Molecular Insights into the Thrombotic and Microvascular Injury in Placental Endothelium of Women with Mild or Severe COVID-19

Abstract

Clinical manifestations of coronavirus disease 2019 (COVID-19) in pregnant women are diverse, and little is known of the impact of the disease on placental physiology. Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has been detected in the human placenta, and its binding receptor ACE2 is present in a variety of placental cells, including endothelium. Here, we analyze the impact of COVID-19 in placental endothelium, studying by immunofluorescence the expression of von Willebrand factor (vWf), claudin-5, and vascular endothelial (VE) cadherin in the decidua and chorionic villi of placentas from women with mild and severe COVID-19 in comparison to healthy controls. Our results indicate that: (1) vWf expression increases in the endothelium of decidua and chorionic villi of placentas derived from women with COVID-19, being higher in severe cases; (2) Claudin-5 and VE-cadherin expression decrease in the decidua and chorionic villus of placentas from women with severe COVID-19 but not in those with mild disease. Placental histological analysis reveals thrombosis, infarcts, and vascular wall remodeling, confirming the deleterious effect of COVID-19 on placental vessels. Together, these results suggest that placentas from women with COVID-19 have a condition of leaky endothelium and thrombosis, which is sensitive to disease severity.

Keywords: COVID-19; VE-cadherin; claudin-5; endothelium; placenta; von Willebrand factor.

Figure 1

The expression of vWf augments in decidual endothelium of placentas of women with COVID-19. Paraffin blocks of placenta from women with COVID-19 and controls were cut in 1 µm slices, deparaffinized and processed for immunofluorescence with antibodies against vWf. (a) Representative images of vWf in decidua. DNA of nuclei was stained with DAPI. Bar, 100 μm. (b) Quantification of mean fluorescent intensity done on three independent images from each condition. Data are expressed as mean ± SD; F ANOVA test followed by Duncan’s multiple comparison test, *** p < 0.001. Cnt, control; mCV, mild COVID-19; sCV, severe COVID-19.

Figure 2

The expression of vWf increases in the endothelium of chorionic villi in placentas of women with COVID-19. Paraffin blocks of placentas derived from women with COVID-19 and controls were cut in 1 µm slices, deparaffinized and processed for immunofluorescence with antibodies against vWf. (a) Representative images of vWf in chorionic villi. DNA of nuclei was stained with DAPI. Bar, 100 μm. (b) Quantification of mean fluorescent intensity done on three independent images from each condition. Data are expressed as mean ± SD; F ANOVA test followed by Duncan’s multiple comparison test, *** p < 0.001. Cnt, control; mCV, mild COVID-19; sCV, severe COVID-19.

Figure 3

The expression of claudin-5 decreases in decidual endothelium of women with severe COVID-19. Paraffin blocks of placentas derived from women with COVID-19 and controls were cut in 1 µm slices, deparaffinized, and processed for immunofluorescence with antibodies against claudin-5. (a) Representative images of claudin-5 in decidua. DNA of nuclei was stained with DAPI. Bar, 100 μm. (b) Quantification of mean fluorescent intensity done on three independent images from each condition. Data are expressed as mean ± SD; F test with Welch correction followed by the multiple comparison tests of Bonferroni and Dunnett, *** p < 0.001. Cnt, control; mCV, mild COVID-19; sCV, severe COVID-19.

Figure 4

Claudin-5 expression diminishes in the endothelium of chorionic villi of placentas from women with severe COVID-19. Paraffin blocks of placentas derived from women with COVID-19 and controls were cut in 1 µm slices, deparaffinized, and processed for immunofluorescence with antibodies against claudin-5. (a) Representative images of claudin-5 in chorionic villi. DNA of nuclei was stained with DAPI. Bar, 100 μm. (b) Quantification of mean fluorescent intensity done on three independent images from each condition. Data are expressed as mean ± SD; One way ANOVA F test followed by the multiple comparison tests of Bonferroni and Dunnett, *** p < 0.001. Cnt, control; mCV, mild COVID-19; sCV, severe COVID-19.

Figure 5

VE-cadherin expression in decidual endothelium decreases in placentas from women with severe COVID-19 but no in those with mild disease. Paraffin blocks of placental tissue derived from women with COVID-19 and controls were cut in 1 µm slices, deparaffinized, and processed for immunofluorescence with antibodies against VE-cadherin. (a) Representative images of VE-cadherin in decidua. DNA of nuclei was stained with DAPI. Bar, 100 μm. (b) Quantification of mean fluorescent intensity done on three independent images from each condition. Data are expressed as mean ± SD; F test with Welch correction followed by the multiple comparison tests of Bonferroni and Dunnett, *** p < 0.001. Cnt, control; mCV, mild COVID-19; sCV, severe COVID-19.

Figure 6

VE-cadherin expression diminishes in the endothelium of chorionic villi of placentas from women with severe COVID-19. Paraffin blocks of placentas derived from women with COVID-19 and controls were cut in 1 µm slices, deparaffinized, and processed for immunofluorescence with antibodies against VE-cadherin. (a) Representative images of VE-cadherin in chorionic villi. DNA of nuclei was stained with DAPI. Bar, 100 μm. (b) Quantification of mean fluorescent intensity done on three independent images from each condition. Data are expressed as median ± SD; Kruskal–Wallis test and its multiple comparison test, ** p = 0.02, *** p < 0.001. Cnt, control; mCV, mild COVID-19; sCV, severe COVID-19.

Figure 7

Histopathology of placentas from women with mild and severe COVID-19 shows signs of maternal and fetal vascular malperfusion and decidual vasculopathy. Representative placental sections stained with H&E (3 µm). Villous placental parenchyma from control women with no histological alterations ((A); 10×). Placental tissue from women with mild COVID 19 showing decidual vasculopathy characterized by fibrinoid necrosis (arrows), and karyorrhexis (*) ((B,C); 40× and 10×, respectively), avascular villous (*), and accelerate villous maturation (small villi not expected for gestational age) (arrow) ((D); 20×), and chorangiosis (arrow) ((E); 20×). Placental tissue from women with severe COVID 19 showing extensive parenchymal infarcts with accumulation of fibrin (*) ((F); 10×), acute and chronic inflammation in villous space (villitis, black arrow) and villous (intervillositis, white arrow) (F); magnification, 20×); subacute thrombosis of the intermediate villi with remodeling of the vascular wall (*) ((G); 10×) and vessel obliteration (arrow) ((G); magnification, 20×); avascular villi (*) ((H); magnification, 20×), and severe maternal vascular malperfusion with accelerate villous maturation (small villi not expected for gestational age) ((I); 10×) and increased syncytial knots (arrows) ((I); magnification, 20×).

Figure 8

Placental chorionic villi from women with severe COVID-19 display a higher number of Hofbauer cells. Hofbauer cells (arrows) were detected with an antibody against CD68 and counterstained with hematoxylin. Representative 3 µm placental sections (20×) from control women (A,D), women with mild COVID-19 (B,E), and women with severe COVID-19 (C,F). (G) Quantification of Hofbauer cells in placentas from control women (Cnt), with mild COVID-19 (mCV), and severe COVID-19 (sCV). The number of Hofbauer cells was obtained by counting CD68+ positive cells in five optical fields per placenta. Each dot represents the mean of Hofbauer cells in five fields per placenta of each condition. Data are expressed as mean ± SD; F ANOVA test followed by Duncan’s multiple comparison test, ** p < 0.01.