The arterial pole of the heart is the region where the ventricular myocardium continues as the vascular smooth muscle tunics of the aorta and pulmonary trunk. It has been shown that the arterial pole myocardium derives from the secondary heart field and the smooth muscle tunic of the aorta and pulmonary trunk derives from neural crest. However, this neural crest-derived smooth muscle does not extend to the arterial pole myocardium leaving a region at the base of the aorta and pulmonary trunk that is invested by vascular smooth muscle of unknown origin. Using tissue marking and vascular smooth muscle markers, we show that the secondary heart field, in addition to providing myocardium to the cardiac outflow tract, also generates prospective smooth muscle that forms the proximal walls of the aorta and pulmonary trunk. As a result, there are two seams in the arterial pole: first, the myocardial junction with secondary heart field-derived smooth muscle; second, the secondary heart field-derived smooth muscle with the neural crest-derived smooth muscle. Both of these seams are points where aortic dissection frequently occurs in Marfan's and other syndromes.

Nonalcoholic steatohepatitis (NASH) is a leading cause of cirrhosis. Recently, we showed that NASH-related cirrhosis is associated with Hedgehog (Hh) pathway activation. The gene encoding osteopontin (OPN), a profibrogenic extracellular matrix protein and cytokine, is a direct transcriptional target of the Hh pathway. Thus, we hypothesize that Hh signaling induces OPN to promote liver fibrosis in NASH. Hepatic OPN expression and liver fibrosis were analyzed in wild-type (WT) mice, Patched-deficient (Ptc 1/2 ) (overly active Hh signaling) mice, and OPNdeficient mice before and after feeding methionine and choline-deficient (MCD) diets to induce NASH-related fibrosis. Hepatic OPN was also quantified in human NASH and nondiseased livers. Hh signaling was manipulated in cultured liver cells to assess direct effects on OPN expression, and hepatic stellate cells (HSCs) were cultured in medium with different OPN activities to determine effects on HSC phenotype. When fed MCD diets, Ptc 1/2 mice expressed more OPN and developed worse liver fibrosis (P < 0.05) than WT mice, whereas OPN-deficient mice exhibited reduced fibrosis (P < 0.05). In NASH patients, OPN was significantly up-regulated and correlated with Hh pathway activity and fibrosis stage. During NASH, ductular cells strongly expressed OPN. In cultured HSCs, SAG (an Hh agonist) up-regulated, whereas cyclopamine (an Hh antagonist) repressed OPN expression (P < 0.005). Cholangiocyte-derived OPN and recombinant OPN promoted fibrogenic responses in HSCs (P < 0.05); neutralizing OPN with RNA aptamers attenuated this (P < 0.05). Conclusion: OPN is Hh-regulated and directly promotes profibrogenic responses. OPN induction correlates with Hh pathway activity and fibrosis stage. Therefore, OPN inhibition may be beneficial in NASH (HEPATOLOGY 2011;53:106-115) N onalcoholic steatohepatitis (NASH) is a potentially serious form of chronic liver injury because it increases the risk of developing cirrhosis and primary liver cancer. The mechanisms that lead to these outcomes have not been fully elucidated, but they appear to involve responses triggered

Hedgehog signaling through its receptor, Patched, activates transcription of genes, including Patched, that regulate the fate of various progenitors. Although Hedgehog signaling is required for endodermal commitment and hepatogenesis, the possibility that it regulates liver turnover in adults had not been considered because mature liver epithelial cells lack Hedgehog signaling. Herein, we show that this pathway is essential throughout life for maintaining hepatic progenitors. Patched-expressing cells have been identified among endodermally lineage-restricted, murine embryonic stem cells as well as in livers of fetal and adult Ptc-lacZ mice. An adult-derived, murine hepatic progenitor cell line expresses Patched, and Hedgehog-responsive cells exist in stem cell compartments of fetal and adult human livers. In both species, manipulation of Hedgehog activity influences hepatic progenitor cell survival. Therefore, Hedgehog signaling is conserved in hepatic progenitors from fetal development through adulthood and may be a new therapeutic target in patients with liver damage.

The Hedgehog (Hh) signaling pathway mediates several processes that are deregulated in patients with the metabolic syndrome (e.g., fat mass regulation, vascular/endothelial remodeling, liver injury and repair, and carcinogenesis). The severity of nonalcoholic fatty liver disease (NAFLD) and the metabolic syndrome generally correlate. Therefore, we hypothesized that the level of Hh pathway activation would increase in parallel with the severity of liver damage in NAFLD. To assess potential correlations between known histologic and clinical predictors of advanced liver disease and Hh pathway activation, immunohistochemistry was performed on liver biopsies from a large well-characterized cohort of NAFLD patients (n=90) enrolled in the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) Database 1 study. Increased Hh activity (evidenced by accumulation of Hh-ligand producing cells and Hh-responsive target cells) strongly correlated with portal inflammation, ballooning, and fibrosis stage (each p<0.0001), supporting a relationship between Hh pathway activation and liver damage. Pathway activity also correlated significantly with markers of liver repair, including numbers of hepatic progenitors and myofibroblastic cells (both p<0.03). In addition, various clinical parameters that have been linked to histologically-advanced NAFLD, including increased patient age (p<0.005), BMI (p<0.002), waist circumference (p<0.0007), homeostatic model assessment of insulin resistance (HOMA-IR) (p<0.0001) and hypertension (p<0.02), correlated with hepatic Hh activity. Conclusion: In NAFLD patients, the level of hepatic Hh pathway activity is highly correlated with the severity of liver damage and with metabolic syndrome parameters that are known to be predictive of advanced liver disease. Hence, deregulation of the Hh signaling network may contribute to the pathogenesis and sequelae of liver damage that develops with the metabolic syndrome.

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In cardiac neural-crest-ablated embryos, the secondary heart field fails to add myocardial cells to the outflow tract and elongation of the tube is deficient. Since that study, we have shown that the secondary heart field provides both myocardium and smooth muscle to the arterial pole. The present study was undertaken to determine whether addition of both cell types is disrupted after neural crest ablation. Marking experiments confirm that the myocardial component fails to be added to the outflow tract after neural crest ablation. The cells destined to go into the outflow myocardium fail to migrate and are left at the junction of the outflow myocardium with the nascent smooth muscle at the base of the arterial pole. In contrast, the vascular smooth muscle component is added to the arterial pole normally after neural crest ablation. When the myocardium is not added to the outflow tract, the point where the outflow joins the pharynx does not move caudally as it normally should, the aortic sac is smaller and fails to elongate resulting in abnormal connections of the outflow tract with the caudal aortic arch arteries.

Hepatic stellate cells (HSC) have a complex phenotype that includes both neural and myofibroblastic features. The Hedgehog (Hh) pathway has been shown to direct the fate of neural and myofibroblastic cells during embryogenesis and during tissue remodeling in adults. Therefore, we hypothesized that Hh signaling may regulate the fate of HSC in adults. In this study, we find that freshly isolated stellate cells from adult PatchedlacZ transgenic mice exhibit b-galactosidase activity, indicating Hh pathway activity. Transcripts of Hh ligands, the Hh pathway receptor, and Hh-regulated transcription factors are expressed by stellate cells from mice, rats, and humans. Transfection experiments in a cell line using a Hh-inducible luciferase reporter demonstrate constitutive Hh pathway activity. Moreover, neutralizing antibodies to Hh increase apoptosis, while viability is restored by treatment with Hh ligand. In vitro treatment of primary stellate cells with cyclopamine (Cyc), a pharmacologic inhibitor of the Hh pathway, inhibits activation and slightly decreases cell survival, while a single injection of Cyc into healthy adult mice reduces activation of HSC by more than 50% without producing obvious liver damage. Our findings reveal a novel mechanism, namely the Hh pathway, that regulates the activation and viability of HSC.

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