Activity and expression of soluble and particulate guanylate cyclases in myometrium from nonpregnant and pregnant women: down-regulation of soluble guanylate cyclase at term.

The role of cGMP in the regulation of human myometrial smooth muscle contractility is at present unclear. cGMP can be synthesized by a cytoplasmic, soluble guanylate cyclase (sGC), which is stimulated by nitric oxide and carbon monoxide, and by particulate membrane-bound GC, which are activated by natriuretic peptides. The aim of this study was to determine whether sGC or pGC are present in nonpregnant and pregnant human myometrium, and whether the activity and expression of these enzymes and the cGMP content change during pregnancy and with labor. Myometrium was obtained from nonpregnant women (n = 12) and pregnant women who were preterm (25-34 wk gestation; n = 12), term (>38 wk) not in labor (n = 14), or term in active labor (n = 12). The cGMP content in myometrium obtained from preterm deliveries was significantly higher than that in tissue obtained from nonpregnant women and decreased at term, especially in laboring groups. Protein and mRNA for sGC, particulate GC-A, GC-B, and the clearance receptor were detected in human myometrium. cGMP in pregnant human myometrium, however, appears to be produced predominantly by sGC and possibly by GC-B, as GC-A was only weakly expressed. sGC activity was greater in myometrium from preterm (nonlabor) deliveries compared those taken at term (in labor), but was down-regulated compared with activity in nonpregnant myometrium. Neither atrial natriuretic peptide nor C-type natriuretic peptide (agonists for GC-A and GC-B, respectively) altered contractility in vitro of myometrium from women at term (not in labor). We conclude that the cGMP/guanylate cyclase system in human myometrium is gestationally regulated and potentially plays an important role in mediating quiescence during early pregnancy. A reduction in cGMP availability may contribute to the switch to contractile activity at term.

Normal expression of cell adhesion molecules in placentae from women with systemic lupus erythematosus and the antiphospholipid syndrome.

Pregnant women with active systemic lupus erythematosus (SLE) and/or the antiphospholipid syndrome (APS) are prone to recurrent miscarriage, pre-eclampsia, intrauterine growth restriction and premature delivery. Placental dysfunction may account for these complications yet the mechanisms remain uncertain. Amongst these, an inflammatory response in the placental vasculature could play a role, involving recruitment of neutrophils and platelets and the increased endothelial expression of cell adhesion molecules (CAM), central to the recruitment process. The aim of this study was primarily to investigate CAM expression in the fetoplacental vasculature in women with SLE/APS. Circulating maternal concentrations of soluble CAM were also elucidated. There were no differences in CAM immunostaining in placentae from patients with SLE and/or APS compared with controls. In both patients and controls moderate immunostaining for the intercellular adhesion molecule-1 (ICAM-1) was observed in placental vascular endothelium and mild immunostaining was present in the placental villous stroma. Strong immunostaining for platelet endothelial CAM (PECAM) occured in the placental vascular endothelium whereas P-selectin was mildly expressed in the stem vessel endothelium only. Vascular CAM-1 (VCAM-1) and E-selectin were undetectable in either study or control placentae. In contrast, ICAM-1 and VCAM-1 but not E-selectin, as assessed by immunoassay (ELISA), were elevated in maternal serum from SLE/APS patients compared with controls. This study suggests that upregulation of CAM expression and subsequent activation of neutrophil and/or platelet activity within the placental villous tree is unlikely to be a mechanism by which the adverse pregnancy outcome arises in SLE/APS pregnancies. However, maternal endothelial cell activation (ECA) may play a more important role.

Expression of nitric oxide synthase isoforms in pregnant human myometrium.

1. Endogenous nitric oxide has been proposed to play a role in the control of myometrial contractility in pregnancy. In this study, the expression, localisation and regulation of nitric oxide synthase (NOS) isoforms have been examined in human pregnant myometrium and cultured human myometrial smooth muscle cells, by immunoblotting, immunohistochemistry and reverse transcription-polymerase chain reaction. 2. Immunoblotting of extracts from freshly isolated myometrial tissue, affinity-enriched for NOS proteins by precipitation with ADP-sepharose, revealed expression of endothelial NOS (eNOS or NOS3) in tissues from preterm, term non-labour and active labour at term. Inducible NOS (iNOS or NOS2) and neuronal NOS (nNOS or NOS1) proteins were not detected at any stage of pregnancy. 3. Immunohistochemical detection showed that expression of eNOS protein was restricted to the endothelium of the myometrial vasculature, with no staining detected in myometrial smooth muscle cells. 4. Messenger RNA for all three NOS isoforms was detected, although iNOS and nNOS mRNAs were detectable only with high cycle number, implying a low copy number. 5. NOS isoforms were not detectable in human myometrial smooth muscle cells cultured from term non-labour pregnancies. Cytokine stimulation of cultured myometrial cells did not induce iNOS expression or nitrite accumulation in the culture medium, although both iNOS protein and nitrite release were detected in the human pulmonary epithelial cell line A549. 6. Levels of eNOS protein and of NOS mRNA expression were not correlated with gestational stage, suggesting that endogenously produced NO is not likely to be a modulator of myometrial tone during human pregnancy.

Expression of cyclo-oxygenase types-1 and -2 in human myometrium throughout pregnancy.

Human labour is associated with increased prostaglandin synthesis within the uterus. The aim of this study was to examine the expression of the two isoforms of the central prostaglandin synthetic enzyme, cyclo-oxygenase (COX-1 and COX-2) in human myometrium throughout pregnancy and to test the hypothesis that COX in the myometrium may play a role in labour onset. Expression of COX-1 and COX-2 at the mRNA level was analysed using reverse transcriptase-polymerase chain reaction (RT-PCR) and at the protein level using Western blotting. No significant changes of COX-1 RNA or protein expression were observed either with gestational age or labour. COX-2 mRNA and protein expression increased at term with significant up-regulation occurring prior to the onset of labour (P < 0.005). These data would suggest that up-regulation of COX-2, rather than COX-1, mediates increased prostaglandin synthesis in human myometrium at term. The increased COX-2 expression observed preceded labour onset, suggesting that COX-2 has a role in labour onset, rather than its presence merely a consequence of labour.

Effects of corticotrophin releasing hormone on contractile activity of myometrium from pregnant women.

OBJECTIVE: To determine whether corticotrophin releasing hormone plays a role in the regulation of tone in term nonlabouring human myometrium. SETTING: A teaching hospital research laboratory. SAMPLE: Thirty-seven women undergoing elective nonlabour caesarean section under regional anaesthesia. METHODS: Human corticotrophin releasing hormone (1, 10, 100 nmol/L) was added to strips of term, nonlabouring myometrium mounted in an organ bath, and the effect on spontaneous, oxytocin (1 nmol/L) or prostaglandin F2alpha (100 nmol/L) stimulated contractions determined. Cyclic adenosine monophosphate (cAMP) content of the tissue was also determined by enzyme immunoassay. RESULTS: Corticotrophin releasing hormone did not affect myometrial tension development in any of the experimental protocols. cAMP increased transiently after addition of corticotrophin releasing hormone (166.7 +/- 12.7% at 1 minute) but this was not reflected by any change in tension. CONCLUSIONS: This study suggests that despite high maternal plasma concentrations of corticotrophin releasing hormone in pregnancy at term, this peptide is unlikely to play a direct role in the control of myometrial contractility in nonlabouring myometrium.

Normal expression of tissue factor, thrombomodulin, and annexin V in placentas from women with antiphospholipid syndrome.

OBJECTIVE: Placentas from pregnancies complicated by antiphospholipid syndrome often show thromboses and infarction, which may result from aberrations in placental coagulant pathways. We tested the hypothesis that alterations in tissue factor, thrombomodulin, and annexin V expressions contribute to poor pregnancy outcome associated with antiphospholipid syndrome. STUDY DESIGN: Frozen sections from random biopsy samples of the basal plates of placentas from patients with primary antiphospholipid syndrome (n = 9), patients with secondary antiphospholipid syndrome (n = 3), and gestational age-matched control subjects (n = 10) were immunostained for tissue factor, thrombomodulin, and annexin V. Intensity of immunostaining was assessed by means of quantitative image analysis. Annexin V protein expression was evaluated with Western blotting techniques. RESULTS: Tissue factor was expressed in the perivascular cells of the villous vasculature. Thrombomodulin and annexin V immunostaining was localized to the syncytiotrophoblast. There were no differences in the intensity of immunostaining for tissue factor, thrombomodulin, and annexin V between placentas from women with antiphospholipid syndrome and those from control subjects. Western blot analysis of annexin V expression showed no differences between study patients and control subjects. CONCLUSION: Alterations in placental coagulant pathways involving tissue factor, thrombomodulin, and annexin V do not contribute to poor pregnancy outcome associated with antiphospholipid syndrome.

Changes in collagen metabolism in response to endothelin-1: evidence for fibroblast heterogeneity.

Endothelin-1 (Et-1) is a 21-amino acid peptide primarily synthesized by endothelial cells. It was originally classified as a potent vasoconstrictor but recent evidence suggests that it also possesses a wide variety of non-vascular actions. It stimulates fibroblast and smooth muscle cell proliferation and it has been shown to stimulate fibroblast collagen metabolism. However, studies on its ability to regulate collagen production remain incomplete, and its effect on post-translational processing of procollagen has not been studied. This report details the effect of Et-1 on the rates of procollagen synthesis and degradation in two fibroblast cell lines; human foetal lung (HFL-1) and whole foetal rat fibroblasts (Rat 2). Fibroblast cultures were incubated for 24 hr in the presence or absence of Et-1 before procollagen metabolism was determined by measuring hydroxyproline. Non-collagen metabolism was also determined in these cultures from the uptake of tritiated phenylalanine. Et-1 stimulated procollagen synthesis in HFL-1 fibroblasts and reduced synthesis in Rat 2 cells. The response was dose dependent with the greatest effect at 1.10(-6) M Et-1 for both cell types (155 +/- 6% of control (mean +/- SD, n = 6, P < 0.01) and 61 +/- 4% of control (n = 4, P < 0.01) for HFL-1 and Rat 2 fibroblasts, respectively). Non-collagen protein synthesis was increased to 148 +/- 5% of control (P < 0.05) at 1.10(-6) M Et-1. Non-collagen protein synthesis remained unaffected in the HFL-1 fibroblast cultures. Procollagen degradation, expressed as a proportion of total procollagen synthesis, was decreased in HFL-1 fibroblasts (control, 29 +/- 2%; Et-1, 1.10(-6) M; 21 +/- 2%; P < 0.01), and increased in Rat 2 fibroblasts (control 42 +/- 1%; Et-1, 1.10(-6) M; 49 +/- 1%; P < 0.01). Blocking of the EtA receptor for Et-1, using the receptor antagonist-BQ123, abolished the effect of Et-1 on procollagen metabolism in both cell types. These results suggest that different populations of fibroblasts exhibit heterogeneous responses to Et-1. It is concluded that Et-1 may play an important role in the extent and distribution of fibrosis seen in diseases associated with the overproduction of Et-1.

Age-related alterations in collagen and total protein metabolism determined in cultured rat dermal fibroblasts: age-related trends parallel those observed in rat skin in vivo.

The cultured fibroblast has been extensively used as a model system to study aging. However, few studies have examined the veracity of observations obtained in cultured fibroblasts aged in vitro to those made in animal tissues in vivo. This paper compares age-related alterations in collagen metabolism measured in cultured cells with previously reported results in the aging rat (Mays et al. (1991) Biochem. J. 276, 307-313). Age-related changes in collagen synthesis in rat skin fibroblasts in vitro over 30 population doublings were determined based on the production of hydroxy-[14C]proline. Degradation of newly synthesized collagen was based on the appearance of free hydroxy-[14C]proline in the culture system. Total protein synthesis rates were based on the incorporation of [14C]proline into proteins. In vitro rates of collagen synthesis decreased 5-fold over 30 population doublings (P < 0.05). Degradation of newly synthesized collagen increased from 33.0 +/- 0.8% (n = 4, SEM) to 45.2 +/- 1.1% (n = 4; P < 0.05) over the same period, with a maximum after 25 population doublings of 55.8 +/- 1.1% (n = 4). Total protein synthesis rates decreased by one-half over 30 population doublings (P < 0.05). The results indicated that collagen production decreased as cells aged in vitro and that this was due to both changes in synthesis and degradation. The results demonstrate that age-related alterations in collagen and total protein metabolism of skin fibroblasts in culture were similar to those reported previously for skin in vivo, suggesting that for studies of these processes, fibroblasts in culture provide an appropriate model.

Cadmium selectively inhibits fibroblast procollagen production and proliferation.

Chronic inhalation of cadmium fumes has been associated with the development of emphysema, a disease characterized by extensive disruption of lung connective tissue. Cadmium is also an important contaminant of tobacco and may play a role in cigarette smoking-related emphysema. In this paper we investigated the effect of nontoxic doses of cadmium chloride (CdCl2) on fibroblast procollagen production and proliferation, key features of connective tissue repair following injury. CdCl2 inhibited fibroblast procollagen production in a dose-dependent manner in two different cell lines. For fetal rat fibroblasts, maximal effects were observed at 10 microM CdCl2, with values reduced by 82 +/- 6% (mean +/- SE, n = 6, P < 0.01) relative to control cells. In contrast, noncollagen protein synthesis by these cells was enhanced in the presence of CdCl2. In human fetal lung fibroblasts (HFL1), maximal inhibition of procollagen production (83 +/- 2%, P < 0.01) was observed at 40 microM CdCl2, whereas noncollagen protein synthesis was unaffected. In both cell lines the inhibition of procollagen production was shown to be due to decreased procollagen synthesis and an increase in the proportion of newly synthesized procollagen degraded. Cadmium also affected fibroblast proliferation in response to 2% serum, with values for fetal rat cells depressed by 17 +/- 4, 72 +/- 2, and 86 +/- 4% (all P < 0.01) compared with controls at 1, 5, and 10 microM CdCl2, respectively. These data show that cadmium selectively inhibits fibroblast procollagen production and also attenuates their mitogenic response to serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related changes in collagen synthesis and degradation in rat tissues. Importance of degradation of newly synthesized collagen in regulating collagen production.

During developmental growth, collagens are believed to be continuously deposited into an extracellular matrix which is increasingly stabilized by the formation of covalent cross-links throughout life. However, the age-related changes in rates of synthetic and degradative processes are less well understood. In the present study we measured rates of collagen synthesis in vivo using a flooding dose of unlabelled proline given with [14C]proline and determining production of hydroxy[14C]proline. Degradation of newly synthesized collagen was estimated from the amount of free hydroxy [14C]proline in tissues 30 min after injection. Collagen fractional synthesis rates ranged from about 5%/day in skeletal muscle to 20%/day in hearts of rats aged 1 month. At 15 months of age, collagen fractional synthesis rates had decreased markedly in lung and skin, but in skeletal muscle and heart, rates were unchanged. At 24 months of age, synthesis rates had decreased by at least 10-fold in all tissues, compared with rates at 1 month. The proportion of newly synthesized collagen degraded ranged from 6.4 +/- 0.4% in skin to 61.6 +/- 5.0% in heart at 1 month of age. During aging the proportion degraded increased in all tissues to maximal values at 15 months, ranging from 56 +/- 7% in skin to 96 +/- 1% in heart. These data suggest that there are marked age-related changes in rates of collagen metabolism. They also indicate that synthesis is active even in old animals, where the bulk of collagens produced are destined to be degraded.

The effect of transforming growth factor beta on rates of procollagen synthesis and degradation in vitro.

Transforming growth factor beta (TGF beta) is known to stimulate procollagen production and steady-state levels of procollagen mRNAs, but its ability to affect post-translational processing of procollagen has been little studied. This paper demonstrates the application of recently developed ultrasensitive methods for measuring hydroxyproline to assess rates of procollagen synthesis and degradation in vitro with and without TGF beta. Foetal rat fibroblasts synthesized 8.63 +/- 0.21 pmol hydroxyproline/micrograms DNA per h, which corresponds to approx. 40 molecules of procollagen/cell per s. Addition of TGF beta to cultures increased total amounts of procollagen synthesized and degraded by 112% and 82%, respectively, but there was a significant decrease in the proportion of procollagen degraded (control, 38.0 +/- 1.1%; TGF beta, 32.3 +/- 0.9%; P less than 0.005). This study demonstrates a novel mechanism which may contribute to the TGF beta-induced increase in procollagen production by fibroblasts.

Application of high-pressure liquid chromatography to studies of collagen production by isolated cells in culture.

Techniques for assessing collagen production by cells in culture are usually based on evaluation of uptake of radiolabeled proline into collagen. Although simple in theory, this approach is often flawed because of uncertainties concerning the specific activity of labeled proline in the precursor pool for collagen synthesis. An alternative approach is to assess collagen production directly by measuring hydroxyproline in proteins secreted by cultured cells, although this has been difficult, due to the insensitivity of the methods available. Here we apply high-pressure liquid chromatography using reverse-phase elution of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole derivatives of hydroxyproline to measure collagen production by fibroblasts. The method is easy to perform and allows quantitation of hydroxyproline down to 5 pmol, making it applicable to fibroblasts in 12-well culture plates. Collagen production was shown to be constant over a period of 24 h, with a mean rate of 391 +/- 18 (SE n = 14) ng collagen/10(6) cells/h. Similar values were obtained using thin-layer chromatography and an enzyme-linked immunosorbent assay for type I collagen, but these techniques were judged to be less convenient and required additional assumptions compared with the technique described here in full.

Collagen in abdominal aortic aneurysm: typing, content, and degradation.

The collagen content of the media of infrarenal aorta has been compared in age- and sex-matched normal aorta and dilated and nondilated atherosclerotic aorta. The proportion of collagen was increased in aneurysmal aorta from 62% to 84% and appears to be the result of preferential elastin degradation. The ratio of type I to type III collagen, estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of specific cyanogenbromide peptides, did not vary significantly from 2:1 in any of the three groups of aortas. There was no evidence of increased collagenase activity in unruptured aneurysmal aorta. Collagenase activity was increased in ruptured aneurysmal aorta but could only be satisfactorily measured after resolution from the endogenous tissue inhibitor of metalloproteinases. We suggest that only limited collagen turnover occurs in the media of abdominal aortic aneurysms before rupture. A subgroup of three patients with a significant family history of aneurysm had lower amounts of type III collagen in the aortic media, suggesting that abnormalities in type III collagen may be one of the genetic factors contributing to familial clustering of aneurysms.

Elastin degradation in abdominal aortic aneurysms.

Histological sections through the walls of abdominal aortic aneurysms showed scarce and disrupted elastic tissue. The elastin content of the aneurysmal aortic media was only 8.1 +/- 3.2% dry defatted weight (n = 11). The elastin content of grossly normal age and anatomically matched aortic media was 35.0 +/- 3.2% dry weight (n = 4) and the elastin content of severely atherosclerotic, stenosed infrarenal aortic media was 22.0 +/- 7.2% dry weight (n = 6). There was an inverse correlation of elastin content with the elastinolytic activity of aortic media homogenates, r = -0.78. Elastase activity, measured by the hydrolysis of [3H]elastin, was highest in aneurysmal aortic homogenates, 92.1 +/- 43.7 U/mg protein (n = 18), falling to 46.9 +/- 13.3 U/mg protein (n = 13) in severely stenosed atherosclerotic aortic homogenates and 35.5 +/- 11.9 U/mg (n = 6) in grossly normal aortic homogenates. The elastinolytic activity of stenotic aorta contained leukocyte elastase as an important component. In aneurysmal homogenates leukocyte elastase was also found but the increased elastase activity resulted from a protease(s) (Mr 95,000) extracted in 2 M urea, having minimal specificity for alanyl bonds and no immunological cross-reactivity with leukocyte elastase.