Physiological pathway of differentiation of hematopoietic stem cell population into mural cells.

Endothelial cells (ECs), which are a major component of blood vessels, have been reported to develop in adulthood from hematopoietic cell populations, especially those of the monocyte lineage. Here we show that mural cells (MCs), another component of blood vessels, develop physiologically during embryogenesis from a hematopoietic stem cell (HSC) population, based on the in vitro culture of HSCs and histological examination of acute myeloid leukemia 1 mutant embryos, which lack HSCs. As in the embryo, HSCs in adult bone marrow differentiate into CD45+CD11b+ cells before differentiating into MCs. Moreover, CD45+CD11b+ cells are composed of two populations, CD11bhigh and CD11blow cells, both of which can differentiate into MCs as well as ECs. Interestingly, in a murine ischemia model, MCs and ECs derived from the CD11blow population had a long-term potential to contribute to the formation of newly developed blood vessels in vivo compared with the CD11high population, which could not. Moreover, injection of the CD11bhigh population induced leaky blood vessels, but the CD11blow population did not. With respect to the permeability of vessels, we found that angiopoietin 1, which is a ligand for Tie2 receptor tyrosine kinase expressed on ECs and is suggested to induce cell adhesion between ECs and MCs, is produced by the CD11blow population and plays a critical role in the formation of nonleaky vessels. These observations suggested that the CD11low cell population serves as a good source of cells for in vivo blood vessel regeneration.

Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size.

Stem cells reside in a specialized niche that regulates their abundance and fate. Components of the niche have generally been defined in terms of cells and signaling pathways. We define a role for a matrix glycoprotein, osteopontin (OPN), as a constraining factor on hematopoietic stem cells within the bone marrow microenvironment. Osteoblasts that participate in the niche produce varying amounts of OPN in response to stimulation. Using studies that combine OPN-deficient mice and exogenous OPN, we demonstrate that OPN modifies primitive hematopoietic cell number and function in a stem cell-nonautonomous manner. The OPN-null microenvironment was sufficient to increase the number of stem cells associated with increased stromal Jagged1 and Angiopoietin-1 expression and reduced primitive hematopoietic cell apoptosis. The activation of the stem cell microenvironment with parathyroid hormone induced a superphysiologic increase in stem cells in the absence of OPN. Therefore, OPN is a negative regulatory element of the stem cell niche that limits the size of the stem cell pool and may provide a mechanism for restricting excess stem cell expansion under conditions of niche stimulation.

Angiotensin II promotes development of the renal microcirculation through AT1 receptors.

Pharmacologic or genetic deletion of components of the renin-angiotensin system leads to postnatal kidney injury, but the roles of these components in kidney development are unknown. To test the hypothesis that angiotensin II supports angiogenesis during postnatal kidney development, we quantified CD31(+) postglomerular microvessels, performed quantitative PCR analysis of vascular growth factor expression, and measured renal blood flow by magnetic resonance. Treating rats with the angiotensin II type 1 receptor antagonist candesartan for 2 weeks after birth reduced the total length, volume, and surface area of capillaries in both the cortex and the medulla and inhibited the organization of vasa recta bundles. In addition, angiotensin II type 1 antagonism inhibited the transcription of angiogenic growth factors vascular endothelial growth factor, angiopoietin-1, angiopoietin-2, and the angiopoietin receptor Tie-2 in cortex and medulla. Similarly, Agtr1a(-/-);Agtr1b(-/-) mouse kidneys had decreased angiopoietin-1, angiopoietin-2, and Tie-2 mRNAs at postnatal day 14. To test whether increased urinary flow leads to microvascular injury, we induced postnatal polyuria with either lithium or adrenalectomy, but these did not alter vascular endothelial growth factor expression or vasa recta organization. Compared with vehicle-treated rats, renal blood flow was significantly (approximately 20%) lower in candesartan-treated rats even 14 days after candesartan withdrawal. Taken together, these data demonstrate that angiotensin II promotes postnatal expansion of postglomerular capillaries and organization of vasa recta bundles, which are necessary for development of normal renal blood flow.

In vivo imaging shows abnormal function of vascular endothelial growth factor-induced vasculature.

Although the angiogenic effect of vascular endothelial growth factor (VEGF) is widely recognized, a central question concerns whether the vessels formed on its overexpression effectively increase tissue perfusion in vivo. To explore this issue, here we exploit AAV vectors to obtain the prolonged expression of VEGF and angiopoietin-1 (Ang1) in rat skeletal muscle. Over a period of 6 months, muscle blood flow (MBF) and vascular permeability were measured by positron emission tomography and single-photon emission computed tomography, respectively. All measurements were performed under resting conditions and after electrically induced muscle exercise. Despite the potent angiogenic effect of VEGF, documented by vessel counting and intravascular volume assessment, the expression of this factor did not improve resting MBF, and it even decreased perfusion after exercise. This deleterious effect was related to the formation of leaky vascular lacunae, which accounted for the occurrence of arteriovenous shunts that excluded the downstream microcirculation. These effects were significantly counteracted by the coinjection of VEGF and Ang1, which determined a marked increase in resting MBF and, most notably, a significant improvement after exercise that persisted over time. Taken together, these results challenge the effectiveness of VEGF as a sole factor to induce angiogenesis and suggest the use of factor combinations to achieve competent vessel formation.

Cerebral angiogenic factors, angiogenesis, and physiological response to chronic hypoxia differ among four commonly used mouse strains.

Angiogenesis is a critical element for adaptation to low levels of oxygen and occurs following long-term exposure to mild hypoxia in rats. To test whether a similar response in mice occurs, CD1, 129/Sv, C57Bl/6, and Balb/c mice were exposed to 10% oxygen for up to 3 wk. All mice showed significant increases in the percentage of packed red blood cells, and CD1 and 129/Sv mice showed increased respiration frequency and minute volume, common physiological measures of hypoxia. Significant angiogenesis was observed in all strains except Balb/c following 3-wk exposure to chronic hypoxia. CD1 hypoxic mice had the largest increase (88%), followed by C57Bl/6 (48%), 129/Sv (41%), and Balb/c (12%), suggesting that some mice undergo more remodeling than others in response to hypoxia. Protein expression analysis of vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1 and Ang2, and Tie2 were examined to determine whether regulation of different angiogenic proteins could account for the differences observed in hypoxia-induced angiogenesis. CD1 mice showed the strongest upregulation of VEGF, Ang2, Ang1, and Tie2, whereas Balb/c had only subtle increases in VEGF and no change in the other proteins. C57Bl/6 mice showed a regulatory response that fell between the CD1 and Balb/c mice, consistent with the intermediate increase in angiogenesis. Our results suggest that genetic heterogeneity plays a role in angiogenesis and regulation of angiogenic proteins and needs to be accounted for when designing and interpreting experiments using transgenic mice and when studying in vivo models of angiogenesis.

Pathological but not physiological retinal neovascularization is altered in TNF-Rp55-receptor-deficient mice.

PURPOSE: Tumor necrosis factor (TNF)-alpha is one of the major cytokines in inflammation and apoptosis. It has been demonstrated that inhibition of TNFalpha can reduce leukocyte adhesion, vascular leakage, and apoptotic endothelial cell death in diabetes. This study was conducted to investigate the effect of TNF-Rp55 and TNF-Rp75 on retinal development in oxygen-induced retinopathy. METHODS: TNF-Rp55- and TNF-Rp75-deficient mice, as well as their respective wild-type controls, were exposed to 75% oxygen from postnatal day P7 to P12. Retinal vascularization was investigated in flatmount preparations after concanavalin A labeling of endothelial cells on days P6, P14, P17, and P20. Retinal mRNA expression of VEGF, angiopoietin-1 and -2, and PDGF was examined at days P14 and P20. RESULTS: TNF-Rp55- and TNF-Rp75-deficient mice demonstrated similar retinal development and vascularization under normoxic conditions. In comparison to wild-type mice, the vascularized area remained stable during the observation time, although the gene expression of VEGF, angiopoietin (ang)-1 and -2, and PDGFb changed. Compared with that in the wild type mice, the relative expression of VEGF, ang-1, ang-2, and PDGFb changed 5.14-, 1.7-, 0.39-, and 0.36-fold in Rp55(-/-) mice and 4.1-, 9.5 x 10(-5)-, 0.12-, and 2975-fold in Rp75(-/-) mice, respectively. Treatment with oxygen resulted in a significantly reduced vascularization in Rp55(-/-) but not Rp75(-/-) mice on postnatal day (P)20. CONCLUSIONS: Inhibition of TNFalpha via TNF-Rp55 can alter retinal development and angiogenesis in a model of oxygen-induced retinopathy. The data underscore the potential effectiveness of TNF-inhibitory treatments as modulators in oxygen-induced retinopathy.

The role of PDGF receptor inhibitors and PI3-kinase signaling in the pathogenesis of corneal neovascularization.

PURPOSE: Corneal neovascularization remains an unsolved therapeutic problem. Platelet-derived growth factor (PDGF) is directly linked to vessel formation and stabilization. This study was undertaken to elucidate the mechanisms by which PDGF exerts its effects on corneal angiogenesis. METHODS: Corneal neovascularization was induced in C57 mice by removal of the limbal epithelium. When mature vessels appeared after 7 days, mice were treated with the PDGF receptor-beta inhibitor AG 1296 or the phosphatidylinositol 3-kinase (PI3-K)-inhibitors wortmannin and LY294002, respectively, using an intraperitoneally implanted miniosmotic pump. At day 14 after scraping, corneas of treated and untreated (control) mice were dissected and immunostained with FITC-CD31 antibody for endothelial cells and with Cy3-SMA (smooth muscle actin) for pericytes. VEGF (vascular endothelial growth factor), ang1/2 (angiopoietin 1 and 2), and PDGF mRNA levels of treated and untreated corneas were determined by real-time RT-PCR. RESULTS: Mice treated with the PDGF inhibitor AG 1296 showed an inhibition of corneal neovascularization of 21.1% and a reduction of pericytes of 52% in the newly formed vessels compared with untreated animals. VEGF, ang1, ang2, and PDGF mRNA expression was reduced in the corneas of AG 1296-treated mice compared with the respective control. Treatment with the PI3-K inhibitors wortmannin and LY29002 had similar effects, inducing a decrease in corneal neovascularization and a reduction of VEGF, ang1, ang2, and PDGF mRNA levels. CONCLUSIONS: Inhibition of the PDGF signal pathway results in loss of pericytes and a reduction in vessel density in the neovascularized cornea that correlates with reduced expression of PDGF, ang1/2, and VEGF mRNA. Furthermore, PI3-K was shown to be involved in the regulation of VEGF, ang1, and PDGF, as the PI3-K inhibitors wortmannin or LY294002 had similar effects. Because PDGF is a known stimulus for PI3-K activation, it can be postulated that the observed decrease in VEGF, ang1/2, and PDGF mRNA levels on administration of the PDGF inhibitor is caused by the decreased activation of the PI3-K signaling cascade.

Effects of angiopoietin-1 on vascular endothelial growth factor-induced angiogenesis in the mouse brain.

A better understanding of angiogenic factors and their effects on angiogenesis in brain is necessary to treat cerebral vascular disorders such as ischemic brain injury. Vascular endothelial growth factor (VEGF) induces angiogenesis and increases blood-brain barrier (BBB) permeability in adult mouse brain. The effect of angiopoietin-1 on BBB leakage during the angiogenesis process is unclear. We sought to identify the effects of combining VEGF with angiopoietin-1 on cerebral angiogenesis and BBB. Adult male CD-1 mice underwent AdFc (adenoviral vector control), AdAng-1, VEGF protein, VEGF protein plus AdAng-1, or saline (negative control) injection. Brain microvessels were counted using lectin staining on tissue sections after 2 weeks of adenoviral gene transfer. The presence of zonula occludens-1 (ZO-1) was determined by Western blot analysis and immunohistochemistry. Microvessel count and augmented capillary diameter increased in mice treated with either VEGF protein or AdAng-1 plus VEGF protein compared to saline, AdFc, or AdAng-1 alone (p < 0.05). Double-labeled immunostaining demonstrated that ZO-1-positive staining was more complete on the microvessel wall in the AdAng-1 and AdAng-1 plus VEGF protein treated group compared to VEGF protein group. The results of ZO-1 expression from Western blot analysis paralleled that from immunohistochemistry (p < 0.05). We conclude that focal VEGF and angiopoietin-1 hyperstimulation in mouse brain increases microvessel density while maintaining ZO-1 protein expression, suggesting that angiopoietin-1 plays a role in synergistically inducing angiogenesis and BBB integrity.

Bradycardia induces angiogenesis, increases coronary reserve, and preserves function of the postinfarcted heart.

BACKGROUND: We tested the hypothesis that induction of chronic bradycardia would trigger an upregulation of key growth factors and receptors, which would then lead to angiogenesis and improve coronary reserve in the left ventricle after myocardial infarction. METHODS AND RESULTS: Bradycardia was induced in rats by administering alinidine via osmotic pumps beginning 1 day after coronary artery ligation. Echocardiographic analysis was conducted before and after treatment. Morphometric analysis was used in perfusion-fixed hearts to document arteriolar and capillary growth. Western blots were used to evaluate growth factor and receptor changes. During the first week of treatment, vascular endothelial growth factor (VEGF), VEGF receptor 1 (Flt-1), and basic fibroblast growth factor proteins were higher in the treated group, whereas VEGF receptor 2 (Flk-1), angiopoietin-1, and angiopoietin-2 were not affected by treatment. After 3 weeks, VEGF protein remained elevated, and bradycardia was associated with a higher capillary length density in the border (40%) and remote (14%) regions and a higher arteriolar length density in the septum (62%), despite a greater increase in left ventricular mass. Although arteriolar length density increased in all size classes, the greatest increase occurred in the smallest (terminal) arterioles. This vascular growth was associated with a 23% greater coronary reserve. Echocardiography revealed a smaller increase in ventricular volume and a greater preservation of ejection fraction in rats treated with bradycardia. CONCLUSIONS: Pharmacologic induction of bradycardia enhances vascularity and coronary reserve, preserves function of surviving myocardium, and therefore, is a noninvasive, therapeutic avenue that provides an alternative to gene therapy.

Altered levels of angiopoietin 1 and tie 2 are associated with androgen-regulated vascular regression and growth in the ventral prostate in adult mice and rats.

The involution of the rat ventral prostate gland after castration could be caused by primary changes in the vasculature. To explore the mechanisms, we studied the effects of castration and testosterone treatment on the vasculature in the ventral prostate in adult rats and mice. Androgen receptor expression, vascular morphology, and the expression of angiopoietin (ang) 1 and 2 and their receptor tie 2 were examined 1, 3, and 7 d after castration and after testosterone treatment of castrated animals using stereological methods, immunohistochemistry, laser capture microdissection, and Western blotting. One day after castration, the percentage of blood vessels covered with smooth muscle actin, endothelial cell proliferation, and vascular volume had decreased, whereas endothelial cell apoptosis had increased. Simultaneously, ang 1 and tie 2 protein levels decreased. Nuclear expression of androgen receptor was observed not only in glandular and stroma smooth muscle cells but also in the mural cells of prostate arteries and veins and was markedly down-regulated already 1 d after castration. Testosterone administration of castrated mice and rats reversed all the observed effects. At the mRNA level, tie 2 was exclusively, but ang 1 predominantly, expressed in the stroma, compared with the epithelial compartment. Local delivery of soluble tie 2 during testosterone-stimulated growth, inhibited vascular maturation and increased vascular volume and leukocyte infiltration compared with controls. We conclude that androgens may regulate the prostate vasculature by direct effects on mural vascular cells and by influencing the secretion of the angiopoietins, in above all, the stroma cells.

Vascular endothelial growth factor and angiopoietins regulate sinusoidal regeneration and remodeling after partial hepatectomy in rats.

AIM: To study the regulatory mechanisms of sinusoidal regeneration after partial hepatectomy. METHODS: We investigated the expression of angiopoietin (Ang)-1, Ang-2, Tie-2, and vascular endothelial growth factor (VEGF) in regenerating liver tissue by quantitative reverse-transcription polymerase chain reaction (RT-PCR) using a LightCycler (Roche Diagnostics) and also immunohistochemical staining after 70% hepatectomy in rats. In the next step, we isolated liver cells (hepatocytes, sinusoidal endothelial cell (SEC), Kupffer cell, and hepatic stellate cells (HSC)) from regenerating liver tissue by in situ collagenase perfusion and counterflow elutriation, to determine potential cellular sources of these angiogenic factors after hepatectomy. Proliferation and apoptosis of SECs were also evaluated by proliferating cell nuclear antigen (PCNA) staining and the terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling (TUNEL) assay, respectively. RESULTS: VEGF mRNA expression increased with a peak at 72 h after hepatectomy, decreasing thereafter. The expression of Ang-1 mRNA was present at detectable levels before hepatectomy and increased slowly with a peak at 96 h. Meanwhile, Ang-2 mRNA was hardly detected before hepatectomy, but was remarkably induced at 120 and 144 h. In isolated cells, VEGF mRNA expression was found mainly in the hepatocyte fraction. Meanwhile, mRNA for Ang-1 and Ang-2 was found in the SEC and HSC fractions, but was more prominent in the latter. The PCNA labeling index of SECs increased slowly, reaching a peak at 72 h, whereas apoptotic SECs were detected between 120 h and 144 h. CONCLUSION: Ang-Tie system, together with VEGF, plays a critical role in regulating balance between SEC proliferation and apoptosis during sinusoidal regeneration after hepatectomy. However, the VEGF system plays a more important role in the early phase of sinusoidal regeneration than angiopoietin/Tie system.

A single local injection of recombinant VEGF receptor 2 but not of Tie2 inhibits retinal neovascularization in the mouse.

PURPOSE: The purpose of this study was to develop pharmacological therapeutic alternatives for ischemia-induced proliferative retinopathy. METHODS: C57BL/6J mice were placed in 76% oxygen on postnatal day 7 (P7) for 5 days. On P12 recombinant, chimeric vascular endothelial growth factor (sVEGF-R2) or sTie2 was injected intravitreally in one eye. The fellow eye received a control injection. On P17, retinal wholemounts were prepared after perfusion with fluorescein-dextran to quantify the retinopathy. RESULTS: A single intravitreal injection of sVEGF-R2 reduced pathologic vascular changes significantly (p = 0.02). No significant effect was observed after intravitreal application of sTie2 (p = 0.07), although Ang-2 was upregulated in control animals without treatment as neovascularization developed and Ang-1 was constantly transcribed (ratio PCR). CONCLUSIONS: sVEGF-R2 interferes with VEGF signaling via VEGF-R2 receptor. Thus, local application of soluble receptors for angiogenic factors is a possible therapy for proliferative retinopathy. Receptors with a wide range of ligands might prove more useful for local application than those binding few or antagonistic ligands.

Localization of angiopoietin-1 and Tie2 immunoreactivity in rodent ependyma and adjacent blood vessels suggests functional relationships.

Angiopoietin-1 (Angpt1; previously Ang-1) participates in vascular maintenance and remodeling. In the current study, we investigated the distribution of Angpt1 protein in rat brain. We detected Angpt1 immunoreactivity (IR) in cerebral blood vessels, cuboidal ependyma, and tanycytes, which are specialized hypothalamic bipolar ependymal cells. We also evaluated patterns of IR of endothelium-specific receptor tyrosine kinase 2 (Tie2, the receptor for Angpt1). Tie2 IR was present in Angpt1-immunoreactive cuboidal ependyma in a membranous pattern, suggesting an autocrine or paracrine role for Angpt1-Tie2. Tie2 IR was also associated with peri-ependymal blood vessels, some of which were contacted by tips of Angpt1-immunoreactive tanycyte processes, implying a potential functional ligand-receptor interaction mediating communication between the cerebrospinal fluid and vascular compartments. Because we previously found that cerebral Angpt1 expression was modulated by 17beta-estradiol (E2), and because some tanycyte functions are modulated by E2, we tested the hypothesis that E2 affects ependymal and tanycyte Angpt1 expression in vivo. No gross E2 effect on the ependymal pattern of Angpt1 IR or cerebral Angpt1 protein content was observed.

Peritoneal morphologic changes in a peritoneal dialysis rat model correlate with angiopoietin/Tie-2.

The angiopoietin/Tie-2 system plays an important role in the initiation of angiogenesis. However, the role of angiopoietin/Tie-2 in peritoneal angiogenesis and fibrosis is unclear. In our study we investigated the peritoneal morphologic changes in a uremic peritoneal dialysis (PD) rat model, focusing on the relationship between angiopoietin/Tie-2 and peritoneal angiogenesis. We subjected uremic (subtotal nephrectomy) rats to dialysis, using a standard PD solution, for 10 days, 28 days, or 56 days, and compared them with uremic rats that had not undergone dialysis and control rats. Functional [dialysate-to-plasma (D/P) creatinine; ultrafiltration (UF)] and structural (vessel density and thickness of the submesothelial extracellular matrix) changes of the peritoneum were quantified. Levels of angiopoietin (Ang)-1, Ang-2, Tie-2 and vascular endothelial growth factor (VEGF) were examined in the peritoneum by real-time quantitative polymerase chain reaction (PCR) and related to angiogenesis. The uremic group that had not undergone dialysis was characterized by increased vessel density in the peritoneum compared with that of the control, which correlated with decreased UF and increased D/P creatinine. Progressive angiogenesis and fibrosis were found in the PD groups when compared with the uremic non-dialyzed or control group, accompanied by an increased D/P creatinine that occurred in the PD group after 56 days, while UF decreased. Furthermore, Ang-2 and VEGF levels increased, while Tie-2 level decreased significantly in the uremic non-dialyzed group compare with the control. This tendency was more obvious in the PD groups than in the uremic non-dialyzed or control group, but no difference was found among the PD groups. Both VEGF and Ang-2 correlated positively with vessel density, while Tie-2 correlated negatively. We confirmed angiogenesis and fibrosis changes of the peritoneum as a result of uremic status and PD therapy in the uremic PD rat model. An increased level of Ang-2 and a reduced level of Tie-2 in conditions of uremia and PD therapy correlated with peritoneal angiogenesis and functional deterioration.

Enalapril alters expression of key growth factors in experimental diabetic retinopathy.

PURPOSE: Angiogenic factors such as vascular endothelial growth factor (VEGF), erythropoietin, and angiopoietin play important roles in the development of diabetic retinopathy. However, the suppression of a single factor does not inhibit angiogenesis completely. This study simultaneously evaluated the expression of several angiogenic factors in the retinas of diabetes-induced rats and determined the effects of an angiotensin-converting enzyme inhibitor (enalapril) on the expression of angiogenic factors. METHODS: Diabetes was chemically induced by injecting 14 of 21 Sprague-Dawley rats with streptozotocin. After induction of diabetes, enalapril (10 mg/kg) was administered orally to seven rats. The rats were divided into normal, diabetes mellitus (DM), and enalapril-treated groups (each group, n = 7). The eyeballs were removed at 8 weeks after the induction of diabetes, and the retinal expression of VEGF, the signal transducer and activator of transcription (STAT)3/5, erythropoietin, and angiopoietin were examined using immunohistochemistry, RT-PCR, and Western blotting. RESULTS: RT-PCR revealed that the expression of VEGF, VEGF receptors, STAT3, erythropoietin, erythropoietin receptor, STAT5, angiopoietin 2, and Tie2 mRNA increased in the DM group, whereas angiopoietin 1 expression decreased. The enalapril-treated group showed no increase in mRNA expression of angiogenic factors. Immunohistochemical staining and Western blotting showed that the expression of VEGF, STAT3, and erythropoietin receptor proteins increased in the DM group but not in the enalapril-treated group. Erythropoietin and angiopoietin proteins were not detected by immunohistochemical staining or Western blotting. STAT5 protein expression was detected only in the DM group using immunohistochemical staining and Western blotting. The mRNA expression of the angiogenic factors VEGF, erythropoietin, and angiopoietin 2 increased in the DM group but not in the enalapril-treated group. In contrast, angiopoietin 1 mRNA expression decreased in the DM group. CONCLUSIONS: Enalapril treatment prevented increased angiogenic factor levels in the retinas of experimentally induced diabetic rats.

Angiopoietin-1 induces neurite outgrowth of PC12 cells in a Tie2-independent, beta1-integrin-dependent manner.

Overexpression of angiopoietin (Ang) 1 in the brain results in increased vascularization and altered neuronal dendrite configuration. We hypothesized that Ang1 acts directly on neurons inducing neurite outgrowth. We stimulated PC12 cells with Ang1 and observed outgrowth levels comparable to nerve growth factor (NGF). Western blotting and RT-PCR demonstrated the absence of the Ang1 receptor, Tie2 and the presence of beta1-integrin. Downstream of beta1-integrin, Ang1 stimulation led to a approximately 2.6 fold increase in focal adhesion kinase (FAK) phosphorylation and no change in the activation of mitogen-activated protein kinase (MAPK) nor c-Jun N-terminal kinase (JNK). Conversely, NGF stimulation had no effect on FAK phosphorylation but led to a approximately 3.1 and approximately 2 fold increase in phosphorylation of MAPK and JNK. Ang1, but not NGF-mediated outgrowth was attenuated following functional inhibition of beta1-integrin and FAK, and Wortmannin inhibited neurite outgrowth mediated by both. Our results suggest that Ang1 induces neurite outgrowth in PC12 cells in a Tie2-independent, beta1-integrin-FAK-PI3K-Akt-dependent manner and that NGF and Ang1 mediate neurite outgrowth via two independent signaling mechanisms.

Strengthening tight junctions of retinal microvascular endothelial cells by pericytes under normoxia and hypoxia involving angiopoietin-1 signal way.

PURPOSE: To determine the effects of pericytes and angiopoietin-1 on the expression of occludin and zonula occludens-1 (ZO-1) in retinal endothelial cells (ECs) under both normoxic and hypoxic conditions. METHODS: Rat primary retinal microvascular ECs were cultured under normoxia or hypoxia in either absence or presence of pericytes conditioned medium (PCM). PCM was pretreated with or without angiopoietin-1 neutralizing antibody. Immuofluorescent staining, Western blot and RT-PCR were used to detect the alterations of occludin and ZO-1 expression. RESULTS: Under normoxia, PCM strengthened occludin and ZO-1 immunofluorescent staining at cytomembrane as well as increased their expression at both protein and mRNA level. When pretreated with angiopoietin-1 neutralizing antibody, occludin upregulation induced by PCM was significantly blocked at protein level (62%) and mRNA level (34%). Under hypoxia, the continuity of occludin and ZO-1 staining at cell boundaries was disrupted consistent with a decrease of their protein level by 31 and 27%, respectively. Also occludin and ZO-1 mRNA level decreased by 46 and 57%, respectively. PCM was observed to partially increase expression of occludin at protein and mRNA level. Angiopoietin-1 antibody slightly inhibited (16%) PCM induced occludin mRNA increase under hypoxia. CONCLUSION: Pericytes improved the integrity of endothelial barrier through inducing occludin and ZO-1 expression at protein and mRNA level under normoxia. Under hypoxia, pericytes could partially reverse occludin decrease. These protecting effects of pericytes on endothelial barrier were at least in part mediated by angiopoietin-1.

Vascular endothelial growth factor and angiopoietin are required for prostate regeneration.

BACKGROUND: The regulation of the prostate size by androgens may be partly the result of androgen effects on the prostatic vasculature. We examined the effect of changes in androgen levels on the expression of a variety of angiogenic factors in the mouse prostate and determined if vascular endothelial growth factor (VEGF)-A and the angiopoietins are involved in the vascular response to androgens. METHODS: Expression of angiogenic factors in prostate was quantitated using real-time PCR at different times after castration and after administration of testosterone to castrated mice. Angiopoietins were localized in prostate by immunohistochemistry and in situ hybridization. The roles of VEGF and the angiopoietins in regeneration of the prostate were examined in mice inoculated with cells expressing soluble VEGF receptor-2 or soluble Tie-2. RESULTS: Castration resulted in a decrease in VEGF-A, VEGF-B, VEGF-C, placenta growth factor, FGF-2, and FGF-8 expression after 1 day. In contrast, VEGF-D mRNA levels increased. No changes in angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), hepatocyte growth factor, VEGF receptor-1, VEGF receptor-2, or tie-2 mRNA levels were observed. Administration of testosterone to castrated mice had the opposite effect on expression of these angiogenic factors. Ang-2 was expressed predominantly in prostate epithelial cells whereas Ang-1 was expressed in epithelium and smooth muscle. Inoculation of mice with cells expressing soluble VEGF receptor-2 or Tie-2 blocked the increase in vascular density normally observed after administration of testosterone to castrated mice. The soluble receptors also blocked the increase in prostate weight and proliferation of prostatic epithelial cells. CONCLUSION: VEGF-A and angiopoietins are required for the vascular response to androgens and for the ability of the prostate to regenerate in response to androgens.

Localization of Ang-1, -2, Tie-2, and VEGF expression at endothelial-pericyte interdigitation in rat angiogenesis.

Endothelial cells and pericytes play critical role in angiogenesis, which is controlled, in part, by the angiopoietin (Ang)/Tie-2 system and vascular endothelial growth factor (VEGF). Here, we investigated Ang, Tie-2, and VEGF expression within endothelial cells and pericyte interdigitations (EPI), which consist of cytoplasmic projections of pericytes and corresponding endothelial indentations. After subcutaneous implantation of a thermoreversible gelation polymer disc in rats, the capillary density was low on day 5, increased to a peak on day 7, and then decreased on days 10-20. A small number of EPI were observed on day 5, then increased sharply to a peak on day 10, but had decreased on day 20. Light and electron microscopy immunohistochemical and RNA in situ hybridization analyses revealed that Tie-2 localized at endothelial cells, and Ang-2 localized at endothelial cells and pericytes, while Ang-1 and VEGF localized at pericytes, and Ang-1 was most intensely observed at EPI of pericytes. Conventional quantitative RT-PCR and Western blot analyses revealed that the level of Ang-1 was low on days 5-7, then increased on days 10-20, while the level of VEGF was high on days 5-10, but had decreased on day 20. The level of Ang-2 remained high and Tie-2 remained at the level of the control on days 5-20. The present study showed that the angiogenic phase might be initiated by increases in Ang-2 and VEGF, while the microvessel maturation phase might be initiated by a relative increase in Ang-1 and a decrease in VEGF. Moreover, EPI might serve as a pathway for the Ang-1/Tie-2 system, with VEGF promoting pericyte recruitment for microvascular integrity.

Angiopoietin 1 causes vessel enlargement, without angiogenic sprouting, during a critical developmental period.

Early in development, endothelial cells proliferate, coalesce, and sprout to form a primitive plexus of undifferentiated microvessels. Subsequently, this plexus remodels into a hierarchical network of different-sized vessels. Although the processes of proliferation and sprouting are well studied and are dependent on the angiogenic growth factor VEGF, the factors involved in subsequent vessel remodeling are poorly understood. Here, we show that angiopoietin 1 can induce circumferential vessel enlargement, specifically on the venous side of the circulation. This action is due to the ability of angiopoietin 1 to promote endothelial cell proliferation in the absence of angiogenic sprouting; vessel growth without sprouting has not been ascribed to other vascular growth factors, nor has specificity for a particular segment of the vasculature. Moreover, angiopoietin 1 potently mediates widespread vessel enlargement only during a brief postnatal period, in particular, prior to the fourth postnatal week, corresponding to stages in which VEGF inhibition causes widespread vessel regression. These findings show that angiopoietin 1 has a potentially unique role among the vascular growth factors by acting to enlarge blood vessels without inducing sprouting, and also define a critical window of vascular plasticity in neonatal development. Finding the key molecular factors that regulate this plasticity may prove crucial to the further development of pro- and anti-angiogenic therapies.