PAI-1 Regulates the Cytoskeleton and Intrinsic Stiffness of Vascular Smooth Muscle Cells.

BACKGROUND: Plasma concentration of PAI-1 (plasminogen activator inhibitor-1) correlates with arterial stiffness. Vascular smooth muscle cells (SMCs) express PAI-1, and the intrinsic stiffness of SMCs is a major determinant of total arterial stiffness. We hypothesized that PAI-1 promotes SMC stiffness by regulating the cytoskeleton and that pharmacological inhibition of PAI-1 decreases SMC and aortic stiffness. METHODS: PAI-039, a specific inhibitor of PAI-1, and small interfering RNA were used to inhibit PAI-1 expression in cultured human SMCs. Effects of PAI-1 inhibition on SMC stiffness, F-actin (filamentous actin) content, and cytoskeleton-modulating enzymes were assessed. WT (wild-type) and PAI-1-deficient murine SMCs were used to determine PAI-039 specificity. RNA sequencing was performed to determine the effects of PAI-039 on SMC gene expression. In vivo effects of PAI-039 were assessed by aortic pulse wave velocity. RESULTS: PAI-039 significantly reduced intrinsic stiffness of human SMCs, which was accompanied by a significant decrease in cytoplasmic F-actin content. PAI-1 gene knockdown also decreased cytoplasmic F-actin. PAI-1 inhibition significantly increased the activity of cofilin, an F-actin depolymerase, in WT murine SMCs, but not in PAI-1-deficient SMCs. RNA-sequencing analysis suggested that PAI-039 upregulates AMPK (AMP-activated protein kinase) signaling in SMCs, which was confirmed by Western blotting. Inhibition of AMPK prevented activation of cofilin by PAI-039. In mice, PAI-039 significantly decreased aortic stiffness and tunica media F-actin content without altering the elastin or collagen content. CONCLUSIONS: PAI-039 decreases intrinsic SMC stiffness and cytoplasmic stress fiber content. These effects are mediated by AMPK-dependent activation of cofilin. PAI-039 also decreases aortic stiffness in vivo. These findings suggest that PAI-1 is an important regulator of the SMC cytoskeleton and that pharmacological inhibition of PAI-1 has the potential to prevent and treat cardiovascular diseases involving arterial stiffening.

Nupr1-mediated vascular smooth muscle cell phenotype transformation involved in methamphetamine induces pulmonary hypertension.

AIMS: Nuclear protein 1 (Nupr1) is a multifunctional stress-induced protein involved in the regulation of tumorigenesis, apoptosis, and autophagy. However, its role in pulmonary hypertension (PH) after METH exposure remains unexplored. In this study, we aimed to investigate whether METH can induce PH and describe the role and mechanism of Nupr1 in the development of PH. METHODS AND RESULTS: Mice were made to induce pulmonary hypertension (PH) upon chronic intermittent treatment with METH. Their right ventricular systolic pressure (RVSP) was measured to assess pulmonary artery pressure. Pulmonary artery morphometry was determined by H&E staining and Masson staining. Nupr1 expression and function were detected in human lungs, mice lungs exposed to METH, and cultured pulmonary arterial smooth muscle cells (PASMCs) with METH treatment. Our results showed that chronic intermittent METH treatment successfully induced PH in mice. Nupr1 expression was increased in the cultured PASMCs, pulmonary arterial media from METH-exposed mice, and METH-ingested human specimens compared with control. Elevated Nupr1 expression promoted PASMC phenotype change from contractile to synthetic, which triggered pulmonary artery remodeling and resulted in PH formation. Mechanistically, Nupr1 mediated the opening of store-operated calcium entry (SOCE) by activating the expression of STIM1, thereby promoting Ca2+ influx and inducing phenotypic conversion of PASMCs. CONCLUSIONS: Nupr1 activation could promote Ca2+ influx through STIM1-mediated SOCE opening, which promoted METH-induced pulmonary artery remodeling and led to PH formation. These results suggested that Nupr1 played an important role in METH-induced PH and might be a potential target for METH-related PH therapy.

Enhanced Reendothelialization and Thrombosis Prevention with a New Drug-Eluting Stent.

PURPOSE: The objective of the study is to test the efficacy of cyclopentenyl cytosine (CPEC)-coated stents on blocking artery stenosis, promoting reendothelialization, and reducing thrombosis. METHODS: Scanning electron microscopy was employed to observe the morphological characteristics of stents coated with a mixture of CPEC and poly(lactic-co-glycolic acid) (PLGA) copolymer. PLGA has been used in various Food and Drug Administration (FDA)-approved therapeutic devices. In vitro release of CPEC was tested to measure the dynamic drug elution. Comparison between CPEC- and everolimus-coated stents on neointimal formation and thrombosis formation was conducted after being implanted into the human internal mammary artery and grafted to the mouse aorta. RESULTS: Optimization in stent coating resulted in uniform and consistent coating with minimal variation. In vitro drug release tests demonstrated a gradual and progressive discharge of CPEC. CPEC- or everolimus-coated stents caused much less stenosis than bare-metal stents. However, CPEC stent-implanted arteries exhibited enhanced reendothelialization compared to everolimus stents. Mechanistically, CPEC-coated stents reduced the proliferation of vascular smooth muscle cells while simultaneously promoting reendothelialization. More significantly, unlike everolimus-coated stents, CPEC-coated stents showed a significant reduction in thrombosis formation even in the absence of ongoing anticoagulant treatment. CONCLUSIONS: The study establishes CPEC-coated stent as a promising new device for cardiovascular interventions. By enhancing reendothelialization and preventing thrombosis, CPEC offers advantages over conventional approaches, including the elimination of the need for anti-clogging drugs, which pave the way for improved therapeutic outcomes and management of atherosclerosis-related medical procedures.

Adipsin in the pathogenesis of cardiovascular diseases.

Adipsin is an adipokine predominantly synthesized in adipose tissues and released into circulation. It is also known as complement factor-D (CFD), acting as the rate-limiting factor in the alternative complement pathway and exerting essential functions on the activation of complement system. The deficiency of CFD in humans is a very rare condition. However, complement overactivation has been implicated in the etiology of numerous disorders, including cardiovascular disease (CVD). Increased circulating level of adipsin has been reported to promote vascular derangements, systemic inflammation, and endothelial dysfunction. Prospective and case-control studies showed that this adipokine is directly associated with all-cause death and rehospitalization in patients with coronary artery disease. Adipsin has also been implicated in pulmonary arterial hypertension, abdominal aortic aneurysm, pre-eclampsia, and type-2 diabetes which is a major risk factor for CVD. Importantly, serum adipsin has been recognized as a unique prognostic marker for assessing cardiovascular diseases. At present, there is paucity of experimental evidence about the precise role of adipsin in the etiology of CVD. However, this mini review provides some insight on the contribution of adipsin in the pathogenesis of CVD and highlights its role on endothelial, smooth muscle and immune cells that mediate cardiovascular functions.

ADAR1 Is Essential for Smooth Muscle Homeostasis and Vascular Integrity.

Vascular smooth muscle cells (VSMCs) play a critical role in maintaining vascular integrity. VSMC dysfunction leads to numerous vascular diseases. Adenosine deaminases acting on RNA 1 (ADAR1), an RNA editing enzyme, has shown both RNA editing and non-editing functions. Global deletion of ADAR1 causes embryonic lethality, but the phenotype of homozygous ADAR1 deletion specifically in SMCs (ADAR1sm-/-) remains to be determined. By crossing ADAR1fl/fl mice with Myh11-CreERT2 mice followed by Tamoxifen induction, we found that ADAR1sm-/- leads to lethality in adult mice 14 days after the induction. Gross examination revealed extensive hemorrhage and detrimental vascular damage in different organs. Histological analyses revealed destruction of artery structural integrity with detachment of elastin laminae from VSMCs in ADAR1sm-/- aortas. Furthermore, ADAR1sm-/- resulted in severe VSMC apoptosis and mitochondrial dysfunction. RNA sequencing analyses of ADAR1sm-/- aorta segments demonstrated profound transcriptional alteration of genes impacting vascular health including a decrease in fibrillin-1 expression. More importantly, ADAR1sm-/- disrupts the elastin and fibrillin-1 interaction, a molecular event essential for artery structure. Our results indicate that ADAR1 plays a critical role in maintaining SMC survival and vascular stability and resilience.

Surfactant protein A promotes western diet-induced hepatic steatosis and fibrosis in mice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) remains the most common cause of liver disease in the United States due to the increased incidence of metabolic dysfunction and obesity. Surfactant protein A (SPA) regulates macrophage function, strongly binds to lipids, and is implicated in renal and idiopathic pulmonary fibrosis (IPF). However, the role of SPA in lipid accumulation, inflammation, and hepatic fibrosis that characterize MASLD remains unknown. SPA deficient (SPA-/-) and age-matched wild-type (WT) control mice were fed a Western diet for 8 weeks to induce MASLD. Blood and liver samples were collected and used to analyze pathological features associated with MASLD. SPA expression was significantly upregulated in livers of mice with MASLD. SPA deficiency attenuated lipid accumulation along with downregulation of genes involved in fatty acid uptake and reduction of hepatic inflammation as evidenced by the diminished macrophage activation, decreased monocyte infiltration, and reduced production of inflammatory cytokines. Moreover, SPA-/- inhibited stellate cell activation, collagen deposit, and liver fibrosis. These results highlight the novel role of SPA in promoting fatty acid uptake into hepatocytes, causing excessive lipid accumulation, inflammation, and fibrosis implicated in the pathogenesis of MASLD.

Digital light 4D printing of bioresorbable shape memory elastomers for personalized biomedical implantation.

Four-dimensional (4D) printing unlocks new potentials for personalized biomedical implantation, but still with hurdles of lacking suitable materials. Herein, we demonstrate a bioresorbable shape memory elastomer (SME) with high elasticity at both below and above its phase transition temperature (Ttrans). This SME can be digital light 3D printed by co-polymerizing glycerol dodecanoate acrylate prepolymer (pre-PGDA) with acrylic acid monomer to form crosslinked Poly(glycerol dodecanoate acrylate) (PGDA)-Polyacrylic acid (PAA), or PGDA-PAA network. The printed complex, free-standing 3D structures with high-resolution features exhibit shape programming properties at a physiological temperature. By tuning the pre-PGDA weight ratios between 55 wt% and 70 wt%, Ttrans varies between 39.2 and 47.2 ℃ while Young's moduli (E) range 40-170 MPa below Ttrans with fractural strain (εf) of 170 %-200 %. Above Ttrans, E drops to 1-1.82 MPa which is close to those of soft tissue. Strikingly, εf of 130-180 % is still maintained. In vitro biocompatibility test on the material shows > 90 % cell proliferation and great cell attachment. In vivo vascular grafting trials underline the geometrical and mechanical adaptability of these 4D printed constructs in regenerating the aorta tissue. Biodegradation of the implants shows the possibility of their full replacement by natural tissue over time. To highlight its potential for personalized medicine, a patient-specific left atrial appendage (LAA) occluder was printed and implanted endovascularly into an in vitro heart model. STATEMENT OF SIGNIFICANCE: 4D printed shape-memory elastomer (SME) implants particularly designed and manufactured for a patient are greatly sought-after in minimally invasive surgery (MIS). Traditional shape-memory polymers used in these implants often suffer from issues like unsuitable transition temperatures, poor biocompatibility, limited 3D design complexity, and low toughness, making them unsuitable for MIS. Our new SME, with an adjustable transition temperature and enhanced toughness, is both biocompatible and naturally degradable, particularly in cardiovascular contexts. This allows implants, like biomedical scaffolds, to be programmed at room temperature and then adapt to the body's physiological conditions post-implantation. Our studies, including in vivo vascular grafts and in vitro device implantation, highlight the SME's effectiveness in aortic tissue regeneration and its promising applications in MIS.

Cell-specific polymerization-driven biomolecular condensate formation fine-tunes root tissue morphogenesis.

Formation of biomolecular condensates can be driven by weak multivalent interactions and emergent polymerization. However, the mechanism of polymerization-mediated condensate formation is less studied. We found lateral root cap cell (LRC)-specific SUPPRESSOR OF RPS4-RLD1 (SRFR1) condensates fine-tune primary root development. Polymerization of the SRFR1 N-terminal domain is required for both LRC condensate formation and optimal root growth. Surprisingly, the first intrinsically disordered region (IDR1) of SRFR1 can be functionally substituted by a specific group of intrinsically disordered proteins known as dehydrins. This finding facilitated the identification of functional segments in the IDR1 of SRFR1, a generalizable strategy to decode unknown IDRs. With this functional information we further improved root growth by modifying the SRFR1 condensation module, providing a strategy to improve plant growth and resilience.

Chronic Intermittent Hypoxia Facilitates the Development of Angiotensin II-Induced Abdominal Aortic Aneurysm in Male Mice.

Obstructive sleep apnea (OSA), characterized by episodes of intermittent hypoxia (IH), is highly prevalent in patients with abdominal aortic aneurysm (AAA). However, whether IH serves as an independent risk factor for AAA development remains to be investigated. Here, we determined the effects of chronic (6 months) IH on angiotensin (Ang II)-induced AAA development in C57BL/6J male mice, and IH underlying mechanisms in cultured vascular smooth muscle cells (SMCs). IH increased abdominal aortic diameter and the incidence of AAA in mice infused with Ang II as assessed by transabdominal ultrasound imaging. Importantly, IH with Ang II augmented aortic elastin degradation and expression of matrix metalloproteinase (MMP)s, mainly MMP8, MMP12 and a disintegrin and metalloproteinase-17 (ADAM17) as measured by histology and immunohistochemistry. Mechanistically, IH increased the activities of MMP2, MMP8, MMP9, MMP12, and ADAM17, while reducing the expression of the MMP regulator, reversion inducing cysteine rich protein with kazal motifs (RECK) in cultured SMCs. Aortic samples from human AAA were associated with decreased RECK and increased expression of ADAM17 and MMPs. These data suggest that IH promotes the development of AAA in association with an increased expression of MMPs and ADAM17, while decreased expression of RECK may be responsible for the increased protease activity. These findings support a potential causal link between OSA and AAA and provide a better understanding of the molecular mechanisms underlying the pathogenesis of AAA.

KCNH2 mutation c.3099_3112del causes congenital long QT syndrome type 2 with gender differences

Abstract Introduction: Long QT Syndrome (LQTS) is an inherited disease with an abnormal electrical conduction system in the heart that can cause sudden death as a result of QT prolongation. LQT2 is the second most common subtype of LQTS caused by loss of function mutations in the potassium voltage-gated channel subfamily H member 2 (KCNH2) gene. Although more than 900 mutations are associated with the LQTS, many of these mutations are not validated or characterized. Methods and results: Sequencing analyses of genomic DNA of a family with LQT2 identified a putative mutation. i.e., KCNH2(NM_000238.3): c.3099_3112del, in KCNH2 gene which appeared to be a definite pathogenic mutation. The family pedigree information showed a gender difference in clinical features and T-wave morphology between male and female patients. The female with mutation exhibited recurring ventricular arrhythmia and syncope, while two male carriers did not show any symptoms. In addition, T-wave in females was much flatter than in males. The female proband showed a positive reaction to the lidocaine test. Lidocaine injection almost completely blocked ventricular arrhythmia and shortened the QT interval by ≥30 ms. Treatment with propranolol, mexiletine, and implantation of cardioverter-defibrillators prevented the sustained ventricular tachycardia, ventricular fibrillation, and syncope, as assessed by a 3-year follow-up evaluation. Conclusions: A putative mutation c.3099_3112del in the KCNH2 gene causes LQT2 syndrome, and the pathogenic mutation mainly causes symptoms in female progeny.