HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Bernd Schumacher Rainald Seitelberger Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fasol, R. Articles by Seitelberger, R. Search for Related Content PubMed PubMed Citation Articles by Fasol, R. Articles by Seitelberger, R.

J Thorac Cardiovasc Surg 1994;107:1432-1439
© 1994 Mosby, Inc.

SURGERY FOR ACQUIRED HEART DISEASE

Experimental use of a modified fibrin glue to induce site-directed angiogenesis from the aorta to the heart

Freiburg, Germany

Supported by the Division of Surgical Research, Department of Surgery, University of Freiburg (Prof. Dr. v.Spect).

Received for publication June 9, 1993. Accepted for publication Sept. 27, 1993. Address for reprints: Roland Fasol, MD, FACA, Department of Cardiovascular Surgery, University of Freiburg, Hugstetterstrasse 55, 79106 Freiburg, Germany.

Abstract

From 10 cultures of manipulated Escherichia coli bacteria expressing the class I heparin-binding growth factor polypeptide -endothelial cell growth factor, 11.2 ± 0.7 mg -endothelial cell growth factor was eluted by heparin-sepharose affinity chromatography. Analysis of molecular weight (17,000 kD) was done by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and purification of the growth factor was done by high-performance liquid chromatography. The harvested -endothelial cell growth factor was proved by protein blotting. To assess the growth-promoting activity, we did an endothelial cell growth assay by comparing adult human endothelial cell control cultures, without adding growth factor to the culture medium, with adult human endothelial cell cultures with 0.02 to 20.0 ng/ml -endothelial cell growth factor and 1.0 ng/ml heparin and with adult human endothelial cell cultures with -endothelial cell growth factor but without heparin. Tritiated thymidine counts proved the significant growth-promoting activity of -endothelial cell growth factor. In 10 experimental animals modified fibrin glue containing 1 µg -endothelial cell growth factor was implanted between the aorta and the myocardium of the left ventricle and results were compared with those in five control animals that received normal fibrin glue without growth factor. After 9 weeks of implantation, angiography and histologic investigation showed newly grown vascular structures between the aorta and the myocardium in all experimental animals, but none in the control animals. Our study proved the feasibility of initiating site-directed formation of new blood vessel structures to the heart by a modified fibrin glue implant containing angiogenic growth factor -endothelial cell growth factor. (J THORAC CARDIOVASC SURG 1994;107:1432-9)

In 1935 Beck, Tichy, and Moritz, ¹ in Cleveland, poured asbestos powder into the pericardium, and in 1946, Vineberg, ² in Montreal, reported the direct implantation of the mammary artery into the myocardium. Both techniques resulted in new blood supply to the myocardium, years before the direct surgical approach to coronary artery disease was begun. ³ Nevertheless, these sporadic surgical efforts were almost blind and the new blood flow was too small in amount to be really effective. In 1988, Maciag's group in Rockville, Maryland, proved that applying angiogenic growth factor allows site-directed neovessel formation in vivo. ⁴

In vivo administration of angiogenic growth factors as a possible therapeutic intervention in the management of advanced coronary artery obstructive disease has remained more or less unexplored. We have now surveyed, in the experimental animal, the possibility of inducing controlled, site-directed growth of new blood vessel structures from the aorta to the heart by a modified fibrin glue implant.

MATERIALS AND METHODS

Bacterial culture and isolation of growth factor
Bacteria expressing the class I heparin-binding growth factor (HBGF-I) polypeptides - and ß-endothelial cell growth factor (- and ß-ECGF) were kindly donated by Tom Maciag (American Red Cross, Rockville, Md.) for our experiments. The manipulation of Escherichia coli was done by the Laboratory of Molecular Biology, American Red Cross, Rockville, Md. Growth of bacterial cultures and the isolation of the biologic activity from the homogenized and dialyzed bacterial pellet were done by eluting the proteins with the use of heparin-sepharose affinity chromatography as previously described. ⁵ This was followed by a Bio-Rad assay (Bio-Rad Laboratories, Richmond, Calif.) to assess the concentration of growth factor and its purification. ⁵ Characterization of - and ß-ECGF was done by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, done as described by Laemmli. ⁶ Further purification of the growth factor was done by high-performance liquid chromatography, done essentially as described by Gospodarowicz and associates. ⁷ Only -ECGF was used for the surgical experiments of this study.

Western blot study
The antigenic structure of the isolated growth factors (- and ß-ECGF) was proved by protein blotting with the use of anti--/ß-ECGF immunoglobulin G antibodies (Laboratory of Molecular Biology, American Red Cross) according to the previously described Western blot technique. ⁸

Endothelial cell growth assay
After approval by the ethics committee of our University adult human saphenous vein endothelial cells were harvested and grown from vein remnants (2.4 ± 1.3 cm²) that are usually discarded during coronary artery bypass grafting. Starch-free gloves (Biogel, Regent, Mönchengladbach, Germany) were used for operation to avoid the cytotoxic effect of glove powder. ⁹ Endothelial cells were isolated and cultivated according to the method of Watkins and associates, ¹⁰ with slight modifications. According to the previously described technique the cells were harvested by in situ vessel cannulation and cultured as reported earlier. ¹¹ First passage cultures were commenced by seeding1.0 by 10 ⁴ endothelial cells/cm² into each well (9.6 cm²) of six-well plates (Falcon Medical Instruments, Inc., Wilmington, Del.), precoated with 28 µg/ml human fibronectin (Biozol, Eching, Germany). Endothelial cells were grown to confluence with different concentrations of growth factor (-ECGF, with and without heparin) added to the culture medium. Cell feeding was done every second day by replacing only 50% of the medium, and the cell density was determined by microgrid. ¹¹

Three different groups of endothelial cell cultures were studied. Group 1 comprised control cultures, cultivated with complete medium (CM; medium 199 containing 2 mmol/L L-glutamine [Gibco, Gaithersburg, Md.], 20% fetal calf serum [Biozol], 80 U/ml penicillin [Gibco], and 80 µg/ml streptomycin sulfate [Gibco]). In group 2 0.02 ng/ml, 0.1 ng/ml, 0.2 ng/ml, 1.0 ng/ml, 2.0 ng/ml, 10.0 ng/ml, or 20.0 ng/ml -ECGF and 1.0 ng/ml preservative-free heparin (Seromed, Vienna, Austria) were added to the CM. Group 3 differed from group 2 only in that no heparin was added with -ECGF to the CM. Endothelial cell cultures in all three groups were grown to confluence, the endothelial cell density was counted, and growth activity measured by thymidine incorporation.

Thymidine incorporation
Details of the proliferation assay, based on the incorporation of ³H-thymidine into macromolecules by subconfluent, actively replicating cells in the log phase of growth, has been described elsewhere. ¹² Separate experiments for thymidine incorporation were done for the three groups and the different concentrations of -ECGF, with and without heparin. Counts were obtained on the day of reaching confluence. The different endothelial cell cultures were incubated with 5 µCi/ml methyl-³H-thymidine per well (185 GBec, 5.0 Ci/mmol, approximately 5 ml Amersham International Ltd., Little Chalford, Bucks, United Kingdom, plc. TRA 120 batch 278) for 24 hours, followed by a rinsing procedure with 20% fetal calf serum in phosphate-buffered saline without Ca++ and Mg+, repeated three times within 30 minutes. Trypsin was then added for 30 minutes, and counting done with a 1211 Rackbeta liquid scintillation counter (LKB Scintillation Products, Wallac, Inc., Gaithersburg, Md.).

Endothelial cells were identified by factor VIII–related antigen staining and by biochemically testing the rapid receptor-mediated uptake of acetylated low-density lipoprotein. ¹³

Fibrin glue
In all experiments a clinically approved but modified fibrin glue (Immuno, Heidelberg, Germany) was used. The fibrin compound contained 3.5 to 5.5 mg/ml fibrinogen, 0.1 to 0.45 mg/ml human fibronectin, 0.05 to 0.25 U/ml factor VIII, 1.4 µg/ml plasminogen, 10,000 U/ml aprotinin (Bayer, Leverkusen, Germany), and 24 mg/ml tranexamic acid (Kabi-Vitrum, Vienna, Austria). The thrombin compound contained 20 U/ml thrombin, 475 ng/ml Ca-gluconium, 5000 U/ml aprotinin, and 1.0 ng/ml preservative-free heparin (Seromed). In all 15 animals 0.5 ml fibrin glue was implanted. For the implantation of fibrin glue in the 10 experimental animals 1 µg of -ECGF was added to the 0.25 ml of the thrombin compound and stored at 37° C.

Animals and anesthesia
Fifteen Lewis rats (280 to 300 gm, age 4 to 5 months) were used. General anesthesia was induced by 100 mg/kg ketamin (Parke-Davis, Berlin, Germany) and 5 mg/kg xylazine hydrochloride (Rompun; Bayer) and ventilation of the lungs after intubation through a 23-gauge catheter (Abbocath; Abboth, Sligo, Ireland). All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985). This study was approved by the ethical committee of the University of Freiburg, Germany.

Surgical procedure
With all surgical implantations only starch-free gloves were used to avoid cytotoxic effects of glove powder and uncontrolled tissue reactions caused by powder particles. ⁹ The thoracic aorta was exposed through a left-sided thoracotomy under aseptic and sterile conditions. Access to the myocardium of the left ventricle was obtained through a small incision into the pericardium. The fibrin glue was applied by syringe from the aorta to the left ventricular myocardium and care was taken to allow the fibrin glue to set (Fig. 1). A black nonabsorbable silk suture was placed at the site of implantation to allow exact identification of the fibrin glue implant at the time of explanation. Ten animals received the fibrin glue containing 1 µg -ECGF (0.5 ml) and the five control animals received normal fibrin glue (0.5 ml) without growth factor.

View larger version (73K): [in this window] [in a new window]   Fig. 1. Experimental setup: after left-sided thoracotomy, thoracic aorta and myocardium of left ventricle were exposed. In experimental animals 0.5ml fibrin glue containing 1 µg angiogenic -ECGF was applied by syringe from aorta to myocardium (arrow). In control animals fibrin glue without growth factor was applied. Black nonabsorbable silk suture was placed to allow identification of implant at explantation.

Statistics
Statistical analysis was done by unpaired, two-tailed t test with the commercially available computer software package Stat View II (Abacus Concepts, Inc., Calabasas, Calif.). All values are expressed as mean plus or minus the standard deviation. Statistical significance was assumed for a p value less than 0.05.

RESULTS

Bacterial culture and growth factor isolation
Twenty cultures of manipulated Escherichia coli bacteria were processed for our experiments, 10 cultures containing the -ECGF plasmid and 10 the ß-ECGF plasmid. All 20 cultures grew successfully. We only used -ECGF for this study. The harvested bacterial pellet was 9.3 ± 0.7 ml/culture (10¹⁰ to10¹² bacteria). A total of 11.2 ± 0.7 mg -ECGF was eluted by heparin-sepharose affinity chromatography. All performed chromatography investigations proved to contain growth factor in their eluted samples. The concentrations were 61 ± 5 µg/100 ml -ECGF. Analysis of the molecular weight and characterization of -ECGF was done by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ⁵ All samples proved to contain -ECGF with a molecular weight of 17,000 kD. Purification of the growth factor was done by high-performance liquid chromatography. The polypeptide -ECGF was eluted after 70 to 80 minutes. A total of 7 by 10 ml -ECGF was yielded. Western blot test results are shown in Fig. 2.

View larger version (100K): [in this window] [in a new window]   Fig. 2. Western blot assay, using anti--/ß-ECGF immunoglobulin G antibodies for assessing growth factor eluted by high-performance liquid chromatography: (1) -ECGF and ß-ECGF (mixture of 1:1); (2) ß-ECGF, molecular weight 20,000 kD; (3) -ECGF, molecular weight 17,000 kD; (4) control sample with anti--/ß-ECGF immunoglobulin G only.

View larger version (34K): [in this window] [in a new window]   Fig. 3. Endothelial cell growth assay. Group 1: control cultures, no growth factor was added to culture medium. Group 2: 0.02 to 20.0 ng/ml -ECGF with 1.0 ng/ml preservative-free heparin was added. Group 3: same growth factor concentrations as in group 2, but no heparin was added to culture medium. Tritiated thymidine counts (cpm x 10-3 ) show significant growth-promoting efficacy of angiogenic -ECGF on adult human saphenous vein endothelial cells in culture (groups 2 and 3 compared with control cultures, p < 0.005). Statistically significant difference was found between groups 2 and 3, with and without additional heparin in culture medium (**p < 0.005; *p < 0.01).

View larger version (100K): [in this window] [in a new window]   Fig. 4. Aortic angiograms of experimental animals. A, Injection of contrast media into descending aorta clearly shows dense area of contrast media-filled new blood vessel structures from aorta into myocardium (arrow). B, Aortic root angiogram, showing clearly newly grown blood vessel structures (double asterisks), filled with contrast media by native coronary arteries (nca). There is also contrast media seen in implantation area to thoracic aorta (single asterisk).

Tissue samples were harvested for further histologic examinations, which clearly showed newly grown blood vessels in the experimental animals (Fig. 5, A and B). We observed the formation of new intact blood vessels along the fibrin glue implants treated with HBGF-I from the aorta to the left ventricle. The vascular bridge was continuous between the organs and histologic study suggested that the neovascular response was composed of cells recruited from the host organs. In control animals no vascular structures could be observed and histologic study did not show any vascular or inflammatory cells in the investigated implants.

View larger version (94K): [in this window] [in a new window]   Fig. 5. Histologic examination showing new blood vessel growth. A, Sample taken from vascular bridge along HBGF-I-treated fibrin glue implant between aorta and myocardium, showing formations of new blood vessels of significant diameter (arrows). In left corner is cross section of suture material placed during operation. B, Clearly visible group of four new blood vessels (asterisk) very close to native coronary arteries (arrows). Note significant inner diameter of these newvascular structures compared with native coronary arteries.

The results of this study confirm previous observations that site-directed neovessel formation is possible by applying angiogenic growth factor. ^4,5 The endothelial cell growth assay proved -ECGF to have a significant growth-promoting effect on adult human saphenous vein endothelial cells in culture, and heparin significantly increased this mitogenic effect (Fig. 3). These results confirm previous observations that heparin obviously potentiates the mitogenic effects of HBGF-I in vitro, enhances HBGF-I binding to intact endothelium, and helps prevent a decrease in the bioactivity of HBGF-I by protection from proteolytic inactivation in vivo ^14,15 Furthermore, our results show that the surgical implantation of modified fibrin glue containing -ECGF between the aorta and the myocardium of the heart induces site-directed angiogenesis, resulting in new blood vessel growth to the heart that was detected by angiography, macroscopically, and by histologic study (Figs. 4 and 5).

Angiogenesis is the growth and formation of new capillary structures up to greater vessels, involving the orderly migration, proliferation, and differentiation of vascular cells. ¹⁶ The HBGF-I polypeptide-ECGF exerts the biologic activity through high-affinity cell surface receptors to initiate an angiogenic response and site-directed neovessel formation in vivo. ^17,18 HBGF-I produces a significant increase in vascular cell replication by direct stimulation of endothelial cell proliferation, producing activation of quiescent vessel wall–derived cells to induce cellular migration and differentiation in situ. ^4,16,19,20

Ischemic tissue injury causes the release of endogenous biochemical agents, including growth factors, that stimulate angiogenesis and growth of preexisting collateral vessels. ^21,22 It is also known that under pathologic situations, when blood flow is limited by coronary artery stenosis, the heart is capable of growing new arteries, provided the vascular pathologic condition does not proceed too fast. ²³ However, these physiologic responses to pathophysiologic processes are often inadequate to prevent clinical manifestation of ischemic disease.

Our experiments were designed to investigate the possibility of inducing controlled, site-directed growth of new blood vessels from the aorta to the heart by a modified fibrin glue implant while avoiding other possible factors stimulating angiogenesis. ²⁴ We therefore used only starch-free gloves during operations to avoid uncontrolled tissue reactions as a result of powder particles. ^1,9 Furthermore, we did not choose the model of induced angiogenesis in ischemic myocardial tissue beds to avoid possible angiogenic effects of endogenous biochemical agents released by such ischemia-injured tissue. ^23-25 Our study proved the possibility of growing new blood vessels to the heart by directly implanting the angiogenic growth factor to the target organ and by ruling out the possibility that the growth of these new blood vessels was induced by other angiogenic influences. In addition, we do not believe that the growth of these new vascular structures was a result of a nonspecific inflammatory or immune response to HBGF-I. No such response has been reported in animal models. ⁴ Nevertheless, we are doing subsequent experiments with ischemic myocardial tissue to verify the influence of ischemia on induced, site-directed blood vessel growth to the heart by a surgically implanted modified fibrin glue containing -ECGF.

We conclude from our results that a modified fibrin glue meets the conditions of being an easily available substance for applying the angiogenic growth factor to the target organs and has the additional advantage of clinical applicability. Because endothelial cells could produce fibrinolytic substances, we previously attempted to inhibit fibrinolytic activity with aprotinin, an unspecific inhibitor of fibrinolysis. ^26,27 Because aprotinin was not sufficient, we added tranexamic acid to the fibrin compound for specific inhibition of fibrinolysis, to avoid a possible fibrinolysis of the growth factor–containing fibrin glue implant by endothelial cells. Furthermore, we added heparin to the thrombin compound to prevent a decrease in the bioactivity of HBGF-I by protection from proteolytic inactivation in vivo. ¹⁵

Recent reports have focused on the enhanced angiogenesis and growth of collaterals and salvage of infarcted myocardium by in vivo administration of angiogenic growth factors. ^25,28,29 It was shown that intracoronary injection of basic fibroblast growth factor (FGF) improved cardiac systolic function and reduced infarct size and increased numbers of arterioles in a canine myocardial infarct model. ²⁸ Baffour and associates ²⁵ showed that treatment with exogenous basic FGF in an acute ischemic rabbit lower limb enhanced angiogenesis and collateral vessel growth. This model had some limitations, inasmuch as the administration of heparin and ischemia itself could have affected angiogenesis and growth of collaterals. ²³ Banai and colleagues ²⁹ used a canine model based on the Vineberg technique to investigate the effects of exogenous acidic FGF on ischemic myocardium. Their data suggested that acidic FGF (100 to 800 µg) administered locally by epicardial sponge to ischemic myocardium (18 to 35 kg dogs) resulted in no evidence of angiogenesis, but caused subendocardial necrosis. These results are in contrast to our findings, which showed significant growth of new blood vessels to the heart by applying modified fibrin glue containing the HBGF-I polypeptide -ECGF (1 µg) and which showed no evidence of subendocardial infarction. We believe this discrepancy may be explained by the very high dose of acidic FGF the other investigators used.

Considering a possible implication of this new technique on future therapeutic concepts to treat cardiovascular disease, one may see some sort of a revival of the procedures of Beck, Tichy, and Moritz ¹ or Vineberg. ² Nevertheless, it seems not to be very realistic to think that any of these new techniques may be capable of replacing modern myocardial revascularization in future procedures. Even with an optimistic outlook on future research developments, the only possible implication may be to attempt to apply angiogenic growth factors to patients with advanced coronary artery obstructive disease in whom, unfortunately, surgical and endovascular interventional therapy is unsuccessful or cannot be done because of the extent of the disease.

In summary, we have shown that we could induce significant site-directed formation of new blood vessel structures to the heart by a modified fibrin glue implant containing the angiogenic growth factor HBGF-I. However, to date we do not have any information concerning the hemodynamic significance of these new blood vessels in supplying blood to the heart, if any, but we are assessing the possible hemodynamic influence by a method previously described. ³⁰ Application of HBGF-I has the potential for being of possible therapeutic value and therefore merits some further evaluation.

Acknowledgments

We offer our very special thanks to T. Maciag, Laboratory of Molecular Biology, American Red Cross, Rockville, Md., for his support and for providing the bacteria for this study.

References

1. Beck CS, Tichy VL, Moritz AR. Production of a collateral circulation to the heart. Proc Soc Exp Biol Med 1935;32:759-63.
   
2. Vineberg AM. Development of anastomosis between the coronary vessels and a transplanted internal mammary artery. Can Med Assoc J 1946;55:117-9.
   
3. Effler DB, Sones FM, Groves LK, Suarez E. Myocardial revascularization by Vineberg's internal mammary artery implant: evaluation of postoperative results. J THORAC CARDIOVASC SURG 1965;50:527-30. [Medline]
   
4. Thompson JA, Anderson KD, DiPietro JM, et al. Site-directed neovessel formation in vivo. Science 1988;241:1349-52. [Abstract/Free Full Text]
   
5. Schumacher B, v.Specht BU, Seitelberger R, Fasol R. Growth of "new" coronary vascular structures by angiogenetic growth factors. Eur J Cardiothorac Surg 1993 (In press).
   
6. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5. [Medline]
   
7. Gospodarowicz D, Cheng J, Kui GM, et al. Isolation of brain fibroblast growth factor by heparin-sepharose affinity chromatography: identity with pituitary fibroblast growth factor. Proc Natl Acad Sci U S A 1984;81:6963-7. [Abstract/Free Full Text]
   
8. Bjerrum OJ, Heegaard NHH, eds. Handbook of immunoblotting of proteins. Vol. I: technical descriptions. Boca Raton, Fla.: CRC Press, 1988.
   
9. Sharefkin JB, Fairchild KD, Albus RA, Creuss DF, Rich NM. The cytotoxic effects of surgical glove powder particles on adult human vascular endothelial cell cultures: implications for clinical use of tissue culture techniques. J Surg Res 1986;41:463-72. [Medline]
   
10. Watkins MB, Sharefkin JB, Zajtchuk R, et al. Adult human saphenous vein endothelial cells: assessment of their reproductive capacity for use in endothelial cell seeding of vascular prostheses. J Surg Res 1984;36:588-96. [Medline]
    
11. Fasol R, Zilla P, Preiss P, Odell J, Reichart B. Allogenic, multidonor in vitro endothelialization of small diameter PTFE grafts in baboons. Vasc Surg 1991;25:64-71.
    
12. Morgan DML. Oxidised polyamines and the growth of human vascular endothelial cells: prevention of cytotoxic effects by selective acetylation. Biochem J 1987;242:347-52. [Medline]
    
13. Voyta JC, Via DP, Butterfield CE, Zetter BR. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 1984;99:2034-40. [Abstract/Free Full Text]
    
14. Rosengart TK, Kupferschmid JP, Casscells W, Maciag T. Pharmacokinetics and distribution of endothelial cell growth factor with and without heparin in the rat. Clin Res 1987;35:321A.
    
15. Rosengart TK, Johnson WV, Friesel R, Clark R, Maciag T. Heparin protects heparin-binding growth factor-I from proteolytic inactivation in vitro. Biochem Biophys Res Commun 1988;152:432-40. [Medline]
    
16. Folkman J, Klagsbrun M. Angiogenetic factors. Science 1987;235:442-7. [Abstract/Free Full Text]
    
17. Friesel R, Burgess WH, Mehlman T, Maciag T. The characterization of the receptor for endothelial cell growth factor by covalent ligand attachment. J Biol Chem 1986;261:7581-4. [Abstract/Free Full Text]
    
18. Huang SS, Huang JS. Association of bovine brain-derived growth factor receptor with protein tyrosine kinase activity. J Biol Chem 1986;261:9568-71. [Abstract/Free Full Text]
    
19. Thomas KA. Fibroblast growth factors. FASEB J 1987;1:434-40. [Abstract]
    
20. Rosengart TK, Kupferschmid JP, Ferrans VJ, et al. Heparin-binding growth factor-I (endothelial cell growth factor) binds to endothelium in vivo. J Vasc Surg 1988;7:311-7. [Medline]
    
21. Pasyk S, Schaper W, Schaper J, Pasyk K, Miskiewicz G, Steinseifer B. DNA synthesis in coronary collaterals after coronary occlusion in the conscious dog. Am J Physiol 1982;242:H1031-7.
    
22. Kumar S, West D, Shahabuddin S, et al. Angiogenesis factor from human myocardial infarcts. Lancet 1983;13:364-8.
    
23. Schaper W. Coronary collaterals. In: Zilla P, Fasol R, Callow A, eds. Applied cardiovascular biology 1990-91. Basel: Karger, 1992:25-30.
    
24. D'Amore PA, Thompson RW. Collateralization in peripheral vascular disease. In: Strandness D, Didisheim P, Clowes A, Watson J, eds. Vascular disease. Orlando, Fla.: Grune and Stratton, 1987:319-33.
    
25. Baffour R, Berman J, Garb JL, Rhee SW, Kaufman J, Friedmann P. Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor. J Vasc Surg 1992;16:181-91. [Medline]
    
26. Esnard F, Dupuy E, Dosne AM, Bodevin E. Partial characterization of a fibrinolytic inhibitor produced by cultured endothelial cells derived from human umbilical vein. Thromb Haemost 1982;47:128-31. [Medline]
    
27. Zilla P, Fasol R, Preiss P, et al. Use of fibrin glue as a substrate for in vitro endothelialization of PTFE vascular grafts. Surgery 1989;105:515-22. [Medline]
    
28. Yanagisawa-Miwa A, Uchida Y, Nakamura F, et al. Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor. Science 1992;257:1401-2. [Abstract/Free Full Text]
    
29. Banai S, Jaklitsch MT, Casscells W, et al. Effects of acidic fibroblast growth factor on normal and ischemic myocardium. Circ Res 1991;69:76-85. [Abstract/Free Full Text]
    
30. Seitelberger R, Raberger G. A canine model of transient myocardial dysfunction: regional shortening in the presence of critical stenosis and cardiac stimulation. J Pharmacol Methods 1984;12:233-46.[Medline]
    

This article has been cited by other articles:

				A. Nguyen and H. Cai Netrin-1 induces angiogenesis via a DCC-dependent ERK1/2-eNOS feed-forward mechanism PNAS, April 25, 2006; 103(17): 6530 - 6535. [Abstract] [Full Text] [PDF]

				M. Simons Angiogenesis: Where Do We Stand Now? Circulation, March 29, 2005; 111(12): 1556 - 1566. [Full Text] [PDF]

				V. S Chekanov, M. Zargarian, I. Baibekov, P. Karakozov, G. Tchekanov, J. Hare, V. Nikolaychik, T. Bajwa, and M. Akhtar Deferoxamine-fibrin accelerates angiogenesis in a rabbit model of peripheral ischemia Vascular Medicine, August 1, 2003; 8(3): 157 - 162. [Abstract] [PDF]

				V. S. Chekanov, V. Nikolaychik, M. A. Maternowski, R. Mehran, M. B. Leon, M. Adamian, J. Moses, G. Dangas, N. Kipshidze, and M. Akhtar Deferoxamine enhances neovascularization and recovery of ischemic skeletal muscle in an experimental sheep model Ann. Thorac. Surg., January 1, 2003; 75(1): 184 - 189. [Abstract] [Full Text] [PDF]

				P. Pecher and B. A. Schumacher Angiogenesis in ischemic human myocardium: clinical results after 3 years Ann. Thorac. Surg., May 1, 2000; 69(5): 1414 - 1419. [Abstract] [Full Text] [PDF]

				J. J. Lopez, E. R. Edelman, A. Stamler, M. G. Hibberd, P. Prasad, K. A. Thomas, J. Disalvo, R. P. Caputo, J. P. Carrozza, P. S. Douglas, et al. Angiogenic potential of perivascularly delivered aFGF in a porcine model of chronic myocardial ischemia Am J Physiol Heart Circ Physiol, March 1, 1998; 274(3): H930 - H936. [Abstract] [Full Text] [PDF]

				B. Schumacher, P. Pecher, B. U. von Specht, and Th. Stegmann Induction of Neoangiogenesis in Ischemic Myocardium by Human Growth Factors : First Clinical Results of a New Treatment of Coronary Heart Disease Circulation, February 24, 1998; 97(7): 645 - 650. [Abstract] [Full Text] [PDF]

				V. Chekanov, V. Nikolaychik, and G. Tchekanov THE USE OF BIOLOGIC GLUE FOR BETTER ADHESIONS BETWEEN THE SKELETAL MUSCLE FLAP AND THE MYOCARDIUM AND FOR INCREASING CAPILLARY INGROWTH J. Thorac. Cardiovasc. Surg., March 1, 1996; 111(3): 678 - 680. [Full Text]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Bernd Schumacher Rainald Seitelberger Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fasol, R. Articles by Seitelberger, R. Search for Related Content PubMed PubMed Citation Articles by Fasol, R. Articles by Seitelberger, R.