Journal: Acta biomaterialia

Article Title: Ultrasound-guided photoacoustic imaging-directed re-endothelialization of acellular vasculature leads to improved vascular performance

doi: 10.1016/j.actbio.2015.12.029

Figure Lengend Snippet: Effects of nanoparticle incubation on HDMEC morphology. HDMECs incubated with gold nanoparticles overnight, then allowed proliferate near confluency (A–C) displayed similar morphology as HDMECs plated without nanoparticles (D–F). The same field of view was captured in phase-contrast, dark-field and bright-field images. Phase-contrast imaging reveals cobblestone morphology in nanoparticle and control groups (A, D) and a departure from spindle-like morphology. Dark-field and bright-field imaging demonstrate the presence of nanoparticles (light in dark-field imaging, black in bright-field imaging) (B, C) compared to control (E, F). Scale bar = 50μm. HDMECs injected into OA decellularized scaffolds with staining for CD-31 (G), VE-Cadherin (I). Tissue was not extensively cellularized, intimating at the need for more optimization. CD-31 positive staining matched up with VE-Cadherin with an association with nanoparticles (H). Scale bar = 200 μm. MTS assay for HDMECs incubated with gold nanotracers (J). Following incubation with gold nanotracers for 24 hours, HDMECs were maintained for 24 and 72 hours then incubated with MTS for four hours. The absorbance of the culture media and MTS dye were then measured on a 96-well plate in triplicate at 490 nm, n = 3. HDMECs incubated with NPs did not exhibit abnormal metabolic activity when compared to HDMECs without NPs.

Article Snippet: Endothelial cell culture and incubation with gold nanotracers Human dermal microvascular endothelial cells (HDMECs) were obtained from Promega (Heidlberg, Germany).

Techniques: Incubation, Imaging, Control, Injection, Staining, MTS Assay, Activity Assay

Journal: Acta biomaterialia

Article Title: Ultrasound-guided photoacoustic imaging-directed re-endothelialization of acellular vasculature leads to improved vascular performance

doi: 10.1016/j.actbio.2015.12.029

Figure Lengend Snippet: Ultrasound-guided photoacoustic imaging of an optimized acellular scaffold devoid of cells (A–C, G–I) and re-endothelialized with HDMECs (D–F; J–R). Images A–F represent a top down view of the overlay of photoactoustic signal and ultrasound; whereas G-R represent individual cross-sections of the lung. Three injections of 3.3×105 HDMECs, with gold nanoparticles in 3.3 ml media over two hours were delivered to an OA scaffold. Cells were allowed to adhere overnight before gradually increasing perfusion flow was administered (3.2 dyn/cm2 for 24 hours, 8.7 dyn/ cm2 for 12 hours, 19.6 dyn/ cm2 for 24 hours). Photoacoustic signal from cells at 750 nm indicates the retention of cells distributed within the entirety of an OA scaffold at the conclusion of the culture, whereas barren OA scaffolds presented no signal. Media was rinsed out then tissue was fixed prior to US/PA imaging at 750 nm. A void in the scaffold representing the bronchus can be seen in cross-sections of the ultrasound images (arrow).

Article Snippet: Endothelial cell culture and incubation with gold nanotracers Human dermal microvascular endothelial cells (HDMECs) were obtained from Promega (Heidlberg, Germany).

Techniques: Imaging

Journal: Acta biomaterialia

Article Title: Ultrasound-guided photoacoustic imaging-directed re-endothelialization of acellular vasculature leads to improved vascular performance

doi: 10.1016/j.actbio.2015.12.029

Figure Lengend Snippet: Vascular corrosion casts of fresh (A, D), decellularized (B, E) and re-endothelialized scaffolds following perfusion culture for 60 hours (C, F). Media was rinsed from scaffolds then fixed before vascular corrosion casting performed. Capillary networks from re-endothelialized scaffolds (C) closely resembled those of fresh tissue (A), compared to decellularized scaffolds alone (B). In fresh (D) and re-endothelialized (F) scaffolds, the larger vessels appear to be irregularly formed (arrowheads), denoting cellularity, whereas decellularized tissues have smooth vessels (asterisk) (E). Scale bar = 20 μm. Total extravasation volume was calculated per mm2 by cubic micron for VCCs of fresh, decellularized, and re-endothelialized lung (G), n = 2. Re-endothelialization with HDMECs clearly rescues some of the vascular patency of the OA tissue.

Article Snippet: Endothelial cell culture and incubation with gold nanotracers Human dermal microvascular endothelial cells (HDMECs) were obtained from Promega (Heidlberg, Germany).

Techniques:

Journal: Acta biomaterialia

Article Title: Ultrasound-guided photoacoustic imaging-directed re-endothelialization of acellular vasculature leads to improved vascular performance

doi: 10.1016/j.actbio.2015.12.029

Figure Lengend Snippet: OA scaffolds re-endothelialized with HDMECs help decrease leakage of whole blood. Following re-endothelialization and whole-blood perfusion, scaffolds were perfused with PBS to clear blood. Sections were stained for CD-31 (A, C), staining positively for endothelial cells and platelets (red), and DAPI (blue). The majority of positive staining for nuclei was likely from white blood cells from blood. Extensive leakage of blood unable to be cleared was seen (A, B). Blood leakage can be visualized in (A) and (B) around the top of the vessel (demarked with a dashed line). Although occlusion failed to occur, the resultant leakage can be traced. An overlay with brightfield imaging (B) shows the presence of red blood cells (arrow). (C) and (D) demonstrate a non-occluded vascular pair. Some damage in the form of cell aggregation can be seen (asterisk) which failed to occur in some of the regions containing nanoparticles (arrowhead). In contrast, occlusions were present in other vessels (dagger). Scale bar = 200 μm

Article Snippet: Endothelial cell culture and incubation with gold nanotracers Human dermal microvascular endothelial cells (HDMECs) were obtained from Promega (Heidlberg, Germany).

Techniques: Staining, Imaging

Journal: The Journal of Cell Biology

Article Title: Vav3-induced cytoskeletal dynamics contribute to heterotypic properties of endothelial barriers

doi: 10.1083/jcb.201706041

Figure Lengend Snippet: High levels of barrier resistance correlate with continuous intercellular junctions and cortical arrangement of the actin cytoskeleton. (A) Confocal images of confluent endothelial monolayers displayed in order of low (HAEC, HBMVEC, and HUVEC) and high levels of barrier resistance (HLMVEC and HDMEC) left to right (bar, 20 µm; representative of n = 3). HLMVEC and HDMEC exhibit a linear and organized junctional staining of VE-cadherin, claudin-5, and ZO-1 compared with an irregular junctional pattern in cell types with lower levels of resistance. Phalloidin staining reveals a strict cortical arrangement of the actin cytoskeleton in HLMVEC and HDMEC, whereas cells with low resistance exhibit more stress fibers. Localization of cortactin is more peripheral and less cytoplasmic in HLMVECs and HDMECs. F-actin fibers aligned along the cell periphery, minimizing radial tension forces at cell junctions, indicated by linear junctional pattern of VE-cadherin/phalloidin overlay images (arrows) and lower presence of pMLC2 in HLMVECs and HDMECs. ( B–F) Graphs presenting mean values of fluorescence intensity across multiple cell–cell junctions per cell type (Fig. S2 B) for each barrier protein shown in A. Junctional components as well as phalloidin and cortactin are concentrated along the cell borders in HLMVECs and HDMECs compared with cell types with low barrier resistance.

Article Snippet: Specifically, we obtained HUVECs (C2519A; Lonza; C-12203; PromoCell), HSaVECs (HSVEC/A; VEC-Technologies; cAP-0019; Angio-Proteomie), HAECs (PCS-100-011; ATCC; 6100; ScienCell), HIAECs (CC-2545; Lonza; cAP-0020; Angio-Proteomie), HBMVECs (ACBRI 376 V; Cell-Systems; cAP-0002; Angio-Proteomie), HUMVECs (C-12295; PromoCell; 7000; ScienCell), HLMVECs (3000; ScienCell; C-12281; PromoCell), HAMVECs (7200; ScienCell), and HDMECs (C-12212; PromoCell; 2000; ScienCell).

Techniques: Staining, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Vav3-induced cytoskeletal dynamics contribute to heterotypic properties of endothelial barriers

doi: 10.1083/jcb.201706041

Figure Lengend Snippet: Knockdown of VAV3 reduces barrier strength and alters cytoskeletal arrangement in HDMECs and HLMVECs. (A) siRNA knockdown of three candidate genes with a strong correlation of expression to barrier resistance ( BAIAP2, VAV3, and SORBS2 ) validates the effect of VAV3 as an important regulator. (B) Efficiency of siRNA knockdown for BAIAP2, VAV3, and SORBS2 . (C and E) Effect of VAV3 silencing on barrier resistance compared with combined siRNA knockdown of VAV2 and VAV3 or CLDN5 (positive control) in HDMECs and HLMVECs. (D and F) Efficiency of siRNA knockdown for VAV2 , VAV3 , and CLDN5 (ECIS dataset C and E) in HDMECs and HLMVECs versus control siRNA. (G and H) Bar graphs of barrier resistance levels at 48 h for HDMECs and HLMVECs (ECIS dataset C and E). Error bars show mean ± SEM; *, P < 0.05; n = 3. (I) Table presenting ECIS data modeling values of R b , α , and C m at 48 h for HDMECs and HLMVECs with siRNA knockdown for VAV3 , VAV2/3 , and CLDN5 versus control siRNA, respectively (mean ± SEM; n = 3). (J) Immunofluorescence staining of VE-cadherin, ZO-1, F-actin (phalloidin), and cortactin in HDMEC monolayers subjected to either siRNA knockdown of VAV3 and VAV2/3 or siRNA control (bars, 20 µm). Magnification of phalloidin staining highlights loss of cortical actin and gain of stress fibers upon VAV3 and VAV2/3 knockdown. Translocation of cortactin to the cell periphery (filled arrows) is reduced in monolayers with siRNA knockdown of either VAV3 alone or VAV2/3 (open arrows). (K) Fluorescence intensity across cell–cell junctions (mean of n = 6) in cells exposed to siRNA control, siRNA VAV3 , and siRNA VAV2/3 (for VE-cadherin, ZO-1, phalloidin, and cortactin; as shown in J).

Article Snippet: Specifically, we obtained HUVECs (C2519A; Lonza; C-12203; PromoCell), HSaVECs (HSVEC/A; VEC-Technologies; cAP-0019; Angio-Proteomie), HAECs (PCS-100-011; ATCC; 6100; ScienCell), HIAECs (CC-2545; Lonza; cAP-0020; Angio-Proteomie), HBMVECs (ACBRI 376 V; Cell-Systems; cAP-0002; Angio-Proteomie), HUMVECs (C-12295; PromoCell; 7000; ScienCell), HLMVECs (3000; ScienCell; C-12281; PromoCell), HAMVECs (7200; ScienCell), and HDMECs (C-12212; PromoCell; 2000; ScienCell).

Techniques: Knockdown, Expressing, Positive Control, Control, Immunofluorescence, Staining, Translocation Assay, Fluorescence

Journal: bioRxiv

Article Title: Cross-family Signaling: PDGF Mediates VEGFR Activation and Endothelial Function

doi: 10.1101/2025.02.27.640684

Figure Lengend Snippet: HDMECs ( PDGFRA −/− and PDGFRB −/− ) were treated with PDGF-AA, -AB, or -BB at the concentrations of 5 and 50 ng/mL for 15 min. The effects of PDGF treatment on phosphorylating PLCγ1 at tyrosine site 783 (A-D) , Akt at serine site 473 (E-H) , and FAK at tyrosine site 397 (I-L) were assessed via immunoblotting.

Article Snippet: Primary human dermal microvascular endothelial cells (HDMECs) (ATCC, CRL-4060) PDGFRA and PDGFRB double knock out (DKO) (to eliminate PDGFRα and β expression, respectively) via CRISPR/Cas9 at the Genome Engineering & Stem Cell Center at Washington University in St. Louis, MO.

Techniques: Western Blot