Maturation of the fetal human choriocapillaris

Abstract

Purpose: The purpose of this study was to examine the structural and functional maturation of the choriocapillaris (CC) and to determine when fenestrations form, the capillaries are invested with pericytes, and the endothelial cells (ECs) became functional.

Methods: Immunohistochemistry was performed on cryopreserved sections of embryonic/fetal human eyes from 7 to 22 weeks' gestation (WG), using antibodies against PAL-E, PV-1 (fenestrations), carbonic anhydrase IV (CA IV), eNOS, and alpha-smooth muscle actin (alphaSMA) and NG2 (two pericyte markers) and the EC marker (CD31). Alkaline phosphatase (APase) enzymatic activity was demonstrated by enzyme histochemistry. Transmission electron microscopy (TEM) was performed on eyes at 11, 14, 16, and 22 WG. Adult human eyes were used as the positive control.

Results: All EC markers were present in the CC by 7 WG. PAL-E, CA IV, and eNOS immunoreactivities and APase activity were present in the CC by 7 to 9 WG. TEM analysis demonstrated how structurally immature this vasculature was, even at 11 WG: no basement membrane, absence of pericytes, and poorly formed lumens that were filled with filopodia. The few fenestrations that were observed were often present within the luminal space in the filopodia. Contiguous fenestrations and significant PV-1 were not observed until 21 to 22 WG. alphaSMA was prominent at 22 WG, and the maturation of pericytes was confirmed by TEM.

Conclusions: It appears that ECs and their precursors express enzymes present in adult CC well before they are structurally mature. Although ECs make tight junctions early in development, contiguous fenestrations and mature pericytes occur much later in development.

Figure 1

Immunohistochemical localization of endothelial cell markers (A–C) and PV-1 (D) in 12 WG choroid. Endothelial cell markers label the choriocapillaris (CC) and developing deeper vessels while no PV-1 immunolabeling is seen. The structure of the choroid is shown with H&E (E) and a nonimmune IgG control for PV-1 is shown in “F”. (arrowhead, RPE; scale bar 30µm; A–D & F, APase immunoreactivity)

Figure 2

Ultrastructure of CC at 11 WG in periphery (A), equator (B), and posterior pole (C). In peripheral choroid, undifferentiated angioblasts (pericyte and endothelial cell precursors) had dense chromatin and formed loosely arranged aggregates with slit-like lumens (L). One angioblast appears to have assumed the posxition of a pericyte (arrow). Immature endothelial cells at the equator (B) had numerous mitochondria (M) with some tight junctions present (J). Complex membranous infoldings resembling filopodia (arrow F) extended into the developing lumen (F) and when immature erythrocytes (E) were present, their membranes appeared to fuse. In posterior pole (C), pericyte-like cells were observed and appeared more differentiated, with dispersed chromatin in their nuclei (PN) and numerous mitochondria (M) in close proximity with rough endoplasmic reticulum (RER) and some Golgi (arrowhead). Endothelial cells had condensed chromatin in their nuclei (EN), made tight junctions with one another and displayed some mitochondria (M). Bruch’s membrane (asterisk) was starting to form in the central choroid, posterior to the RPE basement membrane (BMb). (Scale bar in A&B 2 µm and C 1 µm)

Figure 3

Comparison of endothelial cell marker localization and PV-1 immunolabeling in 17 WG (A, C, E, G) and 21 WG fetal choroids (B, D, F, H). CD31 (A&B) and PAL-E (C&D) show labeling of both the CC and deeper vessels. Patchy weak PV-1 staining is seen in the 17 WG CC (arrow in “E”) while the reaction product is more intense and uniform in the 21 WG CC (paired arrows in “F”). H&E staining shows the structure of the choroid and RPE (arrowheads in all) with rudimentary inner segments (IS) are present at 21 WG (H). (scale bar = 30µm; A–F, APase immunoreactivity)

Figure 4

16 WG fetal choroid TEM showing an occasional scattered fenestration (arrow) in the CC endothelium in periphery and more numerous fenestrations in posterior pole (arrow in B). Very few filopodia (arrowhead) are present in the lumen (L) at this age.(scale bar = 1 µm)

Figure 5

TEM of a 22 WG fetal choroid showing scattered fenestrations (arrow) in the CC endothelium in periphery (arrow in A) and more numerous fenestrations in posterior pole (arrow in B). Membranous infoldings were fewer and sometimes had fenestration-like structures (arrowhead). (scale bar = 1 µm)

Figure 6

Comparison of endothelial cell marker staining and PV-1 immunolabeling in 73-yr-old. CD34 (A), CD31 (B) and PAL-E (C) show labeling of both the CC and larger vessels while PV-1 is restricted to the CC and most intense on the retinal side of CC (D).(arrowheads = RPE, scale bar = 30 µm) (E) TEM from a 58 yr-old shows contiguous fenestrations in the inner CC wall. (Scale bar = 1 µm)

Figure 7

Smooth muscle actin (αSMA) (A, C &E) and NG2 immunostaining (B, D &F) in choroid at 7 (A,B), 12 (C,D) and 22 WG (E,F). At 7 WG, no immunoreactivity for αSMA (A) was observed but NG2 (B) labeled cells were associated with the CC. At 12 WG, some scattered αSMA positive cells (C) were associated with CC and NG2 staining (D) was more intense. By 22 WG, αSMA immunoreactivity (E) was associated with CC, medium and large choroidal vessels. NG2 also intensely immunolabeled all vessels but the pattern was more uniform and similar to the pattern observed with endothelial cell markers (Figure 3B).(Scale bar = 30 µm)

Figure 8

Maturation of pericytes in CC. At 11 WG (A), poorly differentiated pericytes (PN) were associated with immature endothelial cells in peripheral choroid. The nuclei were irregularly shaped and had condensed chromatin. The cell cytoplasm contained rough endoplasmic reticulae (RER) and mitochrondria (M). Junctions (J) were present between endothelial cells (EN) and the developing lumen contained numerous endothelial cell projections. No basement membrane was apparent around the pericyte or endothelial cells. (B) A capillary from peripheral choroid at 14 WG. The pericyte was more compact and its processes form peg-in-socket formations with endothelial cell processes (double arrow in “B” shown at higher magnification in inset). There are many vesicles present in the endothelial cell processes. Both the pericyte and endothelial cell nuclei (EN) have condensed chromatin but this forming vessel had a more open lumen than at 11 WG. (Scale bar 2 mm)

Figure 9

At 22 WG, capillary walls were much thinner, tight junctions (TJ) were obvious, and basement membrane was being formed but still was limited. A pericyte was well developed (PN) and clearly abluminal but in close contact with endothelial cell processes (EP). (Scale bar in A 1 µm and B 2 µm)

Figure 10

Carbonic anhydrase IV (CA IV)(A, B, C) and eNOS (D, E, F) immunolocalization in 8 (A, D), 14 (B, E) and 21 WG choroid (C, F). Forming CC shows weak staining for CA IV and eNOS at 8 WG that increases with age with all choroidal vessels showing prominent immunoreactivity at 21 WG. (arrowhead, RPE). (Scale bar 50 µm; APase immunohistochemical reaction product in all)

Figure 11

Enzyme histochemistry for alkaline phosphatase (APase) and counterstaining with PAS. At 7 WG, forming CC already had APase activity (A). At 12 WG, the CC and intermediate connecting vessel had APase activity (B). At 16 WG, two layers of choroidal vessels showed APase activity (C). At 20 WG, all three layers of choroidal vasculature had APase activity (D). The limited PAS staining suggests that neither Bruch’s membrane nor capillary basement membranes were completely formed. (arrowhead, unbleached RPE).(Scale bar 50 µm)