Freeze-dried platelets promote hepatocyte proliferation in mice

In recent years, platelets are reported to promote liver, as well as bone regeneration and dermal wound healing. Platelets are required not only for thrombocytopenia treating but also for regenerative therapy. Platelets cannot be stored beyond three days, therefore, shortage of fresh platelets sometimes occurs. To preserve platelets for a long duration without degrading growth factors, a freeze-dried technique is required. We report here that platelets can be preserved by freeze-drying, using a programmed freezing method to avoid intracellular ice crystal formation. Freeze-dried platelets kept their morphological countenance and response with the agonist of thrombin was well maintained. Freeze-dried platelets stored adenine nucleotides, PDGF, and IGF-1 the same as those of fresh platelets. Freeze dried platelets also preserved their proliferative effect on hepatocytes identical to that of fresh platelets. These results of our study suggest that freeze dried platelets will obviate the storage problem of fresh platelets.     

Exendin-4 improves hepatocyte injury by decreasing proliferation through blocking NGF/TrkA in diabetic mice

The hepatocytes express nerve growth factor (NGF) and its high affinity receptor tyrosine kinase A (TrkA). However, the link between NGF/TrkA system and hepatocyte proliferation in diabetic animals and the effects of exendin-4, a glucagon like peptide-1 (GLP-1) receptor agonist, on this system are not known. BALB/c male mice were divided into four groups. The first group was given citrate buffer only, the second group was administered exendin-4 alone, the third group received streptozotocin (STZ), and the fourth group was given both STZ and exendin-4. Exendin-4 (3 μg/kg) was administered by subcutaneous injection daily for 30 days after the animals were rendered diabetic by administration of STZ (200 mg/kg). With treatment of exendin-4 to the diabetic mice the following results were noted (i) NGF, TrkA and proliferating cell nuclear antigen positive hepatocytes were decreased; (ii) p75 neurotrophin receptor and caspase-3 positive hepatocyte could not be detected; (iii) liver alanine transaminase and aspartate transaminase activities, lipid peroxidation, protein carbonyl and myeloperoxidase levels were decreased; (iv) liver catalase, superoxide dismutase, glutathione peroxidase activities and glutathione levels were increased. These data suggest that exendin-4 might exerts its anti-proliferative action through blocking NGF/TrkA system and stimulating oxidative defense system in liver of diabetic mice.     

Biological properties of a hepatocyte growth factor from rat platelets

In an accompanying communication we demonstrated that about half of the potency of rat serum to stimulate DNA synthesis in cultured adult rat hepatocytes resides in a polypeptidelike substance from the platelets. A lysate of rat platelets was able to restore the potency of platelet-poor rat serum, whereas a lysate of human platelets inhibited thymidine incorporation by the hepatocytes. Moreover, addition to these cultures of either highly purified human platelet-derived growth factor (PDGF) or human platelet factor 4 (PF-4) failed to influence DNA synthesis either alone or in the presence of rat or human platelet-poor serum, which is required for expression of PDGF activity. Unlike the human platelet factors, rat platelet lysate (RPL) was moderately active by itself and was augmented equally well by platelet-poor serum from either source. At concentrations below 5%, platelet-poor serum from hypophysectomized rats was as potent as that from normal rats in augmenting RPL activity. This suggests that, unlike PDGF, which is not activated by hypophysectomized rat serum, the hepatotrophic component of RPL does not require the presence of exogenous somatomedins for activity, but interacts instead with other plasma constituents or with somatomedins produced by the hepatocytes in vitro. Rat platelets do, however, appear to contain PDGF or its rat equivalent in addition to the hepatocyte growth factor, since if they are heated to 100 degrees C for 10 min, their ability to stimulate nuclear labeling in confluent BALB/c 3T3 cells is not impaired, while their ability to stimulate DNA synthesis in rat hepatocytes is destroyed. These studies indicate that the hepatocyte growth factor from rat platelets differs from PDGF in its biological as well as physical characteristics, but that rat platelets also contain PDGF or an equivalent substance.     

Characteristic effects of rabbit liver extract on hepatocyte proliferation in the regenerating liver of mice of various ages

Chalone isolated by the Verly method was injected into animals of different age (2 groups) under partial hepatectomy. The percentage of hepatocytes in S-, G2-, M- and post-M-periods was determined by morphoautoradiography. These parameters were used for the reconstruction of the extent and dynamics of cell proliferation during 36-48 hours of regeneration. The suppressing effect of chalone on hepatocyte proliferation was more pronounced in young mice. The lack of complete inhibition of DNA synthesis and of the effect of proliferative synchronization was observed in both groups of mice.     

Partial characterization of a hepatocyte growth factor from rat platelets

Rat serum has been shown to stimulate DNA synthesis in primary cultures of adult rat hepatocytes 2-3 times more potently than serum from several other mammalian sources, including humans. Parallel to its stimulation of thymidine incorporation into DNA, rat serum increased the total DNA content of the hepatocyte cultures over time, and also increased the frequency of nuclear labeling and mitosis. Moreover, normal rat serum, derived from whole blood (NRS), stimulated DNA synthesis in hepatocytes twice as effectively as platelet-poor rat serum, derived from plasma (ppNRS). Addition of a rat platelet lysate (RPL) to ppNRS restored the activity to equal that of NRS. The avid binding of the active principle to CM Sephadex and its sensitivity to trypsin digestion suggest that it is a cationic polypeptide with an apparent molecular weight of about 65,000, as determined by gel filtration. It was inactivated by reduction of disulfide bonds, or by exposure to pH below 5.5, to NaCl concentration below 0.05 M, to 65 degrees C for 30 min, or to 100 degrees C for 10 min. Although it resembles the human platelet-derived mitogen platelet-derived growth factor (PDGF) in several of its properties, it differs in others. Hence the hepatocyte growth factor from rat platelets, which accounts for 50% of the DNA synthesis-stimulatory activity of rat serum, appears to be a distinct entity.     

Change in hepatocyte growth factor concentration promote mesenchymal stem cell-mediated osteogenic regeneration

Mesenchymal stem cells (MSCs) play a crucial role in tissue repair by secretion of tissue nutrient factors such as hepatocyte growth factor (HGF). However, studies examining the effects of HGF on the proliferation and differentiation of MSCs used different concentrations of HGF and reported conflicting conclusions. This study aimed to determine the mechanisms by which different concentrations of HGF regulate MSC proliferation and osteogenic differentiation, and validate the mechanism in an animal model of early stage avascular necrosis of femoral head (ANFH). Our results demonstrate that a low concentration of HGF (20 ng/ml) preferentially promotes MSC osteogenic differentiation through increased c-Met expression and phosphorylation, Akt pathway activation, and increased expression of p27, Runx2 and Osterix. In contrast, a high concentration of HGF (100 ng/ml) strongly induced proliferation by inducing strong activation of the ERK1/2 signalling pathway. As validated by animal experiments, high localized expression of HGF achieved by transplantation of HGF transgenic MSCs into ANFH rabbits increased the number of MSCs. Subsequently, 2 weeks after transplantation, HGF levels decreased and MSCs differentiated into osteoblasts and resulted in efficient tissue repair. Our results demonstrate that sequential concentration changes in HGF control the proliferation and osteogenic differentiation of MSCs in vivo. This phenomenon can be exploited therapeutically to induce bone regeneration and, in turn, improve the efficacy of pharmacological intervention for ANFH treatment.     

Purification and subunit structure of hepatocyte growth factor from rat platelets

A hepatocyte growth factor (HGF) that stimulates DNA synthesis of adult rat hepatocytes in primary culture was purified as a homogeneous material from platelets of 1000 rats by a four-step procedure: stimulation of its release from platelets by thrombin, cation-exchanger fast protein liquid chromatography (FPLC) on a Mono S column, heparin-Sepharose CL-6B chromatography, and reverse-phase HPLC on a C4 column. The purified HGF stimulated DNA synthesis of adult rat hepatocytes in primary culture at 1 ng/ml and was maximally effective at 5 ng/ml, being about twice as potent as EGF at this concentration. HGF did not stimulate DNA synthesis of Swiss 3T3 cells. It was found to be a heat- and acid-labile protein that was inactivated by reduction with dithiothreitol. The purified HGF had a molecular mass of 82 kDa, as estimated by SDS-PAGE, and was found to be a heterodimer which dissociated into a large subunit of 69 kDa and a small one of 34 kDa by SDS-PAGE under reducing conditions. These biological and chemical properties showed that HGF was not identical with any known growth factors, including platelet-derived growth factor (PDGF).     

Angiopoietin-like 3 regulates hepatocyte proliferation and lipid metabolism in zebrafish

Loss-of-function mutations in angiopoietin-like 3 (ANGPTL3) cause familial hypobetalipoproteinemia type 2 (FHBL2) in humans. ANGPTL3 belongs to the angiopoietin-like family, the vascular endothelial growth factor family that is structurally similar to angiopoietins and is known for a regulator of lipid and glucose metabolism, although it is unclear how mutations in ANGPTL3 lead to defect in liver development in the vertebrates. We report here that angptl3 is primarily expressed in the zebrafish developing liver and that morpholino (MO) knockdown of Angptl3 reduces the size of the developing liver, which is caused by suppression of cell proliferation, but not by enhancement of apoptosis. However, MO knockdown of Angptl3 did not alter angiogenesis in the developing liver. Additionally, disruption of zebrafish Angptl3 elicits the hypocholesterolemia phenotype that is characteristic of FHBL2 in humans. Together, our findings propose a novel role for Angptl3 in liver cell proliferation and maintenance during zebrafish embryogenesis. Finally, angptl3 morphants will serve as a good model for understanding the pathophysiology of FHBL2.     

窖蛋白-1在细胞增殖和肝再生中的作用

窖蛋白-1(caveolin-1,Cav-1)是组成胞膜窖(caveolae)的主要功能蛋白.作为质膜上的独立结构,胞膜窖参与多种细胞活动,如胆固醇运输、信号转导以及细胞膜的组装等.通常,窖蛋白-1可以通过其N端的窖蛋白脚手架区(caveolin scaffolding domain,CSD)寡聚细胞外信号激酶(Erk1/2)的上游蛋白,抑制Erk1/2的活化,从而抑制细胞增殖和肿瘤转移.新近研究表明,窖蛋白-1通过促进甘油三酯的储存和利用而对肝再生起重要的调控作用.因此,窖蛋白-1可能是调控肝实质细胞增殖的关键蛋白.     

Selective proliferation of rat hepatocyte progenitor cells in serum-free culture

This protocol details a method of obtaining selectively proliferated hepatocyte progenitor cells using hyaluronic acid (HA)-coated dishes and serum-free medium. A small hepatocyte (SH) is a hepatocyte progenitor cell of adult livers and has many hepatic functions. When the rat SH begins to proliferate, CD44 is specifically expressed. To define the purification of SH, CD44 and cytokeratin 8 are used as marker proteins. The growth of SHs is faster on HA-coated dishes than on other extracellular matrix-coated ones. The use of both DMEM/F12 medium and HA-coated dishes allows the selective proliferation of SHs in culture. The purification of SHs is approximately 85% at day 10.     

Low venular shear rates promote leukocyte-dependent recruitment of adherent platelets

The influence of reductions in venular shear rate on platelet-endothelial (P/E) cell adhesion has not been previously addressed. The objectives of this study were to define the effects of reductions in venular shear rate on P/E cell adhesion and to determine the interdependence of P/E cell adhesion and leukocyte-endothelial (L/E) cell adhesion at low shear rates. Intravital videomicroscopy was used to quantify P/E and L/E cell adhesion in rat mesenteric venules exposed to shear rates ranging between 118 +/- 9 and 835 +/- 44 s(-1). Shear rate was altered in postcapillary venules by rapid, graded blood withdrawal, without retransfusion of shed blood. Reducing shear rate from >600 s(-1) to <200 s(-1) resulted in an eightfold increase in L/E cell adhesion, whereas P/E cell adhesion increased 18-fold. A blocking antibody directed against P-selectin blunted both the P/E and L/E cell adhesion elicited by low shear rates. Immunoneutralization of CD11/CD18 on leukocytes or rendering animals neutropenic also blocked the shear rate-dependent recruitment of both platelets and leukocytes. These findings indicate that 1) low shear rates promote P/E and L/E cell adhesion in mesenteric venules, and 2) adherent neutrophils (mediated by CD11/CD18) create a platform onto which platelets can bind to the venular wall at low shear rates.     

Genetic abolishment of hepatocyte proliferation activates hepatic stem cells

Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.     

Does chemically induced hepatocyte proliferation predict liver carcinogenesis?

Cell proliferation has long been recognized as having an important role in chemically induced carcinogenesis. Based on findings that certain nongenotoxic chemical carcinogens induced cell proliferation in the same organ that had an increased incidence of tumors, it has been hypothesized that a chemically induced response of enhanced DNA synthesis and cellular division causes cancer by increasing the rate of spontaneous mutations. It was further suggested that there would be no increased human risk of cancer by non-DNA-reactive compounds at doses that do not cause a proliferative response. An evaluation of the literature on the relationship between chemically induced cell proliferation and liver carcinogenesis reveals that very few systematic cell proliferation studies have been conducted over periods of extended exposure, and in many cases the exposure concentrations were not similar to those used in the cancer studies. The proliferative response resulting from exposure to many nongenotoxic carcinogens is not well sustained, whereas the carcinogenic response by these chemicals often requires prolonged exposure. The available literature leads to the conclusion that quantitative correspondences between cellular proliferation and carcinogenic responses have not been demonstrated and do not support the hypothesis that chemically induced cell proliferation is the primary mechanism by which nongenotoxic chemicals cause liver cancer. Studies of liver carcinogenesis in two-stage models point out the need to better understand chemical effects on cell loss as well as on cell replication, and demonstrate that measurements of cell proliferation alone are not sufficient to elucidate mechanisms of tumor development.     

Urothelial proliferation in growing mice

Developing murine urothelium undergoes pronounced proliferation until at least 10 days after birth. Thereafter, both mitotic and [3H]TdR-labelling indices fall sharply with age. The ratio of labelling to mitotic indices also alters dramatically during development, which is probably due to both endoreduplication and changes in the relative durations of the DNA synthesis and mitotic phases. This ratio reaches stability at 5 weeks of age. The adult labelling and mitotic indices were 0.11 and 0.019% respectively, indicating a very slow turnover.