Hyperglycemia-Induced Vasculopathy in the Murine Conceptus Is Mediated via Reductions of VEGF-A Expression and VEGF Receptor Activation
2001; Elsevier BV; Volume: 158; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64069-2
ISSN1525-2191
AutoresEmese Pinter, Jody J. Haigh, András Nagy, Joseph A. Madri,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoMajor congenital malformations, including those affecting the cardiovascular system, remain the leading cause of mortality and morbidity in infants of diabetic mothers. Interestingly, targeted mutations of several genes (including VEGF and VEGF receptors) and many teratogenic agents (including excess D-glucose) that give rise to embryonic lethal phenotypes during organogenesis are associated with a failure in the formation and/or maintenance of a functional vitelline circulation. Given the similarities in the pathology of the abnormal vitelline circulation in many of these conditions, we hypothesized that the hyperglycemic insult present in diabetes could cause the resultant abnormalities in the vitelline circulation by affecting VEGF/VEGF receptor signaling pathway(s). In this study we report that hyperglycemic insult results in reduced levels of VEGF-A in the conceptus, which in turn, leads to abnormal VEGF receptor signaling, ultimately resulting in embryonic (vitelline) vasculopathy. These findings and our observation that addition of exogenous rVEGF-A165 within a defined concentration range blunts the hyperglycemia-induced vasculopathy in the conceptus support the concept that VEGF levels can be modulated by glucose levels. In addition, these findings may ultimately lead to novel therapeutic approaches for the treatment of selected congenital cardiovascular abnormalities associated with diabetes. Major congenital malformations, including those affecting the cardiovascular system, remain the leading cause of mortality and morbidity in infants of diabetic mothers. Interestingly, targeted mutations of several genes (including VEGF and VEGF receptors) and many teratogenic agents (including excess D-glucose) that give rise to embryonic lethal phenotypes during organogenesis are associated with a failure in the formation and/or maintenance of a functional vitelline circulation. Given the similarities in the pathology of the abnormal vitelline circulation in many of these conditions, we hypothesized that the hyperglycemic insult present in diabetes could cause the resultant abnormalities in the vitelline circulation by affecting VEGF/VEGF receptor signaling pathway(s). In this study we report that hyperglycemic insult results in reduced levels of VEGF-A in the conceptus, which in turn, leads to abnormal VEGF receptor signaling, ultimately resulting in embryonic (vitelline) vasculopathy. These findings and our observation that addition of exogenous rVEGF-A165 within a defined concentration range blunts the hyperglycemia-induced vasculopathy in the conceptus support the concept that VEGF levels can be modulated by glucose levels. In addition, these findings may ultimately lead to novel therapeutic approaches for the treatment of selected congenital cardiovascular abnormalities associated with diabetes. Offspring of diabetic mothers (both humans and experimental animals) experience a two- to fourfold increase in congenital anomalies.1Eriksson UJ Hakan-Borg LA Forsberg H Styrud J Diabetic embryopathy: studies with animal and in vitro models.Diabetes. 1991; 40: 94-98Crossref PubMed Google Scholar, 2Kitzmiller JL Buchanan TA Kjos S Combs AC Ratner RE Preconception care of diabetes, congenital malformations, and spontaneous abortions.Diabetes Care. 1996; 19: 514-541PubMed Google Scholar Although no particular organ system or tissue seems to be specifically targeted, a variety of cardiovascular anomalies are frequently observed.3Ferencz C Rubin JD McCarter RJ Clark EB Maternal diabetes and cardiovascular malformations: predominance of double outlet right ventricular and truncus arteriosus.Teratology. 1990; 41: 319-326Crossref PubMed Scopus (124) Google Scholar In addition to the increased incidence of congenital anomalies noted in the live births and stillbirths of diabetic mothers, these mothers experience difficulties in becoming pregnant and exhibit an increased incidence of fetal resorption.1Eriksson UJ Hakan-Borg LA Forsberg H Styrud J Diabetic embryopathy: studies with animal and in vitro models.Diabetes. 1991; 40: 94-98Crossref PubMed Google Scholar, 4Schwarz R Teramo KA Effects of diabetic pregnancy on the fetus and newborn.Semin Perinatol. 2000; 24: 120-135Abstract Full Text PDF PubMed Scopus (163) Google Scholar, 5Lee AT Plump A DeSimone C Cerami A Bucala R A role for DNA mutations in diabetes-associated teratogenesis in transgenic embryos.Diabetes. 1995; 44: 20-24Crossref PubMed Scopus (51) Google Scholar, 6Baker L Piddlington R Diabetic embryopathy: a selective review of recent trends.J Diabetes Comp. 1993; 7: 404-412Abstract Full Text PDF Scopus (35) Google Scholar, 7Pampfer S Vanderheyden I McCracken JE Vesela J DeHertogh R Increased cell death in rat blastocysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor-α in vitro.Development. 1997; 124: 4827-4836PubMed Google Scholar, 8Ellington SK Effects of excess glucose on mammalian post-implantation embryos.Int J Dev Biol. 1997; 41: 299-306PubMed Google Scholar After implantation and before placentation, embryonic growth is dependent on proper development of the yolk sac vasculature and the vitelline circulation. Arrested development or maldevelopment of this vasculature would lead to fetal demise early resulting in the termination of pregnancy; whereas arrest and/or maldevelopment of vasculature(s) at later times, associated with specific organ or tissue development, would contribute to congenital abnormalities in a wide variety of organs and tissues. It was previously shown that in vitro culture of rat and mouse conceptuses at the primitive streak stage is possible and that conceptuses develop nearly normally for 48 hours during the initial stages of organogenesis. During this period they are nourished via the vitelline circulation, after which further growth and development would require a placental circulation.9Pinter E Reece EA Leranth CZ Sanyal MK Hobbins JC Mahoney MJ Naftolin F Yolk sac failure in embryopathy due to hyperglycemia: ultrastructural analysis of yolk sac differentiation associated with embryopathy in rat conceptuses under hyperglycemic conditions.Teratology. 1986; 33: 73-84Crossref PubMed Scopus (97) Google Scholar, 10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar The addition of 20 mmol/L D-glucose (a plasma concentration of glucose often observed in diabetic mothers of humans and of experimental animals) to these cultures results in significant yolk sac and embryonic vasculopathy and abnormal embryonic development.10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar In light of these findings and the data illustrating the crucial roles of VEGF and VEGF receptors (VEGFRs) during early vasculogenesis and angiogenesis,11Ferrara N Carvermoore K Chen H Dowd M Lu L Oshera KS Powellbraxton L Hillan KJ Moore MW Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene.Nature. 1996; 380: 439-442Crossref PubMed Scopus (3003) Google Scholar, 12Carmeliet P Ferreira V Breier G Pollefeyt S Kieckens L Gertenstein M Fahrig M Vandenhoeck A Harpal K Eberhardt C Declercq C Powling J Moons L Collen D Risau W Nagy A Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.Nature. 1996; 380: 435-439Crossref PubMed Scopus (3403) Google Scholar, 13Shalaby F Rossant J Yamaguchi TP Gertsenstein M Wu XF Breitman ML Schuh AC Failure of blood island formation and vasculogenesis in Flk-1-deficient mice.Nature. 1995; 376: 66-70Crossref PubMed Scopus (3308) Google Scholar, 14Shalaby F Ho J Stanford WL Fisher KD Schuh AC Schwartz L Bernstein A Rossant J A requirement for Flk-1 in primitive and definitive hematopoiesis and vasculogenesis.Cell. 1997; 89: 981-990Abstract Full Text Full Text PDF PubMed Scopus (736) Google Scholar, 15Dumont DJ Jussila L Taipale J Lyboussaki A Mustonen T Pajusola K Breitman M Alitalo K Cardiovascular failure in mouse embryos deficient in VEGF receptor-3.Science. 1998; 282: 946-949Crossref PubMed Scopus (683) Google Scholar, 16Soker S Takashima S Mial J Neufeld G Kalagsbrun M Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2049) Google Scholar, 17Fong GH Rossant J Gertsenstein M Breitman ML Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium.Nature. 1995; 376: 66-70Crossref PubMed Scopus (2185) Google Scholar, 18Breier G Clauss M Risau W Coordinate expression of vascular endothelial growth factor receptor-1 (flt-1) and its ligan suggests a paracrine regulation of murine vascular development.Dev Dyn. 1995; 204: 228-239Crossref PubMed Scopus (260) Google Scholar we hypothesized that the vasculopathy observed in our cultured conceptuses and in embryos of streptozotocin-induced diabetic pregnant mice10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar was due to abnormalities in the VEGF signaling pathways. In this report we demonstrate that hyperglycemic insult results in reduced levels of VEGF-A, which in turn, leads to abnormal VEGFR signaling, resulting in embryonic vasculopathy. These findings and our observation that addition of exogenous VEGF-A blunts the hyperglycemia-induced vasculopathy may ultimately lead to novel therapeutic approaches for the prevention and treatment of congenital abnormalities associated with diabetes. Murine conceptuses 7.5, 8.5, and 9.5 days post-coitus (dpc) were harvested from pregnant CD1 mice (Charles River, Wilmington, MA) as described19New DAT Whole-embryo culture and the study of mammalian embryos during organogenesis.Biol Rev. 1976; 53: 81-122Crossref Google Scholar, 20Pinter E Barreuther M Lu TT Imhof BA Madri JA Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) tyrosine phosphorylation state changes during vasculogenesis in the murine conceptus.Am J Pathol. 1997; 150: 1523-1530PubMed Google Scholar and used for morphological and biochemical studies directly or after defined periods of culture.10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 20Pinter E Barreuther M Lu TT Imhof BA Madri JA Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) tyrosine phosphorylation state changes during vasculogenesis in the murine conceptus.Am J Pathol. 1997; 150: 1523-1530PubMed Google Scholar, 21Ilan N Cheung L Pinter E Madri JA Platelet-endothelial cell adhesion molecule-1 (CD31), a scaffolding molecule for selected catenin family members whose binding is mediated by different tyrosine and serine/threonine phosphorylation.J Biol Chem. 2000; 275: 21435-21443Crossref PubMed Scopus (100) Google Scholar VEGF-A-LacZ knock-in mice were generated as described22Miguerol L Gertsenstein M Harpal K Rossant J Nagy A Multiple developmental roles of VEGF suggested by a LacZ-tagged allele.Dev Biol. 1999; 212: 307-322Crossref PubMed Scopus (232) Google Scholar by inserting an internal ribosome entry site (IRES)-LacZ cassette into the 3′UTR (exon 8) of VEGF-A and using a sequence encoding the β-galactose reporter (LacZ cassette) inserted into the noncoding region of exon 8 (3′ end) of the VEGF-A stop codon. An IRES preceded the LacZ coding sequence. This strategy permitted the production of two functional proteins, VEGF-A and LacZ, from a single bicistronic transcript. In vitro murine conceptuses were cultured as described, in the presence of pooled, heat-inactivated, undiluted rat serum, and oxygenated using a series of gas mixtures with increasing oxygen concentrations as previously described.9Pinter E Reece EA Leranth CZ Sanyal MK Hobbins JC Mahoney MJ Naftolin F Yolk sac failure in embryopathy due to hyperglycemia: ultrastructural analysis of yolk sac differentiation associated with embryopathy in rat conceptuses under hyperglycemic conditions.Teratology. 1986; 33: 73-84Crossref PubMed Scopus (97) Google Scholar, 10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Hyperglycemic culture conditions were achieved by addition of α-D-glucose (Sigma Chemical Co., St. Louis, MO) to a final concentration of 20 mmol/L as described.10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar VEGF/placental growth factor (PlGF) supplementation of control and hyperglycemic cultures was accomplished by addition of 0.2 to 20 pg/ml rVEGF-A165, rVEGF-A120, or rPlGF (Chemicon International, Inc., Temecula, CA; R&D Systems, Inc., Minneapolis, MN). Addition of growth factors or vehicle alone was made either at the start of culture or 3 hours after initiation of culture. Conceptuses were assessed for the presence or absence of major structural and functional defects including heartbeat, yolk sac circulation, vitelline vessel branching, neural tube closure, and completion of axial rotation. Growth and development were scored by their organ primordia development as described.23Kaufman MH Atlas of Mouse Development. Academic Press, London1990: 5-85Google Scholar Heartbeat and blood flow through the vitelline circulation was assessed visually by an Olympus dissecting microscope (Olympus Optical Co., Ltd., Japan) in each group of conceptuses at the termination of the culture period, as recorded and summarized in Table 1.Table 1Effects of VEGF Supplementation on Yolk Sac Vascular Arborization and on the Development of a Functional Vitelline CirculationCondition/doseNormalAbnormalArborizationFunctional circulationArborizationFunctional circulationNormoglycemic−VEGF120/126 (96%)120/126 (96%)120/126 (4%)120/126 (4%)+VEGF165:0.2 pg/ml9/10 (90%)9/10 (90%)1/10 (10%)1/10 (10%)2.0 pg/ml49/54 (91%)49/54 (91%)5/54 (9%)5/54 (9%)20.0 pg/ml0/20 (0%)0/20 (0%)20/20 (100%)20/20 (100%)200.0 pg/ml0/10 (0%)0/10 (0%)10/10 (100%)10/10 (100%)Hyperglycemic−VEGF0/20 (0%)0/20 (0%)20/20 (100%)20/20 (100%)+VEGF165:0.2 pg/ml2/10 (20%)2/10 (20%)8/10 (80%)8/10 (80%)1.0 pg/ml8/22 (36%)8/22 (36%)14/22 (64%)14/22 (64%)2.0 pg/ml*Optimal VEGF-A165 concentration.72/80 (90%)72/80 (90%)8/80 (10%)8/80 (10%)10.0 pg/ml48/62 (77%)48/62 (77%)12/62 (23%)12/62 (23%)+VEGF165 @ 3 hours2.0 pg/ml 10.0 pg/ml0/34 (0%)0/34 (0%)34/34 (100%)34/34 (100%)+VEGF 120:0.2 pg/ml8/15 (53%)0/15 (0%)7/15 (47%)15/15 (100%)10.0 pg/ml12/20 (60%)5/20 (25%)8/20 (40%)15/20 (75%)+PGF:0.2 pg/ml0/19 (0%)0/19 (0%)19/19 (100%)19/19 (100%)10.0 pg/ml0/19 (0%)0/19 (0%)19/19 (100%)19/19 (100%)* Optimal VEGF-A165 concentration. Open table in a new tab Conceptuses harvested from VEGF-A-LacZ knock-in mice were fixed in 0.2% glutaraldehyde, 2% paraformaldehyde, 2 mmol/L EGTA, and 2 mmol/L MgCl2 in Pipes buffer, pH 6.9. Staining was performed at 37°C in 0.02% X-Gal, 5 mmol/L K3Fe(CN)6, 2 mmol/L MgCl2 in phosphate-buffered saline overnight. Stained conceptuses were washed, postfixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. Sections were mounted on glass slides and counterstained with Nuclear Fast Red as described.22Miguerol L Gertsenstein M Harpal K Rossant J Nagy A Multiple developmental roles of VEGF suggested by a LacZ-tagged allele.Dev Biol. 1999; 212: 307-322Crossref PubMed Scopus (232) Google Scholar Whole mount staining of conceptuses with anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) was performed as described.10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 20Pinter E Barreuther M Lu TT Imhof BA Madri JA Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) tyrosine phosphorylation state changes during vasculogenesis in the murine conceptus.Am J Pathol. 1997; 150: 1523-1530PubMed Google Scholar Approximately 20 to 25 randomly selected in vivo grown and cultured conceptuses were evaluated for each experimental group. Light level semithin and TEM level thin sections of 8.5 and 9.5 dpc conceptuses were examined using a Zeiss Axiophot light microscope (Carl Zeiss, Oberkochen, Germany) and a Zeiss EM910 electron microscope (Carl Zeiss, Oberkochen, Germany), respectively, as described elsewhere.10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Preparation of yolk sac and embryo lysates and subsequent immunoprecipitation with anti-VEGFR-2/Flt-1 and Western blotting with anti-VEGF, anti-VEGFR-2/Flt-1 and anti-PY (PY 99) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were performed as described.10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 20Pinter E Barreuther M Lu TT Imhof BA Madri JA Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) tyrosine phosphorylation state changes during vasculogenesis in the murine conceptus.Am J Pathol. 1997; 150: 1523-1530PubMed Google Scholar, 21Ilan N Cheung L Pinter E Madri JA Platelet-endothelial cell adhesion molecule-1 (CD31), a scaffolding molecule for selected catenin family members whose binding is mediated by different tyrosine and serine/threonine phosphorylation.J Biol Chem. 2000; 275: 21435-21443Crossref PubMed Scopus (100) Google Scholar, 24Ilan N Mahooti S Rimm DL Madri JA PECAM-1 (CD31) functions as a reservoir for and a modulator of tyrosine-phosphorylated beta-catenin.J Cell Sci. 1998; 112: 3005-3014Google Scholar All blots were scanned into a Macintosh G3 computer (Apple Computer, Brea, CA) using an Arcus II scanner (Agfa-Gevaert, N.V.) and Photoshop 5.0 software (Adobe Systems, Inc. San Jose, CA). All experiments were repeated at least three times using independently prepared lysates. Although it is recognized that hyperglycemia elicits embryonic vasculopathy, the mechanism(s) responsible for this embryopathy remain unclear.3Ferencz C Rubin JD McCarter RJ Clark EB Maternal diabetes and cardiovascular malformations: predominance of double outlet right ventricular and truncus arteriosus.Teratology. 1990; 41: 319-326Crossref PubMed Scopus (124) Google Scholar, 5Lee AT Plump A DeSimone C Cerami A Bucala R A role for DNA mutations in diabetes-associated teratogenesis in transgenic embryos.Diabetes. 1995; 44: 20-24Crossref PubMed Scopus (51) Google Scholar, 8Ellington SK Effects of excess glucose on mammalian post-implantation embryos.Int J Dev Biol. 1997; 41: 299-306PubMed Google Scholar, 10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 25Natrajan R Bai W Lanting L Gonzales N Nadler J Effect of high glucose on vascular endothelial factor expression in vascular smooth muscle cells.Am J Physiol. 1997; 273: H2224-H2231PubMed Google Scholar, 26Duh E Aiello LP Perspectives in diabetes: vascular endothelial growth factor and diabetes.Diabetes. 1999; 48: 1899-1906Crossref PubMed Scopus (280) Google Scholar We have reported earlier that hyperglycemic insult results in arrest of vascular development at the primary capillary plexus stage in the yolk sacs of the conceptuses of streptozotocin-induced diabetic mothers and in cultured murine conceptuses.10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar From this observation we hypothesized that perturbations in VEGF-A expression, VEGFR expression, and/or VEGFR signaling may be crucial to the observed vasculopathy. To assess VEGF-A levels in the murine conceptus, two approaches were taken. In an earlier project a β-galactosidase (LacZ) reporter gene with a preceding IRES was introduced by gene targeting into the 3′ UTR region of VEGF-A. This modification allows the production of both VEGF-A and LacZ from the same bicistronic mRNA created. Therefore the LacZ and VEGF-A expression are transcriptionally coupled.22Miguerol L Gertsenstein M Harpal K Rossant J Nagy A Multiple developmental roles of VEGF suggested by a LacZ-tagged allele.Dev Biol. 1999; 212: 307-322Crossref PubMed Scopus (232) Google Scholar In the first approach, these VEGF-A-LacZ knock-in heterozygous male mice were mated with CD1 mothers. Conceptuses recovered at 7.5 dpc were placed in normoglycemic and hyperglycemic culture as described.10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Conceptuses were harvested after 24 (Figure 1, a and b)and 48 hours (Figure 1, d and e) in culture. Yolk sacs were isolated and 5-μm sections prepared and stained for activity. As illustrated in Figure 1, a, b, d, and e, we observed a reduced LacZ activity in the hyperglycemia-exposed yolk sacs compared with the normoglycemic yolk sacs, consistent with reduced levels of VEGF-A mRNA after hyperglycemic insult. Specifically, by 8.5 dpc the primitive capillary plexus formed from fused blood islands in the yolk sac. Abundant LacZ expression was observed in the visceral endodermal yolk sac cells, in extraembryonic mesoderm-derived cells and mesothelial cells forming the inner yolk sac layer. Hematopoietic and endothelial cells were unstained (Figure 1a). At 9.5 dpc an arborizing vascular network had developed and LacZ/VEGF-A was extensively expressed in the yolk sac endodermal cells and mesenchymal/mesothelial cells (Figure 1d). In hyperglycemia-exposed conceptuses, reductions in LacZ/VEGF-A expression were noted (Figure 1, b and e), being more pronounced in the visceral endodermal cells compared to mesenchymal/mesothelial cells in the inner layer of the yolk sacs. In the second approach, we performed Western blots on yolk sac lysates at 8.5 and 9.5 dpc. VEGF-A expression was noted throughout all stages of yolk sac vascularization, being barely detectable at 7.5 dpc (data not shown); increased at 8.5 dpc, when the primary capillary plexus has formed but arborization is not yet manifested; and decreased at 9.5 dpc, when there has been completion of branching morphogenesis and establishment of the vitelline circulation. Figure 1, c and f, illustrates the reduction in VEGF-A protein in the 8.5 and 9.5 dpc hyperglycemic cultures compared to the normoglycemic cultures. In light of our findings of reduced levels of VEGF-A in the hyperglycemic cultures, we hypothesized that perhaps treatment of these cultured embryos with exogenous VEGF-A would abrogate or blunt the effects of the hyperglycemic insult. Because VEGF-A levels are known to be tightly regulated during vasculogenesis,11Ferrara N Carvermoore K Chen H Dowd M Lu L Oshera KS Powellbraxton L Hillan KJ Moore MW Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene.Nature. 1996; 380: 439-442Crossref PubMed Scopus (3003) Google Scholar, 12Carmeliet P Ferreira V Breier G Pollefeyt S Kieckens L Gertenstein M Fahrig M Vandenhoeck A Harpal K Eberhardt C Declercq C Powling J Moons L Collen D Risau W Nagy A Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.Nature. 1996; 380: 435-439Crossref PubMed Scopus (3403) Google Scholar, 27Ferrara N Davis-Smith T The biology of vascular endothelial growth factor.Endocrinol Rev. 1997; 18: 4-25Crossref PubMed Scopus (3667) Google Scholar, 28Ferrara N Endothelial growth factor: molecular and biological aspects.Curr Topics Microbiol Immunol. 1999; 237: 1-30Crossref PubMed Scopus (505) Google Scholar, 29Carmeliet P Collen D Vascular development and disorders: molecular analysis and pathogenetic insights.Kidney Int. 1998; 53: 1519-1549Crossref PubMed Scopus (87) Google Scholar, 30Miquerol L Langille BL Nagy A Embryonic development is disrupted by modest increases in vascular endothelial growth factor gene expression.Development. 2000; 127: 3941-3946Crossref PubMed Google Scholar we determined the effects of exogenous VEGF-A165 on yolk sac vascularization. Concentrations of 0.2 to 2.0 pg/ml of VEGF-A165had no detectable adverse effects on vascular morphology. However, concentrations higher than 20.0 pg/ml elicited abnormalities in yolk sac vascular development, manifested as edematous conceptuses (data not shown). This abnormal yolk sac vasculature was characterized by the presence of an enlarged, tortuous, ectatic vasculature. Our findings are consistent with known VEGF concentration-dependent effects on vascular development and function.11Ferrara N Carvermoore K Chen H Dowd M Lu L Oshera KS Powellbraxton L Hillan KJ Moore MW Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene.Nature. 1996; 380: 439-442Crossref PubMed Scopus (3003) Google Scholar, 12Carmeliet P Ferreira V Breier G Pollefeyt S Kieckens L Gertenstein M Fahrig M Vandenhoeck A Harpal K Eberhardt C Declercq C Powling J Moons L Collen D Risau W Nagy A Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.Nature. 1996; 380: 435-439Crossref PubMed Scopus (3403) Google Scholar, 31Drake CJ Little CD Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization.Proc Natl Acad Sci USA. 1995; 92: 7657-7661Crossref PubMed Scopus (283) Google Scholar, 32Drake CJ Larue A Ferrara N Little CD VEGF regulates cell behavior during vasculogenesis.Dev Biol. 2000; 224: 178-188Crossref PubMed Scopus (102) Google Scholar When 2 or 10 pg/ml of VEGF-A165 were added to hyperglycemia-exposed cultures at the start of the culture period a marked improvement in the branching morphology of yolk sac vessels was noted (see Table 1). Figure 2 illustrates the effects of exogenous VEGF-A165 on conceptuses exposed to hyperglycemic culture conditions at 8.5 and 9.5 dpc. At 8.5 dpc the effects of hyperglycemia and VEGF-A165treatment are not apparent on low power examination of intact conceptuses (Figure 2, a–c). However, at 9.5 dpc the effects of hyperglycemic insult are readily observable (Figure 2, d, e, g, and h). An arborized yolk sac vasculature, an actively beating heart and blood flow in the vitelline vessel were noted in the normoglycemic conceptuses (Figure 2, d and g). In contrast, the yolk sacs of the hyperglycemia-exposed conceptuses displayed an ectatic vascular plexus with no signs of arborization (Figure 2, e and h) and large, non-fused blood islands toward the ectoplacental cone (Figure 2h). The embryos were malformed, with slowly beating hearts and no appreciable blood flow. However, when exogenous VEGF-A165 was added to the cultures (0.2 to 10 pg/ml), the hyperglycemia-exposed conceptuses displayed arborizing yolk sac vascular networks (Figure 2, f and i). A striking difference between this group and the hyperglycemia-exposed group was the presence of a faster beating heart and a vigorous blood flow through the yolk sac vasculature. It is important to note that this significant improvement in yolk sac vascularization and circulation was achieved when exogenous VEGF-A and D-glucose were added to the medium at the start of the culture period and were present for the duration of the culture. To ascertain whether VEGF-A treatment could be preventive, we exposed 7.5 dpc conceptuses to 20 mmol/L D-glucose for an initial 3-hour period (known to elicit yolk sac vasculopathy10Pinter E Mahooti S Wang V Imhof BA Madri JA Hyperglycemia-induced vasculopathy in the murine vitelline vasculature: correlation with PECAM-1/CD31 tyrosine phosphorylation state.Am J Pathol. 1999; 154: 1367-1379Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and then transferred the conceptuses into VEGF-A165-supplemented normoglycemic media for the remaining 45 hours of culture. We observed that VEGF-A165 added at this time failed to correct the vasculopathy, indicating that VEGF-A is preventive rather than corrective and its effects are time-dependent. PECAM-1 staining of whole mounts of yolk
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