Clonally expanded memory CD8+ T cells accumulate in atherosclerotic plaques and are pro-atherogenic in aged mice.

Aging is a strong risk factor for atherosclerosis and induces accumulation of memory CD8+ T cells in mice and humans. Biological changes that occur with aging lead to enhanced atherosclerosis, yet the role of aging on CD8+ T cells during atherogenesis is unclear. In this study, using femle mice, we found that depletion of CD8+ T cells attenuated atherogenesis in aged, but not young, animals. Furthermore, adoptive transfer of splenic CD8+ T cells from aged wild-type, but not young wild-type, donor mice significantly enhanced atherosclerosis in recipient mice lacking CD8+ T cells. We also characterized T cells in healthy and atherosclerotic young and aged mice by single-cell RNA sequencing. We found specific subsets of age-associated CD8+ T cells, including a Granzyme K+ effector memory subset, that accumulated and was clonally expanded within atherosclerotic plaques. These had transcriptomic signatures of T cell activation, migration, cytotoxicity and exhaustion. Overall, our study identified memory CD8+ T cells as therapeutic targets for atherosclerosis in aging.

Age-associated adipose tissue inflammation promotes monocyte chemotaxis and enhances atherosclerosis.

Although aging enhances atherosclerosis, we do not know if this occurs via alterations in circulating immune cells, lipid metabolism, vasculature, or adipose tissue. Here, we examined whether aging exerts a direct pro-atherogenic effect on adipose tissue in mice. After demonstrating that aging augmented the inflammatory profile of visceral but not subcutaneous adipose tissue, we transplanted visceral fat from young or aged mice onto the right carotid artery of Ldlr-/- recipients. Aged fat transplants not only increased atherosclerotic plaque size with increased macrophage numbers in the adjacent carotid artery, but also in distal vascular territories, indicating that aging of the adipose tissue enhances atherosclerosis via secreted factors. By depleting macrophages from the visceral fat, we identified that adipose tissue macrophages are major contributors of the secreted factors. To identify these inflammatory factors, we found that aged fat transplants secreted increased levels of the inflammatory mediators TNFα, CXCL2, and CCL2, which synergized to promote monocyte chemotaxis. Importantly, the combined blockade of these inflammatory mediators impeded the ability of aged fat transplants to enhance atherosclerosis. In conclusion, our study reveals that aging enhances atherosclerosis via increased inflammation of visceral fat. Our study suggests that future therapies targeting the visceral fat may reduce atherosclerosis disease burden in the expanding older population.

Homologous cardiac calcium pump regulators phospholamban and sarcolipin adopt distinct oligomeric states in the membrane.

Phospholamban (PLN) and Sarcolipin (SLN) are homologous membrane proteins that belong to the family of proteins that regulate the activity of the cardiac calcium pump (sarcoplasmic reticulum Ca2+-ATPase, SERCA). PLN and SLN share highly conserved leucine zipper motifs that control self-association; consequently, it has been proposed that both PLN and SLN assemble into stable pentamers in the membrane. In this study, we used molecular dynamics (MD) simulations and Western blot analysis to investigate the precise molecular architecture of the PLN and SLN oligomers. Analysis showed that the PLN pentamer is the predominant oligomer present in mouse ventricles and ventricle-like human iPSC-derived cardiomyocytes, in agreement with the MD simulations showing stable leucine zipper interactions across all protomer-protomer interfaces and MD replicates. Interestingly, we found that the PLN pentamer populates an asymmetric structure of the transmembrane region, which is likely an intrinsic feature of the oligomer in a lipid bilayer. The SLN pentamer is not favorably formed across MD replicates and species of origin; instead, SLN from human and mouse atria primarily populate coexisting dimeric and trimeric states. In contrast to previous studies, our findings indicate that the SLN pentamer is not the predominant oligomeric state populated in the membrane. We conclude that despite their structural homology, PLN and SLN adopt distinct oligomeric states in the membrane. We propose that the distinct oligomeric states populated by PLN and SLN may contribute to tissue-specific SERCA regulation via differences in protomer-oligomer exchange, oligomer-SERCA dynamics, and noise filtering during ß-adrenergic stimulation in the heart.

Aging Impairs Mitochondrial Function and Mitophagy and Elevates Interleukin 6 Within the Cerebral Vasculature.

Background The blood-brain barrier (BBB) is critical for cerebrovascular health. Although aging impairs the integrity of the BBB, the mechanisms behind this phenomenon are not clear. As mitochondrial components activate inflammation as mitochondria become dysfunctional, we examined how aging impacts cerebrovascular mitochondrial function, mitophagy, and inflammatory signaling; and whether any alterations correlate with BBB function. Methods and Results We isolated cerebral vessels from young (2-3 months of age) and aged (18-19 months of age) mice and found that aging led to increases in the cyclin-dependent kinase inhibitor 1 senescence marker with impaired mitochondrial function, which correlated with aged mice exhibiting increased BBB leak compared with young mice. Cerebral vessels also exhibited increased expression of mitophagy proteins Parkin and Nix with aging. Using mitophagy reporter (mtKeima) mice, we found that the capacity to increase mitophagy from baseline within the cerebral vessels on rotenone treatment was reduced with aging. Aging within the cerebral vessels also led to the upregulation of the stimulator of interferon genes and increased interleukin 6 (IL-6), a cytokine that alters mitochondrial function. Importantly, exogenous IL-6 treatment of young cerebral vessels upregulated mitophagy and Parkin and impaired mitochondrial function; whereas inhibiting IL-6 in aged cerebral vessels reduced Parkin expression and increased mitochondrial function. Furthermore, treating cerebral vessels of young mice with mitochondrial N-formyl peptides upregulated IL-6, increased Parkin, and reduced Claudin-5, a tight junction protein integral to BBB integrity. Conclusions Aging alters the cerebral vasculature to impair mitochondrial function and mitophagy and increase IL-6 levels. These alterations may impair BBB integrity and potentially reduce cerebrovascular health with aging.

Age-Associated Mitochondrial Dysfunction Accelerates Atherogenesis.

Rationale: Aging is one of the strongest risk factors for atherosclerosis. Yet whether aging increases the risk of atherosclerosis independently of chronic hyperlipidemia is not known. Objective: To determine if vascular aging before the induction of hyperlipidemia enhances atherogenesis. Methods and Results: We analyzed the aortas of young and aged normolipidemic wild type, disease-free mice and found that aging led to elevated IL (interleukin)-6 levels and mitochondrial dysfunction, associated with increased mitophagy and the associated protein Parkin. In aortic tissue culture, we found evidence that with aging mitochondrial dysfunction and IL-6 exist in a positive feedback loop. We triggered acute hyperlipidemia in aged and young mice by inducing liver-specific degradation of the LDL (low-density lipoprotein) receptor combined with a 10-week western diet and found that atherogenesis was enhanced in aged wild-type mice. Hyperlipidemia further reduced mitochondrial function and increased the levels of Parkin in the aortas of aged mice but not young mice. Genetic disruption of autophagy in smooth muscle cells of young mice exposed to hyperlipidemia led to increased aortic Parkin and IL-6 levels, impaired mitochondrial function, and enhanced atherogenesis. Importantly, enhancing mitophagy in aged, hyperlipidemic mice via oral administration of spermidine prevented the increase in aortic IL-6 and Parkin, attenuated mitochondrial dysfunction, and reduced atherogenesis. Conclusions: Before hyperlipidemia, aging elevates IL-6 and impairs mitochondrial function within the aorta, associated with enhanced mitophagy and increased Parkin levels. These age-associated changes prime the vasculature to exacerbate atherogenesis upon acute hyperlipidemia. Our work implies that novel therapeutics aimed at improving vascular mitochondrial bioenergetics or reducing inflammation before hyperlipidemia may reduce age-related atherosclerosis.

CD146 (Cluster of Differentiation 146).

CD146 (cluster of differentiation 146) is an adhesion molecule that is expressed by different cells constituting vessels, particularly endothelial cells. The last 30 years of research in this field have shown that CD146 plays a key role in the control of several vessel functions. Three forms of CD146 have been described, including 2 transmembrane isoforms and a soluble protein that is detectable in the plasma. These CD146 forms mediate pleiotropic functions through homophilic and heterophilic interactions with proteins present on surrounding partners. Several studies used neutralizing antibodies, siRNA, or genetically modified mice to demonstrate the involvement of CD146 in the regulation of angiogenesis, vascular permeability, and leukocyte transmigration. In this review, we will focus on the current knowledge of the roles of CD146 in vascular homeostasis and diseases associated with endothelial dysfunction.

CD146 deficiency promotes plaque formation in a mouse model of atherosclerosis by enhancing RANTES secretion and leukocyte recruitment.

AIMS: The progression of atherosclerosis is based on the continued recruitment of leukocytes in the vessel wall. The previously described role of CD146 in leukocyte infiltration suggests an involvement for this adhesion molecule in the inflammatory response. In this study, we investigated the role of CD146 in leukocyte recruitment by using an experimental model of atherogenesis. METHODS AND RESULTS: The role of CD146 was explored in atherosclerosis by crossing CD146-/- mice with ApoE-/- mice. CD146 -/-/ApoE -/- and ApoE -/- mice were fed a Western diet for 24 weeks and were monitored for aortic wall thickness using high frequency ultrasound. The arterial wall was significantly thicker in CD146-deficient mice. After 24 weeks of Western diet, a significant increase of atheroma in both total aortic lesion and aortic sinus of CD146-null mice was observed. In addition, atherosclerotic lesions were more inflammatory since plaques from CD146-deficient mice contained more neutrophils and macrophages. This was due to up-regulation of RANTES secretion by macrophages in CD146-deficient atherosclerotic arteries. This prompted us to further address the function of CD146 in leukocyte recruitment during acute inflammation by using a second experimental model of peritonitis induced by thioglycollate. Neutrophil recruitment was significantly increased in CD146-deficient mice 12 h after peritonitis induction and associated with higher RANTES levels in the peritoneal cavity. In CD146-null macrophages, we also showed that increased RANTES production was dependent on constitutive inhibition of the p38-MAPK signaling pathway. Finally, Maraviroc, a RANTES receptor antagonist, was able to reduce atherosclerotic lesions and neutrophilia in CD146-deficient mice to the same level as that found in ApoE -/- mice. CONCLUSIONS: Our data indicate that CD146 deficiency is associated with the upregulation of RANTES production and increased inflammation of atheroma, which could influence the atherosclerotic plaque fate. Thus, these data identify CD146 agonists as potential new therapeutic candidates for atherosclerosis treatment.

CD146 mediates VEGF-induced melanoma cell extravasation through FAK activation.

CD146 is an adhesion molecule expressed by both melanoma and endothelial cells and thus is well positioned to control melanoma extravasation. Nevertheless, during melanoma metastasis, the involvement of CD146 expressed within tumor microenvironment has never been analyzed. To investigate whether host CD146 mediates the extravasation of melanoma cells across the endothelium, we generated CD146 KO mice. We demonstrated that host CD146 did not affect melanoma growth or tumor angiogenesis but promoted hematogenous melanoma metastasis to the lung. Accordingly, the survival of CD146-deficient mice was markedly prolonged during melanoma metastasis. Interestingly, vascular endothelial growth factor-induced vascular permeability was significantly decreased in CD146 KO mice. We also provided evidence that VEGF-induced transendothelial migration of melanoma cells was significantly reduced across CD146 KO lung microvascular endothelial cells (LMEC). CD146 deficiency decreased the expression of VEGFR-2/Ve-cadherin and altered focal adhesion kinase (FAK) activation in response to VEGF. In addition, inhibition of FAK phosphorylation reduced transmigration of B16 melanoma cells across WT LMEC at the same level that across CD146 KO LMEC. Altogether, we propose a novel mechanism involving the VEGF/CD146/FAK/Ve-cadherin network in melanoma extravasation across the vessel barrier that identifies CD146-targeted therapy as a potential strategy for the treatment of melanoma metastasis.