A metabolomic approach shows sphingosine 1-phosphate and lysophospholipids as mediators of the therapeutic effect of liver growth factor in emphysema.

Tobacco smoke exposure is the principal cause of lung tissue destruction, which in turn results in emphysema that leads into shortness of breath. Liver growth factor (LGF, a cell and tissue regenerating factor with therapeutic activity in several organs) has antifibrotic and antioxidant properties that could be useful to promote lung tissue regenerating capacity in damaged lungs. The current study has examined differences in metabolite profiles (fingerprints) of plasma from mice (strain C57BL/6J, susceptible to develop emphysema) exposed to tobacco smoke during six months. One group of mice received a treatment with Liver Growth Factor (LGF) after emphysema was established, whereas the other group did not receive the treatment. Age and sex-matched mice not exposed to smoke were also maintained with or without treatment as controls. Metabolic fingerprints (untargeted analysis) of plasma after protein precipitation were obtained by LC-QTOF-MS. The signals were processed and a large number of possible metabolites were found (23944). Multivariate data analysis provided models that highlighted the differences between control and smoke exposed mice in both conditions. Accurate masses of features (possible compounds) representing significant differences were searched using online public databases. Lipid mediators, related to intracellular signaling in inflammation, were found among the metabolites putatively identified as markers of the different conditions and among them, sphingosine, sphingosine 1-phosphate and lysophospholipids point at the relevance of such metabolites in the regulation of the processes related to tissue regeneration mediated by LGF. These results also suggest that metabolomic fingerprinting could potentially guide the characterization of relevant metabolites leading the regeneration of lungs in emphysema disease.

Liver growth factor induces testicular regeneration in EDS-treated rats and increases protein levels of class B scavenger receptors.

The aim of the present work was to determine the effects of liver growth factor (LGF) on the regeneration process of rat testes after chemical castration induced by ethane dimethanesulfonate (EDS) by analyzing some of the most relevant proteins involved in cholesterol metabolism, such as hormone sensitive lipase (HSL), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), scavenger receptor SR-BI, and other components of the SR family that could contribute to the recovery of steroidogenesis and spermatogenesis in the testis. Sixty male rats were randomized to nontreated (controls) and LGF-treated, EDS-treated, and EDS + LGF-treated groups. Testes were obtained on days 10 (T1), 21 (T2), and 35 (T3) after EDS treatment, embedded in paraffin, and analyzed by immunohistochemistry and Western blot. LGF improved the recovery of the seminiferous epithelia, the appearance of the mature pattern of Leydig cell interstitial distribution, and the expression of mature SR-BI. Moreover, LGF treatment resulted in partial recovery of HSL expression in Leydig cells and spermatogonia. No changes in serum testosterone were observed in control or LGF-treated rats, but in EDS-castrated animals LGF treatment induced a progressive increase in serum testosterone levels and 3ß-HSD expression. Based on the pivotal role of SR-BI in the uptake of cholesteryl esters from HDL, it is suggested that the observed effects of LGF would facilitate the provision of cholesterol for sperm cell growth and Leydig cell recovery.

Improvement in liver fibrosis, functionality and hemodynamics in CCI4-cirrhotic rats after injection of the Liver Growth Factor.

BACKGROUND/AIMS: Most substances used in experimental models of cirrhosis are chosen either as protectors of lipid peroxidation, as antifibrogenic agents or as vitamins, among others. In this report, we analyze the improvement produced, in established cirrhosis (CCl4 plus phenobarbital) in rats, by intraperitoneal injection of Liver Growth Factor, a hepatic mitogen with activity both in vivo and in vitro. METHODS: Following confirmation of CCl4-induced cirrhosis, Liver Growth Factor (4.5 microg per ratx2 injections/week for 3 weeks) was administered to one group of rats (Cirr+LGF). The remaining rats (Cirr) received saline. The groups were compared in terms of serum enzymes, tissue damage, total liver collagen, collagenase activity, microsomal enzyme activities, splanchnic and systemic hemodynamics and portosystemic shunting. RESULTS: Treatment of rats presenting CCl4-induced cirrhosis with Liver Growth Factor decreased serum aminotransferase levels and increased levels of serum albumin and total protein. The Liver collagen content was lower in rats treated with Liver Growth Factor (2.96 vs. 4.32 mg/g liver, p<0.01). Microscopic studies revealed that the livers of rats receiving Liver Growth Factor showed decreases in fibrosis, necrosis and inflammatory infiltration, as well as a recovery of architectural integrity. Liver function was improved after treatment with Liver Growth Factor, as indicated by the rate constant for elimination of aminopyrine, which increased from 0.0063 to 0.0170 (p<0.05). This increase was accompanied by a higher total amount of cytochrome P-450 as well as of certain P-450 isoenzymes, especially those that are hormone-dependent, such as P-450 3A. The improved liver histology and function observed in Cirr+LGF rats was associated with decreases in portal pressure (14.4 vs. 9.4 mm Hg, p<0.01) and portosystemic shunting (55.8 vs. 11.5%, p<0.01), as well as increases in mean arterial pressure and systemic vascular resistance, and a reduction in ascites. CONCLUSIONS: Administration of the hepatic mitogen, Liver Growth Factor, to CCl4-cirrhotic rats decreased liver collagen and reorganized the hepatic extracellular matrix, resulting in an improvement in liver function, reduced portal pressure and amelioration of ascites.

Na+ :H+ exchange inhibition induces intracellular acidosis and differentially impairs cell growth and viability of human and rat hepatocarcinoma cells.

Amiloride and its more potent analog, hexamethylene amiloride (HMA), inhibits Na+ :H+ exchange and decreases intracellular pH in a concentration-dependent way in two human hepatocarcinoma cell lines and in a rat hepatocarcinoma cell line that differs in its phenotypic characteristics, resembling the clinical situation encountered in human hepatocarcinomas. After 24 h of exposure, DNA synthesis and cell protein content of the cultures decreases according to the concentration of the drugs and in parallel to Na+ exchange inhibition and the drop in pHi promoted. RNA and protein syntheses are less sensitive to its action. The above effects induced by HMA are accompanied by an abrupt decrease in cell viability and lysosomal integrity at 24 h. These effects develop gradually with the exposure time as does the increase in free radical production. Decreased viability is totally or partially restored by N-acetylcysteine or deferoxamine, but the degree of intracellular acidification produced is not. These results tend to suggest that intracellular acidification can diminish cell growth and provoke cytotoxic cell death by diminishing reduced glutathione (GSH) levels and impairing lysosomal integrity, reflecting the sensitivity of hepatocarcinoma cells to Na+ exchange inhibition and intracellular acidosis.

Hepatic growth induced by injection of the liver growth factor into normal rats.

Normal Wistar rats injected with the liver growth factor (LGF), a mitogen specific for liver cells, experienced hepatic growth. LGF shows two peaks of activity in vivo, both of them mitogenic. Rats injected either with 6.8 ng or 3.9 micrograms LGF/rat every 3-4 days experienced liver growth showing a see-saw profile. Dry liver weight usually peaked at day 2 (microgram doses) or at day 3 (ng doses) after each injection, with increases of about 30% over controls. Liver DNA synthesis, measured by [3H]-thymidine incorporation, peaked 24 h after LGF injection at both doses. Liver protein synthesis, measured by [14]C-leucine incorporation, usually peaked 24 h after DNA synthesis maximums. Mitogen-stimulated cells were also assessed by immunohistochemical staining for proliferating cell nuclear antigen in livers of LGF-injected rats. Rats injected with rat serum albumin purified from normal rats to serve as controls showed a 6% increase in dry liver weight, but when serum albumin from 3-day fasted rats was injected instead, the increase was not statistically significant. The mild effect of rat serum albumin could be due to the lipid content of the solutions injected, but the level of lipids/mg protein in LGF solutions was half that determined with serum albumin from 3-day fasted rats. From the microscopic and ultramicroscopic studies carried out in rat livers injected with LGF at each dose, we observed: (1) an increase in the number of hepatocytes undergoing mitosis; (2) transient increases in lipid and glycogen contents, as occur after liver resection; (3) no signs of degeneration, such as the appearance of amyloids or fibrosis; (4) no increase in lysosome number, as in hepatotoxicity; (5) no alterations in endothelial or Kupffer cells; and (6) no ultrastructural signs of degeneration either in cytoplasmic organelles (rough endoplasmic reticulum, mitochondria) or in nuclei. One year after LGF injection, rat liver, pancreas, kidneys and spleen were normal, with no signs of degeneration or onset of fibrosis.

Liver growth factor purified from human plasma is an albumin-bilirubin complex.

We have reported that a liver growth factor isolated from plasma of partially hepatectomized rats is an albumin-bilirubin complex. In this paper, we characterize the liver growth factor purified from subjects with hepatitis (h-LGF). This factor increases synthesis of DNA in a dose-dependent manner both in vivo in mouse hepatocytes, with a dose of maximal stimulation of 150 ng of h-LGF/mouse, and in vitro in rat liver cell culture, with maximal effect at 7.5 to 10 ng of h-LGF/ml. In vivo, h-LGF increases the mitotic index of mouse hepatocytes, its action being organ-specific, acting on liver, but not on spleen, kidney, lung or brain. In vitro, h-LGF stimulates the uptake of 22Na+ by hepatocytes. In addition, we carried out a study comparing it with human serum albumin in terms of absorbance, fluorescence, circular dichroism spectra, amino acid composition, tryptic maps and antigenic determinants (Ouchterlony immunodiffusion). All these tests suggested that human serum albumin is a constituent of h-LGF. Moreover, when albumin isolated from humans without hepatic pathology is incubated with bilirubin, the albumin-bilirubin complex formed mimics the activity of the human liver growth factor with respect to stimulation of DNA synthesis and the effects on the mitotic index of mouse hepatocytes in vivo. We propose that this human liver growth factor is an albumin-bilirubin complex.

Identification of biliprotein as a liver growth factor.

We determined the concentration of biliprotein in plasma of rats at different times after partial hepatectomy. From the same plasma samples, we purified a liver growth factor previously characterized by our group. When we plotted the 14 points studied, a linear relationship was obtained (r = 0.999; p less than 0.001). This result, in addition to our group's recent identification of this liver growth factor as an albumin-bilirubin complex, strongly suggests that biliprotein is a liver growth factor.

A liver DNA synthesis promoter induced in rat plasma by injection of dimethylnitrosamine (DMNA) or thioacetamide.

The appearance of a liver DNA synthesis promoter (HP) in rat plasma after dimethylnitrosamine (DMNA) or thioacetamide injection was investigated. After 48 h, DMNA (30 mg kg-1 body weight) produced liver (centrilobular) necrosis and intense hepatic regeneration, as assessed by microscopic observations of liver slices, as well as augmented transaminase levels; HP was detectable under these conditions. After 5 days, transaminases and HP returned to normal values (the latter undetectable), coinciding with a lack of necrotic zones. At 60 mg DMNA kg-1 body weight, necrotic areas were more marked and transaminases and HP levels higher after 48 h than with the lower dose; these increases were even more pronounced at 90 mg DMNA kg-1 body weight. After thioacetamide injection (200 mg kg-1 body wt) the situation at 48 h was very similar, with focal, centrilobular necrosis, frequent regenerative signs, high transaminases and detectable HP. Rats recovered after 7 days in a similar fashion as with DMNA. At 400 mg thioacetamide kg-1 body weight, necrotic areas and regeneration zones were more widespread and transaminases and HP higher after 48 h than with the lower dose. On account of the differing modes of action of DMNA and thioacetamide in rat liver, it is proposed that the appearance of HP activity in plasma could be related to the regenerative process that follows hepatotoxic damage.

Identification of a liver growth factor as an albumin-bilirubin complex.

We have reported the purification and characterization of a protein that behaves as a liver growth factor, showing activity either in vivo or in vitro [Díaz-Gil et al. (1986) Biochem. J. 235, 49-55]. In the present paper, we identify this liver growth factor (LGF) as an albumin-bilirubin complex. This conclusion is supported by the results of chemical and spectroscopic characterization of this protein as well as by experiments in vivo. Incubation of albumin isolated from normal rats with bilirubin/albumin molar ratios (r) resulted (when r = 1 or 2) in a complex with liver DNA synthesis promoter activity identical with that of LGF. The exact amount of bilirubin bound to albumin was assessed by fluorescence and c.d. spectra. This albumin-bilirubin complex showed the same dose-dependence profile as LGF either at low or high dose of protein injected per mouse. Both LGF and albumin-bilirubin complex produced similar increases in the mitotic index of mouse hepatocytes in vivo. A new mechanism for the onset of the hepatic regenerative process is proposed.

Serum and cerebrospinal fluid antibodies against myelin basic protein and their IgG subclass distribution in multiple sclerosis.

IgG class antibodies reactive with myelin basic protein (MBP) were determined by enzyme-linked immunosorbent assay (ELISA) in serum and cerebrospinal fluid (CSF) of 37 patients with multiple sclerosis and a control group of 32 patients with tension headache or psychoneurosis. Using standardised amounts of IgG from CSF and serum in ELISA, significantly higher mean antibody levels were found in CSF as well as in serum from the patients with multiple sclerosis. Ten (27%) of the multiple sclerosis CSF samples and 15 (41%) of the multiple sclerosis sera revealed anti MBP antibody levels exceeding 2 SD of the control group. Seven patients (19%) showed exclusive or higher levels of anti MBP antibodies in CSF, suggesting synthesis within the central nervous system. Analysis by ELISA for IgG subclasses of anti MBP antibodies revealed that they were restricted to IgG 1 in four patients and IgG 3 in one.

Liver DNA synthesis promoter activity detected in human plasma from subjects with hepatitis.

A liver DNA synthesis promoter activity was detected in human plasma from subjects with hepatitis. The assay procedure consisted of intraperitoneal injection into mice of aliquots of plasma, previously chromatographed on Sephadex G-25. After 24 hr, [3H]thymidine was injected and its incorporation into liver DNA measured. The increase in [3H]thymidine uptake of injected mice was not detected in those administered plasma from normal subjects (basal [3H]thymidine incorporation was that corresponding to saline-injected mouse values). At a maximal effective dose (0.3 mg protein per mouse), plasma from subjects with hepatitis increased the mitotic index of mouse liver hepatocytes; at the same dose, plasma from normal subjects had no effect. This DNA synthesis promoter activity appears to be a protein, as it is sensitive to trypsin digestion and heat.

Purification of a liver DNA-synthesis promoter from plasma of partially hepatectomized rats.

A protein was isolated from plasma of partially (70%) hepatectomized rats that, injected in mice, increases the uptake of [3H]thymidine by liver DNA by 200-300% over that by injected control saline. The purification procedure consists essentially of three chromatography steps, employing Sephadex G-75, DEAE-cellulose and hydroxyapatite. The hepatic promoter (HP) preparation shows a single band in SDS/polyacrylamide (15%)-gel electrophoresis (silver stained), with an Mr of 64 000; its activity is suppressed by trypsin or pepsin and is unaffected by deoxyribonuclease or ribonuclease. On injection into mice (150 ng/mouse), it increases the mitotic index of the liver. It shows organ-specificity, acting on liver but not on spleen, kidney, lung or brain. In primary liver cultures, it produces an increase in uptake of [3H]thymidine into DNA in the range 1-10 ng/ml. In this system in vitro, it increases the uptake of 22Na+ immediately after addition.

[Diminution of the rate of growth of HeLa cells caused by thioproline (Tp) and 2-aminothiazoline HCl (2-AT). Effect of L-proline]. / Disminución de la velocidad de crecimiento de células HeLa causada por tioprolina (Tp) y 2-amino-tiazolina-HCl (2-AT). Efecto de L-prolina.

Some aspects of the effect of thioproline and 2-amino-thiazoline-HCl on HeLa cell cultures are studied. Both drugs, at 1 mM concentrations, produce morphological changes that become stabilized after four days. The morphology changes depend on the microtubules, microfilaments, and de novo protein synthesis. Both drugs diminish the growth rate of HeLa cells, the effect being partially reverted by high concentrations (2 mM) of L-proline.