Screening for pre-eclampsia with competing-risks model using placental growth factor measurement in blood samples collected before 11 weeks' gestation.

OBJECTIVES: To describe the distributional properties and assess the performance of placental growth factor (PlGF) measured in blood samples collected before 11 weeks' gestation in the prediction of pre-eclampsia (PE). METHODS: The study population consisted of pregnant women included in the Pre-eclampsia Screening in Denmark (PRESIDE) study with a PlGF measurement from the routine combined first-trimester screening (cFTS) blood sample collected at 8-14 weeks' gestation. PRESIDE was a prospective multicenter study investigating the predictive performance of the Fetal Medicine Foundation (FMF) first-trimester screening algorithm for PE in a Danish population. In the current study, serum concentration of PlGF in the cFTS blood samples was analyzed in batches between January and June 2021. RESULTS: A total of 8386 pregnant women were included. The incidence of PE was 0.7% at < 37 weeks' gestation and 3.0% at ≥ 37 weeks. In blood samples collected at 10 weeks' gestation, PlGF multiples of the median (MoM) were significantly lower in pregnancies with preterm PE < 37 weeks compared to unaffected pregnancies. However, PlGF MoM did not differ significantly between pregnancies with PE and unaffected pregnancies in samples collected before 10 weeks' gestation. CONCLUSIONS: The gestational-age range for PlGF sampling may be expanded from 11-14 to 10-14 weeks when assessing the risk for PE using the FMF first-trimester screening model. There is little evidence to support the use of PlGF in blood samples collected before 10 weeks' gestation. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Pre-eclampsia screening in Denmark (PRESIDE): national validation study.

OBJECTIVES: To investigate the predictive performance of the Fetal Medicine Foundation (FMF) first-trimester screening algorithm for pre-eclampsia in a Danish population and compare screening performance with that of the current Danish strategy, which is based on maternal risk factors. METHODS: This was a prospective study of women with a singleton pregnancy attending for their first-trimester ultrasound scan and screening for aneuploidies at six Danish university hospitals between May 2019 and December 2020. Prenatal data on maternal characteristics and medical history were recorded, and measurements of mean arterial pressure (MAP), uterine artery pulsatility index (UtA-PI), serum pregnancy-associated plasma protein-A (PAPP-A) and serum placental growth factor (PlGF) were collected without performing a risk assessment for pre-eclampsia. Information on acetylsalicylic acid use was recorded. After delivery, pregnancy outcome, including gestational age at delivery and pre-eclampsia diagnosis, was recorded. Pre-eclampsia risk assessment for each woman was calculated blinded to outcome using the FMF screening algorithm following adjustment to the Danish population. Detection rates (DRs) of the FMF algorithm were calculated for a fixed screen-positive rate (SPR) of 10% and for the SPR achieved in the current Danish screening. RESULTS: A total of 8783 pregnant women were included, with a median age of 30.8 (interquartile range (IQR), 28.1-33.9) years. The majority were white (95%), naturally conceiving (90%), non-smokers (97%) and had no family history of pre-eclampsia (96%). The median body mass index was 23.4 (IQR, 21.2-26.6) kg/m2 . A complete risk assessment including maternal characteristics, MAP, UtA-PI, PlGF and PAPP-A was available for 8156 women (92.9%). In these women, UtA-PI was measured bilaterally with a median value of 1.58 (IQR, 1.27-1.94) and the median resting MAP of 80.5 (IQR, 76.1-85.4) mmHg in two consecutive measurements. Among these, 303 (3.7%) developed pre-eclampsia, including 55 (0.7%) cases of pre-eclampsia with delivery < 37 weeks of gestation and 16 (0.2%) cases of pre-eclampsia with delivery < 34 weeks. At a SPR of 10%, combined screening using the FMF algorithm based on maternal characteristics, MAP, UtA-PI, PlGF and PAPP-A had a DR of 77.4% (95% CI, 57.6-97.2%) for pre-eclampsia with delivery < 34 weeks, 66.8% (95% CI, 54.4-79.1%) for pre-eclampsia with delivery < 37 weeks and 44.1% (95% CI, 38.5-49.7%) for pre-eclampsia with delivery at any gestational age. The current Danish screening strategy using maternal risk factors detected 25.0% of women with pre-eclampsia with delivery < 34 weeks and 19.6% of women with pre-eclampsia with delivery < 37 weeks at a SPR of 3.4%. When applying the FMF algorithm including maternal characteristics, MAP, UtA-PI and PlGF at the fixed SPR of 3.4%, the DRs were 60.5% (95% CI, 36.9-84.1%) for PE with delivery < 34 weeks and 45.2% (95% CI, 32.0-58.5%) for PE with delivery < 37 weeks. CONCLUSION: In this large Danish multicenter study, the FMF algorithm based on maternal characteristics, MAP, UtA-PI, PlGF and PAPP-A predicted 77.4% of cases with pre-eclampsia with delivery < 34 weeks and 66.8% of cases with pre-eclampsia with delivery < 37 weeks of gestation at a SPR of 10%, suggesting that the performance of the algorithm in a Danish cohort matches that in other populations. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

The brown bear as a translational model for sedentary lifestyle-related diseases.

Sedentary lifestyle accelerates biological ageing, is a major risk factor for developing metabolic syndrome and is associated with cardiovascular disease, diabetes mellitus, kidney failure, sarcopenia and osteoporosis. In contrast to the linear path to worsening health in humans with metabolic syndrome, brown bears have developed a circular metabolic plasticity enabling these animals to tolerate obesity and a 'sedentary lifestyle' during hibernation and exit the den metabolically healthy in spring. Bears are close to humans physiology wise, much closer than rodents, the preferred experimental animals in medical research, and may better serve as translational model to develop treatments for lifestyle-related diseases. In this review, aspects of brown bear hibernation survival strategies are outlined and conceivable experimental strategies to learn from bears are described.

Antimicrobial peptide CAP18 and its effect on Yersinia ruckeri infections in rainbow trout Oncorhynchus mykiss (Walbaum): comparing administration by injection and oral routes.

The antimicrobial peptide CAP18 has been demonstrated to have a strong in vitro bactericidal effect on Yersinia ruckeri, but its activity in vivo has not been described. In this work, we investigated whether CAP18 protects rainbow trout Oncorhynchus mykiss (Walbaum) against enteric red mouth disease caused by this pathogen either following i.p. injection or by oral administration (in feed). It was found that injection of CAP18 into juvenile rainbow trout before exposure to Y. ruckeri was associated with lowered mortality compared to non-medicated fish although it was less effective than the conventional antibiotic oxolinic acid. Oral administration of CAP18 to trout did not prevent infection. The proteolytic effect of secretions on the peptide CAP18 in the fish gastrointestinal tract is suggested to account for the inferior effect of oral administration.

A robust immunoassay for pregnancy-associated plasma protein-A2 based on analysis of circulating antigen: establishment of normal ranges in pregnancy.

Pregnancy-associated plasma protein-A (PAPP-A) and PAPP-A2, two homologous metzincin metalloproteases, are both tightly linked to regulation within the insulin-like growth factor (IGF) system because of their specific cleavage of IGF binding proteins. Recent studies suggest that PAPP-A may be involved in clinical conditions related to unwanted cellular growth, and the circulating levels of PAPP-A is an established biomarker in prenatal screening for chromosomal abnormalities. Microarray data indicate that PAPP-A2 has potential as a biomarker for pre-eclampsia. However, well-characterized immunological methods of quantification are not available. We therefore developed monoclonal antibodies against recombinant PAPP-A2. The antibodies were epitope mapped against recombinantly expressed chimeras between PAPP-A2 and PAPP-A. Furthermore, circulating PAPP-A2 was immunoaffinity purified and characterized by sequence analysis and mass spectrometry. Unlike PAPP-A, PAPP-A2 is a noncovalent dimer in which each subunit of 1558 amino acids originates from all of the 22 predicted coding exons. A previously hypothesized variant (PAPP-E) does not exist, but low amounts of a C-terminally truncated PAPP-A2 variant was detected. A sensitive and robust ELISA for full-length PAPP-A2 was developed and used to establish normal ranges of PAPP-A2 through pregnancy. The functional sensitivity of this ELISA at 20% CV was 0.08 ng/ml, and the serum concentration of PAPP-A2 was found to increase during pregnancy in agreement with placental synthesis. The existence of this assay will enable an assessment of the biomarker potential of PAPP-A2 in pre-eclampsia as well as other clinical conditions.

Tamoxifen-independent Cre-activity in SMMHC-CreER T2 mice.

Background and aims: Recent technological advances have established vascular smooth muscle cells (SMCs) as central players in atherosclerosis. Increasingly complex genetic mouse models have unveiled that 30-70% of cells in experimentally induced atherosclerotic lesions derive from a handful of medial SMCs, and that these can adopt a broad range of plaque cell phenotypes. Most of these models are based on the SMMHC-CreER T2 mouse line as Cre-driver. Importantly, Cre-activation can be controlled in time (by administration of tamoxifen, TAM), which is critical to avoid unwanted effects of premature recombination events. The aim of this study was to scrutinize an unexpected observation of TAM-independent Cre-activity in this mouse line. Methods: Cre-activity was assessed by PCR in tissues from SMMHC-CreER T2 mice crossed with mice homozygous for loxP-flanked (floxed) exon 4 of Ccn2 (our gene-of-interest), and Ccn2 protein was measured in aortas by targeted mass spectrometry. Results: We observed spontaneous near-complete excision of floxed Ccn2 in aortas from adult mice that were not treated with TAM. As a result, Ccn2 protein was significantly reduced in aortas from these mice, but not to the same extent as TAM-treated littermates. Remarkably, most of the excision was completed in 4-week-old mice. Excision was Cre-dependent, as knockout bands were negligible in heart and liver (dominated by non-SMCs) of these mice, and undetectable in the aorta in the absence of Cre. Conclusion: Our observations warrant caution, and we advocate inclusion of appropriate controls (i.e., TAM-untreated mice) in future studies.

Basement membrane collagen IV deficiency promotes abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) is a complex disease which is incompletely accounted for. Basement membrane (BM) Collagen IV (COL4A1/A2) is abundant in the artery wall, and several lines of evidence indicate a protective role of baseline COL4A1/A2 in AAA development. Using Col4a1/a2 hemizygous knockout mice (Col4a1/a2+/-, 129Svj background) we show that partial Col4a1/a2 deficiency augmented AAA formation. Although unchallenged aortas were morphometrically and biomechanically unaffected by genotype, explorative proteomic analyses of aortas revealed a clear reduction in BM components and contractile vascular smooth muscle cell (VSMC) proteins, suggesting a central effect of the BM in maintaining VSMCs in the contractile phenotype. These findings were translated to human arteries by showing that COL4A1/A2 correlated to BM proteins and VSMC markers in non-lesioned internal mammary arteries obtained from coronary artery bypass procedures. Moreover, in human AAA tissue, MYH11 (VSMC marker) was depleted in areas of reduced COL4 as assessed by immunohistochemistry. Finally, circulating COL4A1 degradation fragments correlated with AAA progression in the largest Danish AAA cohort, suggesting COL4A1/A2 proteolysis to be an important feature of AAA formation. In sum, we identify COL4A1/A2 as a critical regulator of VSMC phenotype and a protective factor in AAA formation.

Reduced muscle activation during exercise related to brain oxygenation and metabolism in humans.

Maximal exercise may be limited by central fatigue defined as an inability of the central nervous system to fully recruit the involved muscles. This study evaluated whether a reduction in the cerebral oxygen-to-carbohydrate index (OCI) and in the cerebral mitochondrial oxygen tension relate to the ability to generate a maximal voluntary contraction and to the transcranial magnetic stimulated force generation. To determine the role of a reduced OCI and in central fatigue, 16 males performed low intensity, maximal intensity and hypoxic cycling exercise. Exercise fatigue was evaluated by ratings of perceived exertion (RPE), arm maximal voluntary force (MVC), and voluntary activation of elbow flexor muscles assessed with transcranial magnetic stimulation. Low intensity exercise did not produce any indication of central fatigue or marked cerebral metabolic deviations. Exercise in hypoxia (0.10) reduced cerebral oxygen delivery 25% and decreased 11+/-4 mmHg (P<0.001) together with OCI (6.2+/-0.7 to 4.8+/-0.5, P<0.001). RPE increased while MVC and voluntary activation were reduced (P<0.05). During maximal exercise declined 8+/-4 mmHg (P<0.05) and OCI to 3.8+/-0.5 (P<0.001). RPE was 18.5, and MVC and voluntary activation were reduced (P<0.05). We observed no signs of muscular fatigue in the elbow flexors and all control MVCs were similar to resting values. Exhaustive exercise provoked cerebral deoxygenation, metabolic changes and indices of fatigue similar to those observed during exercise in hypoxia indicating that reduced cerebral oxygenation may play a role in the development of central fatigue and may be an exercise capacity limiting factor.

Tissue microarrays compared with whole sections and biochemical analyses. A subgroup analysis of DBCG 82 b&c.

INTRODUCTION: The tissue microarray (TMA) technique comprises the potential of significantly reducing time and tissue spent on slicing and performing immunohistochemical (IHC) stainings of paraffin-embedded tumor tissue. Tissue heterogeneity is an argument against using TMAs, which has been dealt with by increasing the size and number of cores punched from each tumor. No consensus exists on the most optimal size, number, and position of TMA cores in the donor paraffin block and no information exist regarding agreement between TMA cores from two different paraffin blocks from the same tumor or between TMA cores and biochemical analyses. PATIENTS AND METHODS: A central and a peripheral 1mm core and a whole section from each of 54 paraffin blocks from 27 breast cancers included in a one-institution cohort, and a single 1mm central TMA core, from each breast tumor from 1000 patients included in the DBCG82 b&c trials, were IHC stained for ER, PgR and HER2. In addition, ER and PgR were measured in the DBCG82 b&c trials by a biochemical analysis. Statistical analyses included Kappa statistics, Kaplan-Meier survival curves, Log-rank tests, and Cox regression hazards analyses. RESULTS AND CONCLUSION: IHC stainings for ER, PgR, and HER2 showed a substantial agreement between a single 1mm TMA core and the corresponding whole section, between central and peripheral cores, and between cores from two different paraffin blocks from the same tumor. In addition, a fine agreement was found for ER and PgR between IHC stainings of TMA cores and biochemical analyses. Divergence between IHC and biochemical analyses was predominantly due to the chosen thresholds. IHC staining of one 1mm core from each tumor revealed a significant independent prognostic value of PgR and HER2 on overall survival. In conclusion, IHC stainings for ER, PgR, and HER2 of just a single 1mm TMA core seems to be sufficient, as no significant heterogeneity was noticed.

Impact of BCL2 and p53 on postmastectomy radiotherapy response in high-risk breast cancer. A subgroup analysis of DBCG82 b&c.

PURPOSE: To examine p53 and BCL2 expression in high-risk breast cancer patients randomized to postmastectomy radiotherapy (PMRT). PATIENTS AND METHODS: The present analysis included 1 000 of 3 083 high-risk breast cancer patients randomly assigned to PMRT in the DBCG82 b&c studies. Tissue microarray sections were stained with immunohistochemistry for p53 and BCL2. Median potential follow-up was 17 years. Clinical endpoints were locoregional recurrence (LRR), distant metastases (DM), overall mortality, and overall survival (OS). Statistical analyses included Kappa statistics, chi(2) or exact tests, Kaplan-Meier probability plots, Log-rank test, and Cox univariate and multivariate regression analyses. RESULTS: p53 accumulation was not significantly associated with increased overall mortality, DM or LRR probability in univariate or multivariate Cox regression analyses. Kaplan-Meier probability plots showed reduced OS and improved DM and LRR probabilities after PMRT within subgroups of both p53 negative and p53 positive patients. Negative BCL2 expression was significantly associated with increased overall mortality, DM and LRR probability in multivariate Cox regression analyses. Kaplan-Meier probability plots showed a significantly improved overall survival after PMRT for the BCL2 positive subgroup, whereas practically no survival improvement was seen after PMRT for the BCL2 negative subgroup. In multivariate analysis of OS, however, no significant interaction was found between BCL2 and randomization status. Significant reductions in LRR probability after PMRT were recorded within both the BCL2 positive and BCL2 negative subgroups. CONCLUSION: p53 was not associated with survival after radiotherapy in high-risk breast cancer, but BCL2 might be.

Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.

Histological evaluation of endometrium has been the gold standard for clinical diagnosis and management of women with endometrial disorders. However, several recent studies have questioned the accuracy and utility of such evaluation, mainly because of significant intra- and interobserver variations in histological interpretation. To examine the possibility that biochemical or molecular signatures of endometrium may prove to be more useful, we have investigated whole-genome molecular phenotyping (54,600 genes and expressed sequence tags) of this tissue sampled across the cycle in 28 normo-ovulatory women, using high-density oligonucleotide microarrays. Unsupervised principal component analysis of all samples revealed that samples self-cluster into four groups consistent with histological phenotypes of proliferative (PE), early-secretory (ESE), mid-secretory (MSE), and late-secretory (LSE) endometrium. Independent hierarchical clustering analysis revealed equivalent results, with two major dendrogram branches corresponding to PE/ESE and MSE/LSE and sub-branching into the four respective phases with heterogeneity among samples within each sub-branch. K-means clustering of genes revealed four major patterns of gene expression (high in PE, high in ESE, high in MSE, and high in LSE), and gene ontology analysis of these clusters demonstrated cycle-phase-specific biological processes and molecular functions. Six samples with ambiguous histology were identically assignable to a cycle phase by both principal component analysis and hierarchical clustering. Additionally, pairwise comparisons of relative gene expression across the cycle revealed genes/families that clearly distinguish the transitions of PE-->ESE, ESE-->MSE, and MSE-->LSE, including receptomes and signaling pathways. Select genes were validated by quantitative RT-PCR. Overall, the results demonstrate that endometrial samples obtained by two different sampling techniques (biopsy and curetting hysterectomy specimens) from subjects who are as normal as possible in a human study and including those with unknown histology, can be classified by their molecular signatures and correspond to known phases of the menstrual cycle with identical results using two independent analytical methods. Also, the results enable global identification of biological processes and molecular mechanisms that occur dynamically in the endometrium in the changing steroid hormone milieu across the menstrual cycle in normo-ovulatory women. The results underscore the potential of gene expression profiling for developing molecular diagnostics of endometrial normalcy and abnormalities and identifying molecular targets for therapeutic purposes in endometrial disorders.

Risk of radiation-induced subcutaneous fibrosis in relation to single nucleotide polymorphisms in TGFB1, SOD2, XRCC1, XRCC3, APEX and ATM--a study based on DNA from formalin fixed paraffin embedded tissue samples.

PURPOSE: In two previously published studies, associations with risk of radiation-induced subcutaneous fibrosis were found for single nucleotide polymorphisms (SNP) in TGFB1 (transforming growth factor beta 1 gene), XRCC1 (X-ray repair cross-complementing 1 gene), XRCC3 (X-ray repair cross-complementing 3 gene), SOD2 (manganese superoxide dismutase gene) and ATM (gene of ataxia telangiectasia mutated). The present study was conducted to seek a confirmation of these findings. MATERIALS AND METHODS: Like the 41 patients previously investigated, the 120 subjects included in the present study were accrued from a historical cohort of 319 post-mastectomy radiotherapy patients. All patients received hypo-fractionated radiotherapy. The TGFB1 position--509, codons 10 and 25, XRCC1 codons 194, 280 and 399, XRCC3 codon 241, SOD2 codon 16, ATM codon 1853 and APEX (apurinic/apyrimidinic exonuclease gene) codon 148 polymorphisms were assessed based on archival histological material. Differences in fibrosis risk were quantified from dose-response assessments. RESULTS: For none of the investigated polymorphisms, significant associations with risk of subcutaneous fibrosis were observed. A detailed analysis did not reveal any obvious explanation for the discrepancy between the previous and the present study. CONCLUSION: The previously observed associations with risk of radiation-induced subcutaneous fibrosis could not be replicated in the present study. Further studies are needed to elucidate the influence of genetic variation upon normal tissue radiosensitivity.

Radiotherapy-related lung fibrosis enhanced by tamoxifen.

BACKGROUND: Tamoxifen is an anti-estrogen with proven efficacy and low toxicity in the treatment of breast cancer. However, tamoxifen has been shown to exert a number of nonhormonal as well as hormonal effects. One nonhormonal effect of tamoxifen is the induction of transforming growth factor-beta (TGF-beta) secretion. TGF-beta has been implicated in the pathogenesis of radiation-induced fibrosis. PURPOSE: We investigated the development of lung fibrosis in breast cancer patients who were treated after mastectomy with radiotherapy, with or without simultaneous adjuvant treatment with tamoxifen. METHODS: Data from 196 women were included in the analysis. Eighty-four women were postmenopausal patients who participated in a randomized trial testing tamoxifen as an adjuvant to postmastectomy radiotherapy. The radiotherapy technique employed an 8-MV photon field covering the axillary and the infraclavicular and supraclavicular regions of the affected side of the chest; the chest wall was treated with an abutted electron field. Optical density changes in pretreatment and post-treatment chest x-ray films were used to monitor the development of lung fibrosis; lung reactions were assessed in the photon-irradiated field only. Logistic regression analysis was used to explore relationships between radiation dose and the development of lung fibrosis in patients either receiving or not receiving tamoxifen. All P values are from two-sided tests. RESULTS: Among the 84 women who participated in the randomized trial of radiotherapy plus tamoxifen (n = 38) versus radiotherapy alone (n = 46), there was a significant association between tamoxifen treatment and the incidence of marked lung fibrosis (relative risk = 2.0; 95% confidence interval [CI] = 1.2-3.5; P = .01). When logistic regression analysis was used to evaluate data from all 196 patients, a highly significant relationship was found between the incidence of lung fibrosis and total radiation dose (P = .0005). In the full analysis, an increased risk of marked lung fibrosis was found again for patients who received tamoxifen simultaneously with radiotherapy (with patients receiving radiotherapy alone as the referent, odds ratio = 2.9; 95% CI = 1.3-6.3; P = .007). Patient age and menopausal status did not significantly influence the results. CONCLUSION: Tamoxifen treatment during postmastectomy radiotherapy enhances the risk of radiation-induced lung fibrosis. IMPLICATIONS: In view of pre-existing data, we hypothesize that tamoxifen mediates the enhancement of radiation-induced lung fibrosis through the induction of TGF-beta secretion. If this hypothesis is correct, new strategies might be devised for preventing or reducing radiation-induced fibrosis. Because we studied a relatively small portion of the irradiated lung, we cannot recommend changes in current therapeutic measures; however, we strongly encourage additional studies of lung fibrosis in patients receiving tamoxifen and radiotherapy.

Enhancement by Corynebacterium parvum of the normal and tumor tissue response to hyperthermia.

The effect of Corynebacterium parvum treatment on the response of tumor and normal tissue to hyperthermia (43.5 degrees) was studied. Animals were C3Hf/Sed mice from our defined flora mouse colony. The time at hyperthermia that achieved control of one-half of methylcholanthrene-induced fibrosarcomas and the foot reaction were examined after treatment. C. parvum, if given 3 to 32 days before hyperthermia, enhanced the reaction to local hyperthermia of normal tissue. No enhancement was observed if C. parvum was given after hyperthermia. This enhancement was more dramatic for tumor response resulting in a therapeutic gain factor of congruent to 2.3 (3.7/1.6). Comparative studies on combined Corynebacterium and radiation failed to demonstrate the enhancement to normal tissue.

Differences in risk factors for local and distant recurrence after breast-conserving therapy or mastectomy for stage I and II breast cancer: pooled results of two large European randomized trials.

PURPOSE: Risk factors for local and distant recurrence after breast-conserving therapy and mastectomy were compared to define guidelines for the decision making between both treatments. PATIENTS AND METHODS: The data of two randomized clinical trials for stage I and II breast cancer patients were pooled. The total number of patients in the study was 1,772, of whom 879 underwent breast conservation, and 893, modified radical mastectomy. Representative slides of the primary tumor were available for histopathologic review in 1,610 cases (91%). RESULTS: There were 79 patients with local recurrence after breast-conservation and 80 after mastectomy, the 10-year rates being 10% (95% confidence interval [CI], 8% to 13%) and 9% (95% CI, 7% to 12%), respectively. Age no more than 35 years (compared with age >60: hazard ratio [HR], 9.24; 95% CI, 3.74 to 22.81) and an extensive intraductal component (HR, 2.52; 95% CI, 1.26 to 5.00) were significantly associated with an increased risk of local recurrence after breast-conserving therapy. Vascular invasion was predictive of the risk of local recurrence, irrespective of the type of primary treatment (P <.01). Tumor size, nodal status, high histologic grade, and vascular invasion were all highly significant predictors of distant disease after breast-conserving therapy and mastectomy (P <.01). Age no more than 35 years and microscopic involvement of the excision margin were additional independent predictors of distant disease after breast-conserving therapy (P <.01). CONCLUSION: Age no more than 35 years and the presence of an extensive intraductal component are associated with an increased risk of local recurrence after breast-conserving therapy. Vascular invasion causes a higher risk of local recurrence after mastectomy as well as after breast-conserving therapy and should therefore not be used for deciding between the two treatments.

Insulin-like growth factor binding protein-4 protease produced by smooth muscle cells increases in the coronary artery after angioplasty.

Insulin-like growth factor (IGF)-I stimulates vascular smooth muscle cell (VSMC) migration and proliferation, which are fundamental to neointimal hyperplasia in postangioplasty restenosis. IGF-I action is modulated by several high-affinity IGF binding proteins (IGFBPs). IGFBP-4 is the predominant IGFBP produced by VSMCs and is a potent inhibitor of IGF-I action. However, specific IGFBP-4 proteases can cleave IGFBP-4 and liberate active IGF-I. In this study, we document IGFBP-4 protease produced by human and porcine coronary artery VSMCs in culture as pregnancy-associated plasma protein-A (PAPP-A). This was shown by a distinctive IGFBP-4 cleavage pattern, specific inhibition of IGFBP-4 protease activity with PAPP-A polyclonal antibodies, and immunorecognition of PAPP-A by monoclonal antibodies. Furthermore, we found a 2-fold increase in IGFBP-4 protease activity in injured porcine VSMC cultures in vitro (P<0.05). We also evaluated IGFBP-4 protease/PAPP-A expression in vivo after coronary artery balloon injury. Twenty-five immature female pigs underwent coronary overstretch balloon injury, and vessels were examined at defined time points after the procedure. Abundant PAPP-A expression was observed in the cytoplasm of medial and neointimal cells 7, 14, and 28 days after angioplasty (P<0.01 vs control). The highest PAPP-A labeling indices were located in the neointima (36.1+/-2.1%) and the media (31.7+/-1.2%) 28 days after injury. Western blot analysis confirmed increased PAPP-A in injured vessels. PAPP-A, a regulator of IGF-I bioavailability through proteolysis of IGFBP-4, is thus expressed by VSMCs in vitro and in restenotic lesions in vivo. These results suggest a possible role for PAPP-A in neointimal hyperplasia.

Pregnancy-associated plasma protein-A (PAPP-A) in ovine, bovine, porcine, and equine ovarian follicles: involvement in IGF binding protein-4 proteolytic degradation and mRNA expression during follicular development.

IGF binding protein-4 (IGFBP-4) proteolytic degradation is a common feature of preovulatory follicles from human, ovine, bovine, porcine, and equine ovary. In all these species, the protease is a zinc-dependent metalloprotease and its ability to degrade IGFBP-4 is IGF dependent. The human intrafollicular IGFBP-4-degrading protease has recently been identified as pregnancy-associated plasma protein-A (PAPP-A). The aim of this study was to investigate whether PAPP-A is also involved in IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles and to study the expression of PAPP-A mRNA in bovine and porcine granulosa cells from different classes of follicles. Immunoneutralization and immunoprecipitation with polyclonal antibodies raised against human PAPP-A inhibited IGFBP-4 proteolytic degradation in preovulatory follicular fluid from the four species studied. As previously reported for the intrafollicular proteolytic activity degrading IGFBP-4, recombinant human PAPP-A generated in vitro 17- and 10-kDa IGFBP-4-proteolytic fragments. Recombinant PAPP-A activity was also shown to be IGF dependent and was inhibited by heparin-binding domain-containing peptides. In all mammalian species studied, the PAPP-A sequences showed high degree of identity. Moreover, the PAPP-A gene was localized on porcine chromosome 1 (1q29-1q213), in agreement with the localization of human PAPP-A gene on human chromosome 9q33.1. In bovine and porcine ovaries, real-time quantitative RT-PCR showed that PAPP-A mRNA expression in granulosa cells was maximal in fully differentiated follicles and was positively correlated with expression of P450 aromatase and LH receptor mRNAs. Overall, these data show that PAPP-A is responsible for IGFBP-4 degradation in ovine, bovine, porcine, and equine preovulatory follicles. The high expression of PAPP-A mRNA in granulosa cells from large, differentiated follicles suggest that it is a new functional marker of follicular development.

Pregnancy-associated plasma protein-a is the insulin-like growth factor binding protein-4 protease secreted by human ovarian granulosa cells and is a marker of dominant follicle selection and the corpus luteum.

Insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and IGFBP proteases are important in ovarian function. IGFs stimulate granulosa steroidogenesis, an effect that is inhibited by IGFBP-4 and augmented by IGFBP-4 proteolysis. We have recently identified the IGFBP-4 protease in human ovarian follicular fluid (FF) as pregnancy-associated plasma protein-A (PAPP-A). In the current study, we identify the IGFBP-4 protease secreted by cultured human ovarian granulosa cells as PAPP-A, based on specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with PAPP-A polyclonal antibodies and immunorecognition by PAPP-A monoclonal antibodies in ELISA. PAPP-A was barely detectable in conditioned media (CM) from granulosa derived from /=9 mm, coincident with dominant follicle selection, and by luteinizing granulosa. PAPP-A levels in CM from the latter did not change in response to IGF-II or hCG (100 ng/mL). A naturally occurring inhibitor of PAPP-A, proform of eosinophil major basic protein (proMBP), was detected by ELISA in estrogen-dominant follicular fluid FF, but not in CM from granulosa or luteinizing granulosa cells treated with IGF-II (0-200 ng/mL), FSH (0-100 ng/mL) or hCG (0-100 ng/mL), suggesting an alternative source (other than granulosa) for proMBP, compared to PAPP-A. The data demonstrate granulosa cells as a source of PAPP-A in human ovary and suggest that PAPP-A is a marker of ovarian follicle selection and corpus luteum formation. In addition the data suggest complex regulation of this system in human ovary.

Evidence that the insulin-like growth factor binding protein-4 protease in human ovarian follicular fluid is pregnancy associated plasma protein-A.

Ovarian insulin-like growth factor binding protein-4 (IGFBP-4) proteolysis is involved in the regulation of follicular development, but until now the identity of the responsible enzyme was unknown. In this study, we identify the IGFBP4 protease in human follicular fluid as pregnancy associated plasma protein-A (PAPP-A) based on distinctive IGFBP-4 cleavage pattern, the same protease inhibitor profile, specific inhibition and immunodepletion of IGFBP-4 protease activity with PAPP-A polyclonal antibodies, and immunorecognition by PAPP-A monoclonal antibodies in ELISA. Furthermore, PAPP-A levels in estrogen-dominant and androgen-dominant follicular fluids reflect their IGFBP-4 proteolytic activity. PAPP-A was also secreted by human granulosa cells, the reputed source of IGFBP-4 protease activity in follicular fluid. We have the molecular and biochemical tools to begin to delineate the regulation and biological function of PAPP-A in normal and dysregulated follicular development and atresia.

Identification and regulation of the IGFBP-4 protease and its physiological inhibitor in human trophoblasts and endometrial stroma: evidence for paracrine regulation of IGF-II bioavailability in the placental bed during human implantation.

The IGF family plays an important role in implantation and placental physiology. IGF-II is abundantly expressed by placental trophoblasts, and IGF binding protein (IGFBP)-4, a potent inhibitor of IGF actions, is the second most abundant IGFBP in the placental bed, expressed exclusively by the maternal decidua. Proteolysis of IGFBP-4 results in decreased affinity for IGF peptides, thereby enhancing IGF actions. In the current study, we have identified the IGFBP-4 protease and its inhibitor in human trophoblast and decidualized endometrial stromal cell cultures, and we have investigated their regulation in an effort to understand control of IGF-II bioavailability at the placental-decidual interface in human implantation. IGFBP-4 protease activity was detected in conditioned media (CM) from human trophoblasts and decidualized endometrial stromal cells using (125)I-IGFBP-4 substrate. Identification of the IGFBP-4 protease as pregnancy-associated plasma protein-A (PAPP-A) was confirmed by specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with specific PAPP-A antibodies. The IGFBP-4 protease activity was IGF-II-dependent in trophoblast CM. In decidualized stromal CM, PAPP-A/IGFBP-4 protease activity was also IGF-II-dependent, but was evident only when IGF-II was added in molar excess of the predominant IGFBP in decidualized stromal cell CM, IGFBP-1, supporting bioavailable IGF-II as a key cofactor of IGFBP-4 proteolysis by PAPP-A. Cultured first and second trimester human trophoblasts (n = 5) secreted PAPP-A into CM with mean +/- SEM levels of 172.4 +/- 32.8 mIU/liter.10(5) cells, determined by specific ELISA. PAPP-A in trophoblast CM (n = 3) and did not change in the presence of IGF-II (1-100 ng/ml). Cultured human endometrial stromal cells (n = 4) secreted low levels of PAPP-A (6.25 +/- 3.6 mIU/liter.10(5) cells). A physiological inhibitor of PAPP-A, the proform of eosinophil major basic protein (proMBP), was detected in trophoblast CM at levels of 1853 +/- 308 mIU/liter.10(5) cells, determined by specific ELISA, and was nearly undetectable in CM of human endometrial stromal cells. Upon in vitro decidualization of endometrial stromal cells with progesterone, PAPP-A levels in CM increased nearly 9-fold without a concomitant change in proMBP. In contrast to the experiments with trophoblasts, IGF-II and the IGF analogues, Leu(27) IGF-II, and Des (1-6) IGF-II, resulted in a dose-dependent decrease of PAPP-A levels in decidualized endometrial stromal CM by 70-90%, and a dose-dependent increase in proMBP of 14- to 41-fold. The data demonstrate conclusively that the IGF-II-dependent IGFBP-4 protease of human trophoblast and decidual origin is PAPP-A. Furthermore, the differential regulation of decidual PAPP-A and proMBP by insulin-like peptides supports a role for trophoblast-derived IGF-II as a paracrine regulator of these maternal decidual products that have the potential to regulate IGF-II bioavailability at the trophoblast-decidual interface. Overall, the data underscore potential roles for a complex family of enzyme (PAPP-A), substrate (IGFBP-4), inhibitor (proMBP), and cofactor (IGF-II) in the placental bed during human implantation.