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ALK1 belongs to the type I receptor family for transforming growth factor-␤ family ligands. Heterozygous ALK1 mutations cause hereditary hemorrhagic telangiectasia type 2 (HHT2), a multisystemic vascular disorder. Based largely on in vitro studies, TGF-␤1 has been considered as the most likely ALK1 ligand related to HHT, yet the identity of the physiologic ALK1 ligand remains controversial. In cultured endothelial cells, ALK1 and another TGF-␤ type I receptor, ALK5, regulate angiogenesis by controlling TGF-␤ signal transduction, and ALK5 is required for ALK1 signaling. However, the extent to which such interactions between these 2 receptors play a role in pathogenesis of HHT is unknown. We directly addressed these issues in vivo by comparing the phenotypes of mice in which the Alk1, Alk5, or Tgfbr2 gene was conditionally deleted in restricted vascular endothelia using a novel endothelial Cre transgenic line. Alk1-conditional deletion resulted in severe vascular malformations mimicking all pathologic features of HHT. Yet IntroductionHereditary hemorrhagic telangiectasia (HHT) is an autosomaldominant vascular disorder characterized by recurrent nosebleeds, mucocutaneous telangiectases, and arteriovenous malformations (AVMs) in the brain, lungs, liver, and gastrointestinal tract. 1,2 It has been shown that heterozygous mutations in ENDOGLIN (ENG) and Activin receptor-like kinase 1 (ALK1) cause HHT1 and HHT2, respectively. 2-4 Both of these genes are expressed predominantly in endothelial cells. 5,6 Because ENG and ALK1 are transforming growth factor-␤ (TGF-␤) type III and type I receptors, respectively, it has been postulated that HHT is caused by impaired signaling of a common TGF-␤ family ligand that interacts with these 2 receptors. Recent finding of mutations in the common downstream mediator of TGF-␤ family signals, SMAD4, in a subset of HHT patients also support this hypothesis. 7 Despite the identification of these genes responsible for HHT, the underlying mechanisms for the pathogenesis of HHT remain obscure. One of the chief contributing factors underlying this obscurity is the complexity of the transduction pathway of ENG, ALK1, and SMAD4. The TGF-␤ superfamily consists of more than 40 ligands that can be classified into several subfamilies, including TGF-␤, Activin, and bone morphogenetic protein (BMP). 8 TGF-␤ family cytokines exert their effects by binding to heteromeric complexes of 2 types of transmembrane serine/threonine kinase receptors. 9 The type II receptors function primarily as the binding receptors. On binding their ligand(s), type II receptors associate with and phosphorylate the type I receptors, which in turn activate downstream SMAD proteins. Each TGF-␤ ligand interacts with one or more type II and type I receptors, but TGFBR2 is the only type II receptor that has been shown to interact with TGF-␤ subfamily ligands (TGF-␤1, -␤2, and -␤3).ENG can interact with multiple TGF-␤ family members, such as TGF-␤1/␤3, Activin-A, BMP2, and BMP7, in the presence of a suitable ligand-binding type II...

Background Pulmonary arterial hypertension (PAH) is a rare but fatal lung disease of diverse etiologies. PAH is now further subclassified as idiopathic (IPAH), familial (FPAH) and associated (APAH) varieties. Heterozygous mutations in BMPR2 can be detected in 50-70% of patients with FPAH and 10-40% of patients with IPAH. Although endothelial cells have been suspected as the cellular origin of PAH pathogenesis, no direct in vivo evidence has been clearly presented. The present study was designed to investigate whether endothelial Bmpr2 deletion can predispose to PAH. Methods and Results The Bmpr2 gene was deleted in pulmonary endothelial cells (pECs) using Bmpr2 conditional knockout mice and a novel endothelial Cre transgenic mouse line. Wide ranges of right ventricular systolic pressure (RVSP) were observed in mice with heterozygous (21.7 - 44.1 mmHg, median: 23.7 mmHg) and homozygous (20.7- 56.3 mmHg, median: 27 mmHg) conditional deletion of Bmpr2 in pECs in comparison with control mice (19.9 - 26.7 mmHg, median: 23 mmHg) at two to seven months of age. A subset of mice with RVSP greater than 30 mmHg exhibited right ventricular hypertrophy and an increase in the number and wall thickness of muscularized distal pulmonary arteries. In the lungs of these high RVSP mice, expression of proteins involved in the pathogenesis of PAH, such as serotonin transporter and tenacin-C, were elevated in distal arteries, and had a high incidence of perivascular leukocyte infiltration and in situ thrombosis. Conclusions Conditional hetero or homozygous Bmpr2 deletion in pECs predisposes mice to develop PAH.

Vertebral bodies are segmented along the anteroposterior (AP) body axis, and the segmental identity of the vertebrae is determined by the unique expression pattern of multiple Hox genes. Recent studies have demonstrated that a transforming growth factor ␤ (TGF-␤) family protein, Gdf11 (growth and differentiation factor 11), and the activin type II receptor, ActRIIB, are involved in controlling the spatiotemporal expression of multiple Hox genes along the AP axis, and that the disruption of each of these genes causes anterior transformation of the vertebrae. Skeletal defects are more severe in Gdf11-null mice than in ActRIIB-null mice, however, leaving it uncertain whether Gdf11 signals via ActRIIB. Here we demonstrate using genetic and biochemical studies that ActRIIB and its subfamily receptor, ActRIIA, cooperatively mediate the Gdf11 signal in patterning the axial vertebrae, and that Gdf11 binds to both ActRIIA and ActRIIB, and induces phosphorylation of Smad2. In addition, we also show that these two receptors can functionally compensate for one another to mediate signaling of another TGF-␤ ligand, nodal, during left-right patterning and the development of anterior head structure.

Objective-Hereditary hemorrhagic telangiectasia is a genetic disorder characterized by visceral and mucocutaneous arteriovenous malformations (AVMs). Clinically indistinguishable hereditary hemorrhagic telangiectasia 1 and hereditary hemorrhagic telangiectasia 2 are caused by mutations in ENG and ALK1, respectively. In this study, we have compared the development of visceral and mucocutaneous AVMs in adult stages between Eng-and Alk1-inducible knockout (iKO) models. Approach and Results-Eng or Alk1 were deleted from either vascular endothelial cells (ECs) or smooth muscle cells in adult stages using Scl-CreER and Myh11-CreER lines, respectively. Latex perfusion and intravital spectral imaging in a dorsal skinfold window chamber system were used to visualize remodeling vasculature during AVM formation. Global Eng deletion resulted in lethality with visceral AVMs and wound-induced skin AVMs. Deletion of Alk1 or Eng in ECs, but not in smooth muscle cells, resulted in wound-induced skin AVMs. Visceral AVMs were observed in EC-specific Alk1-iKO but not in Eng-iKO. Intravital spectral imaging revealed that Eng-iKO model exhibited more dynamic processes for AVM development when compared with Alk1-iKO model. In this study, we examined the effect of wounding in the development of mucocutaneous telangiectases and investigated the cellular origin of de novo AVMs in both HHT1 and HHT2 mouse models. Conclusions-Both Materials and MethodsMaterials and Methods are available in the online-only Supplement. ResultsTo investigate the role of ENG in adult mice, Eng was globally deleted using the tamoxifen-inducible ROSA26 CreER mouse strain (R26 CreER ). 10 We found that 3 consecutive day injection of tamoxifen at 2.5 mg/25 g body weight was the most effective regimen for R26CreER/+ ;Eng 2f/2f mice (Figure I in the online-only Data Supplement). In 3 to 4 days after the first tamoxifen injection, the R26 CreER/+ ;Eng 2f/2f mutant mice displayed signs of illness, such as slow movement, diarrhea, and dehydration, and died around day 4 to 10 (n>30). To analyze the subdermal vessels in the back skin, vascular casting with blue latex was performed at days 5 to 8 by infusing it into the left ventricle. Subdermal vessels were unaffected in the intact back skins of tamoxifen-injected adult R26CreER/+ ;Eng 2f/2f mice (data not shown) and also wounded skin of tamoxifen-injected adult R26CreER/+ ;Eng 2f/+ mice ( Figure IIA in the online-only Data Supplement; n=6). However, areas of wounds in mid-dorsum and ear of tamoxifen-injected R26 CreER/+ ;Eng 2f/2f mice showed dilated and tortuous vessels, and the latex dye was found in both arteries and veins, indicating the presence of AV shunts ( Figure IIB in the online-only Data Supplement; n=13). However, blood vessels away from the wound in Eng-iKOs had normal morphology and latex only in arterial branches.To determine the vascular cell type where ENG plays a critical role for the development of the vascular network at adult stages, we used 2 cell-type-specific inducible Cre lines: Scl-CreER 11 fo...

Arteriovenous malformation (AVM) refers to a vascular anomaly where arteries and veins are directly connected through a complex, tangled web of abnormal AV fistulae without a normal capillary network. Hereditary hemorrhagic telangiectasia (HHT) types 1 and 2 arise from heterozygous mutations in endoglin (ENG) and activin receptor-like kinase 1 (ALK1), respectively. HHT patients possess AVMs in various organs, and telangiectases (small AVMs) along the mucocutaneous surface. Understanding why and how AVMs develop is crucial for developing therapies to inhibit the formation, growth, or maintenance of AVMs in HHT patients. Previously, we have shown that secondary factors such as wounding are required for Alk1-deficient vessels to develop skin AVMs. Here we present evidences that AVMs establish from nascent arteries and veins rather than from remodeling of a preexistent capillary network in the wound-induced skin AVM model. We also show that VEGF can mimic the wound effect on skin AVM formation, and VEGF neutralizing antibody can prevent skin AVM formation and ameliorate internal bleeding in Alk1-deficient adult mice. With topical applications at different stages of AVM development, we demonstrate that the VEGF blockade can prevent the formation of AVM and cease the progression of AVM development. Taken together, the presented experimental model is an invaluable system for precise molecular mechanism of action of VEGF blockades as well as for preclinical screening of drug candidates for epistaxis and gastrointestinal bleedings.

Recent studies have suggested that changes in serum phosphate levels influence pathological states associated with aging such as cancer, bone metabolism, and cardiovascular function, even in individuals with normal renal function. The causes are only beginning to be elucidated but are likely a combination of endocrine, paracrine, autocrine, and cell autonomous effects. We have used an integrated quantitative biology approach, combining transcriptomics and proteomics to define a multi-phase, extracellular phosphate-induced, signaling network in pre-osteoblasts as well as primary human and mouse mesenchymal stromal cells. We identified a rapid mitogenic response stimulated by elevated phosphate that results in the induction of immediate early genes including c-fos. The mechanism of activation requires FGF receptor signaling followed by stimulation of N-ras and activation of AP-1 and serum response elements. A distinct long-term response also requires FGF receptor signaling and results in N-ras activation and expression of genes and secretion of proteins involved in matrix regulation, calcification, and angiogenesis. The late response is synergistically enhanced by addition of FGF23 peptide. The intermediate phase results in increased oxidative phosphorylation and ATP production and is necessary for the late response providing a functional link between the phases. Collectively, the results define elevated phosphate, as a mitogen and define specific mechanisms by which phosphate stimulates proliferation and matrix regulation. Our approach provides a comprehensive understanding of the cellular response to elevated extracellular phosphate, functionally connecting temporally coordinated signaling, transcriptional, and metabolic events with changes in long-term cell behavior.

Due to its specificity, fluorescence microscopy has become a quintessential imaging tool in cell biology. However, photobleaching, phototoxicity, and related artifacts continue to limit fluorescence microscopy’s utility. Recently, it has been shown that artificial intelligence (AI) can transform one form of contrast into another. We present phase imaging with computational specificity (PICS), a combination of quantitative phase imaging and AI, which provides information about unlabeled live cells with high specificity. Our imaging system allows for automatic training, while inference is built into the acquisition software and runs in real-time. Applying the computed fluorescence maps back to the quantitative phase imaging (QPI) data, we measured the growth of both nuclei and cytoplasm independently, over many days, without loss of viability. Using a QPI method that suppresses multiple scattering, we measured the dry mass content of individual cell nuclei within spheroids. In its current implementation, PICS offers a versatile quantitative technique for continuous simultaneous monitoring of individual cellular components in biological applications where long-term label-free imaging is desirable.