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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 6, 2000 as doi:10.1096/fj.00-0490fje. |
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2
* Institute of Anatomy, University of Cologne, D-50931 Köln, Germany;
Institute of Cell Biology, Swiss Federal Institute of Technology, CH-8093 Zürich, Switzerland; and
Max-Planck Institute of Biochemistry, D-82152 Martinsried, Germany
2Correspondence: Institute of Cell Biology, Swiss Federal Institute of Technology, ETH Hönggerberg, CH-8093 Zürich, Switzerland. E-mail: Sabine.werner{at}cell.biol.ethz.ch
SPECIFIC AIMS
In this study we determined the effect of the tumor angiogenesis inhibitor endostatin on the healing of full-thickness excisional wounds in mice. We studied the overall wound-healing process, the density and morphological appearance of the wound vessels, and the expression of angiogenesis regulators and of major wound matrix proteins.
PRINCIPAL FINDINGS
1. Endostatin does not affect wound closure, wound contraction, and
wound reepithelialization, but reduces granulation tissue formation
Recombinant mouse endostatin was purified to homogeneity, and a
dose of 0.3 mg/(kg · day) was injected subcutaneously in the belly.
Treatment was started 2 days before wounding and continued during the
wound-healing process. The wounded tissue was analyzed at day 5 (15
endostatin-treated mice and 15 control mice) and day 7 (5
endostatin-treated mice and 5 control mice).
No obvious abnormalities in wound closure or wound appearance were observed in the endostatin-treated mice. Histological analysis of wound sections revealed a normal inflammatory response as well as normal reepithelialization, contraction, and overall wound closure in the endostatin-treated mice. However, the extent of granulation tissue was reduced and the connective tissue appeared less dense.
2. Endostatin treatment causes hemorrhage in the wound tissue and
severe narrowing of the wound vessels, but does not reduce the vessel
density in the wound
Histological analysis revealed no significant difference in vessel
density between endostatin-treated mice and control mice, indicating
that endostatin does not affect the extent of blood vessel formation in
the wound. However, severe hemorrhage was observed in the granulation
tissue of the endostatin-treated mice as demonstrated by the large
number of extravasated erythrocytes. This seems to be the result of a
defect in vessel maturation. As shown by electron microscopy
(Fig. 1A
, B
), a normal lumen was seen in the vessels of control
animals. The vessels were filled with blood cells (Fig. 1A
),
and even the sprouting vessels contained erythrocytes (Fig. 1B
). In the endostatin-injected mice, however, the vessels
were narrowed (Fig. 1C
) or closed (Fig. 1D
) and
they had an irregular luminal surface. The endothelial cells of such
vessels were enlarged and revealed a large number of vacuoles and short
cytoplasmic protrusions (Fig. 1E
). Furthermore, the contrast
of the endothelial cells appeared different, indicating severe cell
damage. In contrast to the abnormalities seen in the newly formed wound
vessels, the preexisting vessels in the dermis adjacent to the wound
appeared normal (Fig. 1F
).
|
3. Endostatin treatment improves the quality of the healed wound
To determine the effect of endostatin treatment on the quality of
the healed wound, we performed an additional wound-healing experiment
where the endostatin treatment was stopped at day 7 after injury. One
week later, the mice were killed and the wounds were analyzed
histologically. As shown by Masson trichrome staining (Fig. 2
, top panel), both endostatin-treated and control wounds were fully
healed at this time. The extent of the remaining scar was slightly
reduced in the endostatin-treated mice, and the deposited collagen
appeared less dense (Fig. 2
, top and middle panel), demonstrating that
endostatin treatment even improves the quality of the healed wound.
Similar as in early wounds, no differences in vessel density were
observed in either treatment group, as demonstrated by immunostaining
with the endothelial cell marker PECAM (Fig. 2
, lower panel). Most
vessels appeared normal at this time, but a few vessels that revealed
the characteristic abnormalities remained.
|
4. Endostatin treatment does not affect expression and/or
localization of the endogenous collagen XVIII
Since endostatin is a cleavage product of collagen XVIII, we
determined the expression of this type of collagen in the wounds of
control mice and of endostatin-treated mice. The mRNA expression levels
of collagen XVIII did not significantly change after skin injury.
In situ hybridization and immunohistochemistry revealed the
presence of collagen XVIII mRNA and protein in epidermal keratinocytes
and hair follicle keratinocytes of nonwounded skin, but not in blood
vessels. In the wounded tissue, expression of collagen XVIII was
detected throughout the hyperproliferative epithelium and in
endothelial cells of microvessels in control mice and
endostatin-treated mice. Neither expression levels nor distribution of
collagen XVIII transcripts were affected by the endostatin treatment.
5. Endostatin treatment does not affect expression of vascular
endothelial growth factor (VEGF), angiopoietin-1, and angiopoietin-2
during the angiogenic phase of wound repair
To gain insight into the mechanisms that might underlie the
phenotype observed in the endostatin-treated wounds, we analyzed the
expression of VEGF, angiopoietin-1 and angiopoietin-2. These factors
are major players involved in the angiogenic response. As previously
demonstrated in our laboratory, expression of VEGF increased in wounded
skin during the period when angiogenesis occurs. This time course of
expression was not affected by endostatin treatment. Furthermore, no
differences in the expression of the corresponding protein were
detected by immunostaining with a VEGF-specific antiserum.
A slight and transient decline in angiopoietin-1 expression was observed after wounding, and a second decline followed at day 14 after injury. Angiopoietin-2 expression increased after skin injury, with maximal levels being found between days 3 and 7 after injury. At day 14 after wounding, angiopoietin-2 mRNA levels had declined to basal levels. This time course of angiopoietin-2 expression correlated with the one observed for VEGF. No differences in angiopoietin-1 and -2 expression were observed in the endostatin-treated mice during the period when angiogenesis occurs.
6. Endostatin treatment reduces the expression of major wound
matrix proteins
To determine the reason for the less dense appearance of the
connective tissue in the wounds of the endostatin-treated mice, we
analyzed the expression of major extracellular matrix molecules by
RNase protection assay. The mRNA levels of collagen 
1 (I) chain and
fibronectin were significantly reduced in the wounds of the
endostatin-treated animals at all times. Furthermore, reduced
expression of collagen III was observed at day 14 after injury. These
results demonstrate that endostatin treatment leads to suppression of
the expression of major wound matrix proteins. This finding is likely
to provide an explanation for the less dense connective tissue seen in
the wounds of the endostatin-treated mice.
CONCLUSIONS
Inhibition of angiogenesis is one of the most promising novel approaches for the treatment of cancer. Thus, several anti-angiogenic substances have been shown to inhibit the growth of primary tumors and metastases in animals models; some of these drugs are already being tested in patients. In contrast to conventional chemotherapy, development of resistance has as yet not been observed with anti-angiogenic therapy, indicating that long-term treatment with these drugs should be effective. In addition to this advantage, the almost complete lack of angiogenesis in the adult organism should minimize the side effects of anti-angiogenic therapy. An important exception, however, is the wound-healing process, which strongly depends on the growth of new blood vessels in the injured tissue. Since tumor patients often undergo extensive surgery, inhibition of the wound-healing response might create a serious problem in patients treated with anti-angiogenic molecules.
Therefore, we determined the effect of endostatin on the healing process of full-thickness excisional wounds in mice. Endostatin was chosen, since it is one of the most promising anti-angiogenic molecules currently being tested in clinical trials. We did not observe abnormalities in wound closure, contraction, inflammation, and reepithelialization, demonstrating that wounds can heal under endostatin treatment.
The most interesting finding of our study was the severe blood vessel abnormalities observed in the endostatin-treated animals. We saw many extravasated erythrocytes, indicating impaired blood vessel integrity. Indeed, ultrastructural analysis revealed that a large proportion of the newly formed vessels in the granulation tissue were narrowed or closed, thereby inhibiting the normal blood flow. Thus a significant reduction in the number of functional vessels was observed in the wounds of endostatin-treated mice. The endothelial cells of these vessels were enlarged and vacuolized. Their shape was strikingly changed and multiple small protrusions were observed. These morphological changes are a typical sign of cell damage that might eventually lead to cell death. This finding is consistent with the effect of endostatin on endothelial cell apoptosis in vitro.
In contrast to the abnormal blood vessel morphology, no obvious reduction in the density of newly formed vessels was observed in the endostatin-treated mice. This demonstrates that endothelial cell migration, proliferation and angiogenesis can occur in the wounds of endostatin-treated mice. Most important, the strong mitogenic activity of wound endothelial cells seems to compensate for the endothelial cell loss caused by endostatin as demonstrated by the normal vessel density. By contrast, the balance between endothelial cell proliferation and endostatin-induced cell death might be different in tumors, possibly due to their lower angiogenic activity. This hypothesis is supported by results of a recent study, demonstrating that even in highly angiogenic tumors the angiogenesis is 4- to 20-fold less intense as compared with the physiological angiogenesis seen in the growing ovarian corpus rubrum. Similar differences might exist between wound angiogenesis and tumor angiogenesis.
A series of previous studies have documented the important role of VEGF and of angiopoietins in angiogenesis. Whereas VEGF is required for various processes involved in angiogenesis, including endothelial cell proliferation, angiopoietin-1 is an important factor for the stabilization of blood vessels. This stabilizing effect of angiopoietin-1 is antagonized by angiopoietin-2, a naturally occurring antagonist. Consistent with the destabilizing function of angiopoietin-2, it is expressed at sites of vascular remodelling in the adult, such as in the female reproductive tract and in certain areas of tumors where angiogenesis occurs. Expression of angiopoietin-2 together with VEGF seems to induce the angiogenic response, whereas the presence of angiopoietin-2 in the absence of VEGF leads to vessel regression. The results described in our study provide the first indication for a similar role of angiopoietin-2 and VEGF in wound angiogenesis, since expression of both factors was induced with a similar kinetics after skin injury. However, this expression pattern was not modified by endostatin during the period when wound angiogenesis occurs, indicating that abnormalities in VEGF or angiopoietin expression are not involved in the phenotypic abnormalities seen in the endostatin-treated mouse wounds. However, it seems possible that endostatin inhibits the biological activity of at least some of these factors in the wound, since it has been shown to inhibit VEGF-induced endothelial cell migration in vitro and basic fibroblast growth factor-induced angiogenesis in the chorioallantoic membrane assay.
Despite the normal wound closure seen in the endostatin-treated mice, we observed a reduced connective tissue density in early and late wounds of these animals, indicating that endostatin treatment even improves the quality of the healed wound. This effect on the matrix is at least partially regulated at the mRNA level, since expression of the major wound matrix molecules collagen types I and III, and fibronectin was reduced in the endostatin-treated mice. Although we cannot exclude the possibility that endostatin directly affects the expression of these genes, an indirect effect based on the reduced number of functional blood vessels seems more likely.
Taken together, we demonstrated striking and unexpected effects of
endostatin on blood vessel maturation during wound healing (summarized
in Fig. 3
). Our results suggest that the large number of
newly formed functional vessels in the wound is not required for
healing under normal circumstances. Thus, inhibition of angiogenesis to
a certain extent seems to be tolerable for the wound-healing process,
an observation that is likely to be important when use of endostatin is
considered in tumor patients who undergo surgery.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0490fje To cite this article, use (October 6, 2000) FASEB J. 10.1096/fj.00-0490fje 
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