Alteration of thrombospondin-1 and -2 in rat brains following experimental intracerebral hemorrhage

Laboratory investigation

Full access

Object

Spontaneous intracerebral hemorrhage (ICH) is among the most intractable forms of stroke. Angiogenesis, an orchestrated balance between proangiogenic and antiangiogenic factors, is a fundamental process to brain development and repair by new blood vessel formation from preexisting ones and can be induced by ICH. Thrombospondin (TSP)–1 and TSP-2 are naturally occurring antiangiogenic factors. The aim of this study was to observe their expression in rat brains with ICH.

Methods

Intracerebral hemorrhage was induced in adult male Sprague-Dawley rats by stereotactic injection of collagenase VII or autologous blood into the right globus pallidus. The expression of TSP-1 and -2 was evaluated by immunohistochemistry and quantitative real-time reverse transcription–polymerase chain reaction analysis.

Results

After the induction of ICH, some TSP1- or TSP2-immunoreactive microvessels resided around the hematoma for ~ 7 days and extended into a clot thereafter. Cerebral endothelial cells expressed the TSPs. The expression of TSP-1 and TSP-2 mRNA peaked at 4 and 14 days after collagenase-induced ICH, respectively.

Conclusions

Findings in this study suggest that ICH can alter the expression of TSP-1 and TSP-2, which may be involved in modulating angiogenesis in brains following ICH.

Abbreviations used in this paper: Ang-1 = angiopoietin-1; EC = endothelial cell; ICH = intracerebral hemorrhage; MMP-2 = matrix metalloproteinase-2; RT-PCR = reverse transcription–polymerase chain reaction; TSP = thrombospondin; VEGF = vascular endothelial growth factor; vWF = von Willebrand factor.

Spontaneous ICH represents 15% of all strokes in Western populations, with a higher proportion (20–30%) in Asian and black populations, and is the most intractable form of stroke with high morbidity and mortality rates.20 Currently, no effective medical therapy is available for victims, except for supportive care or invasive neurosurgical evacuation of a hematoma, although considerable efforts have been devoted to developing treatments.1,18 A controlled trial has indicated that patients with ICH show no overall benefit from early surgery in comparison with medical treatment.18 Hence, to improve neurological recovery from an attack, alternative approaches are eagerly awaited.6

As blood is the only source of glucose and oxygen for the brain, angiogenesis is fundamental to brain development and repair. Clinical data have suggested that angiogenesis is beneficial to the functional recovery of victims of ischemic stroke.11 It is well known that angiogenesis, which refers to the expansion or remodeling of preexisting blood vessels,30 depends on the net effect of a variety of proangiogenic and antiangiogenic molecules, including growth factors and components of extracellular matrix.7 Moreover, antiangiogenic factors participate in several indispensible steps to form new vasculature, such as the initiation of angiogenesis by an angiogenic switch, the inhibition of excessive growth by apoptosis induction, and the remodeling of vasculature by pruning.5,22

Our recent studies have proved that ICH can induce angiogenesis in rat brains with the upregulation of proangiogenic factors including VEGF, Ang-1, and their receptors.26,32 To our knowledge, however, the expression of antiangiogenic factors following ICH has rarely been studied. Among the major antiangiogenic factors, TSP-1 and TSP-2 have gained increasing importance in recent studies. Therefore, to understand the mechanisms of angiogenesis induced by ICH further, in the present study we determined whether the expression of TSP-1 and TSP-2 mRNA was altered in rat brains with collagenaseinduced ICH.

Methods

Animal Preparation

Studies were performed on adult male Sprague-Dawley rats (250–300 g, 8–10 weeks of age) obtained from the Experimental Animal Science Center of Central South University. All animals were housed under identical conditions (room temperature 25°C, 12-hour light-dark cycle) and allowed free access to food and water. The experimental protocol complied with guidelines of Central South University and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80–23) and was approved by the Institutional Animal Care and Use Committee of Central South University. Rats undergoing collagenase-induced ICH were randomly assigned to a sham-operated control group (8 rats per time point) or an ICH group (8 rats per time point); and rats undergoing autologous blood–induced ICH were randomly assigned to a sham-operated control group (3 rats per time point) and an ICH group (3 rats per time point).

Induction of ICH

Intracerebral hemorrhage was induced with collagenase as described elsewhere.26 After fasting for a night, animals were anesthetized intraperitoneally with chloral hydrate (400 mg/kg) and then fixed pronely on a stereotactic frame (Stoelting Co.). Following a scalp incision, a small cranial bur hole was drilled near the right coronal suture 3.2 mm lateral to the midline. Bacterial collagenase (0.5 U, Type VII, Sigma Aldrich Co.) in 2.5 μl 0.9% sterile saline was injected into the right globus pallidus (1.4 mm posterior and 3.2 mm lateral to the bregma, 5.6 mm ventral to the cortical surface) over 5 minutes by using a 5-μl Hamilton syringe, and the needle was left there for 5 minutes. The bone hole was sealed with bone wax, the scalp wound was sutured, and each animal was placed in a warm box to recover. In the sham group, 2.5 μl 0.9% sterile saline without the collagenase was injected into the same site. During the procedure, each animal's rectum temperature was monitored and maintained at 37.5°C with a feedback-controlled heating pad.

For autologous blood–induced ICH, fresh nonheparinized blood (100 μl) from the femoral artery was injected into the right globus pallidus over 2 minutes using a 26-gauge needle, and the needle was slowly removed over another 10 minutes to prevent backflow. Needle insertion alone was performed in the sham controls.16,17

Specimen Preparation

Randomly chosen animals from the 2 ICH-induction groups were deeply anesthetized with chloral hydrate (800 mg/kg) at 2, 4, 7, 14, 21, and 28 days postoperation. For immunohistochemistry, either collagenase-induced or autologous blood–induced ICH animals (3 rats per time point) were transcardially perfused with 0.9% saline followed by 250 ml ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Removed brains were postfixed in the same fixative for 2 hours and sequentially transferred to 20% and then 30% sucrose in 0.1 M phosphate buffer (pH 7.4) at 4°C until the brains sank in the solution. Brain tissue sections (30 μm) at −20°C were coronally cut with a cryostat (CM1900, Leica Co.); some sections were collected in 0.01 M phosphate-buffered saline (pH 7.4) and stored at 4°C. For RT-PCR, collagenase-induced ICH rats (5 animals per time point) were killed by decapitation, their brains were immediately removed, and the tissues in the striatum adjacent to the hematoma and without the needle track were dissected and frozen at –196°C in liquid nitrogen.

Immunohistochemical Analysis

Sections were brought to room temperature and incubated in 3% H2O2 for 15 minutes. After washing 3 times in phosphate-buffered saline for 5 minutes each, nonspecific binding was blocked in 5% bovine serum albumin (Sigma Aldrich Co.) for 1 hour at 37°C. Sections were then incubated overnight at 4°C with goat anti–TSP-1 (1:200, Santa Cruz Biotech) or goat anti–TSP-2 (1:200, Santa Cruz Biotech), with a biotinylated anti–goat immunoglobulin G (1:200) for 1 hour, and then with avidin-biotin-peroxidase complex (1:100, Vector Laboratories) for 1 hour at 37°C. Immunoreactivity was visualized with diaminobenzidine (Boster Biotech Co.).

To determine whether TSPs were expressed in ECs, immunofluorescence double labeling was performed. Tissue sections were first incubated at 4°C for 48 hours with a mixture of 2 primary antibodies: rabbit anti–vWF (a marker for ECs, 1:200, Chemicon International) and either of the goat anti–TSPs (1:100). The following secondary antibodies were used: fluorescein isothiocyanate–conjugated donkey anti–rabbit antibody (1:50, Santa Cruz Biotechnology) for vWF detection and rhodamine-conjugated donkey anti–goat antibody (1:100, Santa Cruz Biotechnology) for TSP detection. These sections were scanned using a laser confocal microscope (LSM-510, Zeiss).

For a negative control, 1% bovine serum albumin was used instead of the primary antibody.

Quantitative Real-Time RT-PCR

Total RNA was purified from 100 mg of tissue near the hematoma in each animal group by using TRIzol reagent (Invitrogen Co.). The integrity of the total RNA was detected using agarose gel; the purity and concentration were detected using a spectrophotometer (UV-1201, Shimadzu). Reverse transcription was performed on 2 μg of total RNA using 1 μg/μl oligo(dT)-18 (1 μl), 10 mM dNTPs Mix (2 μl), RNase inhibitor (1 μl), and 200 U/μl Moloney murine leukemia virus (M-MuLV) reverse transcriptase (1 μl) at 70°C for 5 minutes, 37°C for 5 minutes, 42°C for 60 minutes, and 70°C for 10 minutes consecutively, according to the manufacturer's instructions (Fermentas), and then cDNA was stored at –20°C. Amplification was performed using a SYBR Premix Ex Taq PCR kit (4 μl of 1:2 cDNA dilution was used, Takara Bio Inc.) in a LightCycler Real-Time Detection System (Roche Diagnostics Limited), and the following protocol was used: 10 seconds at 95°C, 30–40 cycles of 5 seconds at 95°C, 20 seconds at 52°C, 10 seconds at 72°C, and a melting temperature of 60°C. Primers for TSP-1, TSP-2, and β-actin were designed with Primer Premier 5.0 software for rats (Premier Biosoft International), listed as follows: TSP-1, sense 5′-AACGTGGATCAGAGGGACAC-3′ and antisense 5′-GTCATCGTCATGGTCACAGG-3′; TSP-2, sense 5′-CGTCACCAAGGCAAAGAG-3′ and antisense 5′-CACCAGAGTAGCCGTAAGC-3′; β-actin, sense 5′-CGTTGACATCCGTAAAGAC-3′ and antisense 5′-TGGAAGGTGGACAGTGAG-3′. Melting curves of all samples were always performed as controls for specificity. All gene expression data were calculated as 2–ΔΔCT, which indicated an n-fold change in gene expression relative to the sham control sample.15

Statistical Analysis

All data were expressed as the means ± SDs. An ANOVA was used to compare the means.

Results

Temporospatial Profile of TSP1- and TSP2-Immunoreactive Microvessels After ICH

To determine where the expression of TSP-1 and TSP-2 was changed in ICH-affected brains, immunohistochemistry was performed. In sham-operated animals, few slim microvessels of immunoreactivity were observed in either hemisphere (Fig. 1A and B). After ICH induction by collagenase, many TSP1- and TSP2-immunoreactive microvessels with a dilated outline were detected in perihematomal tissue (Fig. 1C and D), and the vessels extended into the clot from the surrounding area at ~ 7 days (Fig. 1E and F) and then appeared in the clot thereafter (Fig. 1G and H).

Fig. 1.
Fig. 1.

Immunohistochemistry for TSP-1 and TSP-2 after ICH. Photomicrographs demonstrating a few slim microvessels of TSP-1 or TSP-2 immunoreactivity in sham-operated animals (A, B, I, and J). After ICH induction by either collagenase or autologous blood, many enlarged and thin-walled microvessels of TSP-1 and TSP-2 immunoreactivity were detected around the hematoma (C, D, K, and L), and the vessels appeared along the border of the clot at ~ 7 days (E, F, M, and N), then approached the core afterward (G, H, O, and P). By the double-labeling method, it was revealed that immunoreactivity (Q) for TSP-1 (red) and TSP-2 (red) was overlapped with vWF (green). Arrows indicate microvessels of corresponding immunoreactivity. Inset: The red disc represents the hematoma; the 3 rectangles indicate where the pictures were taken in relation to the hematoma; and the dashed lines mark the border of the hematoma. Diaminobenzidene stain (A–P), fluorescence of fluorescein isothiocyanate (green, Q), and rhodamine (red, Q). Bar = 100 μm (A–P), 50 μm (Q).

To make clear whether the changes could be attributed to the hemorrhage, we investigated them in autologous blood–induced ICH animals as well. In sham controls, slim microvessels with slight staining were occasionally found in both hemispheres (Fig. 1I and J). After autologous blood infusion at the basal ganglion, a few enlarged and thin-walled microvessels of TSP-1 and TSP-2 immunoreactivity appeared in the perihematomal tissue (Fig. 1K and L), and the microvessels resided along the border of the clot at ~ 7 days (Fig. 1M and N), which gradually approached the core afterward (Fig. 1O and P).

By the double-labeling method, the expression of TSP-1 and TSP-2 was primarily located in the vWF-immunoreactive ECs (Fig. 1Q).

Expression of TSP-1 and TSP-2 mRNA After ICH

Because it was demonstrated through immunohistochemistry that the expression of TSP-1 and TSP-2 changed mainly around the hematoma, we evaluated by RT-PCR their mRNA expression at the basal ganglion ipsilateral to the collagenase-induced ICH. In sham-operated rats, TSP-1 and TSP-2 mRNA were hardly detected. However, TSP-1 (Fig. 2A) and TSP-2 (Fig. 2B) mRNA expression increased continuously after ICH induction; TSP-1 mRNA peaked at 4 days, whereas TSP-2 mRNA peaked at 14 days after ICH induction.

Fig. 2.
Fig. 2.

Bar graphs demonstrating quantitative analysis of TSP-1 and TSP-2 mRNA after collagenase-induced ICH. Both TSP-1 (A) and TSP-2 (B) mRNA expression rose continuously; TSP-1 mRNA peaked at 4 days, whereas TSP-2 mRNA peaked at 14 days. Data are presented as mean ± SD, with 5 rats per time point. *p < 0.01.

Discussion

We have proved that ICH induces angiogenesis, particularly in the perihematomal region.26 In the present study, histological analysis showed that TSP1- and TSP2-immunoreactive microvessels surrounded and then extended into the clot after the stroke, and that ECs expressed the TSPs. Moreover, the temporospatial profile is quite similar to that of the newborn vessels or the proangiogenic factors like VEGF and Ang-1 after ICH.26,32 Therefore, the data support the notion that the alteration of TSP-1 and TSP-2 probably has a close relationship with angiogenesis in rat brains following hemorrhagic stroke.

Thrombospondin-1, a major component of platelet α-granules,2,3 is a 450-kD multifunctional extracellular matrix glycoprotein produced by a variety of cells, including ECs, monocytes/macrophages, and smooth-muscle cells.21,24 It is known as the first discovered and characterized endogenous inhibitor of angiogenesis.8 After ICH, the striking increase of TSP-1 mRNA at the early stage may reflect an initial attempt of antiangiogenic drives to impair EC growth and migration and to act on cell cycle progression and apoptosis.14,27 Besides, it has been reported that TSP-1 could block VEGF-induced angiogenesis,10,25 so the change of TSP-1 in hemorrhagic brains may confer a negative-feedback mechanism in angiogenesis.12 However, the phenotypic analysis of TSP-1 knockout animals displayed prolonged healing in response to wounding as well as susceptibility to pulmonary infections,14 which suggested that TSP-1 played a vital role not only in inhibiting angiogenesis but also in controlling infections. As far as the present study is concerned, TSP-1 may increase the recruitment of monocytes into the clot and promote the transformation of monocytes into macrophages, and thus cleaning up the degenerated materials.23

Thrombospondin-2, a close relative of TSP-1, shares a similar domain structure with TSP-1 but has different temporal and spatial distributions in mice.9,28 Thrombospondin-2 is also regarded as an endogenous inhibitor of angiogenesis. Experiments in vitro have documented that TSP-2 can also inhibit EC migration and proliferation,19,29 and that TSP-2 knockouts display an increased density of blood vessels, prolonged bleeding time, increased wound neovascularization, and abnormal tensile strength of the skin.12 The upregulation of TSP-2 mRNA may promote newly formed vessel regression to prevent them from hyperplasia by inhibiting migration and mitogenesis of ECs and antagonizing the proangiogenic effects of some angiogenic factors such as basic fibroblast growth factor.19,29 Our results showed that there were massive newborn vessels in the hemorrhagic basal ganglion at 14 days after ICH.26 In our experiment, TSP-1 mRNA increased dramatically during the early stage of ICH when a low density of new vessels was observed, whereas TSP-2 mRNA expression reached peak at 14 days. This evidence may suggest that the expression of TSP-1 and TSP-2 mRNA appears inversely coupled with the regression of ICHrelated angiogenesis in the late stage. Thrombospondin-2, as a matricellular protein, also plays a primary role in modulating cell-matrix interactions and maintaining matrix integrity. It has been reported that TSP-2 could eliminate proteases from the pericellular environment by binding with MMP-2.31 Conversely, an increased vascularity of subcutaneously implanted sponges associated with increased levels of MMP-2 was detected in TSP2-null mice.13 Accordingly, the upregulation of TSP-2 might reduce the degradation of the extracellular matrix by attenuating levels of MMPs. Therefore, the upregulation of TSP-2 by ICH may contribute to the stabilization of newly formed vessels.

But there is a concern as to whether it is ICH itself that triggered the TSP changes, so we conducted an immunohistological study in rat brains following autologous blood infusion, another widely used ICH model in rodents.17 Our data indicated that both collagenase- and blood-induced ICH share the very similar spatiotemporal profiles of TSPs, which supports that blood plays a pivotal role in TSP expression following ICH.4

Conclusions

Our results demonstrated for the first time that ICH can lead to the upregulation of TSP-1 and TSP-2 with different temporal profiles. But because they belong to multifunctional molecules of angiogenesis, much work may be required to make clear the exact effects of their expression in different time windows during ICH-induced angiogenesis. Moreover, since angiogenesis, a dynamic balance between proangiogenic and antiangiogenic factors, is a moving target, the clinical implications of our findings may be that the modulation of TSP's expression can be taken into account to develop the angiogenesisrelated therapy for ICH, and that a proper therapy should frequently weigh the contributions of both proangiogenic and antiangiogenic factors at certain a stage to enhance the formation of functional vessels.4

Disclosure

The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

The work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 30400581 and 30873221 to T.T.), Chinese Postdoctoral Science Foundation (Grant No. 2005038224 to T.T.), Hunan Provincial Natural Science Foundation (Grant No. 07JJ5007 to T.T.), Hunan Provincial Project of Traditional Chinese Medicine (Grant No. 2008016 to T.T.), and graduate degree thesis Innovation Foundation of Central South University (Grant No. 2008yb048 to H.J.Z.).

Author contributions to the study and manuscript preparation include the following. Conception and design: T Tang. Acquisition of data: HJ Zhou, JH Zhong, Y Qi, JK Luo, Y Lin, QD Yang. Analysis and interpretation of data: HJ Zhou, HN Zhang, Y Qi, XQ Li. Drafting the article: HJ Zhou, JH Zhong. Critically revising the article: T Tang, HN Zhang, JK Luo, Y Lin, QD Yang, XQ Li. Reviewed final version of the manuscript and approved it for submission: T Tang, HJ Zhou, HN Zhang, JH Zhong, Y Qi, JK Luo, Y Lin, QD Yang, XQ Li. Statistical analysis: HJ Zhou, Y Qi. Study supervision: T Tang.

References

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    Andaluz NZuccarello M: Recent trends in the treatment of spontaneous intracerebral hemorrhage: analysis of a nationwide inpatient database. J Neurosurg 110:4034102009

  • 2

    Baenziger NLBrodie GNMajerus PW: Isolation and properties of a thrombin-sensitive protein of human platelets. J Biol Chem 247:272327311972

  • 3

    Baenziger NLBrodie GNMajerus PW: A thrombin-sensitive protein of human platelet membranes. Proc Natl Acad Sci U S A 68:2402431971

  • 4

    Carmeliet P: Manipulating angiogenesis in medicine. J Intern Med 255:5385612004

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    Chavakis EDimmeler S: Regulation of endothelial cell survival and apoptosis during angiogenesis. Arterioscler Thromb Vasc Biol 22:8878932002

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    Christensen MCValiente RSampaio Silva GLee WCDutcher SGuimarães Rocha MS: Acute treatment costs of stroke in Brazil. Neuroepidemiology 32:1421492009

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    Eliceiri BPCheresh DA: Adhesion events in angiogenesis. Curr Opin Cell Biol 13:5635682001

  • 8

    Good DJPolverini PJRastinejad FLe Beau MMLemons RSFrazier WA: A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci U S A 87:662466281990

  • 9

    Iruela-Arispe MLLiska DJSage EHBornstein P: Differential expression of thrombospondin 1, 2, and 3 during murine development. Dev Dyn 197:40561993

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    Iruela-Arispe MLLombardo MKrutzsch HCLawler JRoberts DD: Inhibition of angiogenesis by thrombospondin-1 is mediated by 2 independent regions within the type 1 repeats. Circulation 100:142314311999

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    Krupinski JKaluza JKumar PKumar SWang JM: Role of angiogenesis in patients with cerebral ischemic stroke. Stroke 25:179417981994

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    Kyriakides TRZhu YHSmith LTBain SDYang ZLin MT: Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis. J Cell Biol 140:4194301998

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    Kyriakides TRZhu YHYang ZHuynh GBornstein P: Altered extracellular matrix remodeling and angiogenesis in sponge granulomas of thrombospondin 2-null mice. Am J Pathol 159:125512622001

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    Lawler J: The functions of thrombospondin-1 and-2. Curr Opin Cell Biol 12:6346402000

  • 15

    Livak KJSchmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(–ΔΔC(T)) Method. Methods 25:4024082001

  • 16

    Lu HShi JXChen HLHang CHWang HDYin HX: Expression of monocyte chemoattractant protein-1 in the cerebral artery after experimental subarachnoid hemorrhage. Brain Res 1262:73802009

  • 17

    MacLellan CLSilasi GPoon CCEdmundson CLBuist RPeeling J: Intracerebral hemorrhage models in rat: comparing collagenase to blood infusion. J Cereb Blood Flow Metab 28:5165252008

  • 18

    Mendelow ADGregson BAFernandes HMMurray GDTeasdale GMHope DT: Early surgery versus initial conservative treatment in patients with spontaneous supratentorial intracerebral haematomas in the International Surgical Trial in Intracerebral Haemorrhage (STICH): a randomised trial. Lancet 365:3873972005

  • 19

    Panetti TSChen HMisenheimer TMGetzler SBMosher DF: Endothelial cell mitogenesis induced by LPA: inhibition by thrombospondin-1 and thrombospondin-2. J Lab Clin Med 129:2082161997

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    Qureshi AITuhrim SBroderick JPBatjer HHHondo HHanley DF: Spontaneous intracerebral hemorrhage. N Engl J Med 344:145014602001

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    Raugi GJMumby SMAbbott-Brown DBornstein P: Thrombospondin: synthesis and secretion by cells in culture. J Cell Biol 95:3513541982

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    Risau W: Mechanisms of angiogenesis. Nature 386:6716741997

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    Savill JHogg NRen YHaslett C: Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J Clin Invest 90:151315221992

  • 24

    Sheibani NFrazier WA: Down-regulation of platelet endothelial cell adhesion molecule-1 results in thrombospondin-1 expression and concerted regulation of endothelial cell phenotype. Mol Biol Cell 9:7017131998

  • 25

    Suzuma KTakagi HOtani AOh HHonda Y: Expression of thrombospondin-1 in ischemia-induced retinal neovascularization. Am J Pathol 154:3433541999

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    Tang TLiu XJZhang ZQZhou HJLuo JKHuang JF: Cerebral angiogenesis after collagenase-induced intracerebral hemorrhage in rats. Brain Res 1175:1341422007

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    Tolsma SSVolpert OVGood DJFrazier WAPolverini PJBouck N: Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. J Cell Biol 122:4975111993

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    Tooney PASakai TSakai KAeschlimann DMosher DF: Restricted localization of thrombospondin-2 protein during mouse embryogenesis: a comparison to thrombospondin-1. Matrix Biol 17:1311431998

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    Volpert OVTolsma SSPellerin SFeige JJChen HMosher DF: Inhibition of angiogenesis by thrombospondin-2. Biochem Biophys Res Commun 217:3263321995

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    Ward NLDumont DJ: The angiopoietins and Tie2/Tek: adding to the complexity of cardiovascular development. Semin Cell Dev Biol 13:19272002

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    Yang ZKyriakides TRBornstein P: Matricellular proteins as modulators of cell-matrix interactions: adhesive defect in thrombospondin 2-null fibroblasts is a consequence of increased levels of matrix metalloproteinase-2. Mol Biol Cell 11:335333642000

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    Zhou HTang TGuo CZhang HZhong JZheng J: Expression of Angiopoietin-1 and the receptor Tie-2 mRNA in rat brains following intracerebral hemorrhage. Acta Neurobiol Exp (Wars) 68:1471542008

Article Information

Address correspondence to: Tao Tang, Ph.D., M.D., Institute of Integrative Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, People's Republic of China 410008. email: falcontang@126.com.

Please include this information when citing this paper: published online February 5, 2010; DOI: 10.3171/2010.1.JNS09637.

© AANS, except where prohibited by US copyright law."

Headings

Figures

  • View in gallery

    Immunohistochemistry for TSP-1 and TSP-2 after ICH. Photomicrographs demonstrating a few slim microvessels of TSP-1 or TSP-2 immunoreactivity in sham-operated animals (A, B, I, and J). After ICH induction by either collagenase or autologous blood, many enlarged and thin-walled microvessels of TSP-1 and TSP-2 immunoreactivity were detected around the hematoma (C, D, K, and L), and the vessels appeared along the border of the clot at ~ 7 days (E, F, M, and N), then approached the core afterward (G, H, O, and P). By the double-labeling method, it was revealed that immunoreactivity (Q) for TSP-1 (red) and TSP-2 (red) was overlapped with vWF (green). Arrows indicate microvessels of corresponding immunoreactivity. Inset: The red disc represents the hematoma; the 3 rectangles indicate where the pictures were taken in relation to the hematoma; and the dashed lines mark the border of the hematoma. Diaminobenzidene stain (A–P), fluorescence of fluorescein isothiocyanate (green, Q), and rhodamine (red, Q). Bar = 100 μm (A–P), 50 μm (Q).

  • View in gallery

    Bar graphs demonstrating quantitative analysis of TSP-1 and TSP-2 mRNA after collagenase-induced ICH. Both TSP-1 (A) and TSP-2 (B) mRNA expression rose continuously; TSP-1 mRNA peaked at 4 days, whereas TSP-2 mRNA peaked at 14 days. Data are presented as mean ± SD, with 5 rats per time point. *p < 0.01.

References

1

Andaluz NZuccarello M: Recent trends in the treatment of spontaneous intracerebral hemorrhage: analysis of a nationwide inpatient database. J Neurosurg 110:4034102009

2

Baenziger NLBrodie GNMajerus PW: Isolation and properties of a thrombin-sensitive protein of human platelets. J Biol Chem 247:272327311972

3

Baenziger NLBrodie GNMajerus PW: A thrombin-sensitive protein of human platelet membranes. Proc Natl Acad Sci U S A 68:2402431971

4

Carmeliet P: Manipulating angiogenesis in medicine. J Intern Med 255:5385612004

5

Chavakis EDimmeler S: Regulation of endothelial cell survival and apoptosis during angiogenesis. Arterioscler Thromb Vasc Biol 22:8878932002

6

Christensen MCValiente RSampaio Silva GLee WCDutcher SGuimarães Rocha MS: Acute treatment costs of stroke in Brazil. Neuroepidemiology 32:1421492009

7

Eliceiri BPCheresh DA: Adhesion events in angiogenesis. Curr Opin Cell Biol 13:5635682001

8

Good DJPolverini PJRastinejad FLe Beau MMLemons RSFrazier WA: A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci U S A 87:662466281990

9

Iruela-Arispe MLLiska DJSage EHBornstein P: Differential expression of thrombospondin 1, 2, and 3 during murine development. Dev Dyn 197:40561993

10

Iruela-Arispe MLLombardo MKrutzsch HCLawler JRoberts DD: Inhibition of angiogenesis by thrombospondin-1 is mediated by 2 independent regions within the type 1 repeats. Circulation 100:142314311999

11

Krupinski JKaluza JKumar PKumar SWang JM: Role of angiogenesis in patients with cerebral ischemic stroke. Stroke 25:179417981994

12

Kyriakides TRZhu YHSmith LTBain SDYang ZLin MT: Mice that lack thrombospondin 2 display connective tissue abnormalities that are associated with disordered collagen fibrillogenesis, an increased vascular density, and a bleeding diathesis. J Cell Biol 140:4194301998

13

Kyriakides TRZhu YHYang ZHuynh GBornstein P: Altered extracellular matrix remodeling and angiogenesis in sponge granulomas of thrombospondin 2-null mice. Am J Pathol 159:125512622001

14

Lawler J: The functions of thrombospondin-1 and-2. Curr Opin Cell Biol 12:6346402000

15

Livak KJSchmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(–ΔΔC(T)) Method. Methods 25:4024082001

16

Lu HShi JXChen HLHang CHWang HDYin HX: Expression of monocyte chemoattractant protein-1 in the cerebral artery after experimental subarachnoid hemorrhage. Brain Res 1262:73802009

17

MacLellan CLSilasi GPoon CCEdmundson CLBuist RPeeling J: Intracerebral hemorrhage models in rat: comparing collagenase to blood infusion. J Cereb Blood Flow Metab 28:5165252008

18

Mendelow ADGregson BAFernandes HMMurray GDTeasdale GMHope DT: Early surgery versus initial conservative treatment in patients with spontaneous supratentorial intracerebral haematomas in the International Surgical Trial in Intracerebral Haemorrhage (STICH): a randomised trial. Lancet 365:3873972005

19

Panetti TSChen HMisenheimer TMGetzler SBMosher DF: Endothelial cell mitogenesis induced by LPA: inhibition by thrombospondin-1 and thrombospondin-2. J Lab Clin Med 129:2082161997

20

Qureshi AITuhrim SBroderick JPBatjer HHHondo HHanley DF: Spontaneous intracerebral hemorrhage. N Engl J Med 344:145014602001

21

Raugi GJMumby SMAbbott-Brown DBornstein P: Thrombospondin: synthesis and secretion by cells in culture. J Cell Biol 95:3513541982

22

Risau W: Mechanisms of angiogenesis. Nature 386:6716741997

23

Savill JHogg NRen YHaslett C: Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J Clin Invest 90:151315221992

24

Sheibani NFrazier WA: Down-regulation of platelet endothelial cell adhesion molecule-1 results in thrombospondin-1 expression and concerted regulation of endothelial cell phenotype. Mol Biol Cell 9:7017131998

25

Suzuma KTakagi HOtani AOh HHonda Y: Expression of thrombospondin-1 in ischemia-induced retinal neovascularization. Am J Pathol 154:3433541999

26

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