Fluoride impairs vascular smooth muscle A7R5 cell lines via disrupting amino acids metabolism.

Given the insidious and high-fatality nature of cardiovascular diseases (CVDs), the emergence of fluoride as a newly identified risk factor demands serious consideration alongside traditional risk factors. While vascular smooth muscle cells (VSMCs) play a pivotal role in the progression of CVDs, the toxicological impact of fluoride on VSMCs remains largely uncharted. In this study, we constructed fluorosis model in SD rats and A7R5 aortic smooth muscle cell lines to confirm fluoride impaired VSMCs. Fluoride aggravated the pathological damage of rat aorta in vivo. Then A7R5 were exposed to fluoride with concentration ranging from 0 to 1200 µmol/L over a 24-h period, revealing a dose-dependent inhibition of cell proliferation and migration. The further metabolomic analysis showed alterations in metabolite profiles induced by fluoride exposure, notably decreasing organic acids and lipid molecules level. Additionally, gene network analysis underscored the frequency of fluoride's interference with amino acids metabolism, potentially impacting the tricarboxylic acid (TCA) cycle. Our results also highlighted the ATP-binding cassette (ABC) transporters pathway as a central element in VSMC impairment. Moreover, we observed a dose-dependent increase in osteopontin (OPN) and α-smooth muscle actin (α-SMA) mRNA level and a dose-dependent decrease in ABC subfamily C member 1 (ABCC1) and bestrophin 1 (BEST1) mRNA level. These findings advance our understanding of fluoride as a CVD risk factor and its influence on VSMCs and metabolic pathways, warranting further investigation into this emerging risk factor.

Nur77 induced by HIF-1α mediates vascular remodeling in hypoxic pulmonary hypertension.

Nur77 is a member of the NR4A subfamily of orphan nuclear receptors that is expressed and has a function within the immune system. This study aimed to investigate the role of Nur77 in hypoxic pulmonary hypertension. SPF male SD rats were exposed in hypobaric chamber simulating 5000 m high altitude for 0, 3, 7, 14, 21 or 28 days. Rat pulmonary artery smooth muscle cells (RPASMCs) were cultured under normoxic conditions (5% CO2-95% ambient air) or hypoxic conditions (5% O2 for 6 h, 12 h, 24 h, 48 h). Hypoxic rats developed pulmonary arterial remodeling and right ventricular hypertrophy with significantly increased pulmonary arterial pressure. The levels of Nur77, HIF-1α and PNCA were upregulated in pulmonary arterial smooth muscle from hypoxic rats. Silencing of either Nur77 or HIF-1α attenuated hypoxia-induced proliferation. Silencing of HIF-1α down-regulated Nur77 protein level, but Nur77 silence did not reduce HIF-1α. Nur77 was not con-immunoprecipitated with HIF-1α. This study demonstrated that Nur77 acted as a downstream regulator of HIF-1α under hypoxia, and plays a critical role in the hypoxia-induced pulmonary vascular remodeling, which is regulated by HIF-1α. Nur77 maybe a novel target of HPH therapy.

Elevated plasma levels of factor VIII enhance arterial thrombus formation on erosive smooth muscle cell-rich neointima but not normal intima in rabbits.

BACKGROUND: Plaque erosion, a type of coronary atherothrombosis, involves superficial injury to smooth muscle cell (SMC)-rich plaques. Elevated levels of coagulation factor VIII (FVIII) correlate with an increased ischemic heart disease risk. FVIII may contribute to thrombus formation on eroded plaques. AIMS: We aimed to elucidate the role of elevated FVIII in arterial thrombus formation within SMC-rich neointima in rabbits. METHODS AND RESULTS: We assessed the effect of recombinant human FVIII (rFVIII) on blood coagulation in vitro and platelet aggregation ex vivo. An SMC-rich neointima was induced through balloon injury to the unilateral femoral artery. Three weeks after the first balloon injury, superficial erosive injury and thrombus formation were initiated with a second balloon injury of the bilateral femoral arteries 45 min after the administration of rFVIII (100 IU/kg) or saline. The thrombus area and contents were histologically measured 15 min after the second balloon injury. rFVIII administration reduced the activated partial thromboplastin time and augmented botrocetin-induced, but not collagen- or adenosine 5'-diphosphate-induced, platelet aggregation. While rFVIII did not influence platelet-thrombus formation in normal intima, it increased thrombus formation on SMC-rich neointima post-superficial erosive injury. Enhanced immunopositivity for glycoprotein IIb/IIIa and fibrin was observed in rFVIII-administered SMC-rich neointima. Neutrophil count in the arterial thrombus on the SMC-rich neointima correlated positively with thrombus size in the control group, unlike the rFVIII group. CONCLUSIONS: Increased FVIII contributes to thrombus propagation within erosive SMC-rich neointima, highlighting FVIII's potential role in plaque erosion-related atherothrombosis.

Exosomes enriched by miR-429-3p derived from ITGB1 modified Telocytes alleviates hypoxia-induced pulmonary arterial hypertension through regulating Rac1 expression.

BACKGROUND: Recent studies have emphasized the critical role of Telocytes (TCs)-derived exosomes in organ tissue injury and repair. Our previous research showed a significant increase in ITGB1 within TCs. Pulmonary Arterial Hypertension (PAH) is marked by a loss of microvessel regeneration and progressive vascular remodeling. This study aims to investigate whether exosomes derived from ITGB1-modified TCs (ITGB1-Exo) could mitigate PAH. METHODS: We analyzed differentially expressed microRNAs (DEmiRs) in TCs using Affymetrix Genechip miRNA 4.0 arrays. Exosomes isolated from TC culture supernatants were verified through transmission electron microscopy and Nanoparticle Tracking Analysis. The impact of miR-429-3p-enriched exosomes (Exo-ITGB1) on hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs) was evaluated using CCK-8, transwell assay, and inflammatory factor analysis. A four-week hypoxia-induced mouse model of PAH was constructed, and H&E staining, along with Immunofluorescence staining, were employed to assess PAH progression. RESULTS: Forty-five miRNAs exhibited significant differential expression in TCs following ITGB1 knockdown. Mus-miR-429-3p, significantly upregulated in ITGB1-overexpressing TCs and in ITGB1-modified TC-derived exosomes, was selected for further investigation. Exo-ITGB1 notably inhibited the migration, proliferation, and inflammation of PASMCs by targeting Rac1. Overexpressing Rac1 partly counteracted Exo-ITGB1's effects. In vivo administration of Exo-ITGB1 effectively reduced pulmonary vascular remodeling and inflammation. CONCLUSIONS: Our findings reveal that ITGB1-modified TC-derived exosomes exert anti-inflammatory effects and reverse vascular remodeling through the miR-429-3p/Rac1 axis. This provides potential therapeutic strategies for PAH treatment.

Inhibition of Slc39a14/Slc39a8 reduce vascular calcification via alleviating iron overload induced ferroptosis in vascular smooth muscle cells.

BACKGROUND: Vascular calcification (VC) is an independent risk factor for cardiovascular diseases. Recently, ferroptosis has been recognised as a novel therapeutic target for cardiovascular diseases. Although an association between ferroptosis and vascular calcification has been reported, the role and mechanism of iron overload in vascular calcification are still poorly understood. Specifically, further in-depth research is required on whether metalloproteins SLC39a14 and SLC39a8 are involved in ferroptosis induced by iron overload. METHODS: R language was employed for the differential analysis of the dataset, revealing the correlation between ferroptosis and calcification. The experimental approaches encompassed both in vitro and in vivo studies, incorporating the use of iron chelators and models of iron overload. Additionally, gain- and loss-of-function experiments were conducted to investigate iron's effects on vascular calcification comprehensively. Electron microscopy, immunofluorescence, western blotting, and real-time polymerase chain reaction were used to elucidate how Slc39a14 and Slc39a8 mediate iron overload and promote calcification. RESULTS: Ferroptosis was observed in conjunction with vascular calcification (VC); the association was consistently confirmed by in vitro and in vivo studies. Our results showed a positive correlation between iron overload in VSMCs and calcification. Iron chelators are effective in reversing VC and iron overload exacerbates this process. The expression levels of the metal transport proteins Slc39a14 and Slc39a8 were significantly upregulated during calcification; the inhibition of their expression alleviated VC. Conversely, Slc39a14 overexpression exacerbates calcification and promotes intracellular iron accumulation in VSMCs. CONCLUSIONS: Our research demonstrates that iron overload occurs during VC, and that inhibition of Slc39a14 and Slc39a8 significantly relieves VC by intercepting iron overload-induced ferroptosis in VSMCs, providing new insights into the VC treatment.

Expression of glucocorticoid receptor and HDACs in airway smooth muscle cells is associated with response to steroids in COPD.

BACKGROUND: Steroid insensitivity in Chronic Obstructive Pulmonary Disease (COPD) presents a problem for controlling the chronic inflammation of the airways. The glucocorticoid receptor (GR) mediates the intracellular signaling of inhaled corticosteroids (ICS) by interacting with transcription factors and histone deacetylases (HDACs). The aim of this study was to assess if COPD patients' response to ICS in vivo, may be associated with the expression of GR, the complex of GR with transcription factors, and the expression of various HDACs in vitro. METHODS: Primary airway smooth muscle cells (ASMC) were established from endobronchial biopsies obtained from patients with asthma (n = 10), patients with COPD (n = 10) and subjects that underwent diagnostic bronchoscopy without pathological findings and served as controls (n = 6). ASMC were also established from 18 COPD patients, 10 responders and 8 non-responders to ICS, who participated in the HISTORIC study, an investigator-initiated and driven clinical trial that proved the hypothesis that COPD patients with high ASMC in their endobronchial biopsies respond better to ICS than patients with low ASMC. Expression of GR and its isoforms GRα and GRß and HDACs was investigated in primary ASMC in the absence or in the presence of dexamethasone (10- 8M) by western blotting. The complex formation of GR with transcription factors was assessed by co-immunoprecipitation. RESULTS: Expression of GR and its isoform GRα but not GRß was significantly reduced in ASMC from COPD patients as compared to controls. There were no significant differences in the expression of GR, GRα and GRß between responders and non-responders to ICS. However, treatment with dexamethasone upregulated the expression of total GR (p = 0.004) and GRα (p = 0.005) after 30 min in responders but not in non-responders. Τhe formation of the complex GR-c-Jun was increased 60 min after treatment with dexamethasone only in responders who exhibited significantly lower expression of HDAC3 (p = 0.005) and HDAC5 (p < 0.0001) as compared to non-responders. CONCLUSIONS: These data suggest that ASMC from COPD patients who do not respond to treatment with ICS, are characterized by reduced GR-c-Jun complex formation and increased expression of HDAC3 and HDAC5. TRIAL REGISTRATION: ISRCTN11017699 (Registration date: 15/11/2016).

Single-Cell Transcriptome Analysis Reveals Dynamic Populations of Vascular Cells in Neointimal Hyperplasia.

BACKGROUND: Neointimal hyperplasia (NIH) is the pathological basis of vascular injury disease. Vascular cells are the dominant cells in the process of NIH, but the extent of heterogeneity amongst them is still unclear. METHODS: A mouse model of NIH was constructed by inducing carotid artery ligation. Single-cell sequencing was then used to analyze the transcriptional profile of vascular cells. Cluster features were determined by functional enrichment analysis, gene set scoring, pseudo-time analysis, and cell-cell communication analysis. Additionally, immunofluorescence staining was conducted on vascular tissues from fibroblast lineage-traced (PdgfraDreER-tdTomato) mice to validate the presence of Pecam1+Pdgfra+tdTomato+ cells. RESULTS: The left carotid arteries (ligation) were compared to right carotid arteries (sham) from ligation-induced NIH C57BL/6 mice. Integrative analyses revealed a high level of heterogeneity amongst vascular cells, including fourteen clusters and seven cell types. We focused on three dominant cell types: endothelial cells (ECs), vascular smooth muscle cells (vSMCs), and fibroblasts. The major findings were: (1) four subpopulations of ECs, including ECs4, mesenchymal-like ECs (ECs1 and ECs2), and fibro-like ECs (ECs3); (2) four subpopulations of fibroblasts, including pro-inflammatory Fibs-1, Sca1+ Fibs-2, collagen-producing Fibs-3, and mesenchymal-like Fibs-4; (3) four subpopulations of vSMCs, including vSMCs-1, vSMCs-2, vSMCs-3, and vSMCs-3-derived vSMCs; (4) ECs3 express genes related to extracellular matrix (ECM) remodeling and cell migration, and fibro-like vSMCs showed strong chemokine secretion and relatively high levels of proteases; (5) fibro-like vSMCs that secrete Vegfa interact with ECs mainly through vascular endothelial growth factor receptor 2 (Vegfr2). CONCLUSIONS: This study presents the dynamic cellular landscape within NIH arteries and reveals potential relationships between several clusters, with a specific focus on ECs3 and fibro-like vSMCs. These two subpopulations may represent potential target cells for the treatment of NIH.

Divergent and Compensatory Effects of BMP2 and BMP4 on the VSMC Phenotype and BMP4's Role in Thoracic Aortic Aneurysm Development.

Vascular smooth muscle cells (VSMCs) play a key role in aortic aneurysm formation. Bone morphogenetic proteins (BMPs) have been implicated as important regulators of VSMC phenotype, and dysregulation of the BMP pathway has been shown to be associated with vascular diseases. The aim of this study was to investigate for the first time the effects of BMP-4 on the VSMC phenotype and to understand its role in the development of thoracic aortic aneurysms (TAAs). Using the angiotensin II (AngII) osmotic pump model in mice, aortas from mice with VSMC-specific BMP-4 deficiency showed changes similar to AngII-infused aortas, characterised by a loss of contractile markers, increased fibrosis, and activation of matrix metalloproteinase 9. When BMP-4 deficiency was combined with AngII infusion, there was a significantly higher rate of apoptosis and aortic dilatation. In vitro, VSMCs with mRNA silencing of BMP-4 displayed a dedifferentiated phenotype with activated canonical BMP signalling. In contrast, BMP-2-deficient VSMCs exhibited the opposite phenotype. The compensatory regulation between BMP-2 and BMP-4, with BMP-4 promoting the contractile phenotype, appeared to be independent of the canonical signalling pathway. Taken together, these results demonstrate the impact of VSMC-specific BMP-4 deficiency on TAA development.

A comparative study of smooth muscle cell characteristics and myofibroblasts in processus vaginalis of pediatric inguinal hernia, hydrocele and undescended testis.

BACKGROUND: Congenital inguinal hernia, hydrocele and undescended testis (UDT) are associated with patent processus vaginalis. The smooth muscles present in the processus vaginalis aid in the descent of the testis and undergo programmed cell death after testicular descent leading to obliteration. The persisting amount of smooth muscle in the processus vaginalis influences the clinical outcome as inguinal hernia, hydrocele or UDT. Therefore, a study was conducted to evaluate the processus vaginalis in these three conditions to observe the presence and phenotype of smooth muscle cells and the presence of myofibroblasts. MATERIALS AND METHODS: The processus vaginalis sacs in patients with inguinal hernia, hydrocele and UDT were examined using light microscopy for the presence and distribution of smooth muscle cells and immunohistochemical staining for vimentin, desmin, and α-smooth muscle actin (SMA) to identify the smooth muscle phenotype. Transmission electron microscopy was also performed in all the sacs to observe the presence of myofibroblasts. RESULTS: Seventy-eight specimens of processus vaginalis (from seventy-four patients), distributed as 47%, 27%, and 26% as inguinal hernia, hydrocele and UDT respectively, were included in the study. The sacs from inguinal hernia and hydrocele had significantly more presence of smooth muscles distributed as multiple smooth muscle bundles (p < 0.001). Desmin and SMA staining of smooth muscle cells was observed in significantly more sacs from hydrocele, followed by inguinal hernia and UDT (p < 0.001). The sacs from UDT had a significant presence of striated muscles (p = 0.028). The sacs from inguinal hernia had a significant presence of myofibroblasts, followed by hydrocele and UDT (p < 0.001) and this significantly correlated with the light microscopy and immunohistochemical features. The processus vaginalis sacs from four female patients did not differ statistically from the male inguinal hernia sacs in any of the above parameters. CONCLUSION: The processus vaginalis sacs in pediatric inguinal hernia, hydrocele and undescended testis differ in the presence, distribution and phenotype of smooth muscles and the presence of myofibroblasts. The clinical presentations in these entities reflect these differences.

Unveiling cellular and molecular aspects of ascending thoracic aortic aneurysms and dissections.

Ascending thoracic aortic aneurysm (ATAA) remains a significant medical concern, with its asymptomatic nature posing diagnostic and monitoring challenges, thereby increasing the risk of aortic wall dissection and rupture. Current management of aortic repair relies on an aortic diameter threshold. However, this approach underestimates the complexity of aortic wall disease due to important knowledge gaps in understanding its underlying pathologic mechanisms.Since traditional risk factors cannot explain the initiation and progression of ATAA leading to dissection, local vascular factors such as extracellular matrix (ECM) and vascular smooth muscle cells (VSMCs) might harbor targets for early diagnosis and intervention. Derived from diverse embryonic lineages, VSMCs exhibit varied responses to genetic abnormalities that regulate their contractility. The transition of VSMCs into different phenotypes is an adaptive response to stress stimuli such as hemodynamic changes resulting from cardiovascular disease, aging, lifestyle, and genetic predisposition. Upon longer exposure to stress stimuli, VSMC phenotypic switching can instigate pathologic remodeling that contributes to the pathogenesis of ATAA.This review aims to illuminate the current understanding of cellular and molecular characteristics associated with ATAA and dissection, emphasizing the need for a more nuanced comprehension of the impaired ECM-VSMC network.

Bioengineered vascular grafts with a pathogenic TGFBR1 variant model aneurysm formation in vivo and reveal underlying collagen defects.

Thoracic aortic aneurysm (TAA) is a life-threatening vascular disease frequently associated with underlying genetic causes. An inadequate understanding of human TAA pathogenesis highlights the need for better disease models. Here, we established a functional human TAA model in an animal host by combining human induced pluripotent stem cells (hiPSCs), bioengineered vascular grafts (BVGs), and gene editing. We generated BVGs from isogenic control hiPSC-derived vascular smooth muscle cells (SMCs) and mutant SMCs gene-edited to carry a Loeys-Dietz syndrome (LDS)-associated pathogenic variant (TGFBR1A230T). We also generated hiPSC-derived BVGs using cells from a patient with LDS (PatientA230T/+) and using genetically corrected cells (Patient+/+). Control and experimental BVGs were then implanted into the common carotid arteries of nude rats. The TGFBR1A230T variant led to impaired mechanical properties of BVGs, resulting in lower burst pressure and suture retention strength. BVGs carrying the variant dilated over time in vivo, resembling human TAA formation. Spatial transcriptomics profiling revealed defective expression of extracellular matrix (ECM) formation genes in PatientA230T/+ BVGs compared with Patient+/+ BVGs. Histological analysis and protein assays validated quantitative and qualitative ECM defects in PatientA230T/+ BVGs and patient tissue, including decreased collagen hydroxylation. SMC organization was also impaired in PatientA230T/+ BVGs as confirmed by vascular contraction testing. Silencing of collagen-modifying enzymes with small interfering RNAs reduced collagen proline hydroxylation in SMC-derived tissue constructs. These studies demonstrated the utility of BVGs to model human TAA formation in an animal host and highlighted the role of reduced collagen modifying enzyme activity in human TAA formation.

Pentamethylquercetin attenuates angiotensin II-induced abdominal aortic aneurysm formation by blocking nuclear translocation of C/EBPß at Lys253.

BACKGROUND: Pentamethylquercetin (PMQ) is a natural polymethyl flavonoid that possesses anti-apoptotic and other biological properties. Abdominal aortic aneurysm (AAA), a fatal vascular disease with a high risk of rupture, is associated with phenotypic switching and apoptosis of medial vascular smooth muscle cells (VSMCs). This study aimed to investigate the protective effects of PMQ on the development of AAA and the underlying mechanism. METHODS: ApoE-/- mice were continuously infused with angiotensin II (Ang II) for 4 weeks to develop the AAA model. Intragastric administration of PMQ was initiated 5 days before Ang II infusion and continued for 4 weeks. In vitro, VSMCs were cultured and pretreated with PMQ, stimulated with Ang II. Real-time PCR, western blotting, and immunofluorescence staining were used to examine the roles and mechanisms of PMQ on the phenotypic switching and apoptosis of VSMCs. RESULTS: PMQ dose-dependently reduced the incidence of Ang II-induced AAA, aneurysm diameter enlargement, elastin degradation, VSMCs phenotypic switching and apoptosis. Furthermore, PMQ also inhibited phenotypic switching and apoptosis in Ang II-stimulated VSMCs. PMQ exerted protective effects by regulating the C/EBPß/PTEN/AKT/GSK-3ß axis. AAV-mediated overexpression of PTEN reduced the therapeutic effects of PMQ in the AAA model mice, suggesting that the effects of PMQ on Ang II-mediated AAA formation were related to the PTEN/AKT/GSK-3ß axis. PMQ inhibited VSMCs phenotypic switching and apoptosis by bounding to C/EBPß at Lys253 with hydrogen bond to regulate C/EBPß nuclear translocation and PTEN/AKT/GSK-3ß axis, thereby inhibiting Ang II-induced AAA formation. CONCLUSIONS: Pentamethylquercetin inhibits angiotensin II-induced abdominal aortic aneurysm formation by bounding to C/EBPß at Lys253. Therefore, PMQ prevents the formation of AAA and reduces the incidence of AAA.

LncRNA-LncDACH1 mediated phenotypic switching of smooth muscle cells during neointimal hyperplasia in male arteriovenous fistulas.

Arteriovenous fistulas (AVFs) are the most common vascular access points for hemodialysis (HD), but they have a high incidence of postoperative dysfunction, mainly due to excessive neointimal hyperplasia (NIH). Our previous studies have revealed a highly conserved LncRNA-LncDACH1 as an important regulator of cardiomyocyte and fibroblast proliferation. Herein, we find that LncDACH1 regulates NIH in AVF in male mice with conditional knockout of smooth muscle cell-specific LncDACH1 and in male mice model of AVF with LncDACH1 overexpression by adeno-associated virus. Mechanistically, silence of LncDACH1 activates p-AKT through promoting the expression of heat shock protein 90 (HSP90) and serine/arginine-rich splicing factor protein kinase 1 (SRPK1). Moreover, LncDACH1 is transcriptionally activated by transcription factor KLF9 that binds directly to the promoter region of the LncDACH1 gene. In this work, during AVF NIH, LncDACH1 is downregulated by KLF9 and promotes NIH through the HSP90/ SRPK1/ AKT signaling axis.

Linarin Ameliorates Restenosis After Vascular Injury in Type 2 Diabetes Mellitus via Regulating ADAM10-Mediated Notch Signaling Pathway.

Vascular lesions frequently arise as complication in patients diagnosed with diabetes mellitus (DM). Presently, percutaneous coronary intervention (PCI) and antithrombotic therapy serve as primary treatments. However, in-stent restenosis persists as a challenging clinical issue following PCI, lacking sustained and effective treatment. Linarin (LN) exhibits diverse pharmacological activities and is regarded as a potential drug for treating various diseases, including DM. But its specific role in restenosis after vascular injury in DM patients remains unclear. A rat model of diabetes-related restenosis was established to evaluate the role of LN on neointimal hyperplasia. Vascular smooth muscle cells (VSMCs) stimulated by high glucose (HG, 30 mM) underwent LN treatment. Additionally, an overexpression plasmid of A disintegrin and metalloproteinases (ADAM10) was constructed to transfect VSMCs. We employed CCK-8, Brdu, wound-healing scratch, and transwell migration assays to evaluate the proliferation and migration of VSMCs. Furthermore, western blot and immunofluorescence assays were utilized to investigate the expressions of ADAM10 and the downstream Notch signaling pathway in vivo and in vitro models. LN notably alleviated intimal hyperplasia after vascular injury in DM rats and reduced the protein expression of ADAM10, alongside its downstream Notch1 signaling pathway-related proteins (Notch1, NICD and Hes1) in rat carotid artery tissues. LN effectively suppressed the proliferation and migration of VSMCs induced by HG, downregulating the protein expression of ADAM10, Notch1, NICD and Hes1. Moreover, our findings indicated that ADAM10 overexpression significantly reversed LN's effects on proliferation, migration, and the expression of Notch1 signaling pathway-related proteins in HG-treated VSMCs. LN demonstrates potential therapeutic efficacy in addressing restenosis after diabetic-related vascular injury, with the ADAM10 mediated Notch signaling pathway playing a pivotal role.

IL-1ß Inhibition Partially Negates the Beneficial Effects of Diet-Induced Atherosclerosis Regression in Mice.

BACKGROUND: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. METHODS: Smooth muscle cell lineage-tracing Apoe-/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1ß or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis. RESULTS: Lipid lowering by switching Apoe-/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1ß antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1ß antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap. CONCLUSIONS: Taken together, IL-1ß appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

BAG3 promotes proliferation and migration of arterial smooth muscle cells by regulating STAT3 phosphorylation in diabetic vascular remodeling.

BACKGROUND: Diabetic vascular remodeling is the most important pathological basis of diabetic cardiovascular complications. The accumulation of advanced glycation end products (AGEs) caused by elevated blood glucose promotes the proliferation and migration of vascular smooth muscle cells (VSMCs), leading to arterial wall thickening and ultimately vascular remodeling. Therefore, the excessive proliferation and migration of VSMCs is considered as an important therapeutic target for vascular remodeling in diabetes mellitus. However, due to the lack of breakthrough in experiments, there is currently no effective treatment for the excessive proliferation and migration of VSMCs in diabetic patients. Bcl-2-associated athanogene 3 (BAG3) protein is a multifunctional protein highly expressed in skeletal muscle and myocardium. Previous research has confirmed that BAG3 can not only regulate cell survival and apoptosis, but also affect cell proliferation and migration. Since the excessive proliferation and migration of VSMCs is an important pathogenesis of vascular remodeling in diabetes, the role of BAG3 in the excessive proliferation and migration of VSMCs and its molecular mechanism deserve further investigation. METHODS: In this study, BAG3 gene was manipulated in smooth muscle to acquire SM22αCre; BAG3FL/FL mice and streptozotocin (STZ) was used to simulate diabetes. Expression of proteins and aortic thickness of mice were detected by immunofluorescence, ultrasound and hematoxylin-eosin (HE) staining. Using human aorta smooth muscle cell line (HASMC), cell viability was measured by CCK-8 and proliferation was measured by colony formation experiment. Migration was detected by transwell, scratch experiments and Phalloidin staining. Western Blot was used to detect protein expression and Co-Immunoprecipitation (Co-IP) was used to detect protein interaction. RESULTS: In diabetic vascular remodeling, AGEs could promote the interaction between BAG3 and signal transducer and activator of transcription 3 (STAT3), leading to the enhanced interaction between STAT3 and Janus kinase 2 (JAK2) and reduced interaction between STAT3 and extracellular signal-regulated kinase 1/2 (ERK1/2), resulting in accumulated p-STAT3(705) and reduced p-STAT3(727). Subsequently, the expression of matrix metallopeptidase 2 (MMP2) is upregulated, thus promoting the migration of VSMCs. CONCLUSIONS: BAG3 upregulates the expression of MMP2 by increasing p-STAT3(705) and decreasing p-STAT3(727) levels, thereby promoting vascular remodeling in diabetes. This provides a new orientation for the prevention and treatment of diabetic vascular remodeling.

Neutrophil extracellular traps promote proliferation of pulmonary smooth muscle cells mediated by CCDC25 in pulmonary arterial hypertension.

BACKGROUND: Previous studies have indicated that neutrophil extracellular traps (NETs) play a pivotal role in pathogenesis of pulmonary arterial hypertension (PAH). However, the specific mechanism underlying the impact of NETs on pulmonary artery smooth muscle cells (PASMCs) has not been determined. The objective of this study was to elucidate underlying mechanisms through which NETs contribute to progression of PAH. METHODS: Bioinformatics analysis was employed in this study to screen for potential molecules and mechanisms associated with occurrence and development of PAH. These findings were subsequently validated in human samples, coiled-coil domain containing 25 (CCDC25) knockdown PASMCs, as well as monocrotaline-induced PAH rat model. RESULTS: NETs promoted proliferation of PASMCs, thereby facilitating pathogenesis of PAH. This phenomenon was mediated by the activation of transmembrane receptor CCDC25 on PASMCs, which subsequently activated ILK/ß-parvin/RAC1 pathway. Consequently, cytoskeletal remodeling and phenotypic transformation occur in PASMCs. Furthermore, the level of NETs could serve as an indicator of PAH severity and as potential therapeutic target for alleviating PAH. CONCLUSION: This study elucidated the involvement of NETs in pathogenesis of PAH through their influence on the function of PASMCs, thereby highlighting their potential as promising targets for the evaluation and treatment of PAH.

Curcumol reduces lower limb arteriosclerosis in rats by inhibiting human arterial smooth muscle cell activity.

Cardiovascular diseases, particularly those involving arterial stenosis and smooth muscle cell proliferation, pose significant health risks. This study aimed to investigate the therapeutic potential of curcumol in inhibiting platelet-derived growth factor-BB (PDGF-BB)-induced human aortic smooth muscle cell (HASMC) proliferation, migration and autophagy. Using cell viability assays, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays and Western Blot analyses, we observed that curcumol effectively attenuated PDGF-BB-induced HASMC proliferation and migration in a concentration-dependent manner. Furthermore, curcumol mitigated PDGF-BB-induced autophagy, as evidenced by the downregulation of LC3-II/LC3-I ratio and upregulation of P62. In vivo experiments using an arteriosclerosis obliterans model demonstrated that curcumol treatment significantly ameliorated arterial morphology and reduced stenosis. Additionally, curcumol inhibited the activity of the KLF5/COX2 axis, a key pathway in vascular diseases. These findings suggest that curcumol has the potential to serve as a multi-target therapeutic agent for vascular diseases.

Spatiotemporal ATF3 Expression Determines VSMC Fate in Abdominal Aortic Aneurysm.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy, likely due to the limited understanding of the mechanisms underlying AAA development and progression. ATF3 (activating transcription factor 3) has been increasingly recognized as a key regulator of cardiovascular diseases. However, the role of ATF3 in AAA development and progression remains elusive. METHODS: Genome-wide RNA sequencing analysis was performed on the aorta isolated from saline or Ang II (angiotensin II)-induced AAA mice, and ATF3 was identified as the potential key gene for AAA development. To examine the role of ATF3 in AAA development, vascular smooth muscle cell-specific ATF3 knockdown or overexpressed mice by recombinant adeno-associated virus serotype 9 vectors carrying ATF3, or shRNA-ATF3 with SM22α (smooth muscle protein 22-α) promoter were used in Ang II-induced AAA mice. In human and murine vascular smooth muscle cells, gain or loss of function experiments were performed to investigate the role of ATF3 in vascular smooth muscle cell proliferation and apoptosis. RESULTS: In both Ang II-induced AAA mice and patients with AAA, the expression of ATF3 was reduced in aneurysm tissues but increased in aortic lesion tissues. The deficiency of ATF3 in vascular smooth muscle cell promoted AAA formation in Ang II-induced AAA mice. PDGFRB (platelet-derived growth factor receptor ß) was identified as the target of ATF3, which mediated vascular smooth muscle cell proliferation in response to TNF-alpha (tumor necrosis factor-α) at the early stage of AAA. ATF3 suppressed the mitochondria-dependent apoptosis at the advanced stage by upregulating its direct target BCL2. Our chromatin immunoprecipitation results also demonstrated that the recruitment of NFκB1 and P300/BAF/H3K27ac complex to the ATF3 promoter induces ATF3 transcription via enhancer activation. NFKB1 inhibitor (andrographolide) inhibits the expression of ATF3 by blocking the recruiters NFKB1 and ATF3-enhancer to the ATF3-promoter region, ultimately leading to AAA development. CONCLUSIONS: Our results demonstrate a previously unrecognized role of ATF3 in AAA development and progression, and ATF3 may serve as a novel therapeutic and prognostic marker for AAA.

Single-Cell Gene-Regulatory Networks of Advanced Symptomatic Atherosclerosis.

BACKGROUND: While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remains poorly understood. METHODS: Single-cell RNA sequencing data generated with SmartSeq2 (≈8000 genes/cell) in 16 588 single cells isolated during atherosclerosis progression in Ldlr-/-Apob100/100 mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease patients in the STARNET (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task) study. RESULTS: Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by 3 smooth muscle cells (SMCs), and 3 macrophage subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type proinflammatory/Trem2-high lipid-associated (macrophage) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of 3 arterial wall GRNs: GRN33 (macrophage), GRN39 (SMC), and GRN122 (macrophage) with major contributions to coronary artery disease heritability and strong associations with clinical scores of coronary atherosclerosis severity. The presence and pathophysiological relevance of GRN39 were verified in 5 independent RNAseq data sets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM in cultured human coronary artery SMCs. CONCLUSIONS: By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a coronary artery disease framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced, symptomatic atherosclerosis.