CDK6-mediated endothelial cell cycle acceleration drives arteriovenous malformations in hereditary hemorrhagic telangiectasia.

Increased endothelial cell (EC) proliferation is a hallmark of arteriovenous malformations (AVMs) in hereditary hemorrhagic telangiectasia (HHT). The underlying mechanism and disease relevance of this abnormal cell proliferative state of the ECs remain unknown. Here, we report the identification of a CDK6-driven mechanism of cell cycle progression deregulation directly involved in EC proliferation and HHT vascular pathology. Specifically, HHT mouse liver ECs exhibited defects in their cell cycle control characterized by a G1/S checkpoint bypass and acceleration of cell cycle speed. Phosphorylated retinoblastoma (p-RB1)-a marker of G1/S transition through the restriction point-significantly accumulated in ECs of HHT mouse retinal AVMs and HHT patient skin telangiectasias. Mechanistically, ALK1 loss of function increased the expression of key restriction point mediators, and treatment with palbociclib or ribociclib, two CDK4/6 inhibitors, blocked p-RB1 increase and retinal AVMs in HHT mice. Palbociclib also improved vascular pathology in the brain and slowed down endothelial cell cycle speed and EC proliferation. Specific deletion of Cdk6 in ECs was sufficient to protect HHT mice from AVM pathology. Thus, CDK6-mediated endothelial cell cycle acceleration controls EC proliferation in AVMs and is a central determinant of HHT pathogenesis. We propose that clinically approved CDK4/6 inhibitors have repurposing potential in HHT.

ANG2 Blockade Diminishes Proangiogenic Cerebrovascular Defects Associated With Models of Hereditary Hemorrhagic Telangiectasia.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder characterized by arteriovenous malformations and blood vessel enlargements. However, there are no effective drug therapies to combat arteriovenous malformation formation in patients with HHT. Here, we aimed to address whether elevated levels of ANG2 (angiopoietin-2) in the endothelium is a conserved feature in mouse models of the 3 major forms of HHT that could be neutralized to treat brain arteriovenous malformations and associated vascular defects. In addition, we sought to identify the angiogenic molecular signature linked to HHT. METHODS: Cerebrovascular defects, including arteriovenous malformations and increased vessel calibers, were characterized in mouse models of the 3 common forms of HHT using transcriptomic and dye injection labeling methods. RESULTS: Comparative RNA sequencing analyses of isolated brain endothelial cells revealed a common, but unique proangiogenic transcriptional program associated with HHT. This included a consistent upregulation in cerebrovascular expression of ANG2 and downregulation of its receptor Tyr kinase with Ig and EGF homology domains (TIE2/TEK) in HHT mice compared with controls. Furthermore, in vitro experiments revealed TEK signaling activity was hampered in an HHT setting. Pharmacological blockade of ANG2 improved brain vascular pathologies in all HHT models, albeit to varying degrees. Transcriptomic profiling further indicated that ANG2 inhibition normalized the brain vasculature by impacting a subset of genes involved in angiogenesis and cell migration processes. CONCLUSIONS: Elevation of ANG2 in the brain vasculature is a shared trait among the mouse models of the common forms of HHT. Inhibition of ANG2 activity can significantly limit or prevent brain arteriovenous malformation formation and blood vessel enlargement in HHT mice. Thus, ANG2-targeted therapies may represent a compelling approach to treat arteriovenous malformations and vascular pathologies related to all forms of HHT.

Tissue-Resident PDGFRα+ Progenitor Cells Contribute to Fibrosis versus Healing in a Context- and Spatiotemporally Dependent Manner.

PDGFRα+ mesenchymal progenitor cells are associated with pathological fibro-adipogenic processes. Conversely, a beneficial role for these cells during homeostasis or in response to revascularization and regeneration stimuli is suggested, but remains to be defined. We studied the molecular profile and function of PDGFRα+ cells in order to understand the mechanisms underlying their role in fibrosis versus regeneration. We show that PDGFRα+ cells are essential for tissue revascularization and restructuring through injury-stimulated remodeling of stromal and vascular components, context-dependent clonal expansion, and ultimate removal of pro-fibrotic PDGFRα+-derived cells. Tissue ischemia modulates the PDGFRα+ phenotype toward cells capable of remodeling the extracellular matrix and inducing cell-cell and cell-matrix adhesion, likely favoring tissue repair. Conversely, pathological healing occurs if PDGFRα+-derived cells persist as terminally differentiated mesenchymal cells. These studies support a context-dependent "yin-yang" biology of tissue-resident mesenchymal progenitor cells, which possess an innate ability to limit injury expansion while also promoting fibrosis in an unfavorable environment.

Correcting Smad1/5/8, mTOR, and VEGFR2 treats pathology in hereditary hemorrhagic telangiectasia models.

Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1/ENG/Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K/Akt/mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs - including in HHT patient blood outgrowth ECs - and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in patients with HHT.

CD90 Identifies Adventitial Mesenchymal Progenitor Cells in Adult Human Medium- and Large-Sized Arteries.

Mesenchymal stem cells (MSCs) reportedly exist in a vascular niche occupying the outer adventitial layer. However, these cells have not been well characterized in vivo in medium- and large-sized arteries in humans, and their potential pathological role is unknown. To address this, healthy and diseased arterial tissues were obtained as surplus surgical specimens and freshly processed. We identified that CD90 marks a rare adventitial population that co-expresses MSC markers including PDGFRα, CD44, CD73, and CD105. However, unlike CD90, these additional markers were widely expressed by other cells. Human adventitial CD90+ cells fulfilled standard MSC criteria, including plastic adherence, spindle morphology, passage ability, colony formation, and differentiation into adipocytes, osteoblasts, and chondrocytes. Phenotypic and transcriptomic profiling, as well as adoptive transfer experiments, revealed a potential role in vascular disease pathogenesis, with the transcriptomic disease signature of these cells being represented in an aortic regulatory gene network that is operative in atherosclerosis.

Mouse Model of Wire Injury-Induced Vascular Remodeling.

We introduced the vascular remodeling mouse system induced by the wire injury to investigate the molecular and cellular mechanisms of cardiovascular diseases. Using these models, we focus on the adventitial cell population in the outermost layer of the adult vasculature as a vascular progenitor niche. Firstly we used the standard wire injury approach, leaving the wire for 1 min in the artery and retracting the wire by twisting out to expand the artery and denude the inner layer endothelial cells in the both peripheral artery and femoral artery. This method leads to adventitial lineage cell accumulation on the medial-adventitial border, but no contribution into the hyperplastic neointima. Since advanced atherosclerotic plaques in the mouse models and human clinical specimens show the elastic lamina in the media broken, we hypothesized that adventitial lineage cells contribute to acute neointima formation induced by the mechanical damage in both endothelial and medial layers. To make this intensive damage, next, we used the bigger diameter wire with no hydrophilic coating and repeated the ten-times insertion and retraction of the wire after leaving for 1 min in the femoral artery. The additional ten-times intensive movements of the wire lead to breakdown and rupture of the elastic lamina together with a contribution of adventitial lineage cells to the hyperplastic neointima. Here we describe these two different wire injury methods to induce different types of vascular remodeling at the point of adventitial lineage cell contribution to the hyperplastic neointima by targeting two separate locations of hind limb artery, the peripheral artery and femoral artery, and using two different diameter wires.

Endothelial to mesenchymal transition is common in atherosclerotic lesions and is associated with plaque instability.

Endothelial to mesenchymal transition (EndMT) plays a major role during development, and also contributes to several adult cardiovascular diseases. Importantly, mesenchymal cells including fibroblasts are prominent in atherosclerosis, with key functions including regulation of: inflammation, matrix and collagen production, and plaque structural integrity. However, little is known about the origins of atherosclerosis-associated fibroblasts. Here we show using endothelial-specific lineage-tracking that EndMT-derived fibroblast-like cells are common in atherosclerotic lesions, with EndMT-derived cells expressing a range of fibroblast-specific markers. In vitro modelling confirms that EndMT is driven by TGF-ß signalling, oxidative stress and hypoxia; all hallmarks of atherosclerosis. 'Transitioning' cells are readily detected in human plaques co-expressing endothelial and fibroblast/mesenchymal proteins, indicative of EndMT. The extent of EndMT correlates with an unstable plaque phenotype, which appears driven by altered collagen-MMP production in EndMT-derived cells. We conclude that EndMT contributes to atherosclerotic patho-biology and is associated with complex plaques that may be related to clinical events.

Mesodermal Nkx2.5 is necessary and sufficient for early second heart field development.

The vertebrate heart develops from mesoderm and requires inductive signals secreted from early endoderm. During embryogenesis, Nkx2.5 acts as a key transcription factor and plays essential roles for heart formation from Drosophila to human. In mice, Nkx2.5 is expressed in the early first heart field, second heart field pharyngeal mesoderm, as well as pharyngeal endodermal cells underlying the second heart field. Currently, the specific requirements for Nkx2.5 in the endoderm versus mesoderm with regard to early heart formation are incompletely understood. Here, we performed tissue-specific deletion in mice to dissect the roles of Nkx2.5 in the pharyngeal endoderm and mesoderm. We found that heart development appeared normal after endodermal deletion of Nkx2.5 whereas mesodermal deletion engendered cardiac defects almost identical to those observed on Nkx2.5 null embryos (Nkx2.5(-/-)). Furthermore, re-expression of Nkx2.5 in the mesoderm rescued Nkx2.5(-/-) heart defects. Our findings reveal that Nkx2.5 in the mesoderm is essential while endodermal expression is dispensable for early heart formation in mammals.

Coronary artery calcification is inversely related to body morphology in patients with significant coronary artery disease: a three-dimensional intravascular ultrasound study.

AIMS: Emerging data have indicated unexpected complexity in the regulation of vascular and bone calcification. In particular, several recent studies have challenged the concept of a universally positive relationship between body morphology [weight, height, body mass index (BMI), body surface area (BSA)] and the extent of vascular calcification. We sought to clarify these discrepancies and investigated the relationship between index lesion coronary artery calcification (CAC) and body morphology in patients undergoing percutaneous coronary intervention (PCI) using three-dimensional intravascular ultrasound (IVUS). METHODS AND RESULTS: We analysed CAC in patients who underwent PCI with pre-intervention IVUS imaging. The main outcome measure was the calcium index (CalcIndex); a three-dimensional IVUS-derived measure of total calcification per obstructive coronary lesion. A total of 346 patients (65.3 ± 10.6 years; 29.5% females) underwent PCI with IVUS-based CAC assessment. CalcIndex was categorized as zero-low (0-0.1399; n = 152) or intermediate-high (0.1400-1.2541; n = 194). All measures of body morphology were lower in patients with intermediate-high CalcIndex (height, P = 0.024; weight, P = 0.008; BMI, P = 0.064; BSA, P = 0.005). In adjusted multivariable models, weight and BSA were independent inverse predictors of intermediate-high CalcIndex [weight: odds ratio (OR) 0.986, P = 0.017; BSA: OR 0.323, P = 0.012] while CalcIndex also trended towards an inverse association with both height (P = 0.068) and BMI (P = 0.064). These independent inverse associations were consistent across multiple clinical subgroups, including stratification by age, race, gender, diabetes, and renal impairment. CONCLUSION: Using three-dimensional IVUS to assess vascular calcification, these data confirm an independent, inverse relationship between body size and index lesion CAC in patients with obstructive coronary artery disease.

Induction of a hemogenic program in mouse fibroblasts.

Definitive hematopoiesis emerges during embryogenesis via an endothelial-to-hematopoietic transition. We attempted to induce this process in mouse fibroblasts by screening a panel of factors for hemogenic activity. We identified a combination of four transcription factors, Gata2, Gfi1b, cFos, and Etv6, that efficiently induces endothelial-like precursor cells, with the subsequent appearance of hematopoietic cells. The precursor cells express a human CD34 reporter, Sca1, and Prominin1 within a global endothelial transcription program. Emergent hematopoietic cells possess nascent hematopoietic stem cell gene-expression profiles and cell-surface phenotypes. After transgene silencing and reaggregation culture, the specified cells generate hematopoietic colonies in vitro. Thus, we show that a simple combination of transcription factors is sufficient to induce a complex, dynamic, and multistep developmental program in vitro. These findings provide insights into the specification of definitive hemogenesis and a platform for future development of patient-specific stem and progenitor cells, as well as more-differentiated blood products.

Myocardial Tbx20 regulates early atrioventricular canal formation and endocardial epithelial-mesenchymal transition via Bmp2.

During early embryogenesis, the formation of the cardiac atrioventricular canal (AVC) facilitates the transition of the heart from a linear tube into a chambered organ. However, the genetic pathways underlying this developmental process are poorly understood. The T-box transcription factor Tbx20 is expressed predominantly in the AVC of early heart tube. It was shown that Tbx20 activates Nmyc1 and suppresses Tbx2 expression to promote proliferation and specification of the atrial and ventricular chambers, yet it is not known if Tbx20 is involved in early AVC development. Here, we report that mice lacking Tbx20 in the AVC myocardium fail to form the AVC constriction, and the endocardial epithelial-mesenchymal transition (EMT) is severely perturbed. Tbx20 maintains expression of a variety of genes, including Bmp2, Tbx3 and Hand1 in the AVC myocardium. Intriguingly, we found Bmp2 downstream genes involved in the EMT initiation are also downregulated. In addition, re-expression of Bmp2 in the AVC myocardium substantially rescues the EMT defects resulting from the lack of Tbx20, suggesting Bmp2 is one of the key downstream targets of Tbx20 in AVC development. Our data support a complex signaling network with Tbx20 suppressing Tbx2 in the AVC myocardium but also indirectly promoting Tbx2 expression through Bmp2. The spatiotemporal expression of Tbx2 in the AVC appears to be balanced between these two opposing signals. Overall, our study provides genetic evidence that Tbx20 has essential roles in regulating AVC development that coordinate early cardiac chamber formation.

Outflow tract cushions perform a critical valve-like function in the early embryonic heart requiring BMPRIA-mediated signaling in cardiac neural crest.

Neural crest-specific ablation of BMP type IA receptor (BMPRIA) causes embryonic lethality by embryonic day (E) 12.5, and this was previously postulated to arise from a myocardial defect related to signaling by a small population of cardiac neural crest cells (cNCC) in the epicardium. However, as BMP signaling via cNCC is also required for proper development of the outflow tract cushions, precursors to the semilunar valves, a plausible alternate or additional hypothesis is that heart failure may result from an outflow tract cushion defect. To investigate whether the outflow tract cushions may serve as dynamic valves in regulating hemodynamic function in the early embryo, in this study we used noninvasive ultrasound biomicroscopy-Doppler imaging to quantitatively assess hemodynamic function in mouse embryos with P0-Cre transgene mediated neural crest ablation of Bmpr1a (P0 mutants). Similar to previous studies, the neural crest-deleted Bmpr1a P0 mutants died at approximately E12.5, exhibiting persistent truncus arteriosus, thinned myocardium, and congestive heart failure. Surprisingly, our ultrasound analyses showed normal contractile indices, heart rate, and atrioventricular conduction in the P0 mutants. However, reversed diastolic arterial blood flow was detected as early as E11.5, with cardiovascular insufficiency and death rapidly ensuing by E12.5. Quantitative computed tomography showed thinning of the outflow cushions, and this was associated with a marked reduction in cell proliferation. These results suggest BMP signaling to cNCC is required for growth of the outflow tract cushions. This study provides definitive evidence that the outflow cushions perform a valve-like function critical for survival of the early mouse embryo.

Endoglin is dispensable for angiogenesis, but required for endocardial cushion formation in the midgestation mouse embryo.

Vascular patterning depends on precisely coordinated timing of endothelial cell differentiation and onset of cardiac function. Endoglin is a transmembrane receptor for members of the TGF-beta superfamily that is expressed on endothelial cells from early embryonic gestation to adult life. Heterozygous loss of function mutations in human ENDOGLIN cause Hereditary Hemorrhagic Telangiectasia Type 1, a vascular disorder characterized by arteriovenous malformations that lead to hemorrhage and stroke. Endoglin null mice die in embryogenesis with numerous lesions in the cardiovascular tree including incomplete yolk sac vessel branching and remodeling, vessel dilation, hemorrhage and abnormal cardiac morphogenesis. Since defects in multiple cardiovascular tissues confound interpretations of these observations, we performed in vivo chimeric rescue analysis using Endoglin null embryonic stem cells. We demonstrate that Endoglin is required cell autonomously for endocardial to mesenchymal transition during formation of the endocardial cushions. Endoglin null cells contribute widely to endothelium in chimeric embryos rescued from cardiac development defects, indicating that Endoglin is dispensable for angiogenesis and vascular remodeling in the midgestation embryo, but is required for early patterning of the heart.

Optimized beta-galactosidase staining method for simultaneous detection of endogenous gene expression in early mouse embryos.

beta-Galactosidase (beta-gal) is one of the popular reporters for detecting the expression of endogenous or exogenous genes. Here we report 6-chloro-3-indoxyl-beta-D-galactopyranoside (S-gal) is more sensitive for beta-gal activity than 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal), particularly during the early developmental stages of mouse embryos. Further, we successfully combined beta-gal staining with S-gal and in situ hybridization using DIG-labeled probes in both whole and sections of early stage embryos due to the sensitivity and color compatibility of S-gal.

Hypomorphic Mesp allele distinguishes establishment of rostrocaudal polarity and segment border formation in somitogenesis.

A bHLH-type transcription factor, Mesp2, plays an essential role in somite segmentation in mice. Zebrafish mespb (mesp-b), a putative homologue of mouse Mesp2, is transiently expressed in the rostral presomitic mesoderm similarly to Mesp2. To determine whether zebrafish mespb is a functional homologue of mouse Mesp2, zebrafish mespb was introduced into the mouse Mesp2 locus by homologous recombination. Introduced mespb almost rescued the Mesp2 deficiency in the homozygous mespb knockin mouse, indicating that mespb is a functional homologue of mouse Mesp2. Segmented somites were clearly observed although the partial fusion of the vertebral columns still occurred. Interestingly, however, the nature and dosage of the mespb gene affected the rescue event. A mouse line, which has a hypomorphic Mesp2 allele generated by the introduction of neo-mespb, gave rise to an epithelial somite without normal rostrocaudal (RC) polarity. RC polarity was also lacking in the presomitic mesoderm. The defects in RC polarity were determined by the altered expressions of Uncx4.1 and Dll1 in the segmented somites and presomitic mesoderm, respectively. In contrast, the expression of EphA4 (Epha4), lunatic fringe or protocadherin, thought to be involved in segment border formation, was fairly normal in hypomorphic mutant embryos. These results suggest that the Mesp family of transcription factors is involved in both segment border formation and establishment of RC polarity through different genetic cascades.