Muscle Cells Research Articles

Integrating Network Pharmacology and Experimental Verification to Explore the Pharmacological Mechanisms of Cordycepin against Pulmonary Arterial Hypertension in Rats.

Pulmonary Arterial Hypertension (PAH) is a fatal disease with high morbidity and mortality. Cordycepin has anti-inflammatory, antioxidant and immune enhancing effects. However, the role of Cordycepin in the treatment of PAH and its mechanism is not clear. The Cordycepin structure and PAH-related gene targets were obtained from public databases. The KEGG and GO enrichment analysis of common targets was performed in DAVID. PPI networks were also mapped using the STRING platform. AutoDock Vina, AutoDockTools, ChemBio3D and Pymol tools were selected for molecular docking of key targets. The therapeutic effects of Cordycepin on PAH were observed in Monocrotaline (MCT)-induced PAH rats and platelet-derived growth factor BB (PDGFBB)-induced rat pulmonary artery smooth muscle cells (PASMCs). The right ventricular systolic pressure (RVSP) was detected. HE staining, Western Blot, Scratch assay, EDU and TUNEL assays were used, respectively. Through Network Pharmacology and molecular docking, the Cordycepin-PAH core genes were found to be TP53, AKT1, CASP3, BAX and BCL2L1. In MCT-induced PAH rats, the administration of Cordycepin significantly reduced RVSP, and inhibited pulmonary vascular remodeling. In PDGFBB-induced PASMCs, Cordycepin reduced the migration and proliferation of PASMCs and promoted apoptosis. After the Cordycepin treatment, the protein expressions of TP53, Cleaved CASP3 and BAX were significantly increased, while the protein expressions of p-AKT1 and BCL2L1 were significantly decreased in MCT-PAH rats and PDGFBB-induced PASMCs. This study identified that TP53, AKT1, CASP3, BAX, and BCL2L1 were the potential targets of Cordycepin against PAH by ameliorating pulmonary vascular remodeling, inhibiting the abnormal proliferation and migration of PASMCs and increasing apoptosis of PASMCs. which provided a new understanding of the pharmacological mechanisms of Cordycepin in the treatment of PAH.

Pyridoxal-5'-phosphate: A cost-effective treatment candidate for hypertensive patients?

Because angiotensin (Ang) II is an essential vasoconstrictive peptide, we analyzed the impact of its post-translational modification to pyruvamide-Ang II (Ang P) by pyridoxal-5'-phosphate (PLP) on blood pressure. PLP is a less expensive vitamin B6 derivative and, therefore, could be a cost-effective drug against hypertension. Effect of Ang P on calcium ion entry into vascular smooth muscle cells (VSMCs) was analyzed. Binding affinity of Ang P to angiotensin II type 1 receptor (AT1R) was measured. Vasoconstrictive effect of Ang P was investigated using the bioassay of isolated perfused rat kidneys. Spontaneously hypertensive rats (SHR) were administered PLP. Additionally, Wistar Kyoto rats (WKY) received Ang II and PLP. Blood pressure was measured time-dependently. Ang II, incubated with PLP, was post-translationally modified to Ang P. Calcium ion entry in VSMCs was significantly lower with Ang P compared to Ang II. Binding affinity of Ang P to AT1R was lower compared to Ang II. Perfusion pressure of isolated perfused rat kidneys increased less by Ang P than by Ang II. Blood pressure of SHR treated with PLP decreased significantly. Blood pressure of WKY rats treated with Ang II was increased to hypertensive values, whereas blood pressure of WKY rats cotreated with AngII and PLP was not. PLP induces a post-translational modification of Ang II decreasing blood pressure in rats. Assuming that increased PLP intake in the form of vitamin B6 might reduce blood pressure in hypertensive patients, PLP might be a cost-effective drug against hypertension.

Liver function tests in patients receiving high-intensity care after hospital admission for acute heart failure: the STRONG-HF trial

Abstract Background/Introduction Episodes of acute heart failure (AHF) unfavorably affect extra-cardiac organs such as the liver due to altered hemodynamics and neurohormonal activation. In AHF, elevated levels of levels of alanine aminotransferase (ALT) and total bilirubin (tBil) may reflect congestion and impaired liver function, while lower levels of ALT (which is also produced in muscle cells) correlate with malnutrition and sarcopenia. Thus, the clinical importance of these markers in AHF and the post-discharge period is unclear. Purpose To assess circulating concentrations of ALT and tBil in patients with AHF before discharge and during high-intensity care (HIC) vs usual care follow-up in association with clinical outcomes. Methods ALT and tBil concentrations were measured 1-2 days before discharge in patients hospitalized for AHF (baseline), and again after 90 days of either HIC or usual care according to the STRONG-HF protocol. Baseline values and changes in ALT and tBil were analyzed in association with the primary outcome of all-cause death or HF readmission by day 180. Results At baseline, the median (Q1-Q3) concentrations of ALT and tBil were 21 (15-32) U/L and 14 (10-21) umol/L, respectively. Lower levels of both ALT and tBil were associated with female sex, Black race, lower BMI, higher LVEF, lower hemoglobin, lower creatinine and less frequent atrial fibrillation/flutter, while there were no significant associations with age. Patients with lower ALT, but not tBil, were more likely to have edema, elevated JVP and orthopnea, and used higher loop diuretic doses and less beta-blockers, pre-randomization (Figure, panel A). ALT was inversely associated with risk for the primary outcome (HR 0.81 [95%CI 0.65,1.00) per log increment, p=0.05), while there was no association with risk for tBil (HR 1.04 [95%Ci 0.84-1.28], p=0.73) (Figure, panel B). After 90 days, ALT and tBil concentrations were lower than at baseline, with a greater reduction in tBil in the HIC group vs usual care group (mean -1.7 [95%CI -3.0,-0.4] umol/L, p=0.01), while there was no significant between-group differences in ALT changes (mean -0.6 [95%CI -4.7,3.4] U/L, p=0.75) (Figure, panel C). The treatment effect of HIC over usual care on the primary outcome was consistent across the range of baseline ALT (P-interaction=0.30) and tBil (P-interaction=0.80) (Figure, panel D). Conclusion Contrary to our hypothesis of higher liver function tests with hepatic congestion in AHF, lower ALT was associated with more congestion and worse outcomes among patients hospitalized for AHF in STRONG-HF. There was no prognostic value from tBil levels. This suggests that ALT may be a marker of sarcopenia in these patients. Importantly, the beneficial effect of HIC on clinical outcomes is consistent irrespective of ALT and tBil levels at discharge.Figure

Krill oil supplementation in vivo promotes increased fuel metabolism and protein synthesis in cultured human skeletal muscle cells

IntroductionKrill oil is a dietary supplement derived from Antarctic krill; a small crustacean found in the ocean. Krill oil is a rich source of omega-3 fatty acids, specifically eicosapentaenoic acid and docosahexaenoic acid, as well as the antioxidant astaxanthin. The aim of this study was to investigate the effects of krill oil supplementation, compared to placebo oil (high oleic sunflower oil added astaxanthin), in vivo on energy metabolism and substrate turnover in human skeletal muscle cells.MethodsSkeletal muscle cells (myotubes) were obtained before and after a 7-week krill oil or placebo oil intervention, and glucose and oleic acid metabolism and leucine accumulation, as well as effects of different stimuli in vitro, were studied in the myotubes. The functional data were combined with proteomic and transcriptomic analyses.ResultsIn vivo intervention with krill oil increased oleic acid oxidation and leucine accumulation in skeletal muscle cells, however no effects were observed on glucose metabolism. The krill oil-intervention-induced increase in oleic acid oxidation correlated negatively with changes in serum low-density lipoprotein (LDL) concentration. In addition, myotubes were also exposed to krill oil in vitro. The in vitro study revealed that 24 h of krill oil treatment increased both glucose and oleic acid metabolism in myotubes, enhancing energy substrate utilization. Transcriptomic analysis comparing myotubes obtained before and after krill oil supplementation identified differentially expressed genes associated with e.g., glycolysis/gluconeogenesis, metabolic pathways and calcium signaling pathway, while proteomic analysis demonstrated upregulation of e.g., LDL-receptor in myotubes obtained after the krill oil intervention.ConclusionThese findings suggest that krill oil intervention promotes increased fuel metabolism and protein synthesis in human skeletal muscle cells, with potential implications for metabolic health.

Association of polarimetric signatures with coronary plaque composition in patients with acute and chronic coronary syndrome: an intravascular polarization-sensitive optical coherence tomography study

Abstract Background Intravascular polarimetry with polarization-sensitive (PS-) optical coherence tomography (OCT) measures polarization properties, birefringence and depolarization, in parallel with structural features available with conventional OCT (Figure 1). Birefringence is elevated in collagen and collagen-synthesizing smooth muscle cells, which imparts mechanical integrity to fibrous caps. Depolarization is elevated in the presence of macrophages and lipid/necrotic core. Purpose This study aimed to investigate polarimetric signatures of coronary lesions in patients with acute coronary syndrome (ACS) and chronic coronary syndrome (CCS). Furthermore, we aimed to investigate diagnostic value for birefringence and depolarization of ACS culprit lesions discriminating from CCS target lesions. Methods This was the first-in-human study of intracoronary PS-OCT in Japan, consisting of 17 patients with ACS (n = 5) or CCS (n = 12) who underwent intravascular PS-OCT. ACS culprit lesions (ACS-lesions) and CCS target lesions (CCS-lesions) were included in the analysis, resulting in a total of 862 cross-sections. In the cross-sectional images, lumen segmentation and guidewire artifact were automatically segmented. The median value of birefringence and depolarization were automatically analyzed using depolarization cut-off value (cross-sectional level). Birefringence and depolarization of ACS- and CCS-lesions were compared by using a generalized estimating equation model. Receiver operating characteristic (ROC) analysis was used to investigate diagnostic performance of polarimetric signatures for the identification of ACS-lesions. Results Compared to CCS-lesions, ACS-lesions featured higher lipid-burden index and maximum lipid arc (both p < 0.05). Compared to the CCS-lesions, ACS-lesions exhibited significantly lower birefringence (p < 0.05) and higher depolarization (p < 0.05) (Figure 2). In the ROC analysis for differentiating ACS-lesions from CCS-lesions, area under the curves (AUC) for birefringence and depolarization were 0.712 and 0.672, respectively. In the multivariable ROC analysis in diagnosing ACS lesions, combination of birefringence with depolarization improved the AUC to 0.755 (p ＝ 0.025). Conclusions Intravascular PS-OCT provides quantitative compositional assessment of coronary lesions. Decreased birefringence and pronounced depolarization within the ACS-lesions indicate increased collagenolytic activity and the presence of macrophage infiltration, respectively. These results suggest that polarization properties may serve as quantitative imaging markers for assessing natural history of coronary atherosclerosis.

Lipoprotein(a) induces spontaneous aortic dissection via increasing monocyte mediated vascular smooth muscle cell phenotype transformation in human Lp(a) transgenic mice

Abstract Background Elevated lipoprotein(a) [Lp(a)] is a prevalent, independent risk factor of atherosclerotic cardiovascular disease and aortic valve stenosis. Recent studies have reported that high levels of Lp(a) are strongly associated with aortic dissection (AD), independent of other cardiovascular risk factors. However, orthologues of Lp(a) are only found in humans, Old World monkeys, apes, and hedgehogs. The lack of a proper animal model has hindered elucidation of the true pathophysiological mechanisms of Lp(a). Here, we conducted the first humanized Lp(a) transgenic mice experiment to verify the pathological mechanism of Lp(a) in aortic dissection. Methods We conducted ApoE knock out and human ApoA transgenic mice via CRISPR-Cas9 and then crossed with human ApoB transgenic mice to achieve high levels of human-like Lp(a) particles in plasma. Plasma was assayed by ELISA for the concentrations of each lipoprotein. Echocardiography was performed to detect aortic morphology in vivo. Single cell transcriptome analysis was performed on the dissection lesion. Cell interaction was examined in macrophage from Lp(a) transgenic mice and vascular smooth muscle cell (VSMC) coculture system. Result Our results indicate that the concentration of ApoA and ApoB in plasma were increased in transgenic mice fed a chow diet. Low density lipoprotein (LDL) levels were increased and shows that LDL is the major cholesterol carrier in these mice. We were surprised to find that the dissection of the ascending aortic arch could be seen by echocardiography in transgenic mice as early as 2 months, and transgenic mice showed significant increase in aortic enlargement, dissection, and rupture in both the thoracic and abdominal aortic regions, resulting in a survival rate of only 58% after 12 months. Single cell transcriptome analysis revealed that Lp(a) transgene induced the inflammatory response, dedifferentiation and pathological VSMC phenotypical switch, and matrix metalloproteinase expression in macrophages. Monocytes isolated from transgenic mice with elevated Lp(a) remain in a long-lasting primed state, as evidenced by an increased capacity to transmigrate and produce proinflammatory cytokines on stimulation. Monocytes isolated from wild type mice cultured in media with high level Lp(a) for 24h also showed the ability to facilitated cocultured VSMC phenotype transformation, which was reversed by E06, a monoclonal antibody blocking the oxidized phospholipids in Lp(a). Conclusion These findings demonstrate that Lp(a) induces spontaneous aortic dissection via increasing monocyte trafficking to the arterial wall, mediating proinflammatory responses and VSMC phenotype transformation through its OxPL content. The study presents a rodent model of spontaneous aortic dissection and provide a novel mechanism by which Lp(a) mediates aortic dissection.Graphical abstract

Ultrasound findings of ovarian intravenous leiomyomatosis: a case report

Intravenous leiomyomatosis (IVL), an abnormal growth pattern of uterine leiomyomas, is a rare tumor characterized by masses of smooth muscle cells appearing histologically benign and proliferating within the blood vessels but not invading the tissue. Currently, there have been limited reports of early cases of IVL, and the imaging characteristics of IVL remain uncertain, resulting in frequent misdiagnosis prior to surgery. The present study utilized a case of early IVL detected through conventional ultrasound and subsequently confirmed via contrast-enhanced ultrasound (CEUS) to further investigate ultrasound’s diagnostic efficacy for early IVL detection. Here, a case of a 49-year-old woman was reported who presented with uterine leiomyoma and an echogenic mass in the left adnexal region on physical examination. Subsequent transvaginal CEUS examination revealed a left ovarian venous leiomyoma. The patient underwent resection of tumors in the uterus, bilateral ovaries, and left ovarian vein under general anesthesia. A venous plexus was identified above the left broad ligament close to the left ovary, and a myoma-like growth was detected in the posterior uterine wall during the operational procedure. Reports on pathology and immunohistochemistry verified leiomyomatosis with fatty metaplasia in the left broad ligament and uterine wall vein. The prognosis of patients with IVL is determined based on the appropriate surgical methods and the timely diagnosis of the condition. In this case, conventional ultrasound aided in the early identification of IVL, which was later verified by a CEUS examination, resulting in a successful surgical treatment. This example highlights the importance of ultrasound technology in diagnosing this uncommon condition and presents a new method for preoperative detection of IVL.

Long-term clinical-pathologic results of enzyme replacement therapy in pre-hypertrophic fabry disease cardiomyopathy

Abstract Background Limited ability of ERT in removing Gb-3 from cardiomyocytes is recognized for advanced FDCM. Pre-hypertrophic FDCM is believed to be cured or stabilized by ERT. However, no pathologic confirmation is available. We report here in the long-term clinical-pathologic impact of ERT on pre-hypertrophic FDCM. Purpose Long-term clinical-pathologic impact of ERT on pre-hypertrophic FDCM. Methods Fifteen Fabry patients with LVMWT ≤ 10.5 mm at CMR required EMB because of angina and/or ventricular arrhythmias. EMB showed coronary small vessel disease in angina cohort and vacuoles in smooth muscle cells and cardiomyocytes around 20% of cell surface containing myelin bodies at electron microscopy. Patients received α-agalsidase in 8 and β-agalsidase in 7 cases. Both groups referred symptoms improvement except 2 patients treated with α- and β-agalsidase respectively. After ERT administration ranging from 4 and 20 years all patients had control CMR and LVEMB because of persistence of symptoms or patients enquiry on disease resolution. Results In 13 asymptomatic FDCM patients, LVMWT and LV-mass, cardiomyocyte diameter, vacuole surface/cell surface ratio and vessels remained unchanged or minimally increased (LV-mass augmented by < 2%) even after 20 years observation and storage material was still present at electron microscopy. In 2 symptomatic patients, FDCM progressed with larger and more engulfed by Gb-3 myocytes being associated to myocardial virus-negative lymphocytic inflammation. Conclusions ERT stabilizes storage deposits and myocyte dimensions in 87% of patients with pre-hypertrophic FDCM. Gb-3 is never completely removed even after long-term treatment. Immune-mediated myocardial inflammation can overlap limiting ERT activity. Figure Legend Patient 11 (24-year-old man) with genetic and biopsy-proven diagnosis of FDCM before and after 20 years ERT. CineMR (A,B) and LGE (C,D) images, performed at baseline (A,C) and 20 years after ERT (B,D) show normal LV wall thickness (8mm and 10 mm) and absence of fibrotic areas. Panel E and F represent semithin section of Epon-embedded EMB samples, stained with basic fuchsin, showing mildly hypertrophied cardiomyocytes containing storage material that result unmodified after long-term ERT administration. Panel G and H are ultrastructural evidence of storage material to consist of myelin bodies (panel G) that remain unchanged after ERT (Panel H).

The novel role of complement 3 in Wnt5a-mediated vascular smooth muscle cell calcification

Abstract Background Although vascular calcification (VC) is a risk factor for cardiovascular and all-cause mortality, a therapy is not yet available. VC involves the transdifferentiation of vascular smooth muscle cells (VSMC) towards an osteochondrogenic lineage that results in calcium phosphate salts deposition in the arterial wall. We have previously shown an association between plasma WNT5A levels and new onset and progression of coronary artery calcification. Purpose We aimed to study the poorly understood role of Wnt5a in VSMC calcification. Methods Study 1 included 18 patients with coronary artery disease (CAD) undergoing cardiac surgery; VSMC were isolated from their saphenous veins to perform in vitro assays and microarrays. Study 2 included 11 patients with CAD; IMA were treated ex vivo with Wnt5a recombinant protein (rWnt5a) to perform western blot (WB) analyses. Study 3 included 647 patients with CAD; RNA sequencing was performed on samples from their internal mammary arteries (IMA), IMA perivascular adipose (PVAT), and thoracic adipose tissue (ThAT). VSMC and human aortic smooth muscle cells (HASMC) from healthy donors were grown in osteogenic medium in the presence or absence of rWnt5a with or without specific protein inhibitors. Calcium deposition and expression of VC markers (i.e. RUNX2, BMP2, and MSX2) were assessed with a colorimetric assay and by RT-qPCR, respectively. Phosphorylation/activation of non-canonical Wnt pathway effectors was evaluated by WB analyses. Results rWnt5a treatment significantly increased calcium deposition in both VSMC and HASMC at a concentration that activates both JNK and CaMKII, that are effectors of the non-canonical planar cell polarity pathway and the calcium-dependent pathway, respectively. Inhibition of either JNK or CaMKII could not counteract rWnt5a-mediated increase in VSMC calcium deposition; whereas, inhibition of both non-canonical pathway effectors (JNK-i and CaMKII-i) prevented rWnt5a-mediated increase in calcium deposition by VSMC (A) and HASMC. Inhibition of JNK and CaMKII also prevented rWnt5a-induced gene expression of VC markers, such as RUNX2 (B). rWnt5a treatment in IMA increased the activation of JNK and CaMKII. Patients with higher WNT5A levels in IMA had significantly higher RUNX2 levels. By intersecting RNA sequencing and microarray data complement 3 (C3) resulted as the most upregulated gene in rWnt5a-treated VSMC among those genes whose levels positively correlated in IMA with both WNT5A and RUNX2 expression (C). Patients with higher WNT5A levels in IMA, their PVAT, and ThAT had significantly higher C3 levels (D), and patients with higher levels of C3AR1, that is the gene coding for the C3a receptor, had higher VC markers expression. C3 inhibition (C3-i) with a C3AR1 antagonist counteracted Wnt5a-induced VSMC calcium deposition (E). Conclusions Non-canonical Wnt signalling promotes VSMC calcification through C3. Wnt5a may be a therapeutic target in VC.Abstract Figure

Locally increased dysferlin expression improves left-ventricular function by stabilizing membrane nanodomains of cardiomyocytes in the myocardial infarction border zone

Abstract Introduction Dysferlin, a transmembrane protein with multiple Ca2+-binding C2-domains, is expressed in the sarcolemma and vesicle membranes of striated muscle cells. Upon Ca2+ entry, Dysferlin is thought to mediate vesicle fusion and membrane repair events in order to rapidly seal sarcolemmal lesions. Dysferlin mutations are associated with progressive muscular dystrophies and dilated cardiomyopathy. However, the precise role of Dysferlin in protection of membrane nanodomains in ventricular myocytes (VMs) after myocardial infarction (MI) remains elusive. Purpose We hypothesized that Dysferlin is crucial to prevent VMs from loss-of-function and cell death by stabilizing membrane nanodomains like the transverse-axial tubule (TAT) system and the intercalated disc (ICD) cell-cell contact sites post-MI. Methods and Results 1 week post-MI induced by LAD artery ligation, investigator-blinded echocardiography revealed increased infarct sizes and decreased left-ventricular (LV) ejection fraction of Dysferlin-knockout (KO) vs. WT mice (mean±SEM: 21±2 vs. 28±2%, n=20/30 mice, P<0.05). Immunoblotting of WT LV tissue lysates showed an upregulated Dysferlin expression to 148% compared to sham-operated controls. Importantly, immunohistology identified a maximal Dysferlin expression of 230% in the MI border zone that faces a high ischemic and mechanical stress. Hence, we used data-independent mass spectrometry (DIA-MS) to analyse the proteomic profile of the infarction zone, border zone and remote zone from WT vs. KO hearts 1 week post-MI (each n = 5 mice). Overall, we reproducibly quantified 4360 proteins across all LV samples, identifying local genotype-specific proteotypes post-MI. In WT vs. KO samples, DIA-MS revealed 725 differentially abundant proteins in the zone pairwise comparison, highlighting a vital role of Dysferlin for LV remodelling post-MI. In addition, superresolution stimulated emission depletion (STED) microscopy was applied to study the subcellular localization and function of Dysferlin in the MI border zone. Here, we observed VMs with disrupted TAT endomembrane networks and significantly extended ICD regions. Interestingly, STED imaging revealed Dysferlin clusters that highly decorated residual TAT structures and interdigitated ICD membrane folds in the MI border zone. DIA-MS analysis of anti-Dysferlin co-imunoprecipitation experiments from WT vs. KO LV tissues characterized the cardiac interactome of Dysferlin, confirming interactions with proteins of the TAT system and the gap junction hemichannel Connexin-43. Conclusions Our data demonstrate Dysferlin as an endogenous membrane repair protein necessary to stabilize sarcolemmal nanodomains in the MI border zone. Dysferlin not only prevents from loss of TAT structures, but also compensates for increased mechanical stress at the ICDs. Hence, Dysferlin emerges as a novel therapeutic target to control membrane integrity in VMs to prevent from loss-of-function and cell death post-MI.

The role of cellular senescence in aortic dissection

Abstract Background Aortic dissection (AD) is a catastrophic disease with high mortality. Survived patients frequently suffer from serious complications due to progressive destruction of the aortic walls. Recently we have reported that cell proliferation precedes the inflammatory response which is important in AD development and progression. Since cell proliferation promotes cellular senescence that can induce inflammatory response through SASP (senescence-associated secretary phenotype), we hypothesized that cellular senescence plays a critical role in AD pathogenesis. Purpose To investigate if cellular senescence contributes to AD pathogenesis in mouse AD model. Methods and Results A mouse AD model was created by the continuous infusion of beta-aminopropionitrile and angiotensin II (BAPN+AngII) for 14days. BAPN+AngII infusion induced senescence markers and senescent cells in mouse AD model. Senescent cells, as demonstrated by the expression of senescence-associated beta-galactosidase, were evident in intimal endothelial cells, medial smooth muscle cells, adventitial macrophages, and fibroblasts. Rapamycin, an inhibitor of mTOR pathway, suppressed the expression of senescent cells on the aortic tissue and the senescent marker protein expression. To examine the role of cellular senescence in AD, we orally administrated ABT263 known as "senolytics" that eliminates senescent cells. ABT263 treatment prevented cellular senescence in mouse AD model. The AD mortality of ABT263 group was 35%, which was lower than that of vehicle group (66.7%, P < 0.05 by log-rank test). The severity of AD, as assessed by the lesion length in vehicle group was 33.2 ± 3.1mm, whereas that in ABT263 group was 24.6 ± 1.8mm (P < 0.05). Transcriptome analysis revealed that ABT263 treatment suppressed the immune and inflammatory response in the aorta before AD onset. Quantitative RT-PCR confirmed that ABT263 treatment prevented the induction of p21Cip1, interleukin-6, several chemokines and M1 macrophage marker CD80 and CD86. It is known that Jnk activation induces SASP and accelerates cellular senescence. Western blotting confirmed that ABT263 reduced the ratio of pJnk to total Jnk, which was induced by BAPN + AngⅡ. Moreover, aortic smooth muscle cells（SMCs）remained contractile phenotype in ABT263 group, whereas BAPN+AngII reduced that phenotype in vehicle group. Conclusions Cell proliferation via mTOR pathway causes cellular senescence in mouse AD model. Jnk activation was confirmed in mouse AD model. Cellular senescence contributes to AD progression and death associated with inflammatory responses including SASP, the differentiation to M1 macrophage, and the phenotype change of SMCs.Cellular senescence represents a potential predictor and a therapeutic target for AD.

Long noncoding RNA Sngh20 promotes vascular smooth muscle cell senescence and abdominal aortic aneurysm growth by interacting with dead-box helicase 5

Abstract Background Development of non-surgical treatment of human abdominal aortic aneurysm (AAA) has clinical significance. Long noncoding RNAs (lncRNAs) are emerging as powerful mediators participated in cellular senescence process that a critical pathological alteration prompting AAA development. Purpose To investigate the role and underlying mechanism of lncRNA Small nucleolar RNA host gene-20 (Snhg20) in AAA formation. Methods Experimental AAA mice models were produced by peri-aortic CaPO4 injury in C57bl/6J mice or Subcutaneousangiotensin-II (Ang-II) infusion in Apoe–/– mice and gapmeR was used to knockdown Snhg20 expression. Adeno-associated virus and anti-IL-6 monoclonal antibody was used to knockdown dead-box helicase 5 (DDX5) and neutralize IL-6, respectively.Immunohistochemical and immunofluorescent staining, ELISA and real-time PCR were performed to determine the vascular structure and inflammation, cellular senescence and senescence-associated secretory phenotype (SASP) content in human and mouse AAA lesions. Human aortic SMC cell line was used to investigate the effect of Snhg20 knockdown on senescence and SASP in vitro. LncRNA pulldown and mass spectrometry were used to identify potential targets that interact with Snhg20. p53 DNA binding affinity was examined by EMSA. Results The expression of Snhg20 was downregulated in both human and mice AAA lesion. Snhg20 knockdown promoted SMC senescence and loss, vascular inflammation and AAA growth in both experimental AAA mice models. Mechanistically, Snhg20 interacted with DDX5, an important transcriptional coactivator of p53. The absence of snhg20 increased DDX5 and p53 interaction and thereby promoted p53 DNA binding affinity to transcriptionally initiate p21expression, resulting in increased cellular senescence and senescence-associated secretory phenotype (SASP) secretion, including IL-6. Moreover, DDX5 silencing or anti-IL-6 therapy rescued the SMC senescence and loss, vascular inflammation and AAA growth mediated by Snhg20 knockdown. Furthermore, human AAA lesions showed increased DDX5 co-localization with p53 and abundant senescent vascular SMC. Conclusion These findings identify an important role of Snhg20 in controlling vascular SMC senescence and SPAP expression and suggest that Snhg20 is a potential target for AAA treatment aimed at reducing VSMCs senescence.Schematic summary

PI 3-kinase/AKT/FOXO1 signaling in smooth muscle cells protects against abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs). Growth factors induce proliferation and cell survival of SMCs. PI 3-kinase (PI3K) is an important downstream mediator in growth factor signaling. Objective This project is based on the hypothesis that PI3K-mediated SMC proliferation can compensate for the loss of SMCs in AAA and thus inhibit AAA progression. Therefore, we determined effects of reduced PI3K activity in SMCs on AAA progression, cell proliferation as well as vascular remodeling. Methods To investigate the role of PI3K, we analyzed mice and aortic SMCs harboring a smooth muscle-specific deficiency of the PI3K catalytic subunit p110α (SM-p110α-/-). AAA development in SM-p110α-/- mice and wild-type (WT) littermates was induced by perfusion of the infrarenal aortic segment with porcine pancreatic elastase (PPE). AAA diameter was determined by echocardiography. Structural changes in the aorta were identified by (immuno)histochemical staining. SMC proliferation was measured in vitro by BrdU incorporation or real-time cell analysis. p110α induced signal transduction pathways were characterized by Western blot and transcriptome analysis. Results PPE treatment led to a massive loss of SMCs in the compromised aortic segment after 3 days in both SM-p110α-/- and WT mice. The aortic diameter was enlarged after 28 days in all PPE-treated mice. However, the increase was significantly higher in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10) (p<0.05). A similar effect was obtained in aged (untreated) mice: In 24-month-old SM-p110α-/- mice, abdominal aortic lumen diameter was increased by 42% compared to 3-month-old animals, but only by 15% in corresponding WT mice. PPE-treated SM-p110α-/- mice were characterized by reduced vascular remodeling with less PCNA-positive proliferating cells compared to WT animals. In addition, proliferation of aortic p110α-/- SMCs in serum-containing medium was impaired. Mechanistic analyses showed that PDGF- and insulin-induced activation of AKT as well as AKT-dependent phosphorylation and inactivation of the transcription factors FOXO-1, -3 and -4 were significantly reduced in p110α-/- SMCs compared to WT cells. Further analyses revealed that specific inhibition of FOXO1 by AS1842856 rescued PDGF or serum stimulated proliferation of p110α-/- SMCs. In addition, expression analysis of FOXOs in patients revealed significantly increased FOXO1 expression in AAA compared to aortic samples from healthy individuals. Conclusion Deficiency of the catalytic PI3K isoform p110α in SMCs promotes the development and progression of AAA. p110α/AKT-dependent inactivation of FOXO1 induces SMC proliferation, which can ameliorate aortic wall damage. As FOXO1 inhibitors and a recently developed p110α activator are available, the PI3K/AKT/FOXO1 signaling cascade may provide a therapeutic target to influence the stability of the aortic wall in AAA.

RGS5 balances macrophage vs vascular smooth muscle cell origin of foam cells in apoE-deficient plaques

Abstract Background G Protein Coupled Receptors and regulators of G-protein signalling (RGS), play a pivotal role in the signalling processes associated with atherosclerosis [1]. RGS5 is highly expressed in aortic tissue [2]. Development of atherosclerotic plaque is a complex process that involves foam cells originating from macrophages (Mϕ) and/or vascular smooth muscle cells (VSMCs). Differentiation of Mϕ and VSMC into various intermediary cells of varying inflammatory potency affects atherosclerotic plaque composition and vulnerability [3]. We hypothesize that RGS5 is involved in signalling of foam cell transition and thus, plaque biology. Purpose To evaluate the role of RGS5 on atherosclerotic plaque complexity and shifts of foam cell origin. Methods ApoE-/-RGS5+/+ and ApoE-/-RGS5-/- mice were fed a high fat Western Diet for 14 weeks. Cryosections bearing aortic valve cusps were used for staining. Lesion size was quantified on hematoxylin and eosin staining. Plaque composition was assessed by CD68, CD45, Oil red O and collagen staining. Triple staining was performed for CD45, CD68 and aSMA. Double immunofluorescent staining for CD45 and pro- and anti-inflammatory markers was carried out. Co-Localisation was analysed by Zeiss Zen. Data were analysed using Mann-Whitney-U test. Results Lesion size was significantly reduced in RGS5-deficient mice in comparison to control (in mm2: 0.398±0.02 vs 0.191±0.09, p=0.0001, n=15). Relative areas positive for Oil Red O and collagen did not differ between genotypes. Area positive for leukocyte marker CD45 increased significantly by 30.8% (p<0.0001, n=15), whereas area positive for CD68 was reduced by 10.3% (p=0.0292, n=15) in RGS5-deficient mice. Absence of RGS5 decreased co-localization of Mϕ-derived foam cells by 23% (p=0.03734, n=4-5) while leading to an 5.8% increase in aSMA-derived foam cells compared to control. Area positive for pro-inflammatory M1 Mϕ marker CD86 and iNOS increased by 36.3% and 36.8% after RGS5 knock-out (p<0.0001, n=15). No difference in M2 Mϕ positive area was observed when RGS5 was lacking. Conclusion RGS5 promotes plaque formation. The number of Mϕ-derived foam cells is increased by RGS5, while it reduces inflammatory M1 Mϕ. Interestingly, there were no differences in numbers of M2 Mϕ, indicating RGS5 inhibits VSMCs transition to M1 Mϕ resulting in an overall decrease of CD68-positive foam cells. Further study is required to determine molecular signalling of RGS5 that modulates the phenotype of Mϕ- and VSMC-derived cells. RGS5 could be useful as therapeutic target to suppress undesirable plaque phenotypes and to reduce acute cardiovascular events.

The safety and efficacy of treprostinil for the treatment of pulmonary arterial hypertension: a systematic review and meta-analysis of randomized controlled trials

Abstract Introduction Pulmonary arterial hypertension (PAH) is a severe and life-threatening condition characterized by increased pulmonary vascular resistance and elevated mean pulmonary artery pressure. Treprostinil, a prostacyclin analog, has vasodilatory and anti-remodeling effects on pulmonary arterial smooth muscle cells, indicating promising benefits in enhancing exercise capacity, improving hemodynamics, and reducing morbidity in patients with PAH. Aim The primary objective of this systematic review and Meta-analysis is to assess the efficacy and safety of Treprostinil in patients with PAH. Methods We systematically searched PubMed, Scopus, Cochrane CENTRAL, and ClinicalTrials.gov from inception until July 2023. Randomized controlled trials comparing Treprostinil with Placebo were selected. We used the mean values to compare continuous data and odds ratio (OR) for dichotomous outcomes with their 95% confidence interval (CI). Results We included 7 studies with a total of 2,447 patients. The Treprostinil group was favored over the control group in terms of 6-minute walk distance (6MWD) (Mean difference (MD), meters (m)) (MD = 15.16 m, 95% confidence interval (CI) (10.42 – 19.90 )). Subgroup analysis based on the route of administration revealed a 6MWD of (MD = 13.55 m (95% CI (7.82 – 19.28)) for oral administration and (MD = 17.49 m, 95% CI (6.01 – 28.89)) for intravenous (IV)/subcutaneous (SC) administration, indicating enhanced exercise capacity. When evaluating the efficacy of Treprostinil for oral administration, there was a slight but non-significant trend toward functional improvement (Odds Ratio (OR): 1.36; 95% CI: 0.95 to 1.93) and no significant change in maintaining functional status (OR: 0.94; 95% CI: 0.59 to 1.51). In terms of functional deterioration, the risk was also not significantly reduced (OR: 0.84; 95% CI: 0.36 to 1.98). Safety assessment of the drug was based on clinical, mortality, and adverse events outcomes. Clinically, Treprostinil demonstrated a protective effect against disease progression, evidenced by a reduced risk of clinical worsening (OR: 0.69; 95% CI: 0.52 to 0.91). Mortality rates were not significantly impacted by Treprostinil addition (OR: 0.95; 95% CI: 0.57 to 1.58 for oral, 0.80; 95% CI: 0.32 to 1.98 for IV/SC). However, the incidence of adverse events was notably higher among Treprostinil-treated patients, particularly with oral administration. Conclusion The addition of Treprostinil therapy for patients with PAH significantly improved exercise capacity, as evidenced by an increase in the 6MWD compared to control groups and demonstrated a reduced risk of clinical worsening. The incidence of adverse events was higher among Treprostinil-treated patients, especially with oral administration, potentially impacting quality of life.Forest plot of the 6MWD

Suppression of Fibulin 5 Attenuated Pulmonary Arterial Hypertension Associated with Congenital Heart Disease via Mitigating Pulmonary Arterial Smooth Muscle Cell Dysfunction

Abstract Backgrounds: The specific molecular pathogenesis of pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH) and the reversal still remain elusive. Fibulin 5 was involved in the regulation of cardiovascular system while little is known about the expression and functions of fibulin 5 in the development of CHD-PAH. This study aimed to investigate the role of fibulin 5 in the pathogenesis of the disease. Methods Lung samples were obtained both from patients with CHD-PAH and aortocaval shunt-associated PAH rat models. TGF-β1was used to induce human pulmonary artery smooth muscle cells (hPASMCs) dysfunction and lentiviruses were responsible for alteration of fibulin 5 expression. In an effort to explore the specific role of fibulin 5, an inhibitor of phosphatidylinositol-3-kinases (PI3K) /protein-serine-threonine kinase (AKT) which was defined as the TGF-β1-related downstream signaling was applied for further exploration. Results Fibulin 5 was pronouncedly elevated in the PASMCs of the remodeled pulmonary arterioles from patients with CHD-PAH and shunt-associated PAH rats. We found that over-expression of fibulin 5 could promote hPASMCs dysfunction induced by TGF-β1 and it could be reversed by the PI3K/AKT inhibitor LY294002 suggesting the essential role of TGF-β1/PI3K/AKT signaling activation in phenotypic switch, proliferation and migration of PASMCs. Moreover, an adeno-associated virus vector encoding fibulin 5 by intratracheally instillation with rats improve the hemodynamic parameters in shunt-associated PAH rats and its related pulmonary artery remodeling. Conclusion Over-expression of fibulin 5 could significantly promote hPASMCs dysfunction and pulmonary artery remodeling via reinforcing TGF-β1/PI3K/AKT signaling activation, which highlighted the potential valuable pharmacological target for the treatment of CHD-PAH.Fibulin 5 was over-expressed in PAHInhibition of fibulin 5 attenuated PAH

Arrhythmogenic effects of DEHP exposure: insights into cardiac electrophysiology and myocardial cells

Abstract Introduction Hemodialysis patients, who are exposed to plasticizers through the hemodialysis circuit, face a high risk of cardiovascular disease (CVD). Plasticizers, such as the endocrine-disrupting compound Di(2-ethylhexyl) phthalate (DEHP), have emerged as potential contributors to CVD. This study explores the impact of DEHP on cardiac electrophysiology and myocardial cells, providing insights into its cardiotoxic effects. Methods We employed optical mapping techniques to assess cardiac electrophysiology in Langendorff-perfused rat hearts exposed to DEHP, comparing them to a control group. Additionally, we evaluated the effect of DEHP on cardiac muscle cells using H9C2 rat cardiomyoblasts. Results Prolonged DEHP exposure resulted in elevated heart rate, increased electrocardiographic variability, and higher low-frequency values, indicative of sympathetic nervous system overactivity. Action potential duration at APD80 increased, leading to greater action potential triangulation. Electrical alternans observed in the DEHP-treated group heightened the risk of arrhythmias. Single-cell analysis revealed severe mitochondrial damage, myocardial cell apoptosis, and disrupted calcium ion balance. Conclusion This study highlights DEHP's cardiotoxicity, suggesting its potential role in arrhythmogenesis and cardiac dysfunction. Environmental pollutants like DEHP warrant consideration as contributors to cardiovascular diseases, emphasizing the need for further research on preventive strategies.

Itga9 protects against thoracic aortic aneurysm or dissection

Abstract Background The thoracic aortic aneurysm or dissection (TAAD) is a fatal cardiovascular disease with unclear pathogenic mechanisms. Integrins are important in coordinating the transmembrane connections between the extracellular matrix (ECM) and cytoskeleton, contributing to pathological processes of various diseases. Integrin α9 (Itga9), one of the integrin family subunits, has emerged as critical for vascular homeostasis. However, the contribution of Itga9 to TAAD remains elusive. Purpose The present study aimed to determine the role of Itga9 in the development of TAAD and its underlying mechanisms. Methods Western blotting and immunofluorescence staining were used to evaluate ITGA9 expression in the human Standford-A AAD tissues and rodent model of TAAD. Beta-aminopropionitrile monofumarate (BAPN) was used to induce TAAD mouse model. Vascular smooth muscle cell (VSMC)-specific Itga9 knockout mice or adeno-associated virus (AAV)-treated Itga9 overexpressed mice were generated to detect the role of Itga9 in the progression of TAAD. RNA-sequencing analysis was performed to discern possible transcriptome changes in the aorta tissues of VSMC-specific Itga9 knockout mice after BAPN treatment. Primary mouse aortic VSMC culture, flow cytometry analysis, EdU incorporation assay, transmission electron microscopy, and autophagy flux experiments were performed to evaluate the function of Itga9 on VSMCs and uncover its downstream target. Results ITGA9 expression levels were reduced in the aorta tissues of Standford-A AAD patients and BAPN-induced TAAD mice. VSMC-specific deletion of Itga9 exacerbated the incidence of BAPN-induced TAAD in mice. Itga9 deficiency downregulated Valosin-containing protein (Vcp) expression in mice aorta and caused reduced autophagy biomarkers of LC3 after BAPN treatment. In the primary aortic VSMCs, Itga9 deficiency resulted in repressed autophagy, accelerated apoptosis, and reduced proliferation ability. Mechanistically, Itga9 recruited the transcription factor Pre-B-cell leukemia transcription factor 1 (Pbx1) to activate the Vcp and promote VSMC autophagy. Consistently, overexpression of Itga9 in vitro and in vivo rescued the autophagic change and protected against the TAAD progression by upregulating the expression of Pbx1 and Vcp. Conclusions Our findings indicate a critical role of Itga9 in the regulation of VSMC autophagy by controlling Pbx1-Vcp. The Itga9-Pbx1-Vcp axis may represent a novel therapeutic target against TAAD.

PEDF upregulated in Ngfr-positive cells suppresses proliferation of pulmonary artery smooth muscle cells

Abstract Background Pulmonary arterial hypertension (PAH) is a disease with poor prognosis that causes right heart failure due to progressive pulmonary artery remodeling. Although existing pulmonary vasodilators contribute to pulmonary artery reverse remodeling by reducing share stress, the development of therapeutic agents that directly target pulmonary vascular remodeling is desired. Nerve growth factor receptor (Ngfr) is involved in the inflammatory reaction and repair process of damaged tissues. We have previously reported that Ngfr-positive cells are increased in the peripheral blood (PB) of PAH patients and are associated with disease progression. We also found that Ngfr inhibited the pathogenetic progression of pulmonary hypertension in a model of hypoxia-induced pulmonary hypertension. However, it remains unclear how Ngfr positive cells are involved in the pathology of PAH. Purpose In this study, we investigate how Ngfr-positive cells affect the pathogenesis of PAH. Methods & Results To evaluate the localization of Ngfr-positive cells in lung tissue, WT mice (C57BL/6) transplanted with bone marrow (BM) of GFP-Tg mice were kept under hypoxia for 3 weeks to create hypoxia-induced pulmonary hypertension (PH) model. Immunostaining of lung tissue sections indicated that double positive cells (Ngfr+GFP+) were present in the interstitial tissue around the small pulmonary artery. To examine the cellular characteristics of Ngfr-positive cells, RNA sequencing of Ngfr-positive and Ngfr-negative cells in PB was performed. Significantly 78 genes were upregulated in Ngfr-positive cells, including 11 secreted proteins. Among these, we focused on pigment epithelium-derived factor (PEDF), a secreted protein involved in angiogenesis. PEDF mRNA expression was decreased in the lung of Ngfr-KO mice compared to WT mice. We next examined the effect of PEDF on pulmonary artery smooth muscle cells (PASMCs). Platelet-derived growth factor (PDGF)-induced cell proliferation of PASMC was inhibited by PEDF stimulation. Furthermore, PEDF stimulation significantly inhibited PDGF-induced mRNA expressions of TNF and MMP9 in PASMCs. Conclusion In this study, we show that PEDF expression is upregulated in Ngfr-positive cells and that PEDF inhibits PDGF-induced hPASMC proliferation, suggesting that NGFR-positive cells may suppress pulmonary vascular remodeling through PEDF-mediated paracrine effects. PEDF might be a new therapeutic target for PH.

Engineering Nanoclusters of Cell Adhesive Ligands on Biomaterial Surfaces: Superior Cell Proliferation and Myotube Formation for Skeletal Muscle Tissue Regeneration.

Engineering biointerfaces with nanoscale clustering of integrin-binding cell adhesive peptides is critical for promoting receptor redistribution into signaling complexes. Skeletal muscle cells are exquisitely sensitive to integrin-mediated signaling, yet biomaterials supporting myogenesis through control of the density and nanodistribution of ligands have not been developed. Here, materials are developed with tailorable cell adhesive ligands distribution at the interface by independently controlling their global and local density to enhance myogenesis, by promoting myoblast growth and myotube formation. To this end, RGD-functionalized low-fouling polymer surfaces with global ligand densities (G) from 0-7µg peptide/mg polymer and average local ligand densities (L) from 1-6.3 ligands/cluster, are generated and characterized. Cell studies demonstrate improvements in cell adhesion, spreading, growth, and myotube formation up to a density of 7µg peptide/mg polymer with 4 ligands/cluster. Optimizing ligand density and distribution also promotes early myofiber maturation, identified by increased MF20 marker protein expression and sarcomere-forming myotubes. At higher ligand densities, these cell properties are decreased, indicating that ligand multivalency is a critical parameter for tailoring cell-material interactions, to a certain threshold. The findings provide new insights for designing next-generation biomaterials and hold promise for improved engineering of skeletal muscle.