Suk Paul Oh

Gene Dosage–Dependent Embryonic Development and Proliferation Defects in Mice Lacking the Transcriptional Integrator p300

The transcriptional coactivator and integrator p300 and its closely related family member CBP mediate multiple, signal-dependent transcriptional events. We have generated mice lacking a functional p300 gene. Animals nullizygous for p300 died between days 9 and 11.5 of gestation, exhibiting defects in neurulation, cell proliferation, and heart development. Cells derived from p300-deficient embryos displayed specific transcriptional defects and proliferated poorly. Surprisingly, p300 heterozygotes also manifested considerable embryonic lethality. Moreover, double heterozygosity for p300 and cbp was invariably associated with embryonic death. Thus, mouse development is exquisitely sensitive to the overall gene dosage of p300 and cbp. Our results provide genetic evidence that a coactivator endowed with histone acetyltransferase activity is essential for mammalian cell proliferation and development.

Lack of collagen XVIII/endostatin results in eye abnormalities

Mice lacking collagen XVIII and its proteolytically derived product endostatin show delayed regression of blood vessels in the vitreous along the surface of the retina after birth and lack of or abnormal outgrowth of retinal vessels. This suggests that collagen XVIII/endostatin is critical for normal blood vessel formation in the eye. All basement membranes in wild‐type eyes, except Descemets membrane, showed immunogold labeling with antibodies against collagen XVIII. Labeling at sites where collagen fibrils in the vitreous are connected with the inner limiting membrane and separation of the vitreal matrix from the inner limiting membrane in mutant mice indicate that collagen XVIII is important for anchoring vitreal collagen fibrils to the inner limiting membrane. The findings provide an explanation for high myopia, vitreoretinal degeneration and retinal detachment seen in patients with Knobloch syndrome caused by loss‐of‐function mutations in collagen XVIII.

Evidence for angiotensin-converting enzyme 2 as a therapeutic target for the prevention of pulmonary hypertension.

RATIONALE It has been proposed that an activated renin angiotensin system (RAS) causes an imbalance between the vasoconstrictive and vasodilator mechanisms involving the pulmonary circulation leading to the development of pulmonary hypertension (PH). Recent studies have indicated that angiotensin-converting enzyme 2 (ACE2), a member of the vasoprotective axis of the RAS, plays a regulatory role in lung pathophysiology, including pulmonary fibrosis and acute lung disease. Based on these observations, we propose the hypothesis that activation of endogenous ACE2 can shift the balance from the vasoconstrictive, proliferative axis (ACE-Ang II-AT1R) to the vasoprotective axis [ACE2-Ang-(1-7)-Mas] of the RAS, resulting in the prevention of PH. OBJECTIVES We have taken advantage of a recently discovered synthetic activator of ACE2, XNT (1-[(2-dimethylamino) ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one), to study its effects on monocrotaline-induced PH in rats to support this hypothesis. METHODS The cardiopulmonary effects of XNT were evaluated in monocrotaline-induced PH rat model. MEASUREMENTS AND MAIN RESULTS A single subcutaneous treatment of monocrotaline in rats resulted in elevated right ventricular systolic pressure, right ventricular hypertrophy, increased pulmonary vessel wall thickness, and interstitial fibrosis. These changes were associated with increases in the mRNA levels of renin, ACE, angiotensinogen, AT1 receptors, and proinflammatory cytokines. All these features of PH were prevented in these monocrotaline-treated rats by chronic treatment with XNT. In addition, XNT caused an increase in the antiinflammatory cytokine, IL-10. CONCLUSIONS These observations provide conceptual support that activation of ACE2 by a small molecule can be a therapeutically relevant approach for treating and controlling PH.

Prevention of Pulmonary Hypertension by Angiotensin-Converting Enzyme 2 Gene Transfer

In spite of recent advancements in the treatment of pulmonary hypertension, successful control has yet to be accomplished. The abundant presence of angiotensin-converting enzyme 2 (ACE2) in the lungs and its impressive effect in the prevention of acute lung injury led us to test the hypothesis that pulmonary overexpression of this enzyme could produce beneficial outcomes against pulmonary hypertension. Monocrotaline (MCT) treatment of mice for 8 weeks resulted in significant increases in right ventricular systolic pressure, right ventricle:left ventricle plus septal weight ratio, and muscularization of pulmonary vessels. Administration of a lentiviral vector containing ACE2, 7 days before MCT treatment prevented the increases in right ventricular systolic pressure (control: 25±1 mm Hg; MCT: 44±5 mm Hg; MCT+ACE2: 26±1 mm Hg; n=6; P<0.05) and right ventricle:left ventricle plus septal weight ratio (control: 0.25±0.01; MCT: 0.31±0.01; MCT+ACE2: 0.26±0.01; n=8; P<0.05). A significant attenuation in muscularization of pulmonary vessels induced by MCT was also observed in animals overexpressing ACE2. These beneficial effects were associated with an increase in the angiotensin II type 2 receptor:angiotensin II type 1 receptor mRNA ratio. Also, pulmonary hypertension–induced increases in proinflammatory cytokines were significantly attenuated by lentiviral vector–containing ACE2 treatment. Furthermore, ACE2 gene transfer in mice after 6 weeks of MCT treatment resulted in a significant reversal of right ventricular systolic pressure. These observations demonstrate that ACE2 overexpression prevents and reverses right ventricular systolic pressure and associated pathophysiology in MCT-induced pulmonary hypertension by a mechanism involving a shift from the vasoconstrictive, proliferative, and fibrotic axes to the vasoprotective axis of the renin-angiotensin system and inhibition of proinflammatory cytokines.

Perinatal Loss of Nkx2-5 Results in Rapid Conduction and Contraction Defects

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+ channel pore-forming &agr;-subunit (Nav1.5-&agr;), the largest ion channel in the heart responsive for rapid depolarization of the action potential, which leads to increased intracellular Ca2+ for contraction (conduction–contraction coupling). In addition, expression of ryanodine receptor 2, through which Ca2+ 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.

B-Cell Translocation Gene 2 (Btg2) Regulates Vertebral Patterning by Modulating Bone Morphogenetic Protein/Smad Signaling

ABSTRACT Btg2 is a primary p53 transcriptional target gene which may function as a coactivator-corepressor and/or an adaptor molecule that modulates the activities of its interacting proteins. We have generated Btg2-null mice to elucidate the in vivo function of Btg2. Btg2-null mice are viable and fertile but exhibit posterior homeotic transformations of the axial vertebrae in a dose-dependent manner. Consistent with its role in vertebral patterning, Btg2 is expressed in the presomitic mesoderm, tail bud, and somites during somitogenesis. We further provide biochemical evidence that Btg2 interacts with bone morphogenetic protein (BMP)-activated Smads and enhances the transcriptional activity of BMP signaling. In view of the genetic evidence that reduced BMP signaling causes posteriorization of the vertebral pattern, we propose that the observed vertebral phenotype in Btg2-null mice is due to attenuated BMP signaling.

The mouse α1(XII) and human α1(XII)-like collagen genes are localized on mouse chromosome 9 and human chromosome 6

Type XII collagen is a member of the FACIT (fibril-associated collagens with interrupted triple helices) group of extracellular matrix proteins. Like the other members of this group, collagen types IX and XIV, type XII has alternating triple-helical and non-triple-helical domains. Because of its structure, its association with collagen fibrils, and its distribution in dense connective tissues, type XII is thought possibly to act as a cross-bridge between fibrils and resist shear forces caused by tension. A portion of the ffuse gene was isolated by screening a genomic library with a chicken alpha 1 (XII) cDNA probe, followed by subcloning and sequence analysis. Comparison of exon sequences with the sequence of a mouse cDNA clone allowed the mouse gene to be identified as the alpha 1 (XII) collagen gene. In the mouse, Col12a1 is located on chromosome 9, as determined by linkage analysis using DNA from interspecific backcrosses with Mus spretus. Screening of a human genomic library also allowed the isolation of a human alpha 1(XII)-like gene (CoL12A1). This gene was mapped to chromosome 6 by blot hybridization to DNA from human/hamster hybrid cell lines. This information should prove useful in determining the role of type XII collagen genes as candidate genes in inheritable connective tissue diseases.

Vascular smooth muscle Jak2 mediates angiotensin II-induced hypertension via increased levels of reactive oxygen species.

AIMS Angiotensin II (Ang II) type AT(1) receptors expressed on vascular smooth muscle cells (VSMCs) couple to the Jak2 signalling pathway. However, the importance of this tissue-specific coupling is poorly understood. The purpose of this investigation was to determine the importance of VSMC-derived Jak2 in angiotensin II-mediated hypertension. METHODS AND RESULTS The Cre-loxP system was used to conditionally eliminate Jak2 tyrosine kinase expression within the smooth muscle cells of mice. Following chronic Ang II infusion, the resulting increase in mean arterial pressure (MAP) was significantly attenuated in the Jak2 null mice when compared with littermate controls. The VSMC Jak2 null mice were also protected from the Ang II-induced vascular remodelling. Aortic rings from the VSMC Jak2 null mice exhibited reduced Ang II-induced contraction and enhanced endothelial-dependent relaxation via increased nitric oxide (NO) bioavailability. When compared with controls, the VSMC Jak2 nulls also had lower levels of hydrogen peroxide, Rho kinase activity, and intracellular Ca(2+) in response to Ang II. CONCLUSIONS The data indicate that VSMC Jak2 expression is involved in the pathogenesis of Ang II-dependent hypertension due to the increased presence of reactive oxygen species (ROS). As such, VSMC-derived Jak2 tyrosine kinase modulates overall vascular tone via multiple, non-redundant mechanisms.

Mitochondrial ATP transporter Ant2 depletion impairs erythropoiesis and B lymphopoiesis.

Adenine nucleotide translocases (ANTs) transport ADP and ATP through mitochondrial inner membrane, thus playing an essential role for energy metabolism of eukaryotic cells. Mice have three ANT paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. While knockout mice have been characterized with Ant1 and Ant4 genes, which resulted in exercise intolerance and male infertility, respectively, the role of the ubiquitously expressed Ant2 gene in animal development has not been fully demonstrated. Here, we generated Ant2 hypomorphic mice by targeted disruption of the gene, in which Ant2 expression is largely depleted. The mice showed apparently normal embryonic development except pale phenotype along with a reduced birth rate. However, postnatal growth was severely retarded with macrocytic anemia, B lymphocytopenia, lactic acidosis and bloated stomach, and died within 4 weeks. Ant2 depletion caused anemia in a cell-autonomous manner by maturation arrest of erythroid precursors with increased reactive oxygen species and premature deaths. B-lymphocyte development was similarly affected by Ant2 depletion, and splenocytes showed a reduction in maximal respiration capacity and cellular ATP levels as well as an increase in cell death accompanying mitochondrial permeability transition pore opening. In contrast, myeloid, megakaryocyte and T-lymphocyte lineages remained apparently intact. Erythroid and B-cell development may be particularly vulnerable to Ant2 depletion-mediated mitochondrial dysfunction and oxidative stress.

Essential role for TMEM100 in vascular integrity but limited contributions to the pathogenesis of hereditary haemorrhagic telangiectasia

AIMS TMEM100 was previously identified as a downstream target of activin receptor-like kinase 1 (ALK1; ACVRL1) signalling. Mutations on ALK1 cause hereditary haemorrhagic telangiectasia (HHT), a vascular disorder characterized by mucocutaneous telangiectases and visceral arteriovenous malformations (AVMs). The aims of this study are to investigate the in vivo role of TMEM100 at various developmental and adult stages and to determine the extent to which TMEM100 contributed to the development of AVMs as a key downstream effector of ALK1. METHODS AND RESULTS Blood vasculature in Tmem100-null embryos and inducible Tmem100-null neonatal and adult mice was examined. We found that TMEM100 deficiency resulted in cardiovascular defects at embryonic stage; dilated vessels, hyperbranching, and increased number of filopodia in the retinal vasculature at neonatal stage; and various vascular abnormalities, including internal haemorrhage, arteriovenous shunts, and weakening of vasculature with abnormal elastin layers at adult stage. However, arteriovenous shunts in adult mutant mice appeared to be underdeveloped without typical tortuosity of vessels associated with AVMs. We uncovered that the expression of genes encoding cell adhesion and extracellular matrix proteins was significantly affected in lungs of adult mutant mice. Especially Mfap4, which is associated with elastin fibre formation, was mostly down-regulated. CONCLUSION These results demonstrate that TMEM100 has essential functions for the maintenance of vascular integrity as well as the formation of blood vessels. Our results also indicate that down-regulation of Tmem100 is not the central mechanism of HHT pathogenesis, but it may contribute to the development of vascular pathology of HHT by weakening vascular integrity.