The TMEM16A channel as a potential therapeutic target in vascular disease.

PURPOSE OF REVIEW: The transmembrane protein 16A (TMEM16A) Ca 2+ -activated Cl - channel constitutes a key depolarising mechanism in vascular smooth muscle and contractile pericytes, while in endothelial cells the channel is implicated in angiogenesis and in the response to vasoactive stimuli. Here, we offer a critical analysis of recent physiological investigations and consider the potential for targeting TMEM16A channels in vascular disease. RECENT FINDINGS: Genetic deletion or pharmacological inhibition of TMEM16A channels in vascular smooth muscle decreases artery tone and lowers systemic blood pressure in rodent models. Inhibition of TMEM16A channels in cerebral cortical pericytes protects against ischemia-induced tissue damage and improves microvascular blood flow in rodent stroke models. In endothelial cells, the TMEM16A channel plays varied roles including modulation of cell division and control of vessel tone through spread of hyperpolarisation to the smooth muscle cells. Genetic studies implicate TMEM16A channels in human disease including systemic and pulmonary hypertension, stroke and Moyamoya disease. SUMMARY: The TMEM16A channel regulates vascular function by controlling artery tone and capillary diameter as well as vessel formation and histology. Preclinical and clinical investigations are highlighting the potential for therapeutic exploitation of the channel in a range of maladaptive states of the (micro)circulation.

The activator protein-1 complex governs a vascular degenerative transcriptional programme in smooth muscle cells to trigger aortic dissection and rupture.

BACKGROUND AND AIMS: Stanford type A aortic dissection (AD) is a degenerative aortic remodelling disease marked by an exceedingly high mortality without effective pharmacologic therapies. Smooth muscle cells (SMCs) lining tunica media adopt a range of states, and their transformation from contractile to synthetic phenotypes fundamentally triggers AD. However, the underlying pathomechanisms governing this population shift and subsequent AD, particularly at distinct disease temporal stages, remain elusive. METHODS: Ascending aortas from nine patients undergoing ascending aorta replacement and five individuals undergoing heart transplantation were subjected to single-cell RNA sequencing. The pathogenic targets governing the phenotypic switch of SMCs were identified by trajectory inference, functional scoring, single-cell regulatory network inference and clustering, regulon, and interactome analyses and confirmed using human ascending aortas, primary SMCs, and a ß-aminopropionitrile monofumarate-induced AD model. RESULTS: The transcriptional profiles of 93 397 cells revealed a dynamic temporal-specific phenotypic transition and marked elevation of the activator protein-1 (AP-1) complex, actively enabling synthetic SMC expansion. Mechanistically, tumour necrosis factor signalling enhanced AP-1 transcriptional activity by dampening mitochondrial oxidative phosphorylation (OXPHOS). Targeting this axis with the OXPHOS enhancer coenzyme Q10 or AP-1-specific inhibitor T-5224 impedes phenotypic transition and aortic degeneration while improving survival by 42.88% (58.3%-83.3% for coenzyme Q10 treatment), 150.15% (33.3%-83.3% for 2-week T-5224), and 175.38% (33.3%-91.7% for 3-week T-5224) in the ß-aminopropionitrile monofumarate-induced AD model. CONCLUSIONS: This cross-sectional compendium of cellular atlas of human ascending aortas during AD progression provides previously unappreciated insights into a transcriptional programme permitting aortic degeneration, highlighting a translational proof of concept for an anti-remodelling intervention as an attractive strategy to manage temporal-specific AD by modulating the tumour necrosis factor-OXPHOS-AP-1 axis.

Endothelial-Smooth Muscle Cell Interactions in a Shear-Exposed Intimal Hyperplasia on-a-Dish Model to Evaluate Therapeutic Strategies.

Intimal hyperplasia (IH) represents a major challenge following cardiovascular interventions. While mechanisms are poorly understood, the inefficient preventive methods incentivize the search for novel therapies. A vessel-on-a-dish platform is presented, consisting of direct-contact cocultures with human primary endothelial cells (ECs) and smooth muscle cells (SMCs) exposed to both laminar pulsatile and disturbed flow on an orbital shaker. With contractile SMCs sitting below a confluent EC layer, a model that successfully replicates the architecture of a quiescent vessel wall is created. In the novel IH model, ECs are seeded on synthetic SMCs at low density, mimicking reendothelization after vascular injury. Over 3 days of coculture, ECs transition from a network conformation to confluent 2D islands, as promoted by pulsatile flow, resulting in a "defected" EC monolayer. In defected regions, SMCs incorporated plasma fibronectin into fibers, increased proliferation, and formed multilayers, similarly to IH in vivo. These phenomena are inhibited under confluent EC layers, supporting therapeutic approaches that focus on endothelial regeneration rather than inhibiting proliferation, as illustrated in a proof-of-concept experiment with Paclitaxel. Thus, this in vitro system offers a new tool to study EC-SMC communication in IH pathophysiology, while providing an easy-to-use translational disease model platform for low-cost and high-content therapeutic development.

JNJ0966 inhibits PDGF-BB-induced airway smooth muscle cell proliferation and extracellular matrix production by regulating MMP-9.

The increased proliferation and extracellular matrix (ECM) production of airway smooth muscle cells (ASMCs) are crucial factors in asthma progression. JNJ0966, one of the metalloproteinase-9 (MMP-9)-specific inhibitors, has been demonstrated to be involved in the progression and development of diversified diseases. Nevertheless, the function of JNJ0966 in ASMCs remains unclear. This study aimed at investigating the effects of JNJ0966 on asthma progression. In our study, the platelet-derived growth factor BB (PDGF-BB) was first utilized to stimulate the cell model for asthma. Results demonstrated that the cell viability of ASMCs was increased by PDGF-BB (0, 10, 20, and 30 ng/mL) in a dose-dependent manner. Further investigation revealed that JNJ0966 inhibited the cell activity and migration ability of PDGF-BB-induced ASMCs. In addition, JNJ0966 relieved ECM deposition in PDGF-BB-induced ASMCs. Finally, through rescue assays, the results showed that overexpression of MMP-9 reversed the inhibitory effects of JNJ0966 on cell viability and ECM deposition in ASMCs. In conclusion, our findings suggested that JNJ0966 inhibited PDGF-BB-induced ASMC proliferation and ECM production by modulating MMP-9. These findings might provide novel insight for the treatment of asthma.

GATA6 is a crucial factor for Myocd expression in the visceral smooth muscle cell differentiation program of the murine ureter.

Smooth muscle cells (SMCs) are a crucial component of the mesenchymal wall of the ureter, as they account for the efficient removal of the urine from the renal pelvis to the bladder by means of their contractile activity. Here, we show that the zinc-finger transcription factor gene Gata6 is expressed in mesenchymal precursors of ureteric SMCs under the control of BMP4 signaling. Mice with a conditional loss of Gata6 in these precursors exhibit a delayed onset and reduced level of SMC differentiation and peristaltic activity, as well as dilatation of the ureter and renal pelvis (hydroureternephrosis) at birth and at postnatal stages. Molecular profiling revealed a delayed and reduced expression of the myogenic driver gene Myocd, but the activation of signaling pathways and transcription factors previously implicated in activation of the visceral SMC program in the ureter was unchanged. Additional gain-of-function experiments suggest that GATA6 cooperates with FOXF1 in Myocd activation and SMC differentiation, possibly as pioneer and lineage-determining factors, respectively.

Computational analysis of the role of mechanosensitive Notch signaling in arterial adaptation to hypertension.

Arteries grow and remodel in response to mechanical stimuli. Hypertension, for example, results in arterial wall thickening. Cell-cell Notch signaling between vascular smooth muscle cells (VSMCs) is known to be involved in this process, but the underlying mechanisms are still unclear. Here, we investigated whether Notch mechanosensitivity to strain may regulate arterial thickening in hypertension. We developed a multiscale computational framework by coupling a finite element model of arterial mechanics, including residual stress, to an agent-based model of mechanosensitive Notch signaling, to predict VSMC phenotypes as an indicator of growth and remodeling. Our simulations revealed that the sensitivity of Notch to strain at mean blood pressure may be a key mediator of arterial thickening in hypertensive arteries. Further simulations showed that loss of residual stress can have synergistic effects with hypertension, and that changes in the expression of Notch receptors, but not Jagged ligands, may be used to control arterial growth and remodeling and to intensify or counteract hypertensive thickening. Overall, we identify Notch mechanosensitivity as a potential mediator of vascular adaptation, and we present a computational framework that can facilitate the testing of new therapeutic and regenerative strategies.

Polarized light retardation analysis allows for the evaluation of tension in individual stress fibers.

The tension in the stress fibers (SFs) of cells plays a pivotal role in determining biological processes such as cell migration, morphological formation, and protein synthesis. Our previous research developed a method to evaluate the cellular contraction force generated in SFs based on photoelasticity-associated retardation of polarized light; however, we employed live cells, which could have caused an increase in retardation and not contraction force. Therefore, the present study aimed to confirm that polarized light retardation increases inherently due to contraction, regardless of cell activity. We also explored the reason why retardation increased with SF contractions. We used SFs physically isolated from vascular smooth muscle cells to stop cell activity. The retardation of SFs was measured after ATP administration, responsible for contracting SFs. The SFs were imaged under optical and electron microscopes to measure SF length, width, and retardation. The retardation of isolated SFs after ATP administration was significantly higher than before. Thus, we confirmed that retardation increased with elevated tension in individual SFs. Furthermore, the SF diameter decreased while the SF length remained almost constant. Thus, we conclude that a contraction force-driven increase in the density of SFs is the main factor for the rise in polarized light retardation.

JNJ0966 inhibits PDGF-BB-induced airway smooth muscle cell proliferation and extracellular matrix production by regulating MMP-9

The increased proliferation and extracellular matrix (ECM) production of airway smooth muscle cells (ASMCs) are crucial factors in asthma progression. JNJ0966, one of the metal-loproteinase-9 (MMP-9)-specific inhibitors, has been demonstrated to be involved in the pro-gression and development of diversified diseases. Nevertheless, the function of JNJ0966 in ASMCs remains unclear. This study aimed at investigating the effects of JNJ0966 on asthma progression. In our study, the platelet-derived growth factor BB (PDGF-BB) was first utilized to stimulate the cell model for asthma. Results demonstrated that the cell viability of ASMCs was increased by PDGF-BB (0, 10, 20, and 30 ng/mL) in a dose-dependent manner. Further investigation revealed that JNJ0966 inhibited the cell activity and migration ability of PDGF-BB-induced ASMCs. In addition, JNJ0966 relieved ECM deposition in PDGF-BB-induced ASMCs. Finally, through rescue assays, the results showed that overexpression of MMP-9 reversed the inhibitory effects of JNJ0966 on cell viability and ECM deposition in ASMCs. In conclusion, our findings suggested that JNJ0966 inhibited PDGF-BB-induced ASMC proliferation and ECM pro-duction by modulating MMP-9. These findings might provide novel insight for the treatment of asthm (AU)

Generation of Embryonic Origin-Specific Vascular Smooth Muscle Cells from Human Induced Pluripotent Stem Cells.

Vascular smooth muscle cells (VSMCs), a highly mosaic tissue, arise from multiple distinct embryonic origins and populate different regions of our vascular network with defined boundaries. Accumulating evidence has revealed that the heterogeneity of VSMC origins contributes to region-specific vascular diseases such as atherosclerosis and aortic aneurysm. These findings highlight the necessity of taking into account lineage-dependent responses of VSMCs to common vascular risk factors when studying vascular diseases. This chapter describes a reproducible, stepwise protocol for the generation of isogenic VSMC subtypes originated from proepicardium, second heart field, cardiac neural crest, and ventral somite using human induced pluripotent stem cells. By leveraging this robust induction protocol, patient-derived VSMC subtypes of desired embryonic origins can be generated for disease modeling as well as drug screening and development for vasculopathies with regional susceptibility.

Utilizing the Precision-Cut Lung Slice to Study the Contractile Regulation of Airway and Intrapulmonary Arterial Smooth Muscle.

Smooth muscle cells (SMC) mediate the contraction of the airway and the intrapulmonary artery to modify airflow resistance and pulmonary circulation, respectively, hence playing a critical role in the homeostasis of the pulmonary system. Deregulation of SMC contractility contributes to several pulmonary diseases, including asthma and pulmonary hypertension. However, due to limited tissue access and a lack of culture systems to maintain in vivo SMC phenotypes, molecular mechanisms underlying the deregulated SMC contractility in these diseases remain fully identified. The precision-cut lung slice (PCLS) offers an ex vivo model that circumvents these technical difficulties. As a live, thin lung tissue section, the PCLS retains SMC in natural surroundings and allows in situ tracking of SMC contraction and intracellular Ca2+ signaling that regulates SMC contractility. Here, a detailed mouse PCLS preparation protocol is provided, which preserves intact airways and intrapulmonary arteries. This protocol involves two essential steps before subjecting the lung lobe to slicing: inflating the airway with low-melting-point agarose through the trachea and infilling pulmonary vessels with gelatin through the right ventricle. The PCLS prepared using this protocol can be used for bioassays to evaluate Ca2+-mediated contractile regulation of SMC in both the airway and the intrapulmonary arterial compartments. When applied to mouse models of respiratory diseases, this protocol enables the functional investigation of SMC, thereby providing insight into the underlying mechanism of SMC contractility deregulation in diseases.

A molecular complex of Cav1.2/CaMKK2/CaMK1a in caveolae is responsible for vascular remodeling via excitation-transcription coupling.

Elevation of intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+/calmodulin-dependent kinases (CaMK) and promotes gene transcription. This signaling pathway is referred to as excitation­transcription (E-T) coupling. Although vascular myocytes can exhibit E-T coupling, the molecular mechanisms and physiological/pathological roles are unknown. Multiscale analysis spanning from single molecules to whole organisms has revealed essential steps in mouse vascular myocyte E-T coupling. Upon a depolarizing stimulus, Ca2+ influx through Cav1.2 voltage-dependent Ca2+ channels activates CaMKK2 and CaMK1a, resulting in intranuclear CREB phosphorylation. Within caveolae, the formation of a molecular complex of Cav1.2/CaMKK2/CaMK1a is promoted in vascular myocytes. Live imaging using a genetically encoded Ca2+ indicator revealed direct activation of CaMKK2 by Ca2+ influx through Cav1.2 localized to caveolae. CaMK1a is phosphorylated by CaMKK2 at caveolae and translocated to the nucleus upon membrane depolarization. In addition, sustained depolarization of a mesenteric artery preparation induced genes related to chemotaxis, leukocyte adhesion, and inflammation, and these changes were reversed by inhibitors of Cav1.2, CaMKK2, and CaMK, or disruption of caveolae. In the context of pathophysiology, when the mesenteric artery was loaded by high pressure in vivo, we observed CREB phosphorylation in myocytes, macrophage accumulation at adventitia, and an increase in thickness and cross-sectional area of the tunica media. These changes were reduced in caveolin1-knockout mice or in mice treated with the CaMKK2 inhibitor STO609. In summary, E-T coupling depends on Cav1.2/CaMKK2/CaMK1a localized to caveolae, and this complex converts [Ca2+]i changes into gene transcription. This ultimately leads to macrophage accumulation and media remodeling for adaptation to increased circumferential stretch.

Differential Regulation by Mechanical Stretch of the Expressions of Large-Conductance Ca2+-Activated K+ Channel and L-Type Voltage-Dependent Ca2+ Channel in Rat Uterine Smooth Muscle Cells.

Large-conductance Ca2+-activated K+ (BKCa) channel and L-type voltage-dependent Ca2+ channel (L-VDCC) play important roles in regulating uterine contractility. The uterus stretch, occurring during pregnancy, is a critical factor to trigger uterine contraction. However, how mechanical stimuli impact the two channels remains unknown. Here we investigated the effects of exposure to mechanical stretches with varying magnitudes and durations on expressions of the two channels in rat uterine smooth muscle cells. Our results show that stretch down-regulates the BKCa channel expression but upregulates the L-VDCC expression. These findings are helpful to better understand the roles of L-VDCC and BKCa channel in stretch-triggered uterine contraction.

Succinate: A Novel Mediator to Promote Atherosclerotic Lesion Progression.

Succinate is an important intermediate product of mitochondrial energy metabolism. Recent studies revealed that beyond its known traditional metabolic functions, succinate plays important roles in signal transduction, immunity, inflammation, and posttranslational modification. Recent studies showed that patients and mouse models with cardiovascular disease have high levels of serum succinate and succinate accumulation. Atherosclerosis (As) is the pathological basis of cardiovascular and peripheral vascular diseases, such as coronary heart disease, cerebral infarction, and peripheral vascular disease, and is a major factor affecting human health. This article reviews the progression of succinate in As diseases and its underlying mechanisms.

Impact of Notch3 Activation on Aortic Aneurysm Development in Marfan Syndrome.

BACKGROUND: The leading cause of mortality in patients with Marfan syndrome (MFS) is thoracic aortic aneurysm and dissection. Notch signaling is essential for vessel morphogenesis and function. However, the role of Notch signaling in aortic pathology and aortic smooth muscle cell (SMC) differentiation in Marfan syndrome (MFS) is not completely understood. METHODS: RNA-sequencing on ascending aortic tissue from a mouse model of MFS, Fbn1mgR/mgR , and wild-type controls was performed. Notch 3 expression and activation in aortic tissue were confirmed with real-time RT-PCR, immunohistochemistry, and Western blot. Fbn1mgR/mgR and wild-type mice were treated with a γ-secretase inhibitor, DAPT, to block Notch activation. Aortic aneurysms and rupture were evaluated with connective tissue staining, ultrasound, and life table analysis. RESULTS: The murine RNA-sequencing data were validated with mouse and human MFS aortic tissue, demonstrating elevated Notch3 activation in MFS. Data further revealed that upregulation and activation of Notch3 were concomitant with increased expression of SMC contractile markers. Inhibiting Notch3 activation with DAPT attenuated aortic enlargement and improved survival of Fbn1mgR/mgR mice. DAPT treatment reduced elastin fiber fragmentation in the aorta and reversed the differentiation of SMCs. CONCLUSIONS: Our data demonstrated that matrix abnormalities in the aorta of MFS are associated with increased Notch3 activation. Enhanced Notch3 activation in MFS contributed to aortic aneurysm formation in MFS. This might be mediated by inducing a contractile phenotypic change of SMC. Our results suggest that inhibiting Notch3 activation may provide a strategy to prevent and treat aortic aneurysms in MFS.

Yes-associated protein reacts differently in vascular smooth muscle cells under different intensities of mechanical stretch.

Vascular smooth muscle cells (VSMCs) are stromal cells of the vascular wall and are continually exposed to mechanical signals. The loss of VSMCs is closely related to the occurrence of many vascular diseases, such as aortic aneurysms and aortic dissection. The proliferation and apoptosis of VSMCs are mechanically stimulated. Yes-associated protein (YAP), one of the core components of the Hippo pathway, plays a key role in the response of VSMCs to mechanical signals. In this study, we tested the impact of different intensities of mechanical stretch on the proliferation and apoptosis of VSMCs, as well as YAP. We tested VSMCs' proliferation and apoptosis and YAP reaction via immunocytochemistry, western blotting, CCK-8 and flow cytometric analysis. We found that 10% elongation could increase the phosphorylation of YAP and prevent it from entering the nucleus, as well as inhibit cell proliferation and promote apoptosis. However, 15% elongation reduced YAP phosphorylation and promoted its nuclear entry, thereby promoting cell proliferation and inhibiting apoptosis. Accordingly, YAP knockdown suppressed the phenotype of VMSCs induced by 15% elongation. Taken together, YAP regulates proliferation and apoptosis of VSMCs differently under different intensity of mechanical stretch. Mechanical stretch with appropriate intensity can promote the proliferation and inhibit apoptosis of VSMCs by activating YAP.

TRPV1 in arteries enables a rapid myogenic tone.

Arterioles maintain blow flow by adjusting their diameter in response to changes in local blood pressure. In this process called the myogenic response, a vascular smooth muscle mechanosensor controls tone predominantly through altering the membrane potential. In general, myogenic responses occur slowly (minutes). In the heart and skeletal muscle, however, tone is activated rapidly (tens of seconds) and terminated by brief (100 ms) arterial constrictions. Previously, we identified extensive expression of TRPV1 in the smooth muscle of arterioles supplying skeletal muscle, heart and fat. Here we reveal a critical role for TRPV1 in the rapid myogenic tone of these tissues. TRPV1 antagonists dilated skeletal muscle arterioles in vitro and in vivo, increased coronary flow in isolated hearts, and transiently decreased blood pressure. All of these pharmacologic effects were abolished by genetic disruption of TRPV1. Stretch of isolated vascular smooth muscle cells or raised intravascular pressure in arteries triggered Ca2+ signalling and vasoconstriction. The majority of these stretch-responses were TRPV1-mediated, with the remaining tone being inhibited by the TRPM4 antagonist, 9-phenantrol. Notably, tone developed more quickly in arteries from wild-type compared with TRPV1-null mice. Furthermore, the immediate vasodilation following brief constriction of arterioles depended on TRPV1, consistent with a rapid deactivation of TRPV1. Pharmacologic experiments revealed that membrane stretch activates phospholipase C/protein kinase C signalling combined with heat to activate TRPV1, and in turn, L-type Ca2+ channels. These results suggest a critical role, for TRPV1 in the dynamic regulation of myogenic tone and blood flow in the heart and skeletal muscle. KEY POINTS: We explored the physiological role of TRPV1 in vascular smooth muscle. TRPV1 antagonists dilated skeletal muscle arterioles both ex vivo and in vivo, increased coronary perfusion and decreased systemic blood pressure. Stretch of arteriolar myocytes and increases in intraluminal pressure in arteries triggered rapid Ca2+ signalling and vasoconstriction respectively. Pharmacologic and/or genetic disruption of TRPV1 significantly inhibited the magnitude and rate of these responses. Furthermore, disrupting TRPV1 blunted the rapid vasodilation evoked by arterial constriction. Pharmacological experiments identified key roles for phospholipase C and protein kinase C, combined with temperature, in TRPV1-dependent arterial tone. These results show that TRPV1 in arteriolar myocytes dynamically regulates myogenic tone and blood flow in the heart and skeletal muscle.

Atomic Force Microscopy Stiffness Mapping in Human Aortic Smooth Muscle Cells.

Aortic smooth muscle cells (SMCs) play a vital role in maintaining mechanical homeostasis in the aorta. We recently found that SMCs of aneurysmal aortas apply larger traction forces than SMCs of healthy aortas. This result was explained by the significant increase of hypertrophic SMCs abundance in aneurysms. In this study, we investigate whether the cytoskeleton stiffness of SMCs may also be altered in aneurysmal aortas. For that, we use atomic force microscopy (AFM) nano-indentation with a specific mode that allows subcellular-resolution mapping of the local stiffness across a specified region of interest of the cell. Aortic SMCs from a commercial human lineage (AoSMCs, Lonza) and primary aneurysmal SMCs (AnevSMCs) are cultured in conditions promoting the development of their contractile apparatus, and seeded on hydrogels with stiffness properties of 12 kPa and 25 kPa. Results show that all SMCs exhibit globally a lognormal stiffness distribution, with medians in the range 10-30 kPa. The mean of stiffness distributions is 16 kPa in aneurysmal SMCs and 12 kPa in healthy cells, but the differences are not statistically significant due to the large dispersion of AFM nano-indentation stiffness. We conclude that the possible alterations previously found in aneurysmal SMCs do not affect significantly the AFM nano-indentation stiffness of their cytoskeleton.