Fibronectin/α5 Integrin Contribute to Hypertension-Associated Arterial Ageing and Calcification through Affecting BMP2/MGP Imbalance and Enhancing Vascular Smooth Muscle Cell Phenotypic Transformation.

INTRODUCTION: Hypertension can accelerate and aggravate the process of arterial ageing and calcification. However, the mechanism behind has yet to be well elucidated. METHODS: Here, we monitored the dynamic changes of fibronectin (FN)/α5 integrin, bone morphogenetic protein 2/matrix Gla protein (BMP2/MGP), and Runx2 in the aorta of spontaneously hypertensive rats (SHRs) and thoracic aortic vascular smooth muscle cells (VSMCs), also the phenotypic transformation of VSMCs during the process of arterial ageing and calcification. Further, study on arterial ageing and calcification through antagonist experiments at the molecular level was explored. RESULTS: We found extracellular FN and its α5 integrin receptor expressions were positively associated with arterial ageing and calcification in SHR during ageing, as well in VSMCs from SHR in vitro. Integrin receptor inhibitor of GRGDSP would delay this arterial ageing and calcification process. Moreover, the elevated FN and α5 integrin receptor expression evoked the disequilibrium of BMP2/MGP, where the expression of BMP2, a potent osteogenic inducer, increased while MGP, a calcification inhibitor, decreased. Furthermore, it was followed by the upregulation of Runx2 and the phenotypic transformation of VSMCs from the contractile phenotype into the osteoblast-like cells. Notably, BMP2 antagonist of rmNoggin was sufficient to ameliorate the ageing and calcification process of VSMCs and exogenous BMP2-adding accelerate and aggregate the process. CONCLUSION: Our study revealed that hypertension-associated arterial ageing and calcification might be a consequence that hypertension up-regulated FN and its high binding affinity integrin α5 receptor in the aortic wall, which in turn aggravated the imbalance of BMP2/MGP, promoted the transcription of Runx2, and induced the phenotypic transformation of VSMCs from the contractile phenotype into the osteoblast-like cells. Our study would provide insights into hypertension-associated arterial ageing and calcification and shed new light on the control of arterial calcification, especially for those with hypertension.

Loss of Endothelial Annexin A1 Aggravates Inflammation-Induched Vascular Aging.

Chronic inflammation is increasingly considered as the most important component of vascular aging, contributing to the progression of age-related cardiovascular diseases. To delay the process of vascular aging, anti-inflammation may be an effective measure. The anti-inflammatory factor annexin A1 (ANXA1) is shown to participate in several age-related diseases; however, its function during vascular aging remains unclear. Here, an ANXA1 knockout (ANXA1-/-) and an endothelial cell-specific ANXA1 deletion mouse (ANXA1△EC) model are used to investigate the role of ANXA1 in vascular aging. ANXA1 depletion exacerbates vascular remodeling and dysfunction while upregulates age- and inflammation-related protein expression. Conversely, Ac2-26 (a mimetic peptide of ANXA1) supplementation reverses this phenomenon. Furthermore, long-term tumor necrosis factor-alpha (TNF-α) induction of human umbilical vein endothelial cells (HUVECs) increases cell senescence. Finally, the senescence-associated secretory phenotype and senescence-related protein expression, rates of senescence-ß-galactosidase positivity, cell cycle arrest, cell migration, and tube formation ability are observed in both ANXA1-knockdown HUVECs and overexpressed ANXA1-TNF-α induced senescent HUVECs. They also explore the impact of formyl peptide receptor 2 (a receptor of ANXA1) in an ANXA1 overexpression inflammatory model. These data provide compelling evidence that age-related inflammation in arteries contributes to senescent endothelial cells that promote vascular aging.

Adropin inhibits the progression of atherosclerosis in ApoE-/-/Enho-/- mice by regulating endothelial-to-mesenchymal transition.

Adropin, a secreted protein, coded by energy homeostasis-associated gene (Enho), is recently reported to modulate atherogenesis, with endothelial-to-mesenchymal transition (EndMT) involved in the early process. We explored whether adropin may alleviate atherosclerosis by regulating EndMT. We found that an intraperitoneal injection of adropin [105 µg/(kg·d) for 13 weeks] inhibited the progression of high-fat diet (HFD)-induced aortic atherosclerosis in apolipoprotein E-deficient mice (ApoE-/-) and those with double gene deletion (ApoE-/-/Enho-/-), as detected by Oil Red O and haematoxylin-eosin staining. In the aortas of ApoE-/- mouse, adropin treatment ameliorated the decrease in the mRNA expression of endothelial cell markers (leukocyte differentiation antigen 31, CD31, and vascular endothelial cadherin, VE-cadherin), but increased that of EndMT markers (alpha smooth muscle actin, α-SMA, and fibroblasts specific protein-1). In vitro, an adropin treatment (30 ng/ml) arrested the hydrogen peroxide (H2O2)-induced EndMT in human umbilical vein endothelial cells (HUVECs), attenuated the morphological changes of HUVECs, reduced the number of immunofluorescence-positive α-SMA, increased the mRNA and protein expressions of CD31 and VE-cadherin, and decreased those of α-SMA. Furthermore, the adropin treatment decreased the mRNA and protein expressions of transforming growth factor (TGF)-ß1 and TGF-ß2, and suppressed the phosphorylation of downstream signal protein Smad2/3 in HUVECs. These mitigative effects of adropin on H2O2-induced EndMT were reversed by the transfection of TGF-ß plasmid. The findings signify that adropin treatment may alleviate the atherosclerosis in ApoE-/-/Enho-/- mice by inhibiting EndMT via the TGF-ß/Smad2/3 signaling pathway.

p38 and ERK1/2-Dependent Activation of c-Jun Is Required for the Downregulation of Oxidative Stress-Induced ERα in Hypothalamic Astrocytes.

INTRODUCTION: Gonadotropin-releasing hormone (GnRH) is a hypothalamic neuropeptide that plays important roles in the female fertility. Accumulating evidence suggests that ERα present in the astrocytes of the hypothalamus region is essential for production of GnRH. The astrocytes display age-related senescence associated to oxidative stress induced by the estrogen metabolites. However, it is still unclear whether and how ERα expression changes during astrocyte aging. METHODS: Immunofluorescence was performed to analyze the ERα gene levels in hypothalamic astrocytes of naturally aging C57BL/6J female mice. We employed an oxidative stress cell model receiving 2-hydroxyestradiol (2OH-E2) intervention to confirm the downregulation of ERα expression in primary astrocytes. Western blot analysis was used to explore which oxidative stress signaling pathways induced loss of the ERα gene. Finally, ChIP-qPCR was employed to evaluate whether the c-Jun protein is able to regulate ERα gene expression. RESULTS: Compared to young mice, we found that the ERα expression of mid-aged mice was significantly decreased. In hypothalamic astrocytes, 2OH-E2 treatment significantly reduced the expression of the ERα gene. Moreover, we observed that transcription factor c-Jun could directly inhibit transcriptional ERα gene expression and might also reduce it by decreasing H3K27 acetylation at promoter regions. Administration of the antioxidants Rg1 and astaxanthin significantly attenuated the decrease in ERα gene expression induced by oxidative stress. CONCLUSIONS: The current data demonstrate that oxidative stress leads to loss of ERα involving the activation of the p38 and ERK1/2 pathways and the induction of the c-Jun protein in hypothalamic astrocytes. C-Jun protein regulates ERα gene expression via direct transcriptional repression or involving histone acetylation modifications at ERα gene promoter sites.

HDL and Lipid Metabolism.

Mediating reverse cholesterol transport (RCT) is the most classic function of HDL. HDL and HDL-C participate in the entire process of RCT, including cholesterol removal from cells, cholesterol transport in circulation, and cholesterol excretion. As cholesterol is a component of lipid rafts and lipid droplets in cells, HDL and RCT can influence cell activity. HDL has also been shown to be related to the metabolism of some other biological lipids, such as S1P and ox-PL. Here we will introduce in detail the molecular mechanism of HDL participation in RCT and its significance.

Single-Cell RNA Sequencing and Assay for Transposase-Accessible Chromatin Using Sequencing Reveals Cellular and Molecular Dynamics of Aortic Aging in Mice.

OBJECTIVE: The impact of vascular aging on cardiovascular diseases has been extensively studied; however, little is known regarding the cellular and molecular mechanisms underlying age-related vascular aging in aortic cellular subpopulations. Approach and Results: Transcriptomes and transposase-accessible chromatin profiles from the aortas of 4-, 26-, and 86-week-old C57/BL6J mice were analyzed using single-cell RNA sequencing and assay for transposase-accessible chromatin sequencing. By integrating the heterogeneous transcriptome and chromatin accessibility data, we identified cell-specific TF (transcription factor) regulatory networks and open chromatin states. We also determined that aortic aging affects cell interactions, inflammation, cell type composition, dysregulation of transcriptional control, and chromatin accessibility. Endothelial cells 1 have higher gene set activity related to cellular senescence and aging than do endothelial cells 2. Moreover, construction of senescence trajectories shows that endothelial cell 1 and fibroblast senescence is associated with distinct TF open chromatin states and an mRNA expression model. CONCLUSIONS: Our data provide a system-wide model for transcriptional and epigenetic regulation during aortic aging at single-cell resolution.

CX3C-chemokine receptor 1 modulates cognitive dysfunction induced by sleep deprivation.

BACKGROUND: Microglia plays an indispensable role in the pathological process of sleep deprivation (SD). Here, the potential role of microglial CX3C-chemokine receptor 1 (CX3CR1) in modulating the cognition decline during SD was evaluated in terms of microglial neuroinflammation and synaptic pruning. In this study, we aimed to investigat whether the interference in the microglial function by the CX3CR1 knockout affects the CNS's response to SD. METHODS: Middle-aged wild-type (WT) C57BL/6 and CX3CR1-/- mice were either subjected to SD or allowed normal sleep (S) for 8 h to mimic the pathophysiological changes of middle-aged people after staying up all night. After which, behavioral and histological tests were used to explore their different changes. RESULTS: CX3CR1 deficiency prevented SD-induced cognitive impairments, unlike WT groups. Compared with the CX3CR1-/- S group, the CX3CR1-/- SD mice reported a markedly decreased microglia and cellular oncogene fos density in the dentate gyrus (DG), decreased expression of pro-inflammatory cytokines, and decreased microglial phagocytosis-related factors, whereas increased levels of anti-inflammatory cytokines in the hippocampus and a significant increase in the density of spines of the DG were also noted. CONCLUSIONS: These findings suggest that CX3CR1 deficiency leads to different cerebral behaviors and responses to SD. The inflammation-attenuating activity and the related modification of synaptic pruning are possible mechanism candidates, which indicate CX3CR1 as a candidate therapeutic target for the prevention of the sleep loss-induced cognitive impairments.

Development and Validation of a Prediction Model for Elevated Arterial Stiffness in Chinese Patients With Diabetes Using Machine Learning.

BACKGROUND: Arterial stiffness assessed by pulse wave velocity is a major risk factor for cardiovascular diseases. The incidence of cardiovascular events remains high in diabetics. However, a clinical prediction model for elevated arterial stiffness using machine learning to identify subjects consequently at higher risk remains to be developed. METHODS: Least absolute shrinkage and selection operator and support vector machine-recursive feature elimination were used for feature selection. Four machine learning algorithms were used to construct a prediction model, and their performance was compared based on the area under the receiver operating characteristic curve metric in a discovery dataset (n = 760). The model with the best performance was selected and validated in an independent dataset (n = 912) from the Dryad Digital Repository (https://doi.org/10.5061/dryad.m484p). To apply our model to clinical practice, we built a free and user-friendly web online tool. RESULTS: The predictive model includes the predictors: age, systolic blood pressure, diastolic blood pressure, and body mass index. In the discovery cohort, the gradient boosting-based model outperformed other methods in the elevated arterial stiffness prediction. In the validation cohort, the gradient boosting model showed a good discrimination capacity. A cutoff value of 0.46 for the elevated arterial stiffness risk score in the gradient boosting model resulted in a good specificity (0.813 in the discovery data and 0.761 in the validation data) and sensitivity (0.875 and 0.738, respectively) trade-off points. CONCLUSION: The gradient boosting-based prediction system presents a good classification in elevated arterial stiffness prediction. The web online tool makes our gradient boosting-based model easily accessible for further clinical studies and utilization.

Adropin-based dual treatment enhances the therapeutic potential of mesenchymal stem cells in rat myocardial infarction.

Both weak survival ability of stem cells and hostile microenvironment are dual dilemma for cell therapy. Adropin, a bioactive substance, has been demonstrated to be cytoprotective. We therefore hypothesized that adropin may produce dual protective effects on the therapeutic potential of stem cells in myocardial infarction by employing an adropin-based dual treatment of promoting stem cell survival in vitro and modifying microenvironment in vivo. In the current study, adropin (25 ng/ml) in vitro reduced hydrogen peroxide-induced apoptosis in rat bone marrow mesenchymal stem cells (MSCs) and improved MSCs survival with increased phosphorylation of Akt and extracellular regulated protein kinases (ERK) l/2. Adropin-induced cytoprotection was blocked by the inhibitors of Akt and ERK1/2. The left main coronary artery of rats was ligated for 3 or 28 days to induce myocardial infarction. Bromodeoxyuridine (BrdU)-labeled MSCs, which were in vitro pretreated with adropin, were in vivo intramyocardially injected after ischemia, following an intravenous injection of 0.2 mg/kg adropin (dual treatment). Compared with MSCs transplantation alone, the dual treatment with adropin reported a higher level of interleukin-10, a lower level of tumor necrosis factor-α and interleukin-1ß in plasma at day 3, and higher left ventricular ejection fraction and expression of paracrine factors at day 28, with less myocardial fibrosis and higher capillary density, and produced more surviving BrdU-positive cells at day 3 and 28. In conclusion, our data evidence that adropin-based dual treatment may enhance the therapeutic potential of MSCs to repair myocardium through paracrine mechanism via the pro-survival pathways.

Hypertension accelerates age-related intrarenal small artery (IRSA) remodelling and stiffness in rats with possible involvement of AGEs and RAGE.

OBJECTIVES: To study changes in morphology, advanced glycation end products (AGEs) and the AGEs receptor, RAGE, that occur with ageing in intrarenal small arteries (IRSAs) of spontaneously hypertensive rats (SHRs) and to investigate the possible roles of hypertension, AGEs and RAGE in the progression of IRSA remodelling and stiffness with ageing in rats. METHODS: Ageing SHRs and ageing normotensive Wistar Kyoto (WKY) rats were studied. The minimal renal vascular resistance (minRVR) was measured. Renal arcuate arteries (RAAs) and interlobular arteries (RILAs), the expression of α-smooth muscle actin, proliferating cell nuclear antigen, AGEs, RAGE and the plasma concentrations of AGEs were also examined. RESULTS: The IRSA minRVR, wall thickening, cell proliferation and collagen deposition in RILAs and RAAs gradually increased with age in SHRs and were much higher in 24-week-old SHRs than in age-matched WKY rats (p<0.05); these indexes in WKY rats were only elevated in the 72-week group (p<0.05). The expression of RAGE in the RAA and RILA tunica media in SHRs was upregulated by 24 weeks and 12 weeks (p<0.05), respectively, while AGEs levels in the plasma and in the IRSA tunica media were increased by 48 weeks (p<0.05) and increased gradually with age. The levels of both RAGE and AGEs in WKY rats were increased only at 72 weeks (p<0.05). CONCLUSION: Hypertension accelerates the development of age-related IRSA remodelling and stiffness in rats, which may be related to upregulation of RAGE in the IRSA tunica media and increased expression of AGEs at the late stage.

Trimethylamine-N-oxide promotes brain aging and cognitive impairment in mice.

Gut microbiota can influence the aging process and may modulate aging-related changes in cognitive function. Trimethylamine-N-oxide (TMAO), a metabolite of intestinal flora, has been shown to be closely associated with cardiovascular disease and other diseases. However, the relationship between TMAO and aging, especially brain aging, has not been fully elucidated. To explore the relationship between TMAO and brain aging, we analysed the plasma levels of TMAO in both humans and mice and administered exogenous TMAO to 24-week-old senescence-accelerated prone mouse strain 8 (SAMP8) and age-matched senescence-accelerated mouse resistant 1 (SAMR1) mice for 16 weeks. We found that the plasma levels of TMAO increased in both the elderly and the aged mice. Compared with SAMR1-control mice, SAMP8-control mice exhibited a brain aging phenotype characterized by more senescent cells in the hippocampal CA3 region and cognitive dysfunction. Surprisingly, TMAO treatment increased the number of senescent cells, which were primarily neurons, and enhanced the mitochondrial impairments and superoxide production. Moreover, we observed that TMAO treatment increased synaptic damage and reduced the expression levels of synaptic plasticity-related proteins by inhibiting the mTOR signalling pathway, which induces and aggravates aging-related cognitive dysfunction in SAMR1 and SAMP8 mice, respectively. Our findings suggested that TMAO could induce brain aging and age-related cognitive dysfunction in SAMR1 mice and aggravate the cerebral aging process of SAMP8 mice, which might provide new insight into the effects of intestinal microbiota on the brain aging process and help to delay senescence by regulating intestinal flora metabolites.

Ageing-related aorta remodelling and calcification occur earlier and progress more severely in rats with spontaneous hypertension.

The effects of hypertension on vascular remodelling, ageing and calcification are not fully understood. In this study, we monitored the dynamic changes of aorta remodelling, senescence and calcification in spontaneously hypertensive rats (SHRs) during ageing. RESULTS: Vascular remodelling and senescence cells occurred in SHR aortas at 24 weeks. The calcium content and calcium deposition of the aorta increased in SHRs at 48 weeks. All of these changes became increasingly significant with ageing. In contrast, these pathologic changes appeared in Wistar-Kyoto (WKY) normotensive rats at a much later stage (72 weeks). These data showed that the ageing-related aorta remodelling, senescence and calcification in SHRs occurred earlier and progressed more severely than in WKY rats. CONCLUSION: Ageing-related vascular remodelling and calcification were accelerated and augmented in SHR aortas.

Gut flora-dependent metabolite Trimethylamine-N-oxide accelerates endothelial cell senescence and vascular aging through oxidative stress.

Trimethylamine-N-oxide (TMAO), gut microbiota-dependent metabolites, has been shown to be associated with cardiovascular diseases. However, little is known about the relationship between TMAO and vascular aging. Here, we observed a change in TMAO during the aging process and the effects of TMAO on vascular aging and endothelial cell (EC) senescence. We analyzed age-related plasma levels of TMAO in young adults (18-44 years old), older adults (≥ 65 years old), and 1-month-old, 3-month-old, 6-month-old and 10-month-old senescence-accelerated mouse prone 8 (SAMP8) and age-matched senescence-accelerated mouse resistance 1 (SAMR1) models. We found that circulating TMAO increased with age both in humans and mice. Next, we observed that a TMAO treatment for 16 weeks induced vascular aging in SAMR1 mice and accelerated the process in SAMP8 mice, as measured by an upregulation of senescence markers including senescence-associated ß-galactosidase (SA-ß-gal), p53, and p21, vascular dysfunction and remodeling. In vitro, we demonstrated that prolonged TMAO treatment induced senescence in human umbilical vein endothelial cells (HUVECs), characterized by reduced cell proliferation, increased expressions of senescence markers, stagnate G0/G1, and impaired cell migration. Furthermore, TMAO suppressed sirtuin 1 (SIRT1) expression and increased oxidative stress both in vivo and in vitro and then activated the p53/p21/Rb pathway resulting in increased p53, acetylation of p53, p21, and decreased CDK2, cyclinE1, and phosphorylation of Rb. In summary, these data suggest that elevated circulating TMAO during the aging process may deteriorate EC senescence and vascular aging, which is probably associated with repression of SIRT1 expression and increased oxidative stress, and, thus, the activation of the p53/p21/Rb pathway.