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• W133774038 abstract "Transformation of uterine spiral arteries is critical for healthy human pregnancy. We recently proposed a role for maternal leukocytes in decidual spiral artery remodeling and suggested that matrix metalloprotease (MMP) activity contributed to the destruction of the arterial wall. In the current study we used our first trimester placental-decidual co-culture (PDC) model to define the temporal relationship and test the mechanistic aspects of this process. PDC experiments were assessed by image analysis over a six-day time-course for degree of vascular transformation and leukocyte distribution around progressively remodeled arterioles. We observed rapid transformation in PDCs associated with loss of vascular smooth muscle cells, widening of the vessel lumen, and significant accumulation of uterine Natural Killer cells and macrophages within the vascular wall (P < 0.001) before trophoblast presence in the vessel lumens. These events did not occur in decidua-only cultures. Active MMP-9 was detected in leukocytes and vascular cells of remodeling arterioles, and inhibition of MMP-2/9 activity in PDC resulted in failure of decidual vascular remodeling compared with vehicle-treated PDCs. Apoptosis of vascular cells, macrophage-mediated phagocytosis, and vascular smooth muscle cell dedifferentiation contributed to the remodeling observed. The PDC model indicates that placental presence is required to initiate decidual spiral artery remodeling but that uterine Natural Killer cells and macrophages mediate the early stages of this process at the cellular level. Transformation of uterine spiral arteries is critical for healthy human pregnancy. We recently proposed a role for maternal leukocytes in decidual spiral artery remodeling and suggested that matrix metalloprotease (MMP) activity contributed to the destruction of the arterial wall. In the current study we used our first trimester placental-decidual co-culture (PDC) model to define the temporal relationship and test the mechanistic aspects of this process. PDC experiments were assessed by image analysis over a six-day time-course for degree of vascular transformation and leukocyte distribution around progressively remodeled arterioles. We observed rapid transformation in PDCs associated with loss of vascular smooth muscle cells, widening of the vessel lumen, and significant accumulation of uterine Natural Killer cells and macrophages within the vascular wall (P < 0.001) before trophoblast presence in the vessel lumens. These events did not occur in decidua-only cultures. Active MMP-9 was detected in leukocytes and vascular cells of remodeling arterioles, and inhibition of MMP-2/9 activity in PDC resulted in failure of decidual vascular remodeling compared with vehicle-treated PDCs. Apoptosis of vascular cells, macrophage-mediated phagocytosis, and vascular smooth muscle cell dedifferentiation contributed to the remodeling observed. The PDC model indicates that placental presence is required to initiate decidual spiral artery remodeling but that uterine Natural Killer cells and macrophages mediate the early stages of this process at the cellular level. After human blastocyst implantation, extravillous trophoblasts (EVTs) arise from placental villi and invade the decidualizing maternal endometrium (decidua) where they participate in the remodeling of spiral arteries. During remodeling, the spiral arteries undergo extensive changes including loss of their vasoactive medial vascular smooth muscle cells (VSMCs) and most of their intimal endothelial monolayer. This transforms the muscular, tightly coiled decidual spiral arteries into dilated sinusoids capable of increasing uterine blood volume to perfuse the placenta. This process is essential for successful establishment of utero-placental circulation and a healthy pregnancy. These changes are thought to be induced by the EVTs, which invade the spiral arteries, eventually reline the vessels, and acquire an endothelial-like phenotype.1Dunk C Huppertz B Kingdom J Development of the placenta and its circulation.in: Rodeck CH Whittle MJ Churchill Livingstone Elsevier., London2009: 69-96Google Scholar Failure of appropriate remodeling in the myometrial portions of these vessels has been described in patients with preeclampsia and intrauterine growth restriction.2Brosens JJ Pijnenborg R Brosens IA The myometrial junctional zone spiral arteries in normal and abnormal pregnancies: a review of the literature.Am J Obstet Gynecol. 2002; 187: 1416-1423Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar Before embryo implantation, the high progesterone levels of the late secretory phase initiate the first stages of decidualization in the endometrium including angiogenesis of the spiral arteries and a large infiltration of innate immune cells.3Benirschke K Kaufmann P Baergen R Pathology of the Human Placenta. Springer, New York, NY2006Google Scholar By early pregnancy, leukocytes comprise 40% of all decidual cells. Specialized uterine Natural Killer (uNK) cells and macrophages constitute 70% and 20% of decidual leukocytes, respectively.4Bulmer JN Kurpisz M Fernandez N Immune cells in decidua. Oxford Bios Scientific Publishers, Oxford1995: 334Google Scholar, 5King A Burrows T Verma S Hiby S Loke YW Human uterine lymphocytes.Hum Reprod Update. 1998; 4: 480-485Crossref PubMed Scopus (181) Google Scholar Both decidual macrophages and uNK cells produce angiogenic factors, including vascular endothelial growth factor, placental growth factor, and angiopoetin-2, which are proposed to contribute to decidual vascular remodeling.6Hanna J Goldman-Wohl D Hamani Y Avraham I Greenfield C Natanson-Yaron S Prus D Cohen-Daniel L Arnon TI Manaster I Gazit R Yutkin V Benharroch D Porgador A Keshet E Yagel S Mandelboim O Decidual NK cells regulate key developmental processes at the human fetal-maternal interface.Nat Med. 2006; 12: 1065-1074Crossref PubMed Scopus (1275) Google Scholar, 7Tabiasco J Rabot M Aguerre-Girr M El Costa H Berrebi A Parant O Laskarin G Juretic K Bensussan A Rukavina D Le Bouteiller P Human decidual NK cells: unique phenotype and functional properties – a review.Placenta. 2006; 27: S34-S39Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar Similarly, a specific M2 tumor-associated macrophage population is thought to be the precipitating factor in tumor-mediated angiogenesis and metastasis as they possess many protumor activities including secretion of growth factors, matrix remodeling, and suppression of adaptive immunity.8Davies MJ Pathology of arterial thrombosis.Br Med Bull. 1994; 50: 789-802PubMed Google Scholar, 9Porta C Subhra Kumar B Larghi P Rubino L Mancino A Sica A Tumor promotion by tumor-associated macrophages.Adv Exp Med Biol. 2007; 604: 67-86Crossref PubMed Scopus (84) Google Scholar We suggest that the decidual macrophage may play a similar role in decidual angiogenesis and spiral artery remodeling. Multiple studies have identified an essential role for uNK cells in the murine implantation site. Mice deficient in either uNK or interferon-γ signaling exhibit implantation abnormalities and defects of maternal artery remodeling.10Croy BA Di Santo JP Greenwood JD Chantakru S Ashkar AA Transplantation into genetically alymphoid mice as an approach to dissect the roles of uterine natural killer cells during pregnancy–a review.Placenta. 2000; 21: S77-S80Abstract Full Text PDF PubMed Scopus (32) Google Scholar, 11Croy BA Esadeg S Chantakru S van den Heuvel M Paffaro VA He H Black GP Ashkar AA Kiso Y Zhang J Update on pathways regulating the activation of uterine Natural Killer cells, their interactions with decidual spiral arteries and homing of their precursors to the uterus.J Reprod Immunol. 2003; 59: 175-191Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 12Ashkar AA Black GP Wei Q He H Liang L Head JR Croy BA Assessment of requirements for IL-15 and IFN regulatory factors in uterine NK cell differentiation and function during pregnancy.J Immunol. 2003; 171: 2937-2944PubMed Google Scholar, 13Zhang JH He H Borzychowski AM Takeda K Akira S Croy BA Analysis of cytokine regulators inducing interferon production by mouse uterine natural killer cells.Biol Reprod. 2003; 69: 404-411Crossref PubMed Scopus (55) Google Scholar In humans, communication between uNK cell receptors and interstitial EVTs is believed to dictate depth of trophoblast invasion.14King A Hiby SE Gardner L Joseph S Bowen JM Verma S Burrows TD Loke YW Recognition of trophoblast HLA class I molecules by decidual NK cell receptors–a review.Placenta. 2000; 21: S81-S85Abstract Full Text PDF PubMed Scopus (136) Google Scholar However, no conclusive evidence exists to implicate uNK cells directly in human vascular transformation. We recently reported an intimate relationship between uNK cells, macrophages, and remodeling arteries in biopsies of first trimester decidua basalis.15Smith SD Dunk CE Aplin JD Harris LK Jones RL Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy.Am J Pathol. 2009; 174: 1959-1971Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar Leukocytes were observed in close proximity to early and mid-stage remodeling arterial walls, in the absence of either interstitial EVTs (inEVTs) or endovascular EVTs (enEVTs).15Smith SD Dunk CE Aplin JD Harris LK Jones RL Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy.Am J Pathol. 2009; 174: 1959-1971Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar Moreover, we demonstrated that uNK cells and macrophages within the vascular wall expressed matrix metalloprotease (MMP)-7 and -9. MMPs are key proteases in the reproductive system and are known to be important for processes such as trophoblast invasion16Lash GE Otun HA Innes BA Bulmer JN Searle RF Robson SC Inhibition of trophoblast cell invasion by TGFB1, 2, and 3 is associated with a decrease in active proteases.Biol Reprod. 2005; 73: 374-381Crossref PubMed Scopus (133) Google Scholar, 17Bischof P Meisser A Campana A Control of MMP-9 expression at the maternal-fetal interface.J Reprod Immunol. 2002; 55: 3-10Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 18Cohen M Meisser A Bischof P Metalloproteinases and human placental invasiveness.Placenta. 2006; 27: 783-793Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar and focal degradation of the endometrial extracellular matrix during menstruation.19Salamonsen LA Matrix metalloproteinases and endometrial remodelling.Cell Biol Int. 1994; 18: 1139-1144Crossref PubMed Scopus (46) Google Scholar We suggested that leukocyte-derived MMPs contribute to vascular remodeling, consistent with their reported roles in tumor angiogenesis20Handsley MM Edwards DR Metalloproteinases and their inhibitors in tumor angiogenesis.Int J Cancer. 2005; 115: 849-860Crossref PubMed Scopus (236) Google Scholar and metastasis.21Coussens LM Tinkle CL Hanahan D Werb Z MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis.Cell. 2000; 103: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1138) Google Scholar However, in these in vivo specimens we were unable to conclusively describe the direct temporal relationship of the leukocytes with progression of vascular remodeling or directly test mechanistic functionality. We have developed a placenta-decidua co-culture (PDC) model, adapted from previous work by Vicovac et al (1995),22Vicovac L Jones CJ Aplin JD Trophoblast differentiation during formation of anchoring villi in a model of the early human placenta in vitro.Placenta. 1995; 16: 41-56Abstract Full Text PDF PubMed Scopus (144) Google Scholar which allows us to study the dynamic processes of vascular remodeling in intact decidual explants. Using this model, we have previously reported EVT invasion into arterioles, endothelial cell and VSMC loss, and relining of arteries, but not veins, by endovascular EVTs.23Dunk C Petkovic L Baczyk D Rossant J Winterhager E Lye S A novel in vitro model of trophoblast-mediated decidual blood vessel remodeling.Lab Invest. 2003; 83: 1821-1828Crossref PubMed Scopus (55) Google Scholar We hypothesized that uNK cells and macrophages participate in artery remodeling through specific mechanisms inducing vascular cell priming, destruction, and clearance. We used our PDC model to define the temporal, spatial, and mechanistic relationships of uNK cells and macrophages with spiral arteries during remodeling. Placentae and decidua parietalis (without prior invasion) were obtained, following written informed consent from patients undergoing first trimester elective terminations at the Morgantaler Clinic and Mount Sinai Hospital (Toronto, Canada) or the Whitworth Clinic at St. Mary’s Hospital (Manchester, UK). The Mount Sinai Hospital Research Ethics Board (Toronto) and the North West Research Ethics Committee (Manchester) approved collections of human tissues. Tissue was collected in cold PBS and dissected at 6 to 9 weeks (n = 16) according to the criteria of the Carnegie classification evaluating characteristics of embryonic/fetal parts. PDCs were established by placement of placental villi on the apical epithelial surface of patient-matched decidual explants. Briefly, small fragments of placental villi (15–20 mg wet weight) were dissected from the placenta, teased apart, and selected for the presence of EVT cell columns ensuring rapid attachment of the placental explant to the decidual surface. Thickness and integrity of decidua parietalis was assessed and dissected into 2 to 3 mm2 cubes. Decidual explants were placed with the apical epithelial surface uppermost in Millicell-CM culture dish inserts pore size 0.4 μm (Fisher Scientific, Ottawa, Canada), pre-coated with 0.2 ml undiluted phenol red-free Matrigel (Becton Dickinson, Mississauga, Canada). The Matrigel was allowed to solidify before the placement of the corresponding placental villous explant in contact with the decidual epithelial surface. Explants were cultured overnight with no media to allow attachment to occur followed by the addition of serum-free DMEM-Ham’s F-12 media (Invitrogen, Burlington, Canada) supplemented with 20 ng/ml progesterone (Sigma, Oakville, Canada), 300 pg/ml 17β-estradiol (Sigma), and 100 μg/ml normocin (Cedarlane laboratories, Burlington, Canada) at 3% O2/5% CO2. Culture media was changed every 48 hours. PDCs (from a single patient) were cultured in triplicate for each treatment point. Adjacent explants of decidua parietalis were cultured in the absence of placenta to confirm that there was no trophoblast invasion before the establishment of the culture and no degradation of blood vessels due to the culture conditions. Decidua-only controls from adjacent tissues and PDC were maintained in culture for 3 or 6 days. PDC replicates and matching decidua-only controls were fixed in 4% paraformaldehyde for 1 hour at room temperature and rinsed 3× in cold Ca2+- and Mg2+-free PBS on a shaker for 20 minutes and stored in PBS at 4°C until processing. Explants were dehydrated by a gradient of ethanol in PBS solutions from 70% to 100%. Experiments were cleared in xylene for 1 hour, excess xylene was removed by paraffin infiltration for 3 hours and embedded in paraffin wax using an embedding machine. Only explants that had attached to the decidual epithelial surface by day 1 and remained attached at the time of collection and through fixation were processed. Occasionally the multiple solution changes during processing and paraffin-embedding resulted in the loss of the placental explant, as observed in some photomicrographs. Immunohistochemistry was performed on PDCs and decidua-only controls for vascular (α-smooth muscle actin [α-SMA], CD31), EVT (cytokeratin), leukocyte (CD45, CD56, CD68), and phagocytic (lysozyme muramidase) markers. Paraffin-embedded explants were sectioned to 5 μm using a microtome, adhered to Superfrost++ slides (Fisher Scientific) using a warm water bath and dried in an oven at 60°C overnight. Slides were deparaffinized in xylene and rehydrated through a gradient series of ethanol in PBS. Endogenous peroxidase activity was blocked by incubation of the sections in 3% hydrogen peroxide (Fisher Scientific) in methanol for 40 minutes. Antigen retrieval methods specific for each antibody are summarized in Table 1. All slides were incubated with Dako protein blocking solution (Dako, Mississauga, Ontario, Canada) for 1 hour at room temperature to block nonspecific binding. Usage conditions, source, and specificity for all immunohistochemistry primary and secondary antibodies are provided in Table 1. Slides were developed using the labeled streptavidin biotin - horseradish peroxidase (1 hour) (Dako) and 3,3-diaminobenzidine+ in diluting solution (Dako). After counterstaining with Harris Hematoxylin Solution (Sigma) slides were dehydrated in an ascending ethanol series, cleared in xylene, and mounted with Permount (Fisher Scientific).Table 1Details of Antibodies Used for ImmunohistochemistryAntibodySpeciesSourceDilutionAntigen retrievalSpecificityCytokeratinMouseDako0.17 μg/mlMicrowave: 10 mmol/L Sodium (Na) Citrate pH6Epithelial cells incl. trophoblastCD31 (PECAM-1)MouseDako1.3 μg/mlMicrowave: NaCitrateEndothelial cellsα-SMAMouseDako0.035 μg/mlMicrowave: NaCitrateSmooth muscle cellsCD45MouseDako0.35 μg/mlMicrowave: NaCitrateAll leukocytesCD56MouseDako0.3 μg/mlMicrowave: 1 mmol/L EDTAuNK cellsCD68MouseNovocastra Laboratories0.5 μg/mlMicrowave: NaCitrateMacrophagesMMP-9MouseCalbiochem4 μg/mlMicrowave: NaCitrateMMP-9Lysozyme muramidaseRabbitBiomedia1 μg/mlMicrowave: NaCitratePhagocytic cellsAnti-mouse IgG-biotinGoatDako0.024 μg/mlMatched to primary antibodyMouse IgGMouse IgG IsotypeMouseDakoMatched to primaryMatched to primary antibodyAll IgG Open table in a new tab To determine the identity of MMP-9+ cells, dual immunofluorescence was performed using combinations of antibodies as described in Table 2. Sections were dewaxed, rehydrated, and antigens retrieved as indicated in Table 1. Autofluorescence was blocked using sodium borohydride (Sigma, Gillingham, UK) applied at 0.1% in TBS for 3 × 10 minutes. Nonimmune block (10% goat serum [Sigma, UK] and 2% human serum [in-house] in 0.1% Tween-20 [BioRad, Hemel Hempstead, UK] in Tris buffered saline) was applied before incubation with primary antibody for 1 hour at 37°C. Antigen was detected by rabbit anti-mouse FITC conjugate (Dako, Ely, UK) at 46 μg/ml. Unlabeled goat anti-mouse IgGs (Dako, UK) were applied at 15.2 μg/ml for 1 hour to saturate binding site for the first primary antibody. Nonimmune block was reapplied before incubation with the second primary antibody for 1 hour at 37°C, which was then detected by application of rabbit anti-mouse Alexa Fluor-568 conjugate (Molecular Probes, Paisley, UK) at 40 μg/ml. Sections were mounted using Vectashield containing DAPI (Vector Laboratories, Burlingame, CA). Negative controls included combinations of mouse and rabbit IgGs to match concentration of primary antibodies, omission of all antibody to control for autofluorescence, or omission of the first secondary antibody to control for cross reactivity between the first primary and the second secondary antibodies.Table 2Combinations of Antibodies Used for Dual ImmunofluorescenceFirst primaryFirst secondarySecond primarySecond secondaryTUNEL reagentN/Aα-SMAAnti-mouse Alexa Fluor-568TUNEL reagentN/ACD31Anti-mouse Alexa Fluor-568MMP-9Anti-mouse-FITCCD56Anti-mouse Alexa Fluor-568MMP-9Anti-mouse-FITCα-SMAAnti-mouse Alexa Fluor-568MMP-9Anti-mouse-FITCPan cytokeratinAnti-mouse Alexa Fluor-568MMP-9Anti-mouse-FITCCD31Anti-mouse Alexa Fluor-568N/A, not applicable. Open table in a new tab N/A, not applicable. To determine whether remodeling vessels contained apoptotic cells, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assays were performed using the In Situ Cell Death detection kit with FITC detection (Roche, Welwyn Garden City, UK) as described previously.15Smith SD Dunk CE Aplin JD Harris LK Jones RL Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy.Am J Pathol. 2009; 174: 1959-1971Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar A positive control was treated with DNase I for 10 minutes at 37°C, while a negative control was generated by the omission of the terminal deoxynucleotidase enzyme. To determine the identity of apoptotic cells, sections were TUNEL stained, followed by immunofluorescent detection of anti–α-SMA or anti-CD31 using rabbit anti-mouse Alexa Fluor-568 conjugate. In situ zymography to detect gelatinase activity in PDC was performed as previously described.15Smith SD Dunk CE Aplin JD Harris LK Jones RL Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy.Am J Pathol. 2009; 174: 1959-1971Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar Unfixed PDCs (n = 3) were embedded in OCT and snap frozen in liquid nitrogen. Ten micrometer sections were cut on a cryostat (Leica, Milton Keynes, UK), mounted on Superfrost++ slides, fixed with 10% normal buffered formalin for 30 minutes, and stained with α-SMA and CD45 to identify remodeling arteries with leukocytic infiltration. In situ zymography was then performed on serial sections to examine MMP activity. Freshly cut air-dried 10 μm sections were fixed in 10% normal buffered formalin for 5 minutes at 4°C. Slides were washed and counterstained with propidium iodide for 8 minutes. One hundred microliters of the substrate, DQ gelatin (25 μg/ml, Invitrogen, Paisley, UK) which fluoresces when cleaved, was layered over the tissue section, covered with a coverslip, and incubated for 16 hours at 37°C. Negative and positive controls were included; 1,10 phenanthroline (Invitrogen, UK) or collagenase was applied to control sections for 1 hour at 37°C before counterstaining. Given the MMP-9 expression in VSMCs, endothelial cells, uNK cells, and macrophages, as well as the active MMP-2/9 detected in VSMCs and leukocytes in both in vivo decidua basalis and PDC model specimens, we decided to use the PDC model to investigate MMP-2/9 function. This was performed by manipulating culture conditions with addition of a specific MMP-2/9 inhibitor, (2R)-[(4-Biphenylylsulfonyl)amino]-N-hydroxy-3-phenylproprionamide (Calbiochem, Darmstadt, Germany), more commonly referred to as MMP-2/9 Inhibitor II, to determine the role of these proteases on vascular remodeling processes. To minimize damage to the decidual epithelial surface decidual explants were immobilized in a Petri dish and injected at 2 basal stromal sites with 50 nmol/L MMP-2/9 Inhibitor II or with vehicle (0.1% DMSO; Sigma) (50 μl/site) using a 700 series Microliter Hamilton syringe with cemented needle gauge 22s (VWR, Mississauga, Ontario, Canada) followed by a 30 minute incubation at 37°C before establishment of PDC as described above (MMP Inh: n = 3, vehicle: n = 3). The concentration used in this study more selectively inhibits MMP-9 over MMP-2. Photomicrographs of immunohistochemical staining were captured using an Olympus BX61 Upright Microscope and an Olympus DP70 12.5 megapixel camera with accompanying Olympus software (Olympus America Inc., Center Valley, PA). Images of immunofluorescent staining were captured using a Zeiss fluorescence microscope with an AxioCam MRn (Zeiss, Welwyn Garden City, UK). Serial sections of PDCs and corresponding decidua-only controls from day 3 and 6 of culture immunostained for α-SMA, CD45, CD56, and CD68 were scanned at ×16 and ×100 magnification (Visiopharm Integrator System Version 3.0.8.0, Visiopharm, Horsholm, Denmark). Adjacent digital photomicrographs were stitched together automatically by the Visiopharm software to create composite images of the entire PDC tissue section. Image analysis was conducted using Visiopharm’s Visiomorph analysis software which established pixel classification parameters based on pigment for stroma, positive immunohistochemical stain, and vessel lumen. These parameters were then applied to each image and PDC decidua were divided into 3 depths (0–500, 500-1000, and 1000–2000 μm) from the epithelial surface. Veins and glands were excluded from the analysis based on morphological assessment after immunostaining for the vascular markers CD31 and α-SMA. To identify changes in the decidual arterioles, areas of VSMCs and vessel lumens were calculated by Visiomorph within each depth and expressed as a ratio (α-SMA/lumen). For each patient sample, triplicate α-SMA/lumen measurements from replicate PDCs were calculated and averaged. In addition, we determined leukocyte association with arterioles in decidua-only controls and in PDCs before and during remodeling. Decidual arterioles were designated by morphology as unremodeled (multiple, well-organized layers of smooth muscle, intact endothelium, and narrow lumen), actively remodeling (disrupted VSMC and/or disorganized medial VSMC and endothelial desquamation and/or loss), or advanced remodeling (complete loss of VSMC, few/no endothelial cells, and dilated lumen) arterioles. On average the medial wall thickness of unremodeled arterioles from both PDCs and decidua-only controls was ∼15 μm. A line was drawn to denote the lumen of each vessel and concentric rings were automatically executed at 15 μm distances from this line by the Visiopharm software. The concentric zones 30 to 60 μm from vessel lumens were examined during optimization, and no significant differences in area of CD45+ leukocytes was found between the 15 to 30 μm zone and those further from vessels. Measurements taken >30 μm from the vessel lumen frequently included leukocytes clustered around neighboring vessels. Therefore, only the two 15 μm concentric distances closest to the arterioles were used in the comparisons presented. This ensured that the area examined accounted for the muscular arterial wall and excluded leukocytes not directly associated with the vessel under investigation. Immunohistochemical staining was classified by pixel pigment (as described above) to calculate area of leukocyte staining (CD45, CD56, and CD68), which was then expressed as a proportion of each concentric ring area (ie, area of CD45+/0 to 15 μm concentric ring area) and excluded the arteriole lumen area. Because of the nonparametric nature of our data, statistical analyses were performed using Kruskal-Wallis Analysis of Variance with Dunn’s post hoc test. Data are presented as medians and interquartile ranges in box and whisker plots. Whiskers of vascular remodeling quantification include 100% of the data. Whiskers of leukocyte quantification represent 10% to 90% of data inclusive. Additional comparisons were made using Mann–Whitney t-tests as indicated. The time course of decidual vascular changes induced by presence of placental explants was examined (Figure 1A). At the site of placental-decidual contact, cytokeratin+ trophoblast were consistently observed in anchoring columns arising from the tips of placental villi and attaching to the decidual epithelial surface (Figure 1B). Serial sections of a representative PDC at day 6 demonstrated progressive remodeling along a vessel from the point of placental contact (Figure 1, D: CD31; E: α–SMA; and F: cytokeratin). Superficial portions of arterioles (within the first 500 μm from the decidual epithelial surface) showed features of more advanced remodeling (Figure 1: right of Su) than deeper portions of the same vessels, evidenced by little or no remaining endothelium, disorganized, sporadic VSMCs, and intraluminal EVTs. Mid-portions of the arterioles showed evidence of active remodeling in the absence of intraluminal EVTs (Figure 1: Mi). Active remodeling is characterized by endothelial desquamation and shedding into the vessel lumen (Figure 1D: *), as well as disruption and loss of VSMCs (Figure 1E: ^). Deep arterioles (Figure 1: De) in PDCs remained largely untransformed, with intact VSMCs and endothelial layers, and no EVT invasion, similar to decidua-only control arterioles (Figure 1, G and H). Veins were excluded based on morphological features such as thin layers of VSMCs, intact flattened endothelium, and dilated lumens (Figure 1, I and J). All arterioles in decidua-only control cultures were intact and unremodeled. Quantification of vascular remodeling in PDCs at day 3 and 6 along with corresponding decidua-only controls is presented as median and interquartile ranges in Figure 2. Decreased α–SMA/lumen ratios resulted from the loss of VSMCs and dilation of the remodeling arteriole lumens. No statistical differences in α-SMA/lumen ratios for decidua-only controls were observed between 3 and 6 days, so the data were pooled for graphical representation. Within 500 μm of the decidual epithelial surface, a tenfold decrease in the α-SMA/lumen ratio of PDCs at both day 3 (P < 0.05) and 6 (P < 0.05) was observed compared with decidua-only controls. No significant difference existed between day 3 and 6 PDCs at this first depth, indicating that maximal VSMC loss occurs within the first 3 days in culture. The α-SMA/lumen ratio between 500 to 1000 μm was also significantly decreased at both day 3 (P < 0.05) and 6 (P < 0.01) compared with decidua-only controls, with no significant difference between time points. Beyond 1000 μm there was a trend toward a decrease in α-SMA/lumen ratio with time in culture, using pooled decidua-only controls. Interestingly, when α-SMA/lumen ratios from day 6 PDCs were compared with their internal experiment-matched day 6 controls at the 1000 to 2000 μm depth, a significant decrease in the PDC α-SMA/lumen ratio was found (P < 0.05). Similar" @default.
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• W133774038 title "Vascular-Leukocyte Interactions" @default.
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• W133774038 doi "https://doi.org/10.2353/ajpath.2010.091105" @default.
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