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CRISPR/Cas9, as a new genome-editing tool, offers new approaches to understand and treat diseases, which is being rapidly applied in various areas of biomedical research including sepsis field. The type II prokaryotic CRISPR/Cas system uses a single-guide RNA (sgRNA) to target the Cas9 nuclease to a specific genomic sequence, which is introduced into disease models for functional characterization and for testing of therapeutic strategies. This incredibly precise technology can be used for therapeutic research of gene-related diseases and to program any sequence in a target cell. Most importantly, the multifunctional capacity of this technology allows simultaneous editing of several genes. In this review, we focus on the basic principles, advantages and limitations of CRISPR/Cas9 and the use of the CRISPR/Cas9 system as a powerful tool in sepsis research and as a new strategy for the treatment of sepsis.

Background: Aortic atherosclerosis and dissection cause significant morbidity and mortality. Although atherosclerosis is associated with aortic dissection, points of interaction are unclear. Regulator-of-G-protein signaling 5 (RGS5) is still insufficiently characterized regarding its role in aortic atherosclerosis and dissection. Objectives: We investigated Rgs5-deficiencie’s effects on aortic atherosclerosis and dissection. Methods: ApoE -/- Rgs5 GFP/GFP double knockout mice (rraa, n=15) and WT littermates (RRaa, n=17) were fed a cholesterol-rich diet for 14weeks. Plaque size, plaque morphology (composition and a validated vulnerability score based on Stary criteria), macrophage phenotype and aortic dissection were evaluated. Results: rraa mice displayed more aortic dissections (5/15) compared to WT (0/17; p2 -test). Surprisingly, rraa showed significantly smaller (rraa plaque size 190 657 μm 2 ± 93184 vs. control 398 279 μm 2 ± 153339) and less complicated plaques (5 of 15 mice with >2 vulnerability features) compared to RRaa (14 of 15 with >2 vulnerability features, p+ -myeloid cells revealed increased proportions of macrophages staining positive for pro-inflammatory markers (Cd86/Cd45: 24.71 area% ± 3.75 for RRaa; 60.95 area% ± 5.909, for rraa and iNOS/Cd45: 37.22 area% ± 6.37, for RRaa; 73.99 area% ± 3.997 for rraa, p Conclusions: rraa mice are more prone to aortic dissection despite smaller and less complex plaques with reduced anti-inflammatory but increased numbers of pro-inflammatory macrophages. We hypothesize that pro-inflammatory macrophage polarization along with evasion of anti-inflammatory macrophages in rraa mice could be causative for higher dissection numbers. Our data warrant further studies into the diverging pathomechanistic roles of Rgs5 in aortic dissection and atherosclerosis.

Myocardial infarction and stroke are frequent after surgical procedures and consume a considerable amount of benefit of surgical therapy. Perioperative stress, induced by surgery, is composed of hemodynamic and inflammatory reactions. The effects of perioperative stress on atherosclerotic plaques are ill-defined. Murine models to investigate the influence of perioperative stress on plaque stability and rupture are not available. We developed a model to investigate the influence of perioperative stress on plaque growth and stability by exposing apolipoprotein E-deficient mice, fed a high cholesterol diet for 7 weeks, to a double hit consisting of 30 minutes of laparotomy combined with a substantial blood loss (20% body weight; 400µl). The innominate artery was harvested 72 hours after the intervention. Control groups were sham and baseline controls. Interleukin-6 (IL-6) and Serum Amyloid A plasma levels were determined. Plaque load VSMC- and macrophage-content were quantified. Plaque stability was assessed using the Stary score and frequency of signs of plaque rupture. High-dose atorvastatin (80 mg/kg body weight/day) was administered for 6 days starting 3 days prior to double hit. A single dose of an IL-6-neutralizing antibody or the fusion protein sgp130-Fc selectively targeting IL-6 trans-signaling was subcutaneously injected. IL-6 plasma levels increased peaking at 6h after the intervention. SAA levels peaked at 24 hours (n=4, p Using this model, researchers will be able to further investigate the pathophysiology of perioperative plaque stability, which can result in myocardial infarction, and additionally, to test potential protective strategies.

GAP-inhibitor, RGS5 has been described as a VSMC marker and is associated with plaque stability. Its role in endothelial cells (EC) is unclear. We examined RGS5 expression patterns in human vasculature and hypothesized that loss of RGS5 alters EC biology and crosstalk with VSMC such, that it could promote atherogenesis. Methods: Human internal mammary artery (IMA), saphenous vein (SV) and failing myocardium (MYO) samples were stained for RGS5, αSMA and vWF. RGS5 or scrambled siRNA knocked down (RGS5-KD) HUVECs were used. oxLDL or TNFα induced THP1-adhesion to HUVECs was assessed. The 2h diffusion rate of Evans-blue bound to 0.1%-BSA/DPBS through a HUVEC monolayer was spectrophotometrically measured in the presence of PTX. HUVECs were also subjected to spheroid sprouting in collagen/methylcellulose. qRT-PCR using the Δ(ΔCT) method with HPRT as house-keeper was used to assess MMP-9, VEGF-A and caveolin-1 expression in transfected cells. To test RGS5-dependent paracrine effects of EC-secreted factors on VSMC, isotonic buffer was conditioned for 2h by transfected HUVECs (CB). CB was added to VSMC during a scratch assay. Results: RGS5 signal was enhanced in the endothelium of SV compared to IMA (63±9 vs 22±4 %vessel perimeter, p Conclusion: In conclusion, RGS5 levels could be involved in vascular differentiation. Its effects on permeability and VSMC migration point towards a prominent role in angiogenesis and atherogenesis. Atherosclerosis studies in RGS5-deficient animals are warranted.

Purpose: Plaque stability is governed by it's collagen content and VSMC phenotype1. RGS5, a GPCR modulator, has been shown to be associated with plaque stability3. RGS5 may moderate VSMC/endothelial cell interactions4. We examined if RGS5 expression is associated with plaque stability and detailed its role in VSMC/HUVEC behavior in vitro. Methods: With IRB permission, samples from 91 patients undergoing carotid thrombendarterectomy were collected and RNA was extracted. Target-PCRs for RGS5 were normalized for PCR efficiency and 3 housekeeping genes (GAPDH, ATPsynthase, Tubulin) using qBase software. RGS5 was downregulated in human VSMC or HUVEC by siRNA. After 24h, cells were incubated with mytomicin-C for 1 hour, and the confluent monolayer was wounded. Wound closure percentage after 20h, of scrambled vs. RGS5 siRNA transfected VSMCs was measured (ImageJ). HUVECs conditioned medium was used to perform VSMC scratch assays. RGS5 expression in resident vs. migrated VSMCs was assessed by collecting the cells that had migrated off a confluent coverslip. RNA was extracted for qRT-PCR. RGS5 expression was normalized by HPRT using the Δ(ΔCT) method. Scrambeled and RGS5 siRNA transfected HUVECs were cocultured with VSMCs to produce spheroids in a 96 well plate (1:1 ratio, 3000 cell/well). Spheroids structure was scored 24 hours later for HUVEC coverage, spheroid shape and size. Results: RGS5-RNA was increased in stable compared to unstable plaques (3.1±0.5 vs 1.6±0.3 CNRQ, n=91, p