Magnetic Resonance Imaging Reveals Distinct Roles for Tissue Transglutaminase and Factor XIII in Maternal Angiogenesis During Early Mouse Pregnancy

Abstract

Objective: The early embryo implantation is characterized by enhanced uterine vascular permeability at the site of blastocyst attachment, followed by extracellular-matrix remodeling and angiogenesis. Two TG (transglutaminase) isoenzymes, TG2 (tissue TG) and FXIII (factor XIII), catalyze covalent cross-linking of the extracellular-matrix. However, their specific role during embryo implantation is not fully understood. Approach and Results: For mapping the distribution as well as the enzymatic activities of TG2 and FXIII towards blood-borne and resident extracellular-matrix substrates, we synthetized selective and specific low molecular weight substrate analogs for each of the isoenzymes. The implantation sites were challenged by genetically modifying the trophoblast cells in the outer layer of blastocysts, to either overexpress or deplete TG2 or FXIII, and the angiogenic response was studied by dynamic contrast-enhanced-magnetic resonance imaging. Dynamic contrast-enhanced-magnetic resonance imaging revealed a decrease in the permeability of decidual vasculature surrounding embryos in which FXIII were overexpressed in trophoblast cell. Reduction in decidual blood volume fraction was demonstrated when either FXIII or TG2 were overexpressed in embryonic trophoblast cell and was elevated when trophoblast cell was depleted of FXIII. These results were corroborated by histological analysis.

Conclusions: In this study, we report on the isoenzyme-specific roles of TG2 and FXIII during the early days of mouse pregnancy and further reveal their involvement in decidual angiogenesis. Our results reveal an important magnetic resonance imaging-detectable function of embryo-derived TG2 and FXIII on regulating maternal angiogenesis during embryo implantation in mice.Visual Overview: An online visual overview is available for this article.

Keywords: blood volume; embryo implantation; factor XIII; magnetic resonance imaging; transglutaminase.

Figure 1.. TG2 and FXIII activities, detected by SAs distribution, resemble their localization in ISs retrieved from E5.5 pregnant C57bl/6J mice.

Myr-Venus homozygote males) were mated with C57bl/6J female mice, producing hemizygote Myr-Venus embryos. A. TG2 localization in paraffin sections of embryo IS. Red represents antibody staining for TG2 (4 dams, 20 ISs); B, C. TG2 activity, detected by T29-B distribution 45 min after it was injected IV (B, 4 dams, 14 ISs) or applied in situ on live sections (C, 4 dams, 16 ISs). For images B and C, red represents T29-B distribution, followed by Cy3-streptavidin staining; D. Negative control sections stained with Cy3-streptavidin staining without T29-B SA (5 dams, 20 ISs); E. FXIII distribution in paraffin sections of embryo IS (4 dams, 20 ISs). Red represents antibody staining for FXIII; F, G. FXIII activity, detected by F11-B distribution 45 min after it was injected IV (F, 5 dams, 17 ISs) or applied in situ on live sections (G, 4 dams, 11 ISs). For images F and G, red represents F11-B distribution, following Cy3-streptavidine staining; H. Negative control sections stained with Cy3-streptavidin staining without F11-B SA (5 dams, 20 ISs). For all images: green represents hemizygote Myr-Venus embryos; blue DAPI staining. Image scale bars are 200 μm.

Figure 2.. TG2 and FXIII specific activities matched their localization on the feto-maternal interface of ISs retrieved from E6.5 pregnant C57bl/6J mice.

Myr-Venus homozygote males were mated with C57bl/6J female mice, producing hemizygote Myr-Venus embryos. A. Left, TG2 localization in paraffin sections of embryo IS. Red represent antibody for TG2. * Estimated embryonic region. Right, magnification of the marked region (3 dams, 24 ISs); TG2 activity, detected by T29-B distribution 45 min after it was injected IV (B, 3 dams, 13 ISs) or applied in situ on live sections (C, 4 dams, 18 ISs); D. TG2 specific activity, detected in situ on live sections after T29-B was applied in the presence of TG inhibitor, Iodoacetamide. For images B, C and D, red represents T29-B distribution, following Cy3-streptavidin staining (4 dams, 16 ISs); E. Left, FXIII localization in paraffin sections of embryo IS. Red represents antibody for FXIII. * Estimated embryonic region. Right, magnification of the marked region (4 dams, 28 ISs); FXIII activity, detected by F11-B distribution 45 min after it was injected IV (F, 5 dams, 24 ISs) or applied in situ on live sections (G, 5 dams, 20 ISs); H. FXIII specific activity, detected in situ on live sections after F11-B was applied in the presence of Iodoacetamide (4 dams, 16 ISs). For images F, G and H, red represents F11-B distribution, following Cy3-streptavidin staining. For all images: green represents hemizygote Myr-Venus embryos; DAPI in blue. Images scale bars are 100 or 200 μm.

Figure 3.. Impaired decidual vascular function of ISs with genetically modified TC overexpressing TG2 or FXIII.

T1 weighted gradient-echo images acquired from surrogate E6.5 pregnant ICR mice carrying transgenic embryonic TC overexpressing TG2 (A, D; TG2-LV-GFP), overexpressing FXIII (B, E; FXIII-LV-GFP) or expressing the control vector (C, F; Control-LV-GFP), 3 min (A, B, C) or 40 min (D, E, F) after biotin-BSA-GdDTPA injection. Individual ISs are indicated by orange circles. Right, magnification of the adjacent left image; Validation of the decidual blood vessels permeability functions by visualizing biotin-BSA-GdDTPA distribution in paraffin sections of ISs with transgenic embryonic TC overexpressing TG2 (G, Left), overexpressing FXIII (H, Left) or expressing the control vector (I, Left). Green represent contrast agent distribution, 40 min after it was injected, using streptavidin-Cy2 staining; Validation of functional decidual blood vessels by detecting BSA-ROX distribution in paraffin sections of ISs with transgenic embryonic TC overexpressing TG2 (G, Right), overexpressing FXIII (H, Right) or expressing the control vector (I, Right). Red represents the distribution of BSA-ROX, 2 min after it was injected; Quantitative analysis of vessel density and vascular permeability in the ISs using the MRI parameters: permeability surface area product (J) and fraction blood volume (K). The values are calculated as mean ± SD of transgenic embryos overexpressing TG2 (4 dams, 12 ISs), overexpressing FXIII (5 dams, 19 ISs) or expressing the control vector (5 dams, 13 ISs). * p<0.05 vs Control-LV-GFP. Image scale bars are 200 μm.

Figure 4.. Decidual blood vessel function increased in ISs with TG2- or FXIII-depleted embryonic TC.

T1 weighted gradient-echo images acquired from surrogate pregnant ICR mice carrying embryos with genetically modified TC depleted from TG2 (A, D; TG2-CRISPR-V2), depleted from FXIII (B, E; FXIII-CRISPR-V2) or expressing the control vector (C, F; Control-CRISPR-V2), 3 min (A, B, C) or 40 min (D, E, F) after biotin-BSA-GdDTPA injection. Individual ISs are indicated by orange circles. Right side, magnification of the adjacent left image; Validation of the decidual blood vessels permeability by visualizing biotin-BSA-GdDTPA distribution in paraffin sections of ISs with embryonic TC depleted from TG2 (D, Left; TG2-CRISPR-V2), depleted from FXIII (E, Left; FXIII-CRISPR-V2) or expressing the control vector (F, Left; Control-CRISPR-V2), Green represent the distribution of the contrast agent 40 min after injection using streptavidin-Cy2 staining; Validation of functional decidual blood vessels by detecting BSA-ROX distribution in paraffin sections of ISs with embryonic TC depleted from TG2 (G, Right; TG2-CRISPR-V2), depleted from FXIII (H, Right; FXIII-CRISPR-V2) or expressing the control vector (I, Right; Control-CRISPR-V2), Red represents the distribution of BSA-ROX injected 2 min before sacrificing the mouse; Quantitative analysis using the MRI parameters: permeability surface area product (J) and fraction blood volume (K). The values are calculated as mean ± SD of transgenic embryo models depleted from TG2 ( 3 dams, 10 ISs), FXIII (4 dams, 12 ISs) or expressing the control vector (4 dams, 9 ISs), PS: * p<0.05 vs Control-CRISPR-V2; ** p<0.05 vs TG2-CRISPR-V2. Images scale bars are 200 μm.

Figure 5.. Microvascular evaluation of the ISs with transgenic embryos.

Immunostaining for CD34 in ISs of genetically modified TC expressing the control vector (LV-GFP, A and E; CRISPR-V2, C and G, both 4 dams, 10 ISs), overexpressing TG2 (TG2-LV-GFP; B, 4 dams, 12 ISs), depleted from TG2 (D, 3 dams, 10 ISs), overexpressing FXIII (F, 5 dams, 19 ISs) or depleted from FXIII (H, 4 dams, 12 ISs). CD34 was visualized by cyan fluorescence channel after labeled with Cy5-avidin. Similar exposure time was used for each section and its appropriate control. Quantitative analysis of % CD34 staining in relation to IS area of genetically modified TC overexpressing TG2 (I), depleted from TG2 (J), overexpressing FXIII (K) or depleted from FXIII (L). Image scale bars are 200 μm. * p<0.05 vs Control-vector.

Figure 6.. The effect of TC derived TG2 or FXIII on the decidual vascular function.

A. Decidual blood vessels permeability and blood volume; Embryo TC infected by lenti virus to overexpressing TG2 (B, Left) or FXIII (C, Left), display a decrease in decidual blood volume. Overexpressing of FXIII also shows a significant decrease in decidual blood vessels permeability (C, Left); Decidual vasculature properties did not change when embryonic TC were depleted of TG2 (B, Right), while depletion of FXIII (C, Right) shows a significant increase in decidual blood volume. Red branched lines represent blood vessels density; ovals represent blood vessel permeability.