Vascular Smooth Muscle Cells In Culture Research Articles

Circulating miR-6821-5p levels and coronary calcification in asymptomatic familial hypercholesterolemia patients

Background and aimsPremature atherosclerotic cardiovascular disease (CVD) is a clinic characteristic of familial hypercholesterolemia (FH). Coronary calcium score (CCS) is a highly used imaging modality to evidence atherosclerotic plaque burden. microRNAs (miRNAs) are non-coding RNAs that epigenetically regulate gene expression. Here, we investigated whether CCS associates with a specific miRNA-signature in FH-patients. MethodsPatients with genetic diagnosis of FH (N = 86) from the nationwide SAFEHEART-cohort were investigated by computed tomography angiography imaging and classified depending on the presence of coronary calcification in FH-CCS (+) and FH-CCS (−) groups by the Agatston score. Differential miRNA profiling was performed in two stages: first by Affymetrix microarray technology (high-throughput differential profiling-studies) and second by RT-PCR using TaqMan-technology (analytical RT-qPCR study) in plasma of the two patient groups. ResultsmiR-193a-5p, miR-30e-5p and miR-6821-5p levels were significantly higher in FH-CCS (+) compared to FH-CCS (−). miR-6821-5p was the best miRNA to discriminate FH-patients CCS(+), according to receiver operating characteristic (ROC) analysis (AUC: 0.70 ± 0.06, p = 0.006). High miR-6821-5p levels were associated with older age (p = 0.03) and high LDL-burden (p = 0.014) using a ROC-derived cut-off value. However, miR-6821-5p did not correlate with age in either the CCS- or CCS + group. Genes involved in calcification processes were identified by in silico analysis. The relation of cell-calcification and expression levels of miR-6821-5p, BMP2 and SPP1 was validated experimentally in human vascular smooth muscle cell cultures. ConclusionsUp-regulated levels of miR-6821-5p are found in the plasma of asymptomatic FH-patients with coronary calcified atherosclerotic plaques, as well as in isolated human vascular smooth muscle cells expressing the pro-calcification genes BMP2 and SPP1. These findings highlight the impact of epigenetic regulation on the development of subclinical atherosclerosis.

Vasculoprotective Potential of Baicalein in Angiotensin II-Infused Abdominal Aortic Aneurysms through Inhibiting Inflammation and Oxidative Stress.

Aortic wall inflammation, abnormal oxidative stress and progressive degradation of extracellular matrix proteins are the main characteristics of abdominal aortic aneurysms (AAAs). The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome dysregulation plays a crucial role in aortic damage and disease progression. The first aim of this study was to examine the effect of baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) on AAA formation in apolipoprotein E-deficient (ApoE-/-) mice. The second aim was to define whether baicalein attenuates aberrant vascular smooth muscle cell (VSMC) proliferation and inflammation in VSMC culture. For male ApoE-/- mice, a clinically relevant AAA model was randomly divided into four groups: saline infusion, baicalein intraperitoneal injection, Angiotensin II (Ang II) infusion and Ang II + baicalein. Twenty-seven days of treatment with baicalein markedly decreased Ang II-infused AAA incidence and aortic diameter, reduced collagen-fiber formation, preserved elastic structure and density and prevented smooth muscle cell contractile protein degradation. Baicalein inhibited rat VSMC proliferation and migration following the stimulation of VSMC cultures with Ang II while blocking the Ang II-inducible cell cycle progression from G0/G1 to the S phase in the synchronized cells. Cal-520 AM staining showed that baicalein decreased cellular calcium in Ang II-induced VSMCs; furthermore, a Western blot assay indicated that baicalein inhibited the expression of PCNA and significantly lowered levels of phospho-Akt and phospho-ERK, along with an increase in baicalein concentration in Ang II-induced VSMCs. Immunofluorescence staining showed that baicalein pretreatment reduced NF-κB nuclear translocation in Ang II-induced VSMCs and furthered the protein expressions of NLRP3 while ASC and caspase-1 were suppressed in a dose-dependent manner. Baicalein pretreatment upregulated Nrf2/HO-1 signaling in Ang II-induced VSMCs. Thus, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining showed that its reactive oxygen species (ROS) production decreased, along with the baicalein pretreatment. Our overall results indicate that baicalein could have therapeutic potential in preventing aneurysm development.

ER stress induced immunopathology involving complement in CADASIL: implications for therapeutics

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by NOTCH3 mutations. Typical CADASIL is characterised by subcortical ischemic strokes due to severe arteriopathy and fibrotic thickening of small arteries. Arteriolar vascular smooth muscle cells (VSMCs) are the key target in CADASIL, but the potential mechanisms involved in their degeneration are still unclear. Focusing on cerebral microvessels in the frontal and anterior temporal lobes and the basal ganglia, we used advanced proteomic and immunohistochemical methods to explore the extent of inflammatory and immune responses in CADASIL subjects compared to similar age normal and other disease controls. There was variable loss of VSMC in medial layers of arteries in white matter as well as the cortex, that could not be distinguished whether NOTCH3 mutations were in the epidermal growth factor (EGFr) domains 1–6 or EGFr7-34. Proteomics of isolated cerebral microvessels showed alterations in several proteins, many associated with endoplasmic reticulum (ER) stress including heat shock proteins. Cerebral vessels with sparsely populated VSMCs also attracted robust accrual of perivascular microglia/macrophages in order CD45+ > CD163+ > CD68+cells, with > 60% of vessel walls exhibiting intercellular adhesion molecule-1 (ICAM-1) immunoreactivity. Functional VSMC cultures bearing the NOTCH3 Arg133Cys mutation showed increased gene expression of the pro-inflammatory cytokine interleukin 6 and ICAM-1 by 16- and 50-fold, respectively. We further found evidence for activation of the alternative pathway of complement. Immunolocalisation of complement Factor B, C3d and C5-9 terminal complex but not C1q was apparent in ~ 70% of cerebral vessels. Increased complement expression was corroborated in > 70% of cultured VSMCs bearing the Arg133Cys mutation independent of N3ECD immunoreactivity. Our observations suggest that ER stress and other cellular features associated with arteriolar VSMC damage instigate robust localized inflammatory and immune responses in CADASIL. Our study has important implications for immunomodulation approaches to counter the characteristic arteriopathy of CADASIL.

Epidermal growth factor receptor inhibition prevents vascular calcifying extracellular vesicle biogenesis.

Chronic kidney disease (CKD) increases the risk of cardiovascular disease, including vascular calcification, leading to higher mortality. The release of calcifying extracellular vesicles (EVs) by vascular smooth muscle cells (VSMCs) promotes ectopic mineralization of vessel walls. Caveolin-1 (CAV1), a structural protein in the plasma membrane, plays a major role in calcifying EV biogenesis in VSMCs. Epidermal growth factor receptor (EGFR) colocalizes with and influences the intracellular trafficking of CAV1. Using a diet-induced mouse model of CKD followed by a high-phosphate diet to promote vascular calcification, we assessed the potential of EGFR inhibition to prevent vascular calcification. Furthermore, we computationally analyzed 7,651 individuals in the Multi-Ethnic Study of Atherosclerosis (MESA) and Framingham cohorts to assess potential correlations between coronary artery calcium and single-nucleotide polymorphisms (SNPs) associated with elevated serum levels of EGFR. Mice with CKD developed widespread vascular calcification, associated with increased serum levels of EGFR. In both the CKD mice and human VSMC culture, EGFR inhibition significantly reduced vascular calcification by mitigating the release of CAV1-positive calcifying EVs. EGFR inhibition also increased bone mineral density in CKD mice. Individuals in the MESA and Framingham cohorts with SNPs associated with increased serum EGFR exhibit elevated coronary artery calcium. Given that EGFR inhibitors exhibit clinical safety and efficacy in other pathologies, the current data suggest that EGFR may represent an ideal target to prevent pathological vascular calcification in CKD.NEW & NOTEWORTHY Here, we investigate the potential of epidermal growth factor receptor (EGFR) inhibition to prevent vascular calcification, a leading indicator of and contributor to cardiovascular morbidity and mortality. EGFR interacts and affects the trafficking of the plasma membrane scaffolding protein caveolin-1. Previous studies reported a key role for caveolin-1 in the development of specialized extracellular vesicles that mediate vascular calcification; however, no role of EGFR has been reported. We demonstrated that EGFR inhibition modulates caveolin-1 trafficking and hinders calcifying extracellular vesicle formation, which prevents vascular calcification. Given that EGFR inhibitors are clinically approved for other indications, this may represent a novel therapeutic strategy for vascular calcification.

Circulating Extracellular Vesicle-Propagated microRNA Signature as a Vascular Calcification Factor in Chronic Kidney Disease.

Chronic kidney disease (CKD) accelerates vascular calcification via phenotypic switching of vascular smooth muscle cells (VSMCs). We investigated the roles of circulating small extracellular vesicles (sEVs) between the kidneys and VSMCs and uncovered relevant sEV-propagated microRNAs (miRNAs) and their biological signaling pathways. We established CKD models in rats and mice by adenine-induced tubulointerstitial fibrosis. Cultures of A10 embryonic rat VSMCs showed increased calcification and transcription of osterix (Sp7), osteocalcin (Bglap), and osteopontin (Spp1) when treated with rat CKD serum. sEVs, but not sEV-depleted serum, accelerated calcification in VSMCs. Intraperitoneal administration of a neutral sphingomyelinase and biogenesis/release inhibitor of sEVs, GW4869 (2.5 mg/kg per 2 days), inhibited thoracic aortic calcification in CKD mice under a high-phosphorus diet. GW4869 induced a nearly full recovery of calcification and transcription of osteogenic marker genes. In CKD, the miRNA transcriptome of sEVs revealed a depletion of 4 miRNAs, miR-16-5p, miR-17~92 cluster-originated miR-17-5p/miR-20a-5p, and miR-106b-5p. Their expression decreased in sEVs from CKD patients as kidney function deteriorated. Transfection of VSMCs with each miRNA-mimic mitigated calcification. In silico analyses revealed VEGFA (vascular endothelial growth factor A) as a convergent target of these miRNAs. We found a 16-fold increase in VEGFA transcription in the thoracic aorta of CKD mice under a high-phosphorus diet, which GW4869 reversed. Inhibition of VEGFA-VEGFR2 signaling with sorafenib, fruquintinib, sunitinib, or VEGFR2-targeted siRNA mitigated calcification in VSMCs. Orally administered fruquintinib (2.5 mg/kg per day) for 4 weeks suppressed the transcription of osteogenic marker genes in the mouse aorta. The area under the curve of miR-16-5p, miR-17-5p, 20a-5p, and miR-106b-5p for the prediction of abdominal aortic calcification was 0.7630, 0.7704, 0.7407, and 0.7704, respectively. The miRNA transcriptomic signature of circulating sEVs uncovered their pathologic role, devoid of the calcification-protective miRNAs that target VEGFA signaling in CKD-driven vascular calcification. These sEV-propagated miRNAs are potential biomarkers and therapeutic targets for vascular calcification.

Mitochondrial Dysfunction and Increased DNA Damage in Vascular Smooth Muscle Cells of Abdominal Aortic Aneurysm (AAA-SMC).

There is increasing evidence for enhanced oxidative stress in the vascular wall of abdominal aortic aneurysms (AAA). Mitochondrial damage and dysfunction are hypothesized to be actors in altered production of reactive oxygen species (ROS) and oxidative stress. However, the role of mitochondria and oxidative stress in vascular remodelling and progression of AAA remains uncertain. We here addressed whether mitochondrial dysfunction is persistently increased in vascular smooth muscle cells (VSMCs) isolated from AAA compared to healthy VSMC. AAA-derived VSMC cultures (AAA-SMC, n = 10) and normal VSMC cultures derived from healthy donors (n = 7) were grown in vitro and analysed for four parameters, indicating mitochondrial dysfunction: (i) mitochondrial content and morphology, (ii) ROS production and antioxidative response, (iii) NADP+/NADPH content and ratio, and (iv) DNA damage, in the presence or absence of angiotensin II (AngII). AAA-SMC displayed increased mitochondrial circularity (rounded shape), reduced mitochondrial area, and reduced perimeter, indicating increased fragmentation and dysfunction compared to healthy controls. This was accompanied by significantly increased O2 - production, reduced NADP+/NADPH levels, a lower antioxidative response (indicated by antioxidative response element- (ARE-) driven luciferase reporter assays), more DNA damage (determined by percentage of γ-H2A.X-positive nuclei), and earlier growth arrest in AAA-SMC. Our data suggest that mitochondrial dysfunction and oxidative stress are persistently increased in AAA-SMC, emphasizing their implication in the pathophysiology of AAA.

The Effects of Angiotensin II, Olmesartan and PD123,319 on Protein Kinase C in STZ Induced Rat Vascular Smooth Muscle Cell Culture

Protein kinase C (PKC) is a promoter enzyme that plays a vital role in signal transduction of vascular smooth muscle cells (VSMCs). It has numerous vascular functions, such as vascular cell growth, cytokine activation and angiogenesis. But, these mechanisms are deteriorating in diabetes mellitus. Angiotensin II (Ang II) effects on vascular structure binding Ang II type-1 and type-2 receptors (ATR1 and ATR2) and stimulates PKC mostly through ATR1s. We aim to investigate the effects of Ang II, Olmesartan and PD123,319 on PKC levels of healthy and streptozotocin (STZ) induced rat VSMCs (H-VSMCs and STZ-VSMCs) in this study. The primary culture of VSMCs were isolated from thoracic aorta of healthy and STZ (45 mg/kg, given via the tail vein) induced Wistar rats. PKC levels were measured in H-VSMCs and STZ-VSMCs by enzyme-linked immunosorbent assay (ELISA). In H-VSMCs, Ang II group compared to the control group, PKC levels decreased significantly (P = 0.000); whereas, Ang II group compared to control, PKC levels were higher, but not significantly in STZ-VSMCs (P = 0.088). PKC levels were increased in Ang II+Olmesartan (P = 0.000) and Ang II+PD123,319 (P = 0.000) groups compared to Ang II group in H-VSMCs, but in STZ-VSMCs, PKC levels in Ang II+Olmesartan (P = 0.001) and Ang II+PD123,319 (P = 0.000) groups compared to Ang II group were decreased significantly. Ang II, its receptors and PKC seem to modulate each other and may have a relationship in hyperglycemic conditions. Also, considering that, ATR1 blocker (ATR1B) Olmesartan and ATR2 blocker (ATR2B) PD123,319 may be protective against vascular injury by reducing PKC levels in STZ-VSMCs.

Tracing G-Protein-Mediated Contraction and Relaxation in Vascular Smooth Muscle Cell Spheroids.

Analyses of G-protein-mediated contraction and relaxation of vascular smooth muscle cells (VSMCs) are usually hampered by a rigid growth surface and culture conditions promoting cell proliferation and a less contractile phenotype. Our studies indicated that mouse aortic VSMCs cultured in three-dimensional spheroids acquire a quiescent contractile status while decreasing the baseline G-protein-dependent inositolphosphate formation and increasing the expression of endothelin receptor type A (Ednra). Endothelin-1 (ET-1) promoted inositolphosphate formation in VSMC spheroids, but not in VSMCs cultured under standard conditions. To trace ET-1-mediated contraction of VSMC spheroids, we developed an assay by adhering them to collagen hydrogels and recording structural changes by time-lapse microscopy. Under these conditions, mouse and human VSMC spheroids contracted upon treatment with ET-1 and potassium chloride or relaxed in response to caffeine and the prostacyclin analogue Iloprost. ET-1 activated AKT-, MKK1-, and MKK3/6-dependent signaling cascades, which were inhibited by an overexpressing regulator of G-protein signaling 5 (Rgs5) to terminate the activity of Gα subunits. In summary, culture of VSMCs in three-dimensional spheroids lowers baseline G-protein activity and enables analyses of both contraction and relaxation of mouse and human VSMCs. This model serves as a simple and versatile tool for drug testing and investigating G-protein-depending signaling.

Proteins secreted by brain arteriolar smooth muscle cells are instructive for neural development

Intercellular communication between vascular and nerve cells mediated by diffusible proteins has recently emerged as a critical intrinsic program for neural development. However, whether the vascular smooth muscle cell (VSMC) secretome regulates the connectivity of neural circuits remains unknown. Here, we show that conditioned medium from brain VSMC cultures enhances multiple neuronal functions, such as neuritogenesis, neuronal maturation, and survival, thereby improving circuit connectivity. However, protein denaturation by heating compromised these effects. Combined omics analyses of donor VSMC secretomes and recipient neuron transcriptomes revealed that overlapping pathways of extracellular matrix receptor signaling and adhesion molecule integrin binding mediate VSMC-dependent neuronal development. Furthermore, we found that human arterial VSMCs promote neuronal development in multiple ways, including expanding the time window for nascent neurite initiation, increasing neuronal density, and promoting synchronized firing, whereas human umbilical vein VSMCs lack this capability. These in vitro data indicate that brain arteriolar VSMCs may carry direct instructive information for neural development through intercellular communication in vivo.

Vascular response to stress: Protective action of the bisphosphonate alendronate.

Since several additional actions of bone bisphosphonates have been proposed, we studied the effect of the bisphosphonate alendronate (ALN) on the vascular response to environmental stress. Primary cultures of endothelial cells (EC) and vascular smooth muscle cells (VSMC) exposed to strained conditions were employed for experimental evaluation. After ALN treatment, cell migration, proliferation, and angiogenesis assays were performed. The participation of signal transduction pathways in the biochemical action of ALN was also assessed. In VSMC cultures, ALN counteracted the stimulation of cellular migration elicited by the proinflammatory agent lipopolysaccharide (LPS) or by high levels of calcium and phosphorus (osteogenic medium). Indeed, ALN reduced the increase of VSMC proliferation evoked by the stressors. When LPS and osteogenic medium were added simultaneously, the enhancement of cell proliferation dropped to control values in the presence of ALN. The mechanism of action of ALN involved the participation of nitric oxide synthase, mitogen-activated protein kinase (MAPK), and protein kinase C (PKC) signaling pathways. The study revealed that ALN exhibits a proangiogenic action. On EC, ALN enhanced vascular endothelial growth factor (VEGF) synthesis, and induced capillary-like tube formation in a VEGF-dependent manner. The presence of vascular stress conditions (LPS or osteogenic medium) did not modify the proangiogenic action elicited by ALN. The findings presented suggest an extra-bone biological action of ALN, which could contribute to the maintenance of vascular homeostasis avoiding cellular damage elicited by environmental stress.

Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4

Prevention of phenotype switching of vascular smooth muscle cells is an important determinant of normal vascular physiology. Hydrogen peroxide (H2O2) promotes osteogenic differentiation of vascular smooth muscle cells through expression of Runt related transcription factor 2 (Runx2). In this study, an increase in dietary NaCl increased endothelial H2O2 generation through NOX4, a NAD(P)H oxidase. The production of H2O2 was sufficient to increase Runx2, osteopontin and osteocalcin in adjacent vascular smooth muscle cells from control littermate mice but was inhibited in mice lacking endothelial Nox4. A vascular smooth muscle cell culture model confirmed the direct involvement of the activation of protein kinase B (Akt) with inactivation of FoxO1 and FoxO3a observed in the control mice on the high NaCl diet. The present study also showed a reduction of catalase activity in aortas during high NaCl intake. The findings demonstrated an interesting cell-cell communication in the vascular wall that was initiated with H2O2 production by endothelium and was regulated by dietary NaCl intake. A better understanding of how dietary salt intake alters vascular biology may improve treatment of vascular disease that involves activation of Runx2.

Vascular hyperacetylation is associated with vascular smooth muscle dysfunction in a rat model of non-obese type 2 diabetes

BackgroundAdvanced type 2 diabetes mellitus (T2DM) accelerates vascular smooth muscle cell (VSMC) dysfunction which contributes to the development of vasculopathy, associated with the highest degree of morbidity of T2DM. Lysine acetylation, a post-translational modification (PTM), has been associated with metabolic diseases and its complications. Whether levels of global lysine acetylation are altered in vasculature from advanced T2DM remains undetermined. We hypothesized that VSMC undergoes dysregulation in advanced T2DM which is associated with vascular hyperacetylation.MethodsAged male Goto Kakizaki (GK) rats, a non-obese murine model of T2DM, and age-matched male Wistar rats (control group) were used in this study. Thoracic aortas were isolated and examined for measurement of global levels of lysine acetylation, and vascular reactivity studies were conducted using a wire myograph. Direct arterial blood pressure was assessed by carotid catheterization. Cultured human VSMCs were used to investigate whether lysine acetylation participates in high glucose-induced reactive oxygen species (ROS), a crucial factor triggering diabetic vascular dysfunction.ResultsThe GK rats exhibited marked glucose intolerance as well as insulin resistance. Cardiovascular complications in GK rats were confirmed by elevated arterial blood pressure and reduced VSMC-dependent vasorelaxation. These complications were correlated with high levels of vascular global lysine acetylation. Human VSMC cultures incubated under high glucose conditions displayed elevated ROS levels and increased global lysine acetylation. Inhibition of hyperacetylation by garcinol, a lysine acetyltransferase and p300/CBP association factor (PCAF) inhibitor, reduced high glucose-induced ROS production in VSMC.ConclusionThis study provides evidence that vascular hyperacetylation is associated with VSMC dysfunction in advanced T2DM. Understanding lysine acetylation regulation in blood vessels from diabetics may provide insight into the mechanisms of diabetic vascular dysfunction, and opportunities for novel therapeutic approaches to treat diabetic vascular complications.

Stressor-Induced "Inflammaging" of Vascular Smooth Muscle Cells via Nlrp3-Mediated Pro-inflammatory Auto-Loop.

Calcification of the vessel wall as one structural pathology of aged vessels is associated with high cardiovascular mortality of elderly patients. Aging is linked to chronic sterile inflammation and high burden of reactive oxygen species (ROS), leading to activation of pattern recognition receptors (PRRs) such as Nlrp3 in vascular cells. The current study investigates the role of PRR activation in the calcification of vascular smooth muscle cells (VSMCs). Therefore, in vitro cell culture of primary rat VSMCs and ex vivo aortic stimulations were used to analyze osteogenic, senescence and inflammatory markers via real-time PCR, in situ RNA hybridization, Western Blot, photometric assays and histological staining. Induction of ROS and DNA-damage by doxorubicin induces a shift of VSMC phenotype toward the expression of osteogenic, senescence and inflammatory proteins. Induction of calcification is dependent on Nlrp3 activity. Il-1β as a downstream target of Nlrp3 induces the synthetic, pro-calcifying VSMC phenotype. Inhibition of PRR with subsequent reduction of chronic inflammation might be an interesting target for reduction of calcification of VSMCs, with subsequent reduction of cardiovascular mortality of patients suffering from vessel stiffness.

Cold-pressed oil from Citrus aurantifolia inhibits the proliferation of vascular smooth muscle cells via regulation of PI3K/MAPK signaling pathways.

Vascular occlusive disease is a chronic disease with significant morbidity and mortality. Although a variety of therapies and medications have been developed, the likelihood of disease re-emergence is high and this can be life-threatening. Based on a previous screening experiment related to vascular obstructive diseases using 34 types of essential oils, cold-pressed oil (CpO) from Citrus aurantifolia (lime) has been demonstrated to have the best effect for the inhibition of vascular smooth muscle cells (VSMCs) proliferation. The aim of the present study was to evaluate the effect of lime CpO on the pathological changes of VSMCs. To determine this, the effect of lime CpO on VSMC proliferation, a major cause of vascular disease, was investigated. To determine the safe concentration interval for toxicity of CpO during VSMC culture, a dilution of 1x10-5 was determined using Cell Counting Kit-8 assay, which was confirmed to be non-toxic using a lactate dehydrogenase assay. To examine the effect of lime CpO in cellular signaling pathways, changes in phosphorylation of both the PI3K/AKT/mTOR and extracellular signal-regulated MEK/ERK signaling pathways with serum were investigated. Furthermore, lime CpO with FBS also significantly decreased the expression levels of the cell cycle regulators cyclin D1 and proliferating cell nuclear antigen. Additionally, lime CpO with FBS significantly inhibited the sprouting of VSMCs in an ex vivo culture system. These results suggested that lime CpO inhibited the abnormal proliferation of VSMCs and can be developed as a nature-based therapeutic agent for obstructive vascular disease.

Abstract P218: Ume ( Prunus Mume ) Attenuates Er Stress And Inflammation In Rat Vascular Smooth Muscle Cells

Ume ( Prunus mume ), is a traditional and familiar food in Asian countries, and is most often pickled or used to make wine and juice concentrate. Recently, Ume is attracting attention as a health food. We previously reported that Ume attenuates the activation of EGFR-ERK cascade and ROS production induced by angiotensin II (Ang II) in vascular smooth muscle cells (VSMC) of thoracic aorta. However, how Ume works on vascular inflammation and ER stress still remains unknown. AngII may enhance inflamm-aging via ER stress contributing to arterial stiffness and hypertension.We used primary culture of rat VSMC for experiment. We obtained 5 fractions (methanol, hexane, dichloromethane, ethyl acetate, and water) extracted from Ume juice. VSMC were pretreated with each fraction for 30 minutes followed by Ang II or tumor necrosis factor (TNF)-α stimulation. Stimulation period was 24 and 48 hours for Ang II, 6 hours for TNF-α. As the preliminary screening, we tested the cell response and solubility of each fraction (n=1~2). Pretreatment with 500μg/mL hexane fraction (PMF-H) showed the tendency to decrease VCAM1 expression under Ang II condition. PMF-H was completely soluble in the media at the concentration of 150 μg/mL. Based on this result, we focused on 150μg/mL PMF-H. DMSO was used as vehicle. In Western blot analysis, PMF-H attenuates ATF6 expression under both AII (p=0.0351, n=5) and TNF-α (p=0.0199, n=5) condition compared to vehicle. VCAM1 expression was significantly enhanced by Ang II stimulation (p=0.0471, n=5), and attenuated by PMF-H. PMF-H also reduced VCAM1 expression under TNF-α stimulation compared to vehicle (p=0.0377, n=5). GRP78 and Crystallin AB did not show any significant change. ATF6 and VCAM1 are key modulators of ER stress and vascular inflammation that may lead to cellular aging. Our results indicate PMF-H attenuates ATF6 and VCAM1 induced by Ang II or TNF-α stimulation. Thus, identification of the active ingredient in hexane fraction of Ume is desired as it may constitute a good candidate for a supplement or drug for anti-inflamm-aging therapy.

Efficacy and limitations of senolysis in atherosclerosis.

AimsTraditional markers of cell senescence including p16, Lamin B1, and senescence-associated beta galactosidase (SAβG) suggest very high frequencies of senescent cells in atherosclerosis, while their removal via ‘senolysis’ has been reported to reduce atherogenesis. However, selective killing of a variety of different cell types can exacerbate atherosclerosis. We therefore examined the specificity of senescence markers in vascular smooth muscle cells (VSMCs) and the effects of genetic or pharmacological senolysis in atherosclerosis.Methods and resultsWe examined traditional senescence markers in human and mouse VSMCs in vitro, and in mouse atherosclerosis. p16 and SAβG increased and Lamin B1 decreased in replicative senescence and stress-induced premature senescence (SIPS) of cultured human VSMCs. In contrast, mouse VSMCs undergoing SIPS showed only modest p16 up-regulation, and proliferating mouse monocyte/macrophages also expressed p16 and SAβG. Single cell RNA-sequencing (scRNA-seq) of lineage-traced mice showed increased p16 expression in VSMC-derived cells in plaques vs. normal arteries, but p16 localized to Stem cell antigen-1 (Sca1)+ or macrophage-like populations. Activation of a p16-driven suicide gene to remove p16+ vessel wall- and/or bone marrow-derived cells increased apoptotic cells, but also induced inflammation and did not change plaque size or composition. In contrast, the senolytic ABT-263 selectively reduced senescent VSMCs in culture, and markedly reduced atherogenesis. However, ABT-263 did not reduce senescence markers in vivo, and significantly reduced monocyte and platelet counts and interleukin 6 as a marker of systemic inflammation.ConclusionsWe show that genetic and pharmacological senolysis have variable effects on atherosclerosis, and may promote inflammation and non-specific effects respectively. In addition, traditional markers of cell senescence such as p16 have significant limitations to identify and remove senescent cells in atherosclerosis, suggesting that senescence studies in atherosclerosis and new senolytic drugs require more specific and lineage-restricted markers before ascribing their effects entirely to senolysis.

Delivery of Cinnamic Aldehyde Antioxidant Response Activating nanoParticles (ARAPas) for Vascular Applications.

Selective delivery of nuclear factor erythroid 2-related factor 2 (Nrf2) activators to the injured vasculature at the time of vascular surgical intervention has the potential to attenuate oxidative stress and decrease vascular smooth muscle cell (VSMC) hyperproliferation and migration towards the inner vessel wall. To this end, we developed a nanoformulation of cinnamic aldehyde (CA), termed Antioxidant Response Activating nanoParticles (ARAPas), that can be readily loaded into macrophages ex vivo. The CA-ARAPas-macrophage system was used to study the effects of CA on VSMC in culture. CA was encapsulated into a pluronic micelle that was readily loaded into both murine and human macrophages. CA-ARAPas inhibits VSMC proliferation and migration, and activates Nrf2. Macrophage-mediated transfer of CA-ARAPas to VSMC is evident after 12 h, and Nrf2 activation is apparent after 24 h. This is the first report, to the best of our knowledge, of CA encapsulation in pluronic micelles for macrophage-mediated delivery studies. The results of this study highlight the feasibility of CA encapsulation and subsequent macrophage uptake for delivery of cargo into other pertinent cells, such as VSMC.

Differentiation of adipose tissue-derived stem cells towards vascular smooth muscle cells on modified poly(L-lactide) foils

The aim of our research was to study the behaviour of adipose tissue-derived stem cells (ADSCs) and vascular smooth muscle cells (VSMCs) on variously modified poly(L-lactide) (PLLA) foils, namely on pristine PLLA, plasma-treated PLLA, PLLA grafted with polyethylene glycol (PEG), PLLA grafted with dextran (Dex), and the tissue culture polystyrene (PS) control. On these materials, the ADSCs were biochemically differentiated towards VSMCs by a medium supplemented with TGFβ1, BMP4 and ascorbic acid (i.e. differentiation medium). ADSCs cultured in a non-differentiation medium were used as a negative control. Mature VSMCs cultured in both types of medium were used as a positive control. The impact of the variously modified PLLA foils and/or differences in the composition of the medium were studied with reference to cell adhesion, growth and differentiation. We observed similar adhesion and growth of ADSCs on all PLLA samples when they were cultured in the non-differentiation medium. The differentiation medium supported the expression of specific early, mid-term and/or late markers of differentiation (i.e. type I collagen, αSMA, calponin, smoothelin, and smooth muscle myosin heavy chain) in ADSCs on all tested samples. Moreover, ADSCs cultured in the differentiation medium revealed significant differences in cell growth among the samples that were similar to the differences observed in the cultures of VSMCs. The round morphology of the VSMCs indicated worse adhesion to pristine PLLA, and this sample was also characterized by the lowest cell proliferation. Culturing VSMCs in the differentiation medium inhibited their metabolic activity and reduced the cell numbers. Both cell types formed the most stable monolayer on plasma-treated PLLA and on the PS control. The behaviour of ADSCs and VSMCs on the tested PLLA foils differed according to the specific cell type and culture conditions. The suitable biocompatibility of both cell types on the tested PLLA foils seems to be favourable for vascular tissue engineering purposes.

Vitamin D Promotes Trophoblast Cell Induced Separation of Vascular Smooth Muscle Cells in Vascular Remodeling via Induction of G-CSF.

Vitamin D deficiency is associated with complications of pregnancy such as pre-eclampsia, fetal growth restriction, and miscarriage, all of which are also associated with incomplete spiral artery (SpA) remodeling. We have previously shown that both uterine natural killer (uNK) cells and extravillous trophoblast cells (EVT) are required for successful SpA remodeling, but whether their activity in this process is modulated by vitamin D is not known. In the current study, we use a previously described chorionic plate artery (CPA) ex vivo model of vascular remodeling to determine the effects of 1,25(OH)2D treated uNK cell, placental explant (PEx), and uNK/PEx conditioned medium (CM) on vascular smooth muscle cell (VSMC) disorganization and phenotypic switching. Significant results were followed up in VSMCs in vitro. We demonstrate that 1,25(OH)2D can enhance the ability of PEx to induce SpA remodeling, via a mechanism associated with increased secretion of granulocyte-colony stimulating factor (G-CSF). G-CSF appears able to increase VSMC disorganization and phenotypic switching in both an ex vivo vascular model and in vitro VSMC cultures. The clinical relevance of these findings are still to be determined. G-CSF may have differential effects depending on dose and vascular bed, and vitamin D may play a role in potentiating these actions. G-CSF may be an interesting potential therapeutic target for facilitating physiological vascular remodeling for the prevention of adverse obstetric outcomes.

Heat Shock Protein 70 Is Associated With Cardioversion Outcome and Recurrence of Symptomatic Recent Onset Atrial Fibrillation in Hypertensive Patients.

Accumulating evidence indicates that heat shock proteins (HSPs) may represent a suitable biomarker to predict atrial fibrillation (AF). We investigated the relation of circulating serum HSP70 (sHSP70) with inflammatory cytokines and recurrence of symptomatic recent onset AF (ROAF). We enrolled 90 patients with ROAF (the duration from onset of symptoms ≤24 hours) and 30 controls. Patients received amiodarone for cardioversion and rhythm control. The association of serum HSP70, serum interleukin-2 (sIL-2), and serum interleukin-4 (sIL-4) with the presence of cardioversion and AF recurrence within a year was investigated. Toll-like receptor 4 (TLR4) signaling dependence for IL-2 and IL-4 induction in response to stimulation with HSP70 was tested in rat aortic vascular smooth muscle cell cultures. Patients had higher sHSP70 and sIL-2 and lower sIL-4 compared with controls. Serum HSP70 was independently associated with ROAF (P = 0.005) and correlated with sIL-2 (r = 0.494, P < 0.001) and sIL-4 (r = -0.550, P < 0.001). By 48 hours, 71 of the 90 patients were cardioverted, with noncardioverted patients having higher sHSP70 and sIL-2 and lower sIL-4, which were the only independent factors associated with cardioversion. AF recurred in 38 of the 71 cardioverted patients in 1 year. A cutoff value of sHSP70 ≥0.65 ng/mL and sIL-2 ≥0.21 pg/mL was the only independent factor associated with AF recurrence (hazard ratio: 3.311, 95% confidence interval: 1.503-7.293, P = 0.003 and hazard ratio: 3.144, 95% confidence interval: 1.341-7.374, P = 0.008, respectively). The exposure of smooth muscle cell to HSP70 in vitro increased the expression of IL-2 (5×) and IL-4 (1.5×) through TLR4-dependent and receptor-independent mechanisms. In conclusion, sHSP70 and sIL-2 might constitute a prognostic tool for determining the cardioversion and recurrence likelihood in ROAF.