Journal: Nature Cardiovascular Research

Article Title: Pharmacological blocking of neutrophil extracellular traps attenuates immunothrombosis and neuroinflammation in cerebral cavernous malformation

doi: 10.1038/s44161-024-00577-y

Figure Lengend Snippet: ( a ) Representative images of HBMVECs (shScramble (shScr), top and shCCM3 , bottom) stimulated with 500 ng/mL NET-enriched supernatant or control (Ctrl) media for 24 h showing changes in cell morphology (n = 3). ( b–g ) Representative western blot images of HBMVECs (shscramble or shCCM3) stimulated with 500 ng/mL NET-enriched supernatant or control media for 24 h (n = 3 biological replicates) ( B - C ) and quantified ( D - G ; in E-G normalization was done to α-tubulin or GAPDH). ( h ) Representative images of HBMVECs (shScramble, top and shCCM3 , bottom) stimulated with 500 ng/mL NET-enriched supernatant or control (Ctrl) media for 48 h showing showing changes in cell morphology (n = 6 biological replicates). ( i–n ) Representative western blot images of HBMVECs (shscramble or shCCM3) stimulated with 500 ng/mL NET-enriched supernatant or control media for 48 h (n = 6) ( J ) and quantified ( J - N ). ( o ) Representative images of HBMVECs (shScramble, left and shCCM3 , right) stimulated with NET-enriched supernatant (bottom) or control (Ctrl; top) media for 48 h stained for VE-cadherin (red; junction) and DAPI (blue; nuclei). White arrow head highlights areas with no VE-cadherin staining. The stains were repeated independently three times with similar results. ( p ) Representative images of HBMVECs (shScramble, top and shCCM3 , bottom) stimulated with 500 ng/mL NET-enriched supernatant or control (Ctrl) media for 72 h. The stains were repeated independently two times with similar results. In the graphs, the bar indicates the mean of each group, and the error bars represent the standard deviation. Statistical significance was determined using one-way ANOVA followed by a post-hoc Holm-sidak’s test.

Article Snippet: Endothelial-specific Ccm3 -deficient C57BL/6J mice ( Cdh5 (PAC)-Cre-ER T2 / Ccm3 flox/flox ) were generated by crossing Cdh5 (PAC)-Cre-ER T2 mice with Ccm3 fl/fl mice (Taconic Artemis).

Techniques: Control, Western Blot, Staining, Standard Deviation

Journal: Nature Communications

Article Title: Endothelial Piezo1 channel mediates mechano-feedback control of brain blood flow

doi: 10.1038/s41467-024-52969-0

Figure Lengend Snippet: a Crossbreeding scheme used to generate inducible, EC-specific Piezo1-GOF mice. Tamoxifen-treated Cre+ mice are used as GOF, and littermate Cre- mice are the control. b Representative traces of Piezo1 currents recorded from freshly isolated ECs from the somatosensory cortices of control and GOF mice. The cell-attached configuration was used, where EC patches were held at -50 mV in the absence or presence of Yoda1 (5 µM) in the pipette solution. C denotes closed channel. c Averaged data of the open probability (NP O ) of Piezo1 in the absence (n = 11 ECs/6 control; 10 ECs/4 GOF mice) or presence of Yoda1 (n = 10 ECs/6 control; 7 ECs/4 GOF mice). d Schematic of the LSCI setup for imaging blood flow through a thinned skull. ( e ) The Allen mouse brain atlas highlighting the somatosensory barrel cortex. f, g Differential maps of blood flow responses in a control ( f ) and a GOF ( g ) mouse. Whisker stimulation using air puffs (5 Hz, 30 s) evoked hyperemia in the contralateral somatosensory cortex (arrows). h Representative traces and summary data of the hyperemic response to 30 s-whisker stimulations in control ( n = 8) and GOF ( n = 9) mice. Right: area under the curve analyses ( n = 19 whisker stimulations/6 control; 20 stimulations/8 GOF mice). i Representative responses and scatter plots of FH evoked by 5 s air puffs (n = 7 control, n = 9 GOF mice). Area under the curve analysis was calculated from 41 and 43 stimulations from 7 control and 9 GOF mice, respectively. j Representative images demonstrating the staining of ECs (caveolin-1), basement membranes (collagen IV), and nuclei (DAPI) in brain slices of the cortex of GOF and control mice. Scale bar: 100 µm. Vascular densities and string vessel densities in Piezo1 cx/cx ; Cdh5 -Cre+ ( n = 12) and control ( n = 10) mice. k Representative images and averaged data as in j from brain slices of the hippocampus of Piezo1 cx/cx ; Cdh5 -Cre+ ( n = 12) and control ( n =10) mice. All statistical tests were unpaired Student’s t test (two-sided, * P < 0.05, ** P < 0.01, *** P < 0.001). All error bars are SEM. Scatter plot data in c, h, I, j, k are presented as mean ± SEM. Area under the curve data in h and i are presented as histograms. Source data are provided as a Source Data file.

Article Snippet: Tamoxifen-inducible, Piezo1 gain-of-function (Piezo1 cx/cx ; Cdh5 -Cre+) mice were engineered by crossing Cdh5 -CreER T2 and knock-in Piezo1 cx/cx mice (Taconic Biosciences, Model No. 21617) in which floxed exons 45-51 are followed by a mutant Piezo1 copy (R2482H mouse mutation- equivalent to R2456H in humans) , .

Techniques: Control, Isolation, Transferring, Imaging, Whisker Assay, Staining

Journal: Nature Communications

Article Title: Endothelial Piezo1 channel mediates mechano-feedback control of brain blood flow

doi: 10.1038/s41467-024-52969-0

Figure Lengend Snippet: a Left : Example FH response to whisker stimulation showing the increase in CBF shortly after the stimulus starts (upstroke) and the return to baseline at the end of the stimulation (downstroke). Right : A schematic showing that neuronal activity increases blood flow (FH), and the resulting change in forces activates cationic influx by opening Piezo1 channels, thus acting as a mechano-feedback system to attenuate FH. b Experimental setup illustrating impalement of a freshly isolated brain EC using a sharp microelectrode. ECs were obtained from a Pdgfrb -Cre-TdTomato mouse, where pericytes are TdTomato+. Scale bar = 10 µm. c Traces and summary data of V m measurements showing the effect of Yoda1 (5 µM) on endothelial V m (-Yoda1 [n = 16 ECs/5 mice]; +Yoda1 [n = 23 ECs/4 mice]). d, e Representative traces and scatter plots of functional hyperemia responses to whisker stimulations in Piezo1 cx/cx ; Cdh5 -Cre+ (GOF) and Piezo1 flox/flox ; Cdh5 -Cre+ (knockout) mice, and their respective controls ( d : n = 6 GOF, 6 controls; e : n = 7 knockout, 5 controls). f Representative traces and plateau-followed-by-exponential fittings ( dotted ) to obtain time constant for downstroke (Tau), in a control mouse and a GOF mouse. Right : Scatter plot of downstroke Tau values in control ( n = 5) and GOF ( n = 6) mice. g Similar to f in knockout and control mice (n = 5 each). h Representative traces and plateau-followed-by-exponential fittings ( dotted ) to obtain upstroke Tau, in a control mouse and a GOF mouse. Right : Scatter plot of upstroke Tau values in control and GOF ( n = 6 mice each). i Representative traces and averaged data of upstroke Tau in control and knockout mice (n = 5 mice each). FH responses were measured through a cranial window using laser Doppler flowmetry. Statistical tests were unpaired Student’s t test (two-sided, c ), and Mann-Whitney test (one-sided in d – e , two-sided in f – i ; * P < 0.05, ** P < 0.01, *** P < 0.001). Error bars in c , d and e are SEM. Data in c , d , e are presented as mean values ± SEM, and data in f , g , h , i are presented as violin scatter plots. Source data are provided as a Source Data file.

Article Snippet: Tamoxifen-inducible, Piezo1 gain-of-function (Piezo1 cx/cx ; Cdh5 -Cre+) mice were engineered by crossing Cdh5 -CreER T2 and knock-in Piezo1 cx/cx mice (Taconic Biosciences, Model No. 21617) in which floxed exons 45-51 are followed by a mutant Piezo1 copy (R2482H mouse mutation- equivalent to R2456H in humans) , .

Techniques: Whisker Assay, Activity Assay, Isolation, Functional Assay, Knock-Out, Control, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial Piezo1 channel mediates mechano-feedback control of brain blood flow

doi: 10.1038/s41467-024-52969-0

Figure Lengend Snippet: a Novel object recognition test in control and Piezo1 cx/cx ; Cdh5 -Cre+ mice. Heat maps demonstrate the cumulative time spent with familiar (F) and novel (N) objects. b Scatter plot shows the discrimination index (DI%-calculated based on duration) for the two groups (n = 7 control, 9 Piezo1 cx/cx ; Cdh5 -Cre+ mice). DI of 50% ( dotted ) indicates equal time spent with novel and familiar objects. c Velocities of control ( n = 7) and Piezo1 cx/cx ; Cdh5 -Cre+ mice (n = 9) during the NOR test. d, e Similar to a and b , test results from Piezo1 cx/cx ; Slco1c1 -Cre+ and their controls ( n = 10 each). f Motor activity of Piezo1 cx/cx ; Slco1c1 -Cre+ mice and their controls (n = 10 each) during the NOR test. g Percent of alternation over all 6 trials during the spontaneous alternation T-maze was calculated as [(number of correct alternations/6)*100)] in Piezo1 cx/cx ; Slco1c1 -Cre+ and their controls ( n = 11 each). The dotted line represents the 50% chance level. h Choice latency in Piezo1 cx/cx ; Slco1c1 -Cre+ and control mice over trials 1 to 6. Asterisks highlight significantly higher choice latency in Piezo1 cx/cx ; Slco1c1 -Cre+ mice at T4, T5 and T6. Data from individual mice are shown, and bold lines and transparent shades are means and SEM, respectively (n = 11 mice each). Statistical tests were unpaired Student’s t test (two-sided, b, c, e, f, g ) and two-way ANOVA test in h (* P < 0.05, ** P < 0.01, *** P < 0.001). All error bars are SEM. Data in b, c, e-g are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Tamoxifen-inducible, Piezo1 gain-of-function (Piezo1 cx/cx ; Cdh5 -Cre+) mice were engineered by crossing Cdh5 -CreER T2 and knock-in Piezo1 cx/cx mice (Taconic Biosciences, Model No. 21617) in which floxed exons 45-51 are followed by a mutant Piezo1 copy (R2482H mouse mutation- equivalent to R2456H in humans) , .

Techniques: Control, Activity Assay

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet:

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Recombinant, Reverse Transcription, Multiplex Assay, Software, Microscopy

Journal: Current opinion in hematology

Article Title: Mouse models of vascular development and disease

doi: 10.1097/MOH.0000000000000649

Figure Lengend Snippet: Genetic mouse models useful to study vascular malformations. Non-exhaustive list of mouse models of Arteriovenous Malformations (AVMs), Brain AVMs (BAVMs), Lymphatic malformations (LMs), Hereditary Hemorrhagic Telangiectasias (HHTs) and Cerebral Cavernous Malformations (CCMs). Table adapted from Nielsen et al., 2016 [ 49 ], Tual-Chalot et al, 2015 [ 34 ], Zeng et al., 2019[ 50 ] and McDonald et al., 2011[ 39 ]. (Endothelial cell, EC; lymphatic endothelial cell, LEC; overexpression, OE; vascular endothelial growth factor, VEGF; vascular smooth muscle cell, vSMC).

Article Snippet: Cdh5-CreER T2 (VE-cad) , Tg(Cdh5-cre/ERT2) 1Rha Taconic # 13073-M , Pan EC , E9.5 , yes , [ 8 ].

Techniques: Over Expression, Mutagenesis, Knock-Out, Expressing, Knock-In