Figure 1. Dimorphism of the human umbilical artery and vein.

(a) Synchrotron imaging of umbilical vessels at birth illustrates a bilayered arterial wall comprising an inner buckled tunica media (TM) (red) and outer TM (purple) but no distinct inner layer or buckling in the vein. X-Y and Y-Z image planes are indicated by red dashed lines (n = 3 umbilical cords). (b) Quantitation of luminal cross-sectional area at birth shows that the umbilical arteries are occluded whereas the veins remain patent (top) and have significantly thicker walls (bottom) (n = 20 cords, error bars indicate mean ± S.E.M., whiskers indicate minimum and maximum values. ***, p<0.001). (c) Alcian blue, eosin (pink) and nuclear fast red staining of umbilical vessel cross-sections shows a proteoglycan-rich (blue) inner TM in the umbilical artery but not the vein. Quantified staining intensity is shown on the right (n = 6 umbilical cords, whiskers indicate minimum and maximum values, ***, p<0.001). (d) Chondroitin sulfate (CS), heparan sulfate (HS), aggrecan and versican immunofluorescence (n = 4 cords for each antibody) showing that CS staining corresponds with aggrecan and versican staining and alcian blue in (c). (e) Volcano plots illustrating differential gene expression between human umbilical artery (red) and vein (green) (top, n = 4 umbilical arteries and veins) and differential gene expression between human umbilical artery inner TM (red) and the outer TM (green) (bottom, n = 2). (f) RNA in situ hybridization shows robust ACAN and VCAN expression (red signal) in the inner artery TM and weak expression in the vein (n = 3 umbilical cords for each in situ probe). * marks the vessel lumen. Brackets in c,d,f mark the TM. Wj, Wharton’s jelly. Scale bars = 100 μm in c,d,f.

Figure 1—figure supplement 1. Morphological and cellular characteristics of the human umbilical arteries and veins at birth.

(a) Synchrotron image from Figure 1a without red and purple shading illustrates contrast between inner and outer arterial tunica media (TM) as well as inner TM buckling, compared with a uniform appearance of venous TM and lack of buckling (n = 3 umbilical cords). (b) Hematoxylin and eosin stained human umbilical artery cross-sections collected from the placental and fetal ends show occlusion of the umbilical artery with buckling of its interior, while the vein remains patent and lacks buckling (n = 25 umbilical cords). (c) Hematoxylin and eosin stained cross-sections show multiple cell layers composed of alternating circumferentially (C) or longitudinally (L) oriented smooth muscle cells (SMC) in the umbilical artery. Curved white arrows indicate internal protrusion of the inner TM of the umbilical artery arising from buckling. (d) α-SMA (α-smooth muscle actin, red) and DAPI (blue) staining of the umbilical artery and vein shows distinct orientation of the SMC layers as in (c) (n = 3 umbilical cords). (e) (Left) Alcian blue staining shows intense staining of the inner arterial TM, with radially-oriented rounded cells contrasting with outer TM cells having elongated morphology. Eosin (pink) and nuclear fast red counterstaining. (Right) Sox9 immunostaining shows nuclear staining of SMCs of the inner umbilical artery TM (n = 6 umbilical cords, * marks the vessel lumen in panels (c,d,e). White brackets in (c,d and e) mark the TM. Tm, tunica media). Scale bars = 200 μm in (b) and c, 100 μm in d and 50 μm in e.

Figure 1—figure supplement 2. Transcriptome comparison and pathway analysis of differences in the human umbilical artery and vein.

(a) Heat map of a subset of differentially expressed genes (1.9 fold-change) from umbilical artery vs vein (n = 4 umbilical cords). (b) Ingenuity pathway analysis (IPA) summary of the most significantly different pathways (n = 4 umbilical cords).

Figure 1—figure supplement 3. Transcriptome comparison and pathway analysis of differences in the human inner umbilical artery tunica media (TM) vs the outer tunica media.

(a) Heat map of a subset of differentially expressed genes (1.9 fold-change) (n = 2 umbilical arteries, two inner TM samples matched to two outer TM samples). (b) Ingenuity pathway analysis (IPA) showing significantly different pathways in the umbilical artery inner and outer tunica media (n = 2 umbilical arteries, two inner TM samples matched to two outer TM samples).

Figure 1—video 1. Synchrotron image stack of an umbilical artery.

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A human umbilical artery was imaged cross-sectionally and the images reconstructed along the luminal axis using Amira.

Figure 1—video 2. Synchrotron image stack of an umbilical vein.

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A human umbilical vein was imaged cross-sectionally and the images reconstructed along the luminal axis using Amira.