Hepatic stellate cells' involvement in progenitor-mediated liver regeneration.

Earlier studies conducted by our laboratory have shown that suppression of transforming growth factor-beta (TGFbeta)-mediated upregulation of connective tissue growth factor (CTGF) by iloprost resulted in a greatly diminished oval cell response to 2-acetylaminofluorene/partial hepatectomy (2AAF/PH) in rats. We hypothesized that this effect is due to decreased activation of hepatic stellate cells. To test this hypothesis, we maintained rats on a diet supplemented with 2% L-cysteine as a means of inhibiting stellate cell activation during the oval cell response to 2AAF/PH. In vitro experiments show that L-cysteine did, indeed, prevent the activation of stellate cells while exerting no direct effect on oval cells. Desmin immunostaining of liver sections from 2AAF/PH animals indicated that maintenance on the L-cysteine diet resulted in an 11.1-fold decrease in the number of activated stellate cells within the periportal zones. The total number of cells proliferating in the periportal zones of livers from animals treated with L-cysteine was drastically reduced. Further analyses showed a greater than fourfold decrease in the magnitude of the oval cell response in animals maintained on the L-cysteine diet as determined by immunostaining for both OV6 and alpha-fetoprotein (AFP). Global liver expression of AFP as measured by real-time PCR was shown to be decreased 4.7-fold in the L-cysteine-treated animals. These data indicate that the activation of hepatic stellate cells is required for an appropriate oval cell response to 2AAF/PH.

A detailed characterization of the adult mouse model of glycogen storage disease Ia.

Glycogen storage disease type Ia (GSDIa) is caused by a genetic defect in the hepatic enzyme glucose-6-phosphatase (G6Pase-alpha), which manifests as life-threatening hypoglycemia with related metabolic complications. A G6Pase-alpha knockout (KO) mouse model was generated to study potential therapies for correcting this disorder. Since then, gene therapy studies have produced promising results, showing long-term improvement in liver histology and glycogen metabolism. Under existing protocols, however, untreated KO pups seldom survived weaning. Here, we present a thorough characterization of the G6Pase-alpha KO mouse, as well as the husbandry protocol for rearing this strain to adulthood. These mice were raised with only palliative care, and characterized from birth through 6 months of age. Once KO mice have survived the very frail weaning period, their size, agility, serum lipids and glycemic control improve dramatically, reaching levels approaching their wild-type littermates. In addition, our data reveal that adult mice lacking G6Pase-alpha are able to mate and produce viable offspring. However, liver histology and glycogen accumulation do not improve with age. Overall, the reliable production of mature KO mice could provide a critical tool for advancing the GSDIa field, as the availability of a robust enzyme-deficient adult offers a new spectrum of treatment avenues that would not be tolerated by the frail pups. Most importantly, our detailed characterization of the adult KO mouse provides a crucial baseline for accurately gauging the efficacy of experimental therapies in this important model.

Connective tissue growth factor with a novel fibronectin binding site promotes cell adhesion and migration during rat oval cell activation.

UNLABELLED: Oval cell activation, as part of the regenerative process after liver injury, involves considerable cell-matrix interaction. The matricellular protein, connective tissue growth factor (CTGF), has been shown to be critical for oval cell activation during liver regeneration following N-2-acetylaminofluorene/partial hepatectomy. To understand the mode of action of CTGF during this process, N-terminal CTGF was used as bait to screen a yeast two-hybrid complementary DNA library specific for regenerating livers with massive oval cell presence. Fibronectin (FN), a prominent component of hepatic extracellular matrix (ECM), was found to specifically bind to a new site on CTGF. In addition to module IV, this study showed that module I of CTGF was sufficient for binding to FN in both solid-phase in vitro binding assays and immunoprecipitation. Immunofluorescent staining revealed a dynamic ECM remodeling characterized by an FN-concentrated provisional matrix during oval cell-aided liver regeneration. Abundant CTGF protein was colocalized with FN in the provisional matrix. When expressed as recombinant proteins and immobilized on plastic surfaces, modules I and IV of CTGF were selectively adhesive to thymus cell antigen 1-positive (Thy1(+)) oval cells, stellate cells, and sinusoidal endothelial cells but not to hepatocytes. The adhesion of these two modules on Thy1(+) oval cells required heparan sulfate proteoglycan and integrin alpha(5)beta(1). Recombinant CTGF promoted an integrin alpha(5)beta(1)-dependent migration but not proliferation on Thy1(+) oval cells. CONCLUSION: Modules I and IV enabled the linkage of CTGF to FN and activated hepatic cells. Through these bindings, CTGF on the FN-concentrated provisional matrix promoted cell adhesion and migration, thereby facilitating oval cell activation.

Inhibition of Notch signaling affects hepatic oval cell response in rat model of 2AAF-PH.

BACKGROUND AND AIMS: Activation of the oval cell compartment occurs in the liver when hepatocytes are functionally compromised and/or unable to divide. Our goal was to investigate the systemic signals responsible for determining the efficiency of oval cell-mediated liver regeneration, focusing on the Notch signaling cascade. METHODS: The established oval cell induction protocol of 2-acetylaminofluorine (2-AAF) implantation followed by 70% surgical resection of the liver (partial hepatectomy, PH) was employed in a rat model. This oval cell induction model was further combined with injections of a γ-secretase inhibitor (GSI XX) to examine the effects of Notch inhibition on oval cell-aided regeneration of the liver. RESULTS: Notch signaling was found to be upregulated at the peak of oval cell induction during 2AAF-PH alone. Treatment with GSI XX led to interruption of the Notch signal, as shown by a decrease in expression of Hes1. While there was a robust oval cell response seen at day 11 post-PH, there was a measurable delay in differentiation when Notch was inhibited. This was confirmed morphologically as well as by immunohistochemistry for the oval cell markers, α-fetoprotein, OV-6, and CK19. The hepatocytes seen at day 22 demonstrated an enhanced hepatocellular mitoinhibition index (p21(Waf1)/Ki67), suggestive of dysregulated proliferation and cell cycle progression. Moreover, these hepatocytes exhibited decreased expression of hepatocyte functional markers, such as cytochrome P450 and glucose-6-phosphatase-α. CONCLUSIONS: Taken together, these results identify the Notch signaling pathway as a potent regulator of differentiation and proliferation in oval cells, which is necessary for functional for repair of the liver by oval cells.

[Cardiac amyloidosis. A case report]. / Amiloidoza cardiaca. Prezentare de caz.

The paper presents the morphoclinical picture in cardiac amyloidosis to a 50 years old man admitted at Iasi Cardiology Center with progressive chronic cardiac failure, the patient having recent history of restrictive cardiomyopathy. It was made a complete cardiovascular evaluation including the right cardiac catheterization for endomyocardial biopsy. The biopsy specimens were fixed in buffered 10 % formalin, followed by routine paraffin embedding, and were stained with haematoxylin-eosin, elastic Van Gieson and sulphated blue Alcian for amyloid evaluation. The amyloid deposits were evidentiated in the interstitium and into vascular walls of the biopsy, pointing the importance of the morphological exam for amyloidosis diagnosis.

[Systemic amyloidosis. Case report]. / Amiloidoza sistemica. Prezentare de caz.

The amyloid deposition in skeletal muscle is well known but a rare occurrence. The study reflects the morphoclinical picture in cardiac amyloidosis in a 51 years old woman, having progressive cardiac failure and sinus node disease. A complete clinical evaluation of the patient showed a concomitant malignancy, plasma-cell myeloma. Muscle-cutaneous biopsy and a sulfated blue alcian staining was routinely performed to screen for amyloid. Histologically, amyloid was confirmed by the presence of deposits in the interstitium around perivascular region, or rarely, in the endomysial region. Focally, the muscles showed a small group atrophy and scattered regenerating muscle fibers and some degenerating myofibers. Generally, is known that prevalence rate of amyloid myopathy in muscle biopsy specimens is low (0.004%), and, as in our case, only a minority of patients have multiple myeloma, as well.

[Characteristics of anastomoses between the celiac trunk and the superior mesenteric artery]. / Particularitatile anastomozelor dintre trunchiul celiac si artera mezenterica superioara la nivelul complexului duodeno-pancreatic.

Duodenum and pancreas are two deep abdominal viscerae with multiple arterial sources and complex vascular relations, that impose peculiar surgical techniques. The dissection of 120 corpses and the examination of selective angiographies revealed the variants of the anastomoses between the superior pancreaticoduodenal arteries (that receive blood from the celiac trunk) and the inferior pancreaticoduodenal arteries (belonging to the collateral subsystem of the superior mesenteric artery). The pancreaticoduodenal arches are the main anastomosis around the pancreatic head, and supply the duodeno-pancreatic complex. During this study I found the absence of this anastomosis of the antero-inferior duodenopancreatic arch 5 cases (4.16%), and the lack of anastomosis of the postero-superior duodenopancreatic arch in 11 cases (9.16%). In these situations, the superior and the inferior duodenopancreatic arteries give off the vasa recta and the pancreatic branches, as normally. I found different variants of accessory duodenopancreatic arches, and I analyzed their impact on the surgical techniques on duodenum and pancreas. This study pinpoints the importance of the anastomotic subsystem in the arterial supply of the duodeno-pancreatic complex, reveals the necessity of the preoperative angiographic exam in order to choose the most opportune surgical technique.