A non-genetic model of vascular shunts informs on the cellular mechanisms of formation and resolution of arteriovenous malformations.

AIMS: Arteriovenous malformations (AVMs), a disorder characterized by direct shunts between arteries and veins, are associated with genetic mutations. However, the mechanisms leading to AV shunt formation and how shunts can be reverted are poorly understood. METHODS AND RESULTS: Here, we report that oxygen-induced retinopathy (OIR) protocol leads to the consistent and stereotypical formation of AV shunts in non-genetically altered mice. OIR-induced AV shunts show all the canonical markers of AVMs. Genetic and pharmacological interventions demonstrated that changes in the volume of venous endothelial cells (EC)-hypertrophic venous cells-are the initiating step promoting AV shunt formation, whilst EC proliferation or migration played minor roles. Inhibition of the mTOR pathway prevents pathological increases in EC volume and significantly reduces the formation of AV shunts. Importantly, we demonstrate that ALK1 signalling cell-autonomously regulates EC volume in pro-angiogenic conditions, establishing a link with hereditary haemorrhagic telangiectasia-related AVMs. Finally, we demonstrate that a combination of EC volume control and EC migration is associated with the regression of AV shunts. CONCLUSION: Our findings highlight that an increase in the EC volume is the key mechanism driving the initial stages of AV shunt formation, leading to asymmetric capillary diameters. Based on our results, we propose a coherent and unifying timeline leading to the fast conversion of a capillary vessel into an AV shunt. Our data advocate for further investigation into the mechanisms regulating EC volume in health and disease as a way to identify therapeutic approaches to prevent and revert AVMs.

Localized conditional induction of brain arteriovenous malformations in a mouse model of hereditary hemorrhagic telangiectasia.

BACKGROUND: Longitudinal mouse models of brain arteriovenous malformations (AVMs) are crucial for developing novel therapeutics and pathobiological mechanism discovery underlying brain AVM progression and rupture. The sustainability of existing mouse models is limited by ubiquitous Cre activation, which is associated with lethal hemorrhages resulting from AVM formation in visceral organs. To overcome this condition, we developed a novel experimental mouse model of hereditary hemorrhagic telangiectasia (HHT) with CreER-mediated specific, localized induction of brain AVMs. METHODS: Hydroxytamoxifen (4-OHT) was stereotactically delivered into the striatum, parietal cortex, or cerebellum of R26CreER; Alk12f/2f (Alk1-iKO) littermates. Mice were evaluated for vascular malformations with latex dye perfusion and 3D time-of-flight magnetic resonance angiography (MRA). Immunofluorescence and Prussian blue staining were performed for vascular lesion characterization. RESULTS: Our model produced two types of brain vascular malformations, including nidal AVMs (88%, 38/43) and arteriovenous fistulas (12%, 5/43), with an overall frequency of 73% (43/59). By performing stereotaxic injection of 4-OHT targeting different brain regions, Alk1-iKO mice developed vascular malformations in the striatum (73%, 22/30), in the parietal cortex (76%, 13/17), and in the cerebellum (67%, 8/12). Identical application of the stereotaxic injection protocol in reporter mice confirmed localized Cre activity near the injection site. The 4-week mortality was 3% (2/61). Seven mice were studied longitudinally for a mean (SD; range) duration of 7.2 (3; 2.3-9.5) months and demonstrated nidal stability on sequential MRA. The brain AVMs displayed microhemorrhages and diffuse immune cell invasion. CONCLUSIONS: We present the first HHT mouse model of brain AVMs that produces localized AVMs in the brain. The mouse lesions closely resemble the human lesions for complex nidal angioarchitecture, arteriovenous shunts, microhemorrhages, and inflammation. The model's longitudinal robustness is a powerful discovery resource to advance our pathomechanistic understanding of brain AVMs and identify novel therapeutic targets.

BMP10 functions independently from BMP9 for the development of a proper arteriovenous network.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic vascular disorder characterized by the presence of arteriovenous malformation (AVM) in multiple organs. HHT is caused by mutations in genes encoding major constituents for transforming growth factor-ß (TGF-ß) family signaling: endoglin (ENG), activin receptor-like kinase 1 (ALK1), and SMAD4. The identity of physiological ligands for this ENG-ALK1 signaling pertinent to AVM formation has yet to be clearly determined. To investigate whether bone morphogenetic protein 9 (BMP9), BMP10, or both are physiological ligands of ENG-ALK1 signaling involved in arteriovenous network formation, we generated a novel Bmp10 conditional knockout mouse strain. We examined whether global Bmp10-inducible knockout (iKO) mice develop AVMs at neonatal and adult stages in comparison with control, Bmp9-KO, and Bmp9/10-double KO (dKO) mice. Bmp10-iKO and Bmp9/10-dKO mice showed AVMs in developing retina, postnatal brain, and adult wounded skin, while Bmp9-KO did not display any noticeable vascular defects. Bmp10 deficiency resulted in increased proliferation and size of endothelial cells in AVM vessels. The impaired neurovascular integrity in the brain and retina of Bmp10-iKO and Bmp9/10-dKO mice was detected. Bmp9/10-dKO mice exhibited the lethality and vascular malformation similar to Bmp10-iKO mice, but their phenotypes were more pronounced. Administration of BMP10 protein, but not BMP9 protein, prevented retinal AVM in Bmp9/10-dKO and endothelial-specific Eng-iKO mice. These data indicate that BMP10 is indispensable for the development of a proper arteriovenous network, whereas BMP9 has limited compensatory functions for the loss of BMP10. We suggest that BMP10 is the most relevant physiological ligand of the ENG-ALK1 signaling pathway pertinent to HHT pathogenesis.

Emerging pathogenic mechanisms in human brain arteriovenous malformations: a contemporary review in the multiomics era.

A variety of pathogenic mechanisms have been described in the formation, maturation, and rupture of brain arteriovenous malformations (bAVMs). While the understanding of bAVMs has largely been formulated based on animal models of rare hereditary diseases in which AVMs form, a new era of "omics" has permitted large-scale examinations of contributory genetic variations in human sporadic bAVMs. New findings regarding the pathogenesis of bAVMs implicate changes to endothelial and mural cells that result in increased angiogenesis, proinflammatory recruitment, and breakdown of vascular barrier properties that may result in hemorrhage; a greater diversity of cell populations that compose the bAVM microenvironment may also be implicated and complicate traditional models. Genomic sequencing of human bAVMs has uncovered inherited, de novo, and somatic activating mutations, such as KRAS, which contribute to the pathogenesis of bAVMs. New droplet-based, single-cell sequencing technologies have generated atlases of cell-specific molecular derangements. Herein, the authors review emerging genomic and transcriptomic findings underlying pathologic cell transformations in bAVMs derived from human tissues. The application of multiple sequencing modalities to bAVM tissues is a natural next step for researchers, although the potential therapeutic benefits or clinical applications remain unknown.

Suppression of BMP signaling by PHD2 deficiency in Pulmonary Arterial hypertension.

BMP signaling deficiency is evident in the lungs of patients with pulmonary arterial hypertension. We demonstrated that PHD2 deficiency suppresses BMP signaling in the lung endothelial cells, suggesting the novel mechanisms of dysregulated BMP signaling in the development of pulmonary arterial hypertension.

Genetics and Emerging Therapies for Brain Arteriovenous Malformations.

Brain arteriovenous malformations (AVMs) are characterized by a high-pressure, low-resistance vascular nidus created by direct shunting of blood from feeding arteries into arterialized veins, bypassing intervening capillaries. AVMs pose a risk of spontaneous rupture because the vessel walls are continuously exposed to increased shear stress and abnormal flow phenomena, which lead to vessel wall inflammation and distinct morphologic changes. The annual rupture rate is estimated at 2%, and once an AVM ruptures, the risk of rerupture increases 5-fold. The ability of AVMs to grow, regress, recur, and undergo remodeling shows their dynamic nature. Identifying the underlying cellular and molecular pathways of AVMs not only helps us understand their natural physiology but also allows us to directly block vital pathways, thus preventing AVM development and progression. Management of AVMs is challenging and often necessitates a multidisciplinary approach, including neurosurgical, endovascular, and radiosurgical expertise. Because many of these procedures are invasive, carry a risk of inciting hemorrhage, or are controversial, the demand for pharmacologic treatment options is increasing. In this review, we introduce novel findings of cellular and molecular AVM physiology and highlight key signaling mediators that are potential targets for AVM treatment. Furthermore, we give an overview of syndromes associated with hereditary and nonhereditary AVM formation and discuss causative genetic alterations.

Bone Marrow-Derived Alk1 Mutant Endothelial Cells and Clonally Expanded Somatic Alk1 Mutant Endothelial Cells Contribute to the Development of Brain Arteriovenous Malformations in Mice.

We have previously demonstrated that deletion of activin receptor-like kinase 1 (Alk1) or endoglin in a fraction of endothelial cells (ECs) induces brain arteriovenous malformations (bAVMs) in adult mice upon angiogenic stimulation. Here, we addressed three related questions: (1) could Alk1- mutant bone marrow (BM)-derived ECs (BMDECs) cause bAVMs? (2) is Alk1- ECs clonally expended during bAVM development? and (3) is the number of mutant ECs correlates to bAVM severity? For the first question, we transplanted BM from PdgfbiCreER;Alk12f/2f mice (EC-specific tamoxifen-inducible Cre with Alk1-floxed alleles) into wild-type mice, and then induced bAVMs by intra-brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor and intra-peritoneal injection of tamoxifen. For the second question, clonal expansion was analyzed using PdgfbiCreER;Alk12f/2f;confetti+/- mice. For the third question, we titrated tamoxifen to limit Alk1 deletion and compared the severity of bAVM in mice treated with low and high tamoxifen doses. We found that wild-type mice with PdgfbiCreER;Alk12f/2f BM developed bAVMs upon VEGF stimulation and Alk1 gene deletion in BMDECs. We also observed clusters of ECs expressing the same confetti color within bAVMs and significant proliferation of Alk1- ECs at early stage of bAVM development, suggesting that Alk1- ECs clonally expanded by local proliferation. Tamoxifen dose titration revealed a direct correlation between the number of Alk1- ECs and the burden of dysplastic vessels in bAVMs. These results provide novel insights for the understanding of the mechanism by which a small fraction of Alk1 or endoglin mutant ECs contribute to development of bAVMs.

Novel experimental model of brain arteriovenous malformations using conditional Alk1 gene deletion in transgenic mice.

OBJECTIVE: Hereditary hemorrhagic telangiectasia is the only condition associated with multiple inherited brain arteriovenous malformations (AVMs). Therefore, a mouse model was developed with a genetics-based approach that conditionally deleted the causative activin receptor-like kinase 1 (Acvrl1 or Alk1) gene. Radiographic and histopathological findings were correlated, and AVM stability and hemorrhagic behavior over time were examined. METHODS: Alk1-floxed mice were crossed with deleter mice to generate offspring in which both copies of the Alk1 gene were deleted by Tagln-Cre to form brain AVMs in the mice. AVMs were characterized using MRI, MRA, and DSA. Brain AVMs were characterized histopathologically with latex dye perfusion, immunofluorescence, and Prussian blue staining. RESULTS: Brains of 55 Tagln-Cre+;Alk12f/2f mutant mice were categorized into three groups: no detectable vascular lesions (group 1; 23 of 55, 42%), arteriovenous fistulas (AVFs) with no nidus (group 2; 10 of 55, 18%), and nidal AVMs (group 3; 22 of 55, 40%). Microhemorrhage was observed on MRI or MRA in 11 AVMs (50%). AVMs had the angiographic hallmarks of early nidus opacification, a tangle of arteries and dilated draining veins, and rapid shunting of blood flow. Latex dye perfusion confirmed arteriovenous shunting in all AVMs and AVFs. Microhemorrhages were detected adjacent to AVFs and AVMs, visualized by iron deposition, Prussian blue staining, and macrophage infiltration using CD68 immunostaining. Brain AVMs were stable on serial MRI and MRA in group 3 mice (mean age at initial imaging 2.9 months; mean age at last imaging 9.5 months). CONCLUSIONS: Approximately 40% of transgenic mice satisfied the requirements of a stable experimental AVM model by replicating nidal anatomy, arteriovenous hemodynamics, and microhemorrhagic behavior. Transgenic mice with AVFs had a recognizable phenotype of hereditary hemorrhagic telangiectasia but were less suitable for experimental modeling. AVM pathogenesis can be understood as the combination of conditional Alk1 gene deletion during embryogenesis and angiogenesis that is hyperactive in developing and newborn mice, which translates to a congenital origin in most patients but an acquired condition in patients with a confluence of genetic and angiogenic events later in life. This study offers a novel experimental brain AVM model for future studies of AVM pathophysiology, growth, rupture, and therapeutic regression.

Overexpression of Activin Receptor-Like Kinase 1 in Endothelial Cells Suppresses Development of Arteriovenous Malformations in Mouse Models of Hereditary Hemorrhagic Telangiectasia.

RATIONALE: Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease caused by mutations in ENG, ALK1, or SMAD4. Since proteins from all 3 HHT genes are components of signal transduction of TGF-ß (transforming growth factor ß) family members, it has been hypothesized that HHT is a disease caused by defects in the ENG-ALK1-SMAD4 linear signaling. However, in vivo evidence supporting this hypothesis is scarce. OBJECTIVE: We tested this hypothesis and investigated the therapeutic effects and potential risks of induced-ALK1 or -ENG overexpression (OE) for HHT. METHODS AND RESULTS: We generated a novel mouse allele (ROSA26Alk1) in which HA (human influenza hemagglutinin)-tagged ALK1 and bicistronic eGFP expression are induced by Cre activity. We examined whether ALK1-OE using the ROSA26Alk1 allele could suppress the development of arteriovenous malformations (AVMs) in wounded adult skin and developing retinas of Alk1- and Eng-inducible knockout (iKO) mice. We also used a similar approach to investigate whether ENG-OE could rescue AVMs. Biochemical and immunofluorescence analyses confirmed the Cre-dependent OE of the ALK1-HA transgene. We could not detect any pathological signs in ALK1-OE mice up to 3 months after induction. ALK1-OE prevented the development of retinal AVMs and wound-induced skin AVMs in Eng-iKO as well as Alk1-iKO mice. ALK1-OE normalized expression of SMAD and NOTCH target genes in ENG-deficient endothelial cells (ECs) and restored the effect of BMP9 (bone morphogenetic protein 9) on suppression of phosphor-AKT levels in these endothelial cells. On the other hand, ENG-OE could not inhibit the AVM development in Alk1-iKO models. CONCLUSIONS: These data support the notion that ENG and ALK1 form a linear signaling pathway for the formation of a proper arteriovenous network during angiogenesis. We suggest that ALK1 OE or activation can be an effective therapeutic strategy for HHT. Further research is required to study whether this therapy could be translated into treatment for humans.

TMEM100 is a key factor for specification of lymphatic endothelial progenitors.

BACKGROUND: TMEM100 is identified as a downstream gene of bone morphogenetic protein 9 (BMP9) signaling via activin receptor-like kinase 1 (ALK1), which is known to participate in lymphangiogenesis as well as angiogenesis. TMEM100 has been shown to be important for blood vessel formation and maintenance, but its role in the development of lymphatic vasculature remains unknown. The objective is to investigate the role of TMEM100 in development of the lymphatic system. METHODS AND RESULTS: Global Tmem100 gene deletion was induced by tamoxifen on 10.5 days post-coitus. Tmem100-inducible knockout (iKO) embryos in embryonic days (E)14.5-16.5 exhibited edema and blood-filled enlarged lymphatics with misconnections between veins and lymphatic vessels. For a reciprocal approach, we have generated a novel mouse line in which TMEM100 overexpression (OE) can be induced in endothelial cells by intercrossing with Tie2-Cre driver. TMEM100-OE embryos at E12.5-14.5 exhibited edema with small size and number of lymphatic vessels, the exact opposite phenotypes of Tmem100-iKOs. In Tmem100-iKO embryos, the number of progenitors of lymphatic endothelial cells (LECs) in the cardinal vein was increased, while it was decreased in TMEM100-OE embryos. The activity of NOTCH signaling, which limits the number of progenitors of LECs in the cardinal vein, was decreased in Tmem100-iKO embryos, whereas it was increased in TMEM100-OE embryos. CONCLUSION: TMEM100 plays an important role in the specification of LECs in the cardinal veins, at least in part, by regulating the NOTCH signaling.

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.

Recent Advances in Basic Research for Brain Arteriovenous Malformation.

Arteriovenous malformations (AVMs) are abnormal connections of vessels that shunt blood directly from arteries into veins. Rupture of brain AVMs (bAVMs) can cause life-threatening intracranial bleeding. Even though the majority of bAVM cases are sporadic without a family history, some cases are familial. Most of the familial cases of bAVMs are associated with a genetic disorder called hereditary hemorrhagic telangiectasia (HHT). The mechanism of bAVM formation is not fully understood. The most important advances in bAVM basic science research is the identification of somatic mutations of genes in RAS-MAPK pathways. However, the mechanisms by which mutations of these genes lead to AVM formation are largely unknown. In this review, we summarized the latest advance in bAVM studies and discussed some pathways that play important roles in bAVM pathogenesis. We also discussed the therapeutic implications of these pathways.

Pazopanib may reduce bleeding in hereditary hemorrhagic telangiectasia.

Pazopanib (Votrient) is an orally administered tyrosine kinase inhibitor that blocks VEGF receptors potentially serving as anti-angiogenic treatment for hereditary hemorrhagic telangiectasia (HHT). We report a prospective, multi-center, open-label, dose-escalating study [50 mg, 100 mg, 200 mg, and 400 mg], designed as a proof-of-concept study to demonstrate efficacy of pazopanib on HHT-related bleeding, and to measure safety. Patients, recruited at 5 HHT Centers, required ≥ 2 Curacao criteria AND [anemia OR severe epistaxis with iron deficiency]. Co-primary outcomes, hemoglobin (Hgb) and epistaxis severity, were measured during and after treatment, and compared to baseline. Safety monitoring occurred every 1.5 weeks. Seven patients were treated with 50 mg pazopanib daily. Six/seven showed at least 50% decrease in epistaxis duration relative to baseline at some point during study; 3 showed at least 50% decrease in duration during Weeks 11 and 12. Six patients showed a decrease in ESS of > 0.71 (MID) relative to baseline at some point during study; 3/6 showed a sustained improvement. Four patients showed > 2 gm improvement in Hgb relative to baseline at one or more points during study. Health-related QOL scores improved on all SF-36 domains at Week 6 and/or Week 12, except general health (unchanged). There were 19 adverse events (AE) including one severe AE (elevated LFTs, withdrawn from dosing at 43 days); with no serious AE. In conclusion, we observed an improvement in Hgb and/or epistaxis in all treated patients. This occurred at a dose much lower than typically used for oncologic indications, with no serious AE. Further studies of pazopanib efficacy are warranted.

SMAD4 Deficiency Leads to Development of Arteriovenous Malformations in Neonatal and Adult Mice.

Background Hereditary hemorrhagic telangiectasia ( HHT ) is a rare genetic vascular disorder caused by mutations in endoglin ( ENG ), activin receptor-like kinase 1 ( ACVRL 1; ALK 1), or SMAD 4. Major clinical symptoms of HHT are arteriovenous malformations ( AVM s) found in the brain, lungs, visceral organs, and mucosal surface. Animal models harboring mutations in Eng or Alk1 recapitulate all of these HHT clinical symptoms and have been useful resources for studying mechanisms and testing potential drugs. However, animal models representing SMAD 4 mutations have been lacking. The goal of this study is to evaluate Smad4-inducible knockout ( iKO ) mice as an animal model of HHT and compare the phenotypes with other established HHT animal models. Methods and Results Global Smad4 deletion was induced at neonatal and adult stages, and hemoglobin levels, gastrointestinal hemorrhage, and presence of aberrant arteriovenous connections were examined. Neonatal Smad4- iKO mice exhibited signs of gastrointestinal bleeding and AVM s in the brain, intestine, nose, and retina. The radial expansion was decreased, and AVM s were detected on both distal and proximal retinal vasculature of Smad4- iKO s. Aberrant smooth muscle actin staining was observed in the initial stage AVM s and their connecting veins, indicating abnormal arterial flow to veins. In adult mice, Smad4 deficiency caused gastrointestinal bleeding and AVM s along the gastrointestinal tract and wounded skin. HHT -related phenotypes of Smad4- iKO s appeared to be comparable with those found in Alk1- iKO and Eng- iKO mice. Conclusions These data further confirm that SMAD signaling is crucial for normal arteriovenous network formation, and Smad4- iKO will be an alternative animal model of AVM research associated with HHT .

CXCL12-CXCR4 signalling plays an essential role in proper patterning of aortic arch and pulmonary arteries.

AIMS: Chemokine CXCL12 (stromal derived factor 1: SDF1) has been shown to play important roles in various processes of cardiovascular development. In recent avian studies, CXCL12 signalling has been implicated in guidance of cardiac neural crest cells for their participation in the development of outflow tract and cardiac septum. The goal of this study is to investigate the extent to which CXCL12 signalling contribute to the development of aortic arch and pulmonary arteries in mammals. METHODS AND RESULTS: Novel Cxcl12-LacZ reporter and conditional alleles were generated. Using whole mount X-gal staining with the reporter allele and vascular casting techniques, we show that the domain branching pattern of pulmonary arteries in Cxcl12-null mice is completely disrupted and discordant with that of pulmonary veins and airways. Cxcl12-null mice also displayed abnormal and superfluous arterial branches from the aortic arch. The early steps of pharyngeal arch remodelling in Cxcl12-null mice appeared to be unaffected, but vertebral arteries were often missing and prominent aberrant arteries were present parallel to carotid arteries or trachea, similar to aberrant vertebral artery or thyroid ima artery, respectively. Analysis with computed tomography not only confirmed the results from vascular casting studies but also identified abnormal systemic arterial supply to lungs in the Cxcl12-null mice. Tie2-Cre mediated Cxcr4 deletion phenocopied the Cxcl12-null phenotypes, indicating that CXCR4 is the primary receptor for arterial patterning, whereas Cxcl12 or Cxcr4 deletion by Wnt1-Cre did not affect aortic arch patterning. CONCLUSION: CXCL12-CXCR4 signalling is essential for the correct patterning of aortic arches and pulmonary arteries during development. Superfluous arteries in Cxcl12-null lungs and the aortic arch infer a role of CXCL12 in protecting arteries from uncontrolled sprouting during development of the arterial system.

Reduced activin receptor-like kinase 1 activity promotes cardiac fibrosis in heart failure.

INTRODUCTION: Activin receptor-like kinase 1 (ALK1) mediates signaling via the transforming growth factor beta-1 (TGFß1), a pro-fibrogenic cytokine. No studies have defined a role for ALK1 in heart failure. HYPOTHESIS: We tested the hypothesis that reduced ALK1 expression promotes maladaptive cardiac remodeling in heart failure. METHODS AND RESULTS: In patients with advanced heart failure referred for left ventricular (LV) assist device implantation, LV Alk1 mRNA and protein levels were lower than control LV obtained from patients without heart failure. To investigate the role of ALK1 in heart failure, Alk1 haploinsufficient (Alk1+/-) and wild-type (WT) mice were studied 2 weeks after severe transverse aortic constriction (TAC). LV and lung weights were higher in Alk1+/- mice after TAC. Cardiomyocyte area and LV mRNA levels of brain natriuretic peptide and ß-myosin heavy chain were increased similarly in Alk1+/- and WT mice after TAC. Alk-1 mice exhibited reduced Smad 1 phosphorylation and signaling compared to WT mice after TAC. Compared to WT, LV fibrosis and Type 1 collagen mRNA and protein levels were higher in Alk1+/- mice. LV fractional shortening was lower in Alk1+/- mice after TAC. CONCLUSIONS: Reduced expression of ALK1 promotes cardiac fibrosis and impaired LV function in a murine model of heart failure. Further studies examining the role of ALK1 and ALK1 inhibitors on cardiac remodeling are required.

Conditional knockout of activin like kinase-1 (ALK-1) leads to heart failure without maladaptive remodeling.

Activin like kinase-1 (AlK-1) mediates signaling via the transforming growth factor beta (TGFß) family of ligands. AlK-1 activity promotes endothelial proliferation and migration. Reduced AlK-1 activity is associated with arteriovenous malformations. No studies have examined the effect of global AlK-1 deletion on indices of cardiac remodeling. We hypothesized that reduced levels of AlK-1 promote maladaptive cardiac remodeling. To test this hypothesis, we employed AlK-1 conditional knockout mice (cKO) harboring the ROSA26-CreER knock-in allele, whereby a single dose of intraperitoneal tamoxifen triggered ubiquitous Cre recombinase-mediated excision of floxed AlK-1 alleles. Tamoxifen treated wild-type (WT-TAM; n = 5) and vehicle treated AlK-1-cKO mice (cKO-CON; n = 5) served as controls for tamoxifen treated AlK-1-cKO mice (cKO-TAM; n = 15). AlK-1 cKO-TAM mice demonstrated reduced 14-day survival compared to cKO-CON controls (13 vs 100%, respectively, p < 0.01). Seven days after treatment, cKO-TAM mice exhibited reduced left ventricular (LV) fractional shortening, progressive LV dilation, and gastrointestinal bleeding. After 14 days total body mass was reduced, but LV and lung mass increased in cKO-TAM not cKO-CON mice. Peak LV systolic pressure, contractility, and arterial elastance were reduced, but LV end-diastolic pressure and stroke volume were increased in cKO-TAM, not cKO-CON mice. LV AlK-1 mRNA levels were reduced in cKO-TAM, not cKO-CON mice. LV levels of other TGFß-family ligands and receptors (AlK5, TBRII, BMPRII, Endoglin, BMP7, BMP9, and TGFß1) were unchanged between groups. Cardiomyocyte area and LV levels of BNP were increased in cKO-TAM mice, but LV levels of ß-MHC and SERCA were unchanged. No increase in markers of cardiac fibrosis, Type I collagen, CTGF, or PAI-1, were observed between groups. No differences were observed for any variable studied between cKO-CON and WT-TAM mice. Global deletion of AlK-1 is associated with the development of high output heart failure without maladaptive remodeling. Future studies exploring the functional role of AlK-1 in cardiac remodeling independent of systemic AVMs are required.

Mitochondrial ATP transporter depletion protects mice against liver steatosis and insulin resistance.

Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder in obese individuals. Adenine nucleotide translocase (ANT) exchanges ADP/ATP through the mitochondrial inner membrane, and Ant2 is the predominant isoform expressed in the liver. Here we demonstrate that targeted disruption of Ant2 in mouse liver enhances uncoupled respiration without damaging mitochondrial integrity and liver functions. Interestingly, liver specific Ant2 knockout mice are leaner and resistant to hepatic steatosis, obesity and insulin resistance under a lipogenic diet. Protection against fatty liver is partially recapitulated by the systemic administration of low-dose carboxyatractyloside, a specific inhibitor of ANT. Targeted manipulation of hepatic mitochondrial metabolism, particularly through inhibition of ANT, may represent an alternative approach in NAFLD and obesity treatment.