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Abstract-Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by epistaxis, mucocutaneous telangiectases, and arteriovenous malformations (AVM). Two genes are linked to HHT: endoglin (ENG) in HHT1 and activin receptor-like kinase 1 (ACVRL1; ALK1) in HHT2. Although both genes are involved in the transforming growth factor ␤ signaling pathways, the pathogenetic mechanisms for HHT remain elusive. It was shown that mutations in the Alk1 gene in mice and zebrafish resulted in an embryonic lethal phenotype due to severe dilation of blood vessels. We created a novel null mutant mouse line for Alk1 (Alk1 lacZ ) by replacing its exons, including the one that encodes the transmembrane domain, with the ␤-galactosidase gene. Using Alk1 lacZ mice, we show that Alk1 is predominantly expressed in developing arterial endothelium. Alk1 expression is greatly diminished in adult arteries, but is induced in preexisting feeding arteries and newly forming arterial vessels during wound healing and tumor angiogenesis. We also show that hemodynamic changes, which require vascular remodeling, may regulate Alk1 expression. Our studies suggest the role of Alk1 signaling in arterialization and remodeling of arteries. Contrary to the current view of HHT as venous disease, our findings suggest that the arterioles rather than the venules are the primary vessels affected by the loss of an Alk1 allele, and that blood vessels with reduction in Alk1 expression may harbor defects in responding to demands for vascular remodeling. , also known as Osler-Weber-Rendu syndrome, is an autosomaldominant vascular disorder that affects more than 1 in 10 000 individuals. 1 HHT is characterized by recurrent epistaxis, localized mucocutaneous telangiectases in the nasal septum, oral mucosa, and gastrointestinal tract, as well as arteriovenous malformations (AVM) in the lungs, liver, gastrointestinal tract, and brain, which can cause severe ischemic injuries or stroke. 2 Multiple mutations in endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1; ALK1) have been identified for HHT1 and HHT2, respectively. 1,3,4 Previous studies have shown that haploinsufficiency of ENG or ALK1 causes HHT. 5,6 The earliest clinically detectable telangiectasia is focal dilation of postcapillary venules. 7,8 However, the pathogenetic mechanisms by which reduced expression of Endoglin or ALK1 causes dilation of venules are not clearly understood. Furthermore, the questions of why only limited vascular beds in a HHT patient are affected and what determines the age of onset and the severity of disease manifestation among HHT patients remain to be answered.Both endoglin and Alk1 are involved in TGF-␤ signaling. TGF-␤ family cytokines exert their effects by binding to heteromeric complexes of two 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 a...

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...

Six distinct genes have been identified as belonging to the type IV collagen gene family. They can be organized into three sets, i.e., COL4A1/COL4A2, COL4A3/COL4A4, and COL4A5/COL4A6, which are localized on three different chromosomes in humans, 13, 2, and X, respectively. Within each set the genes are aligned head-to-head and their expression is regulated by bidirectional promoters between the genes. Transcriptional regulation of the COL4A1/COL4A2 set has been well characterized. The transcription of COL4A6 seems to be controlled by two alternative promoters. While collagen IV molecules composed of alpha1 and alpha2 chains are broadly distributed, molecules comprising combinations of the other four chains, alpha3-alpha6, are important components of specialized basement membranes. The precise chain composition of triple-helical molecules assembled from the alpha3-alpha6 chains is not entirely clear, but it is hypothesized that alpha3-alpha5 chains and alpha5 and alpha6 chains form heterotrimeric molecules. Several pieces of evidence indicate that alpha3/alpha4/alpha5 molecules and alpha5/alpha6 molecules are components of the basement membrane network. This helps explain the observation that the kidney and skin basement membranes from patients with Alport syndrome caused by mutations in the alpha5 coding gene, COL4A5, are defective in the alpha3, alpha4, and alpha6 chains together with the alpha5 chain. Large deletions involving the COL4A5 and COL4A6 genes have been found in rare cases of diffuse leiomyomatosis associated with Alport syndrome.

Transforming growth factor b (TGF-b) transmits signals through a heterotetrameric cell-surface complex of type II (TGFBR2) and type I (activin receptor-like kinase 5, ALK5; TGFBR1) serine/threonine kinase receptors, as well as Smad2/3. We have previously shown that another type I receptor, ALK1 (ACVRL1), can also mediate TGF-b signals via BMP-activated Smads in vascular endothelial cells (ECs). Our group and others have proposed the hypothesis that two TGF-b signaling pathways via ALK1 and ALK5 in vascular ECs may play a balancing role for controlling the proliferation and migration of ECs during angiogenesis. To address in vivo roles of this balance in vascular development, we have created a knockin mouse line that carries a lacZ reporter in the Alk5 gene locus (Alk5 lacZ ). Throughout development, a well-defined, nonubiquitous expression pattern of Alk5 expression was observed in multiple tissues, and organs. Overall, a high level of Alk5 expression was found in perichondria, periostea, and the mesenchymal layers underlying epithelia in the kidney, lung, and gallbladder. In blood vessels, contrasting to predominant Alk1 expression in arterial endothelium, Alk5 expression was localized in the medial and adventitial layers of blood vessels, but was undetectable in the intimal layer. In addition, although Alk5-null embryos exhibit a defect in the formation of vascular smooth muscle layers, the lumens of blood vessels are generated properly, which stands in contrast to the severe dilation of the vascular lumens in Alk1-null mice. These mutually exclusive expression patterns of Alk1 and Alk5 in blood vessels, as well as the undisturbed formation of the vascular lumens in Alk5-null embryos, suggest that each type I receptor has its own unique functions in vascular development. The Alk5 lacZ mice will be a valuable resource in identifying the in vivo cellular targets of TGF-b family signals mediated by Alk5, both during embryonic development as well as in diverse pathological conditions.

The adenine nucleotide translocases (Ant) facilitate the transport of ADP and ATP by an antiport mechanism across the inner mitochondrial membrane, thus playing an essential role in cellular energy metabolism. We recently identified a novel member of the Ant family in mouse, Ant4, of which gene configuration as well as amino acid homology is well conserved among mammals. The conservation of Ant4 in mammals, along with the absence of Ant4 in nonmammalian species, suggests a unique and indispensable role for this ADP/ATP carrier in mammalian development. Of interest, in contrast to its paralog Ant2, which is encoded by the X chromosome and ubiquitously expressed in somatic cells, Ant4 is encoded by an autosome and selectively expressed in testicular germ cells. Immunohistochemical examination as well as RNA expression analysis using separated spermatogenic cell types revealed that Ant4 expression was particularly high in spermatocytes. When we generated Ant4-deficient mice by targeted disruption, a significant reduction in testicular size was observed without any other distinguishable abnormalities in the mice. Histological examination as well as stage-specific gene expression analysis in adult and neonatal testes revealed a severe reduction of spermatocytes accompanied by increased apoptosis. Subsequently, the Ant4-deficient male mice were infertile. Taken together, these data elucidated the indispensable role of Ant4 in murine spermatogenesis. Considering the unique conservation and chromosomal location of the Ant family genes in mammals, the Ant4 gene may have arisen in mammalian ancestors and been conserved in mammals to serve as the sole and essential mitochondrial ADP/ATP carrier during spermatogenesis where the sex chromosome-linked Ant2 gene is inactivated.

Abstract-Homeobox transcription factor Nkx2-5, highly expressed in heart, is a critical factor during early embryonic cardiac development. In this study, using tamoxifen-inducible Nkx2-5 knockout mice, we demonstrate the role of Nkx2-5 in conduction and contraction in neonates within 4 days after perinatal tamoxifen injection. Conduction defect was accompanied by reduction in ventricular expression of the cardiac voltage-gated Na ϩ channel pore-forming ␣-subunit (Na v 1.5-␣), the largest ion channel in the heart responsive for rapid depolarization of the action potential, which leads to increased intracellular Ca 2ϩ for contraction (conduction-contraction coupling). In addition, expression of ryanodine receptor 2, through which Ca 2ϩ is released from sarcoplasmic reticulum, was substantially reduced in Nkx2-5 knockout mice. These results indicate that Nkx2-5 function is critical not only during cardiac development but also in perinatal hearts, by regulating expression of several important gene products involved in conduction and contraction. (Circ Res. 2008;103:580-590.) Key Words: conduction Ⅲ contraction Ⅲ gene targeting Ⅲ transcription C oordinated conduction and contraction is critical for cardiac function. Cardiac conduction is initiated by a spontaneous wave of electricity (action potential) that arises from sinoatrial (SA) nodal cells located in the upper right atrium, followed by sequential spreading of action potential to the atria, atrioventricular (AV) node, His bundles, peripheral Purkinje fiber, and the ventricles. In the SA and AV nodes, inward Ca 2ϩ currents are primarily responsible for slow depolarization of the action potential, whereas Na ϩ currents are primarily responsible for rapid depolarization of the action potential in other cardiomyocytes. [1][2][3][4][5] In both neonatal and adult mouse cardiomyocytes, robust Na ϩ currents producing the rapid upstroke action potential are tetrodotoxin-resistant. 6,7 In the mouse heart, tetrodotoxin-resistant Na v 1.5 is the primary isoform of the Na ϩ channel mRNA throughout development and is most highly expressed in adults; embryonic day (ED)12.5 50% to 100%, neonatal 150% to 200%, and adult 500% to 550%, in comparison to a house keeping gene. 8 Other Na ϩ channels, mostly tetrodotoxin-sensitive, are also expressed at lower levels in the heart at the 3 stages. 8 Among them, the highest expression was demonstrated for Na v 2.3 and Na v 1.4 in the adult heart but at 25% to 30% at most compared to the house keeping gene. 8 In adult mouse ventricular cardiomyocytes, 16% of Na ϩ channel transcripts are other than Na v 1.5, expressed in the order of Na v 1.4ϾNa v 1.3ϾNa v 1.2ϾNa v 1.1ϾNa v 1.6. 4 After membrane depolarization, L-type Ca 2ϩ channels are activated, followed by Ca 2ϩ release from intracellular Ca 2ϩ stores in sarcoplasmic reticulum (SR) through the cardiac isoform of the ryanodine receptor (RyR2) (Ca 2ϩ -induced Ca 2ϩ release). Sufficient intracellular Ca 2ϩ allows actomyosin interactions resulting in cardiac contraction (excitation-contr...

A novel aldosterone-producing pathology, pAATL that causes unilateral PA, was detected in the adrenals of two patients. Next-generation sequencing analyses of the large pAATLs suggested that the introduction of APA-associated mutations in the ion channel/pump genes may be involved in the development of mAPA from existing APCCs.