Journal: Nature Cardiovascular Research

Article Title: Semaphorin-3A regulates liver sinusoidal endothelial cell porosity and promotes hepatic steatosis

doi: 10.1038/s44161-024-00487-z

Figure Lengend Snippet: a , Schematic illustration of SEMA3A signaling. Upon SEMA3A binding to NRP1, NRP1 forms a holoreceptor complex with a plexin, which acts as the signal-transducing unit. Through a signaling cascade, LIMK1 is activated and catalyzes the phosphorylation of cofilin-1. Cofilin-1 is an actin depolymerization factor, which is de-activated upon phosphorylation at its serine 3 (S3). Thus, less actin is depolymerized, resulting in a less dynamic actin network and, subsequently, fewer fenestrae. b , Western blots of mouse LSEC protein lysates ( n = 5 independent LSEC isolations). LSECs were pretreated with either DMSO or LIMKi 3, a LIMK1 inhibitor, and then treated with either SEMA3A-Fc or IgG2a-Fc. For the analysis, cofilin-1 and p-S3-cofilin-1 were normalized to GAPDH and then put into relation of each other (p-S3-cofilin-1 to cofilin-1). c , Representative SEM images of mouse LSECs pretreated with either DMSO or LIMKi 3 and then treated with either SEMA3A-Fc or IgG2a-Fc. The fenestrae were colorized with a digital charcoal pencil for better visualization. Scale bar, 1 µm. Brightness and contrast have been adjusted to enhance visibility in b , c . d – f , Analyses of fenestrae frequency ( d ) and diameter ( e ) as well as porosity ( f ) of mouse LSECs pretreated with LIMKi 3 or DMSO and subsequently treated with SEMA3A-Fc or IgG2a-Fc, as indicated. For each condition, ten images (taken from different LSECs) were analyzed ( n = 5 LSEC isolations). For statistical analysis, a one-way ANOVA with multiple comparisons (Tukey’s post hoc test) was performed in b , d – f . In all graphs, data points and mean ± s.e.m. are presented.

Article Snippet: After addition of the antibodies, the cells were incubated at 37 °C and 5% CO 2 for 1 h. If LSECs were to be pretreated with the LIMK1 inhibitor LIMKi 3 (Tocris, 4745), they were allowed to grow 4 h and then incubated with LIMKi 3 for 1 h at 37 °C and 5% CO 2 .

Techniques: Binding Assay, Phospho-proteomics, Western Blot

Journal: Nature Cardiovascular Research

Article Title: Semaphorin-3A regulates liver sinusoidal endothelial cell porosity and promotes hepatic steatosis

doi: 10.1038/s44161-024-00487-z

Figure Lengend Snippet: Left side: in the setting of low physiological SEMA3A levels (as is the case at low concentrations of saturated fatty acids and normal BW without T2D), active cofilin-1 and normal F-actin cytoskeleton dynamics contribute to maintain a high frequency of fenestrae in LSECs. LSEC porosity facilitates bidirectional exchange of lipids between bloodstream and hepatocytes, such as the release of VLDL particles from hepatocytes into the blood circulation. Right side: in the setting of high SEMA3A levels (as is the case at high concentrations of FFAs and in DIO with or without T2D), the angiocrine signal SEMA3A acts via NRP1 on LSECs to activate multiple STKs, including LIMK1, which phosphorylates cofilin-1 to reduce F-actin cytoskeleton dynamics and fenestrae frequency as well as LSEC porosity. The reduced LSEC porosity lowers VLDL export from the hepatocytes into the blood and might contribute to lipid retention and macrovesicular steatosis in the hepatocytes. The resulting hepatic steatosis is an early event in MASLD that can subsequently (in concert with hepatic stellate cells; HSCs) progress to severe hepatic and cardiometabolic diseases. The figure was created with BioRender.com .

Article Snippet: After addition of the antibodies, the cells were incubated at 37 °C and 5% CO 2 for 1 h. If LSECs were to be pretreated with the LIMK1 inhibitor LIMKi 3 (Tocris, 4745), they were allowed to grow 4 h and then incubated with LIMKi 3 for 1 h at 37 °C and 5% CO 2 .

Techniques:

Journal: Frontiers in Physiology

Article Title: Cofilin activation in pancreatic acinar cells plays a pivotal convergent role for mediating CCK-stimulated enzyme secretion and growth

doi: 10.3389/fphys.2023.1147572

Figure Lengend Snippet: Effect of two LIMK inhibitors, LIMKi3 and SR7826, on the ability of CCK-8 (0.3 and 100 nM) to alter activation of LIMK (A) and cofilin (B) . Isolated pancreatic acini were incubated in the absence or presence of LIMKi3 (10 µM) or SR7826 (10 µM) for 3 h and then incubated with no addition (control), CCK-8 (0.3 or 100 nM) for 3 min, and then lysed. Western blots were analyzed using anti-pT508 LIMK and anti-pS3 cofilin. Bands were visualized using chemiluminescence and quantified by densitometry. Top : Results of a representative blot of four independent experiments are shown. Bottom : Means ± S.E. of at least 4 independent experiments. Results are expressed as % of basal stimulation of the control group. *, p < 0.05 compared to the control group; ∞, p < 0.05 compared to stimulants without inhibitors.

Article Snippet: LIMKi3 and SR7826 were from TOCRIS bioscience (Bristol, UK).

Techniques: CCK-8 Assay, Activation Assay, Isolation, Incubation, Control, Western Blot