Departments of 1 Anatomy, 2 Histology and 3 Laboratory of Scanning Electron Microscopy, Jagiellonian University Medical College, Kopernika 12, 31-034 Kraków, Poland
4 To whom correspondence should be addressed. e-mail: mburbani{at}cyf-kr.edu.pl
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Abstract |
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Key words: angiogenesis/blood vessels/corrosion casting/electron microscopy/leiomyoma
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Introduction |
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In recent years, angiogenesis and vascularization have been regarded as significant factors controlling the growth of tumours, especially malignant ones. However, the information concerning fibroid vasculature is not abundant and to some extent controversial. Since the early paper of Sampson (1912), classical injection studies using colour or radio-opaque dyes demonstrated the arrangement of arteries and veins in the leiomyomata. There has been a consensus that small fibroids are significantly less vascular than the surrounding myometrium, but as far as large leiomyomata were concerned, some authors reported increased density of blood vessels (Sampson, 1912
; Faulkner, 1944
), whereas others observed the opposite (Farrer-Brown et al., 1970
). More recent studies of blood flow in leiomyomata (Forssman, 1976a
;b; Kurjak et al., 1992
; Huang et al., 1996
; Sosic et al., 1996
), as well as quantitative assessment of the vascular density in the immunocytochemically stained fibroid sections (Casey et al., 2000
; Hague et al., 2000
), yielded ambiguous results.
The corrosion casting technique combined with scanning electron microscopy (SEM) is the best currently available method for morphological examination of the vascular networks (Lametschwandtner et al., 1990). The injected resin fills all blood vessels including capillaries, and SEM offers high resolution and quasi-three-dimensional image. Since this method has not been employed in the studies of fibroid vasculature, the present study was undertaken to examine the vascular architecture of leiomyomata.
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Materials and methods |
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Immediately after removal, the uteri were perfused via the afferent arteries with prewarmed (37°C), heparinized saline (12.5 IU/ml heparin; Polfa, Poland) containing 3% dextrane (70 kDa) and 0.025% lidocaine (Lignocaine; Polfa), until the fluid outflowing via the veins was completely transparent (5 min). Next, perfusion was continued using a solution of 0.66% paraformaldehyde/0.08% glutaraldehyde (Sigma) in 0.1 mol/l cacodylate buffer, pH 7.4, supplemented with 0.2% lidocaine. Finally, the vascular system was injected with 60 80 ml of Mercox CL-2R resin (Vilene Comp. Ltd, Japan) containing 0.0625 mg/ml methyl acrylate polymerization initiator (Vilene Comp. Ltd) and the uteri were left in a warm water bath (56°C) for several hours to allow polymerization and tempering of the resin.
When the polymerization was completed, the uterine tissues were macerated for 56 days by repeated baths in 10% potassium hydroxide at 37°C followed by washing with warm (5055°C) running tap water. The obtained vascular casts were washed for the next 45 days in multiple changes of distilled water under mild vacuum conditions, cleaned in 5% trichloroacetic acid for 12 days, washed again in distilled water for 23 days and freeze-dried in a lyophilizer (Liovag G2; Aqua Fina, Germany).
The freeze-dried casts were examined macroscopically, gently dissected to expose the vasculature of myomata and stored in an exiccator containing phosphorus pentoxide until the microscopic examination. They were then mounted onto copper plates using colloidal silver and conductive bridges (Lametschwandtner et al., 1980) and coated with gold. The casts were examined using a JEOL SEM 35-CF scanning electron microscope at 2025 kV.
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Results |
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Discussion |
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In his classic paper, Faulkner (1944) described the vascular system of myoma as a mass of proliferating arteries. The present observations have not confirmed such a view, showing that arteries are not the predominant vessel type in the myomata (the technique used by Faulkner did not visualize capillaries) and that the vascular density of myomata demonstrated by SEM seems to be lower than or similar to that of unchanged myometrium. However, it seems possible that Faulkers description refers to the vascular capsule, a zone of very high vascular density revealed by corrosion casting around the tumour. Such structure has not been explicitly described in the previous publications, although venous plexus (Farrer-Brown et al., 1970
) or vascular plexus (Awataguchi, 1982
) were mentioned to occur around the periphery of myomata. In a recent immunocytochemical/morphometric study, Casey et al. (2000
) reported significantly higher microvascular density in the adjacent myometrium than in small and large myomata. In our material, vascular capsule was a constant feature of all myomata except for the smallest ones and it reached the highest density of blood vessels in large tumours.
The corrosion casting technique did not reveal extremely dilated veins in the myometrium and inner layer of myometrium described by Farrer-Brown et al. (1970) in their dye-injection study, although flattening of venous vessels surrounding the tumours, also reported by these authors, was observed in our material and seemed to result from the compression exerted by the tumour. Since these authors injected the veins, the reported dilatation might have been artificially produced by the injection pressure, whereas in the present study the resin was injected via arteries and filled the veins under low pressure after passing through the capillary bed.
Although some differences in blood flow revealed by colour Doppler sonography have been demonstrated between outer myometrial and submucosal leiomyomata (Tsuda et al., 1998), no differences in the vascular pattern of tumours originating from those two locations have been observed in this study.
The capillaries present in myomata had typical appearance. We did not encounter thick capillary vessels with irregular profiles, observed in corrosion casts of some malignant tumours investigated earlier in our laboratory (Bugajski et al., 1989; Miodo
ski et al., 1998
). The relatively low vascular density of myomata points to lower intensity of angiogenesis in this benign tumour, as compared with that of rapidly growing malignant neoplasms in which the accelerated angiogenesis may lead to formation of morphologically abnormal capillary vessels. As recently reported (Hong et al., 2001
), angiogenic growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor show significantly stronger expression in leiomyosarcoma that in leiomyoma. In our study on urinary bladder cancer (Miodo
ski et al., 1998
), we suggested that VEGF may promote formation of wide, irregular capillaries observed in the exophytic part of that tumour.
As can be concluded from the vascular architecture of the myomatous uteri, the most intense angiogenesis occurs around the periphery of the myomata. The fibroids have been shown to produce a variety of angiogenic growth factors, including epidermal growth factor, VEGF, platelet-derived growth factor, transforming growth factor- and -
, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) and adrenomedullin (ADM) (Harrison-Woolrych et al., 1994
, 1995; Mangrulkar et al., 1995
; Vollenhoven et al., 1995
; Arici and Sozen, 2000
; Dixon et al., 2000
; Hague et al., 2000
; Hong at al., 2001
). These factors, released from the tumour, influence angiogenesis not only inside the myoma, where the process may be obstructed by the compact character of tumour tissue, but also in the surrounding myometrium. Among the angiogenic factors, bFGF and ADM are the likely candidates to promote angiogenesis around myomata, since they were found to occur in larger quantites in myomata than in the unchanged myometrial areas (Mangrulkar et al., 1995
; Hague et al., 2000
).
ADM is produced upon hypoxiaa natural consequence of the avascularity of small myomataand its expression correlates with the vascular density of leiomyomata and the myometrium of leiomyoma-bearing uteri. Its release from the tumour was suggested to stimulate angiogenesis in the surrounding myometrium (Hague et al., 2000). The possible concentration gradient of the centrifugally released factor (the highest concentration at the tumour periphery) might be responsible for the formation of the vascular capsule around the tumour. Large quantities of bFGF are stored in the extracellular matrix of leiomyomata (Mangrulkar et al., 1995
), hence its action in larger tumours, containing connective tissue septa and surrounded by connective tissue capsule, could also significantly contribute, respectively, to the development of new vessels inside the tumour and at its periphery.
The vascular patterns observed in leiomyomata of various size allow us to propose a pathway of vascular system development in these tumours. The small myomatous foci compress the pre-existing blood vessels, induce their regression, leading to formation of transiently avascular regions inside such tumours. Subsequently, the density of blood vessels increases in the direct surroundings of the myoma, and, as it grows in size, new blood vessels penetrate the tumour from its periphery where the vascular capsule is being formed, and provide origin to vascular network observed inside the larger leiomyomata. Small avascular areas observed in the latter tumours probably represent either necrotic or newly developing myomatous foci.
This concept does not quite agree with the earlier suggestion (Farrer-Brown et al., 1970) that the arterial pattern of a myoma represents an expansion of the pre-existing supply to that area of myometrium. It does correspond, however, with the recently proposed model of angiogenesis in growing tumours (Holash et al., 1999a
,b): the pre-existing vasculature is first co-opted by the tumour, then undergoes regression and the angiogenesis starts at the periphery of the tumour leading to invasion of new vessels into the tumour which promote its further growth. Such sequence of events was also considered in the case of myomata (Casey et al., 2000
).
In our material, the earliest stage of vessel co-option by newly developing myomatous foci cannot be visualized, because such areas would not differ in the vascular architecture from the surrounding normal myometrial tissue. However, large, branchless arteries and veins occasionally found inside small, almost avascular leiomyomata probably represent co-opted vessels which, due to their size, are more resistant to tumour-induced regression than smaller, thin-walled vessels.
The role of vascular abnormalities in the development of clinical symptoms associated with leiomyomata, such as abnormal uterine bleeding, was first proposed nearly a century ago (Sampson, 1912). Contemporary studies demonstrated dysregulation of various growth factors and their receptors in the myomatous uteri (Stewart and Nowak, 1996
). Further research aimed at disclosing the successive angiogenic events in the course of leiomyoma growth and also at explaining the involvement of specific growth factors in that process will help not only to elucidate the pathogenesis of this common tumour, but also to design new therapeutic strategies.
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Acknowledgements |
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References |
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Awataguchi, K. (1982) Studies on the angioarchitecture of uterine myoma [in Japanese]. Nippon Ika Daigaku Zasshi, 49, 225232.[Medline]
Banya, Y., Ushiki, T., Takagane, H., Aoki, H., Kubo, T., Ohhori, T. and Ide, C. (1989) Two circulatory routes within the human corpus cavernosum penis: a scanning electron microscopic study of corrosion casts. J. Urol., 142, 879883.[ISI][Medline]
Bugajski, A., Nowogrodzka-Zagórska, M., Leñko, J. and Miodoski, A.J. (1989) Angiomorphology of the human renal clear cell carcinoma. A light and scanning electron microscopic study. Virchows Arch. A, 415, 103113.[ISI]
Casey, R., Rogers, P.A.W. and Vollenhoven, B.J. (2000) An immuno histochemical analysis of fibroid vasculature. Hum. Reprod., 15, 14691475.
Dixon, D., He, H. and Haseman, J.K. (2000) Immunohistochemical localization of growth factors and their receptors in uterine leiomyomas and matched myometrium. Environ. Health Perspect., 108 (Suppl. 5), 797802.[ISI][Medline]
Farrer-Brown, G., Beilby, J.O.W. and Tarbit, M.H. (1970) The vascular patterns in myomatous uteri. J. Obstet. Gynecol., 77, 967975.
Faulkner, R.L. (1944) The blood vessels of the myomatous uterus. Am. J. Obstet. Gynecol., 47, 185197.
Forssman, L. (1976a) Blood flow in myomatous uteri as measured by intraarterial 133Xenon. Acta Obstet. Gynecol. Scand., 55, 2124.[ISI][Medline]
Forssman, L. (1976b) Distribution of blood flow in myomatous uteri as measured by locally injected 133Xenon. Acta Obstet. Gynecol. Scand., 55, 101104.[ISI][Medline]
Gentry, C.C., Okolo, S.O., Fong, L.F.W.T., Crow, J.C., Maclean, A.B. and Perrett, C.W. (2001) Quantification of vascular endothelial growth factor-A in leiomyomas and adjacent myometrium. Clin. Sci., 101, 691695.[CrossRef][ISI][Medline]
Hague, S., Zhang, L., Oehler, M.K., Manek, S., MacKenzie, I.Z., Bicknell, R. and Rees, M.C.P. (2000) Expression of hypoxically regulated angiogenic factor adrenomedullin correlates with uterine leiomyoma vascular density. Clin. Cancer Res., 6, 28082814.
Harrison-Woolrych, M.L., Charnock-Jones, D.S. and Smith, S.K. (1994) Quantification of messenger ribonucleic acid for epidermal growth factor in human myometrium and leiomyomata. J. Clin. Endocrinol. Metab., 78, 11791184.[Abstract]
Harrison-Woolrych, M.L., Sharkey, A.M., Charnock-Jones, D.S. and Smith, S.K. (1995) Localization and quantification of vascular endothelial growth factor messenger ribonucleic acid in human myometrium and leiomyomata. J. Clin. Endocrinol. Metab., 80, 18531858.[Abstract]
Holash, J., Masonpierre, P.C., Compton, D., Boland, P., Alexander, C.R., Zagzag, D., Yancopoulos, G.D. and Wiegand, S.J. (1999a) Vessel co-option, regression and growth in tumors mediated by angiopoietins and VEGF. Science, 284, 19941998.
Holash, J., Wiegand, S.J. and Yancopoulos, G.D. (1999b) New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene, 18, 53565362.[CrossRef][ISI][Medline]
Hong, T., Shimada, Y., Uchida, S., Itami, A., Li, Z., Ding, Y., Kaganoi, J., Komoto, I., Sakurai, T. and Imamura, M. (2001) Expression of angiogenic factors and apoptotic factors in leiomyosarcoma and leiomyoma. Int. J. Mol. Med., 8, 141148.[ISI][Medline]
Huang, S.C., Yu, C.H., Huang, R.T., Hsu, K.F., Tsai, Y.C. and Chou, C.Y. (1996) Intratumoral blood flow in uterine myoma correlated with a lower tumor size and volume but not correlated with cell proliferation or angiogenesis. Obstet. Gynecol., 87, 10191024.
Kurjak, A., Kupesic-Urek, S. and Miric, D. (1992) The assesment of benign uterine tumor vascularization by transvaginal color Doppler. Ultrasound Med. Biol., 18, 645649.[CrossRef][ISI][Medline]
Lametschwandtner, A., Miodoski, A. and Simonsberger, P. (1980) On the prevention of specimen charging in scanning electron microscopy of vascular corrosion casts by attaching conductive bridges. Mikroskopie, 36, 270273.[ISI][Medline]
Lametschwandtner, A., Lametschwandtner, U. and Weiger, T. (1990) Scanning electron microscopy of vascular corrosion caststechnique and application: updated review. Scanning Microsc., 4, 889941.[ISI][Medline]
Mangrulkar, R.S., Ono, M., Ishikawa, M., Takashima, S., Klagbrun, M. and Nowark, R.A. (1995) Isolation and characterization of heparin-binding growth factors in human leiomyomas and normal myometrium. Biol. Reprod., 53, 636646.[Abstract]
Miodoski, A.J., Bugajski, A., Litwin, J.A. and Piasecki, Z. (1998) Vascular architecture of human urinary bladder carcinoma: a SEM study of corrosion casts. Virchows Arch., 433, 145151.[CrossRef][ISI][Medline]
Murakami, T., Fujita, T., Tanaka, T., Tsubouchi, M., Tsubouchi, Y., Taguchi, T., Ohtsuka, A. and Kikuta, A. (1994) Microcirculatory patterns in human pancreas: supplementary observations of vascular casts by scanning electron microscopy. Arch. Histol. Cytol., 57, 916.[ISI][Medline]
Sampson, J.A. (1912) The influence of myomata on the blood supply of the uterus, with special reference to abnormal uterine bleeding. Surg. Gynecol. Obstet., 16, 144180.
Sosic, A., Skupski, D.W., Streltzoff, J., Yun, H. and Chervenak, F.A. (1996) Vasculariy of uterine myomas: assessment by color and pulsed Doppler ultrasound. Int. J. Gynaecol. Obstet., 4, 245250.[CrossRef]
Stewart, E.A. and Nowak, R.A. (1996) Leiomyoma-related bleeding: a classic hypothesis updated for the molecular era. Hum. Reprod. Update, 2, 295306.
Tsuda, H., Kawabata, M., Nakamoto, O. and Yamamoto, K. (1998) Clinical predictors in the natural history of uterine leiomyoma: preliminary study. J. Ultrasound Med., 17, 1720.
Vollenhoven, B.J., Pearce, P., Herington, A.C. and Healy, D.L. (1995) Messenger ribonuclein acid expression of the insulin-like growth factors and their binging proteins in uterine fibroids and myometrium. J. Clin. Endocrinol. Metab., 76, 11061110.
Submitted on December 9, 2002; accepted on January 21, 2003.