Advanced Atherosclerosis Research Articles

Results of the tibioperoneal trunk repair in patients with advanced atherosclerosis

Objective: To study immediate and long-term results of the tibioperoneal trunk repair (plastic reconstruction or prosthetic repair) during femoropopliteal bypass.Materials and methods: In our retrospective cohort study we analyzed surgical treatment results of 109 patients with lower extremities atherosclerosis who were treated in the Vascular Surgery Unit of Interregional Clinical Diagnostic Center (Kazan, Russian Federation) from 2018 to 2020. 26 (23.8%) patients were hospitalized for stage IB-IIA acute arterial insufficiency with atherothrombosis, and 83 (76.1%) patients were admitted with critical limb ischemia (CLI) as a result of advanced atherosclerosis. Among the CLI patients, 43 (39.4%) of them had stage III chronic arterial insufficiency, and 40 (36.7%) patients had stage IV chronic arterial insufficiency. The study selection criteria included extensive femoral and popliteal arteries disease and significant stenosis or occlusion of the tibioperoneal trunk. TASC II type C lesions were detected in 2 (1.8%) patients, while 107 (98.2%) patients were diagnosed with TASC II type D lesions. Based on the GLASS classification, all 109 (100%) patients had FP grade 3-4 femoropopliteal lesions with concomitant IP grade 1-4 tibial lesion. The main group included 24 (22%) patients who underwent femoropopliteal bypass with plastic or prosthetic repair of the tibioperoneal trunk using an original technique. The control group included 85 (78%) patients who underwent bypass surgery without the tibioperoneal trunk repair. Isolated femorotibial bypass was performed in 7 (8.2%) patients, and 78 (91.7%) patients underwent femoropopliteal bypass with a reversed autogenous vein.Results: We followed up patients for 2 years after surgery. Immediate technical success was 97.24% (106/109). Graft thrombosis was reported in 3 cases. No difference between the groups was observed. Long-term graft patency was significantly higher in the main group (P = .044) and significantly associated with age (RR = 0.96; 95% CI of 0.92 to 1.00, P = .03), type 2 diabetes mellitus (RR = 2.10; 95% CI of 1.10 to 4.10, P = .03), and history of the tibioperoneal trunk repair (RR = 0.43; 95% CI of 0.18 to 1.00, P = .06). Variables associated with patency in the univariate regression at a significance level P ≤ .1 were included in a multivariate model that demonstrated the combined effect of predictors on the outcome.Conclusions: Femoropopliteal bypass with the tibioperoneal trunk repair improves treatment results in patients with extensive peripheral artery disease and immediately threatened limbs or a threat to a limb within 2 years.

Protocatechuic acid boosts continual efferocytosis in macrophages by derepressing KLF4 to transcriptionally activate MerTK.

Macrophages clear apoptotic cells through a process called continual efferocytosis. We found that protocatechuic acid (PCA), a polyphenolic compound abundant in fruits and vegetables, increased the continual efferocytic capacity of macrophages and inhibited the progression of advanced atherosclerosis. PCA reduced the intracellular amounts of microRNA-10b (miR-10b) by promoting its secretion in extracellular vesicles, which led to an increase in the abundance of the miR-10b target Krüppel-like factor 4 (KLF4). In turn, KLF4 transcriptionally induced the gene encoding Mer proto-oncogene tyrosine kinase (MerTK), an efferocytic receptor for the recognition of apoptotic cells, resulting in increased continual efferocytic capacity. However, in naive macrophages, the PCA-induced secretion of miR-10b did not affect KLF4 and MerTK protein abundance or efferocytic capacity. In mice, oral administration of PCA increased continual efferocytosis in macrophages residing in the peritoneal cavities, thymi, and advanced atherosclerotic plaques through the miR-10b-KLF4-MerTK pathway. In addition, pharmacological inhibition of miR-10b with antagomiR-10b also increased the efferocytic capacity of efferocytic but not naive macrophages in vitro and in vivo. Together, these data describe a pathway that promotes continual efferocytosis in macrophages through miR-10b secretion and a KLF4-dependent increase in MerTK abundance, which can be activated by dietary PCA and which has implications for understanding the regulation of continual efferocytosis in macrophages.

Baseline modern medical management in the BEST-CLI trial

ObjectivesThe use of optimal medical therapy (OMT) in patients with chronic limb-threatening ischemia (CLTI) has not been well-studied. The Best Endovascular vs Best Surgical Therapy in Patients with CLTI study (BEST-CLI) is a multicenter, randomized, controlled trial sponsored by the National Institutes of Health comparing revascularization strategies in patients with CLTI. We evaluated the use of guideline-based OMT among patients with CLTI at the time of their enrollment into the trial. MethodsA multidisciplinary committee defined OMT criteria related to blood pressure and diabetic management, lipid-lowering and antiplatelet medication use, and smoking status for patients enrolled in BEST-CLI. Status reports indicating adherence to OMT were provided to participating sites at regular intervals. Baseline demographic characteristics, comorbid medical conditions, and use of OMT at trial entry were evaluated for all randomized patients. A linear regression model was used to identify the relationship of predictors to the use of OMT. ResultsAt the time of randomization (n = 1830 total enrolled), 87% of patients in BEST-CLI had hypertension, 69% had diabetes, 73% had hyperlipidemia, and 35% were currently smoking. Adherence to four OMT components (controlled blood pressure, not currently smoking, use of one lipid-lowering medication, and use of an antiplatelet agent) was modest. Only 25% of patients met all four OMT criteria; 38% met three, 24% met two, 11% met only one, and 2% met none. Age ≥80 years, coronary artery disease, diabetes, and Hispanic ethnicity were positively associated, whereas Black race was negatively associated, with the use of OMT. ConclusionsA significant proportion of patients in BEST-CLI did not meet OMT guideline-based recommendations at time of entry. These data suggest a persistent major gap in the medical management of patients with advanced peripheral atherosclerosis and CLTI. Changes in OMT adherence over the course of the trial and their impact on clinical outcomes and quality of life will be assessed in future analyses.

Anti-inflammation nanomedicine shots through atherosclerotic plaques for targeted treatment and precise diagnosis

Atherosclerosis, formed by fibrofatty lesions in the artery wall, underpins myocardial infarctions, strokes and disabling peripheral artery diseases. Inflammation with multiple maladaptive roles runs through atherosclerotic inception, propagation, and complications. Given degree of inflammation differing among atherosclerotics, inflammation-targeted nanomedicine may produce individualized monitoring and manipulation of inflammatory regulators beyond traditionally slowing down atherosclerosis progression. Equipped with biomimetic membrane, microenvironment-responsive and targeting structures, nanomedicines could escape from the innate immune system and autonomously navigate to targeted lesions, thereby muting inflammation in atherosclerotic sites. Moreover, nanoparticles’ advantages go beyond advancing pharmacology. The optimal physicochemical properties of nanoparticles favor noninvasive imaging function to provide diagnostic and prognostic information of atherosclerotic plaques. Despite efficacious ability of the preclinical anti-inflammation nanotherapeutics to reduce atherosclerotic risks, future translation of inflammation-targeted nanomedicines requires to comprehensively refine the strategies that the nanomedicines forestall inflammatory pathways via systemic exposure, drug-cell interactions, and endothelial barrier reconstruction. In this review, we outline the development of diverse nanomedicines to specifically identify pathophysiological changes and combat advanced atherosclerosis along with dynamically tracing inflammatory markers. Finally, challenges and opportunities of inflammation-targeted nanomedicines are highlighted to pave avenues toward personalized medicine for atherosclerosis to stem the growing global burden of cardiovascular diseases.

Gene deletion of Interleukin-1α reduces ER stress-induced CHOP expression in macrophages and attenuates the progression of atherosclerosis in apoE-deficient mice

The pathophysiology of atherosclerosis initiation and progression involves many inflammatory cytokines, one of them is interleukin (IL)-1α that has been shown to be secreted by activated macrophages. We have previously shown that IL-1α from bone marrow-derived cells is critical for early atherosclerosis development in mice. It is known that endoplasmic reticulum (ER) stress in macrophages is involved in progression to more advanced atherosclerosis, but it is still unknown whether this effect is mediated through cytokine activation or secretion. We previously demonstrated that IL-1α is required in ER stress-induced activation of inflammatory cytokines in hepatocytes and in the associated induction of steatohepatitis. In the current study, we aimed to examine the potential role of IL-1α in ER stress-induced activation of macrophages, which is relevant to progression of atherosclerosis. First, we demonstrated that IL-1α is required for atherosclerosis development and progression in the apoE knockout (KO) mouse model of atherosclerosis. Next, we showed that ER stress in mouse macrophages results in the protein production and secretion of IL-1α in a dose-dependent manner, and that IL-1α is required in ER stress-induced production of the C/EBP homologous protein (CHOP), a critical step in ER stress-mediated apoptosis. We further demonstrated that IL-1α-dependent CHOP production in macrophages is specifically mediated through the PERK-ATF4 signaling pathway. Altogether, these findings highlight IL-1α as a potential target for prevention and treatment of atherosclerotic cardiovascular disease.

Identification of Functionally Significant Polymorphic Variants in miRNA Genes in Carotid Atherosclerosis

miRNAs are vital molecules of gene expression. They are involved in the pathogenesis of various common diseases, including atherosclerosis, its risk factors and complications. A detailed characterization of the spectrum of functionally significant polymorphisms of miRNA genes of patients with advanced carotid atherosclerosis is an actual research task. We analyzed miRNA expression and exome sequencing data of carotid atherosclerotic plaques of the same male patients (n = 8, 66–71 years of age, 67‒90% degree of carotid artery stenosis). For further study and analysis of the association between rs2910164 polymorphism of the MIR146A gene and advanced carotid atherosclerosis, we recruited 112 patients and 72 relatively healthy Slavic residents of Western Siberia. 321 and 97 single nucleotide variants (SNVs) were detected in the nucleotide sequences of pre- and mature miRNAs in carotid atherosclerotic plaques. These variants were located in 206 and 76 miRNA genes, respectively. Integration the data of exome sequencing and miRNA expression revealed 24 SNVs of 18 miRNA genes which were processed to mature form in carotid atherosclerotic plaques. SNVs with the greatest potential functional significance for miRNA expression predicted in silico were rs2910164:CG (MIR146A), rs2682818:AC (MIR618), rs3746444:AG (MIR499A), rs776722712:CT (MIR186), rs199822597:GA (MIR363). The expression of miR-618 was lower in carotid atherosclerotic plaques of patients with the AC rs2682818 genotype of the MIR618 gene compared with the CC genotype (log2FC = 4.8; p = 0.012). We also found the association of rs2910164:C (MIR146A) with the risk of advanced carotid atherosclerosis (OR = 2.35; 95% CI: 1.43–3.85; p = 0.001). Integrative analysis of polymorphism in miRNA genes and miRNA expression is informative for identifying functionally significant polymorphisms in miRNA genes. The rs2682818:AC (MIR618) is a candidate for regulating miRNA expression in carotid atherosclerotic plaques. The rs2910164:C (MIR146A) is associated with the risk of advanced carotid atherosclerosis.

Abstract 107: CaMK4 is a Novel Regulator of Myeloid Phenotype and Function in Atherosclerosis

Background: Chronic inflammation is a major driver of atherosclerotic cardiovascular disease, and therapeutics that target inflammation reduce clinical cardiac events beyond levels seen with conventional strategies targeting cholesterol alone. Recent work by our group and others has demonstrated that advanced atherosclerosis is also characterized by the failure of an active repair process termed ‘inflammation resolution’. Strategies that boost resolution and break the cycle of chronic inflammation promotes plaque stability. Hypothesis: Our review of publicly available RNAseq data revealed an increase in expression of Calcium/calmodulin-dependent protein kinase 4 (CaMK4) in advanced/unstable regions of plaque within human carotid arteries as well as in myeloid cells derived from atherosclerotic murine aortas. Therefore, we hypothesized that CaMK4 promotes inflammation and impairs resolution. Results and Methods: Control and Camk4 -/- mice were injected with AAV-8 PCSK9 virus and fed a high-fat high-cholesterol Western diet for 12 weeks. Cross-sectional analysis of aortic roots showed that Camk4 -/- mice had significantly less plaque burden than Controls. Flow cytometric analysis revealed elevated monocyte numbers in Camk4 -/- mice compared to Control mice. Further analysis demonstrated increased skewing of Camk4 -/- monocyte populations toward a Ly6c low subset, indicating a more pro-reparative phenotype compared to Control mice. We considered whether CaMK4 may regulate a gene signature driving monocyte conversion and found that Camk4 -/- monocytes expressed higher levels of Nr4a1, Cebpb, and Klf2, which have been shown to promote conversion to Ly6c low monocytes. As Ly6c low monocytes give rise to pro-reparative macrophages, we generated bone marrow-derived macrophages and found that loss of CaMK4 in vitro led to higher levels of pro-reparative cytokines being produced in response to LPS stimulation as well as more efficient clearance of apoptotic cells when compared to Controls. Conclusions: Overall, these findings suggest that CaMK4 regulates myeloid phenotype in vitro and in vivo and that loss of CaMK4 promotes pro-resolving macrophage function. Therefore, targeting CaMK4 may offer a unique way to target atheroprogression.

Abstract 460: Catalase Overexpression Modulates Plaque Fibrosis And Vascular Contractile Function In Aged Hyperlipidemic Mice

Background: Plaque fibrosis and re-differentiation of vascular smooth muscle cells (VSMCs) from a contractile to a “synthetic” phenotype are hallmarks of advanced atherosclerosis. While in vitro studies demonstrated a clear role of oxidative stress in the loss of VSMC contractile phenotypes, the roles of specific antioxidant mechanisms and their therapeutic utility remain unclear. Here we test the hypothesis that overexpression of catalase protects against excess plaque fibrosis and losses in contractile phenotypes in vivo . Methods: We used littermate-matched, hypercholesteremic (ldlr -/- /ApoB 100/100 ; LA) mice lacking a transgenic construct (LA-CAT 0/0 ) or harboring a single copy of the human catalase gene (LA-CAT Tg/0 ), which were fed a Western Diet for 3 or 12 months. Isolated organ bath chambers, histopathological analyses, and quantitative RT-PCR were used to assess vasomotor function, structure, and molecular changes in aorta. Results: Oxidative stress increased with disease progression in aortic tissue from LA-CAT 0/0 mice and was modestly reduced in LA-CAT Tg/0 mice. Collagen deposition and fiber thickness increased with disease progression in LA-CAT 0/0 mice, and these increases in fiber thickness were attenuated in LA-CAT Tg/0 mice at 12 months and more pronounced in male mice. In parallel, we observed favorable rightward shifts in the vascular stress-strain relationship in LA-CAT Tg/0 mice that were more pronounced in aged male mice. Interestingly, these structural changes were associated with improved contractile responses to either PGF 2α or serotonin in male LA-CAT Tg/0 mice at 12 months. Consistent with these changes, the contractile marker calponin-1 was profoundly reduced with increasing age in LA-CAT 0/0 mice, with these changes being partially attenuated in aging, male LA-CAT Tg/0 mice. In vitro, treatment of VSMCs with PEG-catalase attenuated BMP2-dependent contractile protein expression. Conclusion: These results demonstrate that moderate overexpression of catalase can attenuate deleterious fibrotic and contractile changes associated with prolonged progression of atherosclerosis. Future mechanistic investigation exploring the context-dependence of these changes (e.g., sex dependence) is warranted.

Abstract 212: Transcriptional Profiling Of Vascular Smooth Muscle Cell-foam Cells In Atherosclerotic Lesions

Background: One of the hallmarks of atherosclerosis progression is the uptake of oxidized lipids by cells to become Foam/Foamy cells - the most prominent being vascular smooth muscle cells (VSMC)-foam cells. Although studies have identified the ability of VSMC to become VSMC-foam cells, their transcriptomic profile and the phenotypic modulations which distinguish them from contractile VSMC remain poorly defined and understood. Hypothesis: We hypothesize that VSMC-foamy cells differ from contractile VSMC not only by modulation of genes shared by both cell types, but also by the expression of a unique set of genes which contribute to their formation and maintenance within atherosclerotic lesions. Method: To address this, we performed bulk RNA sequencing of atherosclerotic plaques from conditional VSMC-lineage tracing mice models on high fat diet. VSMC and VSMC-foam cells were isolated and sequenced at specific time points reflective of late and advanced atherosclerosis disease progression. Computational analysis enabled us identify differentially expressed genes, biological pathways and molecular functions which distinguished VSMC from VSMC-foam cells. Result and Conclusion: Our computational analysis identified differential genes and pathways unique to VSMC-foam cells which might be associated with disease progression. From our analysis, VSMC showed a lower expression of contractile VSMC marker genes and a higher expression of genes in synthetic/intermediary multipotent state, we term “SEM” state. Our study also showed that VSMC-foam cells also expressed unique genes which distinguished them from contractile VSMC. These genes not only serve as potential genetic markers, but might further explain the nuclear composition, as well as the inflammatory and metabolic profile of VSMC-foam cells. Defining and understanding the molecular mechanisms and drivers which promote VSMC-foam cell formation and maintenance in atherosclerosis, would aid in our understanding of the disease with potentials for more effective therapeutic solutions.

PD1 blockade promotes IFN-γ production by progenitor exhausted PD1-expressing T cells in atherosclerotic plaques

Abstract Objective: Interferon-γ (IFN-γ) is a key cytokine regulating the development of atherosclerosis, but little is known regarding the phenotype and regulation of T cells producing IFN-γ in plaques. Methods: To identify and characterize IFN-γ-producing T cells in the development atherosclerosis, we utilized IFN-γ-YFP and Nur77-GFP reporter mice crossed onto an atherosclerotic background (Apoe−/−IFN-γ YFP/YFP; Apoe−/−Nur77GFP/wt), adoptive transfer of transgenic ApoB-reactive T-cells into mice containing humanized ApoB100 (HuBL mice), and treatment with immune checkpoint inhibitors in atherosclerotic mice. Results: Aortic CD4 +and CD8 +T cells producing IFN-γ were a complex set of cells comprised predominantly of “progenitor exhausted” PD1-intermediate (PD1 int) SLAMF6 +IL7R +T cells and a minor population of “terminally exhausted” PD1 hiTOX +T cells. However, both PD1 intand PD1 hiplaque T cells were Ki67 +and displayed elevated levels of recent TCR signaling (Nur77-GFP +). ApoB-specific transgenic CD4 +T cells adoptively transferred to a hypercholesterolemic HuBL mice showed sign of early exhaustion, upregulating PD1, TOX, and Tim3. Finally, PD1 blockade in mice with advanced atherosclerosis resulted in increased levels of IFN-γ production of plaque-infiltrating CD4 +and CD8 +T cells. Conclusion: IFN-γ-producing T cells in the atherosclerotic plaque display markers classically associated with an exhausted T cell phenotype but still produce cytokines, proliferate, and are responsive to anti-PD1 treatment. Our study suggests that PD1 signaling regulates T cell IFN-γ production in plaques and reveals a potential mechanism by which immune checkpoint blockade therapy aggravates cardiovascular disease. Supported by grants from Swedish Heart-Lung Foundation (#20200522, #20190449) sourced by Daniel Engelbertsen.

Abstract 468: Nano-targeting Epsin In Cholesterol Uptake-efflux Fortifies Atheroma Regression

Excess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In our study, we determined the role of Epsins in macrophage-mediated metabolic regulation and developed a method to target macrophage Epsins with specially-designed S2P-conjugated lipid nanoparticles (NPs), which encapsulate siRNAs to suppress Epsins.We used scRNA-seq with our newly developed algorithm MEBOCOST to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. The NPs targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis.We revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing macrophage-specific NP delivery of Epsin siRNAs, we showed that silencing of macrophage Epsins markedly diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport.Our findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis.

Effect of erythrophagocytosis-induced ferroptosis during angiogenesis in atherosclerotic plaques.

Intraplaque (IP) angiogenesis is a key feature of advanced atherosclerotic plaques. Because IP vessels are fragile and leaky, erythrocytes are released and phagocytosed by macrophages (erythrophagocytosis), which leads to high intracellular iron content, lipid peroxidation and cell death. In vitro experiments showed that erythrophagocytosis by macrophages induced non-canonical ferroptosis, an emerging type of regulated necrosis that may contribute to plaque destabilization. Erythrophagocytosis-induced ferroptosis was accompanied by increased expression of heme-oxygenase 1 and ferritin, and could be blocked by co-treatment with third generation ferroptosis inhibitor UAMC-3203. Both heme-oxygenase 1 and ferritin were also expressed in erythrocyte-rich regions of carotid plaques from ApoE-/- Fbn1C1039G+/- mice, a model of advanced atherosclerosis with IP angiogenesis. The effect of UAMC-3203 (12.35mg/kg/day) on atherosclerosis was evaluated in ApoE-/- Fbn1C1039G+/- mice fed a western-type diet (WD) for 12 weeks (n = 13 mice/group) or 20 weeks (n = 16-21 mice/group) to distinguish between plaques without and with established IP angiogenesis, respectively. A significant decrease in carotid plaque thickness was observed after 20 weeks WD (87 ± 19μm vs. 166 ± 20μm, p = 0.006), particularly in plaques with confirmed IP angiogenesis or hemorrhage (108 ± 35μm vs. 322 ± 40μm, p = 0.004). This effect was accompanied by decreased IP heme-oxygenase 1 and ferritin expression. UAMC-3203 did not affect carotid plaques after 12 weeks WD or plaques in the aorta, which typically do not develop IP angiogenesis. Altogether, erythrophagocytosis-induced ferroptosis during IP angiogenesis leads to larger atherosclerotic plaques, an effect that can be prevented by ferroptosis inhibitor UAMC-3203.

Bruton's Tyrosine Kinase inhibition by Acalabrutinib does not affect early or advanced atherosclerotic plaque size and morphology in Ldlr-/- mice

Atherosclerosis is characterized by the accumulation of lipids and immune cells, including mast cells and B cells, in the arterial wall. Mast cells contribute to atherosclerotic plaque growth and destabilization upon active degranulation. The FcεRI-IgE pathway is the most prominent mast cell activation route. Bruton's Tyrosine Kinase (BTK) is involved in FcεRI-signaling and may be a potential therapeutic target to limit mast cell activation in atherosclerosis. Additionally, BTK is crucial in B cell development and B-cell receptor signaling. In this project, we aimed to assess the effects of BTK inhibition on mast cell activation and B cell development in atherosclerosis.In human carotid artery plaques, we showed that BTK is primarily expressed on mast cells, B cells and myeloid cells. In vitro, BTK inhibitor Acalabrutinib dose-dependently inhibited IgE mediated activation of mouse bone marrow derived mast cells. In vivo, male Ldlr−/− mice were fed a high-fat diet for eight weeks, during which mice were treated with Acalabrutinib or control solvent. In Acalabrutinib treated mice, B cell maturation was reduced compared to control mice, showing a shift from follicular II towards follicular I B cells. Mast cell numbers and activation status were not affected. Acalabrutinib treatment did not affect atherosclerotic plaque size or morphology. In advanced atherosclerosis, where mice were first fed a high-fat diet for eight weeks before receiving treatment, similar effects were observed.Conclusively, BTK inhibition by Acalabrutinib alone did neither affect either mast cell activation nor early- and advanced atherosclerosis, despite the effects on follicular B cell maturation.

Both hyperglycemia and hyperuricemia aggravate acute kidney injury during cholesterol embolism syndrome despite opposite effects on kidney infarct size

Kidney cholesterol crystal embolism (CCE) occurs in advanced atherosclerosis and induces a thrombotic (micro)angiopathy, a drop in the glomerular filtration rate (GFR), and an ischemic kidney infarction with necroinflammation. We speculated that common metabolic comorbidities such as diabetes or hyperuricemia would independently modulate each of these distinct pathophysiological processes. To test this, experimental CCE was induced by injecting cholesterol crystals into the left kidney artery of mice and thrombotic angiopathy, GFR drop, and infarct size were analyzed after 24 hours in the presence of hyperglycemia (about 500 mg/dL) or hyperuricemia (about 8 mg/dL) or their absence. In healthy mice, unilateral CCE caused diffuse thrombotic angiopathy in interlobar, arcuate and interlobular arteries, followed by a 50% or less drop in GFR compared to baseline and a variable degree of ischemic kidney necrosis. Hyperglycemia but not hyperuricemia aggravated thrombotic angiopathy although both caused a GFR decline, albeit via different mechanisms. Hyperglycemia aggravated GFR loss by increasing necroinflammation and infarct size, while the antioxidative effects of hyperuricemia reasonably attenuated necroinflammation and infarct size but induced a diffuse vasoconstriction in affected and unaffected kidney tissue. Thus, both hyperglycemia or hyperuricemia aggravate CCE-induced acute kidney failure despite having opposite effects on ischemic necroinflammation and infarction.

Mast cells participate in smooth muscle cell reprogramming and atherosclerotic plaque calcification

BackgroundCalcification, a key feature of advanced human atherosclerosis, is positively associated with vascular disease burden and adverse events. We showed that macrocalcification can be a stabilizing factor for carotid plaque molecular biology, due to inverse association with immune processes. Mast cells (MCs) are important contributors to plaque instability, but their relationship with macrocalcification is unexplored. With a hypothesis that MC activation negatively associates with carotid plaque macrocalcification, we aimed to investigate the link between MCs and carotid plaque vulnerability, and study MC role in plaque calcification via smooth muscle cells (SMCs). MethodsPre-operative computed tomography angiographies of patients (n = 40) undergoing surgery for carotid stenosis were used to characterize plaque morphology. Plaque microarrays (n = 40 and n = 126) were used for bioinformatic deconvolution of immune cell populations. Tissue microarrays (n = 103) were used to histologically validate the contribution of activated and resting MCs in plaques. ResultsActivated MCs and their typical markers were negatively correlated with macrocalcification. The ratio of activated vs. resting MCs was increased in low-calcified plaques from symptomatic patients. There was no modulating effect of medication on MC ratios. In vitro experiments showed that SMC calcification attenuated MC activation, while both active and resting MCs stimulated SMC calcification and induced dedifferentiation towards a pro-inflammatory-, osteochondrocyte-like phenotype, without modulating their migro-proliferative function. ConclusionsIntegrative analyses from human plaques showed that MC activation is inversely associated with macrocalcification and positively with parameters of plaque vulnerability. Mechanistically, MCs induce SMC osteogenic reprograming, while matrix calcification in turn attenuates MC activation, offering new therapeutic avenues for exploration.

A comparative gene expression matrix in Apoe-deficient mice identifies unique and atherosclerotic disease stage-specific gene regulation patterns in monocytes and macrophages

Background and aimsAtherosclerosis is a systemic and chronic inflammatory disease propagated by monocytes and macrophages. Yet, our knowledge on how transcriptome of these cells evolves in time and space is limited. We aimed at characterizing gene expression changes in site-specific macrophages and in circulating monocytes during the course of atherosclerosis. MethodsWe utilized apolipoprotein E-deficient mice undergoing one- and six-month high cholesterol diet to model early and advanced atherosclerosis. Aortic macrophages, peritoneal macrophages, and circulating monocytes from each mouse were subjected to bulk RNA-sequencing (RNA-seq). We constructed a comparative directory that profiles lesion- and disease stage-specific transcriptomic regulation of the three cell types in atherosclerosis. Lastly, the regulation of one gene, Gpnmb, whose expression positively correlated with atheroma growth, was validated using single-cell RNA-seq (scRNA-seq) of atheroma plaque from murine and human. ResultsThe convergence of gene regulation between the three investigated cell types was surprisingly low. Overall 3245 differentially expressed genes were involved in the biological modulation of aortic macrophages, among which less than 1% were commonly regulated by the remote monocytes/macrophages. Aortic macrophages regulated gene expression most actively during atheroma initiation. Through complementary interrogation of murine and human scRNA-seq datasets, we showcased the practicality of our directory, using the selected gene, Gpnmb, whose expression in aortic macrophages, and a subset of foamy macrophages in particular, strongly correlated with disease advancement during atherosclerosis initiation and progression. ConclusionsOur study provides a unique toolset to explore gene regulation of macrophage-related biological processes in and outside the atheromatous plaque at early and advanced disease stages.

Large unstained cell (LUC) count as a predictor of carotid artery occlusion.

Carotid artery stenosis is often considered a stable clinical condition, and the underlying atherosclerosis is thought to have an inflammatory background. The aim of the study was to assess the value of different parameters obtained from whole blood counts for the prediction of advanced carotid artery atherosclerosis, including vessel occlusion, irrespective of symptom occurrence. The study group comprised 290 patients (84 (29%) females and 206 (71%) males) with a mean age of 68 ±8 years, who were admitted to the Vascular Surgery Department due to significant carotid artery disease. Patients were retrospectively divided into 2 subgroups regarding the presence or absence of artery occlusion. The demographic, clinical and laboratory preoperative data were compared between both groups. We found significant differences in preoperative large unstained cell (LUC) counts between patients with and without carotid artery occlusion (p = 0.003), when analyzed with the Mann-Whitney test for independent samples. The receiver operating characteristic (ROC) curve showed that LUC count has prognostic properties for carotid artery occlusion, with an area under the curve (AUC) of 0.637 (p = 0.033), yielding a 69.70% sensitivity and a 51.75% specificity. Large unstained cells represent an acute inflammatory state related to artery occlusion. An LUC count below the cutoff value of 0.16×109/L may be a predictor of carotid artery occlusion. Therefore, carotid artery occlusion should not be regarded as a chronic state, but as a clinical challenge being promoted by active inflammatory processes.

Plaque morphology and distribution in patients with and without myocardial bridge – an intravascular ultrasound study

Background Myocardial bridging (MB) is a common congenital cardiovascular anomaly. There are reported associations of MB with different clinical presentations like effort angina, acute coronary syndromes (ACS) and sudden cardiac death. Acceleration of atherosclerosis in proximal vessel is reported in patients with MB, while bridged segments are reported to be free of atherosclerosis Methods We assessed patients who underwent intravascular ultrasound (IVUS) guided percutaneous intervention (PCI) of left anterior descending (LAD) artery. Plaque characteristics derived from IVUS analysis were compared between those who displayed myocardial bridge versus those who did not harbour the anomaly. Results One hundred and forty-seven (147) patients underwent IVUS guided PCI. Incidence of MB was 44/147 (29.9%). Mean age of patients who had MB {+} was higher (62.1 ± 10.3 vs. 57.8 ± 11.2 (p = .03). 142/147 (96.6%) patients presented with ACS. ST elevation myocardial infarction (STEMI) was the most common presenting diagnosis (110/147 to 74.8%). There were no differences in qualitative plaque characteristics – attenuated plaque, calcification or calcium score between two groups. Plaque burden and length of the lesion in the proximal vessel were not different. Among patients with MB {+}, atheromatous extension to segments underlying the bridge was seen in 31/44 (70.5%) cases. Conclusions In a series of patients who presented with advanced clinical atherosclerosis, plaque characteristics were not different in patients who harboured myocardial bridge vs. those who did not have the anomaly. Atheromatous involvement was seen extending into bridged segment contrary to previous reports.

Clinical significance of atherosclerotic risk factors differs in early and advanced stages of plaque formation: A longitudinal study in the general population

BackgroundCarotid plaque is a well-known prognostic factor for cardiovascular diseases. It is unclear which risk factors are associated with the transformation of carotid plaque over time. In this longitudinal study, we examined the risk factors related to carotid plaque progression. MethodsWe enrolled 738 men without medication (mean age: 55 ± 10 years) who underwent the first and second health examinations. We measured carotid plaque thickness (PT) at three points of the right and left carotid artery. Plaque score (PS) was calculated by summing all the PTs. We divided the PS into three groups: None-group (PS <1.1), Early-group (1.1 ≤ PS <5.1), and Advanced-group (PS ≥5.1). We analyzed the relationship between PS progression and parameters such as age, body mass index, systolic blood pressure (SBP), fasting blood sugar, low-density lipoprotein cholesterol (LDL-C), and smoking and exercise habits. ResultsIn multivariable logistic regression analysis, age and SBP were independent factors for PS progression from none to early stages (age, OR 1.07, p = 0.002; SBP, 10 mmHg, OR 1.27, p = 0.041). Age, follow-up period and LDL-C were independently associated factors for PS progression from early to advanced stages (age, OR 1.08,p < 0.001; follow-up period OR1.19, p = 0.041; LDL-C, 10 mg/dL, OR 1.10, p = 0.049). ConclusionsSBP was independently associated with the progress of early atherosclerosis, while LDL-C was independently associated with the progression of advanced atherosclerosis in the general population. Further studies are needed to assess whether early control of SBP and LDL-C levels can reduce the occurrence of future cardiovascular events.

Pairing of single-cell RNA analysis and T cell antigen receptor profiling indicates breakdown of T cell tolerance checkpoints in atherosclerosis

Atherosclerotic plaques form in the inner layer of arteries triggering heart attacks and strokes. Although T cells have been detected in atherosclerosis, tolerance dysfunction as a disease driver remains unexplored. Here we examine tolerance checkpoints in atherosclerotic plaques, artery tertiary lymphoid organs and lymph nodes in mice burdened by advanced atherosclerosis, via single-cell RNA sequencing paired with T cell antigen receptor sequencing. Complex patterns of deteriorating peripheral T cell tolerance were observed being most pronounced in plaques followed by artery tertiary lymphoid organs, lymph nodes and blood. Affected checkpoints included clonal expansion of CD4+, CD8+ and regulatory T cells; aberrant tolerance-regulating transcripts of clonally expanded T cells; T cell exhaustion; Treg–TH17 T cell conversion; and dysfunctional antigen presentation. Moreover, single-cell RNA-sequencing profiles of human plaques revealed that the CD8+ T cell tolerance dysfunction observed in mouse plaques was shared in human coronary and carotid artery plaques. Thus, our data support the concept of atherosclerosis as a bona fide T cell autoimmune disease targeting the arterial wall.