A vein wall cell atlas of murine venous thrombosis determined by single-cell RNA sequencing.

Deep vein thrombosis (DVT) is a common clinical problem, but its cellular and molecular mechanisms remain incompletely understood. In this study, we performed single-cell RNA sequencing on mouse inferior vena cava (IVC) 24 h after thrombus-inducing IVC ligation or sham operation. 9 cell types composed of multiple subpopulations were identified. Notable transcriptomic changes induced by DVT included a marked inflammatory response, elevated hypoxia, and globally reduced myogenesis. Analysis of individual cell populations revealed increased inflammation and reduced extracellular matrix production across smooth muscle cells and fibroblasts, juxtaposed against an early phenotypic shift in smooth muscle cell populations away from a contractile state. By characterizing the transcriptomic changes in the vein wall during acute venous thrombosis at the single-cell level, this work provides novel insights into early pathological events in the vein wall that may potentiate thrombus formation and result in long term adverse venous remodeling.

Investigating the role of receptor interacting protein kinase 3 in venous thrombosis.

Objective: Venous thromboembolism is a disease that encompasses both deep vein thrombosis and pulmonary embolism. Recent investigations have shown that receptor interacting protein kinase 3 (RIPK3), a protein known for its role in the programmed form of cell death necroptosis, may play a role in thrombosis. Specifically, RIPK3 has been shown to promote platelet activation in arterial thrombosis and mixed lineage kinase domain-like pseudokinase (MLKL), a protein downstream of RIPK3 in the necroptosis pathway, has been shown to promote neutrophil extracellular trap formation in deep vein thrombosis. This investigation sought to comprehensively investigate the role of RIPK3 in deep vein thrombogenesis. Methods: The inferior vena cava ligation and stenosis models of deep vein thrombosis were used in C57BL/6J, RIPK3 wild-type (Ripk3 +/+ ) and RIPK3-deficient (Ripk3 -/- ) mice. Downstream tissue analyses included measurement of thrombus weight and histological and Western blot analysis of tissues for markers of necroptosis and cell death. A subset of C57BL/6J mice were treated with a RIPK3 inhibitor to determine the effect on venous thrombosis. Results: C57BL/6J mice showed significant increases in thrombus weight from 6 to 48 hours. During the same time frame, RIPK3 progressively accumulated in the vein wall (a 35-fold increase from 0 to 48 hours). RIPK3 was present in the thrombus; however, it decreased with time. Although present in the thrombus, MLKL was nearly undetectable in the vein wall by Western blot at any timepoint. Immunostaining confirmed the high accumulation of RIPK3 in the vein wall, primarily colocalized to endothelial and smooth muscle cells. Phosphorylated MLKL, the active form of MLKL and executioner of necroptotic cell death, was detectable by immunostaining in the thrombus, but was present at low to undetectable levels in the vein wall. Propidium iodide and terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed a high burden of necrotic and apoptotic cells within the thrombus at 48 hours, but a relatively lower burden within the vein wall. Despite robust accumulation of RIPK3 within the vessel wall and the thrombus, knockout and inhibition of RIPK3 failed to impact thrombus incident or weight at 48 hours after inferior vena cava ligation. Neutrophil extracellular trap burden did not differ between Ripk3 +/+ and Ripk3 -/- mice. Conclusions: In mice, the vein wall responded to deep vein thrombosis induction with elevation of RIPK3 without showing markers of necroptosis and apoptosis. Studies using genetic or pharmacological inhibition of RIPK3 suggest that this cell death mediator may not have a major role in the acute phase of venous thrombogenesis. Further investigation is needed to determine if RIPK3 plays a potentially non-necroptotic role within the vein wall during later stages of thrombus resolution and vein wall remodeling.

Endothelial Dysfunction in the Pathogenesis of Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA), defined as a focal dilation of the abdominal aorta beyond 50% of its normal diameter, is a common and potentially life-threatening vascular disease. The molecular and cellular mechanisms underlying AAA pathogenesis remain unclear. Healthy endothelial cells (ECs) play a critical role in maintaining vascular homeostasis by regulating vascular tone and maintaining an anti-inflammatory, anti-thrombotic local environment. Increasing evidence indicates that endothelial dysfunction is an early pathologic event in AAA formation, contributing to both oxidative stress and inflammation in the degenerating arterial wall. Recent studies utilizing single-cell RNA sequencing revealed heterogeneous EC sub-populations, as determined by their transcriptional profiles, in aortic aneurysm tissue. This review summarizes recent findings, including clinical evidence of endothelial dysfunction in AAA, the impact of biomechanical stress on EC in AAA, the role of endothelial nitric oxide synthase (eNOS) uncoupling in AAA, and EC heterogeneity in AAA. These studies help to improve our understanding of AAA pathogenesis and ultimately may lead to the generation of EC-targeted therapeutics to treat or prevent this deadly disease.

MLKL and CaMKII Are Involved in RIPK3-Mediated Smooth Muscle Cell Necroptosis.

Receptor interacting protein kinase 3 (RIPK3)-mediated smooth muscle cell (SMC) necroptosis has been shown to contribute to the pathogenesis of abdominal aortic aneurysms (AAAs). However, the signaling steps downstream from RIPK3 during SMC necroptosis remain unknown. In this study, the roles of mixed lineage kinase domain-like pseudokinase (MLKL) and calcium/calmodulin-dependent protein kinase II (CaMKII) in SMC necroptosis were investigated. We found that both MLKL and CaMKII were phosphorylated in SMCs in a murine CaCl2-driven model of AAA and that Ripk3 deficiency reduced the phosphorylation of MLKL and CaMKII. In vitro, mouse aortic SMCs were treated with tumor necrosis factor α (TNFα) plus Z-VAD-FMK (zVAD) to induce necroptosis. Our data showed that both MLKL and CaMKII were phosphorylated after TNFα plus zVAD treatment in a time-dependent manner. SiRNA silencing of Mlkl-diminished cell death and administration of the CaMKII inhibitor myristoylated autocamtide-2-related inhibitory peptide (Myr-AIP) or siRNAs against Camk2d partially inhibited necroptosis. Moreover, knocking down Mlkl decreased CaMKII phosphorylation, but silencing Camk2d did not affect phosphorylation, oligomerization, or trafficking of MLKL. Together, our results indicate that both MLKL and CaMKII are involved in RIPK3-mediated SMC necroptosis, and that MLKL is likely upstream of CaMKII in this process.

Single-Cell RNA Sequencing Reveals Heterogeneity of Vascular Cells in Early Stage Murine Abdominal Aortic Aneurysm-Brief Report.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease characterized by smooth muscle cell depletion, ECM (extracellular matrix) degradation, and infiltration of immune cells. The cellular and molecular profiles that govern the heterogeneity of the AAA aorta are yet to be elucidated. Approach and Results: We performed single-cell RNA sequencing on mouse AAA tissues. AAA was induced in C57BL/6J mice by perivascular application of CaCl2. Unbiased clustering identified 12 distinct populations of 8 cell types. Percentages of each population and gene expression were compared between sham and AAA tissue. Furthermore, we characterized the transcriptional profiles and potential functional features of populations in smooth muscle cells, fibroblasts, and macrophages and revealed the unique regulons in each cell type. CONCLUSIONS: Together, these data provide high-resolution insight into the complexity and heterogeneity of mouse AAA and indicate that populations within major cell types such as smooth muscle cells, fibroblasts, and macrophages may contribute differently to AAA pathogenesis. Graphic Abstract: A graphic abstract is available for this article.

Pathophysiology of Aortic Aneurysms: Insights from Animal Studies.

Aortic aneurysms are defined as dilations of the aorta greater than 50 percent. Currently, the only effective treatment for aortic aneurysms is surgical repair, which is recommended only to those that meet criteria. There is no available pharmaceutical therapy to slow aneurysm growth and thus prevent lethal rupture. The development of a number of murine models has allowed in depth studies of various cellular and extracellular components of aneurysm pathophysiology. The identification of key therapeutic targets has resulted in several clinical trials evaluating pharmaceutical candidates to treat aneurysm progression. In this review, we focus on providing recent updates on developments in murine models of aortic aneurysm. In addition, we discuss recent studies of the various cellular and extracellular components of the aorta along with the abutting aortic structures that contribute to aneurysm development and progression.

Role of FOXM1 in vascular smooth muscle cell survival and neointima formation following vascular injury.

BACKGROUND: Accelerated smooth muscle cell (SMC) proliferation is the primary cause of intimal hyperplasia (IH) following vascular interventions. Forkhead Box M1 (FOXM1) is considered a proliferation-associated transcription factor. However, the presence and role of FOXM1 in IH following vascular injury have not been determined. OBJECTIVE: We examined the expression of FOXM1 in balloon-injured rat carotid arteries and investigated the effect of FOXM1 inhibition in SMCs and on the development of IH. METHODS AND RESULTS: FOXM1 was detected by immunofluorescent staining in balloon-injured rat carotid arteries where we observed an upregulation at day 7, 14, and 28 compared to uninjured controls. Immunofluorescence staining revealed FOXM1 coincided with proliferating cell nuclear antigen (PCNA). FOXM1 was also detectable in human carotid plaque samples. Western blot showed an upregulation of FOXM1 protein in serum-stimulated SMCs. Inhibition of FOXM1 using siRNA or chemical inhibition led to the induction of apoptosis as measured by flow cytometry and western blot for cleaved caspase 3. Perturbations in survival signaling were measured by western blot following FOXM1 inhibition, which showed a decrease in phosphorylated AKT and ß-catenin. The chemical inhibitor thiostrepton was delivered by intraperitoneal injection in rats that underwent balloon injury and led to reduced intimal thickening compared to DMSO controls. CONCLUSIONS: FOXM1 is an important molecular mediator of IH that contributes to the proliferation and survival of SMCs following vascular injury.

GSK2593074A blocks progression of existing abdominal aortic dilation.

OBJECTIVE: Receptor interacting proteins kinase 1 and 3 (RIPK1 and RIPK3) have been shown to play essential roles in the pathogenesis of abdominal aortic aneurysms (AAAs) by mediating necroptosis and inflammation. We previously discovered a small molecular inhibitor GSK2593074A (GSK'074) that binds to both RIPK1 and RIPK3 with high affinity and prevents AAA formation in mice. In this study, we evaluated whether GSK'074 can attenuate progression of existing AAA in the calcium phosphate model. METHODS: C57BL6/J mice were subjected to the calcium phosphate model of aortic aneurysm generation. Mice were treated with either GSK'074 (4.65 mg/kg/day) or dimethylsulfoxide (DMSO) controls starting 7 days after aneurysm induction. Aneurysm growth was monitored via ultrasound imaging every 7 days until harvest on day 28. Harvested aortas were examined via immunohistochemistry. The impact of GSK'074 on vascular smooth muscle cells and macrophages were evaluated via flow cytometry and transwell migration assay. RESULTS: At the onset of treatment, mice in both the control (DMSO) and GSK'074 groups showed similar degree of aneurysmal expansion. The weekly ultrasound imaging showed a steady aneurysm growth in DMSO-treated mice. The aneurysm growth was attenuated by GSK'074 treatment. At humane killing, GSK'074-treated mice had significantly reduced progression in aortic diameter from baseline as compared with the DMSO-treated mice (83.2% ± 13.1% [standard error of the mean] vs 157.2% ± 32.0% [standard error of the mean]; P < .01). In addition, the GSK'074-treated group demonstrated reduced macrophages (F4/80, CD206, MHCII), less gelatinase activity, a higher level of smooth muscle cell-specific myosin heavy chain, and better organized elastin fibers within the aortic walls compared with DMSO controls. In vitro, GSK'074 inhibited necroptosis in mouse aortic smooth muscle cells; whereas, it was able to prevent macrophage migration without affecting Il1b and Tnf expression. CONCLUSIONS: GSK'074 is able to attenuate aneurysm progression in the calcium phosphate model. The ability to inhibit both vascular smooth muscle cell necroptosis and macrophage migration makes GSK'074 an attractive drug candidate for pharmaceutical treatment of aortic aneurysms. CLINICAL RELEVANCE: Previous clinical trials evaluating pharmaceutical treatments in blocking aneurysm progression have failed. However, most agents used in those trials focused on inhibiting only one mechanism that contributes to aneurysm pathogenesis. In this study, we found GSK'074 is able to attenuate aneurysm progression in the calcium phosphate model by inhibiting both vascular smooth muscle cell necroptosis and macrophage migration, which are both key processes in the pathogenesis of aneurysm progression. The ability of GSK'0474 to inhibit multiple key pathologic mechanisms makes it an attractive therapeutic candidate for aneurysm progression.