the PI3K/Akt/mTOR signal transduction pathway.     

Galectin-3 protein modulates cell surface expression and activation of vascular endothelial growth factor receptor 2 in human endothelial cells

Angiogenesis is heavily influenced by VEGF-A and its family of receptors, particularly VEGF receptor 2 (VEGF-R2). Like most cell surface proteins, VEGF-R2 is glycosylated, although the function of VEGF-R2 with respect to its glycosylation pattern is poorly characterized. Galectin-3, a glycan binding protein, interacts with the EGF and TGFβ receptors, retaining them on the plasma membrane and altering their signal transduction. Because VEGF-R2 is glycosylated and both galectin-3 and VEGF-R2 are involved with angiogenesis, we hypothesized that galectin-3 binds VEGF-R2 and modulates its signal transduction as well. Employing a Western blot analysis approach, we found that galectin-3 induces phosphorylation of VEGF-R2 in endothelial cells. Knockdown of galectin-3 and Mgat5, an enzyme that synthesizes high-affinity glycan ligands of galectin-3, reduced VEGF-A mediated angiogenesis in vitro. A direct interaction on the plasma membrane was detected between galectin-3 and VEGF-R2, and this interaction was dependent on the expression of Mgat5. Using immunofluorescence and cell surface labeling, we found an increase in the level of internalized VEGF-R2 in both Mgat5 and galectin-3 knockdown cells, suggesting that galectin-3 retains the receptor on the plasma membrane. Finally, we observed reduced suture-induced neovascularization in the corneas of Gal3(-/-) and Mgat5(-/-) mice. These findings are consistent with the hypothesis that, like its role with the EGF and TGFβ receptors, galectin-3 contributes to the plasma membrane retention and proangiogenic function of VEGF-R2.     

Six placenta permeability-related genes: molecular characterization and expression analysis in pigs

The nutrient transportation ability of placenta depends on placental size, vascular density and permeability. Regulation of angiogenesis in the placenta is critical for successful gestation. Placenta vascularity exhibits disparity in different gestation stages and different pig breeds. To investigate the expression of genes related to permeability in the porcine placenta of different gestation stages and breeds, molecular cloning and gene expression analysis of six porcine genes, vascular endothelial growth factor (VEGF), VEGF-R1, VEGF-R2, endothelial nitric oxide synthase (eNOS), vascular endothelial cadherin (CDH5) and β-arrestin2 (Arrb2), were performed in this study. The results demonstrated that from gestation day 33 to day 90, Landrace exhibited significant increase (P < 0.05) in placental VEGF and Arrb2 mRNA expression. Moreover, expression levels of VEGF, VEGF-R1, VEGF-R2 and eNOS mRNA were higher (P < 0.01) in the placenta of Erhualian than those in Landrace on day 90 of gestation. In contrast, CDH5 placental mRNA expression level exhibited significant decrease (P < 0.05) from day 33 to day 90 gestation in Landrace. Erhualian placental CDH5 and Arrb2 expression levels were significantly lower (P < 0.01) than those in Landrace conceptuses on day 90 of gestation. Our study offered new data on the expression of genes in VEGF signal transduction pathway in porcine placenta.     

Complete structure of an increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom. ICPP is angiogenic via vascular endothelial growth factor receptor signalling

The partial sequence of the increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom revealed a strong homology to vascular endothelial growth factor (VEGF)-A. We now report its complete amino acid sequence determined by Edman degradation and its biological effects on mouse and human vascular endothelial cells. ICPP is a homodimeric protein linked by cysteine disulfide bonds of 25115 Da revealed by mass spectrometry. Each monomer is composed of 110 amino acids including eight cysteine residues and a pyroglutamic acid at the N-terminal extremity. ICPP shares 52% sequence identity with human VEGF but lacks the heparin binding domain and Asn glycosylation site. Besides its strong capillary permeability activity, ICPP was found to be a potent in vitro angiogenic factor when added to mouse embryonic stem cells or human umbilical vein endothelial cells. ICPP was found to be as potent as human VEGF165 in activating p42/p44 MAPK, in reinitiation of DNA synthesis in human umbilical vein endothelial cells, and in promoting in vitro angiogenesis of mouse embryonic stem cells. All these biological actions, including capillary permeability in mice, were fully inhibited by 1 microm of a new specific VEGF receptor tyrosine kinase inhibitor (ZM317450) from AstraZeneca that belongs to the anilinocinnoline family of compounds. Indeed, up to a 30 times higher concentration of inhibitor did not affect platelet-derived growth factor, epidermal growth factor, FGF-2, insulin, alpha-thrombin, or fetal calf serum-induced p42/p44 MAPK and reinitiation of DNA synthesis. Therefore, we conclude that this venom-derived ICPP exerts its biological action (permeability and angiogenesis) through activation of VEGF receptor signaling (VEGF-R2 and possibly VEGF-R1).     

An impedimetric vascular endothelial growth factor biosensor-based PAMAM/cysteamine-modified gold electrode for monitoring of tumor growth

In the present work, we have developed a new biosensor based on fourth-generation (G4) PAMAM dendrimers for the analysis of vascular endothelial growth factor (VEGF). First, the PAMAM dendrimers were covalently attached to a cysteamine-modified Au electrode by glutaraldehyde. With the help of the amino groups located on its surface, vascular endothelial growth factor receptor-1 (VEGF-R1) was immobilized via glutaraldehyde cross-linking. VEGF-R1 loading was investigated to identify the optimal VEGF-R1 immobilization conditions for the best sensitivity of the new biosensor. In addition, Kramers-Kronig transforms were also analyzed for immobilization and measurement processes. The biosensor had a linear range of 5 to 125 pg/mL VEGF. The fabricated biosensor had good repeatability and reproducibility. Finally, the results for artificial serum samples measured by the present biosensor showed a good recovery for VEGF detection.     

Vascular endothelial growth factor, vascular endothelial growth factor receptor-3 and cyclooxygenase-2 expression in psoriasis

OBJECTIVE: To investigate expression patterns and relationship of vascular endothelial growth factor (VEGF), vascular endothelial receptor-3 (VEGF-R3) (FLT-4) and cyclooxygenase-2 (COX-2) in psoriasis. STUDY DESIGN: Forty-three patients were included in this study. The clinical severity of psoriasis was assessed using the psoriasis area and severity index (PASI). Punch biopsy samples both from psoriatic and nonlesional skin were taken and VEGF, VEGF-R3 and COX-2 expressions determined. RESULTS: VEGF, VEGF-R3 and COX-2 expressions were detected in 90.9%, 78.0% and 86.4% of psoriatic and 84.1%, 71.8%, and 84.1% of nonlesional skin, respectively. Epidermal VEGF, VEGF-R3 and COX-2 expressions were detected in 56.8%, 77.8% and 34.1 of psoriatic and 75%, 78.1% and 65.9% of nonlesional skin, respectively. In dermis, VEGF, VEGF-R3 and COX-2 expression was observed in 88.6%, 77.5% and 84.1% of psoriatic and 81.8%, 64.1% and 77.3% of nonlesional skin, respectively. Among the PASI subgroups no statistically significant differences were detected for VEGF, VEGF-R3 and COX-2 expression. CONCLUSION: Our study demonstrated that VEGF, VEGF-R3 and COX-2 expression in psoriatic and nonlesional skin is significantly high in epidermis and dermis. Although there was significant concordance between VEGF and VEGF-R3 expressions in psoriatic lesions, there seems to be no concordance between the others.     

Osteoblasts and osteoclasts express PEDF, VEGF-A isoforms, and VEGF receptors: possible mediators of angiogenesis and matrix remodeling in the bone

Pigment epithelial derived factor (PEDF) is one of the most effective inhibitors of angiogenesis described so far, especially in controlling the growth of blood vessels in the eye. We now describe the localization of PEDF in regions of active bone formation in the mid-gestation mouse embryo and its specific and high levels of secretion by osteoblasts. PEDF is detected to a lesser extent in osteoclasts as well. The proangiogenic factors, VEGF and its receptors VEGF-R1 and VEGF-R2, are also expressed by both osteoblasts and osteoclasts. These findings suggest that bone angiogenesis and matrix remodeling may be mediated both by PEDF and by VEGF.     

Comparison of Trophic Factors Changes in the Hippocampal CA1 Region Between the Young and Adult Gerbil Induced by Transient Cerebral Ischemia

In the present study, we investigated neuronal death/damage in the gerbil hippocampal CA1 region (CA1) and compared changes in some trophic factors, such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), in the CA1 between the adult and young gerbils after 5?min of transient cerebral ischemia. Most of pyramidal neurons (89?%) were damaged 4?days after ischemia?Creperfusion (I?CR) in the adult; however, in the young, about 59?% of pyramidal neurons were damaged 7?days after I?CR. The immunoreactivity and levels of BDNF and VEGF, not GDNF, in the CA1 of the normal young were lower than those in the normal adult. Four days after I?CR in the adult group, the immunoreactivity and levels of BDNF and VEGF were distinctively decreased, and the immunoreactivity and level of GDNF were increased. However, in the young group, all of their immunoreactivities and levels were much higher than those in the normal young group. From 7?days after I?CR, all the immunoreactivities and levels were apparently decreased compared to those of the normal adult and young. In brief, we confirmed our recent finding: more delayed and less neuronal death occurred in the young following I?CR, and we newly found that the immunoreactivities of trophic factors, such as BDNF, GDNF, and VEGF, in the stratum pyramidale of the CA1 in the young gerbil were much higher than those in the adult gerbil 4?days after transient cerebral ischemia.     

VEGF111b,a new member of VEGFxxxb isoforms and induced by mitomycin C,inhibits angiogenesis

Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA_3.1 empty vector, pcDNA_3.1-VEGF111b or pcDNA_3.1-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.     

Acute ethanol exposure disrupts VEGF receptor cell signaling in endothelial cells

Physiological angiogenesis is regulated by various factors, including signaling through vascular endothelial growth factor (VEGF) receptors. We previously reported that a single dose of ethanol (1.4 g/kg), yielding a blood alcohol concentration of 100 mg/dl, significantly impairs angiogenesis in murine wounds, despite adequate levels of VEGF, suggesting direct effects of ethanol on endothelial cell signaling (40). To examine the mechanism by which ethanol influences angiogenesis in wounds, we employed two different in vitro angiogenesis assays to determine whether acute ethanol exposure (100 mg/dl) would have long-lasting effects on VEGF-induced capillary network formation. Ethanol exposure resulted in reduced VEGF-induced cord formation on collagen and reduced capillary network structure on Matrigel in vitro. In addition, ethanol exposure decreased expression of endothelial VEGF receptor-2, as well as VEGF receptor-2 phosphorylation in vitro. Inhibition of ethanol metabolism by 4-methylpyrazole partially abrogated the effect of ethanol on endothelial cell cord formation. However, mice treated with t-butanol, an alcohol not metabolized by alcohol dehydrogenase, exhibited no change in wound vascularity. These results suggest that products of ethanol metabolism are important factors in the development of ethanol-induced changes in endothelial cell responsiveness to VEGF. In vivo, ethanol exposure caused both decreased angiogenesis and increased hypoxia in wounds. Moreover, in vitro experiments demonstrated a direct effect of ethanol on the response to hypoxia in endothelial cells, as ethanol diminished nuclear hypoxia-inducible factor-1alpha protein levels. Together, the data establish that acute ethanol exposure significantly impairs angiogenesis and suggest that this effect is mediated by changes in endothelial cell responsiveness to both VEGF and hypoxia.     

Differential expression of VEGF isoforms and VEGF(164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF(164) in vascular permeability and angiogenesis during implantation

The mechanism(s) by which localized vascular permeability and angiogenesis occur at the sites of implantation is not clearly understood. Vascular endothelial growth factor (VEGF) is a key regulator of vasculogenesis during embryogenesis and angiogenesis in adult tissues. VEGF is also a vascular permeability factor. VEGF acts via two tyrosine kinase family receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). Recent evidence suggests that neuropilin-1 (NRP1), a receptor involved in neuronal cell guidance, is expressed in endothelial cells, binds to VEGF(165) and enhances the binding of VEGF(165) to VEGFR2. We examined the spatiotemporal expression of vegf isoforms, nrp1 and vegfr2 as well as their interactions in the periimplantation mouse uterus. We observed that vegf(164) is the predominant isoform in the mouse uterus. vegf(164) mRNA accumulation primarily occurred in epithelial cells on days 1 and 2 of pregnancy. On days 3 and 4, the subepithelial stroma in addition to epithelial cells exhibited accumulation of this mRNA. After the initial attachment reaction on day 5, luminal epithelial and stromal cells immediately surrounding the blastocyst exhibited distinct accumulation of vegf(164) mRNA. On days 6-8, the accumulation of this mRNA occurred in both mesometrial and antimesometrial decidual cells. These results suggest that VEGF(164) is available in mediating vascular changes and angiogenesis in the uterus during implantation and decidualization. This is consistent with coordinate expression of vegfr2, and nrp1, a VEGF(164)-specific receptor, in uterine endothelial cells. Their expression was low during the first 2 days of pregnancy followed by increases thereafter. With the initiation and progression of implantation (days 5-8), these genes were distinctly expressed in endothelial cells of the decidualizing stroma. Expression was more intense on days 6-8 at the mesometrial pole, the presumptive site of heightened angiogenesis and placentation. However, the expression was absent in the avascular primary decidual zone immediately surrounding the implanting embryo. Crosslinking experiments showed that (125)I-VEGF(165) binds to both NRP1 and VEGFR2 present in decidual endothelial cells. These results suggest that VEGF(164), NRP1 and VEGFR2 play a role in VEGF-induced vascular permeability and angiogenesis in the uterus required for implantation. genesis 26:213-224, 2000.     

Angiogenesis and diabetes: different responses to pro-angiogenic factors in the chorioallantoic membrane assay

Hyperglycemia induces defects in angiogenesis without alteration in the expression of major vascular growth factors in the chicken chorioallantoic membrane (CAM) model. A direct negative effect of hyperglycemia on angiogenesis may participate in failures of "therapeutic angiogenesis" trials. Here, we tested the hypothesis that the response to pro-angiogenic molecules such as angiotensin-converting enzyme (ACE), endothelin-1 (ET-1), and vascular endothelial growth factor-A (VEGF) is altered by hyperglycemia. Transfected (Chinese hamster ovary [CHO] or human embryonic kidney [HEK]) cells overexpressing ACE, ET-1, or VEGF were deposed onto the CAM of hyperglycemic or control embryos. The proangiogenic effect was evaluated 3 d later by angiography and histological analyses. Gene expression in response to these factors was assessed by in situ hybridization. Only VEGF overexpression evoked a proangiogenic response in the CAM from hyperglycemic embryos, upregulating the expression of endogenous VEGF, VEGF-R2, and Tie-2, all of them related to activation of endothelial cells. In conclusion, in a model where hyperglycemia does not alter the major vascular growth factor expression, the negative effect of diabetes on capillary density was overcome only by VEGF overexpression, whereas responses to other vasoactive peptides were practically abolished under hyperglycemic conditions.     

Expression of Vascular Endothelial Growth Factor A and Its Type 1 Receptor in Supratentorial Neoplasm

Background:Vascular endothelial growth factor (VEGF) is one of the primary angiogenesis regulators in solid cancers. Brain solid tumors are life-threatening diseases in which angiogenesis is an important phase of tumor development and progression. In the present study, VEGF-A and VEGF receptor (VEGF-R1) gene expression was evaluated in CNS brain tumors.Methods:VEGF-A and VEGF-R1 expression was quantified using real-time PCR on fresh biopsies of 38 supratentorial brain tumors compared to 30 non-tumoral tissues. Then, the correlations were investigated with clinic-pathological and demographic factors of the patients.Results:PCR product sequencing confirmed the validity of qRT-PCR. Although VEGF-A and VEGF-R1 expression showed increasing trends with the progression of cell proliferation in different stages of astrocytoma, VEGF-R1 did not meet the 95% confidence interval in other brain tumors. An increasing trend in VEGF-A expression and a declining trend in VEGF-R1 expression from Stage I to II were observed in meningioma. VEGF-A and VEGF-R1 expression had no significant correlation with age and gender. Although peritumoral brain edema (PTBE) in astrocytoma was significantly associated with tumor stages, VEGF-A and VEGF-R1 were not correlated with PTBE in meningioma and metastasis.Conclusion:VEGF-A is a valuable factor for the prognosis of PTBE and malignancy in astrocytoma and is helpful in monitoring treatment approaches.Key Words: Angiogenesis, Brain edema, Brain neoplasm, Peritumoral brain, VEGF, VEGFR1     

VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis

Vascular endothelial growth factor-A (VEGF-A) signaling directs both vasculogenesis and angiogenesis. However, the role of VEGF-A ligand signaling in the regulation of epithelial-mesenchymal interactions during early mouse lung morphogenesis remains incompletely characterized. Fetal liver kinase-1 (Flk-1) is a VEGF cognate receptor (VEGF-R2) expressed in the embryonic lung mesenchyme. VEGF-A, expressed in the epithelium, is a high affinity ligand for Flk-1. We have used both gain and loss of function approaches to investigate the role of this VEGF-A signaling pathway during lung morphogenesis. Herein, we demonstrate that exogenous VEGF 164, one of the 3 isoforms generated by alternative splicing of the Vegf-A gene, stimulates mouse embryonic lung branching morphogenesis in culture and increases the index of proliferation in both epithelium and mesenchyme. In addition, it induces differential gene and protein expression among several key lung morphogenetic genes, including up-regulation of BMP-4 and Sp-c expression as well as an increase in Flk-1-positive mesenchymal cells. Conversely, embryonic lung culture with an antisense oligodeoxynucleotide (ODN) to the Flk-1 receptor led to reduced epithelial branching, decreased epithelial and mesenchymal proliferation index as well as downregulating BMP-4 expression. These results demonstrate that the VEGF pathway is involved in driving epithelial to endothelial crosstalk in embryonic mouse lung morphogenesis.