Background and Purpose Occlusion in cerebral vessels results in ischemic stroke and is followed by proliferation of microvessels, ie, angiogenesis. The process is particularly marked in the border zone of the infarct, known as the ischemic penumbra. This increase in vascularization is likely to be caused by the action of angiogenic factors, such as TGF-β1, which is a powerful regulator of angiogenesis.
Methods In this study we examined 10 brain samples from patients who suffered from ischemic stroke for the expression of mRNA encoding TGF-β1.
Results The ischemic penumbra contained the highest levels of TGF-β1 mRNA, whereas the normal contralateral hemispheres had the least (P<.001, Mann-Whitney U test). Unlike those from normal brain, protein extracts from infarcted tissue contained active TGF-β1 as a 25-kD band in Western blot analysis. Extracts from the penumbra also contained a 12.5-kD isoform of TGF-β1. Both penumbra and infarct contained TGF-β1 immunoreactive products as assessed with immunohistochemistry, whereas very weak staining was observed in the contralateral hemisphere.
Conclusions These results suggest that TGF-β1 is important in the pathogenesis of the angiogenic response in ischemic brain tissue and its modulation may be used for therapeutic purposes.
In most industrialized countries, stroke is a leading cause of death in adults, following coronary heart disease and cancer. Occlusion of a cerebral blood vessel is responsible for the cerebral infarction.1 There is a rim of mild to moderately ischemic tissue (ischemic penumbra) between the normally perfused brain and the evolving infarct in which poorly understood mechanisms either suppress or completely block normal synaptic transmission.2 3
Ischemic brain tissues possess angiogenic activity, with large numbers of proliferating endothelial cells in the penumbra.4 5 The same areas contain activated microglia/macrophages and reactive astrocytes, which represent an immunologic response of the brain tissue to damage. Endothelial cells may be activated by angiogenic factors from other cells (macrophages, microglial cells, and astrocytes) of the infarcted area. Activated macrophages are potentially angiogenic and appear to mediate blood vessel growth through the secretion of a number of growth factors (eg, TGF-β), cytokines, and other extracellular matrix components.6
The pleiotropic actions of TGF-β on cultured neural cells are promotion of neuronal survival and neuritic growth, regulation of Schwann cell and inhibition of astroglial cell division, stimulation of protease and protease-inhibitor secretion in astroglia, and autoinduction of TGF-β mRNA.7 TGF-β1 treatment of three-dimensional cultures of microvascular endothelial cells elicits rapid tube formation, ie, in vitro angiogenesis associated with an increase in junctional complexes and zonula occludens mRNA.8 The purpose of this study was to evaluate the distribution of TGF-β1 mRNA and protein in stroke tissue.
Materials and Methods
Source of Tissues
Brains were obtained within 24 hours of death from 10 patients with stroke (as defined by World Health Organization criteria) aged 49 to 89 years who had survived for various lengths of time after their stroke (Table⇓). Infarcted areas and representative control tissues from the contralateral uninvolved brain hemisphere were fixed in 10% buffered formalin and embedded in paraffin.
Ischemic stroke tissue comprising infarct plus penumbra was taken from four patients (Fig 1⇓, lanes 1 through 8). Penumbra alone was dissected from the brains of another two patients (lanes 9 through 12).
Human brain cortical slices were homogenized in 6 vol of a homogenization buffer containing sodium deoxycholate, 1% vol/vol Triton X-100, 100 μmol/L EDTA, 2 μmol/L E-64, 1 μmol/L leupeptin, 1 μmol/L pepstatin, 200 μmol/L PMSF, and 100 μmol/L TLCK. Samples were centrifuged at 560g for 5 minutes and stored in aliquots at −20°C. The protein concentration of each sample was determined with a BioRad assay9 to ensure loading of equal amounts during Western blotting. Samples containing 50 mg of protein were stabilized with 5× sample buffer containing 3.6 g Tris, 10 g SDS, 25 mg bromophenol blue, 35 mL distilled water, and 10 mL glycerol, adjusted to pH 6.8. Mercaptoethanol was added (30 μL/mL) immediately before use, and SDS-polyacrylamide gel electrophoresis was carried out with a running gel. Proteins were transferred using a Hoefer electroblotting apparatus (1 hour, 0.8 mA/cm2 gel) to polyvinylidene difluoride (PVDF) membrane prewetted in methanol and equilibrated in Towbin buffer containing 192 mmol/L glycine, 25 mmol/L Tris-HCl, 1% SDS, and 20% methanol, pH 8.3. After blotting, membranes were blocked with 7.5% Marvel in TBS-Tween 20 blocking buffer, pH 7.4, for 1 hour and stained with polyclonal anti–TGF-β1 antibody (1:100 in blocking buffer) for 2 hours. Membranes were subsequently rinsed in blocking buffer (five 3-minute washes) and stained with peroxidase-conjugated, anti-chicken secondary antibody in blocking buffer for 1 hour, after which they were rinsed in TBS-Tween 20, pH 7.4 (five 3-minute washes), and the proteins were visualized with an ECL kit (Amersham).
Tissue sections were cut at 5 μm, dewaxed for 1 hour, rehydrated, and rinsed in 0.1 mol/L TBS, pH 7.6. They were incubated with 3% hydrogen peroxide for 5 minutes, rinsed in distilled water, and washed. This and all subsequent washes were in TBS for 5 minutes each. Nonspecific binding was blocked with normal rabbit serum (1:200, vol/vol; DAKO) for 20 minutes followed by overnight incubation at 4°C with primary antibody (1:40, chicken polyclonal anti–human TGF-β1, kindly provided by Dr P. Brenchley, Manchester, UK). Tissue sections were washed and incubated with biotinylated porcine anti-chicken secondary antibody (1:200; R&D Systems) for 1 hour; they were then washed again, followed by incubation with streptavidin-peroxidase (1:1000; DAKO) for 1 hour and another washing. The DAKO AEC substrate system was used as chromogen, with incubation for 15 minutes. Slides were washed in cold tap water, counterstained with Mayer’s hematoxylin, blued in warm water, and mounted in gelatin gel.
In Situ Hybridization
A human cDNA antisense TGF-β1 cDNA probe (1100 bp), a kind gift of Dr J. Hoyland, was used for in situ hybridization according to Hoyland et al.10 The probe was random prime–labeled to a specific activity of approximately 1×108 cpm/mg using [35S]α-dCTP (Megaprime Labelling kit, Amersham). Duplicate slides were treated with RNase A to estimate the contribution of nonspecific signal to the overall labeling and a probe with negative controls omitted.
In situ hybridization reactions were performed with other labeled cDNA probes (eg, for platelet-derived growth factors AA and BB) and gave different distributions (J. Krupinski, P. Kumar, S. Kumar, J. Kaluza, R. Samoail-Issa, T. Bujny, unpublished data, 1996). Thus, they might be considered as controls for possible nonspecific binding of TGF-β1.
Aseptic conditions and diethyl pyrocarbonate–treated H2O were used throughout to maintain RNase-free conditions. The paraffin-embedded tissue sections were mounted on RNase-free slides and coated with 3% amino-silane in acetone to increase adhesiveness. Slides were dewaxed, hydrated, and transferred to diethyl pyrocarbonate–treated H2O. The prehybridization treatments included sequential immersion in 0.2 mol/L HCl for 20 minutes; 2× SSC (0.9% sodium chloride, 0.015 mol/L sodium citrate) twice for 3 minutes each; 1 mg/mL proteinase K in 10 mmol/L Tris-HCl pH 7.4 for 1 hour at 37°C; and 2 mg/mL glycine in PBS for 3 minutes. After proteinase treatment, duplicate serial sections were incubated with 1 mg/mL RNase A in PBS for 1 hour at 37°C and then treated with 0.4% paraformaldehyde in PBS for 20 minutes, followed by freshly prepared 0.25% vol/vol acetic anhydride in 0.1 mol/L triethanolamine, pH 8.0, for 10 minutes. Prehybridization buffer was prepared with 50% formamide, 1 mg/mL bovine serum albumin, 0.02% wt/vol Ficoll, 0.02% wt/vol polyvinyl pyrrolidone, 0.2 mg/mL sheared salmon sperm DNA, 0.6 mol/L NaCl, 10 mmol/L Tris pH 7.4, 0.5 mol/L EDTA pH 8.0, 10 mmol/L dithiothreitol, and 10% wt/vol dextran sulfate. Heat-denatured 35S-labeled probe at 100 ng/mL in prehybridization buffer was hybridized to sections in an incubator overnight at 37°C. Aliquots of 50 μL were applied to each slide and covered with Parafilm (American National Can). After hybridization, slides were washed with a series of high-stringency washes: 0.5× SSC, 1 mmol/L EDTA, and 10 mmol/L dithiothreitol for 5 minutes two times; 0.5× SSC and 1 mmol/L EDTA for 5 minutes two times; 50% formamide, 0.15 mol/L NaCl, 0.5 mmol/L EDTA, and 5 mmol/L Tris pH 7.5 for 15 minutes; and 0.5× SSC at 55°C for 5 minutes four times, followed by 0.5× SSC at 20°C for 5 minutes. Slides were dehydrated and air dried. Autoradiography was performed with Ilford K5 emulsion. Slides were exposed at 4°C (all for the same length of time, approximately 7 days), developed in Kodak D-19 developer, rinsed, fixed, and counterstained with hematoxylin and eosin. Hybridization grains were counted at a magnification of ×400 with an image analysis system (Seescan PLC) by placing a manually drawn gate around the perceived cell boundary. Forty to fifty cells of each type were counted in the penumbra, infarct, and contralateral normal hemisphere, and counts were expressed as the median number of grains per cell. Counts were obtained after subtraction of background areas among the cells being analyzed. The signal obtained on RNase-treated controls was almost zero.
Quantification of TGF-β1 Protein in Brain Tissues
Western blot analysis demonstrated the presence of a 25-kD band corresponding to TGF-β1 in the extracts from stroke brains and very weak or no expression in the contralateral hemisphere (Fig 1⇑, lanes 1 through 8). There was also a 12.5-kD band present in the penumbra, but this was absent in normal contralateral tissue (Fig 1⇑, lanes 9 through 12)
Tissue Distribution of TGF-β1 Protein
Cells within penumbra and infarct stained with anti–TGF-β1 antibody, but there was hardly any TGF-β1 in the contralateral hemispheres of the 10 patients. Cells within penumbra stained more strongly than cells in infarcted tissue (Fig 2a⇓ and 2b⇓).
Synthesis of TGF-β1 mRNA by Brain Cells
All neurons and glial cells, especially oligodendrocytes, expressed TGF-β1 mRNA (Fig 2c⇑ and 2d⇑). When oligodendrocytes were close to neurons, they contained many grains. TGF-β1 mRNA expression was also quite strong in macrophages surrounding microvessels and in endothelial cells in stroke tissue. Monocytes within microvessels did not express any TGF-β1 mRNA. Furthermore, macrophages/monocytes attached to microvessels had fewer grains than those within brain parenchyma. This suggests that macrophages seem to be activated once they reach brain tissue. The Table⇑ and Fig 3⇓ show the numbers of grains per cell in various cell types in the infarcted core, surrounding penumbra, and contralateral hemisphere of the 10 patients. Grain counts per cell are the medians of 50 neurons, astrocytes, oligodendrocytes, microglial cells, macrophages, or endothelial cells: a total of 300 cells per tissue. The expression of TGF-β1 mRNA in contralateral hemispheres was very weak, stronger in infarct, and highest in penumbra for most brain cell types (Table⇑). The Mann-Whitney U test demonstrated significant differences between contralateral hemisphere versus infarct and infarct versus penumbra (P<.001). Cell types that strongly expressed TGF-β1 mRNA in one area presented very weak signal in another, whereas those around which the signal was hardly detected in the first area contained more grains in the second area. Neurons showed the greatest difference in grain counts between the penumbra and the ischemic infarct (P<.05). Macrophages in the infarct were synthesizing less TGF-β1 mRNA than those in the penumbra (P<.05), possibly because of failure of other dying cells to stimulate them.
Astrocytes were cells with a rather regular pattern of TGF-β1 mRNA expression, as demonstrated by smaller range values in grain counts. The penumbra also demonstrated heterogeneity. In one patient, sections taken from contralateral cerebellum showed a very strong signal for TGF-β1 mRNA in Purkinje cells (Fig 2e⇑) compared with weaker signal in ipsilateral hemisphere (Fig 2f⇑). Individual patients differed; comparisons were made among infarct, penumbra, and contralateral hemisphere of the same patient for all 10 patients.
Western blotting demonstrated that TGF-β1 was increased in infarct or penumbra versus normal contralateral hemisphere. Immunohistochemistry also demonstrated that both infarcts and penumbras contained TGF-β1–immunoreactive protein in all cell types, but there was very little TGF-β1 in contralateral hemispheres.
We have demonstrated that in human brain after ischemic stroke there is an upregulation of TGF-β1 mRNA synthesis, which is translated into 25-kD TGF-β1 protein. The presence of TGF-β1 mRNA in contralateral hemispheres, albeit in very small amounts, is contrary to a previous report in which no signal was detected in the unaffected hemisphere of rats with gliomas.11 An explanation for the high levels of TGF-β1 mRNA in the penumbra might be that hypoxia-induced stress of neuronal and astroglial cells increases growth factor expression. If the stress is too severe, cells might not respond as efficiently (ie, lower levels observed in infarcts). Similar explanations were given by Shweiki et al,12 who studied the upregulation of vascular endothelial growth factor after hypoxia.
Monocytes/macrophages actively participate in reperfusion injury in different organs after ischemia.13 14 15 They can interact with endothelial and glial cells and thus play a key role in stroke pathogenesis. In brain tumors at least, macrophages are usually observed in the vicinity of the blood vessels from which they seem to come.16 The possibility exists that, once the blood-brain barrier is broken, monocytes/macrophages infiltrate into ischemic tissue, having been activated by cytokines such as interleukin-1 secreted by activated astrocytes or other glial cells. In turn, macrophages secrete TGF-β1 and other growth factors.
TGF-β1, which is capable of acting on both immune and neural cells, seems to be important for the part it plays in regulating tissue damage and promoting repair. The former can occur because of increasing BCL-2 production and decreasing Ca2+ concentration.7 The longer survival of neurons and higher grain counts in the penumbra might be explained by the fact that TGF-β can protect neurons from glutaminergic cytotoxicity and oxidative stress by increasing BCL-2 protein expression.17
An important finding is heterogeneity in TGF-β1 mRNA expression within the penumbra. This implicates TGF-β1 as a general regulatory factor that can be switched on and off in pathological situations in the brain. Once it is released, it seems to promote both its own production by microglia and the astrocytic response.18 The role of oligodendrocytes where the TGF-β1 mRNA signal was significant is unclear. It is possible that TGF-β1 plays a role in remyelination, involving other growth factors such as nerve or brain-derived growth factors.19 Morris et al20 studied the acute phases of demyelinating diseases and found marked activation of oligodendrocytes with hyperplasia. However, in chronic disease the numbers of oligodendrocytes were reduced and none were proliferating, suggesting that the reactive potential of oligodendrocytes may be limited. It is possible that oligodendrocytes are activated after acute stroke also.
Stronger expression of TGF-β1 mRNA in Purkinje cells of the contralateral hemisphere compared with ipsilateral hemisphere seems to confirm the earlier observed phenomenon of crossed cerebellar diaschisis, which is demonstrated by a decrease in cerebellar activity contralateral to cerebral supratentorial infarct. Such diaschisis is correlated with both stroke severity and size.21
Ischemia induces endothelial cell proliferation in human brains after stroke, especially in the penumbra.4 5 Angiogenesis may occur by the action of growth factors, proteolytic enzymes, or extracellular matrix factors on endothelial cells.8 The observed higher expression of TGF-β1 mRNA in penumbras and infarcts versus the normal contralateral hemispheres should be considered together with the angiogenesis reported earlier by us.
TGF-β1 induces angiogenesis in vivo but inhibits endothelial cell proliferation and migration in vitro.22 TGF-β increases synthesis of α-2, α-5, and β-1 integrins. Altered integrin profiles might influence microvessel endothelial cell interaction with the extracellular matrix during neovascularization.23 TGF-β1 has no effect on expression by endothelial cells of intracellular adhesion molecule-1 or vascular cell adhesion molecule-1, although it decreases adhesion, probably via its action on E-selectin.24
Injection of a mixture of antibodies to TGF-β1 and TGF-β2 decreased scarring during skin wound healing, as did injection of TGF-β3, suggesting that TGF-β1, -β2, and -β3 directly or indirectly influence collagen synthesis during matrix remodeling.25 26 TGF-β1 appears to have a similar effect on glial scar formation after brain injury or stroke, as it has during collagen scar formation in other tissues.
TGF-β1 has been suggested to act in neuroprotection and as an organizer of responses in neurodegeneration (involving cytoskeletal gene expression) and anti-inflammatory effects, chemotaxis, antiproliferation, and extracellular matrix remodeling. It upregulates brain-derived neurotrophic factor and ciliary neuronal trophic factor18 and also induces autosynthesis of TGF-β1 in neurons. This may further explain the existence of the ischemic penumbra, where neurons survive longer because of a protective action of TGF-β1.
In a population of patients with advanced atherosclerosis, the serum levels of active as opposed to latent TGF-β are severely depressed, and this failure to activate otherwise normal amounts of TGF-β is a negative prognostic factor.27 Our studies do not include an evaluation of TGF-β1 activity. The 25-kD protein demonstrable on Western blots may or may not be biologically active. Thus, further studies are warranted to determine whether the detectable TGF-β1 is active and to evaluate whether experimental activation of TGF-β1 after ischemic stroke could be of therapeutic value.
Selected Abbreviations and Acronyms
|SSC||=||saline sodium citrate|
|TGF-β||=||transforming growth factor-β|
- Received October 16, 1995.
- Revision received January 18, 1996.
- Accepted January 29, 1996.
- Copyright © 1996 by American Heart Association
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