1 Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria 3168, Australia
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Abstract |
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Key words: alpha smooth muscle actin/endometrium/menorrhagia/myosin heavy chains/vascular smooth muscle
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Introduction |
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Menorrhagia is excessive menstrual blood loss, >80 ml/cycle, and affects up to 15% of all women, increasing to 20% during the perimenopause (Fraser, 1992; McKinley et al., 1992
). 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., 1979
; Fraser, 1992
). 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., 1999
). These results agree with previous studies (Haynes et al., 1979
), 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, 1940
; 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, 1940), 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., 1988
; Holycross et al., 1992
; Owens, 1995
). 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, 1995). 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 (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., 1986
; Owens, 1995
). It is also reduced in VSMC undergoing mitosis (Clowes et al., 1988
). Myosin heavy chain proteins (MHC) appear towards the end of VSMC differentiation (Schwartz and Liaw, 1993
; Price et al., 1994
; Owens, 1995
). This paper aims to investigate the amounts of the differentiation markers
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.
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Materials and methods |
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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 (SMA) immunostaining
Serial sections adjacent to those used for the MHC staining were selected for SMA immunolocalization following a similar protocol. Non-specific background staining was much lower on sections stained for
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-
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, 12 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.
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Results |
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Straight arteriole staining
Across the cycle
When comparing control samples across the menstrual cycle, immunolocalization of SMA and MHC did not vary significantly across the cycle (Figure 3A, B
), although MHC expression appeared to be slightly increased in the mid-secretory phase of the cycle (Figure 3B
). In menorrhagic tissues,
SMA expression was apparently decreased at the early/mid-secretory phases though not significantly when compared with the rest of the cycle (Figure 3A
).
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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 SMA (Figure 4
) 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.
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Discussion |
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Although the MHC antibody does not differentiate between MHC1 and MHC2, both these proteins are expressed after SMA in the differentiation and maturation cycle of VSMC (Aikawa et al., 1993
; Miano et al., 1994
; Owens, 1995
). 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., 1999
), 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 midlate secretory stages of the cycle in the spiral arterioles of menorrhagic women. As it had been reported that SMA concentrations were decreased in proliferating VSMC (Desmoulière et al., 1981
; Aikawa et al., 1993
; Shanahan et al., 1993
) 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
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., 1988
; Hansson et al., 1989
) and in vitro (Holycross et al., 1992
), 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., 1993
).
The lack of change in contractile protein expression in the VSMC of the spiral arterioles in the midlate 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., 1992; Marsh et al., 1997
), angiotensin II (Li and Ahmed, 1996
), transforming growth factor alpha (TGF
)/epidermal growth factor (Giudice, 1994
; Stewart and Nowak, 1996
), TGFß (Gold et al., 1994
) and platelet derived growth factor (PDGF). Of these factors TGF
and PDGF regulate
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 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.
SMA levels did vary across the cycle in menorrhagic tissues but not significantly.
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 3A
). The variations in
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 SMA compared with non-perimenopausal women, particularly in perimenopausal menorrhagic women (Abberton et al., 1996
), 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
SMA as a VSMC marker only, and the differences found between groups reflected a change in arteriole numbers between perimenopausal and non-perimenopausal women.
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
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
SMA than non-perimenopausal women, but the individual vessels chosen at random may not be expressing higher amounts of
SMA.
As with many studies using human tissue, intersample variability was quite large and may have masked some cyclic differences in MHC and 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 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
SMA expression may represent differing phenotype profiles in the VSMC in all women, some of which are related to menorrhagia.
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Notes |
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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
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References |
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Submitted on April 26, 1999; accepted on September 22, 1999.