This Article Abstract Full Text (PDF) All Versions of this Article: 36/2/337    most recent 01.STR.0000153795.38388.72v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ardelt, A. A. Articles by Hurn, P. D. Search for Related Content PubMed PubMed Citation Articles by Ardelt, A. A. Articles by Hurn, P. D. Pubmed/NCBI databases Gene GEO Profiles HomoloGene Protein UniGene Compound via MeSH Substance via MeSH Hazardous Substances DB ESTRADIOL Medline Plus Health Information Stroke Related Collections Angiogenesis Animal models of human disease Brain Circulation and Metabolism Other Vascular biology

(Stroke. 2005;36:337.)
© 2005 American Heart Association, Inc.

Original Contributions

Estradiol Regulates Angiopoietin-1 mRNA Expression Through Estrogen Receptor- in a Rodent Experimental Stroke Model

Agnieszka A. Ardelt, MD, PhD; Louise D. McCullough, MD, PhD; Kenneth S. Korach, PhD; Michael M. Wang, MD, PhD; Diane H. Munzenmaier, PhD Patricia D. Hurn, PhD

From the Departments of Neurology (A.A.A., L.D.M., M.M.W.) and Anesthesiology and Critical Care Medicine (A.A.A., P.D.H.), Johns Hopkins Medical Institutions, Baltimore, Md; National Institutes of Health (K.S.K.), Bethesda, Md; and Department of Physiology (D.H.M.), Medical College of Wisconsin, Milwaukee.

Reprint requests to Agnieszka A. Ardelt, MD, PhD, Division of Neurosciences Critical Care, Meyer 8-140, Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287-7840. E-mail aardelt1{at}jhmi.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background and Purpose— Female, compared with male, animals are protected from cerebral ischemic injury. Physiological concentrations of 17ß-estradiol (E2) reduce damage in experimental stroke. E2 augments angiogenesis in reproductive organs and noncerebral vascular beds. We hypothesized that E2 protects brain in stroke through modulation of angiogenesis. We quantified molecular markers of angiogenesis and capillary density before and after unilateral middle cerebral artery occlusion (MCAO).

Methods— Female animals were ovariectomized, treated with 25 µg E2 or placebo implants, and subjected to 2-hour MCAO and 22 hours of reperfusion. Brain angiopoietin-1 (Ang-1), Ang-2, Tie-1, Tie-2, vascular endothelial growth factor (VEGF), VEGF R1, and VEGF R2 mRNA levels were determined by RNAse protection assays, and CD31-positive vessels were counted.

Results— E2, but not ischemia, upregulated cerebral Ang-1 mRNA by 49%. Capillary density was higher in the brains of E2-treated animals. In estrogen receptor- knockout (ERKO) mice, E2-mediated induction of Ang-1 mRNA was absent relative to wild-type littermates.

Conclusions— These results suggest that E2 increases Ang-1 and enhances capillary density in brain under basal conditions, priming the MCA territory for survival after experimental focal ischemia.

Key Words: angiogenesis • cerebrovascular accident • endothelial growth factors

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Women are protected from stroke relative to men; this advantage is lost with menopause. In 2002, the combined estrogen–progestin arm of the Women’s Health Initiative was stopped because of lack of efficacy in cardiovascular disease prevention, including stroke.¹ The estrogen-alone arm of the trial was discontinued because of increased stroke risk in hormone users.² Therefore, data from animals and cultured cells are under intense scrutiny to elucidate biological pathways of the effects of 17ß-estradiol (E2) in brain and cerebral vasculature. The cellular and molecular pathophysiology of cerebral ischemia is strongly influenced by gender and female sex steroids. Although the preventative role of E2 is unclear, it does reduce stroke sensitivity (ie, attenuates neuronal damage once ischemia occurs).^3–6 The mechanism of protection is likely multifactorial.⁴ E2 signals through estrogen receptor- (ER) and ERß to alter gene expression.⁴ Rodent brains express both receptors, but ER functions in E2-mediated protection in stroke.⁷ A candidate mechanism of cerebral protection is the ability of E2 to modulate angiogenesis.^8–11 E2-mediated angiogenesis occurs in normal tissues (eg, female reproductive tract^8,9,12, retina,¹⁰ and other nonreproductive tissues)¹¹ as well as pathologic conditions (eg, experimental limb ischemia,¹³ tumors,⁸ and aging brain).¹⁴

Enhancement of angiogenesis is an attractive property. Improvement of collateral blood flow through angiogenesis or other endothelial-based adaptive mechanisms may occur after transient ischemic attacks, before stroke, in patients with intracranial stenosis.¹⁵ Such patients may be protected from infarction after vessel closure, similar to phenomena in coronary vasculature.^16–19 Angiogenesis also occurs after stroke and may improve outcome.^20–26 Current imaging modalities are of insufficient resolution to visualize the smallest collateral vessels, and therefore, molecular markers of angiogenesis are sometimes used to provide spatial and temporal quantification of angiogenesis.^{15,20,22,26–30}

Several angiogenic factors are involved in the production and maintenance of vascular networks.^31,32 The angiopoietin family includes angiopoietin-1 (Ang-1) and Ang-2.³² Ang-1 signals through the Tie-2 receptor, reducing vascular permeability and stabilizing blood vessels.^32–34 Ang-2 competitively inhibits the Ang-1/Tie-2 interaction, leading to blood vessel disruption.^35,36 Tie-1 modulates Tie-2 by direct intramembrane interactions.³⁷ The vascular endothelial growth factor (VEGF) family includes VEGF-A,³⁸ which signals through VEGF R1 (flt-1) and VEGF R2 (kinase insert domain-containing receptor [KDR] or flk-1).³⁹ The 2 families are linked functionally: the effect of VEGF is dependent on the Ang-1/Ang-2 ratio. When Ang-2 is abundant and the ratio is low, VEGF promotes robust remodeling of the vascular network.³⁶

In this study, we investigated whether E2 modulates expression of angiogenesis markers and alters brain capillary density in an established model of experimental stroke. Using reversible middle cerebral artery occlusion (MCAO),⁴⁰ we evaluated the effect of E2 on angiopoietin and VEGF family gene expression and determined whether these transcriptional changes were mediated by ER.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Middle Cerebral Artery Occlusion
All methods were described previously.^41–44 Animal experiments were performed according to institutional guidelines. Female Wistar rats (weighing 200 to 250 g; Harlan Breeders) were ovariectomized and subcutaneously implanted with placebo or E2 pellets (25 µg; 21-day release; Innovative Research of America). One week later, animals underwent sham surgery or 2-hour MCAO, followed by 22 hours of reperfusion. Animals were anesthetized with halothane (induction 2.5%; maintenance 1%); femoral arterial lines were placed for blood sampling and mean arterial pressure (MAP) monitoring; and the skull overlying the right MCA territory was thinned for laser Doppler flow (LDF) monitoring (Moor Instruments). A 4.0 monofilament suture was introduced into the internal carotid artery and advanced until LDF dropped to <45% of baseline. MAP and rectal and temporalis temperatures were monitored and controlled, and arterial blood gases, blood glucose, and hemoglobin concentrations were monitored before occlusion and 1 hour into ischemia. After 2 hours of ischemia, the suture was removed and reperfusion documented by LDF. After 22 hours of reperfusion, the animal was euthanized, the brain removed, and blood obtained for E2 radioimmunoassay (Diagnostic Products).⁴⁰ For mRNA quantification, brains were separated into hemispheres and frozen in 2-methylbutane (Sigma-Aldrich) on dry ice.

ERKO mice (weighing 22 to 26 g) were studied using a 2-hour MCAO protocol with a 6.0 suture.⁴² Seven days before ischemia, ERKO and age- and weight-matched wild-type (WT) mice were ovariectomized and treated with subcutaneous E2 or vehicle (Silastic capsule; 0.062 ID, 0.125 OD), as described previously.^7,42,43 Intraischemic neurologic deficit was scored as follows: 0, no deficit; 1, forelimb weakness and torso turning to the ipsilateral side when held by the tail; 2, circling to affected side; 3, unable to bear weight on affected side; and 4, no spontaneous locomotor activity or barrel rolling.^43,44 If no deficit was observed, the animal was removed from further study. After 22 hours of reperfusion, mice were processed as described above.

mRNA Quantification
RNA was extracted from brains using Trizol (Invitrogen) and chloroform. mRNA levels were determined by RNAse protection assay (RPA; RPA III kit; Ambion) using previously characterized probes for Ang-1, Ang-2, Tie-1, and Tie-2.⁴⁵ VEGF, VEGF R1, and VEGF R2 RNA probes were subcloned into the EcoRI/BamHI site of pBluescript II SK (+) using the following primers: VEGF: sense 5'-AACAAAGCCAGAAAAAAAATCA-3'; antisense 5'-TCACCGCCTTGGCTTGTCACA-3'; VEGF R1: sense 5'-ACGGA-CAGTTAACAACAGGA-3'; antisense 5'-GACTCTTTC-AATAAACAGCGT-3'; VEGF R2: sense 5'-CTCACACCAGT-TTGCAAGAA-3'; and antisense 5'-AGTACAATGCCTAATC-TTCT-3'.

Probes were labeled with ³²P-UTP using T7 polymerase (Ambion).⁴⁵ After hybridization, samples were processed on polyacrylamide gels, and mRNA species were quantified relative to actin using a phosphorimager (Molecular Dynamics).

Capillary Counts
Cerebral microvessels were counted in placebo- and E2- treated nonischemic and ischemic rats. Brains were removed, placed in embedding-medium-filled plastic molds, and frozen in 2-methylbutane on dry ice. In each animal, 8 5-µm-thick coronal sections were collected every 50 µm, 1.7 mm to –0.8 mm from bregma. The sampled area corresponded to plates 11 through 16 in Paxinos and Watson’s The Rat Brain in Stereotactic Coordinates.⁴⁶ Sections were processed with anti-CD31 antibody (Chemicon) at 1:100 dilution, and labeling was visualized with diaminobenzidine (DAB; Vector Laboratories). Three regions per hemisphere per slice, corresponding to basal ganglia, parasaggital cortex, and parietal cortex (Figure 1), were digitally photographed at x200. DAB-positive structures were quantified using Metamorph Offline version 4.6r4 software (Universal Imaging). Capillary counts from all 8 slices were averaged to derive 1 capillary count number per region per animal. In nonischemic animals, left hemisphere capillary counts were quantified and compared between placebo- and E2-treated animals. In postischemic animals, ischemic/nonischemic hemisphere capillary count ratios were derived and compared between placebo- and E2-treated animals.

View larger version (139K): [in this window] [in a new window]   Figure 1. A, A representative 2-mm-thick section of rat brain stained with a vital dye reveals the infarct (pale tissue), and demonstrates the parasaggital cortex (1), basal ganglia (2), and parietal cortex (3) regions in which capillaries were counted in digital micrographs of 5-µm-thick sections. Nonischemic hemisphere regions were mirror images. Bar=1 mm. B and C, Micrographs of a representative area of the basal ganglia in a 5-µm-thick frozen rat brain section processed with anti-CD31 antibody. Each region in which CD31-positive structures (arrows) were counted totaled 0.26 mm2. B, Placebo-treated rat. C, E2-treated rat. Bar=0.1 mm.

Statistical Analysis
RPA data were analyzed using ANOVA, and average capillary counts were compared with Student t tests. Sigmastat 3.0 (SPSS) was used for analysis. Data are expressed as mean±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
MAP, PCO₂, PO₂, and temperature were controlled; only animals with normal values were included in subsequent analyses. Ischemic LDF was <45% of baseline in all rat protocols, confirming adequate occlusion.⁴⁰ In mice, intraischemic neurologic deficit scores were similar in all groups (P=0.37; ANOVA): E2-treated ERKO mice 2.6±0.5; vehicle-treated ERKO mice 2.7±0.2; E2-treated WT mice 2.0±0; vehicle-treated WT mice 2.3±0.2. Plasma E2 levels were 4±1 pg/mL in placebo-treated and 19±1 pg/mL in E2-treated rats (P<0.001), consistent with metestrus/diestrus values.⁴⁷ In WT mice, plasma E2 was 18±3 pg/mL and 263±79 pg/mL in vehicle- and E2-treated animals, respectively (P=0.021). In ERKO mice, plasma E2 was 14±1 pg/mL and 344±239 pg/mL in vehicle- and E2-treated animals, respectively (P=0.295).

E2 Selectively Increases Gene Expression in the Angiopoietin Family Through ER
Ang-1 mRNA was induced by E2 but not by ischemia (Figure 2). In E2-treated rats, Ang-1 mRNA levels were decreased in ischemic hemispheres. Ang-2 mRNA was induced by ischemia but not by E2 (Figure 3). Other angiopoietin and VEGF family members were not affected by E2 or ischemia (Table 1). To determine whether Ang-1 induction was mediated by ER, we evaluated Ang-1 expression in ERKO mice. E2 increased Ang-1 expression in WT ovariectomized mice. However, E2-mediated Ang-1 expression was not observed in ERKO females (Figure 4).

View larger version (29K): [in this window] [in a new window]   Figure 2. Brain Ang-1 mRNA was quantified in placebo- vs E2-treated ovariectomized female rats. RNA was isolated from nonischemic (left) and ischemic (right) brain hemispheres after 22 hours of reperfusion; both hemispheres were pooled for shams. *P=0.011; E2-treated sham vs placebo-treated sham; 2-factor ANOVA; Holm–Sidak multiple comparison method.

View larger version (28K): [in this window] [in a new window]   Figure 3. Brain Ang-2 mRNA was quantified in placebo- vs E2-treated ovariectomized female rats. RNA was isolated from nonischemic (left) and ischemic (right) brain hemispheres after 22 hours of reperfusion; both hemispheres were pooled for shams. *P=0.001; ischemic vs nonischemic; 1-way ANOVA.

View this table: [in this window] [in a new window]   TABLE 1. Tie-1, Tie-2, VEGF, VEGF R1, and VEGF R2 Are Unaffected by E2 or Ischemia/Reperfusion in Ovariectomized Female Rats

View larger version (28K): [in this window] [in a new window]   Figure 4. Whole-brain Ang-1 mRNA was quantified in placebo- vs E2-treated ovariectomized female WT and ERKO mice. *P=0.005; WT/E2 vs all other conditions; 1-way ANOVA.

Brain Capillary Density Is Higher in Rats Treated With E2
The brain regions selected for study (basal ganglia and parasaggital and parietal cortex) are directly involved in, or are in proximity to, regions injured in MCAO (Figure 1).

In nonischemic rats, E2 treatment resulted in increased capillary density in the basal ganglia and parietal cortex but not in the parasaggital cortex (Table 2). Ischemic hemisphere/nonischemic hemisphere capillary density ratio in postischemic rats was 1 in placebo-treated and E2-treated animals 22 hours after stroke (data not shown).

View this table: [in this window] [in a new window]   TABLE 2. Cerebral Microvessel Density in Nonischemic Rats 1 Week After Ovariectomy and Implantation of Placebo- or E2-Releasing Pellets

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study demonstrates 3 important findings. First, therapy with E2 selectively increases Ang-1 mRNA in ovariectomized female rat brain. Second, induction of Ang-1 is mediated by ER. Third, capillary density is higher in brains of nonischemic animals treated with E2 compared with placebo. Ang-1 stabilizes microvascular networks and reduces vascular permeability. We speculate that E2-mediated augmentation of Ang-1 expression and enhanced microvascular structure and function before experimental stroke may prime the brain for survival after stroke.

E2 modulates angiogenesis (ie, alters microvascular network anatomy, endothelial cell function, and expression of angiogenesis markers) in uterine and nonreproductive tissues.⁸ E2 enhances endothelial cell proliferation, migration, and survival in vitro.^48,49 Some of the angiogenic effects of E2 are ER dependent.⁵⁰ In the brain, E2 enhances cerebral capillary density in senescent animals.¹⁴ Our finding that E2 increases brain Ang-1 mRNA through ER and enhances capillary density in young adult rodent brain before ischemia compliments this large body of evidence. The higher capillary density in E2-treated animals may result from new capillary generation or stabilization of existing capillaries through Ang-1.³⁴

Numerous reports indicate that VEGF transcription is sensitive to E2 and ischemia.^8,9,39,51,52 We did not detect a VEGF response to E2. VEGF induction is E2 dose dependent,⁵¹ and it is plausible that the metestrus plasma E2 levels in the rats were not sufficiently high to affect VEGF expression. We also did not observe a VEGF response to ischemia. We may have missed the peak of induction because VEGF increases at 3 hours after cerebral ischemia.^27,53 Timing may also explain our not detecting ischemic induction of Tie-1 and Tie-2.²⁹ An additional explanation is the low sensitivity of our approach for detecting molecular changes in small brain regions (eg, the ischemic penumbra). By harvesting mRNA from the entire hemisphere, we dilute the mRNA, and only the most robust changes, such as the ischemic induction of Ang-2, are detectable.^29,54

E2-mediated angiogenesis and protection in stroke are absent in ERKO mice.^7,55 Our finding that E2-mediated induction of Ang-1 is attenuated in ERKO mice suggests that this angiogenic factor is important for the ability of the steroid to promote or maintain vascularization and may be an important link between E2, angiogenesis or vascular stability, and protection from stroke. Zhang et al introduced Ang-1 acutely into male mice and observed attenuated cerebral edema because of a reduction in vascular permeability and decreased stroke size.³³ We hypothesize that chronic E2 treatment before stroke stabilizes or augments the cerebral vasculature via elevation of Ang-1 and enhancement of tissue resistance to ischemic damage through augmentation of perfusion within the ischemic territory during experimental stroke.⁴⁰

Based on the results of the current experiments, Ang-1 emerges as a candidate molecule for E2-mediated neuroprotection in experimental transient focal cerebral ischemia. This hypothesis must be tested directly because the results may be applicable to the clinical arena, specifically to patients with symptomatic intracranial vascular stenosis not amenable to standard treatments.

	Acknowledgments

 
This research was supported by the following grants: NIH NS33668 and NR03521 to P.D.H. The authors wish to thank Judith Klaus, Leslie Spencer, and Kathleen Kibler for expert technical assistance, and Tzipora Sofare, MA, for editorial assistance with an earlier version of this manuscript.

	Footnotes

 
L.D.M. is now at the Department of Neurology, University of Connecticut Health Center, Farmington, Conn; M.M.W. is now at the Department of Neurology and Physiology, University of Michigan, Ann Arbor; and P.D.H. is now at the Department of Anesthesiology and Perioperative Medicine, Oregon Health Sciences University, Portland.

Received August 11, 2004; revision received October 26, 2004; accepted November 15, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the women’s health initiative randomized controlled trial. J Am Med Assoc. 2002; 288: 321–333.[Abstract/Free Full Text]

2. Anderson GL, Limacher M, Assaf AR, Bassford T, Beresford SA, Black H, Bonds D, Brunner R, Brzyski R, Caan B, Chlebowski R, Curb D, Gass M, Hays J, Heiss G, Hendrix S, Howard BV, Hsia J, Hubbell A, Jackson R, Johnson KC, Judd H, Kotchen JM, Kuller L, LaCroix AZ, Lane D, Langer RD, Lasser N, Lewis CE, Manson J, Margolis K, Ockene J, O’Sullivan MJ, Phillips L, Prentice RL, Ritenbaugh C, Robbins J, Rossouw JE, Sarto G, Stefanick ML, Van Horn L, Wactawski-Wende J, Wallace R, Wassertheil-Smoller S. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the women’s health initiative randomized controlled trial. J Am Med Assoc. 2004; 291: 1701–1712.[Abstract/Free Full Text]

3. Hurn PD, Brass LM. Estrogen and stroke: a balanced analysis. Stroke. 2003; 34: 338–341.[Free Full Text]

4. Hurn PD, Macrae IM. Estrogen as a neuroprotectant in stroke. J Cereb Blood Flow Metab. 2000; 20: 631–652.[CrossRef][Medline] [Order article via Infotrieve]

5. McCullough LD, Hurn PD. Estrogen and ischemic neuroprotection: an integrated view. Trends Endocrinol Metab. 2003; 14: 228–235.[CrossRef][Medline] [Order article via Infotrieve]

6. Murphy S, McCullough L, Littleton-Kearney M, Hurn P. Estrogen and selective estrogen receptor modulators: neuroprotection in the women’s health initiative era. Endocrine. 2003; 21: 17–26.[CrossRef][Medline] [Order article via Infotrieve]

7. Dubal DB, Zhu H, Yu J, Rau SW, Shughrue PJ, Merchenthaler I, Kindy MS, Wise PM. Estrogen receptor alpha, not beta, is a critical link in estradiol-mediated protection against brain injury. Proc Natl Acad Sci U S A. 2001; 98: 1952–1957.[Abstract/Free Full Text]

8. Losordo DW, Isner JM. Estrogen and angiogenesis. Arterioscl Thromb Vasc Biol. 2001; 21: 6–12.[Abstract/Free Full Text]

9. Rubanyi GM, Johns A, Kauser K. Effect of estrogen on endothelial function and angiogenesis. Vasc Pharmacol. 2002; 38: 89–98.[CrossRef]

10. Suzuma I, Mandai M, Takagi H, Suzuma K, Otani A, Oh H, Kobayashi K, Honda Y. 17 beta-estradiol increases vegf receptor-2 and promotes DNA synthesis in retinal microvascular endothelial cells. Invest Ophthalmol Visual Sci. 1999; 40: 2122–2129.[Abstract/Free Full Text]

11. Ye F, Florian M, Magder SA, Hussain SNA. Regulation of angiopoietin and Tie-2 receptor expression in non-reproductive tissues by estrogen. Steroids. 2002; 67: 305–310.[CrossRef][Medline] [Order article via Infotrieve]

12. Perrot-Applanat M, Ancelin M, Buteau-Lozano H, Meduri G, Bausero P. Ovarian steroids in endometrial angiogenesis. Steroids. 2000; 65: 599–603.[CrossRef][Medline] [Order article via Infotrieve]

13. Kyriakides Z, Petinakis P, Kaklamanis L, Sbarouni E, Karayannakos P, Iliopoulos D, Dontas I, Kremastinos D. Intramuscular administration of estrogen may promote angiogenesis and perfusion in a rabbit model of chronic limb ischemia. Cardiovasc Res. 2001; 49: 626–633.[Abstract/Free Full Text]

14. Jesmin S, Hattori Y, Sakuma I, Liu M-Y, Mowa CN, Kitabatake A. Estrogen deprivation and replacement modulate cerebral capillary density with vascular expression of angiogenic molecules in middle-aged female rats. J Cereb Blood Flow Metab. 2003; 23: 181–189.[CrossRef][Medline] [Order article via Infotrieve]

15. Liebeskind DS. Collateral circulation. Stroke. 2003; 34: 2279–2284.[Abstract/Free Full Text]

16. Fujita M, Tambara K. Recent insights into human coronary collateral development. Heart. 2004; 90: 246–250.[Abstract/Free Full Text]

17. Koyama T, Xie Z, Gao M, Suzuki J, Batra S. Adaptive changes in the capillary network in the left ventricle of rat heart. Jpn J Physiol. 1998; 48: 229–241.[CrossRef][Medline] [Order article via Infotrieve]

18. Tyagi SC. Vasculogenesis and angiogenesis: extracellular matrix remodeling in coronary collateral arteries and the ischemic heart. J Cell Biochem. 1997; 65: 388–394.[CrossRef][Medline] [Order article via Infotrieve]

19. Schaper W. Angiogenesis in the adult heart. Basic Res Cardiol. 1991; 86 (suppl 2): 51–56.[Medline] [Order article via Infotrieve]

20. Issa R, Krupinski J, Bujny T, Kumar S, Kaluza J, Kumar P. Vascular endothelial growth factor and its receptor, KDR, in human brain tissue after ischemic stroke. Lab Invest. 1999; 79: 417–425.[Medline] [Order article via Infotrieve]

21. Krupinski J, Kaluza J, Kumar P, Kumar S, Wang JM. Role of angiogenesis in patients with cerebral ischemic stroke. Stroke. 1994; 25: 1794–1798.[Abstract]

22. Lin T, Sun S, Cheung W, Li F, Chang C. Dynamic changes in cerebral blood flow and angiogenesis after transient focal cerebral ischemia in rats. Evaluation with serial magnetic resonance imaging. Stroke. 2002; 33: 2985–2991.[Abstract/Free Full Text]

23. Szpak G, Lechowicz W, Lewandowska E, Bertrand E, Wierzba-Bobrowicz T, Dymecki J. Border zone neovascularization in cerebral ischemic infarct. Folia Neuropathol. 1999; 37: 264–268.[Medline] [Order article via Infotrieve]

24. Wei L, Erinjeri JP, Rovainen CM, Woolsey TA. Collateral growth and angiogenesis around cortical stroke. Stroke. 2001; 32: 2179–2184.[Abstract/Free Full Text]

25. Zhang ZG, Zhang L, Jiang Q, Zhang R, Davies K, Powers C, Bruggen Nv, Chopp M. VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain. J Clin Invest. 2000; 106: 829–838.[Medline] [Order article via Infotrieve]

26. Zhang Z, Zhang L, Tsang W, Soltanian-Zadeh H, Morris D, Zhang R, Goussev A, Powers C, Yeich T, Chopp M. Correlation of VEGF and angiopoietin expression with disruption of blood-brain barrier and angiogenesis after focal cerebral ischemia. J Cereb Blood Flow Metab. 2002; 22: 379–392.[Medline] [Order article via Infotrieve]

27. Hayashi T, Abe K, Suzuki H, Itoyama Y. Rapid induction of vascular endothelial growth factor gene expression after transient middle cerebral artery occlusion in rats. Stroke. 1997; 28: 2039–2044.[Abstract/Free Full Text]

28. Lin TN, Nian GM, Chen SF, Cheung WM, Chang C, Lin WC, Hsu CY. Induction of Tie-1 and Tie-2 receptor protein expression after cerebral ischemia-reperfusion. J Cereb Blood Flow Metab. 2001; 21: 690–701.[CrossRef][Medline] [Order article via Infotrieve]

29. Lin T-N, Wang C-K, Cheung W-M, Hsu C-Y. Induction of angiopoietin and Tie receptor mRNA expression after cerebral ischemia-reperfusion. J Cereb Blood Flow Metab. 2000; 20: 387–395.[CrossRef][Medline] [Order article via Infotrieve]

30. Zhang ZG, Chopp M, Lu D, Wayne T, Zhang RL, Morris D. Receptor tyrosine kinase Tie 1 mRNA is upregulated on cerebral microvessels after embolic middle cerebral artery occlusion in the rat. Brain Res. 1999; 847: 338–342.[CrossRef][Medline] [Order article via Infotrieve]

31. Risau W. Mechanisms of angiogenesis. Nature. 1997; 386: 671–674.[CrossRef][Medline] [Order article via Infotrieve]

32. Gale NW, Yancopoulos GD. Growth factors acting via endothelial cell-specific receptor kinases: VEGFs, angiopoietins, and ephrins in vascular development. Genes Dev. 1999; 13: 1055–1066.[Free Full Text]

33. Zhang Z, Zhang L, Croll S, Chopp M. Angiopoietin-1 reduces cerebral blood vessel leakage and ischemic lesion volume after focal cerebral embolic ischemia in mice. Neuroscience. 2002; 113: 683–687.[CrossRef][Medline] [Order article via Infotrieve]

34. Thurston G. Complementary actions of VEGF and angiopoietin-1 on blood vessel growth and leakage. J Anat. 2002; 200: 575–580.[CrossRef][Medline] [Order article via Infotrieve]

35. Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClain J, Aldrich TH, Papadopoulos N, Daly TJ, Davis S, Sato TN, Yancopoulos GD. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science. 1997; 277: 55–60.[Abstract/Free Full Text]

36. Asahara T, Chen D, Takahashi T, Fujikawa K, Kearney M, Magner M, Yancopoulos GD, Isner JM. Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization. Circ Res. 1998; 83: 233–240.[Abstract/Free Full Text]

37. Marron MB, Hughes DP, Edge MD, Forder CL, Brindle NP. Evidence for a heterotypic interaction between the receptor tyrosine kinases TIE-1 and TIE-2. J Biol Chem. 2000; 275: 39741–39746.[Abstract/Free Full Text]

38. Clauss M. Molecular biology of the VEGF and the VEGF receptor family. Semin Thromb Hemostasis. 2000; 26: 561–569.[CrossRef][Medline] [Order article via Infotrieve]

39. Breier G. Functions of the VEGF/VEGF receptor system in the vascular system. Semin Thromb Hemostasis. 2000; 26: 553–559.[CrossRef][Medline] [Order article via Infotrieve]

40. Alkayed NJ, Harukumi I, Kimes AS, London ED, Traystman RJ, Hurn PD. Gender-linked brain injury in experimental stroke. Stroke. 1998; 29.

41. Rusa R, Alkayed N, Crain B, Traystman R, Kimes A, London E, Klaus J, Hurn P. 17beta-estradiol reduces stroke injury in estrogen-deficient female animals. Stroke. 1999; 30: 1665–1670.[Abstract/Free Full Text]

42. Sampei K, Goto S, Alkayed N, Crain B, Korach K, Traystman R, Demas G, Nelson R, Hurn P. Stroke in estrogen receptor-alpha-deficient mice. Stroke. 2000; 31: 738–744.[Abstract/Free Full Text]

43. McCullough LD, Blizzard K, Simpson ER, Oz OK, Hurn PD. Aromatase cytochrome p450 and extragonadal estrogen play a role in ischemic neuroprotection. J Neurosci. 2003; 23: 8701–8705.[Abstract/Free Full Text]

44. Longa E, Weinstein P, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989; 20: 84–91.[Abstract/Free Full Text]

45. Wang MM, Klaus JA, Joh H-D, Traystman RJ, Hurn PD. Postischemic angiogenic factor expression in stroke-prone rats. Exp Neurol. 2002; 173: 283–288.[CrossRef][Medline] [Order article via Infotrieve]

46. Paxinos G, Watson C. The Rat Brain in Stereotactic Coordinates. Sydney, Australia: Academic Press; 1982.

47. Brown-Grant K, Exley D, Naftolin F. Peripheral plasma oestradiol and luteinizing hormone concentrations during the oestrus cycle of the rat. J Endocrinol. 1970; 48: 295–296.[Abstract/Free Full Text]

48. Razandi M, Pedram A, Levin ER. Estrogen signals to the preservation of endothelial cell form and function. J Biol Chem. 2000; 275: 38540–38546.[Abstract/Free Full Text]

49. Alvarez RJ, Gips SJ, Moldovan N, Wilhide CC, Milliken EE, Hoang AT, Hruban RH, Silverman HS, Dang CV, Goldschmidt-Clermont PJ. 17beta-estradiol inhibits apoptosis of endothelial cells. Biochem Biophys Res Commun. 1997; 237: 372–381.[CrossRef][Medline] [Order article via Infotrieve]

50. Morales DE, McGowan KA, Grant DS, Maheshwari S, Bhartiya D, Cid MC, Kleinmna HK, Schnapper HW. Estrogen promotes angiogenic activity in human umbilical vein endothelial cells in vitro and in a murine model. Circulation. 1995; 91: 755–763.[Abstract/Free Full Text]

51. Hyder SM, Huang J-C, Nawaz Z, Boettger-Tong H, Makela S, Chiappetta C, Stancel GM. Regulation of vascular endothelial growth factor expression by estrogens and progestins. Environ Health Perspect. 2000; 108: 785–790.[Medline] [Order article via Infotrieve]

52. Mueller MD, Vigne JL, Minchenko A, Lebovic DI, Leitman DC, Taylor RN. Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors alpha and beta. Proc Natl Acad Sci U S A. 2000; 97: 10972–10977.[Abstract/Free Full Text]

53. Plate K, Beck H, Danner S, Allegrini P, Wiessner C. Cell type specific upregulation of vascular endothelial growth factor in an MCA-occlusion model of cerebral infarct. J Neuropathol Exp Neurol. 1999; 58: 654–666.[Medline] [Order article via Infotrieve]

54. Beck H, Acker T, Wiessner C, Allegrini P, Plate K. Expression of angiopoietin-1, angiopoietin-2, and Tie receptors after middle cerebral artery occlusion in the rat. Am J Pathol. 2000; 157: 1473–1483.[Abstract/Free Full Text]

55. Johns A, Freay AD, Fraser W, Korach KS, Rubanyi GM. Disruption of estrogen receptor gene prevents 17beta estradiol-induced angiogenesis in transgenic mice. Endocrinology. 1996; 137: 4511–4513.[Abstract]

This article has been cited by other articles:

				D. P. Schafer, S. Jha, F. Liu, T. Akella, L. D. McCullough, and M. N. Rasband Disruption of the Axon Initial Segment Cytoskeleton Is a New Mechanism for Neuronal Injury J. Neurosci., October 21, 2009; 29(42): 13242 - 13254. [Abstract] [Full Text] [PDF]

				A. Cignarella, C. Bolego, V. Pelosi, C. Meda, A. Krust, C. Pinna, R. M. Gaion, E. Vegeto, and A. Maggi Distinct Roles of Estrogen Receptor-{alpha} and {beta} in the Modulation of Vascular Inducible Nitric-Oxide Synthase in Diabetes J. Pharmacol. Exp. Ther., January 1, 2009; 328(1): 174 - 182. [Abstract] [Full Text] [PDF]

				O. V. Glinskii, T. W. Abraha, J. R. Turk, L. J. Rubin, V. H. Huxley, and V. V. Glinsky Microvascular network remodeling in dura mater of ovariectomized pigs: role for angiopoietin-1 in estrogen-dependent control of vascular stability Am J Physiol Heart Circ Physiol, August 1, 2007; 293(2): H1131 - H1137. [Abstract] [Full Text] [PDF]

				D. N. Krause, S. P. Duckles, and D. A. Pelligrino Influence of sex steroid hormones on cerebrovascular function J Appl Physiol, October 1, 2006; 101(4): 1252 - 1261. [Abstract] [Full Text] [PDF]

				C. Bolego, E. Vegeto, C. Pinna, A. Maggi, and A. Cignarella Selective Agonists of Estrogen Receptor Isoforms: New Perspectives for Cardiovascular Disease Arterioscler Thromb Vasc Biol, October 1, 2006; 26(10): 2192 - 2199. [Abstract] [Full Text] [PDF]

				D. B. Dubal, S. W. Rau, P. J. Shughrue, H. Zhu, J. Yu, A. B. Cashion, S. Suzuki, L. M. Gerhold, M. B. Bottner, S. B. Dubal, et al. Differential Modulation of Estrogen Receptors (ERs) in Ischemic Brain Injury: A Role for ER{alpha} in Estradiol-Mediated Protection against Delayed Cell Death Endocrinology, June 1, 2006; 147(6): 3076 - 3084. [Abstract] [Full Text] [PDF]

				A. Alonso, R. Fernandez, M. Moreno, P. Ordonez, H. Gonzalez-Pardo, N. M. Conejo, F. Diaz, and C. Gonzalez Positive Effects of 17{beta}-Estradiol on Insulin Sensitivity in Aged Ovariectomized Female Rats. J. Gerontol. A Biol. Sci. Med. Sci., May 1, 2006; 61(5): 419 - 426. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 36/2/337    most recent 01.STR.0000153795.38388.72v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ardelt, A. A. Articles by Hurn, P. D. Search for Related Content PubMed PubMed Citation Articles by Ardelt, A. A. Articles by Hurn, P. D. Pubmed/NCBI databases Gene GEO Profiles HomoloGene Protein UniGene Compound via MeSH Substance via MeSH Hazardous Substances DB ESTRADIOL Medline Plus Health Information Stroke Related Collections Angiogenesis Animal models of human disease Brain Circulation and Metabolism Other Vascular biology