Emerging role of YAP/TAZ in vascular mechanotransduction and disease

Cells have an incredible ability to physically interact with neighboring cells and their environment. They can detect and respond to mechanical forces by converting mechanical stimuli into biochemical signals in a process known as mechanotransduction. This is a key process for the adaption of vascular smooth muscle and endothelial cells to altered flow and pressure conditions. Mechanical stimuli, referring to a physical force exerted on cells, are primarily sensed by transmembrane proteins and the actin cytoskeleton, which initiate a cascade of intracellular events, including the activation of signaling pathways, ion channels, and transcriptional regulators. Recent work has highlighted an important role of the transcriptional coactivators YAP/TAZ for mechanotransduction in vascular cells. Interestingly, the activity of YAP/TAZ decreases with age, providing a potential mechanism for the detrimental effects of aging in the vascular wall. In this review, we summarize the current knowledge on the functional role of YAP and TAZ in vascular endothelial and smooth muscle cells for mechanotransduction in homeostasis and disease. In particular, the review is focused on in vivo observations from conditional knockout (KO) models of YAP/TAZ and the potential implications these studies may have for our understanding of vascular disease development.

muscle cells in small arteries contract in response to mechanical stress, reducing vascular diameter and increasing wall thickness, both of which mitigate wall stress according to the law of Laplace.Over the long term, altered mechanical stimuli result in remodeling of the vascular wall, defined as a permanent change in vascular structure. 30rthermore, adaptation to elevated mechanical stimuli promotes strengthening of the smooth cytoskeletal network.This network encompasses contractile actin filaments, intermediate filaments, and adhesion sites on the cell membrane. 1,2Moreover, smooth muscle cells produce extracellular matrix proteins that lend structural support to the vascular cells and alleviate mechanical tension.
Similar to smooth muscle cells, endothelial cells rely on mechanotransduction for many of their functions.Structurally, endothelial cells adapt to shifting mechanical forces by aligning in relation to shear and stretch stimuli. 18Furthermore, endothelial cells maintain the physical barrier between the blood and the extravascular environment through adhesion sites connected to the intracellular cytoskeleton.Functionally, endothelial cells produce vasoactive substances such as nitric oxide in response to shear stress and play a central role in angiogenesis, the formation of new blood vessels.
In summary, mechanotransduction is required for several key processes in both smooth muscle and endothelial cells, and a reduced ability to detect and adapt to mechanical stimuli increases the risk of vascular dysfunction.

| YAP/ TA Z TR ANSCRIP TIONAL COAC TI VATO R S
The YAP (yes-associated protein; YAP1) and TAZ (WW domaincontaining transcription regulator 1; WWTR1) proteins are mechanosensitive transcriptional coactivators and effectors of the Hippo signaling pathway, regulating the TEAD family of transcription factors.
The Hippo pathway is a critical regulator of various cellular processes, including cell proliferation, organ size, and tissue regeneration (reviewed in 54 ).Anchored in a conserved kinase cascade, which involves the large tumor suppressor 1/2 (LATS1/2) and mammalian sterile 20-like 1/2 (MST1/2) kinases, the Hippo pathway principally controls the activity and intracellular localization of YAP and TAZ (Figure 1).When the Hippo pathway is activated, YAP and TAZ are phosphorylated, leading to their cytoplasmic retention and degradation.Conversely, inhibition of the Hippo pathway results in nuclear translocation of YAP/TAZ and activation of transcriptional programs, including genes coding for components of the intracellular cytoskeleton and extracellular matrix proteins.
In addition to the kinases of the Hippo pathway, various phosphatases are involved in regulating the activity of YAP and TAZ.By dephosphorylating different components of the Hippo pathway, phosphatases can increase the transcriptional coactivity of YAP and TAZ.An example of such a phosphatase is protein phosphatase type 2A (PP2A).Together with its regulatory subunit PR55α, PP2A promotes YAP activity by (1) dephosphorylating YAP, (2) destabilizing LATS, and (3) inhibiting mob kinase activator 1 (MOB1)-mediated LATS autophosphorylation and subsequent activation (Figure 1). 16P and TAZ are expressed in various cell types, and their expression levels likely vary during development.However, data from the GTEx Portal and the Human Protein Atlas strongly support the enrichment of YAP in vascular smooth muscle cells of human adults.
In contrast, TAZ appears more ubiquitously expressed and is preferentially found in the endothelial cells of human blood vessels. 41nerally, YAP/TAZ exert their primary biological functions by binding to and forming a complex with one of the four TEAD members (TEAD1-4), thus resulting in the translation of their target genes. 33,56,58All members of the TEAD family share a highly conserved DNA binding domain called the TEA domain.This DNAbinding domain has a consensus DNA sequence 5'-CATTCCA/T-3' called the MCAT element.TEAD-dependent transcription may also have cell-specific effects.For example, it has been shown that TEAD regulates the expression of myocardin, a transcriptional coactivator of serum response factor (SRF), which is specifically expressed in cardiac muscle and smooth muscle cells and is a critical regulator of smooth muscle-specific genes. 6,7,26Since myocardin is suggested to promote its own splicing, reduced myocardin expression may also affect the activity of the expressed myocardin. 22sregulation of the Hippo pathway has been associated with various diseases, including vascular disease. 43,47Understanding the molecular mechanisms and cellular processes involved in disease progression due to YAP/TAZ dysregulation can lead us to identify novel therapeutic targets.This may enable precision medicine approaches that target dysregulated factors in the vascular wall following loss of cellular mechanotransduction.

| ROLE OF YAP/ TA Z IN MECHANOS ENS ING
Both in vitro and in vivo studies have recognized YAP and TAZ as pivotal regulators of cellular mechanotransduction. 41 This was first reported by Dupont et al., suggesting that YAP and TAZ activation occurs, independent of the Hippo pathway, in response to various mechanical stimuli, including extra cellular matrix substrate stiffness, cell geometry, and cytoskeletal tension. 11 and others have previously demonstrated that mechanical stretch promotes actin polymerization by activating the Rho/Rhokinase signaling pathway. 2,55A depleted pool of monomeric G-actin results in the release of G-actin-bound myocardin-dependent transcription factors (MRTFs), which then translocate to the nucleus.In the nucleus, MRTFs interact with SRF to induce transcription of cytoskeletal genes. 27triguingly, YAP/TAZ-dependent mechanosensing also requires Rho-activation, and is regulated by the actin cytoskeleton, but it is not directly controlled by G-actin. 11Instead, the mechanosensitive activation of YAP/TAZ is suggested to be modulated via angiomotin (AMOT), which binds competitively to F-actin and YAP/TAZ. 12,57hus, a low abundance of actin filaments promotes the interaction between YAP/TAZ and AMOT, inhibiting YAP/TAZ activity and expression (Figure 1).Conversely, under conditions of elevated mechanical strain, actin polymerization increases AMOT binding to actin filaments, consequently releasing the YAP/TAZ inhibition. 12,57 the vascular wall, recent studies have demonstrated that YAP/ TAZ mechanosensing is paramount for maintaining vascular integrity. 9,41Furthermore, YAP/TAZ activity decreases with age in mice, which results in cGAS-STING-induced vascular inflammation. 39This provides a potential link between aging, loss of mechanotransduction, and development of vascular disease, although the effects in human vessels are not yet known.Furthermore, while we and others have demonstrated a critical role of YAP/TAZ in maintaining vascular integrity (discussed below), the potential role of reduced YAP/TAZ expression in the aging vasculature has not yet been investigated.
Nonetheless, it is worth noting that forced expression of constitutively active YAP in cardiomyocytes in vivo can stimulate cardiac regeneration and improve contractile function following myocardial infarction. 52Similar results have been reported in liver regeneration, which is impaired in aged mice, potentially due to reduced YAP activation.While young livers regenerated after injury, knockdown of MST1/2 to enhance YAP-activation was required for liver regeneration in aged mice. 23

| IMP ORTAN CE OF YAP/ TA Z IN S MOOTH MUSCLE CELL S
Initial reports in the field clearly demonstrated the critical importance of YAP during embryonic development.Yap1 −/− mouse embryos presented vascular defects in the yolk sac and a subsequent developmental arrest at E8.5. 28Thus, tissue-specific deletion of YAP/TAZ is required to evaluate the in vivo importance.However, work by Wang et al. demonstrated that even conditional deletion of YAP in cardiac and smooth muscle during development resulted in perinatal lethality.This was associated with severe cardiovascular abnormalities, including myocardial hypoplasia, dilated cardiomyopathy, and aortic aneurysms, potentially caused by reduced cardiomyocyte and smooth muscle cell proliferation via induction of cell cycle arrest proteins. 49These studies suggest the critical importance F I G U R E 1 Dual mechanisms leading to activation of YAP/TAZ.Nuclear translocation of YAP/TAZ can be induced by mechanical stretch or other stimuli leading to Rho activation, Actin polymerization, and sequestration of AMOT to filamentous Actin.In addition, reduced activation of the Hippo-signaling pathway involving MST and LATS kinases leads to reduced degradation of YAP/TAZ.AMOT, angiomotin; ANKRD1, ankyrin repeat domain-containing protein 1; CTGF, connective-tissue growth factor; CYR61, cysteine-rich angiogenic inducer 61; LATS1/2, large tumor suppressor kinase 1/2; LIMK, lim domain kinase; MOB1, mob kinase activator 1; MST1/2, macrophage stimulating 1/2; PPA2, protein phosphatase type 2A; PR55α, PPA2 subunit B isoform alpha; Rho, rhodopsin; ROCK, rho associated coiled-coil containing protein kinase; SAV1, Salvador family WW domain-containing protein 1; TAZ, WW domain-containing transcription regulator 1; TEAD, tea domain transcription factor; YAP, yes-associated protein.
of YAP for smooth muscle development but preclude any investigation of its role in vascular function and disease in adult mice.
To circumvent the embryonic lethality, we and others have exploited the tamoxifen-inducible smooth muscle myosin heavy chain (Myh11)-driven Cre generated by Offermanns and coworkers 51 crossed with mice that possess loxP sites on either side of exon 2 of the WWTR1 and YAP1 gene. 34Tamoxifen-inducible deletion of smooth muscle YAP/TAZ using this mouse model (that we denote M11-YT-KO) results in a remarkably rapid gastrointestinal phenotype, including loss of peristalsis and development of lethal colonic pseudo-obstruction 12-14 days after the first tamoxifen injection. 10The loss of contractile function was preceded by decreased expression of contractile markers and muscarinic receptors in the large intestine.Notably, the uniquely rapid and severe phenotype observed in M11-YT-KO mice suggests that even a minor dysregulation of Hippo-related proteins in smooth muscle can result in detrimental consequences. 9It is therefore important to clarify whether dysregulation of the Hippo-signaling pathway and/or YAP/TAZ activity is associated with human vascular disease states.
The short lifespan of the M11-YT-KO is a limitation for studies of the vascular phenotype, which generally requires longer time to develop.
Even so, inducible deletion of smooth muscle YAP/TAZ has been shown to result in rapid loss of vascular smooth muscle contractile differentiation and function 8,44 (Figure 2).Studies by Wang et al. also demonstrated that the deletion of smooth muscle YAP/TAZ promotes a shift from a contractile to an osteogenic phenotype and vascular calcification. 44This effect is potentially mediated through the release and nuclear translocation of Dishevelled 3 (DVL3), a mediator of the Wnt signaling pathway.
To determine the importance of smooth muscle YAP/TAZ in vascular mechanotransduction, we investigated myogenic responses and stretch-induced contractile differentiation in vessels from M11-YT-KO mice. 8Myogenic responses are typically evaluated using a pressure myograph, where small mesenteric arteries from mice are cannulated and pressurized ex vivo.The diameter of the vessel is recorded at various intraluminal pressure levels.In wild-type mesenteric arteries, an increased intraluminal pressure initially results in passive dilatation, but at pressure levels above ~70 mmHg, the arteries contract in response to the increased wall stress.However, deletion of smooth muscle YAP/TAZ, even for only 9-11 days, results in a complete loss of this myogenic response. 8Furthermore, stretch-dependent contractile differentiation was diminished in M11-YT-KO.This was evaluated in an organ culture model where a load was applied to the vessel to maintain mechanical stretch.
The main purpose of the adaptation to mechanical forces in the vascular wall is to maintain wall stress within an adequate range to prevent tissue damage.Wall stress is directly proportional to the diameter of the vessel and inversely proportional to the wall thickness.During contraction, the cross-sectional area of the vessel wall is unchanged, and wall thickness will, therefore, increase when the diameter decreases.Thus, the myogenic response effectively reduces wall stress by reducing diameter and increasing wall thickness.
Although the acute response only temporarily alleviates wall stress, sustained contraction of arteries can eventually lead to a permanent change in diameter, a process known as inward remodeling. 40terestingly, passive force-circumference and pressure circumference relationships under nominally calcium-free conditions revealed F I G U R E 2 Effects of YAP/TAZ deletion in smooth muscle.In vivo, reduced expression of YAP/TAZ in vascular smooth muscle cells results in (A) downregulation of myocardin expression and/or activity, decreased contractile differentiation and function, and subsequent elevated wall stress resulting from an increased vessel diameter and decreased wall thickness, (B) activation of chondrogenic and osteogenic transdifferentiation of smooth muscle due to increased expression of SOX9, and (C) activation of vascular inflammation and aging through induction of the cGAS-STING pathway.ACTR2, actin-related protein 2; cGAS-STING, cyclic GMP-AMP synthase stimulator of interferon response CGAMP interactor; LMNB1, lamin B1; SMC, smooth muscle cells; SOX9, SRY-box transcription factor 9; TAZ, WW domaincontaining transcription regulator 1; YAP, yes-associated protein.
that deletion of smooth muscle YAP/TAZ for 9-11 days resulted in an increased diameter of both tail and mesenteric arteries at physiological tension and pressure levels. 8Thus, not only are the M11-YT-KO unable to counteract increased wall stress by myogenic contraction, but they are also subject to higher wall stress in the relaxed state of the vessel wall.In addition, reduced contractile responses to various vasoactive agonists likely contribute to the elevated wall stress levels in YAP/TAZ KO mice. 4,8,44Taken together, these results suggest that YAP and TAZ in smooth muscle are critically important to sense and adapt to variations in blood pressure by contractile differentiation, myogenic contraction, and inward remodeling (Figure 2).The inability of the vascular wall to adapt to elevated pressure levels was further investigated in hypertensive M11-YT-KO mice.It was demonstrated that deletion of smooth muscle YAP/TAZ in hypertensive mice resulted in severe pathological consequences, including arterial lesions characterized by neointimal hyperplasia, elastin fragmentation, vascular inflammation, and adventitial thickening. 8 mentioned above, a limitation of the M11-YT-KO mouse is the shortened lifespan attributed to the intestinal phenotype.This is also evident in Myh11-Cre-driven deletion of other genes critical for smooth muscle function, such as SRF. 3,14,25,36To circumvent this issue, a novel Credriver based on the integrin α8 (Itga8) promoter, which is highly expressed in vascular but not intestinal smooth muscle, 20 was recently developed. 50 generate tamoxifen-inducible and vascular smooth muscle-specific deletion of YAP/TAZ, we crossed Itga8-Cre mice with YAP/TAZ floxed mice (denoted I8-YT-KO).These mice did not display an overt gastrointestinal phenotype, but within 2-4 weeks, I8-YT-KO mice developed spontaneous abdominal aortic aneurysms with complete penetrance.At later time points, widespread pathological changes were also observed in smaller vessels of I8-YT-KO, including cerebral and mesenteric arteries.
The aortic aneurysms of I8-YT-KO share several characteristics of human aneurysmal pathology, including loss of contractile differentiation, proteoglycan accumulation, elastin disarray, medial apoptosis, and adventitial cell proliferation. 4Furthermore, the aortic aneurysms display a marked activation and infiltration of inflammatory cells.Interestingly, deletion of vascular smooth muscle YAP/ TAZ resulted in an almost immediate activation of the transcription factor Sox9, a master regulator of chondrocyte differentiation, which is continuously repressed in normal smooth muscle cells to maintain the contractile phenotype 5 (Figure 2).Activation of Sox9 expression subsequently caused the induction of Sox9-regulated genes, medial accumulation of proteoglycans, including aggrecan, and, in rare cases, even true cartilage formation in the aortic wall. 4filtration of inflammatory cells plays a critical role in the development of aneurysms and is one of the most striking events in I8-YT-KO aorta.A suggested mechanism for triggering the inflammatory response in YAP/TAZ KO mice is activation of the cGAS-STING pathway 4,39 (Figure 2).cGAS-STING is an innate immune signaling cascade that detects cytoplasmic DNA and activates transcription of inflammatory genes via interferon regulatory factor 3 (IRF3). 29Notably, cGAS-STING activation has been shown to drive age-related inflammation. 15nsidering that YAP/TAZ activity declines with age, maintaining YAP/TAZ activity could potentially prevent vascular aging. 39

| IMP ORTAN CE OF YAP/ TA Z IN ENDOTHELIAL CELL S
In endothelial cells, YAP/TAZ are involved in the regulation of several processes, including flow-sensing angiogenesis and cell adhesions. 19,43,45,53Both embryonic and inducible endothelial YAP/TAZ deletion have been shown to cause lethality.Embryonic deletion causes severe morphological defects throughout the embryo, resulting in developmental arrest. 46In adult KO mice, postmortem examinations identified pleural effusion as the cause of lethality. 42chanotransduction in endothelial cells involves sensing fluid shear stress through the glycocalyx, a carbohydrate-rich gel-like structure located on the luminal side of the endothelium. 35The glycocalyx senses fluid shear stress and triggers regulation of signaling pathways, cytoskeletal remodeling, and ion channels, resulting in various adaptive responses, including the release of nitric oxide, changes in endothelial cell morphology, and altered permeability. 13,48 addition to the glycocalyx, shear stress is sensed by primary cilia anchored to the endothelial surface.Activation of primary cilia has been identified to trigger Ca 2+ influx and nitric oxide production in endothelial cells following changes in fluid shear stress. 17For more detailed information on endothelial mechanotransduction, see the review by Wang et al. 48 the normal vasculature, shear stress detection in endothelial cells is essential for local blood flow regulation.Local blood flow patterns can also determine the propensity for atherosclerosis at specific sites of the vasculature, such as bifurcations associated with turbulent flow.These sites are typically prone to atherosclerosis, whereas areas with laminar flow are protected.Interestingly, unidirectional shear stress results in the phosphorylation and inhibition of YAP/TAZ, whereas disturbed flow increases YAP/TAZ activity. 21,45rthermore, in contrast to smooth muscle cells, inhibition of endothelial YAP/TAZ suppresses inflammation and delays atherosclerosis (Figure 3).These results suggest that, in contrast to smooth muscle cells, endothelial YAP/TAZ expression accelerates vascular disease.However, conflicting reports suggest that deletion of endothelial YAP alone enhances vascular inflammation, similar to what we have observed in smooth muscle cells. 24Thus, the effects of YAP/TAZ are highly dependent on the cell type and biological situation.
Transcriptional control of changes in endothelial cells during angiogenesis is only beginning to be understood.Angiogenesis is partly controlled by mechanical factors, and several studies have recently suggested an important role of YAP/TAZ in this process.
During sprouting angiogenesis, YAP/TAZ positively regulate the formation and maintenance of filopodia via actin cytoskeleton remodeling and cell division control protein 42 (CDC42) activation. 19,37YAP/TAZ also regulate the formation of tight and adherence junctions between endothelial cells.The activation of YAP/ TAZ during angiogenesis is thought to be mediated via vascular endothelial growth factor (VEGF)-dependent effects on actin dynamics and LATS1/2 inhibition, as well as mechanical stimulation at cell-cell junctions. 19,31,46Deletion of YAP/TAZ using inducible endothelial cell-specific KO mice results in stunted sprouting, branching abnormalities, and vascular network impairments leading to hemorrhage in the brain and retinal microvasculature 19,31,32,37,46 (Figure 3).Curiously, YAP and TAZ have been proposed to inhibit angiogenesis in bones specifically, possibly due to the hypoxic environment of bone tissue. 38

| CON CLUS I ON S AND PER S PEC TIVE S
It is increasingly clear that mechanical factors such as YAP/TAZ play a crucial role in the development, function, and pathologies of the vascular system.Mechanisms involved in this process are continuously being discovered.It is likely that future precision medicine approaches will target mechanically activated signaling pathways to modulate mechanosensing in vascular cells.As evident from this review, the biological effects of YAP/ TAZ are context-dependent, but it is clear that both excessive and insufficient activation of YAP/TAZ can be detrimental to vascular development and function.In future work, it will be important to clarify the role of YAP/ TAZ signaling in specific human vascular dysfunctions and vascular aging.
Furthermore, vascular dysfunction also leads to secondary diseases such as cognitive disorders, cancer, and metabolic disease.However, the importance and therapeutic implications of YAP/TAZ in these situations are not well understood and warrant further investigation.

R E FE R E N C E S
F I G U R E 3 Effects of YAP/TAZ deletion in endothelial cells.In vivo, reduced expression of YAP/TAZ in endothelial cells results in (A) decreased or abnormal angiogenesis due to altered actomyosin remodeling and decreased proliferation/ sprouting in developing vessels and (B) increased proliferation and inflammation contributing to the development of atherosclerosis when deleted in larger arteries.CDC42, cell division control protein 42; MYC, myelocytomatosis oncogene; TAZ, WW domain-containing transcription regulator 1; YAP, yesassociated protein.