ARTICLE |
Correspondence to: May J. Reed, Box 359755, University of Washington, Seattle, WA 98104. E-mail: mjr@u.washington.edu
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Summary |
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It is generally accepted that angiogenesis is delayed in aging. To define the effects of age on the neovascular response, polyvinyl alcohol sponges were implanted SC in young (68 months old, n=11) and aged (2325 months old, n=13) mice and sampled at 14 and 19 days. Angiogenic invasion was significantly delayed in aged mice at 14d relative to young at 14d (% area of invasion 9.0 ± 3.7 vs 19.0 ± 5.6; p=0.02). Although microvessel morphology and basement membrane composition were similar between the age groups, a significant decrease in capillary density was noted in aged tissues at 14d (7.5 ± 4.1) and 19d (12.1 ± 2.8) relative to young at 14d (18.7 ± 2.3) (p<0.01 A14d vs Y14d). In comparison to young at 14d, the inflammatory response was decreased by 43 ± 2.9% and 36 ± 7.8% in aged mice at 14d and 19d, respectively. Tissues of aged mice showed less newly deposited collagen. There was a lack of expression of transforming growth factor-ß1 (TGF-ß1) and vascular endothelial growth factor (VEGF) in aged mice at 14d (0.63 ± 0.3) and 19d (1.14 ± 0.5) vs young at 14d (1.92 ± 0.5) (p0.01 A14d vs Y14d for VEGF). However, similar production of VEGF receptor2 was observed. In contrast to young mice, there was significantly increased expression of thrombospondin-2 (TSP-2) in aged mice from 14d (14.6 x 103 ± 7.3 x 103) to 19d (34.9 x 103 ± 17 x 103). We conclude that angiogenesis in aging is not merely delayed, but is altered due to multiple impairments.
(J Histochem Cytochem 51:11191130, 2003)
Key Words: angiogenesis, aging, collagen, VEGF, TSP-2, capillary density
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Introduction |
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ANGIOGENESIS, the development of new vessels from pre-existing vasculature, is delayed in aging (
Although much is known about angiogenesis in general, the changes that occur during angiogenesis in aging are not well defined. Delayed neovascularization in aged tissues has been noted and proposed to contribute to slowed wound repair (
Concurrent studies in vitro have confirmed that the age-related delay in angiogenesis is associated with several deficits in cell functions. The latter include slowed migration of aged microvascular endothelial cells (
In a previous study of the angiogenic response in healthy aged mice, our laboratory demonstrated a delay in fibrovascular invasion in aged mice at 14 d relative to young mice at 14 d that was coincident with decreased levels of type 1 collagen mRNA and deficient expression of TGF-ß1 protein (
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Materials and Methods |
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Animal Model
An established model of angiogenesis in mice using SC PVA sponges was applied (
Histology and Invasion Index
Five-µm sections of paraffin-embedded sponges were dewaxed in xylene, hydrated in a graded series (100%70%) of ethanol solutions, and stained with Masson's Trichrome. For quantification of invasion, magnified images were viewed on a Leica microscope and were captured by a spot camera. Images were imported into NIH Image software program (US National Institutes of Health; http://rsb.info.nih.gov/nih-image/). Fibrovascular invasion into the sponges was clearly visible on the imported images and was defined by the presence of vessels that contained red blood cells (
BrdU Injection
A subset of mice (n=47 in each group) were injected IP with BrdU (2 µg/g body weight) 8 hr before sacrifice. Sponges were then removed, fixed in neutral buffered formalin, and embedded in paraffin. Tissue sections were examined for the incorporation of BrdU as previously described (
Immunohistochemistry
For immunohistochemistry (IHC), sections were dewaxed in xylene and hydrated in a graded series (100%70%) of ethanol solutions. Slides were blocked overnight in PBS with 5% normal goat serum and incubated with specified primary antibodies (at 525µg/ml) for 1 hr at room temperature. The following primary antibodies were used: a rabbit polyclonal antibody against laminin (Sigma Chemical; St Louis, MO), a mouse monoclonal antibody against smooth muscle -actin (Dako; Carpinteria, CA), an affinity-purified rabbit polyclonal antibody against VEGF-165 (Santa Cruz Biotechnology; Santa Cruz, CA), a rabbit polyclonal antibody against VEGR receptor2 (a gift from Dr. Rolf Brekken), and a rabbit polyclonal antibody against mouse TSP-2 (a gift from Dr. Paul Bornstein, U. of Washington). For assessment of basement membrane components, slides were dewaxed, hydrated, and then incubated with Jones silver stain. For immunostaining, slides were exposed to the primary antibody, incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase (15 µg/ml) (Jackson Immuno Research; West Grove, PA) for 1 hr at RT, followed by uniformly timed detection with 3,3-diaminobenzidine (Vector; Burlingame, CA). In some cases, an amplification with strepavidin/biotin was performed (Dako). Immunostained sections were counterstained with toluidine blue, dehydrated with a graded series of ethanol, cleaned with xylene, mounted, and visualized by light microscopy. In all experiments, pre-immune serum and secondary antibody alone served as negative controls.
Assessment of Vessel Morphology and Capillary Density
Slides were prepared as indicated previously and stained with Masson's Trichrome. Vessels were identified by the presence of hematopoetic cells in their lumens and positive staining for VEGFR2 (to confirm the presence of a lining of endothelial cells). Vessels were counted by three different investigators at x200 magnification in at least six randomly selected fields per section.
Picrosirius Red Staining for Collagen
Slides were dewaxed, hydrated, and stained with Picrosirius Red (PSR). PSR is an anionic dye that differentiates collagen fiber thickness and density by the color emitted under polarized light. Whereas thin, loosely packed fibers are green-yellow, thicker, tightly packed fibers emit longer wavelength colors, such as orange and red (
Quantification of the Inflammatory Response
Measurement of the presence of macrophages/monocytes was performed by staining tissue with an antibody against F4/80 antigen (10 µg/ml), a specific marker for these cell types (Serotec; Raleigh, NC). The number of macrophages/monocytes was counted and analyzed in five to eight random fields by two different observers.
Computer-assisted Morphometric Analysis
TSP-2-immunostained sections were captured with a spot digital camera (Diagnostic Instruments; Sterling Heights, MI). Morphometric analysis of digital images was done by applying Metamorph Software (Universal Image; Westchester, PA). For quantification, a threshold value was set representing the maximal background intensity observed in control tissues (no primary antibody). Relative values above background readings were measured in young and aged tissues. Only sections immunostained in the same experiment were evaluated to avoid interexperiment variability (
Statistical Analysis
All comparisons between groups were analyzed for significance by a two-tailed unpaired t-test. All data are presented as the mean ± SD.
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Results |
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The Angiogenic Response is Delayed in Aged Mice
To quantify the delay in angiogenesis in the aged mice, we measured the amount of fibrovascular invasion into a PVA sponge by analysis of digital images. There was minimal invasion at 9d in either age group. There was maximal difference in the young and aged mice at 14d. Whereas sponges in aged mice at 19d were still undergoing the angiogenic response, fibrovascular invasion into the sponges was completed by 19d in the young mice. Therefore, the data to be presented will use young mice at 14d (n=7) as a reference point and will focus on the response of aged mice at 14d (n=6) and 19d (n=7).
The angiogenic response in aged mice was delayed at 14d (Fig 1B) but reached the value of young mice at 14d (Fig 1A) by 19d (Fig 1C). Fig 4A shows a bar graph of the percent area of invasion into the sponge obtained from young mice at 14d and aged mice at 14d and 19d. The percent area of angiogenic invasion was significantly delayed in aged mice at 14d (9.0 ± 3.7) relative to the young at 14d (19 ± 5.6). By 19d, sponges from the aged mice showed a similar amount of angiogenesis (20 ± 7.6) as sponges from young mice at 14d.
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The Proliferative Response Is Not Significantly Decreased in Aged Animals
Angiogenesis requires the proliferation and migration of endothelial cells and their supporting cells. To assess the relative contribution of proliferation to neovascular invasion into the sponge, we measured the incorporation of BrdU by all cells in the sponges from animals injected with BrdU 8 hr before sacrifice. The absolute number of cells stained for BrdU in each section, as well as the number of cells divided by the percent area of fibrovascular invasion, was determined. The number of BrdU-positive cells in the tissues was low (Fig 2). Comparison of the number of BrdU-positive cells in sponges in young 14d and aged mice at 14d and 19d showed that fewer than 15% of the cells were proliferating at either time point. Note that the absolute number of BrdU-positive cells in the sponges, when corrected for the percent area of fibrovascular invasion, was similar in both groups of mice at 14d.
The Appearance of Large Vessels Is Delayed in the Sponges of Aged Mice
The size of newly formed vessels in the area of fibrovascular invasion was measured via a micrometer. As shown in Fig 3A3C, whereas capillaries (<30 µm wide; small arrows) were present at all time points in young and aged sponges (Fig 3A3C), only the sponges from aged at 19d (Fig 3C) showed the larger blood vessels (>40 µm wide; large arrows) that were present in the tissues of young mice at 14d (Fig 3A).
Vessel Density Is Significantly Decreased in Aged Mice
Although sponges from aged mice reached a comparable percent area of invasion at 19d as those of young mice at 14d, tissues from aged mice never attained the vessel density of sponges from young mice. Both 14d (7.5 ± 4.1) and 19d (12.1 ± 2.8) sponges from aged mice showed significantly decreased vessel density relative to that of young tissue at 14d (18.7 ± 2.3) (Fig 4B). The endothelial cell marker VEGFR2 was applied to confirm that the vessels were lined with endothelial cells (Fig 8A inset).
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Basement Membrane Composition Is Similar in Young and Aged Mice
Jones silver stain and an antibody against laminin showed no detectable difference in the distribution of basement membrane proteins in the vessels of young and aged mice at any time point (data not shown). However, immunofluorescence for laminin deposition supported our finding that there was a significant decrease in vessel density in the sponges from aged mice relative to those from young mice. Immunostaining for actin, representing the presence of mural cells (smooth muscle cells and pericytes) (
Newly Deposited Collagen Is Decreased in Sponges of Aged Mice
The amount of collagen mRNA in the PVA sponge is decreased during angiogenesis in aged mice (
Sponges from Aged Mice Have Decreased Infiltration of Macrophages/Monocytes
It is well known that the immune response is decreased in aged tissues during infection and wound repair (
VEGF Protein, but Not Its Receptor, Is Decreased in Aged Tissues
Others have shown that the expression of VEGF is decreased during the response to ischemic injury in aged muscle (
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Aged Tissues Express High Levels of TSP-2
Although it is accepted that aged tissues have less access to both circulating and locally secreted angiogenic growth factors, little is known about their expression of inhibitors of angiogenesis. In this context, we wished to examine the expression of TSP-2, a potent inhibitor of angiogenesis (
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Discussion |
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Angiogenesis is requisite for many physiological and pathological processes. With few exceptions (
Angiogenesis was delayed in sponges obtained from aged mice at 14d compared to sponges from young mice at 14d. However, by 19d the percent area of fibrovascular invasion in the aged tissue was similar to that of their young counterparts at 14d. Of note, despite the similar area of invasion into the sponge by 19d in aged animals, the density of vessels in the sponge remained significantly decreased in the aged tissues relative to the young tissues. Previous reports have noted that capillary density in aged animals is less than that of young animals in the myocardium and brain (
Neovascular invasion requires the proliferation of endothelial cells and their supporting cells (
To further define the features of fibrovascular invasion in aged mice, we evaluated sponge tissues from young and aged mice for vessel morphology, matrix composition, and the presence of mural cells. We utilized Jones silver stain and an antibody against laminin to visualize basement membrane composition, and an antibody against actin to define the presence of mural cells. The morphology of vessels in the aged mice at 19d was similar to that of young at 14d with respect to vessel size. In that context, larger vessels that may represent the coalescence of smaller vessels were present at 14d in the young and at 19d in the aged tissues. Others have examined the shape of blood vessels induced in normal adult tissues by VEGF and have noted that vessel size can change significantly within a few days during the neovascular response (
We have previously reported that the delay in angiogenesis was coincident with decreased transcripts for type 1 collagen (
During wound repair in aged animals, an impaired inflammatory response precedes the delay in angiogenesis (
Others have documented that a deficiency in VEGF secretion by connective tissue cells contributes directly to altered angiogenesis in aging in ischemic vascular disease (
An additional novel finding in this study was the increased expression of TSP-2 in the tissues of the aged mice. TSP-2, a matricellular protein, has been reported to regulate angiogenesis by direct influences on endothelial cell behavior as well as modulation of cellmatrix interactions (
In summary, this study demonstrates that delayed angiogenesis in aged tissues does not merely reflect a slower model of angiogenesis in young tissues. Whereas the neovascular response in the aged mice approximates that of young mice in isolated features such as deposition of basement membrane proteins, other aspects of angiogenesis are truly impaired. The changes in aging include decreased vessel density, less newly deposited collagen, altered inflammatory response, reduced expression of proangiogenic factors, and increased expression of TSP-2, an inhibitor of angiogenesis. The many components of the neovascular response that are altered with age indicate that those interventions that affect multiple pathways will have the greatest impact on enhancing or inhibiting angiogenesis in aged tissues.
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Acknowledgments |
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Supported by the National Institutes of Health AG015837 and the Paul Beeson Physician Faculty Scholars in Aging Research Program.
We wish to thank Drs E. Helene Sage, Paul Vernon, Pauli Puolakkainen, Teruhiko Koike, Paul Bornstein, Azin Agah, and Themis Kyriakides for thoughtful discussions, and Nancy Ferara for technical assistance.
Received for publication December 5, 2002; accepted March 26, 2003.
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