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(Stroke. 1996;27:1865-1873.)
© 1996 American Heart Association, Inc.

Articles

VEGF and flt

Expression Time Kinetics in Rat Brain Infarct

Zsombor Kovacs, MD; Kiyonobu Ikezaki, MD, PhD; Ken Samoto, MD, PhD; Takanori Inamura, MD, PhD Masashi Fukui, MD, PhD

the Department of Neurosurgery, Neurological Institute, Kyushu University Faculty of Medicine, Fukuoka, Japan.

Correspondence to Kiyonobu Ikezaki, MD, PhD, Department of Neurosurgery, Neurological Institute, Kyushu University Faculty of Medicine, Fukuoka 812-82, Japan. E-mail nobu@ns.med.kyushu-u.ac.jp.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Background and Purpose Vascular endothelial growth/vascular permeability factor (VEGF) is a candidate for an angiogenic and hyperpermeability inducing factor in an infarct because it is a secretable mitogen specific for endothelial cells and is upregulated by hypoxia. Our study attempts to clarify the chronological expression of VEGF and its receptor (flt) system in experimental cerebral infarction.

Methods With the use of a reproducible middle cerebral artery occlusion model in rats, VEGF expression was identified by Western blotting with anti-VEGF antibody. The chronological expression of the VEGF/flt system was analyzed semiquantitatively by immunohistochemical means in infarcts with different time courses from 3 hours to 3 weeks.

Results VEGF and flt were expressed exclusively in the ischemic brain. The bands obtained on the immunoblot at 38 and 45 kD are related to those of VEGF₁₂₁ and VEGF₁₆₅ isoforms. Macrophages, neurons, and glial cells chronologically expressed VEGF immunoreactivity in a different fashion. Both VEGF (bound) and flt were detected in endothelial cells along with the development of angiogenesis.

Conclusions In the ischemic brain the macrophages, neurons, and glial cells appear to contain VEGF. The VEGF receptor flt was induced in endothelial cells along with the progression of angiogenesis in infarct. The VEGF/flt system is thus considered to be involved in the healing process of brain infarct.

Key Words: cerebral infarction • endothelium • growth factors • immunohistochemistry • rats

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Vascular endothelial growth/vascular permeability factor consists of a family of polypeptide isoforms that specifically regulate endothelial cell function, including enhancement of angiogenesis,^{1 2 3 4} enhancement of capillary permeability,^{5 6} and induction of transient uptake of calcium in endothelial cells.⁷ Alternative splicing has been implicated in the formation of multiple VEGF polypeptides consisting of 121, 165, 189, and 206 amino acids.^{8 9} VEGF has a hydrophobic signal peptide required for extracellular transport; in terms of this feature, VEGF is unique among heparin-binding, angiogenesis-inducing proteins.^{10 11 12} On the other hand, there is strong experimental evidence that angiogenesis requires the release of diffusible factors.^{13 14 15 16}

VEGF, examined by in situ hybridization in rat normal tissue, revealed organ-specific expression patterns. In the normal adult rat brain, VEGF expression was observed in the granule cell layer of the cerebellum.¹⁷ During brain development, VEGF transcript levels are abundant in the ventricular neuroectoderm of the embryonic and postnatal brain when endothelial cells proliferate rapidly, but they are reduced in adults when endothelial cell proliferation ceases.¹⁸

The fms-like tyrosine kinase receptor (flt) and KDR have been identified as VEGF receptors.^{19 20} Both flt and KDR are membrane-spanning receptors and belong to the class III tyrosine kinase receptor family.^{21 22} The expression of transcripts encoding flt has been localized to endothelial cells in glioma-associated vasculature but not to endothelial cells in the normal adult brain.^{23 24} To the best of our knowledge, no evidence has yet been shown for VEGF/VEGF receptor activation in brain infarct in vivo.

Hypoxia is an important inducer for biological processes in infarct. Several experiments have found VEGF to be distinctly induced by hypoxia.^{25 26 27 28} In a brain infarct, edema formation precedes angiogenesis.²⁹ VEGF has also been shown to play a role in mediating the increase of both capillary permeability and angiogenesis.^{30 31}

The present report describes the involvement of the VEGF/flt system in the process of brain infarct by studying the chronological sequence of VEGF/flt expression in infarcts at different times.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
MCA Occlusion
Fifty-five adult male Wistar rats weighing approximately 250 g were used for this study. The rats were anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg) and divided into three groups. The first group of 11 rats was used for TTC staining for the detection of infarct. In the second group, 33 rats were subjected to an MCA occlusion, with 3 rats for each different duration of infarct. The third group included 11 sham-operated animals in which the MCA was surgically exposed without ligation.

A left MCA occlusion was performed according to the technique of Tamura et al.³² After the infratemporal fossa was exposed, a small craniectomy was made, approximately 3 mm anterior and 1 mm lateral to the foramen ovale. The trunk of the MCA was occluded by ligation, from the origin of the lateral striate arteries to the inferior cerebral vein, and was then divided to ensure a complete occlusion. In addition, the main branches emerging from the MCA within the ligated sequence were cauterized. The craniectomy was covered with the temporal muscle, and the skin incision was closed with wound clips. Normothermia was maintained by external heating. All procedures were performed under sterile conditions with the use of an operating microscope.

Detection of the Infarct
To detect and quantify cerebral infarction of different durations, staining with TTC was performed. The rats from the first group were killed under diethyl ether anesthesia, and the brains were removed. Two coronal slices were made 5 and 7 mm from the frontal pole and were immersed into a 2% solution of TTC in normal saline at 37°C for 30 minutes. The sections were fixed in 10% phosphate-buffered formalin for photography.

Tissue Processing
Under diethyl ether anesthesia, the animals were decapitated after post–MCA occlusion survival times of 3, 6, or 18 hours; 1, 2, 3, 5, 7, 10, or 14 days; and 3 weeks. The brains were then removed and snap-frozen in liquid nitrogen. The specimens were cut into 6-µm slices, between 5 to 7 mm from the frontal pole, with a cryostat and then were mounted onto silane-coated slides. After they were dried at 37°C for 60 minutes, the sections were stored at -80°C until processing or stained with hematoxylin-eosin for histological evaluation.

Immunoblotting
Frozen tissue samples (2 mm³) from the infarct cortex were obtained between 7 and 9 mm from the frontal pole. The specimens of day 1 and day 3 infarcts as well as the contralateral cortex of day 3 infarct were processed. Samples were then ground into fine powder and homogenized in a lysis buffer (0.1 mol/L NaCl, 0.01 mol/L Tris-HCl, 0.001 mol/L EDTA, 1 µg/mL aprotinin). Assays to determine the protein concentration of the lysate were subsequently performed by comparison with known concentrations of bovine serum albumin. SDS-gel electrophoresis was performed in 10% polyacrylamide gels under nonreducing conditions. Lysate equivalent to 15 µg of protein from brain tissue samples of day 1 and day 3 infarcts and the contralateral cortex was run on each gel, together with prestained low-molecular-weight markers (BioRad). The electrophoresis running buffer contained 25 mmol/L Tris base, 250 mmol/L glycine, and 0.1% SDS (pH 8.3). The proteins on the gel were subsequently transferred to the Immobillon PVDF transfer membrane (Millipore Corp) in buffer containing 20% methanol, 39 mmol/L glycine, 48 mmol/L Tris base, and 0.4% SDS (pH 8.3).

The membrane was placed in 5% powdered milk in 25 mmol/L Tris-buffered saline for 1 hour to block nonspecific binding. The membrane was then incubated for 2 hours with a rabbit polyclonal antibody to VEGF (Santa Cruz Biotechnology) at a concentration of 50 µg/mL, diluted in Tris-buffered saline with 5% powdered milk. The membranes were washed in TBST (50 mmol/L Tris-HCl [pH 7.6], 150 mL NaCl, and 0.05% Tween-20), and an anti-rabbit immunoglobulin, biotinylated secondary antibody (Amersham) was applied for 60 minutes. An additional series of washes was followed by incubation in the preformed horseradish peroxidase–streptavidin complex and then by detection with the use of DAB (Wako Pure Chemical Industries, Ltd). Membranes were then washed in distilled water and air dried.

Immunohistochemistry
To determine cell specificity, the following primary monoclonal antibodies were used: anti-macrophages (BMA), anti–glial fibrillary acidic protein from clone G-A-5 (Boehringer Mannheim Biochemica), neuron-specific enolase (Dacopatts), and factor VIII (Dacopatts). A primary rabbit polyclonal antibody to VEGF was used at a concentration of 30 µg/mL for VEGF staining, diluted with 5% milk in TBST. A primary rabbit polyclonal antibody to flt (Santa Cruz Biotechnology) was used for VEGF receptor staining. As a nonimmune primary antibody, mouse normal serum (Dacopatts) was used for a negative control study. For the endogenous peroxidase block, the frozen sections of the brain tissue were immersed in 3% hydrogen peroxide in methanol for 30 minutes. The specimens were incubated with each antibody at 4°C overnight. The streptavidin-biotin method was performed with a Histofine Sab (M) and (R) kit (Nichirei Co) for polyclonal antibodies. The sections were incubated with 300 µg/mL of DAB in 0.003% hydrogen peroxide for 5 minutes and counterstained lightly with hematoxylin.

For double-staining procedures, the specimens were incubated with a rabbit polyclonal primary antibody for VEGF and subsequently with a peroxidase-conjugated anti-rabbit IgG antibody (Dacopatts). VEGF immunoreactivity was finally visualized with DAB-nickel-cobalt. A mouse monoclonal primary antibody for cell-specific antigen was then added, and alkaline phosphatase–conjugated anti-mouse IgG antibody (Dacopatts) was also applied. Cell-specific antigens were finally visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium. Double-stained sections were counterstained with methyl green.

Histopathological Classification of Ischemic Tissue
We based our classification of the ischemic lesion into two histological zones on hematoxylin-eosin staining and double-staining findings.

By day 5, the core, characterized by a marked decrease in the stainability of neuropil and vacuolation around ischemic neurons, corresponds to the central zone defined by Garcia and Kamijiyo.³³ The periphery, where infiltration of inflammatory cells and a large number of capillaries with thickened endothelial cells as well as astrogliosis can be observed, corresponds to the reactive and marginal zone.

In later stages, from day 7 to week 3, the line determined by the appearance of the first cells adjacent to the necrotic area represents a border between the core and the periphery. The reactive glia defined the extent of the periphery, marking the border between normal brain and infarct.

Cell Counting
To chronologically examine and semiquantitatively analyze the VEGF/flt expression, the double-stained sections were examined for macrophages, neurons, and glial cells. Three animals were used at each infarct time point, and measurements were made on three consecutive sections of each brain. Endothelial cells were counted on VEGF and flt immunostained sections and correlated with adjacent sections immunostained for factor VIII, at a total magnification of x400, with the use of a x10 eyepiece with a square lattice grid. The grid was randomly oriented over the infarct area to examine the core of the infarct. To count the cells in the periphery, the grid was moved along the band located at the periphery and adjacent to the core. Data were expressed as the mean±SE number of immunoreactive cells per high-power field (n=20).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
For each infarct time from 3 hours to 3 weeks, we found an obvious correlation between the localization of VEGF or flt expression and the space of the infarct area detected by TTC (Fig 1).

View larger version (42K): [in this window] [in a new window]   Figure 1. Series of 2-mm-thick coronal slices in a rat brain 3 days after MCA occlusion. The sections were stained with TTC. The infarct appears as a pale area (arrows).

Immunoblotting
A Western blot analysis of the infarct brain lysates and the contralateral brain incubated with VEGF antibody revealed distinct VEGF expression in infarct. The Western blots on day 1 and day 3 infarct brain lysates revealed two significant bands, at 38 and 45 kD, which may correspond to VEGF₁₂₁ and VEGF₁₆₅ amino acid homodimers. The contralateral brain lysates did not show any VEGF expression (Fig 2).

View larger version (49K): [in this window] [in a new window]   Figure 2. Western blot analysis of brain lysates incubated with VEGF antibody. The 1-day-old and 3-day-old infarct tissue as well as the contralateral (contra lat) cortex lysates were run on SDS-PAGE, under nonreducing conditions. Two bands are observed at molecular weights of 38 and 45 kD on the blot of the infarct tissue lysates.

Immunohistochemistry and Cell Counting
VEGF was detected selectively in infarct areas (Fig 3A). Staining with nonimmune serum was negative (Fig 3B). In the contralateral hemisphere, VEGF immunoreactivity was not observed (Fig 3C).

View larger version (1698K): [in this window] [in a new window]   Figure 3. Photomicrographs show sections counterstained with hematoxylin-eosin. A, Immunostaining with VEGF antibody in a 5-day-old infarct. Arrows are oriented toward the core and point to the border between the infarct and normal brain (original magnification x40). B, Contralateral hemisphere of the 5-day-old infarct shows negative immunoreactivity (original magnification x40). C, Immunostaining with mouse normal serum in a 5-day-old infarct shows no immunoreactivity. Arrows indicate the necrotic core (original magnification x40). On double-stained sections, which were counterstained with methyl green, thin straight arrows point to cells stained only with cell-specific antibody (purple contour), thick straight arrows indicate cells stained only with VEGF antibody (black), and curved arrows show those immunostained with both (black with purple contour). D, Double staining with anti-neuron and anti-VEGF in the core (left lower side), periphery (between dotted lines), and normal brain (right upper side) of a 3-day-old infarct (original magnification x120). E, Double staining with anti-glia and anti-VEGF in the core (left lower corner), periphery (between dotted lines), and normal brain (right upper corner) of a 5-day-old infarct (original magnification x120). F, Double staining with anti-macrophages and anti-VEGF in the periphery of an 18-hour-old infarct. Most of the double-stained macrophages have penetrated the capillary wall (original magnification x275). G, Double staining with anti-neuron and anti-VEGF in the periphery of a 3-day-old infarct. Neurons with dendrites are immunostained (original magnification x275). H, Double staining with anti-glia and anti-VEGF in the periphery of a 7-day-old infarct (original magnification x275).

From 18 hours after the lesion until day 5, the cells in the core, presenting more evident ischemic features, were well distinguishable by their morphology from those in the periphery (Fig 3D). After day 5 the extent of the periphery could be well defined by the immunostained glial cells (Fig 3E).

Macrophages in the periphery and the core of infarct represented the major source of VEGF during the early stage from hour 18 to day 2. Double-immunostained macrophages were observed around a capillary, represented almost identically in the core and in the periphery during these early stages (Fig 3F). The number of immunoreactive macrophages decreased gradually from day 2 in both the periphery and core.

VEGF was detected in neurons from 18 hours after the lesion to day 5 in the core and to day 10 in the periphery. In the early stages, intracytoplasmic granular immunolabeling in the neurons and dendrites was observed (Fig 3G). A few days later the immunostained neurons in the core appeared to be shrunken and surrounded by vacuoles, while the nuclei had almost disappeared.

Immunostaining in glial cells was observed during the entire active period from 18 hours after the lesion to day 14. However, their peak activity was noted from day 5 to day 7 in the periphery. Glial cells with VEGF were seen as small, dark cells, either elongated or triangular, which sometimes had short and fine processes (Fig 3H).

By week 3, we could not detect any more immunolabeled macrophages, neurons, or glial cells in the infarct. A semiquantitative analysis of VEGF immunostaining revealed the VEGF-expressing cell types to change chronologically in relation to time after infarct. A peak in the number of immunostained cells was noted at day 1 in the core (Fig 4). In the periphery, first the macrophages followed by the neurons and finally the glial cells were found to exhibit peak expression of VEGF (Fig 5).

View larger version (36K): [in this window] [in a new window]   Figure 4. VEGF expression patterns in different cell types in the infarct core. Macrophages and neurons showed immunoreactivity as early as 18 hours after the lesion. Glial cells showed peak expression until day 5.

View larger version (49K): [in this window] [in a new window]   Figure 5. VEGF expression patterns in different cell types in the infarct periphery. Macrophages exhibited peak expression from 18 hours after the lesion to day 2, followed by neurons from day 3 to day 5; glial cells exhibited peak expression from day 7 to day 10.

flt immunostaining was observed distinctly in the infarct area starting from day 1 (Fig 6A). An increase of flt-immunostained cells in the later stages was observed (Fig 6B). Vascular changes in infarct were evaluated in sections immunostained for factor VIII. A chronological observation of the endothelial cells in infarct revealed thin capillary walls in the early stages (Fig 6C) and a significant thickening in the later stages (Fig 6D). On the serial sections stained with flt, we could detect immunoreactivity specifically in these endothelial cells. (Fig 6E and 6F) Thereafter, flt immunoreactivity ceased completely by week 3.

View larger version (1395K): [in this window] [in a new window]   Figure 6. Immunostaining with flt antibody in a 1-day-old infarct (A) and a 5-day-old infarct (B). The arrows in each photomicrograph show the margin of the infarct area (original magnification x120). Immunostaining with factor VIII antibody shows capillaries in the periphery of a 1-day-old infarct (C) and a 5-day-old infarct (D). Note thickening of endothelial cells in the later stage (original magnification x120). Thicker endothelial cells in the later stage are also observed on sections immunostained with flt antibody in the periphery of a 1-day-old infarct (E) and a 5-day-old infarct (F) (original magnification x120).

Almost the same expression pattern was observed in endothelial cells on the sections immunostained for VEGF. VEGF expression in endothelial cells was observed simultaneously with the development of thickened endothelial cell capillaries, with a peak between days 3 and 14 (Fig 7).

View larger version (29K): [in this window] [in a new window]   Figure 7. Chronological change of flt, factor VIII, and VEGF immunostained endothelial cells. A peak number of cells were noted for all three antibodies in a 7-day-old infarct. A similar spatiotemporal expression pattern of flt and VEGF in endothelial cells was noted.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
A Western blot analysis was performed to examine whether the infarcted brain distinctly contains VEGF. All four VEGF isoforms were found to stimulate the growth of endothelial cells in culture, to induce angiogenesis, and to promote vascular permeability.^{30 34 35 36} Differences between them have been noted in their affinity to heparin, which affects the behavior of each isoform after secretion from the cell. VEGF₁₂₁ and VEGF₁₆₅ can diffuse from the producing cells into the surrounding microenvironment, while VEGF₁₈₉ and VEGF₂₀₆ are tightly bound to the extracellular matrix. Our immunohistochemical data, which showed VEGF to be detected in other cells rather than in their target capillaries, support the hypothesis that diffusible VEGF isoforms play an important role in brain infarct. The two secretable isoforms, the predominantly expressed VEGF₁₆₅ polypeptide and the shortest VEGF₁₂₁, have been detected by Western blot at molecular weights of 45 kD and approximately 40 kD, respectively.^{9 34} We observed two almost identical bands at 45 and 38 kD in the blot of infarct brain lysates, and subsequently we related these bands to the secretable VEGF₁₆₅ and VEGF₁₂₁ isoforms. We could not detect any bands on the immunoblot of brain lysates of the contralateral cortex. These results are thus considered to be consistent with the hypothesis that VEGF is only present in an ischemic brain.

The aim of the present study was also to define which cell types have the potential to produce VEGF in infarct. Macrophages have been found to express VEGF in normal tissues and in different pathological conditions as well.^{37 38 39} Our data revealed that macrophages in the periphery and in the core of the early stage infarct become the first main source of VEGF. These macrophages, according to their localization, seem to originate directly from the blood flow. We could observe that macrophages located extraluminally were all VEGF positive. This finding suggests that conditions other than those existing in the blood flow may stimulate macrophages to express VEGF. Their early appearance in infarct indicates the capacity to become the primary source of VEGF in ischemic brain.

A remarkable observation of our study is that ischemic neurons also contain VEGF. To the best of our knowledge, there have been no previous reports describing neurons expressing VEGF. From day 2 to day 3, a simultaneous decrease of the immunoreactive macrophages with an increase of immunopositive neurons was observed, while the number of the total immunoreactive cells did not change significantly. This finding thus would indicate that the VEGF expression is taken over by ischemic neurons. In the core their activity seem to last as long as they survive in the infarct. There is experimental evidence in the literature that ischemic conditions in the brain induce biological activities in neurons.^{40 41} This functional aspect also suggests that neurons could secrete VEGF under hypoxic conditions. We speculate that the range of VEGF immunoreactivity in neurons throughout the infarct may be related to their grade of hypoxia.

Gliomas from many different cell lines^{42 43 44 45} have been demonstrated to express VEGF under hypoxic conditions. The peak number of immunoreactive glial cells was detected in the period from day 5 to day 7. This can be temporally and spatially correlated with the peak occurrence of reactive gliosis in rat brain infarct.⁴⁶

Endothelial cells within the infarct were found to express VEGF immunoreactivity from day 1 to day 14. A very similar spatiotemporal expression pattern was also observed for flt. Endothelial cells are known to express high-affinity receptors for VEGF. Accordingly, after VEGF is secreted it also appears to be bound to its receptor. Some studies have identified VEGF as an autocrine growth factor for endothelial cells and demonstrated transcripts for VEGF in different clones of cultured brain capillaries as well.^{4 47} Nevertheless, it would require an in situ hybridization examination to determine whether the capillaries in brain infarct express transcripts of VEGF.

Our findings are consistent with the hypothesis that there is a continuous demand for VEGF during the entire active period of an infarct. In the periphery we found three chronologically distinguishable VEGF expression patterns related to different cell types. It would thus seem that the VEGF immunostaining is superseded by a different cell type once the immunoreactivity in the previous one has decreased. Whether VEGF-producing cells have a common induction factor or different precursors play a role in stimulating them remains a question to be answered by future VEGF experiments.

One possible reason for the particular VEGF expression pattern we detected is the fact that VEGF has been found to exhibit two kinds of activities when assayed in animals: induction of capillary permeability and endothelial cell proliferation.^{48 49 50 51} A determination of how the brain capillary endothelial cells change their response to a bifunctional VEGF remains a major challenge for researchers.

Following the time kinetics of VEGF and its receptor immunoreactivity, we attempted to speculate on the biological role of VEGF in brain infarct. A study with a proliferating cell nuclear antigen, in which the same rat infarction model was used, has demonstrated that day 3 after infarct is the period of most intensive angiogenesis.⁵² Our cell-counting study revealed an increase in the number of thickened endothelial cells with both factor VIII and flt immunoreactivity between day 3 and day 14 (Fig 7). These results suggest that the chronological changes of flt immunoreactivity were comparable to those detected by factor VIII immunoreactivity in endothelial cells. Furthermore, this phenomenon also appeared to explain the involvement of the VEGF/flt system in angiogenesis after brain infarct.

In conclusion, our results suggest that in brain infarct there is a communication system between macrophages, ischemic neurons, and glial cells on one side and endothelial cells on the other side through VEGF and flt. Since each of the responses of endothelial cells is elicited by many other growth factors and cytokines, we do not intend to overestimate the role of VEGF in the infarct. Nevertheless, because of its distinct expression in the ischemic brain, the VEGF/flt system is considered to merit more attention in further stroke studies.

	Selected Abbreviations and Acronyms

 

DAB = diaminobenzidine tetrahydrochloride KDR = kinase insert domain–containing receptor MCA = middle cerebral artery PAGE = polyacrylamide gel electrophoresis SDS = sodium dodecyl sulfate TTC = 2,3,5-triphenyltetrazolium chloride VEGF = vascular endothelial growth/vascular permeability factor

	Acknowledgments

 
This study was supported by grants from the Ministry of Education, Science, and Culture (Japan) and the Ministry of Health and Welfare (Japan). The authors thank Dr Tooru Iwaki (Department of Neuropathology) for helpful comments and Masaru Yoneda for help in providing the photographs.

Received December 8, 1995; revision received May 24, 1996; accepted June 4, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1. Millauer B, Wizigmann-Voos S, Schnurch H, Martinez R, Moller NP, Risau W, Ullrich A. High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell.. 1993;72:835-846.[Medline] [Order article via Infotrieve]

2. Samoto K, Ikezaki K, Ono M, Shono T, Kohno K, Kuwano M, Fukui M. Expression of vascular endothelial growth factor and its possible relation with neovascularization in human brain tumors. Cancer Res.. 1995;55:1189-1193.[Abstract/Free Full Text]

3. Plate KH, Breier G, Weich HA, Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature.. 1992;359:845-848.[Medline] [Order article via Infotrieve]

4. Ladoux A, Frelin C. Expression of vascular endothelial growth factor by cultured endothelial cells from brain microvessels. Biochem Biophys Res Commun.. 1993;194:799-803.[Medline] [Order article via Infotrieve]

5. Risau W. Molecular biology of blood-brain barrier ontogenesis and function. Acta Neurochir Suppl.. 1994;60:109-112.[Medline] [Order article via Infotrieve]

6. Criscuolo GR. The genesis of peritumoral vasogenic brain edema and tumor cysts: a hypothetical role for tumor-derived vascular permeability factor. Yale J Biol Med.. 1993;66:277-314.[Medline] [Order article via Infotrieve]

7. Brock TA, Dvorak HF, Senger DR. Tumor-secreted vascular permeability factor increases cytosolic Ca2+ and von Willebrand factor release in human endothelial cells. Am J Pathol.. 1991;138:213-221.[Abstract]

8. Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, Abraham JA. The human gene for vascular endothelial growth factor: multiple protein forms are encoded through alternative exon splicing. J Biol Chem.. 1991;266:1194711954.

9. Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol.. 1991;5:1806-1814.[Abstract/Free Full Text]

10. Morii K, Tanaka R, Washiyama K, Kumanishi T, Kuwano R. Expression of vascular endothelial growth factor in capillary hemangioblastoma. Biochem Biophys Res Commun.. 1993;194:749-755.[Medline] [Order article via Infotrieve]

11. Olander JV, Connolly DT, DeLarco JE. Specific binding of vascular permeability factor to endothelial cells. Biochem Biophys Res Commun.. 1991;175:68-76.[Medline] [Order article via Infotrieve]

12. Jakeman LB, Winer J, Bennett GL, Altar CA, Ferrara N. Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues. J Clin Invest.. 1992;89:244-253.

13. Ershler WB. Explanations for reduced tumor proliferative capacity with age. Exp Gerontol.. 1992;27:551-558.[Medline] [Order article via Infotrieve]

14. Gleave ME, Hsieh JT, Wu HC, Hong SJ, Zhau HE, Guthrie PD, Chung LW. Epidermal growth factor receptor-mediated autocrine and paracrine stimulation of human transitional cell carcinoma. Cancer Res.. 1993;53:5300-5307.[Abstract/Free Full Text]

15. Lu C, Kerbel RS. Cytokines, growth factors and the loss of negative growth controls in the progression of human cutaneous malignant melanoma. Curr Opin Oncol.. 1994;6:212-220.[Medline] [Order article via Infotrieve]

16. Rak JW, Hegmann EJ, Lu C, Kerbel RS. Progressive loss of sensitivity to endothelium-derived growth inhibitors expressed by human melanoma cells during disease progression. J Cell Physiol.. 1994;159:245-255.[Medline] [Order article via Infotrieve]

17. Monacci WT, Merrill MJ, Oldfield EH. Expression of vascular permeability factor/vascular endothelial growth factor in normal rat tissues. Am J Physiol.. 1993;264:C995-C1002.[Abstract/Free Full Text]

18. Breier G, Albrecht U, Sterrer S, Risau W. Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development.. 1992;114:521-532.[Abstract]

19. Waltenberger J, Claesson-Welsh L, Siegbahn A, Shibuya M, Heldin CH. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem.. 1994;269:26988-26995.[Abstract/Free Full Text]

20. Quinn TP, Peters KG, De Vries C, Ferrara N, Williams LT. Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium. Proc Natl Acad Sci U S A.. 1993;90:7533-7537.[Abstract/Free Full Text]

21. Barleon B, Hauser S, Schollmann C, Weindel K, Marme D, Yayon A, Weich HA. Differential expression of the two VEGF receptors flt and KDR in placenta and vascular endothelial cells. J Cell Biochem.. 1994;54:56-66.[Medline] [Order article via Infotrieve]

22. Neufeld G, Tessler S, Gitay-Goren H, Cohen T, Levi BZ. Vascular endothelial growth factor and its receptors. Prog Growth Factor Res.. 1994;5:89-97.[Medline] [Order article via Infotrieve]

23. Plate KH, Breier G, Weich HA, Mennel HD, Risau W. Vascular endothelial growth factor and glioma angiogenesis: coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms. Int J Cancer.. 1994;59:520-529.[Medline] [Order article via Infotrieve]

24. Plate KH, Breier G, Millauer B, Ullrich A, Risau W. Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis. Cancer Res.. 1993;53:5822-5827.[Abstract/Free Full Text]

25. Ladoux A, Frelin C. Hypoxia is a strong inducer of vascular endothelial growth factor mRNA expression in the heart. Biochem Biophys Res Commun.. 1993;195:1005-1010.[Medline] [Order article via Infotrieve]

26. Mukhopadhyay D, Tsiokas L, Zhou XM, Foster D, Brugge JS, Sukhatme VP. Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature.. 1995;375:577-581.[Medline] [Order article via Infotrieve]

27. Banai S, Shweiki D, Pinson A, Chandra M, Lazarovici G, Keshet E. Upregulation of vascular endothelial growth factor expression induced by myocardial ischaemia: implications for coronary angiogenesis. Cardiovasc Res.. 1994;28:1176-1179.[Abstract/Free Full Text]

28. Brogi E, Wu T, Namiki A, Isner JM. Indirect angiogenic cytokines upregulate VEGF and bFGF gene expression in vascular smooth muscle cells, whereas hypoxia upregulates VEGF expression only. Circulation.. 1994;90:649-652.[Abstract/Free Full Text]

29. Liu HM. Neovasculature and blood-brain barrier in ischemic brain infarct. Acta Neuropathol (Berl).. 1988;75:422-426.[Medline] [Order article via Infotrieve]

30. Detmar M, Yeo KT, Nagy JA, Van de Water L, Brown LF, Berse B, Elicker BM, Ledbetter S, Dvorak HF. Keratinocyte-derived vascular permeability factor (vascular endothelial growth factor) is a potent mitogen for dermal microvascular endothelial cells. J Invest Dermatol.. 1995;105:44-50.[Medline] [Order article via Infotrieve]

31. Brown LF, Olbricht SM, Berse B, Jackman RW, Matsueda G, Tognazzi KA, Manseau EJ, Dvorak HF, Van de Water L. Overexpression of vascular permeability factor (VPF/VEGF) and its endothelial cell receptors in delayed hypersensitivity skin reactions. J Immunol.. 1995;154:2801-7.[Abstract]

32. Tamura A, Graham DI, McCulloch J, Teasdale GM. Focal cerebral ischemia in the rat, II: regional cerebral blood flow determined by [14C] iodoantipyrine autoradiography following middle cerebral artery occlusion. J Cereb Blood Flow Metab. 1981;61-69.

33. Garcia JH, Kamijiyo Y. Cerebral infarction: evolution of histopathological changes after occlusion of a middle cerebral artery in primates. J Neuropathol Exp Neurol.. 1974;33:408-421.[Medline] [Order article via Infotrieve]

34. Kondo S, Matsumoto T, Yokoyama Y, Ohmori I, Suzuki H. The shortest isoform of human vascular endothelial growth factor/vascular permeability factor (VEGF/VPF121) produced by Saccharomyces cerevisiae promotes both angiogenesis and vascular permeability. Biochim Biophys Acta.. 1995;1243:195-202.[Medline] [Order article via Infotrieve]

35. Levy AP, Levy NS, Loscalzo J, Calderone A, Takahashi N, Yeo KT, Koren G, Colucci WS, Goldberg MA. Regulation of vascular endothelial growth factor in cardiac myocytes. Circ Res.. 1995;76:758-766.[Abstract/Free Full Text]

36. Simon M, Grone HJ, Johren O, Kullmer J, Plate KH, Risau W, Fuchs E. Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney. Am J Physiol.. 1995;268:F240-250.[Abstract/Free Full Text]

37. Fava RA, Olsen NJ, Spencer-Green G, Yeo KT, Yeo TK, Berse B, Jackman RW, Senger DR, Dvorak HF, Brown LF. Vascular permeability factor/endothelial growth factor (VPF/VEGF): accumulation and expression in human synovial fluids and rheumatoid synovial tissue. J Exp Med.. 1994;180:341-346.[Abstract/Free Full Text]

38. Berse B, Brown LF, Van-de-Water L, Dvorak HF, Senger DR. Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors. Mol Biol Cell.. 1992;3:211-220.[Abstract]

39. Sharkey AM, Chamock-Jones DS, Boocock CA, Brown KD, Smith SK. Expression of mRNA for vascular endothelial growth factor in human placenta. J Reprod Fertil.. 1993;99:609-615.[Abstract/Free Full Text]

40. Romijn HJ, Janszen AW, Van den Bogert C. Permanent increase of immunocytochemical reactivity for gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, mitochondrial enzymes, and glial fibrillary acidic protein in rat cerebral cortex damaged by early postnatal hypoxia-ischemia. Acta Neuropathol (Berl).. 1994;87:612-627A.[Medline] [Order article via Infotrieve]

41. Martinou J, Dubois-Dauphin M, Staple JK, Rodriguez I. Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia. Neuron.. 1994;13:1017-1030.[Medline] [Order article via Infotrieve]

42. Ikezaki K, Samoto K, Inamura T, Shono T, Fukui M, Ono M, Kuwano M. Expression of vascular endothelial growth factor in human brain tumors in vivo. In: Nagai M, eds. Brain Tumor Research and Therapy. Tokyo, Japan: Springer; 1996:237-245.

43. Hatva E, Kaipainen A, Mentula P, Jaaskelainen J, Paetau A, Haltia M, Alitalo K. Expression of endothelial cell-specific receptor tyrosine kinases and growth factors in human brain tumors. Am J Pathol.. 1995;146:368-378.[Abstract]

44. Goldman CK, Kim J, Wong WL, King V, Brock T, Gillespie GY. Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells: a model of glioblastoma multiforme pathophysiology. Mol Biol Cell.. 1993;4:121-133.[Abstract]

45. Tsai JC, Goldman CK, Gillespie GY. Vascular endothelial growth factor in human glioma cell lines: induced secretion by EGF, PDGF-BB, and bFGF. J Neurosurg.. 1995;82:864-873.[Medline] [Order article via Infotrieve]

46. Nordborg C, Sokrab TE, Johansson BB. Oedema-related tissue damage after temporary and permanent occlusion of the middle cerebral artery. Neuropathol Appl Neurobiol.. 1994;20:56-65.[Medline] [Order article via Infotrieve]

47. Uchida K, Uchida S, Nitta K, Yumura W, Maruno F, Nihei H. Glomerular endothelial cells in culture express and secrete vascular endothelial growth factor. Am J Physiol.. 1994;266:F81-F88.[Abstract/Free Full Text]

48. Connolly DT. Vascular permeability factor: a unique regulator of blood vessel function. J Cell Biochem.. 1991;47:219-223.[Medline] [Order article via Infotrieve]

49. Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, Conolly DT. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science.. 1989;246:1309-1312.[Abstract/Free Full Text]

50. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science.. 1989;246:1306-1309.[Abstract/Free Full Text]

51. Senger DR, Van-de-Water L, Brown LF, Nagy JA, Yeo KT, Yeo TK, Berse B, Jackman RW, Dvorak AM, Dvorak HF. Vascular permeability factor (VPF, VEGF) in tumor biology. Cancer Metastasis Rev.. 1993;12:303-324.[Medline] [Order article via Infotrieve]

52. Liu HM, Chen HH. Correlation between fibroblast growth factor expression and cell proliferation in experimental brain infarct: studied with proliferating cell nuclear antigen immunohistochemistry. J Neuropathol Exp Neurol.. 1994;53:118-126.[Medline] [Order article via Infotrieve]

Editorial Comment

Expression Time Kinetics in Rat Brain Infarct

Takakazu Kawamata, MD, Guest Editor Seth P. Finklestein, MD, Guest Editor

CNS Growth Factor Research LaboratoryDepartment of NeurologyMassachusetts General HospitalBoston, Mass

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
VEGF is the quintessential angiogenic factor. This member of the platelet-derived growth factor superfamily is a selective endothelial mitogen in vitro and promotes new capillary growth (angiogenesis) in several in vivo models.^1R VEGF is expressed at very low levels in the mature mammalian brain, but its expression is markedly unregulated in parallel with angiogenesis occurring during brain development or within brain neoplasms.^2R The accompanying article by Kovacs et al reports that VEGF expression is also upregulated after focal cerebral infarction in rats and postulates that VEGF may play an important role in new capillary growth occurring after infarction.

Indeed, the report by Kovacs et al suggests that VEGF is an excellent candidate for a key molecular signal regulating angiogenesis after stroke. Levels of VEGF are increased in neurons, glia, and macrophages, and levels of its high-affinity receptor (flt) are upregulated on endothelial cells in tissue surrounding focal infarcts with a time course that parallels new vascular growth. Since at least two isoforms of VEGF are freely secreted, it is easy to visualize how newly synthesized VEGF might reach its receptors, thus stimulating angiogenesis. However, it must be noted that the observations by Kovacs et al are purely correlative and by no means prove that VEGF is the sole or even the most important stimulant of angiogenesis after infarction. In particular, another potent angiogenic factor, basic fibroblast growth factor, is also upregulated after focal brain infarction with a time course that also parallels angiogenesis.^{3R 4R} Further studies of animals that are genetically deficient in one or the other of these growth factors may be required to settle this issue.

The identification of VEGF, basic fibroblast growth factor, and other factors with potent angiogenic effects in brain may ultimately pave the way for "therapeutic angiogenesis" of cerebrovascular disease, ie, the use of angiogenic factors to "revascularize" ischemic brain tissue at risk for stroke. Such treatment may conceivably be useful to prevent stroke in patients with repeated transient ischemic attacks in whom there is a surgically inaccessible stenosis of a major intracerebral artery. Indeed, the concept of therapeutic angiogenesis (in the form of intravascular VEGF gene therapy) is currently being applied to the clinical treatment of peripheral vascular disease^5R and may be appropriate for cerebrovascular disease as well.

	Selected Abbreviations and Acronyms

 

DAB = diaminobenzidine tetrahydrochloride KDR = kinase insert domain–containing receptor MCA = middle cerebral artery PAGE = polyacrylamide gel electrophoresis SDS = sodium dodecyl sulfate TTC = 2,3,5-triphenyltetrazolium chloride VEGF = vascular endothelial growth/vascular permeability factor

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1R. Thomas KA. Vascular endothelial growth factor, a potent and selective angiogenic agent. J. Biol. Chem.. 1996;271:603-606.[Free Full Text]

2R. Plate KH, Breier G, Weich HA, Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature.. 1992;359:845-848.

3R. Chen HH, Chien C-H, Liu HM. Correlation between angiogenesis and basic fibroblast growth factor expression in experimental brain infarct. Stroke.. 1994;25:1651-1657.[Abstract]

4R. Speliotes EK, Caday CG, Do T, Weise J, Kowall NW, Finklestein SP. Increased expression of basic fibroblast growth factor (bFGF) following focal cerebral infarction in the rat. Mol. Brain Research.. 1996;39:31-42.[Medline] [Order article via Infotrieve]

5R. Isner JM, Walsh K, Symes J, Pieczek A, Takeshita S, Lowry J, Rossow S, Rosenfield K, Weir L, Brogi E, Schainfeld R. Arterial gene therapy for therapeutic angiogenesis in patients with peripheral artery disease. Circulation.. 1995;91:2687-2692.[Free Full Text]

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