Smooth muscle alpha actin and myosin heavy chain expression in the vascular smooth muscle cells surrounding human endometrial arterioles

K.M. Abberton1,2,3, D.L. Healy1 and P.A.W. Rogers1

1 Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria 3168, Australia


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endometrial spiral arterioles are believed to play a major role in controlling menstruation. These arterioles coil and grow through the secretory stages of the cycle, unlike the `straight' endometrial arterioles that remain uncoiled. We postulate that alterations in the growth and development of spiral arterioles, in particular the vascular smooth muscle cells (VSMC), may contribute to menorrhagia. We examined smooth muscle alpha actin ({alpha}SMA) and myosin heavy chains (MHC), two VSMC differentiation markers, in the endometrial arterioles of 64 women, comparing them in controls, menorrhagic tissues and across the menstrual cycle. {alpha}SMA and MHC expression were determined immunohistochemically then evaluated using computer-aided image analysis. {alpha}SMA expression in the straight arterioles of menorrhagic women was reduced in the early secretory stage of the cycle and significantly decreased at the mid-secretory stage of the cycle (0.67 ± 0.03 versus 0.55 ± 0.04, P <= 0.05). No other significant differences were observed in {alpha}SMA and MHC expression in straight arterioles. MHC expression was significantly reduced in the spiral arterioles of menorrhagic tissues at the early secretory stage (0.57 ± 0.01 versus 0.38 ± 0.04, P <= 0.05). Our results demonstrate differences in the VSMC of menorrhagic women compared with controls, with delayed MHC expression in the spiral arterioles and reduced {alpha}SMA expression in straight arterioles during the mid-secretory stage of the cycle.

Key words: alpha smooth muscle actin/endometrium/menorrhagia/myosin heavy chains/vascular smooth muscle


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The human endometrium differs from other parts of the body in that it is shed/absorbed and regenerated every 28 days (Sheppard and Bonner, 1979Go; Fraser, 1992Go). This requires cyclic regeneration and remodelling of the vascular bed, involving angiogenesis and development of mature arterioles. In particular, during the latter half of the cycle, specialized arterioles known as spiral arterioles form (Markee, 1940Go). Spiral arterioles differ from other small arterioles in having a coiled shape and developing over a 14 day period every 28 day menstrual cycle. Their vascular smooth muscle cells (VSMC) are reported to be larger than normal VSMC (Ramsey, 1977Go) and the arterioles lack elastin. It has been suggested that spiral arterioles play a crucial role in menstruation, being involved in the initiation and cessation of bleeding. Given their short maturation time (Markee, 1940Go; Ramsey, 1977Go; Sheppard and Bonner, 1979Go), it is possible that relatively minor deviations from the normal process of spiral arteriole growth and maturation could affect menstrual blood loss.

Menorrhagia is excessive menstrual blood loss, >80 ml/cycle, and affects up to 15% of all women, increasing to 20% during the perimenopause (Fraser, 1992Go; McKinley et al., 1992Go). Menorrhagic women suffer varying and often severe side effects such as pelvic pain and anaemia. In 50% of cases, menorrhagia has no identifiable cause and is termed idiopathic (Haynes et al., 1979Go; Fraser, 1992Go). Previous work in our laboratory has shown that women with perimenopausal menorrhagia lose blood at three times the rate of women with normal bleeding patterns (Abberton et al., 1999Go). These results agree with previous studies (Haynes et al., 1979Go), suggesting that while there may be a slight increase in length of menses in menorrhagic women, the primary problem is increased rate of bleeding. With 50% of menstrual blood loss occurring from the spiral arterioles (Markee, 1940Go; Sheppard and Bonnar, 1979), improved understanding of the development of these vessels may be an important step in understanding pathological conditions such as menorrhagia.

Based on the studies of Markee, using endometrial explants from Rhesus monkeys (Markee, 1940Go), menstruation appears to be initiated and ended by the contraction of spiral arterioles, although this mechanism may differ in humans. Aberrations in the growth/development of the VSMC surrounding these vessels may play a role in menorrhagia. Phenotypic change in, or even number of, differentiated VSMC may lead to the arterioles responding inappropriately during the process of menstruation. Classically, VSMC were said to exist in two main states; contractile, where the muscle cell has high levels of contractile proteins and does not proliferate, and secretory or synthetic. In the latter state the contractile proteins are reduced, the cell appears to de-differentiate and is able to divide and secrete basement membrane proteins. VSMC may alternate between the two states, de-differentiating to the secretory phenotype when proliferation is required. It is probable that these states are not absolute and that different VSMC populations exist in varying stages between the two extremes (Clowes et al., 1988Go; Holycross et al., 1992Go; Owens, 1995Go). It is likely that in all arterioles a certain number of VSMC exist in varying states of differentiation, so that they can respond rapidly to events such as injury.

Smooth muscle cells are known to lose contractile proteins and myofilaments in culture depending on culture conditions (Owens, 1995Go). Since exposure to local control mechanisms and mechanical factors such as fluid pressure in vivo are not always recreated in culture, responses found in cultured cells are not necessarily those seen in vivo. Using immunolocalization methods it is possible to look at the proteins that mark phenotypic changes in different smooth muscle cell populations in fixed tissue sections, thus allowing an understanding of in-vivo events.

We postulate that structural/functional differences in the endometrial arterioles, in particular the VSMC, may contribute to idiopathic menorrhagia. In particular, that the endometrial arterioles of menorrhagic women may have a varying proportion of differentiated VSMC compared with women with normal bleeding patterns, causing the arterioles to respond inappropriately to the signals causing menstruation. Smooth muscle alpha actin ({alpha}SMA) is a contractile protein that is one of the earliest markers of smooth muscle lineage in cell differentiation and the most abundant contractile protein present in VSMC (Skalli et al., 1986Go; Owens, 1995Go). It is also reduced in VSMC undergoing mitosis (Clowes et al., 1988Go). Myosin heavy chain proteins (MHC) appear towards the end of VSMC differentiation (Schwartz and Liaw, 1993Go; Price et al., 1994Go; Owens, 1995Go). This paper aims to investigate the amounts of the differentiation markers {alpha}SMA and MHC proteins in the arterioles of the human endometrium across the menstrual cycle and compare their expression in women with or without menorrhagia.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Seventy-five endometrial blocks were selected from archival cryostat blocks. The subjects from whom these samples were collected had the following criteria established: objective menstrual blood loss measurement, cycle stage, lack of gross uterine pathology, peripheral sex steroid concentrations and presence/absence of perimenopausal symptoms as described in previous studies (Abberton et al., 1996Go, 1999Go). Biopsies were routinely fixed in Tissue-Tek O.C.T (Sakura Finetek, Torrance, CA, USA) mounting compound immersed in propandiol resting on dry ice and stored at –80°C. Each biopsy was dated by an experienced histopathologist using established criteria (Noyes et al., 1950Go), into six cycle stages: menstrual, early proliferative, mid-proliferative, early-, mid- and late secretory. Eleven samples were excluded due to inability to recognize the same vessel profiles in serial sections, leaving a total of 64 samples (see Table IGo for details). The average age of women from whom control samples had come was 46.3 ± 0.5 years and that for women from whom the menorrhagic samples had come was 44.8 ± 0.8 years. Institutional ethics approval from Monash Medical Centre was received for all experiments.


View this table:
[in this window]
[in a new window]
 
Table I. Details of endometrial samples used for this study
 
Immunohistochemistry
Serial 5 µm frozen sections were cut from each biopsy and immunostained for {alpha}SMA (Clone 1A4; Dako, Denmark) or MHC 1 and 2 (Clone M-7786; Sigma, St Louis, MO, USA). Both primary antibodies used were commercially available mouse anti-human monoclonal antibodies.

Myosin heavy chain (MHC) immunostaining
Air-dried sections were dipped in acetone for 5 min at –20°C, then rinsed in phosphate buffered saline (PBS). Slides were rinsed after each step in the staining process (PBS pH 7.2, used for all rinses unless otherwise stated); 3% H2O2 was applied to block endogenous peroxidase (20 min, room temperature, RT), followed by a 5% bovine serum albumin (BSA) protein block (1 h, RT) with four drops of an endogenous avidin D block added (Vector Laboratories, Burlingame, CA, USA). The primary antibody, anti-MHC (1:400 in PBS with 1% BSA and four drops endogenous biotin block), was incubated at 37°C for 2 h. A biotinylated secondary antibody (rabbit anti-mouse) was applied for 10 min. Horseradish peroxidase/streptavadin complex was applied for 10 min, then amino-ethyl carbazole red chromogen was applied (5 min, RT). After rinsing with distilled H2O, samples were mounted in Clearmount® (Zymed Laboratories, San Francisco, CA, USA)

Smooth muscle alpha actin ({alpha}SMA) immunostaining
Serial sections adjacent to those used for the MHC staining were selected for {alpha}SMA immunolocalization following a similar protocol. Non-specific background staining was much lower on sections stained for {alpha}SMA compared to sections stained for MHC so the avidin-biotin blocking and the addition of a separate protein blocking step was not required. The concentration of the anti-{alpha}SMA primary antibody used was 1:200 in 1% BSA. In all immunohistochemical studies, appropriate non-immune immunoglobulins (IgG) were used as negative controls.

Quantitative image analysis
Sections were examined under an Axioskop® stereo microscope (Ziess, Germany) and four images containing straight arterioles were captured using a Fujix® HC-1000 3CCD digital camera. Straight arterioles were identified as single vessel profiles surrounded by a layer of VSMC. Images from the same regions on each section were captured to enable the same vessels to be scored for each protein. Spiral arterioles were identified morphologically as several profiles in cluster, 1–2 layers of smooth muscle cells and distortion of the stroma showing the convolutions of the arteriole. Images of each spiral arteriole were captured and scored separately from the images containing other vessels. The camera was linked to a Pentium 200 running AIS (analytical imaging station) image analysis software (Berthold Australia, Bundoora, Victoria, Australia).

Each blood vessel in the four areas chosen was measured for the visible vessel wall area as defined by a hand drawn line surrounding the vessel profile, with a second line excluding the lumen. Other factors measured were the intensity of the immunostaining and proportional area of vessel wall immunostained. Vessel wall area was determined by measuring the total number of pixels scanned for the vessel and converting the area measured to µm2. A score was assigned to each vessel based on the intensity of the immunostainingxthe proportional area of the vessel wall stained. As the image analysis system measured intensity using the transmission of light through the section, this led to a scale of results where 0 = intense staining and 1 = little/no staining. For analysis this data was transformed so that 0 = little or no staining and 1 = intense staining using the formula 1-IOD (integrated optical density)xproportional area. Staining for vessels and vessel size (for straight arterioles) were averaged across the section, while the staining for the spiral arterioles, when present, was scored separately. Statistical analysis was performed using one-way analysis of variance and Student's t-test using the statistical package SPSS 6.1.3 (SPSS, Chicago, IL, USA) for Windows.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
General staining patterns
The antibody to MHC stained the cytoplasm of the VSMC strongly, with some background present in the stromal cells. In comparison the {alpha}SMA immunostaining, also located in the VSMC cytoplasm, was more variable in appearance with some vessels staining lighter than others (Figure 1Go). Several vessels of various sizes stained in each section, with between 1–2 layers of VSMC. Since sections taken from each sample were 5 µm apart, vessel profiles could change size and shape; however, vessels were always recognizable between the two sections. Straight arteriole numbers ranged from 1–4 per section and 7–12 measured per sample. On average there were more smaller vessels than larger ones per section. Examples of different staining intensities for both MHC and {alpha}SMA in straight arterioles can be seen in Figure 1Go. Spiral arteriole profiles immunostained strongly for the {alpha}SMA antibody, while MHC expression was more variable. Examples of spiral arteriole immunostaining can be seen in Figure 2Go.



View larger version (147K):
[in this window]
[in a new window]
 
Figure 1. Examples of differing patterns of smooth muscle alpha actin ({alpha}SMA) and myosin heavy chains (MHC) immunostaining in straight arterioles. Late secretory control sample, showing (A) moderate {alpha}SMA staining with (B) strong MHC staining. Early secretory control sample, showing (C) strong {alpha}SMA staining and (D) moderate MHC staining. Late secretory menorrhagic sample showing (E) strong {alpha}SMA staining and (F) strong MHC staining. All serial sections 5 µm apart. Scale bar = 10 µm.

 


View larger version (144K):
[in this window]
[in a new window]
 
Figure 2. Examples of {alpha}SMA and MHC staining showing different patterns of staining in spiral arterioles. A sample from a mid-secretory control, showing (A) strong {alpha}SMA staining with (B) weak MHC staining, and a sample from a late secretory menorrhagic (the same as Figure 1E and FGo) showing (C) strong {alpha}SMA staining and (D) strong MHC staining. All serial sections 5 µm apart. Scale bar = 10 µm.

 
Spiral arterioles were found predominantly in the secretory and menstrual stages of the cycle as expected, with two examples being found in the proliferative stage of the cycle.

Straight arteriole staining
Across the cycle
When comparing control samples across the menstrual cycle, immunolocalization of {alpha}SMA and MHC did not vary significantly across the cycle (Figure 3A, BGo), although MHC expression appeared to be slightly increased in the mid-secretory phase of the cycle (Figure 3BGo). In menorrhagic tissues, {alpha}SMA expression was apparently decreased at the early/mid-secretory phases though not significantly when compared with the rest of the cycle (Figure 3AGo).



View larger version (23K):
[in this window]
[in a new window]
 
Figure 3. Expression of (A) {alpha}SMA and (B) MHC immunolocalization in straight arterioles across the menstrual cycle. IOD = integrated density, MEN = menstrual, EP = early proliferative, MP = mid-proliferative, ES = early secretory, MS = mid-secretory, LS = late secretory. {blacksquare} = controls, {square} = menorrhagics. *P <= 0.05 representing a significant decrease in menorrhagic {alpha}SMA expression.

 
Control compared with menorrhagic
{alpha}SMA expression was significantly decreased in menorrhagic tissues compared with control tissues at the mid-secretory stage (0.67 ± 0.03 versus 0.55 ± 0.04, P <= 0.05). {alpha}SMA expression also appeared to be reduced in the early secretory stage in menorrhagic women compared with controls, but this difference was not statistically significant (Figure 3AGo). No significant differences in MHC expression were observed between control and menorrhagic women.

Total vessel wall/VSMC area
Straight arterioles
No significant difference was found between the vessel wall area measured in menorrhagic tissues compared with controls with either {alpha}SMA (Figure 4Go) or MHC (data not shown). No significant difference in total vessel wall area measured per section occurred between cycle stages, or in comparisons of arterioles between control and menorrhagic tissues.



View larger version (21K):
[in this window]
[in a new window]
 
Figure 4. Total vascular smooth muscle cell (VSMC) area of straight arterioles stained for {alpha}SMA per section. For abbreviations, see Figure 3Go. {blacksquare} = controls, {square} = menorrhagics.

 
Spiral arteriole staining
MHC expression was significantly reduced in the VSMC of spiral arterioles in menorrhagic women compared with controls at the early secretory stage (Figure 5BGo; 0.57 ± 0.01 versus 0.38 ± 0.04, P <= 0.05). No differences were observed in {alpha}SMA expression when comparing control tissues with menorrhagics (Figure 5AGo). No significant differences were found across the menstrual cycle stages for either MHC or {alpha}SMA in the spiral arterioles.



View larger version (16K):
[in this window]
[in a new window]
 
Figure 5. Expression of (A) {alpha}SMA and (B) MHC immunolocalization in spiral arterioles across the menstrual cycle. For abbreviations, see Figure 3Go. M/P = menstrual/proliferative. {blacksquare} = controls, {square} = menorrhagics. *P <= 0.05.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Using the immunolocalization methods described, we have examined {alpha}SMA and MHC chain expression across the cycle in both straight and spiral arterioles. We observed a significant reduction in MHC expression in the VSMC of endometrial spiral arterioles in menorrhagic women compared to controls at the early secretory stage. No significant differences were observed in {alpha}SMA expression in spiral arterioles between the two groups. In the straight arterioles, we found that {alpha}SMA expression levels were significantly lower in menorrhagic tissues at the mid-secretory stage and appeared to be reduced in the early secretory stage. In contrast, no significant differences were observed in MHC expression across the menstrual cycle or between control and menorrhagic tissues in the straight arterioles.

Although the MHC antibody does not differentiate between MHC1 and MHC2, both these proteins are expressed after {alpha}SMA in the differentiation and maturation cycle of VSMC (Aikawa et al., 1993Go; Miano et al., 1994Go; Owens, 1995Go). The reduction in MHC expression in the VSMC of spiral arterioles at the early secretory stage in menorrhagic tissues suggests that these VSMC may be slower to differentiate into a mature VSMC phenotype than those in control tissues. Although MHC expression in the spiral arterioles of menorrhagic women has reached similar levels to controls by the mid- and late secretory cycle stages, it is possible that the later maturation of the VSMC may affect the structural integrity of the arteriole wall. For example, extracellular matrix proteins such as collagen and elastin are synthesized by VSMC and these may be reduced or compromised, though further study is required to determine if such a reduction occurs. Synthesis rates vary between phenotypic states in VSMC, being decreased in the more contractile phenotype. It is possible that the delay in differentiation and maturation could affect the secretion of the extracellular matrix of the vessel wall. A change in the structure of the extracellular matrix could weaken the spiral arterioles, affecting contractility, or offer an altered or weakened target for the matrix metalloproteinases active at menstruation. Together with a reduction in proliferation in the VSMC of spiral arterioles from menorrhagic women reported in our previous study (Abberton et al., 1999Go), the data suggest that a less competent or functional population of VSMC may exist in the spiral arterioles of menorrhagic women.

Previous work in our laboratory has shown that VSMC proliferation is significantly decreased in the mid–late secretory stages of the cycle in the spiral arterioles of menorrhagic women. As it had been reported that {alpha}SMA concentrations were decreased in proliferating VSMC (Desmoulière et al., 1981Go; Aikawa et al., 1993Go; Shanahan et al., 1993Go) in vitro, we expected to see this reflected in our current results, i.e. that menorrhagic women, having less spiral arteriole VSMC proliferation, would have more {alpha}SMA and MHC expression. This did not prove to be the case, suggesting that in the endometrium the reduction of VSMC contractile proteins is not required for proliferation. This finding is supported by other studies in vivo (Clowes et al., 1988Go; Hansson et al., 1989Go) and in vitro (Holycross et al., 1992Go), suggesting that the loss of contractile proteins is independent of proliferation. Compared to other vascular beds in the adult, the endometrial vasculature has a high level of VSMC proliferation. It is possible that the lack of change in contractile protein expression indicates that spiral arterioles possess a population of VSMC that can proliferate without de-differentiating fully. This may be necessary when proliferation is occurring at the same time that contractile activity is required. The situation is similar to the developing blood vessels in the embryo, where VSMC can express contractile proteins and proliferate at the same time (Shanahan et al., 1993Go).

The lack of change in contractile protein expression in the VSMC of the spiral arterioles in the mid–late secretory stages of the cycle, when VSMC proliferation is occurring, may simply be due to the number and type of VSMC mitogens present in the endometrium. Although it is unknown which mitogens control VSMC development in the spiral arterioles, possible candidates found around the spiral arterioles include endothelin (Salamonsen et al., 1992Go; Marsh et al., 1997Go), angiotensin II (Li and Ahmed, 1996Go), transforming growth factor alpha (TGF{alpha})/epidermal growth factor (Giudice, 1994Go; Stewart and Nowak, 1996Go), TGFß (Gold et al., 1994Go) and platelet derived growth factor (PDGF). Of these factors TGF{alpha} and PDGF regulate {alpha}SMA and MHC expression independently of their mitogenic activity. The consistent expression of these contractile proteins suggests that these factors are not activated at this time and that other factors such as endothelin may be controlling VSMC mitogenesis.

In the straight arterioles, there were no significant changes in MHC or {alpha}SMA levels across the cycle in either controls or menorrhagic tissues, though MHC levels did increase in the mid-proliferative stage for menorrhagics and the mid-secretory stage for control tissues. {alpha}SMA levels did vary across the cycle in menorrhagic tissues but not significantly. {alpha}SMA expression was significantly lower in menorrhagic women compared to controls at the mid-secretory stage of the cycle and varied from the controls (lower in early secretory, higher in proliferative stages) at other stages, though not significantly (Figure 3AGo). The variations in {alpha}SMA expression in the straight arterioles does not appear to fit a pattern. It is possible that this reflects the normal arteriole growth and differentiation during the cycle. Alternatively, this may reflect differences in VSMC populations in different vessel types. The high variability found in samples in this and previous studies and the lack of significant difference in the total vessel wall area stained in control and menorrhagic samples suggests VSMC heterogeneity may influence arteriole development. Markee (1940) estimated that up to 25% of menstrual blood loss (MBL) was lost from arterioles other than the spiral arterioles. It is possible that some VSMC populations are less competent to act in arteriole growth and function than others, and that the arterioles in menorrhagic women are compromised, contributing in part to menorrhagia.

In a previous study we observed that more vessels in perimenopausal women expressed {alpha}SMA compared with non-perimenopausal women, particularly in perimenopausal menorrhagic women (Abberton et al., 1996Go), and suggested that this may be an age-related phenomenon as the perimenopausal women were significantly older than the reproductive age group (mean age 49 versus 43 years). The first study used {alpha}SMA as a VSMC marker only, and the differences found between groups reflected a change in arteriole numbers between perimenopausal and non-perimenopausal women. {alpha}SMA intensity was used as a semi-quantitative score to identify the amount of VSMC surrounding a vessel. Rather than total arteriole numbers or the amount of VSMC surrounding a blood vessel, the current study used objective quantification of MHC and {alpha}SMA expression to study the VSMC phenotypes in control and menorrhagic tissues by looking at the relative amounts of these proteins in the vessel wall rather than the number of vessels they are expressed in. Lower sample numbers in this study did not allow separation into perimenopausal and non-perimenopausal groups and the average ages of the control and menorrhagic women were not significantly different (mean age 44.8 versus 46.4 years). Preliminary analysis suggested there were no significant differences between the samples in this study, allowing them to be combined. The lack of age-related difference in this study may be due to the fact that rather than total vessels with VSMC being counted and analysed, a representative number was analysed from each section, i.e. the perimenopausal sections may have more vessels expressing {alpha}SMA than non-perimenopausal women, but the individual vessels chosen at random may not be expressing higher amounts of {alpha}SMA.

As with many studies using human tissue, intersample variability was quite large and may have masked some cyclic differences in MHC and {alpha}SMA expression. Changes in vessel shapes between sections can affect the area of vessel showing immunostaining, thus increasing variability between samples. It is also possible that the immunolocalization methods are not sensitive enough to pick up minor changes in expression levels of the contractile proteins. However, use of the computerized image analysis increased the sensitivity and objectivity of the study. The presence of some spiral arterioles in the proliferative stages of the cycle is not surprising, though they are usually shorter and less coiled than those in the secretory phase of the cycle. As not all of the endometrium is shed at menstruation, it is possible that some highly coiled arterioles may not have been shed and thus continue to grow through the following proliferative phase of the cycle.

In conclusion we have found a significant reduction of MHC expression in the spiral arterioles of menorrhagic women at the early secretory stage suggesting that these VSMC are maturing later than spiral arterioles in control tissues, which may affect MBL control and hence menorrhagia. Variations in {alpha}SMA expression in straight arterioles also occurred, with the only significant finding being a decrease in expression in menorrhagic tissues at the mid-secretory phase of the cycle. No significant difference was found in the total vessel wall area stained and scored between control and menorrhagic women, suggesting that the variations in MHC and {alpha}SMA expression may represent differing phenotype profiles in the VSMC in all women, some of which are related to menorrhagia.


    Notes
 
2 Present address: Department of Pharmacology and Physiology, University of Rochester Medical Center, Box 711, University of Rochester, Rochester, NY 14642, USA Back

3 To whom correspondence should be addressed at: Department of Pharmacology and Physiology, University of Rochester Medical Center, Box 711, University of Rochester, Rochester, NY 14642, USA Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Abberton, K.M., Taylor, N.H., Healy, D.L. et al. (1996) Vascular smooth muscle {alpha}-actin distribution around endometrial arterioles during the menstrual cycle: increased expression during menopause and lack of correlation with menorrhagia. Hum. Reprod., 11, 204–211.[Abstract]

Abberton, K.M., Taylor, N.H., Healy, D.L. and Rogers, P.A.W. (1999) Vascular smooth muscle cell proliferation in arterioles of the human endometrium. Hum. Reprod., 14, 1072–1079.[Abstract/Free Full Text]

Aikawa, M., Sivam, P.N., Kuro-o, M. et al. (1993) Human smooth muscle myosin heavy chain isoforms as molecular markers for vascular development and atherosclerosis. Circ. Res., 73, 1000–1012.[Abstract]

Clowes, A.W., Clowes, M.M., Kocher, O. et al. (1988) Arterial smooth muscle cells in vivo: Relationship between actin isoform expression and mitogenesis and their modulation by heparin. J. Cell. Biol., 107, 1939–1945.[Abstract]

Desmouliere, A., Rubbia-Brandt, L. and Gabbiani, G. (1981) Modulation of actin isoform expression in cultured arterial smooth muscle cells by heparin and culture conditions. Arterioscl. Thromb., 11, 244–253.[Abstract]

Fraser, I.S. (1992) Treatment of menorrhagia. In Drife, O.J. and Calder, A.A. (eds), Prostaglandins and the Uterus. Springer-Verlag, London, pp. 67–92.

Giudice, L.C. (1994) Growth factors and growth modulators in human uterine endometrium: their potential relevance to reproductive medicine. Fertil. Steril., 61, 1–17.[ISI][Medline]

Gold, L.I., Saxena, B. and Mittal, K.R. et al. (1994) Increased expression of transforming growth factor {alpha} isoforms and basic fibroblast growth factor in complex hyperplasia and adenocarcinoma of the endometrium: evidence for paracrine and autocrine action. Cancer Res., 54, 2347–2358.[Abstract]

Hansson, G.K., Hellstrand, M., Rymo, L. et al. (1989) Interferon {alpha} inhibits both proliferation and expression of differentiation specific {alpha}-smooth muscle actin in arterial smooth muscle cells. J. Exp. Med., 170, 1595–1608.[Abstract]

Haynes, P.J., Anderson, A.B.M. and Turnball, A.C. (1979) Patterns of menstrual blood loss in menorrhagia. Res. Clin. Forums, 1, 73–78.

Holycross, B.J., Blank, R.S., Thompson, M.M. et al. (1992) Platelet-derived growth factor-BB induced suppression of smooth muscle cell differentiation. Circ. Res., 71, 1525–1532.[Abstract]

Li, X.F. and Ahmed, A. (1996) Dual role of angiotensin II in the human endometrium. Hum. Reprod., 11 (Suppl. 2), 95–108.[Abstract]

Markee, J.E. (1940) Menstruation in intraocular endometrial transplants in the Rhesus monkey. Cont. Embryol., 28, 219–308.

Marsh, M.M., Malakooti, N., Taylor, N.H. et al. (1997) Endothelin and neutral endopeptase in the endometrium of women with menorrhagia. Hum. Reprod., 12, 2036–2040.[Abstract]

McKinley, S.M., Brambilla, D.J. and Posner, J.G. (1992) The normal menopause transition. Maturitas, 14, 103–115.[ISI][Medline]

Miano, J.M., Cserjesi, P., Ligon, K.L. et al. (1994) Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ. Res., 75, 803–812.[Abstract]

Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3–25.[ISI][Medline]

Owens, G.K. (1995) Regulation of differentiation of vascular smooth muscle cells. Am. J. Physiol., 75, 487–517.

Price, R.J., Owens, G.K. and Skalak, T.C. (1994) Immunohistochemical identification of arteriolar development using markers of smooth muscle differentiation: evidence that capillary arterialization proceeds from terminal arterioles. Circ. Res., 75, 520–527.[Abstract]

Ramsey, E. (1977) Vascular anatomy. In Wynn, R.M. (ed.), Biology of the Uterus. Plenum Press, New York, pp. 59–76.

Salamonsen, L.A., Butt, A.R., Macpherson, A.M. et al. (1992) Immunolocalisation of the vasoconstrictor endothelin in the human endometrium during the menstrual cycle and in umbilical cord at birth. Am. J. Obstet. Gynaecol., 167, 163–167.[ISI][Medline]

Schwartz, S.M. and Liaw, L. (1993) Growth control and morphogenesis in the development and pathology of arteries. J. Cardiovasc. Pharm., 21 (Suppl. 1), S31–S49.[ISI][Medline]

Shanahan, C.M., Weissberg, P.L. and Metcalfe, J.C. (1993) Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells. Circ. Res., 73, 193–208.[Abstract]

Sheppard, B.L. and Bonner, J. (1979) The development of vessels of the endometrium during the menstrual cycle. In Diczfalusy, E., Fraser, I.S., Webb, F.T.G. (eds), Endometrial Bleeding and Steroidal Contraception. Pitman Press, Bath, pp. 65–85.

Skalli, O., Ropraz, P., Trzeciak, A. et al. (1986) A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell Biol., 103, 2787–2796.[Abstract]

Stewart, E.A. and Nowak, R.A. (1996) Leiomyoma-related bleeding: a classic hypothesis updated for the molecular era. Hum. Reprod. Update, 2, 295–306.[Abstract/Free Full Text]

Submitted on April 26, 1999; accepted on September 22, 1999.