Isolation and Characterization of Heparin-Binding Growth Factors in Human Leiomyomas and Normal Myometrium1

Abstract

Uterine leiomyomas (fibroids) are benign, smooth muscle cell (SMC) tumors of the myometrium containing abundant extracellular matrix (ECM). Heparin-binding growth factors present in leiomyoma and normal myometrial fresh tissue were isolated using heparin-affinity fast protein liquid chromatography. Purification of these growth factors was monitored by the stimulation of [3H]thymidine incorporation into BALBc-3T3 cells and myometrial SMC. Western blot analysis confirmed that two consistent peaks of growth factor activity (eluting at 0.5 M NaCl and 1.7 M NaCl) were platelet-derived growth factor (PDGF), 31 kDa, and basic fibroblast growth factor (bFGF), 18 kDa, respectively. Northern blot analysis of leiomyoma and myometrial tissue revealed three RNA transcripts (2.8, 2.3, and 1.9 kb) for PDGF-A chain, one RNA transcript (4.0 kb) for PDGF-B chain, and two RNA transcripts (3.7 and 3.5 kb) for bFGF. RNase protection assay showed elevated expression of the bFGF mRNA transcript in leiomyomas in 3 out of 5 patients. Immunoperoxidase staining of paraffin-embedded tissue showed that PDGF was predominantly intracellular in both vascular and myometrial SMC. Basic FGF, by contrast, was found primarily bound to the ECM of myometrium and fibroids. Leiomyomas showed much stronger staining for bFGF due to the large areas of ECM in these tumors. A third mitogenic peak eluting at 1.1 M NaCl was also seen in both myometrial and leiomyoma tissue. This peak was not definitively identified by Western blotting. However, Northern analysis for heparin binding-epidermal growth factor (HBEGF), which also elutes at 1.1 M NaCl, detected one RNA transcript for HBEGF (2.5 kb) in normal myometrium but little or no expression in the corresponding leiomyoma tissue. Immunoperoxidase staining showed that HBEGF was a cell-membrane-associated protein in both normal myometrial and leiomyoma SMC with more intense staining in normal myometrium. These results show that both leiomyomas and myometrium synthesize a number of heparin-binding growth factors. The enhanced growth of leiomyomas may be due, in part, to the presence of large quantities of bFGF that are stored in the ECM of these tumors. In addition, the level of HBEGF mRNA declines during the transformation of myometrial SMC into leiomyomas.

Differential Expression of Receptor Tyrosine Kinases (RTKs) and IGF-I Pathway Activation in Human Uterine Leiomyomas

“…The growth factor bFGF also induces proliferation of smooth muscle cells in both leiomyomas and myometrial tissue (23). The enhanced growth of leiomyomas may be due partially to the presence of large quantities of bFGF stored in the extracellular matrix (ECM) of these tumors (24). The expression of FGF receptor protein was also reported to be more intense in leiomyomas than in the myometrium (25).…”

Etiology and pathogenesis of uterine leiomyomas: a review.

“…Certainly, morphologic similarities exist, as fibroids exhibit both an increased proliferative rate and the synthesis of extracellular fibrous matrix. After vascular injury, basic fibroblast growth factor (bFGF) is critical to smooth muscle proliferation, and this factor is also overexpressed in leiomyomas (Lindner and Reidy 1991;Mangrulkar et al 1995). Finally, injury related to menses is worthy of consideration in view of the universality of menstruation and the commonality of fibroids.…”

“…The growth-promoting effects of estrogen and progesterone upon the myometrium and uterine myomas may be mediated through the mitogenic effects of growth factors produced locally by smooth muscle cells and fibroblasts (Mangrulkar et al 1995;Rein and Nowak 1992). Growth factors are polypeptides or proteins that are secreted by a number of cell types, have a wide range of biologic effects, and generally act over short distances either in an autocrine or paracrine manner (Pusztai et al 1993).…”

“…bFGF causes proliferation of smooth muscle cells, including leiomyoma and myometrial cells (Stewart and Nowak 1996), and also promotes angiogenesis. This factor can also bind to a component of the extracellular matrix (Dixon et al 2000;Mangrulkar et al 1995). In one study there was much stronger immunohistochemical staining for bFGF in fibroids than in the myometrium because of the large amount of extracellular matrix in uterine myomata; this finding led the authors to conclude that large quantities of bFGF are stored in the extracellular matrix of these tumors (Mangrulkar et al 1995).…”

“…This factor can also bind to a component of the extracellular matrix (Dixon et al 2000;Mangrulkar et al 1995). In one study there was much stronger immunohistochemical staining for bFGF in fibroids than in the myometrium because of the large amount of extracellular matrix in uterine myomata; this finding led the authors to conclude that large quantities of bFGF are stored in the extracellular matrix of these tumors (Mangrulkar et al 1995). In addition, increased expression of bFGF mRNA was found in the leiomyomas compared with the myometrium.…”