Ang-2 mRNA Research Articles

Retinal overexpression of angiopoietin-2 mimics diabetic retinopathy and enhances vascular damages in hyperglycemia

Our previous data suggested that angiopoietin-2 (Ang-2) is linked to pericyte loss, thereby playing an important role in diabetic retinopathy. In this study, we investigated the effect of retinal overexpression of human Ang-2 (mOpsinhAng2 mouse) on vascular morphology in non-diabetic and streptozotozin-induced diabetic animals. Pericyte (PC) coverage and acellular capillary (AC) formation were quantitated in retinal digest preparations after 3 and 6 months of diabetes duration. The degree of retinopathy in non-diabetic mOpsinhAng2 mice at 3 months (-21% PC, +49% AC) was comparable to age-matched diabetic wild type mice. Diabetic mOpsinhAng2 mice exhibited significantly worse vascular pathology than wild type counterparts at 6 months. Quantitative PCR revealed that human Ang-2 mRNA was highly overexpressed in retinas of transgenic mice. Our data demonstrate that overexpression of Ang-2 in the retina enhances vascular pathology, indicating that Ang-2 plays an essential role in diabetic vasoregression via destabilization of pericytes.

Expression of Ang-1, Ang-2 and Tie-2 receptor mRNAs and their implications in hemangioma by RT-PCR

Objective To detect the expression level of Ang-1, Ang -2 and Tie-2 receptor mRNAs in the hemangioma tissuses and to explore the effects of Ang-1, Ang-2, and Tie-2 receptor on the angio-genesis in the hemangioma. Methods By use of semiquantinative reverse transcription polymerase chain reaction, we detected the expression level of Ang-1 mRNA, Ang-2 mRNA and Tie-2 mRNA in 30 cases of hemangioma (17 specimens of proliferating hemangioma, and 13 involuting hemangioma )together with 10 cases of normal skin as control, and the expression level of Ki-67 protein was also detected by an im-munohistochemica method. Results The expression of Ang-2 mRNA and Tie-2 mRNA in the prolifera-ting hemangiomas was higher than that in involuting hemangiomas (P<0.01) . The expression of Ang-1 mRNA in hemangiomas was negative. Conclusion The unbalance of Ang-Tie-2 system is probably impor-tant for the pathogenesis and development in hemangiomas. Key words: Hemangiomas; Angiopoietin; Tie-2 reeeptor; Cell proliferation associated pro-tein Ki-67

Efficacy of topical application of eosin for ulcerated hemangiomas

To the Editor: Ulceration is the most common complication of infantile hemangiomas.1Chamlin S.L. Haggstrom A.N. Drolet B.A. Baselga E. Frieden I.J. Garzon M.C. et al.Multicenter prospective study of ulcerated hemangiomas.J Pediatr. 2007; 151: 684-689Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar Recent studies suggest that ulceration may arise because of an imbalance between angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF), because Ang-2 promotes angiogenesis in the presence of VEGF but causes endothelial cell death in the absence of VEGF.2Reiss Y. Scharpfenecker M. Grunow V. Koidl S. Thurston G. Gale N.W. et al.Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation.Nat Med. 2006; 12: 235-239Crossref PubMed Scopus (713) Google Scholar Agents that decrease the production of Ang-2 might promote ulcer healing. Recently, we showed that triphenylmethane dyes including gentian violet and brilliant green are potent inhibitors of nicotinamide adenine dinucleotide phosphate oxidases, which are highly expressed in endothelial cells and downregulate Ang-2 production.3Perry B.N. Govindarajan B. Bhandarkar S.S. Knaus U.G. Valo M. Sturk C. et al.Pharmacologic blockade of angiopoietin-2 is efficacious against model hemangiomas in mice.J Invest Dermatol. 2006; 126: 2316-2322Crossref PubMed Scopus (106) Google Scholar Eosin, which is also a triphenylmethane dye, is used in many countries for the treatment of diaper dermatitis and as a topical antibiotic.4Arad A. Mimouni D. Ben-Amitai D. Zeharia A. Mimouni M. Efficacy of topical application of eosin compared with zinc oxide paste and corticosteroid cream for diaper dermatitis.Dermatology. 1999; 199: 319-322Crossref PubMed Scopus (30) Google Scholar We tested whether eosin is efficacious in the treatment of ulcerated hemangiomas of infancy. Approval was obtained from the Institutional Review Board of the Schneider Children's Medical Center. Eosin 2% solution was applied three times a day unoccluded for superficial ulcers and once a day under hydrocolloid wound dressings for deep ulcers. Eighteen patients who had 21 well-documented photos of their hemangiomas were included in the study. The mean duration of treatment was 7 weeks (range, 3-14 weeks). Four ulcerated hemangiomas needed adjuvant therapy for complete reepithelialization (pulsed dye laser treatment in two cases and hydrocolloid dressings, with or without topical antibiotics, in four cases). One hemangioma required surgical excision. Complete ulcer healing was achieved in 16 hemangiomas after 4 weeks of treatment (Fig 1). There was no recurrence in any case. In order to determine whether eosin has the same mechanism of activity as gentian violet and brilliant green, we treated bend3 endothelial cells in tissue culture with eosin and demonstrated that eosin causes a significant suppression of Ang-2 mRNA in bend3 cells3Perry B.N. Govindarajan B. Bhandarkar S.S. Knaus U.G. Valo M. Sturk C. et al.Pharmacologic blockade of angiopoietin-2 is efficacious against model hemangiomas in mice.J Invest Dermatol. 2006; 126: 2316-2322Crossref PubMed Scopus (106) Google Scholar (Fig 2). In this study, we show that eosin inhibits production of Ang-2 in endothelial cells and may be efficacious in accelerating the healing of ulcerated hemangiomas of infancy. While eosin has also been shown to have photodynamic properties, it is unlikely that a photodynamic effect played a role, because the sites were not exposed to therapeutic light sources and were often occluded. Eosin offers several advantages in terms of treatment of infantile hemangiomas. First, it has a long record of human safety, including in infants.4Arad A. Mimouni D. Ben-Amitai D. Zeharia A. Mimouni M. Efficacy of topical application of eosin compared with zinc oxide paste and corticosteroid cream for diaper dermatitis.Dermatology. 1999; 199: 319-322Crossref PubMed Scopus (30) Google Scholar Second, in addition to being antiangiogenic, it has antibacterial properties. Third, it is less expensive than many other therapies, and is therefore amenable for use in the developing and the developed world. We believe that our pilot study demonstrates the efficacy of eosin in a pilot study of human hemangiomas of infancy, and provides a rationale for larger scale clinical trials of eosin in the treatment of hemangiomas of infancy and other angiogenic disorders.

Reduced expression of angiopoietin-1 in Hantaan virus-infected human umbilical vein endothelial cell increases their permeability

Hemorrhagic fever with renal syndrome (HFRS) caused by Hantaan virus (HTNV) is characterized by vascular hemorrhage and acute renal failure. Angiopoietin-1 (Ang-1) is a glycoprotein that maintains vessel integrity and reduces endothelial permeability. We found that in HTNV-infected human umbilical vein endothelial cells (HUVECs) the levels of Ang-1 mRNA and protein were reduced on days 2 and 3 post-infection (p.i.), when endothelial permeability was increased. The HTNV-stimulated permeability was reduced by treatment of HUVECs with Ang-1. The plasma Ang-1 level was lower in HFRS patients than in healthy persons. Paired plasma samples of HFRS patients revealed markedly lower Ang-1 levels during the acute phase of HFRS as compared to the convalescent phase. These findings suggested that HTNV reduced the Ang-1 expression in endothelial cells that might play an important role in the increase of vascular permeability in HFRS.

Sildenafil‐mediated neovascularization and protection against myocardial ischaemia reperfusion injury in rats: role of VEGF/angiopoietin‐1

Sildenafil citrate (SC), a drug for erectile dysfunction, is now emerging as a cardiopulmonary drug. Our study aimed to determine a novel role of sildenafil on cardioprotection through stimulating angiogenesis during ischaemia (I) reperfusion (R) at both capillary and arteriolar levels and to examine the role of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) in this mechanistic effect. Rats were divided into: control sham (CS), sildenafil sham (SS), control + IR (CIR) and sildenafil + IR (SIR). Rats were given 0.7 mg/kg, (i.v) of SC or saline 30 min. before occlusion of left anterior descending artery followed by reperfusion (R). Sildenafil treatment increased capillary and arteriolar density followed by increased blood flow (2-fold) compared to control. Treatment with sildenafil demonstrated increased VEGF and Ang-1 mRNA after early reperfusion. PCR data were validated by Western blot analysis. Significant reduction in infarct size, cardiomyocyte and endothelial apoptosis were observed in SC-treated rats. Increased phosphorylation of Akt, eNOS and expression of anti-apoptotic protein Bcl-2, and thioredoxin, hemeoxygenase-1 were observed in SC-treated rats. Echocardiography demonstrated increased fractional shortening and ejection fraction following 45 days of reperfusion in the treatment group. Stress testing with dobutamine infusion and echocardiogram revealed increased contractile reserve in the treatment group. Our study demonstrated for the first time a strong additional therapeutic potential of sildenafil by up-regulating VEGF and Ang-1 system, probably by stimulating a cascade of events leading to neovascularization and conferring myocardial protection in in vivo I/R rat model.

Angiopoietin-2 mRNA Expression Is Elevated in Chronic Lymphocytic Leukemia Patients with Poor Prognostic Features

Several studies have demonstrated potential prognostic importance of angiogenesis in chronic lymphocytic leukemia (CLL). Elevated expression of angiogenic activator angiopoietin-2 (Ang-2) was recently reported in CLL but data regarding prognostic significance of Ang-2 in CLL is limited. Therefore, we evaluated potential relationship between Ang-2 expression and other prognostic factors in patients with CLL. Ang-2 mRNA was quantified by real-time quantitative PCR in purified mononuclear cells of 33 untreated CLL patients. Ang-2 transcript levels were further analyzed with regard to clinical stage, disease course, IgVH mutation status, CD38 and ZAP-70 expression. Elevated Ang-2 mRNA was detected in 12 cases; twenty-one patients had very low or undetectable levels of Ang-2 transcript. High Ang-2 mRNA levels were significantly associated with unmutated IgVH genes (p=0.010) and high CD38 expression (p=0.011) but not with ZAP-70 expression (p=0.784), Rai stage (p=0.305) or stable vs. progressive clinical course (p=0.443). Interestingly, there was a trend towards shorter progression-free survival in patients with high Ang-2 expression, but it did not reach statistical significance (median 15 months vs. not reached; p=0.090). In conclusion, our data show that Ang-2 mRNA is differentially expressed in patients with CLL and its increased expression appears to be associated with poor prognostic features.

Vascular endothelial growth factor and angiopoietin-2 play a major role in the pathogenesis of vascular leakage in cirrhotic rats

Background and aims:The extent and molecular mechanisms governing plasma extravasation and formation of ascites in cirrhosis are unknown. Vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Ang-2) are endogenous substances with...

Remodeling Retinal Neovascularization by ALK1 Gene Transfection In Vitro

To explore a novel strategy for balancing retinal neovascularization by assessing the role activin-like kinase receptor 1 (ALK1) plays in neovascularization in vascular endothelial growth factor (VEGF)-stimulated human retinal capillary endothelial cells (HRCECs). HRCECs were transfected with an ALK1 gene-encoding plasmid or a pSIREN-ALK1 RNAi vector and stimulated with VEGF. The mRNA and protein expression levels of ALK1, occludin, ANG2, and ALK5 were evaluated by real-time PCR and/or Western blot analysis. Microscopy techniques and flow cytometry were used to assess the effects of enhanced levels of ALK1 on migration and proliferation and the formation of tubelike structures of HRCECs. The level of ALK1 in ALK1-transfected cells was significantly increased compared with that in control cells. ALK1-transfected cells exhibited increased expression of occludin and decreased expression of ANG2 and ALK5, compared with expression in the control cells. HRCECs transfected with pSIREN-ALK1 RNAi exhibited decreased expression of ALK1 and occludin and increased expression of ANG2 and ALK5 compared with the control cells. Transfection with ALK1 affected the migration and proliferation of VEGF-stimulated HRCECs. ALK1 also inhibited the formation of endothelial tubelike structures, but did allow the formation of entire vessel structures. Overexpression of ALK1 promoted remodeling of newly formed blood vessels and prevented further angiogenesis. These findings provide insight into the control of retinal neovascularization and demonstrate a novel strategy for maintaining a stable phase of vessel formation, allowing for effective retinal neovascularization without the common adverse effects seen in patients with diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion.

Expressions of Angiopoietin-1, Angiopoietin-2, and Tie2 and Their Roles in Rat Renal Allografts With Chronic Allograft Nephropathy

Objective Angiopoietin-1 (Ang1) and −2(Ang2) are 2 ligands for the endothelium–specific tyrosine kinase Tie2. Previous studies have shown that reciprocal regulation of Ang1, Ang2, and Tie2 plays an important role in chronic cardiac allograft vasculopathy. This study investigated the expressions of Ang1, Ang2, and Tie2 in rat renal allografts undergoing chronic allograft nephropathy (CAN). Materials and Methods Renal transplantations following the procedure of Kamada with our modification were orthotopically performed using Fisher (F344, RT1 1v1) rats as both donors and recipients in the autograft group. Fisher and Lewis (LEW, RT1 1) rats were used as donors and recipients in the allograft group, respectively, which was treated with cyclosporine (CsA; 10 mg/kg/d × 10 d). At 4w, 8w, and 12 weeks posttransplantation, serum creatinine (SCr) was measured and pathologic changes assessed according to the Banff 97 criteria. The mRNA (Δct) and protein expressions of Ang1, Ang2, and Tie2 were localized by real-time fluorescence quantitative polymerase chain reaction (PCR) and by immunohistochemistry. Results The elevation in SCr and the pathologic changes in CAN were observed in all allografts at 8 and 12 weeks. The expressions of Ang1 and Ang2 were localized to epithelial cells and endothelium of the vascular bundles of the glomeruli; Tie2 was specifically expressed in endothelium of vessels both in auto- and allografts at all time points posttransplantation. At 4 weeks, the differences in mRNA expression of Ang1, Ang2, and Tie2 between the 2 groups were not significant ( P > .05). Compared with autografts, the mRNA expression of Ang1 decreased significantly ( P = .008 and .003 for 8 and 12 weeks, respectively), and the mRNA expressions of Ang2 and Tie2 significantly increased ( P = .001/.006 and .005/.001 for 8 and 12 weeks, respectively). The changes in expression of all 3 genes showed significant correlation with the Banff score in the allografts. Conclusion This study suggested that the abnormal expression and reciprocal regulation of Ang1, Ang2, and Tie2 may play important roles in the development of CAN in rat renal allografts.

Angiopoietin-2 Stimulates Blood Flow Recovery After Femoral Artery Occlusion by Inducing Inflammation and Arteriogenesis

Recently, we have shown that shear stress regulates the angiogenic potential of endothelial cells in vitro by an Angiopoietin-2 (Ang2)-dependent mechanism; however its pathophysiological significance in vivo was not clear. We hypothesized that Ang2 plays an important role in blood flow recovery after arterial occlusion in vivo by regulating angiogenesis and arteriogenesis. C57Bl/6J mice underwent femoral artery ligation and were injected with a specific Ang2 inhibitor, L1-10, or vehicle for 10 days. Ang2 mRNA was upregulated at day 2, and Ang2 protein was upregulated at day 2, 5, and 7 in the ligated hindlimb. L1-10 treatment significantly blunted blood flow recovery. L1-10 decreased smooth muscle cell coverage of neovessels without affecting capillary density, suggesting a specific role for Ang2 in arteriogenesis. Mechanistically, L1-10 decreased expression of intercellular and vascular cell adhesion molecules as well as infiltrating monocytes/macrophages in the ischemic tissue. Although L1-10 had no effect on the number of CD11b+ cells (monocytes/macrophages) mobilized in the bone marrow, it maintained elevated numbers of circulating CD11b+ cells in the peripheral blood. These results suggest that Ang2 induced in ischemic tissue plays a critical role in blood flow recovery by stimulating inflammation and arteriogenesis.

Angiopoietin‐2 levels in the hepatic vein as a useful predictor of tumor invasiveness and prognosis in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is characteristically a hypervascular tumor and its progression is known to be closely related to angiogenesis. In this study, we investigated angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) levels in the hepatic vein draining from HCC, as well as in the peripheral vein, to evaluate their relation to clinicopathological features and prognosis. To obtain hepatic venous blood samples, a catheter was placed into the main branch of the hepatic vein draining from HCC in 21 patients. The Ang-2 and VEGF levels in both the hepatic and peripheral veins were investigated. Furthermore, Ang-2 mRNA expression in surgically resected HCC was evaluated by quantitative reverse-transcription polymerase chain reaction (RT-PCR), as well as microvessel density (MVD) by CD34 immunostaining. Ang-2 levels in the hepatic vein significantly correlated with Ang-2 mRNA expression in HCC, but Ang-2 levels in the peripheral vein did not correlate. Furthermore, a significant correlation was found between hepatic venous Ang-2 and MVD levels, whereas there was no significant correlation with hepatic venous VEGF levels. When hepatic venous Ang-2 levels were compared with clinicopathological features, a significant relationship was found between high Ang-2 levels and portal vein invasion. The survival for patients in the high hepatic venous Ang-2 group was significantly poorer when compared with the low group. Preoperative hepatic venous Ang-2 levels may be a good predictor for portal vein invasion and also prognosis in patients with HCC.

Expression of Angiopoietin-1 and the receptor Tie-2 mRNA in rat brains following intracerebral hemorrhage.

Angiopoietin-1 (Ang-1) belongs to a novel family of endothelial growth factors that function as ligands for an endothelialspecific receptor tyrosine kinase (Tie-2). The Ang-/Tie-2 system may contribute to angiogenesis and vascular remodeling by mediating interactions of endothelial cells with smooth muscle cells and pericytes. The spatial distribution and temporal expression of Ang-1 and Tie-2 in the rat brain were studied following collagenase-induced intracerebral hemorrhage (ICH) by immunohistochemistry and reverse transcription-polymerase chain (RT-PCR) analysis, respectively. Immunohistochemical analysis revealed that some Ang-1 or Tie-2-positive dilated vessels resided around the hematoma and extended into the clot. RT-PCR analysis showed that Ang-1 and Tie-2 mRNA signal was detected at 2 days and persisted for 28 days after ICH. These findings suggest that ICH could lead to upregulation of Ang-1 and the receptor Tie-2 mRNA.

The Periphery of the Human Fetal Adrenal Gland Is a Site of Angiogenesis: Zonal Differential Expression and Regulation of Angiogenic Factors

Although the inner fetal zone (FZ) of the mid-gestation human fetal adrenal (HFA) produces dehydroepiandrosterone sulfate, the function of the outer definitive zone (DZ) remains less clear. We have proposed that the DZ phenotype is that of a pool of progenitor cells, many of which are mitotically active. Recently, we studied HFA expression of a family of vascular endothelial cell-specific angiogenic factors, the angiopoietins (Angs), and demonstrated that Ang2 was localized predominantly in the periphery of the gland. Ang1 stabilizes, whereas Ang2 destabilizes, vessels, increasing responsiveness to angiogenic stimuli such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factor (FGF)-2. Our objective was to test the hypothesis that the periphery of the HFA is a site of angiogenesis. Studies were conducted involving RNA, frozen sections, and primary cell cultures from midgestation HFAs. Immunofluorescence, laser capture microdissection, and real-time quantitative RT-PCR were used. Double immunostaining demonstrated that proliferating endothelial cells were limited to the DZ and DZ/FZ border. Ang2 mRNA was primarily expressed in the DZ, whereas Ang1 mRNA was primarily in the FZ. VEGF-A and FGF-2 mRNA levels were higher in the DZ. FGF-2 (10 ng/ml) induced Ang2 mRNA by 4-fold in both zones of cells (P < 0.01, at 24 h), but not Ang1 or VEGF-A mRNA. Data suggest that angiogenesis occurs at the periphery of the HFA. The DZ-predominant expression of Ang2 may be explained, in part, by the parallel pattern of FGF-2 expression.

Wenyang Huoxue Recipe up-regulates the expression of angiopoietin-1 mRNA in rats with chronic aristolochic acid nephropathy

To investigate the effects of Wenyang Huoxue Recipe (WRHXR), a compound traditional Chinese herbal medicine for warming yang and promoting blood flow, on the expression of angiopoietin mRNA in rats with chronic aristolochic acid nephropathy induced by Caulis Aristolochia Manshuriensis (CAM) decoction, and to explore the protection mechanism of WYHXR against kidney damage. Twenty-eight male SD rats were randomly divided into normal control group, CAM group and WYHXR-treated group. Rats in the normal control group (n=8) and CAM group (n=10) were intragastrically administered with normal saline 10 ml/(kg.d) or CAM decoction 10 ml/(kg.d) respectively. Rats in the WYHXR-treated group (n=10) were intragastrically administered with WYHXR 30 g/(kg.d) and CAM decoction 10 ml/(kg.d). The expressions of Ang-l and Ang-2 mRNAs were detected by real-time reverse transcription polymerase chain reaction (RT-PCR) after 20-week treatment. Compared with the normal control group, the expression of Ang-l mRNA was significantly decreased, and the expression of Ang-2 mRNA was significantly increased in the CAM group (P<0.01). Compared with the CAM group, the expression of Ang-l mRNA was increased in the WYHXR-treated group (P<0.01). The expression of Ang-2 mRNA had no significant difference between the CAM group and the WYHXR-treated group (P>0.05). Renal pathology showed that renal damage in WYHXR-treated group was significantly reduced as compared with the CAM group. WYHXR can up-regulate the expression of Ang-l mRNA, which may be its action mechanism in protecting the kidneys.

Influence of Maternal Nutrition on Placental mRNA Expression of Angiogenic Factors (AF) and Their Receptors (AFR) in Adolescent Sheep at Days 50 and 75 of Pregnancy.

Placental and fetal growth are severely compromised by late pregnancy in adolescent ewes overnourished throughout gestation. Switching ewes from a high to a moderate-control intake at d 50 of gestation prevents these negative pregnancy outcomes (Wallace et al. 1999, BOR 61:101-110). Moreover, recent analyses suggest that placental proliferation rate and vascularity are perturbed in high intake pregnancies as early as d 50. The aim here was to determine placental AF/AFR mRNA expression in control (C) and high-intake (H) ewes at d 50 and 75, and in ewes switched from a high to a control intake (H-C) at d 50 and euthanized on d 75. Singleton pregnancies (single sire) were established by embryo transfer to maximize feto-placental homeogeneity. Whole placentomes, collected at d 50 or 75 of gestation, were separated into fetal cotyledon (COT) and maternal caruncle (CAR) and frozen for quantitative real-time RT-PCR determination of placental AF/AFR mRNA. Maternal diet did not affect fetal or total placentome mass (g) at d 50 (103 ± 10 vs 94 ± 8 in H vs C; P>0.1) or at d 75 (537 ± 35 vs 557 ± 30 in H vs C; P>0.1). However, switching H ewes to C at d 50 increased placentome mass at d 75 (681 ± 46; P<0.05). Of 14 AF/AFR measured for mRNA expression, only the following differences were found. By d 50 in CAR, H-intakes decreased by one-third basic fibroblast growth factor receptor-2 (P<0.01) and angiopoietin (Ang)-1 (P<0.07) mRNA expression. By d 50 in COT, H-intakes increased by nearly two-fold Ang-2 (P=0.07) and vascular endothelial growth factor (VEGF) receptor-2 (KDR; P<0.05) mRNA expression. At d 75 for CAR, H-C increased endothelial nitric oxide synthase (eNOS) mRNA expression by over 50% (P<0.05), while H-intakes increased neuropilin-2 (NP2; P<0.05) and H-C returned NP2 to control levels. At d 75 for COT, H-intakes increased VEGF (P<0.05) and decreased placental growth factor (P<0.05) mRNA expression. Thus a complex picture of AF/AFR involved in the nutritional mediation of placental vascular growth is emerging. Future studies examining protein expression for AF/AFR are requisite to an improved understanding of the mechanisms by which placental vascular growth can be altered by maternal dietary intake during pregnancy. Funded by the Scottish Government and NIH HD045784.

N-(4-hydroxyphenyl) Retinamide Augments Laser-Induced Choroidal Neovascularization in Mice

To evaluate the effect of N-4-hydroxyphenyl retinamide (4-HPR) on experimental laser-induced choroidal neovascularization (CNV) and on the expression and secretion of relevant growth factors by cultured human retinal pigment epithelial (RPE) cells. CNV was induced by laser photocoagulation in C57BL/6 mice. 4-HPR (0.2 or 1 mg) or vehicle, was injected intraperitoneally twice daily for 14 days. Plasma and tissue levels of 4-HPR were measured by HPLC. CNV was evaluated by fluorescein angiography, histology, and quantitative confocal analysis of isolectin B4 histochemistry on days 7 and 14. Induction of apoptosis and expression and secretion of growth factors was studied in 4-HPR-treated RPE cultures. Mice treated with 4-HPR exhibited time- and dose-dependent increases in plasma and tissue 4-HPR levels. CNV lesions showed increased volume with increased vascular leakage and contained fewer lesion-associated RPE in treated versus untreated mice. Treatment of nonpolarized RPE cultures with 4-HPR in the presence of serum resulted in RPE apoptosis; however, apoptosis was minimal in similarly treated highly polarized RPE. Treatment of RPE cells with 4-HPR resulted in the upregulation of VEGF-A and -C (P < 0.05) and Ang-1 (P < 0.01) mRNA and increased secretion of VEGF-A and -C (P < 0.05), whereas pigment epithelium-derived growth factor (PEDF) and thrombospondin (TSP)-1 mRNA expression and secretion were downregulated (P < 0.05). 4-HPR increases lesion size and leakage in laser-induced CNV and is associated with the upregulation of key proangiogenic factors and the downregulation of antiangiogenic factors. Consistent with the preferential loss of RPE in CNV lesions in vivo, 4-HPR induces apoptosis of nonpolarized RPE in the presence of serum.

Regulation of angiopoietin expression by bacterial lipopolysaccharide

Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-kappaB. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.

Role of uterine natural killer cells in angiogenesis of human decidua of the first-trimester pregnancy

Decidualization is accompanied by extensive angiogenesis, which is an essential step in the maturation of new blood vessels in mammalian pregnancy. The purpose of this study was to determine a distribution of uNK cells (CD56(+) uNK or CD56(bright) cells) in human decidua of the first-trimester pregnancy, and investigate whether uNK cells in human decidua could express vascular endothelial growth factor (VEGF-A) and/or angiopoietin2 (Ang2). Our immunohistochemical staining results demonstrated that a great amount of uNK (CD56(+)) cells scattered throughout the decidual stroma and near endometrial gland and spiral vessels in human decidua. The protein expression of VEGF-A and Ang2 was detected in decidual stroma cells, capillary endothelial cells and glandular cells in tissue specimens. There was a positive correlation between microvessel density (MVD) and the number of the CD56-positive uNK cells in decidual stroma, and also between the number of the CD56-positive uNK cells and VEGF-A protein expression in the tissue. In addition, we found that uNK cells in human decidua could express VEGF-A mRNA, but not Ang2 mRNA, in isolated uNK cells in human decidua of the first-trimester gestation by combination of LCM and Nested-PCR. Our study indicated that uNK cells, through expressing VEGF-A, may play an important role in the angiogenic response at the time of human decidualization and early placenta development.

Angiopoietin-2 Deficiency Decelerates Age-Dependent Vascular Changes in the Mouse Retina

Retinae of aged humans show signs of vascular regression. Vascular regression involves a mismatch between Angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) expression. We used heterozygous Ang-2 deficient (Ang2LacZ) mice to evaluate murine retinal vascular changes and gene expression of growth factors. Vascular changes were assessed by quantitative retinal morphometry and gene expression levels of growth factors were measured by quantitative PCR. The numbers of endothelial cells and pericytes did not change in the Ang2LacZ retinae with age, whereas they decreased throughout the age spectrum studied in the wild type retinae. Moreover, vascular regression significantly decelerated in the heterozygous Ang2LacZ retinae (200% to 1 month), while the formation of acellular capillaries was significantly increased at 13 months in the wild type retinae (340% to 1 month). Gene expression analysis revealed that VEGF, Ang-1, PDGF-B and Ang2 mRNA levels were decreased in the wild type retinae at 9 month of age. However, the decrease of Ang-2 was smaller compared with other genes. While VEGF levels dropped in wild type mice up to 60% compared to 1 month, VEGF increased in heterozygous Ang-2 deficient retinae at an age of 9 months (141% to 1 month). Similarly, Ang-1 levels decreased in wild type mice (45% to 1 month), but remained stable in Ang2LacZ mice. These data suggest that Ang-2 gene dose reduction decelerates vasoregression in the retina with age. This effect links to higher levels of survival factors such as VEGF and Ang-1, suggesting that the ratio of these factors is critical for capillary cell survival.

Stage-specific Integration of Maternal and Embryonic Peroxisome Proliferator-activated Receptor δ Signaling Is Critical to Pregnancy Success

Successful pregnancy depends on well coordinated developmental events involving both maternal and embryonic components. Although a host of signaling pathways participate in implantation, decidualization, and placentation, whether there is a common molecular link that coordinates these processes remains unknown. By exploiting genetic, molecular, pharmacological, and physiological approaches, we show here that the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) delta plays a central role at various stages of pregnancy, whereas maternal PPARdelta is critical to implantation and decidualization, and embryonic PPARdelta is vital for placentation. Using trophoblast stem cells, we further elucidate that a reciprocal relationship between PPARdelta-AKT and leukemia inhibitory factor-STAT3 signaling pathways serves as a cell lineage sensor to direct trophoblast cell fates during placentation. This novel finding of stage-specific integration of maternal and embryonic PPARdelta signaling provides evidence that PPARdelta is a molecular link that coordinates implantation, decidualization, and placentation crucial to pregnancy success. This study is clinically relevant because deferral of on time implantation leads to spontaneous pregnancy loss, and defective trophoblast invasion is one cause of preeclampsia in humans.