Alterations in TGF-beta1 expression in lambs with increased pulmonary blood flow and pulmonary hypertension

The mechanisms responsible for pulmonary vascular remodeling in congenital heart disease with increased pulmonary blood flow remain unclear. We developed a lamb model of congenital heart disease and increased pulmonary blood flow utilizing an in utero placed aortopulmonary vascular graft (shunted lambs). Morphometric analysis of barium-injected pulmonary arteries indicated that by 4 wk of age, shunts had twice the pulmonary arterial density of controls (P < 0.05), and their pulmonary vessels showed increased muscularization and medial thickness at both 4 and 8 wk of age (P < 0.05). To determine the potential role of TGF-beta1 in this vascular remodeling, we investigated vascular changes in expression and localization of TGF-beta1 and its receptors TbetaRI, ALK-1, and TbetaRII in lungs of shunted and control lambs at 1 day and 1, 4, and 8 wk of life. Western blots demonstrated that TGF-beta1 and ALK-1 expression was elevated in shunts compared with control at 1 and 4 wk of age (P < 0.05). In contrast, the antiangiogenic signaling receptor TbetaRI was decreased at 4 wk of age (P < 0.05). Immunohistochemistry demonstrated shunts had increased TGF-beta1 and TbetaRI expression in smooth muscle layer and increased TGF-beta1 and ALK-1 in endothelium of small pulmonary arteries at 1 and 4 wk of age. Moreover, TbetaRI expression was significantly reduced in endothelium of pulmonary arteries in the shunt at 1 and 4 wk. Our data suggest that increased pulmonary blood flow dysregulates TGF-beta1 signaling, producing imbalance between pro- and antiangiogenic signaling that may be important in vascular remodeling in shunted lambs.     

Ovarian steroids in endometrial angiogenesis

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is fundamental for human endometrial development and differentiation, which are necessary for implantation. This vascular process is supposed to be mainly mediated by the vascular endothelial growth factor (VEGF), also named vascular permeability factor (VPF). We report here the expression and modulation of VEGF and its receptors, Flk-1/KDR and Flt-1, in the functionalis throughout the menstrual cycle. Using immunocytochemistry, VEGF is localized in glandular epithelial cells and in the surrounding stroma, as well as in capillaries and spiral arterioles. The localization of VEGF on the endothelium correlates with the presence of Flt-1 and Flk-1/KDR receptors on vascular structures, including capillary strands that have not yet formed a lumen and that have been previously described in tumors as angiogenic capillaries. The strongest immunoreactivity for both VEGF and Flk-1/KDR receptor on endothelial cells is detected in the proliferative and midsecretory phases. Enhanced expression of VEGF and its Flk-1 receptors on narrow capillary strands during the proliferative phase may account for the rapid capillary growth associated with endometrial regeneration from the residual basal layer following menstrual shedding of the functionalis. The vascular expression of Flt-1 is more important in the secretory than in the proliferative phase, associated with a high microvascular density and an increase in vascular permeability in the implantation period. Consistently with these in vivo observations, the treatment of isolated endometrial stromal cells with estradiol (E(2)), or E(2) + progesterone, significantly increased VEGF mRNA over the control value in a dose-dependent manner. These results demonstrate that the expression of VEGF and its receptors is cyclically modulated by ovarian steroids, and that this endothelial growth factor acts on the endothelium in a paracrine fashion to control endometrial angiogenesis and permeability.     

Expression of vascular endothelial growth factor and its receptors in the porcine corpus luteum during the estrous cycle and early pregnancy

The present study was undertaken to determine the expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in the porcine corpus luteum (CL) during the estrous cycle and early pregnancy. Immunohistochemical studies localized proteins of VEGF ligand-receptor system in the cytoplasm of luteal cells and in some blood vessels. Western blot analysis revealed significantly higher levels of VEGF protein during early and mid-luteal phase (vs. late luteal phase; P<0.001 and P<0.01, respectively). Quantification of VEGF mRNA in the CL showed increased mRNA levels during entire luteal phase (vs. Days 16-17; P<0.05). Expression of Flt-1 protein remained high during luteal phase (P<0.001), but the mRNA levels tended to increase from the early to the late luteal phase. Elevated protein expression of Flk-1/KDR was found in the mid-luteal phase (vs. Days 16-17; P<0.05). However, induction of Flk-1/KDR mRNA expression occurred earlier, in early luteal phase. The lowest VEGF, Flt-1 and Flk-1/KDR mRNA and protein levels were observed in regressed CL (P<0.001). During pregnancy, VEGF, Flt-1 and Flk-1/KDR mRNA and protein expression was comparable to the mid-luteal phase. In conclusion, the present study has demonstrated dynamic expression of VEGF and its receptors in the porcine CL during the estrous cycle and early pregnancy. These data suggest that the VEGF ligand-receptor system may play an important role in the development and maintenance of the CL in pigs.     

Expression of vascular endothelial growth factor receptors in the human endometrium: modulation during the menstrual cycle

Angiogenesis is fundamental for human endometrial development and differentiation necessary for implantation. These vascular changes are thought to be mediated by the vascular endothelial growth factor (VEGF), whose specific receptors have not been examined in detail thus far. We conducted the present study to determine, by immunocytochemistry and computerized image analysis of the functionalis, the expression and modulation of the receptors Flk-1/KDR and Flt-1, which mediate VEGF effects on endothelial mitogenicity, chemotaxis, and capillary permeability. VEGF receptors are expressed mainly in endometrial endothelial cells, with variations of intensity and number of stained capillaries related to the phase of the cycle. The number of capillaries immunostained for Flk-1/KDR was maximal in the proliferative phase (ratio Flk-1/CD34: 1), twice as high as the number of Flt-1-expressing capillaries (ratio Flt-1/CD34: 0.47). The staining intensity for Flk-1 decreased during the late proliferative and early secretory phases, to increase again in the midsecretory period. The number of Flt-1-labeled capillaries was about 2-fold higher in the secretory than in the proliferative phase; however, the proportion of Flt-1-positive cells did not change, owing to the associated increase in vascular density that characterizes progression of the functionalis from the proliferative to the secretory stage. The staining intensity for Flt-1 was higher during the late proliferative and secretory phases (especially in the midsecretory phase) and the premenstrual period. In contrast, the proportion of capillaries expressing Flk-1/KDR decreased in the secretory phase (ratio Flk-1/Von Willebrand factor: 0.55). Enhanced expression of Flk-1/KDR, and of Flt-1, on narrow capillary strands at the beginning of and during the proliferative phase may account for the rapid capillary growth associated with endometrial regeneration following menstrual shedding. The high coexpression of Flk-1/KDR and Flt-1 observed on capillaries during the midsecretory period correlates with an increase of endometrial microvascular density and of permeability characteristic of this phase of the cycle, which is a prerequisite for implantation. Finally, strong expression of Flt-1, but not Flk-1/KDR, was observed on dilated capillaries during the premenstrual period and the late proliferative phase, suggesting preferential association of Flt-1 with nonproliferating capillaries at those times; activation of this receptor by VEGF could be involved in premenstrual vascular hyperpermeability, edema, and extravasation of leukocytes. In addition to the endothelial localization, we found that epithelial cells expressed Flt-1 and Flk-1/KDR. We conclude that Flt-1 and Flk-1/KDR in the functionalis are modulated in parallel or independently according to the phase of the cycle, and that these changes are responsible for VEGF actions on endometrial vascular growth and permeability. The molecular mechanisms concerning these regulations will require further investigation.     

Expression of vascular endothelial growth factor (VEGF) and its cognate receptors in human pheochromocytomas

Pheochromocytomas are well-vascularized tumors, suggesting that a potent angiogenic factor may be involved in the mechanism of their formation. As vascular endothelial growth factor (VEGF) is a potent mitogen for vascular endothelial cells, here we have investigated the mRNA and protein expression of VEGF and the mRNA expression of its two receptors (Flt-1 and Flk-1/KDR) in pheochromocytomas tissue. An increase in VEGF mRNA (mainly isoforms VEGF(121) and VEGF(165)) and in VEGF protein expression were observed by semi-quantitative RT-PCR and Western blot, respectively, compared to normal adrenomedullary tissue. Flk-1/KDR, and Flt-1 levels of mRNA were also increased markedly in tumors and correlated with levels of VEGF mRNA. Therefore, we speculate that upregulation of VEGF expression and its receptors might be important in the pathogenesis of pheochromocytomas.     

sGC and PDE5 are elevated in lambs with increased pulmonary blood flow and pulmonary hypertension

Utilizing aortopulmonary vascular graft placement, we established a lamb model of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. We previously demonstrated that endothelial nitric oxide synthase (eNOS) is increased in lambs at age 4 wk. However, these lambs display a selective impairment of endothelium-dependent pulmonary vasodilation that is suggestive of a derangement downstream of NO release. Thus our objective was to characterize potential alterations in the expression and activity of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5) induced by increased pulmonary blood flow and pulmonary hypertension. Late-gestational fetal lambs (n = 10) underwent in utero placement of an aortopulmonary vascular graft (shunt). Western blotting analysis on lung tissue from 4-wk-old shunted lambs and age-matched controls showed that protein for both subunits of sGC was increased in shunted lamb lungs compared with age-matched controls. Similarly, cGMP levels were increased in shunted lamb lungs compared with age-matched controls. However, PDE5 expression and activity were also increased in shunted lambs. Thus although cGMP generation was increased, concomitant upregulation of PDE5 expression and activity may have (at least partially) limited and accounted for the impairment of endothelium-dependent pulmonary vasodilation in shunted lambs.     

Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow: a role for oxidative stress

Cardiac defects associated with increased pulmonary blood flow result in pulmonary vascular dysfunction that may relate to a decrease in bioavailable nitric oxide (NO). An 8-mm graft (shunt) was placed between the aorta and pulmonary artery in 30 late gestation fetal lambs; 27 fetal lambs underwent a sham procedure. Hemodynamic responses to ACh (1 microg/kg) and inhaled NO (40 ppm) were assessed at 2, 4, and 8 wk of age. Lung tissue nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), and heat shock protein 90 (HSP90), lung tissue and plasma nitrate and nitrite (NO(x)), and lung tissue superoxide anion and nitrated eNOS levels were determined. In shunted lambs, ACh decreased pulmonary artery pressure at 2 wk (P < 0.05) but not at 4 and 8 wk. Inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). In control lambs, ACh and inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). Total NOS activity did not change from 2 to 8 wk in control lambs but increased in shunted lambs (ANOVA, P < 0.05). Conversely, NO(x) levels relative to NOS activity were lower in shunted lambs than controls at 4 and 8 wk (P < 0.05). eNOS protein levels were greater in shunted lambs than controls at 4 wk of age (P < 0.05). Superoxide levels increased from 2 to 8 wk in control and shunted lambs (ANOVA, P < 0.05) and were greater in shunted lambs than controls at all ages (P < 0.05). Nitrated eNOS levels were greater in shunted lambs than controls at each age (P < 0.05). We conclude that increased pulmonary blood flow results in progressive impairment of basal and agonist-induced NOS function, in part secondary to oxidative stress that decreases bioavailable NO.     

Upregulation of vascular endothelial growth factor receptors Flt-1 and Flk-1 following acute spinal cord contusion in rats.

To investigate the possible role of vascular endothelial growth factor (VEGF) in the injured spinal cord, we analyzed the distribution and time course of the two tyrosine kinase receptors for VEGF, Flt-1 and Flk-1, in the rat spinal cord following contusion injury using a weight-drop impactor. The semi-quantitative RT-PCR analysis of Flt-1 and Flk-1 in the spinal cord showed slight upregulation of these receptors following spinal cord injury. Although mRNAs for Flt-1 and Flk-1 were constitutively expressed in neurons, vascular endothelial cells, and some astrocytes in laminectomy control rats, their upregulation was induced in association with microglia/macrophages and reactive astrocytes in the vicinity of the lesion within 1 day in rats with a contusion injury and persisted for at least 14 days. The spatiotemporal expression of Flt-1 in the contused spinal cord mirrored that of Flk-1 expression. In the early phase of spinal cord injury, upregulation of Flt-1 and Flk-1 mRNA occurred in microglia/macrophages that infiltrated the lesion. In addition, the expression of both receptors increased progressively in reactive astrocytes within the vicinity of the lesion, predominately in the white matter, and almost all reactive astrocytes coexpressed Flt-1 or Flk-1 and nestin. These results suggest that VEGF may be involved in the inflammatory response and the astroglial reaction to contusion injuries of the spinal cord via specific VEGF receptors.     

Alterations in ET-1, not nitric oxide,in 1-week-old lambs with increased pulmonary blood flow

Altered pulmonary vascular reactivity is a source of morbidity and mortality for children with congenital heart disease and increased pulmonary blood flow. Nitric oxide (NO) and endothelin (ET)-1 are important mediators of pulmonary vascular reactivity. We hypothesize that early alterations in endothelial function contribute to the altered vascular reactivity associated with congenital heart disease. The objective of this study was to characterize endothelial function in our lamb model of increased pulmonary blood flow at 1 wk of life. Eleven fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt) and were studied 7 days after delivery. The pulmonary vasodilator response to both intravenous ACh (endothelium dependent) and inhaled NO (endothelium independent) was similar in shunted and control lambs. In addition, tissue NO(x), NO synthase (NOS) activity, and endothelial NOS protein levels were similar. Conversely, the vasodilator response to both ET-1 and 4Ala-ET-1 (an ET(B) receptor agonist) were attenuated in shunted lambs, and tissue ET-1 concentrations were increased (P < 0.05). Associated with these changes were an increase in ET-converting enzyme-1 protein and a decrease in ET(B) receptor protein levels (P < 0.05). These data demonstrate that increased pulmonary blood flow induces alterations in ET-1 signaling before NO signaling and suggest an early role for ET-1 in the altered vascular reactivity associated with increased pulmonary blood flow.     

Vascular endothelial growth factor and its receptors

Vascular endothelial growth factor (VEGF) is a prime regulator of endothelial cell proliferation, angiogenesis, vasculogenesis and vascular permeability. Its activity is mediated by the high affinity tyrosine kinase receptors, KDR/Flk-1 and Flt-1. In this article, recently discovered structural, molecular and biological properties of VEGF are described. Among the topics discussed are VEGF and VEGF receptor structure and bioactivity, the regulation of VEGF expression, the role of VEGF and its receptors in vascular development, and the involvement of VEGF and its receptors in normal and pathological (ocular and tumor) angiogenesis.     

Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) peceptor-1 down-modulates VPF/VEGF receptor-2-mediated endothelial cell proliferation, but not migration, through phosphatidylinositol 3-kinase-dependent pathways

Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) achieves its multiple functions by activating two receptor tyrosine kinases, Flt-1 (VEGF receptor-1) and KDR (VEGF receptor-2), both of which are selectively expressed on primary vascular endothelium. To dissect the respective signaling pathways and biological functions mediated by these receptors in primary endothelial cells with these two receptors intact, we developed a chimeric receptor system in which the N terminus of the epidermal growth factor receptor was fused to the transmembrane domain and intracellular domain of KDR (EGDR) and Flt-1 (EGLT). We observed that KDR, but not Flt-1, was responsible for VPF/VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration. Moreover, Flt-1 showed an inhibitory effect on KDR-mediated proliferation, but not migration. We also demonstrated that the inhibitory function of Flt-1 was mediated through the phosphatidylinositol 3-kinase (PI-3K)-dependent pathway because inhibitors of PI-3K as well as a dominant negative mutant of p85 (PI-3K subunit) reversed the inhibition, whereas a constitutively activated mutant of p110 introduced the inhibition to HUVEC-EGDR. We also observed that, in VPF/VEGF-stimulated HUVECs, the Flt-1/EGLT-mediated down-modulation of KDR/EGDR signaling was at or before intracellular Ca(2+) mobilization, but after KDR/EGDR phosphorylation. By mutational analysis, we further identified that the tyrosine 794 residue of Flt-1 was essential for its antiproliferative effect. Taken together, these studies contribute significantly to our understanding of the signaling pathways and biological functions triggered by KDR and Flt-1 and describe a unique mechanism in which PI-3K acts as a mediator of antiproliferation in primary vascular endothelium.     

Green tea extract inhibits angiogenesis of human umbilical vein endothelial cells through reduction of expression of VEGF receptors

Epidemiological and animal studies have indicated that consumption of green tea is associated with a reduced risk of developing certain forms of cancer. However, the inhibitory mechanism of green tea in angiogenesis, an important process in tumor growth, has not been well established. In the present study, green tea extract (GTE) was tested for its ability to inhibit cell viability, cell proliferation, cell cycle dynamics, vascular endothelial growth factor (VEGF) and expression of VEGF receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/Kinase insert domain containing receptor (Flk-1/KDR) in vitro using human umbilical vein endothelial cells (HUVECs). GTE in culture media did not affect cell viability but significantly reduced cell proliferation dose-dependently and caused a dose-dependent accumulation of cells in the G1 phase. The decrease of the expression of Flt-1 and KDR/Flk-1 in HUVEC by GTE was detected with immunohistochemical and Western blotting methods. These results suggest that GTE may have preventive effects on tumor angiogenesis and metastasis through reduction of expression of VEGF receptors.     

Hemochorial placentation in the primate: expression of vascular endothelial growth factor, angiopoietins, and their receptors throughout pregnancy

Vascular development and its transformation are necessary for successful hemochorial placentation, and vascular endothelial growth factor (VEGF), angiopoietins, and their receptors may be involved in the molecular regulation of this process. To determine the potential role of these putative regulators in a widely studied primate, the common marmoset, we investigated their mRNA expression and protein location in the placenta throughout pregnancy using in situ hybridization, Northern blot analysis, and immunocytochemistry. VEGF was localized in decidual and cytotrophoblast cells, and its highest expression was found in the maternal decidua. The Flt receptor was exclusively detected in the syncytial trophoblast with increasing expression in placentae from 10 wk to term. Soluble Flt (sFlt) was also detectable by Northern blot analysis. KDR receptor expression was restricted to mesenchymal cells during early placentation and to the fetoplacental vasculature during later placentation. KDR expression increased throughout pregnancy. Angiopoietin-1 (Ang-1) was localized in the syncytial trophoblast, being highly expressed in the second half of gestation. Ang-2 mRNA localized exclusively to maternal endothelial cells, and was highly expressed in 10-wk placentae. The Tie-2 receptor was found in cytotrophoblast cells and in fetal and maternal vessels. High Tie-2 levels were detected in the wall of chorion vessels at 14-wk, 17-wk, and term placentae. These results suggest that the processes of trophoblast invasion, maternal vascular transformation, and fetoplacental vascular differentiation and development are regulated by the specific actions of angiogenic ligand-receptor pairs. Specifically, 1) VEGF/Flt and Ang-1/Tie-2 may promote trophoblast growth, 2) VEGF/KDR and Ang-1/Tie-2 may support fetoplacental vascular development and stabilization, 3) sFlt may balance VEGF actions, and 4) Ang-2/Tie-2 may remodel the maternal vasculature.     

Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants

Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability. VEGF mutants that bind to either KDR or Flt-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase Cgamma in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF. We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.     

Altered endothelium-dependent relaxations in lambs with high pulmonary blood flow and pulmonary hypertension

Congenital heart disease associated with increased pulmonary blood flow produces pulmonary hypertension. To characterize vascular alterations in the nitric oxide (NO)-cGMP cascade induced by increased pulmonary blood flow and pulmonary hypertension, 10 fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). When the lambs were 4-6 wk of age, we assessed responses of pulmonary arteries (PAs) and pulmonary veins (PVs) isolated from lungs of control and shunted lambs. PVs from control and shunted lambs relaxed similarly to exogenous NO (S-nitrosyl-acetyl-penicillamine), to NO produced endogenously (zaprinast and A-23187), and to cGMP (atrial natriuretic peptide). In contrast, relaxations to A-23187 and zaprinast were blunted in PAs isolated from shunted lambs relative to controls. Inhibitors of NO synthase (NOS) and soluble guanylate cyclase constricted control but not shunt PAs, indicating reduced basal NOS activity in shunt PAs. Pretreatment of shunt PAs with the substrates L-arginine and sepiapterin, a precursor for tetrahydrobiopterin synthesis, did not augment A-23187 relaxations. However, pretreatment with superoxide dismutase and catalase significantly enhanced A-23187 relaxations in shunt PAs. We conclude that increased pulmonary blood flow induces an impairment of endothelium-dependent relaxation that is selective to PAs. The impaired relaxation may be mediated in part by excess superoxide production.