Abrogation of ALK5 in hepatic stellate cells decreases hepatic fibrosis and ameliorates liver damage in mice following treatment with thioacetamide

While transforming growth factor-β (TGF-β) is known to be a key inducer of hepatic stellate cell (HSC) activation during liver fibrosis but it is unclear which TGF-β receptor is required for this HSC-mediated fibrogenesis. Here, we report that abrogation of TGF-β type I receptor ALK5 in HSC activation led to reduced collagen deposition and a decreased number of myofibroblasts in livers of mutant mice lacking ALK5 in HSC (Alk5/GFAP-Cre mice) following thioacetamide (TAA) exposure. The reduced fibrosis was accompanied by decreased expression of HSC activation markers in livers. In addition, Alk5/GFAP-Cre mice exhibited decreased immune cell infiltration and reduced production of inflammatory cytokines. Associated with reduced fibrosis and inflammation, amelioration of liver injury was observed in Alk5/GFAP-Cre mice after TAA treatment. In conclusion, our results indicated that TGF-β signaling via ALK5 in HSC enhanced liver fibrogenesis and inflammation led to amplification of hepatic injury in mice exposed to TAA

Atrioventricular cushion transformation is mediated by ALK2 in the developing mouse heart

AbstractDevelopmental abnormalities in endocardial cushions frequently contribute to congenital heart malformations including septal and valvular defects. While compelling evidence has been presented to demonstrate that members of the TGF-β superfamily are capable of inducing endothelial-to-mesenchymal transdifferentiation in the atrioventricular canal, and thus play a key role in formation of endocardial cushions, the detailed signaling mechanisms of this important developmental process, especially in vivo, are still poorly known. Several type I receptors (ALKs) for members of the TGF-β superfamily are expressed in the myocardium and endocardium of the developing heart, including the atrioventricular canal. However, analysis of their functional role during mammalian development has been significantly complicated by the fact that deletion of the type I receptors in mouse embryos often leads to early embryonal lethality. Here, we used the Cre/loxP system for endothelial-specific deletion of the type I receptor Alk2 in mouse embryos. The endothelial-specific Alk2 mutant mice display defects in atrioventricular septa and valves, which result from a failure of endocardial cells to appropriately transdifferentiate into the mesenchyme in the AV canal. Endocardial cells deficient in Alk2 demonstrate decreased expression of Msx1 and Snail, and reduced phosphorylation of BMP and TGF-β Smads. Moreover, we show that endocardial cells lacking Alk2 fail to delaminate from AV canal explants. Collectively, these results indicate that the BMP type I receptor ALK2 in endothelial cells plays a critical non-redundant role in early phases of endocardial cushion formation during cardiac morphogenesis

Deficient Signaling via Alk2 (Acvr1) Leads to Bicuspid Aortic Valve Development

Bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly in humans. Despite recent advances, the molecular basis of BAV development is poorly understood. Previously it has been shown that mutations in the Notch1 gene lead to BAV and valve calcification both in human and mice, and mice deficient in Gata5 or its downstream target Nos3 have been shown to display BAVs. Here we show that tissue-specific deletion of the gene encoding Activin Receptor Type I (Alk2 or Acvr1) in the cushion mesenchyme results in formation of aortic valve defects including BAV. These defects are largely due to a failure of normal development of the embryonic aortic valve leaflet precursor cushions in the outflow tract resulting in either a fused right- and non-coronary leaflet, or the presence of only a very small, rudimentary non-coronary leaflet. The surviving adult mutant mice display aortic stenosis with high frequency and occasional aortic valve insufficiency. The thickened aortic valve leaflets in such animals do not show changes in Bmp signaling activity, while Map kinase pathways are activated. Although dysfunction correlated with some pro-osteogenic differences in gene expression, neither calcification nor inflammation were detected in aortic valves of Alk2 mutants with stenosis. We conclude that signaling via Alk2 is required for appropriate aortic valve development in utero, and that defects in this process lead to indirect secondary complications later in life

Hepatocyte-specific expression of constitutively active Alk5 exacerbates thioacetamide-induced liver injury in mice

While Transforming growth factor-βs (Tgf-βs) have been known to play an important role in liver fibrosis through an activation of Hepatic Stellate Cells (HSC), their fibrotic role on hepatocytes in liver damage has not been addressed thoroughly. To shed more light on the hepatocyte-specific role of Tgf-β signaling during liver fibrosis, we generated transgenic mice expressing constitutively active Tgf-β type I receptor Alk5 under the control of albumin promoter. Uninjured mice with increased Tgf-β/Alk5 signaling in hepatocytes (caAlk5/Alb-Cre mice) did not show characteristics related to hepatocyte death, fibrosis and inflammation. When subjected to thioacetamide (TAA) treatment, caAlk5/Alb-Cre mice exhibited more severe liver injury, when compared to control littermates. After TAA administration for 12 weeks, an increase in pathological changes was evident in caAlk5/Alb-Cre livers, with higher number of infiltrating cells in the portal and periportal area. Immunohistochemistry for F4/80, myeloperoxidase and CD3 showed that there was an increased accumulation of macrophages, neutrophils and T-lymphocytes, respectively, in caAlk5/Alb-Cre livers. Coincidently, we observed an exacerbated liver damage as seen by increases in serum aminotransferase level and number of apoptotic hepatocytes in caAlk5/Alb-Cre mice. Sirius staining of collagen demonstrated that the fibrotic response was worsened in caAlk5/Alb-Cre mice. The enhanced fibrosis in mutant livers was associated with marked production of α-SMA-positive myofibroblast. Hepatic expression of genes indicative of HSC activation was greater in caAlk5/Alb-Cre mice. In conclusion, our data indicated that elevation of Tgf-β signaling via Alk5 in hepatocytes is not sufficient to induce liver pathology but plays an important role in amplifying TAA-induced liver damage

Signaling via the Tgf-beta type I receptor Alk5 in heart development

Trophic factors secreted both from the endocardium and epicardium regulate appropriate growth of the myocardium during cardiac development. Epicardially-derived cells play also a key role in development of the coronary vasculature. This process involves transformation of epithelia] (epicardial) cells to mesenchymal cells (EMT). Similarly, a subset of endocardial cells undergoes EMT to form the mesenchyme of endocardial cushions, which function as primordia for developing valves and septa. While it has been suggested that transforming growth factor-beta s (Tgf-beta) play an important role in induction of EMT in the avian epi- and endocardium, the function of Tgf-beta s in corresponding mammalian tissues is still poorly understood. In this study, we have ablated the Tgf-beta type I receptor Alk5 in endo-, myo- and epicardial lineages using the Tie2-Cre, Nkx2.5-Cre, and Gata5-Cre driver lines, respectively. We show that while Alk5-mediated signaling does not play a major role in the myocardium during mouse cardiac development, it is critically important in the endocardium for induction of EMT both in vitro and fit vivo. Moreover, loss of epicardial Alk5-mediated signaling leads to disruption of cell-cell interactions between the epicardium and myocardium resulting in a thinned myocardium. Furthermore, epicardial cells lacking Alk5 fail to undergo Tgf-beta-inducecl EMT in vitro, Late term mutant embryos lacking epicardial Alk5 display defective formation of a smooth Muscle cell layer around coronary arteries, and aberrant formation of capillary vessels in the myocardium suggesting that AIk5 is controlling vascular homeostasis during cardiogenesis. To conclude, Tgf-beta signaling via AIk5 is not required in myocardial cells during mammalian cardiac development, but plays an irreplaceable cell-autonomous role regulating cellular communication, differentiation and proliferation in endocardial and epicardial cells. (c) 2008 Elsevier Inc, All rights reserved

<i>Sox9</i> and <i>Tbx20</i> expression and less cleaved versican and tenascin-C in endocardial cushions and valve leaflets in controls and <i>Alk2/Gata5-Cre</i> mutants.

<p>In situ hybridization for <i>Sox9</i> (A–D) and <i>Tbx20</i> (E–J) on sagittal (A–H) and transverse (I, J) sections of E11 control and <i>Alk2/Gata5-Cre</i> mutant hearts. Prominent expression of both genes in atrioventricular (AVJ) and outflow tract (OFT) cushions in both control and mutant samples. Stronger staining detectable in certain regions of endocardium (<i>Tbx20</i>: red arrows in I, J) in equivalent areas of both control and mutant. Although staining for both <i>Sox9</i> and <i>Tbx20</i> in cushion mesenchyme also varied between adjacent cells (illustrated in J: short arrow lower vs double arrowhead higher), mutant but not control had groups of adjacent groups cells of lower (such as red asterisk in H) and higher (red ellipse) <i>Tbx20</i> signal. Immunostaining for total versican on sagittal E11 control (K) and <i>Alk2/Gata5-Cre</i> mutant (L) sections (green: versican; red: MF20). M–P, immunostaining for cleaved versican using anti-DPEAAE antibody on control (M,O) and <i>Alk2/Gata5-Cre</i> (N,P) sections. M–N, sagittal sections at E11; O–P, transverse sections at E14; green: cleaved versican; red: MF20. Staining in control AVJ mesenchyme (arrow in M,O) more extensive than mutant (arrowhead in N,P), though both similar in OFT. Immunostaining for tenascin-C on control (Q,S) and <i>Alk2/Gata5-Cre</i> mutant (R,T) sections; Q,R, sagittal sections at E11; S,T, transverse sections at E14. Green, tenascin-C (TNC); red, MF20. Staining in control mesenchyme (arrow) more extensive than mutant (arrowhead). AVJ, atrioventricular cushions; OFT, outflow tract cushions; IVS, ventricular septum; myo, OFT myocardium; RV, right ventricle.</p

<i>Gata5-Cre</i>-induced deletion of <i>Alk2</i> function leads to defective development of aortic valves.

<p><b>A–F</b>: Histological comparison of representative control (A, C, E) and mutant (B, D, F) aortic valves at E14; approximately transverse sections of the same valves at three levels. A, B: lower magnification at the level of coronary orifices (red arrow; coronary vessel: red arrowhead), where formed, distal to aortic valve leaflets showing normal development and relationship of aortic-side structures relative to pulmonary valve. All three control aortic leaflet cushions show evidence of excavation forming sinuses (green *): left (L) in panel C, right (R) and non-coronary (N) in panel E. N cushion occupies a similar segment of the circumference (black lines) as each of R and L (in E). In mutant valve, L (in D) and R (in F) leaflet cushions show normal excavation. Cushion ‘R’, although in the same position as N in control, is continuous with tissue R (panel D), not simply apposed as in control (in C). Nearer the base of the leaflet cushions (in E, F), mutant R is larger than control R. A small cushion (‘N’) is present between L and ‘R’ (in D, F), but shows no evidence of excavation and occupies a much smaller segment of the circumference (black lines in F) than N in control, so the valve is functionally bicuspid (bifoliate), with two excavating leaflet cushions, L and R/‘R’, apposing one another distally (in D). <b>G–R</b>: Histological comparison of representative control (A, J, M, P) and two functionally bicuspid mutant (H, K, N, Q and I, L, O, R) aortic valves at E17; approximately transverse sections of the same valves at four levels. G-I, at the level of coronary orifices showing their consistent position above right and left coronary leaflets, and consistent positioning of the leaflets in relation to adjacent tissues, in control and mutants. All three control aortic leaflet walls lie adjacent to well-developed sinuses (green *) left (L) in panel J, right (R) and non-coronary (N) in panel M. N leaflet occupies a similar segment of the circumference (black lines) as R and L (in J). Two examples of functionally bicuspid mutant valves are shown to illustrate key details of their morphology at this stage. The example in H, K, N, Q has features consistent with a fusion of R and N leaflets. Distally (K), only two leaflets, R and L, appose each other, with no small leaflet between them in the non-coronary position, each occupying about half the circumference (black lines). More proximally, the sinus of the R leaflet is divided into two (green *) by a raphe (black arrowhead) in N, but the leaflet does not then divide into two separate bases (arrowhead, Q). Distally the mutant valve shown in I, L, O, R also consists of two leaflets (in panel L; as R and ‘R’ are joined more proximally) that are not separated by a small cushion, but more proximally, one (‘N’) present. An area representing an interleaflet triangle (red curved line) can be identified between L and R leaflets in control (in M) and mutant (K, L) valves, and between L and N (control, in M) but the bases of the R and N leaflets even in control remain adjacent (in P, red X, black arrow) though not continuous (unlike Q). <b>S,T</b>: 6 month old adult control aortic valve with normal left (L) right (R) and non-coronary (N) leaflets (black arrows) and three sinuses (green*), and partially dissected functionally bicuspid aortic valve in <i>Alk2/Gata5-Cre</i> mutant, demonstrating two leaflets (black arrows) which appose only with each other across the entire lumen, and two sinuses (green *). Aor Tr, Aortic trunk; Pul Val, Pulmonary Valve.</p

Regional patterns of BAV-associated gene expression do not differ between control and <i>Alk2/Gata5-Cre</i> mutant outflow tracts.

<p>Sections transverse to distal (A–F), mid (G–L) and proximal (M–R) outflow tracts of control (A–C; G–I, M–O) and mutant (D–F; J–L; P–R) embryos showing expression patterns of Nos3 (Immunostaining: Nos3, pink; myocardium, blue-purple; nuclei, yellow; images color-inverted so that signal in single layer of endocardium is visible), (A, D, G, J, M, P); <i>Gata5</i> (ISH: B, E, H, K, N, Q) and <i>Notch1</i> (ISH: C, F, I, L, O, R). Staining for Nos3 and <i>Notch1</i> present in virtually all endocardial cells in the OFT; <i>Gata5</i> (e.g., arrow heads) detected above background in endocardium lateral to rather than over the two main cushions (septal, parietal: *) where <i>Notch1</i> and Nos3 also more strongly expressed (arrows). Non-coronary leaflet develops from the intercalated cushion on the left in these images, right coronary from the adjacent part of the parietal cushion (* in the more anterior position). <i>Gata5</i> also detected in second heart field (SHF) cells/most distal OFT wall. OFT, myo: outflow tract myocardium; OFT end, OFT endocardium; AV myo, atrioventricular myocardium; AVJ, atrioventricular cushion; RV, right ventricle; LV, left ventricle.</p

Less proliferation in endocardial cushion mesenchyme in <i>Alk2/Gata5-Cre</i> mutants.

<p>Representative images of anti-BrdU immunostaining (green-yellow) of control (A, C, E, G) and <i>Alk2/Gata5-Cre</i> mutant (B, D, F, H) sagittal sections at E11 (A, B, E, F) and E12 (C, D, G, H). A–D, OFT cushions; E–H, AV cushions. Mutant OFT cushion shows patchy incidence of BrdU- labeled nuclei (e.g., less in myocardium-adjacent region ringed by white ellipse). Bar graphs (I, J) illustrate differences in proportion of BrdU-labeled nuclei in the OFT (I) and AVJ (J) mesenchyme at E11 (left columns) and E12 (right columns) (n = 3) OFT, Outflow tract cushions; AV, atrioventricular cushions; C, control; M, mutant; Error bars, SEM; *p<0.05.</p

The <i>Gata5-Cre</i> transgene induces recombination in the endocardial cushion mesenchyme after EMT.

<p>Comparison of areas of <i>Gata5-Cre (A), Tie2-Cre</i> (B) and <i>Wnt1-Cre</i> (C) recombination (blue) in OFT region (arrowhead) at E11.5 (wholemount <i>R26R</i>-driven βgal stain, viewed from the left side, distal OFT to the right). βgal staining of sagittal sections at E10 (D) and at E11 (E) shows extensive <i>Gata5-Cre</i>-induced recombination in OFT cushion mesenchyme by E11 (arrow), but not in endocardium (arrowhead). Comparison of <i>Gata5-Cre</i> (F, H and J) and <i>Tie2-Cre</i> (G, I and K) recombination patterns (<i>R26R</i>-driven β-gal stain in blue) at three levels of the OFT, from proximal (J, K) to distal (F, G). <i>Tie2-Cre</i> (arrowheads, G, I) but not <i>Gata5-Cre</i> (arrowheads F, H) drives recombination (blue) in endocardium; recombined mesenchymal cells (blue) in both lines (arrows, J, K) only in proximal OFT. Comparison of <i>Gata5-Cre</i> and (L, N and P) <i>Tie2-Cre</i> (M, O and Q) recombination at AV junction: sagittal sections (L–O) at E10 (L, M) and E11 (N, O) and transverse sections at E11 (P, Q). At E10 (L, M) endocardium (arrowhead) recombined (blue) by <i>Tie2-Cre</i> but not <i>Gata5-Cre</i>. At E11 (N–P) AV mesenchymal cells (arrows) recombined (blue) by both <i>Tie2-Cre</i> and <i>Gata5-Cre</i>. R, In situ hybridization for <i>Alk2</i> RNA showing expression (blue) in OFT cushion mesenchyme (black arrows) and endocardium (arrowhead) at E11.5. LV, left ventricle; RV, right ventricle; OFT myo, OFT myocardium; AV, AV cushion; AVJ, atrioventricular junction.</p