Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: BMP6 signaling enhancement compensates for shallow trophoblast invasion in PE. An examination of clinical samples from PE patients and their gestational age-matched controls revealed that BMP6 was upregulated in preeclamptic placentas. BMP6 enhances trophoblast invasion via ID1-mediated upregulation of SERPINE2 and PlGF. Moreover, BMP6 also intensifies trophoblast vascular mimicry through the ID1-mediated upregulation of PlGF. Both canonical (p-SMAD1/5/9) and noncanonical (p-SMAD2/3) pathways participate in BMP6-induced ID1 expression. Our findings indicate that the increase in BMP6 signaling during late gestation could serve as a compensatory response to shallow trophoblast invasion in PE, which in light of BMP6 and its downstream targets could serve as diagnostic markers and therapeutic target applications in the clinical management of PE. This schematic diagram was created with Biorender.com

Article Snippet: Rat plasma was measured with a sFlt-1 ELISA kit (Proteintech, KE10069), a BMP6 ELISA Kit (Novus Biologicals, NBP2-69999, Centennial, CO, USA), and a PlGF ELISA kit (CUSABIO, CSB-E07400r, Wuhan, China).

Techniques: Expressing, Diagnostic Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: BMP6 facilitates human trophoblast invasion and vascular mimicry and increases the expression of ID1 , which is associated with PE. A , BMP6 is expressed at the maternal-fetal interface. Immunohistochemistry localization of BMP6 in first-trimester villi, first-trimester decidua, and third-trimester placenta. Scale bar, 100 μm. B-C , BMP6 facilitates human trophoblast invasion and vascular mimicry. B , HTR8/SVneo cells were treated with or without BMP6, followed by analysis of cell invasion. Scale bar, 100 μm. C , HTR8/SVneo cells were treated with or without BMP6, followed by analysis of endothelial-like tube formation. Scale bar, 100 μm. D - I , Bulk RNA-Seq analysis reveals that BMP6 treatment significantly upregulates ID1 , ID2 , and ID3 in HTR8/SVneo cells. D - F , Heatmap ( D ), volcano plots ( E ), and waterfall plots ( F ) obtained from RNA-Seq analysis of HTR8/SVneo cells with or without BMP6 treatment for 6 h. G - I , Heatmap ( G ), volcano plots ( H ), and waterfall plots ( I ) obtained from RNA-Seq analysis of HTR8/SVneo cells treated with or without BMP6 for 24 h. J , Purity of the primary human EVTs. Primary human EVTs were stained by CK7 (left panel) and HLA-G (right panel). Scale bar, 50 μm. K-P , single-cell analysis of the placenta shows ID1 is predominantly expressed in invasive EVTs. K and M , Analysis of previously published single-cell transcriptomes of human placentas collected during early pregnancy (6–12 gestational weeks, n = 5). L , N - P , Analysis of previously published single-cell transcriptomes of placentas collected during late pregnancy: three control placentas at 38 gestational weeks and three preeclamptic placentas at 34‒35 gestational weeks. K and L , UMAP visualization of all captured cell types in the placenta during early pregnancy ( K ) and late pregnancy ( L ), respectively. M and N , UMAP plot displaying the ID1 expression levels in all cell types in the early placenta ( M ) and the late placenta ( N ), respectively. O , UMAP visualization of all captured cell types in control women and PE patients. P , Violin plot displaying the expression level of ID1 in the EVT cell type. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. One-way ANOVA was used for analyses in B and C , and Student’s t-test was used for comparisons between two groups in P . Groups without common letters are significantly different from each other ( P < 0.05)BMP6, bone morphogenetic protein 6; GE, glandular epithelium; SC, stromal cell; CK7, cytokeratin-7; HLA-G, human leukocyte antigen G; UMAP, uniform manifold approximation and projection; EVT, extravillous cytotrophoblast; SCT, syncytiotrophoblast; VCT, villous cytotrophoblast; ID1, inhibitor of DNA-binding 1; PE, preeclampsia

Article Snippet: Rat plasma was measured with a sFlt-1 ELISA kit (Proteintech, KE10069), a BMP6 ELISA Kit (Novus Biologicals, NBP2-69999, Centennial, CO, USA), and a PlGF ELISA kit (CUSABIO, CSB-E07400r, Wuhan, China).

Techniques: Expressing, Immunohistochemistry, RNA Sequencing, Staining, Single-cell Analysis, Control, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: ID1 mediates BMP6-induced trophoblast invasion and vascular mimicry. A-D , BMP6 upregulated ID1 mRNA and protein levels in HTR8/SVneo cells. A and B , HTR8/SVneo cells were treated with different concentrations (0, 6.25, 12.5, 25, 50 or 100 ng/mL) of BMP6, and the ID1 mRNA levels after 6 h of treatment ( A ) and the ID1 protein levels after 24 h of treatment ( B ) were examined by RT‒qPCR and Western blot analysis, respectively. C - D , ID1 protein levels in HTR8/SVneo cells ( C ) and primary EVTs ( D ) after treatment with vehicle (Ctrl) or 50 ng/mL BMP6 for different durations. E‒I , ID1 mediates BMP6-promoted human trophoblast invasion and vascular mimicry. HTR8/SVneo cells or primary human EVTs were transfected for 48 h with 20 nM control non-targeting siRNA (si-Ctrl) or siRNA targeting ID1 (si- ID1 ) before treatment with or without 50 ng/mL BMP6. E and F , ID1 mRNA levels were examined by qPCR after BMP6 treatment for 6 h in HTR8/SVneo cells ( E ) and primary EVTs ( F ), with GAPDH used as the reference gene. G and H , Transwell assays were employed to examine the invasiveness of HTR8/SVneo cells ( G ) and primary EVTs ( H ) with or without BMP6 treatment for 36 h. Representative images from the invasion assay are displayed in the upper panel, while the summarized quantitative results of the invasion assay are shown in the lower panel. Scale bar, 100 μm. I , Endothelial-like tube formation assays were used to assess the acquisition of the endothelial-like phenotype of HTR8/SVneo cells with or without BMP6 treatment for 12 h. Representative images from the endothelial-like tube formation assay are displayed in the upper panel, while the summarized quantitative results of the endothelial-like tube formation assay are shown in the lower panel. Scale bar, 100 μm. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. One-way ANOVA was used for analyses in A and B . Two-way ANOVA was used for grouped analyses in C - I . Groups without letters are significantly different from each other ( P < 0.05). BMP6, bone morphogenetic protein 6; ID1, inhibitor of DNA-binding 1; Ctrl, control

Article Snippet: Rat plasma was measured with a sFlt-1 ELISA kit (Proteintech, KE10069), a BMP6 ELISA Kit (Novus Biologicals, NBP2-69999, Centennial, CO, USA), and a PlGF ELISA kit (CUSABIO, CSB-E07400r, Wuhan, China).

Techniques: Western Blot, Transfection, Control, Invasion Assay, Tube Formation Assay, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: ID1 mediates BMP6-induced SERPINE2 and PlGF upregulation in human trophoblasts. A-C , BMP6 upregulates SERPINE2 protein levels in trophoblasts. A , HTR8/SVneo cells were treated with different concentrations (0, 6.25, 12.5, 25, 50, or 100 ng/mL) of BMP6, and the SERPINE2 protein levels after 24 h of treatment were examined by Western blot analysis. The upper panel shows a representative Western blot image, and the lower panel shows the summarized quantitative results. B , SERPINE2 protein levels in HTR8/SVneo cells after treatment with vehicle (Ctrl) or 50 ng/mL BMP6 for different durations (24, 48, and 72 h). The upper panel shows a representative Western blot image, and the lower panel shows the summarized quantitative results. C , SERPINE2 protein levels in human primary EVTs. The left panel shows a representative Western blot image, and the right panel shows the summarized quantitative results. D-E , BMP6 promotes PlGF accumulation in the conditioned medium of trophoblasts. D , HTR8/SVneo cells were treated with or without 50 ng/mL BMP6 for 24–48 h. PlGF accumulation in conditioned medium was measured using ELISA. E , PlGF accumulation in conditioned medium was assayed by ELISA 48 h after BMP6 treatment in primary EVTs. F-J , ID1 mediates BMP6-induced SERPINE2 and PlGF upregulation in human trophoblasts. HTR8/SVneo cells were transfected for 48 h with 20 nM control non-targeting siRNA (si-Ctrl) or siRNA targeting ID1 (si- ID1 ) before treatment with or without 50 ng/mL BMP6. F , ID1 mRNA levels were examined by qPCR 6 h after BMP6 (50 ng/mL) treatment in HTR8/SVneo cells, with GAPDH used as the reference gene. G and H , SERPINE2 and ID1 protein levels in HTR8/SVneo cells ( G ) and human primary EVTs ( H ) after transfection with siRNA targeting ID1 , followed by treatment with or without BMP6 for 24 h, as assessed by Western blot. The left panel shows a representative Western blot image, and the right panel shows the summarized quantitative results. I , PGF mRNA levels were examined by qPCR 6 h after BMP6 treatment in HTR8/SVneo cells, with GAPDH used as the reference gene. J , PlGF accumulation in conditioned medium was assayed by ELISA 24 h after BMP6 treatment in HTR8/SVneo cells. K , SMAD4 mediates BMP6-induced upregulation of PlGF in human trophoblasts. HTR8/SVneo cells were transfected for 48 h with 20 nM control non-targeting siRNA (si-Ctrl) or siRNA targeting SMAD4 (si- SMAD4 ) before treatment with or without 50 ng/mL BMP6. PlGF accumulation in conditioned medium was assayed by ELISA 24 h after BMP6 treatment in HTR8/SVneo cells. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. One-way ANOVA was used for analyses in A , and two-way ANOVA was used for comparisons in B-K . Groups without common letters are significantly different from each other ( P < 0.05). BMP6, bone morphogenetic protein 6; SERPINE2, serpin family E member 2; EVT, extravillous cytotrophoblast; Ctrl, control; PlGF, placental growth factor; ID1, inhibitor of DNA-binding 1

Article Snippet: Rat plasma was measured with a sFlt-1 ELISA kit (Proteintech, KE10069), a BMP6 ELISA Kit (Novus Biologicals, NBP2-69999, Centennial, CO, USA), and a PlGF ELISA kit (CUSABIO, CSB-E07400r, Wuhan, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Control, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: Both SERPINE2 and PlGF mediate BMP6-induced trophoblast invasion. A-J , HTR8/SVneo cells or primary EVTs were transfected for 48 h with 20 nM control nontargeting siRNA (si-Ctrl), 20 nM siRNA targeting SERPINE2 (si- SERPINE2 ) or PGF (si- PGF ) before treatment with or without 50 ng/mL BMP6 for 24 h. A and B , The protein levels of SERPINE2 in HTR8/SVneo cells ( A ) and human primary EVTs ( B ) after 24 h of BMP6 treatment. The upper panel shows a representative Western blot image, and the lower panel shows the summarized quantitative results. C , PlGF accumulation in conditioned medium with or without BMP6 treatment for 24 h was assayed by ELISA in HTR8/SVneo cells. D , PGF mRNA levels in human primary EVTs with or without BMP6 treatment for 6 h were examined by RT‒qPCR, with GAPDH as the reference gene. E - H , Transwell assays were employed to examine the invasiveness of HTR8/SVneo cells ( E and G ) and primary EVTs ( F and H ) with or without BMP6 treatment for 36 h. I and J , Endothelial-like tube formation assays were used to assess vascular mimicry of HTR8/SVneo cells with or without BMP6 treatment for 12 h. Representative images from the endothelial-like tube formation assay are displayed in the above panel; the summarized quantitative results are displayed in the lower panel. Scale bar, 100 μm. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. Two-way ANOVA was used for data comparison. Groups without common letters are significantly different from each other ( P < 0.05). BMP6, bone morphogenetic protein 6; SERPINE2, serpin family E member 2; PGF, placental growth factor; Ctrl, control; EVT, extravillous cytotrophoblast

Article Snippet: Rat plasma was measured with a sFlt-1 ELISA kit (Proteintech, KE10069), a BMP6 ELISA Kit (Novus Biologicals, NBP2-69999, Centennial, CO, USA), and a PlGF ELISA kit (CUSABIO, CSB-E07400r, Wuhan, China).

Techniques: Transfection, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Tube Formation Assay, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: BMP6 is elevated in patients with PE and in PE model rats. A-C , BMP6 is elevated in patients with PE. A , RT‒qPCR analysis of BMP6 mRNA expression levels in the placentas of control women ( n = 10) and PE patients ( n = 10), with GAPDH as the reference gene. B , Western blot analysis of BMP6 protein expression levels in the placentas of control women ( n = 4) and PE patients ( n = 4). C , Spearman correlation analysis between the placental BMP6 mRNA levels and the value of log10 (SBP) of the corresponding patients. The gray area represents the 95% CI. Each dot represents one sample. D , The animal experimental protocol. E and F , BMP6 is elevated in the plasma of PE model rats. Rat plasma levels of BMP6 ( E ) and PlGF ( F ) in the Ad Fc + PBS group ( n = 4) and Ad Flt1 + PBS group ( n = 4). G-M , BMP6 is elevated in the placenta of PE model rats at G13. RNA-seq analysis of rat placentas at G13 in the Ad Fc + PBS group ( n = 3) and Ad Flt1 + PBS group ( n = 3). G , Heatmap depicting DEGs in the two groups. H , Dot plots of significantly enriched GO terms; the dot size represents the number of DEGs associated with a particular GO term. I , KEGG hierarchical network plot of pathways. J , GSEA-KEGG Ridge plot of pathways. K , GSEA plots of cytokine-cytokine receptor interaction pathway. L , Volcano plot of RNA-seq data showing DEGs between the Ad Fc + PBS group and the Ad Flt1 + PBS group. M , Circos graph displaying the coexpression networks of five genes in rat placenta samples. Each sector of the circle represents one gene, and its width indicates the total amount of co-occurrence that connects one gene to the other. The width of each link represents the total number of coexpressed genes among the linked genes. Student’s t-test was used for comparisons between two groups in A , B , E , and F . Groups without common letters are significantly different from each other ( P < 0.05). SD, Sprague–Dawley; BMP6, bone morphogenetic protein 6; PlGF, placental growth factor; PBS, phosphate-buffered saline; Ad Flt1, adenovirus expressing fms-like tyrosine kinase-1; Ad Fc, adenovirus-expressing control IgG2a Fc fragment; FC, fold change; Serpine2, serpin family E member 2; Id1, inhibitor of DNA-binding 1

Article Snippet: Rat plasma was measured with a sFlt-1 ELISA kit (Proteintech, KE10069), a BMP6 ELISA Kit (Novus Biologicals, NBP2-69999, Centennial, CO, USA), and a PlGF ELISA kit (CUSABIO, CSB-E07400r, Wuhan, China).

Techniques: Expressing, Control, Western Blot, Clinical Proteomics, RNA Sequencing, Saline, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: Supplementation with recombinant BMP6 alleviates PE-related phenotypes in the Ad Flt1-induced PE rat model. An SD rat model of PE was established as described in Fig. A. Pregnant rats were randomly divided into four groups: the Ad Fc + PBS, Ad Fc + BMP6, Ad Flt1 + PBS, and Ad Flt1 + BMP6 groups. A , BMP6 supplementation rescues the increased blood pressure in the Ad Flt1-induced PE rat model. SBP and MAP of pregnant rats in each group ( n = 4). B-C , BMP6 supplementation rescues fetal growth restriction and placental hypoefficiency in the Ad Flt1-induced PE rat model. B , Representative images of fetal rats and placentas from each group at G19 ( n = 7). C , Weights of fetal rats and corresponding placental efficiency at G19 in the Ad Fc + PBS group ( n = 33), Ad Fc + BMP6 group ( n = 39), Ad Flt1 + PBS group ( n = 32), and Ad Flt1 + BMP6 group ( n = 47). D-F , BMP6 supplementation alleviates placental damage in the Ad Flt1-induced PE rat model. D , HE staining of rat placentas was performed to observe the placental labyrinth and junction areas. Representative images are presented in the left panel, and the placental labyrinth/junction ratios in each group were quantified and summarized in the right panel ( n = 8). Scale bar, 3 mm. E , Immunohistochemistry localization of CD34 in rat placenta on G19. Representative images are presented in the left panel, and the ratios of CD34-positive area were quantified and are summarized in the right panel. Scale bar, 100 μm. F , Immunohistochemistry localization of α-SMA in the rat uterus on G19. Scale bar, 100 μm. Representative images are presented in the left panel, and the ratios of un-remodeled blood vessels were quantified and are summarized in the right panel. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. Two-way ANOVA was used for data comparison. Groups without common letters are significantly different from each other ( P < 0.05). SD, Sprague–Dawley; SBP, systolic blood pressure; MAP, mean arterial pressure; PBS, phosphate-buffered saline; Ad Fc, adenovirus-expressing control IgG2a Fc fragment; Ad Flt1, adenovirus expressing fms-like tyrosine kinase-1; LZ, labyrinth zone; JZ, junction zone

Article Snippet: Rat plasma was measured with a sFlt-1 ELISA kit (Proteintech, KE10069), a BMP6 ELISA Kit (Novus Biologicals, NBP2-69999, Centennial, CO, USA), and a PlGF ELISA kit (CUSABIO, CSB-E07400r, Wuhan, China).

Techniques: Recombinant, Staining, Immunohistochemistry, Comparison, Saline, Expressing, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: BMP6 signaling enhancement compensates for shallow trophoblast invasion in PE. An examination of clinical samples from PE patients and their gestational age-matched controls revealed that BMP6 was upregulated in preeclamptic placentas. BMP6 enhances trophoblast invasion via ID1-mediated upregulation of SERPINE2 and PlGF. Moreover, BMP6 also intensifies trophoblast vascular mimicry through the ID1-mediated upregulation of PlGF. Both canonical (p-SMAD1/5/9) and noncanonical (p-SMAD2/3) pathways participate in BMP6-induced ID1 expression. Our findings indicate that the increase in BMP6 signaling during late gestation could serve as a compensatory response to shallow trophoblast invasion in PE, which in light of BMP6 and its downstream targets could serve as diagnostic markers and therapeutic target applications in the clinical management of PE. This schematic diagram was created with Biorender.com

Article Snippet: SMAD1 (D59D7) rabbit monoclonal antibody (#6944), phospho-SMAD1/5/9 (D5B10) rabbit monoclonal antibody (#13820), SMAD2 (D43B4) rabbit monoclonal antibody (#5339), phospho-SMAD2 (E8F3R) rabbit monoclonal antibody (#18338), SMAD3 (C67H9) rabbit monoclonal antibody (#9523), phospho-SMAD3 (C25A9) rabbit monoclonal antibody (# 9520), SMAD4 (D3R4N) rabbit monoclonal antibody (#46535), horseradish peroxidase (HRP)-linked anti-mouse IgG (#7076), and anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA); the mouse monoclonal antibody HLA-G (#11–499-C100) was purchased from EXBIO (Vestec, Czech Republic); CK7-specific rabbit polyclonal antibody (#17513-1-AP), BMP6 rabbit polyclonal antibody (#55421-1-AP), SERPINE2 rabbit polyclonal antibody (#11303-1-AP), and α-Tubulin mouse monoclonal antibody (#66031-1-Ig) were purchased from Proteintech (Wuhan, China); ID1 mouse monoclonal antibody (#sc-133104) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); CD34 rabbit monoclonal antibody (#A19015) and α-SMA rabbit monoclonal antibody (#A2235) were obtained from ABclonal (Wuhan, China); Goat anti-Rabbit IgG secondary antibody, Alexa Fluor 594 (#A-11012) and goat anti-Mouse IgG secondary antibody, Alexa Fluor 488 (#A-11011) were purchased from Thermo Fisher (NY, USA).

Techniques: Expressing, Diagnostic Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: BMP6 facilitates human trophoblast invasion and vascular mimicry and increases the expression of ID1 , which is associated with PE. A , BMP6 is expressed at the maternal-fetal interface. Immunohistochemistry localization of BMP6 in first-trimester villi, first-trimester decidua, and third-trimester placenta. Scale bar, 100 μm. B-C , BMP6 facilitates human trophoblast invasion and vascular mimicry. B , HTR8/SVneo cells were treated with or without BMP6, followed by analysis of cell invasion. Scale bar, 100 μm. C , HTR8/SVneo cells were treated with or without BMP6, followed by analysis of endothelial-like tube formation. Scale bar, 100 μm. D - I , Bulk RNA-Seq analysis reveals that BMP6 treatment significantly upregulates ID1 , ID2 , and ID3 in HTR8/SVneo cells. D - F , Heatmap ( D ), volcano plots ( E ), and waterfall plots ( F ) obtained from RNA-Seq analysis of HTR8/SVneo cells with or without BMP6 treatment for 6 h. G - I , Heatmap ( G ), volcano plots ( H ), and waterfall plots ( I ) obtained from RNA-Seq analysis of HTR8/SVneo cells treated with or without BMP6 for 24 h. J , Purity of the primary human EVTs. Primary human EVTs were stained by CK7 (left panel) and HLA-G (right panel). Scale bar, 50 μm. K-P , single-cell analysis of the placenta shows ID1 is predominantly expressed in invasive EVTs. K and M , Analysis of previously published single-cell transcriptomes of human placentas collected during early pregnancy (6–12 gestational weeks, n = 5). L , N - P , Analysis of previously published single-cell transcriptomes of placentas collected during late pregnancy: three control placentas at 38 gestational weeks and three preeclamptic placentas at 34‒35 gestational weeks. K and L , UMAP visualization of all captured cell types in the placenta during early pregnancy ( K ) and late pregnancy ( L ), respectively. M and N , UMAP plot displaying the ID1 expression levels in all cell types in the early placenta ( M ) and the late placenta ( N ), respectively. O , UMAP visualization of all captured cell types in control women and PE patients. P , Violin plot displaying the expression level of ID1 in the EVT cell type. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. One-way ANOVA was used for analyses in B and C , and Student’s t-test was used for comparisons between two groups in P . Groups without common letters are significantly different from each other ( P < 0.05)BMP6, bone morphogenetic protein 6; GE, glandular epithelium; SC, stromal cell; CK7, cytokeratin-7; HLA-G, human leukocyte antigen G; UMAP, uniform manifold approximation and projection; EVT, extravillous cytotrophoblast; SCT, syncytiotrophoblast; VCT, villous cytotrophoblast; ID1, inhibitor of DNA-binding 1; PE, preeclampsia

Article Snippet: SMAD1 (D59D7) rabbit monoclonal antibody (#6944), phospho-SMAD1/5/9 (D5B10) rabbit monoclonal antibody (#13820), SMAD2 (D43B4) rabbit monoclonal antibody (#5339), phospho-SMAD2 (E8F3R) rabbit monoclonal antibody (#18338), SMAD3 (C67H9) rabbit monoclonal antibody (#9523), phospho-SMAD3 (C25A9) rabbit monoclonal antibody (# 9520), SMAD4 (D3R4N) rabbit monoclonal antibody (#46535), horseradish peroxidase (HRP)-linked anti-mouse IgG (#7076), and anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA); the mouse monoclonal antibody HLA-G (#11–499-C100) was purchased from EXBIO (Vestec, Czech Republic); CK7-specific rabbit polyclonal antibody (#17513-1-AP), BMP6 rabbit polyclonal antibody (#55421-1-AP), SERPINE2 rabbit polyclonal antibody (#11303-1-AP), and α-Tubulin mouse monoclonal antibody (#66031-1-Ig) were purchased from Proteintech (Wuhan, China); ID1 mouse monoclonal antibody (#sc-133104) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); CD34 rabbit monoclonal antibody (#A19015) and α-SMA rabbit monoclonal antibody (#A2235) were obtained from ABclonal (Wuhan, China); Goat anti-Rabbit IgG secondary antibody, Alexa Fluor 594 (#A-11012) and goat anti-Mouse IgG secondary antibody, Alexa Fluor 488 (#A-11011) were purchased from Thermo Fisher (NY, USA).

Techniques: Expressing, Immunohistochemistry, RNA Sequencing, Staining, Single-cell Analysis, Control, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: ID1 mediates BMP6-induced trophoblast invasion and vascular mimicry. A-D , BMP6 upregulated ID1 mRNA and protein levels in HTR8/SVneo cells. A and B , HTR8/SVneo cells were treated with different concentrations (0, 6.25, 12.5, 25, 50 or 100 ng/mL) of BMP6, and the ID1 mRNA levels after 6 h of treatment ( A ) and the ID1 protein levels after 24 h of treatment ( B ) were examined by RT‒qPCR and Western blot analysis, respectively. C - D , ID1 protein levels in HTR8/SVneo cells ( C ) and primary EVTs ( D ) after treatment with vehicle (Ctrl) or 50 ng/mL BMP6 for different durations. E‒I , ID1 mediates BMP6-promoted human trophoblast invasion and vascular mimicry. HTR8/SVneo cells or primary human EVTs were transfected for 48 h with 20 nM control non-targeting siRNA (si-Ctrl) or siRNA targeting ID1 (si- ID1 ) before treatment with or without 50 ng/mL BMP6. E and F , ID1 mRNA levels were examined by qPCR after BMP6 treatment for 6 h in HTR8/SVneo cells ( E ) and primary EVTs ( F ), with GAPDH used as the reference gene. G and H , Transwell assays were employed to examine the invasiveness of HTR8/SVneo cells ( G ) and primary EVTs ( H ) with or without BMP6 treatment for 36 h. Representative images from the invasion assay are displayed in the upper panel, while the summarized quantitative results of the invasion assay are shown in the lower panel. Scale bar, 100 μm. I , Endothelial-like tube formation assays were used to assess the acquisition of the endothelial-like phenotype of HTR8/SVneo cells with or without BMP6 treatment for 12 h. Representative images from the endothelial-like tube formation assay are displayed in the upper panel, while the summarized quantitative results of the endothelial-like tube formation assay are shown in the lower panel. Scale bar, 100 μm. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. One-way ANOVA was used for analyses in A and B . Two-way ANOVA was used for grouped analyses in C - I . Groups without letters are significantly different from each other ( P < 0.05). BMP6, bone morphogenetic protein 6; ID1, inhibitor of DNA-binding 1; Ctrl, control

Article Snippet: SMAD1 (D59D7) rabbit monoclonal antibody (#6944), phospho-SMAD1/5/9 (D5B10) rabbit monoclonal antibody (#13820), SMAD2 (D43B4) rabbit monoclonal antibody (#5339), phospho-SMAD2 (E8F3R) rabbit monoclonal antibody (#18338), SMAD3 (C67H9) rabbit monoclonal antibody (#9523), phospho-SMAD3 (C25A9) rabbit monoclonal antibody (# 9520), SMAD4 (D3R4N) rabbit monoclonal antibody (#46535), horseradish peroxidase (HRP)-linked anti-mouse IgG (#7076), and anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA); the mouse monoclonal antibody HLA-G (#11–499-C100) was purchased from EXBIO (Vestec, Czech Republic); CK7-specific rabbit polyclonal antibody (#17513-1-AP), BMP6 rabbit polyclonal antibody (#55421-1-AP), SERPINE2 rabbit polyclonal antibody (#11303-1-AP), and α-Tubulin mouse monoclonal antibody (#66031-1-Ig) were purchased from Proteintech (Wuhan, China); ID1 mouse monoclonal antibody (#sc-133104) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); CD34 rabbit monoclonal antibody (#A19015) and α-SMA rabbit monoclonal antibody (#A2235) were obtained from ABclonal (Wuhan, China); Goat anti-Rabbit IgG secondary antibody, Alexa Fluor 594 (#A-11012) and goat anti-Mouse IgG secondary antibody, Alexa Fluor 488 (#A-11011) were purchased from Thermo Fisher (NY, USA).

Techniques: Western Blot, Transfection, Control, Invasion Assay, Tube Formation Assay, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: ID1 mediates BMP6-induced SERPINE2 and PlGF upregulation in human trophoblasts. A-C , BMP6 upregulates SERPINE2 protein levels in trophoblasts. A , HTR8/SVneo cells were treated with different concentrations (0, 6.25, 12.5, 25, 50, or 100 ng/mL) of BMP6, and the SERPINE2 protein levels after 24 h of treatment were examined by Western blot analysis. The upper panel shows a representative Western blot image, and the lower panel shows the summarized quantitative results. B , SERPINE2 protein levels in HTR8/SVneo cells after treatment with vehicle (Ctrl) or 50 ng/mL BMP6 for different durations (24, 48, and 72 h). The upper panel shows a representative Western blot image, and the lower panel shows the summarized quantitative results. C , SERPINE2 protein levels in human primary EVTs. The left panel shows a representative Western blot image, and the right panel shows the summarized quantitative results. D-E , BMP6 promotes PlGF accumulation in the conditioned medium of trophoblasts. D , HTR8/SVneo cells were treated with or without 50 ng/mL BMP6 for 24–48 h. PlGF accumulation in conditioned medium was measured using ELISA. E , PlGF accumulation in conditioned medium was assayed by ELISA 48 h after BMP6 treatment in primary EVTs. F-J , ID1 mediates BMP6-induced SERPINE2 and PlGF upregulation in human trophoblasts. HTR8/SVneo cells were transfected for 48 h with 20 nM control non-targeting siRNA (si-Ctrl) or siRNA targeting ID1 (si- ID1 ) before treatment with or without 50 ng/mL BMP6. F , ID1 mRNA levels were examined by qPCR 6 h after BMP6 (50 ng/mL) treatment in HTR8/SVneo cells, with GAPDH used as the reference gene. G and H , SERPINE2 and ID1 protein levels in HTR8/SVneo cells ( G ) and human primary EVTs ( H ) after transfection with siRNA targeting ID1 , followed by treatment with or without BMP6 for 24 h, as assessed by Western blot. The left panel shows a representative Western blot image, and the right panel shows the summarized quantitative results. I , PGF mRNA levels were examined by qPCR 6 h after BMP6 treatment in HTR8/SVneo cells, with GAPDH used as the reference gene. J , PlGF accumulation in conditioned medium was assayed by ELISA 24 h after BMP6 treatment in HTR8/SVneo cells. K , SMAD4 mediates BMP6-induced upregulation of PlGF in human trophoblasts. HTR8/SVneo cells were transfected for 48 h with 20 nM control non-targeting siRNA (si-Ctrl) or siRNA targeting SMAD4 (si- SMAD4 ) before treatment with or without 50 ng/mL BMP6. PlGF accumulation in conditioned medium was assayed by ELISA 24 h after BMP6 treatment in HTR8/SVneo cells. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. One-way ANOVA was used for analyses in A , and two-way ANOVA was used for comparisons in B-K . Groups without common letters are significantly different from each other ( P < 0.05). BMP6, bone morphogenetic protein 6; SERPINE2, serpin family E member 2; EVT, extravillous cytotrophoblast; Ctrl, control; PlGF, placental growth factor; ID1, inhibitor of DNA-binding 1

Article Snippet: SMAD1 (D59D7) rabbit monoclonal antibody (#6944), phospho-SMAD1/5/9 (D5B10) rabbit monoclonal antibody (#13820), SMAD2 (D43B4) rabbit monoclonal antibody (#5339), phospho-SMAD2 (E8F3R) rabbit monoclonal antibody (#18338), SMAD3 (C67H9) rabbit monoclonal antibody (#9523), phospho-SMAD3 (C25A9) rabbit monoclonal antibody (# 9520), SMAD4 (D3R4N) rabbit monoclonal antibody (#46535), horseradish peroxidase (HRP)-linked anti-mouse IgG (#7076), and anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA); the mouse monoclonal antibody HLA-G (#11–499-C100) was purchased from EXBIO (Vestec, Czech Republic); CK7-specific rabbit polyclonal antibody (#17513-1-AP), BMP6 rabbit polyclonal antibody (#55421-1-AP), SERPINE2 rabbit polyclonal antibody (#11303-1-AP), and α-Tubulin mouse monoclonal antibody (#66031-1-Ig) were purchased from Proteintech (Wuhan, China); ID1 mouse monoclonal antibody (#sc-133104) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); CD34 rabbit monoclonal antibody (#A19015) and α-SMA rabbit monoclonal antibody (#A2235) were obtained from ABclonal (Wuhan, China); Goat anti-Rabbit IgG secondary antibody, Alexa Fluor 594 (#A-11012) and goat anti-Mouse IgG secondary antibody, Alexa Fluor 488 (#A-11011) were purchased from Thermo Fisher (NY, USA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Control, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: Both SERPINE2 and PlGF mediate BMP6-induced trophoblast invasion. A-J , HTR8/SVneo cells or primary EVTs were transfected for 48 h with 20 nM control nontargeting siRNA (si-Ctrl), 20 nM siRNA targeting SERPINE2 (si- SERPINE2 ) or PGF (si- PGF ) before treatment with or without 50 ng/mL BMP6 for 24 h. A and B , The protein levels of SERPINE2 in HTR8/SVneo cells ( A ) and human primary EVTs ( B ) after 24 h of BMP6 treatment. The upper panel shows a representative Western blot image, and the lower panel shows the summarized quantitative results. C , PlGF accumulation in conditioned medium with or without BMP6 treatment for 24 h was assayed by ELISA in HTR8/SVneo cells. D , PGF mRNA levels in human primary EVTs with or without BMP6 treatment for 6 h were examined by RT‒qPCR, with GAPDH as the reference gene. E - H , Transwell assays were employed to examine the invasiveness of HTR8/SVneo cells ( E and G ) and primary EVTs ( F and H ) with or without BMP6 treatment for 36 h. I and J , Endothelial-like tube formation assays were used to assess vascular mimicry of HTR8/SVneo cells with or without BMP6 treatment for 12 h. Representative images from the endothelial-like tube formation assay are displayed in the above panel; the summarized quantitative results are displayed in the lower panel. Scale bar, 100 μm. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. Two-way ANOVA was used for data comparison. Groups without common letters are significantly different from each other ( P < 0.05). BMP6, bone morphogenetic protein 6; SERPINE2, serpin family E member 2; PGF, placental growth factor; Ctrl, control; EVT, extravillous cytotrophoblast

Article Snippet: SMAD1 (D59D7) rabbit monoclonal antibody (#6944), phospho-SMAD1/5/9 (D5B10) rabbit monoclonal antibody (#13820), SMAD2 (D43B4) rabbit monoclonal antibody (#5339), phospho-SMAD2 (E8F3R) rabbit monoclonal antibody (#18338), SMAD3 (C67H9) rabbit monoclonal antibody (#9523), phospho-SMAD3 (C25A9) rabbit monoclonal antibody (# 9520), SMAD4 (D3R4N) rabbit monoclonal antibody (#46535), horseradish peroxidase (HRP)-linked anti-mouse IgG (#7076), and anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA); the mouse monoclonal antibody HLA-G (#11–499-C100) was purchased from EXBIO (Vestec, Czech Republic); CK7-specific rabbit polyclonal antibody (#17513-1-AP), BMP6 rabbit polyclonal antibody (#55421-1-AP), SERPINE2 rabbit polyclonal antibody (#11303-1-AP), and α-Tubulin mouse monoclonal antibody (#66031-1-Ig) were purchased from Proteintech (Wuhan, China); ID1 mouse monoclonal antibody (#sc-133104) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); CD34 rabbit monoclonal antibody (#A19015) and α-SMA rabbit monoclonal antibody (#A2235) were obtained from ABclonal (Wuhan, China); Goat anti-Rabbit IgG secondary antibody, Alexa Fluor 594 (#A-11012) and goat anti-Mouse IgG secondary antibody, Alexa Fluor 488 (#A-11011) were purchased from Thermo Fisher (NY, USA).

Techniques: Transfection, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Tube Formation Assay, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: BMP6 is elevated in patients with PE and in PE model rats. A-C , BMP6 is elevated in patients with PE. A , RT‒qPCR analysis of BMP6 mRNA expression levels in the placentas of control women ( n = 10) and PE patients ( n = 10), with GAPDH as the reference gene. B , Western blot analysis of BMP6 protein expression levels in the placentas of control women ( n = 4) and PE patients ( n = 4). C , Spearman correlation analysis between the placental BMP6 mRNA levels and the value of log10 (SBP) of the corresponding patients. The gray area represents the 95% CI. Each dot represents one sample. D , The animal experimental protocol. E and F , BMP6 is elevated in the plasma of PE model rats. Rat plasma levels of BMP6 ( E ) and PlGF ( F ) in the Ad Fc + PBS group ( n = 4) and Ad Flt1 + PBS group ( n = 4). G-M , BMP6 is elevated in the placenta of PE model rats at G13. RNA-seq analysis of rat placentas at G13 in the Ad Fc + PBS group ( n = 3) and Ad Flt1 + PBS group ( n = 3). G , Heatmap depicting DEGs in the two groups. H , Dot plots of significantly enriched GO terms; the dot size represents the number of DEGs associated with a particular GO term. I , KEGG hierarchical network plot of pathways. J , GSEA-KEGG Ridge plot of pathways. K , GSEA plots of cytokine-cytokine receptor interaction pathway. L , Volcano plot of RNA-seq data showing DEGs between the Ad Fc + PBS group and the Ad Flt1 + PBS group. M , Circos graph displaying the coexpression networks of five genes in rat placenta samples. Each sector of the circle represents one gene, and its width indicates the total amount of co-occurrence that connects one gene to the other. The width of each link represents the total number of coexpressed genes among the linked genes. Student’s t-test was used for comparisons between two groups in A , B , E , and F . Groups without common letters are significantly different from each other ( P < 0.05). SD, Sprague–Dawley; BMP6, bone morphogenetic protein 6; PlGF, placental growth factor; PBS, phosphate-buffered saline; Ad Flt1, adenovirus expressing fms-like tyrosine kinase-1; Ad Fc, adenovirus-expressing control IgG2a Fc fragment; FC, fold change; Serpine2, serpin family E member 2; Id1, inhibitor of DNA-binding 1

Article Snippet: SMAD1 (D59D7) rabbit monoclonal antibody (#6944), phospho-SMAD1/5/9 (D5B10) rabbit monoclonal antibody (#13820), SMAD2 (D43B4) rabbit monoclonal antibody (#5339), phospho-SMAD2 (E8F3R) rabbit monoclonal antibody (#18338), SMAD3 (C67H9) rabbit monoclonal antibody (#9523), phospho-SMAD3 (C25A9) rabbit monoclonal antibody (# 9520), SMAD4 (D3R4N) rabbit monoclonal antibody (#46535), horseradish peroxidase (HRP)-linked anti-mouse IgG (#7076), and anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA); the mouse monoclonal antibody HLA-G (#11–499-C100) was purchased from EXBIO (Vestec, Czech Republic); CK7-specific rabbit polyclonal antibody (#17513-1-AP), BMP6 rabbit polyclonal antibody (#55421-1-AP), SERPINE2 rabbit polyclonal antibody (#11303-1-AP), and α-Tubulin mouse monoclonal antibody (#66031-1-Ig) were purchased from Proteintech (Wuhan, China); ID1 mouse monoclonal antibody (#sc-133104) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); CD34 rabbit monoclonal antibody (#A19015) and α-SMA rabbit monoclonal antibody (#A2235) were obtained from ABclonal (Wuhan, China); Goat anti-Rabbit IgG secondary antibody, Alexa Fluor 594 (#A-11012) and goat anti-Mouse IgG secondary antibody, Alexa Fluor 488 (#A-11011) were purchased from Thermo Fisher (NY, USA).

Techniques: Expressing, Control, Western Blot, Clinical Proteomics, RNA Sequencing, Saline, Binding Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: BMP6 as a therapeutic target for preeclampsia: enhancing trophoblast invasion and vascular mimicry

doi: 10.1007/s00018-025-06040-w

Figure Lengend Snippet: Supplementation with recombinant BMP6 alleviates PE-related phenotypes in the Ad Flt1-induced PE rat model. An SD rat model of PE was established as described in Fig. A. Pregnant rats were randomly divided into four groups: the Ad Fc + PBS, Ad Fc + BMP6, Ad Flt1 + PBS, and Ad Flt1 + BMP6 groups. A , BMP6 supplementation rescues the increased blood pressure in the Ad Flt1-induced PE rat model. SBP and MAP of pregnant rats in each group ( n = 4). B-C , BMP6 supplementation rescues fetal growth restriction and placental hypoefficiency in the Ad Flt1-induced PE rat model. B , Representative images of fetal rats and placentas from each group at G19 ( n = 7). C , Weights of fetal rats and corresponding placental efficiency at G19 in the Ad Fc + PBS group ( n = 33), Ad Fc + BMP6 group ( n = 39), Ad Flt1 + PBS group ( n = 32), and Ad Flt1 + BMP6 group ( n = 47). D-F , BMP6 supplementation alleviates placental damage in the Ad Flt1-induced PE rat model. D , HE staining of rat placentas was performed to observe the placental labyrinth and junction areas. Representative images are presented in the left panel, and the placental labyrinth/junction ratios in each group were quantified and summarized in the right panel ( n = 8). Scale bar, 3 mm. E , Immunohistochemistry localization of CD34 in rat placenta on G19. Representative images are presented in the left panel, and the ratios of CD34-positive area were quantified and are summarized in the right panel. Scale bar, 100 μm. F , Immunohistochemistry localization of α-SMA in the rat uterus on G19. Scale bar, 100 μm. Representative images are presented in the left panel, and the ratios of un-remodeled blood vessels were quantified and are summarized in the right panel. The quantitative results are expressed as the means ± SEMs of at least three independent experiments. Two-way ANOVA was used for data comparison. Groups without common letters are significantly different from each other ( P < 0.05). SD, Sprague–Dawley; SBP, systolic blood pressure; MAP, mean arterial pressure; PBS, phosphate-buffered saline; Ad Fc, adenovirus-expressing control IgG2a Fc fragment; Ad Flt1, adenovirus expressing fms-like tyrosine kinase-1; LZ, labyrinth zone; JZ, junction zone

Article Snippet: SMAD1 (D59D7) rabbit monoclonal antibody (#6944), phospho-SMAD1/5/9 (D5B10) rabbit monoclonal antibody (#13820), SMAD2 (D43B4) rabbit monoclonal antibody (#5339), phospho-SMAD2 (E8F3R) rabbit monoclonal antibody (#18338), SMAD3 (C67H9) rabbit monoclonal antibody (#9523), phospho-SMAD3 (C25A9) rabbit monoclonal antibody (# 9520), SMAD4 (D3R4N) rabbit monoclonal antibody (#46535), horseradish peroxidase (HRP)-linked anti-mouse IgG (#7076), and anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA); the mouse monoclonal antibody HLA-G (#11–499-C100) was purchased from EXBIO (Vestec, Czech Republic); CK7-specific rabbit polyclonal antibody (#17513-1-AP), BMP6 rabbit polyclonal antibody (#55421-1-AP), SERPINE2 rabbit polyclonal antibody (#11303-1-AP), and α-Tubulin mouse monoclonal antibody (#66031-1-Ig) were purchased from Proteintech (Wuhan, China); ID1 mouse monoclonal antibody (#sc-133104) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); CD34 rabbit monoclonal antibody (#A19015) and α-SMA rabbit monoclonal antibody (#A2235) were obtained from ABclonal (Wuhan, China); Goat anti-Rabbit IgG secondary antibody, Alexa Fluor 594 (#A-11012) and goat anti-Mouse IgG secondary antibody, Alexa Fluor 488 (#A-11011) were purchased from Thermo Fisher (NY, USA).

Techniques: Recombinant, Staining, Immunohistochemistry, Comparison, Saline, Expressing, Control