Loss of Wnt Secretion by Macrophages Promotes Hepatobiliary Injury after Administration of 3,5-Diethoxycarbonyl-1, 4-Dihydrocollidine Diet.

Exposure of mice to a diet containing 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) induces porphyrin accumulation, cholestasis, immune response, and hepatobiliary damage mimicking hepatic porphyria and sclerosing cholangitis. Although ß-catenin signaling promotes hepatocyte proliferation, and macrophages are a source of Wnts, the role of macrophage-derived Wnts in modulating hepatobiliary injury/repair remains unresolved. We investigated the effect of macrophage-specific deletion of Wntless, a cargo protein critical for cellular Wnt secretion, by feeding macrophage-Wntless-knockout (Mac-KO) and wild-type littermates a DDC diet for 14 days. DDC exposure induced Wnt11 up-regulation in macrophages. Mac-KO mice on DDC showed increased serum alkaline phosphatase, aspartate aminotransferase, direct bilirubin, and histologic evidence of more cell death, inflammation, and ductular reaction. There was impaired hepatocyte proliferation evidenced by Ki-67 immunostaining, which was associated with decreased hepatocyte ß-catenin activation and cyclin-D1 in Mac-KO. Mac-KO also showed increased CD45, F4/80, and neutrophil infiltration after DDC diet, along with increased expression of several proinflammatory cytokines and chemokines. Gene expression analyses of bone marrow-derived macrophages from Mac-KO mice and F4/80+ macrophages isolated from DDC-fed Mac-KO livers showed proinflammatory M1 polarization. In conclusion, this study shows that a lack of macrophage Wnt secretion leads to more DDC-induced hepatic injury due to impaired hepatocyte proliferation and increased M1 macrophages, which promotes immune-mediated cell injury.

Elimination of Wnt Secretion From Stellate Cells Is Dispensable for Zonation and Development of Liver Fibrosis Following Hepatobiliary Injury.

Alterations in the Wnt signaling pathway including those impacting hepatic stellate cells (HSCs) have been implicated in liver fibrosis. In the current study, we first examined the expression of Wnt genes in human HSC (HHSCs) after treatment with a profibrogenic factor TGF-ß1. Next, we generated HSC-specific Wntless (Wls) knockout (KO) using the Lrat-cre and Wls-floxed mice. KO and littermate controls (CON) were characterized for any basal phenotype and subjected to two liver fibrosis protocols. In vitro, TGF-ß1 induced expression of Wnt2, 5a and 9a while decreasing Wnt2b, 3a, 4, and 11 in HHSC. In vivo, KO and CON mice were born at normal Mendelian ratio and lacked any overt phenotype. Loss of Wnt secretion from HSCs had no effect on liver weight and did not impact ß-catenin activation in the pericentral hepatocytes. After 7 days of bile duct ligation (BDL), KO and CON showed comparable levels of serum alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, total and direct bilirubin. Comparable histology, Sirius red staining, and immunohistochemistry for α-SMA, desmin, Ki-67, F4/80, and CD45 indicated similar proliferation, inflammation, and portal fibrosis in both groups. Biweekly administration of carbon tetrachloride for 4 or 8 weeks also led to comparable serum biochemistry, inflammation, and fibrosis in KO and CON. Specific Wnt genes were altered in HHSCs in response to TGF-ß1; however, eliminating Wnt secretion from HSC did not impact basal ß-catenin activation in normal liver nor did it alter the injury-repair response during development of liver fibrosis.

In vitro and in vivo SAR of pyrido[3,4-d]pyramid-4-ylamine based mGluR1 antagonists.

The SAR of a series of novel pyrido[3,4-d]pyramid-4-ylamine mGluR1 antagonists is described. The multiple of the unbound K(i) in cerebrospinal fluid necessary to give morphine like analgesic effects in an electromyograph pinch model in rodents is determined and the effect of structure on CNS penetration examined.

Identification of the alpha2-delta-1 subunit of voltage-dependent calcium channels as a molecular target for pain mediating the analgesic actions of pregabalin.

Neuropathic pain is a debilitating condition affecting millions of people around the world and is defined as pain that follows a lesion or dysfunction of the nervous system. This type of pain is difficult to treat, but the novel compounds pregabalin (Lyrica) and gabapentin (Neurontin) have proven clinical efficacy. Unlike traditional analgesics such as nonsteroidal antiinflammatory drugs or narcotics, these agents have no frank antiinflammatory actions and no effect on physiological pain. Although extensive preclinical studies have led to a number of suggestions, until recently their mechanism of action has not been clearly defined. Here, we describe studies on the analgesic effects of pregabalin in a mutant mouse containing a single-point mutation within the gene encoding a specific auxiliary subunit protein (alpha2-delta-1) of voltage-dependent calcium channels. The mice demonstrate normal pain phenotypes and typical responses to other analgesic drugs. We show that the mutation leads to a significant reduction in the binding affinity of pregabalin in the brain and spinal cord and the loss of its analgesic efficacy. These studies show conclusively that the analgesic actions of pregabalin are mediated through the alpha2-delta-1 subunit of voltage-gated calcium channels and establish this subunit as a therapeutic target for pain control.