Journal: Nature Cardiovascular Research

Article Title: The thrombin receptor PAR1 orchestrates changes in lymphatic endothelial cell junction morphology to augment lymphatic drainage during lung injury

doi: 10.1038/s44161-025-00681-7

Figure Lengend Snippet: a , Graphical depiction of buttoned lymphatic capillaries draining lymphatic fluid and cells to zippered lymphatic collecting vessels with valves. b , Representative PCLS image of pulmonary lymphatic vessels relative to lung structures ( Prox1-EGFP , green; VE-cadherin, blue; smooth muscle actin (SMA), red). The boxes indicate areas shown in higher magnification in c – f . c – f , Visualization of LEC junctions in lung lymphatic collectors ( c and d ) and initial lymphatic capillaries ( e and f ) ( Prox1-EGFP , green; VE-cadherin, black). c , GFP labeling of a lung lymphatic collector vessel. d , VE-cadherin staining of the lung lymphatic shown in c demonstrating continuous zipper junction morphology. e , GFP labeling of a lung initial lymphatic. f , VE-cadherin staining of the lung lymphatic shown in e with discontinuous button morphology. g , Quantification of junction morphology among visualized lymphatics ( n = 5). Each dot represents junction quantification for an individual mouse, with 8–12 images used for each mouse. A total of 507 unique junctions were annotated for quantification. A one-way analysis of variance (ANOVA) was used; **** P < 0.0001. h – k , Representative PCLS images of LEC junctions in lymphatic collecting vessels from mice exposed to hyperoxia-induced lung injury or control room air ( Prox1-EGFP , green; VE-cadherin, black). h , GFP labeling of a lung lymphatic vessel from a room air-exposed mouse. i , VE-cadherin staining of the lung lymphatic vessel shown in h with zippered junction morphology. j , GFP labeling of a lung lymphatic vessel from a mouse exposed to hyperoxia. k , VE-cadherin staining of the lung lymphatic vessel shown in j with buttoned morphology. l , Quantification of junction types in room air mice ( n = 3) compared to mice exposed to hyperoxia for 84 h ( n = 2). m , Percentage of button junctions in the experiment presented in l . Each dot represents junction quantification for an individual mouse, with 8–12 images used for each mouse. A total of 1,038 unique junctions were annotated for quantification. n , Representative in vivo imaging system (IVIS) images of ICG fluorescence detected over the chest of mice initially (0 h) or 6 h after intratracheal ICG instillation. o , Decrease in ICG fluorescence intensity over time relative to peak ICG fluorescence in room air mice ( n = 9, solid line) compared to mice exposed to hyperoxia for 72 h ( n = 4, dashed line). p , Quantification of ICG signal at 6 h relative to peak ICG fluorescence. Each dot represents an individual mouse and reports fluorescence intensity at 6 h relative to peak ICG fluorescence intensity for that same mouse. An unpaired, two-tailed t -test was used; * P = 0.0184. Data are representative of at least two independent experiments. All mice used for this experiment were control PAR1 loxP / loxP mice in a mixed genetic background. The black arrows point to zipper junctions. The black arrowheads point to button junctions. All error bars represent the s.e.m. h – k , Scale bars, 20 μm.

Article Snippet: Mice remained anesthetized during imaging with the IVIS Spectrum CT (PerkinElmer, CitiGroup Biomedical Imaging Center at Weill Cornell Medicine); images were collected using the Living Image software (PerkinElmer).

Techniques: Labeling, Staining, Control, In Vivo Imaging, Fluorescence, Two Tailed Test

Journal: Nature Cardiovascular Research

Article Title: The thrombin receptor PAR1 orchestrates changes in lymphatic endothelial cell junction morphology to augment lymphatic drainage during lung injury

doi: 10.1038/s44161-025-00681-7

Figure Lengend Snippet: a , Top: immunoblot for phospho-PKC (pPKC), pan-PKC and actin in lysates of lung-derived human microvascular endothelial cells (HMVEC-Ls) grown in culture and treated with 1 U ml −1 thrombin for 10 min with or without pretreatment with 50 nM of the pan-PAR1 inhibitor vorapaxar for 1 h. Bottom: quantification performed using relative densitometric intensity of pPKC relative to pan-PKC. b , Schematic depiction of the hyperoxia experiments. Mice for the IVIS experiments underwent 72 h of hyperoxia to maximize survival during IVIS imaging. c , Top: immunoblot of whole-lung tissue from hyperoxia-injured mice ( PAR1 loxP / loxP , n = 6) and room air mice ( PAR1 loxP / loxP , n = 3) to detect the 55-kDa fibrin fragment. Bottom: quantification using the relative densitometric intensity of fibrin relative to actin. An unpaired two-tailed t- test was used; ** P = 0.0045. d – k , Representative PCLS images of lung lymphatic junctions ( Prox1-EGFP , green; VE-cadherin, black) from PAR1 loxP / loxP and PAR1 iLEC knockout (KO) mice exposed to room air ( d – g ) and PAR1 loxP / loxP and PAR1 iLEC KO mice exposed to hyperoxia ( h – k ). d , GFP labeling of a lung lymphatic from a PAR1 loxP/loxP mouse exposed to room air. e , VE-cadherin staining of the lung lymphatic shown in d with zippered junctions. f , GFP labeling of a lung lymphatic from a PAR1 iLEC KO mouse exposed to room air. g , VE-cadherin staining of the lung lymphatic shown in f with zippered junctions. h , GFP labeling of a lung lymphatic from a PAR1 loxP/loxP mouse exposed to hyperoxia. i , VE-cadherin staining of the lung lymphatic shown in h with buttoned morphology. j , GFP labeling of a lung lymphatic from a PAR1 iLEC KO mouse exposed to hyperoxia. k , VE-cadherin staining of the lung lymphatic shown in j with zippered morphology. The black arrowheads indicate button junctions. The black arrows indicate zipper junctions. l , Quantification of junction morphology in PAR1 loxP / loxP room air mice ( n = 5), PAR1 iLEC KO room air mice ( n = 3), PAR1 loxP / loxP hyperoxia mice ( n = 3) and PAR1 iLEC KO hyperoxia mice ( n = 4). m , Proportion of button junctions; 2,084 unique junctions were annotated for quantification in l and m . A one-way ANOVA was used; **** P = 0.0001, **** P < 0.0001. n , Representative IVIS images of ICG fluorescence detected after intratracheal ICG instillation. o , Decrease in ICG fluorescence intensity over time relative to peak ICG fluorescence in PAR1 loxP / loxP room air mice ( n = 5, black solid line), PAR1 iLEC KO room air mice ( n = 5, gray solid line), PAR1 loxP / loxP hyperoxia mice ( n = 6, black dashed line) and PAR1 iLEC KO hyperoxia mice ( n = 9, gray dashed line). p , Individual quantification of ICG retention at 6 h after peak ICG fluorescence. * P = 0.097. Data are representative of at least two independent experiments. All error bars represent the s.e.m. d – k , Scale bars, 20 μm.

Article Snippet: Mice remained anesthetized during imaging with the IVIS Spectrum CT (PerkinElmer, CitiGroup Biomedical Imaging Center at Weill Cornell Medicine); images were collected using the Living Image software (PerkinElmer).

Techniques: Western Blot, Derivative Assay, Imaging, Two Tailed Test, Knock-Out, Labeling, Staining, Fluorescence

Journal: Nature Cardiovascular Research

Article Title: The thrombin receptor PAR1 orchestrates changes in lymphatic endothelial cell junction morphology to augment lymphatic drainage during lung injury

doi: 10.1038/s44161-025-00681-7

Figure Lengend Snippet: a , Schematic depiction of LPS-induced lung injury with PM2, a selective allosteric inhibitor of PAR1/G q , in Prox1 - EGFP lymphatic reporter mice. Mice for the IVIS experiments were shaved and allowed to recover an additional 24 h after LPS instillation to maximize mouse survival during IVIS imaging. b – g , Representative PCLS images of lung lymphatic junctions ( Prox1-EGFP , green; VE-cadherin, black) mice treated with PBS, intratracheal LPS, or intratracheal LPS with preinjection of PM2. b , GFP labeling of a lung lymphatic vessel of a mouse treated with PBS. c , VE-cadherin staining of the lung lymphatic shown in b with zippered junctions. d , GFP labeling of a lung lymphatic from a mouse treated with LPS. e , VE-cadherin staining of the lung lymphatic shown in d with buttoned junctions. f , GFP labeling of a lung lymphatic from a mouse treated with LPS with preinjection of PM2. g , VE-cadherin staining of the lung lymphatic shown in f with zippered junctions. The black arrows indicate zipper junctions. The black arrowheads indicate button junctions. h , Quantification of junction types in PBS control mice ( Prox1-EGFP mice, n = 4) compared to LPS-treated mice ( Prox1-EGFP mice, n = 5) and LPS-treated mice preinjected with PM2 ( Prox1 - EGFP mice, n = 6). i , Proportion of the button junctions presented in h . A total of 2,252 unique junctions were annotated for h and i . A one-way ANOVA was used; **** P < 0.0001. j , Representative IVIS images of ICG fluorescence detected over the chest of mice after intratracheal ICG instillation, demonstrating the retention of ICG in PM2-treated mice. k , Decrease in ICG fluorescence intensity over time relative to peak ICG fluorescence in PBS control mice ( Prox1 - EGFP mice, n = 5, solid line), LPS-treated mice ( Prox1-EGFP mice, n = 6, black dashed line) and LPS-treated mice preinjected with PM2 ( Prox1-EGFP mice, n = 6, gray dashed line). l , Quantification of ICG retention 6 h after peak ICG fluorescence in k . A one-way ANOVA was used; from left to right, * P = 0.0386 and P = 0.0324. m , Left: proposed model of PAR1-dependent plasticity of pulmonary lymphatic collecting vessel junctions after lung injury. Right: lymphatic-endothelial-cell-specific loss of PAR1 ( PAR1 iLEC KO) and inhibition of PAR1/G q signaling with PM2 prevented zipper-to-button junction transformation in lymphatic collecting vessels and blunt lymphatic drainage. Data are representative of at least two independent experiments. All error bars represent the s.e.m. b – g , Scale bars, 20 μm.

Article Snippet: Mice remained anesthetized during imaging with the IVIS Spectrum CT (PerkinElmer, CitiGroup Biomedical Imaging Center at Weill Cornell Medicine); images were collected using the Living Image software (PerkinElmer).

Techniques: Imaging, Labeling, Staining, Control, Fluorescence, Inhibition, Transformation Assay