Genes That Escape X Chromosome Inactivation Modulate Sex Differences in Valve Myofibroblasts.

BACKGROUND: Aortic valve stenosis is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain underexplored. METHODS: Hydrogel biomaterials were designed to recapitulate key aspects of the valve tissue microenvironment and to serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to profibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA sequencing was used to define pathways involved in driving sex-dependent activation. Interventions with small molecule inhibitors and siRNA transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors. RESULTS: In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker α-smooth muscle actin compared with male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-smooth muscle actin stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than in male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase signaling as a potential driver of this sex-dependent myofibroblast activation. Furthermore, we found that genes that escape X-chromosome inactivation such as BMX and STS (encoding for Bmx nonreceptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation through Rho-associated protein kinase signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation through Rho-associated protein kinase signaling. CONCLUSIONS: Together, in vivo and in vitro results confirm sex dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent aortic valve stenosis progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of aortic valve stenosis and to help guide sex-based precision therapies.

Discovery of an Experimental Model of Unicuspid Aortic Valve.

BACKGROUND: The epithelial growth factor receptor family of tyrosine kinases modulates embryonic formation of semilunar valves. We hypothesized that mice heterozygous for a dominant loss-of-function mutation in epithelial growth factor receptor, which are EgfrVel/+ mice, would develop anomalous aortic valves, valve dysfunction, and valvular cardiomyopathy. METHODS AND RESULTS: Aortic valves from EgfrVel/+ mice and control mice were examined by light microscopy at 2.5 to 4 months of age. Additional EgfrVel/+ and control mice underwent echocardiography at 2.5, 4.5, 8, and 12 months of age, followed by histologic examination. In young mice, microscopy revealed anatomic anomalies in 79% of EgfrVel/+ aortic valves, which resembled human unicuspid aortic valves. Anomalies were not observed in control mice. At 12 months of age, histologic architecture was grossly distorted in EgfrVel/+ aortic valves. Echocardiography detected moderate or severe aortic regurgitation, or aortic stenosis was present in 38% of EgfrVel/+ mice at 2.5 months of age (N=24) and in 74% by 8 months of age. Left ventricular enlargement, hypertrophy, and reversion to a fetal myocardial gene expression program occurred in EgfrVel/+ mice with aortic valve dysfunction, but not in EgfrVel/+ mice with near-normal aortic valve function. Myocardial fibrosis was minimal or absent in all groups. CONCLUSIONS: A new mouse model uniquely recapitulates salient functional, structural, and histologic features of human unicuspid aortic valve disease, which are phenotypically distinct from other forms of congenital aortic valve disease. The new model may be useful for elucidating mechanisms by which congenitally anomalous aortic valves become critically dysfunctional.

Fibrotic Aortic Valve Stenosis in Hypercholesterolemic/Hypertensive Mice.

OBJECTIVE: Hypercholesterolemia and hypertension are associated with aortic valve stenosis (AVS) in humans. We have examined aortic valve function, structure, and gene expression in hypercholesterolemic/hypertensive mice. APPROACH AND RESULTS: Control, hypertensive, hypercholesterolemic (Apoe(-/-)), and hypercholesterolemic/hypertensive mice were studied. Severe aortic stenosis (echocardiography) occurred only in hypercholesterolemic/hypertensive mice. There was minimal calcification of the aortic valve. Several structural changes were identified at the base of the valve. The intercusp raphe (or seam between leaflets) was longer in hypercholesterolemic/hypertensive mice than in other mice, and collagen fibers at the base of the leaflets were reoriented to form a mesh. In hypercholesterolemic/hypertensive mice, the cusps were asymmetrical, which may contribute to changes that produce AVS. RNA sequencing was used to identify molecular targets during the developmental phase of stenosis. Genes related to the structure of the valve were identified, which differentially expressed before fibrotic AVS developed. Both RNA and protein of a profibrotic molecule, plasminogen activator inhibitor 1, were increased greatly in hypercholesterolemic/hypertensive mice. CONCLUSIONS: Hypercholesterolemic/hypertensive mice are the first model of fibrotic AVS. Hypercholesterolemic/hypertensive mice develop severe AVS in the absence of significant calcification, a feature that resembles AVS in children and some adults. Structural changes at the base of the valve leaflets include lengthening of the raphe, remodeling of collagen, and asymmetry of the leaflets. Genes were identified that may contribute to the development of fibrotic AVS.

Valve Endothelial Cell-Derived Tgfß1 Signaling Promotes Nuclear Localization of Sox9 in Interstitial Cells Associated With Attenuated Calcification.

OBJECTIVE: Aortic valve disease, including calcification, affects >2% of the human population and is caused by complex interactions between multiple risk factors, including genetic mutations, the environment, and biomechanics. At present, there are no effective treatments other than surgery, and this is because of the limited understanding of the mechanisms that underlie the condition. Previous work has shown that valve interstitial cells within the aortic valve cusps differentiate toward an osteoblast-like cell and deposit bone-like matrix that leads to leaflet stiffening and calcific aortic valve stenosis. However, the mechanisms that promote pathological phenotypes in valve interstitial cells are unknown. APPROACH AND RESULTS: Using a combination of in vitro and in vivo tools with mouse, porcine, and human tissue, we show that in valve interstitial cells, reduced Sox9 expression and nuclear localization precedes the onset of calcification. In vitro, Sox9 nuclear export and calcific nodule formation is prevented by valve endothelial cells. However, in vivo, loss of Tgfß1 in the endothelium leads to reduced Sox9 expression and calcific aortic valve disease. CONCLUSIONS: Together, these findings suggest that reduced nuclear localization of Sox9 in valve interstitial cells is an early indicator of calcification, and therefore, pharmacological targeting to prevent nuclear export could serve as a novel therapeutic tool in the prevention of calcification and stenosis.

Atherosclerosis stabilization with PCSK-9 inhibition: An evolving concept for cardiovascular prevention.

Monoclonal antibodies (mAbs) to proprotein convertase subtilisin/kexin type 9 (PCSK-9) can further lower LDL-C by ≥60% in statin-treated patients. Preliminary data suggest they may reduce cardiovascular (CVD) events. Ongoing PCSK-9 mAb cardiovascular outcomes trials could provide the opportunity to determine whether a "legacy effect" similar to that observed for statins will occur over the post-trial observation period. We hypothesize these trials could demonstrate that (1) very aggressive LDL-C lowering with PCSK-9 mAbs added to background statin therapy will induce extensive atherosclerosis stabilization and regression in the large majority of treated patients, and (2) continued maintenance therapy with high intensity statin therapy (with or without ezetimibe) should then inhibit new plaque formation, with a long-term prevention of CVD events. The necessity of expensive lifetime treatment with PCSK-9 inhibitors could then be avoided in all but a small subset of patients who could benefit from longer treatment.

Paradoxical Increase in Mortality and Rupture of Intracranial Aneurysms in Microsomal Prostaglandin E2 Synthase Type 1-Deficient Mice: Attenuation by Aspirin.

BACKGROUND: Inflammation plays an important role in formation and rupture of intracranial aneurysms. Expression of microsomal prostaglandin E2 (PGE2) synthase type 1 (mPGES-1) is increased in the wall of intracranial aneurysms in humans. PGE2, a by-product of mPGES-1, is associated with inflammation and cerebrovascular dysfunction. OBJECTIVE: To test the hypothesis that deletion of mPGES-1 decreases the formation and rupture of intracranial aneurysms in a murine model. METHODS: Intracranial aneurysms were induced in wild-type and mPGES-1 knockout (mPGES-1 KO) mice by using a combination of deoxycorticosterone acetate-salt-induced hypertension and intracranial injection of elastase in the basal cistern. Prevalence of aneurysms, subarachnoid hemorrhage, and mortality were assessed. We also tested the effects of administration of aspirin (6 mg/kg/d) by gavage and PGE2 (1 mg/kg/d) by subcutaneous infusion. RESULTS: Systolic blood pressure and prevalence of aneurysm were similar in wild-type and mPGES-1 KO mice. However, mortality and the prevalence of subarachnoid hemorrhage were markedly increased in mPGES-1 KO mice (P < .05). Bone marrow reconstitution studies suggest that mPGES-1 derived from leukocytes does not appear to increase rupture of intracranial aneurysms. Aspirin, but not PGE2, attenuated the increased mortality in mPGES-1 KO mice (P < .05). CONCLUSION: Vascular mPGES-1 plays a protective role in blood vessels and attenuates rupture of cerebral aneurysms. In contrast to effects on abdominal aneurysms, mPGES-1 deficiency is associated with an increase in rupture of cerebral aneurysms and mortality, which are attenuated by low-dose aspirin.

Spontaneous Aortic Regurgitation and Valvular Cardiomyopathy in Mice.

OBJECTIVE: We studied the mechanistic links between fibrocalcific changes in the aortic valve and aortic valve function in mice homozygous for a hypomorphic epidermal growth factor receptor mutation (Wave mice). We also studied myocardial responses to aortic valve dysfunction in Wave mice. APPROACH AND RESULTS: At 1.5 months of age, before development of valve fibrosis and calcification, aortic regurgitation, but not aortic stenosis, was common in Wave mice. Aortic valve fibrosis, profibrotic signaling, calcification, osteogenic markers, lipid deposition, and apoptosis increased dramatically by 6 and 12 months of age in Wave mice. Aortic regurgitation remained prevalent, however, and aortic stenosis was rare, at all ages. Proteoglycan content was abnormally increased in aortic valves of Wave mice at all ages. Treatment with pioglitazone prevented abnormal valve calcification, but did not protect valve function. There was significant left ventricular volume overload, hypertrophy, and fetal gene expression, at all ages in Wave mice with aortic regurgitation. Left ventricular systolic function was normal until 6 months of age in Wave mice, but became impaired by 12 months of age. Myocardial transverse tubules were normal in the presence of left ventricular hypertrophy at 1.5 and 3 months of age, but became disrupted by 12 months of age. CONCLUSIONS: We present the first comprehensive phenotypic and molecular characterization of spontaneous aortic regurgitation and volume-overload cardiomyopathy in an experimental model. In Wave mice, fibrocalcific changes are not linked to valve dysfunction and are epiphenomena arising from structurally incompetent myxomatous valves.

Smooth Muscle Peroxisome Proliferator-Activated Receptor γ Plays a Critical Role in Formation and Rupture of Cerebral Aneurysms in Mice In Vivo.

Vascular inflammation plays a critical role in the pathogenesis of cerebral aneurysms. Peroxisome proliferator-activated receptor γ (PPARγ) protects against vascular inflammation and atherosclerosis, whereas dominant-negative mutations in PPARγ promote atherosclerosis and vascular dysfunction. We tested the role of PPARγ in aneurysm formation and rupture. Aneurysms were induced with a combination of systemic infusion of angiotensin-II and local injection of elastase in (1) mice that received the PPARγ antagonist GW9662 or the PPARγ agonist pioglitazone, (2) mice carrying dominant-negative PPARγ mutations in endothelial or smooth muscle cells, and (3) mice that received the Cullin inhibitor MLN4924. Incidence of aneurysm formation, rupture, and mortality was quantified. Cerebral arteries were analyzed for expression of Cullin3, Kelch-like ECH-associated protein 1, nuclear factor (erythroid-derived 2)-like 2, NAD(P)H dehydrogenase (quinone)1 (NQO1), and inflammatory marker mRNAs. Neither pioglitazone nor GW9662 altered the incidence of aneurysm formation. GW9662 significantly increased the incidence of aneurysm rupture, whereas pioglitazone tended to decrease the incidence of rupture. Dominant-negative endothelial-specific PPARγ did not alter the incidence of aneurysm formation or rupture. In contrast, dominant-negative smooth muscle-specific PPARγ resulted in an increase in aneurysm formation (P<0.05) and rupture (P=0.05). Dominant-negative smooth muscle-specific PPARγ, but not dominant-negative endothelial-specific PPARγ, resulted in significant decreases in expression of genes encoding Cullin3, Kelch-like ECH-associated protein 1, and nuclear factor (erythroid-derived 2)-like 2, along with significant increases in tumor necrosis factor-α, monocyte chemoattractant protein-1, chemokine (C-X-C motif) ligand 1, CD68, matrix metalloproteinase-3, -9, and -13. MLN4924 did not alter incidence of aneurysm formation, but increased the incidence of rupture (P<0.05). In summary, endogenous PPARγ, specifically smooth muscle PPARγ, plays an important role in protecting from formation and rupture of experimental cerebral aneurysms in mice.

Myeloperoxidase is increased in human cerebral aneurysms and increases formation and rupture of cerebral aneurysms in mice.

BACKGROUND AND PURPOSE: Cerebral aneurysm (CA) affects 3% of the population and is associated with hemodynamic stress and inflammation. Myeloperoxidase, a major oxidative enzyme associated with inflammation, is increased in patients with CA, but whether myeloperoxidase contributes to CA is not known. We tested the hypotheses that myeloperoxidase is increased within human CA and is critical for formation and rupture of CA in mice. METHODS: Blood was drawn from the lumen of CAs and femoral arteries of 25 patients who underwent endovascular coiling of CA, and plasma myeloperoxidase concentrations were measured with ELISA. Effects of endogenous myeloperoxidase on CA formation and rupture were studied in myeloperoxidase knockout mice and wild-type (WT) mice using an angiotensin II-elastase induction model of CA. In addition, effects of myeloperoxidase on inflammatory gene expression in endothelial cells were analyzed. RESULTS: Plasma concentrations of myeloperoxidase were 2.7-fold higher within CA than in femoral arterial blood in patients with CA. myeloperoxidase-positive cells were increased in aneurysm tissue compared with superficial temporal artery of patients with CA. Incidence of aneurysms and subarachnoid hemorrhage was significantly lower in myeloperoxidase knockout than in WT mice. In cerebral arteries, proinflammatory molecules, including tumor necrosis factor-α, cyclooxygenase-2 (COX2), chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C motif) ligand (XCL1), matrix metalloproteinase (MMP) 8, cluster of differentiation 68 (CD68), and matrix metalloproteinase 13, and leukocytes were increased, and α-smooth muscle actin was decreased, in WT but not in myeloperoxidase knockout mice after induction of CA. Myeloperoxidase per se increased expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in endothelial cells. CONCLUSIONS: These findings suggest that myeloperoxidase may contribute importantly to formation and rupture of CA.

Multimodality and molecular imaging of matrix metalloproteinase activation in calcific aortic valve disease.

UNLABELLED: Calcific aortic valve disease (CAVD) is the most common cause of aortic stenosis. Matrix metalloproteinases (MMPs) are upregulated in CAVD and contribute to valvular remodeling and calcification. We investigated the feasibility and correlates of MMP-targeted molecular imaging for detection of valvular biology in CAVD. METHODS: Apolipoprotein E-deficient (apoE(-/-)) mice were fed a Western diet (WD) for 3, 6, and 9 mo (n = 108) to induce CAVD. Wild-type mice served as the control group (n = 24). The development of CAVD was tracked with CT, echocardiography, MMP-targeted small-animal SPECT imaging using (99m)Tc-RP805, and histologic analysis. RESULTS: Key features of CAVD­leaflet thickening and valvular calcification­were noted after 6 mo of WD and were more pronounced after 9 mo. These findings were associated with a significant reduction in aortic valve leaflet separation and a significant increase in transaortic valve flow velocity. On in vivo SPECT/CT images, MMP signal in the aortic valve area was significantly higher at 6 mo in WD mice than in control mice and decreased thereafter. The specificity of the signal was demonstrated by blocking, using an excess of nonlabeled precursor. Similar to MMP signal, MMP activity as determined by in situ zymography and valvular inflammation by CD68 staining were maximal at 6 mo. In vivo (99m)Tc-RP805 uptake correlated significantly with MMP activity (R(2) = 0.94, P < 0.05) and CD68 expression (R(2) = 0.98, P < 0.01) in CAVD. CONCLUSION: MMP-targeted imaging detected valvular inflammation and remodeling in a murine model of CAVD. If this ability is confirmed in humans, the technique may provide a tool for tracking the effect of emerging medical therapeutic interventions and for predicting outcome in CAVD.

Angiotensin 1-7 reduces mortality and rupture of intracranial aneurysms in mice.

Angiotensin II (Ang II) stimulates vascular inflammation, oxidative stress, and formation and rupture of intracranial aneurysms in mice. Because Ang 1-7 acts on Mas receptors and generally counteracts deleterious effects of Ang II, we tested the hypothesis that Ang 1-7 attenuates formation and rupture of intracranial aneurysms. Intracranial aneurysms were induced in wild-type and Mas receptor-deficient mice using a combination of Ang II-induced hypertension and intracranial injection of elastase in the basal cistern. Mice received elastase+Ang II alone or a combination of elastase+Ang II+Ang 1-7. Aneurysm formation, prevalence of subarachnoid hemorrhage, mortality, and expression of molecules involved in vascular injury were assessed. Systolic blood pressure was similar in mice receiving elastase+Ang II (mean±SE, 148±5 mm Hg) or elastase+Ang II+Ang 1-7 (144±5 mm Hg). Aneurysm formation was also similar in mice receiving elastase+Ang II (89%) or elastase+Ang II+Ang 1-7 (84%). However, mice that received elastase+Ang II+Ang 1-7 had reduced mortality (from 64% to 36%; P<0.05) and prevalence of subarachnoid hemorrhage (from 75% to 48%; P<0.05). In cerebral arteries, expression of the inflammatory markers, Nox2 and catalase increased similarly in elastase+Ang II or elastase+Ang II+Ang 1-7 groups. Ang 1-7 increased the expression of cyclooxygenase-2 and decreased the expression of matrix metalloproteinase-9 induced by elastase+Ang II (P<0.05). In Mas receptor-deficient mice, systolic blood pressure, mortality, and prevalence of subarachnoid hemorrhage were similar (P>0.05) in groups treated with elastase+Ang II or elastase+Ang II+Ang 1-7. The expression of Mas receptor was detected by immunohistochemistry in samples of human intracranial arteries and aneurysms. In conclusion, without attenuating Ang II-induced hypertension, Ang 1-7 decreased mortality and rupture of intracranial aneurysms in mice through a Mas receptor-dependent pathway.

Aortic valve sclerosis in mice deficient in endothelial nitric oxide synthase.

Risk factors for fibrocalcific aortic valve disease (FCAVD) are associated with systemic decreases in bioavailability of endothelium-derived nitric oxide (EDNO). In patients with bicuspid aortic valve (BAV), vascular expression of endothelial nitric oxide synthase (eNOS) is decreased, and eNOS(-/-) mice have increased prevalence of BAV. The goal of this study was to test the hypotheses that EDNO attenuates profibrotic actions of valve interstitial cells (VICs) in vitro and that EDNO deficiency accelerates development of FCAVD in vivo. As a result of the study, coculture of VICs with aortic valve endothelial cells (vlvECs) significantly decreased VIC activation, a critical early phase of FCAVD. Inhibition of VIC activation by vlvECs was attenuated by N(G)-nitro-l-arginine methyl ester or indomethacin. Coculture with vlvECs attenuated VIC expression of matrix metalloproteinase-9, which depended on stiffness of the culture matrix. Coculture with vlvECs preferentially inhibited collagen-3, compared with collagen-1, gene expression. BAV occurred in 30% of eNOS(-/-) mice. At age 6 mo, collagen was increased in both bicuspid and trileaflet eNOS(-/-) aortic valves, compared with wild-type valves. At 18 mo, total collagen was similar in eNOS(-/-) and wild-type mice, but collagen-3 was preferentially increased in eNOS(-/-) mice. Calcification and apoptosis were significantly increased in BAV of eNOS(-/-) mice at ages 6 and 18 mo. Remarkably, these histological changes were not accompanied by physiologically significant valve stenosis or regurgitation. In conclusion, coculture with vlvECs inhibits specific profibrotic VIC processes. In vivo, eNOS deficiency produces fibrosis in both trileaflet and BAVs but produces calcification only in BAVs.

The role of valvular endothelial cell paracrine signaling and matrix elasticity on valvular interstitial cell activation.

The effects of valvular endothelial cell (VlvEC) paracrine signaling on VIC phenotype and nodule formation were tested using a co-culture platform with physiologically relevant matrix elasticities and diffusion distance. 100 µm thin poly(ethylene glycol) (PEG) hydrogels of 3-27 kPa Young's moduli were fabricated in transwell inserts. VICs were cultured on the gels, as VIC phenotype is known to change significantly within this range, while VlvECs lined the underside of the membrane. Co-culture with VlvECs significantly reduced VIC activation to the myofibroblast phenotype on all gels with the largest percent decrease on the 3 kPa gels (~70%), while stiffer gels resulted in approximately 20-30% decrease. Additionally, VlvECs significantly reduced αSMA protein expression (~2 fold lower) on both 3 and 27 kPa gels, as well as the number (~2 fold lower) of nodules formed on the 27 kPa gels. Effects of VlvECs were prevented when nitric oxide (NO) release was inhibited with l-NAME, suggesting that VlvEC produced NO inhibits VIC activation. Withdrawal of l-NAME after 3, 5, and 7 days with restoration of VlvEC NO production for 2 additional days led to a partial reversal of VIC activation (~25% decrease). A potential mechanism by which VlvEC produced NO reduced VIC activation was studied by inhibiting initial and mid-stage cGMP pathway molecules. Inhibition of soluble guanylyl cyclase (sGC) with ODQ or protein kinase G (PKG) with RBrcGMP or stimulation of Rho kinase (ROCK) with LPA, abolished VlvEC effects on VIC activation. This work contributes substantially to the understanding of the valve endothelium's role in preventing VIC functions associated with aortic valve stenosis initiation and progression.

Fibrocalcific aortic valve disease: opportunity to understand disease mechanisms using mouse models.

Studies in vitro and in vivo continue to identify complex-regulated mechanisms leading to overt fibrocalcific aortic valve disease (FCAVD). Assessment of the functional impact of those processes requires careful studies of models of FCAVD in vivo. Although the genetic basis for FCAVD is unknown for most patients with FCAVD, several disease-associated genes have been identified in humans and mice. Some gene products which regulate valve development in utero also protect against fibrocalcific disease during postnatal aging. Valve calcification can occur via processes that resemble bone formation. But valve calcification can also occur by nonosteogenic mechanisms, such as formation of calcific apoptotic nodules. Anticalcific interventions might preferentially target either osteogenic or nonosteogenic calcification. Although FCAVD and atherosclerosis share several risk factors and mechanisms, there are fundamental differences between arteries and the aortic valve, with respect to disease mechanisms and responses to therapeutic interventions. Both innate and acquired immunity are likely to contribute to FCAVD. Angiogenesis is a feature of inflammation, but may also contribute independently to progression of FCAVD, possibly by actions of pericytes that are associated with new blood vessels. Several therapeutic interventions seem to be effective in attenuating the development of FCAVD in mice. Therapies which are effective early in the course of FCAVD, however, are not necessarily effective in established disease.